Aluminium oxide

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Seven‐day chronic toxicity tests were conducted with the cladoceran C, dubia, one of the taxa that provided the basis for the MLR model for Al toxicity (Deforest et al. 2018). Tests followed ASTM International (2015) and US Environmental Protection Agency (2002) standard methods unless otherwise noted.

GLP compliance:

not specified

Specific details on test material used for the study:

it is unclear whether the hexahydrate (CAS 7784-13-6) or the anhydrous form (CAS 7446-70-0) of aluminium chloride was used

Analytical monitoring:

yes

Details on sampling:

Samples for analyses of total Al,filtered Al, and major cations (sodium, magnesium, potassium, calcium, strontium, iron, and manganese) were preserved by addition of nitric acid (in‐house sub‐boiling distilled from BDH Aristar ACS; VWR International) to a final acid content of 2% (v/v).

Details on test solutions:

Modified field-collected waters (Beaver Brook, near Royalston MA, USA) was used as test medium.
Test solutions prepared 1 d before the start of the test were used for water replacements for the entire test, based on stability studies demonstrating that measured total‐Al concentrations remained stable over a 7‐d period. Test solutions were prepared in glass jars and stored in a walk‐in cooler at 4 °C between uses. The highest Al concentration for each test was prepared by dissolving Al chloride hexahydrate salt (reagent grade; Sigma‐Aldrich) directly into each test water, adjusting the pH as needed, and preparing 3 additional Al concentrations by 0.5× serial dilutions of the high‐Al test water with unspiked, pH‐adjusted test water. The volumes of test solutions prepared on day 0 were enough to conduct an entire 7‐d test.

Test organisms (species):

Ceriodaphnia dubia

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Hardness:

Test 1: 20 mg/L as CaCO3
Test 2: 22 mg/L as CaCO3
Test 3: 21 mg/L as CaCO3
Test 4: 20 mg/L as CaCO3
Test 5: 23 mg/L as CaCO3
Test 6: 28 mg/L as CaCO3
Test 7: 36 mg/L as CaCO3
Test 8: 35 mg/L as CaCO3
Test 9: 36 mg/L as CaCO3
Test 10: 37 mg/L as CaCO3
Test 11: 35 mg/L as CaCO3

Test temperature:

25°C

pH:

Test 1: 5.98
Test 2: 5.60
Test 3: 6.19
Test 4: 6.20
Test 5: 6.20
Test 6: 6.23
Test 7: 6.46
Test 8: 6.40
Test 9: 6.48
Test 10: 6.53
Test 11: 6.44

Details on test conditions:

Tests were started with<24‐h‐old neonates (parthenogenic females), which were cultured at a hardness of approximately 20 mg/L for approximately 8 wk before the start of the tests.
On each subsequent day of the test, the unused portions of Al‐spiked test waters were used tofill additional sets of 10 cups for each treatment for equilibration, and each test organism was transferred into a cup with freshly equilibrated test water.
Exposure cups with test water were equilibrated in a temperature‐controlled (25 °C) incubator for 24 h before animals were stocked and fed to start the test.
Each test consisted of 5 Al concentrations in a 0.5× dilution series of one of the test waters, plus a control without added Al.
One neonate was stocked in each of 10 replicate 30‐mL exposure cups/treatment, with 20 mL of test water/cup.
The organisms in each cup were fed daily with 0.1‐mL portions of 2 diet suspensions: 1) yeast–cerophyll–trout chow (1800 mg/L stock solution), and 2) algae (Pseudokirchneriella subcapitata, 3.0 × 107cell/mL; Aquatic Bio Systems).
Tests were ended after 7 or 8 d, with the end date determined by when at least 60% of stocked adults in the control group had produced at least 3 broods averaging 15 or more young/female.

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

20 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Basis for effect:

immobilisation

Remarks on result:

other: Test 1

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

2.7 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Basis for effect:

immobilisation

Remarks on result:

other: Test 2

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

18 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Basis for effect:

immobilisation

Remarks on result:

other: Test 3

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

43 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Basis for effect:

immobilisation

Remarks on result:

other: Test 4

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 6.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Basis for effect:

immobilisation

Remarks on result:

other: Test 5

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

1.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Basis for effect:

immobilisation

Remarks on result:

other: Test 6

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

76 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Basis for effect:

immobilisation

Remarks on result:

other: Test 7

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 12 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Basis for effect:

immobilisation

Remarks on result:

other: Test 8

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 5.6 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Basis for effect:

immobilisation

Remarks on result:

other: Test 9

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 2.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Basis for effect:

immobilisation

Remarks on result:

other: Test 10

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 2.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Basis for effect:

immobilisation

Remarks on result:

other: Test 11

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

3.6 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: Test 3

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.87 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: Test 4

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

2.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: Test 5

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.58 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: Test 6

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

14 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: Test 7

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

4.6 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: Test 8

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

3.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: Test 9

Details on results:

EC20- values for reproduction were also reported in the publication, as well as CL ranges for all reported effet levels

Reported statistics and error estimates:

Chronic effect concentrations for effects of total Al on reproduction (e.g., 20% effect concentration [EC20]) were determined using the Toxicity Relationship Analysis Program (TRAP), Ver 1.30a (Erickson 2010). Median effect concentrations (EC50s) for lethality or immobilization of test organisms were estimated using trimmed Spearman–Karber or probit procedures (Sebaugh 1998).

Executive summary:

We conducted a series of 7‐d toxicity tests with Ceriodaphnia dubia in dilutions of low‐hardness natural waters, which contained dissolved organic carbon (DOC) concentrations up to 10 mg/L. Stream waters were mixed with well water to achieve 2 target hardness levels (20 and 35 mg/L) and 4 DOC concentrations. Tests with aluminum (Al)‐spiked waters were conducted in a controlled CO2 atmosphere to maintain the pH at a range of 6.0 to 6.5. The results were used to estimate effect concentrations for survival and reproduction, expressed as total (unfiltered) Al concentrations. There were small differences in total‐Al thresholds between waters with 20 and 35 mg/L hardness, but effect concentrations for C. dubia survival (median lethal concentrations) and reproduction (effect concentrations, 20%) increased log‐linearly with increasing DOC concentrations in the range, 0.3 to 6 mg/L. Slopes of these regressions were similar to slopes from data used to revise the US Environmental Protection Agency water quality criterion for Al, but toxic effects in the present study occurred at total‐ Al concentrations 8‐ to 10‐fold greater than toxicity values used for criteria development. This difference probably reflects the long equilibration (aging) times of Al test waters used in the present study (up to 192 h) compared with short (3‐h) equilibration times in other studies used for criteria development. These results confirm the importance of DOC as a control on Al toxicity in low‐hardness waters, but they also demonstrate that total‐Al concentrations are not predictive of Al toxicity, except under defined water quality (pH, hardness, DOC) and exposure conditions (e.g., aging of test waters). Environ Toxicol Chem 2019;38:2121–2127. Published 2019 Wiley Periodicals, Inc. on behalf of SETAC. This article is a US government work, and as such, is in the public domain in the United States of America.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

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:

no guideline followed

Principles of method if other than guideline:

Daphnids were individually exposed to 15 mL of renewed solution of AlCl3 at mean pH of 7.58 and fed for 7 days. Endpoints were mortality and reproduction.

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 exposures including control
- Sampling method: 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: Exposure and control tanks were prepared a minimum of 48 hr in advance of organism introduction to the tanks. The water volume was measured, then added to each exposure tank, and aluminum introduced from a 50,000 mg/l stock solution. Daily adjustments of pH were necessary in many of the exposures. Tests run at pH values above neutrality declined in pH in proportion to the total aluminum present. Controls dropped the least or not at all. Tests run at a pH below neutrality (6.5 nominal) rose in pH in all test chambers.
- 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):

Ceriodaphnia sp.

Details on test organisms:

TEST ORGANISM
- Common name: Cladoceran
- Age: < 16 hrs old at start of test
- Feeding during test
- Food type: Food consisted of a mixture of yeast, Cerophyll, and fermented trout chow
- Amount: 2 drops per beaker
- Frequency: every 2 days when solutions were renewed

ACCLIMATION: Not reported

QUARANTINE: Not applicable

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Not reported

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

8 d

Post exposure observation period:

Not reported

Hardness:

47.4 mg/L as CaCO3

Test temperature:

20-22 degrees C (room temperature)

pH:

mean measured pH was 7.58 ± 0.07

Dissolved oxygen:

mean measured DO level was 6.92 ± 0.57 mg/l

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal Al (mg/L): 0, 1.56, 3.12, 6.25, 12.5, 25.0
Measured Al (mg/L): <0.3, 1.0, 3.0, 4.9, 12.1, 23.7

Details on test conditions:

TEST SYSTEM
- Test vessel: beaker
- Material, size, headspace, fill volume: 30 mL beakers with 15 mL of water
- Aeration: not aerated
- Renewal rate of test solution: solutions were renewed on days 2, 4, and 6
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): not applicable
- All other template details: Not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Lake Superior water collected from a tap on the City of Cloquet, MN raw water intake in the City of Superior, WI. The source of the water was from approximately 3200 meters offshore on the lake bottom.
- Total solids: 61 ± 5 mg/L
- Particulate matter (total suspended solids): <1.0 mg/L
- Chloride: 1.5 ± 0.42 mg/L
- Alkalinity: 41.6 ± 1.27 mg/L as CaCO3
- Culture medium different from test medium: no
- Intervals of water quality measurement: measured on each batch of water used
- All other template details: Not reported

OTHER TEST CONDITIONS
- Adjustment of pH: Exposure solutions were pH adjusted using either NaOH or HCl of ACS grade purity if the pH differed from the desired test pH by more than 0.2 units.
- Photoperiod: not reported
- Light intensity: not reported

EFFECT PARAMETERS MEASURED: mortality, reproduction

VEHICLE CONTROL PERFORMED: not applicable

RANGE-FINDING STUDY: not reported

Reference substance (positive control):

no

Duration:

8 d

Dose descriptor:

LC50

Effect conc.:

8.6 mg/L

Nominal / measured:

not specified

Conc. based on:

element

Basis for effect:

mortality

Remarks on result:

other: 95% CI: 5.2 - 14.2 mg/L

Duration:

8 d

Dose descriptor:

NOEC

Effect conc.:

4.9 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

reproduction

Remarks:

: days to first brood, mean number of broods surviving, and mean total number of offspring per surviving adult

Duration:

8 d

Dose descriptor:

LOEC

Effect conc.:

12.1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

reproduction

Remarks:

: days to first brood, mean number of broods surviving, and mean total number of offspring per surviving adult

Duration:

8 d

Dose descriptor:

EC10

Effect conc.:

4.909 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: 95% CL: 1.834-13.140

Details on results:

- Mortality of parent animals: See Table 2
- No. of offspring produced per day per female: See Table 2
- Time to first brood release or time to hatch: See Table 2
- All other template details: Not reported

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

NOEC and LOEC values were statistically determined with a 99.0% confidence level (p = 0.01).

Table 2. Effects of Al3+Upon Survival and Reproduction in an Eight-Day Chronic Test with Ceriodaphnia sp.

Al3+ Concentration (mg/L)
Nominal	0.0	1.56	3.12	6.25	12.5	25.0
Total Measured 1	<0.3	1.0	3.0	4.9	12.1	23.7
Number of Survivors	7	9	6	9	5	1
Days to First Brood (± S.D.)	5.3 (±0.8)	5.8 (±1.5)	5.0 (±0.9)	5.6 (±0.5)	>8.0 ** (±0.0)	>8.0 (±0.0)
Mean Number of Broods per Surviving Adult (± S.D.)	2.9 (±2.2)	1.9 (±1.0)	2.3 (±0.5)	2.3 (±0.9)	0.2 ** (±0.4)	0.0 (±0.0)
Mean Number of Total Offspring per Surviving Adult (± S.D.)	6.6 (±5.4)	3.5 (±2.2)	4.8 (±2.3)	4.3 (±1.7)	0.2 ** (±0.4)	0.0 (±0.0)

** Significantly different from controls at 99.0% confidence level.

1 - total measured Al is the amount present in the water column upon acidification to a pH where no precipitate was observed

Conclusions:

The 8-day LC50 was calculated as 8.6 mg/L (95% CI 5.2-14.2 mg/L) for Ceriodaphnia sp. exposed to AlCl3 in freshwater. The NOEC and LOEC (measured) values for all reproductive endpoints were 12.1 and 23.7 mg/L, respectively.

Reference 3

Endpoint:

long-term toxicity to aquatic invertebrates

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: USEPA 2002, method 1002.0 Daphnid, Ceriodaphnia dubia, survival and reproduction test in EPA-821-R-02-013 "Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Water to Freshwater Organisms."

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: control and all test concentrations
- Sampling method: not reported
- 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 (MOPS, pK 7.2 for pH 7.0; HEPES, pK 7.5 for pH 8.0). Test conditions were filtered and non filtered. For filtered experiments, the test solutions were filtered trough 0.2 μm pore diameter membrane, three hours after equilibrate the medium with Aluminium.
- All other template details: Not reported

Test organisms (species):

Ceriodaphnia dubia

Details on test organisms:

TEST ORGANISM
- Age of parental stock (mean and range, SD): less than 24-hr old
- Feeding during test
- Food type: YCT and algae suspension
- Amount: 0.1 ml each
- Frequency: daily
- All other template details: Not reported

ACCLIMATION: Not reported

QUARANTINE: Not reported

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Not reported

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Post exposure observation period:

Not reported

Hardness:

10.6 mg/L

Test temperature:

25 ± 1 degree C

pH:

Nominal: 7, 8
Measured: see Table 1

Dissolved oxygen:

Not reported

Salinity:

Not reported

Nominal and measured concentrations:

Nominal for pH 8 (μg/L): 0, 10, 100, 500, 1000, 5000
Nominal for pH 7 (μg/L): 0, 10, 100, 1000, 5000, 10000
Measured: see Table 1

Details on test conditions:

TEST SYSTEM
- Material, size, headspace, fill volume: 30 mL plastic beakers filled with 25 mL of test solution
- Aeration: none
- Renewal rate of test solution: daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- All other template details: Not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: EPA very soft water
- KCl (mg/L): 0.5
- Ca/mg ratio: 1:1
- NaHCO3: 12.0 mg/L
- All other template details: Not reported

OTHER TEST CONDITIONS
- Adjustment of pH: not reported
- Photoperiod: 16 hr light:8 hr dark
- Light intensity: 10-20 μE/m2/s (50-100 ft candles)

EFFECT PARAMETERS MEASURED: reproduction (number of juveniles produced per adult female) and survival measured at 7 days

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY: Not reported

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

IC50

Effect conc.:

399.8 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Basis for effect:

reproduction

Remarks on result:

other: pH 8, non-filtered; 95% CL 378.5-439.5 µg/L; Nominal IC50 613.2 µg/L (95% CL 484.6-682.3 µg/L)

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

10 µg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: pH 8, non-filtered

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

100 µg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: pH 8, non-filtered

Duration:

7 d

Dose descriptor:

IC50

Effect conc.:

424.1 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Basis for effect:

reproduction

Remarks on result:

other: pH 8, filtered; nominal IC50 942.6 µg/L

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

500 µg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: pH 8, filtered

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

1 000 µg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: pH 8, filtered

Duration:

7 d

Dose descriptor:

IC50

Effect conc.:

25.9 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Basis for effect:

reproduction

Remarks on result:

other: pH 7, non-filtered; 95% CL 25.3-26.5 µg/L; Nominal IC50 649.5 µg/L (95% CL 404.8-917.5 µg/L)

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

100 µg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: pH 7, non-filtered

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

1 000 µg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: pH 7, non-filtered

Duration:

7 d

Dose descriptor:

IC50

Effect conc.:

14.3 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Basis for effect:

reproduction

Remarks on result:

other: pH 7, filtered; 95% CL 8.8-23.7 µg/L; Nominal IC50 4217.4 µg/L (95% CL 854.7-6174.2)

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

100 µg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: pH 7, filtered

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

1 000 µg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: pH 7, filtered

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.058 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: 95% CL: 0.002-1.607, pH7

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.386 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: 95%CL: 0.197-0.757, pH8

Details on results:

- Mortality of parent animals: 90-100%
- Total number of produced juveniles in control after 7 days: 179-216
- All other template details: Not reported

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

IC50 values, with a 95% confidence interval, were calculated from the experimental data using linear interpolation from ICp software v2.0 (EPA). The IC50 was calculated for “Nominal Al concentrations” using the software already mentioned, but in the case of “Dissolved Al” and “Monomeric Al” concentrations, the IC50 were determined by a simple proportionality calculation based in the Nominal Al concentration values closest to the EC50 or IC50.

Not applicable

Conclusions:

The 7-d IC50 for reproduction of Ceriodaphnia dubia in non-filtered and filtered waters at pH 8 were 613.2 and 942.6 µg/L nominal Al, respectively. The 7-d IC50 for reproduction of Ceriodaphnia dubia in non-filtered and filtered waters at pH 7 were 649.5 and 4217.4 µg/L nominal Al, respectively.

Reference 4

Endpoint:

long-term toxicity to aquatic invertebrates

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: USEPA 2002, method 1002.0 Daphnid, Ceriodaphnia dubia, survival and reproduction test in EPA-821-R-02-013 "Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Water to Freshwater Organisms."

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: control and all test concentrations measured at 0 and 24 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 laboratory media with synthetic buffer to reduce pH drift (MES 5mM)
- All other template details: Not reported

Test organisms (species):

Ceriodaphnia dubia

Details on test organisms:

TEST ORGANISM
- Age of parental stock: less than 24-hr old
- Feeding during test
- Food type: YCT and algae suspension
- Amount: 0.1 ml each
- Frequency: daily
- All other template details: Not reported

ACCLIMATION: Not reported

QUARANTINE: Not reported

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Not reported

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Post exposure observation period:

Not reported

Hardness:

25, 60, 120 mg/L

Test temperature:

25 ± 1 degree C

pH:

Nominal: 6.3
Measured: see Table 1

Dissolved oxygen:

Not reported

Salinity:

Not reported

Nominal and measured concentrations:

Nominal (μg/L): 0-8000
See Table 1 for nominal and measured concentrations for each test

Details on test conditions:

TEST SYSTEM
- Material, size, headspace, fill volume: 30 mL plastic beakers filled with 25 mL of test solution
- Aeration: none
- Renewal rate of test solution: daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- All other template details: Not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: EPA synthetic standard moderately hard water
- Alkalinity (mg/L): 17, 40, 81 for tests with hardness of 25, 60, and 120 mg/L
- KCl (mg/L): 1.2, 2.9, 5.7 for tests with hardness of 25, 60, and 120 mg/L
- Ca/Mg ratio: 1:1 for all tests
- NaHCO3 (mg/L): 28.6, 68.6, 137.1 for tests with hardness of 25, 60, and 120 mg/L
- Dissolved organic carbon (mg/L): 0, 2, 4
- All other template details: Not reported

OTHER TEST CONDITIONS
- Adjustment of pH: not reported
- Photoperiod: 16 hr light:8 hr dark
- Light intensity: 10-20 μE/m2/s (50-100 ft candles), ambient laboratory illumination

EFFECT PARAMETERS MEASURED: reproduction (number of juveniles produced per adult female) and survival measured at 7 days

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY: Not reported

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

7.6 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Basis for effect:

reproduction

Remarks on result:

other: pH 6.3; 95% CL 6.9-8.3 µg/L; Nominal EC50 286 µg/L (95% CL 148.9-428.4 µg/L); H 25, DOC 0

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

62.5 µg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: pH 6.3, H 25, DOC 0: LOEC 125 µg/L

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

14.4 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Basis for effect:

reproduction

Remarks on result:

other: pH 6.3; 95% CL 14.3-14.6 µg/L; Nominal EC50 382.1 µg/L (95% CL 289.8-429.3 µg/L); H 60, DOC 0

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

125 µg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: pH 6.3, H 60, DOC 0: LOEC 250 µg/L

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

7.7 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Basis for effect:

reproduction

Remarks on result:

other: pH 6.3; 95% CL 7.2-8.8 µg/L; Nominal EC50 582.0 µg/L (95% CL 369.7-693.6 µg/L); H 120, DOC 0

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

100 µg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: pH 6.3, H 120, DOC 0: LOEC 200 µg/L

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.021 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: 95% CL: 0.011-0.037, Hardness 25mg/L, DOC: 0 mg/L

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.11 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: 95% CL: 0.029-0.409, Hardness: 60 mg/L, DOC: 0 mg/L

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.195 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: 95% CL: 0.040-0.948, Hardness: 120 mg/L, DOC: 0mg/L

Details on results:

- Mortality of parent animals: > 80%
- Total number of produced juveniles in control after 7 days: 202-312
- All other template details: Not reported

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Not reported, assumed same as CIMM 2009 studies

Not applicable

Conclusions:

The 7-d nominal EC50 for reproduction of Ceriodaphnia dubia exposed to Aluminium increased from 286 - 1619.1 μg/L as the hardness increased from 25 to 120 mg/L and DOC increased from 0 to 4 mg/L.

Reference 5

Endpoint:

long-term toxicity to aquatic invertebrates

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: USEPA 2002, method 1002.0 Daphnid, Ceriodaphnia dubia, survival and reproduction test in EPA-821-R-02-013 "Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Water to Freshwater Organisms."

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: control and all test concentrations measured at 0 and 24 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 laboratory media with synthetic buffer to reduce pH drift (MES 5mM)
- All other template details: Not reported

Test organisms (species):

Ceriodaphnia dubia

Details on test organisms:

TEST ORGANISM
- Age of parental stock: less than 24-hr old
- Feeding during test
- Food type: YCT and algae suspension
- Amount: 0.1 ml each
- Frequency: daily
- All other template details: Not reported

ACCLIMATION: Not reported

QUARANTINE: Not reported

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Not reported

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Post exposure observation period:

Not reported

Hardness:

25, 60, 120 mg/L

Test temperature:

25 ± 1 degree C

pH:

Nominal: 6.3
Measured: see Table 1

Dissolved oxygen:

Not reported

Salinity:

Not reported

Nominal and measured concentrations:

Nominal (μg/L): 0-8000
See Table 1 for nominal and measured concentrations for each test

Details on test conditions:

TEST SYSTEM
- Material, size, headspace, fill volume: 30 mL plastic beakers filled with 25 mL of test solution
- Aeration: none
- Renewal rate of test solution: daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- All other template details: Not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: EPA synthetic standard moderately hard water
- Alkalinity (mg/L): 17, 40, 81 for tests with hardness of 25, 60, and 120 mg/L
- KCl (mg/L): 1.2, 2.9, 5.7 for tests with hardness of 25, 60, and 120 mg/L
- Ca/Mg ratio: 1:1 for all tests
- NaHCO3 (mg/L): 28.6, 68.6, 137.1 for tests with hardness of 25, 60, and 120 mg/L
- Dissolved organic carbon (mg/L): 0, 2, 4
- All other template details: Not reported

OTHER TEST CONDITIONS
- Adjustment of pH: not reported
- Photoperiod: 16 hr light:8 hr dark
- Light intensity: 10-20 μE/m2/s (50-100 ft candles), ambient laboratory illumination

EFFECT PARAMETERS MEASURED: reproduction (number of juveniles produced per adult female) and survival measured at 7 days

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY: Not reported

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

56.7 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Basis for effect:

reproduction

Remarks on result:

other: pH 6.3; 95% CL 52.3-66.7 µg/L; Nominal EC50 723.7 µg/L (95% CL 570.8-791.2 µg/L); H 25, DOC 2

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

80.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Basis for effect:

reproduction

Remarks on result:

other: pH 6.3; 95% CL 67.4-86.5 µg/L; Nominal EC50 932.9 µg/L (95% CL 821.4-1152.3 µg/L); H 60, DOC 2

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

21.4 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Basis for effect:

reproduction

Remarks on result:

other: pH 6.3; 95% CL 20-23.1 µg/L; Nominal EC50 1356.5 µg/L (95% CL 1171.8-1497.5 µg/L); H 120, DOC 2

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

500 µg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: pH 6.3, H 120, DOC 2: LOEC 1000 µg/L

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.266 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: 95% CL: 0.087-0.819, Hardness: 25mg/L, DOC: 2mg/L

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.504 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: 95% CL: 0.226-1.126, Hardnes: 60 mg/L, DOC: 2ml/L

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.997 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: 95% CL: 0.612-1.623, Hardness: 120 mg/L, DOC: 2mg/L

Details on results:

- Mortality of parent animals: > 80%
- Total number of produced juveniles in control after 7 days: 202-312
- All other template details: Not reported

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Not reported, assumed same as CIMM 2009 studies

Not applicable

Conclusions:

The 7-d nominal EC50 for reproduction of Ceriodaphnia dubia exposed to Aluminium increased from 286 - 1619.1 μg/L as the hardness increased from 25 to 120 mg/L and DOC increased from 0 to 4 mg/L.

