2-hydroxy-3-(prop-2-enoyloxy)propyl 2-methyl-2-propylhexanoate

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 July 12, 2016 to July 19, 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

but uncritical or assessed

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

but uncritical or assessed

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch no.: JBGJ0045R
Purity: 100% (UVCB)
Appearance: clear yellowish liquid

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant in D-67435 NW-Lachen-Speyerdorf. Upon arrival in the test facility, the sludge was filtrated, washed with tap water 3 times and re-suspended in tap water. The activated sludge was aerated until usage in the test and fed daily with 50 mL synthetic sewage feed /L.
First experiment. The dry matter was determined as 2.9g suspended solids/L, giving a concentration of 1.45g suspended solids/L in the test.
Second experiment. The dry matter of the inoculum was determined as 2.96 g suspended solids/L, giving a concentration of 1.48g suspended solids/L in the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

-

Test temperature:

20.7 - 22.2°C

pH:

7.5 ± 0.5

Dissolved oxygen:

-

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

1000, 320, 100, 32, 10 and 1 mg/L nominal concentration

Details on test conditions:

cfr "Any other information on materials and methods incl. tables"

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

54 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.:

1 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: extrapolated

Details on results:

- For evaluation of the results the nominal concentrations were used because the difference between real concentration and nominal concentration can be stated as not significant.
- Two valid experiments were performed. In the first experiment (pre-test) the test substance was tested using 4 concentrations: 1000, 100, 10 and 1 mg/L nominal concentration. Because significant inhibition was observed, one additional experiment was performed under the same test conditions. In the second experiment (main test) the test substance was tested using 5 concentrations: 1000, 320, 100, 32 and 10 mg/L nominal concentration. Significant inhibition was observed at all concentration with the exception of the lowest concentrated treatment. EC50 lies outside the tested range of concentrations and was extrapolated using linear regression. In some replicates oxygen concentration in the test solution at the beginning of oxygen measurement was unexpected low. Therefore the measurement period was partly less than 5 minutes. Because linearity of all regression curves was given and the correlation of the inhibition values within the test replicates was very good, this can be stated as uncritical. Inhibition at concentration 1000 mg/L in the pre-test was slightly lower than inhibition at the same concentration in the main test. The apparent difference between the experiments was related to the used sludge. Taking into account the results of the positive control (higher EC50 in the first experiment), the sludge which was used in the first experiment may have been slightly less sensitive than the sludge which was used in the second experiment. Nevertheless, the differences in the inhibition values and oxygen uptake rates in the control replicates were within the normal range of a biological system. All validity criteria were met. For the estimation of the EC50 of the positive control, the fits showed good statistical correspondence of the data with the dose-response-equation. The positive control gave an EC50 of 9.3 mg/L (first experiment) and 8.5 mg/L (second experiment) which lie within the recommended range of 2 – 25 mg/L. The coefficient of variation of oxygen uptake rate in control replicates was below 30% at the end of the test. The oxygen uptake rate of the blank controls was above 20 mg O2 per gram activated sludge in 1 hour. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.
Based on the results, the values for the test substance were determined: :
3 h NOEC: 10 mg/L
3 h EC10: 54 mg/L
3 h EC50: 1500 mg/L (extrapolated)

Results with reference substance (positive control):

The positive control gave an EC50 of 9.3 mg/L (first experiment) and 8.5 mg/L (second experiment) which lie within the recommended range of 2 – 25 mg/L.

Reported statistics and error estimates:

- Statistical determination of the NOEC and LOEC:
For the treatments with the test substance concentrations 32 and 10 mg/L, it was tested whether the differences between treatment and control were significant. For this determination, the values of the O2 consumption were used. In order to select a suitable test for significance, it was checked whether equality of variance was given. The difference between treatment 10 mg/L and the control can be considered as not significant as the calculated t-value lay below the significance level and O2 consumption in the treatment was higher than in the control. Therefore, the concentration 10 mg/L was stated as NOEC. As the difference between treatment 32 mg/L and the control can be considered as significant because the calculated t-value lay above the significance level, the concentration 32 mg/L was stated as LOEC.
- EC50 lies outside the tested range of concentrations and was extrapolated using linear regression. As good correlation of the tested concentrations was observed (r2 = 0.874), and EC50 lies above 1000 mg/L, this was considered as uncritical. For the calculation of the EC10 and EC50, the percentage inhibition was plotted versus concentration in a Gauß-logarithmic diagram. EC10 and EC50 were determined from the x-values of the regression line at y = 10% and y = 50%.

Deviations from the Study Plan

The following deviations from the study plan were recorded:

- The temperature in the first experiment was in a range of 21.2 – 22.2 °C instead of 20.0 ± 2.0 °C. This deviation was stated as uncritical, as normal respiration activity of the control

could be observed.

- The measurement period was partly less than 5 minutes. Because linearity of all regression curves was given and the correlation of the inhibition values within the test replicates was good, this was stated as uncritical.

- The test item was stored from the date of receipt to last usage for the study at 20 ± 5 °C except of 8 h at 25.8 °C. This is uncritical as the test item was stored only for 8 h above 25 °C. It is assumed that this marginal deviation has no impact on the test item.

Deviations from the Guideline

The following deviations from the guideline were recorded:

- The temperature in the first experiment was in a range of 21.2 – 22.2 °C instead of 20.0 ± 2.0 °C. This deviation was stated as uncritical, as normal respiration activity of the control

could be observed.

- The measurement period was partly less than 5 minutes. Because linearity of all regression curves was given and the correlation of the inhibition values within the test replicates was good, this was stated as uncritical.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 3 h NOEC, EC10 and EC50 values were determined to be at 10, 54 and 1500 mg/L (extrapolated using linear regression) (respiration inhibition test).

