mecoprop-P (ISO); (R)-2-(4-chloro-2-methylphenoxy)propionic acid

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:

20 February 2013 to 06 March 2013

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 (Carbon and Ammonium Oxidation))

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test mixtures containing water, synthetic wastewater and the test material were prepared to obtain different nominal concentrations of the test material. All test material concentrations were weighed directly into the test vessels and were dispersed by intense aeration.
Synthetic wastewater and the corresponding test solutions were filled up with water to a final volume of approximately 250 mL. For each nitrification control, another 2.5 mL of a 2.32 g/L ATU stock solution was added. The reference assay with the different concentrations (e.g. 1, 3, 9, 25 mg/L) was set up in the same way as the test material.
The nitrification controls were prepared with ATU using 2.5 mL of a 2.32 g/L stock solution for each nitrification control. These controls were performed for each concentration of the test assay and for the reference assay.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Activated sludge from the municipal wastewater treatment plant of Pforzheim, Germany, collected from the aeration tank, was used as the microbial inoculum for the test. This plant was predominantly treating domestic sewage.
- Method of cultivation: The sludge was used one day after collection. It was settled for about 10 minutes and the upper layer with finer solids was decanted. Before starting the test, it was washed three times with chlorine free tap water by centrifugation (10 minutes at 1 000 rpm). After centrifuging, the supernatant was decanted and discarded and the sludge was re-suspended in chlorine free tap water. The mixed liquor suspended solids (MLSS) were adjusted to a concentration of 3.0 g/L (± 10 %). The activated sludge was continuously aerated at the test temperature, the solids did not settle down.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

18.2 - 20.7 °C

pH:

6.01 - 7.72

Nominal and measured concentrations:

Nominal concentrations: 10.0, 32.0, 100, 320 and 1 000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 1 L glass beakers
- Material, size, headspace, fill volume: 500 mL
- Aeration: Aeration was performed for some minutes to saturate the solution with 0 2 above 60 - 70 %. At to = 0 hours microbial inoculum was added. The total oxygen consumption rate was measured 3 hours after test start. The measurements were started at approximately 5 min. intervals. There was no aeration during the measurement.
- No. of vessels per concentration: Three replicates
- No. of vessels per control: Six replicates
- No. of vessels per abiotic control: The abiotic control, which was only performed in the range-finding test, was prepared without microbial inoculum but with the highest test material concentration and filled up with water to a final volume. The abiotic control was prepared with the highest test material concentration, water and synthetic wastewater. Due to the fact that no effects were determined in the abiotic control in the range-finding test, this control was excluded from the main test.
- Nutrients provided for bacteria: The test was performed in a synthetic wastewater that was prepared with the following amounts of substances in 1 L of deionised water:
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2 x 2 H2O
0.2 g MgSO4 X 7 H2O
2.8 g K2HPO4
The solution was sterilised prior to storage. If the preparation was not used immediately it was stored in the dark at a temperature of between 0 – 4 °C for up to one week.
- Nitrification inhibitor used: N-allylthiourea (ATU)

EFFECT PARAMETERS MEASURED:
Measurements were taken 3 hours after test start (t0). The test start (t0) was defined as the initial contact of the activated sludge inoculurn with the other constituents of the final mixture. The test solution was filled into a BOD-flask and stirred, and the oxygen consumption was measured and recorded over a period of about 5 minutes. All treatments were measured in the same way at approximately 5-minute intervals.

TEST CONCENTRATIONS
- Range finding study
The control and 1 000 mg/L were prepared with three replicates whereas the 10 and 100 mg/L treatments were prepared as single replicates. Additionally, for each concentration and replicate of the test material assay and of the reference concentrations a nitrification control with ATU was prepared. The test solutions were mixed with synthetic wastewater and were aerated. The test was started by adding activated sludge. Measurements of the oxygen uptake of the activated sludge were performed 3 hours after starting the respiration.
- Test concentrations: This range-finding test was performed in a limit test design with the concentrations: 10, 100 and 1 000 mg/L.
- Results used to determine the conditions for the definitive study: A range-finding test was performed in order to determine the range of concentrations for the definitive test. Since statistically significant effects were observed in the range-finding test at the highest test material concentration a main test was performed with the following concentrations: 10.0, 32.0, 100, 320 and 1 000 mg/L.

