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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:
2017-01-09 - 2017-03-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
OECD Guideline for Testing of Chemicals No. 201, “Freshwater Alga and Cyanobacteria, Growth Inhibition Test” adopted 23 March 2006, Annex 5 corrected: 28 July 2011.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
LUBW Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg, Postfach 10 01 63, 76231 Karlsruhe

Test material

Constituent 1
Chemical structure
Reference substance name:
3-[(aminoiminomethyl)thio]propanesulphonic acid
EC Number:
244-520-8
EC Name:
3-[(aminoiminomethyl)thio]propanesulphonic acid
Cas Number:
21668-81-5
Molecular formula:
C4H10N2O3S2
IUPAC Name:
3-(carbamimidoylsulfanyl)propane-1-sulfonic acid
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient (≤ + 30 °C), dark, dry

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling method: Analytical data are required by the guidelines for verification of test item concentrations as well as the stability of the test item over the entire test period. Analytical samples were taken at 0 hours (initial value) from fresh test solutions and after 72 hours from aged solutions from the highest test item concentration and from the control. For each sampling also a retain sample was taken.
- Sample storage conditions before analysis: After sampling, the test medium samples (10 mL) were stored deep-frozen (≤ - 18 °C) until analysis. In the analytical laboratory, the samples were thawed to ambient temperature, mixed with 10 mL acetonitrile and shaken well using a Vortex-Mixer. If necessary, the samples were further diluted with acetonitrile/water (1:1, v/v) prior to analysis by HPLC-MS/MS.

Test solutions

Vehicle:
no
Remarks:
Test medium was used.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The necessary amount of test item for preparing the stock solution S1 (100 mg/l) was weighed on a weighing scoop and transferred to a volumetric flask. Test medium was added up to the bench mark and the solution was homogenised by shaking. Afterwards the solution was clear and transparent. Lower test solutions were prepared by dilution of appropriate solutions with test medium. All stock and test solutions were prepared with AAP-medium containing algae cells with a density of 0.5 × 10exp4 cells per mL.
- Controls: medium only
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none stated

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: Desmodesmus subspicatus Chodat (Sphaeropleales: Scenedesmaceae), strain: SAG 86.81
- Source (laboratory, culture collection): purchased from a commercial supplier, e.g. MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany
- Age of inoculum (at test initiation): 3 to 4 days before start of the test, test medium was inoculated with the test organism and held under test conditions in order to produce a pre-culture in the state of exponential growth.
- Method of cultivation: The algae are grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state. Stock cultures are ordered regularly from the commercial supplier.

ACCLIMATION
- Culturing media and conditions (same as test or not): same
- Any deformed or abnormal cells observed: none stated

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Remarks on exposure duration:
none
Post exposure observation period:
none

Test conditions

Test temperature:
22.5 – 23.0 °C
pH:
7.43 – 8.43 (pH of control
Nominal and measured concentrations:
The following nominal concentrations were tested in the limit test (range-finding test in limit test design) (spaced by a factor of 10) (nominal): 100, 10.0, 1.00, 0.100, 0.0100 mg/L and control.
Concentrations of nominal 100 & 0 mg/l were analytically determined to:
95.7, n.d. mg/l (main test, fresh medium, measured)
97.3, n.d. mg/l (main test, 72h aged medium, measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks (100 mL) with aluminium caps were filled up with ~ 50 mL test solution.
- Aeration: CO2 was supplied by continuous agitation: Test vessels were placed in an incubator on a pivoted bogie which turns around and induces shaking by regular sudden stops
- Initial cells density: 0.5 x 10exp4 cells/mL
- Control end cells density: 36.19 x 10exp4 cells/mL
- No. of vessels per concentration (replicates): 3 (6 for 100 mg/l)
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: Not specified; the algae are grown semi-continuously in sterile cultures in the laboratory.
- Adjustment of pH: The pH was adjusted to 7.5 ± 0.1 with NaOH or HCl, if necessary.
- Photoperiod: Continuous
- Light intensity and quality: Continuously from the side, 79.7 µ Em-2 s-1 (mean)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The daily fluorescence measurements were performed with a fluorescence microplate reader (infinite 200Pro) with an emission wavelength of 670 nm and evaluated with Tecan i-control (Software for Tecan Readers Tecan i-control, 1.11.1.0).
At defined dates (24, 48 and 72 hours), the number of cells in each replicate was determined in duplicate. The determination was performed by fluorescence measurement.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline: Results were determined in a preliminary range-finding test. No statistically significant inhibitory effects on any parameter (growth rate, yield) were observed at any of the test item concentrations, so the range-finding test was considered sufficient to serve a the full test.
- Range finding study
- Test concentrations: 100, 10.0, 1.00, 0.100, 0.0100 mg/L and control.
- Results used to determine the conditions for the definitive study: n/a
Reference substance (positive control):
yes
Remarks:
Routine Toxic Reference Test with Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other:
Remarks:
growth rate & yield
Duration:
72 h
Dose descriptor:
LOEC
Remarks on result:
not determinable
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other:
Remarks:
growth rate & yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other:
Remarks:
growth rate & yield
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:
Remarks:
growth rate & yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none stated
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: none
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: ErC50 = 0.977 mg/l; EyC50 = 0.464 mg/l, EbC50 = 0.536 mg/l
- Other: NOEC = 0.192 mg/l

