Registration Dossier

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:
2016-04-04 to 2016-05-21
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:
2016

Materials and methods

Test guidelineopen allclose all
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study reports): JNJ-117676-AAA (T000263)
- Physical state: solid (powder)
- Appearance: White, slight beige amorphous, crystalline, micropowder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I15BB0491
- Expiration date of the lot/batch: 2021-02-04 (retest date)
- Purity test date: 2015-02-06

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

OTHER SPECIFICS:
- Name of test material (as cited in study reports): JNJ-117676-AAA (T000263)
- Physical state: solid (powder)
- Appearance: white to slightly grey powder

specific details on test material used for the study:
- Analytical purity: 99.8% (base titration: >=98.0%)

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling method: samples for possible analysis were taken form all test concentrations and the control at t=0 and t=72h. 3.0 mL of volume. At the end of the exposure period, the replicates were pooled at each concentration before sampling
- Sample storage conditions before analysis: the samples were stored in a freezer. 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: The batch of JNJ-117676-AAA (T000263) tested was a white to slightly grey powder with a purity of 99.8% and completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item. Preparation of test solutions started with the highest concentration of 100 mg/L applying ultrasonic waves for a period of approximately 10 minutes followed by 49-72 minutes of magnetic stirring to accelerate the dissolution of the test item in test medium. For the combined limit/range-finding test, the pH was adjusted from 9.6 to 8.5 and for the final test, from 9.7 to 8.5 with 1 M HCl (Merck, Darmstadt, Germany). The 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. Six replicates of exponentially growing algal cultures were exposed to a control and a concentration of 100 mg/L. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
*Three replicates per concentration were exposed to 0.10, 1.0 and 10 mg/L.
*pH was only measured in the control and the highest test concentration.
*Samples for possible analysis were taken from the control and all test concentrations at the start, after 24 hours of exposure and at the end of the test period.
*Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.
*At the end of the test, algae were not observed to verify a normal and healthy appearance.
- Controls: yes

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Method of cultivation: Algae 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.

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions (same as test or not): same as the test. 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

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:
8.0-8.2
Dissolved oxygen:
not reported
Salinity:
not applicable
Nominal and measured concentrations:
nominal test concentrations final test: 0, 1.0, 3.2, 10, 32 and 100 mg/L
measured test concentration final test t= 0 h: 0.010, 1.11, 3.65, 11.3, 11.5, 35.5, 115 mg/L
measured test concentration final test t= 72h: 0.0970, 1.13, 3.67, 11.6, 10.6, 36.9, 118 mg/L
Details on test conditions:
TEST SYSTEM
- 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
- 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: 143,9 x 10,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

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: no
- Intervals of water quality measurement: temperature measured continuously, pH at the beginning and the end of the test.

OTHER TEST CONDITIONS
- Sterile test conditions: no information
- Adjustment of pH: none
- Photoperiod: continuous illumination
- Light intensity and quality: 86 to 93 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: 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: 3.2
* Replicates 6 replicates of the control.
* 3 replicates of each test concentration.
* 1 or 2 replicate of each test concentration without algae.

- Range finding study
- Test concentrations: 0.1, 1.0 and 10 mg/L
- Results used to determine the conditions for the definitive study: yes.
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.:
12 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% C.L.: 11-12 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.2 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:
- EC50 (yield) = 4.9 mg T000263 /L
- EC10 (growth rate) = 6.2 mg T000263 / L (95% C.L. 5.8-6.6 mg/L).
- EC10 (yield) = 2.0 mg T000263 / L (95% C.L. 1.7-2.2 mg/L).
- NOEC (yield) = 1.0 mg T000263 / L.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 72-h EC50 (growth rate) = 1.0 mg/L (95% C.L.1.0 to 1.1 mg/L)
Reported statistics and error estimates:
Calculation of ECx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding TWA concentrations of the test item. The calculations were performed with ToxRat Professional v. 3.0.0 (ToxRat Solutions® GmbH, Germany).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed wit the test substance T000263 according to the OECD guideline 201. Under the test conditions, T000263 inhibited growth rate of this fresh water algae species significantly. The 72-h ECr50 was determined to be 12 mg/L.
The results of the test can be considered reliable without restrictions.