Registration Dossier

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:
2018-07-17 - 2018-07-20 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
OECD Guideline for the Testing of Chemicals No. 201, adopted 23. Mar. 2006, Annex 5 corrected: 28 July 2011 “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”
Deviations:
yes
Remarks:
but not affecting the integrity or validity of the study
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Regulation EU No. 2016/266 amending Regulation EC No. 440/2008, Annex IV, Method C.3: “FRESHWATER ALGAE AND CYANOBACTERIA, GROWTH INHIBITION TEST,” adopted 07. December 2015
Deviations:
yes
Remarks:
but not affecting the integrity or validity of the study
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
The test item was stored in the test facility in a closed vessel at room temperature (20 ± 5°C).

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 9.2, 20, 44, 92, 200 mg/L (test item)
- Sample storage conditions before analysis: no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution containing 200.4 mg/L test item (= 100.2 mg/L dry weight test item) in algal medium (demineralised water enriched with minerals but without algae) was prepared.
Lower test concentrations were prepared by dilution of the stock solution in algal medium.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

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). 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:
23.1 – 26.2 °C
pH:
7.6 - 8.0
Nominal and measured concentrations:
Nominal concentration: 0 / 9.2 / 20 / 44 / 92 / 200 mg/L nominal of test item wet weight / 0 / 4.6 / 10 / 22 / 46 / 100 mg/L nominal of test item dry weight
Measured concentration, t = 0 h: n.d. / n.d. / 25.57 / 53.80 / 97.86 / 203.42 mg/L test item wet weight
Measured concentration, t = 72 h: n.d. / n.d. / 33.98 / 56.10 / 102.09 / 242.90 mg/L test item wet weight
Details on test conditions:
TEST SYSTEM
- Test vessel: flasks
- Type (delete if not applicable): open (covered with perforated plastic foil acting as a stopper)
- Material, size, headspace, fill volume: glass flasks total volume 65 mL, fill volume 45 ±1 mL
- Renewal rate of test solution (frequency/flow rate): none
- Initial cells density: 2960 cells/mL
- Control end cells density: 850253 cells/mL
- No. of vessels per concentration (replicates): 3 replicates for each treatment
- No. of vessels per control (replicates): 6 replicates for the blank control
- No. of vessels per vehicle control (replicates): n/a

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 (daily)
- Chlorophyll measurement: no
- Other: pH values were determined at 0h and 72h

TEST CONCENTRATIONS
The concentrations to be tested are based on non GLP pre-tests
- Range finding study
- Test concentrations / Results used to determine the conditions for the definitive study: No growth inhibition occurred at 2 / 20 / 200 mg/L test item wet weight (= 1 / 10 / 100 mg/L test item dry weight).
Reference substance (positive control):
yes
Remarks:
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a separate reference test.

