Naphthenic acids, lithium salts

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study is GLP compliant and follows OECD guideline 201. Therefore, it has been given a reliability score of 1.

Justification for type of information:

See IUCLID section 13 for read across justification

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

Full chemical analysis was not conducted in the range finding test but as the results are based on a final definitive test with full supporting analysis this was not considered to affect the validity of the test.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

Full chemical analysis was not conducted in the range finding test but as the results are based on a final definitive test with full supporting analysis this was not considered to affect the validity of the test.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Analytical monitoring:

yes

Details on sampling:

The confimatory chemical analysis was based on the concentration of the fatty acid component (adipate) and on the concentration of the lithium ion. The concentration of total organic carbon (TOC) was also measured.
Samples for analysis were taken from all test concentrations at the start, after 24 and 72 hours of exposure (lithium and adipate analysis) and at the test start and after 72 hours of exposure (TOC analysis). Samples were stored in a refrigerator (TOC) or freezer (lithium and adipate) until analysis.
At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at an intermediate substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
For the determination of the test substance based on adipate, the samples were diluted in a 1:1 (v:v) ratio with 2% formic acid in acetonitrile and analysed. If necessary, the samples were further diluted with 1% formic acid in 50/50 (v/v) acetonitrile/M2-medium to obtain concentrations within the calibration range.
For the determination of the test substance based on lithium the samples were diluted in a 24:1 (v:v) ratio with HNO3 and analysed. If necessary, the samples were further diluted with 4% HNO3 in water to obtain concentrations within the calibration range.

Vehicle:

no

Details on test solutions:

Nominal concentrations 1.0, 3.2, 10, 32 and 100 mg dilithium adipate/L.
Medium: M2
Solubility: The test item was completely soluble in test medium at the concentrations tested.
Preparation of test solutions: A solution with a concentration of 100 mg/L was prepared by vigorous shaking. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium.
Appearance: The final test solutions were all clear and colourless.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Strain: NIVA CHL 1 from an in-house laboratory culture
Final test inoculation: Cells taken from stock culture
Stock culture: M1 growth medium inoculated with algal cells from pure culture on agar. Suspensions were continuously aerated and exposed to light at 21-24°C.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

24 mg CaCO3/L

Test temperature:

21-22°C

pH:

7.8-8.2

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg dilithium adipate per litre. For measured concentrations, please see additional information box.
The nominal concentrations of dilithium adipate were measured at 0h and 72 h.
The actual concentrations based on lithium were at the level of 92-103% of the nominal, whereas the concentrations based on adipate were between 83-114% of nominal.

Details on test conditions:

Controls: Blank (test medium without test substance)
Solutions: 50 mL test solutions with 1 mL algae suspension (cell density of 10 (4) cells/mL)
Replicates: 3 per test concentration, 6 for control, 1 for turbidity control, 2 per group without algae for TOC analysis
Test vessels: 100 mL, all glass
Illumination: Continuous
Incubation: Vessels were randomly distributed in the incubator and daily repositioned.
Agitation: Continuous shaking.
Observations: At the beginning of the test, cells were counted using a microscope and counting chamber and thereafter, cell densities were determined by spectrographic measurement, with algal medium as the blank. Microscopic observations were performed at the nominal concentration of 10 mg/L at the end of the test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Lower 95% CL: 5.0 mg/L. Upper 95% CL: 6.6 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Lower 95% CL: 22mg/L. Upper 95% CL: 25mg/L

Key result

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

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: Lower 95% CL: 2.0 mg/L. Upper 95% CL: 5.2 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: Lower 95% CL: 6.8 mg/L. Upper 95% CL: 9.4 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

Growth rates and yield were in the range of the controls at the two lowest concentrations but statistically significant inhibition was observed at concentrations of 10 mg/L and higher during the 72-hour test period. The NOEC for both growth rate inhibition and yield was set at 3.2 mg/L. Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

Results with reference substance (positive control):

Potassium dichromate inhibited growth rate of this fresh water algae species at nominal concentrations of 0.56 mg/L and higher. The growth rate 72h-EC50 was 1.3 mg/L (95% confidence interval: 1.2 to 1.5 mg/L). The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L, so result corresponds with this range. The yield inhibition 72h-EC50 was 0.46 mg/L (95% confidence interval: 0.43 to 0.49 mg/L). The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L so result was within the low end of this range.

