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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-10-08 to 2013-10-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study performed under GLP, no deficiencies

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 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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

Details on test material:

- Physical state: Colourless powder
- Sum formula: C15H31NO6 (C8-Glucamide) C17H35NO6 (C10-Glucamide)
- Molecular weight:
C8-Glucamide: 321.42 g/Mol
C10-Glucamide 349.47 g/Mol
- Storage condition of test material: Room temperature, protected from light, in original container.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L
- Sampling method:The concentrations of the test item were analytically determined from the limit concentration and the control at the start of the exposure (0 h) and at the end of the exposure (72 h). Samples at the start of the exposure were taken from an additional replicate without algae. At the end of the exposure samples were taken directly from the pooled test replicates.

- Sample storage conditions before analysis: All original samples were stored at room temperature until sample preparation. Prepared samples were stored in an autosampler at room temperature until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:A stock solution of 100 mg/L was freshly prepared with dilution water. Agitation
- Eluate: Dilution water
- Differential loading: Limit test concentration of 100 mg/L
- Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Hardness:

Dilution water: 0.24 mmol Ca+mg/L.

Test temperature:

min.: 22.5 max.: 23.5, mean value: 23.0

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
100 7.91 8.81
Control 7.90 9.02

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 103 cells/mL, Actual: mean 4864 cells/mL
- Control end cells density: 1634634 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6



GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1, mean value: 72.7



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.



TEST CONCENTRATIONS
- Range finding study:
Results of the Range Finding Test (0-72 hours)
Nominal Test Item Concentration
[mg/L] Growth Rate Inhibition [%] Yield Inhibition [%]
100 -5 -27
10.0 -3 -16
1.00 -3 -17
0.100 1 4

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: highest concentration of test item used in the test

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes

Current Study Valid Range (average ± 3 x SD)
Growth rate inhibition
ErC50 0.805 0.684 ± 0.436
95 % confidence interval 0.733 - 0.867
Yield inhibition
EyC50 0.334 0.314 ± 0.135
95 % confidence interval 0.296 - 0.378
SD = Standard deviation

Reported statistics and error estimates:

EC-values of the growth rate and yield inhibition were estimated empirically based on the results of the only treatment level (limit test design).


NOEC/LOEC were determined by calculation of statistical significance of growth rate and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

Any other information on results incl. tables

Cell Densities

	Nominal Test Item Concentration		Replicate		Cell Density [cells/mL]
[mg/L]		No.		0 hours		24 hours	48 hours	72 hours
100		1		4864		47161	440810	1877060
		2		4864		42693	423410	1760425
		3		4864		48321	438458	1769493
		4		4864		42332	404696	1633013
		5		4864		44894	372209	1739923
		6		4864		48784	451981	1941259
		Mean		4864		45698	421927	1786862
Control		1		4864		38678	336975	1607321
		2		4864		25333	319365	1505404
		3		4864		33678	299757	1575123
		4		4864		38560	341720	1566580
		5		4864		30304	336174	1744786
		6		4864		44658	395431	1808590
		Mean		4864		35202	338237	1634634

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

Nominal Test Item Concentration	Replicate	Growth Rate		Growth Rate Inhibition	Yield		Yield Inhibition
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		1.99	-2		1872196	-15
	2		1.96	-1		1755561	-8
	3		1.97	-1		1764629	-8
	4		1.94	0		1628149	0
	5		1.96	-1		1735059	-6
	6		2.00	-3		1936395	-19
	Mean	(+)^1	1.97	-2	(+)^1	1781998	-9
Control	1		1.93			1602457
	2		1.91			1500540
	3		1.93			1570259
	4		1.93			1561716
	5		1.96			1739922
	6		1.97			1803726
	Mean		1.94			1629770

Section-by-Section and Average Specific Growth Rates of the Control Group (0 - 72 hours)

	Replicate No.	Specific Growth Rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		Section-by-Section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	2.07	2.17	1.56	1.93	0.325	16.8	19.5
	2	1.65	2.53	1.55	1.91	0.542	28.3
	3	1.94	2.19	1.66	1.93	0.264	13.7
	4	2.07	2.18	1.52	1.92	0.353	18.3
	5	1.83	2.41	1.65	1.96	0.397	20.2
	6	2.22	2.18	1.52	1.97	0.392	19.9
				Mean	1.94
				SD ±	0.02
				CV [%]	1.21

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study no inhibition on the growth of the freshwater green alga Desmodesmus subspicatus was found after 72 hours exposure to the test item C8/C10-Glucamide AC93/13. The NOEC for inhibition of specific growth rate and yield after 72 hours was 100 mg/L.
C8/C10-Glucamide AC93/13 caused a slight stimulation on the algae growth. All effect levels are based on the nominal concentration of C8/C10-Glucamide AC93/13.

Executive summary:

The toxicity of C8/C10-Glucamide AC93/13 (batch number: AC93/13) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2011) at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany from 2013-10-08 to 2013-10-28 with the definitive exposure phase from 2013-10-22 to 2013-10-25. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 4864 cells/mL.Based on a preliminary test a limit test with one concentration level of 100 mg/L was carried out.Six replicates were tested for the limit concentration and for the control.Environmental conditionswere determined to be within the acceptable limits.

The concentrations ofthetest item C8/C10-Glucamide AC93/13 were analytically verified by LC-MS/MS at the start and the end of the exposure of the limit concentration and the control.
The measured concentration of the test item at the start of the exposure (0 h) was 109 % of the fraction C8-Glucamid and 107 % of the fraction C10-Glucamid of the nominal value. The measured concentrations of the test item at the end of the exposure (72 h) was 93 % of the fraction C8-Glucamid and 100 % of the fraction C10-Glucamid of the nominal value.

Therefore, all effect values given are based on the nominal test item concentration ofC8/C10-Glucamide AC93/13.

NOEC, LOEC, EC - values of C8/C10-Glucamide AC93/13 (0-72 hours)

                   based on nominal test item concentrations [mg/L]

	Growth Rate Inhibition Nominal test item concentration [mg/L]
NOEC	100
LOEC	> 100
ErC10	> 100
ErC20	> 100
ErC50	> 100
	Inhibition of Yield Nominal test item concentration [mg/L]
NOEC	100
LOEC	> 100
EyC10	> 100
EyC20	> 100
EyC50	> 100