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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-08-26 to 2011-10-24
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Method; For dose confirmation, 10-mL aliquots of test water were transferred to tubes containing 1-mL aliquot of internal standard extraction solution in 14-mL glass tubes. The tubes were capped and vortexed for 2 minutes, then centrifuged for to separate the aqueous and solvent phase. A portion of the hexane layer was transferred to an autosampler vial for analysis.
Vehicle:
yes
Details on test solutions:
Method: The nominal test concentrations was 0.21 mg/L A stock solution was prepared by adding 2.104 g of test article to a 1-L volumetric flask. The flask was brought to volume with dimethylformamide (DMF) and inverted to mix.

The stock solution was delivered to a mixing chamber (at a rate of 0.015 mL/minute) where it was mixed with dilution water (at a rate of 150 mL/minute) to achieve the desired test concentration (1:10,000 dilution factor). The solvent control was prepared using DMF without test article. The concentration of DMF in the treatment group and the solvent control was 0.1 mL/L. All test solutions appeared clear and colorless throughout the test.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
Source: Rainbow trout used in this test were obtained from Thomas Fish Company, Anderson, California on September 21, 2011.

Holding conditions: The rainbow trout were held for 27 days in water from the same source as used during the test. During the 7-day holding period preceding the test, water temperatures ranged from 11.8 to 12.6°C.

Feeding: During holding, rainbow trout were fed finfish starter #1 obtained from Zeigler Brothers, Gardners, PA. The trout chow is analyzed annually for selected contaminants. No contaminants have been shown to be present in the trout chow at concentrations that might affect the outcome of this study. The trout were not fed for two days prior to test initiation and were not fed during the test.

Pre-test mortality: No mortality was observed during the 7-Day acclimation period prior to the test.

Test initiation: At test initiation, rainbow trout were collected from the holding tank and transferred to the test chambers. All fish used in the test were from the same source and the length of the longest fish was no more than twice the length of the shortest. The average total length of 10 negative control fish sampled at test termination was 41 mm with a range of 40 to 43 mm. The average wet weight was 0.50 grams with a range of 0.41 to 0.58 grams.

Biomass loading: The flow-through loading rate was 0.046 g of fish/L of water passing through the test chambers in 24 hours. Instantaneous loading was 0.33 g/L of test water present in the test chambers at any one time.
Test type:
flow-through
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
137 mg/L as CaCO3
Test temperature:
11.8 to 12.5°C
pH:
7.1 to 7.5
Dissolved oxygen:
≥8.0 mg/L (76% of saturation)
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal test concentration: 0.21 mg/L

Mean measured concentration: 0.13 mg/L (62% of nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: Test vessels were 25-L polyethylene aquaria containing approximately 15-L of test solution. Test vessels were covered with clear Plexiglas to prevent the fish from escaping. Test vessels were placed in a temperature-controlled water bath set to maintain a temperature of 12+/-1°C. Test vessels were labelled with the study number, test concentration and replicate.

- Type of flow-through (e.g. peristaltic or proportional diluter): The test article was mixed with dilution water using a continuous-flow diluter system. Materials that came into contact with the test article were constructed of glass, Teflon, stainless steel or polyethylene. Syringe pumps (KD Scientific Inc, Holliston, MA) were used to deliver the neat test article, working stock solutions and solvent for the solvent control to mixing chambers where they were mixed with dilution water. Syringe pumps were verified prior to the test and daily during the test. Dilution water was delivered using rotameters (Porter Instrument Company, Inc., Hatfield, PA). Rotameters were calibrated/verified prior to the test. After mixing, the test solutions were split into two replicate test chambers. The proportion of water split to the two replicates was checked prior to the test to ensure that the flow rate to each replicate was +/-10% of the mean of the two replicates. The diluter flow rates were maximized to maximize the recovery of the test article. Flow rates were adjusted to provide approximately 7.2 volume additions per day to each replicate. The general operation of the diluter was checked and recorded twice per day during the test. The diluter system was operated for approximately 13 days prior to test initiation to allow equilibrium of the solutions in the test chambers.

