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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
17 Oct 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg, Karlsruhe, Germany
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 586 µl of the test item were added into the test vessels to obtain final concentrations of 1000 mg/L. Three parallel vessels of the test item were prepared.
- Eluate: no
- Differential loading: yes
- Controls: yes, blank control
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: Activated sludge from the municipal wastewater treatment plant Staufener Bucht (Germany) was used as test system. The treatment plant clarifies predominantly domestic wastewater of the region of Staufen and has a capacity of 140 000 inhabitant equivalents. Sampling date of activated sludge was 16 Oct 2012.
- Laboratory culture: no
- Preparation of inoculum for exposure: The activated sludge was kept aerated and fed with synthetic sewage (50 mL per litre and day) until use. Dry solids of the activated sludge was determined as 5.3 g/L by weight measurements before and after 3 h drying at 105°C (mean of triplicate measurements). The activated sludge was washed twice by settling the sludge, decanting the supernatant, and re-suspending the sludge in aerated tap water. Before using, the activated sludge was diluted to 3 g/L dry solids with tap water, in order to obtain a final concentration of 1.5 g/L dry solids in the test.
- Initial biomass concentration: 1.5 g/L (dry solids)
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
18 - 20 °C
pH:
7.7
Nominal and measured concentrations:
nominal: blank, 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: BOD bottles
- Fill volume: 500 mL
- Aeration: All vessels were stirred and aerated throughout the contact time with room air at a rate of 0.5 - 1 L/min using a membrane pump.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline (demineralised water)

EFFECT PARAMETERS MEASURED: respiration rate was measured every 15 min to allow a contact time of 3 h
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
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
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 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 h): 22.48 mg/L. The EC50 was calculated with ToxRat version 2.09 (ToxRat Solutions GmbH, Alsdorf, Germany) by probit analysis using linear max. likelihood regression.

The validity criteria set out in the Guideline are met.

Table 1: Respiration rate of activated sludge after application of the test substance of the definite test.

Vessel

O2 concentration [mg/L]

Consumption rate [mg/(L*h)]

Inhibition [%]

First measurement

Last measurement

Blank 1

7.77

6.15

38.9

 

Blank 2

7.50

6.07

34.3

 

Blank 3

7.88

6.66

29.3

 

Blank 4

7.44

6.15

31.0

 

Blank 5

7.80

6.53

30.5

 

Blank 6

8.59

7.77

19.7

 

Test item 1

5.22

3.65

37.7

-23

Test item 2

5.85

4.55

31.2

-2

Test item 3

8.09

3.75

32.2

-5

Coefficient of variation of blanks: 0.19

The results of a first test with a concentration of 1000 mg/L showed high negative inhibitions (until -53%) and a relative low start value of oxygen at the beginning of the measurement. Therefore, the aeration of the test was optimized and the test was repeated. The data of the first test are not shown in detail in the report. In the repetition the values of oxygen at the start of measurement were considerably higher and the evaluation showed conclusive results.

Validity criteria fulfilled:
yes
Conclusions:
The NOEC of dodecyl oleate (CAS No 36078-10-1), a constituent of the registration substance, is >/= 1000 mg/L, the EC50 > 1000 mg/L.
Executive summary:

The present study with dodecyl oleate (CAS No 36078 -10 -1), constituent of the registration substance, was performed according to OECD 209 under GLP conditions. A limit test with a concentration of 1000 mg/L did not result in inhibition of respiration rate of activated sludge microorganisms (obtained from a sewage treatment plant treating predominantly domestic wastewater). An increase of respiration was observed in all three test vessels. Thus, a NOEC (3 h) of ≥ 1000 mg/L was derived.

Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Data based on a ready biodegradability study. This approach is in accordance with the Guidance on information requirements and chemical safety assessment (Chapter R.7b: Endpoint specific guidance, ECHA 2008).
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Circular on Test Methods of New Chemical Substances (Japan), biodegraadation test
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Vehicle:
no
Test organisms (species):
other: mixture of activated sludge from STP with freshwater from rivers and lakes, marine water and marine sediment
Details on inoculum:
- Source of inoculum/activated sludge (location, sampling depth): location: 10 sites (4 sewage plants, 3 rivers, 1 lake and 2 bays) around Japan, sampling depth: return sludge in sewage plants, surface water and surface soil of foreshore
- Laboratory culture: Thirty minutes after stopping of aeration, discard about one third of the whole volume of supernatant and add an equal volume of a solution (pH adapted to 7.0 with NaOH) containing 0.1% each of glucose, peptone and potasium orthophosphate, to the settled material and re-commence aeration. This procedure was repeated once per day.
- Temperature: 25 ± 2 °C
- Storage conditions: appearance and general condition of sludge were observed with a light microscope, sediments, pH, temperature and dissolved oxygen
- Storage length: started to use: 15 Oct 1991
- Concentration of sludge: 5500 mg/L
- Water filtered: no
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
28 d
Test temperature:
25 ± 2 °C
Nominal and measured concentrations:
nominal: 100 mg/L
Details on test conditions:
TEST CONDITIONS
- Composition of medium: recommended medium in the guideline
- Test temperature: 25 ± 1 °C
- Suspended solids concentration: 30 mg/L
- Continuous darkness: no data

TEST SYSTEM
- Culturing apparatus: closed oxygen consumption measuring machine
- Number of culture flasks/concentration: 1 (water + test substance), 3 (sludge + test sub.), 1 (sludge + reference sub.), 1 (sludge)
- Measuring equipment: Coulometer
- Details of trap for CO2 and volatile organics if used: soda lime

SAMPLING
- Sampling frequency: continuously until day 28
- Sterility check if applicable: no
- Sample storage before analysis: no data

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
Reference substance (positive control):
yes
Remarks:
aniline
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Result from a ready biodegradability test
Results with reference substance (positive control):
The oxygen consumption for aniline in the above used test system reached 58% and 75% degradation after 7 days and 14 days.

The test substance was degraded to an extent of 67% (O2 consumption) and 94% (Test material analysis) after 28 d.Since the substance was readily biodegradable it is with high probability not toxic to aquatic microorganisms. The test item concentration in the test vessels of 100 mg/L can be used as NOEC value for the toxicity to aquatic microorganisms.

Validity criteria fulfilled:
yes
Conclusions:
The NOEC ot tetra decyl myristate (CAS No 3234-85-3), a constituent of the registration substance, is derived at >/= 100 mg/L (nominal).
Executive summary:

The toxicity of tetradecyl myristate (CAS 3234-85-3), a constituent of the registration substance, to activated sludge microorganisms was investigated in a biodegradation study conducted according to OECD 301C under GLP conditions (Ministry of Economy, Trade and Industry, Japan, 1992). A test concentration of 100 mg/L (nominal) did not inhibit the biodegradation process of the test substance. Therefore, the test substance is not toxic to activated sludge microorganisms and the test concentration can be used as a NOEC value for the toxicity to activated sludge microorganisms. Thus, a NOEC (28 d) of ≥ 100 mg/L resulted.

Description of key information

NOEC (28 d) of ≥ 100 mg/L for activated sludge microorganisms (OECD 301C).

NOEC (3 h) of ≥ 1000 mg/L for activated sludge microorganisms (OECD 209).

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

Two GLP Guideline studies conducted according to OECD 301 C and 209 are available investigating the effects of tetradecyl myristate (CAS 3234-85-3) and dodecyl oleate (CAS No 36078-10-1), constituents of the registration substance. Microorganisms were exposed to test item concentrations of up to 1000 mg/L (nominal).

1. Result of study with tetradecyl myristate:

A test concentration of 100 mg/L (nominal) did not inhibit the biodegradation process of the test substance. Therefore, the test substance is not toxic to activated sludge microorganisms and the test concentration can be used as a NOEC value for the toxicity to activated sludge microorganisms. Thus, a NOEC (28 d) of ≥ 100 mg/L resulted.

.

2. Result with dodecyl oleate:

A limit test with a concentration of 1000 mg/L did not result in inhibition of respiration rate of activated sludge microorganisms (obtained from a sewage treatment plant treating predominantly domestic wastewater). An increase of respiration was observed in all three test vessels. Thus, a NOEC (3 h) of ≥ 1000 mg/L was derived.