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

Sediment toxicity

Link to relevant study record(s)

Reference

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 May - 2 Jul 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: U.S. EPA, Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms. Fourth Edition. Office of Water, U.S. Environmental Protection Agency, Washington, D.C. EPA/821/R-02-013 (2000)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

SEDIMENT
- Additional four replicates were established for analytical and pore water quality measurements on test day 0 and were not initiated with any larvae.
- Concentrations: all treatment levels and the controls (replicate vessels Q, R, S and T respectively)
- Sampling interval: day 0, 20 and 61

PORE WATER
- Concentrations: all treatment levels and the controls (replicate vessels Q, R and S, respectively)
- Sampling interval: day 0, 20 and 61

OVERLYING WATER
- Concentrations: all treatment levels and the controls (replicate vessels Q, R and S, respectively)
- Sampling interval: day 0, 20 and 61

Vehicle:

yes

Details on sediment and application:

PREPARATION OF SPIKED SEDIMENT
- Sediment preparation according to OECD Guideline No. 218 (2004)
- Pooling or mixing of different substrates: mixing of 2.9 kg sphagnum peat (soaked for seven days in laboratory well water and CaCO3 added), 10.6 kg kaolin clay and 39.5 kg fine sand (i.e., 5.5, 20 and 74.5%, respectively)
- Method of mixing: blended together in a large-scale laboratory mixer together with 10.6 liters of water from the same source used in the toxicity test
- Details of spiking: 100 µg/mL primary stock solution with acetone was prepared and was further dissolved with different volumes acetone to achieve final dosing stock solutions (final volume: 25 mL). 10-mL volume of each dosing stock solution was applied to 0.050 kg of fine silica sand. The dry sand, containing the test substance, was then added to 3.5 kg of wet sediment (2.296 kg total dry weight based on a percent solids of 64.16%) in individual glass jars. The jars were sealed and positioned horizontally on the rolling mill. Each jar was then rolled for four hours at room temperature at approximately 15 rpm. Following four hours of rolling, the
jars were stored upright in the dark at 2 to 8°C. The sediments were allowed to equilibrate for a 7-day period in the refrigerator. Once during the 7-day equilibration period and again prior to allocating the treated sediment to the replicate test vessels, the jars were mixed on the rolling mill for an additional two hours at room temperature to ensure the sediment was homogeneous.
- Equilibration time: 1 day
- Equilibration conditions: same as test conditions
- Controls: solvent control sample was prepared in a similar manner as the treated sediment by adding 10 mL of acetone, containing no test substance, control sediment consisted of 3.5 kg of unadulterated wet sediment without the addition of solvent, test substance or the additional 0.050 kg of fine silica sand
- Evaporation of vehicle before use: The solvent was allowed to evaporate for 50 minutes, leaving only the test substance adhered to the sand.

Test organisms (species):

Chironomus dilutus (previous name: Chironomus tentans)

Details on test organisms:

TEST ORGANISM
- Common name: dipteran midge
- Source: Smithers Viscient’s culture
- Details on collection: Hatched midge larvae were transferred to a shallow glass bowl containing approximately 1 L of culture water and were fed 2.5 mL of Ankistrodesmus falcatus, a unicellular green algae (4 × 10^7 cells/mL)
- Handling of egg masses and larvae: Newly oviposited midge egg masses were placed in 250-mL crystallizing dishes containing approximately 200 mL of laboratory well water at a temperature of 22 - 24 °C.
- Age of animals at beginning of exposure: 3 days after hatching
- Feeding during test
- Food type: finely ground flaked fish food suspended in laboratory well water
- Amount: 1.5 mL (4.0 mg food/mL) per test vessel per day

Study type:

laboratory study

Test type:

flow-through

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

16 d

Exposure phase:

larvae from first generation (P)

Remarks:

Exposure to test item: until emergence from test vessels starting on day 16

Hardness:

60 - 68 mg CaCO3/L

Test temperature:

22 - 24°C

pH:

6.6 - 7.3 (overlying water), 6.8 - 7.6 (pore water), 7.2 (sediment)

Dissolved oxygen:

2.6 - 8.5 mg/L

Ammonia:

≤ 10 - 1.6 mg N/L (overlying water), 0.26 - 7.6 (pore water)

