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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:
The study was conducted between 18 May 2009 and 19 June 2009.
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 the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
Standard solutions of test material were prepared in tetrahydrofuran at a nominal concentration of 10 mg/l.

- Sampling method:
Water samples were taken from the solvent control and each replicate test vessel at 0 (fresh media), 24, 48, 72 (old and fresh media) and 96 hours (old media) for quantitative analysis.

Two samples of the solvent control and each replicate were taken at each occasion.One sample was analysed untreated and one sample after centrifugation (40000 g for approx. 30 minutes.

- Sample storage conditions before analysis:
Used immediately upon sampling. Further samples (in duplicate) were taken and stored at approximately 20°C for further analysis, if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
the test material fell into the category of a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore media preparation trials were conducted in order to determine the solubility of the test material under test conditions.

Saturated solution preparation:
An amount of test material (2250 mg) was dispersed in 22.5 litres of dechlorinated tap water with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 14°C. After the stirring period samples were taken for chemical analysis after the following pre-treatments:
• Untreated
• Centrifugation at 10000 g for 30 minutes
• Centrifugation at 40000 g for 30 minutes
• Filtration through a 0.2 µm Sartorius Sartopore filter (initial approximate 1 litre discarded)
• Filtration through a 0.2 µm Sartorius Sartopore filter (initial approximate 2 litres discarded)
The above was prepared in duplicate with samples being taken after stirring for 24 hours and 48 hours.

Solvent spike preparation:
An amount of test material (300 mg) was dissolved in dimethylformamide and the volume adjusted to 10 ml to give a 300 mg/10 ml solvent stock solution. An aliquot (2000 µl) of this solvent stock solution was dispersed in 20 litres of dechlorinated tap water with the aid of magnetic stirring for approximately 10 minutes to give the 3.0 mg/l test concentration.
Samples were taken for chemical analysis after the following pre-treatments:
• Untreated
• Centrifugation at 10000 g for 30 minutes
• Centrifugation at 40000 g for 30 minutes
• Filtration through a 0.2 µm Gelman AcroCap filter (initial approximate 100 ml discarded)
• Filtration through a 0.2 µm Gelman AcroCap filter (initial approximate 500 ml discarded)
Following sampling, the remainder of the test concentration was stirred at approximately 14°C using a magnetic stirrer with samples being taken after stirring for 24 hours and 48 hours.



- Controls:
The control and solvent control groups were maintained under identical conditions but not exposed to the test material. The solvent control group was exposed to 100 µl/l of dimethylformamide. Data from the control group was shared with similar concurrent studies.

- Evidence of undissolved material:
The results from the media preparation trial indicated that the test material adsorbed to the filter matrix. Therefore it was considered that filtration was not a suitable method for the removal of undissolved test material. The results of the centrifuged samples from the saturated solution method of preparation showed measured concentrations significantly in excess of the water solubility value thereby indicating that centrifugation did not remove all the undissolved test material when an excess was used.

The results of the solvent spike media preparation trial showed the measured concentration of the centrifuged samples to be approximately 0.20 and 0.10 mg/l after centrifugation at 10000 and 40000 g respectively. It was therefore considered that centrifugation at 10000 g may not remove all the undissolved test material. The use of prolonged stirring did not result in an increase in the amount of dissolved test material present.

It was therefore considered that the most appropriate method of preparation for the test material was as a solvent spike at an initial 3.0 mg/l. Due to the volume required it was not possible to centrifuge the test media prior to the exposure of the test organisms. Therefore the samples were analysed untreated and following centrifugation at 40000 g for 30 minutes in order to give an indication of the dissolved and hence bioavailable test material concentration.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name:
Rainbow Trout

- Source:
Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK.

- Age at study initiation: juvenile

- Length at study initiation:
5.0 cm (sd = 0.1)

- Weight at study initiation:
1.67 g (sd = 0.06)

- Feeding during test:
Received no food during exposure.


ACCLIMATION
- Acclimation period:
Fish were acclimatised to test conditions from 3 June 2009 to 15 June 2009.
- Acclimation conditions:
Fish were maintained in a glass fibre tank with a "single pass" water renewal system.

- 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):
There was zero mortality in the 7 days prior to the start of the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
Not applicable.
Hardness:
Test water: approximately 140 mg/l as CaCO3.
Test temperature:
The water temperature was recorded daily throughout the test. The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
Temperature was maintained at approximately 14°C throughout the test.
pH:
The pH was recorded daily throughout the test. The pH was measured using a WTW pH/Oxi 340I pH meter.
Dissolved oxygen:
The dissolved oxygen concentrations was recorded daily throughout the test. The dissolved oxygen concentration was measured using a WTW pH/Oxi 340I pH meter.
Salinity:
Not applicable.
Nominal and measured concentrations:
In the range-finding test fish were exposed to a series of nominal test concentrations of 0.30 and 3.0 mg/l.
Based on the results of the range-finding test a "Limit test" was conducted at a nominal concentration of 3.0 mg/l to confirm that at the highest attainable test concentration (by visual inspection), no mortalities or sub-lethal effects of exposure were observed.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: closed. The test vessels were then covered to reduce evaporation
- Material, size, headspace, fill volume: 20 litre glass exposure vessels
- Aeration: The test vessels were aerated via narrow bore glass tubes.

