Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-07-08 to 2005-01-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: approximately 4°C, in the dark (Date received: 24 May 2004)
- Stability under test conditions: The test samples have been shown to be unstable in the test medium in light and dark conditions. A range of preliminary stock solutions was prepared, analysed initially and then after storage in sealed glass vessels at ambient temperature in light conditions for approximately 24 hours (equivalent to the period of stock solution renewal). The stock solutions have been shown to be stable in the test medium.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: dissolved directly in deionised reverse osmosis water.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Range finding test (static): 1.0, 10 and 100 mg/L; Second range finding test (static): 1000 mg; Third range finding test (dynamic): 3.2, 32 and 100 mg/L, Final range finding test (dynamic): 1.0 and 10 mg/L test concentrations. Definitive test: 100 mg/mL
-- Physico-chemical measurements: The water temperature, pH and dissolved oxygen concentrations were 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 WTW pH 320 pH meter, the dissolved oxygen concentration was measured using a YSI 550 dissolved oxygen meter and the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
-- Verification of test concentrations: Water samples were taken from the control and all surviving test groups at 0, 24, 48, 72 and 96 hours for quantitative analysis. The concentration of the test material in the fresh aqueous stock solutions was also verified by chemical analysis at -24, 0, 24, 48 and 72 hours. Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
-- Rangefinding tests: see below.
-- Definitive Test: For the purpose of the definitive test the test material was dissolved directly in deionised reverse osmosis water. Amounts of test material (219.6, 390.4, 683.2, 1220 and 2196 mg) were each separately dissolved in deionised reverse osmosis water with the aid of ultrasonication for approximately 1.5 hours and the volume of each adjusted to 2 litres to give the 109.8, 195.2, 341.6, 610 and 1098 mg/L aqueous stock solutions. The stock solutions were freshly prepared each day prior to dosing by the dynamic, continuous flow apparatus (see Figure 1) to give the 1.8, 3.2, 5.6, 10 and 18 mg/L test concentrations. Each of the stock solutions was inverted several times to ensure adequate mixing and homogeneity.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): A test sample was tested for stability without prior mixing (sonication) the test sample bottle to assess for losses due to adsorption and/or insolubility.The unsonicated stability vessel showed no evidence of insolubility or adherence to glass.

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: Fish were obtained from 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): mean standard length of 4.5 cm (sd = 0.2) at the end of the definitive test.
- Weight at study initiation (mean and range, SD): mean weight of 1.12 g (sd = 0.21) at the end of the definitive test.
- Maintenance of the brood fish: in-house since 6 January 2005. Fish were maintained in a glass fibre tank with a "single pass" water renewal system.

ACCLIMATION
- Acclimation period: Fish were acclimatised to test conditions from 12 January 2005 to 24 January 2005
- 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 test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.
- Type and amount of food during acclimation: The stock fish were fed commercial trout pellets which was discontinued 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

FEEDING DURING TEST
none

Study design

Test type:
flow-through
Water media type:
freshwater
Remarks:
Laboratory tap water, dechlorinated and softened
Limit test:
no
Total exposure duration:
96 h

Test conditions

Hardness:
total hardness of approximately 100 mg/L as CaCO3
Test temperature:
approximately 14°C
pH:
Typical Water Quality Characteristics: mean pH = 7.6 prior to dechlorination and softening. There was no treatment related difference for pH.
Dissolved oxygen:
there was no treatment related difference for oxygen concentration
Conductivity:
Typical Water Quality Characteristics: mean 439 µS/cm prior to dechlorination and softening
Nominal and measured concentrations:
Verification of test concentrations
Analysis of the aqueous stock solutions used to prepare the test concentrations was performed at each dosing occasion. The results showed measured concentrations of the aqueous stock solutions to range from 103% to 114% of nominal.
Analysis of the test preparations at 0, 24, 48, 72 and 96 hours (see Appendix 1) showed measured test concentrations to range from 105% to 139% of nominal with the majority of measured values being within the acceptance limits of 80% to 120% of nominal. The mean measured test concentrations were shown to range from 114% to 123% of the nominal test concentrations with only the 18 mg/L test group showing a mean measured value in excess of 120% of nominal. Given that the single value in excess of 120% was close to this value, it was considered appropriate to base the results on nominal test concentrations only.
Details on test conditions:
TEST SYSTEM
- Test vessel: see below
- Type (delete if not applicable): The test vessels were covered to reduce evaporation
- Material, size, headspace, fill volume: 20 litre glass exposure vessels
- Aeration: The test vessels received no auxiliary aeration; the diluent supply only was aerated.
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate): Dynamic, continuous flow test conditions were employed in the test. The diluent supply was pumped at 60 ml/minute by a Watson Marlow 505S peristaltic pump with the aqueous stock solutions dosed by a Watson Marlow 205U peristaltic pump at a rate of 1.0 ml/minute. The diluent and the aqueous stock solutions were mixed in glass mixing vessels by magnetic stirrer prior to delivery to the test vessels. The dosing apparatus was set up approximately 24 hours prior to the start of the test to allow equilibration of the test concentrations.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 1
- Biomass loading rate: Based on the mean weight value the loading rate was 0.56 g bodyweight/litre (static volume), 0.13 g of bodyweight/litre (volume in 24 hours) and 4.4 tank volumes per day.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution 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 100 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: 1.700 mg C/L prior to dechlorination and softening
- Pesticides: <0.010 µg/l prior to dechlorination and softening
- Chlorine: 27.000 mg Cl/L prior to dechlorination and softening
- Alkalinity: 113.000 mg HCO3/L prior to dechlorination and softening
- Ca/Mg ratio: 51.000 mg Ca/L; 8.000 mg Mg/L prior to dechlorination and softening
- 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.
- Intervals of water quality measurement: The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the study. The pH was measured using a WTW pH 320 pH meter and the dissolved oxygen concentration was measured using a YSI 550 dissolved oxygen meter and a WTW pH/Oxi 340I dissolved oxygen meter.

