Caesium fluoride

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:

2021-03-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Cesium fluoride
Physical state/Appearance: white, crystalline powder
Batch: 2101021139
Water Solubility: >1000 g/L
Purity: 99.9 %
Expiry Date: 07 Dec 2022
Storage Conditions: Room temperature (15-25°C), protected from humidity

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Balatonfüred, Hungary
- Method of cultivation: continuously aerated (2 L/minute) at the test temperature (20 ± 2 °C, actual temperature range: 20.0-21.0 oC) for about 24 hours (one day) and fed with 50 mL synthetic sewage/L activated sludge
- Preparation of inoculum for exposure: The coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. The activated sludge used for this study was washed by centrifugation and the
supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed (5.533 g wet weight), dried and the ratio of wet sludge to dry weight (1.3962 g dry weight) determined. Based on this ratio, calculated amount of wet sludge (30 g dry weight that was equivalent to 119 g wet sludge) was suspended in isotonic saline solution (ad. 10 L) to yield a concentration equivalent to about 3 g/L (on dry weight basis). (In the test containers (300 mL final volume) the final concentration of suspended solids, containing 150 mL inoculum was 1.5 g/L on dry weight basis.)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

minimum: 18.0 °C, maximum: 20.4 °C

pH:

7-8

Nominal and measured concentrations:

nominal: 3.2, 10, 32, 100, 320, 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer bottles
- Material, fill volume: glass, 300 mL volume
- Aeration: with compressed air (0.5 L/minute)
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 8 (4 at the start and 4 at the end of the test series)
- No. of vessels per reference control (replicates): 5
- No. of vessels per nitrification control (replicates): 3
- Sludge concentration (weight of dry solids per volume): 3 g/L on a dry weight basis
- Nitrification inhibitor used: N-allylthiourea

TEST MEDIUM / WATER PARAMETERS
- in accordance to the guideline

OTHER TEST CONDITIONS
- Adjustment of pH: not needed

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approx. 3.2

- Range finding study
- Test concentrations: 10, 100, 1000 mg/L
- Results used to determine the conditions for the definitive study:
The nitrification respiration was not significant in the pre-test and it can therefore be assumed that the heterotrophic oxygen uptake equals the total uptake and no significant nitrification was occurring. In the pre-test no abiotic oxygen consumption was noticed. The respiration rates were slightly inhibited by 8.38 % at the lowest examined concentration of 10 mg/L, and by 12.83 % at 100 mg/L. 25.72 % inhibition (revealing statistical significance) was noticed at the highest test item concentration of 1000 mg/L. The pre-test results revealed a slight but statistically significant inhibition of oxygen consumption by the test substance at the highest examined concentration of 1000 mg/L; therefore, a definite test is required according to OECD 209. The pre-test was performed without pH adjustment, as the test item did not have any influencing effect on the pH within the test system and additional neutralization step of test item containing mixtures before inoculum addition was thus not necessary. Therefore, no pH adjustment was foreseen for the main test either.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

16.17 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.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No
- Adsorption (e.g. of test material to the walls of the test container): No

The observed oxygen consumption rates remained in the range of the blank controls at the lowest examined concentration of 3.2 mg/L. The slight deviation of 2.33 % is within the biological variability range of the applied test system. The respiration rates were inhibited by 10.52 % at the concentration of 10 mg/L. 15.28 % inhibition was noticed at the test item concentration of 32 mg/L, 18.29 % at 100 mg/L, 22.47 % at 320 mg/L and 26.55 % at 1000 mg/L.
The obtained oxygen consumption values and calculated specific respiration rates show a concentration related inhibitory effect of the test item.
The examined concentrations did not cover the appropriate inhibition range for an exact EC50 calculation as even at the top (limit) concentration of 1000 mg/L less than 50% inhibition was noted.
At the nitrification control the differentiation between the total, heterotrophic and nitrification respiration was possible. The total respiration (RT) was 45.74 mg/Lh, the heterotrophic respiration (RH) was 45.09 mg/Lh, the nitrification respiration (RN): 0.65 mg/Lh was calculated according to the following equation: RN = RT - RH.
The obtained 0.65 mg/Lh was considered as not significant difference within a biological variability range of the applied test system, and lower than the 5 % of RT (2.29 mg/Lh) in blank controls. The above calculation confirmed the assumption, that the heterotrophic oxygen uptake equals the total uptake.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50 = 6.53 mg/L
- Other:
The following concentrations of the positive reference control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as the test item: 2, 7 and 24.5 mg/L. In comparison to the blank controls the oxygen consumption rate of the activated sludge was inhibited by 25.23 % at the lowest concentration of 2 mg/L and at the nominal concentrations of 7 and 24.5 mg/L, the oxygen consumption rate was inhibited by 38.74 % and 89.37 %, respectively

Reported statistics and error estimates:

The 3-hour ECx values of the test and reference items and their 95 %-confidence limits were calculated by Probit analysis by IBM® SPSS® Statistics (Version 25 (2017) statistical software program).
For the determination of the NOEC the calculated specific respiration rate values were checked for normality (Kolmogorov-Smirnov Test) and for homogeneity of variance (Levene’s Test).
After these analyses for the determination of the NOEC the parametric method Dunnett t-Test was used for testing of significant differences to the control values.
The statistical analysis was performed by IBM® SPSS® Statistics, Version 25 (2017) statistical software program

Validity criteria fulfilled:

yes

Conclusions:

Based on the measured oxygen consumption values and the calculated specific
respiration rates, a slight, but concentration-dependent and unequivocal test item
related inhibition of the respiration rates was obtained. Based on the statistically significant differences (Dunnett t-Test (α=0.05)) between the blank control and different test item concentration values the NOEC was determined as 3.2 mg/L.
The 3-hour EC10 value of the test item is 16.17 mg/L (95 % conf. limits: 4.04–35.80 mg/L) and the 3-hour EC50 and EC80 values are significantly higher than 1000 mg/L.

