Reaction mass of aluminium hydroxide and aluminium nitrate and aluminium sulphate

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to fish

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented publication which meets basic scientific principles.

Principles of method if other than guideline:

Early life stages of Salomo salar were continuously exposed to Al concentrations ranging from 33 to 264 ug/L at pH 5.5. Tests were conducted with eyed eggs and conducted until 60 d post-hatch. Hatching success, mortality, growth, behavior, and tissue residues of aluminum were determined during the tests.

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

At 14-days intervals Al was measured in exposure water

Vehicle:

no

Test organisms (species):

Salmo salar

Details on test organisms:

TEST ORGANISM
- Common name: eyyed eggs of Atlantic salmon
- Source: Craig Brook National Fish Hatchery, East Orland, Maine, USA


METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Subsequent handling of eggs: after obtaining eggs were held overnight in experimental control water at pH 7.2 and 8°C before initiation of study

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

60

Hardness:

252 - 256 ueq/L

Test temperature:

8°C

pH:

5.6 - 5.8

Nominal and measured concentrations:

< 2.0, 33.0, 71.0, 124.0, and 264.0 ug/L (measured total Al)

Details on test conditions:

- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass aquaria
- Type of flow-through: proportional diluter
- Renewal rate of test solution (frequency/flow rate): 1L/15 min, thus 2.8 volume replacements per day
- Hatching success: No. of fertilized eggs/embryos per vessel: 4 groups of 20 eggs per vessel
- Hatching success: No. of vessels per concentration (replicates): 2
- Hatching success: No. of vessels per control (replicates): 2
- Growth determination: No. of vessels per concentration (replicates): 2
- Growth determination: No. of larvae per vessel: 2 groups of 15 larvae
- Remaining larvae were placed into another compartment within each vessel for behavioral observations and tissue residue determinations


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:reconstituted water with a pH of 7.2, low hardness (200 to 280 ueq/L), low alkalinity (240 ueq/L), and about 3.0 mg/L calcium


OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: 16:8 (light:dark)

Reference substance (positive control):

no

Duration:

60 d

Dose descriptor:

NOEC

Effect conc.:

71 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

Al

Basis for effect:

mortality

Duration:

60 d

Dose descriptor:

LOEC

Effect conc.:

124 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

Al

Basis for effect:

mortality

Duration:

60

Dose descriptor:

EC10

Effect conc.:

124 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

Al

Basis for effect:

length

Remarks on result:

other: EC10 value not stated in original reference. Re-calculated by submitter based on data provided in original reference

Duration:

60 d

Dose descriptor:

EC50

Effect conc.:

240 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

Al

Basis for effect:

mortality

Remarks on result:

other: EC50 value not stated in original reference. Re-calculated by submitter based on data provided in original reference.

Duration:

60 d

Dose descriptor:

EC50

Effect conc.:

765 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

Al

Basis for effect:

length

Remarks on result:

other: EC50 value not stated in original reference. Re-calculated and extrapolated by submitter based on data provided in original reference.

Reported statistics and error estimates:

A randomized block experimental design was used for each study, which included 6 duplicated treatments. Each duplicate treatment contained 2 replicates from which mortality and growth data were obtained. Analysis of variance (ANOVA) was used to analyze the data. Arcsine tgransformation for binomial proportions was performed on percent data (hatchability, survival) to normalize the variance of values near 0 and 100 before ANOVA. Data for swimming activity and prey-strike frequency were square root-transformed before ANOVA. Treatment effects were identified by use of the least squares method of mean comparison. A propability level of <= 0.005 was used to determine statistical significance. All analyses were performed with SAS programs on the mainframe computer system at the University of Missouri, Columbia.

Table1: Cumulative mortality (%) ± standard deviation with n (in parentheses) of Atlantic salmon continuously exposed to acidic pH with aluminum for 60 d.

	Days of post-hatch exposure
Nominal treatments	15	30	45	60
pH / Al(ug/L)
5.5 / 0	0 ± 0 (8)	0 ± 0 (4)	2 ± 4 (4)	4 ± 4 (4)
5.5 / 33	0 ± 0 (8)	2 ± 4 (4)	5 ± 4 (4)	5 ± 4 (4)
5.5 / 71	4 ± 4 (8)	5 ± 4 (4)	9 ± 7 (4)	9 ± 7 (4)
5.5 /124	0 ± 0 (8)	3 ± 6 (4)	10 ± 9 (4)	15 ± 12(a) (4)
5.5 /264	2 ± 4 (8)	15 ± 8(a) (4)	44 ± 16(a) (4)	63 ± 22(a) (4)

(a) significantly different from pH 7.2 control treatment (p<= 0.05).

Table2: Total length (mm) ± standard deviation with n (in parentheses) of Atlantic salmon continuously exposed to acidic pH with aluminum for 60 d.

