4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Toxicity to terrestrial arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to non-target arthropods on inert substrate (NTA other than pollinators)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Nov 2014 to 22 Apr 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 232 (Collembolan Reproduction Test in Soil)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes

Application method:

soil

Analytical monitoring:

no

Vehicle:

yes

Remarks:

acetone

Details on preparation and application of test substrate:

For the test-item treatments, the product was dissolved in acetone. For each concentration of the test item in the test, the preparation of 200 g dry weight of soil was carried out as follows: a 2 mL volume of the appropriate acetone solution of the test item was mixed with 31.61 g of sand in a glass dish and the acetone was allowed to evaporate away over a period of 40 - 100 minutes. Once the treated sand was dry, it was then mixed
with the other ingredients of the artificial soil (i.e. 169.0 g of a sand + peat + clay + CaCO3 mix), to achieve 200 g dry weight of soil (actually corresponded to 200.61 g, as soil components already contained small amount of moisture). Mixing of the treated sand and the other soil components was carried out in a clean glass container and in a thorough manner, in order to achieve what was considered to be a homogeneous distribution of the test item throughout the soil. Purified water (37.497 mL) was then mixed with each 200 g dry weight of treated artificial soil to wet it to 50% of its water-holding capacity
For the untreated control treatment (purified water only), 47.42g sand (untreated) was mixed with 253.49 g of a peat + clay + sand + CaCO3 mix in order to make 300 g dry weight of soil (actually corresponded to 301.91 g, as soil components already contained small amount of moisture) and this was then thoroughly mixed with 56.246 mL of purified water to give a final moisture content of 50% of the soil’s WHC
For the solvent-treated control, 47.42 g sand was treated with 3.0 mL acetone and this was allowed to evaporate away for approx. 25 mins. When dry, the treated sand was mixed with 253.49 g of a peat + clay + sand + CaCO3 mix in order to make 300 g dry weight of soil (actually corresponded to 301.91 g, as soil components already contained small amount of moisture) and this was then thoroughly mixed with 56.246 mL of purified water to give a final moisture content of 50% of the soil’s WHC.
For the toxic reference treatment, 0.222g product was diluted in 100mL purified water and then a sufficient amount of the solution was added to the artificial soil, in combination with a volume of purified water, to achieve 50% WHC. The applied concentration equivalent to 200 mg Betosip 114/kg dry soil substrate.
Once the treatments had been fully mixed in, the soil had a naturally crumbly structure, which allowed the springtails to migrate into the substrate. Care was taken to avoid compressing the soil following its treatment. Once treated, 30 g of the soil was transferred into each replicate glass jar. An additional jar was also prepared per treatment. The pH of spare soil was measured (in the presence of 1 mol/L KCl) at the start of the test The additional jar was maintained alongside those used in the bioassay and the pH of the soil was measured at the end of the study. In addition, the starting weight of each test arena was recorded.

Test organisms (species):

Folsomia candida

Animal group:

Collembola (soil-dwelling springtail)

Details on test organisms:

TEST ORGANISM
- Common name: Springtails
- Source: In-house culture maintained at the Test Facility, since 2005
- Age at test initiation: 10-day-old (range-finding bioassay); 11-day-old (definitive bioassay)

CULTIVATION
- Culture conditions: Adult and juvenile springtails were kept in clear polystyrene boxes (27 cm x 15 cm x 10 cm deep) with tight-fitting lids. Each box contained a substrate to a depth of ca. 1 cm. The substrate consisted of the following: 1) 11.1% w/w activated pulverised charcoal and 2) 88.9% w/w plaster of Paris. Purified water was added to the mixture to produce a paste, which was allowed to set hard before being used.
- Hatching: The springtails were obtained by transferring egg clusters from the breeding containers to 9-cm-diameter plastic Petri dishes (each contained a 0.5-cm-deep layer of the culturing substrate). These egg clusters were collected from the culture arenas by use of a scalpel and transferred to filter paper in the Petri dish to hatch. Once hatching had started, any unhatched eggs and the associated filter papers were transferred to a fresh Petri dish and t he remaining juveniles were provided with approximately 2 mg of food. This was carried out on consecutive days during the hatching period so that all juveniles in a single Petri dish were of the same age in days.
- Food type: Dried granulated baker’s yeas
- Amount and Frequency: Approximately 30 mg and 2-3 times per week
- Food providing method: The food was placed on a filter paper square (4 cm x 4 cm). The filter paper and food were removed and replaced when necessary, to avoid spoilage by fungal growth. Relative humidity was maintained by keeping the substrate permanently moist by addition of purified water to it, 2-3 times per week.

