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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:
16 May 2011 to 18 July 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant. Guideline study. Acceptable without restriction.
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All test concentrations and control were sampled..
- Sampling method: All samples were taken from the approximate centre of the aquaria without mixing. For the determination of the actual test item concentrations, duplicate samples were taken from each treatment just before the start of the first and last renewal periods (Days 0 and 3). At the end of these renewal periods (Days 1 and 4), additional samples were taken from the aged test media for determination of the stability of the test item during the renewal periods of 24 hours. However, from the highest nominal test concentration (dilution 1:3.2), the last samples were taken after 24 hours, since all fish at the concentration were dead after 48 hours.
- Sample storage condition before analysis: Samples were deep-frozen immediately after sampling and stored at about -20˚C until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low solubility of the test substance in test water, a dispersion with the loading rate of 100 mg/L was prepared at the start of the test by dispersing 800 mg of the test substance (dosing range: 800.0 - 800.4 mg) in 8 L of test water. This preparation was subjected to ultrasonic treatment for 15 minutes and intense stirring on a magnetic stirrer over 96 hours in the dark, to dissolve a maximum amount of the test item in the dispersion. After the 96-hour stirring period, the dispersion of the test substance was filtered through a membrane filter (pore size 0.45 μm). The undiluted filtrate was used as a stock solution for preparation of the test media for all test concentrations. The test media were prepared just before the start of the exposure and before each daily test medium renewal.
- Controls: Reconstituted test water, consisting of analytical grade salts dissolved in purified water.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Particles at bottom after 24 hours for undiluted filtrate (100 mg/L). Noticeable colouration of new test solution for 1:3.2 dilution. Clear solution after 72 hours for 1:10 and higher dilutions.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Source: Breeding culture at test laboratory
- Length at study initiation (length definition, mean, range and SD): 4.3 ± 0.09 cm (Mean ± SD)
- Weight at study initiation (mean and range, SD): 0.71 ± 0.12 g (Mean ± SD)
- Feeding during test: None

ACCLIMATION
- Acclimation period: Eight days
- Acclimation conditions (same as test or not): Same as test conditions
- Type and amount of food: Commercial fish diet
- Feeding frequency: During holding and acclimatization until two days before the start of the test
- Health during acclimation (any mortality observed): No mortalities and all fish were healthy

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
125 mg CaCO3/L
Test temperature:
14ºC
pH:
7.3 to 7.4
Dissolved oxygen:
9.1 to 9.8 mg/L (≥ 88% oxygen saturation)
Salinity:
Not applicable
Nominal and measured concentrations:
The following concentrations of test substance were tested; 1:320, 1:100, 1:32, 1:10 and 1:32 of the undiluted filtrate with the loading rate of 100 mg/L. Additionally, a control (test water without test item) was tested in parallel. Measured concentration for dilutions 1:32, 1:10 and 1:3.2 at the start of the first renewal period (Days 0) were 0.288, 1.01 and 3.49 mg test substance/L, respectively. At the start of the last renewal periods (Day 3) the values were 0.343 and 0.99 mg test substance/L for dilutions 1:32 and 1:10, respectively. At the highest test concentration (1:3.2 dilution) measurements were only performed for the first test medium renewal period due to 100% fish mortality after 48 hours. At the end of the first test renewal period the measured test substance concentrations were 0.303, 1.02 and 3.62 mg test substance/L for dilution 1:32, 1:10 and 1:3.2 dilutions and at the end of the last renewal period they were 0.319 and 1.17 mg test substance/L for dilutions 1:32 and 1:10, respectively. The results show that the test substance was stable during the test renewal periods of 24 hours. The samples from the dilutions 1:320 and 1:100 were not analyzed, since these concentrations were below the NOEC determined in the test. The biological results are based to the mean measured test substance concentration calculated as the arithmetic means of the concentrations measured at the start and end of the test medium renewal periods. (see table below).

Details on test conditions:
TEST SYSTEM
- Test vessel: glass test vessel
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass test vessel (length: 35 cm; width: 23 cm; height 25 cm), height of water level: 19 cm, fill volume: 15 litres
- Aeration: Slightly aerated
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable, semi-static test
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.33 g fish wet weight/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water, consisting of analytical grade salts dissolved in purified water:
- Alkalinity: 0.4 mmol/L
- Ca/Mg ratio: 4:1
- Culture medium different from test medium: No
- Intervals of water quality measurement: The water temperature, pH and dissolved oxygen concentrations were measured at the start of the test and once every day during the test at each test concentration and in the control in freshly prepared and old test media. At the same occasions the appearance of the test media was recorded.

