1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Dec 2014 - 15 Jan 2015

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)

Version / remarks:

draft version, 1994

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control, Solvent Control, 0.313, 0.625, 1.25, 2.50, and 5.00 mg a.i./L (nominal)
- Sampling method: Two alternating replicates (a 60 mL from the mid-depth in each aquarium sampled) for each level (A and B, C and D) were taken on a weekly basis. An additional set of samples was taken on day 4 from 2.50 mg a.i./L replicates A and B, because a sampling error occurred on day 0.

Vehicle:

yes

Remarks:

Dimethylformamide (DMF)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The stock solutions were prepared bi-weekly. The appropriate amount of compound was weighed into a volumetric flask (corrected for percent a.i.). The flask was brought to volume with dimethylformamide (DMF) and inverted several times. The stock solutions were placed on a magnetic stirrer. The test solutions were prepared by diluting the stock solution with artificial sea salt mixed with processed water to achieve the appropriate test concentration. Detailed description is reported in section " Any other information on material and methods incl tables"
- Controls: yes (control and solvent control)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 100 µL

Test organisms (species):

Cyprinodon variegatus

Details on test organisms:

TEST ORGANISM
- Common name: Sheepshead minnow
- Source: Aquatic Biosystems Inc., Fort Collins, CO, USA; Parental lot number: ABS121114
- Age at study initiation (mean and range, SD): 24 - 48 h old eggs in the neurula stage
- Method of breeding: no brood, Sheepshead minnow eggs were received on the morning of the experimental start
- Feeding during test: starting on day 4
- Food type: brine shrimp (Artemia salina)
- Amount: Equal distribution at 0.5 - 2.5 mL per feeding (feeding rate was increased as fish grew during the exposure)
- Frequency: Twice daily on weekends and one to three times daily on weekdays until approximately 24 h prior to study termination.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
After acclimation, eggs were placed in dishes containing dilution water. Microscopic observations on the eggs were made to determine the stage of development, and eggs that appeared to be in poor health were not used. Eggs appropriate for testing were pooled for impartial placement into randomly positioned egg cups.

ACCLIMATION
- Acclimation period: Upon receipt, the eggs were acclimated over a period of 2 h.
- Acclimation conditions: the eggs were acclimated from 15 °C - 25 °C

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

35 d

Test temperature:

24.3 - 26.0 °C

pH:

8.0 - 8.3

Dissolved oxygen:

5.8 - 7.0 mg/L (saturated range: 72 to 95%)

Salinity:

17 - 21‰ (mean: 19‰)

Nominal and measured concentrations:

Control, Solvent Control, 0.313, 0.625, 1.25, 2.50, and 5.00 mg a.i./L (nominal)
Control (< 0.025), Solvent control (< 0.25), 0.306 ,0.531, 1.13, 2.41, and 4.21 mg a.i./L (mean measured test concentration)

Details on test conditions:

TEST SYSTEM
- Egg cups: One oscillating egg cup per replicate placed in each replicate through the egg hatching phase
- Size of vessel: 8.4 L (21.6 x 12.7 x 30.5 cm)
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: All glass; Volume: 7 L (21.6 x 12.7 x 25.5 cm); loading: 0.12 g mean wet weight based on controls, 0.041 g/L/day flow-through (mean wet weight based on controls); in a test bath with liquid circulated temperature control
- Egg distribution: On day 0, impartially placed 5 eggs into each egg cup until 35 eggs were in each, egg cups were then placed in test chambers based on randomization sequence.
- Type of flow-through (e.g. peristaltic or proportional diluter): All glass 2 L modified Mount-Brungs proportional diluter system used for intermittent delivery of test solutions to test chambers. Splitter cups were used to equally divide (± 10%) test solutions between replicates at each level. One Hamilton Microlab 600 Series pump was usedd for toxicant and solvent control injections and one Hamilton Microlab 500 Series pump for a solvent equalizing injection. Dilution monitoring: Visually checked twice daily during the week and at least once on weekends and holidays
- Renewal rate of test solution (frequency/flow rate): Approximately 8 turnover per vessel per 24 h, checked visually twice daily during the week and at least once on weekends and holidays.
- No. of organisms per vessel: 35 eggs at initiation, thinned to 20 alevin after hatching phase
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: The biomass loading factor estimate for 20 fish in an aquarium was 0.041 g/L during a 24-hour period.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Soft processed water (dechlorinated municipal and reverse osmosis waters); Water used for estuarine organism studies and culturing in the SynTech Research Laboratory Services Aquatic Toxicology Laboratory consists of artificial sea salts (Crystal Sea Marinemix) mixed with process water to produce a salinity of 20 ± 5 parts per thousand (‰). Process water is made by blending reverse osmosis water with dechlorinated municipal water and/or spring water. Blended water is stored in polypropylene or PVC holding tanks and intensely aerated before use.
- Total organic carbon: soft water: 1.2 mg/L; hard water: 0.55 mg/L
- Metals: soft water: Aluminium: 3.4 - 6.9 µg/L; Calcium: 19000 - 76000 µg/L; Copper 1.3 - 4.9 µg/L; Iron <10 - 10 µg/L; Magnesium: 1100 - 3700 µg/L; Molybdenum: 1.0 - 1.6 µg/L; Potassium: 730 - 3100 µg/L; Silver: <0.10 - 0.23 µg/L; Sodium: 14000 - 24000 µg/L; Zinc: <1.0 - 27 µg/L
- Pesticides: <0.05 µg/L
- Chlorine: Total Residual Chlorine: < 0.003 mg/L
- Culture medium different from test medium: All test levels, including the controls, were handled and held under similar test conditions with the exception of the application of test material
- Intervals of water quality measurement: At experimental start, weekly (± 2 d) thereafter including experimental finish (in two replicates per level).

