Hatcol 1760

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 14 May 2008 and 20 June 2008.

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:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). Date of inspection: 21/08/2007. Date of signature: 11/02/2009

Test material

Test material information

Constituent 1

Details on test material:

Sponsor's identification :Hatcol 1760
Description : clear colourless slightly viscous liquid
Batch number : A25983
Date received : 4 February 2008
Storage conditions :room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- 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.
Definitive test: nominal loading rate of 100 mg/l.

The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 (fresh media), 24 (old media), 72 (fresh media) and 96 hours (old media)

- Sampling method: The test material concentration in the test samples was determined by high performance liquid chromatography (HPLC) using an external standard. The test material gave a chromatographic profile consisting of 10 peaks. The results have been calculated using the total peak area associated with the test material.
The method was developed by the Department of Analytical Services, Safepharm Laboratories Limited.

- Sample preparation: Following the addition of sodium chloride (150 g) to the test sample (500 ml) it 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 (10 ml).

- Sample storage conditions before analysis: -20°C

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Validation of mixing period
Pre-study work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF. A WAF with a nominal loading rate of 100 mg/l was prepared, in duplicate, in algal culture medium. 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 aqueous phases were then removed by siphon and the concentration of the test material in the 100 mg/l loading rate WAFs was verified by chemical analysis. Observations made on the WAF which had stirred for a period of 95 hours indicated that gross mixing of the test material had occurred. It was therefore considered appropriate to filter the aqueous phase through a glass wool plug prior to sampling for chemical analysis.

Range finding test
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. The test material was prepared as a Water Accommodated Fraction.
Amounts of test material (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 material, 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. These were stirred for 23 hours. 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 WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test material 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 material.
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 - experimental preparation
Due to the low aqueous solubility and complex nature of the test material for the purposes of the definitive test the test material was prepared as a Water Accommodated Fraction (WAF).
An amount of test material (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 material, 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. This was stirred for 23 hours. 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 material to be present.
This method of preparation was conducted in duplicate to give replicates R1 and R2.
The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 (fresh media), 24 (old media), 72 (fresh media) and 96 hours (old media)

- Eluate:
Test 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.

- Differential loading: no data.

- Controls: The control groups 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): none

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 (mean and range, SD): juvenile
- Length at study initiation (length definition, mean, range and SD): mean standard length of 4.9 cm (sd = 0.2)
- Weight at study initiation (mean and range, SD): mean weight of 1.70 g (sd = 0.21)
- Method of breeding: asexual
- Feeding during test - received no food during exposure.


ACCLIMATION
- Acclimation period:Fish were acclimatised to test conditions from 4 June 2008 to 16 June 2008.
- Acclimation conditions (same as test or not): yes
- Type and amount of food: ommercial trout pellets
- Feeding frequency: discontinued approximately 24 hours prior to the start of the definitive test.
- Health during acclimation (any mortality observed): no motality 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:

140 mg/l as CaCO3

Test temperature:

14°C to 16ºC in a temperature controlled room

pH:

Average of 7.655. (Range = 7.220 - 7.960)

Dissolved oxygen:

Greater than or equal to 9.7 mg O2/l.

Salinity:

No data

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: closed
- Material, size, headspace, fill volume:
- Aeration: The test vessels were 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
- 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: not stated in report


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: laboratory tap water.
- Total organic carbon: Range: 1.090 to 1.900 mg/l. Average: 1.429 mg/l
- Particulate matter:
- Metals:no total value for metals provided in report.
- Pesticides: Range: 0.000 to 0.092 µg/l. Mean: 0.019 µg/l
- Chlorine:
Free chlorine: Range: 0.010 to 0.460 mg/l. Mean: 0.197 mg/l.
Total chlorine: Range: 0.020 to 0.510 mg/l. Mean 0.0274 mg/l.
- Alkalinity:
- Ca/mg ratio: not stated in report
- Conductivity: Range: 289.000 to 504.000 µS/cm at 20°C. Mean: 403.577 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 conducted during study
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours.
- Light intensity: not stated in report


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.(with observation intervals if applicable).


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: 10 and 100 mg/l loading rates
- 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 concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

mortality (fish)

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: none noted
- Observations on body length and weight: body length and weight at end 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: Analysis of the test preparations at 0 (fresh media), 24 (old media), 72 (fresh media) and 96 hours (old media) showed the measured concentrations to be less than the limit of quantitation (LOQ) of the analytical method with the exception of a measured concentration of 0.864 mg/l at 96 hours for replicate R2.

