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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 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material: 85% w/w 2,6-dimethyl-4-heptanone and 15% w/w 4,6-dimethyl-2-heptanone
(Diisobutyl Ketone)
- Physical state: transparent colorless liquid
- Analytical purity: 91.4%
- Lot/batch No.: SL3055T643

Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control - 1.49 - 4.77 - 15.3 - 48.8 - 156 - 500 mg DIBK/L
- Sampling method: 4-mL aliquots were transferred to vials (control - 1.49 - 4.77 - 15.3 mg DIBK/L); 2-mL aliquots were transferred to vials (48.8 - 156 - 500 mg DIBK/L)
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct addition of test material to the medium to reduce the volatilization of the test substance
- Controls: test medium without the addition of test material
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: UTCC 37
- Source: University of Toronto, Culture Collection at the University of Toronto, Toronto, Ontario, Canada
- Method of cultivation: Cultured in axenic conditions in 500 mL Erlenmeyer flasks with a volume of 200 mL (Algal assay medium); pH: 7.0 - 7.5; Temperature: 24 +/- 2°C; Light: 4300 +/- 650 lux
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24.3 - 24.5°C
pH:
7.7 -7.8 at test initiation; 7.8 - 8.0 in sacrifice replicates with algae at 24h; 7.9 - 8.6 in sacrifice replicates with algae at 48h; 8.0 - 9.3 in pooled replicates with algae and 7.7 to 7.9 in blank replicates without algae at test termination
Nominal and measured concentrations:
Nominal concentrations: control - 1.49 - 4.77 - 15.3 - 48.8 - 156 - 500 mg DIBK/L
Measured concentrations: Measured concentrations of the nominal concentrations 1.49 - 4.77 - 15.3 mg DIBK/L were within +/- 20%. In the highest test levels the recovery rate ranged from 232 - 420%. The day 3 recovery rates were lower (36.8 - 86.3%).
Details on test conditions:
TEST SYSTEM
- Test vessel: 125 mL glass serum bottles
- Type: crimp sealed with an aluminium seal containing a Teflon-lined rubber septa to prevent volatilization of test material
- Material, fill volume: glass, approx. 160 mL of total volume (void of headspace)
- Initial cells density: approx. 5000 cells/mL
- Agitation: continuous (100 rpm)
- No. of vessels per concentration (replicates): 7 (5 with algae, 2 without algae) for high doses; 6 (5 with algae, 1 without algae) for low doses
- No. of vessels per control (replicates): 7 (5 with algae, 2 without algae)

GROWTH MEDIUM
- Standard medium used: yes (Algal Assay Medium (AAM) with 20x concentration of NaHCO3 designated for the EPA algal assay bottle test)


TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: Culture medium is without the addition of NaHCO3
- Intervals of water quality measurement: every day


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous
- Light intensity and quality: 5200 +/- 780 lux


EFFECT PARAMETERS MEASURED: Cell density, growth rate and biomass area
- Determination of cell concentrations: Determined by electron particle counting using a Coulter Multisizer 3 (24, 48, and 72 h). Three cell counts were made per replicate. The readings for the blank replicates were used to correct for background in daily calculations. The adjusted cell counts were converted to cells x 10000/mL (cell density) for statistical analysis and reporting.
- Other: Morphological malformations were observed under a microscope


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
46.9 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 27.0 - 66.8 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.55 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
26.3 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 24.1 - 28.4 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.55 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
37.3 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 35.4 - 39.2 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
12.2 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
biomass
Reported statistics and error estimates:
The data (untransformed, square root transform for cell density, and the log transform for biomass and growth rate) were tested for normality using the Shapiro-Wilk’s Test and for homogeneity of variance using the Levene’s Test. If the assumptions of normality and homogeneity were met for the untransformed data or one of the transformations, no observed-effect concentrations (NOECs) were determined using a one-tailed Dunnett's mean comparison procedure. If the assumptions of normality and/or homogeneity were not met and a nonparametric analysis was required, the ranks of the values were determined and then the above analysis was performed on these ranks. The 72-hour ErC50 value, the 72-hour EbC50 value, and the 72-hour EC50 value were determined. Two sigmoid-shaped nonlinear models were used for calculation of ECx values and 95% confidence intervals, if possible. One model used to describe the response to increasing concentrations was the four-parameter logistic model (percent inhibition versus concentration). The second model used was the cumulative normal model (response compared to log concentration).

The test exhibited an 72h ErC50 of 46.9 mg/L and an 72h EbC50 of 37.3 mg/L. The effect concentrations are based on the mean measured concentrations because the recoveries in the highest test concentrations ranged from 36.8 - 86.3% of nominal concentrations on day 3. Microscopic evaluation of algal cells at each exposure concentration ant the control revealed no abnormal observations at any test level.

Validity criteria fulfilled:
yes
Executive summary:

The acute toxicity of 2,6 -dimethylheptan-4 -one (diisobutyl ketone or DIBK) to the freshwater algae, Pseudokirchneirella subcapitata was evaluated following OECD guidelines (OECD 201) over a 72 -hour exposure period. These data were used to calculate 72 -hour EC50 and NOEC values for growth rate, cell number and biomass. The 72-hour EC50 values for growth rate was 46.9 mg/L with a 95% confidence interval of 27.0 -66.8 mg/L. The 72-hour NOEC for growth rate was 12.2 mg/l.

Description of key information

A 72-hour static test evaluated the toxicity of DIBK to the green algae, Psuedokirchneirella subcapitata.  The test followed OECD 201 and meet GLP requirements.  The reported 72-hour EC50 value for growth rate was 46.9 mg/L while the 72-hour NOEC for growth was 12.2 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
46.9 mg/L

Additional information

The acute toxicity of 2,6-dimethylheptan-4-one (diisobutyl ketone or DIBK) to the freshwater algae, Pseudokirchneirella subcapitata was evaluated following OECD guidelines (OECD 201) over a 72 -hour exposure period. These data were used to calculate 72 -hour EC50 and NOEC values for growth rate, cell number and biomass. The 72-hour EC50 values for growth rate was 46.9 mg/L with a 95% confidence interval of 27.0 -66.8 mg/L. The 72-hour NOEC for growth rate was 12.2 mg/l.