Fatty acids C18-(unsaturated) lithium salts

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 February 2010 to 05 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 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

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
Date received : 21 January 2010
Expiry date : 21 January 2012
Storage conditions : room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control and the 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l loading rate WAF test groups (replicates R1 – R2
pooled) at 0 and 48 hours for quantitative 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)

Method:
Amounts of test item (10, 18, 32, 56, 100, 180, 320, 560 and 1000 mg) were each separately added to the surface of 10 litres of reconstituted water to give the 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l loading rates respectively. After the addition of the test item, the reconstituted 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. Microscopic 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 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 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l loading rate WAFs. 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. Particles of test item may have passed through that 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 to 100 mg/l loading rates were observed to be cloudy dispersions.

The test preparations were not renewed during the exposure period. Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

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

At the start of the mixing period the 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l loading rates were observed to have white particles of test item dispersed throughout the water column and floating at the surface. After 23 hours stirring the 1.0, 1.8, 3.2, 5.6, 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. After a 1-Hour standing period the 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l loading rates were observed to have white particles of test item dispersed throughout, floating at the surface and settled at the bottom of the vessels. 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 a glass wool plug (2-4 cm in length). Microscopic examination after filtering showed the glass wool plug had removed all the test item particles. During the test the 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l loading rates were observed to be clear, colourless solutions.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 20 deg C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a suspension of algae (Chlorella sp.). Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.

The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.

Stock Solutions
a) CaCl2.2H2O 11.76 g/l
b) MgSO4.7H2O 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l

Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test temperature:

Temperature was maintained at approximately 21°C throughout the test.

The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

pH:

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl.
The pH was measured using a WTW pH/Oxi 340I pH meter.
There were no treatment related differences for pH.
See Appendix 4 for results- see sction ay other information on results.

Dissolved oxygen:

Dissolved oxygen concentrations were recorded at the start and termination of the test. The dissolved oxygen concentration was measured using a
dissolved oxygen meter.
See Appendix 4 for results - see section any other information on results.

Salinity:

freshwater used.

Nominal and measured concentrations:

In an initial range-finding test 100% immobilisation was observed at 10 mg/l loading rate WAF. However, during the initial experiment less than 50% immobilisation was observed at the highest test loading rate of 10 mg/l.

Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the second range-finding test are given in Table 1 - see Section any other information on results.

No immobilisation was observed at 1.0 and 10 mg/l loading rate WAF. However, immobilisation was observed at 100 mg/l loading rate WAF.

Based on the information obtained from the initial tests and the second range-finding test, loading rates of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, were selected for the definitive test.

Details on test conditions:

TEST SYSTEM

As in the range-finding test 250 ml glass jars containing approximately 200 ml of test preparation were used. At the start of the test 10 daphnids were placed in each test and control vessel at random, in the test preparations. Duplicate test vessels were used for each test and control group. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at approximately 20ºC with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control group was maintained under identical conditions but not exposed to the test item.

The test preparations were not renewed during the exposure period. Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 95% CL of 14 - 19 mg/l.

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

5.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: Not applicable

Details on results:

Range-finding Test

Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the second range-finding test are given in Table 1 - (see section any other information on results).

Definitive Test
immobilisation data
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2 (see section any other information on results). The relationship between percentage immobilisation and loading rate at 24 and 48 hours is given in Figures 1 and 2 -
see attached

Analysis of the immobilisation data by the probit method (Finney 1971) at 24 and 48 hours based on the nominal loading rates gave the following results:

EL*50 (24 h) : 50 mg/l loading rate WAF; 95% confidence limits 32 - 105 mg/l loading rate WAF
EL*50 (48 h) : 16 mg/l loading rate WAF; 95% confidence limits 14 - 19 mg/l loading rate WAF

The No Observed Effect Loading rates after 24 and 48 hours exposure were 18 and 5.6 mg/l loading rate WAF respectively. The No Observed Effect Loading rate is based upon zero immobilisation at this loading rate.

The slopes and their standard errors of response curves at 24 and 48 hours were 2.7 (SE = 0.7) and 5.6 (SE = 1.0) respectively.