OTHER TEST CONDITIONS
- Photoperiod: 16 h light/ 8 h dark (with 30 min dawn/dusk transition period)
- Light intensity: 700 to 856 lux (mean: 771 lux)
- Light Source: Cool white fluorescent light

EFFECT PARAMETERS MEASURED:
Fish hatchability, sublethal effects, survival, and growth (length and dry weight for all surviving fish on day 35) via visual observations, total lengths and dry weights, Interval: Hatching observations made daily during hatching phase, observations for sublethal effects and survival made daily, growth determinations made at the end of the exposure

Egg Observations: Daily for mortality (nonviable eggs removed) and hatching
Thinning Phase: When at least 90% of all viable control eggs had hatched, observations were made, and the alevin were impartially thinned to 20 per replicate by releasing them from the egg cup to the test vessel in which the egg cup had been suspended. Thinning day was on day 7.

POST-HATCH DETAILS
- Begin of post-hatch period: Day 7
- Post Hatch Phase Observations: Daily for abnormal behavior, abnormal physical changes and mortality (dead organisms were removed)
- Length Measurements: Total length (nearest 0.5 mm) via a millimeter scale (all surviving fish on day 35) after sacrificing fish via MS-222 solution (200 mg/L)
- Wet Weight Measurements: Taken at termination for control fish to establish biomass loading estimate (nearest 0.01 g)
- Dry Weight Measurements: All surviving fish at day 35 (nearest 0.0001 g); Drying period: Fish were placed in a drying oven (Thelco 6547 Oven) set to approximately 60 °C for approximately 4 d. The dried fish were cooled to ambient temperature in a desiccator for approximately 4.5 h, and were then individually weighed.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0

Reference substance (positive control):

no

Duration:

8 d

Dose descriptor:

NOEC

Effect conc.:

>= 4.21 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: alevin survival

Duration:

8 d

Dose descriptor:

LOEC

Effect conc.:

> 4.21 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: alevin survival

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 4.21 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: fry survival

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

> 4.21 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: fry survival

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 4.21 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: percent Hatch

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

> 4.21 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: percent Hatch

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 4.21 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

time to hatch

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

> 4.21 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

time to hatch

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 4.21 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

length

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

> 4.21 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

length

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 4.21 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

weight

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

> 4.21 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

weight

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 4.21 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

morphology

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

> 4.21 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

morphology

Details on results:

- Observed effects at each concentration for each observation time: Details of analytical and biological results are summarized within tables 1 to 4 in section "Any other information on results incl. tables"
- Cumulative mortality at each concentration and for each recommended observation time if possible: 0.306 mg a. i./L = 5 dead; 0.531 mg a. i./L = 2 dead; 1.13 mg a. i./L= 4 dead; 2.41 mg a. i./L= 5 dead; 4.21 mg a. i./L= 7 dead
- Mortality in the controls: One fish from 80 (for control and solvent control, respectively)
- Behavioural observation of the fish: There was one small fish in replicate B, test level 0.306 mg a.i./L and in replicate A, test level 2.41 mg a.i./L, respectively. All remaining fish including the controls appeared normal during the course of the study.
Day 6 mean percent hatch: Control (84.3%), Solvent Control (81.4%), 0.306 (80.7%), 0.531 (82.9%), 1.13 (80.7%), 2.41 (72.1%) and 4.21 (74.3%) mg a.i./L
Day 7 mean percent hatch: Control (87.1%), Solvent Control (83.6%), 0.306 (83.6%), 0.531 (86.4%), 1.13 (85.0%), 2.41 (82.1%) and 4.21 (82.9%) mg a.i./L
Day 8 mean percent alevin survival: Control (87.9%), Solvent Control (85.0%), 0.306 (85.7%), 0.531 (86.4%), 1.13 (85.7%), 2.41 (82.1%) and 4.21 (82.9%) mg a.i./L
Day 35 mean percent fry survival: Control (98.8%), Solvent Control (98.8%), 0.306 (92.5%), 0.531 (97.5%), 1.13 (95.0%), 2.41 (93.8%) and 4.21 (91.3%) mg a.i./L
Mean Total Length (mm): Control (20.4), Solvent Control (20.9), 0.306 (20.8), 0.531 (20.8), 1.13 (20.9), 2.41 (20.8) and 4.21 (20.7) mg a.i./L
Mean Dry Weights (mg): Control (27.1), Solvent Control (29.1), 0.306 (29.1), 0.531 (29.6), 1.13 (30.3), 2.41 (30.6) and 4.21 (29.9) mg a.i./L

Results with reference substance (positive control):

No reference substance was tested.

Reported statistics and error estimates:

Raw data from the control and solvent control groups were compared for equal variance using the Equal Variance Two-Sample t test to determine if the data sets were poolable. Appropriate tests were used to determine if the data had equal variances and normal distribution (i.e. Variance Ratio F test, Bartlett’s Test, and Shapiro-Wilk’s test). If normality and homogeneity of variance were demonstrated for the raw or transformed values, then parametric analyses were conducted using analysis of variance (ANOVA) followed by Dunnett’s test (p= 0.05). If normality and/or homogeneity of variance were not demonstrated on raw or transformed values, non-parametric procedures were used. ECx values were calculated where applicable using linear interpolation. All data analysis was conducted using CETIS statistical software

Table 1: Measured Test Concentrations of the test substance During the Sheepshead Minnow Early Life Stage Toxicity Test

