United States Prevention, Pesticides EPA712-C-98-084
Environmental Protection and Toxic Substances January 1998
Agency (7101)
&EPA Fate, Transport and
Transformation Test
Guidelines
OPPTS 835.3200
Zahn-Wellens/EMPA
Test
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-0132 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from EPA's World Wide Web site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS Harmonized Test
Guidelines."
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OPPTS 835.3200 Zahn-Wellens/EMPA test.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline are 40 CFR 796.3360 Inherent
Biodegradability: Modified Zahn-Wellens Test and OECD guideline 302
B Inherent Biodegradability: Zahn-Wellens/EMPA Test.
(b) General concepts. The original Zahn-Wellens test under para-
graph (1)(7) of this guideline was adopted in 1981 as OECD Guideline
302 B for determining inherent biodegradability. Later proposals were
made by Switzerland and Germany to modify this guideline by merging
it with elements contained in a test developed by EMPA (Swiss Federal
Laboratories for Materials Testing and Research) under paragraph (1)(6)
of this guideline, hence the change in name of the test. The merged version
of the test was further changed with respect to the mineral medium used.
The medium retained is identical with that which is used in the DOC Die-
Away, CO2 Evolution, Manometric Respirometry, and Modified OECD
screening methods of for determining ready biodegradability (835.3110).
(c) Principle of the test. (1) A mixture containing the test substance,
mineral nutrients, and a relatively large amount of activated sludge in
aqueous medium is agitated and aerated at 20-25 °C in the dark or in
diffuse light for up to 28 days. Blank controls, containing activated sludge
and mineral nutrients but no test substance, are run in parallel. The biodeg-
radation process is monitored by determination of DOC (dissolved organic
carbon) (or COD (chemical oxygen demand)) in filtered samples taken
at daily or other time intervals. The ratio of eliminated DOC (or COD),
corrected for the blank, after each time interval, to the initial DOC value
is expressed as the percent biodegradation at the sampling time. The per-
cent biodegradation is plotted against time to give the biodegradation
curve.
(2) Specific analysis of the test substance may be useful in cases
where molecular changes, caused by biochemical reactions (primary bio-
degradation) are to be detected.
(d) Information on the test substance. It is necessary to know the
water solubility and vapor pressure of the test substance and it is also
advisable to know its foaming properties. The chemical structure should
be known if the measured values of DOC or COD are to be checked.
Information on the toxicity of the test substance to bacteria is useful for
selecting appropriate test concentrations and in interpreting results showing
poor biodegradability under paragraph (1)(5) of this guideline. The test is
usually performed only after failure to pass a test for ready
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biodegradability. Thus, the physical and inhibitory properties may have
already been ascertained.
(e) Applicability of the method. Chemicals which are nonvolatile
and are soluble in water to at least 50 mg DOC/L may be assessed by
this method, provided also that they do not significantly adsorb, are not
lost by foaming and do not inhibit bacteria at the concentration tested.
(f) Sensitivity. The limits of sensitivity are given by the sensitivity
of the DOC determination (normally 0.5-1 mg C/L) or the COD deter-
mination (15 mg O2/L) and also by the variability of the blank. The rel-
atively high concentration of test substance (50-400 mg DOC/L) gives
the advantage of greater analytical reliability.
(g) Reference compounds. In order to check the functional capability
of the activated sludge, a test using a reference compound of known
biodegradability should be run in parallel with each series. For this pur-
pose, ethylene glycol, diethylene glycol, lauryl sulfonate, and aniline are
recommended. Biodegradation of these compounds must reach at least 70
percent (DOC or COD) within 14 days.
(h) Reproducibility. The test has been shown to have good reproduc-
ibility in ring tests.
(i) Description of the method—(1) Apparatus, (i) Cylindrical glass
vessels with a volume of 1-5 L, each equipped with a stirrer made of
inert material rotating about 5 to 10 cm above the bottom of the vessel
(a magnetic stirrer with a 7-10 cm long rod can also be used) and a glass
tube of 2-4 mm i.d. to introduce air at about 1 cm above the bottom
of the vessel, or vessels of the same size equipped with a glass frit at
the bottom, permitting aeration and agitation.
