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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07-03-2018 to 12-06-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was a brown powder and was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to the dark brown test bottles containing 200 mL Milli-RO water (tap water purified by reverse osmosis). The test item – Milli-RO water mixtures were magnetically stirred for a short period and subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added resulting in the required concentrations. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring. The deployed concentrations were: Range Finder concentrations: 10, 100, 1000 mg/L ; Definitive Test concentrations: 0 (Control), 3.2, 10, 32, 100, 320 and 1000 mg/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None in the prepared solutions. See comments above.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: Micro-organism in aerobic activated sludge
- Name and location of sewage treatment plant where inoculum was collected: aerobic activated sludge from sewage treatment plant at Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, which treats predominantly domestic sewage (dates provided in the full study report).
- Method of cultivation: Not applicable.
- Preparation of inoculum for exposure: 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 7.6 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.
- Pretreatment: See above.
- Initial biomass concentration: Determination of the suspended solids level of the activated sewage sludge was carried out. The suspended solids concentration was equal to 3.0 g/L prior to use. The amount of suspended solids in the final test mixture was 1.5 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Remarks on exposure duration:
During 3 hour exposure aeration and stirring took place.
Post exposure observation period:
The synthetic medium (16 mL) made up to 50 mL with Milli- RO and 200 mL test item solution were mixed (total volume 250 mL). The pH was determined. Thereafter, 250 mL activated sludge was added. This was the start of the test. After the 3-hour contact time the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference item concentrations and controls. The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference item concentrations and controls. The medium temperature was recorded continuously in temperature control vessels.
Test temperature:
20 ± 2 °C
pH:
0 hours: control: 7.3 and test groups: 7.0 - 7.2 and reference item: 7.3 - 7.5
3 hours: control: 7.6 - 7.8 and test groups: 7.8 - 8.2 and reference item: 7.8 - 8.2
Dissolved oxygen:
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.
Nominal and measured concentrations:
Range Finder concentrations: 10, 100, 1000 mg/L (in single vessels, three replicates for 1000 mg/L and three for nitrification control, single vessel for abiotic control and six for blank control).
Definitive Test concentrations: 3.2, 10, 32, 100, 320 and 1000 mg/L (in five replicates; six for control)
Reference item was completed: nominal: 1.0, 3.2, 10, 32 mg/L (in single vessels replicates)
Details on test conditions:
TEST SYSTEM
- Test vessel: 0.5 L glass bottle
- Type (delete if not applicable): Open, vessel continuously aerated
- Aeration: The aeration was adjusted in such a way that the dissolved oxygen concentration at the start is above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not reported.
- Renewal rate of test solution (frequency/flow rate): None.
- No. of vessels per concentration (replicates): Five
- No. of vessels per control (replicates): Six (negative) and single (reference item)
- Nitrification inhibitor used (delete if not applicable): The final test was a standard test without nitrification inhibitor and abiotic conditions. In the combined limit/range finder test: There was no oxygen uptake from abiotic processes and the result at 1000 mg/L with a nitrification inhibitor showed that heterotrophic inhibition of the respiration rate was slightly lower than total inhibition. Based on the results of total and heterotrophic respiration, nitrification inhibition was calculated to be 100%.
Since there is a clear dose-response curve in the combined range finding, (no bi-phasic or distorted dose-response curve) the final study was performed without determination of heterotrophic and nitrification inhibition.
- Biomass loading rate: See table.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for the range-finding and definitive tests was deionised reverse osmosis water containing less than 1 mg/L Total Organic Carbon (TOC). Synthetic sewage was subsequently prepared.
- Culture medium different from test medium: Not applicable. Adjusted ISO-medium, formulated using RO water was used as the medium.
- Intervals of water quality measurement: pH and temperature were determined in all test media and controls; prior to and at the end of the 3-hour incubation period.

OTHER TEST CONDITIONS
- Adjustment of pH: No. The pH in all test vessels, before addition of sludge, was between 7.0 and 7.5. After the 3 hour exposure period the pH was between 7.6 and 8.2.
- Light intensity: The test was conducted under normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Respiration Rates. Monitor the oxygen consumed by the test and control mixtures following a 3-hour exposure phase. Specifically measuring: Respiration Rate and % Inhibition of the Respiration Rate.

