N-ethyl-N-[2-[1-(2-methylpropoxy)ethoxy]ethyl]-4-(phenylazo)aniline

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 January 2018 to 8 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study, conducted to GLP and current methodology.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

yes

Details on sampling:

Test item quantification was performed according to a validated analytical method (RRCo-000072-02).
Full study report under Section 8 - Analytical methods.

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

Since the test item is poorly soluble under the test conditions, a saturated solution of test item (limit of solubility LS) was prepared. A stock solution in acetone was prepared and mixed with glass beads. The solvent was eliminated with rotary evaporator and the glass beads were rinsed with water. Then, the saturated solution was prepared by agitation of the glass beads in test medium during at least 24h with a loading rate approximately 2g/L.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Fertilised eggs of Danio rerio produced from the laboratory genitors were used as soon as possible after fertilisation. At the laboratory, male and female genitors were separated in aquariums with a continuous renewal of the water.
The day before the experiment, male and females were added in the nest box. Egg laying and fertilisation occurred at « dawn », simulated with lighting. Eggs were harvested and a first selection was done in order to eliminate not fertilised eggs.
Breeding conditions for genitors were the following:
Medium: water from the water system
Temperature: 23 °C ± 2
Light/dark cycle: 16 h/8 h
Dissolved oxygen concentration: ≥ 80 % of the air saturation value
Feeding: daily

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

30 d

Remarks on exposure duration:

As per guideline.

Post exposure observation period:

No post exposure observation period specified.

Hardness:

Not specified

Test temperature:

24 - 25 °C

pH:

7.4 - 7.8

Dissolved oxygen:

81.0 - 95.3 %

Salinity:

Not applicable.

Conductivity:

Not specified

Nominal and measured concentrations:

5 concentrations at approximately 2.56 – 6.4 – 16 – 40 and 100 μg/L were prepared in ASTM medium.

Details on test conditions:

Based on the results of the range-finder, 5 concentrations at approximately 2.56 – 6.4 – 16 – 40 and 100 μg/L were prepared in ASTM medium. A control was set up with the test medium only. There were 4 replicates for each treatment with 20 eggs per replicate.
The test was performed in an experimental room maintained at 26° C ± 1.5 with a day / night cycle of 16h / 8h.
pH and oxygen of test solutions were recorded once a week.
Before hatching, the number of coagulated eggs was noted, and after hatching, the number of deaths in each replicate was recorded daily. The test was performed during 30 days post hatching.
At the end of the test, fish were measured individually and then weighed in batches. Means of lengths and weights were calculated by replicate.
Two days post hatching, fish were fed with Nobilfluid ® and live rotifera (Brachionus plicatilis) twice or three times a day. Eight days post hatching, Nobilfluid ® was stopped and live artemia (Artemia salina) were added.
Fish were euthanized in order to carry out the measurements of weight and length. For this, fish were placed for about two minutes in a crystallizer containing sodium methanesulfonate or MS222 (CAS number 886-86-2) at a lethal concentration of 300 mg.L-1.

Reference substance (positive control):

no

Key result

Duration:

30 d

Dose descriptor:

EC50

Effect conc.:

> 607.7 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Key result

Duration:

30 d

Dose descriptor:

EC50

Effect conc.:

20.81 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

1.66 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

607 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: hatching

Key result

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

35.04 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Key result

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

35.04 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: wet weight

Key result

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

35.04 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: dry weight

Details on results:

The effects of test item solution diluted in ASTM medium at the definitive measured concentrations of 1.66; 8.55; 35.04; 211.7; and 607.70 μg.L-1 to fertilised eggs of Danio rerio were investigated under laboratory conditions.
The study is valid since:
- the hatching rate in the control was higher than 70 % (actual value: 97.5 % of hatching in the control),
- the post-hatching survival rate in the control was higher than 75 % (actual value : 89.8 % of survival in the control),
- the dissolved oxygen during the test was higher than 60 % of the air saturation,
- the water temperature has not varied by more than 1.5°C.

Results with reference substance (positive control):

Not required

Reported statistics and error estimates:

The hatching, mortality and abnormality percentage were determined for each concentration. EC10 (Effect Concentration for 10% effect), EC50 and confidence limits were obtained by calculation according to Regtox® Macro (“Vindimian”), using the Hill model.
The equation of Hill (1910) was described to model the binding of oxygen to haemoglobin. It was shown to be relevant for many other mechanisms like binding of a ligand to a receptor, Michaelian or non Michaelian enzymatic kinetics, dose or concentration response in toxicology and ecotoxicology (Vindimian 1983, Garric 1990). The logistic model is also used which is similar to the Hill model.

