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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14/03/2018 to 04/04/2018

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 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

See "Any other information" for details

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 details can be found in 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. The test solutions were prepared continuously by dilution of this saturated solution with peristaltic pump.

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia, Daphnia magna Strauss clone 5, between 6 and 24 hours old and produced from parthenogenically reproducing brood female population were used. Daphnia are issued from the breeding lab. The juvenile stemming at least from the second litter were used to start the test.
Culture conditions:
Medium: Elendt M4
Temperature: 20 °C ± 2
Light/dark cycle: 16 h/8 h
pH: Between 6 and 9
Hardness (CaCO3): Between 140 and 250 mg.L-1
Daily feeding: Pseudokirchneriella subcapitata and Chlorella vulgaris suspensions (about 10 mL/replicates/day)

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Remarks on exposure duration:

As per the guideline.

Post exposure observation period:

No post exposure observations period specified in the study report.

Hardness:

(CaCO3): Between 140 and 250 mg.L-1

Test temperature:

20.4 - 21.1 °C

pH:

7.8 - 8.1

Dissolved oxygen:

8.6 - 9.0 %

Salinity:

Not applicable.

Conductivity:

Not specified

Nominal and measured concentrations:

definitive measured concentrations of 0.52; 2.67; 9.87; 27.14 and 375.25 μg.L-1

Details on test conditions:

Based on the results of the range-finder, 5 concentrations were prepared in Elendt-M4 medium. A control was set up with the test medium only. There were 4 replicates for each treatment with 10 daphnia per replicate.
Organisms were maintained at temperature of 20°C ± 2°C and light intensity between 1000 and 1500 lux with a light:dark cycle of 16 hours light and 8 hours dark.
4 to 5 times a week, the number of living offspring produced in each replicate were recorded. pH dissolved oxygen and temperature were recorded once a week.
Every day, the daphnias were fed with Pseudokirchneriella subcapitata and Chlorella vulgaris suspensions (about 10 mL per day).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

2.65 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

22.8 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.68 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: mortality & reproduction

Details on results:

The study is valid since:
-the mortality of the parent animals (female Daphnia) was not exceed 20% at the end of the test.
-the mean number of living offspring produced per parent animal surviving at the end of the test was ≥ 60.
-EC50 with reference item over a period of 24 hours must be between 0.6 and 2.1 mg.L-1.
Based on the reproduction rate, the EC50 after a 21 days exposure period was calculated to be 2.65 μg.L-1. Based on the survival rate, the LC50 after a 21 days exposure period was calculated to be 22.8 μg.L-1.
The lowest NOEC was estimated to be at 0.68 μg.L-1.

Results with reference substance (positive control):

24-hour EC50 (mg.L-1): 1.428. See "Any other information on results incl. tables" below.

Reported statistics and error estimates:

The LOEC, the NOEC and the ECx were determined using the following data:
-The total number of living offspring per parent animal not accidentally or fortuitously dead at the end of the test.
-The total number of living offspring per parent animal alive at the end of the test.
The NOEC (No Observed Effect Concentration) were determined by statistical test from replicate data on survival and reproduction. Statistical calculations were made using R statistical software and the R script MOSAIC repro and surv.

POTASSIUM DICHROMATE RESULTS

24 hours after exposure

ID	Rate [mg reference item.L^-1]	Immobilized daphnids 24 hours after exposure				Total of immobilized daphnids	Mean of immobilized daphnids (%)
		Replicates
		A	B	C	D
T	0	0	0	0	0	0	0
C1	0.5	0	0	0	0	0	0
C2	0.9	0	1	0	0	1	5
C3	1.35	3	1	2	2	8	40
C4	2.025	5	4	4	5	18	90
C5	3.0375	5	5	5	5	20	100

 

24-hour EC50

24-hour EC50 (mg.L^-1)	95% confidence limits (mg.L^-1)	Starting date
1.428	13.48 – 1.509	21 February 2018

 

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

pH	C11	C12	C13	C14	C15	C16
14/03/18	7.9	7.9	7.9	7.9	7.9	8.0
23/03/18	7.9	7.9	7.8	8.0	7.9	8.1
29/03/18	8.0	8.1	8.1	8.1	8.0	8.0
04/04/18	7.9	8.0	8.0	7.9	8.0	8.1

