[No public or meaningful name is available]

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

yes

Remarks:

[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: Sample 1 >99%; Sample 2 >95%
- Purity test date: Sample 1 2007-02-26; Sample 2 2007-10-04
- Lot/batch No.: Sample 1 JYC0367; Sample 2 51V007D7
- Expiration date of the lot/batch: Sample 1 2007-08-26; Sample 2 2008-04-04
- Storage condition of test material: ambient temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: - Sampling method: Duplicate 10 mL samples were collected from the control, vehicle control, and test substance treatment at 0 and 48 hours. The 0 hour samples were collected directly from the parent solutions. The 48 hour samples were comprised of pooled 5 mL volumes collected from
each A and B replicate and pooled 5 mL volumes collected from each C and D replicate. QC samples were prepared by fortifying 10 mL of dilution water with C-4000 at concentrations of 0.0400 and 0.0572 mg whole product/L at 0 hour and at concentrations of 0.286 and 0.400 mg whole product/L at 48 hours. A 6 mL volume of each 10 mL sample was then diluted with 4 mL of methanol and a 1 mL sample of the methanol diluted test substance treatment and QC samples were further diluted with 1 mL 40:60 methanol:ABC reagent water.
- 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: A 100 mg whole product/mL primary standard was prepared by diluting 1.0001 g of C-4000 with acetone to a 10 mL volume. A 0.18 mL volume of the primary standard was added to 1.8 L of dilution water in a 2-L glass aspirator bottle to prepare the test substance treatment. Each 1.8 L volume sealed with parafilm and stirred with a teflon stir bar for approximately 19 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 one hour. Following the settling period, the solutions were clear and colorless and an oily-like surface film was present in the 10 mg whole product/L solution. Beginning with the control, followed by the vehicle control and test substance treatment, the lower aqueous phase of each solution (i.e., water-accommodated fraction) was drained from the bottom outlet of each aspirator bottle 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 1,000 mL was collected in a glass beaker. The collected volumes were allowed to acclimate to test temperature then transferred to appropriately labeled test chambers.
- Differential loading: Nominal Loading Rates: 0 (control), 0 (vehicle control; 0.10 mL acetone/L), and 10 mg whole product/L
- Controls: 0 (control), 0 (vehicle control; 0.10 mL acetone/L)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
- Concentration of vehicle in test medium (stock solution and final test solution): not exceeding 100 uL/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): When stirring was terminated, the phases were allowed to separate for approximately one hour. Following the settling period, the solution was clear and colorless with an oily-like surface film and a few small-spherical globules of material present on the bottom of the aspirator bottle.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Fruit fly
- Strain: Daphnia magna
- Source: Obtained from an in-house culture.
- Age at study initiation (mean and range, SD): Neonates, approximately 17 days old
- Method of breeding: The adult daphnids were cultured in a temperature-controlled waterbath at approximately 20°C.
- Feeding during test: Test daphnids were not fed during the test.


ACCLIMATION
- Acclimation period: Culturing and testing environmental parameters were equivalent, no acclimation period was necessary.
- Acclimation conditions (same as test or not): Since the culturing and testing environmental parameters were equivalent (i.e. temperature, dilution water, and lighting), no acclimation period was necessary.
- Type and amount of food: The daphnids were fed a suspension of the algal species, Pseudokirchneriella subcapitata (formerly Selenastrumcapricornutum) supplemented by an artificail diet
- Feeding frequency: At least once a day
- Health during acclimation (any mortality observed): No adult mortality occurred during the 48 hour period immediately prior to production of neonates used at test initiation.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

140 mg CaCO3/L

Test temperature:

19.8 to 20.9 degrees C

pH:

8.3 to 8.5

Dissolved oxygen:

5.7 to 8.4 mg/L (66 to 99% saturation)

Salinity:

356 uS

Nominal and measured concentrations:

Nominal loading rate: 10 mg whole product/L
Mean measured concentration: 0.290 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250-mL glass beakers
- Type (delete if not applicable): Open
- Material, size, headspace, fill volume: glass, 250-mL containing 200 -mL of test or control solution
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Static
- No. of organisms per vessel: five
- No. of vessels per concentration (replicates): Four
- No. of vessels per control (replicates): Four
- No. of vessels per vehicle control (replicates): Four



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was an aged laboratory freshwater prepared by blending naturally hard well water with well water that was demineralized by reverse osmosis. These waters were blended to yield a total hardness of approximately 130 to 160 mg CaCO3/L and biologically aged (held in a tank containing aquatic organisms). The water was then filtered through a sediment filter and UV irradiated prior to use.
- Metals: within range
- Pesticides: within range
- Alkalinity: 144 mg CaCO3/L
- Ca/mg ratio: NA
- Conductivity: 356 uS

- Culture medium different from test medium: no
- Intervals of water quality measurement: initiation and termination


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16-hr light:8-hr dark, with 30 minute dawn/dusk transition periods
- Light intensity: 537 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable): immobilization/mortality and sublethal responses at 24 and 48 hours of the test.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test
- Justification for using less concentration than requested by guideline: limit test
- Range finding study
- Test concentrations: The nominal loading rates for the range-finding test were 0 (control), 0 (vehicle control; 0.10 mL acetone/L), 0.0010, 0.010, 0.10, 1.0, and 10 mg whole product/L.
- Results used to determine the conditions for the definitive study: After 48 hours of exposure, immobilization in the controls and all test substance treatments was 0% and there were no sublethal effects. Based upon the biological results of this test, the nominal loading rates of 0 (control), 0 (vehicle control; 0.10 mL acetone/L), and 10 mg whole product/L were selected for the definitive test.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.29 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.29 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

other: sublethal effects

Details on results:

- Behavioural abnormalities: In this study no abnormal effects or immobility occurred at the highest concentration tested.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test substance, C-4000, was not soluble in the dilution water. The nominal loading rate concentration of C-4000 used in the dilution water was 10 mg/L.

Reported statistics and error estimates:

Statistical analysis of the mean measured concentration versus immobility data was not performed since <10% immobility occurred in the treatment tested. In addition, the slope of the concentration-response line could not be calculated.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

After 48 hours, immobility was 0% in the 0 (control), 0 (vehicle control), and 0.290 mg/L treatments. There were no sublethal effects observed during the exposure. Based on the mean measured concentration, the 48-hour EC50 for Daphnia magna exposed to C-4000 was estimated to be >0.290 mg/L, the highest achievable concentration tested. The 48-hour noobserved- effect concentration was the mean measured concentration of 0.290 mg/L, the highest test substance treatment with no abnormal effects or immobility.

Executive summary:

A test was conducted to estimate the potential acute toxicity of C-4000 to the water flea, Daphnia magna. Daphnids were exposed for 48 hours under static conditions to nominal loading rates of 0 (control), 0 (vehicle control; 0.10 mL acetone/L), and 10 mg whole product/L C-4000. The guideline followed was the OECD guideline 202. A single test substance treatment was prepared on a weight/volume basis based on whole product. The mean measured concentration of C-4000 in the test substance treatment was 0.290 mg/L which represented 2.9% of the nominal loading rate and 87% of the initial mean measured concentration. No residues of C-4000 were detected in the control or vehicle control solutions above the minimum quantifiable limit of 0.00289 mg/L. Water quality characteristics of temperature, dissolved oxygen concentration, and pH were measured at initiation and termination and remained within acceptable limits throughout the exposure. All test solutions appeared clear and colorless throughout the exposure with no visible particulates, surface film, undissolved test substance, or precipitate. After 48 hours, immobility was 0% in the 0 (control), 0 (vehicle control), and 0.290 mg/L treatments. There were no sublethal effects observed during the exposure. Based on the mean measured concentration, the 48-hour EC50 for Daphnia magna exposed to C-4000 was estimated to be >0.290 mg/L, the highest achievable concentration tested. The 48-hour noobserved- effect concentration was the mean measured concentration of 0.290 mg/L, the highest test substance treatment with no abnormal effects or immobility.