(E)-3-(benzyl(4-((4-nitrophenyl)diazenyl)phenyl)amino)propanenitrile

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

28 October 1997 to 06 February 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 C (Bioaccumulation: Test for the Degree of Bioconcentration in Fish)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Chemical Substances Control Law (Japanese law 117, 1974)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Radiolabelling:

no

Details on sampling:

Test water and fish were analysed on the following dates:
Day 0 Analysis of water
Day 7 Analysis of water and fish
Day 14 Analysis of water and fish
Day 21 Analysis of water and fish
Day 28 Analysis of water and fish
Day 35 Analysis of water
Day 42 Analysis of water and fish
Day 49 Analysis of water
Day 56 Analysis of water and fish
Analysis of control fish: Fish were collected and analysed at the beginning and the end of the exposure period (Days 0 and 56).

Vehicle:

yes

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

Dispersion method
Dispersing agent: FDPS (Naphthalene sulfonic acid - formaldehyde condensation product)
Procedure: The test substance (T-9601), 2.52463 g, and FDPS which is three times as much as the test substance were ground in a mortar with a pestle (An automatic apparatus was used (SOP/TNS/4.30)). Pure water was added drop wise into the mortar when the substances began to be coagulated. After the test substance was dispersed uniformly, the water dispersion was diluted with additional water to 2.5 L (Concentration of the test substance: 1000 ppm).
Temperature: 23.7±0.1°C
Test water: De-chlorinated water (prepared from the Tokyo metropolitan tap water)

Test organisms (species):

Cyprinus carpio

Details on test organisms:

Test Fish Carp (Cyprinus carpio L.)
Source; Saku-yoshoku Co., 3-5-14 Setagaya, Setagaya-ku, Tokyo, Japan
Date of purchase: September 17, 1997 (Lot No. 70917)
Acclimation: Carp of 20-40 g and about 10 cm were sorted out when received in Institute of Ecotoxicology, Gakushuin University (SOP 2.5), and they were acclimated in Aquarium No. 1 (SOP 2.5). On November 25, 1997, they were sorted out again and transferred to Aquarium No. 7. They were on medication (New Green F®, Shin Fuso Pharmaceutical Co., Ltd.) in water before the testing (SOP 2.3). Water temperature during the acclimation was gradually raised to 25°C by the start of the test (SOP 2.5).
Carp: Average body weight: 25.9 ± 4.5 g (SOP 2.8)
Average body length: 9.9 ± 0.6 cm (SOP 2.8)
Average fat content: 4.1 ± 0.5% (SOP 2.9 (2))
(measured by CHCl3-MeOH Extraction Method)

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

8 wk

Hardness:

No data

Test temperature:

24.8 – 25.2°C

pH:

7.1 - 7.6

Dissolved oxygen:

7.0 - 7.6 ppm

TOC:

No data

Salinity:

Not applicable.

Details on test conditions:

Aquarium:
Aquarium for flow-through test. Made of glass, Test volume of 100l Flow rate: 300ml/min. (432L/day) Aquarium No, 8 was used for high exposure level, No. 9 for low exposure level, and No. 7 for control.
Water: De-chlorinated water (prepared from the Tokyo metropolitan tap water).
Number of fish: High and low exposure levels: 25 fish each; Control: 6 fish
Temperature 25.1±0.3°C
Concentrations: The test substance in water was set at concentrations as low as possible with consideration given to both the analytical sensitivity and the 43-hr LC5O value.
Concentration of the test substance Concentration of FDPS
High exposure level 0.1 ppm (w/v) 0.3 ppm (w/v)
Low exposure level 0.01 ppm (w/v) 0.03 ppm (w/v)
Control ---- 0.3 ppm (w/v)


