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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:
2018-08-29 to 2019-05-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
adopted March 2006, corrected July 28, 2011
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material:
AH17.001
- Expiration date of the lot/batch:
2019-06-30
- Aggregate state at room temperature: Liquid
- Color: Brown-Orange
- Storage conditions at Test Facility: 4°C +/- 4°C
Analytical monitoring:
yes
Details on sampling:
Sample Preparation Procedure
- The samples were allowed to thaw to room temperature.
- They were then shaken well and treated with ultrasound for 1 minute to obtain homogenous samples
- The samples were already diluted with acetonitrile by factor 2 directly after sampling. The samples were diluted further with acetonitrile / test water (1/1, v/v) to match the calibration range, if necessary.
- The samples were centrifuged (13,000 rpm, 3 minutes) before analysis to remove the algae. The samples were filtered (0.2 µm cellulose acetate/0.45 µm PTFE) before analysis.

Fortified samples and analytical blank control samples:
- The samples were shaken well and were diluted with acetonitrile by factor 2. They were diluted further with acetonitrile / test water (1/1, v/v) to match the calibration range, if necessary. The samples were centrifuged (13,000 rpm, 3 minutes) before analysis.
- The samples were filtered (0.2 µm cellulose acetate/0.45 µm PTFE) before analysis.
Vehicle:
no
Details on test solutions:
Test Concentrations: 10, 5, 2.5, 1.25 and 0.63 mg test item/L (spacing factor 2) nominal loading rate and a control.

Dosage of Test Item:
- A defined amount of the test item was added directly to the test water for each test concentration and was carefully stirred for 24 hours in the dark to dissolve as much of the test item as possible.
- The highest test item concentration of 10 mg test item/L was prepared by mixing 10.98 µL test item into 1050 mL test water, for the test item concentration of 5 mg test item/L, 12.29 µL test item were mixed into 2350 mL test water, for the concentration of 2.5 mg test item/L, 6.15 µL were mixed into 2350 mL test water.
- The concentration of 1.25 mg test item/L was prepared by mixing 7.45 µL into 5700 mL test. After cessation of mixing and a following period (0.5 hour) of settling to allow phase separation, the aqueous phase, i.e. the water accommodated fraction, was drawn off carefully and used as the test medium of the corresponding nominal test concentration. Each concentration was prepared separately, except the lowest concentration of 0.63 mg test item/L.
- Adequate volumes of the water accommodated fraction of 1.25 mg test item/L (302.4 mL) were mixed into 600 mL test water to obtain the desired dilutions of nominal 0.63 mg test item/L.
- The test media were prepared just before introduction of the algae (= start of the test).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (KORSHIKOV)
- Strain: 61.81 SAG
- Source (laboratory, culture collection): originally supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzen-wissenschaften, Universität Göttingen", 37073 Göttingen, Germany
- Method of cultivation: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
no
Hardness:
Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3.
Test temperature:
22.7 to 23.6 °C
pH:
pH values in the test item treatments at test start: 8.2,
pH values in the test item treatments at test end: 9.2 to 9.6;
Nominal and measured concentrations:
nominal loading rate: 10, 5, 2.5, 1.25 and 0.63 mg test item/L (spacing factor 2)
Details on test conditions:
Water Temperature:
The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
Water temperature was: 22.7 to 23.6 °C

Light Regime: Continuous illumination
Light Intensity: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media.
Mean light intensity: 4988 lux (range: 4460 to 5530 lux)

1.1 Test Water
Reconstituted Water (OECD Medium):

Analytical grade salts were added at the following nominal concentrations in deionised water:
Macro-nutrients:
NaHCO3 50.0 mg/L
CaCl2 * 2 H2O 18.0 mg/L
NH4Cl 15.0 mg/L
MgSO4 * 7 H2O 15.0 mg/L
MgCl2 * 6 H2O 12.0 mg/L
KH2PO4 1.6 mg/L

Trace Elements:
Na2EDTA * 2 H2O 100.0 µg/L
FeCl3 * 6 H2O 64.0 µg/L
MnCl2 * 4 H2O 415.0 µg/L
H3BO3 185.0 µg/L
Na2MoO4 * 2 H2O 7.0 µg/L
ZnCl2 3.0 µg/L
CoCl2 * 6 H2O 1.5 µg/L
CuCl2 * 2 H2O 0.01 µg/L

- The test media was enriched with 300 mg NaHCO3 to enable algae growth in closed vessels. Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3.
- The culture medium was prepared 1-2 days before test start to allow pH to stabilise.
- The stock solutions and the test water were sterilized by membrane filtration (mean pore size 0.2 µm or by autoclaving (120 °C, 15 min.) before addition of the test item.


Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
4.42 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.85 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Aggregation of algal cells: no
Reported statistics and error estimates:
Based on the calculated cell densities, the 72 hour ErC50 and the 72 hour EyC50, the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by Probit analysis.

For the determination of the 72 hour LOEC and the 72 hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Williams t-test (yield and growth rate).
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

The test is valid according to OECD Test Guideline 201 (23 March 2006). The initial measured test item concentrations in the test item treatments were 10, 5, 2.5, 1.25 and 0.63 mg test item/L as nominal loading rates.

Table 1: Biological results

 Parameter  Yield [mg test item/L]  Growth rate [mg test item/L]

 72 -hour EC50

95 % conf. interval

 > 10

n.d.

n.d.

n.d.

72 -hour EC20

95 % conf. interval

 4.42

2.99 - 6.54

n.d.

n.d.

 72 -hour EC10

95 % conf. interval

2.85

1.55 - 5.22 

6.91

4.49 - > 10 

 72 -hour NOEC

 5

 5

 72 -hour LOEC

 10

 10

Validity criteria fulfilled:
yes
Conclusions:
The 72-hour EC50 (growth rate) for Pseudokirchneriella subcapitata towards jasmine absolute was determined with > 10 mg/L
Executive summary:

In a 72-hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata were exposed to jasmine absolute at nominal concentrations of 10, 5, 2.5, 1.25 and 0.63 mg/L under static conditions in accordance with the OECD test guideline 201. The NOEC and EC50 values based on growth rate were 5 and 10 mg/L, respectively. 

This toxicity study is classified as acceptable and satisfies the guideline requirements for freshwater alga and cyanobacteria, Growth Inhibition Tests.

Based on the results of this study, jasmine absolute would be classified as toxic to aquatic algae in accordance with the EU CLP Regulation. 

Description of key information

A 72-h static toxicity study with to Pseudokirchneriella subcapitata was conducted according to the OECD test guideline 201 and in accordance with the principles of GLP. The derived 72 h EC50 (yield) was > 10 mg/L based on the nominal concentration. The 72-hr NOEC was 5 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
10 mg/L
EC10 or NOEC for freshwater algae:
2.85 mg/L

Additional information