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

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:

29 April 2022 - 22 August 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

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), 0.10, 0.29, 0.81, 2.28, 6.38, 17.86 and 50.0 mg/L.
- Sampling method: no 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, 50 and 100 mg/L were prepared separately. Test item quantities of 1.0, 50.0 and 100 mg were dispensed in a separate beaker, to this 1000 mL of reconstituted 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 90 min, hence 90 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: 2.8 (main test)
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: daphnid
- Strain/clone: Daphnia magna Straus
- Age at study initiation (mean and range, SD): less than 24 hours old
- Method of breeding: 25 and 50 pregnant female Daphnids were acclimatized in the reconstituted water for 48 h prior to test during dose range finding study and main study respectively. Daphnids had been previously maintained in reconstituted water in the test facility. Daphnids were fed with algal cells at the beginning and on Day 2 of acclimatization. Offspring produced on the first day were not used. Offspring produced on day two (less than 24 h old, derived from healthy stock showing no signs of stress) was collected and used for the test.
- Source: Environmental and Molecular Toxicology Division, Department of Zoology, Karnataka University, Dharwad, Karnataka, India.
- Feeding during test: yes
- Food type: live algal cells (Pseudokirchneriella subcapitata)
- Amount: Feeding was done within the recommended range of 0.1 to 0.2 mg C/Daphnia/day at all times.

ACCLIMATION
- Acclimation period: 48 hours
- Acclimation conditions: same as test
- Type and amount of food: Daphnids were fed with algal cells at the beginning and on Day 2 of acclimatization. Daphnids were fed with live algal cells (Pseudokirchneriella subcapitata) 0.1 to 0.2 mg of organic carbon (C) for each parent daphnid per day.
- Feeding frequency: daily
- Health during acclimation: daphnids derived from healthy stock showing no signs of stress, such as high mortality rate, discolored daphnids, presence of male and ephippia or delay in the production of the first brood, were selected for the treatment. No mortality reported.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

173.2 - 187.4 mg/L as CaCO3 (fresh) and 167.8 - 174.9 mg/L as CaCO3 (spent)

Test temperature:

20.1 to 20.6ºC (fresh and spent)

pH:

6.72 to 7.46 (fresh) and 7.09 to 7.35 (spent)

Dissolved oxygen:

7.31 to 8.45 mg/L (fresh) and 7.22 to 8.32 mg/L (spent)

Nominal and measured concentrations:

Nominal loading rates: 0 (control), 0.10, 0.29, 0.81, 2.28, 6.38, 17.86 and 50.0 mg/L.
Measured concentrations: see table included in 'Any other information on results incl. tables'.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers
- Material, size, headspace, fill volume: glass, 100 mL fill volume
- Aeration: The dilution water was aerated prior to use so that the dissolved oxygen concentration reached saturation.
- Renewal rate of test solution (frequency): 24 h
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate: 100 mL of test media per daphnid.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO reconstituted water as described in the OECD 202.
- Culture medium different from test medium: no
- Intervals of water quality measurement: Total hardness was recorded at the beginning and once a week in fresh and spent media. Temperature, pH, and dissolved oxygen was recorded daily in fresh and spent media in control and in all test item nominal loading until termination of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours dark cycle
- Light intensity: 1050.0 to 1391.0 lux (main test)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Each test vessel was checked for immobilized Daphnids, mortality, and offspring daily. The offspring produced by each parent animal was removed and counted daily from the appearance of the first brood. Those Daphnids not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile. In addition to immobility, any abnormal behavior or appearance was recorded. Observations of daphnids were made by placing the test vessel on a light box. Mortality of the parent animal was recorded daily and counted in every test vessel. Nominal loading-response mortality pattern was not observed during definitive test. Reproductive output per surviving parent animal (i.e. the number of living offspring produced during the test per surviving parent) was measured. The measurement of the length of the parent animals (i.e. body length excluding the anal spine) was recorded at the end of the test.
Other parameters like production of first brood, number and size of broods per animal, number of aborted broods, presence of male neonates and possibly the intrinsic rate of population increase were measured.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.8
- Range finding study: A dose range finding study with 6 nominal loading rates plus control with 10 replicates per dose group, under semi-static conditions was performed. At the end of the dose range finding study, reproduction of the Daphnids was assessed. The number of parents and the occurrence of offspring was recorded.
- Test concentrations: 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L
- Results used to determine the conditions for the definitive study: No clinical signs were observed in control and at the nominal loading rates of 0.01, 0.1, 1.0, 10.0 and 50.0 mg/L during the 21 days of exposure period. Complete mortality of parent daphnids were observed at the loading rate of 100.0 mg/L, hence 100 mg/L loading rate was not considered during main study.

