United States
Environmental Protection
Office of Chemical Safety
and Pollution Prevention
January 2012
        Ecological Effects
        Test Guidelines

        OCSPP 850.3300:
        Modified Activated
        Sludge, Respiration
        nhibition  Test


     This guideline is one of a series of test guidelines established by the United States
Environmental Protection Agency's Office of Chemical Safety and Pollution Prevention
(OCSPP) for use in testing pesticides and chemical substances to develop data for
submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601,
et seq.), 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).
Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides and Toxic
Substances (OPPTS). To distinguish these guidelines from guidelines issued by other
organizations, the numbering convention adopted in 1994 specifically included OPPTS as
part of the guideline's number.  Any test guidelines developed after April 22, 2010 will use
the new acronym (OCSPP)  in their title.

     The OCSPP harmonized test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform regulatory decisions
under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the
test, and is also  used  by EPA, the public, and the companies that are subject to data
submission requirements under TSCA, FIFRA, and/or the FFDCA.  As a guidance
document, these guidelines are not binding on either EPA or any outside parties, and the
EPA may depart from the guidelines where circumstances warrant and without prior notice.
At places in this  guidance, the Agency uses the word "should."  In this guidance, the use of
"should" with regard to an action means that the action is recommended rather than
mandatory. The procedures contained in this guideline are strongly recommended for
generating the data that are the subject of the guideline, but EPA recognizes that departures
may be appropriate in specific situations. You may propose alternatives to the
recommendations described in these guidelines, and the Agency will assess them for
appropriateness on a  case-by-case basis.

     For additional information about these test guidelines and to access these guidelines
electronically, please go to http://www.epa.gov/ocspp and select "Test Methods &
Guidelines" on the left side navigation menu.  You may also access the guidelines in
http://www.requlations.qov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-
0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.

OCSPP 850.3300:  Modified activated sludge, respiration inhibition test.

(a) Scope—

       (1) Applicability. This guideline is intended to be used to help develop data to submit to
       EPA under the Toxic Substances Control Act (TSCA)  (15  U.S.C. 2601, et  seq.), the
       Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C.  136, et  seq.), and
       the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 346a).

       (2) Background.  The source materials used in developing this harmonized OCSPP test
       guideline include the OPPT guideline under 40 CFR 795.170 (proposed in the Federal
       Register of May 15, 1986 (51 FR 17880)); and OECD guideline 209, Activated Sludge,
       Respiration Inhibition Test.  This guideline was formerly Public Draft OCSPP 850.6800
       (April, 1996).

(b) Purpose. This guideline is intended for use in developing  data on the effect of chemical
substances and mixtures ("test chemicals" or "test substances") subject to environmental effects
test regulations.  This guideline describes procedures for measuring the effect of test substances
upon  the respiration  rate  of microorganisms  found in  sewage treatment plants.    The
Environmental Protection Agency  will use data from  this test to assess the hazard  a test
substance may present in the environment.

(c) Definitions.  The definitions in the OCSPP 850.3000 guideline apply to this  guideline.  In
addition, the more specific definition in this paragraph also applies:

       Respiration rate refers  to  the oxygen consumption of aerobic  sludge or wastewater
       microorganisms, generally expressed as milligrams of oxygen  (Q^) per liter per hour.

(d) General considerations—

       (1) Summary of the test.  The method described in this test guideline assesses the effect
       of a test substance on microorganisms by measuring the respiration rate under defined
       conditions in  the presence of different concentrations of the test substance.  The method
       is based  on  that  described by the Ecological and Toxicological Association of the
       Dyestuffs  Manufacturing Industry (see paragraph (j)0) °f this  guideline),  in  which
       activated sludge obtained from a sewage treatment plant is used as the microbial source.
       The purpose of this test guideline is to provide a rapid screening method whereby test
       substances  which  may  adversely affect  aerobic  microbial  treatment plants can  be
       identified and to indicate suitable non-inhibitory  concentrations of test substances to be
       used in biodegradability tests.  The test is designed to  determine the quantity of test
       substance required to cause a 50 percent inhibition or reduction (ICso) in respiration.
       Note historically in OCSPP pesticide and industrial chemical guidelines the term ECX was
       used  to  cover  both the   current  OCSPP  850.3000  guideline  definition  of ECX
       (concentration where x percent (x%) of the population exhibit the effect (e.g.,  survival))
       and 1C* (concentration resulting in a x% decrease or inhibition effect on an attribute of
       the test population (e.g., respiration)).