OTHER TEST CONDITIONS
- 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) :
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
- Range finding study test concentrations: see below
- Definitive test test concentration: 1.8, 3.2, 5.6, 10 and 18 mg/L
- Results used to determine the conditions for the definitive study: see below
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
LC50
Effect conc.:
> 18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
6 h
Dose descriptor:
LC50
Effect conc.:
> 18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
> 18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
16 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other:
Remarks:
Analysis of the mortality data by the trimmed Spearman-Karber method (Hamilton et al 1977).
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other:
Remarks:
Analysis of the mortality data by the trimmed Spearman-Karber method (Hamilton et al 1977)
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
8.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other:
Remarks:
Analysis of the mortality data by the the probit method (Finney 1971). Nominal values are analyticall verified.
Details on results:
- Behavioural abnormalities: Sub-lethal effects of exposure were observed at test concentrations of 5.6 mg/L and above. These responses were fish swimming at the bottom, swimming at the surface and increased pigmentation
- Observations on test material solubility: The test preparations and the stock solutions were observed to be clear, colourless solutions throughout the duration of the test.
- Verification of test concentrations: See above (Nominal and measured concentrations)
Reported statistics and error estimates:
The LC 50 values and associated confidence limits at 48 and 72 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al 1977) using the ToxCalc computer software package (ToxCalc 1999) and at 96 hours by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999).
Probit analysis is used where two or more partial responses to exposure are shown.
When only one partial response is shown the trimmed Spearman-Karber method is appropriate.

Any other information on results incl. tables

Range-finding Tests

The results of the initial range-finding test conducted under static test conditions showed no mortalities at the test concentrations of 1.0 and 10 mg/L. However, mortalities were observed at 100 mg/L and sub-lethal effects of exposure (swimming at the bottom and increased pigmentation) were observed at 10 mg/L.

The results of the second range-finding test conducted under static test conditions showed no mortalities at 3.2 mg/L. However, mortalities were observed at 32 mg/L.

The results of the third range-finding test conducted under dynamic test conditions showed mortalities at all test concentrations of 3.2, 32 and 100 mg/L.

The results of the final range-finding test conducted under dynamic test conditions showed no mortalities at 1.0 mg/L. However, mortalities were observed at 10 mg/L.

Definitive Test

Cumulative Mortality Data in the Definitive Test

 Cumulative Mortality (Initial Population =10)

 % Mortality

  Nominal Concentration (mg/L)

 3 Hours

 6 Hours

 24 Hours

 48 Hours

 72 Hours

 96 Hours

 

 Control

 0

 0

 0

 0

 0

 0

 0

 1.8

 0

 0

 0

 0

 0

 0

 0

 3.2

 0

 0

0

 0

 0

 0

 5.6  0  0  0  0  0  1  10
 10  0  0  0  0  1  6  60
 18  0  0  0  6  10  10  100

Sub-lethal Effects of Exposure in the Definitive Test

 Sub-lethal Effects

 Time (Hours)

  Nominal Concentration (mg/L)

 3

 6

 24

 48

 72

 96

 Control

 No abnormalities detected

 

 

 

 

 

 

 1.8

 No abnormalities detected

 

 

 

 

 

 

 3.2

 No abnormalities detected

 

 

 

 

 

 5.6

 Swimming at the bottom

 

 

 

 

 9/9

 10

  Swimming at the bottom 

Swimming at the surface

Swimming at the bottom with increased pigmentation

 

 

 

 7/9

2/9

 

4/4

 18

 

4/4

 A/D

 

A/D = All fish dead

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In a flow-through test according to OECD 203 with rainbow trout (Oncorhynchus mykiss) the test substance showed a 96h-LC50 =8.8 mg/L (nominal, analytically verified) and 96h-EC0: 3.2 mg/L (nominal, analytically verified).