Violet sodium polysulfide aluminosilicate with a SOD-type framework structure

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:

27 August 2020 - 27 January 2021

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: 0 (control), 0.60, 0.95, 1.53, 2.44, 3.91, 6.25 and 10.0 mg/L.
- Sampling method: In order to verify the actual test concentrations, 15 mL samples were collected from low and highest test concentrations in duplicates at regular intervals. Additionally, at 0, 24, 48 and 72 hours, three samples (about 15 ml each) were collected from test media replicates in duplicates.
- 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: Stock solution was freshly prepared prior to exposure by weighing required quantity of test item in a beaker; then, a small volume of reconstituted water was added and mixed well using a glass rod. After complete solubility of the test item, test formulation was transferred into a measuring cylinder and the beaker was rinsed with Reconstituted water and transferred again to the measuring cylinder, rinsing process was repeated until the complete transfer of test contents. Stock solution prepared was kept on magnetic stirrer to maintain homogeneity for 30 minutes. Test medium of chosen concentration was prepared by dilution of stock solution.
- Test concentration separation factor: 1.6
- 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 45 pregnant female Daphnids were acclimatized in the reconstituted water for 48 h prior to test during dose range finding study and main study. Daphnids had been previously maintained in reconstituted water in the test facility. Daphnids were fed with algal cells at the beginning and on Day 1 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 1 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:

212 - 216 mg/L as CaCO3

Test temperature:

20.0 to 20.7ºC

pH:

7.91 to 8.44

Dissolved oxygen:

7.81 to 8.99 mg/L

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 0.60, 0.95, 1.53, 2.44, 3.91, 6.25 and 10.0 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 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): 48 hours, based on based on the results of stability of test item performed during analytical method validation the test media
- 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 of the diluent water (reconstituted water) prior to its use for exposure was analysed. Water hardness, Temperature, pH, and dissolved oxygen was recorded at daily where applicable in (fresh and spent media) in control(s) and in all the test item concentrations until termination of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours dark cycle
- Light intensity: 1000 to 1500 lux

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. The mortality of the parent animal was recorded daily and counted in every test vessel. Since mortality did not follow a concentration-response pattern, replicates with parental mortality were excluded from the analysis of the test result. 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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.6
- Range finding study: A dose range finding study with 5 concentrations plus control with two replicates per dose group, under semi-static conditions. At the end of 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, 5.0 and 10.0 mg/L
- Results used to determine the conditions for the definitive study: No clinical signs of toxicity or immobility of parent daphnids was observed in the control and at the tested concentrations of 0.01, 0.1, 1.0 and 5.0 mg/L during the 21 days of exposure period. Clinical signs of lethargy and immobility in parent daphnids were observed at the parent daphnids at the tested concentration of 10.0 mg/L. The total no. of offspring produced per surviving adult was 274 in control and 275, 276, 177, 88 and 0.0 at the tested concentrations of 0.01, 0.1, 1.0, 5.0 and 10.0 mg/L, respectively.
Reduction in reproductive output was found to be -0.4, -0.7, 35.4, 67.9 and 100% was observed at the tested concentrations of 0.01, 0.1, 1.0, 5.0 and 10.0 mg/L, respectively.
The first offspring was observed after 7 days in control, similarly on day 7, at the test concentrations of 0.01 and 0.1 mg/L, on day 9 and 10 at the test concentrations of 1.0 and 5.0 mg/L. No offspring was seen and parent daphnids were observed dead on day 12 and 13 after exposure at the test concentration of 10.0 mg/L.
Inhibition in growth of adult length was 0.6, 1.2, 1.3, 6.3 and 100% at the tested concentrations of 0.01, 0.1, 1.0, 5.0 and 10.0 mg/L, respectively.
Based on the results of dose range finding study, main study was performed at the test concentrations of 0.60, 0.95, 1.53, 2.44, 3.91, 6.25 and 10.0 mg/L.

