United States Prevention, Pesticides EPA712-C-08-007
Environmental Protection And Toxic Substances October 2008
Agency (7101)
4>EPA Fate, Transport and
Transformation Test
Guidelines
OPPTS 835.3420
Anaerobic
Biodegradability of
Organic Compounds in
Digested Sludge: By
Measurement of Gas
Production
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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 835.3420: Anaerobic biodegradability of organic compounds
in digested sludge: by measurement of gas production.
(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. This OPPTS test guideline is based on the OECD
Guideline for the Testing of Chemicals no. 311, Anaerobic biodegradability of
organic compounds in digested sludge: by measurement of gas production,
(adopted 23 March 2006), available from Source OECD at
http://masetto.sourceoecd.org/.
(b) Purpose. This guideline describes a screening method for the
evaluation of potential anaerobic biodegradability of organic chemicals in typical
anaerobic digesters. Sludge is exposed to the test substance for up to 60 days,
which is longer than the normal sludge retention time (25 to 30 days) in anaerobic
digesters, though at industrial sites retention times may be much longer. Because
a diluted sludge is used with a relatively high concentration of test substance and
the duration of the test is longer than typical retention times, the conditions of the
test do not necessarily correspond to the conditions in real anaerobic digesters.
The objective is to provide a screening-level indication of methanogenic
anaerobic biodegradation potential. No inferences about anaerobic biodegradation
with other electron acceptors (e.g. sulfate or nitrate) can be made based on this
test. Moreover, the test is not necessarily applicable to other anoxic environmental
compartments even if methanogenic.
(c) Overview—(1) Various proportions of water-insoluble chemicals, as
well as of those that adsorb onto sewage solids, are treated aerobically, since they
are present in settled sewage. However, a large fraction of these chemicals is
bound to the primary settled sludge, which is separated from raw sewage in
settlement tanks before the settled, or supernatant, sewage is treated aerobically.
The sludge, also containing some of the soluble compounds in the interstitial
liquid, is then passed to heated digesters for anaerobic treatment. There are a
number of screening tests for assessing aerobic biodegradability of organic
chemicals (OECD Test Guidelines 301 A-F; 302 A-C; 303 A) (see paragraphs
(1)(1) and (1)(2) of this guideline), and the results of applying these have been
successfully used to predict the fate of chemicals in aerobic environments
including wastewater treatment. However, as yet there are no tests in this series
for assessing biodegradability under methanogenic conditions in anaerobic
digesters. This test is targeted to fill this gap.
(2) Respirometric techniques that measure the amounts of gas produced,
mainly methane (CH/t) and carbon dioxide (CO2) under anaerobic conditions,
have been used successfully for assessing anaerobic biodegradability. Birch et al.
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(see paragraph (1)(3) of this guideline) reviewed these procedures and concluded
that the work of Shelton and Tiedje (see paragraph (1)(4) of this guideline), based
on earlier studies (see paragraphs (1)(5), (1)(6) and (1)(7) of this guideline), was the
most comprehensive. The method (see paragraph (1)(4) of this guideline), which
was further developed by others (see paragraph (1)(8) of this guideline) and has
become the American standard (see paragraphs (1)(9) and (1)(10) of this
guideline), did not resolve problems related to the differing solubilities of CC>2
and CH4 in the test medium and to the calculation of the theoretical gas
production of a test substance. The ECETOC report (see paragraph (1)(3) of this
guideline) recommended the additional measurement of the dissolved inorganic
carbon (DIG) content of the supernatant liquid, which made the technique more
widely applicable. The ECETOC method was subjected to an international
calibration exercise (or ring test) and became the ISO Standard, ISO 11734 (see
paragraph (1)(11) of this guideline).
(3) Predictions from the results of this test cannot be made as convincingly
as they can be made in the case of aerobic biodegradation. The evidence accrued
on the behavior of test substances in ready aerobic tests and in simulation tests
and the aerobic environment is sufficient to be confident that there is a
connection, but little similar evidence exists for the anaerobic environment.
Complete anaerobic biodegradation can be assumed to occur if 75%-80% of
theoretical gas production is achieved. The high ratios of chemical-to-biomass
used in these tests mean that a chemical that passes is more likely to be degraded
in an anaerobic digester. Additionally, substances that fail to be converted to gas
in the test may not necessarily persist at more environmentally realistic substance-
to-biomass ratios. Also, other anaerobic reactions occur by which substances may
be at least partially degraded, e.g. by dechlorination, but this test does not detect
such reactions. However, by applying specific analytical methods for determining
the test substance, its disappearance (see paragraph (j)(5) of this guideline) may
be monitored.
(d) Principle of the test. (1) Washed digested sludge, containing low
(<10 mg/L) concentrations of inorganic carbon (1C), is diluted about ten-fold to a
total solids concentration of 1 g/L to 3 g/L and incubated at 35 ± 2°C in sealed
vessels with the test substance at 20 to 100 mg C/L for up to 60 days. Allowance
is made for measuring the activity of the sludge by running parallel blank controls
with sludge inoculum in the medium but without test substance. Digested sludge is
a mixture of the settled phases of sewage and activated sludge, which have been
incubated in an anaerobic digester at about 35°C to reduce biomass and odor problems
and to improve the dewater-ability of the sludge. It consists of an association of anaerobic
fermentative and methanogenic bacteria producing carbon dioxide and methane (see
paragraph (1)(10)).
(2) The increase in headspace pressure in the vessels resulting from the
production of carbon dioxide and methane is measured. Much of the CO2
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produced will be dissolved in the liquid phase or transformed into carbonate or
hydrogen carbonate under the conditions of the test. This inorganic carbon is
measured at the end of the test.
