2,3-Epoxypropyl neodecanoate, oligomeric reaction products with toluene-4-sulfonic acid

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-05-16 to 2019-11-13

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)

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test organisms: Four fish samples were taken from each tank on days 0.3, 1, 2, 4, 8, 14 and 28 of the uptake phase as well as on days 0.3, 1, 2, 4, 8 and 14 of the depuration phase.
- Sampling intervals/frequency for test medium samples: Aqueous phase samples were taken and analysed on uptake phase days 0.3, 1, 2, 4, 8, 14, 28 and on days 0.3 and 1 of the depuration phase. Sampling and analysis of water samples after depuration day 1 was stopped, since measured concentrations on depuration day 1 were < LCL.
- Sample storage conditions before analysis: All samples were stored at room temperature. Fish samples were prepared directly after sampling or stored at ca. –18 °C until analysis, if necessary. Prepared samples were stored on an autosampler until analysis.
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods): 4 Fish were taken from control groups and exposure groups tank at each sampling day.
Appropiate volumes were taken on each sampling interval from the middle of the water phase from each aquarium.
The samples were analysed by LC-MS on a reserved phase column in gradient mode. A dual jetstream electrospray ionization source was used for ionization. A high-resolution quadrupole time of flight mass spectrometer operating in positive ion mode was used as detector for the quantitative and qualitative analysis.

Vehicle:

yes

Remarks:

DMF (dimethylformamide; Merck, purity: ≥ 99.8%, batch: K49038953728) was used as solvent with 0.100 mL/L test medium.

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

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 10 mg test item /L was prepared in dimethylformamide and mixed with appropriate volume of dilution water in a mixing chamber by magnetic stirring to a final test concentration of 1 mg/test item/L. Precision syringe pumps were used for the introduction of the stock solution and Membrane piston pumps provided the water flow-through.
- Controls: solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): DMF (dimethylformamide; Merck, purity: ≥ 99.8%, batch: K49038953728) was used as solvent.
- Concentration of vehicle in test medium (stock solution and final test solution(s) at different concentrations and in control(s)): 0.100 mL/L test medium.
An equilibration period of 26 days was carried out prior to exposure.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): not indicated.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Danio rerio (zebrafish), Vertebrata, Gnathostomata, Pisces, Osteichthyes, Teleostei, Cypriniformes, Cyprinidae
- Strain: not indicated
- Source: All fish used in the test were gained at the test facility from a single brood stock (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin).
- Age at study initiation (mean and range, SD): 17 to 23 weeks (date of hatch: calendar week 13 to 19, 2018)
- Length at study initiation (length definition, mean, range and SD): Mean size at test start was 2.91 cm. The smallest fish was not smaller than two-third of the weight of the largest fish. A sub-sample of 10 fish of the stock of fish was weighed prior to experimental starting in order to estimate the initial mean fish weight. Three of these fish were used for lipid analysis at start of the test.
- Weight at study initiation (mean and range, SD): initial mean wet weight 182 mg
- Weight at termination (mean and range, SD): mean wet weight of solvent control 333 mg
- Method of breeding: Holding was performed at the test facility at 20 - 25 °C, diffuse
light (7 – 750 lux, 16 h / 8 h photoperiod daily) and under flow-through conditions. The water exchange was about 1 - 2 times the water volume per day. Tap water of local origin was used for holding. The water was filtered on activated charcoal and aerated for at least 24 hours to remove chlorine.
- Lipid content at test initiation (mean and range, SD):
- Health status: Only zebrafish with at least 12 days of acclimation in the laboratory and mortality < 5 % within the last 7 days before start of the exposure were used in the test.
- Description of housing/holding area: Holding was performed at the test facility at 20 - 25 °C, diffuse light (7 – 750 lux, 16 h / 8 h photoperiod daily) and under flow-through conditions
- Feeding during test
- Food type: Sera Vipan; SERA GMBH, 52518 Heinsberg, Germany. This product is composed of fish products, oils and fats, cereal grains, yeast, byproducts, minerals and vitamins.
- Amount: The given feed amount (1.5 % of body weight) was documented. The amount of food was adjusted accordingly to the weights of sacrificed fish to account for growth during the experiment.
- Frequency: The fish were fed daily in two feedings.

ACCLIMATION
- Acclimation period: Only zebrafish with at least 12 days of acclimation in the laboratory before start of the exposure were used in the test.
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: same as test
- Feeding frequency: 1.5% of fish body weight per feeding day. Same as test
- Health during acclimation (any mortality observed): Only Fish with mortality < 5 % within the last 7 days before start of the exposure were used in the test.

Route of exposure:

aqueous

Justification for method:

aqueous exposure method used for following reason: The method was discussed with ECHA and is justified based on the substance properties and the fact that result of bioaccumulation test via aqueous exposure (i.e. BCF value) is directly comparable to the REACH Annex XIII criteria for the B/vB assessment

Test type:

flow-through

Water / sediment media type:

other: Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 hours to remove chlorine.

