Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 21 March 2010 and 13 April 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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
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 :Lanolin Fatty Acids
Description : brown waxy solid
Batch number : 0000371886
Date received : 8 February 2010
Expiry date : 21 September 2010
Storage conditions :room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Loading rate of 100 mg/l in definitive test

- Sampling method: Analysis of the WAFs was carried out by Total Organic Carbon (TOC) analysis. Water samples were taken from the control and each replicate test vessel at 0 (fresh media), 24 (old media), 72 (fresh media) and 96 hours (old media)

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

Test solutions

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

Validation of mixing period
Pre-study work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic substances in the WAF. A WAF of nominal loading rate of 100 mg/l was prepared, in duplicate, in deionised reverse osmosis water. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and samples taken for Total Organic Carbon analysis (see Appendix 3).

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 single nominal loading rate of 100 mg/l. A single loading rate was used as results from the range-finding test for the Acute Toxicity to Daphnia magna study indicated that toxicity was not expected at this loading rate.
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. 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 100 mg/l loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
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 control group was maintained under identical conditions but not exposed to the test item.
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.

Definitive test
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.

Experimental preparation
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. 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 100 mg/l loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
This method of preparation was conducted in duplicate to give replicates R1 and R2.
Total Organic Carbon (TOC) analysis was performed on the test solutions at 0 (fresh media), 24 (old media), 72 (fresh media) and 96 hours (old media).

Test organisms

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Strain: not applicable
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK
- Age at study initiation: Juvenile
- Length at study initiation: 4.8 cm (sd = 0.2)
- Weight at study initiation (mean and range, SD): 1.42 g (sd = 0.19)
- Method of breeding: asexual
- Feeding during test: not fed during test period

ACCLIMATION
- Acclimation period: Fish were acclimatised to test conditions from 24 March 2010 to 6 April 2010.
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test.
- Feeding frequency: not stated in report
- Health during acclimation (any mortality observed): zero mortality in the 7 days prior to the start of the test

QUARANTINE (wild caught)
- not applicabe

Study design

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

Test conditions

Hardness:
Approximately 140 mg/l as CaCO3
Test temperature:
The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
pH:
The pH was measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter.

Range of 7.9 to 8.6
Dissolved oxygen:
The dissolved oxygen concentration were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter.

The oxygen concentration in some of the test vessels at 0, 48 and 72 hours (fresh media) was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed.
Salinity:
Not applicable, as freshwater study
Nominal and measured concentrations:
Nominal loading rate of 100 mg/l only in definitive test
Details on test conditions:
TEST SYSTEM
- Test vessel: 20 litre glass exposure vessels
- Type: closed
- Material, size, headspace, fill volume:
- 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: Seven
- No. of vessels per concentration (replicates): Two
- No. of vessels per control (replicates): Two
- No. of vessels per vehicle control (replicates): Two
- Biomass loading rate: not stated in report

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 then passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

- Total organic carbon: Range: 0.740 to 1.390 mg/l. Mean: 1.018 mg/l
- Metals: no total value for metals provided in report
- Pesticides: Range: 0.000 to 0.005 µg/l. Mean: 0.001 µg/l
- Chlorine:
Free chlorine: Range: 0.040 to 0.480 mg/l. Mean: 0.248 mg/l
Total chlorine: Range: 0.090 to 0.560 mg/l. Mean: 0.307 mg/l
- Conductivity: Range: 288.000 to 493.000 µS/cm. Mean: 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
- Light intensity: not stated in report


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
- Spacing factor for test concentrations: 100 mg/l loading rate only concentration tested in main study
- 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: Single nominal loading rate of 100 mg/l . A single loading rate was used as results from the range-finding test for the Acute Toxicity to Daphnia magna study (ref Section 6.1.3) indicated that toxicity was not expected at this loading rate.
- Results used to determine the conditions for the definitive study: There were no sub-lethal effects of exposure during the range-finding test.
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
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
dissolved
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: none noted
- Observations on body length and weight: body length and weight described only at start of study
- 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: None
- Other adverse effects control: none
- 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:Not applicable, as no effects observed
Results with reference substance (positive control):
Not applicable

Any other information on results incl. tables

Validation of Mixing Period

Pre-study work indicated that there was no significant increase in the amount of total organic carbon by extending the preparation period for longer than 24 hours. As such, for the purposes of testing, the WAF was prepared using a stirring period of 23 hours followed by a 1-Hour standing period.

  Range-finding Test

Cumulative mortality data from the exposure of rainbow trout to the test item during the range-finding test are given in Table 1. There were no sub-lethal effects of exposure during the range-finding test.

The results showed no mortalities at the 100 mg/l loading rate WAF.

Based on this information, a single loading rate, in duplicate, of 100 mg/l using a stirring period of 23 hours followed by a 1-Hour standing period was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that no mortalities or sub-lethal effects of exposure were observed.

 Vortex depth measurements

The vortex depth was recorded at the start and end of each mixing period and was observed to be a dimple at the water surface on each occasion.

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 each mixing period, and after the 1-Hour settlement period the 100 mg/l loading rate was observed to be a clear colourless water column with lumps of waxy test item floating at the surface. After siphoning and for the duration of the test, the 100 mg/l loading rate was observed to be a clear, colourless solution. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.

Total organic carbon analysis

Samples of the control and 100 mg/l loading rate WAFs were taken at 0 (fresh media), 24 (old media), 72 (fresh media) and 96 hours (old media) for Total Organic Carbon (TOC) analysis. Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that the results gave no evidence of the presence of test item in the WAF.

Therefore, given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, and the dissolved test item was close to or below the quantifiable limit of the analytical method, 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.
It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.
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) No. 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*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.

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

Samples of the control and 100 mg/l loading rate WAF were taken at 0 (fresh media), 24 (old media), 72 (fresh media) and 96 hours (old media) for Total Organic Carbon (TOC) analysis. Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that the results gave no evidence of the presence of test item in the WAF.

Therefore, given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, and the dissolved test item was close to or below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.


*LL = Lethal Loading rate