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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-08-21 to 2016-09-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study reports): JNJ-7928531-AAA (T001481)
- Physical state: liquid
- Appearance: Colourless, light yellow to brown liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M16AB0419
- manufacture date: 2016-01-26
- Expiration date of the lot/batch: 2016-09-23 (retest date)
- Purity test date: 2016-08-09

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

OTHER SPECIFICS:
- purity: 102.5% GC
- appearance: colourless to light yellow liquid
- Water solubility: 1.6 g/L

Sampling and analysis

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 each test concentrations and from the control. 3.0 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 3.0 mL were taken for possible analysis

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with the highest concentration of 100 mg/L applying an overnight period of magnetic stirring to accelerate the dissolution of the test item in test medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration 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:
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)

Study design

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

Test conditions

Hardness:
24 mg CaCO3/L
Test temperature:
21-23°C
pH:
t=0h: 8.1
t=72h: 7.8-8.0
Dissolved oxygen:
not reported
Salinity:
not relevant
Conductivity:
not applicable
Nominal and measured concentrations:
Nominal concentrations: 1.0, 3.2, 10, 32, 100 mg/L
Measured concentrations:
after 24h: 0.926, 2.93, 8.79, 29.2, 89.1 mg/L
after 72h: 0.603, 2.04, 5.64, 22.3, 83 mg/L

The measured concentrations were in agreement with the nominal concentrations (90-94%) at the start of the test. Measured concentrations decreased to 62-96% of initially measured at the end of exposure. Therefore, the Time Weight Average (TWA) concentrations were calculated:
TWA concentrations: 0.81, 2.6, 7.7, 27, 88 mg/L
Details on test conditions:
TEST SYSTEM
- 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):
6 replicates of the control;
3 replicates of each test concentrations;
2 replicates of 10 mg/L without algae;
1 replicate of each concentration for sampling purposes after 24 hours of exposure.

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 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 (control and 100% filtrate). Temperature was continuously measured in a control vessel. At the end of the final test microscopic observations were performed on the control and 32 mg/L treatments to observe for any abnormal appearance of the algae.

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- illumination: TLD-lamps with a light intensity within the range of 80 to 89 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

TEST CONCENTRATIONS
- Range finding study: combined limit/range finding study was performed
- Range finding study concentrations: 0.10, 1.0, 10 and 100 mg/L
- Spacing factor for final test concentrations: 3.2
- Results used to determine the conditions for the definitive study: yes, results of limit range test were used to determine definitive study concentrations
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
51 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.:
7.7 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) = 19 mg / L
- 72-h EC10 (growth rate) =17 mg / L
- 72-h EC10 (yield) = 6.9 mg / L
- 72-h NOEC (yield) = 2.6 mg / L
--> based on TWA concentrations
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 72h-ErC50 was 1.0 mg/L (95% confidence interval ranging from 0.97 to 1.0 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:
Statistical analysis of the data is shown in APPENDIX 2 and APPENDIX 3.
The effects on the growth rate were statistically significant in the three highest test groups. However, at the TWA concentration of 7.7 mg/L the inhibition was biologically not relevant, i.e. <10%, and therefore, the NOEC was set at a TWA concentration of 7.7 mg/L.
The inhibition of yield increased from 7.0% at 2.6 mg/L to 98% at the highest concentration tested, i.e. 88 mg/L. The inhibition of yield was statistically significant at the three highest concentrations tested.
The calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
A 72-h growth inhibition test with the unicellular algal species Pseudokirchneriella subcapitata was performed with the test substance JNJ-7928531-AAA (T001481) according to the OECD guideline 201 (GLP conditions). It can be concluded that the test item had a significant inhibitory effect on the growth of Pseudokirchneriella subcapitata at a TWA concentrations of 27 mg/L and higher, after the test period of 72 hours, i.e. the 72-hour NOEC for growth rate was determined to be 7.7 mg/L. The EC50 for growth rate inhibition was determined to be 51 mg/L.
The results of the test can be considered reliable without restrictions.