Reference 6

Endpoint:

long-term toxicity to aquatic invertebrates

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: USEPA 2002, method 1002.0 Daphnid, Ceriodaphnia dubia, survival and reproduction test in EPA-821-R-02-013 "Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Water to Freshwater Organisms."

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: control and all test concentrations measured at 0 and 24 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 laboratory media with synthetic buffer to reduce pH drift (MES 5mM)
- All other template details: Not reported

Test organisms (species):

Ceriodaphnia dubia

Details on test organisms:

TEST ORGANISM
- Age of parental stock: less than 24-hr old
- Feeding during test
- Food type: YCT and algae suspension
- Amount: 0.1 ml each
- Frequency: daily
- All other template details: Not reported

ACCLIMATION: Not reported

QUARANTINE: Not reported

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Not reported

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Post exposure observation period:

Not reported

Hardness:

25, 60, 120 mg/L

Test temperature:

25 ± 1 degree C

pH:

Nominal: 6.3
Measured: see Table 1

Dissolved oxygen:

Not reported

Salinity:

Not reported

Nominal and measured concentrations:

Nominal (μg/L): 0-8000
See Table 1 for nominal and measured concentrations for each test

Details on test conditions:

TEST SYSTEM
- Material, size, headspace, fill volume: 30 mL plastic beakers filled with 25 mL of test solution
- Aeration: none
- Renewal rate of test solution: daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- All other template details: Not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: EPA synthetic standard moderately hard water
- Alkalinity (mg/L): 17, 40, 81 for tests with hardness of 25, 60, and 120 mg/L
- KCl (mg/L): 1.2, 2.9, 5.7 for tests with hardness of 25, 60, and 120 mg/L
- Ca/Mg ratio: 1:1 for all tests
- NaHCO3 (mg/L): 28.6, 68.6, 137.1 for tests with hardness of 25, 60, and 120 mg/L
- Dissolved organic carbon (mg/L): 0, 2, 4
- All other template details: Not reported

OTHER TEST CONDITIONS
- Adjustment of pH: not reported
- Photoperiod: 16 hr light:8 hr dark
- Light intensity: 10-20 μE/m2/s (50-100 ft candles), ambient laboratory illumination

EFFECT PARAMETERS MEASURED: reproduction (number of juveniles produced per adult female) and survival measured at 7 days

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY: Not reported

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

500 µg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: pH 6.3, H 25, DOC 2 and DOC 4: LOEC 1000 µg/L

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

170.4 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Basis for effect:

reproduction

Remarks on result:

other: pH 6.3; 95% CL 141.7-192.8 µg/L; Nominal EC50 1042.3 µg/L (95% CL 867.6-1265.5 µg/L); H 25, DOC 4

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

151.5 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Basis for effect:

reproduction

Remarks on result:

other: pH 6.3; 95% CL 140.9-162.6 µg/L; Nominal EC50 1117.6 µg/L (95% CL 899.5-1346.6 µg/L); H 60, DOC 4

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

59.5 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Basis for effect:

reproduction

Remarks on result:

other: pH 6.3; 95% CL 58.8-60.5 µg/L; Nominal EC50 1619.1 µg/L (95% CL 1498.8-1810.8 µg/L); H 120, DOC 4

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

1 000 µg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: pH 6.3, H 120, DOC 4: LOEC 2000 µg/L

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.478 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: 95% CL: 0.402-0.567, Hardness 25mg/L, DOC: 4 mg/L

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.55 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: 95% CL: 0.436-0.694, Hardness: 60 mg/L, DOC: 4 mg/L

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.6 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: 95% CL: 0.440-0.619, Hardness: 120 mg/L, DOC: 4mg/L

Details on results:

- Mortality of parent animals: > 80%
- Total number of produced juveniles in control after 7 days: 202-312
- All other template details: Not reported

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Not reported, assumed same as CIMM 2009 studies

Not applicable

Conclusions:

The 7-d nominal EC50 for reproduction of Ceriodaphnia dubia exposed to Aluminium increased from 286 - 1619.1 μg/L as the hardness increased from 25 to 120 mg/L and DOC increased from 0 to 4 mg/L.

Reference 7

Endpoint:

long-term toxicity to aquatic invertebrates

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: USEPA 2002, method 1002.0 Daphnid, Ceriodaphnia dubia, survival and reproduction test in EPA-821-R-02-013 "Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Water to Freshwater Organisms."

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: control and all test concentrations measured at 0 and 24 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 laboratory media with synthetic buffer to reduce pH drift (HEPES 5 mM)
- All other template details: Not reported

Test organisms (species):

Ceriodaphnia dubia

Details on test organisms:

TEST ORGANISM
- Age of parental stock: less than 24-hr old
- Feeding during test
- Food type: YCT and algae suspension
- Amount: 0.1 ml each
- Frequency: daily
- All other template details: Not reported

ACCLIMATION: Not reported

QUARANTINE: Not reported

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Not reported

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Post exposure observation period:

Not reported

Hardness:

10.6 mg/L

Test temperature:

25 ± 1 degree C

pH:

Nominal: 8
Measured (range): 7.94-8.19

Dissolved oxygen:

Not reported

Salinity:

Not reported

Nominal and measured concentrations:

Nominal (μg/L): 0, 62.5, 125, 250, 500, 1000
Measured total Al (μg/L): -, 59.1, 141.8, 287.7, 585.3, 1084.9
Measured mean dissolved Al (μg/L): -, 56.4, 121.7, 166.9, 355.5, 700.1

Details on test conditions:

TEST SYSTEM
- Material, size, headspace, fill volume: 30 mL plastic beakers filled with 25 mL of test solution
- Aeration: none
- Renewal rate of test solution: daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- All other template details: Not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: EPA synthetic standard moderately hard water
- Alkalinity (mg/L): 7.5
- KCl (mg/L): 0.5
- Ca/Mg ratio: 1:1
- NaHCO3 (mg/L): 12.0
- Dissolved organic carbon (mg/L): 0
- All other template details: Not reported

OTHER TEST CONDITIONS
- Adjustment of pH: not reported
- Photoperiod: 16 hr light:8 hr dark
- Light intensity: 10-20 μE/m2/s (50-100 ft candles), ambient laboratory illumination

EFFECT PARAMETERS MEASURED: reproduction (number of juveniles produced per adult female) and survival measured at 7 days

VEHICLE CONTROL PERFORMED: Not reported

RANGE-FINDING STUDY: Not reported

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

550 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Basis for effect:

reproduction

Remarks on result:

other: 95% CL 459.4-655.9 µg/L; Nominal EC50 782.3µg/L (95% CL 650.7-935.8 µg/L); pH 8, H 10.6, DOC 0

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

250 µg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: pH 8, H 10.6, DOC 0

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

500 µg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: pH 8, H 10.6, DOC 0

Details on results:

- Mortality of parent animals: > 80%
- Total number of produced juveniles in control after 7 days: 186
- All other template details: Not reported

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Not reported, assumed same as CIMM 2009 studies

Not applicable

Conclusions:

The 7-d nominal EC50 for reproduction of Ceriodaphnia dubia exposed to Aluminium was calculated as 550 μg/L with pH 8, hardness of 10.6 mg/L and DOC of 0 mg/L.

Reference 8

Endpoint:

long-term toxicity to aquatic invertebrates

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 211 (Daphnia magna Reproduction 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: control and all test concentrations measured weekly
- 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 (MES 5mM)
- All other template details: Not reported

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Age of parental stock: less than 24-hr old
- Feeding during test
- Food type: algae suspension of P. subcapitata and Ch. reinhardtti
- Amount: 4x10^6 and 12x10^6 cells/test chamber of each, respectively
- Frequency: daily
- All other template details: Not reported

ACCLIMATION: Not reported

QUARANTINE: Not reported

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Not reported

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

Not reported

Hardness:

140 mg/L

Test temperature:

20 ± 1 degree C

pH:

Nominal: 6.3
Measured mean range: 6.27-6.32

Dissolved oxygen:

Not reported

Salinity:

Not reported

Nominal and measured concentrations:

Nominal (μg/L): 0, 150, 300, 600, 1200, 2400
Measured total Al (μg/L): 2.1, 127.5, 349.0, 539.3, 1106.4, 2401.6
Measured mean dissolved Al (μg/L): 0.2, 39.1, 40.7, 39.6, 20.2, 13.0

Details on test conditions:

TEST SYSTEM
- Material, size, headspace, fill volume: 70 mL plastic beakers filled with 50 mL of test solution
- Aeration: none
- Renewal rate of test solution: three times per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- All other template details: Not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: EPA synthetic standard water of 25 mg/L hardness
- Alkalinity (mg/L): 93
- KCl (mg/L): 6.7
- Ca/Mg ratio: 1:1
- NaHCO3 (mg/L): 160
- Dissolved organic carbon (mg/L): 2
- All other template details: Not reported

OTHER TEST CONDITIONS
- Adjustment of pH: not reported
- Photoperiod: 16 hr light:8 hr dark
- Light intensity: 10-20 μE/m2/s (50-100 ft candles), ambient laboratory illumination

EFFECT PARAMETERS MEASURED: reproduction (number of juveniles produced per adult female) and survival measured at 7 days

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY: Not reported

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

27.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Basis for effect:

reproduction

Remarks on result:

other: 95% CL 23.9-28.8 µg/L; Nominal EC50 986.5 µg/L (95% CL 935.8-1087.6 µg/L); H 140, DOC 2, pH 6.3

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

600 µg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: pH 6.3, H 140, DOC 2: LOEC 1200 µg/L

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.703 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: 95% CL: 0.672-0.736

Details on results:

- Mortality of parent animals: > 80%
- Total number of produced juveniles in control after 21 days: 1012
- All other template details: Not reported

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Not reported, assumed same as CIMM 2009 studies

Not applicable

Conclusions:

The 21-d nominal EC50 for reproduction of Daphnia magna exposed to Aluminium was calculated as 986.5 μg/L at pH 6.3, hardness of 140 mg/L, and 2 mg/L DOC.

Reference 9

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Documentation insufficient for assessment; too few details on methods, exposure conditions, and test animals

Reason / purpose for cross-reference:

reference to same study

Specific details on test material used for the study:

- Name of test material: alum effluent from a drinking water treatment plant
- Molecular formula: Al2(SO4)3

Test organisms (species):

Ceriodaphnia dubia

Details on test organisms:

Not reported

Reference 10

Endpoint:

long-term toxicity to aquatic invertebrates

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:

no guideline followed

Principles of method if other than guideline:

Static renewal test in 100 ml beakers with one control and 6 treatments for 28 days. Solution was renewed three times per week, and survival, molting, and reproduction was recorded. The range of treatment means was 0.19 - 4.26 µg/L Al.

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material: aluminum sulfate hexadecahydrate
- Molecular formula: (Al)2(SO4)3 x 16H2O
- Analytical purity: reagent grade

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all concentrations
- Sampling method: sample collection timing not reported. Samples were most likely drawn into 15 ml tubes and acidified with 1 drop of 1.0 N HNO3.
- Sample storage conditions before analysis: Not reported

Vehicle:

not specified

Details on test solutions:

Not reported

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: daphnid
- Source: commercially available source, Aquatic Life Company, Minneapolis, MN.
- Age at study initiation: neonates 12 ± 12 hrs old
- Method of breeding: used a series of 250 ml beakers containing 200 ml water with food at a concentration of 30 mg/L in each beaker. Water was totally renewed three times per week with approximately 5 adult daphnia per beaker transferred to the new solution.
- Feeding during test: details not reported
- All other template details: Not reported

ACCLIMATION: Not reported

QUARANTINE (wild caught): Not applicable

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

28 d

Post exposure observation period:

Not reported

Hardness:

Hard, value not reported

Test temperature:

19.9 degrees C

pH:

8.30

Dissolved oxygen:

7.81 mg/L

Salinity:

Not applicable

Nominal and measured concentrations:

Range of measured treatment means: 0.19-4.26 µg/L total Al and 0.14-0.71 mg/L supernatant

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 100 ml beakers filled with 50 ml solution
- No. of organisms per vessel: 1 x 10 vessels
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- All other template details: Not reported or Not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: well water tapping the Jordan sandstone stratum underlying the Minneapolis-St. Paul metropolitan area, with iron catalytically removed
- Alkalinity: 211 mg/L
- Culture medium different from test medium: no
- All other template details: Not reported

OTHER TEST CONDITIONS: Not reported

EFFECT PARAMETERS MEASURED: survival, neonates, and adult molts were counted every three days with renewal of solution

VEHICLE CONTROL PERFORMED: Not applicable

RANGE-FINDING STUDY
- Test concentrations: 0, 0.7, 1.5, 2.4, 3.3, 8.4, 17 mg/L
- Results used to determine the conditions for the definitive study: reproduction NOEC 0.7 mg/L, LOEC 1.5 mg/L

Reference substance (positive control):

not specified

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

1.61 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

1.89 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

reproduction

Remarks:

as young per female

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

4.26 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

reproduction

Remarks:

as young per female

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

2.518 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: 95% CL: 0.506-12.528

Details on results:

- No. of offspring produced per day per female: in controls -5.26
- All other template details: Not reported

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

All chronic test data were examined by one-way analysis of variance with treatment differences separated by Dunnett's procedure (one-tailed) at an alpha = 0.05 (Steele and Torre, 1960).

Survival and reproduction of daphnia in 28 day chronic test

Concentration (mg/L total)	Percentage Survival	Mean young per female per day	Mean young per female
0	70	5.26	101.5
0.19	60	5.96	118.1
0.29	70	4.67	93.3
0.54	70	4.83	88.0
1.02	50	5.71	100.3
1.89	40	4.29	68.5
4.26	20	2.07 *	40.3 *

* - Significantly different at p = 0.05

 

Conclusions:

Daphnia magna were exposed to a series of aluminium concentrations for 28 days and observed for effects on mortality and reproduction. The estimated 28-d LC50 was 1.61 mg/L. For Daphnia reproduction, the NOEC and LOEC was 1.89 and 4.26 mg/L, respectively.

Reference 11

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Significant methodological deficiencies: statistics were not used to determine effect levels

Specific details on test material used for the study:

- Molecular formula: (Al)2(SO4)3 x 18H2O
- Physical state: crystals
- Analytical purity: reagent grade

Test organisms (species):

other aquatic arthropod: Tanytarsus dissimilis

Reference 12

Endpoint:

long-term toxicity to aquatic invertebrates

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:

no guideline followed

Principles of method if other than guideline:

Chronic exposures were conducted with ten replicates for each Al concentration and control. Single C. dubia neonates less than 16 hours old were added to a beaker via a fire-polished, wide-mouth pipette and observed for 7 days. Organisms were fed a mix fermented trout chow, green algae, and yeast daily during the exposures. Test waters were renewed every other day, and checked for deaths or reproduction.

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material: aluminum chloride
- Source: MCB Chemicals
- Analytical purity: 99.8 %
- Lot/batch No.: B7N14

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all concentrations
- Sampling method: samples were collected at the initiation, prior to and after solution renewal, and at the termination of the tests
- Sample storage conditions before analysis: samples were immediately acidified with HNO3 to 0.1% (v/v, pH range 1.5 to 2.0), mixed, and allowed to stand (>1 hr) before aliquots were withdrawn and composited.

Vehicle:

not specified

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: aliquots of 5000 mg/L Al stock was added to filtered raw Lake Superior or dechlorinated tap water.
- All other template details: Not reported

Test organisms (species):

Ceriodaphnia dubia

Details on test organisms:

TEST ORGANISM
- Common name: cladoceran
- Source: initially from USEPA Research Laboratory, Duluth, MN stock culture - then cultured at the University of Wisconsin-Superior culture facility for several generations before use in these tests
- Age at study initiation: less than 16 hours old
- Method of breeding: adult cladocerans were cultured individually in 30 mL containers and were transferred to new water every Monday, Wednesday, and Friday. They were fed a 1:1:1 ratio of fermented trout chow solution, yeast solution, and green algae (Selenastrum capricornutum) one daily.
- Feeding during test
- Food type: 1:1:1 ratio of fermented trout chow solution, yeast solution, and green algae (Selenastrum capricornutum)
- Amount: 100 µL
- Frequency: daily
- All other template details: Not reported

ACCLIMATION: none

QUARANTINE: Not applicable

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Post exposure observation period:

Not reported

Hardness:

50-50.5 mg CaCO3/L

Test temperature:

25 ± 2 degrees C

pH:

Nominal: 7.2-7.4, 7.6-7.9
Measured: 7.15 ± 0.05, 7.61 ± 0.11

Dissolved oxygen:

Measured DO for the 3 pH levels (mg/L): 7.1 ± 0.1, 7.3 ± 0.1

Salinity:

Not applicable

Nominal and measured concentrations:

Measured in pH 7.15 test (mg/L): control, 1.4, 2.6, 5.0, 10.0, 17.2
Measured in pH 7.86 test (mg/L): control, 1.1, 2.4, 5.7, 10.1, 17.8

Details on test conditions:

TEST SYSTEM
- Test vessel: 30 mL beakers
- Material, size, headspace, fill volume: 30 mL containers with 16 mL of test solution
- Aeration: test solutions were aerated for at least 24 hours before test initiation, none during testing
- Renewal rate of test solution: every other day
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- All other template details: Not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dilution water was either raw Lake Superior water for pH 7.15 and UW-Superior Laboratory water for pH 7.61
- Total solids (mg/L at 105 degrees C): 80, 70
- Particulate matter (suspended solids, mg/L): 2, <1
- Chloride (mg Cl/L): 2.8, 3.8
- Alkalinity (mg CaCO3/L): 46.0, 39.6
- All other measured characteristics presented in Table 1
- All other template details: Not reported

OTHER TEST CONDITIONS
- Adjustment of pH: addition of reagent grade HCl or NaOH prior to test initiation
- Photoperiod: not reported
- Light intensity: not reported

EFFECT PARAMETERS MEASURED: mortality and reproduction (number of young produced per adult surviving 7 days) checked every other day

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2x
- All other template details: Not reported

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

1.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other: acid exchangeable

Basis for effect:

reproduction

Remarks on result:

other: pH 7.15

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

2.6 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other: acid exchangeable

Basis for effect:

reproduction

Remarks on result:

other: pH 7.15

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

1.1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other: acid exchangeable

Basis for effect:

reproduction

Remarks on result:

other: pH 7.61

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

2.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other: acid exchangeable

Basis for effect:

reproduction

Remarks on result:

other: pH 7.61

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

1.495 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: 95% CL: 1.184-1.889, pH: 7.15, hardness:50 mg/L

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.796 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: 95% CL: 0.306-2.073, pH: 7.61, Hardness: 50.5 mg/L

Details on results:

- Mortality of parent animals: see Table 2
- No. of offspring produced per day per female in the control exposures: 2.5 for pH 7.15 and 6.3 for pH 7.61
- There was a test performed at pH 8.1, but 100% of organisms died in the control and all exposure concentrations.
- All other template details: Not reported

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Chronic exposure effects were determined by one-way analysis of variance (ANOVA) tests at p ≤ 0.05 significance. Specific concentrations causing significant (p ≤ 0.05) reductions in reproduction were identified using the Dunnett's one-tailed procedure (Steele and Torrie 1980).

Table 2. Percent survival of Ceriodaphnia dubia exposed to a range of aluminium in water with 2 different pH levels for 7 days.

	Aluminium Exposure (mg/L)
pH level	Control	1.4	2.6	5.0	10.0	17.2
7.15	90	80	60	20	40	80
	Control	1.1	2.4	5.7	10.1	17.8
7.61	80	60	70	60	0	0

Conclusions:

The NOEC and LOEC for the mean cumulative number of young per starting adult Ceriodaphnia dubia in raw Lake Superior water at mean pH 7.15 for 7 days was 1.4 and 2.6 mg Al/L, respectively. The NOEC and LOEC for the mean cumulative number of young per starting adult Ceriodaphnia dubia in raw Lake Superior water at mean pH 7.61 for 7 days was 1.1 and 2.4 mg Al/L, respectively.

Reference 13

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

February 15 2000 - March 14 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 211 (Daphnia magna Reproduction 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: 99.75
- 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 test concentrations and controls
- Sampling method: not reported
- Sample storage conditions before analysis: preserved by freezing at -20 degrees C

Vehicle:

not specified

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: the aluminium in solution was from a 28-d transformation/dissolution (T/D-test) of aluminium powder at pH 8.5 according to an OECD protocol
- All other template details: Not reported

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: fresh water crustacean
- Strain/clone: Strain A (Baird et al. 1992)
- Justification for species other than prescribed by test guideline: not applicable
- Source: University of Sheffield
- Age of parental stock (mean and range, SD): not reported
- Feeding during test
- Food type: Selenastrum capricornutum
- Amount: 75-180 µg C / animal / day
- Frequency: 6 days / week

ACCLIMATION: Not reported

QUARANTINE: Not reported

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: adult D.magna were isolated 24 hours prior to initiation of test. Young daphnia's produced overnight (less than 24 hours) were used in the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

Not reported

Hardness:

Not reported (other than CaCl and MgSO4 concentrations in test medium)

Test temperature:

20 ± 2 degrees C

pH:

7.6-8.1

Dissolved oxygen:

7.12 - 9.30 ppm

Salinity:

Not applicable

Nominal and measured concentrations:

nominal (µg/L): 0, 19, 35, 61, 108, 195, 347, 606
measured (µg/L): see table 1

Details on test conditions:

TEST SYSTEM
- Test vessel: flat bottom flask
- Material, size, headspace, fill volume: 100 mL flat bottom flasks filled with 50 mL culture solution and covered with inverted plastic beakers
- Renewal rate of test solution (frequency): 3 times per week (days 3,6,8,10,13,15,17)
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- All other template details: not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted standard water in accordance with ISO 6341 medium with components the same as recommended in the OECD 211 guideline, with glass-distilled water as the dilution water
- All other template details: Not reported

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to 8.0 by addition of 1N hydrochloric acid
- Photoperiod: 16h light: 8h dark
- Light intensity: Not reported

EFFECT PARAMETERS MEASURED: animals observed daily and immobilised animals recorded. The number of live offspring was counted and removed.

VEHICLE CONTROL PERFORMED: not applicable

RANGE-FINDING STUDY: not reported

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

76 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

137 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Basis for effect:

immobilisation

Details on results:

- Mortality of parent animals: 0 control animals died
- No. of offspring produced per day per female: 102-184 in the control group
- Time to first brood release or time to hatch: 9-10 days for first brood
- Average measured concentrations were not provided for the LOECs
- All other template details: Not reported

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

The NOEC for effect on the reproductive output was estimated after calculation of the mean reproductive output across replicates for each concentration and the pooled residual standard deviation by analysis of variance (ANOVA). The mean for each concentration was compared with the control mean using Dunnet's test performed with JMP version 3.2.2.

Table 2. The number of young produced per surviving adult in the different groups.

Concentration (nominal)	Minimum	Maximum	Average	Standard Deviation
Control A	106	184	137	25.2
Control B	102	157	130	19.7
1.8% T/D	104	173	139	25.3
3.2% T/D	85	143	117	19.7
5.6% T/D	79	162	123	32.9
10% T/D	68	162	112	25.9
18% T/D	36	109	91	21

Conclusions:

The NOECs for Daphnia magna exposed to a series of dissolved Aluminium concentrations for 21 days were 76 µg Al/L and 137 µg Al/L, respectively. The average measured concentrations were not reported for the LOEC concentrations.

Reference 14

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01/2011-02/2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: Bhargava S(1992). General behaviour and mortality of Lymnaea stagnalis and their egg mass undert the stress of thiourea and DDT. J. Fresh Biol., 4, 129-134.

Qualifier:

according to guideline

Guideline:

other: Grosell M, Gerdes R, Brix KV (2006). Chronic toxicity of lead to three freshwater invertebates (Brachionus cayciflorus, Chironomus tentans and Lymnaea stagnalis. Environmental Toxicology Chemistry, 25, 97-104

Qualifier:

according to guideline

Guideline:

other: Slooff W, Canton, J.H. (1983). Comparison of the susceptibility of 11 freshwater species to 8 chemical compounds. II. (semi)chronic toxicity tests. Aquat Toxicol. 4, 277-282.

Principles of method if other than guideline:

/

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): Aluminum nitrate nonahydrate
- Molecular formula (if other than submission substance): Al(NO3))3.9H2O
-Source: J.T. Baker, Philipsburg, NJ, USA
- Analytical purity:100.6%
- Impurities (identity and concentrations): Ca = 0.001%, Cl = 0.001%, Mg = 0.0002%, K = 0.0005%, Na = 0.003%, Pb = 0.0006%, Fe = 0.001%
- Purity test date: 20/10/2009
- Lot/batch No.:H43606
- Storage condition of test material:stored and sealed in its original container at room temperature

Analytical monitoring:

yes

Details on sampling:

- Analytical samples from each treatment were collected

- Sampling method:

TOTAL ALUMINUM:
“New” waters were taken directly from the cubitainer using a 20 mL syringe after the 3-hour equilibrium period. Approximately 5 mL was drawn into the syringe to rinse the inside of the syringe and then expunged. Then 15 mL of sample was drawn into the syringe and injected into a 15 mL polypropylene conical tube. Samples of “old” waters were taken from a composite of each replicate from each concentration. “Old” water was poured off from the top half/layer of the water column within each beaker.

