[No public or meaningful name is available]

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:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

Test flasks were not inoculated within 30 minutes of the final test solution preparation. They were inoculated 45 minutes after final test soution preparation.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

yes

Remarks:

Samples were collected from a single biotic analytical replicate of test substance solutions at each sampling period. Single samples were then analyzed in triplicate rather than triplicate samples collected from the test substance replicate and analyzed

GLP compliance:

yes

Remarks:

OECD [C(97)186/Final]

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): C-4000
- Physical state: liquid
- Analytical purity: first sample: >95%; second sample: 86.3 +/- 0.7%
- Purity test date: first sample: 2007-10-04; second sample: 2008-01-01 through 2008-1-25
- Lot/batch No.: first sample: 51V007D7; second sample: 51V034K7
- Expiration date of the lot/batch: first sample: 2008-04-04; second sample: 2008-02-01
- Storage condition of test material: first sample: ambient temperature; second sample: refrigerated
The first sample was used to prepare analytical matrix spikes and analytical standard solutions.
The second sample was used to prepare test solutions during the range-finding and limit tests.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: geometric mean concentrations: 0, 0 (vehicle control), 0.266 mg C-4000/L at 72 hours, 0.228 mg C-4000/L at 96 hours; nominal loading rate: 10 mg whole product/L
- Sampling method: Ten mL samples were collected from the control, vehicle control , and the test substance treatment at each time point. The 0-hour samples were collected directly from the parent solutions. The 24, 48, 72 and 96-hour samples were collected from a single biotic analytical repliate for each time period. An abiotic replicate of the lowest test concentration was prepared for sampling at 96-hours. All samples were centrifuged for approximately 10 minutes after collection.
- Sample storage conditions before analysis: Directly analyzed by LC-MS/MS

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: prepared as water accommodated fraction solutions. A 100 mg whole product/mL primary standard was prepared by diluting 0.5001 g C-4000 with acetone to a 5 mL volume. A 0.18 mL volume of the primary standard was added to 1.8 L of freshwater algal medium in a 2-L glass aspirator bottle to prepare the 10 mg whole product/L treatment. Following addition of the acetone-diluted C-4000 to freshwater algal medium, the test substance treatment was covered with parafilm and stirred with a Teflon stir bar for approximately 20 hours. The stirring was adjusted to provide a vortex <25% of the solution depth. When stirring was terminated, the phases were allowed to separate for approximately one hour. Following the settling period, the solution was cleaer and colorless. The bottom aqueous phase was drained from the aspirator bottles through a medium porosity glass filter tube. A volume of approximately 100 mL was first drained into a waste bucket and the next volume of approximately 1,500 mL was collected in an autoclaved 2-L volumetric flask. The collected volume was then allowed to acclimate to test temperature. The collected solution was used as the highest test substance treatment, and the three lower test substance treatments were prepared individually using appropriate volumes of the collected solution and freshwater algal medium.
- Controls: blank control, vehicle control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): maximum concentration 100 uL/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): none

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Strain: UTEX 1648
- Source (laboratory, culture collection): Obtained from an established ABC Laboratory culture which originated from an inoculum (formerly Selenastrum capricornutum) from Department of Botany, Culture Collection of Algae, University of Texas at Austin.
- Age of inoculum (at test initiation): 3 days old
- Method of cultivation: peridoically, new Selenastrum cultures were cloned from an existing culture derived from the parent stock. Cultured in freshwater algal nutrient medium under continuous illumination.


ACCLIMATION
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: none

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test conditions

Hardness:

258 mg CaCO3/L (reagent water used to prepare algal growth medium)

Test temperature:

22.3 to 24.4 degrees C

pH:

7.4 to 8.8

Dissolved oxygen:

not given

Salinity:

not given

Nominal and measured concentrations:

10 mg whole product/L; geometric mean concentrations: 0.266 mg C-4000/L at 72-hours; 0.228 mg C-4000/L at 96-hours

Details on test conditions:

TEST SYSTEM
- Test vessel: 250-mL Erlenmeyer flasks with foam stoppers
- Material, size, headspace, fill volume: glass, 250 mL
- Initial cells density: 1.0e4 cells/mL for each flask
- Control end cells density: 72-hours control: 38e4 cells/mL, 72-hour vehicle control: 40e4 cells/mL; 96-hour contorl: 136e4 cells/mL, 96-hour vehicle control 143e4 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes; freshwater algal nutrient medium prepared in ABC reagent water



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: filtered freshwater algal growth medium prepared with ABC reagent water
- Metals: within range
- Pesticides: within range
- Alkalinity: 304 mg CaCO3/L (reagent water used to prepare algal growth medium)
- Ca/mg ratio:
- Conductivity: 597 uS (reagent water used to prepare algal growth medium)
- Culture medium different from test medium: no
- Intervals of water quality measurement: temperature and pH measured at 0 and 96 hours. Temperature in the environmental chamber was measured continuously.


