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

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 Aug - 17 Sep 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 72-4 (Fish Early Life-Stage and Aquatic Invertebrate Life-Cycle Studies)

Version / remarks:

adopted 1987

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: During the first two renewal periods, samples were collected at t = 0 from bulk preparations of each test concentration, dilution water control and solvent control and at 48 or 72 h from pooled samples of each test concentration, dilution water control and solvent control. Thereafter, samples were analysed once per week from bulk preparations of each concentration level and from the corresponding pooled samples at the end of the relevant exposure period. Each sample was collected from the approximate midpoint of the test vessel with a pipet.
- 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: A stock solution of the highest test substance concentration was prepared by dissolution of 72 mg of the test substance in 2 mL of DMF. Stock solutions of the five lower test substance concentrations were prepared by serial dilution of the first stock solution in DMF. Test solutions were prepared by dilution of the stock solution with dilution water. Fresh stock and test solutions were prepared for each renewal of test media.
- 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
- Source: Laboratory culture, original source: clone 5 from INERIS Laboratory, 91710 Vert-le-petit, France
- Feeding during test
- Type and amount of food: A combination of 0.2 mL of yeast suspension (7.8 mg/L), 0.2 mL of Chlorella vulgaris (1.25E08 cells/mL), 0.8 mL of seaweed extract, Marinure 30 (8 mL/L) and 0.2 mL of a combination of meat extract and fish food, Tetramin (2 g/L and 10 g/L respectively).
- Frequency: The food was added to each fresh 200 mL of test solution before daphnids were transferred.

ACCLIMATION
- Acclimation period: 22 days
- Acclimation conditions (same as test or not): same as test

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

164 - 175 mg/L CaCO3

Test temperature:

19.7 - 22.1 °C

pH:

7.3 - 8.0

Dissolved oxygen:

≥ 7.3 mg/L

Nominal and measured concentrations:

Nominal: 0, 0.015, 0.044, 0.13, 0.40, 1.20 and 3.60 mg/L
Mean measured: < LOQ, 0.016, 0.046, 0.14, 0.40, 1.30 and 3.80 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: beakers
- Type (delete if not applicable): covered with plastic sheets
- Material, size, fill volume: glass, 250 mL, 200 mL
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): Surviving first generation daphnids were transferred into fresh test solutions each Monday, Wednesday and Friday.
- No. of organisms per vessel: 7 daphnids were maintained individually for data on survival, growth and reproduction. 3 groups of 5 daphnids were maintained for data on survival only. Thus, 22 individuals were exposed per concentration level.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water
- Metals (mg/L): Ca 44.0, Mg 14.8, K 17.7, Na 22.9, other metals < 0.005 to 0.5
- Pesticides: < 0.01 to 10 µg/L
- Chlorine: 96 mg/L
- Ca/Mg ratio: 3:1
- Conductivity: 539 - 554 µS/cm
- Salinity: Culture medium different from test medium: no
- Intervals of water quality measurement: At test initiation and at each media renewal, total hardness, conductivity, temperature, dissolved oxygen and pH were measured in the bulk preparations of each test concentration and in the control. At each renewal (after transfer of daphnids), temperature, dissolved oxygen and pH were measured from one replicate of old test solutions, at each concentration level.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light/8 h dark
- Light intensity: 560 - 616 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): At days 2, 5, 7, 9, 12, 14, 16 and 19 before transfer of the parent animals to fresh test solutions, the following parameters were recorded: (1) Survival of first generation daphnids, (2) any males or winter eggs, (3) the number of offspring both alive and dead, (4) the presence of eggs in the brood pouch, (5) abnormal appearance or behaviour. At test end, the total body length and wet weight were measured.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- No detailed information given.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: mortality, growth, reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: mortality, growth, reproduction

Duration:

21 d

Dose descriptor:

other: MATC

Effect conc.:

0.72 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: mortality, growth, reproduction

Remarks on result:

other: geometric mean of the range 0.4 - 1.3 mg/L

Details on results:

- Mortality of parent animals: No mortality was observed in the dilution water control, 14% mortality was observed in the solvent control.
- The mean number of offspring produced by individually held control daphnids was 152.9 per female in the dilution water control group and 159.6 in the solvent control group. The minimum number produced by any one individual surviving at Day 21 was 135.
- The first offspring in the control group appeared at day 7.
- No physical or behavioral abnormalities were observed among first generation daphnids during the test period.
- Analytical monitoring: All starting concentrations were 75 - 108% of nominal values, after 48 or 72 h 74 - 153% of nominal values were detected.

Table 1: Survival and reproduction of live offspring by first generation daphnids during the 21 day exposure to RPA 107382.

(*), a significant reduction in total reproductive output was observed at the mean measured concentrations of 1.3 and 3.8 mg/L (α = 0.05). A, individually held daphnids, B, daphnids held in 3 replicate groups of 5 individuals.

Mean measured concentrations (mg/L)	A/B	% Survival at day 21	Live offspring from parent animals alive on day 21
			Total number	Mean number (SD)
Solvent Control	A	71	1070	152.9 (5.52)
	B	93
Control < LOQ	A	100	798	159.6 (17.64)
	B	100
0.016	A	86	939	156.5 (3.83)
	B	80
0.046	A	71	780	156.0 (9.67)
	B	87
0.14	A	86	914	152.3 (4.76)
	B	93
0.40	A	100	1010	144.3 (10.08)
	B	60 *
1.3	A	86	692	115.3 * (17.77)
	B	93
3.8	A	57	256	64.0 * (24.90)
	B	73

Table 2: Mean lengths and wet weights of surviving individually exposed first generation daphnids at test termination.

(**), a significant effect was observed on length and weight of first generation daphnids exposed to 1.3 and 3.8 mg/L of RPA 107382 (α = 0.01).

Mean measured concentrations (mg/L)	n	Mean length (SD) (mm)	Mean weight (SD) (mg)
Solvent Control	7	4.29 (0.12)	9.52 (1.27)
Control < LOQ	5	4.36 (0.09)	9.74 (0.75)
0.016	6	4.30(0.14)	9.70 (0.79)
0.046	5	4.30(0.17)	9.55 (0.66)
0.14	6	4.25(0.12)	8.77 (0.73)
0.4	7	4.17(0.13)	8.53 (0.85)
1.3	6	4.00(0.18)**	7.75(1.29)**
3.8	4	3.40(0.17)**	5.69(0.61)**

Table 3: Validity criteria for OECD 211.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality of the parent animals in the control (female Daphnia) does not exceed 20% at the end of the test.	Control: 0% Solvent control: 14%	yes
The mean number of living offspring produced per parent animalsurvivingat the end of the test is ≥ 60 in the control.	Control: 152.9 Solvent control: 159.6	yes

Note: The same validity criterion (20%) can be used for accidental and inadvertent parental mortality for the controls as well as for each of the test concentrations.

Validity criteria fulfilled:

yes

Remarks:

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

Description of key information

NOEC (21d) = 0.4 mg/L (mean measured, Daphnia magna, EPA OPP 72-4)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

0.4 mg/L

Additional information

The long-term effects of the test substance towards Daphnia magna were investigated in a 21-day semi-static life cycle test following GLP and EPA OPP 72-4 guidelines. Based on mean measured concentrations of the test substance (measured values were in the range of 74 – 153% of nominal values), a NOEC (21d) of 0.4 mg/L and a LOEC of 1.3 mg/L were determined.