N-(2-oxo-1,2-dihydropyrimidin-4-yl)benzamide

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 Starting Date: 29 November 2012. Experimental Completion Date: 28 December 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

Please see details on test solution section

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

Please see details on test solution section.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N4-Benzoylcytosine

EC Number:

628-907-2

Cas Number:

26661-13-2

Molecular formula:

C11H9N3O2

IUPAC Name:

N4-Benzoylcytosine

Specific details on test material used for the study:

Identification: N-Benzoyl Cytosine
Description: off white powder
Batch: NBC-5-11001
Purity: 100%
Expiry Date: not provided
Storage conditions: room temperature over silica gel in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and the 100% v/v saturated solution test group (replicates
Ri - R6 pooled) at 0 and 72 hours for quantitative analysis. All samples were stored at
approximately -20 °C prior to analysis. Duplicate samples were taken at 0 and 72 hours and
stored at approximately -20 °C for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water
soluble test items, a modification of the standard method for the preparation of aqueous media
was performed. An approach endorsed by several important regulatory authorities in the EU and
elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a saturated solution of the
test item in cases where the test item is of high purity and is poorly soluble in water and in the
permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was
prepared by stirring an excess (50 mg/L) of test item in culture medium for a period of 24 hours
prior to removing any undissolved test item present by filtration (0.2 pm Gelman Acrocap, first
approximate 100 mL discarded in order to pre-condition the filter) to give a saturated solution of
the test item.

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 categorised 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.

An amount of test item (550 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 pretreatments:
• Centrifugation at 10000 g for 30 minutes
• Centrifugation at 40000 g for 30 minutes
• Filtration through a 0.2 pm Gelman Acrocap filter (approximately 100 mL discarded in
order to pre-condition the filter)
• Filtration through a 0.2 pm Gelman Acrocap filter (approximately 500 mL discarded in
order to pre-condition the filter)

Based on this information the test item was prepared using a saturated solution method of
preparation at an initial loading rate of 50 mg/L, stirred for a period of 24 hours prior to the
removal of any undissolved test item by filtration through a 0.2 pm Gelman Acrocap filter (first
approximate 100 mL discarded) to give a nominal test concentration of approximately 1.8 mg/L.

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

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 ± 1 °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 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 10E4 - 10E5 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

Temperature was maintained at 24 ± 1 °C throughout the test.

pH:

The pH of the definitive test ranged from 7.8 to 8.0
The pH value of the control cultures was measured to be pH 8.0 at 0 and 72 hours.
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.

Salinity:

not applicable freshwater test

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 of 100% v/v saturated solution. Mean measured test concentration was 0.94 mg/L.

Details on test conditions:

Range-finding test
The results obtained from the pre-study media preparation trial conducted indicated that a
dissolved test item concentration of approximately 1.8 mg/L could be obtained using a saturated
solution method of preparation.

The test concentration to be used in the definitive test was 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.
An amount of test item (550 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 pm Gelman Acrocap filter
(first approximate 100 mL 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 10, 1.0 and 0.10% 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 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 (1NFORS 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.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle
Counter.
Samples were taken for chemical analysis from each test concentration at 0 and 72 hours in order
to determine the stability of the test item under test conditions. All samples were stored at
approximately -20 °C prior to analysis.

Definitive test
Based on the result of the pre-study media preparation trial and range-finding test a "limit test"
was conducted at a concentration of 100% v/v saturated solution to confirm that at highest
attainable concentration, no effect on algal growth was observed.

Experimental Preparation
An amount of test item (550 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 pm Gelman Acrocap filter
(first approximate 100 mL discarded in order to pre-condition the filter) to give a 100% v/v
saturated solution. An aliquot (1 liter) of the stock solution was inoculated with 7.4 mL of algal
suspension to give the required test concentration of 100% v/v saturated solution.
The stock solution and the prepared concentration were inverted several times to ensure adequate
mixing and homogeneity.
The concentration and stability of the test item in the test solutions were verified by chemical
analysis at 0 and 72 hours

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 100% v/v saturated solution 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 6.79E5 cells per mL. Inoculation of 1 liter of test medium with 7.4 mL of this algal
suspension gave an initial nominal cell density of 5E3cells 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.
Samples were taken at 0, 23, 48 and 72 hours and the cell densities determined using a Coulter®
Multisizer Particle Counter.

