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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2017-12-06 to 2018-05-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
Qualifier:
according to
Guideline:
other: OCSPP 850.3300, "Modified Activated Sludge, Respiration Inhibition Test"
Version / remarks:
January, 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
- Just before the start of the test defined amounts (5 x 1; 5 x 3; 5 x 10; 5 x 30; 5 x 96 and 5 x 300 mg) of the test item corresponding to 3.2, 10, 32, 100, 320 and 1000 mg/L concentrations were administered directly into empty containers that were filled up with water and synthetic sewage (to a final volume of 300 mL), just before the inoculation. Concentrations in excess of nominal 1000 mg test item/L were not tested.
- Based on the preliminary experience, the test item affects adversely the pH within the test system; therefore, a second series of test containers were investigated at the concentrations of 100, 320 and 1000 mg/L (with five replicates each). In these additional containers pH adjustment (a neutralization step with 1N HCl) was performed prior to inoculum addition.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary on 21 March 2018 (one day before the main test)
- Preparation of inoculum for exposure:
The coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined. Based on this ratio, calculated amount of wet sludge was suspended in isotonic saline solution to yield a concentration equivalent to 3 g per litre (on dry weight basis).
(In the test containers the final concentration of suspended solids was 1.5 g per litre.)
The activated sludge was not used on the day of the collection but it was continuously aerated (2 L/minute) at the test temperature for about 24 hours (1 day) and was fed once with 50 mL synthetic sewage/L activated sludge.
- Initial biomass concentration: 3 g/L (on dry weight basis)
- pH of the activated sludge: 7.53
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
19.5 - 22.0 °C
pH:
before pH adjustment: 8.54 - 8.76 at 100 mg/L, between 9.90 - 9.96 at 320 mg/L and it varied between 10.88 - 10.90 at 1000 mg/L
after pH adjustment: 7.74 - 7.89
Dissolved oxygen:
7.07 - 8.90 mg/L
Nominal and measured concentrations:
without pH adjustment: 3.2, 10, 32, 100, 320 and 1000 mg/L
with pH adjustment: 100, 320 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer bottles
- Material, size, headspace, fill volume: glass, 300 mL volume
- Aeration: yes, with compressed air (0.5 litre per minute)
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 5 at the start and 5 at the end
- No. of vessels per abiotic control (replicates): 3 (only tested in preliminary experiment)
- Sludge concentration (weight of dry solids per volume): 3 g per litre (on dry weight basis)
- Nutrients provided for bacteria: synthetic sewage feed
- Nitrification inhibitor used: yes; N-allylthiourea, 3 replicates

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water

OTHER TEST CONDITIONS
- Adjustment of pH: yes, the test item was tested with and without pH adjustment

TEST CONCENTRATIONS
- Range finding study
Two preliminary range-finding tests were performed - with and without pH adjustment.
In the first pre-experiment the test item was investigated at the nominal concentrations of 10, 100 and 1000 mg/L. The pH of the solution of the test item (without inoculum) at concentration of 10 mg/L was in the acceptable range (pH 7 - 8); however, higher pH values (8.6 at 100 mg/L and 10.7 - 10.9 at 1000 mg/L) were noticed at the concentrations of 100 and 1000 mg/L (in test item treatments and abiotic controls). The test item treatments were not neutralized prior to inoculum addition.
To investigate the influence of higher pH values on the test system an additional pre-experiment was performed with the concentration levels of 1, 10, 100 and 1000 mg/L. Two parallel series were investigated: a series of test containers with pH setting and a second series without pH setting. Similarly to first pre-experiment the pH of the test item solutions (without inoculum) at the concentrations of 1 and 10 mg/L were in the acceptable pH 7 - 8 range and higher (pH 8.8 at 100 mg/L and 10.8 at 1000 mg/L) pH values were noticed at the concentrations of 100 and 1000 mg/L. The pH was adjusted to the range of pH 7 - 8 (in the series with pH setting, only) at the concentrations of 100 and 1000 mg/L before inoculation.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: without pH adjustment
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: with pH adjustment
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
248.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: without pH adjustment
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
1 261.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: with pH adjustment
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
618.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: without pH adjustment
Results with reference substance (positive control):
The 3-hour EC50 of 3,5-dichlorophenol was calculated to be 13.03 mg/L, (95 % confidence limits: 10.95 - 15.81 mg/L).
Reported statistics and error estimates:
The inhibition of the oxygen consumption rates allowed the calculation of the appropriate ECx values in the test series and without pH adjustment. Based on the available data the 3-hour EC10; EC50 and the EC80 values were calculated by Probit analysis using IBM® SPSS® Statistics, Using the same method the EC50 with pH adjustment was calculated.