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 4159 to 4603 lux; cool, white fluorescent bulbs


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell density: hemacytometer and an optical microscope; 24, 48, 72 and 96 hours (+/- 1 hour from test initiation) to evaluate algal growth (inhibition or enhancement). In addition, microscopic examinations were conducted to determine if there are any morphological or physical effects on algal cells.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test
- Justification for using less concentrations than requested by guideline: limit test
- Range finding study- an abbreviated range-finding test was conducted concurrently with the definitive test. The test substance was anticipated to be of low toxicity at the nominal loading rate of the WAF.
- Test concentrations: 0, 0(vehicle control), 0.10%, 1.0%, and 10% of a 10 mg whole product/L
- Results used to determine the conditions for the definitive study: all test solutions were clear with no visible particulates, surface film, undissolved test substance, or precipitate throughtout the test. The control cell density at termination was 136e4 cells/mL.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.266 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: could not be determined

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.266 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: could not be determined

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.266 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: cell density

Remarks on result:

other: could not be determined

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.266 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.266 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.266 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: cell density

Effect concentrations 7

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 0.225 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: could not be determined

Effect concentrations 8

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 0.225 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: could not be determined

Effect concentrations 9

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 0.225 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: cell density

Remarks on result:

other: could not be determined

Effect concentrations 10

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.225 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 11

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.225 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 12

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.225 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: cell density

Details on results:

- Exponential growth in the control (for algal test): yes, the number of algal cells was at least 16 times the initial inoculum density in the control after 2 hours
- Observation of abnormalities (for algal test): clear with no visible particulates, surface film, undissolved test substance, or precipitate throughout the exposure.
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
- Effect concentrations exceeding solubility of substance in test medium: no

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 te 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 96-hour area under the growth curve data. Non-parametric analses were performed on the 24-, 48- and 96-hour cell density, 24-hour under the growth curve, and 24-, 48- and 96-hour growth rate data.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The number of algal cells in the control after the intial 72 hours of the test were at least 16 times the initial inoculum density and the final density was approximately 1e6 cells/mL after 96 hours of testing to verify logarithmic phase growth during the test duration. The coefficient of variation for daily growth rates in the control replicates during the course of the test did not exceed 35% during the first 72 hours of testing for the OECD requirements. The coefficient of variation of average specific growth rates during the whole test period in control replicates did not exceed 7% and therefore met validity criteria for less than 7% variation. In addition, the coefficicent of variation for cell numbers in the control replicates at 96 hours did not vary by more than 20% to satisfy the OPPTS requirements. Since the study was conducted as a limit test, no decrease in growth was required for the test substance treatment relative to the control. The pH in the control did not increase moe than 1.5 units after the initial 72 hours to satisfy the OECD acceptability requirements for pH.

Executive summary:

A growth inhibition test was conducted to estimate the potential toxicity if C-4000 to the unicellular green alga, Pseudokirchneriella subcapitata. Algal cells were exposed for 96 hours to nominal loading rate concentrations of 0 (control), 0 (vehicle control), and 10 mg whole product/L of C-4000.

A single test substance treatment was prepared as a WAF on a weight/volume basis on whole product. For the purposes of the analytical verification, it was assumed that the whole product was >99% pure and therefore the analytical verification of the active ingredient (a.i.) concentrations reflect the whole product concentrations at 96 hours. The 72 -and 96 -hour geometric mean measured concentrations of C-4000 in the test substance treatment were 0.266 and 0.225 mg a.i./L, respectively. This represented 2.7% and 2.3% of the nominal loading rate of 10 mg whole product/L and 69% and 59% of the initial mean measured concentration, which was 0.383 mg a.i./L, respectively. No residues of C-4000 were detected in the control or vehicle control solutions above the minimum quantifiable limit of 0.00282 mg/L. All water quality parameters remained within acceptable limits throughout the 96 -hour exposure. Test solution temperatures, measured at 0 and 96 hours, ranged from 22.3 to 24.4 degrees C. The continuous temperature recording indicated a periodic average temperature of 23.5 +/- 0.1 degrees C ranging from 22.9 to 24.0 degrees C. Test solution pH ranged from 7.4 to 8.8 during the 96 hours.

Percent inhibition in cell density, as compared to the pooled control, was -9% at the geometric mean concentration of 0.225 mg/L after 96 hours of exposure. One-way analysis of variance showed no significant (p = 0.05) reduction in cell density in the test substance treatment at 72 and 96 hours, as compared to the pooled control. Due to the lack of a statistically significant reduction in cell density, the NOECs at 72 and 96 hours were 0.266 and 0.225 mg C-4000/L, respectively. Based on cell density, the 72 - a nd 96 -hour EC50 values were >0.266 and >0.225 mg C-4000/L, respectively.

Percent inhibition in growth rate from 0 hour, as compared to the pooled control, was -2% at the geometric mean concentration of 0.225 mg C-4000/L after 96 hours of exposure. One-way analysis of variance showed no significant (p = 0.05) reduction of growth rate from 0 hour in the test substance treatment at 72 and 96 hours, as compared to the pooled control. The NOECs at 72 and 96 hours were 0.266 and 0.225 mg C-4000/L. Due to the lack of statistically significant reductions in growth rate from 0 hour, the 72- and 96 -hour EC50 values were >0.266 and >0.225 mg C-4000/L, respectively.

Percent inhibition in yield, as compared to the pooled control,was -10% at the geometric mean concentration of 0.225 mg C-4000/L after 96 hours of exposure. One-way analysis of variance showed no significant reduction in yield (p = 0.05), for 0.266 and 0.225 mg C-4000/L at 72 and 96 hours, compared to the pooled controls. At 72 and 96 hours the NOECs were 0.266 and 0.225 mg C-4000/L, respectively. Based on yield, the 72- and 96- hour EC50 values were >0.266 and >0.225 mg C-4000/L, respectively.