Benzaldehyde, 4-[(3-fluorophenyl)methoxy]-

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 FEB 2015 - 17 AUG 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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: Alga, Growth Inhibition Test, adopted on March 23, 2006; Annex 5 corrected: July 28, 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Council Regulation (EC) No. 761/2009 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and the council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). C.3. Freshwater algae and cyanobacteria, growth inhibition test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 1.0, 3.2, 10.0, 31.6 and 100.0 mg/L
- Sampling method: Samples of the test medium and the control medium were taken directly after preparation and after 24, 48 and 72 hours and were subjected to chemical analysis. Before analysis, the algae were removed from the medium by centrifugation at a low g-force, sufficient to settle the algae. Additionally, analysis of the highest test item preparation (100 mg/L) not inoculated with algae was performed daily.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was prepared freshly before the exposure. Therefore, the calibrated flask with test medium was treated in an ultrasonic device for 1 hour. Subsequently, the preparation was stirred with a magnetic stirrer for further 23 hours. Then the formulation was passed through a filter membrane (pore size: 0.2 µm). The filtrate was used for the study.
- Control: Reconstituted water with algae.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: strain 86.81 SAG
- Source (laboratory, culture collection): Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen, Germany. This strain was further cultivated in the laboratories of Non-Clinical Safety.
- Age of inoculum (at test initiation): The algae were taken from an exponentially growing preculture (growth rate 100.2) which was set up 3 days prior to the experimental part under the same conditions as in the final study.

ACCLIMATION
- Culturing media and conditions (same as test or not): The preculture was cultured under the same conditions as in the final study.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

The water temperature was measured in an Erlenmeyer flask containing reconstituted water, kept under experimental conditions using MCPS.
The water temperature in the control flask (containing reconstituted water) during the experimental part was 22.7-23.4°C and therefore in the range of 21.0-24.0°C and constant within ± 2°C.

pH:

The pH-values were measured in the control group and the test item groups at the start (0 hours) and at the end (72 hours) of the experimental part. Furthermore, one flask of each group was used without algae for measurement of pH.
pH test start: 7.49 - 7.56
pH test end (with and without algae): 7.25 - 7.49

Nominal and measured concentrations:

nominal: 0, 1.0, 3.2, 10.0, 31.6 and 100.0 mg/L
geometric mean measured: < LOD (0.013 mg/L), 0.019, 0.044, 0.092, 0.638, 9.057 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Preparations of 100 mL each were continuously shaken in 300 mL Erlenmeyer flasks at about 120 rpm by an orbital shaker. The flasks were covered with air permeable steristoppers.
- Aeration: No.
- Initial cells density: 10000 cells / mL
- Control end cells density: 1243587 cells / mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: No. Reconstituted water was used as growth medium.
- Detailed composition if non-standard medium was used:
Nutrients in mg/L:
NH4CI: 15.0
MgCh*6H2O: 12.0
CaCl2*2H2O: 18.0
MgSO4*7H2O: 15.0
KH2PO4: 1.60
FeCl3*6H2O: 0.0640
Na2EDTA*2H2O: 0.100
H3BO3: 0.185
MnCI2*4H2O: 0.415
ZnCl2: 0.00300
CoCI2*6H2O: 0.00150
CuCl2*2H2O: 0.00001
Na2MoO4*2H2O: 0.00700

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: The flasks were continuously illuminated for the exposure period.
- Light intensity and quality: The anticipated light intensity was between 4440 and 8880 Lux and within ± 15% from the average. The illumination was achieved by fluorescent tubes (Lumilux T5 nws FLH1 HO 80W/840, Osram GmbH, Munich, Germany) installed above the orbital shaker with the flasks.
- Other: The study was performed in an air-conditioned room.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The duration of exposure was 72 hours. During this time, the cell density was measured daily. In addition, the morphological effect of the test item on the algal cells was determined after 72 hours by microscopical observation of the morphological appearance of the treated algal cells in comparison to the controls.
- Determination of cell concentrations: The cell densities in the samples were determined with an electronic particle counter (Coulter-Counter Z2, Beckman Coulter GmbH, Krefeld, Germany). Volumes of the algal suspension not impairing the study were taken from the test item preparations and the controls with algae after exposure times of 24 (± 1h), 48 (± 1h), and 72 (± 1h) hours. The required volume depended on the cell density. The withdrawn volumes were not substituted. Additionally, the blank values in the Erlenmeyer flasks without algae were measured at the same time as the test item concentrations and the control group. The blank value was subtracted from the measured value of the respective replicate of the control group and the test item groups.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Test concentrations: One control and five test item concentrations of 1.0, 3.2, 10.0, 31.6 and 100.0 mg/L.
- Results used to determine the conditions for the definitive study: The test item concentrations used in this study were selected on the basis of the results of a preliminary study.

