Lithium bis(oxalato)borate

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:

2011-12-06 to 2011-12-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

22nd July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

20th May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)

Version / remarks:

April 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

- Concentrations: 3 g activated sludge/L (on dry weight basis)
- Sample storage conditions before analysis: The activated sludge was not used on the day of the collection but continuously aerated (2 L/minute) at the test temperature for about 24 hours (1 day) and was fed once with 50 mL synthetic sewage/L activated sludge.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Based on the results of performed preliminary range-finding tests, in the main test five concentrations in a geometric series with five replicates were tested with neutralisation. One additional series (five concentrations in a geometric series), was tested without pH adjustment.

The five concentrations with pH setting (neutralisation):
655, 410, 256, 160, 100 mg/L (factor: 1.6),
The five concentrations without pH adjustment:
410, 256, 160, 100, 62.5 mg/L (factor: 1.6).

Just before the start of the test defined amounts of (5 x 196.6; 5 x 123; 5 x 76.8; 5 x 48 mg and 5 x 30 mg furthermore 5 x 123; 5 x 76.8; 5 x 48; 5 x 30 and 5 x 18.8 mg) test item were weighted into a small inert plastic dish (“plate”: to allow the contact between the inoculum and the whole test item amount) near to the test container. This plastic dish containing the adequate test item amount was thrown immediately into each test flask just before the inoculation.
Thereafter, pH measurement (and at one series the pH adjustment) was carried out and finally the test item solutions were inoculated.

- Controls:
Blank Control (CB): In the main test eight controls (containing water, synthetic sewage and inoculum, but without addition of the test or reference item), four at the start and four at the end of the test series were investigated. The number of the blank control containers was set up to the number of the O2 electrodes.
Reference Control (R): In the main test the reference item 3,5-Dichlorophenol was tested at three concentrations with three parallels (at the nominal test concentrations of 2, 7 and 24.5 mg/L).
Nitrification Control (CN): In order to check whether the sludge nitrifies and at what rate, mixtures (same as the blank controls, however containing 11.6 mg/L N-allylthiourea) were included (with three parallels) in the preliminary experiment and in the main experiment.

- Evidence of undissolved material (e.g. precipitate, surface film, etc): None

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge, microorganisms were taken from a domestic waste water treatment plant. The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary on 06 December 2011 (one day before the main test).
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, dried and the ratio of wet sludge to dry weight determined. Based on this ratio, calculated amount of wet sludge was suspended in isotonic saline solution to yield a concentration equivalent to 3 g per litre (on dry weight basis). The activated sludge was not used on the day of the collection but continuously aerated (2 L/minute) at the test temperature for about 24 hours (1 day) and was fed once with 50 mL synthetic sewage/L activated sludge. The pH of the activated sludge inoculum was checked after preparation and before use. The pH of the activated sludge inoculum after preparation was 7.53, before use: 7.55. The pH adjustment before use was considered as not necessary.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

None.

Test temperature:

20 ± 2 °C

pH:

without pH adjustment during the 3-hour exposure period:
0 mg/L: 7.58 - 8.18
62.5 mg/L: 6.84 - 8.20
100 mg/L: 6.73 - 8.14
160 mg/L: 6.87 - 8.13
256 mg/L: 6.24 - 7.76
410 mg/L: 6.50 - 7.62

with pH adjustment during the 3-hour exposure period: 7.33 - 8.49

Dissolved oxygen:

without adjustment of pH:
at start of 3-hour aeration: 6.83 - 8.69 mg O2/L
at end of 3-hour aeration: 7.03 - 8.28 mg O2/L

with adjustment of pH:
at start of 3-hour aeration: 6.83 - 8.79 mg O2/L
at end of 3-hour aeration: 7.03 - 8.81 mg O2/L

Nominal and measured concentrations:

nominal:
62.5, 100, 160, 256, 410 mg/L without pH adjustment
100, 160, 256, 410, 655 mg/L with pH adjustment

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer bottles
- Type (delete if not applicable): closed
- Material, fill volume: glass, 300 mL volume
- Aeration: with compressed air (0.5 L per minute)
- No. of vessels per concentration (replicates): five replicates
- No. of vessels per control (replicates): Reference control: three replicates; Nitrification control: 8 controls were investigated four at the start and four at the end
- No. of vessels per vehicle control (replicates): Blank control: 8 controls were investigated four at the start and four at the end
- Biomass loading rate: 3 g activated sludge/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water

