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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 Nov 2015 to 2 Feb 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
2010
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
2008
Principles of method if other than guideline:
Because the test substance solution in the test vessel were foaming during aeration 25 μL/test vessel of Triisobutylphosphate was added to all test vessels.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Name of test material (as cited in study report): Fatty acids, C12-14, a-sulfo, disodium salts
- Batch No.: Ra-He 2014-054
- Purity: 100 % UVCB
- Identity: confirmed by NMR, IR and UV spectroscopies
- Homogeneity: Homogeneous
- Expiry date: Oct 2016
- Date of production: Oct 2014
- Physical state/apperance: Solid / beige
- Storage conditions: Ambient at room temperature
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The test substance was weighed in the required amounts for the test concentrations directly to the test vessels.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Origin: aeration tank of the wastewater treatment plant of Mannheim, Germany. Sludge was collected at 20 Jan 2016 and arrived in the test facility on the same date.
- Preparation of inoculum for exposure: After arrival of the activated sludge suspension in the test facility the suspension was sieved with a fine woven mesh (mesh size about 1 mm). This suspension was pre-aerated over night at room temperature. At the next day the sludge suspension was washed once with drinking water and the suspension was adjusted to 3 g/L Dw.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
19.8 - 20.5 °C
pH:
7.1 - 7.9
Dissolved oxygen:
- Dissolved O2 at start of the test: 5.5 - 9.2 mg/L
- Dissolved O2 at termination of the test: 0.9 - 8.9 mg/L
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 62.5, 125, 250, 500 and 1000 mg/L (based on test guidelines)
- Test concentrations were not analytically verified.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glas-beakers (nominal volume 1L)
- Volume: 500 mL, 16 mL/test vessel of 100-fold concentrated OECD medium
- Aeration: Yes
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per concentrations of the reference substance (replicates): 2
- Sludge concentration in the test vessel: 1.5 g/L (dry weight)

PREPARATION OF TEST
The test substance was added in the required amounts according to the test concentrations directly to the test vessels with 234 mL deionized water. Aliquots of the reference substance stock solution were dosed to the test vessels and made up with deionized water to a volume of 234 mL. 16 mL synthetic medium were dosed to each test vessel with test substance and reference substance afterwards. To prepare the blank control assays 234 mL of deionized water and 16 mL synthetic medium were mixed. The pH-values in all test vessels were checked and adjusted. After addition of 250 mL of inoculum suspension (3 g/L DW) the incubation was started by aeration of the test vessels with pressured air. The vessels for the blank control assays were prepared to the same procedure without addition of test- or reference substance.

EFFECT PARAMETERS MEASURED: respiration rate (oxygen consumption)
After 3 hours incubation at 20 ± 2°C the mixtures in the test vessels were placed subsequently for oxygen measuring. The temperature was measured for seven times in a separate vessel filled with deionized water. The content of oxygen at the start of the measurements was > 7 mg/L. No abiotic control was tested. The values of oxygen consumption of two test assays were set as outliers and were not used for test result calculation. The oxygen consumption was determined. Duration of the measurement was 8 - 10 minutes with some exceptions (see 'Any other information on materials and methods incl. tables'). Furthermore, the pH was determined.

Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
260 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% C.L.: 154.1 - 451.3 mg/L
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
Inhibition of the respiration rate was 1% in the lowest test concentration and increased in a dose-dependent manner up to 41% inhibition at 1000 mg/L. See 'Any other information on results incl. tables'.
Results with reference substance (positive control):
The 3-h EC50 value for the reference substance was determined to be 6.8 mg/L. See 'Any other information on results incl. tables'.
Reported statistics and error estimates:
The consumption rates were used for the determination of the ECx by the probit method based on Finney (Finney, D.J., Probit Analysis; Cambr. Univ. Press, 3rd ed., 1971) with the software TOXRAT Professional 2.10. The effect concentrations weregiven with an accuracy of 2 significant digits.

Table: Measured data of oxygen content of the test assays at the start and end of the evaluation time

Test substance concentration [mg/L]

0 (mean blank control)

62.5

125

250

500*

1000*

mean O2 conc. Start [mg/L]

6.7

6.4

6.8

6.4

6.6

7.0

mean O2 conc. End [mg/L]

1.6

1.8

2.0

1.5

2.4

3.9

mean O2 consumption rate [mg/L]

5.2

5.4

6.0

6.2

6.2

6.0

mean O2 consumption in minutes

6.0

4.5

4.8

4.9

4.3

3.1

mean O2 consumption rate [mg/L*h], RT

51.5

51.0

47.7

48.0

41.5

30.5

mean specific O2 consumption rate [mg/g*h]

34.5

 

34.0

31.7

32.0

27.5

20.5

Calculation of mean inhibition respiration [%], IT

 

1.0

7.7

7.0

19.0

41.0

IT= inhibition of the total respiration [%]

RT = oxygen consumption in the test assay [mg O2/L×h]

* The result of one of three test vessels was identified as outlier and not included in the calculation.

Table: Measured data of oxygen content of the reference subsstance

Reference substance concentration [mg/L]

1

10

100

Mean O2 conc. Start [mg/L]

6.85

8.65

9.1

Mean O2 conc. End [mg/L]

2.2

6.65

8.75

Mean O2 consumption rate [mg/L]

4.65

2

0.35

Mean O2 consumption in minutes

6

6

5.75

Mean O2 consumption rate [mg/L*h], RT

46.5

20

3.5

Mean specific O2 consumption rate [mg/g*h]

31

13

2.5

Calculation of mean inhibition respiration [%], IT

10

61

93

IT= inhibition of the total respiration [%]

RT = oxygen consumption in the test assay [mg O2/L×h]

ADDITIONAL TEST RESULTS TEST SUBSTANCE

- EC20: 680 mg/L

- EC80: >1000 mg/L

- EC90: 2600 mg/L

Validity criteria fulfilled:
yes
Remarks:
See 'Any other information on materials and methods incl. tables'

Description of key information

The 3-h EC50 and 3-h EC10 values are 1000 and 260 mg/L, respectively, as observed in a respiration inhibition study with activated sludge of a predominantly domestic sewage.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
260 mg/L

Additional information

The toxicity of the substance to microorganisms was investigated in an activated sludge respiration inhibition study according to OECD TG 209 and in compliance with GLP criteria. In this study activated sludge from a municipal wastewater treatment plant was exposed to nominal test substance concentrations of 0 (control), 62.5, 125, 250, 500 and 1000 mg/L for 3 hours. Test concentrations were not analytically verified. Oxygen consumption was measured over a period of 8 -10 minutes. The respiration rate was calculated using the measured oxygen consumption and compared to an unexposed control. Inhibition of the respiration rate was 1% in the lowest test concentration and increased in a dose-dependent manner up to 41% inhibition at 1000 mg/L. Based on these results, 3-h EC50 and EC10 values of > 1000 mg/L and 260 mg/L were derived, respectively.