	Days of post-hatch exposure
Nominal treatments	30	45	60
pH / Al(ug/L)
5.5 / 0	17.9 ± 1.1 (b) (60)	23.0 ± 1.7 (59)	25.1 ± 1.7 (58)
5.5 / 33	18.1 ± 1.6 (58)	21.8 ± 1.7 (a) (57)	24.3 ± 1.8 (57)
5.5 / 71	17.3 ± 0.9 (42)	21.4 ± 1.4 (a) (53)	23.3 ± 1.5 (a) (54)
5.5 /124	17.1 ± 0.8 (58)	21.2 ± 1.2 (a) (54)	22.6 ± 1.3 (a) (50)
4.5 / 264	16.9 ± 1.0 (a) (51)	22.6 ± 1.2 (33)	20.0 ± 0.8 (a) (14)

(a) significantly different from pH 7.2 control treatment (p<= 0.05).

(b) Comparison of pH 7.2 and pH 5.5 controls indicates that only the 30 d values are significantly different (p<= 0.05).

Table3: Swimming and feeding activity of Alantic salmon continuously exposed to acidic pHs with aluminum for 60 d.

	Variables
Nominal treatments	Swimming activity (movements / 2 min)	Feeding activity (prey-strikes / 5 min)
pH / Al(ug/L)
5.5 / 0	37.2 (4.5)	4.8 (1.6)
5.5 / 33	27.2 (4.5) (a)	0.8 (1.6) (a)
5.5 / 71	22.1 (4.5) (ab)	1.6 (1.6) (a)
5.5 /124	6.3 (9.0) (ab)	0 (0) (ab)
4.5 / 264	NS	NS

Values are means with standard error in parentheses. Sample size (n) was 12 for all except the 124 ug Al/L treatment for which n was 3.

(a) significantly different from pH 7.2 control treatment (p<= 0.05).

(b) significantly different from pH 5.5 control treatment (p<= 0.05).

NS, no samples were tested due to poor survival

Reference 2

Endpoint:

fish early-life stage toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented study which meets basic scientific principles.

Qualifier:

according to guideline

Guideline:

other: Cleveland, L., E.E. Little, 8J. Hamilton, D.R. Buckler, and J.B. Hunn. "Interactive toxicity of aluminum and increased acidity to early life stages of brook trout," Trans. Am. Fish. Soc. 115:610-620 (1986).

Qualifier:

according to guideline

Guideline:

other: Ingle, S.E., J.A. Keniston, and D.W. Schultz. "Aqueous chemical equilibrium computer program," Unites States Environmental Protection Agency. EPA-60013-80-049 (May 1980).

Principles of method if other than guideline:

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

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

Water samples were collected at the beginning of the exposures and weekly
thereafter to determine aluminum concentrations. Our analytical approach involved
separating the "fast reacting" or labile aluminum species (inorganic and organic) from
the "slow reacting" polymeric forms. A m.odification of the oxine/methyl isobutyl
ketone (MIBK) extraction procedure of Barnes16 was used to separate the two forms of
aluminum.

- Concentrations: all exposures and controls were measured
- Sampling method: water samples were collected at the beginning of the exposures and weekly thereafter

Vehicle:

no

Details on test solutions:

The following four steps were incorporated into the method: (1)
determination oftotal aluminum in acidified unfiltered samples (weekly determinations
for each treatment); (2) determination of total aluminum in filtered acidified samples
(two determinations per treatment); (3) determination of extr"dctable aluminum in
filtered samples (twodeterminations pertreatment); and (4) determination ofextractable
aluminum in unfiltered samples (3-4 determinations per treatment). The commercial
. in-line apparatus used to filter water samples contained 0.1 um mixed cellulose acet.ate
and nitrate filters. The filters were rinsed with 50 mL of 1% nitric acid and then with
50 mL of reverse osmosis (RO) water before use. Aluminum stock solutions were
prepared in RO water obtained from a commercial RO water purification system and
anionic, cationic, and mixed-bed exchange resins. In addition, the REDEQL-EPAK
chemical equilibrium model of Ingle et al., 1980 was used to estimate aluminum speciation
during the two exposures.
To determine filterability of aluminum at a nominal pH of 5.5, we used filters of
several types and pore sizes to filter 100-ml water samples that contained nominal
aluminum concentrations of 150 Jlg/L.
Atomic absorption standards (1000 ug/L: J.T. Baker Chemical Company,
Phillipsburg, NJ) were used for the preparation of standard solutions of aluminum. All
standards were preserved in 1% nitric acid solutions

Test organisms (species):

Salvelinus fontinalis

Details on test organisms:

TEST ORGANISM
- Common name: brook trout
- 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

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

60 d

Hardness:

about 246 µeq/L; see table 1 in "any other information" below

Test temperature:

12 °C

pH:

about 6.5; see table 1 in "any other information" below

Nominal and measured concentrations:

nominal: 0, 38, 50, 75, 100, 150, 200, 300, 400 ug/L
measured: 5 (background), 4, 8, 29, 57, 68, 88, 142, 169, 292, 350 ug/L (total aluminium)

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

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)
- Intervals of water quality measurement: weekly for Al and monthly for organic carbon

EFFECT PARAMETERS MEASURED: egg mortality, hatch, incomplete hatch recorded daily until hatching was complete. After hatching, mortality of fis

