5-hydroxyiminobarbituric acid

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:

2016.11.04-2016.11.07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Following GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

5-hydroxyiminobarbituric acid

EC Number:

201-741-4

EC Name:

5-hydroxyiminobarbituric acid

Cas Number:

87-39-8

Molecular formula:

C4H3N3O4

IUPAC Name:

5-(hydroxyimino)-1,3-diazinane-2,4,6-trione

Test material form:

solid: particulate/powder

Details on test material:

Lot/batch No.: 201713807

Specific details on test material used for the study:

Chemical name (IUPAC): 5-Hydroxyiminobarbuturic acid
CAS: 87-39-8
Lot no: 201616909T
Purity: ≥ 98%
Color: off whit to light yellow or light pink
Production date: 2016.07.01
Date of expiry: 2018.07.01

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Concentrations: 0.9, 1.7, 3.2, 6.1, 11.6, 22.0 aqnd 42 mg/L
- Sampling method: Samples were collected at the start (0h) and the end (72h) of the test.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Violuric acid was directly suspended in algal medium.
- Controls: algal culture medium and 3,5-Dichlorophenol (positive control, directly dissolved in algal medium)

Test organisms

Test organisms (species):

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

Details on test organisms:

initial concentrationof test culture 5638 cells/mLTEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection
- Method of cultivation: Alga stock was periodically subcultured at least once in a week and maintained with illumination and temperature of 4440 to 8880 lux and 21-24 degrees celsius. From healthy axenic laga culture, preculture was prepared.

ACCLIMATION
- Culturing media and conditions (same as test or not): yes

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

21.3-22.0°C

pH:

The measured pH values in control group flasks after 72H were 7.93 - 8.13

Nominal and measured concentrations:

Nominal concentration:0; 0.9; 1.7; 3.2; 6.1; 11.6; 22.0 and 42.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterilized glass conical flasks (Erlenmeyer flasks)
- Type (delete if not applicable): open
- Material, size, fill volume: Glass, 250 mL, 100 mL
- Initial cells density: 5638 cells/mL
- Control end cells density:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes (OECD alga medium)


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterilised deionized water


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Light intensity and quality: 5420, universal white type fluorescent lamp

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: By manual counting under a microscope using a hemocytometer (Neubauer improved).


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.9

- Range finding study: Yes
- Test concentrations: 0.9, 1.7, 3.2, 6.1, 11.6, 22.0 and 42.0 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

3,5 Dichlorophenol (99.7%)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

12.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

11.77 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

not specified

Effect concentrations 4

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

not specified

Details on results:

Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Any stimulation of growth found in any treatment: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

Reference substance: 3,5-Dichlorophenol
EC50 (growth rate) = 3.18 mg/L
EC50 (yield) = 1.45 mg/L

Reported statistics and error estimates:

EC50 values: Probit analysis (Finney, 1971)
LOEC and NOEC values: SPSS statistical procedure for multiple comparison using individual replicate values of the specific growth rate

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

From the test conducted according to the standard conditions the growth rat inhibition ErC50 of Violuric acid was found to be 12.20 mg/L. The No Observed Effect Concentration over 72 h test was 0.9 mg/L and the Lowest Observed Effect Concentration was 1.7 mg/L.

Executive summary:

The test item Violuric Acid was assessed for Alga (Pseudokirchneriella subcapitata) Growth Inhibition Test as per the OECD TG 201 and in compliance with GLP.

A range finding studywith 6 concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L (each concentration with 2 replicates except control possessing 6 replicates) . The algal growth was assessed at 24, 48 and 72h exposure during the test.

During range finding study exponetially growing alga cells (5775 cells/mL9 were exposed to range of selected concentrations. There was a concentration dependent percent growth inhibition of 0.69, 1.60, 8.02, 13.76, 100 and 100% observed in treated algal groups of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L Violuric Acid, respectively, compared to control group.

Based on the results of the range finding test the main study was conducted with following concentrations: 0.9, 1.7, 3.2, 6.1, 11.6, 22.0 and 42.0 mg/L violuric acid. Each concentration was tested in triplicates except for the control, which was tested in 6 replicates. The algal growth density was assessed at 24, 48 and 72 hours. Exponentially growing alga cells were exposed to the range of selected concentrations.

Percent growth inhibition of 0.96, 4.55, 10.55, 12.71, 15.83, 18.22 and 100% was observed in treated concentrations

0.9, 1.7, 3.2, 6.1, 11.6, 22.0 and 42.0 mg/L violuric acid, respectively, compared to control algae.

The present alga growth inhibition study fulfills the validity criteria of the test and is thus considered valid.

Analytical results showed that test concentrations at the beginning (0h) and the end (72h) of the test were in the acceptable range of +/-20% recovery of the nominal concentrations.

Under the conditions of the test the ErC50 (72h) value was determined to be 12.2 mg/L and the No Observed Effect Concentration (NOEC) (72h) to be 0.9 mg Violuric Acid/L.