1-(2,2,4-trimethyl-1-quinolyl)ethanone

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28.08.2020 - 16.02.2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-(2,2,4-trimethyl-1,2-dihydroquinolin-1-yl)ethan-1-one

EC Number:

665-911-3

Cas Number:

5855-23-2

Molecular formula:

C14 H17 N O

IUPAC Name:

1-(2,2,4-trimethyl-1,2-dihydroquinolin-1-yl)ethan-1-one

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

Measurement of Respiration Rate:
For the measurement of the respiration rate a well-mixed sample of test medium from each flask was poured into a Karlsruher flask after exactly 3 hours incubation time and was not further aerated during measurement. The oxygen concentration was then measured with an oxygen electrode and recorded for approximately ten minutes. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption (in mg O2 L-1 minute-1) was determined over periods up to 10 minutes.

Measurement of pH, Dissolved Oxygen and Water Temperature:
The oxygen concentrations were determined at the start and at the end of the incubation period in at least one replicate of all test concentrations and controls. The pH-value was determined at the start and at the end in at least one replicate of the test concentrations and controls. The water temperature was measured in one control medium at the start and the end of the incubation period.

Test solutions

Vehicle:

yes

Details on test solutions:

Dosage of Test Item:
Appropriate amounts of test item were directly weight into the test vessels and test water was added. The composition was stirred intensively.

Test Concentrations:
Nominal 10, 32, 100, 320 and 1000 mg test item/L
Control: Six controls (pure water, synthetic sewage feed and inoculum, but without addition of the test item) were tested in parallel.

Stock Solution of 3,5-Dichlorophenol:
The reference item 3,5-dichlorophenol was tested at the nominal test concentrations of 1, 4, and 16 mg/L (five replicates for each test concentration) under otherwise identical test conditions.

A stock solution of 3,5-dichlorophenol was prepared according to the OECD Guideline No. 209: 0.5 g of 3,5-dichlorophenol was dissolved in 500 mL pure water. Warm water was used to accelerate the dissolution. The solution was filled up to volume when it had cooled to room temperature. The final pH was 7.0 and therefore in the range of 7 to 8. NaOH was used for the adjustment of the pH.
Stock Solution of N-allylthiourea (ATU): In parallel a nitrification inhibitor N-allylthiourea (ATU) was tested in the same way with six separate controls and at the identical nominal concentrations of the test item and also the reference item 3,5-dichlorophenol under otherwise identical test conditions.
A stock solution of N-allylthiourea was prepared according to the OECD Guideline No. 209: a stock solution of 2.32 g/L N-allylthiourea was prepared. 2.5 mL of this stock solution were added to an incubation mixture of final volume of 500 mL. This resulted in a final concentration of 11.6 mg ATU/L.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Species / Origin: Activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the municipal sewage treatment plant Bensheim, Germany.
Conditioning:
The activated sludge used for this study was used as collected, but coarse particles were removed by settling for a short period (15 minutes) and then the upper layer decanted. During holding prior to use the sludge was fed with 50 mL synthetic sewage feed (see below) per litre and kept aerated at room temperature overnight.

An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight determined. Based on the sludge dry matter, calculated amounts of wet sludge were suspended in pure water to yield a concentration equivalent to 3 g/L on dry weight basis. This level gives a concentration of 1.5 g/L suspended solids in the test medium. The pH of the activated sludge inoculum was 6.5 and therefore no adjustment necessary.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Test temperature:

20°C +- 2°C

pH:

6.5 - 7.9

Dissolved oxygen:

6.6 - 8.9

Nominal and measured concentrations:

10, 32, 100, 320 and 1000 mg/L

Details on test conditions:

Temperature: 20°C ± 2°C (for the limit study and for the dose response study),
Aeration: With compressed air (1.017 litre per minute)
Recording: The room temperature was constantly recorded by a software controlled temperature recording system (AMR Wincontrol).

