Diammonium dihydrogenpyrophosphate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 13 November 2015 and 16 December 2015

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 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

The proposed source chemical (diammonium hydrogenorthophosphate) is highly soluble in water (> 10000 mg/L). In aqueous media soluble inorganic orthophosphates will dissociate to their ionic constituents; in this case ammonium and orthophosphate ions. Diammonium dihydrogenpyrophosphate will dissociate to ammonium cations and pyrophosphate anions. The pyrophosphate anions are unstable in aqueous solutions with the degree of instability varying according to pH. In distilled water they will hydrolyse slowly via abiotic mechanisms to orthophosphate. In natural waters a number of different processes can occur; abiotic hydrolysis, biotic degradation (as a result of the action of phosphatases which cleave pyrophosphates into orthophosphate subunits) and assimilation by organisms in the water. Thus the target substance (diammonium dihydrogenpyrophosphate) and the source substance (diammonium hydrogenorthophosphate) will be primarily absorbed as the same inorganic ions: ammonium and orthophosphate and are expected to behave in a similar manner under test conditions.
All (bio) transformation products of the source chemical are common to the target chemical and as such the data is considered to be adequate and reliable for use in the assessment of diammonium dihydrogenpyrophosphate for the ecotoxicity hazard assessment.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report attached.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report attached.

4. DATA MATRIX
See read-across justification report attached.

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: 17 June 2015 Date of Signature on Certificate: 24 September 2015

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Water samples were taken from the control and each test group (6.25, 12.5, 25, 50 and 100 mg/L) from fresh media and at 24 and 96 hours from old media for quantitative analysis.
- Sample storage conditions before analysis: The samples were stored frozen prior to analysis.
- Duplicate samples at 24 (fresh media), 48 (old and fresh media) and 72 hours (old media) were taken and stored frozen for further analysis if necessary

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Preliminary solubility work conducted indiciated that the test item was practically insoluble in water using traditional method of preparation e.g. ultrasonication and high shear mixing. Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.

In the definitive test, a nominal amount of test item (2200 mg) was dissolved, in duplicate, in 22 litres of test water with the aid of the propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and as a precautionary measure, any undissolved test item was removed by filtration through a 0.2 μm Sartorious Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) prior to pooling to give the 100 mg/L test concentration. A series of dilutions was made from this test solution to give further test concentrations of 6.25, 12.5, 25 and 50 mg/L.

Each solution was mixed with a flat bladed stirrer for approximately 1 minute to ensure adequate mixing and homogeneity.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): The vehicle was the test 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. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK
- Age at study initiation (mean and range, SD): No data; fish described as 'juvenile'
- Length at study initiation (length definition, mean, range and SD): No data; mean standard length at the end of definitive test: 5.2 cm (sd = 0.20)
- Weight at study initiation (mean and range, SD): No data; mean standard weight at the end of definitive test: 1.2g (sd = 0.16)
- Method of breeding: No data
- Feeding during test: None

ACCLIMATION
- Acclimation period: From 29 November 2015 to 07 December 2015
- 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 approximately 25.5 hours prior to the start of the definitive test.
- Health during acclimation (any mortality observed): There were no mortalities in the 7 days prior to the start of the test

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

140 mg/L as CaCO3

Test temperature:

13 to 15 °C

pH:

7.5 to 8.1

Dissolved oxygen:

>=9.7 mg O2/L

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal: 6.25, 12.5, 25 and 50 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 25-30 L glass vessel
- Aeration: The test vessels were aerated via narrow bore glass tubes
- Renewal rate of test solution (frequency/flow rate): Daily renewal
- No. of organisms per vessel: Seven
- No. of vessels per concentration (replicates): Seven
- No. of vessels per vehicle control (replicates): Seven
- Biomass loading rate: 0.41 g bodyweight/litre (volume in 24 hours)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.
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. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Intervals of water quality measurement: The water temperatures, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24,48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-hour old test preparations.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods


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.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 1000 mg/L
- Results used to determine the conditions for the definitive study: The results showed a single mortality at the test concentration of 100 mg/L. After approximately 46 hours exposure 1 out of 3 fish at 100 mg/L was observed to have a loss of equilibrium. Due to animal welfare implications this fish was killed and classed as mortalities for the 48 hour time point.
Based on this information test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L were selected for the definitive test.

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 (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Observations on body length and weight: The fish had a mean standard length of 5.2 cm (sd = 0.20) and a mean weight of 1.2 g (sd = 0.16) at the end of the definitive test.
- Other biological observations:
- Mortality of control: None
- Abnormal responses: Sub-lethal effects of exposure were observed at test concentrations of 50 and 100 mg/L. These responses were sitting at the bottom and increased pigmentation (see Table 4).
After approximately 72 hours exposure 2 out of 7 fish at 100 mg/L were observed to be moribund. Due to animal welfare implications (Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities for the following 72 hour observational time point.
- Effect concentrations exceeding solubility of substance in test medium: The test item preparations were observed to be clear colorless solutions throughout the test.

