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REACH

Sodium polysulfide aluminosilicate with a SOD-type framework structure

EC number: 701-340-9 | CAS number: -

Life Cycle description
Uses advised against

Ecotoxicological information

Long-term toxicity to fish

Currently viewing:

Administrative data

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:

02 March 2021 - 03 April 2021

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:

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:

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 6.0, 9.5, 15.3, 24.4, 39.1, 62.5 and 100.0 mg/L.
- Sampling method: During the main study, samples from all the test concentrations were collected. Fresh media samples were collected at 0 hours on days 6, 8, 14, 16 and 18; spent media were be collected on days 8, 10, 16, 18 and 20, and they were analysed for active content. Before collection, the test media was mixed slowly (without affecting the test system) and then, sampling was made; 10 mL water samples were collected from the central point of the tank in duplicates from all the test concentrations. Additionally, at 0, 24, and 48 hours, three samples (about 15 ml each) were collected in duplicates from test media replicates for analytical verification of saturation concentration.
- Sample storage conditions before analysis: One set was analysed on the same day of collection and the other set was stored at 26 ± 1.5ºC as backup sample.

Vehicle:

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item stock solution was prepared by weighing required quantity of the test item in beaker. To this small volume of reverse osmosis water was added and stirred well, after complete solubility, the test formulation was transferred to measuring cylinder and beaker was rinsed with reverse osmosis water and transferred again to measuring cylinder, the rinsing procedure was repeated until the complete transfer of test contents, finally the volume made up to required volume using reverse osmosis water. The solution prepared was kept on magnetic stirrer to maintain homogeneity. The stock solution prepared was introduced to know volume of dilution medium to obtain desired concentration. The preparation of test media on day 0 during main study are presented in ''Any other information on materials and methods incl. tables.
- Test concentration separation factor: 1.6
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The test media was clear with no fine particles on the day of preparation (fresh) and the appearance of test media was blue in all the test concentration. However, at 24 and 48 hours (spent) observation fine particles were settled at the bottom of the test vessels at the test concentrations of 92.5 and 100.0 mg/L.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish
- Strain: Danio rerio
- Source: Fertilized eggs of Danio rerio were procured from Whizbang Bioresearch Private Limited, Tamil Nadu, India.

POST-HATCH FEEDING
- Type/source of feed: flake food (commercial larvae food or BSN48 flake food for juvenile fish as appropriate)
- Frequency of feeding: twice daily

Test type:

semi-static

Water media type:

freshwater

Limit test:

Total exposure duration:

30 d

Hardness:

140 to 250 mg/L as CaCO3

pH:

6.08 to 6.94

Dissolved oxygen:

81.20 to 98.30%

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 6.0, 9.5, 15.3, 24.4, 39.1, 62.5 and 100.0 mg/L.
Measured concentrations: see table in 'any other information on results incl. tables'. The test concentrations were maintained at ± 20% recovery to nominal concentration throughout the exposure period.

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): 48 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: Maximum loading of fish embryos did not exceed 0.5 g/L wet weight per 24 hours solution, and not exceeding 5 g/L of solution at any time, as recommended.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis water (from tap water)
- Culture medium different from test medium: no
- Intervals of water quality measurement: Dissolved oxygen and pH were measured in all the test vessels at the beginning and at every renewal period, hardness at the beginning and end of the test. Temperature was monitored daily until the termination of the test from all the test vessels.

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.

RANGE-FINDING STUDY (n.a.)
- Results used to determine the conditions for the definitive study: The test concentrations for the main study were selected based on the available results from Acute Toxicity Test of C.I. Pigment Blue 29 to Oryzias latipes by MITI (Blue Sodium polysulfide aluminosilicate with a SOD-type framework structure), 96 hours LC50 is >90 mg/L.