       (2) General test guidance.  General guidance applicable to this guideline can be found in
       the OCSPP 850.3000 guideline.
                                       Page 1 of 12

       (3)  Range-finding  test.   A  range-finding test  is usually conducted to establish the
       appropriate test substance concentrations  for the definitive test.   Activated  sludge  is
       exposed to a series of widely-spaced (e.g.,  log interval) concentrations of test substance.
       The details of the range-finding test do not have to be the same as for definitive testing.
       Treatment replication is  not  needed and  nominal concentrations of the chemical are
       acceptable. However, the range-finding test will be more useful the greater the similarity
       between the  range-finding test and the definitive test.  Report results of range-finding
       tests along with the results of the definitive  test, if range-finding tests are conducted.

       (4) Definitive test.   The goal of the  definitive test is to  determine  the concentration-
       response curve and ICso value  (and the 95% confidence intervals) for  microbial
       respiration at 3 hours.  In addition, the slope of the  concentration-response  curve, its
       associated standard error and the 95% confidence interval  should  be determined,  if
       possible. For ICso determination, a minimum of five concentrations of the test chemical,
       plus appropriate controls,  are tested. Analytical confirmation of the concentration of test
       substance  in the stock  solutions may  be performed as described  in  OCSPP  850.3000.
       Elements of an acceptable definitive test are given in Table 1.

       (5) Limit test. In some situations, it is only necessary to ascertain that the ICso is above a
       certain limit.  In a limit test, activated sludge is exposed to a single  "limit concentration",
       with the appropriate controls.  The multiple-definitive test may be waived if the limit
       concentration has not  caused greater than 50% reduction  in  respiration.   For most
       chemicals, 1000 milligrams  per liter  (mg/L)  (based upon 100%  active ingredient for
       pesticides), or the limits of water solubility or dispersion, are considered appropriate  as
       the  limit concentration.  Except  for the  number of test  concentrations and endpoint
       determinations, an acceptable  limit test follows the same test procedures and is the same
       duration as the multi-concentration definitive test (Table 1).

(e) Test standards—

       (1) Test substance.  The substance to be tested should be technical grade, unless the test
       is designed to test a specific formulation, mixture, or end-use product.   The OCSPP
       850.3000  guideline  lists the type of information that should be known about the test
       substance before testing, and discusses methods for preparation of test solutions.

       (2) Test duration.  The respiration rate  of the activated sludge is  typically measured  at
       30 minutes and at 3 hours after exposure  to the test substance.  However, if only one
       measurement is to be done, then the 3 hour  measurement should be done.

       (3) Test species—

             (i) Activated sludge.  Activated sludge from a sewage treatment plant is normally
             used  as the microbial  inoculum  for the test.   Where possible activated  sludge
             should be obtained from  a  sewage  works  treating predominantly  domestic
             sewage.  If this is not possible, the activated sludge may be obtained from sewage
             works treating predominantly  industrial waste water but  used  only  following
             deadaptation (activated sludge solids could be maintained in modified SCAS (see
             the OCSPP  835.3210 guideline)  or  Porous  Pot (see  the  OCSPP  835.3220
             guideline)  systems being fed domestic  sewage for at least 3 sludge retention
                                       Page 2 of 12

       times).  Even so, results obtained with  activated sludge from  works treating
       industrial waste waters may be atypical.  Activated sludge may contain potentially
       pathogenic organisms and should be handled with care.

       (ii) Microbial inoculum. Activated sludge (collected from  a sewage treatment
       plant) is washed, if necessary, with tap  water or an isotonic solution.   After
       centrifuging the supernatant is decanted.  This procedure is repeated three times.
       A  small amount of the  washed sludge  is weighed and dried.  From this  result
       calculate the amount  of wet sludge to suspended in water in order to obtain an
       activated sludge with a mixed liquor suspended solids level of 4  grams per liter
       (g/L) (+ 10%).  This level gives a concentration of 1.6 g/L in the test medium if
       200 milliliters (mL)  of inoculum is used in a  final volume  of 500 mL.  If the
       sludge cannot be used on the day  of collection,  50 mL  synthetic sewage is added
       to  each  liter of the activated sludge prepared as  described; this  is then aerated
       overnight at 20 + 2 degrees Celsius (°C).  It is then kept aerated for use during the
       day. Before use the pH is checked and buffered, if necessary to pH 6.0 to 8.0
       using sodium bicarbonate solution. Determine the mixed liquor suspended solids.
       If the same batch of sludge is to be used on subsequent days (maximum 4 days), a
       further 50 mL of synthetic sewage feed is added at the end of each working day.

