[No public or meaningful name is available]

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

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 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes

Remarks:

[C(97)186/Final]

Test material

Test material information

Constituent 1

Details on test material:

Sample 1: used to prepare analytical matrix spikes and analytical standard solutions
- Name of test material (as cited in study report): C-4000
- Physical state: liquid
- Analytical purity: >95%
- Purity test date: 2007-10-04
- Lot/batch No.: 51V007D7
- Expiration date of the lot/batch: 2008-04-04
- Storage condition of test material: ambient temperature

Sample 2: used to prepare test solutions during the range finding and definitive tests.
- Name of test material (as cited in study report): C-4000
- Physical state: liquid
- Analytical purity: 86.3 +/- 0.7%
- Purity test date: 2008-01-01 through 2008-01-25
- Lot/batch No.: 51V034K7
- Expiration date of the lot/batch: 2008-02-01
- Storage condition of test material: refrigerated

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: WAF loading rate concentration 10 mg/L
- Sampling method: Duplicate 10 mL sample were collected from the control, vehicle control, and test substance treatment at 0 and 96 hours. QC samples were prepared by fortifying 10 mL of dilution water with C-4000 at concentrations of 286 and 400 ng whole product/mL. A 6 mL volume of each sample was then diluted with 4 mL of methanol. A 0.5 mL sample of the methanol diluted test substance treatment and QC fortification samples were further diluted with 9.5 mL of 40:60 methanol:ABC reagent water.
- Sample storage conditions before analysis: All samples were analyzed by LC-MS/MS directly after sampling

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
-The water accommodated fraction (WAF) solution for the testing was prepared at a nominal loading rate concentration of 10 mg whole product/L. A 100 mg whole product/mL primary standard was prepared by diluting 0.9998 g of C-4000 with acetone to a 10 mL volume. An 8.0 mL volume of the primary standard was added to 80 L of dilution water in a 50 gallon stainless steel barrel to prepare the 10 mg whole product/L treatment. Each 80 L volume was stirred with a stainless steel mixer 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. Beginning with the vehicle control and test substance treatment, the lower aqeous phase of each solution (i.e. water accommodated fraction) was drained from the outlet of each barrel through a medium porosity glass filter tube. A volume of approximately 100 mL was first drained into a waste bucket and a total volume of approximately 39 L was collected by filling several 20-L glass jars. The collected volumes were allowed to acclimate to test temperature.
- Controls: dilution water; vehicle control: acetone/dilution water
- 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 of 100 uL/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no evidence; clear and colorless

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: Trout Lodge, Sumner, Washington
- Length at study termination (length definition, mean, range and SD): The fish in Control Replicate A ranged from 44 to 53mm in total lenth (mean and standard deviation: 49 +/- 3.1 mm); The fish in Control Replicate B ranged from 42 to 54 mm in total length (mean and standard deviation: 49 +/- 3.9 mm).
- Weight at study termination (mean and range, SD): The fish in Control Replicate A ranged from 0.660 to 1.28 g in blotted wet weight (mean and standard deviation: 0.957 +/- 0.204 g); The fish in Control Replicate B ranged from 0.648 to 1.30 g in blotted wet weight (mean and standard deviation: 0.976 +/- 0.217 g).
- Feeding during test: fish were not fed during the test



ACCLIMATION
- Acclimation period: the entire culture was placed in acclimation 2 days prior to test initiation
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: Salmon starter
- Feeding frequency: Daily; food was withheld 2 days prior to test initiation.
- Health during acclimation (any mortality observed): No mortality was observed during the 48-hour acclimation period

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test conditions

Hardness:

132 mg CaCO3/L

Test temperature:

14.3 to 15.5 degrees C

pH:

7.7 to 8.5

Dissolved oxygen:

9.4 to 10.3 mg/L (97 to 106% of saturation) at test initiation
5.7 to 9.2 mg/L (58 to 93% of saturation) throughout the remiander of the test
Gentle aeration was provided to all treatment solutions at 24-hours after the DOC fell below 60% saturation in the vehicle control solution. DOC levels remained above 60% in all treatment levels for the remainder of the test.

