Registration Dossier

Environmental fate & pathways

Bioaccumulation: aquatic / sediment

Currently viewing:

Administrative data

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:
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. certificate)
Radiolabelling:
not specified
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:
other: 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 24hours 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 test item is an UVCB substance with a complex mixture of isomers and homologue components. 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 (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.
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:
12.7 %
Time point:
other: Uptake ; Mean value : days 1; 8
Remarks on result:
other: Control group
Remarks:
Lipid content as Fat weight/Body weight
Lipid content:
11.4 %
Time point:
other: Uptake, mean value : days 0.3; 2; 4; 14
Remarks on result:
other: Test group
Remarks:
Lipid content as Fat weigth/Body weight
Lipid content:
10.8 %
Time point:
other: Depuration; Day 1
Remarks on result:
other: Control group
Remarks:
Lipid content as Fat weight/Body weight
Lipid content:
12.6 %
Time point:
other: Depuration, mean value: days 0.3; 2; 4; 8; 14
Remarks on result:
other: test group
Remarks:
Lipid content as Fat weight/Body weight
Type:
other: BCFKg calculated from kinetic rate constants (k1/k2)
Value:
250 L/kg
Basis:
total lipid content
Calculation basis:
kinetic, corrected for growth
Remarks on result:
other:
Remarks:
Component C15H16O2S
Key result
Type:
other: BCFKgL calculated from kinetic bioconcentration factors; lipid normalized and growth corrected
Value:
104 L/kg
Basis:
normalised lipid fraction
Calculation basis:
kinetic, corrected for growth
Remarks on result:
other:
Remarks:
Component C15H16O2S
Type:
other: BCFKg calculated from kinetic rate constants (k1/k2)
Value:
482 L/kg
Basis:
total lipid content
Calculation basis:
kinetic, corrected for growth
Remarks on result:
other:
Remarks:
Component C16H18O2S
Key result
Type:
other: BCFGL calculated from kinetic rate constants, lipid normalized and growth corrected
Value:
201 L/kg
Basis:
normalised lipid fraction
Calculation basis:
kinetic, corrected for growth
Remarks on result:
other:
Remarks:
Component C16H18O2S
Elimination:
yes
Parameter:
other: depuration phase
Depuration time (DT):
14 d
Rate constant:
growth rate constant (d-1)
Value:
0.009
Rate constant:
overall uptake rate constant (L kg-1 d-1)
Value:
676.1
Remarks on result:
other: Compound C15H16O2S
Rate constant:
overall uptake rate constant (L kg-1 d-1)
Value:
802.7
Remarks on result:
other: Compound C16H18O2S
Rate constant:
growth-corrected depuration rate constant (d-1)
Value:
2.704
Remarks on result:
other: Compound C15H16O2S
Rate constant:
growth-corrected depuration rate constant (d-1)
Value:
1.666
Remarks on result:
other: Compound C16H18O2S
Details on kinetic parameters:
- Uptake rate constant k(s): C15H16O2S; K1= 676.1 [L * kg-1 * day-1]; C16H18O2S= 802.7 [L * kg-1 * day-1]
- Depuration rate constant k(e): C15H16O2S; K1= 2.713 [L * kg-1 * day-1]; C16H18O2S= 1.675 [L * kg-1 * day-1]
- Indication of bi- or multiphasic kinetics: no indication
- Computation / data analysis: yes
Metabolites:
The test item is an UVCB substance with a complex mixture of isomers and homologue components. The following relevant compounds 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.
Details on results:
- Mortality of test organisms: one dead fish in the test group at day 3.
- Behavioural abnormalities: No
- Observations on feeding behavior: No
- Observations on body length and weight: No abnormality observed
- Reproduction during test period: not indicated
- Other biological observations:
- Organ specific bioaccumulation: No
- Bound residues forming a plateau: not indicated
- Mortality and/or behavioural abnormalities of control: No
- Loss of test substance during test period: No
- Non-eliminated residues (NER) at the end of elimination phase: not applicable
- Results with vehicle control: available as solvent control

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 Uptake 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

Validity criteria fulfilled:
yes
Conclusions:
Since an increase of concentrations in fish was observed for the compounds C15H16O2S and C16H18O2S 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 both compounds are 104 L./Kg and 201 L/Kg respectively.
These results are below the regulatory threshold value of concern regarding bioaccumulation.
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, following relevant compounds of the test item were analytically verified:

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

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 fishwas observedonly for the compounds C15H16O2S and C16H18O2S, uptake / depuration rates and kinetic bioconcentration factors were calculated for these compounds.

The kinetic bioconcentration factors (growth/lipid normalized) determined for both compounds are 104 L./Kg and 201 L/Kg respectively. These results are below the regulatory threshold value of concern regarding bioaccumulation.

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, following relevant compounds of the test item were analytically verified:


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


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 fishwas observedonly for the compounds C15H16O2S and C16H18O2S, uptake / depuration rates and kinetic bioconcentration factors were calculated for these compounds.


The kinetic bioconcentration factors (growth/lipid normalized) determined for both compounds are 104 L./Kg and 201 L/Kg respectively. These results are below the regulatory threshold value of concern regarding bioaccumulation.


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):
305 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 bioconcentration factors of two relavant constituents of the test item. Results show that the test item does not bioaccumulate.