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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
1,1,3,3-tetramethylbutylamine
EC Number:
203-491-1
EC Name:
1,1,3,3-tetramethylbutylamine
Cas Number:
107-45-9
Molecular formula:
C8H19N
IUPAC Name:
2,4,4-trimethylpentan-2-amine
Details on test material:
- Name of test material (as cited in study report): Primene(TM) TOA
- Physical state: liquid
- Analytical purity: 97.45%
- Lot/batch No.: 2377627

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: all test concentrations
- Sampling method: For each treatment sample, a 15-mL sample was collected
- Sample storage conditions before analysis: no data

Test solutions

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A 0.20-mg whole product/mL primary standard was prepared by weighing 0.0501 g of the test substance into a 250-mL volumetric flask and bringing the flask to volume with dilution medium. The primary standard was used to prepare the parent test solutions by diluting appropriate aliquots of the primary standard solution to 500 mL with dilution medium.
- Controls: dilution medium
- Evidence of undissolved material (e.g. precipitate, surface film, etc): none

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): Department of Botany, Culture Collection of Algae, University of Texas at Austin
- Age of inoculum (at test initiation): four days
- Method of cultivation: The prepared cultures were maintained in a temperature-controlled environmental chamber under continuous light. Periodically, new Selenastrum cultures were cloned from an existing culture derived from the parent stock.

ACCLIMATION
- Acclimation period: no data
- Culturing media and conditions (same as test or not): same
- Any deformed or abnormal cells observed: none

Study design

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

Test conditions

Test temperature:
22.5 - 25.0 deg C
pH:
7.5 to 9.8
Nominal and measured concentrations:
0.65, 1.3, 2.5, 5.0, 10, and 20 mg whole product/L nominal
Details on test conditions:
TEST SYSTEM
- Test vessel: 250-mL Erlenmeyer flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250-mL Erlenmeyer
- Initial cells density: 1.0 x 10e4 cells/mL
- No. of vessels per concentration (replicates): triplicate
- No. of vessels per control (replicates): six
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: laboratory reagent water
- Metals: recovery below limit of detection
- Culture medium different from test medium: no
- Intervals of water quality measurement: periodically

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 8,569 to 8,963 lux
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: direct microscopic counting with a hemacytometer
- Chlorophyll measurement: no data
- Other: At 24,48, and 72 (+/-1) hours, cell density was measured

TEST CONCENTRATIONS
- Range finding study: two range-finding studies were conducted with different concentrations; Due to the unpredicted results and the poor cell growth in the first definitive study, a third range-finding test was deemed necessary.
- Test concentrations: first RF: 0.10, 1.0, 10, 100, and 1,000 mg whole product/L; second RF: 0.50, 5.0, and 50 mg whole product/L; third RF: 0.20, 2.0, and 20 mg whole product/L
- Results used to determine the conditions for the definitive study: Based on the results of the range-finding tests, nominal concentrations were selected for the definitive exposure

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
13.3 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 13.1 to 13.5 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.93 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: cell density
Remarks on result:
other: 95% CL 7.47 to 8.58 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.4 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: Area under the growth curve
Remarks on result:
other: 95% CL 7.96 to 8.84 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.27 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
All statistical analyses were performed using SAS software. The NOEC values, based on cell density, area under the growth curve, and growth rate, were estimated using a one-way analysis of variance (ANOVA) procedure and a one-tailed Dunnett's test. The alternate hypothesis was the mean for the growth parameter was reduced in comparison to the control mean. Prior to the Dunnett's test, a Shapiro-Wilk's test and a Levene's test were conducted to test for normality and homogeneity of variance, respectively, over treatments at each time point. If the results from the Shapiro-Wilk's and Levene's tests indicated normality and insignificant heterogeneity, the analysis was performed on the non-transformed raw data. In instances of non-normality or heterogeneity, a square root transformation was performed. If both the non-transformed raw data and the transformed data exhibited non-normality or inequality of variance, a non-parametric analysis of variance was performed on the ranks of the raw data values. Parametric analyses were performed on the 48- and 72-hour area under the growth curve data. Non-parametric analyses were performed on the 24-, 48-, and 72-hour cell density, 24-hour area under the growth curve, and 24-, 48-, and 72-hour growth rate data.

