N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl-aminocarbonyl]-2,6-difluorobenzamide

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:

11 Feb 2003 to 11 Mar 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ISO 11267 (Inhibition of Reproduction of Collembola by Soil Pollutants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Application method:

mixed into soil

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

An exactly weighed amount of the test item was dispersed in deionised water to make a stock, without addition of solubility mediators, immediately before application. This stock mixture was diluted with deionised water such that each dispersion contained the amount of test item in 25 mL required to dose 250 g dry weight equivalent of artificial soil (at 25 % dry weight water content). Each test item dispersion (25 mL) was thoroughly mixed with the artificial soil using a mixing machine, achieving a final water content of nominally 35 % of dry weight.
The control was treated with deionised water only.

Test organisms (species):

Folsomia candida

Animal group:

Collembola (soil-dwelling springtail)

Details on test organisms:

TEST ORGANISM
- Comon name: Springtail
- Source: The test organisms were originally purchased and then reared in the lab of the test facility under ambient laboratory conditions:
> Breeding medium: Mixture of plaster for stucco, activated charcoal and water (8:1:9)
> Breeding conditions: breeding in plastic vessel of Bellaplast with a transparent lid (9.5 cm x 9.5 cm x 6 cm); moistening automatically by means of an absorbent wick; aeration in combination with feeding; fed with granulated dry yeast, supplied twice a week; light : dark cycle = 16 h : 8 h (diffuse light); temperature nominally at 20 +/-2 °C
- Age at test initiation: Juvenile Collembola (10-12 days) from synchronised cohort
- Food: granulated dry yeast (purchased)
- Feeding regime: 2 mg granulated dry yeast at the start of the test and after 14 days

Study type:

laboratory study

Limit test:

no

Total exposure duration:

4 wk

Test temperature:

18 - 21 °C

pH (if soil or dung study):

- Test initiation: 6.04 - 6.20
- Test termination: 6.38 - 6.42

Humidity:

- WHC: 65.1%
- Test initiation: 35.27 - 35.49% (equivalent to 54.2 - 54.5 % WHC)
- Test termination: 34.33 - 34.81% (equivalent to 52.7 - 53.5 % WHC)

Photoperiod and lighting:

- Photoperiod: 16 hours light and 8 hours darkness
- Light intensity: 600 lux

Details on test conditions:

TEST SYSTEM
- Test container: Glass beakers (about 150 ml) covered with a glass lid
- Amount of substrate/vessel: 30 g wet weight of articial crumbly structured soil
- No. of organisms per container: 10
- No. of replicates per treatment group: 5 (+ 2 replicates not loaded with Collembola for measurement purposes)
The test vessels were set up at random in the test room.

PROPERTIES OF SUBSTRATE (artificial soil)
- Composition of the artificial soil: 10 % Sphagnum peat; 20 % kaolinite clay; 0.5 % calcium carbonate; 69.5 % industrial quartz sand
(more than 50 % of the particles between 0.05 mm and 0.2 mm); and deionised water

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness
- Light intensity: 600 lux

EFFECT PARAMETERS MEASURED
- At the start of the test, physico-chemical parameters of the articial soil (water holding capacity, water content, pH) were determined.
- The following parameters were analysed 8 weeks after application: the number of parental and juvenile Collembola per test vessel; physico-chemical parameters of the articial soil (water content, pH).

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 mg test substance/kg dry weight of soil

Reference substance (positive control):

yes

Remarks:

Betosip (Phenmedipham 157 g/L)

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.2 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

0.59 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Details on results:

An overview of the results is provided in Table 1 – Table 3 in ‘Any other information on results incl. tables’
In the control group, the mean mortality of the parental Collembola was 8%. The mean mortality levels were 4%, 6%, 8%, 6%, 4%, 14% and 44% in 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 mg/kg dry weight of soil treatment groups, respectively. No statistically significant effects on parental mortality were found for concentrations up to and including 3.2 mg/kg dry weight of artificial soil substrate. The test item concentrations including and above 0.4 mg/kg d.w. resulted in a statistically significant decrease in the number of juvenile Collembola compared to control (46% - 73% decreased in reproduction). According to these results the NOEC was determined to be 0.2 mg/kg dry weight of artificial soil substrate. The EC50 for number of juvenile Collembola was calculated to be 0.59 mg/kg dry artificial soil weight of substrate.

