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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-10-07 to 2004-12-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

adopted 04 April 1984

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

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:

Because of the low water solubility (<0.03 mg/L) and the fact that the study was performed as a limit test, the test material concentration was not analyzed during the course of the study.

Details on sampling:

not applicable

Test solutions

Vehicle:

no

Details on test solutions:

Before exposure of the daphnia, the test medium (reconstituted water and test material) was freshly prepared: The calibrated flask with test material and reconstituted water was treated in an ultrasonic device for 1 hour. Subsequently the preparation was aerated, and stirred with a magnetic stirrer for further 23 hours. After 24 hours the formulation was given through a nutsch filter (pore size 0.2 µm). The filtrate was used for the study.

Reconstituted water according to ELENDT (1990):
Macro nutrients (mg/L):
CaCI2 x 7 H20 293.80
MgSO4 x 7 H20 123.30
NaHCO3 64.80
KCI 5.80
Na2SiO3 x 9H20 10.00
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18

Trace elements (mg/L):
B 0.5000
Fe 0.2000
Mn 0.1000
Li, Rb and Sr 0.0500
Mo 0.0250
Br 0.0125
Cu and Zn 0.0063
Co and I 0.0025
Se 0.0010
V 0.0003

Macro nutrients (mg/L):
Na2EDTA x 2H20 2.50

Vitamins (µg/L):
Thiamine 75.00
B12 1.00
Biotin 0.75


After preparation, the reconstituded water was aerated for 24 hours.

- Hardness: about 14° dH (about 250 mg/L CaCO3)
- pH: 7.9 ± 0.3, after an aeration for 24 hours.



References:

ELENDT, B.-P. Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus.
Protoplasma 154, 25-33, 1990

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Straus)
- Justification for species: internationally accepted for this type of study
- Age at study initiation (mean and range, SD): not older than 24 hours
- Source: Merck KGaA

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

14 ° dH (about 250 mg/L CaCO3)

Test temperature:

21-22 °C

pH:

7.9 +/- 0.1

Dissolved oxygen:

at least 91.4 %

Nominal and measured concentrations:

Nominal 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 25 mL glass beaker
- Type (delete if not applicable): open
- Volume of solution: 10 mL
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light - 8 hours dark

RANGE-FINDING STUDY
- Test concentrations: 100 mg/L
- Results used to determine the conditions for the definitive study: no immobilization

Further Information:
-- Preparation
The test medium (reconstituted water and test material) was freshly prepared. Therefore, the calibrated flask with test material and vehicle, reconstituted water, was treated in an ultrasonic device for 1 hour. Subsequently the preparation was aerated, and stirred with a magnetic stirrer for further 23 hours. After 24 hours the formulation was given through a nutsch filter (pore size >10 - <16 µm). The filtrate was used for the study.

-- Analysis
The solubility in water was examined. The solubility of the test material could not be determined but was < 0.02 mg/L. Further refinement of the analytical method would be extremely cost- and time consuming. Thus, no further analysis could be performed during this study.

-- Administration
At the start of the experimental phase, 5 Daphnia magna were placed into 10 ml of reconstituted water (control group) or test medium (test material group). The Daphnia magna were not fed, and the control medium and test medium were not aerated during the test.
The test vessels were labeled to assure an unequivocal identification.

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

-- Observation schedule
The motility was determined by visual control and recorded after 24 and 48 hours. Those animals, not able to swim within 15 seconds after gentle agitation of the test container, are considered to be immobile.

-- pH and oxygen concentration
The pH values and dissolved oxygen concentration (O2) were measured in the control and test material concentration at the beginning and at the end of the experimental part.

-- Temperature
During the experimental part, the temperature was registered in a control vessel with an electronic thermometer containing a maximum and minimum memory display.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Clinical findings
The nominal concentration of the preparation of the test substance and the immobilization data are shown in the following table:

--------------------------------------------------------------------
Nominal concentration
[mg/L J Immobilized/exposed Daphnia magna
---------------------------
24 hours 48 hours
--------------------------------------------------------------------
0 0/20 0/20
100 0/20 0/20
--------------------------------------------------------------------

A dose group with nominal 100 mg/L of the test substance, maximal water solubility < 0.03 mg/L, revealed no aquatic toxicity in this test system.

No remarkable observations were made concerning the appearance of the solution of the test material preparation. The test medium was a clear preparation and stayed unchanged throughout the study.


EC50 value
An aqueous solution of nominal 100 mg/L of the test substance revealed no aquatic toxicity in the test system. The 24 hours and 48 hours EC50 to Daphnia magna exceeded the maximal water solubility (< 0.03 mg/L) of the test material and, thus, was not determined in this test.
Under the given experimental conditions, the test material showed an analytical 24 h and 48 h EC50 value > 0.03 mg/L.

Analysis
pH, oxygen concentrations, and temperature during the study were inconspicuous.

------------------------------------------------------------------------------------------------------------------------------------------
Nominal concentration
[mg/L J Start of the experiment End of the experiment
---------------------------
0 hour 48 hours
pH O2 [%] pH O2 [%]
-------------------------------------------------------------------------------------------------------------------------------------------
0 7.92 94.8 7.89 92.7
100 7.81 93.5 7.85 91.4
-------------------------------------------------------------------------------------------------------------------------------------------

The temperature in the control vessel was 21 °C.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

A dose group with nominal 100 mg/L of test item (maximal water solubility < 0.03 mg/L) revealed no aquatic toxicity in this test system.
No remarkable observations were made concerning the appearance of the solution of the test material preparation. The test medium was a clear preparation and stayed unchanged throughout the study

Executive summary:

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

Study Design
For this purpose, juvenile daphnids were exposed to an aqueous test material solution over 48 hours, under defined conditions. The study comprised of four test vessels per concentration and control group containing five daphnids each, i.e. 20 daphnids per concentration (test medium group) and control group.
Daphnids were exposed to a nominal test material concentration of 100 mg/L (limit test) in an open static system. The method followed that described in the OECD Guidelines for Testing of Chemicals No 202 Daphnia sp., Acute Immobilisation Test.

Results
After exposure of Daphnia magna to a saturated aqueous solution of nominal 100 mg/L for 48 hours, the following results were obtained:

Nominal concentration	Immobilized/exposed daphnids
[mg/L]	24 hours	48 hours
0	0/20	0/20
100	0/20	0/20

Daphnia magna exposed to an aqueous preparation of the nominal concentration of 100 mg test item /L were not affected.

The analysis revealed, that the water solubility of the test substance was 0.03 mg/L. Because of this low water solubility and the fact that the study was performed as a limit test, the test material concentration in the aqueous medium at the start and the end of this study was not quantified.

For the test material, the following EC50 values for Daphnia magna were determined:

24 h EC50 > 100 mg/L (nominal)
48 h EC50 > 100 mg/L (nominal)


Conclusion
An aqueous solution of nominal 100 mg/L of the test item revealed no aquatic toxicity in the test system. The analytical 48 hours EC50 to Daphnia magna exceeded the maximal solubility of the test material in reconstituted water (< 0.03 mg/L) and, thus, could not be determined in this test.