(4) Administration of test  substance.  The sludge microorganisms are exposed under
static conditions to aqueous solutions of the test substance. Each test vessel should be
inoculated with 200 mL  of the microbial inoculum prepared as described  in paragraph
(e)(3)(ii) of this guideline.

       (i) Test substance solutions.

             (A) At least five concentrations, spaced by a constant factor preferably not
             exceeding  3.2, plus appropriate control(s) are tested.  Each control group
             is prepared in two  sets  (see paragraphs  (e)(4)(i)(C), (e)(5)(i)(A) and
             (e)(5)(i)(B) of this guideline) to  bracket the start and end  of treatment
             solution microbial preparations).   Solutions  of the  test substance are
             freshly prepared at the start of  the study  using a stock  solution.   The
             preparation of test  solutions is  described in  the  OCSPP  850.3000

             (B)  If the test  substance is not sufficiently soluble to allow preparation of a
             concentrated stock solution  in water, it should be added  directly to test
             vessels or,  alternatively, as  a concentrated  stock solution in  an organic
             solvent.  Direct addition is recommended.  If an organic solvent is  used,
             for  an acceptable test the  solvent should neither significantly inhibit nor
             contribute  to respiration (i.e., solvent control respiration should be within
              15% of control respiration).

             (C)  A suggested experimental procedure which may be followed for the 3-
             hour contact period is:

                    (1) At time "0"  16  mL of the synthetic sewage feed is made  up to
                    300 mL with water.  Microbial inoculum (200 mL)  is added and
                                Page 3 of 12

the total mixture  (500 mL) poured into the first set  of control
vessels (first control set, Cl; see paragraph (e)(6) of this guideline
for number of replicates).  Aeration at  0.5 to 1 liter per minute
(L/min) is commenced using a Pasteur pipet as an aeration device.

(2) At  time  "15  minutes"  (15  minutes is an  arbitrary,  but
convenient,  interval)  the   process  described   in  paragraph
(e)(4)(i)(C)(7j of this guideline is repeated, except that  100 mL of
the test substance stock solution is added to the 16 mL of synthetic
sewage  feed  before adding  water to  300  mL  and  microbial
inoculum (200 mL) to make a volume of 500 mL.  This mixture is
then poured into the first treatment level, Tl, set of vessels  (see
paragraph  (e)(6) of this guideline for number of replicates)  and
aerated as  described in paragraph (e)(4)(i)(C)(7j of this guideline.
This process  is repeated  at  15-minute  intervals  with  different
volumes of the test substance stock solution to a series of treatment
vessels  containing  different concentrations of the test  substance
(i.e., second treatment level (T2) through the last (T5)).  Finally, a
second control set (C2) (see paragraph (e)(6)  of this  guideline for
number of replicates) are prepared.

(3) If the test substance is not sufficiently soluble to allow addition
by  aqueous stock  solution,  the appropriate proportion of the  100
mL volume of test substance stock solution is  replaced with water.
For example,  if 10  mL of an insoluble liquid test  substance or
solvent  containing test substance  is added  directly to the  test
vessels, 90 mL of water is added.  If insoluble solid test substance
is added to the test vessel, 100 mL of water is added.

(4) After 3 hours the contents of the first  control vessel (performed
for each replicate), Cl,  are poured into a measuring apparatus and
the respiration rate is measured over a period of up to 10 minutes;
the measuring can  also be carried out directly in each vessel.

(5) This  determination  is  repeated  on  the contents  of  each
treatment vessel set (i.e., first treatment level (Tl) through the last
treatment level (T5)) at 15-minute intervals, in such a way that the
contact time in each treatment vessel is  three hours.  Finally, the
determination on the  second control  set (C2) is carried out, with a
resultant contact time of three hours for this control set.

(6) A different regime (e.g., more than one oxygen meter) should
be used when measurements are to also be made after 30 minutes
of contact.

(7) For  measurement of  the chemical oxygen  consumption,
additional vessels are prepared containing test substance, synthetic
sewage feed and water, but no activated sludge.

