5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-(ethylsulfinyl)-1H-pyrazole-3-carbonitrile

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

04 Nov - 08 Nov 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Insoluble test substance was visible at the highest test concentration.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

adopted 1992

Deviations:

yes

Remarks:

test temperature below 13 °C

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

adopted 1992

Qualifier:

according to guideline

Guideline:

EPA OPP 72-1 (Fish Acute Toxicity Test)

Version / remarks:

adopted 1985

Deviations:

yes

Remarks:

higher concentration series factor

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples of each concentration and the control(s) were analysed at test initiation and termination
- Sample storage conditions before analysis: Samples were analysed immediately after sampling.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide (DMF)
- Method: For each test concentration, a concentrated stock solution of test substance in a fixed volume of DMF was prepared.
- Concentration of vehicle in test medium (stock solution and final test solution): 0.1 mL/L

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: batch no. 96T091 from Aquatic Research Organisms, Hampton NH, USA
- Length at study initiation (mean and range): 5.32 cm (5.0 cm - 5.6 cm)
- Weight at study initiation (mean and range): 1.31 g (1.03 g - 1.50 g)
- Feeding during test: none

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions (same as test or not): same as test; prior to acclimation and testing, the fish were held in 450 L glass tanks providing at least one litre of dilution water per fish.
- Type and amount of food: commercial pelleted trout food
- Feeding frequency: twice daily except during 24 h before test start
- Health during acclimation (any mortality observed): no mortality during 7 days before test start

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

43 mg/L CaCO3

Test temperature:

11.4 - 12.0 °C

pH:

7.2 - 7.6

Dissolved oxygen:

≥ 8.2 mg/L

Nominal and measured concentrations:

Nominal: 0, 0.34, 0.75, 1.65, 3.64 and 8.00 mg/L
Mean measured: < LOQ, 0.35, 0.78, 1.64, 3.65 and 6.99 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: container
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: clear glass, 20 L
- Aeration: None in the test vessels, but dilution water was air-saturated by bubbling prior to test start
- Type of flow-through (e.g. peristaltic or proportional diluter): constant flow system consisting of peristaltic pumps providing fixed flow rates of dilution water (50 mL/min) and syringe pumps providing fixed flow rates of test substance stock solutions (5 µL/min)
- Renewal rate of test solution (frequency/flow rate): approximately 7 test aquarium volumes per day of newly prepared test solution
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2
- Biomass loading rate: 0.18 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water filtered through active charcoal and diluted with varying proportions of deionised water
- Metals (mg/L): Ca 36.2, Mg 12.8, K 18.7, Na 18.4, other metals < 0.005 - 0.5
- Pesticides: < 0.01 - 1.0 µg/L
- Chlorine: 74 mg/L
- Ca/Mg ratio: 2.8:1
- Conductivity: 129.9 µS/cm
- Test medium: 80% Dutch Standard Water, 20% LC oligo medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: Dissolved oxygen concentration, temperature and pH of test solutions were measured at test initiation and once daily at each treatment level and in the controls throughout the exposure period. The specific conductance and the total hardness of the dilution water was measured at test initiation.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light, 8 h dark
- Light intensity: 383 - 583 lux (35.4 - 53.9 footcandles)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortalities and physical or behavioural alterations were monitored 3h18min after test start and at each 24 h interval until test end.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Results used to determine the conditions for the definitive study: based on available toxicity information

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 6.99 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 6.99 mg/L was the visual limit of solubility

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.35 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Mortality of control: No mortalities or sublethal toxic effects were observed in the control groups throughout the test period.
- LC50 exceeds solubility of substance in test medium
- Analytical monitoring: 77 - 108% of nominal values at test start and 91 - 113% of nominal values at test end.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: presence of particles in test solutions
- Effect concentrations exceeding solubility of substance in test medium: yes

Reported statistics and error estimates:

Statistics: LC50 values are estimated by means of a computer program using one of the three statistical methods: probit analysis, moving average method or binomial probability. The method selected is determined by the data set. LC50 values are presented with 95% confidence limits when the methods of probit analysis or moving average are used to perform the calculation.

