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REACH

Quaternary ammonium compounds, C20-22-alkyltrimethyl, chlorides

EC number: 271-756-9 | CAS number: 68607-24-9

Life Cycle description
Uses advised against

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:

from 2019-03-12 to 2020-01-23 with the definitive exposure period from 2019-03-12 to 2019-05-21

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

Deviations:

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch number DEG4440803

CAS number 68607-24-9

Purity Analyzed for c-chain distribution:
C16 0.09%
C18 6.21%
C20 12.89%
C22 78.15%
C24 2.66%

Active ingredient N,N,N-trimethyl-C20-22-(even numbered)-alkyl-1-aminium chloride

Active content (certified) 79.3%

Appearance White to slightly yellowish pellets

Water solubility 0.01 g/L (cmc)

Density Approx. 0.9 g/cm3 (20 °C, DIN 51757)

pH 5 - 7 (20 °C)

Stability under test Not specified
conditions

Expiry date 2020-02-06

Recommended storage Room temperature, protected from direct sunlight and temperatures above 40 °C; in the original container, tightly closed container in a dry, cool and well-ventilated place; test item is hygroscopic.
Receipt 2018-06-05

Identification parameters Name, batch number, state and color

Retention sample At least 1 g of the test item has been retained on 2018-06-05 and stored at 6 ± 2 °C.

Storage conditions 18 - 25 °C, dark, in the tightly closed original container in a desiccator

Radiolabelling:

Details on sampling:

Analysis of Fish
Fish samples were taken from each tank as stated in the sampling schedule.
At given sampling periods, 4 fish per test concentration and control group were euthanized according to laboratory standard operating procedure.
The fish were rinsed with water, blotted dry and weighed and stored at approx. –18 °C until analysis, if necessary.

Analysis of Water
Water samples were taken from each tank as stated in the sampling schedule from the middle of the water phase from each aquaria. Analysis of water samples after depuration day 2 was stopped, since measured concentrations on depuration day 1 were < LOQ. Further samples from depuration day 4 were not analysed.

Uptake Phase Sampling Schedule

Sampling interval
Uptake day
Water samples
Number of
fish per sample
Control group Exposure groups each Control group Exposure groups each
1st 0.3 X X 4 4
2nd 1 X X 4 4
3rd 2 X X 4 4
4th 3 X X Not sampled
5th 4 X X 4 4
6th 7 X X 4 4
7th 14 X X 4 4
8th 28 X X 4 4
9th 35 X X 4 4
10th 42 X X 4 4
X = appropriate aliquot

Depuration Phase Sampling Schedule

Sampling Interval
Depuration Day
Water Samples Number of
fish per sample
Control group Exposure groups each Control group Exposure groups each
1st 0.3 X X 4 4
2nd 1 X X 4 4
3rd 2 X X 4 4
4th 4 X X 4 4
5th 7 - - 4 4
6th 14 - - 4 4
7th 28 - - 4 4
X = appropriate aliquot

Analysis of Food
Samples of the food were analyzed before the beginning and at the end of the uptake phase of the study to demonstrate that bioaccumulation of Genamin KDMP via nutrition can be ruled out.

Vehicle:

yes

Remarks:

Methanol

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

Preparation of Standards and Samples – Aqueous Phase

Dilution Medium
The dilution medium was acetonitrile : ultrapure water (50 : 50), containing 1% formic acid.

Preparation of Standards
A stock solution of 500 mg test item/L in methanol was prepared. This solution was further diluted to at least 6 calibration standards in the range from 0.2 – 2 µg/L with dilution medium.

Preparation of Samples – Aqueous Phase
The control and the samples (20 mL) were stabilized factor 2 with acetonitrile, containing 2% formic acid directly after sampling. Samples of the control group were measured without further dilution as well as samples of the both exposure groups during the depuration phase. Further dilution steps were conducted with dilution medium, if necessary.

Additional Dilution Steps
Nominal
test item concentration
[µg/L] Final Dilution
factor
Sample
volume
[mL] Final
volume
[mL]
5.0 4 0.5 1.0
5.0 1) 20 0.1 1.0
20.0 20 0.1 1.0
20.0 3) 50 0.2 5.0
20.0 2) 2 20 40
1)= dilution factor only used for samples on day 3 of uptake phase
2)= dilution factor only used for samples of depuration phase
3)= dilution factor only used for samples on day 4 of uptake phase

Preparation of Standards and Samples – Matrix Fish

Media
The extraction medium was acetonitrile containing 2% formic acid.
The dilution medium A was extract of unspiked matrix fish.
The dilution medium B was acetonitrile : ultrapure water (50 : 50), containing 1% formic acid.
The matrix dilution medium was a mixture of dilution medium A and dilution medium B in a ratio of 10:90.

Preparation of Standards
A stock solution of 500 mg test item/L in methanol was prepared. The stock solution was further diluted to at least 5 calibration standards in the range from 0.3 – 6 µg/L with matrix dilution medium. The calibration standards were filtered with syringe filters after dilution.

Preparation of Samples
An euthanized and weighed fish was homogenized with a spatula in centrifugation tubes. Afterwards the samples were shaken for 45 min at least at 175 rpm with 3 mL extraction medium and centrifuged for 5 min at 3000 rpm. The supernatant was transferred into a 10 mL graduated flask. The procedure of extraction was repeated twice and the combined supernatants were filled up to the mark with extraction medium. The samples were diluted with dilution medium A, if necessary. Finally, the samples were diluted factor 10 with dilution medium B and then filtrated with syringe filters (Chromafil RC-20/15) prior to analysis.

Preparation of Quality Controls – Matrix Fish
One fish of the control group was spiked ≤ 1 x LOQ on each day of analysis and processed to investigate the robustness of the analytical method.

Preparation of Standards and Samples – Food
Three replicates of 250 mg fish food were weighed in centrifugation tubes and prepared as described in the report.

Determination of Lipid Content
Each fish was first homogenized with a spatula in a 50 mL centrifugation tube and after addition of 8 mL propane-2-ol and 10 mL cyclohexane homogenized for 2 min with an Ultraturrax (9 x 1000 rpm). 11 mL ultrapure water were added and the samples were homogenized again for 1 min (9 x 1000 rpm). After centrifugation for 10 min at 4000 rpm, a few drops of concentrated hydrochloric acid were added. The samples were homogenized and centrifuged again for 10 min at 4000 rpm. The organic supernatant was transferred into a pear shape flask (dried for 60 min at 105 °C in a drying cabinet, cooled down in a desiccator 15 min and weighed before use) after centrifugation.
20 mL cyclohexane containing 13 % propane-2-ol (w/w) were added to the samples and after homogenization for 1 min (5 x 1000 rpm) they were centrifuged again for 10 min at 4000 rpm. The organic supernatant was transferred again into the corresponding pear shape flask.
The extracts were evaporated to dryness at 40 °C with a rotary evaporator. The pre-weighed pear flasks were dried for 60 min at 105 °C in a drying cabinet, cooled down for 15 min in a desiccator and weighed again.

Sample Storage
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.