Total exposure / uptake duration:

28 d

Total depuration duration:

14 d

Hardness:

68 mg CaCO3/L at test start in solvent control and test group.

Test temperature:

Solvent control :21.6 - 22.4 °C; Mean value (3 times per week) = 22.4 °C
Test group :22.1 - 23.0 °C; Mean value (3 times per week) = 22.7 °C

pH:

Solvent control :7.08 - 7.88; Mean value (3 times per week) = 7.53
Test group: 7.00 - 7.84; Mean value (3 times per week) = 7.46

Dissolved oxygen:

Mean value = 96% in solvent control
Mean value = 94 % in test group

TOC:

Uptake phase:
Mean measured value in solvent control = 55.1 mgC/L
Mean measured value in test group = 55.3 mgC/L
Depuration phase:
Mean measured value in solvent control < LOQ (2mgC/L)
Mean measured value in test group < LOQ (2mgC/L)

Conductivity:

162 µS/cm (Dilution water)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria.
- Type (delete if not applicable): covered by glass tops
- Material, size, headspace, fill volume: 35 L
- Aeration: yes
- Type of flow-through (e.g. peristaltic or proportional diluter): Membrane piston pumps provided the water flow-through
- Renewal rate of test solution (frequency/flow rate): Water exchange in the test aquaria (approx. Vol. 35 L) was 5 times per day (7.29 L/h).
- No. of organisms per vessel: 85
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1
- Biomass loading rate: The loading did not exceed a range of 0.1 to 1.0 g of fish (wet weight) per liter per day
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 hours to remove chlorine.
- Particulate matter: No. Water was filtered
- Chlorine: Water was filtered on activated charcoal to remove chlorine. Residual chlorine, measured once per week from the dilution water supply tank on study days 0, 7, 14, 23, 29 and 36 was < 0.01 mg/L.
- Alkalinity: 0.8 mmol/L
- Conductance: 162 µS/cm
- Holding medium different from test medium: No
- Intervals of water quality measurement: Dissolved oxygen concentration and pH were measured 3 times a week and the temperature was recorded continuously (once per hour) with a data logger.
- Intervals of test medium replacement: 5 times per day
OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: A daily photoperiod of 16 h light / 8 h dark was provided
- Light intensity: 7 – 750 Lux
RANGE-FINDING / PRELIMINARY STUDY
- Test concentrations: No
- Results used to determine the conditions for the definitive study: not applicable
- Other justification for choice of test concentrations: The test concentration was 1.00 mg test item/L. The concentration was based on the toxicity data of an acute toxicity test with zebrafish (LL100 (96 h) > 100 mg/L, based on nominal test item loadings.

Nominal and measured concentrations:

The concentrations of test item in the aqueous phase were measured during the uptake and depuration phase via LC-MS. An electrospray tandem mass spectrometer operating in positive ion mode was used as detector. External standards were used for calibration. Further details on the evaluation method are provided in the IUCLID section relative to analytical methods.
During the uptake phase, the nominal aqueous concentration to be verified for all relevant components was 1 mg/L. The measured concentrations were slightly above the nominal concentration of 1 mg/L. Therefore, a time weighted average mean concentration was calculated for each relevant component and used for BCF calculation. In the depuration phase of the exposure group the measured concentrations were < LOQ (see tables below).
In the control group the measured concentrations in the uptake and depuration phase were < LOQ.

Mean Concentrations and Standard Deviation of the test item in Water during Uptake Phase
Nominal concentration 1 mg test item/L
Compound Mean meas. [%]
Conc.[mg/L]
C10H14O5S 0.6 ± 0.1 64
C13H24O3 0.9 ± 0.2 91
C15H16O2S 1.2 ± 0.1 117
C16H18O2S 1.1 ± 0.1 109
C20H32O6S 0.8 ± 0.1 75
C21H34O6S 0.9 ± 0.2 85
C26H48O6 1.4 ± 0.6 138
C26H50O7 2.3 ± 1.1 230
C33H56O9S 1.4 ± 0.5 145
Meas. conc. = Measured concentration of the test item; weighing factor, dilution factor and standard deviation taken
into account
[%] = Percent of nominal concentration

Mean Concentrations of testitem in Water during Depuration Phase
Nominal concentration 1 mg test item/L
Sampling Control Group Exposure Group
day Mean.conc.[mg/L] Meas. conc. [mg/L]
[d]
0.3 / 1 < LOQ < LOQ
Meas. conc. = Measured concentration of the test item, weighing of the calibration and dilution factor taken into account
LOQ = Limit of quantification (0.7 mg test item/L)

Reference substance (positive control):

no

Details on estimation of bioconcentration:

BASIS INFORMATION
- Measured/calculated logPow: 4.2
- Results from toxicokinetic study: Not available. Toxicokinetic properties of the substance are based on physico-chemical properties of the compound and on toxicological data. (see section 7.1 of IUCLID)
BASIS FOR CALCULATION OF BCF
- Estimation software: Microsoft Excel, MICROSOFT CORPORATION; GraphPad Prism, GRAPHPAD SOFTWARE, INC
Bioconcentration Factor (BCF)
Kinetic bioconcentration factors (BCFK and BCFKGL)

The kinetic bioconcentration factors have been calculated based on fish body weight and time-weighted average mean concentration of the test item in water during uptake phase. The lipid normalized (and growth corrected) BCFKgL is based on the mean lipid content of fish (12%).

Prior to the analysis, the fish data were transformed with the Box-Cox power transformation (Y = IF(Y=0, ln(Y), (Y^lambda-1)/lambda)) for optimal fitting of the data to the curve. The best fitting lambda values were λ = 0.7 for C15H16O2S data and λ = 0.8 for C16H18O2S data.
The kinetic bioconcentration factors for the test item compounds C15H16O2S and C16H18O2S were calculated as given in the test guideline 305 and OECD guidance document 264 by non-linear regression with simultaneous fitting of the uptake and depuration rates using the following formulas:

〖BCF〗_K= k_1/k_2


〖BCF〗_(Kg )= k_1/(k_2 - k_g )


〖BCF〗_KgL= 0.05/L_n · 〖BCF〗_Kg

k1 = uptake rate constant
k2 = depuration rate constant
kg = overall growth rate
Ln = Mean lipid content of fish

Lipid content:

12 %

Time point:

other: During uptake and depuration phases.

Remarks on result:

other: Overall mean lipid content of test group determined according to SMEDES (1999).

Conc. / dose:

1 mg/L

Type:

BCF

Value:

249 L/kg

Basis:

whole body w.w.

Calculation basis:

kinetic

Remarks on result:

other: CI ((212 – 286); Component C15H16O2S

Key result

Conc. / dose:

1 mg/L

Type:

BCF

Value:

104 L/kg

Basis:

normalised lipid fraction

Calculation basis:

kinetic, corrected for growth

Remarks on result:

other:

Remarks:

Component C15H16O2S

Conc. / dose:

1 mg/L

Type:

BCF

Value:

479 L/kg

Basis:

whole body w.w.

Calculation basis:

kinetic

Remarks on result:

other: CI (408 – 550); Component C16H18O2S

Key result

Conc. / dose:

1 mg/L

Type:

BCF

Value:

201 L/kg

Basis:

normalised lipid fraction

Calculation basis:

kinetic, corrected for growth

Remarks on result:

other:

Remarks:

Component C16H18O2S

Conc. / dose:

1 mg/L

Type:

BCF

Value:

233 L/kg

Basis:

whole body w.w.

Calculation basis:

kinetic

Remarks on result:

other: CI (188 – 277); Component C26H48O6

Key result

Conc. / dose:

1 mg/L

Type:

BCF

Value:

109 L/kg

Basis:

normalised lipid fraction

Calculation basis:

kinetic, corrected for growth

Remarks on result:

other:

Remarks:

Component C26H48O6

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

0.256 d

Remarks on result:

other: Component C15H16O2S

Key result

Elimination:

yes

Parameter:

DT90

Depuration time (DT):

0.852 d

Remarks on result:

other: Component C15H16O2S

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

0.416 d

Remarks on result:

other: Component C16H18O2S

Key result

Elimination:

yes

Parameter:

DT90

Depuration time (DT):

1.382 d

Remarks on result:

other: Component C16H18O2S

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

9.24 d

Remarks on result:

other: Component C26H48O6

Key result

Elimination:

yes

Parameter:

DT90

Depuration time (DT):

30.7 d

Remarks on result:

other: Component C26H48O6

Key result

Rate constant:

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

Value:

676.1

Remarks on result:

other: Compound C15H16O2S

Key result

Rate constant:

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

Value:

802.7

Remarks on result:

other: Compound C16H18O2S

Key result

Rate constant:

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

Value:

19.6

Remarks on result:

other: Compound C26H48O6

Key result

Rate constant:

growth-corrected depuration rate constant (d-1)

Value:

2.704

Remarks on result:

other: Compound C15H16O2S

Key result

Rate constant:

growth-corrected depuration rate constant (d-1)

Value:

1.666

Remarks on result:

other: Compound C16H18O2S

Key result

Rate constant:

growth-corrected depuration rate constant (d-1)

Value:

0.075

Remarks on result:

other: Compound C26H48O6

Details on kinetic parameters:

The above given rate constants were calculated for compounds for which a significant uptake in fish was observed during the uptake phase compared to the other compounds of the test item followed during the present study. Nonlinear regression with simultaneous fitting of the uptake and depuration phase was used. Since the measured concentrations were partly above the nominal concentration (1 mg/L), all calculations were based on the time-weighted mean average concentration per test item compound [L * kg-1]. Where possible to estimate, 95% confidence intervals are given in brackets. Results are summarized in a table and provided in the section on any other information on results.

