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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10-19 January 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
This study was performed according to OECD Guideline 209 with GLP statement: average oxygen uptake rate for the control replicates was slightly lower than the guideline
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
average oxygen uptake rate for the control replicates was slightly lower than the guideline
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
18 March 2010
Test material information:
Composition 1
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
Nopol was weighed directly in the incubation vessels.
Test organisms (species):
other: Unadapted secondary activated sludge
Details on inoculum:
Unadapted secondary activated sludge was obtained (17-01-2012) from the WWTP Nieuwgraaf in Duiven. The WWTP Duiven is an activated sludge plant treating predominantly domestic wastewater. Prior to use the activated sludge was homogenized with a syringe. The dry weight of the homogenized activated sludge was determined and subsequently concentrated by settlement to the required dry weight concentration. The dry weight of the activated sludge in the incubation vessels was 1.5 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
None
Hardness:
No data
Test temperature:
20.0-20.6 °C
pH:
6.9-7.3
Dissolved oxygen:
Average oxygen uptake rate for the control replicates of 19 mg O2/g dry weight/hour on the first day and 23 mg O2/g dry weight/hour on the second day.
Salinity:
No data
Conductivity:
No data
Nominal and measured concentrations:
19.3, 38.7, 77.4, 154.7, 309.4 and 618.9 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Test was performed in 250 mL Erlenmeyer flasks closed with Teflon coated lids, with a working volume of 50 mL.
- Type: closed
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Synthetic Sewage: 16 g peptone; 11 g meat extract; 3 g urea; 0.7 g NaCl; 0.3 g CaCl2; 0.2 g MgSO4 · 7H2O; 2.8 g K2HPO4 - filled up to 1 litre with deionised water.
- In the incubation vessels 1.6 mL of the synthetic sewage feed is added in a total volume of 50 mL.

TEST PROCEDURE
- Activated sludge with test substances was incubated in a shaking bath (100 rpm/min) for 3 hours.

EFFECT PARAMETERS MEASURED
Determination of respiration rates, pH, dry weight and temperature: The respiration rates of the activated sludge were measured in a Biological Oxygen Monitor YSI 5300. The BOM consisted of a thermostated vessel with a magnetic stirrer and an oxygen electrode. The electrode to measure the oxygen depletion tightly closed the vessel. The volume of the vessel was 10 mL and the temperature in the vessel was 20 °C. The pH was measured using an EUTECH pH meter. The temperature was measured with a Keithley 871A digital thermometer. The dry weight of the inoculum was determined by filtrating 30 mL of the activated sludge over a preweighed 12 µm cellulose nitrate filter. This filter was dried for minimal 1.5 hours at 104 ± 5 °C and weighed after cooling. Dry weight was calculated by subtracting the weight of the filters and dividing the difference by the filtered volume.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
106 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% confidence limits of 44 and 247 mg/L
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
48 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
234 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The inhibition percentage of test substance was 3, 18, 28, 64, 94 and 96 at 19.3, 38.7, 77.4, 154.7, 309.4 and 618.9 mg/L, respectively.
Results with reference substance (positive control):
The inhibition percentage of positive control was 8, 26, 39, 80 and 88% at 2.5, 5.1, 10.2, 20.3 and 40.6 mg/L, respectively. Test is valid as shown by the EC50 of the reference compound, 3,5-dichlorophenol is 11 mg/L.
Reported statistics and error estimates:
The EC values were computed from the best-fitted line (least square method) through the points given by the probit of the percentage inhibition and the logarithm of the nominal concentration of the substance. All computations were performed with the program TOXCALCTM(Tidepool Scientific Software 1994-1999).

Table 6.1.7/1: Respiration rates of the activated sludge, inhibition percentages and pH at various concentrations of test substance after 3 hours contact time

 

Concentration

(mg/L)

Activity

(mg O2/g/min)

Inhibition

(%)

pH at start test

pH at end test

Temperature

end test (°C)

Control

0.4083

-

7.0

7.2

20.0

Control

0.4000

-

7.0

7.3

20.4

Control*

0.3833

-

7.0

7.3

20.5

Control*

0.3833

-

7.0

7.3

20.5

19.3*

0.3704

3

n.d

7.2

20.1

38.7

0.3333

18

n.d

7.2

20.1

77.4

0.2917

28

n.d

7.2

20.6

154.7

0.1458

64

n.d

7.2

20.5

309.4

0.0250

94

n.d

7.2

20.5

618.9

0.0167

96

n.d

7.2

20.2

* = corresponding control and reference concentration

n.d. = not determined, the pH was not measured at the start to prevent evaporation of the test substance.

