Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
11 May to 30 May 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Recently conducted GLP compliant study using the most recent test methods

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): JKY-214
- Physical state: white solid
- Analytical purity: No information

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 2.0mg/l
- Sampling method: untreated in duplicate
- Sample storage conditions before analysis: c. -20C

Test solutions

Vehicle:
yes
Details on test solutions:
Information provided by the manufacturer indicated that the test material was insoluble in water. Pre-study solubility work conducted indicated that the test material was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. A test concentration of 2.0 mg/l (by visual inspection) was obtained using a preliminary solution in dimethylformamide.

Based on this information the test material was categorised as being a 'difficult substance' as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.

Saturated solution preparation

An amount of test material (1125 mg) was dispersed, in duplicate in 22.5 litres of reconstituted water with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21 °C for periods of either 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:

1) Centrifugation at 10000 g for 30 minutes
2) Centrifugation at 40000 g for 30 minutes
3) Filtration through a 0.2 urn Sartorius Sartopore filter (approximate 1 litre discarded in order to pre-condition the filter)
4) Filtration through a 0.2 urn Sartorius Sartopore filter (approximate 2 litres discarded in order to pre-condition the filter)

Solvent spike preparation

An amount of test material (200 mg) was dissolved in dimethylformamide and the volume adjusted to 10 ml to give a 200 mg/10 ml solvent stock solution. An aliquot (200 ul) of this 200 mg/10 ml solvent stock solution was dispersed, in triplicate, in 2 litres of reconstituted water with the aid of magnetic stirring at approximately 21 °C for approximately 10 minutes, 24 hours or 48 hours prior to taking samples for chemical analysis after the following pre-treatments:
1) Untreated
2) Centrifugation at 10000 g for 30 minutes
3) Centrifugation at 40000 g for 30 minutes
4) Filtration through a 0.2 urn Gelman Acrocap filter (approximate 100 ml discarded in order to pre-condition the filter)
5) Filtration through a 0.2 urn Gelman Acrocap filter (approximate 500 ml discarded in order to pre-condition the filter)

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 test concentration of 2.0 mg/l as the results of the Acute Toxicity to Daphnia magna test (Harlan Laboratories Ltd Project Number 0696/0114) indicated that toxicity was not expected at this concentration. The test material was prepared using a preliminary solution in dimethylformamide.

An amount of test material (200 mg) was dissolved in dimethylformamide and the volume adjusted to 10 ml to give a 200 mg/10 ml solvent stock solution. An aliquot (2.0 ml) of this solvent stock solution was dispersed in a final volume of 20 litres of dechlorinated tap water with the aid of stirring with a flat bladed mixer for approximately 1 minute to give the 2.0 mg/l test concentration.

The solvent 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 solvent control group was maintained under identical conditions but not exposed to the test material. The solvent control group was exposed to 100 ul/l of dimethylformamide. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.

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 ul/l of dimethyformamide.

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.


Verification of test concentrations

Water samples were taken from the solvent control and each replicate test vessel at 0 (fresh media) 24 (old media) and 96 (old media) hours for quantitative analysis.

Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

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 approximately 30 minutes). Further samples (in duplicate) at 24 (fresh media), 48 and 72 hours (fresh and old media) were taken and stored at approximately -20°C for further analysis, if necessary.

Test organisms

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
- Length at study initiation (length definition, mean, range and SD): 6.5 cm (sd = 0.8)
- Weight at study initiation (mean and range, SD): 3.68 g (sd = 0.94)
- Feeding during test discontinued approximately 24 hours prior to the start of the definitive test
- Food type: commercial trout pellets

ACCLIMATION
- Acclimation period: 14 May 2009 to 26 May 2009
- Acclimation conditions (same as test or not): same as test
- Type of food: commercial trout pellets
- Health during acclimation (any mortality observed): none

QUARANTINE (wild caught)
- Duration: n/a
- Health/mortality: n/a

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h

Test conditions

Hardness:
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
Test temperature:
The water temperature was 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 temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

