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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: protocol used by the EPA Office of Toxic Substances
Deviations:
yes
Remarks:
nutrient solution was four times as concentrated as guideline recommended
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Specific details on test material used for the study:
- Name of test material: anhydrous AlCl3
- Analytical purity: 99.7%
- Source: MCB Manufacturing Chemists, Inc.
- Lot/batch No.: B7N14
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all test concentrations and controls
- Sampling method: samples were collected before, at the beginning of, and at the end of the exposure period. Samples (15-20 mL) were collected midway between the surface and bottom of the water column in the center of each exposure chamber. A 30 mL adjustable-volume glass-teflon syringe was rinsed three times with the exposure water prior to collecting a sample.
- Sample storage conditions before analysis: All samples were immediately acidified to 1% (v/v) with concentrated nitric acid (Baker Instra analyzed Lot No: 340040). Two mL of potassium chloride solution (95 g KCl/L) was added per 100 mL sample, as suggested in method 202.1. Methods for Chemical Analysis of Water and Wastewater (U.S. EPA, 1979).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Al stock solution (about 10,000 mg/L) was added to the nutrient medium in appropriate volume to achieve the desired nominal test concentrations.
- Evidence of undissolved material: a precipitate was readily apparent at concentrations of 3.12 mg/L or above
- All other template details: Not reported
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Source: stock culture was obtained from the EPA Environmental Research Laboratory-Duluth and sub-cultured in nutrient solution until utilized in the test
- All other template details: Not reported

ACCLIMATION: Not reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
Not reported
Hardness:
Not reported
Test temperature:
25 ± 3 degrees C
pH:
two pH levels: 7.6 ± 0.05 and 8.2 ± 0.05
Dissolved oxygen:
Not reported
Salinity:
Not reported
Nominal and measured concentrations:
Nominal (mg/L): 0, 0.4, 0.8, 1.6, 3.2 and 6.4
Measured (mg/L): <0.2, 0.4, 0.7, 1.4, 2.8, 5.8
Details on test conditions:
TEST SYSTEM
- Test vessel: erlenmeyer flasks
- Type: closed
- Material, size, headspace, fill volume: 125 mL erlenmeyer flasks filled with 50 mL of solution
- Aeration: none
- Type of flow-through: not applicable
- Renewal rate of test solution: not reported
- Initial cells density: 10,000 cells/mL
- Control end cells density: not reported
- No. of organisms per vessel: not applicable
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates):not applicable

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS: no parameters reported for the nutrient solutions

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: the pH was adjusted to the appropriate level and re-adjusted the following morning
- Photoperiod: not reported
- Light intensity and quality: 400 ± 40 ft candles
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED: biomass measured at end of exposure period
- Other: solutions were acidified to 1% nitric acid and filtered through prewashed and preweighed 0.45 µm Millipore membrane filters. The filters were then dried at 70 degrees C and reweighed to determine the biomass of algae present in each sample. To determine the actual biomass of algae, a correction for the weight of Al floc on the filters was made by measuring the Al floc in flasks at each test concentration which had not been inoculated with algae for 96 hrs.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2x previous concentration
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study
- Test concentrations: 0, 3.12, 6.25, 12.5, 25.0, 50 mg/L at pH of 7.6 and 8.2
- Results used to determine the conditions for the definitive study: inhibitions greater than 50% for all concentrations tested
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
0.57 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
biomass
Remarks on result:
other: pH 7.6, 95% CI: 0.39 - 0.83 mg/L
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
0.46 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
biomass
Remarks on result:
other: pH 8.2, 95% CI: 0.41 - 0.52 mg/L
Details on results:
- Other: the pH in most exposure water samples dropped during the 96 hrs. The 96 hr Al concentrations were at or below the detection limit (0.2 mg/L) in all but one sample (an uninoculated flask).
- All other template details: Not reported
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
The initial total Al concentration and 96 hr percent inhibition data were tested with a Spearman-Karber statistical program to estimate the concentration that would result in a 50% reduction in mean biomass (96 hr EC50).

TABLE 4. Biomass and Growth Inhibition of Green Algae (Selenastrum capricornutum) After 96 hr of Exposure to Various Concentrations of Al3+.

Al3+ Concentration (mg/L)

 

Nominal

0

0.4

0.8

1.6

3.2

6.4

 

Total Measured 1

<0.2

0.4

0.7

1.4

2.8

5.8

pH 7.6

Mean Corrected Algal Biomass (mg) (± SD)

5.12 ± 0.67

3.48 ± 0.42

1.20 ± 0.26

1.87 ± 0.21

2.35 ± 1.01A

1.73 ± 0.32

 

Percent Effect

-

32.0

76.6

63.7

54.1

66.2

pH 8.2

Mean Corrected Algal Biomass (mg) (± SD)

5.08 ± 0.25

3.07 ± 0.45

1.10 ± 0.20

0.33 ± 0.06

1.38 ± 0.06

1.23 ± 0.15

 

Percent Effect

-

39.6

78.3

93.5

72.8

75.8

 

1- Total measured Al is the initial amount present in the water column prior to pH adjustment and the start of the test. No precipitate was observed.

A- Median and standard deviation from the median.

Conclusions:
96 hr exposure of Selenastrum capricornutum to aluminium chloride concentrations up to 5.8 mg/L resulted in EC50s for reduction in biomass at pHs of 7.6 of 0.57 mg/L and at pH 8.2 of 0.46 mg/L.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
not specified
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Remarks:
The testing laboratory is operating according to GLP specifications but is not cerfied according to GLP.
Specific details on test material used for the study:
- Molecular formula: Al(NO3)3 x 9H2O
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all exposure concentrations and controls
- Sampling method: measured at beginning, middle, and end of each test
- Sample storage conditions before analysis: the extraction aliquot can be stored at 4 degrees C in an Ependorf tube
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Al was dissolved in standard laboratory media with synthetic buffers to reduce pH drift (MES, pK 6.1, for pH 6.2; MOPS, pK 7.2 for pH 7.0; HEPES, pK 7.5 for pH 8.0)
- All other template details: Not reported
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Age of inoculum (at test initiation): 4 days
- All other template details: Not reported

ACCLIMATION: Not reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not reported
Hardness:
24.3, 60, 120 mg/L as CaCO3
Test temperature:
23 ± 1 degree C
pH:
nominal: 6, 7, 8
measured (range of means): 6.09-6.29, 6.94-7.25, 7.74-8.12
Dissolved oxygen:
not reported
Salinity:
not reported
Nominal and measured concentrations:
Nominal (μg/L): range from 12.5-6,400
Measured mean dissolved Al (μg/L): range from 0.3-1019.4
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, headspace, fill volume: 250 mL test chamber filled with 100 mL solution, loosely stoppered and placed in an orbital shaker at 100 cycles per minute
- Initial cells density: 10,000 cells/mL
- Control end cells density: 487,000-2,093,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- All other template details: Not reported

GROWTH MEDIUM
- Standard medium used: Yes, see Table 20 for composition

TEST MEDIUM / WATER PARAMETERS: see Table 20 for composition
- Dissolved organic carbon: 0,2,4 mg/L
- All other template details: Not reported

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: 86 ± 8.6 μE/m2/s, "Cool white" fluorescent lighting
- All other template details: Not reported

EFFECT PARAMETERS MEASURED: growth monitored daily
- Determination of cell concentrations: counting chamber- Improved Neubauer Chamber
- All other template details: Not reported

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2x
- All other template details: Not reported
Reference substance (positive control):
not specified
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
16.9 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: pH 7, Hardness 24.3 mg/L, DOC 0 mg/L: Nominal EC50 345.6 μg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
350 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: pH 8, Hardness 24.3 mg/L, DOC 0 mg/L: Nominal EC50 1351.8 μg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
20 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: pH 7, Hardness 60 mg/L, DOC 0 mg/L: Nominal EC50 584 μg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
265.7 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: pH 8, Hardness 60 mg/L, DOC 0 mg/L: Nominal EC50 1607.2 μg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
19 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: pH 7, Hardness 120 mg/L, DOC 0 mg/L: Nominal EC50 550.1 μg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
321.5 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: pH 8, Hardness 120 mg/L, DOC 0 mg/L: Nominal EC50 889.1 μg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.123 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.073-0.226, pH7, hardness: 22 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.172 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.131-0.226, pH 7, hardness: 60 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.184 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.115-0.294, pH7, Hardness: 120 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.709 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.572-0.880, pH8, Hardness: 22 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.382 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.281-0.519, pH 7, hardness: 60 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.249 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.138-0.448, pH 8, Hardness: 120 mg/L
Details on results:
Results based on biomass are presented in Table 1 below
Results based on waters with pH 6 are presented in Table 2 below
- All other template details: Not reported
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
EC50 values, with a 95% confidence interval, were calculated from the experimental data using the Probit method (EPA) and ICp software v2.0 (EPA). The EC50 was calculated for “Nominal Al concentrations” using the software already mentioned, but in the case of “Dissolved Al” and “Monomeric Al” concentrations, the EC50 were determined by a simple proportionality calculation based in the Nominal Al concentration values closest to the EC50 or IC50.

Table 1. P.subcapitata 72 -h Al growth inhibition test results expressed as biomass with OECD medium at different conditions of pH

(6, 7 and 8), Hardness (24.3, 60 and 120 mg/L as CaCO3) and DOC (0, 2, 4 mg/L).

