Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

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:

16 Sep 2005 to 13 Jan 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 218 (Sediment-Water Chironomid Toxicity Test Using Spiked Sediment)

Version / remarks:

adopted 13th April 2004

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: Proposal for a BBA-Guideline

Version / remarks:

1995

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Test item concentrations were verified by analysing for the main isomer of the test substance, Syn-isomer. Concentrations were determined in the overlying water and the sediment phase. For the verification of test concentrations at the start of the study, one additional replicate per treatment (control, solvent control and all the test concentrations) was prepared. These additional replicates were used exclusively for analytical measurements of the test item concentrations in the water column and in the sediment. At test termination, the first replicate vessel from the biological phase of the test of each test concentration and control was used to provide analytical samples.

Vehicle:

yes

Remarks:

acetone

Details on sediment and application:

- Composition of sediment: 4.5% sphagnum peat (air dried and finely ground), 20% kaolin clay (kaolinite content >30%), 75.5% industrial sand (fine sand will predominate with more than 50% of the particles between 50 and 200 microns). Organic carbon content of the mixture 2.5% and pH 6.9 (adjusted by addition of calcium carbonate).
- Sediment preparation: Finely ground peat was moistened and mixed with deionised water and then gently stirred for three days at room temperature. The pH of the peat suspension was adjusted by addition of calcium carbonate to 6.0 ± 0.5 (measured 5.61). All constituents were then thoroughly blended in a laboratory mixer to give homogeneous wet sediment. Deionised water was added to obtain a nominal moisture content of the final mixture of 40% of dry weight of the sediment. Samples of the sediment were taken to determine the dry weight and the organic carbon content. The mean moisture content of the sediment (triplicate samples were taken) was 39.6%. The wet sediment was then left for preconditioning at room temperature for 6 days, until it was transferred into the test beakers.
- Stock and application solution preparation: Due to the low water solubility of the test item in water, a stock solution in acetone was prepared. 1600.38 mg of the test substance was made up to 10 mL with acetone (to give a nominal concentration of 160 g/L). Aliquots of this stock solution were diluted with acetone to give the application solutions for the lower test concentrations (i.e. 80, 40, 20, 10 and 5 g/L).
- Spiking of water-sediment systems: A series of application solutions of the test substance in acetone were made up. An aliquot of 25 µL of each respective application solution (i.e. nominal concentrations of 5, 10, 20, 40, 80 and 160 µ/L acetone) was spiked into the water column of each of the test vessels to receive the desired nominal test concentrations of 2.5, 5.0, 10, 20, 40 and 80 mg/kg dry weight of sediment. The solvent control test vessels were spiked with 25 µL of acetone only. After spiking, all the test vessels were sealed, shaken and transferred to a rolling mill for two hours at room temperature to ensure even mixing throughout the sediment. The test vessels were then left to stand undisturbed for three days to allow the sediment to settle. The vessels contained a sediment layer of approximately 1.5 cm and a water column of approximately 6 cm depth. The water level was marked outside on the test vessel. It was not necessary to top up the water columns with deionised water during the study to the original starting volume since there was little evaporation.

Test organisms (species):

Chironomus riparius

Details on test organisms:

TEST ORGANISM
- Justification for species: Characteristics which make this organism suitable for toxicity testing are its ease of culturing and that the sediment-dwelling larvae are common to most freshwater environments. They go through four instar stages, pupate and emerge as adults in approximately 13-25 days at 20°C.
- Source: From in-house cultures
- In-house cultures: Egg masses of Chironomus riparius for the start of the cultures were originally received on July 01, 2004.
- Details on collection: First instar larvae were obtained by collecting fresh egg ropes from in-house cultures three to six days prior to the start of the study. Larvae were then allowed to hatch from the egg ropes. The day before addition of the larvae to the test vessels, egg ropes were removed to ensure that no further hatching would occur and larvae were at least one day old.

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Hardness:

260, 265 and 272 mg/L as CaCO3 for the three media batches used at start of test.
300 mg/L as CaCO3 for control
305 mg/L as CaCO3 for highest test concentration at the end of the test

Test temperature:

19.6 - 20.7°C

pH:

7.56 - 8.50

Dissolved oxygen:

64 - 118% air saturation

Nominal and measured concentrations:

- Nominal concentrations: 0 (negative control), 0 (solvent control), 2.5, 5.0, 10, 20, 40 and 80 mg/kg sediment dw
- Initial measured concentrations: 0 (negative control), 0 (solvent control), 1.3, 1.9, 7.0, 9.3, 28 and 56 mg/kg sediment dw, respectively.