Reference substance (positive control):

not required

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

1.66 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Reproductive output of offspring of F0 parent

Remarks on result:

other: 95% CL: 1.27 - 2.17 mg/L

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

1.63 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Reproductive output of live offspring per surviving F0 parent

Remarks on result:

other: 95% CL: 1.28 - 2.08 mg/L

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

4.09 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Reproductive output of offspring of F0 parent

Remarks on result:

other: 95% CL: 3.43 - 4.88 mg/L

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

3.99 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Reproductive output of live offspring per surviving F0 parent

Remarks on result:

other: 95% CL: 3.41 - 4.66 mg/L

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.95 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Reproductive output of live offspring per surviving F0 parent

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

2.44 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Details on results:

- Behavioural abnormalities: none reported
- Observations on body length and weight: no abnormalities in control group. Inhibition in growth of adult length was 1.0, 1.7, 6.8, 7.2, 10.3, 11.1 and 16.1% at the tested concentrations of 0.60, 0.95, 1.53, 2.44, 3.91, 6.25 and 10.0 mg/L, respectively.
- 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, the limit of saturation of the test item in the test media was confirmed to be 8.2 to 9.6 mg/L.

Reported statistics and error estimates:

Data were analysed by appropriate statistical methods (Finney’s probit analysis) to calculate EC10, EC20 and EC50 with confidence limits for reproductive output. NOEC/LOEC for reproductive output and daphnid length was determined using appropriate statistical test (One-way ANOVA by Dunnett t-test).

Table 1. Summary of results. 

Response Variable		21 Day Endpoints [mg/L]
F0 adult parent survival	95%-CL	EC10	nd	EC20	nd	EC50	nd	NOEC	LOEC
		Lower	-	Lower	-	Lower	-	nd	nd
		Upper	-	Upper	-	Upper	-
Reproductive output of offspring of F0 parent	95%-CL	EC10	1.66	EC20	2.26	EC50	4.09	nd	nd
		Lower	1.27	Lower	1.83	Lower	3.43
		Upper	2.17	Upper	2.8	Upper	4.88
Reproductive output of live offspring per surviving F0 parent	95%-CL	EC10	1.63	EC20	2.22	EC50	3.99	0.95	1.53
		Lower	1.28	Lower	1.83	Lower	3.41
		Upper	2.08	Upper	2.69	Upper	4.66
Growth                  (Length)	95%-CL	EC10	nd	EC20	nd	EC50	nd	2.44	3.91
		Lower	-	Lower	-	Lower	-
		Upper	-	Upper	-	Upper	-

 

Table 2. Surviving Adults F0 parent daphnids at the end of the test (DRF)

Group	Test Conc. (mg/L)	Initial No. of Parent Daphnids released	Surviving (F0) parent Daphnid at the end of the test	Percent Inhibition	Clinical signs
G1	0.0	2	2	-	N
G2	0.01	2	2	0.0	N
G3	0.1	2	2	0.0	N
G4	1.0	2	2	0.0	N
G5	5.0	2	2	0.0	N
*G6	10.0	2	0	100.0	L

^{N: Normal; L: Lethargy; -: Not applicable; *: Immobility observed in parent daphnids.}

 

Table 3. Reproductive output of parent daphnids (DRF)

Group	Test Conc. (mg/L)	Total no. of off spring produced		Total no. of off spring produced per surviving adult		Day on first brood release	F0 production rate first brood release
		Total no. of off spring produced	Reduction in Reproductive Output (%)	Total no. of off spring produced	Reduction in Reproductive Output (%)
G1	0.0	274	-	274	-	7	0.1538
G2	0.01	275	-0.4	275	-0.4	7	0.1538
G3	0.10	276	-0.7	276	-0.7	7	0.1538
G4	1.00	177	35.4	177	35.4	9	0.1176
G5	5.00	88	67.9	88	67.9	10	0.1053
G6	10.00	0	100.0	0	100.0	-	0.0000

 

Table 4. Comparison of response variable (DRF)

Group	Test Conc. (mg/L)	Response Variable - Percent Inhibition
		Adult Mortality	F0 Production Rate first brood release	Reproductive Output of live offspring per  adult	No. of live offspring per surviving F0 adult	Mean Surviving F0 adult length
G1	0.00	-	-	-	-	-
G2	0.01	0.0	0.0	-0.4	-0.4	0.6
G3	0.10	0.0	0.0	-0.7	-0.7	1.2
G4	1.00	0.0	23.5	35.4	35.4	1.3
G5	5.00	0.0	31.6	67.9	67.9	6.3
G6	10.00	100.0	100.0	100.0	100.0	100.0

