N-(5-chloro-2-methoxyphenyl)-2-[(2-methoxy-4-nitrophenyl)azo]-3-oxobutyramide

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:

27 September 2022 - 29 October 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD Series on Testing and Assessment no. 23

Version / remarks:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations (loading rates): 0 (control), 0.24, 0.51, 1.08, 2.27, 4.76 and 10.0 mg/L.
- Sampling method: Not specified.
- Sample storage conditions before analysis: all samples were analysed on the same day of collection from the respective test vessels

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
At the date of performing the present study specific OECD guidance documents are under development for aquatic toxicity tests of test chemicals considered as nanomaterials. According to OECD GD 23 on for testing of difficult substances, for nanomaterials both dissolved and undissolved fractions could be included in the exposure. In tests with nanomaterials, particulate uptake has the potential to exert toxic effects that are not solely physical (Petersen E, 2015). For this reason, it was decided to perfom the study in the test medium without implementing further separation technique to remove particule/dispersed fraction. Thus, the test is performed in the test medium with dissolved and undissolved fraction of the test item. The approach described above is conceptually similar to water-accommodated fraction (WAF) used for multicomponent substances in OECD GD 23. Thus, WAF approach was used for the preparation of test solutions.

WAF equilibration trials were performed prior to exposure to determine the optimum mixing duration to be used for WAF preparation:

For time points of 30, 60, 90, 120 minutes, 4, 6 and 24 hours, loading rates of 1, 5 and 10 mg/L were prepared separately. Test item quantities of 1.0, 5.0 and 10.0 mg were dispensed in a separate beaker, to this 1000 mL of reverse osmosis water was added and stirred well by using glass rod and kept on magnetic stirrer at 100 rpm. At respective time points (30, 60, 90, 120 minutes, 4, 6 and 24 hours), stirring was stopped and allowed the solution to equilibrate for one hour and then the samples were collected from the central part of test vessel and analysed for TOC.

After WAF preliminary trials, it was determined that maximum equilibrium was achieved at 60 min, hence 60 min of slow stirring at 100 rpm was selected for preparation of test solutions.

Additionally, samples of this test medium were collected and filtered through 0.22 μm pore size filter in order to determine the actual dissolved fraction present in the exposure medium.

- Test concentration separation factor: 2.1
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish
- Source: Fertilized eggs of Danio rerio were procured from the lab maintained breeding facility.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): not reported
- Method of collection of fertilised eggs: spawn traps were placed into the spawning tank before the onset of darkness on the day prior to test or before the onset of light on day of the test. Mating, spawning and fertilization take place within 30 minutes after the onset of light and the spawn traps with the collected eggs were carefully removed. Viable fertilized eggs were separated from unfertilized eggs, viable fertilized eggs were collected and rinsed with reconstituted water.
- Subsequent handling of eggs: For viable fertilized eggs collected, fertilization rate was calculated and exposed to test solutions.

POST-HATCH FEEDING
- Type/source of feed: flake food. Commercial larvae food for newly hatched larvae fish (3-10 days post-hatch) and BSN48 flake food for juvenile fish.
- Amount given: sufficient level to support normal growth.
- Frequency of feeding: twice daily

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

30 d

Hardness:

169.9 to 208.8 mg CaCO3/L

Test temperature:

25.0 to 25.9°C

pH:

7.14 to 7.78

Dissolved oxygen:

85.40 to 97.70 %

Nominal and measured concentrations:

Nominal loading rates: 0 (control), 0.24, 0.51, 1.08, 2.27, 4.76 and 10.0 mg/L.
Measured concentrations: see table included in 'Any other information on results incl. tables'.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquarium vessels
- Material, size, headspace, fill volume: glass, 3-7L fill volume
- Renewal rate of test solution (frequency/flow rate): 24 hours
- No. of fertilized eggs/embryos per vessel: 20 eggs per vessel, 80 eggs per group
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: loading of fish embryos was low enough in order that a dissolved oxygen concentration of at least 60% of the air saturation value was maintained without aeration during the egg and larval stage, as recommended.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis water
- Culture medium different from test medium: no
- Intervals of water quality measurement: Dissolved oxygen, temperature and pH were measured in all the test vessels at the beginning and at every renewal period, hardness at the beginning and once a weak until the termination of test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 12 - 16 hours (constant light regime)
- Light intensity: n.a.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The embryonic stage at the beginning of exposure to the test item was verified using a representative sample of eggs suitably preserved and cleared. Observation for Hatching and survival was made daily, and numbers were recorded. Dead embryos, larvae and juvenile fish were removed as soon as observed. If fungus were detected on eggs in early embryonic development, those eggs were counted and removed. Number of larvae or fish showing abnormality of body form was recorded at 2 days intervals. Abnormal fish was removed from the test vessels on death. Abnormalities (atypical quiescence and loss of balance) was recorded at 2 days intervals. Wet weight (blotted dry) and individual length (total length) of all surviving fish was recorded on replicates basis at the end of the test.

