Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31st May 2010 to 23 July 2010
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 relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 15th September 2009 Date of signature; 26th November 2009

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification :DVS005u (aka Weston 705)
Description : clear colourless viscous liquid
Batch number : MW9F23A901
Date received : 05 November 2009
Expiry date : 15 January 2011
Storage conditions :approximately 4°C in the dark under nitrogen

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Final thoeretical concentrations of between 6 mg/l (0.10 mg/l spiked sample) and 10 mg/l (for 1.0 and 10 mg/l spiked samples)

- Sampling method: A volume of test sample was extracted with dichloromethane (3 x 50 ml). The extracts were filtered through anhydrous sodium sulphate. The combined extracts were evaporated to dryness and the residue re-dissolved in acetonitrile to give a final theoretical concentration of between 6 and 10 mg/l.

- Sample storage conditions before analysis: Duplicate samples were taken and stored at approximately -20°C for further analysis, if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:

Range finding test:
Due to the low aqueous solubility and complex nature of the test item for the purposes of the range-finding test the test item was prepared as a Water Accommodated Fraction (WAF).
The loading rate 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 loading rates of 10 and 100 mg/l.
Amounts of test item (210 and 2100 mg) were each separately added to the surface of 21 litres of dechlorinated tap water to give the 10 and 100 mg/l loading rates respectively. After the addition of the test item, the dechlorinated tap water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 10 and 100 mg/l loading rate WAF. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.

Definitive test
An amount of test item (2100 mg) was added to the surface of 21 litres of dechlorinated tap water to give the 100 mg/l loading rate. After the addition of the test item, the dechlorinated tap water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. Microscopic observations made on the WAF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAF by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first approximate 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic observations of the WAF were performed after filtering through glass wool showed no evidence of particles or micro-dispersions of test item. This differs from the range-finding test where filtration through a glass wool plug was not required as microscopic observation showed no micro-dispersions of test item in the WAF. This was considered to be due to possible slight differences in water quality and variations in stirring.

- Eluate: same as culture media

- Differential loading: Not in the definitive test

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

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable

- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Not applicable

- Evidence of undissolved material (e.g. precipitate, surface film, etc): Microscopic observations of the WAF were performed after filtering through glass wool showed no evidence of particles or micro-dispersions of test item.

Test organisms

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Strain: not stated in report
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK
- Age at study initiation (mean and range, SD): Juveniles
- Length at study initiation (length definition, mean, range and SD): No length at study initiation provided. Length at end of definitive test = mean standard length of 4.7 cm (sd = 0.2)
- Weight at study initiation (mean and range, SD): No weight at study initiation provided. Weight at end of defintive study - mean weight of 1.23 g (sd = 0.10)
- Method of breeding: not applicable
- Feeding during test: - no feeding during exposure

ACCLIMATION
- Acclimation period: acclimatised to test conditions from 7 July 2010 to 19 July 2010.
- Acclimation conditions (same as test or not): yes
- Type and amount of food: commercial trout pellets which was discontinued 24 hours prior to the start of the definitive test.
- Feeding frequency: not stated in report
- Health during acclimation (any mortality observed): no mortality observed

QUARANTINE (wild caught)
- not applicable

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
Not applicable

Test conditions

Hardness:
total hardness of approximately 140 mg/l as CaCO3.
Test temperature:
Record daily throughout the test.
Temperature was maintained at approximately 14ºC throughout the test.
pH:
Recorded daily throughout the test using a WTW pH/Oxi 3401 pH meter.
Dissolved oxygen:
Recorded daily throughout the test using a WTW pH/Oxi 3401 dissolved oxygen meter.
Salinity:
Not applicable as freshwater study.
Nominal and measured concentrations:
In the range-finding test fish were exposed to a series of nominal loading rates of 10 and 100 mg/l. The test material was prepared as a Water Accommodated Fraction.

Based on the results of the range-finding test a "Limit test" was conducted at a single loading rate of 100 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass jars
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 20 l glass vessels
- Aeration: aerated via narrow bore glass tubes
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 7 fish
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): not applicable, as no vehicle
- Biomass loading rate: Based on the mean weight value this gave a loading rate of 0.43 g bodyweight/litre.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: laboratory tap water (dechlorinated)
- Total organic carbon: Range: 0.740 to 1.390 mg/l. Average: 1.018 mg/l
- Metals: no total value for metals provided in report
- Pesticides: Range; 0.000 to 0.005 µg/l. Average: 0.001 µg/ml
- Chlorine:
Free chlorine: Range: 0.040 to 0.480 mg/l Average: 0.248 mg/l
Total chlorine: Range: 0.090 to 0.580 mg/l. Average: 0.307 mg/l
- Conductivity: Range: 288.000 to 493.000 µS/cm at 20°C. Average: 386.192 µS/cm at 20°C.
- Culture medium different from test medium: not applicable
- Intervals of water quality measurement: 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.

