2-ethylhexyl acrylate

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11.11.2019 - 05.03.2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Non-Radiolabeled
- Identification: 2-Ethylhexyl Acrylate
- Batch (Lot) Number: B114192061
- Expiry date: 09 May 2020 (expiry date)
- Physical Description: Clear colourless liquid
- Purity/Composition: 99.79%
- Storage Conditions: At room temperature protected from light

Additional information
- Test Facility test item number: 209708/B
- Purity/Composition correction factor: No correction factor required
- Test item handling: Use amber glassware or wrap container in aluminum foil
- Chemical name (IUPAC, synonym or trade name): 2-ethylhexyl acrylate
- CAS number: 103-11-7
- Molecular structure: Not indicated
- Molecular formula: C11H20O2
- Molecular weight: 184.28
- Irritant or corrosive: Yes
- Highly flammable: Yes
- Highly reactive to water: Not indicated
- Highly reactive to oxygen: Not indicated
- Volatile: Yes, vapour pressure: 0.24 hPa at 25°C
- Specific gravity / density: 0.88 at 20°C
- Solubility in water: 9.6 mg/L at 25°C
- Stability in water: Not indicated

Radiolabeled
- Identification: 2-Ethyl[1-14C]hexyl acrylate
- Batch (Lot) Number: 6455SXD008-3
- Expiry Date: Not indicated
- Physical Description: Colourless solution in acetonitrile
- Radiochemical Purity: 94.6%
- Chemical Purity: 87.6%
- Specific Activity: 4.96 MBq/mg
- Storage Conditions: In freezer (≤ -15°C) protected from light
- Supplier: Selcia Limited, Fyfield Business & Research park, Fyfield Road, Ongar, Essex, CM5 OGS, UK
- Total activity received: 185.9 MBq

Additional information
- Test Facility test item number: 209707/A (file 209708)
- Concentration: 33.19 MBq/mL
- Purity/Composition correction factor: No correction factor required
- Test item handling: Use amber glassware or wrap container in aluminum foil
- Molecular formula: C11H20O2
- Molecular weight: 185.08 g/mol(at this specific activity)

Radiolabelling:

yes

Details on sampling:

During the bioconcentration test, sample were taken from the test medium and from the fish for several purposes and at several time points.

Determination of test item in test medium

- Method of sampling: Test solutions were sampled by siphoning through inert tubing from a central point in the test chamber.
- Initial water samples (Day -1): Samples were taken from the control and the test concentration and quantitatively analysed before introduction of the fish to check if the actual test concentrations were in agreement with the expected concentrations.
- Volume : 250 mL
- Handling: Three sub-samples of 10 mL were taken from the 250 mL-sample for quantitative analysis. The remaining volume was used for qualitative analysis.

- Quantitative analysis:
Total radioactivity in the sub-samples was determined in an appropriate amount of Ultima Gold XR cocktail (Perkin Elmer Life and Analytical Sciences, Boston MA, USA) using a Liquid Scintillation Counter (Tri-Carb 2910 TR Packard, Perkin Elmer Life and Analytical Sciences, Boston MA, USA).

- Qualitative analysis:
The test item concentration was determined in samples of the test medium taken after 1, 3, 7, 14, 21 and 28 days of exposure (20 µg/L). At t=21 days, also a blank sample was analysed. All the water samples were extracted, concentrated and if not analysed at the day of sampling, the extracts were stored in the freezer (≤ -15°C). Storage stability of samples under these conditions was demonstrated in Test Facility Reference No. 20179864.
To the ISO medium samples (approximately 220 mL), samples were extracted with 15 mL n-hexane for 1 minute and from day 3 onwards first 500 µL acetonitrile was added and thereafter samples were extracted with 15 mL n-hexane for 1 minute. At t=3 days, the sample was extracted a second time with 15 mL n-hexane for 1 minute. The percentage of applied radioactivity in the extract was determined by analysis of a weighed 1 mL aliquot, using a Liquid Scintillation Counter (LSC). The concentrated extracts were analysed by GC-FID.


Determination of test item in fish tissue

- Handling: Fish were sampled with a small fishing net, rinsed quickly with untreated water, blotted dry and then instantly killed. Subsequently, fish were weighed and measured for total length.
- Number of replicates: 2 for the control; 4 for the test concentration
- Number of fish sampled per replicate: 1
- Homogenisation: Each replicate was homogenized (1:1) in Milli-RO water using a blender (Turrax).
- Handling: Three sub-samples of 400 mg homogenate (containing approximately 200 mg fish each) were taken and transferred to LSC-vials for quantitative analysis. The remaining volume was used for qualitative analysis and/or lipid extraction.

- Treatment for quantitative analysis:
Fish tissues in each sub-sample were dissolved overnight in Solvable (Perkin Elmer Life and Analytical Sciences, Boston MA, USA) at approximately 60°C. Thereafter, the dissolved tissues were bleached with 30% H2O2 (Merck, Darmstadt, Germany). The vials were incubated for at least 30 minutes at room temperature and then further for at least 2 hours at approximately 60°C. Subsequently, scintillation liquid (Hionic Fluor; Perkin Elmer Life and Analytical Sciences, Boston MA, USA) was added to each vial and samples were left to stabilize for at least 4 hours in the dark before analysis.

