United States      Prevention, Pesticides     EPA712-C-98-084
         Environmental Protection   and Toxic Substances     January 1998
         Agency       (7101)
&EPA   Fate, Transport and
         Transformation Test
         OPPTS 835.3200

     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

     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, 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

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

     (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

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.

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-

     (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

     (A) One or two containing test substance and inoculum (test suspen-

     (B) One or two containing inoculum alone (inoculum blank).

     (C) One containing reference compound and inoculum  (procedure

     (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).

     (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-

     (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


     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-

     (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.

     (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

     (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.




  -y 70

  Z 60


  5 50

  a 40

  O 30




                                •—*  PEPTONE

                                i^^r  ALIPHATIC AMINE

                                •—•  GLYCOLETHER
                            TIME (DAYS)












                             TIME (DAYS)