Metabolites:

The test item is a UVCB substance consisting of oligomeric reaction products of 2,3-epoxypropyl neodecanoate and toluene-4-sulfonic acid. An overview about the components of the test item is given in the following table:
Component No. MW MF Intensity
1 172.2 C7H8O3S 0.16% (GPC)
2 228.3 C13H24O3 12.9% (GPC)
3 400.5 C20H32O6S 1.3% (HPLC)
4 414.6 C21H34O6S 0.5% (HPLC)
5 456.6 C26H48O6 6.5 (HPLC)
6 474.7 C26H50O7 5.8 (HPLC), 54.4 (GPC)
7 628.9 C33H56O9S 1 2.3 (HPLC)
8 703.0 C39H74O10 18.3 (HPLC), 15.1 (GPC)
9 777.1 C42H80O12 1.4 (HPLC)
10 857.2 C46H80O12S 11.3 (HPLC)
11 931.3 C52H98O13 17.7 (HPLC), 17.3 (GPC)
12 1005.4 C55H104O15 1.4 (HPLC)
13 1085.5 C59H104O15S 2.7 (HPLC)
14 1159.6 C65H122O16 4.3 (HPLC)
For the analytical monitoring of the test item components during the bioconcentration study, a LC-MS/MS-method based on electrospray ionization (ESI) in positive ion mode, was implemented. The test item components are chromatographically separated by a gradient method using mixtures of methanol and HPLC water as mobile phase. Most test item components were considered during method development. An overview about the selected components are given the table below. The BCF potential of each component was either calculated based on EPI Web 4.1 Web (regression-based method RB or Arnot-Gobas AG method were used) or experimental data were considered.
Only components 1, 4, 9 and 14 were so far not considered for analytical monitoring. Component 1 has no bioaccumulation potential. Components 4 and 9 could not be detected when using a test concentration of 1 mg/L. Higher concentrations are not possible because toxic effects of the test item on fish cannot be ruled out. Component 14 was not considered due to poor chromatographic behaviour.
Component No. BCF Potential Selected? Y/N Reason
1 not bioaccumulative acc. to ECHA (J. Pestic. Sci. 13, 85 - 92) n no B or vB
2 62 (RB), 372 (AG) L/kg y --
3 8.4 (RB), 44.3 L/kg (AG) y --
4 44.0 (RB), 125 L/kg (AG) n not detectable
5 2018 (RB), 11.2 L/kg (AG) y --
6 282 (RB), 9.2 L/kg (AG) y --
7 611 (RB), 3.3 L/kg (AG) y --
8 126 (RB), 0.9 L/kg (AG) y --
9 356 (RB), 0.9 L/kg (AG) n not detectable
10 10.4 (RB), 0.9 L/kg (AG) y --
11 8.4 (RB), 0.9 L/kg (AG) y --
12 23.9(RB), 0.9 L/kg (AG) y --
13 3.2 (RB), 0.9 L/kg (AG) y --
14 3.2 (RB), 0.9 L/kg (AG) n poor chromatography

After the analytical method implementation for the components of the test item, the suitability of the flow-through system to reliably dose each component of the test item was tested over a time period of fourteen days. Finally, the following relevant metabolites of the test item were analytically verified for the issue of bioaccumulation in Fish:
C10H14O5S, C13H24O3, C15H16O2S, C16H18O2S, C20H32O6S, C21H34O6S, C26 H48O6, C26H50O7 and C33H56O9S.

Results with reference substance (positive control):

No reference substance was used during this study.

Details on results:

- Mortality of test organisms: For both groups the mortality was less than 10% at the end of the test. In the test concentration one fish died on study day 3 (uptake phase).
- Behavioural abnormalities: No behavioural abnormalities were observed in the test item group and in the solvent control group.
- Observations on feeding behavior: No anomalous feeding behavior was observed throughout the study in any of the test groups.
- Observations on body length and weight: The initial mean wet weight and length of 10 representative fish sampled from the stock fish at test start were 182 mg and 2.91 cm, respectively. At each sampling date wet weight and length of the sampled fish were determined. Until the end of the study the mean wet weight of the solvent control group increased up to 333 mg and the mean length increased up to 3.31 cm. Weight data of the control and test item group were converted to natural logs and plotted vs. time. A linear correlation was calculated for the ln (fish wet weight) vs. time. The variances in the slopes of the test and control lines were calculated and used to evaluate the statistical significance of the difference in the slopes (growth rates). Since there was no significant difference, the test and control data were pooled and an overall fish growth rate for the study (kgrowth= 0.00935) was calculated as the overall slope of the linear correlation. For results see table below
- Reproduction during test period: not indicated
- Other biological observations: No other effects (morphological and behavioural) were observed in the test item group and in the solvent control group.
- Organ specific bioaccumulation: Not indicated
- Bound residues forming a plateau: not indicated
- Mortality and/or behavioural abnormalities of control: No behavioural abnormalities were observed in the solvent control group.
- Loss of test substance during test period: Not indicated. Analytical monitoring was performed and reported in the section to analytic.
- Non-eliminated residues (NER) at the end of elimination phase: not applicable
- Results with vehicle control: No morphological and behavioural effects were observed in the solvent control group. Until the end of the study the mean wet weight of the solvent control group increased up to 333 mg and the mean length increased up to 3.31 cm. The initial mean wet weight and length of 10 representative fish sampled from the stock fish at test start were 182 mg and 2.91 cm, respectively.

Control 1 mg/L Overall
Growth rate kg
(=Slope) 0.00724 0.0115 0.00935
95% CI 0.00132 – 0.0132 0.00427 – 0.0187 0.00477 – 0.0139
Intercept 5.43 5.43 5.43
Total number of values
(fish) for evaluation 61 58 119
Statistical difference No Not applicable

Mean Concentrations and Standard Deviation of the test item in Water during Uptake Phase

Nominal concentration 1 mg test item/L

Compound	Mean meas. Conc. [mg/L]	[%]
C10H14O5S	0.6 ± 0.1	64
C13H24O3	0.9 ± 0.2	91
C15H16O2S	1.2 ± 0.1	117
C16H18O2S	1.1 ± 0.1	109
C20H32O6S	0.8 ± 0.1	75
C21H34O6S	0.9 ± 0.2	85
C26H48O6	1.4 ± 0.6	138
C26H50O7	2.3 ± 1.1	230
C33H56O9S	1.4 ± 0.5	145

Meas. conc.= Measured concentration of the test item; weighing factor, dilution factor and standard deviation taken

                        into account

[%]                = Percent of nominal concentration

Mean Concentrations and Standard Deviation of the test item in Water during Depuration Phase

Nominal concentration 1 mg test item/L

Sampling		Control Group	Exposure Group
day [d]		Meas. conc.   [mg/L]	Meas. conc.   [mg/L]
0.3	I	< LOQ	< LOQ
	II
	III
1	I
	II
	III

Meas. conc. = Measured concentration of the test item, weighing of the calibration   and dilution factor taken into account

LOQ        = Limit of quantification (0.7 mg test item/L)

Exposure Group     = Exposure Group contains the following compounds:C₁₀H₁₄O₅S, C₁₃H₂₄O₃, C₁₅H₁₆O₂S, C₁₆H₁₈O₂S, C₂₀H₃₂O₆S, C₂₁H₃₄O₆S, C₂₆H₄₈O₆, C₂₆H₅₀O₇,  C₃₃H₅₆O₉S

The lipid content was determined according to Smedes (1999). The overall mean of the test item group was calculated and used for calculation on the lipid normalized BCF_KG

Results are presented in the table below:

Mean Lipid Contents of the Fish

Sampling Phase	Study Day	Sample	FW/BW [%]	Mean [%]	Test Item-Mean [%]
Uptake	Day 1	Control Group	14.2	12.7	-
	Day 8		11.3
Depuration	Day 1		10.8	10.8
Uptake	Day 0.3	Test Item Group	10.2	11.4	12.0
	Day 2		10.6
	Day 4		9.60
	Day 14		15.1
Depuration	Day 0.3		12.6	12.6
	Day 2		10.5
	Day 4		11.6
	Day 8		14.6
	Day 14		13.6

FW = Fat weight                                    BW = Body weight

Measured test item concentrations in fish

During the uptake and depuration phase, fish samples were taken from the tanks as stated in the sampling schedule (see section to any other information on materials and methods). At given sampling periods, 3 fish of the test concentration and 2 fish of the control group were sampled. All measured concentrations in fish from the control group were < LOQ,apart from the control group on day 14 of the uptake phase. As the results for fish of the test item concentration were in the range and below LOQ, respectively, it was assumed that interferences within the measured ions led to these results. The results of measured concentrations in fish from exposure group are summarized in following tables:

Overview of measured Test Item relevant Components in Fish during Uptake Phase

Test item relevant Components	Uptake phase: Fish matrix (Mean value; CFish[mg/kg])
	(days)	0.3	1	2	4	8	14	28
	C10H14O5S	48.8*	46.2*	32.6*	42.2*	36.0*	33.5*	31.6*
	C13H24O3	39.4*	-	29.0*	34.6*	-	30.9*	-
	C15H16O2S	166	319	394	377	177	312	314
	C16H18O2S	223	414	637	698	327	619	558
	C20H32O6S	33.8*	-	34.4*	37.5*	-	40.2*	-
	C21H34O6S	38.8*	41.4*	48.4*	47.4*	46.6*	47.8*	46.8*
	C26H48O6	-	-	110*	101*	173*	334	515
	C26H50O7	73.6*	-	-	-	-	-	-
	C33H56O9S	-	-	-	-	-	-	-

C_Fish                             =  Concentration of the test item related to fish body weight during uptake phase

Bold font                        =   Concentrations of components with significant uptake. The kinetic bioconcentration factors were calculated for these components.

