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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 March 2018 to 26 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: Rubidium Oxide
Batch: S19D031
Purity: 45.8 wt% in water
Physical state/Appearance: Pale yellow liquid
Expiry Date: 20 October 2018
Storage Conditions: Room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control and each test group for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
A nominal amount of test item (109 mg) was dissolved in test water and the volume adjusted to 500 mL to give the 100 mg a.i./L test concentration from which a series of dilutions was made to give further test concentrations of 0.10, 1.0 and 10 mg a.i./L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (3.1 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg ai/L. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity. At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ±1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours. After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg ai/L. All test concentrations were corrected for a test item purity value of 45.8%.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1°C.

Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10^4 to 10^5 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
Standard duration
Post exposure observation period:
None, not a standard test requirement
Hardness:
Not reported
Test temperature:
24 ± 1°C
pH:
0h: 7.6 (control), 7.6, 8.1, 9.3, 10.3 and 11.1 in rubidium oxide treatments in ascending concentration order.
72h: 7.5 (control), 7.8, 8.0, 7.8, 7.8 and 8.1 in rubidium oxide treatments in ascending concentration order.
Dissolved oxygen:
NA
Salinity:
NA
Conductivity:
NA
Nominal and measured concentrations:
Nominal test concentrations: 1.0, 3.2, 10, 32 and 100 mg a.i./L
Details on test conditions:
Exposure Conditions:
In the definitive test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group. Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 1.23 x 10^6 cells per mL. Inoculation of 500 mL of test medium with 2.0 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL and had no significant dilution effect on the final test concentration. The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ±1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Test Organism Observations:
Samples were taken at 27, 50 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density. To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Water Quality Criteria:
The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily. The appearance of the test media was recorded daily.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
40 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
9.2 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Remarks on result:
other: 95% Confidence Limits: 8.7 - 9.6 mg a.i./L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Details on results:
After 72 hours there were no abnormalities detected in the control or test cultures at 1.0, 3.2 and 10 mg a.i./L, however cell debris and very few healthy cells were observed to be present in the test cultures at 32 mg a.i./L whilst cell debris and some enlarged cells were observed to be present in the 100 mg a.i./L test cultures.
Results with reference substance (positive control):
The positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L, with exposure conditions being similar to those in the definitive test. Exposure of Pseudokirchneriella subcapitata to the reference item resulted in 72 hour ErC50 and EyC50 values of 1.6 mg/L (95% confidence limits: 1.4 to 1.8 mg/L) and 0.77 mg/L (95% confidence limits: 0.68 to 0.87 mg/L), respectively. Resulting 72 hour NOEC and LOEC values were 0.25 mg/L and 0.5 mg/L respectively, based on both growth rate and yield. The results from the positive control were within the normal range for this reference item.
Reported statistics and error estimates:
Growth rate:
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences (P≥0.05), between the control, 1.0 and 3.2 mg a.i./L test concentrations, however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 3.2 mg a.i./L. Correspondingly the LOEC based on growth rate was 10 mg a.i./L.

Inhibition of Yield:
There were no statistically significant differences (P≥0.05), between the control, 1.0 and 3.2 mg a.i./L test concentrations however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on yield was 3.2 mg a.i./L. Correspondingly the LOEC based on yield was 10 mg a.i./L.

Inhibition of growth rate and yield in the definitive test

Nominal concentration (mg a.i./L)

Growth rate (cells/mL/hour)

Yield (cells/mL)

0 to 72 hour

% inhibition

0 to 72 hour

% inhibition

Control

R1

0.074

-

1.02E+06

-

R2

0.075

1.07E+06

R3

0.073

9.83E+05

R4

0.076

1.15E+06

R5

0.076

1.20E+06

R6

0.075

1.12E+06

Mean

0.075

1.09E+06

SD

0.001

8.24E+04

1.0

R1

0.073

3

9.37E+05

 

R2

0.073

3

9.71E+05

 

R3

0.074

1

1.04E+06

 

Mean

0.073

2

9.83E+05

10

SD

0.001

 

5.43E+04

 

3.2

R1

0.073

3

9.78E+05

 

R2

0.073

3

9.31E+05

 

R3

0.080

[7]

1.56E+06

 

Mean

0.075

[0]

1.16E+06

[6]

SD

0.004

 

3.52E+05

 

10

R1

0.063

16

4.63E+05

 

R2

0.055

27

2.57E+05

 

R3

0.054

28

2.44E+05

 

Mean

0.057

24

3.21E+05

71

SD

0.005

 

1.23E+05

 

32

R1

0.043

43

1.03E+05

 

R2

0.040

47

8.16E+04

 

R3

0.039

48

7.56E+04

 

Mean

0.041

46

8.66E+04

92

SD

0.002

 

1.42E+04

 

100

R1

0.026

65

2.68E+04

 

R2

0.022

71

1.86E+04

 

R3

0.022

71

1.92E+04

 

Mean

0.023

69

2.15E+04

98

SD

0.002

 

4.58E+03

 

Validity criteria fulfilled:
yes
Remarks:
The study fulfilled all of the validity described by the OECD Guideline.
Conclusions:
Exposure of the freshwater algae Pseudokirchneriella subcapitata to the test item (rubidium oxide) over a 72 hour period resulted in nominal EC50 values of 40 mg a.i./L and 9.2 mg a.i./L based on growth rate and yield, respectively. The resulting 72 hour NOEC and LOEC values were 3.2 mg a.i./L and 10 mg a.i./L for both growth rate and yield.
Executive summary:

The toxicity of Rubidium Oxide to the freshwater algae, Pseudokirchneriella subcapitata, was assessed via an OECD Guideline 201 (‘Freshwater Alga and Cyanobacteria, Growth Inhibition Test’) study. The study was performed at a GLP accredited laboratory, fulfilled the validity criteria described by the guideline and is well documented and is therefore considered to be reliable.  

  

Exposures of P. subcapitata to the test item resulted in nominal 72 hour EC50 values of 40 mg a.i./L and 9.2 mg a.i./L based on growth rate and yield, respectively. The resulting 72 hour NOEC and LOEC values were 3.2 mg a.i./L and 10 mg a.i./L for both growth rate and yield.

Description of key information

The toxicity of Rubidium Oxide to the freshwater algae, Pseudokirchneriella subcapitata, was assessed via an OECD Guideline 201 (‘Freshwater Alga and Cyanobacteria, Growth Inhibition Test’) study. The study was performed at a GLP accredited laboratory, fulfilled the validity criteria described by the guideline and is well documented and is therefore considered to be reliable.  

  

Exposures of P. subcapitata to the test item resulted in nominal 72 hour EC50 values of 40 mg a.i./L and 9.2 mg a.i./L based on growth rate and yield, respectively. The resulting 72 hour NOEC and LOEC values were 3.2 mg a.i./L and 10 mg a.i./L for both growth rate and yield.

Key value for chemical safety assessment

EC50 for freshwater algae:
9.2 mg/L
EC10 or NOEC for freshwater algae:
3.2 mg/L

Additional information