- Quantitative analysis:
The test item concentration was determined in fish tissue after 1, 3, 7, 14, 21 and 28 days of exposure (20 µg/L). At t=21 days, also blank samples were analysed to determine possible matrix effects in the fish samples. All the fish tissue samples were extracted, and if not analysed at the day of sampling, the extracts were stored in the freezer (≤ -15°C).
Four fish tissue samples were treated as followed:
Approximately 4 g fish tissue (approximately 2 g fish and 2 g ISO medium) was homogenised and weighed. The homogenised fish tissue was extracted three times with 15 mL n-hexane, which was added to the fish tissue and put on a vortex for 10 minutes. After 5 minutes of centrifugation at approximately 1400 g and 20°C the supernatant was collected. At t=1 day, phase separation was not complete and therefore the samples were centrifuged for an extra 5 minutes at 2561 g and 20°C. At further time points (from day 3 onward), 370 µL acetonitrile was added to the homogenised fish tissue before extraction with 15 mL n-hexane to achieve better phase separation.
The three extracts were weighed and radioactivity was determined by LSC of a weighed 1 mL aliquot of each extract. GC-FID analysis was used for qualitative analysis in n-hexane after the first extraction cycle.

- Qualitative analysis:
GC-FID analysis was used for qualitative analysis in n-hexane after the first extraction cycle.

- Lipid Extraction:
Handling: Lipid extraction was performed on the remaining part of the homogenized fish already sampled for quantitative analysis, with the exception of the fish sampled at the start of the test.
Storage: The remaining part of the homogenized fish were stored in a freezer (≤ -15°C) until extraction. The fish sampled at the start of the test was homogenized using a blender and then stored in a freezer until extraction.
Lipid extraction: A modified Bligh and Dyer method (BD-method*) where samples were centrifuged instead of filtered.

* Jensen, S.; Häggberg, L.; Jörundsdóttir, H.; Odham, G. "A Quantitative Lipid Extraction Method for Residue Analysis of Fish Involving Nonhalogenated Solvents". J. Agric Food Chem., 2003, 51, 5607-5611.

Vehicle:

yes

Remarks:

Acetonitrile (ACN)

Details on preparation of test solutions, spiked fish food or sediment:

Stock and spike solutions were prepared in acetonitrile (ACN). For determination of the radiochemical purity, a stock solution St_RCZ was prepared on 14 Nov 2019 by dissolving an aliquot of 2-Ethyl[1-14C]hexyl acrylate in 0.99 mL acetonitrile. Based on LSC (liquid scintillation counting) of three 10 µL aliquots of the stock solution St-RCZ, the solution contained 0.352 MBq/mL (RSD 0.55%), which is equivalent to 71 mg 2-Ethyl[1-14C]hexyl acrylate /L.
A spike solution containing 200 mg/L was prepared on 14 Nov 2019 by pipetting 1494 µL of 2-Ethyl[1-14C]hexyl acrylate and 190.55 mg unlabeled 2-EHA into a 1 L volumetric flask. Then the volumetric flask was filled with acetonitrile to obtain a total concentration of 200 mg/L.
A second spike solution containing 200 mg/L was prepared on 02 Dec 2019 by pipetting 1494 µL of 2-Ethyl[1-14C]hexyl acrylate and 190.26 mg unlabeled 2-EHA into a 1 L volumetric flask. Then the volumetric flask was filled with acetonitrile to obtain a total concentration of 200 mg/L.
The concentration in the spike solutions was a factor 10,000 higher than the respective test concentration in water, i.e. concentration in the spike solution was 200 mg/L. The test item concentration consisted of 95% 2-Ethylhexyl-Acrylate and 5% 2-Ethyl[1-14]hexyl-acrylate. No correction was made for the purity/composition of the test item.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST SYSTEM
- Species: Juvenile male and female carp, i.e. less than 1 year old (Cyprinus carpio, Teleostei Cyprinidae) Linnaeus, 1758
- Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands.
- Initial fish length: 9.4 ± 0.4 cm, based on measurement of all fish used for this test
- Initial fish weight: 10.47 ± 1.10 g, based on weighing of all fish used for this test
- Characteristics: F1 from a single parent-pair bred in UV-treated water.
- Reason for selection: This system has been selected as an internationally accepted species.

HOLDING
- Quarantine/Acclimatisation: At least 12 days after delivery
- Medium: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L

- Measurements: Conductivity, pH, nitrate, nitrite and ammonia concentration: once a week. Temperature: continuous. In addition, pH and temperature were measured before transferring the fish to the test system.
- Water quality parameters: Were kept within the optimum limits for the tested fish species.
- Feeding: Daily with pelleted fish food (Essence 0.5-0.8 mm, Coppens International B.V., Helmond, The Netherlands)
- Validity of batch: In the batch of fish used for the test, mortality during seven days prior to the start of the test was less than 5%.

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

28 d

Total depuration duration:

56 d

Hardness:

Total hardness ranged from 196 to 214 mg calcium carbonate per litre.

Test temperature:

Average water temperature in the control and test item concentration over the study period (84 days) was at 23 +/- 0.18 °C and 23 +/- 0.43 °C respectively. Temperature remained within the range prescribed by the Study Plan (20-25°C, constant within ± 2°C).

pH:

The pH ranged from 7.3 to 8.0 during the test period, with an average value of 7.7.

Dissolved oxygen:

Oxygen concentrations ranged from 5.8 to 9.3 mg/L during the test period.

TOC:

During the test period, T(D)OC in plain test medium was low (i.e. ≤ 2 mg/L). During the uptake phase, T(D)OC in the ACN-control (test medium + ACN) ranged from 57 to 64 mg C/L. Considering an ACN concentration of 100 mg/L, T(D)OC was expected to be above 2 mg/L but below 59 mg C/L. The T(D)OC measured in the ACN-control group during the uptake phase was consequently for the largest part attributed to the presence of ACN. A slightly higher carbon concentration was related to the presence of fish. During the depuration phase, T(D)OC was not measured in the control because no ACN was applied anymore during this phase.

