Trialuminium bismuth hexaoxide

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: 0, 10, 18, 32, 56 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 quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary. An additional sample of each loading rate WAF containing no algal cells was incubated alongside the test to provide samples for uninoculated analysis at 72 hours.

Vehicle:

no

Details on test solutions:

Preliminary work:
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000).

Definitive test preparation:
Nominal amounts of test item (20, 36, 64, 112 and 200 mg) were each separately added to the surface of 2 liters of culture medium to give the 10, 18, 32, 56 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1-Hour.

Visual observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the insoluble test article by filtering through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded). Further filtration through one sheet of filter paper was conducted to remove as much undissolved test item as possible. Microscopic observations of the WAFs were performed after filtering and showed no micro-dispersions (insoluble test article) of test item to be present, visual inspection of the 100 mg/L loading rate WAF did however show it to be a slightly hazy dispersion.

An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (4.6 mL) to give the required test concentrations of 10, 18, 32, 56 and 100 mg/L loading rate WAF.

Controls: Media without test substance.

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): Obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation):
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1°C.

ACCLIMATION
- Acclimation period: Acclimated until the algae cell density was approximately 10000 to 100000 cells/mL.
- Culturing media and conditions (same as test or not): Same conditions used
- Any deformed or abnormal cells observed: None noted

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

Standard as per OECD guideline

Hardness:

Not specified

Test temperature:

Maintained at 24 +/- 1°C throughout the test

pH:

Test substance: pH ranged from 7.4 to 8.6.
Controls: The pH value of the control cultures was observed to increase from pH 7.3 at 0 hours to pH 8.8 at 72 hours. The pH deviation in the control cultures was less than or equal to 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal loading rates: 0, 10, 18, 32, 56 and 100 mg/L.
Measured concentrations: Varied based on both Bismuth and Aluminium ion calculations

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flask
- Type (delete if not applicable): open although plugged with polyurethane foam bungs
- Material, size, headspace, fill volume: 250 mL containing 100 mL of test or control solutions
- Aeration: No
- Initial cells density: Nominally 5000 cells/mL
- Control end cells density: 1.02 x 10^6 cells/mL
- No. of vessels per concentration (replicates): 3 replicates
- No. of vessels per control (replicates): 6 replicates
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes as per OECD guideline

OTHER TEST CONDITIONS
- Sterile test conditions: Not specified
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: Approximately 7000 lux provided by warm white lighting (380 to 730 nm).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 23, 47 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally 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: Acceptable as per OECD guideline
- Range finding study
- Test concentrations: 0, 1, 10 and 100 mg/L loading rates
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at 1.0 and 10 mg/L loading rate WAF. However, growth was observed to be reduced at 100 mg/L loading rate WAF. Based on this information loading rates of 10, 18, 32, 56 and 100 mg/L were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

53 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

< 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): 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 10, 18 and 32 mg/L loading rate WAF, however only a few cells were observed to be present in the test cultures at 56 mg/L loading rate WAF and cell debris observed to be present in the test cultures at 100 mg/L loading rate WAF.
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At both the start and end of the mixing period, and following a 1-Hour standing period, all loading rate WAFs were observed to have formed cloudy white homogenous dispersions, the opacity of which increased with increasing loading rate. Given the presence of dispersed test item all loading rates were filtered through one glass wool plug and one sheet of filter paper to remove as much dispersed test item as possible. Microscopic examination of the WAFs after filtration showed there to be no micro-dispersions of test item present, however, the 100 mg/L loading rate WAF appeared slightly hazy.

At the start of the test all control, 10, 18, 32 and 56 mg/L loading rate WAF test cultures were observed to be clear colorless solutions while the 100 mg/L loading rate WAF test cultures were very slightly hazy dispersions. After the 72-Hour test period all control, 10 and 18 mg/L loading rate WAF test cultures were observed to be green dispersions. The 32 mg/L loading rate WAF test cultures were light green dispersions, the 56 mg/L loading rate WAF test cultures were very pale green dispersions and the 100 mg/L loading rate WAF test cultures were very slightly hazy dispersions.

Results with reference substance (positive control):

- Results with reference substance valid: The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
- EC50: 1.6 mg/L (72 hour, growth rate).
- NOEC: 0.25 mg/L (72 hour, growth rate)

Reported statistics and error estimates:

Statistical analysis of the growth rate data was carried out for the control and all loading rates using one way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control.

Inhibition of growth rate and yield:

Nominal Loading Rate (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.073	-	9.74E+05	-
	R2	0.075		1.07E+06
	R3	0.074		9.91E+05
	R4	0.073		9.79E+05
	R5	0.074		1.06E+06
	R6	0.074		1.01E+06
	Mean	0.074		1.01E+06
	SD	0.001		4.06E+04
10	R1	0.073	1	9.24E+05
	R2	0.072	3	9.14E+05
	R3	0.072	3	8.76E+05
	Mean	0.072	2	9.05E+05	11
	SD	0.001		2.56E+04
18	R1	0.069	7	7.10E+05
	R2	0.069	7	6.95E+05
	R3	0.068	8	6.82E+05
	Mean	0.069	7	6.96E+05	31
	SD	0.001		1.44E+04
32	R1	0.056	24	2.78E+05
	R2	0.055	26	2.53E+05
	R3	0.056	24	2.69E+05
	Mean	0.056	25	2.67E+05	74
	SD	0.001		1.24E+04
56	R1	0.041	45	9.25E+04
	R2	0.041	45	9.38E+04
	R3	0.043	42	1.02E+05
	Mean	0.042	44	9.61E+04	91
	SD	0.001		5.18E+03
100	R1	0.000	100	-1.01E+02
	R2	-0.005	107	-1.57E+03
	R3	-0.003	104	-1.07E+03
	Mean	-0.003	104	-9.13E+02	100
	SD	0.003		7.46E+02

Chemical analysis:

Time Point	Nominal Loading Rate cnom	Sample Preparation Factor F	Determined Concentration of Aluminium in Test Sample c	Determined Concentration of Bismuth in Test Sample c	Equivalent Concentration of Test Item in Test Sample (based on Bismuth) c
[hours]	[mg/L]		[mg/LAl]	[mg/LBi]	[mg/L]
0	Control	1.02	<LOQ	<LOQ	<LOQ
	10	1.02	<LOQ	0.0387	0.0892
	18	1.02	<LOQ	0.0492	0.113
	32	1.02	<LOQ	0.0576	0.133
	56	1.02	<LOQ	0.0819	0.189
	100	1.02	0.238	1.63	3.77
72	Control	1.02	<LOQ	<LOQ	<LOQ
	10	1.02	<LOQ	0.0234	0.0540
	18	1.02	<LOQ	0.0309	0.0713
	32	1.02	<LOQ	0.0286	0.0659
	56	1.02	0.254	0.0278	0.0641
	100	1.02	0.0584	0.548	1.26
72	10	1.02	0.0206	0.0655	0.151
(no algae)	18	1.02	0.0221	0.0507	0.117
	32	1.02	0.0244	0.0533	0.123
	56	1.02	0.0259	0.139	0.321
	100	1.02	0.376	2.36	5.44

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata was investigated over a 72 Hour period and provided an EL50 value of 53 mg/L (WAF) based on nominal loading rates.

Description of key information

A reliable OECD guideline study assessing the toxicity of the substance to green unicellular algae is available. This provides a 72 hour ErL50 value of 53 mg/L and a NOEL of 10 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

53 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L