Reference substance (positive control):

not required

Duration:

30 d

Dose descriptor:

EC10

Effect conc.:

13.867 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Remarks on result:

other: 95% CL 9.43 - 20.37

Duration:

30 d

Dose descriptor:

EC10

Effect conc.:

15.259 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% CL: 10.260 - 23.223

Duration:

30 d

Dose descriptor:

EC10

Effect conc.:

9.832 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Remarks on result:

other: 95% CL: 6.542 - 14.777

Key result

Duration:

30 d

Dose descriptor:

EC10

Effect conc.:

7.599 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Remarks on result:

other: 95% CL: 5.090 - 11.344

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

9.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

9.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Details on results:

- Mortality/survival at embryo, larval, juvenile, and adult stages: Post hatch larval survival until day 30 in the control group was 91.%. Post-hatch larval survival across the test concentrations ranges from 90.4, 84.1, 83.9, 78.0, 76.5, 45.9 and 26.7% at the tested concentrations of 6.0, 9.5, 15.3, 24.4, 39.1, 62.5 and 100.0 mg/L, respectively.
- Days to hatch or time to release of young: First egg hatch in control and treatment vessels occurred at 48 hour period.
- Numbers hatched, Numbers of offspring produced, or Number of offspring per live female per day: Hatching success of 91.3, 86.3, 77.5, 73.8, 63.8, 46.3 and 18.8% was observed at the tested concentrations of 6.0, 9.5, 15.3, 24.4, 39.1, 62.5 and 100.0 mg/L, respectively.
- Number of fish in swim-up stage at one or more time periods (e.g., day x1, x2):
- Observations on body length and weight of young and/or exposed parents at one or more time periods: The length was measured for the surviving fish of each replicate at the termination of the test and the mean fish length measured for found to be 2.6 cm in control, 2.6, 2.4, 2.1, 1.7, 1.4, 1.1 and 0.8 cm at the tested concentrations of 6.0, 9.5, 15.3, 24.4, 39.1, 62.5 and 100.0 mg/L, respectively.
- Number of healthy fish at end of test: not reported
- Type of and number with morphological abnormalities: No abnormal appearance was observed in the control and the tested concentration of 6.0 mg/L. Abnormal appearance of bent tail (tail malformations) was observed at the tested concentrations of 15.3, 24.4, 39.1 and 62.5 mg/L. Abnormal appearance of bent tail (tail malformation) and darkened coloration was observed at the test concentration of 100.0 mg/L. Tail malformation (bent tail) was considered as sub-lethal effects of test item.
- Type of and number with behavioural abnormalities: No abnormal behaviour was observed in the control. Abnormal behaviour of fish localized on the bottom of the test vessel was observed in the 6.0 mg/L group. Lethargy and fish localized on the bottom of the test vessel with no active movement were observed at the concentrations of 9.5, 62.5 and 100.0 mg/L. Lethargy, erratic behaviour and fish localized on the bottom of the test vessel with no active movement was observed at the tested concentrations of 15.3, 24.4, and 39.1 mg/L.
- Type and number of developmental effects: Embryonic stage was verified from the representative samples using a microscope, stage was in the before cleavage of the blastodiscs commences eggs were clear with any abnormalities.
- Effect concentrations exceeding solubility of substance in test medium: no. The limit of saturation of test item in test media was determined to be 17.375 to 20.869 mg/L.
- Incidents in the course of the test which might have influenced the results: none reported.

Reported statistics and error estimates:

Statistical analysis was performed to determine the NOEC and LOEC for cumulative mortality, numbers of healthy fish at end of test, length and weight of surviving fishes, numbers of deformed larvae, number of survival larvae and numbers of fish exhibiting abnormal behaviour. In order to make a multiple comparison between the results at the individual concentrations and those for the controls Dunnett’s method was employed.
EC10, EC20 and EC50 was calculated using Probit Finney’s method for larvae hatching success, larvae survival, fish length and fish wet weight.