DISSOLVED ALUMINUM/
Approximately 20 mL was drawn into the syringe of which 5 mL was pushed through the filter and discarded; the remaining 15 mL was collected into a 15-mL polypropylene conical test tube for subsequent Al analysis.

MONOMERIC ALUMINUM/
Following collection of a sample for dissolved aluminum analysis, the same filter was used to filter approximately 15 mL aliquots for monomeric aluminum analysis.

- Sample storage conditions before analysis:

TOTAL ALUMINUM-DISSOLVED ALUMINUM: Samples were preserved with trace metal grade nitric acid (AR-ACS grade, Mallinckrodt Chemical, Hazelwood, MO, USA) to pH < 2 and refrigerated (0 - 4 °C) prior to analysis.

MONOMERIC ALUMINUM: Monomeric samples were submitted for immediate analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:A stock solution of 1000 mg Al/L was prepared by addition of 6.96 grams of Al(NO3)3·9H2O to 500 mL of Milli-Q water in a volumetric flask. The stock solution was then stored in a plastic container in the dark at 0 - 4°C. The pH of the 1000 mg Al/L stock solution was measured at 3.6 - 3.8.
The dilution/ control water used in the toxicity test was reconstituted laboratory water prepared by adding the appropriate reagent grade salts to Milli-Q water to achieve the desired nominal hardness and alkalinity values of 120 and 80 mg/L as CaCO3, respectively. Reconstituted water was prepared as detailed in standard USEPA methods (USEPA 2002).
Test concentrations were prepared by adding the appropriate volume of stock solution (1000 mg/L Al) to the dilution (reconstituted) water. A volumetric flask was filled to approximately 80% of its capacity with the respective dilution (reconstituted) water. Next, the aluminum stock solution was added to the volumetric flask with a micro-pipette to achieve the desired nominal concentration. The volume was then completed to 1500 mL with the respective dilution water. The pH was checked and adjusted to a pH of 6.0 using dilute HCl or NaOH. Each individual concentration was poured into a cubitainer which was then placed (with its cap off) in an “air-tight” enclosure and this enclosure was injected with a pre-determined volume (2.3 – 2.6%) of CO2 to maintain the pH near 6.0. Solutions were held as described for a 3-hour equilibration period in an environmental chamber at test temperature.
- Controls:dilution water control

Test organisms (species):

other aquatic mollusc: Lymnaea stagnalis

Details on test organisms:

TEST ORGANISM
- Common name: Great Pond Snail
- Justification for species other than prescribed by test guideline: L. stagnalis is a common representative of gastropod molluscs and is an important herbivore/detritivore in many freshwater food webs. Use of this test organism qualifies as a level of organization (Phylum) not otherwise represented as part of the minimum taxonomic requirements for calculation of a predicted no effect concentration (PNEC) for the freshwater aquatic compartment, within the context of a species sensitivity distribution (SSD) approach (European Commission 2003).
- Source: L. stagnalis are cultured at OSU AquaTox. Stock of L. stagnalis egg masses were originally obtained from Dr. Martin Grosell, Rosenstiel School of Marine and Atmospheric Science, University of Miami (Florida, USA).
- Feeding during test: Each test chamber contained one very small piece of lettuce, carrot, and sweet potato.
- Food type: vegetables
- Amount: each vegetable 2-4 mm³ in size
- Frequency: Uneaten food was removed at each water change (onday, Wednesday, and Friday) and replaced with fresh food to maintain water quality.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

30 d

Post exposure observation period:

/

Hardness:

117 mg/L as CaCO3

Test temperature:

25 ± °C

pH:

6.0-6.2

Dissolved oxygen:

> 40%

Salinity:

/

Nominal and measured concentrations:

Nominal concentration: 0, 125, 250, 500,1000, 2000 μg/L
Average measured total Al concentration: <10, 142, 271, 546, 1053, 2063 μg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 120mL polypropylene Soufflé cups. Because the lid was not air-tight and therefore exchange of CO2 between the chamber and atmosphere would take place, the chambers were placed within an air-tight enclosure injected with CO2 to maintain the pH near 6.0.
- Material, size, headspace, fill volume: fill volume: 120 mL, no headspace
- Renewal rate of test solution (frequency/flow rate):Water changes were performed on a Monday, Wednesday, and Friday schedule with at least 80 - 90% replacement of test solution.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates):10
- No. of vessels per control (replicates):10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution/ control water used in the toxicity test was reconstituted laboratory water prepared by adding the appropriate reagent grade salts to Milli-Q water to achieve the desired nominal hardness and alkalinity values of 120 and 80 mg/L as CaCO3, respectively. Reconstituted water was prepared as detailed in standard USEPA methods (USEPA 2002). Three batches of reconstituted laboratory water were used for the duration of the study.
- Alkalinity:48mg/L as CaCO3
- Ca/mg ratio:3.02
-DOC: <0.50 mg/L
- Total residual chlorine: <0.05mg/L
- Ammonia: <1.0mg/L
- Calcium: 29.3mg/L
- Magnesium: 9.7mg/L
- Sodium: 34.8mg/L
- Potassium: 7.03mg/L
- Chloride: 51.5 mg/L
- Sulfate: 114mg/L
- Culture medium different from test medium:no
- Intervals of water quality measurement:Certain water quality parameters (i.e., temperature, dissolved oxygen [DO], and pH) were measured in each concentration at test initiation, at renewal, (in both freshly prepared renewal waters and discarded waters), and at test termination. Conductivity was also measured in the freshly-prepared renewal water at test initiation and renewal. Hardness, alkalinity, total residual chlorine, and total ammonia were measured in the dilution water at test initiation and when a newly prepared batch of control/dilution water was used.

OTHER TEST CONDITIONS
- Adjustment of pH: /
- Photoperiod:16:8 hour light:dark cycle
- Light intensity:100 foot candles

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :Observations of live and dead organisms were conducted three times a week (Monday, Wednesday, and Friday). Dead organisms were recorded and immediately removed. At test termination, each surviving organism was blot-dried with a Kimwipe® and weighed to within 0.01 mg.

Reference substance (positive control):

no

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

2 099.2 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

1 092 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

other: wet weight

Duration:

30 d

Dose descriptor:

LOEC

Effect conc.:

> 2 099.2 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

30 d

Dose descriptor:

LOEC

Effect conc.:

2 099.2 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

other: wet weight

Duration:

30 d

Dose descriptor:

LC50

Effect conc.:

> 2 099.2 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

30 d

Dose descriptor:

EC10

Effect conc.:

860.7 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

other: wet weight

Remarks on result:

other: CL: 733.1-1010.5 μg/L

Duration:

30 d

Dose descriptor:

other: EC20

Effect conc.:

1 148.5 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

other: wet weight

Remarks on result:

other: CL: 1026.7-1284.7 μg/L

Duration:

30 d

Dose descriptor:

EC50

Effect conc.:

2 036 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

other: wet weight

Remarks on result:

other: CL: 1944.0-2131.0 μg/L

Results with reference substance (positive control):

/

Reported statistics and error estimates:

STATISTICAL ANALYSIS
Statistical analysis was performed using measured average total Al concentrations of newly prepared solutions. Differences in the number of individuals and the intrinsic growth rate and growth at test termination were evaluated using a statistical computer package (Comprehensive Environmental Toxicity Information System [CETIS], Tidepool Scientific Software, McKinleyville, CA, USA and Toxicity Relationship Analysis Program [TRAP], Duluth, MN, USA). If the data met the assumptions of normality and homogeneity, the NOEC and LOEC were estimated using an analysis of variance to compare (p = 0.05) organism performance in the experimental treatments with that observed in the control. The median-lethal concentration (LC50) was estimated using linear interpolation. The effective concentrations to reduce growth by 10%, 20%, or 50% relative to control performance (EC10/EC20/EC50) were estimated using threshold sigmoid regression analysis. Exposure concentrations were log-transformed before determination of the EC10, EC20, and EC50 values.

Table: Summary of biological results

Average total aluminum (μg/l)	Proportion survived - Average ± Std Dev	Wet weight - Average ± Std. Dev
5	1.00 ± 0	32.39 ±9.22
175.2	0.90 ± 0.32	31.34 ± 14.67
309.6	1.00 ± 0	31.68 ± 16.62
569.6	0.90 ± 0.32	31.96 ± 10.52
1092.0	0.67 ± 0.50	26.17 ± 8.94
2099.2	1.00 ± 0	15.45 ± 5.78

Conclusions:

Total measured exposure concentrations ranged from 5 to 2099 μg Al/L and resulted in no effect on organism survival; however, a significant effect on snail growth was observed at the highest treatment. The study resulted in a no observable effect concentration [NOEC] of 1092 μg/L total Al and a lowest observable effect concentration [LOEC] of 2099 μg/L total Al for growth. The effective concentrations to reduce growth by 10% and 20% relative to control performance (EC10 and EC20 with 95% confidence intervals) was 860.7 (733.1 – 1010.5) and 1148.5 (1026.7 – 1284.7) μg/L total Al, respectively.

Executive summary:

As part of an environmental program designed to provide data for the setting of water quality standards, data describing the chronic toxicity of aluminum to a variety of aquatic organisms are needed. Aluminum toxicity is a function of the chemical species of aluminum present in the water and this speciation is a function of the physico/chemical properties (e.g., pH) of the water. To this end, efforts are underway to develop data describing the chronic toxicity of aluminum to aquatic organisms at hydrogen ion concentrations (i.e., pH) typical of natural environmental conditions (i.e., pH of 6.0).

The study reported herein determines the chronic toxicity of aluminum, at a pH of 6.0, to the great pond snail, Lymnaea stagnalis. Use of this test organism qualifies as a level of organization (Phylum) that is not currently represented as part of the minimum taxonomic requirements for calculation of a predicted no effect concentration (PNEC) for the freshwater aquatic compartment, within the context of a species sensitivity distribution approach (European Commission 2003). In this study, L. stagnalis were exposed to a series of aluminum concentrations for thirty days, starting as newly hatched organisms (<24 hours old). To allow for possible changes in aluminum speciation, exposure solutions were aged for a 3-hour equilibrium period prior to organism exposure. Nominal test concentrations ranged from 0 to 2000 μg Al/L and total, dissolved, and monomeric aluminum were measured throughout the test. As the formation of insoluble chemical species was apparent through low dissolved and monomeric measurements (all concentrations measuring below 100 μg Al/L), total Al was used to interpret the biological data in this study. Total measured exposure concentrations ranged from 5 to 2099 μg Al/L and resulted in no effect on organism survival; however, a significant effect on snail growth was observed at the highest treatment. The study resulted in a no observable effect concentration [NOEC] of 1092 μg/L total Al and a lowest observable effect concentration [LOEC] of 2099 μg/L total Al for growth. The effective concentrations to reduce growth by 10% and 20% relative to control performance (EC10 and EC20 with 95% confidence intervals) was 860.7 (733.1 – 1010.5) and 1148.5 (1026.7 – 1284.7) μg/L total Al, respectively.

Reference 15

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

04/2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: Grosell, M.; Gerdes, R.; Brix, K.V. (2006). Chronic toxicity of lead to three freshwater invertebrates - Brachionus calyciflorus, Chironomus tentans, and Lymnea stagnalis. Environ. Toxicol. Chem., 25, 97-104.

Qualifier:

according to guideline

Guideline:

other: Snell, T.; Moffat, B. (1992). A 2-d life cycle test with the rotifer, Brachionus calyciflorus. Environ. Toxicol. Chem., 11, 1249-1257.

Principles of method if other than guideline:

/

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): Aluminum nitrate nonahydrate
- Molecular formula (if other than submission substance): Al(NO3))3.9H2O
-Source: J.T. Baker, Philipsburg, NJ, USA
- Analytical purity:100.6%
- Impurities (identity and concentrations): Ca = 0.001%, Cl = 0.001%, Mg = 0.0002%, K = 0.0005%, Na = 0.003%, Pb = 0.0006%, Fe = 0.001%
- Purity test date: 20/10/2009
- Lot/batch No.:H43606
- Storage condition of test material:stored and sealed in its original container at room temperature

Analytical monitoring:

yes

Details on sampling:

- Analytical samples from each treatment were collected

- Sampling method:

TOTAL ALUMINUM
“New” waters were taken directly from the 1-Liter beakers using a 20 mL syringe after the 3 hour equilibrium period. Approximately 5 mL was drawn into the syringe to rinse the inside of the syringe and then expunged. Then 15 mL of sample was drawn into the syringe and injected into a 15 mL polypropylene conical tube. Samples of “old” waters were taken from a composite of each replicate from each concentration. “Old” water was poured off from the top half/layer of the water column within each test tube and composited.

DISSOLVED ALUMINUM
Approximately 20 mL was drawn into the syringe of which 5 mL was pushed through the filter and discarded; the remaining 15 mL was collected into a 15-mL polypropylene conical test tube.

MONOMERIC ALUMINUM
Following collection of a sample for dissolved aluminum analysis, the same filter was used to filter a 20 mL aliquot for monomeric aluminum analysis.

- Sample storage conditions before analysis:

TOTAL AND DISSOLVED ALUMINUM:Samples were then preserved with trace metal grade nitric acid (AR-ACS grade, Mallinckrodt Chemical, Hazelwood, MO, USA) to pH < 2 and refrigerated (0 - 4 °C) prior to analysis.

MONOMERIC ALUMINUM:Samples were immediately analyzed for monomeric Al content.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
1. A stock solution of 1000 mg Al/L was prepared by addition of 6.95 grams of Al(NO3)3·9H2O to 500 mL of Milli-Q water in a volumetric flask. The stock solution was then stored in a plastic container in the dark at 0 - 4°C. The pH of the 1000 mg Al/L stock solution was measured at 3.6 - 3.8.
2. Test concentrations were prepared by adding the appropriate volume of stock solution (1000 mg/L Al) to the dilution (reconstituted) water. A volumetric flask of 1000 mL was filled to approximately 80% of its capacity with dilution water. The aluminum stock solution was then added with a micro-pipette to achieve the nominal concentration. The volume was then completed to 1000 mL with the dilution water. The pH was checked and adjusted to a pH of 6.0 using dilute HCl or NaOH, as appropriate. Each individual concentration was placed in an “air-tight” enclosure which was injected with a pre-determined percent volume (2.3 – 2.6%) of CO2). Solutions were held, as described, for a 3-hour equilibration period in an environmental chamber maintained at test temperature.

Test organisms (species):

other: Brachionus calyciflorus

Details on test organisms:

TEST ORGANISM
- Common name:/
- Justification for species other than prescribed by test guideline:Rotifers play a large role in freshwater ecosystems and also have a very short life-cycle. Use of this test organism qualifies as a “level of organization (Phylum) not otherwise represented” as part of the minimum taxonomic requirements for calculation of a predicted no effect concentration (PNEC) for the freshwater aquatic compartment, within the context of a species sensitivity distribution approach (European Commission 2003).
- Source:cysts from Florida Aqua Farms, Dade City, Florida, USA
- Feeding during test: yes
- Food type:Nannochloropsis occulata. lgal cells were obtained on an agar base (from Florida Aqua Farms, Dade City, FL, USA) and rinsed off in moderately-hard reconstituted lab water (dilution water). Algal density was then determined using a hemacytometer. The algal suspension was diluted with dilution water to obtain the appropriate feeding concentration.
- Amount:3.0 x 10^6 cells per milliliter.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

/

Hardness:

100 mg/L as CaCO3

Test temperature:

25 ± 2 °C

pH:

6.1-6.5

Dissolved oxygen:

> 60%

Salinity:

/

Nominal and measured concentrations:

nominal concentrations: 0, 100, 200, 400, 800, 1600 μg/L
Average measured total Al concentration: <10, 97, 200, 405, 820, 1636 μg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:15mL glass tube
- Type (delete if not applicable): open in a sealed chamber
- Material, size, headspace, fill volume: glass filled with 12mL of test solution
- No. of organisms per vessel:6
- No. of vessels per concentration (replicates):4
- No. of vessels per control (replicates):4


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution/control water used in the toxicity test was moderately hard reconstituted laboratory water prepared by adding the appropriate reagent grade salts to Milli-Q water to achieve the desired nominal hardness value of 100 mg/L as CaCO3. Reconstituted water was
prepared as detailed in standard USEPA methods (USEPA 2002). Reconstituted water was warmed to test temperature (25 ± 2°C) prior to use in the toxicity test.
- Alkalinity: 16mg/L as CaCO3
- Conductivity: 384 μmhos/cm
- Total residual chlorine: <0.50 mg/L
- Ammonia: <1.0 mg/L
- Adjustment of pH: The pH was checked and adjusted to a pH of 6.0 using dilute HCl or NaOH, as appropriate
- Photoperiod:dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations of live and dead rotifers were conducted at test termination. From the final numbers, intrinsic growth rate was calculated.

VEHICLE CONTROL PERFORMED: no

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

405 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

other: number of individuals

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

820 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

other: number of individuals

Duration:

48 h

Dose descriptor:

LC10

Effect conc.:

303.7 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

other: number of individuals

Remarks on result:

other: CL: 84.7-1088.6 μg/L

Duration:

48 h

Dose descriptor:

other: LC20

Effect conc.:

431 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

other: number of individuals

Remarks on result:

other: CL: 177.2-1048.4 μg/L

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

863.5 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

other: number of individuals

Remarks on result:

other: CL: 545.1-1367.7 μg/L

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

405 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

other: intrinsic growth rate

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

820 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

other: intrinsic growth rate

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

441.2 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

other: intrinsic growth rate

Remarks on result:

other: CL: 284.8-683.5 μg/L

Duration:

48 h

Dose descriptor:

other: EC20

Effect conc.:

523.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

other: intrinsic growth rate

Remarks on result:

other: CL: 373.8-734.0 μg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

736.4 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

other: intrinsic growth rate

Remarks on result:

other: CL: 581.1-933.4 μg/L

Results with reference substance (positive control):

/

Reported statistics and error estimates:

STATISTICAL ANALYSIS
Statistical analysis was performed using measured average total Al concentrations. Differences in the number of individuals and the instrinsic rate of growth at test termination were evaluated using a statistical computer package (Comprehensive Environmental Toxicity Information System [CETIS], Tidepool Scientific Software, McKinleyville, CA, USA and Toxicity Relationship Analysis Program [TRAP], Duluth, MN, USA). Intrinsic growth rates were calculated as r = (lnNt – lnN0)/T, where r = intrinsic rate of growth, ln Nt = natural logarithm of number of rotifers after 48 hours, ln N0 = natural logarithm of rotifers at test initiation (6), and T = time (48 hrs) (Snell and Moffat, 1992). If the data met the assumptions of normality and homogeneity, the NOEC and LOEC were estimated using an analysis of variance to compare (p = 0.05) organism performance in the experimental treatments with that observed in the control. The effective concentrations to reduce population by 10%, 20%, or 50% relative to control performance (EC10/EC20/EC50) were estimated using threshold sigmoid regression analysis. Exposure concentrations were log-transformed before determination of the EC10, EC20, and EC50 values.

Conclusions:

The test had measured exposure concentrations ranging from 5 to 1636 μg total Al/L and resulted in a significant growth effect at the two highest treatments. The study resulted in a no observable effect concentration [NOEC] of 405 μg/L total Al and a lowest observable effect concentration [LOEC] of 820 μg/L total Al for growth (based upon both final number of individuals and growth rate). The effective concentrations to reduce growth (most sensitively as the final number of individuals) by 10% and 20% relative to control performance (EC10 and EC20 with 95% confidence intervals) was 304 (85 – 1089) and 431 (177 – 1048) μg/L total Al, respectively.

Executive summary:

As part of an environmental program designed to provide data for the setting of water quality standards, data describing the chronic toxicity of aluminum to a variety of aquatic organisms are needed. Aluminum toxicity is a function of the chemical species of aluminum present in the water and this speciation is a function of the physico/chemical properties (e.g., pH, temperature, hardness) of the water. Much of the extant aluminum toxicity data was generated under “acid” water conditions (pH 4.5-5.5); therefore, efforts are underway to develop data describing the chronic toxicity of aluminum to aquatic organisms at hydrogen ion concentrations (i.e., pH) typical of natural environmental conditions. The freshwater rotifer provides a 48-hour life-cycle testing opportunity. In this study, the freshwater rotifer, Brachionus calyciflorus, was exposed to a series of aluminum concentrations for 48 hours starting as freshly hatched cysts. Use of this test organism qualifies as a level of organization (Phylum) not currently represented in the species sensitivity distribution used in the derivation of a predicted no-effect concentration (PNEC) for the freshwater aquatic compartment (European Commission 2003). To allow for potential changes in aluminum speciation, exposure solutions were aged for a 3-hour equilibrium period. Nominal test concentrations ranged from 0 to 1600 μg/L Al and total, dissolved, and monomeric aluminum were measured throughout the test. As the formation of insoluble chemical species was apparent through low dissolved and monomeric measurements, total Al was used to interpret the biological data in this study. The test had measured exposure concentrations ranging from 5 to 1636 μg total Al/L and resulted in a significant growth effect at the two highest treatments. The study resulted in a no observable effect concentration [NOEC] of 405 μg/L total Al and a lowest observable effect concentration [LOEC] of 820 μg/L total Al for growth (based upon both final number of individuals and growth rate). The effective concentrations to reduce growth (most sensitively as the final number of individuals) by 10% and 20% relative to control performance (EC10 and EC20 with 95% confidence intervals) was 304 (85 – 1089) and 431 (177 – 1048) μg/L total Al, respectively.

Reference 16

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

12/2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

other: Newman, J.P., Jr. (1975). The effects of heavy metals on the asexual reproduction of the annelid Aeolosoma headleyi Beddard (1888). M.S. thesis. Virginia Polytechnic Institute and State University, Blacksburg, V.A.

Qualifier:

equivalent or similar to guideline

Guideline:

other: Niederlehner, B.R., Buikema, A.L., Jr., Pittinger, C.A., and Cairns, J., Jr. (1984). Effects of cadmium on the population size of a benthic invertebrate Aeolosoma headleyi (Oligochaeta): Environmental Toxicology and Chemistry 2, 255-262.

Principles of method if other than guideline:

/

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): Aluminum nitrate nonahydrate
- Molecular formula (if other than submission substance): (Al(NO3))3.9H2O
-Source: J.T. Baker, Philipsburg, NJ, USA
- Analytical purity:100.6%
- Impurities (identity and concentrations): Ca = 0.001%, Cl = 0.001%, Mg = 0.0002%, K = 0.0005%, Na = 0.003%, Pb = 0.0006%, Fe = 0.001%
- Purity test date: 20/10/2009
- Lot/batch No.:H43606
- Storage condition of test material:stored and sealed in its original container at room temperature

Analytical monitoring:

yes

Details on sampling:

- Analytical samples from each treatment were collected

- Sampling method:

TOTAL ALUMINUM
“New” waters were taken directly from the beaker using a 20 mL syringe after the 3-hour equilibrium period. Approximately 5 mL was drawn into the syringe to rinse the inside of the syringe and then expunged. Then 15 mL of sample was drawn into the syringe and injected into a 15 mL polypropylene conical tube. Samples of “old” waters were taken from a composite of each replicate from each concentration. “Old” water was pulled off from the top half/layer of the water column within each test chamber.

DISSOLVED ALUMINUM
Approximately 20 mL was drawn into the syringe of which 5 mL was pushed through the filter to waste and the remaining 15 mL was collected into a 15-mL polypropylene conical test tube.

MONOMERIC ALUMINUM
Following collection of a sample for dissolved aluminum analysis, the same filter was used to filter approximately 15 mL aliquots for monomeric aluminum analysis. Due to reduced sample volumes, no monomeric metals samples were taken from “old” waters.

- Sample storage conditions before analysis:

TOTAL AND DISSOLVED ALUMINUM:Samples were then preserved with trace metal grade nitric acid (AR-ACS grade, Mallinckrodt Chemical, Hazelwood, MO, USA) to pH < 2 and refrigerated (0 - 4 °C) prior to analysis.

