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Bioaccumulation: aquatic / sediment

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Reference
Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study period: September 22, 2017 to March 5, 2018; Exposure period: September 22, 2017 till December 5, 2017
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:
OECD Guidelines for Testing of Chemicals No.305 "Bioaccumulation in Fish: Aqueous and Dietary Exposure" (2012)
GLP compliance:
yes (incl. certificate)
Radiolabelling:
no
Details on sampling:
Analysis of Test Substance and measurement of lipid content
1) The test substance in the test water
The test water from each concentration level was sampled on days 0 (before fish were introduced),7, 14, 21, 28, 35 ,48, 54 and 61 of the exposure period, and analyzed for the test substance.

Pretreatment Method for Analysis of Test water
The test substance in the test water was analyzed according to the following procedures:
Test water 50 mL (high concentration level), 500 mL (low concentration level)
Pass sample throngh an Empore disk Anion_SR (*1)+SDB_XD (*'2) 47mm with aspiration (Pre-conditioned with approx, 5 mL tetrahydrofuran and approx, 10 mL ultrapure water)
Discard the filtered water phase.
Extract substance from Empore disk using: Tetrahydrofuran /2% formic acid solution ('3) = 1/1 (v/v) (4*) approx, 4.8 mL x 4
Fill extract upt to 20 mL with Tetrahydrofuran /2% formic acid solution('3) = 1/1 (v/v)4*
Perform LC-MS/MS analysis

* 1: Empore disk Anion-SR: 3M, Empore Extraction Disk Anion-SR
*2: Empore disk SDB-XD: 3M, Empore Extraction Disk Styrenedivinylbenzene-XD
*3: 2% formic acid solution: 5 mL of formic acid is added to 245 mL of ultrapure water.
*4: Tetrahydrofuran I 2% formic acid solution =111 (v/v):
Tetrahydrofuran is mixed with same amount of 2% formic acid solution,

2) The test substance in the test fish
Four fish were randomly sampled from each concentration level on days 7, 14, 28, 48 and 61 of the exposure period, and analyzed two by two.
The fish remaining from analysis at the end of the exposure period and back-up samples were stored in a freezer for three months, and may be discarded after that period, unless additional analysis is necessary.

Pretreatment Method for Analysis of Test Fish
The test substance in the test fish was analyzed according to the following procedures:
Weigh two fish and Cut into pieces with scissors
Homogenize 4 times for apnrox. 1 min at approx. 8000 rpm.
2 g portion of the fish slurry (Back up sample taken * 1 (> 1 g, stored in a freezer))
4% formic acid tetrahydrofuran solution (*2) 20 mL
Homogenize for approx. 3 min at approx. 8000 rpm.
Filter with suction through a filter paper in a 40-mm Kiriyama funnel
4% formic acid tetrahydrofurull solution layer
The residue is extracted again with 4% formic acid tetrahydrofuran solulion ('2) 20 mL by Homogenizing for approx. 3 min at approx. 8000 rpm and Filtering with suction through a filter paper in a 40-mm Kiryama funnel
Fill up to 50 mL with 4% formic acid tetrahydrofuran solution
From this solution 2.5 mL (Sampling factor: 20) and dilute to 10 mL with with ultra pure waler
Pass through Inertsep SAX (*3) + Inelisep PLS-2 (*4) (pre-conditioned with approx. 5 mL tetrahydrofuran and apprrox. 10 mL ultra pure water
Elute with Tetrahydrofuran / 2% fonnic acid solution~5=1/1(v/v) (*6) appox.4.8 mL
Fill up to 5 mL with Tetrahydrofuran /2% formic acid solution (*5) = 1/1 (v/v) (*6)
and perform LC-MS/MS analysis

* 1: Back up samples were preserved for the pmpose ofusillg in case where additiollDl measurement of the lipid content or analysis of the test substance is required.
*2: 4% fonnic acid tetrahydrofiuan solution: 8 mL of ibm ric acid is added to 192 mL of tetrahydrofuran.
*3: InertSep SAX: GL Sciences, InertSep Slim SAX Cartridge
*4: InertSep PLS-2: GL Sciences. InertSep Slim PLS-2 Cartridge
*5: 2% formic acid solution: 5 mL formic acid is added to 245 mL of ultrapure water.
*6: Tetrahydrofuran / 2% formic acid solution = 1/1 (v/v): Tetrahydrofurac is mixed with same amount of 2% formic acid solulion.

