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Toxicity to microorganisms

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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 - 08 Nov 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
(2010)
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Rheinland Pfalz, Germany
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: test substance was warmed up in a water bath at 39.6 °C. Container was then shaken. Direct addition by initial weight into the test flasks followed.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: municipal wastewater treatment plant of Mannheim, Germany
- 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). Suspension was pre-aerated over night at room temperature. At the next day sludge suspension was washed once with drinking water and adjusted to 3 g/L dw
- Initial biomass concentration: 1.5 g/L dw
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
21.2 - 21.5 °C
pH:
7.4 - 7.5 (initial pH)
7.9 - 8.0 (pH at the end)
Dissolved oxygen:
7.2 - 8.0 mg/L (test start)
Nominal and measured concentrations:
0 (control), 182, 364, 729, 1458, 2915 mg/L (nominal test concentrations)
0 (control), 62.5, 125, 250, 500 and 1000 mg/L (a.i.)
Details on test conditions:
TEST SYSTEM
- Test vessel: 1 L glas beakers
- Fill volume: 500 mL (16 mL 100-fold concentrated synthetic medium with either test substance or reference substance + 234 mL deionized water + 250 mL inoculum suspension)
- Aeration: after preparation of the vessels incubation started by aeration of the vessels with pressured air
- Sludge concentration: 1.5 g/L dw
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per positive control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Intervals of water quality measurement: temperature was measured 8 times during the incubation in a separate test vessel with deionized water

EFFECT PARAMETERS MEASURED: oxygen uptake was measured for a period of about 8 to 10 min. Due to foaming of the test substance solution in the test vessel during aeration, 25 μL of triisobutyl sulfate was added in all test vessels as antifoam agent
Reference substance (positive control):
yes
Remarks:
3,5 dichlorophenol (1, 10, 100 mg/L)
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 2 915 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
140 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
410 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Results with reference substance (positive control):
- Results with reference substance valid: yes
- Relevant effect levels: EC50 = 3.5 mg/L
Reported statistics and error estimates:
O2 consumption rates were used for the determination of the ECx by the probit method with the software TOXRAT Professional 2.10.

EC80 > 1000 mg/L based on active ingredient (2915 mg/L based on nominal effect concentration)

Due to technical problems the measured values of the oxygen consumption after 3 hours of incubation of blank control 2 were lost.

Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
14 - 15 Mar 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Pseudomonas putida
Details on inoculum:
- Preparation of inoculum for exposure: before the batches were inoculated, the extinction of the bacterial preliminary culture solution was measured and this was diluted with test culture nutrient medium to an extinction of 0.15 approximately (original bacterial suspension: extinction 0.152 at 436 nm.
- Initial biomass concentration: extinction 0.15
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
17 h
Remarks on exposure duration:
approximately
Test temperature:
21.7 °C
pH:
7.2 - 8.3
Nominal and measured concentrations:
nominal: 1250, 2500, 3750, 5000, 7500, 10000 mg a.i./L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size: sterile 250 mL Erlenmeyer flasks, plugged with cotton wool
- No. of vessels per concentration (replicates): 3 parallels for each of the 6 test substance batches
- No. of vessels per control (replicates): 6 growth controls, 6 control batches, 1 pH control for each of the 6 test substance batches.

Reference substance (positive control):
no
Duration:
16 h
Dose descriptor:
EC10
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth inhibition
Duration:
16 h
Dose descriptor:
EC50
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth inhibition

Concentration [mg a.s./L]

Extinction mean values at 436 nm

Inhibition (%)

Control*

1.660

--

1250

1.686

-1.6

2500

1.646

0.9

3750

1.687

-1.6

5000

1.656

0.3

7500

1.588

4.4

10000

1.572

5.4

*cell multiplication: 1.644

Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
14 - 15 Oct 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Pseudomonas putida
Details on inoculum:
- Initial biomass concentration: extinction of 0.140 at a wave length of 436 nm
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
16 h
Remarks on exposure duration:
16 +/- 1 hour
Test temperature:
21.8 °C
pH:
before incubation: 5.6 - 6.2
after incubation: 6.8
Nominal and measured concentrations:
nominal content of active substance: 313, 626, 1251, 2502.5, 5005 and 10010 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL Erlenmeyer flasks filled with 100 mL test medium
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

Reference substance (positive control):
no
Duration:
16 h
Dose descriptor:
EC50
Effect conc.:
> 10 g/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth inhibition
Duration:
16 h
Dose descriptor:
EC10
Effect conc.:
> 10 g/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth inhibition

Test Concentration [mg a.s./L]

Inhibition [%]

313

-6.9

626

-7.3

1251

-4.7

2502.5

-2.9

5005

-0.9

10010

7.4

Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to the Category Approach Justification provided in Section 13
Reason / purpose:
read-across source
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other:
Remarks:
Source, key, 157627-95-7, 2016
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 2 915 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other:
Remarks:
Source, key, 157627-95-7, 2016
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
140 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other:
Remarks:
Source, key, 157627-95-7, 2016
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
410 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other:
Remarks:
Source, key, 157627-95-7, 2016

Description of key information

EC50 (3 h) activated sludge > 1000 mg/L a.i. (nominal, OECD 209)

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

For the toxicity to microorganisms no studies of AES (C12-14, 1-2.5 EO) TEA (CAS 157627-94-6) are available. Therefore, a read-across approach from AES (C16-18 and C18-unsaturated, 1-2.5 EO) Na (CAS 157627-95-7), AES (C12-14, 1-2.5EO) Na (CAS 68891-38-3) and AES (C12-14, 1-2.5EO) MIPA (CAS 1187742-72-8) was applied.

The key study investigated the toxicity of AES (C16-18 and C18-unsaturated, 1-2.5 EO) Na (CAS 157627-95-7) to activated sludge microorganisms (BASF, 2016). This GLP study was performed according to OECD guideline 209 with activated sludge from a municipal wastewater treatment plant. The respiration inhibition of the microorganisms was measured after 3 hours incubation to five test item concentrations (62.5, 125, 250, 500 and 1000 mg/L a.i.). The EC50 (3h) was determined to be > 1000 mg/L a.i. (nominal).

Two further studies confirms the low effect on microorganisms. Both tests were performed following the procedures of DIN 38412, part 8 and GLP. Based on growth inhibition EC10 values > 10000 mg a.i./L (nominal) for Pseudomonas putida were observed in both tests after 16 hours (Sasol, 1993d and Sasol, 1999d).