Reference substance (positive control):

yes

Remarks:

An independent study was performed with the reference item potassium dichromate to check the sensitivity of the test system as recommended by guidelines (Study No.: 14-DA479-N0, Start of experiment: 2014-12-01, End of experiment: 2015-02-07)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.244 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.323 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

Effect conc.:

0.553 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.149 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.186 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.285 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Other: No morphological changes of the treated algae in comparison to the control algae were observed.
- Any stimulation of growth found in any treatment: No significant stimulation of growth compared to the control.
- 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: no

Results with reference substance (positive control):

- Results with reference substance valid? Yes.
- EC50:
EC50 (72h) growth rate = 0.71 mg/L (95%-Confidence intervals: 0.67 - 0.74 mg/L)
EC50 (72h) biomass integral = 0.39 mg/L (95%-Confidence interval: 0.37 - 0.41 mg/L)
Under the given experimental conditions, the test item Art. 104864 (potassium dichromate) showed 72h EC values which are within the recommended range (Biomass integral EC50 = 0.20 - 0.75 mg/L and Growth rate EC50 = 0.60 - 1.03 mg/L).

Reported statistics and error estimates:

The evaluation and calculation was performed with the validated program ToxRat (ToxRat Professional Version 2.10, ToxRat Solutions GmbH, Alsdorf, Germany).

Validity criteria fulfilled:

yes

Conclusions:

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 the test substance towards algae.
Based on the geometric mean measured concentrations the 72 -hour ErC50 was calculated to be 0.553 mg/L and the 72 -hour EyC50 value was calculated to be 0.285 mg/L. The 72-hour ErC10 was determined to be 0.244 mg/L and the 72-hour EyC10 0.149 mg/L, respectively.

Executive summary:

The study was conducted under GLP according to the valid OECD Guideline for Testing of Chemicals 201 and the Council Regulation (EC) No. 761/2009 on the registered substance itself.

The objective of this study was to evaluate the influence of the test item on the growth and growth rate of the green algal species Desmodesmus subspicatus.

The study design included one control group with six replicates and five test item groups with three replicates, each containing 100 mL reconstituted water or test medium and 10000 cells/mL at the start of the experimental phase.

The study was performed under static conditions with nominal concentrations of 1.0, 3.2, 10.0, 31.6 and 100.0 mg/L in reconstituted water. The study was performed in open vessels.

The growth of the algae was calculated after 24, 48, and 72 hours exposure in the test item and control groups.

The concentrations of the test item were analytically determined by HPLC-UV. Due to the results of a stability determination before start of study, analytical controls of the test item preparations were performed at the start of the study and then daily.

The concentrations of the test item in test media without algae exceeded the values analyzed from the preparations with algae after 24, 48 and 72 hours, respectively. Therefore, based on the results of the stability determination and the analytical results it can be assumed that the decrease of the test item in the preparation during the study is a combination of instability and adsorption of the test item to the algae.

During the experimental phase of the study, the test item concentrations could not be maintained within ± 20% of the initial analytical concentration. Therefore, the EC values were calculated with the geometric mean exposure concentration. In cases where no test item could be detected, the limit of detection of the analytical method was used for calculation as recommended in OECD Guidance Document No. 23. In cases where the test item is detected but not quantified, the half of the limit of quantification was used for calculation as recommended in OECD Guidance Document No. 23.

