Aluminium oxide

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish early-life stage toxicity

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: Cleveland, L., E.E. Little, S.J. Hamilton, D.R. Buckler, and J.B. Hunn. 1986. Interactive toxicity of aluminum and increased acidity to early life stages of brook trout. Trans.Am.Fish.Soc. 115:610-620.

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Specific details on test material used for the study:

- Analytical purity: 98%
- Composition of test material, percentage of components: 8.56% aluminium
- Source: Fisher Scientific Company, Fairlawn, NJ, USA

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all exposures and controls were measured
- Sampling method: water samples were collected at the beginning of the exposures and weekly thereafter
- Sample storage conditions before analysis: Not reported

Vehicle:

not specified

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION: Not reported

Test organisms (species):

Salvelinus fontinalis

Details on test organisms:

TEST ORGANISM
- Common name: brook trout
- Strain: not reported
- Source: eyed eggs were obtained from Beity's Resort, Valley, Washington

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): not applicable
- Method of collection of fertilised eggs: not applicable
- Subsequent handling of eggs: eggs were held overnight in control water at pH 7.2 and 12 degrees C before the study was initiated

POST-HATCH FEEDING
- Type/source of feed: Daphnia magna or Artemia salina
- All other template details: Not reported

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

60 d

Post exposure observation period:

Not reported

Hardness:

Means for each exposure concentration are shown in Table 1; about 246 µeq/L

Test temperature:

12 degrees C

pH:

Means for each exposure concentration are shown in Table 1; about 6.5

Dissolved oxygen:

Not reported

Salinity:

Not reported

Nominal and measured concentrations:

Nominal (µg/L): 0, 0, 50, 100, 200, 400
Measured, total (µg/L): 5, 4, 57, 88, 169, 350

Details on test conditions:

TEST SYSTEM
- Material, size, headspace, fill volume: covered 177 mL glass hatching chambers for eyed eggs, other growth chambers were used in the larger replicate tanks
- Aeration: aeration provided continuous circulation of water
- Type of flow-through: proportional diluter
- No. of fertilized eggs/embryos per vessel: 50
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- All other template details: Not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: used methods of Cleveland et al. (1986) to prepare the experimental water
- Total organic carbon: average of 2.1 mg/L (dissolved OC: 1.9 mg/L and particulate OC: 0.21 mg/L)
- Alkalinity: see Table 1
- Intervals of water quality measurement: weekly for Al and monthly for organic carbon
- All other template details: Not reported

OTHER TEST CONDITIONS: Not reported

EFFECT PARAMETERS MEASURED: egg mortality, hatch, incomplete hatch recorded daily until hatching was complete. After hatching, mortality of fish was recorded daily, lengths and weights were recorded at 30, 45, and 60 days after the median hatching date. Behavioral evaluation on 10 fish from each duplicate included measurements of locomotory activity, feeding and swimming capacity, and buoyancy of individual fish at 30 and 60 days of exposures.

VEHICLE CONTROL PERFORMED: not applicable

RANGE-FINDING STUDY: Not reported

POST-HATCH DETAILS: Not reported

FERTILIZATION SUCCESS STUDY: Not applicable

Reference substance (positive control):

no

Duration:

60 d

Dose descriptor:

NOEC

Effect conc.:

169 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Remarks:

: fry mortality

Remarks on result:

other: NOEC same at 30 d

Duration:

60 d

Dose descriptor:

LOEC

Effect conc.:

350 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Remarks:

: fry mortality

Remarks on result:

other: LOEC same at 30 d

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

57 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

length

Duration:

30 d

Dose descriptor:

LOEC

Effect conc.:

88 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

length

Duration:

60 d

Dose descriptor:

NOEC

Effect conc.:

88 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

length

Duration:

60 d

Dose descriptor:

LOEC

Effect conc.:

169 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

length

Duration:

60 d

Dose descriptor:

NOEC

Effect conc.:

88 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

weight

Remarks on result:

other: NOEC same as 30 d

Duration:

60 d

Dose descriptor:

LOEC

Effect conc.:

169 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

weight

Remarks on result:

other: LOEC same as 30 d

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

57 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

number hatched

Duration:

30 d

Dose descriptor:

LOEC

Effect conc.:

88 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

number hatched

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

88 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

behaviour

Remarks:

: swimming capacity

Duration:

30 d

Dose descriptor:

LOEC

Effect conc.:

169 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

behaviour

Remarks:

: swimming capacity

Duration:

60 d

Dose descriptor:

NOEC

Effect conc.:

> 350 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

behaviour

Remarks:

: swimming capacity

Duration:

60 d

Dose descriptor:

EC10

Effect conc.:

0.1 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Basis for effect:

weight

Remarks on result:

other: 95% CL: 0.045-0.221

Details on results:

- Mortality/survival at embryo, larval, juvenile, and adult stages: embryo mortality not significantly different than control at 350 µg/L (6%)
- Numbers hatched, Numbers of offspring produced, or Number of offspring per live female per day: control, about 46 (93% hatch rate of 50 embryos)
- Observations on body length and weight of young and/or exposed parents at one or more time periods: see Table 5a for length data and Table 5b for weights
- All other template details: Not reported

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

One-way analysis of variance was used to compare treatment effects on survival, growth, and behavior. Percent data were arcsine transformed, and square root transformations were made on strike frequency and water column position data prior to statistical analysis. The Least Significant Different means comparison test was used to distinguish differences among treatment means (p <= 0.05). Swimming capacity data were also analyzed by simple regression. Statistical Analysis Systems programs were used to perform all analyses on the mainframe computer system of the University of Missouri, Columbia, Missouri, USA.

Table 5a. Mean length (mm) and standard deviation (SD) of brook trout during exposure to aluminum at acidic pH.

		Exposure duration
Mean Al (µg/L)	Mean pH	Day 15		Day 30		Day 45		Day 60
		Mean	SD	Mean	SD	Mean	SD	Mean	SD
Exposure B
5	6.9	16.5	1.7	22.1	2.3	29.7	2.4	36.8	3.2
4	6.5	15.7	2.0	20.4	2.5	29.5	1.5	35.3	2.3
57	6.5	16.1	2.0	19.5	2.6	29.2	2.0	35.1	3.1
88	6.5	16.3	2.0	18.2	2.9	28.9	1.6	34.8	2.9
169	6.6	15.8	2.1	15.9	2.9	28.0	1.8	33.4	2.7
350	6.6	15.3	1.9	14.4	2.5	24.1	4.2	30.1	4.6

 

Table 5b. Mean weight (mg) and standard deviation (SD) of brook trout during exposure to aluminum at acidic pH.

		Exposure duration
Mean Al (µg/L)	Mean pH	Day 15		Day 30		Day 45		Day 60
		Mean	SD	Mean	SD	Mean	SD	Mean	SD
Exposure B
5	6.9	--	--	--	--	248	48	439	142
4	6.5	--	--	--	--	224	37	376	105
57	6.5	--	--	--	--	224	47	372	110
88	6.5	--	--	--	--	210	34	361	100
169	6.6	--	--	--	--	182	36	286	83
350	6.6	--	--	--	--	112	60	204	84

Conclusions:

The 60 day NOECs for S. fontialis exposed to Aluminium sulphate dissolved in the water ranged from 88 to >350 µg/L.
The 30 day NOECs for S. fontialis exposed to Aluminium sulphate dissolved in the water ranged from 57 to 169 µg/L.

Reference 2

Endpoint:

long-term toxicity to fish

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: authors did not statistically calculate effect concentrations

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Reference 3

Endpoint:

long-term toxicity to fish

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: only one exposure concentration

Specific details on test material used for the study:

- CAS no. from report: 7446-70-0
- Source: Aldrich Chemical Co., Milwaukee, WI

Test organisms (species):

Salmo salar

Reference 4

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

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:

Tests began with fathead minnow eggs 16-40 hr old and placed into hatching baskets. All hatched fry were counted and up to ten fry of normal development were taken and released into the exposure aquaria and fed brine shrimp 3-5 times daily for the next 27 days. Observations were made on mortality, length, and weight.

GLP compliance:

not specified

Specific details on test material used for the study:

Analytical purity: reagent grade

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all concentrations
- Sampling method: samples were taken from aquaria centers three times per week. Samples were 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:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Concentrated stock solutions were made for each test. Aliquots were diluted to concentrations of the highest toxicant level tested mechanically on each cycle by the diluters
- All other template details: Not reported

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: fathead minnow
- Strain: Not reported
- Source: Duluth-Newtown laboratory strain, maintained in the Fisheries Laboratories

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish: Not reported
- Method of collection of fertilised eggs: removed from hatchery tiles and viable ones used for testing
- Subsequent handling of eggs: placed in hatching baskets at rate of 20 per basket

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

28 d

Remarks on exposure duration:

posthatch

Post exposure observation period:

Not reported

Hardness:

Hard, value not reported

Test temperature:

24.8 degrees C (overall mean)

pH:

7.27 (overall mean)

Dissolved oxygen:

6.98 mg/L (overall mean)

Salinity:

Not reported

Nominal and measured concentrations:

Range of treatment means (mg/L): 2.32-53.81

Details on test conditions:

TEST SYSTEM
- Embryo cups: not applicable
- Test vessel:
- Type (open vs. closed): Not reported
- Material, size, headspace, fill volume: glass aquaria approximately 5.1 L with a stainless steel screen restricting the fish to a 4.1 L volume.
- Aeration: Diluent water was initially heated and oxygenated in a head tank prior to entering the diluter
- Type of flow-through (e.g. peristaltic or proportional diluter): the diluter is a modification of that of Mount and Brungs (1967). The solenoid switch trip bucket was composed of a 250 ml beaker counter-balanced on a fulcrum. The beaker had a drainage hole which regulated the rate at which water drained out. The stock concentration was held in a stoppered marriot bottle.
- Renewal rate of test solution: The diluter was designed to deliver 2 liters of treatment water to each concentration per cycle, with the 95% turnover time in the test chambers of 5.8 hours.
- No. of fertilized eggs/embryos per vessel: 20, 10 fry selected for further exposure
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: Not reported

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: 233 mg/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: daily from random exposures
- All other template details: Not reported

OTHER TEST CONDITIONS: Not reported

EFFECT PARAMETERS MEASURED: survival determined every 7 days, lengths and weights measured at day 28 posthatch

VEHICLE CONTROL PERFORMED: not applicable

RANGE-FINDING STUDY: Not reported

Reference substance (positive control):

not specified

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

7.1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Remarks:

(fry)

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

11.9 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Remarks:

(fry)

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

4.7 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

weight

Remarks:

(mean)

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

7.1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

weight

Remarks:

(mean)

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

4.7 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

length

Remarks:

(mean)

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

7.1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

length

Remarks:

(mean)

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

23.1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

number hatched

Remarks:

as percent

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

53.8 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

number hatched

Remarks:

as percent

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

5.184 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Basis for effect:

other: Mean dry biomass

Remarks on result:

other: 95% CL: 3.844-6.991

Details on results:

- Fish weights and lengths (mean values) on day 28: see Table 6
- Effect concentrations exceeding solubility of substance in test medium: all metals remained in solution
- 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).

Table 6. Mean terminal lengths (mm) and weights (gm) of the fathead minnow embryo-larval tests.

Mean Al (mg/L)	Mean Length (mm)	Mean Weight (g)
Control	25.4	0.140
2.3	24.9	0.130
4.7	24.8	0.124
7.1	23.1 *	0.098 *
11.9	19.7 *	0.055 *
23.1	- *	- *
53.3	- *	- *

* - significantly different at p = 0.05

Conclusions:

The fathead fry had significant mortality from days 7-28 at the 11.9 mg/L concentration compared to the controls, but not at 7.1 mg/L. The mean weight and length of fry at 28 days was significantly lower than controls at 4.7 mg/L but not at 7.1 mg/L. The number of hatched fry (as percent) was decreased compared to the controls at 53.8 mg/L, but not at 23.1 mg/L.

Reference 5

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01 June 2010- -22 September 2010

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:

equivalent or similar to guideline

Guideline:

other: EPA 2002. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms. Fourth Edition. Office of Water, USEPA, Washington, DC. EPA-821-R-02-013.

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Molecular formula (if other than submission substance): (Al(NO3))3.9H2O
- 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
- Source: J.T. Baker (Philipsburg, NJ, USA)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: samples from each treatment were collected
- Sampling method:

TOTAL ALUMINUM
Analytical samples from each treatment were collected for total recoverable aluminum analysis from “new” renewal waters on Day 0 and 3 and from “old” test waters on Days 4 and 7. 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 beaker. Exceptions to this sampling regime occurred at brief intervals during the test if all organisms within a whole concentration died.

DISSOLVED ALUMINUM
Analytical samples from each treatment were collected for dissolved (filtered through a 0.20 μm PTFE filter with glass microfiber (GMF) pre-filter, Whatman, Maidstone, England #6874-2502) aluminum analysis. 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 collect two 15-20 mL aliquots into a 50 mL polypropylene conical tube 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
- Method:Stock solutions of 1000 mg Al/L were 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 solutions were 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 are expressed as micrograms total Al per liter (μg/L).
The synthetic buffer, MES (2-(n-morpholino)ethanesulfonic acid, monohydrate) was used to control pH during the testing. The buffers (CAS# 145224-94-8, Lot #H08590 and #J24639) were received from J.T. Baker (Phillipsburg, NJ, USA) on 02 February and 21 July 2010, respectively. Two kilograms of MES were supplied in two plastic bottles and stored sealed and in their original containers. The certificates of analysis are reported in Appendix B.
Stock solutions of 200 mM MES was prepared by addition of 85.3 g of substance to 2 Liters of Milli-Q water. The stock solutions were then adjusted to a pH of 5.9 – 6.0 through addition of 10 M NaOH and stored in plastic containers in the dark at 0 - 4°C.
- Controls: dilution water control
- All other template details: Not applicable

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: fathead minnow
- Source: laboratory in-house culture (OSU AquaTox)
- Age at study initiation: less than 24 hours

FEEDING DURING TEST
- Food type: brine shrimp (Artemia salina) nauplii (Brine Shrimp Direct, Ogden, UT, USA)
- Amount: 0.15 mL per chamber of a concentrated suspension
- Frequency: twice per day

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Post exposure observation period:

Not reported

Hardness:

12 mg/L as CaCO3

Test temperature:

25 +/- 2 °C

pH:

6.0 - 6.1

Dissolved oxygen:

> 60%

Salinity:

/

Nominal and measured concentrations:

Nominal concentrations:0, 20, 40, 80, 160, 320 μg/L
Average measured total Al concentrations: 5.0, 25.1, 44.4, 95.3, 175.5, 306.6 μg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass beakers
- Material, size, headspace, fill volume: 250 mL beaker filled with 200 mL of solution
- Renewal rate of test solution: daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS (measured for unfiltered, filtered controls waters)
- Source/preparation of dilution water: Dilution water will be reconstituted laboratory water made from deionized water and amended with the appropriate reagent grade salts (CaSO4 • 2H2O, MgSO4, KCl, and NaHCO3) to achieve the respective nominal hardness values of 10, 25, 60, 120 mg/L as CaCO3. Hardness will be adjusted through the addition of Ca and Mg salts, while alkalinity, Na, K, Cl¯, and HCO3 will remain constant. Reconstituted water will be prepared as detailed in standard USEPA methods (USEPA 2002). DOC will be added as Suwannee River Natural Organic Matter (NOM; obtained from International Humic Substances Society) based on a composition of 52.47% carbon in the NOM to DOC.
- Intervals of water quality measurement:Certain water quality parameters (i.e., temperature, dissolved oxygen [DO], pH, conductitivty) were measured in each concentration on a daily basis. Hardness, alkalinity, total residual chlorine, and total ammonia were measured in the dilution water at test initiation and weekly thereafter.
- Hardness: 12 mg/L as CaCO3
- Alkalinity: 136 mg/L as CaCO3
- Calcium: 1.78 mg/L
- Magnesium: 1.50 mg/L
- Sodium: 3.16 mg/L
- Potassium: 0.282 mg/L
- Chloride: 0.18 mg/L
- Sulfate: 9.62 mg/L
- DOC: <0.08 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to a pH of 6.0 using NaOH and/or HCl, mixed and equilibrated for 3 hours
- Photoperiod: 16:8 light:dark cycle
- Light intensity: 100 foot candles

EFFECT PARAMETERS MEASURED: Observations of live and dead fish were conducted on a daily basis and dead fish were removed immediately. At test termination, the fish in each test chamber were counted, examined for grossly-observable abnormalities, and sacrificed via immersion in ethyl alcohol. The fish from each replicate were then placed into a pre-weighed pan and dried at 105°C for over 12 hours and then re-weighed to the nearest 0.01 mg to obtain a dry weight.

VEHICLE CONTROL PERFORMED: Not applicable

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

95.3 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

175.5 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LC50

Effect conc.:

187.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Remarks on result:

other: CL: 169.2-208.4 μg/L

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

25.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

44.4 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

116.9 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: undeterminable

Duration:

7 d

Dose descriptor:

other: EC20

Effect conc.:

127.2 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: undeterminable

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

150.5 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: undeterminable

Details on results:

/

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Statistical analysis was performed using measured average total Al concentrations. Total Al concentrations have been observed to correlate best with toxicity tests with daphnids, algae, and fathead minnows and have been used consistently for reporting toxicity measures in all Oregon State University studies. Differences in survival 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 or Trimmed Spearman-Karber. The effective concentrations to reduce growth by 10%, 20%, or 50% relative to control performance (EC10/EC20/EC50) were estimated using threshold sigmoid or piecewise linear 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	Mean dry biomass - Average ± Std Dev
5.0	0.90 ± 0.08	0.173 ± 0.01
25.1	1.00 ± 0.00	0.237 ± 0.02
44.1	0.93 ± 0.06	0.129 ± 0.01
95.3	1.00 ± 0.00	0.222 ± 0.03
175.5	0.58 ± 0.22	0.037 ± 0.02
306.6	0.05 ± 0.10	0.003 ± 0.006

Validity criteria fulfilled:

yes

Conclusions:

The NOEC (7d) for Pimephales promelas exposed to a series of aluminum concentrations was 95.3 μg/L Al based on mortality and25.1 μg/L Al based on biomass (based on total Al). The EC10 (7 d) for Pimephales promelas exposed to a series of aluminum concentration was 116.9 μg/L All based on biomass (based on total Al).

Reference 6

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01 June 2010- -22 September 2010

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:

equivalent or similar to guideline

Guideline:

other: EPA 2002. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms. Fourth Edition. Office of Water, USEPA, Washington, DC. EPA-821-R-02-013.

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Molecular formula (if other than submission substance): (Al(NO3))3.9H2O
- 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
- Source: J.T. Baker (Philipsburg, NJ, USA)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: samples from each treatment were collected
- Sampling method:

TOTAL ALUMINUM
Analytical samples from each treatment were collected for total recoverable aluminum analysis from “new” renewal waters on Day 0 and 3 and from “old” test waters on Days 4 and 7. 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 beaker. Exceptions to this sampling regime occurred at brief intervals during the test if all organisms within a whole concentration died.

