Bis(2-ethylhexyl) adipate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Well documented peer-reviewed publication, which meets basic scientific principles. However, undissolved test material was not removed from the test system. Emulsions were tested. Since toxic effects were observed far above the solubility of DEHA in water it cannot be excluded (and is most likely) that the adverse effects are caused by physical interactions rather than systemic toxicity. Therefore, the study is assessed to be invalid and the result is not used for the hazard assessment of DEHA.

Qualifier:

no guideline followed

Principles of method if other than guideline:

96h static bioassay

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Aliquots were teken from the top test concentration at the start and the end of the bioassay.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Stock emulsions were prepared by emulsifying measured quantities of DEHA in the test medium using a ultrasonic mixer.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): emulsion was tested

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout

ACCLIMATION
- Acclimation period: 2 weeks

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

70 - 80 mg/L

Test temperature:

15 +- 1 °C

pH:

7.4 - 7.8

Dissolved oxygen:

> 6 mg/L

Nominal and measured concentrations:

Nominal: 54, 110, 220, 320 and 420 mg/L
Measured: Top concentration: 420 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 40 L unplasticized polyethylene lined buckets
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Intervals of water quality measurement: Dissolved oxygen and ph were measured at 24h intervals. Temperature was monitored at 12h intervals.

EFFECT PARAMETERS MEASURED: Mortality was recorded after 0,25, 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 60, 72, 84 and 96h exposure time.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

54 - 110 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Validity criteria fulfilled:

not applicable

Conclusions:

The study is considered to be invalid, since undissolved test material was not removed from the test system. The effects observed at test concentrations far above the solubility of DEHA in water are assumed 9/to be caused by physical interactions rather than systemic toxicity of DEHA

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented peer-reviewed publication, which meets basic scientific principles

Qualifier:

according to guideline

Guideline:

other: EPA-66013-75-009: Methods for acute toxicity tests with fish, macroinvertebrates, and amphibians.

GLP compliance:

not specified

Analytical monitoring:

not specified

Vehicle:

no

Test organisms (species):

other: Oncorhynchus mykiss, Lepomis macrochirus, Pimephales promelas

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

255 - 368 mg/L as CaCO3

Test temperature:

25 +- 1 °C

pH:

8.0

Dissolved oxygen:

9.4 mg/L

Nominal and measured concentrations:

Different concentrations up to 1000 times the water solubility

Details on test conditions:

no details given

Reference substance (positive control):

not specified

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.78 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: for all tested fish species, value is based on the water solubility given in the publication

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

> 0.78 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: for all tested fish species, value is based on the water solubility given in the publication

Details on results:

Concentrations in excess of 100 times the water solubility (0.78 +/- 0.16 mg/L) were tested without producing mortality.

Validity criteria fulfilled:

not applicable

Conclusions:

A 96h acute toxicity test with three different fish species (Oncorhynchus mykiss, Lepomis macrochirus, Pimephales promelas) revealed that DEHA is not acutely harmful to freshwater fish up to the saturation limit in water.

Executive summary:

A 96h acute toxicity test of DEHA with three different fish species (Oncorhynchus mykiss, Lepomis macrochirus, Pimephales promelas) was performed according to US-EPA guideline 66013-75-009: Methods for acute toxicity tests with fish, macroinvertebrates, and amphibians. Different test concentrations up to 1000 times the water solubility were tested. No toxicity for any of the fish species was observed in any control or treatment. Thus, the 96h-LC50 value is determined to be > 0.78 mg/L, which was measured to be the solubility of the substance in water in this publication. Even if the true water solubility is considered to be lower, the results clearly indicate, that DEHA is not acutely toxic to freshwater fish up to its saturation limit in water.

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Scientifically acceptable 48h acute toxicity to freshwater fish scrrening test

Principles of method if other than guideline:

Method: 48h Screening test for the acute lethal toxicity to fish

GLP compliance:

no

Analytical monitoring:

not specified

Vehicle:

no

Test organisms (species):

Leuciscus idus

Details on test organisms:

TEST ORGANISM
- Length at study initiation (length definition, mean, range and SD): 6.5 cm
- Weight at study initiation (mean and range, SD): 2.88 g

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

pH:

7.0 - 7.6

Nominal and measured concentrations:

Nominal: 10, 100, 1000, 1600, 2500, 4000, 6300 and 10000 mg/L

Details on test conditions:

TEST CONDITIONS
- 3 resp. 10 animals per test
- determination of mortality and symptoms after 4, 24, 48h

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 10 000 mg/L

Sublethal observations / clinical signs:

RESULT MORTALITY
LC 50 (4h): > 10000 mg/l
LC 50 (24h): > 10000 mg/l
LC 50 (48h): > 10000 mg/l

RESULTS MORTALITY
Nominal conc.
mg/l number of fish 4h 24h 48h
0 (control) 10 - - -
10 3 - - -
100 3 - - -
1000 3 - - -
1600 3 - - -
2500 3 - - -
4000 3 - - -
6300 3 - - -
10000 3 - - -

REMARK
Effect concentration is greater than the solubility of the substance in the test medium.

