Bis(acetato-O)hydroxyaluminium

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:

2019-02-22 to 2019-04-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

updated on 1st March 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OCSPP 850.3300 (Modified Activated Sludge, Respiration Inhibition Test)

Version / remarks:

January 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Remarks:

In this study no analytical measurements were performed. Because of the directly added test item the obtained results were referred to the nominal test item concentration.

Vehicle:

no

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary on 25 February 2019 (two days before the test).
- Preparation of inoculum for exposure:
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 (5.394 g wet weight), dried and the ratio of wet sludge to dry weight (0.4222 g dry weight) determined. Based on this ratio, calculated amount of wet sludge (21 g dry weight that was equivalent to 268 g wet sludge) was suspended in isotonic saline solution (ad. 7 L) to yield a concentration equivalent to about 3 g per litre (on dry weight basis). (In the test containers (300 mL final volume) the final concentration of suspended solids, containing 150 mL inoculum was 1.5 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 48 hours (2 days) and was fed daily with 50 mL synthetic sewage/L activated sludge. The pH of the activated sludge inoculum was checked after preparation (pH: 7.38), additional pH adjustment of the inoculum was considered not necessary.
- Initial biomass concentration: 3 g/L (on dry weight basis)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Hardness:

No data

Test temperature:

20 ± 2 °C

pH:

4.72 - 8.20

Dissolved oxygen:

No data

Salinity:

Not applicable

Conductivity:

No data

Nominal and measured concentrations:

Nonimal: 0, 10, 100 and 1000 mg/L
No measured test item concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel (type and size): Erlenmeyer bottles of approximately 300 mL volume.
- Aeration: With compressed air (0.5 litre per minute)
- No. of vessels per concentration (replicates): According to the guideline requirements, triplicates were examined at the highest tested concentration of 1000 mg/L and additionally two lower concentration levels of 10 and 100 mg/L were examined with one vessel each.
- No. of vessels per control (replicates): Eight blank 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; a reference control (reference item 3,5-Dichlorophenol) was tested at three concentrations; a nitrification control was included in two parallels.
- No. of vessels per abiotic control (replicates): The abiotic control was tested at the highest test item concentration of 1000 mg/L in three parallels

TEST MEDIUM / WATER PARAMETERS
- in accordance with guideline

OTHER TEST CONDITIONS
- Adjustment of pH: No

EFFECT PARAMETERS MEASURED: Respiration rate after 3 hours

TEST CONCENTRATIONS
- Range finding study : Yes
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: This pre-test demonstrated the absence of inhibition of oxygen consumption of the test item up to and including the limit concentration of 1000 mg/L, therefore in line with OECD guideline 209 an additional definite test is not required.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC10

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.:

> 900 mg/L

Nominal / measured:

nominal

Conc. based on:

other: calculated value for anhydrous substance

Basis for effect:

inhibition of total respiration

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:

EC50

Effect conc.:

> 900 mg/L

Nominal / measured:

nominal

Conc. based on:

other: calculated value for anhydrous substance

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

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:

NOEC

Effect conc.:

900 mg/L

Nominal / measured:

nominal

Conc. based on:

other: calculated value for anhydrous substance

Basis for effect:

inhibition of total respiration

Details on results:

The observed oxygen consumption rates and consequently the specific respiration rates in all examined test item concentrations remained in the range of the blank controls, no inhibitory effect of the test item was observed. The specific respiration rates did not differ statistically significantly from that of the control at the concentration of 1000 mg/L (2 Sample t-Test; (α=0.05)).
The test was performed including abiotic controls. The abiotic controls were investigated at the test item concentration of 1000 mg/L and no remarkable abiotic oxygen consumption was noticed.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: The 3-hour EC50 of the reference item 3,5-dichlorophenol (for the used activated sludge batch) was 14.6 mg/L and 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:

The 3-hour EC50 value of the reference item and its 95 %-confidence limits were calculated by appropriate statistical test (Probit analysis by IBM® SPSS® Statistics, Version 25 (2017) statistical software program).
The specific respiration rates of the highest test item concentration level were compared to the blank control values using the 2 Sample t-Test (2-sided, α=0.05) by IBM® SPSS® Statistics, Version 25 (2017) software.

Validity of the Study:

The study was considered as valid because the specific respiration rate of the blank controls (without test item or reference item) was 28.25 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (higher than 20 mg/gh) with a coefficient of variation of 3.64 %.

The 3-hour EC50 of the reference item 3,5-dichlorophenol (for the used activated sludge batch) was 14.6 mg/L and 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).