Results with reference substance (positive control):

Not applicable

Any other information on results incl. tables

Sublethal observations / clinical signs:

Validation of mixing period

Pre-study work indicated that there was a significant increase in the measured test concentrations by extending the preparation period from 24 to 96 hours. However, observations made on the WAF stirred for a period of 95 hours indicated that gross mixing of the test material had occurred. Whilst the aqueous phase was removed and filtered through a glass wool plug it was considered that some undissolved test material may have remained which may have resulted in excessively high measured test concentrations. As such it was considered that a preparation period of 24 hours was sufficient to ensure equilibration between the test material and water phase.

Range finding test

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.

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.

Definitive test

Mortality data

There were no mortalities in 14 fish exposed to a 100 mg/l loading rate WAF for a period of 96 hours. Inspection of the mortality data gave the following results:

Time (h)	LL50*(mg/l)	95% Confidence Limits (mg/l)
3	>100	-
6	>100	-
24	>100	-
48	>100	-
72	>100	-
96	>100	-

The results of the definitive test showed the highest loading rate resulting in 0% mortality to be greater than or equal to 100 mg/l loading rate WAF, the lowest loading rate resulting in 100% mortality to be greater than 100 mg/l loading rate WAF and the No Observed Effect Loading rate (NOEL) to be 100 mg/l loading rate WAF. The No Observed Effect Loading rate is based upon zero mortalities and the absence of any sub-lethal effects of exposure at this loading rate

*LL = Lethal Loading rate

Sub-lethal effects

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.

Physico-chemical measurements

Temperature was maintained at 14°C to 16ºC throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Some of the temperatures during the definitive test were observed to be slightly in excess of the range given in the protocol of 14 ± 1°C. This deviation was considered not to have affected the outcome or the validity of the test as the fish showed no sub-lethal effects or mortality over the test period.

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 material 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 colourless oily globules of test material 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 material to be present.

Chemical analysis of test loading rates

Analysis of the test preparations at 0 (fresh media), 24 (old media), 72 (fresh media) and 96 hours (old media) showed the measured concentrations to be less than the limit of quantitation (LOQ) of the analytical method with the exception of a measured concentration of 0.864 mg/l at 96 hours for replicate R₂.

This does not infer that no test material was in solution but that the dissolved concentration (i.e. bioavailable to the test organisms) was generally below the limit of quantitation which was assessed down to 0.414 mg/l.

The dissolved test material may have been one or several components of the test material. Given that toxicity cannot be attributed to a single component or mixture of components but to the test material as a whole and the dissolved test material was generally below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

1.                            

Sample	Nominal Loading Rate (mg/l)	Concentration Found (mg/l)
0 hours	Control	<LOQ
(Fresh Media)	100 R1	<LOQ
	100 R2	<LOQ
24 hours	Control	<LOQ
(Old Media)	100 R1	<LOQ
	100 R2	<LOQ
72 hours	Control	<LOQ
(Fresh Media)	100 R1	<LOQ
	100 R2	<LOQ
96 hours	Control	<LOQ/<LOQ^*
(Old Media)	100 R1	2.26/<LOQ^*
	100 R2	<LOQ/0.864^*

LOQ= Limit of quantitation

*duplicate samples, frozen prior to analysis

R₁- R₂= replicates 1 and 2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 96-Hour LL50 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 material 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 Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods.Following a preliminary range-finding test fish were exposed, in two groups of seven, to a Water Accommodated Fraction (WAF) of the test material, at a single nominal loading rate of 100 mg/l for a period of 96 hours at a temperature of 14°C to 16ºC 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 LL₅₀*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.

Analysis of the test preparations at 0 (fresh media), 24 (old media), 72 (fresh media) and 96 hours (old media) showed the measured concentrations to be less than the limit of quantitation (LOQ) of the analytical method with the exception of a measured concentration of 0.864 mg/l at 96 hours for replicate R₂.

This does not infer that no test material was in solution but that the dissolved concentration (i.e. bioavailable to the test organisms) was generally below the limit of quantitation which was assessed down to 0.414 mg/l.

The dissolved test material may have been one or several components of the test material. Given that toxicity cannot be attributed to a single component or mixture of components but to the test material as a whole and the dissolved test material was generally below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

*LL = Lethal Loading rate