MONOMERIC ALUMINUM:Samples were immediately analyzed for monomeric Al content.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
A stock solution of 1000 mg Al/L was prepared by addition of 6.96 grams of Al(NO3)3·9H2O to 500 mL of Milli-Q water in a volumetric flask. The stock solution was then stored in a plastic container in the dark at 0 - 4°C. The pH of the 1000 mg Al/L stock solution was measured at 3.6 - 3.8. All concentrations within this report are expressed as micrograms Al per liter (μg/L Al).
Test concentrations were prepared by adding the appropriate volume of stock solution (1000 mg/L Al) to the dilution (reconstituted) water. A volumetric flask was filled to approximately 80% of its capacity with dilution water. Next, the aluminum stock solution was added to the volumetric flask to achieve the desired nominal concentration. The volume was then completed to 250 mL with the dilution water. The pH was checked and adjusted to a pH of 6.0 using dilute HCl or NaOH, as appropriate. The waters were placed in an enclosed box (injected with a pre-determined percent volume (2.3 – 2.6%) of CO2) for 3 hours equilibration period in an environmental chamber at test temperature.

Test organisms (species):

other aquatic worm: Aeolosoma sp.

Details on test organisms:

TEST ORGANISM
- Justification for species other than prescribed by test guideline:Use of this test organism qualifies as a level of organization (Phylum) not otherwise represented as part of the minimum taxonomic requirements for calculation of a predicted no effect concentration (PNEC) for the freshwater aquatic compartment, within the context of a species sensitivity distribution (SSD) approach (European Commission 2003). The choice of Aeolosoma sp. as a testing species was based upon its commercial availability and its inclusion in the latest update to the USEPA’s cadmium ambient water quality criteria.
- Source: Carolina Biological Supply (Burlington, NC, USA).
- Feeding during test: yes
- Food type:nfusoria (slurry made from 3-5 rabbit pellets [Animal House, Corvallis, OR, USA]
- Amount:200 μL Infusoria in 300-400 mL control/dilution water)

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

17 d

Post exposure observation period:

/

Hardness:

48 mg/L as CaCO3

Test temperature:

25± 2°C

pH:

5.9-6.1

Dissolved oxygen:

/

Salinity:

/

Nominal and measured concentrations:

nominal concentrations: 0, 100, 500, 1000, 2000, 4000 μg/L
Average measured total Al concentrations: 0.5, 84.5, 480.6, 962.5, 2156.9, 4460.6 μg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:six-well polystyrene culture plates
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume:polystyrene, 100.2 mL filled with test solution
- Renewal rate of test solution (frequency/flow rate): Water renewals on a Monday, Wednesday, Friday
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates):4
- No. of vessels per control (replicates):4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution/ control water used in the toxicity test was soft reconstituted laboratory water prepared by adding the appropriate reagent grade salts (CaSO4, MgSO4, KCl, and NaHCO3; USEPA 2002) to Milli-Q water to achieve the desired nominal hardness and alkalinity values of 50 and 30 mg/L as CaCO3, respectively. Reconstituted water was prepared as detailed in standard USEPA methods (USEPA 2002). Two batches of reconstituted laboratory water were used for the duration of the study.
- Chlorine:<0.05mg/L
- Alkalinity: 8mg/L as CaCo3
- Ca/mg ratio: 2.72
- DOC: <0.50mg/L
- Ammonia: <1.0mg/L
- Calcium: 12.5 mg/L
- Magnesium: 4.6mg/L
- Sodium: 13.3mg/L
- Chloride: 14.7mg/L
- Sulfate: 48.7mg/L
- Intervals of water quality measurement: Certain water quality parameters (i.e., temperature, dissolved oxygen [DO], and pH) were measured in each concentration at test initiation, at renewal, (in both freshly prepared renewal waters and old waters), and at test termination. Conductivity was also measured in the freshly-prepared renewal water at test initiation and renewal.

OTHER TEST CONDITIONS
- Adjustment of pH:The pH was checked and adjusted to a pH of 6.0 using dilute HCl or NaOH, as appropriate.
- Photoperiod:6:8 hour light:dark cycle
- Light intensity:cool-white fluorescent lights at ~50 foot candles.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Observations of the number of organisms were made on days 3, 5, 7, 10, 12, 14, and 17. On those days, organisms were counted and transferred from their old chambers into newly- prepared test chambers. No observations regarding the different stages of pygidial budding occurred during the test and the total of all organisms at test termination were used in the summary of total population size.

Reference substance (positive control):

no

Duration:

17 d

Dose descriptor:

NOEC

Effect conc.:

962.5 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

other: population size

Duration:

17 d

Dose descriptor:

LOEC

Effect conc.:

2 156.9 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

other: population size

Duration:

17 d

Dose descriptor:

EC10

Effect conc.:

987.9 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

other: population size

Remarks on result:

other: CL: 336.6-2899.4 μg/L

Duration:

17 d

Dose descriptor:

other: EC20

Effect conc.:

1 235.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

other: population size

Remarks on result:

other: CL: 649.7-2347.9 μg/L

Duration:

17 d

Dose descriptor:

EC50

Effect conc.:

1 923.9 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

other: population size

Remarks on result:

other: CL : 1368.1-2705.3 μg/L

Details on results:

/

Results with reference substance (positive control):

/

Reported statistics and error estimates:

STATISTICAL ANALYSIS
Statistical analysis was performed using measured average total Al concentrations of newly prepared solutions. Differences in the population sizes at test termination were evaluated using statistical computer packages (Comprehensive Environmental Toxicity Information System [CETIS], Tidepool Scientific Software, McKinleyville, CA, USA and Toxicity Relationship Analysis Program [TRAP], Duluth, MN, USA). If the data met the assumptions of normality and homogeneity, the no-observable effect concentration (NOEC) and lowest- observable effect concentration (LOEC) were estimated using an analysis of variance to compare (p = 0.05) organism performance in the experimental treatments with that observed in the control. The effective concentrations to reduce population size by 10%, 20%, or 50% relative to control performance (EC10/EC20/EC50) were estimated using threshold sigmoid regression analysis. Exposure concentrations were log-transformed before determination of the EC10, EC20, and EC50 values.

Table: Population size data

Average Total Aluminum (μg/l)	Average population (± Std Dev)
0.5	65.3 ± 5.3
84.5	66.0 ± 13.8
480.6	78.3 ± 9.2
962.5	65.0 ± 15.9
2156.9	27.5 ± 6.8
4460.6	3.3 ± 2.6

Conclusions:

Total measured exposure concentrations ranged from 0.5 to 4461 μg Al/L and resulted in a significant effect on organism population growth at the highest two treatments. The study resulted in a no observable effect concentration [NOEC] of 963 μg/L total Al and a lowest observable effect concentration [LOEC] of 2157 μg/L total Al for growth. The effective concentrations to reduce growth by 10% and 20% relative to control performance (EC10 and EC20 with 95% confidence intervals) was 988 (337 – 2899) and 1235 (650 – 2348) μg/L total Al, respectively.

Executive summary:

As part of an environmental program designed to provide data for the setting of water quality standards, data describing the chronic toxicity of aluminum to a variety of aquatic organisms are needed. Aluminum toxicity is a function of the chemical species of aluminum present in the water and this speciation is a function of the physico/chemical properties (e.g., pH, temperature, hardness) of the water. Much of the existing aluminum toxicity data was conducted under “acid” water conditions (pH 4.5-5.5); therefore, efforts are underway to develop data describing the chronic toxicity of aluminum to aquatic organisms at hydrogen ion concentrations (i.e., pH) typical of natural environmental conditions. The study reported herein determines the chronic toxicity of aluminum, at a pH of 6.0, to an aquatic oligochaete, Aeolosoma sp. Aeolosoma sp. is a representative of family Oligochaeta. Use of this test organism qualifies as a level of organization (Phylum) not currently represented in the species sensitivity distribution used in the derivation of a predicted no effect concentration (PNEC) for the freshwater aquatic compartment, within the context of a species sensitivity distribution approach (European Commission 2003). In this study, Aeolosoma sp. were exposed to a series of aluminum concentrations for seventeen days, starting as < 24 hour old organisms. To allow for possible changes in aluminum speciation, exposure solutions were aged for a 3-hour equilibrium period prior to organism exposure. Nominal test concentrations ranged from 0 to 4000 μg Al/L and total, dissolved, and monomeric aluminum were measured at test initiation. As the formation of insoluble chemical species was apparent through low dissolved and monomeric measurements (all concentrations measuring below 10 μg Al/L), total Al was used to interpret the biological data in this study. Total measured exposure concentrations ranged from 1 to 4461 μg Al/L and resulted in a significant effect on organism population growth at the highest two treatments. The study resulted in a no observable effect concentration [NOEC] of 963 μg/L total Al and a lowest observable effect concentration [LOEC] of 2157 μg/L total Al for growth. The effective concentrations to reduce growth by 10% and 20% relative to control performance (EC10 and EC20 with 95% confidence intervals) was 988 (337 – 2899) and 1235 (650 – 2348) μg/L total Al, respectively.

Reference 17

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

10/2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: ASTM. 2002. Standard Test Methods for Measuring the Toxicity of Sediment-Associated Contaminants with Fresh Water Invertebrates. E1706-00. In: 2002 ASTM Annual Book of Standards, Biological Effects and Environmental Fate.

Qualifier:

according to guideline

Guideline:

other: USEPA. 2000. Methods for Measuring the Toxicity and Bioaccumulation of Sediment- associated Contaminants with Freshwater Invertebrates. EPA-600-R-99-064. Office of Research and Development, Duluth, MN.

Qualifier:

according to guideline

Guideline:

other: Organisation of Economic Co-operation and Development (OECD). 2004. Sediment- Water Chironomid Toxicity Test Using Spiked Waters. OECD guidelines for the testing of chemicals. Method 219. Paris, France.

Principles of method if other than guideline:

/

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report):reagent grade aluminum nitrate nonahydrate
- Molecular formula (if other than submission substance): (Al(NO3))3.9H2O
-Source: J.T. Baker, Philipsburg, NJ, USA
- Analytical purity: 100.6%
- Lot/batch No.: K34588
- Storage condition of test material: the test substance was stored sealed and in its original container at room temperature.

Analytical monitoring:

yes

Details on sampling:

- Sampling method:
TOTAL ALUMINUM:Analytical samples from each treatment were collected for total recoverable aluminum analysis from “diluter” waters once weekly and from “chamber” test waters once weekly. “Diluter” waters were taken directly from the proportional diluter. “Chamber” waters were taken directly from the test chamber, starting with one replicate during the first sampling event and rotating through replicates as the test continued. Approximately 5 mL was drawn into the syringe to rinse the inside of the syringe and then expunged. Then 15 mL of sample was drawn into the syringe and injected into a 15 mL polypropylene conical tube.
DISSOLVED ALUMINUM:
Analytical samples from each treatment were collected for dissolved (filtered through a 0.22 μm Millex GV (PVDF) filter, (Millipore; Billerica, MA, USA) aluminum analysis at the same time as total samples were taken. Approximately 20 mL was drawn into the syringe of which 5 mL was pushed through the filter to waste and the remaining 15 mL was collected into a 15-mL polypropylene conical test tube.
MONOMERIC ALUMINUM:
Following collection of a sample for dissolved aluminum analysis, the same filter was used to filter approximately 15 mL aliquots for monomeric aluminum analysis.
- Sample storage conditions before analysis:
TOTAL AND DISSOLVED ALUMINUM:
amples were preserved with trace metal grade nitric acid (AR-ACS grade, Mallinckrodt Chemical, Hazelwood, MO, USA) to pH < 2 and refrigerated (0 - 4 °C) prior to analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The highest aluminum exposure concentration was prepared in a 1000 liter holding tank, holding 800 Liter of solution. The tank was filled with laboratory (heated blend) water to 500 Liter, followed by the addition of dilute HCl (25 mL HCl/L deionized water). Next a concentrated stock of aluminum (5.005 grams Al(NO3)3 x 9H20 in 4 L deionized water) with a pH of approximately 3.7 was added and the remaining laboratory water was added (topped at 800 Liters).

Test organisms (species):

other aquatic arthropod: Chironomus riparius

Details on test organisms:

TEST ORGANISM
- Common name:Hyalella azteca
- Justification for species other than prescribed by test guideline: Use of this test organism qualifies as a level of organization (Phylum) to be used as part of the minimum taxonomic requirements for calculation of a predicted no effect concentration (PNEC) for the freshwater aquatic compartment, within the context of a species sensitivity distribution (SSD) approach (European Commission, 2003).
- Source: Aquatic Biosystems, Fort Collings, CO, USA
- Age of parental stock (mean and range, SD): 5-7 days
- Feeding during test: Day 0: 1.0 mL of 1800mg/L YTC (Yeast, Trout Chow, Cereal Leaves) was fed to each chamber. Day 14: feeding was increased to 2.0 mL YTC per test chamber
- Food type: YTC (Yeast, Trout Chow, Cereal Leaves)
- Amount: 1.0 mL of 1800mg/L per day
- Frequency: once a day

ACCLIMATION
- Acclimation period: 2 days
- Acclimation conditions (same as test or not): In order to help acclimate the organisms to test conditions, specifically the pH 6 water, water renewals on the first day occurred with a pH 7 water and on the second day occurred with a pH 6 water.
- Health during acclimation (any mortality observed):Based upon visual observation, the organisms appeared healthy at test initiation.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

28 d

Post exposure observation period:

/

Hardness:

91 ± 8 mg/L as CaCO3

Test temperature:

23 ± 2°C (range: 21-24 °C)

pH:

range: 6.0 - 6.7

Dissolved oxygen:

range: 7.7 - 8.3 mg/L

Salinity:

/

Nominal and measured concentrations:

Nominal concentrations: 312.5, 625, 1250, 2500, 5000 µg/L Al
Measured concentrations: 213, 509, 1158, 2323, 4695 µg/l Al

Details on test conditions:

TEST SYSTEM
- Test vessel:glass jars
- Material, size, headspace, fill volume: 00-mL glass jars containing 20 mL of sediment and approximately 350 mL of overlying water.
- Type of flow-through (e.g. peristaltic or proportional diluter): proportional diluter
- Renewal rate of test solution (frequency/flow rate): 5 mL/min providing approximately 14 volume exchanges per replicate chamber per day.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates):12
- No. of vessels per control (replicates):12

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution/control water used for this study was laboratory blended water (well water blended with reverse osmosis-treated water with a targeted hardness of 80 – 120 mg/L as CaCO3). The dilution/control water was initially adjusted from an approximate pH of 8.2 to 7.0 – 7.2 using a Mariotte drip of dilute HCl. The water was then pumped into a secondary holding tank where the water was adjusted to a pH of 6.0 by control of a pH pump control system with proportional output (Etatron DLX pH-Rx/MBB, Rome Italy). The final holding tank of dilution/control water was continuously mixed with a recirculating pump prior to it being delivered to the flow-through diluter system.
- Dissolved organic carbon: 0.51 mg/L
- Alkalinity: 19 ± 5 mg/L as CaCO3
- Ca/mg ratio: 2.21
- Total residual chlorine: <0.05 mg/L
- Ammonia: <1.0 mg/L
- Calcium: 21.9 mg/L
- Magnesium: 9.90 mg/L
- Sodium: 29.7 mg/L
- Potassium: 0.916 mg/L
- Chloride: 102 mg/L
- Sulfate: 1.07 mg/L
- Culture medium different from test medium: Culture medium has pH 7, while the test medium pH 6
- Intervals of water quality measurement: Certain water quality parameters (temperature, dissolved oxygen [DO], pH, conductivity) were measured in each concentration, prior to entering the test chambers, at test initiation and daily thereafter. These measurements were classified as “diluter” chemistries. “Chamber” chemistries were measured directly from the exposure chamber. Monitoring of “chamber” chemistries was conducted daily in one alternating replicate from each treatment. Hardness, alkalinity, total residual chlorine, and total ammonia were measured in the dilution water at test initiation and weekly thereafter.

OTHER TEST CONDITIONS
- Adjustment of pH: The solution was then continuously mixed using a recirculating pump while the solution pH was automatically monitored with a pH pump control system with proportional output (Etatron DLX pH-Rx/MBB, Rome Italy) and additional dilute (1 M) HCl was injected to reach and maintain a pH of 6.0 following mixing.
- Photoperiod:16:8 hour light:dark cycle
- Light intensity: ~50 foot candles

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations of live and dead organisms were conducted once daily from initiation until Day 10. At test Day 10, four replicates (replicates I, J, K, L) were taken down and sieved through a 250 μm mesh to obtain a 10-day survival and growth endpoint. In these four replicates, organisms from each replicate were placed into a pre-weighed ceramic crucible (which had been ashed) and dried at 95°C for over 12 hours, and then re-weighed to the nearest 0.01 mg to obtain a dry weight. Following the dry-weight measurement, each crucible was ashed in a muffle furnace (1 hour at 550 °C) to obtain an ash-free dry weight.
From day 10 to day 28: Daily monitoring for emergence. Pair emerged females with available males. Daily monitoring of ovi- position chambers for laying of egg masses. Daily monitoring of egg hatchability. Each concentration taken down separately following 5 days of no emergence.

Reference substance (positive control):

no

Duration:

10 d

Dose descriptor:

NOEC

Effect conc.:

4 281.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

4 281.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

10 d

Dose descriptor:

NOEC

Effect conc.:

1 100.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

growth

Remarks:

Ash free dry weight

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

4 281.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

reproduction

Duration:

10 d

Dose descriptor:

LOEC

Effect conc.:

> 4 281.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 4 281.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

10 d

Dose descriptor:

LOEC

Effect conc.:

2 132.7 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

growth

Remarks:

Ash free dry weight

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 4 281.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

reproduction

Duration:

10 d

Dose descriptor:

EC10

Effect conc.:

2 923 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Remarks on result:

other: CL: 857.0-9997.0 µg/l

Duration:

10 d

Dose descriptor:

EC10

Effect conc.:

971.6 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

growth

Remarks:

Ash free dry weight

Remarks on result:

other: CL: 220.8-4276.0 µg/l

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

1 271.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

reproduction

Remarks on result:

other: CL: 134.7 - 14715.5 µg/l

Duration:

10 d

Dose descriptor:

other: EC20

Effect conc.:

4 691 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Remarks on result:

other: CL: 547.0-40191.0 µg/l

Duration:

10 d

Dose descriptor:

other: EC20

Effect conc.:

1 933.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

growth

Remarks:

Ash free dry weight

Remarks on result:

other: CL: 798.3 - 4681.4 µg/l

Duration:

28 d

Dose descriptor:

other: EC20

Effect conc.:

3 387 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

reproduction

Remarks on result:

other: CL: 1010.0 - 13438.0 µg/l

Details on results:

/

Results with reference substance (positive control):

/

Reported statistics and error estimates:

STATISTICAL ANALYSIS
Statistical analysis was performed using measured average total Al concentrations. Differences in survival (at Day 10), growth (at Day 10), adult emergence, and reproduction (as number of eggs per egg case) were evaluated using a statistical computer package (Comprehensive Environmental Toxicity Information System [CETIS], Tidepool Scientific Software, McKinleyville, CA, USA and Toxicity Relationship Analysis Program [TRAP], Duluth, MN, USA). If the data met the assumptions of normality and homogeneity, the NOEC and LOEC were estimated using an analysis of variance to compare (p = 0.05) organism performance in the experimental treatments with that observed in the control. The effective concentrations to reduce survival, growth, or reproduction by 10% or 20% relative to control performance (EC10/EC20) were estimated using threshold sigmoid regression analysis. Exposure concentrations were log-transformed before determination of the EC10 and EC20 values. Initial parameters input into the TRAP database for logIC50, Y-intercept, and steepness were: 4.2, 335.0, and 0.50, respectively.

Table 1: Summary of total metal analyses (µg/L Al)

Nominal conc.	Total Aluminum
	Total average	Total Std Dev	Total average	Total Std Dev
	with day 0		without day 0
0	7.5*	-	7.5*	-
312.5	233.0	136.3	254.2	136.1
625	490.4	144.5	527.0	122.4
1250	1100.2	295.9	1184.0	230.3
2500	2132.7	436.4	2282.3	255.7
5000	4281.8	752.5	4568.2	294.5

* For calculation of average concentrations, the control water has been given a value of 7.5 μg/L Al (half the detection limit).

Table 2: Summary of dissolved metal analysis (µg/L Al)

Nominal conc.	Dissolved Al (µg/L)
	Dissolved average*	Dissolved Std Dev
0	<15	-
312.5	<15	-
625	<15	-
1250	28	26
2500	21	15
5000	17	21

* For average and standard deviation determinations, samples with values below the detection limit (< 15) are given a value of 7.5 ug/L Al (half the detection limit).

Table 3: Summary of monomeric metal analyses (µg/L Al)

Nominal conc.	Monomeric aluminum (µg/L)
	Monomeric average*	Monomeric Std Dev
0	< 10.8	-
312.5	< 10.8	3.1
625	< 10.8	3.8
1250	< 10.8	4.1
2500	14.8	6.4
5000	38.2	17.5

* For average and standard deviation determinations, samples with values below the detection limit (< 10.8) are given a value of 5.4 ug/L Al (half the detection limit).

Table 4: Survival

Average measured total Al conc. (µg/L)	Proportion survided (±SD) ^1
	Day 10	Day 28 (as adult emergence)
7.5	0.8500 ± 0.06383	0.6833 ± 0.1168
233.0	0.9333 ± 0.07698	0.7667 ± 0.1182
490.4	0.8333 ± 0.1388	0.6833 ± 0.1944
1100.2	0.8667 ± 0.1089	0.8583 ± 0.09718
2132.7	0.8333 ± 0.08607	0.7167 ± 0.1054
4281.8	0.7167 ± 0.2396	0.6167 ± 0.1543

1 Survival is reported as the average survival within a treatment (combination of all replicates within a treatment).

SD = Standard Deviation

Table 5: Organism growth data - Ash free dry weight¹

Average measured total Al concentration (µg/L)	Ash free dry weight (average weight per surviving organism) (in mg ± SD))
7.5	1.149 ± 0.1199
233	1.118 ± 0.1064
490.4	1.279 ± 0.2599
1100.2	0.9965 ± 0.1593
2132.7	0.8677 ± 0.1205
4281.8	0.7413 ± 0.0943

¹ Ash-Free Dry Weight is reported as the average weight per organism (combination of all replicates within a treatment).

* Significantly less than control (p <= 0.05)

SD =standard deviation

Table 6: Organism reproduction data

Average measured total Al conc. (µg/L)	Average reproduction (± SD) (number of eggs/case)
7.5	337.8 ± 125.8
233.0	340.8 ± 137.0
490.4	349.3 ± 83.77
1100.2	288.7 ± 108.7
2132.7	298.2 ± 53.62
4281.8	269.6 ± 42.48

Validity criteria fulfilled:

yes

Conclusions:

The endpoints analyzed were: 10- day survival and growth, adult emergence, and reproduction. No effect on survival was observed at Day 10 or Day 28. Growth at Day 10 (as ash-free dry weight) was determined to be the most sensitive growth endpoint. The study resulted in a no observable effect concentration [NOEC] of 1100.2 μg/L total Al and a lowest observable effect concentration [LOEC] of 2132.7 μg/L total Al for growth (for Day 10 growth). The effective concentrations to reduce growth by 10% and 20% relative to control performance (EC10 and EC20 with 95% confidence intervals) were 971.6 (220.8 – 4276.0) and 1933.2 (798.3 – 4681.4) μg/L total Al, respectively.

Executive summary:

As part of an environmental program designed to provide data for the setting of water quality standards, data describing the chronic toxicity of aluminum to a variety of aquatic organisms is needed. Aluminum toxicity is a function of the chemical species of aluminum present in the water and this speciation is a function of the physico/chemical properties (e.g., pH) of the water. Efforts are underway to develop data describing the chronic toxicity of aluminum to aquatic organisms at hydrogen ion concentrations (i.e., pH) typical of natural environmental conditions (i.e., pH of 6). The study reported herein describes the chronic toxicity of aluminum, at a pH of 6.0, to the midge, Chironomus riparius. Use of this test organism qualifies as a level of organization (Phylum) to be used as part of the minimum taxonomic requirements for calculation of a predicted no effect concentration (PNEC) for the freshwater aquatic compartment, within the context of a species sensitivity distribution approach (European Commission 2003). C. riparius were exposed to a series of aluminum concentrations for 28 days, starting as 1st instar larvae (3 days old). To allow for potential changes in aluminum speciation, exposure solutions were aged for a 3-hour equilibrium period. As the formation of insoluble chemical species was apparent through low dissolved and monomeric measurements, total Al was used to interpret the biological data in this study. Measured pH values in the test averaged 6.0 – 6.1 in waters immediately prior to organism exposure and averaged 6.5 – 6.7 within the test chambers. Nominal test concentrations ranged from 0 to 5,000 μg Al/L and total, dissolved, and monomeric aluminum were measured throughout the test. As the formation of insoluble chemical species was apparent through low dissolved measurements (all concentrations measuring below 66 μg Al/L), total Al was used to interpret the biological data in this study. The test had measured exposure concentrations ranging from 7.5 to 4281.8 μg total Al/L. A short-term endpoint of survival and growth was measured at day 10. The endpoints analyzed for the entire life-cycle test were adult emergence and reproduction. Additional short-term data of survival and growth was quantified at Day 10. There was no effect on organism short-term survival (at Day 10). The short-term growth endpoint at Day 10 (as ash-free dry weight) was affected at the two highest concentrations. Upon comparison of the full life-cycle chronic endpoints of adult emergence and reproduction (hypothesis testing), there was no statistically significant effect in any of the concentrations, resulting in a no-observable effect concentration (NOEC) and a lowest observable effect concentration (LOEC) of 4281.8 μg/L total Al and > 4281.8 μg/L total Al, respectively. Based upon point estimate analysis of the life-cycle endpoints, reproduction was the most sensitive endpoint, resulting in effective concentrations to reduce reproduction by 10% and 20% relative to control performance (EC10 and EC20 with 95% confidence intervals) of 1271.5 (109.4 – 14776.4) and 3387.0 (858.0 – 13373.0) μg/L total Al, respectively.

