Aluminium triethanolate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control, 1, 3.2, 10, 32 and 100 mg/L
- Sampling method: Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for immediate quantitative analysis.
- Sample storage conditions before analysis: A set of duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary. An additional sample of each test concentration containing no algal cells was incubated alongside the test to provide samples for analysis at 24 and 48 hours. These samples were stored frozen.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Nominal amounts of test item (100 and 32 mg) were dissolved in culture medium with the aid of ultrasonication and the volumes adjusted to 1 liter to give 100 or 32 mg/L stock dispersions from which series of dilutions were made to give further stock dispersions of 1.0, 3.2, 10 and 32 mg/L. An aliquot (600 mL) of each of the stock dispersions was separately inoculated with algal suspension (2.1 mL) to give an initial nominal cell density of 5.00 x 10^3 cells/mL. The stock dispersions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity. As the substance was expected to hydrolyse rapidly with ethanol as a product, normal approaches for difficult test substances (e.g. WAFs) were not considered appropriate owing to the prolonged stirring time required.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Liquid cultures of Raphidocelis subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Method of cultivation: Approximately 3 to 4 days before the start of the test, inoculum cultures of algae was set up at an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1°C until the algal cell density was approximately 10^5 to 10^6 cells/mL.

:

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

Standard as per OECD guidelines

Hardness:

Not specified

Test temperature:

Temperature was maintained at 24 ±1 ºC throughout the test.

pH:

The pH value of the control cultures was observed to increase from pH 7.9 at 0 hours to pH 8.9 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.

Dissolved oxygen:

Not applicable

Salinity:

Not applicable

Conductivity:

Not applicable

Nominal and measured concentrations:

Nominal: Control, 1, 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): open
- Material, size, headspace, fill volume: 250 mL flasks each containing around 100 mL of test preparations
- Aeration: No
- Initial cells density: 5000 cells/mL
- Control end cells density: 4.37 x 10^5 to 6.50 x 10^5
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Standard as per OECD guidelines

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Continuous light
- Light intensity and quality: Approximately 7000 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 24, 49 and 72 hours and the cell densities determined using a haemocytometer and light microscope. Three determinations were made for each sample. The nominal inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density. It was not possible to monitor algal cells using a Coulter® Multisizer Particle Counter due to presence of undissolved test item degradant.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: Standard as per OECD guideline
- Range finding study
- Test concentrations: 0.1, 1, 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 test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (test not run concurrantly)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

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:

Range-finder results: 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. Chemical analysis of the test preparations at 0 and 72 hours showed measured test concentrations as aluminium of 0.0050 to 9.5 mg/L and 0.091 to 2.7 mg/L respectively indicating hydrolysis of the test item under test conditions.

Definitive test:
Verification of test concentrations: Analysis of the test preparations showed measured test concentrations as aluminium to range from 0.064 to 15.8 mg/L at 0 hours and from 0.12 to 1.2 mg/L at 72 hours. A low level of test item, 0.011 mg/L as aluminium was detected in the control sample at 72 hours. As the amount detected was near to the LOQ for the method (0.01 mg/L) this was considered not to impacted on the study. The measured aluminium concentrations do not directly indicate the aluminium species present and in some cases are variable. The only aluminium source should be the parent substance with a good correlation between nominal 0 hour parent concentrations and the increase in aluminium content. Ethanol is also rapidly produced as a hydrolysis product as evidenced through trial work. Accordingly, based on the intrinsic properties of the substance, effect concentrations are based on nominal values.

Valididty criteria: The following data show that the cell concentration of the control cultures increased by a factor of 10^6 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.
Nominal cell density of control at 0 hours 5000 cells/mL and mean cell density of control at 72 hours 5.3 x 10^5 cells/mL.
The mean coefficient of variation for section by section specific growth rate for the control cultures was 29% 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 to 72 hour) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Observations on cultures: All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 1.0 and 3.2 mg/L. Some cell clumping was observed to be present in the test culture 10 mg/L. Enlarged cells were observed to be present in the test cultures 32 and 100 mg/L with cell debris also present in the 100 mg/L test culture.

Observations on test item solubility: At the start of the test all control, 1.0, 3.2 and 10 mg/L test cultures were observed to be clear colorless solutions whilst the 32 and 100 mg/L test cultures were very slightly or slightly cloudy dispersions. After the 72-Hour test period all control, 1.0 and 3.2 mg/L test cultures were green dispersions. The 10 mg/L test cultures were pale green dispersions, with a white precipitate on the bottom on the vessel. The 32 and 100 mg/L test cultures were slightly or very slightly cloudy dispersions, with a white precipitate on the bottom of the vessel.

Results with reference substance (positive control):

- Results with reference substance valid?
- EC50: 1.3 mg/L (in house data over a 5 year period shows a mean ErC50 value of 1.4 mg/L indicating validity of the test procedure.

Reported statistics and error estimates:

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 (P<0.05), between the control and 1.0 and 3.2 mg/L test groups; however, all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 3.2 mg/L. Correspondingly the LOEC based on growth rate was 10 mg/L.

Inhibition of growth rate in the definitive test

Nominal concentration (mg/L)		Growth rate (cells/mL/hour)
		0 to 72 hours	% inhibition
Control	R1	0.065
	R2	0.065
	R3	0.064
	R4	0.065
	R5	0.062
	R6	0.068
	Mean	0.065
	SD	0.002
1.0	R1	0.062	5
	R2	0.066	[2]
	R3	0.08	[5]
	Mean	0.065	[1]
	SD	0.003
3.2	R1	0.063	3
	R2	0.066	[2]
	R3	0.063	3
	Mean	0.064	1
	SD	0.002
10	R1	0.049	25
	R2	0.046	29
	R3	0.047	28
	Mean	0.047	27
	SD	0.002
32	R1	0.010	85
	R2	0.036	45
	R3	0.012	82
	Mean	0.019	71
	SD	0.014
100	R1	-0.006	109
	R2	0.000	100
	R3	-0.006	109
	Mean	-0.004	106
	SD	0.003

[ ] = Increase in growth rate compared with controls

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Raphidocelis subcapitata has been investigated over a 72-Hour period and gave a ErC50 value of 18 mg/L, a NOEC (growth rate) of 3.2 mg/L and an ErC10 value of 5.7 mg/L.

Description of key information

The effect of the test item on the growth of Raphidocelis subcapitata has been investigated over a 72-Hour period and gave a ErC50 value of 18 mg/L, a NOEC (growth rate) of 3.2 mg/L and an ErC10 of 5.7 mg/L. No marine water algae studies are available.

Key value for chemical safety assessment

EC50 for freshwater algae:

18 mg/L

EC10 or NOEC for freshwater algae:

5.7 mg/L