Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented GLP study, according to international guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
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
Remarks:
Periodic analyses of well water for contaminants were not performed according to GLP Standards, but were performed using a certified laboratory and standard US EPA analytical methods.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Test material: Fyrquel EHC Plus
- Physical state: liquid
- Lot No.: 1113-84-4
- Composition: 4.0% triphenyl phosphate, 76% C4, 17% C8, 1.5% C12
- Source: Bromine Compounds Ltd.
- Date received: 2012-01-16
- Expiration date: 2015-01-01

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 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.
- Sampling method: Algal cells present in samples collected at exposure termination were removed prior to analysis by centrifugation. At each interval, samples were collected directly into glass vessels containing 10.0 mL of acetonitrile and processed immediately for analysis.

Test solutions

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A primary stock solution was prepared by dissolving 0.2002 g of 3 G FF in 100 mL of N,N-dimethylformamide (DMF) to achieve a nominal concentration of 2.0 mg/mL. The primary stock was mixed by inversion and appeared clear and colorless and was otherwise unremarkable. The 2.0 mg/mL stock was serially diluted with DMF to prepare four additional stock solutions at target nominal concentrations of 51.2, 128, 320 and 800 μg/mL. All stock solutions were mixed by inversion. The test solutions were prepared by diluting 50 μL of each respective stock solution into 500 mL of freshwater AAP medium.
- Controls: negative control solution consisted of freshwater AAP medium without test substance or solvent added
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N,N-dimethylformamide
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 0.1 mL/L
- Evidence of undissolved material: all test solutions were inverted to mix and appeared clear and colorless at the time of preparation, and there was no evidence of surface slicks or precipitation at exposure termination

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Source: Original algal cultures were obtained from the University of Toronto, and have been maintained in culture medium at Wildlife International, Ltd., Easton, Maryland since March 2000.

ACCLIMATION
- Acclimation period: Algal cells used in this test were obtained from Wildlife International cultures that had been actively growing in culture medium under the same environmental conditions as used in this test for at least two weeks prior to test initiation.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Test temperature:
24 ± 2°C
pH:
- pH day 0: 7.3-7.4
- pH day 4: 8.5-9.0
- The pH of the medium in each treatment and control group was measured at test initiation and exposure termination using a Thermo Orion Model 4Star plus pH/ISE meter. At test initiation, pH was measured in the individual batches of test solution prepared for each treatment and control group. At exposure termination, pH was measured in pooled samples of test solution collected from each of the replicates of each treatment and control group.
Nominal and measured concentrations:
- Nominal: negative control, solvent control, 5.1, 13, 32, 80, 200 μg/L
- Measured day 0: < LOQ, < LOQ, 4.56, 11.2, 31.2, 81.9, 220 μg/L
- Measured geometric mean: < LOQ, < LOQ, 1.12, 1.71, 4.06, 14.8, 133 μg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: sterile 250 mL glass Erlenmeyer flasks plugged with sterile foam stoppers
- Initial cells density: 10000 cells/mL
- No. of organisms per vessel:
- No. of vessels per concentration (replicates):
- No. of vessels per control (replicates):
- No. of vessels per vehicle control (replicates):

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH: pH of the medium was adjusted to 7.5 ± 0.1 using 10% hydrochloric acid
- Photoperiod: continuous cool-white fluorescent lighting
- Light intensity and quality: target light intensity was 6,000 lux ± 10%. Light intensity was measured at test solution level at five locations surrounding the test flasks at test initiation using a SPER Scientific 840006C light meter.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter (Coulter Electronics, Inc.), samples at approximately 24 h intervals during 96 h exposure
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study: Test concentrations: negative control, 2.0, 20, 200 μg/L
- Results used to determine the conditions for the definitive study:
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 220 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: area under the growth curve
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 220 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 220 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
220 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: area under the growth curve
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
220 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
220 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
cell number
Details on results:
- Exponential growth in the control (for algal test): yes/no
- Observation of abnormalities: no morphological deformities in any of the treatment groups
- Flocculation: not observed in the negative control or any of the treatment groups
- Adherence to test vessels: not observed in the negative control or any of the treatment groups
- Aggregation of algal cells: not observed in the negative control or any of the treatment groups

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
(1) mean cell density control increase ok (2) coefficicent of variation of average specific growth rates and yield in the negative control replicates from 0-96 h ok
Conclusions:
The freshwater alga, Pseudokirchneriella subcapitata, was exposed to a geometric series of five treatment levels of 3 G FF ranging from 4.56 to 220 μg/L, based on day 0 measured concentrations of 3 G FF. Toxicity of 3 G FF 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 h NOEC values were determined to be 220 μg/L for both intervals. The 72 and 96-h 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, 220 μg/L.
Executive summary:

Measurement of test concentrations

Nominal test concentrations were 5.1, 13, 32, 80 and 200 μg/L. The test solutions appeared clear and colorless at the time of preparation, and there was no evidence of surface slicks or precipitation at exposure termination. Measured concentrations on day 0 ranged from 86 to 110% of nominal. Measured concentrations of 3 G FF in the test solutions on day 4 ranged from 2 to 40% of nominal. The lack of test material present in the biotic test solutions at test termination may have been caused by the test substance adhering to the algae. The algae were removed in the day 4 samples through centrifugation and subsequently any material adhering to the algae would have also been removed. The results of the study are based on day 0 measured concentrations of 4.56, 11.2, 31.2, 81.9 and 220 μg/L, representing 89, 86, 98, 102 and 110% of the target nominal test concentrations.

Measured concentrations of TPP in the nominal 5.1, 13, 32, 80 and 200 μg/L test solutions at test initiation were 0.156, 0.427, 1.17, 3.25and 8.26 μg a.i./L, respectively. Measured concentrations of TPP at test termination were 0.157, 0.466, 1.04, 2.92 and 7.83 μg a.i./L.

Observations and measurements

Temperatures remained within the 24 ± 2°C range established for the test. The pH of the test solutions ranged from 7.3 to 7.4 at test initiation and ranged from 8.5 to 9.0 in the test solutions at test termination. The observed increase in pH is typical for tests conducted with P. subcapitata and is attributed to the photosynthetic activity of the algae. The light intensity ranged from 5230 to 6340 lux.

The toxicity of 3 G FF to P. subcapitata was determined by evaluating changes in cell density over a 96 h exposure period. Cell densities were used to determine area under the growth curve and growth rates for each 24 h interval of exposure and yields at 72 and 96 h of exposure. Exponential growth, characterized by the linear section of the growth curve, occurred throughout the duration of the test in the negative control replicates.

After 72 hours of exposure, inhibition of area under the growth curve in the 4.56, 11.2, 31.2, 81.9 and 220 μg/L treatment groups was -22, -21, 3, -13 and -9%, respectively, relative to the negative control (negative values indicate stimulation). Inhibition of growth rate in the 4.56, 11.2, 31.2, 81.9 and 220 μg/L treatment groups was -3, -4, 0, -2 and -1%, respectively, relative to the negative control. Inhibition of yield in the 4.56, 11.2, 31.2, 81.9 and 220 μg/L treatment groups was -18, -19, 3, -11 and -7%, respectively, relative to the negative control. The 72 h EC50 values and 95% confidence intervals for area under the growth curve, yield and growth rate could not be determine based on the lack of a dose-response and were empirically estimated to be greater than the highest concentration tested, 220 μg/L. None of the treatment group means for area under the growth curve, yield and growth were significantly reduced (Dunnett’s test; p > 0.05) relative to the mean control response. The 72 h NOEC was determined to be 220 μg/L.

After 96 h of exposure, inhibition of area under the growth curve in the 4.56, 11.2, 31.2, 81.9 and 220 μg/L treatment groups was -11, -15, 2, -7 and -6%, respectively, relative to the negative control. Inhibition of growth rate in the 4.56, 11.2, 31.2, 81.9 and 220 μg/L treatment groups was -1, -2, 0, -1 and -1%, respectively, relative to the negative control. Inhibition of yield in the 4.56, 11.2, 31.2, 81.9 and 220 μg/L treatment groups was -5, -12, 2, -4 and -5%, respectively, relative to the negative control. The 96 h EC50 values and 95% confidence intervals for area under the growth curve, yield and growth rate were unable to be calculated due to the lack of a dose-response and were empirically estimated to be greater than the highest concentration tested, 220 μg/L. Statistically significant reductions (Dunnett’s test; p < 0.05) were not observed in any of the treatment groups when the mean treatment group responses for area under the growth curve, yield and growth rate were compared to the mean control response. Consequently, the 96 h NOEC was determined to be 220 μg/L.

After 96 hours of exposure, flocculations or aggregations of cells were not observed in the negative control or any of the treatment groups. Adherence of cells to the test chambers was not observed in the negative control or any of the treatment groups. Cells in the treatment groups appeared normal when compared to cells present in the negative control. No morphological deformities were observed in any of the treatment groups.

Conditions for the validity of the test

The mean cell density in the control flasks increased by a factor of 401 after four days, exceeding the 100X growth criterion. The coefficient of variation of average specific growth rates and yield in the negative control replicates from 0-96 hours was 2.15 and 12.4%, respectively, achieving the control growth criteria specified in the OCSPP 850.4500 guideline of less than 15% for both endpoints.