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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-02-26 to 2016-03-24
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
according to guideline
EU Method C.3 (Algal Inhibition test)
according to guideline
other: 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:
EC Number:
Cas Number:
Molecular formula:
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study reports): JNJ-17299412-AAA (T001599)
- Physical state: solid (powder)
- Appearance: white powder
Specific details on test material used for the study:
- Batch n°: I15HB2951
- Analytical purity: 100.2% (base titration: >=98.0%)

- Expiration date: 2017-08-09 (re-test date)
- Storage condition: at room temperature
- Stability under storage conditions: Analysis of stability, homogeneity and concentration of the test item under test conditions were not performed as part of this study.

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Sampling method: samples were taken before the start of the test and after 24 and 72 hours from all test concentrations and from the control. A volume of 3.5 ml was taken. 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 of 3.5 ml were taken from all test solutions for possible analysis.
- Sample storage conditions before analysis: stored in a freezer (< -15°C) until analysis.

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to an expected low water solubility of the test item, Saturated Solutions (SS) were prepared. The preparation of test solutions started with a loading rate of 100 mg/L, applying a 10 minute period of ultrasonic waves, followed by three days of magnetic stirring at room temperature to ensure maximum dissolution of the test item in the test medium. This resulted in a hazy mixture that contained undissolved floating material and precipitate. The obtained mixture was subsequently 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.

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

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- 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 period: not relevant (except pre-culture 3 days before start of the test)

Study design

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

Test conditions

24 mg CaCO3/L
Test temperature:
Dissolved oxygen:
not reported
not applicable
Nominal and measured concentrations:
Range finder:
Nominal concentrations: 1.0 and 10% of the SS
Measured concentrations (mg/L) at t=0h: 0.18, 1.85
Measured concentrations (mg/L) at t=72h: 0.14, 1.83

Final test
nominal test concentrations final test: 0, 0.46, 1.0, 2.2, 4.6 and 10 % of the SS
measured test concentration final test t= 0 h: n.d., 0.103, 0.237, 0.527, 0.583, 1.07, 2.40 mg/L
measured test concentration final test t= 24h: n.d., 0.0996, 0.238, 0.531, 0.538, 1.06, 2.42 mg/L
measured test concentration final test t= 72h: n.d., 0.037, 0.167, 0.436, 0.475, 0.972, 2.44 mg/L
Details on test conditions:
- 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
- Renewal rate of test solution (frequency/flow rate): no renewal
- Initial cells density: 10,000 cells/mL
- Control end cells density: 164.7 x 10,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

- 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

- 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.

- 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 680 nm using a spectrophotometer with immersion probe.
- effect calculated parameters: specific growth rate and yield

- Spacing factor for test concentrations: 2.2
- Range finding study
- Test concentrations: 1.00 and 10% of the SS
- Results used to determine the conditions for the definitive study: yes.
Reference substance (positive control):
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
72 h
Dose descriptor:
Effect conc.:
1.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% C.L.: 0.99-1.5
72 h
Dose descriptor:
Effect conc.:
0.5 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:
- EC50 (yield) = 0.70 mg T001599 /L
- EC10 (growth rate) = 0.34 mg T001599 / L (95% C.L. 0.23-0.45 mg/L).
- EC10 (yield) = 0.26 mg T001599 / L (95% C.L. 0.20-0.31 mg/L).
- NOEC (yield) = 0.074 mg T001599 / L.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 72-h EC50 (growth rate) = 1.0 mg/L
Reported statistics and error estimates:
Calculation of ECx values was based on Weibull (growth rate) and probit (yield) analysis using linear maximum likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding nominal concentrations of the test item. Methods with best fit were used.

Applicant's summary and conclusion

Validity criteria fulfilled:
A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed wit the test substance T001599 according to the OECD guideline 201 (GLP conditions).
Under the test conditions with Pseudokirchneriella subcapitata, T001599 had a statistically significant inhibitory effect on the growth of Pseudokirchneriella subcapitata at TWA concentrations of 1.0 mg/L and higher, after the test period of 72 hours. Based on biological significance, however, the 72-hour NOEC for growth rate was determined to be 0.50 mg/L.
The results of the test can be considered reliable without restrictions.