Executive summary:

The purpose of the test was to evaluate the influence of the cesium fluoride on the activity of the activated sludge by measuring the respiration rate under defined conditions for a 3-hour exposure. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode for a contact time of 3 hours. The test item was investigated in a pre-test. The pre-test results revealed a slight but statistically significant inhibition of oxygen consumption by the test item at the highest examined concentration of 1000 mg/L; therefore, in line with OECD 209 Guideline a definite test was performed. Based on the effects noted in the preliminary study on the activated sludge inoculum, in the present main test the test item was investigated at the nominal concentrations of 3.2, 10, 32, 100, 320 and 1000 mg/L. Defined amounts of the test item were added directly into the test vessels. Analytical concentration verification of the actual test item concentrations was not performed as the test item was directly added to the sludge. Based on the preliminary experience, the test item does not have any influencing effect on the pH within the test system and therefore an additional neutralization step of test item containing mixtures before inoculum addition was not necessary. Consequently, the main test was performed without pH adjustment. In parallel with the test item treatments 3,5-Dichlorophenol as positive reference control was examined in concentrations of 2, 7 and 24.5 mg/L; furthermore, a blank (inoculum) control and nitrification controls were investigated. The main test was performed without abiotic controls, based on the results of the preliminary test where abiotic controls were tested at the test item concentration of 1000 mg/L and no remarkable abiotic oxygen consumption was noticed. All validity criteria of the study were met. The average specific respiration rate of the blank was 30.49 mg O2/g activated sludge (based on dry weight) in an hour (unequivocally higher than 20 mg/gh) with a coefficient of variation of 6.66 % (clearly below the 30 % as required in the validity criteria). The 3-hour EC50 of the reference item 3,5-Dichlorophenol was 6.53 mg/L, thus within the range of 2 mg/L to 25 mg/L, that is required for total respiration (in this study the differentiation between heterotrophic respiration and nitrification was not necessary as the parallel investigated nitrification control, containing N-allylthiourea confirmed the equality of heterotrophic and total oxygen uptake). The observed oxygen consumption rates remained in the range of the blank controls at the lowest examined concentration of 3.2 mg/L. The slight deviation of 2.33 % is within the biological variability range of the applied test system.. The obtained oxygen consumption values and calculated specific respiration rates show a concentration related inhibitory effect of the test item with 10.52 % to 26.55% inhibition in the examined concentration range of 10-1000 mg/L.

The examined concentrations did not cover the appropriate inhibition range for an exact EC50 calculation as even at the top (limit) concentration of 1000 mg/L less than 50% inhibition was noted. The degree of inhibition of oxygen consumption rates allowed the calculation of the 3-hour EC10 value and estimation of EC50 and EC80 values. Based on the available data the 3-hour EC10, value is 16.17 mg/L (95 % conf. limits: 4.04–35.80 mg/L), within the range of >10 mg/L, and <100 mg/L. The EC50 (estimated as 22031 mg/L) and EC80 (estimated as 2519772 mg/L) values are significantly higher than 1000 mg/L (>>1000 mg/L). The specific respiration rates of test item treatments were compared with the blank control values using Dunnett t-Test (α=0.05). The specific respiration rates of test item treatments did not differ statistically significantly from the blank control at the concentration of 3.2 mg/L and statistically significant differences were obtained at the concentration range of 10-1000 mg/L (Dunnett t-Test (α=0.05)).

Based on the measured oxygen consumption values and the calculated specific respiration rates, a slight, but concentration-dependent and unequivocal test item related inhibition of the respiration rates was obtained. Based on the statistically significant differences (Dunnett t-Test (α=0.05)) between the blank control and different test item concentration values the NOEC was determined as 3.2 mg/L.

The 3-hour EC10 value of the test item is 16.17 mg/L (95 % conf. limits: 4.04–35.80 mg/L); and the 3-hour EC50 and EC80 values are significantly higher than 1000 mg/L.

Description of key information

The 3-hour EC10 value of cesium fluoride is 16.17 mg/L  and the 3-hour EC50 and EC80 values are significantly higher than 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

16 mg/L

Additional information

The purpose of the test was to evaluate the influence of the cesium fluoride on the activity of the activated sludge by measuring the respiration rate under defined conditions for a 3-hour exposure according to OECD guideline 209. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode for a contact time of 3 hours. In the present main test the test item was investigated at the nominal concentrations of 3.2, 10, 32, 100, 320 and 1000 mg/L. Defined amounts of the test item were added directly into the test vessels. Analytical concentration verification of the actual test item concentrations was not performed as the test item was directly added to the sludge. The main test was performed without pH adjustment. In parallel with the test item treatments 3,5-Dichlorophenol as positive reference control was examined; furthermore, a blank (inoculum) control and nitrification controls were investigated. The main test was performed without abiotic controls.

All validity criteria of the study were met. Based on the measured oxygen consumption values and the calculated specific respiration rates, a slight, but concentration-dependent and unequivocal test item related inhibition of the respiration rates was obtained. Based on the statistically significant differences (Dunnett t-Test (α=0.05)) between the blank control and different test item concentration values the NOEC was determined as 3.2 mg/L. The 3-hour EC10 value of the test item is 16.17 mg/L (95 % conf. limits: 4.04–35.80 mg/L); and the 3-hour EC50 and EC80 values are significantly higher than 1000 mg/L.