Details on test conditions:

TEST SYSTEM
- Test container: Glass jars (500 mL, approximately 7.5 cm in diameter)
- Type: During the test, the vessels were covered with Parafilm to reduce evaporation and prevent the escape of emerged adults.
- Aeration: Yes (During the test (with exception of the period of approximately 24 hours after the introduction of the larvae) the water in the watersediment systems was gently aerated through a glass Pasteur pipette, fixed above the sediment layer.)
- Preparation of test container: Test vessels were prepared by weighing out the equivalent of 50 g dry weight sediment (i.e. approximately 70 g wet sediment) into each glass jar and then adding 250 ml of test medium.

EXPOSURE REGIME
- No. of organisms per container; 20
- No. of replicates per treatment group: 4
- No. of replicates per control / vehicle control: 4
- Feeding of the larvae: The larvae were fed with a Tetramin fish food suspension (2.0 g of finely ground fish food suspended in 40 ml of test medium) on a daily basis, except for days 2, 3, 9, 23, 24, 25, 26, 27 and 28 where larvae were not fed. The food ration from day 0 to day 23 was between 0.5 and 1.0 mg Tetramin fish food per larvae, per day. From day 25 of the test onwards no more food was supplied to the test vessels since enough seemed to be available at the surface of the sediment. The following amounts were fed:
Days 0/4/5/6/7: 0.2 mL/vessel (equivalent to 10 mg Tetramin/vessel or 0.5 mg Tetramin/larvae/day);
Day 1 and 22: 0.6 mL/vessel (equivalent to 30 mg/vessel or 0.5 mg Tetramin/larvae/day*); * e.g. 0.6mL given on day 1 was the supply for days 2 and 3 also.
Days 8/10-21 : 0.4 mL/ vessel (equivalent to 20 mg/vessel or 1 mg Tetramin/larvae/day).

OVERLYING WATER CHARACTERISTCS
- Type of water: The aqueous test medium used in this study was a reconstituted water ("M7-medium", OECD guideline 218, 2004).
- pH: pH should remain within the required range 6.0 - 9.0. The pH of the overlying water of each test vessel was recorded at the start and the end of the test and on a weekly basis during the test.
- Oxygen concentration: Dissolved oxygen concentration should be maintained at the required ≥ 60% air saturation. Oxygen concentrations of the overlying water of each test vessel were recorded at the start and the end of the test and on a weekly basis during the test.
- Hardness of the overlying water: Hardness of the test media used to set up the vessels was measured prior to the test and the hardness of the overlying water was measured in the control and the highest test concentration at the end of the test.

CHARACTERIZATION OF ARTIFICIAL SEDIMENT
- 4.5% sphagnum peat, air dried and finely ground
- 20% kaolin clay (kaolinite content >30%)
- 75.5% industrial sand (fine sand will predominate with more than 50% of the particles between 50 and 200 microns)
- The organic carbon content of the final sediment mixture was 2.5%
- pH: The pH of the final mixture of the sediment was 6.9. The pH was adjusted by the addition of calcium carbonate (pulverized and of
recognized analytical grade).

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours dark with a 30 minute transition period between light and darkness.
- Light intensity: Approximately 500 to 1000 Lux

EFFECT PARAMETERS MEASURED
- Observations/ Emergence:The test vessels were observed at least three times per week for any abnormal behaviour compared to the control and any other observations. During the period of expected emergence, from day 11 of the test, a daily count of emerged midges was made. The sex (males were distinguished by their plumose antennae) and the number of fully emerged midges were recorded daily. After identification, the adult midges were removed from the test vessels and discarded. Any visible pupae that failed to emerge were counted and recorded. Any other signs of intoxication of the larvae, pupae or emerged midges were recorded. Egg masses deposited during the test were removed to prevent re-introduction of new larvae into the sediment.
- Measured endpoints: Total number of male and female adults emerged and the time to emergence. From this the emergence ratio and the development rate were calculated.