- Renewal rate of test solution:
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.

- No. of organisms per vessel:
7

- No. of vessels per concentration (replicates):
2

- No. of vessels per control (replicates):
1

- Biomass loading rate:
Based on the mean weight value this gave a loading rate of 0.58 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) 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:
1.041 mg/l

- Pesticides:
0 µg/l

- Chlorine:
0.297 mg/l

- Conductivity:
421.385 µS/cm at 20°C

- Culture medium different from test medium:
The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
An estimate of the LC50 values was given by inspection of the mortality data.



TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline:
A study to determine the general physico-chemical properties of the test material determined the water solubility of the test material to be less than 1.1 mg/l. Pre-study solubility work showed that the highest attainable test concentration (by visual inspection) that could be prepared was 3.0 mg/l with an aliquot (100 µl/l) of a solvent stock solution prepared in dimethylformamide. At higher test concentrations precipitation of the test material was observed on addition of the test material solvent stock solution to water.

Based on this information the test material fell into the category of a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore media preparation trials were conducted in order to determine the solubility of the test material under test conditions.

- Range finding study
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 0.30 and 3.0 mg/l. The test material was prepared using a preliminary solution in dimethylformamide.

An amount of test material (300 mg) was dissolved in dimethylformamide and the volume adjusted to 10 ml to give a 300 mg/10 ml solvent stock solution. A dilution from this solvent stock solution was prepared in dimethylformamide to give a further solvent stock solution of 30 mg/10 ml. An aliquot (2.0 ml) of the 30 and 300 mg/10 ml solvent stock solutions were each separately dispersed in a final volume of 20 litres of dechlorinated tap water, and stirred using a flat bladed mixer for approximately 1 minute, to give the 0.30 and 3.0 mg/l test concentrations respectively.
Each of the solvent stock solutions 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 solvent control group was maintained under identical conditions but not exposed to the test material. The solvent control group was exposed to 100 µl/l of dimethylformamide.

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.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 0.26 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: The dissolved test material concentration of 0.26 mg/l (based on mean measured test concentrations) was the highest attainable test concentration that could be prepared
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.26 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: NOEC based on zero mortalities and the absence of any sub-lethal effects
Details on results:
RESULTS:
Range-finding Test:
Cumulative mortality data from the exposure of rainbow trout to the test material during the range-finding test are give in Table 1. There were no sub-lethal effects of exposure during the range-finding test.

The results showed no mortalities at the test concentrations of 0.30 and 3.0 mg/l.

Based on this information, a single test concentration, in duplicate, of 3.0 mg/l was selected for the definitive test. This experimental design conforms to a "Limit Test" to confirm that at the highest attainable test concentration (by visual inspection), no mortalities or sub-lethal effects of exposure were observed.

Definitive Test:
Verification of test concentrations:
The results of the media preparation trials indicated that at a test concentration of 3.0 mg/l prepared using a preliminary solution in auxiliary solvent to spike test medium, a significant proportion of undissolved/dispersed test material would be present. Samples were therefore analysed untreated and after centrifugation (40000 g, 30 minutes) in order to give an indication of the dissolved and hence bioavailable test material concentration.

Analysis of the untreated samples showed measured concentrations of 80% to 112% except on eight occasions where the measured concentration was outside the 80% to 120% acceptance limits. Analysis of the centrifuged samples showed measured concentrations to range from 0.0694 to 0.745 mg/l. This variability was considered to be due to the poor water solubility of the test material.

Given that measured test concentrations in the centrifuged samples were shown to be relatively constant over each 24-Hour dosing period, it was considered justifiable to base the results on the mean measured test concentrations of the centrifuged test media to give a "worst case" analysis of the data. The mean measured concentration was calculated to be 0.26 mg/l.

Mortalitiy Data:
Cumulative mortality data from the exposure of rainbow trout to the test material during the definitive test are given in Table 2.

There were no mortalities in 14 fish exposed to a mean measured test concentration of 0.26 mg/l for a period of 96 hours.

The results of the definitive test showed the highest test concentration resulting in 0% mortality to be greater than or equal to 0.26 mg/l, the lowest test concentration resulting in 100% mortality to be greater than 0.26 mg/l and the No Observed Effect Concentration (NOEC) to be 0.26 mg/l. The NOEC is based upon zero mortalities and the absence of any sub-lethal effects of exposure at this concentration.

The dissolved test material concentration of 0.26 mg/l (based on mean measured test concentrations) was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and having due regard to the amount of auxiliary solvent permitted in the study under the OECD Guidelines. Other various recognised auxiliary solvents were used during preliminary solubility work, however, dimethylformamide was found to give the best testable dispersion of the test material in water.

At higher test concentrations there was a marked precipitation of the test material on addition of the solvent stock solution to water.

