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Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-Mar-22 - 2021-Mar-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Due to UCVB substance with constituents of different water solubility, guidance documents OECD 23 and ASTM d6081-19 were used

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

28th July 2011

Deviations:

yes

Remarks:

according to OECD No. 23 and ASTM D6081 – 19, because of poorly soluble test item

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: information provided by sponsor, Batch no. 0320SA017,
- Purity, including information on contaminants, isomers, etc.: not applicable, UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (20± 5 °C) in tightly closed vessel under dry, dark conditions
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: homogeneous, stable under storage conditions
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: not stated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): preparation of water-accommodated fractions: nominal loading (100 / 32 / 10 / 3.2 / 1 mg/L)added to cellulose ester filter. Filters were put on decanting bottles filled with algal medium. Stirring for 23h20 min on magnetic stirrer under closed conditions. Settling period of 1h, lower phase was used without filtration/centrifugation, first few mL were discarded
- Final concentration of a dissolved solid, stock liquid or gel: 101.3 / 32.0 / 10.6 / 3.1 / 1.0 mg/L

Analytical monitoring:

yes

Remarks:

Only the component C16 was used for quantitative evaluation

Details on sampling:

750 µL aqueous sample were mixed with 250 µL THF in a vial. If necessary, further dilutions were performed using THF/ water 25/75 (% v/v) with a maximum dilution factor of 10667

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: direct application via cellulose ester filter and 24 h stirring under closed conditions
- Eluate:
- Differential loading:
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):-
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): -
- Test concentration separation factor: default, nominal loadings 100 / 32 / 10 / 3.2 / 1.0 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.):At the 2 highest concentrations, the resulting solutions were clearly turbid, but homogeneous.
- Other relevant information:

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: 86.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen)
- Age of inoculum (at test initiation): a pre-culture was made by using an aliqout of permanent culture (made from stock culture) into nutrient medium and incubated under continuous lighting for 4 days under test conditions (Lighting: within the specified range of 4440 – 8880 lux; 22.1 – 22.8 °C)

ACCLIMATION
- Acclimation period: settling period of 1h for permanent culture before making pre-culture
- Culturing media and conditions (same as test or not): same
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.1 – 22.8 °C

pH:

7.4 - 7.9

Nominal and measured concentrations:

1 / 3.2 / 10 / 32 / 100 mg/L nominal concentrations based on non GLP pre-test,
1.0/ 3.1/ 10.6/ 32.0/ 101.3 mg/L measured concentrations

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks, total volume 65 mL
- Type (delete if not applicable): open (covered with perforated plastic foil acting as stopper)
- Material, size, headspace, fill volume: glass, 65mL size, filled up to 45 ± 1 mL
- Aeration: shaking on orbital shaker to ensure suspension
- Initial cells density: 2.6 *10^3 cells/mL
- Control end cells density: after 1:20 dilution 24013 - 27757 cells/mL
- No. of vessels per concentration (replicates): 3 each
- No. of vessels per control (replicates): 6 each
- No. of vessels per analytical determination (replicates): 1 each for treatment and control

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: not indicated
- Photoperiod: continuous light for sufficient period
- Light intensity and quality: 4440 – 8880 Lux, checked monthly

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter, every 24 ± 1 hours
- Chlorophyll measurement: not indicated

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: not indicated
- Test concentrations: 100 / 32 / 10 / 3.2 / 1.0 mg/L (nominal)

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7, under GLP conditions, May 2020

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

71.12 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks:

95% confidence interval: 27.27 - 185.50 mg/L (extrapolated by program)

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

32 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

32 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Deviations from study plan: turbidity in higher concentrations (32 and 100mg/L), cell numbers were also measured in test solutions and control without algae; measurement of test item at beginning and after 48, 72h was intended but 24h measurement was forgotten. Both deviations were deemed non-critical
- Deviations from Guideline: pre-culture was prepared 5 days before test start; stock solutions were autoclaved 20 instead of 15 minutes. Both deviations were deemed non-critical

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 72h ErC50 1.00 mg/L (c.i. 0.97 – 1.03 mg/L), 72h EyC50 0.35 mg/L (c.i. 0.32 – 0.39 mg/L)
- Other:

Reported statistics and error estimates:

Microsoft Excel, ToxRat Professional version 3.3.0

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings in this study a NOEL of 10 mg/L (nominal) was determined for the growth rate of Desmodesmus subspicatus.

Executive summary:

The valid test was performed with the poorly soluble UVCB substance with constituents of different water solubility, so water-accomodated fractions were prepared. Five loading rates ranging from 1 - 100mg/L were used. Due to turbidity, the measurement of cell numbers were falsified and also measured in solutions without algae.

For results for both endpoints (growth rate and yield), the measured numbers at the beginning in the control solution was used as start cell concen-tration for all replicates and the turbidity-based cell number in the solutions without algae was subtracted from the measured cell numbers in the test solutions with algae in the three highest tested concentrations at all measurement points.
Slight but statistically significant inhibition of algal growth was observed at the two high-est concentrations 100 mg/L and 32 mg/L.

At the start, after 48 hours and at the end of the test, the content of the test item in the test solutions was determined using LC-MS-MS. The measured concentrations lay between 63 % and 90 % of the nominal concentrations.

Description of key information

The test item is a poorly soluble UVCB substance with constituents of different water solubility. Therefore, water-accommodated fractions (WAF) were prepared for the test.

The study was performed in accordance with OECD guideline 201 under static conditions and is considered reliable without restriction (Klimisch 1). Five loading rates ranging from 1 - 100mg/L were used. Due to turbidity, the measurement of cell numbers could not be directly determined. For both endpoints (growth rate and yield), the measured numbers at the beginning in the control solution was used as start cell concentration for all replicates and the turbidity-based cell number in the solutions without algae was subtracted from the measured cell numbers in the test solutions with algae in the three highest tested concentrations at all measurement points.

Slight but statistically significant inhibition of algal growth was observed at the two highest concentrations. Therefore, the EL10 and EL50 (growth rate) were determined to be >100 mg/L and the NOEL (growth rate) was determined to be 10 mg/l.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L