RANGE-FINDING STUDY
- Test concentrations: nominal loading rates of 0 (control), 0.01, 0.1, 0.5, 1, 5 and 10 mg/L.
- Results used to determine the conditions for the definitive study: The results obtained were the following:
Hatching success of 92.5, 88.8, 83.8, 78.8, 61.3 and 18.8% was observed at the nominal loading rates of 0.01, 0.1, 0.5, 1.0, 5.0 and 10.0 mg/L, respectively.
Larval survival until day 30 in the control group was 82.4%. Larval survival of 79.7, 77.5, 73.1, 66.7, 38.8 and 0.0 % was observed at the loading rates of 0.01, 0.1, 0.5, 1.0, 5.0 and 10.0 mg/L, respectively.
No abnormal appearance was observed in the control and the nominal loading rate of 0.01 mg/L.
Abnormal appearance of bent tail (tail malformations) was observed at the loading rate of 0.1 mg/L. Abnormal appearance of bent tail (tail malformations), darkened coloration and scoliosis were observed at the loading rates of 0.5, 1.0 and 5.0 mg/L.
Abnormal appearance of darkened coloration and scoliosis were observed at the loading rate of 10.0 mg/L.

Reference substance (positive control):

not required

Key result

Duration:

30 d

Dose descriptor:

EL50

Effect conc.:

4.59 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Key result

Duration:

30 d

Dose descriptor:

EL50

Effect conc.:

1.562 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

30 d

Dose descriptor:

EL50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Key result

Duration:

30 d

Dose descriptor:

EL50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Details on results:

Details on tables included in 'Any other information on results incl. tables'.

Reported statistics and error estimates:

Statistical analysis for NOELR and LOELR was made for larvae hatching, post emergence survival, fish length and fish weight. In order to make a multiple comparison between the results at the individual concentrations and those for the controls Dunnett’s method was employed.
EL10, EL25 and EL50 was calculated using SYS STAT Version No. 13 method for larvae hatching success, larvae survival, fish length and fish wet weight.

Table 1. Hatching Success, fish larval survival and percent mortality – dose range finding study

Group	Nominal Loading Rate  (mg/L)	Replicate	Number Of Eggs Added at The Start Of The Test		Number of Hatched Larvae		Percentage of Hatched Larvae		Number of Larvae Surviving at 30 Days Post-Hatch		Percentage of Larvae Surviving at 30 Days Post Hatch		Mortality Percent
			Individual Vessels	Polled                     vessels	Individual Vessels	Polled vessels	Individual Vessels	Polled vessels	Individual Vessels	Polled                 vessels	Individual Vessels	Polled vessels	No. of Mortality	Polled vessels	% Mortality
G1	0.0	1	20	80	18	74	90	92.5	16	61	89	82.4	2	13	17.6
		2	20		19		95		15		79		4
		3	20		18		90		16		89		2
		4	20		19		95		14		74		5
G2	0.01	1	20	80	19	74	95	92.5	16	59	84	79.7	3	15	20.3
		2	20		18		90		14		78		4
		3	20		19		95		14		74		5
		4	20		18		90		15		83		3
G3	0.1	1	20	80	17	71	85	88.8	14	55	82	77.5	3	16	22.5
		2	20		18		90		12		67		6
		3	20		18		90		13		72		5
		4	20		18		90		16		89		2
G4	0.5	1	20	80	18	67	90	83.8	15	49	83	73.1	3	18	26.9
		2	20		17		85		10		59		7
		3	20		15		75		12		80		3
		4	20		17		85		12		71		5

