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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:
2018-08-20 to 2108-12-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Remarks:
TOC
Details on sampling:
- Nominal concentrations: control, 0.954, 3.05, 9.77, 31.3 and 100 mg/L
- Sampling method: after 0 (fresh), 24, 48 and 72h from aged solutions
- Sample storage conditions before analysis: refrigerated until transfer to analytical lab
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The test item has a low water solubility and is hydrologically unstable, therefore a degraded solution of the test item was prepared before application based on OECD Series on Testing and Assessment No. 23.
- Method:
Degraded solutions were prepared by directly weighing appropriate test item amounts in test medium. The stock solutions were stirred in the dark at room temperature for 24 h. At the highest concentration (100 mg/L) not the entire test item went into solution as particles were observed floating at the surface of the solution and at the bottom of the glass vessel. Subsequently the undissolved test item was allowed to sediment and/or float for a period until the phases had separated. After the settling of 30 minutes the necessary volumes for the test were withdrawn via a Teflon tube from the medium level of the degraded stock solutions. Algae were added to each solution individually, resulting in target cell densities of 0.5 × 104 cells per mL in each solution. Approximately 50 mL of the prepared solutions were transferred to each test vessel.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Test species: Pseudokirchneriella subcapitata (Korshikov) Hindák (Sphaeropleales: Selenastraceae) , formerly known as Selenastrum capricornutum, and renamed as Raphidocelis subcapitata Korshikov
- Strain: SAG 61.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH, Germany
- Age of inoculum (at test initiation): algae kept in exponential growth state

ACCLIMATION
- Acclimation period: 3-4 days before test start, test medium was inoculated with test organisms and held under test conditions in order to produce a pre-culture in state of exponential growth
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
/
Test temperature:
22.6 - 23.2°C
pH:
7.48-8.05
Dissolved oxygen:
/
Salinity:
/
Conductivity:
/
Nominal and measured concentrations:
- Nominal concentrations: control, 0.954, 3.05, 9.77, 31.3 and 100 mg/L
- Measured concentrations (TOC): < LOD, < LOD, 1.65, 5.18, 15.3 and 80 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks (100 mL)
- Type (delete if not applicable): closed with aluminium caps
- Material, size, headspace, fill volume: 50 mL test solution
- Aeration: continuous agitation
- Initial cells density: 0.5 x 10^4 cells/mL
- Control end cells density: 38 x 10^ 4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, AAP medium

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuously
- Light intensity and quality: 88.1 μEm-2s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorescence measurement - microplate reader at wavelenght 670 nm. Calibration curve set-up by fluorescence signals and couns in Neubauer chamber
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.19
- Range finding study
- Test concentrations: 100, 10, 3.3, 1.1 and control
- Results used to determine the conditions for the definitive study:
Two non-GLP range-finding tests were performed comparing the effects of non-degraded (treated with 5 minutes of ultrasonication) and degraded solutions (treated with 24 hours of stirring in the dark at room temperature) to choose an appropriate application design. In the range finding test using ultrasonication for 5 minutes the inhibition of yield was 19.4 % and the inhibition of growth rate was 5.2 % at 100 mg/L test item concentration. In the range finding test using degraded solutions the inhibition of yield was 25.6 % and the inhibition of growth rate was 5.7 % at 100 mg/L test item concentration. The results of both non-GLP range-finding tests were similar. Therefore, for the main test 24h stirring was selected as pre-treatment.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
55.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
35.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: TOC
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
31.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
15.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: TOC
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
55.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
35.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: TOC
Basis for effect:
biomass
Remarks:
yield
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
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: yes, increased growth in test vesels 0.954, 3.05, 9.77 and 31.3. Only at highest concentration 100 mg/L decrease in growth compared to control.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Particles were observed floating at the surface of the solution and at bottom of the glass vessel at the highest test concentration 100 mg/L
- Effect concentrations exceeding solubility of substance in test medium: no
- EC 10 values could not be determined since only the 2 highest test concentrations were within the concentration-response relation.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 1.60 mg/L (growth rate) and 0.990 mg/L (yield)
Reported statistics and error estimates:
The data was inappropriate for the calculation of EC10, 20-values, which were thus not determined. Due to statistical reasons inhibition-values above 100 % were set to 100 and values below 0 % were set to zero. Only test item concentrations within a clear concentration response relation were used for calculations.

