Sodium selenite

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-05-08 to 2012-07-05

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:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: For each experiment, a stock solution of an appropriate concentration in deionised water was prepared.
- Controls:
- 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 1.60 g/L, second experiment 1.50 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:

19.8 - 20.8 °C (first experiment)
21.3 - 22.3 °C (second experiment)
20.3 - 21.8 °C (third experiment)

pH:

pH values for test item solutions are in the range of 8.4 to 7.4, depending on concentration

Nominal and measured concentrations:

first experiment: 0, 1, 10.2, 102.3, 1022.7 mg/L
second experiment: 0, 3.2, 10, 32.1, 100.4, 321.4 mg/L
third experiment: 0, 0.32, 1 mg/L

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 solution 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 minutest. The following vessels were measured likewise in five minute intervals.

The first experiment showed 60 % inhib ition at 1000 mg/L test item concentration , no inhibition at 1 mg/L.
The second experiment was designed as full test ; the range 320 - 3.2 mg/L showed inhibition values ranging from 54 - 18 %.
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:

EC50

Effect conc.:

180 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 = 160 - 220 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 estimation 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:7.6 mg/L
second experiment: 8.3 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.

Because inhibition values in the second experiment ranged from 18 % to 54 % EC10 had to be determined via extrapolation. The extrapolated EC10 of 0.45 mg/L lies below the statistically determined NOEC in the third experiment. Even though for safety purposes, the lower value is normally used, in this case, it should be kept in mind that the EC10 was extrapolated whereas the NOEC was determined in an unequivocal experiment.

The apparent difference between the second and the third experiment may also be related to the used sludge. Taking into account the results of the positive control (lower EC 50 in the third experiment), the sludge which was used in the third experiment may have been slightly more sensitive than the sludge which was used in the second . Nevertheless, the differences in the inhibition values and oxygen uptake rates in the control replicates were within the normal range of a biological system.

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 EC50s of 7.6 mg/L (first experiment), 8.3 mg/L (second experiment) and 7.6 mg/L (third experiment); all values lie within the recommended range of 2 - 25mg/L. In all experiments, the coeffic ient 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.

Validity criteria fulfilled:

yes

Conclusions:

The following results for the test item Sodium selenite were determined:
3h NOEC =1.0 mg/L
3h EC50 = 180 mg/L (95% confid. interv.: 160 - 220 mg/L)

Executive summary:

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

In the first experiment, a 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 3.2 g suspended solids/L, giving a concentration of 1.6 g suspended solids/L in the test. As inhibition > 20% was observed, a full test was performed.

The second experiment, a full test, was performed likewise using five concentrations, ranging from 320 to 3.2 mg/L. The dry matter of the activated sludge was determined as 3.0 g suspended solids/L, giving a concentration of 1.5 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, 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 Sodium selenite were determined:

3h NOEC =1.0 mg/L

3h EC10 = 0.45 mg/L (95% confid. interv.: 0.37 - 0.61 mg/L)

3h EC50 = 180 mg/L (95% confid. interv.: 160 - 220 mg/L)

 

Because inhibition values in the second experiment ranged from 18 % to 54 % EC10 had to be determined via extrapolation. The extrapolated EC10 of 0.45 mg/L lies below the statistically determined NOEC in the third experiment. Even though for safety purposes, the lower value is normally used, in this case, it should be kept in mind that the EC10 was extrapolated whereas the NOEC was determined in an unequivocal experiment.

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 EC50s of 7.6 mg/L (first experiment), 8.3 mg/L (second experiment) and 7.6 mg/L (third experiment); all values lie within the recommended range of 2 - 25mg/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 conclusion the three experiments showed good correlation of the inhibitory effects of the test item. 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 selenite, reporting a NOEC and EC50 of 1 and 180 mg sodium selenite /L, respectively (corresponding to 0.46 and 82 mg Se/L).

Key value for chemical safety assessment

EC50 for microorganisms:

180 mg/L

EC10 or NOEC for microorganisms:

1 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 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.

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).

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 inorganic hexavalent Se substances.