Temperature was maintained at 13.4 deg C to 15.1 deg C throughout the test
pH:
The pH was 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 was measured using a either a WTW pH 320 pH meter or a WTW pH/Oxi 340I pH and dissolved oxygen meter. Average pH was measured to be 7.719
Dissolved oxygen:
9.9-10.0 mg/l
Salinity:
27.25 mg/l (total sodium)
Nominal and measured concentrations:
Chemical analysis of the freshly prepared test media at 0, 24, 48 and 72 hours (see Appendix 2) showed measured concentrations to range from 118% to 140% of nominal (2.37 to 2.81 mg/l) for the untreated samples. Chemical analysis of the freshly prepared test media at 24, 48 and 72 hours showed measured concentrations to range from 14% to 21% of nominal (0.278 to 0.413 mg/l) for the centrifuged samples (indicating the dissolved and hence bioavailable test material concentration). Chemical analysis of the freshly prepared test media at 0 hours showed measured concentrations to range from 65% to 68% of nominal (1.30 to 1.36 mg/l) for the centrifuged samples. These values were considered to be erroneously high when compared to other fresh media centrifuged samples therefore frozen duplicate samples were analysed. This analysis showed measured concentrations to range from 8% to 11% of nominal (0.160 to 0.228 mg/l) which was closer to what was expected and confirmed the original analysis to be erroneous.
Analysis of the test media at 24, 48, 72 and 96 hours (old media) showed measured concentrations to range from 103% to 130% of nominal (2.06 to 2.60 mg/l) for the untreated samples and from 14% to 36% of nominal (0.277 to 0.722 mg/l) for the centrifuged samples.
Given that the measured concentrations of the centrifuged samples were consistently lower than the nominal concentration of 2.0 mg/l and that there was no significant decline in measured test concentrations over each 24-Hour test media renewal period it was considered justifiable to base the results on the mean measured test concentrations of the centrifuged test media.
The mean measured test concentration of the centrifuged test media was determined to be 0.39 mg/l.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass aquaria holding 20 l of test media
- Renewal rate of test solution (frequency/flow rate): Medium renewed daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates):two
- No. of vessels per control (replicates): two
- Biomass loading rate: 0.86 g bodyweight/ litre


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised laboratory water
- Total organic carbon: mean 4.52 mg/l
- Particulate matter: No information
- Metals: No information
- Pesticides: No information
- Chlorine: mean 0.09 mg/l
- Alkalinity: mean 215.22 mg/l (calcium carbonate)
- Ca/mg ratio: mean Ca: Mg ratio = 16:1
- Conductivity: mean 905.31
- Culture medium different from test medium: No information
- Intervals of water quality measurement: 3 monthly intervals.


OTHER TEST CONDITIONS
- Adjustment of pH: no adjustment
- Photoperiod: 16 h light : 8 h dark
- Light intensity: no information


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations made at intervals of 3, 6, 24, 48, 72 and 96 hours

Reference substance (positive control):
no

Results and discussion

Effect concentrations
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 0.39 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: None observed
- Observations on body length and weight: no abnormal effects
- Other biological observations: None observed
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none recorded
Results with reference substance (positive control):
Not applicable

Any other information on results incl. tables

Mortality data

There were no mortalities in fish exposed to a mean measured dissolved test material concentration of 0.39 mg/lfor a period of 96 hours. Inspection of the mortality data gave the following results:

Time (h)

LC50(mg/l)

95% Confidence limits (mg/l)

3

>0.39

-

6

>0.39

-

24

>0.39

-

48

>0.39

-

72

>0.39

-

96

>0.39

-

 

The results of the definitive test showed the highest test concentration resulting in 0% mortality to be greater than or equal to 0.39 mg/l, the lowest test concentration resulting in 100% mortality to be greater than 0.39 mg/l and the No Observed Effect Concentration (NOEC) to be 0.39 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.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity to fish is considered to be >0.39 mg/l
Executive summary:

The acute toxicity to fish has been assessed by means of exposure to rainbow trout according to EU method C1in compliance with GLP. Due to the extremely low water solubility the maximum attainable concentration was 0.39 mg/l. As no mortality or clinical signs were observed at the highest attainable test concentration the substance is considered not acutely toxic to fish.