Sublethal observations / clinical signs:

Cumulative mortality of O. mykiss after 96 h of exposure to RPA 107382:

No mortality up to 1.64 mg/L

10 % mortality at 3.65 mg/L

20% mortality at 6.99 mg/L

Table 1: Sublethal alterations observed during the exposure period. R1/R2, replicates

Mean measured concentrations (mg/L)	Sublethal alterations	24 h		48 h		72 h		96 h
		R1	R2	R1	R2	R1	R2	R1	R2
Solvent Control	none	0	0	0	0	0	0	0	0
Control < LOQ	none	0	0	0	0	0	0	0	0
0.35	Lethargy	0	0	0	0	1	0	0	0
	Pigmentation disorders	0	0	0	0	0	0	1	0
0.78	Muscular contraction	0	2	2	3	2	4	2	4
	Lethargy	1	2	1	3	2	2	5	4
	Pigmentation disorders	0	0	0	0	0	0	1	0
	Accelerated respiration	1	2	0	0	0	0	0	0
1.64	Muscular contraction	10	10	10	10	10	10	10	10
	Lethargy	5	9	5	8	6	10	7	10
	Pigmentation disorders	3	1	2	1	3	1	3	0
	Accelerated respiration	4	2	1	1	2	1	0	1
3.65	Muscular contraction	10	10	9	9	9	9	9	9
	Lethargy	2	1	0	0	0	0	0	0
	Moribund	0	0	9	9	9	9	9	9
	Pigmentation disorders	8	6	7	0	7	4	6	4
	Accelerated respiration	10	10	9	9	9	9	7	5
6.99	Muscular contraction	10	10	10	10	10	9	9	7
	Lethargy	0	0	0	0	0	0	0	0
	Moribund	10	10	10	10	10	9	9	7
	Pigmentation disorders	6	10	6	10	6	9	5	5
	Accelerated respiration	10	10	10	10	7	5	9	3

Table 1: Validity criteria for OECD 203.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality in the control(s) should not exceed 10% (or one fish if less than ten are used) at the end of the test	0%
The dissolved oxygen concentration must have been at least 60 per cent of the air saturation value throughout the test	≥ 8.2 mg/L	yes
There must be evidence that the concentration of the substance being tested has been satisfactorily maintained, and preferably it should be at least 80 per cent of the nominal concentration throughout the test. If the deviation from the nominal concentration is greater than 20 per cent, results should be based on the measured concentration.	Recovery: 77 -113%  Results are based on the measured concentrations.	yes

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

01 - 05 May 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Controls, solvent controls, 0.313, 0.625, 1.25, 2.50 and 5.00 mg/L (at day 0 and 4)
- Sampling method: 10 mL samples were taken

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solution for the highest test concentrations was prepared by diluting 2.5935 g of test substance in 50 mL solvent. From this stock solution dilution series in DMF were prepared for each single test concentration. Test solutions were prepared by adding the appropriate amounts (3.0 mL) of stock solution to dilution water.
- Differential loading: no
- Controls: Dilution water only (control), dilution water and solvent (solvent control)
- Chemical name of vehicle: Dimethylformamide (DMF)
- Concentration of vehicle in test medium: stock solution: 100%, final test solutions: 0.01%, solvent control: 0.01%
- Evidence of undissolved material: no

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: fathead minnow
- Lot no: EG031914
- Source: in house culture
- Length at study termination: mean: 25.8 mm, SD: 1.1 mm, range: 24.5 - 27.5 mm
- Weight at study termination: mean:0.1257 d, SD: 0.017 g, range: 0.1010 - 0.1535 g

ACCLIMATION
- Acclimation period: > 14 d
- Acclimation conditions: same as test
- Type and amount of food during acclimation: Tetramin flakes and live brine shrimp
- Feeding frequency during acclimation: daily
- Health during acclimation: no mortalities during 48 hours prior to testing, no treatments for disease

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

44 - 52 mg/L

Test temperature:

21.5 - 22.3°C

pH:

7.4 - 7.9

Dissolved oxygen:

7.3 - 8.4 mg/L (83 - 96% saturation)

Nominal and measured concentrations:

Nominal: 0 (control), 0 (solvent control), 0.313, 0.625, 1.25, 2.50 and 5.00 mg a.i./L
Mean measured: < LOQ (control), < LOQ (solvent control), 0.307, 0.615, 1.18, 2.44 and 4.81 mg a.i./L

Details on test conditions:

