D-Glucitol, 1-deoxy-1-(methylamino)-, N-C8-10 acyl derivs.

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-10-31 to 2013-11-06

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

D-Glucitol, 1-deoxy-1-(methylamino)-, N-[C8-10(even numbered) acyl] derivs.

EC Number:

940-284-1

Cas Number:

1591782-62-5

Molecular formula:

C15H31NO6 C17H35NO6

IUPAC Name:

D-Glucitol, 1-deoxy-1-(methylamino)-, N-[C8-10(even numbered) acyl] derivs.

Constituent 2

Reference substance name:

Glucamide 810

IUPAC Name:

Glucamide 810

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Molecular formula (if other than submission substance): C15H31NO6 (C8-Glucamide), C17H35NO6 (C10-Glucamide)
- Molecular weight (if other than submission substance): C8-Glucamide: 321.42 g/Mol, C10-Glucamide: 349.47 g/Mol
- Physical state: Coulourless powder

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Respective amount directly weighed into each test vessel
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loading: Extended limit test to determine the NOEC: 500 - 1000 mg/L
- Controls: test medium without test or reference item; two replicates at the beginning of the test + four replicates at the end of the test

Test organisms

Test organisms (species):

activated sludge

Details on inoculum:

- Culture: Non adapted activated sludge from the sewage plant at Hildesheim (Municipal sewage treatment plant of 31137 Hildesheim, Germany)
- Pretreatment: The sludge was washed twice with chlorine free tap water. An adjustment to a dry sludge concentration of 3.0 g/L ± 10 % was not necessary (actual dry sludge concentration: 3.00 g/L).
The sludge was used within 24 h after sampling.
- Dry sludge concentration: 3.00 g/L corresponding to 1.50 g/L in the test vessel

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Hardness:

Not determined

Test temperature:

21.6 °C

pH:

pH-value of the activated sludge 7.24
pH-value of the synthetic waste water 7.31

Salinity:

Not determined

Details on test conditions:

TEST SYSTEM
- Test vessel: 1000 mL Erlenmeyer flask, DIN 12380
- Aeration: Permanent; shaking of flasks at 150 rpm
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 2 at test start, 4 at test end
- Composition of test medium:
Test item concentration: the respective amount directly weighed into each test vessel

Test item concentration [mg/L]
500 1000
Inoculum [mL] 250
Synthetic waste water [mL] 16
Demineralised water [mL] 234



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
immediately after transferring the solution to the measuring cell. The oxygen depletion was recorded for at least 3 minutes on a flat bed recorder.

TEST CONCENTRATIONS
- Range finding study:
Inhibitions were as follows:

Nominal Test Item Concentration [mg/L] pH (test item in demin. water) pH (adjusted to) pH (test item in medium) Inhibition [%]
10 6.21 - 7.47 -9
6.13 - 7.44 -10
100 6.88 - 7.41 -6
6.66 - 7.46 -9
1000 8.94 - 7.58 4
8.81 7.12* 7.48 15

*) adjusted with 0.05 mol/L HCl

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Effect concentrations 2

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

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 97.3 mg/L

Reported statistics and error estimates:

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

The EC50-value of the reference item was calculated by linear regression using software GraphPad Prism. Calculation of the confidence intervals for the EC-values was carried out using standard procedures.

Any other information on results incl. tables

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 with the oxygen uptake rates of the control.

Raw Data and Oxygen Uptake Rates of the Control, the Test Item and the Reference Item

Test / Reference Item Concentration		Repl.	Oxygen Concentration* [mg O2/L]	Measured distance** [mm/3 min]	Oxygen uptake rate [mg O2/L×h]
Control		1	5.64	64	25.6
		2	5.01	64	25.6
		3	5.54	55	22.0
		4	5.02	54	21.6
		5	5.09	56	22.4
		6	5.06	55	22.0
Test item	500	1	5.19	57	22.8
		2	5.15	54	21.6
		3	5.13	54	21.6
		4	6.24	59	23.6
		5	5.24	55	22.0
	1000	1	6.72	45	18.0
		2	7.10	47	18.8
		3	6.77	45	18.0
		4	5.02	44	17.6
		5	6.81	37	14.8
Reference item	58	1	6.02	43	17.2
		2	6.20	43	17.2
		3	5.98	43	17.2
	100	1	6.78	30	12.0
		2	6.72	28	11.2
		3	6.92	28	11.2
	180	1	7.81	15	6.0
		2	7.92	11	4.4
		3	7.86	10	4.0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of the test item C8/C10-Glucamide AC93/13 is 500 mg/L, the EC50 of the test item is > 1000 mg/L.
The test item is not toxic at the concentration of 500 mg/L to activated sludge of a municipal sewage treatment plant.

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item C8/C10-Glucamide AC93/13 (batch number: AC93/13) from2013-10-31 to 2013-11-06, with the definitive experimental phase on2013-11-06 at the Dr.U.Noack-Laboratorien, 31157Sarstedt, Germany. Test system was activated sludge of the municipal sewage treatment plant of 31137 Hildesheim, Germany. The study was carried out under static conditions with the test item concentrations of 500 and 1000 mg/L. 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 respiration for the test item replicates were 4 % and 25 % .

 

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 as reference item and the reference toxicity was determined. The EC₅₀-value for the reference item was 97.3 mg/L.

 

NOEC and EC-ValueofC8/C10-Glucamide AC93/13

		EC-value [mg/L]
NOEC*		500
EC50		> 1000

                *) No statistically significant effect (p < 0.05), ANOVA, Dunnett’s Method