 

 

 

EC50values based on biomass

pH

Hardness (mg/L)

DOC (mg/L)

Nominal (μg/L)

Mean Dissolved Al (μg/L)

Mean Monomeric Al (μg/L)

6

24.3

0

28.3

0.9

0.2

7

24.3

0

155.5

14.6

11.9

8

24.3

0

851.4

310.2

368.0

6

60

0

76.4

3.3

0.7

7

60

0

232.9

24.6

18.3

8

60

0

516.2

196.8

148.5

6

120

0

74.2

4.5

3.5

7

120

0

226.3

11.1

9.0

8

120

0

366.9

208.8

204.5

 

 

 

 

 

 

6

24.3

2

669.9

260

38.1

7

24.3

2

1815.8

128.6

72.5

8

24.3

2

2157

864.7

397.6

6

60

2

1030.1

44.1

21.9

7

60

2

2266.7

70.5

44.1

8

60

2

927.1

441.4

344.7

6

120

2

1451.5

54.9

29.3

7

120

2

2591.7

87

51.6

8

120

2

774.2

342.7

285.8

 

 

 

 

 

 

6

24.3

4

778.2

490.8

113.1

7

24.3

4

2630.2

550.5

181.4

8

24.3

4

2229.7

696.7

370.9

6

60

4

1273.7

98.9

52.1

7

60

4

2736.4

436.7

108.7

8

60

4

1660.2

536.7

393.6

6

120

4

1576.2

97.1

83.6

7

120

4

3546.2

82.4

84.9

8

120

4

1521.2

450.1

336.2

 

 

 

 

 

 

Table 2. P. subcapitata 72-h Al growth inhibition test results expressed as growth rate with OECD medium at pH 6 and different conditions of Hardness (24.3, 60 and 120 mg/L as CaCO3) and DOC (0, 2, 4 mg/L).

 

 

 

EC50values based on growth rate

pH

Hardness (mg/L)

DOC (mg/L)

Nominal (μg/L)

Mean Dissolved Al (μg/L)

Mean Monomeric Al (μg/L)

6

24.3

0

72.1

0.6

0.0

6

60

0

206.2

4.8

0.3

6

120

0

323.4

2.6

0.2

6

24.3

2

1181.1

20.0

8.1

6

60

2

1473.5

14.0

9.4

6

120

2

2100.1

9.5

5.3

6

24.3

4

1443.9

176.9

79.9

6

60

4

1890.7

30.2

22.0

6

120

4

2429.3

26.4

25.5

Conclusions:
The 72-h EC50 values for dissolved Al that inhibited growth rate in Pseudokirchneriella subcapitata in OECD medium at pH 6, 7, and 8, Hardness of 24.3,60,120 mg/L and DOC of 0,2,4 mg/L ranged from 0.6-176.9 μg/L at pH 6, 16.9-136.6 μg/L at pH 7, and 265.7-699.9 μg/L at pH 8.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
not specified
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Remarks:
The testing laboratory is operating according to GLP specifications but is not cerfied according to GLP.
Specific details on test material used for the study:
- Molecular formula: Al(NO3)3 x 9H2O
Analytical monitoring:
no
Remarks:
The testing laboratory is operating according to GLP specifications but is not cerfied according to GLP.
Details on sampling:
- Concentrations: all exposure concentrations and controls
- Sampling method: measured at beginning, middle, and end of each test
- Sample storage conditions before analysis: the extraction aliquot can be stored at 4 degrees C in an Ependorf tube
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Al was dissolved in standard laboratory media with synthetic buffers to reduce pH drift (MES, pK 6.1, for pH 6.2; MOPS, pK 7.2 for pH 7.0; HEPES, pK 7.5 for pH 8.0)
- All other template details: Not reported
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Age of inoculum (at test initiation): 4 days
- All other template details: Not reported

ACCLIMATION: Not reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not reported
Hardness:
24.3, 60, 120 mg/L as CaCO3
Test temperature:
23 ± 1 degree C
pH:
nominal: 6, 7, 8
measured (range of means): 6.09-6.29, 6.94-7.25, 7.74-8.12
Dissolved oxygen:
not reported
Salinity:
not reported
Nominal and measured concentrations:
Nominal (μg/L): range from 12.5-6,400
Measured mean dissolved Al (μg/L): range from 0.3-1019.4
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, headspace, fill volume: 250 mL test chamber filled with 100 mL solution, loosely stoppered and placed in an orbital shaker at 100 cycles per minute
- Initial cells density: 10,000 cells/mL
- Control end cells density: 487,000-2,093,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- All other template details: Not reported

GROWTH MEDIUM
- Standard medium used: Yes, see Table 20 for composition

TEST MEDIUM / WATER PARAMETERS: see Table 20 for composition
- Dissolved organic carbon: 0,2,4 mg/L
- All other template details: Not reported

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: 86 ± 8.6 μE/m2/s, "Cool white" fluorescent lighting
- All other template details: Not reported

EFFECT PARAMETERS MEASURED: growth monitored daily
- Determination of cell concentrations: counting chamber- Improved Neubauer Chamber
- All other template details: Not reported

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2x
- All other template details: Not reported
Reference substance (positive control):
not specified
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
42.8 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: pH 7, Hardness 24.3 mg/L, DOC 2 mg/L: Nominal EC50 2896 μg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
430.3 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: pH 8, Hardness 24.3 mg/L, DOC 2 mg/L: Nominal EC50 4980.9 μg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
38.7 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: pH 7, Hardness 60 mg/L, DOC 2 mg/L: Nominal EC50 4332.3 μg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
434.6 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: pH 8, Hardness 60 mg/L, DOC 2 mg/L: Nominal EC50 2000 μg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
55.3 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: pH 7, Hardness 120 mg/L, DOC 2 mg/L: Nominal EC50 3645.8 μg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
451 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: pH 8, Hardness 120 mg/L, DOC 2 mg/L: Nominal EC50 1639.9 μg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.293 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.078-1.108, pH6, hardness: 22 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.851 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.672-1.077, pH6, Hardness: 60 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.389 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 1.172-1.647, pH6, Hardness: 120 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.012 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 1.639-2.471, pH7, hardness: 22 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.969 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 1.767-2.195, pH7, hardness: 60mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.432 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 1.832-3.227, pH7, Hardness: 120 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.57 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 1.357-1.817, pH8, Hardness: 22 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.774 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.582-1.030, pH8, Hardness: 60mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.673 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.602-0.752, pH 8, Hardness: 120mg/L
Details on results:
Results based on biomass are presented in Table 1 below
Results based on waters with pH 6 are presented in Table 2 below
- All other template details: Not reported
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
EC50 values, with a 95% confidence interval, were calculated from the experimental data using the Probit method (EPA) and ICp software v2.0 (EPA). The EC50 was calculated for “Nominal Al concentrations” using the software already mentioned, but in the case of “Dissolved Al” and “Monomeric Al” concentrations, the EC50 were determined by a simple proportionality calculation based in the Nominal Al concentration values closest to the EC50 or IC50.

Table 1. P.subcapitata 72 -h Al growth inhibition test results expressed as biomass with OECD medium at different conditions of pH

(6, 7 and 8), Hardness (24.3, 60 and 120 mg/L as CaCO3) and DOC (0, 2, 4 mg/L).

 

 

 

EC50values based on biomass

pH

Hardness (mg/L)

DOC (mg/L)

Nominal (μg/L)

Mean Dissolved Al (μg/L)

Mean Monomeric Al (μg/L)

6

24.3

0

28.3

0.9

0.2

7

24.3

0

155.5

14.6

11.9

8

24.3

0

851.4

310.2

368.0

6

60

0

76.4

3.3

0.7

7

60

0

232.9

24.6

18.3

8

60

0

516.2

196.8

148.5

6

120

0

74.2

4.5

3.5

7

120

0

226.3

11.1

9.0

8

120

0

366.9

208.8

204.5

 

 

 

 

 

 

6

24.3

2

669.9

260

38.1

7

24.3

2

1815.8

128.6

72.5

8

24.3

2

2157

864.7

397.6

6

60

2

1030.1

44.1

21.9

7

60

2

2266.7

70.5

44.1

8

60

2

927.1

441.4

344.7

6

120

2

1451.5

54.9

29.3

7

120

2

2591.7

87

51.6

8

120

2

774.2

342.7

285.8

 

 

 

 

 

 

6

24.3

4

778.2

490.8

113.1

7

24.3

4

2630.2

550.5

181.4

8

24.3

4

2229.7

696.7

370.9

6

60

4

1273.7

98.9

52.1

7

60

4

2736.4

436.7

108.7

8

60

4

1660.2

536.7

393.6

6

120

4

1576.2

97.1

83.6

7

120

4

3546.2

82.4

84.9

8

120

4

1521.2

450.1

336.2

 

 

 

 

 

 

Table 2. P. subcapitata 72-h Al growth inhibition test results expressed as growth rate with OECD medium at pH 6 and different conditions of Hardness (24.3, 60 and 120 mg/L as CaCO3) and DOC (0, 2, 4 mg/L).

