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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-01-15 to 2016-03-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I15FC2164
- Expiration date of the lot/batch: 2017-06-23 (retest date)
- Purity test date: 2015-07-01

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: Not indicated
- Solubility and stability of the test substance in the solvent/vehicle: Not indicated
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not indicated
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 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.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The preparation of test solutions started with a loading rate of 100 mg/L, applying two days of magnetic stirring at room temperature in the dark to ensure maximum dissolution of the test item in the test medium. This resulted in a hazy dispersion that contained undissolved floating material and precipitate. The obtained dispersion was consequently filtered through a 0.45 µm membrane filter (Whatman; RC55) to remove any non-dissolved material. The filter was pre-conditioned with a small volume of test solution that was discarded. The resulting SS was used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All final test solutions were clear and colourless.
- Controls: yes
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)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
22-23°C
pH:
t=0h: 8.2-8.3
t=72h: 8.0-8.2
Dissolved oxygen:
not reported
Salinity:
not applicable
Nominal and measured concentrations:
Combined limit/range-finding test:
Nominal test concentrations: 1.0, 10% and 100% of the SS (10% SS not analysed)
Measured test concentrations: 0.010 and 0.94 mg/L
The measured concentrations remained stable during the test period (79-102% of the initial concentration at the end of the exposure)

Final test:
Nominal test concentrations: 10, 18, 32 and 100% SS prepared at 100 mg/L
Measured test concentration t= 0 h: 0.105, 0.178, 0.331, 0.573, 0.984 mg/L
Measured test concentration t= 24h: 0.101, 0.186, 0.290, 0.567, 0.994 mg/L
Measured test concentration t= 72h: 0.0758, 0.148, 0.276, 0.542, 0.972 mg/L
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: 283.6 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 89 to 93 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 680 nm using a spectrophotometer with immersion probe.
- effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study
- Test concentrations: 1.00, 10 and 100 % of the SS
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.98 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.29 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 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: clear test solution
- EC50 (yield) = 0.94 mg T002488 /L
- EC10 (growth rate) = 0.67 mg T002488 / L (95% CL 0.60-0.76 mg/L).
- EC10 (yield) = 0.057 mg T002488 / L (95% CL 0.042-0.073 mg/L).
- NOEC (yield) = <0.039 mg T002488 / L.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 72-h EC50 (growth rate) = 1.5 mg/L (95% CL 1.4 to 1.6 mg/L)
- EC50: 72-h EC50 (yield) = 0.51 mg/L (95% CL 0.49 to 0.53 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, JNJ-4754724-AAA (T002488) reduced the growth rate of this fresh water algae species significantly at analytically confirmed concentrations of 0.56 mg/L and higher. The EC50 for growth rate inhibition (72h-ErC50) was beyond the range tested, i.e. exceeded a TWA concentration of 0.98 mg/L, being considered the maximum solubility of the test item in medium at a loading rate of 100 mg/L.
The EC50 for yield inhibition (72h-EyC50) was 0.94 mg/L with a 95% confidence interval ranging from 0.82 to 1.1 mg/L. The 72h-NOEC for growth rate inhibition was 0.29 mg/L, while no NOEC for yield inhibition could be determined.

Description of key information

The acute toxicity of T002488 to freshwater algae was determined in a 72-hour static test according to the OECD guideline 201 (Tobor-Kaplon, 2016). Based on the results obtained during the present study, the 72-h EC50 was beyond the range tested, i.e. exceeded a measured concentration (based on a Time Weighted Average) of 0.98 mg/L, being considered the maximum solubility of the test item in test medium.Therefore, the highest concentration causing no effects to freshwater algae was determined to be the loading rate of 100 mg/L. The 72-h EC50 was clearly higher than the loading rate of 100 mg/L.The results of the test can be considered reliable without restriction.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
0.67 mg/L

Additional information