Table 1. Summary of results

Parameters	End Points (mg/L)
Larvae Hatching Success	EC₁₀	13.867	EC₂₀	22.223	EC₅₀	54.788
	Lower limit	9.436	Lower limit	15.123	Lower limit	37.285
	Upper Limit	20.376	Upper Limit	32.656	Upper Limit	80.509

Larvae Surviving	EC₁₀	15.259	EC₂₀	25.685	EC₅₀	69.555
	Lower limit	10.026	Lower limit	16.876	Lower limit	45.701
	Upper Limit	23.223	Upper Limit	39.091	Upper Limit	105.858

Fish Length	EC₁₀	9.832	EC₂₀	16.782	EC₅₀	46.674
	Lower limit	6.542	Lower limit	11.166	Lower limit	31.055
	Upper Limit	14.777	Upper Limit	25.223	Upper Limit	70.149

Fish fresh weight	EC₁₀	7.599	EC₂₀	13.068	EC₅₀	36.868
	Lower limit	5.090	Lower limit	8.754	Lower limit	24.697
	Upper Limit	11.344	Upper Limit	19.508	Upper Limit	55.038

Parameters	End Points (mg/L)
Parameters	NOEC	LOEC
Larvae Hatching Success	9.5	15.3
Larvae Surviving	9.5	15.3
Fish Length	6.0	9.5
Fish Fresh Weight	6.0	9.5

Table 2. Post-hatch observed larval fish mortality

Group

Test Conc. (mg/L)

