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

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 Starting Date: 20 November 2012. Experimental Completion Date: 20 December 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

Please see details on test solutions section

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

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: not supplied
Expiry / retest date: not supplied
Storage conditions: room temperature in the dark over silica gel

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Verification of Test Concentrations
Water samples were taken from the control and the 100% v/v saturated solution test group
(replicates Ri - R4 pooled) at 0 and 48 hours for quantitative analysis. Samples were stored at
approximately -20 °C prior to analysis.
Duplicate samples were taken 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 reconstituted water for a period of
24 hours prior to removing any undissolved test item present by filtration (0.2 pm Gelman
Acrocap filter, first approximate 500 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 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.

Saturated Solution Preparation
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 via propeller stirrer for 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 in order to pre-condition the filter).

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory
cultures.
Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium
in a temperature controlled room at approximately 20 °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:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/L as
CaC03.

Test temperature:

20 - 22°C
the temperature was measured using a Hanna Instruments HI 93510 digital thermometer

pH:

7.6 - 8.1
The pH and was measured using a Hach HQ30d Flexi handheld meter

Dissolved oxygen:

8.5 - 8.8 mg O2/L
The dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter

Salinity:

Not applicable as freshwater study

Conductivity:

<5 µS cm-1

Nominal and measured concentrations:

Range fiinding: 0.10, 1.0, 10 and 100% v/v saturated solution
Definitive test: 100% v/v saturated solution

Details on test conditions:

Procedure

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% v/v saturated solution.
An amount of test item (1125 mg) was added to 22.5 liters of reconstituted water and stirred
using a propeller stirrer for 24 hours at approximately 1500 rpm. After stirring, any undissolved
test item was removed by filtration through a 0.2 pm Gelman Acrocap filter (initial 500 mL
discarded) to give the 100% v/v saturated solution. Serial dilutions were prepared from the
100% v/v saturated solution to give the remainder of the test concentrations.
Each stock solution and prepared concentration was inverted several times to ensure adequate
mixing and homogeneity.
In the range-finding test 10 daphnids were placed in each test and control vessel and maintained
in a temperature controlled room at 20 °C to 21 °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. Each
250 mL test and control vessel contained 200 mL of test media and was covered to reduce
evaporation. After 24 and 48 hours the number of immobilized Daphnia magna 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 at
approximately -20 °C prior to analysis. Only concentrations within the range to be used for the
definitive test were analyzed.

Definitive Test
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of
100% v/v saturated solution to confirm that at the highest attainable test concentration, no
immobilization or adverse reactions to exposure were observed.
Experimental Preparation
For the purpose of the definitive test the test item was prepared as a saturated solution.
An amount of test item (1125 mg) was added to 22.5 liters of reconstituted water and stirred
using a propeller stirrer at approximately 1500 rpm for 24 hours. After stirring, any undissolved
test item was removed by filtration through a 0.2 pm Gelman Acrocap filter (initial 500 mL
discarded) to give the 100% v/v saturated solution test concentration.
The concentration and stability of the test item in the test preparations were verified by chemical
analysis at 0 and 48 hours.
Exposure Conditions
In the definitive test 250 mL glass jars containing approximately 200 mL of test preparation were
used. At the start of the test 5 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 at
21 °C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk
transition periods with a light intensity ranging from 647 to 703 lux. 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. 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 for approximately 15 seconds after gentle agitation.

Physico-Chemical Measurements
Water temperature and light intensity were 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 HQ30d Flexi handheld meter
whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 1.6 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1.6 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range-finding Test
No immobilization was observed 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 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.
Chemical analysis of the 100% v/v saturated solution test preparations at 0 and 48 hours
showed that measured concentrations of 1.66 and 1.55 mg/L were obtained
respectively indicating that the test item was stable under test conditions.
The analytical methodology was not validated prior to the analysis of range-finding samples; full
validation was later conducted specifically at the concentration employed in the definitive test.

Definitive Test
Verification of Test Concentrations
Analysis of the 100% v/v saturated solution test concentration at 0 and 48 hours
showed that measured concentrations of 1.60 and 1.67 mg/L were obtained respectively.

Immobilization Data
There was no immobilization in 20 daphnids exposed to a test concentration of 100% v/v
saturated solution for a period of 48 hours.
The No Observed Effect Concentration after 24 and 48 hours exposure was 1.6 mg/L.

Observations on Test Item Solubility
Throughout the duration of the test, the test media was observed to be a clear colorless solution.

Physico-Chemical Measurements
Temperature was maintained at approximately 21 °C throughout the test, while there were no treatment related
differences for oxygen concentration or pH.
The oxygen concentration in some of the test vessels was observed to have an air saturation
value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic
air bubbles in the media super-saturating the diluent and was considered not to have had an
impact on the outcome or integrity of the test as no adverse effects were observed in the control
group.

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.
The No Observed Effect Concentrations after 24 and 48 hours were 0.56 and 0.32 mg/L
respectively. The No Observed Effect Concentration is based upon zero 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

Definitive Test

Inspection of the immobilization data gave the

following results:

Time (h)	EC50 (mg/L)
24	>1.6
48	>1.6

Positive Control

Analysis of the immobilization data by the geometric mean method at 24 hours and the trimmed

Spearman-Karber method (Hamilton et al 1977 ) at 48 hours based on the nominal test

concentrations gave the following results:

Time (h)	EC50 (mg/L)	95% Confidence limits EC50 (mg/L)
24	0.75	0.56 - 1.0
48	0.45	0.42 - 0.48

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 of greater than 1.6 mg/L based on the mean measured
concentration. The No Observed Effect Concentration was 1.6 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, twenty 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 approximately 21 °C under static test conditions. The test item

solution was prepared by stirring an excess (50 mg/L) of test item in test 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 500 mL discarded in order to pre-condition the filter) to produce a

100% v/v saturated solution of the test. Immobilization and any adverse reactions to exposure

were recorded after 24 and 48 hours.

Results

Analysis of the 100% v/v saturated solution test concentration at 0 and 48 hours showed that

measured concentrations of 1.60 and 1.67 mg/L were obtained respectively.

The 48-Hour EC50 for the test item to Daphnia magna based on the mean measured test

concentration was greater than 1.6 mg/L. The No Observed Effect Concentration was 1.6 mg/L.