(3) The amount of carbon (inorganic plus methane) resulting from the
biodegradation of the test substance is calculated from the net gas production and
net 1C formation in the liquid phase in excess of blank control values. The extent
of biodegradation is calculated from total 1C and methane-C produced as a
percentage of the measured or calculated amount of carbon added as test
substance. The course of biodegradation can be followed by taking intermediate
measurements of gas production only. Additionally the primary biodegradation
can be determined by specific analyses at the beginning and end of the test.
(e) Information on the test substance. The purity, water solubility,
volatility and adsorption characteristics of the test substance should be known to
enable correct interpretation of results to be made. The organic carbon content (%
w/w) of the test substance needs to be known either from its chemical structure or
by measurement. For volatile test substances, a measured or calculated Henry's
law constant is helpful in deciding whether the test is applicable. Information on
the toxicity of the test substance to anaerobic bacteria is useful in selecting an
appropriate test substance concentration, and for interpreting results showing poor
biodegradability. It is recommended to include the inhibition control unless it is
known that the test substance is not inhibitory to anaerobic microbial activities:
see paragraph (i)(9) of this guideline and ISO 13641-1 (paragraph (1)(12) of this
guideline).
(f) Applicability of the method. The test may be applied to water-soluble
chemicals; it may also be applied to poorly soluble and insoluble chemicals,
provided that a method of exact dosing is used (e.g. see ISO 10634; paragraph
(1)(13) of this guideline). In general, a case-by-case decision is necessary for
volatile substances. Special steps may have to be taken, for example, to avoid
releasing gas during the test.
(g) Reference substances. To check the procedure, a reference substance
is tested by setting up appropriate vessels in parallel as part of normal test runs.
Phenol, sodium benzoate and polyethylene glycol 400 are examples and would be
expected to be degraded by more than 60% theoretical gas production (i.e.
methane and inorganic carbon) within 60 days (see paragraphs (1)(3) and (1)(14)
of this guideline).
(h) Reproducibility of test results. (1) In an international ring test (see
paragraph (1)(15) of this guideline) there was good reproducibility in gas pressure
measurements between triplicate vessels. The relative standard deviation
(coefficient of variation, COV) was mainly below 20%, although this value often
increased to >20% in the presence of toxic chemicals or towards the end of the 60
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d incubation period. Higher deviations were also found in vessels of volume <150
ml. Final pH values of the test media were in the range 6.5-7.0.
Table 1. Ring test results for two test substances.
Test
Substance
Palmitic acid
Polyethylene
Glycol 400
Total
data
36
38
Mean
degradation
(for all data)
68.7 ±30.7
79.8 ±28.0
Relative
Standard
deviation
(for all
data)
fO/\
(/o)
45
35
Valid
data
n2
27
29
Mean
degradation
(of valid
data) (%)
72.2 ±18.8
77.7 ±17.8
Standard
deviation
(of valid
data) (%)
26
23
Data>60%
degradation
in valid
tests n3
19 = 70%*
24 = 83 %*
* Proportion of n2
(2) Table 1 presents ring test results for two substances. The coefficients
of variation of the mean for all values obtained with palmitic acid and
polyethylene glycol 400 were as high as 45% (n = 36) and 35% (n = 38),
respectively. When values of <40% and >100% were omitted (the former being
assumed to be due to suboptimal conditions, the latter due to unknown reasons),
the COVs were reduced to 26% and 23%, respectively. The proportions of valid
values attaining at least 60% degradation were 70% for palmitic acid and 83% for
polyethylene glycol 400. The proportions of the percentage biodegradation
derived from DIG measurements were relatively low but variable. For palmitic
acid the range was 0-35%, mean 12%, with COV of 92%; and for
polyethyleneglycol 400 the range was 0-40%, mean 24%, with COV of 54%.
(i) Description of the test method—(1) Apparatus, (i) The usual
laboratory equipment and that listed in paragraphs (i)(l)(ii) through (i)(l)(iv) of
this guideline are used:
(ii) Incubator - spark-proof and controlled at 35°C ± 2°C;
(iii) Pressure-resistant glass test vessels of an appropriate nominal size,
each fitted with a gaslight septum, capable of withstanding about 2 bar of
pressure. The recommended size is 0.1 liter to 1 liter. The headspace volume should
be about 10% to 30% of the total volume. If biogas is released regularly, about
10% headspace volume is appropriate, but if the gas release is made only at the
end of the test 30% is appropriate. Glass serum bottles, of nominal volume 125
ml, total volume ca. 160 ml, sealed with serum septa and crimped aluminum
rings, are recommended when the pressure is released at each sampling time. The
use of gas-tight silicone septa is recommended. It is further recommended that the gas-
tightness of caps, especially butyl rubber septa, be tested, because several commercially
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available septa are not sufficiently gas-tight against methane and some septa do not stay
tight when they are pierced with a needle under the conditions of the test.
(iv) Pressure-measuring device adapted to enable measurement and
venting of the gas produced; for example, a hand-held precision pressure meter
connected to a suitable syringe needle. A 3-way gas-tight valve facilitates the
release of excess pressure (see Figure 1). It is necessary to keep the internal
volume of the pressure transducer tubing and valve as low as possible, so that
errors introduced by neglecting the volume of the equipment are insignificant.
Figure 1. Example of an apparatus to measure biogas production by gas
pressure
Pressure
meter
\xvs
3-way gas-tight valve
Syringe needle
Gaslight seal (crimp cap and septum)
Head space
Digested sludge inoculum (Vf)
(test vessels in an environment of 35°C ± 2°C)
(A) The device should be used and calibrated at regular intervals,
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according to the manufacturer's instructions. If a pressure meter of the prescribed
quality is used (e.g. capsulated with a steel membrane), no calibration is necessary
in the laboratory. The accuracy of the calibration can be checked at the laboratory
with a measurement at 1 x 105 Pa against a pressure meter with a mechanical
display. When this point is measured correctly, the linearity will also be unaltered.
