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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:
16 August 2018 to 13 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test
Version / remarks:
2012
Deviations:
no
Qualifier:
according to
Guideline:
other: Method for Testing the Degree of Accumulation of Chemical Substances in Fish Body
Version / remarks:
stipulated in the "Testing Methods for New Chemical Substances" (March 31, 2011, No. 0331-7, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; March 29, 2011, No. 5, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 110331009, Environmental Policy Bureau, Ministry of the Environment, Japan; latest revision, March 29, 2018, No. 0329-13, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare;
No. 20180326-1, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 1803293, Environmental Policy Bureau, Ministry of the Environment, Japan)
Deviations:
no
GLP compliance:
yes
Radiolabelling:
no
Details on sampling:
TEST WATER ANALYSIS
- Test water of Levels 1 and 2 were analysed once before the uptake phase and at the same time as analysis of test fish.
- The test water of Control was analysed only at the end of uptake phase. The test water analysis before uptake phase was not conducted since no interfering peak was detected in the blank test. The results of blank test were regarded as that of Control before uptake phase.
- One sample was analysed at each sampling time.

TEST FISH ANALYSIS
- The test fish of Levels 1 and 2 were analysed five times during uptake phase. Four fish per treatment level were taken out at each sampling time and divided into two groups (two fish per group) because one fish was not enough for the analytical sensitivity of the test material. The test fish analysis was carried out at intervals of at least two days, and the final analysis was conducted at 28 days of uptake phase.
- The test fish of Control was analysed only after uptake phase. Four fish were taken out and divided into two groups (two fish per group). The test fish analysis before uptake phase was not conducted since no interfering peak was detected in the blank test. The result of blank test was regarded as that of Control before uptake phase.
Vehicle:
yes
Details on preparation of test solutions, spiked fish food or sediment:
PREPARATION AND APPLICATION OF TEST SOLUTION
- N,N-dimethylformamide was used as a solubilising agent to prepare the stock solutions.
> Level 1: The test material (100 mg) was dissolved in N,N-dimethylformamide to prepare 1000 mg/L solution of the test material (100 mL). This solution (25 mL) was taken out by transfer pipette and was diluted with N,N-dimethylformamide to prepare 50.0 mg/L stock solution (500 mL).
> Level 2: The solution of the test item (5 mL) prepared in a) was taken out by transfer pipette and was diluted with N,N-dimethylformamide to prepare 10.0 mg/L stock solution (500 mL).
> Control: N,N-dimethylformamide was used for stock solution.
Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
- Common name: Common carp
- Source: CERI Kurume
- Lot No. TFC-180123
- Age at study initiation: One-year-old fish
- Length: 6.8 - 8.2 cm (at the beginning of uptake phase 6.8 - 7.2 cm)
- Feeding during test: yes
- Food type: Feed for coloured carp from KYORIN FOOD INDUSTRIES LTD. (Composition: Proteins content ≥ 30 % and Lipid content ≥ 4.0 %)
- Amount and Frequency: Amount corresponding to 2 % of total body weight was fed. The daily dose of feed was split into two feedings (once a day during weekends and holidays). The fish were starved for 24 hours before the sampling.

ACCLIMATION
- The external disinfection was carried out in an aqueous solution containing OTC for fisheries (oxytetracycline hydrochloride, Kyoritsu Seiyaku) and sodium chloride (The Salt Industry Center of Japan). Thereafter fish were acclimatised in the following conditions. Test fish were moved from the acclimatisation tank (No. 8-6) to the test tank after the acclimatisation.
Period: 75 days
Temperature: 25 ± 2 °C
Mortality during acclimatisation was less than 5 %.
Route of exposure:
aqueous
Justification for method:
aqueous exposure method used for following reason: Test nominal concentration of Level 2 (0.4 µg/L) was set five times lower than that of Level 1 (2 µg/L) taking into account the analytical sensitivity of Peak 1.
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
28 d
Hardness:
15.6 - 16.6 mg CaCO3/L
Test temperature:
24.6 - 25.6 °C
pH:
7.9 - 8.1
Dissolved oxygen:
7.5 - 7.9 mg/L
TOC:
17.6 - 20.2 mg C/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 70-L glass tank
- Aeration: The test tanks were supplied with air.
- Type of flow-through: A flow-through system assembled at the laboratory was used. The stock solution (0.04 mL/min) and the dilution water (1000 mL/min), namely 1440 L/day of test water was supplied to the test tank from August 13, 2018.
- Renewal rate of test solution: For the test material groups the renewal was once or twice per week and for the control group it was once per month.
- No. of organisms per vessel: 26
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1 (16 fish)
- Duration of uptake: 28 days. Peak 1: Although the calculable bio-concentration factors of the targeted components were more than 100 L/kg, it was evaluated that a steady-state was reached after 28 days because obvious uptake of the test item was not observed. Peak 2: Because bio-concentration factors of the targeted components were less than 100 L/kg, it was evaluated that a steady-state was reached after 28 days.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Groundwater from the premises of CERI Kurume was used as dilution water for the test.
- Intervals of water quality measurement: Water temperature was measured once a day before uptake phase and during uptake phase. Flow rate of test water was measured once a day before uptake phase and during uptake phase. Dissolved oxygen was measured once before uptake phase, and once a week during uptake phase. pH was measured once before uptake phase, and twice during uptake phase. Total organic carbon was measured twice before uptake phase (24 h and 48 h before uptake phase), and once a week during uptake phase. Total hardness was measured once during uptake phase for Control and Level 1.

