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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:
2018/2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth 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:
Dry powder, white to slightly yellow
Specific details on test material used for the study:
Appearance: White to slightly yellow powder
Batch: Q0926501
Purity/Composition: 99.3%
Test item storage: At room temperature protected from light
Stable under storage conditions until: 30 November 2019 (expiry date)
Solubility in water: Approximately 83 g/100g at 25°C

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
* Sampling for Analysis of Test Concentrations:
Frequency: at t=0 h, t=24 h and t=72 h.
Volume: 1.0 mL from the approximate centre of the test vessels.
Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
Analysis was based on the analytical method validated for the test item in project 20152801.
Instrument: Acquity UPLC system (Waters, Milford, MA, USA)
Detector: Acquity Refractive Index detector (Waters)
Column: Acquity UPLC BEH Amide, 50 mm x 2.1 mm i.d., dp =1.7 μm (Waters)
Column temperature: 60°C +/- 1°C
Injection volume: 20 μL
Mobile phase: 80/20 (v/v) 0.4% ammonia solution in acetonitrile/0.4% ammonia solution in water
Flow: 0.250 mL/min
RI detection: Temperature = 30°C +/- 5°C; Time constant = 0.2 seconds; Polarity = positive
Test samples were stored in the freezer (≤ -15°C). Storage stability of samples under these conditions was demonstrated in project 20152801.
On the day of analysis, the test samples were thawed at room temperature. The samples were diluted in a 1:4 (v:v) ratio with acetonitrile and analyzed.

* Sampling for TOC Analysis of Test Concentrations:
Samples for possible analysis were taken from the limit test concentration and the control from solutions incubated without algae according to the schedule below.
Frequency: at t=0 h and t=72 h.
Volume: 40 mL from the approximate centre of the test vessels.
Storage: Samples were stored in a refrigerator (2-8°C) until analysis.
Apparatus: Samples were injected into a Shimadzu TOC-L total organic carbon analyser combined with a Shimadzu ASI-V auto sampler (Shimadzu Kyoto, Japan). Each sample was analysed in triplicate. TOC values were determined as the Total Carbon values in the pre-treated solutions minus the measured Inorganic Carbon (TOC = TC- IC).
Calibration solution(s): TC standard: Potassium hydrogen phthalate solution (C8H5KO4, Merck, Darmstadt, Germany); IC standards: Sodium carbonate (Na2CO3, p.a., Merck, Darmstadt, Germany) and Sodium Bicarbonate (NaHCO3, p.a.Merck, Darmstadt, Germany).
Analysis set-up: No. of repeats: at least 3

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), strain: NIVA CHL 1

Source: In-house laboratory culture.

Reason for selection: This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

Fresh water algae stock culture: 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.

Pre-culture 3 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

Test temperature:
Temperature was continuously recorded in a temperature control vessel.
pH:
ph was :
- for control: 8.2 at t=0 and 8.3 at t=72h
- for the 100 nominal concentration: 8.0 at t=0 and 8.2 at t=72h
Nominal and measured concentrations:
Measured concentrations reached 96% to nominal concentrations.
Details on test conditions:
A range-finding test was performed to provide information about the range of concentrations to be used in the final test. Test procedure and conditions were similar to those applied in the limit test with the following exceptions:
- Three replicates of exponentially growing algal cultures were exposed to a range of 0.10
to 100 mg/L increasing by a factor of 10 and to a control.
- Cell densities were recorded only at the end of the exposure period.
- One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval.
- pH was only measured in the control and the highest test concentration.
- No sampling for determination of actual concentrations was performed.

* Control(s): Test medium without test item or other additives.
* Replicates: 6 replicates of the test concentration, 6 replicates of the control, 2 or 3 replicates of the test group without algae. In addition, a solution of 1000 mg/L without algae was added to the test for sampling purposes at the start, after 24 hours and at the end of the test.
* Test vessels: 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution
* Test Medium: M2
* Cell density: An initial cell density of 1 x 10^4 cells/mL.
* Illumination: Continuously using TLD-lamps with a light intensity within the range of 81 to 83 μE.m-2.s-1.
* Incubation: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The 72h-EC50 for growth rate inhibition (ERC50) was beyond the range tested, i.e. exceeded the regulatory limit concentration of nominally 100 mg/L.
The 72h-EC50 for yield inhibition (EYC50) beyond the range tested, i.e. exceeded the regulatory limit concentration of nominally 100 mg/L.
The 72h-NOEC for growth rate inhibition was 100 mg/L based on statistical significance.
The 72h-NOEC for yield inhibition was 100 mg/L based on statistical significance.
Executive summary:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata, no inhibition of growth rate or inhibition of yield was recorded at any of the concentrations of D-ribose.