Azelaic acid, calcium salt (1:1)

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:

Please see attached read across justification for full details of the read across approach.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

The validity criteria were not fulfilled in the first definitive so it was repeated. The results are based on the results of the valid second definitive test.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

The validity criteria were not fulfilled in the first definitive so it was repeated. The results are based on the results of the valid second definitive test.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: A058/99
- Expiration date of the lot/batch: 6 March 2018
- Purity test date: Not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Stability under test conditions: Not specified
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None
- Preliminary purification step (if any): None
- Final dilution of a dissolved solid, stock liquid or gel: None
- Final preparation of a solid: None

Analytical monitoring:

yes

Details on sampling:

The confimatory chemical analysis was based on the concentration of the fatty acid component (azelate) 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 (2.0 mL for lithium, 4.8 mL for azelate and 40 mL for TOC analysis). Samples were stored in a freezer (lithium and azelate) or refrigerator (TOC) 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 azelate, 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/ M2-medium 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 azelate/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 magnetic stirring for 5-26 minutes. 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:

22-24°C

pH:

7.8-8.1

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations 1.0, 3.2, 10, 32 and 100 mg dilithium azelate per litre. For measured concentrations, please see additional information box. The actual concentrations based on lithium were at the level of 91-115% of the nominal during the exposure period, whereas the concentrations based on azelate were at the level of 80-126% of nominal during the first 24 hours of exposure but at the end of the test, the actual concentrations could not be quantified.

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 replicate of each test concentration for sampling at 24 hours, 1 replicate of each test concentration for turbidity control, 4 per group without algae for TOC analysis
Test vessels: 100 mL, all glass, capped
Illumination: Continuous
Incubation: Vessels were randomly distributed in the incubator and daily repositioned.
Agitation: Continuous shaking.
Observations: At the beginning of the second definitive test solutions were clear and colorless. At the end of the exposure it was observed that the solutions without algae became slightly hazy at the concentration of 10 mg/L and higher.
Counting: At the beginning of the test, cells were counted using a microscope and a counting chamber.In the second full test cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe (pathlength =20 mm). At the end of the test algal density was also determined by use of a microscope and a counting chamber because the highest test concentration was hazy and the effects measured with the spectrophotometer deviated from the results obtained in the preceding tests. The effects are reported based on the results from the microscope counting which were slightly more conservative.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

19 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: Lower 95% CL: 0.11 mg/L. Upper 95% CL: 39 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

For the first test the mean coefficient of variation for the section by section specific growth rate was higher than 35%. Therefore, it did not meet the validity criterion and the study was repeated. Despite the fact that the concentrations of the test substance were similar between both full test, the effect observed in the second test were less pronounced. The reason for this is not known.
Growth rates were in the range of the controls at the three lowest concentrations during the 72-hour test period, whereas the growth rate of algae exposed to the two highest concentrations was slightly reduced. Statistically significant inhibition of growth rate was found at test concentrations of 32 mg/L and higher. However, this effect was considered biologically irrelevant (<10%) and therefore, the NOEC was set at nominally 100 mg/L.Yield was not inhibited at the three lowest concentrations tested. At the two highest concentrations a significant inhibition was observed. At the highest concentration yield was inhibited by 27%.
At the end of the test, cell densities were determined both with spectrophotometer and counted direct under microscope (at the end of the test). Since the effect determined with the microscopic counting were stronger, the effect parameters were expressed based on this method (worst case scenario). Microscopic observations at the end of the test revealed normal and healthy appearance of the exposed cells when compared to the control.
The fact that the test concentrations based on azelate measured in the solution incubated without algae (32 mg/L) were stable during exposure indicates that azelate was adsorbed during the entire exposure indicates that azelate was adsorbed by the algal biomass rather than degraded. This means that the algae were exposed to azelate during the entire exposure period and it is justified to express the effect parameters based on nominal concentrations of dilithium azelate. To cover the worst-case scenario, effect parameters based on TWA concentrations were also calculated.

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 EC50 for growth rate inhibition (72h-ERC50) was 1.2 mg/L with a 95% confidence interval ranging from 1.1 to 1.2 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.

The EC50 for yield inhibition (72h-EYC50) was 0.38 mg/L with a 95% confidence interval ranging from 0.38 to 0.39 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L. Hence, the 72h-EYC50 for the algal culture tested was just below the low end of this range.

Reported statistics and error estimates:

Calibration curves for the lithium analysis were constructed using five concentrations. For each concentration, two responses were used. Linear regression analysis was performed using the least squares method with a 1/concentration squared as the weighting factor. The coefficient of correlation (r) was > 0.99 for each curve.

Measured and calculated TWA concentrations based on azelate in the second definitive test
Dilithium azelate Nominal concentration (mg/L)	Measured concentration (mg/L)			TWA concentration (mg/L)
	t=0h	t=24h	t=72h
1	1.17	0.803	0.004*	0.36
3.2	3.66	3.11	0.004*	1.2
10	12.2	9.32	0.02*	3.8
32	36.1	32.8	0.04*	12
32 (WA)	42.7	32.5	50.5	39
100	126	97.9	0.4*	41
WA – without algae
* - half of the lowest calibration standard
Measured Total Organic Carbon (TOC) concentrations in the second definitive test
Dilithium azelate Nominal concentration (mg/L)	Measured concentration (mg TOC/L) at t (h)
	0	24	72
Control	0.1335	0.157	0.2447
1	0.1732	0.2247	0.3875
3.2	0.5805	0.5408	0.3297
10	3.585	1.019	0.6294
32	13.05	1.94	1.182
100	41.37	27.74	5.068

