Aluminum magnesium oxide

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

Study period:

21 September 2018 - 31 October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006 (Annex 5 corrected 28 July 2011)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals, OECD series on testing and assessment number 23

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Remarks:

ICP-MS (based on aluminium detection)

Details on sampling:

- Concentrations: all test concentrations and the control
- Sampling method: 4.8 mL from the approximate centre of the test vessels, at t=0 h, t=24 h and t=72 h (range-finding test) and at t=0 h and t=72 h (final test) .
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
- 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 checked by running a test vessel at at a WAF prepared at a loading rate of 4.6 mg/L 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.
Additionally, reserve samples of 4.8 mL were taken from all test solutions and stored were stored in a freezer (≤-15°C) for possible analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: direct addition of test item to test medium yielding individually prepared loading rates; 3-day magnetic stirring period to ensure maximum dissolution of the test item in medium, followed by filtration through a 0.45 μm membrane filter (RC55, Whatman) to give clear and colourless aqueous Water Accommodated Fractions (WAFs), which were used as test concentrations
- Differential loading: yes - loading rates between 1.0 and 100 mg/L
- Controls: Test medium without test item or other additives
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no (based on visual inspection)

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: in-house laboratory culture.
- Age of inoculum at test initiation: 3 days
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Stock culture medium: M1, according to NPR 6505 (Nederlandse Praktijk Richtlijn no. 6505).
- Pre-culture medium and test medium: M2, according to OECD 201.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

23 - 24°C

pH:

At t=0 h: 7.6 - 8.9
At t=72 h: 7.9 - 8.1

Nominal and measured concentrations:

Nominal concentrations (final test): untreated control and WAFs individually prepared at loading rates of 1.0, 2.2, 4.6, 10 and 22 mg/L (corrected for purity).
Measured concentrations (final test; based on Al detection and expressed in terms of test item): at test start - 0.60, 1.5, 4.7, 6.5 and 2.3 mg/L, resp.; measured concentrations changed to 100, 99, 50, 48 and 92% of initial at the end of the test.

Average exposure concentrations (geometric means) were calculated to be 0.60, 1.5, 3.3, 4.5 and 2.2 mg/L, resp.

See 'Any other information on materials and methods' for calculation of average exposure concentrations and 'Any other information on results' for details on measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution
- Aeration: no
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 195 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- 1 or 2 replicates of each test concentration without algae.

GROWTH MEDIUM
- Standard medium used: yes, M2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2 according to OECD 201, formulated using water purified by reverse osmosis.
- Intervals of water quality measurement: pH: at beginning and end of test; temperature of medium: continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod and light intensity: Continuously using TLD-lamps with a light intensity within the range of 92 to 94 μE.m-2.s-1.
- Incubation: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

EFFECT PARAMETERS MEASURED
- Cell density was measured at t=24 h, t=48 h and t=72 h.
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
- Appearance of the cells: At the end of the final test, microscopic observations were performed on the WAF prepared at a nominal loading rate of 1.0 mg/L to observe for any abnormal appearance of the algae compared to the control.

TEST CONCENTRATIONS
- Test concentrations in Range-Finding Test: WAFs prepared at loading rates of 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes, ErC50 is estimated to be between WAFs prepared at a loading rate of 1.0 and 10 mg/L. Also, exposure concentrations were found to remain generally stable during the exposure period (97-124% of initial at the end of the test), thus in the final test only samples taken at test start and test end were analysed.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (performed Nov 2018)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 2.1 - 2.3 mg/L; test material concentration based on aluminium response

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.34 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 0.29 - 0.39 mg/L (extrapolated values); test material concentration based on aluminium response

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the WAF prepared at a loading rate of 1.0 mg/L when compared to the control.
- Inhibition of growth rate (and yield) was observed in all tested WAFs resulting in 18 – 74% growth rate inhibition (and 61 – 98% yield inhibition). All inhibitions were statistically significant. NOECs could therefore not be determined, and the EC10 was calculated and reported instead.
- Measured concentrations: a small response was observed in the control after 72h of exposure and evaluated to have no impact on the results as it was not measured in the control at the start of the test.
- The concentrations measured in the samples taken from the solutions incubated with algae were comparable to those measured in their counterparts incubated without algae. Therefore, it was concluded that the presence of the algae had no effect on the concentrations of the test item in test medium.
- All water quality parameters remained within the requirements as laid down in the study plan throughout the test.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 72h-ErC50: 1.05 mg/L (95% confidence interval: 1.04-1.06 mg/L)
- Other: The historical ranges for growth rate inhibition (observed at the Test Facility) lie between 0.82 and 2.6 mg/L. Hence, the 72h-ErC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

Statistical significance: Step-down Jonckheere-Terpstra Test Procedure, α=0.05, one-sided, smaller.
Calculation of ECx-values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield reduction versus the logarithms of the corresponding geometric mean measured concentrations of the test item.