 

 

 

EC50values based on growth rate

pH

Hardness (mg/L)

DOC (mg/L)

Nominal (μg/L)

Mean Dissolved Al (μg/L)

Mean Monomeric Al (μg/L)

6

24.3

0

72.1

0.6

0.0

6

60

0

206.2

4.8

0.3

6

120

0

323.4

2.6

0.2

6

24.3

2

1181.1

20.0

8.1

6

60

2

1473.5

14.0

9.4

6

120

2

2100.1

9.5

5.3

6

24.3

4

1443.9

176.9

79.9

6

60

4

1890.7

30.2

22.0

6

120

4

2429.3

26.4

25.5

Conclusions:
The 72-h EC50 values for dissolved Al that inhibited growth rate in Pseudokirchneriella subcapitata in OECD medium at pH 6, 7, and 8, Hardness of 24.3,60,120 mg/L and DOC of 0,2,4 mg/L ranged from 0.6-176.9 μg/L at pH 6, 16.9-136.6 μg/L at pH 7, and 265.7-699.9 μg/L at pH 8.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
not specified
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Remarks:
The testing laboratory is operating according to GLP specifications but is not cerfied according to GLP.
Specific details on test material used for the study:
- Molecular formula: Al(NO3)3 x 9H2O
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all exposure concentrations and controls
- Sampling method: measured at beginning, middle, and end of each test
- Sample storage conditions before analysis: the extraction aliquot can be stored at 4 degrees C in an Ependorf tube
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Al was dissolved in standard laboratory media with synthetic buffers to reduce pH drift (MES, pK 6.1, for pH 6.2; MOPS, pK 7.2 for pH 7.0; HEPES, pK 7.5 for pH 8.0)
- All other template details: Not reported
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Age of inoculum (at test initiation): 4 days
- All other template details: Not reported

ACCLIMATION: Not reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not reported
Hardness:
24.3, 60, 120 mg/L as CaCO3
Test temperature:
23 ± 1 degree C
pH:
nominal: 6, 7, 8
measured (range of means): 6.09-6.29, 6.94-7.25, 7.74-8.12
Dissolved oxygen:
not reported
Salinity:
not reported
Nominal and measured concentrations:
Nominal (μg/L): range from 12.5-6,400
Measured mean dissolved Al (μg/L): range from 0.3-1019.4
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, headspace, fill volume: 250 mL test chamber filled with 100 mL solution, loosely stoppered and placed in an orbital shaker at 100 cycles per minute
- Initial cells density: 10,000 cells/mL
- Control end cells density: 487,000-2,093,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- All other template details: Not reported

GROWTH MEDIUM
- Standard medium used: Yes, see Table 20 for composition

TEST MEDIUM / WATER PARAMETERS: see Table 20 for composition
- Dissolved organic carbon: 0,2,4 mg/L
- All other template details: Not reported

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: 86 ± 8.6 μE/m2/s, "Cool white" fluorescent lighting
- All other template details: Not reported

EFFECT PARAMETERS MEASURED: growth monitored daily
- Determination of cell concentrations: counting chamber- Improved Neubauer Chamber
- All other template details: Not reported

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2x
- All other template details: Not reported
Reference substance (positive control):
not specified
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
118.7 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: pH 7, Hardness 24.3 mg/L, DOC 4 mg/L: Nominal EC50 3845.9 μg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
699.9 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: pH 8, Hardness 24.3 mg/L, DOC 4 mg/L: Nominal EC50 4716.1 μg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
136.6 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: pH 7, Hardness 60 mg/L, DOC 4 mg/L: Nominal EC50 4260 μg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
478.4
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: pH 8, Hardness 60 mg/L, DOC 4 mg/L: Nominal EC50 2905 μg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
45.5 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: pH 7, Hardness 120 mg/L, DOC 4 mg/L: Nominal EC50 4930 μg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
476.1 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: pH 8, Hardness 120 mg/L, DOC 4 mg/L: Nominal EC50 3556.3 μg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.582 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.394-0.859, pH6, Hardness: 22 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.11 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.929-1.305, pH6, Hardness: 60 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.258 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 1.130-1.372, pH6, Hardness: 120 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.478 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 2.011-3.054, pH7, Hardness: 22 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.653 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 2.279-3.090, pH8, Hardness: 60mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3.155 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 2.955-3.368, pH7, Hardness: 120mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.342 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.773-2.330, pH8, Hardness: 22 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.509 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 1.356-1.680, pH8, Hardness: 60 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.086 mg/L
Nominal / measured:
estimated
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 1.029-1.147, pH8, Hardness: 120 mg/L
Details on results:
Results based on biomass are presented in Table 1 below
Results based on waters with pH 6 are presented in Table 2 below
- All other template details: Not reported
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
EC50 values, with a 95% confidence interval, were calculated from the experimental data using the Probit method (EPA) and ICp software v2.0 (EPA). The EC50 was calculated for “Nominal Al concentrations” using the software already mentioned, but in the case of “Dissolved Al” and “Monomeric Al” concentrations, the EC50 were determined by a simple proportionality calculation based in the Nominal Al concentration values closest to the EC50 or IC50.

Table 1. P.subcapitata 72 -h Al growth inhibition test results expressed as biomass with OECD medium at different conditions of pH

(6, 7 and 8), Hardness (24.3, 60 and 120 mg/L as CaCO3) and DOC (0, 2, 4 mg/L).

 

 

 

EC50values based on biomass

pH

Hardness (mg/L)

DOC (mg/L)

Nominal (μg/L)

Mean Dissolved Al (μg/L)

Mean Monomeric Al (μg/L)

6

24.3

0

28.3

0.9

0.2

7

24.3

0

155.5

14.6

11.9

8

24.3

0

851.4

310.2

368.0

6

60

0

76.4

3.3

0.7

7

60

0

232.9

24.6

18.3

8

60

0

516.2

196.8

148.5

6

120

0

74.2

4.5

3.5

7

120

0

226.3

11.1

9.0

8

120

0

366.9

208.8

204.5

 

 

 

 

 

 

6

24.3

2

669.9

260

38.1

7

24.3

2

1815.8

128.6

72.5

8

24.3

2

2157

864.7

397.6

6

60

2

1030.1

44.1

21.9

7

60

2

2266.7

70.5

44.1

8

60

2

927.1

441.4

344.7

6

120

2

1451.5

54.9

29.3

7

120

2

2591.7

87

51.6

8

120

2

774.2

342.7

285.8

 

 

 

 

 

 

6

24.3

4

778.2

490.8

113.1

7

24.3

4

2630.2

550.5

181.4

8

24.3

4

2229.7

696.7

370.9

6

60

4

1273.7

98.9

52.1

7

60

4

2736.4

436.7

108.7

8

60

4

1660.2

536.7

393.6

6

120

4

1576.2

97.1

83.6

7

120

4

3546.2

82.4

84.9

8

120

4

1521.2

450.1

336.2

 

 

 

 

 

 

Table 2. P. subcapitata 72-h Al growth inhibition test results expressed as growth rate with OECD medium at pH 6 and different conditions of Hardness (24.3, 60 and 120 mg/L as CaCO3) and DOC (0, 2, 4 mg/L).

 

 

 

EC50values based on growth rate

pH

Hardness (mg/L)

DOC (mg/L)

Nominal (μg/L)

Mean Dissolved Al (μg/L)

Mean Monomeric Al (μg/L)

6

24.3

0

72.1

0.6

0.0

6

60

0

206.2

4.8

0.3

6

120

0

323.4

2.6

0.2

6

24.3

2

1181.1

20.0

8.1

6

60

2

1473.5

14.0

9.4

6

120

2

2100.1

9.5

5.3

6

24.3

4

1443.9

176.9

79.9

6

60

4

1890.7

30.2

22.0

6

120

4

2429.3

26.4

25.5

Conclusions:
The 72-h EC50 values for dissolved Al that inhibited growth rate in Pseudokirchneriella subcapitata in OECD medium at pH 6, 7, and 8, Hardness of 24.3,60,120 mg/L and DOC of 0,2,4 mg/L ranged from 0.6-176.9 μg/L at pH 6, 16.9-136.6 μg/L at pH 7, and 265.7-699.9 μg/L at pH 8.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: EPA. 2002. Short-term methods for estimating the chronic toxicity of effluents and receiving water to freshwater organisms. EPA-821-R-02-013.
Deviations:
not specified
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Remarks:
The testing laboratory is operating according to GLP specifications but is not cerfied according to GLP.
Specific details on test material used for the study:
- Molecular formula: Al(NO3)3 x 9H2O
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all exposure concentrations and controls
- Sampling method: measured at beginning, middle, and end of each test
- Sample storage conditions before analysis: the extraction aliquot can be stored at 4 degrees C in an Ependorf tube
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Al was dissolved in standard laboratory media with synthetic buffers to reduce pH drift (MES, pK 6.1, for pH 6.2; MOPS, pK 7.2 for pH 7.0; HEPES, pK 7.5 for pH 8.0)
- All other template details: Not reported
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Age of inoculum (at test initiation): 4 days
- All other template details: Not reported

ACCLIMATION: Not reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
Not reported
Hardness:
see Table 11
Test temperature:
25 ± 1 degree C
pH:
nominal: 6, 7, 8
measured (range of means): 6.09-6.10, 6.95-7.01, 7.98-8.02
Dissolved oxygen:
not reported
Salinity:
not reported
Nominal and measured concentrations:
Nominal for pH 6 (μg/L): control, 0, 12.5, 25, 50, 100
Measured mean dissolved Al for pH 6 (μg/L): 0.0, 0.1, 8.4, 21.1, 46.7, 101.0

Nominal for pH 7 (μg/L): control, 0, 100, 200, 400, 800
Measured mean dissolved Al for pH 7 (μg/L): 0.6, 1.0, 4.9, 6.0, 8.1, 13.2

Nominal for pH 8 (μg/L): control, 0, 50, 100, 200, 400
Measured mean dissolved Al for pH 8 (μg/L): 1.8, 1.5, 43.9, 87.4, 135.6, 161.2
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, headspace, fill volume: 250 mL test chamber filled with 100 mL solution, loosely stoppered and placed in an orbital shaker at 100 cycles per minute
- Initial cells density: 10,000 cells/mL
- Control end cells density: 2,650,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- All other template details: Not reported

GROWTH MEDIUM
- Standard medium used: Yes, see Table 11 for composition

TEST MEDIUM / WATER PARAMETERS: see Table 11 for composition

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: 86 ± 8.6 μE/m2/s, "Cool white" fluorescent lighting
- All other template details: Not reported

EFFECT PARAMETERS MEASURED: growth monitored daily
- Determination of cell concentrations: counting chamber- Improved Neubauer Chamber
- All other template details: Not reported

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2x
- All other template details: Not reported
Reference substance (positive control):
not specified
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
20.1 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
biomass
Remarks on result:
other: pH 6, 95% CI 15.5-31.3 µg/L
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
5.4 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
biomass
Remarks on result:
other: pH 7, 95% CI 5.1-5.6 µg/L
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
150.6 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
biomass
Remarks on result:
other: pH 8, 95% CI 147.9-152.4 µg/L
Details on results:
Not reported
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
EC50 values, with a 95% confidence interval, were calculated from the experimental data using the Probit method (EPA) and ICp software v2.0 (EPA). The EC50 was calculated for “Nominal Al concentrations” using the software already mentioned, but in the case of “Dissolved Al” and “Monomeric Al” concentrations, the EC50 were determined by a simple proportionality calculation based in the Nominal Al concentration values closest to the EC50 or IC50.