Analysis of Test Fish
Pre-treatment
Three fish were collected with a dip net from high or low exposure level or control on their respective dates of analysis, and led to apparent death by hitting the back of the head, the surface of which was then wiped softly with a piece of gauze, and weighed in tenths of a gram with a top-loading balance. The body length, which is defined as a distance from the tip of mouth to the base of caudal fin, was measured in tenths of a centimetre. Two of three fish each were used for analysis, and the other was wrapped with a piece of aluminium foil, and then packed in a plastic bag and placed in a plastic case before stored in a freezer.
The individual test fish was cut into pieces and placed in a homogenizer cup, to which 150 ml of dichloromethane / acetone (2/1 (v/v)) was added, and homogenized at 10000 rpm for 10 minutes. The homogenate was transferred into a 250 ml centrifuge tube. The inside of the cup was washed with six 30 ml of dichloromethane / acetone (2/1 (v/v)) and the six washings were transferred into the centrifuge tube, which was then centrifuged at 8000 rpm for 15 minutes at 15 °C. The supernatant was filtered with Advantec No. 2 filter paper, of which the filtrate was collected in a 500 ml rounded-bottom flask. After that, the filtrate was evaporated to dryness under a reduced pressure with a rotary evaporator (water bath. 25°C). The residue was dissolved with divisions of 80 ml of n-hexane and 140 ml of acetonitrile successively, and its solutions were transferred into a 300 ml separatory funnel. The funnel was shaken for 15 minutes with a shaker and left stand for a while. The acetonitrile layer (lower layer) was transferred to a 500 ml rounded-bottom flask and evaporated to dryness under a reduced pressure with the rotary evaporator (water bath: 25°C).
This extract from fish was dissolved with 2 ml of n-hexane/ acetone (9/1) and the solution was injected into a Sep-Pack Silica® cartridge column (conditioned with 5 ml of n-hexane / acetone (9/1) in advance). The inside of the flask was washed with two 1 ml of the solvent mixture, and the two washings were also injected into the column (the test substance is adsorbed to the Sep-Pack Silica). Seven millilitres of n-hexane/ acetone (8/2) was gradually added to the column, and the eluate was pooled in a 50 ml rounded-bottom flask (the test substance is eluted in this fraction). After that, the eluate was evaporated to dryness under a reduced pressure with the rotary evaporator (water bath: 25°C).
The residue was dissolved with 2 ml of acetonitrile, and the solution was transferred into a 10 ml volumetric flask. Three millilitres of pure water was also added into the flask. The inside of the rounded-bottom flask was washed with several portions of acetonitrile, and each washings was transferred into the volumetric flask, which was then filled with acetonitrile (an acetonitrile solution containing 30% water). An aliquot of this final solution was filtered with LC disk 13 CR, and the filtrate was analysed by high performance liquid chromatography (HPLC).
Analysis of Test Water
Pre-treatment
About 500 ml (about 2000 ml) of test water of high exposure level was withdrawn from the middle of the aquarium with a conical flask. The supernatant water, 200 mL (800 ml) was measured with a graduated cylinder.
The supernatant measured was transferred into a 300 ml (1l) separatory funnel. The inside of the graduated cylinder was washed with a small amount of pure water and the washings was transferred into the funnel. Dichloromethane, 80 ml (120 ml), was added into the funnel, which was then shaken for 15 minutes with the shaker and left stand for a while. The lower layer was transferred into a 300 ml rounded-bottom flask, and concentrated under a reduced pressure with the rotary evaporator. This concentration was stopped immediately before dryness, and the residual solvent was removed by forced ventilation. The residue was dissolved in a small amount of acetonitrile, and the solution was transferred into a 10 ml (5 ml) volumetric flask, into which 3 ml (1.5 ml) of pure water was then added. The inside of the rounded-bottom flask was washed with several portions of acetonitrile, and each washings was transferred into the volumetric flask, which was then filled with acetonitriie (an acetonitrile solution containing 30% water). The final solution was filtered with LC disk 13 CR, and the filtrate was analyzed by HPLC.

Nominal and measured concentrations:

Concentration of the test substance Concentration of FDPS
High exposure level 0.1 ppm (w/v) 0.3 ppm (w/v)
Low exposure level 0.01 ppm (w/v) 0.03 ppm (w/v)
Control ---- 0.3 ppm (w/v)

Reference substance (positive control):

not required

Details on estimation of bioconcentration:

The 48-hr LC50 of the test substance to orange killifish was more than 186 ppm. Accordingly, the nominal concentrations to be used in the bioconcentration test were set at 0.1 ppm and 0.01 ppm. The exposure period of the test was set at 8 weeks. We obtained the bioconcentration factors (BCFs) ranging from 15 to 58 for the 0.1 ppm exposure level and from 62 to 124 for the 0.01 ppm exposure level.

Type:

BCF

Value:

> 15 - < 58 dimensionless

Basis:

whole body w.w.

Time of plateau:

56 d

Calculation basis:

steady state

Remarks on result:

other: Conc.in environment / dose:0.1 ppm (w/v)

Type:

BCF

Value:

> 62 - < 124 dimensionless

Basis:

whole body w.w.

Time of plateau:

56 d

Calculation basis:

steady state

Remarks on result:

other: Conc.in environment / dose:0.01 ppm (w/v)

Details on kinetic parameters:

Not calculated.

Metabolites:

Not assessed.

Results with reference substance (positive control):

Not applicable.

Details on results:

Further information in table form submitted under any other information

Reported statistics:

No data

Bioconcentration factors

Day	High exposure level		Low exposure level
	A	BCFs	A	BCFs
	B		B
7	A	25	A	114
	B	15	B	82
14	A	43	A	77
	B	29	B	88
21	A	42	A	62
	B	37	B	105
28	A	31	A	11
	B	33	B	104
42	A	40	A	98
	B	44	B	96
56	A	51	A	121
	B	58	B	124

(Note 1) Fish were given the letter A or B for identification when they were collected for analysis.

