Lithium nickel potassium oxide

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:

11th June 2019 to August 1st 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

no

GLP compliance:

yes

Details on sampling:

Preparation of Inoculum:

The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC overnight prior to use in the test. Determination of the mixed liquor suspended solids level (MLSS or SS) of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed Whatman GF/A filter paper* using a Buchner funnel which was then rinsed 3 times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC for at least one hour and allowed to cool before weighing. This process was repeated until two consecutive dry weights within 4% were attained. The SS concentration was equal to 3.0 g dry weight/L prior to the activated sewage sludge (9.9 liters) being fed an aliquot of synthetic sewage (495 mL).

The pH of the sample on the day of the test was 7.5, measured using a Hach HQ40d Flexi handheld meter. The level of the MLSS was checked prior to use to ensure the suspended solids concentration was equal to 3.0 g/L so that the level of the MLSS in the final test medium after dilution was equivalent 1.5 g dry weight/L.

Vehicle:

yes

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Preparation of Inoculum:

The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC overnight prior to use in the test. Determination of the mixed liquor suspended solids level (MLSS or SS) of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed Whatman GF/A filter paper* using a Buchner funnel which was then rinsed 3 times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC for at least one hour and allowed to cool before weighing. This process was repeated until two consecutive dry weights within 4% were attained. The SS concentration was equal to 3.0 g dry weight/L prior to the activated sewage sludge (9.9 liters) being fed an aliquot of synthetic sewage (495 mL).

The pH of the sample on the day of the test was 7.5, measured using a Hach HQ40d Flexi handheld meter. The level of the MLSS was checked prior to use to ensure the suspended solids concentration was equal to 3.0 g/L so that the level of the MLSS in the final test medium after dilution was equivalent 1.5 g dry weight/L.

Test type:

not specified

Water media type:

other:

Remarks:

Test Water The test water used for the test was deionized reverse osmosis water containing less than 1 mg/L Total Organic Carbon (TOC).

Total exposure duration:

3 h

Remarks on exposure duration:

Observations were made on the test preparations throughout the test period. Observations of the test item vessels at 0 hours were made prior to addition of activated sewage sludge.

Test temperature:

Activated sewage sludge was exposed to an aqueous dispersion of the test item at nominal concentrations of 10, 100 and 1000 mg/L (three replicates of the 1000 mg/L test concentration) for a period of 3 hours at measured temperatures of between 20 °C and 21 °C with the addition of a synthetic sewage as a respiratory substrate. A reference item, 3,5-dichlorophenol, and control group were incubated concurrently and the rates of respiration were determined following the 3-hour exposure period.

pH:

The pH of test preparations was measured at the test start (i.e. after the addition of activated sludge) and at the end of the 3-hour incubation period using a Hach HQ40d Flexi handheld meter.

Dissolved oxygen:

The oxygen concentrations in all vessels were measured after 30 minutes contact time.

As each vessel reached the 3-Hour contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between 7 mg O2/L and 2 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over a period of up to 10 minutes.

Nominal and measured concentrations:

Activated sewage sludge was exposed to an aqueous dispersion of the test item at nominal concentrations of 10, 100 and 1000 mg/L (three replicates of the 1000 mg/L test concentration) for a period of 3 hours at measured temperatures of between 20 °C and 21 °C with the addition of a synthetic sewage as a respiratory substrate. A reference item, 3,5-dichlorophenol, and control group were incubated concurrently and the rates of respiration were determined following the 3-hour exposure period.

Details on test conditions:

Experimental Design and Study Conduct

Range-Finding Test

Test Item Preparation
In the range-finding test, activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L (three replicates for the test concentration of 1000 mg/L). The test item was dispersed directly in water.
Nominal amounts of test item (5, 50 and 500 mg (in triplicate for 500 mg)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and sealed with Nescofilm prior to being subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at room temperature, to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing.

Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (three replicates for the 1000 mg/L test concentration). A single test vessel was prepared for each of the two lower concentrations and three replicates were prepared for the highest test item concentration.
The pH of the test item dispersions was measured after stirring using a Hach HQ40d Flexi handheld meter and adjusted to between pH 7.0 and 8.0 as necessary.


As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
The control group (four replicates) was maintained concurrently under identical conditions, but was not exposed to the test item.

Reference Item Preparation
A reference item, 3,5-Dichlorophenol, was included in the range-finding test at concentrations of 3.2, 10 and 32 mg/L to confirm the suitability of the inoculum. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 20 minutes. The pH of this stock solution was measured to be pH 5.8 and was adjusted to pH 7.1 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d Flexi handheld meter. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to a final volume of 500 mL to give the required concentrations of 3.2, 10 and 32 mg/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.