Evaluation
Concentrations of the test item in the different matrices were calculated by matrix matched external standard calibrations.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Test species
Danio rerio (zebrafish): Vertebrata, Gnathostomata, Pisces, Osteichthyes, Teleostei, Cypriniformes, Cyprinidae

Reason for the selection of the test species
Danio rerio is one of the recommended species according to the guideline.

Origin All fish used in the test were gained at the test facility from a single brood stock (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin).

Husbandry and acclimation 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.
The dissolved oxygen concentration was more than 80 % of the air saturation value.
No disease treatments were carried out 2 weeks preceding the start of the test.
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. The fish were held in water of the quality to be used in the test. The stock population were fed with the same type of food to be used in the test.

Water
Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated.
Water parameters:
Total hardness 10 - 250 mg CaCO3 / L
pH-value: 6.0 - 8.5
Acidity: 0.1 mmol/L (recent measurement 2019-02-14)
Alkalinity: 0.5 mmol/L (recent measurement 2019-02-14)
Conductivity: 142 µS/cm (recent measurement 2019-02-14)

Feeding
Feeding was carried out daily, with an amount of 1.5 % of the fish body weight per feeding day over approximately 14 weeks before start of the uptake phase.

Food
Sera Vipan; SERA GMBH, 52518 Heinsberg, Germany
This product is composed of fish products, oils and fats, cereal grains, yeast, byproducts, minerals and vitamins.

- Age at study initiation (mean and range, SD): 14 weeks (date of hatch: 2018-11-30)
- Length at study initiation (length definition, mean, range and SD): Mean size at test start was 2.72 cm
- Weight at study initiation (mean and range, SD): 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 of 159.6 mg.
- Weight at termination (mean and range, SD): 256.2 - 273.3 mg
- Lipid content at test initiation (mean and range, SD): 10 %
- Health status: healthy

Route of exposure:

aqueous

Justification for method:

aqueous exposure method used for following reason: Requested by authority

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

ca. 42 d

Total depuration duration:

ca. 28 d

Hardness:

Total hardness of the test media was measured from the solvent control as well as the test concentrations of 3.97 and 15.9 µg a.i./L at the start of the study (study day 0). The measured total hardness was 60 mg CaCO3/L in the solvent control and 59 mg CaCO3/L for both test item concentrations of 3.97 and 15.9 µg a.i./L, respectively.

Test temperature:

Temperature (Continuous Measuring) in the Solvent Control
Period of Measurements 2019-03-12 to 2019-05-21
Minimum Temperature [°C] 22.4
Maximum Temperature [°C] 23.4
Mean Temperature [°C] 23.2

For the meaured temperature values in the test vesels see the following both windows "pH" and "Dissolved oxygen".

pH:

Water Parameters in the Solvent Control Group
Study Day
O2-saturation
[%] pH value
Temperature
[°C]
0 96 7.49 23.0
1 95 7.45 23.0
3 94 7.66 23.2
6 93 7.90 23.0
8 91 7.78 23.0
10 90 7.64 23.5
13 90 7.69 23.6
15 92 7.73 23.0
17 93 7.80 23.0
20 94 7.95 23.1
22 92 7.68 22.8
24 94 7.85 22.8
27 90 7.82 23.3
29 96 7.74 23.2
31 95 7.88 23.2
34 94 7.74 23.1
36 96 7.76 22.9
37 95 7.78 23.0
42 100 7.61 22.6
43 100 7.65 22.9
45 98 7.89 23.0
48 100 7.79 23.0
49 100 7.78 23.0
52 99 7.71 23.2
55 97 7.28 23.3
57 95 7.25 23.4
59 96 7.86 23.4
62 98 7.45 23.3
64 96 7.35 23.3
66 96 7.23 23.4
69 98 7.26 23.4
Mean 95 7.66 23.1
SD 3.01 0.21 0.23
Min. 90 7.23 22.6
Max. 100 7.95 23.6

Water Parameters in the Test Item Concentration 3.97 µg a.i./L
Study Day
O2-saturation
[%] pH value
Temperature
[°C]
0 95 7.48 22.8
1 94 7.46 22.8
3 94 7.48 22.7
6 92 7.70 23.0
8 90 7.76 22.9
10 92 7.53 23.1
13 90 7.68 23.1
15 92 7.71 23.0
17 92 7.71 22.9
20 93 7.84 22.8
22 92 7.62 23.0
24 93 7.80 22.8
27 93 7.83 22.8
29 94 7.68 23.0
31 92 7.77 22.9
34 92 7.72 22.9
36 93 7.74 22.9
37 92 7.75 22.9
42 100 7.51 22.6
43 100 7.41 22.9
45 98 7.83 22.8
48 99 7.76 23.1
49 99 7.72 22.9
52 98 7.69 22.9
55 97 7.41 23.0
57 97 7.43 23.1
59 97 7.75 23.0
62 98 7.61 23.2
64 96 7.49 23.1
66 97 7.41 23.2
69 99 7.44 23.1
Mean 95 7.64 22.9
SD 3.07 0.14 0.14
Min. 90 7.41 22.6
Max. 100 7.84 23.2

Mean 95 7.62 22.8
SD 2.58 0.10 0.16
Min. 92 7.42 22.5
Max. 100 7.81 23.1
SD = Standard deviation
Min. = Minimum value
Max. = Maximum value

For the measured values of temperature, oxygen and pH of the test concentration 15.9 µg a.i./L see the following window "DIssolved oxygen".

Dissolved oxygen:

Water Parameters in the Test Item Concentration 15.9 µg a.i./L
Study Day
O2-saturation
[%] pH value
Temperature
[°C]
0 95 7.49 22.9
1 93 7.42 22.7
3 93 7.48 22.5
6 93 7.62 22.8
8 92 7.60 22.7
10 92 7.51 22.9
13 92 7.68 22.9
15 93 7.63 22.8
17 95 7.66 22.6
20 95 7.77 22.8
22 94 7.57 22.7
24 95 7.73 22.7
27 94 7.76 22.7
29 94 7.63 22.6
31 93 7.63 22.8
34 93 7.64 22.7
36 94 7.69 22.7
37 93 7.68 22.7
42 100 7.60 22.8
43 100 7.51 22.8
45 98 7.81 23.1
48 99 7.74 22.8
49 99 7.69 23.0
52 98 7.71 23.0
55 97 7.47 23.1
57 96 7.47 23.0
59 97 7.70 23.0
62 98 7.60 23.0
64 97 7.57 23.0
66 98 7.47 23.1
69 99 7.60 23.1
Mean 95 7.62 22.8
SD 2.58 0.10 0.16
Min. 92 7.42 22.5
Max. 100 7.81 23.1
SD = Standard deviation
Min. = Minimum value
Max. = Maximum value

For the measured oxygen values in the solvent control and the concentration 3.97 µg a.i./L see the window "pH" above.

TOC:

Total Organic Carbon (TOC) and Dissolved organic Carbon

The total organic carbon (TOC) and the dissolved organic carbon (DOC) were sampled and analyzed per test group and solvent control once per week on study days -2, -1, 3, 7, 14, 21, 27, 34, 43, 51, 56 and 63.
Throughout the uptake phase the mean measured TOC values were 15.1 mg C/L for the solvent control, 12.8 mg C/L for the concentration level of 3.97 µg a.i./L and 16.5 mg C/L for the concentration level of 15.9 µg a.i./L, respectively. The nominal carbon content of the solvent methanol in the test media was 14.8 mg C/L (corresponding to 0.050 mL/L) during uptake phase. The measured TOC/DOC values came from the application of the solvent.
Measured TOC/DOC values during depuration phase (no additional solvent) were nearby or below the limit of quantification (2 mg C/L). For results, see the
table below.

