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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 December 2014
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Not GLP Compliant

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
The study procedures described in this report are a simplified version of the Organization for Economic Co-operation and Development (OECD), OECD guidelines for Testing of Chemicals, Section 2: Effects on biotic systems, Guideline no. 209, "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation), adopted 22 July 2010.
Principles of method if other than guideline:
A concentration of 2.0 mg/l was tested instead of 1.0 mg/l. To the weighed test substance 400 ml Milli-RO should have been added and after stirring half of the suspension should be removed. However, 200 ml Milli-RO was added and no test suspension was removed after the 24 hour stirring period.
Evaluation: No biologically relevant effects were observed at any of the tested loading rates and therefore the results were not influenced.
GLP compliance:
Study not done for EU Reach as an intermediate under SCC but to evaluate the test substance for its ability to adversely affect aerobic microbial treatment plants.

Test material

Constituent 1
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Purity 99.7%

Sampling and analysis

Analytical monitoring:
Details on sampling:
The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference substance concentrations and controls.
The medium temperature was recorded continuously in a temperature control vessel(s). The temperature control vessel(s) was/were identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room.

Oxygen recording Determination of oxygen was performed with multiple oxygen probes connected to a BlueBox (GOSystemelektronik GmbH, Germany), a multichannel measuring and controlling system.

5.6. Electronic data capture
Observations/measurements in the study were recorded electronically using the following programmes:
• REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA): Temperature.
• BlueBox Software version (GO-Systemelektronik GmbH, Germany): Oxygen.

Test solutions

Details on test solutions:
The test substance was not sufficiently soluble to allow the preparation of a 10 g/l stock solution in water. Therefore, 1-Litre test bottles were filled with 200 ml of test substance mixtures in Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for 25½ hours. Subsequently, 16 ml synthetic medium made up to 50 ml with Milli-RO water and 250 ml sludge were added resulting in the required loading rates (see also paragraph 5.3). Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage

Study design

Test type:
other: Screening
Water media type:
other: Tap-water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA).
Limit test:
Total exposure duration:
3 h
Remarks on exposure duration:
3 hours, during which aeration and stirring took place.
Post exposure observation period:
After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 10 minutes

Test conditions

Test temperature:
21.4 - 22.1 °C
The pH in all test vessels, before addition of sludge was between 7.5 and 7.6. After the 3 hour exposure period the pH was between 7.2 and 8.0.
Dissolved oxygen:
The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/l at 20°C) and to maintain the sludge flocs in suspension.
In the synthetic medium there was 0.7 g NaCl, 0.4 g CaCl2.2H2O, 0.2 g MgSO4.7H2O, and 2.8 g K2HPO4 dissolved in Milli-RO water
Details on test conditions:
Preparation of the sludge:
The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/l of sludge, as used for the test). The pH was 6.0 and adjusted to 7.1 using a sodium bicarbonate solution (70 g/l, Sigma Aldrich, St. Louis, USA) on the day of testing. The batch of sludge was used one day after collection; therefore 50 ml of synthetic medium (=sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated
at test temperature until use.

Adjusted ISO medium, formulated using RO-water (tapwater purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/l
MgSO4.7H2O 88.8 mg/l
NaHCO3 46.7 mg/l
KCl 4.2 mg/l
Reference substance (positive control):
The batch of activated sludge was checked for sensitivity by testing the reference substance 3,5-dichlorophenol.

Results and discussion

Effect concentrations
3 h
Dose descriptor:
Effect conc.:
5.04 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Results with reference substance (positive control):
Replicate Loading rate pH Respiration rate % Inhibition respiration
(mg/l) Start End (mg O2/l.h) (mg O2/g.h)¹ rate(mean value)

R1 2.0 7.6 7.9 31 21 30
R2 5.0 7.6 8.0 23 15 48
R3 12 7.6 8.0 13 9 71
Reported statistics and error estimates:
Table 4 Probit analysis 3,5-dichlorophenol
Parameter Value
Computation runs: 4.0000
Slope b: 1.3693
Intercept a: -0.96209
Variance of b: 4.3890
Goodness of Fit
Chi²: 0.0030
Degrees of freedom: 1.0000
p(Chi²): 0.9567
Log EC50: 0.7026
SE Log EC50: 0.4614
g-Criterion: 1.1166
Residual Variance (Chi²/df): 0.0030
r²: 0.9930
F: 144.5900
p(F) (df: 1;1): 0.0530

Any other information on results incl. tables

Replicate              Loading rate              pH                     Respiration rate                            % Inhibition respiration

                                  (mg/l)              Start       End       (mg O2/l.h)       (mg O2/g.h)¹              rate(mean value)

T1                            2.0                    7.5       7.4       45 (=RT)              30                                   -2 (=IT)

T2                            10                     7.5       7.4       47 (=RT)              31                                   -6 (=IT)

T3                            100                   7.5       7.3       44 (=RT)              29                                   1 (=IT)

T4                            1000                 7.5       7.2       47 (=RT)              31                                   -6 (=IT)

Applicant's summary and conclusion

Validity criteria fulfilled:
Under the conditions of this present test PF-05029508 was not toxic to waste water bacteria (activated sludge) at a loading rate of 1000 mg/l.
The EL50 was above 1000 mg/l.