Menadione

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 03 July 2017. Experimental completion date 03 November 2017.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Menadione

EC Number:

200-372-6

EC Name:

Menadione

Cas Number:

58-27-5

Molecular formula:

C11H8O2

IUPAC Name:

2-methyl-1,4-dihydronaphthalene-1,4-dione

Specific details on test material used for the study:

Identification: Beta-Menadione
Physical state/Appearance: Light yellow powder
Batch: J17022301
Purity: 99.4%
Expiry Date: 22 February 2018
Storage Conditions: Room temperature in the dark
Intended use/Application: Synthesis of beta menadione derivatives

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.

Definitive Test
Samples were taken from the control and each 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 and stored frozen for further analysis if necessary. Only samples of the No Observed Effect Concentration (NOEC) and above were sent for analysis.

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.
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.
Based on the results the test item was prepared using a saturated solution method of preparation at an initial loading rate of 100 mg/L, stirred for a period of 24 hours prior to the removal of any undissolved test item by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 1 liter discarded) to give a nominal test concentration of approximately 79 mg/L.

Range-finding Test
A nominal amount of test item (1100 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 1 liter 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 (1100 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 1 liter discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A dilution was made from the 100% v/v saturated solution stock solution to give a further stock solution of 10% v/v saturated solution, from which a series of dilutions was made to give the test concentrations of 0.10, 0.18, 0.32, 0.56 and 1.0% v/v saturated solution.
Each 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 (see Annex 2) 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:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Test temperature:

Temperature was maintained at 22 °C throughout the test

pH:

7.9 at 0 hours and 8.0-8.1 at 48 hours
there were no treatment related differences for pH.

Dissolved oxygen:

8.5-8.7 mg O2/L at 0 hours and 8.5-8.6 mg O2/L at 48 hours.
there were no treatment related differences for oxygen concentration.

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 test concentrations 0.10, 0.18, 0.32, 0.56 and 1.0% v/v saturated solution.
measured test concentrations0.202, 0.362, 0.698 mg/L for the 0.32, 0.56 and 1.0% v/v saturated solution respectively.

Details on test conditions:

Range-finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 79 mg/L could be obtained using a saturated solution method of preparation.
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.
In the range-finding test 5 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.
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.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 0.10, 0.18, 0.32, 0.56 and 1.0% v/v saturated solution.

Exposure Conditions
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.
The test preparations were not renewed during the exposure period.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.36 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.42 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range-finding Test
No immobilization was observed at the test concentration of 0.10% v/v saturated solution, however, immobilization was observed at 1.0, 10 and 100% v/v saturated solution.
No sub-lethal effects of exposure were observed throughout the test.
Based on this information test concentrations of 0.10, 0.18, 0.32, 0.56 and 1.0% v/v saturated solution were selected for the definitive test.
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.79 to 74 mg/L. There was no significant change in the measured concentrations at 48 hours indicating that the test item was stable under test conditions.

Definitive Test
Verification of Test Concentrations
Analysis of the 0.32, 0.56 and 1.0% v/v saturated solution test preparations at 0 hours showed measured test concentrations to range from 0.23 to 0.80 mg/L. There was no significant change in the measured concentrations at 48 hours and so the results are based on 0-Hour measured test concentrations only.

Sub-Lethal Effects
There were no sub-lethal effects observed in 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
Throughout the test the light intensity was observed to be in the range 754 to 791 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, however, throughout the positive control test the temperature range was recorded between 19 and 22 °C, therefore outside of the range quoted in the study plan of 18 to 22 °C with a maximum deviation of ±1 °C during the test. This deviation was considered not to have adversely affected the results of the test.
Analysis of the immobilization data was carried out using the Binomial Distribution method at 24 hours and the Trimmed Spearman-Karber 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:

24 hours
EC50: 1.3 mg/L; 1.0-1.8 (95% confidence limits mg/L)
NOEC: 1.0 mg/L
LOEC: 1.8 mg/L

48 hours
EC50: 1.2 mg/L; 1.1-1.3 (95% confidence limits 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

Immobilization Data

Analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood regression method at 24 and 48 hours based on the 0-Hour measured test concentrations gave the following results:

Time (Hour)	EC50 (mg/L)	95% Confidence limits (mg/L)
24	0.36	Not determined
48	0.31	Not determined

The No Observed Effect Concentration after 24 and 48 hours exposure was 0.23 mg/L.

The Lowest Observed Effect Concentration after 24 and 48 hours exposure was 0.42 mg/L.

The slopes and their standard errors of the response curves at 24 and 48 hours were 26 (SE = 0.73) and 46 (SE = 13) respectively.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test item has been investigated and gave the following results based on the 0-Hour measured test concentrations:
Time point (hours): 48
EC50 (mg/L): 0.31
95% Confidence Limits (mg/L): Not determined due to the nature of the data

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

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 79 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, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 0.10, 0.18, 0.32, 0.56 and 1.0% v/v saturated solution for 48 hours at a temperature of 22 °C under static test conditions. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 μm Sartorius Sartopore filter, first approximate 1 liter discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the required test concentrations. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results

Chemical analysis of the 0.32, 0.56 and 1.0% v/v saturated solution test preparations at 0 hours showed measured test concentrations to range from 0.23 to 0.80 mg/L. There was no significant change in the measured concentrations at 48 hours and so the results are based on 0-Hour measured test concentrations only.

Exposure of Daphnia magna to the test item gave the following results based on the 0-Hour measured test concentrations:

Time (Hour)	EC50 (mg/L)	95% Confidence limits (mg/L)
48	0.31	Not determined due to the nature of the data