Fatty acids C18-(unsaturated) lithium salts

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 25 April 2010 and 06 June 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

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 the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

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

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. Pre-study work showed that the maximum amount of dissolved test item was achieved after a preparation period of 24 hours. At the completion of mixing, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

LiTOFA

IUPAC Name:

LiTOFA

Details on test material:

Sponsor's identification: LiTOFA
Description : off white solid
Batch number : SF 173
Expiry date : 21/01/2012
Date received : 21/01/2010
Storage conditions: room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
10, 18, 32, 56 and 100 mg/l loading rate WAF
- Sampling method:
Water samples were taken from the control and all surviving test groups at 0 (fresh media), 24 (old media), 72 (fresh media) and 96 (old media) hours for quantitative analysis.
Duplicate samples and samples at 24 (old media), 48 (fresh and old media) and 72 hours (old media) were taken and stored at approximately -20°C for further analysis, if necessary. The method of analysis, recovery and test solution analyses are described in the attached Appendix 3.

Test solutions

Vehicle:

no

Details on test solutions:

- Method:
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).
Amounts of test item (210, 378, 672, 1176 and 2100 mg) were each separately added to the surface of 21 litres of dechlorinated tap water to give the 10, 18, 32, 56 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. These were stirred for 23 hours. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Visual observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through two 0.2 µm retention size Postlip filter papers. 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 WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) and filtered through two 0.2 µm retention size Postlip filter papers to give the 10, 18, 32, 56 and 100 mg/l loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no particles of test item to be present.


Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start of the mixing period the 10, 18, 32, 56 and 100 mg/l loading rates were observed to be clear water columns with white particles of test item dispersed throughout and floating at the surface. After 23 hours stirring period and a 1-Hour standing period the 10, 18, 32, 56 and 100 mg/l loading rates were observed to have white particles of test item dispersed throughout and floating at the surface. Microscopic examination of the WAFs showed particles of test item to be present and therefore it was considered justifiable to remove the WAFs by filtering through two Postlip filter papers. Microscopic examination after filtering showed no particles of test item present, however, this does not infer that no test item was present in the media. Given the retention size of the filter paper the size of particles that passed through could possibly be too small to be detected in small quantities but are observed in a large amount, such as was required in this study. During the test the 10 mg/l loading rate was observed to be a clear colourless solution, the 18 and 32 mg/l loading rates were observed to be pale homogenous dispersions and the 56 and 100 mg/l loading rates were observed to be cloudy homogenous dispersions.

As the test item was observed to form a dispersion in the test diluent, microscopic inspection was performed on the gill filaments of the dead fish observed during the test. This examination revealed no test item adhered to the gill filaments indicating that physical toxicity as not a factor.

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name:
Rainbow Trout

- Strain:
Not stated

- 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):
Fish had a mean standard length of 4.2 cm (sd = 0.3)

- Weight at study initiation (mean and range, SD):
And a mean weight of 0.90 g (sd = 0.16) at the end of the definitive test
Based on the mean weight value this gave a loading rate of 0.32 g bodyweight/litre.

- Method of breeding:
Not stated

- Feeding during test
- Food type:
The stock fish were fed commercial trout pellets which was discontinued 24 hours prior to the start of the definitive test

- Amount:
Not stated

- Frequency:
Not stated


ACCLIMATION
- Acclimation period:
Fish were acclimatised to test conditions from 12 May 2010 to 24 May 2010.

- Acclimation conditions (same as test or not):
Same as test conditions.

- Type and amount of food:
The stock fish were fed commercial trout pellets which was discontinued 24 hours prior to the start of the definitive test

- Feeding frequency:
Not stated

- Health during acclimation (any mortality observed):
Zero mortality in the 7 days prior to the start of the test.


QUARANTINE (wild caught)
- Duration:
Not applicable

- Health/mortality:
Not applicable

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

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 conditions

Hardness:

Total hardness of approximately 140 mg/l as CaCO3.

Test temperature:

The test vessels were covered and maintained at approximately 14ºC

pH:

The pH was measured using a WTW pH/Oxi 340I pH.
pH range of 7.6-8.5.
Please see Physico-Chemical Measurements appendix 4(attached background material).

Dissolved oxygen:

The dissolved oxygen concentration was measured using a dissolved oxygen meter.
Please see Physico-Chemical Measurements appendix 4 (attached background material) for dissolved oxygen results.