TEST SYSTEM
- Test vessel: 38 L glass aquaria
- Material, size: 49.5 x 25.4 x 30.5 cm, headspace: 6.7 cm, fill volume: 30 L
- Aeration: no
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10
- Biomass loading rate: 0.042 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated municipal water (dechlorinated with sodium metabisulfite, passed through a multimedia filter and granular activated carbon units, then blended with municipal water that has been dechlorinated with sodium metabisulfite, passed through a multimedia filter, granular activated carbon units, demineralized by conventional softeners, passed through a 5-micron cartridge filter, treated by double pass reverse osmosis and passed through an ultraviolet
sterilizer) blended with reverse osmosis water
- Culture medium different from test medium: no
- Intervals of water quality measurement: temperature: continuously in control test vessel, dissolved oxygen, pH: daily, alkanility: day 0 and 4

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light, 8 hours dark
- Light intensity: 700 - 868 lux (cool white fluorescents)

EFFECT PARAMETERS MEASURED: survival and sublehal effects: (after 0, 24, 48, 72 and 96 h)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 0 (control), 0 (solvent control), 0.040, 0.20, 1.0, and 5.0 mg a.s./L
- Results used to determine the conditions for the definitive study: Various sublethal effects and 20% mortality in the 5.0 mg/L and 1.0 mg/L test levels.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 4.81 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Details on results:

- Other biological observations: Sublethal effects were observed in all fish exposed to 1.18, 2.44 and 1.18 mg/L, in 9 of 10 fish at 0.615 mg/L and in 1 of 10 fish exposed to 0.307 mg/L. No sublethal effects were observed in the control groups.
- Mortality of control: 0%
- Other adverse effects control: No

Sublethal observations / clinical signs:

Table 1: Mortality of P. promelas after 96 h

Mortality
Mean Measured Concentration (mg a.i./L)	Percent Dead at 96-Hours
Control	0 %
Solvent Control	0 %
0.307	0 %
0.615	0 %
1.18	0 %
2.44	0 %
4.81	0 %

Table 2: Measured concentrations of the test item during the 96-hour static exposure of P. promelas

NominalConcentration(mg a.i./L)	Day 0MeasuredConc. (mg a.i./L)	Day 0 %Nominal	Day 4MeasuredConc. (mg a.i./L)	Day 4 %Nominal	MeanMeasuredConc. (mg a.i./L)	Percent MeanMeasuredConc.
Control	< 0.025	NA	< 0.025	NA	NA	NA
Solvent control	< 0.025	NA	< 0.025	NA	NA	NA
0.313	0.284	91%	0.329	105%	0.307	98%
0.625	0.598	96%	0.631	101%	0.615	98%
1.25	1.16	93%	1.20	96%	1.18	94%
2.50	2.39	96%	2.49	100%	2.44	98%
5.00	4.61	92%	5.01	100%	4.81	96%

LOQ = (0.025 mg a.i./L)

NA = Not Applicable

Calculations for mean and percent of nominal concentration are based on recoveries from Day 0 and Day 4.

Table 3: Validity criteria for OECD 203.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality in the control(s) should not exceed 10% (or one fish if less than ten are used) at the end of the test	0%	yes
The dissolved oxygen concentration must have been at least 60 per cent of the air saturation value throughout the test	83 - 96%	yes
There must be evidence that the concentration of the substance being tested has been satisfactorily maintained, and preferably it should be at least 80 per cent of the nominal concentration throughout the test. If the deviation from the nominal concentration is greater than 20 per cent, results should be based on the measured concentration.	94 -105%	yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Feb - 12 Feb 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Fish were 1 cm longer than allowed by the required range

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

adopted 1992

Deviations:

yes

Remarks:

Fish were 1 cm longer than allowed by the required range

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

adopted 1992

Qualifier:

according to guideline

Guideline:

EPA OPP 72-1 (Fish Acute Toxicity Test)

Version / remarks:

adopted 1985

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples of each concentration and the control(s) were analysed at test initiation and termination. Additional sampling: 1.0 mg/L after 48 h and 0.48 mg/L after 72 h.
- Sample storage conditions before analysis: Samples were analysed immediately after sampling.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide (DMF)
- Method: For each test concentration, a concentrated stock solution of test substance in a fixed volume of DMF was prepared just before test start. Test solutions were homogenised by magnetic stirring.
- Concentration of vehicle in test medium (stock solution and final test solution): 0.1 mL/L

