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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
12 April 2010 to 30 April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In water aluminium orthophosphates will dissociate into their ionic forms (Al3+ and PO43-, which will further associate with the ionic forms of H2O). It is therefore considered acceptable to assess the aluminium and phosphate ions as separate entities.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
No additional information.
3. ANALOGUE APPROACH JUSTIFICATION
This study is submitted as part of a weight of evidence approach in accordance with REACH Annex XI, Section 1.2. When considered alongside the additional data provided for this endpoint it is considered that the ecotoxicity of the substance to be registered is adequately assessed.
4. DATA MATRIX
See full read-across justification attached under Section 13 of this dossier and summary of data included in the endpoint summary under Section 6.1 of this dossier.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of GLP inspection: 15/09/2009 Date of Signature on GLP certificate:26/11/2209
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Chemical name: Phosphoric acid, potassium salt (2:3), dihydrate.
Synonyms (IUPAC name): Tripotassium trihydrogen diphosphate dihydrate.
Chemical formula: H3 O4 P . H2 O . 3/2 K
CAS number: 6922-99-4
Molecular weight: 346.29
Melting range: No complete melting up to 573 K
Solubility in water ( g/L, at 20 °C): 705 g/L (pH 7)
Analytical monitoring:
yes
Details on sampling:

- Concentrations:
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 (fresh media), 24 (old and fresh media), and 96 hours (old media) (SEE ATTACHED APPENDIX)

- Sampling method:
Water samples were taken from the control and each replicate test vessel at 0 (fresh media), 24 and 96 hours (old media) for quantitative analysis.
Duplicate samples and samples at 24 (fresh media), 48 and 72 hours (fresh and old media) were taken and stored at approximately -20C for further analysis if necessary.
The method of analysis, stability, recovery and test preparation analyses are described in attached Appendix.

- Sample storage conditions before analysis:
Storage conditions: room temperature in the dark
Vehicle:
no
Details on test solutions:
For the purpose of the definitive test the test item was dissolved directly in test media
An amount of test item (4000 mg) was dissolved in dechlorinated tap water and the volume adjusted to 2 litres to give a 2000 mg/l stock solution. An aliquot (1000 ml) of this stock solution was added separately to a final volume of 20 litres of dechlorinated tap water 9in duplicate) to give the 100 mg/l test concentration (replicates R1 and R2).The stock solution was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 (fresh media), 24 (old and fresh media), and 96 hours (old media).
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name:
Rainbow Trout

- Strain:
Not stated

- Source:
Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in house since 16 March 2010.

- Age at study initiation (mean and range, SD):
Juvenile.

- Length at study initiation (length definition, mean, range and SD):
Fish had a mean standard length of 6.0 cm (sd = 0.4)

- Weight at study initiation (mean and range, SD):
And a mean weight of 2.82 g (sd = 0.47) at the end of the definitive test

- Method of breeding:
Not stated

- Feeding during test
- Food type:
The stock fish were fed commercial trout pellets which was discontinued 24 hours prior to the start of the definitive test

- Amount:
Not stated

- Frequency:
Not stated


ACCLIMATION
- Acclimation period:
Fish were acclimatised to test conditions from 14 April to 26 April 2010.

- Acclimation conditions (same as test or not):
Same as test conditions.

- Type and amount of food:
The stock fish were fed commercial trout pellets which was discontinued 24 hours prior to the start of the definitive test

- Feeding frequency:
Not stated

- Health during acclimation (any mortality observed):
Zero mortality in the 7 days prior to the start of the test.


Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.
Hardness:
Total hardness of approximately 140 mg/l as CaCO3.
Test temperature:
Approximately 14ºC
pH:
The pH was measured using a WTW pH/Oxi 340I pH.
pH range of 7.2-7.9.
Please see Physico-Chemical Measurements Appendix 2(attached background material).
Dissolved oxygen:
The dissolved oxygen concentration was measured using a dissolved oxygen meter.
Please see Physico-Chemical Measurements Appendix 4 (attached background material) for dissolved oxygen results.
Salinity:
No Data
Nominal and measured concentrations:
RANGE-FINDER:

In the range-finding test fish were exposed to a single test concentration of 100 mg/l as the Acute Toxicity to Daphnia magna Test (Harlan Laboratories Ltd Project Number: 3113/0003) indicated that toxicity is not expected at this level.

