Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15/12/2008 to 30/01/2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with recognised guideline

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling method:
Water samples were taken from the control and the 1000 mg/l test group at 0 (fresh media), 24, 48, 72 (fresh and old media) and 96 hours (old media) for quantitative analysis.

- Sample storage conditions before analysis:
Room temperature in the dark
Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

- Sampling for analytical method
A volume of test sample was diluted with water to give a final theoretical concentration of 5.0 mg/L

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
For the purpose of the definitive test the test material was dissolved directly in dechlorinated tap water. An amount of test material (20 g) was dissolved in dechlorinated tap water, the volume adjusted to 20 litres and stirred with a flat bladed mixer for approximately 1 minute, to ensure adequate mixing and homogeneity, to give the required test concentration of 1000 mg/l.

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

- Evidence of undissolved material (e.g. precipitate, surface film, etc):
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, nr. Skipton, Yorkshire, UK and maintained in-house since 11 November 2008.
- Age at study initiation (mean and range, SD): nda
- Length/weight at study initiation (length definition, mean, range and SD):
The fish had a mean standard length of 6.0 cm (sd = 0.6) and a mean weight of 3.05 g (sd = 0.86) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 1.5 g bodyweight/litre (static volume). This loading rate was in excess of the maximum value stated in the protocol of 0.8 g/l. This deviation was considered not to have adversely affected the results of the test as the fish showed no mortalities or sub-lethal effects of exposure and was considered not to have affected the validity or integrity of the test.

- Method of breeding: nda
- Feeding during test: The fish received no food during exposure.

ACCLIMATION
- Acclimation period: fish were acclimatised to test conditions from 12 January 2009 to 26 January 2009.

- Type and amount of food: the stock fish were fed commercial trout pellets which was discontinued approximately 48 hours prior to the start of the definitive test.

- Feeding frequency: nda

- Health during acclimation (any mortality observed):
There was less than 1% 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:
n/a

Test conditions

Hardness:
At the start of the study the diluent had a total water hardness of 120 mg/l as CaCO3. The water hardness weas determined using the methods described in Field and On-site Methods for the Analysis of Water (British Standard Institution, 1993) and the COD by the HACH reactor digestion method (Hach).
Test temperature:
Temperature was maintained at 14°C to 15°C throughout the test.

The temperature in the control and test vessels was measured to be above the 12 ± 2 °C range given in the protocol at 0 (fresh media), 24 (old media), 48 (old media), 72 (old media) and 96 hours (old media). This was considered not to have affected the validity or integrity of the test as no adverse effects of exposure were observed in the fish throughout the test. Temperature was measured using Hanna Instruments HI 93510 digital thermometer.
pH:
The pH was seen to be between 7.1-7.7 during the study. The pH was measured using a WTW pH/Oxi 3401 pH and dissolved oxygen meter
Dissolved oxygen:
Dissolved oxygen content of greater than or equal to 9.8 mg O2/l. The dissolved oxygen was measured using a WTW pH/Oxi 3401 pH and dissolved oxygen meter
Salinity:
n/a
Nominal and measured concentrations:
Given that the overall mean measured concentration was 97% of nominal it was considered justifiable to estimate the LC50 values in terms of the nominal test concentrations only.

Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: Each vessel was covered to reduce evaporation
- Material, size, headspace, fill volume: 20 litre glass exposure vessels were used for each test concentration
- Aeration: The test vessels received no auxiliary aeration

- Renewal rate of test solution:
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.

- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 2

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)
- Total organic carbon: 0.21 mg C/l
- Particulate matter: 0.3 mg/l
- Metals: Various metals are present in the water at differing concentrations
- Pesticides: Maximum 0.0001
- Chlorine: Ranged from 0.010 to 0.024 mg Cl2/l
- Alkalinity: Alkalinity of 84 mg/l as CaCO3
- Conductivity: 339 µS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test.

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:
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. No abnormalities detected.

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. Water samples were taken from the control and the 1000 mg/l test group at 0 (fresh media) 24, 48, 72 (fresh and old media) and 96 hours (old media) for quantitative analysis. Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
The test concentrations to be used in the definative test were determined by preliminary range-finding tests.