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The study was conducted under GLP according to OECD guideline 201 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of UPS towards algae.
No statistically significant inhibitory effects on any parameter (growth rate, yield) were observed at any of the test item concentrations, including the highest test item concentration of 100 mg/L (nominal) at test end. Thus the overall LOEC was not determinable and the overall NOEC was observed to be at 100 mg/L (nominal).
The EC10-, EC20- and EC50-value for growth rate (ErC10, 20, 50) and the EC10-, EC20- and EC50-value for yield (EyC10, 20, 50) were >100 mg/L (nominal).
Based on the obtained results, 3-[(amino-iminomethyl)-thio]-1-propanesulphonic acid does not need to be classified as hazardous to the aquatic environment, neither acute nor chronic, according to the Regulation (EC) No. 1272/2008.
Executive summary:

The objective of this study according to OECD 201 under GLP was to determine the effects of 3-[(amino-iminomethyl)-thio]-1-propanesulphonic acid on the growth of the single cell green alga Desmodesmus subspicatus, to determine the no observed effect concentration (NOEC), to determine the lowest observed effect concentration (LOEC) and to determine the effect concentration (EC10, 20, 50), where possible.

 

Material and methods:

Test species: Desmodesmus subspicatus.

Test design: Initial cell densities of 0.5 × 104 cells/mL were employed for the individual replicates. The increase of cell numbers was assessed over a test period of 72 hours.

Endpoints: Inhibition of growth was assessed by the determination of NOEC/LOEC and EC10, 20, 50 for growth rate and yield after 72 hours.

Test rates: A static limit test (range-finding test in limit test design) with nominal concentrations of 100, 10.0, 1.00, 0.100, 0.0100 mg/L and control was performed.

Test conditions: Six replicates were employed for the control and the highest test item concentration and three for the other test item concentrations. The test was performed in 100 mL Erlenmeyer flasks each containing ~ 50 mL test solution. The pH was recorded at test start and test end. Temperature was measured continuously over the whole test period and recorded daily. Light intensity of the continuous illumination was measured at test start.

Samples analysed: Analytical samples were taken and analysed at 0 hours (initial value) from fresh test solutions and after 72 hours from aged solutions from the highest test item concentration and from the control

Statistics: Where possible NOEC and LOEC were determined by using a multiple comparison method and EC10, 20, 50-values were determined by probit analysis.

 

Findings:

Validity criteria: Biomass: Cell numbers, measured in the controls between 0 h and 72 hours, were found to increase by a factor of 55.68 for the control, which exceeds the threshold of 16. It corresponds to a growth rate of 1.34149 d-1.

Coefficient of Variation (section by section): The mean coefficient of variation for the section-by-section specific growth rates (hours 0 - 24, 24 - 48 and 48 - 72) in the control cultures was 19 % for the control and did not exceed 35 %.

Coefficient of Variation: (average growth): The coefficient of variation of average growth in replicate control cultures was 1.3 % for the control and did not exceed 7 % for the whole test period.

Test conditions: The pH-value of the control ranged from 7.43 to 8.43 during the test period, the temperature was measured to be 22.5 – 23.0 °C during the test and the mean light intensity was 79.7 µEm-2s-1 at cell culture level.

Analytical Results: The measured initial concentration of UPS was 96 % of nominal. In the aged samples the measured content was 98 % of nominal. Therefore toxicological endpoints were evaluated using the nominal concentrations of the test item.

Statistical Results: EC10, 20, 50- and NOEC-values of the test organism exposed to the test item evaluated using nominal concentrations

 

Test item [mg/L]

 

nominal

ErC10 (Growth rate)

> 100

ErC20

> 100

ErC50

> 100

EyC10 (Yield)

> 100

EyC20

> 100

EyC50

> 100

NOEC 1)

100

LOEC 1)

-

1) Following Dunnetts-t-test (left-sided, p<0.05) for growth rate and for yield

- No NOEC was observed

 

Conclusions: No statistically significant inhibitory effects on any parameter (growth rate, yield) were observed at any of the test item concentrations, including the highest test item concentration of 100 mg/L (nominal) at test end. Thus the overall LOEC was not determinable and the overall NOEC was observed to be at 100 mg/L (nominal).

The EC10-, EC20- and EC50-value for growth rate (ErC10, 20, 50) and the EC10-, EC20- and EC50-value for yield (EyC10, 20, 50) were >100 mg/L (nominal).

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