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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
24 MARCH 2010 AND 17 MAY 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

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. 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.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of GLP inspection: 15/09/2009 Date of Signature on GLP certificate: 26/11/2009
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 (pH 7)
Analytical monitoring:
yes
Details on sampling:
- Sampling method:
Water samples were taken from the control (replicates R1 – R4 pooled) and the 100 mg/l test group (replicates R1 – R2 and R3 – R4 pooled) at 0 and 48 hours for quantitative analysis.

- Sample storage conditions before analysis:
Duplicate samples 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 Appendix 1 (attached).
Vehicle:
no
Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method:
A positive control (Harlan Laboratories Ltd Project No: 0039/1133) conducted approximately every six months used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.
An amount of reference item (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give a 100 mg/l stock solution. An aliquot (50 ml) of this stock solution was diluted in reconstituted water and the volume adjusted to 500 ml to give a 10 mg/l stock solution. Aliquots (16, 28, 50, 90 and 160 ml) of the 10 mg/l stock solution were each separately dispersed in a final volume of 500 ml of reconstituted water to give the test series of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.
Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Exposure conditions for the positive control were similar to those used in the definitive test.

The temperature was maintained at approximately 20°C.

Evaluation of data for the positive control

The EC50 value and associated confidence limits at 24 hours and the slope of the response curve and standard error were calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999). The EC50 value and associated confidence limits at 48 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al 1977) using the ToxCalc computer software package (ToxCalc 1999).
Probit analysis is used where two or more partial responses to exposure are shown.
When only one partial response is shown the trimmed Spearman-Karber method is appropriate.

Test organisms (species):
Daphnia magna
Details on test organisms:

TEST ORGANISM
- Common name:
Water Flea

- Source:
Derived from in-house laboratory cultures.

- Age at study initiation:
Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old

- Feeding during test:
Received no food during exposure

ACCLIMATION
- Acclimation period:
Not stated


- Acclimation conditions:
Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 20 degC.


- Type and amount of food:
Each culture was fed daily with a suspension of algae (Chlorella sp.).
The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

- Health during acclimation:
No mortality observed


Test Water:
The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.


Reconstituted Water
i) Stock Solutions
a) CaCl2.2H2O 11.76 g/l
b) MgSO4.7H2O 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l
ii) Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.
Test temperature:
Temperature was maintained at 20°C throughout the test.
The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
pH:
The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl.
The pH was measured using a WTW pH/Oxi 340I pH meter.
There were no treatment related differences for pH.
See Appendix 2 for results.
Dissolved oxygen:
The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.
Dissolved oxygen concentrations were recorded at the start and termination of the test. The dissolved oxygen concentration was measured using a dissolved oxygen meter.
See Appendix 2 for results.
Salinity:
not applicable
Nominal and measured concentrations:
The test concentration to be used in the definitive test was determined by a preliminary range-finding test.
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.010, 0.10, 1.0, 10 and 100 mg/l. The test item was dissolved directly in water.
An amount of test item (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give the 100 mg/l test concentration. Serial dilutions from this were then performed to give further test concentrations of 10, 1.0, 0.10 and 0.010 mg/l.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
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 immobilisation or adverse reactions to exposure were observed.

Details on test conditions:

TEST SYSTEM
- Test vessel:
As in the range-finding test 250 ml glass jars containing approximately 200 ml of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at approximately 20C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were not renewed during the exposure period. Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

- Type (delete if not applicable):
closed

- Material, size, headspace, fill volume:
The test vessels were then sealed, with minimal headspace to prevent losses of the test material due to its volatile nature,

- Aeration:
None.

- Type of flow-through (e.g. peristaltic or proportional diluter):
None.

- Renewal rate of test solution (frequency/flow rate):
None.

- No. of organisms per vessel:
10 daphnids were placed in each test and control vessel at random, in the test preparations

- No. of vessels per concentration (replicates):
Duplicate test vessels were used for each test and control group.

- No. of vessels per control (replicates):
Duplicate test vessels were used for each test and control group.

- No. of vessels per vehicle control (replicates):
Duplicate test vessels were used for each test and control group.

- Biomass loading rate:
Not recorded.

- Source/preparation of dilution water:

Appendix 2 Reconstituted Water
i) Stock Solutions
a) CaCl2.2H2O 11.76 g/l
b) MgSO4.7H2O 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l
ii) Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

- Total organic carbon:
Not recorded

- Particulate matter:
Not recorded

- Metals:
Not recorded

- Pesticides:
Not recorded

- Chlorine:
Not recorded

- Alkalinity:
Not recorded

- Ca/mg ratio:
Not recorded

- Conductivity:
Not recorded

- Culture medium different from test medium:
The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.

