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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-11-12 to 2013-12-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, no deficiencies

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 5.00 - 10.0 - 20.0 - 40.0 - 80.0 mg/L, factor 2


- Sampling method: All test item concentrations, the pH-control and the control were analytically verified at the start of the exposure (0 hours) and at the end of the exposure (72 hours). 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 and diluted samples were stored at room temperature until sample preparation and until analysis, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:A stock solution of 80.0 mg/L was prepared with dilution water.Agitation.
- Eluate: Dilution water
- Differential loading: 5.00 - 10.0 - 20.0 - 40.0 - 80.0 mg/L, factor 2
- Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates.

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): 3 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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Dilution water: 0.24 mmol Ca+mg/L.

Test temperature:

min.: 23.0 max.: 24.0, mean value: 23.5

pH:

Environmental Conditions

Nominal Concentration of the pH-value
Test item Main component
C18/C18 (sum of glucamides)
unsatd.- [mg/L]
Glucamide
[mg/L]
Start; 0 hours End; 72 hours
80.0 64.3 8.13 8.21
40.0 32.2 8.12 8.24
20.0 16.1 8.14 8.28
10.0 8.04 8.17 8.30
5.00 4.02 8.20 8.38
Control 8.24 8.59

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: 1473744 cells/mL
- No. of vessels per concentration (replicates): 3
- 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: 68.0



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 [%]
40.0 4 19
20.0 3 17
10.0 3 14

*) Concentration related slight turbidity was observed in all concentration levels

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 80 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 80 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

80 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: highest tested conc.

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:

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.

EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 hours could not be calculated due to the results.

Cell Densities

Nominal Concentration of the		Replicate No.	Cell density [cells/mL]
Test item C18/C18 unsatd.- Glucamide [mg/L]	Main component (sum of glucamides) [mg/L]		0 hours	24 hours	48 hours	72 hours
80.0	64.3	1	4864	26088	229198	1463192
		2	4864	22040	199983	1366664
		3	4864	22372	204964	1312125
		Mean	4864	23500	211382	1380660
40.0	32.2	1	4864	21393	186683	1182808
		2	4864	22129	198511	1125574
		3	4864	22349	204110	1371724
		Mean	4864	21957	196435	1226702
20.0	16.1	1	4864	20930	174974	1156524
		2	4864	21452	183529	1333612
		3	4864	25116	225334	1386509
		Mean	4864	22499	194612	1292215
10.0	8.04	1	4864	23243	184173	1164475
		2	4864	24012	223494	1316922
		3	4864	29121	217423	1544541
		Mean	4864	25459	208363	1341979
5.00	4.02	1	4864	23907	181361	1191120
		2	4864	24071	186499	1022804
		3	4864	22776	228580	1389991
		Mean	4864	23585	198813	1201305
Control		1	4864	35330	327224	1527588
		2	4864	28529	317302	1464572
		3	4864	27301	255889	1587318
		4	4864	24183	315081	1287089
		5	4864	29906	334176	1625102
		6	4864	32370	325489	1350795
		Mean	4864	29603	312527	1473744

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

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

Nominal Concentration of the		Replicate	Growth rate		Growth rate inhibition	Yield		Inhibition of yield
Test item C18/C18 unsatd.- Glucamide [mg/L]	Main component (sum of glucamides) [mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
80.0	64.3	1		1.90	0		1458328	1
		2		1.88	1		1361800	7
		3		1.87	2		1307261	11
		Mean	(-)	1.88	1	(-)	1375796	6
40.0	32.2	1		1.83	4		1177944	20
		2		1.81	5		1120710	24
		3		1.88	1		1366860	7
		Mean	(-)	1.84	3	(-)	1221838	17
20.0	16.1	1		1.82	4		1151660	22
		2		1.87	2		1328748	10
		3		1.88	1		1381645	6
		Mean	(-)	1.86	2	(-)	1287351	12
10.0	8.04	1		1.83	4		1159611	21
		2		1.87	2		1312058	11
		3		1.92	-1		1539677	-5
		Mean	(-)	1.87	2	(-)	1337115	9
5.00	4.02	1		1.83	4		1186256	19
		2		1.78	6		1017940	31
		3		1.89	1		1385127	6
		Mean	(-)	1.83	4	(-)	1196441	19
Control		1		1.92			1522724
		2		1.90			1459708
		3		1.93			1582454
		4		1.86			1282225
		5		1.94			1620238
		6		1.88			1345931
		Mean		1.90			1468880

