Ethanol, 1-[2-butyne-1,4-diylbis(oxy)][2,2,2-trichloro-

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Definitions to explain the test parameters chosen:
Yield (according to OECD Guideline 201) is defined as the biomass at the end of the exposure period minus the biomass at the start of the exposure period.
Growth and growth inhibition are quantified from measurements of the algal biomass as a function of time. Algal biomass is defined as the dry weight per volume, e.g. mg algae/L test solution. However, dry weight is difficult to measure and therefore surrogate parameters are used. Of these surrogates, cell counts are most often used.

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP-Conformity All procedures according to the principles of GLP (Chemikaliengesetz §19a and §19b and annexes 1 and 2 from 28. Aug. 2013, published in Federal Law Gazette, Germany (BGBl) No. 55/2013 as of 06. Sep. 2013, and further revisions).

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: The test material is representative of the registered substance, and no significant differences in the purity profile exist
- Expiration date of the lot/batch: not relevant (stable)
- Purity test date: not relevant, the test material is representative of the registered substance

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble
- Reactivity of the test substance with the solvent: none (contains water)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: The test material is representative of the registered substance
- Expiration date of the lot/batch: not relevant
- Purity test date: not relevant

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

At the beginning and at the end of the experiment, the DOC in the test solution is analytically determined.
The following instruments and devices will be used in the test. Specifications will be stated in the final report.
• Data logger for temperature
• Analytical scales
• Luxmeter
• Adjustable pipettes with one-way tips
• pH-Meter
• Cell Counter
• Microscope
• Autoclave
• Orbital shaker
• Carbon analyser
Standard laboratory equipment will also be used.
Usage and, if applicable, calibration of all instruments following the corresponding SOP in the current edition.

Test solutions

Vehicle:

no

Details on test solutions:

The tabulated test solutions are reported in the tabulatable text feields.
The weighted mineral concentrations may vary within ± 5 %.
The given volumes are exemplary for the composition of the medium. The real volumes depend on the needed final volume and are stated in the raw data.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

SAG Strain Number 86.81
Taxonomic position Chlorophyta - Chlorophyceae
Origin and Culture
The culture of Desmodesmus subspicatus was obtained from 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.
Pre-culture
3 or 4 days before the start of each experiment, an aliquot of the permanent culture is brought into nutrient medium. The mixture is incubated for 72 - 96 hours at 21.0 –24.0 °C and 4440 - 8880 Lux. The resulting pre-culture is growing exponentially.
Before usage, the culture will be checked for the absence of cell aggregates.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

72 ± 3 hours

Test conditions

Hardness:

Composition of the Solutions (all solutions are used sterilised):
3.4.1 Stock Solution I
NH4Cl 1500 mg
MgCl2*6H2O 1200 mg
CaCl2*2H2O 1800 mg
MgSO4*7H2O 1500 mg
KH2PO4 160 mg
H2O deionised ad 1000 mL
3.4.2 Stock Solution II
FeCl3*6H2O 64 mg
Na2EDTA*2H2O 100 mg
H2O deionised ad 1000 mL
3.4.3 Stock Solution III
H3BO3 185 mg
MnCl2*4H2O 415 mg
ZnCl2 3 mg
CoCl2*6H2O 1.5 mg
CuCl2*2H2O 0.01 mg
Na2MoO4*2H2O 7 mg
H2O deionised ad 1000 mL
3.4.4 Stock Solution IV
NaHCO3 50 g
H2O deionised ad 1000 mL
3.4.5 Algal medium
stock solution I 10.0 mL
stock solution II 1.0 mL
stock solution III 1.0 mL
stock solution IV 1.0 mL
H2O deionised ad 1000 mL

The weighted mineral concentrations may vary within ± 5 %.
The given volumes are exemplary for the composition of the medium. The real volumes depend on the needed final volume and are stated in the raw data.