- Intervals of water quality measurement:
Not recorded

OTHER TEST CONDITIONS

- Adjustment of pH:
The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.

- Photoperiod:
a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.

- Light intensity:
Not recorded

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

TEST CONCENTRATIONS

- Spacing factor for test concentrations:
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 immobilisation or adverse reactions to exposure were observed.

- Justification for using less concentrations than requested by guideline:
Not applicable

RANGE FINDING STUDY

- Test concentrations:

test concentrations of 0.010, 0.10, 1.0, 10 and 100 mg/l.

Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:

- Behavioural abnormalities: Not recorded
- Other biological observations: None recorded

- Immobilisation of control:
Cumulative immobilisation data from the exposure of Daphnia magna to the reference material (Safepharm Laboratories Project No: 0039/0977) during the positive control are given in the following table. The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 3 and 4 (see attached background material).

Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the trimmed Spearman-Karber method (Hamilton et al 1977) at 24 hours and the probit method (Finney 1971) at 48 hours based on the nominal test concentrations gave the following results:

Time (h) EL50 (mg/l loading rate WAF) 95% Confidence limits
(mg/l loading rate WAF)
3 >3.2* -
24 0.97 0.85 - 1.1
48 0.70 0.62 - 0.80

The No Observed Effect Concentrations after 24 and 48 hours were 0.56 and 0.32 mg/l respectively. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
* A 3-Hour EC50 value was not calculated as less than 50% immobilisation occurred at this time point


- Other adverse effects control: No other effects observed.

- Abnormal responses: None recorded

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None recorded

- Effect concentrations exceeding solubility of substance in test medium: Observations on the test media were carried out during the mixing and testing of the WAFs.
At both the start and end of the mixing period and following the 1-Hour standing period WAFs were observed to have formed clear colourless water columns with an oily slick of test material at the water surface. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test material to be present.
Throughout the duration of the test all loading rates were observed to be clear, colourless solutions.
Results with reference substance (positive control):

- Results with reference substance valid?
Yes

- Mortality:
No mortalities recorded.

- EC50/LC50:

Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2.
There was no immobilisation in 20 daphnids exposed to a test concentration of 100 mg/l for a period of 48 hours. Inspection of the immobilisation data gave the following results:
Time (h) EC50 (mg/l)
24 > 100
48 > 100
The No Observed Effect Concentration after 24 and 48 hours exposure was 100 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

DEFINITIVE TEST

Table 1.  Cumulative Immobilisation Data in the Range-finding Test

Nominal
Concentration
(mg/l)

Cumulative ImmobilisedDaphnia
(Initial Population: 10 Per Replicate)

24 Hours

48 Hours

Control

0

0

0.010

0

0

0.10

0

0

1.0

0

0

10

0

0

100

0

0

 


Table 2. Cumulative Immobilisation Data in the Definitive Test

Nominal
Concentration
(mg/l)

Cumulative Immobilised Daphnia
(Initial Population: 5 Per Replicate)

24 Hours

48 Hours

No. Per

Replicate

Total

%

No. Per

Replicate

Total

%

Control

R1

0

0

0

0

0

0

R2

0

0

R3

0

0

R4

0

0

100

R1

0

0

0

0

0

0

R2

0

0

R3

0

0

R4

0

0

 

 


Table 3. Cumulative Immobilisation Data in the Positive Control

Nominal
Concentration
(mg/l)

Cumulative ImmobilisedDaphnia
(Initial Population: 10 Per Replicate)

24 Hours

48 Hours

R1

R2

Total

%

R1

R2

Total

%

Control

0

0

0

0

0

0

0

0

0.32

0

0

0

0

0

0

0

0

0.56

1

1

2

10

3

2

5

25

1.0

7

7

14

70

10

10

20

100

1.8

10

10

20

100

10

10

20

100

3.2

10

10

20

100

10

10

20

100

 


R1– R4= Replicates 1 to 4

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 of greater than 100 mg/l. Correspondingly the No Observed Effect Concentration was 100 mg/l.

