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Diss Factsheets

Administrative data

Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2014

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2-ethylhexyl) fumarate
EC Number:
205-448-2
EC Name:
Bis(2-ethylhexyl) fumarate
Cas Number:
141-02-6
Molecular formula:
C20H36O4
IUPAC Name:
bis(2-ethylhexyl) but-2-enedioate
Details on test material:
Identification: Dioctylfumarate
Batch: LEDF1C7047
Purity: 99.2 %

Sampling and analysis

Analytical monitoring:
yes

Test solutions

Vehicle:
yes
Details on test solutions:
The application solutions used for the dosage of the test media were renewed every 7 days during the test period. From the freshly prepared application solutions, duplicate samples were taken on 2 preparation dates (days -2 and 26). To confirm the stability of the test item in the application solutions during its renewal periods, duplicate samples were taken from the aged application solutions on days 5 and 33, corresponding to the renewal periods of 7 days.
From the test media, duplicate samples were taken from all test concentrations and the solvent control at 11 dates during the test period, starting from day 0 until test end.
During the first period of the test, when 200-mL beakers were used as test vessels, the test medium samples were collected from separate flow through vessels. Sampling from the small replicate test vessels was not performed to avoid damage of the eggs, larvae and fish.
Due to the high water exchange per day in the 200-mL test vessels, it can be considered that the test item concentrations in the separate vessels and in the 200-mL test vessels were essentially the same. Later during the test, when larger test vessels were used samples were taken from the test vessel itself. Thereby, the samples were taken alternately from the different replicates.
Immediately after sampling, all samples were stored deep-frozen (at about -20 °C). In pre-experiments for investigation of the storage stability of the samples, the test item proved to be stable under these storage conditions.
The nominal test concentrations of 0.47 and 1.5 mg/L were determined in this test as the overall NOEC and LOEC (NOEC and LOEC of the most sensitive test parameter), and therefore, were analytically verified after the end of the test.
From these test concentrations, the duplicate samples from all sampling times distributed over the test period were analyzed.
From the corresponding application solutions, one of the duplicate samples was analyzed from both stability controls.
The samples from the nominal test concentrations of 0.15 to 0.015 mg/L were not analyzed, since these concentrations were below the overall NOEC or LOEC determined in this test, and thus, not relevant for the interpretation of the biological results.
From the solvent control, one of the duplicate samples was analyzed from three sampling dates during the test period.

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
The study was performed with newly fertilized eggs of zebra fish, Danio rerio (Hamilton-Buchanan 1822, Teleostei, Cyprinidae). The origin of the strain of zebra fish is West Aquarium GmbH, 37431 Bad Lauterberg / Germany. At Harlan Laboratories, a brood batch of at least 200 individuals of this strain was held.

Study design

Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
35 d
Post exposure observation period:
Test duration was in total 35 days (hatching period from day 0 to day 5 plus a 30-day post hatch period, whereby day 5 post fertilization equals day 0 post hatch).

Test conditions

Hardness:
1.25 mmol/L; 125 mg/L as CaCO3
Test temperature:
26.5–26.6 °C
pH:
The pH values in the controls and at the test concentrations ranged between 7.1 and 7.2
Dissolved oxygen:
7.7 mg/L, corresponding to an oxygen saturation of at least 94 %.
Salinity:
Not applicable
Nominal and measured concentrations:
The following nominal concentrations were tested: 0.015, 0.047, 0.15, 0.47, and 1.5 mg/L.
Additionally, a control and a solvent control were tested in parallel.
Details on test conditions:
The parental fish were held in an aquarium in reconstituted water (same water quality as described in section 3.3). A 16 hour light to 8 hour dark photoperiod with a 30 minute transition period was applied. The fish were fed with a commercial fish diet (TETRA MIN Hauptfutter, TETRA-Werke, 49304 Melle / Germany) and brine shrimps (Artemia salina).
The brood batch was regularly visually checked for abnormal behavior, diseases or mortality. No visible abnormalities were observed in the fish during the three months prior to test start and no medication was applied.
Glass dishes covered with a mesh were placed on the bottom of the aquarium one day prior to test start for collecting the newly fertilized eggs for the test. The mesh separated fertilized eggs (which sank to the bottom into the glass dishes) from the parent fish. Spawning started in the morning on day 0 after turning on the light. The eggs were exposed to the test media within about 3 hours after fertilization.
The method of exposure and also the fish species Danio rerio is recommended by the testing guideline.