If other measurement devices are used (without certified calibration by the
manufacturer), calibration is recommended over the total range at regular
intervals.
(B) The pressure readings are used directly to calculate the amount of
carbon produced in the headspace (see paragraphs (k)(l)(i), (k)(2)(i) and (k)(2)(ii)
of this guideline). Alternatively, the pressure readings may be converted to
volumes (at 35°C, atmospheric pressure) of gas produced using a conversion
graph. This graph is constructed from data obtained by injecting known volumes
of nitrogen gas into a series of test vessels (e.g. serum bottles) at 35° +/-2°C, and
recording the resulting stabilized pressure readings, as shown below in paragraph
(i)(2) of this guideline. The calculation is shown in the Note in paragraph
(k)(2)(ii) of this guideline.
(v) Carbon analyzer, suitable for the direct determination of inorganic
carbon in the range of 1 mg/L to 200 mg/L;
(vi) Syringes of high precision for gaseous and liquid samples;
(vii) Magnetic stirrers and followers (optional);
(viii) Glove box (recommended).
(2) Conversion of the pressure meter. The pressure meter readings may
be related to gas volumes by means of a standard curve produced by injecting
known volumes of air at 35°C ± 2°C into serum bottles containing a volume of
water equal to that of the reaction mixture, PR, as in paragraphs (i)(2)(i) through
(i)(2)(viii) of this guideline:
(i) Dispense PR mL aliquots of water, kept at 35°C ± 2°C, into five serum
bottles. Seal the bottles and place in a water bath at 35°C for 1 hour to equilibrate;
(ii) Switch on the pressure meter, allow to stabilize, and adjust to zero;
(iii) Insert the syringe needle through the seal of one of the bottles, open
the valve until the pressure meter reads zero and close the valve;
(iv) Repeat the procedure with the remaining bottles;
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(v) Inject 1 mL of air at 35°C ± 2°C into each bottle. Insert the needle (on
the meter) through the seal of one of the bottles and allow the pressure reading to
stabilize. Record the pressure, open the valve until the pressure reads zero and then
close the valve;
(vi) Repeat the procedure for the remaining bottles;
(vii) Repeat the total procedure above (see paragraphs (i)(2)(i) through
(i)(2)(vi) of this guideline) using 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 8 mL, 10 mL,
12 mL, 16 mL, 20 mL and 50 mL of air;
(viii) Plot a conversion curve of pressure (Pa) against volume of air
injected (mL). The response of the instrument is linear over the range 0 Pa to 70
000 Pa, and 0 mL to 50 mL of gas production.
(3) Reagents. Use analytical grade reagents throughout.
(4) Water. Distilled or deionized water (deoxygenated by sparging with
nitrogen gas containing less than 5 |iL/L oxygen), containing less than 2 mg/L
DOC.
(5) Test medium, (i) Prepare the dilution medium to contain the
constituents in this paragraph at the stated amounts:
Anhydrous potassium dihydrogen phosphate (KH2PO4) 0.27 g
Disodium hydrogen phosphate dodecahydrate ... (Na2HPO4.12H2O) 1.12 g
Ammonium chloride (NH^C!) 0.53 g
Calcium chloride dihydrate (CaCl2.2H2O) 0.075g
Magnesium chloride hexahydrate (MgCl2.6H2O) 0.10 g
Iron (II) chloride tetrahydrate (FeCl2.4H2O) 0.02 g
Resazurin (oxygen indicator) O.OOlg
Sodium sulfide nonahydrate (Na2S.9H2O) 0.10 g
Stock solution of trace elements (optional, see paragraph (i)(6)) 10 ml
Add deoxygenated water (see paragraph (i)(4)) to 1 liter
Freshly supplied sodium sulfide should be used or it should be washed and
dried before use, to ensure sufficient reductive capacity. The test may be
performed without using a glove box. In this case, the final concentration of
sodium sulfide in the medium should be increased to 0.20 g of Na2S.9H2O per
liter. Sodium sulfide may also be added from an appropriate anaerobic stock
solution (prepared in deoxygenated water; see paragraph (i)(4) of this guideline)
through the septum of the closed test vessels as this procedure will decrease the
risk of oxidation. Sodium sulphide may be replaced by titanium (III) citrate,
which is added through the septum of closed test vessels at a final concentration
of 0.8 to 1.0 mmol/L. Titanium (III) citrate is a highly effective and low-toxicity
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reducing agent, which is prepared as follows: Dissolve 2.94 g of trisodium citrate
dihydrate in 50 ml of deoxygenated water (to result in a solution of 200 mmol/L)
and add 5 ml of a 15% (w/v) titanium (III) chloride solution. Neutralize to pH 7 ±
0.2 with mineral alkali and dispense to an appropriate vessel under a stream of
nitrogen. The concentration of titanium (III) citrate in this stock solution is 164
mmol/L.
(ii) Mix the components of the test medium except the reducing agent
(sodium sulfide or titanium citrate) and sparge the solution with nitrogen gas for
about 20 min immediately before use to remove oxygen. Then add the appropriate
volume of freshly prepared solution of the reducing agent just before use of the
medium. Adjust the pH of the medium, if necessary, with dilute mineral acid or
alkali to 7 ± 0.2.
(6) Stock solution of trace elements (optional). It is recommended that
the test medium should contain the trace elements in this paragraph to improve
anaerobic degradation processes, especially if low concentrations (e.g. Ig/L) of
inoculum are used (see paragraph (1)(11) of this guideline).