Other:
We placed 50 eyed brook trout eggs into each of four 177-mL glass hatching containers, which were suspended in each
duplicate exposure tank. The tanks were covered to shield the eggs from light, and
aeration provided continuous circulation of the exposure water. Egg mortality, hatch,
and incomplete hatch were recorded dailyuntil hatching was complete. Dead eggs were
removed and discarded and newly hatched larvae were transferred to duplicate
containers within each exposure tank. Eggs yielding larvae that were unable to
complete the hatching process and had the chorion attached were considered to be
incompletely hatched. The median hatching date for each treatment was considered to
be day 0 of the larval exposure. On day 15, two groups of 20 larvae were randomly
selected from the hatching containers, photographed for length determination,14 and
placed in the smaller growth chambers within each duplicate tank. The remaining
larvae were placed in the larger chamber of each tank for behavioral, biochemical, and
aluminum bioconcentration measurements.
Mortality offish in the growth chambers was recorded daily and analyzed for days
15, 30,45 and 60 of the exposures. These fish were photographed again on day 30 for
length determinations. At day 30, each small chamber was thinned to 10 fish each to
avoid the effects of over-crowding on growth. Lengths and weights were detetmined
from direct measurements on days 45 and 60. Behavioral evaluation on 10 fish from each duplicate treatment included measurements of locomotory activity, feeding and
swimming capacity, and buoyancy of individual fish at 30 and 60 days of exposures A
and B. Frequency of movement was the number of times a fish changed position as it
began movementor changed directionduring a 2-min interval. Feedingwas the number
of strikes directed atprey-- Daphnia magna or Artemia salina-- during a 5-min period.
Swimming capacity was measured in a stamina tunnel as fish were subjected to
incremental increases in water velocity until fatigue occurred. The buoyancy of fish
was assessed by assigning a score of 3 to fish that remained on the aquaria bottoms, 2
to fish that swam in midwater, and 1 to fish that swam at the water surface,
Whole-body RNA and DNA were determined8 at 15 and 30 days of exposure Aand B.
Whole-body aluminum residues were determined on a wet-weight basis for 10 fish
per duplicate treatment on day 15 and 5 fish per duplicate on days 30, 45, and 60.15
Tissue residue data were statistically analyzed to derive surface response plots based on
whole-body aluminum residues, aluminum exposure concentrations. and days of
exposure.

Reference substance (positive control):

no

Duration:

60 d

Dose descriptor:

NOEC

Effect conc.:

24 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Remarks:

Aluminium

Basis for effect:

mortality

Remarks:

in Fry

Remarks on result:

other: pH 5.6-5.7

Duration:

60 d

Dose descriptor:

NOEC

Effect conc.:

26 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Remarks:

Aluminium

Basis for effect:

mortality

Remarks:

in Fry

Remarks on result:

other: pH 6.5-6.6

Duration:

60 d

Dose descriptor:

NOEC

Effect conc.:

15 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Remarks:

aluminium

Basis for effect:

weight

Remarks on result:

other: pH 5.6-5.7

Duration:

60 d

Dose descriptor:

NOEC

Effect conc.:

26 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Remarks:

Aluminium

Basis for effect:

weight

Remarks on result:

other: pH 6.5-6.6

Duration:

60 d

Dose descriptor:

NOEC

Effect conc.:

13 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Remarks:

Aluminium

Basis for effect:

number hatched

Remarks on result:

other: pH 6.5-6.6

Duration:

60 d

Dose descriptor:

NOEC

Effect conc.:

14 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Remarks:

Aluminium

Basis for effect:

other: swimming capacity

Remarks on result:

other: pH 5.6-5.7

Duration:

60 d

Dose descriptor:

NOEC

Effect conc.:

22 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Remarks:

Aluminium

Basis for effect:

other: Swimming capacity

Remarks on result:

other: pH 6.5-6.6

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)


Other:
Total dissolved aluminum measured in filtered acidified
samples ranged from 13 to 21 ug/L in exposure A and from 12 to 22 ug/L in exposure
B; total extractable aluminum in samples that were extracted immediately without
filtration ranged from 39 to 211 ug/L in exposure Aand from 27 to 194 ug/L in B; and
total dissolved aluminum in filtered extracted samples ranged from 14 to 27 ug/L in exposure A and from 13 to 26 µg/L in B. Results from the REDEQL-EPAK modeling suggest that a large portion of the total aluminum present in these studies was
in the solid, or undissolved, form. This was confirmed by the results obtained
from our filtration procedures which showed that concentrations ofdissolved aluminum
were consistently low. Filtration of exposure water with 3.0-um Teflon,
0.4 um polycarbonate, 0.2 um nylonand Fluoropore (Millipore Corporation, Bedford,
MA), and 0.1 um mixed cellulose-acetate and nitrate filters gave similar results. The
filtration results, coupled with the REDEQL-EPAK estimates, suggest the presence of
particulate or polymeric aluminum species larger than 3.0 um.

Reported statistics and error estimates:

One-way analysis of variance was used to compare treatment effects on survival,
growth, behavior, and nucleic acid content in both exposures. Percent data were arcsine
transformed, and square root transformations were made on strike frequency and water
column position data prior to statistical analyses. The Least Significant Difference
means comparison test was used to distinguish differences among treatment means (p
::;; 0.05). Swimming capacity data were also analyzed by simple regression to determine
the relation betweenswimming capacity and aluminum exposure concentration. Wholebody
residues ofaluminum were analyzed as a completely randomized design in which
the treatments were arranged factorially. The linear statistical model contained the
effects of aluminum exposure concentrations, days of exposure, and interactions of
exposure concentrations and days of exposure. Linear and quadratic polynomial
orthogonal contrasts were computed to produce surface response plots for whole-body
residues of aluminum. Statistical Analysis Systems20 programs were used to perform
aU analyses on the mainframe computer system of the University of Missouri,
Columbia, Missouri.