 

Table 5. Physico-chemical parameters of test medium (DRF)

Dissolved Oxygen (mg/L)
Group	Test Conc.	Fresh		Spent
		Min	Max	Min	Max
G1	0.0	7.39	7.85	7.22	7.71
G2	0.01	7.34	7.87	7.13	7.70
G3	0.1	7.32	7.70	7.21	7.63
G4	1.0	7.29	7.56	7.15	7.44
G5	5.0	7.28	7.86	7.19	7.71
G6	10.0	7.32	7.51	7.19	7.47
Min		7.28		7.13
Max		7.87		7.71

pH
Group	Test Conc.	Fresh		Spent
		Min	Max	Min	Max
G1	0.0	8.17	8.43	8.10	8.35
G2	0.01	8.26	8.47	8.20	8.40
G3	0.1	8.31	8.56	8.28	8.49
G4	1.0	8.34	8.56	8.26	8.50
G5	5.0	8.20	8.46	8.09	8.27
G6	10.0	8.22	8.54	8.15	8.46
Min		8.17		8.09
Max		8.56		8.50

Temperature (°c)
	Fresh	Spent
Mean	20.3	19.4
±SD	0.1	3.9
Min	20.1	20.0
Max	20.5	20.5

Measurement on	Total Hardness [mg/L (as CaCO3)]
Acclimatization	216
Day 0 (Fresh)	212
Day 7 (Spent)	216
Day 7 (Fresh)	220
Day 14 (Spent)	220
Day 14 (Fresh)	212
Day 20 (Spent)	212
Day 20 (Fresh)	216

 

Table 6. Surviving Adults F0 parent daphnids at the end of the test (main study)

Group	Test Conc. (mg/L)	Initial No. of Parent Daphnids released	Surviving (F0) parent Daphnid at the end of the test	Percent Inhibition	Clinical Signs
G1	0.00	10	10	-	N
G2	0.60	10	10	0.0	N
G3	0.95	10	10	0.0	N
G4	1.53	10	10	0.0	N
G5	2.44	10	10	0.0	N
G6	3.91	10	8	20.0	L
G7	6.25	10	8	20.0	L, B
G8	10.00	10	7	30.0	L, B

^{-: Not applicable; N: Normal; L: Lethargy; B: On Bottom (localized on the bottom  of container)}

 

Table 7. Reproductive output of parent daphnids (main study)

Group	Test Conc. (mg/L)	Total no. of offspring produced		Total no. of offspring produced per surviving adult		Day on first brood release	F0 production rate first brood release
		Total no. of offspring produced	Reduction in Reproductive Output (%)	Total no. of offspring produced	Reduction in Reproductive Output (%)
G1	0.00	1191	-	1191	-	7	0.1538
G2	0.60	1189	0.2	1189	0.2	7	0.1538
G3	0.95	1146	3.8	1146	3.8	7	0.1538
G4	1.53	1035	13.1	1035	13.1	7	0.1538
G5	2.44	1002	15.9	1002	15.9	8	0.1333
G6	3.91	775	34.9	687	42.3	9	0.1176
G7	6.25	413	65.3	413	65.3	10	0.1053
G8	10.00	61	94.9	58	95.1	14	0.0741

 

Table 8. Comparison of the response variable (main study)

Group	Test Conc. (mg/L)	Response Variable - Percent Inhibition
		Adult Mortality	F0 Production Rate first brood release	Reproductive Output of live offspring per  adult	No. of live offspring per surviving F0 adult	Mean Surviving F0 adult length
G1	0.00	-	-	-	-	-
G2	0.60	0.0	0.0	0.2	0.2	1.0
G3	0.95	0.0	0.0	3.8	3.8	1.7
G4	1.53	0.0	0.0	13.1	13.1	6.8
G5	2.44	0.0	13.3	15.9	15.9	7.2
G6	3.91	20.0	23.5	34.9	42.3	10.3
G7	6.25	20.0	31.6	65.3	65.3	11.1
G8	10.00	30.0	51.9	94.9	95.1	16.1

