phosphonic acid, [2-(4-aminophenyl)-1-hydroxyethylidene]bis-, monosodium salt

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 05 December 2018 and 02 February 2019.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

There was no effect on validity of the study

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

yes

Remarks:

There was no effect on validity of the study

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

phosphonic acid, [2-(4-aminophenyl)-1-hydroxyethylidene]bis-, monosodium salt

EC Number:

828-215-2

Cas Number:

172796-84-8

Molecular formula:

C8H12NO7P2Na

IUPAC Name:

phosphonic acid, [2-(4-aminophenyl)-1-hydroxyethylidene]bis-, monosodium salt

impurity 1

Reference substance name:

4-aminophenylacetic acid

EC Number:

214-828-7

EC Name:

4-aminophenylacetic acid

Cas Number:

1197-55-3

Molecular formula:

C8H9NO2

IUPAC Name:

4-aminophenylacetic acid

impurity 2

Reference substance name:

EBP dimer

Molecular formula:

C16H22N2O12P4

IUPAC Name:

EBP dimer

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Identification: Phosphonic acid, [2-(4-aminophenyl)-1-hydroxyethylidene]bis-,monosodium salt (“EBP”)
CAS Number: 172796-84-8
Batch: CHPC071917EBP
Purity: >98% pure
Physical State/Appearance: Beige colored powder
Expiry Date: 01 July 2019
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Remarks:

high performance liquid chromatography

Details on sampling:

Samples were taken from the control and the 100 mg/L test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for immediate quantitative analysis. Duplicate sets of samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

Preliminary Media Preparation Trial
Preliminary solubility work indicated that the test item was soluble in reverse osmosis (R.O.) water at 300 mg/L with the aid of ultrasonication but solubility testing in Elendt M7 media showed the test item was not fully soluble using traditional methods of preparation such as ultrasonication and high shear mixing. Further trials to obtain a suitable preparation in Elendt M7 media were then conducted as part of this study.
As the test item was soluble in R.O. water it was decided to investigate whether an initial high concentration stock solution could be made in R.O. water and then diluted into range in Elendt M7 to give the test solutions. A solubility test was also conducted in order to determine the preparation method with and without pH adjustment. The trial was conducted by making two 1000 mg/L stock solutions in R.O. water. One stock solution was sonicated for 15 minutes then the pH was adjusted from 5.5 to 7.6 with 1M NaOH before sonication for a further 5 minutes. The other stock solution was sonicated for 15 minutes and shaken by hand but the pH was not adjusted, giving a pH of 4.68 in the final solution. A clear yellow solution was obtained for both stock solutions. The stock solutions were then both further diluted in Elendt M7 test media to make 100 mg/L solutions. Both 100 mg/L solutions were clear and very pale yellow in color. The pH adjusted stock had a final pH of 6.94 once diluted and the unadjusted stock had a final pH of 6.32 once diluted. Both 100 mg/L solutions had a final pH within the range of 6-9.
From this trial it was decided that for the Daphnia test, preparation of the test item as a 1000 mg/L stock solution in R.O. water without pH adjustment would be a suitable method. It was decided not to adjust the pH of the stock solution as the final solution at 100 mg/L was within an acceptable pH range. The 100 mg/L test concentration would then be diluted down to make the full concentration range. In order to compensate for any reduction of water hardness by using a stock solution made in R.O. water, the control was treated in a similar manner as the 100 mg/L stock solution by adding the same amount of R.O. water to the control media as the volume of stock solution used to make up the 100 mg/L test solution.

Range-finding Test
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 0.10, 1.0, 10 and 100 mg/L.
A nominal amount of test item (500 mg) was used to make a 1000 mg/L stock preparation in R.O. water, which was dissolved with aid of ultrasonication for approximately 20 minutes and shaking by hand. A series of dilutions was made from the 1000 mg/L stock preparation into Elendt M7 test medium to give the test concentrations of 0.10, 1.0, 10 and 100 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 (500 mg) was dissolved in R.O. water with the aid of ultrasonication for approximately 15 minutes and shaking by hand. The volume was adjusted to 500 mL to give the 1000 mg/L stock preparation from which a 1:10 dilution was made in Elendt M7 test medium to produce the test concentration of 100 mg/L.
The test preparation 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 first instar Daphnia magna derived from in house laboratory cultures.
Adult daphnids were maintained in 150 mL glass vessels containing 100 mL 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 GM300 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:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Test temperature:

18 to 22 °C with a maximum deviation of ± 1 °C

pH:

6.4-7.9

Dissolved oxygen:

8.6-9.3 mg O2/L

Nominal and measured concentrations:

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.

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.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations of 87 mg/L and 80 mg/L, respectively; therefore test results were based on the nominal test concentration.

Details on test conditions:

Range-finding Test
The test concentration to be used in the definitive test was determined by a preliminary range finding test.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
In the range finding test five 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. Two replicate test and control vessels were prepared. 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 numbers of immobilized daphnids were recorded.
The control group was maintained under identical conditions but not exposed to the test item. The control media was prepared with 50 mL R.O. water in 500 mL Elendt M7, giving the same proportion of R.O. water to Elendt M7 that was used in the 100 mg/L stock solution. This was carried out to ensure comparability of the media between control and test groups and confirm that the use of R.O water in the initial stock solution would not cause any adverse effects to the Daphnia.
A sample of each test concentration was taken for immediate chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. A duplicate set of samples was taken on each sampling occasion and stored frozen for further analysis if required.