ACCLIMATION
- The hatched juveniles were maintained in a controlled environment cabinet for a further 9 days (range-finding bioassay) and 10 days (definitive bioassay) after hatching and before being used for the test. Conditions recorded during culture of the juveniles were 18.5 - 20.5 ºC (range-finding bioassay) and 18.5 - 20.6 ºC (definitive bioassay).

FEEDING DURING TEST
- One day prior to starting the bioassays, yeast was provided as food and a check was made to ensure that there were ten healthy springtails in the container, prior to being added to the test arenas.
- At the beginning of the test, approximately 6 mg of granulated dry yeast was added to the soil surface of each test arena and was also replenished at 14 DAT.

Study type:

laboratory study

Limit test:

no

Total exposure duration:

28 d

Test temperature:

19.6 - 21.2 ºC

pH (if soil or dung study):

6 ± 0.5

Humidity:

50% WHC

Details on test conditions:

TEST SYSTEM
- Test container: Glass jars (approximately 125 mL capacity and 4.5 cm in diameter).
- Ventilation: The close-fitting lids of the arenas were then securely fastened. To allow ventilation, the arena lids were opened for brief periods, every 3 - 4 days.
- Amount of soil or substrate:
- No. of organisms per container (treatment):10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- No. of replicates toxic reference treatment: 5

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- The test substrate was an artificial soil comprising the following dry-mass fractions. The information on preparation and properties of the substrate is provided in Test substrate in "Any other information on materials and methods". The springtails were picked up from the breeding containers using a low-suction aspirator and then dislodged from the collection tip onto the surface of the substrate in each container. Within 60 minutes of the soil being treated, moistened and placed into each test arena, 10 juvenile springtails were placed in each replicate. The weight of each test arena was noted at the start of the test. At 14 DAT the arenas were reweighed; the change in the mean weight (through water loss from the soil) was calculated to be > 2% of the soil’s original water content for the bioassay, and so additional purified water was added to restore the arenas’ original weights. The weight of each arena was also measured at the end of the test.

OTHER TEST CONDITIONS
- Photoperiod: 12h
- Light intensity: 400 - 710 lux

BIOASSAY PROCEDURES
- One day prior to starting the bioassays, the ten test springtails for each replicate were transferred to holding-containers. These were stoppered glass-vials (50 mm in height x 25 mm in diameter) with a 0.5 mm deep layer of plaster-of-Paris/charcoal substrate set in the base

EFFECT PARAMETERS MEASURED
- The numbers both of surviving adults and F1 progeny in each test arena were recorded at 28 DAT. Water (approx. 150 - 200 mL) was then added to the substrate and stirred gently and frequently, so that the soil sank and the springtails floated to the surface. Any adult springtails floating on the water were counted and removed. The water-filled arenas were left for a period of > 2 h and any further adult springtails that had surfaced were recorded. Black ink was then added to the water and the numbers of any nymphs (smaller in size to adults) left in each arena were assessed. The ink darkened the water so that it contrasted with the light-coloured springtails floating on the water surface. A grill was held over the surface to aid with counting, and a binocular microscope was also used to facilitate observation of the juvenile springtails. It was assumed that any adult springtails that were recovered would have been those originally introduced and that any shortfall in the original number was an indication that they had died during the bioassay.