 

Table 9. Physico-chemical parameters of test medium (main study)

Dissolved Oxygen (mg/L)
Group	Test Conc.	Fresh		Spent
		Min	Max	Min	Max
G1	0.0	8.05	8.94	7.94	8.89
G2	0.60	8.31	8.94	8.20	8.90
G3	0.95	8.29	8.99	8.15	8.90
G4	1.53	8.25	8.89	8.10	8.81
G5	2.44	8.30	8.81	8.19	8.76
G6	3.91	8.25	8.74	8.01	8.64
G7	6.25	7.99	8.93	7.81	8.84
G8	10.00	8.25	8.93	8.10	8.74
Min		7.99		7.81
Max		8.99		8.90

pH
Group	Test Conc.	Fresh		Spent
		Min	Max	Min	Max
G1	0.0	8.14	8.34	8.04	8.27
G2	0.60	8.15	8.34	7.98	8.31
G3	0.95	8.05	8.37	7.91	8.30
G4	1.53	8.15	8.34	8.01	8.30
G5	2.44	8.17	8.41	8.01	8.34
G6	3.91	8.17	8.44	8.01	8.36
G7	6.25	8.14	8.39	8.05	8.33
G8	10.00	8.14	8.44	8.05	8.31
Min		8.05		7.91
Max		8.44		8.36

Temperature (°C)
	Fresh	Spent
Mean	20.4	20.2
±SD	0.2	0.1
Min	20.2	20.0
Max	20.7	20.6

Measurement on	Total Hardness [mg/L (as CaCO3)]
Acclimatization	216
Day 0 (Fresh)	212
Day 7 (Spent)	212
Day 7 (Fresh)	216
Day 14 (Spent)	212
Day 14 (Fresh)	212
Day 20 (Spent)	216
Day 20 (Fresh)	212

 

Table 10. Analytical verification of saturation concentration

Day 0 Analysis
Sample Code	Conc. Obtained (mg/L)	Nominal Conc.(mg/L)	% Recovery
Sample G1	-	-	-
Sample G2 As such formulation	8.695	10.000	86.95
Sample G2 G Filtered formulation	8.351	10.000	83.51
Sample G2 Centrifuged formulation	8.558	10.000	85.58
Day 1 Analysis
Sample Code	Conc. Obtained (mg/L)	Nominal Conc.(mg/L)	% Recovery
Sample G1	-	-	-
Sample G2 As such formulation	9.641	10.000	96.41
Sample G2 G Filtered formulation	8.236	10.000	82.36
Sample G2 Centrifuged formulation	8.494	10.000	84.94
Day 2 Analysis
Sample Code	Conc. Obtained (mg/L)	Nominal Conc.(mg/L)	% Recovery
Sample G1	-	-	-
Sample G2 As such formulation	8.583	10.000	85.83
Sample G2 G Filtered formulation	8.243	10.000	82.43
Sample G2 Centrifuged formulation	8.288	10.000	82.88

 

Table 11. Test concentration analysis phase report

Particulars	Nominal Conc. (mg/L)	Obtained  Conc. (mg/L)	Recovery (%)
Day - 0 Fresh (Date: 06/01/2021)
Sample G1	0.0	-	-
Sample G2	0.600	0.557	92.79
Sample G8	10.000	10.005	100.05
Day - 2 Spent (Date: 08/01/2021)
Sample G1	0.0	-	-
Sample G2	0.600	0.590	98.33
Sample G8	10.000	10.516	105.16
Day - 10  Fresh (Date: 16/01/2021)
Sample G1	0.0	-	-
Sample G2	0.600	0.637	106.18
Sample G8	10.000	9.870	98.70
Day 12 Spent sample (Date: 18/01/2021)
Sample Spent G1	0.0	-	-
Sample Spent G2	0.600	0.581	96.78
Sample Spent G8	10.000	10.555	105.55
Day 12 Fresh (Date: 18/01/2021)
Sample Fresh G1	0.0	-	-
Sample Fresh G2	0.600	0.621	103.47
Sample Fresh G8	10.000	10.594	105.94
Day 14 Spent (Date: 20/01/2021)
Sample Spent G1	0.0	-	-
Sample Spent G2	0.600	0.639	106.44
Sample Spent G8	10.000	10.806	108.06
Day 14 Fresh (Date: 20/01/2021)
Sample Fresh G1	0.0	-	-
Sample Fresh G2	0.600	0.631	105.15
Sample Fresh G8	10.000	10.661	106.61
Day 16 Spent (Date: 22/01/2021)
Sample G1	0.0	-	-
Sample G2	0.600	0.636	105.92
Sample G8	10.000	9.242	92.42
Day - 20 Fresh (Date: 26/01/2021)
Sample Fresh G1	0.0	-	-
Sample Fresh G2	0.600	0.656	109.27
Sample Fresh G8	10.000	11.144	111.44
Day - 21 Spent (Date: 27/01/2021)
Sample Spent G1	0.0	-	-
Sample Spent G2	0.600	0.646	107.72
Sample Spent G8	10.000	10.473	104.73