*EL = Effective Loading Rate

Results with reference substance (positive control):

Positive Control
A positive control (Harlan Laboratories Ltd Project No: 0039/1133) conducted approximately every six months used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.
An amount of reference item (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give a 100 mg/l stock solution. An aliquot (50 ml) of this stock solution was diluted in reconstituted water and the volume adjusted to 500 ml to give a 10 mg/l stock solution. Aliquots
(16, 28, 50, 90 and 160 ml) of the 10 mg/l stock solution were each separately dispersed in a final volume of 500 ml of reconstituted water to give the test series of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.
Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Exposure conditions for the positive control were similar to those used in the definitive test.
The temperature was maintained at approximately 20°C.

Analysis of the immobilisation data by the probit method (Finney 1971) at 24 hours and the trimmed Spearman-Karber method at 48 hours based on the nominal ltest concentrations gave the following results:

EC50 (24 h) : 0.81 mg/l; 95% confidence limits 0.72 - 0.97 mg/l
EC50 (48 h) : 0.65 mg/l; 95% confidence limits 0.58 - 0.72 mg/l

The No Observed Effect Concentration after 24 and 48 hours exposure was 0.32 mg/l. The No Observed Effect Loading rate is based upon zero immobilisation at this loading rate.

The slope and its standard error of response curvesat 24 hours was 7.7 (SE = 1.6). Due to the unsuitable nature of the data it was not possible to calculate the slope and error of response curve at 48 hours.

The results from the positive control with potassium dichromate were within the normal range for this reference item. The mean 48-Hour EC50 value calculated from all positive controls was 0.77 mg/l (sd = 0.20).

Reported statistics and error estimates:

The EL*50 values and associated confidence limits at 24 and 48 hours were calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999).

Probit analysis is used where two or more partial responses to exposure are shown.

*EL = Effective Loading Rate

Any other information on results incl. tables

Table1              Cumulative Immobilisation Data in the Range-finding Test

Nominal Loading Rate (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
1.0	0	0
10	0	0
100	5	5

Table 2              Cumulative Immobilisation Data in the Definitive Test

Nominal Loading Rate (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
1.0	0	0	0	0	0	0	0	0
1.8	0	0	0	0	0	0	0	0
3.2	0	0	0	0	0	0	0	0
5.6	0	0	0	0	0	0	0	0
10	0	0	0	0	1	3	4	20
18	0	0	0	0	4	5	9	45
32	6	5	11	55	10	10	20	100
56	7	7	14	70	10	10	20	100
100	4	8	12	60	10	10	20	100

 

R₁– R₂= Replicates 1 and 2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 48-Hour EL*50 for the test item to Daphnia magna based on nominal loading rates was 16 mg/l loading rate WAF with 95% confidence limits of 14 - 19 mg/l loading rate WAF. The No Observed Effect Loading rate was 5.6 mg/l loading rate WAF.

*EL = Effective Loadign Rate

Executive summary:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Following preliminary range-finding tests and an initial experiment, twenty daphnids (2 replicates of 10 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l for 48 hours under at a temperature of approximately 20°C under static test conditions. The number of immobilised Daphnia were recorded after 24 and 48 hours.

The 48-Hour EL*₅₀for the test item to Daphnia magnia based on nominal loading rates was 16 mg/l loading rate WAF with 95% confidence limits of 14 - 19 mg/l loading rate WAF. The No Observed Effect Loading rate was 5.6 mg/l loading rate WAF.

Analysis of the test loading rates at 0 hours showed a concentration dependant increase in measured values ranging from 0.0475 mg/l to 15.6 mg/l.

Analysis of the test loading rates at 48 hours showed a decline in measured values ranging from less than the limit of quantitation (LOQ) of the analytical method which was determined to be 0.0095 mg/l, to 9.67 mg/l.

The decline in measured concentrations over the 48 hour period is in line with stability analysis which showed the test item to be unstable particularly at the lower levels employed.

Given that 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.

*EL = Effective Loading rate