Not applicable

Conclusions:
The 96-h EC50 values for reduction of biomass of Pseudokirchneriella subcapitata in AAP-Medium at pH 6, 7, and 8 were estimated as 20.1, 5.4, and 150.6 μg/L, respectively, for dissolved Al.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
not specified
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Remarks:
The testing laboratory is operating according to GLP specifications but is not cerfied according to GLP.
Specific details on test material used for the study:
- Molecular formula: Al(NO3)3 x 9H2O
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all exposure concentrations and controls
- All other template details: Not reported
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Al was dissolved in standard laboratory media with synthetic buffer to reduce pH drift (HEPES 5mM)
- All other template details: Not reported
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- All other template details: Not reported

ACCLIMATION: Not reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not reported
Hardness:
24.3 mg/L as CaCO3
Test temperature:
Not reported
pH:
nominal: 7.7, 8
measured (range of means): 7.65-7.7, 7.99-8.08
Dissolved oxygen:
not reported
Salinity:
not reported
Nominal and measured concentrations:
Nominal (μg/L): 0, 100, 200, 400, 800, 1600
Measured mean dissolved Al pH 7.7 (μg/L): 0.0, 29.8, 77.2, 151.5, 226.1, 230.8
Measured mean dissolved Al pH 8 (μg/L): 0.4, 75.2, 147.3, 256.0, 399.4, 536.0
Details on test conditions:
TEST SYSTEM: Not reported, assumed same as CIMM 2009 (250 mL Erlenmeyer flasks)

GROWTH MEDIUM
- Standard medium used: Yes, standard OECD medium
- All other template details: Not reported
Reference substance (positive control):
not specified
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
370 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
biomass
Remarks on result:
other: pH 8; 95% CI- 340.5-395.2 μg/L; Nominal EC50- 717.9 μg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
182 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
biomass
Remarks on result:
other: pH 7.7; 95% CI- 167.1-195.8 μg/L; Nominal EC50- 563.3 μg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
515 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: pH 8; 95% CI- 494.2-537.6 μg/L; Nominal EC50- 1476.6 μg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
229.8 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: pH 7.7; 95% CI- 229.2-230.4 μg/L; Nominal EC50- 1417.9 μg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
200 µg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
biomass
Remarks on result:
other: pH 7.7
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
400 µg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
biomass
Remarks on result:
other: pH 7.7
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
400 µg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: pH 7.7
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
800 µg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: pH 7.7
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
400 µg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
biomass
Remarks on result:
other: pH 8
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
800 µg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
biomass
Remarks on result:
other: pH 8
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
400 µg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: pH 8
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
800 µg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: pH 8
Details on results:
Not reported
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Not reported, assumed same as CIMM 2009:
"EC50 values, with a 95% confidence interval, were calculated from the experimental data using the Probit method (EPA) and ICp software v2.0 (EPA). The EC50 was calculated for “Nominal Al concentrations” using the software already mentioned, but in the case of “Dissolved Al” and “Monomeric Al” concentrations, the EC50 were determined by a simple proportionality calculation based in the Nominal Al concentration values closest to the EC50 or IC50. "

Not applicable

Conclusions:
The 72-h EC50 values for dissolved Al that inhibited growth rate in Pseudokirchneriella subcapitata in OECD medium were 229.8 μg/L at pH 7.7 and 515.0 μg/L at pH 8. The 72-h EC50 values for dissolved Al that inhibited biomass in P.subcapitata in OECD medium were 182.0 μg/L at pH 7.7 and 370.0 μg/L at pH 8.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
not specified
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Remarks:
The testing laboratory is operating according to GLP specifications but is not cerfied according to GLP.
Specific details on test material used for the study:
- Name of test material (as cited in study report): alkaline aluminate solution
- Molecular formula: NaAlO2
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all exposure concentrations and controls measured at 0 and 72 hours
- All other template details: Not reported
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Al was dissolved in standard OECD media with synthetic buffer to reduce pH drift (HEPES 5mM)
- All other template details: Not reported
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- All other template details: Not reported

ACCLIMATION: Not reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not reported
Hardness:
10.6 mg/L as CaCO3
Test temperature:
23 ± 1 degree C
pH:
nominal: 8
measured (range): 7.92-8.29
Dissolved oxygen:
not reported
Salinity:
not reported
Nominal and measured concentrations:
Nominal (μg/L): 0, 200, 600, 1000, 1400, 1800
Measured total Al (μg/L): 4.4, 197.3, 468.5, 984.5, 1254.9, 1839.6
Measured mean dissolved Al (μg/L): 5.5, 210.3, 558.6, 1016.8, 1456.2, 1897.5
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 250 mL flask filled with 100 mL of test solution
- Renewal rate of test solution: none
- Initial cells density: 10000 cells/mL
- Control end cells density: 753000 cells/mL
- No. of vessels per concentration (replicates): 3
- All other template details: Not reported

GROWTH MEDIUM
- Standard medium used: yes
- All other template details: Not reported

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: 86 ± 8.6 μE/m2/s from "cool white" fluorescent lighting
- All other template details: Not reported

EFFECT PARAMETERS MEASURED: biomass and growth rate calculated every 24 hours
- All other template details: Not reported

TEST CONCENTRATIONS: Not reported
Reference substance (positive control):
not specified
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
991.8 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
biomass
Remarks on result:
other: pH 8; 95% CI- 908.5 - 1082.8 μg/L; Nominal EC50- 970.9 μg/L (95% CI- 878.6-1073.0 μg/L)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1 799 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: 95% CI- 1666.7-1941.8 μg/L; Nominal EC50- 1703.8 μg/L (95% CI- 1563.7-1856.5 μg/L)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
600 µg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1 000 µg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
600 µg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1 000 µg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
growth rate
Details on results:
Not reported
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Not reported, assumed same as CIMM 2009:
"EC50 values, with a 95% confidence interval, were calculated from the experimental data using the Probit method (EPA) and ICp software v2.0 (EPA). The EC50 was calculated for “Nominal Al concentrations” using the software already mentioned, but in the case of “Dissolved Al” and “Monomeric Al” concentrations, the EC50 were determined by a simple proportionality calculation based in the Nominal Al concentration values closest to the EC50 or IC50. "

Not applicable

Conclusions:
The 72-h EC50 values for dissolved Al in Pseudokirchneriella subcapitata in OECD medium was 991.8 μg/L for inhibition of biomass and 1799 μg/L for inhibition of growth rate.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Principles of method if other than guideline:
/
GLP compliance:
no
Remarks:
The testing laboratory is operating according to GLP specifications but is not cerfied according to GLP.
Analytical monitoring:
yes
Details on sampling:
/
Vehicle:
not specified
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:static non-renewal
- Controls: One control
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM

- Age of inoculum (at test initiation): 4 days


Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
/
Hardness:
24,3-120 mg/L as CaCO3
Test temperature:
24°C
pH:
6,20-6,21 with hardness 24,3 mg/L
6,17-6,19 with hardness 60 mg/L
6,15-6,17 with hardness 120 mg/L
Dissolved oxygen:
/
Salinity:
/
Nominal and measured concentrations:
Hardness 24,3 mg/L Nominal concentrations: 25,50, 100, 200, 400 µg/L Measured concentrations: 32, 63, 129, 250, 504 µg/L Hardness 60 mg/L Nominal concentrations: 50, 100, 200, 400, 800 µg/L Measured concentrations: 63, 119, 237, 485, 982 µg/L Hardness 120 mg/L Nominal concentrations: 100, 200, 400, 800, 1600 µg/L Measured concentrations: 122, 244, 492, 956, 1945 µg/L
Details on test conditions:
1. Test type: Static non-renewal
2. Test duration: 72 h
3. Temperature: 24 °C ± 1°C
4. Light quality: Uniform fluorescent cool daylight type
5. Light Intensity: 86 ± 8.6 µE/m2/s
6. Photoperiod: Continuous illumination
7. Test chamber size: 250 ml
8. Test solution volume: 100 ml
9. Renewal of test solutions: None
10. Age of test organisms: 4 days
11. Initial cell density 10.000 cells/ml
12. No. replicate chambers per conc.: 3
13. Aeration None
14. Dilution water: OECD medium
15. Test acceptability criterion: Control cultures should have increased by a factor of at least 16 within the test period.
Coefficient of variation daily growth in the control cultures during the course of the test must not exceed 35%.
Coefficient of variation of average growth in replicate control cultures must not exceed 7%.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.153 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0,098-0,239, pH6, Hardness: 24,3 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.203 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.126-0.326, pH6, Hardness: 60 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.403 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.197-0.823, pH6, Hardness: 120 mg/L
Details on results:
/
Results with reference substance (positive control):
/
Reported statistics and error estimates:
/
Validity criteria fulfilled:
yes
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Principles of method if other than guideline:
No reference to a standard guideline with limited information in the publication
GLP compliance:
not specified
Specific details on test material used for the study:
Titrisol standard solutions of AlCl3 (1000 mg Al L1) (Merck, Germany).
Analytical monitoring:
yes
Details on sampling:
no details provided
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Raphidocelis subcapitata was cultured in half-strength CHU-10 medium (Nalewajko and O’Mahony, 1989) at 25 ± 2 C in a 12 h/12 h of light (y 3900 lux, fluorescent light)/dark photoperiod. Culture medium was autoclaved (121 C, 1 atm above standard pressure, 20 min) prior to its use. The cultures in exponential growth were inoculated at 5 104 cells mL1 in 500 mL polycarbonate flasks filled with 250 mL of test solutions.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Nominal and measured concentrations:
control, 14.82, 22.24, 29.65 and 37.06 mM Al
Duration:
96 h
Dose descriptor:
IC50
Effect conc.:
0.739 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.4 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
0.6 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
growth rate
Reported statistics and error estimates:
For statistically significant differences and NOEC (no observed effect concentration) and LOEC (lowest observed effect concentration) values, normal data were tested using one-way analysis of variance (ANOVA) and Dunnett’s post-hoc test, while non-normal data were compared with Kruskal-Wallis test and Dunn’s post hoc test. The normality of residuals was assessed using R version 3.4.1 (R Development Core Team, 2017) with Shapiro-Wilk test and using a half-normal graph of probabilities with a simulation envelope (Moral et al., 2018).. The IC50 values were calculated using specific growth rates, by non-linear regression, with a sigmoidal three parameter logistic curve.
Conclusions:
No information on test method and test conditions; this may be important as a standard solutions was used for preparing test solutions, and pH of this stock may affect the test outcome.
Reported EC50 is below the provided detection limit/limis of quantitation
Executive summary:

A large number of metals is present in aquatic ecosystems, often occurring simultaneously, however, the isolated toxicity of them are better well known than their mixtures. Based on that, for the first time we aimed to test the effects of zinc (Zn) and aluminum (Al) mixtures to the microalgae Raphidocelis sub- capitata. Regarding isolated toxicity, the 96 h IC50 of Zn and Al based on specific growth rates occurred, respectively, at 0.40 and 27.40 mM, thus Zn was z70-fold more toxic than Al. Both Zn and Al altered the cell size and complexity of R. subcapitata at the highest concentrations, although only during Zn exposure was the chlorophyll a fluorescence significantly diminished. Microalgae exposed to Al produced more ROS than during Zn exposure. Moreover, algae produced less ROS at the highest Zn concentration than in the lower concentrations. According to species sensitivity curves (SSD), R. subcapitata was the most sensitive organism to Zn and one of the most sensitive to Al. With respect to mixture toxicity tests, there were significant deviations for both CA (concentration addition) and IA (independent action) models, although data best fitted the CA model and DL (dose level-dependence) deviation, in which metals showed synergic effects at low concentrations and antagonist effects at higher concentrations.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
Dissolved (<0.45 􏰁m) aluminium was defined as that which passed through a 0.45 􏰁m filter and included colloidal as well as the low molecular weight aluminium complexes. The term total aluminium refers to aluminium in all forms (from truly soluble species through to precipitated forms.
Sub-samples were preserved with 5% and 2% HNO3 v/v (Tracepur, Merck) for total and dissolved (<0.45 􏰁m and <0.025 􏰁m filtered) aluminium, respectively.
Details on test solutions:
Algal culture media and aluminium bioassay solutions were prepared with natural seawater collected from a rock platform at Cronulla, NSW, Australia.
Aluminium stock solutions (1 and 10 g Al/L) were prepared on the day of toxicity test initiation by the addition of aluminium chloride to sodium hydroxide solution, to counteract the pH decrease in spiked test solutions caused by aluminium hydrolysis.
Test organisms (species):
other: Ceratoneis closterium (formerly Nitzschia closterium)
Details on test organisms:
Algae were obtained from the CSIRO Collection of Living Microalgae, Marine and Atmospheric Research, Hobart.
C. closterium (formerly Nitzschia closterium (Ehrenb.) W. Smith (strain CS-5, temperate)) was cultured in natural seawater medium with the trace metal concentrations halved.
Cultures were maintained in a temperature-controlled room at 21 ◦ C on a 12:12h light:dark cycle (Sylvania F20W/154-RS daylight fluo- rescent tubes; 70 􏰁mol photons/m2 /s) and transferred into fresh media weekly.
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
72 h
pH:
8.2 +/- 0.05
Nominal and measured concentrations:
range: 10-10000µg/L
Details on test conditions:
Bioassays were carried out in acid-washed 250 mL borosilicate glass Erlenmeyer flasks, coated with a silanising solution to reduce adsorption of aluminium to the flask walls. For each bioassay, five different aluminium treatments (10–10,000 􏰁g Al/L) and a control were prepared in triplicate. The test volume was increased from the standard 50 mL to 100 mL per flask to provide sufficient volume of test solution to sample for metal size-fractionation at multiple time points

Preliminary experiments confirmed no effect of volume change on population growth rate or sensitivity
Cells in the exponential growth phase (5–6 days old) were washed 3 times with 0.45 􏰁m filtered seawater by centrifuging to remove residual culture media.
Flasks were inoculated with 2–4 × 103 cells/mL, covered with transparent lids, placed randomly in a growth cabinet and incubated for 72 h at 21 ◦ C on a 12:12 h light/dark cycle (Phillips TLD 30W/33; 100–120 􏰁mol photons/m2/s).
Test flasks were shaken manually and randomly repositioned twice daily.
Reference toxicity tests using copper (CuSO4 ·5H2 0, AR Grade, Ajax Finechem, Australia) were conducted with each test.
Each bioassay was repeated on at least three occasions.

Duration:
72 h
Dose descriptor:
IC10
Effect conc.:
80 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
element (total fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95%CL: 55-100 µg Al/L
Duration:
72 h
Dose descriptor:
IC50
Effect conc.:
807 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
element (total fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95%CL: 780-810 µg Al/L
Duration:
72 h
Dose descriptor:
IC10
Effect conc.:
69 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95%CL: 44-89 µg Al/L
Duration:
72 h
Dose descriptor:
IC50
Effect conc.:
680 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95%CL: 570-710 µg Al/L
Reported statistics and error estimates:
The IC10 and IC50 values (i.e. the aluminium concentration to reduce growth rate by 10% and 50%, respectively, after 72h compared to the control) were calculated using linear interpo- lation of pooled data from individual bioassays (ToxCalc Version 5.0.23C, Tidepool Scientific Software, McKinleyville, CA, USA). The data were tested for normality using Kolmogorov D Test (p > 0.01) and equal variances using Bartlett’s test (p > 0.01). Effect concen- trations were based on measured dissolved (<0.45 􏰁m) and total aluminium concentrations at the time of test initiation and com- pared to effect concentrations based on time averaged measured dissolved (<0.45􏰁m) and total aluminium. Time-averaged alu- minium concentrations were calculated by averaging aluminium concentrations measured at 24-h intervals over the bioassay (0, 24, 48 and 72 h), assuming linear change between each time point, according to Angel et al. (2015).
Executive summary:

Localised aluminium contamination can lead to high concentrations in coastal waters, which have the potential for adverse effects on aquatic organisms. This research investigated the toxicity of 72-h exposures of aluminium to three marine diatoms (Ceratoneis closterium (formerly Nitzschia closterium), Minutocellus polymorphus and Phaeodactylum tricornutum) by measuring population growth rate inhibition and cell membrane damage (SYTOX Green) as endpoints. Toxicity was correlated to the time-averaged concentrations of different aluminium size-fractions, operationally defined as <0.025 􏰁m filtered, <0.45 􏰁m filtered (dissolved) and unfiltered (total) present in solution over the 72-h bioassay. The chronic population growth rate inhibition after aluminium exposure varied between diatom species. C. closterium was the most sensitive species (10% inhibition of growth rate (72-h IC10) of 80 (55–100) 􏰁g Al/L (95% confidence limits)) while M. polymorphus (540 (460–600) 􏰁g Al/L) and P. tricornutum (2100 (2000–2200) 􏰁g Al/L) were less sensitive (based on measured total aluminium). Dissolved aluminium was the primary contributor to toxicity in C. closterium, while a combination of dissolved and precip- itated aluminium forms contributed to toxicity in M. polymorphus. In contrast, aluminium toxicity to the most tolerant diatom P. tricornutum was due predominantly to precipitated aluminium. Preliminary investigations revealed the sensitivity of C. closterium and M. polymorphus to aluminium was influenced by initial cell density with aluminium toxicity significantly (p < 0.05) increasing with initial cell density from 103 to 105 cells/mL. No effects on plasma membrane permeability were observed for any of the three diatoms suggesting that mechanisms of aluminium toxicity to diatoms do not involve compromis- ing the plasma membrane. These results indicate that marine diatoms have a broad range in sensitivity to aluminium with toxic mechanisms related to both dissolved and precipitated aluminium.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
Dissolved (<0.45 􏰁m) aluminium was defined as that which passed through a 0.45 􏰁m filter and included colloidal as well as the low molecular weight aluminium complexes. The term total aluminium refers to aluminium in all forms (from truly soluble species through to precipitated forms.
Sub-samples were preserved with 5% and 2% HNO3 v/v (Tracepur, Merck) for total and dissolved (<0.45 􏰁m and <0.025 􏰁m filtered) aluminium, respectively.
Details on test solutions:
Algal culture media and aluminium bioassay solutions were prepared with natural seawater collected from a rock platform at Cronulla, NSW, Australia.
Aluminium stock solutions (1 and 10 g Al/L) were prepared on the day of toxicity test initiation by the addition of aluminium chloride to sodium hydroxide solution, to counteract the pH decrease in spiked test solutions caused by aluminium hydrolysis.
Test organisms (species):
other: Minutocellus polymorphus
Details on test organisms:
Algae were obtained from the CSIRO Collection of Living Microalgae, Marine and Atmospheric Research, Hobart.
The algae M. polymorphus (Hargraves and Guillard) Hasle, Von Stosch and Syvertsen (strain CS-3) was cultured in f/2 growth medium (half strength f medium, Guillard and Ryther (1962)).
Cultures were maintained in a temperature-controlled room at 21 ◦ C on a 12:12h light:dark cycle (Sylvania F20W/154-RS daylight fluo- rescent tubes; 70 􏰁mol photons/m2 /s) and transferred into fresh media weekly.
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
72 h
pH:
8.2 +/- 0.05
Nominal and measured concentrations:
range: 10-10000µg/L
Details on test conditions:
Bioassays were carried out in acid-washed 250 mL borosilicate glass Erlenmeyer flasks, coated with a silanising solution to reduce adsorption of aluminium to the flask walls. For each bioassay, five different aluminium treatments (10–10,000 􏰁g Al/L) and a control were prepared in triplicate. The test volume was increased from the standard 50 mL to 100 mL per flask to provide sufficient volume of test solution to sample for metal size-fractionation at multiple time points