Executive summary:

A study was conducted to determine the toxicity of the test substance to microorganisms according to OECD Guideline 209 and EU Method C.11 (respiration inhibition test), in compliance with GLP. In a preliminary test, non-adapted activated sludge was exposed to the test substance at 4 nominal concentrations: 0, 1, 10, 100 and 1000 mg/L for 3 h. Because of significant inhibition at all the tested concentrations, the main test was conducted with the test substance at 5 nominal concentrations: 0, 10, 32, 100, 320 and 1000 mg/L under similar test conditions. In addition, two controls and three different concentrations of the reference substance 3,5-dichlorophenol (5, 16 and 50 mg/L) were tested. The respiration rate (oxygen consumption) of the aerobic activated sludges fed with a standard amount of synthetic wastewater was measured. The inhibitory effect of the test substance at the particular concentrations was expressed as a percentage of the mean respiration rate of the controls. Significant inhibition was observed at all concentrations with the exception of the lowest tested concentration. Exposure to the highest tested concentration lead to about 42% inhibition. Therefore, the EC50 was considered to lie outside the tested range of concentrations and was extrapolated using linear regression. In some replicates oxygen concentration in the test solution at the beginning of oxygen measurement was unexpected low. Therefore the measurement period was partly less than 5 minutes. Because linearity of all regression curves was given and the correlation of the inhibition values within the test replicates was very good, this can be stated as uncritical. Inhibition at concentration 1000 mg/L in the pre-test was slightly lower than inhibition at the same concentration in the main test. The apparent difference between the experiments was related to the used sludge. Taking into account the results of the positive control (higher EC50 in the first experiment), the sludge which was used in the first experiment may have been slightly less sensitive than the sludge which was used in the second experiment. Nevertheless, the differences in the inhibition values and oxygen uptake rates in the control replicates were within the normal range of a biological system. All validity criteria were met. For the estimation of the EC50 of the positive control, the fits showed good statistical correspondence of the data with the dose-response-equation. The positive control gave an EC50 of 9.3 mg/L (first experiment) and 8.5 mg/L (second experiment) which lie within the recommended range of 2 – 25 mg/L. The coefficient of variation of oxygen uptake rate in control replicates was below 30% at the end of the test. The oxygen uptake rate of the blank controls was above 20 mg O2 per gram activated sludge in 1 h. No observations were made which might cause doubts concerning the validity of the study outcome. Therefore, results of the test can be considered valid. The results of the nominal concentrations were used because the difference between real and and nominal concentration were considered not significant. Under the study conditions, the 3 h NOEC, EC10 and EC50 values were determined to be at 10, 54 and 1500 mg/L (extrapolated using linear regression) (Muckle, 2016).

Description of key information

The 3 h EC50 and EC10 values were determined to be 1500 and 54 mg/L (nominal) respectively.

Key value for chemical safety assessment

EC50 for microorganisms:

1 500 mg/L

EC10 or NOEC for microorganisms:

54 mg/L

Additional information

A study was conducted to determine the toxicity of the test substance to microorganisms according to OECD Guideline 209 and EU Method C.11 (respiration inhibition test), in compliance with GLP. In a preliminary test, non-adapted activated sludge was exposed to the test substance at 4 nominal concentrations: 0, 1, 10, 100 and 1000 mg/L for 3 h. Because of significant inhibition at all the tested concentrations, the main test was conducted with the test substance at 5 nominal concentrations: 0, 10, 32, 100, 320 and 1000 mg/L under similar test conditions. In addition, two controls and three different concentrations of the reference substance 3,5-dichlorophenol (5, 16 and 50 mg/L) were tested. The respiration rate (oxygen consumption) of the aerobic activated sludges fed with a standard amount of synthetic wastewater was measured. The inhibitory effect of the test substance at the particular concentrations was expressed as a percentage of the mean respiration rate of the controls. Significant inhibition was observed at all concentrations with the exception of the lowest tested concentration. Exposure to the highest tested concentration lead to about 42% inhibition. Therefore, the EC50 was considered to lie outside the tested range of concentrations and was extrapolated using linear regression. In some replicates oxygen concentration in the test solution at the beginning of oxygen measurement was unexpected low. Therefore the measurement period was partly less than 5 minutes. Because linearity of all regression curves was given and the correlation of the inhibition values within the test replicates was very good, this can be stated as uncritical. Inhibition at concentration 1000 mg/L in the pre-test was slightly lower than inhibition at the same concentration in the main test. The apparent difference between the experiments was related to the used sludge. Taking into account the results of the positive control (higher EC50 in the first experiment), the sludge which was used in the first experiment may have been slightly less sensitive than the sludge which was used in the second experiment. Nevertheless, the differences in the inhibition values and oxygen uptake rates in the control replicates were within the normal range of a biological system. All validity criteria were met. For the estimation of the EC50 of the positive control, the fits showed good statistical correspondence of the data with the dose-response-equation. The positive control gave an EC50 of 9.3 mg/L (first experiment) and 8.5 mg/L (second experiment) which lie within the recommended range of 2 – 25 mg/L. The coefficient of variation of oxygen uptake rate in control replicates was below 30% at the end of the test. The oxygen uptake rate of the blank controls was above 20 mg O2 per gram activated sludge in 1 h. No observations were made which might cause doubts concerning the validity of the study outcome. Therefore, results of the test can be considered valid. The results of the nominal concentrations were used because the difference between real and and nominal concentration were considered not significant. Under the study conditions, the 3 h NOEC, EC10 and EC50 values were determined to be at 10, 54 and 1500 mg/L (extrapolated using linear regression) (Muckle, 2016).