Reaction product of propylidynetrimethanol, propoxylated, reaction products with ammonia and 2,2-Dimethyl-3-(4-morpholinyl)propanal

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:

2014-05-12-2014-06-23

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

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

- Sampling method: The test item was directly added.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Just before the start of the test defined amounts of (5 x 300; 5 x 150; 5 x 75; 5 x 38 and 5 x 19 mg) test item were weighted into a small inert plastic dish (plate) that were put into each test container. Thereafter a pH measurement was carried out and the test item solutions were inoculated.
- 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.
- Reference control: 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: 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):

activated sludge, domestic

Details on inoculum:

- Laboratory culture: Activated Sludge, microorganisms from a domestic waste water treatment plant. The sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary on 28. May 2014.
- 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 resuspended 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). 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 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 before use. The pH of the activated sludge inoculum was checked just before use: 7.87. pH adjustment of the inoculum was considered as not necessary.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

not measured

Test temperature:

20 ± 2 °C

pH:

7 - 8

Dissolved oxygen:

2 - 7 mg O2/L

Salinity:

not applicable

Nominal and measured concentrations:

1000, 500, 250, 125 and 62.5 mg/L (Nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer bottles of approximately 300 mL volume
- Material, size, headspace, fill volume : Normal laboratory equipments were used in the study, additionally: a complex measuring system with O2 electrode and pH electrode and built in temperature probe; Aeration system, Orbital shaker, Min/Max. Thermometer, Balance, Centrifuge and Moisture analyzer.
- Aeration: With compressed air (0.5 litre per minute)

OTHER TEST CONDITIONS
- Adjustment of pH: no adjustement necessary
- Surrounding Type: Controlled environment room

EFFECT PARAMETERS MEASURED:
- Measurement of Respiration Rate: The measurement of the respiration rate in a well-mixed aerated sample of each treatment was performed after exactly 3 hours incubation time. The treatment sample was not further aerated. The oxygen concentration was measured with a O2 electrode (working based on LDO method) under stirred conditions and was recorded for about 6 - 10 minutes. The measurement was carried out in completely filled Winkler bottles. For practical reason more O2 electrodes were used. Simultaneous (four vessels were investigated in parallel) measurements were performed; the test vessels were investigated in 12 cycles with the available four O2 electrodes. The oxygen consumption rate (in mg O2 L-1 hour-1) was determined from the linear part of the respiration curve, in the range between approximately 2 - 7.
- Measurement of pH, dissolved Oxygen and water Temperature: The pH and the oxygen concentrations were determined at the start and at the end of the incubation period in all test concentrations, reference item concentrations and controls. The temperature was measured in the controlled environment room with a min/max thermometer during the incubation period. The water temperature was recorded during the oxygen measurement in all test bottles.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

Reference substance (positive control):

yes

Remarks:

3.5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

125.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 1 000 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

62.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Results with reference substance (positive control):

The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 15.51 mg/L.

Reported statistics and error estimates:

The degree of inhibition (31.93 %) at the highest examined concentration level of 1000 mg/L did not allow the calculation of the 3-hour exact EC50 and EC80 values. Based on the available data the 3-hour EC10 and the theoretical EC50 and EC80 values were calculated by Probit analysis using TOXSTAT software.

Validity criteria fulfilled:

yes

Conclusions:

The EC10 value was 125.8 mg/L after 3 hours. The EC50 and EC80 values were determined as > 1000 mg/L. Based on the statistical evaluation in this test the NOEC was 62.5 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 nominal concentrations of 62.5; 125; 250; 500 and 1000 mg/L. Defined amounts of the test item were added directly into the test vessels. 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 test was performed without pH adjustment. All validity criteria of the study were met. The observed-calculated specific respiration rates remained within the range of the specific respiration rates of the blank controls at the concentration of 62.5 mg/L, where not significant 3 % inhibition was obtained. The inhibition of the specific respiration rates compared to the respiration rates of the blank controls showed clear dose-related tendency from ~12 % at 125 mg/L to ~ 32 % obtained at 1000 mg/L. The degree of inhibition (31.93 %) at the highest examined concentration level of 1000 mg/L did not allow the calculation of the 3 -hour exact EC50 and EC80 values. Based on the available data the 3-hour EC10 and the theoretical EC50 and EC80 values were calculated by Probit analysis using TOXSTAT software. The values are the following: EC10: 125.8 mg/L(79.6–198.7) EC50: 2522.2 mg/L(1188.0–5355.0) EC80: 18066.8 mg/L(4497.3–72578.1) The EC50 and EC80 values were determined as > 1000 mg/L. The specific respiration rates at the concentration of 62.5 mg/L did not differ statistically significantly from the blank control (Bonferroni t-Test (alpha=0.05)) consequently the NOEC was statistically and biologically determined as 62.5 mg/L.

Description of key information

The EC10 value was 125.8 mg/L after 3 hours. The EC50 and EC80 values were determined as > 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

125.8 mg/L

Additional information

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 nominal concentrations of 62.5; 125; 250; 500 and 1000 mg/L. Defined amounts of the test item were added directly into the test vessels. 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 test was performed without pH adjustment. All validity criteria of the study were met. The observed-calculated specific respiration rates remained within the range of the specific respiration rates of the blank controls at the concentration of 62.5 mg/L, where not significant 3 % inhibition was obtained. The inhibition of the specific respiration rates compared to the respiration rates of the blank controls showed clear dose-related tendency from ~12 % at 125 mg/L to ~ 32 % obtained at 1000 mg/L. The degree of inhibition (31.93 %) at the highest examined concentration level of 1000 mg/L did not allow the calculation of the 3 -hour exact EC50 and EC80 values. Based on the available data the 3-hour EC10 and the theoretical EC50 and EC80 values were calculated by Probit analysis using TOXSTAT software. The values are the following: EC10: 125.8 mg/L (79.6 – 198.7) EC50: 2522.2 mg/L (1188.0 – 5355.0) EC80: 18066.8 mg/L (4497.3 – 72578.1) The EC50 and EC80 values were determined as > 1000 mg/L. The specific respiration rates at the concentration of 62.5 mg/L did not differ statistically significantly from the blank control (Bonferroni t-Test (alpha = 0.05)) consequently the NOEC was statistically and biologically determined as 62.5 mg/L.