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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:
2009-09-04 to 2009-09-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Method: 47 µl of the test substance, Milli-RO water, synthetic sewage feed (16 ml) and activated sludge (200 ml) were mixed and made up to 500 ml with Milli-RO water in a 1 litre bottle.

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Preparation of inoculum for exposure: The sludge was coarsely sieved to remove large particles. The sludge was then washed by centrifuging at 1000 g for 10 minutes. The supernatant was decanted and replaced with ISO-medium. The washing step was performed three times. The pH was 7.8 on the day of testing. The batch of sludge was used one day after collection; therefore 50 ml of synthetic sewage feed was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

- Initial biomass concentration: 3.8 g/L
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
Oxygen consumption was measured and recorded for approximately 10 minutes.
Test temperature:
18.7 - 19.3°C.
pH:
8.4
Nominal and measured concentrations:
100 mg/l (loading rate).
Details on test conditions:
TEST SYSTEM

- Test vessel:

- Material, size, headspace, fill volume: glass, 300 ml oxygen bottles, 1L test bottles

- Aeration: continuous during 3 hour test period

- No. of vessels per concentration (replicates): two

- No. of vessels per control (replicates): two

- Biomass loading rate: 100 mg/L


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges (Milli-Q)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After the 3-hour contact time, a well mixed sample of the contents of the reaction mixture was poured into a 300 ml oxygen bottle, and the flask was sealed with an oxygen electrode connected to a recorder, forcing the air out of the vessel. WTW inolab Oxi 730 supplied with a WTW CellOx 325 oxygen electrode, electrolyte type ELY/G

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 100 mg/L only

- Justification for using less concentrations than requested by guideline: No significant inhibition of respiration rate of the sludge was recorded for the test substance at a loading rate of 100 mg/L in both replicates and therefore no further testing was required.
Reference substance (positive control):
yes
Remarks:
3, 5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 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:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Details on results:
The two controls had a difference in respiration rate of 9%, which is less than the 15% limit required by the guideline for a valid study.
Results with reference substance (positive control):
A positive control test with 3, 5-dichlorophenol yielded an EC50 of 7.2 mg/L, which is within the accepted range of 5 to 30 mg/l required by the guideline for a valid study.

Table 1: Results of the activated sludge respiration inhibition test

Test sample

Loading rate (mg/l)

Oxygen conc. At start (mg O2/l)

Oxygen consumption (mg O2/l/h)

% Inhibition

respiration rate

pH

 

 

Initial control

0

7.0

34

-

8.3

Final control

0

7.2

37

-

8.4

[3-(2,3-epoxypropoxy)propyl]trimethoxysilanereplicate (1)

100

7.7

35

1

8.4

[3-(2,3-epoxypropoxy)propyl]trimethoxysilanereplicate (2)

100

8.0

33

7

8.4

 

 

3,5-dichlorophenol

5

-

-

43

-

12

-

-

61

-

30

-

-

67

-

Validity criteria fulfilled:
yes
Conclusions:
An activated sludge respiration inhibition 3hr EC50 value of >100 mg/l (loading rate) was determined in a reliable study conducted according to an appropriate test protocol, and in compliance with GLP. This indicates that the test substance was not toxic to waste water (activated sludge) bacteria at a loading rate of 100 mg/l. Whilst this was a limit test the very low levels of inhibition indicate to the present reviewers that the EC10 can be concluded to be =100 mg/l.

Description of key information

Toxicity to microorganisms: ASRI 3 hour EC50 >100 mg/l (loading rate) (OECD 209). This was a limit test, but the very low inhibition seen indicates that the EC10 can be taken to be =100 mg/l.

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L
EC10 or NOEC for microorganisms:
100 mg/L

Additional information

A 3 hour activated sludge respiration inhibition EC10 value of >100 mg/l (loading rate) was determined in a reliable study conducted according to an appropriate test protocol, and in compliance with GLP. This indicates that the test substance was not toxic to waste water (activated sludge) bacteria at a loading rate of 100 mg/l.

An EC10 of 1520 mg/l for toxicity to microorganisms (Pseudomonas putida) was determined in a reliability 2 study (reliable with restrictions) conducted according to generally accepted scientific principles and in compliance with GLP (Schoberl, 1993). The restrictions were that nonylphenol ethoxylate propoxylate (EO 10 Mol and PO 5 Mol) was used as a solubilising agent, which could potentially have affected the results, and in addition, no reference substance was used to check the activity of the inoculum. This study is selected as supporting study.

An IC50 >2500 mg/l is reported in secondary literature (handbook, Chandra 1997). The original reference was not available for review and no further information is available. The reliability of this data cannot be assigned.