Esterification product of sunflower-oil fatty acids, with 1,4:3,6-dianhydro-D-glucitol

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:

From 7 Decembre to 10 December 2018

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

(EU) 2017/735 of 14 February 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

Adopted 28th July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Remarks:

There is no suitable analytical method to measure the test item in the OECD medium.

Details on sampling:

There was no sampling since the test item cannot be measured in the OECD medium.

Vehicle:

no

Details on test solutions:

Saturated test item solution, 100 mg/L nominal loading rate (WAF) was prepared by dispersing/dissolving the amount of test item into the test medium (OECD Medium) two days before the start of the test. This solution was stirred (slow stirring using magnetic stirrer) for about 48 hours at test temperature and then equilibrated for about 2 hours at the same temperature. Afterwards the WAF was taken from the homogeneous liquid phase (the approximate centre of the glass flask) and then this solution was filtered through a fine (0.22 µm) sterile filter. The filtered solution was used for the test.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source (laboratory, culture collection): SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY.
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines.
The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium (see OECD 201), incubated under the same conditions as the test and used when still growing exponentially, after an incubation period of three days.
- Any deformed or abnormal cells observed: When the algal cultures contain deformed or abnormal cells, they were discarded.

Test type:

static

Water media type:

freshwater

Remarks:

OECD medium

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Not reported.

Test temperature:

Between 22.4 – 22.7 °C measured in the flasks

pH:

Between 7.56 – 8.93 measured at the beginning and at the end of the test in each test vessel.
The pH of the control medium was not increased by more than 1.5 units during the test

Dissolved oxygen:

Not relevant.

Salinity:

Not relevant.

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal concentration is 100 mg/L nominal loading rate WAF.
Measured concentration : despite all efforts made to improve the analytical method, it has not been possible to measure the test item in the OECD medium. The sensitivity of the analytical method was not enough in view of the poorly solubility of the test item in this aquatic medium.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml sterile Erlenmeyer flasks covered with air-permeable stoppers
- Material, size, headspace, fill volume: 100 ml test solutions
- Aeration: continuously shaken by a laboratory orbital shaker
- Renewal rate of test solution : static
- Initial cells density: 10000 cells/ml
- Control end cells density: not reported
- No. of vessels per concentration (replicates): 6 replicates
- No. of vessels per control (replicates): 6 replicates

GROWTH MEDIUM
- Standard medium used: yes (OECD 201 medium)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuously illuminated
- Light intensity and quality: 7338 lux (equivalent to ~99 µE/m2/s), ensured with fluorescent lamps (with a spectral range of 400-700 nm). Differences in light intensity between the test vessels did not exceed ± 15 %


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: manual cell counting using a microscopic method with a counting chamber. Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: only one concentration tested (limit test) at 100 mg/L nominal loading rates WAF
- Range finding study : yes
- Test concentrations: 0.1; 1; 10 and 100 mg/L nominal loading rates WAF
- Results used to determine the conditions for the definitive study: No inhibition observed at any of the tested concentrations.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Average specific growth rate : the 0-72h average specific growth rate of the test group was not statistically significantly different from the control group (t-test; (α = 0.05);
Areas under the growth curve : the 0-72h areas of the test group was not statistically significantly different from the control group (t-test; (α = 0.05);

Cell Number (× 10⁴cell/mL) determined in the Main Experiment

 

Test group		Number of cells
		0 h	24 h	48 h	72 h
Control		1	4	21	76
		1	4	17	78
		1	3	18	70
		1	3	20	77
		1	4	21	78
		1	5	20	74
	Mean	1.00	3.83	19.50	75.50
	SD	0.0	0.8	1.6	3.1
100 mg/L nominal loading rate WAF		1	4	20	78
		1	4	19	76
		1	4	17	70
		1	5	20	68
		1	4	19	76
		1	3	17	78
	Mean	1.00	4.00	18.67	74.33
	SD	0.0	0.6	1.4	4.3

 

 

Growth Rates and Percentage Inhibition of growth rates during the Test Period

Test group	Growth ratemand % inhibition ofm
	0–24 h		0–48 h		0–72 h
	m	%	m	%	m	%
Control	0.0553^+	0.0	0.0618	0.0	0.0600	0.0
100 mg/L nominal loading rate WAF	0.0573	-3.6	0.0609	1.4	0.0598	0.4

⁺at this value the rounding of the EXCEL and TOXSTAT software was different.

The table contains the values calculated with EXCEL.