(ii) A supply of compressed air passed through a cotton wool strainer
and a wash-bottle containing water, or from an aeration pump delivering
air free from dust, oil, and organic impurities.
(iii) Normal laboratory equipment, especially a centrifuge (capable
of at least 1,000 g), pH meter, dissolved oxygen measuring apparatus, and
membrane filters (pore size 0.2-0.45 (im).
(iv) Analytical equipment for determining DOC (refer to paragraph
(1)(1) of this guideline) or COD (refer to paragraph (1)(2) of this guideline).
(2) Reagents. Use analytical grade reagents throughout.
(3) Water. Deionized or distilled water, free from inhibitory con-
centrations of toxic substances (e.g. Cu2+ ions) is used. It should contain
only minimal amounts of organic carbon so that high blank values are
eliminated. Contamination may result from inherent impurities and also
from the ion-exchange resins and lysed materials from bacteria and algae.
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For each test series use only one batch of water, previously checked by
DOC analysis.
(4) Stock solutions for mineral medium, (i) Prepare the following
stock solutions:
(A) Dissolve 8.5 g potassium dihydrogen orthophosphate,
21.75 g dipotassium hydrogen orthophosphate, K^HPCU, 33.4 g disodium
hydrogen orthophosphate dihydrate, Na2HPO4 2H2O, and 0.5 g ammonium
chloride, NH4C1, in water and make up to 1 L. The pH of the solution
should be 7.4.
(B) Dissolve 27.5 g calcium chloride, anhydrous, CaCk or 36.4 g
calcium chloride dihydrate, CaCl2-2H2O, in water and make up to 1 L.
(C) Dissolve 22.5 g magnesium sulphate heptahydrate, MgS04-7H2O,
in water and make up to 1 L.
(D) Dissolve 0.25 g iron(III) chloride hexahydrate, FeCl3-6H2O, in
water and make up to 1 L.
(ii) Note: In order to avoid having to prepare this solution imme-
diately before use, add one drop of concentrated HC1 or add 0.4 g
ethylenediaminetetraacetic acid (EDTA, disodium salt) per liter. If a pre-
cipitate forms in a stock solution, replace with a freshly made solution.
(5) Preparation of mineral medium. Mix 10 mL of solution (A)
with 800 mL water, add 1 mL each of solutions (B), (C) and (D) and
make up to 1 L.
(6) Inoculum. Collect a fresh sample of activated sludge from a sew-
age treatment works (BOD5 of effluent should be <25 mg/L) and wash
twice with mineral medium or tap water. Separate the sludge by
centrifuging for 3-5 min at about 1,000 g or by allowing the sludge to
settle. In special cases, to get as many different species and strains as
possible, mix samples from different sources (e.g. other treatment plants,
soil extracts, river water, etc.) and treat the mixture as above. Use the
sludge within 6 h of sampling, otherwise disperse it in mineral medium
and aerate until required. Check the activity of the sludge with the proce-
dural control using a reference compound, as described under paragraph
(i)(7)(iv) of this guideline.
(7) Preparation of vessels, (i) Before starting the test, make certain
with appropriate methods that no inhibition of sludge occurs at the chosen
concentration of test substance if this is not already known (see paragraphs
(1)(3) and (4) of this guideline). If an inhibitory effect is found, reduce
the concentration of test substance to a level which is unlikely to be inhibi-
tory.
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(ii) To an appropriate number of test vessels introduce 500 mL min-
eral medium and the appropriate amounts of test substance and inoculum
to reach respectively between 50 and 400 mg DOC/L (between 100 and
1,000 mg COD/L) and 0.2-1.0 g dry matter/L in the final volume. Ensure
that the ratio between inoculum and test compound (as DOC) lies between
2.5:1 and 4:1. Make up to the required volume with mineral medium. The
final volume, between 1 and 5 L, depends on the number of samples to
be taken for DOC or COD determinations and the volumes necessary for
the analytical procedures. Normally a volume of 2 L is satisfactory.
(iii) Set up one or two blank vessels in parallel to contain only acti-
vated sludge and mineral medium with volumes identical to those of the
test suspensions.