 

Table 12. Survival and reproduction of adult daphnids (main study)

Group: G1, Test Conc.: 0.0 mg/L
	Replicate
Days	1	2	3	4	5	6	7	8	9	10
1	0	0	0	0	0	0	0	0	0	0
2	0	0	0	0	0	0	0	0	0	0
3	0	0	0	0	0	0	0	0	0	0
4	0	0	0	0	0	0	0	0	0	0
5	0	0	0	0	0	0	0	0	0	0
6	0	0	0	0	0	0	0	0	0	0
7	0	0	0	0	0	1	7	5	7	7
8	2	4	1	3	2	5	8	10	5	4
9	8	3	9	5	9	9	10	7	10	12
10	5	2	10	7	10	10	5	3	8	8
11	10	4	0	1	12	11	3	9	9	7
12	9	10	1	6	8	14	10	5	12	4
13	7	9	9	10	7	12	9	4	10	11
14	8	7	10	12	10	8	7	7	8	10
15	10	12	8	8	8	7	4	12	10	14
16	12	8	12	7	9	10	3	10	14	8
17	8	9	10	10	12	14	8	9	10	7
18	10	11	7	8	15	10	10	8	12	5
19	7	8	12	7	0	10	12	7	8	10
20	5	7	8	12	12	8	11	10	9	11
21	8	5	8	10	10	12	8	12	14	10
Surviving F0 adult	1	1	1	1	1	1	1	1	1	1
No. Offspring per surviving F0 adult	109	99	105	106	124	141	115	118	146	128
Total No. of Offspring’s	1191.0
±SD	15.6
CV%	13.1
Total No. Offspring per surviving F0 adult	1191.0
Average No. Offspring per parent	119.1

Group: G2, Test Conc.: 0.60 mg/L
Replicate
Days	1	2	3	4	5	6	7	8	9	10
1	0	0	0	0	0	0	0	0	0	0
2	0	0	0	0	0	0	0	0	0	0
3	0	0	0	0	0	0	0	0	0	0
4	0	0	0	0	0	0	0	0	0	0
5	0	0	0	0	0	0	0	0	0	0
6	0	0	0	0	0	0	0	0	0	0
7	2	3	1	4	5	8	9	4	7	8
8	5	9	0	5	6	9	8	8	8	10
9	7	10	10	3	9	5	11	10	9	4
10	10	8	8	0	10	8	12	7	8	8
11	7	8	9	8	8	9	10	4	13	9
12	5	9	5	12	7	10	7	4	8	7
13	9	10	3	7	5	8	8	0	5	7
14	12	11	4	8	9	9	5	12	5	5
15	11	8	2	3	8	7	7	4	9	8
16	8	12	8	5	6	8	5	8	10	8
17	10	11	12	12	4	10	4	4	8	11
18	11	14	10	7	8	8	9	5	14	16
19	7	8	8	7	9	9	10	10	10	8
20	10	10	7	10	10	7	8	12	8	10
21	12	12	5	13	11	10	7	8	9	14
Surviving F0 adult	1	1	1	1	1	1	1	1	1	1
No. Offspring per surviving F0 adult	126	143	92	104	115	125	120	100	131	133
Total No. of Offspring’s	1189.0
±SD	16.1
Total No. Offspring per surviving F0 adult	1189
Average No. Offspring per parent	118.9

