Bis(2-ethylhexyl) 2-(4-hydroxy-3,5-dimethoxybenzylidene)malonate

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:

2004-10-07 to 2005-01-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted 07 June 1984

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

29 December 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Sampling and analysis

Analytical monitoring:

no

Remarks:

The solubility in reconstituted water was examined using a HPLC method. The solubility of the test material could not be determined but was < 0.03 mg/L in the reconstituted water used in this test. Thus, no analysis was performed during this study.

Details on sampling:

not performed

Test solutions

Vehicle:

no

Details on test solutions:

The test medium (reconstituted water as vehivle and test material) was freshly prepared. The calibrated flask with test material and vehicle, reconstituted water, was treated in an ultrasound bath for 1 hour.
Subsequently, the preparation was stirred with a magnetic stirrer for further 23 hours. Afterwards, the preparation was filtered through a filter funnel with a pore size of 0.2 µm. The filtrate was used for the study.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The test system used for this study was a green unicellular algae species, Desmodesmus subspicatus, strain No. SAG 86.81, supplied by the "Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universitat Göttingen". This strain is recommended by international guidelines.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

250 mg CaCO3

Test temperature:

23+/-1°C

pH:

Concentration pH without algae pH with algae
([mg/L] Start End Start End
0 7.96 7.82 7.96 9.74
100 7.94 7.71 7.94 8.73

Dissolved oxygen:

80 %

Nominal and measured concentrations:

The solution of a nominal test material concentration of 100 mg/L was tested in an open static system.

Details on test conditions:

The study was located in an air-conditioned room. Preparations of 100 mL each were continuously shaken in 300 mL Erlenmeyer flasks by a pulsating panel. The flasks were completely sealed with sterilized stoppers. The flasks were continuously illuminated for the exposure period by fluorescent tubes installed above the panel containing the flasks.

The test medium (reconstituted water and test material) was freshly prepared. The calibrated flask with test material and vehicle, reconstituted water, was treated in an ultrasound bath for 1 hour. Subsequently, the preparation was stirred with a magnetic stirrer for further 23 hours. Afterwards, the formulation was given to a separating funnel for 6 hours. The middle phase of the preparation was filtered through a filter funnel with a pore size of 0.2 µm. The filtrate was used for the study.

Volumes of 100 mL reconstituted water or test medium (reconstituted water with test material) were freshly prepared before the algae suspensions were added. The control and the test medium were placed into 300 mL Erlenmeyer flasks and covered with sterilized cellulose stoppers.

The flasks were incubated under standardized conditions, in an air-conditioned room under continuous rotating and continuous illumination. The light intensity was approx. 7500 to 9500 Lux. The illumination was achieved by fluorescent tubes (Philips Master tubes TL5 80W/840 1-10) instaIled above a rotating panel with the flasks.

The experimental part was started by inoculation of algae to the different groups so that the flasks contained about 10,000 cells/mL. The algae were taken from an exponentially growing preculture (growth rate 184) which was set up 3 days prior to the experimental part under the same conditions as in the final study.

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks
- Type: open
- Material, size, headspace, fill volume: 100 mL
- Initial cells density: 10,000 cells/mL
- Control end cells density: 1,840,401 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes
- Details on composition of the reconstituted water:
Macro-nutrients:
50.00 mg/L NaHCO3
18.00 mg/L CaCl2 x 2 H2O
15.00 mg/L NH4Cl
15.00 mg/L MgSO4 x 7 H2O
12.00 mg/L MgCl2 x 6 H2O
1.60 mg/L KH2PO4

Trace elements:
100.00 µg/L Na2EDTA x 2 H2O
80.00 µg/L FeCl3 x 6 H2O
415.00 µg/L MnCl2 x 4 H2O
185.00 µg/L H3BO3
7.00 µg/L Na2MoO4 x 2 H2O
3.00 µg/L ZnCl2
1.50 µg/L CoCl2 x 6 H2O
0.01 µg/L CuCl2 x 2 H2O

The pH of the reconstituted water after aeration is approximately 8.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuously
- Light intensity and quality: 7500 to 9500 Lux

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

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: Limit test
- Range finding study: yes
- Test concentrations: 100 mg/L (nominal)

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 2

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

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: --
- Any stimulation of growth found in any treatment: slight stimulation
- 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

Any other information on results incl. tables

see attached table

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

An aqueous preparation of nominal 100 mg/L of the test substance revealed no toxic effect in the test system. Therefore, the 72 hours EC50 could not be determined, because it exceeded the maximum solubility of the test material in reconstituted water (EC50> 0.03 mg/L).

Executive summary:

Purpose
The purpose of this assay was to identify the aquatic toxicity potential of the test item in algea to provide a rational basis for hazard estimation for the test item in aquatic environments.

Study Design
The study was performed according to OECD TG 201. Briefly, the study design included one control and one test material group with three replicates, each containing 100 mL reconstituted water or test medium and about 10,000 cells/mL at the start of the experimental phase.
The study was performed as a limit test in an open static test system. The algae were exposed to a filtrate of nominal 100 mg/L.
The growth of the algae was calculated after 24, 48, and 72 hours exposure in the test medium.

Results

The analysis of the saturated aqueous preparation revealed that the water solubility of the test item in reconstituted water was < 0.03 mg/L. An exact value could not be determined.

After exposure of Desmodesmus subspicatus the following results were obtained after 24, 48 and 72 hours, respectively:

	Mean cell density per mL					Growth inhibition (0-72 h)
Nom. mg/L		0 hour	24 hours	48 hours	72 hours	IA	Iµt
0		10000	49578	310114	1840401
100		10000	57381	383373	2124360
	IA		-19,7	-23,9	-16,8	-17,8	-2,8

I_A= inhibition of growth in percent
I_µt= inhibition of growth rate in percent
Nom. = nominal concentration

For the test material, the following EC50 values and no effect concentration for Desmodesmus subspicatus were determined:

72 h E_bC₅₀> 100 mg/L (nominal)
72 h E_rC₅₀  > 100 mg/L (nominal)
NOEC =  100 mg/L (nominal)


Conclusion
An aqueous preparation of nominal 100 mg/L of the test substance revealed no toxic effect in the test system. Therefore, the 72 hours EC50 could not be determined, because it exceeded the maximum solubility of the test material in reconstituted water (EC50> 0.03 mg/L).