Physical-chemical parameters (pH, Oxygen, Temperature) (mean of 4 replicates):

 

pH	C01	C02	C03	C04	C05	C06
2018-01-26	7.5	7.5	7.5	7.5	7.6	7.6
2018-02-02	7.8	7.8	7.8	7.8	7.8	7.8
2018-02-07	7.5	7.5	7.5	7.5	7.6	7.6
2018-02-15	7.5	7.5	7.5	7.5
2018-02-22	7.4	7.5	7.5	7.4
2018-03-01	7.4	7.4	7.4	7.4
2018-03-05	7.4	7.5	7.5	7.4

 

Dissolved Oxygen (%)	C01	C02	C03	C04	C05	C06
2018-01-26	87.0	85.0	84.8	81.0	82.5	79.5
2018-02-02	95.3	96.8	99.5	90.2	94.1	96.2
2018-02-07	93.5	93.6	94.2	92.0	93.2	92.2
2018-02-15	93.1	93.9	93.7	93.6
2018-02-22	92.1	90.5	90.4	89.1
2018-03-01	90.8	90.0	87.5	91.2
2018-03-05	90.5	88.9	90.8	88.1

 

Temperature (°C)	C01	C02	C03	C04	C05	C06
2018-01-26	25.4	25.4	25.4	25.4	25.4	25.4
2018-02-02	25.2	25.2	25.1	25.1	25.2	25.2
2018-02-07	24.4	24.5	24.5	24.4	24.4	24.5
2018-02-15	25.0	25.0	25.0	25.0
2018-02-22	24.9	24.9	24.9	25.0
2018-03-01	24.8	24.9	24.8	24.8
2018-03-05	25.0	25.1	25.0	24.9

 

Hatching and survival rates

	Replicate	Number of hatches on 2018/02/03	Hatching rate (%)	Hatching rate (%)	Survivors at the end of the test	Survival rate (%)	Survival rate (%)
C01	R1	19	95	97.5	18	94.7	89.8
	R2	19	95		17	89.5
	R3	20	100		16	80.0
	R4	20	100		19	95.0
C02	R1	19	95	86.25	16	74.2	90.9
	R2	19	95		17	89.5
	R3	20	100		19	95.0
	R4	19	95		18	94.7
C03	R1	19	95	96.25	14	73.7	71.6
	R2	19	95		14	73.7
	R3	20	100		12	60.0
	R4	19	95		15	78.9
C04	R1	20	100	100	6	30.0	28.8
	R2	20	100		6	30.0
	R3	20	100		4	20.0
	R4	20	100		7	35.0
C05	R1	20	100	95	0	0.0	0.0
	R2	18	90		0	0.0
	R3	19	95		0	0.0
	R4	19	95		0	0.0
C06	R1	19	95	96.25	0	0.0	0.0
	R2	20	100		0	0.0
	R3	19	95		0	0.0
	R4	19	95		0	0.0

 

Weight and length

	Replicate	Length (mm)	Average length (mm)	Wet weight (g)	Average wet weight (g)	Dry weight (g)	Average dry weight (g)
C01	R1	12.08	12.91	7.67	8.71	1.50	1.56
	R2	13.27		8.88		1.56
	R3	13.30		9.32		1.53
	R4	12.98		8.97		1.64
C02	R1	13.17	12.43	8.16	7.72	1.43	1.28
	R2	12.70		8.48		1.40
	R3	11.74		7.20		1.23
	R4	12.14		7.06		1.07
C03	R1	12.02	11.90	7.61	7.73	1.46	1.31
	R2	11.60		9.11		1.09
	R3	12.63		8.02		1.35
	R4	11.35		6.16		1.34
C04	R1	12.67	12.88	9.77	8.31	1.72	1.60
	R2	12.63		8.05		1.42
	R3	13.97		7.95		1.60
	R4	12.25		7.47		1.67

 

Validity criteria fulfilled:

yes

Conclusions:

The effects of test item solution diluted in ASTM medium at the definitive measured concentrations of 1.66; 8.55; 35.04; 211.7; and 607.70 μg.L-1 to fertilised eggs of Danio rerio were investigated under laboratory conditions.
Based on the hatching rate, the EC50 was estimated to be higher than 607.70 μg.L-1 of test item.L-1 and based on survival rate, the EC50 was calculated to be at 20.81 μg test item.L-1.
The lowest NOEC was estimated to be at 1.66 μg.L-1 corresponding to the NOEC Survival.

Executive summary:

The objective of this study was to assess the effects of test item (C.I. Solvent Yellow 124) on the hatching and survival of fertilized eggs of Danio rerio over a period of 30 days after hatching according to OECD 210 Guideline. Zebrafish (Danio rerio) is a cold freshwater fish recommended by the OECD 210 Guideline.