Total Organic Carbon in the Test Vessels
Uptake Phase
Study Day TOC [mg/L] DOC [mg/L]
Solvent Control 5 µg/L 20 µg/L Solvent Control 5 µg/L 20 µg/L
-2 15.5 10.4 16.3 16.1 11.4 17.2
-1 16.2 11.4 17.3 15.6 10.2 16.1
3 15.5 14.8 16.7 15.3 14.5 16.6
7 15.1 12.4 15.9 15.7 12.3 16.3
14 14.2 11.3 17.2 14.5 11.6 17.3
21 16.9 10.2 16.9 16.8 10.1 16.7
27 15.1 15.5 15.4 15.1 15.5 15.4
34 12.4 16.7 16.4 12.0 16.4 15.9
Mean 15.1 12.8 16.5 15.1 12.8 16.4
SD 1.35 2.50 0.67 1.45 2.42 0.65
Min. 12.4 10.2 15.4 12.0 10.1 15.4
Max. 16.9 16.7 17.3 16.8 16.4 17.3
Depuration Phase
Study Day TOC [mg/L] DOC [mg/L]
Solvent Control 5 µg/L 20 µg/L Solvent Control 5 µg/L 20 µg/L
43 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ
51 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ
56 < LOQ < LOQ 2.16 2.26 2.02 2.02
63 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ
LOQ = Limit of Quantification (2 mg C/L) SD = Standard deviation
Min. = Minimum value Max. = Maximum value

Details on test conditions:

Test design, test concentration
A flow-through exposure design was carried out. Three groups of fish were tested: two concentrations of the test item and one solvent control group. The test concentrations were 5 and 20 µg/L. The concentrations are based on the toxicity data of an acute toxicity test with zebrafish (LC50 (96 h) = 3.48 mg/L, based on geometric mean measured concentrations) SCHEERBAUM, 2009.
Membrane piston pumps provided the water flow-through. Precision syringe pumps were used for the introduction of the stock solution. The stock solution and the dilution water were mixed in a mixing chamber by magnetic stirring before passing the test aquaria. The accuracy of the water flow-through was checked prior to the start of the exposure. The variation of flow rates was less than ± 10 %.
Water exchange in the test aquaria (approx. Vol. 35 L) was 5 times per day (7.29 L/h).

Stock solutions The concentrations of the stock solutions for preparation of the test concentrations and flow rate data are listed in the table below.

Dilution Table of the Stock Solutions
Nominal
test item conc. Dilution water flow rate Stock solution
Flow rate Concentration
[µg/L] [L/h] [µL/h] [mg/L]
20 7.29 364.6 400
5 7.29 364.6 100
Solvent control 7.29 364.6 ---

Solvent
Methanol (Sigma, purity ≥ 99.9 % for HPLC, gradient grade; batch: STBH 9138) was used as solvent with 0.050 mL per L test medium.

Solvent control
A solvent control was tested with one replicate containing the same solvent concentration as the test concentration. A control with test medium only was not tested for animal welfare reasons. The concentration of the solvent (50 µL, which is below the maximum concentration of 100 µL) was not expected to have an effect on the fish.
Equilibration period An equilibration period of about 6 weeks days was carried out prior to start of the exposure.

Number of test fish
85 fish per group were inserted. The fish were taken from a pool larger than needed for the study to ensure quality of the organisms. For the whole study, 255 fish were used.

Loading The loading did not exceed a range of 0.1 to 1.0 g of fish (wet weight) per liter per day.

Introduction of fish The fish were introduced randomly to the test groups.

Feeding of test fish The fish were fed daily: two feedings on working days and one feeding on non-working days. 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.

Uptake phase Over a period of 42 days the fish were exposed to the test item.

Depuration phase A depuration phase of 28 days was carried out after the uptake phase.

Test vessel Glass aquaria covered by glass tops were used.

Test volume About 35 L

Replicates One replicate per test group

Aeration The test vessels were aerated.

Dilution water Same as used for holding.

Water temperature 20 – 25 °C

Dissolved oxygen Not less than 60 % of air saturation value.
concentration

Light intensity 7 – 750 Lux

Photoperiod A daily photoperiod of 16 h light / 8 h dark was provided.

Cleaning Uneaten food and feces were siphoned from the test vessels based upon visual inspection: twice on working days and once on non-working days. Cleaning was waived if no fecal matter or uneaten food was visible. Care was taken not to injure the test organisms.

Nominal and measured concentrations:

During the uptake phase, two groups of fish were exposed to the test item at a nominal concentration of 5 and 20 µg test item/L, corresponding to 3.97 and 15.9 µg a.i./L. After 42 days of uptake the fish were then transferred to a medium free of the test item (depuration phase) for 28 days. In parallel a solvent control was tested containing the same solvent concentration as the test concentrations during uptake.
The test item with a certified amount of the active ingredient of 79.3% (c-chain distribution: C16_ATQ 0.09 %, C18_ATQ 6.21 %, C20_ATQ 12.89 %, C22_ATQ 78.15 % and C24_ATQ 2.66%) was quantitatively analyzed for the main compounds C18_ATQ, C20_ATQ and C22_ATQ.
Uptake phase:

C18_ATQ:
The aqueous concentrations of the test concentration of nominal 3.97 µg a.i./L were in the range of 44 – 119 % of the nominal value throughout the uptake phase. Since the measured concentrations were partly below the nominal concentration, a time weighted average mean concentration was calculated (2.85 µg C18_ATQ/L) and used for BCF calculations.
The aqueous concentrations of the test concentration of nominal 15.9 µg a.i./L were in the range of 87 – 163 % of the nominal value throughout the uptake phase. Since the measured concentrations were partly above the nominal concentration, a time weighted average mean concentration was calculated (15.8 µg C18_ATQ/L) and used for BCF calculations.
An increase of the C18_ATQ concentration in fish was observed at this test concentration, a mean value of 61.6 mg C18_ATQ / kg fish was measured at uptake day 42. No steady-state phase was reached. Uptake rate constants were calculated.

For C20_ATQ and C22_ATQ see "Any other information on materials and methods incl. tables".

Reference substance (positive control):

Details on estimation of bioconcentration:

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 compounds C18_ATQ, C20_ATQ and C22_ATQ in water during uptake phase. The lipid normalized (and growth corrected) BCFKgL is based on the mean lipid content of fish per test group (solvent control 10%, 3,97 µg a.i./L 10.2% and 15.9 µg a.i./L 11.0%).

Prior to the analysis the fish data were transformed with the Box-Cox power transformation for fitting of the data to the curve (see part 5.4). The optimal lambda values were estimated by observation of best fitting of fish data to the uptake and depuration curve.
The best fitting lambda values were estimated to be:
C18_ATQ: λ = 0.3 (3.97 µg a.i./L) and λ = 0.7 (15.9 µg a.i./L)
C20_ATQ: λ = 0.5 (3.97 µg a.i./L) and λ = 0.8 (15.9 µg a.i./L)
C22_ATQ: λ = 0.3 (3.97 µg a.i./L) and λ = 0.4 (15.9 µg a.i./L)
Further details are given in part 14.2 to 14.7.