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

56 mg/kg sediment dw

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

other: Emergence ratio and development Rate

Details on results:

An overview of the results is provided in Table 1 – Table 3 in ‘Any other information on results incl. tables’
- Analytical results: Based on the measured concentrations of the syn-isomer, this is equivalent to a % nominal concentration between 39 and 71% of the test substance applied and corresponds to initial measured concentrations of 1.3, 1.9, 7.0, 9.3,28 and 56 mg/kg sediment dw.
- Emergence Ratio: The emergence ratios of the two controls were compared using a two-tailed test with a 5% type one error rate. Since emergence ratios of the two controls were not statistically different to each other, controls were pooled for subsequent comparisons with the test item treatments. The mean emergence ratios for the control group and the 1.3, 1.9, 7.0, 9.3, 28 and 56 mg/kg sediment dw treated groups were 0.74, 0.75, 0.78, 0.72, 0.58, 0.74 and 0.63, respectively.
- Development Rate: The mean development rate of males, females, and males and females together, for the two controls were compared using a two-tailed t test with a 5% type one error rate. Since development rates of the two controls were not statistically different to each other, controls were pooled for subsequent comparisons with the test item treatments. The mean development rate (males and females pooled) for the control group and the 1.3, 1.9, 7.0, 9.3, 28 and 56 mg/kg sediment dw treated groups were 0.0607, 0.0610, 0.0619, 0.0600, 0.0599, 0.0568 and 0.0609, respectively.
- Effect values: The 28 day NOEC of the test substance for emergence ratio and for development rate was 56 mg/kg sediment dw based on initial measured concentrations of the test substance in the sediment. The 28 day LOEC for emergence ratio and development rate could not be calculated. The 28 day EC50 for emergence ratio and development rate could also not be determined. A small number of dead pupa, midges and half emerged pupa, midges were observed in all treatments. The occurrence of dead pupa, midges and half emerged pupa, midges seems to be consistent in numbers throughout the treatments.

Table 1. Analytical results of the test substance

Nominal concentrations (mg/kg)	% of nominal measured in the sediment Day 0	% of nominal measured in the sediment Day 28	Initial measured concentration in the sediment (mg/kg)
Control	<LOQ	<LOQ	-
Solvent control	<LOQ	<LOQ	-
2.5	53	78	1.3
5.0	39	63	1.9
10	70	67	7.0
20	46	56	9.3
40	71	94	28
80	70	100	56

Table 2. Effects of the test substance on emergence and development of Chironomus riparius after 28 days exposure

Nominal concentration mg/kg	Number emerged	Mean emergence ratio (ERa)	Mean development rate (males and females pooled)
Pooled Control	117b	0.73	0.0607
2.5	60	0.75	0.0610
5.0	62	0.78	0.0619
10	43c	0.72	0.0600
20	46	0.58	0.0599
40	59	0.74	0.0568
80	50	0.63	0.0609

 

Table 3.Summary of the effects of the test substance on Chironomus riparius after 28 days exposure 

Endpoint	NOEC (mg/kg)	LOEC (mg/kg)	EC50 (mg/kg)	95% confidence limits (mg/kg)
Emergence ratio	56	>56	>56	N/A
Development rate (males)	56	>56	>56	N/A
Development rate (females)	56	>56	>56	N/A
Development rate (pooled)	56	>56	>56	N/A

Validity Criteria

Emergence of adult midges in the pooled control and solvent control was 74% (required at least 70% at the end of the test). pH and dissolved oxygen concentrations of the overlying water were in the range of 7.56 to 8.50 (required range 6 to 9) and 64 to 118% air saturation (required at least 60% saturation) in all test vessels, respectively. During the test the water temperature in the test vessels ranged from 19.6 to 20.7˚C (required to not differ by more than ± 1.0˚C). Emergence in both control and solvent control occurred between 14 and 23 days after introduction of larvae into the test vessel, apart from a single midge in the dilution water control which emerged on day 27. However, the effect of this one midge emerging five days late is so minimal that the study can still be deemed valid.

Validity criteria fulfilled:

yes

Remarks:

See Validity Criteria in 'Any other information on results incl. tables'

Conclusions:

In a toxicity test on the sediment dwelling organism Chironomus riparius, performed in accordance with OECD TG 218 and a proposal for BBA-Guideline, the 28-d NOEC for emergence ratio and development rate was determined to be at the highest concentration of 56 mg/kg sediment dw, based on initial measured concentrations when spiking the sediment.

