Lithium naphthalene-2-carboxylate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 July 2008 and 4 October 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). These Regulations are in accordance with GLP standards published as OECD Principles on

Test material

Test material information

Constituent 1

Reference substance name:

Lithium naphthalene-2-carboxylate

EC Number:

700-040-5

Cas Number:

674347-28-5

Molecular formula:

C11 H7 Li O2

IUPAC Name:

Lithium naphthalene-2-carboxylate

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:An aliquot (250 ml) of each of the stock solutions was separately mixed with algal suspension (250 ml) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/l.
- 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 a single peak. The test samples were analysed following filtration through glass wool to remove the algal cells.
- Sample storage conditions before analysis:
Sponsor's identification : NA-Li
Description : off white crystalline solid
Batch number : 7001
Date received : 12 May 2008
Storage conditions : room temperature in the dark

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For the purpose of the definitive test, the test material was dissolved directly in culture medium.
- Eluate: Same as culture media
- Controls: The control group was maintained under identical conditions but not exposed to the test material.
A positive control (Safepharm Laboratories Project Number: 0039/0994) used potassium dichromate as the reference material. An amount of reference material (100 mg) was dissolved in culture medium and the volume adjusted to 1 litre to give a 100 mg/l stock solution from which a series of dilutions was made to give further stock solutions of 10, 2.0, 1.0, 0.50, 0.25 and 0.125 mg/l. An aliquot (250 ml) of each of the 0.125, 0.25, 0.50, 1.0 and 2.0 mg/l stock solutions was separately mixed with algal suspension (250 ml) to give the required test concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.
The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Six replicate flasks were prepared for the control and three replicate flasks prepared for each test concentration.
The flasks were incubated (INFORS Multitron Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: CCAP 276/20.
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): Not available
- Method of cultivation:Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1C until the algal cell density was approximately 104 - 105 cells/ml.


ACCLIMATION
- Acclimation period:Not available
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l
The culture medium was prepared using reverse osmosis purified deionised water* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

- Culturing media and conditions (same as test or not): Same as test.
- Any deformed or abnormal cells observed:None recorded

Study design

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Post exposure observation period:

All test and control cultures were inspected microscopically at 72 hours.

Test conditions

Hardness:

Not available

Test temperature:

Temperature was maintained at 24 ± 1ºC throughout the test.The temperature within the incubator was recorded daily.

pH:

The pH values of the control cultures (see Table 2) were observed to increase from pH 7.5 – 7.6 at 0 hours to pH 7.9 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter.

Dissolved oxygen:

Not available

Salinity:

Not available

Nominal and measured concentrations:

The range-finding test; nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l
definitive test: 1.0, 3.2, 10, 32 and 100 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel:250 ml glass conical flasks
- Type (delete if not applicable): closed, plugged with polyurethane foam bungs to reduce evaporation.
- Material, size, headspace, fill volume: 250ml glass conical flasks each containing 100ml of test preparation.
- Aeration: None
- Type of flow-through (e.g. peristaltic or proportional diluter):Not applicable
- Renewal rate of test solution (frequency/flow rate):Not applicable
- Initial cells density: 4 x 10^3 cells per ml.
- Control end cells density: 5.68 x 10^5 cells per ml
- No. of organisms per vessel:Not applicable
- No. of vessels per concentration (replicates):Three flasks each containing 100 ml
- No. of vessels per control (replicates):Six flasks each containing 100 ml of test preparation



GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: For the purpose of the definitive test, the test material was dissolved directly in culture medium.


TEST MEDIUM / WATER PARAMETERS
For the purpose of the definitive test, the test material was dissolved directly in culture medium.
Amounts of test material (100 and 32 mg) were each separately dissolved in culture medium and the volumes adjusted to 500 ml to give 200 and 64 mg/l stock solutions respectively. A series of dilutions was made from these stock solutions to give further stock solutions of 20, 6.4 and 2.0 mg/l. An aliquot (250 ml) of each of the stock solutions was separately mixed with algal suspension (250 ml) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/l.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 72 hours

Culture Medium

NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l
The culture medium was prepared using reverse osmosis purified deionised water* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.



OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod:Under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm)
- Light intensity and quality: Under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm)



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 0, 23, 49 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100 mg/l for a period of 72 hours.
- Results used to determine the conditions for the definitive study: The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test material during the range-finding test are given in below.