The kinetic bioconcentration factors for the test item compounds C18_ATQ, C20_ATQ and C22_ATQ were calculated as given in the test guideline and OECD guidance document 264 by non-linear regression with simultaneous fitting of the uptake and depuration rates (see part 5.4), 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 [L*kg-1*d-1]
k2 = depuration rate constant [d-1]
kg = overall growth rate[d-1]
Ln = Mean lipid content of fish / Lipid normalization factor

BCFK and BCFKGL of C18_ATQ (3.97 and 15.9 µg a.i./L)
3.97 µg a.i./L

〖BCF〗_(K )= 574.8/0.08534=6735 L*〖kg〗^(-1)

〖BCF〗_(Kg )= 574.8/(0.08534 - 0.00963)= 7592 L*〖kg〗^(-1)

〖BCF〗_KgL=0.05/0.102 ·7592=3722 L*〖kg〗^(-1)
15.9 µg a.i./L
〖BCF〗_(K )= 455.0/0.1134=4012 L*〖kg〗^(-1)

〖BCF〗_(Kg )= 455.0/(0.1134 - 0.00963)= 4385 L*〖kg〗^(-1)

〖BCF〗_KgL=0.05/0.110 ·4385=1993 L*〖kg〗^(-1)

BCFK and BCFKGL of C20_ATQ (3.97 and 15.9 µg a.i./L)

3.97 µg a.i./L

〖BCF〗_(K )= 277.7/0.07968=3486 L*〖kg〗^(-1)

〖BCF〗_(Kg )= 277.7/(0.07968 - 0.00963)= 3964 L*〖kg〗^(-1)

〖BCF〗_Kgl=0.05/0.102 ·3964=1943 L*〖kg〗^(-1)
15.9 µg a.i./L

〖BCF〗_(K )= 248.8/0.08089=3076 L*〖kg〗^(-1)

〖BCF〗_(Kg )= 248.8/(0.08089 - 0.00963)= 3491 L*〖kg〗^(-1)

〖BCF〗_Kgl=0.05/0.110 ·3491=1587 L*〖kg〗^(-1)
BCFK and BCFKGL of C22_ATQ (3.97 and 15.9 µg a.i./L)

3.97 µg a.i./L

〖BCF〗_(K )= 130.3/0.09012=1446 L*〖kg〗^(-1)

〖BCF〗_(Kg )= 130.3/(0.09012 - 0.00963)= 1619 L*〖kg〗^(-1)

〖BCF〗_Kgl=0.05/0.102 ·3964=794 L*〖kg〗^(-1)
15.9 µg a.i./L

〖BCF〗_(K )= 79.52/0.08161=974 L*〖kg〗^(-1)

〖BCF〗_(Kg )= 79.52/(0.08161 - 0.00963)= 1105 L*〖kg〗^(-1)

〖BCF〗_Kgl=0.05/0.110 ·1105=502 L*〖kg〗^(-1)

Lipid content:

ca. 12.1 %

Time point:

start of exposure

Remarks on result:

other: Stock population

Lipid content:

ca. 9.62 %

Time point:

end of exposure

Remarks on result:

other: Control fish

Lipid content:

ca. 8.45 %

Time point:

other: End of depuration

Remarks on result:

other: Control fish

Lipid content:

ca. 10.8 %

Time point:

end of exposure

Remarks on result:

other: 3.97 µg/L

Lipid content:

ca. 9.65 %

Time point:

other: End of depuration

Remarks on result:

other: 3.97 µg/L

Lipid content:

ca. 11.6 %

Time point:

end of exposure

Remarks on result:

other: 15.9 µg/L

Lipid content:

ca. 10.5 %

Time point:

other: End of depuration

Remarks on result:

other: 15.9 µg/L

Key result

Conc. / dose:

ca. 15.9 µg/L

Type:

other: BCF KGL

Value:

ca. 351 L/kg

Basis:

other: filet

Calculation basis:

other: kinetic, normalized for growth and lipid

Remarks on result:

other: C22_ATQ

Key result

Conc. / dose:

ca. 15.9 µg/L

Type:

other: BCF KGL

Value:

ca. 920 L/kg

Basis:

other: filet

Calculation basis:

other: kinetic, normalized for growth and lipid

Remarks on result:

other: C20_ATQ

Key result

Conc. / dose:

ca. 15.9 µg/L

Type:

other: BCF KGL

Value:

ca. 957 L/kg

Basis:

other: filet

Calculation basis:

other: kinetic, normalized for growth and lipid

Remarks on result:

other: C18_ATQ

Key result

Conc. / dose:

ca. 3.97 µg/L

Type:

other: BCF KGL

Value:

ca. 691 L/kg

Basis:

other: filet

Calculation basis:

other: kinetic, normalized for growth and lipid

Remarks on result:

other: C22_ATQ

Key result

Conc. / dose:

ca. 3.97 µg/L

Type:

other: BCF KGL

Value:

ca. 1 263 L/kg

Basis:

other: filet

Calculation basis:

other: kinetic, normalized for growth and lipid

Remarks on result:

other: C20_ATQ

Key result

Conc. / dose:

ca. 3.97 µg/L

Type:

other: BCF KGL

Value:

ca. 1 787 L/kg

Basis:

other: Filet

Calculation basis:

other: kinetic, normalized for growth and lipid

Remarks on result:

other: C18_ATQ

Conc. / dose:

ca. 3.97 µg/L

Type:

other: BCF KGL

Value:

ca. 3 722 L/kg

Basis:

whole body w.w.

Calculation basis:

other: kinetic, normalized for growth and lipid

Remarks on result:

other: C18_ATQ

Conc. / dose:

ca. 15.9 µg/L

Type:

other: BCF KGL

Value:

ca. 1 993 L/kg

Basis:

whole body w.w.

Calculation basis:

other: kinetic, normalized for growth and lipid

Remarks on result:

other: C18_ATQ

Conc. / dose:

ca. 3.97 µg/L

Type:

other: BCF KGL

Value:

ca. 1 943 L/kg

Basis:

whole body w.w.

Calculation basis:

other: kinetic, normalized for growth and lipid

Remarks on result:

other:

Remarks:

C20_ATQ

Conc. / dose:

ca. 15.9 µg/L

Type:

other: BCF KGL

Value:

ca. 1 587 L/kg

Basis:

whole body w.w.

Calculation basis:

other: kinetic, normalized for growth and lipid

Remarks on result:

other:

Remarks:

C20_ATQ

Conc. / dose:

ca. 3.97 µg/L

Type:

other: BCF KGL

Value:

ca. 794 L/kg

Basis:

whole body w.w.

Calculation basis:

other: kinetic, normalized for growth and lipid

Remarks on result:

other: C22_ATQ

Conc. / dose:

ca. 15.9 µg/L

Type:

other: BCF KGL

Value:

ca. 502 L/kg

Basis:

whole body w.w.