Preliminary experiments confirmed no effect of volume change on population growth rate or sensitivity
Cells in the exponential growth phase (5–6 days old) were washed 3 times with 0.45 􏰁m filtered seawater by centrifuging to remove residual culture media.
Flasks were inoculated with 2–4 × 103 cells/mL, covered with transparent lids, placed randomly in a growth cabinet and incubated for 72 h at 21 ◦ C on a 12:12 h light/dark cycle (Phillips TLD 30W/33; 100–120 􏰁mol photons/m2/s).
Test flasks were shaken manually and randomly repositioned twice daily.
Reference toxicity tests using copper (CuSO4 ·5H2 0, AR Grade, Ajax Finechem, Australia) were conducted with each test.
Each bioassay was repeated on at least three occasions.

Duration:
72 h
Dose descriptor:
IC10
Effect conc.:
540 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
element (total fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95%CL: 460-600 µg Al/L
Duration:
72 h
Dose descriptor:
IC50
Effect conc.:
1 370 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
element (total fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95%CL: 1300-1400 µg Al/L
Duration:
72 h
Dose descriptor:
IC10
Effect conc.:
440 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95%CL: 420-8470 µg Al/L
Duration:
72 h
Dose descriptor:
IC50
Effect conc.:
778.5 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95%CL: 778.2-778.8 µg Al/L
Reported statistics and error estimates:
The IC10 and IC50 values (i.e. the aluminium concentration to reduce growth rate by 10% and 50%, respectively, after 72h compared to the control) were calculated using linear interpo- lation of pooled data from individual bioassays (ToxCalc Version 5.0.23C, Tidepool Scientific Software, McKinleyville, CA, USA). The data were tested for normality using Kolmogorov D Test (p > 0.01) and equal variances using Bartlett’s test (p > 0.01). Effect concen- trations were based on measured dissolved (<0.45 􏰁m) and total aluminium concentrations at the time of test initiation and com- pared to effect concentrations based on time averaged measured dissolved (<0.45􏰁m) and total aluminium. Time-averaged alu- minium concentrations were calculated by averaging aluminium concentrations measured at 24-h intervals over the bioassay (0, 24, 48 and 72 h), assuming linear change between each time point, according to Angel et al. (2015).
Executive summary:

Localised aluminium contamination can lead to high concentrations in coastal waters, which have the potential for adverse effects on aquatic organisms. This research investigated the toxicity of 72-h exposures of aluminium to three marine diatoms (Ceratoneis closterium (formerly Nitzschia closterium), Minutocellus polymorphus and Phaeodactylum tricornutum) by measuring population growth rate inhibition and cell membrane damage (SYTOX Green) as endpoints. Toxicity was correlated to the time-averaged concentrations of different aluminium size-fractions, operationally defined as <0.025 􏰁m filtered, <0.45 􏰁m filtered (dissolved) and unfiltered (total) present in solution over the 72-h bioassay. The chronic population growth rate inhibition after aluminium exposure varied between diatom species. C. closterium was the most sensitive species (10% inhibition of growth rate (72-h IC10) of 80 (55–100) 􏰁g Al/L (95% confidence limits)) while M. polymorphus (540 (460–600) 􏰁g Al/L) and P. tricornutum (2100 (2000–2200) 􏰁g Al/L) were less sensitive (based on measured total aluminium). Dissolved aluminium was the primary contributor to toxicity in C. closterium, while a combination of dissolved and precip- itated aluminium forms contributed to toxicity in M. polymorphus. In contrast, aluminium toxicity to the most tolerant diatom P. tricornutum was due predominantly to precipitated aluminium. Preliminary investigations revealed the sensitivity of C. closterium and M. polymorphus to aluminium was influenced by initial cell density with aluminium toxicity significantly (p < 0.05) increasing with initial cell density from 103 to 105 cells/mL. No effects on plasma membrane permeability were observed for any of the three diatoms suggesting that mechanisms of aluminium toxicity to diatoms do not involve compromis- ing the plasma membrane. These results indicate that marine diatoms have a broad range in sensitivity to aluminium with toxic mechanisms related to both dissolved and precipitated aluminium.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
not specified
Specific details on test material used for the study:
Al2O3 (99% purity) micro particles were procured from Merck, and used without any further purification.
Analytical monitoring:
yes
Details on sampling:
Dissolved (<0.45 􏰁m) aluminium was defined as that which passed through a 0.45 􏰁m filter and included colloidal as well as the low molecular weight aluminium complexes. The term total aluminium refers to aluminium in all forms (from truly soluble species through to precipitated forms.
Sub-samples were preserved with 5% and 2% HNO3 v/v (Tracepur, Merck) for total and dissolved (<0.45 􏰁m and <0.025 􏰁m filtered) aluminium, respectively.
Details on test solutions:
Algal culture media and aluminium bioassay solutions were prepared with natural seawater collected from a rock platform at Cronulla, NSW, Australia.
Aluminium stock solutions (1 and 10 g Al/L) were prepared on the day of toxicity test initiation by the addition of aluminium chloride to sodium hydroxide solution, to counteract the pH decrease in spiked test solutions caused by aluminium hydrolysis.
Test organisms (species):
Phaeodactylum tricornutum
Details on test organisms:
Algae were obtained from the CSIRO Collection of Living Microalgae, Marine and Atmospheric Research, Hobart.
The algae P. tricornutum Bohlin (strain CS-29/4) ) was cultured in f/2 growth medium (half strength f medium, Guillard and Ryther (1962)).
Cultures were maintained in a temperature-controlled room at 21 ◦ C on a 12:12h light:dark cycle (Sylvania F20W/154-RS daylight fluo- rescent tubes; 70 􏰁mol photons/m2 /s) and transferred into fresh media weekly.
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
72 h
pH:
8.2 +/- 0.05
Nominal and measured concentrations:
range: 10-10000µg/L
Details on test conditions:
Bioassays were carried out in acid-washed 250 mL borosilicate glass Erlenmeyer flasks, coated with a silanising solution to reduce adsorption of aluminium to the flask walls. For each bioassay, five different aluminium treatments (10–10,000 􏰁g Al/L) and a control were prepared in triplicate. The test volume was increased from the standard 50 mL to 100 mL per flask to provide sufficient volume of test solution to sample for metal size-fractionation at multiple time points

Preliminary experiments confirmed no effect of volume change on population growth rate or sensitivity
Cells in the exponential growth phase (5–6 days old) were washed 3 times with 0.45 􏰁m filtered seawater by centrifuging to remove residual culture media.
Flasks were inoculated with 2–4 × 103 cells/mL, covered with transparent lids, placed randomly in a growth cabinet and incubated for 72 h at 21 ◦ C on a 12:12 h light/dark cycle (Phillips TLD 30W/33; 100–120 􏰁mol photons/m2/s).
Test flasks were shaken manually and randomly repositioned twice daily.
Reference toxicity tests using copper (CuSO4 ·5H2 0, AR Grade, Ajax Finechem, Australia) were conducted with each test.
Each bioassay was repeated on at least three occasions.