Days

0.0

H-19

N-19

N-18, D-1

N-18

H-18

N-18

N-17, D-1

N-17

H-17

N-17

N-17,D-2

N-15

H-19

N-19

N-17, D-2

N-17

6.0

H-19

N-19

N-18, D-1

N-18

H-18

N-18

N-16, B-2

N-16, D-2

N-16

H-18

N-18

N-17, D-1

N-17

H-18

N-18

N-15, B-3

N-15, D-3

N-15

9.5

H-19

N-19

N-14, L, B-5

N-14, L, B-3, D-2

N-14, L,B-1, D-2

N-14, L,B-1

H-17

N-17

N-15, B-2

N-15, B-1, D-1

N-15, B-1

H-17

N-17

N-13 ,L, B-4

N-13, L,B-2, D-2

N-11, L,B-3,D-2

N-11, L, B- 2

H-16

N-16

N-10, L,B-6

N-10, L,B-4, D-2

N-10, L,B-4

15.3

H-14

N-14

N-9, LB-5

N-9, B-4, D-1

N-9, B-4

N-8, B-4, BT-1

H-16

N-16

N-9, L,B-7

N-9, D-2, B-5

N-9, B-5

N-8, B-5, BT-1

H-17

N-17

N-8, EB-3, LB- 4, D-2

N-8, EB-3, LB- 2, D-2

N-8, EB-3, LB- 2,

H-15

N-15

N-9, L-6

N-9, L-3, D-3

N-9, L-3

N-8, L-2, EB-2

24.4

H-15

N-15

N-8, L-5, EB-2

N-6, L-5, EB-2, D-2

N-6, L-5, EB-2

N-4, L-5, EB-2, BT-2

H-16

N-16

N-2, L-7, EB-4, D-3

N-2, L-7, EB-4

L-6, EB-4, D-3

L-6, EB-4

L-3, BT-3, EB-4

H-14

N-14

N-10, L,B-4

N-7, L,B-4, D-3

N-2, L-B-4, BT-5

N-2, B-4, BT-5

N-2,B-4, BT-5

N-2, B-4, BT-5

H-14

N-14

N-8, L-6

N-5, EB-1, L-6, D-2

N-5, EB-1, L-6

B-3, EB-1, L-6, BT-2

39.1

H-14

N-14

N-10, L-4

N-5, L-4, EB-2, D-3

N-5, L-4, EB-2

B-4, L-4, EB-2, BT-1

H-13

N-13

N-3, EB-4, L-4,D-2

N-1, EB-4, L-4,D-2

B-1, EB-4, L-4

H-11

N-11

EB-4, L-4, D-3

EB-4, L-4

EB-3, L-4, BT-1

H-13

N-13

N-2, L-5,B-4, D-2

N-2, L-5,B-4

BT-2, L-5,B-4

62.5

H-8

N-8

L-8

L-6, D-2

L-5, D-1

L-5

L,B-5

L,B-4, BT-1

L,B-4, DC, BT-1

H-8

N-8

L-5, D-3

L-5

L-3, D-2

L,B-3

BT, L,B-3

H-10

N-10

L-8, D-2

L-6, D-2

L-4, D-2

L-4

L, B-4

H-11

L-11

L-8, D-3

L-5,D-3

L-5

B,L-5

L-3, BT-2

100.0

H-4

B-4

B-1, D-3

B-1

BT, B-1

H-4

B-4

L, B-4

L, B-1, D-3

L,B-1

BT, DC, L,B-1

H-3

L-3

L,B-1, D-2

L,B-1

H-4

B-4

B-1, D-3

B-1

L,B-1

BT, L,B-1

^{R: Replicate; H: Hatched; N: Normal; L: Lethargy; B: On bottom; BT: Bent Tail; EB: Erratic behaviour; D: Dead; DC: Dark coloration; Alphabet followed by a hyphen – indicates no. of larvae exhibiting a particular symptom}

Table 3. Hatching success, fish larval survival and percent mortality

Group	Test Conc. (mg/L)	R	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
Group	Test Conc. (mg/L)	R	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	73	95	91.3	18	67	95	91.8	1	6	8.2
		2	20		18		90		17		94		1
		3	20		17		85		15		88		2
		4	20		19		95		17		89		2
G2	6.0	1	20	80	19	73	95	91.3	18	66	95	90.4	1	7	9.6
		2	20		18		90		16		89		2
		3	20		18		90		17		94		1
		4	20		18		90		15		83		3
G3	9.5	1	20	80	19	69	95	86.3	15	58	79	84.1	4	11	15.9
		2	20		17		85		16		94		1
		3	20		17		85		13		76		4
		4	20		16		80		14		88		2
G4	15.3	1	20	80	14	62	70	77.5	13	52	93	83.9	1	10	16.1
		2	20		16		80		14		88		2
		3	20		17		85		13		76		4
		4	20		15		75		12		80		3
G5	24.4	1	20	80	15	59	75	73.8	13	46	87	78.0	2	13	22.0
		2	20		16		80		10		63		6
		3	20		14		70		11		79		3
		4	20		14		70		12		86		2

G6	39.1	1	20	80	14	51	70	63.8	11	39	79	76.5	3	12	23.5
		2	20		13		65		9		69		4
		3	20		11		55		8		73		3
		4	20		13		65		11		85		2
G7	62.5	1	20	80	8	37	40	46.3	5	17	63	45.9	3	20	54.1
		2	20		8		40		3		38		5
		3	20		10		50		4		40		6
		4	20		11		55		5		45		6
G8	100.0	1	20	80	4	15	20	18.8	1	4	25	26.7	3	11	73.3
		2	20		4		20		1		25		3
		3	20		3		15		1		33		2
		4	20		4		20		1		25		3

Table 4. Physico-chemical parameters during exposure

Group	Test Conc. (mg/L)	Dissolved Oxygen (%)				Temperature (°C)				pH
		Fresh		Spent		Fresh		Spent		Fresh		Spent
		Min	Max	Min	Max	Min	Max	Min	Max	Min	Max	Min	Max
G1	0.0	85.60	98.30	81.20	93.40	25.0	25.9	24.9	25.9	6.34	6.89	6.08	6.94
G2	6.0	89.30	97.70	82.00	93.60	25.0	25.9	25.1	25.9	6.33	6.94	6.18	6.89
G3	9.5	86.90	97.40	81.60	96.10	25.1	25.9	25.0	25.9	6.40	6.88	6.21	6.89
G4	15.3	88.30	98.20	81.20	95.50	25.1	25.9	25.0	25.9	6.40	6.92	6.21	6.94
G5	24.4	88.70	96.90	83.10	94.50	25.0	25.9	25.0	25.9	6.33	6.90	6.13	6.86
G6	39.1	87.40	96.20	82.30	95.20	25.0	25.9	25.0	25.9	6.46	6.86	6.17	6.80
G7	62.5	89.70	98.10	81.70	93.10	25.0	25.9	25.1	25.9	6.15	6.89	6.21	6.84
G8	100.0	87.60	96.10	82.10	94.40	25.0	25.9	25.1	25.9	6.39	6.86	6.14	6.86
Min		81.20				24.9				6.08
Max		98.30				25.9				6.94

Table 5. Percent inhibition in larvae hatching, survival, length and fresh weight

