3-(2-chloroethyl)(1H,3H)quinazoline-2,4-dione

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:

2015-10-05 to 2015-10-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, 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:

3-(2-chloroethyl)(1H,3H)quinazoline-2,4-dione

EC Number:

225-795-3

EC Name:

3-(2-chloroethyl)(1H,3H)quinazoline-2,4-dione

Cas Number:

5081-87-8

Molecular formula:

C10H9ClN2O2

IUPAC Name:

3-(2-CHLOROETHYL)QUINAZOLINE-2,4(1H,3H)-DIONE

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): T001201
- Physical state: off-white powder
- Analytical purity: 99.8 % (Chlorine (relative))
- Lot/batch No.: I15CB1185
- Expiration date of the lot/batch: 2017-03-15 (retest date)
- Stability under test conditions: not stable
- Storage condition of test material: at room temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: samples were taken before the start of the test and after 72 hours from all test concentrations and from the control. The filter used for preparation of the SS was kept for possible analysis of the residue. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling. Reserve samples were taken from all test solutions for possible analysis.
- Sample storage conditions before analysis: stored in a freezer (< -15°C) until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium of the highest nominal test item concentration of 100 mg/L was prepared applying 2 days of magnetic stirring in the dark followed by a filtration through a preconditioned 0.45 µm membrane filter. Adequate volumes of the highest test concentration were diluted with test medium to prepare the test media with the lower test item concentrations.
- Controls: yes

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): same as the test. 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

21-24°C

pH:

7.6 - 8.0

Dissolved oxygen:

not reported

Salinity:

not applicable

Nominal and measured concentrations:

nominal test concentrations final test: 1, 3.2, 10, 32 and 100 %SS prepared at 100 mg/L
measured test concentration final test t= 0 h: 0.611, 1.96, 5.87, 18.4 and 54.2 mg/L
measured test concentration final test t= 24h: 0.448, 1.43, 4.14, 14.1 and 42.0 mg/L
measured test concentration final test t= 72h: 0.226, 0.790, 2.31, 6.78 and 25.7 mg/L
TWA (mg/L) = 0.39, 1.3, 3.7, 12 and 38 mg/L

Actual concentrations decreased to 37-47% of initial at the end of the test period.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks
- Type (delete if not applicable): capped vessels
- Material, size, headspace, fill volume: 100 mL all-glass flasks filled with 50 mL test solution
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): no flow-through system applied
- Renewal rate of test solution (frequency/flow rate): no renewal
- Initial cells density: 10,000 cells/mL
- Control end cells density: 205.5 x 10,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2 according to the OECD 201 guideline, formulated using Milli-RO water
- Culture medium different from test medium: no
- Intervals of water quality measurement: temperature measured continuously, pH at the beginning and the end of the test.

OTHER TEST CONDITIONS
- Sterile test conditions: no information
- Adjustment of pH: none
- Photoperiod: continuous illumination
- Light intensity and quality: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm, using TLD-lamps.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] At the beginning, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe.
- effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x 3.2

- Range finding study
- Test concentrations: 1.00, 10 and 100 % of the SS
- Results used to determine the conditions for the definitive study: yes.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.L.: 8.6-9.4 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Additional peaks were observed in chromatograms at a different retention time than the test item. Intensity of the peaks increased with time. The appearance of the additional peaks occurred possibly due to degradation or reaction.
- EC50 (yield) = 1.9 mg T001201 /L
- EC10 (growth rate) = 1.7 mg T001201 / L (95% C.L. 1.5-1.8 mg/L).
- EC10 (yield) = 0.50 mg T001201 / L (95% C.L. 0.41-0.59 mg/L).
- NOEC (yield) = 0.39 mg T001201 / L.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 72-h EC50 (growth rate) = 0.39 mg/L

Reported statistics and error estimates:

Calculation of ECx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding TWA concentrations of the test item. The calculations were performed with ToxRat Professional v. 3.0.0 (ToxRat Solutions® GmbH, Germany).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed wit the test substance T001201 according to the OECD guideline 201 (GLP conditions).
Under the test conditions with Pseudokirchneriella subcapitata, T001201 inhibited growth rate of this fresh water algae species significantly. The 72-h ECr50 was determined to be 9 mg/L (calculated as a Time Weighted Average concentration).
The results of the test can be considered reliable without restrictions.