C-M5

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:

The range-finding test was conducted between 10 April 2007 and 14 April 2007 and the definitive test between 23 April 2007 and 27 April 2007.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994))

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/l in range finding test and definative test.

- Sampling method: Verification of test concentrations - 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
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.

- Sample storage conditions before analysis: Duplicate samples and samples at 24 (fresh media), 48 and 72 hours (fresh and old media) were taken and stored at approximately -20 deg C for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

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 single nominal concentration of 100 mg/l. A single test concentration was used as results from the Acute Toxicity to Daphnia magna study (Safepharm Laboratories Project Number: (0345/0899) indicated that toxicity was not expected at this concentration.
An amount (2000 mg) of test material was dissolved directly 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 give the 100 mg/l test concentration.
The stock solution was inverted several times to ensure adequate mixing and homogeneity.
In the range-finding test 3 fish were added to each 20 litre 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 material.
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.

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/EEC Test Guidelines, no mortalities or sub-lethal effects of exposure were observed.

Experimental Preparation-
For the purpose of the definitive test the test material was dissolved directly in dechlorinated tap water.
An amount (4000 mg) of test material was dissolved in dechlorinated tap water and the volume adjusted to 1 litre to give a 4000 mg/l stock solution. An aliquot (500 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.
The concentration and stability of the test material in the test preparations samples were verified by chemical analysis at 0 (fresh media), 24 and 96 hours (old media)

The control group was maintained under identical conditions but not exposed to the test material.

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Strain: Oncorhynchus mykiss
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK
- Age at study initiation (mean and range, SD): The test was carried out using juvenile rainbow trout (<12 months)
- Length at study initiation (length definition, mean, range and SD): 4.9 cm (sd = 0.3)
- Weight at study initiation (mean and range, SD): 1.56 g (sd = 0.24)
- Method of breeding: Not recorded.
- Feeding during test
- Food type: Commercial trout pellets
- Amount: Not recorded.
- Frequency: ad/lib on a daily basis but discontinued approximately 24 hours prior to the start of the definitive test.
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 acclimatised to test conditions from 11 April 2007 to 23 April 2007
- Acclimation conditions (same as test or not): 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 deg C with a dissolved oxygen content of greater than or equal to 9.1 mg O2/l.
- Type and amount of food: Commercial trout pellets
- Feeding frequency: ad/lib on a daily basis but discontinued approximately 24 hours prior to the start of the definitive test
- Health during acclimation (any mortality observed): one mortality in the 7 days prior to the start of the test

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

A semi-static test regime was employed in the test involving a daily renewal of the test preparations to ensure that the concentrations of the test material remained near nominal and to prevent the build up of nitrogenous waste products.
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 conditions

Hardness:

approximately 140 mg/l as CaCO3

Test temperature:

Recorded daily throughout the test using a Hanna Instruments HI 93510 digital thermometer.
Maintained at approximately 14deg C in a temperature controlled room

pH:

Recorded daily throughout the test using a WTW pH/Oxi 340I pH and dissolved oxygen meter.
7.8 - 8.2

Dissolved oxygen:

The water dissolved oxygen concentrations were recorded daily throughout the test using a WTW pH/Oxi 340I pH and dissolved oxygen meter
>9.0 mg O2/l.

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.

Salinity:

No data.

Nominal and measured 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.
Nominal concentration: 100 mg/l for both range-finding and definitive study.

Details on test conditions:

TEST SYSTEM
- Test vessel:20 litre glass exposure vessels
- Type (delete if not applicable): sealed, to reduce evaporation
- Material, size, headspace, fill volume: 20 litre
- Aeration: The test vessels were aerated via narrow bore glass tubes
- Type of flow-through (e.g. peristaltic or proportional diluter): semi-static
- Renewal rate of test solution (frequency/flow rate): daily renewal of the test preparations
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 1




TEST MEDIUM / WATER PARAMETERS
Dechlorinated tap 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.

OTHER TEST CONDITIONS
- Adjustment of pH: no treatment related differences for pH.
- Photoperiod: 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) :
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. Phsico-chemical measurments were also made.



TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 100 mg/l
- Results used to determine the conditions for the definitive study: A single test concentration (100 mg/l) was used as results from the Acute Toxicity to Daphnia magna study (Safepharm Laboratories Project Number: (0345/0899) indicated that toxicity was not expected at this concentration.

There were no sub-lethal effects of exposure during the range-finding.
The results showed no mortalities at single test concentrations of 100 mg/l.
Based on this information, a single test concentration, in duplicate, of 100 mg/l was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the maximum test concentration given in the OECD/EEC Test Guidelines, no mortalities or sub lethal effects of exposure were observed.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL not recorded

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks:

plus sub-lethal effects

Remarks on result:

other: 95% Cl not recorded

Details on results:

Range-finding test: The results showed no mortalities at single test concentrations of 100 mg/l.

Definitive Test:
Mortalitiy data: There were no mortalities in 20 fish exposed to a test concentration of 100 mg/l for a period of 96 hours
Sub-lethal effects: 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.

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

95% confidence limits not applicable.

Any other information on results incl. tables

Sublethal observations / clinical signs:

The results of the definitive test showed the highest test concentration resulting in 0% mortality to be greater than or equal to 100 mg/l, the lowest test concentration resulting in 100% mortality to be greater than 100 mg/l and the No Observed Effect Concentration (NOEC) to be 100 mg/l. The No Observed Effect Concentration is based upon zero mortalities and the absence of any sub-lethal effects of exposure at this concentration.

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

Observations on test material solubility:

Throughout the duration of the test the test concentrations were observed to be dark red clear solutions.

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 LC₅₀values 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 LC50of 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 LC₅₀based 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.