Group	Test Conc. (mg/L)	Percent Inhibition in Larvae Hatching	Percent Inhibition in Post hatch Larvae Surviving	Percent Inhibition of Larvae Length	Percent Inhibition of Fish Fresh Weight
G1	0.0	-	-	-	-
G2	6.0	0.00	1.49	3.6	5.40
G3	9.5	5.48	8.41	7.9	18.28
G4	15.3	15.07	8.62	20.8	26.60
G5	24.4	19.18	15.05	35.6	32.62
G6	39.1	30.14	16.68	47.5	39.48
G7	62.5	49.32	49.94	58.6	75.18
G8	100.0	79.45	70.95	68.8	98.69

^{-: Not applicable; Percent inhibition calculated in comparison with the control group}

Table 6. Summary of test concentration analysis during main study

Fresh (Day 6)
Group	Nominal concentration (mg/L)	Conc. obtained (mg/L)	Recovery (%)
G1	0.0	-
G2	6.0	5.865	97.76
G3	9.5	9.377	98.71
G4	15.3	14.977	97.39
G5	24.4	24.822	101.73
G6	39.1	38.575	98.66
G7	62.5	63.593	101.75
G8	100.0	100.813	100.81

Spent (Day 8)
Group	Nominal concentration (mg/L)	Conc. obtained (mg/L)	Recovery (%)
G1	0.0	-	-
G2	6.0	5.936	98.94
G3	9.5	9.364	98.56
G4	15.3	14.675	95.91
G5	24.4	25.016	102.52
G6	39.1	38.728	99.05
G7	62.5	65.415	104.66
G8	100.0	100.737	100.74

Fresh (Day 8)
Group	Nominal concentration (mg/L)	Conc. obtained (mg/L)	Recovery (%)
G1	0.0	-	-
G2	6.0	6.253	104.21
G3	9.5	9.373	98.66
G4	15.3	14.948	97.70
G5	24.4	25.153	103.09
G6	39.1	38.384	98.17
G7	62.5	65.415	104.66
G8	100.0	102.210	102.21

Spent (Day 10)
Group	Nominal concentration (mg/L)	Conc. obtained (mg/L)	Recovery (%)
G1	0.0	-	-
G2	6.0	6.022	100.37
G3	9.5	9.410	99.05
G4	15.3	15.181	99.22
G5	24.4	25.247	103.47
G6	39.1	37.857	96.82
G7	62.5	65.561	104.90
G8	100.0	102.515	102.58

Fresh (Day 14)
Group	Nominal concentration (mg/L)	Conc. obtained (mg/L)	Recovery (%)
G1	0.0	-	-
G2	6.0	5.871	97.86
G3	9.5	9.252	97.39
G4	15.3	14.763	96.49
G5	24.4	25.091	102.83
G6	39.1	38.183	97.65
G7	62.5	63.187	101.10
G8	100.0	101.524	101.52

Spent (Day 16)
Group	Nominal concentration (mg/L)	Conc. obtained (mg/L)	Recovery (%)
G1	0.0	-	-
G2	6.0	6.072	101.21
G3	9.5	9.489	99.88
G4	15.3	14.841	97.00
G5	24.4	25.460	104.34
G6	39.1	39.206	100.27
G7	62.5	67.593	108.15
G8	100.0	104.878	104.88

Fresh (Day 16)
Group	Nominal concentration (mg/L)	Conc. obtained (mg/L)	Recovery (%)
G1	0.0	-	-
G2	6.0	5.876	97.93
G3	9.5	9.078	95.56
G4	15.3	14.235	93.04
G5	24.4	26.048	106.75
G6	39.1	39.417	100.81
G7	62.5	66.846	106.95
G8	100.0	103.176	103.18

Spent (Day 18)
Group	Nominal concentration (mg/L)	Conc. obtained (mg/L)	Recovery (%)
G1	0.0	-	-
G2	6.0	5.762	96.03
G3	9.5	9.164	96.46
G4	15.3	14.690	96.01
G5	24.4	24.966	102.32
G6	39.1	38.317	98.00
G7	62.5	65.301	104.48
G8	100.0	103.633	103.63

