C-C1

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Definitive test between 23rd April 2007 and 27th April 2007.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes

Remarks:

Date of inspection: 30th August 2005 Date of signature: 21st November 2005

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Sponsor's identification :C-C1
Description : dark blue powder
Date received : 5 March 2007
Lot Number : MB-1
Storage conditions :room temperature in the dark over silica gel

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Standard solutions of test material were prepared in water* at a nominal concentration of 100 mg/l.

- Sampling method: The test material concentration in the test samples was determined by high performance liquid chromatography (HPLC) using an external standard. The test material gave a chromatographic profile consisting of a single peak.
The method was based on a procedure supplied by the Sponsor.

- Sample storage conditions before analysis: -20°C

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

Range-finding test
An amount (2000 mg) of test material was dissolved in dechlorinated tap water and the volume adjusted to 1 litre to give a 2000 mg/l stock solution. The entire volume of the 2000 mg/l stock solution was added to a final volume of 20 litres of dechlorinated tap water to the 100 mg/l test concentration.
The stock solution was inverted several times to ensure adequate mixing and homogeneity.

Definitive test - Experimental Preparation
For the purpose of the definitive test the test material was prepared using a preliminary solution.
An amount (4000 mg) of test material was dissolved in dechlorinated tap water and the volume adjusted to 2 litres to give a 2000 mg/l stock solution. An aliquot (1000 ml) of this stock solution was added to a final volume of 20 litres of dechlorinated tap water to the 100 mg/l test concentration.
This method of preparation was conducted in duplicate to give replicates R1 and R2.
The stock solution was inverted several times to ensure adequate mixing and homogeneity.

- Controls: The control groups was maintained under identical conditions but not exposed to the test material.

- Evidence of undissolved material (e.g. precipitate, surface film, etc): none

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Strain: Not applicable
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK
- Age at study initiation (mean and range, SD): Juvenile
- Length at study initiation (length definition, mean, range and SD): fish had a mean standard length of 4.9 cm (sd = 0.3)
- Weight at study initiation (mean and range, SD): mean weight of 1.56 g (sd = 0.24)
- Method of breeding: Asexual
- Feeding during test: Animals not fed during the test

ACCLIMATION
- Acclimation period: acclimatised to test conditions from 11 April 2007 to 23 April 2007
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: The stock fish were fed commercial trout pellets which was discontinued 24 hours prior to the start of the definitive test.
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): There was one mortality in the 7 days prior to the start of the test

QUARANTINE (wild caught)
- Not applicable

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

None

Test conditions

Hardness:

ca. 140 mg CaCO3/L

Test temperature:

The water temperature was controlled at approximately 14°C.

Recorded daily using a Hanna Instruments HI 93510 digital thermometer.

pH:

Recorded daily using a WTW pH/Oxi 340I pH meter.

7.8 - 8.3

Dissolved oxygen:

Dissolved oxygen content of greater than or equal to 9.0 mg O2/l.

Recorded daily a dissolved oxygen meter.

Salinity:

Not applicable, as freshwater study

Nominal and measured concentrations:

100 mg/l in defnitive test

Details on test conditions:

TEST SYSTEM
- Test vessel: glass vessel
- Type (delete if not applicable): closed - covered to reduce evaporation
- Material, size, headspace, fill volume: 20 litre glass vessels
- Aeration: aeratedd via narrow bore glass tubes
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): Daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: not stated in report

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) 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.

- Total organic carbon:
Average: 1.573 mg/l. Range: 1.380 - 2.040 mg/l

- Metals: no total value for metals provided in report

- Pesticides: no total value for ppesticides provided in report

- Chlorine:
Free chlorine:
Average: 0.211 mg/l. Range: 0.010 to 0.480 mg/l

Total chlorine:
Average: 0.287 mg/l. Range: 0.080 to 0.550 mg/l

- Ca/mg ratio: not stated in report

- Conductivity:
Average: 410.769 µS/cm at 20°C
Range: 331.000 to 539.000 µS/cm at 20°C

- Culture medium different from test medium: not applicable

- Intervals of water quality measurement: Water samples were taken from the control and each replicate test vessel at 0 (fresh media), 24 and 96 hours (old media) for quantitative analysis.

OTHER TEST CONDITIONS
- Adjustment of pH: not required

- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods

- Light intensity: not stated in report

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
- Spacing factor for test concentrations: not applicable, as 100 mg/l only test concentration

- Justification for using less concentrations than requested by guideline: based on the results of the range-finding study a limit test was conducted

- Range finding study
- Test concentrations: 100 mg/l

- Results used to determine the conditions for the definitive study: There were no sub-lethal effects of exposure during the range-finding test.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Basis for effect:

mortality (fish)

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Details on results:

- Behavioural abnormalities: none noted

- Observations on body length and weight: body length and weight at end of study not described in study report.

- Other biological observations: There were no sub-lethal effects of exposure observed in 20 fish exposed to a test concentration of 100 mg/l for a period of 96 hours.

- Mortality of control: not applicable

- Other adverse effects control: not applicable

- Abnormal responses: none recorded

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Throughout the duration of the test the test concentrations were observed to be dark blue coloured solutions.

- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

None

Any other information on results incl. tables

Sublethal observations / clinical signs:

The oxygen concentration in the control vessel at 0-Hours 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.

Verification of test concentrations

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

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LC50 of greater than 100 mg/l. Correspondingly the No Observed Effect Concentration was 100 mg/l.

Executive summary:

Introduction. A study was performed to assess the acute toxicity of the test material to rainbow trout (Oncorhynchus mykiss). The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods. Following a preliminary range-finding test fish were exposed, in two groups of ten, to an aqueous solution of the test material, at a single concentration of 100 mg/l for a period of 96 hours at a temperature of approximately 14ºC 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 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results. The 96-Hour LC₅₀ based on nominal test concentrations was greater than 100 mg/l and correspondingly the No Observed Effect Concentration was 100 mg/l.

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

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/l.