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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Mar 2018 – 25 Apr 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
All validity criteria were met
Justification for type of information:
Test data are required for classification purposes.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Adopted 23 March 2006, Annex 5 corrected: 28 July 2011
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Specific details on test material used for the study:
- Expiration date of the batch: 24 April 2019
- Appearance / colour: solid / white
- Purity analysed: 97.6% w/w
- Storage condition of test material: ambient (5 °C - 30 °C), dark, dry

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Analytical samples were taken from all test item concentrations and control at test start and after 72 hours from aged solutions. For each sampling a retained sample was also taken. All samples were stored refrigerated until they were transferred to the analytical laboratory.
After receipt in the analytical laboratory the test medium samples (21 mL) were stored between 1-10 °C until analysis. On the day of analysis, the samples were brought to room temperature. Samples were diluted with 5% nitric acid by the following dilution factors:
Control- dilution factor 1
0.0954 mg test item/L- dilution factor 1
0.305 mg test item/L- dilution factor 1
0.977 mg test item/L- dilution factor 2
3.13 mg test item/L- dilution factor 10
10.0 mg test item/L- dilution factor 20

Test solutions

Vehicle:
no
Details on test solutions:
AAP-medium (according to Annex 3 of OECD 201) was used as test medium.
The following nominal concentrations were tested: control, 0.0954, 0.305, 0.977, 3.13 and 10.0 mg/L.
The necessary amount of test item for preparing the stock solution was weighed on a weighing scoop and transferred to a volumetric flask. Test medium was added up to the benchmark and the solution was homogenised by shaking and treated with 3 minutes of ultrasonication. Lower test concentrations were prepared by dilution of the stock solution with test medium. All stock and test solutions were prepared with AAP-medium containing algae cells with a target density of 5000 cells per mL.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Strain: Pseudokirchneriella subcapitata Hindák (Sphaeropleales: Selenastraceae) strain SAG 61.81
- Source: Commercial supplier, MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany.
- Age of inoculum (at test initiation): 3 to 4 days before start of the test, test medium was inoculated with the test organism and held under test conditions in order to produce a pre-culture in the state of exponential growth.
- Method of cultivation: The algae are grown semi-continuously in sterile cultures in the laboratory. Cells from this semi-continuous liquid stock culture were used for the test. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state. Stock cultures are ordered regularly from our commercial supplier.
- Culturing conditions: Continuous illumination approx. 4440 - 8880 lux at cell culture level; temperature 21 – 24 °C; Culture flasks 100 mL Erlenmeyer flasks; CO2 supply by shaking on a rotating shaker, approximately 105 rpm.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not applicable

Test conditions

Hardness:
Not specified
Test temperature:
22.6 – 23.2 °C
pH:
Control: 7.47 – 8.23
Test groups: 7.52 – 8.22
Dissolved oxygen:
Not specified
Salinity:
Not applicable
Conductivity:
Not applicable
Nominal and measured concentrations:
Nominal (barium selenate): 10.0, 3.13, 0.977, 0.305, 0.0954 mg barium selenate/L, and control.

Measured (based on barium: 0 h fresh sample): 7.08, 2.46, 0.740, 0.237, 0.0754 mg barium selenate/L and not detectable in control (73, 81, 78, 80, 81% of nominal, respectively).
Measured (based on barium: 72 h aged sample): 7.03, 2.26, 0.775, 0.232, 0.0777 mg barium selenate/L and not detectable in control (72, 74, 81, 78 and 83% of nominal, respectively).

Measured (based on selenium: 0 h fresh sample): 7.57, 2.59, 0.760, 0.241, 0.0770 mg barium selenate/L and not detectable in control (78, 85, 80, 81 and 83% of nominal, respectively).
Measured (based on selenium: 72 h aged sample): 7.76, 2.49, 0.809, 0.252, 0.0833 mg barium selenate/L and not detectable in control (80, 82, 85, 85 and 89% of nominal, respectively).
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL Erlenmeyer flasks with aluminium caps, with approximately 50 mL of test solution.
- Aeration: CO2 supplied by continuous agitation. Test vessels were placed in an incubator on a pivoted bogie which turns around and induces shaking by regular sudden stops.
- Initial cell density: Target of 5000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 3 (separate study 15 Jan 2018 – 25 Jan 2018)

GROWTH MEDIUM
- Standard medium used: yes (AAP-Medium according to Annex 3 of OECD 201)

