Reaction products of N,N'-ethane-1,2-diylbis(1,3-propanediamine), cyclohexane, peroxidized 4-butylamino-2,2,6,6-tetramethylpiperidine and 2,4,6-trichloro-1,3,5-triazine

Bioaccumulation: aquatic / sediment

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:

2000-11-30 - 2001-04-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (OECD test guideline 305)

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)

Qualifier:

according to guideline

Guideline:

other: Testing methods for new chemical substances, as prescribed in Chemical Substances Control Law of Japan

GLP compliance:

yes

Radiolabelling:

yes

Details on sampling:

Test water: On days 0, 7, 13, 21, 28, 35, 42, 49 and 60 of the exposure period, a sample of each test water was analyzed
Test fish: On days 7, 13, 28, 42 and 60 of the exposure period and day 1, 3, 7 and 14 of elimination period, four fish were sampled, and each analyzed

Vehicle:

yes

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

PREPARATION OF TEST WATER
- Exposure level: The unlabeled compound, 500 mg, was dissolved with THF and diluted to 10 mL (Solution 1). 20 g dispersant was dissolved with purified water and diluted to 200 mL (Solution 2). One mL of the solution 1 and 25 mL of solution 2 added dropwise to purified water with stirring,and diluted to 50 mL, resulting in a dispersion with 1000 mg/L test substance and 50000 mg/L dispersant. 20 mL of this solution was diluted to 2 L with dilution water, resulting in test water with 10 mg/L test substance and 500 mg/L dispersant.
- Control level: Ten milliliters of solution 2 (dispersant l00000 mg/L) described above) and 0.4 mL of THF added dropwise to dilution water with stirring, and diluted to 2 L, resulting in test water with 500 mg/L dispersant.

- Preparation of feed solution:
- Unlabeled 750 mg - dissoved in 200 mL THF = Stock solution 1, Conc. 3750 mg/L;
- 14C-labeled 196 mg - dissoved in 52.5 mL THF = Stock solution 2, Conc. 3750 mg/L
- Dispersant (37 . 5 mL of 100000 mg/L solution in water)
- HCL: 19 mL Stock solution 1 + 1 mL Stock solution 2 + 3750 mg dispersant ad 1000 mL (purified water)
[Conc. of test substance: 75 mg/L; Specific activity: 9.45 kBq/mg]
- LCL: 1 mL Stock solution 1 + 1 mL Stock solution 2 + 375 mg dispersant ad 1000 mL (purified water)
[Conc. of test substance: 7.5 mg/L; Specific activity: 94.5 kBq/mg]

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Source: Sankyo Suisan Co., Japan
- Lot number: 00-H-0807
- Age at study initiation: about one and half year after hatching
- Body length: 4 – 8 cm
- Body weight: about 4 g
- Lipid content: 4.4 % (at the beginning of the exposure period)
- Lipid content: 5.8% (at the end of the exposure period)
- Weight at termination: 3.79 - 7.18 g
- Feeding during test: yes, 1% of the fish weight everyday
- Health status: Observations were made on the external appearance of fish and the swimming and feeding behavior.

ACCLIMATION
- Acclimation period: ≥ 2 weeks
- Water temperature: 24 +/- 2 °C
- Type and amount of food: Babygold from Kyorin
- Feeding frequency: 1% of the body weight everyday.
- Health during acclimation: Mortality <5% per 1 week before the test

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

60 d

Total depuration duration:

14 d

Hardness:

65 - 70 mg/L

Test temperature:

22.0 - 24.4°C

pH:

7.9 - 8.3

Dissolved oxygen:

7.2 - 8.3 mg/L

TOC:

- 1.034 mgC/L in control at the beginning of the test
- 1.579 mgC/L in control during the test
- 11.26 mgC/L in test water prepared by the only unlabeled compound

Details on test conditions:

EXPOSURE LEVEL:
- 0.05 mg/L in high concentration level (HCL) [Dispersant 2.5 mg/L, THF 13.3 ppm (v/v)]
- 0.005 mg/L in low concentration level (LCL) [Dispersant 0.25 mg/L, THF 1.33 ppm (v/v)]
- Control: 0 mg/L [(Dispersant 0 mg/L, THF 0 ppm (v/v)]