RANGE FINDING STUDY
- Test concentrations: 1000, 100, 10, 1 and 0.1 mg a.s./kg soil dry weight, untreated control and a acetone control.
- Results used to determine the conditions for the definitive study: The number of adult springtail found at 21 DAT was 10.0, 10.0, 9.5, 3.0, 2.0, 10.0 and 8.0 for 1000, 100, 10, 1 and 0.1 mg a.s./kg soil dry weight, untreated control and a acetone control, respectively. The number of springtail progeny at at 21 DAT was 159.0, 172.5, 143.0, .0, 0.0, 167.0 and 118.5 for 1000, 100, 10, 1 and 0.1 mg a.s./kg soil dry weight, untreated control and a acetone control, respectively. The test item was then evaluated in a definitive bioassay at nine concentrations, equivalent to 1.000, 0.556, 0.309, 0.171, 0.095, 0.053, 0.029, 0.016 and 0.009 a.s./kg soil dry weight, based on the measured purity of 98.4% w/w.

Nominal and measured concentrations:

Nominal concentration (test substance): 1.000, 0.556, 0.309, 0.171, 0.095, 0.053, 0.029, 0.016 and 0.009 a.s./kg soil dry weight
Nominal concentration (toxic reference): 114 g/L phenmedipham

Reference substance (positive control):

yes

Remarks:

Betosip 114 (Sipcam)

Key result

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

0.112 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks on result:

other: 95% C.L.: 0.089 to 0.133 mg/kg soil dw

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.095 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: mortality and reproduction

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

0.235 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks on result:

other: 95% C.L.: 0.208 to 0.265 mg/kg soil dw

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

0.271 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 95% C.L.: 0.232 to 0.312 mg/kg soil dw

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

0.171 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: reproduction and mortality

Details on results:

An overview of the results is provided in Table 1 - Table 4 in 'Any other information on results incl. tables'.

MORTALITY
At 28 days, the percentage mortality in the untreated control was 8.8%, and in the acetone-treated control was 6.3%. When compared statistically (Fisher’s Exact Test, α = 0.05), the two controls did not differ significantly, and therefore the two datasets (with a mean mortality of 7.5%) were combined for subsequent comparisons with the test-item treatments.
At 28 days, the percentage mortality was 100%, 92.5%, 65.0%, 22.5%, 5.0%, 10.0%, 12.5%, 17.5% and 7.5% in the 1.000, 0.556, 0.309, 0.171, 0.095, 0.053, 0.029, 0.016 and 0.009 mg a.s./kg soil dry weight treatment concentrations of the test substance, respectively. When adjusted for the combined control treatment losses, the respective test-item treatments resulted in 100%, 91.9% 62.2%, 16.2%, 0.0%, 2.7%, 5.4%, 10.8% and 0.0% corrected mortality. When compared statistically, only the results for the 1.000, 0.556, 0.309 and 0.171 mg a.s./kg soil dry weight treatment concentrations differed significantly from the combined control data (Fisher’s Exact Test, α = 0.05). Therefore, the LOEC (mortality) was taken to be 0.171 mg a.s./kg soil dry weight and the NOEC (mortality) 0.095 mg a.s./kg soil dry weight.
The 28-day LC50 for the test substance was calculated to be 0.271 mg a.s./kg soil dry weight, with 95% confidence limits of 0.232 and 0.312 mg a.s./kg soil dry weight.

REPRODUCTION
The mean number of progeny produced per replicate was 640.6 in the untreated control and 640.0 in the acetone-treated control. Statistically, the two control treatments did not differ (Mann-Whitney U-test, α = 0.05), and so the two datasets were combined (mean control progeny = 640.3) for subsequent comparisons with the test-item treatments.
The mean number of progeny produced per replicate was 16.3, 36.5, 168.0, 466.5, 628.8, 747.3, 686.8, 662.0 and 742.0 in the 1.000, 0.556, 0.309, 0.171, 0.095, 0.053, 0.029, 0.016 and 0.009 mg a.s./kg soil dry weight treatment concentrations of the test substance, respectively. When compared statistically, only the results for the 1.000, 0.556, 0.309 and 0.171 mg a.s./kg soil concentrations showed a significant reduction from the combined control (MannWhitney U-test, α = 0.05). The LOEC (reproduction) was therefore 0.171 mg a.s./kg soil dry weight, and the NOEC (reproduction) was 0.095 mg a.s./kg soil dry weight.
The 28-day EC50, EC20 and EC10 values for the test substance were calculated to be 0.235, 0.144 and 0.112 mg a.s./kg soil dry weight, respectively.

