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:
Novembre 2017 to April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
yes
Remarks:
see below for adaptation of the OECD 201 guideline.
Principles of method if other than guideline:
A closed-bottle test system was used to reduce losses of volatile components of the test item. The test medium was supplemented with sodium bicarbonate to increase the CO2 concentration in the test medium to achieve maximum algal growth rates without pH drift (Mayer et al. (2000), ISO 14442 (2006)).

Deviating from the guideline the temperature during the exposure period dropped below the required range of 21–24 °C by for about 16 hours at a maximum of 0.2 °C. This deviation has no influence on the integrity of the study results, since all cell growth-related validity criteria were fulfilled.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of 1-chloro, 2-(difluoromethoxy) -1,1,2,3,3,3-hexafluoropropane and 2-difluoromethoxy-1,1,1,2,3,3,3-eptafluoro-propane
EC Number:
947-520-2
Cas Number:
60632-00-0
Molecular formula:
Cl-(C3F6O)-CF2-H and C4F9HO
IUPAC Name:
Reaction mass of 1-chloro, 2-(difluoromethoxy) -1,1,2,3,3,3-hexafluoropropane and 2-difluoromethoxy-1,1,1,2,3,3,3-eptafluoro-propane
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Solvent N1
- Physical state: liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Manufacturer / Batch 24/02/2016
- Expiration date of the lot/batch: 31 July 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In a dry, well ventilated dark location at ambient temperature/10–30°C
- Stability under test conditions: Chemically stable. Due to the presumed high volatility of the test substanceitem, specific measures for avoiding volatilizsation were applied during all the phases of the experimental work.
- Solubility and stability of the test substance in the solvent/vehicle: Not Applicable / No solvent used.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not Applicable

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:
Samples of stock solution (time 0) and test solutions (time 24h, 48h, 72h) were taken to determine the actual test item concentrations in comparison to the nominally applied concentrations.

Control and test solutions were sampled in duplicate of a defined volume (20 mL). The duplicate samples were kept separately as a reserve. The volume of each sample was recorded.

The samples for the analytical determination after 24 and 48 hours exposure of each test concentration were taken from additional test vessels. This measure was applied in order to provide sufficient volume for potential analytical measurements and to avoid removal of high amounts of test solutions from the test replicates used for biological evaluation, which would lead to significantly increased headspace and consequently enhanced losses of the test item from the test vessels while opening the test vessels due to determination of cell density.

At the end of the exposure period (day 3) the samples for the analytical determination of the test concentrations were taken immediately before determination of the biological and physico-chemical parameters, the test solutions of the replicates of a given concentration were mixed

After sampling and before shipment, all samples were stored in glass bottles in the dark at a temperature of ≤ -18°C. A record was kept for each sample.

Samples of untreated test medium were provided to the analytical laboratory in order to determine the recovery of the analytical method with the specific test medium. Samples were transferred to the test site for chemical analysis (address see section 1.3) under the required storage conditions. The storage conditions were established in non-GLP pre-tests.
According to the results of such non-GLP pre-tests the samples are demonstrated to be stable if maintained at a temperature of ≤ -18°C for a storage period of up to 10 weeks. Details are shown in the analytical phase report.

Test solutions

Vehicle:
no
Remarks:
No vehicle used
Details on test solutions:
Medium was OECD-Medium (modified) + additional NaHCO3

PREPARATION AND APPLICATION OF TEST SOLUTIONS
As the test item is of limited water-solubility and assumed to be highly volatile, the preparation procedure was set taking into account aspects of OECD 2000, Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. OECD Series on Testing and Assessment Number 23. ENV/JM/MONO(2000)6.

A saturated stock solution was prepared at a nominal loading rate of 100 mg/L by adding 154 µL of the test item to 2057 g of the test medium. Therefore, a 2 L volumetric flask was filled-up with test medium (2057 g) in excess of the calibration mark to minimize the headspace. The test item was added to the preparation flask using a piston stroke pipette (154 µL) and the time of unlooked preparation flask was keep as short as possible. This stock solution (S1) was slowly stirred at 400 rpm for 22.5 hours at ambient temperature in the dark in a completely closed system with a minimized headspace. Thereafter this stock solution was left to settle for 50 min. The stock solution was visually examined for undissolved/particulate matter in the free water column. At the bottom of the measuring flask some droplets of the test item were still visible.