Reference substance (positive control):

not required

Key result

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

1.08 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Key result

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

5.37 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Details on results:

- Behavioural abnormalities: none reported
- Observations on body length and weight: no abnormalities in control group.
- Mortality of control: The mortality of the parent animals in control group (female Daphnia) was 0% at the end of the test (validity criteria does not exceed 20% at the end of the test).
- Other adverse effects control: no
- Immobilisation of control: no
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no.

Reported statistics and error estimates:

NOELR/LOELR for the response variables was determined using One-way ANOVA by student t-test and EL10 and EL50 was calculated by Finney’s Probit method. The results along with 95% confidence limits were reported.

Table 1. Surviving adults parent daphnids at the end of test- main study

Group	Nominal Loading Rate (mg/L)	Initial No. of Parent Daphnids Released	No. of Surviving Parent Daphnids	Percent Survival	Clinical signs
G1	0.00	10	10	100	N
G2	0.10	10	10	100	N
G3	0.29	10	10	100	N
G4	0.81	10	10	100	N
G5	2.28	10	10	100	N
G6	6.38	10	10	100	N
G7	17.86	10	10	100	N
G8	50.00	10	7	70	B, L

N: Normal; L: Lethargy; B: On bottom.

 

Table 2. Reproductive output of parent daphnids – main study

Group	Nominal Loading Rate (mg/L)	Parameters
		Total Off Springs	Total Off Spring per Surviving Parent Daphnids	Average Off Springs Per Surviving Parent	Day of First Brood Release	F0 Production Rate of First Brood	Mean Brood Size	Mean Body Length (µm)
G1	0.00	1439	1439	143.9	7	0.1538	9.6	2147.490
G2	0.10	1456	1456	145.6	7	0.1538	9.7	2138.821
G3	0.29	1428	1428	142.8	7	0.1538	9.5	2153.720
G4	0.81	1335	1335	133.5	7	0.1333	9.2	2098.835
G5	2.28	932	932	93.2	8	0.1176	6.66	2044.524
G6	6.38	730	730	73	8	0.1333	5.21	2009.412
G7	17.86	410	410	41	9	0.1176	3.2	1114.534
G8	50.00	27	27	3.9	13	0.08	0.4	995.5586

 

Table 3. Comparison of response variable during main study

Group	Nominal Loading Rate (mg/L)	Response Variable - Percent Inhibition
		Portion of Surviving Parent Daphnids	F0 Production Rate First Brood Release	Total number of Live Offspring Per Surviving Parent Daphnids	Mean Surviving Parent Daphnids Length	Mean Brood Size
G1	0.00	-	-	-	-	-
G2	0.10	0.0	0.0	-1.2	0.40	-1.2
G3	0.29	0.0	0.0	0.8	-0.29	0.8
G4	0.81	0.0	13.3	7.2	2.27	4.0
G5	2.28	0.0	23.5	35.2*	4.79*	31.0
G6	6.38	0.0	13.3	49.3*	6.43*	45.6
G7	17.86	0.0	23.5	71.5*	48.10*	67.1
G8	50.00	30.0	48.0	98.1*	53.64*	95.5

-: Not applicable; Response variable calculated by comparing values of nominal loading rate with control group.