Conductivity:

370 - 440 µS/cm (overlying water)

Nominal and measured concentrations:

Nominal (sediment): 0 (control), 0 (solvent control), 3.8, 12, 39, 130 and 400 µg/kg
Mean measured (sediment): 1.2, 6.6, 16, 45 and 140 µg/kg
Mean measured (pore water): 0.15, 0.69, 2.0, 6.1 and 19 µg/L

Details on test conditions:

TEST SYSTEM
- Test container: 300-mL glass vessels with two openings on the top edge of the beaker which was covered with 40-mesh Nitex® screen for drainage
- Sediment volume: 100 mL (approx. 4 cm layer)
- Weight of wet sediment with and without pore water: 151 g wet weight, 96.7 g dry weight
- Overlying water volume: 175 mL
- Aeration: no

EXPOSURE REGIME
- No. of organisms per container (treatment): 12
- No. of replicates per treatment group: 12 (Four replicates (Q through T) were established and designated for chemical analysis)
- No. of replicates per control / vehicle control: 12 (Four replicates (Q through T) were established and designated for chemical analysis)
- Type and preparation of food: finely ground flaked fish food suspended in laboratory well water
- Amount of food: 1.5 mL (4.0 mg food/mL) per test vessel per day

RENEWAL OF OVERLYING WATER
- Details on volume additions: two volume additions of water (i.e., 350 mL) per test vessel per day using an intermittent delivery system in combination with a calibrated water-distribution system
- Flow-rate: 50 mL of water per cycle was provided to each replicate test vessel, cycle of 7 times per day, providing approximately 350 mL per vessel every 24 hours (i.e., approximately two overlying volume replacements per vessel per day), after test day 10: flow rate in the system was doubled so that the water delivery system cycled approximately 14 times per day, providing approximately 700 mL per vessel every 24 hours (i.e., approximately four overlying volume replacements per vessel per day) due to low dissolved oxygen measurements.

OVERLYING WATER CHARACTERISTCS
- Type of water: well water (unadulterated water from a 100-meter bedrock well supplemented on demand with untreated Town of Wareham, USA well water)
- Alkalinity: 20 - 24 mg CaCO3/L
- Periodic analysis of representative samples of the overlying water source will be conducted using U.S. EPA standard methods (U.S. EPA, 1997) by GeoLabs, Inc., Braintree, Massachusetts, in accordance with Smithers Viscient's standard operating procedures, to ensure the absence of potential toxicants, including pesticides, PCBs and selected toxic metals, at concentrations that may be harmful to the test organisms.

CHARACTERIZATION OF ARTIFICIAL SEDIMENT
- % dry weight of sphagnum moss peat: 5.5%
- Particle size distribution
- % sand: 79%
- % silt: 2%
- % clay: 19%
- Composition: 2.9 kg sphagnum peat, 10.6 kg kaolin clay and 39.5 kg fine sand
- Method of preparation: according to OECD Guideline No. 218 (2004)
- pH pore water: 6.8 - 7.6
- pH dry matter and/or whole sediment: 7.2
- Ammonia content of pore water: 0.26 - 7.6
- Total organic carbon (%): 2.6%

OTHER TEST CONDITIONS
- Light quality: fluorescence light
- Photoperiod: 16 hours light, 8 hours darkness
- Light intensity: 200 - 420 lux

