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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study conducted under GLP. The presence of a precipitate in the test solution (most likely insoluble tin oxide) is an anticipated occurrence based on the known characteristics of the test material, however this precipitate may have resulted in physical toxicity toward the test organism.
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
U.S. EPA OPPTS # 850.1075
Deviations:
no
Qualifier:
according to
Guideline:
other: ASTM Standard E729-96 (1996)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Primary stock solution 120 mg/L
- Sampling method: Samples of the 120 mg/L primary stock solution were collected on Days 0 and 1 of the test to confirm concentration. On Day 0, samples of the test solutions were collected from each test chamber and pooled by treatment group to determine concenetrations of the test substance. Samples also were collected from each test chamber prior to renewal on Day 1 and at test termination to determine test substance concentrations. Samples were collected at mid-depth, placed in glass vials, and analyzed as soon as possible without storage. Additional samples were collected from the primary stock solution and the newly prepared test solutions on Days 1, 2, and 3 of the test, and from the 24-hour old test solutions prior to renewal on Days 2 and 3. These samples were placed in plastic storage containers and were stored under refrigeration for possible future analysis
- Sample storage conditions before analysis: Samples were placed in plastic storage containers and were stored under refrigeration for possible future analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A primary stock solution was prepared at a nominal concentration of 120 mg/L, the highest concentration tested, by mixing 18 g of test substance into 150 L of dilution water in a stainless steel tank. The stock solution was stirred with an electric top-down mixer and sonicated overnight- Eluate: no data
- Differential loading: Loading was defined as the total wet weight of fish per liter of test water and was 0.3 g fish/L.
- Controls: Negative control (dilution water)
- Chemical name of vehicle: none
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Measured concentrations in the newly prepared 120 mg/L stock solutions at 0 and 24 hours were 85 and 93% of nominal. Measured concentrations in the newly prepared test solutions at the beginning of the test ranged from 96 to 101% of nominal. Measured concentrations at 24 and 96 hours ranged from 90 to 99% of nominal concentrations. When the measured concentrations of the test solution samples collected at 0, 24, and 96 hours were averaged, the mean measured concentrations for the study were 15, 25, 40, 68 and 116 mg/L, representing 94, 96, 93, 94 and 97% of nominal concentrations, respectively. The results of the study were based on the mean measured test concentrations.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): After mixing, the stock appeared cloudy white with a yellow precipitate present. The test solutions were prepared in individual test chambers by dilution of the primary stock solution, and were mixed for approximately two to three minutes with an electric top-down mixer. After mixing, the solutions were cloudy white in appearance, increasing in intensity with increasing concentration. New test solutions were prepared daily.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Strain: Oncorhynchus mykiss
- Source: Thomas Fish Company, Anderson, California
- Length at study initiation (length definition, mean, range and SD): The average total length of 10 negative control fish measured at the end of the test was 4.8 cm with a range of 4.5 to 5.2 cm.
- Weight at study initiation (mean and range, SD): The average wet weight (blotted dry) of 10 negative control fish measured at the end of the test was 0.89 grams, with a range of 0.67 to 1.3 grams.
- Method of breeding: No data
- Feeding during test: No
- Food type: Commercially-prepared diet supplied by Zeigler Brothers, Inc., Gardners, PA
- Amount: No data
- Frequency: Daily during 14 day holding period

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions (same as test or not): Fish were held for at least 14 days prior to the test in water from the same source and at approximately the same temperature as used during the test.
- Type and amount of food: Commercially-prepared diet supplied by Zeigler Brothers, Inc., Gardners, PA/ No amount fed documented
- Feeding frequency: Daily during 14 day holding period
- Health during acclimation: During the 14 day holding period prior to the test the fish showed no signs of disease or stress.

QUARANTINE (wild caught)
- Duration: Not applicable
- Health/mortality: Not applicable
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Remarks on exposure duration:
96 hr LC50 >116 mg/L
Post exposure observation period:
Observations were made approximately 3.5, 24, 48, 72 and 96 hours after test initiation.
Hardness:
134 (mg /L as CaCO3)
Test temperature:
Water temperatures were within 12 +\- 1 deg C range established for the test. The temperature of the test water ranged from 11.4 to 12.4 across all test concentrations at 96 hours.
pH:
Measurements of pH ranged from 8.1 to 8.6.
Dissolved oxygen:
Negative control- 96 hours 8.6-9.0 (First reading represents replicate A and second number represents replicate B)
15 (mg/L) 8.5-8.3
25 (mg/L) 8.6-8.2
40 (mg/L) 8.3-8.4
68 (mg/L) 8.3-8.6
116 (mg/L) 8.5-8.3
Nominal and measured concentrations:
Nominal concntrations: Negative control (0.0), 16, 26, 43, 72, 120, mg/L
Mean measured concentrations: Negative control (0.0), 15, 25, 40, 68, 116 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Test chambers were 38-L stainless steel aquaria, alternating with 54 =-L stainless steel aquareia ant each renewal cycle.
- Aeration: No data
- Renewal rate of test solution: semi static
- No. of organisms per vessel: 20
- No. of vessels per concentration: two
- No. of vessels per control: two
- No. of vessels per vehicle control (replicates): no data
- Biomass loading rate: no data