DISSOLVED ALUMINUM
Analytical samples from each treatment were collected for dissolved (filtered through a 0.20 μm PTFE filter with glass microfiber (GMF) pre-filter, Whatman, Maidstone, England #6874-2502) aluminum analysis. 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 collect two 15-20 mL aliquots into a 50 mL polypropylene conical tube 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
- Method:Stock solutions of 1000 mg Al/L were 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 solutions were 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 are expressed as micrograms total Al per liter (μg/L).
The synthetic buffer, MES (2-(n-morpholino)ethanesulfonic acid, monohydrate) was used to control pH during the testing. The buffers (CAS# 145224-94-8, Lot #H08590 and #J24639) were received from J.T. Baker (Phillipsburg, NJ, USA) on 02 February and 21 July 2010, respectively. Two kilograms of MES were supplied in two plastic bottles and stored sealed and in their original containers. The certificates of analysis are reported in Appendix B.
Stock solutions of 200 mM MES was prepared by addition of 85.3 g of substance to 2 Liters of Milli-Q water. The stock solutions were then adjusted to a pH of 5.9 – 6.0 through addition of 10 M NaOH and stored in plastic containers in the dark at 0 - 4°C.
- Controls: dilution water control
- All other template details: Not applicable

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: fathead minnow
- Source: laboratory in-house culture (OSU AquaTox)
- Age at study initiation: less than 24 hours

FEEDING DURING TEST
- Food type: brine shrimp (Artemia salina) nauplii (Brine Shrimp Direct, Ogden, UT, USA)
- Amount: 0.15 mL per chamber of a concentrated suspension
- Frequency: twice per day

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Post exposure observation period:

Not reported

Hardness:

12 mg/L as CaCO3

Test temperature:

25 +/- 2 °C

pH:

6.0 - 6.1

Dissolved oxygen:

> 60%

Salinity:

/

Nominal and measured concentrations:

Nominal concentrations: 0, 37.5, 75, 150, 300, 600 μg/L
Average measured total Al concentrations: 6.0, 56.6, 117.9, 235.4, 442.6, 564.0 μg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass beakers
- Material, size, headspace, fill volume: 250 mL beaker filled with 200 mL of solution
- Renewal rate of test solution: daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS (measured for unfiltered, filtered controls waters)
- Source/preparation of dilution water: Dilution water will be reconstituted laboratory water made from deionized water and amended with the appropriate reagent grade salts (CaSO4 • 2H2O, MgSO4, KCl, and NaHCO3) to achieve the respective nominal hardness values of 10, 25, 60, 120 mg/L as CaCO3. Hardness will be adjusted through the addition of Ca and Mg salts, while alkalinity, Na, K, Cl¯, and HCO3 will remain constant. Reconstituted water will be prepared as detailed in standard USEPA methods (USEPA 2002). DOC will be added as Suwannee River Natural Organic Matter (NOM; obtained from International Humic Substances Society) based on a composition of 52.47% carbon in the NOM to DOC.
- Intervals of water quality measurement:Certain water quality parameters (i.e., temperature, dissolved oxygen [DO], pH, conductitivty) were measured in each concentration on a daily basis. Hardness, alkalinity, total residual chlorine, and total ammonia were measured in the dilution water at test initiation and weekly thereafter.
- Hardness: 12 mg/L as CaCO3
- Alkalinity: 110 mg/L as CaCO3
- Calcium: 1.68 mg/L
- Magnesium: 1.45 mg/L
- Sodium: 3.06 mg/L
- Potassium: 0.705 mg/L
- Chloride: 1.23 mg/L
- Sulfate: 10.9 mg/L
- DOC: 0.92 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to a pH of 6.0 using NaOH and/or HCl, mixed and equilibrated for 3 hours
- Photoperiod: 16:8 light:dark cycle
- Light intensity: 100 foot candles

EFFECT PARAMETERS MEASURED: Observations of live and dead fish were conducted on a daily basis and dead fish were removed immediately. At test termination, the fish in each test chamber were counted, examined for grossly-observable abnormalities, and sacrificed via immersion in ethyl alcohol. The fish from each replicate were then placed into a pre-weighed pan and dried at 105°C for over 12 hours and then re-weighed to the nearest 0.01 mg to obtain a dry weight.

VEHICLE CONTROL PERFORMED: Not applicable

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

235.4 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

442.6 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LC50

Effect conc.:

491.5 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Remarks on result:

other: CL: 455.1-530.8 μg/L

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

235.4 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

442.6 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

402.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 372.1-436.1 μg/L

Duration:

7 d

Dose descriptor:

other: EC20

Effect conc.:

425.7 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 402.1-450.6 μg/L

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

475 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 457.4-493.3 μg/L

Details on results:

/

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Statistical analysis was performed using measured average total Al concentrations. Total Al concentrations have been observed to correlate best with toxicity tests with daphnids, algae, and fathead minnows and have been used consistently for reporting toxicity measures in all Oregon State University studies. Differences in survival 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 or Trimmed Spearman-Karber. The effective concentrations to reduce growth by 10%, 20%, or 50% relative to control performance (EC10/EC20/EC50) were estimated using threshold sigmoid or piecewise linear 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	Mean dry biomass - Average ± Std Dev
6.0	1.00  ± 0.00	0.267  ± 0.03
56.6	0.98  ± 0.05	0.282 ± 0.04
117.9	0.95  ± 0.06	0.280 ± 0.04
235.4	0.98  ± 0.05	0.277 ± 0.02
442.6	0.85  ± 0.10	0.195 ± 0.04
564.0	0.10  ± 0.12	0.025 ± 0.03

Conclusions:

The NOEC (7d) for Pimephales promelas exposed to a series of aluminum concentrations was 235.4 μg/L Al based on mortality and 235.4 μg/L Al based on biomass (based on total Al). The EC10 (7 d) for Pimephales promelas exposed to a series of aluminum concentration was 402.8 μg/L All based on biomass (based on total Al).

Reference 7

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01 June 2010- -22 September 2010

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:

equivalent or similar to guideline

Guideline:

other: EPA 2002. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms. Fourth Edition. Office of Water, USEPA, Washington, DC. EPA-821-R-02-013.

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Molecular formula (if other than submission substance): (Al(NO3))3.9H2O
- 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
- Source: J.T. Baker (Philipsburg, NJ, USA)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: samples from each treatment were collected
- Sampling method:

TOTAL ALUMINUM
Analytical samples from each treatment were collected for total recoverable aluminum analysis from “new” renewal waters on Day 0 and 3 and from “old” test waters on Days 4 and 7. 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 beaker. Exceptions to this sampling regime occurred at brief intervals during the test if all organisms within a whole concentration died.

DISSOLVED ALUMINUM
Analytical samples from each treatment were collected for dissolved (filtered through a 0.20 μm PTFE filter with glass microfiber (GMF) pre-filter, Whatman, Maidstone, England #6874-2502) aluminum analysis. 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 collect two 15-20 mL aliquots into a 50 mL polypropylene conical tube 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
- Method:Stock solutions of 1000 mg Al/L were 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 solutions were 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 are expressed as micrograms total Al per liter (μg/L).
The synthetic buffer, MES (2-(n-morpholino)ethanesulfonic acid, monohydrate) was used to control pH during the testing. The buffers (CAS# 145224-94-8, Lot #H08590 and #J24639) were received from J.T. Baker (Phillipsburg, NJ, USA) on 02 February and 21 July 2010, respectively. Two kilograms of MES were supplied in two plastic bottles and stored sealed and in their original containers. The certificates of analysis are reported in Appendix B.
Stock solutions of 200 mM MES was prepared by addition of 85.3 g of substance to 2 Liters of Milli-Q water. The stock solutions were then adjusted to a pH of 5.9 – 6.0 through addition of 10 M NaOH and stored in plastic containers in the dark at 0 - 4°C.
- Controls: dilution water control
- All other template details: Not applicable

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: fathead minnow
- Source: laboratory in-house culture (OSU AquaTox)
- Age at study initiation: less than 24 hours

FEEDING DURING TEST
- Food type: brine shrimp (Artemia salina) nauplii (Brine Shrimp Direct, Ogden, UT, USA)
- Amount: 0.15 mL per chamber of a concentrated suspension
- Frequency: twice per day

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Post exposure observation period:

Not reported

Hardness:

12 mg/L as CaCO3

Test temperature:

25 +/- 2 °C

pH:

6.1

Dissolved oxygen:

> 60%

Salinity:

/

Nominal and measured concentrations:

Nominal concentrations:0, 62.5, 125, 250, 500, 1000 μg/L
Average measured total Al concentrations: 8.6, 77.2, 149.6, 264.0, 534.8, 833.6 μg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass beakers
- Material, size, headspace, fill volume: 250 mL beaker filled with 200 mL of solution
- Renewal rate of test solution: daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS (measured for unfiltered, filtered controls waters)
- Source/preparation of dilution water: Dilution water will be reconstituted laboratory water made from deionized water and amended with the appropriate reagent grade salts (CaSO4 • 2H2O, MgSO4, KCl, and NaHCO3) to achieve the respective nominal hardness values of 10, 25, 60, 120 mg/L as CaCO3. Hardness will be adjusted through the addition of Ca and Mg salts, while alkalinity, Na, K, Cl¯, and HCO3 will remain constant. Reconstituted water will be prepared as detailed in standard USEPA methods (USEPA 2002). DOC will be added as Suwannee River Natural Organic Matter (NOM; obtained from International Humic Substances Society) based on a composition of 52.47% carbon in the NOM to DOC.
- Intervals of water quality measurement:Certain water quality parameters (i.e., temperature, dissolved oxygen [DO], pH, conductitivty) were measured in each concentration on a daily basis. Hardness, alkalinity, total residual chlorine, and total ammonia were measured in the dilution water at test initiation and weekly thereafter.
- Hardness: 12 mg/L as CaCO3
- Alkalinity: 150 mg/L as CaCO3
- Calcium: 1.67 mg/L
- Magnesium: 1.55 mg/L
- Sodium: 3.28 mg/L
- Potassium: 0.327 mg/L
- Chloride: 0.42 mg/L
- Sulfate: 10.6 mg/L
- DOC: 1.73 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to a pH of 6.0 using NaOH and/or HCl, mixed and equilibrated for 3 hours
- Photoperiod: 16:8 light:dark cycle
- Light intensity: 100 foot candles

EFFECT PARAMETERS MEASURED: Observations of live and dead fish were conducted on a daily basis and dead fish were removed immediately. At test termination, the fish in each test chamber were counted, examined for grossly-observable abnormalities, and sacrificed via immersion in ethyl alcohol. The fish from each replicate were then placed into a pre-weighed pan and dried at 105°C for over 12 hours and then re-weighed to the nearest 0.01 mg to obtain a dry weight.

VEHICLE CONTROL PERFORMED: Not applicable

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

534.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

833.6 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LC50

Effect conc.:

734.3 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Remarks on result:

other: CL:680.7-792.1 μg/L

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

534.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

833.6 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

589.2 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 435.7-796.9 μg/L

Duration:

7 d

Dose descriptor:

other: EC20

Effect conc.:

632.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 497.2-805.3 μg/L

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

729 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 645.2-823.7 μg/L

Details on results:

/

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Statistical analysis was performed using measured average total Al concentrations. Total Al concentrations have been observed to correlate best with toxicity tests with daphnids, algae, and fathead minnows and have been used consistently for reporting toxicity measures in all Oregon State University studies. Differences in survival 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 or Trimmed Spearman-Karber. The effective concentrations to reduce growth by 10%, 20%, or 50% relative to control performance (EC10/EC20/EC50) were estimated using threshold sigmoid or piecewise linear 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	Mean dry biomass - Average ± Std Dev
8.6	1.00 ± 0.00	0.284 ± 0.05
77.2	1.00 ± 0.00	0.344 ± 0.02
149.6	1.00 ± 0.00	0.329 ± 0.02
264.0	1.00 ± 0.00	0.325 ± 0.03
534.8	1.00 ± 0.00	0.325 ± 0.03
833.6	0.30 ± 0.10	0.070 ± 0.01

Conclusions:

The NOEC (7d) for Pimephales promelas exposed to a series of aluminum concentrations was 534.8 μg/L Al based on mortality and 534.8 μg/L Al based on biomass (based on total Al). The EC10 (7 d) for Pimephales promelas exposed to a series of aluminum concentration was 589.2 μg/L All based on biomass (based on total Al).

Reference 8

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01 June 2010- -22 September 2010

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:

equivalent or similar to guideline

Guideline:

other: EPA 2002. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms. Fourth Edition. Office of Water, USEPA, Washington, DC. EPA-821-R-02-013.

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Molecular formula (if other than submission substance): (Al(NO3))3.9H2O
- 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
- Source: J.T. Baker (Philipsburg, NJ, USA)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: samples from each treatment were collected
- Sampling method:

TOTAL ALUMINUM
Analytical samples from each treatment were collected for total recoverable aluminum analysis from “new” renewal waters on Day 0 and 3 and from “old” test waters on Days 4 and 7. 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 beaker. Exceptions to this sampling regime occurred at brief intervals during the test if all organisms within a whole concentration died.

DISSOLVED ALUMINUM
Analytical samples from each treatment were collected for dissolved (filtered through a 0.20 μm PTFE filter with glass microfiber (GMF) pre-filter, Whatman, Maidstone, England #6874-2502) aluminum analysis. 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 collect two 15-20 mL aliquots into a 50 mL polypropylene conical tube 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
- Method:Stock solutions of 1000 mg Al/L were 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 solutions were 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 are expressed as micrograms total Al per liter (μg/L).
The synthetic buffer, MES (2-(n-morpholino)ethanesulfonic acid, monohydrate) was used to control pH during the testing. The buffers (CAS# 145224-94-8, Lot #H08590 and #J24639) were received from J.T. Baker (Phillipsburg, NJ, USA) on 02 February and 21 July 2010, respectively. Two kilograms of MES were supplied in two plastic bottles and stored sealed and in their original containers. The certificates of analysis are reported in Appendix B.
Stock solutions of 200 mM MES was prepared by addition of 85.3 g of substance to 2 Liters of Milli-Q water. The stock solutions were then adjusted to a pH of 5.9 – 6.0 through addition of 10 M NaOH and stored in plastic containers in the dark at 0 - 4°C.
- Controls: dilution water control
- All other template details: Not applicable

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: fathead minnow
- Source: laboratory in-house culture (OSU AquaTox)
- Age at study initiation: less than 24 hours

FEEDING DURING TEST
- Food type: brine shrimp (Artemia salina) nauplii (Brine Shrimp Direct, Ogden, UT, USA)
- Amount: 0.15 mL per chamber of a concentrated suspension
- Frequency: twice per day

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Post exposure observation period:

Not reported

Hardness:

16 mg/L as CaCO3

Test temperature:

25 +/- 2 °C

pH:

6.0

Dissolved oxygen:

> 60%

Salinity:

/

Nominal and measured concentrations:

Nominal concentrations: 0, 150, 300, 600, 1200, 2400μg/L
Average measured total Al concentrations: 18.8, 224.8, 390.0, 770.5, 1762.7, 1908.5 μg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass beakers
- Material, size, headspace, fill volume: 250 mL beaker filled with 200 mL of solution
- Renewal rate of test solution: daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS (measured for unfiltered, filtered controls waters)
- Source/preparation of dilution water: Dilution water will be reconstituted laboratory water made from deionized water and amended with the appropriate reagent grade salts (CaSO4 • 2H2O, MgSO4, KCl, and NaHCO3) to achieve the respective nominal hardness values of 10, 25, 60, 120 mg/L as CaCO3. Hardness will be adjusted through the addition of Ca and Mg salts, while alkalinity, Na, K, Cl¯, and HCO3 will remain constant. Reconstituted water will be prepared as detailed in standard USEPA methods (USEPA 2002). DOC will be added as Suwannee River Natural Organic Matter (NOM; obtained from International Humic Substances Society) based on a composition of 52.47% carbon in the NOM to DOC.
- Intervals of water quality measurement:Certain water quality parameters (i.e., temperature, dissolved oxygen [DO], pH, conductitivty) were measured in each concentration on a daily basis. Hardness, alkalinity, total residual chlorine, and total ammonia were measured in the dilution water at test initiation and weekly thereafter.
- Hardness: 16 mg/L as CaCO3
- Alkalinity: 120 mg/L as CaCO3
- Calcium: 2.13 mg/L
- Magnesium: 1.57 mg/L
- Sodium: 3.26 mg/L
- Potassium: 2.43 mg/L
- Chloride: 2.34 mg/L
- Sulfate: 9.68 mg/L
- DOC: 3.350 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to a pH of 6.0 using NaOH and/or HCl, mixed and equilibrated for 3 hours
- Photoperiod: 16:8 light:dark cycle
- Light intensity: 100 foot candles

EFFECT PARAMETERS MEASURED: Observations of live and dead fish were conducted on a daily basis and dead fish were removed immediately. At test termination, the fish in each test chamber were counted, examined for grossly-observable abnormalities, and sacrificed via immersion in ethyl alcohol. The fish from each replicate were then placed into a pre-weighed pan and dried at 105°C for over 12 hours and then re-weighed to the nearest 0.01 mg to obtain a dry weight.

VEHICLE CONTROL PERFORMED: Not applicable

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

770.5 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

1 762.7 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LC50

Effect conc.:

1 738 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Remarks on result:

other: CL:1590-1900 μg/L

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

390 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

770.5 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

726.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL:547.6-962.8 μg/L

Duration:

7 d

Dose descriptor:

other: EC20

Effect conc.:

828.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL:690.6-994.7 μg/L

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

1 077.5 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 931.3-1246.7 μg/L

Details on results:

/

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Statistical analysis was performed using measured average total Al concentrations. Total Al concentrations have been observed to correlate best with toxicity tests with daphnids, algae, and fathead minnows and have been used consistently for reporting toxicity measures in all Oregon State University studies. Differences in survival 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 or Trimmed Spearman-Karber. The effective concentrations to reduce growth by 10%, 20%, or 50% relative to control performance (EC10/EC20/EC50) were estimated using threshold sigmoid or piecewise linear 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	Mean dry biomass - Average ± Std Dev
18.8	1.00 ± 0.00	0.362 ± 0.03
224.8	1.00 ± 0.00	0.354 ± 0.01
390.0	1.00 ± 0.00	0.345 ± 0.02
770.5	1.00 ± 0.00	0.303 ± 0.01
1762.7	0.75 ± 0.17	0.144 ± 0.06
1908.5	0.10 ± 0.08	0.006 ± 0.005

Conclusions:

The NOEC (7d) for Pimephales promelas exposed to a series of aluminum concentrations was 770.5 μg/L Al based on mortality and 390 μg/L Al based on biomass (based on total Al). The EC10 (7 d) for Pimephales promelas exposed to a series of aluminum concentration was 726.1 μg/L All based on biomass (based on total Al).

Reference 9

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01 June 2010- -22 September 2010

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:

equivalent or similar to guideline

Guideline:

other: EPA 2002. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms. Fourth Edition. Office of Water, USEPA, Washington, DC. EPA-821-R-02-013.

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Molecular formula (if other than submission substance): (Al(NO3))3.9H2O
- 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
- Source: J.T. Baker (Philipsburg, NJ, USA)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: samples from each treatment were collected
- Sampling method:

TOTAL ALUMINUM
Analytical samples from each treatment were collected for total recoverable aluminum analysis from “new” renewal waters on Day 0 and 3 and from “old” test waters on Days 4 and 7. 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 beaker. Exceptions to this sampling regime occurred at brief intervals during the test if all organisms within a whole concentration died.

DISSOLVED ALUMINUM
Analytical samples from each treatment were collected for dissolved (filtered through a 0.20 μm PTFE filter with glass microfiber (GMF) pre-filter, Whatman, Maidstone, England #6874-2502) aluminum analysis. 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 collect two 15-20 mL aliquots into a 50 mL polypropylene conical tube 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
- Method:Stock solutions of 1000 mg Al/L were 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 solutions were 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 are expressed as micrograms total Al per liter (μg/L).
The synthetic buffer, MES (2-(n-morpholino)ethanesulfonic acid, monohydrate) was used to control pH during the testing. The buffers (CAS# 145224-94-8, Lot #H08590 and #J24639) were received from J.T. Baker (Phillipsburg, NJ, USA) on 02 February and 21 July 2010, respectively. Two kilograms of MES were supplied in two plastic bottles and stored sealed and in their original containers. The certificates of analysis are reported in Appendix B.
Stock solutions of 200 mM MES was prepared by addition of 85.3 g of substance to 2 Liters of Milli-Q water. The stock solutions were then adjusted to a pH of 5.9 – 6.0 through addition of 10 M NaOH and stored in plastic containers in the dark at 0 - 4°C.
- Controls: dilution water control
- All other template details: Not applicable

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: fathead minnow
- Source: laboratory in-house culture (OSU AquaTox)
- Age at study initiation: less than 24 hours

FEEDING DURING TEST
- Food type: brine shrimp (Artemia salina) nauplii (Brine Shrimp Direct, Ogden, UT, USA)
- Amount: 0.15 mL per chamber of a concentrated suspension
- Frequency: twice per day

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Post exposure observation period:

Not reported

Hardness:

116 mg/L as CaCO3

Test temperature:

25 +/- 2 °C

pH:

6.0 - 6.1

Dissolved oxygen:

> 60%

Salinity:

/

Nominal and measured concentrations:

Nominal concentrations:0, 125, 250, 500, 1000, 2000 μg/L
Average measured total Al concentrations: 3.8, 142.5, 253.9, 446.1, 946.7, 1561.5 μg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass beakers
- Material, size, headspace, fill volume: 250 mL beaker filled with 200 mL of solution
- Renewal rate of test solution: daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS (measured for unfiltered, filtered controls waters)
- Source/preparation of dilution water: Dilution water will be reconstituted laboratory water made from deionized water and amended with the appropriate reagent grade salts (CaSO4 • 2H2O, MgSO4, KCl, and NaHCO3) to achieve the respective nominal hardness values of 10, 25, 60, 120 mg/L as CaCO3. Hardness will be adjusted through the addition of Ca and Mg salts, while alkalinity, Na, K, Cl¯, and HCO3 will remain constant. Reconstituted water will be prepared as detailed in standard USEPA methods (USEPA 2002). DOC will be added as Suwannee River Natural Organic Matter (NOM; obtained from International Humic Substances Society) based on a composition of 52.47% carbon in the NOM to DOC.
- Intervals of water quality measurement:Certain water quality parameters (i.e., temperature, dissolved oxygen [DO], pH, conductitivty) were measured in each concentration on a daily basis. Hardness, alkalinity, total residual chlorine, and total ammonia were measured in the dilution water at test initiation and weekly thereafter.
- Hardness: 116 mg/L as CaCO3
- Alkalinity: 148 mg/L as CaCO3
- Calcium: 19.6 mg/L
- Magnesium: 18.2 mg/L
- Sodium: 3.62 mg/L
- Potassium: 0.746 mg/L
- Chloride: 1.28 mg/L
- Sulfate: 107 mg/L
- DOC: 0.088 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to a pH of 6.0 using NaOH and/or HCl, mixed and equilibrated for 3 hours
- Photoperiod: 16:8 light:dark cycle
- Light intensity: 100 foot candles

EFFECT PARAMETERS MEASURED: Observations of live and dead fish were conducted on a daily basis and dead fish were removed immediately. At test termination, the fish in each test chamber were counted, examined for grossly-observable abnormalities, and sacrificed via immersion in ethyl alcohol. The fish from each replicate were then placed into a pre-weighed pan and dried at 105°C for over 12 hours and then re-weighed to the nearest 0.01 mg to obtain a dry weight.

