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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:
August 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
Please refer to "Principles of Method if other than Guideline"
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
yes
Remarks:
Please refer to "Principles of Method if other than Guideline"
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Deviations:
yes
Remarks:
Please refer to "Principles of Method if other than Guideline"
Principles of method if other than guideline:
While performing the measurements for the second reference substance concentration, the recorder was turned off. Therefore, oxyen depletion of the second reference substance concentration was not recorded. Since repitition of one single concentration did not result in a valid series, the complete reference substance series was repeated. The invalid reference substance series is not reported.

All criteria for acceptability of the final reference substance series were met.

The study integrity was not advsersely affected by the deviation.
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Analytical monitoring:
no
Details on sampling:
- Concentrations: 100 mg/l (nominal conentration)

- Sampling method:
After 30 minutes a well mixed sample of the contents 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. Oxygen consumption was measured and recorded for approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH and temperature were determined in the remaining part of the reaction mixture.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Since the test substance is poorly water soluble, it was added quantitatively to the test vessels. A nominal concentration of 100 mg/l was tested in duplicate. Optimal contact between the test substance and the test medium was ensured by applying continuous aeration and stirring.


- Controls: A 3,5-dichlorophenol solution with a final concentration of 0.5 g/l was prepared. A weighed amount of 250.4 mg was dissolved in 5 ml NaOH and subsequently diluted to approximately 15 ml with Milli-Q water. Then, while stirring 1N H2SO4 was added to the point of incipient precpitation, and finally Milli-Q water was added to make the volume up to 500 ml. The pH was 8.0. aliquots of 50 ml were stored in a freezer until use. On the day of testing the reference substance solution was defrosted at room temperature. Three concentrations were tested: 3.2, 20 and 32 mg/l.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Source: Municipal sewage treatment plant, receiving predominantly domestic sewage.

Number of micro-organisms: Number of micro-organisms was determined as the amount of Mixed Liquor Suspended SOlids (MLSS) per litre test medium.

Preparation of the sludge:
The sludge was coarsely sieved, washed and diluted with tap water. A small amount of the sludge was weighed and dried at ca. 105°C to determine the amount of suspended solids (2.2 g/l of sludge, as used for the test). The pH was 7.5 on the day of sampling and 8.0 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.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
0.5 h
Post exposure observation period:
Not applicable
Hardness:
Not stated in report
Test temperature:
The temperature of the test medium was between 20.4 and 20.8ºC
pH:
8.2 for control flasks
8.1 for test substance flasks
Dissolved oxygen:
6.5 and 7.2 mg O2/l at start of experiment
Salinity:
Not applicable as freshwater study
Nominal and measured concentrations:
Nominal concentration: 100 mg/l
Details on test conditions:
Test procedure and conditions:
Contact time: 30 minutes, during which aeration and stirring took place.

Vessels: All glass, approximately 300 ml oxygen bottles and 1 litre test bottles.

Milli-RO/Milli-Q water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges.

Synthetic sewage feed:
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
Dissolved in Milli-Q water, made up to 1 litre and filtered. The pH was 6.8 and adjusted with 1 M NaOH to 7.1

Air supply: Clean, oil-free air.

Oxygen meter: WTW inolab Oxi Level 2 supplied with a WTW CellOX 325 oxygen electrode, electrolyte type ELY/G.

Recorder: Flatbed recorder SE 102

Perfomance of the test:
The synthetic sewage feed (16 ml) and an adequate amount of the test substance were mixed and made up to 300 ml with Milli-RO water. Activated sludge (200 ml) was added to provide a final volume of 500 ml. The mixture was then aerated in a 1 litre bottle during the contact time, using a pipette as an aeration device.

Then a well mixed sample of the contents 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. Oxygen consumption was measured and recorded for approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH and temperature were determined in the remaining part of the reaction mixture.

This procedure was repeated for the duplicate concentration. Two controls without test substance were tested in each test series, one at the start and one at the end.
Reference substance (positive control):
yes
Remarks:
3,5 dichlorophenol
Duration:
0.5 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
Duration:
0.5 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: None
Results with reference substance (positive control):
The EC50 of dichlorophenol was in the accepted range of 5 - 30 mg/l.
See appendix 1 - EC-values (attached background material).