            Page 4 of 12

       (iii) Abiotic oxygen uptake.  A preliminary test (or can be conducted at same
       time as biotic  measurements)  should be  conducted to determine  if the test
       substance causes measurable abiotic oxygen consumption.  Vessels are prepared
       as  described  in (e)(4)(i) of this guideline containing test  substance, synthetic
       sewage feed and water, but no activated sludge.  Note that an inhibitor may need
       to  be added to prevent biological  oxygen  consumption.  After the appropriate
       contact time  (should be the  same as that used or to be used in the limit  or
       definitive  test),  measurements of oxygen consumption  are made for each
       treatment and control vessel.

(5) Controls—

       (i) Negative controls.

              (A)  Every  test  includes two  sets  of negative  controls  (Cl and  C2)
              consisting of the same synthetic sewage feed, conditions, procedures, and
              microbial inoculum from the same source, except that none  of the test
              substance is added.   One  set of the controls  (Cl) is prepared  at the
              beginning of the test before any other test solutions are prepared and the
              other (C2)  at the end after all other test solutions have been prepared.
              Two sets of a vehicle (solvent) control (SCI and SC2) are also tested if a
              solvent is used.  The volume of solvent added should be less than 0.1%  of
              the total volume.

              (B)  In  the  suggested  experimental  design described  in   paragraph
              (e)(4)(i)(C)  of this guideline, the first solvent control set (SCI)  is prepared
              immediately after the first negative control set (Cl) and the second solvent
              control set (SC2) is prepared immediately before the last negative control
              set (C2) is prepared.  Where the volume of solvent used is not the same
              between treatment  levels, for a satisfactory test the maximum volume  of
              solvent should be used to  prepare  the solvent control set  (i.e.., SCI and

              (C)  For an  acceptable definitive or limit test,  control respiration rates  of
              Cl and C2  should be within 15% of each other and SCI and SC2  should
              be within 15% of each other and the negative controls (Cl and C2).

       (ii) Reference substance.  A  positive  control (3,  5-dichlorophenol, a  known
       inhibitor of respiration) is also tested to  determine the ICso on  each batch  of
       activated sludge as a means of checking that the sensitivity of the sludge is not
       abnormal.   For the positive control, a solution  of 3, 5-dichlorophenol  can be
       conveniently prepared by dissolving 0.5 g of 3, 5-dichlorophenol in 10 mL of a 1
       normal (IN)  sodium hydroxide (NaOH) solution, diluting to approximately 30
       mL with reagent water, adding, while stirring, IN sulfuric acid (H2SO4 ) solution
       to  the point of incipient precipitation (approximately  8 mL), and finally diluting
       the mixture to 1 L with reagent water.  The pH should then be in the range 7 to 8.
       The reference substance is tested on each batch of microbial inoculum in the same
       way as the test substance (see paragraph (e)(4) of this  guideline).  For an

                                Page 5 of 12

       acceptable definitive test, the  3-hour  ICso of 3, 5-dichlorophenol  should  range
       between 5 to 30 mg/L.

(6) Number of replicates.  At a minimum three replicates for each concentration of test
substance and each negative control group set (Cl and C2), and solvent control set (SCI
and SC2), if a solvent was used, are tested.

(7) Facilities, apparatus and supplies—

       (i) Apparatus.   Normal laboratory  equipment  and  especially  that  listed  in
       paragraphs (e)(7)(i)(A) through (e)(7)(i)(D) of this guideline is used:

             (A) Measuring apparatus (flat-bottom flask, stirrer bar, magnetic stirrer,
             oxygen electrode, and recorder).  There should be no head space and the
             oxygen  probe should fit tightly  in  the neck  of the  measuring  flask;
             biological oxygen demand (BOD) bottles are suitable for this purpose.

             (B) Aeration device.

             (C) pH electrode and measuring equipment.

             (D)  For  measuring oxygen  concentration,  a  polarographic  oxygen
             electrode, connectable to a potentiometric recorder (200 milivolt range).

       (ii)  Test containers.  Glass beakers,  1  L  capacity,  are  recommended as test

       (iii) Cleaning.  Test containers should be cleaned before each test.  See OCSPP

       (iv) Synthetic sewage feed.

             (A)  A synthetic  sewage feed is made by dissolving  the  amounts  of
             reagent-grade  substances listed in paragraphs (e)(7)(iv)(A)(7j  through
             (e)(7)(iv)(A)(^7y) of this guideline in 1 L of reagent water:

                    (1) 16 g peptone.