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 aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
06-05-2003 to 08-02-2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

As aluminium orthophosphate will dissociate in their ionic forms in water (Al3+ and PO43- ions which will further associate with the ionic forms of H2O) it is justified to assess the toxicity of the Al3+ ion and the PO43- ion as separate entities. 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 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 extrapolation of the results on aluminium sulphate can be done based on the aluminium content and relative molecular weights. Calculations have been based on aluminium orthophosphate (EC No. 236-875-2) on the basis that this substance contains the greatest amount of aluminium (%w/w) in comparison to the following aluminium phosphates: - aluminium tris(dihydrogen phosphate) (EC No. 236-875-2) - sodium aluminium phosphate (EC No. 232-090-4) The water solubility of aluminium sulphate-14-hydrate is stated as being approximately 600 g/L, therefore as aluminium orthophosphate has a solubility of approximately 6.92 x 10-3 g/L (aluminium tris(dihydrogen phosphate) and sodium aluminium phosphate are closer in solubility to aluminium sulphate-14-hydrate; 522 g/L and 294 g/L at 20±0.5°C respectively) the results of an extrapolation to aluminium orthophosphate would give the worst case in terms of aluminium content for read across and solubility as all substance are less soluble and hence less bioavailable than aluminium sulphate-14-hydrate.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
(reconstitution water according to the OECD 203 guideline)
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Water solubility: very soluble, approx. 600g/L
Vapour pressure: not applicable
Specific density (20 °C): approx. 1.7 g/cm³
Analytical monitoring:
yes
Details on sampling:
Water phase samples were taken from the freshly prepared test solutions before filling the individual beakers and from old media after decanting and pooling the water per concentration by using a plastic syringe and transferred into disposable 20 mLpolyethylene vials. One subsample of 20 mL was acidified with 0.2 mL 14 M nitric acid, thoroughly mixed and analysed 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):
Daphnia magna
Details on test organisms:
Justification for the use of the test organism:
Daphnia magna (Crustacea, Phyllopoda, Cladocera) was chosen by OECD-experts (ref 1) as test organism representing aquatic invertebrates.

Specification:
Species: Daphnia magna STRAUS, Crustacea, Cladocera (clone V).
Age: 4 - 24 hours old.
Origin: Umweltbundesamt, Institut für Wasser-, Boden- und Lufthygiene, bred in the laboratory of the Fraunhofer-IME.

Breeding and holding conditions:
Adult Daphnia, at least 3 weeks old, were separated from the stock population by sieving. Batches of 30 to 50 animals were held at room temperature in ca. 1800 mL purified drinking water. During the week the daphnids are fed daily with an algal suspension (Scenedesmus subspicatus) and LiquizellR (HOBBY) according to the EEC Guideline. Algae growing in the log phase, are centrifuged and the pellet is suspended in a few mL of medium. 30 mL of this suspension is given to 1 L Daphnia
medium. The water was changed once per week. Newborn daphnids were separated by sieving, the first generation was discarded.

Sensitivity
The sensitivity of the test clone was checked by using K2Cr2O7 as reference substance. Between 04.04.2003 and 13.05.2003 the EC50 ranged from 1.0 to 1.3 mg/L.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
20.0- 20.1°C
pH:
7.5-8.0
Dissolved oxygen:
94-100% saturation
Nominal and measured concentrations:
nominal: 10, 20, 40, 80, 160 mg/L
nominal Al: 0.9, 1.8, 3.6, 7.2, 14.4 mg/L
measured Al: 1.11, 1.89, 4.02, 7.38, 14.1 mg/L (geometric mean)
measured Al (dissolved):0.172, 0.176, 0.166, 0.107, 0.115 mg/L (geometric mean)
Details on test conditions:
Daphnids (Daphnia magna) were exposed to 5 concentrations of the test item in 4 replicates under semi-static conditions for a period of 48 hours. 60 mL glass beakers were used as test vessels. To each beaker 50 mL test solution and 5 daphnids, not older than 24 hours, were added. They were transferred to freshly prepared test liquids in new test vessels after 24 h. No feeding and no aeration occurred throughout the test. The controls were kept under the same conditions in reconstituted water.
Immobility and abnormal behaviour were recorded after 24 h and 48 h. Immobile animals were eliminated from the vessels as soon as they were discovered. The daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of the test vessels. The temperature during the test was 20.0 ± 0.1 °C. The beakers were covered with glass panes and subjected to a light/dark cycle of 16/8 h with light intensities of less than 1000 Lux. In the freshly prepared test liquids at test start and after 24 h (before adding the daphnids), and in the old liquids after 24 h and at test end pH-values (WTW Microprocessor pH-Meter pH 196) and oxygen concentrations (WTW Microprocessor Oximeter OXI 196) of the decanted and pooled test solutions and control water were measured.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
> 160 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Results with reference substance (positive control):
EC50 ranged from 1.0 to 1.3 mg/L.

Measured concentrations appeared to be very pH dependent and reached maximum solubility. Therefore the nominal and measured (dissolved) concentration) are very different. The result is that the NOEC exceeds the water solubility at the tested pH.

