D-Glucitol, 1-deoxy-1-(dimethylamino)-

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:

2016-02-22 to 2016-02-25

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
Analytical evaluation of the limit concentration of Genamin DMG 75 and the control was carried out via LC-MS/MS after 0 (start of the exposure) and 72 hours (end of the exposure) with algae.

Vehicle:

no

Details on test solutions:

Stock solution, preparation of the test media
A stock solution with a concentration of 100 mg/L was freshly prepared with dilution water and agitated until the test item was completely dissolved.

Test concentrations
A limit concentration level of 100 mg/L was tested.

Control
Six replicates (without test item) were tested under the same conditions as the test concentration.

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:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

not specified

Test temperature:

min.: 21.0 max.: 22.0, mean value: 21.5 °C

pH:

Nominal test item concentration
[mg/L] pH-value
Start; 0 hours End; 72 hours
100 8.76 8.47
Control 7.96 8.43

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

See window "Any other information on materials and methods incl. tables"

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 10^3 cells/mL, Actual: mean 4847 cells/mL
- Control end cells density: Mean 756300 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:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
Na2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 2 H2O 1 x 10^-5
NaHCO3 50
pH 8.1 ± 0.2



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: 70.5 µE x m^-2 x s^-1,



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.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

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):

The toxicity of potassium dichromate (SIGMA, batch number MKBQ9179V, purity 100.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Desmodesmus subspicatus was determined over a period of 72 hours from 2015-10-19 to 2015-10-22. For results see Table 10. The reference item toxicity is in the valid range following test facility SOPS.

EC50-Values of the Reference Item based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate Inhibition
ErC50 0.685 0.671 ± 0.401
95 % confidence interval 0.603 - 0.794
Yield Inhibition
EyC50 0.362 0.320 ± 0.139
95 % confidence interval 0.328 - 0.398
SD = Standard deviation

Reported statistics and error estimates:

EC-values and statistical analyses
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- and LOEC-values
The NOEC / LOEC was determined by calculation of statistical significance of biomass integrals, growth rate and yield. The following statistical tests were conducted: Shapiro-Wilk’s test on normal distribution was done with a significance level of 0.01. Levene’s test on variance homogeneity was done with a significance level of 0.01. Two-sample t-test was done with a significance level of 0.05.

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
• Excel, MICROSOFT CORPORATION
•SigmaPlot, SPSS INC.
•ToxRat Pro Version 3.2.1

Cell Densities

 

Nominal test item concentration [mg/L]	Replicate	Cell density [cells/mL]
	No.	0 hours	24 hours	48 hours	72 hours
100	1	4847	21029	131060	790526
	2	4847	25633	142947	855661
	3	4847	28072	127062	787977
	4	4847	24964	133446	747232
	5	4847	23699	143173	814194
	6	4847	24291	127697	812895
	Mean	4847	24615	134231	801414
Control	1	4847	20490	142071	873075
	2	4847	22078	128376	632886
	3	4847	26807	123690	690035
	4	4847	29606	129535	690949
	5	4847	25094	135101	819919
	6	4847	35769	136770	830935
	Mean	4847	26641	132591	756300

         

 

 

 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 [mg/L]	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		1.70	-1		785679	-5
	2		1.73	-3		850814	-13
	3		1.70	-1		783130	-4
	4		1.68	0		742385	1
	5		1.71	-2		809347	-8
	6		1.71	-2		808048	-8
	Mean	(-)	1.70	-1	(-)	796567	-6
Control	1		1.73			868228
	2		1.62			628039
	3		1.65			685188
	4		1.65			686102
	5		1.71			815072
	6		1.72			826088
	Mean		1.68			751453

 

 

Section-by-Section and Average Specific Growth Ratesof 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	1.44	1.94	1.82	1.73		0.258	14.9	10.6
	2	1.52	1.76	1.60	1.62		0.125	7.67
	3	1.71	1.53	1.72	1.65		0.107	6.49
	4	1.81	1.48	1.67	1.65		0.168	10.1
	5	1.64	1.68	1.80	1.71		0.0828	4.84
	6	2.00	1.34	1.80	1.72		0.338	19.7
				Mean	1.68
				SD ±	0.04
				CV [%]	2.57

CV= Coefficient of variation

SD= Standard deviation

Validity criteria fulfilled:

yes

Conclusions:

In this study, Genamin DMG 75 was found not to inhibit the growth rate of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-value of the test item for both inhibition of growth rate and inhibition of yield after 72 hours was 100 mg/L. The EC50-values of the test item for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were both > 100 mg/L, respectively. All effect levels are given based on the nominal test item concnetartion of Genamin DMG 75.

Executive summary:

The toxicity of Genamin DMG 75  to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2011) and Council Regulation (EC) No.761/2009 Method C.3 (2009) at the test facility from
2016-02-22 to 2016-02-25.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 4847 cells/mL. A stock solution with a nominal concentration of 100 mg/L was tested. Sixr eplicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of the test item Genamin DMG 75 in the limit concentration and the control were analytically verified via LC/MS-MS at the start and the end of exposure. The measured concentration of the test item at the start of the exposure (0 hours) was 106 % of the nominal value at the tested limit concentration. The measured concentration in the old media (72 h) was 109 % of the nominal value.

All effect values of Genamin DMG 75 are given based on the nominal concentration.

 

NOEC, LOEC, EC_x-values of Genamin DMG 75 (0-72 hours)

              based on the nominal test item concentrations [mg/L]

 

	Growth Rate Inhibition
NOEC	100
LOEC	> 100
ErC10	> 100
ErC20	> 100
ErC50	> 100
	Inhibition of Yield
NOEC	100
LOEC	> 100
EyC10	> 100
EyC20	> 100
EyC50	> 100

 

Description of key information

In this study, Genamin DMG 75  was found not to inhibit the growth rate of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-value of the test item for both inhibition of growth rate and inhibition of yield after 72 hours was 100 mg/L. The EC50-values of the test item for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were both > 100 mg/L, respectively. All effect levels are given based on the nominal test item concnetartion of Genamin DMG 75.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L