Ammonium N-methyl-N-(1-oxododecyl)glycinate

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:

28 Jan 2016

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:

22 Jul 2010

Deviations:

yes

Remarks:

The temperature in the first experiment was 21.4 - 22.7 °C instead of 18.0 - 22.0 °C. This deviation was stated as uncritical, as normal respiration activity of the control could be observed.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Rheinland Pfalz, Germany

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: For the first and second experiment, a stock solution of an appropriate concentration in deionised water was prepared. Then, the stock solution was used to prepare the treatments. For the third experiment the test item, an aqueous solution was added directly with a pipette based on a density of 1.0 ± 0.1 g/mL (stated in the MSDS).
- Controls: 2 replicates before and two after measuring positive control and test item, respectively.
- Evidence of undissolved material: The solubility of the test item is sufficient.

Test organisms (species):

activated sludge

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: The sludge was taken from the activation basin of ESN (Stadtentsorgung Neustadt) sewage treatment plant in NW-Lachen-Speyerdorf, Germany.
- Preparation of inoculum for exposure: Upon arrival in the test facility, the sludge was filtered, washed with tap water three times and re-suspended in tap water.
- Pretreatment: The activated sludge was aerated until use in the test and fed daily with 50 mL synthetic sewage feed/L.
- Initial biomass concentration: 3.14 g suspended solids/L (experiment 1), 3.18 g suspended solids/L (experiment 2), 2.84 g suspended solids/L (experiment 3)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

1.03 mmol/L (dilution water)

Test temperature:

21.4 - 22.7 °C (1st experiment)
18.4 - 19.9 °C (2nd experiment)
18.1 - 20.3 °C (3rd experiment)

pH:

7.3 - 7.5 (1st experiment)
7.2 - 7.6 (2nd experiment)
7.2 - 7.7 (3rd experiment)

Conductivity:

242 µS/cm (25 °C, dilution water)

Nominal and measured concentrations:

Control, 3.4, 34, 345, and 3448, mg/L (nominal, 1st experiment)
Control, 2.2, 6.8, 22, 68, and 220 mg/L (nominal, 2nd experiment)
Control, 345, 759, 1586, 3448, and 7586 mg/L (nominal, 3rd experiment)

Details on test conditions:

TEST SYSTEM
- Test vessel: 2000 mL Schott-flasks were used as test vessels (1st experiment). Glass beakers were used as test vessels (2nd and 3rd experiments). Narrow-neck glass bottles with flat bottoms (250 mL) were used as measuring flasks.
- Type: The respiration rates of samples of activated sludge fed with synthetic sewage were mesured in an enclosed cell.
- Material, size, headspace, fill volume: Material: glass; Fill volume: 500 mL
- Aeration: Purified air, using Pasteur pipettes (2nd and 3rd experiment).
- No. of vessels per concentration (replicates): 5 replicates/treatment in all three experiments, except in the 1st experiment at the treatment levels 3.4, 34, and 345 mg/L.
- No. of vessels per control (replicates): 2 replicates
- Sludge concentration (weight of dry solids per volume): 3.14 g suspended solids/L (1st experiment), 3.18 g suspended solids/L (2nd experiment), 2.84 g suspended solids/L (3rd experiment)
- Weight of dry solids per volume of reaction mixture per unit of time: 1.57 g suspended solids/L (1st experiment), 1.59 g suspended solids/L (2nd experiment), 1.42 g suspended solids/L (3rd experiment)
- Nutrients provided for bacteria: Synthetic sewage.

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

OTHER TEST CONDITIONS
- Other: The test vessels were prepared by first adding nutrient solution and dilution water and then by adding inoculum solution in 5 minute intervals.
- Details on termination of incubation: After 3 h, the content of the 1st test vessel was poured into a 250 mL narrow-neck bottle and the respiration rate was determined by measurement of the O2-concentration over a period of max. 5 min. The following vessels were measured likewise, in 5 min intervals. The respiration rates of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode.