Manganese chloride tetrahydrate (MnCl2.4H2O) 50 mg
Boric acid(H3BO3) 5 mg
Zinc chloride (ZnCb) 5 mg
Copper (II) chloride (CuC^) 3 mg
Disodium molybdate dihydrate (Na2MoO4.2H2O) 1 mg
Cobalt chloride hexahydrate (CoCl2.6H2O) 100 mg
Nickel chloride hexahydrate (NiCl2.6H2O) 10 mg
Disodium selenite (Na2SeC>3) 5 mg
Add deoxygenated water (see paragraph (i)(4)) to 1 liter
(7) Test substance. Add the test substance as a stock solution, suspension,
emulsion, or directly as solid or liquid, or absorbed onto glass-fiber filter, to give
a concentration of no more than 100 mg/L as organic carbon. If stock solutions
are used, prepare a suitable solution in water prepared as in paragraph (i)(4) of
such a strength that the volume added is less than 5% of the total volume of
reaction mixture. Adjust the pH of the stock solution to pH 7 ± 0.2 if necessary.
For test substances that are insufficiently soluble in water, consult ISO 10634 (see
paragraph (1)(13) of this guideline). If a solvent is used, prepare an additional
control, with the solvent only added to the inoculated medium. Organic solvents
that are known to inhibit methane production, such as chloroform and carbon
tetrachloride, should be avoided.
(8) Reference substance. Reference substances such as sodium benzoate,
phenol and polyethylene glycol 400 have been used successfully to check the
procedure, being biodegraded by more than 60% within 60 days. Prepare a stock
solution (in deoxygenated water; see paragraph (i)(4) of this guideline) of the
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chosen reference substance in the same way as for the test substance and adjust to
pH 7 ± 0.2 if necessary.
(9) Inhibition control (conditional). In order to obtain information on the
toxicity of the test substance to anaerobic microorganisms in order to find the
most appropriate test substance concentration, add the test substance and
reference substance to a vessel containing the test medium: see paragraph (i)(5)(i)
of this guideline); also ISO 13641-1 (paragraph (1)(12) of this guideline).
(10) Digested sludge, (i) Collect digested sludge from a digester at a
wastewater treatment plant which treats predominantly domestic sewage. The
sludge should be fully characterized and its background information should be
reported (see paragraph (k)(9)(i) of this guideline). If use of adapted inoculum is
intended, digested sludge from an industrial sewage treatment plant may be
considered. Use wide-necked bottles constructed from high-density polyethylene
or a similar material, which can expand, for the collection of the digested sludge.
Add sludge to within about 1 cm of the top of the bottles and seal tightly,
preferably with a safety valve. After transport to the laboratory, the collected
sludge may be used directly or placed in a laboratory-scale digester. Release
excess biogas by opening bottles of sludge carefully. Alternatively, laboratory-
grown anaerobic sludge may be used as a source of inoculum; but its spectrum of
activity may be lower.
(ii) In order to reduce background gas production and to decrease the
influence of the blank controls, predigestion of the sludge may be considered. If
predigestion is required, the sludge should be allowed to digest without the
addition of any nutrients or substrates at 35°C ± 2°C for up to 7 days. It has been
found that predigestion for about 5 days usually gives an optimal decrease in gas
production of the blank without unacceptable increases in either lag or incubation
periods during the test phase, or loss of activity.
(iii) For test substances that are, or are expected to be, poorly
biodegradable, consider preexposure of the sludge to the test substance to obtain
an inoculum that is better adapted. In such a case, add the test substance at an
organic carbon concentration of 5 to 20 mg/L to the digested sludge and incubate
for up to 2 weeks. Wash the pre-exposed sludge carefully before use (see
paragraph (i)(l 1) of this guideline) and indicate in the test report the conditions of
the preexposure.
(11) Inoculum. Wash the sludge (see paragraph (i)(10) of this guideline)
just prior to use, to reduce the 1C concentration to less than 10 mg/L in the final
test suspension. Centrifuge the sludge in sealed tubes (e.g. 3,000 x g during 5
min) and discharge the supernatant. Suspend the resulting pellet in deoxygenated
medium (see paragraph (i)(5) of this guideline), re-centrifuge the suspension and
discharge the supernatant liquid. If the 1C has not been sufficiently lowered, the
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washing procedure of the sludge can be repeated up to two times. This does not
appear to affect the microorganisms adversely. Finally, suspend the pellet in the
requisite volume of test medium and determine the concentration of total solids
(e.g. see ISO 11923; paragraph (1)(15) of this guideline). The final concentration
of total solids in the test vessels should be in the range of 1 g/L to 3 g/L (or about
10% of that in undiluted digested sludge). Inoculum should be prepared in such a
way that the sludge has minimal contact with oxygen (e.g. use a nitrogen
atmosphere).
(j) Test procedure—(1) General. Perform the initial procedures in
paragraphs (j)(2) and (j)(3) of this guideline using techniques to keep the contact
between digested sludge and oxygen as low as practicable; for example, it may be
necessary to work within a glove box in an atmosphere of nitrogen and/or purge
the bottles with nitrogen (see paragraph (1)(4) of this guideline).
(2) Preparation of test and control assays, (i) Prepare at least triplicate
test vessels (see paragraph (i)(l)(ii) of this guideline) for the test substance, blank
controls, reference substance, inhibition controls (conditional) and pressure
control chambers (optional procedure). Additional vessels for the purpose of
evaluating primary biodegradation using test substance-specific analyses may also
be prepared. The same set of blank controls may be used for several test
substances in the same test as long as the headspace volumes are consistent.