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol (A solution of 1.00 g of 3,5-dichlorophenol in 1 000 mL of water was prepared. The pH of the solution was checked and adjusted, if necessary, with NaOH or H2SO4 to pH 7 - 8 after preparation).

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

767 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Oxygen uptake

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Oxygen uptake

Duration:

3 h

Dose descriptor:

LOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Oxygen uptake

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Heterotrophic oxygen uptake

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Heterotrophic oxygen uptake

Duration:

3 h

Dose descriptor:

LOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Heterotrophic oxygen uptake

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

319 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: oxygen uptake due to nitrification

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: oxygen uptake due to nitrification

Duration:

3 h

Dose descriptor:

LOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: oxygen uptake due to nitrification

Details on results:

Range-finding test
- Statistically significant differences to the control were determined at 1 000 mg/L after 3 hours for the total oxygen uptake and oxygen uptake due to nitrification.

Main test
- The total oxygen uptake was measured up to a test material concentration of 1 000 mg/L. After 3 hours, statistically significant effects were observed at 320 and 1 000 mg/L for the total oxygen uptake, respectively. The NOEC is therefore determined to be 100 mg/Land the LOEC 320 mg/L. The EC50 was determined to be 767 mg/L.
- For the heterotrophic oxygen uptake, after 3 hours, statistically significant effects were observed at 1 000 mg/L. The NOEC is therefore determined to be 320 mg/L and the LOEC 1 000 mg/L. The EC50 was determined to be 1 000 mg/L.
- For the oxygen uptake due to nitrification statistically significant effects were observed at 320 and 1 000 mg/L, respectively. The NOEC is therefore determined to be 100 mg/L and the LOEC 320 mg/L. The EC50 was determined to be 319 mg/L.

Results with reference substance (positive control):

The EC50 of the total oxygen uptake for DCP was estimated within a range of 3 to 9 mg/L after 3 hours. This is within the recommended range of 2 to 25 mg/L. For the heterotrophic oxygen uptake for DCP, the EC50 was calculated without the inhibition value at 1.0 mg/L DCP, since this value was without the clear dose-response range of 3 to 25 mg/L DCP. The EC50 was determined to be within a range of 9 and 25 mg/L after 3 hours. The recommended range of 5 to 40 mg/L was fulfilled. For the oxygen uptake due to nitrification the EC50 was estimated without the inhibition value at 1.0 mg/L DCP, since this value was without the clear dose-response range of 3 to 25 mg/L DCP. On the base of extrapolation, the EC50 was estimated to be at 1.26 mg/L after 3 hours. This was also within the recommended range of 0.1 to 10 mg/L.

Reported statistics and error estimates:

The statistical evaluation was performed for specific respiration rate of the control and the test material concentrations using SAS® (2002 - 2008). The calculation was performed using SAS Software service pack 9.2. A test for normality of the data was performed by calculating the Shapiro-Wilk's statistic. A test for homogeneity of variance for the data was performed using the Levene-test. The NOEC was determined using Dunnett's, left sided. P-values below 0.05 showed statistically significant differences to the control. The EC50 values for the test material were calculated using the probit analysis following the Gompertz distribution. In addition, the EC50 values for DCP were performed with probit procedure following the Gompertz distribution for the total oxygen uptake and the oxygen uptake due to nitrification. In case of the nitrification the calculation had to be extrapolated. No statistical evaluation was performed for the heterotrophic oxygen uptake due to the fact that 50 % inhibition was observed at 1000 mg/L.