VEHICLE CONTROL PERFORMED: Not applicable

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

1 561.5 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 1 561.5 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LC50

Effect conc.:

> 1 561.5 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Remarks on result:

other: CL: not applicable

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

446.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

946.7 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

481.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL:279.5-828.2 μg/L

Duration:

7 d

Dose descriptor:

other: EC20

Effect conc.:

624.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 409.8-950.5 μg/L

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

1 046.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 845.6-1294.0 μg/L

Details on results:

/

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Statistical analysis was performed using measured average total Al concentrations. Total Al concentrations have been observed to correlate best with toxicity tests with daphnids, algae, and fathead minnows and have been used consistently for reporting toxicity measures in all Oregon State University studies. Differences in survival 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 or Trimmed Spearman-Karber. The effective concentrations to reduce growth by 10%, 20%, or 50% relative to control performance (EC10/EC20/EC50) were estimated using threshold sigmoid or piecewise linear 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	Mean dry biomass - Average ± Std Dev
3.8	1.00 ± 0.00	0.260 ± 0.06
142.5	0.95 ± 0.10	0.302 ± 0.04
253.9	0.98 ± 0.05	0.290 ± 0.03
446.1	1.00 ± 0.00	0.255 ± 0.03
946.7	0.98 ± 0.05	0.180 ± 0.04
1561.5	0.98 ± 0.05	0.072 ± 0.01

Conclusions:

The NOEC (7d) for Pimephales promelas exposed to a series of aluminum concentrations was 1561.5 μg/L Al based on mortality and 446.1 μg/L Al based on biomass (based on total Al). The EC10 (7 d) for Pimephales promelas exposed to a series of aluminum concentration was 481.1 μg/L All based on biomass (based on total Al).

Reference 10

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01 June 2010- -22 September 2010

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:

equivalent or similar to guideline

Guideline:

other: EPA 2002. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms. Fourth Edition. Office of Water, USEPA, Washington, DC. EPA-821-R-02-013.

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Molecular formula (if other than submission substance): (Al(NO3))3.9H2O
- 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
- Source: J.T. Baker (Philipsburg, NJ, USA)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: samples from each treatment were collected
- Sampling method:

TOTAL ALUMINUM
Analytical samples from each treatment were collected for total recoverable aluminum analysis from “new” renewal waters on Day 0 and 3 and from “old” test waters on Days 4 and 7. 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 beaker. Exceptions to this sampling regime occurred at brief intervals during the test if all organisms within a whole concentration died.

DISSOLVED ALUMINUM
Analytical samples from each treatment were collected for dissolved (filtered through a 0.20 μm PTFE filter with glass microfiber (GMF) pre-filter, Whatman, Maidstone, England #6874-2502) aluminum analysis. 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 collect two 15-20 mL aliquots into a 50 mL polypropylene conical tube 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
- Method:Stock solutions of 1000 mg Al/L were 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 solutions were 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 are expressed as micrograms total Al per liter (μg/L).
The synthetic buffer, MES (2-(n-morpholino)ethanesulfonic acid, monohydrate) was used to control pH during the testing. The buffers (CAS# 145224-94-8, Lot #H08590 and #J24639) were received from J.T. Baker (Phillipsburg, NJ, USA) on 02 February and 21 July 2010, respectively. Two kilograms of MES were supplied in two plastic bottles and stored sealed and in their original containers. The certificates of analysis are reported in Appendix B.
Stock solutions of 200 mM MES was prepared by addition of 85.3 g of substance to 2 Liters of Milli-Q water. The stock solutions were then adjusted to a pH of 5.9 – 6.0 through addition of 10 M NaOH and stored in plastic containers in the dark at 0 - 4°C.
- Controls: dilution water control
- All other template details: Not applicable

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: fathead minnow
- Source: laboratory in-house culture (OSU AquaTox)
- Age at study initiation: less than 24 hours

FEEDING DURING TEST
- Food type: brine shrimp (Artemia salina) nauplii (Brine Shrimp Direct, Ogden, UT, USA)
- Amount: 0.15 mL per chamber of a concentrated suspension
- Frequency: twice per day

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Post exposure observation period:

Not reported

Hardness:

116 mg/L as CaCO3

Test temperature:

25 +/- 2 °C

pH:

6.1

Dissolved oxygen:

> 60%

Salinity:

/

Nominal and measured concentrations:

Nominal concentrations: 0, 250, 500, 1000, 2000, 4000 μg/L
Average measured total Al concentrations: 5.7, 270.8, 533.4, 833.7, 1470.7, 2493.8 μg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass beakers
- Material, size, headspace, fill volume: 250 mL beaker filled with 200 mL of solution
- Renewal rate of test solution: daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS (measured for unfiltered, filtered controls waters)
- Source/preparation of dilution water: Dilution water will be reconstituted laboratory water made from deionized water and amended with the appropriate reagent grade salts (CaSO4 • 2H2O, MgSO4, KCl, and NaHCO3) to achieve the respective nominal hardness values of 10, 25, 60, 120 mg/L as CaCO3. Hardness will be adjusted through the addition of Ca and Mg salts, while alkalinity, Na, K, Cl¯, and HCO3 will remain constant. Reconstituted water will be prepared as detailed in standard USEPA methods (USEPA 2002). DOC will be added as Suwannee River Natural Organic Matter (NOM; obtained from International Humic Substances Society) based on a composition of 52.47% carbon in the NOM to DOC.
- Intervals of water quality measurement:Certain water quality parameters (i.e., temperature, dissolved oxygen [DO], pH, conductitivty) were measured in each concentration on a daily basis. Hardness, alkalinity, total residual chlorine, and total ammonia were measured in the dilution water at test initiation and weekly thereafter.
- Hardness: 116 mg/L as CaCO3
- Alkalinity: 120 mg/L as CaCO3
- Calcium: 19.7 mg/L
- Magnesium: 18.1 mg/L
- Sodium: 3.49 mg/L
- Potassium: 0.356 mg/L
- Chloride: 1.75 mg/L
- Sulfate: 110 mg/L
- DOC: 0.88 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to a pH of 6.0 using NaOH and/or HCl, mixed and equilibrated for 3 hours
- Photoperiod: 16:8 light:dark cycle
- Light intensity: 100 foot candles

EFFECT PARAMETERS MEASURED: Observations of live and dead fish were conducted on a daily basis and dead fish were removed immediately. At test termination, the fish in each test chamber were counted, examined for grossly-observable abnormalities, and sacrificed via immersion in ethyl alcohol. The fish from each replicate were then placed into a pre-weighed pan and dried at 105°C for over 12 hours and then re-weighed to the nearest 0.01 mg to obtain a dry weight.

VEHICLE CONTROL PERFORMED: Not applicable

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

2 493.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 2 493.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LC50

Effect conc.:

> 2 494 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Remarks on result:

other: CL: not applicable

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

533.4 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

833.7 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

626.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL:433.1-907.1 μg/L

Duration:

7 d

Dose descriptor:

other: EC20

Effect conc.:

773.4 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 559.1-1069.8 μg/L

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

1 452.6 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 1126.7-1872.9 μg/L

Details on results:

/

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Statistical analysis was performed using measured average total Al concentrations. Total Al concentrations have been observed to correlate best with toxicity tests with daphnids, algae, and fathead minnows and have been used consistently for reporting toxicity measures in all Oregon State University studies. Differences in survival 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 or Trimmed Spearman-Karber. The effective concentrations to reduce growth by 10%, 20%, or 50% relative to control performance (EC10/EC20/EC50) were estimated using threshold sigmoid or piecewise linear 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	Mean dry biomass - Average ± Std Dev
5.7	1.00 ± 0.00	0.325 ± 0.02
270.8	1.00 ± 0.00	0.327 ± 0.04
533.4	1.00 ± 0.00	0.330 ± 0.02
833.7	0.98 ± 0.05	0.232 ± 0.02
1470.7	0.73 ± 0.15	0.090 ± 0.07
2493.8	0.90 ± 0.12	0.085 ± 0.02

Conclusions:

The NOEC (7d) for Pimephales promelas exposed to a series of aluminum concentrations was 2493.8 μg/L Al based on mortality and 533.4 μg/L Al based on biomass (based on total Al). The EC10 (7 d) for Pimephales promelas exposed to a series of aluminum concentration was 626.8 μg/L All based on biomass (based on total Al).

Reference 11

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01 June 2010- -22 September 2010

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:

equivalent or similar to guideline

Guideline:

other: EPA 2002. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms. Fourth Edition. Office of Water, USEPA, Washington, DC. EPA-821-R-02-013.

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Molecular formula (if other than submission substance): (Al(NO3))3.9H2O
- 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
- Source: J.T. Baker (Philipsburg, NJ, USA)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: samples from each treatment were collected
- Sampling method:

TOTAL ALUMINUM
Analytical samples from each treatment were collected for total recoverable aluminum analysis from “new” renewal waters on Day 0 and 3 and from “old” test waters on Days 4 and 7. 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 beaker. Exceptions to this sampling regime occurred at brief intervals during the test if all organisms within a whole concentration died.

DISSOLVED ALUMINUM
Analytical samples from each treatment were collected for dissolved (filtered through a 0.20 μm PTFE filter with glass microfiber (GMF) pre-filter, Whatman, Maidstone, England #6874-2502) aluminum analysis. 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 collect two 15-20 mL aliquots into a 50 mL polypropylene conical tube 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
- Method:Stock solutions of 1000 mg Al/L were 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 solutions were 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 are expressed as micrograms total Al per liter (μg/L).
The synthetic buffer, MES (2-(n-morpholino)ethanesulfonic acid, monohydrate) was used to control pH during the testing. The buffers (CAS# 145224-94-8, Lot #H08590 and #J24639) were received from J.T. Baker (Phillipsburg, NJ, USA) on 02 February and 21 July 2010, respectively. Two kilograms of MES were supplied in two plastic bottles and stored sealed and in their original containers. The certificates of analysis are reported in Appendix B.
Stock solutions of 200 mM MES was prepared by addition of 85.3 g of substance to 2 Liters of Milli-Q water. The stock solutions were then adjusted to a pH of 5.9 – 6.0 through addition of 10 M NaOH and stored in plastic containers in the dark at 0 - 4°C.
- Controls: dilution water control
- All other template details: Not applicable

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: fathead minnow
- Source: laboratory in-house culture (OSU AquaTox)
- Age at study initiation: less than 24 hours

FEEDING DURING TEST
- Food type: brine shrimp (Artemia salina) nauplii (Brine Shrimp Direct, Ogden, UT, USA)
- Amount: 0.15 mL per chamber of a concentrated suspension
- Frequency: twice per day

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Post exposure observation period:

Not reported

Hardness:

108 mg/L as CaCO3

Test temperature:

25 +/- 2 °C

pH:

6.0

Dissolved oxygen:

> 60%

Salinity:

/

Nominal and measured concentrations:

Nominal concentrations: 0, 312.5, 625, 1250, 2500, 5000 μg/L
Average measured total Al concentrations: 9.4, 364.3, 568.8, 1362.2, 2019.2, 3202.3 μg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass beakers
- Material, size, headspace, fill volume: 250 mL beaker filled with 200 mL of solution
- Renewal rate of test solution: daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS (measured for unfiltered, filtered controls waters)
- Source/preparation of dilution water: Dilution water will be reconstituted laboratory water made from deionized water and amended with the appropriate reagent grade salts (CaSO4 • 2H2O, MgSO4, KCl, and NaHCO3) to achieve the respective nominal hardness values of 10, 25, 60, 120 mg/L as CaCO3. Hardness will be adjusted through the addition of Ca and Mg salts, while alkalinity, Na, K, Cl¯, and HCO3 will remain constant. Reconstituted water will be prepared as detailed in standard USEPA methods (USEPA 2002). DOC will be added as Suwannee River Natural Organic Matter (NOM; obtained from International Humic Substances Society) based on a composition of 52.47% carbon in the NOM to DOC.
- Intervals of water quality measurement:Certain water quality parameters (i.e., temperature, dissolved oxygen [DO], pH, conductitivty) were measured in each concentration on a daily basis. Hardness, alkalinity, total residual chlorine, and total ammonia were measured in the dilution water at test initiation and weekly thereafter.
- Hardness: 108 mg/L as CaCO3
- Alkalinity: 124 mg/L as CaCO3
- Calcium: 18.8 mg/L
- Magnesium: 17.3 mg/L
- Sodium: 3.31 mg/L
- Potassium: 0.275 mg/L
- Chloride: 0.67 mg/L
- Sulfate: 106 mg/L
- DOC: 1.56 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to a pH of 6.0 using NaOH and/or HCl, mixed and equilibrated for 3 hours
- Photoperiod: 16:8 light:dark cycle
- Light intensity: 100 foot candles

EFFECT PARAMETERS MEASURED: Observations of live and dead fish were conducted on a daily basis and dead fish were removed immediately. At test termination, the fish in each test chamber were counted, examined for grossly-observable abnormalities, and sacrificed via immersion in ethyl alcohol. The fish from each replicate were then placed into a pre-weighed pan and dried at 105°C for over 12 hours and then re-weighed to the nearest 0.01 mg to obtain a dry weight.

VEHICLE CONTROL PERFORMED: Not applicable

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

1 362.2 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

2 019.2 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LC50

Effect conc.:

2 513 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Remarks on result:

other: CL: 2156-2930 µg/L

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

364.3 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

586.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

1 388.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL:996.6-1933.4 μg/L

Duration:

7 d

Dose descriptor:

other: EC20

Effect conc.:

1 493.7 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 1115.9-1999.4 μg/L

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

1 861.2 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 1382.7-2505.3 μg/L

Details on results:

/

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Statistical analysis was performed using measured average total Al concentrations. Total Al concentrations have been observed to correlate best with toxicity tests with daphnids, algae, and fathead minnows and have been used consistently for reporting toxicity measures in all Oregon State University studies. Differences in survival 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 or Trimmed Spearman-Karber. The effective concentrations to reduce growth by 10%, 20%, or 50% relative to control performance (EC10/EC20/EC50) were estimated using threshold sigmoid or piecewise linear 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	Mean dry biomass - Average ± Std Dev
9.4	1.00 ± 0.00	0.337 ± 0.05
364.3	1.00 ± 0.00	0.371 ± 0.03
586.8	0.88 ± 0.10	0.223 ± 0.05
1362.2	1.00 ± 0.00	0.315 ± 0.02
2019.2	0.75 ± 0.13	0.132 ± 0.03
3202.3	0.23 ± 0.15	0.016 ± 0.01

Conclusions:

The NOEC (7d) for Pimephales promelas exposed to a series of aluminum concentrations was 1362.2 μg/L Al based on mortality and 364.3 μg/L Al based on biomass (based on total Al). The EC10 (7 d) for Pimephales promelas exposed to a series of aluminum concentration was 1388.1 μg/L All based on biomass (based on total Al).

Reference 12

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01 June 2010- -22 September 2010

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:

equivalent or similar to guideline

Guideline:

other: EPA 2002. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms. Fourth Edition. Office of Water, USEPA, Washington, DC. EPA-821-R-02-013.

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Molecular formula (if other than submission substance): (Al(NO3))3.9H2O
- 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
- Source: J.T. Baker (Philipsburg, NJ, USA)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: samples from each treatment were collected
- Sampling method:

TOTAL ALUMINUM
Analytical samples from each treatment were collected for total recoverable aluminum analysis from “new” renewal waters on Day 0 and 3 and from “old” test waters on Days 4 and 7. 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 beaker. Exceptions to this sampling regime occurred at brief intervals during the test if all organisms within a whole concentration died.

DISSOLVED ALUMINUM
Analytical samples from each treatment were collected for dissolved (filtered through a 0.20 μm PTFE filter with glass microfiber (GMF) pre-filter, Whatman, Maidstone, England #6874-2502) aluminum analysis. 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 collect two 15-20 mL aliquots into a 50 mL polypropylene conical tube 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
- Method:Stock solutions of 1000 mg Al/L were 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 solutions were 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 are expressed as micrograms total Al per liter (μg/L).
The synthetic buffer, MES (2-(n-morpholino)ethanesulfonic acid, monohydrate) was used to control pH during the testing. The buffers (CAS# 145224-94-8, Lot #H08590 and #J24639) were received from J.T. Baker (Phillipsburg, NJ, USA) on 02 February and 21 July 2010, respectively. Two kilograms of MES were supplied in two plastic bottles and stored sealed and in their original containers. The certificates of analysis are reported in Appendix B.
Stock solutions of 200 mM MES was prepared by addition of 85.3 g of substance to 2 Liters of Milli-Q water. The stock solutions were then adjusted to a pH of 5.9 – 6.0 through addition of 10 M NaOH and stored in plastic containers in the dark at 0 - 4°C.
- Controls: dilution water control
- All other template details: Not applicable

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: fathead minnow
- Source: laboratory in-house culture (OSU AquaTox)
- Age at study initiation: less than 24 hours

FEEDING DURING TEST
- Food type: brine shrimp (Artemia salina) nauplii (Brine Shrimp Direct, Ogden, UT, USA)
- Amount: 0.15 mL per chamber of a concentrated suspension
- Frequency: twice per day

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Post exposure observation period:

Not reported

Hardness:

112 mg/L as CaCO3

Test temperature:

25 +/- 2 °C

pH:

6.0 - 6.1

Dissolved oxygen:

> 60%

Salinity:

/

Nominal and measured concentrations:

Nominal concentrations: 0,500, 1000, 2000, 4000, 8000 μg/L
Average measured total Al concentrations: 14.1, 648.8, 1548.7, 2534.4, 3941.1, 4711.9 μg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass beakers
- Material, size, headspace, fill volume: 250 mL beaker filled with 200 mL of solution
- Renewal rate of test solution: daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS (measured for unfiltered, filtered controls waters)
- Source/preparation of dilution water: Dilution water will be reconstituted laboratory water made from deionized water and amended with the appropriate reagent grade salts (CaSO4 • 2H2O, MgSO4, KCl, and NaHCO3) to achieve the respective nominal hardness values of 10, 25, 60, 120 mg/L as CaCO3. Hardness will be adjusted through the addition of Ca and Mg salts, while alkalinity, Na, K, Cl¯, and HCO3 will remain constant. Reconstituted water will be prepared as detailed in standard USEPA methods (USEPA 2002). DOC will be added as Suwannee River Natural Organic Matter (NOM; obtained from International Humic Substances Society) based on a composition of 52.47% carbon in the NOM to DOC.
- Intervals of water quality measurement:Certain water quality parameters (i.e., temperature, dissolved oxygen [DO], pH, conductitivty) were measured in each concentration on a daily basis. Hardness, alkalinity, total residual chlorine, and total ammonia were measured in the dilution water at test initiation and weekly thereafter.
- Hardness: 112 mg/L as CaCO3
- Alkalinity: 128 mg/L as CaCO3
- Calcium: 18.5 mg/L
- Magnesium: 16.4 mg/L
- Sodium: 53.3 mg/L
- Potassium: 0.442 mg/L
- Chloride: 0.35 mg/L
- Sulfate: 104 mg/L
- DOC: 3.62 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to a pH of 6.0 using NaOH and/or HCl, mixed and equilibrated for 3 hours
- Photoperiod: 16:8 light:dark cycle
- Light intensity: 100 foot candles

EFFECT PARAMETERS MEASURED: Observations of live and dead fish were conducted on a daily basis and dead fish were removed immediately. At test termination, the fish in each test chamber were counted, examined for grossly-observable abnormalities, and sacrificed via immersion in ethyl alcohol. The fish from each replicate were then placed into a pre-weighed pan and dried at 105°C for over 12 hours and then re-weighed to the nearest 0.01 mg to obtain a dry weight.