Reference 4

Endpoint:

short-term toxicity to fish

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Data from reliable source. Though no detailed test report is available, all tests from the Japanese Authorities are performed according to an OECD guideline and GLP incl. analytics.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

not specified

Remarks:

probably yes

Analytical monitoring:

yes

Vehicle:

not specified

Test organisms (species):

Oryzias latipes

Test type:

other: probably semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Details on test conditions:

Test vessel and equipment
The following test vessel and equipment are used for the study.

2-1. Test vessel
The test vessel or other instrument that contacts the test solution must be made of glass or other chemically inert materials. Use a test vessel of an appropriate size in regard to the recommended volume. Cap the vessel loosely to prevent evaporation and dust contamination.
If the test substance is volatile, perform the test in a sealed system. Use a sufficiently large vessel to prevent shortage of dissolved oxygen.

2-2. Instrument
Use a dissolved oxygen meter and an appropriate instrument or apparatus for controlling the temperature for the test.


Material water
Use water suitable for culturing and testing the fish. It can be natural water (surface water or groundwater), dechlorinated tap water or artificially prepared water (see the note). The recommended total hardness is 10 - 250 mg/L in terms of calcium carbonate concentration, and the recommended pH is 6.0 - 8.5. Prepare the artificially prepared water using reagents of analytical grade and deionized or distilled water having an electric conductivity of 10 μS/cm or lower.

Acclimatization
All test fish must be acquired at least 12 days before the test and acclimatized. After monitoring the fish for 48 hr, acclimatize them in water used for the test under the following conditions for at least 7 days before beginning the exposure. If possible, avoid using medicated bath after the monitoring period.
Illumination: 12 - 16 hr light per day
Temperature: Appropriate temperature for the species tested (see Appendix Table 1 under VI.)
Oxygen concentration: At least 80% of the saturated oxygen concentration
Feeding: Daily or three times per week until 24 hr before beginning the exposure.
Record the mortality during the acclimatization period and apply the following conditions to the test fish.

If the overall mortality for seven consecutive days during the acclimatization period exceeds 10%, do not use them for the test.
If the overall mortality for seven consecutive days during the acclimatization period falls within 5 - 10%, extend the acclimatization period for 7 days.
If the overall mortality for seven consecutive days during the acclimatization period is below 5%, use them for the test.

Test solution
To prepare a test solution of each concentration, directly dissolve the required amount of the test substance in the material water, or prepare a stock solution of the test substance at an appropriate concentration and dilute it with the material water. Follow the descriptions in "Preparation of the test solution" under "III. General rules."
Perform the test without adjusting the pH. If the pH of the material water is greatly changed by the addition of the test substance, it is recommended to perform an additional test after adjusting the pH to that observed prior to the addition of the test substance. Perform the pH adjustment through a method causing no change in the concentration, chemical reaction or precipitation of the test substance. Preferably, use HCl or NaOH for the pH adjustment.

Test conditions
6-1. Test method
The test should be performed under a flow-through or semi-static condition. If the test substance concentration is unstable, a flow-through test is recommended.

6-2. Exposure period
Perform the exposure period for 96 hr.

6-3. Volume and number of test fish

Volume: A maximum density of 1.0 g fish weight/L is recommended for a semi-static test. The volume can be larger in a flow-through test.
Number of test fish: Use at least 7 fish for each of the test concentrations and the control.

6-4. Test concentrations
Adopt a concentration range comprising at least 5 concentrations that are setup in a geometric progression, preferably at a geometric ratio of within 2.2. The highest test concentration preferably gives fatal effects on all individuals tested, but concentrations of 100 mg/L or higher do not need to be tested. Preferably, no effect is observed at the lowest concentration.
Perform a control, and additionally an auxiliary control if using any auxiliary.

6-5. Culture method
Temperature: The temperature is set within the appropriate range for the species tested (see Appendix Table 1 under VI.), and kept constant with a variation of ± 2°C.
Illumination: The photoperiod is set to 12 - 16 hr light per day.
Dissolved oxygen concentration: It must be kept at 60% of the saturated oxygen concentration or higher. Aeration may be performed unless it causes a great loss of the test substance.
Feeding: Do not feed the organisms.
Disturbing factor: Avoid any factor that should disturb the fish behavior.