Pre-experiments

In the first preliminary experiment the test item showed significant inhibitory effect: 88.3 % inhibition when compared to the blank control values, at the nominal concentration of 1000 mg/L. 32.4 % inhibition was noticed at the nominal concentration of 100 mg/L and 23.3 % at the lowest examined concentration of 10 mg/L.

In the second preliminary experiment the test item showed significant inhibitory effect without pH setting: 82.9 % inhibition when compared to the blank control values, at the nominal concentration of 1000 mg/L, in average 6.5 % inhibition was noticed at the nominal concentration of 100 mg/L and inhibition was not noticed at 10 mg/L and 1 mg/L concentrations. At 1000 mg/L concentration with pH adjustment 19.0 % inhibition was observed, the obtained inhibition/stimulation values at lower concentrations remained within the biological variability range of the applied test system.

Main test - test item

Inhibition was not noticed at the two lowest examined concentrations of 3.2 and 10 mg/L. At these concentrations slight stimulation (within the biological variability range of the applied test system) was noticed. The obtained 2.03 % inhibition at the concentration of 32 mg/L remained within the biological variability range of the applied test system.

Without pH setting, 4.69 % inhibition (within the biological variability range) was obtained at 100 mg/L, 23.63 % at 320 mg/L and 88.01 % at 1000 mg/L. With pH setting, inhibition was not noticed (the noticed slight stimulation remained within the biological variability range of the applied test system) at 100 and 320 mg/L and 28.04 % inhibition was detected at 1000 mg/L.

Table 1. The Percentage Inhibition (IT) of Total Oxygen Consumption

Identification

Test group

Concentration
(mg/L)

Inhibition (IT), of total oxygen consumption (%)

CB (A-J)

Blank Control

0.00

CN (A-C)

Nitrification Control

11.6 mg ATU/L

1.68

R1(A-C)

Reference Item

2 mg 3,5-DCP/L

3.17

R2 (A-C)

Reference Item

7 mg 3,5-DCP/L

25.95

R3 (A-C)

Reference Item

24.5 mg 3,5-DCP/L

74.09

T1 (A-E)

Test Item

3.2 mg Test Item/L

-0.83 #

T2 (A-E)

Test Item

10 mg Test Item/L

-0.73 #

T3 (A-E)

Test Item

32 mg Test Item/L

2.03

T4 (A-E)

Test Item

100 mg Test Item/L

4.69

T5 (A-E)

Test Item

320 mg Test Item/L

23.63

T6 (A-E)

Test Item

1000 mg Test Item/L

88.01

T4pH (A-E)

Test Item

100 mg Test Item/L

-1.54 #

T5pH (A-E)

Test Item

320 mg Test Item/L

-0.72 #

T6pH (A-E)

Test Item

1000 mg Test Item/L

28.04

#: In the subsequent calculations the negative value was taken into consideration as zero.

3,5-DCP:  3,5-dichlorophenol

ATU:          N-allylthiourea

Specific Respiration Rates - test item concentrations

Without pH adjustment the specific respiration rates did not differ statistically significantly from the blank control in the concentration range of 3.2 - 100 mg/L (Dunnett t-test, α=0.05). Without pH adjustment the NOEC can be statistically and biologically determined as 100 mg/L.

With pH adjustment the specific respiration rates did not differ statistically significantly from the blank control additionally at the concentration of 320 mg/L (Dunnett t-test, α=0.05). With an additional pH adjustment, neutralization step, the NOEC can be statistically and biologically determined as 320 mg/L.