Calculation basis:

other: kinetic, normalized for growth and lipid

Remarks on result:

other: C22_ATQ

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

28 d

Remarks on result:

other: 9.15 / 3,97 µg/L C18_ATQ

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

28 d

Remarks on result:

other: 6.68 / 15.9 µg/L C18_ATQ

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

28 d

Remarks on result:

other: 9.89 / 3.97 µg/L C20_ATQ

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

28 d

Remarks on result:

other: 9.73 / 15.9 µg/L C20_ATQ

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

28 d

Remarks on result:

other: 8.61 / 3.97 µg/L C22_ATQ

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

28 d

Remarks on result:

other: 9.63 / 15.9 µg/L C22_ATQ

Key result

Rate constant:

growth rate constant (d-1)

Value:

0.01

Key result

Rate constant:

growth-corrected depuration rate constant (d-1)

Value:

0.076

Remarks on result:

other: 3.97 µg/L; C18_ATQ

Key result

Rate constant:

growth-corrected depuration rate constant (d-1)

Value:

0.104

Remarks on result:

other: 15.9 µg/L; C18_ATQ

Key result

Rate constant:

growth-corrected depuration rate constant (d-1)

Value:

0.07

Remarks on result:

other: 3.97 µg/L; C20_ATQ

Key result

Rate constant:

growth-corrected depuration rate constant (d-1)

Value:

0.071

Remarks on result:

other: 15.9 µg/L; C20_ATQ

Key result

Rate constant:

growth-corrected depuration rate constant (d-1)

Value:

0.081

Remarks on result:

other: 3.97 µg/L; C22_ATQ

Key result

Rate constant:

growth-corrected depuration rate constant (d-1)

Value:

0.072

Remarks on result:

other: 15.9 µg/L; C22_ATQ

Details on kinetic parameters:

- Uptake rate constant k1 [L*kg-1*day-1]:
C18_ATQ:
3.97 µg/L: 574.8
15.9 µg/L 455.0
C20_ATQ:
3.97 µg/L 277.7
15.9 µg/L 248.8
C22_ATQ:
3.97 µg/L 130.3
15.9 µg/L 79.52

- Depuration rate constant k(2) [day-1]:
C18_ATQ:
3.97 µg/L 0.08534
15.9 µg/L 0.1134
C20_ATQ:
3.97 µg/L 0.07968
15.9 µg/L 0.08089
C22_ATQ:
3.97 µg/L 0.09012
15.9 µg/L 0.08161

Details on results:

Fish Observations, Mortality and non-lethal Effects

For all test groups the mortality was less than 10% at the end of the test. One dead fish was observed and removed on day 40 of the uptake phase in the nominal concentration of 15.9 µg a.i./L. At the end of the study the number of remaining fish was counted. In the nominal concentration of 15.9 µg a.i./L two fish were missing. After removing the test aquaria and the flow through system two fish were found dead outside the aquarium. Probably these fish skipped out of the aquaria through to a small opening of covering lid. Nevertheless the consideration of these fish in mortality calculations would result in 3.53% (< 10%) mortality for this test concentration.
No non-lethal effects (morphological and behavioral) were found in the test item group and in the solvent control group.

Feeding Behavior

No anomalous feeding behavior was observed throughout the study in any of the test groups.

Growth Rates

The initial mean wet weight and length of 10 representative fish sampled from the stock fish at test start were 160 mg and 2.72 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 256 mg and the mean length increased up to 3.06 cm.
Weight data of the control and test item groups were converted to natural logs and plotted vs. time (days). 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) was calculated as the overall slope of the linear correlation.

Reported statistics:

Evaluation

All calculations were done according to the test guideline OECD 305 and the OECD guidance document No. 264.

Test item (compound) The individual fish concentration data were transformed for calculation of uptake and elimination rates using the
Box-Cox power transformation with the following lambdas:
C18_ATQ: lambda 0.3 (3.97 µg a.i./L) and 0.7 (15.9 µg a.i./L)
C20_ATQ: lambda 0.5 (3.97 µg a.i./L) and 0.8 (15.9 µg a.i./L)
C22_ATQ: lambda 0.3 (3.97 µg a.i./L) and 0.4 (15.9 µg a.i./L)
The optimal lambda values were estimated by observation of best fitting of fish data to the uptake and depuration curve.

The Box-Cox power transformation is defined as:

y^((λ) )= {█((y^((λ))-1)/y if λ ≠0@ln⁡〖(y) if〗 λ=0)┤

Uptake / Elimination rates The uptake (k1) and elimination rate (k2) constants as a function
(k1, k2 and k2g) of the total wet weights were determined (via non-linear parameter estimation methods).
A growth correction was included for calculation of the growth-corrected depuration rate (k2g).

Bioconcentration factors

BCFK and BCFKGL
The kinetic bioconcentration factors were calculated by non-linear regression with simultaneous fitting of the uptake and depuration rates per test item compound.

〖BCF〗_K= k_1/k_2

k1 = uptake rate constant
k2 = depuration rate constant

C_(fish ) (t)= {█(C_(water )·〖BCF〗_K ·(1- e^(-k_2·t) ), t≤ t_dep@C_water·〖BCF〗_K ·(e^(k_2·t_dep )-1)· e^(-k_2·t),t≥ t_dep )┤

Cfish = measured concentration in fish
Cwater = time-weighted average mean concentration during uptake

〖BCF〗_(Kg )= k_1/(k_2 - k_g )

BCFKg =growth corrected BCF
kg =growth corrected depuration rate k2

〖BCF〗_KgL= 0.05/L_n · 〖BCF〗_Kg

Ln = Mean lipid content of fish
BCFKgL =growth corrected and lipid normalized BCFK

Total Hardness

Residual Chlorine

Residual chlorine, measured once per week from the dilution water supply tank on study days 0, 7, 14, 21, 28, 35, 42, 49, 56 and 63 was < 0.01 mg/L.

Light intensity

Light intensity was measured at experimental starting on the surface of all test vessels and ranged from 104 to 134 lux (mean: 122 lux).

Calculations

Overview

A significant uptake of the analyzed compounds C18_ATQ, C20_ATQ and C22_ATQ of test item in fish was observed. The uptake / depuration rates and kinetic bioconcentration factors were calculated for these compounds, respectively. Nonlinear regression with simultaneous fitting of the uptake and depuration phase was used. Since no steady-state phase was reached during uptake, no steady-state bioconcentration factors were calculated. The kinetic bioconcentration factors have been calculated based on fish body weight and time-weighted average mean measured concentrations of the test item compounds C18_ATQ, C20_ATQ and C22_ATQ in water during uptake phase and were corrected for growth of the fish and the measured lipid content of fish (see part 9.4).

All calculations were done according to the test guideline OECD 305 and the OECD guidance document No. 264.

Uptake Phase

C18_ATQ:

The aqueous concentrations of the test concentration of nominal 3.97 µg a.i./L were in the range of 44 – 119 % of the nominal value throughout the uptake phase. Since the measured concentrations were partly below the nominal concentration, a time weighted average mean concentration was calculated (2.85 µg C18_ATQ/L) and used for BCF calculations.

