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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:
01 September 2015 to 18 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
As a result of the extended test solution preparation time the 6-hour fish observations were conducted after 5 hours. Considered not to adversely affect the results of the test.
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
yes
Remarks:
The time between the test media being added to the test vessels and the fish being added to the test solutions exceeding 30 mins. Considered not to adversely affect the results of the test.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Physical state: Pale yellow paste
- Analytical purity: 100 %
- Expiration date of the lot/batch: 24 April 2017
- Storage condition of test material: Room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
- Water samples were taken from the control and 100 mg/L test vessel at 0 and 72 hours (fresh media) and at 24 and 96 hours (old media) for quantitative analysis.
- Samples were stored frozen prior to analysis.
- Duplicate samples at 24 (fresh media), 48 (old and fresh media) and 72 hours (old media) were stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
TEST WATER
- The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.
- Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3.
- After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
-Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening are given in Appendix 2 (attached).
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
- The test was carried out using juvenile rainbow trout.
- Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in-house since 04 August 2015.
- Fish were maintained in a glass fibre tank with a 'single pass' water renewal system.
- Fish were acclimatised to test conditions from 28 September 2015 to 5 October 2015.
- Lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 min dawn and dusk transition periods..
- Water temperature was controlled at 14 °C with a dissolved oxygen content of ≥ 8.1 mg O2/L. These parameters were recorded daily.
- The stock fish were fed commercial trout pellets, which were discontinued approximately 24 hours prior to the start of the definitive test.
- There was no mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 5.9 cm (sd = 0.3) and a mean weight of 1.74 g (sd = 0.3) at the end of the definitive test. Based on mean weight value this gave a loading rate of 0.61 g bw/L.
- The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
Not applicable
Hardness:
Approximately 140 mg/L as CaCO3
Test temperature:
14-15 °C (see Table 3, attached)
pH:
7.5 to 8.2 ( see Table 3, attached)
Dissolved oxygen:
8.8 to 9.8 mg O2/L ( see Table 3 attached)
Salinity:
Not applicable
Nominal and measured concentrations:
100 mg/L nominal
Details on test conditions:
RANGE FINDING-TEST
-Test concentration to be used in the definitive test was determined by a preliminary range-finding test.
- Fish were exposed to a series of nominal test concentrations of 1.0, 10 and 100 mg/L. Test item was dispersed directly in test water.
-- A nominal amount of test item (2200 mg) was dispersed in test water with the aid of sonication for approximately 50 mins and the volume adjusted to 22 L to give the 100 mg/L test concentration
- After sonication, temperature and dissolved oxygen of the test solution was checked and adjusted to 15 ± 2°C and ≥ 6.1 mg O2/L before preparation of the required test concentration of 1.0 and 10 mg/L.
- Each of the test solutions was mixed with a flat bladed stirrer for 1 min to ensure adequate mixing and homogeneity
- Three fish were placed in each test and control vessel and maintained in a temperature controlled room at 14 to 16°C 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.
- Each 20-30 L test and control vessel contained 20 L of test media and 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 fish.
- The control group was maintained under identical conditions but not exposed to the test item.


DEFINITIVE TEST
- Based on the results of the range-finding test a ‘Limit test” was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines, no mortalities or sub-lethal effects of exposure were observed.