Salinity:

-

Conductivity:

-

Details on test conditions:

- Control: Test medium without test item but including ACN in the same amount as used in the treated solution.
- Test duration: 84 days, i.e. an uptake phase of 28 days and a depuration phase of 56 days.
- Test vessels: Uptake phase: 64 liters (40x40x40 cm) consisting of stainless steel and covered by a removable Perspex plate;
- Test design: Flow-through
- Introduction of fish: After test concentrations were allowed to stabilise for 4 days following the start of the dosing.
- Maximum loading: 1.6 g fish (wet weight)/litre/day
- Illumination: 16 hours photoperiod daily
- Aeration: The test media were aerated continuously
- Feeding: Pelleted fish food. Ration was 2% of body weight per day. Recalculation of the feeding rate was performed after each sampling point based on mean fish weight.
- Cleaning: Uneaten food and fecal debris were siphoned daily from the test chambers about an hour after feeding.
- Depuration phase until day 30: 30 liters (50 x 30 x 20 cm) consisting of glass and covered by a removable Perspex plate;
- Depuration phase until day 56: 10 liters (43 x 21 x 12 cm) consisting of glass and covered by a removable Perspex plate;
- Test medium: Adjusted ISO medium with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3.
- Total number of fish: 67
- Number of fish per test group: 23 for the control
44 for the test concentration

- Fish appearance and behaviour: Although the test concentrations were thought to be well below levels that might induce toxic effects, fish were observed daily for any adverse effects.
- Dissolved oxygen content and pH: Prior to the introduction of the fish and three times a week during both the uptake and the depuration phase in each vessel.
- Temperature of medium: Continuously in each vessel starting from one day before the introduction of the fish.
- Total hardness: At the start and the end of both the uptake and the depuration phase in each vessel.
- T(D)OC analysis: Total (dissolved) organic carbon in ISO medium and the control was measured once before the start of exposure and thereafter once a week.

DOSING SYSTEM

During the uptake phase, the spike solutions were dosed via a computer-controlled system (DaVinci) consisting of micro-dispensers (Gilson). Via this system the dosed volume from the spike entered a mixing flask separately from the medium supply. The medium was supplied via a flow meter and the flow rate was 20 L/h, allowing 7.5 volume replacements (64 litres) through each aquarium each day from day -4 until day -1. Due to the relatively low concentration in water on day -1, the water flow was lowered from 20 to 12L/h which resulted in a higher water concentration. However, it also resulted in a higher fish loading rate of 1.6 g fish (wet weight)/litre/day as a maximum. The temporarily higher maximum loading rate was accepted because the required concentration of test item in medium was maintained within ± 20% limits and the concentration of dissolved oxygen did not fall below 60% saturation. Therefore, the flow rate was 12 L/h. allowing 4.5 volume replacements (64 liters) through each aquarium each day. In the mixing flask the dosed spike volume and the medium were mixed under continuous stirring at a ratio of 1 : 10,000. The whole system was checked at least daily.
During the depuration phase the medium was supplied directly via the same flow meters at a rate of 12 L/h without the dosing of spike solution. The flow allowed for 9.6 volume replacements (30 liters) through each aquarium each day until day 30 of the depuration phase. Thereafter, the flow allowed for 28.8 volume replacements (10 liters) through each aquarium each day. The system was checked at least daily.

Nominal and measured concentrations:

Nominal test item concentration: 20 µg/L (This concentration was taken as a factor of 100 below the 96h-LC50 value for fish)
Mean measured test item concentration: 16 +/- 0.86 µg/L

Reference substance (positive control):

no

Details on estimation of bioconcentration:

The depuration and the uptake rate constants were estimated following a sequential optimization procedure. The coefficients of determination (r2) were 0.67 and 0.72 when the rate constants were derived by sequential fitting of the data, indicating that the obtained model better described the measured results than the results of the simultaneous fitting approach (r2 = 0.55). Based on a poor fit of the lipid-normalized data in both the simultaneous and sequential models, along with the understanding that the parent substance is likely readily metabolised, lipid-normalization of the kinetic BCF was not further pursued with this dataset (Additional information can be founf under “Details on kinetic parameters

BASIS FOR CALCULATION OF BCF (full calculation model can be found under "Other information on materials and methods)
- Result based on calculated log Pow of: 4
- When sequentially determining the k(d) (depuration rate constant) and the k(u) (uptake rate constant), the following approach was applied: The k(d) (depuration rate constant) was calculated first and then used for the calculation of the k(u) (uptake rate constant).

Lipid content:

2.6 %

Time point:

start of exposure

Remarks on result:

other: Lipid extraction performed on unexposed fish samples from the control group

Lipid content:

>= 1.3 - <= 2.5 %

Time point:

other: During 28 days uptake phase and the following 56 days depuration phase

Key result

Conc. / dose:

16 µg/L

Temp.:

ca. 23 °C

pH:

7.7

Type:

BCF

Value:

347 L/kg

Basis:

other: Based on measured total radioactivity (no parent 2EHA detectable)

Calculation basis:

kinetic

Remarks on result:

other: Not normalized for 5% lipid content

Remarks:

No growth correction performed

Elimination:

no

Parameter:

DT50

Depuration time (DT):

19 d

Rate constant:

overall depuration rate constant (d-1)

Value:

0.036

Rate constant:

overall uptake rate constant (L kg-1 d-1)

Value:

13

Details on kinetic parameters:

- Uptake rate constant k(s): 13 +/- 0.61
- Depuration rate constant k(e): 0.036 +/- 0.0068

- The coefficient of determination (r2) was 0.55 when both the depuration rate constant (k(d)) and the uptake rate constant (k(u)) were derived by simultaneous fitting of the data, indicating that the model was not very suitable to describe the observed study results. Especially for the depuration phase, the model tended to underestimate the measured test item concentrations in fish tissue.

