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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2012-08-16
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Qualifier:
according to
Guideline:
OECD Guideline 209 (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: A stock solution of 1 g/L was prepared and agitated.
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loading: 1 - 3.2 - 10 - 32 - 100 mg/L
- Controls: Test medium without test or reference item.
Test organisms (species):
activated sludge
Details on inoculum:
- Preparation of inoculum for exposure: Non adapted activated sludge from the sewage plant at Hildesheim (Municipal sewage treatment plant of 31137 Hildesheim, Germany)
- Pretreatment: Washed twice with chlorine free tap water. Adjustment to a dry sludge concentration of 3.9 g/L +/- 10 % was not necessary. The sludge was used within 24 h after sampling.
- Dry sludge concentration: 2.97 g/L corresponding to 1.49 g/L suspended solids in the test vessel.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
Not determined
Test temperature:
20.9 °C
pH:
7.99 (activated sludge)
7.55 (synthetic waste water)
Salinity:
None, freshwater
Details on test conditions:
TEST SYSTEM
- Test vessel: 1000 mL Erlenmeyer flask, DIN 12380
- Aeration: Permanent
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6 (3 at the beginning of the test and 3 at the end of the test)
- Composition of test medium:
Test concentration [mg a.i./L]
0.4 1.28 4.0 12.8 40.0
Inoculum 250
Synthetic waste water 16
Stock solution [mL] 0.5 1.6 5 16 50
Demineralised water 233.5 232.4 229 218 184

Reference item concentration [mg/L]
58 100 180
Inoculum 250
Synthetic waste water 16
Stock solution [µL] 96.7 166.7 300
Demineralised water [mL] 233.9 233.83 233.7



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After 3 h incubation, the oxygen concentration of each control. reference item and test item replicate was determined by means of oximeter immediately after transferring the solution to the measuring cell and the oxygen depletion was recorded for at least 3 minutes on a flat bed recorder.

TEST CONCENTRATIONS
- Range finding study
Inhibition of Respiration (Preliminary Tests)
Nominal Test Item Concentration Inhibition [%]
[mg/L]
10 16
100 95
1000 89

















Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate p.a.
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
5.95 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: (5.22-6.73)
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 115 mg/L
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 -value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

The EC-values of the test item were calculated by sigmoidal dose-response regression and the EC50-value of the reference item by linear regression using software GraphPadPrism. Calculations of the confidence intervals for the EC-values were carried out using standard procedures.

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration
of the Control, Test and Reference Item Concentrations

Test Item
Concentration

Repl.

Oxygen Uptake Rate
R
[mg O2/L
×h]

Specific Respiration Rate Rs
[mg O2/g
×h]

Inhibition


[%]

Mean Inhibition

[%]

[mg/L]

[mg a.i./L]

Control

1

32.4

21.7

2

32.4

21.7

3

32.4

21.7

4

30.4

20.4

5

28.8

19.3

6

28.4

19.1

Test Item

1

0.4

1

32.0

-4

-4

2

32.0

-4

3

32.4

-5

4

31.6

-3

5

31.6

-3

3.2

1.28

1

31.6

-3

-1

2

30.8

0

3

30.8

0

4

31.2

-1

5

31.2

-1

10

4.0

1

25.6

17

6

2

29.2

5

3

32.0

-4

4

30.0

3

5

28.0

9

32

12.8

1

24.4

21

33

2

20.0

35

3

19.6

36

4

19.2

38

5

19.6

36

100

40.0

1

3.2

90

90

2

3.2

90

3

3.2

90

4

2.8

91

5

3.2

90

Reference Item

58

1

24.8

19

19

2

24.8

19

3

24.8

19

100

4

16.8

45

45

5

17.2

44

2

16.8

45

180

3

10.8

65

69

4

10.4

66

5

7.2

77

EC-Values with Confidence Interval of DDAC

EC-values

[mg a.i./L]

Confidence interval
P = 95 [%]
[mg a.i./L]

NOEC*

4.0

EC10

5.95

5.22 - 6.73

EC20

8.90

8.06 - 9.89

EC50

17.9

16.4 - 19.5

Validity criteria fulfilled:
yes
Conclusions:
Under the test conditions, the EC10, EC20, EC50 and NOEC of the test substance for activated sludge respiration inhibition were determined at 5.95; 8.90; 17.9 and 4 mg a.i./L respectively.
Executive summary:

A study was conducted to determine the toxicity to microorganisms of the read across substance, DDAC, in an activated sludge respiration inhibition test according to OECD 209 Guideline, in compliance with GLP. The test system was activated sludge of a municipal treatment plant. The test was carried out under static conditions with substance concentrations of 0, 1.0 - 3.2 - 10 - 32 - 100 mg/L, corresponding to 0, 0.40 - 1.28 - 4.0 - 12.8 - 40.0 mg a.i./L. The respiration rates of the control, reference and test substance replicates were measured after a contact time of 3 h and the inhibitory effects of the test and reference substance were determined in comparison to the control respiration rates.The EC50 of the reference compound, copper (II) sulphate pentahydrate, was determined to be 115 mg/L, indicating validity of the test system.The mean inhibition of respiration for the test substance replicates ranged from -4% to 90%. The EC10, EC20 and EC50 of the test substance for activated sludge respiration inhibition were determined at 5.95 mg a.i./L, 8.90 mg a.i./L and 17.9 mg a.i./L respectively. No toxic effects were found at the lowest test concentration leading to the establishment of the NOEC at 4 mg a.i./L(Fiebig, 2012).Based on the results of the read across study with DDAC, the 3 h EC10 of C12-14 TMAC for activated sludge respiration inhibition can be considered to be equivalen to 5.95 mg a.i./L.

Description of key information

Based on the results of the read across study, the 3 h EC10 value of 5.95 mg a.i./L for activated sludge respiration inhibition has been considered further for hazard assessment of C12-14 TMAC.

Key value for chemical safety assessment

EC50 for microorganisms:
17.9 mg/L
EC10 or NOEC for microorganisms:
5.95 mg/L

Additional information

A study was conducted to determine the toxicity to microorganisms of the read across substance, DDAC, in an activated sludge respiration inhibition test according to OECD 209 Guideline, in compliance with GLP. The test system was activated sludge of a municipal treatment plant. The test was carried out under static conditions with substance concentrations of 0, 1.0 - 3.2 - 10 - 32 - 100 mg/L, corresponding to 0, 0.40 - 1.28 - 4.0 - 12.8 - 40.0 mg a.i./L. The respiration rates of the control, reference and test substance replicates were measured after a contact time of 3 h and the inhibitory effects of the test and reference substance were determined in comparison to the control respiration rates. The EC50 of the reference compound, copper (II) sulphate pentahydrate, was determined to be 115 mg/L, indicating validity of the test system. The mean inhibition of respiration for the test substance replicates ranged from -4% to 90%. The EC10, EC20 and EC50 of the test substance for activated sludge respiration inhibition were determined at 5.95 mg a.i./L, 8.90 mg a.i./L and 17.9 mg a.i./L respectively. No toxic effects were found at the lowest test concentration leading to the establishment of the NOEC at 4 mg a.i./L (Fiebig, 2012). Based on the results of the read across study with DDAC, the 3 h EC10 of C12-14 TMAC for activated sludge respiration inhibition can be considered to be equivalen to 5.95 mg a.i./L.