5-chloro-1,3-dihydro-1-(4-piperidinyl)-2H-benzimidazol-2-one

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:

2015-08-14 to 2015-09-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Analytical purity: >98.5% (based on chromatographic purity HPLC: 99.96%, base titration: 98.5%, acid titration: 100.4%)
- Source and lot/batch No.of test material: A14JB3414
- Expiration date of the lot/batch: 2015-10-01 (retest date)
- Purity test date: 2014-12-22 (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: 0.5 g/L

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Samples were taken before the start of the test and after 72 hours from each test medium and from the control. Additionally, reserve samples of 1.7 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.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low water solubility of the test item, a saturated solution (SS) was prepared at a loading rate of 100 mg/L applying a 2-day period of magnetic stirring followed by filtration through a 0.45 µm membrane filter (RC55, Whatman). The pH of the obtained filtrate was set with 1M HCl from 9.1 to 8.0 in the combined limit/range-finding test and from 9.3 to 8.3 in the final test. This SS was used as the highest concentration. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colourless. Note that blank M2 test medium was also filtered to exclude any treatment related effects. 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): final test solutions were clear and colourless

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:

22-23 °C

pH:

t=0h: 8.2
t=72h: 7.8

Dissolved oxygen:

not applicable

Salinity:

not relevant

Conductivity:

not relevant

Nominal and measured concentrations:

Range-finder:
nominal test concentrations: 1.0 and 100% SS (10% SS not analysed)
measured test concentration t = 0 h: 1.02 and 98.1 mg/L
measured test concentration t = 72 h: 1.00 and 101 mg/L

Final test:
nominal test concentrations: 1.0, 3.2, 10, 32 and 100 % of the SS prepared at 100 mg/L
measured test concentration t = 0 h: 1.02, 3.29, 9.94, 10.3, 31.8, 101 mg/L
measured test concentration t = 72 h: 1.00, 3.27, 10.1, 10.3, 32.1, 101 mg/L

Measured concentrations remained stable until the end of the exposure (98-102% of the initial concentrations).

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: 101.1 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: yes, the obtained filtrate of the 100% saturated solution was set with 1M HCl from 9.1 to 8.0 in the combined range finder/limit test and from 9.3 to 8.3 in the final test
- Photoperiod: continuous illumination
- Light intensity and quality: using TLD-lamps (with a light intensity within the range of 78 to 80 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 720 nm using a spectrophotometer with immersion probe (pathlength = 20mm).
- Effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- 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

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

37 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.L. 36-38 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 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) = 17 mg T000990/L (95% C.L. 16-18 mg/L)
- 72-h EC10 (growth rate) = 21 mg T000990/L (95% C.L. 19-22 mg/L)
- 72-h EC10 (yield) = 8.8 mg T000990/L (95% C.L. 8.0-10 mg/L)
- 72-h NOEC (yield) = 3.2 mg T000990 /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) = 0.38 mg/L (95% C.L. 0.38 to 0.39 mg/L)

Validity criteria fulfilled:

yes

Conclusions:

A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed with the test substance T000990 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 up to and including the test concentration of 10 mg/L, after the test period of 72 hours. The analytically confirmed nominal EC50 for growth rate inhibition (72h-ErC50) was 37 mg/L with a 95% confidence interval ranging from 36 to 38 mg/L. The results of the test can be considered reliable without restrictions.

Description of key information

The study of Tobor-Kaplon (2016), investigating the acute toxicity of T000990 to algae according to OECD guideline 201, was considered as the key study for endpoint coverage. The 72-h NOEC and 72-h EC50 for growth rate were determined to be 10 mg/L and 37 mg/L respectively. The substance can be considered as harmful to algae.

Key value for chemical safety assessment

EC50 for freshwater algae:

37 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L