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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-05-24 to 2012-07-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item was added to the test vessels in the highest concentrations directly.For the third experiment, a stock solution of an appropriate concentration in deionised water was prepared. The stock solution was slightly clouded but homogenous. It was used to prepare the treatments.

- Controls: 3.5-Dichlorophenol (CAS 591-35-5) was used as positive control. A stock solution in deionised water containing 500 mglL (nominal) was freshly prepared for each experiment.

- Evidence of undissolved material (e.g. precipitate, surface film, etc): no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: Upon arrival in the test facility , the sludge was filtrated , washed with tap water and resuspended in tap water.
- Method of cultivation: The activated sludge was aerated until usage in the test and fed with 50 ml /L synthetic sewage feed .
- Preparation of inoculum for exposure: On the day before the experiment, the inoculum was taken from its source, washed, aerated and the dry
matter was determined . Volume was adapted to the desired content of dry matter.
The nutrient solution was thawed and the sludge was fed with 50 mL nutrient solution/L sludge.
- Initial biomass concentration: first experiment 1.59 g/L, second experiment 1.40 g/L, third experiment 1.55 g/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
1.08 mmmol/L
Test temperature:
21.4 - 22.5 °C (first experiment)
21.7 - 23.5 °C (second experiment)
20.8 - 21.8 °C (third experiment)
pH:
first experiment: 7.8 - 8.0
second experiment: 7.8 - 8.0
third experiment: 7.5
Nominal and measured concentrations:
first experiment: 0, 1, 10.6 102, 1032 mg/L nominal
second experiment: 0, 3.2, 10, 32, 100, 320 mg/L nominal
third experiment: 0, 0.32, 1 mg/L nominal
Details on test conditions:
Duration: three hours
Replicates: one replicate/treatment (positive control all experiments, test item experiment 1); five replicates/treatment (test item experiments 2 and 3)
Control: two replicates before and two after measuring positive control and test item, respectively

In the control vessels, 16 mL nutrient solution was mixed with 234 mL water. The positive control vessels and the treatments were prepared by putting the, appropriate amount of positive control solution respectively test item (Exp.1 und Exp 2.) or test item solution (Exp. 3) into the respective test vessel, adding 16 mL nutrient solution and water to give 250 mL.
Then, 250 mL inoculum was added in five minute intervals and the mixtures were aerated. After three hours, the content of the first vessel was poured in a 250 mL narrow-neck bottle and the respiration rate was determined by measurement of the O2-concentration over a period of max. five minutes. The following vessels were measured likewise in five minute intervals.


The second experiment was designed as full test ; the range 320 - 3.2 mg/L. No NOEC was determinable. Therefore, a third experiment was
performed using the concentrations 1.0 and 0.32 mg/L for NOEC determination .
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenole
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
3.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL: 2.7 - 5.1 mg/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL: 230 - 430 mg/L
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
For the calculation of EC10 and EC50, the percentage inhibition was plotted versus concentrat ion in a Gauß-loqarithrnic diagram. EC10 and EC50
were determined from the x values of the regression line at y = 10% and y = 50%.
Results with reference substance (positive control):
positive control 3,5-dichlorophenole
first experiment:8.1 mg/L
second experiment: 11 mg/L
third experiment: 7.6 mg/L
Reported statistics and error estimates:
Statistical Determination of the NOEC:
It was tested whether the differences between O2 consumption of treatment and control were significant.
In order to select a suitable test for significance, it was checked whether equality of variance was given .
Equal ity of variance is tested using the following equation: F = s1²/ s2²
with S1 being the greater variance and S2 the smaller one.
The calculated value F is compared with the F-test table (level of significance 95%).
If equality of variance is given, the t-test is used; else, the WEIR test is used .

EC50 was estimated using the software Origin. The data were evaluated using linear fit on a probability-logarithmic scale.

All validity criteria were met. For the estimation of the EC50s of test item and positive control, the fits showed good statistical correspondence of the data with the dose-responseequation. The positive control gave EC50s of 8.1 mg/L (first experiment), 11 mg/L (second experiment) and 7.6 mg/L (third experiment); all values lie within the recommended range of 2 - 25 mg/L. In all experiments, the coefficient of variation of oxygen uptake rate in control replicates was below 30 % at the end of the test. The oxygen uptake rate of the blank controls was above 20 mg O2 per gram activated sludge in all three experiments.