*                  =   Measured concentrations in sampled fish were partly below the limit of quantification, but higher than the lowest calibration level or > 70 % of the LOQ. To achieve a better fitting to the uptake and depuration curves, corresponding values were used for calculations.

-                  =  Calculated mean measured concentrations in fish were below the limit of quantification (LOQ 200 mg test item/kg body weight)

Overview of measured Test Item relevant Components in Fish during Depuration Phase

Test item relevant Components	Depuration phase: Fish matrix (Mean value; CFish[mg/kg])
	(days)	0.3	1	2	4	8	14
	C10H14O5S	-	-	-	-	-	-
	C13H24O3	-	-	-	-	-	-
	C15H16O2S	105*	-	-	-	-	-
	C16H18O2S	268*	161*	47.9*	-	-	-
	C20H32O6S	-	-	-	-	-	-
	C21H34O6S	-	-	-	-	-	-
	C26H48O6	198*	358*	417	179*	122*	195*
	C26H50O7	35.6*	-	-	-	75.0*	-
	C33H56O9S	-	-	-	-	-	-

C_Fish                          =  Concentration of the test item related to fish body weight during depuration phase

Bold font                      =   Concentrations of components with significant uptake. The kinetic bioconcentration factors were calculated for these components.

*                =   Measured concentrations in sampled fish were partly below the limit of quantification, but higher than the lowest calibration level or > 70 % of the LOQ. To achieve a better fitting to the uptake and depuration curves, corresponding values were used for calculations.

-                 =  Calculated mean measured concentrations in fish were below the limit of quantification (LOQ 200 mg test item/kg body weight)

Bioconcentration factors per test item compound:

All calculations were based on the time-weighted mean average concentration per test item compound [L * kg^-1]. 

Where possible to estimate, 95% confidence intervals are given in brackets.

Test item compound	Body Weight	Body Weight, Growth and Lipid Normalized
	BCFK	BCFKGL
C15H16O2S	249 (212 – 286)	104
C16H18O2S	479 (408 – 550)	201
C26H48O6	233 (188 – 277)	109

Overview of uptake and depuration rates, growth rate, growth corrected depuration Rates and Lipid normalized factor:

All calculations were based on the time-weighted mean average concentration per test item compound [L * kg^-1]. Where possible to estimate, 95% confidence intervals are given in brackets.

Test item compound	Growth rate kg [day^-1]	Uptake rate k1 [L * kg^-1 * day^-1]	Depuration rate k2 [day^-1]	Growth-corrected depuration Rate K2g [day^-1]	Lipid normalization factor	DT50 [days]	DT90 [days]
C15H16O2S	0.00935 (0.00477 – 0.0139)	676.1 (320.9 – 1031)	2.713 (1.307 – 4.120)	2.7037	0.12	0.256	0.852
C16H18O2S		802.7 (403.4 – 1202)	1.675 (0.8494 – 2.501)	1.6657		0.416	1.382
C26H48O6		19.6 (12.4 – 26.9)	0.08437 (0.05008 – 0.1187)	0.07502		9.24	30.7

Validity criteria fulfilled:

yes

Conclusions:

Since an increase of concentrations in fish was observed for the metabolites C15H16O2S, C16H18O2S and C26H48O6 of the test item, uptake / depuration rates and kinetic bioconcentration factors were calculated only for these compounds, respectively.
The kinetic bioconcentration factors (growth/lipid normalized) determined for these compounds are 104 L/Kg, 201 L/Kg and 109 L/Kg, respectively.
These results are below the regulatory threshold value of concern regarding bioaccumulation as set in ECHA guidance R11 relative to PBT assessment.
For all other relevant components investigated in the present study, no increase of concentrations in fish was determined during the uptake phase. Measured values in fish were clearly below the limit of quantification of the test item in fish.

Executive summary:

An aqueous bioaccumulation study with zebrafish (Danio rerio) was conducted according to OECD Guideline 305 (2012) to determine the bioconcentration potential of the test item; an UVCB substance with a complex mixture of isomers and homologue components.

