Aluminium oxide

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:

26 – 29 Jul 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted March 2006
corrected July 2011

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Aluminium oxide

EC Number:

215-691-6

EC Name:

Aluminium oxide

Cas Number:

1344-28-1

Molecular formula:

Al2O3

IUPAC Name:

oxo[(oxoalumanyl)oxy]alumane

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 and 100 mg/L
- Sampling method: The treatments were sampled at the start and the end of the experiment. Another solution was sampled separately from the preparation container at the start of exposure. Equal volumes of the test solution were taken out from the test vessels in each test level (at the end of exposure). For the chemical analysis algae were removed subsequently by centrifugation (3000 rpm, 10 min, at the end of exposure).

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item (0.100 g) and test medium (1000 mL) were mixed for 48 hours using a magnetic stirrer to produce a suspension with a nominal concentration of 100 mg/L. Afterwards, the suspension was filtrated with a membrane filter (PVDF, 0.45 μm pore size, Merck) by suction. The filtrate (test solution) was filled into each test vessel.
For saturation purposes the filter was treated with ca. 50 mL test solution beforehand. It was confirmed that there was no adsorption to the filter. These operations were performed along OECD Guidance Document No. 23. This was investigated during non-GLP preliminary tests (for further information please see the attached document under section 'Overall remarks, attachments').

- Controls: Dilution water (M4)
- Test concentration separation factor: Only one concentration (at the limit of the test item solubility) was tested
- Evidence of undissolved material: Only the dissolved fraction was tested, the undissolved fraction was removed by filtration as described before.
- Other relevant information: In non- GLP pretests the preparation and application of the test solution was assessed before. Accordingly, an optimized filter size was evaluated (for further information please see the attached document under section 'Overall remarks, attachments' ). Further, the influence of EDTA within the OECD test medium on the availability of aluminum was tested. There was no significant difference as the result of comparing the presence or absence of chelate agents(EDTA) contained in OECD medium. Therefore, the definitive study was conducted in normal OECD medium including EDTA.

Test organisms

Test organisms (species):

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

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

22.2 °C

pH:

7.8 -7.9

Nominal and measured concentrations:

Nominal: 0 and 100 mg/L (the test item was tested at its' solubility limit, please see section 'Details on test solution')
Measured:
Start: < LOQ (control) and 0.108 mg/L
End: < LOQ (control) and 0.0983 mg/L
Geometric mean: 0.103 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Type: Closed with gas-permeable Silicosen
- Material, size, fill volume: Glas, 300 mL, 100 mL
- Initial cells density: 0.75x10^4 cells/mL
- Control end cells density: (Mean) 64.71 x10^4 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes, OECD medium according to OECD 201

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium was used. Since the test item was a metal compound, potential differences depending on the presence or absence of the chelating agent EDTA (as part of the OECD medium) was tested and compared (for further details please see the attached document under section 'Overall remarks, attachments'). As a result, the regular OECD medium for the pre-culture and algae growth inhibition study was used and prepared with purified water.
- Culture medium different from test medium: No
- Intervals of water quality measurement (pH, temperature, light intensity): pH, at the start and end of the experiment; temperature and light intensity: daily

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination
- Light intensity and quality: 90-91 μmol・m-2-s-1 in wavelength range of 400-700 nm within ±20% of nominal (within ±15% from the average light intensity) provided by alternative LED light to fluorescent lamp.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Cell growth and yield in terms of biomass (cell concentration), every 24 h;
- Observation for deformed and/or abnormal cells, at the start and the end of the experiment
- Determination of cell concentrations: Electronic particle counter (a blank correction was used by measuring the value of blank solution (without algae addition) each test level.)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable. A limit approach was used.
- Justification for using less concentrations than requested by guideline: A non-GLP pretest revealed no effects at the max. solubility of the test item in the test medium.
- Range finding study: Yes, static for 72 h testing the max. soluble fraction of the test item by using its filtrate (aging for 48 h in OECD medium then filtering with 0.45 µm PTFE filter) of the 100 mg/L test solution.
- Test concentrations: 0 and filtrate of 100 mg test item/L (0.45 µm PTFE filter)
- Results used to determine the conditions for the definitive study: Yes, results were used for the final setup as no effects (Growth inhibition rate based on the growth rate was 2.2% after 72 h) occurred when testing the filtrate of 100 mg test item/L during the range finder.

CULTURING APPARATUS
-Details on culturing apparatus used: Cultured under sterile conditions in this test facility Culture medium; OECD medium. Temperature and light intensity same as for the testing.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.103 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

Filtrate of a nominal 100 mg test item/L loading rate. Before filtering (0.45 µm PTFE) the suspension was aged for 48 h (under stirring) in OECD media.

Basis for effect:

other: growth rate, yield

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.103 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

Filtrate of a nominal 100 mg test item/L loading rate. Before filtering (0.45 µm PTFE) the suspension was aged for 48 h (under stirring) in OECD media.

