Menadione

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 29 June 2017. Experimental completion date 02 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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

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 72 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, from samples run alongside the test at 24 and 48 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 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.
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.

Preliminary Media Preparation Trial
Methods
A nominal amount of test item (1100 mg) was dispersed, in duplicate, in 11 liters of deionized reverse osmosis water with the aid of propeller stirring at approximately 1500 rpm for periods of either 24 or 48 hours. After stirring samples were taken for chemical analysis
after the following pre-treatments:
· Centrifugation at 10000 g for 30 minutes
· Centrifugation at 40000 g for 30 minutes
· Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 1 liter discarded in order to pre-condition the filter)
· Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 2 liters discarded in order to pre-condition the filter)
Discussion
Based on this information 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
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution for a period of 72 hours.
A nominal amount of test item (1100 mg) was dispersed in 11 liters of culture medium 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 stock solutions of 0.10, 1.0 and 10% v/v saturated solution. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (5.6 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
The stock solutions and each of the prepared concentrations 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 culture medium 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 Sartorious Sartopore filter (first approximate 1 liter discarded in order to precondition the filter) to give a 100% v/v saturated solution.
A series of dilutions was made from this saturated solution to give stock solutions of 0.10, 0.32, 1.0, 3.2 and 10% v/v saturated solution. An aliquot (900 mL) of each of the stock solutions was separately inoculated with 4.7 mL of algal suspension to give the required test concentrations of 0.10, 0.32, 1.0, 3.2 and 10% v/v saturated solution.
The stock solutions and each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 2 °C.
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 - 10^5 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

Temperature was maintained at 24 ± 1 ºC throughout the test.
The temperature within the incubator was recorded daily.

pH:

The pH value of the test cultures ranged from pH 7.3 at 0 hours to pH 8.3 at 72 hours.
The pH value of the control cultures was observed to increase from pH 7.3 at 0 hours to pH 7.9 at 72 hours.
The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter.

Nominal and measured concentrations:

Range finding test
0.10, 1.0, 10 and 100% v/v

Definitve test
Nominal: 0.10, 0.32, 1.0, 3.2 and 10% v/v saturated solution.
Measured: 0.0093, 0.029, 0.11, 0.39, 1.3 mg/L

Details on test conditions:

Range-Finding Test
The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration.
The control group was maintained under identical conditions but not exposed to the test item. At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours
After 72 hours the cell density of each flask was determined using a haemocytometer and light microscope. It was not possible to monitor algal growth using a Coulter® Multisizer Particle Counter due
to undissolved test item which had precipitated out in the 100% v/v saturated solution test group.
A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis.

Definitive Test
Exposure Conditions
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of solution were used for the control and three flasks each containing 100 mL were used for each treatment group.
The control group was maintained under identical conditions but not exposed to the test item. Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 9.52 x 10^5 cells per mL. Inoculation of 900 mL of test medium with 4.7 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Assessments
Test Organism Observations
Samples were taken at 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.
Water Quality Criteria
The appearance of the test media was recorded daily.

Culture Medium
NaNO3: 25.5 mg/L
MgCl2.6H2O: 12.16 mg/L
CaCl2.2H2O: 4.41 mg/L
MgSO4.7H2O: 14.6 mg/L
K2HPO4:1.044 mg/L
NaHCO3: 15.0 mg/L
H3BO3: 0.186 mg/L
MnCl2.4H2O: 0.415 mg/L
ZnCl2: 0.00327 mg/L
FeCl3.6H2O: 0.160 mg/L
CoCl2.6H2O: 0.00143 mg/L
Na2MoO4.2H2O: 0.00726 mg/L
CuCl2.2H2O: 0.000012 mg/L
Na2EDTA.2H2O: 0.30 mg/L
The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 with 0.1N NaOH or HCl.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.064 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.009 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.029 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.029 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.009 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.029 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

Range-finding Test
The results showed no effect on growth at the test concentration of 0.10% v/v saturated solution. However, growth was observed to be reduced at 1.0, 10 and 100% v/v saturated solution.
Based on this information test concentrations of 0.10, 0.32, 1.0, 3.2, and 10% v/v saturated solution were selected for the definitive test.
Chemical analysis of the test preparations at 0 hours showed measured concentrations to range from less than the LOQ of the analytical method, determined to be 0.012 mg/L, to 66 mg/L. A significant decline in measured concentration was observed after 72 hours, indicating that the test item was not stable under test conditions.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.045 to 6.6 mg/L. A decline in measured test concentration was observed at each time point in the range of less than the LOQ of the analytical method, determined to be 0.012 mg/L, to 1.3 mg/L at 24 hours, less than the LOQ to 0.37 mg/L at 48 hours and less than the LOQ to 0.21 mg/L at 72 hours.
Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC50 values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. In cases where the measured concentration was less than the LOQ of the analytical method following current regulatory advice a value of half the LOQ (i.e. 0.0060 mg/L) was used to enable calculation of the geometric mean measured concentrations.