3) Lipid content
At the start and end of the exposure period, lipid contents of the fish were measured. Five fish were sampled from the acclimation aquarium at the start of the exposure period, and three fish each were sampled from the test aquariums at the end of the exposure period. In these samples, lipid contents of three fish were sampled from the acclimation aquarium and the control aquarium were measured individually (n=3).
The two fish remaining from analysis that were sampled upon initiation of exposure and three fish each sampled from each concentration level upon completion of exposure were used as back-up samples.

Lipid content procedure:
Weigh one fish and cut into pieces with scissors.
add Sodium sulfate anhydrous 6-fold of fish weight and more in case of incomplete dehydration
Mix and dehydrate
Leave it for more than 30 min (mix as necessary)
add approx 80 mL n-hexane and homogenize for approx. 5 min at approx. 8000 rpm
Filter with suction through filter paper in a 60-mm Kiriyama funnel
collect the filtrate in rotary evaporation vessel and repeat the extraction step with the Residue using again approx. 80 mL n-hexane
combine the filtrates in vessel and repeat the extraction step with the Residue using again approx. 20 mL n-hexane and discard the residue
combine the filtrates in vessel and reduce the volume in rotary evaporator at 45 deg C to about 10 mL
Transfer to a preweighed* smaller rotary evaporation flask and rinse the rotary evaporation vessel with 5 mL n-hexane twice and transfer to the preweighed flask
Evaporate to dryness at 45 deg C.
Purge the content of the small rotary evaporation flask with nitrogen.
Dry in desiccator for 4 hours and weigh** to quantify the fatt content.

Equations: lipid weight (g) = lipid and flask weight (**) - flask weight (*)
lipid content (%) = lipid weight (g) / fish wet weight (g) x 100
Vehicle:
yes
Remarks:
Tetrahydrofuran
Details on preparation of test solutions, spiked fish food or sediment:
1) Supply of test water: Continuous flow-through dilution system
2) Exposure level (nominal concentrations of the test substance)
High concentration level: 0.05 mg/L
[Vehicle concentration: Tetrahydrofuran 25 ppm (v/v)]
Low concentration level: 0.005 mg/L
[Vehicle concentration: Tetrahydrofuran 25 ppm (v/v)]
Control: 0 mg/L
[Vehicle concentration: Tetrahydrofuran 25 ppm (v/v)]
3) Dilution water supply: 1600 L/day (Turnover rate: 32 volumes/day)
4) Feed solution supply: 40 mL/day

2) Preparation of Test Water
Each feed solution prepared in step 1) and the dilution water were delivered to a mixing glass tube through individual metering pumps with individually determined flow rates, which were then supplied continuously to respective test aquariums.
Test organisms (species):
Cyprinus carpio
Details on test organisms:
BCF study
Carp was selected for the test fish as it is recommended in the test guideline and could be satisfactorily maintained under the laboratory conditions such as water temperature, feeding and handling. Besides, a large number of healthy fish with convenient size were available in one batch.

Species: Carp (Cyprinus carpio)
Source: Kitamura-yourijou 12-388, Gunchiku, Yatsushiro, Kumamoto 866-0001, Japan
Date of purchase:April20, 2017
Lot number: 17-K-0420
Pre-acclimation:At the start of acclimation, abnormal fish were removed upon visual inspection. The remaining normal fish were then reared in an aquarium with flowing water.
Total length: 8±4cm
Body weight: Approximately 5 g
Age: Yearling fish

Acclimation
Aquarium number: C-6
Water temperature: 24±2°C
Period: September 4, 2017 to October 4, 2017
Food: Babygold, Kyorin
Feeding:

Type offood Floating and pellet type
Crude protein >32%
Crude fat ;::4.0%
Crude fiber <3.0%
Moisture <10%
Ash <12%
Phosphorus >0.7%

Mortality 1 week before testing: <5%
Medication during acclimation: None

Introduction of Fish
After ensuring the test substance concentrations in both test aquariums, the fish were introduced into the test system to start the exposure.
Management of Fish
Daily observations were made on the appearance, swimming and feeding behavior of the fish. Babygold, Kyorin was given daily equivalent to 1.5 % of the fish weight.