Reference 18

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

04-05/2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: ASTM. 2002. Standard Test Methods for Measuring the Toxicity of Sediment-Associated Contaminants with Fresh Water Invertebrates. E1706-00. In: 2002 ASTM Annual Book of Standards, Biological Effects and Environmental Fate.

Qualifier:

according to guideline

Guideline:

other: USEPA. 2000. Methods for Measuring the Toxicity and Bioaccumulation of Sediment- associated Contaminants with Freshwater Invertebrates. EPA-600-R-99-064. Office of Research and Development, Duluth, MN.

Principles of method if other than guideline:

/

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report):reagent grade aluminum nitrate nonahydrate
- Molecular formula (if other than submission substance): (Al(NO3))3.9H2O
-Source: J.T. Baker, Philipsburg, NJ, USA
- Analytical purity: 100.6%
- Lot/batch No.: K34588
- Storage condition of test material: the test substance was stored sealed and in its original container at room temperature.

Analytical monitoring:

yes

Details on sampling:

- Sampling method:
TOTAL ALUMINUM: Analytical samples from each treatment were collected for total recoverable aluminum analysis from “new” waters once weekly and from “old” test waters once weekly. “New” waters were taken directly from the proportional diluter. “Old” waters were taken directly from the test chamber, starting with one replicate during the first sampling event and rotating through replicates as the test continued. Approximately 5 mL was drawn into the syringe to rinse the inside of the syringe and then expunged. Then 15 mL of sample was drawn into the syringe and injected into a 15 mL polypropylene conical tube.
DISSOLVED ALUMINUM:
Analytical samples from each treatment were collected for dissolved (filtered through a 0.22 μm Millex GV (PVDF) filter, (Millipore; Billerica, MA, USA) aluminum analysis at the same time as total samples were taken. Approximately 20 mL was drawn into the syringe of which 5 mL was pushed through the filter to waste and the remaining 15 mL was collected into a 15-mL polypropylene conical test tube.
MONOMERIC ALUMINUM:
Following collection of a sample for dissolved aluminum analysis, the same filter was used to filter approximately 15 mL aliquots for monomeric aluminum analysis.
- Sample storage conditions before analysis:
TOTAL AND DISSOLVED ALUMINUM:
amples were preserved with trace metal grade nitric acid (AR-ACS grade, Mallinckrodt Chemical, Hazelwood, MO, USA) to pH < 2 and refrigerated (0 - 4 °C) prior to analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The highest aluminum exposure concentration was prepared in a 1000 liter holding tank, holding 800 Liter of solution. The tank was filled with laboratory (heated blend) water to 500 Liter, followed by the addition of dilute HCl (25 mL HCl/L deionized water). Next a concentrated stock of aluminum (5.005 grams Al(NO3)3 x 9H20 in 4 L deionized water) with a pH of approximately 3.7 was added and the remaining laboratory water was added (topped at 800 Liters).

Test organisms (species):

other aquatic crustacea: Hyalella azteca

Details on test organisms:

TEST ORGANISM
- Common name:Hyalella azteca
- Justification for species other than prescribed by test guideline: Use of this test organism qualifies as a level of organization (Phylum) to be used as part of the minimum taxonomic requirements for calculation of a predicted no effect concentration (PNEC) for the freshwater aquatic compartment, within the context of a species sensitivity distribution (SSD) approach (European Commission, 2003).
- Source: Aquatic Biosystems, Fort Collings, CO, USA
- Age of parental stock (mean and range, SD): 5-7 days
- Feeding during test: Day 0: 1.0 mL of 1800mg/L YTC (Yeast, Trout Chow, Cereal Leaves) was fed to each chamber. Day 14: feeding was increased to 2.0 mL YTC per test chamber
- Food type: YTC (Yeast, Trout Chow, Cereal Leaves)
- Amount: 1.0 mL of 1800mg/L per day
- Frequency: once a day

ACCLIMATION
- Acclimation period: 2 days
- Acclimation conditions (same as test or not): In order to help acclimate the organisms to test conditions, specifically the pH 6 water, water renewals on the first day occurred with a pH 7 water and on the second day occurred with a pH 6 water. Based upon visual observation, the organisms appeared healthy at test initiation.
- Health during acclimation (any mortality observed):Based upon visual observation, the organisms appeared healthy at test initiation.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

42 d

Post exposure observation period:

/

Hardness:

95 ± 6 mg/L as CaCO3

Test temperature:

23 ± 2°C (range: 22-25 °C)

pH:

range: 5.9 - 6.4

Dissolved oxygen:

range: 8.1 - 8.2 mg/L

Salinity:

/

Nominal and measured concentrations:

Nominal concentrations: 28.1, 56.3, 112.5, 225, 450 µg/L Al
Measured concentrations: 29.7, 55.1, 129.7, 243.2, 485.6 µg/l Al

Details on test conditions:

TEST SYSTEM
- Test vessel:glass jars
- Material, size, headspace, fill volume: 500-mL glass jars containing 20 mL of sediment and approximately 350 mL of overlying water.
- Type of flow-through (e.g. peristaltic or proportional diluter): proportional diluter
- Renewal rate of test solution (frequency/flow rate): 5 mL/min providing approximately 14 volume exchanges per replicate chamber per day.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates):12
- No. of vessels per control (replicates):12

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution/control water used for this study was laboratory blended water (well water blended with reverse osmosis-treated water with a targeted hardness of 80 – 120 mg/L as CaCO3). The dilution/control water was initially adjusted from an approximate pH of 8.2 to 7.0 – 7.2 using a Mariotte drip of dilute HCl. The water was then pumped into a secondary holding tank where the water was adjusted to a pH of 6.0 by control of a pH pump control system with proportional output (Etatron DLX pH-Rx/MBB, Rome Italy). The final holding tank of dilution/control water was continuously mixed with a recirculating pump prior to it being delivered to the flow-through diluter system.
- Dissolved organic carbon: 0.51 mg/L
- Alkalinity: 19 ± 5 mg/L as CaCO3
- Ca/mg ratio: 2.21
- Total residual chlorine: <0.05 mg/L
- Ammonia: <1.0 mg/L
- Calcium: 21.9 mg/L
- Magnesium: 9.90 mg/L
- Sodium: 29.7 mg/L
- Potassium: 0.916 mg/L
- Chloride: 1.2 mg/L
- Sulfate: 1.07 mg/L
- Culture medium different from test medium: Culture medium has pH 7, while the test medium pH 6
- Intervals of water quality measurement: Certain water quality parameters (temperature, dissolved oxygen [DO], pH, conductivity) were measured in each concentration, prior to entering the test chambers, at test initiation and daily thereafter. These measurements were classified as “new” chemistries. “Old” chemistries were measured directly from the exposure chamber. Monitoring of “old” chemistries was conducted daily in one alternating replicate from each treatment. Hardness, alkalinity, total residual chlorine, and total ammonia were measured in the dilution water at test initiation and weekly thereafter.

OTHER TEST CONDITIONS
- Adjustment of pH: The solution was then continuously mixed using a recirculating pump while the solution pH was automatically monitored with a pH pump control system with proportional output (Etatron DLX pH-Rx/MBB, Rome Italy) and additional dilute (1 M) HCl was injected to reach and maintain a pH of 6.0 following mixing.
- Photoperiod:16:8 hour light:dark cycle
- Light intensity: ~50 foot candles

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations of live and dead organisms were conducted once daily from initiation to termination on Day 42. At day 35 and 42 adults and young were quantified.

Reference substance (positive control):

no

Duration:

42 d

Dose descriptor:

NOEC

Effect conc.:

453.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

53.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

growth

Remarks:

biomass

Duration:

42 d

Dose descriptor:

NOEC

Effect conc.:

232.6 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

growth

Remarks:

biomass

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

123.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

growth

Remarks:

dry weight

Duration:

42 d

Dose descriptor:

NOEC

Effect conc.:

232.6 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

growth

Remarks:

dry weight

Duration:

42 d

Dose descriptor:

NOEC

Effect conc.:

232.6 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

reproduction

Duration:

42 d

Dose descriptor:

LOEC

Effect conc.:

> 453.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

123.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

growth

Remarks:

biomass

Duration:

42 d

Dose descriptor:

LOEC

Effect conc.:

453.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

growth

Remarks:

biomass

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

232.6 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

growth

Remarks:

dry weight

Duration:

42 d

Dose descriptor:

LOEC

Effect conc.:

453.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

growth

Remarks:

dry weight

Duration:

42 d

Dose descriptor:

LOEC

Effect conc.:

453.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

142.6 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

growth

Remarks:

biomass

Remarks on result:

other: CL: 66.8-304.2 µg/L

Duration:

42 d

Dose descriptor:

EC10

Effect conc.:

227.7 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

growth

Remarks:

biomass

Remarks on result:

other: CL: 59.2-875.9 µg/L

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

152.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

growth

Remarks:

dry weight

Remarks on result:

other: CL: 128.3 - 375.1 µg/L

Duration:

42 d

Dose descriptor:

EC10

Effect conc.:

219.4 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

growth

Remarks:

dry weight

Remarks on result:

other: CL: 128.3-375.1 µg/L

Duration:

42 d

Dose descriptor:

EC10

Effect conc.:

170.6 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

reproduction

Remarks on result:

other: CL: 126.8-229.6 µg/L

Details on results:

/

Results with reference substance (positive control):

/

Reported statistics and error estimates:

Statistical analysis was performed using measured average total Al concentrations. Differences in survival (at Days 28, 35, and 42), growth (at Days 28 and 42), and reproduction (at Days 35 and 42) were evaluated using a statistical computer package (Comprehensive Environmental Toxicity Information System [CETIS], Tidepool Scientific Software, McKinleyville, CA, USA and Toxicity Relationship Analysis Program [TRAP], Duluth, MN, USA). If the data met the assumptions of normality and homogeneity, the NOEC and LOEC were estimated using an analysis of variance to compare (p = 0.05) organism performance in the experimental treatments with that observed in the control. The median-lethal concentration (LC50) was estimated using linear interpolation. The effective concentrations to reduce survival, growth, or reproduction by 10%, 20%, or 50% relative to control performance (EC10/EC20/EC50) were estimated using threshold sigmoid regression analysis. Exposure concentrations were log-transformed before determination of the EC10, EC20, and EC50 values.

Table 1: Summary of statistics (continued)

Endpoint	Growth-Biomass		Growth - Dry weight		Reproduction
	28 -day	42 -day	28 -day	42-day	42 -day
EC20 (95% CI)	199.3 (116.7 - 340.2)	291.2 (119.9 - 706.8)	207.0 (179.9 -238.2)	277.7 (191.3 -403.0)	197.7 (154.9 -252.3)
EC50 (95% CI)	387.2 (293.0 -511.7)	474.0 (350.2 -641.5)	380.6 (339.0 -427.3)	443.3 (367.9 -534.1)	264.8 (225.6 -310.8)

Table 2: Summary of Total Metal Analyses (μg/L Al)

Nominal conc.	Total Al (µg/L)
	Total average	Total Std Dev
0	2.2*	-
28.1	30.9	6.8
56.3	53.1	11.8
112.5	123.2	23.9
225	232.6	28.0
450	453.8	66.0

* For calculation of average concentrations, the control water has been given a value of 2.2 μg/L Al (half the detection limit).

Table 3: Summary of Dissolved Metal Analyses (μg/L Al)

Nominal conc.	Dissolved aluminum (µg/L)
	Dissolved average	Dissolved Std Dev.
0	2.2*	-
28.1	3.7	2.1
56.3	6.5	3.4
112.5	11.0	3.0
225	15.6	5.4
450	36.0	19.3

* For calculation of average concentrations, samples below the detection limit are given a value of 2.2 μg/L Al (half the detection limit).

Table 4: Survival

Average measured Al conc. (µg/L)	Proportion survived (±SD)
	Day 28	Day 35	Day 42
2.2	0.92 ± 0.10	0.84 ± 0.11	0.79 ± 0.15
30.9	0.83 ± 0.07	0.71 ± 0.15	0.69 ± 0.16
53.1	0.88 ± 0.07	0.86 ± 0.07	0.85 ± 0.08
123.2	0.79 ± 0.22	0.78 ± 0.27	0.75 ± 0.28
232.6	0.92 ± 0.07	0.80 ± 0.09	0.79 ± 0.11
453.8	0.92 ± 0.08	0.90 ± 0.11	0.88 ± 0.14

Table 5: Organism growth data (Biomass)

Average measured total Al conc (µg/L)	Average dry weight per original organism in mg ( ±SD)^1
	Day 28	Day 42
2.2	0.419  ± 0.05	0.446  ± 0.09
30.9	0.379  ±   0.03	0.409  ± 0.07
53.1	0.367  ± 0.04	0.507  ± 0.05
123.2	0.321  ± 0.04*	0.486  ± 0.14
232.6	0.295  ± 0.03*	0.415  ± 0.09
453.8	0.155  ± 0.02*	0.248  ± 0.04

1 Biomass (average dry weight per original organism) is reported as an average of all replicates within a treatment.

* Significantly less than control (p <= 0.05). SD = Standard Deviation

Table 6: Organism growth data

Average measured total Al conc. (µg/L)	Dry weight in mg ( ±SD)^1
	Day 28	Day 42
2.2	0.474  ± 0.09	0.569  ± 0.07
30.9	0.446  ± 0.03	0.606  ± 0.07
53.1	0.435  ±   0.06	0.598 ± 0.06
123.2	0.442  ± 0.04	0.692 ± 0.15
232.6	0.338  ± 0.01*	0.525 ± 0.07
453.8	0.183  ± 0.03*	0.288 ± 0.06

1 Dry Weight is reported as the average weight per organism (combination of all replicates within a treatment).

* Significantly less than control (p <= 0.05)

Table 7: Organism reproduction data

Average measured total Al conc. (µg/L)	Average reproduction (± SD) (young/female)^1
2.2	9.30 ± 3.69
30.9	9.36 ± 4.69
53.1	9.16 ± 4.02
123.2	7.89 ± 3.95
232.6	5.85 ± 3.02
453.8	0.46 ± 0.37*

1 Reproduction is reported as the average number of young per female (sum of Day 35 and 42 divided by # of females; combination of all replicates within a treatment).

* Significantly less than control (p <= 0.05)

Validity criteria fulfilled:

yes

Conclusions:

No effect on survival was observed at 28, 35, or 42-days. Growth (as mean dry biomass) at Day 28 was determined to be most sensitive endpoint. The study resulted in a no observable effect concentration [NOEC] of 53.1 μg/L total Al and a lowest observable effect concentration [LOEC] of 123.2 μg/L total Al for growth (as Day 28 mean dry biomass). The effective concentrations to reduce growth by 10% and 20% relative to control performance (EC10 and EC20 with 95% confidence intervals) were 142.6 (66.8 – 304.2) and 199.3 (116.7 – 340.2) μg/L total Al, respectively.

Executive summary:

As part of an environmental program designed to provide data for the setting of water quality standards, data describing the chronic toxicity of aluminum to a variety of aquatic organisms is needed. Aluminum toxicity is a function of the chemical species of aluminum present in the water and this speciation is a function of the physico/chemical properties (e.g., pH) of the water. Efforts are underway to develop data describing the chronic toxicity of aluminum to aquatic organisms at hydrogen ion concentrations (i.e., pH) typical of natural environmental conditions (i.e., pH of 6). The study reported herein describes the chronic toxicity of aluminum, at a pH of 6.0, to the amphipod, Hyalella azteca. Use of this test organism qualifies as a level of organization (Phylum) to be used as part of the minimum taxonomic requirements for calculation of a predicted no effect concentration (PNEC) for the freshwater aquatic compartment, within the context of a species sensitivity distribution approach (European Commission 2003). H. azteca were exposed to a series of aluminum concentrations for forty-two days, starting as 7-9 day old juveniles. To allow for potential changes in aluminum speciation, exposure solutions were aged for a 3-hour equilibrium period. As the formation of insoluble chemical species was apparent through low dissolved and monomeric measurements, total Al was used to interpret the biological data in this study. Measured pH values in the test averaged 5.7 – 6.3 in waters immediately prior to organism exposure and averaged 6.2 – 6.6 within the test chambers. Nominal test concentrations ranged from 0 to 450 μg Al/L and total, dissolved, and monomeric aluminum were measured throughout the test. The test had measured exposure concentrations ranging from 2.2 to 453.8 μg total Al/L and the endpoints analyzed were: 28-day survival and growth, 35- survival and reproduction, and 42-day survival, growth, and reproduction. Out of the full chronic life-cycle (42 day) endpoints of survival, growth, and reproduction, reproduction was the most sensitive endpoint for the entire life-cycle study, resulting in a no observable effect concentration [NOEC] of 232.6 μg/L total Al and a lowest observable effect concentration [LOEC] of 453.8 μg/L total Al. The effective concentrations to reduce reproduction by 10% and 20% relative to control performance (EC10 and EC20 with 95% confidence intervals) were 170.6 (126.8 –229.6) and 197.7 (154.9 – 252.3) μg/L total Al, respectively.

Reference 19

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline available

Principles of method if other than guideline:

Although no standard chronic testing methodology is currently available from the U.S. Environmental Protection Agency (USEPA), ASTM, or the Organisation of Economic Co-operation and Development (OECD) for B. calyciflorus, test methods have been developed and reported in the scientific literature (Snell and Moffat 1992, Grosell et al. 2006, Cardwell et al. 2018). Test were conducted in accordance with these testing procedures

GLP compliance:

yes

Remarks:

The study was carried out in the spirit of US EPA and OECD Good Laboratory Practice (GLP) standards

Specific details on test material used for the study:

The test substance was reagent grade aluminum nitrate nonahydrate (Al(NO3)3x9H2O; CAS number 7784-27-2, Lot #K34588) from J.T. Baker (Center Valley, PA, USA). The test substance had a reported assay purity of 98.7%. Following receipt at OSU AquaTox, the test substance was stored sealed in its original container at room temperature.

Analytical monitoring:

yes

Details on sampling:

Total Metals: Analytical samples from each treatment were collected for total Al analysis from newly prepared waters (after the 3-hr equilibrium period; “new”) at test initiation and from surrogate chemistry vials at test termination “old”. Approximately 5 mL of sample was drawn into the syringe to rinse the inside of the syringe and then discarded. Next, 15 mL of sample was drawn into the syringe and injected into a 15 mL polypropylene conical tube. Samples were preserved with trace metal grade nitric acid (BDH Aristar® Plus; VWR Analytical, Mississauga, ON, Canada) to pH < 2 and refrigerated (1 - 4 °C) prior to analysis.

Dissolved Metals: Analytical samples from each treatment were collected for dissolved (filtered through a 0.20 μm PVDF filter, PALL Life Sciences, Ann Arbor, MI, USA) Al analysis from newly prepared solutions at test initiation. In addition, dissolved Al samples were measured from a subset of the chemistry vials at test termination. Due to the difficulty (filter clogging) in filtering solutions containing the algae food source through 0.2 μm, only a subset was sampled. Approximately 20 mL was drawn into the syringe of which 5 mL was pushed through the filter to waste and the remaining 15 mL was collected into a 15 mL polypropylene conical test tube. Samples were then preserved with trace metal grade nitric acid (BDH Aristar® Plus; VWR Analytical, Mississauga, ON, Canada) to pH < 2 and refrigerated (1 - 4 °C) prior to analysis.

Vehicle:

no

Details on test solutions:

The dilution and control water used in the toxicity tests was reconstituted laboratory water prepared by adding the reagent grade salts (CaSO4·2H2O, MgSO4, KCl, NaHCO3) to deionized water to achieve the desired nominal values. Reconstituted water was prepared as detailed in standard USEPA methods (USEPA 2002). DOC was added as Suwannee River Natural Organic Matter (NOM; catalogue #1R101N; obtained from International Humic Substances Society, St. Paul, MN, USA) to achieve nominal concentrations of 1 mg/L (for hardness 50 mg/L as CaCO3 test) and 2 mg/L (for hardness 100 mg/L as CaCO3 test) based on a composition of 52.47% carbon in the NOM. The dilution water was aerated for the duration of the study, prior to each day’s use. Water quality parameters were measured and are reported. The hardness, alkalinity, total residual chlorine (TRC), and ammonia (NH3) were measured at test initiation in the control/dilution water. Reconstituted water was warmed to test temperature (25 ± 2°C) prior to use in the toxicity tests.
The toxicity tests were pH controlled to achieve the target pH of 6.0. After preparation of each exposure concentration, the pH of each test solution was adjusted, if necessary, using dilute NaOH and/or HCl to the desired pH. The solutions were then equilibrated for 3-hrs.

Test organisms (species):

other: Brachionus calyciflorus

Details on test organisms:

The tests were initiated with newly hatched B. calyciflorus (< 2 hrs old). Due to commercial supply availability, resting cysts were obtained from multiple sources: Florida Aqua Farms (Dade City, Florida, USA) and MicroBio tests (Gent, Belgium). Approximately 16 hours prior to testing, rotifer cysts were placed in 100 mL of the respective laboratory test water (Section 2.4) in glass petri dishes for hatching. The cysts were incubated in a 25°C environmental chamber continuously illuminated at 2150-3770 lux (200-350 ft. candles). At 16 hours incubation time, the cysts were checked and any hatched organisms were removed. The incubation dishes were re-checked every hour until sufficient hatched organisms were available for testing. If an insufficient number of organisms had hatched at each 1-hr observation period, any hatched organisms were removed and the cysts continued to incubate until full hatching occurred. This ensured all organisms were < 2 hrs old prior to test initiation. Test organisms were used immediately upon hydrating and hatching of resting cysts (< 2 hrs old), therefore, no acclimation of the test organisms to the varying control waters occurred.
Test acceptability criteria, appropriate concentration-response relationships, and test sensitivity were achieved in the reference toxicant tests verifying that organism health was optimal prior to the conduct of the tests. Organism health was also assessed prior to use (by visible active movement) and deemed to be in excellent condition.

Test type:

static

Water media type:

freshwater

Remarks:

The dilution/control waters used in the toxicity tests were reconstituted laboratory water prepared by adding the reagent grade salts (CaSO4·2H2O, MgSO4, KCl, NaHCO3) to deionized water to achieve the desired nominal values.

