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Ecotoxicological information

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:
From July 05, 2009 to July 10, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPP 72-1 (Fish Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Samples were collected from each test chamber of each treatment and control group at approximately 0, 48 and 96 hours of the test to measure concentrations of the test substance. All samples were collected at mid-depth, placed in glass vials, and processed immediately without storage.
Vehicle:
no
Details on test solutions:
Individual test solutions were prepared directly in each test chamber. Solutions were prepared by mixing calculated amounts of test substance in dilution water with a final volume of 45 L to achieve the nominal concentrations of 9.9, 15, 22, 33 and 50 mg/L. The solutions were mixed with electric top-down mixers for approximately 1 to 2 minutes. The negative control solution was dilution water only.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
The rainbow trout, Oncorhynchus mykiss, was selected as the test species for this study. Rainbow trout used in the definitive test were hatched on May 11, 2009 and were obtained from Thomas Fish Company, Anderson, California. Identification of the species was verified by the supplier. All fish used in the test were from the same source and year class, and the length of the longest fish measured was no more than twice the length of the shortest. The average total length per fish of 10 negative control fish measured at the end of the test was 5.0 cm, with a range of 4.7 to 5.5 cm. The average wet weight (blotted dry) per fish of 10 negative control fish measured at the end of the test was 1.3 grams, with a range of 1.0 to 1.7 grams. Loading was defined as the total wet weight of fish per liter of test water and was 0.29 g fish/L. The rainbow trout 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.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
144 mg CaCO3/L (at test initiation)
Test temperature:
11.5 – 12.6oC (during holding); 12.6 – 12.9o C (during the test)
pH:
7.7 – 8.0 (during holding); 8.2 – 8.8 (during the test)
Dissolved oxygen:
8.2 – 9.3 mg/L (during holding); 6.9 – 9.6 mg/L (during the test)
Conductivity:
301 μS/cm (at test initiation)
Nominal and measured concentrations:
Nominal: Negative Control; 9.9 mg/L; 15 mg/L; 22 mg/L; 33 mg/L; 50 mg/L
Mean Measured: < LOQ; 9.5 mg/L; 15 mg/L; 21 mg/L; 34 mg/L; 50 mg/L
Details on test conditions:
During the 2-week period immediately preceding the test, water temperatures in the cultures ranged from 11.5 to 12.6°C, measured with a hand-held liquid-in-glass thermometer. The pH of the water ranged from 7.7 to 8.0, measured with a Fisher Scientific Accumet Model 915 pH meter. Dissolved oxygen concentrations ranged from 8.2 to 9.3 mg/L (≥76% of saturation), measured with a Yellow Springs Instruments Model 85 dissolved oxygen meter. Daily during the holding period, the fish were fed a commercially-prepared diet supplied by Zeigler Brothers, Inc., Gardners, Pennsylvania. The fish were not fed for at least two days prior to the test or during the test. During the 14-day holding period prior to the test, no mortalities occurred and the fish showed no signs of disease or stress. At test initiation, the rainbow trout were collected from the holding tank and impartially distributed one and two at a time to the test chambers until each contained 10 fish.

