[No public or meaningful name is available]

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Remarks:

use of copper(II)sulphate pentahydrate instead of 3,5-dichlorophenol

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

The test item was crushed using a mortar, weighed out directly into Erlenmeyer flasks, filled with 250 mL autoclaved tap water and stirred for 24 h.

Test organisms

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected:
Municipal sewage treatment plant of Sarstedt, Germany
- Preparation of inoculum for exposure:
The sludge was washed with autoclaved tap water and diluted corresponding to a dry weight of 4.0 g/L +/- 10%
Nutrient solution: synthetic waste water according to OECD 209
- Pretreatment:
non adopted activated sludge
- Initial biomass concentration:
4.0 g/L +/- 10%; 3.63 g/L (dry sludge concentration)

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

no

Test conditions

Test temperature:

21°C

pH:

6.96

Dissolved oxygen:

7.26 mgO2/L (mean at study start)

Nominal and measured concentrations:

nominal: 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask, DIN 12380
- Type (delete if not applicable): open
- fill volume: 500 mL
- Aeration: permanent ventilation
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Sludge concentration (weight of dry solids per volume): 3.63 g/L
- Nutrients provided for bacteria: synthetic waste water according to OECD 209

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: autoclaved tap water

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : oxygen depletion

TEST CONCENTRATIONS
limit test: 1000 mg/L

- Range finding study
- Test concentrations: 10, 100 and 1000 mg/L

Reference substance (positive control):

yes

Remarks:

Copper(II) sulphate pentahydrate

Results and discussion

Effect concentrations

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 heterotrophic respiration

Details on results:

1. Solubility
Before stirring, the test item was dispersed and partly set on the side of the flask. After stirring, the test item was completely dispersed.

2. Respiration rates and inhibition of the respiration
test item (1000 mg/L) 20.4 mgO2/L x h (respiration rate), 10 % inhibition
control (1) 22.4 mgO2/L x h (respiration rate), 0 % inhibition
control (2) 22.8 mgO2/L x h (respiration rate), 0 % inhibition
reference item (180 mg/L) 8.0 mgO2/L x h (respiration rate), 65 % inhibition
reference item (100 mg/L) 14.0 mgO2/L x h (respiration rate), 38 % inhibition
reference item (58 mg/L) 16.4 mgO2/L x h (respiration rate), 27 % inhibition

3. EC-values
An inhibition of of less than 20% is biologically not significant. The EC-values were than the tested limit concentration (> 1000 mg/L).

4. Biotic and abiotoc factors
pH-avlue of the activated sludge: 6.96
Dry sludge concentration: 3.63 g/L
Test Temperature: 21°C

5. Reference test
In the reference test the EC50-value was determined by probit anaöysis. The EC50-value of the reference item is 124 mg/L. The EC50-value of the reference item copper(II) sulfaphate pentahydrate is in the confidence interval of 54-150 mg/L.

6. Validity criteria
Validity criteria were fulfilled:
- The two control respiration rates differ not more than 15% in the study
- The EC50 (3 h) of copper(II) sulphate pentahydrate is in the accepted range of 54-150 mg/L

Results with reference substance (positive control):

Results are valid
reference item (180 mg/L) 8.0 mgO2/L x h (respiration rate), 65 % inhibition
reference item (100 mg/L) 14.0 mgO2/L x h (respiration rate), 38 % inhibition
reference item (58 mg/L) 16.4 mgO2/L x h (respiration rate), 27 % inhibition

Reported statistics and error estimates:

EC50-value was calculated for the reference item with probit analysis using software SgmaPlot.
Probit values were taken from Weber (1996). Confidence intervals were calculated according to a standard procedure (Breitig & Tümpling 1982)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test substance is not toxic at concentrations

Executive summary:

A respiration inhibition test with activated sludge according to OECD guideline 209 was performed for 3 with the test substance. Test system was activated sludge of the municipal treatment plant of Sarstedt, Germany. The test was performed under static conditions as limit test with the concentration: 1000 mg/L. The respiration rates of control, reference and test item concentrations were measured after a contact time of 3 h, and the inhibitory effects of the test and reference concentrations were determined in comparison to the control respiration rates. An inhibition of 10% was observed at the tested limit concentration. In order to check the activity of the test system and the test conditions a reference test is carried out with copper(II) sulphate pentahydrate as reference item and the reference toxicity is detewrmined. The EC50 -value for the reference item was 124 mg/L. Validity criteria of the test guideline were fulfilled.

EC50 (test item): > 1000 mg/L