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Environmental fate & pathways

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:
2013-08-26 to 2014-02-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed for a Japanese notification according to OECD Guideline 305 with GLP compliance. All validity criteria were fulfilled and no deviations were observed.
Qualifier:
according to
Guideline:
OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Remarks:
GLP statement (February 28, 2014)
Specific details on test material used for the study:
No additional information
Details on sampling:
Test group:
- Sampling intervals/frequency for test organisms: Day 0, 7, 14, 21, 26 and 28 after exposure
- Sampling intervals/frequency for test medium samples: Day 0, 7, 14, 21, 26 and 28 after exposure
Control group:
- Analysis of water, analysis of fish and lipid contents (Day 0); analysis of fish and lipid contents (Day 28 after exposure)
- Sample storage conditions before analysis: In the test group, two fish were used as one sample and analyzed in duplicate, and other sample was put into a plastic bag and stored in a freezer after the fish were wrapped with aluminum-foil and packed in a plastic bag.

- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):

Analytical method for fish
Each six fish in the Concentration Level 1 and Level 2 and two fish in the control group were sampled using a landing net on the analysis day. The fish were sliced and put into a homogenizer cup with addition of anhydrous sodium sulfate (15 g) and acetonitrile (50 mL). The mixture was homogenized at 9000 r.p.m. for 6 min; the lid and the interior of the homogenizer cup were rinsed three times with acetonitrile (4 mL) and the washings and the content were transferred to a centrifugal tube. After centrifugal separation, the supernatant was filtered into a 100 mL volumetric flask. A portion (50 mL) of the constant volume solution was transferred to a 300 mL round-bottom flask. The mixture was evaporated in vacuo by using a rotary evaporator (water bath 35 °C) and the residue was dried by blowing air. The dried residue was dissolved in n-hexane (1 mL). Separately, n-hexane/acetone (8:2) 5 mL and n-hexane 15 mL were passed in this order through what two Sep-Pak Diol (Nihon Waters K.K.) were connected for conditioning. The said n-hexane solution was passed through the connected Sep-Pak Diol. n-hexane 8 mL was divided into three portions 2 mL, 2 mL and 4 mL and passed through the connected Sep-Pak Diol while rinsing the interior of the flask. Next, 5 mL of n-hexane was passed through the connected Sep-Pak Diol. The eluates were received in a 50 mL round-bottom flask. The flow rate through the connected Sep-Pak Diol was controlled at about 3 mL/min. The eluates in the round-bottom flask were evaporated in vacuo by using the rotary evaporator (water bath 35 °C) and the residue was dried by blowing air. The dried residue was dissolved in a small amount of acetone and was transferred to a measuring flask to obtain a constant volume of 0.5 mL. This constant volume solution was analyzed by GC.

Analytical method for test water
Pretreatment: Test water was sampled (approx. 300 mL from the Concentration Level 1 and approx.1000 mL from the Concentration Level 2) from the central area of each aquarium with an Erlenmeyer flask. After leaving it standing, the supernatant (100 mL in the concentration Level 1 and 600 mL in the Concentration Level 2) was measured using a measuring cylinder. Separately, acetone 5 mL and ultra pure water 20 mL were passed in this order through what two Sep-Pak C18 (Nihon Waters K.K.) were connected for conditioning. The said test water was passed through the connected Sep-Pak C18. The connected Sep-Pak C18 was dried using a water aspirator. Acetonitrile 15 mL was passed through the connected Sep-Pak C18 to elute the test substance. The eluate was received in a 50 mL round-bottom flask. The flow rate through the connected Sep-Pak C18 was controlled at about 3 mL/min. The eluate in the round-bottom flask was evaporated in vacuo by using the rotary evaporator (water bath 35 °C) and the residue was dried by blowing air. The dried residue was dissolved in a small amount of acetone and was transferred to a volumetric flask (1 mL in Concentration Level 1 and 0.5 mL in Concentration Level 2). The interior of the round-bottom flask was rinsed several times with acetone and the washings were also added to the said volumetric flask. The volume was adjusted with acetone to 1 mL in Concentration Level 1 and 0.5 mL in Concentration Level 2. The constant volume solution was analyzed by GC.