VEHICLE CONTROL PERFORMED: Not applicable

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

4 711.9 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 4 711.9 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LC50

Effect conc.:

> 4 711.9 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Remarks on result:

other: CL: not applicable

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

2 534.4 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

3 941.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

2 729 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL:2048-3636 μg/L

Duration:

7 d

Dose descriptor:

other: EC20

Effect conc.:

2 938 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL:2288-3772 μg/L

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

3 666 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 3182-4224 μg/L

Details on results:

/

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Statistical analysis was performed using measured average total Al concentrations. Total Al concentrations have been observed to correlate best with toxicity tests with daphnids, algae, and fathead minnows and have been used consistently for reporting toxicity measures in all Oregon State University studies. Differences in survival 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 or Trimmed Spearman-Karber. The effective concentrations to reduce growth by 10%, 20%, or 50% relative to control performance (EC10/EC20/EC50) were estimated using threshold sigmoid or piecewise linear 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	Mean dry biomass - Average ± Std Dev
14.1	0.93 ± 0.05	0.315 ± 0.06
648.8	0.98 ± 0.05	0.292 ± 0.06
1548.7	0.98 ± 0.05	0.356 ± 0.04
2534.4	0.98 ± 0.05	0.035 ± 0.03
3941.1	0.88 ± 0.05	0.132 ± 0.04
4711.9	0.95 ± 0.06	0.055 ± 0.01

Conclusions:

The NOEC (7d) for Pimephales promelas exposed to a series of aluminum concentrations was 4711.9 μg/L Al based on mortality and 2534.4 μg/L Al based on biomass (based on total Al). The EC10 (7 d) for Pimephales promelas exposed to a series of aluminum concentration was 2729 μg/L All based on biomass (based on total Al).

Reference 13

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01 June 2010- -22 September 2010

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:

equivalent or similar to guideline

Guideline:

other: EPA 2002. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms. Fourth Edition. Office of Water, USEPA, Washington, DC. EPA-821-R-02-013.

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Molecular formula (if other than submission substance): (Al(NO3))3.9H2O
- 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
- Source: J.T. Baker (Philipsburg, NJ, USA)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: samples from each treatment were collected
- Sampling method:

TOTAL ALUMINUM
Analytical samples from each treatment were collected for total recoverable aluminum analysis from “new” renewal waters on Day 0 and 3 and from “old” test waters on Days 4 and 7. 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 beaker. Exceptions to this sampling regime occurred at brief intervals during the test if all organisms within a whole concentration died.

DISSOLVED ALUMINUM
Analytical samples from each treatment were collected for dissolved (filtered through a 0.20 μm PTFE filter with glass microfiber (GMF) pre-filter, Whatman, Maidstone, England #6874-2502) aluminum analysis. 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 collect two 15-20 mL aliquots into a 50 mL polypropylene conical tube 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
- Method:Stock solutions of 1000 mg Al/L were 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 solutions were 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 are expressed as micrograms total Al per liter (μg/L).
The synthetic buffer, MES (2-(n-morpholino)ethanesulfonic acid, monohydrate) was used to control pH during the testing. The buffers (CAS# 145224-94-8, Lot #H08590 and #J24639) were received from J.T. Baker (Phillipsburg, NJ, USA) on 02 February and 21 July 2010, respectively. Two kilograms of MES were supplied in two plastic bottles and stored sealed and in their original containers. The certificates of analysis are reported in Appendix B.
Stock solutions of 200 mM MES was prepared by addition of 85.3 g of substance to 2 Liters of Milli-Q water. The stock solutions were then adjusted to a pH of 5.9 – 6.0 through addition of 10 M NaOH and stored in plastic containers in the dark at 0 - 4°C.
- Controls: dilution water control
- All other template details: Not applicable

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: fathead minnow
- Source: laboratory in-house culture (OSU AquaTox)
- Age at study initiation: less than 24 hours

FEEDING DURING TEST
- Food type: brine shrimp (Artemia salina) nauplii (Brine Shrimp Direct, Ogden, UT, USA)
- Amount: 0.15 mL per chamber of a concentrated suspension
- Frequency: twice per day

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Post exposure observation period:

Not reported

Hardness:

24 mg/L as CaCO3

Test temperature:

25 +/- 2 °C

pH:

6.0 - 6.1

Dissolved oxygen:

> 60%

Salinity:

/

Nominal and measured concentrations:

Nominal concentrations: 0, 31.3, 62.5, 125, 250, 500 μg/L
Average measured total Al concentrations: 10.1, 38.3, 90.7, 181.4, 275.9, 527.6 μg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass beakers
- Material, size, headspace, fill volume: 250 mL beaker filled with 200 mL of solution
- Renewal rate of test solution: daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS (measured for unfiltered, filtered controls waters)
- Source/preparation of dilution water: Dilution water will be reconstituted laboratory water made from deionized water and amended with the appropriate reagent grade salts (CaSO4 • 2H2O, MgSO4, KCl, and NaHCO3) to achieve the respective nominal hardness values of 10, 25, 60, 120 mg/L as CaCO3. Hardness will be adjusted through the addition of Ca and Mg salts, while alkalinity, Na, K, Cl¯, and HCO3 will remain constant. Reconstituted water will be prepared as detailed in standard USEPA methods (USEPA 2002). DOC will be added as Suwannee River Natural Organic Matter (NOM; obtained from International Humic Substances Society) based on a composition of 52.47% carbon in the NOM to DOC.
- Intervals of water quality measurement:Certain water quality parameters (i.e., temperature, dissolved oxygen [DO], pH, conductitivty) were measured in each concentration on a daily basis. Hardness, alkalinity, total residual chlorine, and total ammonia were measured in the dilution water at test initiation and weekly thereafter.
- Hardness: 24 mg/L as CaCO3
- Alkalinity: 184 mg/L as CaCO3
- Calcium: 4.1 mg/L
- Magnesium: 3.78 mg/L
- Sodium: 3.18 mg/L
- Potassium: 0.631 mg/L
- Chloride: 0.53 mg/L
- Sulfate: 25.0 mg/L
- DOC: 0.19 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to a pH of 6.0 using NaOH and/or HCl, mixed and equilibrated for 3 hours
- Photoperiod: 16:8 light:dark cycle
- Light intensity: 100 foot candles

EFFECT PARAMETERS MEASURED: Observations of live and dead fish were conducted on a daily basis and dead fish were removed immediately. At test termination, the fish in each test chamber were counted, examined for grossly-observable abnormalities, and sacrificed via immersion in ethyl alcohol. The fish from each replicate were then placed into a pre-weighed pan and dried at 105°C for over 12 hours and then re-weighed to the nearest 0.01 mg to obtain a dry weight.

VEHICLE CONTROL PERFORMED: Not applicable

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

527.6 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 527.6 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LC50

Effect conc.:

> 527.6 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Remarks on result:

other: CL: not applicable

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

90.7 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

181.4 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

93 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL:61.9-139.9 μg/L

Duration:

7 d

Dose descriptor:

other: EC20

Effect conc.:

135.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL:98.4-187.5 μg/L

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

287.6 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 206.7-400.1 μg/L

Details on results:

/

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Statistical analysis was performed using measured average total Al concentrations. Total Al concentrations have been observed to correlate best with toxicity tests with daphnids, algae, and fathead minnows and have been used consistently for reporting toxicity measures in all Oregon State University studies. Differences in survival 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 or Trimmed Spearman-Karber. The effective concentrations to reduce growth by 10%, 20%, or 50% relative to control performance (EC10/EC20/EC50) were estimated using threshold sigmoid or piecewise linear 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	Mean dry biomass - Average ± Std Dev
10.1	1.00 ± 0.00	0.275 ± 0.03
38.3	1.00 ± 0.00	0.298 ± 0.02
90.7	1.00 ± 0.00	0.263 ± 0.01
181.4	0.97 ± 0.06	0.205 ± 0.03
275.9	0.98 ± 0.05	0.184 ± 0.06
527.6	0.85 ± 0.17	0.064 ± 0.03

Conclusions:

The NOEC (7d) for Pimephales promelas exposed to a series of aluminum concentrations was 527.6 μg/L Al based on mortality and 90.7 μg/L Al based on biomass (based on total Al). The EC10 (7 d) for Pimephales promelas exposed to a series of aluminum concentration was 93.0 μg/L All based on biomass (based on total Al).

Reference 14

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01 June 2010- -22 September 2010

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:

equivalent or similar to guideline

Guideline:

other: EPA 2002. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms. Fourth Edition. Office of Water, USEPA, Washington, DC. EPA-821-R-02-013.

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Molecular formula (if other than submission substance): (Al(NO3))3.9H2O
- 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
- Source: J.T. Baker (Philipsburg, NJ, USA)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: samples from each treatment were collected
- Sampling method:

TOTAL ALUMINUM
Analytical samples from each treatment were collected for total recoverable aluminum analysis from “new” renewal waters on Day 0 and 3 and from “old” test waters on Days 4 and 7. 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 beaker. Exceptions to this sampling regime occurred at brief intervals during the test if all organisms within a whole concentration died.

DISSOLVED ALUMINUM
Analytical samples from each treatment were collected for dissolved (filtered through a 0.20 μm PTFE filter with glass microfiber (GMF) pre-filter, Whatman, Maidstone, England #6874-2502) aluminum analysis. 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 collect two 15-20 mL aliquots into a 50 mL polypropylene conical tube 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
- Method:Stock solutions of 1000 mg Al/L were 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 solutions were 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 are expressed as micrograms total Al per liter (μg/L).
The synthetic buffer, MES (2-(n-morpholino)ethanesulfonic acid, monohydrate) was used to control pH during the testing. The buffers (CAS# 145224-94-8, Lot #H08590 and #J24639) were received from J.T. Baker (Phillipsburg, NJ, USA) on 02 February and 21 July 2010, respectively. Two kilograms of MES were supplied in two plastic bottles and stored sealed and in their original containers. The certificates of analysis are reported in Appendix B.
Stock solutions of 200 mM MES was prepared by addition of 85.3 g of substance to 2 Liters of Milli-Q water. The stock solutions were then adjusted to a pH of 5.9 – 6.0 through addition of 10 M NaOH and stored in plastic containers in the dark at 0 - 4°C.
- Controls: dilution water control
- All other template details: Not applicable

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: fathead minnow
- Source: laboratory in-house culture (OSU AquaTox)
- Age at study initiation: less than 24 hours

FEEDING DURING TEST
- Food type: brine shrimp (Artemia salina) nauplii (Brine Shrimp Direct, Ogden, UT, USA)
- Amount: 0.15 mL per chamber of a concentrated suspension
- Frequency: twice per day

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Post exposure observation period:

Not reported

Hardness:

60 mg/L as CaCO3

Test temperature:

25 +/- 2 °C

pH:

6.0

Dissolved oxygen:

> 60%

Salinity:

/

Nominal and measured concentrations:

Nominal concentrations: 0, 50, 100, 200, 400, 800 μg/L
Average measured total Al concentrations: <5.9, 61.6, 117.1, 240.6, 363.3, 614.8 μg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass beakers
- Material, size, headspace, fill volume: 250 mL beaker filled with 200 mL of solution
- Renewal rate of test solution: daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS (measured for unfiltered, filtered controls waters)
- Source/preparation of dilution water: Dilution water will be reconstituted laboratory water made from deionized water and amended with the appropriate reagent grade salts (CaSO4 • 2H2O, MgSO4, KCl, and NaHCO3) to achieve the respective nominal hardness values of 10, 25, 60, 120 mg/L as CaCO3. Hardness will be adjusted through the addition of Ca and Mg salts, while alkalinity, Na, K, Cl¯, and HCO3 will remain constant. Reconstituted water will be prepared as detailed in standard USEPA methods (USEPA 2002). DOC will be added as Suwannee River Natural Organic Matter (NOM; obtained from International Humic Substances Society) based on a composition of 52.47% carbon in the NOM to DOC.
- Intervals of water quality measurement:Certain water quality parameters (i.e., temperature, dissolved oxygen [DO], pH, conductitivty) were measured in each concentration on a daily basis. Hardness, alkalinity, total residual chlorine, and total ammonia were measured in the dilution water at test initiation and weekly thereafter.
- Hardness: 60 mg/L as CaCO3
- Alkalinity: 130 mg/L as CaCO3
- Calcium: 10.0 mg/L
- Magnesium: 8.70 mg/L
- Sodium: 3.13 mg/L
- Potassium: 0.371 mg/L
- Chloride: 0.32 mg/L
- Sulfate: 55.8 mg/L
- DOC: 0.22 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to a pH of 6.0 using NaOH and/or HCl, mixed and equilibrated for 3 hours
- Photoperiod: 16:8 light:dark cycle
- Light intensity: 100 foot candles

EFFECT PARAMETERS MEASURED: Observations of live and dead fish were conducted on a daily basis and dead fish were removed immediately. At test termination, the fish in each test chamber were counted, examined for grossly-observable abnormalities, and sacrificed via immersion in ethyl alcohol. The fish from each replicate were then placed into a pre-weighed pan and dried at 105°C for over 12 hours and then re-weighed to the nearest 0.01 mg to obtain a dry weight.

VEHICLE CONTROL PERFORMED: Not applicable

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

614.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 614.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LC50

Effect conc.:

> 614.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Remarks on result:

other: CL: not applicable

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

363.3 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

614.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

241.3 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL:156.3-372.4 μg/L

Duration:

7 d

Dose descriptor:

other: EC20

Effect conc.:

314.3 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL:199.6-494.9 μg/L

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

694.7 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 255.2-1890.7 μg/L

Details on results:

/

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Statistical analysis was performed using measured average total Al concentrations. Total Al concentrations have been observed to correlate best with toxicity tests with daphnids, algae, and fathead minnows and have been used consistently for reporting toxicity measures in all Oregon State University studies. Differences in survival 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 or Trimmed Spearman-Karber. The effective concentrations to reduce growth by 10%, 20%, or 50% relative to control performance (EC10/EC20/EC50) were estimated using threshold sigmoid or piecewise linear 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	Mean dry biomass - Average ± Std Dev
<5.9	1.00 ± 0.00	0.240 ± 0.02
61.6	1.00 ± 0.00	0.309 ± 0.03
117.1	0.98 ± 0.05	0.307 ± 0.01
240.6	1.00 ± 0.00	0.264 ± 0.002
363.3	1.00 ± 0.00	0.230 ± 0.02
614.8	0.98 ± 0.05	0.156 ± 0.02

Conclusions:

The NOEC (7d) for Pimephales promelas exposed to a series of aluminum concentrations was 614.8 μg/L Al based on mortality and 363.3 μg/L Al based on biomass (based on total Al). The EC10 (7 d) for Pimephales promelas exposed to a series of aluminum concentration was 241.3 μg/L All based on biomass (based on total Al).

Reference 15

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01 June 2010- -22 September 2010

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:

equivalent or similar to guideline

Guideline:

other: EPA 2002. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms. Fourth Edition. Office of Water, USEPA, Washington, DC. EPA-821-R-02-013.

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Molecular formula (if other than submission substance): (Al(NO3))3.9H2O
- 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
- Source: J.T. Baker (Philipsburg, NJ, USA)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: samples from each treatment were collected
- Sampling method:

TOTAL ALUMINUM
Analytical samples from each treatment were collected for total recoverable aluminum analysis from “new” renewal waters on Day 0 and 3 and from “old” test waters on Days 4 and 7. 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 beaker. Exceptions to this sampling regime occurred at brief intervals during the test if all organisms within a whole concentration died.

DISSOLVED ALUMINUM
Analytical samples from each treatment were collected for dissolved (filtered through a 0.20 μm PTFE filter with glass microfiber (GMF) pre-filter, Whatman, Maidstone, England #6874-2502) aluminum analysis. 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 collect two 15-20 mL aliquots into a 50 mL polypropylene conical tube 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
- Method:Stock solutions of 1000 mg Al/L were 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 solutions were 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 are expressed as micrograms total Al per liter (μg/L).
The synthetic buffer, MES (2-(n-morpholino)ethanesulfonic acid, monohydrate) was used to control pH during the testing. The buffers (CAS# 145224-94-8, Lot #H08590 and #J24639) were received from J.T. Baker (Phillipsburg, NJ, USA) on 02 February and 21 July 2010, respectively. Two kilograms of MES were supplied in two plastic bottles and stored sealed and in their original containers. The certificates of analysis are reported in Appendix B.
Stock solutions of 200 mM MES was prepared by addition of 85.3 g of substance to 2 Liters of Milli-Q water. The stock solutions were then adjusted to a pH of 5.9 – 6.0 through addition of 10 M NaOH and stored in plastic containers in the dark at 0 - 4°C.
- Controls: dilution water control
- All other template details: Not applicable

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: fathead minnow
- Source: laboratory in-house culture (OSU AquaTox)
- Age at study initiation: less than 24 hours

FEEDING DURING TEST
- Food type: brine shrimp (Artemia salina) nauplii (Brine Shrimp Direct, Ogden, UT, USA)
- Amount: 0.15 mL per chamber of a concentrated suspension
- Frequency: twice per day

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Post exposure observation period:

Not reported

Hardness:

60 mg/L as CaCO3

Test temperature:

25 +/- 2 °C

pH:

6.0 - 6.1

Dissolved oxygen:

> 60%

Salinity:

/

Nominal and measured concentrations:

Nominal concentrations:0,75, 150, 300, 600, 1200 μg/L
Average measured total Al concentrations: 7.4, 95.0, 177.7, 378.7, 744.0, 1122.0 μg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass beakers
- Material, size, headspace, fill volume: 250 mL beaker filled with 200 mL of solution
- Renewal rate of test solution: daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS (measured for unfiltered, filtered controls waters)
- Source/preparation of dilution water: Dilution water will be reconstituted laboratory water made from deionized water and amended with the appropriate reagent grade salts (CaSO4 • 2H2O, MgSO4, KCl, and NaHCO3) to achieve the respective nominal hardness values of 10, 25, 60, 120 mg/L as CaCO3. Hardness will be adjusted through the addition of Ca and Mg salts, while alkalinity, Na, K, Cl¯, and HCO3 will remain constant. Reconstituted water will be prepared as detailed in standard USEPA methods (USEPA 2002). DOC will be added as Suwannee River Natural Organic Matter (NOM; obtained from International Humic Substances Society) based on a composition of 52.47% carbon in the NOM to DOC.
- Intervals of water quality measurement:Certain water quality parameters (i.e., temperature, dissolved oxygen [DO], pH, conductitivty) were measured in each concentration on a daily basis. Hardness, alkalinity, total residual chlorine, and total ammonia were measured in the dilution water at test initiation and weekly thereafter.
- Hardness: 60 mg/L as CaCO3
- Alkalinity: 120 mg/L as CaCO3
- Calcium: 9.95 mg/L
- Magnesium: 8.78 mg/L
- Sodium: 3.35 mg/L
- Potassium: 0.422 mg/L
- Chloride: 0.32 mg/L
- Sulfate: 55.9 mg/L
- DOC: 0.86 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to a pH of 6.0 using NaOH and/or HCl, mixed and equilibrated for 3 hours
- Photoperiod: 16:8 light:dark cycle
- Light intensity: 100 foot candles

EFFECT PARAMETERS MEASURED: Observations of live and dead fish were conducted on a daily basis and dead fish were removed immediately. At test termination, the fish in each test chamber were counted, examined for grossly-observable abnormalities, and sacrificed via immersion in ethyl alcohol. The fish from each replicate were then placed into a pre-weighed pan and dried at 105°C for over 12 hours and then re-weighed to the nearest 0.01 mg to obtain a dry weight.