Table: 1: Highest no-observed effect concentration (µg/L) for biological responses of brook trout:

Variable	pH 5.6-5.7		pH 6.5-6.6
	30d	60d	30d	60d
Movement/2min	68(15)	142(24)	169(26)	>350 (22)
Strike frequentie	8(-)	142 (24)	4d(-)	>350 (22)
Buoyancy	68(15)	--	169(26)	--
Swimming capacity	>292 (27)	29 (14)	88(26)	>350 (26)
Incomplete hatch^a	8b(-)	--	57(13)	--
Fry mortality	142 (24)	142(24)	169 (26)	169 (26)
Length	142(24)	142 (24)	57(13)	88(26)
Weight	142c (24)	68 (15)	88c (26)	88(26)
RNA Content	142 (24)	--	>350 (22)	--
DNA Content	>292 (27)	--	>350 (22)	--

Note: Values represent total aluminumand(in parentheses) total dissolved monomeric aluminum

aEmbryo responses were determined after all embryos had hatched or died.

bBackground aluminum measured in pH 5.7 control treatment

cDetermined at 45 days of exposure.

Table 2. Mean length (mm) and standard deviation (SD) of brook trout during exposure to aluminum at acidic pH

Mean Al (µg/L)	Mean pH	Exposure duration
		Day 15		Day 30		Day 45		Day 60
		Mean	SD	Mean	SD	Mean	SD	Mean	SD
ExposureB
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 3. Mean weight (mg) and standard deviation (SD) of brook trout during exposure to aluminum at acidic pH

Mean Al (µg/L)	Mean pH	Exposure duration
		Day 15		Day 30		Day 45		Day 60
		Mean	SD	Mean	SD	Mean	SD	Mean	SD
ExposureB
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

Validity criteria fulfilled:

not applicable

Conclusions:

The lowest NOEC was determined to be 13 ug/L (measured dissolved Al) for both the endpoint " incomplete hachting". The test is valid and the method has gone trough peer review. Therefore the data can be used in a risk assessment as key data.

Executive summary:

Two partial life-cycle studies were conducted for 60 days in a modified flow-through proportional diluter.n In the first exposure (exposure A), eyed eggs of brook trout and the resultant larvae and juveniles were exposed to nominal aluminum concentrations of 38, 75, 150, and 300 ug/L at pH5.5; controls (no aluminum added) were held at pH 5.5 and 7.2. In the second exposure (exposure B), eyed eggs and the resultant larvae and juveniles were exposed to nominal aluminum concentrations of 50, 100,200, and 400 ug/L at pH 6.5; controls were held at pH 6.5 and 7.2. Because aluminum is less soluble under less acidic conditions and its toxicity to fish decreases, we used slightly higher aluminum exposure concentrations in exposure B than in A. The exposures were conducted in soft water containing about 3.0 mg/L calcium. Aluminum sulfate hexadecahydrate (98% pure: 8.56% allminum) was purchased from Fisher Scientific Company, Fairlawn, New Jersey. The eyed eggs were obtained from Beity's Resort, Valley, Washington. and held overnight in control water at pH 7.2 and 12°C before the study was initiated.

The lowest NOEC was determined for incomplete hachting to be 13 ug/L dissolved Al.

Reference 3

Endpoint:

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

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented publication which meets basic scientific principles.

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:

no

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.

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

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: fathead minnow
- Source: Duluth-Newtown laboratory strain, maintained in the Fisheries Laboratories

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- 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

Hardness:

Hard, value not reported

pH:

7.27 (mean)

Dissolved oxygen:

6.98 mg/L (mean)

Nominal and measured concentrations:

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

Details on test conditions:

TEST SYSTEM
- Test vessel:
- 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

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

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

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

7.1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

Al

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

Remarks:

Al

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

Remarks:

Al

Basis for effect:

weight

Remarks:

and length

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

7.1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

Al

Basis for effect:

weight

Remarks:

and length

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

23.1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

Al

Basis for effect:

number hatched

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

53.8 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

Al

Basis for effect:

number hatched

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

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 1. 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

Reference 4

Endpoint:

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

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

23-11-1988 to 18-04-1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Acceptable, well documented study which meets basic scientific principles. Data on disturbing factors is partial missing, like feeding data. however when looking at the difference between the 28 and 42 day endpoints this argument is considered as having no significant impact on the reliability. Data on the method for mortality score is not clear, however, this may be considered as a minor flaw. Endpoints are usable for risk assessments, eventhough this is no GLP study. Therefore it is rated as reliable with restrictions.

Qualifier:

according to guideline

Guideline:

other: Stoner, J.H, A S Gee and K.R. Wade, 1984. The effects of acidification on the ecology of streams in the Upper Tywi catchment in West Wales. Environ Pollut Ser A 35, 125-157.