OTHER TEST CONDITIONS
- Adjustment of pH: This was done due to the fact that the test item affected the pH within the test system. Therefore the test item treatments were investigated in duplicate series: with and without pH adjustment.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.6 (geometric scaling)
- Range finding study
Pre-experiments were performed to investigate the test item effect on the pH within the test system, to select the suitable method for the appropriate dosage of the test item in the test medium and to select the concentration range to be examined in the main test. In the preliminary experiments the test item showed a significant inhibitory effect and had remarkable influence on the pH.
Based on the preliminary results it was concluded that the test substance adversely affects the pH within the test system and the solutions, suspensions of the test substance in water needed to be neutralized prior to inoculum addition. However, since neutralization may change the chemical properties of the test substance, further testing was performed to assess the effect of the test substance on the sludge without pH adjustment.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

228.69 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: without pH adjustment

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

421.62 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: with pH adjustment

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: overall NOEC of the neutralised and unadjusted treatments

Details on results:

In this test analytical concentration measurements did not follow the actual test item concentrations, because of the test items direct addition. Based on the results of the preliminary experiments neither inclusion of abiotic controls nor differentiation between heterotrophic respiration and nitrification was considered as necessary. The observed-calculated specific respiration rates remained within the range of the specific respiration rates of the blank controls at the concentrations of 62.5 and 100 mg/L, without pH adjustment and at 100 and 160 mg/L (not significant: 6 % inhibition –in average- was noticed at 160 mg/L), with pH adjustment. The occurring negative values were considered and evaluated as zero inhibition. The inhibition of the specific respiration rates compared to the respiration rates of the blank controls showed dose-related tendencies with and also without neutralisation. At 160 mg/L 30 % inhibition (in average) was observed without and slight inhibition (6 %) with pH adjustment. At 256 mg/L the 61 % and 23 % and at 410 mg/L the 86 % and 31 % inhibition values showed the significant differences between the results observed when the test item was investigated without and with pH adjustment. The specific respiration rates were compared with the blank control values using Bonferroni t-Test (α=0.05). The specific respiration rates did not differ statistically significantly from the blank control at the concentrations of 62.5 and 100 mg/L, without pH adjustment and at 100 and 160 mg/L, with pH adjustment (Bonferroni t-Test, α=0.05), consequently the NOEC can be statistically and biologically determined as 100 mg/L, without pH adjustment and 160 mg/L, with pH adjustment.

Results with reference substance (positive control):

Validity Criteria: The 3 hour EC50 of the reference item 3,5-Dichlorophenol (for the used activated sludge batch) should be in the range of 2 mg/L to 25 mg/L for total respiration.

The 3-hour EC50 of the reference item 3,5-Dichlorophenol (for the used activated sludge batch) was 12.17 mg/L within the range of 2 mg/L to 25 mg/L, that was required for total respiration (in this study the differentiation between heterotrophic respiration and nitrification was considered as not necessary).

Reported statistics and error estimates:

Based on measured inhibition rates the 3 hour EC10, EC50 and EC80 and their 95 %-confidence limits were calculated by Probit analysis using TOXSTAT software.

Validity criteria fulfilled:

yes

Conclusions:

Lithium bis(oxalato)borate (LiBOB) was tested for toxicity to microorganisms by determination of inhibition of the respiration of activated sludge. The 3h NOEC was determined at 100 mg/L.

Executive summary:

The purpose of the 3 hour test was to evaluate the influence of the test item lithium bis(oxalato)borate (LiBOB) on the activity of the activated sludge by measuring the respiration rate under defined conditions. 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 after a contact time of 3 hours. The test item affected the pH within the test system, therefore the test item treatments were investigated in duplicate series: with and without pH adjustment. Based on the preliminary information about the test item, lithium bis(oxalato)borate (LiBOB) was investigated at the nominal concentrations of 62.5; 100; 160; 256 and 410 mg/L, without pH adjustment and at the concentrations of 100; 160; 256; 410 and 655 mg/L, with pH adjustment. Defined amounts of the test item were weighed directly into the test vessels in both cases. 2, 7 and 24.5 mg of 3,5-Dichlorophenol /L served as positive reference control. Further blank (inoculum) controls and nitrification controls were examined. 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. Based on measured inhibition rates the 3 hour EC10, EC50 and EC80 and their 95 %-confidence limits were calculated by Probit analysis using TOXSTAT software.

The results without pH adjustment:

EC10: 122.37 mg/L (109.25–137.06)

EC50: 228.69 mg/L (212.73–245.85)

EC80: 344.83 mg/L (311.54–381.68)

The EC50 value was determined as 228.69 mg/L, consequently: EC50 > 100 mg/L.

