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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

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 211 (Daphnia magna Reproduction Test)

Version / remarks:

adopted 2nd October 2012

Deviations:

no

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. 25 mL acetonitrile per 50 mL water sample). The samples were defrosted and vigorously homogenised on a Vortex mixer (at least 1 min). An aliquot of approx. 1 mL was transferred into HPLC vials for determination by HPLC-MS/MS.

Vehicle:

no

Details on test solutions:

Based on the results of preliminary non-GLP range finding tests, the following nominal
concentrations in a geometrical series (spacing factor: 2.0) will be tested in the definitive test:
100, 50.0, 25.0, 12.5 and 6.25% of a filtrate at a loading rate of 10 mg test item/L.

A saturated stock solution was prepared in test medium at a nominal loading rate of 10 mg test item/L as described below.

Stock solutions (S) of the test item in test medium were prepared by weighing amounts equivalent to 10 mg of the test item per 1 L of dilution medium into a volumetric glass flask of appropriate volume, containing less than its nominal volume. The resulting solution (>> 10 mg test item/L) was intensely stirred on a magnetic stirrer at 1300 rpm at room temperature in the dark for 15 - 21 minutes to ensure a homogeneous distribution of the test item in the test medium.

Thereafter the volumetric flask, containing the pre-dissolved test item, was filled up to the mark with test medium used to prepare the final stock solution at a nominal loading rate of 10 mg test item/L. This stock solution was termed S1, and was then stirred for 24±3 hours at 750 rpm at ambient temperature in the dark.

Subsequently the stock solution S1 was filtered using a glass fibre filter with an average retention capacity of 0.4 µm (Macherey-Nagel, MN GF-5). The filtrate (stock solution S2) was visually examined for un-dissolved/particulate matter (using the Tyndall effect).

The filtrate was termed stock solution (S2) and was used to produce the desired test item concentrations. All further calculations of the nominal concentration are expressed in % of filtrate at a loading rate of 10 mg test item/L.

Test organisms (species):

Daphnia magna

Details on test organisms:

The daphnids were held in a specific medium (Elendt Medium M4 as specified in the test guideline). The vessels containing the stock culture were on a 16/8 hours light/dark cycle at a target temperature of 20±2 °C. The daphnids were fed with green algae (Desmodesmus subspicatus) and yeast.

Material of stock vessel: glass
Volume of medium per stock vessel: 1.8 L
Depth of medium in the stock vessel: approximately 14.5 cm
Number of daphnids kept as stock per culture vessel (batch size): approximately 20
Renewal of medium: batchwise, twice per week
Light intensity: 50–1000 lx
Aeration: none
Feeding frequency: 3-4 times per week

The selected holding vessels designated for production of the neonates for the test were cleared of all mixed-aged young daphnids on the day before test start. These vessels had a record of more than one brood and contained parental daphnids aged less than 28 days. On the day of test start (day 0) all newly produced neonates of these vessels were < 24 hours old. These neonates were collected for the test.

Test type:

semi-static

Water media type:

other: Elendt medium M4 was used for the test

Limit test:

no

Total exposure duration:

21 d

Hardness:

Water hardness in mg CaCO3/L was calculated by multiplication of the hardness-value in °dH by 17.86. Water hardness [°dH] minimum: 13.6 maximum: 14.5. Water hardness (mg CaCO3/L] minimum: 243 maximum: 259

Test temperature:

manual measurements (n=12): 19.9 - 21.6 °C
automatic measurements (n=511): 20.2 - 22.0 °C

pH:

pH: minimum 7.5 maximum 9.0

Dissolved oxygen:

dissolved oxygen [% of air sat. value]: minimum 88 maximum 132
dissolved oxygen [mg/L]: minimum 7.7 maximum 11.5

Salinity:

The medium consists of trace elements, vitamins and macro-nutrients in the following final concentrations (in mg/L):

Trace elements: Macronutrients:
H3BO3 2.86 CaCl2 × 2 H2O 293.8
MnCl2 × 4 H2O 0.361 MgSO4 × 7 H2O 123.3
LiCl 0.306 KCl 5.8
RbCl 0.071 NaHCO3 64.8
SrCl2 × 6 H2O 0.152 Na2SiO3 × 9 H2O 10.0
NaBr 0.016 NaNO3 0.274
Na2MoO4 × 2 H2O 0.063 KH2PO4 0.143
CuCl2 × 2 H2O 0.017 K2HPO4 0.184
ZnCl2 0.013
CoCl2 × 6 H2O 0.010 Vitamins:
KI 0.0033 Thiamine hydrochloride 0.075
Na2SeO3 0.0022 Cyanocobalamin 0.001
NH4VO3 0.00058 Biotin 0.00075
Na2EDTA × 2 H2O 2.5
FeSO4 × 7 H2O 1.0

Conductivity:

The conductivity [µS/cm] of the medium M4 batches was determined before use:
02 Oct 2020: 636
19 Oct 2020: 647
29 Oct 2020: 678

Nominal and measured concentrations:

Nominal concentrations were given as a percentage of the filtrate of a stock solution with a concentration of
10 mg test item/L:

6.25% corresponding to 0.625 mg test item/L
12.5% corresponding to 1.25 mg test item/L
25.0% corresponding to 2.5 mg test item/L
50.0% corresponding to 5.0 mg test item/L
100% corresponding to 10 mg test item/L

Concentrations were measured over the course of time. Initially measured concentrations for the various nominal concentrations are:

Nominal 6.25% (0.625 mg test item/L), measured 0.0199 mg test item/L
Nominal 12.5% (1.25 mg test item/L), measured 0.0146 mg test item/L
Nominal 25.0% (2.5 mg test item/L), measured 0.0473 mg test item/L
Nominal 50.0% (5.0 mg test item/L), measured 0.112 mg test item/L
Nominal 100% (10 mg test item/L), measured 0.203 mg test item/L
Stock Solution: Nominal 100% (10 mg test item/L), measured 0.231 mg test item/L

Details on test conditions:

Material and methods:

Test system: Daphnia magna (Straus)
Medium: Elendt Medium M4
Endpoints: NOEC/LOEC, ECx for reproduction and mortality
Biological parameters: - reproduction as assessed by fecundity (total number of living offspring (F1));
- mortality/survival as assessed by immobility of parental (F0) daphnids
Test duration: 21 days
Temperature: 19.9–21.6 °C (manual measurement; n = 12);
20.2–22.0 °C (automatic measurement in test medium; n = 511)
Photoperiod: 16 h light/8 h dark
Aeration: no aeration during exposure
Test units: 100 mL glass beakers, covered by glass lids

Volume of test solution per test vessel: 50–60 mL
Renewal of test solution during exposure: three times per week
Number of daphnids per test vessel: one at start of exposure

Feeding during exposure: three times per week, fresh algae suspension of Desmodesmus subspicatus; 0.15–0.20 mg C/daphnia × d

Test item concentrations (nominal): 100, 50.0, 25.0, 12.5 and 6.25% of a filtrate at a loading rate of 10 mg test item/L plus a control

Number of replicates per test concentration and control: 10
Chemical analysis of test concentrations: once per week (fresh and aged solutions)

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

8.68 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

4.34 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Key result

Duration:

21 d

Dose descriptor:

other: EC20

Effect conc.:

10.45 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

4.05 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 26.6 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks:

parental

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

26.6 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks:

parental

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Duration:

21 d

Dose descriptor:

other: EC20

Effect conc.:

3.138 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

1.029 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks:

parental

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks:

parental

Details on results:

Sublethal effects as observed in the living parental daphnids were recorded throughout the treatments. A slight increase of the frequency of sublethal observations (e.g. daphnids trapped at the water surface) was noted towards higher concentration levels. Detailed observations are tabulated below

Treatment [% of filtrate]: Control 6.25 12.5 25.0 50.0 100
Observation Code frequency of observation
Daphnid temporarily
floating on water surface1 a 2 3 10 11 8 8
Daphnid lay on the bottom
of the test vessel b 0 3 0 0 1 2
Daphnid showed reduced
swimming activity c 0 3 0 0 1 0

Aborted eggs d 0 3 2 4 3 6

Algae on antennae/carapace e 0 2 2 1 2 3

Immobile offspring f 2 0 0 0 1 0

Immobile parents M 0 1 0 0 1 0

1: upon observation, floating daphnids were gently pushed under the water surface; afterwards they showed normal swimming behaviour.

For further information on biological endpoints refer to tables 4 – 6 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 Chronic Toxicity to Daphnia magna (Egeler, Witte, 2021) Study No. 20AN1DB"

 

Validity criteria fulfilled:

yes

Conclusions:

In a “Daphnia Magna, reproduction test" according OECD TG 211 the toxicity of the Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate on daphnia magna was assessed. Over the course of 21 d no concentration-response relationship was observed for parental daphnid survival/immobility resulting in a NOEC = 26.6 µg/L and a LOEC >26.6 µg/L. For the endpoint "reproduction based on surviving F0 daphnids" the following effect concentrations were observed: EC10 = 4.05 µg/L, EC20 = 10.45 µg/L, NOEC 4.34 µg/L and LOEC = 8.68 µg/L

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.

Executive summary:

In a “Daphnia Magna, reproduction test" according OECD TG 211 the toxicity of the Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate on daphnia magna was assessed.

 

Young female Daphnia, aged less than 24 hours at the start of the test, were exposed to a range of concentrations, nominally in percentages of a filtrate of a stock solution with a concentration of 10 mg/L. The resulting nominal test concentrations are: 0.625 mg/L, 1.25 mg/L, 2.5 mg/L, 5.0 mg/L and 10 mg/L.

 

Measured concentrations over the course of the study revealed much lower concentrations resulting in effect concentrations in the lower µg/L range.

 

Over the course of 21 d no concentration-response relationship was observed for parental daphnid survival/immobility resulting in a NOEC = 26.6 µg/L and a LOEC >26.6 µg/L.

 

For the endpoint "reproduction based on surviving F0 daphnids" the reproductive output of the Daphnia exposed to the test item was compared to that of the control in order to determine the effect concentrations according to Williams Multiple Sequential-t Test Procedure.

 

Following effect concentrations were observed for "reproduction based on surviving F0 daphnids": EC10 = 4.05 µg/L, EC20 = 10.45 µg/L, NOEC 4.34 µg/L and LOEC = 8.68 µg/L

Description of key information

In a “Daphnia Magna, reproduction test" according OECD TG 211 the toxicity of the Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate on daphnia magna was assessed. Over the course of 21 d no concentration-response relationship was observed for parental daphnid survival/immobility resulting in a NOEC = 26.6 µg/L and a LOEC >26.6 µg/L. For the endpoint "reproduction based on surviving F0 daphnids" the following effect concentrations were observed: EC10 = 4.05 µg/L, EC20 = 10.45 µg/L, NOEC 4.34 µg/L and LOEC = 8.68 µg/L

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.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Remarks:

The derived EC10 lies slightly below the NOEC. As the NOEC is an observed value, this value is chosen for the assessment.

Effect concentration:

4.34 µg/L