D-Glucitol, 1-deoxy-1-(methylamino)-, N-[C18-C18 (unsaturated) acyl] derivs.

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:

2013-11-27 to 2013-12-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, no deficiencies

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:The stock solution of 12500 mg/L was stirred overnight.
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loading: 10 - 32 - 100 - 320 - 1000 - 5000 mg/L were diluted from a stock solution
- Controls: Test medium without test or reference item. Triplicates at the beginning of the test + triplicates at the end of the test

Test organisms (species):

activated sludge

Details on inoculum:

- Preparation of inoculum for exposure: 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 tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %. The sludge was used within 24 h after sampling.
- Dry sludge concentration: 2.96 g/L, corresponding to 1.48 g/L in the test vessel

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

Not determined

Test temperature:

21.5 °C

pH:

pH-value of the activated sludge 7.39
pH-value of the synthetic waste water 7.33

Salinity:

Not determined

Details on test conditions:

TEST SYSTEM
- Test vessel: 1000 mL Erlenmeyer flask
- Aeration: Shaking of flasks at 150 rpm to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension.

- 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.)
- Composition of test medium:

Table for the Test Item compositions
Test item concentration [mg/L]
10 32 100 320 1000 5000
Inoculum [mL] 250
Synthetic waste water [mL] 16
Stock solution [mg/L] 1250* 12500
Stock solution [mL] 4.0 12.8 40 128 40 200
Demineralised water [mL] Filled up to 234 mL
* Diluted from the stock solution with 12500 mg/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 immediately after transferring the solution to the measuring cell and the oxygen depletion was recorded for at least 3 minutes on a flat bed recorder.

TEST CONCENTRATIONS
Inhibition of Respiration (Preliminary Tests)

Inhibitions in the Preliminary Tests
Nominal Test
Item
Concentration
[mg/L] pH(test item in demin. water) pH (adjusted to) pH (test item in medium) Inhibition [%]
1st Preliminary test

1
(diluted from
stock
solution) - - - 11
10* - - - 11
- - - 13
100* - - - 25
- - - 27
1000* 8.70 7.30 - 25
- - - 25
1st Preliminary test with stirring over night
1 (diluted from
stock
solution) - - - 7
- - - 5
10* - - - 3
- - - 5
100* - - - 5
- - - 11
1000* - - 7.46 25
- - - 25



2nd Preliminary test with stirring overnight
1 (diluted from
stock
solution) - - - -3
10* - - - 1
- - - -3
1000* 7.98 - - 17
8.03 7.73 - 32
3rd Preliminary test diluted from a stock solution
3.2 - - - -2
- - - -1
10 - - - -7
- - - 4
32 - - - 7
- - - 6
100 - - - 18
- - - 19
4th Preliminary test *
1000* - - - 25
- - - 22
2500* 9.08 - 7.68 22
8.40 - 7.53 30
5000* 9.48 - 7.90 23
9.41 - 7.88 23
*) direct weighing

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

36.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: (22.9-59.8)

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 5 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 5 000 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: The test solutions were milky, turbid and foamed slightly.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 104 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) and DUNNETT’s test 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 EC-values of the test item and the reference item were calculated by sigmoidal dose-response regression and linear regression, respectively, using software GraphPadPrism. Calculations of the confidence intervals for the EC-values were carried out using standard procedures.

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.

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration of the Control and Test Item Concentrations

	Test Item Concentration [mg/L]	Repl.	Oxygen Uptake Rate R [mg O2/L×h]	Specific Respiration Rate Rs [mg O2/g×h]	Inhibition [%]	Mean Inhibition [%]
	Control	1	25.6	17.3	–	–
		2	26.0	17.6
		3	26.0	17.6
		4	25.2	17.0
		5	24.0	16.2
		6	24.0	16.2
Test Item	10	1	28.0		-12	2
		2	24.4		3
		3	24.4		3
		4	24.4		3
		5	22.4		11
	32	1	23.6		6	9
		2	22.4		11
		3	23.2		8
		4	22.8		9
		5	22.8		9
	100	1	18.4		27	21
		2	19.2		24
		3	20.0		20
		4	21.2		16
		5	20.4		19
	320	1	20.4		19	20
		2	20.4		19
		3	20.0		20
		4	19.6		22
		5	20.0		20
	1000	1	21.6		14	13
		2	21.6		14
		3	22.4		11
		4	22.0		12
		5	21.6		14
	5000	1	22.4		11	12
		2	23.2		8
		3	22.0		12
		4	21.2		16
		5	21.6		14

Repl. = Replicate

            Mean specific respiration rate of the control: 17.0 mg O₂/g×h

Coefficient of variation of oxygen uptake rates of the control: 3.7%

Suspended solids in the test vessel: 1.48 g/L

Oxygen Uptake Rates and Inhibition of the Respiration of the Reference Item Concentrations

	Reference Item Concentration [mg/L]	Repl.	Oxygen Uptake Rate R [mg O2/L×h]		Inhibition   [%]	Mean Inhibition [%]
Reference Item	58	1	21.2		16	21
		2	19.6		22
		3	18.8		25
	100	1	12.8		49	50
		2	12.4		51
		3	12.4		51
	180	1	6.0		76	75
		2	6.4		75
		3	6.4		75

 

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of C18/C18 unsatd.-Glucamide is 10 mg/L, which corresponds to 8.04 mg/L active components Glucamides.
The EC10 is 36.8 mg/L, which corresponds to 29.6 mg/L active components Glucamides.
The EC50 is > 5000 mg/L, corresponding to 4023 mg/L active components Glucamides.

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item C18/C18 unsatd.-Glucamide (batch number: SN 119/13/3-8) from 2013-11-27 to 2013-12-12, with the definitive exposure on 2013-12-12 at the Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany. Test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the test item concentrations 10 - 32 - 100 - 320 - 1000 - 5000 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.

 

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 104 mg/L.

 

The mean inhibition of respiration for the test item replicates ranged from 2 % to 21 % (Table 5). A concentration effect relationship was determined only for thethree lowest test item concentrations. Therefore, only the EC₁₀was determinable.No inhibition > 21 % was determined up to 5000 mg/L, as a result theEC₅₀and EC₈₀are > 5000 mg/L.

NOEC and EC-Values with Confidence Interval ofC18/C18 unsatd.-Glucamide

		EC-values [mg/L]		Confidence interval P = 95 [%] [mg/L]
NOEC*		10		–
EC10		36.8		22.9 – 59.8
EC20		n.d		n.d
EC50		> 5000		–
EC80		> 5000		–

*) No statistically significant inhibition (p < 0.05), ANOVA, Dunnett`s Method

n.d. = not determinable

 

 

The NOEC of C18/C18 unsatd.-Glucamideis 10mg/L, which corresponds to 8.04 mg/L active components Glucamides. The EC10is 36.8 mg/L, which corresponds to 29.6 mg/L active components Glucamides. The EC50is > 5000 mg/L, corresponding to 4023 mg/L active components Glucamides.

Description of key information

The NOEC of C18/C18 unsatd.-Glucamide is 10 mg/L, which corresponds to 8.04 mg/L active components Glucamides.

The EC10 is 36.8 mg/L, which corresponds to 29.6 mg/L active components Glucamides.

The EC50 is > 5000 mg/L, corresponding to 4023 mg/L active components Glucamides.

Key value for chemical safety assessment

EC50 for microorganisms:

5 000 mg/L

EC10 or NOEC for microorganisms:

36.8 mg/L