Group: G3 , Test Conc.: 0.95 mg/L
Replicate
Days	1	2	3	4	5	6	7	8	9	10
1	0	0	0	0	0	0	0	0	0	0
2	0	0	0	0	0	0	0	0	0	0
3	0	0	0	0	0	0	0	0	0	0
4	0	0	0	0	0	0	0	0	0	0
5	0	0	0	0	0	0	0	0	0	0
6	0	0	0	0	0	0	0	0	0	0
7	5	0	0	0	0	0	0	0	0	0
8	6	2	5	0	2	5	5	10	3	8
9	1	5	7	1	10	8	8	9	10	10
10	0	7	9	5	8	7	15	5	8	8
11	8	6	8	8	9	10	5	10	14	8
12	7	4	8	9	12	8	12	8	10	10
13	10	5	11	7	7	9	8	8	12	14
14	7	8	9	5	8	10	10	9	10	12
15	15	10	8	8	11	7	4	7	15	10
16	10	7	7	10	13	15	11	10	7	8
17	8	8	8	4	9	12	9	14	9	9
18	9	5	5	5	13	7	7	10	10	7
19	7	7	7	7	8	8	5	12	7	5
20	12	8	9	5	9	10	4	10	14	7
21	14	7	5	7	8	4	7	9	10	8
Surviving F0 adult	1	1	1	1	1	1	1	1	1	1
No. Offspring per surviving F0 adult	119	89	106	81	127	120	110	131	139	124
Total No. of Offspring’s	1146.0
±SD	18.4
Total No. Offspring per surviving F0 adult	1146
Average No. Offspring per parent	114.6

Group: G4                                                                                                                    Test Conc.: 1.53 mg/L
Replicate
Days	1	2	3	4	5	6	7	8	9	10
1	0	0	0	0	0	0	0	0	0	0
2	0	0	0	0	0	0	0	0	0	0
3	0	0	0	0	0	0	0	0	0	0
4	0	0	0	0	0	0	0	0	0	0
5	0	0	0	0	0	0	0	0	0	0
6	0	0	0	0	0	0	0	0	0	0
7	4	0	2	2	0	0	0	0	0	0
8	3	4	4	8	4	8	7	5	7	5
9	7	5	5	7	5	8	5	3	9	8
10	10	9	7	4	8	9	9	7	10	10
11	12	7	8	3	10	12	10	8	4	7
12	8	10	10	4	7	14	7	10	8	8
13	9	8	4	5	5	17	5	7	7	7
14	10	7	3	7	9	10	4	8	7	5
15	11	6	8	2	11	8	8	7	8	7
16	7	9	1	9	7	5	3	5	9	9
17	5	10	7	10	8	7	9	10	10	10
18	8	7	8	7	4	4	12	8	12	7
19	10	8	10	8	5	10	7	9	7	4
20	7	9	8	9	7	7	5	10	8	3
21	5	10	7	5	3	3	7	7	7	8
Surviving F0 adult	1	1	1	1	1	1	1	1	1	1
No. Offspring per surviving F0 adult	116	109	92	90	93	122	98	104	113	98
Total No. of Offspring	1035.0
±SD	11.1
Total No. Offspring per surviving F0 adult	1035
Average No. Offspring per parent	103.5

