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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Nov - 07 Nov 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Insoluble test substance was visible during the test.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

adopted 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

adopted 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 72-2 (Aquatic Invertebrate Acute Toxicity Test)

Version / remarks:

adopted 1985

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples of test solutions were taken for analysis from each bulk preparation at test start and from each concentration level (pooled replicates) at the termination of each cycle of 24 hours.
- Sampling method: Each sample volume was collected from the approximate midpoint of the container with a pipet.
- Sample storage conditions before analysis: Samples were analysed within 24 h.

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: A stock solution of the highest test substance concentration was prepared by dissolution of 160 mg of test substance in 2 mL of the solvent DMF. Stock solutions of the four lower test substance concentrations were prepared by serial dilutions of the first stock solution submitted to magnetic agitation until used. Test solutions were prepared by dilution of the stock solutions with known volumes of dilution water. Volumes of 500 mL (bulk preparation) for each fixed concentration were prepared. Then each bulk preparation was distributed between the test vessels to provide final test volumes of 200 mL. An appropriate volume of solvent was added to the stock solutions of each of the four lower test substance concentrations so that the final concentration of solvent in each test solution was 0.1 mL/L.
- Concentration of vehicle in test medium (stock solution and final test solution): 0.1 mL/L

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain: Straus
- Source: Laboratory culture at the Aquatic Ecotoxicology facilities of Rhône-Poulenc Agrochimie; clone 5 originally obtained from INERIS Laboratory, Vert-le-petit, France.
- Age at study initiation (mean and range, SD): < 24 h
- Feeding during test: none

CULTURE CONDITIONS
- Animals were maintained in covered glass vessels of 4 to 8 L capacities. Each culture medium was continuously aerated (air bubbling) and did not contain more than 50 daphnids per liter.
- Type and amount of food: flake fish food (Tetramin), nutrient broth, yeast suspension, seaweed extract (Marinure 30), Chlorella vulgaris
- Feeding frequency: three times weekly

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

164 mg/L CaCO3

Test temperature:

19.7 - 21.7 °C

pH:

7.8 - 7.9

Dissolved oxygen:

≥ 8.4 mg/L

Nominal and measured concentrations:

Nominal: 0, 0.96, 1.63, 2.77, 4.71 and 8.0 mg/L
Mean measured: < LOQ, 0.97, 1.66, 2.91, 4.85 and 8.33 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: beakers
- Type (delete if not applicable): covered
- Size, fill volume: 250 mL, 200 mL
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): After 24 h, daphnids were gently transferred into fresh test solutions
- 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

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 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: 551 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, test solution temperature and pH were measured in each bulk preparation at the initiation and in each test vessel at the termination of each 24 h cycle of exposure. Total hardness and conductivity were measured at test start.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light/8 h dark
- Light intensity: 621 - 638 lux (57.7 - 59.3 footcandles)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Immobilisation and behavioural alterations were recorded after 3, 24 and 48 h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.7
- Results used to determine the conditions for the definitive study: available toxicity information

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 8.33 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 8.33 mg/L was the visual limit of solubility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.66 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Mortality of control: During the test period, no immobilisation was observed in the control groups and no daphnid was found entrapped at the surface of the control solutions.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The concentration of 8 mg/L exhibited particles in the test solutions.
- LC50 exceeds solubility of substance in test medium.
- Other observations: Several daphnids were found entrapped at the surface or coated in test material at the bottom of test solutions at 1.66, 2.91, 4.85 and 8.33 mg/L (number of coated organisms increasing with the test concentrations), which did not allow observations on further sublethal effects. This was considered to be more a mechanical effect than a toxic effect.
- Analytical monitoring: 96 - 110% of nominal values at test start and 93 - 105% of nominal values at the end of each 24 h exposure period.

Reported statistics and error estimates:

Statistics: EC50 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. EC50 values are presented with 95% confidence limits when the methods of probit analysis or moving average are used to perform the calculation.

Table 1: Cumulative number of immobilised daphnids after 24 and 48 h of exposure to RPA 107382.

R1/R2, replicates.

Mean measured concentrations (mg/L)	24 h			48 h
	R1	R2	Mean %	R1	R2	Mean %
Solvent Control	0	0	0	0	0	0
Control < LOQ	0	0	0	0	0	0
0.97	0	0	0	0	0	0
1.66	0	0	0	0	0	0
2.91	1	0	5	3	1	20
4.85	0	1	5	1	2	15
8.33	0	0	0	2	0	10

Table 2: Validity criteria for OECD 202.