Validity criteria fulfilled:

yes

Conclusions:

In this study no inhibition on growth of the freshwater green alga Desmodesmus subspicatus was found after 72 hours exposure to the test item C18/C18 unsatd.-Glucamide. The NOEC for inhibition of specific growth rate and yield after 72 hours was 80.0 mg test item/L, corresponding to 64.3 mg main component (sum of glucamides)/L.
All effect levels are based on the nominal concentration of C18/C18 unsatd.-Glucamide and the main component (sum of glucamides).

Executive summary:

The toxicity of C18/C18 unsatd.-Glucamide (batch number:SN 119/13/3-8) 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 2013-11-12 to 2013-12-19 with the definitive exposure phase from 2013-12-16 to 2013-12-19.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 range finding test five concentration levels weretested in a geometrical series with a dilution factor of 2 (nominal):5.00 - 10.0 - 20.0 - 40.0 - 80.0 mg/L.Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of the unsatd. C18 fraction of the test itemC18/C18 unsatd.-Glucamide were analytically verified by LC-MS/MS at the start and the end of the exposure of all test concentrations and the control.
The measured concentrations of the unsatd. C18 fraction of the test item C18/C18 unsatd.-Glucamideat the start of the exposure (0 hours) were in the range of 101 to 108 % of the nominal values. The measured concentrations of the unsatd. C18 fraction of thet est item C18/C18 unsatd.-Glucamide at the end of the exposure (72 hours) were 72 to 103 % of the nominal value. The three top concentrations were in the range of 99 to 103 % of the nominal values. In the two lower concentration levels the measured concentrations slightly decreased. The measured concentrations of the unsatd. C18 fraction of the test item C18/C18 unsatd.-Glucamide in aqueous phase and at the glass wall of the test vessels at the end of the exposure (72 hours) were 86 – 107 % and 0 – 2 %, respectively.

All effect values given are based on the nominal test item and main component (sum of glucamide) concentrations ofC18/C18 unsatd.-Glucamide.

NOEC, LOEC, EC - values and 95 % Confidence Intervals of C18/C18 unsatd.-Glucamide (0-72 hours)

                   based on nominal test item and main component concentrations [mg/L]

Endpoints	Nominal concentration of the
	Test item C18/C18 unsatd.- Glucamide [mg/L]	Main component (sum of glucamides 80.4 % w/w) [mg/L]
	Growth Rate Inhibition
NOEC	80.0	64.3
LOEC	> 80.0	> 64.3
ErC10	> 80.0	> 64.3
ErC20	> 80.0	> 64.3
ErC50	> 80.0	> 64.3
	Yield Inhibition
NOEC	80.0	64.3
LOEC	> 80.0	> 64.3
EyC10	> 80.0	> 64.3
EyC20	> 80.0	> 64.3
EyC50	> 80.0	> 64.3

Description of key information

In this study no inhibition on growth of the freshwater green alga Desmodesmus subspicatus was found after 72 hours exposure to the test item C18/C18 unsatd.-Glucamide. The NOEC for inhibition of specific growth rate and yield after 72 hours was 80.0 mg test item/L, corresponding to 64.3 mg main component (sum of glucamides)/L. All effect levels are based on the nominal concentration of C18/C18 unsatd.-Glucamide and the main component (sum of glucamides).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

80 mg/L