Nominal concentrations [mg a.i./L]	Replicate	Day 0	Day 4	Day 7	Day 13	Day 20	Day 28	Day 35	Mean	SD	Percent of nominal
Control	A	< 0.025	---	---	< 0.025	---	< 0.025	---	NA	NA	NA
	B	< 0.025	---	---	< 0.025	---	< 0.025	---
	C	---	---	<0.025	---	< 0.025	---	< 0.025
	D	---	---	<0.025	---	< 0.025	---	< 0.025
Solvent Control	A	< 0.025	---	---	< 0.025	---	< 0.025	---	NA	NA	NA
	B	< 0.025	---	---	< 0.025	---	< 0.025	---
	C	---	---	<0.025	---	< 0.025	---	< 0.025
	D	---	---	<0.025	---	< 0.025	---	< 0.025
0.313	A	0.314	---	---	0.332	---	0.352	---	0.306	0.0219	98
	B	0.321	---	---	0.295	---	0.306	---
	C	---	---	0.284	---	0.300 (b)	---	0.308
	D	---	---	0.275	---	0.283	---	0.297
0.625	A	0.540	---	---	0.528	---	0.546	---	0.531	0.0238	85
	B	0.557	---	---	0.556	---	0.569	---
	C	---	---	0.491	---	0.530	---	0.513
	D	---	---	0.496	---	0.522 (b)	---	0.528
1.25	A	1.14	---	---	1.20	---	1.13	---	1.13	0.0673	90
	B	1.18	---	---	1.18	---	1.24	---
	C	---	---	1.06	---	1.13	---	1.03
	D	---	---	1.05	---	1.13	---	1.04
2.50	A	4.10 (a)	2.49	---	2.48	---	2.47	---	2.41	0.1624	96
	B	1.19 (a)	2.32	---	2.65	---	2.47	---
	C	---	---	2.29	---	2.54	---	2.05
	D	---	---	2.36	---	2.55	---	2.25
5.00	A	4.24	---	---	4.72	---	4.15	---	4.21	0.382	84
	B	4.11	---	---	4.73	---	4.44	---
	C	---	---	4.20	---	4.32	---	3.60
	D	---	---	4.19	---	4.40	---	3.44

(a) = Sampling error, recoveries not used. An additional set of samples was taken on day 4.

(b) = Mean value retain sample

NA = Not applicable

SD = Standard Deviation

--- No samples taken at this interval

LOQ = 0.025 mg a.i/L

Calculations for mean, standard deviation, and percent of nominal concentration are based on recoveries from days 0, 4, 7, 13, 20, 28, and 35. Calculations are made in Microsoft Excel using unrounded data. Manual calculations may vary slightly.

Table 2: Time to hatch during the Sheepshead Minnow Early Life Stage Toxicity Test

Mean Measured Concentration [mg a.i./L]	Replicate	Day 4a	Day 5a	Day 6 a	Day 7 a
Control	A	2.86	40.0	94.3	94.3
	B	0.00	45.7	68.6	80.0
	C	5.71	62.9	88.6	88.6
	D	2.86	71.4	85.7	85.7
	Mean	2.86	55.0	84.3	87.1
	S.D.	2.33	14.6	11.1	5.95
Solvent Control	A	0.00	37.1	77.1	77.1
	B	0.00	11.4	85.7	85.7
	C	0.00	2.86	85.7	91.4
	D	0.00	5.71	77.1	80.0
	Mean	0.00	14.3	81.4	83.6
	S.D.	0.00	15.6	4.95	6.34
0.306	A	2.86	20.0	85.7	85.7
	B	0.00	28.6	88.6	91.4
	C	0.00	45.7	74.3	74.3
	D	0.00	11.4	74.3	82.9
	Mean	0.71	26.4	80.7	83.6
	S.D.	1.43	14.6	7.51	7.14
0.531	A	0.00	2.86	62.9	77.1
	B	0.00	5.71	94.3	94.3
	C	0.00	14.3	82.9	82.9
	D	2.86	37.1	91.4	91.4
	Mean	0.71	15.0	82.9	86.4
	S.D.	1.43	15.5	14.2	7.87
1.13	A	0.00	31.4	71.4	77.1
	B	0.00	62.9	85.7	85.7
	C	2.86	31.4	74.3	85.7
	D	0.00	8.57	91.4	91.4
	Mean	0.71	33.6	80.7	85.0
	S.D.	1.43	22.3	9.44	5.89
2.41	A	0.00	11.4	91.4	91.4
	B	2.86	8.57	68.6	85.7
	C	0.00	17.1	71.4	85.7
	D	0.00	14.3	57.1	65.7
	Mean	0.71	12.9	72.1	82.1
	S.D.	1.43	3.69	14.3	11.3
4.21	A	0.00	57.1	80.0	88.6
	B	0.00	28.6	77.1	77.1
	C	2.86	22.9	62.9	80.0
	D	0.00	17.1	77.1	85.7
	Mean	0.71	31.4	74.3	82.9
	SD	1.43	17.8	7.74	5.22

a Percent Hatch = (# of alevin / # of eggs on day 0) * 100

Calculations done in Excel using unrounded numbers; manual calculations may vary.

S.D. = Standard deviation

Table 3: Fry Survival During Sheepshead Minnow Early Life Stage Toxicity Test

Mean Measured Concentration		Percent Survivorship
[mg a.i./L]	Replicate	Day 7a	Day 35b
Control	A	94.3	95.0
	B	82.9	100
	C	88.6	100
	D	85.7	100
	Mean (S.D.)	87.9 (4.88)	98.8 (2.50)
Solvent Control	A	82.9	100
	B	85.7	100
	C	91.4	95.0
	D	80.0	100
	Mean (S.D.)	85.0 (4.88)	98.8 (2.50)
0.306	A	85.7	95.0
	B	91.4	95.0
	C	82.9	80.0
	D	82.9	100
	Mean (S.D.)	85.7 (4.04)	92.5 (8.66)
0.531	A	77.1	100
	B	94.3	100
	C	82.9	90.0
	D	91.4	100
	Mean (S.D.)	86.4 (7.87)	97.5 (5.00)
1.13	A	80.0	100
	B	85.7	100
	C	85.7	80.0
	D	91.4	100
	Mean (S.D.)	85.7 (4.67)	95.0 (10.0)
2.41	A	91.4	95.0
	B	85.7	90.0
	C	85.7	90.0
	D	65.7	100
	Mean (S.D.)	82.1 (11.3)	93.8 (4.79)
4.21	A	88.6	100
	B	77.1	95.0
	C	80.0	85.0
	D	85.7	85.0
	Mean (S.D.)	82.9 (5.22)	91.3 (7.50)

a Percent Survivorship before thinning= (# of alevin and eggs on Day 7/# of eggs on Day 0) * 100

b Percent Survivorship after thinning = (# of fish on day 35/# of fish at thinning) * 100

Calculations done in Excel using unrounded numbers; manual calculations may vary.