Condition of the Fish during the test
Surviving fish showed no abnormality in appearance, swimming or feeding behavior.
Therefore, the test fish were confirmed as being reared appropriately. No occurrence of mortality or abnonnality was observed.
Route of exposure:
aqueous
Justification for method:
aqueous exposure method used for following reason: The water solubility of the three constituent used for Quantification of the BCF were measured to be 11 mg/L for component D, 9.3 mg/L for component E and 5.7 mg/L for component F.
Remarks:
Nominal concentration: High concentration level: 0.05 mg/L (1 % of 96hr-LC50, less than water solubility) Low concentration level: 0.005 mg/L (0.1 % of 96hr-LC50, less than water solubility)
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
62 d
Hardness:
48~50 mg CaCO3/L
Test temperature:
23.9~24.4°C
pH:
7.1
Dissolved oxygen:
7.5 to 8.3 mg/L
TOC:
13.0~ 17.2 mg/L corrected for THF content of 16.8 mg/L
Salinity:
Fresh water
Conductivity:
-
Details on test conditions:
1) Preparation of Feed Solutions
High concentration level: Test substance. 2 g dissolved in Tetrahydrofuran
Dissolve in THF and fill up to 1000 mL ==> Concentration of test substance: 2000 mg/L
Low concentration level
100 mL of high concentration level diluted to 1000 mL with THF ==> Concentration of test substance: 200 mg/L
Control solution
Tetrahydrofuran

It was confirmed that the test substance in the feed solution is stable for 20 days.

Dilution Water
Dechlorinated water processed by treating Yokohama city tap water with an activated charcoal filter and sodium thiosulfate was used as the dilution water. Water quality
parameters of the dechlorinated water have been measured periodically (about every 6 months) by MC Evolve Technologies Corporation. As each measurement value met the criteria specified in the standard operating procedure of the testing facility, the quality of this water was considered to be acceptable for use as the dilution water.

5) Number offish (at the start of the exposure period):
High concentration level: 28 fish in 50-L test water (::0:1 L/day/g)
Low concentration level: 28 fish in 50-L test water (d L/day/g)
Control: 12 fish in 50-L test water (::0:1 L/day/g)
6) Water temperature: 24±2°C
7) Dissolved oxygen: >60% of the saturation (>5 mg/L at 24°C)
8) Aeration: continuous
9) pH: 6.0 - 8.5
l0) Lighting: Approx. 16 hr/day, Hf fluorescent lamp (wavelength 400-700 run)
11) Exposure period: 62 days
Reason: The exposure test was performed for 61 days that satisfied the maximum length of the period (60 days) prescribed in the test guidelines and the analysis results of the test fish sampled on the final day were obtained on day 62.
Nominal and measured concentrations:
Nominal Concentration Levels
In the test guideline, the nominal concentration of the high concentration level should be set at less than 1 % of the 96-hr 50% lethal concentration (LCso), and the nominal concentration of the low concentration level should be set at 1/10 of the high concentration level (0.1 % of the 96-hr LCso). Also these concentration levels should be less than water solubility. The nominal concentration for the bioconcentration test was determined, considering the following information.
96hr-LC50: >5 mg/L (Measured value in the preliminary toxicity test by the testing laboratory)

Nominal concentration:
High concentration level 0.05 mg/L (1 % of 96hr-LCso, less than water solubility)
Low concentration level 0.005 mg/L (0.1 % of 96hr-LCso, less than water solubility)

The mean recovery rates, and ranges of variation in two separate runs are shown below.
Although the mean recovery rate was low for the low concentration level, the range of variation was small, that indicated satisfactory accuracy. Therefore, it was judged that the bioconcentration potential could be evaluated using this analytical method. The measured values of the test substance concentration in the test water were corrected with the mean recovery rates of the respective components.