G5	1.0	1	20	80	17	63	85	78.8	10	42	59	66.7	7	21	33.3
		2	20		15		75		12		80		3
		3	20		16		80		10		63		6
		4	20		15		75		10		67		5
G6	5.0	1	20	80	10	49	50	61.3	4	19	40	38.8	6	30	61.2
		2	20		14		70		5		36		9
		3	20		12		60		7		58		5
		4	20		13		65		3		23		10
G7	10.0	1	20	80	4	15	20	18.8	0	0	0	0.0	4	15	100.0
		2	20		3		15		0		0		3
		3	20		4		20		0		0		4
		4	20		4		20		0		0		4

 

Table 2. Percent inhibition in larvae hatching, surviving, length and fresh weight – dose range finding study

Group	Nominal Loading Rate  (mg/L)	No. of Embryo at the Start Day 0	Percent Inhibition in Larvae Hatching	Percent Inhibition in Post Larvae Survival	Percent Inhibition of Larvae Length	Percent Inhibition of Fish Fresh Weight
G1	0	80	-	-	-	-
G2	0.01	80	0.00	3.28	1.83	0.07
G3	0.1	80	4.05	9.84	5.67	14.91
G4	0.5	80	9.46	19.67	8.45	23.20
G5	1.0	80	14.86	31.15	14.91	32.95
G6	5.0	80	33.78	68.85	32.71	59.18
G7	10.0	80	79.73	100.00	-	100.00

Percent inhibition calculated in comparison with the control group

 

Table 3. Hatching Success, fish larval survival and percent mortality – Main study

Group	Nominal Loading Rate  (mg/L)	Replicate	Number of eggs added at the start of the test		Number of hatched larvae		Percentage of hatched larvae		Number of larvae surviving at 30 days post-hatch		Percentage of larvae surviving at 30 days post hatch		Mortality Percent
			Individual Vessels	Polled                     vessels	Individual Vessels	Polled vessels	Individual Vessels	Polled Vessels	Individual Vessels	Polled                 Vessels	Individual Vessels	Polled vessels	No. of Mortality	Polled vessels	% Mortality
G1	0.0	1	20	80	19	72	95	90.0	18	62	95	86.1	1	10	13.9
		2	20		18		90		17		94		1
		3	20		17		85		14		82		3
		4	20		18		90		13		72		5
G2	0.24	1	20	80	18	71	90	88.8	15	59	83	83.1	3	12	16.9
		2	20		16		80		15		94		1
		3	20		18		90		16		89		2
		4	20		19		95		13		68		6
G3	0.51	1	20	80	18	68	90	85.0	14	50	78	73.5	4	18	26.5
		2	20		17		85		11		65		6
		3	20		16		80		12		75		4
		4	20		17		85		13		76		4
G4	1.08	1	20	80	16	59	80	73.8	10	39	63	66.1	6	20	33.9
		2	20		15		75		11		73		4
		3	20		14		70		9		64		5
		4	20		14		70		9		64		5

Group	Nominal Loading Rate  (mg/L)	Replicate	Number of Eggs Added at the Start of the Test		Number of Hatched Larvae		Percentage of Hatched Larvae		Number of Larvae Surviving at 30 Days Post-Hatch		Percentage of Larvae Surviving at 30 days Post Hatch		Mortality Percent
			Individual Vessels	Polled                     vessels	Individual Vessels	Polled vessels	Individual Vessels	Polled vessels	Individual Vessels	Polled                 vessels	Individual Vessels	Polled vessels	No. of Mortality	Polled vessels	% Mortality
G5	2.27	1	20	80	12	48	60	60.0	7	28	58	58.3	5	20	41.7
		2	20		14		70		8		57		6
		3	20		10		50		7		70		3
		4	20		12		60		6		50		6
G6	4.76	1	20	80	8	37	40	46.3	3	10	38	27.0	5	27	73.0
		2	20		10		50		2		20		8
		3	20		9		45		2		22		7
		4	20		10		50		3		30		7
G7	10.0	1	20	80	5	21	25	26.3	0	0	0	0.0	5	21	100.0
		2	20		6		30		0		0		6
		3	20		5		25		0		0		5
		4	20		5		25		0		0		5

 