S.D. = Standard Deviation

Table 4: Day 35 Total Length and Dry Weight for Sheepshead Minnows Exposed to the test item in the Early Life Stage Toxicity Test

Mean Measured Concentration [mg a.i./L]	Replicate	Mean Wet Weight [g]	Mean Length [mm]	Mean Dry Weight [mg]
Control	A	0.14	21.5	32.6
	B	0.11	19.9	25.7
	C	0.10	20.0	24.2
	D	0.11	20.3	25.9
	Mean (S.D.)	0.12 (0.016)	20.4 (0.754)	27.1 (3.74)
Solvent Control	A	NA	21.1	30.0
	B	NA	20.9	28.4
	C	NA	20.8	30.0
	D	NA	20.7	28.1
	Mean (S.D.)	NA	20.9 (0.197)	29.1 (1.01)
0.306	A	NA	21.2	30.5
	B	NA	21.0	28.8
	C	NA	20.3	28.6
	D	NA	20.6	28.5
	Mean (S.D.)	NA	20.8 (0.398)	29.1 (0.960)
0.531	A	NA	21.3	31.9
	B	NA	21.1	29.6
	C	NA	20.6	29.0
	D	NA	20.4	27.8
	Mean (S.D.)	NA	20.8 (0.432)	29.6 (1.72)
1.13	A	NA	21.1	31.0
	B	NA	20.9	30.1
	C	NA	21.4	34.0
	D	NA	20.2	26.0
	Mean (S.D.)	NA	20.9 (0.527)	30.3 (3.33)
2.41	A	NA	20.7	31.5
	B	NA	21.3	31.5
	C	NA	20.7	30.2
	D	NA	20.4	29.2
	Mean (S.D.)	NA	20.8 (0.390)	30.6 (1.13)
4.21	A	NA	20.7	30.0
	B	NA	20.5	29.3
	C	NA	20.5	28.5
	D	NA	21.3	31.9
	Mean (S.D.)	NA	20.7 (0.394)	29.9 (1.47)

NA = not applicable        

S.D. = Standard deviation

Calculations done in Excel using unrounded numbers; manual calculations may vary.

Validity criteria fulfilled:

yes

Reference 2

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 May - 29 Aug 2014

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:

adopted 2013

Deviations:

yes

Remarks:

A short temperature decrease in the control over 2 h to a minimum of 23.1 °C. This deviation had no influence on the biological outcome of the study.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)

Version / remarks:

1996

Deviations:

yes

Remarks:

A short temperature decrease in the control over 2 h to a minimum of 23.1 °C. This deviation had no influence on the biological outcome of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control, solvent control, 0.313, 0.625, 1.25, 2.50 and 5.00 mg a.s./L. (nominal)
- Sampling method: at timepoints: on days -2, 0, 7, 14, 21, 28 and 33, additionally samples on day -2 were also analysed for internal verification. Freshly and aged samples were analysed.The sampling dates for the stock solutions were on study day -3, 4, 11, 18, 25 and 33. On day 24 additional water samples were taken at 0.625 and 2.50 mg a.s./L to assure correct dosing after a short and directly corrected water flow decrease to 6.5 L/h and 6.0 L/h respectively (instead of 8 L/h).
- Sample storage conditions before analysis: The samples were stored in a freezer until they were analysed.

Vehicle:

yes

Remarks:

dimethylformamide

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Five stock solutions were prepared by dissolving the test item into the dimethylformamide p.a. (DMF) by intensely stirring for at least 1 h at room temperature. Afterwards the stock solution was sonicated for at least 15 min. The test solutions were prepared by diluting the stock solution to achieve the appropriate test concentration (detailed information is presented in table 1 "Any other information on material and methods incl tables").
- Controls: yes (control and solvent control)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 100 µL

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: fathead minnow
- Source: Bayer CropScience AG
- Age at study initiation (mean and range, SD): freshly fertilised eggs (< 24 hours old)
- Method of breeding: adult fathead minnows used as breeding pairs for the egg production; twenty-four breeding groups (1 male and 2 female per group) were used to produce a sufficient number of fertilized eggs for starting the study.
- Feeding during test: Start: on the day the first larvae had hatched.
- Food type: The larvae were fed ad libitum with living freshly hatched brine shrimp nauplii (< 48 h old).
- Amount: Each aquarium received an equal quantity of food. In cases the numbers of fish were reduced due to mortalities the amount of food was reduced accordingly.
- Frequency: Food was added to the aquaria 3 times daily, except on weekends when food was added 2 times per day. Feeding was stopped one day prior study termination.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

33 d

Remarks on exposure duration:

28.0 d post-hatch

Hardness:

2.24 - 3.09 °dH (control)
2.23 - 3.07 °dH (solvent control)
2.25 - 3.00 °dH (test item concentrations)

Test temperature:

23.8 - 24.7 °C (control)
24.2 - 24.7 °C (solvent control)
24.1 - 24.8 °C (test item concentrations)

pH:

6.8 - 7.2 (control)
7.0 - 7.4 (solvent control)
7.0 - 7.4 (test item concentrations)

Dissolved oxygen:

93 - 101% (control)
80 - 101% (solvent control)
86 - 104% (test item concentration)

Conductivity:

120.3 µS/cm² (mean)

Nominal and measured concentrations:

control, solvent control, 0.313, 0.625, 1.25, 2.50 and 5.00 mg a.s./L (nominal)
< LOQ,

Details on test conditions:

TEST SYSTEM
- Test vessel: Eggs were placed in incubation cups; incubation cups were placed in each chamber; cups were positioned on an oscillating rocker arm assembly to facilitate the circulation of test solution in the egg cups.
- Size of vessel: cup size: 5.5 cm diameter stainless steel pipes with a stainless steel bottom perforated with holes (hole diameter: 0.8 mm)
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass aquaria 15.5 (width) x 16.0 (depth) cm; water height of approximately 16.0 cm
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): ProMinent® mikro g/5a pumps and flow-meters were used to funnel the intended amount of the stock solutions and test water into mixing chambers to prepare the different test media. The accuracy of the test solution splits was checked at test start. The accuracy of the splits and stock solution dispensers/pumps was within the acceptable range (± 10% and ± 3% of nominal, respectively). The diluter system and syringe pump function were visually checked and documented twice each working day and once daily during the weekend. The flow-through device was initiated 3 days prior to the start of the study.
- Renewal rate of test solution (frequency/flow rate): aerated reconstituted test water was 8 L/h and the stock solutions (prepared with dimethylformamide) were transferred at a rate of 0.8 mL/hour resulting in a dilution factor of 10000 and a solvent concentration of 100 μL dimethylformamide/L test water; 12 exchanges of test solution per day in all replicate test chambers
- No. of organisms per vessel: 30
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: ranged between 0.006 and 0.007 g fish/L/day

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water was prepared by adding stock solutions prepared of analytical grade salts to deionised water (conductivity < 0.10 μS/cm²) to yield the following nominal ionic concentrations (based to ISO guidance).
- Total organic carbon: < 2 mg/L
- Chemical oxygen demand: < 5 mg/L
- Particulate matter (total solids): < 5 mg/L
- Inorganic Contaminants: < 1 μg/L: Al, As, Cr, Cu,Co, Pb, Ni, Zn; < 0.1 μg/L: Cd, Hg, Ag; < 100 μg/L F-, Bor; < 1.8 μg/L: Fe, NH4+ < 0.03 mg/L
- Cations: 15.0 mg/L: Ca++; 2.40 mg/L: Mg++; 2.80 mg/L: Na+; 0.59 mg/L: K+
-Organochlorine Contaminants: < 0.01 µg/L: HCB ; α-HCH ; β-HCH ; γ-HCH ; Heptachlor ; Heptachlorepoxid ; Aldrin ; Pentachlorbenzol; Quintozen ; α-Endosulfan ; β-Endosulfan ; Dieldrin ; 2,4-DDE ; 4,4-DDE ; 2,4-DDD ; 4,4-DDD ; 2,4-DDT ; 4,4-DDT ; Endrin ; PCB 28 ; PCB 52 ; PCB 101 ; PCB 138 ; PCB 153 ; PCB 180
- Pesticides: < 0.05 μg/L: 2,4-D Linuron ;Anilazine; MCPA; Atrazine ; Mecoprop ; Azinphos Ethyl ; Mercaptodimethur ; Azinphos Methyl ; Metamitron ; Bentazon ; Metazachlor ; Bitertanol ; Methabenzthiazuron ; Bromacil ; Metoxuron ; Buturon ; Metribuzin ; Carbofuran ; Monolinuron ; Chlorphoxim ; Parathion Ethyl ; Chlortoluron ; Parathion Methyl ; Coumaphos ; Phoxim ; Coumatetralyl ; Promecarb ; Cyanazin ; Prometryn ; Dichlorprop ; Propanil ; Dinosep ; Propazin ; Dinosepacetat ; Propoxur ; Disulfoton ; Simazin ; Diuron ; 2,4,5-T ; Ethidimuron ; Tebuconazole ; Fenitrothion ; Terbutylazin ; Folpet ; Thiram ; Hexazinon ; Triadimefon ; Isofenphos ; Triadimenol ; Isomethiozin ; Trichlorfon ; Isoproturon ; Lenacil
- Chlorine (residual): < 0.01 mg/L
- Conductivity: ranged between 109 and 138 μS/cm² during the whole test period
- Culture medium different from test medium: no
- Intervals of water quality measurement: residue-analysis of artificially fish testing water is performed at least two times per year (ASTM Standard E 729).

OTHER TEST CONDITIONS
- Photoperiod: 16 h light and 8 h dark
- Light intensity: 398 - 803 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Egg Observations: Daily for mortality (non-viable eggs removed) and hatching. The hatching of larvae was observed and started on day 4 and lasted until day 5, Larval survival was observed between day 6 and test termination on day 33; behavioural and morphological abnormalities were observed for the whole test period and larval growth, expressed as total length, wet weight and dry weight, was measured at test termination. The biomass loading factor for the study was determined using the wet weights of the control and solvent control fish at study termination.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0

RANGE-FINDING STUDY
no range-finding study was performed

Reference substance (positive control):

no

Duration:

4 d

Dose descriptor:

NOEC

Effect conc.:

>= 5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

number hatched

Remarks on result:

other: hatching success after day 4

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

>= 5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

larval development

Remarks:

and larval survival

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

length

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

>= 5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

weight

Remarks:

Growth (wet weight)

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

>= 5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

weight

Remarks:

Growth (dry weight)

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

>= 5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

morphology

Remarks:

and behavioral effects

Duration:

33 d

Dose descriptor:

LOEC

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

number hatched

Duration:

33 d

Dose descriptor:

LOEC

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

larval development

Remarks:

; Larval survival day 33

Duration:

33 d

Dose descriptor:

LOEC

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

length

Remarks:

Growth (total length)

Duration:

33 d

Dose descriptor:

LOEC

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

weight

Remarks:

Growth (wet weight)

Duration:

33 d

Dose descriptor:

LOEC

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

length

Remarks:

Growth (wet weight)

Duration:

33 d

Dose descriptor:

LOEC

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

morphology

Remarks:

and behavioral effects

Details on results:

- Observed effects at each concentration for each observation time: see Table 1 to 6 in section "Any other additional information on results incl. Tables"

- Mortality in the controls: Larval survival was observed between day 6 and test termination on day 33 (post hatch day 28). The statistical data analysis for this parameter resulted in no significant differences compared to the pooled control. Mean larval survival at test termination ranged from 98 to 100% in all treatment groups including pooled controls. Data analysis showed a statistically significant decrease in total length in comparison to the pooled control data at the highest test concentration of 5.00 mg a.s./L. No statistically significances were observed for wet and dry weight in comparison to the pooled control data.