EFFECT PARAMETERS MEASURED:
- Midge larvae survival and growth (day 20): sieving the sediment to remove all surviving midges, and drying midges at 60 to 61 °C for 22 hours.
- Midge emergence (emergence rate): daily
- Reproduction: merged male and female midges were collected on a replicate basis from the emergence traps and placed in reproductive/oviposit chambers. Reproductive/oviposit chambers consisted of a 3.5-cm tall Plexiglass® tube (inside diameter of
6 cm) covered on the top with wide mesh Nitex® screen placed within a 100 x 20-mm Petri dish. Once the midges were placed in the reproductive/oviposit chambers, approximately 50 mL of laboratory well water was added to the Petri dish. Males from different replicates within the same treatment level were paired with females of replicates where no males had emerged. Reproductive/oviposit chambers for each treatment level and control were checked daily for dead adults and egg masses.
- Egg counts: using the ring method: Five rings of eggs in each egg mass were selected at about equal distances along the length of the egg mass and the number of eggs in these five rings was then counted using a dissecting microscope. The mean number of eggs per ring was then multiplied times the number of rings in the egg mass to estimate the total number of eggs.
- Hatch determination: Egg masses were then incubated at test temperature in 30-mL plastic cups containing approximately 20-mL of laboratory well water. The number of unhatched eggs was counted following six days of incubation. Hatching success
was determined by subtracting the number of unhatched eggs from the original estimate of egg numbers from that egg mass.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.01 - 3.33
- Range finding study
- Test concentrations: 0 (control), 0 (solvent control), 0.0051, 0.034, 0.23, 1.5 and 10 mg/kg dw
- Results used to determine the conditions for the definitive study: Survival in control and solvent control: 56% and 83%, respectively, NOEC (day 21, survival): 0.23 mg/kg dw, NOEC (day 45, emergence): 0.034 mg/kg dw

Reference substance (positive control):

no

Duration:

20 d

Dose descriptor:

NOEC

Effect conc.:

45 µg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: mortality, midge larval growth

Remarks on result:

other: based on sediment measured

Duration:

61 d

Dose descriptor:

NOEC

Effect conc.:

45 µg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: percent emergence, emergence rate (males and females), egg masses per mated female, number of eggs per egg mass, percent hatch, days to oviposition

Remarks on result:

other: based on sediment measured concentrations

Details on results:

- Mortality of test animals at end of exposure period (day 20): Control mortality: 92%, solvent control mortality: 90%
- Mean percent emergence: control: 83%, solvent control: 79%, midges exposed to 3.8, 12, 39, 130 and 400 µg/kg treatment levels: 83, 67, 82, 77 and 8%
- Mean development rate of fully emerged midges (per replicate and treatment rate; male and female midges pooled):
- Mean individual dry weight of larvae (per larvae and per instar):

Reported statistics and error estimates:

All statistical analyses were conducted at the 95% level of certainty except in the case of Shapiro-Wilk’s Test for normality and Bartlett's Test or Variance Ratio F Test for homogeneity, in which the 99% level of certainty was applied.
An Equal Variance Two-Sample t-Test or Unequal Variance TwoSample t-Test was used to compare the performance of the negative control organisms with that of the solvent control organisms in order to determine if there were any statistically significant positive or negative effects. If the negative control and
solvent control data were statistically similar, these data were pooled for further comparison to the treatment data. If the negative control and solvent control data were not statistically similar, the solvent control data were used for comparison to the treatment data. During this exposure, the negative control and solvent control data were similar for all endpoints with the exception of larval growth, male emergence rate, and male days to death. Solvent control was deemed a more appropriate control comparator in these instances.
Shapiro-Wilk’s Test for normality (U.S. EPA, 2002) was conducted to compare the observed sample distribution with a normal distribution for all endpoints. The
assumption that observations are normally distributed must be validated before subsequent analyses, following parametric procedures, can be performed. If the data are not normally distributed, then a non-parametric procedure is used for subsequent analyses. Analysis of the data for all endpoints with the exception of days to oviposition met the assumption for normality.
As a check on the assumption of homogeneity of variance implicit in parametric statistics, data for each endpoint were analyzed using Bartlett's Equality of Variance Test or a Variance Ratio F Test.

Table 1: Test item concentrations measured in the sediment samples throughout the definitive exposure

Nominal Concentration (µg/kg)	Day 0 Measured Concentration (µg/kg)	Day 20 Measured Concentration (µg/kg)	Day 61 Measured Concentration (µg/kg)
Control	< 1.1	< 0.33	< 0.33
Solvent control	< 1.1	< 0.33	< 0.33
3.8	2.3	0.97	0.37
12	12	4.9	2.4
39	35	9.3	3.2
130	91	32	11
400	310	79	30

Table 2: Test item concentrations measured in the pore water samples throughout the definitive exposure

Nominal Concentration (µg/kg)	Day 0 Measured Concentration (µg/L)	Day 20 Measured Concentration (µg/L)	Day 61 Measured Concentration (µg/L)
Control	< 0.20	< 0.022	< 0.020
Solvent Control	< 0.20	< 0.022	< 0.020
3.8	0.33	0.095	0.027
12	1.7	0.37	0.047
39	5.0	0.92	0.13
130	15	3.2	0.38
400	47	8.3	0.97