*: Statistically significance (<0.05)

 

Table 4. Physico-chemical parameters of test medium during main study

Dissolved Oxygen (mg/L)
Group	Test Conc.	Fresh		Spent
		Min	Max	Min	Max
G1	0	8.21	8.45	8.13	8.29
G2	0.10	8.27	8.43	8.12	8.31
G3	0.29	7.35	8.41	7.22	8.32
G4	0.81	8.19	8.38	8.11	8.28
G5	2.28	8.15	8.39	8.08	8.31
G6	6.38	8.18	8.38	8.05	8.32
G7	17.86	8.14	8.41	8.02	8.26
G8	50.00	7.31	8.39	8.00	8.29
Min		7.31		7.22
Max		8.45		8.32

pH
Group	Test Conc.	Fresh		Spent
		Min	Max	Min	Max
G1	0.0	6.93	7.45	7.14	7.35
G2	0.10	6.85	7.43	7.14	7.29
G3	0.29	6.91	7.42	7.11	7.34
G4	0.81	6.88	7.42	7.12	7.32
G5	2.28	6.84	7.43	7.10	7.28
G6	6.38	6.88	7.43	7.15	7.34
G7	17.86	6.72	7.44	7.09	7.31
G8	50.00	6.86	7.46	7.12	7.32
Min		6.72		7.09
Max		7.46		7.35

Temperature (°C)
Group	Test Conc.	Fresh		Spent
		Min	Max	Min	Max
G1	0.0	20.3	20.6	20.2	20.5
G2	0.10	20.3	20.6	20.1	20.5
G3	0.29	20.3	20.6	20.1	20.5
G4	0.81	20.2	20.6	20.1	20.6
G5	2.28	20.2	20.6	20.1	20.5
G6	6.38	20.2	20.6	20.1	20.5
G7	17.86	20.2	20.6	20.1	20.5
G8	50.00	20.1	20.6	20.1	20.5
Min		20.1		20.1
Max		20.6		20.6

Measurement on Day	Total Hardness [mg/L (as CaCO3)]
	Fresh	Spent
Day 0	173.2	-
Day 1	-	167.8
Day 7	176.7
Day 8	-	174.9
Day 14	187.43	-
Day 15	-	174.9
Day 20	176.7	-
Day 21	-	173.15
Min	173.2	167.8
Max	187.4	174.9

Light Intensity (Lux)
Mean	1297.2
±SD	66.7
Min	1050.0
Max	1391.0

 

Table 5. Complete listing of the statistical evaluations for individual endpoints - main study

Response Variable		21 Day Endpoints [mg/L]
Portion of Surviving Parent Daphnids	95%-CL	EL10	nd	EL50	nd	NOEC	LOEC
		Lower	-	Lower	-	-	-
		Upper	-	Upper	-
F0 Production Rate First Brood Release	95%-CL	EL10	nd	EL50	nd	-	-
		Lower	-	Lower	-
		Upper	-	Upper	-
Total Number of Live Offspring Per Surviving Parent Daphnids	95%-CL	EL10	1.08	EL50	5.37	0.81	2.28
		Lower	0.67	Lower	3.93
		Upper	1.73	Upper	7.34
Mean Brood Size	95%-CL	EL10	1.26	EL50	6.76	-	-
		Lower	0.76	Lower	4.89
		Upper	2.09	Upper	9.35
Mean Surviving Parent Daphnids Length	95%-CL	EL10	3.32	EL50	52.56	0.81	2.28
		Lower	1.45	Lower	23.01
		Upper	7.56	Upper	120.04

nd: Not determined [(Data obtained is not sufficient), no 50% mortality in parent daphnids and no 50% inhibition in first brood release and mean brood size was observed.

 

Table 6. 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	0.7733	0.3180	0.3343	0.4134	0.7594	1.237	0.8140
50	3.290	3.100	3.244	2.754	2.808	2.961	2.384
100	5.634	5.567	6.611	6.401	6.283	4.889	3.440

Maximum TOC values obtained at 90 minutes, hence stirring of 90 minutes was followed during test media formulation.