Sublethal observations / clinical signs:

Definitive Test

Verification of Test Concentrations

Analysis of the freshly prepared test media at 0 and 72 hours showed measured concentrations to range from 90% to 106%. There was no significant change in the measured concentrations obtained from the old or expired test media at 24 and 96 hours and therefore the results are based on nominal test concentrations only.

Mortality Data

Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 3. Analysis of the mortality data by Probit analysis using Linear Maximum-Likelihood regression 3, 6, 24, 48, 72 and 96 hours based on the nominal test concentrations gave the following results:

Time (h)	LC50
3	> 100
6	> 100
24	> 100
48	> 100
72	> 100
96	> 100

The No Observed Effect Concentration (NOEC) was 100 mg/L.

Sub-lethal Effects

Sub-lethal effects of exposure were observed at test concentrations of 50 and 100 mg/L. These responses were sitting at the bottom and increased pigmentation (see Table 4).

After approximately 72 hours exposure 2 out of 7 fish at 100 mg/L were observed to be moribund. Due to animal welfare implications (Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities for the following 72 hour observational time point.

 

Table 3: Cumulative Mortality Data in the Definitive Test

Nominal concentration (mg/L)	Cumulative Mortality (Initial Population = 7)						% Mortality
	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours
Control	0	0	0	0	0	0	0
	0	0	0	0	0	0	0
	0	0	0	0	0	0	0
	0	0	0	0	0	0	0
	0	0	0	0	0	0	0
	0	0	0	0	2*	0	29

* Fish found to be moribund, classed as mortality at 72 hours

 

Table 4: Sub-lethal effects of exposure in the definitive test

Nominal concentration (mg/L)	Sub-lethal effects	Time (Hours)
		3	6	24	48	72	96
Control	No abnormalities detected	7	7	7	7	7	7
6.25	No abnormalities detected	7	7	7	7	7	7
12.5	No abnormalities detected	7	7	7	7	7	7
25	No abnormalities detected	7	7	7	7	7	7
50	No abnormalities detected	7	7	7	-	-	-
	Increased pigmentation	-	-	-	7	7	7
100	No abnormalities detected	7	7	6	-	-	-
	Sitting at the bottom	-	-	1	-	-	-
	Increased pigmentation	-	-	-	7	5	5
	Moribund	-	-	-	-	2*	-

* Observation of sub-lethal effects recorded at approximately 72 hours, 2 fish from 100% v/v saturated solution found to be moribund, classed as mortality at 72 hours

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and based on the nominal test concentrations gave a 96-Hour LC50 of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

Methods

Following a preliminary range-finding test, fish were exposed, in groups of seven, to an aqueous solution of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/L for a period of 96 hours at a temperature of 13 to 15 °C under semi-static test conditions. The test item solution was prepared by stirring an excess (100 mg/L) of test item in test water using a prolonged stir at approximately 1500 rpm for 24 hours. As a pre caution, after the stirring period any undissolved test item was removed by filtration (0.2 ìm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100 mg/L stock solution of the test item. This stock solution was then further diluted as necessary to provide the remaining test concentrations. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results

Analysis of the freshly prepared test media at 0 and 72 hours showed measured concentrations to range from 90% to 106%. There was no significant change in the measured concentrations obtained from the old or expired test media at 24 and 96 hours and therefore the results are based on nominal test concentrations only. Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave the following results based on the nominal test concentrations:

Time Point (hours)	LC50 (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)
96	>100	100

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

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

The proposed source chemical (diammonium hydrogenorthophosphate) is highly soluble in water (> 10000 mg/L). In aqueous media soluble inorganic orthophosphates will dissociate to their ionic constituents; in this case ammonium and orthophosphate ions. Diammonium dihydrogenpyrophosphate will dissociate to ammonium cations and pyrophosphate anions. The pyrophosphate anions are unstable in aqueous solutions with the degree of instability varying according to pH. In distilled water they will hydrolyse slowly via abiotic mechanisms to orthophosphate. In natural waters a number of different processes can occur; abiotic hydrolysis, biotic degradation (as a result of the action of phosphatases which cleave pyrophosphates into orthophosphate subunits) and assimilation by organisms in the water. Thus the target substance (diammonium dihydrogenpyrophosphate) and the source substance (diammonium hydrogenorthophosphate) will be primarily absorbed as the same inorganic ions: ammonium and orthophosphate and are expected to behave in a similar manner under test conditions.
All (bio) transformation products of the source chemical are common to the target chemical and as such the data is considered to be adequate and reliable for use in the assessment of diammonium dihydrogenpyrophosphate for the ecotoxicity hazard assessment.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report attached.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report attached.