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

55.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Effect concentrations 2

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

148.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Total respiration rate:
The respiration rates of all test item concentrations (10 mg/L, 32 mg/L, 100 mg/L, 320 mg/L and 1000 mg/L were significantly different from the control.
The test item showed an increasing inhibiting effect on the total respiration rate of the activated sludge with increasing test item concentrations. The inhibitions were 13.9%, 26.8%, 22.2%, 77.0% and 83.8% at test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively.
The 3-hour EC50 value for total respiration was determined to be 148.3 mg/L; the NOEC was determined to be lower than a test item concentration of 10 mg/L.

The 3-hour EC10, EC20 and EC50 and their 95 % confidence limits were calculated. The results are listed below:
Parameter EC10 EC20 EC50
Value [mg/L] 14.9 32.8 148.3
lower 95%-cl 5.3 12.1 45.9
upper 95%-cl 42.0 88.1 487.9
The NOEC was established to be below the lowest test item concentration of 10 mg/L.


Heterotrophic respiration rate:
The respiration rates of all test item concentrations (10 mg/L, 32 mg/L, 100 mg/L, 320 mg/L and 1000 mg/L) were significantly different from the control.
In comparison to the inoculum controls the respiration was inhibited for all test item concentrations. The inhibitions were 40.7%, 37.4%, 43.8%, 74.4% and 83.1% at test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively.

The 3-hour EC10, EC20 and EC50 and their 95 % confidence limits of the test item were calculated. The results are listed below:
Parameter EC10 EC20 EC50
Value [mg/L] 0.6 2.9 55.3
lower 95%-cl 0.2 0.8 11.1
upper 95%-cl 2.5 10.9 268.4
The NOEC was established to be below the lowest test item concentration of 10 mg/L.

Nitrification Respiration:
The respiration rates of the two highest test item concentrations (320 mg/L and 1000 mg/L) were significantly different from the control.
In comparison to the inoculum controls the nitrification respiration rate of the activated sludge was only slightly inhibited for test item concentrations up to and including 100 mg/L. The two highest test item concentrations showed a strong inhibiting effect on the respiration. The inhibitions were -16.7%, 14.6%, -2.4%, 80.0% and 84.6% at test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively.

The 3-hour EC10 and the 95 % confidence limits of the test item were calculated. The results are listed below:
Parameter EC10 EC20 EC50
Value [mg/L] 241.8 255.9 285.2
lower 95%-cl 0.0 0.0 0.0
upper 95%-cl n.d. n.d. n.d.

n.d.: not determined due to mathematical reasons.
As the confidence limits could not be determined, the value has to be treated with care. The NOEC was determined to be at a test item concentration of 100 mg/L.

Results with reference substance (positive control):

The inhibition of the activated sludge treated with the reference item was in the range of 28% to 79% for the test concentrations from 1 until 16 mg/L.

The 3-hour EC10, EC20 and EC50 and their 95 % confidence limits of 3,5-Dichlorophenol were calculated. The results are listed below:
Parameter EC10 EC20 EC50
Value [mg/L] 0.6 1.3 5.8
lower 95%-cl 0.2 0.5 1.8
upper 95%-cl 1.6 3.4 18.6

Reported statistics and error estimates:

ECx Estimation:
Regression Analysis: 3-Parametric normal CDF (cumulative distribution function), non-linear regression without weighting;
Optimization method: Levenberg-Marquardt
3-Parametric normal CDF R2:
0.873 (a significant amount of variance is explained by the regression model, total respiration)
0.888 (a significant amount of variance is explained by the regression model, heterotrophic respiration)
0.856 (a significant amount of variance is explained by the regression model, nitrification respiration)
NOEC estimation:
Test on Normal Distribution: Shapiro Wilk’s test (α= 0.01) (total, heterotrophic and nitrification respiration)
Variance Homogeneity: Levene’s Test (α= 0.01) (total. heterotrophic and nitrification respiration)
T-test Procedure for treatment comparison and NOEC estimation: Williams Multiple Sequential t-test (total and heterotrophic respiration) (α= 0.05, one-sided smaller)
Multiple Sequentially-rejective Welsh t-test (nitrification respiration) (α= 0.05, one-sided smaller)