Results of the Main Test, Specific Respiration Rates

Test Assay (Sample)	Specific Respiration Rates [mg O2 / (g x h)]
	Total	Hetertrophic	Due to Nitrification
	3 h	3 h	3 h
Control (1)	25.98	14.23	8.91*
Control (2)	22.19	14.57
Control (3)	20.67	12.59
Control (4)	20.99	13.26
Control (5)	24.07	14.11
Control (6)	20.97	12.69
10 mg/L (1)	23.21	13.66	8.04*
10 mg/L (2)	21.39	14.55
10 mg/L (3)	21.22	13.49
32 mg/L (1)	21.31	14.67	7.29*
32 mg/L (2)	22.17	14.79
32 mg/L (3)	23.30	15.45
100 mg/L (1)	21.84	12.39	7.91*
100 mg/L (2)	22.17	13.27
100 mg/L (3)	19.80	14.43
320 mg/L (1)	17.09	13.23	3.32*
320 mg/L (2)	17.36	13.80
320 mg/L (3)	15.91	13.05
1000 mg/L (1)	7.19	7.15	2.22*
1000 mg/L (2)	9.71	5.41
1000 mg/L (3)	9.89	7.57
DCP 1 mg/L	17.51	18.92	-1.41
DCP 3 mg/L	15.59	13.58	2.01
DCP 9 mg/L	8.94	8.55	0.39
DCP 25 mg/L	2.69	2.97	-0.28

 * These values are the difference between the mean total and the mean of the heterotrophic rates.

Main Test Inhibition Results

Test Assay Nominal [mg/L]	Mean Inhibition [%]***
	Total	Heterotrophic	Due to Nitrification
	3 h	3 h	3 h
10	2.4	-2.4	9.7
32	1.0	-10.2	18.1
100	5.4	1.6	11.1
320	25.3*	1.6	61.5*
1000	60.3*	50.6*	75.1*
DCP 1	22.1	-39.3**	115.8**
DCP 3	30.6	0.0	77.4
DCP 9	60.2	37.0	95.6
DCP 25	88.0	78.1	103.1

* Statistically significant different to control.

** Values excluded from the calculation due to measuring errors.

*** Negative inhibition values indicate stimulating effects.

Validity Criteria

The test fulfills the criteria of validity, since:

• The EC50 of the total oxygen uptake for DCP was estimated within a range of 3 to 9 mg/L after 3 hours. This is within the recommended range of 2 to 25 mg/L. For the heterotrophic oxygen uptake for DCP, the EC50 was determined to be within a range of 9 and 25 mg/L after 3 hours. The recommended range of 5 to 40 mg/L was fulfilled. For the oxygen uptake due to nitrification the EC50 was estimated to be 1.26 mg/L after 3 hours. This was also within the recommended range of 0.1 to 10 mg/L.

• The coefficients of variation of the control for total oxygen uptake were 9 % after 3 hours and did not exceed 30 %.

• The oxygen uptake rates of the blank controls were determined to be on average 22 mg O2/(g x h) dry matter after 3 hours. There was not less than 20 mg oxygen uptake per g activated sludge (dry weight of suspended solids) in 1 hour.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the test material had an inhibitory effect on the total and nitrifying organisms at 320 and 1 000 mg/L and on heterotrophic microorganisms at 1 000 mg/L.
The EC50 for total oxygen uptake was determined to be 767 mg/L.
The EC50 for heterotrophic oxygen uptake was determined to be 1 000 mg/L.
The EC50 for oxygen uptake due to nitrification was determined to be 319 mg/L.

Executive summary:

Inhibition to activated sludge respiration by the test material was assessed according to OECD Test Guideline 209 and in compliance with GLP.

The respiration rate of an activated sludge, fed with a standard amount of synthetic wastewater feed, was measured 3 hours after application of the test material. The inhibitory effects of the test material in particular concentrations were expressed as a percentage of the mean respiration rate of the controls.

Activated sludge from the municipal wastewater treatment plant of Pforzheim, Germany, was used as microbial inoculum for the test. This plant predominantly is treating domestic sewage. The sludge was adjusted to a content of 1.5 g/L dry matter and was exposed to the test material under continuous aeration. After stopping the aeration, the O2 consumption was measured for approx. 5 minutes. The slope of the O2 consumption straight line is an indication for toxic effects on respiration activity of microorganisms.