- Subsequently, the depuration and the uptake rate constants were estimated following a sequential optimization procedure. The coefficients of determination (r2) were 0.67 and 0.72 when the rate constants were derived by sequential fitting of the data, indicating that the obtained model better described the measured results than the results of the simultaneous fitting approach. The sequential approach provided a model that tends to slightly overestimate the measured test item concentrations in fish tissue, which results in a more conservative study interpretation.

- Generally, it is preferred to fit the uptake and depuration rate constants simultaneously as both processes occur also at the same time during the uptake phase of the study and these parameters thus have an influence on each other. Taking into account the lipid normalization, the coefficient of determination (r2) was 0.23 when both the depuration rate constant (k(d)) and the uptake rate constant (k(u)) were derived by simultaneous fitting of the data, indicating that the model was not suitable to describe the observed study results..

- Subsequently, the depuration and the uptake rate constants were estimated following a sequential optimization procedure. The coefficient of determination (r2) were 0.51 and 0.52 when the rate constants were derived by sequential fitting of the data, indicating that the obtained model better described the measured results than the results of the simultaneous fitting approach. However, the lipid-normalized sequential model approach was also not a good fit for the data. The model overestimates the measured test item concentrations in fish tissue, which results in a more conservative study interpretation.

- To better understand the observed mismatch of the model results of the simultaneous fitting approach, the same approach was implemented and investigated using R again (R 3.6.3, R Foundation for Statistical Computing, Vienna, Austria), following the steps laid out in the Guidance Document on Aspects of OECD TG 305 on Fish Bioaccumulation Series on Testing & Assessment No. 264. Data transformation did not improve the overall fit of model results with the measured data, i.e. ln-transformation or Box-Cox optimization as laid out in section 3.4. of the above cited Guidance Document. Additionally, the model diagnostics indicated that the errors, i.e. the differences between model and measured values, were not entirely random. This can be caused when the observed uptake and depuration kinetics do not precisely follow first-order kinetics. In the present study some process seems to occur which delays the depuration of the total radioactivity from fish tissue. The identity of this process, however, cannot be identified with the available informationThese observations were slightly stronger than observed for the non-lipid normalised data. Also, with the lipid normalised fish tissue concentrations, it did not become clear which process caused the uptake and depuration kinetics to deviate from first-order kinetics.

- 2-Ethyhexyl Acrylate has been demonstrated to be quickly metabolized via hydrolysis to acrylic acid and/or glutathione conjugation in rats (*). When substances are rapidly cleared by biotransformation or are known to interact significantly with proteins, the simple distribution models predicted by partitioning to tissue lipids becomes increasingly uncertain (** / ***). Thus, the lack of model fit for the lipid-normalized data is not entirely unexpected. Based on a poor fit of the lipid-normalized data in both the simultaneous and sequential models, along with the understanding that the parent substance is likely readily metabolised, lipid-normalization of the kinetic BCF was not further pursued with this dataset.

- Furthermore, no other modelling was tried. Consequently, the study interpretation will be based on the sequential modelling approach following a worst-case assumption. This approach is supported by the fact that total radioactivity was cleared slowly from fish tissues and was still present in fish tissue after 56 days of depuration.


* Zhang F, Erskine TC, McClymont EL. 2018. "In vitro hydrolysis and glutathione conjugation of acrylates. Dow Chemical Company Study ID 180018. 20 November 2018." Report ID DR-0190-7966.

** Nichols JW, Bonnell M, Dimitrov SD, Escher BI, Han X, Kramer NI. 2009. "Bioaccumulation assessment using predictive approaches. Integrated Environmental Assessment and Management" 5:577-597.

*** Arnot JA, Gobas FAPC. 2006. "A review of bioconcentration factor (BCF) and bioaccumulation factor (BAF) assessments for organic chemicals in aquatic organisms." Environ. Rev. 14:257-297.

Metabolites:

Since the measurement was based on total radioactivity, possible metabolites were not individually considered for the calculation of the BCF.

2-Ethyhexyl Acrylate has been demonstrated to be quickly metabolized via hydrolysis to acrylic acid and/or glutathione conjugation in rats.

Details on results:

VALIDITY CRITERIA

- The water temperature variation was less than ± 2°C for the test concentration and the control.
- The oxygen concentration was maintained at least at 60% of the air saturation value throughout the test (>5 mg/L at 22 °C).
- The analytical results showed that the variation of the actual test item concentrations (total radioactivity) in water was maintained within ± 20% of the mean of the measured values during the uptake phase.
- Test concentrations were below the limit of solubility.
- Mortality or other adverse effects in the control and the target concentration was less than 10% at the end of the test.


ANALYTICAL RESULTS

- The radiochemical purity of the 200 mg/L spike solutions was determined to be 97-98%.
- The extraction was considered successful as the recoveries of total radioactivity during the uptake phase were in the range of 80-101%. Only 23-52% of the radioactivity partitioned to the organic phase, i.e. could be extracted. The remaining radioactivity was measured in the aqueous residue.
- The extraction of total radioactivity from fish tissue was within the range of 24-38%.
- GC-FID analysis of the extracts of the fish tissues showed no signal, indicating that 2- Ethylhexyl Acrylate was not detectable in fish tissue. The limit of detection of 2- Ethylhexyl Acrylate in fish tissue was 15 µg/kg. Therefore, if parent 2-Ethylhexyl Acrylate was present in the fish, it was at a concentration below 15 µg/kg. This is a maximum of <1% of the concentration of total radioactivity present in the fish at day 28 of the uptake phase.
- The low extraction recovery and the absence of test item signal in the GC chromatogram are both an indication that the radioactivity that is present in the fish is not 2-Ethylhexyl Acrylate, but degradation products of 2-Ethylhexyl Acrylate and/or assimilated carbon.