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for culturing and testing was freshwater obtained from a well approximately 40 meters deep located on the Wildlife International, Ltd. site. The well water is characterized as moderately -hard water. The well water was passed through a sand filter to remove particles greater than approximately 25 um, and pumped into 37,800-L storage tank where the water was aerated with spray nozzles. Prio to use, the water was filtered to 0.45 um to remove fine particles.
- Alkalinity: 180 mg/L as CaC03
- Conductivity: 285 (umhos/cm)
- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature, dissolved oxygen and pH were measured in each test chamber at the beginng and end of the test and at approximately 24 hour intervals during the test, including before and after each renewal. Specific conductance, hardness and alkalinity were measured in the dilution water at the beginning of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: No data
- Photoperiod: A Photoperiod of 16 hours of light and 8 hours of darkness was controlled with an automatic timer.
- Light intensity: Light intensity was measured using a SPER Scientific Model 840006C light meter, Fluorescent light bulbs that emit wavelengths similar to natural sunlight (Colortone 50) were used for illumination of the cultures and test chambers. A Photoperiod of 16 hours of light and 8 hours of darkness was controlled with an automatic timer. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting. Light intensity at test initiation was 154 lux at the surface of the water of one represenative test chamber.

EFFECT PARAMETERS MEASURED:
Mortality and clinical observations were made at 3.5, 24, 48, 72, and 96 hours. There was no mortality at any time point. All negative control fish appeared normal throughout the test period. One fish in the 116 mg/L treatment group appeared lethargic at test termination. All other fish in the treatment groups appeared normal throughout the test. The no-mortality concentration was 116 mg/L and the NOEC was 68 mg/L. LC50 values at 24, 48, 72 and 96 hours were estimated to be >116 mg/L, the highest concnetration tested.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approximately 1.67.
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: yes
- Test concentrations: Test concentrations selected were 16, 26, 43, 72, and 120 mg test substance/L
- Results used to determine the conditions for the definitive study: No data
Reference substance (positive control):
not specified
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
68 mg/L
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 116 mg/L
Details on results:
- Behavioural abnormalities: None
- Observations on body length and weight: None
- Other biological observations: None
- Mortality of control: None
- Other adverse effects control: None
- Abnormal responses: Only observation was lethargy in one fish at 116 mg/L at 96 hours
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
The absence of mortality in all treatment groups precluded the statistical calculation of LC50 values. Therefore, the 24, 48, 72, and 96 hour LC50 values were estimated to be greater than the highest concentration tested. The no-mortality concentration (NOEC) were determined by visual intrepretation of the mortality and observation data.
Validity criteria fulfilled:
yes
Conclusions:
Rainbow trout (Oncorhynchus mykiss) were exposed for 96 hours under static-renewal conditions to five mean measured concentrations of Tin bis (2-ethylhexanoate) ranging from 15 to 116 mg/L. The 96-hour LC50 value was >116 mg/L, the highest concentration tested. The no-mortality concentration was 116 mg/L and the no-observed -effect concentration (NOEC) was 68 mg/L.
Executive summary:

The objective of this study was to evaluate the acute toxicity of Tin bis (2 -ethylhexanoate) to the rainbow trout during a 96 -hour exposure period under static-renewal test conditions. Rainbow trout were exposed to a geometeric series of five test concentrations and a negative (dilution water) control for 96 hours. Two replicate test chambers were maintained in each treatment and control group, with 10 fish in each test chamber for a total of 20 fish per concentration. Surviving fish were transferred to freshly prepared test solutions at approximately 24 -hour intervals. Nominal test concentrations were selected in consultation with the Sponsor, and were based upon the results of an exploratory range finding toxicity test. Nominal test concentrations selected were determined from samples of test water collected from each treatment and control group at the beginning of the test, prior to renewal at 24 hours, and at the end of the test. Rainbow trout were impartially assigned to exposure chambers at test initiation. Observations of mortality and other signs of toxicity were made approximately 3.5, 24, 48, 72 and 96 hours after test initiation. The cumulative percent mortality observed in the treatment groups was used to determine LC50 values at 24, 48, 72, and 96 hours. The no-mortality concentration and the no-observed -effect concentration (NOEC) were determined by visual interpretation of the mortality and observation data.

24, 48, 72, and 96 hours LC50 >116 mg/L - 95% confidence limits could not be calculated from the data. A statistical method was not applicable; the absence of mortality precluded statistical calculation of an LC50 value.

Description of key information

The 96-hour LC50 value of >116 mg/L for tin bis(2-ethylhexanoate) was taken from a valid exposure study with rainbow trout (Oncorhynchus mykiss).

Key value for chemical safety assessment

LC50 for freshwater fish:
116 mg/L

Additional information

One acute freshwater fish study with rainbow trout (Oncorhynchus mykiss) was assessed for this endpoint. This study was determined to be acceptable for use in risk assessment (Klimisch score = 2). Rainbow trout were exposed to a geometric series of five test concentrations and a negative (dilution water) control for 96 hours in a static-renewal exposure system. Test solutions were renewed every 24 hours. Observations of mortality and other signs of toxicity were made approximately 3.5, 24, 48, 72 and 96 hours after test initiation. The 24, 48, 72, and 96 hour LC50 values were >116 mg/L tin bis(2-ethylhexanoate).