DEFINITIVE TEST:
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100 mg/l to confirm that at the maximum concentration given in the OECD/EEC Test Guidelines, no mortalities or sub-lethal effects of exposure were observed.
Details on test conditions:

TEST SYSTEM
- Test vessel:
As in the range-finding test 20 litre completely filled and sealed glass round-bottomed vessels were used for each test concentration.The test vesselswere aerated via narrow bore glass tubes.

- Renewal rate of test solution (frequency/flow rate): Daily

- No. of organisms per vessel: At the start of the test 7 fish were placed in each test vessel at random, in the test preparations.

-No. of vessels per concentration (replicates): 1

- No. of vessels per control (replicates): 1


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/l as CaCO3.

-Total Organic Carbon Analysis:
Please see Appendix 2 (attached background material).

- Particulate matter:
Not measured

- Metals: Not Stated
- Pesticides: Not Stated
- Chlorine: Not Stated
- Alkalinity: Not Stated
- Ca/mg ratio: Not Stated
- Conductivity: Not Stated
- Culture medium different from test medium: Not Stated
- Intervals of water quality measurement: Not Stated


OTHER TEST CONDITIONS
The test vessels were then sealed and maintained at approximately 14ºC in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
potassium
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
potassium
Basis for effect:
mortality (fish)
Sublethal observations / clinical signs:

 Range-finding Test

Cumulative mortality data from the exposure of rainbow trout to the test item during the range-finding test are given in Table 1. There were no sub-lethal effects of exposure during the range-finding test.

Based on this information, a single test concentration, in duplicate, of 100 mg/l was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the maximum test concentration given in the OECD/Test Guidelines, no mortalities or sub‑lethal effects of exposure were observed.

Table1. Cumulative Mortality Data in the Range-findingTest

Nominal

Concentration

(mg/l)

Cumulative Mortality

(Initial Population = 3)

3 Hours

6 Hours

24 Hours

48 Hours

72 Hours

96 Hours

Control

0

0

0

0

0

0

100

0

0

0

0

0

0

 

DefinitiveTest

 Mortality data

Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 2.

 Table 2.  Cumulative Mortality Data in the DefinitiveTest

Nominal

Concentration

(mg/l)

Cumulative Mortality
(Initial Population =7)

%

Mortality

3 Hours

6 Hours

24 Hours

48 Hours

72 Hours

96 Hours

96 Hours

Control

0

0

0

0

0

0

0

100 R1

0

0

0

0

0

0

0

100 R2

0

0

0

0

0

0

0

There were no mortalities in 14 fish exposed to a test concentration of 100 mg/l for a period of 96 hours. Inspection of the mortality data gave the following results:

Time (h)

LC50(mg/l)

3

>100

6

>100

24

>100

48

>100

72

>100

96

>100

The results of the definitive test showed the highest test concentration resulting in 0% mortality to be greater than or equal to 100 mg/l, the lowest test concentration resulting in 100% mortality to be greater than 100 mg/l and the No Observed Effect Concentration (NOEC) to be 100 mg/l. The No Observed Effect Concentration is based upon zero mortalities and the absence of any sub-lethal effects of exposure at this concentration.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/l. 

Sub-lethal effects

There were no sub-lethal effects of exposure observed in the test.

 Observations on test item solubility

The test preparations were observed to be clear, colourless solutions throughout the duration of the test.

Physico-chemical measurements

The results of the physico-chemical measurements are given in Appendix 3. Temperature was maintained at approximately 14°C throughout the test. While there were no treatment related differences for oxygen concentration, there was a slight difference observed between the pH of the control and 100 mg/l test groups in the fresh media.

 Verification of test concentrations

Analysis of the test preparations at 0, 24 and 96 hours (see Appendix 2) showed measured test concentrations to range from 109% to 118% of nominal and so it was considered justifiable to estimate the LC50values in terms of the nominal test concentrations only.

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LC50 of greater than 100 mg/l. Correspondingly the No Observed Effect Concentration was 100 mg/l.