Results and discussion

Effect concentrationsopen allclose all
Duration:
35 d
Dose descriptor:
LOEC
Effect conc.:
ca. 0.86 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
ca. 0.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Details on results:
The test item concentration in the freshly prepared and aged application solutions ranged from 85 to 117 % of the nominal values. This shows the correct preparation of the application solutions and stability of the test item in the application solutions during the renewal periods of 7 days.

The test item concentrations in the analyzed test medium of nominal 0.47 mg/L varied in the range from 54 to 91% of the nominal value. In the test medium of nominal 1.5 mg/L the range was 15 to 143% of the nominal value. The reason for the high variation of the measured test concentrations at the highest test concentration of nominal 1.5 mg/L is considered to be the water solubility of the test item in the test water. The water solubility was determined in a pre-experiment to be approximately 1.1 mg/L. Therefore, slightly above the solubility limit of the test item in test water was tested to result with the mean measured concentration as close as possible at the water solubility limit of 1.1 mg/L.

The mean measured test item concentrations (calculated as the arithmetic mean of all measurements of each test concentration) were 0.30 mg/L and 0.86 mg/L, corresponding to 65 and 58% of the nominal values of 0.47 mg/L and 1.5 mg/L, respectively.

The biological results are related to the mean measured test item concentrations.

Hatching Success
In the control, solvent control and at all test concentrations, hatching was completed on Day 5 post fertilization (Table 1). The mean hatching success in the control and solvent control was 98.3 ± 3.3% and 100%, respectively. The hatching success in the solvent control was not statistically significant different to the hatching success in the control (Fisher exact binomial test, two-sided, α = 0.05) and was sufficiently high under the test conditions.

At the test concentrations up to and including 0.30 mg/L (mean measured), the mean hatching success was not statistically significantly different compared to the solvent control (mortality of eggs was tested by a Fisher’s exact binomial test one-sided greater, α = 0.05) and no concentration-effect relationship was observed. At the highest test concentration of 0.86 mg/L, the mean hatching success was statistically significantly reduced compared to the solvent control. The reduction of 13.3% compared to the solvent control was estimated to be a toxic effect of the test item.

Therefore, the NOEC for hatching success was determined to be 0.30 mg/L. The LOEC was 0.86 mg/L.

Development Rate of Embryos
The results for the development rate of the embryos are shown in Table 2. No statistically significant difference was determined between control and solvent control (results of a Student t-test,  = 0.05, two-sided).

The development rate of the embryos was at all test concentrations nearly identical to or even slightly higher than in the solvent control and no concentration-effect relationship was observed (not statistically significantly different compared to the solvent control according to a Dunnett t-test, two-sided, α = 0.05).

The NOEC for the embryonic development rate was determined to be ≥0.86 mg/L. The LOEC for development rate could not be determined since it was above the highest concentration tested (>0.86 mg/L).


Survival of Larvae and Juvenile Fish
The mean survival rate of the fish was 83.2 ± 8.4% in the control and 75.0 ± 11.4% in the solvent control, demonstrating the suitability of the test conditions (the validity limit for post hatch success is ≥70%). No statistically significant difference was determined between both controls for this parameter (results of a Student t-test,  = 0.05, two-sided).

During the test, sporadic, non concentration effect-related mortality of larvae and fish was observed at all test concentrations. Since identical low mortality rate was also observed in the control and solvent control it was not regarded as a toxic effect of the test item but as a natural mortality rate.

All fish, which survived until the end of the test were healthy and showed normal behavior. The larvae and fish found to be dead showed no previous visible abnormalities.