Qualifier:

according to guideline

Guideline:

other: Whitehead, P G, S Bird, M Hornung, J. Cosby, C. Neal and P. Paricos, 1988 Stream acidification trends in the Welsh Uplands — A modelling study of the Llyn Brianne catchments. J. Hydrology 101, 191-212.

Qualifier:

according to guideline

Guideline:

other: Drinscoll, C.T, J P. Baker, J.J. Bisognilsogm and C L. Schofield, 1980. Effect of aluminium speciation on fish in dilute acidified watersEffect of ahimimum speciation on fish m dilute acidified waters. Nature 284, 161-164.

Qualifier:

according to guideline

Guideline:

other: Goenaga, X. and D.J.A Williams, 1988. Alumimum speciation in surface waters from a Welsh upland area. Environ. Pollut. 52, 131-149

Qualifier:

according to guideline

Guideline:

other: Finney, D.J, 1971. Probit analysis. Cambridge University Press.

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

monomeric aluminium (Alm) was measured in samples pressure filtered through a 0.015 um membrane
filter; organic monomeric aluminium (Alom) was the fraction of a filtered sample
which passed through a cation exchange column of Amberlite resin (preconditioned
for pH and conductivity); inorganic monomeric aluminium (Al1) was Alm-Alom and
adsorbed aluminium (Alad) was Altm-Alm.

Vehicle:

no

Details on test solutions:

Natural water according to Stoner et al., 1984 and Whitehead et al., 1988

Test organisms (species):

Salmo trutta

Details on test organisms:

To ensure comparability, all life stages were derived from the same strain of brown
trout, and reared in the hatchery (pH 6.2, 22 mg CaCO31-1) until required. Hatchery
temperatures were about 3°C warmer than those in the study streams and fish held
here developed faster. This led to some overlap in the exposure periods of different
life stages.
eggs:
On 23 November 1988, eggs and milt were stripped from the parent fish and transported
separately to the study site. In order to maximise fertilisation success in the
field, milt and eggs were mixed dry in a plastic tray and left for 2 min. The ova were
divided into six batches, each placed in 5 1 of water from one of the six study streams.
After leaving to harden for 1 h, 300 fertilized ova from each batch were divided equally
amongst six egg boxes.
alevins:
Alevin experiments started on the 25 January 1989, approximately 17 days after
hatching.

Test type:

other: natural streams

Water media type:

freshwater

Limit test:

no

Hardness:

< 20 mg CaCO3/L

pH:

4.7-6.9

Nominal and measured concentrations:

measured Al monomeric concentrations: 3, 5.2, 5.6, 12, 34, 56, 88, 377, 397 ug/L

Details on test conditions:

eggs:
The ova were
divided into six batches, each placed in 5 1 of water from one of the six study streams.
After leaving to harden for 1 h, 300 fertilized ova from each batch were divided equally
amongst six egg boxes. Egg boxes were cylindrical, 13 cm high by 7 cm diameter,
and constructed from 2 mm plastic mesh reinforced with 5 mm mesh. They were filled
with gravel 1-3 cm in diameter in order to reduce egg contact and siltation. Boxes
were buried 8-10 cm deep (to the box centre) in artificial redds located in gravels
typical of those used for natural spawning.
alevin:
Alevin cages were of similar construction to the egg boxes, measuring 20
cm high by 12 cm diameter. These were filled with approximately 16 cm depth of
gravel and placed in buckets of stream water. Eighteen alevins were added to each
cage and allowed several minutes to disperse into the gravel. The cages were then
buried in similar locations to the eggs, leaving the top 4 cm exposed. This allowed
for the emergence of the 'swim-up' stage. Six cages, three for both pre- and post-
'swim-up' alevins were used in each of nine streams
Parr:
Parr of age 80 days were held in 10 streams for a maximum exposure
period of 21 days In each stream, four cages (2 mm
mesh) each holding five fry were secured to the bed by steel stakes. Survival was recorded
every 3-4 days. The limitation in exposure time was imposed to minimise any
confounding stress through reduced ability to feed.

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

19 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Remarks:

aluminium

Basis for effect:

mortality

Remarks:

pH 5.8-5.9 effects on Alevins

Duration:

42 d

Dose descriptor:

LC50

Effect conc.:

15 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Remarks:

Aluminium

Basis for effect:

mortality

Remarks:

pH 5.8-5.9 effects on alevins

Details on results:

Apart from LI6, survival of eggs to hatching ranged from 71.1% to 84.0% (means
back-transformed from arcsine values), and was apparently independent of variations
in aluminium concentration between streams.
Mean percentage alevin mortality increased with aluminium concentration and exposure
time.

