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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-10-08 to 2004-12-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

adopted 17 July 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

29 December 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

Vehicle:

no

Details on test solutions:

Before exposure of the fish, 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 >10 - <16 µm). The filtrate was used for the study.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Selection and adaptation
The fish were acclimatized to the conditions of the laboratory for more than 14 days and were held in reconstituted water for at least for 7 days before testing. During the acclimatization, until one day before the experimental part, the fish were fed with flakes (Tetra Min® diet; Tetra GmbH, Melle). During the week before testing, the mortality of the test fish batch was lower than 5%.
At the start of the test, 10 fish per group were randomly introduced into aquaria filled with test medium or reconstituted water, respectively.

Assignment
Numbers of zebra fish
Pretest: 4 zebra fish
Control group: 10 fish;
Test material group: 10 fish.
A total of 24 zebra fish were used in this study.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

about 267 mg CaCO3

Test temperature:

23-25 °C

pH:

7.16 - 7.93

Dissolved oxygen:

The dissolved oxygen concentration was > 60 % oxygen saturation throughout the study.

Nominal and measured concentrations:

nominal 100 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: Aquarium
- Type: open
- Material, size, fill volume: glass, 30cm x 20cm x 15cm, 9 liter
- Aeration: open conditions
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: fully demineralized water

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 12 hours light - 12 hours dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Lethality, behaviour and general conditions, Twice at the first day and then daily

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: limit test based on results of a prestudy
- Range finding study: yes
- Test concentrations: 100 mg/L (nominal
- Results used to determine the conditions for the definitive study: no toxicity observed

Environmental conditions
During the study, the zebra fish were kept in all-glass aquaria. The study was located in an air-conditioned room. Lighting was controlled by a timer provide a 12 hours light - 12 hours dark regime. The fish were not fed during the study.

Preparation
Before exposure of the fish, 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 ultrasonic device for 1 hour. Subsequently the preparation was stirred with a magnetic stirrer for further 23 hours. Thereafter the formulation was given in a separating funnel for 6 hours. To remove small test material droplets the formulation was subsequently given through a nutsch filter (pore size >10 - <16 µm). The filtrate was used for the study.

Administration
At the start of the experimental phase, 10 zebra fish per group were inserted into an aquarium (all-glass) with about 9 liters of reconstituted water (control group) or test medium (test material group). The aquaria were labeled to assure an unequivocal identification.

Observation schedule
The behavior and general condition of all fish were checked immediately after the introduction into the test medium or reconstituted water, after three hours, and then daily.

Laboratory tests
Dissolved oxygen (O2) concentration in percent and pH values were measured in the control and test material groups at the beginning and at the end of the exposure period.
During the experimental part, the temperature was registered in the control vessel with an electronic thermometer containing a maximum and minimum memory display.

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Clinical findings
No clinical findings were observed in the control and the test material group during the experimental part. All fish survived the observation period.

LC50 value
A dose group with nominal 100 mglL of the test item (maximal solubility <0.03 mg/L analytically) revealed no aquatic toxicity in this test system. The analytical 24, 48, 72, and 96 h LC50 values exceed the maximum solubility of the test material.
No remarkable observations were made concerning the appearance of the test material solution. The test medium was a clear preparation and stayed unchanged throughout the study.


Laboratory Tests
Analysis
The solubility in water was examined using a HPLC method. The solubility of the test material could not be determined but was < 0.03 mg/L. The same low solubility can be expected for reconstituted water for fish.

Temperature
The temperature during the main study was 22 to 23 °C.

pH and oxygen
The dissolved oxygen concentrations were > 60 % oxygen saturation throughout the study. The pH values (~7.7) during the final study were inconspicuous.

Validity criteria fulfilled:

yes

Conclusions:

An aqueous solution of the test substance, of a nominal concentration of 100 mg/L, revealed no aquatic toxicity in the test system. The 96 h LC50 value to zebra fish exceeded the maximal solubility of < 0.03 mg/L (as established analytically) of the test material in reconstituted water and, thus, could not be determined in this test.

Executive summary:

Purpose

The objective of this aquatic toxicity study in zebra fish (Danio rerio) was to provide information on environmental hazards likely to arise from exposure of the test material and to serve as a basis for classification and labeling purposes.

Study Design

For this purpose, 10 fish (test material group) were exposed to the test material over 96 hours, under defined conditions in a limit test. Further, 10 fish were used as a control group. The fish were observed for signs of toxicity or death for 96 hours. Zebra fish of the test material group were exposed to a saturated aqueous test material solution of nominal 100 mg/L (limit test) in an open static system. The study was conducted according to Good Laboratory Practice (GLP) and followed the OECD Guidelines for the Testing of Chemicals, No. 203 Fish Acute Toxicity Test.

Results
The following results were obtained:
Mortality                                   24h       48h        72h        96 h

Control Group                           0/10      0/10       0/10       0/10

Test item 100 mg/L nominal       0/10     0/10        0/10       0/10

Zebra fish exposed to an aqueous preparation of the test item, with a nominal concentration of 100 mg/L were not affected.

The solubility of the test item in reconstituted water for daphnia was established as < 0.03 mg/L (separate study) . Due to this low water solubility and the fact that the study was performed as a limit test, the test material concentrations in the aqueous medium at the start and the end of this study were not quantified.

For the test material, the following LC50 values for zebra fish were determined.

24 h LC50    >    100 mg/L (nominal)
48 h LC50    >    100 mg/L (nominal)
72 h LC50    >    100 mg/L (nominal)
96 h LC50    >    100 mg/L (nominal)

Conclusion

An aqueous solution of the test substance, of a nominal concentration of 100 mg/L, revealed no aquatic toxicity in the test system. The 96 h LC50 value to zebra fish exceeded the maximal solubility of < 0.03 mg/L (as established with reconstituted water for daphnia, separate study) of the test material in reconstituted water and, thus, could not be determined in this test.

Description of key information

In an acute toxicity test in fish, no mortality or adverse effects have been observed in zebrafish exposed to test medium with nominal 100 mg/L test material for a time period of 96 hours. The 96 h LC50 value exceeds the maximum solubility of the test material in the test medium. The study was performed according to international accepted guidelines and under GLP regulation, i.e. of very high quality.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L