 

Table 7. Average TOC values in fresh and spent during the exposure period – main study

TOC Values (ppm) - Dissolved and Undissolved
Day 0	Day 1	Day 8	Day 9	Day 11	Day 12	Day 13	Day 14	Day 17	Day 18
Fresh	Spent	Fresh	Spent	Fresh	Spent	Fresh	Spent	Fresh	Spent
0.625	0.1953	0.285	0.223	0.2854	0.263	0.2423	0.241	0.2541	0.02653

TOC (ppm) in control group during the exposure period

 

Average TOC values for the exposure period
Group	Nominal Loading Rate (mg/L)	Dissolved and Undissolved
		TOC Values (ppm)
		Fresh	Spent
G1	0.00	0.338	0.190
G2	0.10	*1.063	*0.248
G3	0.29	*0.227	*0.431
G4	0.81	*0.567	*0.445
G5	2.28	*0.228	*0.186
G6	6.38	*0.271	*0.758
G7	17.86	*0.827	*0.650
G8	50.00	*1.972	*1.520

*: Values presented are the subtracted values from the control.
There are some anomalous cases such as the high value of 1.063 mg/L for 0.1 mg/L Loading rate. 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.

 

TOC Values (ppm) - Filtered
Day 0	Day 1	Day 7	Day 8	Day 11	Day 12	Day 13	Day 14	Day 17	Day 18
Fresh	Spent	Fresh	Spent	Fresh	Spent	Fresh	Spent	Fresh	Spent
0.345	0.265	0.265	0.241	0.254	0.235	0.214	0.241	0.223	0.215

TOC (ppm) in control group during the exposure period

 

Average TOC values for the exposure period
Group	Nominal Loading Rate (mg/L)	Filtered
		TOC Values (ppm)
		Fresh	Spent
G1	0.00	0.260	0.239
G2	0.10	*0.198	*0.344
G3	0.29	*0.472	*0.404
G4	0.81	*0.676	*0.622
G5	2.28	*0.716	*0.464
G6	6.38	*0.543	*0.432
G7	17.86	*0.787	*0.613
G8	50.00	*1.726	*0.962

*: 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 EL10 for reproductive output of live offspring per surviving F0 parent was 1.08 mg/L. The EL50 for the same response variable was 5.37 mg/L. Mortality was only observed at 30% in the highest loading rate of 50.0 mg/L. No accidental and inadvertent parent mortality was observed.

Executive summary:

A Daphnia magna reproduction test was performed on the test item, over a period of 21 days in semi-static conditions, according to OECD TG 211.

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 90 min, hence 90 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, 1, 10, 50 and 100 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), 0.10, 0.29, 0.81, 2.28, 6.38, 17.86 and 50.0 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.

24-hour old daphnids were exposed to the test item in ISO reconstituted water under semi-static conditions, test media was renewed once in 24 hours until the termination of the test. Daphnids were observed for signs of toxicity, immobility of parent daphnids, and reproductive output of the surviving parent daphnids. The test design included ten replicates per concentration and for the untreated control, containing one parent animal per test vessel. 

Each test vessel was checked for immobilized Daphnids, mortality, and offspring daily. The offspring produced by each parent animal was removed and counted daily from the appearance of the first brood. The mortality of the parent animal was recorded daily and counted in every test vessel. Reproductive output per surviving parent animal (i.e., the number of living offspring produced during the test per surviving parent) was recorded. The measurement of the length of the parent animals (i.e., body length excluding the anal spine) was recorded at the end of the test. The other parameters were measured or calculated including time to production of first brood, number, and size of broods per animal, number of aborted broods, presence of male neonates, and possibly the intrinsic rate of population increase.

Nominal loading-response mortality pattern was not observed during definitive test. Mortality was only observed at 30% in the highest loading rate of 50.0 mg/L. No accidental and inadvertent parent mortality was observed. All validity criteria of the test were fulfilled.

Based on the results obtained under test conditions, the EL10 for reproductive output of live offspring per surviving F0 parent was 1.08 mg/L. The EL50 for the same response variable was 5.37 mg/L.

Description of key information

Key study. Method according to OECD 211. The 21-day EL50 of the test item in Daphnia Magna was found to be 5.37 mg/L (nominal concentration, based on reproductive output of live offspring per surviving F0 parent).