Additionally, the observed difference between start and the end blank control values was minimal (~5 %), within the biological variability of the applied system.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of test item was determined as higher than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was 1000 mg/L. This pre-test demonstrated the absence of inhibition of oxygen consumption of the test item up to and including the limit concentration of 1000 mg/L, therefore in line with OECD guideline 209 an additional definite test is not required. Based on the molecular weight the recalculated EC10 and EC50 for the anhydrous substance is higher than 900 mg/L and the NOEC for the anhydrous substance is 900 mg/L.

Executive summary:

The influence of the test item Aluminium hydroxide diacetate hydrate on the activity of the activated sludge was evaluated by measuring the respiration rate under defined conditions according to OECD guideline 209 and EU method C.11. 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. Three test item concentrations (10, 100 and 1000 mg/L) were selected in accordance with the referred OECD 209 Guideline to demonstrate a possible significant inhibition of oxygen consumption to necessitate a subsequent definite test or demonstrate the absence of significant toxic effect (examined by 2 Sample t-Test) up to the highest examined test item concentration of 1000 mg/L. Defined amounts of the test item were added (measured) directly into the test vessels. In parallel with the test item treatments 3,5-dichlorophenol as positive reference control in concentrations of 2, 7 and 24.5 mg/L; furthermore blank (inoculum) control, nitrification controls and abiotic controls were investigated. The observed oxygen consumption rates and consequently the specific respiration rates in all examined test item concentrations remained in the range of the blank controls, no inhibitory effect of the test item was observed. The specific respiration rates did not differ statistically significantly from that of the control at the concentration of 1000 mg/L (2 Sample t-Test; (α=0.05)). The test was performed including abiotic controls. The abiotic controls were investigated at the test item concentration of 1000 mg/L and no remarkable abiotic oxygen consumption was noticed.

Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of test item was determined as higher than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was 1000 mg/L. This pre-test demonstrated the absence of inhibition of oxygen consumption of the test item up to and including the limit concentration of 1000 mg/L, therefore in line with OECD guideline 209 an additional definite test is not required. Based on the molecular weight the recalculated EC10 and EC50 for the anhydrous substance is higher than 900 mg/L and the NOEC for the anhydrous substance is 900 mg/L.

Description of key information

Under the conditions of the performed Activated Sludge Respiration Inhibition Test (reference 6.1.7-1), the EC10 and EC50 values of test item was determined as higher than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was 1000 mg/L. This pre-test demonstrated the absence of inhibition of oxygen consumption of the test item up to and including the limit concentration of 1000 mg/L, therefore in line with OECD guideline 209 an additional definite test is not required. Based on the molecular weight the recalculated EC10 and EC50 for the anhydrous substance is higher than 900 mg/L and the NOEC for the anhydrous substance is 900 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

900 mg/L

Additional information

The influence of the test item Aluminium hydroxide diacetate hydrate on the activity of the activated sludge was evaluated by measuring the respiration rate under defined conditions according to OECD guideline 209 and EU method C.11 (reference 6.1.7-1). 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. Three test item concentrations (10, 100 and 1000 mg/L) were selected in accordance with the referred OECD 209 Guideline to demonstrate a possible significant inhibition of oxygen consumption to necessitate a subsequent definite test or demonstrate the absence of significant toxic effect (examined by 2 Sample t-Test) up to the highest examined test item concentration of 1000 mg/L. Defined amounts of the test item were added (measured) directly into the test vessels. In parallel with the test item treatments 3,5-dichlorophenol as positive reference control in concentrations of 2, 7 and 24.5 mg/L; furthermore blank (inoculum) control, nitrification controls and abiotic controls were investigated. The observed oxygen consumption rates and consequently the specific respiration rates in all examined test item concentrations remained in the range of the blank controls, no inhibitory effect of the test item was observed. The specific respiration rates did not differ statistically significantly from that of the control at the concentration of 1000 mg/L (2 Sample t-Test; (α=0.05)). The test was performed including abiotic controls. The abiotic controls were investigated at the test item concentration of 1000 mg/L and no remarkable abiotic oxygen consumption was noticed.

Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of test item was determined as higher than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was 1000 mg/L. This pre-test demonstrated the absence of inhibition of oxygen consumption of the test item up to and including the limit concentration of 1000 mg/L, therefore in line with OECD guideline 209 an additional definite test is not required. Based on the molecular weight the recalculated EC10 and EC50 for the anhydrous substance is higher than 900 mg/L and the NOEC for the anhydrous substance is 900 mg/L.