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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)
Deviations:
yes
Remarks:
see Principles and method other than guideline
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control, 0.930, 2.14, 4.93, 11.4, 26.1, 60.0 mg/L test item corresponding to control, 0.341, 0.786, 1.81, 4.17, 9.58, 22.0 mg/L a.s.
- Sample storage conditions before analysis: Specimens were stabilised with methanol (1:1) and were stored deep frozen (≤ -18°C) until they were analysed. Concentrations of the test item C18 unsaturated fatty acids, reaction products with 1-aminopropan-2-ol, maleic anhydride and sodium bisulfite in test solutions at test start (0 h), at 24 and 48 hours after test start and at test end (72 hours) were analysed.
Vehicle:
yes
Remarks:
triethylene glycol
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 to test medium. The stock solution was stirred for 10 minutes on a magnetic stirrer. Stock solution and test vessels were prepared in the following way: a stock solution (stock A) was prepared by weighing 64.3 mg test item into a graduated flask and volume to 1000 mL (= 64.3 mg/L test item) 0.276 mL algal inoculum (measured 181.5 x 104 cells/mL in the stock culture) were added to a 100 mL test volume to result in an initial biomass of 5 x 103 cells/mL

- Controls: blank control

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none
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
- Source (laboratory, culture collection): MBM ScienceBridge GmbH, Hans-Adolf-Krebs-Weg 1, 37077 Göttingen, Germany
- Method of cultivation: liquid

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.6 – 22.7°C
pH:
7.84 to 8.61
Nominal and measured concentrations:
Nominal Control, 0.930, 2.14, 4.93, 11.4, 26.1, 60.0 mg/L test item corresponding to control, 0.341, 0.786, 1.81, 4.17, 9.58, 22.0 mg/L a.s.
Geometric mean measured: Control (0), 0.648, 1.773, 4.287, 10.992, 25.209 and 61.077 mg test item/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flask with air-permeable stoppers
- Type (delete if not applicable): air-permeable stoppers
- Material, size, headspace, fill volume: 100 mL test volume
- Aeration: no
- Initial cells density: 5 x 10^3 cells/mL test solution in each replicate
- Control end cells density: Biomass in control cultures increased exponentially by a factor of 117.6 (16 is required).
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, OECD medium


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: continuous cold white fluorescent light; light intensity was measured once before test start: light intensity: on average 63 μE·m-2·s-1 measured at 400-700 nm differences from the selected light intensity over the test area did not exceed the range ± 15%

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Neubauer counting chambers
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
7.74 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.74 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 colour differences of algae cells were noticed between the control vessels and the test vessels containing the test item during the test.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Reported statistics and error estimates:
Regression analysis was performed using individual replicate responses, not treatment group means. From average specific growth rates and yield, recorded in a series of test solutions, effect concentrations of ErC10, ErC20 and ErC50, (average specific growth rate) and EyC10, EyC20 and EyC50 (yield) was determined using concentrations-response modelling (non-linear regression, 3 parameters Normal). To determine a LOEC and to derive a NOEC for effects on growth rate, it was necessary to compare treatment means using analysis of variance (ANOVA) techniques. Shapiro-Wilk´s Test on Normal Distribution was performed. The mean for each concentration was compared with control means using an appropriate multiple comparison test method. Williams’s t-test was used if variance-homogeneity requirements are fulfilled. The Welch-t-test After Bonferroni-Holm for non-homogeneous variances with Bonferroni-Holmadjustment was used. As a test for homogeneity of variances, Levene’s test was used.

Statistical analysis was performed using the software ToxRat Professional (Version 3.3, 20.10.2018).
Validity criteria fulfilled:
yes
Conclusions:
72h-ErC50: 7.74 mg a.i./L
72h ErC10: 1.74 mg a.i./L
Executive summary:

 

In the Klimisch 1 GLP study from Juckeland (2019) the toxicity of C18 unsaturated fatty acids, reaction products with 1-aminopropan-2-ol, maleic anhydride and sodium bisulfite to Pseudokirchneriella subcapitata was determined in a static 72-Hour Algal Growth Inhibition Test. The test was performed according to OECD 201. The nominal concentrations of the test item of Control, 0.930, 2.14, 4.93, 11.4, 26.1, 60.0 mg/L test item corresponding to control, 0.341, 0.786, 1.81, 4.17, 9.58, 22.0 mg/L a.s.. Dose verification analysis was performed. The geometric mean measured concentrations were control (0), 0.648, 1.773, 4.287, 10.992, 25.209 and 61.077 mg test item/L. The biological results were based on geometric mean measured test concentrations. The cell density was determined at 0, 24, 48 and 72 hours. The growth of the control cultures fulfilled the validity criteria from OECD 201 (2006). The 72 -hour ErC10 and ErC50 are 1.74 and 7.74 mg a.i./L based on geometric mean measured test concentrations.

 

This information is considered to be relevant and reliable for the further risk assessment.

Description of key information

The 72 -hour ErC10 and ErC50 are 1.74 and 7.74 mg a.i./L based on geometric mean measured test concentrations.

 

Key value for chemical safety assessment

EC50 for freshwater algae:
7.74 mg/L
EC10 or NOEC for freshwater algae:
1.74 mg/L

Additional information

In the Klimisch 1 GLP study from Juckeland (2019) the toxicity of C18 unsaturated fatty acids, reaction products with 1-aminopropan-2-ol, maleic anhydride and sodium bisulfite to Pseudokirchneriella subcapitata was determined in a static 72-Hour Algal Growth Inhibition Test. The test was performed according to OECD 201. The nominal concentrations of the test item of Control, 0.930, 2.14, 4.93, 11.4, 26.1, 60.0 mg/L test item corresponding to control, 0.341, 0.786, 1.81, 4.17, 9.58, 22.0 mg/L a.s.. Dose verification analysis was performed. The geometric mean measured concentrations were control (0), 0.648, 1.773, 4.287, 10.992, 25.209 and 61.077 mg test item/L. The biological results were based on geometric mean measured test concentrations. The cell density was determined at 0, 24, 48 and 72 hours. The growth of the control cultures fulfilled the validity criteria from OECD 201 (2006). The 72 -hour ErC10 and ErC50 are 1.74 and 7.74 mg a.i./L based on geometric mean measured test concentrations.

 

This information is considered to be relevant and reliable for the further risk assessment.