Executive summary:

The effects of the test substance on the development of larvae of Chironomus riparius were determined under static conditions. The study was conducted according to OECD TG 218 and a proposal for BBA-Guideline, and in compliance with GLP criteria. The test organisms (20 larvae per vessel; 4 verssel per concentration) were exposed to the test item at nominal concentrations of 2.5, 5, 10, 20, 40 and 80 mg/kg sediment dw (initial measured concentrations: 1.3, 1.9, 7.0, 9.3, 28 and 56 mg/kg sediment dw) in glass jars filled with sediment (50 g dry weight) and test medium (250 mL) for 28 days. The test vessels were spiked three days before the larvae were introduced by adding 25 μL of the application solutions to the water-sediment slurry to give the nominal concentrations. The test vessels were then shaken and placed on a rolling mill for two hours to ensure the even distribution of test material throughout the water sediment. The test vessels were then left to stand for three days and aerated for approximately 24 hours before larvae were introduced. The larvae were randomly distributed amongst the test vessels. Throughout the test, the larvae were fed daily and from day one the vessels were gently aerated. The test conditions were as follows: 19.6 - 20.7 °C, pH 7.56 - 8.50, and the oxygen concentrations of the overlaying water is 64 to 118% air saturation. From day 11 of the test, a daily count of emerged midges was made and their sex was recorded. Once emerged, the adults were discarded. Visible pupae that failed to emerge were counted and recorded. Any signs of chemical effect on the larvae, pupae or emerged midges were recorded. The emergence ratio and development rate were calculated from the total numbers of emerged male and female adults, and from the time ofemergence. The concentrations of test material were determined at day 0 and day 28 in the sediment and overlying water by analysing for the syn-isomer using a GC-MSD method.

After 28 days exposure, since the two controls were not statistically significantly different to each other, controls were pooled for all subsequent comparisons with the test item treatments. The mean emergence ratios for the control group, the 1.3, 1.9, 7.0, 9.3, 28 and 56 mg/kg sediment dw treated groups were 0.74, 0.75, 0.78, 0.72, 0.58, 0.74 and 0.63, respectively. The mean development rate (males and females pooled) for the control group, the 1.3, 1.9, 7.0, 9.3, 28 and 56 mg/kg sediment dw treated groups were 0.0607, 0.0610, 0.0619, 0.0600, 0.0599, 0.0568 and 0.0609, respectively. Based on these findings, the 28-d NOEC was determined to be 56 mg/kg sediment dw, based on initial measured concentrations for emergence ratio and development rate when spiking the sediment.

Description of key information

All available data was assessed. The study representing the worst-case effects was included here and its effect value was used as the key value. Another study is included as supporting information.

28-d, NOEC = 56 mg/kg sediment dw (based on initial measured concentrations), Spiking of sediment, Chironomus riparius, emergence ratio and development rate, OECD TG 218 and proposal of BBA guideline, Pfeifle 2007

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:

56 mg/kg sediment dw

Additional information

Two studies are available on the toxicity to the sediment dwelling organism Chironomus riparius, both following standard guidelines and GLP compliant. In the first study, the application of the test substance was via spiking of the sediment. The test organisms were exposed to the test item at nominal concentrations of 2.5, 5, 10, 20, 40 and 80 mg/kg sediment dw (initial measured concentrations: 1.3, 1.9, 7.0, 9.3, 28 and 56 mg/kg sediment dw) in glass jars filled with sediment (50 g dry weight) and test medium (250 mL) for 28 days. In addition, a negative control and a solvent (acetone) were included in the test. The test conditions were as follows: 19.6 - 20.7 °C, pH 7.56 - 8.50, and the oxygen concentrations of the overlaying water is 64 to 118% air saturation. The 28-d NOEC for emergence ratio and development rate was determined to be 56 mg/kg sediment dw based on the initial measured concentrations (Pfeifle 2007). This result is selected for the CSA, since sediment-spiking is preferred for sediment toxicity endpoint, according to Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7b: Endpoint specific guidance (version June 2017).

In the second study, the application of the test substance was via spiking of the water. The test organisms were exposed to the test item at nominal concentrations of 0.125, 0.25, 0.50 and 1.0 mg/L. In addition, a negative control and a solvent control (DMF) were included as well. The 27-d NOEC for emergence ratio and development rate was determined to be ≥ 1.0 mg/L based on nominal concentrations (Memmert 2008).