Duration:
72 h
Dose descriptor:
IC10
Effect conc.:
2 100 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
element (total fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95%CL: 2000-2200 µg Al/L
Duration:
72 h
Dose descriptor:
IC50
Effect conc.:
> 9 500 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
element (total fraction)
Basis for effect:
growth rate
Remarks on result:
other:
Duration:
72 h
Dose descriptor:
IC10
Effect conc.:
920 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95%CL: 910-940 µg Al/L
Duration:
72 h
Dose descriptor:
IC50
Effect conc.:
> 1 200 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other:
Reported statistics and error estimates:
The IC10 and IC50 values (i.e. the aluminium concentration to reduce growth rate by 10% and 50%, respectively, after 72h compared to the control) were calculated using linear interpo- lation of pooled data from individual bioassays (ToxCalc Version 5.0.23C, Tidepool Scientific Software, McKinleyville, CA, USA). The data were tested for normality using Kolmogorov D Test (p > 0.01) and equal variances using Bartlett’s test (p > 0.01). Effect concen- trations were based on measured dissolved (<0.45 􏰁m) and total aluminium concentrations at the time of test initiation and com- pared to effect concentrations based on time averaged measured dissolved (<0.45􏰁m) and total aluminium. Time-averaged alu- minium concentrations were calculated by averaging aluminium concentrations measured at 24-h intervals over the bioassay (0, 24, 48 and 72 h), assuming linear change between each time point, according to Angel et al. (2015).
Executive summary:

Localised aluminium contamination can lead to high concentrations in coastal waters, which have the potential for adverse effects on aquatic organisms. This research investigated the toxicity of 72-h exposures of aluminium to three marine diatoms (Ceratoneis closterium (formerly Nitzschia closterium), Minutocellus polymorphus and Phaeodactylum tricornutum) by measuring population growth rate inhibition and cell membrane damage (SYTOX Green) as endpoints. Toxicity was correlated to the time-averaged concentrations of different aluminium size-fractions, operationally defined as <0.025 􏰁m filtered, <0.45 􏰁m filtered (dissolved) and unfiltered (total) present in solution over the 72-h bioassay. The chronic population growth rate inhibition after aluminium exposure varied between diatom species. C. closterium was the most sensitive species (10% inhibition of growth rate (72-h IC10) of 80 (55–100) 􏰁g Al/L (95% confidence limits)) while M. polymorphus (540 (460–600) 􏰁g Al/L) and P. tricornutum (2100 (2000–2200) 􏰁g Al/L) were less sensitive (based on measured total aluminium). Dissolved aluminium was the primary contributor to toxicity in C. closterium, while a combination of dissolved and precip- itated aluminium forms contributed to toxicity in M. polymorphus. In contrast, aluminium toxicity to the most tolerant diatom P. tricornutum was due predominantly to precipitated aluminium. Preliminary investigations revealed the sensitivity of C. closterium and M. polymorphus to aluminium was influenced by initial cell density with aluminium toxicity significantly (p < 0.05) increasing with initial cell density from 103 to 105 cells/mL. No effects on plasma membrane permeability were observed for any of the three diatoms suggesting that mechanisms of aluminium toxicity to diatoms do not involve compromis- ing the plasma membrane. These results indicate that marine diatoms have a broad range in sensitivity to aluminium with toxic mechanisms related to both dissolved and precipitated aluminium.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
January - February 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
growth medium had reduction in Fe-EDTA concentrations
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Name of test material: Aluminium hydroxide
- Analytical purity: 99.8%
- Impurities: Na2O, Fe2O3, CaO
- Storage condition of test material: room temperature
Analytical monitoring:
yes
Details on sampling:
- Concentrations: test concentration and control
- Sampling method: concentration of aluminium in solution was recorded during the equilibrium after 1,2,3,4,7 days and at the start and end of the toxicity test
- Sample storage conditions before analysis: samples were preserved by addition of 1 mL 6M HNO3 per 100 mL sample
Vehicle:
not specified
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 100 mg aluminium hydroxide was added to 1000 mL of algal growth medium in a 2 L erlenmeyer flask which was placed in the dark on a reciprocal shaker.
- Eluate: after 9 days, the soluble fraction was siphoned out and used in the toxicity test
- All other template details: Not reported
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: NIVA strain CHL1
- Source: NIVA laboratory
- Age of inoculum (at test initiation): 1-3 days
- Method of cultivation: cultured in 10% Z8 medium in reciprocal shaker and continuous light at 20 degrees C

ACCLIMATION: Not reported
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
Not reported
Hardness:
Not reported
Test temperature:
21 ± 1 degree C
pH:
7.8
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal (mg/L): 0, 100
Measured dissolved Al (µg/L): 3 at start and 5 at end, mean = 4
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL flat bottom round glass flask
- Type: closed with a perforated plastic cup
- Material, size, headspace, fill volume: 100 mL flat bottom round glass flasks filled with 50 mL of test medium
- Aeration: none
- Initial cells density: 5000 cells/mL
- Control end cells density: not reported
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: yes, modified with reduced iron chloride and Na2EDTA
- Detailed composition if non-standard medium was used: both iron chloride and sodium EDTA were reduced 30%. See Table 1 for detailed composition.

TEST MEDIUM / WATER PARAMETERS: distilled water
- Culture medium different from test medium: 10% Z8 medium in stock culture
- Intervals of water quality measurement: pH was measured at the start and after 72 hours
- All other template details: Not reported

OTHER TEST CONDITIONS
- Sterile test conditions: not reported
- Adjustment of pH: growth medium was not pH adjusted after equilibrium with aluminium hydroxide
- Photoperiod: continuous
- Light intensity and quality: approximately 80 µE/m2-s
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: coulter counter after 24, 48, 72 hours
- Chlorophyll measurement: not reported
- Other: not reported

TEST CONCENTRATIONS
- Spacing factor for test concentrations: only one test concentration for limit test
Reference substance (positive control):
not specified
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 4 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
growth rate
Details on results:
- There were no significant decreases in growth rate or area under the growth curve in the 100 mg/L concentration.
- All other template details: Not reported
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Not reported

Not applicable

Conclusions:
There was no significant reduction in growth rate in the 100 mg/L limit test with S. capricornutum. The dissolved aluminium concentrations at the start of the test was 5 µg/L and 3 µg/L at the end of the test, with an average of 4 µg/L.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
January - February 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
not specified
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Name of test material: Aluminium oxide
- Analytical purity: 99.3%
- Impurities: Fe2O3, Na2O, CaO
- Expiration date of the lot/batch: none
- Storage condition of test material: room temperature
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all concentrations and control
- Sampling method: concentration of aluminium in solution was recorded during the equilibrium after 1,2,3,4,7 days and at the start and end of the toxicity test
- Sample storage conditions before analysis: samples were preserved by addition of 1 mL 6M HNO3 per 100 mL sample
Vehicle:
not specified
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: aluminium oxide was added to 1000 mL of algal growth medium in a 2 L erlenmeyer flask which was placed in the dark on a reciprocal shaker.
- Eluate: after 7-9 days, the soluble fraction was siphoned out and used in the toxicity test
- All other template details: Not reported
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: NIVA strain CHL1
- Source: NIVA laboratory
- Age of inoculum (at test initiation): 1-3 days
- Method of cultivation: cultured in 10% Z8 medium in reciprocal shaker and continuous light at 20 degrees C

ACCLIMATION: Not reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not reported
Hardness:
Not reported
Test temperature:
21 ± 1 degree C
pH:
7.8 ± 0.2
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal (mg/L): 0, 0.05, 0.5, 5.0, 20, 100
Measured dissolved Al at start of test (µg/L): 4, 3, 6, 8, 25, 52
Measured dissolved Al at end of test (µg/L): 3, 3, 7, 4, 8, 34
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL flat bottom round glass flasks
- Type: closed with a perforated plastic cup
- Material, size, headspace, fill volume: 100 mL flat bottom round glass flasks filled with 50 mL of test medium
- Aeration: none
- Initial cells density: 5000 cells/mL
- Control end cells density: 3.73 x 10^8 - 6.87 x 10^8 cells/L
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: yes, modified with reduced iron chloride and Na2EDTA
- Detailed composition if non-standard medium was used: both iron chloride and sodium EDTA were reduced 30%. See Table 1 for detailed composition.

TEST MEDIUM / WATER PARAMETERS: distilled water
- Culture medium different from test medium: 10% Z8 medium in stock culture
- Intervals of water quality measurement: pH was measured at the start and after 72 hours
- All other template details: Not reported

OTHER TEST CONDITIONS
- Sterile test conditions: not reported
- Adjustment of pH: growth medium was not pH adjusted after equilibrium with aluminium oxide
- Photoperiod: continuous
- Light intensity and quality: approximately 80 µE/m2-s
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: coulter counter after 24, 48, 72 hours
- Chlorophyll measurement: not reported
- Other: not reported

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10x
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study
- Test concentrations: 100 mg/L limit test
- Results used to determine the conditions for the definitive study: reduced cell counts with 100 mg/L compared to controls
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 52 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
growth rate
Details on results:
- Other: the limit test with 100 mg/L aluminium showed an 86% growth rate inhibition compared to controls at 72 hours, and a 57% area under the growth curve reduction. However, the definitive test with 100 mg/L only showed a 93% and 73% decrease for growth rate and area under the growth curve, respectively. These were not significantly different than the control response.
- All other template details: not reported
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
t-statistics were used to check for significance of the endpoints.

Not applicable

Conclusions:
The reduction in green algae Selenastrum capricornutum growth rate that was observed in the limit test with 100 mg/L aluminium oxide could not be reproduced in a definitive test with 5 concentrations. The unbounded NOEC was 52 µg/L for growth inhibition compared to controls.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
January - February 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
growth medium had reduction in Fe-EDTA concentrations
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Name of test material: Aluminium metal
- Analytical purity: 97.7%
- Impurities: Fe, Si
- Storage condition of test material: room temperature
Analytical monitoring:
yes
Details on sampling:
- Concentrations: test concentration and control
- Sampling method: concentration of aluminium in solution was recorded during the equilibrium after 1,2,3,4,7 days and at the start and end of the toxicity test
- Sample storage conditions before analysis: samples were preserved by addition of 1 mL 6M HNO3 per 100 mL sample
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 100 mg aluminium was added to 1000 mL of algal growth medium in a 2 L erlenmeyer flask which was placed in the dark on a reciprocal shaker.
- Eluate: after 9 days, the soluble fraction was siphoned out and used in the toxicity test
- All other template details: Not reported
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: NIVA strain CHL1
- Source: NIVA laboratory
- Age of inoculum (at test initiation): 1-3 days
- Method of cultivation: cultured in 10% Z8 medium in reciprocal shaker and continuous light at 20 degrees C

ACCLIMATION: Not reported
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
Not reported
Hardness:
Not reported
Test temperature:
21 ± 1 degree C
pH:
7.8
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal (mg/L): 100
Measured dissolved Al start of test (µg/L): 53
Measured dissolved Al end of test (µg/L): 34
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL flat bottom round glass flask
- Type: closed with a perforated plastic cup
- Material, size, headspace, fill volume: 100 mL flat bottom round glass flasks filled with 50 mL of test medium
- Aeration: none
- Initial cells density: 5000 cells/mL
- Control end cells density: not reported
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: yes, modified with reduced iron chloride and Na2EDTA
- Detailed composition if non-standard medium was used: both iron chloride and sodium EDTA were reduced 30%. See Table 1 for detailed composition.