Reported statistics and error estimates:

Quantification of cell densities was based on a calibration curve. The average specific growth rate for a specific period is calculated as the logarithmic increase in the biomass, which is compared with the control value and the percentage inhibition in growth rate is calculated.
An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure for growth rate and Welch t-test for inhomogeneous variances with Bonferroni-Holm adjustment for yield, both α=0.05, one-sided, smaller). Calculation of ECx values was based on probit analysis using linear maximum likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding nominal concentrations of the test substance.
The calculations were performed with ToxRat Professional v. 3.0.0. (ToxRat Solutions® GmbH, Germany).

Validity criteria fulfilled:

yes

Remarks:

Control cell density increased by an average factor of >16 in 2 days, the mean coefficient of variation was 26% and the coefficient of variation of average specific growth rates in the control over the whole test did not exceed 7%

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, Dilithium adipate reduced growth rate and inhibited the yield of this fresh water algae species significantly at 10 mg/L and higher. The EC50 for growth rate inhibition (72h-ERC50) was 23 mg/L. The EC50 for yield inhibition (72h-EYC50) was 8.2 mg/L. The 72h-NOEC for both, growth rate and yield inhibition was 3.2 mg/L.

Executive summary:

The effect of dilithium adipate on the growth of the algae (Pseudokirchneriella subcapitata) was investigated according to an OECD 201 guideline and EC method C3. A 72 hour test was conducted at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L dilithium adipate. All test solutions were clear and colourless. Analysis of the fatty acid (adipate) was between 83 and 114% nominal and the lithium concentration was between 92-103% of nominal throughout the test. The total organic carbon (TOC) was stable at all test concentrations. The test concentrations were considered to be maintained and the results are reported based on nominal concentrations. The study met all the validity criteria for the test. Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control. Statistically significant inhibition of growth was observed at concentrations of 10 mg/L dilithium adipate and higher and the NOEC based on both growth rate and yield was 3.2 mg/L dilithium adipate. The EC50 based on growth rate (72h-ErC50) was 23 mg/L and on yield (72h-EyC50) was 8.2 mg/L.

Description of key information

Lithium naphthenate consists of lithium salts of naphthenic acids. Therefore, data have been presented for both the organic anion and the lithium cation. Studies are included for naphthenic acids as well as lithium salts of docosanoic acid (C22) and dilithium adipate (C6). For naphthenic acids, the ErLC50 is 41.3 mg/L (load) and ErC50 is 29.6 mg/L (measured) for 72 hours of exposure. For growth rate, the 72 hour NOELR was approximately 10 mg/L and the NOEC was approximately 7.38 mg/L. In addition, the toxicity of naphthenic acids to populations of the freshwater diatom, Navicula seminulum, gave a 96-h EC50 for growth of 26.0 - 80.5 mg/L. The toxicity of lithium docosanoate to algal growth gave an EL50 of greater than 100 mg/L loading rate WAF and a NOELR of 100 mg/L loading rate WAF. The toxicity of dilithium adipate to algal growth gave an EC50 of 23 mg/L and a NOEC of 3.2 mg/L. Taking a worst-case approach for the results of the key studies, the toxicity of lithium naphthenate to algal growth is determined to give a 72 hour EC50 of 23 mg/L and a 72 hour NOEC of 3.2 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

23 mg/L

EC10 or NOEC for freshwater algae:

3.2 mg/L

Additional information

Lithium naphthenate consists of lithium salts of naphthenic acids. Therefore, data have been presented for both the organic anion and the lithium cation. Studies are included for naphthenic acids as well as lithium salts of docosanoic acid (C22) and dilithium adipate (C6).

Lithium naphthenate is produced through the reaction of naphthenic acids with lithium hydroxide. Naphthenic acids consist of a main acidic (naphthenic) fraction (70-95%) and a smaller non-acidic (petroleum) fraction (5-30%). The acidic fraction contains typically C8-C20 with 0-3 rings and the non-acidic fraction has a carbon range mainly of C12-C22 and no single constituent >10%. The lithium hydroxide added to the naphthenic acids will neutralise the components within the acid fraction to form lithium salts while any components present in the non-acidic fraction of naphthenic acids (the unsaponifiable matter) will be unchanged by the manufacturing process. Therefore, lithium naphthenate is expected to consist of 70 - 95% lithium salts of naphthenic acids (C8-C20, 0-3 rings), with 5 - 30% unsaponifiable matter unchanged from the naphthenic acids starting material.