Reference item
Test on Normal Distribution:
Shapiro Wilk’s test (α= 0.01) (total, heterotrophic and nitrification respiration)
Variance Homogeneity:
Levene’s Test (α= 0.01) (total, heterotrophic and nitrification respiration)
T-test Procedure for treatment comparison:
Williams Multiple Sequential t-test (α= 0.05, one-sided smaller) (total, heterotrophic and nitrification respiration)

ECx Estimation:
Regression Analysis: 3-Parametric normal CDF (cumulative distribution function), non-linear regression without weighting;
Optimization method: Levenberg-Marquardt
3-Parametric normal CDF R2:
0.828 (total respiration)
0.973 (heterotrophic respiration)
0.834 (nitrification respiration)
The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ® ToxRat Solutions GmbH.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test item had inhibiting effects for test item concentrations up to and including 1000 mg/L for total, heterotrophic and nitrification respiration.
The EC50 values were determined to be 148.3 mg/L, 55.3 mg/L and 285.2 mg/L.
The NOEC was established to be below the lowest test item concentration of 10 mg/L for total and heterotrophic respiration. The NOEC was determined to be at a test item concentration of 100 mg/L for nitrification respiration.

Executive summary:

Title:	[trade name]: Toxicity to Activated Sludge in a Respiration Inhibition Test
Guidelines/Recommendations:	-      Commission Regulation (EC) No 440/2008, Method C.11: "Activated Sludge Respiration Inhibition Test", Official Journal of the European Union No. L 142/559-563, dated May 30, 2008 -      OECD Guideline for Testing of Chemicals, No. 209: "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)", adopted July 22, 2010 -      Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation (ISO 8192:2007); German version EN ISO 8192:2007
Purpose:	The influence of the test item on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage feed was measured in the presence of various concentrations of the test item after an incubation period of 3 hours.
Test Concentrations:	10, 32, 100, 320 and 1000 mg test item/L; 1, 4 and 16 mg 3,5-Dichlorophenol/L and Six inoculum controls
Results:
Toxicity of Test Item:	The test item was directly dosed into each test flask and pure water was added. The test item was dissolved into the pure water as homogeneously as possible.   Total respiration: The test item showed an increasing inhibiting effect on the total respiration rate of the activated sludge with increasing test item concentrations. The inhibitions were 13.9%, 26.8%, 22.2%, 77.0% and 83.8% at test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively. Concentrations exceeding 1000 mg/L nominal were not tested. The 3-hour EC50 value for total respiration was determined to be 148.3 mg/L; the NOEC was determined to be lower than a test item concentration of 10 mg/L.  Heterotrophic Respiration: In comparison to the inoculum controls the respiration was inhibited for all test item concentrations. The inhibitions were 40.7%, 37.4%, 43.8%, 74.4% and 83.1% at test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively. The 3-hour EC50 value for total respiration was determined to be 55.3 mg/L; the NOEC was determined to be lower than a test item concentration of 10 mg/L.    Respiration based on nitrification: In comparison to the inoculum controls the nitrification respiration rate of the activated sludge was only slightly inhibited for test item concentrations up to and including 100 mg/L. The two highest test item concentrations showed a strong inhibiting effect on the respiration. The inhibitions were -16.7%, 14.6%, -2.4%, 80.0% and 84.6% at test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively. The 3-hour EC50 values for oxygen uptake due to nitrification was determined to be 285.2 mg/L; As the confidence limits could not be determined, the value has to be treated with care. The NOEC was determined to be lower than a test item concentration of 100 mg/L.
Toxicity of 3,5-Dichlorophenol:	The positive control 3,5-Dichlorophenol was tested in the same way as the test item. The 3-hour EC50 for total respiration was found to be 5.8 mg/L thus lying in the range of 2 - 25 mg/L recommended by the test guidelines. The 3-hour EC50 for heterotrophic respiration was found to be 13.2 mg/L thus lying in the range of 5 - 40 mg/L recommended by the test guidelines For the oxygen uptake due to nitrification the 3-hour EC50 was 1.0 mg/L, lying in the range of 0.1 - 10 mg/L recommended by the test guidelines. The result confirms the suitability of the activated sludge used.