Benzonitrile, 4-[(1,6-dihydro-4-hydroxy-6-oxo-2-pyrimidinyl)amino]-

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-06-31 to 2015-08-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: samples were taken before the start of the test, after 24 hours and after 72 hours from all test concentrations and from the control. The filter containing undissolved residue 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.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Based on a pre-test performed at WIL Research and available information provided by the Sponsor preparation of test solutions started with a loading rate of 100 mg/L applying a two-day period of magnetic stirring at room temperature, mostly in the dark , followed by a filtration through a preconditioned 0.45 µm membrane filter (RC55, Whatman) to remove the undissolved fraction. The obtained saturated solution (SS) was used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of this SS in test medium. The final test solutions were all clear and colourless.
- Controls: yes

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

21-22°C

pH:

t=0h: 7.4 - 8.0
t=72h: 8.0 - 8.1

Dissolved oxygen:

not reported

Salinity:

not applicable

Nominal and measured concentrations:

Combined limit/range-finder test:
nominal test concentrations: 1, 10 and 100 %SS prepared at 100 mg/L
measured test concentration t= 0 h: 100% SS: 58,4 mg/L (with algae), 59.9 mg/L (without algae)
measured test concentration t= 72h: 100% SS: 58.2 mg/L (with algae), 55.1 mg/L (without algae)

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: 171.0 x 10,000 cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- 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: none
- Photoperiod: continuous illumination
- Light intensity and quality: using TLD-lamps with a light intensity within the range of 78 to 82 µE/m²/s

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

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 58 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 other: % v/v saturated solution

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: clear test solution
- 72-h ErC50 and 72-h ErC10 (based on measured concentrations): > 58 mg/L
- 72-h NOErC (based on measured concentrations): >= 58 mg/L

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 72-h EC50 (growth rate) = 1.2 mg/L (95% CL 1.1 to 1.2 mg/L)
- EC50: 72-h EC50 (yield) = 0.38 mg/L (95% CL 0.38 to 0.39 mg/L)

Reported statistics and error estimates:

No EC50-values could be calculated because the test item proved to be non-toxic (EC50 > maximum soluble concentration tested)

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, no biologically significant inhibition of growth rate or inhibition of yield was recorded at any of the concentrations of T002326 tested. The 72h-ErC50 was beyond the concentration range tested, i.e. exceeding a measured concentration of 58 mg/L being considered as the maximum soluble concentration in the test medium. The EC50 values could therefore not be quantified. In conclusion, the test item T002326 clearly had no inhibitory effect to Pseudokirchneriella subcapitata up to a measured concentration of 58 mg/L obtained at a loading rate of 100 mg/L.

Description of key information

Under the conditions of the present study with Pseudokirchneriella subcapitata, no biologically significant inhibition of growth rate or inhibition of yield was recorded at any of the concentrations of T002326 tested. The 72h-ErC50 was beyond the concentration range tested, i.e. exceeding a measured concentration of 58 mg/L being considered as the maximum soluble concentration in the test medium. The EC50 values could therefore not be quantified. In conclusion, the test item T002326 clearly had no inhibitory effect to Pseudokirchneriella subcapitata up to a measured concentration of 100 % v/v saturated solution.