The stock solution (S1) was prepared once to prepare the test solutions.
The volume of the stock solution (S1) was large enough to prepare the test solutions and all analytical samples and solutions used for conditioning.

The stock solution was used immediately to prepare the desired test item concentrations from the free water column of S1 using volumetric pipettes for removal of S1 solution which was transferred to the respective volumetric flasks (C1–C5) that had been pre-filled as far as operationally possible with an appropriate volume of test medium prior to addition of the required volume of S1.

The stock solution (S1) was removed from the free water column of S1 using volumetric pipettes (30, 40 and 100 mL volumetric glass pipettes), to minimized swirl up of test item particles, and added to the test medium of each test solution volumetric flask. The stock solution and test solution flask was closed with a lid immediately after each removal or addition of the required S1 aliquot, respectively.

The steps involving transfer and dilution of stock solution to the test solutions until closing of the test vessels were performed as quickly as possible in order to minimise air contact for the solutions during the manipulation steps; between removals of stock and test solutions, the respective bottles were closed.

After preparation the test solutions were stirred in closed vessels for 5 minutes on a magnetic stirrer. The vessels containing the test solutions were placed into a temperature controlled room for temperature adaptation.

After temperature adaptation of the test solutions, 0.434 or 0.313 mL of the algal pre-culture (Pseudokirchneriella subcapitata) were added to each of the volumetric flasks containing 500 or 360 mL of the test solutions, respectively to achieve an algal cell concentration of approximately 0.3×104 cells/mL. These manipulations were performed under a laminar flow box. The test solutions were homogenised by manual shaking 15 times overhead, before adding 60±5 mL of the test media to each test vessel to ensure a homogeneous distribution of the algal cells.
The air-tightly closed test vessels (screw-cap with PTFE-lining) with a minimised headspace were placed onto a shaker under light and temperature controlled conditions.

The medium was sterilised by sterile filtration (pore size 0.2 µm) before use. All glassware and materials used for testing purposes were sterilised for at least 3 hours at ≥150 °C using a heating furnace or autoclaved for 20 minutes at 120°C, except preparation flask (volumetric flask) used for preparation of test solution.


Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
To adapt the algae to the test conditions a pre-culture was inoculated by a liquid algal culture and incubated under test conditions.

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (SAG 61.81) (Raphidocelis subcapitata)
- Source (laboratory, culture collection): Sammlung von Algenkulturen, Albrecht-von-Haller-Institut, Universität Göttingen, Germany
- Culturing media and conditions (same as test or not): yes

- Method of cultivation:
pH-value of the algal medium: 7.9
Culture medium: Mod. OECD medium (OECD 201, EN ISO 8692)
Volume of liquid stock culture per pre-culture vessel: 100±5 mL
Pre-culture vessels: 300 mL Erlenmeyer flasks
Number of replicates: 3
Light: Permanent illumination (24/0 h light/dark); Econlux LED Sunstrip “daylight”
Light intensity: Target: 60–120 µE m-2s-1, measured: 75.3–78.6 µE m-2s-1
Shaker: 100±5 oscillations/min
Temperature in the test room: Target: 21–24 °C, measured: 21.7 – 23.0


Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
//
Post exposure observation period:
not applicable

Test conditions

Hardness:
//
Test temperature:
Temperature (target): 21–24 °C, controlled at ±2 °C
Temperature was recorded once per hour throughout the test in a separate test vessel placed on the shaker containing approximately 60 mL of deionised water.
Temperature (measured):
Minimum value: 20.8 °C
Maximum value: 22.8 °C
Mean value: 21.8 °C
Number of measurements: 78

The temperature fell below 21 °C for approximately 16 hours at a maximum of 0.2 °C. This deviation has no influence on the integrity of the study results, since all cell growth-related validity criteria were fulfilled
pH:
After temperature adaptation of the test solutions, the pH was measured and recorded for each test concentration and the control.
At the end of the test, the pH was measured and recorded in the pooled replicates per test concentration and control