 

Dissolved Oxygen (mg.L^-1)	C11	C12	C13	C14	C15	C16
14/03/18	8.8	8.8	8.7	8.6	8.9	8.7
23/03/18	8.7	8.7	8.7	8.7	8.7	8.7
29/03/18	8.9	9.0	8.8	8.9	8.9	8.8
04/04/18	8.7	8.7	8.6	8.7	8.8	8.7

 

	Temperature (°C)
14/03/18	21.1
23/03/18	20.4
29/03/18	21.1
04/04/18	20.9

 

Reproduction and survival

	Replicates	Number of juveniles							Total juveniles per replicate	Number juveniles per adult	Number of adults of the end of the test	Total juveniles per surviving adult	Mean juveniles per surviving parent	Coefficient of variation for control fecundity
		Day 12	Day 13	Day 14	Day 15	Day 16	Day 20	Day 21
Control C11	R1	62	20	116	56	129	265	111	759	75.9	10	75.9	86.3	-
	R2	69 (1 dead)	35	81	49	140	248	178	800	80	9	88.9
	R3	72	28 (1 dead)	104	52	165	279	124	824	82.4	9	91.6
	R4	119 (1 dead)	32	136	57	125	251	78	798	79.8	9	88.7
0.68 μg. L^-1  C12	R1	76 (1 dead day 9)	41	81 (1 dead)	46	89	307	73	713	71.3	8	89.1	84.7	2%
	R2	95	29	102	55	112	294	138	825	82.5	10	82.5
	R3	73	37 (1 dead)	131	58	106 (1 dead)	245	33	683	68.3	8	85.4
	R4	74 (1 dead)	28	138	50	99	282	64	735	73.5	9	81.7
2.67 μg.L^-1  C13	R1	0 (2 dead (days 7 and 8))	10	61	28	52	156	10	317	31.7	8	39.6	45.3	47%
	R2	16 (2 dead (days 5 and 8))	17	35	24	33	142	51	318	31.8	8	39.8
	R3	0 (2 dead day 8)	12	69	29	84	161	18	373	37.3	8	46.6
	R4	37 (2 dead day 7)	15	57 (1 dead)	31	38	139 (1 dead)	14	331	33.1	6	55.2
9.87 μg.L^-1  C14	R1	0 (2 dead day 7 and 1 dead day 8)	0	1	2	0	4	5	12	1.2	7	1.7	1.5	97%
	R2	0	0	1	1	0	6	0	8	0.8	10	0.8
	R3	0 (1 dead day 6)	0	0	0	0	10	0	10	1	9	1.1
	R4	2 (1 dead day 8)	0	0	2	0	8	9	21	2.1	9	2.3
27.14 μg.L^-1  C15	R1	0 (3 dead day 5, 2 dead days 6 and 7, 3 dead day 9)	0	0	0	0	0	0	0	0	0	0	0.0	100%
	R2	0 (5 dead day 5, 4 dead day 6, 1 dead day 8)	0	0	0	0	0	0	0	0	0	0
	R3	9 (2 dead day 5, 4 dead day 6, 3 dead day 7, 1 dead day 8)	0	0	0	0	0	0	0	0	0	0
	R4	0 (2 dead days 6 and 7, 6 dead day 8)	0	0	0	0	0	0	0	0	0	0
375.25 μg.L^-1  C16	R1	0 (10 dead day 2)	0	0	0	0	0	0	0	0	0	0	0.0	100%
	R2	0 (10 dead day 2)	0	0	0	0	0	0	0	0	0	0
	R3	0 (9 dead day 2, 1 dead day 3)	0	0	0	0	0	0	0	0	0	0
	R4	0 (8 dead day 2, 2 dead day 3)	0	0	0	0	0	0	0	0	0	0

 

Validity criteria fulfilled:

yes

Conclusions:

The effects of C.I. Solvent Yellow 124 diluted in OECD test medium at the definitive measured concentrations of 0.68; 2.67; 9.87; 27.14 and 375.25 µg.L-1 to Daphnia magna were investigated under laboratory conditions.
Based on the reproduction rate, the EC50 after a 21 days exposure period was calculated to be 2.65 µg.L-1. Based on the survival rate, the LC50 after a 21 days exposure period was calculated to be 22.8 µg.L-1.
The lowest NOEC was estimated to be at 0.68 µg.L-1.