OTHER TEST CONDITIONS
- Photoperiod: 14 hours light /10 hours dark (artificial light of white fluorescent lamp)
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall daily feeding rate used in the study: 2 % of total body weight
- For OECD 305 part III (dietary exposure fish bioaccumulation), number of feeds per day (number of feeds daily ration split between): two feedings (once a day during weekends and holidays)
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall lipid content of spiked food before test start taking into account the contribution from the corn or fish oil vehicle, if used: ≥ 4.0 %

OBSERVATIONS
- Observations of test fish were performed twice a day (once a day at weekends and during holidays) during uptake phase.
Nominal and measured concentrations:
Nominal: Level 1: 2 µg/L and Level 2: 0.4 µg/L
Details on estimation of bioconcentration:
CALCULATION OF BIOCONCENTRATION FACTOR (BCF)
- Calculation of bioconcentration factor:
BCF = Cf / Cw
BCF: Bioconcentration factor (L/kg)
Cf: Concentration of test material in test fish (subtracting the FB) (ng/g)
Cw: Average concentration of test material in test water during uptake phase (μg/L)
FB: Average concentration of fish blank in analysis of Control (ng/g)
The results of blank test and analysis of Control (after uptake phase) were regarded as the average concentration of fish blank.

- Average bioconcentration factor in m-th analysis:
BCFm = (BCFa + BCFb) / n
BCFm: Average BCF in m-th analysis (number of group 2 (a,b)) (L/kg)
BCFa,b: Each BCF in m-th analysis of test fish (L/kg)
n: Number of group in m-th analysis of test fish
BCFm was not calculated when one or more concentrations of the test material in test fish at m-th analysis were not higher than LOQ.

DEFINITION OF STEADY STATE
The steady-state is defined to reach when the four successive analyses of concentration of the test material in test fish made on samples taken at intervals of at least two days are within ± 20 % of each other, and there is no significant increase of concentration of the test material in test fish in time between the first and last successive analyses.
Criterion for the steady-state: V(m-3), V(m-2), V(m-1), V(m) ≤ 20 (%)
V(m-3) = [(Cf(m-3) –Cf) / Cf] x100
V(m-2) = [(Cf(m-2) –Cf) / Cf] x100
V(m-1) = [(Cf(m-1) –Cf) / Cf] x100
V(m) = [(Cf(m) –Cf) / Cf] x100
V(m-3), V(m-2), V(m-1), V(m): Variation rate of concentration of test material in test fish from average value (%)
Cf (m-3), Cf (m-2), Cf (m-1), Cf (m): Average concentration of test material in test fish in m-3, m-2, m-1, m-th analysis (ng/g)
Cf: {Cf(m-3)+ Cf(m-2) + Cf(m-1) + Cf(m)}/ 4

CALCULABLE BCF
On the basis of the LOQ for the test material, calculable BCFs are as follows. They were rounded off to 2 figures.
Level 1: Peak 1: 160 L/kg. Peak 2: 8.7 L/kg.
Level 2: Peak 1: 900 L/kg. Peak 2: 50 L/kg.