Salinity:

No Data

Nominal and measured concentrations:

In the range-finding test fish were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/l.
Definitive test: 10, 18, 32, 56 and 100 mg/l loading rates.

Details on test conditions:

As in the range-finding test 20 litre glass exposure vessels were used for each test concentration. At the start of the test 7 fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at approximately 14ºC 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. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.

The control group was maintained under identical conditions but not exposed to the test item.
A semi-static test regime was employed in the test involving a daily renewal of the test preparations to ensure test concentrations of soluble components of the test item were maintained over the test and to prevent the build up of nitrogenous waste products.
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 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.















TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: In the range-finding test fish were exposed to a series of nominal loading rates of 10 and 100 mg/l.
- Results used to determine the conditions for the definitive study:Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/l.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

24 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% confidence limits of 18 - 32 mg/l loading rate WAF

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: CL not stated

Details on results:

- Behavioural abnormalities:
None recorded.

- Observations on body length and weight:
None recorded

- Other biological observations:
None recorded

- Mortality of control:
None recorded

- Other adverse effects control:
None recorded

- Abnormal responses:
Sub-lethal effects of exposure were observed at the 18 mg/l loading rate WAF and above. These responses were increased pigmentation, lethargy and sitting at the bottom, swimming at the surface, lethargy with increased pigmentation, swimming at the surface with increased pigmentation and loss of equilibrium with increased pigmentation .

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
At the start of the mixing period the 10, 18, 32, 56 and 100 mg/l loading rates were observed to be clear water columns with white particles of test item dispersed throughout and floating at the surface. After 23 hours stirring period and a 1-Hour standing period the 10, 18, 32, 56 and 100 mg/l loading rates were observed to have white particles of test item dispersed throughout and floating at the surface. Microscopic examination of the WAFs showed particles of test item to be present and therefore it was considered justifiable to remove the WAFs by filtering through two Postlip filter papers. Microscopic examination after filtering showed no particles of test item present, however, this does not infer that no test item was present in the media. Given the retention size of the filter paper the size of particles that passed through could possibly be too small to be detected in small quantities but are observed in a large amount, such as was required in this study. During the test the 10 mg/l loading rate was observed to be a clear colourless solution, the 18 and 32 mg/l loading rates were observed to be pale homogenous dispersions and the 56 and 100 mg/l loading rates were observed to be cloudy homogenous dispersions.

- Effect concentrations exceeding solubility of substance in test medium:
None recorded

Results with reference substance (positive control):

- Results with reference substance valid?
Not applicable

- Mortality:
Not applicable

- LC50:
Not applicable

- Other:
Not applicable

Reported statistics and error estimates:

An estimate of the LL50*values was given by inspection of the mortality data at 3 and 6 hours.
The LL50*values and associated confidence limits at 24 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al1977) using the ToxCalc computer software package (ToxCalc 1999) and at 48, 72 and 96 hours the LL50* values were calculated using the geometric mean method as follows:

*LL = Lethal Loading rate

LL50 * value = √L1 x L2


Where:
L1= loading rate showing 0% mortality L2= loading rate showing 100 % mortality
When only one partial response is shown the trimmed Spearman-Karber method is appropriate.
If there are no test concentrations showing mortalities between 0% and 100%, then the geometric mean of the highest loading rate showing no lethality and the lowest loading rate showing 100% lethality is calculated. The loading rates resulting in 0% and 100% mortality will be the 95% confidence limits.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Table1              Cumulative Mortality Data in the Range-finding Test

Nominal Loading Rate (mg/l)	Cumulative Mortality (Initial Population = 3)
	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours
Control	0	0	0	0	0	0
1.0	0	0	0	0	0	0
10	0	0	0	0	0	0
100	0	0	0	3	3	3

 

Table 2              Sub-lethal Effects of Exposure in the Range-finding Test

Nominal Loading Rate (mg/l)	Sub-lethal Effects	Time (Hours)
		3	6	24	48	72	96
Control	No abnormalities detected
1.0	No abnormalities detected
10	No abnormalities detected
100	Lethargic Sitting at bottom and lethargic Lethargic with test item observed adhered to dorsal fin	3/3	2/3     1/3	2/3     1/3	A/D