(iv) Also, set up one vessel in parallel with each test series as a proce-
dural control, using one of the reference compounds in place of the test
substance. If information on abiotic degradation is required, a sterile
uninoculated solution of the test chemical can be prepared.
(8) Number of vessels, (i) The following vessels are used in a typical
run:
(A) One or two containing test substance and inoculum (test suspen-
sion).
(B) One or two containing inoculum alone (inoculum blank).
(C) One containing reference compound and inoculum (procedure
control).
(ii) It is mandatory to follow DOC in the test suspension and
inoculum blanks in parallel. It is advisable to follow DOC in the other
vessel in parallel as well but this may not always be possible.
(j) Procedure. (1) For practical reasons, do not start the test imme-
diately before a week-end. Run the test, normally for up to 28 days, in
the dark or in diffuse light at 20-25 °C. Aerate the suspensions with puri-
fied, humidified air and, if necessary, stir to ensure that sludge does not
settle and that the concentration of dissolved oxygen does not fall below
mg/L. Check the pH value at regular intervals (e.g. on each day of sam-
pling) and adjust to pH 6.5-8.0 with NaOH (40 g/L) or H2S04
(50 g/L) if necessary.
(2) Sampling. Follow the biodegradation of the test substance by de-
termining the DOC or COD in samples of suspension taken:
(i) At 3 h + 30 min after addition of the test substance in order to
estimate any adsorption by the activated sludge (see example in Figure
1. under paragraph (m)(l) of this guideline).
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(ii) On at least four occasions in the interval between the 1st and
27th day.
(iii) On the 27th and 28th days, or, if the plateau is attained in less
than 28 days, on the last 2 days of the test run.
(iv) The volume of sample taken depends on the type of carbon ana-
lyzer to be used. Additional sampling may be necessary in order to de-
scribe the reaching of the plateau or if adaptation is to be followed.
(v) Replace losses due to evaporation immediately prior to each sam-
pling.
(4) Adaptation, (i) If adaptation (see curve 1, Figure 2. under para-
graph (m)(2) of this guideline) is to be followed, carry out analyses for
DOC or COD at relatively short intervals (e.g. daily). Prolong the test
beyond 28 days if adaptation occurs in the final days of the test period.
(ii) If more detailed knowledge of the behavior of the adapted sludge
is needed, reexpose the same activated sludge to the test substance. To
do this, stop aeration and agitation and allow the sludge to settle. Draw
off the supernatant liquid, refill the vessel to the original volume with
mineral medium, stir for 15 min and repeat this operation once more. Al-
ternatively, isolate the sludge by centrifuging (refer to paragraph (i)(6) of
this guideliine). Repeat the test using the recovered sludge, which may
be augmented with fresh sludge if insufficient recovered sludge is available
to yield 0.2-1 g dry matter/L.
(5) Analytical methods, (i) Filter the samples of sludge suspensions
(test, blank, and procedure control) as soon as they are taken, discarding
the first 5 mL of filtrate. Use either carefully washed paper filters or mem-
brane filters, which are suitable if they neither release nor adsorb organic
compounds. Otherwise wash the membranes 3 times in deionized or dis-
tilled water at about 60 °C, and store in water. Sludges which are difficult
to separate by filtration should be be separated by centrifugation or other
suitable separation techniques.
(ii) Determine the DOC or COD in duplicate in the filtered or
centrifuged samples by any suitable methods e.g. refer to paragraphs (1)(1)
and (2) of this guideliine. If primary bio degradation is to be followed,
use specific analyses, e.g. UV spectroscopy, in addition to DOC or COD.
If the filtrates cannot be analyzed on the day of sampling, store at
2-4 °C for a maximum of 48 h, or at -18 °C for longer periods. Storage
for long periods is not recommended.
(k) Data and reporting—(1) Treatment of results, (i) Calculate the
percent degradation at time t from
Dt = [1 - (Ct - CB)/CA - CBA)] X 100
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where:
Dt = percent degradation at time t; CA = concentration of DOC or
COD in the test suspension measured after 3 h + 30 min of incubation,
expressed as milligrams per liter; Ct = mean concentration of DOC or
COD in the test suspension at time t, expressed as milligrams per liter;
CBA = mean concentration of DOC or COD in the blanks measured after
3 h + 30 min of incubation, expressed as milligrams per liter; CB = mean
concentration of DOC or COD in the blanks at time t, expressed as milli-
grams per liter.