Limit test:

no

Total exposure duration:

48 h

Hardness:

Hardness was test dependent (see additional information)

Test temperature:

25 ± 2 °C in a temperature-controlled environmental chamber

pH:

6.0 (nominal)

Test #1:
new medium: 5.98-6.02
old medium: 6.41-6.56

Test #2:
new medium: 6.02-6.07
old medium: 6.20-6.55

Test #3:
new medium: 5.98-6.08
old medium: 6.11-6.47

Test #4:
new medium: 5.89-5.93
old medium: 5.99-6.63

Test #5:
new medium: 6.05-6.13
old medium: 6.40-6.54

Dissolved oxygen:

Test #1:
new medium: 8.5 mg/L
old medium: 8.5-8.6 mg/L

Test #2:
new medium: 8.7-8.8 mg/L
old medium: 8.2-8.8 mg/L

Test #3:
new medium: 8.5 mg/L
old medium: 8.4-8.7 mg/L

Test #4:
new medium: 8.6-8.7 mg/L
old medium: 8.3-8.6 mg/L

Test #5:
new medium: 8.3-8.4 mg/L
old medium: 8.3-8.5 mg/L

Conductivity:

Test #1:
new medium: 201-248 µS/cm
old medium: 1001-1076 µS/cm

Test #2:
new medium: 423-428 µS/cm
old medium: 1243-1483 µS/cm

Test #3:
new medium: 411-469 µS/cm
old medium: 452-524 µS/cm

Test #4:
new medium: 398-405 µS/cm
old medium: 1264-1423 µS/cm

Test #5:
new medium: 737-833 µS/cm
old medium: 762-870 µS/cm

Nominal and measured concentrations:

Concentrations at start of test
Test #1: 0, 250, 500, 1000, 2000, 4000, 8000 µg Al/L (nominal)
Test #1: <16, 194, 463, 963, 1992, 4042, 8019 µg Al/L (total measured)
Test #1: <16, 120, < 16, 43, 92, 434, <16 µg Al/L (dissolved measured)

Test #2: 0, 187.5, 375, 750, 1500, 3000, 6000 µg Al/L (nominal)
Test #2: <14, 127, 346, 681, 1368, 2822, 5574 µg Al/L (total measured)
Test #2: <14, 115, 25, 23, 77, 20, 217 µg Al/L (dissolved measured)

Test #3: 0, 500, 1000, 2000, 4000, 8000, 16000 µg Al/L (nominal)
Test #3: <0.7, 517, 1033, 2040, 4052, 8047, 16352 µg Al/L (total measured)
Test #3: <0.7, 151, 53, 47, 232, 940, 2506 µg Al/L (dissolved measured)

Test #4: 0, 250, 500, 1000, 2000, 4000, 8000 µg Al/L (nominal)
Test #4: <16, 225, 476, 1016, 2034, 4140, 8355 µg Al/L (total measured)
Test #4: <16, 196, 410, 439, 147, 150, 750 µg Al/L (dissolved measured)

Test #5: 0, 437.5, 875, 1750, 3500, 7000, 14000 µg Al/L (nominal)
Test #5: <16, 399, 868, 1766, 3592, 7299, 13750 µg Al/L (total measured)
Test #5: <16, <16, <16, 59, 46, 103, 629 µg Al/L (dissolved measured)

Details on test conditions:

Each test concentration (treatment) was prepared in a batch and then equally distributed to the test chambers. Four replicate chambers (+ 5 surrogates for chemical measurements) were prepared for each treatment and control. Test concentrations were prepared by adding the appropriate volume of stock solution (5 g/L Al) to the dilution water. A volumetric flask was filled to approximately 80% of its capacity with the dilution water. The Al stock solution was then added with a micro-pipette to achieve the nominal concentration and the volume was completed with the dilution water. The pH of each test solution was then individually adjusted, if necessary, using dilute NaOH and/or HCl to pH 6. Solutions were held for a 3-hour equilibration period at test temperature prior to organism exposure. After the equilibrium period, but before test initiation, solutions were re-adjusted (only if solutions were outside ± 0.2 SU) to the target pH of 6.0 using small amounts of dilute HCl/NaOH.
Control of the pH throughout the 3-hr equilibrium period and the duration of the tests was achieved by exposure in a CO2-enriched environment. Although test vials were capped and had minimal space, the test was conducted in an “air-tight” enclosure with a pre-determined percent volume (2.3 – 2.6%) of CO2 to ensure pH control (Mount and Mount 1991).
Organisms were tested in 20 mL glass Kimax® tubes with plastic screw caps. Each tube contained 18 mL of test solution. The test was conducted under darkness (i.e., no light cycle). Test vials were continuously rocked on a rocking platform (Model 200; VWR, Radnor, PA, USA) at approximately 60 tilts per minute to maintain a homogenous solution. The test chambers were randomly distributed within the test enclosure at test initiation and on Day 1.

The test diet for the toxicity tests was the green microalga, Nannochloropsis occulata. Algal cells were cultured and obtained from an agar base (Florida Aqua Farms, Dade City, FL, USA) and rinsed off in laboratory reconstituted water. Cells were then concentrated by centrifugation and cell density determined using a hemacytometer. The algal suspension was diluted with dilution water to obtain the appropriate concentration to allow for only a minute addition of volume to each test chamber. Following the addition of the test organisms to the test chambers, a predetermined volume of algae (from 99 to 181 μL based upon concentration of algae suspension) was pipetted into each test tube to obtain a final algal concentration of 3.0 x 106 cells per milliliter.

Reference substance (positive control):

yes

Remarks:

copper

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

1 345 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

other: # of individuals

Remarks on result:

other: Test#1; 95%CL 512-3533 µg/L

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

1 594 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

other: # of individuals

Remarks on result:

other: Test #2: 95% CL 560-4534 µg/L

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

2 225 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

other: # of individuals

Remarks on result:

other: Test#3; 95%CL 1426-3470 µg/L

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

3 983 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

other: # of individuals

Remarks on result:

other: Test#4; 95%CL 2965-5351 µg/L

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

1 145 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

other: # of individuals

Remarks on result:

other: Test#5; 95%CL 486-2695 µg/L

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

2 153 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

other: intrinsic growth rate

Remarks on result:

other: Test #1; 95%CL 1624-2853 µg/L

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

3 068 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

other: intrinsic growth rate

Remarks on result:

other: Test#2; 95%CL 1906-4939 µg/L

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

4 081 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

other: intrinsic growth rate

Remarks on result:

other: Test#3; 95%CL 2409-6915 µg/L

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

4 884 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

other: intrinsic growth rate

Remarks on result:

other: Test #4; 95%CL 4119-5792 µg/L

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

1 839 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

other: intrinsic growth rate

Remarks on result:

other: Test#5; 95%CL 1319-2564 µg/L

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

963 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

other: # of individuals

Remarks on result:

other: Test #1

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

2 822 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

other: # of individuals

Remarks on result:

other: Test #2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

2 040 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

other: # of individuals

Remarks on result:

other: Test #3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

4 140 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

other: # of individuals

Remarks on result:

other: Test #4

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1 766 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

other: # of individuals

Remarks on result:

other: Test #5

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1 992 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

other: intrinsic growth rate

Remarks on result:

other: Test #1

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

2 822 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

other: intrinsic growth rate

Remarks on result:

other: Test #2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

2 040 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

other: intrinsic growth rate

Remarks on result:

other: Test #3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

4 140 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

other: intrinsic growth rate

Remarks on result:

other: Test #4

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1 766 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

other: intrinsic growth rate

Remarks on result:

other: Test #5

Details on results:

Intrinsic rates of growth ranged from 1.219 (69 individuals) to 1.648 (163 individuals) in the control exposures, exceeding the control acceptability criteria of 0.65 set by Snell and Moffat (1992). Although the control responses varied between waters, it is not uncommon to see variability due to the reproduction rate of individual organisms (e.g., one additional offspring early in the study reproducing an additional generation). Due to the exceedance of all controls above acceptability, it is not anticipated that any of the control waters negatively affected results. In all tests, the population growth endpoint of final number of individuals was more sensitive than the endpoint of intrinsic rate of growth.
In addition, 48h-EC20 and EC50-values were also calculated for both endpoints and are provided in the final report.

Results with reference substance (positive control):

In order to assess any variation in organism sensitivity between suppliers, reference toxicant tests were performed exposing B. calyciflorus to copper and no significant differences were observed between effects, therefore it was determined that the health and sensitivity of the organisms from different suppliers was sufficient for their use.

Reported statistics and error estimates:

Statistical analysis was performed for each test using measured total Al concentrations from test initiation. Measured total Al concentrations from the “new” waters were used for the statistical analysis, rather than the old solutions. This was deemed a more appropriate confirmation of concentrations, as samples directly from the test chambers were considered to be under representative of concentrations due to the difficulty in consistently removing a homogenized sample from the narrow test vial. Differences in the number of individuals and the intrinsic rate of growth at test termination were evaluated using a statistical computer package (Comprehensive Environmental Toxicity Information System [CETIS], Tidepool Scientific Software, McKinleyville, CA, USA and Toxicity Relationship Analysis Program [TRAP], Duluth, MN, USA).

Intrinsic growth rates were calculated as (Snell and Moffat, 1992): r = (lnNt – lnN0)/T,
where r = intrinsic rate of growth, ln Nt = natural logarithm of number of rotifers after 2 days (48 hours), ln N0 = natural logarithm of number of rotifers at test initiation (6), and T = time (2 days)

If the data met the assumptions of normality and homogeneity, the NOEC and LOEC were estimated using an analysis of variance to compare (p = 0.05) organism performance in the experimental treatments with that observed in the control. The effective concentrations to reduce population growth by 10%, 20%, or 50% relative to control performance (EC10/EC20/EC50) were estimated using threshold sigmoid regression analysis. Mean data points for each exposure (weighted by standard deviations) were used in the TRAP model. Exposure concentrations were log-transformed before determination of the EC10, EC20, and EC50 values.

Water quality parameters for each control/dilution water are provided in the study report. Dilution water was characterized prior to the preparation of new test solutions without organisms. pH was maintained within 0.2 SU of the target pH 6 in the new solutions after the equilibrium period but increased up to 6.6 in the old test waters. For modeling purposes, an average pH was used (calculated as the average hydrogen ion concentrations, followed by conversion to standard units). Measured conductivity also increased in the old waters, most likely due to the addition of algae as a food source. All tests met test acceptability criteria for temperature (25 ± 2°C) and dissolved oxygen (saturation ≥ 60%).

Validity criteria fulfilled:

yes

Remarks:

In all tests, control acceptability criteria (> 80 % survival and > 60% surviving females having 15 or more neonates) were met. Reproduction is reported as the total number of young per original female.

Conclusions:

The studies reported here represent a validation exercise of the Al bioavailability models (BLM and MLR) using laboratory waters, at a pH of 6, with varying hardness and DOC characteristics. The empirical data reported herein allow a comparison of model predictions with observed effects, using an organism not used in model development: B. calyciflorus. Observed effect concentrations (EC10 values with 95% confidence intervals) ranged from 1145 (486 – 2695) μg/L total Al in the pH 6.3, hardness = 185, DOC = 1.33 mg/L water to 3983 (2965 – 5351) μg/L total Al in the pH 6.1, hardness = 105, DOC = 3.77 mg/L water. The BLM predictions in these waters were 698 μg/L and 435 μg/L total Al, respectively, and the MLR predictions were 880 and 797 μg/L total Al, respectively.
B. calyciflorus is a moderately sensitive organism in the chronic database (ranking 5th most sensitive out of 13 species in the species sensitivity distribution (SSD) [Cardwell et al. 2018]), and is slightly more sensitive than C. dubia and P. promelas. The original toxicity test with B. calyciflorus and used in the current Al SSD was performed in water at a pH 6.3, hardness of 100 mg/L as CaCO3, and a DOC of 0.25 mg/L. This test resulted in an EC10 (with 95% confidence intervals) of 303.7 (84.7 – 1088.6) μg/L total Al for the most sensitive endpoint of the final number of individuals. The results of these validation tests demonstrated that an increase in DOC provided a protective effect on Al toxicity, while a protective effect of hardness was not observed. Both the BLM and MLR over predicted toxicity (lower ECx predictions than those observed) in the majority of the waters tested. The MLR predicted ECx values were predominately closer to observed values than the BLM predictions. When including 95% confidence intervals around observed ECx values, the BLM predicted toxicity values (within a factor of two) for two of the five waters tested, and the MLR predicted toxicity values in four of the five waters (within a factor of 2). It should be noted that for B. calyciflorus, there is only a single observation in the chronic database and for BLM modeling, this requires assigning a calibration value and also adjustment of more than one parameter for the factoring of toxicity due to both dissolved and precipitated Al in the BLM model. Although both models slightly over predicted toxicity values, the model predictions would be adequately protective of B. calyciflorus. In addition, the results from these studies are now available for inclusion in the species sensitivity distribution.

Executive summary:

Bioavailability models, presented as both a biotic ligand model (BLM) and a multiple linear regression model (MLR), have been developed for aluminum (Al). These models account for changes in metal bioavailability to aquatic organisms in waters with varying water quality parameters. The models utilized, in part, empirical data obtained from toxicity tests with the fathead minnow,Pimephales promelas, the cladoceran, Ceriodaphnia dubia, and the green algae, Pseudokirchneriella subcapitata, with exposure to Al concentrations in a range of water quality characteristics (e.g., calcium, magnesium, sodium, pH, dissolved organic carbon [DOC]). In an effort to further extend the application of the Al bioavailability models using organisms not previously considered in their development, a series of chronic toxicity tests were conducted using the rotifer, Brachionus calyciflorus. Rotifers (< 2 hr old) were exposed for 48 hrs to Al, at a pH of 6, in five different laboratory waters, representing a range of hardness and DOC conditions (i.e., nominal hardness: 50–180 mg/L as CaCO3and nominal DOC 1.0–4.0 mg/L). Population growth endpoints (measured as both the final number of individuals and the intrinsic growth rate) were assessed at test termination. The effect concentrations (EC10 with 95% confidence intervals) ranged from 1145 (486–2695) μg/L total Al in the high hardness/low DOC water to 3983 (2965–5351) μg/L total Al in the moderate hardness/high DOC water (most sensitive endpoint: final number of individuals). The results indicate a protective effect from increased DOC, while an increase in hardness did not exhibit a protective effect. The rotifer is a moderately sensitive organism in the chronic database (ranking as the 5thmost sensitive out of 13 species reported in Cardwell et al. 2018). It is less sensitive thanC. dubia,P.promelas, andP. subcapitata. The observed 10% and 20% effect concentrations (EC10s and EC20s) from the B. calyciflorus tests were compared to predicted values from the bioavailability models. These results are presented in Figures 1a and b and demonstrate that while both bioavailability models over predicted toxicity in the majority of waters, the Al MLR had closer predicted effects concentrations than the BLM predictions. It should be noted that for B.calyciflorus, there is only a single observation in the chronic database and for BLM modeling, this requires assigning a calibration value and also adjustment of more than one parameter for the factoring of toxicity due to both dissolved and precipitated Al in the BLM model. Although both models over predicted toxicity, the model predictions would be adequately protective of B.calyciflorus. With the studies reported here, additional data points are now available to expand the chronic database and update the bioavailability modeling for this species.

Reference 20

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

other: US EPA, 2002. Method 1002.0: Daphnid, Ceriodaphnia dubia, survival and reproduction test,; chronic toxicity

Deviations:

yes

Remarks:

modifications for testing with aluminium

GLP compliance:

yes

Remarks:

The study was carried out in the spirit of US EPA and OECD Good Laboratory Practice (GLP) standards

Specific details on test material used for the study:

The test substance was reagent grade aluminum nitrate nonahydrate (Al(NO3)3x9H2O; CAS number 7784-27-2, Lot #K34588) from J.T. Baker (Center Valley, PA, USA). The test substance had a reported assay purity of 98.7%.

Analytical monitoring:

yes

Details on sampling:

Total metals: Analytical samples from each treatment were collected for total Al analysis from newly prepared waters (after the 3-hr equilibrium period; “new”) at test initiation, during the conduct of the tests, and from a composite of replicates at test termination (termed “old”). Approximately 5 mL of sample was drawn into the syringe to rinse the inside of the syringe and then discarded. Next, 15 mL of sample was drawn into the syringe and injected into a 15 mL polypropylene conical tube. Samples were preserved with trace metal grade nitric acid (BDH Aristar® Plus; VWR Analytical, Mississauga, ON, Canada) to pH < 2 and refrigerated (1 - 4 °C) prior to analysis.

Dissolved metals: Analytical samples from each treatment were collected for dissolved (filtered through a 0.45 μm Acrodisc Supor PES filter, [Pall Life Sciences; Ann Arbor, MI, USA]) Al analysis from newly prepared solutions at test initiation and once during the conduct of each test (termed “new”). In addition, dissolved Al samples were measured from a composite of replicates at test terminations (termed “old”). Approximately 20 mL was drawn into the syringe of which 5 mL was pushed through the filter to waste and the remaining 15 mL was collected into a 15 mL polypropylene conical test tube. Samples were then preserved with trace metal grade nitric acid (BDH Aristar® Plus; VWR Analytical, Mississauga, ON, Canada) to pH < 2 and refrigerated (1 - 4 °C) prior to analysis.

Vehicle:

no

Details on test solutions:

Stock solutions of 1 g/L Al and 10 g/L Al were prepared separately by adding Al(NO3)3x9H2O to deionized water (18 Ω) in a volumetric flask. The stock solutions were highly acidic with pHs of < 3.8. The stock solutions were stored in plastic containers in the dark at 1 - 6°C until use. Stock solutions were warmed to test temperature (25 ± 2 °C) prior to spiking dilution waters. Varying concentrated stocks were prepared due to the range of Al exposure concentrations in the studies and the necessity of only spiking small aliquots to the waters. All concentrations are expressed in micrograms Al per liter (μg/L Al) either as total or dissolved (defined as < 0.45 μg/L) Al.

Test organisms (species):

Ceriodaphnia dubia

Details on test organisms:

The tests were initiated with <24 hour old neonates from in-house cultures (see culture acclimation in Section 2.4.3). Original cultures of C. dubia were obtained from Aquatic BioSystems (Fort Collins, CO, USA) and in-house cultures have been successfully maintained at OSU AquaTox for 10+ years. Parental organisms were isolated onto brood boards in order to obtain <24 hr old neonates for testing.
Organism health was assessed prior to use (by visible active movement) and deemed to be in excellent condition.

C. dubia are typically cultured at OSU AquaTox in a “moderately hard” reconstituted water (hardness of 100 mg/L as CaCO3 and pH of 7.8 – 8.0). These cultures were used for testing at the 120 mg/L as CaCO3 hardness tests. Three additional acclimated cultures were used for the testing in softer hardness water (60 mg/L as CaCO3) and the high hardness tests (250 and 400 mg/L as CaCO3). All acclimated cultures were successfully maintained in their respective laboratory water for multiple generations (2+ months). Organism survival and reproduction were excellent and organism health was maintained over the period of acclimation.

Test type:

semi-static

Water media type:

freshwater

Remarks:

The dilution/control waters used in the toxicity tests were reconstituted laboratory water prepared by adding the reagent grade salts (CaSO4·2H2O, MgSO4, KCl, NaHCO3) to deionized water to achieve the desired nominal values.

Limit test:

no

Total exposure duration:

7 d

Hardness:

Hardness was test dependent (see additional information)

Test temperature:

25 ± 2 °C in a temperature-controlled environmental chamber

pH:

pH was test dependent (see additional information). In order to maintain target pH values in the tests, pH control was obtained through the use of non-metal- complexing buffers, through the use of weak acid/bases, and control of CO2 in the headspace of the test chambers.
pH 6.3: MES-buffer
pH 7: MOPS-buffer
pH >8: HEPES-buffer

Dissolved oxygen:

> 60%

Nominal and measured concentrations:

Test #1: 0, 62.5, 125, 250, 500, 1000, 2000 µg Al/L (nominal)
Test #1: 6.5 62.5, 128.5, 264.5, 529.0, 1043, 2066 µg Al/L (total measured)
Test #1: 4.8, 59.0, 124.5, 238, 400, 862, 587 µg Al/L (dissolved measured)

Test #2: 0, 312.5, 625, 1250, 2500, 5000 µg Al/L (nominal)
Test #2: 1.5, 315.3, 706, 1412.7, 2801.3, 5702.3 µg Al/L (total measured)
Test #2: 1.3, 253, 518.5, 654, 942, 736.5 µg Al/L (dissolved measured)

Test #3: 0, 902, 2005, 4455, 9900, 22000 µg Al/L (nominal)
Test #3: 19, 853.7; 2099.3, 4876.7, 10536, 23041 µg Al/L (total measured)
Test #3: 18.0, 747, 1015, 1003.5, 642.5, 25.5 µg Al/L (dissolved measured)

Test #4: 0, 625, 1250, 2500, 5000, 10000 µg Al/L (nominal)
Test #4: 5.5, 690, 1367, 2684.7, 5666.7, 10933.7 µg Al/L (total measured)
Test #4: 1.2, 120.5, 164.5, 257.5, 1045.1, 3616.1 µg Al/L (dissolved measured)

Test #5: 0, 625, 1250, 2500, 5000, 10000 µg Al/L (nominal)
Test #5: 8.3, 681.7, 1331.3, 2804.7, 5406, 10858.7 µg Al/L (total measured)
Test #5: 3.3, 469.5, 701.5, 1060, 278, 93.5 µg Al/L (dissolved measured)

Test #6: 0, 750, 1500, 3000, 6000, 12000 µg Al/L (nominal)
Test #6: 7.3, 840, 1618.7, 3161.3, 6592.3, 12762.7 µg Al/L (total measured)
Test #6: 3.8, 691.5, 916, 245, 187, 89.5 µg Al/L (dissolved measured)

Test #7: 0, 500, 1000, 2000, 4000, 8000 µg Al/L (nominal)
Test #7: 1.6, 530.7, 1058.7, 2116.7, 4491, 8740.3 µg Al/L (total measured)
Test #7: 1.6, 527.5, 844, 905, 944, 946.5 µg Al/L (dissolved measured)

Test #8: 0, 1000, 2000, 4000, 8000, 16000 µg Al/L (nominal)
Test #8: 1.6, 1054.3, 2127.7, 4438.3, 8949, 17544 µg Al/L (total measured)
Test #8: 1.6, 1025.5, 1398, 1406.5, 1321.5, 1255.5 µg Al/L (dissolved measured)

Test #9: 0, 125, 250, 500, 1000, 2000 µg Al/L (nominal)
Test #9: 1.6, 128.3, 267.3, 565.3, 1107.3, 2212 µg Al/L (total measured)
Test #9: 3.3, 129.5, 265.5, 578.5, 1129, 2141.5 µg Al/L (dissolved measured)

Details on test conditions:

Reconstituted water was prepared as detailed in standard USEPA methods (USEPA 2002). All waters were prepared with a calcium:magnesium (Ca:Mg) ratio of 0.70 to be consistent with the historic C.dubia dataset as reported in Gensemer et al (2018). DOC was added as Suwannee River Natural Organic Matter (NOM; catalogue #1R101N; obtained from International Humic Substances Society, St. Paul, MN, USA) to achieve nominal DOC concentrations in each test water based on a composition of 48% carbon in the NOM. Each dilution water was aerated for the entirety of each test. Water quality parameters were measured and reported. The hardness, alkalinity, total residual chlorine (TRC), and ammonia (NH3) were measured at test initiation in the control/dilution water. Reconstituted water was warmed to test temperature (25 ± 2°C) prior to use in the toxicity tests.
Exposure concentrations were selected based on historical response data with C. dubia in other waters to cover the range from no effects concentrations to concentrations anticipated to illicit biological effects. Each test concentration (treatment) was prepared in a batch and then equally distributed to the test chambers. Ten replicate chambers were prepared for each treatment and control. Test concentrations were prepared by filling a graduated cylinder to 80% capacity with dilution water, adding the appropriate volume of buffer (for pH), adding the appropriate volume of stock solution (1 g/L or 10 g/L Al), and then completing the volume with the dilution water. The pH of each test solution was then individually adjusted, if necessary, using dilute NaOH and/or HCl to the target pH. Solutions were held for a 3-hour equilibration period at test temperature prior to organism exposure. pH control during the equilibrium periods and duration of the toxicity tests was reported.
Organisms were tested in 30 mL Souffle cups (Dart, Mason, MI, USA). Each test chamber contained 25 mL of test solution. To initiate the tests, neonates (< 24 hours old) from a single adult from the appropriate acclimated culture were distributed into one row (1 neonate for 1 replicate of each treatment) of randomly ordered test chambers. This process was repeated using a new brood of neonates from a single adult for each row of the entire randomization pattern to initiate testing.
Each test chamber was fed 0.3 mL of an algae (Pseudokirchneriella subcapitata) and yeast/trout chow/cereal leaf (YTC) suspension (1:1) at test initiation (prior to introduction of the test organism) and once daily prior to water renewal. On a daily basis, only the original organism was transferred to a freshly prepared test chamber and neonates were counted daily.
Observations of live and dead organisms were conducted on a daily basis from initiation to termination. The number of young was counted daily. Only original live adult organisms were moved to fresh solution daily.