Nitrification Controls - Inhibition of Respiration Rate

An additional nitrification control was examined in the main test with three parallels to check the possible nitrification potential of the applied activated sludge batch. At the nitrification control the differentiation between the total, heterotrophic and nitrification respiration was possible. The total respiration (RT) was 67.93 mg/Lh, the heterotrophic respiration (RH) was 66.79 mg/Lh, the nitrification respiration (RN): 1.14 mg/Lh was calculated according to the following equation:

RN = RT - RH.

The obtained 1.14 mg/Lh was considered as not significant difference within a biological variability range of the applied test system, and lower than the 5 % of RT (3.40 mg/Lh) in blank controls. According to the above calculations it was assumed that the heterotrophic oxygen uptake equals the total uptake.

Validity of the Study

The study was considered as valid because the specific respiration rate of the blank controls (without the test substance or reference substance) was 45.29 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (higher than 20 mg/gh) with a coefficient of variation of 5.19 %.

The 3-hour EC50 of the reference item 3,5-dichlorophenol (for the used activated sludge batch) was 13.03 mg/L within the range of 2 mg/L to 25 mg/L, that was required for total respiration.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the performed test, a strong test item effect on the pH was established. Without pH adjustment the EC50 value of test item was determined as 618.8 mg/L (95% confidence limits: 514.6 – 757.8 mg/L), (1000 mg/L > EC50 > 100 mg/L) and the NOEC calculated as 100 mg/L. With an additional pH adjustment, neutralization step, the EC50 became higher than 1000 mg/L (EC50 > 1000 mg/L) and the NOEC was determined as 320 mg/L.
Executive summary:

In a 3-hour test the influence of the test item on the activity of the activated sludge was determined according OECD TG 209, EU Method C.11 and EPA OCSSP 850.3300 by measuring the respiration rate under defined conditions.

The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours.

Based on the preliminary information that the test item caused effect on the activated sludge inoculum, the test item was investigated at the nominal concentrations of 3.2, 10, 32, 100, 320 and 1000 mg/L. Defined amounts of the test item were added (measured) directly into the test vessels. These test item concentrations were tested without pH adjustment.

Based on the preliminary experience, the test item affects adversely the pH within the test system; therefore, a second series of test containers were investigated at the concentrations of 100, 320 and 1000 mg/L. In these additional containers pH adjustment (an additional neutralization step to the required pH range of 7 - 8) was performed with 1N HCl prior to inoculum addition.

In parallel with the test item treatments with 3,5-dichlorophenol as positive reference control in a concentrations of 2, 7 and 24.5 mg/L ran; furthermore, blank (inoculum) control and nitrification controls were investigated. The main test was performed without abiotic controls, based on the results of the first preliminary test where abiotic controls were tested at the test item concentration of 1000 mg/L and no remarkable abiotic oxygen consumption was noticed.

Inhibition was not noticed at the two lowest examined concentrations of 3.2 and 10 mg/L. The obtained 2.03 % inhibition at the concentration of 32 mg/L remained within the biological variability range of the applied test system.

Without pH setting 4.69 % inhibition (within the biological variability range) was obtained at 100 mg/L, 23.63 % at 320 mg/L and 88.01 % at 1000 mg/L.

With pH setting, inhibition was not noticed at 100 and 320 mg/L and 28.04 % inhibition was detected at 1000 mg/L.

The degree of inhibitions (23.63 and 88.01 %) obtained without pH adjustment at the concentration levels of 320 and 1000 mg/L allowed the calculation of the 3-hour exact EC10, EC50 and EC80 values. The values are the following:

EC10:   248.5 mg/L        (133.9–346.1 mg/L)

EC50:   618.8 mg/L        (514.6–757.8 mg/L)

EC80:   862.0 mg/L        (728.3–1064.6 mg/L)

Without pH adjustment, the EC50 value was determined as 618.8 mg/L, consequently 1000 mg/L > EC50 > 100 mg/L.

With additional neutralization step 28.04 % inhibition was noticed at 1000 mg/L. In this case the EC50 value was estimated as higher than 1000 mg/L. 