High concentration (0.05 mg/L)
Component Mean recovery rate Range of variation
(%) (%)
D 77.4 0.6
E 78.9 4.2
F 81.8 2.6

Low concentration (0.005 mg/L)
Component Mean recovery rate Range of variation
(%) (%)
D 58.3 10.0
E 78.9 1.1
F 62.7 4.6
Reference substance (positive control):
no
Details on estimation of bioconcentration:
Determination Limit of BCF
The lower limit of quantification in the fish sample was calculated from the results of the blank test to be 0.32 mglkg. This value was 6.4 and 64 times the nominal concentration for the high (0.05 mg/L) and low (0.005 mg/L) concentration levels, respectively.
Thus, the determination limits of the BCF were approximately 6 and 64 for the high and low concentration levels, respectively. However, the actual lower limit might depend on the variation of the test substance concentrations in the test water, fish body weight or the recovery rate in the fish analysis.
Calculations of Concentration in Test Water and BCF
The mean concentrations in the test water and BCF were calculated according to the JIS Handbook "Guide to the handling of numbers".
JIS Z9041-1:1999, Statistical interpretation of data - Part l:Statistical presentation of data
JIS Z8401: 1999, Guide to the rounding of numbers
Lipid content:
4.1 %
Time point:
start of exposure
Remarks on result:
other: n=3, 3.0 to 4.8%
Lipid content:
3.8 %
Time point:
end of exposure
Remarks on result:
other: n=3, 3.1 to 5.0%
Key result
Conc. / dose:
0.05 mg/L
Temp.:
24 °C
pH:
7.1
Type:
BCF
Value:
41 - 164
Basis:
whole body w.w.
Time of plateau:
7 d
Calculation basis:
steady state
Remarks:
Component D: Tetramine 2C16
Remarks on result:
other: Bioaccumulation too low to Quantify depuration
Key result
Conc. / dose:
0.005 mg/L
Temp.:
24 °C
pH:
7.1
Type:
BCF
Value:
110 - 213 L/kg
Basis:
whole body w.w.
Time of plateau:
7 d
Calculation basis:
steady state
Remarks:
Compoent D: Tetramine 2C16
Remarks on result:
other: Bioaccumulation too low to Quantify depuration
Key result
Conc. / dose:
0.05 mg/L
Temp.:
24 °C
pH:
7.1
Type:
BCF
Value:
26 - 107 L/kg
Basis:
whole body w.w.
Time of plateau:
7 d
Calculation basis:
steady state
Remarks:
Component E: Tetramine C16&18
Remarks on result:
other: Bioaccumulation too low to Quantify depuration
Key result
Conc. / dose:
0.005 mg/L
Temp.:
24 °C
pH:
7.1
Type:
BCF
Value:
160 - 225 L/kg
Basis:
whole body w.w.
Time of plateau:
7 d
Calculation basis:
steady state
Remarks:
Component E: Tetramine C16&18
Remarks on result:
other: Bioaccumulation too low to Quantify depuration
Key result
Conc. / dose:
0.05 mg/L
Temp.:
24 °C
pH:
7.1
Type:
BCF
Value:
34 - < 112
Basis:
whole body w.w.
Time of plateau:
7 d
Calculation basis:
steady state
Remarks:
Component F: Tetramine 2C18
Remarks on result:
other: Bioaccumulation too low to Quantify depuration
Key result
Conc. / dose:
0.005 mg/L
Temp.:
24 °C
pH:
7.1
Type:
BCF
Value:
< 170 L/kg
Basis:
whole body w.w.
Time of plateau:
7 d
Calculation basis:
steady state
Remarks:
Component F: Tetramine 2C18
Remarks on result:
other: Bioaccumulation too low to Quantify depuration
Elimination:
no
Remarks on result:
not determinable because of methodological limitations
Remarks:
Due to the limited bioaccumulation observed the depuration could not be monitored
Details on kinetic parameters:
As the all BCF values of analyzed components and monitored component A, B, C and G were below 1000 L/kg and no significant peak was detected from fish sample for monitored component H. The results confirmed that there was not concentration dependence.
The BCF value indicating a low to moderate bioaccumulation potential is below 1000 L/kg according to the Chemical Substances Control Law and 2000 L/kg or below according to the standard for PBT in REACH. Since the bioconcentration determined in this study was well below the regulatory thresholds above, the test substance was judged to have
no significant bioconcentration in fish. Therefore BCF normalized by 5% lipid content was not calculated. In this regard, depuration study was not performed according to the OECD guideline. Because of the insufficient analytical sensitivity and the low BCF values, it was not possible to confirm 95% loss of the concentration of the test substance in fish body if the
depuration test was performed. Therefore, it was difficult to calculate BCFk correctly and the depuration test was not performed.