OBSERVATIONS

- Mortality of test organisms: No mortality or other clinical effects were observed on the fish exposed to the control and the average water concentration of 16 µg/L during the entire test period
- Observations on weight: Mean fish weight at the start and the end of the study was 10.47 and 22.64 g respectively. This corresponds to a fish growth during the study of 116%. During the depuration phase specifically, the growth was 60%. Fish growth during the depuration phase can affect the interpretation of measured test item concentrations in the fish, since the overall depuration rate constant (kd) is determined to be greater than would arise from removal processes alone. However, growth dilution correction of the kinetic BCF (i.e. subtracting out growth rate from the overall depuration rate) is subject to a lack of precision or may not work in cases where there is slow depuration and fast-growing fish (*), as in the present study. Furthermore, there is uncertainty with the biological relevance of the growth dilution correction of kinetic BCF values based on violations of the laws of mass balance (**). It is important to stress that relationships between feeding, respiration, growth, and metabolic transformation are highly complex and perhaps currently insufficiently understood to fully anticipate the effect of growth on bioconcentration and biomagnification. More research may be required to better comprehend the effect of growth on bioaccumulation. Therefore, in this case data were not corrected for fish growth
- Observations on body length: Mean fish body length at the start and the end of the study was 9.3 and 12.1 cm respectively.
- Loss of test substance during test period: The average concentration of total radioactivity in the test medium (i.e. water) during the 28-day uptake phase was 16 ± 0.86 µg/L at the target concentrations of 20 µg/L. The measured concentrations varied within a ± 20% window of the mean concentration
- On day 28, the remaining fish were transferred into dilution medium without 2-Ethylhexyl Acrylate and without ACN to start the depuration phase. After 56 days of depuration, total radioactivity concentrations in fish previously exposed to an average water concentration of 16 µg/L (total radioactivity) was reduced to 21% of the concentration at the start of the depuration phase.

* OECD, 2012. "OECD Guidelines for Testing of Chemicals Bioaccumulation in Fish: Aqueous and Dietary Exposure." OECD 305 Adopted 2 October 2012.
** Frank A.P.C. Gobas and Yung-Shan Lee "Growth-Correcting the Bioconcentration Factor and Biomagnification Factor in Bioaccumulation Assessments." Environmental Toxicology and Chemistry – Volume 38, Number 9 – pp. 2065-2072, 2019.


BIOLOGICAL RESULTS

- When corrected for lipid content, no steady-state concentration in fish was reached.
- A steady-state concentration in fish was not reached in this study. In the three samples taken during Day 14 until Day 28, tissue concentrations were within 20% of the mean concentration, however there was a 34% increase in total radioactivity measured between Days 14 and 28, indicating that steady state was not achieved. Since a steady state was not achieved within 28 days, a BCF(ss) could not be calculated. Therefore, only the kinetic approach was followed.
- The depuration and the uptake rate constants were estimated following a sequential optimization procedure. The coefficient of determination (r2) were 0,67 and 0.72 without lipid normalization and 0.51 and 0.52 with lipid normalization.
- The kinetic BCF based on total radioactivity, obtained with sequential modelling, was 347 L/kg and the half-life (DT50) value was 19 days indicating slow depuration.

Reported statistics:

Optimisations for BCF calculation was performed using non-linear regression with the program ModelMaker 4.0 (AP Benson, Wallingford, Oxfordshire, UK, 2004).

Due to an observed mismatch of the model results of the simultaneous fitting approach, the same approach was implemented in another statistical software (R 3.6.3, R Foundation for Statistical Computing, Vienna, Austria).

Test conditions

Temperature of the test solutions
Average conc. Total radioactivity [µg/L]	Range of temperatures [°C]	Average temperature +/- standard deviation [°C]
Control	22 - 23	23 +/- 0.18
16	21 - 23	23 +/- 0.43

Total hardness measurements [mg/L CaCO3]
Average conc. Total radioactivity [µg/L]	Day 0	Day 28 uptake	Day 28 depuration	Day 84
Control	214	196	196	196
16	214	196	196	196

T(D)OC measurements [mg/L]
Day of measurement	ISO	Control (ISO + ACN)
-1	n.d.	31.62
7	n.d.	63.96
14	n.d.	60.37
21	n.d.	59.61
28	n.d.	57.53
35	n.d.	-
41	n.d.	-
49	n.d.	-
56	n.d.	-
63	n.d.	-
70	n.d.	-
77	n.d.	-
84	n.d.	-
n.d.: not detectable; concentration was below the detection limit of the method (i,e, < 0.1 mg/L)
- : not measured during the depuration phase because the control consisted only of ISO medium

pH-values
Day of measurement	Average conc. Total radioactivity [µg/L]
	Control	16
0	8	8
1	7.8	7.8
3	7.7	7.5
6	7.6	7.5
8	7.8	7.6
10	7.7	7.5
13	7.7	7.5
15	7.8	7.5
17	7.7	7.4
20	7.8	7.4
22	7.7	7.5
24	7.8	7.5
27	7.8	7.5
28 (uptake)	7.8	7.6
28 (depuration)	7.9	7.9
29	7.7	7.3
31	7.6	7.5
34	7.7	7.5
36	7.6	7.4
38	7.7	7.6
41	7.8	7.8
43	7.9	7.7
45	7.8	7.8
48	7.8	7.7
50	7.8	7.7
52	7.8	7.8
55*	7.8	7.8
55**	7.9	7.8
57	7.8	7.8
59	7.7	7.6
62	7.8	7.7
64	7.9	7.8
66	7.8	7.7
69	7.8	7.6
71	7.8	7.8
73	7.8	7.6
76	7.9	7.8
78	7.8	7.6
80	7.8	7.7
83	7.8	7.7
84	8	7.9
* : Measured before the test vessels were changed for cleaning
** : Measured after the test vessels were changed for cleaning