Results with reference substance (positive control):

No reference substance was tested

Reported statistics and error estimates:

Shapiro Wilk’s, Kolmogorov-Smirnov test was used to check the normality of the data set and Levene’s test for homogenecity of variances. If the required assumptions for parametric methods will not be met – non-normal distribution or heterogeneous variance, consideration should be given to transforming the data. Data in percent will be arcsine transformed before analysis. For the evaluation of the NOEC and the LOEC a Williams Multiple Sequential t-test Procedure or the Welch t-test for inhomogeneous variances with Bonferroni adjustment were used. The ECx values and the 95% confidence limits were calculated for each observation time by appropriate dose/response modelling procedures (e.g. probit analysis). All statistical analyses were conducted using a computer program TOXRAT®.

Analytical Results:

The mean measured concentrations of the test item in the treatment groups ranged between 0.329 -0.348, 0.606 -0.708, 1.30 -1.39, 2.62 -2.94 and 4.97 -5.45 mg a.s./L. These overall mean measured values ranged between 97 and 118 % of nominal during the test period for all treatment groups. As the number of analytical samples revealed the succesfull maintenance of nominal concentrations (guideline recommendation 80 – 120% of nominal), it was decided to base the results of this study on nominal concentrations.

Table 1: Biological Results Endpoints

Test Substance
Test Object	Fathead minnow (Pimephales promelas)
Exposure	33 Day, flow-through (ELS)
Results	NOEC [mg a.s./L]	LOEC [mg a.s./L]	EC10 (95%-CL) [mg a.s./L]
Hatching success (day 4)	≥ 5.00	> 5.00	n.d.
Larval Survival (day 33)	≥ 5.00	> 5.00	n.d.
Growth (Total Length)	2.50	5.00	n.d.
Growth (Wet Weight)	≥ 5.00	> 5.00	1.20* (n.d. – n.d.)
Growth (Dry Weight)	≥ 5.00	> 5.00	4.50* (n.d. – n.d.)
Morphological & Behavioral Effects**	≥ 5.00	> 5.00	not applicable

n.d.: not determined due to mathematical reasons or inappropriate data

*: No meaningful concentration/response was found (p(F) > 0.05; i.e. slope of the relationship is not significant different from zero). Due to the lacking concentration/response the shown ECx are not valid.

**: The results of morphological & behavioral effects are based on expert judgment. This type of data is not suitable for a statistical analysis.

Table 2: Biological Results Hatching success (day 4; day 5)

Nominal conc. [mg a.s./L]	Replicate	PHD-0 (day 5)			PHD 0 (day 5)
		Egg Hatch1 [%]	Egg Hatch1 [n]	SD of n	Egg Hatch1 [%]	Egg Hatch1 [n]	SD of n
Control	A	3	1		97	29
	B	3	1		97	29
	C	3	1		87	26
	D	0	0		97	28
	Mean	3	1	0.5	93	28	1.4
SolventControl	A	0	0		90	27
	B	3	1		93	28
	C	3	1		90	27
	D	0	0		93	28
	Mean	2	1	0.6	92	28	0.6
Pooled Controls	Mean	2		0.5	93	28	1.0
0.313	A	7	2	93	28
	B	20	6		93	29
	C	0	0		97	29
	D	0	0		100	30
	Mean	7	2	2.8	97	29	0.8
0.625	A	10	3		93	28
	B	13	4		97	29
	C	7	2		93	28
	D	10	3		100	30
	Mean	10	3	0.8	96	29	1.0
1.25	A	0	0		90	27
	B	3	1	93	28
	C	3	1	87	26
	D	7	2	87	26
	Mean	3	1	0.8	89	27	1.0
2.50	A	0	0		100	30
	B	3	1		97	29
	C	7	2		87	26
	D	3	1		97	29
	Mean	3	1	0.8	95	29	1.7
5.00	A	0	0		97	29
	B	7	2		100	30
	C	7	2		97	29
	D	0	0		100	30
	Mean	3	1	1.2	98	30	0.6

SD: Standard Deviation

PHD: post hatch day

1 Hatch data (cumulative) = (no. of larvae)/( no. of inserted eggs on study day 0)*100; n: number of

Larvae

Table 3: Biological Results: Hatching success (Day 5) based on inserted and living eggs

Nominal conc. [mg a.s./L]	Replicate	PHD 0 (Day 5) Hatching Success (based on inserted eggs)			PHD 0 (Day 5) Hatching Success (based on living eggs)
		Egg Hatch1) [%]	Egg Hatch1) [n]	SD of n	Egg Hatch2) [%]	Egg Hatch2) [n]	SD of n
Control	A	97	29		100	29
	B	97	29	100	29
	C	87	26	100	26
	D	93	28	100	28
	Mean	93	28	1.4	100	28	1.4
Solvent Control	A	90	27		100	27
	B	93	28	100	28
	C	90	27	100	27
	D	93	28	100	28
	Mean	92	28	0.6	100	28	0.6
Pooled Controls	Mean	93	28	1.0	100	28	1.0
0.313	A	93	28		100	28
	B	97	29	100	29
	C	97	29	100	29
	D	100	30	100	30
	Mean	97	29	0.8	100	29	0.8
0.625	A	93	28		100	28
	B	97	29	100	29
	C	93	28	100	28
	D	100	30	100	30
	Mean	96	29	1.0	100	29	1.0
1.25	A	90	27		100	27
	B	93	28	97	28
	C	87	26	100	26
	D	87	26	100	26
	Mean	89	27	1.0	99	27	1.0
2.50	A	100	30		100	30
	B	97	29	100	29
	C	87	26	100	26
	D	97	29	100	29
	Mean	95	29	1.7	100	29	1.7
5.00	A	97	29		100	29
	B	100	30	100	30
	C	97	29	100	29
	D	100	30	100	30
	Mean	98	30	0.6	100	30	0.6