(ii) Prepare the diluted inoculum before adding it to the vessels e.g. by the
means of a wide-mouthed pipette. Add aliquots of well-mixed inoculum (see
paragraph (i)(ll) of this guideline) so that the concentration of total solids is the
same in all vessels (between 1 and 3 g/L). Add stock solutions of the test and
reference substance after adjustment to pH 7 ± 0.2 if necessary. The test substance
and the reference substance should be added using the most appropriate route of
administration (see paragraph (i)(7) of this guideline).
(iii) The concentration of test substance as organic carbon should normally
be between 20 and 100 mg/L (see paragraph (d)(l) of this guideline). If the test
substance is toxic, the test concentration should be reduced to 20 mg C/L, or even
less if only primary biodegradation with specific analyses is to be measured. It
should be noted that the variability of the test results increases at lower test
concentrations.
(iv) For blank vessels, add an equivalent amount of the carrier used to
dose the test substance instead of a stock solution, suspension or emulsion. If the
test substance was administered using glass fiber filters or organic solvents, add to
the blanks a filter or an equivalent volume solvent that has been evaporated.
Prepare an extra replicate with test substance for the measurement of the pH
value. Adjust the pH to 7 ± 0.2, if necessary, with small amounts of dilute mineral
acid or alkali. The same amounts of neutralizing agents should be added to all the
10
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test vessels. These additions should not have to be made since the pH value of the
stock solutions of the test substance and reference substance have already been
adjusted (see paragraphs (i)(7) and (i)(8) of this guideline). If primary
biodegradation is to be measured, an appropriate sample should be taken from the
pH-control vessel, or from an additional test vessel, and the test substance
concentration should be measured using specific analyses. Covered magnets may
be added to all the vessels if the reaction mixtures are to be stirred (optional).
(v) Ensure that the total volume of liquid Vi and the volume of headspace
Vh are the same in all vessels; note and record the values of Vi and Vh. Each
vessel should be sealed with a gas septum and transferred from the glove box into
the incubator.
(3) Insoluble test substances. Add weighed amounts of substances that
are poorly soluble in water directly to the prepared vessels. When the use of a
solvent is necessary, transfer the test substance solution or suspension into the
empty vessels. Where possible, evaporate the solvent by passing nitrogen gas
through the vessels and then add the other ingredients namely diluted sludge and
deoxygenated water, as required. An additional solvent control should also be
prepared. For other methods of adding insoluble substances, ISO 10634 (see
paragraph (1)(13) of this guideline) can be consulted. Liquid test substances may
be dosed with a syringe into the completely prepared sealed vessels, if it is
expected that the initial pH will not exceed 7 ± 1; otherwise dose as described in
paragraph (i)(7) of this guideline).
(4) Incubation and gas pressure measurements, (i) Incubate the
prepared vessels at 35°C ± 2°C for about 1 hr to allow equilibration and release
excess gas to the atmosphere; for example, by shaking each vessel in turn,
inserting the needle of the pressure meter (see paragraph (i)(l)(iii) of this
guideline) through the seal and opening the valve until the pressure meter reads
zero. If at this stage, or when making intermediate measurements, the headspace
pressure is less than atmospheric, nitrogen gas should be introduced to reestablish
atmospheric pressure. Close the valve and continue to incubate in the dark,
ensuring that all parts of the vessels are maintained at the digestion temperature.
Observe the vessels after incubation for 24 to 48h. Reject vessels if the contents
of the vessels show a distinct pink coloration in the supernatant liquid, i.e. if
resazurin has changed color indicating the presence of oxygen. While small
amounts of oxygen may be tolerated by the system, higher concentrations can
seriously inhibit the course of anaerobic biodegradation. The rejection of the
occasional single vessel of a set of triplicates may be accepted, but the incidence
of more failures than this should lead to an investigation of the experimental
procedures and possibly repeating the test.
(ii) Carefully mix the contents of each vessel by stirring or by shaking for
a few minutes at least 2 or 3 times per week and soon before each pressure
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measurement. Shaking resuspends the inoculum and ensures gaseous equilibrium.
All pressure measurements should be taken quickly, since the test vessels could
be subject to lowering of temperature, leading to false readings. While measuring
pressure the whole test vessel including the headspace should be maintained at the
digestion temperature. Measure the gas pressure, for example by inserting through
the septum the syringe needle (see paragraph (i)(l)(iii) of this guideline)
connected to the pressure-monitoring meter. Care should be taken to prevent entry
of water into the syringe needle; if this occurs the wet parts should be dried and a
new needle fitted. The pressure should be measured in millibars. The gas pressure
in the vessels may be measured periodically, e.g. weekly, and (optionally) the
excess gas released to the atmosphere. Alternatively, the pressure is measured
only at the end of the test to determine the amount of biogas produced.
(iii) It is recommended that intermediate readings of gas pressure be made,
since pressure increase provides guidance as to when the test may be terminated
and allows the kinetics to be followed.
(iv) Normally the test should be ended after an incubation period of 60
days unless the biodegradation curve obtained from the pressure measurements
has reached the plateau phase before then; that is, the phase in which the maximal
degradation has been reached and the biodegradation curve has leveled out. If the
plateau value is less than 60%, interpretation is problematic because it indicates
that only part of the molecule has been mineralized or that an error has been
made. If at the end of the normal incubation period, gas is being produced but a
plateau phase obviously has not been reached, then prolonging the test should be
considered.
(5) Measurement of inorganic carbon, (i) At the end of the test, after the
last measurement of gas pressure, allow the sludge to settle. Open each vessel in
turn and immediately take a sample for the determination of the concentration
(mg/L) of inorganic carbon (1C) in the supernatant liquor. Neither centrifugation
nor filtration should be applied to the supernatant liquor, since there would be an
unacceptable loss of dissolved carbon dioxide. If the liquor cannot be analyzed
immediately after sampling, store it in a sealed vial without headspace and cooled
to about 4°C for up to 2 days. After the 1C measurement, measure and record the
pH value.