A range-finding test was performed with concentrations of 10, 100 and 1 000 mg/L in a limit test design. The highest concentration of 1 000 mg/L and a control were tested in three replicates whereas 10 and 100 mg /L were tested in one replicate.

Four concentrations of 3,5-dichlorophenol (DCP) as toxic reference material were also tested to demonstrate the sensitivity of the test system.

To test the differences between total oxygen uptake, heterotrophic oxygen uptake and oxygen uptake due to nitrification, two test assays were performed, one with and one without allylthiourea (ATU). The oxygen uptake rate for the range-finding test was determined after 3 hours.

A main test was performed with concentrations of 10.0, 32.0, 100, 320 and 1 000 mg /L. All test material concentrations were tested in three replicates whereas the control was performed with six replicates. Four concentrations of 3,5-dichlorophenol as toxic reference material were also tested to demonstrate the sensitivity of the test system. To test the differences between total oxygen uptake, heterotrophic oxygen uptake and oxygen uptake due to nitrification, two test assays were performed, one with and one without allylthiourea (ATU).

The total oxygen uptake was measured up to a test material concentration of 1 000 mg/L.

After 3 hours, statistically significant effects were observed at 320 and 1 000 mg/L for the total oxygen uptake. The NOEC is therefore determined to be 100 mg/L and the LOEC 320 mg/L. The EC50 was determined to be 767 mg/L. For the heterotrophic oxygen uptake, after 3 hours, statistically significant effects were observed at 1 000 mg/L.

The NOEC is therefore determined to be 320 mg/L and the LOEC 1 000 mg/L. The EC50 was determined to be 1 000 mg/L.

For the oxygen uptake due to nitrification statistically significant effects were observed at 320 and 1 000 mg/L, respectively. The NOEC is therefore determined to be 100 mg/L and the LOEC 320 mg/L. The EC50 was determined to be 319 mg/L.

The EC50 of the total oxygen uptake for DCP was estimated within a range of 3 to 9 mg/L after 3 hours. This is within the recommended range of 2 to 25 mg/L.

For the heterotrophic oxygen uptake for DCP, the EC50 was calculated without the inhibition value at 1.0 mg/L DCP due to measuring errors. The EC50 was determined to be within a range of 9 and 25 mg/L after 3 hours. The recommended range of 5 to 40 mg/L was therefore fulfilled.

For the oxygen uptake due to nitrification the EC50 was estimated without the inhibition value at 1.0 mg/L DCP due to measuring errors. On the base of extrapolation, the EC50 was estimated to be at 1.26 mg/L after 3 hours. This was also within the recommended range of 0.l to 10 mg/L.

The oxygen uptake rates of the blank controls were determined to be on average 22 mg O2/(g x h) dry matter after 3 hours. There was not less than 20 mg oxygen uptake per g activated sludge (dry weight of suspended solids) in 1 hour.

The coefficients of variation of the control for total oxygen uptake were 9 % after 3 hours and did not exceed 30 %. All validity criteria according to OECD guideline 209 were therefore fulfilled.

Under the conditions of the study the test material had an inhibitory effect on the total and nitrifying organisms at 320 and 1 000 mg/L and on heterotrophic microorganisms at 1 000 mg/L.

The EC50 for total oxygen uptake was determined to be 767 mg/L.

The EC50 for heterotrophic oxygen uptake was determined to be 1 000 mg/L.

The EC50 for oxygen uptake due to nitrification was determined to be 319 mg/L.

Description of key information

Falk (2013)

Under the conditions of the study the test material had an inhibitory effect on the total and nitrifying organisms at 320 and 1 000 mg/L and on heterotrophic microorganisms at 1 000 mg/L.

The EC50 for total oxygen uptake was determined to be 767 mg/L.

The EC50 for heterotrophic oxygen uptake was determined to be 1 000 mg/L.

The EC50 for oxygen uptake due to nitrification was determined to be 319 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

767 mg/L

EC10 or NOEC for microorganisms:

100 mg/L

Additional information

Falk (2013)

Inhibition to activated sludge respiration by the test material was assessed according to OECD Test Guideline 209 and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The respiration rate of an activated sludge, fed with a standard amount of synthetic wastewater feed, was measured 3 hours after application of the test material. The inhibitory effects of the test material in particular concentrations were expressed as a percentage of the mean respiration rate of the controls.