Fresh (Day 18)
Group	Nominal concentration (mg/L)	Conc. obtained (mg/L)	Recovery (%)
G1	0.0	-	-
G2	6.0	5.724	95.39
G3	9.5	9.041	95.16
G4	15.3	14.475	94.61
G5	24.4	24.165	99.04
G6	39.1	36.911	94.40
G7	62.5	63.577	101.72
G8	100.0	99.136	99.14

Spent (Day 20)
Group	Nominal concentration (mg/L)	Conc. obtained (mg/L)	Recovery (%)
G1	0.0	-	-
G2	6.0	5.898	98.30
G3	9.5	9.515	100.15
G4	15.3	14.908	97.44
G5	24.4	24.697	101.22
G6	39.1	38.020	97.24
G7	62.5	65.122	104.20
G8	100.0	101.651	101.65

Table 7. Analytical verification of limit of saturation

Day 0 Analysis
Sample Code	Conc. Obtained (mg/L)	Nominal Conc. (mg/L)	% Recovery
Sample G1	-	-	-
Sample G2 As such formulation	102.819	100.000	102.82
Sample G2 G Filtered formulation	17.375	100.000	17.37
Sample G2 Centrifuged formulation	18.340	100.000	18.34

Day 1 Analysis
Sample Code	Conc. Obtained (mg/L)	Nominal Conc. (mg/L)	% Recovery
Sample G1	-	-	-
Sample G2 As such formulation	101.023	100.000	101.02
Sample G2 G Filtered formulation	18.301	100.000	18.30
Sample G2 Centrifuged formulation	20.869	100.000	20.87

Day 2 Analysis
Sample Code	Conc. Obtained (mg/L)	Nominal Conc. (mg/L)	% Recovery
Sample G1	-	-	-
Sample G2 As such formulation	114.286	100.000	114.29
Sample G2 G Filtered formulation	19.247	100.000	19.25
Sample G2 Centrifuged formulation	19.498	100.000	19.50

Table 8. Total fish length (cm) at the end of the test

Fish Number	Test Conc. (mg/L)
	0.0				6.00				9.5				15.3
	G1				G2				G3				G4
	1	2	3	4	1	2	3	4	1	2	3	4	1	2	3	4
1	2.2	2.4	2.2	2.9	2.5	2.1	2.2	2.4	2.4	2.6	2.8	2.2	2.2	2.2	2.1	1.9
2	2.5	3.0	2.4	2.4	2.6	2.9	2.3	2.3	2.5	2.4	3.0	2.5	1.8	1.9	1.8	2.2
3	2.9	2.8	2.3	3.2	2.4	2.5	2.8	2.8	2.9	2.5	2.7	2.1	2.0	2.1	2.2	2.4
4	3.0	2.9	2.6	2.8	2.9	2.7	2.9	2.9	2.4	2.2	2.5	2.9	2.3	2.0	2.0	2.1
5	2.8	2.5	2.7	2.7	2.4	2.2	2.1	2.1	2.3	2.9	2.4	2.2	2.1	2.3	2.1	2.0
6	2.2	2.4	2.8	2.5	2.9	2.1	2.2	2.3	2.1	2.8	2.6	2.1	2.2	2.5	2.5	1.8
7	2.4	2.3	2.9	2.9	3.1	2.0	2.3	2.6	2.4	2.3	2.3	2.0	2.0	2.1	2.1	1.8
8	2.5	2.4	2.6	2.4	2.8	3.2	2.5	3.1	2.9	2.9	2.4	2.2	1.9	2.2	1.8	2.2
9	2.6	2.8	3.1	2.7	2.9	2.9	2.4	3.3	2.8	2.4	2.2	2.4	2.2	1.9	2.1	2.4
10	2.4	2.7	2.9	2.3	2.8	3.2	2.4	2.2	2.1	2.4	2.4	2.5	2.5	2.3	2.2	1.9
11	2.3	2.9	2.4	2.4	2.7	3.1	2.3	2.4	2.2	2.7	2.5	2.3	2.1	2.4	2.1	1.6
12	2.5	2.8	2.5	2.6	2.3	2.8	2.9	2.8	2.8	2.5	2.4	2.1	2.0	2.1	2.3	2.2
13	2.7	2.4	2.8	2.4	2.8	1.9	2.2	2.3	2.6	2.6	2.2	2.2	2.1	2.0	2.2	-
14	2.9	2.5	2.9	2.9	2.4	2.1	2.5	2.4	2.1	2.4	-	2.0	-	2.1	-	-
15	2.4	2.6	2.9	2.8	2.9	2.2	2.6	2.2	2.2	2.2	-	-	-	-	-	-
16	2.5	2.9	-	2.9	2.4	2.6	2.4	-	-	2.7	-	-	-	-	-	-
17	3.1	3.0	-	2.4	2.8	-	2.3	-	-	-	-	-	-	-	-	-
18	2.8	-	-	-	2.9	-	-	-	-	-	-	-	-	-	-	-
19	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
20	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Individual mean	2.6	2.7	2.7	2.7	2.7	2.5	2.4	2.5	2.4	2.5	2.5	2.3	2.1	2.2	2.1	2.0
Pooled mean	2.6				2.6				2.4				2.1
Individual minimum	2.2	2.3	2.2	2.3	2.3	1.9	2.1	2.1	2.1	2.2	2.2	2.0	1.8	1.9	1.8	1.6
Pooled minimum	2.2				1.9				2.0				1.6
Individual maximum	3.1	3.0	3.1	3.2	3.1	3.2	2.9	3.3	2.9	2.9	3.0	2.9	2.5	2.5	2.5	2.4
Pooled maximum	3.2				3.3				3.0				2.5
Individual ±SD	0.27	0.24	0.26	0.26	0.24	0.47	0.24	0.36	0.29	0.22	0.23	0.24	0.18	0.19	0.19	0.22
Pooled ±SD	0.25				0.34				0.26				0.20