                    (2) 11 g meat extract.

                    (3) 3  g urea.

                    (4) 0.7 g sodium chloride (NaCl).

                    (5) 0.4 g calcium chloride dihydrate (CaCl2.2H2O).

                    (6) 0.2 g magnesium sulfate heptahydrate (MgSO4.7H2O).

                    (7) 2.8 g dipotassium hydrogen phosphate

                                Page 6 of 12

                    (B) This synthetic sewage feed is a 100-fold concentrate of that described
                    in the OECD Technical Report (June 11, 1976) "Proposed method for the
                    determination of the biodegradability of surfactants used in synthetic
                    detergents," with dipotassium hydrogen phosphate added.

       (8) Environmental conditions. Environmental parameters during the test are maintained
       as specified in paragraphs (e)(8)(i) and (e)(8)(ii) of this guideline:

             (i) Temperature.  The test temperature is 20 °C and it should be constant within
             + 2 °C during the test.

             (ii) Aeration.  Test containers are continuously aerated with clean, oil-free air at a
             flow rate of 0.5 to  1 L per minute.

       (9) Observations—

             (i) Measurement of test substance. Analytical confirmation of the concentration
             of test substance in the stock solutions may be performed  as described in the
             OCSPP 850.3000  guideline.  Validated analytical methods are used to measure
             the amount of test  substance in a sample before beginning the test, as described in
             the OCSPP 850.3000 guideline.

             (ii) Environmental conditions—

                    (A)  Temperature.    Temperature  should   be monitored;  preferably
                    continuously but at a  minimum,  the maximum  and  minimum  daily
                    temperature should be recorded.

                    (B) Aeration. The aeration rate should be checked during the study.

             (iii) Measures of effect—

                    (A) Respiration rate. The  respiration rate of an activated sludge fed with
                    a standard amount of synthetic sewage feed is measured and recorded after
                    a contact time of 3 hours, or contact times of both 30 minutes and 3 hours.
                    The respiration rate of the same activated sludge in the presence of various
                    concentrations of the test substance under otherwise identical conditions is
                    also measured and recorded.

                    (B) Abiotic oxygen demand.  The respiration rate  of a synthetic sewage
                    feed is measured and recorded after a contact time  of 3 hours, or contact
                    times of both 30 minutes and  3 hours. The  respiration rate of the  same
                    sewage feed in the presence of various concentrations  of the test substance
                    under otherwise identical conditions is also measured and recorded.

(f) Treatment of results—

       (1) Respiration  rate.  The respiration rate is  calculated  from  the recorder trace  as
       milligrams of oxygen per liter per hour (O2/L/h) between approximately 6.5 mg O2/L and

                                      Page 7 of 12

       2.5 mg C>2/L, or over a 10 minute period when the respiration rate is low. The portion of
       the respiration curve over which the respiration rate is measured should be linear.

       (2) Percent inhibition. In order to calculate the inhibitory effect of a test substance at a
       particular concentration  (e.g.,  Tl), the respiration rate at a given  treatment level is
       expressed as a percentage of the  mean respiration rates of the two control sets (Cl and

              1 - [2Rs/(Rci +RC2)] x 100 = percent inhibition (%I)

              RS = oxygen-consumption rate at test substance concentration (e.g., Tl)

              RCI = oxygen-consumption rate, control 1

              Rc2 = oxygen-consumption rate, control 2

       (3) Concentration-response relationship.  The ICso  and its 95% confidence limits are
       determined for respiration rate at 3 hours (30 min also, if measured) using an  appropriate
       statistical model to establish the concentration-response relationship for the test substance
       and for the reference substance.  Various  statistical procedures for modeling  continuous
       toxicity data are available and can be used. Additional  discussion about  endpoints and
       statistical procedures is found in the OCSPP 850.3000 guideline.

       (4) Correction for abiotic oxygen demand.  If it is  determined that the  test substance
       causes measurable abiotic oxygen consumption, the microbial respiration rate should be
       calculated  by  subtracting  the  abiotic oxygen  uptake  rate from the  respiration rate
       determined when activated sludge was present.  These adjusted respiration values would
       then be used to determine %I in paragraph  (f)(2) of this guideline.
       (5) Interpretation of results. The ICso value for respiration should be regarded merely
       as a  guide to the likely toxicity of the test substance either to activated sludge sewage
       treatment or to wastewater microorganisms, since the complex interactions occurring in
       the environment cannot be accurately simulated in a laboratory test.