Sub-lethal effects:
There were no sub-lethal effects of exposure observed in 14 fish exposed to a mean measured test concentration of 0.26 mg/l for a period of 96 hours.

Observations on test material solubility:
The test preparations were observed to be clear, colourless solutions throughout the duration of the test.

Physico-chemical measurements:
Temperature was maintained at approximately 14°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
Please see attached background material for physico-chemical measurements.
Results with reference substance (positive control):
Not applicable.
Reported statistics and error estimates:
None noted.
Sublethal observations / clinical signs:

Table1              Cumulative Mortality Data in the Range-findingTest

Nominal

Concentration

(mg/l)

Cumulative Mortality

(Initial Population = 3)

3 Hours

6 Hours

24 Hours

48 Hours

72 Hours

96 Hours

Solvent Control

0

0

0

0

0

0

0.30

0

0

0

0

0

0

3.0

0

0

0

0

0

0

 


 Table2             Cumulative Mortality Data in the DefinitiveTest

Mean Measured

Concentration

(mg/l)

Cumulative Mortality
(Initial Population =7)

%

Mortality

3 Hours

6 Hours

24 Hours

48 Hours

72 Hours

96 Hours

96 Hours

Control

0

0

0

0

0

0

0

Solvent Control

0

0

0

0

0

0

0

0.26 R1

0

0

0

0

0

0

0

0.26 R2

0

0

0

0

0

0

0


R1and R2= Replicates 1 and 2

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test material to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and based on the mean measured test concentrations of the centrifuged test media gave a 96-Hour LC50 value of greater than 0.26 mg/l. Correspondingly the No Observed Effect Concentration was 0.26 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 Regulation (EC) No. 440/2008.

Methods.

A Study to Determine the General Physico-Chemical Properties of the test material gave a water solubility value for the test material of less than 1.1 mg/l. A pre-study media preparation trial indicated that the use of a preliminary solution in auxiliary solvent to spike the test medium was the most suitable method of preparation.

Following a preliminary range-finding test fish were exposed, in two groups of seven, to an aqueous solution of the test material, at a single concentration of 3.0 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 results of the media preparation trials indicated that at a test concentration of 3.0 mg/l prepared using a preliminary solution in auxiliary solvent to spike test medium, a significant proportion of undissolved/dispersed test material would be present. Samples were therefore analysed untreated and after centrifugation (40000g, 30 minutes) in order to give an indication of the dissolved and hence bioavailable test material concentration.

Analysis of the untreated samples showed measured concentrations of 80% to 112% except on eight occasions where the measured concentration was outside the 80% to 120% acceptance limits. Analysis of the centrifuged samples showed measured concentrations to range from 0.0694 to 0.745 mg/l. This variability was considered to be due to the poor water solubility of the test material.

Given that measured test concentrations in the centrifuged samples were shown to be relatively constant over each 24-Hour dosing period, it was considered justifiable to base the results on the mean measured test concentrations of the centrifuged test media to give a "worst case" analysis of the data.

The 96-Hour LC50 based on the mean measured test concentrations of the centrifuged test media was greater than 0.26 mg/l and correspondingly the No Observed Effect Concentration was 0.26 mg/l.

Description of key information

To assess short-term toxicity to fish of the substance (SiMAA2), an experimental result has been read-across from a structural analgoue substance (EC 700-043-1).

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 Regulation (EC) No. 440/2008.

Methods.

A Study to Determine the General Physico-Chemical Properties of the test material gave a water solubility value for the test material of less than 1.1 mg/l. A pre-study media preparation trial indicated that the use of a preliminary solution in auxiliary solvent to spike the test medium was the most suitable method of preparation.

Following a preliminary range-finding test fish were exposed, in two groups of seven, to an aqueous solution of the test material, at a single concentration of 3.0 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 results of the media preparation trials indicated that at a test concentration of 3.0 mg/l prepared using a preliminary solution in auxiliary solvent to spike test medium, a significant proportion of undissolved/dispersed test material would be present. Samples were therefore analysed untreated and after centrifugation (40000g, 30 minutes) in order to give an indication of the dissolved and hence bioavailable test material concentration.

Analysis of the untreated samples showed measured concentrations of 80% to 112% except on eight occasions where the measured concentration was outside the 80% to 120% acceptance limits. Analysis of the centrifuged samples showed measured concentrations to range from 0.0694 to 0.745 mg/l. This variability was considered to be due to the poor water solubility of the test material.

Given that measured test concentrations in the centrifuged samples were shown to be relatively constant over each 24-Hour dosing period, it was considered justifiable to base the results on the mean measured test concentrations of the centrifuged test media to give a "worst case" analysis of the data.

The 96-Hour LC50based on the mean measured test concentrations of the centrifuged test media was greater than 0.26 mg/l and correspondingly the No Observed Effect Concentration was 0.26 mg/l.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
0.26 mg/L

Additional information

The dissolved test material concentration of 0.26 mg/l (based on mean measured test concentrations) was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and having due regard to the amount of auxiliary solvent permitted in the study under the OECD Guidelines.