VEHICLE CONTROL PERFORMED: Not applicable

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

744 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

1 122 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LC50

Effect conc.:

951.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Remarks on result:

other: CL: 901.5 - 1003 µg/L

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

378.7 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

744 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

582.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL:273.3-1239.8 μg/L

Duration:

7 d

Dose descriptor:

other: EC20

Effect conc.:

633.9 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 337.6-1190.4 μg/L

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

818.4 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 575.5-1163.9 μg/L

Details on results:

/

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Statistical analysis was performed using measured average total Al concentrations. Total Al concentrations have been observed to correlate best with toxicity tests with daphnids, algae, and fathead minnows and have been used consistently for reporting toxicity measures in all Oregon State University studies. Differences in survival 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 or Trimmed Spearman-Karber. The effective concentrations to reduce growth by 10%, 20%, or 50% relative to control performance (EC10/EC20/EC50) were estimated using threshold sigmoid or piecewise linear 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	Mean dry biomass - Average ± Std Dev
7.4	0.95 ± 0.06	0.234 ± 0.03
95.0	1.00 ± 0.00	0.292 ± 0.03
177.7	1.00 ± 0.00	0.299 ± 0.01
378.7	0.83 ± 0.13	0.241 ± 0.05
744.0	0.95 ± 0.06	0.179 ± 0.03
1122.0	0.23 ± 0.15	0.038 ± 0.04

Conclusions:

The NOEC (7d) for Pimephales promelas exposed to a series of aluminum concentrations was 744 μg/L Al based on mortality and 378.7 μg/L Al based on biomass (based on total Al). The EC10 (7 d) for Pimephales promelas exposed to a series of aluminum concentration was 582.1 μg/L All based on biomass (based on total Al).

Reference 16

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01 June 2010- -22 September 2010

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:

equivalent or similar to guideline

Guideline:

other: EPA 2002. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms. Fourth Edition. Office of Water, USEPA, Washington, DC. EPA-821-R-02-013.

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Molecular formula (if other than submission substance): (Al(NO3))3.9H2O
- 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
- Source: J.T. Baker (Philipsburg, NJ, USA)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: samples from each treatment were collected
- Sampling method:

TOTAL ALUMINUM
Analytical samples from each treatment were collected for total recoverable aluminum analysis from “new” renewal waters on Day 0 and 3 and from “old” test waters on Days 4 and 7. 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 beaker. Exceptions to this sampling regime occurred at brief intervals during the test if all organisms within a whole concentration died.

DISSOLVED ALUMINUM
Analytical samples from each treatment were collected for dissolved (filtered through a 0.20 μm PTFE filter with glass microfiber (GMF) pre-filter, Whatman, Maidstone, England #6874-2502) aluminum analysis. 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 collect two 15-20 mL aliquots into a 50 mL polypropylene conical tube 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
- Method:Stock solutions of 1000 mg Al/L were 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 solutions were 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 are expressed as micrograms total Al per liter (μg/L).
The synthetic buffer, MES (2-(n-morpholino)ethanesulfonic acid, monohydrate) was used to control pH during the testing. The buffers (CAS# 145224-94-8, Lot #H08590 and #J24639) were received from J.T. Baker (Phillipsburg, NJ, USA) on 02 February and 21 July 2010, respectively. Two kilograms of MES were supplied in two plastic bottles and stored sealed and in their original containers. The certificates of analysis are reported in Appendix B.
Stock solutions of 200 mM MES was prepared by addition of 85.3 g of substance to 2 Liters of Milli-Q water. The stock solutions were then adjusted to a pH of 5.9 – 6.0 through addition of 10 M NaOH and stored in plastic containers in the dark at 0 - 4°C.
- Controls: dilution water control
- All other template details: Not applicable

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: fathead minnow
- Source: laboratory in-house culture (OSU AquaTox)
- Age at study initiation: less than 24 hours

FEEDING DURING TEST
- Food type: brine shrimp (Artemia salina) nauplii (Brine Shrimp Direct, Ogden, UT, USA)
- Amount: 0.15 mL per chamber of a concentrated suspension
- Frequency: twice per day

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Post exposure observation period:

Not reported

Hardness:

56 mg/L as CaCO3

Test temperature:

25 +/- 2 °C

pH:

6.0 - 6.1

Dissolved oxygen:

> 60%

Salinity:

/

Nominal and measured concentrations:

Nominal concentrations: 0, 125, 250, 500, 1000, 2000 μg/L
Average measured total Al concentrations: 7.9, 156.2, 278.4, 504.0, 1156.5, 1553.2 μg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass beakers
- Material, size, headspace, fill volume: 250 mL beaker filled with 200 mL of solution
- Renewal rate of test solution: daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS (measured for unfiltered, filtered controls waters)
- Source/preparation of dilution water: Dilution water will be reconstituted laboratory water made from deionized water and amended with the appropriate reagent grade salts (CaSO4 • 2H2O, MgSO4, KCl, and NaHCO3) to achieve the respective nominal hardness values of 10, 25, 60, 120 mg/L as CaCO3. Hardness will be adjusted through the addition of Ca and Mg salts, while alkalinity, Na, K, Cl¯, and HCO3 will remain constant. Reconstituted water will be prepared as detailed in standard USEPA methods (USEPA 2002). DOC will be added as Suwannee River Natural Organic Matter (NOM; obtained from International Humic Substances Society) based on a composition of 52.47% carbon in the NOM to DOC.
- Intervals of water quality measurement:Certain water quality parameters (i.e., temperature, dissolved oxygen [DO], pH, conductitivty) were measured in each concentration on a daily basis. Hardness, alkalinity, total residual chlorine, and total ammonia were measured in the dilution water at test initiation and weekly thereafter.
- Hardness: 56 mg/L as CaCO3
- Alkalinity: 124 mg/L as CaCO3
- Calcium: 9.77 mg/L
- Magnesium: 8.62 mg/L
- Sodium: 3.36 mg/L
- Potassium: 0.333 mg/L
- Chloride: 0.46 mg/L
- Sulfate: 52.5 mg/L
- DOC: 1.74 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to a pH of 6.0 using NaOH and/or HCl, mixed and equilibrated for 3 hours
- Photoperiod: 16:8 light:dark cycle
- Light intensity: 100 foot candles

EFFECT PARAMETERS MEASURED: Observations of live and dead fish were conducted on a daily basis and dead fish were removed immediately. At test termination, the fish in each test chamber were counted, examined for grossly-observable abnormalities, and sacrificed via immersion in ethyl alcohol. The fish from each replicate were then placed into a pre-weighed pan and dried at 105°C for over 12 hours and then re-weighed to the nearest 0.01 mg to obtain a dry weight.

VEHICLE CONTROL PERFORMED: Not applicable

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

1 553.2 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 1 153.2 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LC50

Effect conc.:

> 1 553.2 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Remarks on result:

other: CL: not applicable

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

1 156.5 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

1 553.2 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

1 238.3 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL:999.2-1534.5 μg/L

Duration:

7 d

Dose descriptor:

other: EC20

Effect conc.:

1 325.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 1119.1-1570.7 μg/L

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

1 627.3 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 1380.3-1918.4μg/L

Details on results:

/

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Statistical analysis was performed using measured average total Al concentrations. Total Al concentrations have been observed to correlate best with toxicity tests with daphnids, algae, and fathead minnows and have been used consistently for reporting toxicity measures in all Oregon State University studies. Differences in survival 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 or Trimmed Spearman-Karber. The effective concentrations to reduce growth by 10%, 20%, or 50% relative to control performance (EC10/EC20/EC50) were estimated using threshold sigmoid or piecewise linear 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	Mean dry biomass - Average ± Std Dev
7.9	1.00 ± 0.00	0.319 ± 0.02
156.2	0.98 ± 0.05	0.333 ± 0.03
278.4	1.00 ± 0.00	0.342 ± 0.04
504.0	1.00 ± 0.00	0.370 ± 0.02
1156.5	0.95 ± 0.05	0.345 ± 0.02
1553.2	0.88 ± 0.13	0.195 ± 0.02

Conclusions:

The NOEC (7d) for Pimephales promelas exposed to a series of aluminum concentrations was 1553.2 μg/L Al based on mortality and 1156.5 μg/L Al based on biomass (based on total Al). The EC10 (7 d) for Pimephales promelas exposed to a series of aluminum concentration was 1238.3 μg/L All based on biomass (based on total Al).

Reference 17

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01 June 2010- -22 September 2010

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:

equivalent or similar to guideline

Guideline:

other: EPA 2002. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms. Fourth Edition. Office of Water, USEPA, Washington, DC. EPA-821-R-02-013.

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Molecular formula (if other than submission substance): (Al(NO3))3.9H2O
- 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
- Source: J.T. Baker (Philipsburg, NJ, USA)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: samples from each treatment were collected
- Sampling method:

TOTAL ALUMINUM
Analytical samples from each treatment were collected for total recoverable aluminum analysis from “new” renewal waters on Day 0 and 3 and from “old” test waters on Days 4 and 7. 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 beaker. Exceptions to this sampling regime occurred at brief intervals during the test if all organisms within a whole concentration died.

DISSOLVED ALUMINUM
Analytical samples from each treatment were collected for dissolved (filtered through a 0.20 μm PTFE filter with glass microfiber (GMF) pre-filter, Whatman, Maidstone, England #6874-2502) aluminum analysis. 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 collect two 15-20 mL aliquots into a 50 mL polypropylene conical tube 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
- Method:Stock solutions of 1000 mg Al/L were 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 solutions were 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 are expressed as micrograms total Al per liter (μg/L).
The synthetic buffer, MES (2-(n-morpholino)ethanesulfonic acid, monohydrate) was used to control pH during the testing. The buffers (CAS# 145224-94-8, Lot #H08590 and #J24639) were received from J.T. Baker (Phillipsburg, NJ, USA) on 02 February and 21 July 2010, respectively. Two kilograms of MES were supplied in two plastic bottles and stored sealed and in their original containers. The certificates of analysis are reported in Appendix B.
Stock solutions of 200 mM MES was prepared by addition of 85.3 g of substance to 2 Liters of Milli-Q water. The stock solutions were then adjusted to a pH of 5.9 – 6.0 through addition of 10 M NaOH and stored in plastic containers in the dark at 0 - 4°C.
- Controls: dilution water control
- All other template details: Not applicable

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: fathead minnow
- Source: laboratory in-house culture (OSU AquaTox)
- Age at study initiation: less than 24 hours

FEEDING DURING TEST
- Food type: brine shrimp (Artemia salina) nauplii (Brine Shrimp Direct, Ogden, UT, USA)
- Amount: 0.15 mL per chamber of a concentrated suspension
- Frequency: twice per day

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Post exposure observation period:

Not reported

Hardness:

60 mg/L as CaCO3

Test temperature:

25 +/- 2 °C

pH:

6.0

Dissolved oxygen:

> 60%

Salinity:

/

Nominal and measured concentrations:

Nominal concentrations:0, 200, 400, 800, 1600, 3200 μg/L
Average measured total Al concentrations: 15.0, 307.5, 520.0, 985.0, 1961.8, 3023.8 μg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass beakers
- Material, size, headspace, fill volume: 250 mL beaker filled with 200 mL of solution
- Renewal rate of test solution: daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS (measured for unfiltered, filtered controls waters)
- Source/preparation of dilution water: Dilution water will be reconstituted laboratory water made from deionized water and amended with the appropriate reagent grade salts (CaSO4 • 2H2O, MgSO4, KCl, and NaHCO3) to achieve the respective nominal hardness values of 10, 25, 60, 120 mg/L as CaCO3. Hardness will be adjusted through the addition of Ca and Mg salts, while alkalinity, Na, K, Cl¯, and HCO3 will remain constant. Reconstituted water will be prepared as detailed in standard USEPA methods (USEPA 2002). DOC will be added as Suwannee River Natural Organic Matter (NOM; obtained from International Humic Substances Society) based on a composition of 52.47% carbon in the NOM to DOC.
- Intervals of water quality measurement:Certain water quality parameters (i.e., temperature, dissolved oxygen [DO], pH, conductitivty) were measured in each concentration on a daily basis. Hardness, alkalinity, total residual chlorine, and total ammonia were measured in the dilution water at test initiation and weekly thereafter.
- Hardness: 60 mg/L as CaCO3
- Alkalinity: 124 mg/L as CaCO3
- Calcium: 10.5 mg/L
- Magnesium: 9.36 mg/L
- Sodium: 3.32 mg/L
- Potassium: 0.370 mg/L
- Chloride: 0.46 mg/L
- Sulfate: 56.8 mg/L
- DOC: 3.51 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to a pH of 6.0 using NaOH and/or HCl, mixed and equilibrated for 3 hours
- Photoperiod: 16:8 light:dark cycle
- Light intensity: 100 foot candles

EFFECT PARAMETERS MEASURED: Observations of live and dead fish were conducted on a daily basis and dead fish were removed immediately. At test termination, the fish in each test chamber were counted, examined for grossly-observable abnormalities, and sacrificed via immersion in ethyl alcohol. The fish from each replicate were then placed into a pre-weighed pan and dried at 105°C for over 12 hours and then re-weighed to the nearest 0.01 mg to obtain a dry weight.

VEHICLE CONTROL PERFORMED: Not applicable

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

3 023.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 3 023.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LC50

Effect conc.:

> 3 023.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Remarks on result:

other: CL: not applicable

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

1 961.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

3 023.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

2 164 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL:1841-2543 μg/L

Duration:

7 d

Dose descriptor:

other: EC20

Effect conc.:

2 523 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 1971-3230 μg/L

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

3 999 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 2068-7736 μg/L

Details on results:

/

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Statistical analysis was performed using measured average total Al concentrations. Total Al concentrations have been observed to correlate best with toxicity tests with daphnids, algae, and fathead minnows and have been used consistently for reporting toxicity measures in all Oregon State University studies. Differences in survival 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 or Trimmed Spearman-Karber. The effective concentrations to reduce growth by 10%, 20%, or 50% relative to control performance (EC10/EC20/EC50) were estimated using threshold sigmoid or piecewise linear 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	Mean dry biomass - Average ± Std Dev
15.0	1.00 ± 0.00	0.354 ± 0.04
307.5	1.00 ± 0.00	0.374 ± 0.02
520.0	1.00 ± 0.00	0.389 ± 0.03
985.0	1.00 ± 0.00	0.360 ± 0.02
1961.8	1.00 ± 0.00	0.357 ± 0.02
3023.8	1.00 ± 0.00	0.253 ± 0.05

Conclusions:

The NOEC (7d) for Pimephales promelas exposed to a series of aluminum concentrations was 3023.8 μg/L Al based on mortality and 1961.8 μg/L Al based on biomass (based on total Al). The EC10 (7 d) for Pimephales promelas exposed to a series of aluminum concentration was 2164 μg/L All based on biomass (based on total Al).

Reference 18

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

09/2011-10/2011

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Qualifier:

according to guideline

Guideline:

other: ASTM. 2002. Standard Guide for Conducting Early Life-Stage Toxicity Tests with Fishes. E1241-98. In: 2002 ASTM Annual Book of Standards, Volume 11.05.ASTM.

Principles of method if other than guideline:

/

GLP compliance:

yes

Specific details on test material used for the study:

- 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:

- Concentrations: samples from each treatment and concentration
- Sampling method:
TOTAL ALUMINUM:
Analytical samples from each treatment were collected for total recoverable aluminum analysis from “new” waters twice 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. Monomeric samples were immediately analyzed. Samples for “new” monomeric were typically collected at the same frequency as the dissolved samples. Samples for “old” monomeric were only collected twice during the test.
- Sample storage conditions before analysis:
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.

Vehicle:

no

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name:Fathead minnow
- Source: OSU AquaTox

POST-HATCH FEEDING
- Start date: Day 6
- Feeding: Once fish began to hatch, each chamber was fed 0.15 mL of live brine shrimp (Artemia salina) nauplii. As the test progressed, the amount of brine shrimp fed to all chambers was increased. The volume of brine shrimp added was dependent upon the amount of uneaten brine shrimp remaining in the chambers one hour after feeding. If very little or no brine shrimp was left, the feeding rate was increased 0.05 mL per feeding, accordingly.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

33 d

Post exposure observation period:

/

Hardness:

96 mg/L as CaCo3

Test temperature:

25°C

pH:

5.9-6.5

Dissolved oxygen:

8.0-8.3 mg/L

Salinity:

/

Nominal and measured concentrations:

- Nominal concentrations: 0, 75, 150, 300, 600, 1200 μg/L
- Average measured total Al concentrations (+ Total standard deviation) : 2.2 (-), 79.2 (15.9), 164.3 (24.0), 308.1 (45.3), 558.1 (72.8), 1104.6 (96.4) μg/L

Details on test conditions:

TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): Embryo cups were constructed of glass tubing (approximately 4 cm outer diameter; 7 cm long) with a Nitex® screen bottom (attached with silicone adhesive). Cups were suspended in the test solution within each test chamber, and were oscillated 2.5 to 4.0 cm vertically in the test water by a rocker-arm apparatus at a rate of 2 revolutions per minute (rpm).
- Test vessel:1L rectangular glass aquaria
- Material, size, headspace, fill volume: Glass filled with 700-800mL of testing solution
- Type of flow-through: proportional diluter
- Renewal rate of test solution (frequency/flow rate): The flow rate to each test chamber was approximately 7.5 mL/min providing approximately 18 to 27 volume exchanges per replicate chamber per day.
- No. of organisms per vessel:15
- 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 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).
- Alkalinity:18mg/L as CaCO3
- Ca/mg ratio:2.2
- DOC: <0.50 mg/L
- Total residual chlorine: <0.05mg/L
- Ammonia: <1.0 mg/L
- Calcium: 21.5 mg/L
- Magnesium: 9.77mg/L
- Sodium: 27.2mg/L
- Potassium: 0.877mg/L
- Chloride: 101mg/L
- Sulfate: 0.93mg/L
- Culture medium different from test medium:no
- Intervals of water quality measurementCertain water quality parameters (i.e., temperature, dissolved oxygen [DO], pH, conductitivty) were measured in each concentration on a daily basis. 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 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:using cool-white fluorescent lights at ~50 foot candles

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations of live and dead eggs were conducted on a daily basis from initiation to thinning (Day 3), and then to full hatch (Day 6 in all concentrations but the highest, which had full hatch by Day 7). At test termination, the fish in each test chamber were counted, examined for grossly-observable abnormalities, and sacrificed via immersion in ethyl alcohol. The fish from each replicate were then placed into a pre-weighed pan, dried at 105°C for over 12 hours, and then re-weighed to the nearest 0.01 mg to obtain a dry weight.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: a 14-day static-renewal range finding test with a high exposure concentration of 10000 ug/L Al resulted in a growth effect in the 1000 μg/L and higher Al concentrations.
- Results used to determine the conditions for the definitive study: yes

POST-HATCH DETAILS
- Begin of post-hatch period: 6-7 days
- No. of hatched eggs (alevins)/treatment released to the test chamber: 15
- Release of alevins from incubation cups to test chamber on day no.: day 6 in all concentrations but the highest, which had full hatch by Day 7.

