Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Deviations:

yes

Remarks:

See principles of method if other than guideline

Principles of method if other than guideline:

Deviation from the study plan one batch of stock solution used to prepare the final test solutions was not stirred for 24±3 hours before used (day 4 of exposure). Due to a human failure the designated stock solution has to be discarded and a new batch of stock solution was prepared on the same day of usage. This stock solution was stirred for 2 hours and thereafter filtered and directly used for preparation of the test solution at day 4 of exposure. This minor deviation had no influence on the reliability of the test data and the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Prior to defrosting and analysis of the test samples, acetonitrile was added to the frozen samples (e.g. 50 mL acetonitrile per 50 mL water sample). The samples were defrosted, vigorously homogenised on a Vortex mixer (at least 1 min). 10 g sodium chloride were added to the sample and shaken vigorously. After that, the phases were allowed to separate. About 20 mL of the upper acetonitrile extract was transferred into a 25 mL volumetric cylinder containing a volume of approx. 5 mL sodium sulphate (anhydrous). The cylinder was closed and shaken to remov the water. An aliquot of approx. 1 mL was transferred into HPLC vials for determination by HPLC-MS/MS.
If necessary, the samples were diluted with final extract of the control sample to achieve final concentrations falling within the linear calibrated range of the detector response.

Vehicle:

no

Details on test solutions:

Based on the results of a preliminary non-GLP range finding tests, the following nominal concentrations in a geometrical series (spacing factor: square root of 10) was tested (extended limit test with three concentration levels): 10.0, 31.6, and 100 % of a filtrate at a loading rate of 10 mg test item/L. Test solutions were continuously renewed in a flow-through system.

The range finder-test showed that a storage period of two days for the stock solution showed a comparable decrease of test item as seen in the pre-test 1. Nevertheless, the decline of test concentrations in the test solution during the exposure phase was higher than expected, especially at the later sampling dates (potentially caused by adsorption and/or degradation processes due to the higher amount of organic matter, e.g. addition of food).
As a consequence of the significant losses especially at the later time points during the exposure period, the renewal interval of the stock solution was shortened from two days to every 24 hours for the definitive test. This shortening was chosen to increase the initial test item concentration provided via application of the stock solution (filtrate), and consequently also the content of test item which will be available for preparation of the test solution.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

- Species: Danio rerio Hamilton-Buchanan 1822 (Zebrafish)
- Origin: Fertilised eggs were collected from adult zebrafish from in-house cultures, which have been maintained and bred at ECT since February 2019
- Holding conditions and fertilisation of test organisms: In the culture tanks, the fish were kept in reconstituted water (OECD guideline 203, 1992) diluted with deionised water (1:1; v/v) and supplemented with 1% of artificial seawater (Tropic Marin, Dr. Biener GmbH Aquarientechnik, Wartenberg, Germany; salinity 28 ± 2‰). Fish were held in glass aquaria at 26 ± 2 °C with a 14 h/10 h photoperiod. They were fed a combined diet of newly hatched nauplii of Artemia sp. (Sanders Brine Shrimp Co., Morgan, UT 84050, U.S.A.) and TetraMin (Tetra Werke, Melle, Germany)
-Test Medium: Reconstituted water according to OECD guideline No. 203 was used to dilute the test item and to keep the fish before and during the period of the test. Local tap water was treated by reverse osmosis and ion exchange to prepare deionised water. Therefore, a contamination with heavy metals, pesticides and total organic carbon is excluded. The required amount of reconstituted water was prepared within four weeks before use. During storage reconstituted water was aerated. Prior to use, the reconstituted water was diluted with deionised water (1:1; v/v) and supplemented with 1% of artificial seawater.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

35 d

Hardness:

Total hardness in mg CaCO3/L was calculated by multiplication of the hardness-value in °dH by 17.86:
Total hardness [°dH]: minimum 9.8, maximum 10.2
Total hardness [mg CaCO3/L]: minimum 175, maximum 182

Test temperature:

T [°C]: minimum 25.2, maximum 26.4

pH:

pH: minimum 7.3, maximum 7.6

Dissolved oxygen:

O2 [mg/L]: minimum 5.5, maximum 9.0
O2 [%]: minimum 70, maximum 105

Salinity:

In the culture tanks, the fish were kept in reconstituted water (OECD guideline 203, 1992) diluted with deionised water (1:1; v/v) and supplemented with 1% of artificial seawater (Tropic Marin, Dr. Biener GmbH Aquarientechnik, Wartenberg, Germany; salinity 28 ± 2‰).

Conductivity:

Conductivity [µS/cm]: minimum 808, maximum 810

Nominal and measured concentrations:

Nominal concentrations were given as a percentage of the stock solution with a concentration of

10 mg test item/L:
10% corresponding to 1mg test item/L
31.6 % corresponding to 3.16 mg test item/L
100% corresponding to 10 mg test item/L

Concentrations were measured over the course of time (see table below). Initially measured concentrations for the various nominal concentrations are:

Nominal 10% (1 mg test item/L), measured 0,0301 mg test item/L
Nominal 31.6% (3.16 mg test item/L), measured 0.163 mg test item/L
Nominal 100% (10 mg test item/L), measured 0,515 mg test item/L

Details on test conditions:

Material and methods:
Test organism: Zebrafish (Danio rerio, Hamilton-Buchanan 1822)
Test medium: Reconstituted water (OECD guideline No. 203) mixed with deionised water (1:1; v/v), supplemented with 1% artificial seawater
Endpoints: NOEC, LOEC
Biological parameters: – Hatching success;
– Number of larvae hatching each day;
– Time to start of hatching and end of hatching;
– Cumulative mortality (Post-Hatch Success (Survival));
– Number of deformed or abnormally appearing larvae;
– Number of organisms exhibiting abnormal behaviour;
– Length of the surviving fish;
– Wet and dry weight of the surviving fish.
Test duration: 35 days
Temperature (target): 26 ± 1.5 °C
Photoperiod: 14/10 hours light/dark cycle
Light intensity (target): 500 - 1000 lx
Test units: Glass vessels, 0.8 L volume
Test concentration
(limit concentration): 10.0, 31.6, and 100 % of a filtrate at a loading rate of 10 mg test item/L plus a control
No. of replicates per control: 4
No. of replicates per treatment: 4

Reference substance (positive control):

not required

Duration:

35 d

Dose descriptor:

EC10

Effect conc.:

70.4 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

wet weight

Remarks on result:

other: no reliable ECx values were calculated for the parameter fresh weight of fish larvae at the end of the exposure period. Nevertheless, the EC10 was determined to be 107 % of a filtrate at a loading rate of 10 mg test item/L based on nominal concentrations,

Duration:

35 d

Dose descriptor:

other: EC20

Effect conc.:

> 72.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

wet weight

Duration:

35 d

Dose descriptor:

EC50

Effect conc.:

> 72.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

wet weight

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

> 72.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

wet weight

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

> 72.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

length

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

> 72.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Post-hatch success (Survival)

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

> 72.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 72.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

Basis for effect wet weight

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 72.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

length

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 72.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Post-hatch success (survival)

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 72.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

For detailed information on biological endpoints refer to tables 2 – 5 and further explanations in the attached document "Summary of relevant tables for Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate: A Study on the Toxicity to Early-Life Stages of Zebrafish (Gilberg, Witte, 2020) Study No. 20AN2FV"

 

Validity criteria fulfilled:

yes

Conclusions:

In a “Fish, Early-life Stage Toxicity Test” (FELS test) according OECD TG 210 the toxicity of the Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate on Zebrafish (danio rerio) was assessed. Over the course of 35 d no adverse effect was found on the biological parameters: hatching success, post-hatch success (survival), number of healthy fish, length of surviving fish and wet weight of the surviving fish.
The test was conducted with a nominal test concentration of max. 10 mg test item/L. The test-item concentration was also measured over the course of the test. The NOEC for all relevant biological parameters was determined to be ≥72.5 µg test itel/L.

Executive summary:

In a “Fish, Early-life Stage Toxicity Test” (FELS test) according OECD TG 210 the toxicity of the Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate on Zebrafish (danio rerio) was assessed.

Caused by the poor solubility and low stability of the substance mixture in aqueous solutions, the study was conducted in flow-through approach. For the test organism zebrafish (danio rerio) the following biological paramets were determined over the course of 35 d: Hatching success, number of larvae hatching each day, time to start of hatching and end of hatching, post-hatch success (survival), number of deformed or abnormally appearing larvae, number of organisms exhibiting abnormal behaviour, length of the surviving fish and wet weight of the surviving fish. In addition the dry weight of the surviving fish was determined but according to OECD Guideline for Testing of Chemicals, No. 210 "Fish, Early-life Stage Toxicity Test" wet weight is the preferred parameter.

 

The test concentrations were prepared as the following percentages of a filtrate of 10 mg/L stock solution: 10.0, 31.6 and 100%. Analytical determinations revealed the following measured geometric mean concentrations in the test solution: <LOQ, 15.6 µg/L and 72,5 µg/L respectively. To calculate the biological endpoints (NOEC/LOEC and EC_x) based on measured concentrations, a theoretical measured concentration was calculated for the lowest concentration level (10%). The resulting mean measured concentrations for the lowest concentration level was 4.93 µg test item/L, for details see section 19.2.

 

The test fulfilled all validity criteria and resulted in the following effect concentrations:

 

	Endpoints (measured) [µg test item/L]
Parameter	EC10	EC20	EC50	NOEC	LOEC
Hatching success	n.d.	n.d.	n.d.	≥72.5	>72.5
Post-hatch success (Survival)	n.d.	n.d.	n.d.	≥72.5	>72.5
Number of healthy fish^1	n.d.	n.d.	n.d.	≥72.5	>72.5
Length of the surviving fish	n.d.	n.d.	n.d.	≥72.5	>72.5
Wet weight of the surviving fish	70.4	>72.5	>72.5	≥72.5	>72.5

 

No distinct correlation was observed between the concentration of the test item and the wet weight of fish. Therefore, no reliable EC_x values were calculated for the parameter fresh weight of fish larvae at the end of the exposure period. Nevertheless, the EC₁₀ was determined to be 107 % of a filtrate at a loading rate of 10 mg test item/L based on nominal concentrations, equivalent to 70.4 µg test item/L based on measured concentrations.

 

Concluding, no effect on zebrafish (danio rerio) was determined for the reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate at the chosen test concentrations.

Description of key information

In a “Fish, Early-life Stage Toxicity Test” (FELS test) according OECD TG 210 the toxicity of the Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate on Zebrafish (danio rerio) was assessed. Over the course of 35 d no adverse effect was found on the biological parameters: hatching success, post-hatch success (survival), number of healthy fish, length of surviving fish and wet weight of the surviving fish.
The test was conducted with a nominal test concentration of max. 10 mg test item/L. The test-item concentration was also measured over the course of the test. The NOEC for all relevant biological parameters was determined to be ≥72.5 µg test itel/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

>= 72.5 µg/L