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Table 1: Test Samples Analysis Results (Final test)

Time of sampling [hours]	Date of sampling	Date of analysis^1	Loading rate^2 [mg/L]	Concentration analyzed [mg/L]	Relative to initial [%]
0	22 Oct 2018	30 Oct 2018	0	n.d.^4
			1.0	0.599
			2.2	1.52
			4.6	4.69
			4.6^3	4.63
			10	6.47
			22	2.27
72	25 Oct 2018	30 Oct 2018	0	0.0050^5	n.a.
			1.0	0.600	100
			2.2	1.50	99
			4.6	2.37	50
			4.6^3	2.16	47
			10	3.08	48
			22	2.09	92

¹      Samples were stored in the freezer (≤ -15°C) until the day of analysis.

²      Awater accommodated fraction (WAF)prepared at the loading rate.

³      Without algae.

⁴     A negative concentration at m/z 27 (same as the Al isotope m/z used for quantification of the test item) was detected in the control sample. This negative concentration was below the intercept of the calibration curve and at approximately the same level of the analytical blanks. Therefore, this was considered as not detected (n.d.).

⁵      Estimated value, calculated by extrapolation of the calibration curve.

n.a.Not applicable.

n.d.     Not detected.

Table 2: Average Exposure Concentration Versus Loading Rate

KW-2200 Loading rate (mg/L)	Measured concentrations (mg/L)		Average exposure conc. (mg/L)
	t=0h	t=72h
1.0	0.60	0.60	0.60
2.2	1.5	1.5	1.5
4.6	4.7	2.4	3.3
4.6^WA	4.6	2.2	3.2
10	6.5	3.1	4.5
22	2.3	2.1	2.2

WA – Without Algae.

Table 3: Growth Rate and Percentage Inhibition for the Total Test Period

KW-2200 Average exposure conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.758	0.0078	6
0.60	1.444	0.0073	3	18*
1.5	1.317	0.0238	3	25*
3.3	0.640	0.0179	3	64*
4.5	0.732	0.0186	3	58*
2.2	0.465	0.1226	3	74*

* - effect was statistically significant.

Table 4: Growth Rate and Percentage Inhibition at Different Time Intervals

KW-2200 Average exposure conc. (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	1.806		1.748		1.719
0.60	3	1.822	-0.89	1.271	27	1.240	28
1.5	3	1.875	-3.9	1.288	26	0.788	54
3.3	3	1.205	33	0.482	72	0.233	86
4.5	3	1.170	35	0.546	69	0.481	72
2.2	3	1.051	42	0.395	77	-0.050	103

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' section for details on validity criteria.

Conclusions:

Statistically significant inhibition of growth rate (and yield) was observed in all test item solutions. The 72-h ErC50 and ErC10 for Pseudokirchneriella subcapitata was 2.2 mg/L and 0.34 mg/L, resp. (based on geometric mean measured average exposure concentrations).

Executive summary:

In a 72 h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to individually prepared Water Accommodated Fractions of the test item prepared at nominal loading rates of 1.0, 2.2, 4.6, 10 and 22 mg/L (3 replicates per concentration), and an untreated control (6 replicates).

Measured concentrations of the test item (based on aluminium) were found to change throughout the exposure period (100, 99, 50, 47, 48 and 92% of initial at the end of the test). Therefore average exposure concentrations (geometric means) were calculated: 0.60, 1.5, 3.3, 4.5 and 2.2 mg/L.

Inhibition of growth rate (and yield) was observed at all KW-2200 concentrations tested resulting in 18 – 74% growth rate inhibition (and 61 – 98% yield inhibition). Statistically significant inhibition of both growth rate (and yield) was found at all test concentrations. Therefore, the NOEC for growth rate (and yield) inhibition could not be determined. Instead, theEC10 was calculated as an equivalent alternative.

The EC50 for growth rate inhibition (72h-ErC50) and 72h-ErC10 (extrapolated value) were 2.2 and 0.34 mg/L, respectively.

The study met all validity criteria, and is considered reliable without restriction.

Description of key information

The 72-h ErC50 and ErC10 for Pseudokirchneriella subcapitata was 2.2 mg/L and 0.34 mg/L, resp. (based on geometric mean measured average exposure concentrations).

Key value for chemical safety assessment

EC50 for freshwater algae:

2.2 mg/L

EC10 or NOEC for freshwater algae:

0.34 mg/L

Additional information

In a 72 h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to individually prepared Water Accommodated Fractions of the test item prepared at nominal loading rates of 1.0, 2.2, 4.6, 10 and 22 mg/L (3 replicates per concentration), and an untreated control (6 replicates).

Measured concentrations of the test item (based on aluminium) were found to change throughout the exposure period (100, 99, 50, 47, 48 and 92% of initial at the end of the test). Therefore average exposure concentrations (geometric means) were calculated: 0.60, 1.5, 3.3, 4.5 and 2.2 mg/L.

Inhibition of growth rate (and yield) was observed at all KW-2200 concentrations tested resulting in 18 – 74% growth rate inhibition (and 61 – 98% yield inhibition). Statistically significant inhibition of both growth rate (and yield) was found at all test concentrations. Therefore, the NOEC for growth rate (and yield) inhibition could not be determined. Instead, the EC10 was calculated as an equivalent alternative.

The EC50 for growth rate inhibition (72h-ErC50) and 72h-ErC10 (extrapolated value) were 2.2 and 0.34 mg/L, respectively.

The study met all validity criteria, and is considered reliable without restriction.