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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-10-15 to 2016-11-11
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:

Test material

Test material form:
Details on test material:
- Name of test material (as cited in the report): JNJ-47961173-AAA
- Physical state: Liquid
- Colour: Colourless
Specific details on test material used for the study:
- Source and lot/batch No.of test material: M15IB3251
- Expiration date of the lot/batch: 07 April 2017 (retest date)
- Purity: 100% (based on gas chromatography)

- Storage condition of test material: at room temperature
- Stability under test conditions: stable

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 all test concentrations and from the control. A volume of 16 mL was taken.
- Sample storage conditions before analysis: All samples were transferred to a vessel containing 4 mL of n-Hexane (SupraSolv Batch: I845971631; Merck, Darmstadt, Germany) immediately after sampling. At the end of the exposure period, the replicates with algae were not pooled at each concentration before sampling.

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with a loading rate of 100 mg/L applying two days of magnetic stirring at room temperature to ensure maximum dissolution of the test item in test medium. For the subsequent second combined limit/range-finding test, the resulting aqueous mixture was left to stabilize for approximately 1.5 hours since undissolved material was observed in the test medium. The Saturated Solution (SS) was subsequently siphoned off and used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All final test solutions were clear and colourless.
After preparation of the test solutions, volumes of 50 mL in the 1st combined limit/range-finding test and 120 mL in the 2nd combined limit/range-finding test were added to each replicate of the respective test concentration. Subsequently, 1.0 and 2.4 mL of an algal suspension were added to each replicate, providing a cell density of 104 cells/mL in the 1st combined limit/range-finding test and in the 2nd combined limit/range-finding test, respectively.
- 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:
t=0h: 7.4-7.6
t=72h: 7.5-7.7
Dissolved oxygen:
not reported
not relevant
not relevant
Nominal and measured concentrations:
Nominal test concentrations final test: 1.0, 10 and 100% of a SS prepared at 100 mg/L

Combined limit/range-finding test 1:
measured test concentration t= 0 h: n.d., 0.0977, 1.07, 14.5, 16.5 mg/L
measured test concentration t= 72h: n.d., n.d., n.d., n.d., n.d. mg/L
The analysis of the samples taken during the 1st combined limit/range-finding test showed no detectable concentrations of the test item at any of the concentrations tested after 24 hours of exposure. Information obtained after performance of this study indicated that the test item is volatile. Since the volatility of the test item was suspected to be the cause for the observed decrease and no effects were observed in the 1st combined limit/range-finding test, a 2nd combined limit/range-finding test with air-tight closed vessels and minimum headspace was performed to confirm the biological results.

Combined limit/range-finding test 2:
measured test concentration t= 0 h: n.d. 0.102, 0.871, 8.76, 9.09 mg/L
measured test concentration t= 24h: 0.448, n.d., n.d., 0.137, 1.40, 1.43 mg/L
measured test concentration t= 72h: n.d., n.d., n.d., 0.162, 0.122 mg/L
Details on test conditions:
- Test vessel: glass flasks
- Type (delete if not applicable): capped vessels
- Material, size, headspace, fill volume: 120 mL all-glass flasks containing 120 mL of test solution,
airtightly closed with no headspace
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): no flow-through system applied
- Renewal rate of test solution (frequency/flow rate): no renewal
- Initial cells density: 10,000 cells/mL
- Control end cells density: 129.9 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: 10
- Range finding study
- Test concentrations: 1.00, 10 and 100 % 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.:
> 1.5 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: CL 95% could not be determined
Key result
72 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
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:
- EC50 (yield) = >1.5 mg/L
- EC20 (growth rate) = >1.5 mg/L
- EC20 (yield) = >1.5 mg/L
- NOEC (yield) = 1.5 mg/L
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 72-h EC50 (growth rate) = 1.3 mg/L
Reported statistics and error estimates:
For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the highest test concentration compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (STUDENT-t test for Homogeneous Variances, α=0.05, one-sided, smaller). No EC50-values could be calculated because the test item proved to be non-toxic (EC50 > maximum
concentration tested). 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 green alga Pseudokirchneriella subcapitata was performed wit the test substance 2-bromobutane according to the OECD guideline 201 (GLP conditions).
Under the test conditions with Pseudokirchneriella subcapitata, there was no significant inhibition of growth rate of this fresh water algae species. The 72-h ECr50 exeeded 1.5 mg/L being the average measured concentration in a saturated solution prepared at a loading rate of 100 mg/L (calculated as a Time Weighted Average concentration).
The results of the test can be considered reliable without restrictions.