TEST SYSTEM
- Test vessel: 27 L-glass aquarium (W45 cm x L24 cm x D30cm)
- Type: closed
- Fill volume: 20 L
- Aeration: Continuous
- Supply of test water: 210 L/day (Turnover rate: about 10 times per day)
- Supply of feed solution: 140 mL/day (except control)
- Population density at the introduction of fish:
- High concentration level: 40 fish in 20 L test water (> 1 L/day/g) [exposure period/elimination period]
- Low concentration level: 40 fish in 20 L test water (> 1 L/day/g) [exposure period/elimination period]
- Control: 20 fish in 20 L test water (> 1 L/day/g)
- No. of vessels per concentration: 1
- No. of vessels per control / vehicle control: 1
- Test substance is introduced continuously to maintain constant concentration

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Yokohama municipal tap water was treated with activated charcoal and aerated. This dilution water was verified to satisfy the Water Quality Criteria for the Protection of Aquatic Living Resources.
- Chlorine: < 0.01 mg/L
- Alkalinity: 40 mg/L
- Ca/Mg ratio: ~4.2:1
- Conductance: 140 µS/cm

OTHER TEST CONDITIONS
- pH: 6.0-8.5
- Photoperiod: About 16 hr/day (fluorescent lamp with wavelengths of 400-700 nm
- Water temperature: 24 +/- 2°C
- Dissolved oxygen: > 60 % of the saturation (^ 5 mg/L at 24°C)

RANGE-FINDING / PRELIMINARY STUDY
Acute Toxicity Test (OECD TG 203): An acute toxicity test with Medaka was conducted for 96 hours and a 50% lethal concentration (LC50) was determined. Then the bioconcentration test was carried out at lower exposure levels not exceeding a thousandth of the 96-hour LC50 and as low as it could be analyzed, and higher level differing from the one by a factor of ten (not exceeding a hundredth of the 96-hour LC50).
96-hr LC50 to Medaka: >10 mg/L
The test water was prepared by dispersing the test substance with Tetrahydrofuran (THF) and HCO-60 (dispersant, Polyoxyethylene hydrogenated castor oil) in water. Where the concentration of the test substance exceeds 10 mg/L, the concentrations of the solubilizing agent increase to raise the possibility of having an effect on fish. The upper limit of the test concentration was accordingly set at 10 mg/L. The concentration of the test substance was not measured, and the result was calculated by the nominal concentration.
Preparation of test water:
- Exposure level: The unlabeled compound, 500 mg, was dissolved with THF and diluted to 10 mL (Solution 1). 20 g dispersant was dissolved with purified water and diluted to 200 mL (Solution 2). One mL of the solution 1 and 25 mL of solution 2 added dropwise to purified water with stirring,and diluted to 50 mL, resulting in a dispersion with 1000 mg/L test substance and 50000 mg/L dispersant. 20 mL of this solution was diluted to 2 L with dilution water, resulting in test water with 10 mg/L test substance and 500 mg/L dispersant.
- Control level: 10 mL of solution 2 (dispersant 100000 mg/L) described above) and 0.4 mL of THF added dropwise to dilution water with stirring, and diluted to 2 L, resulting in test water with 500 mg/L dispersant.

Nominal and measured concentrations:

Mean measured concentration of test item in test water: During the exposure period of 60 days, test water in HCL and LCL had mean concentrations of 0.0508 and 0.00515 mg/L, respectively. The coefficients of variation were 4.6% and 6.4%.

Reference substance (positive control):

not specified

Details on estimation of bioconcentration:

Calculation of Bioconcentration Factor (BCFk): The uptake rate constant (k1) and the depuration rate constant (k2) were obtained to use non-linear parameter estimation methods on a computer, and kinetic bioconcentration factor (BCFk) was calculated.

Lipid content:

>= 3.7 - <= 5.4 %

Time point:

start of exposure

Remarks on result:

other: mean: 4.4 % (n=3)

Lipid content:

>= 4.5 - <= 6.7 %

Time point:

end of exposure

Remarks on result:

other: mean: 5.8 % (n=3)

Type:

BCF

Value:

>= 6 - <= 17 dimensionless

Basis:

whole body w.w.