OTHER TEST CONDITIONS
- Adjustment of pH: None reported
- Photoperiod: 16:8 light/dark cycle with 30 minute transition period.
- Light intensity: Approximately between 180-480 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The test fish were observed after approximately 3, 24, 48, 72 and 96 hours test duration for mortality and visible abnormalities.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: Yes
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL: 0.69 - 1.9 mg test substance/L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.31 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: In the control and at the test concentrations up to and including the mean measured concentration of 0.31 mg/L, all fish survived until the end of the test and no visible abnormalities were observed in the test fish. At the next higher test concentration of mean measured 1.0 mg/L, five test fish showed one or several visible abnormalities. At the end of the test, two of the seven test fish died at this test concentration. At the highest test concentration of mean measured 3.6 mg/L, all fish were dead already after 48 hours of test duration.
- Mortality of control: None
- Other adverse effects control: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None observed
- Effect concentrations exceeding solubility of substance in test medium: no

Also see table below.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
The 96-hour LC50 and the 95%-confidence were calculated by Probit Analysis. The NOEC and LOEC were determined directly from the raw data.

The biological results are based to the mean measured test substance concentration calculated as the arithmetic means of the concentrations measured at the start and end of the test medium renewal periods. 
  Treatment/Dilution Mean measured concentration of the test item (arithmetic mean)
mg test substance /L
Dilution 1:320 ---
Dilution 1:100 ---
Dilution 1:32 0.31
Dilution 1:10 1
Dilution 1:3.2 3.6
--- : not analyzed, since these concentrations were below the NOEC determined in the test.  

Mortality and Visible Abnormalities in the Test Fish
Dilution (of the loading rate 100 mg test substance/L) Mean measured concentration Number of abnormal and dead fish / number of dead fish Observation time Type of visible abnormalities
 

 (mg test substance/L)

3 hours 24 hours 48 hours 72 hours  96 hours
Control '---          
Dilution 1:320 n.a. 0 / 0 0 / 0 0 / 0 0 / 0 0 / 0
Dilution 1:100 n.a. 0 / 0 0 / 0 0 / 0 0 / 0 0 / 0
Dilution 1:32 0.31 0 / 0 0 / 0 0 / 0 0 / 0 0 / 0
Dilution 1:10 1.0 0 / 0 0 / 0 5 / 0 AP, VF 4 / 1AP, VF 5 / 2 AP, VF
Dilution 1:3.2 3.6 4 / 0 AP 7 / 2 AP 7 / 7 - / - - / -
LC50* (mg test substance/L) > 3.6 > 3.6 1.9 1.2 1.1
95% C. I.* n.d. n.d. 1.0 - 3.6  0.75 - 2.0  0.69 - 1.9
95% C .I.: 95% confidence interval of the LC50 
 n.a : not analysed
n.d. : not determined
*: based on measured concentration
AP: Apathy
VF: Change body colour
Validity criteria fulfilled:
yes
Remarks:
The test was considered to be valid, since no mortality in the control was observed and the validity criterion on at least 60% oxygen saturation was fulfilled.
Conclusions:
The 96-hour LC50 for Oncorhynchus mykiss exposed to the test substance was determined to be 1.1 mg test substance/L (95% confidence interval: 0.69 - 1.9 mg test substance/L). The 96-hour NOEC was determined to be 0.31 mg test substance/L and the 96-hour LOEC was determined to be 1.0 mg test substance/L. The results are based on the mean measured test substance concentration calculated as the arithmetic means of the concentrations measured at the start and end of the test medium renewal periods.
Executive summary:

The sensitivity of Oncorhynchus mykiss to the test substance was determined in a GLP-compliant test performed to standard guidelines. The 96-hour LC50 for Oncorhynchus mykiss exposed to the test substance was determined to be 1.1 mg test substance/L (95% confidence interval: 0.69 - 1.9 mg test substance/L). The 96-hour NOEC was determined to be 0.31 mg test substance/L and the 96-hour LOEC was determined to be 1.0 mg test substance/L. The fish were exposed for up to 96 hours to 5 concentrations of the test substance. As the test substance has low solubility in the test media a dispersion was formed by mixing the test substance in the test media for a prolonged period of time. After filtration this stock solution was used to prepare a series of dilutions to form the other test concentrations. Analytical monitoring of the exposure concentrations was undertaken and the results are based on the mean measured test substance concentration calculated as the arithmetic means of the concentrations measured at the start and end of the test medium renewal periods.

Description of key information

96-h LC50 = 1.1 mg/l (95% confidence interval: 0.69 - 1.9 mg test substance/L), OECD 203, semi-static, Oncorhynchus mykiss, Liedtke 2011

The 96-hour NOEC was determined to be 0.31 mg test substance/L and the 96-hour LOEC was determined to be 1.0 mg test substance/L. The results are based on the mean measured test substance concentration calculated as the arithmetic means of the concentrations measured at the start and end of the test medium renewal periods.

Key value for chemical safety assessment

LC50 for freshwater fish:
1.1 mg/L

Additional information

The acute toxicity of the test substance to Rainbow Trout (Oncorhynchus mykiss) was determined. Fish were exposed to test item concentrations of 0.31, 1.0 and 3.6 mg/l alongside a water control. The 96h LC50 was determined to be 1.1 mg/l and the 96h NOEC was determined to be 0.31 mg/l. The study is considered reliable and adequate for the purpose of risk assessment, classification and labelling.