Beginning of the exposure to the test substance
Start the exposure by transferring a specified number of acclimatized fish established in 6-3 to each test vessel.

Observation
Observe the appearance and behavior of the fish at least at 24, 48, 72 and 96 hr after the beginning of the exposure. Organisms showing no motion (e.g., opercular movements) and no response when touched at their peduncles are considered as being dead. Remove dead organisms and record the mortality. Additional observations at 3 and 6 hr after the beginning of the exposure are also recommended. Record any anomaly in balance, swimming behavior, respiratory function or body color.

Measurements of test substance concentration, etc.
9-1. Test substance concentration measurement
At the beginning and end of the exposure, measure the test substance concentrations at least in the lowest and highest test concentration groups. If it has been predicted that the test substance concentration decreases from the initial concentration by 20% or more during the exposure period, it is recommended to take measurements for all test concentration groups at the beginning and end of the exposure. Furthermore, for volatile or adsorptive substances or those that are likely to be greatly decreased during the exposure period, additional measurements are recommended at 24 hr intervals during the exposure period.
In a semi-static test, perform at least two sets of measurements, provided that the measurements taken immediately after the water renewal and immediately before the next renewal are counted as one set.

9-2. Test condition measurement
Measure the pH, dissolved oxygen concentration and water temperature at least once daily.

Limiting test
If the test substance concentration of 100 mg/L or at the water solubility limit, whichever is lower, is predicted to show no fatality, etc., a limiting test can be performed at this concentration to demonstrate that LC50 is higher than this concentration. Use at least 7 organisms for the limiting test and also for the control. If any dead fish is observed by the end of the exposure, perform a regular test. Record any sublethal effects observed.


Validity of the test
The test is considered valid if it satisfies the following conditions.

The mortality in the control group does not exceed 10% (no more than one fish dead when using less than 10 individuals).
The dissolved oxygen concentration is kept at 60% of the saturated oxygen concentration or higher throughout the exposure period.
A sufficient level of the test substance concentration is kept throughout the exposure period.

Reference substance (positive control):

not specified

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Description of key information

No toxicity is observed within the limit of water solubility.

Additional information

Three studies investigating the short-term toxicity of Bis(2-ethylhexyl) adipate (CAS 103-23-1) to freshwater fish are available. The key study was performed according to EPA-66013-75-009 using Lepomis macrochirus, Pimephales promelas and Oncorhynchus mykiss as test organisms (Felder et al. 1986). Different concentrations up to 100 times the water solubility were tested. No toxicity for any of the fish species was observed in any control or treatment. Thus, the 96h-LC50 value is determined to be > 0.78 mg/L, which was measured to be the solubility of the substance in water in this publication. Even if the true water solubility is considered to be lower (see chapter 4.8), the results clearly indicate, that DEHA is not acutely toxic to freshwater fish up to its saturation limit in water.

This result is confirmed by supporting studies with DEHA and the relevant metabolite 2-ethylhexanoic acid.

A 48h acute toxicity to fish screening test with Leuciscus idus tested concentrations of 10 to 10000 mg DEHA/L (BASF 1976). No toxicity was observed in the control and any treatment. A study with sodium ethylhexanoate was used to assess the acute toxicity of the relevant metabolite 2-ethylhexanoic acid to fish. The study was performed according to OECD guideline 203 using Oryzias latipes as test organism (NITE 2001). No toxicity was observed up to tested concentration of 100 mg/L based on measured concentrations. Thus the 96h-LC50 value is determined to be > 100 mg/L.

 

One study reports acute effects above the water solubility limit (Hrudey et al. 1976). In this study a 96h static acute toxicity test was performed with rainbow trout. DEHA was exposed to emulsions of bis(2-ethylhexyl) adipate. A 96h-LC50 was observed to be between 54 -110 mg/L. However, undissolved test material was not removed from the test system. Emulsions were tested. Since toxic effects were observed far above the solubility of DEHA in water it cannot be excluded (and is most likely) that the adverse effects are caused by physical interactions rather than systemic toxicity. Therefore, the study is assessed to be invalid and the result is not used for the hazard assessment of DEHA (see respective robust study summary for details).

 

The availabe information on short-term toxicity on fish will be supported by the results of a planned long-term toxicity study according to OECD Guidline 210 (see section 6.1.2 of the dossier). In Annex VIII to REACH (Section 9.1.3, Column 2) it is stated that assessment of the short-term toxicity to fish is not mandatory if a aquatic long-term toxicity study on fish is available.