Reference substance (positive control):

no

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

558.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

time to hatch

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

308.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

558.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

33 d

Dose descriptor:

LOEC

Effect conc.:

1 104.6 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

time to hatch

Duration:

33 d

Dose descriptor:

LOEC

Effect conc.:

558.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

33 d

Dose descriptor:

LOEC

Effect conc.:

1 104.6 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

33 d

Dose descriptor:

LC50

Effect conc.:

507.3 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Remarks on result:

other: CL: 463.0-55.8 μg/L

Duration:

33 d

Dose descriptor:

LC10

Effect conc.:

389.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Remarks on result:

other: CL: undeterminable

Duration:

33 d

Dose descriptor:

EC10

Effect conc.:

417.4 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 150.6-1157.0 μg/L

Duration:

33 d

Dose descriptor:

other: LC20

Effect conc.:

428.6 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Remarks on result:

other: CL: undeterminable

Duration:

33 d

Dose descriptor:

other: EC20

Effect conc.:

500.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 237.2-1057.2 μg/L

Duration:

33 d

Dose descriptor:

LC50

Effect conc.:

519.3 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Remarks on result:

other: CL: undeterminable

Duration:

33 d

Dose descriptor:

EC50

Effect conc.:

719 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Remarks on result:

other: CL: 432.6-1194.9 μg/L

Details on results:

- Mortality/survival at embryo, larval, juvenile, and adult stages: control survival from the thinning of eggs until test termination was 83%
Average proportion survived at test termination:
at 2.2 μg/L Al: 0.83 ± 0.13
at 79.2 μg/L Al: 0.78 ± 0.10
at 164.3 μg/L Al: 0.75 ± 0.14
at 308.1 μg/L Al: 0.83 ± 0.26
at 558.1 μg/L Al: 0.30 ± 0.16
at 1104.6 μg/L Al: 0 ± 0
- Days to hatch or time to release of young:
at 2.2 μg/L Al: 3.6 ± 0.3
at 79.2 μg/L Al: 3.6 ± 0.3
at 164.3 μg/L Al: 3.6 ± 0.3
at 308.1 μg/L Al: 3.3 ± 0.3
at 558.1 μg/L Al: 3.4 ± 0.2
at 1104.6 μg/L Al: 5.9 ± 0.7
- Observations on body length and weight of young and/or exposed parents at one or more time periods:
Average dry weight per organism:
at 2.2 μg/L Al: 1.35 ± 0.13mg
at 79.2 μg/L Al: 1.57 ± 0.15 mg
at 164.3 μg/L Al: 1.57 ± 0.49 mg
at 308.1 μg/L Al: 1.37 ± 0.49 mg
at 558.1 μg/L Al: 4.14 ± 1.94 mg
at 1104.6 μg/L Al: -

Results with reference substance (positive control):

/

Reported statistics and error estimates:

STATISTICAL ANALYSIS
Statistical analysis was performed using measured average total Al concentrations of both “new” and “old” test solutions. Differences in the embryo median day to hatch, fry survival and growth 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 or 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. Initial parameters input into the TRAP database for logIC50, Y-intercept, and steepness were: 2.8, 1.17, and 0.78, respectively. TRAP output error summary stated that maximum iterations were reached without convergence and steepness was at a maximum/minimum limit.

Validity criteria fulfilled:

yes

Remarks:

survival:83%

Conclusions:

The NOEC (33d) for Pimephales promelas exposed to a series of aluminum concentrations was 558.1 μg/L Al based on time to hatch and biomass and 308.1 μg/L Al based on mortality (based on total Al). The EC10 (33 d) for Pimephales promelas exposed to a series of aluminum concentration was 389.1 μg/L Al based on mortality, 417.4 μg/L Al based on biomass (based on total Al).

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 fathead minnow, Pimephales promelas. Use of this test organism qualifies as a level of organization (Phylum) that is 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). P. promelas were exposed to a series of aluminum concentrations for 33 days, starting as freshly-fertilized embryos. To allow for possible changes in aluminum speciation, exposure solutions were aged for a minimum of a 3-hour equilibrium period prior to organism exposure. Nominal test concentrations ranged from 0 to 1200 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 1104.6 μg total Al/L and resulted in a significant effect on egg hatchability and growth (as mean dry biomass) at the highest exposure concentration. Larval/fry survival was effected at the two highest concentrations and was calculated to be the most sensitive endpoint. The study resulted in a no observable effect concentration [NOEC] of 308.1 μg/L total Al and a lowest observable effect concentration [LOEC] of 558.1 μg/L total Al for survival. The effective concentrations to reduce survival by 10% and 20% relative to control performance (EC10 and EC20) was 389.1 and 428.6 μg/L total Al, respectively.

Reference 19

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

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:

according to guideline

Guideline:

other: US EPA, 2002.

Deviations:

yes

Remarks:

modifications for specific water types, pH control, and testing with Al

Principles of method if other than guideline:

This research was conducted according to the protocol, “Short-term chronic toxicity of aluminum to the fathead minnow (Pimephales promelas) - expansion of toxicity testing under variable water quality conditions” OSU AquaTox Protocol No. Al-PP-CSR7d-035. Appendix A (Protocol) of the Study Report contains a copy of the protocol. The studies were conducted according to standard USEPA methodology (USEPA 2002), with modifications for specific water types and pH control.

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 #P21D039) from Alfa Aesar (Ward Hill, MA, USA). The test substance had a reported assay purity of 98.3%. . The manufacturer’s safety data sheet reported a water solubility of the test material to be 640 g/L at 25°C. 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, twice 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”). In order to reduce the chance of filter clogging and potential elevated concentrations from breaching of the filter membrane, approximately 12 mL was drawn into the syringe of which 4 mL was pushed through the filter to waste and the remaining 8 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.

Details on test solutions:

Stock solutions of 24 g/L Al and 40 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):

Pimephales promelas

Details on test organisms:

The tests were initiated with <24 hour old larval fish obtained from in-house cultures. Original cultures of P. promelas were obtained from Aquatic BioSystems (Fort Collins, CO, USA) and in-house cultures have been successfully maintained at OSU AquaTox for 10+ years.
For certain tests the adult broodstock of P .promelas were cultured at OSU AquaTox (Albany, OR lab) in laboratory blended water (well water blended with reverse osmosis-treated water with a targeted hardness of 100-120 mg/L as CaCO3 and a pH of 7.8 – 8.0). Following a laboratory move (to Corvallis, OR lab), adult broodstock were moved to the new laboratory location and reared for a period of three months in well water with a hardness of 132 mg/L as CaCO3 and pH of 6.6 – 6.8. Larval fish from this adult broodstock were used for tests.
Fertilized eggs were hatched out in moderately hard reconstituted lab water with a hardness, alkalinity, and pH of 100 mg/L as CaCO3, 70 mg/L as CaCO3, and 8.0, respectively. For the higher hardness tests (250 and 400 mg/L as CaCO3 tests), embryos were acclimated from the moderately hard water to the respective hardness of the test water upon hatching (period of approximately 4 days).

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Hardness:

Test #1:
128 mg/L as CaCO3 (titration)
134 mg/L as CaCO3 (Ca/Mg calculated)

Test #2:
120 mg/L as CaCO3 (titration)
131 mg/L as CaCO3 (Ca/Mg calculated)

Test #3:
404 mg/L as CaCO3 (titration)
442 mg/L as CaCO3 (Ca/Mg calculated)

Test #4:
128 mg/L as CaCO3 (titration)
135 mg/L as CaCO3 (Ca/Mg calculated)

Test #5:
124 mg/L as CaCO3 (titration)
125 mg/L as CaCO3 (Ca/Mg calculated)

Test #6:
268 mg/L as CaCO3 (titration)
288 mg/L as CaCO3 (Ca/Mg calculated)

Test #7:
424 mg/L as CaCO3 (titration)
396 mg/L as CaCO3 (Ca/Mg calculated)

Test temperature:

25 ± 2 °C (temperature-controlled environmental chamber)

pH:

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 (see additional information) .

Test #1 (average):
new medium: 6.03 (6.00-6.10)
old medium: 6.05 (6.01-6.10)

Test #2 (average):
new medium: 6.04 (5.96-6.09)
old medium: 6.05 (5.99-6.10)

Test #3 (average):
new medium: 6.75 (6.69-6.81)
old medium: 6.92 (6.84-7.13)

Test #4 (average):
new medium: 7.01 (6.88-7.12)
old medium: 6.99 (6.86-7.12)

Test #5 (average):
new medium: 6.97 (6.83-7.08)
old medium: 6.95 (6.85-7.07)

Test #6 (average):
new medium: 8.00 (7.87-8.06)
old medium: 8.14 (8.07-8.20)

Test #7 (average):
new medium: 8.11 (7.95-8.27)
old medium: 8.14 (8.03-8.33)

Dissolved oxygen:

Test #1 (average):
new medium: 8.4 mg/L (7.8-8.7 mg/L)
old medium: 7.7 mg/L (7.0-8.2 mg/L)

Test #2 (average):
new medium: 8.5 mg/L (8.2-8.7 mg/L)
old medium: 7.6 mg/L (6.9-8.0 mg/L)

Test #3 (average):
new medium: 8.4 mg/L (8.1-8.9 mg/L)
old medium: 7.2 mg/L (6.5-7.7 mg/L)

Test #4 (average):
new medium: 8.6 mg/L (8.3-8.9 mg/L)
old medium: 7.6 mg/L (5.0-8.3 mg/L)

Test #5 (average):
new medium: 8.5 mg/L (8.0-8.8 mg/L)
old medium: 7.6 mg/L (7.1-8.1 mg/L)

Test #6 (average):
new medium: 8.5 mg/L (8.4-8.7 mg/L)
old medium: 7.7 mg/L (7.0-8.1 mg/L)

Test #7 (average):
new medium: 8.6 mg/L (8.5-8.9 mg/L)
old medium: 6.9 mg/L (5.3-8.2 mg/L)

Conductivity:

Test #1 (average):
new medium: 483 µS/cm (422-579 µS/cm)
old medium: 501 µS/cm (444-614 µS/cm)

Test #2 (average):
new medium: 511 µS/cm (428-650 µS/cm)
old medium: 526 µS/cm (436-677 µS/cm)

Test #3 (average):
new medium: 1150 µS/cm (1087-1314 µS/cm)
old medium: 1145 µS/cm (1069-1290 µS/cm)

Test #4 (average):
new medium: 548 µS/cm (423-777 µS/cm)
old medium: 567 µS/cm (444-809 µS/cm)

Test #5 (average):
new medium: 597 µS/cm (434-1006 µS/cm)
old medium: 616 µS/cm (421-1024 µS/cm)

Test #6 (average):
new medium: 1176 µS/cm (1017-1541 µS/cm)
old medium: 1199 µS/cm (1025-1572 µS/cm)

Test #7 (average):
new medium: 1514 µS/cm (1370-1768 µS/cm)
old medium: 1151 µS/cm (1391-1778 µS/cm)

Nominal and measured concentrations:

Concentrations at start of test

Test #1: 0, 750, 1500, 3000, 6000, 12000 µg Al/L (nominal)
Test #1: 1.6, 886.0, 1744.3, 3350.0, 6964.7, 13736.7 µg Al/L (total measured)
Test #1: 0.2, 544.5, 661.0, 645.5(*), 449.0(*), 0.2 µg Al/L (dissolved measured)

Test #2: 0, 1050, 2100, 4200, 8400, 16800 µg Al/L (nominal)
Test #2: 6.3, 1176.0, 2295.0, 4872.0, 9102.7, 17427.0 µg Al/L (total measured)
Test #2: 0.2, 419.0, 452.5, 226.5, 4.5, 0.2 µg Al/L (dissolved measured)

Test #3: 0, 1100 2200, 4400, 8800, 17600 µg Al/L (nominal)
Test #3: 1.6, 1142.7, 2252.3, 4927.0, 9493.7, 19586.7 µg Al/L (total measured)
Test #3: 0.2, 125.5,97.0, 25.5, 780.0(*), 4507.5(*) µg Al/L (dissolved measured)

Test #4: 0, 1875, 3750, 7500, 15000, 30000 µg Al/L (nominal)
Test #4: 14.7, 2123.0, 4117.3, 8500.0, 16963.3, 32876.0 µg Al/L (total measured)
Test #4: 15.3, 1245.0, 1480.0, 205.5, 71.5, 67.0 µg Al/L (dissolved measured)

Test #5: 0, 2625, 5250, 10500, 21000, 42000 µg Al/L (nominal)
Test #5: 8.4, 2711.7, 5772.3, 11593.7, 22189.7, 49955.7 µg Al/L (total measured)
Test #5: 8.8, 1776.5, 2502.0, 3858.5(*), 8857.0(*), 22093.5(*) µg Al/L (dissolved measured)

Test #6: 0, 2500, 5000, 10000, 20000, 40000 µg Al/L (nominal)
Test #6: 3.7, 2775.0, 5470.3, 10606.7, 20393.7, 42384.3 µg Al/L (total measured)
Test #6: 0.5, 1026.5, 865.5, 776.0, 2921.0(*), 14035.0(*) µg Al/L (dissolved measured)

Test #7: 0, 1875, 3750, 7500, 15000, 30000 µg Al/L (nominal)
Test #7: 1.2, 1924.7, 4044.0, 8128.0, 15621.0, 31132.3 µg Al/L (total measured)
Test #7: 1.2, 1183.0, 1160.5, 1042.0, 1060.0, 7099.5 (*) µg Al/L (dissolved measured)

(*): possible elevated measurement due to breach of filter membrane

Details on test conditions:

Each test included five Al exposure concentrations and a dilution water control. Test concentrations were prepared with a 0.5 dilution factor. Exposure concentrations were selected based on historical response data with P. promelas in other reconstituted waters to cover the range from no effects concentrations to concentrations anticipated to illicit biological effects. In addition to considering historical data for selection of exposure concentrations, nominal water quality characteristics were input into the bioavailability models to predict effects. Each test concentration (treatment) was prepared in a batch and then equally distributed to the test chambers. Four replicate chambers were prepared for each treatment and control. Test concentrations were prepared by filling a graduated cylinder or volumetric flask to 80% capacity with dilution water, adding the appropriate volume of buffer, adding the appropriate volume of stock solution (24 g/L or 40 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 or water renewal.
Organisms were tested in 250 mL glass beakers. Each test chamber contained 200 mL of test solution. The test chambers were housed in a temperature-controlled environmental chamber that maintained test temperature at 25 ± 2 °C. The test was conducted under a 16 hr light: 8 hr dark light cycle under cool white fluorescent light.
To initiate the tests, ten larval fathead minnows (< 24 hours old) were distributed using a 10-mL glass pipette into each test chamber that had been randomly assigned to a location within the environmental chamber. Organisms were fed 0.15 mL per chamber of a concentrated suspension of brine shrimp (Artemia salina) nauplii (Brine Shrimp Direct, Ogden, UT, USA) twice a day. The amount of food added to the test chambers was documented and all chambers containing live fish were fed an equal amount. On a daily basis, an 80% renewal of control and treatment solutions occurred by siphoning out 80% of the old water, removing uneaten food and feces, and pouring freshly prepared water into the chamber to achieve the desired volume.

Observations of live and dead fish were conducted on a daily basis and dead fish were removed from test containers immediately. At test termination, the fish in each test chamber were counted, examined for grossly-observable abnormalities, and sacrificed via immersion in an overdose of MS-222. The fish from each replicate were then placed into a pre-weighed pan and dried at 105°C for over 12 hours and then re- weighed to the nearest 0.01 mg to obtain a dry weight.

Reference substance (positive control):

yes

Remarks:

sodium chloride

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

3 611 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

weight

Remarks:

mean dry biomass

Remarks on result:

other: Test #1; 95%CL 2345-5560 µg/L)

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

8 375 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

weight

Remarks:

mean dry biomass

Remarks on result:

other: Test #2; 95%CL 6074-11549 µg/L)

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

1 648 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

weight

Remarks:

mean dry biomass

Remarks on result:

other: Test #3; 95%CL 947-2868 µg/L)

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

6 164 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

weight

Remarks:

mean dry biomass

Remarks on result:

other: Test #4; 95%CL 4199-9049 µg/L)

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

11 064 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

weight

Remarks:

mean dry biomass

Remarks on result:

other: Test #5; 95%CL 5057-24206 µg/L)

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

3 031 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

weight

Remarks:

mean dry biomass

Remarks on result:

other: Test #6; 95%CL 642-14310 µg/L)

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

7 363 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

weight

Remarks:

mean dry biomass

Remarks on result:

other: Test #7; 95%CL 2646-20489 µg/L)

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

3 350 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

weight

Remarks:

mean dry biomass

Remarks on result:

other: Test #1

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

9 102.7 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

weight

Remarks:

mean dry biomass

Remarks on result:

other: Test #2

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

2 252.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

weight

Remarks:

mean dry biomass

Remarks on result:

other: Test #3

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

4 417.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

weight

Remarks:

mean dry biomass

Remarks on result:

other: Test #4

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

5 772.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

weight

Remarks:

mean dry biomass

Remarks on result:

other: Test #5

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

2 775 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

weight

Remarks:

mean dry biomass

Remarks on result:

other: Test #6

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

8 128 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

weight

Remarks:

mean dry biomass

Remarks on result:

other: Test #7

Duration:

7 d

Dose descriptor:

LC10

Effect conc.:

10 148 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

mortality

Remarks on result:

other: Test #4; 95%CL 7897-13041 µg/L

Duration:

7 d

Dose descriptor:

LC10

Effect conc.:

11 670 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

mortality

Remarks on result:

other: Test #5; 95%CL 8015-16993 µg/L

Details on results:

In all tests, control acceptability criteria (minimum of 80% survival and an average dry weight of surviving fish in control chambers of > 0.25 mg; USEPA 2002) were met. Mean dry biomass is reported as the total dry weight of all surviving organisms divided by the original number of organisms.
The majority of tests did not result in significant effects on survival from Al exposures. The exception to this was tests Al 1997 PPC and Al 1204 PPC, which resulted in an effect at the highest and second highest exposures, respectively. With this survival data, Al 1197 PPC and Al 1204 PPC were the two tests 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, survival is not typically affected, rather, growth has been consistently found to be the most sensitive endpoint in P. promelas tests with Al. Nonetheless, in all tests (including Al 1197 PPC and Al 1204 PPC), growth was the most sensitive endpoint resulting in the lowest ECx).
The report also provides EC20 and EC50-values for the growth endpoint.

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. No differences were observed between offspring from broodstock cultured in the two laboratory waters. Organism health was also assessed prior to use (by visible active movement) and deemed to be in excellent condition.

Reported statistics and error estimates:

Statistical analysis was performed for each test using measured total Al concentrations from “new” waters. Measured total Al concentrations from the “new” waters were used for the statistical analysis, rather than the “old” solutions for a few reasons. 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 test chamber. Differences in survival and growth (as mean dry biomass: final weight of composited organisms per chamber divided by original number of exposed organisms) 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 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 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% and 50% relative to control performance (LC10/LC20/LC50 and EC10/EC20/EC50). Exposure concentrations were log-transformed before determination of the LCx/ECx values. Only two tests (Al 1197 PPC and Al 1204 PPC) had sufficient mortality to calculate LCx values, which were determined by tolerance distribution analysis. ECx statistical analyses of growth were conducted using a weighted regression analysis (mean dry biomass [total dry weight divided by initial number of exposed organisms] weighted by standard deviation). Growth endpoints were estimated using threshold sigmoid regression analysis or piecewise linear regression analysis.

Dilution water was characterized prior to the preparation of Al test solutions (without buffer, pH adjustment, or food). Water hardness was measured by titration (APHA 2012) at the initiation of testing and was also calculated based upon measured Ca and Mg concentrations. Because the BLM utilizes Ca and Mg (in mg/L) as input parameters and the MLR uses hardness (as mg/L CaCO3), the calculated hardness values were used in the MLR analysis to maintain consistency between model input values. In all cases, the titration measurements were close to the calculated values. Measured DOC concentrations ranged from 1.10–1.58 mg/L for the nominally added 1 mg/L, 7.00 and 7.20 mg/L for the nominally added 8 mg/L, and 11.45 and 11.58 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 and variability among the batches may have contributed to the observed DOC variability. 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, in two cases, slightly adjusting the CO2 atmosphere limited observed pH drift. For modeling purposes, an average pH from all measurements was used. The observed range of conductivity values was wide, with values increasing as the Al exposures increased. This may have been artifact from the need for increased pH adjustments in the higher exposures, which required addition of HCl and/or NaOH to achieve target pH values. All tests met test acceptability criteria for temperature (25 ± 2°C) and dissolved oxygen(saturation ≥ 60%).

Validity criteria fulfilled:

yes

Conclusions:

The reported studies 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 P. promelas toxicity tests reflecting a range of water chemistry: pH (6.0 – 8.0), hardness (10 – 127), and DOC (0.08 – 5.0 mg/L). The data reported herein permit an expansion of the dataset up to a pH of 8.12, hardness up to 422 mg/L and DOC up to 11.58 mg/L. This current data will allow for a comparison of current model predicted effect concentrations with observed effect concentrations, for water types outside the range of model development. 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. Almost 10 years in duration, a major portion of this research program was recently completed and the results published in peer-reviewed journals. Included in this effort were a series of chronic toxicity tests with three different organisms, the fathead minnow (Pimephales promelas), a cladoceran (Ceriodaphnia dubia), and a 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 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 provided in publications by Santore et al (2018) and DeForest et al (2018), respectively. The dataset for the two models included P.promelas toxicity tests with the following range of values: pH (6.0–8.0), hardness (10–127 mg/L as CaCO3) and dissolved organic carbon (DOC) (0.1–4.0 mg/L). Additional field data for low pH waters have been used for the BLM. 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 allow for an expansion of theP.promelasdataset up to a pH of 8.12, hardness up to 422 mg/L, and DOC up to 11.58 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 20

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

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:

according to guideline

Guideline:

other: US EPA, 2002.

