2-(4-chloro-2,6-dimethylphenyl)acetic acid

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Feb - 22 Feb 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

Version / remarks:

german version 8192:2007

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-chloro-2,6-dimethylphenyl)acetic acid

EC Number:

858-362-8

Cas Number:

186748-50-5

Molecular formula:

C10H11ClO2

IUPAC Name:

2-(4-chloro-2,6-dimethylphenyl)acetic acid

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
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 (five replicates each)
Control: Six controls (pure water, synthetic sewage feed and inoculum, but without addition of the test item) were tested in parallel.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

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 7.1 and therefore no adjustment necessary.

Study design

Test type:

static

Water media type:

other: pure water

Limit test:

no

Total exposure duration:

3 h

Test conditions

Test temperature:

20 °C ± 2 °C during pre-incubation (3 hours) and evaluation period

Nominal and measured concentrations:

Nominal 10, 32, 100, 320 and 1000 mg test item/L (five replicates each)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass flasks of approximately 1 litre volume and Karlsruher flasks of 300 mL volume
- Aeration: Compressed air (1.017 litre per minute)
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per abiotic control (replicates): 0
- Sludge concentration (weight of dry solids per volume): 1.5 g/L suspended solids in the test medium.
- Nutrients provided for bacteria: Synthetic Sewage feed
- Nitrification inhibitor used: N-allylthiourea

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: pure water

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
For the measurement of the respiration rate a well-mixed sample of each test medium was poured into a Karlsruher flask after 3 hours incubation time. The oxygen concentration was then measured with an oxygen electrode and recorded for about 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 from the most linear part of the respiration curve in the range between 9.3 and 0.2 mg O2/L for total respiration and between 10.3 and 4.6 mg O2/L for heterotrophic respiration. In case of low oxygen consumption the values over a period of 10 minutes were used. In case of a faster drop off below 2 mg O2/L, shorter time intervals were used.
The dissolved 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 CONCENTRATIONS
- Range finding study: no

Reference substance (positive control):

yes

Remarks:

The reference item 3,5-dichlorophenol was tested at the nominal test concentrations of 1, 4, and 16 mg/L

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: total respiration rate

Effect concentrations 2

Key result

Duration:

3 h

Dose descriptor:

EC50

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: respiration rate

Remarks on result:

not determinable

Details on results:

Total Respiration Rate:
The respiration rate of the highest test item concentration of 1000 mg/L was not significantly different from the control; the other test item concentrations were significantly different from the control.
In comparison to the inoculum controls, the total respiration rates of the activated sludge were not dose-response inhibited for test item concentrations up to and including 1000 mg/L, i.e. the respiration rates changes were significant at 10 mg/L, 32 mg/L, 100 mg/L and 320 mg/L, but not significant at 1000 mg/L. Overall, there was no trend of respiration inhibition. The inhibition was 25.5%, 23.9%, 28.5%, 23.6% and 13.8% for test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively.
3-hour EC10, EC20 and 3-hour EC50 and NOEC based on Total Respiration:
No meaningful statistical ECx, LOEC or NOEC values could be calculated. As no dose-response inhibiting effect was measured and the lowest inhibition was measured in the highest test item concentration of 1000 mg/L,
the NOEC can be set to be ≥ 1000 mg/L.

Heterotrophic Respiration Rate:
In comparison to the inoculum controls, the heterotrophic respiration rates of the activated sludge were not dose-response inhibited for test item concentrations up to and including 1000 mg/L. The test item has no statistically significant inhibiting effect on the heterotrophic respiration for test item concentrations of 10 mg/L, 32 mg/L and 1000 mg/L. The inhibition was 2.0%, 9.4%, 18.2%, 19.4% and 2.3% for test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively.
3-hour EC10, EC20 and 3-hour EC50 and NOEC based on Heterotrophic Respiration: No meaningful statistical ECx, LOEC or NOEC values could be calculated. As no dose-response inhibiting effect was measured and the lowest inhibition was measured in the highest test item concentration of 1000 mg/L ,
the NOEC can be set to be ≥ 1000 mg/L.

Respiration Rate based on Nitrification:
In comparison to the inoculum controls, the respiration rates based on nitrification of the activated sludge were not dose-response inhibited for test item concentrations up to and including 1000 mg/L. The test item has no statistically significant inhibiting effect on the nitrification respiration for test item concentrations between 32 mg/L and 1000 mg/L. The inhibitions were between 22% and 36% for all test item concentrations between 32 mg/L and 1000 mg/L.
3-hour EC10, EC20 and 3-hour EC50 and NOEC based on Nitrification Respiration: No meaningful statistical ECx, LOEC or NOEC values could be calculated. As no dose-response inhibiting effect was measured and the lowest inhibition was measured in the highest test item concentration of 1000 mg/L ,
the NOEC can be set to be ≥ 1000 mg/L.

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 43% to 88% 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:
EC10 0.1 mg/, lower 95%-cl 0.0, upper 95%-cl 0.8
EC20 0.3 mg/, lower 95%-cl 0.1, upper 95%-cl 1.9
EC50 2.4 mg/, lower 95%-cl 0.3, upper 95%-cl 19.1

Heterotrophic Respiration Rate: The inhibition of the activated sludge treated with the reference item was in the range of -31% to 69% 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:
EC10 11.7 mg/, lower 95%-cl 0.0, upper 95%-cl n.d.
EC20 12.6 mg/, lower 95%-cl n.d., upper 95%-cl n.d.
EC50 14.5 mg/, lower 95%-cl n.d., upper 95%-cl n.d.