pH at time 0h = 8.2
pH at time 72h = 9.2

The pH-values in the control exceeded the required upper limit (pH = 9) by 0.2 pH units. This deviation has no influence on the integrity of the study results, since all cell growth-related validity criteria were fulfilled, and the pH drift throughout all well growing treatment levels did not exceed 1.0 pH unit.
Dissolved oxygen:
//
Salinity:
//
Conductivity:
//
Nominal and measured concentrations:
Test concentrations (nominal): 1.00, 3.16, 10.0, 31.6 and 100 % of a saturated solution at a loading rate of 100 mg/L, and a control.
Geometric mean measured concentration over 72 hours: 0.015, 0.019, 0.071, 0.092, 0.134 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 60 mL glass vials, filled with the test solutions up to the rim of the vessels and were tightly closed by a screwcap with PTFE lining, 60±5 mL of the appropriate test solution with algae were added to each test vessel.
- Type (delete if not applicable): Closed, air-tightly closed by a screw-cap with PTFE-lining
- Headspace: minimised headspace
- Aeration: none
- Renewal rate of test solution: none
- Initial cells density: Nominal initial cell number = 0.3×10E4 cells per mL. At start of the test, the cell numbers were determined microscopically using a counting chamber (Thoma chamber). Occurrence of morphological deviations of cells was checked.
- Control end cells density: 37.43 ×10E4 cells/mL (STD = 1.162 ×104 cells/mL, CV = 3.1%)

The test vessels were placed on a horizontal shaker, so that the test vessels rotated around its own axis, located in a temperature-controlled room at the desired temperature.

- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not applicable

Additional vessels were prepared for chemical analysis verification and stability check (without algae):
- No. of replicates per test item concentration used for chemical analyses after 24 and 48 hours (with algae):2 (all concentration levels)
- No. of replicates for stability check (without algae): 1 (highest concentration level)

GROWTH MEDIUM
- Standard medium used: no
The growth medium was OECD medium as described in the test guideline, modified to optimise growth of Pseudokirchneriella subcapitata:
The OECD growth medium was prepared with a 10-fold concentrated iron (Fe)-EDTA complex (see bold font in recipe below) compared to the original recipe according to guideline OECD 201. The growth rates of the algae in pre-culture were not sufficient in original OECD medium but satisfactory in the modified medium, which is fully described below. Deviating from this SOP, the test medium was supplemented with sodium bicarbonate (in addition to the normal content; 250 mg/L NaHCO3), marked below in bold (Mayer et al. (2000), ISO 14442 (2006)).

Justification for modification of growth medium: The steady and sufficient supply of trace elements (e.g. iron) is essential for algal growth. In this context, the introduction of chelating agents (e.g. EDTA) is very valuable, since by using these compounds, trace element cations easily form reversible complexes. The splitting of these complexes steadily supplies the algae with the amounts of trace elements required for growth. There was no indication of interaction between this medium and the test item. The use of a sealed system result in culture growth being limited by CO2 depletion, thus additional NaHCO3 is used to serve as a source of CO2

OTHER TEST CONDITIONS
- Sterile test conditions: Yes. The algal medium was sterilised (filtered, pore size 0.2 µm) before use. The vessels used were sterilised by autoclaving for 20 minutes at 120°C.
- Adjustment of pH: Yes. Before use, the pH of the medium was adjusted to 8.1±0.2.
- Photoperiod: Permanent illumination (24/0 h light/dark); Econlux LED Sunstrip “daylight”
- Light intensity and quality during exposure: 84.5–91.5 µE m–2s–1; mean 87.6 µE m–2s–1

EFFECT PARAMETERS MEASURED
Growth inhibition was expressed as the effect of the test item concentration that reduced the cell number compared to the control by a certain percentage. The biological results, i.e. growth rate (µ), and yield (Y), were evaluated statistically.

At start of the test, the cell numbers were determined microscopically using a counting chamber (Thoma chamber). Occurrence of morphological deviations of cells was checked.
After 24, 48 and 72 hours, the cell numbers were determined by measuring the fluorescence intensity in 4 samples of 200 µL of test solution per replicate using a fluorometer (Multiple Reader Tecan ULTRA). The results [RFU, relative fluorescence units] were converted into biomass concentration [cells/mL] using a calibration curve (Figure 1), which was generated by the data analysis software Dell Statistica (Dell Inc. 2016). A dilution series of the pre-culture was used to determine the calibration line by comparison of cell numbers (counted using a microscope) and fluorescence intensity (measured using a fluorometer) in consideration of background fluorescence of the blank algal medium.