TEST CONCENTRATIONS
- Test concentrations: Definitive study: 0 (control), 3.2, 10, 32, 100, 320 and 1000 mg/L (nominal) (in five replicates; six for control)
- Range finding study: 0, (control) 10, 100, 1000 mg/L (nominal) (in single vessels, three replicates for 1000 mg/L and six for control).
- Results used to determine the conditions for the definitive study: Yes.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
104 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other:
Remarks:
C.I. 96 - 114 mg/L
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
21 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other:
Remarks:
C.I. 19 - 24 mg/L
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
9.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other:
Remarks:
C.I. 7.6 - 11 mg/L
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
Validity Criteria:
1. The controls oxygen uptake rate exceeded 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour. Actual: 31 mg oxygen per one gram of activated sludge in the final test
2. The coefficient of variation of oxygen uptake in control replicates did not exceed 30% at the end of the definitive test. Actual: 14%.
3. The reference substance results were valid, the EC50 for 3,5-dichlorophenol was : total respiration: actual 3.7 (C.I. 1.7 – 8.1) mg/L. This was within the expected range: 2 to 25 mg/L.
Therefore, the validity criteria was met.
Results with reference substance (positive control):
- Results with reference substance valid?: Yes.
- Relevant effect levels: the EC50 for 3,5-dichlorophenol was: total respiration: actual 3.7 (C.I. 1.7 – 8.1) mg/L. This was within the expected range: 2 to 25 mg/L. Full information is provided in the full study report.
Reported statistics and error estimates:
For the test item, calculation of the ECx value was based on probit analysis using linear maximum likelihood regression, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the test item.

For the reference item, calculation of the EC50 value was based on a 3-parameter normal cumulative distribution function (CDF) using non-linear regression analysis, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item.

NOEC: An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the control revealed significant inhibition of the respiration rate (Step-down Jonckheere-Terpstra Test Procedure, α=0.05, one-sided, smaller).

Software packaage: ToxRat Professional v. 3.2.1

Table 1.0 : Definitive Test – summarised study results

Treatment

Concentration

(mg/L)

pH before sludge addition

pH after 3 h contact time

Mean respiration rate

% Inhibition of the respiration rate (mean value)

(mg O2/L h)

(mg O2/g h)¹

Control

 

7.3

7.6 – 7.8

47.21

31.47

 

T1

3.2

7.2 – 7.3

7.8

42.50

28.34

9.97

T2

10

7.2

7.8 – 7.9

41.04

27.36

13.06

T3

32

7.2

7.8 – 7.9

37.27

24.85*

21.05

T4

100

7.2

8.1

23.55

15.70*

50.12

T5

320

7.1

8.2

12.41

8.27*

73.72

T6

1000

7.0

8.1

5.44

3.63*

88.48

¹) The amount of suspended solids in the final test mixture was 1.5 g/L.

* Statistically significantly different compared to control.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the 3-hour EC50 for total inhibition was 104 (C.I. 96 - 114) mg/L. The EC20 was 21 (C.I. 19 – 24) mg/L, the EC10 was 9.2 (C.I. 7.6 – 11) mg/L. The NOEC was 10 mg/L with no significant inhibition of respiration rates below this concentration determined under the conditions of the study. All effect levels were based on nominal test item concentrations.
Executive summary:

The effect on respiration rate of activate sludge was examined using a method according to OECD TG 209 in accordance with GLP. Following a preliminary combined limit/range finding test at 0 (control), 10, 100 and 1000 mg/L concentrations, a definitive test was conducted examining total respiration only was carried out under static conditions. The Definitive Test concentrations were 0 (control), 3.2, 10, 32, 100, 320 and 1000 mg/L (in five replicates per test item concentration and six for control) for a period of three hours at a temperature of 20 ± 2 °C with the addition of synthetic sewage as respiratory substrate. The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to the test bottles containing Milli-RO water. The test item – Milli-RO water mixtures were magnetically stirred for a short period and subsequently, synthetic medium, sludge and Milli - RO water were added resulting in the required concentrations. Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the 3 hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes. The respiration rates of the control, reference item (3,5-dichlorophenol) and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The coefficient of variation of oxygen uptake in the control vessels was < 30 % and the specific respiration rate of the controls was 31 mg oxygen per gram dry weight of sludge per hour. The reference substance results were valid, the EC50 for 3,5-dichlorophenol was: total respiration: actual 3.7 (C.I. 1.7 – 8.1) mg/L. This was within the expected range: 2 to 25 mg/L. All validity criteria were considered to have been satisfied. No statistically significant inhibition of the respiration rate of the sludge was recorded at or below a loading rate of 10 mg/L. At higher loading rates the inhibitory effect of test item on aerobic waste water (activated sludge) bacteria increased with increasing loading rate, ranging from 21% inhibition at a loading rate of 32 mg/L to 88% at a loading rate of 1000 mg/L (mean values). Under the conditions of the study, the 3-hour EC50 for total inhibition was 104 (C.I. 96 - 114) mg/L. The EC20 was 21 (C.I. 19 – 24) mg/L, the EC10 was 9.2 (C.I. 7.6 – 11) mg/L. The NOEC was 10 mg/L with no significant inhibition of respiration rates below this concentration determined under the conditions of the study. All effect levels were based on nominal test item concentrations.

Description of key information

ASRIT: EC50-3h = 104 mg/L (C.I. 96 - 114 mg/L) (nominal), 20 °C, OECD TG 209, 2018

ASRIT: EC20-3h = 21 mg/L (C.I. 19 - 24 mg/L) (nominal), 20°C, OECD TG 209, 2018

ASRIT: EC10-3h = 9.2 mg/L (C.I. 7.6 - 11 mg/L) (nominal), 20°C, OECD TG 209, 2018

ASRIT: NOEC-3h = 10 mg/L (nominal), 20 °C, OECD TG 209, 2018

Key value for chemical safety assessment

EC50 for microorganisms:
104 mg/L
EC10 or NOEC for microorganisms:
9.2 mg/L

Additional information

Key study: OECD TG 209, 2018 : The effect on respiration rate of activate sludge was examined using a method according to OECD TG 209 in accordance with GLP. Following a preliminary combined limit/range finding test at 0 (control), 10, 100 and 1000 mg/L concentrations, a definitive test was conducted examining total respiration only was carried out under static conditions. The Definitive Test concentrations were 0 (control), 3.2, 10, 32, 100, 320 and 1000 mg/L (in five replicates per test item concentration and six for control) for a period of three hours at a temperature of 20 ± 2 °C with the addition of synthetic sewage as respiratory substrate. The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to the test bottles containing Milli-RO water. The test item – Milli-RO water mixtures were magnetically stirred for a short period and subsequently, synthetic medium, sludge and Milli - RO water were added resulting in the required concentrations. Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the 3 hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes. The respiration rates of the control, reference item (3,5-dichlorophenol) and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The coefficient of variation of oxygen uptake in the control vessels was < 30 % and the specific respiration rate of the controls was 31 mg oxygen per gram dry weight of sludge per hour. The reference substance results were valid, the EC50 for 3,5-dichlorophenol was: total respiration: actual 3.7 (C.I. 1.7 – 8.1) mg/L. This was within the expected range: 2 to 25 mg/L. All validity criteria were considered to have been satisfied. No statistically significant inhibition of the respiration rate of the sludge was recorded at or below a loading rate of 10 mg/L. At higher loading rates the inhibitory effect of test item on aerobic waste water (activated sludge) bacteria increased with increasing loading rate, ranging from 21% inhibition at a loading rate of 32 mg/L to 88% at a loading rate of 1000 mg/L (mean values). Under the conditions of the study, the 3-hour EC50 for total inhibition was 104 (C.I. 96 - 114) mg/L. The EC20 was 21 (C.I. 19 – 24) mg/L, the EC10 was 9.2 (C.I. 7.6 – 11) mg/L. The NOEC was 10 mg/L with no significant inhibition of respiration rates below this concentration determined under the conditions of the study. All effect levels were based on nominal test item concentrations.