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

TEST ORGANISM
- Common name: Bluegill sunfish
- Source: batch 99B011 from Osage Catfisheries, Osage Beach MO, USA
- Length at study initiation (mean and range): 4.04 cm (3.50 cm - 4.40 cm)
- Weight at study initiation (mean and range): 0.67 g (0.50 g - 0.81 g)

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions (same as test or not): same as test; prior to acclimation and testing, the fish were held in 450 L glass tanks providing at least one litre of dilution water per fish.
- Type and amount of food: pelleted trout food
- Feeding frequency: twice daily except during 24 h before test start
- Health during acclimation (any mortality observed): no mortality during 7 days before test start

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

45 mg/L CaCO3

Test temperature:

21.7 - 23.0 °C

pH:

7.2 - 7.5

Dissolved oxygen:

≥ 7.4 mg/L

Nominal and measured concentrations:

Nominal: 0, 0.05, 0.11, 0.23, 0.48 and 1.0 mg/L
Mean measured: < LOQ, 0.05, 0.11, 0.24, 0.50 and 1.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: container
- Type (delete if not applicable): open
- Material, fill volume: clear glass, 10 L
- Aeration: dilution water was saturated by air-bubbling prior to test start
- Renewal rate of test solution (frequency/flow rate): approx. 7 test aquarium volumes per day of newly prepared test solution via a constant flow system consisting of peristaltic pumps
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2
- Biomass loading rate: 0.093 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water filtered through active charcoal and diluted with deionised water
- Total organic carbon: < 1 mg/L
- Particulate matter: < 1 mg/L
- Metals (mg/L): Ca 12.1, Mg 3.6, K 0.2, Si 0.6, Na 16.9, all other metals tested < 0.005 to 0.1
- Pesticides: < 0.02 to 10 µg/L
- Chloride: 5.8 mg/L
- Conductivity: 133 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: Dissolved oxygen concentration, temperature and pH of each test solution was measured at test initiation, once daily at each treatment level and in the control throughout the exposure period. In addition water temperature was continuously recorded from one reference aquarium. Specific conductance, pH and total hardness of the dilution water were measured at test initiation.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light/8 h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Biological observations of test fish were made approx. 3h25 after test initiation and at each 24 h interval until test termination. Mortalities and abnormalities were recorded every 24 h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.1
- Range finding study
- Test concentrations (nominal): 0.1, 1.0, 1.5 and 6.0 mg/L
- Results used to determine the conditions for the definitive study: 100% mortality at 1.5 and 6.0 mg/L and 90% mortality at 1.0 mg/L after 24 h.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.26 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL = 0.21 - 0.31 mg/L (probit method)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.05 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Mortality of control: No mortalities or significant sublethal toxic effects were observed in the solvent control group throughout the test period. 5% mortality (one fish) was recorded in the dilution water control group after 48 h of exposure.
- The test substance was visually soluble in the dilution water throughout the range of concentrations tested.
- Analytical monitoring: 91 - 120% of nominal values at test start and 100 - 110% of nominal values at test end.

Sublethal observations / clinical signs:

Table 1: Additional effect concentrations

Duration	Endpoint	Effect conc.	Nom./Meas.	Conc. based on	Basis for effect	Remarks
24 h	EC50	0.72 mg/L	meas. (arithm. mean)	test mat.	mortality	95% CL = 0.58 - 0.96 mg/L (probit method)
48 h	EC50	0.38 mg/L	meas. (arithm. mean)	test mat.	mortality	95% CL = 0.32 - 0.47 mg/L (moving average method)
72 h	EC50	0.31 mg/L	meas. (arithm. mean)	test mat.	mortality	95% CL = 0.26 - 0.37 mg/L (probit method)

Table 2: Cumulative mortality of L. macrochirus exposed to the test item

Mean measured concentrations (mg/L)	Mean cumulative mortality (%) after
	24 h	48 h	72 h	96 h
Solvent Control	0	5	5	5
Control < LOQ	0	0	0	0
0.05	0	0	0	0
0.11	0	0	0	0
0.24	0	15	25	45
0.50	30	65	90	95
1.00	70	100	100	100

Table 3: Sublethal alterations of L. macrochirus exposed to the test item. AD, all dead