Criterion from the guideline	Outcome	Validity criterion fulfilled
In the control, including the control containing the solubilising agent, not more than 10% of the daphnids should have been immobilized.	0%	yes
The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in control and test vessels.	≥ 8.4 mg/L	yes

Validity criteria fulfilled:

yes

Remarks:

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

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Sep - 21 Nov 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 235 (Chironomus sp., Acute Immobilisation Test)

Version / remarks:

2011

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: all freshly prepared test levels on day 0 (including control(s)), and in all aged test levels on day 2 at the end of exposure period including control(s)
- Sampling method: freshly prepared test levels: sampled from the prepared volume of each test treatment level, aged test media: contents of all six replicate vessels of day 2 were combined

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 1036.9 mg test item added to 10.0 mL Dimethylformamide. The stock solution (S1) was treated in an ultra-sonicbath for 3 minutes and well agitated on a magnetic stirrer for 10 minutes before further use. From this stock solution dilution series in DMF (S2 – S5) were prepared for each single test concentration. By adding appropriate aliquots of these stock solutions (S1 – S6) to the test medium the nominal test concentrations were reached.
- Differential loading: no
- Controls: Dilution water only (control), dilution water and solvent (solvent control)
- Chemical name of vehicle: Dimethylformamide
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): S1: 1036.9 g/10 mL solvent, final solution: 0.1 mL solvent/L

Test organisms (species):

Chironomus riparius

Details on test organisms:

TEST ORGANISM
- Common name: harlequin fly
- Stage and instar at study initiation: First instar larvae
- Method of breeding: midges are kept in cages (40 x 40 x 40 cm), with a gauze on each side of the cage. A basin is set on the bottom of each cage. Bottom of the basins are covered with a layer of "Kieselgur" (silica) and a layer of reconstituted water. To
start a culture in the cage, 2 - 4 egg masses are placed into the prepared basin. Hatched larvae are fed with green algae and an aqueous suspension of a plant material based fish food (Tetra Phyll®). After 2 to 3 weeks the adults emerge. After mating, female adults will lay egg masses on the water surface where these can be taken to start a new culture or to perform toxicity tests. The culture conditions are 20 ± 2°C and 16 to 8 hours light-dark-cycle (light intensity ca. 500 - 1000 lux).
- Source: Originally obtained from University of Frankfurt am Main (Germany) in 2006
- Feeding during test : no

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS: fresh egg masses were transferred in small dishes with test medium. Two to three days after hatching the L1-larvae were transferred with a blunt pipette to the test vessels. prior to insertion of the larvae into the test vessels, a few drops of an aqueous fish food suspension (50 g Tetra Phyll®/ L deionized water, refrained from the settled fraction) were applied to the culture dishes.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

195.8 mg CaCO3/L

Test temperature:

20.7 - 20.9°C

pH:

7.8 - 7.9

Dissolved oxygen:

8.3 - 8.5 mg/L

Nominal and measured concentrations:

Nominal: 0 (control), 0 (solvent control), 1.00, 1.80, 3.20, 5.60 and 10.0 mg/L
Mean measured: day 0: < 0.001 (control), < 0.001 (solvent control), 0.905, 1.48, 2.69, 5.37 and 8.32 mg/L, day 4: < 0.001 (control), < 0.001 (solvent control), 0.886, 1.45, 2.85, 5.24 and 9.95 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 20 mL glass beakers
- Fill volume: 10 mL
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water: Elendt medium (M7)
- Alkalinity: 53.4 mg CaCO3/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: temperature, pH and oxygen content: at day 0 in all test concentrations and the control and at the end of the exposure period (day 2) in the combined six test vessels of each test concentration and the control (temperature: also continuous measurement in one control vessel by a data logger (non-GLP))

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light/8 h dark
- Light intensity: 500 - 1000 lux

EFFECT PARAMETERS MEASURED: after 24 anf 48 h: number of immobilised larvae (animals showing no swimming movements within 15 seconds after slight agitation of the vessel) and sub-lethal effects

VEHICLE CONTROL PERFORMED: yes (0.1 mL Dimethylformamide /L)

RANGE-FINDING STUDY
- Test concentrations: 0 (solvent control), 0.01, 0.10, 1.0 and 10 mg/L
- Results used to determine the conditions for the definitive study: Immobilisation (48 h): 0% (solvent control), 5% (0.01 mg/L), 0% (0.1 mg/L) , 0% (1.00 mg/L) and 85% (10 mg/L)