Dilithium adipate (C6) has a shorter chain length than the acids present in lithium naphthenate (C8) and is a dicarboxylic acid, while fatty acids present in lithium naphthenate are monocarboxylates. Although no dicarboxylic acid salts were identified in the GC-MS analysis for lithium naphthenate, the dataset for dilithium sebacate shows that the substance follows similar trends in properties to the longer chain monocarboxylic acid lithium salts and supports the read across between lithium salts of different carboxylic acids. Dilithium adipate, as a lithium salt of a carboxylic acid two shorter than those present in lithium naphthenate, presents a worst-case scenario for the shortest chain length present in the target substance, bracketing one end of the range of potential properties of constituents present in the UVCB.

Lithium docosanoate (C22) has a longer chain length as the longest chain length acids present in the acidic fraction of lithium naphthenate (C20). Therefore, lithium docosanoate, as a lithium salt of a carboxylic acid two longer than those present in lithium naphthenate, presents a worst-case scenario for the longest chain length present in the target substance, bracketing one end of the range of potential properties of constituents present in the UVCB.

HPVIS (2010)

The growth rate of the unicellular algae (species:Pseudokirchneriella subcapitata) was evaluated during 72 hours of exposure to the water accommodated fractions (WAF) of naphthenic acids. The test was performed according to GLP principles and OECD TG 201, in a static set up. The results of the study showed that the WAF has inhibiting effects on the growth rate of the unicellular algae Pseudokirchneriella subcapitata. The ErLC50 is 41.3 mg/L (load) and ErC50 is 29.6 mg/L (measured) for 72 hours of exposure. For growth rate, the 72 hour NOELR was approximately 10 mg/L and the NOEC was approximately 7.38 mg/L.

Harlan (2013)

The toxicity of lithium docosanoate to algae was determined in a GLP-compliant, static growth inhibition study following OECD guideline 201. Algae were exposed for 72 hours to a control and a single concentration of 100 mg/L loading rate WAF in media adjusted to a hardness of approximately 150 mg CaCO3/L. The toxicity of lithium docosanoate to algal growth gave an EL50 of greater than 100 mg/L loading rate WAF and a NOELR of 100 mg/L loading rate WAF.

WIL (2015)

The effect of dilithium adipate on the growth of the algae (Pseudokirchneriella subcapitata) was investigated according to an OECD 201 guideline and EC method C3.  The test concentrations were considered to be maintained and the results are reported based on nominal concentrations. The NOEC based on both growth rate and yield was 3.2 mg/L dilithium adipate. The EC50 based on growth rate (72h-ErC50) was 23 mg/L and on yield (72h-EyC50) was 8.2 mg/L.

ANS (1960)

The toxicity of naphthenic acids to populations of the freshwater diatom,Navicula seminulum, has been measured. The 96-h EC50 for growth ranged from 26.0 - 80.5 mg/L. The data have been assigned a Klimisch score of 2, reliable with restriction, as the information was gathered from US EPA Ecotox database, a reliable, peer-reviewed secondary source. No further details on the study methods or conditions are available.

Conclusion

Lithium naphthenate consists of lithium salts of naphthenic acids. Therefore, data have been presented for both the organic anion and the lithium cation. Studies are included for naphthenic acids as well as lithium salts of docosanoic acid (C22) and dilithium adipate (C6).

For naphthenic acids, the ErLC50 is 41.3 mg/L (load) and ErC50 is 29.6 mg/L (measured) for 72 hours of exposure. For growth rate, the 72 hour NOELR was approximately 10 mg/L and the NOEC was approximately 7.38 mg/L. In addition, the toxicity of naphthenic acids to populations of the freshwater diatom,Navicula seminulum, gave a 96-h EC50 for growth of 26.0 - 80.5 mg/L. The toxicity of lithium docosanoate to algal growth gave an EL50 of greater than 100 mg/L loading rate WAF and a NOELR of 100 mg/L loading rate WAF. The toxicity of dilithium adipate to algal growth gave an EC50 of 23 mg/L and a NOEC of 3.2 mg/L.

Taking a worst-case approach for the results of the key studies, the toxicity of lithium naphthenate to algal growth is determined to give a 72 hour EC50 of 23 mg/L and a 72 hour NOEC of 3.2 mg/L.