Deviations:

yes

Remarks:

modifications for specific water types, pH control, and testing with Al

Principles of method if other than guideline:

This research was conducted according to the protocol, “Short-term chronic toxicity of aluminum to the fathead minnow (Pimephales promelas) - expansion of toxicity testing under variable water quality conditions” OSU AquaTox Protocol No. Al-PP-CSR7d-035. Appendix A (Protocol) of the Study Report contains a copy of the protocol. The studies were conducted according to standard USEPA methodology (USEPA 2002), with modifications for specific water types and pH control.

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, reagent grade aluminum nitrate nonahydrate (Al(NO3)3 · 9H2O; CAS number 7784- 27-2, Lot #K34588) was received from J.T. Baker (Fair Lawn, NJ, USA). The test substance had a reported assay purity of 98.7%. The certificate of analysis is provided in Appendix B. The manufacturer’s material safety data sheet reported the water solubility of the test material to be 673 g/L at 20°C. 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 Aluminum: Analytical samples from each treatment were collected for total Al analysis from “new” waters on Days 0, 3, and 6, and from “old” test waters on Day 7 (test termination). After the 3-hour equilibration period, “new” waters were taken directly from the 1-Liter beakers (thoroughly stirred and homogenized) using a 20 mL syringe. Approximately 5 mL 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 of “old” waters were taken from a composite of each replicate from each concentration. “Old” water was removed directly from the water column within each beaker. Although an effort was made to remove a representative sample of all of the solution, due to the test organisms within the chamber and the test concentrations at levels greater than solubility, a completely homogenized sample was unlikely to be obtained from the test chamber. 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 Aluminum: Analytical samples from each treatment were collected for dissolved (filtered through a 0.20 μm PVDF filter, Ann Arbor, MI, USA #4406) Al analysis at the same collection times as total sampling. Sampling for “new” and “old” waters occurred by drawing approximately 20 mL into the syringe of which 5 mL was pushed through the filter to disposal 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.

Details on test solutions:

A stock solution of 5 g/L Al was prepared by addition of 34.758 grams of Al(NO3)3 · 9H2O to 500 mL of deionized water (18 Ω) in a volumetric flask. The stock solution had a pH of < 3.8. The stock solution was then stored in plastic containers in the dark at 1 - 6°C. All concentrations are expressed as micrograms Al per liter (μg/L Al).
The synthetic buffer, MES (2-(n-morpholino) ethanesulfonic acid, monohydrate) was used to control pH in the tests. Stock solutions of 200 mM MES were prepared by addition of the buffer to deionized water, followed by pH adjustment to 5.9 – 6.0 through addition of dilute HCl. The stocks were then stored in plastic containers in the dark at 1 - 6°C. Aliquots of the buffer were then warmed to test chamber on a daily basis prior to use.

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 hardness values of 50 or 100 mg/L as CaCO3. The Ca:Mg (molar) ratio was 1.82 (3:1 by weight). Nominal alkalinity of the dilution water was 7 mg/L as CaCO3 in both tests. This alkalinity was set to maintain consistency between the current tests and previously conducted tests (OSU 2012, OSU 2014). Reconstituted water was prepared as detailed in standard USEPA methods (USEPA 2002). The dilution water was aerated for the duration of the study, prior to each day’s use. DOC was added as Suwannee River Natural Organic Matter (NOM; obtained from International Humic Substances Society) 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. 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.

Test organisms (species):

Pimephales promelas

Details on test organisms:

All tests were initiated with larval (< 24 hours old) P. promelas from the OSU AquaTox in-house cultures. P. promelas are cultured in-house in laboratory blended water (well water blended with reverse osmosis-treated water with a targeted hardness of 100-120 mg/L as CaCO3). Fertilized eggs were hatched out in moderately hard reconstituted lab water with a hardness, alkalinity, and pH of 100 mg/L as CaCO3, 70 mg/L as CaCO3, and 8.0, respectively.
In order to assess organism health prior to the conduct of each test, monthly reference toxicant tests were performed using sodium chloride (NaCl) 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 tests. Organism health was also assessed prior to use (by visible recordings of overall health) and deemed to be in excellent condition.
As newly hatched larval fish (<24 hrs old) were used for test initiation, organisms were not specifically acclimated to each dilution water.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Hardness:

Test #1:
52 mg/L as CaCO3 (titration)
49 mg/L as CaCO3 (Ca/Mg calculated)

Test #2:
104 mg/L as CaCO3 (titration)
94 mg/L as CaCO3 (Ca/Mg calculated)

Test temperature:

25 ± 2 °C (temperature-controlled environmental chamber)

pH:

Test #1:
new medium: 6.0-6.2
old medium: 6.0-6.2

Test #2:
new medium: 6.0-6.1
old medium: 6.0-6.2

Dissolved oxygen:

Test #1:
new medium: 8.3-8.5 mg/L
old medium: 6.2-8.1 mg/L

Test #2:
new medium: 8.1-8.7 mg/L
old medium: 6.5-7.7 mg/L

Conductivity:

Test #1:
new medium: 297-400 µS/cm
old medium: 303-410 µS/cm

Test #2:
new medium: 370-452 µS/cm
old medium: 398-603 µS/cm

Nominal and measured concentrations:

Concentrations at start of test

Test #1: 0, 450, 900, 1800, 3600, 7200 µg Al/L (nominal)
Test #1: 5.5, 422, 883, 1943, 3238, 7553 µg Al/L (total measured)
Test #1: 5.5, 69, 44, 163, 700, 1629 µg Al/L (dissolved measured)

Test #2: 0, 250, 500, 1000, 2000, 4000 µg Al/L (nominal)
Test #2: 5.5, 234, 492, 999, 1978, 4126 µg Al/L (total measured)
Test #2: 5.5, 86, 117, 109, 186, 478 µg Al/L (dissolved measured)

Details on test conditions:

Test concentrations were prepared by adding the appropriate volume of stock solution (5 g/L Al) to the dilution (reconstituted) water. Volumetric flasks of 1000 mL were filled to approximately 80% of their volume capacity with the respective dilution (reconstituted) water. At this stage, the 200 mM buffer (adjusted to testing pH 6) was added to obtain a final concentration of 5 mM buffer in the final solution. Next, the Al stock solution was added with a micro-pipette to achieve the nominal concentration. Finally, the volume was then completed to 1000 mL with the respective dilution water. The flask was mixed, the pH was checked and if needed, adjusted to the appropriate pH using dilute HCl or NaOH. Each individual concentration was allowed to sit for 3 hours equilibration time in an environmental chamber at test temperature.

Each test included five test treatments, a dilution water control, and a concurrent moderately hard reconstituted water control (representative of organism culture media). Each test concentration (treatment) was prepared individually and then distributed to the replicate test chambers. Four replicate chambers were prepared for each treatment and control. Following the 3-hour equilibration period, tests were conducted under ambient conditions. The 3-hour equilibration period was determined to be adequate to stabilize toxicity results. In studies with daphnids (CIMM 2009), shorter time periods were determined to result in increased toxicity due to the presence of low molecular weight aluminum species.
Test chambers for this study were 250 mL glass beakers. The volume of water in each chamber was 200 mL. The test chambers were housed in a temperature-controlled environmental chamber and maintained at the test temperature at 25°C. The tests were conducted under a 16:8 hour light:dark cycle using cool-white fluorescent lights at ~100 foot candles.

At test initiation, ten larval fathead minnows (< 24 hours old) were distributed using a 10-mL glass pipette into each test chamber that had been randomly assigned to a location within the environmental chamber. Organisms were fed 0.15 mL per chamber of a concentrated suspension of brine shrimp (Artemia salina) nauplii (Brine Shrimp Direct, Ogden, UT, USA) twice a day. The amount of food added to the test chambers was documented and all chambers containing live fish were fed an equal amount.
On a daily basis, an 80% renewal of control and treatment solutions occurred by siphoning out 80% of the old water, removing uneaten food and feces, and pouring freshly prepared water into the chamber to achieve the desired volume.

Reference substance (positive control):

yes

Remarks:

sodium chloride

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

560 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

weight

Remarks:

mean dry biomass

Remarks on result:

other: Test #1; 95%CL 322-974 µg/L)

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

1 424 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

weight

Remarks:

mean dry biomass

Remarks on result:

other: Test #2; 95%CL 1028-1974 µg/L)

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

2 195 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

weight

Remarks:

mean dry biomass

Remarks on result:

other: Test #1; 95%CL 1659-2904 µg/L)

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

2 948 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

weight

Remarks:

mean dry biomass

Remarks on result:

other: Test #2; 95%CL 2485-3496 µg/L)

Duration:

7 d

Dose descriptor:

other: EC20

Effect conc.:

885 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

weight

Remarks:

mean dry biomass

Remarks on result:

other: Test #1; 95%CL 574-1365 µg/L)

Duration:

7 d

Dose descriptor:

other: EC20

Effect conc.:

1 817 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

weight

Remarks:

mean dry biomass

Remarks on result:

other: Test #2; 95%CL 1444-2287 µg/L)

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

422 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

weight

Remarks:

mean dry biomass

Remarks on result:

other: Test #1

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

999 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

weight

Remarks:

mean dry biomass

Remarks on result:

other: Test #2

Duration:

7 d

Dose descriptor:

LC50

Effect conc.:

> 7 553 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

mortality

Remarks on result:

other: Test #1

Duration:

7 d

Dose descriptor:

LC50

Effect conc.:

> 4 126 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

mortality

Remarks on result:

other: Test #2

Details on results:

The control acceptability criteria (minimum of 80% survival and an average dry weight of surviving fish in control chambers > 0.25 mg; USEPA 2002) were achieved in both tests (Test Al 983 PPC = 95% survival and average dry weight of 0.432 mg and Test Al 978 PPC = 98% survival and average dry weight of 0.402 mg). In addition to the dilution water control, a concurrent control consisting of moderately hard reconstituted water (without buffer or pH control) was used for comparison. There was no difference in survival or growth in the control/dilution waters and the concurrent unmanipulated control, demonstrating that the buffer and pH control did not negatively impact the fish.
No significant effects in survival between the controls and Al exposures were observed in either test. Growth was affected in the four highest exposure concentrations in Test Al 983 PPC resulting in a NOEC of 422 μg/L total Al and affected in the two highest exposure concentrations in the Test Al 978 PPC resulting in a NOEC of 999 μg/L total Al. In the highest exposure concentrations, clinical observations noted during the tests included changes in relative size of the developing fish (in comparison to control fish) and lethargy in the fish.

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 (NaCl) 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 tests. Organism health was also assessed prior to use (by visible recordings of overall health) and deemed to be in excellent condition.

Reported statistics and error estimates:

Statistical analysis was performed using measured average total Al concentrations of “new” samples. Unlike dissolved Al concentrations which remained low and non-increasing across exposures, total Al concentrations have been observed to correlate best with dose responses within toxicity tests with daphnids, algae, and fathead minnows and have been used consistently for reporting toxicity measures in all Oregon State University Al studies. Differences in survival and growth (mean dry biomass: final dry weight divided by original number of organisms) 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% or 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 ECx values.

Parameter	Test #1 – Al 983 PPC (pH 6, H 50, DOC 1)	Test #2 – Al 978 PPC (pH 6, H 100, DOC 2)
Alkalinity (mg/L as CaCO3)	8	12
Ca (mg/L)	12.6	24.6
Mg (mg/L)	4.15	7.98
Na (mg/L)	3.52	3.51
K (mg/L)	0.949	0.571
Cl (mg/L)	37.8	0.66
SO4^2-(mg/L)	45.3	86.7
DOC (mg/L)	0.80	1.58

Aluminum exposure concentrationsare reported under two categories: “new” and “old”. “New” waterswere sampled directly from the freshly prepared batch of solutions (after 3-hr equilibrium period) at test initiation and each water renewal. Following the equilibration period, solutions were homogenized and metals samples were taken. Any precipitate that may have settled during the equilibration period was captured in the metals sample as it was thoroughly mixed prior to sampling. At test initiation, total Al concentrations were between 95 and 103% of nominal spiked concentrations. At test initiation in the“new” waters, dissolved Al concentrations measured between2 and 36% of total Al concentrations.“Old”waters are classified as solutions in which the organisms have been exposed for a period of approximately24 hours. Samples for “old” metals were poured directly from the top solution layer of the chamber.Apparent losses of Al from the water column (ranging from 17% to 92% in total Al; when comparing“new” to “old” concentrations) most likelyoccurred due to precipitates settling during the exposure period and would not be completelycaptured in the “old” metals sample.Therefore, the measured concentrations of newly prepared solutions are more representative of exposure concentrations, whereas the old solutions show whether any precipitation may have occurred within the test chamber (observed as reduced total Al concentrations). As the formation of insoluble Al species occurred and was observed in the form of visible precipitate in multiple tests and reduced measured dissolved Al, the use of total Al to calculate effect concentrations is appropriate and correlates with toxicity results (Cardwell et al. 2018). Statistical analysis on the biological endpoints was conducted using the total Al concentrations from new solutions. Background concentrations of Al in the control water were below analytical detection limits (< 11 μg/L Al). These control water samples were assigned an estimated Al concentration of half their detection limit (5.5 μg/L Al) 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 (< 11 μg/L Al). 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 92.4-106.2%. The primary standard quality control sample recovery ranged from 99.9- 103.4%. The measured Al concentrations of all quality control samples were within acceptability criteria of 85-115%.

Validity criteria fulfilled:

yes

Conclusions:

Two “confirmatory” tests were conducted using the fathead minnow, a species which was substantially used in the development of the models. This report describes the results of a study to determine the short-term chronic effects of aluminum (tested as aluminum nitrate nonahydrate), on the fathead minnow, Pimephales promelas, in two laboratory waters with varying hardness and DOC concentrations. The tests were conducted according to standard USEPA testing methodology (USEPA 2002) and endpoints for each test were survival and growth. These tests are intended to provide empirical data necessary for the confirmation of Al bioavailability model predictions.

The studies reported here represent a validation exercise of the Al bioavailability models (BLM and MLR). The empirical data permit a comparison with model predictions using an organism that was used in model development: P. promelas. These fathead minnow tests were conducted alongside additional tests with the rotifer, Brachionus calyciflorus (OSU 2018a), the snail, Lymnaea stagnalis (OSU 2018b), and the amphipod, Hyalella azteca (OSU 2018c), three organisms that were not used in the development of the bioavailability models, but were part of the chronic database. For the P. promelas tests, the measured parameters for the models are reported in Table 3-7. Observed effect concentrations (EC10s and EC20s with 95% confidence intervals) were 560 (322 - 974) and 885 (574 - 1365) μg/L total Al, respectively, in the pH 6.10, hardness 49 mg/L as CaCO3, DOC 0.80 mg/L water and were 1424 (1028 - 1974) and 1817 (1444 - 2287) μg/L total Al, respectively, in the pH 6.00, hardness 94 mg/L as CaCO3, DOC 1.58 mg/L water. These results confirm that both hardness and DOC exhibited a protective effect on Al toxicity to P. promelas. For the EC10 endpoint, both the BLM and MLR predicted effect concentrations within a factor of two. For the EC20 endpoint, the MLR also predicted effect concentrations within a factor of two. The BLM was only able to output EC20 predicted values for the longer term chronic fathead minnow early life-stage (ELS) test [output not available for 7-day EC20 predictions]. This is shown (Figure 3-1b) as a lower than observed prediction, with one prediction within a factor of two and the second prediction slightly below a factor of two. The 7-day short-term chronic tests have been shown to be an accurate predictor of the longer term full chronic duration tests and the BLM predicted effect concentrations were quite close to those observed. Overall, both models do an acceptable job at predicting toxicity to P. promelas.

Executive summary:

Toxicity models have been developed to account for the bioavailability and toxicity of Aluminum (Al) to aquatic organisms based upon water quality characteristics of freshwater (e.g., hardness, pH, dissolved organic carbon [DOC]). The two models developed are a biotic ligand model (BLM) and a multiple linear regression (MLR) model. These models were developed using empirical data from toxicity testing of three aquatic organisms: the fathead minnow, Pimephales promelas, the cladoceran, Ceriodaphnia dubia, and the green algae, Pseudokirchneriella subcapitata, with exposure to Al concentrationsin a range of water types (e.g., varying hardness, pH, DOC). A two-phase validation of the Al bioavailability models was conducted. In the studies reported here, tests were performed with P. promelas to use as a check on the model with an organism used in model development. A series of chronic toxicity tests were also conducted using the rotifer Brachionus calyciflorus, the amphipod Hyalella azteca, and the snail Lymnaea stagnalis (reported separately) to further extend the application of the Al models using test organisms not used in model development. The studies reported herein consist of short-term chronic toxicity tests with exposure of P. promelas to varying Al concentrations in two different laboratory waters. Survival and growth following a 7-day exposure period were assessed. The water quality characteristics were, nominally, pH 6, hardness 50 mg/L as CaCO3, DOC 0.5 mg/L and pH 6, hardness 100 mg/L as CaCO3, DOC 2.0 mg/L. The results demonstrated a protective effect at the higher hardness and higher DOC concentrations. The observed effect concentrations from the P. promelas tests were compared to model (both BLM and MLR) predicted effect concentrations. The Al MLR predicted EC10 and EC20 concentrations in both test waters within a factor of two. The Al BLM predicted EC10 effect concentrations in both test waters within a factor of two, while the Al BLM EC20 predictions were reported for the longer term early life-stage (ELS) test and had lower prediction values. Although the 7- day short-term exposures are a good predictor of the longer term full chronic durations (ELS), the slightly lower toxicity values (smaller EC20 predictions) were still close to a factor of two of the observed values. The results in this study demonstrate that both bioavailability models adequately predict toxicity of Al to the fathead minnow.

Reference 21

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

06 January 2009- 13 January 2009

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:

equivalent or similar to guideline

Guideline:

other: EPA 2002. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms. Fourth Edition. Office of Water, USEPA, Washington, DC. EPA-821-R-02-013.