Exploratory regression of mortality probits (Finney, 1971) on mean concentrations of each aluminium species (log transformed), indicated that mortality in both 28- and 42-day exposures was most strongly related to mean total monomeric aluminium (Altm). For alevins exposed for 28 days the following regression explained 81.3 % of the variance in mortality, mortality probit = 1.24 + 2.93 x log[Altm], giving an LC50 of 19 µg/L Altm. For 42 day exposure alevins ('swim-up'), 94.2% of the variance in mortality was explained by the regression equation, mortality probit = 1.35 + 3.12 x log[Altm], with an LC50 of 15 ug/L Altm. LC50 values for Al-filterable were 79 ug/L for 28 -day alevins and 72 ug/L for 42 -day alevins, although this fraction accounted respectively for only 54.8 % and 37.7 % of the variance in mortality. Alevin mortality could also be related to mean pH for both exposure periods. For younger alevins the regression was, mortality probit = 21.5 - 2.85 x pH. (r^2 = 97.4 %) For 'swim-up' alevins, mortality probit = 20.3-2.61 x pH. (r^2= 85.2%) Approximate LC50 values were pH 5.8 and pH 5.9, respectively. Survival of alevins in the limed streams was high, as expected from the low aluminium concentrations and high pH at these sites.

Validity criteria fulfilled:

not applicable

Conclusions:

The long-term LC50s were determined to be 19 and 15 ug/L for 28 and 42 days of exposure in natural waters, respectively. Values are measured values of total monomeric Al. Study is valid and scientific sound. although essential data are missing, these data should not have a impact on the actual endpoint and are therefore considered not significant enough to have an impact on the reliablility. The endpionts can therefore be used in a risk assessment especially when lokking at acidic waters.

Executive summary:

The survival of artificially implanted eggs, alevins and parr of brown trout Salmo trutta (L.) was assessed m streams of different acidity. Chemical analysis included detailed alumlnium speciatmn of surface and interstitial water samples, taken over the duration of lntragravel hfe stages. Egg survival, from two minutes after fertilization to hatching, was usually above 71%, and was independent of the mean concentration of total monomeric aluminium (Altm) over the range 3 -397 ug/L The survival of alevins exposed for 28 days (before 'swim-up') or 42 days ('swim-up') was most strongly related to mean Aim concentration and to pH For 28- and 42-day exposures, LC50 values for Altm were approximately 19 and 15 ug/L, respectively, or 79 and 72 µg/L for 0.45 gm filterable alumlnium (Al-filterable) The 21-day LC50 of parr ca 3 mth old, was between 84 and 105 ug/L mean Al-filerable concentration. During a simulated acid episode of mean pH 4 8 and 880 ug Al/L, parr showed 100 % mortality after only 18 h. We discuss the problems of quantifying toxic responses in soft-water streams. As exemplified in this study, chemical variables show wide and rapid fluctuations, and are often co-correlated. These features confuse interpretation of field experiments or predictions from laboratory data. The reviewer considers the most applicable endpoint to be the LC50 regarding total dissolved monomeric Al to alevin of 19 and 15 ug/L.

Reference 5

Endpoint:

long-term toxicity to fish

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented publication which meets basic scientific principles.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Determination of the effects of humic acid and hardness on subacute aluminum toxicity in flow-through laboratory tests with juvenile rainbow trouts in the neutral alkaline pH range. Mortality and growth were chosen as endpoints of toxicity.

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

Aluminum samples and either hardness or humic acid determinations were taken every 96 h.

Vehicle:

no

Details on test solutions:

Well water was processed by reverse osmosis (RO) reducing total dissolved solids by about 95% (hardness = 8 - 12 mg/L as CaCO3). Prior to toxicity tests, storage tank water was reconstituted to the proper chemical composition.
An aluminium stock was maintained at 16 mg Al/L with a pH of 9.0. The composition of the dilution water was 80 mg/L NaHCO3, 4 mg/L KCl, 10 mg/L CaSO4, and 5 mg/L MgSO4.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout
- Source: Oregon State University marine and Freshwater Biomedical Center Core Facility, USA
- Age at study initiation: juvenile
- Weight at study initiation: 1 - 3 g
- Method of housing: 300 L fiber-glass tanks supplied with running well water; fed Oregon Test Diet (Sinnhuber et al 1977, Neoplasms in rainbow trout, Ann. N.Y. Acad. Sci 298: 389-408) ad libitum until 48 h prior to subacute tests.
- Feeding during test: fed an 8% ration of Oregon Test Diet daily (based on dry weights) and uneaten food was collected each day (siphoned trough a net) to determine net consumption

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

16 d

Hardness:

10 and 100 mg/L as CaCO3 (nominal) in different test series

Test temperature:

14 ± 0.5 °C

pH:

7.3 and 8.3 in different tests

Nominal and measured concentrations:

0, 1, 2, and 4 mg/L (nominal Al) (test with different hardness concentrations)
0, 0.5, 1, and 2 mg/L (nominal Al) (test with different humic acid concentrations)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 20 L aquaria
- Type of flow-through (e.g. peristaltic or proportional diluter): A circulation pump ensured proper mixing of solutions; continuous-flow Chadwick-type diluter
- Renewal rate of test solution (frequency/flow rate): 100 mL / min, resulting in a volume replacement time of 3.3 h
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

Duration:

16 d

Dose descriptor:

NOEC

Effect conc.:

1.49 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

Al

Basis for effect:

growth rate

Remarks on result:

other: average hardness 20.3 mg/L; pH 8.05

Duration:

16 d

Dose descriptor:

NOEC

Effect conc.:

0.14 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Basis for effect:

growth rate

Remarks on result:

other: average hardness 20.3 mg/L; pH 8.05

Duration:

16 d

Dose descriptor:

NOEC

Effect conc.:

1.52 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

Al

Basis for effect:

growth rate

Remarks on result:

other: average hardness 103 mg/L; pH 8.02

Duration:

16 d

Dose descriptor:

NOEC

Effect conc.:

0.16 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Basis for effect:

growth rate

Remarks on result:

other: average hardness 103 mg/L; pH 8.02

Duration:

16 d

Dose descriptor:

LOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

Al

Basis for effect:

growth rate

Remarks on result:

other: average hardness 20.3 mg/L; pH 8.14

Duration:

16 d

Dose descriptor:

LOEC

Effect conc.:

0.26 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Basis for effect:

growth rate

Remarks on result:

other: average hardness 20.3 mg/L; pH 8.14

Duration:

16 d

Dose descriptor:

LOEC

Effect conc.:

2.75 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

Al

Basis for effect:

growth rate

Remarks on result:

other: average hardness 103 mg/L; pH 8.10

Duration:

16 d

Dose descriptor:

LOEC

Effect conc.:

0.28 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Basis for effect:

growth rate

Remarks on result:

other: average hardness 103 mg/L; pH 8.10

Duration:

16 d

Dose descriptor:

LC50

Effect conc.:

1.94 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

Al

Basis for effect:

mortality

Remarks on result:

other: CL: 1.62-2.38; average hardness 20.3 mg/L; pH 8.3

Duration:

16 d

Dose descriptor:

LC50

Effect conc.:

3.91 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

Al

Basis for effect:

mortality

Remarks on result:

other: CL: 2.86-8.57; average hardness 103.0 mg/L; pH 8.3

Duration:

16 d

Dose descriptor:

LC50

Effect conc.:

0.43 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Basis for effect:

mortality

Remarks on result:

other: CL: 0.31-0.61; average hardness 20.3 mg/L; pH 8.3

Duration:

16 d

Dose descriptor:

LC50

Effect conc.:

0.67 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Basis for effect:

mortality

Remarks on result:

other: CL: 0.53-1.19; average hardness 103.0 mg/L; pH 8.3

Reported statistics and error estimates:

Toxicity was analyzed using the trimmed Spearman-Karber method for 16-d LC50 determinations (Hamilton et al. 1977, Trimmed Spearman-Karber method for estaimating median lethal concentrations in toxicity bioassays, Environ. Sci. technol. 11: 714-719) and pairs of LC50s were considered different when confidence limits did not overlap and when the ratio between the greater and lesser LC50s exceeded the calculated f ratio (Litchfield and Wilcoxon 1949, A simplified method of evaluating dose-effect experiments, J. Pharmacol. Exp. Ther. 96: 99-113). Statgraphics (1986) and SAS Institute Inc. (1985) PC software was used for statistical analyses.

Table1: Summary of test conditions in exposure tanks and SGR (= % of initial weight/d), average initial wet weight (g), and consumption (% of food ration consumed) of surviving rainbow trout for the 16-d subacute test at weakly alkaline and near-neutral pH. all reported values are mean ± SE from replicated exposures. The mean pH and aluminum concentrations were not significantly different (p > 0.05) between exposure tanks maintained at different hardness and the same nominal aluminum concentration at each pH tested.

pH	Total hardness (mg CaCO3/L)	Aluminum (mg/L)			% cumulative mortality	Initial wet weight	SGR	Consumption
		Nominal Al	Total Al	Filterable
7.97±0.04	19.9±1.1	0	<0.01	<0.01	0	2.744	1.706±0.242	100
7.94±0.03	103.8±2.3	0	<0.01	<0.01	0	3.191	1.715±0.349	100
7.98±0.04	19.9±1.4	1	0.83±0.14	0.06±0.01	10	2.908	1.570±0.148	100
7.98±0.03	103.5±1.7	1	0.74±0.07	0.06±0.01	5	2.822	1.600±0.173	100
8.05±0.04	20.6±0.8	2	1.49±0.15	0.14±0.02	45	2.776	1.223±0.156	82
8.02±0.04	102.1±1.9	2	1.52±0.25	0.16±0.04	10	2.880	0.852±0.264	93
8.14±0.05	20.3±0.6	4	3.20±0.22	0.26±0.04	75	2.808	0.118±0.515 (a)	69
8.10±0.04	103.4±1.3	4	2.75±0.23	0.28±0.04	45	2.802	0.288±0.268 (a)	66
7.31±0.02	16.8±2.1	0	<0.01	<0.01	0	2.546	1.486±0.156	100
7.30±0.02	87.7±2.3	0	<0.01	<0.01	0	2.232	1.522±0.200	100
7.33±0.02	15.0±1.2	1	0.89±0.10	0.02±0.00	10	2.554	0.088±0.170 (a)	72
7.33±0.01	84.8±3.1	1	0.94±0.06	0.02±0.00	10	2.634	0.756±0.079 (a)	86
7.35±0.02	16.9±1.4	2	2.11±0.10	0.02±0.00	15	2.312	-0.524±0.286 (a)	58
7.33±0.02	85.6±2.6	2	1.88±0.07	0.03±0.01	0	2.704	-0.535±0.286 (a)	70
7.34±0.02	16.3±0.9	4	4.49±0.38	0.02±0.00	15	2.490	-0.380±0.195 (a)	68
7.32±0.02	84.1±2.2	4	4.56±0.42	0.03±0.00	10	2.366	-0.202±0.282 (a)	70

