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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-05-08 to 2012-05-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
For each experiment, a stock solution of 5 giL (nominal) in deionised water was prepared.
The stock solution then was used to prepare the treatments. Only for the highest concentration in the main study, the test item was directly weighed into the test vessels.
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 1.60 g/L, second experiment 1.60 g/L

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
1.08 mmol/L
Test temperature:
20 ± 2 °C
pH:
Pre-experiment: pH 7.7 - 7.5 (1000 mg/L - 1mg/L )
Main experiment: pH 8.2 - 8.0 (3206 - 32 mg/L)
Nominal and measured concentrations:
Pre-experiment: 0, 1, 10, 100, 1000 mg/L, nominal
Main experiment: 0, 32, 100, 320, 1000, 3200 mg/L, nominal
Details on test conditions:
Duration: three hours
Replicates: one replicate/treatment (positive control all experiments, test item pre-test);
five replicates/treatment (test item main study)
Control: two replicates before and two after measuring positive control and test item, respectively


Reference substance (positive control):
yes
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
590 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 440 - 740 mg/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 3 200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: CL not determinable
Duration:
3 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
Remarks on result:
other: CL not determinable
Details on results:
For the calculation of the EC10 and EC50, the percentage inhibition was plotted versus concentration in a Gauß-loqarithrnic diagram. EC10 was
determined from the x values of the regression line at y = 10% and y = 50%. The EC50 lay outside of the tested range and was stated as to be higher than the highest treatment.
Results with reference substance (positive control):
Positive control 3,5-dichlorophenole
Pre-experiment:7.6 mg/L
Main experiment: 9.7 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.
For th is determination, the values of the O2 consumption were used .
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 the calculated value is smaller than the tabular value, equality of variance is given.

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.

In the pre test, the inhibition caused by the test item was higher than the inhibition values in the main study. The difference between the inhibition values of the pre-test and the main study may be related to the used sludge. Taking into account the results of the positive control (lower EC 50 in the pre-test), the sludge which was used in the pre-test may be slightly more sensitive than the sludge which was used in the main study. Nevertheless, the differences in the inhibition values and oxygen uptake rates in the control replicates were within the normal range of a biological system.

In both experiments, all validity criteria were met. For the estimation of the EC50 of the positive control, the fits showed good statistical correspondence of the data with the dose response-equation. The positive control gave an EC50 of 7.6 mg/L in the pre-test and 9.7 mg/L in the main study; both values lie within the recommended range of 2 - 25 mg/L. The coefficient of variation of oxygen uptake rate in control replicates was below 30% in both experiments. The oxygen uptake rate of the controls was above 20 mg O2 per gram activated sludge in both experiments, too.

Validity criteria fulfilled:
yes
Conclusions:
The following results for the test item Sodium selenate were determined:
3h NOEC = 100 mg/L
3h EC10 =590 mg/L (95% confidence interval: 440-740 mg/L)
3h EC50 > 3200 mg/L (95% confidence interval: not determinable)
Executive summary:

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

In the pre-test, the test item was tested using four concentrations (ranging from 1000 to 1 mg/L nominal). 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 3.20 g suspended solids/L, giving a concentration of 1.60 g suspended solids/L in the test.

As the test item caused significant inhibition of the respiration of the activated sludge in the pre-test, a main study was performed.

The main study was performed likewise using five concentrations, ranging from 3200 to 32 mg/L (nominal). The dry matter of the activated sludge was determined as 3.20 g suspended solids/L, giving a concentration of 1.60 g suspended solids/L in the test.

The following results for the test item Sodium selenate were determined:

3h NOEC = 100 mg/L

3h EC10 = 590 mg/L (95% confidence interval: 440-740 mg/L)

3h EC50 > 3200 mg/L (95% confidence interval: not determinable)

 

All validity criteria were met. For the estimation of the EC50 of the positive control, the fits showed good statistical correspondence of the data with the dose response-equation. The positive control gave an EC50 of 7.6 mg/L in the pre-test and 9.7 mg/L in the main study; both values lie within the recommended range of 2 - 25 mg/L. The coefficient of variation of oxygen uptake rate in control replicates was below 30% in both experiments. The oxygen uptake rate of the controls was above 20 mg O2 per gram activated sludge in both experiments, too.

The result of the test can be considered valid.

Description of key information

A GLP-conform study according to the OECD 209 guideline has been performed for sodium selenate, reporting a NOEC, EC10 and EC50 of 100, 590 and >3200 mg sodium selenate /L, respectively (corresponding to 41.4, 244 and >1324 mg Se/L).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information

GLP-conform studies according to the OECD 209 guideline on respiration inhibition of activated sludge have been performed for sodium selenite and sodium selenate. The study for sodium selenate reports a NOEC, EC10 and EC50 of 100, 590 and >3200 mg sodium selenate /L, respectively (corresponding to 41.4, 244 and >1324 mg Se/L).

The study for sodium selenite reports a NOEC and EC50 of 1 and 180 mg sodium selenite /L, respectively (corresponding to 0.46 and 82 mg Se/L). The reported EC10 for sodium selenite was judged not reliable because it was extrapolated out of the tested concentration range.

In addition, three single-species tests with relevant species are available as supporting information. For the highly relevant ciliate Spirostomum ambiguum a 48-h LC50 of 32.3 mg Se/L was obtained from a study with Na2SeO3 (Nalecz-Jawecki and Sawicki, 1998). Next to this value, two 16-h EC50 values were included for growth inhibition of the bacterium Pseudomonas putida. These values are reported in the same study (Schmitz et al., 1998), in which experiments were conducted with both SeO2 and Na2SeO3. The 16-h EC50 values for these substances are 15 and 33.4 mg Se/L, respectively.

Because of the large difference in observed NOEC and EC50 values for the respiration inhibition assays with selenite and selenate, results of sodium selenite are used for the assessment of inorganic tetravalent Se substances and results for sodium selenate are selected for the assessment of inorganic hexavalent Se substances.