Ethylene bis[1,3-dihydro-1,3-dioxoisobenzofuran-5-carboxylate]

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:

Experimental start date: 11 September 2017 Experimental completion date: 17 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ethylene bis[1,3-dihydro-1,3-dioxoisobenzofuran-5-carboxylate]

EC Number:

217-062-1

EC Name:

Ethylene bis[1,3-dihydro-1,3-dioxoisobenzofuran-5-carboxylate]

Cas Number:

1732-96-3

Molecular formula:

C20H10O10

IUPAC Name:

2-(1,3-dioxo-1,3-dihydro-2-benzofuran-5-carbonyloxy)ethyl 1,3-dioxo-1,3-dihydro-2-benzofuran-5-carboxylate

impurity 1

Reference substance name:

Benzene-1,2,4-tricarboxylic acid 1,2-anhydride

EC Number:

209-008-0

EC Name:

Benzene-1,2,4-tricarboxylic acid 1,2-anhydride

Cas Number:

552-30-7

Molecular formula:

C9H4O5

IUPAC Name:

Benzene-1,2,4-tricarboxylic acid 1,2-anhydride

impurity 2

Reference substance name:

not assignable

IUPAC Name:

not assignable

Specific details on test material used for the study:

Identification: RIKACID TMEG-500
Batch: 5259
Purity: 81.1% (HPLC)
Physical state/Appearance: cream colored powder
Expiry Date: 24 April 2018
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. The sample bottles were fortified with 200 µL of formic acid prior to the addition of the test sample. All samples were stored frozen prior to analysis. Only the concentration to be used in the definitive test was analyzed.

The analysis of the range-finding test preparations failed. Therefore, in order to determine the stability of the test item under test conditions a 100% v/v saturated solution of the test item was prepared and a sample of the 100% v/v saturated solution test preparation was taken for immediate analysis. A further sample was then held under test conditions for a period of 48 hours prior to analysis.

Definitive test
Samples were taken from the control and the 100% v/v saturated solution test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate 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 conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. A test concentration of 14 mg/L was obtained using a preliminary solution in reverse osmosis water.
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.

Range-finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 14 mg/L could be obtained using a saturated solution method of preparation.
A nominal amount of test item (550 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 0.10, 1.0 and 10% v/v saturated solution.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
A nominal amount of test item (550 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre condition the filter) to give a 100% v/v saturated solution.
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 first instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnids were maintained in 150 mL glass beakers 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 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.

Test Water
Reconstituted water (Elendt M7 medium) used for both the range-finding and definitive tests.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Test temperature:

20 to 22 ºC

pH:

7.3 - 8.1

Dissolved oxygen:

8.6 - 9.0 mg O2/L

Nominal and measured concentrations:

Range-finding Test
Nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.

Definitive Test
Nominal concentration of 100% v/v saturated solution.
Chemical analysis of the 100% v/v saturated solution at 0 hours showed a measured test concentration of 26 mg/L was obtained. There was no significant change in the measured concentration at 48 hours and therefore the results are based on the 0-Hour measured test concentration only.

Details on test conditions:

Throughout the test the light intensity was observed to be in the range 808 to 859 Lux.

Range-finding Test
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 number of immobilized daphnids were recorded.
The control group was maintained under identical conditions but not exposed to the test item. 


Definitive Test
As in the range‑finding test 150 mL glass jars containing approximately 100 mL of test preparation were used. At the start of the test five 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. 

Reference substance (positive control):

yes

Remarks:

Potassium dichromate. The positive control was conducted between 23 January 2018 and 25 January 2018.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 26 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

26 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 26 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

26 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range-finding Test
No sub-lethal effects of exposure were observed throughout the test.
Based on this information, a single test concentration of four replicates, of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the highest attainable test concentration, no immobilization or adverse reactions to exposure were observed.
Whilst samples were taken from the range-finding test in order to determine the stability of the test item under test conditions the analysis failed.

Preliminary Stability Analyses
Analysis of the 100% v/v saturated solution test preparation at 0 hours showed a measured test concentration of 28 mg/L was obtained. There was no significant decrease in the measured test concentration at 48 hours indicating that the test item was stable under the conditions of the test.

Definitive Test
Verification of Test Concentrations
Chemical analysis of the 100% v/v saturated solution at 0 hours showed a measured test concentration of 26 mg/L was obtained. There was no significant change in the measured concentration at 48 hours and therefore the results are based on the 0-Hour measured test concentration only.

Immobilization Data
There was no immobilization in 20 daphnids exposed to a test concentration of 26 mg/L for a period of 48 hours.

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.

Results with reference substance (positive control):

A positive control (Envigo study number LK67NP) 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 was carried out using the Trimmed Spearman Karber method at 24 hours and the Binomial Distribution method at 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 0.79 0.73 - 0.86 0.56 1.0
48 0.75 0.56 - 1.0 0.56 1.0

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*.

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

Observations on Test Item Solubility

At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Cumulative Immobilization Data and Observations in the Range‑finding Test

Nominal Concentration (% v/v Saturated Solution)	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

 

 

Cumulative Immobilization Data and Observations in the Definitive Test

0-Hour Measured 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
26	0	0	0	0	0	0	5 N	5 N	5 N	5 N

 

0-Hour Measured 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
26	0	0	0	0	0	0	5 N	5 N	5 N	5 N

 

Water Quality Measurements

0-Hour Measured Concentration (mg/L)		0 Hours			24 Hours	48 Hours
		pH	mg O2/L	Temperature °C	Temperature ºC	pH	mg O2/L	Temperature °C
Control	R1	8.1	9.0	20	21	7.8	8.9	21
26	R1	7.3	8.6	22	21	7.7	9.0	21

R_1-R₂    = Replicates 1 to 2

N           = Normal

R_{1 -}R₄    = Replicates 1 to 4

N           = Normal

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 based on the 0 Hour measured test concentration gave a 48 hour EC50 value of greater than 26 mg/L. The NOEC was 26 mg/L.
This study showed that there were no toxic effects at saturation.

Executive summary:

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

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 14 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

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 concentration of 100% v/v saturated solution for 48 hours at a temperature of 20 to 22 °C under static test conditions. The test item solution was prepared by stirring an excess (50 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre‑condition the filter) to produce a 100% v/v saturated solution of the test item. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results

Chemical analysis of the 100% v/v saturated solution at 0 hours showed a measured test concentration of 26 mg/L was obtained. There was no significant change in the measured concentration at 48 hours and therefore the results are based on the 0-Hour measured test concentration only.

Exposure of Daphnia magna to the test item gave EC₅₀values based on the 0‑Hour measured test concentration of greater than 26 mg/L. The No Observed Effect Concentration (NOEC) was 26 mg/L.

This study showed that there were no toxic effects at saturation.