A flow-through test with 2 groups (one solvent control group and one exposure group of nominal 1 mg test item/L) was carried out with an exposure (uptake phase) of 28 days followed with a depuration phase of 14 days. During the uptake phase, one group of fish was exposed to the test item at a nominal concentration of 1 mg test item/L. After 28 days of uptake fish were then transferred to a medium free of the test item (depuration phase). The depuration phase was carried out over a period of 14 days. In parallel a solvent control was tested containing the same solvent concentration as the test concentration during uptake.

Since the test item is an UVCB, preliminary studies have been thoroughly conducted to evaluate suitable lead components for the analytical monitoring that should be of relevance for the B assessment. Measurements were conducted to figure out components which could be detected in aqueous test item samples at a concentration of 0.5 mg test item/L with accurate masses and for which molecular formulas could be derived. This threshold concentration was set because an application concentration of 1 mg/L was envisaged for the conduction of the main study. Higher test concentrations are not allowed because toxic effects of the test item on fish cannot be ruled out. Furthermore, validated analytical methods about the accuracy, precision and specificity could be implemented for these compounds.

Following relevant compounds of the test item were identified for analytical monitoring and verified during the main study:

C10H14O5S, C13H24O3, C15H16O2S, C16H18O2S, C20H32O6S, C21H34O6S, C26 H48O6, C26H50O7 and C33H56O9S.

The choice of these relevant compounds for analytical monitoring during the main study was based on preliminary studies that used HPLC and GPC methods for identification of all components of the test item. The molecular formulas of the compounds, their retention times, the molecular weight, the intensity of the corresponding peaks (height), the number of detected ions relevant for the deduction of the molecular formula and their scores are parameter that were used for the identification. At least three ions were necessary for a reliable deduction of the molecular formula for a compound. These ions include the protonated adducts or other adducts, ions from the¹³C or³⁴S isotope or single and multiple charged ions. Compounds not matching these criteria were not considered for the method implementation and validation because no reliable molecular formula could be derived. The BCF potential of each component of the test item was additionally either calculated based on EPI Web 4.1 Web (regression-based method RB or Arnot-Gobas AG method were used) or experimental data were considered. Components that do not present any bioaccumulation potential as well as components that were not analytically detectable were then no more considered for the monitoring. 

These above-mentioned selected compounds reflected the composition of this UVCB substance as included in IUCLID dossier because reaction products of 2,3-epoxypropanol and toluene-4-sulfonic acid, 2,3-epoxypropanol and neodecanoate or combining all three monomers were used for the analytical monitoring.

For the analytical monitoring during the main study, four fish samples were taken from each tank on days 0.3, 1, 2, 4, 8, 14 and 28 of the uptake phase as well as on days 0.3, 1, 2, 4, 8 and 14 of the depuration phase.

Aqueous phase samples were taken and analyzed on uptake phase days 0.3, 1, 2, 4, 8, 14, 28 and on days 0.3 and 1 of the depuration phase. Sampling and analysis of water samples after depuration day 1 was stopped, since measured concentrations on depuration day 1 were < LCL.

The lipid content of the fish was determined at the start of the study (uptake day 0), at the end of the uptake phase (day 28) and at the end of the depuration phase (depuration day 14). Lipid fish sampling on uptake day 0 was carried out from the stock fish which were used for sub-sample measurements at start of the test.

Since an increase of concentrations in fish was observed for the metabolites C15H16O2S, C16H18O2S and C26H48O6 of the test item, uptake / depuration rates and kinetic bioconcentration factors were calculated only for these compounds, respectively.

Measured concentrations in fish of concerning test item components were partly below the limit of quantification, but higher than the lowest calibration level. To achieve a better fitting to the uptake and the depuration curves, these values were used for calculations. The uptake /depuration rates and kinetic bioconcentration factors were calculated for these components, respectively. Nonlinear regression with simultaneous fitting of the uptake and the depuration phase was used. Since no steady-state phase was reached during uptake, no steady-stade bioconcentration factors were calculated. The kinetic bioconcentration factors have been calculated based on fish body weight and time-weighted average mean concentration of the test item in water during uptake phase.

The kinetic bioconcentration factors (growth/lipid normalized) determined for these compounds are 104 L/Kg, 201 L/Kg and 109 L/Kg, respectively.

These results are below the regulatory threshold value of concern regarding bioaccumulation as set in ECHA guidance R11 relative to PBT assessment.

For all other relevant components investigated in the present study, no increase of concentrations in fish was determined during the uptake phase. Measured values in fish were clearly below the limit of quantification of the test item in fish.

Description of key information

An aqueous bioaccumulation study with zebrafish (Danio rerio) was conducted according to OECD Guideline 305 (2012) to determine the bioconcentration potential of the test item; an UVCB substance with a complex mixture of isomers and homologue components.