The specific respiration rates were compared with the blank control values. Without pH adjustment the specific respiration rates did not differ statistically significantly from the blank control in the concentration range of 3.2 - 100 mg/L. Without pH adjustment the NOEC can be statistically and biologically determined as 100 mg/L. With pH adjustment the specific respiration rates did not differ statistically significantly from the blank control additionally at the concentration of 320 mg/L. With an additional pH adjustment, neutralization step, the NOEC can be statistically and biologically determined as 320 mg/L.

Description of key information

Under the conditions of the performed test, a strong test item effect on the pH was established. Without pH adjustment the EC50 value of test item was determined as 618.8 mg/L (95% confidence limits: 514.6 – 757.8 mg/L), (1000 mg/L > EC50 > 100 mg/L) and the NOEC calculated as 100 mg/L. With an additional pH adjustment, neutralization step, the EC50 became higher than 1000 mg/L (EC50 > 1000 mg/L) and the NOEC was determined as 320 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 261.9 mg/L
EC10 or NOEC for microorganisms:
320 mg/L

Additional information

In a 3-hour test the influence of the test item on the activity of the activated sludge was determined according OECD TG 209, EU Method C.11 and EPA OCSSP 850.3300 by measuring the respiration rate under defined conditions.

The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours.

Based on the preliminary information that the test item caused effect on the activated sludge inoculum, the test item was investigated at the nominal concentrations of 3.2, 10, 32, 100, 320 and 1000 mg/L. Defined amounts of the test item were added (measured) directly into the test vessels. These test item concentrations were tested without pH adjustment.

Based on the preliminary experience, the test item affects adversely the pH within the test system; therefore, a second series of test containers were investigated at the concentrations of 100, 320 and 1000 mg/L. In these additional containers pH adjustment (an additional neutralization step to the required pH range of 7 - 8) was performed with 1N HCl prior to inoculum addition.

In parallel with the test item treatments with 3,5-dichlorophenol as positive reference control in concentrations of 2, 7 and 24.5 mg/L ran; furthermore, blank (inoculum) control and nitrification controls were investigated. The main test was performed without abiotic controls, based on the results of the first preliminary test where abiotic controls were tested at the test item concentration of 1000 mg/L and no remarkable abiotic oxygen consumption was noticed.

Inhibition was not noticed at the two lowest examined concentrations of 3.2 and 10 mg/L. The obtained 2.03 % inhibition at the concentration of 32 mg/L remained within the biological variability range of the applied test system.

Without pH setting 4.69 % inhibition (within the biological variability range) was obtained at 100 mg/L, 23.63 % at 320 mg/L and 88.01 % at 1000 mg/L.

With pH setting, inhibition was not noticed at 100 and 320 mg/L and 28.04 % inhibition was detected at 1000 mg/L.

The degree of inhibitions (23.63 and 88.01 %) obtained without pH adjustment at the concentration levels of 320 and 1000 mg/L allowed the calculation of the 3-hour exact EC10, EC50 and EC80 values. The values are the following:

EC10:   248.5 mg/L        (133.9–346.1 mg/L)

EC50:   618.8 mg/L        (514.6–757.8 mg/L)

EC80:   862.0 mg/L        (728.3–1064.6 mg/L)

Without pH adjustment, the EC50 value was determined as 618.8 mg/L, consequently 1000 mg/L > EC50 > 100 mg/L.

With additional neutralization step 28.04 % inhibition was noticed at 1000 mg/L. In this case the EC50 value was estimated as higher than 1000 mg/L. 

The specific respiration rates were compared with the blank control values. Without pH adjustment the specific respiration rates did not differ statistically significantly from the blank control in the concentration range of 3.2 - 100 mg/L. Without pH adjustment the NOEC can be statistically and biologically determined as 100 mg/L. With pH adjustment the specific respiration rates did not differ statistically significantly from the blank control additionally at the concentration of 320 mg/L. With an additional pH adjustment, neutralization step, the NOEC can be statistically and biologically determined as 320 mg/L.