(mg/l) Cell Densities* (cells per ml) Inhibition Values (%)
0 Hours 72 Hours Growth Rate Yield/Biomass Integral
Control R1 4.37E+03 4.67E+05
R2 4.40E+03 5.48E+05 - -
Mean 4.39E+03 5.08E+05
0.10 R1 4.55E+03 4.99E+05
R2 4.06E+03 4.72E+05 0 4
Mean 4.30E+03 4.85E+05
1.0 R1 4.24E+03 4.98E+05
R2 4.67E+03 5.39E+05 0 [2]
Mean 4.46E+03 5.18E+05
10 R1 4.48E+03 3.88E+05
R2 4.06E+03 4.09E+05 5 22
Mean 4.27E+03 3.99E+05
100 R1 4.62E+03 3.99E+04
R2 4.52E+03 6.08E+04 50 91
Mean 4.57E+03 5.03E+04

The results showed no effect on growth at the test concentrations of 0.10 and 1.0 mg/l. However, growth was observed to be reduced at 10 and 100 mg/l.
Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/l were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 9 - 17 mg/l

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): There were no abnormalities detected in any of the control or test cultures at 72 hours.
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:Not stated
- Effect concentrations exceeding solubility of substance in test medium: At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control, 1.0, 3.2 and 10 mg/l test cultures were observed to be green dispersions. The 32 mg/l test cultures were observed to be pale green dispersions whilst the 100 mg/l test cultures were observed to be brown/green dispersions.

Results with reference substance (positive control):

- Results with reference substance valid?
- EC50:
- Other:

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate, yield and biomass integral data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Any other information on results incl. tables

The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test material during the range-finding test are given in Table 1.

The results showed no effect on growth at the test concentrations of 0.10 and 1.0 mg/l. However, growth was observed to be reduced at 10 and 100 mg/l.

Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/l were selected for the definitive test.

DefinitiveTest

Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rates, yield and biomass integral values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.

The mean cell densities versus time for the definitive test are presented in Figure 1. Percentage inhibition values are plotted against test concentration in Figure 2, Figure 3 and Figure 4.

Validation criteria

The following data show that the cell concentration of the control cultures increased by a factor of 125 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Mean cell density of control at 0 hours                            :   4.55 x 10³ cells per ml
Mean cell density of control at 72 hours                          :   5.68 x 10⁵ cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 30% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Growth data

From the data given in Tables 2 and 4, it is clear that the growth rate (r), yield (y) and biomass integral (b) of Desmodesmus subspicatus (CCAP 276/20) were affected by the presence of the test material over the 72-Hour exposure period.

Accordingly the following results were determined from the data:

Inhibition of growth rate

E_rC₁₀(0 - 72 h)             : 7.2 mg/l
E_rC₂₀(0 - 72 h)             : 24 mg/l
E_rC₅₀(0 - 72 h)             : > 100 mg/l

where E_rC_xis the test concentration that reduced growth rate by x%.

It was not possible to calculate an E_rC₅₀value as no concentration tested resulted in greater than 50% inhibition of growth rate.

Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control, 1.0 and 3.2 mg/l test concentrations (P³0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 3.2 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 10 mg/l.

Inhibition of yield

E_yC₁₀(0 - 72 h)            : 3.5 mg/l
E_yC₂₀(0 - 72 h)            : 5.5 mg/l
E_yC₅₀(0 - 72 h)            : 12 mg/l; 95% confidence limits 9.3 - 17 mg/l

where E_yC_xis the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out as in Section 5.2.2.1. There were no statistically significant differences between the control, 1.0 and 3.2 mg/l test concentrations (P³0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 3.2 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 10 mg/l.

Inhibition of biomass integral

E_bC₁₀(0 - 72 h)            : 4.6 mg/l
E_bC₂₀(0 - 72 h)            : 7.5 mg/l
E_bC₅₀(0 - 72 h)            : 20 mg/l*

where E_bC_xis the test concentration that reduced biomass integral (area under the growth curve) by x%.

Statistical analysis of the biomass integral data was carried out as in Section 5.2.2.1. There were no statistically significant differences between the control, 1.0 and 3.2 mg/l test concentrations (P³0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on biomass integral was 3.2 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on biomass integral was 10 mg/l.

Observationson cultures

All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures at 72 hours.

Observations on test material solubility

At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control, 1.0, 3.2 and 10 mg/l test cultures were observed to be green dispersions. The 32 mg/l test cultures were observed to be pale green dispersions whilst the 100 mg/l test cultures were observed to be brown/green dispersions.

Physico-chemical measurements

The pH values of each test and control flask are given in Table 2. Temperature was maintained at 24 ± 1ºC throughout the test.