STATISTICS
The data were evaluated by the Shapiro-Wilk’s Test for normal distribution, and by Levene's Test for homogeneity of variances. The Williams Multiple Sequential t-test was applied to find out significant differences between the growth of algae in the controls and the algae exposed to the test item concentration. The Dunnett`s Multiple t-Test was additionally performed for interpretation of the results of the Williams Multiple Sequential t-test.
3-parametric normal cumulative distribution function (CDF) analysis was used to determine the concentration-response function.
The statistical software package ToxRat 3.2 Professional (ToxRat Solutions GmbH, Naheweg 15, D-52477 Alsdorf) was used for these calculations.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: square-root of 10
- Range finding study:
The range finder was performed twice:
The test concentrations of the first range finder were 4.0, 20 and 100 % of a saturated solution at a loading rate of 100 mg/L. The range finder was performed using a closed bottle system with individually weight test item per test solution. The volumetric flasks used for preparation of test solutions were not completely filled up with test medium, consequently the preparation was performed with a headspace. The maximum determined inhibition were 3.9% for yield and 0.8% for growth rate at 100 % of a saturated solution at a loading rate of 100 mg/L. A NOEC or ECx could not be determined neither for yield nor for growth rate at the highest test concentration.

The second range finder was performed with the test concentrations of 1.0, 10.0 and 100 % of a saturated solution at a loading rate of 100 mg/L. The range finder was performed using a closed bottle system with dilutions of a saturated stock solution at a loading rate of 100 mg/L. The volumetric flasks used for preparation of stock solution was completely filled up with test medium, to minimize the headspace while preparation, whereas the respective volumetric flasks (1.00 and 10.0 % of a saturated solution at a loading rate of 100 mg/L) were pre-filled as far as operationally possible with an appropriate volume of test medium prior to addition of the required volume of stock solution, comparable to the procedure used in the definitive test.

- Results used to determine the conditions for the definitive study: Second Not-GLP range finding test
EyC10 = 21.7 % (of the nominal 100 mg/L)
EyC50 >100 % (of the nominal 100 mg/L)
NOEC (Yield) : no statistically significant differences obeserved from the control

ErC10 = 147 % (of the nominal 100 mg/L)
EyC50 >100 % (of the nominal 100 mg/L)
NOEC (growth rate) : no statistically significant differences obeserved from the control
Reference substance (positive control):
not required
Remarks:
Two parallel reference tests using potassium dichromate (K2Cr2O7) as reference item were performed in October 2017 in separate studies.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC10
Remarks:
95% CL
Effect conc.:
> 0.134 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Remarks:
95% CL
Effect conc.:
> 0.134 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Remarks:
95% CL
Effect conc.:
> 0.134 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EC50
Remarks:
95% CL
Effect conc.:
> 0.134 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Details on results:
The %-inhibition seen for the parameter yield was <10%. Therefore, the EC10, EC20 and the EC50 was stated to be higher than the highest tested concentration level (100% of a saturated solution at a loading rate of 100 mg/L).

The %-inhibition seen for the parameter growth rate was <2%. Therefore, the EC10, EC20 and the EC50 was stated to be higher than the highest tested concentration level (100% of a saturated solution at a loading rate of 100 mg/L).

The NOEC both for growth rate inhibition and yield based on nominal concentration was calculated to be 3.16% of a saturated solution at a loading of 100 mg/L, equivalent to 0.019 mg Solvent N1/L based on measured concentrations.
However, the biological relevance of the determined statistically significant difference from the control (NOEC/LOEC) using the Williams-t test procedure appears questionable, since the observed inhibition is very low with 2.2–8.1% (yield) and 0.5-1.7% (growth rate). The % Minimum Detectable Difference (MDD) of the statistical method used is also low (4.5–4.6% for yield inhibition, 0.9% for growth rate inhibition). Also a concentration-dependent trend is not evident, since the %-inhibition shows an inverse concentration-response relationship from the intermediate concentration level. Therefore, the Dunnett`s Multiple t-Test was additionally performed taking into account the non-evident concentration-dependent trend. The results of the Dunnett`s Multiple t-Test shows that the two highest concentration levels are not significantly different from the control and support the assumption that the NOEC determined using the Multiple Sequential Williams t Test appears to be questionable. As a consequence a NOEC of 100% of a saturated solution at a loading of 100 mg/L, equivalent to 0.134 mg Solvent N1/L based on measured concentrations can be considered to be the more reliable value.
Results with reference substance (positive control):
No positive control is requested to be used during the main test.