Test Apparatus: test chambers were 54-L stainless steel aquaria containing approximately 45 L of test solution. The depth of water in a representative test chamber was 24.5 cm.
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 μm, and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles. Prior to use, the water was filtered (0.45 μm) again to remove fine particles.
Light: Ambient laboratory light was used to illuminate the test systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight were controlled by an automatic timer to provide a photoperiod of 16 hours of light and 8 hours of darkness.
Temperature: The target test temperature during the study was 12 ± 1°C. Temperature was measured in each test chamber at the beginning of the test, at approximately 24-hour intervals during the test, and at the end of the test using a liquid-in-glass thermometer.
Dissolved oxygen and pH were measured in each test chamber at the beginning and end of the test and at approximately 24-hour intervals during the test.
Specific conductance, hardness and alkalinity in the dilution water were measured at the beginning of the test.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
50 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Confidence intervals
Remarks:
34 mg/L and above
Details on results:
All rainbow trout in the 9.5, 15, 21 and 34 mg/L treatment groups also appeared normal throughout the test with no mortalities or overt signs of toxicity observed. At test termination, ten fish in the 50 mg/L were dead, nine of the surviving fish exhibited signs of toxicity (lethargy) and only one fish was observed to be normal.
Reported statistics and error estimates:
The LC50 value at 96 hours of the test was analyzed using the computer program of C. E. Stephan. The program was designed to calculate the LC50 value and the 95% confidence interval by probit analysis, the moving average method, and binomial probability with nonlinear interpolation.
At a confidence level of 95%, the binomial test shows that the LC50 value at 96 hours was above 34 mg/L and that 50 mg/L can be used to empirically derive an LC50 for the data obtained. The no-mortality concentration and NOEC were determined by visual interpretation of the mortality and observation data.
Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the binomial test showed that the LC50 was above 34 mg/L, at a confidence level of 95%. The 96 h LC50 value was empirically derived to be 50 mg/L, with a 95% confidence interval of 34 mg/L and above. The no-mortality concentration and the NOEC were 34 mg/L.
Executive summary:

A study was conducted to determine the toxicity of the test substance to rainbow trout (Oncorhynchus mykiss) according to OECD Guideline 203, EPA OPPTS Method 850.1075 and EPA OPP Method 72-1, in compliance with GLP. Rainbow trout were exposed to a geometric series of five test concentrations and a negative (well water) control. Two replicate test chambers were maintained in each treatment and control group, with 10 rainbow trout in each test chamber, for a total of 20 fish per test concentration. Nominal concentrations were selected based upon the results of an exploratory range finding toxicity test and the apparent solubility limit of the test substance in the system. Rainbow trout were impartially assigned to exposure chambers at test initiation. Observations of mortality and other signs of toxicity were made approximately 2.5, 24, 48, 72 and 96 h after test start. Nominal test substance concentrations were 9.9, 15, 22, 33 and 50 mg. Mean measured test concentrations were determined from water samples collected from each treatment and control group at the beginning of the study, at approximately 48 h and at test termination. The values corresponded to < LOQ, 9.5, 15, 21, 34 and 50 mg/L. Under the study conditions, the binomial test showed that the LC50 was above 34 mg/L, at a confidence level of 95%. The 96 h LC50 value was empirically derived to be 50 mg/L, with a 95% confidence interval of 34 mg/L and above. The no-mortality concentration and the NOEC were 34 mg/L (Minderhout, 2010).

Description of key information

A study was conducted to determine the toxicity of the test substance to rainbow trout (Oncorhynchus mykiss) according to OECD Guideline 203, EPA OPPTS Method 850.1075 and EPA OPP Method 72-1, in compliance with GLP. Rainbow trout were exposed to a geometric series of five test concentrations and a negative (well water) control. Two replicate test chambers were maintained in each treatment and control group, with 10 rainbow trout in each test chamber, for a total of 20 fish per test concentration. Nominal concentrations were selected based upon the results of an exploratory range finding toxicity test and the apparent solubility limit of the test substance in the system. Rainbow trout were impartially assigned to exposure chambers at test initiation. Observations of mortality and other signs of toxicity were made approximately 2.5, 24, 48, 72 and 96 h after test start. Nominal test substance concentrations were 9.9, 15, 22, 33 and 50 mg. Mean measured test concentrations were determined from water samples collected from each treatment and control group at the beginning of the study, at approximately 48 h and at test termination. The values corresponded to < LOQ, 9.5, 15, 21, 34 and 50 mg/L. Under the study conditions, the binomial test showed that the LC50 was above 34 mg/L, at a confidence level of 95%. The 96 h LC50 value was empirically derived to be 50 mg/L, with a 95% confidence interval of 34 mg/L and above. The no-mortality concentration and the NOEC were 34 mg/L (Minderhout, 2010).

Key value for chemical safety assessment

LC50 for freshwater fish:
50 mg/L

Additional information