Growth Data
From the data it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.
Accordingly the following results were determined from the data:
Inhibition of Growth Rate
ErC10 (0 - 72 h): 0.025 mg/L
ErC20 (0 - 72 h): 0.036 mg/L
ErC50 (0 - 72 h): 0.064 mg/L; 95% confidence limits 0.053 – 0.078 mg/L
Where ErCx is the test concentration that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences between the control and the 0.0093 mg/L test
concentration (P³0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.0093 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 0.029 mg/L.

Inhibition of Yield
EyC10 (0 - 72 h): 0.024 mg/L
EyC20 (0 - 72 h): 0.026 mg/L
EyC50 (0 - 72 h): 0.029 mg/L; 95% confidence limits 0.028 – 0.030 mg/L
Where:
EyCx is the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried. There were no statistically significant differences between the control and the 0.0093 mg/L test concentration (P³0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.0093 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 0.029 mg/L.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 154 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Nominal cell density of control at 0 hours : 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 7.68 x 10^5 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control cultures was 17% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Observations on Cultures
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0.0093 and 0.029 mg/L, however, no intact cells were observed to be present in the test cultures at 0.11, 0.39 and 1.29 mg/L.

Water Quality Criteria
The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Observations on Test Item Solubility
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and 0.0093 mg/L test cultures were observed to be green dispersions, all 0.029 mg/L test cultures were observed to be slightly pale green dispersions, all 0.11 mg/L test cultures were observed to be clear colorless solutions, all 0.39 mg/L test cultures were observed to be very pale orange solutions and all 1.3 mg/L test cultures were observed to be pale orange solutions with some precipitate present.

Results with reference substance (positive control):

A positive control (Envigo Study Number RD71YQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h) : 1.6 mg/L; 95% confidence limits 1.4 – 1.8 mg/L
EyC50 (0 – 72 h) : 0.77 mg/L; 95% confidence limits 0.68 – 0.87 mg/L
No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Any other information on results incl. tables

The geometric mean measured test concentrations were determined to be:

Nominal Test Concentration (% v/v Saturated Solution)	Geometric Mean Measured Test Concentration (mg/L)	Expressed as a Percentage of the 0-Hour Measured Test Concentration
0.1	0.0093	21
0.32	0.029	17
1	0.11	18
3.2	0.39	19
10	1.3	20

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the geometric mean measured test concentrations gave the following results:

Growth Rate
EC50 (mg/L):0.064
95% Confidence Limits (mg/L): 0.053 - 0.078
No Observed Effect Concentration (NOEC) (mg/L): 0.0093
Lowest Observed Effect Concentration (LOEC) (mg/L): 0.029

Yield
EC50 (mg/L): 0.029
95% Confidence Limits (mg/L): 0.028 - 0.030
No Observed Effect Concentration (NOEC) (mg/L): 0.0093
Lowest Observed Effect Concentration (LOEC) (mg/L): 0.029

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD

Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

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, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 0.10, 0.32, 1.0, 3.2 and 10% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in culture medium 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 groups.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Results

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.045 to 6.6 mg/L. A decline in measured test concentration was observed at each time point in the range of less than the Limit of Quantification (LOQ) of the analytical method, determined to be 0.012 mg/L, and 1.3 mg/L at 24 hours, less than the LOQ and 0.37 mg/L at 48 hours and less than the LOQ and 0.21 mg/L at 72 hours and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations only in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.0093, 0029, 0.11, 0.39 and 1.3 mg/L.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:

Response variable	EC50 (mg/L)	95% Confidence Limits (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
growth rate	0.064	0.053 - 0.078	0.0093	0.029
Yield	0.029	0.028 - 0.030	0.0093	0.029