Results with reference substance (positive control):

MORTALITY
In the toxic reference treatment, 80.0% mortality (78.4% corrected mortality) was recorded; this result differed significantly from the control (Fisher’s Exact Test, α = 0.05), and the adults were noted to be smaller than in the controls.

REPROFUCTION
In the toxic reference treatment, the mean number of progeny produced per replicate was 0.4, a value that differed significantly from the combined control (Mann-Whitney U-test, α = 0.05). The response of Collembola exposed in the toxic reference group was within the expectation of the test guideline (OECD, 2009), as there was > 50% reduction in juvenile production. It was considered that this verified that the organisms in the test system were responding at a normal level.

Table 1. Springtail mortality: Mean percentage mortality of original springtails (n = 80 for each control, n = 40 per treatment, n = 50 for toxic reference) in the test at 28 days, both before and after correction of the data for combined control treatment losses.

Concentration (mg a.s./kg soil dry wt.)^a		% mortality^b	Corrected % mortality^c
Control (untreated)	-	8.8	-
Control (acetone)	-	6.3	-
Combined controls	-	7.5	-
Test substance	1.000	100.0*	100.0
	0.556	92.5*	91.9
	0.309	65.0*	62.2
	0.171	22.5*	16.2
	0.095	5.0	0.0
	0.053	10.0	2.7
	0.029	12.5	5.4
	0.016	17.5	10.8
	0.009	7.5	0.0
Toxic reference	-	80.0*	78.4

a Based on a measured purity of 98.4% w/w.

b Mortality amongst springtails originally introduced.Individual test-item treatments were compared to the combined control data using Fisher’s Exact Test (α= 0.05).Values marked with an asterisk differed significantly.

c Corrected for combined control treatment losses using Abbott’s formula.

Note: Only data from what was considered to be the active part of the response curve (shaded values) were included in the Probit regression analysis.

Table 2. Springtail mortality – Probit Analysis: The results of a Probit regression analysis on the numbers of adults found in the arenas treated with the test substance after 28 days.

Regression coefficient (Standard Error)	4.807 (0.688)
Intercept of Probit line (Standard Error)	-11.695 (1.712)
Goodness of fit:χ^2with 33 d.f.	41.063, P > 0.05
Natural Response Rate Estimate (Standard Error)	0.069 (0.018)
LC50 value (95% confidence limits) mg a.s./kg soil	0.271 (0.232 and 0.312)

 

Table 3. Springtail reproduction: Summary of the reproduction of springtails during the bioassay. The data presented are the mean number of juveniles per arena produced over 28 days, and the percentage change in the mean reproduction rate in each treatment, relative to the combined control treatments.

Treatment	Concentration (mg a.s./kg soil dry wt.) a	Mean No. progeny per replicate b	% change relative to the control c
Control (untreated)	-	640.6	-
Control (acetone)	-	640.0	-
Combined controls	-	640.3	-
The test substance	1.000	16.3*	97.5
	0.556	36.5*	94.3
	0.309	168.0*	73.8
	0.171	466.5*	27.1
	0.095	628.8	1.8
	0.053	747.3*	-16.7
	0.029	686.8	-7.3
	0.016	662.0	-3.4
	0.009	742.0	-15.9
Toxic reference	-	0.4*	99.9

a. Based on a measured purity of 98.4% w/w.

b The fecundity data from individual test-item treatments were compared to the combined control data either by Mann-Whitney U-test (α= 0.05). Values marked with an asterisk (*) differed significantly. Note: progeny numbers in the 0.053 mg a.s./kg treatment were significantly higher than the combined control data.

c A positive value indicates a decrease, and a negative value an increase in reproduction relative to the combined control. Note: Only data from what was considered to be the active part of the response curve (shaded values) were included in the Probit regression analysis

 

Table 4. Springtail reproduction - Probit analysis: The results of a Probit regression analysis on the percentage change in numbers of F1 progeny in the test substance treated arenas, relative to the combined control treatments, after 28 days.

