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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:
2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: ARA Ergolz II, Füllinsdorf, Switzerland
- Preparation of inoculum for exposure: Sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted
- Pretreatment: A homogenized aliquot of the final sludge suspension was weighed, dried, and the ratio of wet to dry weight was calculated. Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. During the holding period of two days prior to use, the sludge was fed daily with 50 mL synthetic wastewater per liter and was kept at room temperature under continuous aeration until use. Before use, the dry weight of the activated sludge was measured again in the inoculum used in the test. The pH of the activated sludge inoculum was 6.8.
- Initial biomass concentration: Activated sludge (200 mL) and a total of 284 mL of other components, primarly tap water, was added to each flask, corresponding to about 1.5 g dry material per liter test medium.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 °C at the start and 19 °C at the end of the incubation period
pH:
Inoculum prior to application: pH 6.8
Details on test conditions:
TEST SYSTEM
- Test vessel: 1000 mL glass flasks
- Type: open
- Aeration: Compressed air at a flow of approximately one liter per minute
- No. of vessels per concentration (replicates): Single vessels for each test and reference treatment, duplicate untreated control vessels
- Measuring equipment: Oxygen electrode WTW TriOxmatic® 300 and an oxygen meter WTW Oxi 539, Wissenschaftlich-Technische Werkstaetten WTW, Weilheim/Germany

EFFECT PARAMETERS MEASURED:
Dissolved oxygen concentration

TEST CONCENTRATIONS
- Test concentrations: Nominal: 0 (control), 1.0, 3.2, 10, 32, and 100 mg/L
Reference substance (positive control):
yes
Remarks:
3,5 dichlorophenol
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
52 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
81 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
126 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
47 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other:
Remarks:
NOEC determined as the calculated 3-hour EC15
Results with reference substance (positive control):
- Results with reference substance valid? ves
- Relevant effect levels: EC50: 9 mg/L

 Test results

Test material

Nominal concentration
(mg/L)

Respiration rate
(mg O2/L/min)

Inhibition
 (%)*

Control 1 [a]

0.0

1.186

--

Test substance

1.0

1.188

-1.7

Test substance

3.2

1.184

-1.3

Test substance

10

1.206

-3.2

Test substance

32

1.124

3.8

Test substance

100

0.400

65.8

3,5-dichlorophenol

5

0.872

25.4

3,5-dichlorophenol

16

0.272

76.7

3,5-dichlorophenol

50

0.054

95.4

Control 2 [b]

0.0

1.151

--

[a] = incubation at test initiation; test series incubations started at 15-minute intervals; 
[b] = last incubation in test series; 
* negative values refer to increased oxygen consumption rate relative to control. 

Validity criteria fulfilled:
yes
Conclusions:
Up to and including the concentration of 32 mg/L, the test item ‘MERGAL V615’ had no significant inhibitory effect (<15%) on the respiration rate of activated sludge after the incubation period of three hours.
Executive summary:

A GLP-compliant study was conducted to determine thetoxicity to bacteria of ‘MERGAL V615’ according to OECD Guideline 209 and EU Commission Directive 88/302/EEC, Part C.11.

All of the validity criteria for the study were met. Therefore the study is considered to be valid. ‘MERGAL V615’ showed ‑3.2 to 65.8% inhibition of the respiration of activated sludge at test substance concentrations between 1.0 and 100 mg/L.

Description of key information

The acute toxicity towards bacteria was tested in 3 independent studies, performed according to OECD Guideline 209, by determining the inhibition of respiration in sewage sludge. In the first test, the 30min-EC50 was established at a concentration of 390 mg/L, while the second study resulted in an EC10 of 17 mg/L and an EC50 of 73 mg/L after 3 hours of incubation. In the third test, the 3h-EC50was established at a concentration of 81 mg/L and the 3h-NOEC at 47 mg/L.

The result of the first test seems doubtful, as formaldehyde was found to be significantly more toxic. The result of the second test is used for the PNEC derivation, as the EC10 respresents the worst case.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
17 mg/L

Additional information

Data on formaldehyde

The acute toxicity of formaldehyde towards bacteria was investigated in two studies. The first test is according to OECD guideline 209, determining the inhibition of respiration in a sewage sludge sample. The second test was performed according to Offhaus (1973). The latter also uses respiration inhibition of a sludge sample as the endpoint, but extends contact time to 120 hours. The lowest EC50 was established at a concentration of 20.4 mg/L.

Studies on the toxicity of formaldehyde towards microorganisms in sewage treatment plants demonstrated that this substance was harmful to the test organisms.