An increase of the C18_ATQ concentration in fish was observed at this test concentration, a mean value of 21.2 mg C18_ATQ / kg fish was measured at uptake day 42. No steady-state phase was reached. Uptake rate constants were calculated.

The aqueous concentrations of the test concentration of nominal 15.9 µg a.i./L were in the range of 87 – 163 % of the nominal value throughout the uptake phase. Since the measured concentrations were partly above the nominal concentration, a time weighted average mean concentration was calculated (15.8 µg C18_ATQ/L) and used for BCF calculations.

An increase of the C18_ATQ concentration in fish was observed at this test concentration, a mean value of 61.6 mg C18_ATQ / kg fish was measured at uptake day 42. No steady-state phase was reached. Uptake rate constants were calculated.

C20_ATQ:

The aqueous concentrations of the test concentration of nominal 3.97 µg a.i./L were in the range of 73 – 121 % of the nominal value throughout the uptake phase. Since the measured concentrations were partly not within the nominal range, a time weighted average mean concentration was calculated (3.81 µg C20_ATQ/L) and used for BCF calculations.

An increase of the C20_ATQ concentration in fish was observed at this test concentration, a mean value of 15.0 mg C20_ATQ / kg fish was measured at uptake day 42. No steady-state phase was reached. Uptake rate constants were calculated.

The aqueous concentrations of the test concentration of nominal 15.9 µg a.i./L were in the range of 86 – 182 % of the nominal value throughout the uptake phase. Since the measured concentrations were partly above the nominal concentration, a time weighted average mean concentration was calculated (18.5 µg C20_ATQ/L) and used for BCF calculations.

An increase of the C20_ATQ concentration in fish was observed at this test concentration, a mean value of 55.4 mg C20_ATQ / kg fish was measured at uptake day 42. No steady-state phase was reached. Uptake rate constants were calculated.

C22_ATQ:

The aqueous concentrations of the test concentration of nominal 3.97 µg a.i./L were in the range of 79 – 130 % of the nominal value throughout the uptake phase. Since the measured concentrations were partly not within the nominal range, a time weighted average mean concentration was calculated (4.13 µg C22_ATQ/L) and used for BCF calculations.

An increase of the C22_ATQ concentration in fish was observed at this test concentration, a mean value of 8.62 mg C22_ATQ / kg fish was measured at uptake day 42. No steady-state phase was reached. Uptake rate constants were calculated.

The aqueous concentrations of the test concentration of nominal 15.9 µg a.i./L were in the range of 88 – 168 % of the nominal value throughout the uptake phase. Since the measured concentrations were partly above the nominal concentration, a time weighted average mean concentration was calculated (19.1 µg C22_ATQ/L) and used for BCF calculations.

An increase of the C22_ATQ concentration in fish was observed at this test concentration, a mean value of 19.5 mg C22_ATQ / kg fish was measured at uptake day 42. No steady-state phase was reached. Uptake rate constants were calculated.

Depuration Phase

A depuration phase of 28 days was conducted. Observed concentrations of C18_ATQ, C20_ATQ and C22_ATQ in fish decreased over time and depuration rate constants were calculated

Further analysis for sampling days uptake days 28 and 42 as well as depuration days 1 and 4 were carried out with the remaining sampled fish from the test item groups. One fish per sampling date was exemplarily chosen for preparation. The distribution of the test item in fish was investigated, separating skin, fins and head from the remaining body, i.e. fillet and torso, named thereafter skin and body.

The sample preparation of both parts was executed in the same way as described in section 7.2.4. The volume of the extraction medium was adapted to the weighed amounts of the fish skin and body, respectively.

The measured values for skin and body of C18/C20/C22 ATQ are displayed in Table 60 to Table 62. The distribution is related to the total fish body weight.

The distribution of ATQ differs from the chain length (see Table 60, Table 61, Table 62 and Figure 102):

C18 ATQ: no uniform trend discernible

C20 ATQ: no uniform trend discernible

C22 ATQ: slightly more substance in the body

Table 60: Distribution of C18_ATQ in Skin and Body

a.i. concentrations: 3.97 µg/L and 15.9 µg/L

Sampling day	C18_ATQ
	3.97 µg test item/L (a.i.)				15.9 µg test item/L (a.i.)
		C _{total body weight} [mg/kg]	[%]	Sum C _{total body weight} [mg/kg]		C _{total body weight} [mg/kg]	[%]	Sum C _{total body weight} [mg/kg]
Uptake day 28	Skin	9.65	46	21.0	Skin	44.8	62	72.7
Uptake day 28	Body	11.3	54	21.0	Body	27.9	38	72.7
Uptake day 42	Skin	6.81	52	13.2	Skin	28.5	52	55.0
Uptake day 42	Body	6.37	48	13.2	Body	26.5	48	55.0
Depuration day 1	Skin	8.65	68	12.8	Skin	29.1	52	56.3
Depuration day 1	Body	4.17	32	12.8	Body	27.3	48	56.3
Depuration day 4	Skin	7.57	59	12.8	Skin	14.8	45	33.3
Depuration day 4	Body	5.27	41	12.8	Body	18.5	55	33.3

C _Body: Concentration of the test item (a.i.) in skin and body related to total fish body weight

Table 61: Distribution of C20_ATQ in Skin and Body

a.i. concentrations: 3.97 µg/L and 15.9 µg/L

Sampling day	C20_ATQ
	3.97 µg test item/L (a.i.)				15.9 µg test item/L (a.i.)
		C _{total body weight} [mg/kg]	[%]	Sum C _{total body weight} [mg/kg]		C _{total body weight} [mg/kg]	[%]	Sum C _{total body weight} [mg/kg]
Uptake day 28	Skin	4.49	46	9.72	Skin	17.9	12	156
Uptake day 28	Body	5.23	54	9.72	Body	138	88	156
Uptake day 42	Skin	2.68	35	7.58	Skin	132	42	316
Uptake day 42	Body	4.90	65	7.58	Body	184	58	316
Depuration day 1	Skin	4.93	59	8.29	Skin	158	46	342
Depuration day 1	Body	3.36	41	8.29	Body	184	54	342
Depuration day 4	Skin	3.21	54	5.97	Skin	86.3	43	202
Depuration day 4	Body	2.77	46	5.97	Body	116	57	202

Table 62: Distribution of C22_ATQ in Skin and Body

a.i. concentrations: 3.97 µg/L and 15.9 µg/L

Sampling day	C22_ATQ
	3.97 µg test item/L (a.i.)				15.9 µg test item/L (a.i.)
		C _{total body weight} [mg/kg]	[%]	Sum C _{total body weight} [mg/kg]		C _{total body weight} [mg/kg]	[%]	Sum C _{total body weight} [mg/kg]
Uptake day 28	Skin	0.619	13	4.73	Skin	2.45	34	7.23
Uptake day 28	Body	4.11	87	4.73	Body	4.78	66	7.23
Uptake day 42	Skin	0.489	13	3.72	Skin	2.40	23	10.4
Uptake day 42	Body	3.23	87	3.72	Body	7.96	77	10.4
Depuration day 1	Skin	1.22	31	3.92	Skin	2.61	20	12.8
Depuration day 1	Body	2.70	69	3.92	Body	10.2	80	12.8
Depuration day 4	Skin	0.707	25	2.85	Skin	1.31	29	4.52
Depuration day 4	Body	2.14	75	2.85	Body	3.21	71	4.52

C _Body: Concentration of the test item (a.i.) in skin and body related to total fish body weight

Validity criteria fulfilled:

yes

Conclusions:

A significant uptake of all analyzed compounds C18_ATQ, C20_ATQ and C22_ATQ of the test item in fish was observed. The uptake / depuration rates and kinetic bioconcentration factors were calculated for these compounds. Since no steady-state phase was reached during uptake, no steady-state bioconcentration factors were calculated. The kinetic bioconcentration factors have been calculated based on fish body weight and time-weighted average mean measured concentrations of the test item compounds C18_ATQ, C20_ATQ and C22_ATQ in water during uptake phase.
The kinetic bioconcentration factors BCFK (growth/lipid normalized BCFKGL), determined in this study with the test item Genamin KDMP are given in the report.