Table 3: Test item concentrations measured in the overlying water samples throughout the definitive exposure

Nominal Concentration (µg/kg)	Day 0 Measured Concentration (µg/L)	Day 20 Measured Concentration (µg/L)	Day 61 Measured Concentration (µg/L)
Control	< 0.020	< 0.0024	0.089
Solvent Control	< 0.020	< 0.0024	< 0.0020
3.8	0.023	< 0.0024	< 0.0020
12	0.045	0.0043	< 0.0020
39	0.20	0.0067	< 0.0020
130	0.40	0.054	0.077
400	5.7	0.12	0.022

Table 4: Mean percent survival and mean ash-free dry weightduringthelife-cycle exposure with midge Chironomus dilutus

Test day 20
Initial measured sediment concentration (µg/kg)	Mean percent survival (SD) a	Mean Ash free dry weight per larvae in mg (SD)
Control	92 (7)	2.56 (0.62)
Solvent Control	90 (13)	1.32 (0.55)
Pooled Control	91 (9)	NAb (NA)
2.3	83 (12)	2.07 (1.5)
12	85 (8)	1.08 (0.48)
35	75 (18)	2.05 (0.27)
91	81 (14)	1.66 (0.73)
310	23c (10)	2.10 (1.4)

a: SD = StandardDeviation.

b: NA = Not Applicable. Treatment data were compared to the solvent control to determinetreatmentrelatedeffects for thisendpoint.

c: Significantly reduced compared to the pooled control, based on Bonferroni’s Adjusted t-Test.This treatment level was excluded from further statistical analysis of sublethal endpoints for determination of the NOEC and LOEC values due to the survival effect observed. This treatment level was also excluded from all EC50 estimates, with the exception of the EC50 estimate for percent emergence.

Table 5: Mean percent emergence, mean emergence rate and mean days todeath during the life-cycle exposure to the test item and midge (Chironomus dilutus)

Initial Measured Sediment	Mean Percent	Mean Male	Test Day 61 Mean Female	Mean Male	Mean Female
Concentration	Emergence	Emergence Rate	Emergence Rate	Days to Death	Days to Death
(µg/kg)	(SDa)	(SD)	(SD)	(SD)	(SD)
Control	83 (12)	0.0507 (0.0014)	0.0397 (0.0038)	4.2 (0.81)	4.6 (0.76)
Solvent Control Pooled Control	79 (9)   81 (10)	0.0448 (0.0052) NAb(NA)	0.0326 (0.0048)   0.0380 (0.0045)	2.9 (1.3) NAb(NA)	4.0 (0.65)   4.3 (0.74)
2.3	83 (13)	0.0548 (0.0027)	0.0500 (0.0025)	3.9 (0.63)	3.5 (0.95)
12	67c(7)	0.0521 (0.0028)	0.0472 (0.0028)	4.0 (0.82)	5.0 (1.4)
35	82 (9)	0.0505 (0.0043)	0.0447 (0.0039)	4.2 (0.77)	4.5 (0.51)
91	77 (17)	0.0462 (0.0044)	0.0430 (0.0031)	4.7 (0.54)	4.8 (1.1)
310	8 (12)d	0.0355 (0.0050)e	0.0326 (0.0072)e	3.3 (0.58)e	3.5 (0.71)e

a: SD = Standard Deviation.

b: NA = Not Applicable. Treatment data were compared to the solvent control to determine treatment related effects for this endpoint.

c: Significantly reduced compared to the pooled control, based on Bonferroni’s Adjusted t-Test. Reduction is not considered to be treatment related due to the lack of monotonicity in the data set and the lack of an effect at the next two higher treatment levels.

d: This treatment level was excluded from statistical analysis for determination of the NOEC and LOEC values due to the survival effect observed on test day 20, but was included in an EC50 estimate for this endpoint.

e: This treatment level was excluded from statistical analysis for determination of the NOEC, LOEC and EC50 values due to the survival effect observed on test day20.

