Diammonium phthalate

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 06 December 2016 and 08 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Diammonium phthalate

EC Number:

208-341-9

EC Name:

Diammonium phthalate

Cas Number:

523-24-0

Molecular formula:

C8H6O4.2H3N

IUPAC Name:

diammonium phthalate

Test material form:

solid: crystalline

Specific details on test material used for the study:

Identification: Diammonium phthalate
Physical state/Appearance: White crystalline powder
Batch: 7172002
Purity: 99.3%
Expiry Date: 01 August 2017
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Range-finding test:
A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

Main test:
Samples were taken from the control and the 100 mg/L test group from the freshly prepared bulk test preparation at 0 and 24 hours and from the old or expired pooled replicates at 24 and 48 hours for quantitative analysis. All 0 and 24 hours samples were stored frozen prior to analysis with the 48 hours samples at the test termination.
Duplicate samples were taken and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

Range-finding test:
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L.
A nominal amount of test item (50 mg) was dissolved in test water and the volume adjusted to 500 mL to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 0.10, 1.0 and 10 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

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/EC Test Guidelines no immobilization or adverse reactions to exposure were observed.
A nominal amount of test item (100 mg) was dissolved in test water and the volume adjusted to 1 liter to give the 100 mg/L test concentration.
The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room maintaining the water temperature at 18 to 22 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Test temperature:

19 °C to 21 °C

pH:

7.4 -7.9

Dissolved oxygen:

5.6 - 9.7 mg O2/L

Nominal and measured concentrations:

Nominal concentrations:
Defintive test: 100 mg/L

Measured concentration:
Definitive test: The geometric mean measured test concentration was determined to be 90 mg/L.

Details on test conditions:

Experimental Design and Study Conduct
Range-finding Test
In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded. The control was maintained under identical conditions but not exposed to the test item.

Definitive Test
Exposure Conditions
As in the range-finding test 150 mL glass beakers containing approximately 100 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 maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (between 200 and 1200 Lux) 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.
Semi-static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media.

Assessments
Test Organism Observations
Any immobilization 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 immobilized if they were unable to swim within 15 seconds after gentle agitation.

Water Quality Criteria
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24-Hour old test preparations. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
The light intensity during the light period was measured using an ATP Instrumentation Lux meter.
The appearance of the test media was recorded daily (pre and post media change, where applicable).

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 90 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

> 90 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range-finding Test:
No immobilization was observed at the test concentrations of 0.10, 1.0, 10 and 100 mg/L.
Based on this information, a single test concentration of four replicates, 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/EC Test Guidelines, no immobilization or adverse reactions to exposure were observed.
Chemical analysis of the 100 mg/L test preparation at 0 hours showed a measured test concentration of 97% of nominal. There was a significant decline in the measured concentration at 48 hours to 50% of nominal, indicating that the test item was unstable under test conditions.

Definitive Test:

Verification of Test Concentrations
Analysis of the freshly prepared test samples at 0 and 24 hours showed measured test concentrations of 98% of nominal were obtained. A slight decline in measured concentrations was observed in the old or expired test samples at 24 and 48 hours to 92% and 75% of nominal, respectively.
Given this decline in measured concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentration in order to give a worst case analysis of the data.
The geometric mean measured test concentration was determined to be 90 mg/L.

Immobilization Data
Exposure of Daphnia magna to the test item gave EC50 values of greater than 90 mg/L. The No Observed Effect Concentration was 90 mg/L.
It was considered unnecessary and unrealistic to test at concentrations in excess of 90 mg/L.

Sub-Lethal Effects
No sub-lethal effects of exposure were observed throughout the test.

Validation Criteria
The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

Water Quality Criteria
Temperature was maintained at 19 °C to 21 °C throughout the test, while there were no treatment related differences for pH, concentration dependant differences in the oxygen concentration were observed at 48 hours.
Throughout the test the light intensity was observed to be in the range 525 to 558 Lux.

Observations on Test Item Solubility
At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Exposure conditions for the positive control were similar to those in the definitive test.

Analysis of the immobilization data by the probit analysis using the linear maximum likelihood regression method at 24 and 48 hours using the ToxRat Professional computer software package based on the nominal test concentrations gave the following results:

24 Hour:
EC50: 1.3 mg/L
NOEC: 1.0 mg/L
LOEC: 1.8 mg/L

48 Hours:
EC50: 1.2 mg/L
NOEC: 0.56 mg/L
LOEC: 1.0 mg/L

The No Observed Effect Concentration is based upon equal to or less than 10% immobilization at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item.

Any other information on results incl. tables

Cumulative Immobilization Data and Observations in the Range-finding Test

Nominal Concentration (mg/L)	Observations (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
	Cumulative Immobilized Daphnia	Cumulative Immobilized Daphnia
Control	0	0
0.10	0	0
1.0	0	0
10	0	0
100	0	0

Cumulative Immobilization Data and Observations in the Definitive Test

Nominal Concentration (mg/L)	24 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
100	0	0	0	0	0	0	5 N	5 N	5 N	5 N

 

Nominal Concentration (mg/L)	48 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
100	0	0	0	0	0	0	5 N	5 N	5 N	5 N

R₁– R₄= Replicates 1 to 4

N = No sub-lethal effects observed

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and based on the geometric mean measured test concentration gave a 48-Hour EC50 value of greater than 90 mg/L. The No Observed Effect Concentration was 90 mg/L.

Executive summary:

 Introduction

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a nominal concentration of 100 mg/L for
48 hours at a temperature of 19°C to 21°C under semi-static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results

Analysis of the freshly prepared test samples at 0 and 24 hours showed measured test concentrations of 98% of nominal were obtained. A slight decline in measured concentrations was observed in the old or expired test samples at 24 and 48 hours to 92% and 75% of nominal, respectively.

Given this decline in measured concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentration in order to give a worst case analysis of the data.

Exposure of Daphnia magna to the test item gave EC₅₀ values of greater than 90 mg/L. The No Observed Effect Concentration was 90 mg/L.