CTO

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

exposure-related information

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

yes

Remarks:

Three deviations were noted. These were considered uncritical.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2015

Deviations:

yes

Remarks:

Three deviations were noted. These were considered uncritical.

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document 23 (Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures)

Version / remarks:

2000

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch No.: B0345
Purity: >99%
Homogeneity: Homogeneous

Analytical monitoring:

yes

Details on sampling:

- Concentrations: calibration was performed in the range of 10 – 2000 μg/L Tungsten (W) and 0.5 – 10 mg/L Cesium (Cs) in Daphnia test medium

Vehicle:

no

Details on test solutions:

The saturated solution was prepared for the test. This was done by mixing the nominal load of 220.0 mg/L with the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and shaking vigorously for 24 hours. The resulting solution was filtrated through 0.45 µm nylon filters. The lower treatments were prepared by dilution of this solution with nutrient medium.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: freshwater green algae
- Strain: 86.81
- Source: MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen).
- Method of cultivation: The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

At the end of the test, the treatments were examined microscopically in order to assess the appearance of the algae and detect abnormalities (e.g. caused by the exposure to the test item).

Test temperature:

20.8 – 22.8 °C

Nominal and measured concentrations:

Nominal concentrations: 10, 22 , 46, 100, 220 mg/L

Measured Cesium (Cs) Concentrations:
For nominal test concentration of 10 mg/L: < LOQ at t=0hr; < LOQ at t=72hrs
For nominal test concentration of 22mg/L: < LOQ at t=0hr; 3.10 mg/L at t=72hrs
For nominal test concentration of 46 mg/L: < LOQ at t=0hr; 4.20 mg/L at t=72hrs
For nominal test concentration of 100 mg/L: < LOQ at t=0hr; 2.86 mg/L at t=72hrs
For nominal test concentration of 220 mg/L: 4.50 mg/L at t=0hr; 6.74 mg/L at t=72hrs

Measured Tungsten (W) Concentrations:
For nominal test concentration of 10 mg/L: 0.28 mg/L at t=0hr; 0.26 mg/L at t=72hrs
For nominal test concentration of 22mg/L: 0.60 mg/L at t=0hr; 0.58 mg/L at t=72hrs
For nominal test concentration of 46 mg/L: 1.20 mg/L at t=0hr; 1.19 mg/L at t=72hrs
For nominal test concentration of 100 mg/L: 2.71 mg/L at t=0hr; 2.60 mg/L at t=72hrs
For nominal test concentration of 220 mg/L: 5.56 mg/L at t=0hr; 5.76 mg/L at t=72hrs

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks total volume 65 mL
- Type: open (covered with perforated plastic foil acting as a stopper)
- Material, size, headspace, fill volume: 45 ±1 mL test solution (or algal medium in blank) and algae
- Initial cells density: 3.9*10^3 cells/mL
- No. of vessels per concentration (replicates): 3 replicates
- No. of vessels per control (replicates): 6 replicates

GROWTH MEDIUM
- Standard medium used: Yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Light intensity and quality: 5000 lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: electronic particle counter, cell numbers measured every 24 hours

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Test concentrations: 10, 22, 46, 100, 220 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

31.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.97 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr. (total fraction)

Remarks:

The test item consists of 15.92% Cesium (Cs)

Basis for effect:

growth rate

Remarks on result:

other: based on Cs-determination, best-case

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr. (total fraction)

Remarks:

The test item consists of 66.68% Tungsten (W).

Basis for effect:

growth rate

Remarks on result:

other: based on W-determination, worst-case

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

22.34 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.51 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr. (total fraction)

Remarks:

The test item consists of 15.92% Cesium (Cs)

Basis for effect:

biomass

Remarks on result:

other: based on Cs-determination, best-case

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.88 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr. (total fraction)

Remarks:

The test item consists of 66.68% Tungsten (W)

Basis for effect:

biomass

Remarks on result:

other: based on W-determination, worst-case

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

137.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

21.58 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr. (total fraction)

Remarks:

The test item consists of 15.92% Cesium (Cs)

