Registration Dossier

Diss Factsheets

Ecotoxicological information

Toxicity to microorganisms

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02-06-2014 to 03-09-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met with acceptable minor deviations (the specific oxygen uptake rate in the control was < 20 mg O2/g; however the reference control item gave EC50 effectvalues in required range).
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
The mean specific oxygen uptake rate of the control replicates was 17 mg O2/gh (validity criterion: should be: 20 mg O2/gh). Was not considered to be a significant deviation as the reference item EC50 was in the required range.
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
yes
Remarks:
see above.
Principles of method if other than guideline:
The effect of the test substance on the inhibition of respiration rate of aerobic wastewater microorganisms of activated sludge was investigated. The test organisms were exposed to a series of test concentrations of the test substance for 3 hours and the effects on oxygen up take were measured. Results are reported relative to the reference standard controls: copper (II) sulphate pentahydrate.
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: June 2013; signature: November 2013
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 10 - 32 - 100 - 320 - 1000 mg/l were pipetted directly in Erlenmeyer flasks.
- Eluate:
- Differential loading:
- Controls: Test medium without test or reference item
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): n/a
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): n/a
- Evidence of undissolved material (e.g. precipitate, surface film, etc): not reported
Test organisms (species):
activated sludge
Details on inoculum:
- Laboratory culture: No.
- Method of cultivation: aerobic activated sludge from municipal sewage treatment plant of 31137 Hildesheim, Germany.
- Preparation of inoculum for exposure: The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %.
- Pretreatment: The sludge was used within 24 h after sampling. The dry sludge concentration was 2.81 g/L ; corresponding to 1.41 g/L in the test vessel. The test item was not pretreated. The reference item was ultrasonicated.
- Other: Synthetic waste water was prepared according to OECD 209. The pH value of the activated sludge was determined prior to test
start. The pH value of the synthetic waste water was determined prior to use. Adjustment to 7.5 ± 0.5 was not necessary.
- Initial biomass concentration: Dry sludge concentration 2.81 g/L ; suspended solids 1.41 g per liter (aerobic sludge per litre water).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
nominal 18 - 22 or 20 ± 2°C; actual: 21.7 °C
pH:
The pH value of the activated sludge was determined prior to test start. The pH value of the synthetic waste water was determined prior to use. Adjustment to 7.5 ± 0.5 was not necessary.
pH value of test medium measured during the preliminary study was pH = 7.60 at final (test vessel 1 at 1000 mg/L nominal test item)
pH-value of the activated sludge: 7.77
pH-value of the synthetic waste water: 7.36
Dissolved oxygen:
Dissolved oxygen concentrations: 100 mg/L nominal test item: 24.11 – 26.55 mg O2 /L before incubation and 79.79 – 94.14 mg O2 /L after 3h incubation (preliminary non-GLP test).
During the preliminary and definitive test: aeration was employed by shaking of flasks at 150 rpm to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension.
Nominal and measured concentrations:
Preliminary test: 10, 100 and 1000 mg/L nominal (test item) (non-GLP similar test item; described in the full study report).
The nominal concentrations tested were: 0 (control), 10, 32, 100, 320 and 1000 mg/L (in five replicates per concentration)
The nominal concentrations of reference inhibitor copper (II) sulphate pentahydrate were 58, 100 and 180 mg/L (three replicates per concentration)
Details on test conditions:
TEST SYSTEM
- Test vessel: 1000 ml Erlenmeyer flasks, DIN 12380
- Type (delete if not applicable): Open, vessel continuously aerated
- Material, size, headspace, fill volume: 1000 ml flask; fill volume 500 ml
- Aeration: Shaking of flasks at 150 rpm to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable.
- Renewal rate of test solution (frequency/flow rate): None.
- No. of organisms per vessel: Not applicable.
- No. of vessels per concentration (replicates): Five
- No. of vessels per control (replicates): Five (negative) and one (reference item)
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: dry solids concentration of sludge: 2.81 g/L and dry solid concentration in test solution: 1.41 g/L.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic sewage water was in accordance with OECD TG 209
- Culture medium different from test medium: Yes, chlorine free tap water used for culture medium; synthetic wastewater and RO used for test medium. Replaced during inoculum pretreatment stage and media preparation.
- 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 and dissolved oxygen values was determined in the 100 mg/L test item vessels only and or within preliminary test.

OTHER TEST CONDITIONS
- Adjustment of pH: No.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Monitor the oxygen consumed by the test and control mixtures following a 3-hour exposure phase. The oximeter instrument measured the oxygen depletion for at least 3 minutes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approx. 3.2
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: mean- 7.5% respiration inhibition at 10 mg/l, mean 16 % respiration inhibition at 100 mg/L, mean 73% respiration inhibition at 1000 mg/L.
Reference substance (positive control):
yes
Remarks:
copper (II) sulphate pentahydrate
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
189 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% CL 177-303 mg/l
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
65.7 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% CL 60.3 - 71.4 mg/l
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None reported.
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50 93.5 mg/L
- Other: 58 mg/L: 28% inhibition; 100 mg/L: 50% inhibition; 180 mg/lL 85% inhibition
Reported statistics and error estimates:
The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) and DUNNETT'S test was used for NOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05. The EC-values of the test item and the reference item were calculated by sigmoidal dose-response regression and linear regression, respectively using software. Calculations of the confidence intervals for the EC-values were carried out..

