[No public or meaningful name is available]

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 2019-11-05 to 2019-11-06, with the definitive exposure phase on 2019-11-06 at the test facility

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item Reaction products of DL-methionine and C18 unsaturated fatty acid chloride and isopropanol
Batch number ESD0033040
Purity UVBC substance, ca. 95% dry residue
Composition N-Oleyl-DL-methionine: 57 % (w/w) (100 % minus by-products)
By-products:
N-Oleyl-DL-methionine
isopropylester: 24 % (w/w) (gas chromatography, calculated)
Oleic acid: 12.6 % (w/w) (gas chromatography)
Oleic acid
isopropylester: 1.8 (w/w) (gas chromatography)
Methionine: 1.6 % (w/w) (gas chromatography)
Water: 0.5 % (w/w) (Karl Fischer titration)
Unknown
by-products: 2.5 % (a/a) (gas chromatography)
Acid number: 138 mg KOH/g (titration)
Water solubility <10 g/L (25°C) partially
Density 0.98 g/cm3
Appearance Brownish liquid
Expiry date 2021-08-31
Recommended storage Room temperature

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

Based on the results of the preliminary test (see Annex I for details) the definitive study was carried out with 5 concentrations within the range of 10 to 1000 mg/L (with and without ATU) in a geometrical series and with a dilution factor of approx. 3.2. No pH adjustment was made.
The test item concentrations were weighed out directly in the test vessels.
Triplicates of the control (without ATU) without test item were included at the beginning and at the end of the replicates without ATU.
Triplicates of the control (with ATU) without test item were included at the beginning of the replicates with ATU and at the end of the test.
The reference item Copper (II)-sulfate was tested with three concentrations within the concentration range 58 - 180 mg/L and a dilution factor of approx. 1.8 (without ATU).
The reference item N-Methylanilin was tested with three concentrations within the concentration range 0.1 – 1.8 – 32 mg/L and a dilution factor of 18 (with and without ATU).
A fresh sample of activated sludge taken from the sewage plant Pattensen was used for the test procedure. It was washed twice and adjusted with chlorine free tap water if necessary to a dry sludge concentration of 3.0 g/L ± 10 %.

Test organisms (species):

activated sludge

Details on inoculum:

- Preparation of inoculum for exposure: Municipal sewage treatment plant of 30982 Pattensen, Germany
Non-adapted activated sludge from the sewage plant at Pattensen is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste.
- Receipt: 2019-11-05
- Pretreatment: The sludge was washed twice and adjusted with chlorine free tap water if necessary. The sludge was used within 24 h after sampling.
- Dry sludge concentration: 2.89 g/L, corresponding to 1.45 g/L in the test vessels for replicates without ATU and 2.85 g/L, corresponding to 1.43 g/L in the test vessels for replicates with ATU.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

Not determined

Test temperature:

20.6 °C

pH:

pH-value of the activated sludge for test without ATU 7.29
pH-value of the activated sludge for test with ATU 7.25
pH-value of the synthetic waste water 7.21

Salinity:

Not determined

Details on test conditions:

TEST SYSTEM
- Test vessel: 5
- 500 mL Erlenmeyer flasks, DIN 12380
- Aeration:Permanent, to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension.
- Pretreatment: Stirring overnight in 200 mL ultrapure water at 400 rpm

- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6 (Triplicates at the beginning of the test + triplicates at the end of the test.)
- No. of vessels per reference item Copper-II-Sulfat (replicates): 9 (three concentrations within the range 58 - 180 mg/L and a dilution factor 1.8)
- No. of vessels per reference item N-Methylaniline (replicates): 9 (three concentrations within the range 0.1 - 32 mg/L and a dilution factor 18)
- Composition of test medium:

Test concentrations [mg/L] 10 - 32 - 100 - 320 - 1000 mg/L
The test item will be weighed out directly in test vessels

Dilution Table for the Test Item without ATU
Test item concentration [mg/L] 10 32 100 320 1000
Test item [mg] 5 16 50 160 500
Synthetic waste water [mL] 16
Ultrapure water [mL] Filled up to 250 mL
Inoculum [mL] 250

Dilution Table for the Test Item with ATU
Test item concentration [mg/L] 10 32 100 320 1000
Test item [mg] 5 16 50 160 500
Synthetic waste water [mL] 16
ATU solution [mL]* 2.5
Ultrapure water [mL] Filled up to 250 mL
Inoculum [mL] 250
*) Concentration of the ATU solution: 2.32 g/L
 
The reference item concentration (total respiration) was diluted out of a stock solution.
Dilution Table for the Reference Item (Total Respiration)
Reference item concentration [mg/L] 58 100 180
Stock solution [µL]* 96.7 166.7 300
Synthetic waste water [mL] 16
Demineralized water [mL] Filled up to 250 mL
Inoculum [mL] 250
*) Concentration stock solution: 300 g/L

The reference item concentration (nitrification) was diluted out of a stock solution.