Calculation of results
Calculation of BCF and BCFss
The bioconcentration factor (BCF) was calculated by the following equation:
BCFn = Cf,n / Cw,n

The steady-state bioconcentration factor (BCFss) was calculated by the following
equation:
Where
BCFss = Cf,s / Cw,s
BCFn: BCF after n days (L/kg)
BCFss: The steady-state BCF (L/kg)
Cf,n,: Concentration of the test substance in test fish after n days (mglkg wet weight)
Cw,n,: Mean concentration of the test substance in test water over n days (mgIL)
Cf,s: Mean concentration in test fish at steady-state (mg/kg wet weight)
Cw,s: Mean concentration in test water at steady-state (mg/L)

Steady-state: Steady-state is attained when variations of the test substance concentration in the test fish in four successive measurements conducted at intervals of 48 hrs or longer are within 20%. Cr,s is
calculated from the last four consecutive values. Cw,s is the average of all values during the steady-state period.
Metabolites:
-
Results with reference substance (positive control):
-
Details on results:
Component D, High concentration level
Exposure period Day 7 Day 14 Day 28 Day 48 Day 61
Mean cone. of the test substance in test water (mg/L) 0.0599 0.0582 0.0569 0.0582 0.0575
Cone. of the test substance in test fish (mg/kg wet weight) 1 8.55 3.63 9.33 6.75 5.58
2 2.43 4.24 4.77 6.36 4.13
BCF (L/kg) 1 143 62 164 116 97
2 41 73 84 109 72

Component D, Low concentration level
Exposure period Day 7 Day 14 Day 28 Day 48 Day 61
Mean cone. of the test substance in test water (mg/L) 0.00489 0.00476 0.00499 0.00503 0.00501
Cone. of the test substance in test fish (mg/kg wet weight) 1 0.698 0.793 0.903 1.073 <0.50
2 <0.50 <0.50 0.571 0.619 <0.50
BCF (L/kg) 1 143 167 181 213 <110
2 <110 <110 114 123 <110

Component E, High concentration level
Exposure period Day 7 Day 14 Day 28 Day 48 Day d61
Mean cone. of the test substance in test water (mg/L) 0.0614 0.0579 0.0591 0.0586 0.0587
Cone. of the test substance in test fish (mg/kg wet weight) 1 6.35 2.13 5.16 6.28 2.13
2 1.64 2.48 2.82 4.37 1.5
BCF (L/kg) 1 103 37 87 107 36
2 27 43 48 75 26

Component E, Low concentration level
Exposure period Day 7 Day 14 Day 28 Day 48 Day 61
Mean cone. of the test substance in test water (mg/L) 0.00375 0.00357 0.00366 0.00373 0.00371
Cone. of the test substance in test fish (mg/kg wet weight) 1 <0.58 <0.58 <0.58 0.838 <0.58
2 <0.58 <0.58 <0.58 <0.58 <0.58
BCF (L/kg) 1 <160 <170 <160 225 <160
2 <160 <170 <160 <160 <160


Component F, High concentration level
Exposure period Day 7 Day 14 Day 28 Day 48 Day 61
Mean cone. of the test substance in test water (mg/L) 0.0590 0.0599 0.0570 0.0574 0.0586
Cone. of the test substance in test fish (mg/kg wet weight) 1 6.41 2.77 6.39 5.90 3.97
2 2.00 3.13 2.85 3.80 2.18
BCF (L/kg) 1 109 46 112 103 68
2 34 52 112 103 68

Component F, Low concentration level
Exposure period Day 7 Day 14 Day 28 Day 48 Day 61
Mean cone. of the test substance in test water (mg/L) 0.00400 0.00415 0.00438 0.00442 0.00444
Cone. of the test substance in test fish (mg/kg wet weight) 1 <0.75 <0.75 <0.75 <0.75 <0.75
2 <0.75 <0.75 <0.75 <0.75 <0.75
BCF (L/kg) 1 <190 <190 <180 <170 <170
2 <190 <190 <180 <170 <170


The concentrations in the test water at both concentration levels were close to the nominal concentration, and the variation of the test substance concentrations in the water were within ±20% of the measured mean values for all components.
Rate for the nominal concentration of mean concentration of the test substance in test water are shown below.
Rate for the nominal conc. of mean conc.
Component Rate for the nominal conc. of mean conc. of the test substance in test water (%)
High concentration level Low concentration level
D 115.0 100.2
E 117.4 74.2
F 117.2 88.8

Analyzed components
All components at both concentration levels, the variation of mean concentration of the test substance in the test fish in four consecutive determinations separated by intervals of more than 48 hrs could not be confirmed to be within ±20%, thus, BCFss could not be calculated mathematically and BCF range was described as the result.