Regression coefficient (Standard Error)	3.997 (0.146)
Intercept of Probit line (Standard Error)	-9.479 (0.351)
Goodness of fit:χ2with 18 d.f.	117.322, P < 0.001
EC50value (95% CL)mg a.s/kg soil	0.235 (0.208 and 0.265)
EC20value (95% CL)mg a.s/kg soil	0.145 (0.121 and 0.167)
EC10value (95% CL)mg a.s/kg soil	0.112 (0.089 and 0.133)

 

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the 28-day EC10 for reproduction was determined to be 0.112 mg/kg soil dry weight.

Executive summary:

The aim of this study was to determine under laboratory test conditions whether this test item has harmful effects on the springtail, Folsomia candida. The study was conducted according to OECD TG 232 and in compliance with GLP criteria. Following a preliminary range-finding bioassay, the test substance was evaluated at nine nominal concentrations, equivalent to 1.000, 0.556, 0.309, 0.171, 0.095, 0.053, 0.029, 0.016 and 0.009 mg a.s./kg soil dry weight. Due to its low solubility in water, the test item was diluted in acetone and aliquots of the solutions were mixed with small amounts of sand. The acetone was allowed to evaporate away before the treated dry sand was mixed into the test soil. These variants were compared with a control treatment of just acetone (added to sand aliquots), an untreated control of purified water only, and a toxic reference treatment of Betosip 114 (nominally 114 g/L phenmedipham), applied at a concentration of 200 mg product/kg soil dry weight.

Treatments were incorporated into an artificial soil substrate (containing 5% w/w peat), which was then transferred into small glass jars (n = 8 for each control; n = 4 per test-item treatment concentration; n = 5 for toxic reference). Ten juvenile F. candida (11 days old) were introduced into each jar and dry granulated yeast was provided as food. This food was replenished at 14 days. The jars were ventilated every 3-4 days. At 28 days, the numbers of the springtails originally introduced that still survived and the numbers of their offspring were recorded.

The data for the untreated control and the solvent control were compared statistically, but did not differ significantly (α = 0.05; Fisher’s Exact Test for mortality, t-test for reproduction). Therefore, the data from the two treatments were combined and all subsequent statistical comparisons were made against the combined control treatment data. Statistically significant mortality of adult springtails was observed for the 1.000, 0.556, 0.309 and 0.171 mg a.s./kg soil dry weight concentrations. Thus, the LOEC for assessments of mortality was 0.171 mg a.s./kg soil dry weight, and the NOEC was 0.095 mg a.s./kg soil dry weight. A statistically significant reduction in reproduction was observed for the 1.000, 0.556, 0.309 and 0.171 mg a.s./kg soil dry weight concentrations. Thus, the LOEC for assessments of reproduction was 0.171 mg a.s./kg soil dry weight, and the EC10 was calculated to be 0.112 mg a.s./kg soil dry weight.

Description of key information

28-d EC10 = 0.112 mg a.i./kg dry soil, Folsomia candida, reproduction, OECD TG 232, Geary 2015

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil dwelling arthropods:

0.112 mg/kg soil dw

Additional information

The effects of the substance on the survival and reproduction of terrestrial arthropods were studied under laboratory test conditions and GLP to relevant OECD test guidelines. Test substance was dispersed in acetone and applied to artificial soils, which were moistened to 50% of their water holding capacity. Studies were conducted with springtails (Folsomia candida) and soil mites (Hypoaspis aculeifer), and test organisms were placed in test vessels containing untreated substrate (negative control), soil containing acetone (solvent control), soil containing test substance (treatment groups) or soil containing a suitable toxic control (positive control). Survival and reproduction of test organisms was observed over a period of 14 days. Reproduction was found to be the more sensitive endpoint in the two tested species. The EC10 value for the reproduction of springtail was 0.112 mg/kg soil dry weight and the EC10 value for the reproduction of soil mites was 0.169 mg/kg soil dry weight.