(ii) Alternatively, the 1C in the supernatant may be determined indirectly
by release of the dissolved 1C as carbon dioxide that can be measured in the
headspace. Following the last measurement of gas pressure, adjust the pressure in
each of the test vessels to atmospheric pressure. Acidify the contents of each
vessel to approximately pH 1 by adding concentrated mineral acid (e.g. H2SO4)
through the septum of the sealed vessels. Incubate the shaken vessels at 35°C ±
2°C for approximately 24 hours and measure the gas pressure resulting from the
evolved carbon dioxide by using the pressure meter.
12
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(iii) Make similar readings for the corresponding blank, reference
substance and, if included, inhibition control vessels.
(iv) In some cases, especially if the same control vessels are used for
several test substances, measurements of intermediate 1C concentrations in test
and control vessels should be considered, as appropriate. In this case, a sufficient
number of vessels should be prepared for all the intermediate measurements. This
approach is preferred to taking all samples from a single vessel. The latter can
only be done if the volume needed for DIG analysis is not deemed to be too high.
The DIG measurement should be made after measuring the gas pressure without
release of excess gas, as described in paragraphs (j)(5)(iv)(A) through
(j)(5)(iv)(D) of this guideline:
(A) Using a syringe, remove as small a volume as possible of supernatant
through the septum, without opening the vessels, and determine the 1C in the
sample;
(B) After having taken the sample the excess gas may or may not be
released;
(C) It should be taken into account that even a small decrease in the
supernatant volume (e.g. about 1%) can yield a significant increase in the
headspace gas volume (Vh);
(D) Correct the equations (in paragraph (k)(2)(ii) of this guideline) by
increasing Vh in equation 3, as necessary.
(6) Specific analyses. If primary anaerobic degradation is to be
determined, take an appropriate volume of sample for specific analyses at the
beginning and at the end of the test from the vessels containing the test substance.
If this is done, note the volumes of headspace (Vh) and of the liquid (Vi) will be
changed and take this into account when calculating the results of gas production.
Alternatively samples may be taken for specific analyses from additional test
mixtures previously set up for the purpose.
(k) Data and Reporting—(1) Treatment of results, (i) For practical
reasons, the pressure of the gas is measured in millibars (1 mbar = Ih Pa = 102 Pa;
1 Pa = 1 N/m2), the volume in liters and temperature in degrees Celsius.
(ii) Figures 2 and 3 show sample data sheets that may be helpful for
recording measurements and facilitating calculations.
13
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Figure 2. Example of data sheet for the anaerobic bio degradation test - data sheet for the test substance
Laboratory: Test substrate:
Tcsttcnpcralwc:(°C)
Caibon in test substance Cfn- .(mefl-) m,1:
Test No.:
Volume of liquid (Vs):.,. .(liters)
Day
1C (end)
pH (end)
Pi (i«t)
(rabar)
QC.I
test
(rag)
ft (t«t)
(mbar)
Qc.J
test
(rag)
jjj{(esrt)
(mtwr)
Q;»
test
(rat)
p (test)
mean
(rabar)
CK
test rawn
(rag)
ft
(Wank)
(mhar)
C*K;«
Wank
Cm)
Ps P<
(Wank) (blank)
(mbar) (mbar)
Cits Qc.ii
blank blank
tn>8) . Irai)
p (blank)
mean
(ratoar)
CK
blank
mean
(«««>
/>{aet)
test - Wank
mean
(mbar)
C*K-,B«
test -blank
mean
(rag)
4? (net)
cumulative
(mbar)
mt
liquid C^
(rag)
"h
bcadspacc
C
(ma)
«(
total Cs
(rag)
A,
Biodegrad
atiou1
(%)
A
Biodcpad
ation6
(%)
1 Carton in test vessel, *¥{rag): m, =
2 Carbon in headspace, 1% (mg) at normal incubation temperature (35 °C): mi, - 0,468ApxVb
3 Biodegradalion calculated from headspaee gas, D\, (%): j
4 Carbon in liquid, Bi^mg): mt = Cj^rtXV]
5 Total gasified carbon, ou (mg): tck + wn
s Total biodegratMen, D^ (%): D, - (ia,x 100) / FJ^.
-------
Figure 3. Example of data sheet for the anaerobic bio degradation test - data sheet for the reference substance
Laboratory: Reference substance:..., , Test No,:
Test iernpeimtun^fC) Vaiurac of headspacc (Pj:___(L) Volume of liquid (V,):... (liters)
Cartxm in reference subataaoe Ceft',,. .(mgC) mt.7; (mg)
Day
TC (cad)
pH(ei>d)
Pi (n£)
(jrtw)
Qe, L
rcf.
(i>«)
ft (ret)
(rabar)
Cie,2
rcf.
(rag)
/»j(nsf.)
(irtwr)
P(«r.)
mean
(nsbar)
Cfcj
rcf.
(rag)
cx.
tcf.mean
(rag)
jf4
(inbib.)
(mbar)
ft
(inhib.)
(robai)
!
|
\
1
1
:|
:|
Cic,4
tohJb.
(rag)
Qc,5
inhib.
(™«)
!
5
PIS
(inbib.)