Monitored components
The monitored component A, B, C, G and H were confirmed for the fish samples taken during the exposure period (days 28 to 61). As a significant peak of component A, B, C and G was detected at high concentration level, BCF were calculated using measured concentrations in the test fish and nominal concentrations in the test water. BCF were <50-63 L/kg for component A, <50-68 Llkg for component B, <50-54 L/kg for component C and <50-51 Llkg
for component G. No significant peak was detected for component H at high concentration level and each component at low concentration level.
Reported statistics:
Statistical Methods
Correlation of the Calibration Curve
The correlation coefficient was calculated by the least squares method according to JIS Handbook "General rules for chemical analysis". . JIS K0050, General rules for chemical analysis

Calculations of Concentration in Test Water and BCF
The mean concentrations in the test water and BCF were calculated according to the JIS Handbook "Guide to the handling of numbers". JIS Z904l-1:1999, Statistical interpretation of data - Part l:Statistical presentation of data JIS Z8401: 1999, Guide to the rounding of numbers

Preliminary to the BCF study an acute fish test was performed by lab who performed the BCF study to determine the concentration at which the fish could be exposed during the BCF test.

Acute Toxicity Test

Species: Medaka (Oryzias latipes)

Source: Testing laboratory

Start of egg collection: November 15,2016

Lot number: 16-M-1115B

Pre-acclimation: At the start of acclimation, abnormal fish were removed upon visual inspection. The remaining normal fish were then reared in an aquarium with flowing water.

Total length: 2 ± 1 cm

Body weight: Approximately 0.2 g

Acclimation

Aquarium number: D-22

Water temperature: 24 ± 1°C

Period: September 29,2017 to October 10, 2017

Food:

Tetramin, Tetra Werke

Feeding: The food was given daily equivalent to approximately 2% of the body weight. (Feeding was suspended for 24 hr before testing.)

Mortality 1 week before testing: <5%

Medication during acclimation: None

Validity criteria fulfilled:
yes
Conclusions:
There was no deviation or specific factor that might have affected the reliability of the test results.

Validity of the Test
Validity of the test was assessed according to the following criteria:
The temperature variation is less than ± 2 deg C.

The concentration of dissolved oxygen did not fall below 60% saturation.

The concentrations of the test substance in the aquariums were maintained within ± 20% of the mean of the measured values during the exposure period.

The mortality or abnormality in both control and treated fish was less than 10% at the end of the exposure period.
Executive summary:

A Bioaccumulation study was performed with Tetramine di-C16 -C18 according to the OECD Guidelines for Testing of Chemicals No.305 "Bioaccumulation in Fish: Aqueous and Dietary Exposure" (2012).

Before the start of the test the water solubility of the different constituents of the test item was determined using the flask method. The observed solubilities of the three main constituents Tetramine di-C16, Tetramine C16 -C18 and Tetramine diC18 were 11, 9.3 and 5.7 mg/L respectively. In addition a preliminary acute fish test with Medaca was performed where an 96h LC50 of 4.6 mg/L was observed. The preliminary studies were performed to determine the exposure concentrations during the BCF study. The nominal concentrations were set at 0.05 mg/L (1/100 of 96hr-LC50) and 0.005 mg/L (1/1000 of 96hr-LC50).

Yearling Carp (Cyprinus carpio) of about 5 g were exposed to nominal concentrations of 0.05 and 0.005 mg/L during 62 days. No significant increase of the BCF's was observed after the first sampling point at day 7.

The BCF ranges observed for the three main constituents i.e. Tetramine di-C16, Tetramine C16 -C18 and Tetramine diC18 of the test item were respectively 41 -164, 26 -107 and 34 - 112 L/kg ww for the high concentration level and <110 - 213, <160 - 225 and <190 L/kg ww for the low concentration level.

Lipid content of the Carp was determined at the start (4.1%) and end (3.8%) in triplicate but since the bioaccumulation observed was far below the B and vB thresholds of 2000 and 5000 L/kg ww the BCF normalization to 5% lipid content was not performed. Depuration could not be followed due to the low observed BCF's in relation to the detection limit of the analytical method.

