1-(2,2,4-trimethyl-3,4-dihydroquinolin-l (2H)-yl)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 - 18.02.2021

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-3,4-dihydroquinolin-l (2H)-yl)ethanone

EC Number:

855-228-0

Cas Number:

4497-59-0

Molecular formula:

C14 H19 N O

IUPAC Name:

1-(2,2,4-trimethyl-3,4-dihydroquinolin-l (2H)-yl)ethanone

Test material form:

solid

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:

6.6 - 7.6

Dissolved oxygen:

0.3 - 8.6 mg/L

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.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of respiration due to nitrification

Effect concentrations 2

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 heterotrophic respiration

Effect concentrations 3

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

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. In comparison to the inoculum controls the total respiration rate of the activated sludge was only slightly but similarly inhibited for all test item concentrations. The inhibitions were 10.7%, 8.7%, 7.9%, 11.8% and 16.4% at test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively. In addition, standard deviations of each treatment showed an overlap of the inhibition caused by the other treatments.

3-hour EC10, EC20 and 3-hour EC50 and NOEC based on Total Respiration: Since only minor respiration inhibitions with no concentration-response relationship could be observed, it was not possible to statistically derive ECx values. The NOEC was determined to be lower than a test item concentration of 10 mg/L, as all test item concentrations were statistically significant different from the control.

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 slightly, but similarly, inhibited for all test item concentrations. The inhibitions were 18.8%, 18.8%, 11.0%, 15.2% and 25.4% at test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively. In addition, standard deviations of each treatment showed an overlap of the inhibition caused by the other treatments.

3-hour EC10, EC20 and 3-hour EC50 and NOEC based on Heterotrophic Respiration: Since no concentration-response relationship could be established it was not possible to statistically derive ECx values. The NOEC was determined to be lower than a test item concentration of 10 mg/L, as all test item concentrations were statistically significant different from the control No meaningful 3-hour ECx of the test item could be calculated.
The NOEC was established to be below the lowest test item concentration of
10 mg/L.

Respiration Rate based on Nitrification:
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 not significantly different from the control.
In comparison to the inoculum controls the nitrification respiration rate of the activated sludge was only slightly but similarly inhibited for all test item concentrations. The inhibitions were 1.8%, -2.3%, 4.4%, 8.1% and 6.7% at test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively. In addition, standard deviations of each treatment showed a large overlap of the inhibition caused by the other treatments.

3-hour EC10, EC20 and 3-hour EC50 and NOEC based on Nitrification Respiration: The 3-hour EC10 and the 95 % confidence limits of the test item were calculated. The results are listed below:
Parameter EC10
Value [mg/l] 1571.6
lower 95%-cl n.d.
upper 95%-cl n.d.

Since no concentration-response relationship could be established it was not possible to statistically derive meaningful ECx values. In this case the 3-hour EC10 value could statistically be established to be > 1000 mg/L. However, the confidence limits could not be established, which limits the reliability in this purely statistical finding.
The NOEC was determined to be higher than a test item concentration of
1000 mg/L, as all test item concentrations were statistically not significant different from the control.

Results with reference substance (positive control):

Total Respiration Rate:
The inhibition of the activated sludge treated with the reference item was in the range of 21% to 72% for the test concentrations from 1 until 16 mg/L.

3-hour EC10, EC20 and EC50 of 3,5-Dichlorophenol based on Total Respiration: 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] 0.4 1.0 5.1
lower 95%-cl 0.3 0.8 3.8
upper 95%-cl0.5 1.2 6.8

Heterotrophic Respiration Rate:
The inhibition of the activated sludge treated with the reference item was in the range of 0% to 48% for the test concentrations from 1 until 16 mg/L.

3-hour EC10, EC20 and EC50 of 3,5-Dichlorophenol based on Heterotrophic Respiration: 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] 5.6 8.1 16.7
lower 95%-cl 4.0 5.9 11.0
upper 95%-cl 7.7 11.1 24.7

Respiration Rate based on Nitrification:
The inhibition of the activated sludge treated with the reference item was in the range of 46% to 99% for the test concentrations from 1 until 16 mg/L.

3-hour EC10, EC20 and EC50 of 3,5-Dichlorophenol based on Nitrification Respiration:
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] 0.3 0.5 1.1
lower 95%-cl 0.2 0.3 0.7
upper 95%-cl 0.4 0.7 1.7

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:
0.464 (a significant amount of variance is explained by the regression model, total respiration)
0.627 (a significant amount of variance is explained by the regression model, heterotrophic respiration)
0.075 (the amount of variance explained by the model is not significant, 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)
Dunnett’s Multiple Sequential 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:
0.990(total respiration)
0.964 (heterotrophic respiration)
0.987 (nitrification respiration)


The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ® ToxR
at Solutions GmbH.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In total, the respiration rate of the test item on total and heterotrophic as well as nitrification respiration was assessed. All approaches showed a slight respiration inhibition, but no concentration-response-relationships could be established, and no confidence intervals could be determined. It was not possible to derive ECx values (of sufficient statistical reliability).
NOEC values could be calculated by statistical tests, resulting from the fact that the replicate variation was very low for control and test item concentrations. Taking into account the facts that most reported inhibitions in this study were comparably low, in the same range (between 9% and 16% inhibition for total respiration, between 11% and 25% inhibition for heterotrophic respiration) and showed no dose-response relationship, the suggestion is that the slight reduction in respiration rate is a finding by chance rather than an effect caused by the test item.
It is therefore concluded, that the test item [trade name] had no inhibiting effect on the respiration rate. The NOEC should therefore be considered ≥ 1000 mg/L and the EC10 > 1000 mg/L.

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 [trade name] 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 [trade name]/L; 1, 4 and 16 mg 3,5-Dichlorophenol/L and Six inoculum controls
Results:
Toxicity of Test Item:	The test item [trade name] 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: In comparison to the inoculum controls the total respiration rate of the activated sludge was only slightly but similarly inhibited for all test item concentrations. The inhibitions were 10.7%, 8.7%, 7.9%, 11.8% and 16.4% at test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively. In addition, standard deviations of each treatment showed an overlap of the inhibition caused by the other treatments. Since only minor respiration inhibitions with no concentration-response relationship could be observed, it was not possible to statistically derive ECx values. The NOEC was determined to be lower than a test item concentration of 10 mg/L, as all test item concentrations were statistically significant different from the control.   Heterotrophic Respiration: In comparison to the inoculum controls the respiration was slightly but similarly inhibited for all test item concentrations. The inhibitions were 18.8%, 18.8%, 11.0%, 15.2% and 25.4% at test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively. In addition, standard deviations of each treatment showed an overlap of the inhibition caused by the other treatments. Since no concentration-response relationship could be established, it was not possible to statistically derive ECx values. The NOEC was determined to be lower than a test item concentration of 10 mg/L, as all test item concentrations were statistically significant different from the control.   Respiration based on nitrification: In comparison to the inoculum controls the nitrification respiration rate of the activated sludge was only slightly but similarly inhibited for all test item concentrations. The inhibitions were 1.8%, -2.3%, 4.4%, 8.1% and 6.7% at test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively. In addition, standard deviations of each treatment showed a large overlap of the inhibition caused by the other treatments. Since no concentration-response relationship could be established, it was not possible to statistically derive meaningful ECx values. In this case the 3-hour EC10 value could statistically be established to be > 1000 mg/L. However, the confidence limits could not be established, which limits the reliability in this purely statistical finding. The NOEC was determined to be higher than a test item concentration of 1000 mg/L, as all test item concentrations were statistically not significant different from the control.