 

Analysis of Fish (High exposure level)

Sample fish		A Final volume       (ml)	B Peak area	C Concentration in final solution *   (ppm)	D Mass detected in fish     (μg)	E Fish weight       (g)	F Recovery       (%)	G Concentration in fish     (ppm)	H Concentration in water (cumulative average) (ppm)	I Bio-concentration factor
7	A	10	482191	4.7668	47.668	21.8	89.8	2.43	0.0965	25
7	B	10	282028	2.7921	27.921	21.0	89.8	1.48	0.0965	15
14	A	10#	384425	3.8375	76.750	20.0	89.8	4.27	0.0983	43
14	B	10#	339163	3.3871	67.742	26.4	89.8	2.86	0.0983	29
21	A	10#	530066	5.1702	103.404	27.4	89.8	4.20	0.0992	42
21	B	10#	404617	3.9523	79.046	24.1	89.8	3.65	0.0992	37
28	A	10#	364435	3.6158	72.316	26.1	89.8	3.09	0.0998	31
28	B	10#	385580	3.8250	76.500	25.7	89.8	3.31	0.0998	33
42	A	10#	501163	4.9150	98.300	27.4	89.8	4.00	0.101	40
42	B	10#	491192	4.8172	96.344	23.9	89.8	4.49	0.101	44
56	A	10#	725263	7.1585	143.170	31.2	89.8	5.11	0.101	51
56	B	10#	746819	7.3711	147.422	28.2	89.8	5.82	0.101	58

Calculation equations: Mass detected in fish: D=CXA (X2 (Dilution factor))#

                                  Concentration in fish: G=(DX100)/(EXF)

                                   Bioconcentration factor: I=G/H

*Based on two-point calibration

#On day 14 and after, we diluted the final solutions twofold to determine the test substance concentration within the assay range.

 

Analysis of Fish (Low exposure level)

Sample fish		A Final volume       (ml)	B Peak area	C Concentration in final solution*   (ppm)	D Mass detection in fish   (μg)	E Fish weight       (g)	F Recovery       (%)	G Concentration in fish     (ppm)	H Concentration in water (cumulative average) (ppm)	I Bio-concentration factor
7	A	10	238201	2.3597	23.597	24.2	89.8	1.09	0.00952	114
7	B	10	146567	1.4557	14.557	20.7	89.8	0.783	0.00952	82
14	A	10	160313	1.6075	16.075	24.1	89.8	0.743	0.00959	77
14	B	10	161857	1.6228	16.228	21.3	89.8	0.848	0.00959	88
21	A	10	117668	1.1665	11.665	21.6	89.8	0.601	0.00966	62
21	B	10	234780	2.3035	23.035	25.4	89.8	1.01	0.00966	105
28	A	10	241177	2.3964	23.694	24.7	89.8	1.08	0.00974	111
28	B	10	204985	2.0383	20.383	22.5	89.8	1.01	0.00974	104
42	A	10	227145	2.2270	22.270	26.0	89.8	0.954	0.00971	98
42	B	10	224150	2.1976	21.976	26.2	89.8	0.934	0.00971	96
56	A	10	347790	3.4360	34.360	32.3	89.8	1.18	0.00976	121
56	B	10	330797	3.2685	32.685	30.0	89.8	1.21	0.00976	124

Calculation equations: Mass detected in fish: D=CXA

                                  Concentration in fish: G=(DX100) / (EXF)

                                  Bioconcentration factor: I=G/H

*Based on two-point calibration

 

Analysis of Fish (Control)

Sample fish	A Final volume       (ml)	B Peak area	C Concentration in final solution*   (ppm)	D Mass detected in fish     (μg)	E Fish weight       (g)	F Recovery       (%)	G Concentration in fish     (ppm)	H Concentration in water (cumulative average) (ppm)	I Bio-concentration factor
0 – a	10	N.D.	---	---	21.6	---	---	0	---
0 – b	10	N.D.	---	---	24.9	---	---	0	---
56 – a	10	N.D.	---	---	38.1	---	---	0	---
56 – b	10	N.D.	---	---	37.7	---	---	0	---

(Note) Fish were given the letter “a” or “b” for identification when they were collected from the aquarium.