Test temperature:

21.0 – 24.0 °C during 4440 – 8880 Lux

Dissolved oxygen:

not measured

Salinity:

not measured (see hardness)

Conductivity:

not measured

Nominal and measured concentrations:

3.2 / 10 / 32 / 100 / 320 / 1000 mg/L (nominal)
The concentrations tested were based on non-GLP pre-tests
Treatments: 3 vessels, each filled with 45 ± 1 mL test solution and algae
Blank Control: 6 vessels, each filled with 45 ± 1 mL algal medium and algae

Details on test conditions:

This study is performed in order to evaluate the toxicity of the test item towards Desmodesmus subspicatus. This freshwater green alga is chosen as a typical species of phytoplankton.
The system response is the reduction of growth in a series of algal cultures (test units) exposed to the test item. The response is evaluated as a function of the exposure concentration in comparison with the average growth of replicate, unexposed blank control cultures. For full expression of the system response to toxic effects (optimal sensitivity), the cultures are allowed unrestricted exponential growth under nutrient sufficient conditions and continuous light for a sufficient period of time to measure reduction of the specific growth rate.

A positive control was tested in a separate study to assure that the test conditions are reli-able.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7, CAS No. 7778-50-9)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC10

Remarks:

yield

Effect conc.:

94.61 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks:

yield

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

205.94 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Details on results:

Abbreviations
NOEC Highest concentration of the test item causing no significant inhibition of the respective parameter
LOEC Lowest concentration of the test item causing significant inhibition of the respective parameter
72h-ErC50 Concentration of the test item inhibiting the growth rate by 50% after an exposition time of 72 h.
72h-EyC50 Concentration of the test item inhibiting the yield by 50% after an exposition time of 72 h.
72h-ErC10 Concentration of the test item inhibiting the growth rate by 10% after an exposition time of 72 h.
72h-EyC10 Concentration of the test item inhibiting the yield by 10% after an exposition time of 72 h.

Any other information on results incl. tables

Increase Factor

The cell concentration in the blank control should increase by a factor of at least 16 within 72h.

Daily Growth Rates

Mean coefficient of variation of daily growth rates in the blank controls should be 35% at the most.

Coefficient of variation of average growth rate during the whole test period should be 7% at the most.

Values and assessment

The daily growth rates of the blank controls were calculated. Means, standard deviations and coefficients of variation were determined. Values and assessment can be found in the following tables.

Daily Growth Rates of the blank controls

Growth rates	days 0 – 1	days 1 – 2	days 2 – 3	CV of sectional daily growth rates	days 0 – 3
Replicate 1	1.391	1.845	1.832	15%	1.689
Replicate 2	1.244	2.104	1.853	25%	1.734
Replicate 3	1.456	1.992	1.796	15%	1.748
Replicate 4	1.395	2.002	1.854	18%	1.750
Replicate 5	1.331	2.030	1.798	21%	1.720
Replicate 6	1.212	2.094	1.823	26%	1.710
Mean	1.338	2.011	1.826	20%	1.725
Standard deviation	0.095	0.094	0.025		0.024
CV	7%	5%	1%		1%

CV = Coefficient of Variation

 

Assessment

Parameter	Validity criteria	Observed value	Assessment
Increase factor biomass	factor 16 in 72 h	177	Valid
Mean coefficient of variation of daily growth rates	max. 35%	20%	Valid
Coefficient of variation of average growth rate during the whole test period	max. 7%	1%	Valid

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The preliminary reults reported are
Endpoint NOEC LOEC EC10 EC50
Growth Rate 32.80 mg/L 105.50 mg/L 205.94 mg/L 573.79 mg/L
Yield 32.80 mg/L 105.50 mg/L 94.61 mg/L 224.30 mg/L
The results of NOEC and LOEC are not valid because they are derived form inappropriate concentration data and are in contrast to the conclusions drawn by the Laboratory in regard to biological variability e.g. in the summary.

Executive summary:

The study was performed using 6 concentrations ranging from 3.2 to 1000 mg/L. 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 algae were observed in the incubations at 320 and 1000 mg/L for both growth rate and yield. The EC10 were calculated to be 95 mg/L for yield and 206 mg/L for growth rate.

In the highest concentrations a clear dose-response relationship was observed. The slightly increased inhibition of yield in the lowest concentrations is due to biological variability and is negligible.