Mean measured concentrations (mg/L)	Sublethal alterations	24 h		48 h		72 h		96 h
		R1	R2	R1	R2	R1	R2	R1	R2
Solvent Control	none	0	0	0	0	0	0	0	0
Control < LOQ	Dead	0	0	1	0	0	0	0	0
0.05	none	0	0	0	0	0	0	0	0
0.11	Erratic swimming	0	0	0	0	0	0	3	2
	Haemorrhagic spot	0	0	2	2	4	2	4	2
	Pigmentation disorders	0	0	0	0	0	0	0	1
	Lethargic	0	0	0	0	0	1	0	4
0.24	Haemorrhagic spot	1	2	1	3	2	3	1	1
	Pigmentation disorders	0	0	3	9	3	9	2	2
	Lethargic	5	4	0	0	0	0	0	0
	Moribund	1	1	8	9	6	9	5	6
	Dead	0	0	2	1	2	0	1	3
0.50	Haemorrhagic spot	0	3	0	1	0	1	AD	0
	Pigmentation disorders	0	0	1	3	0	1	AD	0
	Moribund	6	8	2	5	0	2	AD	1
	Dead	4	2	4	3	2	3	AD	1
1.00	Moribund	2	2	0	0	AD	AD	AD	AD
	Dead	6	8	4	2	AD	AD	AD	AD

Table 4: Measured concentration of the test item in test solutions during 96 -h exposure under flow-through conditions

Nominal concentrations (mg/L)	Measured concentrations (mg/L)
	0-hour		96-hour		Mean (a)
	R1	R2	R1	R2
0 (control)	< QL	< QL	< QL	< QL	-
0 (solvent control)	< QL	< QL	< QL	< QL	-
0.05	0.05	0.06	0.05	0.05	0.05
0.11	0.11	0.10	0.12	0.12	0.11
0.23	0.24	0.24	0.24	0.25	0.24
0.48	0.50	0.50	0.49 (c)	0.51	0.50
1.0	1.0	0.95	1.1 (b)	1.1 (b)	1.0

QL: Quantification limit

(a): Mean measured values for reporting, (b): Measured at 48 h (100% mortality), (c): Measured at 72 h (100% mortality)

Table 5: Validity criteria for OECD 203.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality in the control(s) should not exceed 10% (or one fish if less than ten are used) at the end of the test	< 10% in control and solvent control	yes
The dissolved oxygen concentration must have been at least 60 per cent of the air saturation value throughout the test	> 60%	yes
There must be evidence that the concentration of the substance being tested has been satisfactorily maintained, and preferably it should be at least 80 per cent of the nominal concentration throughout the test. If the deviation from the nominal concentration is greater than 20 per cent, results should be based on the measured concentration.	91 -120%	yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

LC50 (96h) = 0.26 mg/L (mean measured, Lepomis macrochirus,OECD 203)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

0.26 mg/L

Additional information

Four GLP-studies according to OECD guideline 203 are available for short-term toxicity testing in fish. All four studies report effect concentrations based on mean measured concentrations which were in the range from 77% to 120% of nominal concentrations throughout the study periods.

In the key study, bluegill sunfish (Lepomis macrochirus) were exposed to five test item concentrations in the range between 0.05 and 1.0 mg/L (nominal), a solvent control (dimethylformamide, DMF) and a dilution water control in a flow-through system for a period of 96 hours. The test substance was visually soluble in the dilution water throughout the range of concentrations tested. The derived LC50 (96h) was 0.26 mg/L and the NOEC (96h) was determined to be 0.05 mg/L. The LC50 of 0.23 mg/L value was the lowest derived effect value from the available short-term fish studies and was therefore used as key value.

In a further study performed according to OECD 203, fathead minnow (Pimephales promelas) were exposed to mean measured test concentrations ranging from 0.307 to 4.81 mg a.i./L and control and solvent control (DMF) in a static system. As there was no mortality at any test level, the 96-hour LC50 value was empirically determined to be > 4.81 mg a.i./L (functional limit of solubility). The NOEC and LOEC were ≥4.81 and > 4.81, respectively.

A further supporting study assessed the effects of the test item under flow-through conditions with rainbow trout (Oncorhynchus mykiss) as test organism. The results of the test were reported in terms of the mean measured concentrations ranging between 0.35 and 6.99 mg/L. At the highest test concentration of 6.99 mg/L, undissolved test substance was visible and a mortality of 20% was recorded. The derived LC50 (96h) was > 6.99 mg/L. The NOEC (96h) was determined to be 0.35 mg/L.