4. DATA MATRIX
See read-across justification report attached.

Reason / purpose for cross-reference:

read-across source

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Observations on body length and weight: The fish had a mean standard length of 5.2 cm (sd = 0.20) and a mean weight of 1.2 g (sd = 0.16) at the end of the definitive test.
- Other biological observations:
- Mortality of control: None
- Abnormal responses: Sub-lethal effects of exposure were observed at test concentrations of 50 and 100 mg/L. These responses were sitting at the bottom and increased pigmentation (see Table 4).
After approximately 72 hours exposure 2 out of 7 fish at 100 mg/L were observed to be moribund. Due to animal welfare implications (Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities for the following 72 hour observational time point.
- Effect concentrations exceeding solubility of substance in test medium: The test item preparations were observed to be clear colorless solutions throughout the test.

Sublethal observations / clinical signs:

Definitive Test

Verification of Test Concentrations

Analysis of the freshly prepared test media at 0 and 72 hours showed measured concentrations to range from 90% to 106%. There was no significant change in the measured concentrations obtained from the old or expired test media at 24 and 96 hours and therefore the results are based on nominal test concentrations only.

Mortality Data

Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 3. Analysis of the mortality data by Probit analysis using Linear Maximum-Likelihood regression 3, 6, 24, 48, 72 and 96 hours based on the nominal test concentrations gave the following results:

Time (h)	LC50
3	> 100
6	> 100
24	> 100
48	> 100
72	> 100
96	> 100

The No Observed Effect Concentration (NOEC) was 100 mg/L.

Sub-lethal Effects

Sub-lethal effects of exposure were observed at test concentrations of 50 and 100 mg/L. These responses were sitting at the bottom and increased pigmentation (see Table 4).

After approximately 72 hours exposure 2 out of 7 fish at 100 mg/L were observed to be moribund. Due to animal welfare implications (Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities for the following 72 hour observational time point.

 

Table 3: Cumulative Mortality Data in the Definitive Test

Nominal concentration (mg/L)	Cumulative Mortality (Initial Population = 7)						% Mortality
	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours
Control	0	0	0	0	0	0	0
	0	0	0	0	0	0	0
	0	0	0	0	0	0	0
	0	0	0	0	0	0	0
	0	0	0	0	0	0	0
	0	0	0	0	2*	0	29

* Fish found to be moribund, classed as mortality at 72 hours

 

Table 4: Sub-lethal effects of exposure in the definitive test

Nominal concentration (mg/L)	Sub-lethal effects	Time (Hours)
		3	6	24	48	72	96
Control	No abnormalities detected	7	7	7	7	7	7
6.25	No abnormalities detected	7	7	7	7	7	7
12.5	No abnormalities detected	7	7	7	7	7	7
25	No abnormalities detected	7	7	7	7	7	7
50	No abnormalities detected	7	7	7	-	-	-
	Increased pigmentation	-	-	-	7	7	7
100	No abnormalities detected	7	7	6	-	-	-
	Sitting at the bottom	-	-	1	-	-	-
	Increased pigmentation	-	-	-	7	5	5
	Moribund	-	-	-	-	2*	-

* Observation of sub-lethal effects recorded at approximately 72 hours, 2 fish from 100% v/v saturated solution found to be moribund, classed as mortality at 72 hours

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and based on the nominal test concentrations gave a 96-Hour LC50 of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

Methods

Following a preliminary range-finding test, fish were exposed, in groups of seven, to an aqueous solution of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/L for a period of 96 hours at a temperature of 13 to 15 °C under semi-static test conditions. The test item solution was prepared by stirring an excess (100 mg/L) of test item in test water using a prolonged stir at approximately 1500 rpm for 24 hours. As a pre caution, after the stirring period any undissolved test item was removed by filtration (0.2 ìm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100 mg/L stock solution of the test item. This stock solution was then further diluted as necessary to provide the remaining test concentrations. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results

Analysis of the freshly prepared test media at 0 and 72 hours showed measured concentrations to range from 90% to 106%. There was no significant change in the measured concentrations obtained from the old or expired test media at 24 and 96 hours and therefore the results are based on nominal test concentrations only. Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave the following results based on the nominal test concentrations:

Time Point (hours)	LC50 (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)
96	>100	100

Description of key information

One key study exists on an analogous substance. This study had been conducted in accordance with a recommended guideline (OECD 203, EU Method C.1) and under the conditions of GLP. As such, no further testing is considered necessary

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

> 100 mg/L