Preparation of Inoculum:

The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC overnight prior to use in the test. Determination of the mixed liquor suspended solids level (MLSS or SS) of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed Whatman GF/A filter paper* using a Buchner funnel which was then rinsed 3 times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC for at least one hour and allowed to cool before weighing. This process was repeated until two consecutive dry weights within 4% were attained. The SS concentration was equal to 3.0 g dry weight/L prior to the activated sewage sludge (9.9 liters) being fed an aliquot of synthetic sewage (495 mL).

The pH of the sample on the day of the test was 7.5, measured using a Hach HQ40d Flexi handheld meter. The level of the MLSS was checked prior to use to ensure the suspended solids concentration was equal to 3.0 g/L so that the level of the MLSS in the final test medium after dilution was equivalent 1.5 g dry weight/L.

Preparation of Test System
At the start of the test (time "0"), 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute. Thereafter, at 15 minute intervals, the procedure was repeated for the second control followed by the reference item vessels (in ascending concentration order) with appropriate amounts of the reference item being added.

Finally, two further control vessels were prepared. The addition of the inoculum to the reaction mixture was considered the start of the 3-hour exposure period for each vessel.
The test was conducted under normal laboratory lighting in a temperature controlled room at measured temperatures of between 20 °C and 21 °C.

Reference substance (positive control):

yes

Remarks:

Identification: 3,5-Dichlorophenol Batch: MKBZ0947V Purity: 97% Physical State/Appearance: Pale brown crystalline solid Expiry: 04 July 2023 Storage Conditions: Room temperature Supplier: Sigma-Aldrich

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Range-Finding Test

No statistically significant toxic effects (p > 0.05) were shown at the test concentrations of 10 and 100 mg/L; however, statistically significant toxic effects (p < 0.05) were shown at the test concentration of 1000 mg/L . Given that the test objectives were met in the preliminary test and the 3-hour EC50 value for the test item was estimated to be greater than 1000 mg/L, it was concluded that further testing to determine a No Observed Effect Concentration (NOEC) was unnecessary.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.
Percentage inhibition was plotted against concentration for the reference and test items.

The temperatures recorded in the first and last control vessels were measured to be 21 °C and 20 °C, respectively.

Results with reference substance (positive control):

Validation Criteria
The EC50 value (3-hour contact time) for the reference item, 3,5-dichlorophenol, was 5.0 mg/L. The specific respiration rate of the controls was 26.33 mg oxygen per gram dry weight of sludge per hour. The coefficient of variation of oxygen uptake in the control vessels was 4.8%.
All validation criteria for the study were therefore satisfied.

Reported statistics and error estimates:

No statistically significant toxic effects (p > 0.05) were shown at the test concentrations of 10 and 100 mg/L; however, statistically significant toxic effects (p < 0.05) were shown at the test concentration of 1000 mg/L . Given that the test objectives were met in the preliminary test and the 3-hour EC50 value for the test item was estimated to be greater than 1000 mg/L, it was concluded that further testing to determine a No Observed Effect Concentration (NOEC) was unnecessary.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.
Percentage inhibition was plotted against concentration for the reference and test items.

The temperatures recorded in the first and last control vessels were measured to be 21 °C and 20 °C, respectively.

Validity criteria fulfilled:

yes

Conclusions:

Based on the observed effects of the test item on the respiration of activated sewage sludge micro-organisms, the 3-hour EC50 value was determined to be greater than 1000 mg/L. It was considered unnecessary to test at concentrations in excess of 1000 mg/L or to determine the No Observed Effect Concentration (NOEC).

Executive summary:

 

Introduction

This study was performed to assess the effect of the test item, Lithium nickel potassium oxide (KDLNO), on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209, "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

Methods

Activated sewage sludge was exposed to an aqueous dispersion of the test item at nominal concentrations of 10, 100 and 1000 mg/L (three replicates of the 1000 mg/L test concentration) for a period of 3 hours at measured temperatures of between 20 °C and 21 °C with the addition of a synthetic sewage as a respiratory substrate. A reference item, 3,5-dichlorophenol, and control group were incubated concurrently and the rates of respiration were determined following the 3-hour exposure period.

Results

Based on the observed effects of the test item on the respiration of activated sewage sludge micro-organisms, the 3-hour EC50 value was determined to be greater than 1000 mg/L. It was considered unnecessary to test at concentrations in excess of 1000 mg/L or to determine the No Observed Effect Concentration (NOEC).

The 3-hour EC50 value for the reference item, 3-5-dichlorophenol, was determined to be 5.0 mg/L, with 95% confidence limits of 3.6 to 6.9 mg/L.

Description of key information

Based on the observed effects of the test item on the respiration of activated sewage sludge micro-organisms, the 3-hour EC50 value was determined to be greater than 1000 mg/L. It was considered unnecessary to test at concentrations in excess of 1000 mg/L or to determine the No Observed Effect Concentration (NOEC).

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L