 

Validity of the test

The reference substance and test substance were measured on two subsequent days. The validity of the test is demonstrated by three criteria.

First, the coefficient of variation of the replicates of the control oxygen uptake rates was 7% on the first day and 4% on the second day, which fulfils the maximum prescribed variation of <30%. Secondly, the EC50 of the reference compound was 11 mg/L which is within the prescribed range of 2 to 25 mg/L.

The third criterion, an oxygen uptake rate of >20 mg O2/g dry weight/hour for the control measurements, was not achieved on the first day (19 mg O2/g dry weight/hour) and was met on the second day (23 mg O2/g dry weight/hour).

The inoculum used was obtained from a treatment plant which is operated at a low organic sludge load and therefore has a natural lower oxygen uptake rate.

According the guideline activated sludge used should be fed with synthetic sewage feed, for a maximum of two additional days, when used after the day of collection. Adding synthetic sewage feed leads to an increase of the oxygen uptake rate of the activated sludge, but does not affect the sensitivity of the activated sludge significantly (AkzoNobel, 2012). Accurate oxygen uptake rate measurements were very well possible, with the slightly lower oxygen uptake rate on the first day, as shown by the very low coefficient of variation. Therefore this is a minor deviation which does not invalidate the test.

Validity criteria fulfilled:
yes
Conclusions:
The EC50 of the test substance for activated sludge after 3 hours contact time is 106 mg/L with 95% confidence limits of 44 and 247 mg/L. The EC10, EC20 and EC80 are 32, 48 and 234 mg/L, respectively. The test substance is therefore considered not harmful to activated sludge.
Executive summary:

An activated sludge respiration inhibition test was performed according to Guideline OECD 209 with GLP compliance. 

Unadapted secondary activated sludge was obtained from activated sludge plant treating predominantly domestic wastewater (WWTP Nieuwgraaf in Duiven). The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage was measured in the presence of various concentrations of the test substance after an incubation period of 3 h.

 

Test Concentrations: 19.3, 38.7, 77.4, 154.7, 309.4 and 618.9 mg/L

 

The test is valid as shown by the EC50 of the reference compound, 3,5-dichlorophenol (11 mg/L), the coefficient of variation of the replicates of the control oxygen uptake rates (7% on first day and 4% on second day) and the average oxygen uptake rate for the control replicates of 19 mg O2/g dry weight/hour on the first day and 23 mg O2/g dry weight/hour on the second day.

 

The inhibition percentage of test substance was 3, 18, 28, 64, 94 and 96 at 19.3, 38.7, 77.4, 154.7, 309.4 and 618.9 mg/L, respectively.

 

Therefore, the EC50 of the test substance for activated sludge after 3 hours contact time is 106 mg/L with 95% confidence limits of 44 and 247 mg/L. The EC10, EC20 and EC80 are 32, 48 and 234 mg/L, respectively. The test substance is therefore considered not harmful to activated sludge.

Description of key information

The EC50 of the test substance for activated sludge after 3 hours contact time is 106 mg/L. The EC10, EC20 and EC80 are 32, 48 and 234 mg/L, respectively. The test substance is therefore considered not harmful to activated sludge.

Key value for chemical safety assessment

EC50 or LC50 for microorganisms:
106 mg/L
EC10, LC10 or NOEC for microorganisms:
32 mg/L

Additional information

An activated sludge respiration inhibition test was available and it was performed according to Guideline OECD 209 under GLP, at the following test concentrations: 19.3, 38.7, 77.4, 154.7, 309.4 and 618.9 mg/L.

The test was valid and the inhibition percentage of the test substance was 3, 18, 28, 64, 94 and 96 at 19.3, 38.7, 77.4, 154.7, 309.4 and 618.9 mg/L, respectively.

The EC50 of the test substance for activated sludge after 3 hours contact time is 106 mg/L with 95% confidence limits of 44 and 247 mg/L. The EC10, EC20 and EC80 are 32, 48 and 234 mg/L, respectively. The test substance is therefore considered not harmful to activated sludge.