Fish Number	Test Conc. (mg/L)
	24.4				39.1				62.5				100.0
	G5				G6				G7				G8
	1	2	3	4	1	2	3	4	1	2	3	4	1	2	3	4
1	1.8	1.5	2.0	1.8	1.5	1.2	1.4	1.4	1.2	1.3	1.0	1.2	0.8	0.9	0.8	0.8
2	1.9	1.9	1.6	2.0	1.4	1.5	1.2	1.3	1.0	1.1	1.1	1.1	-	-	-	-
3	2.0	1.4	1.9	1.9	1.5	1.3	1.1	1.5	1.1	1.2	0.9	1.1	-	-	-	-
4	1.5	1.2	2.1	2.1	1.3	1.4	1.3	1.7	1.2		0.8	1.1	-	-	-	-
5	1.4	2.0	2.0	2.0	1.2	1.2	1.4	1.2	1.2	-		1.0	-	-	-	-
6	1.9	1.9	1.9	1.8	1.6	1.2	1.2	1.6	-	-	-	-	-	-	-	-
7	1.5	1.8	1.5	1.5	1.4	1.3	1.4	1.8	-	-	-	-	-	-	-	-
8	1.3	1.7	1.4	1.7	1.2	1.4	1.3	1.4	-	-	-	-	-	-	-	-
9	2.1	1.8	1.3	1.6	1.1	1.2	-	1.5	-	-	-	-	-	-	-	-
10	1.8	1.5	1.5	1.7	1.2	-	-	1.8	-	-	-	-	-	-	-	-
11	1.9	-	1.9	1.9	1.9	-	-	1.7	-	-	-	-	-	-	-	-
12	1.4	-		1.8	-	-	-		-	-	-	-	-	-	-	-
13	1.9	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
14	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
15	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
16	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
17	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
18	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
19	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
20	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Individual mean	1.7	1.6	1.7	1.8	1.4	1.3	1.3	1.5	1.1	1.2	1.0	1.1	0.8	0.9	0.8	0.8
Pooled mean	1.7				1.4				1.1				0.8
Individual minimum	1.3	1.2	1.3	1.5	1.1	1.2	1.1	1.2	1.0	1.1	0.8	1.0	0.8	0.9	0.8	0.8
Pooled minimum	1.2				1.1				0.8				0.8
Individual maximum	2.1	2.0	2.1	2.1	1.9	1.5	1.4	1.8	1.2	1.3	1.1	1.2	0.8	0.9	0.8	0.8
Pooled maximum	2.1				1.9				1.3				0.9
Individual ±SD	0.27	0.29	0.28	0.17	0.23	0.11	0.11	0.20	0.09	0.10	0.13	0.07	-	-	-	-
Pooled ±SD	0.24				0.20				0.12				0.05