Main test: average cell numbers

conc (mg/L) Average cell numbers (10^4/mL)
0h 24h 48h 72h
control 0.53 1.57 8.66 38.11
0.954 0.53 2.19 11.88 59.78
3.05 0.53 2.45 13.35 60.61
9.77 0.53 2.31 12.31 58.3
31.3 0.53 2.7 16.17 63.44
100 0.53 0.37 0.42 0.47

Main test: percentage inhibition growth rate and yield

nominal conc (mg/L) % inhibition 
growth rate yield
0h-72h 0h-72h
control 0 0
0.954 -10.9 -57.7
3.05 -11.3 -59.9
9.77 -10.4 -53.7
31.3 -12.4 -67.4
100 102.9 100.2

TOC measurements

Test item concentration, nominal TOC content nominal sampling TOC found actual test item concentration
mg/L mg/L   mg/L % of nominal mg/L
control 0 0h fresh < LOD - -
72h aged < LOD
0.954 0,489 0h fresh < LOD - -
72h aged < LOD
3.05 1.56 0h fresh 0.805 52 1.65
  72h aged 0.888 57
9.77 5.01 0h fresh 2.41 48 5.18
  72h aged 2.93 58
31.3 16.1 0h fresh 7.56 47 15.3
  72h aged 8.44 52
100 51.3 0h fresh 40.6 79 80
 

72h aged

41.5

81

Validity criteria fulfilled:
yes
Conclusions:
The toxicity of ODPA to algae has been assessed in a OECD 201 study. At the nominal test concentration of 100 mg/L toxicity was observed. The tested concentrations < 100 mg/L did not show toxicity. The EC50 (growth rate) was derived to be 55.9 mg/L nominal or 35.1 mg/L according to TOC measurement.
Executive summary:

The toxicity of ODPA to algae has been assessed in a OECD 201 study. Pseudokirchneriella subcapitata algae were exposed 72h to ODPA in nominal test concentrations of 0, 0.954, 3.05, 9.77, 31.3 and 100 mg/L.

Since the substance is hydrolytically unstable, aged solutions were used for the tests, i.e. substance dissolved and stirred for 24h in the dark. Verification of test concentrations was done by TOC measurement. The substance was found to be between 47 and 79 % of the nominal test concentration.

At the nominal test concentration of 100 mg/L toxicity was observed. The tested concentrations < 100 mg/L did not show toxicity. The EC50 (growth rate and yield) was derived to be 55.9 mg/L nominal or 35.1 mg/L according to TOC measurement (geometric mean). The NOEC is 31.3 mg/L nominal and 15.3 mg/L according to TOC measurement (geometric mean). EC10 values were not determined since only the 2 highest test item concentrations (31.1 and 100 mg/L, nominal) were within the concentration-response relation after 72h.

Description of key information

There is one key study for the toxicity of ODPA to algae. This is a OECD 201 study in which Pseudokirchneriella subcapitata algae were exposed 72h to ODPA in nominal test concentrations of 0, 0.954, 3.05, 9.77, 31.3 and 100 mg/L.

Since the substance is hydrolytically unstable, aged solutions were used for the tests, i.e. substance dissolved and stirred for 24h in the dark. Verification of test concentrations was done by TOC measurement. The substance was found to be between 47 and 79 % of the nominal test concentration.

At the nominal test concentration of 100 mg/L toxicity was observed. The tested concentrations < 100 mg/L did not show toxicity. The EC50 (growth rate and yield) was derived to be 55.9 mg/L nominal or 35.1 mg/L according to TOC measurement (geometric mean). The NOEC is 31.3 mg/L nominal and 15.3 mg/L according to TOC measurement (geometric mean). EC10 values were not determined since only the 2 highest test item concentrations (31.1 and 100 mg/L, nominal) were within the concentration-response relation after 72h.

Key value for chemical safety assessment

EC50 for freshwater algae:
35.1 mg/L
EC10 or NOEC for freshwater algae:
15.3 mg/L

Additional information

A QSAR has also been generated as supporting information. The corresponding EC50 was 15.1 mg/L, which is in the same order of magnitude of the experimentally observed EC50 values.