A/D

A/D= All fish dead

Table 3              Cumulative Mortality Data in the DefinitiveTest

Nominal Loading Rate (mg/l)	Cumulative Mortality (Initial Population =7)						% Mortality
	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours	96 Hours
Control	0	0	0	0	0	0	0
10	0	0	0	0	0	0	0
18	0	0	0	0	0	0	0
32	0	0	0	7	7	7	100
56	0	0	0	7	7	7	100
100	0	0	4	7	7	7	100

Table 4              Sub-lethal Effects of Exposure in the DefinitiveTest

Nominal Loading Rate (mg/l)	Sub-lethal Effects	Time (Hours)
		3	6	24	48	72	96
Control	No abnormalities detected
10	No abnormalities detected
18	Swimming at surface and increased pigmentation Swimming at surface Increased pigmentation					1/7 1/7 1/7	2/7 2/7
32	Lethargic and sitting at bottom Swimming at surface Swimming at surface and increased pigmentation		3/7 4/7	7/7	A/D
56	Increased pigmentation Lethargic with increased pigmentation Swimming at surface Loss of equilibrium Sitting at bottom	7/7	7/7	7/7     2/7 2/7 3/7	A/D
100	Increased pigmentation Lethargic with increased pigmentation Loss of equilibrium and increased pigmentation	7/7	7/7	3/3	A/D

A/D

A/D= All fish dead

Table 5              Vortex Depth Measurements at the Start and End of Each Mixing Period

FIRST MIXING PERIOD

Nominal Loading Rate (mg/l)

Control

10

18

32

56

100

*

+

*

+

*

+

*

+

*

+

*

+

Height of Water Column (cm)

36.5

36.5

36.5

36.5

36.5

36.5

37.0

37.0

37.0

37.0

37.0

37.0

Depth of Vortex (cm)

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

Observation of Vortex

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

SECOND MIXING PERIOD

Nominal Loading Rate (mg/l)

Control

10

18

32

56

100

*

+

*

+

*

+

*

+

*

+

*

+

Height of Water Column (cm)

36.0

36.0

36.5

36.5

36.0

36.0

36.5

36.5

36.5

36.5

36.5

36.5

Depth of Vortex (cm)

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

Observation of Vortex

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

A/D

Table 5 (continued)           Vortex Depth Measurements at the Start and End of Each Mixing Period

THIRD MIXING PERIOD

Nominal Loading Rate (mg/l)

Control

10

18

32

56

100

*

+

*

+

*

+

*

+

*

+

*

+

Height of Water Column (cm)

35.5

35.5

36.0

36.0

36.0

36.0

36.0

36.0

36.0

36.0

35.5

35.5

Depth of Vortex (cm)

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

Observation of Vortex

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

FOURTH MIXING PERIOD

Nominal Loading Rate (mg/l)

Control

10

18

32

56

100

*

+

*

+

*

+

*

+

*

+

*

+

Height of Water Column (cm)

36.0

36.0

35.5

35.5

36.0

36.0

36.5

A/D

A/D

A/D

A/D

A/D

Depth of Vortex (cm)

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

Observation of Vortex

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

A/D

*= Start of mixing period

+= End of mixing period

A/D= All fish dead

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LL*50 value of 24 mg/l loading rate WAF with 95% confidence limits of 18 - 32 mg/l loading rate WAF. The No Observed Effect Loading rate was 10 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) No.440/2008.

Methods.

Following a preliminary range-finding test, fish were exposed, in groups of seven, to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 10, 18, 32, 56 and 100 mg/l for a period of 96 hours at a temperature of approximately 14ºC 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 24 mg/l loading rate WAF with 95% confidence limits of 18 - 32 mg/l loading rate WAF. The No Observed Effect Loading rate was 10 mg/l loading rate WAF.

Analysis of the test samples at 0 hours showed a concentration dependant increase in measured test concentrations of 0.86 mg/l at 10 mg/l loading rate WAF through to 11.3 mg/l at 100 mg/l loading rate WAF. Analysis of the test preparations at 24 hours showed measured test concentrations in the range of less than the limit of quantitation (LOQ) of the analytical method employed (which was determined to be 0.0486 mg/l) to 14.4 mg/l. Analysis of the test concentrations at 72 hours showed measured test concentrations to be 1.92 mg/l and 3.45 mg/l for the remaining test groups of 10 and 18 mg/l loading rate WAF respectively. At 96 hours measured test concentrations were shown to be less than the LOQ and 0.756 mg/l for the 10 and 18 mg/l loading rate WAFs respectively. 

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