Reaction mass of lauric acid, compound with morpholine (1:1) and 2-ethylhexyl dihydrogen phosphate, compound with morpholine (1:2) and bis(2-ethylhexyl) hydrogen phosphate, compound with morpholine (1:1)

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 2016-07-29 to 2016-11-09

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

No concentrations of the test substance 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 (measured into empty containers) directly. The containers were filled up with water and synthetic sewage, just before the inoculation. Because of the test item direct addition an analytical estimation of the test substance concentration in the test vessels was considered as not necessary, and the subsequent calculations referred to the nominal test item concentration.
- 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.
- Abiotic Control (CA): The investigation of abiotic controls in the main experiment was considered as not necessary based on the preliminary information about the test item and based on the results of the preliminary test, where abiotic controls (with three parallels) were tested at the highest test item concentration (1000 mg/L) and no remarkable abiotic oxygen consumption was noticed.
- 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.

Test organisms (species):

sewage, 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 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, dried and the ratio of wet sludge to dry weight determined. At the concentration calculation the dilution resulted by the fed synthetic sewage was taken into consideration. The activated sludge was not used on the day of the collection but it was 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
- Pretreatment: No pretreatment was performed.
- Initial biomass concentration: 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). In the test containers the final concentration of suspended solids was 1.5 g per litre.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

180 min

Remarks on exposure duration:

None

Post exposure observation period:

Not performed

Hardness:

No data

Test temperature:

20 ± 2 °C. The noticed temperatures in the environmental room varied between: 20.4 - 21.4 °C.

pH:

The pH of the activated sludge inoculum was checked after preparation (pH: 7.25) and before use (pH: 7.69). pH adjustment of the inoculum was considered not necessary.

Dissolved oxygen:

6.38 - 8.09 mg/L

Salinity:

Not applicable

Conductivity:

Not applicable

Nominal and measured concentrations:

- Nominal concentration: 1000 mg/L (Limit test)
- Measured concentration: No concentrations of the test substance were measured.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer bottles
- Fill volume: 300 mL
- Aeration: Yes, with compressed air (0.5 litre per minute)
- No. of vessels per concentration (replicates): 5
- No. of vessels per blank control (replicates): 8
- No. of vessels per toxicity control (replicates): 5
- No. of vessels per nitrification control (replicates): 3
- No. of vessels per reference substance concentration (replicates): 3
- Sludge concentration (weight of dry solids per volume): 1.5 g per litre
- Nitrification inhibitor used: N-allylthiourea

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water
- Adjustment of pH: Yes. The pH of the stock solution was adjusted with 1N NaOH to 7.63.

EFFECT PARAMETERS MEASURED: The purpose of the 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.

TEST CONCENTRATIONS
- Preliminary test: Yes
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: In the preliminary experiment the test item did not show a significant inhibitory effect (~ 2 % inhibition) at the nominal concentration of 1000 mg/L.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

180 min

Dose descriptor:

EC10

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: no adverse effects were observed.

Details on results:

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.

Results with reference substance (positive control):

The 3-hour EC50 of the reference item 3,5-dichlorophenol (for the used activated sludge batch) was 15.54 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:

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 determined as ≥ 1000 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of the test item to the microorganisms of activated sludge after 3 hours was determined according to OECD 209 (2010) under GLP conditions. 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 toxicity of the test item to the microorganisms of activated sludge after 3 hours was determined according to OECD 209 (2010) under GLP conditions. 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. In parallel with the test item treatments 3,5-dichlorophenol as positive reference control in a 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 observed oxygen consumption rates consequently the specific respiration rates were in the range of the blank controls and no inhibitory effect of the test item was observed. The test was performed without pH adjustment. The 3-hour EC50 of the reference item 3,5-dichlorophenol was determined to be 15.54 mg/L within the range of 2 mg/L to 25 mg/L.

All validity criteria of the study were met. Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10 and the EC50 were higher than 1000 mg/L.

Description of key information

The toxicity of the test item to the microorganisms of activated sludge after 3 hours was determined according to OECD 209 (2010) under GLP conditions. 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.

Additional information

The toxicity of the test item to the microorganisms of activated sludge after 3 hours was determined according to OECD 209 (2010) under GLP conditions. 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. In parallel with the test item treatments 3,5-dichlorophenol as positive reference control in a 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 observed oxygen consumption rates consequently the specific respiration rates were in the range of the blank controls and no inhibitory effect of the test item was observed. The test was performed without pH adjustment. The 3-hour EC50 of the reference item 3,5-dichlorophenol was determined to be 15.54 mg/L within the range of 2 mg/L to 25 mg/L.

All validity criteria of the study were met. Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10 and the EC50 were higher than 1000 mg/L.