OTHER TEST CONDITIONS
- Adjustment of pH: pH of the test medium was adjusted to 7.5 ± 0.1 with NaOH or HCl, if necessary.
- Photoperiod: Continuous light
- Light intensity and quality: 88.1 µEm-2s-1 (mean)
Reference substance (positive control):
yes
Remarks:
Potassium dichromate is tested as the toxic reference item in a separate study twice a year to confirm the sensitivity of the test organism against compounds with known effects under the test conditions.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Remarks:
ErC50
Effect conc.:
2.41 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
barium selenate
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits: 2.06 – 2.79 mg/L
Duration:
72 h
Dose descriptor:
EC20
Remarks:
ErC20
Effect conc.:
1.01 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
barium selenate
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits: 0.785 – 1.23 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Remarks:
ErC10
Effect conc.:
0.566 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
barium selenate
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits: 0.401 – 0.734 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.305 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
barium selenate
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Remarks:
EyC50
Effect conc.:
0.651 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
barium selenate
Basis for effect:
cell number
Remarks on result:
other: 95% confidence limits: 0.418-1.43 mg/L
Duration:
72 h
Dose descriptor:
EC20
Remarks:
EyC20
Effect conc.:
0.302 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
barium selenate
Basis for effect:
cell number
Remarks on result:
other: 95% confidence limits: 0.0659 – 0.566 mg/L
Duration:
72 h
Dose descriptor:
EC10
Remarks:
EyC10
Effect conc.:
0.15 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
barium selenate
Basis for effect:
cell number
Remarks on result:
other: 95% confidence limits: 0.0176 – 0.337 mg/L
Details on results:
VALIDITY CRITERIA
All validity criteria were met:
- The biomass in the control cultures increased exponentially by a factor of at least 16 within the 72 hour test period, corresponding to a specific growth rate of 0.92/day (actual increase = factor of 55.27, specific growth rate of 1.33/day)
- The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (actual = 17%).
- The coefficient of variation of average specific growth rates during the whole test period in the control cultures did not exceed 7% (actual = 5.9%).

ANALYTICAL RESULTS
The measured initial concentrations based on barium ranged from 73 to 81% of nominal. In the aged samples the measured concentrations based on barium were between 72 and 83% of nominal. The measured initial concentrations based on selenium ranged from 78 to 85% of nominal. In the aged samples the measured concentrations based on selenium were between 80 and 89% of nominal. Therefore, the ecotoxicological endpoints were evaluated using nominal concentrations of test item.

BIOLOGICAL RESULTS
At test end (72 h), a significant inhibitory effect on growth rate was observed at nominal concentrations of 0.977 mg barium selenate/L and above, with % inhibition relative to the control of 23.2, 55.5 and 117.7% at 0.977, 3.13 and 10.0 mg barium selenate/L, respectively.
At test end (72 h), a significant inhibitory effect on yield was observed at nominal concentrations of 0.977 mg barium selenate/L and above, with % inhibition relative to the control of 62.0, 90.8 and 100.6% at 0.977, 3.13 and 10.0 mg barium selenate/L, respectively.

The morphology of the algae cells was observed microscopically at test end. The cells were considered normal for the control and up to and including a nominal test item concentration of 0.977 mg/L. At 3.13 mg/L only singular algae cells could be seen which appeared to be slightly more voluminous than in the control. No cells were observed at 100 mg/L nominal test item concentration.
Results with reference substance (positive control):
The toxic reference, potassium dichromate, was tested in a separate study at the test facility in January 2018. Test methods and conditions were the same as the main test with the test item. A static test was performed with nominal concentrations of potassium dichromate of 0.0512, 0.128, 0.320, 0.800, 2.00 mg/L and control.

Significant inhibitory effects were determined for all parameters at nominal concentrations of 0.800 mg potassium dichromate/L and above. The NOEC value was determined to be 0.320 mg potassium dichromate/L (nominal).
The ErC50 value was calculated to be 0.971 mg potassium dichromate/L (nominal).

The results demonstrate that the test system was suitable for the determination of ecotoxicological effects of the test item.
Reported statistics and error estimates:
Statistically significant inhibitory effects were determined for growth rate and yield at test item concentrations of 0.977 mg/L (nominal) and above. The overall LOEC value was therefore determined to be 0.977 mg barium selanate/L (nominal). The corresponding NOEC value was set at 0.305 mg barium selanate/L (nominal).

The EC10-value for growth rate (ErC10) was determined to be 0.566 mg barium selanate/L (nominal) (95% confidence limits: 0.401 – 0.734 mg/L).
The EC20-value for growth rate (ErC20) was determined to be 1.01 mg barium selanate/L (nominal) (95% confidence limits: 0.785 – 1.23 mg/L).
The EC50-value for growth rate (ErC50) was determined to be 2.41 mg barium selanate/L (nominal) (95% confidence limits: 2.06 – 2.79 mg/L).

The EC10-value for yield (EyC10) was determined to be 0.150 mg barium selanate/L (nominal) (95% confidence limits: 0.0176 – 0.337 mg/L).
The EC20-value for yield (EyC20) was determined to be 0.302 mg barium selanate/L (nominal) (95% confidence limits: 0.0659 – 0.566 mg/L).
The EC50-value for yield (EyC50) was determined to be 0.651 mg barium selanate/L (nominal) (95% confidence limits: 0.418 – 1.43 mg/L).

