Reaction mixture of hydrogenated tallow alkyl amines with sebacic acid and lithium hydroxide

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:

from 2018-01-17 to 2018-03-01

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))

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

No concentrations were measured

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: In the main test the test item concentration of 1000 mg/L was examined with five replicates. At the start of the test defined amounts of 5 x 300 mg test item were administered directly into empty containers. The containers were filled up with water and synthetic sewage (to a final volume of 300 mL), just before the inoculation. Thereafter a pH measurement was carried out and the test item solutions were inoculated. Concentrations in excess of nominal 1000 mg test item/L were not tested.

One test solution with a final volume of 300 mL was tested per treatment in a glass flask. 9.6 mL synthetic sewage and an adequate volume of the stock solution of the N-allylthiourea adequate volumes of the stock solution of reference item were filled up with water (with deionised water) to 150 mL before the start of the test. At the test item treatments the 300 mg test item was measured directly into the empty test containers. The 150 mL synthetic sewage-water mixture was filled into these test containers just before the inoculation.
At the start of the test 150 mL activated sludge inoculum with a sludge concentration of 3 g/L (on dry weight basis) was added first to the blank controls, then in appropriate time intervals to the nitrification controls, the test solutions of the reference item and the test item.

- Differential loading: No

- Controls:
Blank control: 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.
Nitrification control: N-allylthiourea (ATU); N-allylthiourea solution was prepared by dissolving 0.232 g of N-allylthiourea in 100 mL of water.
Reference Control: 3,5-dichlorophenol (3,5-DCP); In the main experiment 0.1 g 3,5-dichlorophenol was dissolved in 100 mL deionised water in a volumetric flask. The pH of the stock solution was adjusted with 1N NaOH to 7.50.
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).

- Evidence of undissolved material: 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 16 January 2018 (one day before the main 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 (6.244 g wet weight), dried and the ratio of wet sludge to dry weight (0.5126 g dry weight) determined. Based on this ratio, calculated amount of wet sludge (27 g dry weight that was equivalent to 329 g wet sludge) was suspended in isotonic saline solution (ad. 9 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 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 (pH: 7.41), additional pH adjustment of the inoculum was considered not necessary.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

None

Hardness:

Not specified

Test temperature:

The mixtures were aerated at 0.5 L/min in the temperature range of 20 ± 2 °C. The temperature in the test mixtures during the measurements in average: 21.4 °C, the measured minimum: 20.4 °C, maximum: 22.0 °C.

pH:

This test was performed without pH adjustment. The measured pH values in the prepared solutions: 3,5-dichlorophenol reference control stock solution, synthetic sewage, activated sludge inoculum prepared test mixtures etc. were in the acceptable pH 7-8 range at the start of the test.

Dissolved oxygen:

Not applicable

Salinity:

Not applicable

Conductivity:

Not applicable

Nominal and measured concentrations:

Nominal limit concentration: 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer bottles, closed
- Material, size, headspace, fill volume: glass, 300 mL volume
- Aeration: With compressed air (0.5 litre per minute)
- No. of vessels per concentration (replicates): 5
- No. of vessels per blank control: 8
- No. of vessels per nitrification control: 3 (with 11.6 mg/L N-allylthiourea)
- No. of vessels per abiotic control (replicates): none
- Sludge concentration (weight of dry solids per volume): 3 g/L dw
- Nitrification inhibitor used: / N-allylthiourea

TEST MEDIUM / WATER PARAMETERS
- in accordance to guideline

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED
Measurement of Respiration Rate after 3 hours, pH, dissolved oxygen and water temperature

TEST CONCENTRATIONS
- Range finding study
In the preliminary range-finding test three test item concentrations were tested (with one test vessel at the lower and three replicate vessels at the highest test item concentration). Defined amounts of the test item (according to the concentrations of 10, 100 and 1000 mg/L: 3, 30 and 300 mg in a final volume of 300 mL, respectively) were weighted directly into empty test containers, thereafter 140.4 mL water and 9.6 mL synthetic sewage mixture was filled into these test containers just before the inoculation.
In the preliminary experiment the test item did not show a significant inhibitory effect, the obtained 0.64 % inhibition at the concentration of 1000 mg/L remained within the biological variability range of the applied test system.
Based on the preliminary information about the test item caused effect on the activated sludge inoculum, in the main test the test item was investigated at the 1000 mg/L concentration as limit concentration, without pH adjustment.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

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:

NOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

- 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
- Adsorption (e.g. of test material to the walls of the test container): No

Results with reference substance (positive control):

- Results with reference substance valid: Yes
- Relevant effect levels: In comparison to the blank controls the oxygen consumption rate of the activated sludge was inhibited by 12.3 % at the lowest concentration of 2 mg/L, at the nominal concentrations of 7 and 24.5 mg/L, the respiration rate was inhibited by 26.3 % and 79.3 %, respectively. The 3-hour EC50 of 3,5-dichlorophenol was calculated to be 15.5 mg/L, (95 % confidence limits: 7.0 - 44.8 mg/L).

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.

Executive summary:

The purpose of the 3-hour test was to evaluate the influence of the test item 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.

Based on the preliminary information about the test item caused effect on the activated sludge inoculum, the test item was investigated at the concentration of 1000 mg/L as a limit concentration, only. 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 and nitrification controls were investigated. 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.

The test was performed without pH adjustment.

All validity criteria of the study were met.

The test item was investigated at one (limit) concentration level of 1000 mg/L. The observed oxygen consumption rates and consequently the specific respiration rates were in the range of the blank controls, no inhibitory effect of the test item was observed.

Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10, EC50 and EC80 values of the test item are higher than 1000 mg/L.

The EC50 value was determined as: EC50 > 1000 mg/L.

The specific respiration rates were compared with the blank control values using 2 Sample t-Test (α=0.05). No statistical significant differences were observed in the comparison with the blank control values, consequently based on the results of this study the NOEC can be statistically and biologically determined as 1000 mg/L.

Description of key information

Under the conditions of a performed Activated Sludge Respiration Inhibition Test according to OECD TG 209 , the EC10 and EC50 values of the substance was determined as higher than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was ≥ 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

The substance was assessed in an Activated Sludge Respiration Inhibition test, according to the EU-method C. 11 and OECD guideline 209. Based on the preliminary information about the test item caused effect on the activated sludge inoculum, in the main test the substance was investigated at 1000 mg/L test item concentration as limit concentration, without pH adjustment. Based on the results of the preliminary experiment neither inclusion of abiotic controls nor differentiation between heterotrophic respiration and nitrification was considered as necessary. The observed-calculated oxygen consumption rates were in the range of the blank controls, inhibitory effect (the observed in average -0.24 % inhibition (stimulation) was within the biological variability of the applied test system) of the test item at the examined limit concentration of 1000 mg/L was not observed. Based on measured oxygen consumption values it can be stated that the 3-hour EC10, EC50 and EC80 values of the test item are higher than 1000 mg/L. The NOEC can be statistically determined as 1000 mg/L.