OTHER TEST CONDITIONS
- Adjustment of pH: not required
- Photoperiod: 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.
- Light intensity: not stated in report

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mobility, death

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable, as only one concentration used in definitive test
- Justification for using less concentrations than requested by guideline: Based on the results of the range-finding test a "Limit test" was conducted at a single loading rate of 100 mg/l to confirm that no mortalities or sub-lethal effects of exposure were observed.

- Range finding study:
- Test concentrations: 10 and 100 mg/l
- Results used to determine the conditions for the definitive study: There were no sub-lethal effects of exposure during the range-finding test.
The results showed no mortalities at the 10 and 100 mg/l loading rate WAFs.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
dissolved
Remarks:
(loading rate WAF)
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
dissolved
Remarks:
(loading rate WAF)
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: none noted
- Observations on body length and weight: body length and weight at start of study not described in study report.
- Other biological observations: There were no sub-lethal effects of exposure observed in 14 fish exposed to a 100 mg/l loading rate WAF for a period of 96 hours.
- Mortality of control: no
- Other adverse effects control: not applicable
- Abnormal responses: none stated in report
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not applicable, as WAF's used, so measured values not available
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
Not applicable

Any other information on results incl. tables

Observations on test item solubility

Observations on the test media were carried out during the mixing and testing of the WAFs.

At the start and end of the first mixing period the 100 mg/l loading rate Replicates 1 and 2 were observed to be clear colourless water columns with oily globules of test item floating at the surface, after the 1-Hour settlement period the 100 mg/l loading rate Replicates 1 and 2 were observed to be clear colourless water columns with globules of test item in the water column and at the bottom and surface of the mixing vessel. Microscopic examination of the WAF showed particles of test item and therefore it was considered justifiable to remove the WAF by filtering through a glass wool plug (2-4 cm in length). Microscopic observations of the WAF after filtering through glass wool showed no evidence of particles or micro-dispersions of test item. During the test the 100 mg/l loading rate Replicates 1 and 2 were observed to be clear, colourless solutions.

At the start of the remaining mixing periods the 100 mg/l loading rate Replicates 1 and 2 were observed to be clear colourless water columns with oily globules of test item floating at the surface, at the end of the mixing period and after the 1-Hour settlement period the 100 mg/l loading rate Replicates 1 and 2 were observed to be clear colourless water columns with globules of test item suspended in the water column floating at the surface and at the bottom of the mixing vessel. Microscopic examination of the WAF showed particles of test item and therefore it was considered justifiable to remove the WAF by filtering through a glass wool plug (2-4 cm in length). Microscopic observations of the WAF after filtering through glass wool showed no evidence of particles or micro-dispersions of test item. During the test the 100 mg/l loading rate Replicates 1 and 2 were observed to be clear, colourless solutions. The slight differences observed between mixing periods were considered to be due to slight differences in stirring speeds despite every effort to ensure consistency.

Chemical analysis of test loading rates

Analysis of the freshly prepared test media at 0 and 72 hours showed measured test concentrations to range from 0.0054 to 0.192 mg/l. Analysis of the old or expired test media at 24 and 96 hours showed measured concentrations to range from less than the limit of quantitation (LOQ) of the analytical method, (which was determined to be 0.00044 mg/l) to 0.00981 mg/l. 

The slight decline in measured concentrations over the 24 Hour dosing period is contrary to stability analysis which showed the test item to be stable, however, it was considered to be due to possible hydrolysis of the test item. No toxic effects were observed during the test and so this decline over the 24-Hour period was considered to have no impact on the outcome of the test.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The 96-Hour LL*50 based on nominal loading rates was greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading rate was 100 mg/l loading rate WAF.
Executive summary:

Introduction.

A study was performed to assess the acute toxicity of the test item 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) 440/2008).

Methods.

Following a preliminary range-finding test fish were exposed, in two groups of seven, to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/l for a period of 96 hours at a temperature of approximately 14°C 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 LL*50based on nominal loading rates was greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading rate was 100 mg/l loading rate WAF.

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

Analysis of the freshly prepared test media at 0 and 72 hours showed measured test concentrations to range from 0.0054 to 0.192 mg/l. Analysis of the old or expired test media at 24 and 96 hours showed measured concentrations to range from less than the limit of quantitation (LOQ) of the analytical method, (which was determined to be 0.00044 mg/l) to 0.00981 mg/l. 

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.


*LL = Lethal Loading rate