SD: Standard Deviation

PHD: post hatch day

Egg Hatch 1 = hatch data (cumulative) = (no. of larvae)/(no. of inserted eggs on study day 0)*100

Egg Hatch 2 = hatch data (cumulative) =(no. of larvae)/(no. of living and fertilized eggs on day 5)*100

Table 4: Biological Results Larval Survival

Nominal concentration [mg a.s./L]	Replicate	PHD 1 – 28 (Day 6 - 33)
		Larvae at PHD 1 [n]	Larval Survival [%]	Larval Survival corrected [n]	SD of n
Control	A	20	100	20
	B	20	100	20
	C	20	100	20
	D	20	100	20
	Mean	/	100	20	0.0
Solvent Control	A	20	100	20
	B	20	100	20
	C	20	100	20
	D	20	100	20
	Mean	/	100	20	0.0
Pooled Controls	Mean	/	100	20	0.0
0.313	A	20	100	20
	B	20	100	20
	C	20	100	20
	D	20	95	19	*
	Mean	/	99	20	0.5
0.625	A	20	95	19
	B	20	95	19
	C	20	100	20
	D	20	100	20
	Mean	/	98	20	0.6
1.25	A	20	100	20
	B	20	95	19
	C	20	100	20
	D	20	95	19
	Mean	/	98	20	0.6
2.50	A	20	100	20
	B	20	95	19
	C	20	100	20
	D	20	95	19
	Mean	/	98	20	0.6
5.00	A	20	95	19
	B	20	100	20
	C	20	100	20
	D	20	100	20
	Mean	/	99	20	0.5

SD: Standard Deviation; PHD: post hatch day; larval survival data: (no. of fish on study day 33)/(no. of fish (20) after thinning on

study day 5)*100; / not calculated

*: The fish counted at test termination revealed differences with respect to the number of fish at 0.313 mg a.s./L in replicate D. The

respective result was consequently used to correct the number. 19 fish survived in replicate D at 0.313 mg a.s./L instead of 20

fish. Due to the fact that dead larvae cannot allways be detected during a study this procedure is necessary. Larvae dying shortly

after the last observation might be degraded and therefore are invisible at the next observation

Table 5: Biological Results: Mean Total Length, Dry Weight and Wet Weight at Test Termination

Nominal conc. [mg a.s./L]	Repl.	Day 33 (PHD 28)
		Length [mm]	SD	Dry Weight [mg]	SD	Wet weight [mg]	SD
Control	A	19.7	1.99	11.3	4.48	60.9	22.5
	B	19.8	2.27	12.4	5.30	66.9	28.0
	C	19.5	1.92	11.1	4.84	59.8	24.4
	D	20.1	2.39	12.5	4.98	65.5	24.6
	Mean	19.8	2.14	11.8	4.90	63.3	24.9
Solvent Control	A	19.0	2.45	11.6	4.17	65.0	22.4
	B	19.7	2.32	12.4	4.65	66.6	24.3
	C	19.6	2.61	11.9	4.21	66.1	22.0
	D	19.4	2.34	12.0	4.77	64.3	23.8
	Mean	19.4	2.43	12.0	4.45	65.5	23.1
Pooled Controls	Mean	19.6	2.29	11.9	4.68	64.4	24.0
0.313	A	19.9	2.52	10.7	2.25	60.0	13.5
	B	19.1	2.07	9.3	1.91	49.4	10.1
	C	19.6	2.08	13.1	3.35	69.0	17.1
	D	19.7	1.97	12.2	7.29	67.4	40.7
	Mean	19.6	2.16	11.3	3.70	61.5	20.4
0.625	A	19.0	2.07	11.8	4.42	61.5	22.4
	B	19.6	2.39	10.0	1.92	52.7	10.5
	C	19.1	2.68	8.7	4.74	45.6	24.3
	D	19.3	2.20	13.4	5.75	70.5	28.5
	Mean	19.3	2.33	11.0	4.21	57.6	21.4
1.25	A	18.2	2.36	12.8	4.87	65.6	24.3
	B	19.8	1.73	10.9	4.88	56.3	24.7
	C	18.5	2.28	15.4	5.94	77.5	28.6
	D	19.4	2.78	11.4	5.00	61.4	28.7
	Mean	19.0	2.29	12.6	5.17	65.2	26.6
2.50	A	19.1	2.45	11.6	5.97	59.2	29.0
	B	19.4	2.89	12.7	5.66	65.9	28.3
	C	18.8	2.48	10.9	5.35	59.7	28.4
	D	19.6	2.06	9.5	1.98	47.3	9.6
	Mean	19.2	2.47	11.2	4.74	58.0	23.8
5.00	A	19.0	2.47	13.9	5.95	69.8	29.7
	B	18.9	2.52	10.4	3.05	52.7	16.5
	C	18.7	2.25	8.7	2.79	43.2	12.5
	D	18.8	2.59	10.5	3.30	54.9	17.0
	Mean	18.9*	2.46*	10.9	4.50	55.2	23.1

SD: Standard Deviation

PHD: post-hatch day

* Denotes statistically significant difference from pooled control (α=0.05) [Williams-Test]

Table 6: Biological Results Biomass Loading

Nominal concentration in mg a.s./L	Repl.	Total Sum of Wet Weights on Day 33 (Test Termination) in g	Biomass Loading1 on Day 33 (Test Termination) in g/L/d
Control	A	1.2176	0.0063
	B	1.3372	0.0070
	C	1.1957	0.0062
	D	1.3106	0.0068
	Mean	1.2653	0.0066
Solvent Control	A	1.3001	0.0068
	B	1.3328	0.0069
	C	1.3224	0.0069
	D	1.2865	0.0067
	Mean	1.3105	0.0068
Pooled Control	Mean	1.2879	0.0067

1 Biomass Loading in g fish/L/day = total sum of fish weight/ 4 (test solution volume in L)/ 48 (water flow through per day in L)