Any other information on results incl. tables

BIOLOGICAL RESULTS

Nominal test item concentration (mg/L)

Average cell numbers [104/mL]

% Inhibition of growth rate

% Inhibition of yield

0 h

24 h

48 h

72 h

0 h – 24 h

0 h – 48 h

0 h – 72 h

0 h – 24 h

0 h – 48 h

0 h – 72 h

Control

0.52

1.75

8.53

28.74

0.0

0.0

0.0

0.0

0.0

0.0

0.0954

0.52

1.70

8.12

30.69

2.9

2.4

-1.3

4.8

5.1

-6.9

0.305

0.52

2.00

5.68

21.95

-3.6

14.5

6.6

-19.4

35.6

24.1

0.977

0.52

1.05

3.33

11.23

43.9

33.9

23.2*

57.3

64.9

62.0*

3.13

0.52

0.56

2.17

3.13

94.1

49.5

55.5*

96.8

79.4

90.8*

10.0

0.52

0.18

0.67

0.35

209.7

95.6

117.7*

127.4

98.1

100.6*

* Statistically significantly different to the control

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Statistically significant inhibitory effects were determined for growth rate and yield at test item concentrations of 0.977 mg/L (nominal) and above. The overall LOEC value was therefore determined to be 0.977 mg barium selanate/L (nominal). The corresponding NOEC value was set at 0.305 mg barium selanate/L (nominal).

The EC10-value for growth rate (ErC10) was determined to be 0.566 mg barium selanate/L (nominal) (95% confidence limits: 0.401 – 0.734 mg/L).
The EC20-value for growth rate (ErC20) was determined to be 1.01 mg barium selanate/L (nominal) (95% confidence limits: 0.785 – 1.23 mg/L).
The EC50-value for growth rate (ErC50) was determined to be 2.41 mg barium selanate/L (nominal) (95% confidence limits: 2.06 – 2.79 mg/L).

The EC10-value for yield (EyC10) was determined to be 0.150 mg barium selanate/L (nominal) (95% confidence limits: 0.0176 – 0.337 mg/L).
The EC20-value for yield (EyC20) was determined to be 0.302 mg barium selanate/L (nominal) (95% confidence limits: 0.0659 – 0.566 mg/L).
The EC50-value for yield (EyC50) was determined to be 0.651 mg barium selanate/L (nominal) (95% confidence limits: 0.418 – 1.43 mg/L).
Executive summary:

Effects of barium selenate on the growth of the single cell green alga Pseudokirchneriella subcapitata were tested in a 72 hour static dose-response test in accordance with OECD guideline 201 (2011) and to GLP.

Five test item concentrations were tested at nominal concentrations of 0.0954, 0.305, 0.977, 3.13, 10.0 mg/L. Additionally, a control (AAP-Medium) group was tested. Three replicates of each test concentration and six control replicates were tested, containing an initial cell density of 0.5 x 104 cells/mL. A reference item, potassium dichromate, was tested in a separate study at the test facility in January 2018.

The analytical verification of test item concentrations was conducted by analysing the content of barium and selenium in samples taken at test start and from aged solutions at 72 h. The measured initial concentrations based on barium ranged from 73 to 81% of nominal. In the aged samples the measured concentrations based on barium were between 72 and 83% of nominal. The measured initial concentrations based on selenium ranged from 78 to 85% of nominal. In the aged samples the measured concentrations based on selenium were between 80 and 89% of nominal. Therefore, the ecotoxicological endpoints were evaluated using nominal concentrations of test item.

The test was considered valid as all validity criteria were met in accordance with OECD 201 (2011).

At test end (72 h), a significant inhibitory effect on growth rate was observed at nominal concentrations of 0.977 mg barium selenate/L and above, with % inhibition relative to the control of 23.2, 55.5 and 117.7% at 0.977, 3.13 and 10.0 mg barium selenate/L, respectively. The corresponding NOEC value was set at 0.305 mg barium selanate/L (nominal).The EC50-value for growth rate (ErC50) was determined to be 2.41mg barium selanate/L (nominal) (95% confidence limits: 2.06 – 2.79 mg/L).

 

At test end (72 h), a significant inhibitory effect on yield was observed at nominal concentrations of 0.977 mg barium selenate/L and above, with % inhibition relative to the control of 62.0, 90.8 and 100.6% at 0.977, 3.13 and 10.0 mg barium selenate/L, respectively. The corresponding NOEC value was set at 0.305 mg barium selanate/L (nominal).The EC50-value for yield (EyC50) was determined to be 0.651 mg barium selanate/L (nominal) (95% confidence limits: 0.418 – 1.43 mg/L).