Salinity:

324 uS

Nominal and measured concentrations:

Prepared as a water accommodated fraction (WAF)
Mean measured concentration of C-4000 in the test substance was 0.508 mg a.i./L which represented 5 .1% of the nominal loading rate of 10 mg whole product/L and 82% of the initial mean measured concentration

Details on test conditions:

TEST SYSTEM
- Test vessel: 21 L glass jars covered with a plastic lid
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 21 L glass jar
- Aeration: gentle aeration was provided to all control and treatment solutions at 24-hours after the DOC fell below 60% saturation.
- Type of flow-through (e.g. peristaltic or proportional diluter): Static
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 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 CaCO3/L. The water was then filtered through a sediment filter and UV irradiated prior to use.
- Metals: within range
- Pesticides: within range
- Alkalinity: 140 mg CaCO3/L
- Ca/mg ratio: 1.9
- Conductivity: 324 uS
- Intervals of water quality measurement: specific conductivity, total alkalinity and total hardness at test initiation ; temperature- measured in one dilution control test chamber daily and continuously from the waterbath.


OTHER TEST CONDITIONS
- Photoperiod: Fluorescent lighting was maintained on a 16-hour light 8-hour dark photoperiod, with 30-minute simulated dawn and dusk periods.
- Light intensity: 691 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observation for mortality and sublethal responses were made every 24 hours (+/- 1 hour) of the test.


TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: limit test
- Range finding study- run concurrently with definitive limit test
- Test concentrations: 0.010, 0.10 and 1.0 mg whole product/L
- Results used to determine the conditions for the definitive study: after 96 hours of exposure, mortality was 0% and there were no sublethal effects.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.508 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.508 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

other: mortality and sublethal effects

Details on results:

- There were no sublethal effects observed in the control or test substance treatments during the exposure.
- Mortality of control: 0%
- Other adverse effects control: none

Reported statistics and error estimates:

Statistical analysis of the mean measured concentration versus mortality data was not performed since no mortality occurred in the test treatments. In addition, the slope of the concentration-response line could not be calculated.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on mean measured concentration of C-4000, the 96-hour LC50 value for rainbow trout, Oncorhynchus mykiss, was estimated to be >0.508 mg a.i./L. The 96-hour no-observed-effect concentration (NOEC) was 0.508 mg a.i./L, based on the lack of mortality and sublethal effects at this concentration.

Executive summary:

A test was conducted to estimate the potential acute toxicity of C-4000 prepared as a water accommodated fraction (WAF) to the rainbow trout, Oncorhynchus mykiss.  Rainbow trout were exposed for 96 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 . 

A single test substance treatment was prepared as a WAF on a weight/volume basis based 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.  The mean measured concentration of C‑4000 in the test substance treatment was 0.508 mg a.i./L which represented 5.1% of the nominal loading rate of 10 mg whole product/L and 82% 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 daily.  The test solution temperature and pH remained within acceptable limits throughout the exposure.  Due to a drop below 60% oxygen saturation within one of the vehicle control replicates after the initial 24 hours of exposure, all control, vehicle control and treatment replicates received gentle aeration at that time and for the duration of the exposure.  Dissolved oxygen concentration levels remained above 60% in all treatments for the remainder of the test.  Most of the test solutions appeared clear and colorless throughout the exposure with no visible particulates, surface film, undissolved test substance, or precipitate.  Only at test termination was a slight cloudiness observed in the two replicate treatment level test solutions.

After 96 hours, mortality was 0% in the 0 (control), 0 (vehicle control), and 0.508 mg a.i./L treatments.  There were no sublethal effects observed during the exposure.  Based on the mean measured concentration, the 96-hour LC₅₀ for rainbow trout, Oncorhynchus mykiss, exposed to C-4000 was estimated to be >0.508 mg a.i./L, the highest achievable concentration tested.  The 96‑hour no-observed-effect concentration was the mean measured concentration of 0.508 mg a.i./L, the highest test substance treatment with no abnormal effects or mortality.