Recovery test and blank test for the test substance
Recovery test from fish: Fish bodies (two fish paired as one sample) were crashed with a blender after mincing, added an acetonitrile solution (1.0 mL) of the test substance, and analytical sample was prepared according to section “Analytical method for fish" and was analyzed by gas chromatograph. The recovery rates of the test substance were found to be as follows. The amount of an addition of the test substance (3.30 µg) corresponded to 0.408 µg/g relative to the fish 4.04 g (8.08 g/2 fish) and the bioconcentration factor in Concentration Level 2 (0.0025 mg/L) was approximately 163-fold.

Recovery test from test water: Water used for raising fish was sampled and a solution containing the test substance was added. According to Section “Analytical method for test water", an analytical sample was prepared and analyzed by gas chromatograph. The mean recovery rates of the test substance were 92.7 ± 2.5 % in Concentration Level 1 and 87.0 ± 2.1% in Concentration Level 2.
Vehicle:
no
Details on preparation of test solutions, spiked fish food or sediment:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Test substance (32.81 mg) was weighed into a 100 mL sample tube and dissolved in water for test heated to approximately 50 °C while stirring. The solution was transferred into a 5 L reagent bottle. Again, water for test heated to approximately 50 °C was added to the said sample tube while stirring in order to dissolve the remaining test substance, and then the solution was also transferred into the said test aquarium. The dissolution operation was repeated ten times. After cooling down to room temperature while stirring, a stock solution was prepared by adding water for test so that the total volume was 4 L (approx. 8 mg/L). The stock solution was diluted with water for test to obtain test water.
- High exposure level: The stock solution was diluted with water for test to obtain a solution of the test substance (2.5 mg/L). This was further diluted with water for test and continuously flowed through the test aquarium.
- Low exposure level: The stock solution was diluted with water for test to obtain a solution of the test substance (0.25 mg/L). This was further diluted with water for test and continuously flowed through the test aquarium.
- Control: Water for test was continuously flowed through the test aquarium.
Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
- Common name: Carp
- Source: Kitamura Carp Breeding Farm (12-388 Gunchiku, Yatsushiro-shi, Kumamoto Prefecture, Japan)
- Date purchased: August 26, 2013
- Length at study initiation (lenght definition, mean, range and SD): 8.2 ± 0.2 cm
- Weight at study initiation (mean and range, SD): 5.50 ± 0.44 g
- Health status: No mortality of fish during the experiment (0 %)
- Feeding: Mixed feed for carp in pellet form (Swimmy): Crude proteins: 37 %; Crude lipids: 2.5 %
- Feeding method: Fish were fed daily in an amount of approximately 1.5 % of the bodyweight of the fish. However, no feed was given on the day before sampling of the test fish.

ACCLIMATION
Feeding and acclimatization: After receiving the fish, those with a total length of approx. 8.0 ± 4.0 cm were selected. After the fish were treated with Elevage® (4-[2-(5-nitro-2-furanyl)ethenyl]benzoic acid, sodium salt) and rock salt, the process of acclimatization was started. Finally, the fish were acclimatized at water temperature 25 ± 2.0 °C for 7 days and it was confirmed that the mortality was 5% or less before use in the test.
Route of exposure:
aqueous
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
28 d
Hardness:
55 mg/L
Test temperature:
25.3 ± 0.3 °C
pH:
7.6
Dissolved oxygen:
7.28 ± 0.05 mg/L
TOC:
Since solvents or dispersing agents were not used when preparing the solution of the test substance, total organic carbon concentrations in the test water were not measured.
Salinity:
No data
Details on test conditions:
TEST SYSTEM
- Test vessel: Test aquarium: "Flow-through system" (made of glass)
- Capacity: 100 L (amount of test water; 50 L)
- Flow rate: 300 mL/min (432 L/day)
- No. of organisms per vessel: 45
- No. of vessels per concentration (replicates): No. 9 aquarium for high exposure level and No. 8 aquarium for low exposure level were used.
- No. of vessels per control / vehicle control (replicates): No. 7 aquarium for the control was used
- Number of test fish per concentration: High exposure level: 45 fish; Low exposure level: 45 fish
- Number of fish - control: 15 fish