Reference substance (positive control):

yes

Remarks:

sodium chloride

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

678.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

reproduction

Remarks on result:

other: Test#1; 95%CL 298.8-1542.4 µg/L

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

3 494 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

reproduction

Remarks on result:

other: Test #2

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

5 628 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

reproduction

Remarks on result:

other: Test#3; 95%CL 3206-9880 µg/L

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

1 466.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

reproduction

Remarks on result:

other: Test#4; 95%CL 968.5-2221.6 µg/L

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

4 909 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

reproduction

Remarks on result:

other: Test#5; 95%CL 2695-8941 µg/L

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

6 106 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

reproduction

Remarks on result:

other: Test #6

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

2 297 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

reproduction

Remarks on result:

other: Test#7; 95%CL 1575-3350 µg/L

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

2 045 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

reproduction

Remarks on result:

other: Test#8; 95%CL 946-4422 µg/L

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

1 514 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

reproduction

Remarks on result:

other: Test #9

Duration:

7 d

Dose descriptor:

LC10

Effect conc.:

7 554 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

mortality

Remarks on result:

other: Test#8; 95%CL 4165-13701 µg/L

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

264.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

reproduction

Remarks on result:

other: Test #1

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

2 801.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

reproduction

Remarks on result:

other: Test #2

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

4 876.7 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

reproduction

Remarks on result:

other: Test #3

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

690 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

reproduction

Remarks on result:

other: Test #4

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

5 406 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

reproduction

Remarks on result:

other: Test #5

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

3 161.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

reproduction

Remarks on result:

other: Test #6

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

2 116.7 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

reproduction

Remarks on result:

other: Test #7

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

1 054.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

reproduction

Remarks on result:

other: Test #8

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

1 107.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

reproduction

Remarks on result:

other: Test #9

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

8 949 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

mortality

Remarks on result:

other: Test #8

Details on results:

The first toxicity test (Test ID: Al 1185 CDC) performed was conducted as a confirmatory test to the studies performed at the Chilean Mining and Metallurgy Research Center (CIMM; Santiago, Chile) and Universidad Adolfo Ibañez (UAI; Santiago, Chile) and reported in Gensemer et al. (2018). This confirmatory test was performed (nominally) at pH 6.3, hardness 60 mg/L as CaCO3, and DOC 2 mg/L and followed identical testing methods. Following the 7-day exposure period, the resultant EC10 and EC20s (with 95% confidence intervals) were 678.9 (298.8 - 1542.4) and 828.6 (436.8 - 1572.0) μg/L total Al, respectively. The results were quite comparable to those reported in Gensemer et al. (2018) (EC10/EC20 with 95% confidence intervals: 504.4 (226 – 1126) μg/L total Al and 631.3 (362 -1101) μg/L total Al, respectively). A one sample t-test was performed and the values were not statistically different between laboratories. Also, in this test a concurrent moderately hard reconstituted water (MHW; standard culture water) exposure was tested as a comparison to the control/dilution and there was no difference in survival between the two waters, although the reproduction was greater in the MHW. Because the control/dilution water reproduction was significantly higher (average of 33 young/female) than control acceptability criteria, it was deemed that the dilution water and acclimated organisms were acceptable.
No significant effect on survival from Al exposures was observed in the majority of the tests (with the exception of one test (Test ID: Al 1196R CDC) which resulted in an effect at the highest exposure). With this survival data, Al 1196R CDC was the sole test with an observed exposure:response relationship sufficient to calculate 10%, 20%, and 50% lethal effect concentrations (LC10, LC20, LC50). This was an expected outcome as, in our experience, reproduction is consistently found to be the most sensitive endpoint in C. dubia tests with Al. In addition, exposure concentrations were selected to illicit exposure:responses for the calculation of both 10% and 20% effect concentrations for the most sensitive endpoint (reproduction). In all tests, reproduction was the most sensitive endpoint (lowest ECx).
Modeling of reproduction data resulted in qualifiers identified by the TRAP model, in addition to undeterminable confidence intervals. These were identified due to the lack of partial effects concentrations in the datasets and associated steep slopes between the concentration with no effect (NOEC) and the concentration with a reproductive effect (LOEC). According to available TRAP guidance, datasets identified as “exploratory” should be examined on a case-by-case basis to assess the confidence around the result based upon the exposure:response relationship. As the three tests did show a quite significant reproductive effect at the highest concentration, it is believed that these datasets can be included as part of the model refinement effort.
Calculated EC20 and EC50 values are provided in the report.

Results with reference substance (positive control):

In order to assess organism health prior to the conduct of each test, monthly reference toxicant tests were performed using sodium chloride as the toxicant. Test acceptability criteria, appropriate concentration- response relationships, and test sensitivity were achieved in the reference toxicant tests verifying that organism health was optimal prior to the conduct of the tests.

Reported statistics and error estimates:

Statistical analysis was performed using measured total Al concentrations from “new” waters. This was deemed a more appropriate confirmation of concentrations (compared to "old" waters), as samples directly from the test chambers were considered to be under representative of concentrations due to the difficulty in consistently removing a homogenized sample from the test chamber.
Differences in survival and reproduction were evaluated using a statistical computer package (Comprehensive Environmental Toxicity Information System [CETIS], version 1.8.7.4, Tidepool Scientific Software, McKinleyville, CA, USA) following the USEPA statistical decision tree (USEPA 2002). If data met the assumptions of normality/homogeneity, NOEC and LOEC were estimated using an analysis of variance to compare (p = 0.05) organism performance in the experimental treatments with that observed in the control. For the reproduction NOEC/LOEC determinations concentrations in which a survival effect was observed were not included.
The statistical program, Toxicity Relationship Analysis Program [TRAP] (version 1.30a, Duluth, MN, USA) was used for the determination of EC10, EC20 and EC50 (survival or reproduction compared to control). Exposure concentrations were log-transformed before determination of the ECx values. Only one test (Al 1196R CDC) had sufficient mortality to calculate LCx values, which were determined by tolerance distribution analysis. ECx statistical analyses of reproduction were conducted using a weighted regression analysis (mean reproduction weighted by standard deviation) calculated by the total number of young per original female. Reproduction endpoints were estimated using threshold sigmoid regression analysis.
For the initial confirmatory test the control/dilution water was compared to a non-pH MHW laboratory concurrent control water using a Fisher Exact test (survival) and an Equal variance t-test (reproduction).

A summary of all measured water quality parameters is provided in the original report. Measured DOC concentrations ranged from 1.04–1.64 mg/L for the nominally added 1 mg/L, 1.87 mg/L for the nominally added 2 mg/L, 6.57–8.71 mg/L for the nominally added 8 mg/L, and 12.01–12.30 mg/L for the nominally added 14 mg/L. This variability in measured DOC has been observed in the testing laboratory previously, as % DOC in the Suwannee NOM has historically ranged from 48–52.47%. Due to the large quantities of Suwannee NOM necessary for the tests, multiple batches were used. It is also possible that observed differences were due to variability in analytical measurements. Nevertheless, the DOC concentrations are reported as measured and not nominal. pH was maintained within 0.2 SU of the target pH in the new solutions after the equilibrium period. In some studies, an increase in pH occurred in the “old” waters (pH up to 0.3–0.4 SU above the “new” waters) between each 24-hr water renewal. Both the use of the buffer to control pH and also slightly adjusting the CO2 atmosphere limited observed pH drift. For modeling purposes, an average pH from all measurements was used. Average conductivity values remained consistent over the 24-hr period between water renewals. In certain cases the range in conductivity was wide, primarily in the higher DOC tests. The addition of food to the chambers may also be a cause for a slight increase in conductivity. All tests met test acceptability criteria for temperature (25 ± 2°C) and dissolved oxygen (saturation ≥ 60%).

Validity criteria fulfilled:

yes

Remarks:

In all tests, control acceptability criteria (> 80 % survival and > 60% surviving females having 15 or more neonates) were met. Reproduction is reported as the total number of young per original female.

Conclusions:

The studies reported here represent both a validation exercise of the currently available Al bioavailability models (BLM and MLR) and the development of additional empirical data making it possible to extend the applicability of the models to address a wider range of water quality parameters. The current models (Santore et al. 2018 and Deforest et al. 2018) include C. dubia toxicity tests reflecting a range of water chemistry: pH (6.3 – 8.1), hardness (25 – 123), and DOC (0.5 – 4.0 mg/L). The data reported herein permit an expansion of the dataset up to a pH of 8.70, hardness up to 428 mg/L and DOC up to 12.30 mg/L. This current data also allowed for a comparison of current model predicted effect concentrations with observed effect concentrations, for water types outside the range of model development. The water quality parameters measured during the tests and used in the model predictions are provided in the test report. It is anticipated that these data will be used to update the BLM and MLR models.

Executive summary:

In an effort to both improve and expand our understanding of the bioavailability and toxicity of Aluminum (Al) to aquatic organisms in freshwater, a significant research program was undertaken. The results of 10 years of research on Al were recently completed and the results published in the journal Environmental Toxicology and Chemistry (ET&CJanuary 2018, Volume 37, Issue 1). Included in this effort were a series of chronic toxicity tests with three different organisms, the fathead minnow (Pimephales promelas), the cladoceran (Ceriodaphnia dubia), and the green algae (Pseudokirchneriella subcapitata) under varying water quality conditions. The objective of this research was to quantify the effect of water chemistry parameters on Al toxicity and to use these data to develop a bioavailability- based model(s) to predict Al toxicity across a range of water types. A biotic ligand model (BLM) and multiple linear regression model (MLR) are included in the publications of Santore et al. 2018 and DeForest et al. 2018, respectively. The dataset for the two models included C.dubia toxicity tests with the following range of values: pH (6.3–8.1), hardness (25–123 mg/L as CaCO3) and dissolved organic carbon (DOC) (0.5–4.0 mg/L). Although the datasets used in the model development were relatively large, comments received from reviewers suggested that the models, and their regulatory utility, would benefit from an expansion of the range of water quality parameters considered. To address these comments the studies reported here were undertaken and represent both a validation exercise of the Al bioavailability models (BLM and MLR) and an expansion of the available empirical data making it possible to extend the applicability of the models to address a wider range of water quality parameters. The data reported herein will allow for an expansion of the dataset up to a pH of 8.7, hardness up to 428 mg/L, and DOC up to 12.30 mg/L. The new data reported here allows for the refinement of the models to reflect the range of water quality conditions prevalent in waters of Europe and North America.

Reference 21

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline available

Principles of method if other than guideline:

Although no standardized chronic testing methodology is currently available from the U.S. Environmental Protection Agency (USEPA), ASTM, or the Organisation of Economic Co-operation and Development (OECD) for L. stagnalis, test methods have been developed and reported in the scientific literature (Grosell et al, 2006; Cardwell et al, 2018). Hence, the studies were conducted according to the protocol, “Chronic toxicity of aluminum to the Great Pond Snail, Lymnaea stagnalis, under variable dissolved organic carbon (DOC) conditions,” OSU AquaTox Protocol No. Al-LS-CSR30d-032. Appendix A of the final report contains a copy of the protocol. The study was conducted according to methods described by Cardwell et al. (2018) and Grosell et al. (2006) and by reference to Slooff and Canton (1983) and Bhargava (1992).
Survival and growth were the endpoints evaluated after 30-day exposures.

GLP compliance:

yes

Remarks:

The study was carried out in the spirit of US EPA and OECD Good Laboratory Practice (GLP) standards

Specific details on test material used for the study:

The test substance was reagent grade aluminum nitrate nonahydrate (Al(NO3)3x9H2O; CAS number 7784-27-2, Lot #K34588) from J.T. Baker (Center Valley, PA, USA). The test substance had a reported assay purity of 98.7%. Following receipt at OSU AquaTox, the test substance was stored sealed in its original container at room temperature.

Analytical monitoring:

yes

Details on sampling:

Total Metals: Analytical samples from each treatment were collected for total Al analysis from newly prepared waters (“new” waters), following after the 3-hr equilibrium period) at test initiation and at least once weekly. Total Al samples were also taken from “old” samples (following a minimum of 24 hrs exposures) midway through the conduct of the tests and at test termination from the surrogate chemistry replicate. Limited samples were taken from “old” waters as it was difficult to remove a completely homogenized sample from the test chamber without significantly disturbing the test organism. Sampling methods included drawing 5 mL of sample into a plastic syringe to rinse the inside of the syringe and then discarding the rinse water. Next, 15 mL of sample was drawn into the syringe and injected into a 15 mL polypropylene conical tube. Samples were preserved with trace metal grade nitric acid (BDH Aristar® Plus; VWR Analytical, Mississauga, ON, Canada) to pH < 2 and refrigerated (1 - 4 °C) prior to analysis.

Dissolved Metals: Analytical samples from each treatment were collected for dissolved (filtered through a 0.2 μm Acrodisc PVDF filter, [Pall Life Sciences; Ann Arbor, MI, USA]) Al analysis according to the same schedule as the total metals. Sampling occurred by drawing approximately 20 mL into the syringe of which 5 mL was pushed through the filter to waste and the remaining 15 mL was collected into a 15-mL polypropylene conical test tube. Samples were preserved with trace metal grade nitric acid (BDH Aristar® Plus; VWR Analytical, Mississauga, ON, Canada) to pH < 2 and refrigerated (1 - 4 °C) prior to analysis.

Vehicle:

no

Details on test solutions:

One stock solution of 20 g/L Al was prepared by addition of 278.1 grams of Al(NO3)3x9H2O per liter of deionized water (18 Ω) and one stock solution of 40 g/L Al was prepared by addition of 556.1 grams Al(NO3)3x9H2O per liter of deionized water (18 Ω). Both stock solutions were highly acidic with pHs of < 3.5. The stock solutions were stored in plastic containers in the dark at 1 - 4°C until use. All concentrations are expressed as micrograms Al per liter (μg/L Al) either as total or dissolved (<0.20 μm) Al.

The dilution/control water used in the toxicity tests was reconstituted laboratory water prepared by adding the appropriate reagent grade salts (CaSO4 • 2H2O, MgSO4, KCl, and NaHCO3) to deionized water (18 Ω) to achieve the desired nominal hardness and alkalinity values of 120 and 80 mg/L as CaCO3, respectively, with a calcium (Ca): magnesium (Mg) molar ratio of 1.82. Reconstituted water was prepared as detailed in standard USEPA methods (USEPA 2002). Multiple batches of reconstituted laboratory water were used for the duration of each study to maintain freshness. Each batch of water was characterized for hardness, alkalinity, total residual chlorine (TRC), and ammonia (NH3). Reconstituted water was warmed to test temperature (25 ± 2°C) prior to use in the toxicity tests.
The addition of natural organic matter (NOM) to the laboratory dilution water was used in the testing to achieve nominal concentrations of 1, 2, and 4 mg/L DOC for each respective test. The DOC was added as Suwannee River Natural Organic Matter (NOM; obtained from the International Humic Substances Society (IHSS; catalogue #1R101N; St. Paul, MN, USA), based on an expected composition of 52.5% carbon in the NOM.
Test concentrations were prepared by adding the appropriate volume of stock solution (20 g/L Al or 40 g/L, depending upon exposure series) to the dilution (reconstituted) water. A volumetric flask was filled to approximately 80% of its capacity with the respective dilution (reconstituted) water. Next, the Al stock solution was added to the volumetric flask with a micro-pipette to achieve the desired nominal concentration. The total volume was then completed with the respective dilution water. The pH was checked and adjusted to a pH of 6.0 using dilute HCl or NaOH. Each individual concentration was poured into a cubitainer® which was then placed (with its cap off) in an “air-tight” enclosure and this enclosure was injected with a pre-determined volume (2.3 – 2.6%) of CO2 to maintain the pH near 6.0. Solutions were held as described for a 3-hour equilibration period in an environmental chamber at test temperature.

Test organisms (species):

Lymnaea stagnalis

Details on test organisms:

The great pond snail, Lymnaea stagnalis, was used in testing. The snails were <24 hours old at test initiation.
Adult L. stagnalis were cultured at OSU AquaTox in natural well water (hardness and alkalinity of ~180 and 200 mg/L as CaCO3, respectively, pH of 7.4). Cultures have been successfully maintained for over 9 years. Stock of L. stagnalis egg masses were originally obtained from Dr. Martin Grosell, Rosenstiel School of Marine and Atmospheric Science, University of Miami (Florida, USA). Egg masses were collected 10-12 days before test initiation. Newly-hatched snails (< 24 hrs old) from the in-house culture were used to initiate the study. Unhatched egg masses were acclimated to each dilution water over the course of the 10-12 day development period. Newly hatched snails (< 24 hrs old) were hatched into the respective control/dilution water for each test.
In order to assess organism health prior to the conduct of each test, each newly hatched organism was individually inspected for health by shell appearance, soft tissue appearance, and locomotion.

Test type:

semi-static

Water media type:

freshwater

Remarks:

The dilution/control waters used in the toxicity tests were reconstituted laboratory water prepared by adding the reagent grade salts (CaSO4·2H2O, MgSO4, KCl, NaHCO3) to deionized water to achieve the desired nominal values.

Limit test:

no

Total exposure duration:

30 d

Hardness:

Test #1:
120-124 mg/L as CaCO3 (titration)
121-122 mg/L as CaCO3 (Ca/Mg calculated)

Test #2:
116-120 mg/L as CaCO3 (titration)
121-127 mg/L as CaCO3 (Ca/Mg calculated)

Test #3:
108-120 mg/L as CaCO3 (titration)
116-118 mg/L as CaCO3 (Ca/Mg calculated)

Test temperature:

25 ± 2 °C in a temperature-controlled environmental chamber

pH:

6.0 (nominal)

Test #1 (average):
new medium: 6.24-6.45
old medium: 6.08-6.16

Test #2 (average):
new medium: 6.26-6.41
old medium: 6.06-6.20

Test #3 (average):
new medium: 6.06-6.16
old medium: 5.86-6.02

Dissolved oxygen:

Test #1 (average):
new medium: 8.4 mg/L
old medium: 7.1-7.8 mg/L

Test #2 (average):
new medium: 8.2-8.4 mg/L
old medium: 6.7-7.5 mg/L

Test #3 (average):
new medium: 8.4-8.5 mg/L
old medium: 7.5-8.0 mg/L

Conductivity:

Test #1 (average):
new medium: 448-456 µS/cm
old medium: 448-459 µS/cm

Test #2 (average):
new medium: 445-447 µS/cm
old medium: 445-450 µS/cm

Test #3 (average):
new medium: 428-510 µS/cm
old medium: 426-507 µS/cm

Nominal and measured concentrations:

Concentrations at start of test
Test #1: 0, 500, 1000, 2000, 4000, 8000 µg Al/L (nominal)
Test #1: 4.3, 507.5, 1034.0, 2072.8, 4192.0, 8068.2 µg Al/L (total measured)
Test #1: 7.5, 36.2, 58.8, 97.0, 138.2, 363.0 µg Al/L (dissolved measured)

Test #2: 0, 500, 1000, 2000, 4000, 8000 µg Al/L (nominal)
Test #2: 4.0, 510.6, 1059.8, 2107.0, 4221.6, 8130.6 µg Al/L (total measured)
Test #2: 3.2, 80.4, 82.0, 83.2, 188.2, 468.2 µg Al/L (dissolved measured)

Test #3: 0, 1130, 2250, 4500, 9000, 18000 µg Al/L (nominal)
Test #3: 6.0, 1166.6, 2382.8, 4649.6, 9196.6, 18273.4 µg Al/L (total measured)
Test #3: 6.0, 231.4, 214.1, 439.4, 968.8, 545.8 µg Al/L (dissolved measured)

Details on test conditions:

The test included five Al concentrations and a dilution water control. Exposures were selected to cover a range of Al concentrations anticipated to illicit a biological dose response. Each test concentration (treatment) was prepared individually and then distributed among the replicate test chambers. Ten replicate chambers were prepared for each treatment and control. Following the 3-hour equilibrium period, the test was conducted in an “air-tight” enclosed box injected with a pre-determined percent volume (2.3 – 2.6%) of CO2 and the enclosure was maintained in a temperature-controlled environmental chamber. In addition to each control/dilution water, a concurrent control water consisting of natural well water (hardness and alkalinity of ~180 and 200 mg/L as CaCO3, respectively, pH of 7.4) of which the adult organisms were cultured. This concurrent well water control was not pH adjusted or pH controlled and was conducted under the same feeding and water renewal conditions as all of the Al exposures.
The test chambers for the study were 120 mL polypropylene Soufflé cups (Dart, Mason, Michigan, USA). The cups were filled completely full and capped with a plastic lid providing little to no air space within the cup. Cups and lids were pre-soaked in deionized water for 24 hours prior to use. In order to avoid any excess algal or bacteria growth within the test chamber, organisms were transferred to new chambers midway through the test.
The test chambers were housed in a temperature-controlled environmental chamber and maintained at the test temperature of 25°C. The test was conducted under a 16:8 hour light:dark cycle using cool-white fluorescent lights at ~100 foot candles. The test chambers were randomized according to a computer generated randomization. The test was conducted in an enclosed box injected with CO2.

At test initiation, a single newly hatched snail was placed in each 120 mL test chamber, which contained 120 mL of test solution (filled completely). An additional surrogate chamber (chemistry replicate) was also added in which to measure daily water quality directly from the chamber. The test chamber was covered with a snap-on lid so that the snails were forced to remain within the test solution (little to no air existing in chamber). Because the lid was not completely air-tight and therefore exchange of CO2 between the chamber and atmosphere would take place, the chambers were placed within an air-tight enclosure injected with CO2 (as described in Section 2.5) to maintain the pH near 6.0. Each test chamber contained one piece each of lettuce and carrot (each approximately 2-4 mm3 in size). Water changes were performed on a Monday, Wednesday, and Friday schedule with the organisms being transferred to a freshly prepared test chamber with fresh food during each water renewal. Approximately 5 - 10% of old solution was transferred with the organism as it was gently poured into the new chamber. This water renewal/test chamber regime reduced the ability of biofilm to accumulate within the test chamber

Observations of live and dead organisms were conducted three times a week (Monday, Wednesday, and Friday). Dead organisms were recorded and immediately removed. At test termination, each surviving organism was blot-dried with a Kimwipe® and weighed to within 0.01 mg. Each surviving snail from each replicate was then placed into a pre-weighed pan and dried at 105°C for over 12 hrs and then re-weighed to the nearest 0.01 mg to obtain a dry weight.

Key result

Duration:

30 d

Dose descriptor:

LC10

Effect conc.:

2 759 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

mortality

Remarks on result:

other: Test#1

Key result

Duration:

30 d

Dose descriptor:

LC10

Effect conc.:

3 108 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

mortality

Remarks on result:

other: Test #2: 95% CL 1476-6173 µg/L

Key result

Duration:

30 d

Dose descriptor:

LC10

Effect conc.:

8 066 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

mortality

Remarks on result:

other: Test#3; 95%CL 4086-15923 µg/L

Key result

Duration:

30 d

Dose descriptor:

EC10

Effect conc.:

675.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

growth

Remarks:

wet weight basis

Remarks on result:

other: Test#1; 95%CL 303.6-1502.2 µg/L

Key result

Duration:

30 d

Dose descriptor:

EC10

Effect conc.:

1 365.4 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

growth

Remarks:

wet weight basis

Remarks on result:

other: Test#2; 95%CL 747.0-2495.7 µg/L

Key result

Duration:

30 d

Dose descriptor:

EC10

Effect conc.:

1 120.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

growth

Remarks:

wet weight basis

Remarks on result:

other: Test #3; 95%CL 880.8-1425.2 µg/L

Key result

Duration:

30 d

Dose descriptor:

EC10

Effect conc.:

664.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

growth

Remarks:

dry weight basis

Remarks on result:

other: Test#1; 95%CL 325.5-1357.9 µg/L

Key result

Duration:

30 d

Dose descriptor:

EC10

Effect conc.:

1 453.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

growth

Remarks:

dry weight basis

Remarks on result:

other: Test#2; 95%CL 839.8-2515.9 µg/L

Key result

Duration:

30 d

Dose descriptor:

EC10

Effect conc.:

868 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

growth

Remarks:

dry weight basis

Remarks on result:

other: Test #3; 95%CL 568.0-1326.3 µg/L

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

2 073 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

mortality

Remarks on result:

other: Test #1

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

4 221.6 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

mortality

Remarks on result:

other: Test #2

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

18 273 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

mortality

Remarks on result:

other: Test #3

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

1 034 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

growth

Remarks:

wet weight basis

Remarks on result:

other: Test #1

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

1 059.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

growth

Remarks:

wet weight basis

Remarks on result:

other: Test #2

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

1 166.6 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

growth

Remarks:

wet weight basis

Remarks on result:

other: Test #3

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

1 034 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

growth

Remarks:

dry weight basis

Remarks on result:

other: Test #1

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

1 059.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

growth

Remarks:

dry weight basis

Remarks on result:

other: Test #2

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

1 166.6 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

growth

Remarks:

dry weight basis

Remarks on result:

other: Test #3

Details on results:

The control acceptability criteria for the entirety of the 30-day chronic tests (minimum of 70% survival) were achieved in the control/dilution water for each test. In addition to the dilution water control, a concurrent control consisting of well water (from culture conditions) was used. Snail survival and growth in the control/dilution waters was compared to that observed in the concurrent well water exposure. The differences between the lab waters and the well water were hardness, alkalinity, pH, and DOC. The lab waters have lower hardness and alkalinity (nominally at 120/80 mg/L as CaCO3, respectively) and were used at a pH of 6 with pH control, while the well water had a higher hardness/alkalinity (typically around 190/240 mg/L as CaCO3, respectively), and a pH of ~8.5, conducted under ambient conditions. In Test #1, the lab water control organisms were significantly (p=0.05) smaller. The lab water control snails in Test #2 test were also slightly smaller, but not significantly smaller than well-water controls. In Test #2, the concurrent well water snails had low survival with an average of 67%, as compared to the 90% average survival in the lab water controls. In Test #3, the control snails were of comparable size to the concurrent well water snails. The varying water quality characteristics can play a part in shell development. The hardness/alkalinity of the lab waters was prepared at acceptable calcium and magnesium concentrations for shell development. Because the only difference between the lab water between tests was primarily DOC addition, these results demonstrate that the added DOC alone may have been an added benefit to growth and shell development. In all tests, growth was more sensitive to Al exposures than survival. Snails in the nominal 4 mg/L DOC test grew more than twice that of the 1 mg/L DOC test.

