United States

Environmental Protection 

Agency

Office of Chemical Safety

and Pollution Prevention

(7101)

EPA 712-C-014

January 2012

Ecological Effects

Test Guidelines

OCSPP 850.3300:

Modified Activated 

Sludge, Respiration 

Inhibition Test
i

NOTICE

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.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-

0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
Page 1 of 12

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 (O2) 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)(1) of 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 (IC50) 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 ICx (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 2 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 concentrationresponse curve and IC50 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 IC50 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 IC50 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 3 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 (oC). 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 

guideline.

(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 4 of 12

the total mixture (500 mL) poured into the first set of control 

vessels (first control set, C1; 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)(1) 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, T1, set of vessels (see 

paragraph (e)(6) of this guideline for number of replicates) and 

aerated as described in paragraph (e)(4)(i)(C)(1) 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), C1, 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 (T1) 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 5 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 (C1 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 (C1) 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 (SC1 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 (SC1) is prepared 

immediately after the first negative control set (C1) 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., SC1 and 

SC2).

(C) For an acceptable definitive or limit test, control respiration rates of 

C1 and C2 should be within 15% of each other and SC1 and SC2 should 

be within 15% of each other and the negative controls (C1 and C2).

(ii) Reference substance. A positive control (3, 5-dichlorophenol, a known 

inhibitor of respiration) is also tested to determine the IC50 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 (1N) sodium hydroxide (NaOH) solution, diluting to approximately 30 

mL with reagent water, adding, while stirring, 1N 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 6 of 12

acceptable definitive test, the 3-hour IC50 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 (C1 and C2), and solvent control set (SC1 

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 

vessels. 

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

850.3000.

(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)(1) through 

(e)(7)(iv)(A)(7) 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 (K2HPO4).
Page 7 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 oC and it should be constant within 

+ 2 oC 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 8 of 12

2.5 mg O2/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., T1), the respiration rate at a given treatment level is 

expressed as a percentage of the mean respiration rates of the two control sets (C1 and 

C2):

1 - [2RS/(RC1 + RC2)] x 100 = percent inhibition (%I)

where:

RS = oxygen-consumption rate at test substance concentration (e.g., T1)

RC1 = oxygen-consumption rate, control 1

RC2 = oxygen-consumption rate, control 2

(3) Concentration-response relationship. The IC50 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 IC50 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 9 of 12

Table 1.―Summary of Test Conditions for the Modified Activated Sludge, 

Respiration Inhibition Test

Test duration 3 hours

Test matrix Synthetic sewage feed

Temperature 20 oC (constant during test within + 2 oC)

Aeration At a flow rate of 0.5 - 1 L/min.

Test vessel size Glass beakers, 1 L, or similar

Test vessel volume 500 mL

Microbial inoculum Washed activated sludge prepared to give a final 

concentration of 1.6 g/L in test medium 

Test concentrations Minimum of 5 test concentrations plus negative and solvent 

(if solvent used) controls

Number of replicate test vessels per 

treatment

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.

Test concentration preparation Aqueous solution prepared by adding test substance

(directly, or in a water or a vehicle) to synthetic sewage feed

Measure of Effect or Measurement 

Endpoint

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 10 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)(1) 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 11 of 12

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

initiation.

(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 

data).

(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 12 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 IC50 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 IC50 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. Schäfer, 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.

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