Dissolved oxygen concentration [mg/L]
Day of measurement	Average conc. Total radioactivity [µg/L]
	Control	16
0	7.6	7.6
1	8.8	7.6
3	8.4	7.3
6	8.1	7.6
8	8.2	7.1
10	8.3	7.2
13	9.3	8
15	8.5	7.5
17	8.6	7.5
20	8.1	6.7
22	8.3	6.8
24	8.8	7.5
27	8.8	7.2
28 (uptake)	8.6	6.9
28 (depuration)	8.9	8.9
29*	8.3	5.8
29**	-	7.4
31	8.5	8.1
34	8.3	8
36	8.3	7.8
38	8.3	8
41	9	9
43	8.7	8.6
45	8.8	8.7
48	8.8	8.7
50	8.6	8.2
52	8.5	8.4
55	8.3	8.4
57	8.2	8.1
59	9	8.5
62	9	8.6
64	8.8	8.3
66	8.7	8.2
69	8.2	7.8
71	8.7	8.3
73	8.7	8.2
76	8.5	8.1
78	8.5	8
80	9	8.4
83	8.7	8
84	8.7	8.1
* : Measured before extra aeration was applied
** : Measured after extra aeration was applied
- : not measured

Analytical results

Average concentrations in water based on total radioactivity
Time of measurement [days]	Mean measured total radioactivity +/- Standard deviation [µg/L]
	Target concentration 20 µg/L
0 - 1	16 +/- 1.3
0 - 3	16 +/- 1.1
0 - 7	16 +/- 1.1
0 - 14	16 +/- 1.0
0 - 21	15 +/- 0.92
0 - 28	16 +/- 0.86

Determination of test item in n-Hexan extract from test medium: GC-FID
Time of sampling	Date of sampling	Date of	Concentration in		Relative to nominal	Relative to initial
[days]		analysis^1	n-hexane extract		[%]	[%]
			[mg/L]
			Nominal	Analyzed
1	20. Nov 19	03 Dec 2019	0.127	0.0841	66
3	22. Nov 19	03 Dec 2019	0.0542	0.0589	109	70
7	26. Nov 19	03 Dec 2019	0.0934	0.0757	81	90
14	03 Dec 2019	17 Dec 2019	0.0865	0.0807	93	96
21	10 Dec 2019	17 Dec 2019	0	n.d.	n.a.	n.a.
			0.11	0.0759	69	90
28	17 Dec 2019	17 Dec 2019	0.124	0.0883	71	105

¹  Sample extracts were stored in the freezer (≤ -15°C) until the day of analysis.

n.d. = Not detected.

n.a. = Not applicable.

Determination of test item related to concentration in test medium: LSC
Time	Target concentration	Concentrated extract			Aqueous residue		Mass
							balance
	[µg/L]
[days]		Test item related conc.	Recovery n-hexane extract	Test item related conc^2	Test item related conc	Test item related conc,	[%]
			[%]
		[µg/L]^1		[µg/L]^1	[%]	[µg/L]^1
1	20	17.23	49	8.4	43	7.46	92
3	20	15.4	23	3.58	57	8.8	80
7	20	14.53	42	6.14	55	7.96	97
14	20	14.48	40	5.74	47	6.79	87
21	20	15.14	48	7.27	47	7.09	95
28	20	15.87	52	8.2	49	7.82	101

¹  Corresponding concentration in the test medium. Test item related concentration include 2-EHA and/or degradation product(s) and/or assimilated carbon). Calculated concentration is derived fromliquid scintillation radioactivity counts of parent and degradation products (like metabolites and assimilated carbon).
²  GC-FID analyses showed this product to be mainly 2-EHA

Biological results

Effect Parameters based on total radioactivity
Average concentration of total radioactivity in water: 16µg/L	BCF (L/kg)	DT50(d)
16	347	19

Results of the parameter optimization procedure after sequential estimation of uptake and depuration rate constants
Parameter	Average concentration of total radioactivity in water: 16µg/L
k(u) ± optimization error [d-1] (r2)	13 ± 0.61 (0.67)
k(d) ± optimization error [d-1] (r2)	0.036 ± 0.0068 (0.72)
BCFk [L/kg]	347
DT50 [days]	19
k(u): uptake rate constant
k(d): depuration rate constant

Average concentrations in fish based on total radioactivity
Time [days]		Average concentration of total radioactivity in water: 16µg/L
		Concentration +/- SD [µg/kg]	BCF +/- SD [L/kg]
Uptake phase	1	1192 ± 95	73 ± 5.8
	3	1047 ± 76	65 ± 4.7
	7	1399 ± 84	89 ± 5.4
	14	2366 ± 82	152 ± 5.3
	21	2724 ± 413	176 ± 27
	28	3172 ± 119	204 ± 7.7
			Relative to day 28 [%]
Depuration phase	30	1759 ± 140	55
	34	1722 ± 178	54
	41	1380 ± 100	43
	56	989 ± 37	31
	84	674 ± 21	21