(g) Tabular summary of test conditions. Table  1  lists the important conditions that  should
prevail during the definitive test.  Except for the number of test concentrations and endpoint
determinations, Table 1 also lists the important conditions that should prevail during a limit test.
Meeting these test conditions will greatly increase the likelihood that the completed test will be
acceptable or valid.
                                       Page 8 of 12

       Table 1.—Summary of Test Conditions for the Modified Activated Sludge,
Respiration Inhibition Test
Test duration
Test matrix
Test vessel size
Test vessel volume
Microbial inoculum
Test concentrations
Number of replicate test vessels per
Test concentration preparation
Measure of Effect or Measurement
3 hours
Synthetic sewage feed
20 °C (constant during test within + 2 °C)
At a flow rate of 0.5 - 1 L/min.
Glass beakers, 1 L, or similar
Washed activated sludge prepared to give a final
concentration of 1 .6 g/L in test medium
Minimum of 5 test concentrations plus negative and solvent
(if solvent used) controls
Three for each test substance treatment and three for each
negative control set (C1 and C2) and vehicle control set
(SC1 and SC2), if a vehicle was used. For abiotic oxygen
demand, three for each test substance treatment and three
for each negative control set (C1 and C2) and vehicle
control set (SC1 and SC2), if a solvent was used.
Aqueous solution prepared by adding test substance
(directly, or in a water or a vehicle) to synthetic sewage feed
3-h IC50 based upon respiration rate
(h) Test validity. This test would be considered to be unacceptable or invalid if one or more of
the conditions in Table 2 occurred.  This list should not be misconstrued as limiting the reason(s)
that a test could be found unacceptable or invalid.  However, except for the conditions listed in
Table 2 and in the OCSPP 850.3000 guideline, it is unlikely a study will be rejected when there
are slight variations from guideline environmental conditions and study design unless the control
organisms are significantly affected, the precision of the test is reduced, the power of a test to
detect differences is reduced, and/or significant biases are introduced in defining the magnitude
of effect on  measurement  endpoints as compared to guideline conditions.  Before  departing
significantly from this guideline, the investigator should contact the Agency to discuss the reason
for the departure and the effect the change(s) will have on test acceptability. In the test report, all
departures from the guideline should be identified, reasons  for these changes given, and  any
resulting effects on test endpoints noted and discussed.
                                      Page 9 of 12

       Table 2.—Test validity elements for the Modified Activated Sludge Respiration
Inhibition Test
1. Untreated (negative) controls, solvent controls (if a solvent was used) and positive controls were not
included in the test.

2. All test chambers were not identical and did not contain the same amount of activated sludge and
synthetic feed.

3. The control respiration rates of C1 and C2 (beginning and end of exposure period, respectively) were
not within 15% of each other and the solvent control rates SC1 and SC2 (beginning and end of exposure
period, respectively), if a solvent was used, were not within 15% of each other and the negative controls
(C1  and  C2).

4. The 3-h IC50 of 3,5-dichlorophenol (positive control) was not in the accepted range of 5 to 30 mg/L.
(i) Reporting—

       (1)  Background information.  Background information to be supplied in the report
       consists at a minimum of those background information items listed in paragraph (j)0) of
       the  OCSPP 850.3000 guideline.

       (2)  Guideline deviations.  Provide a statement of the guideline or protocol followed.
       Include a description of any deviations from the test guideline or any occurrences which
       may have influenced the results of the test.

       (3)  Test substance.

              (i) Identification of the test substance:  common name, IUPAC and CAS names,
              CAS number, structural formula, source, lot or batch number, chemical  state or
              form of the test substance, and its  purity (i.e.  for  pesticides, the identity and
              concentration of active ingredient(s)).  If radiolabeled substance was used  provide
              the radio purity  and location(s) of the label.

              (ii) Storage conditions of the test chemical or test substance and stability of the
              test chemical or test substance under storage conditions if stored prior to use.

              (iii) Identification of the reference substance: name, source, and its purity.

       (4)  Microbial inoculum.

              (i) Source of activated sludge.

              (ii) Activated sludge collection date and method.

              (iii) Storage duration and conditions  of the activated sludge, any pretreatment or
              addition of synthetic feed to activated sludge or deadaptation before preparation
              of inoculum.