Executive summary:

The objective of this study was to assess the effects of the test item on the reproductive output of Daphnia magna over a period of 21 days according to OECD 211 guideline (2 october 2012).

Daphnia magna is a freshwater crustacean that is recommended by the OECD Guideline 211.

 

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 C.I. Solvent Yellow 124 diluted in OECD test medium at the definitive measured concentrations of 0.68; 2.67; 9.87; 27.14 and 375.25 µg.L^-1to Daphnia magna were investigated under laboratory conditions.

 

Results are the following:

	C.I. Solvent Yellow 124
LC10 Survival (95% confidence limits)	0.431 µg.L^-1 [0.096 µg.L^-1; 1.15 µg.L^-1]
LC50 Survival (95% confidence limits)	22.8 µg.L^-1 [11.6 µg.L^-1; 50.9 µg.L^-1]
EC10 Reproduction (95% confidence limits)	1.29 µg.L^-1 [1.19 µg.L^-1; 1.4 µg.L^-1]
EC50 Reproduction (95% confidence limits)	2.65 µg.L^-1 [2.54 µg.L^-1; 2.76 µg.L^-1]

LC = lethal concentration  

EC = effect concentration

 

	C.I. Solvent Yellow 124
NOEC Survival	0.68 µg.L^-1
NOEC Reproduction	0.68 µg.L^-1

 

The study is valid since:

-the mortality of the parent animals (female Daphnia) was not exceed 20% at the end of the test.

-the mean number of living offspring produced per parent animal surviving at the end of the test was ≥ 60.

-EC50 with reference item over a period of 24 hours must be between 0.6 and 2.1 mg.L^-1.

 

Based on the reproduction rate, the EC50 after a 21 days exposure period was calculated to be 2.65 µg.L^-1. Based on the survival rate, the LC50 after a 21 days exposure period was calculated to be 22.8 µg.L^-1.

The lowest NOEC was estimated to be at 0.68 µg.L^-1.

Description of key information

The lowest NOEC was estimated to be at 0.68 μg.L-¹.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.68 µg/L

Additional information

The objective of this study was to assess the effects of the test item on the reproductive output of Daphnia magna over a period of 21 days according to OECD 211 guideline (2 october 2012).

Daphnia magna is a freshwater crustacean that is recommended by the OECD Guideline 211.

 

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 C.I. Solvent Yellow 124 diluted in OECD test medium at the definitive measured concentrations of 0.68; 2.67; 9.87; 27.14 and 375.25 µg.L^-1to Daphnia magna were investigated under laboratory conditions.

 

Results are the following:

	C.I. Solvent Yellow 124
LC10 Survival (95% confidence limits)	0.431 µg.L^-1 [0.096 µg.L^-1; 1.15 µg.L^-1]
LC50 Survival (95% confidence limits)	22.8 µg.L^-1 [11.6 µg.L^-1; 50.9 µg.L^-1]
EC10 Reproduction (95% confidence limits)	1.29 µg.L^-1 [1.19 µg.L^-1; 1.4 µg.L^-1]
EC50 Reproduction (95% confidence limits)	2.65 µg.L^-1 [2.54 µg.L^-1; 2.76 µg.L^-1]

LC = lethal concentration  

EC = effect concentration

 

	C.I. Solvent Yellow 124
NOEC Survival	0.68 µg.L^-1
NOEC Reproduction	0.68 µg.L^-1

 

The study is valid since:

-the mortality of the parent animals (female Daphnia) was not exceed 20% at the end of the test.

-the mean number of living offspring produced per parent animal surviving at the end of the test was ≥ 60.

-EC50 with reference item over a period of 24 hours must be between 0.6 and 2.1 mg.L^-1.

 

Based on the reproduction rate, the EC50 after a 21 days exposure period was calculated to be 2.65 µg.L^-1. Based on the survival rate, the LC50 after a 21 days exposure period was calculated to be 22.8 µg.L^-1.

The lowest NOEC was estimated to be at 0.68 µg.L^-1.