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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
Dissolved CO2 concentration in the medium was increased by addng 0.3 g NaHCO3/L and subsequent pH adjustment to 7.0. The initial algal density was reduced to 3000 cells/ml, in order to allow sufficient exponential growth period at a limited CO2 supply.
GLP compliance:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Reahim
- Purity: >98%
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: If necessary, poorly soluble chemicals were dissolved over several days in glass-stoppered flasks in a rotary shaker in the dark. In order to minimize losses of volatile chemicals due to evaporation, the dilutions for toxicity testing were prepared on ice, at a temperature between 0 and 1 °C.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Source (laboratory, culture collection): Algal Toxkit F (MicroBioTests Inc., Nazareth, Belgium)
- Age of inoculum (at test initiation): Not specified
- Method of cultivation: Not specified

ACCLIMATION
- Acclimation period: Not specified
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 +/- 1 °C
pH:
7
Nominal and measured concentrations:
Not specified. & or 7 concentrations were tested.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: standard 20-mL glass scintillation vials containing almost 24 mL of algal growth medium when filled up completely
- Aeration: No - Initial cells density: 3000 cells/mL, determined by counting under microscope in a haemocytometer
- Control end cells density: The concentration of algal cells in the control culture increased at least 16 times during 72h
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 8

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: Not specified
- Adjustment of pH: Yes
- Light intensity and quality: Philips TL-D 38W aquarelle fluorescent tubes

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: Spectrophotometer - algal biomass was measured by optical density at 682 nm at least once daily
Reference substance (positive control):
yes
Remarks:
Aniline
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
410.97 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
- Other: The coefficient of variation of biomass density in replicate control cultures did not exceed 5%
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 50.6 ± 6.0 mg/L
Reported statistics and error estimates:
The toxicity values (EC50) and their confidence intervals were determined from dose–response curves by the REGTOX software for Microsoft Excel (Vindimian, 2009) using the Log-normal model. Prism 5 (GraphPad Software Inc., http://www.graphpad.com) was used for calculation of algal growth rate.
Validity criteria fulfilled:
yes
Conclusions:
An algal toxicity test was carried out on Pseudokirchneriella subcapitata according to the guideline OECD 201. A 72h EC50 of 410.97 mg/L was derived.
Executive summary:

An algal toxicity test for cyclohexanol was carried out on Pseudokirchneriella subcapitata according to the guideline OECD 201. The samples were incubated at 24 ± 1 °C for up to 72 h in standard 20-mL glass scintillation vials closed with polypropylene caps and containing almost 24 mL of algal growth medium when filled up completely. Exponentially growing algal cultures were exposed to 6 or 7 concentrations of the test substance under controlled conditions. Aniline was included as a positive control. All samples were run in duplicate with eight controls. Algal biomass was measured by optical density at 682 nm at least once daily. A 72h EC50 of 410.97 mg/L was derived.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
according to guideline
Guideline:
other: DIN 38412 Part 9
Deviations:
yes
Remarks:
No analytical dose verification
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Not specified
- Purity: Not specified
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance was liquidized by heating and a stock solution prepared. Preparation of the concentration range by serial dilution of stock solution.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM- Common name: Desmodesmus subspicatus (former name: Scenedesmus subspicatus)
- Strain: CHODAT SAG 86.81
- Source (laboratory, culture collection): Sammlung für Algenkulturen der Universität Göttingen
- Age of inoculum (at test initiation): The test algae are taken from a 3 days-old continously growing preculture
- Method of cultivation: The test strain is kept in liquid culture in the laboratory.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): Same as test medium (OECD medium)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
20°C
pH:
8.0-9.7
Nominal and measured concentrations:
0.08, 0.03, 0.12, 0.49, 1.95, 7.81, 31.25, 125.0, 500.0 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 20 mL glass tubes with flat bottom, plugged with silicone sponge caps, 10 mL test volume
- Initial cells density: 10000 cells/mL
- Control end cells density: ca. 450000 after 72h
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

GROWTH MEDIUM
- Standard medium used: yes

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

OTHER TEST CONDITIONS
- Adjustment of pH: not during test
- Photoperiod: Continous light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: in vivo chlorophyll-a-fluorescence at 685 nm wavelength (as measure for cell number) after 0, 24, 48, 72 and 96 hours- pH values: after 0 and 72 hours

TEST CONCENTRATIONS
- 0.08, 0.03, 0.12, 0.49, 1.95, 7.81, 31.25, 125.0, 500.0 mg/L (nominal). A control was tested in parallel.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
18.93 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.55 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.12 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.03 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
483.82 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
9.03 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC20
Effect conc.:
35.59 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
- Any stimulation of growth found in any treatment: No
Reported statistics and error estimates:
Mean coefficients of variation in control replicates were 15.5 and 29.4% at 72 and 96h, respectively. The coefficient of variation of average specific growth rates in control cultures was 1.0% after 72h and 1.2% after 96h.
Validity criteria fulfilled:
yes
Conclusions:
A 72h ErC10 of 1.55 mg/L and a 72h ErC50 of >500 mg/L were derived for Desmodesmus subspicatus.
Executive summary:

An algal toxicity test for cyclohexanol was carried out following a method according to DIN 38412 Part 9, which is equivalent or similar to the OECD guideline 201. Green algae (Desmodesmus subspicatus, former name: Scenedesmus subspicatus) were exposed in 4 replicate static vessels to each of 9 nominal concentrations of cyclohexanol (0.08, 0.03, 0.12, 0.49, 1.95, 7.81, 31.25, 125.0, 500.0 mg/L) for up to 96 hours. In vivo chlorophyll-a fluorescence at 685 nm wavelength was measured for the determination of cell number and calculation of growth rates after 0, 24, 48, 72 and 96 hours. The starting cell concentration was 1000 cells/mL. A 72h ErC10 of 1.55 mg/L and a 72h ErC50 of >500 mg/L were derived.

Description of key information

ErC50 >500 mg/L, ErC10 = 1.55 mg/L, Desmodesmus subspicatus, DIN 38412 Part 9 and OECD 201, BASF (1988)

EC50 = 410.97 mg/L, Pseudokirchneriella subcapitata, OECD 201, Aruoja (2014)

Key value for chemical safety assessment

EC50 for freshwater algae:
410.97 mg/L
EC10 or NOEC for freshwater algae:
1.55 mg/L

Additional information

An algal toxicity test for cyclohexanol was carried out following a method according to DIN 38412 Part 9 and equivalent or similar to OECD guideline 201 (BASF 1988). Green algae (Desmodesmus subspicatus, former name: Scenedesmus subspicatus) were exposed in 4 replicate static vessels to each of 9 concentrations of cyclohexanol (0.08, 0.03, 0.12, 0.49, 1.95, 7.81, 31.25, 125.0, 500.0 mg/L; nominal) for up to 96 hours.

An algal toxicity test for cyclohexanol was carried out on green algae (Pseudokirchneriella subcapitata) according to the guideline OECD 201 (Aruoja 2014). The samples were incubated at 24 ± 1 °C for up to 72 h in standard 20-mL glass scintillation vials closed with polypropylene caps and containing almost 24 mL of algal growth medium when filled up completely. Exponentially growing algal cultures were exposed to 6 or 7 concentrations of the test substance under controlled conditions. Aniline was included as a positive control. All samples were run in duplicate with eight controls. Algal biomass was measured by optical density at 682 nm at least once daily.

Both key studies reported complementary results for the purposes of classification and risk assessment.