              (iv) Microbial inoculum preparation  methods and concentration of mixed liquor
              suspended solids in the inoculum.

                                      Page 10 of 12

       (v) Date microbial  inoculum is prepared and age of microbial  inoculum at test

(5) Test system and conditions.  Description of the test system and conditions used in
the definitive  or  limit test, any preliminary  range-finding tests,  and  any reference
substance tests.

       (i) Methods of preparation of the aqueous test solutions in the range-finding and
       definitive test,  or limit test: how  test  substance was introduced into  the test
       medium (e.g., directly mixed,  or as an  aqueous or solvent  stock solution), the
       volume of microbial inoculum and synthetic sewage feed used for each treatment,
       and the volume or mass of test substance added to each treatment.

       (ii)  If stock solution was  used: the name and source of the vehicle, methods of
       stock  solution preparation,  nominal  test  substance  concentrations in  stock
       solution(s), and volume of solvent in each treatment vessel in the definitive test or
       limit test.

       (iii) Test duration.

       (iv) Methods and frequency  of environmental monitoring performed during the
       definitive or limit study and any reference substance study for temperature.

       (v) For the definitive, limit, or reference substance tests, all analytical procedures
       should be described.  The accuracy of the method, method  detection limit, and
       limit of quantification should be given.

(6) Results.

       (i) Environmental monitoring data results (temperature) in tabular form (provide
       raw  data for measurements  not made on a  continuous basis), and descriptive
       statistics (mean, standard deviation, minimum, maximum).

       (ii) For preliminary range-finding test, if conducted, the respiration at each test
       substance level and  in the control(s).

       (iii) For the definitive or limit test, the respiration at each observation period for
       each replicate at each treatment substance level and control(s) (provide  the raw

       (iv) Abiotic oxygen uptake, if any, in each replicate of each test substance  level
       and the control(s) (provide the raw data).

       (v)  For  the  definitive  or limit test and  reference  substance  test,  the %I in
       respiration at each observation period at each treatment substance level.

       (vi) For the definitive test, graph the concentration-response curves at 30  minutes
       (if measured) and at the end of the test.

       (vii) For the definitive study, the slope of the concentration-response curve at 30
                                Page 11 of 12

              minutes (if measured) and the end of the test and their standard error and 95%
              confidence limits.

              (viii) For the definitive and reference substance studies, calculated ICso at end of
              test, and if possible, 95% confidence limits.

              (ix) For a limit test, report the %I at the tested limit concentration.

              (x) Description  of statistical  method used,  including  software  package, for
              determining ICso values and the concentration-response model parameters and the
              basis for the choice of method.  Provide results of any goodness-of-fit tests.

(j) References. The references in this paragraph should be consulted for additional background
material on this test guideline.

       (1)  ETAD (Ecological  and Toxicological  Association  of  Dyestuffs Manufacturing
       Industries) Recommended Method No.  103 (also described in references in paragraphs
       (j)(5) and (j)(6) of this guideline).

       (2) International Organization for  Standardization, 1981.   Activated sludge respiration
       inhibition test.  A method for assessing the inhibition of respiration of activated sludge
       microorganisms by test substance. ISO/TC 147/SC 5/WG 1, N53 No. D, June 1981.

       (3) Broecker, B. and Zahn, R.,  1977.  The performance of activated sludge  plants
       compared  with  the  results of  various bacterial toxicity tests—A study  with 3,5-
       dichlorphenol. Water Research 11:165.

       (4) Brown, D.. H. Hitz and L. Schafer, 1981.   The assessment of the possible inhibitory
       effect of dyestuffs on aerobic waste-water bacteria experience with a screening test.
       Chemosphere 10:245-261.

       (5) Robra, K., 1976. Bewertung toxischer Wasserinhaltsstoffe aus ihrer Inhibitorwirkung
       auf die Substratoxydation von Pseudomonas  Stamm  Berlin mit Hilfe polarographischer
       Sauerstoffmessungen. (Evaluation  of toxic substances contained  in water from their
       inhibitor effect on the substrate oxidation of Pseudomonas Stamm Berlin with the help of
       polarographischer oxygen measurements),  gwf. wasser/abwasser 117(2), 80-86.

       (6) Schefer,  W.,  1977.   Testing  effluent components for toxic activity on  biological
       treatments.  Textilveredlung 6:247-250.
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