Table 4. Physico-chemical parameters during exposure period – Main study

Group	Nominal Loading Rate (mg/L)	Dissolved Oxygen				Temperature				pH
		Fresh		Spent		Fresh		Spent		Fresh		Spent
		Min	Max	Min	Max	Min	Max	Min	Max	Min	Max	Min	Max
G1	0.0	88.10	93.60	90.60	95.70	25.1	25.9	25.1	25.9	7.22	7.69	7.14	7.78
G2	0.24	88.20	93.80	90.50	97.70	25.0	25.9	25.1	25.9	7.22	7.72	7.20	7.76
G3	0.51	89.30	93.40	91.20	96.10	25.1	25.9	25.1	25.9	7.24	7.78	7.21	7.78
G4	1.08	88.10	93.40	90.50	96.20	25.1	25.9	25.0	25.9	7.25	7.72	7.24	7.74
G5	2.27	85.40	93.10	90.80	96.30	25.1	25.9	25.1	25.9	7.24	7.77	7.24	7.76
G6	4.76	88.20	93.60	91.30	96.20	25.1	25.9	25.1	25.9	7.21	7.74	7.25	7.75
G7	10.00	88.70	93.60	91.80	96.30	25.1	25.9	25.1	25.9	7.25	7.73	7.21	7.71
Min		85.40				25.0				7.14
Max		97.70				25.9				7.78

Water Hardness
Days	0	1	7	8	14	15	21	22	27	28
Fresh	169.6		191		183.9		180.3		183.9
Spent		171.4		199.9		208.8		194.6		199.9
Minimum	169.9
Maximum	208.8

 

Table 5. Percent inhibition in larvae hatching, surviving, length and fresh weight – Main study

Group	Nominal Loading (mg/L)	No. of Embryo at the Start Day 0	Percent Inhibition in Larvae Hatching	Percent Inhibition in Post Larvae Survival	Percent Inhibition of Larvae Length	Percent Inhibition of Fish Fresh Weight
G1	0.0	80	-	-	-	-
G2	0.24	80	1.39	4.84	1.78	4.07
G3	0.51	80	5.56	19.35	12.69*	8.00*
G4	1.08	80	18.06*	37.10*	16.53*	14.96*
G5	2.27	80	33.33*	54.84*	17.76*	23.43*
G6	4.76	80	48.61*	83.87*	20.73*	34.96*
G7	10.0	80	70.83*	100.00*	-	-

*: Statistical significance (>0.05).

 

Table 6. Summary of Results for all Endpoints at the End of Exposure Period – Main study

Parameters	End Points  (mg/L)
Larvae Hatching Success	EL10	0.725	EL25	1.824	EL50	4.59
	Lower limit	0.508	Lower limit	1.474	Lower limit	3.831
	Upper Limit	0.946	Upper Limit	2.19	Upper Limit	5.658
	NOELR	0.51
	LOELR	1.08
Larvae Surviving	EL10	0.382	EL25	0.772	EL50	1.562
	Lower limit	0.286	Lower limit	0.636	Lower limit	1.355
	Upper Limit	0.478	Upper Limit	0.907	Upper Limit	1.797
	NOELR	0.51
	LOELR	1.08
Fish Length	EL10	0.628	EL25	5.485	EL50	nd
	Lower limit	0.189	Lower limit	3.001	Lower limit	nd
	Upper Limit	1.082	Upper Limit	24.047	Upper Limit	nd
	NOELR	0.24
	LOELR	0.51
Fish fresh weight	EL10	0.676	EL20	2.57	EL50	nd
	Lower limit	0.373	Lower limit	1.91	Lower limit	nd
	Upper Limit	0.971	Upper Limit	3.725	Upper Limit	nd
	NOELR	0.24
	LOELR	0.51

nd: Not determined  (as there was not percent inhibition of >50%).

 

Table 7. Water accommodation fraction (WAF) equilibrium trail

Loading Rate (mg/L)	Time Points of Analysis
	30 Mins	60 Mins	90 Mins	120 Mins	4 Hour	6 Hour	24 Hour
1	0.426	0.8604	0.6731	0.3485	0.3469	0.389	0.3523
5	0.782	1.151	0.4296	0.4675	0.9578	1.120	0.7601
10	1.505	2.630	1.271	0.7041	0.9854	1.917	0.9935

Maximum TOC values obtained at 60 minutes, hence equilibration time of 60 minutes was selected during test media formulation.