TEST MEDIUM / WATER PARAMETERS: distilled water
- Culture medium different from test medium: 10% Z8 medium in stock culture
- Intervals of water quality measurement: pH was measured at the start and after 72 hours
- All other template details: Not reported

OTHER TEST CONDITIONS
- Sterile test conditions: not reported
- Adjustment of pH: growth medium was not pH adjusted after equilibrium with aluminium
- Photoperiod: continuous
- Light intensity and quality: approximately 80 µE/m2-s
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: coulter counter after 24, 48, 72 hours
- Chlorophyll measurement: not reported
- Other: not reported

TEST CONCENTRATIONS
- Spacing factor for test concentrations: only one test concentration for limit test
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 44 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
growth rate
Details on results:
- There were no significant decreases in growth rate or area under the growth curve in the 100 mg/L concentration.
- All other template details: Not reported
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Not reported

Not applicable

Conclusions:
There was no significant reduction in growth rate in the 100 mg/L limit test with S. capricornutum. The dissolved aluminium concentrations at the start of the test was 53 µg/L and 34 µg/L at the end of the test, with an average of 44 µg/L.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
February 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
not specified
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material: Aluminium powder
- Physical state: powder
- Analytical purity: 99.7 %
- Impurities: Fe 0.054%, Si 0.021%, Cu 0.001%, Mn 0.001%, Pb <0.001%, Mg 0.001%, Zn 0.003%. Ni 0.005%. Ti 0.004%, Zr 0.001%, Cr 0.001%, Ga 0.013%
- Source: ALPOCO, The Aluminium Powder Co.Ltd.
- Storage condition of test material: ambient in dark at room temperature
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all exposure and control test solutions
- Sampling method: unfiltered sample were taken for analysis of aluminium at the end of the test by atomic absorption
- Sample storage conditions before analysis: not reported
Vehicle:
not specified
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: test solutions were prepared by mixing the nutrient spiked T/D medium, test medium, and inoculum culture
- All other template details: Not reported
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: NIVA strain CHL1
- Source: NIVA culture collection
- All other template details: Not reported

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions: cultured in 10% Z8 medium in reciprocal shaker and continuous light at 20 degrees C
- Any deformed or abnormal cells observed: Not reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not reported
Hardness:
Not reported
Test temperature:
20.3 -20.7 degrees C
pH:
measured: 8.0 - 8.1
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal total (µg/L): 6 (control), 88, 158, 280, 490, 875
Measured dissolved (µg/L): 4, 8, 71, 140, 320, 250, 900
Details on test conditions:
TEST SYSTEM
- Test vessel: flat bottom flasks
- Material, size, headspace, fill volume: 100 mL flat bottom flasks filled with 50 mL of culture solution and covered with inverted, perforated plastic beakers
- Initial cells density: 5,000 cells/mL
- Control end cells density: 102% increase
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- All other template details: not reported

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: the test material was the aqueous solution from a 28 day transformation/dissolution test (T/D-test) of aluminium powder at pH 8.5, which was carried out according to OECD 1998 - transformation/dissolution of metals and sparingly soluble metal compounds in aqueous media
- All other template details: Not reported

OTHER TEST CONDITIONS
- Sterile test conditions: not reported
- Adjustment of pH: not reported
- Photoperiod: constant illumination
- Light intensity and quality: 74 µE m-2 s-1 from daylight-type fluorescent tubes suspended 0.6 meters above culture vessels

EFFECT PARAMETERS MEASURED: cell counts at 24, 48, and 72 hours
- Determination of cell concentrations: electronic particle counter, Coulter Multisizer electronic particle counter
- Area under growth curve and growth rate: calculated according to equations presented in report
- Other: Not reported

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2 times
- Range finding study: Not reported
Reference substance (positive control):
not specified
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1 050 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 721 - 1520 µg Al/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
160 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 130 - 210 µg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
280 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
200 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
other: area under growth curve
Remarks on result:
other: 95% CL: 160 - 250 µg Al/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
51 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
other: area under growth curve
Remarks on result:
other: 95% CL: 31 - 82 µg Al/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
158 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
other: area under growth curve
Details on results:
- Exponential growth in the control: yes, close to exponential
- All other template details: Not reported
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
The EC value estimations for growth rate and area under growth curve endpoints were analyzed by a linear regression of probit transformed percentage of inhibition compared to controls. NOEC was determined using Dunnet's test to compare the test endpoints in each treated group with controls (α = 0.05).

Table 2. Calculated endpoints in the algal growth inhibition test (mean values of replicates).

 

Concentration (% of T/D solution)

Concentration of Al (µg/L)

Growth rate (µ) d-1

% of control

Area under growth curve

% of control

Control

6

1.52

100

9725

100

10

88

1.46

96

8129

84

18

158

1.38

91

6010

62

32

280

1.31

86

4553

47

56

490

1.05

69

1862

19

100

875

0.77

51

737

7.6

 

Conclusions:
The 72 hour EC50 for inhibition of growth rate of Selenastrum capricornutum exposed to Al in solution at pH 8 was calculated to be 1050 µg Al/L, with a NOEC of 280 µg Al/L.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Summary of available data used for the endpoint assessment of the target substance
Adequacy of study:
weight of evidence
Justification for type of information:
Refer to analogue justification provided in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
>= 802.1 - <= 4 561 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
other: growth
Remarks on result:
other:
Remarks:
Range is based on normalised data from BLM; lower limit presents Ecoregion #7; upper limit presents Ecoregion #2

Description of key information

Information on the ecotoxicity of aluminium towards algae is available for the green alga P. subcapitata (other names: R. subcapitata, S. capricornutum). Growth rate and biomass growth were the main parameters that were assessed, and reported endpoints were EC50 (short-term endpoint), EC10 and NOEC (long-term endpoint). Most tests were conducted with soluble Al-salts; the Norwegian Institute for Water Research (NIVA), however, conduced and reported a number of algal tests where aluminium substances with low solubility were used as test material. The various studies expressed the observed effects as dissolved aluminum and/or as total/nominal values, depending on the sample treatment and depending whether the exposure concentrations were measured.

 

All available studies data were used in a weight of evidence approach to cover this endpoint. It should be noted that that the majority of studies that investigated the effects of aluminum in the aquatic environment used test solutions with aluminum concentrations above that of its solubility limit. Results of these studies therefore have limited value for the investigation of intrinsic toxicity.

Key value for chemical safety assessment

Additional information

The algal data from the 2009 and 2010 CIMM datasets demonstrate that elevated pH and elevated DOC are protective against aluminium toxicity, whereas hardness appeared to have a minimal effect.  The evidence of both pH and DOC effects are consistent with the Al BLM (attached to IUCLID section 13). Multiple linear regression models (MLRM) based on nominal DOC, and pH were developed to predict nominal EC10 and EC50 values for the algae dataset. The EC50 and EC10 MLRMs performed reasonably well for the dataset. The EC50 MLRM produced an adjusted R2 of 0.747, and the EC10 MLRM produce an adjusted R2 of 0.987.

 

Several chronic toxicity studies to a freshwater microalga (P. subcapitata) were identified in the literature as Klimisch 1 or 2 studies. Additional algal studies with P. subcapitata were performed at CIMM to evaluate acute and chronic toxicity to algae and for evaluation of water chemistry effects for modelling purposes. All endpoints from CIMM (2009; 2010a) were reported on the basis of nominal Al concentrations because total Al was not measured in these studies. However, CIMM (2010b) compared nominal to measured total Al concentrations in an identical set of algal test solutions prepared to match all water quality conditions and nominal Al exposure concentrations as used in the previous studies (2009; 2010a). In these new test solutions, average total Al concentrations were within 10% of nominal Al concentrations. A linear regression between total and nominal Al concentrations demonstrated a strong relationship with an R2 value of 0.99. Therefore, nominal Al concentrations can be considered a reliable estimator of total Al concentrations in these studies. ECr10s were calculated using raw data provided from each study using the statistical program Toxicity Relationship Analysis Program (TRAP) version 1.10 from the US EPA National Health an Environmental Effects Research Laboratory (NHEERL). All other endpoints were as reported in each study. ECr10s and ECr50s ranged from 0.051 to 3.15 mg Al/L and 0.024 to 4.93 mg Al/L, respectively. Water quality data for these studies suggest a direct relationship between toxicity and pH, hardness, and DOC. Studies that experimentally manipulated water quality were reported by CIMM 2009 and 2010a.

 

One reliable toxicity study to a higher plant (L. minor) is included, but no adverse effects were noted in this study. As a consequence, both NOEC and EC10 values were > 45.7 mg Al/L.

For a detailed overview of the data, more information is provided in the Background document "Environmental Effects Assessment of Aluminium" attached to IUCLID section 13.