EFFECT PARAMETERS MEASURED:
- Oxygen consumption: Continuously during 5 min.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: Yes, 1st experiment.
- Test concentrations: 3.4, 34, 345, and 3448 mg/L
- Results used to determine the conditions for the definitive study: Yes, significant inhibition was observed in the 1st experiment.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

68 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other:

Remarks:

corresponding to 20 mg/L active ingredient ammonium lauroyl sarcosinate

Details on results:

Significant inhibition was observed in the 1st experiment.
In the second experiment´, significant inhibition was only observed in the highest treatment.
The third experiment showed a good correlation of the inhibitory effects of the test item.

Reported statistics and error estimates:

For the treatments with the test item concentrations 220 and 68 mg/L, it was tested whether the differences between treatment and control were significant. For this determination, the values of the O2 consumption were used.
EQUALITY OF VARIANCE
In order to select a suitable test for significance, it was checked whether equality of variance was given. The calculated value F is compared with the F-test table. If the calculated value is smaller than the tabular value, equality of variance is given.
If equality of variance is given, the t-test is used; else, the WEIR test its used.
T-TEST
With the t-test, it was checked whether the differences are significant. Significance is given if the calculated t-value is bigger than the limit of significance (t-value taken from the table with degree of freedom: n1 + n2 - 2, level of significance: 95%).
WEIR TEST
If the calculated value t is greater than 2.0, significance is given (error probability 5%).

EC10, EC50
For the calculation of the EC10 and EC50, the percentage inhibition was plotted versus concentration in a Gauss-logarithmic diagram. EC10 and EC50 were determined from the x values of the regression line at y = 10% and y = 50%.

BIOLOGICAL RESULTS

In the first experiment, significant inhibition was observed. In the second experiment, significant inhibition was observed only in the highest treatment. Both main tests (2nd and 3rd experiments) showed good correlation of the inhibitory effects of the test item.

For the determination of the biological results, the inhibition values which were found in the second and third experiment were evaluated (Table 1, and Table 2).

Table 1. Biological Results Test Item.

Parameter	Value	95% confidence interval
NOEC	68 mg/L	--
3 h – EC10	240 mg/L	Not determinable
3 h – EC50	2000 mg/L	1700 – 2300 mg/L

 

Table 2. Biological results based on the active ingredient of the test item (29% ammonium lauroyl sarcosinate).

Parameter	Value	95% confidence interval
NOEC	20 mg/L	--
3 h – EC10	70 mg/L	Not determinable
3 h – EC50	580 mg/L	490 – 607 mg/L

 

Validity criteria fulfilled:

yes

Description of key information

NOEC (3 h) = 68 mg/L (nominal, 20 mg/L active ingredient, OECD 209, activated sludge)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

68 mg/L

Additional information

One study investigating the toxicity to microorganism of Ammonium N-methyl-N-(1-oxododecyl)glycinate (CAS 68003-46-3) is available.

The study was carried out according to OECD 209 under GLP-conditions with non-adapted activated sludge as inoculum. In this static test three valid experiments were performed. In the first experiment, the test item was tested using four concentrations ranging fro 3.4 - 3448 mg/L (nominal), corresponding to 1 - 1000 mg/L active ingredient. As significant inhibition of respiration was observed, two main tests (2nd and 3rd experiments) were conducted.

In the second experiment, the test item was tested using five concentrations ranging from 2.2 – 220 mg/L (nominal), corresponding to 0.64 - 64 mg/L active ingredient. In the third test the test item was tested using five concentrations ranging from 345 - 7586 mg/L (nominal), corresponding to 100 - 2200 mg/L active ingredient. In the first experiment, significant inhibition was observed. In the second experiment, significant inhibition was observed only in the highest treatment. Therefore a third experiment was performed. Both main tests (2nd and 3rd experiments) showed good correlation of the inhibitory effects of the test item. For the determination of the effect concentrations, the inhibition values which were found in the second and third experiment were evaluated. A NOEC (3 h) of 68 mg/L (nominal), corresponding to 20 mg/L based on the active ingredient, was determined.