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 (500 mg) was dissolved in R.O. water with the aid of ultrasonication for approximately 15 minutes and shaking by hand. The volume was adjusted to 500 mL to give the 1000 mg/L stock preparation from which a 1:10 dilution was made in Elendt M7 test medium to produce the test concentration of 100 mg/L.
The test preparation was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparation was verified by chemical analysis at 0 and 48 hours.

Exposure Conditions
As in the range finding test 150 mL glass vessels containing approximately 100 mL of test preparation were used. At the start of the test, five daphnids were placed at random into each test and control vessel containing 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. The room was set to 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 media were not aerated.
The control group was maintained under identical conditions but not exposed to the test item. The control group consisted of Elendt M7 media without a proportion of R.O. water added, as the range finding test showed that the proportion of R.O. water used did not have any adverse effects.
The test preparations were not renewed during the exposure period.

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
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. 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.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range finding Test
No immobilization or sub lethal effects of exposure were observed throughout the test.
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 test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) to 85% of nominal concentrations; although procedural recoveries were below acceptable limits (at 66% of nominal) for the 48 hours analysis this was considered acceptable for range finding purposes. The measured concentrations at 0.1 to 10 mg/L were below 80% of nominal whereas the 100 mg/L and 1000 mg/L (stock) the preparations were close to nominal (85 and 94% of nominal). Since there was no immobilization at 100 mg/L and the measured concentration was close to nominal it was considered that the range finding analysis gave enough information to identify the definitive test conditions. Furthermore, there was no significant change in the measured concentrations for 100 mg/L at 48 hours indicating that the test item was stable under test conditions.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 and 48 hours showed measured test concentrations of 87 mg/L and 80 mg/L, respectively; therefore test results were based on the nominal test concentration.

Immobilization Data
There was no immobilization in 20 daphnids exposed to a test concentration of 100 mg/L for a period of 48 hours. Inspection of the immobilization data gave the following results:
Time (Hour) EC50 (mg/L) 95% Confidence limits(mg/L)
24 >100 Not determined
48 >100 Not determined

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 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 20 °C to 21 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
Throughout the test the light intensity was observed to be in the range 874 to 965 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 (Envigo study number KC41PP) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L. The positive control was conducted from 25 September 2018 to 27 September 2018.
Exposure conditions for the positive control were similar to those in the definitive test with an exception for the light intensity. In the definitive test at 0 and 48 hours, the light intensity measured was in excess of the range specified in the study plan (500 to 1000 Lux), the maximum light intensity recorded was 1037 Lux. Given that the recorded light intensity was only marginally in excess of the specified range and as the daphnids in the control group did not show any mortalities or sub lethal effects, this deviation was considered not to have adversely affected the results of the test.
Analysis of the immobilization data was carried out using the Weibull analysis using linear maximum-likelihood regression at 24 and 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and gave the following results:
Time Point(Hours) EC50(mg/L) 95% Confidence Limits (mg/L) NOEC (mg/L) LOEC(mg/L)
24 1.1 0.95 - 1.3 0.56 1.0
48 0.80 0.12 - 1.5 0.56 1.0

A 20% immobilization was recorded in the 0.32 mg/L test concentration; however, as the immobilization recorded in the 0.56 mg/L test concentration was only 10% immobilization it was considered unlikely that effect observed was as a result of exposure to the reference item and therefore the NOEC was 0.56 mg/L.
The NOEC 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*.

NOEC = No observed effect concentration
LOEC = Lowest observed effect concentration
* The results of the inter laboratory tests and a Technical Corrigendum to ISO 6341 give a 24 Hour EC50 for potassium dichromate within the range 0.6 mg/L to 2.1 mg/L

Any other information on results incl. tables

Cumulative Immobilization Data and Observations in the Range‑finding Test

Nominal Concentration (mg/L)	Observations (Initial Population: 5 Per Replicate)
	24 Hours				48 Hours
	Cumulative Immobilized Daphnia		Observations		Cumulative Immobilized Daphnia		Observations
	R1	R2	R1	R2	R1	R2	R1	R2
Control	0	0	5N	5 N	0	0	5 N	5 N
0.10	0	0	5 N	5 N	0	0	5 N	5 N
1.0	0	0	5 N	5 N	0	0	5 N	5 N
10	0	0	5 N	5 N	0	0	5 N	5 N
100	0	0	5 N	5 N	0	0	5 N	5 N

R =Replicate

N = Normal

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	5N	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

R = Replicate

N = Normal

Water Quality Measurements

Nominal Concentration (mg/L)		0 Hours			24 Hours	48 Hours
		pH	mg O2/L	Temperature °C	Temperature °C	pH	mg O2/L	Temperature °C
Control	R1	7.9	9.3	20	21	7.8	8.6	20
100	R1	6.4	9.2	20	21	7.1	8.7	20

R = Replicate

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 gave a 48 Hour EC50 value of greater than 100 mg/L. The NOEC was 100 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, 20 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 20 °C to 21 °C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be between 80 and 87 mg/L (80-87% of nominal), so the results are based on nominal test concentrations.

No immobilization or sub-lethal effects on the Daphnia were exhibited during the test. Consequently, exposure of Daphnia magna to the test item gave EC₅₀ values of greater than 100 mg/L and a No Observed Effect Concentration (NOEC) of 100 mg/L.

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