Table 1. Oxygen Uptake Rates, Inhibition of the Respiration of the Control and Test Item Concentrations after 3 h Exposure Time (Inhibition of Total Respiration) and reference item

Test Item Concentration [mg/L]

Replicate

Oxygen Uptake Rate (R) [mg O2/L h]

Inhibition [%]

Mean Inhibition [%]

Control

1

21.2

-

-

 

2

25.6

-

 

3

26.0

-

 

4

23.6

-

 

5

25.6

-

 

6

24.8

-

10

1

25.6

-4

-5

 

2

26.8

-9

 

3

24.4

0

 

4

26.0

-6

 

5

25.6

-4

32

1

25.6

-4

-2

 

2

24.8

-1

 

3

25.2

-3

 

4

24.8

-1

 

5

24.4

0

100

1

18.0

27

24

 

2

18.8

23

 

3

19.2

22

 

4

18.4

25

 

5

19.2

22

320

1

8.8

64

66

 

2

9.2

62

 

3

8.0

67

 

4

7.2

71

 

5

8.4

66

1000

1

6.0

76

78

 

2

5.6

77

 

3

5.6

77

 

4

5.2

79

 

5

4.8

80

Reference Item [mg/L]

 

 

 

 

58

1

17.6

28

28

 

2

17.6

28

 

3

18.0

27

100

1

12.8

48

50

 

2

12.0

51

 

3

12.0

51

180

1

4.0

84

85

 

2

3.6

85

 

3

3.6

85

Coefficient of variation of oxygen uptake rates of the control: 7.4%

Suspended solids in the test vessel: 1.41 g/L

 

Table 2. Specific respiration rates of the control replicates

 

Replicate

Specific Respiration Rate (Rs) [mg O2 / g h]

Contraol

1

15

2

18

3

17

4

18

5

18

Mean

 

17

Suspended solids in the test vessel: 1.41 g/L

Validity criteria fulfilled:
yes
Conclusions:
A 3 hour EC50 of 189 mg/L (nominal) was reported. The NOEC was 32 mg/L (nominal) with no significant inhibition of respiration rates below this concentration determined under the conditions of the study.
Executive summary:

The effect on respiration rate of activate sludge was examined using a method according to OECD TG 209 in accordance with GLP. Samples of activated sludge (suspended solids 1.41 g/L, were exposed to dilutions of the test substance for three hours under static conditions with the concentrations: 10, 32, 100, 320 and 1000 mg/L. Their rates of oxygen consumption were determined using an oximeter and were compared with controls that contained activated sludge alone. The nominal test substance concentrations used in the study were 10, 32, 100, 320 and 1000 mg/L; five replicates were prepared for each concentration and six for the control. To check the activity of the test system and the test conditions single triplicates of the reference inhibitor copper (II) sulphate were used at 58, 100 and 180 mg/L. The mean oxygen update rate (Rs) of the control cultures incubated alongside the test mixtures was 17.0 mg O2/gh. The oxygen uptake of activated sludge from municipal treatment plants is influenced by seasonal fluctuations and fluctuations in sewage flow. The lower oxygen uptake rate reflects the range of common uptake rates of activated sludge from municipal treatment plants and was considered to have no impact on quality and integrity of the study. Despite the lower respiration, the EC50-value of the reference item was in the required range. Therefore no impact on the results of the test item was expected through lower respiration within the study. The three-hour 50% effect concentration (EC50) for copper (II) sulphate was 93.5 mg/L. These results show that the sample of activated sludge employed was sensitive to inhibition and that the test was valid. The mean inhibition of respiration for the test item replicates ranged from -5 % to 78 %. The test item was not toxic to activated sludge of a municipal sewage treatment plant at the concentration of 32.0 mg/L. The EC10 was 65.7 mg/L and the EC20 was 90.8 mg/L. The EC50 was 189 mg/L.

Description of key information

EC50 = 189 (C.I 177 - 303) mg/L (nominal), 20 °C, OECD TG 209, 2014

EC10 = 65.7 (C.I. 60.3 -71.4) mg/L (nominal), 20°C, OECD TG 209, 2014

NOEC = 32.0 mg/L (nominal), 20 °C, OECD TG 209, 2014

Key value for chemical safety assessment

EC50 for microorganisms:
189 mg/L
EC10 or NOEC for microorganisms:
32 mg/L

Additional information

The effect on respiration rate of activate sludge was examined using a method according to OECD TG 209 in accordance with GLP. Samples of activated sludge (suspended solids 1.41 g/L, were exposed to dilutions of the test substance for three hours under static conditions with the concentrations: 10, 32, 100, 320 and 1000 mg/L. Their rates of oxygen consumption were determined using an oximeter and were compared with controls that contained activated sludge alone. The nominal test substance concentrations used in the study were 10, 32, 100, 320 and 1000 mg/L; five replicates were prepared for each concentration and six for the control. To check the activity of the test system and the test conditions single triplicates of the reference inhibitor copper (II) sulphate were used at 58, 100 and 180 mg/L. The mean oxygen update rate (Rs) of the control cultures incubated alongside the test mixtures was 17.0 mg O2/gh. The oxygen uptake of activated sludge from municipal treatment plants is influenced by seasonal fluctuations and fluctuations in sewage flow. The lower oxygen uptake rate reflects the range of common uptake rates of activated sludge from municipal treatment plants and was considered to have no impact on quality and integrity of the study. Despite the lower respiration, the EC50-value of the reference item was in the required range. Therefore no impact on the results of the test item was expected through lower respiration within the study. The three-hour 50% effect concentration (EC50) for copper (II) sulphate was 93.5 mg/L. These results show that the sample of activated sludge employed was sensitive to inhibition and that the test was valid. The mean inhibition of respiration for the test item replicates ranged from -5 % to 78 %. The test item was not toxic to activated sludge of a municipal sewage treatment plant at the concentration of 32.0 mg/L. The EC10 was 65.7 mg/L and the EC20 was 90.8 mg/L. The EC50 was 189 mg/L.

Categories Display