Basis for effect:

growth rate

Remarks on result:

other: Cs-determination, best-case

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.37 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr. (total fraction)

Remarks:

The test item consists of 66.68% Tungsten (W)

Basis for effect:

growth rate

Remarks on result:

other: based on W-determination, worst-case

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

46.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.36 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr. (total fraction)

Remarks:

The test item consists of 15.92% Cesium (Cs)

Basis for effect:

biomass

Remarks on result:

other: based on Cs-determination, best-case

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.83 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr. (total fraction)

Remarks:

The test item consists of 66.68% Tungsten (W)

Basis for effect:

biomass

Remarks on result:

other: based on W-determination, worst-case

Details on results:

At the start and at the end of the test, the content of Cesium (Cs) and Tungsten (W) in the test solutions was determined using an ICP-OES-method. The concentration of the dissolved test was calculated from the measured concentrations of these values and the total formula (Cs0.33WO3) provided by the sponsor. The test item consisted of 15.92% Cs and 66.68% W. The solubility behaviour of both metals were different.

For the Cs determination, Cs was detectable only in the highest treatment group at the beginning of the test. At the end of the test, the measured concentrations were strongly scattering and the concentration of Cs in treatment 10 mg/L (nominal) was below the LOQ (LOQ = 2.537 mg/L). This may have been due to the fact that the Cs measurement are generally very inaccurate and fluctuating. Furthermore, a reaction between dissolved Cs and the ingredients of the test medium cannot be excluded. Since the values deviated more than ± 20 % of the nominal values, the geometric means were calculated. Only in the highest concentration was Cs clearly detectable. Therefore, only the highest concentration was used for evaluation based on the measured Cs concentrations. Since the lower concentrations were prepared by dilution of the stock solution, the real concentrations were calculated on the basis of the geometric mean of the measured concentration in the highest concentrated treatment and the respective dilution factor.

For the W determination, the measured concentrations were 4 % of the nominal concentrations at the beginning and at the end of the test. Since the values deviated more than ± 20 % of the nominal values, the geometric means were calculated.

Since the concentrations (calculated via Cs or W concentrations) differed greatly from each another, it demonstrated that the two metals have different solubility characteristics.

Results with reference substance (positive control):

- Results with reference substance valid? Yes

All validity criteria were met. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

 

Three deviations from the study plan were reported. The temperature during pre-culture incubation and during the experiment were not within the desired ranges. However, because normal growth was observed, these were considered uncritical. As well, the QC for Cs at 72hrs was outside the ±15% (76.7%), however no recalibration was conducted. The recovery, though, was taken into account in the calculation. This deviation was thus considered uncritical, as the Cs measurement was generally very unstable and fluctuating.

 

Three deviations from the guidelines were reported. The stock solutions were autoclaved for 20 minutes instead of for 15 minutes. The temperature during pre-culture incubation and during the experiment were not within the desired ranges. However, these deviations were considered uncritical.

Validity criteria fulfilled:

yes

Conclusions:

One valid experiment was performed in order to evaluate the toxicity of Cesium Tungsten Oxide towards “Desmodesmus subspicatus”. Analytical determination of the Cs and W in the test item demonstrated that the concentrations (calculated via Cs or W concentrations) differ greatly from each other.

The 72-hr EC50 for growth rate inhibition based on the nominal values was reported to be 137.25 mg/L. The 72-hr EC50 for growth rate inhibition based on the Cs-determination was reported to be 21.58 mg/L. The 72-hr EC50 for growth rate inhibition based on the W-determination was reported to be 5.37 mg/L.

Executive summary:

One valid experiment was performed in order to evaluate the toxicity of Cesium Tungsten Oxide towards “Desmodesmus subspicatus”. The study was performed using 5 concentrations ranging from 10 to 220 mg/L. Incubation time was 72 hours. Significant inhibition of algal growth was observed at the following concentrations: 46 – 220 mg/L (growth rate); 10 – 220 mg/L (yield). Analytical determination of the Cs and W in the test item demonstrated that the concentrations (calculated via Cs or W concentrations) differ greatly from each other.