Table 7: Analytical Results: Mean Measured Concentrations of the substance in Test Solutions

Study Day (Replicate)	Control	Solvent Control	0.313 mg a.s./L	0.625 mg a.s./L	1.25 mg a.s./L	2.50 mg a.s./L	5.00 mg a.s./L
0 (C)	<0.0125	<0.0125	0.344	0.669	1.31	2.62	5.41
7 (D)	<0.0125	<0.0125	0.345	0.651	1.34	2.63	5.37
14 (A)	<0.0125	<0.0125	0.331	0.630	1.32	2.65	5.18
21 (B)	<0.0125	<0.0125	0.348	0.644	1.39	2.68	5.45
24/1 (D)*	-	-	-	0.630*)	-	2.62*)	-
24/2 (C)*	-	-	-	0.708*)	-	2.94*)	-
28 (A)	<0.0125	<0.0125	0.335	0.632	1.30	2.71	5.18
33 (B)	<0.0125	<0.0125	0.329	0.606	1.33	2.75	4.97
Mean	-	-	0.339	0.646	1.33	2.70	5.26
Standard deviation	-	-	0.008	0.031	0.032	0.107	0.183
Percent of Nominal	-	-	108	103	107	108	105
Minimum	-	-	0.329	0.606	1.3	2.62	4.97
Maximum	-	-	0.348	0.708	1.39	2.94	5.45

- not measured

*) On day 24 a significant deviation of more than 10% of the water flow-through was observed in the treatments 0.625 and 2.50 mg a.s./L. This water flow deviation was manually corrected on the same day. To assure that this short deviation in the water flow did not cause in an increase of the concentration inside these two treatments, two additional water samples for analysis were taken. The first sample was taken immediately after observing the deviation in the flow-through system. The second samples were taken a few hours later to prove if the flow-through system was correctly functioning again. The decrease in water flow observed at 0.625 mg a.s./L and at 2.50 mg a.s./L were 6.5 L/h and 6.0 L/h respectively, instead of 8.0 L/h.

Table 8: Measured Concentrations of the substance in the Stock Solutions

Series	Study Day	3.13 [g a.s./L]	6.25 [g a.s./L]	12.5 [g a.s./L]	25.0 [g a.s./L]	50.0 [g a.s./L]
1	-3 (newly prepared)	3.43	6.78	13.6	27.6	51.1
2	4 (newly prepared)	3.40	6.61	13.3	27.7	49.9
3	11 (newly prepared)	3.50	6.98	13.3	27.5	52.7
4	18 (newly prepared)	3.38	6.79	13.6	25.8	50.4
5	25 (newly prepared)	3.40	6.61	13.9	26.6	51.3
	Mean	3.42	6.75	13.5	27.0	51.1
	% of Nominal	109	108	108	108	102
1	4 (aged, 7 days)	3.29	6.60	13.6	26.6	54.2
2	11 (aged, 7 days)	3.29	6.87	13.6	26.4	54.8
3	18 (aged, 7 days)	3.35	6.87	13.3	26.8	51.9
4	25 (aged, 7 days)	3.36	6.92	13.5	27.0	51.9
5	33 (aged, 8 days)	3.40	6.80	13.8	26.7	49.3
	Mean	3.34	6.81	13.6	26.7	52.4
	% of Nominal	107	109	108	107	105

 

Description of key information

Freshwater

NOEC (33 d) 2.50 mg a.s./L (M-565378-01-1; Pimephales promelas; OECD 210, nominal concentration)

Marine water

NOEC (35 d) ≥ 4.21 mg a.s./L (M-547597-01-1; Cyprinodon variegatus; OECD 210, mean measured concentration)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

2.5 mg/L

Marine water fish

Marine water fish

Effect concentration:

4.21 mg/L

Additional information

Two experimental studies are available investigating the effects of the test item on fish early life stages of the marine species sheepshead minnow (Cyprinodon variegatus) and the freshwater species fathead minnow (Pimephales promelas) (M-547597-01-1 and M-565378-01-1), respectively. The studies were conducted according to GLP and OECD guideline 210 (2013). Due to a low solubility the test item was dissolved with dimethylformamide (0.1 mL/L) in order to prepare the test solutions. In addition, a water control and a solvent control (0.1 mL DMF/L) were tested.

In the first study the eggs and fry of the marine species C. variegatus were exposed in a flow-through system over a period of 35 d to nominal concentrations of 0.313, 0.625, 1.25, 2.50 and 5.00 mg a.s./L (corresponding to mean measured concentrations of 0.306, 0.531, 1.13, 2.41 and 4.21 mg a.s./L). Mean measured recoveries were within the range of 84 to 98% of the nominal concentrations. Hatching rates, sublethal symptoms, fry survival and growth (length and wet and dry weight) were recorded daily. Based on analytical findings the biological endpoints are reported based on mean measured concentrations. The study resulted in a NOEC (35 d) of ≥ 4.21 mg a.s./L and a LOEC (35 d) of > 4.21 mg a.s./L based on mean measured test concentrations.

In the second study eggs and larvae of the freshwater species P. promelas were exposed in a flow-through system over a period of 33 d (28 d post-hatch) to the nominal test concentrations of 0.313, 0.625, 1.25, 2.50 and 5.00 mg a.s./L. The overall mean measured values correspond to recoveries of 103 to 108% of nominal for all test levels. Thus, the effect concentrations were based on nominal values. Hatching rates, sublethal symptoms, fry survival and growth (length and wet and dry weight) were recorded daily. The test fulfilled the validity criteria of the underlying guideline, with the minor exception of a short exceeding of the allowed temperature range. This deviation did not influence the biological outcome of the study, as demonstrated by the overall control data. The 33-day exposure of P. promelas to the test item resulted in an overall NOEC (33 d) of 2.50 mg a.s./L (based on total length) and a LOEC (33 d) of 5.00 mg a.s./L based on nominal test concentrations.