Determination of test item related concentration in fish tissue: LSC
Time	Theoretical activity^1	Total concentration in fish	Total activity extracted	Percentage extracted	Concentration
		test item related concentration			test item related
	[dpm/g]	[µg/kg]^2	[dpm/g]	[%]
[days]					[µg/kg]^2
1	18430	1201	7034	38	458
	20316	1323	7602	37	495
	17345	1130	5953	34	388
	17096	1114	5800	34	378
3	15101	984	3672	24	239
	17009	1108	4424	26	288
	15035	979	3872	26	252
	17140	1117	4601	27	300
7	22610	1473	7280	32	474
	21985	1432	6472	29	422
	21689	1413	6220	29	405
	19640	1279	5553	28	362
14	36696	2391	11248	31	733
	37151	2420	12109	33	789
	34464	2245	9942	29	648
	36984	2409	10044	27	654
21	38165	2389	9327	24	584
	40590	2541	10284	25	644
	53086	3324	17176	32	1075
	42205	2642	10482	25	656
28	53343	3340	14220	27	890
	48945	3064	11767	24	737
	50615	3169	12586	25	788
	49788	3117	12562	25	786

¹             Percentage of radioactivity determined by LSC after solubilisation of the fish tissue homogenate in the main study.

²          Test item related activityinclude 2-EHA and/or degradation products.

Validity criteria fulfilled:

yes

Conclusions:

The test substance 2-Ethylhexyl Acrylate was tested for its ability to accumulate in tissues of Cyprinus carpio according to the OECD Guideline 305 during a uptake phase of 28 days and a depuration phase of 56 days. At a test item concentration of 16 µg/L (measured), the kinetic bioconcentration factor (BCFk) was determinded based on total radioactivity which includes the sum of the parent compound, possible metabolites and assimilated carbon, whereas GC-FID analysis of the extracts of the fish tissues showed no parent 2EHA. Based on total radioactivity the BCFk was 347 L/kg and the half-life (DT50) value of the total radioactivity was 19 days indicating slow depuration.

Executive summary:

The objective of the study was to evaluate 2-Ethylhexyl Acrylate for its ability to accumulate in tissues of carp (Cyprinus carpio) during an uptake phase of 28 days and a depuration phase of 56 days.

The study procedure described in this report was based on the OECD Guidelines for Testing of Chemicals, No. 305, 2012.

For this bioconcentration test, both 2-Ethylhexyl Acrylate and2-Ethyl[1-14C]hexyl acrylate were used. Analysis of water and fish samples was based on the radiochemical test item. Spike solutions were prepared in acetonitrile (ACN) and dosed independently from the dilution water supply (adjusted ISO medium). Test medium was supplied via a flow meter at a rate of 12 L/h and the final target concentration was 20 µg/L.

At the start of the exposure period, 44 fish were exposed to the target concentration and 23 fish were exposed to a solvent control. The uptake phase lasted for 28 days, during which test medium samples and fish samples were taken for both quantitative and qualitative analysis. During the subsequent depuration phase, which lasted for 56 days, test medium and fish were sampled for quantitative analysis.

 

The extraction of radioactivity from the water phase was considered successful as the recoveries of total radioactivity during the uptake phase were in the range of 80-101%. Only 23-52% of the radioactivity partitioned to the organic phase, i.e. could be extracted. The remaining radioactivity was measured in the aqueous residue. GC-FID analysis of the concentrated extracts showed that 66-109% represented the parental compound.

GC-FID analysis of the extracts ofthe fish tissues showed no signal, indicating that 2-Ethylhexyl Acrylate was not detectable in fish tissue. The limit of detection of2-Ethylhexyl Acrylatein fish tissue was 15 µg/kg. Therefore, if parent2-Ethylhexyl Acrylate was present in the fish it was in a concentration below 15 µg/kg. This is a maximum of <1% of the concentration of total radioactivity present in the fish at day 28 of the uptake phase.

The low extraction recovery and the absence of test item signal in the GC chromatogram are both an indication that the radioactivity that is present in the fish is not 2-Ethylhexyl Acrylate but degradation product(s) of 2-Ethylhexyl Acrylate and or/assimilated carbon.

The kinetic bioconcentration factor (BCF_k)based on total radioactivity was 347 L/kg and the half-life (DT₅₀) value of the total radioactivity was 19 days indicating slow depuration.

Reference 2

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

(Q)SAR

Remarks:

Migrated phrase: estimated by calculation

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

See "Attached justification"

Principles of method if other than guideline:

OASIS, Catalogic v5.13.1.156 [BCF base-line model v.03.10]

GLP compliance:

no

Radiolabelling:

no

Details on estimation of bioconcentration:

BASIS INFORMATION
- Calculated logPow: 4.09


BASIS FOR CALCULATION OF BCF
- Estimation software: OASIS Catalogic v5.10.5 [BCF base-line model v.01.02]
- Result based on calculated log Pow of: 4.09

Type:

other: log BCF

Value:

0.81 dimensionless

Type:

BCF

Value:

6.46 L/kg

The BCF value was calculated on the basis of the log Pow = 4.09. Parameters as water solubility, molecular size and basic metabolism are taken into account by this model.

Reference 3

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

see "Attached justification".