Dilution Table for the Reference Item (Nitrification) without ATU
Reference item concentration [mg/L] 0.1 1.8 32
Stock solution [mL]* 0.05 0.9 16
Synthetic waste water [mL] 16
Demineralized water [mL] Filled up to 250 mL
Inoculum [mL] 250
*) Concentration stock solution: 1 g/L



Dilution Table for the Reference Item (Nitrification) with ATU
Reference item concentration [mg/L] 0.1 1.8 32
Stock solution [mL]* 0.05 0.9 16
Synthetic waste water [mL] 16
ATU solution [mL]** 2.5
Demineralized water [mL] Filled up to 250 mL
Inoculum [mL] 250


*) Concentration stock solution: 1 g/L
**) Concentration of the ATU solution: 2.32 g/L



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined by means of oximeter every 5 seconds after transferring the solution to the measuring chamber and the oxygen depletion was recorded for at least 5 minutes with the software PreSens Measurement Studio 2.

TEST CONCENTRATIONS

Inhibitions in the Preliminary Tests
A preliminary range finding test (non GLP) was carried out with the test item concentrations of 1 – 10 – 100 – 1000 mg/L with one replicate each. The pH was measured in one replicate with 1000 mg/L test item. The flasks were aerated for 3 hours.

In the preliminary range finding test, additional replicates were prepared to determine the inhibition of nitrification. One replicate each of 1 – 10 – 100 - 1000 mg/L was measured with the addition of Allylthiourea (ATU).

Inhibitions in the Preliminary Test (with / without ATU)
Nominal Test Item Concentration
[mg/L] pH
Inhibition of total respiration (without ATU)
[%] Inhibition of heterotrophic respiration (with ATU)
[%] Inhibition of nitrification
[%]
1 - 0 1 -2
10 - 5 -3 12
100 - -3 -13 7
1000 6.61 49 48 50

An inhibition of heterotrophic respiration for test item concentration of 1000 mg/L as well as a clear difference between inhibition of nitrification and heterotrophic respiration for test item concentration of 10 mg/L was observed. Due to the request of the sponsor the test was performed with the determination of both endpoints.



A second preliminary range finding test was carried out in the concentrations of 1 - 100 - 1000 mg/L with and without Allylthiourea (ATU) and one replicate
each. Stirring overnight and pH-adjustment were carried out for preparation of the test item solutions. The flasks were aerated for 3 hours.

pH-adjustment in the second Preliminary Test (with / without ATU)
Nominal Test Item Concentration
[mg/L] pH

(test item in ultrapure water) pH

(adjusted with 1 mol/L HCL to) pH

(test item in medium)
1 8.82 - 7.40
100 9.27 7.40 7.44
1000 12.27 7.48 7.42
1 ATU 7.45 - 7.75
100 ATU 11.39 8.88 7.77
1000 ATU 12.28 6.92 7.79
An inhibition of heterotrophic respiration for test item concentration of 1000 mg/L as well as a clear difference between inhibition of nitrification and heterotrophic respiration for test item concentration of 10 mg/L was observed. Due to the request of the sponsor the test was performed with the determination of both endpoints.

Reference substance (positive control):

yes

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

251 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: CI (184 - 359 mg/L)

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

286 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Remarks on result:

other: CI (158 - 462 mg/L)

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

213 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of respiration due to nitrification

Remarks:

respiration rate

Remarks on result:

other: CI (118 -423 mg/L)

Key result

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

497 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration tate

Remarks on result:

other: CI (348 - 697 mg/L)

Key result

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

460 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Remarks:

respiration rate

Remarks on result:

other: CI (323 - 776 mg/L)

Key result

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

470 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of respiration due to nitrification

Remarks:

respiration rate

Remarks on result:

other: CI (247 - > 1000 mg/L)

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 total respiration

Remarks:

respiration rate

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 heterotrophic respiration

Remarks:

respiration rate

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 nitrification rate

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Remarks:

respiration rate

Results with reference substance (positive control):

- Results with reference substance valid? Yes

Reported statistics and error estimates:

The EC-values of the test item and the reference item were calculated by sigmoidal dose-response regression (biphasic regression where necessary) and linear regression, respectively, using software GraphPadPrism. Calculation of the confidence intervals for the EC50-value was carried out using standard procedures. The coefficient of variation of the oxygen uptake rates of the control replicates was calculated with standard methods using Excel.