This study is conducted according to an appropriate guideline and under the conditions of GLP and therefore the study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for this endpoint.
Read-across is justified on the basis detailed in rationale for reliability above. This study is therefore considered to be of sufficient adequacy and reliability to be used as a key study and no further testing is justified.
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
06-05-2003 to 08-08-2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In water aluminium orthophosphates will dissociate into their ionic forms (Al3+ and PO43-, which will further associate with the ionic forms of H2O). It is therefore considered acceptable to assess the aluminium and phosphate ions as separate entities.
The aluminium ion is considered to be toxic to fish under certain circumstances. The bioavailability and hence toxicity of aluminium is predominantly influenced by water quality parameters, in particular pH. The fate of Al3+ ion is of the most importance when considering whether a classification for aquatic toxicity of aluminium orthophosphates is required. In pH conditions of around neutral (as would be maintained via the use of appropriate buffers in a laboratory study) the Al3+ ion would be expected to react with the water molecules forming a weak acid (Al(OH)3)which would ultimately precipitate out of the water column rendering it not bioavailable and thus non-toxic to aquatic organisms. Since aluminium sulphate is soluble in water it is considered that the behaviour in water will be similar to the target substance and as such the data is relevant for assessing the toxicity of aluminium.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The water solubility of aluminium sulphate-14-hydrate is stated as being approximately 600 g/L, therefore the solubility of aluminium tris(dihydrogen phosphate) is very similar (522 g/L) at 20±0.5°C).

3. ANALOGUE APPROACH JUSTIFICATION
On the basis of the discussion presented above the results of acute toxicity studies on aluminium sulphate-14-hydrate have been used and are justified on the basis that Al3+ ion is of concern with regards to potential toxicity and therefore the results of such a study are relevant for read-across to the target substance.
This study is submitted as part of a weight of evidence approach in accordance with REACH Annex XI, Section 1.2. When considered alongside the additional data provided for this endpoint it is considered that the ecotoxicity of the substance to be registered is adequately assessed.

4. DATA MATRIX
See full read-across justification attached under Section 13 of this dossier and summary of data included in the endpoint summary under Section 6.1 of this dossier.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Sampling from the test vessels was performed at middle depth of the water column at test start and after 24 h, 48 h, 72 h and 96 h. At 24 h, 48 h, and 72 h, the medium was exchanged and both, new and old media were sampled. Samples were taken from the test vessels using a plastic syringe and transferred into disposable 20 mL polyethylene vials. One subsample of 20 mL was acidified with 0.2 mL 14 M nitric acid, thoroughly mixed and analyzed without further treatment. The added amount of nitric acid was sufficient to reduce the pH below 2. This fraction is designated as “unfiltered sample” and is assumed to contain the “total aluminium”. A second subsample was passed through a Sartorius 0.45 μm-membrane filter (type Minisart NML, diameter 26 mm). In order to rinse the filter, approx. 2 - 3 mL of the sample water was processed at first. The filtrate from this rinsing was discarded. Then, further 20 mL of the sample water was processed. This filtrate was acidified as described above (20 mL sample plus 0.2 mL 14 M nitric acid; resulting pH below 2). In aquatic chemistry,0.45 μm-membrane filtered samples generally are assumed to contain the “dissolved metal” fractions.
Vehicle:
no
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Species: Danio rerio (Hamilton-Buchanan 1822) (Teleostei, Cyprinidae)
Length: 2 cm + 1 cm
Source: Laboratory bred
The strain of zebra fish was obtained from: West Aquarium GmbH, PB 146, D-37431, Bad Lauterberg. The test fish were taken from cultures of the testing facility.
Holding: Test fish had been reared under study conditions (light, temperature) in purified drinking water. They had been fed daily with Tetra-Min® Hauptfutter (Tetra Werke, Melle, Germany).
Mortality: The criteria of the test guideline (ref 1) were followed. Only healthy fish without diseases and abnormalities were used in the study.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
22.3 +- 0.3 °C
pH:
7.4-8.0
Dissolved oxygen:
86-94% of saturation

Salinity:
not applicable.
Nominal and measured concentrations:
nominal: 62.5, 125, 250, 500, 1000 mg/L
measured (Al total):4.87, 10.4, 21.6, 43.4, 85.9 mg/L
measured (Al dissolved): 0.247, 0.205, 0.166, 0.131, 0.105 mg/L
Details on test conditions:
The test fish were exposed to the test item under semi-static conditions for a period of 96 hours. Each day, the fish were transferred to freshly prepared test liquids in new test vessels. The temperature during the test was 22.3 ± 0.3°C. Mortality and abnormal behaviour were recorded after 24 h, 48 h, 72 h and 96 h. 10 L glass aquaria were used as test vessels. The loading of fish was 0.2 g/L and met the criteria of the OECD-guideline.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Results with reference substance (positive control):
EC50 ranged from 1.0 to 1.3 mg/L.
Sublethal observations / clinical signs:

Results indicate that the toxicity endpoint exceeds the water solubility of the test substance at the tested pH.

