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Ecotoxicological information

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:
2017-03-03 to 2017-03-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Principles of method if other than guideline:
Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(2-hydroxyethyl)-1H,3H-quinazoline-2,4-dione
EC Number:
214-897-3
EC Name:
3-(2-hydroxyethyl)-1H,3H-quinazoline-2,4-dione
Cas Number:
1207-75-6
Molecular formula:
C10H10N2O3
IUPAC Name:
3-(2-hydroxyethyl)-1,2,3,4-tetrahydroquinazoline-2,4-dione
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Analytical purity: >98.0% (based on chromatographic purity HPLC: 99.8%, acid titration: >=98.0%)
- Source and lot/batch No.of test material: I16JB3859
- Expiration date of the lot/batch: 2018-10-15 (retest date)
- Purity test date: 2018-10-28 (certificate of analysis release date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: solubility in water: 0.2 g/L

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling method: Samples were taken before the start of the test and after 24 hours and 72 hours from each test medium and from the control. Additionally, reserve samples of 1.8 mL were taken for possible analysis. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Stored in a freezer until analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: saturated solution (SS) of the test item was prepared starting with the highest concentration of 100 mg/L and applying three days of magnetic stirring to ensure maximum dissolution of the test item in the test medium. The resulting aqueous mixture was filtered through a 0.45 μm membrane filter (Whatman RC55) to remove the fraction of undissolved particles that were present. The filter was preconditioned with a small volume of test solution that was discarded. The resulting SS was used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the SS in test medium. It should be noted that the control received similar treatment. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc): All final test solutions were clear and colourless

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Method of cultivation: Algal 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. M1 medium was used.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in M2 medium at a cell density of 1E+04 cells/mL. The pre-culture was maintained under the same conditions as used in the test. Cell density was measured before use.

ACCLIMATION
- Acclimation period: not relevant (except pre-culture 3 days before start of the test)

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Hardness:
24 mg CaCO3/L
Test temperature:
22-24 °C
pH:
t=0h: 7.8-7.9
t=72h: 8.0
Dissolved oxygen:
not applicable
Salinity:
not relevant
Conductivity:
not relevant
Nominal and measured concentrations:
Range-finder:
nominal test concentrations: 1.0, 10 and 100% of a SS prepared at 100 mg/L (1 and 10% not analysed)
measured test concentration t = 0 h: n.d., 90.1, 91.9 mg/L
measured test concentration t = 24 h: n.d., 92.0, 92.4 mg/L
measured test concentration t = 72 h: 0.0079, 90.4, 80.5 mg/L

n.d. = not detected
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL all-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
- Initial cells density: 10,000 cells/mL
- Control end cells density: 172.0 x 10,000 cells/mL (mean)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis
- Detailed composition if non-standard medium was used:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

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: Yes (M1 versus M2). Three days before the start of the test the algal stock culture (culture in M1) was inoculated in the same culture medium (M2) used in the test. The culture was maintained under the same conditions as used in the test.
- Intervals of measurements: pH was measured at the beginning and at the end of the test. Temperature was continuously measured in a control vessel. At the end of the final test microscopic observations were performed on all test concentrations to observe for any abnormal appearance of the algae.

OTHER TEST CONDITIONS
- Sterile test conditions: no information
- Adjustment of pH: no
- Light intensity and quality: using TLD-lamps (with a light intensity within the range of 84 to 93 µE.m-2.s-1)

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 680 nm using a spectrophotometer with immersion probe (pathlength = 20mm).
- Effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: yes
- Test concentrations: 1.0, 10 and 100% of the SS (setup as combined range finder/limit test)
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
702 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
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: no
- 72-h EC50 (yield) = >100 mg T001200/L
- 72-h EC10 (growth rate) = >100 mg T001200/L
- 72-h EC10 (yield) = n.d. mg T001200/L
- 72-h NOEC (yield) = <100 mg T001200/L
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 72-h EC50 (growth rate) = 1.2 mg/L (95% C.L. 1.1 to 1.2 mg/L)
- 72-h EC50 (yield) = 0.43 mg/L (95% C.L. 0.42 to 0.44 mg/L)
Reported statistics and error estimates:
No EC50-values could be calculated (EC50 > maximum concentration tested).

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 with the test substance T001200 according to the OECD guideline 201 (GLP conditions). It can be concluded that the test item had no significant inhibitory effect on the growth of Pseudokirchneriella subcapitata. Both the EC50 for growth rate inhibition (72h-ErC50) and the EC50 for yield inhibition (72h-EyC50) exceeded 100 mg/L based on analytically confirmed nominal concentrations. The 72h-NOEC for growth rate inhibition was 100 mg/L. The 72h-NOEC for yield inhibition was below 100 mg/L. The results of the test can be considered reliable without restrictions.

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