Basis for effect:

other: growth rate, yield

Details on results:

- Exponential growth in the control: Yes
- Observation of abnormalities: No
- Unusual cell shape: No
- Colour differences: No
- Any stimulation of growth found in any treatment: No
- Any observations that might cause a difference between measured and nominal values: No, only the dissolved fraction was tested

Results with reference substance (positive control):

- Results with reference substance valid? Yes. The found value was within the stipulated range (mean ±2 SD: 0.99—1.5 mg/L (n=11) to background data in the test facility.
- 72 h-ErC50: 1.4 mg/L

Reported statistics and error estimates:

Estimation of EC50
The EC50 was estimated as "> the test concentration" since less than 50% of inhibitionrate was obtained at the exposure level. The EC50 were denoted as ErC50 and EyC50 based on growth rate and yield, respectively.

Estimation of NOEC
Regarding the growth rate and yield, F tests was accomplished to determine the homogeneity of variance for the data. Since variances of the two groups were equal, the Student's t-test was used to estimate a significant difference compared to the control. The statistical analysis was conducted using a computer program (running on Microsoft software "Excel") constructed by our test facility. NOEC was determined by the results of statistical analysis and cell condition. NOEC was estimated as ‘> the test concentration" since the growth inhibition was not observed in the exposure level. NOEC were denoted as NOECr and NOECy based on growth rate and yield, respectively.

Any other information on results incl. tables

Table 1: Nominal and measured concentrations

Nominal concentration (mg/L)	Nominal concentration (mg/L, n=1) [Percentage of measured concentration compared to the start]
	0 h	72 h	Geometric mean
Control	< LOQ	< LOQ	-
100	0.108	0.0983 [90.6 %]	0.103

LOQ: 0.0236 mg/L (as test item concentration)

 

Table 2: Algae biomass over the 72 h time course of the exeriment

Nominal concentration (mg/L)	Measured concentration (dissolved fraction)	Replicate	Cell Concentration (x 10^4 cells mL)
			0 h a)	24 h	48 h	72 h
Control	Control	A	0.75	3.45	13.85	59.63 b)
		B	0.75	3.24	14.86	67.83
		C	0.75	3.34	13.70	62.94
		D	0.75	3.04	13.58	61.76
		E	0.75	3.50	15.20	70.00
		F	0.75	3.37	15.04	66.07
		Mean	0.75	3.32	14.37	64.71
		SD	0.00	0.17	0.73	3.93
100	0.103	A	0.75	3.39	13.17	59.34
		B	0.75	3.39	13.46	58.90
		C	0.75	3.32	13.67	61.32
		D	0.75	3.36	13.94	62.32
		E	0.75	3.34	14.13	63.53
		F	0.75	3.41	14.06	68.27
		Mean	0.75	3.37	13.74	62.28
		SD	0.00	0.035	0.38	3.42

 

a) The value based on the measured value of the pre-culture

b) The minimum cell growth in the control (biomass at the end of exposure/biomass at the start of exposure) 59.63/0.75 = 80

Explanation for the measured concentration: The filtrate of the test item was tested. A nominal 100 mg test item/L loading rate was prepared. Before filtering (0.45 µm PTFE) the suspension was aged for 48 h (under stirring) in OECD media.

 

Table 3: Growth rate, yield and inhibition rate

Nominal concentration (mg/L)	Measured concentration	Replicate	Growth rate (0-3 d)	Growth inhibition rate (%)	Yield (x10^4)	Yield inhibition rate (%)
Control	Control	A	1.46	-	59.00	-
		B	1.50	-	67.00	-
		C	1.48	-	62.00	-
		D	1.47	-	61.00	-
		E	1.51	-	69.00	-
		F	1.49	-	65.00	-
		Mean	1.49	-	64.00	-
		SD	0.0203	-	3.90	-
100	0.103	A	1.46	1.90	59.00	8.40
		B	1.45	2.10	58.00	9.10
		C	1.47	1.20	61.00	5.30
		D	1.47	0.81	62.00	3.70
		E	1.48	0.38	63.00	1.80
		F	1.50	-1.20	68.00	-5.6
		Mean	1.47	0.85	62.00	3.8
		SD	0.018	1.20	3.40	5.3

 

Table 4: See attached background material "Table 1-6"(Content: Findings of the non-GLP range finding experiments including information on the choice of test concentration, test item solubility in the test media and choice of filter size and test media])

 

Table 5: Validity criteria for OECD 201 (2011)

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	≥ 80	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35 %	3.1 %	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7 % in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	1.4 %	Yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to Table 5 under “Any other information on results incl. tables”.

Conclusions:

The ErC50 (72 h) and NOErC (72 h) for R. subcapitata were determined to be > 0.103 mg/L and ≥ 0.103 mg/L for the filtrate of the test item (mean measured) under static conditions.