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:
from June to September 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD, EPA, etc)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): XP-7866
- Substance type: White powder
- Physical state: Solid
- Lot/batch No.: 10188-1 JM
- Expiration date of the lot/batch: 25 February 2015
- Storage condition of test material: under ambient condition
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Details on properties of the test item are availbale in Chapter 4: Physical and chemicals properties.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations:Nominal test concentrations selected were 20, 40, 80, 160 and 320 μg a.i./L.
- Sampling method: Samples of the test solutions of each treatment were collected at approximately 0 and 96 hours. All samples were collected in
glass vials.
- Sample storage conditions before analysis: Processed immediately without storage.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
1) Primary stock solution in methanol, for a nominal concentration of 0.1 mg a.i./mL
2) Appropriate volume of the stock solution was transferred to a clean, empty 500 mL volumetric flask using a Hamilton gas-tight syringe or graduated, glass pipette.
3) The methanol was evaporated off from the flasks under a stream of nitrogen gas.
4) The flasks were brought to volume with freshwater medium to prepare the five nominal test concentrations of 20, 40, 80, 160 and 320 μg a.i./L. The flasks were then sonicated for approximately 45 minutes, stirred on a magnetic stir plate overnight and inverted at least 20 times to enable the test material to go in solution as much as possible.
- Controls: Aliquots of the freshwater medium
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): methanol for the primary stock solution but evaporated in later stage (refer method above)
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no - All test solutions appeared clear and colorless.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): University of Toronto Culture Collection (Original algal cultures) - Algal cells used in this test were obtained from Wildlife International, Ltd. cultures.
- Age of inoculum (at test initiation):
- Method of cultivation: Algal cells obtained from Wildlife International, Ltd. cultures were actively growing in culture medium for at least two weeks prior to test initiation. The culture was last transferred to fresh medium four days prior to test initiation.

Study design

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

Test conditions

Hardness:
Not reported
Test temperature:
Temperatures ranged from 23.9 to 24.8°C and were within the 24 ± 2°C range established for the test.
pH:
The pH of the test solutions at test initiation ranged from 7.4 to 7.5 and at exposure termination ranged from 7.7 to 8.6.
The pH tended to increase over time relative to increases in algal densities, which is typical for tests conducted with Pseudokirchneriella subcapitata.
Dissolved oxygen:
Not reported
Salinity:
Not reported
Nominal and measured concentrations:
* Nominal concentration: 20, 40, 80, 160 and 320 µg a.i/L
* Measured concentration: 24.2, 47.0, 51.4, 131 µg a.i./L
* Geometric mean measured concentration: 5.0, 11, 15, 16, 26 µg a.i/L
Note: Samples collected on Day 4 had measured concentrations all
Details on test conditions:
TEST SYSTEM
- Test vessel: Test chambers were sterile 250-mL Erlenmeyer flasks plugged with foam stoppers and contained 100 mL of test or control medium.
- Type (delete if not applicable): closed
- Initial cells density: 10,000 cells/mL at test initiation
- Control end cells density: 10,000 cell/mL at test initiation
- No. of vessels per concentration (replicates): 3 replicates
- No. of vessels per control (replicates): 6 replicates


GROWTH MEDIUM
- Standard medium used: yes as indicated in American Society for Testing and Materials. 2004. ASTM Standard E1218-04: Standard Guide for Conducting Static Toxicity Tests with Microalgae.
Freshwater AAP Algal Medium composition:
* MgCl2•6H2O = 12.164 mg/L
* CaCl2•2H2O = 4.410 mg/L
* H3BO3 = 0.1855 mg/L
* MnCl2•4H2O = 0.4154 mg/L
* ZnCl2 = 3.27 μg/L
* FeCl3•6H2O = 0.1598 mg/L
* CoCl2•6H2O = 1.428 μg/L
* Na2MoO4•2H2O = 7.26 μg/L
* CuCl2•2H2O = 0.012 μg/L
* Na2EDTA•2H2O = 0.300 mg/L
* NaNO3 = 25.50 mg/L
* MgSO4•7H2O = 14.70 mg/L
* K2HPO4 = 1.044 mg/L
* NaHCO3 = 15.00 mg/L

Note: The pH of the medium was adjusted to 7.5 using 10% HCl.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: NANOpure (R) water
Analyses of pesticides, organics and Metals of water were conducted by Lancaster Laboratories on samples collected on December, 29, 2010. Details are available in the original report.

