5-nitro-2,4,6-triaminopyrimidine

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study according OECD 202

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

5-nitro-2,4,6-triaminopyrimidine

EC Number:

213-742-7

EC Name:

5-nitro-2,4,6-triaminopyrimidine

Cas Number:

1006-23-1

Molecular formula:

C4 H6 N6 O

IUPAC Name:

5-nitropyrimidine-2,4,6-triamine

Details on test material:

- Physical state: Pink powder
- Storage condition of test material: Store in a tightly closed container in a cool (room temperature), dry and weil ventilated area away from incompatible substances.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

At the start of the exposure intervals (0 and 24 hours), sampling was carried out after preparation of the saturated solution of the test item.
At the end of the exposure intervals (24 and 48 hours), samples were taken from additional replicates, which were prepared with test media, but without daphnids. These additional replicates were incubated under test conditions until sampling.

All original samples were stored at 6 ± 2 °C, prepared samples were stored at room temperature until analysis, if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
A dispersion with a nominal loading of 100 mg/L of the test item was prepared with dilution water one day prior to the start of each exposure interval (at -24 and 0 h). 100 mg of the test item were weighed on a glass slide and transferred with 1 L of dilution water into a brown glass bottle. This dispersion was shaken for 24 hours with 20 rpm at room temperature. Then, undissolved test item was removed by membrane filtration (membrane filter 0.45 μm, RC, MACHEREY-NAGEL). The filter was saturated in order to avoid adsorption during the filtration. The first 25 ml of filtrate were discarded. The filtration was interrupted for ca. 15 minutes to allow adsorption and saturation of the filter material with dissolved test item.
Thereafter, the filtration was continued. The next 25 ml were discarded. The resulting filtrate, i.e. the saturated solution, was used in the test. During filtration, the filter was always kept covered.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain: Daphnia magna STRAUS (Clone 5).
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBolu), 14195 Berlin, Germany
- Breeder: DR. U. NOACK-LABORATORIEN, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany
- Culture: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 ± 2°c, in an incubator, 16 h illumination, light intensity of max. 20 μE-m-2. s-1
- Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaC03/L, was used.
- Culture feeding: The culture daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 106 cells/ml. The algae were cultured at the test facility.
- Age at study initiation (mean and range, SD): 2 to 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels latest 22 hours before the start of the exposure and discarded. The juveniles born within this period of max. 22 hours preceding the exposure were used for the test after additional 2 hours after collection. No first brood progeny was used for the test.
- Method of breeding: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 ± 2°c, in an incubator, 16 h illumination, light intensity of max. 20 μE-m-2. s-1
- Feeding during test: No

ACCLIMATION
Acclimatisation was not necessary, because the dilution water and environmental conditions were equivalent to the culture medium.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

160 to 180 mg CaCO3/L

Test temperature:

18 - 22°C, constant within ± 1°C

pH:

8.2 ± 0.8

Dissolved oxygen:

6.09 - 8.59 mg/L

Nominal and measured concentrations:

The effects of the saturated solution with the nominal loading rate of 100 mg/L, which corresponds to a geometric mean measured concentration of 68.1 mg/L, were tested in a limit test.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses
- Test volume: 20 mL
- Renewal of the test solutions: The test solutions were renewed after 24 hours. For this purpose, a second set of test vessels were filled with the freshly prepared test solutions and the daphnids were transferred by pipette
- Aeration: No
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaC03/L, was used
- Culture medium different from test medium: No
- Intervals of water quality measurement:
Dilution water: Prior to the start of the exposure (0 hours) and the water renewal (24 hours), the water quality parameters (i.e. pH value, dissolved
oxygen concentration, temperature, conductivity and total hardness) of the dilution water were measured.
Testmedia: At the start of the exposure intervals (0 and 24 hours), the water quality parameters of the fresh media (i.e. pH value, dissolved oxygen
concentration) were measured in one additional replicate of the saturated solution and the control.
At the end of the exposure intervals (24 and 48 hours), the water parameters of the old media were measured in all replicates of the saturated solution and the control.
Temperature: The incubator temperature was recorded throughout the period of the test.
Quality criteria for the water quality parameters: The dissolved oxygen concentration at the end of the exposure intervals should be ~ 3 mg/L in the test vessels of the saturated solution and the control.
The pH-values should be in the range of 6 - 9. The deviation of the pH-value from the initial value should not be more than 1.5 units.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Light intensity: Diffuse light, light intensity of max. 20 μE·m-2 · s-1 ( < 1340 lx); 16/8 h light/dark cycle

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobilisation was determined in all groups after 24 and 48 hours. An animal was considered to be immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel. Other observations were not made.

TEST CONCENTRATIONS
- Range finding study: A non GLP preliminary range finding test under semi-static conditions over a period of 48 hours was conducted at the test facility with a saturated solution of the test item with a nominal loading of 100 mg/L and further difution levels with nominal loading rates of 1 and 10 mg/L. A semi-static test design was chosen to check the reproducibility of the preparation of the saturated solution and to ensure constant exposure concentrations, because the solubility and stability of the test item under test conditions were unknown. The preliminary range finding test was conducted under diffuse light conditions (light intensity of max. 1340 lx, 16/8 h light/dark cycle ).
The test item dispersion of 100 mg/l was shaken with approximately 20 rpm for 24 hours at room temperature followed by fiftration with a membrane filter (0.45 μm, RC, MACHEREY-NAGEL) to remove the undissolved test item. The filter was saturated in order to avoid adsorption during the filtration. The first 25 ml of filtrate were discarded. The filtration was interrupted for ca. 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 ml were discarded. The resulting filtrate, i.e. the saturated solution, was used in the test. During filtration, the filter was always kept covered. Further loading rates of nominal 1 and 10 mg/L were prepared with the dilution water by diluting the saturated solution.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

mortality

Remarks on result:

other: No acute toxic effects within the range of solubility.

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 68.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: No acute toxic effects within the range of solubility.

Details on results:

The tested solution was visually clear throughout the exposure intervals of 24 hours.

Results with reference substance (positive control):

The percentage immobility for the reference item was determined after 24 hours. The EC50-value with 95 % confidence limits (Cl) was calculated by sigmoidal dose-response regression. The EC50-value for the most recent of the monthly performed reference tests was:
EC50: 1.92 mg/L (Cl 1.71 - 2.29 mg/L)
The EC50-value of the reference item potassium dichromate after 24 h is within the prescribed concentration range of 0.6 - 2.4 mg/L of quality criteria according to AQS P 9/2 (02/2000) for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test substance is with high probability not acutely harmful to aquatic invertebrates. No toxic effects occur within the range of solubility.