The pH values of the control cultures (see Table 2) were observed to increase from pH 7.5 – 7.6 at 0 hours to pH 7.9 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Verification of test concentrations

Analysis of the test preparations at 0 and 72 hours (see Appendix 2) showed measured test concentrations to range from 81% to 96% of nominal and so it was considered justifiable to calculate the EC₅₀values in terms of the nominal test concentrations only.

Positive Control

The cell densities from exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference material during the positive control (Safepharm Laboratories Project No: 0039/0994) are given in Table 5 and Figure 5. Daily specific growth rates for the control cultures are given in Table 6 whilst growth rate, yield and biomass integral values are given in Table 7. Percentage inhibition values are plotted against test concentration in Figure 6, Figure 7 and Figure 8.

Accordingly the following results were determined from the data:

E_rC₅₀(0 - 72 h)             : 0.57 mg/l; 95% confidence limits 0.48 - 0.66 mg/l
E_yC₅₀(0 - 72 h)            : 0.32 mg/l; 95% confidence limits 0.29 - 0.35 mg/l
E_bC₅₀(0 - 72 h)            : 0.31 mg/l; 95% confidence limits 0.28 - 0.35 mg/l

No Observed Effect Concentration (NOEC) based on growth rate                             : 0.25 mg/l
No Observed Effect Concentration (NOEC) based on yield                                        : 0.0625 mg/l
No Observed Effect Concentration (NOEC) based on biomass integral                      : 0.0625 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate            : 0.50 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield                                  : 0.125 mg/l
Lowest Observed Effect Concentration (LOEC) based on biomass integral     : 0.125 mg/l

The results from the positive control with potassium dichromate were within the normal range for this reference material.

*It was not possible to calculate 95% confidence limits for the E_bC₅₀value as the data generated did not fit the models available for the calculation of confidence limits.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated over a 72-Hour period and gave an ErC50 (0 - 72 h) of greater than 100 mg/l, an EyC50 (0 - 72 h) of 12 mg/l; 95% confidence limits 9 - 17 mg/l, and an EbC50 (0 - 72 h) of 20 mg/l. The Lowest Observed Effect Concentration based on growth rate, yield and biomass integral was 10 mg/l, and the No Observed Effect Concentration was 3.2 mg/l.

Executive summary:

Introduction. A study was performed to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods. Following a preliminary range-finding test,Desmodesmus subspicatuswas exposed to an aqueous solution of the test material at concentrations of 1.0, 3.2, 10, 32 and 100 mg/l (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter Multisizer Particle Counter.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Desmodesmus subspicatus was exposed to an aqueous solution of the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter Multisizer Particle Counter.

Results. In terms of growth rate, exposure of Desmodesmus subspicatus to the test material gave an E_rC₅₀(0 - 72 h) value of greater than 100 mg/l*. The Lowest Observed Effect Concentration based on inhibition of growth rate was 10 mg/l and the No Observed Effect Concentration was 3.2 mg/l.

In terms of yield, exposure of Desmodesmus subspicatus to the test material gave an E_yC₅₀ (0 - 72 h) value of 12 mg/l; 95% confidence limits 9.3 - 17 mg/l. The Lowest Observed Effect Concentration based on yield was 10 mg/l and the No Observed Effect Concentration was 3.2 mg/l.

In terms of biomass integral (area under growth curve), exposure of Desmodesmus subspicatus to the test material gave an E_bC₅₀(0 - 72 h) value of 20 mg/l*. The Lowest Observed Effect Concentration based on inhibition of biomass integral was 10 mg/l and the No Observed Effect Concentration was 3.2 mg/l.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 81% to 96% of nominal and so the results are based on nominal test concentrations only.

Exposure of Desmodesmus subspicatus to the reference material, potassium dichromate, gave an E_rC₅₀(0 - 72 h) of 0.57 mg/l; 95% confidence limits 0.48 – 0.66 mg/l, an E_yC₅₀(0 - 72 h) of 0.32 mg/l; 95% confidence limits 0.29 ‑ 0.35 mg/l, and an E_bC₅₀(0 - 72 h) of 0.31 mg/l; 95% confidence limits 0.28 - 0.35 mg/l. The Lowest Observed Effect Concentrations based on inhibition of growth rate, yield and biomass integral were 0.50, 0.125 and 0.125 mg/l respectively and the No Observed Effect Concentrations were 0.25, 0.0625 and 0.0625 mg/l respectively.