Results:

Toxicity of EA-3098

The table below presents:

- pH

- oxygen concentration at the start of the measurement

- the influence of controls and EA-3098 on the oxygen consumption of microbes in activated sludge

- the percentage inhibition of the respiration rate

Flask

Concentration (mg/l)

Oxygen conc. At the start (mg O2/l)

Oxygen consumption mg O2/l/hr

% Inhibition

pH

C1

0

7.9

45

-

8.2

C2

0

6.9

46

-

8.2

Mean C1 + C2

 

 

46

 

 

T1

100

6.5

46

-1*

8.1

T2

100

7.2

46

-1*

8.1

 * Negative values indicate stimulation of respiration rate of the sludge. These values are considered as not significant.

No inhibition of respiration rate of the sludge was recorded at 100 mg EA-3098 per litre. The duplicate measurement confirmed the result of the first measurement. Therefore, no further testing was needed. Hence, the EC50 of EA-3098 exceeded 100 mg/l (based on nominal concentrations).

Toxicity of the reference substance (3,5 -dichlorophenol)

The table below presents:

- pH

- oxygen concentration at the start of the measurement

- the influence of controls and EA-3098 on the oxygen consumption of microbes in activated sludge

- the percentage inhibition of the respiration rate

Flask

Concentration (mg/l)

Oxygen conc. At the start (mg O2/l)

Oxygen consumption mg O2/l/hr

% Inhibition

pH

C2

0

6.9

46

-

8.2

C3

0

7.1

51

-

8.1

Mean C2 + C3

 

 

49

 

 

R1

3.2

7.3

40

18

8.2

R2

10

7.3

31

36

8.1

R3

32

8.6

14

71

8.2

 

The calculated percentages inhibition allowed for a reliable determination of an EC50 -value (see Appendix 1 - attached background material).

Acceptability of the test:

1. The mean respiration rate of control 1 and 2 was 46 mg O2/l/hr. The difference between the controls was within 15% of each other (2%).

2. The mean respiration rate of control 2 and 3 was 49 mg O2/l/hr. The difference between the controls was within 15% of each other (10%).

3. The EC50 of 3 ,5 -dichlorophenol was in the accepted range of 5 -30 mg/l.

Since all criteria were met, the study was considered valid.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this test, the test material was not toxic to activated sludge bacteria at a concentration of 100 mg/l.
Executive summary:

The influence of the test material on the respiration rate of activated sludge was investigated after a contact time of 30 minutes.

The study procedure was based on OECD Guideline No. 209, EEC Directive 67/548 and ISO Standard 8192.

The test material was a white powder and was treated as 100% pure. Since the test material is poorly soluble in water, it was quantitatively added to the test vessels. A nominal concentration of 100 mg/l was tested in duplicate. Optimal contact between the test substance and the test medium was ensured by applying continuous aeration and stirring.

No inhibition of respiration rate of the sludge was recorded at 100 mg/l of test material. The duplicate measurement confirmed the result of the first measurement. Therefore no further testing was needed.

Since all criteria for acceptability of the test were met, the study was considered to be valid.

In conclusion, under the conditions of the test, EA-3098 was not toxic to waste water (activated sludge) bacteria at a concentration of 100 mg/l.

Description of key information

Under the conditions of the test, EA-3098 was not toxic to waste water (activated sludge) bacteria at a concentration of 100 mg/l.

Key value for chemical safety assessment

EC50 or LC50 for microorganisms:
100 mg/L
EC10, LC10 or NOEC for microorganisms:
100 mg/L

Additional information

The influence of the test material on the respiration rate of activated sludge was investigated after a contact time of 30 minutes.

The study procedure was based on OECD Guideline No. 209, EEC Directive 67/548 and ISO Standard 8192.

The test material was a white powder and was treated as 100% pure. Since the test material is poorly soluble in water, it was quantitatively added to the test vessels. A nominal concentration of 100 mg/l was tested in duplicate. Optimal contact between the test substance and the test medium was ensured by applying continuous aeration and stirring.

No inhibition of respiration rate of the sludge was recorded at 100 mg/l of test material. The duplicate measurement confirmed the result of the first measurement. Therefore no further testing was needed.

Since all criteria for acceptability of the test were met, the study was considered to be valid.

In conclusion, under the conditions of the test, EA-3098 was not toxic to waste water (activated sludge) bacteria at a concentration of 100 mg/l.