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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
other: analytical methods
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
GLP compliance:
yes (incl. certificate)
Remarks:
Date of GLP inspection: 19-21 July 2011, Date of Signature on GLP certificate: 31 August 2011
Analytical monitoring:
yes
Details on sampling:
See IUCLID Section 8 for detailed description of analytical method.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: test item dissolved directly in dechlorinated tap water

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: rainbow trout
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK. Fish maintained in-house since 07 March 2012. Fish were maintained in a glass fiber tank with a "single pass" water renewal system.
- Age at study initiation (mean and range, SD):
- Length at study initiation (length definition, mean, range and SD): fish had a mean standard length of 5.1 cm (sd = 0.1) at the end of the definitive test.
- Weight at study initiation (mean and range, SD): mean weight of 2.00 g (sd = 0.14) at the end of the definitive test.
- Method of breeding: no data
- Food: the fish recieved no food during exposure.

The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

ACCLIMATION
- Acclimation period: Fish were acclimatized to test conditions from 18 April 2012 to 29 April 2012.
- Type and amount of food: The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test.
- Health during acclimation (any mortality observed):
The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. The water temperature was controlled at approximately 14 °C with a dissolved oxygen content of greater than or equal to 9.8 mg O2/L. These parameters were recorded daily.

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
140 mg/l at CaCO3
Test temperature:
14°C
pH:
Average: 7.5
Dissolved oxygen:
≥ 9.8 mg O2/l
Salinity:
Not applicable.
Nominal and measured concentrations:
Range-finding study: 10 mg/l and 100 mg/l were tested.
Chemical analysis of the 100 mg/l test preparation at 24 hours showed a measured concentration of 92% of nominal. No result was available for the 100 mg/l test preparation at 0 hours. Analysis of the 10 mg/L test preparation at 0 and 24 hours showed measured concentrations of 65% to 50% of nominal. Although these measured concentrations were low there was no significant decrease in measured concentration over the 24-Hour period. Based on these results it was considered that the test item was stable under test conditions.
Details on test conditions:
RANGE-FINDING TEST:
The test concentration to be used in the definitive test was determined by a preliminary range-finding test.
In the range-finding test fish were exposed to a series of nominal test concentrations of 10 and 100 mg/L. The test item was dissolved directly in dechlorinated tap water.
An amount of test item (2000 mg) was dissolved in dechlorinated tap water and the volume adjusted to 1 liter to give a 2000 mg/L stock solution. Aliquots (50 and 500 mL) of the stock solution were each separately diluted in a final volume of 10 liters of dechlorinated tap water, and stirred using a flat bladed mixer for approximately 1 minute, to give the 10 and 100 mg/L test concentrations respectively.
The stock solution was inverted several times to ensure adequate mixing and homogeneity.
In the range-finding test 3 fish were added to each 10 liter test and control vessel and maintained at approximately 14 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours under static test conditions.
The control group was maintained under identical conditions but not exposed to the test item.
Data from the control group was shared with similar concurrent studies.
Each vessel was covered to reduce evaporation. After 3, 6, 24, 48, 72 and 96 hours any mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish.
A sample of each test concentration was taken for chemical analysis at 0 and 24 hours in order to determine the stability of the test item under test conditions. All samples were stored at approximately -20 °C prior to analysis.


DEFINITIVE TEST :

TEST SYSTEM
- Material, size, headspace, fill volume: 20 litre glass vessel - test vessel was covered to reduce evaporation.
- Aeration: The test vessel was aerated via a narrow bore glass tube.
- Renewal rate of test solution (frequency/flow rate): daily renewal
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- loading rate: Based on the mean weight value this gave a loading rate of 0.7 g bodyweight/litre.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener).
- See attachment 1 for typical parameters.
- Intervals of water quality measurement:
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations. The pH and oxygen were measured using a Hach HQ30d Flexi handheld meter and the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

OTHER TEST CONDITIONS
The test vessel was then covered to reduce evaporation and maintained at approximately 14 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

TEST CONCENTRATIONS
Preparation of test concentrations:
For the purpose of the definitive test the test item was dissolved directly in dechlorinated tap water.
An amount of test item (2000 mg) was dissolved in dechlorinated tap water and the volume adjusted to 1 liter to give a 2000 mg/L stock solution. The entire volume of this stock solution was diluted to a final volume of 20 liters of dechlorinated tap water, and stirred using a flat bladed mixer for approximately 1 minute, to give the 100 mg/L test concentration.
The stock solution was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0 (fresh media), 24 and 96 hours (old media) (see IUCLID Section 8: Analytical methods).

Verification of Test Concentrations:
Water samples were taken from the control and 100 mg/L test vessel at 0 (fresh media), 24 and 96 hours (old media) for quantitative analysis. The samples were stored at approximately -20 oC prior to analysis.
Duplicate samples, and samples at 24 (fresh media), 48 and 72 hours (old and fresh media), were taken and stored at approximately -20 °C for further analysis if necessary.
The method of analysis, recovery and test preparation analyses are described in IUCLID Section 8: Analytical methods.

Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Results of range-finding test: There were no sub-lethal effects of exposure and no mortality at test concentrations of 10 mg/l and 100 mg/l.

Results of the definitive test:

Mortality: No mortality at 100 mg/l.
Sub-lethal effects: No sub-lethal effects of exposure observed in 7 fish exposed to a test concentration of 100 mg/L for a period of 96 hours.

Analysis of the test preparations at 0, 24 and 96 hours showed measured test concentrations to range from 94% to 105% of nominal and so it was considered justifiable to estimate the LC50 values in terms of the nominal test concentrations only.

Observations on Test Item Solubility

The test preparation was observed to be a clear, colorless solution throughout the duration of the test.

Physico-Chemical Measurements (see attachment 2).

Temperature was maintained at approximately 14 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH. The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed in the control group.

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LC50 of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.
This study is considered to be of sufficient adequacy and reliability to be used as a key study in accordance with Regulation (EC) No. 1907/2006 (REACH) and is considered to be sufficient for classification and labelling in accordance with Regulation (EC) No 1272/2008 (EU CLP). Therefore, no further testing is justified.

Description of key information

One key study exists. This study had been conducted in accordance with a recommended guideline (OECD 203, EU Method C.1) and under the conditions of GLP. As such, no further testing is considered necessary.

Key value for chemical safety assessment

LC50 for freshwater fish:
100 mg/L

Additional information