CALCULATION OF THE GROWTH RATE CONSTANT (kg)
- The individual measured fish weight data in each level were used for the calculation of the growth rate constant (kg) in the study. The fish weight data for calculating kg are as follows:
Before uptake phase: Level 1, 2 and control: six test fish for lipid content analysis before uptake phase of Control.
End of uptake phase: Level 1: Four test fish for final test fish analysis + additional two test fish of Level 1. Level 2: Four test fish for final test fish analysis + additional two test fish of Level 2. Control: Six test fish for lipid content analysis after uptake phase of Control.
- The individual measured fish weight data were converted to natural logarithms and ln (fish weight) was plotted vs. time (day), then a linear least squares correlation was calculated for this plot. The kg was calculated as the slope of the linear correlation, and they were rounded off to 3 figures.

CALCULATION OF FISH LIPID CONTENT
- Fish lipid contents were calculated according to the following equation. The calculated fish lipid contents were rounded off to 3 figures.
Fish lipid content (%) = [(T - T0) / S] × 100
T0: Weight of vessel (g)
T: Weight of sample including the vessel after extraction procedure (g)
S: Weight of homogenate sample taken out for analysis of fish lipid content (g)
Lipid content:
4.09 %
Time point:
start of exposure
Lipid content:
3.55 %
Time point:
end of exposure
Key result
Conc. / dose:
2 µg/L
Temp.:
25 °C
pH:
8
Type:
BCF
Value:
ca. 8.7 - < 160 L/kg
Basis:
whole body w.w.
Time of plateau:
28 d
Calculation basis:
steady state
Key result
Conc. / dose:
0.4 µg/L
Temp.:
25 °C
pH:
8
Type:
BCF
Value:
ca. 50 - < 900 L/kg
Basis:
whole body w.w.
Time of plateau:
28 d
Calculation basis:
steady state
Key result
Rate constant:
growth rate constant (d-1)
Value:
0.015
Remarks on result:
other: Level 1
Key result
Rate constant:
growth rate constant (d-1)
Value:
0.014
Remarks on result:
other: Level 2
Key result
Rate constant:
growth rate constant (d-1)
Value:
0.015
Remarks on result:
other: Control
Details on results:
CONCENTRATION OF TEST MATERIAL IN TEST WATER
- The concentrations of the test material were maintained ≥ 70 % of nominal concentrations and the variations were within ± 20 % of the average measured concentrations. Concentrations of the test material in test water before uptake phase were as follows. No interfering peak was detected at the peak positions of the test material in the LC-MS/MS chromatogram for the Control water at the end of uptake phase:
Level 1: Peak 1: 1.76 μg/L, Peak 2: 1.71 μg/L.
Level 2: Peak 1: 0.397 μg/L, Peak 2: 0.410 μg/L

CONCENTRATION OF TEST MATERIAL IN TEST FISH
- No interfering peak was observed at the peak positions of the test material in the LC-MS/MS chromatogram for the Control fish after uptake phase.

BIOCONCENTRATION FACTORS
- BCFs of the test material are as follows.
Level 1: Peak 1: < 160 L/kg. Peak 2: < 8.7 L/kg
Level 2: Peak 1: < 900 L/kg. Peak 2: < 50 L/kg

- BCFs at steady-state (BCFss)
Peak 1: Because the test material concentration in all test fish at four successive analyses (after 12, 14, 21 and 28 days from the initiation of uptake phase) were not more than the LOQ, BCFss could not be calculated. However, because obvious uptake of the test material was not observed, it was evaluated that a steady-state was reached after 28 days although the calculable BCFs of the targeted components were more than 100 L/kg.
Peak 2: Because the test material concentration in all test fish at four successive analyses (after 12, 14, 21 and 28 days from the initiation of uptake phase) were not more than the LOQ, BCFss could not be calculated. However, because all BCFs were less than 100 L/kg, it was evaluated that a steady-state was reached after 28 days.

TEST FISH OBSERVATIONS
- No abnormality in behaviour or appearance was noted.

Table 1: Measured Concentrations of Test Material in Test Water (µg/L)

Level

Peak

After 7 Days

After

12 days

After

14 days

After

21 days

After

28 days

Average

(Standard deviation)

1

1

1.63

1.57

1.89

1.89

2.18

1.83 (0.243)

2

2.32

2.21

1.97

1.76

1.81

2.02 (0.246)

2

1

0.335

0.314

0.349

0.350

0.320

0.334 (0.0162)

2

0.281

0.297

0.387

0.390

0.407

0.352 (0.0585)

 

Table 2: Measured Concentrations of Test Material in Test Fish (ng/g)