Two parallel reference tests using potassium dichromate (K2Cr2O7) as reference item were performed in October 2017 in separate studies. One was performed using a standard test design and the other using a closed-bottle system. The latter was performed to confirm that the use of a modified test system to reduce losses of test item through volatility (completely filled and sealed vessels) is expected to give results consistent with that obtained in a conventional system.  
The results of this reference tests were acceptable and the test conditions were reliable.

Any other information on results incl. tables

Inhibition of Yield in relation to Concentration and Time

Nominal

concen-tration

[%a)]

Measured concen-tration

[mg/Lb)]

Mean yield

[cells]

Std. Dev.

[cells]

n

%Inhibition

Significant difference

from control (Multiple Sequential Williams t Test; p 0.05)

Significant difference from control (Dunnetts t Test; p 0.05)

Control

Control

37.13

1.162

6

0.00

1.00

0.015

37.71

1.862

3

-1.6

No

No

3.16

0.019

37.37

1.684

3

-0.6

No

No

10.0

0.071

34.13

0.981

3

8.1

Yes*

Yes*

31.6

0.092

35.06

0.660

3

5.6

Yes*

No

100

0.134

36.30

1.135

3

2.2

Yes*

No

Inhibition of Growth Rate in Relation to Concentration and Time

Nominal

concen-tration

[%a)]

Measured concen-tration

[mg/Lb)]

Mean

growth rate

[day-1]

Std. Dev.

[day-1]

n

%Inhibition

Significant difference from control (Multiple Sequential Williams t Test; p 0.05)

Significant difference from control (Dunnetts t Test; p 0.05)

Control

Control

1.6087

0.01041

6

 

1.00

0.015

1.6137

0.01625

3

-0.3

No

No

3.16

0.019

1.6107

0.01482

3

-0.1

No

No

10.0

0.071

1.5808

0.00946

3

1.7

Yes*

Yes*

31.6

0.092

1.5898

0.00622

3

1.2

Yes*

No

100

0.134

1.6012

0.01031

3

0.5

Yes*

No

Mean: arithmetic mean; Std. Dev.: standard deviation; n: number of replicates;

a)% of a saturated solution at a loading rate of 100 mg/L

b) given as geometric mean concentrations of each treatment level.

---------------------------------------------------------------------------------

Analytical Results

Conc.
Level

Test period

Nominal concentration

Measured concentration

% of measured initial

(time 0 hours) concentration

Geometric mean measured concentration over
72 hours

 

[h]

[%a)]

[mgSolventN1/L]

[%]

[mgSolventN1/L]

Control

0

0

<LOD

C1

0

1.00

<LOD

C2

0

3.16

<LOQ

C3

0

10.0

0.199

C4

0

31.6

0.341

C5

0

100

0.213

Control

72

0

<LOD

n.d.

C1

72

1.00

<LOD

n.a.

0.015

C2

72

3.16

<LOD

n.a.

0.019

C3

72

10.0

<LOQ

12.6

0.071

C4

72

31.6

<LOQ

7.3

0.092

C5

72

100

0.084

39.4

0.134

LOQ = Limit of Quantification (0.05 mg/L)

LOD =Limit of Detection(0.015 mg/L)

a)% of a saturated solution at a loading rate of 100 mg/L

n.a.: not applicable, since no measured concentrations were determined at both measuring points

 

Samples from the test solutions were analysed after 0 and 72 hours to determine actual levels of the test item in solution during the exposure period. The measured concentrations decreased over the exposure period.

For each concentration level the geometric mean concentration was calculated using the test item concentrations measured during the exposure period (time 0 and 72 hours). Where concentrations were below LOD or LOQ the following values were used for calculation of the geometric mean measured concentrations; <LOD = 0.015 mg/L and <LOQ = 0.025 mg/L.

The test item concentration based on initially measured concentration did not remain stable within±20% throughout the exposure period (Measured concentration at 72 hours ranged between7.3 – 39.4% of the measured initial concentrations)

Decrease in measured concentrations over the exposure period was observed for all the concentration levels. The most probable cause of such decrease in measured concentrations is loss of substance due to volatilisation.

Analyses of the samples at 24 and 48 hours were not analysed, since the biological data/results do not strictly necessitate such analysis, since reliable concentrations were measured at the start and end of the exposure period at the biologically relevant treatment level (C5).

