Lithium bis(oxalato)borate

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:

2008-08-21 to 2008-08-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant study.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: (nominal) 5, 10, 20, 30, 40, 50 mg test item/L
- Sampling method and sample storage conditions before analysis: At the start of the study one sample was taken from each concentration level and at the end one sample was taken from each replicate test vessel. Three samples were taken from the control medium. The samples were analysed directly after sampling.

Vehicle:

no

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Green Algae

- Strain: Pseudokirchneriella subcapitata (formerly: Selenastrum capricornutum)

- Source (laboratory, culture collection): The algae were supplied by the Georg-August-Universität Göttingen, Albrecht-von-Haller Institut für Pflanzenwissenschaften experimentelle Phykologie und Sammlung von Algenkulturen (SAG), Nikolausberger Weg 18, 37073, Göttingen, Germany

- Age of inoculum (at test initiation), method of cultivation, acclimation period and culturing media and conditions:
The test started (0 hours) by inoculation of a biomass of approximately 10,000 algal cells per mL test medium (Algae mineral salts test medium). These cells were taken from an exponentially growing pre-culture, set up three days prior to the test start using the same conditions as in the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24.2 - 24.5 °C +/- 2 °C

pH:

4.91 to 7.58 at test start
4.91 to 8.57 at end test

Nominal and measured concentrations:

The nominal test item concentrations were 5.0; 10.0; 20.0; 30.0; 40.0 and 50.0 mg test item/L.
The measured test item concentrations were in the ±20 % range at the start (85 % - 115 %) and at the end (89 % - 119 %) of the test.

Therefore nominal concentrations were used for calculations.

Details on test conditions:

TEST SYSTEM
The test was performed with three replicates per test concentration and six replicates in the untreated control. On a laboratory orbital shaker, volumes of 100 mL algal suspensions per replicate were continuously shaken in 250 mL Erlenmeyer flasks, covered with air-permeable stoppers. The test was started (0 hours) by inoculation of a biomass of approximately 10,000 algal cells per mL test medium.

GROWTH MEDIUM
- Medium: Standard: Mineral Salts Test Medium (according to OECD 201 and ISO 14442)

- Determination of cell concentrations: The cell numbers were determined at 0, 24, 48 and 72 hours after starting the test by manual cell counting using a microscope with counting chamber.
- Range finding study: The concentration levels are chosen based on the results of a range-finding test. In the non-GLP preliminary study nominal concentrations of 0.01; 0.1; 1.0; 10.0; 25.0; 50.0; 100.0 mg/L were tested. The cell concentration at the beginning of the preliminary study was 10,000 for every concentration and control.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH was checked at the beginning and at the end of the study in the controls and for every test item concentration algae flask. The pH was not adjusted throughout the test period.
- Light intensity and quality: The light intensity at the position occupied by algal culture flasks during the test was about 8166.27 lux, which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The light intensity was checked and recorded at the start of the test. Additional checks were performed regularly throughout the test.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: counting chamber

TEST CONCENTRATIONS
- Range finding study: yes: 0.01, 0.1, 1, 10, 25, 50 and 100 mg/L; 72 hours; growth inhibition started at 25 mg/L
- Test concentrationsin the main study: 0, 5, 10, 20, 30, 40 and 50 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

35.11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL: 33.76-36.46

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

40.69 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 39.48-41.90

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

34.24 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 32.72-35.77

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

30 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

30 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test), e.g. unusal cell shape: No
- Any stimulation of growth found in any treatment: No
- 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

Results with reference substance (positive control):

The date of the last study (Study Code: 08/712-022AL) with the reference item Potassium dichromate is: 20 – 23 June 2008 (valid)
The ErC 50 : 0.90 mg/L, (95 % confidence limits: 0.82 – 0.98 mg/L)
The EbC 50 : 0.60 mg/L, (95 % confidence limits: 0.54 – 0.66 mg/L)
The EyC 50 : 0.48 mg/L, (95 % confidence limits: 0.44 – 0.53 mg/L)

Reported statistics and error estimates:

Mean values and standard deviations of cell concentrations were calculated for each treatment at the start, after 0 h, 24 h, 48 h and at the end of the test (72 hours after the start of the test) using Excel for Windows software.
Percentage inhibition of area A, growth rate r and yield Y were calculated using EXCEL for Windows software.
The EC50 values of the test item and their confidence limits were calculated using Probit analysis. The analysis was done using the statistical software program “TOXSTAT 3.5”.
For the determination of the LOEC and NOEC, the calculated mean biomass b (area under the growth curve), growth rates r and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test and Williams’test by TOXSTAT software. The data were checked for normality by Chi-Square Test, for homogeneity of variance for Levene’s Test.

Validity criteria fulfilled:

yes

Conclusions:

In the 72h algae growth inhibition test on Pseudokirchneriella subcapitata with lithium bis(oxalato)borate (LiBOB), the 72-h EC50 based on growth rate was determined as 40.69 mg/L. Yield was the most sensitive parameter with a 72h EC50 of 34.24 mg/L. The overall NOEC was determined to be 10.0 mg/L.

Executive summary:

The present study examined the influence of the test item lithium bis(oxalato)borate (LiBOB) on the growth of an unicellular green algae- Pseudokirchneriella subcapitata- according to OECD guideline 201 and EU method C.3. The test concentrations (nominal 5.0 to 50.0 mg/L) were selected according to results of a range-finding test. The selected test concentrations were analysed at the beginning and at the end of the study, ensuring that the nominal values could be used for calculations. During the test time, the control algae cell growth was as expected and pH values and temperature were steady.  After exposure for 72 hours, the test item, in the concentration range of 20.0 to 50.0 mg, had a statistically significant inhibitory effect on the growth based on the average specific growth rate, yield and areas under the growth curves of Pseudokirchneriella subcapitata. The 72 h EC50 parameters (EbC50, Er50, EyC50) are detailed as follows: 35.11 mg/L (biomass), 40.69 mg/L (growth rate), 34.24 mg/L (yield). The overall NOEC was determined to be 10.0 mg/L. Additionally, no abnormalities of the algae cells were observed. (LAB, 2008)

Description of key information

In the 72h algae growth inhibition test on Pseudokirchneriella subcapitata with lithium bis(oxalato)borate (LiBOB), the 72h EC50 based on growth rate was determined as 40.69 mg/L. Yield was the most sensitive parameter with a 72h EC50 of 34.24 mg/L. The overall NOEC was determined to be 10.0 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

40.69 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L

Additional information

The present study examined the influence of the test item lithium bis(oxalato)borate (LiBOB) on the growth of an unicellular green algae- Pseudokirchneriella subcapitata- according to OECD guideline 201 and EU method C.3. The test concentrations (nominal 5.0 to 50.0 mg/L) were selected according to results of a range-finding test. The selected test concentrations were analysed at the beginning and at the end of the study, ensuring that the nominal values could be used for calculations. During the test time, the control algae cell growth was as expected and pH values and temperature were steady.  After exposure for 72 hours, the test item, in the concentration range of 20.0 to 50.0 mg, had a statistically significant inhibitory effect on the growth based on the average specific growth rate, yield and areas under the growth curves of Pseudokirchneriella subcapitata. The 72h EC50 parameters (EbC50, Er50, EyC50) are detailed as follows: 35.11 mg/L (biomass), 40.69 mg/L (growth rate), 34.24 mg/L (yield). The overall NOEC was determined to be 10.0 mg/L. Additionally, no abnormalities of the algae cells were observed. (LAB, 2008)