Reaction mass of 2-hydroxyethyl laurate and ethylene dilaurate

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:

24 Sept - 05 Oct 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 23. Mar. 2006, Annex 5 corrected: 28 July 2011

Deviations:

yes

Remarks:

see remarks in section "Any other information on material and methods incl tables"

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

adopted 07. December 2015

Deviations:

yes

Remarks:

see remarks in section "Any other information on material and methods incl tables"

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and all test item concentrations
- Sampling method: Samples were measured at the start and at the end of the test.
- Sample storage conditions before analysis: Samples were stored at - 20 ± 5°C.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Differential loading: The water-accommodated fraction (WAF) was prepared for the test. This was done by weighing the nominal load of 32.0 mg/L, adding the corresponding amount of algal medium and shaking for 23 h The resulting solution was filtrated through 0.45 μm PTFE filters. After filtration, the previously slightly turbid solution was clear. The lower treatments were prepared by dilution of this stock solution with algal medium.
- Controls: medium without test item

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: 86.81
- Source (laboratory, culture collection): The culture was obtained in January 2016 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen).
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions (same as test or not): same

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.0 – 23.9 °C

pH:

7.6 - 8.9 (Control)
7.6 - 9.0 (Test item concentrations)

Nominal and measured concentrations:

Control, 0.32, 1, 3.2, 10 and 32 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open (covered with perforated plastic foil acting as a stopper)
- Material, size, headspace, fill volume: glass vessels 65 mL total volume were filled with 45 ± 1 mL of the test solution
- Initial cells density: 2.3 *10E+03 cells/mL
- Control end cells density: 461.4 *10E+03 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium was used according to OECD guideline 201
- Culture medium different from test medium: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continous illumination
- Light intensity and quality: 5000 lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cell number was determined with an electronic particle counter after 0, 24, 48 and 72 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: Test item concentrations to be tested are based on non GLP pre-tests. No details given in study report.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

243.11 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval

Remarks:

232.06 – 254.93 μg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

176.72 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval

Remarks:

169.27 – 184.49 μg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

92.47 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: At 0.32 mg/L algae cells partly agglomerated and sedimented; at 32 mg/L no cells were visible
- Any stimulation of growth found in any treatment: growth was stimulated at 3.2 mg/L

Results with reference substance (positive control):

ErC50 (72 h): 0.65 mg/L

Table 1: Inhibition Values 

Nominal concentration	Geometric Mean Measured Concentration	% Inhibition
[mg/L]	[μg/L]	Growth Rate (0-72h)	Yield (0-72h)
Blank control	< LOD	0	0
0.32 1)	< LOD	7.12 1)	31.07 1)
1	60.1	0.3	1.7
3.2	92.5	-0.1	-0.4
10	185.4	13.8*	52.4
32	302.7	81.1*	99.1

Negative inhibition values indicate a stimulation of algal growth compared to the blank control.

1 At the lowest concentration 0.32 mg/L the cells were partly agglomerated and sedimented. Therefore, determination of the cell number might be biased. This effect was not caused by the test item. Because in the next two higher concentrations no inhibition of algal growth was observed, the lowest concentration was excluded from evaluation.

* statistically significant compared to the control

Table 2: Cell Number/mL

Nominal Concentration	Geometric Mean Measured Concentration	Parameter	Cell Number/mL
[mg/L]	[μg/L]		0 h	24 h	48 h	72 h
Blank control	< LOD	Mean	2260	11070	63713	461350
Blank control	< LOD	SD	0	220	3013	37739
0.32 1	< LOD	Mean	2260	10013	45853	318720
0.32 1	< LOD	SD	0	230	5715	58341
1	60.1	Mean	2260	10820	60020	453333
1	60.1	SD	0	819	2961	21671
3.2	92.5	Mean	2260	10040	60307	463360
3.2	92.5	SD	0	364	2230	21326
10	185.4	Mean	2260	6727	29753	220667
10	185.4	SD	0	393	5360	7171
32	302.7	Mean	2260	2993	2920	6413
32	302.7	SD	0 81	191	2127

SD = Standard Deviation, LOD = Limit of detection; 1 At the lowest concentration 0.32 mg/L the cells were partly agglomerated and sedimented. Therefore, determination of the cell number might be biased. This effect was not caused by the test item. Because in the next two higher concentrations no inhibition of algal growth was observed, the lowest concentration was excluded from evaluation.

Table 3: Analytical Results: Calculated Test item Concentration (based on 33.3% component 1) and Geometric Mean 0.06 mg/L

Nominal Concentration Test Item	Calculated Concentration Test item t = 0 h	Calculated Concentration Test item t = 72 h	Geometric Mean
mg/L	μg/L	μg/L	μg/L
1	60.2	60	60.1
3.2	142.5	60	92.5
10	572.6	60	185.4
32	1526.8	60	302.7

LOD (Limit of detection) = 0.06 mg/L (33.3 % purity);

Note: All calculations are performed with unrounded values. Therefore, re-calculation with rounded values may lead to slightly different results

Table 4: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	204	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	11%	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	2%	Yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”

Description of key information

ErC50 (72 h): 243.11 µg/L (OECD 201, Desmodesmus subspicatus, based on geometric mean measured concentrations)

ErC10 (72 h): 176.72 µg/L (OECD 201, Desmodesmus subspicatus, based on geometric mean measured concentrations)

Key value for chemical safety assessment

EC50 for freshwater algae:

243.11 µg/L

EC10 or NOEC for freshwater algae:

176.72 µg/L

Additional information

The toxicity of the test substance to aquatic algae was investigated according to OECD guideline 201 under GLP conditions including analytical measurement. In this static test, Desmodesmus subspicatus was exposed to five test concentrations (0.32, 1.0, 2.2, 4.6, 10, 22 and 46 mg/L). Due to the low water solubility the test item concentrations were prepared via water-accommodated fractions. This was done by weighing the nominal load of 32.0 mg/L, adding the corresponding amount of algal medium and shaking vigorously for 23 hours. The resulting solution was filtrated through 0.45 μm PTFE filters. The lower treatments were prepared by dilution of this stock solution with algal medium.

Analytical measurement was done by GC/MSD. The test item consists of two components, ethylene glycol monolaurate (component 1) and ethylene glycol dilaurate (component 2), but only one (component 1) is water soluble. The quantification of the test item was performed using the signal of component 1 only. Since the test item was not stable under test conditions, test item could be measured only at the beginning of the test. After 72 hours incubation all measured values were below the LOD.Therefore, the biological results based on the geometric mean of the measured concentrations at the beginning and the LOD (limit of detection).

In the study report an EC50 (72 h) of 0.243.11 mg/L for growth rate was reported based on the geometric mean measured concentrations and an EC10 of 0.1767 mg/L for growth rate. The NOEC (72 h) was reported as 0.09247 mg/L also based on the geometric mean measured concentrations.

According to the Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7b (2017) the EC10 value is preferred instead of a NOEL as it is statistically derived from the entire dataset. Therefore the EC10 value was used as chronic toxicity endpoint.