 

-------------------------------------------------------------------------------------------------------

All validity criteria were fulfilled:

 

Required:

Found:

Mean biomass increase in the control cultures:factor of at least 16 within the 72-hour test period

124.8

Mean coefficient of variation for section-by-section specific growth rates in the control cultures:up to 35%

29.6%

Coefficient of variation of average specific growth rates during test period in replicate control cultures:up to 7%

0.6%

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
No meaningful concentration-response relationship was observed for the biological parameter yield and growth rate during the exposure period.

The EC10, EC20 and the EC50 for % inhibition compared to the control both for the parameter yield and growth rate were higher than the highest tested concentration level (100% of a saturated solution at a loading rate of 100 mg/L, corresponding to 0.134 mg/L expressed as geometric mean of measured concentrations).
The NOEC is considered equal to the highest tested concentration level (100% of a saturated solution at a loading rate of 100 mg/L, corresponding to 0.134 mg/L expressed as geometric mean of measured concentrations).
Executive summary:

The aim of the study was to determine the toxicity of the test item towards the green algae,Pseudokirchneriella subcapitata.

The study was conducted according to OECD test guideline No. 201 and GLP compliant.

 

A series of concentrations of the test item in aqueous solution were prepared. Due to the volatility of the test item, the test organisms were exposed to these concentrations, as well as to controls without the test item, for a test period of 72 hoursin a completely filled, closed-bottle system. The closed-bottle system is intended to reduce losses of volatile components of the test item during exposure.The study was conducted under static conditions; i.e., the test solutions were not renewed during the test. Test medium was adapted in order to support algal growth in closed system.References tests with potassium dichromate were conducted prior of the main study for evaluating the appropriateness of the test system.

 

The data obtained were analysed in order to estimate the concentration that caused a x% growth inhibition, i.e. ECX(e.g. EC50, EC20or EC10). As additional endpoints the No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) were calculated.

 

As the test item is of limited water-solubility and volatile, the preparation procedure of test solutions was set taking into account aspects of OECD 2000, Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. 

 

Growth was determined daily based on determination of the cell number per volume test solution (cell concentration).

 

To verify the nominally applied concentration, samples were taken from the test solutions and analytically measured. The samples taken from the test vessels were transferred to the analytical test site.

 

In a first pre-screening step, the stability of the aqueous samples at different storage conditions was tested in order to determine the appropriate storage conditions. The stability of stored samples over long-term period was evaluated and found appropriate.

 

Based on the results of a preliminary non-GLP range finding test, the following concentration steps in a geometrical series were tested in the definitive test: 1.00, 3.16, 10.0, 31.6 and 100 % of a saturated solution at a loading rate of 100 mg/L.

 

The measured concentrations at start of the exposure period (0 hours) ranged between <LOD for the lowest concentration level (corresponding to 1.00% of the saturated solution prepared at a loading rate of 100 mg/L) and 0.341 mg Solvent N1/L for the C4 concentration level (corresponding to 31.6% of the saturated solution prepared at a loading rate of 100 mg/L). Decrease in measured concentrations over the exposure period was observed for all the concentration levels. The most probable cause of such decrease in measured concentrations is loss of substance due to volatilisation.

As the test item concentration based on initial measured concentration did not remain stable within ±20% throughout the exposure period the biological results are calculated and reported based on geometric mean measured concentrations at each concentration level.

 

No meaningful concentration-response relationship was observed for the biological parameter yield and growth rate during the exposure period.

The determined effect estimates (% inhibition compared to the control) were slight.

The %-inhibition seen for the parameter yield was <10%, the %-inhibition seen for the parameter growth rate was <2%. Therefore, theEC10, EC20and the EC50both for yield and growth rate inhibition were stated to be higher than the highest tested concentration level (100% of a saturated solution at a loading rate of 100 mg/L, corresponding to 0.134 mg/L expressed as geometric mean of measured concentrations).

 

NOECs were calculated to be 0.019 mg/L using Multiple Sequential Williams t Test but further statistical analysis of data demonstrated the lack of biological relevance of such values therefore the NOEC of 100%of a saturated solution at a loading of 100 mg/L, equivalent to 0.134 mgSolventN1/L based on measured concentrations can be considered to be the more reliable value.