 

Since the test item is poorly soluble under the test conditions, a saturated solution of test item (limit of solubility LS) was prepared. A stock solution in acetone was prepared and mixed with glass beads. The solvent was eliminated with rotary evaporator and the glass beads were rinsed with water. Then, the saturated solution was prepared by agitation of the glass beads in test medium during at least 24h. The test solutions were prepared by dilution of this saturated solution. The test was performed under continuous flow-through condition.

 

Test item quantification was performed according to a validated analytical method using a LC-MS/MS from Thermo (LC Ultimate 3000 and MS Quantiva) as mass spectrometry detection to measure Yellow 124 concentration once a day in all concentrations. As some of the measured concentrations were not within ± 20% of nominal or initial concentrations, all results were based on mean measured concentrations.

 

The effects of test item solution diluted in ASTM medium at the definitive measured concentrations of 1.66; 8.55; 35.04; 211.7; and 607.70 μg.L^-1to fertilised eggs of Danio rerio were investigated under laboratory conditions.

 

Results are the following:

 

	C.I. Solvent Yellow 124
EC10 hatching	>607.7 μg.L^-1
EC20 hatching	>607.7 μg.L^-1
EC50 hatching	>607.7 μg.L^-1
EC10 survival at test item	4.79 μg.L^-1 (95% confidence limits: 3.40 – 7.74 μg.L^-1)
EC20 survival at test item	8.24 μg.L^-1 (95% confidence limits: 6.25 – 11.69 μg.L^-1)
EC50 survival at test item	20.81 μg.L^-1 (95% confidence limits: 17.25 – 24.64 μg.L^-1)

 

	C.I. Yellow 124
NOEC hatching	607.7 μg.L^-1
NOEC survival	1.66 μg.L^-1
NOEC length	35.04 μg.L^-1
NOEC wet weight	35.04 μg.L^-1
NOEC dry weight	35.04 μg.L^-1

 

The study is valid since:

- the hatching rate in the control was higher than 70 % (actual value: 97.5 % of hatching in the control),

- the post-hatching survival rate in the control was higher than 75 % (actual value : 89.8 % of survival in the control),

- the dissolved oxygen during the test was higher than 60 % of the air saturation,

- the water temperature has not varied by more than 1.5 °C.

 

Based on the hatching rate, the EC50 was estimated to be higher than 607.70 μg.L^-1of test item.L^-1and based on survival rate, the EC50 was calculated to be at 20.81 μg test item.L^-1.

The lowest NOEC was estimated to be at 1.66 μg.L^-1corresponding to the NOEC Survival.

Description of key information

The lowest NOEC was estimated to be at 1.66 μg.L^-1 corresponding to the NOEC Survival.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1.66 µg/L

Additional information

The objective of this study was to assess the effects of test item (C.I. Solvent Yellow 124) on the hatching and survival of fertilized eggs of Danio rerio over a period of 30 days after hatching according to OECD 210 Guideline. Zebrafish (Danio rerio) is a cold freshwater fish recommended by the OECD 210 Guideline.

 

Since the test item is poorly soluble under the test conditions, a saturated solution of test item (limit of solubility LS) was prepared. A stock solution in acetone was prepared and mixed with glass beads. The solvent was eliminated with rotary evaporator and the glass beads were rinsed with water. Then, the saturated solution was prepared by agitation of the glass beads in test medium during at least 24h. The test solutions were prepared by dilution of this saturated solution. The test was performed under continuous flow-through condition.

 

The effects of test item solution diluted in ASTM medium at the definitive measured concentrations of 1.66; 8.55; 35.04; 211.7; and 607.70 μg.L^-1to fertilised eggs of Danio rerio were investigated under laboratory conditions.

 

Results are the following:

 

	C.I. Solvent Yellow 124
EC10 hatching	>607.7 μg.L^-1
EC20 hatching	>607.7 μg.L^-1
EC50 hatching	>607.7 μg.L^-1
EC10 survival at test item	4.79 μg.L^-1 (95% confidence limits: 3.40 – 7.74 μg.L^-1)
EC20 survival at test item	8.24 μg.L^-1 (95% confidence limits: 6.25 – 11.69 μg.L^-1)
EC50 survival at test item	20.81 μg.L^-1 (95% confidence limits: 17.25 – 24.64 μg.L^-1)

 

	C.I. Yellow 124
NOEC hatching	607.7 μg.L^-1
NOEC survival	1.66 μg.L^-1
NOEC length	35.04 μg.L^-1
NOEC wet weight	35.04 μg.L^-1
NOEC dry weight	35.04 μg.L^-1

 

Based on the hatching rate, the EC50 was estimated to be higher than 607.70 μg.L^-1of test item.L^-1and based on survival rate, the EC50 was calculated to be at 20.81 μg test item.L^-1.

The lowest NOEC was estimated to be at 1.66 μg.L^-1corresponding to the NOEC Survival.