 

   Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Test group	Areas under the Growth Curves (A) and % inhibition of A
	0–24 h		0–48 h		0–72 h
	A	%	A	%	A	%
Control	34.0	0.0	290.0	0.0	1406.0	0.0
100 mg/L nominal loading rate WAF	36.0	-5.9	284.0	2.1	1376.0	2.1

 

Validity criteria fulfilled:

yes

Conclusions:

No inhibition effect on the growth rate and on the biomass of Pseudokirchneriella subcapitata was observed following 72h of exposure to 100 mg/L nominal loading rate WAF of the test item. The 72h-NOELR ≥ 100 mg/L nominal loading rate WAF.

Executive summary:

The toxicity of the substance "Esterification products of 1,4:3,6-dianhydro-D-glucitol with sunflower oil fatty acids" to Pseudokirchneriella subcapitata was determined according to the EU method C.3 and OECD 201, in compliance with the GLP.

The algae, at the initial concentration of 10000 cells/ml, were exposed during 72h to the test item at the concentration of 100 mg/L nominal loading rate WAF. Since no toxicity was observed in a preliminary range finding test, a limit test was carried out using only one concentration (6 replicates) and one control group (6 replicates).

Despite all efforts made to improve the analytical method, it has not been possible to measure the test item in the OECD medium. The sensitivity of the analytical method was not enough in view of the poorly solubility of the test item in the aquatic medium. Therefore no analytical control was performed and the results are expressed as nominal concentration.

In accordance with the OECD guidance document No. 23 on "Aquatic Toxicity Testing of Difficult Substances and Mixture", a saturated test item solution, 100 mg/L nominal loading rate (WAF), was prepared by dispersing/dissolving the amount of test item into the test medium (OECD Medium) two days before the start of the test. This solution was stirred (slow stirring using magnetic stirrer) for about 48 hours at test temperature and then equilibrated for about 2 hours at the same temperature. Afterwards the WAF was taken from the homogeneous liquid phase (the approximate centre of the glass flask) and then this solution was filtered through a fine (0.22 µm) sterile filter. The filtered solution was used for the test.

No Inhibition effect on the growth rate and on the biomass of Pseudokirchneriella subcapitata was observed following the 72h of exposure to 100 mg/L nominal loading rate WAF by comparison to the controls. So the 72h EL50 value is > 100 mg/L nominal loading rate WAF, and the 72 No-Observed Effect Loading Rate (NOELR) ≥ 100 mg/L nominal loading rate WAF. Based on these results, the substance "Esterification products of 1,4:3,6-dianhydro-D-glucitol with sunflower oil fatty acids" is considered to show no adverse effect to algae in the test conditions.

This toxicity test to Pseudokirchneriella subcapitata is acceptable, and satisfies the requirements of the EU Method C.3 and OECD 201.

Description of key information

No inhibition effect on the growth rate and on biomass of Pseudokirchneriella subcapitata was observed following 72h of exposure to 100 mg/L nominal loading rate WAF of the test item. The 72h-NOELR ≥ 100 mg/L nominal loading rate WAF.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The toxicity of the substance "Esterification products of 1,4:3,6-dianhydro-D-glucitol with sunflower oil fatty acids" to Pseudokirchneriella subcapitata was determined according to the EU method C.3 and OECD 201, in compliance with the GLP.

The algae, at the initial concentration of 10000 cells/ml, were exposed during 72h to the test item at the concentration of 100 mg/L nominal loading rate WAF. Since no toxicity was observed in a preliminary range finding test, a limit test was carried out using only one concentration (6 replicates) and one control group (6 replicates).

Despite all efforts made to improve the analytical method, it has not been possible to measure the test item in the OECD medium. The sensitivity of the analytical method was not enough in view of the poorly solubility of the test item in the aquatic medium. Therefore no analytical control was performed and the results are expressed as nominal concentration.

In accordance with the OECD guidance document No. 23 on "Aquatic Toxicity Testing of Difficult Substances and Mixture", a saturated test item solution, 100 mg/L nominal loading rate (WAF), was prepared by dispersing/dissolving the amount of test item into the test medium (OECD Medium) two days before the start of the test. This solution was stirred (slow stirring using magnetic stirrer) for about 48 hours at test temperature and then equilibrated for about 2 hours at the same temperature. Afterwards the WAF was taken from the homogeneous liquid phase (the approximate centre of the glass flask) and then this solution was filtered through a fine (0.22 µm) sterile filter. The filtered solution was used for the test.

No Inhibition effect on the growth rate and on the biomass of Pseudokirchneriella subcapitata was observed following the 72h of exposure to 100 mg/L nominal loading rate WAF by comparison to the controls. So the 72h EL50 value is > 100 mg/L nominal loading rate WAF, and the 72 No-Observed Effect Loading Rate (NOELR) ≥ 100 mg/L nominal loading rate WAF. Based on these results, the substance "Esterification products of 1,4:3,6-dianhydro-D-glucitol with sunflower oil fatty acids" is considered to show no adverse effect to algae in the test conditions.