Based on the obtained biological and analytical results the following EC-values were determined for the test item:

EC₁₀ (72h) growth rate = 0.244 mg/L

EC₂₀ (72h) growth rate = 0.323 mg/L

EC₅₀ (72h) growth rate = 0.553 mg/L

EC₁₀ (72h) yield = 0.149 mg/L

EC₂₀ (72h) yield = 0.186 mg/L

EC₅₀ (72h) yield = 0.285 mg/L

Based on the obtained results the test substance needs to be classified as acutely toxic to the environment according to Regulation (EC) No 1272/2008 and amendments. The 72h E_rC₅₀ of 0.553 mg/L is below the limit value for classification as aquatic acute Cat. 1 (≤ 1.0 mg/L). Furthermore, the test substance needs to be classified as chronic toxic to the environment according to Regulation (EC) No 1272/2008 and amendments. The 72h E_rC₁₀ of 0.244 mg/L is below the limit value for classification as aquatic chronic Cat. 1 (≤ 0.1 mg/L) and the test material was determined to be not readily biodegradable.

Description of key information

Toxicity to aquatic algae and cyanobacteria: EC₁₀ (72h) growth rate = 0.244 mg/L and EC₅₀ (72h) growth rate = 0.553 mg/L for the freshwater green algae Desmodesmus subspicatus (static, OECD 201, GLP)

Key value for chemical safety assessment

EC50 for freshwater algae:

0.553 mg/L

EC10 or NOEC for freshwater algae:

0.244 mg/L

Additional information

The study was conducted under GLP according to the valid OECD Guideline for Testing of Chemicals 201 and the Council Regulation (EC) No. 761/2009 on the registered substance itself.

The objective of this study was to evaluate the influence of the test item on the growth and growth rate of the green algal species Desmodesmus subspicatus.

The study design included one control group with six replicates and five test item groups with three replicates, each containing 100 mL reconstituted water or test medium and 10000 cells/mL at the start of the experimental phase.

The study was performed under static conditions with nominal concentrations of 1.0, 3.2, 10.0, 31.6 and 100.0 mg/L in reconstituted water. The study was performed in open vessels.

The growth of the algae was calculated after 24, 48, and 72 hours exposure in the test item and control groups.

The concentrations of the test item were analytically determined by HPLC-UV. Due to the results of a stability determination before start of study, analytical controls of the test item preparations were performed at the start of the study and then daily.

The concentrations of the test item in test media without algae exceeded the values analyzed from the preparations with algae after 24, 48 and 72 hours, respectively. Therefore, based on the results of the stability determination and the analytical results it can be assumed that the decrease of the test item in the preparation during the study is a combination of instability and adsorption of the test item to the algae.

During the experimental phase of the study, the test item concentrations could not be maintained within ± 20% of the initial analytical concentration. Therefore, the EC values were calculated with the geometric mean exposure concentration. In cases where no test item could be detected, the limit of detection of the analytical method was used for calculation as recommended in OECD Guidance Document No. 23. In cases where the test item is detected but not quantified, the half of the limit of quantification was used for calculation as recommended in OECD Guidance Document No. 23.

Based on the obtained biological and analytical results the following EC-values were determined for the test item:

EC₁₀(72h) growth rate = 0.244 mg/L

EC₂₀(72h) growth rate = 0.323 mg/L

EC₅₀(72h) growth rate = 0.553 mg/L

EC₁₀(72h) yield = 0.149 mg/L

EC₂₀(72h) yield = 0.186 mg/L

EC₅₀(72h) yield = 0.285 mg/L

Based on the obtained results the test substance needs to be classified as acutely toxic to the environment according to Regulation (EC) No 1272/2008 and amendments. The 72h E_rC₅₀ of 0.553 mg/L is below the limit value for classification as Aquatic Acute Cat. 1 (≤ 1.0 mg/L, H400). Furthermore, the test substance needs to be classified as Aquatic Chronic Cat. 1 (H410) according to Regulation (EC) No 1272/2008 and amendments, due to the 72h E_rC₁₀ of 0.244 mg/L below the limit value for classification as Aquatic Chronic Cat. 1 (≤ 0.1 mg/L) and due to the fact that the test material was determined to be not readily biodegradable.