- Renewal rate of test solution (frequency/flow rate):

- 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: The dilution water was municipal water obtained from Bay City, Michigan. The water was dechlorinated using a carbon filter and aerated prior to use. The pH of the water is periodically adjusted using CO2, as necessary.

- Culture medium different from test medium: No

- Intervals of water quality measurement: Temperature, dissolved oxygen and pH were measured in each test chamber daily.

OTHER TEST CONDITIONS
- Adjustment of pH: No

- Lighting: Lighting used to illuminate the test chambers was provided by cool white fluorescent bulbs with a photoperiod of 16 hours light and 8 hours dark. Light intensity at test initiation was 61 foot-candles.

EFFECT PARAMETERS MEASURED: Observations were made to evaluate the number of mortalities. The numbers of individuals exhibiting clinical signs of toxicity or abnormal behaviour also were evaluated. Observations were made at test initiation and approximately 3, 24, 48, 72 and 96 hours after test initiation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Limit test
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 0.13 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.13 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: None

- Mortality of control: None
Reported statistics and error estimates:
The 24, 48, 72 and 96-hour LC50 values were estimated by visual inspection of the mortality and clinical observation data.

Table 1. Results of analysis of test media

Nominal Concentration (mg/L)

Replicate

Measured Concentration (mg/L)

 

Day 0        Day 2       Day 4

(17Oct2011)   (19Oct2011)   (21Oct2011)

Mean Measured Concentration (mg/L)

Percent of Nominal

RSD

Negative

Control

A

B

-

ND1

ND

-

-

ND

ND

 

 

 

 

 

 

 

 

 

 

Solvent

Control

A

B

-

ND

ND

-

-

ND

ND

 

 

 

 

 

 

 

 

 

 

0.21

A

B

NA

0.136

0.117

NA

NA

0.140

0.131

62%

9.3%

1ND = not detected, there was no observable response at the retention time of TMDS

 

Table 2. Test results

Mean Measured Test Concentration

(mg/L)

 

 

Replicate

0 Hour

 

3 Hours

 

24 Hours

 

48 Hours

 

72 Hours

 

96 Hours

 

 

 

 

Cumulative %

Mortality

No.

Exposed

 

No.

Dead

 

Effects1

 

No.

Dead

 

Effects

 

No.

Dead

 

Effects

 

No.

Dead

 

Effects

 

No.

Dead

 

Effects

Negative Control

 

 

A

B

10

10

 

0

0

10 N

10 N

 

0

0

10 N

10 N

 

0

0

10 N

10 N

 

0

0

10 N

10 N

 

0

0

10 N

10 N

 

 

0

Solvent Control

 

 

A

B

10

10

 

0

0

10 N

10 N

 

0

0

10 N

10 N

 

0

0

10 N

10 N

 

0

0

10 N

10 N

 

0

0

10 N

10 N

 

0

0.13

 

 

A

B

10

10

 

0

0

10 N

10 N

 

0

0

10 N

10 N

 

0

0

10 N

10 N

 

0

0

10 N

10 N

 

0

0

10 N

10 N

 

0

1Effects: N = Normal

Validity criteria fulfilled:
yes
Conclusions:
A 96-hour LC50 value of >0.13 mg/L and NOEC of ≥0.13 mg/L have been determined for the effects of the effects of the registered substance on mortality of Oncorhynchus mykiss. The results were obtained under flow-through exposure conditions are expressed relative to mean measured concentrations of the substance.

Description of key information

Short-term toxicity to fish: 96-h LC50 >0.13 mg/l (measured, arithmetic mean) (highest concentration tested) (OECD Guideline 203)

Key value for chemical safety assessment

Additional information

A 96 hour LC50 value of >0.13 mg/l (mean measured concentration) (highest concentration tested) has been determined for the effects of the registration substance on mortality of Oncorhynchus mykiss (Dow Corning Corporation, 2011). In view of the test media preparation method/exposure regime it is likely that the test organisms were exposed predominantly to the tested substance. Effectively these results indicate no effects at the limit of achievable solubility in test medium.