 

Table 8. Average TOC values in test media during exposure

TOC Values (ppm) – Dissolved and undissolved
Day 0	Day 1	Day 10	Day 11	Day 15	Day 16	Day 22	Day 23	Day 25	Day 26	Day 30	Day 31
Fresh	Spent	Fresh	Spent	Fresh	Spent	Fresh	Spent	Fresh	Spent	Fresh	Spent
0.235	0.345	0.365	0.2001	0.2962	0.1974	0.2153	0.2563	0.246	0.219	0.2376	0.2586

TOC (ppm) in control group during the exposure period

 

Average TOC values for the exposure period
Main Study
Group	Nominal Loading Rate (mg/L)	Dissolved and Undissolved TOC - ppm
		Fresh	Spent
G1	0.00	0.2492	0.2461
G2	0.24	*0.3313	*0.4121
G3	0.51	*0.7971	*0.4488
G4	1.08	*1.0106	*0.5491
G5	2.27	*0.9714	*0.7019
G6	4.76	*1.3480	*1.1319
G7	10.00	*1.6189	*1.0302

*: Values presented are the subtracted values from the control.

 

TOC Values (ppm) – Filtered
Day 0	Day 1	Day 10	Day 11	Day 15	Day 16	Day 22	Day 23	Day 25	Day 26	Day 30	Day 31
Fresh	Spent	Fresh	Spent	Fresh	Spent	Fresh	Spent	Fresh	Spent	Fresh	Spent
0.3057	0.32	0.1443	0.2794	0.2962	0.1974	0.2153	0.2563	0.246	0.219	0.2376	0.2586

TOC (ppm) in control group during the exposure period.

Average TOC values for the exposure period
Main Study
Group	Nominal Loading Rate (mg/L)	Filtered TOC - ppm
		Fresh	Spent
G1	0.00	0.2409	0.2551
G2	0.24	*0.2953	*0.2885
G3	0.51	*0.5410	*0.6078
G4	1.08	*0.9284	*0.6186
G5	2.27	*0.9337	*0.7360
G6	4.76	*1.3030	*1.3642
G7	10.00	*1.4035	*1.1214

*: Values presented are the subtracted values from the control.

Filtered values should be in theory lower than the Dissolved and Undissolved values. However, results do not clearly show this. Variation in the TOC (ppm) values in the nominal loading rates may be due to the nature of test item, as the test item is a nanomaterial; the distribution and agglomeration of nanomaterial may have an effect during the sampling period. This could be the cause of these unexpected TOC values obtained in the nominal loading rates.

 

Validity criteria fulfilled:

yes

Conclusions:

Based on the results obtained under test conditions, the EL50 for larvae surviving and hatching success was found to be 1.562 mg/L and 4.59 mg/L respectively.

Executive summary:

A Fish, Early-life Stage Toxicity Test was performed on the test item, over a period of 30 days in semi-static conditions, according to OECD TG 210, under GLP conditions.

The test was performed in the test medium with dissolved and undissolved fraction of the test item. WAF approach was used for the preparation of test solutions. WAF equilibration trials were performed prior to exposure to determine the optimum mixing duration to be used for WAF preparation. After this preliminary test, it was determined that maximum equilibrium was achieved at 60 min, hence 60 min of slow stirring at 100 rpm was selected for preparation of test solutions. Additionally, samples of this test medium were collected and filtered through 0.22 μm pore size filter in order to determine the actual dissolved fraction present in the exposure medium. Nominal loading rates of 0 (control), 0.01, 0.1, 0.5, 1, 5 and 10 mg/L were used in a dose range finding study. Based on results of this preliminary test, the main study was performed at the nominal loading rates of 0 (control), 0.24, 0.51, 1.08, 2.27, 4.76 and 10.0 mg/L. There is no specific analytical method for identification/quantification of the test substance, thus the test substance was analyzed indirectly by TOC method.

Danio rerio (zebrafish) embryo were exposed to different test concentrations of test item in reverse osmosis water under semi-static conditions, test media was renewed once in 24 hours until the termination of the test. The test design included seven groups with four replicates per concentration and the control, containing 20 fertilized eggs per test vessel. These were observed to establish hatching success, larvae survival, fish weight, fish length, abnormal appearance and abnormal behaviour. 

Based on the results obtained under test conditions, the EL50 for hatching success, larvae survival, fish weight and fish length was found to be 4.59 mg/L, 1.562 mg/L, >10 mg/L and >10 mg/L respectively.

Description of key information

Key study. Method according to OECD 210. The 30-day EL50 of the test item in Danio rerio (zebrafish) embryo was found to be 1.562 mg/L and 4.59 mg/L for larvae surviving and hatching success respectively.