- Justification for using less concentrations than requested by guideline:
The definative study was performed as a limit test.

- Range finding study
- Test concentrations:
An amount of test material (4000 mg) was dissolved in dechlorinated tap water and the volume adjusted to 1 litre to give a 4000 mg/l stock solution. Aliquots of this (50 and 500 ml) were each added separately in a final volume of 20 litres of dechlorinated tap water and stirred with a flat bladed mixer for approximately 1 minute, to ensure adequate mixing and homogeneity, to give the required test concentrations of 10 and 100 mg/l.

Due to the presence of sub-lethal effects in the initial range-finding test, a further range-finding test was conducted at a single nominal test concentration of 100 mg/I. Here an amount of test material (2000 mg) was dissolved in dechlorinated tap water and the volume adjusted to 500 ml to give a 4000 mg/l stock solution. The entire volume of this stock was added to a final volume of 20 litres of dechlorinated tap water and stirred with a flat bladed mixer for approximately 1 minute, to ensure adequate mixing and homogeneity, to give the required test concentration of 100 mg/l.

In order to comply with the maximum concentration recommended to be tested in the test guideline a third range-finding test was performed with a single nominal test concentration of 1000 mg/l. Here an amount of test material (20 g) was dissolved in dechlorinated tap water, the volume adjusted to 20 litres and stirred with a flat bladed mixer for approximately 1 minute, to ensure adequate mixing and homogeneity, to give the required test concentration of 1000 mg/l.

The control groups were maintained under identical conditions but not exposed to the test material.

- Results used to determine the conditions for the definitive study:
The results showed no mortalities at the test concentrations of 10 mg/l in the initial range-finding test, 100 mg/l in the second range-finding test and 1000 mg/l in the third range-finding test. However, a single mortality was observed at 100 mg/l in the initial range-finding test. This mortality was considered to be due to natural causes. Based on this information, a single test concentration, in triplicate, of 1000 mg/l was selected for the definitive test.

- Concentrations:
The test material concentration in the test samples was determined spectrophotometrically using an external standard. In the initial range-finding test fish were exposed to a series of nominal test concentrations of 10 and 100 mg/l. The test material was dissolved directly in dechlorinated tap water. Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 1000 mg/l to confirm that at the maximum concentration given in the US CFR/EPA Test Guidelines, no mortalities or sub-lethal effects of exposure were observed.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: No abnormalities detected.
- Observations on body length and weight: Not measured
- Mortality of control: No mortalities observed in the controls.
Results with reference substance (positive control):
n/a
Reported statistics and error estimates:
Linearity was confirmed in the range 0 to 10.6 mg/l. The method has been considered to be sufficiently accurate for the purposes of this test. The test sample results have not been corrected for recovery. The limit of quantitation has been assessed down to 26.0 mg/l.

Any other information on results incl. tables

The results of the definitive test showed the highest test concentration resulting in 0% mortality to be greater than or equal to 1000 mg/l and the No Observed Effect Concentration (NOEC) to be greater than or equal to 1000 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 of the test material to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LC50 of greater than 1000 mg/l. Correspondingly the No Observed Effect Concentration was greater than or equal to 1000 mg/l.
Executive summary:

In an Acute toxicity to rainbow trout (Oncorhynchus mykiss) study (Harlan project number: 0959/0244) the test material

the test material had an LC50of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) was greater than or equal to 1000 mg/l.

Given that the overall mean measured concentration was 97% of nominal it was considered justifiable to estimate the LC50values in terms of the nominal test concentrations only.

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, US Code of Federal Regulations Title 40, Part 797, Section 1400; US EPA Pesticide Assessment Guidelines, Sub-Division E, Section 72-1 and US EPA Draft Ecological Effects Test Guideline, OPPTS 850.1075.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/l.

There were no sub-lethal effects of exposure observed in 30 fish exposed to a nominal test concentration of 1000 mg/l for a period of 96 hours. Blue staining of fins, mouths and bodies of the fish was observed in the test concentration of 1000 mg/l. These effects were considered to be due to the strong blue colour of the test material and were considered to be physical effects and not toxicological effects of the test material.