The results with pH adjustment:

EC10: 211.29 mg/L (187.86–237.64)

EC50: 421.62 mg/L (388.11–458.03)

EC80: 663.70 mg/L (584.13–754.09)

The EC50 value was determined as 421.62 mg/L, consequently: EC50 > 100 mg/L.

The resulting overall NOEC is 100 mg/L. (Toxicoop, 2012)

Description of key information

1. Lithium bis(oxalato)borate (LiBOB) was tested for toxicity to microorganisms by determination of inhibition of the respiration of activated sludge. The 3h NOEC was determined as 100 mg/L.
2. LiBOB was tested for toxicity to microorganisms by determination of inhibition of the respiration of activated sludge according to OECD guideline 209 revealing a 30 min EC50 of 195.06 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

100 mg/L

EC10 or NOEC for microorganisms:

100 mg/L

Additional information

1. key study

The purpose of the 3 hour test was to evaluate the influence of the test item lithium bis(oxalato)borate (LiBOB) on the activity of activated sludge by measuring the respiration rate under defined conditions. 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 after a contact time of 3 hours. The test item affected the pH within the test system, therefore the test item treatments were investigated in duplicate series: with and without pH adjustment. Based on the preliminary information about the test item, lithium bis(oxalato)borate (LiBOB) was investigated at the nominal concentrations of 62.5; 100; 160; 256 and 410 mg/L without pH adjustment and at the concentrations of 100; 160; 256; 410 and 655 mg/L with pH adjustment. Defined amounts of the test item were weighed directly into the test vessels in both cases. 2, 7 and 24.5 mg of 3,5-Dichlorophenol /L served as positive reference control. Further blank (inoculum) controls and nitrification controls were examined. 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. Based on measured inhibition rates the 3 hour EC10, EC50 and EC80 and their 95 %-confidence limits were calculated by Probit analysis using TOXSTAT software.

The results without pH adjustment:

EC10: 122.37 mg/L (109.25–137.06)

EC50: 228.69 mg/L (212.73–245.85)

EC80: 344.83 mg/L (311.54–381.68)

The EC50 value was determined as 228.69 mg/L, consequently: EC50 > 100 mg/L.

The results with pH adjustment:

EC10: 211.29 mg/L (187.86–237.64)

EC50: 421.62 mg/L (388.11–458.03)

EC80: 663.70 mg/L (584.13–754.09)

The EC50 value was determined as 421.62 mg/L, consequently: EC50 > 100 mg/L. The resulting overall NOEC is 100 mg/L. (Toxicoop, 2012)

2. disregarded study

Lithium bis(oxalato)borate (LiBOB) was tested for toxicity to microorganisms by determination of inhibition of the respiration of activated sludge similar to OECD guideline 209. Three experiments were performed. In the pre-tests, LiBOB was tested using three concentrations (10, 100, 1000 mg/L) for range-finding and 1000 mg/L for the limit test. Duration of the tests was 30 min and 3 hours. The inoculum was taken from a domestic sewage treatment plant and was washed before use. The concentration of activated sludge in the test medium was 1.5 g/L. The EC50 (3 h) of 3,5-Dichlorophenol was 22.68 mg/L, which lies in the recommended range of 2 mg/L to 25 mg/L for total respiration. An inhibition of the respiration was detected in both the range-finding and the limit test. The main test was performed with the following concentrations: 25.6, 64, 180, 400, 1000 mg/L. Duration of the test was 30 min. The following result for LiBOB was determined: 30 min EC50 = 195.06 mg/L (APM, 2011)

Discussion

The study report of APM is exhibiting a few insufficiencies with regards to the used OECD guideline 209, leading to the categorisation of Klimisch 3. The OECD guideline recommends six controls and five treatment concentrations in a geometric series with five replicates in order to obtain both a NOEC and an EC. Only one control group and no replicates had been used in order to validate the observed results. The duration of treatment was three hours and/or 30 min, respectively, in the performed range-finding- as well as in the limit-test. In the definitive test only the 30 min exposure time was tested without any reasonable explanation. The OECD guideline further states that when a substance adversely affects the pH value of the treatment solution a pH adjustment should be implemented in the study record in order to assess the effect of the test substance on the sludge with and without pH adjustment. This was not done in this study although there was a change of pH in the definitive test of up to 20 % after 30 min of exposure. The report of the results in this study was very poor as there were oversights regarding the test substance final concentration in the test suspension of the activated sludge. Instead of doubling the used amount of test substance in order to get the nominal concentration, it was halved . Further in the raw data the concentrations of the range finding test was registered in the table of the definitive test. Taking these insufficiencies into account the results of the TOXICOOP study from 2012 are used for the chemical safety assessment.