Aluminum chloride, basic, reaction products with silica

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

oxo[(oxoalumanyl)oxy]alumane

EC Number:

614-074-2

Cas Number:

675106-31-7

Molecular formula:

UVCB

IUPAC Name:

oxo[(oxoalumanyl)oxy]alumane

Test material form:

solid: fibres

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and the 100% v/v saturated solution test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. Additional samples were run alongside the test to enable sampling of the test group at 24 and 48-Hour time points. An additional uninoculated sample was also run alongside the test and sampled at 72 hours. All 0, 24 and 48-Hour samples were stored frozen prior to analysis. All 72-Hour samples were analyzed on the day of sampling. Duplicate samples were taken at each occasion and stored frozen 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: A nominal amount of test item (550 mg) was dispersed in 11 L of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 10000 g for 30 minutes to give a 100% v/v saturated solution.
An aliquot (1 L) of this saturated solution was inoculated with algal suspension (4.7 mL) to give the required test concentration of 100% v/v saturated solution.
The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): Not specified
- Method of cultivation: Approximately 3 to 4 days before the start of the test, inoculum cultures of algae were 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.

ACCLIMATION
- Acclimation period: 3 to 4 days before the start of test
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: None noted

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

Standard as per OECD test guidelines

Post exposure observation period:

Not required

Test conditions

Hardness:

For testing, 198.4 mg of CaCl2.2H2O was added to each litre of media to increase the hardness due to the test item containing metal.

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 7.6 at 0 hours to pH 8.6 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 Guideline.

In test vessels, this was measured as 7.6 at 0 hours and 8.6 at 72 hours.

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal: Control and 0.051 mg/L as aluminium (loading rate 50 mg/L)
Measured: Control and 0.029 mg/L as aluminium

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type (delete if not applicable): Open (the flasks were plugged with polyurethane foam bungs)
- Material, size, headspace, fill volume: 100 mL of test solution
- Aeration: No
- Initial cells density: 1.07 x 10^6 cells/mL diluted to prepare 5 x 10^3 cells/mL.
- Control end cells density: 5.77 x 10^5 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl. For testing, 198.4 mg of CaCl2.2H2O was added to each litre of media to increase the hardness due to the test item containing metal.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: Not required
- Photoperiod: continuous
- Light intensity and quality: Intensity approximately 7000 lux provided by warm white lighting (380 to 730 nm)
- Salinity (for marine algae): Not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. 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. To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: Limit test
- Justification for using less concentrations than requested by guideline: Not applicable as a limit test was used
- Range finding study
- Test concentrations: Control, 0.1, 1.0, 10 and 100% v/v.
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at the test concentrations of 0.10, 10 and 100% v/v saturated solution. However, growth was observed to be reduced at 1.0% v/v saturated solution. Given that at all other test concentrations there was no effect, it is taken that this result is anomalous. Based on this information a single test concentration of six replicates, of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the highest attainable test concentration no effect on growth was observed.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.020 to 0.081 mg/L as Aluminium. A decline in measured test concentration was observed at 72 hours to between 0.015 and 0.056 mg/L as Aluminium. Therefore, for the definitive test additional samples were run to map the test concentration decline.

CULTURING APPARATUS
-Details on culturing apparatus used: Not specified

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.029 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

Aluminium

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.029 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

Aluminium

Basis for effect:

growth rate

Details on results:

Verification of test concentrations: Analysis of the test preparations at 0 hours showed a measured test concentration of 0.077 mg/L. A decline in measured test concentration was observed at 24, 48 and 72 hours to less than the Limit of Quantification employed (0.020 mg/L as Aluminium), 0.028 and 0.031 mg/L as Aluminium respectively. Samples taken at 72 hours without algae showed a measured test concentration of less than the LOQ, suggesting that the test item did not adhere to algae cells. Due to the inconsistency in measured concentrations, duplicates were analysed. However, the results obtained were considered to be inconsistent in regard to stability and therefore unreliable. As a result, the original samples were taken to be correct.

Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC50 values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. In cases where the measured concentration was less than the LOQ of the analytical method following current regulatory advice a value of half the LOQ (i.e. 0.010 mg/L) was used to enable calculation of the geometric mean measured concentration. As the without algae analysis at 72 hours indicated that the test item was not adhering to algae, concentrations were calculated based on inoculated results.

Growth data: It is clear that the growth rate (r) and yield (y) of Raphidocelis subcapitata (CCAP 278/4) were not affected by the presence of the test item at a geometric mean measured test concentration of 0.029 mg/L as Aluminium over the 72-Hour exposure period. There were no statistically significant differences (P≥0.05), between the control and the 0.029 mg/L test group and therefore the NOEC based on growth rate was 0.029 mg/L.

Validity criteria: The following data show that the cell concentration of the control cultures increased by a factor of 115 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 : 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 5.77 x 10^5 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 11% 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 hours) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Solubility observations: At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and test cultures were observed to be green dispersions.

Observations on 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.

Results with reference substance (positive control):

A positive control (Covance study number 8461000) used potassium dichromate as the reference item at concentrations of 0.125, 0.25, 0.50, 1.0 and 2.0 mg/L. The positive control was conducted between 21 December 2020 and 24 December 2020.

Exposure of Raphidocelis subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour) : 1.3 mg/L; 95% confidence limits 1.2 to 1.5 mg/L
No Observed Effect Concentration based on growth rate: 0.125 mg/L

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

Reported statistics and error estimates:

A Students t-test for homogeneous variances incorporating Levene’s test on Homogeneity and Shapiro-Wilks test on Normal Distribution was carried out on the growth rate and yield data after 72 hours for the control and the 100% v/v saturated solution test concentration to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the ToxRat computer software package (ToxRat® Solutions GMBH).

Any other information on results incl. tables

Table: Inhibition of growth rate and yield in the definitive test:

Geometric Mean Measured Test Concentration (mg/L Aluminium)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 hour	% Inhibition	0 to 72 hour	% Inhibition*
Control	R1	0.065	-	5.44E+05	-
	R2	0.068		6.80E+05
	R3	0.063		4.49E+05
	R4	0.063		4.61E+05
	R5	0.067		6.30E+05
	R6	0.068		6.69E+05
	Mean	0.066		5.72E+05
	SD	0.002		1.03E+05
0.029	R1	0.067	[2]	6.31E+05
	R2	0.067	[2]	6.03E+05
	R3	0.067	[2]	6.29E+05
	R4	0.066	0	5.94E+05
	R5	0.065	2	5.40E+05
	R6	0.069	[5]	6.94E+05
	Mean	0.067	[2]	6.15E+05	[7]
	SD	0.001		5.08E+04

* In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated
R = Replicate
- = Not applicable
SD = Standard Deviation
[ ] = Increase in growth compared to controls

Table: Analytical results for definitive test

Sample description	Time point (hours)	Concentration detected (mg/L Al)
Control	0	< LOQ
100% saturated solution (v/v)		0.0765/<LOQ*
100% saturated solution (v/v)	24	<LOQ/<LOQ*
100% saturated solution (v/v)	48	0.0281/<LOQ*
Control	72	<LOQ
100% saturated solution (v/v)		0.0314/0.0328*
100% saturated solution (v/v)	72 (no algae)	<LOQ/<LOQ*

< LOQ (0.020 mg/L Al)

* Duplicate sample stored frozen prior to analysis

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The validity criteria were met.

Conclusions:

The effect of the test item on the growth of Raphidocelis subcapitata has been investigated and gave EC50 values of greater than 0.029 mg/L based on the geometric mean measured test concentration of aluminium (>50 mg/L as nominal loading rate). The No Observed Effect Concentration (NOEC) was 0.029 mg/L as aluminium (50 mg/L based on nominal loading rate).

This study showed that there were no toxic effects at saturation.