Group: G5,   Test Conc.: 2.44 mg/L
Replicate
Days	1	2	3	4	5	6	7	8	9	10
1	0	0	0	0	0	0	0	0	0	0
2	0	0	0	0	0	0	0	0	0	0
3	0	0	0	0	0	0	0	0	0	0
4	0	0	0	0	0	0	0	0	0	0
5	0	0	0	0	0	0	0	0	0	0
6	0	0	0	0	0	0	0	0	0	0
7	0	0	0	0	0	0	0	0	0	0
8	0	0	0	0	0	0	0	4	2	8
9	2	1	0	0	1	8	7	8	8	7
10	8	8	7	8	5	10	5	10	0	7
11	9	10	8	10	8	12	9	7	1	9
12	7	7	4	8	7	8	5	9	8	10
13	10	5	7	4	10	7	8	7	10	7
14	8	6	9	7	5	5	14	10	12	7
15	4	8	11	5	7	9	12	8	7	9
16	8	7	17	9	9	10	11	7	9	8
17	7	4	7	10	7	15	7	5	7	2
18	10	3	8	7	8	8	8	7	5	7
19	4	8	9	7	10	11	4	8	18	4
20	8	9	10	8	14	7	8	7	0	3
21	7	10	12	10	12	9	5	9	8	8
Surviving F0 adult	1	1	1	1	1	1	1	1	1	1
No. Offspring per surviving F0 adult	92	86	109	93	103	119	103	106	95	96
Total No. of Offspring	1002.0
±SD	9.7
Total No. Offspring per surviving F0 adult	1002
Average No. Offspring per parent	100.2

Group: G6,   Test Conc.: 3.91 mg/L
Replicate
Days	1	2	3	4	5	6	7	8	9	10
1	0	0	0	0	0	0	0	0	0	0
2	0	0	0	0	0	0	0	0	0	0
3	0	0	0	0	0	0	0	0	0	0
4	0	0	0	0	0	0	0	0	0	0
5	0	0	0	0	0	0	0	0	0	0
6	0	0	0	0	0	0	0	0	0	0
7	0	0	0	0	0	0	0	0	0	0
8	0	0	0	0	0	0	0	0	0	0
9	1	0	1	0	0	5	3	4	0	0
10	8	0	10	0	3	10	14	7	0	0
11	4	7	7	0	8	8	8	5	5	0
12	3	10	8	1	10	12	7	8	7	7
13	2	8	5	4	12	9	10	9	7	5
14	0	7	7	7	8	7	7	8	4	7
15	7	5	8	5	7	5	5	4	9	9
16	5	7	0	6	10	10	7	3	10	12
17	1	9	1	-	12	11	8	9	12	7
18	2	8	7	-	8	17	10	7	7	4
19	7	5	8	-	6	8	7	8	8	5
20	8	7	9	-	7	4	5	5	10	9
21	5	6	5	-	5	6	3	7	14	-
Surviving F0 adult	1	1	1	D	1	1	1	1	1	D
No. Offspring per surviving F0 adult	53	79	76	23	96	112	94	84	93	65
Total No. of Offspring	775.0
±SD	17.4
Total No. Offspring per surviving F0 adult	687.0
Average No. Offspring per parent	85.9

Group: G7, Test Conc.: 6.25 mg/L
Replicate
Days	1	2	3	4	5	6	7	8	9	10
1	0	0	0	0	0	0	0	0	0	0
2	0	0	0	0	0	0	0	0	0	0
3	0	0	0	0	0	0	0	0	0	0
4	0	0	0	0	0	0	0	0	0	0
5	0	0	0	0	0	0	0	0	0	0
6	0	0	0	0	0	0	0	0	0	0
7	0	0	0	0	0	0	0	0	0	0
8	0	0	0	0	0	0	0	0	0	0
9	0	0	0	0	0	0	0	0	0	0
10	0	4	7	0	1	0	4	7	0	0
11	0	0	2	0	5	8	2	4	-	-
12	0	2	4	4	7	9	3	3	-	-
13	0	3	1	3	5	4	7	5	-	-
14	7	4	5	4	4	5	5	7	-	-
15	5	7	8	2	8	7	9	-	-	-
16	9	8	7	7	7	8	3	2	-	-
17	4	4	0	5	6	4	4	4	-	-
18	3	4	1	3	5	3	2	8	-	-
19	8	6	8	2	7	2	4	3	-	-
20	7	3	9	4	4	5	3	2	-	-
21	2	7	4	5	3	4	2	7	-	-
Surviving F0 adult	1	1	1	1	1	1	1	1	D	D
No. Offspring per surviving F0 adult	45	52	56	39	62	59	48	52	0	0
Total No. of Offspring	413.0
±SD	7.5
Total No. Offspring per surviving F0 adult	413
Average No. Offspring per parent	51.6

 

Table 13. Individual length of adult daphnids at termination (main study)