Reported statistics and error estimates:

Statistical analysis was performed using measured total Al concentrations from “new” waters. “Old waters were not utilized in the averages, as a completely homogenized solution was difficult to obtain without negatively disturbing the test organism and therefore may have not captured some of the insoluble Al hydroxides. Differences in survival and growth were evaluated using a statistical computer package (Comprehensive Environmental Toxicity Information System [CETIS], version 1.8.4.7, Tidepool Scientific Software, McKinleyville, CA, USA). If the data met the assumptions of normality and homogeneity, the NOEC and LOEC were estimated using an analysis of variance to compare (p = 0.05) survival and growth in the experimental treatments with that observed in the control.

For the determination of effect concentrations (ECx), the statistical program, Toxicity Relationship Analysis Program [TRAP, version 1.30a] (Duluth, MN, USA) was used for the determination of effect concentrations to reduce survival or growth by 10%, 20%, or 50% relative to control performance (LC/EC10, LC/EC20, LC/EC50). Survival endpoints were calculated using tolerance distribution analysis and growth endpoints were estimated using either piecewise linear regression or threshold sigmoid regression analysis. Exposure concentrations were log-transformed before determination of the ECx values. Analysis of toxicity data was conducted using individual replicate data to provide a better fit and to better accommodate for the growth variability. Initially mean growth values (weighted by standard deviations) were selected for ECx determination, but the TRAP identified modeling errors from slope steepness. No model errors were found when entering individual data points and therefore this method was used for all three tests.

Measured water quality characteristics for each control/dilution water are provided in the Study Report (pH, conductivity, and dissolved oxygen). All tests met test acceptability criteria for temperature (25 ± 2°C). TRC and ammonia were all below detection, < 0.05 mg/L and < 1.0 mg/L, respectively.

Dilution water was characterized prior to the preparation of new test solutions without organisms. The cationic and anionic makeup of multiple batches of control/dilution water was measured over the course of the tests and all analytes remained consistent between batches of water. DOC was added to the control/dilution waters based upon a 52.5% carbon makeup of the Suwannee River NOM. Although twice as much NOM was added between the nominal 1 mg/L and 2 mg/L waters, the measured DOC concentrations were very close between the two tests (measured DOC in nominal 1 mg/L test = 1.29–1.45, measured DOC in nominal 2 mg/L = 1.38–1.51 mg/L). The reason for the similarity between the values is unknown, although variability around the DOC measurements is not uncommon (see variability of nominally added 4 mg/L DOC = 3.60, 3.74, 4.20 mg/L). The % DOC in the NOM is calculated based upon historical measurements and ranges from 48-53%, and for these waters was expected to be ~52.5% DOC. The DOC content in the Suwannee River NOM was not measured immediately before preparing the dilution waters and therefore it is a possibility that the batch of NOM was not homogeneous or that the observed differences were due to variability in the analytical measurements. Nevertheless, the DOC concentrations are reported as measured and not nominal.

Variability was observed in the pH control of the test solutions (both after the 3-hr equilibrium period “new” waters and over the course of the exposures between water renewals “old waters”). Averages were within ± 0.2 units between exposures in a test, while instantaneous readings varied to a greater degree at ± 0.7–0.9 SU, ± 0.5–0.7 SU, and ± 0.2–0.6 SU. Average pH measurements between “new” and “old”was within 0.2 units. Because pH is a driver in the bioavailability and toxicity of Al, the pH variability observed in the tests may have affected toxicity, although the changes occurred in the control waters and the pH changes occurred over the course of many hours. Therefore, the pH variability would most not have a significant impact and for the inputs into the bioavailability models, average measurements were used.

D.O. concentrations were above the > 40% saturation acceptability limit, with two deviations where levels dropped to low levels (22% and 23% saturation) in two instantaneous readings on Day 25. Because L.stagnalis is a pulmonate (i.e., has a lung-like organ) and because the levels were for a short duration, this is not expected to have had a negative effect on test acceptability.

Aluminum exposure concentrations are reported under two categories: “new” and “old”. “New” waters were sampled directly from freshly prepared solutions (after 3-hr equilibrium periods). “Old” waters consisted of a sample from the surrogate chemistry sample. Spiked total Al concentrations from “new”samples were between 93 and 112% of nominal spiked concentrations. Dissolved Al in the “new” waters ranged from 0.03% to 33% of their respective total Al concentrations. The greater % dissolved/total Al occurred in the lower Al-spiked exposures and the percent dissolved did not monotonically increase as totals increased. This was a result of the exceedance of solubility limits in the solutions due to dominance of insoluble Al hydroxides. As the tests were performed as static-renewals (removal of most water from test chamber followed by refilling with new waters), analytical samples of “old” waters most likely did not reflect a homogeneous sample (an artifact of sampling technique and being able to remove all of the water from the test chamber without disturbing the organism). This is why measured Al in the”old”samples tended to be more variable and most often lower (i.e., potential for insoluble Al precipitates not captured in the sample). Therefore, use of the homogeneous“new” water measurements is most appropriate for calculating average exposure concentrations. The use of total Al to calculate effect concentrations is appropriate as dissolved Al concentrations did not increase with increasing exposures and were not directly correlated with observed biological responses as reported by Cardwell et al (2018). Background concentrations of Al in the control waters (both the control/dilution water and the concurrent well water) were primarily below detection limits (< 5 to < 12 μg/L Al). The differences in detection limits were due to the instrument variability on the specific days of analysis and correlated to the specific calibration curves of the analytical runs. These control water samples were assigned a value of half their detection limit for determination of biological effect concentrations.

Method blanks were run with each analysis and consisted of deionized water treated identically as the samples through the entire process including acidification. All blank measurements were below the detection limit. Quality control samples were analyzed with a standard concentration and an over-spike of a known addition of Al and analyzed to calculate % recovery for the samples. Standard addition recovery ranged from 97.0-115.4%. The primary standard quality control sample recovery ranged from 95.8- 103.9%. All quality control samples were within acceptability criteria of 85-115%.

Validity criteria fulfilled:

yes

Conclusions:

The studies reported here represent a validation exercise of the Al bioavailability models (BLM and MLR) using laboratory waters, at a pH of 6 and hardness of 120 mg/L as CaCO3, with varying DOC concentrations. The empirical data reported herein allow a comparison of model predictions with observed effects, using an organism not previously included in model development, L. stagnalis. The water quality parameters measured during the tests and used in the models are reported in Table 3-13. Observed effect concentrations (EC10 values with 95% confidence intervals) ranged from 675 (304 – 1502) μg/L total Al in the pH 6.15, hardness = 121, DOC = 1.37 mg/L water to 1365 (747 – 2496) μg/L total Al in the pH 6.17, hardness = 124, DOC = 1.45 mg/L water. The BLM predictions in these waters were 971 μg/L and 1005 μg/L total Al, respectively, and the MLR predictions were 2039 and 2202 μg/L total Al, respectively.
L. stagnalis is a moderately sensitive organism in the chronic database (ranking 8th most sensitive out of 13 species in the species sensitivity distribution (SSD) [Cardwell et al. 2018]) and is slightly less sensitive than C. dubia and P. promelas, but slightly more sensitive than P. subcapitata. The original toxicity test with L. stagnalis used in the current Al SSD was performed in water at a pH 6.1, hardness of 113 mg/L as CaCO3, and a DOC of 0.3 mg/L. This test resulted in an EC10 (with 95% confidence intervals) of 860.7 (733.1 – 1010.5) μg/L μg/L total Al based upon blot-dried wet weight; these results are consistent with the most comparable test reported here (i.e., Al 995 LS). The toxicity tests presented here demonstrate that protectiveness of DOC was somewhat mixed at the concentrations tested and did not consistently provide an increase in protectiveness of Al toxicity to L. stagnalis. The BLM predicted ECx toxicity values within a factor of two for all three tests. The MLR was not quite as accurate as the BLM in predicting ECx toxicity values for all three tests, resulting in slight under predictions of toxicity. The results from these studies can be used for a refinement of the bioavailability models and inclusion in the species sensitivity distribution.

Executive summary:

Aluminum bioavailability models, as both a biotic ligand model (BLM) and a multiple linear regression model (MLR), have been developed to predict Al bioavailability and toxicity to aquatic organisms in freshwaters with varying water quality characteristics. These models predominately used empirical data obtained from toxicity tests with the fathead minnow (Pimephales promelas), a cladoceran (Ceriodaphnia dubia) and the green algae (Pseudokirchneriella subcapitata), with exposure to Al concentrations in a range of water types (e.g., varying hardness, pH, dissolved organic carbon [DOC]). In an effort to extend the application of the Al bioavailability models using organisms not previously considered in their development, a series of chronic toxicity tests (30-day duration) were conducted using the snail,Lymnaea stagnalis. Snails were exposed to Al, at a pH of 6 and hardness of 120 mg/L as CaCO3, in three different laboratory waters representing a range of DOC conditions (nominal DOC 1.0, 2.0, 4.0 mg/L). Survival and growth (as blot-dried wet weight and dry weight) were assessed at the end of the 30-day exposure periods. The effect concentrations (EC10 with 95% confidence intervals) for growth (as wet weight) were 675 (304–1502) μg/L total Al, 1365 (747–2496) μg/L total Al, and 1121 (881–1425) μg/L total Al, in the nominal 1.0, 2.0, and 4.0 mg/L DOC waters, respectively. The growth endpoint for dry weight was slightly more sensitive for two of the tests with EC10s of 665 (326–1358) μg/L total Al, 1454 (840–2416) μg/L total Al, 868 (568–1326) μg/L total Al, in the nominal 1.0, 2.0, and 4.0 mg/L DOC waters, respectively.L.stagnalis is a moderately sensitive organism in the chronic database (ranking 8th most sensitive out of 13 species in the species sensitivity distribution), and is slightly less sensitive than C. dubia and P. promelas and slightly more sensitive than P.subcapitata. The original toxicity test with L.stagnalis that was used in the Al species sensitivity distribution (SSD) was performed in water with a similar hardness and pH, and a DOC of < 0.50 mg/L. This original test resulted in an EC10 (with 95% confidence intervals) of 860.7 (733.1–1010.5) μg/L total Al. The toxicity tests presented here demonstrate that protectiveness of DOC was somewhat mixed at the concentrations tested and did not provide a consistent increase in protectiveness of Al toxicity toL.stagnalis. The observed 10% and 20% effect concentrations (EC10s and EC20s), along with their 95% confidence intervals, from the L. stagnalis tests were compared to predicted values from both the BLM and MLR (shown in figures below for the mean wet weight endpoint). The BLM predicted effect concentrations in all three waters were within a factor of two. It should be noted that for L. stagnalis, there was only a single observation in the chronic database. To obtain predicted effect concentrations using the BLM, the model requires assigning a calibration value and adjustment of more than one parameter for the factoring of toxicity due to both dissolved and precipitated Al. The MLR predicted effect concentrations, within a factor of two, for two of the three waters, although predictions were slightly higher (higher ECx values) than those predicted with the BLM. With the studies reported here, additional data points are now available to expand the chronic toxicity database and future modeling for this species. These results demonstrate that both Al bioavailability models adequately predict toxicity for a species not used in model development.

Reference 22

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

Solutions from Al exposures were sub-sampled unfiltered for analyses of total concentrations, and acidified to 0.13% nitric acid.
Filtered sub- samples (< 0.45 μm fraction representing concentrations of dissolved Al) were taken only for the first four Al exposures. This was discontinued following determination that the required concentration range would exceed the solubility limit of Al in seawater.
For several tests, sub-samples were also taken from 72-h old test solutions at the end of the 14-d test period

Details on test solutions:

Seawater was collected at high tide from Nightcliff jetty, Northern Territory, Australia, and passed through a 0.45-μm filter immediately following transport of the water to the laboratory. Stock solutions of 20 and 2000 mg/L were prepared by dissolving AlCl3·6H2O (Sigma-Aldrich) in Milli-Q water.Test solutions were prepared by adding stock solutions to filtered seawater, and were stored in 5 L HDPE bottles for 24 h prior to test initiation to allow for equilibration. Test solution bottles were stored in a refrigerator throughout the test period and removed only to dispense new test solutions on each water change day.

Test organisms (species):

other aquatic mollusc: Exaiptasia pallida

Details on test organisms:

Exaiptasia pallida was harvested from a population which established naturally in the aquaria at the Australian Institute of Marine Science in Darwin, Northern Territory, Australia. After its potential suitability as a test species was identified, individuals were separated to a specific system for ease of maintenance and selection of individuals for tests. Around 400 adults (~0.3 – 15 mm oral disc diameter) were maintained in 20 L of filtered seawater (FSW, 0.5 μm) in a 30 L HDPE container, connected to a recirculating system in a temperature controlled room. Several air stones were spaced evenly around the tank to provide aeration, and the tank was kept free of rocks or additional substrate so that the anemones could attach only to the tank surface. Broodstock were maintained in this aquaria over the course of testing (~ one year) at pH 8.3 ± 0.1, 29.5 ± 0.6 °C, salinity of 34.6 ± 0.3 PSU (conductivity =51.8 ± 1 mS/cm) and with a 12:12 h day/ night cycle (representative of the diurnal light cycle in northern Australia, indirect 36 W cool white triphosphor ceiling lighting which was measured as ~ 2–3 μmol photons/m2/s inside the enclosed tank). Broodstock were fed live brine shrimp (Artemia salina) each Monday, Wednesday and Friday, harvested from a 1 L Imhoff cone. The A. salina was prepared by adding 1 teaspoon of cysts to 1 L of FSW. The culture was aerated for 24 h under cool white light with a 12:12 h day/night cycle.

Test type:

semi-static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

14 d

Hardness:

6515+/- 15 mg/L as CaCO3

Test temperature:

24°C, 28°C, 31 °C

pH:

8.1 +/- 0.2

Dissolved oxygen:

106% +/- 9%

Salinity:

36 +/- 0.2 PSU

Conductivity:

51 +/- 0.5 mS/cm

Nominal and measured concentrations:

Nominal: 0-10000 µg/L

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

8 870 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: test at 24°C

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

2 270 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: test at 28°C

Duration:

14 d

Dose descriptor:

EC10

Effect conc.:

817 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: test at 28°C

Duration:

14 d

Dose descriptor:

EC10

Effect conc.:

1 500 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: test at 31°C

Reported statistics and error estimates:

In determining the final test method that should be used , the reproduction of anemones was compared using either a t-test or a one-way ANOVA (Minitab 17.0) to identify significant differences between treatments. An alpha level of 0.05 was used so that there was a 5% possibility of detecting a difference where one did not exist.
For each experiment, lacerate numbers were expressed as percent reproduction and presented relative to the control response. Similarly, growth rate was expressed as percent growth and presented relative to the control response. Data points were pooled from two tests at 28 °C and three tests at each of the 24 and 31 °C temperatures. Non-linear (3-parameter sigmoidal or 3-parameter logistic) regression was used to generate concentration-response curves for each metal (SigmaPlot 11.0, Systat Software Inc). The model that provided the best r2 was used in each case. Metal concentrations at which a 10% (EC10) or 50% (EC50) reduction in reproduction or growth rate occurred relative to the control were estimated from the equations of the curve fits, along with the 95% confidence limits for these values.

Executive summary:

The world's most productive bauxite mines and alumina refineries are located in tropical or sub-tropical regions. The discharge water from alumina refineries can contain elevated aluminium (Al, < 0.45 μm fraction), from 30 to 1000 μg/L. There is a need for additional information on the toxicity of Al to aquatic organisms to improve the environmental regulation and management of alumina refinery operations in tropical coastal regions. A 14-d chronic toxicity test was developed for the tropical sea anemone Exaiptasia pallida. Asexual reproduction and growth rates of E. pallida were assessed using the number of lacerates produced and oral disc diameter. The comparative sensitivity of E. pallida was assessed through exposure to a commonly-used reference toxicant, copper (Cu) at 28 °C, with asexual reproduction toxicity estimates of 10% (EC10) and 50% (EC50) effect concentrations, calculated as 8.8 μg/L (95% confidence limits (CL): 1–18 μg/L) and 35 μg/L Cu (95% CL: 30– 39 μg/L), respectively. Growth rate was a suitable additional endpoint (EC50=35 μg/L Cu, 95% CL: 23–49 μg/ L). The EC10 and EC50 for Al (total fraction, based on reproduction) at 28 °C were 817 μg/L (95% CL: 440– 1480 μg/L) and 2270 μg/L (95% CL: 1600–3900 μg/L), respectively. The toxicity of Cu and Al was also assessed at 24 °C and 31 °C, representing average year-round water temperatures for sub-tropical and tropical Australian coastal environments. Changing the temperature from 28 °C to 24 °C or 31 °C resulted in up to 45% less reproduction of anemones and increased their sensitivity to Cu (EC50s at 24 °C=21 μg/L, 95% CL: 17–26 μg/L and at 31 °C=23 μg/L, 95% CL: 21–25 μg/L). Sensitivity to Al was reduced at 24 °C with an EC50 of 8870 μg/L (95% CL: 6200-NC). An EC50 for Al at 31 °C could not be calculated. This test is a reliable and sensitive addition to the suite of standardised tests currently developed for tropical marine species.

Reference 23

Endpoint:

long-term toxicity to aquatic invertebrates

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:

no guideline followed

Principles of method if other than guideline:

The effects of aluminum sulfate on life history characteristics of Daphnia pulex were investigated with three concentrations of Al and a control for 21 days. Daphnia were transferred daily into fresh media, and each individual was measured for body length, egg stage, eggs held in the brood chamber, and the number of neonates.

GLP compliance:

not specified

Analytical monitoring:

no

Details on sampling:

Not reported

Vehicle:

not specified

Details on test solutions:

Not reported

Test organisms (species):

Daphnia pulex

Details on test organisms:

TEST ORGANISM
- Common name: zooplankton
- Strain/clone: OJP1 and OJP2
- Source: clone lineages were established from D.pulex populations taken from 3 lakes in northeastern Washington State in Spokane County, OJP is a small pond with no prior exposure to alum - located on the Turnbull National Wildlife Refuge. The other lakes had pre-exposure to alum, and were considered to be acclimated to the test substance and are not discussed further in this summary.
- Feeding during test
- Food type: algae, Scenedesmus acutus
- Amount: 45000 individuals per day
- Frequency: daily

ACCLIMATION: not reported

QUARANTINE: several months
- All other template details: Not reported

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: offspring were derived from neonates released from ephippial females within these isoclones, and hatched in the laboratory in 1-L jars. After the first broods were dropped, neonates < 8 hrs old were isolated and placed into 60-mL life table jars containing experimental media and algae.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

Not reported

Hardness:

Not reported

Test temperature:

20 ± 1 degree C

pH:

7 ± 1

Dissolved oxygen:

Not reported

Salinity:

Not reported

Nominal and measured concentrations:

Nominal (mg/L): control, 0.05, 0.30, 0.50

Details on test conditions:

TEST SYSTEM
- Test vessel: 60-mL life table jars
- Renewal rate of test solution (frequency): daily
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- All other template details: Not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: well water
- Well water had low concentrations of dissolved materials
- All other template details: Not reported

OTHER TEST CONDITIONS
- Adjustment of pH: pH was maintained at 7 ± 1
- Photoperiod: 16 hrs light : 8 hrs dark
- Light intensity: Not reported

EFFECT PARAMETERS MEASURED: each individual was measured daily for body length, egg stage, eggs held in the brood chamber, and number of neonates

VEHICLE CONTROL PERFORMED: not applicable

RANGE-FINDING STUDY: Not reported

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

< 0.05 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

mortality

Remarks:

: age-specific survivorship

Remarks on result:

other: Unbounded LOEC, effect seen at lowest dose tested compared to controls

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

< 0.05 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks:

: both net reproductive rate and number of offspring

Remarks on result:

other: Unbounded LOEC, effect seen at lowest dose tested compared to controls

Details on results:

- Mortality of parent animals: OJP clone individuals survived only 7 days at the higher concentrations
- Net reproductive rate = total number of offspring produced per female for duration of the life table
- Offspring = total number of offspring produced for those individuals producing five broods
- All other template details: Not reported

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

For age-specific survivorship, a Kruskal Wallis test was used. For the reproductive endpoints, a two-way analysis of variance was used, including a treatment effect, a genotype effect, and treatment/genotype interaction. To see if the clones within a water source were replicates, an analysis by two-way ANOVA tested for clonal effects found that the assumption that clones within a water source were replicates was statistically appropriate.

Not applicable

Conclusions:

This study on the effects of aluminium sulphate on Daphnia pulex from a natural pond (OJP) on survival and reproduction found significant decreases at the lowest concentration tested, 0.05 mg Al/L.

Reference 24

Endpoint:

long-term toxicity to aquatic invertebrates

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

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read-across source

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

>= 694.1 - <= 138 110 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other:

Remarks:

Range is based on normalised data from BLM, 21 and 28 days of exposure data was used; lower limit presents Ecoregion #7; upper limit presents Ecoregion #5

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

>= 653.1 - <= 10 370 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

other: weight

Remarks on result:

other:

Remarks:

Range is based on normalised data from BLM, 28 and 31 days of exposure data was used; lower limit presents Ecoregion #7; upper limit presents Ecoregion #5

Description of key information

Long-term effects for aquatic invertebrates were identified for the following species: Aeilosoma sp., B. calyciflorus, C. dubia, C. riparius, D. magna, D. pulex H. azteca and L. stagnalis. No-effect levels were expressed as total or dissolved Al, and were based on standard endpoint (reproduction, growth/weight, growth rate, population size) .

 

For the standard cladoceran test species D. magna and C. dubia, NOECs and EC10s ranged from 0.076 to 4.9 mg Al/L and 0.021 to 0.997 mg Al/L, respectively. Water quality data for these studies suggest a direct relationship between toxicity and pH, hardness, and DOC. For studies that experimentally manipulated water quality (e.g., CIMM 2009 and 2010a), toxicity decreased with increasing pH, hardness, and DOC.

 

Additional information

Recent studies conducted by the Chilean Mining and Metallurgy Research Center (CIMM) tested aluminium toxicity to C. dubia and D. magna (one data point) across a range of pH, DOC, and hardness values. ECr10s for D. magna and C. dubia 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. 

The reported results demonstrated that increasing DOC concentration has a protective effect on aluminium LC50s for invertebrates. Increasing water hardness also had a protective effect. Aluminium toxicity was reduced at high pH, but a larger reduction was observed when changing pH from 6 to 7 than from 7 to 8. 

 

In order to explain and predict long-term Al-toxicity to fish as a function of water quality parameters, bioavailability models have been developed (Biotic Ligand Models; BLMs). 

The developed Al-BLM for fish. The acute fish BLM that was developed for S. salar was applied to the chronic invertebrate data (CIMM 2009, CIMM 2010) by developing a critical accumulation value appropriate for this organism. In addition, the chronic invertebrate data suggested that overall fit would be improved with a small increase in the Ca binding parameter (i.e. the log K for Ca binding at the biotic ligand was increased from 4.2 to 4.8), which is the same adjusted value used in the chronic fish model. After application of the modified Al BLM, the variability in the response curve data substantially decreased. These data were subsequently used to establish the CA10 (i.e. the critical accumulation level that results in a 10% reduction in reproduction), and likewise, the CA50. The CA10 and CA50 values was then be used to predict EC10 values and EC50 values in various water types.  

 

Additional information on the invertebrate BLM, including an evaluation of the ability of the chronic invertebrate Al BLM to predict EC50 and EC10 values, provided in the report “Background document – Environmental Effects Assessment of Aluminium” which is attached to Section 13 of the IUCLID Dossier. In summary, all of the EC50 values are predicted within 2-fold of the reported EC50 values. Most of the EC10 values are predicted within 2-fold of the reported EC10 values, and all of the predicted EC10 values are within 4-fold of the reported values. These results indicate that the chronic Al BLM performs reasonably well for predicting sublethal effects of Al on invertebrates. It should be noted that in both the fish and the invertebrate tests, saturation index calculations suggested that the majority of the toxicity values exceed Al(OH)3 solubility. However, bioavailability factors (i. e. pH, DOC, and hardness) still are consistent with the trends predicted by the Al BLM.

 

Two additional LC50 values that are not included in this comparison were reported for pH 7 and pH 8 in filtered test media (i. e., filtered before organisms were exposed). The filtered test media were approximately 5-fold less toxic, meaning that their LC50s were approximately 5-fold higher than the results from exposure to unfiltered media. Therefore, toxicity was largely a function of exposure to aluminium hydroxides, which are removed by filtration through these types of filters.

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.