4-(6-acryloyloxyhexyloxy)benzoic acid (4-(trans-4-propylcyclohexyl)phenyl ester)

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:

Nov 20, 2018 - Feb 05, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

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:

Commission Regulation (EU) 2016/266 of 7 December 2015 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Two replicates of each sample were directly taken after preparation (0 hour) and analysed. The samples were analyzed undiluted due to the very low solubility in water. The concentrations of the test item were determined using the calibration curve. The test item concentration directly after preparation (0 hour) was

Vehicle:

yes

Details on test solutions:

Vehicle:

Nutrient (mg/L)
NH4Cl 15.0
MgCl2 * 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
MnCl2 * 4H2O 0.415
ZnCl2 0.00300
CoCl2 * 6H2O 0.00150
CuCl2 * 2H2O 0.00001
Na2MoO4 * 2H2O 0.00700

NaHCO3 50.0

- pH: 7.88
- Total hardness: 24 mg/L as CaCO3 (calculated)

Preparation of the Test Item:
A stock preparation with a test item concentration of 100 mg/L was freshly prepared. For that purpose, the test item was weighed in a calibrated flask and vehicle was added. The preparation was stirred with a magnetic stirrer for 24 hours. Then the formulation was passed through a filter membrane (pore size: 0.2 µm). The filtrate was used for the study. The pH value was not adjusted.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular green algae
- Strain: Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): Pseudokirchneriella subcapitata, strain 61.81 SAG was obtained from „Pflanzenphysiologisches Institut der Universität Göttingen“, Germany.
- Method of cultivation:
The permanent algae cultures, the pre-culture and the algae cultures of the study were cultivated in an air-conditioned room (U9/2110) with a water temperature of 21.0 – 24.0°C, controlled at
± 2°C. For cultivation, 300 mL Erlenmeyer flasks filled with 100 mL algae suspension were covered with air permeable stoppers (Heinz Herenz Medizinalbedarf GmbH, Hamburg, Germany). The cultures were shaken continuously at about 120 rpm (Universalschüttler SM25, Edmund Bühler GmbH, Hechingen, Germany) and lit between 4440 and 8880 Lux. Fluorescent tubes (Lumilux T5 nws FLH1 HO 80W/840, Osram GmbH, München, Germany) installed above the flasks served for lighting.

ACCLIMATION
- Acclimation period: 72h
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

-

Hardness:

24 mg/L as CaCO3 (calculated)

Test temperature:

23.0 to 23.5°C

pH:

7.85 to 9.13

Nominal and measured concentrations:

Nominal concentrations: 100.0 mg/L, measured values are below LOD (0.0056 mg/L)

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks
- Type: open
- Material, size, headspace, fill volume: 300 mL Erlenmeyer glas flasks filled with 100 mL cell suspension
- Aeration: no
- Initial cells density: 10.000 cells per ml
- Control end cells density: 1.940.883 cells per ml
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water according to OECD TG 201
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Light intensity and quality: The mean light intensities were 7535 Lux ± 2.4% and 7299 Lux ± 2.8% prior to and at the end of the exposure period, respectively.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Nominal test concentration: 100 mg/L

Reference substance (positive control):

no

Remarks:

The accuracy and reliability of the test method is demonstrated periodically as recommended by OECD Guideline No. 201 and the Commission Regulation (EU) 2016/266. Therefore, potassium dichromate (Art. 104864) was tested as positive control.

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: > LOD (0.0056 mg/L)

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: > LOD (0.0056 mg/L)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: > LOD (0.0056 mg/L)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: > LOD (0.0056 mg/L)

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: > LOD (0.0056 mg/L)

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: Yield

Remarks on result:

other: >3.389 × 10-8 mg/L

Details on results:

No significant effect on growth of the algae was observed at a nominal concentration of 100 mg/L, i.e. up to the maximum water solubility.

The EC values exceeded the LOD (0.0056 mg/L) ate the nominal concentration of 100 mg/L and, thus, could not be determined in this study.

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

Results with reference substance (positive control):

The accuracy and reliability of the test method is demonstrated periodically as recommended by OECD Guideline No. 201 and the Commission Regulation (EU) 2016/266. Therefore, potassium dichromate was tested as positive control.

Study design:
The study comprised of one control group with six replicates and five test item groups with three replicates each. The test item concentrations of 0.1, 0.2, 0.4, 0.8 and 1.6 mg/L were dissolved in 100 mL reconstituted water. The initial algae cell density in each replicate was 10 000 cells per mL. The growth of the algae was determined over an exposure period of 72 hours and compared with the growth of the control group.

Results:
Thetest item Art. 104864 (Potassium dichromate) showed 72h EC50 of 1.10 mg/L for growth rate and 0.48 mg/L for yield. The EC50 for growth rate and yield obtained in this study are in the range of ± two standard deviation of the current historical means.

Therefore, this study confirms the sensitivity and reliability of the test system used in the test facility.

Objective

The objective of the present study was to determine the effect of on growth rate and yield of the unicellular green alga Pseudokirchneriella subcapitata.

Study Design

The study comprised of one control group and one test item group with six replicates each. A nominal test item concentration of 100 mg/L was prepared in reconstituted water. The initial algae cell density in each replicate was 10 000 cells per mL. The growth of the algae was determined over an exposure period of 72 hours and compared with the growth of the control group.

Samples of the test and control media were taken directly after preparation and the test item concentration was analyzed via HPLC with UV detection.

Results

The test item concentration directly after preparation was <LOD (LOD: 0.0056 mg/L) and, thus, could not be determined. Therefore, no further analysis of the test item concentrations was performed during the study.

No significant effect on growth rate and yield of the algae was observed at a nominal concentration of 100 mg/L, i.e. up to the maximum water solubility.

The following EC values (Growth rate and Yield) were determined:

Parameter (0 -72h)	Growth Rate	Yield
EC10 [mg/L] 95% confidence interval	>0.0056 mg/L (nominal >100 mg/L) n.d.	>0.0056 mg/L (nominal >100 mg/L) n.d.
EC20 [mg/L] 95% confidence interval	>0.0056 mg/L (nominal >100 mg/L) n.d.	>0.0056 mg/L (nominal >100 mg/L) n.d.
EC50 [mg/L] 95% confidence interval	>0.0056 mg/L (nominal >100 mg/L) n.d.	>0.0056 mg/L (nominal >100 mg/L) n.d.

Conclusion

Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of the test item revealed no toxicity to algae.The 72h EC₅₀ for growth rate and yield were >0.0056 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of the test item revealed no toxicity to algae.
The 72h EC50 and EC10 value for growth rate were >0.0056 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.

Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 201. Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of the test item revealed no toxicity to algae.The 72h EC50 and 72h EC10 for growth rate and yield were >0.0056 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.

Description of key information

The 72 h EC50 could not be quantified due to the absence of toxicity at the highest concentration achievable in water. Thus, the EC50 values are greater than 100 mg/L.

The 72h EC50 and EC10 value for growth rate were >0.0056 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study (OECD 201).