*Based on two-point calibration

 

Analysis of Water (High exposure level)

Exposure period (day)	A Final volume       (ml)	B Peak area	C Concentration in final solution*   (ppm)	D Absolute amount in final solution   (μg)	E Volume of sample water     (g)	F Recovery       (%)	G Concentration in water (measured value) (ppm)	H Concentration in water (cumulative average) (ppm)
0	10	177926	1.77673	17.673	200	96.2	0.0919	---
7	10	195487	1.9432	19.432	200	26.2	0.101	0.0965
14	10	198990	1.9682	19.682	200	96.2	0.102	0.0983
21	10	199986	1.9657	19.657	200	96.2	0.102	0.0992
28	10	198010	1.9693	19.693	200	96.2	0.102	0.0998
35	10	195134	1.9409	19.409	200	96.2	0.101	0.100
42	10	203528	1.9953	19.953	200	96.2	0.104	0.101
49	10	204616	2.0041	20.041	200	96.2	0.104	0.101
56	10	201072	1.9635	19.635	200	96.2	0.102	0.101

Calculation equation: Absolute amount in final solution: D=CXA

                                Concentration in water: G=(DX100) /(EXF)

*Based on two-point calibration

 

Analysis of Water (Low exposure level)

Exposure period (day)	A Final volume       (ml)	B Peak area	C Concentration in final solution*   (ppm)	D Absolute amount in final solution   (μg)	E Volume of sample water     (g)	F Recovery       (%)	G Concentration in water (measured value) (ppm)	H Concentration in water (cumulative average) (ppm)
0	5	141866	1.4102	7.0510	800	97.4	0.00905	---
7	5	156212	1.5552	7.7760	800	97.4	0.00998	0.00952
14	5	153464	1.5195	7.5975	800	97.4	0.00975	0.00959
21	5	155382	1.5327	7.6635	800	97.4	0.00984	0.00966
28	5	158286	1.5763	7.8815	800	87.4	0.0101	0.00974
35	5	153818	1.5321	7.6605	800	97.4	0.00983	0.00976
42	5	150251	1.4727	7.3635	800	97.4	0.00945	0.00971
49	5	159355	1.5601	7.8005	800	97.4	0.0100	0.00975
56	5	156923	1.5305	7.6525	800	97.4	0.00982	0.00976

Calculation equations: Absolute amount in final solution: D=CXA

                                  Concentration in water: G=(DX100) / (EXF)

*Based on two-point calibration

Validity criteria fulfilled:

yes

Conclusions:

From the BCFs obtained, it can be concluded that the test substance, T-9601, is not bioaccumulative in carp.

Executive summary:

A bioconcentration study of the test substance, T-9601, with carp (Cyprinus carpio) was performed according to the standard method described in the governmental order, which prescribes the procedure of testing new chemical substances as required by the Chemical Substances Control Law (Japanese law 117, 1974). This method is also known as the "Bioaccumulation, Test for the Degree of Bioconcentration in Fish" in the OECD Guidelines for the Testing of Chemicals No.305C(1981). Study conducted in compliance with GLP.

The 48-hr LC50 of the test substance to orange killifish was more than 186 ppm. Accordingly, the nominal concentrations to be used in the bio-concentration test were set at 0.1 ppm and 0.01 ppm. The exposure period of the test was set at 8 weeks. We obtained the bio-concentration factors (BCFs) ranging from 15 to 58 for the 0.1 ppm exposure level and from 62 to 124 for the 0.01 ppm exposure level.

 

From the BCFs obtained, it can be concluded that the test substance, T-9601, is not bioaccumulative in carp.

 

On the basis of this data, the substance cannot be considered as bioaccumulative under environmentally relevant conditions. The substance is not considered to pose a chronic hazard to the aquatic environment on the basis of this data.

Description of key information

From the BCFs obtained, it can be concluded that the test substance, T-9601, is not bioaccumulative in carp.

Key value for chemical safety assessment

BCF (aquatic species):

124 dimensionless

Additional information

A bioconcentration study of the test substance, T-9601, with carp (Cyprinus carpio) was performed according to the standard method described in the governmental order, which prescribes the procedure of testing new chemical substances as required by the Chemical Substances Control Law (Japanese law 117, 1974). This method is also known as the "Bioaccumulation, Test for the Degree of Bioconcentration in Fish" in the OECD Guidelines for the Testing of Chemicals No.305C(1981). Study conducted in compliance with GLP.

The 48-hr LC50 of the test substance to orange killifish was more than 186 ppm. Accordingly, the nominal concentrations to be used in the bio-concentration test were set at 0.1 ppm and 0.01 ppm. The exposure period of the test was set at 8 weeks. We obtained the bio-concentration factors (BCFs) ranging from 15 to 58 for the 0.1 ppm exposure level and from 62 to 124 for the 0.01 ppm exposure level.

 

From the BCFs obtained, it can be concluded that the test substance, T-9601, is not bioaccumulative in carp.

 

On the basis of this data, the substance cannot be considered as bioaccumulative under environmentally relevant conditions. The substance is not considered to pose a chronic hazard to the aquatic environment on the basis of this data.