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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
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
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

(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric/orthophosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74-/PO43- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-/PO43- anion.
(3) Potassium and Sodium cations are essential micronutrients that are ubiquitous in the environment. As such, their uptake is tightly regulated and is therefore not considered to pose a risk for ecotoxicity. The pyrophosphate anion is unstable in aqueous solutions with the degree of instability varying according to pH. In distilled water pyrophosphates will hydrolyse slowly via abiotic mechanisms to inorganic phosphate. In natural waters a number of different processes can occur; abiotic hydrolysis, biotic degradation (as a result of the action of phosphatases which cleave pyrophosphate into orthophosphate subunits) and assimilation by organisms in the water all resulting in an ultimate breakdown product of orthophosphate. It is therefore deemed scientifically justified to avoid any further vertebrate testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for hazard assessment purposes.

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:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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 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:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality

 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.

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.

Description of key information

One key study to assess the short-term toxicity of disodium dihydrogenpyrophosphate exists, this study is conducted on an analogous substance (see justification below). On this basis sodium and potassium pyrophosphates are not considered to be toxic to fish. This conclusion is supported by the additional literature provided and no further testing is justified. In addition, this read-across value is considered to be conservative (in comparison to the additional data provided) for the purposes of hazard assessment. 

Key value for chemical safety assessment

LC50 for freshwater fish:
100 mg/L

Additional information

Rationale for read across:

 

In accordance with Annex XI, section 1.5 of Regulation (EC) No 1907/2006 (REACH) a read across approach may be used when substances have similarities based on the likelihood of common breakdown products via physical and biological processes, which result in structurally similar chemicals.

 

Sodium and potassium pyrophosphates are ionic in nature and therefore dissociate readily into cations and anions in water the toxicity of the both the cation and the anion must be addressed. Potassium and sodium cations are essential micronutrients that are ubiquitous in the environment. As such, their uptake is tightly regulated and is therefore not considered to pose a risk for ecotoxicity. The pyrophosphate anion is unstable in aqueous solutions with the degree of instability varying according to pH. In distilled water pyrophosphates will hydrolyse slowly via abiotic mechanisms to inorganic phosphate. In natural waters a number of different processes can occur; abiotic hydrolysis, biotic degradation (as a result of the action of phosphatases which cleave pyrophosphate into orthophosphate subunits) and assimilation by organisms in the water all resulting in an ultimate breakdown product of orthophosphate.

It is therefore deemed scientifically justified to avoid any further vertebrate testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for hazard assessment purposes.

The substance tested is essentially a mixture of two potassium orthophosphates: potassium dihydrogenorthophosphate and dipotassium hydrogenorthophosphate.

The substance has the following properties:

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: 66922-99-4 

Molecular weight: 346.29

Melting range: No complete melting up to 573 K

Solubility in water ( g/L, at 20 °C): 705 (pH 7)

 

 

It is therefore considered appropriate; due to the physicochemical nature of the substance tested and its identity, for this data to be used for read-across purposes for the following substances:

 - disodium dihydrogenpyrophosphate

 - trisodium hydrogen diphosphate

 - tetrasodium pyrophosphate

 - tetrapotassium pyrophosphate

Any further testing would not be scientifically justified as all substances would ultimately dissociate to their anionic and cationic forms in natural waters and these ions (Na+, K+ and PO43-(from P2O74-) are all ubiquitous and are not considered to pose a risk of ecotoxicity.