Respiration Rate based on Nitrification: The inhibition of the activated sludge treated with the reference item was in the range of 75% to 103% 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:
EC10 0.7 mg/, lower 95%-cl 0.0, upper 95%-cl n.d.
EC20 0.7 mg/, lower 95%-cl n.d., upper 95%-cl n.d.
EC50 0.9 mg/, lower 95%-cl n.d., upper 95%-cl n.d.

Reported statistics and error estimates:

ECx Estimation of the test item:
Regression Analysis: 3-Parametric normal CDF (cumulative distribution function), non-linear regression without weighting; Optimization method: Levenberg-Marquardt

3-Parametric normal CDF R2:
Total: 0.267 (a significant amount of variance is explained by the regression model, there is no significant lack of fit)
Heterotroph: 0.073 (the amount of variance explained by the regression model is not significant, there is no significant lack of fit)
Nitrification: 0.186 (the amount of variance explained by the regression model is not significant, there is no significant lack of fit)

NOEC Estimation:
Test on Normal Distribution: Shapiro Wilk’s test (α= 0.01)
Variance Homogeneity: Levene’s Test (α= 0.01)
T-test Procedure for treatment comparison and NOEC estimation:
Total: Dunnett’s Multiple Sequential t-test (α= 0.05, one-sided smaller)
Heterotroph: Multiple Sequentially-rejective Welsh t-test after Bonferroni-Holm
(α= 0.05, one-sided smaller)
Nitrification: Dunnett’s Multiple t-test
(α= 0.05, one-sided smaller)

ECx Estimation of the reference item:
Regression Analysis: 3-Parametric normal CDF (cumulative distribution function), non-linear regression without weighting; Optimization method: Levenberg-Marquardt
3-Parametric normal CDF R2: total 0.804, heterotroph 0.813, nitrification 0.822

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

Any other information on results incl. tables

Validity Criteria

Criteria	required range	obtained value	passed
oxygen uptake in the blank controls	not less than 20 mgO2/g/h	21.2 mgO2/g/h	yes
respiration rates of the six controls	< 30%	17.8% for the total respiration and 9.9% for the heterotrophic respiration	yes
Reference Item (Total Respiration):	3-hour EC50 should be 2 - 25 mg/L	3-hour EC50 was 2.4 mg/L	yes
Reference Item (Heterotrophic Respiration):	3-hour EC50 should be 5 - 40 mg/L	3-hour EC50 was 14.5 mg/L	yes
Reference Item (Respiration based on Nitrification):	3-hour EC50 should be 0.1 - 10 mg/L	3-hour EC50 was 0.9 mg/L	yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see above

Conclusions:

The test item had no dose-response inhibiting effect for test item concentrations up to and including 1000 mg/L for total, heterotrophic and nitrification respiration.

For the test item , the NOEC can be set to be ≥ 1000 mg /L for total respiration, heterotrophic respiration and respiration based on nitrification. No meaningful 3-hour ECx values were determined.

Executive summary:

Test Species: Activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the municipal sewage treatment plant Bensheim, Germany.

Test Design: 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 Concentration: 10, 32, 100, 320 and 1000 mg test item/L; 1, 4 and 16 mg 3,5-Dichlorophenol/L and six inoculum controls

Results: 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: In comparison to the inoculum controls, the total respiration rates of the activated sludge were not dose-response inhibited for test item concentrations up to and including 1000 mg/L, i.e. the respiration rates changes were significant at 10 mg/L, 32 mg/L, 100 mg/L and 320 mg/L, but not significant at 1000 mg/L. Overall, there was no trend of respiration inhibition. The inhibition was 25.5%, 23.9%, 28.5%, 23.6% and 13.8% for test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectivel. No meaningful statistical EC_x, LOEC or NOEC values could be calculated. As no dose-response inhibiting effect was measured and the lowest inhibition was measured in the highest test item concentration of 1000 mg/L, the NOEC can be set to be ≥ 1000 mg/L.

Heterotrophic Respiration: In comparison to the inoculum controls, the heterotrophic respiration rates of the activated sludge were not dose-response inhibited for test item concentrations up to and including 1000 mg/L. The test item has no statistically significant inhibiting effect on the heterotrophic respiration for test item concentrations of 10 mg/L, 32 mg/L and 1000 mg/L. The inhibition was 2.0%, 9.4%, 18.2%, 19.4% and 2.3% for test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively. No meaningful statistical EC_x, LOEC or NOEC values could be calculated. As no dose-response inhibiting effect was measured and the lowest inhibition was measured in the highest test item concentration of 1000 mg/L, the NOEC can be set to be ≥ 1000 mg/L.

Respiration based on nitrification: In comparison to the inoculum controls, the respiration rates based on nitrification of the activated sludge were not dose-response inhibited for test item concentrations up to and including 1000 mg/L. The test item has no statistically significant inhibiting effect on the nitrification respiration for test item concentrations between 32 mg/L and 1000 mg/L. The inhibitions were between 22% and 36% for all test item concentrations between 32 mg/L and 1000 mg/L. No meaningful statistical EC_x, LOEC or NOEC values could be calculated. As no dose-response inhibiting effect was measured and the lowest inhibition was measured in the highest test item concentration of 1000 mg/L, the NOEC can be set to be ≥ 1000 mg/L.

Conclusion: The test item had no dose-response inhibiting effect for test item concentrations up to and including 1000 mg/L for total, heterotrophic and nitrification respiration. For the test item, the NOEC can be set to be ≥ 1000 mg /L for total respiration, heterotrophic respiration and respiration based on nitrification. No meaningful 3-hour ECx values were determined.