The mean recovery rates, and ranges of variation in two separate runs was determined and although the mean recovery rate was low for the low concentration level, the range of variation was small, that indicated satisfactory accuracy. Therefore, it was judged that the bioconcentration potential could be evaluated using this analytical method. The measured values of the test substance concentration in the test water were corrected with the mean recovery rates of the respective components.

Description of key information

In the preliminary assessment of the bioaccumulation potential of Tetramine di C16 -C18 based on a calculated log Kow  of >12.95 using a number of calculation models indicated that Tetramine di C16 -C18 would have al ow bioaccumulation potental. A BCF was calculated of 3.162 L/kg with BCFBAF (EPIsuite).

For a definitive assessment of the bioaccumulation potential of Tetramine di C16 -C18 a bioaccumulation study was performed according to the OECD Guidelines for Testing of Chemicals No.305 "Bioaccumulation in Fish: Aqueous and Dietary Exposure" (2012).

Before the start of the BCF test the water solubility of the different constituents of the test item was determined by the BCF test lab using the flask method. The observed solubilities of the three main constituents Tetramine di-C16, Tetramine C16 -C18 and Tetramine diC18 were 11, 9.3 and 5.7 mg/L respectively. In addition a preliminary acute fish test with Medaca was performed by the BCF test lab where an 96h LC50 of 4.6 mg/L was observed. The preliminary studies were performed to determine the exposure concentrations during the BCF study. The nominal concentrations were set at 0.05 mg/L (1/100 of 96hr-LC50) and 0.005 mg/L (1/1000 of 96hr-LC50).

Yearling Carp (Cyprinus carpio) of about 5 g were exposed to nominal concentrations of 0.05 and 0.005 mg/L during 62 days. No significant increase of the BCF's was observed after the first sampling point at day 7.

The BCF ranges observed for the three main constituents i.e. Tetramine di-C16, Tetramine C16 -C18 and Tetramine diC18 of the test item were respectively 41 -164, 26 -107 and 34 - 112 L/kg ww for the high concentration level and <110 - 213, <160 - 225 and <190 L/kg ww for the low concentration level.

Lipid content of the Carp was determined at the start (4.1%) and end (3.8%) in triplicate but since the bioaccumulation observed was far below the B and vB thresholds of 2000 and 5000 L/kg ww the BCF normalization to 5% lipid content was not performed. Depuration could not be followed due to the low observed BCF's in relation to the detection limit of the analytical method.

The mean recovery rates, and ranges of variation in two separate runs was determined and although the mean recovery rate was low for the low concentration level, the range of variation was small, that indicated satisfactory accuracy. Therefore, it was judged that the bioconcentration potential could be evaluated using this analytical method. The measured values of the test substance concentration in the test water were corrected with the mean recovery rates of the respective components.

Key value for chemical safety assessment

BCF (aquatic species):
81 L/kg ww

Additional information

The following is stated in the REACH guidance: At log Kow values between 4 and 5, log BCF increases linearly with log Kow. However, at very high log Kow (>6), a decreasing relationship between the two parameters is observed. Apart from experimental errors in the determination of BCF values for these very hydrophobic chemicals, reduced uptake due to the increasing molecular size may play a role as well. Moreover, the experimental determination of log Kow for very hydrophobic chemicals is normally also very uncertain due to experimental difficulties. The reliability of modelled Kow values > 10 is not known. Ideally the results of several model predictions should be considered. The aquatic BCF of a substance is probably lower than 2000 L/kg if the calculated log Kow is higher than 10. Given that none of the models have experimental information in this range, more than one model should be used to estimate the Kow value and the results evaluated by expert judgement.

Based on the anticipated high log Kow for Tetramine di-C16 -C18 the log Kow was calculated as suggested by the REACH guidance using several Kow estimation models. a calculated log Kow of >12.95 using a number of calculation models a BCF is calculated of 3.162 L/kg ww for tetramine diC16 -C18.

This low predicted BCF was confirmed in a BCF study according to OECD 305 via aquatic exposure where a mean BCF for the higher test concentration was observed of 81 L/kg ww. The BCF was determined from the higher concentration range as several lower than values were observed for test at low concentration level.