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Molecular formula: AlCl3 x 6H2O
- Analytical purity: 99.1%
- Lot/batch No.: 46192708
- Source: EMD Chemicals, Gibbstown, NJ, USA
- Storage condition of test material: stored at room temperature after receipt at the laboratory

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentrations
- Sampling method: Not reported
- Sample storage conditions before analysis: preserved with nitric acid (trace metal grade) to pH < 2 prior to analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: each test concentration was prepared from a stock solution added to MOPS buffered laboratory reconstituted water. The test concentrations were adjusted to pH of 8.0 using 10M NaOH and/or 1M HCl, mixed thoroughly and allowed to sit undisturbed for 3 hours at test temperature. After the equilibration period, each test treatment was divided in half to used as the unfiltered and filtered test solutions.
- Controls: dilution water control
- Evidence of undissolved material: A visual precipitate was observed in test chambers for all of the concentrations spiked with Al stock
- All other template details: Not applicable

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: fathead minnow
- Strain: Not reported
- Source: laboratory in-house culture
- Age at study initiation: less than 24 hours
- Weight at study initiation (mean and range, SD): Not reported
- Method of breeding: Not applicable

FEEDING DURING TEST
- Food type: brine shrimp (Artemia salina) nauplii (Brine Shrimp Direct, Ogden, UT, USA)
- Amount: 0.15 mL per chamber of a concentrated suspension
- Frequency: twice per day

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Post exposure observation period:

Not reported

Hardness:

unfiltered Al control water (mg/L as CaCO3): 48
filtered Al control water (mg/L as CaCO3): 52

Test temperature:

25 degrees C

pH:

7.9-8.2

Dissolved oxygen:

above 60% saturation levels, ranged from 6.2-9.1 mg/L

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal (μg/L): 0, 15000, 30000, 60000, 120000, 240000
Average Measured Total Al (μg/L): <5, 14435, 23297, 56477, 91422, 183802
Average Measured Dissolved Al (μg/L): 6, 1026, 1052, 912, 864, 867
See Table 3-4 for more details

Details on test conditions:

TEST SYSTEM
- Test vessel: 1-liter plastic beakers (Polypropylene, Fisher Brand, Pittsburg, PA, USA)
- Material, size, headspace, fill volume: polypropylene filled with approximately 250 ml of solution
- Aeration: not reported
- Renewal rate of test solution: daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS (measured for unfiltered, filtered controls waters)
- Source/preparation of dilution water: The dilution and control waters in the testing consisted of a soft standard synthetic freshwater using reagent grade salts (CaSO4, MgSO4, KCl, and NaHCO3; USEPA 2002).
- Dissolved organic carbon: 319, 326 mg/L
- Chlorine: <0.05,<0.05 mg/L
- Alkalinity: 40 mg/L nominal
- Ca/mg ratio: 12.4/4.24, 11.9/4.08
- Conductivity: 419, 432 μS/cm
- Intervals of water quality measurement: test initiation, daily
- See table 3-1 for all measured parameters

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to a pH of 8.0 using 10M NaOH and/or 1M HCl, mixed and equilibrated for 3 hours
- Photoperiod: 16:8 light:dark cycle
- Light intensity: cool-white fluorescent lighting

EFFECT PARAMETERS MEASURED: survival monitored daily, weighted at termination of test

VEHICLE CONTROL PERFORMED: Not applicable

RANGE-FINDING STUDY
- Test concentrations: Not reported
- Results used to determine the conditions for the definitive study: effects on survival at the highest concentration of 50 mg/L as unfiltered nominal Al

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

LC50

Effect conc.:

145 190.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Total aluminum

Basis for effect:

mortality

Remarks on result:

other: unfiltered test, 95% CI = 123552.8 - 170616.8)

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

56 476.6 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Total aluminum

Basis for effect:

mortality

Remarks on result:

other: unfiltered test

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

91 421.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Total aluminum

Basis for effect:

mortality

Remarks on result:

other: unfiltered test

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

>= 831 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Total aluminum

Basis for effect:

mortality

Remarks on result:

other: filtered test

Duration:

7 d

Dose descriptor:

other: IC25

Effect conc.:

11 592.2 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Total aluminum

Basis for effect:

weight

Remarks:

: mean dry weight

Remarks on result:

other: unfiltered test, 95% CI = 8384.1 - 109676.8

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

<= 14 434.5 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Total aluminum

Basis for effect:

weight

Remarks:

: mean dry weight

Remarks on result:

other: unfiltered test, significant decrease at lowest test concentration compared to control

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

>= 831.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Total aluminum

Basis for effect:

weight

Remarks:

: mean dry weight

Remarks on result:

other: filtered test

Duration:

7 d

Dose descriptor:

other: IC25

Effect conc.:

10 372.2 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

other: mean dry biomass (weight / number original fish)

Remarks on result:

other: unfiltered test, 95% CI = 7286.8 - 18050.0

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

<= 14 434.5 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Total aluminum

Basis for effect:

other: : mean dry biomass

Remarks on result:

other: unfiltered test, significant decrease at lowest concentration tested

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

751.7 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Total aluminum

Basis for effect:

other: mean dry biomass

Remarks on result:

other: filtered test

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

831.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Total aluminum

Basis for effect:

other: mean dry biomass

Remarks on result:

other: filtered test

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

4.604 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Remarks:

Total aluminum

Basis for effect:

weight

Remarks on result:

other: 95% CL:0.218-97.365

Details on results:

- There were no significant effects at the highest concentrations tested in the filtered toxicity tests for all endpoints measured: survival, mean dry weight, and mean dry biomass
- Fish weights and survival percentages (mean values on day 7): see Table 3-10
- Type of and number with morphological abnormalities: No deformities observed
- Effect concentrations exceeding solubility of substance in test medium: a visual precipitate was observed in test chambers for all of the test concentrations
- All other template details: Not reported

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Differences in survival and growth at test termination were evaluated using a statistical computer package (CETIS, 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 (Dunnett's Multiple Comparison or Steel Many-One Rank) to compare (p≤0.05) organism performance in the experimental treatments with that observed in the control. The LC50 was estimated using a Trimmed Spearman-Karber method (used if > 50% mortality in highest concentration) or Linear Interpolation Method. The effective concentration required to reduce growth by 25% relative to control performance (EC25) was calculated using the Linear Interpolation Method.

Table 3-10. Summary of Biological and Analytical Results

Unfiltered Aluminium Toxicity Test
Nominal (μg/L Al)	Measured Average Total Al (μg/L Al)	% Survival	Mean Dry Weight (mg)	Mean Dry Biomass (mg)
0	< 5.0 ± 0	95 ± 6	0.52 ± 0.06	0.49 ± 0.04
15000	14434.5 ± 871.2	89 ± 12	0.36 ± 0.05 *	0.32 ± 0.06 *
30000	23296.9 ± 3123.3	85 ± 13	0.32 ± 0.04 *	0.27 ± 0.04 *
60000	56476.6 ± 69.2	88 ± 10	0.39 ± 0.05 *	0.34 ± 0.06 *
120000	91421.8 ± 25601.8	77 ± 9 *	0.35 ± 0.07 *	0.27 ± 0.08 *
240000	183802 ± 37488.9	33 ± 10 *	0.14 ± 0.08 *	0.05 ± 0.03 *
Filtered Aluminium Toxicity Test
0	15.8 ± 18.8	100 ± 0	0.57 ± 0.04	0.57 ± 0.04
15000	711.9 ± 60.4	95 ± 10	0.55 ± 0.05	0.52 ± 0.02
30000	831.8 ± 21.4	100 ± 0	0.50 ± 0.04	0.50 ± 0.04 **
60000	751.7 ± 31.5	100 ± 0	0.51 ± 0.03	0.51 ± 0.03
120000	649.5 ± 37.0	98 ± 5	0.55 ± 0.07	0.54 ± 0.06
240000	671.9 ± 153.5	100 ± 0	0.55 ± 0.01	0.55 ± 0.01

* Significantly less than the control (p=0.05) using Dunnett’s Test.

** This treatment had the highest concentrations of both total and dissolved Al.

Conclusions:

In the unfiltered test, significant effects on survival were observed at the two highest concentrations, while no significant effect on survival was observed in the filtered test. Additionally in the unfiltered test, significant effects on organism growth (based upon mean dry weight and mean dry biomass [a combined survival and growth endpoint]) were observed at all of the Al concentrations. In the filtered test, a significant difference in mean dry biomass was observed at the nominal concentration of 30000 μg/L Al (which had the highest measured total and dissolved concentrations). Based upon the results of the total, dissolved and monomeric Al analysis, it appears that toxicity in the unfiltered test was due to high amounts of total or particulate Al. Dissolved and monomeric Al concentrations in the unfiltered test were similar to the filtered toxicity test which showed no toxicity. Therefore, filtering reduced the chronic toxicity of Al on the survival and growth of the fathead minnow.

Reference 22

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

12/2012-01/2013

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:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Qualifier:

according to guideline

Guideline:

other: ASTM. 2002. Standard guide for conducting early life-stage toxicity tests with fishes. E1241-98. In: 2002 ASTM Annual book of standards, Volume 11.05.ASTM

Principles of method if other than guideline:

/

GLP compliance:

yes

Specific details on test material used for the study:

- Analytical purity: 98.1%
- Lot/batch No.: 113078
- Storage condition of test material: sealed in its original container at room temperature

Analytical monitoring:

yes

Details on sampling:

- Concentrations:37,5; 75; 150; 300; 600µg Al/L
- Sampling method:
TOTAL ALUMINUM
“New/Diluter” waters were taken directly from the proportional diluter. “Old/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
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.
- Sample storage conditions before analysis: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.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A continuous-flow proportional diluter (after Benoit et al. 1982) with a dilution factor of 0.5 was used for the test. The diluter system was constructed of glass, silicone adhesive, and silicone stoppers; a diagram of the diluter system is provided in Figure 2-1. Test solutions were delivered to the test chambers through polyethylene tubing. The dilution system was constructed to deliver a dilution water control and nominal concentrations of 37.5, 75, 150, 300, and 600 μg Al/L to the test chambers.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name:Zebrafish
- Strain: Danio rerio
- Source: Oregon State University Sinnhuber Aquatic Research Laboratory (SARL) in Corvallis, OR.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Subsequent handling of eggs: Fertilized eggs less than 8 hours old were brought to the testing facility and held overnight with two water exchanges during this holding period (a 20% and 50% exchange with the dilution water of the test). Freshly fertilized eggs (fertilized within 36 hours) were used to initiate the study.

POST-HATCH FEEDING
Fish were fed according to the SARL feeding protocol for larval fish. Once fish began to hatch and were swimming in the water column (Day 3), each chamber was fed “red powder”. “Red powder” consisted of a combination of Zeigler AP100 (Zeigler Bros., Aquatic Ecosystems) and freeze dried rotifers (OSI, Aquatic Ecosystems). On Day 10, each chamber was switched to “yellow powder” feed. “Yellow powder” consisted of Zeigler AP100 and Golden Pearl fish food (Golden Pearls, Artemia International). On Day 12, each chamber was fed live brine shrimp (Artemia salina) nauplii. As the test progressed, the amount of brine shrimp fed to all chambers was increased. The volume of brine shrimp added was dependent upon the amount of uneaten brine shrimp remaining in the chambers one hour after feeding. If very little or no brine shrimp was left, the feeding rate was increased 0.05 mL per feeding, accordingly. The amount of food added to the test chambers was documented and all chambers containing live fish were fed an equal amount.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

33 d

Post exposure observation period:

/

Hardness:

83 (64-96) mg/L as CaCO3

Test temperature:

26 (25-26) °C

pH:

6,0 (5,6-6,6) - 6,3 (6,2-6,5)

Dissolved oxygen:

8,2 (7,6-8,5) - 8,3 (7,9-8,6)

Salinity:

/

Nominal and measured concentrations:

Nominal: 0, 37.5, 75, 150, 300, 600 µg/L
Measured concentrations: 2.8; 35.5; 71.5; 139.4; 258.0; 548.3 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquaria
- Material, size, headspace, fill volume: glass, headspace: 200-300 mL, fill volume: 700-800mL
- Type of flow-through: proportional diluter
- Renewal rate of test solution (frequency/flow rate):18 to 27 volume changes per replicate per day
- No. of organisms per vessel:25
- No. of vessels per concentration (replicates):4
- No. of vessels per control (replicates):4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:laboratory blended water (well water blended with reverse osmosis-treated water with a targeted hardness of 80 – 120 mg/L as CaCO3).
- 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, daily, (in both water from the diluter and water from the test chamber), and at test termination. Conductivity was also measured in the new/diluter water at test initiation, daily, and at test termination. Hardness, alkalinity, total residual chlorine, and total ammonia were measured in the dilution water at test initiation and weekly thereafter.
-Hardness: 83 (64-96) mg/L as CaCO3
- Alkalinity: 11 (12-20) mg/L as CaCO3
- pH: 5.9-6.3
- DOC: <0.50 mg/L
- Total residual chlorine: <0.05 mg/l
- Ammonia: <1.0 mg/L
- Calcium: 17.5 mg/l
- Magnesium: 8.1 mg/L
- Sodium: 23.6 mg/l
- Potassium: 0.653 mg/L
- Chloride: 90.5 mg/L
- Sulfate: 0.85 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: 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:ddark
- Light intensity:ca. 50 foot candles

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Observations of live and dead eggs were conducted on a daily basis from initiation to thinning (Day 2), and then to full hatch (between Days 3 and 4 in all concentrations). Dead eggs were removed immediately when observed. Upon hatching, fish were released from the egg cups into the test chambers. In addition, the number of live eggs, hatches or partial hatches, and live fish were counted in each test chamber and recorded on a daily basis. Following hatch, mortalities were recorded daily and dead fish were removed immediately. All organisms that were not found during the test were considered to be decomposed and recorded as dead. At test termination, the fish in each test chamber were counted, examined for grossly-observable abnormalities, and sacrificed via immersion in MS-222. The fish from each replicate were then placed into a pre-weighed pan, dried at 70°C for over 36 hours, and then re-weighed to the nearest 0.01 mg to obtain a dry weight.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:2
- Test concentrations:0, 37.5, 75, 150, 300, 600µg/l

Reference substance (positive control):

no

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

548.3 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

time to hatch

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

258 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

71.5 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

growth rate

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

139.4 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

33 d

Dose descriptor:

LOEC

Effect conc.:

> 548.3 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

time to hatch

Duration:

33 d

Dose descriptor:

LOEC

Effect conc.:

548.3 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

33 d

Dose descriptor:

LOEC

Effect conc.:

139.4 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

growth rate

Duration:

33 d

Dose descriptor:

LOEC

Effect conc.:

258 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

weight

Duration:

33 d

Dose descriptor:

LC50

Effect conc.:

> 548.3 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Duration:

33 d

Dose descriptor:

EC10

Effect conc.:

387.8 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Remarks on result:

other: CL: 259.5-579.5 µg/L

Duration:

33 d

Dose descriptor:

EC10

Effect conc.:

98.2 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

growth rate

Remarks on result:

other: CL: 33.0-292.2 µg/L

Duration:

33 d

Dose descriptor:

EC50

Effect conc.:

860.4 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

mortality

Remarks on result:

other: CL: 506.9-1460.6

Duration:

33 d

Dose descriptor:

EC50

Effect conc.:

1 318.7 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

: total Al

Basis for effect:

growth rate

Remarks on result:

other: CL: 352.6-4932.0 µg/L

Details on results:

- Mortality/survival at embryo and larval stages:
Egg survival by treatment:
At 2.8 µg/L:1.00
At 35.5 µg/L: 1.00
At 71,5 µg/L: 0.99
At 139.4 Mg/L:1.00
At 258.0 µg/L:1.00
At 548.3 µg/L:1.00
- Days to hatch and numbers hatched:
Median day to hatch:
At 2.8 µg/L:3.0
At 35.5 µg/L: 3.3
At 71,5 µg/L:3.3
At 139.4 Mg/L:3.8
At 258.0 µg/L:3.5
At 548.3 µg/L:3.5
- Data for length and weight of surviving fish:
Growth - Mean dry weight per organism:
At 2.8 µg/L:1.74
At 35.5 µg/L: 1.57
At 71,5 µg/L: 1.64
At 139.4 Mg/L: 1.56
At 258.0 µg/L:1.42
At 548.3 µg/L:1.45

Growth - Mean dry weight per original organisms (Biomass)
At 2.8 µg/L: 1.654
At 35.5 µg/L: 1.477
At 71,5 µg/L: 1.553
At 139.4 Mg/L:1.433
At 258.0 µg/L:1.309
At 548.3 µg/L:1.044

Results with reference substance (positive control):

/

Reported statistics and error estimates:

Statistical analysis was performed using measured average total Al concentrations. Differences in egg survival, embryo median day to hatch, fry survival and growth 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 or 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.

Validity criteria fulfilled:

yes

Conclusions:

The study resulted in a no observable effect concentration (NOEC) of 71.5 μg/L total Al and a lowest observable effect concentration (LOEC) of 139.4 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) were 98.2 (33.0 – 292.2) and 234.4 (126.3 – 435.2) 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 water and this speciation is a function of the physico/chemical properties (e.g., pH, hardness, dissolved organic carbon) 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 current study determines the chronic toxicity of aluminum (Al), at a target pH of 6.0, to the zebrafish, Danio rerio. Use of this test organism qualifies as a level of organization (Phylum) that is 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). D. rerio were exposed to a series of aluminum concentrations for thirty-three days, starting as freshly- fertilized embryos. To allow for potential changes in aluminum speciation, exposure solutions were aged for a minimum of a 3-hour equilibrium period. Nominal test concentrations ranged from 0 to 600 μg /L Al and total and dissolved aluminum were measured throughout the test. As the formation of insoluble chemical species was apparent through low dissolved measurements, total Al was used to interpret the biological data in this study. Measured pH values in the test averaged 5.9 – 6.1 in waters immediately prior to organism exposure and averaged 6.0 – 6.3 within the test chambers. The test had measured exposure concentrations ranging from 2.8 to 548.3 μg/L total Al and resulted in a survival effect at the highest concentration and a significant growth effect at the three highest concentrations. Growth (as mean dry biomass) was determined to be the most sensitive endpoint. The study resulted in a no observable effect concentration (NOEC) of 71.5 μg/L total Al and a lowest observable effect concentration (LOEC) of 139.4 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) were 98.2 (33.0 – 292.2) and 234.4 (126.3 – 435.2) μg/L total Al, respectively.

Reference 23

Endpoint:

fish early-life stage toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

Summary of available data used for the endpoint assessment of the target substance

Adequacy of study:

weight of evidence

Justification for type of information:

Refer to analogue justification provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

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Reason / purpose for cross-reference:

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Reason / purpose for cross-reference:

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Reason / purpose for cross-reference:

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Reason / purpose for cross-reference:

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Reason / purpose for cross-reference:

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Reason / purpose for cross-reference:

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Reason / purpose for cross-reference:

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Reason / purpose for cross-reference:

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Reason / purpose for cross-reference:

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Reason / purpose for cross-reference:

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Reason / purpose for cross-reference:

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Reason / purpose for cross-reference:

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Reason / purpose for cross-reference:

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Reason / purpose for cross-reference:

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Reason / purpose for cross-reference:

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Reason / purpose for cross-reference:

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Reason / purpose for cross-reference:

read-across source

Duration:

60 d

Dose descriptor:

EC10

Effect conc.:

>= 560.5 - <= 2 321 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

weight

Remarks on result:

other:

Remarks:

Range is based on normalised data from BLM; lower limit presents Ecoregion #7; upper limit presents Ecoregion #3

Duration:

33 d

Dose descriptor:

EC10

Effect conc.:

>= 581.2 - <= 2 487 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

other: biomass

Remarks on result:

other:

Remarks:

Range is based on normalised data from BLM; lower limit presents Ecoregion #7; upper limit presents Ecoregion #3

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

>= 747.4 - <= 5 395 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

other: biomass

Remarks on result:

other:

Remarks:

Range is based on normalised data from BLM; lower limit presents Ecoregion #7; upper limit presents Ecoregion #5

Description of key information

A substantial amount of long-term studies have been conducted by Oregon State Unsiversity (OSU, 2012,2013,2018) with the fathead minnow P. promelas as test species, and using the soluble Aluminium nitrate as test substance. All of these tests were early life-stage exposures that assessed the impact of Aluminium on reproduction and other sub-lethal effects (e.g. weight, biomass, time to hatch, growth rate). Reported EC10/NOEC values ranged from 25 to 11064 µg Al-total/L, but it is important to note significant differences in physicochemical properties of the test media among the various studies. These differences could explain the observed variation.

Kimball (1978) reported comparable effect levels for P. promelas, with NOEC/EC10 values from 4.7 to 23.1 mg Al/L for weight and hatching percentage (test substance: aluminium sulphate).

Effect levels for S. fontinalis and D. rerio were situated below 1 mg Al/L: growth rate (71.5 µg/L; D. rerio) and length/#hatched ( 57 µg Al/L; S. fontinalis) the most sensitive endpoints.

Additional information

From the published literature there were four long-term chronic toxicity studies to two species of fish (P. promelas and S. fontinalis) that were identified as acceptable. NOECs and EC10s ranged from 0.088 to 2.3 mg Al/L and 0.078 to 5.19 mg Al/L, respectively. 

 

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 used gill accumulation data from S. salar which were applied to the chronic P. promelas data that were generate at Oregon State University (OSU, 2012, 2013, 2018). Application of the model to new data required the development of a critical accumulation value appropriate for the exposure duration and toxicity endpoint.

In addition, calibration of the model to these data benefited from two other changes in parameter values. First, since the chronic endpoints for this species and in these test conditions were at much higher aluminium concentrations and saturation of NOM binding sites included in the model was beginning to occur, resulting in a somewhat reduced predicted effect of NOM compared with the observed effect. The binding site density for NOM was increased by two-fold to provide adequate binding sites at these high Al concentrations. In addition, although the effect of hardness on observed aluminium toxicity was consistent in acute and chronic exposures, the predicted effect of hardness could be improved by a small change in the binding strength of Ca (i.e. the log K for binding at the biotic ligand was increased from 4.2 to 4.8. 

 

After application of the Al BLM, the variability in the response curve between effects of aluminium on the biotic ligand was reduced compared with response curve based on total aluminium. Values for critical accumulation were estimated directly from the predicted response curve on the biotic ligand to establish the CA10, or the critical accumulation level that results in a chronic effect of 10% (in this case a reduction in growth). 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. 

More detailed information on the BLM and its application for setting site-specific water quality parameters can be found in the report “Background document – Environmental Effects Assessment of Aluminium” which is attached to Section 13 of the IUCLID Dossier.