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Ecotoxicological information

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:
18/1/2012-4/12/2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
GLP compliance:
yes
Test material information:
Composition 1
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Analytical monitoring:
yes
Details on sampling:
- Concentrations:all
- Sampling method:Samples of the test solutions were collected at approximately 0 and 96 hours to measure concentrations of the test substance. Samples at test initiation were collected from the individual batches of test solution prepared for each treatment and control group prior to distribution into the test chambers. At exposure termination, samples were collected from the pooled replicates from each treatment and control group. Algal cells were removed prior to analysis during extraction of the test substance from test medium into an organic solvent. Samples were collected directly into glass vessels and processed immediately for analysis.
- Sample storage conditions before analysis: No storage
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:A primary stock solution was prepared by dissolving 0.0101 g of brominated epoxy in 200 mL of tetrahydrofuran (THF; Burdick and Jackson; Lot Number: DF758) to achieve a nominal concentration of 50.5 μg/mL. The primary stock was mixed by inversion and appeared clear and colorless and was otherwise unremarkable. The 50.5 μg/mL stock was serially diluted with THF to prepare six additional stock solutions at target nominal concentrations of 2.3, 4.5, 9.0, 18, 27 and 36 μg/mL. All stock solutions were mixed by inversion. The test solutions were prepared by adding 1.0 mL of each respective stock stock solution (except the 50.5 μg/mL primary stock) into 1L volumetric flasks. The solvent (THF) within each flask was then evaporated under a stream of nitrogen until dryness (approximately 5 minutes). The flasks were then brought to a final volume of 1L with freshwater AAP medium (INV#12-167), inverted at least twenty times and sonicated for approximately 5 minutes to mix.
- Controls:The negative control solution consisted of freshwater AAP medium without test substance added.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): tetrahydrofuran (organic solvent)
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 0, the solvent (THF) within each flask was evaporated under a stream of nitrogen until dryness
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Source (laboratory, culture collection): University of Toronto, and have been maintained in culture medium at Wildlife International, Easton, Maryland since March 2000.
- Age of inoculum (at test initiation): at least two weaks
- Method of cultivation: growing in culture medium under the same environmental conditions as used in this test

ACCLIMATION
- Acclimation period: No
- Culturing media and conditions (same as test or not): Freshwater AAP medium.
- Any deformed or abnormal cells observed:No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
/
Hardness:
No data
Test temperature:
24 +/- 2 °C
pH:
Start: 7.45, End: 9.15
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
Nominal concentrations: 2.3; 4.5; 9.0; 18; 27; 36 µg/L
Measured concentrations: 1.8; 3.9; 7.6; 15; 24; 30 µg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:Test chambers were sterile, 250 mL glass Erlenmeyer flasks plugged with sterile foam stoppers, and contained 100 mL of test or control medium.
- Initial cells density:10 000 cells/mL
- Control end cells density: 4 510 000cells/mL
- No. of organisms per vessel: 2020 cells
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates):6
- No. of vessels per vehicle control (replicates):/

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:purified well water (NANOpure® water)

- Metals:See Appendix 4
- Pesticides: See Appendix 4

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: using 10% hydrochloric acid
- Photoperiod:continuous cool-white fluorescent lighting throughout the test
- Light intensity and quality:. The target light intensity was 6,000 lux ± 10%


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations:hemacytometer and microscope

TEST CONCENTRATIONS
- Spacing factor for test concentrations:2
- Test concentrations: 2.3; 4.5; 9.0; 18; 27 and 36 µg/mL

