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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
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
adopted 23. Mar. 2006, Annex 5 corrected: 28 July 2011 “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”
Deviations:
yes
Remarks:
see "Principles of method if other than guideline"
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
adopted 07. December 2015
Deviations:
yes
Remarks:
see "Principles of method if other than guideline"
Qualifier:
according to guideline
Guideline:
other: OECD guidance document no. 23
Version / remarks:
second edition, GUIDANCE DOCUMENT ON AQUATIC TOXICITY TESTING OF DIFFICULT SUBSTANCES AND MIXTURES, adopted 14. Dec. 2000
Deviations:
no
Principles of method if other than guideline:
The following deviations were documented:
- The stock solutions to be autoclaved were autoclaved for 20 minutes instead of the required 15 minutes. This can be considered uncritical, as the goal of sterility is achieved.
- The pre-culture was prepared 5 days before the test start. As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical.
GLP compliance:
yes (incl. QA statement)

Test material

Test material form:
solid: particulate/powder

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 1 / 3.2 / 10 / 32 / 100 mg/L
- Sampling method: A saturated solution was prepared for the test. This was done by mixing 100 mg/L with the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and shaking vigorously for 24 h and 25 min. The resulting solution was filtrated through 0.45 µm PTFE filters.
The lower treatments (1 / 3.2 / 10 / 32 mg/L) were prepared by dilution of the stock solution with algal medium.

- Sample storage conditions before analysis: n.a.

Test solutions

Vehicle:
no
Remarks:
algal medium was used
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
For elimination of the algal cells before analytical determination, samples were centrifuged.
Preparation
A saturated solution was prepared for the test. This was done by mixing 100 mg/L with the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and shaking vigorously for 24 h and 25 min. The resulting solution was filtrated through 0.45 µm PTFE filters.
The lower treatments (1 / 3.2 / 10 / 32 mg/L) were prepared by dilution of the stock solution with algal medium.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Unicellular freshwater green alga
- Strain: Desmodesmus subspicatus, SAG Strain Number 86.81
- Source (laboratory, culture collection): The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen).
- Method of cultivation: The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
none

Test conditions

Test temperature:
21.7 – 22.8 °C
pH:
7.6 - 7.7
Nominal and measured concentrations:
1 / 3.2 / 10 / 32 / 100 mg/L nominal concentration
Because of the very low solubility of the test item the measured concentrations at the start and the end of the test lay in the range of the blank control. Because a significant inhibition was observed, the test item was present in the test solutions but not measurable. Therefore, the nominal concentrations were used for the evaluation of the biological results.
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 a stopper)
- Initial cells density: 2500 cells/mL
- Control end cells density: 350020 cells/mL
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 3 for each treatment
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 5000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
Treatments tested: 1 / 3.2 / 10 / 32 / 100 mg/L nominal concentration
- Range finding study / Test concentrations / Results used to determine the conditions for the definitive study: The concentrations to be tested were based on a non-GLP pre-test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
99.72 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
11.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50:
Parameter Value 95% confidence interval
72h ErC50 0.79 mg/L 0.79 – 0.79 mg/L
72h EyC50 0.39 mg/L 0.35 - 0.45 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The study was conducted under GLP according to OECD 202. The available information allows the conclusion that the test was properly conducted, all criteria for acceptability of the test were met, this study was considered to be valid. The following results for the test item Polyperin Y were determined:
Growth rate
72h-NOEC =32 mg/L
72h-LOEC = 100 mg/L
72h-EC10 =99.72 mg/L
72h-EC50 > 100 mg/L

Yield
72h-NOEC =32 mg/L
72h-LOEC = 100 mg/L
72h-EC10 =11.90 mg/L
72h-EC50 > 100 mg/L

Classification:
- Acute hazard: No acute aquatic toxicity recorded at levels up to the limit of water solubility;
- Long-term hazard: No adequate chronic toxicity data available for all three trophic levels.
Substance is nevertheless of concern based on the following findings:
- Poorly soluble substance (WS < 1 mg/L);
- No acute aquatic toxicity recorded at levels up to the limit of water solubility;
- Not rapidly degradable;
- High potential for bioaccumulation (in absence of BCF data, log Kow > 4);
- No evidence on NOEC being > water solubility for all three trophic levels;
- No other evidence of rapid degradation in the environment.
-> "safety net" classification as aquatic chronic 4, H413
Executive summary:

Determination of the toxicity of Polyperin Y against Des-modesmus subspicatus according to OECD 201 resp. EU C.3 (GLP):

One valid experiment was performed. The study was performed using 5 concentrations ranging from 4.6 to 100 mg/L (nominal). Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield  were determined from the cell number at the respective observation times.  Significant inhibition of algal growth was observed only at the highest test concentration of 100 mg/L (nominal).

At the start and at the end of the test, the content of the test item in the test solutions was estimated by determination of the dissolved organic carbon (DOC) content in the test solutions using a carbon analyser.

Because of the very low solubility of the test item the measured concentrations at the start and the end of the test lay in the range of the blank control. Because significant inhibition was observed, the test item must have been present in the test solutions but wasn’t measurable. Therefore, the nominal concentrations were used for the evaluation of the biological results. Due to the low inhibitory values, the EC50 values can only be given as a range.

The 72h-EC50 values of potassium dichromate (K2Cr2O7, CAS 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

The following results for the test item Polyperin Y were determined:

Endpoint

NOEC

LOEC

EC10

EC50

Growth Rate

32 mg/L

100 mg/L

99.72 mg/L

> 100 mg/L

Yield

32 mg/L

100 mg/L

11.90 mg/L

> 100 mg/L

Note: According to the guideline, NOEC is determined by comparing of the respective treatment with the blank control. Statistically insignificant variation is considered as “no observed effect”, although the EC10 which is read from the graph toxicity vs. concentration may lie lower.