The calculation of the oxygen uptake rates (R) and inhibition of respiration rates (I) was performed according to the guideline.

The NOEC was determined by calculation of statistical significance of the oxygen uptake rate in comparison to the control. One Way Analysis of Variance (ANOVA) and DUNNETT’s test was used for NOEC calculation. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-value for both tests was 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration

From the oxygen depletion the oxygen uptake rates were calculated in mg O2/L/h, additionally the specific respiration rates based on the dry sludge concentration were calculated in mg O2/g/h. The inhibition of the oxygen uptake rates of the test and reference item concentrations in % was calculated in comparison to the oxygen uptake rates of the control. The results are listed in the tables below.

Oxygen Uptake Rates and Specific Respiration Rates of the Total Respiration of the Control

       Repl.       Oxygen

Concentration*       Oxygen Uptake Rate R       Specific Respiration Rate Rs

              [mg O2/L]        [mg O2/(L×h)]        [mg O2/g×h]

Control        1        8.08        23.9        16.5

        2        7.92        24.6        17.0

       3        7.75        24.3        16.8

       4        7.53        21.7        15.0

       5        7.32        22.9        15.8

       6        7.76        22.4        15.4

      Mean value         23.3        16.1

      Standard deviation        1.0       

      CV [%]        4.5       

*) determined at start of the measurement

Repl. = Replicate ; CV = Coefficient of variation

Oxygen Uptake Rates and Inhibition of the Total Respiration of the

             Test Item Concentrations

Test Item Concentration       Repl.       Oxygen

Concentration*       Oxygen Uptake Rate R       Inhibition       Mean Inhibition

[mg/L]              [mg O2/L]       [mg O2/(L×h)]       [%]        [%]

10       1       7.89       22.8       2       -9

      2       7.67       27.0       -16       

      3       7.81       24.2       -4       

      4       7.60       26.5       -14       

      5       7.37       26.5       -14       

32       1       6.39       25.3       -9       -10

      2       6.19       25.8       -11       

      3       5.95       25.1       -8       

      4       6.14       26.2       -12       

      5       5.90       26.1       -12       

100       1       7.03       21.5       8       0

      2       6.28       25.2       -8       

      3       6.37       22.8       2       

      4       7.07       22.2       5       

      5       7.53       24.4       -5       

320       1       6.49       19.7       15       13

      2       6.20       21.5       8       

      3       6.31       20.0       14       

      4       6.77       20.4       12       

      5       5.24       19.5       16       

1000       1       7.66       17.0       27       29

      2       6.94       16.9       27       

      3       7.51       17.9       23       

      4       7.88       16.1       31       

      5       7.48       14.7       37       

*) determined at start of the measurement

Repl. = Replicate

 

Oxygen Uptake Rates of the Heterotrophic Respiration of the Control Replicates with ATU

      Repl.       Oxygen

Concentration*       Oxygen Uptake Rate R

             [mg O2/L]       [mg O2/(L×h)]

Control       1       8.39       10.5

      2       8.27       10.3

      3       8.37       10.5

      4       7.98       10.2

      5       8.30       10.4

      6       8.51       10.5

      Mean value       10.4

      Standard deviation       0.1

      CV [%]       1.1

*) determined at start of the measurement

Repl. = Replicate

CV = Coefficient of variation

 

Oxygen Uptake Rates and Inhibition of the Heterotrophic Respiration of the

Test Item Concentrations

Test Item

Concentration

[mg/L]       Repl.       Oxygen

Concentration*

[mg O2/L]       Oxygen Uptake Rate R

[mg O2/Lh]       Inhibition

[%]       Mean Inhibition

[%]

10 + ATU       1       8.28       10.5       -1       -5

      2       8.26       11.1       -7       

      3       8.35       10.8       -4       

      4#       7.57       21.6       -108       

      5       8.26       11.1       -7       

32 + ATU       1       8.05       11.5       -11       -9

      2       7.80       11.8       -13       

      3       7.32       12.1       -16       

      4       7.82       12.0       -15       

      5       6.86       9.2       12       

100 + ATU       1       7.81       11.6       -12       -4

      2       7.76       10.2       2       

      3       7.84       9.6       8       

      4       7.83       12.5       -20       

      5       7.91       10.1       3       

320 + ATU       1       7.59       8.9       14       12

      2       6.84       8.9       14       

      3       6.69       9.7       7       

      4       8.23       9.2       12       

      5       7.56       8.8       15       

1000 + ATU       1       7.99       7.3       30       30

      2       7.89       7.2       31       

      3       7.58       7.8       25       

      4       8.47       7.3       30       

      5       8.19       7.1       32       

*) determined at start of the measurement

#) Replicate without ATU, determined as an outlier (Grubb´s outlier test)