TEST CONCENTRATIONS RANGE FINDING STUDY
- Spacing factor for test concentrations: 2
- Range finding study: Yes
- Test concentrations: 9.0; 18; 36 µg/L
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
30 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: Area under the growth curve
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 30 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: Area under the growth curve
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
30 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 30 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
30 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 30 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: 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
- Other:/
- Any stimulation of growth found in any treatment: a slight stimulation could be observed of max -4% inhibition
- Effect concentrations exceeding solubility of substance in test medium:No
Results with reference substance (positive control):
/
Reported statistics and error estimates:
The calculation of cell densities, area under the growth curve, yield, growth rates and percent inhibition values, as well as all statistical analyses, were conducted using “The SAS System for Windows, Version 8.2” (5).
Validity criteria fulfilled:
yes
Conclusions:
The freshwater alga, Pseudokirchneriella subcapitata, was exposed to a geometric series of five treatment levels of brominated epoxy ranging from 1.8 to 30 μg/L, based on day 0 measured concentrations of brominated epoxy and one additional level at a day 0 measured concentration of 24 μg/L. Toxicity of brominated epoxy to P. subcapitata was assessed based on effects on area under the growth curve, yield and growth rate relative to the negative control group. None of the treatment groups mean responses for area under the growth curve, growth rate or yield were significantly reduced relative to the mean control responses (Dunnett’s test (p > 0.05). Consequently, the 72 and 96 hour NOEC values were determined to be 30 μg/L for both intervals. The 72 and 96-hour EC50 values for area under the growth curve, yield and growth rate could not be calculated due to the lack of a dose-response and were empirically estimated to be greater than the highest concentration tested, 30 μg/L.
In this study the highest reported water solubility value was lower than the actual value reported in the water solubility study. This difference in solubility values is explained by the hydrophobicity property of the substance and the difference between the experimental media (reagent water vs media with organisms). There is a decrease in concentrations of the test substance along the experiment.
Executive summary:

LENGTH OF EXPOSURE: 96 Hours

TEST ORGANISM: Freshwater Alga (Pseudokirchneriella subcapitata)

SOURCE OF TEST ORGANISMS: Wildlife International Cultures

Easton, Maryland 21601

TEST CONCENTRATIONS:

 Nominal (µg/L) Day 0 measured (µg/L)  Geometric mean measured (µg/L) 
Negative control   < LOQ  < LOQ
 2.3  1.8  0.95
 4.5  3.9  1.4
 9.0  7.6  2.7
 18  15  5.4
 27  24  6.9
 36  30  9.9
 36 (abiotic)  30  21

RESULTS

 Exposure duration (hours)  Area under the growth curve (µg/L)        Growth rate (µg/L)       Yield (µg/L)       
   NOEC EC50  95% CI  NOEC  EC50  95% CI  NOEC  EC50  95% CI 
 72  30  >30 n/a  30   >30  n/a 30  >30  n/a 
 96  30  >30 n/a   30  >30  n/a 30  >30  n/a 

Description of key information

The freshwater alga, Pseudokirchneriella subcapitata, was exposed to a geometric series of five treatment levels of brominated epoxy ranging from 1.8 to 30 μg/L, based on day 0 measured concentrations of brominated epoxy and one additional level at a day 0 measured concentration of 24 μg/L. Toxicity of brominated epoxy to P. subcapitata was assessed based on effects on area under the growth curve, yield and growth rate relative to the negative control group. None of the treatment groups mean responses for area under the growth curve, growth rate or yield were significantly reduced relative to the mean control responses (Dunnett’s test (p > 0.05). Consequently, the 72 and 96 hour NOEC values were determined to be 30 μg/L for both intervals. The 72 and 96-hour EC50 values for area under the growth curve, yield and growth rate could not be calculated due to the lack of a dose-response and were empirically estimated to be greater than the highest concentration tested, 30 μg/L. 30 µg/L is the highest solubility level that could be tested. The NOEC value of 30 μg/L is higher than the measured water solubility value (26.4 μg/L) of F-2200 which is the main component in brominated epory (ca. 70%).

Key value for chemical safety assessment

EC50/LC50 for freshwater algae:
30 µg/L
EC10, LC10 or NOEC for freshwater algae:
30 µg/L

Additional information