The additional analytical data regarding the distrubution in fish body (filet) and skin is available. Based on these data the BCF of all the chain-length are below 2000. A weighted BCF of 835 is used for chemical safety assessment in Chesar.

Executive summary:

Summary

A bioaccumulation study with zebrafish (Danio rerio) was conducted to determine the bioconcentration potential of Genamin KDMP (batch: DEG4440803). An exposure (uptake phase) of 42 days was carried out. The following depuration phase lasted 28 days.

The definitive study was conducted according to OECD Guideline 305 (2012) from 2019-03-12 to 2020-01-23 with the definitive exposure period from 2019-03-12 to 2019-05-21 at the test facility. A flow-through test with 3 groups (one solvent control group and two exposure group of nominal 5 and 20 µg test item/L) was carried out. During the uptake phase, two groups of fish were exposed to the test item at a nominal concentration of 5 and 20 µg test item/L, corresponding to 3.97 and 15.9 µg a.i./L. After 42 days of uptake the fish were then transferred to a medium free of the test item (depuration phase) for 28 days. In parallel a solvent control was tested containing the same solvent concentration as the test concentrations during uptake.

Analytical verification:

The test item with a certified amount of the active ingredient of 79.3% (c-chain distribution: C16_ATQ 0.09 %, C18_ATQ 6.21 %, C20_ATQ 12.89 %, C22_ATQ 78.15 % and C24_ATQ 2.66%) was quantitatively analyzed for the main compounds C18_ATQ, C20_ATQ and C22_ATQ. A validated analytical method according to SANCO 3029/99 rev.4 (2000) was implemented for this purpose. The test item contains further constituents but their amounts in the test item and the application concentrations were too low for a reliable analytical monitoring according to the criteria of SANCO 3029/99 rev.4 (2000). For this reason, an additional qualitative assay was implemented to monitor constituents and possible degradation products showing an uptake in fish during the uptake phase. Identified constituents (abbreviations for them are C24H51N_1, C24H51N_2 and C25H51N) were subsequently analysed with this qualitative assay to monitor the depuration behavior of these compounds. No endpoints according to the guideline could be deduced for these constituents because their presence in the matrix fish were close to the detection limit of the analytical system and no quantitative evaluation was therefore possible. Only the chromatographic areas of these constituents were determined and plotted during the course of the study to give an illustration for their uptake and depuration behavior. Nevertheless, the combination of both assays (quantitative and qualitative) allowed the investigation of constituents of the test item down to 0.1%.

Four fish samples were taken from each tank on days 0.3, 1, 2, 4, 7, 14, 28 and 42 of the uptake phase as well as on days 0.3, 1, 2, 4, 7, 14 and 28 of the depuration phase.

Aqueous phase samples were taken and analyzed on uptake phase days 0.3, 1, 2, 3, 4, 7, 14, 28, 35 and 42 and on days 0.3, 1 and 2 of the depuration phase. Sampling and analysis of water samples after depuration day 2 were stopped, since the measured concentrations on depuration day 2 were already < LOQ. 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 42) and at the end of the depuration phase (depuration day 28). 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.

Evaluation:

Results:

Uptake phase:

C18_ATQ:

C20_ATQ:

C22_ATQ:

Depuration phase:

A depuration phase of 28 days was conducted. Observed concentrations of C18_ATQ, C20_ATQ and C22_ATQ in fish decreased over time and depuration rate constants were calculated.

Bioconcentration factors:

The kinetic bioconcentration factors BCFK (BCFKGL growth and lipid normalized), determined in this study with the test item Genamin KDMP are given in the table below.

All calculated BCFKGL values show a bioaccumulating potential (C20_ATQ and C22_ATQ) as well as a high bioaccumulating potential (C18_ATQ) of the analyzed compounds of the test item.

Bioconcentration Factors of C18_ATQ, C20_ATQ and C22_ATQ of the Test Item

All calculations were based on the time-weighted average mean concentration per test item compound [µg/L]

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

Active ingredient Test concentration BCFK1) BCFKGL2)

Nominal TWA* [L * kg-1] [L * kg-1]

[µg a.i./L] [µg a.i./L]

C18_ATQ 3.97 2.85 6735 (5608 – 7863) 3722

15.9 15.8 4012 (3678 – 4345) 1993

C20_ATQ 3.97 3.81 3486 (2976 – 3995) 1943

15.9 18.5 3076 (2844 – 3307) 1587

C22_ATQ 3.97 4.13 1446 (1179 – 1713) 794

15.9 19.1 974 (853 – 1095) 502

* = Time-weighted-average concentration

1) = Based on body weight of fish

2) = Based on body weight of fish and normalized for growth and lipid content

Overview of Uptake and Depuration Rates, Growth Rate, Growth Corrected Depuration Rates and Lipid Normalization Factor

All calculations were based on the time-weighted average mean concentration per test item compound [µg/L]

Test item compound Nominal test Growth rate Uptake rate Depuration Growth-corrected Lipid normalization

concentration kg k1 rate k2 depuration rate factor

[µg a.i./L] [day-1] [L * kg-1 * day-1] [day-1] k2g [day-1]

C18_ATQ 3.97 0.00963 574.8 0.08534 0.0757 0.102

15.9 455.0 0.1134 0.104 0.110

C20_ATQ 3.97 277.7 0.07968 0.0701 0.102

15.9 248.8 0.08089 0.0713 0.110

C22_ATQ 3.97 130.3 0.09012 0.0805 0.102

15.9 79.52 0.08161 0.0720 0.110

Validity Criteria:

The validity criteria regarding temperature, oxygen saturation and mortality were fulfilled. From uptake day 0 to uptake day 42 the measured concentrations of the active ingredients of the test item were in the range of 44 to 119% and 87 to 163% (C18_ATQ), 73 to 121% and 86 to 182% (C20_ATQ) and 79 to 130% and 88 to 168% (C22_ATQ) of the nominal concentrations, respectively. Since measured concentrations were not constant within the nominal ranges of the test concentrations, the time weighted average mean measured concentrations were calculated for these compounds of both test concentrations and used for BCF calculations.

The measured values for skin and body of C18/C20/C22 ATQ are displayed in Table 60 to Table 62. The distribution is related to the total fish body weight.