The mean survival rates at the test concentrations up to and including the highest test concentration of 0.86 mg/L were in the range of 87.3 and 112.4% of the solvent control value and no concentration-effect relationship was observed.

Therefore, the NOEC for survival of the test fish was determined to be ≥0.86 mg/L. The LOEC for survival of the test fish could not be determined since it was above the highest concentration tested (>0.86 mg/L).

Fish Length and Body Weight
The body length, body wet weight and body dry weight of the test fish at the end of the test were measured. No statistically significant difference was determined between control and solvent control for any of these parameters (results of a Student t-test, α = 0.05, two-sided).

For the measured body length, body wet weight and body dry weight the mean values obtained from all test concentrations up to and including 0.86 mg/L were not statistically significantly reduced compared to the solvent control (Welch t-test, one-sided smaller, α = 0.05) and no concentration effect relationship was observed.

Thus, the NOEC for fish body length, fish wet weight was determined to be ≥0.86 mg/L. The LOEC for these growth parameters could not be determined since it was above the highest concentration tested (>0.86 mg/L).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Fish Early Life Stage Toxicity test: NOEC = 0.3 mg/L; LOEC = 0.86 mg/L (reduced hatching rate)
Executive summary:

The toxicity of the test item Dioctylfumarate to zebra fish (Danio rerio) was investigated in an early-life stage toxicity test according to the OECD Guideline for Testing of Chemicals, No. 210, "Fish, Early-life Stage Toxicity Test", 1992.

Freshly fertilized eggs of zebra fish were exposed to test media containing the test item at nominal concentrations of 0.015, 0.047, 0.15, 0.47, and 1.5 mg/L under flow-through conditions for the test period of 35 days. Additionally, a control and a solvent control were tested in parallel.

The preparation of the test media was based on the OECD series on testing and assessment No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

At the start of the test, 60 eggs each (divided into 4 replicates) were distributed to the test concentrations and control and solvent control. The eggs, larvae and juvenile fish were observed for toxic effects on their development, growth and survival.

The nominal test concentrations of 0.47 and 1.5 mg/L were determined in this test as the overall NOEC and LOEC (NOEC and LOEC of the most sensitive test parameter), and therefore, were analytically verified.

The test item concentrations in the analyzed test medium of nominal 0.47 mg/L varied in the range from 54 to 91% of the nominal value. In the test medium of nominal 1.5 mg/L the range was 15 to 143% of the nominal value. The reason for the high variation of the measured test concentrations at the highest test concentration of nominal 1.5 mg/L is considered to be the water solubility of the test item in the test water. The water solubility was determined in a pre-experiment to be approximately 1.1 mg/L. Therefore, slightly above the solubility limit of the test item in test water was tested to result with the mean measured concentration as close as possible at the water solubility limit of 1.1 mg/L.

The mean measured test item concentrations (calculated as the arithmetic mean of all measurements of each test concentration) were 0.30 mg/L and 0.86 mg/L, corresponding to 65 and 58% of the nominal values of 0.47 mg/L and 1.5 mg/L, respectively.

The biological results are related to the mean measured test item concentrations.

 

 

Mean measured concentration (mg/L)

 

Test parameter

NOEC

LOEC

 

 

Hatching rate:

0.30

0.86

 

 

Development rate of eggs:

≥ 0.86

> 0.86

 

Survival of larvae and juvenile fish:

≥0.86

> 0.86

 

 

Fish length:

≥ 0.86

> 0.86

 

 

Fish wet weight and dry weight:

≥ 0.86

> 0.86

 

 

Conclusion:

Summarizing the NOEC values for each of the test parameters assessed, the overall NOEC of dioctylfumarate for early life stages of zebra fish was determined to be the mean measured test concentration of 0.30 mg/L, since no toxic effect on the eggs, larvae or fish was observed up to and including this concentration. The overall LOEC was determined to be the mean measured test concentration of 0.86 mg/L, due to the statistically significantly reduced hatching rate at that concentration.