A nonlinear modeling procedure was used to estimate the slope (B) and EC values. The distribution of x hat method was used to estimate the 95% confidence lirnits. Two measures of goodness of fit were calculated, R-squared and the root mean square error (RMSE). R-squared was calculated in order to describe the percent of variation about the mean explained by the logistic model. The RMSE describes the mean distance (in units of percent inhibition) between the observed data values and the estimated model fit to these values. It is one measure of the closeness of the data points to the logistic model curve. It is desirable for the value of R-squared to be large (100% being the maximum) and for the value of the RMSE to be small.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The NOEC value at 72 hours was 1.27 mg whole product/L, based on mean measured concentrations. The NOEC value was based on the lack of a statistically significant reduction in growth rate at this and the lowest test substance treatment. Based on growth rate, the 72-hour ErC50 value was 13.3 mg whole product/L with 95% confidence limits of 13.1 to 13.5 mg whole product/L, based on mean measured concentrations.
Executive summary:

A growth inhibition test was conducted to estimate the potential toxicity of the test substance to the unicellular green alga, Pseudokirchneriella subcapitata. Algal cells were exposed for 72 hours under static conditions to nominal concentrations 0 (control), 0.65, 1.3, 2.5, 5.0, 10, and 20 mg whole product/L of PrimeneTMTOA. Analytical confirmation of the test substance exposure concentrations was performed at 0 and 72 hours. Water quality characteristics of temperature, and pH, measured at test initiation and termination, were within acceptable limits. The controls and all treatment solutions were clear with no visible particulates, surface film, undissolved test substance, or precipitate throughout the exposure.

Percent change in cell density, as compared to the control, ranged from +1% at 0.747 mg whole product/L to -98% at 18.3 mg whole product/L after 72 hours of exposure. One-way analysis of variance showed a statistically significant (p = 0.05) reduction of the cell density in the 2.72, 5.26, 8.81 and 18.3 mg whole product/L, test substance treatments at 72 hours as compared to the control. The NOEC value at 72 hours was 1.27 mg whole product/L based on mean measured concentrations. Based on cell density, the 72-hour

EC50 value was 8.03 mg whole product/L with 95% confidence limits of 7.47 to 8.58 mg whole product/L based on mean measured concentrations.

Percent change in area under the growth curve, as compared to the control, ranged from +2% at 0.747 mg whole product/L, to -99% at 18.3 mg whole product/L after 72 hours of exposure to. One-way analysis of variance showed a statistically significant (p = 0.05) reduction of area under the growth curve in the 2.72, 5.26, 8.81, and 18.3 mg whole product/L, test substance treatments at 72 hours as compared to the control. The NOEC value for area under the growth curve at 72 hours was 1.27 mg whole product/L based on the mean measured concentrations. Based on area under the growth curve, the 72-hour EbC50 value was 8.40 mg whole product/L with 95% confidence limits of 7.96 to 8.84 mg whole product/L based on mean measured concentrations.

Percent change in growth rate, as compared to the control, ranged from 0% at 0.747 and 1.27 mg whole product/L to -78% at 18.3 mg whole product/L after 72 hours of exposure. One-way analysis of variance showed a statistically significant (p = 0.05) reduction of growth rate in the 2.72, 5.26, 8.81, and 18.3 mg whole product/L test substance treatments at 72 hours, as compared to the control. The NOEC value for growth rate at 72 hours was 1.27 mg whole product/L based on mean measured concentrations. Based on growth rate, the 72-hour ErC50 value was 13.3 mg whole product/L with 95% confidence limits of 13.1 to 13.5 ug whole product/L, based on mean measured concentrations.