A flow-through test with 2 groups (one solvent control group and one exposure group of nominal 1 mg test item/L) was carried out with an exposure (uptake phase) of 28 days followed with a depuration phase of 14 days. During the uptake phase, one group of fish was exposed to the test item at a nominal concentration of 1 mg test item/L. After 28 days of uptake fish were then transferred to a medium free of the test item (depuration phase). The depuration phase was carried out over a period of 14 days. In parallel a solvent control was tested containing the same solvent concentration as the test concentration during uptake.

Since the test item is an UVCB, preliminary studies have been thoroughly conducted to evaluate suitable lead components for the analytical monitoring that should be of relevance for the B assessment. Measurements were conducted to figure out components which could be detected in aqueous test item samples at a concentration of 0.5 mg test item/L with accurate masses and for which molecular formulas could be derived. This threshold concentration was set because an application concentration of 1 mg/L was envisaged for the conduction of the main study. Higher test concentrations are not allowed because toxic effects of the test item on fish cannot be ruled out. Furthermore, validated analytical methods about the accuracy, precision and specificity could be implemented for these compounds.

Following relevant compounds of the test item were identified for analytical monitoring and verified during the main study:

C10H14O5S, C13H24O3, C15H16O2S, C16H18O2S, C20H32O6S, C21H34O6S, C26 H48O6, C26H50O7 and C33H56O9S.

The choice of these relevant compounds for analytical monitoring during the main study was based on preliminary studies that used HPLC and GPC methods for identification of all components of the test item. The molecular formulas of the compounds, their retention times, the molecular weight, the intensity of the corresponding peaks (height), the number of detected ions relevant for the deduction of the molecular formula and their scores are parameter that were used for the identification. At least three ions were necessary for a reliable deduction of the molecular formula for a compound. These ions include the protonated adducts or other adducts, ions from the¹³C or³⁴S isotope or single and multiple charged ions. Compounds not matching these criteria were not considered for the method implementation and validation because no reliable molecular formula could be derived. The BCF potential of each component of the test item was additionally either calculated based on EPI Web 4.1 Web (regression-based method RB or Arnot-Gobas AG method were used) or experimental data were considered. Components that do not present any bioaccumulation potential as well as components that were not analytically detectable were then no more consider for the monitoring. 

These above-mentioned selected compounds reflected the composition of this UVCB substance as included in IUCLID dossier because reaction products of 2,3-epoxypropanol and toluene-4-sulfonic acid, 2,3-epoxypropanol and neodecanoate or combining all three monomers were used for the analytical monitoring.

For the analytical monitoring during the main study, four fish samples were taken from each tank on days 0.3, 1, 2, 4, 8, 14 and 28 of the uptake phase as well as on days 0.3, 1, 2, 4, 8 and 14 of the depuration phase.

Aqueous phase samples were taken and analyzed on uptake phase days 0.3, 1, 2, 4, 8, 14, 28 and on days 0.3 and 1 of the depuration phase. Sampling and analysis of water samples after depuration day 1 was stopped, since measured concentrations on depuration day 1 were < LCL.

The lipid content of the fish was determined at the start of the study (uptake day 0), at the end of the uptake phase (day 28) and at the end of the depuration phase (depuration day 14). Lipid fish sampling on uptake day 0 was carried out from the stock fish which were used for sub-sample measurements at start of the test.

Since an increase of concentrations in fish was observed for the metabolites C15H16O2S, C16H18O2S and C26H48O6 of the test item, uptake / depuration rates and kinetic bioconcentration factors were calculated only for these compounds, respectively.

Measured concentrations in fish of concerning test item components were partly below the limit of quantification, but higher than the lowest calibration level. To achieve a better fitting to the uptake and the depuration curves, these values were used for calculations. The uptake /depuration rates and kinetic bioconcentration factors were calculated for these components, respectively. Nonlinear regression with simultaneous fitting of the uptake and the depuration phase was used. Since no steady-state phase was reached during uptake, no steady-stade bioconcentration factors were calculated. The kinetic bioconcentration factors have been calculated based on fish body weight and time-weighted average mean concentration of the test item in water during uptake phase.

The kinetic bioconcentration factors (growth/lipid normalized) determined for these compounds are 104 L/Kg, 201 L/Kg and 109 L/Kg, respectively.

These results are below the regulatory threshold value of concern regarding bioaccumulation as set in ECHA guidance R11 relative to PBT assessment.

For all other relevant components investigated in the present study, no increase of concentrations in fish was determined during the uptake phase. Measured values in fish were clearly below the limit of quantification of the test item in fish.

Key value for chemical safety assessment

BCF (aquatic species):

414 L/kg ww

Additional information

The bioaccumulation study was carried out after a decision on a testing proposal set out for the test substance pursuant to Article 40(3) of the REACH Regulation with the decision number TPE-D-2114309016-62-01/F.

The given value is the sum of the kinetic bioconcentration factors growth-corrected and lipid normalised of three relavant metabolites of the test item. Results show that the test item does not bioaccumulate.