(rabar)
CjC.d
inhib,
(rag)
p(iahib.)
mean
(rabar)
Ac
inhib.
meaa
(mg)
p(«f.) j 4K«f.)
reT - blank I cumulative
| (mbar)
(mbar) j
Cr.nfl 1 "*)
ret- inbib, j liquid Cw
(m) I (m)
m* A
ticatoacc Biodcgrad
C* alioii9
(rag) (%)
A
(ottIC Biodcgra4
alion11
(rag) . (%)
' Cwbon in test vessel, «v(mg); m^ =
* Carbon k headspace, wh (m|J M nonm] incubaiiMi temperature (35 T)": 04 - 0.46&&pxVh
9 Bi«tegnwlMi(M caJculaled frem headspace gas, D\, (%}: £H, • (mj,xlOO) / m,,
ls Carbon in liquW, j»j( (nig}: »i|
11 Total gasified carbon, m, (mg):
11 Total biodegraduion, Dt {%): D, - (n^x 106) /
-------
(2) Carbon in the headspace. (i) Since 1 mol of methane and 1 mol
carbon dioxide each contain 12 g of carbon, the mass of carbon in a given volume
of evolved gas may be expressed as:
m = 12 x 103 x n Equation [1]
where:
m = mass of carbon (mg) in a given volume of evolved gas;
12 = relative atomic mass of carbon;
n = number of moles of gas in the given volume.
If a gas other than methane or carbon dioxide (e.g. N2O) is generated in
considerable amounts, the formula [1] should be amended in order to describe the
possibility of effects by gases generated.
(ii) From the gas laws n may be expressed as:
pV
n = —- Equation [2]
Kl
where:
p = pressure of the gas (Pascals);
V = volume of the gas (m3);
R = molar gas constant [8.314J/(mol K)];
T= incubation temperature (degrees Kelvin).
By combination of equations [1] and [2] and rationalizing to allow for blank
control production of gas:
1 x 0. l(Ap • K) Equation [3 ]
BZi = —
RT
where:
nih = mass of net carbon produced as gas in the headspace (mg);
A/> = mean of the difference between initial and final pressures in the
test vessels minus the corresponding mean in the blank vessels
(millibars);
Vh = volume of headspace in the vessel (L);
0.1 = conversion for both newtons/m2 to millibars and m3 to liters.
Equation [4] should be used for the normal incubation temperature of 35°C (308
K):
mh = 0.468 (A/? x Vh) Equation [4]
16
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Alternative volume calculation. Pressure meter readings are converted to
mL of gas produced using the standard curve generated by plotting
volume (mL) injected versus meter reading (see paragraph (i)(2) of this
guideline). The number of moles (n) of gas in the headspace of each vessel
is calculated by dividing the cumulative gas production (ml) by 25,286
ml/mole, which is the volume occupied by one mole of gas at 35°C and
standard atmospheric pressure. Since 1 mole of CH4 and 1 mole of CC>2
each contain 12 g of carbon, the amount of carbon (m, in mg) in the
headspace (mh) is given by Equation [5]:
nih = 12 x 103 Equation [5]
Rationalizing to allow for blank control production of gas:
12QQQ>Ar = o 475AF Equation [6]
25286
where:
mh = mass of net carbon produced as gas in the headspace (mg);
AF = mean of the difference between volume of gas produced in
headspace in the test vessel and blank control vessels;
25286 = volume occupied by 1 mole gas at 35°C, 1 atmosphere.
(iii) The course of biodegradation can be followed by plotting the
cumulated pressure increase A/? (millibars) against time, if appropriate. From this
curve, identify and record the lag phase (days). The lag phase is the time from the
start of the test until significant degradation starts (for example, see Figure 4). If
intermediate samples of supernatant were taken and analyzed (see paragraph
(j)(5)(iv) of this guideline), then the total C produced (in gas plus that in liquid)
may be plotted instead of only the cumulative pressure.
17
-------
Figure 4. Example of a degradation curve (cumulative net pressure
increase)
500 -
0 10 20 30 40
Time (days)
50
60
(3) Carbon in the liquid. The amount of methane in the liquid is ignored
since its solubility in water is known to be very low. Calculate the mass of
inorganic carbon in the liquid of the test vessels using equation [7]:
mi = Cnet X V,
Equation [7]
where:
mi = mass of inorganic carbon in the liquid (mg);
Cnet = concentration of inorganic carbon in the test vessels minus that in the
control vessels at the end of the test (mg/L);
Vi =volume of liquid in the vessel (L).
(4) Total gasified carbon. Calculate the total mass of gasified carbon in
the vessel using equation [8]:
mt =
mi
Equation [8]
18
-------
where:
mt = total mass of gasified carbon (mg);
nih and mi are as defined in this paragraph.
(5) Carbon of test substance. Calculate the mass of carbon in the test
vessels derived from the added test substance using equation [9]:
mv = Ccx Vi Equation [9]
where:
mv= mass of test substance carbon (mg);
Cc = concentration of test substance carbon in the test vessel liquid (mg/L)
Vi= volume of liquid in the test vessel (L).
(6) Extent of biodegradation. (i) Calculate the percentage biodegradation
from headspace gas using equation [10] and the total percentage biodegradation
(i.e. determined from total gasified carbon) using equation [11]:
Dh = (mh I mv ) x 100 Equation [10]
A = (mt I mv ) x 100 Equation [11]
where:
Dh = biodegradation determined from headspace gas (%);
A = total biodegradation (%);
nih, mv and mt are as defined in this paragraph.
(ii) The degree of primary biodegradation is calculated from the (optional)
measurements of the concentration of the test substance at the beginning and end
of incubation, using equation [12]:
Dp = (1 - Se I Si) x 100 Equation [12]
where:
Dp = primary degradation of test substance (%);
Sj = initial concentration of test substance (mg/L);
Se = concentration of test substance at end (mg/L).
