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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch number 8613
Manufacturing date October 25, 2018
Active substances [CAS number]* Lecithins, hydrogenated [92128-87-5]

Test item physical state Slight yellow powder
Test item quantity received at test facility 150 g
Packaging Plastic bag
Storage conditions Ambiant temperature ( 20 ± 5°C), Hydroscopic and keep away from light
Expiry date October 24, 2019
Analytical monitoring:
yes
Details on sampling:
In order to verify the nominal or initial concentrations of the test item and maintenance of the concentrations during the ecotoxicological testing, chemical analysis of the test item in algal test solutions was performed according to the Analytical protocol: Method for the chemical analysis of Carbon in samples containing the test item from algae ecotoxicology solutions by a Total Organic Carbon Analyzer and the following protocol:
- At the beginning of the test (T = 0h)
- At the end of the test (T = 72h)
- for the Control (i.e. algal test medium filtered), the highest and the lowest concentrations and a concentration around expected ErL50.
Vehicle:
no
Details on test solutions:
According to the study monitor, the test item is poorly soluble in the test medium, a special solubilization protocol was carried out before the experimentation, according to the OECD n°23 guidance document on aquatic toxicity testing of difficult substances and mixtures.
Preliminary works (non GLP) were performed to define the solution preparation protocol in order to obtain the maximum achievable dissolved Carbon concentration of the test item in the test medium. Filtration was necessary to obtain the soluble fraction of the test item. A Whatman filter 40 (porosity 8 μm) was used because filtration with lower porosity showed technical issues because of clogging.
A study of stability of the test item was carried out under the testing conditions.
The mixing protocol consisted in stirring each test item nominal concentrations in test medium during 72 hours, in the dark and 20 ± 2°C (the concentrations were prepared by directly adding the test item in flasks containing test medium).
All the test solutions plus Control (test medium) were then filtered (Whatman filter 40) to remove suspended particles: solutions obtained were named WAF (Water Accommodated Fraction).
According to the study monitor and the results of the preliminary test, this study was conducted on five concentrations levels: 10 – 32 – 102 – 328 and 1050 mg/L.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Freshwater unicellular green alga Pseudokirchneriella subcapitata of the chlorococcal order (formerly named Raphidocellis subcapitata) strain number CCAP 278.4 were used in this study.
The algal cultures are stored in the laboratory on a solid medium (LC/agar Petri plates) and in a liquid medium with regular sub-culturing (every week).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.6-23.1°C
pH:
8.1
Nominal and measured concentrations:
Nominal test item concentration (mg/L): 10, 32, 102, 328,1050
Corresponding test item weight (mg): 10.04, 32.05, 102.05, 328.04, 1050.02
Details on test conditions:
In agreement with the study monitor and according to the results of the preliminary test, we proposed to test five concentrations (spacing factor 3.2) for this study: 10 – 32 – 102 – 328 and 1050 mg/L.
A growing exponential phase inoculum of the freshwater unicellular alga Pseudokirchneriella subcapitata was added in order to have an initial concentration of 104 cells/mL at the start of the test:
- 100 mL of algal cultures was grown in 250 mL erlenmeyer flasks which were placed in a cooling incubator at a temperature in the range of 21 to 24°C controlled at ± 2°C and under a constant shaking fixed at 120 rpm and a continuous illumination comprised between 4 440 and 8 880 Lux.
The algal growth inhibition in relation to the control was evaluated over a period of 72 hours. The algal cells per unit volume were measured during the test at 24, 48 and 72 hours with a particle counter and/or under a microscope with a Malassez counting cell.
The pH of each test WAF solutions were measured at the start and at the end of the test. The pH of the Control did not increased by more than 1.5 units during the test.
The final test will contain:
 Control: 6 biotic replicates +1 Abiotic replicate
 Concentration 10 mg/L: 3 biotic replicates+1 Abiotic replicate
 Concentration 32 mg/L: 3 biotic replicates
 Concentration 102 mg/L: 3 biotic replicates
 Concentration 328 mg/l: 3 biotic replicates +1 Abiotic replicate
 Concentration 1050 mg/L: 3 biotic replicates +1 Abiotic replicate


Test duration: 72 hours
Light intensity : 7156 Lux
Test solution temperature: 22.6 °C – 23.1°C
Culturing apparatus: Cooling incubator Infors Multitron (INCU 1001)
Algae measurement: Beckman Coulter Z2 and Malassez counting cell
Dates of the test: August 30 to September 05, 2019

Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7)
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 1 050 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Validity criteria fulfilled:
yes
Conclusions:
An OECD 201 test was performed and presented the following results:
NOEL: 10 mg/L
LOEL: 32 mg/L
EL50 (0 -72h) >1050mg/L
Executive summary:

The aim of this study was to determine the effects of the test item on the growth of the unicellular green alga Pseudokirchneriella subcapitata, according to the OECD 201 guideline (March 2006, corrected July 2011).

The results of this study are summarized below:

Test system : Pseudokirchneriella subcapitata

Exposure duration : 72 hours

Test concentrations : Control - 10- 32 - 102 - 328 and 1050 mg/L

Incubator : Temperature 22.6 °C – 23.1C; shaking at 120 rpm; light intensity: 7156 Lux

Measurement of cell growth : Beckman Coulter Z2 and Malassez counting cell

Results

NOEL: 10 mg/L

LOEL: 32 mg/L

EL50 (0 -72h) >1050mg/L

In the control, the increase of the biomass during 72 hours was determined to be higher than factor of 16.

The coefficients of variation of daily section-by-section specific growth rate and of average specific growth rate were determined respectively to be less than 35% and 7% in the control.

The validity criteria were successfully fulfilled.

Description of key information

The aim of this study was to determine the effects of the test item on the growth of the unicellular green alga Pseudokirchneriella subcapitata, according to the OECD 201 guideline (March 2006, corrected July 2011).

The results of this study are summarized below:

Test system : Pseudokirchneriella subcapitata

Exposure duration : 72 hours

Test concentrations : Control - 10- 32 - 102 - 328 and 1050 mg/L

Incubator : Temperature 22.6 °C – 23.1C; shaking at 120 rpm; light intensity: 7156 Lux

Measurement of cell growth : Beckman Coulter Z2 and Malassez counting cell

Results

NOEL: 10 mg/L

LOEL: 32 mg/L

EL50 (0 -72h) >1050mg/L

In the control, the increase of the biomass during 72 hours was determined to be higher than factor of 16.

The coefficients of variation of daily section-by-section specific growth rate and of average specific growth rate were determined respectively to be less than 35% and 7% in the control.

The validity criteria were successfully fulfilled.

Key value for chemical safety assessment

EC50 for freshwater algae:
1 050 mg/L

Additional information