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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
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)
Version / remarks:
2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DEFM177269
- Expiration date of the lot/batch: 19.09.2022
- Purity test date: 15.10.2018
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control and 25.8, 36.2, 50.7, 70.9, 99.3 mg/L test item
- Sample storage conditions before analysis: Specimens were diluted with methanol (1:1) and were
stored deep frozen (≤ -18°C) until they were analysed.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution was prepared by adding the test item Isobornyl acetate to test medium.
The stock solution was treated with ultrasonic for 30 minutes and stirred for 110 minutes on a magnetic stirrer. The stock solution was visible cloudy.

- Controls: untreated test medium
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not reported
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: 86.81 SAG
- Source (laboratory, culture collection): purchased from MBM ScienceBridge GmbH,Hans-Adolf-Krebs-Weg 1, 37077 Göttingen, Germany
- Age of inoculum (at test initiation): Stock cultures used in the test grew in culturing vessels prior to being used in the test for 4 days
- Method of cultivation: liquid
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.5 – 23.1°C
Nominal and measured concentrations:
Nominal: control, 25.8, 36.2, 50.7, 70.9, 99.3 mg/L test item
geometric mean measured:4.86, 6.31, 8.54, 10.3, 16.6 mg/L Isobornyl acetate (IBA)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flask with air-permeable stoppers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 100 mL test volume
- Aeration: no
- Initial cells density: 5 x 10^3 cells/mL
- Control end cells density: 361 x 10^3 cells/mL
- No. of organisms per vessel: 3
- No. of vessels per concentration (replicates): 3 (+ 1 additional vessel for analysis and retain specimen)
- No. of vessels per control (replicates): 6 (+ 1 additional vessel for analysis and retain specimen)


GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium (according to OECD 201)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: continuous illumination (on average 64 µE m-2 s-1); cold white fluorescent light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.4
- Test concentrations: 25.8, 36.2, 50.7, 70.9, 99.3 mg/L test item
Reference substance (positive control):
yes
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 16.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 16.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No differences such as sedimentation of test solution, cell aggregation or color differences of algae cells were noticed between the control vessels and the test vessels containing the test item during the test.
- Unusual cell shape: not reported
- Colour differences: not reported
- Flocculation: not reported
- Adherence to test vessels: not reported
- Aggregation of algal cells: not reported
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not reported
- Effect concentrations exceeding solubility of substance in test medium: unclear under test c
onditions, but results were based on geometric mean measuired concentrations.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 1.39 mg/L potassium dichromate
Reported statistics and error estimates:
ECx-values: Non-linear Regression (Normal sigmoid 3 Parameters)
LOEC/NOEC: Williams t-test, Dunnett`s t-test p ≤ 0.05, one-sided smaller
statistical program: ToxRat Professional Version 3.2.1 (02.11.2015)

Effects on growth rate and yield of Desmodesmus subspicatus

 

Effect concentration

Isobornyl acetate (IBA), mg/L

Average specific growth rate inhibition

Yield inhibition

0 - 72 h after application

NOEC

 

 

 

 

 

nominal

50.7

50.7

 

 

 

mean measured

8.54

8.54

 

 

 

LOEC

 

 

 

 

 

nominal

70.9

70.9

 

 

 

mean measured

10.3

10.3

 

 

 

EC10

ErC10

EyC10

and 95% confidence intervals

 

 

(lower – upper)

 

 

 

 

 

nominal

> 99.3

63.6

 

(97.0 - > 99.3)

(54.8 – 73.7)

 

 

 

mean measured

> 16.6

9.75

 

(15.9 - > 16.6)

(8.04 – 11.8)

 

 

 

EC20

ErC20

EyC20

and 95% confidence intervals

 

 

(lower – upper)

 

 

 

 

 

nominal

> 99.3

80.2

 

(> 99.3)

(68.8 – 93.9)

 

 

 

mean measured

> 16.6

12.8

 

(>16.6)

(10.5 – 15.7)

 

 

 

EC50

ErC50

EyC50

and 95% confidence intervals

 

 

(lower – upper)

 

 

 

 

 

nominal

> 99.3

> 99.3

 

(> 99.3)

(97.4 - > 99.3)

 

 

 

mean measured

> 16.6

> 16.6

 

(> 16.6)

(15.8 - > 16.6)

 

 

 

Calculations were done using unrounded values

Validity criteria fulfilled:
yes
Conclusions:
Freshwater green algae Desmodesmus subspicatus 72 h-EC50: > 16.6 mg isobornyl acetate/L
Executive summary:

Juckeland (2019) performed an algae growth test according to OECD 201 under GLP to assess the effects of the test item Isobornyl acetate (IBA) to Desmodesmus subspicatus during 72 hours of static expposure.

The growth inhibition test was performed to assess the effects of the test item Isobornyl acetate to green algae (Desmodesmus subspicatus) during 72 hours of exposure.

Measured concentrations of the test item in test solutions were within a range of 86 to 97% of nominal values at test start and after 72 hours the concentrations ranged from 2 to 4 % in spent test solutions. Thus, all endpoints are based on the nominal concentrations and on the geometric mean measured concentration for the test item.

Significant effects on growth rate were found using Williams t-test (p ≤ 0.05) at the nominal test concentrations ≥ 70.9 mg/L test item. As a result, a LOEC of 70.9 mg/L test item (equivalent to 10.3 mg/L IBA, mean measured) was determined. The corresponding NOEC was 50.7 mg/L test item (equivalent to 8.54 mg/L IBA, mean measured).

Effect concentrations for growth rate (ErC10, ErC20 and ErC50) were all higher than 99.3 mg/L test item (equivalent to higher than 16.6 mg/L IBA, mean measured), the highest tested concentration.

All validity criteria were fulfilled.

Description of key information

Isobornyl acetate 72 h EC50 for Green Algae was > 16.6 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
16.6 mg/L
EC10 or NOEC for freshwater algae:
16.6 mg/L

Additional information

The Klimisch 1 study from Juckeland (2019) is the key study. Further Klimisch 2 QSAR information is available, but not considered as relevant as the experimental result and therefore not reported here.

Juckeland (2019) performed an algae growth test according to OECD 201 under GLP to assess the effects of the test item Isobornyl acetate (IBA) to Desmodesmus subspicatus during 72 hours of static expposure.

The growth inhibition test was performed to assess the effects of the test item Isobornyl acetate to green algae (Desmodesmus subspicatus) during 72 hours of exposure.

Measured concentrations of the test item in test solutions were within a range of 86 to 97% of nominal values at test start and after 72 hours the concentrations ranged from 2 to 4 % in spent test solutions. Thus, all endpoints are based on the nominal concentrations and on the geometric mean measured concentration for the test item.

Significant effects on growth rate were found using Williams t-test (p ≤ 0.05) at the nominal test concentrations ≥ 70.9 mg/L test item. As a result, a LOEC of 70.9 mg/L test item (equivalent to 10.3 mg/L IBA, mean measured) was determined. The corresponding NOEC was 50.7 mg/L test item (equivalent to 8.54 mg/L IBA, mean measured).

Effect concentrations for growth rate (ErC10, ErC20 and ErC50) were all higher than 99.3 mg/L test item (equivalent to higher than 16.6 mg/L IBA, mean measured), the highest tested concentration.

The ErC10 and ErC50 of > 16.6 mg isobornyl acetate/L will be used for the risk assessment.