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Diss Factsheets

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:
15 September 2015 - 18 September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles

Data source

Referenceopen allclose all

Reference Type:
other: study amendment
Title:
Unnamed
Year:
2021
Report date:
2021
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
5-[(S)-4-Carboxy-4-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-butyrylamino]-2-nitro-benzoic acid
EC Number:
926-197-1
Cas Number:
2561176-48-3
Molecular formula:
C20 H15 N3 O9
IUPAC Name:
5-[(S)-4-Carboxy-4-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-butyrylamino]-2-nitro-benzoic acid
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identification: P-Glupa-C
Appearance: Yellow powder
Batch: 11328700
Purity/Composition: 86.2 weight-% (average content)
Test substance storage: At room temperature
Stable under storage conditions until: 15 May 2016 (expiry date)

Purity/composition correction factor: No correction factor required
Test substance handling: No specific handling conditions required
Volatile: Not indicated
Solubility in water: Not available
Stability in water: Not available

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.

Frequency: at t=0 h, t=24 h and t=72 h
Volume: 4.0 ml
Storage: Samples were stored in a freezer until analysis.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at an intermediate substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Additionally, reserve samples of 4.0 ml were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis

Test solutions

Vehicle:
no
Details on test solutions:
The batch of P-Glupa-C tested was a yellow powder with a purity of 86.2 weight-% (average content) and completely soluble in test medium at the concentrations tested.

Preparation of test solutions started with a loading rate of 100 mg/l applying a 30 minute treatment period with ultrasonic waves to accelerate the dissolving of the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colourless, except the highest concentration which was clear and slightly yellow.

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 10E4.

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Pseudokirchneriella subcapitata
Strain: NIVA CHL 1
Source: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.
Light intensity : 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
nationally accepted species.
Age of inoculum at test initiation: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
Post exposure observation period:
At the end of the final test microscopic observations were performed on the limit concentration to observe for any abnormal appearance of the algae.

Test conditions

Test temperature:
between 22 and 23°C.
pH:
Start of the test:beween 6.9 and 8.1
End of the test: between 7.6 and 8.0
Nominal and measured concentrations:
Nominal concentrations: control and 0.10, 1.0, 10 and 100 mg/l
Measured concentrations:The actual concentrations at 100 mg/l was at the level of nominal (89% of nominal) at the start of the test and remained stable during the test (95-98% of initial, 85-87% of nominal). Consequently, the effects parameters were reported in terms of analytically confirmed nominal concentration.
Details on test conditions:
Since, no effects on growth rate and yield was observed at the highest concentration the test was used as a limit test.

TEST SYSTEM
Type: Static, open
Test vessels: 100 ml, all-glass, containing 50 ml of test solution
Cell density: An initial cell density of 1 x 10^4 cells/ml.
Control end cells density: 1116000 cells/ml.

Replicates:
6 replicates of the control,
6 replicates of the highest (limit) concentration,
3 replicates of each intermediate test concentration,
1 or 2 replicates of each test concentration without algae,
1 extra replicate of each test group for sampling purposes.

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- llumination: Continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt with a light intensity within the range of 77 to 80 µE.m-2.s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
72 h NOErC, 72 h NOEyC, 72 h ErC50, 72 h EyC50

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Test concentrations: control and 0.1, 1.0, 10 and 100 mg/l
Reference substance (positive control):
yes
Remarks:
(potassium dichromate)

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Details on results:
Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.
Results with reference substance (positive control):
The EC50 for growth rate inhibition (72h-ERC50) was 1.2 mg/l with a 95% confidence interval ranging from 1.1 to 1.2 mg/l.
The EC50 for yield inhibition (72h-EYC50) was 0.38 mg/l with a 95% confidence interval ranging from 0.38 to 0.39 mg/l.
Reported statistics and error estimates:
Statistical analysis of the data was not needed as the effects recorded at 100 mg/l were biologically not significant (<10%).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study with Pseudokirchneriella subcapitata, no inhibition of growth rate or inhibition of yield was recorded at the limit concentration.

Both the EC50 for growth rate (72h-ERC50) and yield inhibition (72h-EYC50) were beyond the range-tested, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/l.