(iii) If the method of analysis indicates significant concentrations of the
test substance in the unamended anaerobic sludge inoculum, use equation [13]:
Dp = - [ 1 - (Se - Seb) / (Si - Sib) x 100 Equation [13]
where:
19
-------
Dp = corrected primary degradation of test substance (%);
Sib = initial 'apparent' concentration of test substance in blank controls
(mg/L);
Seb = 'apparent' concentration of test substance in blank control at end (mg/L).
(7) Validity of results, (i) Pressure readings should be used only from
vessels that do not show pink coloration (see paragraph (j)(4) of this guideline).
Contamination by oxygen is minimized by the use of proper anaerobic handling
techniques.
(ii) It should be considered that the test is valid if the reference substance
reaches a plateau that represents more than 60% biodegradation. This should be
reevaluated if adsorptive and insoluble reference substances are included.
(iii) If the pH at the end of the test has exceeded the range 7 ± 1 and
insufficient biodegradation has taken place, repeat the test with increased buffer
capacity of the medium.
(8) Inhibition of degradation. Gas production in vessels containing both
the test substance and reference substance should be at least equal to that in the
vessels containing only reference substance; otherwise, inhibition of gas
production is indicated. In some cases gas production in vessels containing test
substance without reference substance will be lower than that in the blank
controls, indicating that the test substance is inhibitory.
(9) Test report. The test report should include the information in
paragraphs (k)(9)(i) through (k)(9)(iii) of this guideline.
(i) Test substance: (A) Common name, chemical name, CAS number,
structural formula and relevant physical-chemical properties;
(B) Purity (impurities) of test substance.
(ii) Test conditions: (A) Volumes of diluted digester liquor (Vi) and of the
headspace (Vh) in the vessel;
(B) Description of the test vessels, the main characteristics of biogas
measurement (e.g. type of pressure meter) and of the 1C analyzer;
(C) Application of test substance and reference substance to test system:
test concentration used and any use of solvents;
(D) Details of the inoculum used: name of sewage treatment plant,
description of the source of wastewater treated (e.g. operating temperature, sludge
retention time, predominantly domestic, etc.), concentration, any information
20
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necessary to substantiate this, and information on any pretreatment of the
inoculum (e.g. predigestion, preexposure);
(E) Incubation temperature;
(F) Number of replicates.
(iii) Results: (A) pH and 1C values at the end of the test;
(B) Concentration of test substance at the beginning and end of the test, if
a specific measurement has been performed;
(C) All the measured data collected in the test, blank, reference substance
and inhibition control vessels, as appropriate (e.g. pressure in millibars,
concentration of inorganic carbon (mg/L)) in tabular form (measured data for
headspace and liquid should be reported separately);
(D) Statistical treatment of data, test duration and a diagram of the
biodegradation of test substance, reference substance and toxicity control;
(E) Percentage biodegradation of test substance and reference substance;
reasons for any rejection of the test results;
(F) Discussion of results.
(1) References.
(l)OECD (1981). 302A-C: Inherent Biodegradability and 303A-B:
Simulation Test - Aerobic Sewage Treatment. Organization for Economic
Cooperation Development, Paris.
(2)OECD (1992). Ready Biodegradability 301(A-F) and Inherent
Biodegradability: Zahn-Wellens/EMPA Test 302B. Organization for Economic
Cooperation and Development, Paris.
(3) Birch, R. R., C. Biver, R. Campagna, W.E. Gledhill, U. Pagga, J.
Steber, H. Reust and WJ. Bontinck (1989). WJ. Screening of chemicals for
anaerobic biodegradation. Chemosphere 19, 1527-1550. (Also published as
ECETOC Technical Report No. 28, June 1988).
(4) Shelton, D.R. and J.M. Tiedje (1984). General method for determining
anaerobic biodegradation potential. Appl. Environ. Microbiol. 47, 850-857.
(5) Owen, W.F., D.C. Stuckey, J.B. Healy, Jr., L.Y. Young and P.L.
21
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McCarty (1979). Bioassay for monitoring biochemical methane potential and
anaerobic toxicity. Water Res. 13, 485-492.
(6) Healy, Jr., J.B. and L.Y. Young (1979). Anaerobic biodegradation of
eleven aromatic compounds to methane. Appl. Environ. Microbiol. 38, 84-89.
(7) Gledhill, W.E. (1979). Proposed standard practice for the
determination of the anaerobic biodegradation of organic chemicals. Working
document. Draft 2 no. 35.24. American Society for Testing Materials,
Philadelphia.
(8) Battersby, N.S. and V. Wilson (1988). Evaluation of a serum bottle
technique for assessing the anaerobic biodegradability of organic chemicals under
methanogenic conditions. Chemosphere. 17, 2441-2460.
(9)ASTM El 192-92. Standard Test Method for Determining the
Anaerobic Biodegradation Potential of Organic Chemicals. American Society for
Testing and Materials, Philadelphia.
(lO)OPPTS 796.3140 (1998). Fate, Transport and Transformation Test
Guidelines, Anaerobic Biodegradability of Organic Chemicals. Office of
Prevention, Pesticides and Toxic Substances, U.S. Environmental Protection
Agency.
(11) ISO 11734 (1995). Water Quality - Evaluation of the ultimate
anaerobic biodegradation of organic compounds in digested sludge - Method by
measurement of the biogas production. International Organization for
Standardization, Geneva.
(12) ISO 13641-1 (2003). Water Quality - Determination of inhibition of
gas production of anaerobic bacteria - Part 1 General Test. International
Organization for Standardization, Geneva.
(13) 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.
(14) ISO 11923 (1997). Water Quality - Determination of suspended
solids by filtration through glass-fibre filters. International Organization for
Standardization, Geneva.
(15) Pagga, U. and D.B. Beimborn (1993). Anaerobic biodegradation test
for organic compounds. Chemosphere 27, 1499-1509.
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