EXPERIMENTAL PREPARATION
- A nominal amount of test item (2200 mg) was weighted out onto a microscope and placed in dilution water.
- The test solution was sonicated for 90 to 120 mins to disperse the test item and give the 100 mg/L test concentration.
- After sonication, temperature and dissolved oxygen of test solution was checked and adjusted to 15 ± 2°C and ≥ 6.1 mg O2/L before beginning exposure.
- As in the range-finding test, 25-30 L glass exposure vessels containing 20 L of test media were used for each control and test preparations.
- At the start of the test, seven fish were placed in the test preparations in each test vessel at random.
- Test vessels were then covered to reduce evaporation and maintained at 14 to 15°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.
- Test vessels were aerated via narrow bore glass tubes.
- Fish were not individually identified and received no food during exposure.
- The control group was maintained under identical conditions but not exposed to the test item.
- A semi-static test regime was employed in test involving a daily renewal of the test preparations to prevent build-up of nitrogenous waste products.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
RANGE- FINDING TEST
- Cumulative mortality data from the exposure of rainbow trout to the test item are given in Table 1 (attached).
- Results showed no mortality and no sub-lethal effects of exposure recorded at any test concentrations.
- Chemical analysis of test preparations at 0 and 24 hours showed that were near nominal concentrations were obtained, indicating that test item was stable under test conditions (see Appendix 3, attached).
- Based on this results, a single test concentration of 100 mg/L was selected for the definitive test. Conforms to a ‘Limit test”.

DEFINITIVE TEST- VERIFICATION OF TEST CONCENTRATIONS
- Chemical analysis of fresh test preparation at 0 and 72 hours and old media at 24 and 96 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only (see Appendix 3, attached).

MORTALITY DATA
- Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 2 (attached).
- There were no mortalities in 7 fish exposed to a 100 mg/L for a period of 96 hours.
- Inspection of mortality data gave LC50 values > 100 mg/L at 3, 5, 24, 48, 72 and 96 hours.
- Results of the definitive test showed the highest test concentration resulting in 0% mortality to be 100 mg/L.
-The LOEC was considered to be > 100 mg/L.
- The NOEC was 100 mg/L. It was considered unnecessary and unrealistic to test at concentration in excess of 100 mg/L.

SUB-LETHAL EFFECTS
- No sub-lethal effects of exposure were observed in 7 fish exposed to a test concentration of 100 mg/L for a period of 96 hours.

VALIDATION CRITERIA
- The test was considered to be valid given that none of the control fish died or showed signs of stress during the test and that the oxygen concentration at the end of the test was ≥ 60 % of ASV (6.1 mg O2/L) in the control and test vessels.

WATER QUALITY CRITERIA
- The results of the water quality measurements are given in Table 3 (attached).
- Temperature was maintained at 14-15 °C throughout the test.
- There were no treatment related differences for oxygen concentration or pH.

OBSERVATIONS ON TEST ITEM SOLUBILITY
- At the start and throughout the test, the control was observed to be a clear colourless water solution and the 100 mg/L test concentration to be a slightly hazy homogenous dispersion.
Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater fish rainbow trout has been investigated and based on the nominal test concentration gave a 96-hour LC50 value of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.
Executive summary:

INTRODUCTION

A study was performed to assess the acute toxicity of the test item to rainbow trout. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203 "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

METHODS

Following a preliminary range-finding test, seven fish were exposed to an aqueous dispersion of the test item, at a single concentration of 100 mg/L for a period of 96 hours at a temperature of 14°C to 15°C under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 5 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

RESULTS

Analysis of the fresh test preparations at 0 and 72 hours and old media at 24 and 96 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave LC50 values based on the nominal test concentration of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

Description of key information

The acute toxicity of the test item to the fresh water fish rainbow trout has been investigated and based on analytically confirmed the nominal test concentration, gave at he 96-hour LC50 value was of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L. (OECD 203 and EU Method C.1)

Key value for chemical safety assessment

LC50 for freshwater fish:
100 mg/L

Additional information

INTRODUCTION

A study was performed to assess the acute toxicity of the test item to rainbow trout. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203 "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

METHODS

Following a preliminary range-finding test, seven fish were exposed to an aqueous dispersion of the test item, at a single concentration of 100 mg/L for a period of 96 hours at a temperature of 14°C to 15°C under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 5 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

RESULTS

Analysis of the fresh test preparations at 0 and 72 hours and old media at 24 and 96 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave LC50 values based on the nominal test concentration of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.