Activated sludge from the municipal wastewater treatment plant of Pforzheim, Germany, was used as microbial inoculum for the test. This plant predominantly is treating domestic sewage. The sludge was adjusted to a content of 1.5 g/L dry matter and was exposed to the test material under continuous aeration. After stopping the aeration, the O2 consumption was measured for approx. 5 minutes. The slope of theO2consumption straight line is an indication for toxic effects on respiration activity of microorganisms.

A range-finding test was performed with concentrations of 10, 100 and 1000 mg/L in a limit test design. The highest concentration of 1000 mg/L and a control were tested in three replicates whereas 10 and 100 mg /L were tested in one replicate.

Four concentrations of 3,5-dichlorophenol (DCP) as toxic reference material were also tested to demonstrate the sensitivity of the test system.

To test the differences between total oxygen uptake, heterotrophic oxygen uptake and oxygen uptake due to nitrification, two test assays were performed, one with and one without allylthiourea (ATU). The oxygen uptake rate for the range-finding test was determined after 3 hours.

A main test was performed with concentrations of 10.0, 32.0, 100, 320 and 1000 mg /L. All test material concentrations were tested in three replicates whereas the control was performed with six replicates. Four concentrations of 3,5-dichlorophenol as toxic reference material were also tested to demonstrate the sensitivity of the test system. To test the differences between total oxygen uptake, heterotrophic oxygen uptake and oxygen uptake due to nitrification, two test assays were performed, one with and one without allylthiourea (ATU).

The total oxygen uptake was measured up to a test material concentration of 1000 mg/L.

After 3 hours, statistically significant effects were observed at 320 and 1000 mg/L for the total oxygen uptake. The NOEC is therefore determined to be 100 mg/L and the LOEC 320 mg/L. The EC50 was determined to be 767 mg/L. For the heterotrophic oxygen uptake, after 3 hours, statistically significant effects were observed at 1000 mg/L.

The NOEC is therefore determined to be 320 mg/L and the LOEC 1000 mg/L. The EC50 was determined to be 1000 mg/L.

For the oxygen uptake due to nitrification statistically significant effects were observed at 320 and 1000 mg/L, respectively. The NOEC is therefore determined to be 100 mg/L and the LOEC 320 mg/L. The EC50 was determined to be 319 mg/L.

The EC50 of the total oxygen uptake for DCP was estimated within a range of 3 to 9 mg/L after 3 hours. This is within the recommended range of 2 to 25 mg/L.

For the heterotrophic oxygen uptake for DCP, the EC50 was calculated without the inhibition value at 1.0 mg/L DCP due to measuring errors. The EC50 was determined to be within a range of 9 and 25 mg/L after 3 hours. The recommended range of 5 to 40 mg/L was therefore fulfilled.

For the oxygen uptake due to nitrification the EC50 was estimated without the inhibition value at 1.0 mg/L DCP due to measuring errors. On the base of extrapolation, the EC50 was estimated to be at 1.26 mg/L after 3 hours. This was also within the recommended range of 0.l to 10 mg/L.

The oxygen uptake rates of the blank controls were determined to be on average 22 mg O2/(g x h) dry matter after 3 hours. There was not less than 20 mg oxygen uptake per g activated sludge (dry weight of suspended solids) in 1 hour.

The coefficients of variation of the control for total oxygen uptake were 9 % after 3 hours and did not exceed 30 %. All validity criteria according to OECD guideline 209 were therefore fulfilled.

Under the conditions of the study the test material had an inhibitory effect on the total and nitrifying organisms at 320 and 1000 mg/L and on heterotrophic microorganisms at 1000 mg/L.

The EC50 for total oxygen uptake was determined to be 767 mg/L.

The EC50 for heterotrophic oxygen uptake was determined to be 1000 mg/L.

The EC50 for oxygen uptake due to nitrification was determined to be 319 mg/L.