(a) significantly different (p < 0.05) from corresponding control

Table 2: Summary of test conditions in exposure tanks and SGR (= % of initial weight/d), average initial and final wet weight (g), and consumption (% of food ration consumed) of surviving rainbow trout for the 16-d subacute test at weakly alkaline and near-neutral pH. all reported values are mean ± SE from replicated exposures. The mean pH and aluminum concentrations were not significantly different (p > 0.05) between exposure tanks maintained at different hardness and the same nominal aluminum concentration at each pH tested. Also shown is cumulative mortality in percent.

pH	Total hardness (mg CaCO3/L)	Aluminum (mg/L)			% cumulative mortality	Initial wet weight	SGR	Consumption
		Nominal Al	Total Al	Filterable
8.05±0.02	0	0	<0.01	<0.01	0	1.984	1.291±0.290	100
8.01±0.02	4.60±0.12	0	<0.01	<0.01	0	2.084	1.256±0.198	100
8.05±0.02	0	0.5	0.49±0.10	0.05±0.01	15	1.971	-0.044±0.177(a)	93
8.01±0.02	5.02±0.26	0.5	0.62±0.19	0.06±0.01	0	2.013	1.314±0.133	100
8.07±0.02	0	1	1.20±0.32	0.07±0.01	50	2.093	0.108±0.181(a)	93
8.01±0.02	4.88±0.14	1	1.18±0.28	0.08±0.01	0	2.052	1.238±0.275	100
8.12±0.02	0	2	2.34±0.62	0.13±0.01	80	2.004	-0.657±0.396(a)	91
8.05±0.02	5.20±0.20	2	2.04±0.42	0.13±0.01	15	1.838	1.110±0.308	96
7.18±0.03	0	0	<0.01	<0.01	0	1.917	1.283±0.275	100
7.15±0.03	4.31±0.22	0	<0.01	<0.01	0	1.806	1-196±0.086	100
8.05±0.02	0	0.5	0.55±0.05	0.06±0.01	0	1.808	0.985±0.068	100
8.01±0.02	4.60±0.12	0.5	0.53±0.03	0.07±0.01	0	1.780	1.257±0.068	100
8.05±0.02	0	1	1.09±0.15	0.08±0.02	5	1.875	0.896±0.126(a)	100
8.01±0.02	5.02±0.26	1	1.26±0.05	0.10±0.02	0	1.804	1.196±0.070	100
8.07±0.02	0	2	2.64±0.18	0.09±0.02	0	1.766	0.174±0.078(a)	90
8.01±0.02	4.88±0.14	2	2.56±0.14	0.13±0.03	0	1.848	1.048±0.096	100

(a) significantly different (p < 0.05) from corresponding control

Based on the high mortalities at weakly alkaline pH and filterable aluminium concentrations near the theoretical solubility where Al (OH-)4 is the major soluble form, the authors concluded that soluble Al(OH-)4 was largely responsible for the observed mortalities.

Additional information

No reliable studies on the acute toxicity of reaction mass of aluminium hydroxide and aluminium nitrate and aluminium sulphate to long term fish are available. Therefore, a weight of evidence approach is applied and data are read-across to various aluminium compounds based on an analogue approach.

Cleveland (1989) exposed Salvinelus fontinalis to aluminium sulphate for 60 days in a non-guideline study. The hardness was reported as 246 µq/L and pH was 6.5. NOEC and LOEC values were determined for mortality, length, weight, number hatched, and swimming behaviour, and ranged between 57 to >350 µg/L and 88 to 350 µg/L total Al, respectively.

Buckler et al.(1995) derived NOEC and LOEC values of similar magnitude with 88 and 169 µg/L total Al for the atlantic samon Salmo salar, respectively, based on length and weight after 60 days and pH 5.5..

Another study from Weatherley (1990) was a subchronic test with Salmo trutta in natural water. The survival of artificially implanted eggs, alevins and parr of brown trout Salmo trutta (L.) was assessed m streams of different acidity and chemical variables show wide and rapid fluctuations, and are often co-correlated. It was considers the most applicable endpoint to be the LC50 regarding total dissolved monomeric Al. The LC50 at 28 days was 19 µg/l and at 42 days 15 µg/l. The pH in this study was 5.8 -5.9.

In a 28 day study with Pimephales promelas Kimball (1978) exposed fish to aluminium sulphate at a pH of around 7.3 and under hard water conditions. NOECs and LOECs were determined for mortalities of the fry, weight, length, and number hatched. Based on total Al NOECs were in the range of 4.7 to 23.1 mg/L and LOECs in the range of 7.1 to 53.8 mg/L. In a further non-guideline study Gundersen et al. (1994) exposed rainbow trout for 16 days to AlCl3 at a water hardness of 10 and 100 mg/L as CaCO3 and at pH 7.3 and 8.3. NOECs reported for nominal pH 8 were around 1.5 mg/L (total Al) and 0.15 mg/L (dissolved Al). Similar LOECs were 3.2 and 2.75 mg/L (total Al) and 0.27 mg/L (dissolved Al). There was no significant difference between effect values at different water hardness.