Repl. = Replicate

    

Calculated Oxygen Uptake Rates of the Nitrification of the Control

      Repl.       Oxygen Uptake Rate R

             [mg O2/(L×h)]

Control       1       13.4

      2       14.3

      3       13.8

      4       11.5

      5       12.5

      6       11.9

Mean value       12.9

Standard deviation       1.0

CV [%]       7.8

Repl. = Replicate

CV = Coefficient of variation

Oxygen Uptake Rates and Inhibition of the Nitrification of the Test Item Concentrations

Test Item

Concentration

[mg/L]       Repl.       Oxygen Uptake Rate R

[mg O2/Lh]       Inhibition

[%]       Mean Inhibition

[%]

10       1       11.9       8       -13

      2       16.1       -25       

      3       13.3       -3       

      4       15.6       -21       

      5       15.6       -21       

32       1       14.0       -8       -11

      2       14.5       -12       

      3       13.8       -7       

      4       14.9       -15       

      5       14.8       -15       

100       1       10.7       17       4

      2       14.4       -12       

      3       12.0       7       

      4       11.4       12       

      5       13.6       -5       

320       1       10.6       18       14

      2       12.4       4       

      3       10.9       16       

      4       11.3       12       

      5       10.4       19       

1000       1       9.7       25       29

      2       9.6       26       

      3       10.6       18       

      4       8.8       32       

      5       7.4       43       

Repl. = Replicate

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of Reaction products of DL-methionine and C18 unsaturated fatty acid chloride and isopropanol for Total Respiration is 100 mg/L.
The EC10 for Total Respiration is 251 mg/L and the EC50 is >1000 mg/L.
The NOEC for heterotrophic respiration is 100 mg/L.
The EC10 for heterotrophic respiration is 286 mg/L and the EC50 is >1000 mg/L.
The NOEC for nitrification is 320 mg/L.
The EC10 for nitrification is 213 mg/L and the EC50 is >1000 mg/L.

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item Reaction products of DL-methionine and C18 unsaturated fatty acid chloride and isopropanol (batch number: ESD0033040) from 2019-11-05 to 2019-11-06, with the definitive exposure phase on 2019-11-06 at the test facility. The test system was activated sludge of the municipal treatment plant of 30982 Pattensen, Germany. The test was carried out under static conditions with the test item concentrations 10 - 32 - 100 - 320 - 1000 mg/L with and without ATU. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The mean inhibition of total respiration for the test item replicates ranged from -10 % to 29 %. The mean inhibition of the heterotrophic respiration for the test item replicates ranged from -9 % to 30 %. The mean inhibition of nitrification for the test item replicates ranged from -13 % to 29 %.

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate and N-Methylaniline (with and without ATU) as reference item and the reference toxicity was determined. The EC50-value for the reference item copper (II) sulphate pentahydrate was 104 mg/L. The EC50-value for the reference item N-Methylaniline was

1.92 mg/L.

 

 

Endpoints of Reaction products of DL-methionine and C18 unsaturated fatty acid chloride and isopropanol for Inhibition of Respiration

	Total Respiration		Heterotrophic Respiration		Nitrification
	EC-values [mg/L]	CI  P = 95 [%] [mg/L]	EC-values [mg/L]	CI  P = 95 [%] [mg/L]	EC-values [mg/L]	CI  P = 95 [%] [mg/L]
NOEC	100	–	100	–	320	–
EC10	251	184 – 359	286	158 – 462	213	118 – 423
EC20	497	348 – 697	460	323 – 776	470	247 – > 1000
EC50	> 1000	–	> 1000	–	> 1000	–
EC80	> 1000	–	> 1000	–	> 1000	–

 

 

Description of key information

The NOEC of Reaction products of DL-methionine and C18 unsaturated fatty acid chloride and isopropanol for Total Respiration is 100 mg/L.
The EC10 for Total Respiration is 251 mg/L and the EC50 is >1000 mg/L.
The NOEC for heterotrophic respiration is 100 mg/L.
The EC10 for heterotrophic respiration is 286 mg/L and the EC50 is >1000 mg/L.
The NOEC for nitrification is 320 mg/L.
The EC10 for nitrification is 213 mg/L and the EC50 is >1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

251 mg/L