 

The 72-hr EC50 for growth rate inhibition based on the nominal values was reported to be 137.25 mg/L. The 72-hr EC50 for growth rate inhibition based on the Cs-determination was reported to be 21.58 mg/L. The 72-hr EC50 for growth rate inhibition based on the W-determination was reported to be 5.37 mg/L.

 

These results do not meet the CLP classification for aquatic toxicity. This conclusion is based on the nominal loading rate of the test item (137.25 mg/L) reported in the study. According to Annex IV of ECHA’s CLP guidance version 5.0, it is the loading level which yields the level of metal ion equivalent to the available EC50 or NOEC/EC10 that can then be used to determine the acute or long-term hazard category appropriate for classification. For this algae study, the 72-hr EC50 (growth rate inhibition) reported was >100 mg/L (nominal) which is greater than the CLP cut-off limit (100 mg/L) for both the acute and chronic aquatic hazard classifications.

 

In addition, a full transformation/dissolution (T/D) study was conducted for the test item, Cesium Tungsten Oxide, under separate cover (Study No. 17100406G989). From that 7-day study, a concentration of 0.307 mg/L W was measured from a 1 mg/L (nominal) loading rate. The Cs values lay above the LOQ in flasks 9 – 11 on days 4 and 7 only, therefore the determination of kinetic was not possible for Cs. According to Annex IV of ECHA’s CLP Guidance (ver. 5.0), if the concentration at 1 mg/L loading rate is less than or equal to the acute ecological reference value (EC50) of the dissolved metal ion form, then the substance is not classified for acute hazard. Given that the measured concentration of W (0.307 mg/L) was less than its corresponding 72-hr EC50 (growth rate inhibition) value of 5.37 mg/L, this would further support the conclusion that this test item cannot be classified.

Description of key information

One valid experiment was performed in order to evaluate the toxicity of Cesium Tungsten Oxide towards “Desmodesmus subspicatus”. The study was performed using 5 concentrations ranging from 10 to 220 mg/L. Incubation time was 72 hours. Significant inhibition of algal growth was observed at the following concentrations: 46 – 220 mg/L (growth rate); 10 – 220 mg/L (yield). Analytical determination of the Cs and W in the test item demonstrated that the concentrations (calculated via Cs or W concentrations) differ greatly from each other.

 

The 72-hr EC50 for growth rate inhibition based on the nominal values was reported to be 137.25 mg/L. The 72-hr EC50 for growth rate inhibition based on the Cs-determination was reported to be 21.58 mg/L. The 72-hr EC50 for growth rate inhibition based on the W-determination was reported to be 5.37 mg/L.

 

These results do not meet the CLP classification for aquatic toxicity. This conclusion is based on the nominal loading rate of the test item (137.25 mg/L) reported in the study. According to Annex IV of ECHA’s CLP guidance version 5.0, it is the loading level which yields the level of metal ion equivalent to the available EC50 or NOEC/EC10 that can then be used to determine the acute or long-term hazard category appropriate for classification. For this algae study, the 72-hr EC50 (growth rate inhibition) reported was >100 mg/L (nominal) which is greater than the CLP cut-off limit (100 mg/L) for both the acute and chronic aquatic hazard classifications.

 

In addition, a full transformation/dissolution (T/D) study was conducted for the test item, Cesium Tungsten Oxide, under separate cover (Study No. 17100406G989). From that 7-day study, a concentration of 0.307 mg/L W was measured from a 1 mg/L (nominal) loading rate. The Cs values lay above the LOQ in flasks 9 – 11 on days 4 and 7 only, therefore the determination of kinetic was not possible for Cs. According to Annex IV of ECHA’s CLP Guidance (ver. 5.0), if the concentration at 1 mg/L loading rate is less than or equal to the acute ecological reference value (EC50) of the dissolved metal ion form, then the substance is not classified for acute hazard. Given that the measured concentration of W (0.307 mg/L) was less than its corresponding 72-hr EC50 (growth rate inhibition) value of 5.37 mg/L, this would further support the conclusion that this test item cannot be classified.

Key value for chemical safety assessment

EC50 for freshwater algae:

137.25 mg/L

EC10 or NOEC for freshwater algae:

31.6 mg/L