Validity criteria fulfilled:
yes
Conclusions:
No signs of immobility nor other signs of intoxication were observed during the 48h test. The conclusion is that the NOEC >= 160 mg/L nominal and the NOEC exceeds the water solubility at the tested pH levels.

Description of key information

As Aluminium orthophosphate will dissociate into its ionic forms in water (Al3+and PO43-ions which will further associate with the ionic forms of H2O, see hydrolysis endpoint for further information) it is justified to assess the toxicity of the aluminium ion and the phosphate ion as separate entities. Therefore data are presented on analogous substances aluminium sulphate -14-hydrate (source of Al ions) and tripotassium trihydrogen diphosphate dihydrate (source of PO43- ions) with a view to determining the relative toxicity of the test material.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
65.45 mg/L

Additional information

The toxicity of aluminium orthophosphate is assessed on the basis of the phosphate cation and the aluminium anion separately.

The phosphate anion (PO43 -) is ubiquitous in natural waters and an essential micronutrient for many organisms. Orthophosphates are not anticipated to induce direct acute or long-term adverse effects on aqueous or terrestrial organisms. Because phosphates are a key nutrient for plants, elevated phosphate concentrations in water can lead to excessive growth of algae and other water plants (eutrophication). In many surface waters, algal blooms can have considerable detrimental impacts on fish and other organisms as the increase in primary production leads to increased oxygen consumption, which may reduce the oxygen concentration to critical low levels. However this is not considered to be a toxic effect of the phosphate ion itself. To avoid such undesirable effects, phosphate emissions to surface water via industrial wastewater are regulated in the Council Directive 96/61/EC concerning integrated pollution prevention and control. One of the main ways of reducing phosphorus emissions to water is via the addition of aluminium (or iron) to precipitate the phosphorus out of solution.

The aluminium ion is known to be toxic to aquatic organisms 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 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 extrapolation of the results on aluminium sulphate can be done based on the aluminium content and relative molecular weights. Calculations have been based on the analogous substance aluminium orthophosphate (EC No. 236-875-2) on the basis that this substance contains the greatest amount of aluminium (%w/w) in comparison to the following aluminium phosphates:

-         aluminium tris(dihydrogen phosphate) (EC No. 236-875-2)

-         sodium aluminium phosphate (EC No. 232-090-4)

The water solubility of aluminium sulphate-14-hydrate is stated as being approximately 600 g/L, therefore as aluminium orthophosphate has a solubility of approximately 6.92 x 10-3 g/L (aluminium tris(dihydrogen phosphate) and sodium aluminium phosphate are closer in solubility to aluminium sulphate-14-hydrate; 522 g/L and 294 g/L at 20±0.5°C respectively) the results of an extrapolation to aluminium orthophosphate would give the worst case in terms of aluminium content for read across and solubility as all substance are less soluble and hence less bioavailable than aluminium sulphate-14-hydrate.

Two studies are available for aluminium sulphate-14-hydrate One study was conducted using a static test system without pH adjustment. As the test material concentrations increased the pH of the test media decreased resulting in an increased solubility of the test material and an increase in the toxicity to daphina. It was therefore necessary to perform an additional study to assess the toxicity to daphnia on the basis of the intrinsic toxicity of the test material by using a semi-static test system and maintaining the pH at 7.5 as the effects of the test material on pH would not be so significant in natural waters (depending on the location, initial pH of the watercourse, concentration of the test material amongst other factors). It is therefore considered that the results of this second study are more reliable for the purpose of REACH.

The CoA presented in the study reports state that the analytical content of aluminium in aluminium sulphate-14-hydrate tested was ca. 9%. A molecular weight calculation would give around 10%). Therefore, it is considered appropriate to perform the calculation based on molecular weights.

The molecular weight of aluminium orthophosphate is 122 and the molecular weight of aluminium sulphate-14-hydrate is 246. Therefore, in the study performed on aluminium sulphate-14-hydrate the EC50 was reported as >160 mg test substance/L which would equate to an EC50 for Al of approximately 14.4 mg/L. Therefore, based on molecular weight, aluminium orthophosphate can be considered to contain 22% w/w of Al (therefore 65.45 mg aluminium orthophosphate would contain 14.4 mg/L. On this basis, the extrapolated EC50 for aluminium phosphate would be >65.45 mg test material / L. As no immobility was noted at this level this data alone would not trigger a classification .

In addition, the EC50 for a potassium orthophosphate (see endpoint record) was derived to be >100 mg/L and as such phosphates are not considered to be classified.

For the purpose of a worst-case assessment of toxicity to daphnia the value of >65.45 mg/L will be used when considering the most sensitive species and hence the lowest value for the derivation of PNECS, however it should not be considered as the value at which immobilization and hence toxicity occurs.