Physico-chemical measurements
The pH of each control and test flask was determined at initiation of the test and after 72 hours
exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the
incubator was recorded daily.

Growth medium was a stadium medium used for both range finding and definitive tests.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.94 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Range-finding Test
The results showed no effect on growth at the test concentrations of 0.10, 1.0, 10 and 100% v/v
saturated solution.
Based on this information a single test concentration of six 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 effect on growth was observed.
Chemical analysis of the 100% v/v saturated solution test preparations at 0 and 72 hours
showed measured test concentrations of 1.1 and 0.79 mg/L respectively were
obtained.

The analytical methodology was not validated prior to the analysis of range-finding samples; full
validation was later conducted specifically at the test concentration to be employed in the
definitive test.

Validation criteria
The following data show that the cell concentration of the control cultures increased by a factor of
240 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.
Mean cell density of control at 0 hours: 5.43E3 cells per mL
Mean cell density of control at 72 hours: 1.30E6 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control
cultures was 8% 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 0% and hence satisfied the validation criterion given in the OECD
Guideline which states that this must not exceed 7%.

Verification of test concentrations
Chemical analysis of the 100% v/v saturated solution test preparation at 0 and 72 hours
showed measured test concentrations of 0.85 and 1.0 mg/L respectively were
obtained. As such it was considered appropriate to estimate the results based on the mean
measured test concentration only.

Growth data
From the data, it is clear that the growth rate (r) and yield (y) of
Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item
at a mean measured test concentration of 0.94 mg/L over the 72-Hour exposure period.
The test concentration of 0.94 mg/L was the highest attainable test concentration that could be
prepared due to the limited solubility of the test item in water.
Accordingly the following results were determined from the data based on the mean measured test
concentration:

Inhibition of growth rate
ErC10 (0 - 72 h): >0.94 mg/L
ErC20 (0 - 72 h): >0.94 mg/L
ErC50 (0 - 72 h): >0.94 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 0.94 mg/L test group
using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf
1981). There were no statistically significant differences (P>0.05), between the control and
0.94 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on
growth rate was 0.94 mg/L.

Inhibition of yield
EyC10 (0 - 72 h): >0.94 mg/L
EyC20 (0 - 72 h): >0.94 mg/L
EyC50 (0 - 72 h): >0.94 mg/L
where EyCx is the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out. There were no
statistically significant differences (P>0.05), between the control and 0.94 mg/L test group and
therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.94 mg/L.

Observations on cultures
All test and control cultures were inspected microscopically at 72 hours,
There were no abnormalities detected in any of the control or test cultures.

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 test cultures were observed to be pale green
dispersions.

Physico-chemical measurements
Temperature was maintained at 24 ± 1 °C throughout the test.
The pH value of the control cultures was measured to be pH 8.0 at 0 and 72 hours.
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.

Results with reference substance (positive control):

A positive control 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): 1.1 mg/L; 95% confidence limits 1.0 -1.3 mg/L
EyC50 (0 -72 h): 0.70 mg/L*
No Observed Effect Concentration (NOEC) based on growth rate: 0.50 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 1.0 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.5 mg/L

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

*=It was not possible to calculate 95% confidence limits for the EyC50 value as the data generated did not fit the
models available for the calculation of confidence limits.

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
and based on the mean measured test concentration gave EC50 values of greater than 0.94 mg/L.
The No Observed Effect Concentration was 0.94 mg/L.
This study showed that there were no toxic effects at saturation.

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

Pre-study 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 pre-study media preparation trial indicated that a dissolved test item concentration of

approximately 1.8 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 a

solution of the test item at mean measured concentrations of 0.94 mg/L (six replicate flasks) for

72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item

solution was prepared by stirring an excess (50 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 pm Gelman Acrocap filter, first approximate

100 mL discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of

the test item with a mean measured concentration of 0.94 mg/L.

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

Chemical analysis of the 100% v/v saturated solution test preparation at 0 and 72 hours showed

measured test concentrations of 0.85 and 1.0 mg/L respectively were obtained. As such it was

considered appropriate to estimate the results based on the mean measured test concentration only.

Exposure of Pseudokirchneriella subcapitata to the test item gave EC₅₀ values based on the mean

measured test concentration of greater than 0.94 mg/L. The No Observed Effect Concentration

was determined to be 0.94 mg/L.

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