Group	Test Conc. (mg/L)	Replicates										Mean Length
		1	2	3	4	5	6	7	8	9	10
		Length of Parent Daphnids Measured (µm)
G1	0.00	2441.448	2035.502	2207.08	2590.569	1426.741	2489.002	2398.471	2040.233	1795.129	2182.626	2160.68
G2	0.60	1787.469	1760.321	2437.997	2032.836	2162.400	2197.269	2233.503	2291.763	2226.086	2270.695	2140.03
G3	0.95	2134.462	2000.663	2137.676	2022.879	1716.737	1949.526	2229.142	2896.753	2232.390	1917.798	2123.80
G4	1.53	2124.762	2449.334	2132.571	1481.624	1476.827	1476.827	2365.251	2230.674	2148.163	2256.990	2014.30
G5	2.44	2180.475	2071.188	2107.872	2016.400	2157.598	2078.623	1752.194	1757.007	2168.295	1762.903	2005.26
G6	3.91	2275.339	1802.279	2187.363	1804.028	1774.554	2004.927	1779.454	1884.530	-	-	1939.06
G7	6.25	1744.900	1856.560	1882.394	1929.948	2094.795	1992.416	1985.974	1876.363	-	-	1920.42
G8	10.00	1549.240	1493.218	1892.554	1871.764	1953.880	1868.982	2058.502	-	-	-	1812.59

 

Validity criteria fulfilled:

yes

Remarks:

Mortality of parent animals in controls was 0% at the end of the test (< 20%). Mean no. of offspring per parent animal surviving was 119.1 (>60). CV% around mean no. of offspring was 0.4 (≤ 25%). Dissolved O2 was 7.81 (> 3 mg/L).

Conclusions:

Based on the results obtained under test conditions, the EC10 for reproductive output of live offspring per surviving F0 parent was 1.63 mg/L (95% CL 1.28 - 2.08), and the EC10 for reproductive output of offspring of F0 parent was 1.66 mg/L (95% CL 1.27 - 2.17); the NOEC for daphnid growth was 2.44 mg/L, all based on nominal concentrations.

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, under GLP conditions. 

Based on an in-house solubility test, the test item was found to be soluble in reconstituted water. Based on the performed preliminary range-finding test performed at 0 (control), 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L, the main study was performed at the test concentrations of 0.60, 0.95, 1.53, 2.44, 3.91, 6.25 and 10.0 mg/L (nominal) and one control group. 24-hour old daphnids were exposed to the test item in ISO reconstituted water under semi-static conditions, test media was renewed once in 48 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.

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 pink, and no particles were observed at the bottom of test vessels at 24- and 48-hour observation, at all tested concentrations. Additionally, the limit of saturation of the test item in the test media was confirmed to be 8.2 to 9.6 mg/L in a separate test. All validity criteria were met.

After 21 days of exposure, clinical signs of lethargy, daphnids localized on the bottom of the container, and 20, 20, and 30.0% mobility inhibition were observed at the tested concentrations of 3.91, 6.67, and 10.0 mg/L, respectively. Reduction in reproductive output was found to be -0.4, -0.7, 35.4, 67.9 and 100% was observed at the tested concentrations of 0.01, 0.1, 1.0, 5.0, and 10.0 mg/L, respectively. Inhibition in growth of adult length was 0.6, 1.2, 1.3, 6.3 and 100% at the tested concentrations of 0.01, 0.1, 1.0, 5.0 and 10.0 mg/L, respectively. Since mortality did not follow a concentration-response pattern, replicates with parental mortality were excluded from the analysis of the test result.

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 reproductive output of live offspring per surviving F0 parent was 1.63 mg/L (95% CL 1.28 - 2.08), and the EC10 for reproductive output of offspring of F0 parent was 1.66 mg/L (95% CL 1.27 - 2.17); the NOEC for daphnid growth was 2.44 mg/L, all based on nominal concentrations.

Description of key information

Key study. Method according to OECD 211, GLP study. The 21-day EC10 of the test item in Daphnia Magna was found to be 1.63 mg/L with 95% confidence limits 1.28 - 2.08 mg/L (nominal concentration, based on reproductive performance). 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC10

Effect concentration:

1.63 mg/L