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 222.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
50% aqueous solution
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 222.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
50% aqueous solution
Basis for effect:
biomass
Remarks:
yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 222.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
50% aqueous solution
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 222.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
50% aqueous solution
Basis for effect:
biomass
Remarks:
yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 222.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
50% aqueous solution
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 222.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
50% aqueous solution
Basis for effect:
biomass
Remarks:
yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 222.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
50% aqueous solution
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 222.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
50% aqueous solution
Basis for effect:
biomass
Remarks:
yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 111.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
HBOPS-Na
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 111.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
HBOPS-Na
Basis for effect:
biomass
Remarks:
yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 111.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
HBOPS-Na
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 111.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
HBOPS-Na
Basis for effect:
biomass
Remarks:
yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 111.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
HBOPS-Na
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 111.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
HBOPS-Na
Basis for effect:
biomass
Remarks:
yield
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 111.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
HBOPS-Na
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 111.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
HBOPS-Na
Basis for effect:
biomass
Remarks:
yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none
- Any stimulation of growth found in any treatment: not consistently
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: none
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50:
Parameter Value 95% confidence interval
72h ErC50 0.90 mg/L 0.77 – 1.1 mg/L
72h EyC50 0.42 mg/L 0.35 - 0.51 mg/L
Reported statistics and error estimates:
Calculation of results was performed with the help of validated software (Microsoft Excel ®). However, no statistical evaluation was performed, as no inhibition occured in the highest concentrated treatment.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The study was conducted under GLP according to OECD guideline 201 and EU method C.3 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 2-Butyne-1,4-diol, reaction products with 1,2-oxathiolane 2,2-dioxide and sodium hydroxide (HBOPS-Na) towards algae.
In the lowest concentration no test item was detectable. In the remaining ones, the measured concentrations lay between 102 % and 128 % of the nominal concentrations at the beginning of the test and between 111 % and 170 % of the nominal concentrations at the end of the test. Treatments with 9.2 and 20 mg/L were below the calibration range. The higher deviations from the nominal concentrations were observed only at these lower concentrations. Determinations of the higher ones incl. the top concentration were more accurate. As no inhibition of growth was observed and the NOEC was determined as ≥ 222.3 mg/L (nominal) resp. 115.15 mg/L (measured) of the dry test item, the integrity of the results is given. Therefore, the determination of the results was based on the geometric mean of the measured concentrations.
As no inhibition of algal growth was observed in all tested concentrations up to 111.15 mg/L HBOPS-Na, it does not need to be classified as hazardous to the environment, neither acute nor chronic, according to Regulation 1272/2008 and amendments.
Executive summary:

The study was conducted under GLP according to OECD guideline 201 and EU method C.3 on the registered substance itself.

One valid experiment was performed.

The study was performed using 5 concentrations ranging from 9.2 to 200 mg/L nominal concentrations (= 4.6 – 100 mg/L dry weight test item). 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.

Inhibition occurred only slightly and the values scattered within the different concentrations. In the highest concentration, however, no inhibition occurred, no dose-response was obvious. Therefore, no statistical evaluation was performed. At the start and at the end of the test, the content of the test item in the test solutions was determined using HPLC-UV.

In the lowest concentration no test item was detectable. In the remaining ones, the measured concentrations lay between 102 % and 128 % of the nominal concentrations at the beginning of the test and between 111 % and 170 % of the nominal concentrations at the end of the test. Treatments with 9.2 and 20 mg/L were below the calibration range. The higher deviations from the nominal concentrations were observed only at these lower concentrations. Determinations of the higher ones incl. the top concentration were more accurate.

As no inhibition of growth was observed and the NOEC was determined as ≥ 222.3 mg/L (nominal) resp. 111.15 mg/L (measured) of the dry test item, the integrity of the results is given. Therefore, the determination of the results was based on the geometric mean of the measured concentrations.

Therefore the results are given as a range. In addition, the results are also given in relation to the dry weight (50%) of the substance.

The 72h-EC50 values of potassium dichromate (K2Cr2O7, CAS No. 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 2-Butyne-1,4-diol, reaction products with 1,2-oxathiolane 2,2-dioxide and sodium hydroxide (HBOPS-Na) were determined:

 

Table Results of the test item (based on weighed-in test item)

Endpoint

NOEC

LOEC

EC10

EC50

Growth Rate

≥ 222.3 mg/L

> 222.3 mg/L

> 222.3 mg/L

> 222.3 mg/L

Yield

≥ 222.3 mg/L

> 222.3 mg/L

> 222.3 mg/L

> 222.3 mg/L

 

Table Results of the test item (based on 50% dry weight test item)

Endpoint

NOEC

LOEC

EC10

EC50

Growth Rate

≥ 111.15 mg/L

> 111.15 mg/L

> 111.15 mg/L

> 111.15 mg/L

Yield

≥ 111.15 mg/L

> 111.15 mg/L

> 111.15 mg/L

> 111.15 mg/L

 

HBOPS-Na does not need to be classified as hazardous to the environment, neither acute nor chronic, according to Regulation 1272/2008 and amendments.