Table 9. Fish wet weight (g) at the end of the test

Group	Test Conc. (mg/L)	Replicate	Fish wet weight (g)	Mean Fish weight (g)
Group	Test Conc. (mg/L)	Replicate	Fish wet weight (g)	Mean Fish weight (g)
G1	0.0	1	1.5394	1.6038
		2	1.6141
		3	1.6891
		4	1.5724
G2	6.0	1	1.4321	1.5172
		2	1.5145
		3	1.6241
		4	1.4981
G3	9.5	1	1.3241	1.3105
		2	1.2891
		3	1.3415
		4	1.2874
G4	15.3	1	1.2412	1.1771
		2	1.2154
		3	1.1324
		4	1.1194
G5	24.4	1	1.0241	1.0806
		2	1.1245
		3	1.0987
		4	1.0751
G6	39.1	1	1.0210	0.9706
		2	1.0041
		3	0.8700
		4	0.9874
G7	62.5	1	0.4595	0.3980
		2	0.3241
		3	0.4842
		4	0.3241
G8	100.0	1	0.0325	0.0210
		2	0.0154
		3	0.0214
		4	0.0145

Validity criteria fulfilled:

yes

Remarks:

Dissolved oxygen was 81.2% of saturation throughout the test (> 60%). Water temperature did not differ by more than ± 1.5°C during the exposure. Overall survival of fertilized eggs in control was 91.8% and post-hatch success was 91.3% (> 70% and > 75%).

Conclusions:

Based on the results obtained under test conditions, the EC10 for hatching success was found to be 13.867 mg/L, the EC10 for larvae survival was found to be 15.259 mg/L, the EC10 based on length was 9.832 mg/L and the EC10 based on weight was 7.599 mg/L (worst case scenario), all based on nominal concentrations.

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.

Based on an in-house solubility test, the test item was found to be soluble in reverse osmosis water. Based on based on the available results and from read across data, the main study was performed at the test concentrations of 6.0, 9.5, 15.3, 24.4, 39.1, 62.5 and 100.0 mg/L (nominal) and one control group.

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 48 hours until the termination of the test. The test design included eight 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.

A validated analytical method based on ICP-MS was used to monitor the concentration and stability of the active ingredient in the test solution. The test item was considered stable in the testing conditions after 72 hours of exposure as the measured concentrations in samples were maintained within ± 20 percent of the nominal concentrations. The test media was clear, light blue, and no particles were observed on fresh media. However, at 24 and 48 hours observations, fine particles were settled at the bottom of test vessels at the test concentrations of 62.5 and 100.0 mg/L. Therefore, additional replicates were prepared for analytical monitoring. On analytical verification at the concentration of 100 mg/L, the limit of saturation was found to be 17.37 to 20.869 mg/L. All validity criteria were met.

According to REACH guidance Chapter R.7b: Endpoint specific guidance Version 4.0 – June 2017, EC10 values are preferred as these are statistically derived from the entire dataset, and less dependent on test design considerations than the NOEC. Based on the results obtained under test conditions, the EC10 for hatching success was found to be 13.867 mg/L, the EC10 for larvae survival was found to be 15.259 mg/L, the EC10 based on length was 9.832 mg/L and the EC10 based on weight was 7.599 mg/L (worst case scenario), all based on nominal concentrations.

Description of key information

Key study. Method according to OECD 210, GLP study. The 30-day EC10 of the test item in Danio rerio (zebrafish) embryo was found to be 7.599 mg/L with 95% confidence limits 5.090 - 11.344 mg/L (nominal concentration, based on weight).

Key value for chemical safety assessment

Fresh water fish

Dose descriptor:: EC10
Effect concentration:: 7.599 mg/L

Additional information

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