Section-by-section growth rate – first definitive test - discarded study
Time	Replicate	Dilithium azelate, Nominal concentration (mg/L)
		Control	0.1	0.32	1	3.2	10	32	100
72 h	1	1.422	1.934	1.831	1.774	1.842	1.376	1.555	1.872
	2	1.607	1.785	1.917	1.887	1.574	2.072	0.726	1.386
	3	2.009	1.787	1.808	1.823	1.984	1.381	0.000	1.204
	4	1.874
	5	1.749
	6	1.688
Mean		1.725	1.835	1.852	1.828	1.800	1.610	0.760	1.487
Number		6	3	3	3	3	3	3	3
CV		11.9	4.7	3.1	3.1	11.6	24.9	102.3	23.2

Section-by-section growth rate – second definitive test Dilithium azelate, Nominal concentration (mg/L)
Time	Replicate
		Control	1	3.2	10	32	100
72 h	1	1.254	1.3	1.362	1.189	0.623	0.584
	2	1.275	1.159	1.319	1.14	0.592	0.916
	3	1.329	1.289	1.195	1.059	0.804	0.799
	4	1.334
	5	1.12
	6	1.002
Mean		1.219	1.249	1.292	1.121	0.673	0.766
Number		6	3	3	3	3	3
CV		10.8	6.3	6.7	5.8	17	22

Validity criteria fulfilled:

yes

Remarks:

Control cell density increased by an average factor of >16 in 3 days, the mean coefficient of variation for section-by-section was 14% 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 exposed dilithium azelate, the EC50 for growth rate inhibition (72h-ERC50) was >100 mg/L and the NOEC was 100 mg/L. The EC50 for yield inhibition (72h-EYC50) was >100 mg/L and the NOEC was 10 mg/L.

Executive summary:

The effect of dilithium azelate on the growth of the algae (Pseudokirchneriella subcapitata) was investigated according to an OECD 201 guideline and EC method C3. The results are based on the results of a second definitive study as the first was not a valid test. A 72 hour test was conducted at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L dilithium azelate. All test solutions were clear and colourless at the start of the test but at the end of the exposure the solutions without algae became slightly hazy at concentrations of 10 mg/L and higher. The lithium concentration was between 91- 115% of nominal throughout the test. During the first 24 hours of exposure, in the test solutions containing algae, the fatty acid (azelate) concentration was between 80 and 126% nominal but at the end of the test it was below the limit of quantification for all test concentrations. The azelate concentration was however maintained in the solutions without algae (32 mg/L) being 155% of nominal at the end of the test. The measured TOC concentrations decreased during the exposure with the decrease being proportionally higher at the higher test concentrations. The fact that the concentration of azelate in the solution incubated without algae was maintained during the exposure indicates that azelate was adsorbed by the algal biomass rather than degraded. This means that the algae were exposed to azelate during the entire exposure period and it is justified to express the effect parameters based on nominal concentrations of dilithium azelate. To cover the worst-case-scenario, effect parameters based on time weighted average (TWA) concentrations of azelate were also calculated. The TWA concentrations were 0.36, 1.2, 3.8, 12 and 41 mg/L for the nominal, 1.0, 3.2, 10, 32 and 100 mg/L dilithium azelate, test solutions.

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 rate was found at test concentrations of 32 mg/L and higher but these effects caused a <10% effect on growth rate and were considered to be biologically insignificant. The NOEC for growth rate was therefore 100 mg/L (TWA = 41 mg/L) dilithium azelate. There was a significant effect on yield at 32 and 100 mg/L dilthium azelate. The NOEC based on yield was therefore 10 mg/L (TWA = 3.8 mg/L) dilithium azelate. The ErC50 and the EyC50 were both ≥ 100 mg/L (TWA ≥ 41 mg/L) dilithium azelate.

Description of key information

Calcium azelate is concluded to be not toxic to algae based on data read across from a structural analogue, dilithium azelate. Under the conditions of the study with Pseudokirchneriella subcapitata exposed dilithium azelate, the EC50 for growth rate inhibition (72h-ERC50) was >100 mg/L and the NOEC was 100 mg/L. The EC50 for yield inhibition (72h-EYC50) was >100 mg/L and the NOEC was 10 mg/L.

Additional information

The effect of calcium azelate on the growth of algae (Pseudokirchneriella subcapitata) has been assessed based on data read across from a structural analogue, dilithium azelate. The effect of dilithium azelate was investigated according to OECD 201 guideline in a 72 hour test at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L dilithium azelate. All test solutions were clear and colourless at the start of the test but at the end of the exposure the solutions without algae became slightly hazy at concentrations of 10 mg/L and higher. The lithium concentration was between 91- 115% of nominal throughout the test. During the first 24 hours of exposure, in the test solutions containing algae, the fatty acid (azelate) concentration was between 80 and 126% nominal but at the end of the test it was below the limit of quantification for all test concentrations. The azelate concentration was however maintained in the solutions without algae (32 mg/L) being 155% of nominal at the end of the test. The measured TOC concentrations decreased during the exposure with the decrease being proportionally higher at the higher test concentrations. The fact that the concentration of azelate in the solution incubated without algae was maintained during the exposure indicates that azelate was adsorbed by the algal biomass rather than degraded. This means that the algae were exposed to azelate during the entire exposure period and it is justified to express the effect parameters based on nominal concentrations of dilithium azelate. Statistically significant inhibition of growth rate was found at test concentrations of 32 mg/L and higher but these effects caused a <10% effect on growth rate and were considered to be biologically insignificant. The NOEC for growth rate was therefore 100 mg/L dilithium azelate. There was a significant effect on yield at 32 and 100 mg/L so the NOEC based on yield was therefore 10 mg/L and the ErC50 and the EyC50 were both ≥ 100 mg/L.