Time of plateau:

60 d

Calculation basis:

steady state

Remarks on result:

other: It did not appear that the steady-state was achieved at either concentration level

Remarks:

Conc.in environment / dose:0.05 mg/L in high concentration level (HCL)

Type:

BCF

Value:

>= 7 - <= 30 dimensionless

Basis:

whole body w.w.

Time of plateau:

60 d

Calculation basis:

steady state

Remarks on result:

other: It did not appear that the steady-state was achieved at either concentration level

Remarks:

Conc.in environment / dose:0.005 mg/L in low concentration level (LCL)

Details on results:

There was no dead fish or abnormal condition during the exposure period of 60 days and elimination period of 14 days.

It did not appear that the steady-state was achieved at either concentration level, because three successive analyses of test substance in fish were not within ± 20% of each other. The BCF was ≤ 46 in high concentration level (HCL) and ≤ 98 in low concentration level (LCL).
Because the BCF was more than 10 in either exposure level, a elimination phase was run for 14 days following the 60-day exposure phase. Another kind of BCF (BCFk) was calculated from uptake and depuration rate constants. The BCFk was 26 in HCL and for the experiment in LCL, the BCFk was 19.

It can be concluded that the bioconcentration potential of the test substance in fish is remote.

Concentration of Test Item in Test Fish and BCF:

During the exposure period, test fish in HCL and LCL had concentrations of 0.282-2.333 and <0.0200-0.5042 µg/g, respectively. The bioconcentration factor (BCF) was 6-46 and <4-98, respectively. BCF of three successive analyses (on days 28, 42, 60) were not within ±20% of each other in HCL and LCL, and so steady state could not be confirmed. Therefore BCFss was not calculated.

Executive summary:

In a bioaccumulation study according to OECD TG 305 bioconcentration factors (BCF) for the test item (as described in section 1.2) were determined in Japanese Carp incubated in a continuous flow through system. 14C-labelled test substance was dissolved with the use of dispersant with 60 days of exposure and 14 days of elimination. Of the two concentrations tested, 0.05 mg/L was higher than the water solubility (< 0.04 mg/L), whereas 0.005 mg/L is considered to represent a water soluble concentration. It did not appear that the steady-state was achieved at either concentration level. On a weekly basis test water was analysed during the exposure period and test fish on days 7,13,28,42 and 60 of the exposure period and on day 1, 3, 7 and 14 of the elimination period. There was no dead fish. The appearance of Cyprinus carpio was normal by aquarium-side observations. BCF were calculated as the ratio of the concentration of the test item between fish and the exposure water. The BCF was ≤ 46 for the high concentration level and ≤ 98 for the low concentration level. From the uptake and depuration rate constants, BCF were calculated as 26 for the high and 19 for the low concentration level. It is concluded that the test item is not bioaccumulating in fish.

Description of key information

Does not significantly accumulate in organisms. 

Key value for chemical safety assessment

BCF (aquatic species):

30 dimensionless

Additional information

In a bioaccumulation study according to OECD TG 305 bioconcentration factors (BCF) for the test substance (as described in section 1.2) were determined in Japanese Carp incubated in a continuous flow through system. 14C-labelled test substance was dissolved with the use of dispersant with 60 days of exposure and 14 days of elimination. Of the two concentrations tested, 0.05 mg/L was higher than the water solubility (< 0.04 mg/L), whereas 0.005 mg/L is considered to represent a water soluble concentration. It did not appear that the steady-state was achieved at either concentration level. On a weekly basis test water was analysed during the exposure period and test fish on days 7,13,28,42 and 60 of the exposure period and on day 1, 3, 7 and 14 of the elimination period. There was no dead fish. The appearance of Cyprinus carpio was normal by aquarium-side observations. BCF were calculated as the ratio of the concentration of the test substance between fish and the exposure water. The BCF was ≤ 46 for the high concentration level and ≤ 98 for the low concentration level. From the uptake and depuration rate constants, BCF were calculated as 26 for the high and 19 for the low concentration level. It is concluded that the test substance is not bioaccumulating in fish.