TEST MEDIUM / WATER PARAMETERS
- Residual chlorine: ≤ 0.01 mg/L
- Copper: ≤ 0.005 mg/L
- Dissolved organic concentration: ≤ 2 mg/L
- Holding medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: Yes
- Light type: UV- cut fluorescent lamp
- Photoperiod: About 10 hours

RANGE-FINDING / PRELIMINARY STUDY
- Test concentrations: 1.6, 2.4, 3.6, 4.4 and 7.6 mg/L
- Results used to determine the conditions for the definitive study: The 96 h LC50 value of test item in Dania rerio was 2.5 mg/L and the water solubility is 10.3 mg/L. The setup concentrations for the bioconcentration test were determined to be less than the solubility in water. Based on the 96 h LC50 value, 1/100 and 1/1000 were used as safety factors and the test concentrations for main study were as follows: 0.025 and 0.0025 mg/L.
Nominal and measured concentrations:
High exposure level: 0.025 mg/L
Low exposure level: 0.0025 mg/L
Reference substance (positive control):
no
Details on estimation of bioconcentration:
Not applicable
Lipid content:
4.7 %
Time point:
start of exposure
Remarks on result:
other: n = 2
Lipid content:
5 %
Time point:
end of exposure
Remarks on result:
other: n = 2
Key result
Type:
BCF
Value:
46
Basis:
not specified
Time of plateau:
28 d
Calculation basis:
steady state
Remarks on result:
other: 43 ± 11 (n=10)
Remarks:
Conc.in environment / dose:0.025 mg/L
Key result
Type:
BCF
Value:
60
Basis:
not specified
Time of plateau:
28 d
Calculation basis:
steady state
Remarks on result:
other: 59 ± 7 (n= 9)
Remarks:
Conc.in environment / dose:0.0025 mg/L
Details on kinetic parameters:
No data
Metabolites:
No data
Results with reference substance (positive control):
Not applicable
Details on results:
Concentrations of test substance in test water:
Mean concentration during the test period were as follows
Concentration Level 1: 0.0230 ± 0.00153 mg/L
Concentration Level 2: 0.00224 ± 0.0000765 mg/L

Bioconcentration factors (BCFs):
Since it was confirmed that the steady-state was achieved on the 28th day of exposure in both Concentration Levels, exposure was finished. The mean of the bioconcentration factors of the test substance (BCFs) during the experiment period and the standard deviation are as follows:
Concentration Level 1 BCF = 43 ± 11 (n=10)
Concentration Level 2 BCF = 59 ± 7 (n= 9)

Confirmation of a steady-state
It is confirmed to reach the steady state when a fluctuation of BCFs in three successive analyses made on samples taken at intervals of more than 48 hours is within ±20%. Since it was confirmed that the steady state was achieved within 28 days in both Concentration Levels, exposure was completed on the 28th day.
Concentration Level 1 Fluctuation (%) = (9/46) x 100 = 20 (calculated from BCFs on the 21, 26 and 28th day)
Concentration Level 2 Fluctuation (%) = (8/60) x 100 = 13 (calculated from BCFs on the 21, 26 and 28th day)

Steady state bioconcentration factor (BCFss)
The steady state bioconcentration factor (BCFss) in each Concentration Level is shown below.
Concentration Level 1 BCFss = (1.06 µg/g / 0.0232 mg/L) = 46
Concentration Level 2 BCFss = (0.136 µg/g / 0.00225 mg/L) = 60

Control group: Test fish in the control group were analyzed before exposure Day 0 (n=1) and at the end of exposure Day 28 (n= 1). No background peaks due to fish were detected in the GC chromatograms at the position of the peak of the test substance as in the control group in the added recovery test.