Level

Peak

After 7 Days

After

12 days

After

14 days

After

21 days

After

28 days

1

1

< 300

< 300

< 300

< 300

< 300

< 300

< 300

< 300

< 300

< 300

2

< 18

< 18

< 18

< 18

< 18

< 18

< 18

< 18

< 18

< 18

2

1

< 300

< 300

< 300

< 300

< 300

< 300

< 300

< 300

< 300

< 300

2

< 18

< 18

< 18

< 18

< 18

< 18

< 18

< 18

< 18

< 18

 

Table 3: BCFs (L/kg)

Level

Peak

After 7 Days

After

12 days

After

14 days

After

21 days

After

28 days

1

1

< 160

< 160

< 160

< 160

< 160

< 160

< 160

< 160

< 160

< 160

2

< 8.7

< 8.7

< 8.7

< 8.7

< 8.7

< 8.7

< 8.7

< 8.7

< 8.7

< 8.7

2

1

< 900

< 900

< 900

< 900

< 900

< 900

< 900

< 900

< 900

< 900

2

< 50

< 50

< 50

< 50

< 50

< 50

< 50

< 50

< 50

< 50

Validity criteria fulfilled:
yes
Conclusions:
The target substance has low bioaccumulation potential, with calculated BCFs in the <8.7 to < 900 L/kg range.
Executive summary:

The bioaccumulation of the test material was investigated in accordance with the standardized guideline OECD 305 and other Japanese guidelines, under GLP conditions.

Common carp (Cyprinus carpio) were exposed to the test material at 2 and 0.4 µg/L for an uptake duration of 28 days in a flow-through system. Liquid chromatography-tandem mass spectrometry was used for analysis.

Since this test satisfied all of the validity criteria, it was concluded that the test conditions were reasonable to estimate bioconcentration potential of test material.

The test material concentration in all test fish at four successive analyses (peak 1 after 12, 14, 21 and 28 days from the initiation of uptake phase) were not more than the LOQ, BCFss could not be calculated. However, because obvious uptake of the test material was not observed, it was evaluated that a steady-state was reached after 28 days although the calculable BCFs of the targeted components were more than 100 L/kg.

The test material concentration in all test fish at four successive analyses (peak 2 after 12, 14, 21 and 28 days from the initiation of uptake phase) were not more than the LOQ, BCFss could not be calculated. However, because all BCFs were less than 100 L/kg, it was evaluated that a steady-state was reached after 28 days.

The target substance has low bioaccumulation potential, with calculated BCFs in the <8.7 to < 900 L/kg range.

Description of key information

Two peaks of the test material were observed in the LC-MS/MS chromatogram for each level tested.

Peak 1

The test material concentration in all test fish at four successive analyses (peak 1 after 12, 14, 21 and 28 days from the initiation of uptake phase) were not more than the LOQ, and as a result, BCFss could not be calculated. However, because obvious uptake of the test material was not observed, it was evaluated that a steady-state was reached after 28 days although the calculable BCFs of the targeted components were more than 100 L/kg.

Peak 2

The test material concentration in all test fish at four successive analyses (peak 2 after 12, 14, 21 and 28 days from the initiation of uptake phase) were not more than the LOQ, BCFss could not be calculated. However, because all BCFs were less than 100 L/kg, it was evaluated that a steady-state was reached after 28 days.

The target substance has low bioaccumulation potential, with calculated BCFs in the <8.7 to < 900 L/kg range.

Key value for chemical safety assessment

BCF (aquatic species):
900 L/kg ww

Additional information

The bioaccumulation of the test material was investigated in accordance with the standardised guideline OECD 305 and other Japanese guidelines, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Common carp (Cyprinus carpio) were exposed to the test material at 2 and 0.4 µg/L for an uptake duration of 28 days in a flow-through system. Liquid chromatography-tandem mass spectrometry was used for analysis.

Since this test satisfied all of the validity criteria, it was concluded that the test conditions were reasonable to estimate bioconcentration potential of test material.

The test material concentration in all test fish at four successive analyses (peak 1 after 12, 14, 21 and 28 days from the initiation of uptake phase) were not more than the LOQ, BCFss could not be calculated. However, because obvious uptake of the test material was not observed, it was evaluated that a steady-state was reached after 28 days although the calculable BCFs of the targeted components were more than 100 L/kg.

The test material concentration in all test fish at four successive analyses (peak 2 after 12, 14, 21 and 28 days from the initiation of uptake phase) were not more than the LOQ, BCFss could not be calculated. However, because all BCFs were less than 100 L/kg, it was evaluated that a steady-state was reached after 28 days.

The target substance has low bioaccumulation potential, with calculated BCFs in the <8.7 to < 900 L/kg range.