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.24 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: 95%confidence limits: 1.79 - 2.73 mg/L

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

6.88 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: 95%confidence limits: 5.95 - 8.16 mg/L

Details on results:

- Immobilisation of control: 3% after 48 h

Results with reference substance (positive control):

- Results with reference substance valid? yes
- ECx: EC50 (24 h): 4.2 mg/L, EC50 (48 h): 2.2 mg/L (defined range by OECD 235: 0.50 - 3.6 mg/L

Reported statistics and error estimates:

EC50 values and confidence intervals were calculated for the stated exposure period, using a commercial program ToxRat Professional Software, Vers. 2.10.05, ToxRat Solutions GmbH, Germany.

Table 1: Observation after 48 hours (mortality and behaviour)

Nominal concentration [mg a.s./L]	Replicate No.	No. of larvae introduced d 0	Living larvae showing symptoms after 48 h	Dead larvae after 48 h
control	1	5	-	0
	2	5	-	1
	3	5	-	0
	4	5	-	0
	5	5	-	0
	6	5	-	0
solvent-control	1	5	-	0
	2	5	-	0
	3	5	-	0
	4	5	-	1
	5	5	-	0
	6	5	-	0
1.00	1	5	4 a	1
	2	5	3 a	2
	3	5	3 a	2
	4	5	4 a	1
	5	5	4 a	1
	6	5	4 a	1
1.80	1	5	4 a	1
	2	5	4 a	1
	3	5	4 a	1
	4	5	3 a	2
	5	5	3 a	2
	6	5	2 a	3
3.20	1	5	2 a	3
	2	5	1 a	4
	3	5	2 a	3
	4	5	4 a	1
	5	5	2 a	3
	6	5	-	5
5.60	1	5	2 a	3
	2	5	-	5
	3	5	2 a	3
	4	5	-	5
	5	5	-	5
	6	5	-	5
10.0	1	5	-	5
	2	5	-	5
	3	5	-	5
	4	5	-	5
	5	5	-	5
	6	5	-	5

Symptoms:

a = hardly any movements were observable c = reduced mobility

Table 2: Nominal and measured concentrations of active substance [mg a.s./L]

Nominalconcentrations[mg a.s./L]	Day 0		Day 2
	Analysed conc. mean of two analyses each [mg a.s./L]	Percent of nominal	Analysed conc. mean of two analyses each [mg a.s./L]	Percent of nominal
control	< 0.001	-	< 0.001	-
solvent-control	< 0.001	-	< 0.001	-
1.00	0.905	91	0.886	89
1.80	1.48	82	1.45	81
3.20	2.69	84	2.85	89
5.60	5.37*	96	5.24*	93
10.0	8.32	83	9.98	100
Average		87		90

* mean of four analyses (sample A + B)

Table 3:

Validity criteria for OECD 235

Criterion from the guideline	Outcome	Validity criterion fulfilled
In the control, including the solvent control containing the solubilising agent, not more than 15% of the larvae should have been immobilized or other signs of stress.	3%	yes
The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in control and test vessels.	8.3 - 8.5 mg/L	yes

Validity criteria fulfilled:

yes

Remarks:

For further information, please refer to "Any other information incl. tables".

Description of key information

EC50 (48h) = 2.24 mg/L (nominal, Chironomus riparius, OECD 235)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

2.24 mg/L

Additional information

Two GLP studies testing the short-term toxicity of the test item to aquatic invertebrates are available.

The key study evaluated the acute immobilisation to larvae of Chironomus riparius (1st instar) according to OECD 235. Larvae were exposed for 48 hours in a static test system to concentrations of 1.00, 1.80, 3.20, 5.60 and 10.0 mg a.s./L, a dilution water control and a solvent control containing 100µL/L dimethylformamide. Measured test item concentrations of fresh and old test solutions ranged between 82% and 100%. After 48 hours, a statistically significant reduced mobility was observed in all exposure groups with 24.1% immobility at lowest test concentration and 100% immobility at a nominal test item concentration of 10 mg a.s./L. The derived EC50 (48 h) was 2.24 mg a.s./L (nominal).

Short-term toxicity of the substance to Daphnia magna was investigated in one study according to OECD 202 and EPA OPP 72 -2. An EC50 (48h) > 8.33 mg/L is reported with this concentration being the visual limit of solubility of the test substance, and the NOEC (48h) was 1.66 mg/L. The measured concentrations were 93 - 110% of the nominal values throughout the test period, and the effect concentrations are based on mean measured values.