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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:
2017-01-13 to 2017-02-20
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
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
according to
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)

Test material

Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study reports): T000836 (JNJ-16250351-AAH)
- Physical state: solid
- Appearance: white powder
Specific details on test material used for the study:
- Source and lot/batch No.of test material: I16DB2126
- manufacture date: 2016-04-27
- Expiration date of the lot/batch: 2018-04-27 (retest date)
- Purity test date: 2016-05-27

- Storage condition of test material: at room temperature

- purity: 98.5% (base titration)
- appearance: white powder
- Water solubility: >500 g/L

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Sampling method: samples were taken before the start of the test, after 24 hours and after 72 hours from each test medium and from the control. 2.4 mL of volume was taken. 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. Additionally, reserve samples of 2.4 mL were taken for possible analysis

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: preparation of test solutions started with the highest test concentration of 130 mg/L (100 mg base/L). No special treatment other than careful mixing was needed to fully dissolve the test item in test medium. The resulting clear and colourless solution was used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions in test medium.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): final test solutions were clear and colourless

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:
t=0h: 7.9-8.0
t=72h: 7.6-7.8
Dissolved oxygen:
not reported
not relevant
not applicable
Nominal and measured concentrations:
nominal test concentrations final test: 1.04, 3.12, 9.36, 28.6 and 84.5 mg/L (0.80, 2.4, 7.2, 22 and 65 mg base/L)
measured test concentration final test t= 0 h: 0.846; 2.61; 7.60; 23.0; 68.3 (mg base/L)
measured test concentration final test t= 72h: 0.496; 1.51; 4.41; 13.1; 39.2 (mg base/L)
The measured concentrations were in agreement with the nominal concentrations at the study start (105-109% relative to nominal). Measured concentrations decreased to 86-92% of initial after 24 hours and further to 60-63% of initial at the end of the test. Based on these results the TWA exposure concentrations were calculated. The range tested based on TWA concentrations corresponded with 0.66, 2.0, 5.9, 17 and 53 mg base/L. This corresponded with 0.51, 1.5, 4.5, 13 and 41 mg/L based on the whole test item.
Details on test conditions:
- Test vessel: beaker
- Material, size, headspace, fill volume: 100 ml, all-glass, containing 50 mL of test solution
- Initial cell density: 1 x 10^4 cells/ml
- No. of vessels per concentration and per control (replicates):
* 3 replicates of each test concentration;
* 6 replicates of the control;
* 1 extra replicate of each test group for sampling purposes;
* 1 or 2 repicates of each test concentration without algae;

- 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. Three days before the start of the test the algal stock culture were 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.

- Photoperiod: continuous illumination
- illumination: TLD-lamps with a light intensity within the range of 82 to 84 µE/m²/s.
- Adjustment of pH: no
- Light intensity and quality: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : growth inhibition
- Determination of cell concentrations: 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

- Spacing factor for test concentrations: ca. 2.95
- Range finding study: combined limit/range finding study was performed
- range finding concentrations: 0.13, 1.3 and 13 and 130 mg/L (0.10, 1.0 and
10 and 100 mg base/L).
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
72 h
Dose descriptor:
Effect conc.:
6.4 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
72 h
Dose descriptor:
Effect conc.:
1.5 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: no
- 72-h EC50 (yield) = 2.8 mg/L (2.2 mg base/L)
- 72-h EC10 (growth rate) = 4.0 mg/L (3.1 mg base/L)
- 72-h EC10 (yield) < 0.51 mg/L (<0.39 mg base/L)
- 72-h NOEC (yield) < 0.51 mg/L (<0.39 mg base/L)
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 72h-ErC50 was 1.3 mg/L (95% confidence interval ranging from 1.2 to 1.4 mg/L)
- Other: the historical ranges for growth rate inhibition lie between 0.82 and 2.6 mg/L. The observed 72h-ErC50 for the algal culture tested corresponds with this range
Reported statistics and error estimates:
Growth rates were not biologically significantly reduced (<10%) at the two lowest TWA concentrations of 0.51 and 1.5 mg/L (9.0 and 3.6%, respectively), whereas the growth rate of algae exposed to TWA concentrations of 4.5 mg/L and higher were both biologically and statistically significantly reduced. Despite the statistical significant effect at 0.51 mg/L, the NOEC for growth rate was set at 1.5 mg/L based on biological relevance and the absence of a concentration related increase of effects at the two lowest test groups.

Yield was inhibited by 36.0 and 15.7% at the two lowest TWA concentrations of 0.51 and 1.5 mg/L, respectively. Yield inhibition increased with increasing concentration of JNJ-16250351-AAH (T000836) from 4.5 mg/L upwards resulting in 100% inhibition at a TWA concentration of 41 mg/L. Statistically significant inhibition of yield was found at all tested concentrations. Hence, the NOEC for yield was
< 0.51 mg/L.

The calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

Applicant's summary and conclusion

Validity criteria fulfilled:
A 72-h growth inhibition test with the unicellular algal species Pseudokirchneriella subcapitata was performed with the test substance JNJ-16250351-AAH (T000836) according to the OECD guideline 201 (GLP conditions). The test item reduced growth rate of this fresh water algae species significantly at average measured concentrations of 4.5 mg/L (3.5 mg base/L) and higher. The 72h NOEC for growth rate inhibition was determined to be 1.5 mg/L (1.2 mg base/L). The 72h NOEC for yield inhibition was determined to be <0.51 mg/L (<0.39 mg base/L).
The results of the test can be considered reliable without restrictions.