(ii) Carry out the same calculation for the reference compound. Dis-
play the course of biodegradation graphically (as in Figures 1. and 2. under
paragraph (m) of this guideline) and record all results on data sheets.
(2) Validity and interpretation, (i) The test is considered valid if
the procedural control shows the removal of the reference compound by
at least 70 percent within 14 days and if the removal of DOC (or COD)
in the test suspension took place relatively gradually over days or weeks,
since this indicates biodegradation.
(ii) Physicochemical adsorption can, in some cases, play a role and
this is indicated when there is complete or substantial removal in the first
3 h and the difference between blanks and test solutions remains at an
unexpected low value. In such cases additional information is obtained
from a comparison between the 3-h value, the expected initial value cal-
culated from the amount of test substance added and the value measured
before the inoculum is added. If a more precise distinction between bio-
degradation (or partial degradation) and adsorption is to be drawn, carry
out further testing, preferably running a respirometric test for ready bio-
degradation, using the supernatant of the acclimatized sludge as inoculum.
(iii) Low and 0-values of removal of the test substance may be due
to its inhibition of bacteria; eliminate this possibility by testing for inhibi-
tion at the concentration used if this has not already been done (refer to
paragraph (i)(7)(i) of this guideliine.
(3) Test report. The test report must include the following informa-
tion:
(i) Test substance: (A) Physical nature and, where relevant, physico-
chemical properties.
(B) Identification data.
(ii) Inoculum: Source, concentration, status of adaptation.
(iii) Test conditions: (A) Analytical methods used.
(B) Procedure control and compound used in the control.
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(iv) Results: (A) Biodegradation curve.
(B) Toxicity evaluations.
(C) The degree of biodegradation attained at the end of the test after
28d, or earlier if complete degradation is attained in less than 28 days,
as "inherent biodegradability in the static test after x days."
(D) Any significant difference between the DOC (or COD) in the
first sample at 3 h after starting the test and the value calculated from
the amount of test compound added as "adsorbed by the activated
sludge."
(E) The adaptation phase (days), the biodegradation phase (days) and
the endpoint of biodegradation reached after x days as identified from the
biodegradation curve.
(v) Discussion of the results.
(1) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) DIN 38409, Teil 3. Bestimmung des gelosten organischen
Kohlenstoffgehaltes (DOC) (1983).
(2) ISO Standard 6060 (1986). Water Quality-Determination of
Chemical Oxygen Demand.
(3) ISO Standard 8192. Water Quality-Test for inhibition of oxygen
consumption by activated sludge (1986).
(4) OECD. Activated Sludge, Respiration Inhibition Test. Test Guide-
line 209, Paris (1984).
(5) Reynolds, L. et al. Evaluation of the toxicity of substances to
be assessed for biodegradability. Chemosphere 16:2259 (1987). Chemiker
Zeitung 98:228-232 (1974).
(6) Schefer W. and Walchli O. Priifung der biologischen
Eliminierbarkeit organisch-chemischer Abwasser-Inhaltstoffen. Zur
Wasser- undAbwasserforschung 13, 205-209 (1980).
(7) Zahn R. und Wellens H. Ein einfaches Verfahren zur Prufung
der biologischen Abbaubarkeit von Produkten und Abwasserinhaltsstoffen.
(m) Figures.
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FIGURE i.—EXAMPLES OF BIODEGRADATION CURVES
100
90
80
-y 70
Z 60
2
5 50
a 40
o
O 30
20
10
0
SUBSTANCES:
•—* PEPTONE
i^^r ALIPHATIC AMINE
•—• GLYCOLETHER
10
TIME (DAYS)
FIGURE 2.—EXAMPLE OF SLUDGE-ADAPTATION
O
<
cc
o
Ul
o
100
90
80
70
60
50
40
30
20
10
SUBSTANCE
POLYVINYLALCOHOL
ADAPTED
NON ADAPTED
10
TIME (DAYS)
ADAPTATION PHASE —^DEGRADATION PHASE-f ENDPOINT
15
8
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