bis(2-ethylhexyl) (4-hydroxy-3,5-dimethoxybenzyl)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:

2007-07-19 to 2007-11-27

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

Principles of method if other than guideline:

none

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

N/A

Vehicle:

no

Details on test solutions:

For the reconstituted water the following chemicals (analytical grade) were dissolved in fully demineralized water:
Macro nutrients [mg/L]
CaCI2x2H20 294.00
Mg5O4x7H20 123.25
Na HCO3 64.75
KCI 5.75

Before the start of the experimental part the reconstituted water for fish was prepared. After preparation the water was aerated for 24 hours before using it.
The proportion of Ca to Mg ions was 4:1 and that of Na to K ions 10:1. Hardness: about 15° dH (about 267 mg/L CaCO3).

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). Furthermore, the fish were fed with daphnia, or artemia, or frozen mosquitolarva daily. 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

A total of 24 zebra fish was used in this study.
Pretest: 4 zebra fish Control group: 10 fish
Test material group: 10 fish.


TEST ORGANISM
- Common name: Zebrafish
- Source: Merck KGaA
- Length at study initiation: 2.0 +/- 1 cm

ACCLIMATION
- Acclimation period: > 14 days
- Acclimation conditions: same as test
- Type and amount of food during acclimation: Tetra Min diet (Tetra GmbH, Melle, Germany), daphnia, artemia, frozen mosquito larvae
- Feeding frequency during acclimation: daily
-
FEEDING DURING TEST
- Food type:
- Amount:
- Frequency:

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:

7.29 to 7.79

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 mg/L of the test item (maximal solubility <0.02 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 (MEINERLING, 2006). The solubility of the test material could not be determined but was < 0.02 mg/L. The same low solubility can be expected for reconstituted water for fish.

Temperature
The temperature during the main study was 23 to 25 °C.

pH and oxygen
pH values during the final study were inconspicuous.

Reported statistics and error estimates:

N/A

Validity criteria fulfilled:

yes

Conclusions:

An aqueous solution of the test item at a nominal concentration of 100 mg/L revealed no aquatic toxicity in the test system. The analytical 96 h LC50 value to zebra fish exceeded the maximal solubility of < 0.02 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 over 96 hours, under defined conditions in a limit test.A 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.

Result

The following results were obtained:

	Mortality
	24h	48h	72h	96 h
Cotrol 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 water is < 0.02 mg/L. 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    >    0.02 mg/L (nominal > 100 mg/L)
48 h LC50    >    0.02 mg/L (nominal > 100 mg/L)
72 h LC50    >    0.02 mg/L (nominal > 100 mg/L)
96 h LC50    >    0.02 mg/L (nominal > 100 mg/L)

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 < 0.02 mg/L (as established analytically) of the test material in reconstituted water and, thus, could not be determined in this test.

Description of key information

The 96h LC50 was above the limit concentration of 100 mg/L (nominal) in fish.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

>= 100 mg/L

Additional information

Key study (OECD 203, 2007)

The acute toxicity effect of the test substance to zebrafish (Danio rerio) was determined in a 96-hour static test according to OECD 203. A limit test was performed in accordance with the result of the preliminary test demonstrate that the test substance at a loading rate of 100 mg/L has no acute toxicity effect on the test organisms. Thus, only the treatments at a loading rate of 100 mg/L and the controls were tested.

No aquatic toxicity was observed in this test system up to a nominal concentration of 100 mg/L test material. The 96h LC50 was above the water solubility limit and larger than 100 mg/L (nominal).