OTHER TEST CONDITIONS
- Sterile test conditions: not specified in the report
- Adjustment of pH: the pH of the medium was adjusted to 7.5 using 10% HCl.
- Photoperiod: 24 hours /day
- Light intensity and quality: cool-white fluorescent lighting at a target intensity of 6000 +/- 10% lux.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell counts were performed using an electronic particle counter - Coulter Electronics, Inc.
Prior to conducting cell counts, the linearity of the instrument response was determined. Therefore, primary counting standard containing P. subcapitata cells was prepared, the density was verified using a hemacytometer and a microscope, and the standard was subsequently diluted to provide a
series of seven counting standards for the determination of instrument linearity.
The cell density of the sample was determined by adjusting the mean cell count (cells/mL) obtained using the particle counter, based upon the Y intercept and slope calculated through the regression analysis, and the dilution factor. The following equation was used:
Cell Density of Sample (cells/mL) = (Mean Cell Count (cells/mL) - Y Intercept/Slope) × Dilution Factor

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: No justification given in the report
- Range finding study: No ranfe finding study
- Test concentrations:
* Nominal concentration: 20, 40, 80, 160 and 320 µg a.i/L
* Measured concentration: 24.2, 47.0, 51.4, 131 µg a.i./L
* Geometric mean measured concentration: 5.0, 11, 15, 16, 26 µg a.i/L
Note: Samples collected on Day 4 had measured concentrations all
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 26 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: cell densities
Remarks on result:
other: 95% CL could not be calculated with the data obtained.
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 26 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: cell density
Remarks on result:
other: 95% CL could not be calculated with the data obtained.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 26 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Cell density
Remarks on result:
other: 95% confidence limits could not be calculated with the data obtained.
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 26 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: cell density
Remarks on result:
other: 95% confidence limits could not be calculated with the data obtained.
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 26 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits could not be calculated with the data obtained.
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 26 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits could not be calculated with the data obtained.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 26 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits could not be calculated with the data obtained.
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 26 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits could not be calculated with the data obtained.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 26 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Remarks on result:
other: 95% confidence limits could not be calculated with the data obtained.
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 26 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Remarks on result:
other: 95% confidence limits could not be calculated with the data obtained.
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
26 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Cell density
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
26 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
26 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes - Changes in mean cell density in the negative control replicates over the 96-hour exposure period indicated that exponential growth of cells occurred in those replicates.
- Adherence/Flocculation/Aggregation to test vessels: After 96 hours of exposure, there were no signs of adherence of cells to the test chambers or aggregation/flocculation of algae in the controls or in any treatment group.
- Other: There were no noticeable changes in cell morphology in samples collected from any of the test concentrations when compared to the control.

Results with reference substance (positive control):
No positive control
Reported statistics and error estimates:
- Software: The SAS System for Windows, Version 8.2
- Calculation methodology:
* Growth rate:
μ = (ln Nn - Ln N0)/ tn - t0
where:
μ = Average specific growth rate
N0 = Nominal cell density (cells/mL) at t0
Nn = Measured cell density (cells/mL) at tn
t0 = Time of beginning of test (hours)
tn = Time after beginning of test (hours)
* Yield: Calculated for each treatment and control replicate at 72 and 96 hours as the final biomass (cell density) in the respective exposure period minus the initial biomass (cell density).
* Inhibition:
Percent Inhibition = (Mean Control Response - Mean Treatment Response/ Mean Control Response) × 100
- Statistical test:
Cell density, growth rate and yield data were evaluated for normality and homogeneity of variance (p=0.05) using the Shapiro-Wilk’s and Levene’s tests, respectively. Since the data were normal with homogeneous variances, the treatment groups were compared to the negative control using Dunnett’s test (p=0.05). The results of the statistical analyses, as well as an evaluation of the concentration-response pattern, were used to determine the NOEC relative to each parameter at 72 and 96 hours.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Pseudokirchneriella subcapitata was exposed to five concentrations of XP-7866 at geometric mean, measured concentrations of 5.0 to 26 μg a.i./L and evaluated for effects on cell density, growth rate and yield.
The 72 and 96-hour EC50, ErC50 and EyC50 values, based on cell density, growth rate and yield, respectively, were >26 μg a.i./L, the highest concentration tested. The 72 and 96-hour NOEC for each growth parameter was 26 μg a.i./L.