Table 6: Mean number of eggs per mated female, eggs per eggmass,number of eggs per mated female, percent hatch and days to oviposition during the life-cycle exposure with ethiprole technical and midge (Chironomus dilutus)

Initial Measured Sediment concentration (µg/kg)	Mean Egg Masses per Mated Female (SDa)	Mean Eggs per Egg Mass (SD)	Mean Number of Eggs per Mated Female (SD)	Mean Percent Hatch (SD)	Mean Days to Oviposition (SD)
Control	0.57 (0.26)	619 (289)	379 (223)	91 (9)	1.5 (0.7)
Solvent Control	0.66 (0.19)	770 (187)	508 (198)	98 (1)	1.5 (0.4)
Pooled Control	0.62 (0.23)	695 (248)	436 (212)	94 (7)	1.5 (0.5)
2.3	0.70 (0.23)	1086 (237)	756 (333)	91 (7)	1.5 (0.4)
12	0.62 (0.34)	879 (189)	534 (293)	84b(7)	1.4 (0.4)
35	0.83 (0.11)	922 (131)	767 (148)	84b(9)	1.3 (0.2)
91	0.73 (0.21)	883 (224)	627 (205)	91 (5)	1.2 (0.3)
310	1.0 (0)c	1805 (54)c	1805 (54)c	80 (21)c	1.0 (0)c

a: SD = Standard Deviation.

b: Significantly reduced compared to the pooled control, based on Bonferroni’s Adjusted t-Test. Reduction isnot considered to be treatment related due to the lack of monotonicity in the data set and the lack of an effect at the next highest treatment level.

c: This treatment level was excluded from statistical analysis for determination of the NOEC, LOEC,and EC50 values due to the survival effect observed on test day 20

Table 7: Acceptabiliy criteria for EPA Test Method 100.5

Acceptability criterion from the guideline	Outcome	Validity criterion fulfilled
Average size in the control sediment at 20 d must be at least 0.6 mg/surviving organism as dry weight or 0.48 mg/surviving organism as AFD.	Control: 2.56 mg ash-free dry weight per larvae Solvent control: 1.32 mg ash-free dry weight per larvae	Yes
Emergence should be greater than or equal to 50%.	Control: 83% Solvent control: 79%	Yes
Pupae survival is typically >83% and adult survival is >96%.	Papae: Control: 92% Solvent control: 90%	Yes
Time to death after emergence is <6.5 d for males and <5.1 d for females.	Control females: 4.6 days Solvent control females: 4.0 days   Control males: 4.2 days Solvent control males:2.9 days	Yes
The mean number of eggs/egg case should be greater than or equal to 800	Control: 619 Solvent control:770	No
The percent hatch should begreater than or equal to 80%.	Control: 91% Solvent control: %98	Yes

One acceptability criterion was not met. However, the minimum standard criteria for reproductive endpoints established by EPA based on test method 100.5 and the latest revisions based on discussions with regulatory scientists were met(≥ 800 eggs per egg mass or ≥ 80% hatch).

Validity criteria fulfilled:

yes

Remarks:

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

Description of key information

NOEC (61 d) = 45 µg/kg dw (mean measured sediment, Chironomus dilutus, US EPA 100.5)

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:

0.045 mg/kg sediment dw

Additional information

One toxicity study assessed the effects of the test item applied to the sediment on the life cycle of dipteran midges (Chironomus dilutus) following US EPA test method 100.5. Nominal test item concentrations were in a range of 3.8 and 400 µg/kg dw and test duration was 61 days. Overlying water was renewed by adding two volume additions of water per test vessel per day using an intermittent delivery system in combination with a calibrated water-distribution system.

Analysis of the dosed sediment samples after mixing and prior to allocation into the test vessels resulted in recoveries ranging from 62 to 130% of nominal concentrations. Mean measured sediment concentrations during the test ranged from 32 to 55% of nominal concentrations and defined the treatment levels tested as 1.2, 6.6, 16, 45 and 140 µg/kg dw.

Assessed endpoints were midge larval survival, growth and emergence as well as days to death and reproduction (egg masses per mated female, eggs per egg mass, eggs per female, percent hatch and days to oviposition).

Statistically significant effects on all assessed endpoints were only observed at the highest test concentration of 140 µg/kg (mean measured in sediment), resulting in an overall NOEC of 45 µg/kg dw (mean measured in sediment).