Principles of method if other than guideline:

SRC BCFWIN v3.01

GLP compliance:

no

Radiolabelling:

no

Details on estimation of bioconcentration:

BASIS INFORMATION
- Calculated logPow: 4.09 (SRC KOWWIN)


BASIS FOR CALCULATION OF BCF
- Estimation software: SRC BCFWIN v3.01
- Result based on calculated log Pow of: 4.09

Type:

BCF

Value:

233 dimensionless

Calculation basis:

other: log Pow

Remarks on result:

other: The BCF was calculated based on the log Pow = 4.09.

Type:

other: log BCF

Value:

2.367 dimensionless

SMILES : O=C(OCC(CCCC)CC)C=C

CHEM  : 2-Propenoic acid, 2-ethylhexyl ester

MOL FOR: C11 H20 O2

MOL WT : 184.28

------------------------------ EPI SUMMARY (v4.11) --------------------------

Physical Property Inputs:

Log Kow (octanol-water):  ------

Boiling Point (deg C) :  ------

Melting Point (deg C) :  ------

Vapor Pressure (mm Hg) :  0.18

Water Solubility (mg/L):  9.6

Henry LC (atm-m3/mole) :  ------

Log Octanol-Water Partition Coef (SRC):

Log Kow (KOWWIN v1.68 estimate) = 4.09

Bioaccumulation Estimates (BCFBAF v3.01):

Log BCF from regression-based method = 2.367 (BCF = 232.8 L/kg wet-wt)

Log Biotransformation Half-life (HL) = -0.7619 days (HL = 0.173 days)

Log BCF Arnot-Gobas method (upper trophic) = 1.846 (BCF = 70.15)

Log BAF Arnot-Gobas method (upper trophic) = 1.846 (BAF = 70.15)

log Kow used: 4.09 (estimated)

Description of key information

The estimated log Pow of 4 and the experimentally determined BCF value of 347 indicate a moderate potential for bioaccumulation.

Key value for chemical safety assessment

BCF (aquatic species):

347 L/kg ww

Additional information

1) Partition coefficient: An experimental log Pow of 4.64 (25 °C) was determined using the shake-flask method, but may represent an overestimation of the actual log Pow due to methological deficiencies. Log Pow values calculated by Q(SAR), i.e. Epi Suite SRC KOWWIN v1.67, and regression analysis, were 4.09 (BASF SE, 2008) and 3.67 (Fujisawa & Masuhara, 1981), respectively. Using a weight of evidence approach, the log Pow was estimated to be 4 indicating a potential for bioaccumulation.

 

2) OECD Test Guideline 305 study in Cyprinus carpio:In a study conducted with 2-Ethylhexyl Acrylate (2EHA) according to the OECD Guideline for Testing of Chemicals, No. 305, 2012, the ability of the test substance to accumulate in tissues of carp (Cyprinus carpio) was tested during an uptake phase of 28 days and a depuration phase of 56 days. The kinetic bioconcentration factor (BCF_k) was determined since no steady state was reached over the test duration. A sequential determination model was applied in addition to a normalization for the standard lipid content of 5%, however, based on a poor fit of the lipid-normalized data in both the simultaneous and sequential models, along with the understanding that the parent substance is likely readily metabolised, lipid-normalization of the kinetic BCF was not further pursued with this dataset. The BCF_k was determinded based on total radioactivity which includes the sum of the parent compound, possible metabolites and assimilated carbon, whereas GC-FID analysis of the extracts of the fish tissues showed no parent 2EHA. The BCF_k based on total radioactivity was347 L/kg and the half-life (DT₅₀) value of the total radioactivity was 19 days indicating slow depuration. The experimentally determined BCF is much lower than the cut off criterion of 2000 L/kg.

 

3)  Empirical results and QSAR models that predict significant metabolism of 2-EHA: Significant intrinsic clearance is predicted (via multiple QSAR models) for 2-EHA indicating that the material will be rapidly metabolized and not bioaccumulated (Table 1). Support for this comes from combined evidence of metabolism observed for 2-EHA in rodent toxicokinetic studies. Briefly, metabolism rates 2-EHA in rat liver microsomes and whole rat blood were determined. 2-EHA was hydrolyzed to acrylic acid with a half-life of 2.26 minutes in rat liver microsomes with an intrinsic clearance rate of 306 µL/min/mg (Table 1). Additionally, in whole rat blood, 2-EHA was rapidly metabolized with a half-life of 3.85 minutes and an intrinsic clearance rate of 180 µL/min (The Dow Chemical Company, 2018; Table 1). This in vitro metabolism result indicates that 2-EHA can be quickly metabolized primarily through hydrolysis to acrylic acid and/or glutathione conjugation in vivo.

 

4) PBT criteria: The registered substance and its relevant degradation products are not persistent (readily biodegradable based on OECD 301 F) according to the criteria laid down under annex XIII of REACh regulation. The bioaccumulation study resulted in a BCF of 347 L/kg. Thus, none of them is PBT/vPvB.

 

 

Table 1. Summary of Bioaccumulation QSAR predictions for 2-EHA

 

 

Substance	QSAR EpiWin BCFBAF v 3.01 (regression based method)	QSAR EpiWin BCFBAF v 3.01 (Arnot & Gobas BCF which takes metabolism into consideration)	QSAR BCF Catalogic v.03.10	QSAR EpiWin BCFBAF v 3.01 Predicted Half-life in fish (10 g)	Experimental Rat Liver Half life	Experimental Rat Whole Blood Half-life	Experimental BCF (OECD 305)
Ethylhexyl Acrylate (2-EHA)	233	70	6.46	0.173 days	2.26 minutes	3.85 minutes	347
Ethylhexyl Methacrylate (EHMA)	460	126	-	0.306 days	-	-	37