The distribution of ATQ differs from the chain length (see Table 60, Table 61, Table 62 and Figure 102):

C18 ATQ: no uniform trend discernible

C20 ATQ: no uniform trend discernible

C22 ATQ: slightly more substance in the body

Table 60: Distribution of C18_ATQ in Skin and Body

a.i. concentrations: 3.97 µg/L and 15.9 µg/L

Sampling day	C18_ATQ
	3.97 µg test item/L (a.i.)				15.9 µg test item/L (a.i.)
		C _{total body weight} [mg/kg]	[%]	Sum C _{total body weight} [mg/kg]		C _{total body weight} [mg/kg]	[%]	Sum C _{total body weight} [mg/kg]
Uptake day 28	Skin	9.65	46	21.0	Skin	44.8	62	72.7
Uptake day 28	Body	11.3	54	21.0	Body	27.9	38	72.7
Uptake day 42	Skin	6.81	52	13.2	Skin	28.5	52	55.0
Uptake day 42	Body	6.37	48	13.2	Body	26.5	48	55.0
Depuration day 1	Skin	8.65	68	12.8	Skin	29.1	52	56.3
Depuration day 1	Body	4.17	32	12.8	Body	27.3	48	56.3
Depuration day 4	Skin	7.57	59	12.8	Skin	14.8	45	33.3
Depuration day 4	Body	5.27	41	12.8	Body	18.5	55	33.3

C _Body: Concentration of the test item (a.i.) in skin and body related to total fish body weight

Table 61: Distribution of C20_ATQ in Skin and Body

a.i. concentrations: 3.97 µg/L and 15.9 µg/L

Sampling day	C20_ATQ
	3.97 µg test item/L (a.i.)				15.9 µg test item/L (a.i.)
		C _{total body weight} [mg/kg]	[%]	Sum C _{total body weight} [mg/kg]		C _{total body weight} [mg/kg]	[%]	Sum C _{total body weight} [mg/kg]
Uptake day 28	Skin	4.49	46	9.72	Skin	17.9	12	156
Uptake day 28	Body	5.23	54	9.72	Body	138	88	156
Uptake day 42	Skin	2.68	35	7.58	Skin	132	42	316
Uptake day 42	Body	4.90	65	7.58	Body	184	58	316
Depuration day 1	Skin	4.93	59	8.29	Skin	158	46	342
Depuration day 1	Body	3.36	41	8.29	Body	184	54	342
Depuration day 4	Skin	3.21	54	5.97	Skin	86.3	43	202
Depuration day 4	Body	2.77	46	5.97	Body	116	57	202

Table 62: Distribution of C22_ATQ in Skin and Body

a.i. concentrations: 3.97 µg/L and 15.9 µg/L

Sampling day	C22_ATQ
	3.97 µg test item/L (a.i.)				15.9 µg test item/L (a.i.)
		C _{total body weight} [mg/kg]	[%]	Sum C _{total body weight} [mg/kg]		C _{total body weight} [mg/kg]	[%]	Sum C _{total body weight} [mg/kg]
Uptake day 28	Skin	0.619	13	4.73	Skin	2.45	34	7.23
Uptake day 28	Body	4.11	87	4.73	Body	4.78	66	7.23
Uptake day 42	Skin	0.489	13	3.72	Skin	2.40	23	10.4
Uptake day 42	Body	3.23	87	3.72	Body	7.96	77	10.4
Depuration day 1	Skin	1.22	31	3.92	Skin	2.61	20	12.8
Depuration day 1	Body	2.70	69	3.92	Body	10.2	80	12.8
Depuration day 4	Skin	0.707	25	2.85	Skin	1.31	29	4.52
Depuration day 4	Body	2.14	75	2.85	Body	3.21	71	4.52

C _Body: Concentration of the test item (a.i.) in skin and body related to total fish body weight

Description of key information

Bioconcentration factors:

The kinetic bioconcentration factors BCF_K (BCF_KGL growth and lipid normalized), determined in this study with the test item Genamin KDMP are given.

All calculated BCF_KGL values show a bioaccumulating potential (C20_ATQ and C22_ATQ) as well as a high bioaccumulating potential (C18_ATQ) of the analyzed compounds of the test item.

Further results on uptake and depuration rate constants, growth rate, growth corrected depuration rate, as well as the lipid normalization factor is presented as an overview in Table 2.

Bioconcentration Factors of C18_ATQ, C20_ATQ and C22_ATQ of the Test Item

All calculations were based on the time-weighted average mean concentration per test item compound [µg/L]

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

Active ingredient	Test concentration				BCF_K³⁾ [L * kg^-1]	BCF_KGL⁴⁾[L * kg^-1]
Active ingredient	Nominal test item [µg/L]	Nominal total¹⁾ active ingredient [µg a.i./L]	Nominal single²⁾ active ingredient [µg a.i./L]	TWA* total active ingredient [µg a.i./L]	BCF_K³⁾ [L * kg^-1]	BCF_KGL⁴⁾[L * kg^-1]
C18_ATQ	5.00	3.97	0.246	2.85	6735 (5608 – 7863)	3722
C18_ATQ	20.0	15.9	0.985	15.8	4012 (3678 – 4345)	1993
C20_ATQ	5.00	3.97	0.511	3.81	3486 (2976 – 3995)	1943
C20_ATQ	20.0	15.9	2.04	18.5	3076 (2844 – 3307)	1587
C22_ATQ	5.00	3.97	3.10	4.13	1446 (1179 – 1713)	794
C22_ATQ	20.0	15.9	12.4	19.1	974 (853 – 1095)	502

* = Time-weighted-average concentration

1) = Based on the certified active content 79.3%

2) = Based on the % of c-chain distribution per active ingredient

3) = Based on body weight of fish

4) = Based on body weight of fish and normalized for growth and lipid content

Additionally weighted bioconcentrations factors were calculated for each test concentration. Results are given in Annex II: Further Evaluation.

Calculation of Weighted Bioconcentration Factors

Additionally the weighted bioconcentration factors were calculated for each test concentration.

The basis for the calculation was the given c-chain distribution of the analysed and evaluated main active ingredients C_18ATQ 6.21%, C_20ATQ_12.89% and C22_ATQ 78.15%.

For results see the table below:

: Weighted Bioconcentration Factors of the Test Item

All calculations were based on the time-weighted average mean concentration per test item compound [µg/L]

Nominal test concentration [µg/L]		5.00	20.0
BCF_KGL	C18_ATQ	3722	1993
	C20_ATQ	1943	1587
	C22_ATQ	794	502
Weighted BCF_KGL [L * kg^-1]		1133	741

Based on the analytical Data for Distribution in Fish the following BCF can be calculated.

Detailed information regarding the distribution are listed and named in the robust study summary.

Nominal test concentration [µg/L]		5.00	20.0
BCF_KGL	C18_ATQ	1787	957
	C20_ATQ	1263	920
	C22_ATQ	691	351
Weighted BCF_KGL [L * kg^-1]		835	465

All BCFs of the 3 chain lengths and of the two concentrations are below 2000. The weighted BCF of 835 will be used as Key value for chemical safety assessment.

Key value for chemical safety assessment

BCF (aquatic species):: 835 L/kg ww

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