Validity criteria fulfilled:
yes
Conclusions:
The following results for the test item Tellurium dioxide were determined:
3h NOEC =1.0 mg/L
3h EC10 = 3.7 mg/L (95% confid. interval: 2.7 - 5.1 mg/L)
3h EC50 =320 mg/L (95% confid. interval: 230 - 430 mg/L)
Executive summary:

In a study conducted according to OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test), three valid experiments were performed with Tellurium dioxide, fulfilling all validity criteria.

In the first experiment, a pre-test, the test item was tested using four concentrations (ranging from 1032 to 1 mg/L). Duration of the test was three hours. Activated sludge was used as inoculum. It was taken from a domestic sewage treatment plant and washed before usage.

The dry matter was determined as 3.18 g suspended solids/L, giving a concentration of 1.59 g suspended solids/L in the test.

As inhibition> 20 % was observed, and a full test was performed.

The second experiment, a full test, was performed using five concentrations, ranging from 322.0 to 3.2 mg/L. The dry matter of the activated sludge was determined as 2.8 g suspended solids/L, giving a concentration of 1.40 g suspended solids/L in the test.

Because significant inhibition in the lowest concentrated treatment occurred in the second experiment, a third experiment was performed in the same way using two lower concentrations of the test item, 1.0 and 0.32 mg/L, in order to determine a NOEC. The dry matter of the activated sludge was determined as 3.10 g suspended solids/L, giving a concentration of 1.55 g suspended solids/L in the test.

The following results for the test item Tellurium dioxide were determined:

3h NOEC =1.0 mg/L

3h EC10 = 3.7 mg/L (95% confid. interval: 2.7 - 5.1 mg/L)

3h EC50 =320 mg/L (95% confid. interval: 230 - 430 mg/L)

All validity criteria were met. For the estimation of the EC50s of test item and positive control, the fits showed good statistical correspondence of the data with the dose-responseequation. The positive control gave EC50s of 8.1 mg/L (first experiment), 11 mg/L (second experiment) and 7.6 mg/L (third experiment); all values lie within the recommended range of 2 - 25 mg/L. In all experiments, the coefficient of variation of oxygen uptake rate in control replicates was below 30 % at the end of the test. The oxygen uptake rate of the blank controls was above 20 mg O2 per gram activated sludge in all three experiments.

 

In conclusion the three experiments show good correlation of the inhibitory effects of the test item. No inconsistencies in the dose-response-relationship could be observed. The results of the tests can be considered valid.

Description of key information

Study was conducted according to OECD guideline 209 with GLP, data are reliable without restrictions. 

Key value for chemical safety assessment

EC50 for microorganisms:
320 mg/L

Additional information

In a study conducted according to OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test), three valid experiments were performed with Tellurium dioxide, fulfilling all validity criteria.

In the first experiment, a pre-test, the test item was tested using four concentrations (ranging from 1032 to 1 mg/L). Duration of the test was three hours. Activated sludge was used as inoculum. It was taken from a domestic sewage treatment plant and washed before usage.

The dry matter was determined as 3.18 g suspended solids/L, giving a concentration of 1.59 g suspended solids/L in the test.

As inhibition> 20 % was observed, and a full test was performed.

The second experiment, a full test, was performed using five concentrations, ranging from 322.0 to 3.2 mg/L. The dry matter of the activated sludge was determined as 2.8 g suspended solids/L, giving a concentration of 1.40 g suspended solids/L in the test.

Because significant inhibition in the lowest concentrated treatment occurred in the second experiment, a third experiment was performed in the same way using two lower concentrations of the test item, 1.0 and 0.32 mg/L, in order to determine a NOEC. The dry matter of the activated sludge was determined as 3.10 g suspended solids/L, giving a concentration of 1.55 g suspended solids/L in the test.

The following results for the test item Tellurium dioxide were determined:

3h NOEC =1.0 mg/L

3h EC10 = 3.7 mg/L (95% confid. interval: 2.7 - 5.1 mg/L)

3h EC50 =320 mg/L (95% confid. interval: 230 - 430 mg/L)