Lipid content: The changes in the lipid contents from the beginning of the experiment to the end of the experiment were confirmed to be ± 25 % or less.
Reported statistics:
Number offish analyzed and number repeated:
- 2 fish were paired for a single analysis (n=2).
- 2 fish were paired for analysis of the control group (n=1).

None

Validity criteria fulfilled:
yes
Conclusions:
According to the results of the bioconcentration test, it was confirmed that the steady state in both Concentration Levels was achieved on the 28th day of exposure. The steady-state bioconcentration factors (BCFss) were determined to be 46-fold in Concentration Level 1 and 60-fold in Concentration Level 2.
Executive summary:

The bioaccumulation study was performed for a Japanese notification according to the OECD Guideline No. 305 with GLP compliance. The study was carried out to determine the extent of accumulation of test item by the carp, Cyprinus carpio during the exposure period of 28 days.  

Nominal exposure levels: Level 1: 0.025 mg/L; Level 2: 0.0025 mg/L.

Test was performed at 25.3 ± 0.3 °C and dissolved oxygen was 7.28 ± 0.05 mg/L. Analyses of test water and fish were conducted on Days 0, 7, 14, 21, 26 and 28 after exposure using Gas chromatography. Bioconcentration factor (BCF) was calculated.

 

Concentrations of test substance in test water:

Mean concentration during the test period were as follows

Concentration Level 1: 0.0230 ± 0.00153 mg/L

Concentration Level 2: 0.00224 ± 0.0000765 mg/L

Bioconcentration factors (BCFs): Since it was confirmed that the steady-state was achieved on the 28th day of exposure in both Concentration Levels, exposure was finished. The mean of the bioconcentration factors of the test substance (BCFs) during the experiment period and the standard deviation are as follows: Concentration Level 1 BCF = 43 ± 11 (n=10); Concentration Level 2 BCF = 59 ± 7 (n= 9). 

 

According to the results of the bioconcentration test, it was confirmed that the steady state in both Concentration Levels was achieved on the 28th day of exposure. The steady-state bioconcentration factors (BCFss) were determined to be 46-fold in Concentration Level 1 and 60-fold in Concentration Level 2.

Description of key information

OECD Guideline 305, GLP, Key study, validity 1 (study performed for Japan notification):

BCF = 46-60;

Not bioaccumulable.

Key value for chemical safety assessment

BCF (aquatic species):
60 dimensionless

Additional information

To assess the bioaccumulation potential of the registered substance, one experimental valid study is available.

This study (Institute of Ecotoxicology Co. Ltd., 2014), assessed as the key study, was performed for a Japanese notification on the registered substance, according to OECD Guideline 305 with GLP compliance. The study was carried out to determine the extent of accumulation of the test substance (two exposure levels: 0.025 mg/L and 0.0025 mg/L) by the carp, Cyprinus carpio, during the exposure period of 28 days. The test was performed at 25.3 ± 0.3 °C and dissolved oxygen was 7.28 ± 0.05 mg/L. Analyses of test water and fish were conducted on Days 0, 7, 14, 21, 26 and 28 after exposure using Gas chromatography. Since it was confirmed that the steady-state was achieved on the 28th day of exposure in both Concentration Levels, exposure was terminated. The mean of the bioconcentration factors of the test substance (BCFs) during the experiment period were 46 and 60 for the concentration level 1 (0.025 mg/L) and 2 (0.0025 mg/L), respectively.

In conclusion, the registered substance is not bioaccumulable according to criteria mentionned in the Regulation EC No 1272/2008 (BCF < 500) and Annex XIII of REACH (BCF < 2000; PBT Assessment).