Validity criteria fulfilled:
yes
Conclusions:
During the test period of 96 h, neither mortality occurred nor other signs of intoxication could be observed. Thus, the NOEC of the test item was determined with ≥ 1000 mg test item /L.
This study is conducted according to an appropriate guideline and under the conditions of GLP and therefore the study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for this endpoint.
Executive summary:

A study sponsored by the Aluminium Salts Subgroup of the Cefic Incopa Sector Group was performed at the Fraunhofer-Institute for Molecular Biology and Applied Ecology to evaluate the acute toxicity of aluminium sulphate-14-hydrate to zebra fish (Danio rerio) according to OECD TG 203. Aluminium sulphate-14-hydrate was tested at nominal concentrations of 62.5, 125, 250, 500, and 1000 mg/L under semi-static conditions (daily renewal of the test medium) for 96 hours. Test water was reconstituted water according to OECD TG 203, with pH adjusted at 7.5 at each renewal. The water column was stirred during the test. The mean measured concentrations based on total aluminium differed by less than 20 % from nominal values. Up to the highest test concentration, no mortality could be observed after 96 h. Thus, the NOEC of aluminium sulphate-14-hydrate under the described study conditions was determined as follows: NOEC ≥ 1000 mg aluminium sulphate-14-hydrate / L, and > the water solubility at the tested pH range.

Description of key information

LC50 (96 h)> 100mg/L (read across)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
100 mg/L

Additional information

Data on the acute toxicity of aluminium tris(dihydrogen phosphate) (CAS 13530-50-2) to fish are not available. In accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across to the structurally related substances aluminium sulphate -14-hydrate (CAS 16828-12-9)  and tripotassium trihydrogen diphosphate dihydrate (CAS 66922-99-4) was conducted to fulfil the standard information requirements. In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for aquatic toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across). According to Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 substances can be considered to be similar if their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity. For further details please see chapter 6.1 of the IUCLID file.

The acute toxicity of phosphoric acid, potassium salt (2:3) dihydrate (CAS 66922-99-4) to fish was investigated in a study by Priestly and Mullee (2010). This test was conducted according to OECD 203 under GLP conditions. Test organisms (Oncorhynchus mykiss) were exposed to the test substance for 96 hours, applying a semi-static water regime. Analytical determinations were performed to ensure a proper maintenance of the test concentration throughout the experiment. Observations for lethal and sub-lethal effects were performed at 3, 6, 24, 48, 72 and 96 hours. No effects on survival or fitness of fishes exposed to the test material were reported and therefore, a LC50 above 100 mg/L and a NOEC ≥100 mg/L were determined.

 

The aluminium ion is known to be toxic to fish under certain circumstances. The bioavailability and hence toxicity of aluminium is predominantly influenced by water quality parameters, in particular pH. The fate of Al3+ ion is of the most importance when considering whether a classification for aquatic toxicity of aluminium orthophosphates is required. In pH conditions of around neutral (as would be maintained via the use of appropriate buffers in a laboratory study) the Al3+ ion would be expected to react with the water molecules forming a weak acid (Al(OH)3)which would ultimately precipitate out of the water column rendering it not bioavailable and thus non-toxic to aquatic organisms.

On the basis of the discussion presented above the results of an acute toxicity study on aluminium sulphate-14-hydrate have been used and are justified on the basis that Al3+ ion is of concern with regards to potential toxicity and therefore extrapolation of the results on aluminium sulphate can be done based on the aluminium content.

The water solubility of aluminium sulphate-14-hydrate is stated as being approximately 600 g/L, therefore as aluminium tris(dihydrogen phosphate) has a lower solubility the results would give the worst case in terms of aluminium content for read across.

The semi static short-term study according to OECD guideline 203 and GLP was performed with Danio rerio (Schäfers, 2003). Aluminium sulphate-14-hydrate was tested at nominal concentrations of 62.5, 125, 250, 500, and 1000 mg/L under semi-static conditions (daily renewal of the test medium) for 96 hours, this resulted in dissolved aluminum concentrations of 0.247 to 0.105 mg/L (the concentration decreased with increasing nominal concentrations). Test water was reconstituted water according to OECD 203, with pH adjusted at 7.5 at each renewal. The water column was stirred during the test. Up to the highest test concentration, no mortality could be observed after 96 h. Thus, the NOEC of aluminium sulphate-14-hydrate under the described study conditions was determined as follows: NOEC ≥ 1000 mg aluminium sulphate-14-hydrate / L nominal and > 0.247 mg/L (dissolved Al)), and > the water solubility at the tested pH range. 

Based on the available read across data aluminium tris(dihydrogen phosphate) (CAS 13530-50-2) is considered not acutely toxic to fish.