Results with reference substance (positive control):

For controlling the sensitivity of the test system the reference item is regularly tested at concentrations of 50, 100, 200 and 400 mg/kg dry weight of artificial soil. In the most recent test at the time of the experiment, the determined EC50 (reproduction) was 131 mg Betosip/kg d.w. of artificial soil. The EC50 value for the reduction of reproduction was within the range of 100-150 mg product/kg d.w. of artificial soil determined in the ring test and within the range of 100-200 mg product/kg d.w. specified in ISO 11267 (1999.T), the EC50 therefore showed that the test system was sensitive.

Reported statistics and error estimates:

The parental mortality results were analysed using the DUNNETT-test. The accepted significance level was p ≤ 0.05 (one-sided). The NOEC for reproduction was determined using the DUNNETT-test (p ≤ 0.05, two-sided) which was used to analyse the number of offspring per replicate. The EC50 with respect to the number of juveniles (reproduction rate) was calculated by Logit analysis which was fitted using to the maximum likelihood method. The goodness-of-fit of the model was evaluated by Pearson’s x2 test. All analyses were performed using ToxRat version 2.07 (2002

Table 1. Effects of test item on mortality of parental Collembola

Treatment (mg/kg d.w.)	Number of surviving parental Collembola per replicate (4 weeks after test initiation)					Mean	Coefficient of variation (%)	Standard deviation	Mortality (%)
	1	2	3	4	5
Control	10	9	8	10	9	9.2	9.09	0.8	8
0.1	9	9	10	10	10	9.6	5.71	0.5	4
0.2	10	8	10	10	9	9.4	9.52	0.9	6
0.4	9	8	9	10	10	9.2	9.09	0.8	8
0.8	7	10	10	10	10	9.4	14.27	1.3	6
1.6	9	10	10	9	10	9.6	5.71	0.5	4
3.2	10	6	7	10	10	8.6	22.67	1.9	14
6.4	4	5	7	6	6	5.6 +	20.36	1.1	44

+Statistically significant difference (DINNET T-test; p ≤ 0.05)

Table 2. Effects of test item on number of juvenile Collembola

Treatment (mg/kg d.w.)	Number juveniles per replicate (4 weeks after test initiation)					Mean	Coefficient of variation (%)	Standard deviation	Reduction of Reproduction (%)
	1	2	3	4	5
Control	409	436	326	471	308	390.0	70.5	18.07
0.1	478	457	452	352	350	417.8	61.8	14.78	+7*
0.2	373	448	438	564	485	461.6	70.0	15.17	+18*
0.4	250	139	105	382	176	210.4 +	110.0	52.28	- 46
0.8	102	116	138	213	112	136.2 +	44.9	32.97	- 65
1.6	87	109	154	138	161	129.8 +	31.2	24.03	- 67
3.2	86	106	141	157	150	128.0 +	30.6	23.90	- 67
6.4	107	101	137	87	104	107.2 +	18.3	17.10	- 73

+ Statistically significant difference (Dunnett-test P≤0.05)

* An increase in reproduction occurred relative to the control

 

Table 3. Summary of statistical analysis results

NOEC	0.2 mg/kg dw
LOEC	0.4 mg/kg dw
EC50	0.59 mg/kg dw (95% confidence limits 0.43 to 0.80)
Slope b	2.881

 

 

 Validity criteria

The study is considered to be valid, since the validity criteria were accomplished:

For the control group:

- Parental mortality: ≤ 20 % (observed: 8 %)

- Minimum number of instars/vessel: ≥ 100 (observed: average of 390.0/ vessel)

- Coefficient of variation of juvenile number: ≤ 30 % (observed: 18.07 %)

Validity criteria fulfilled:

yes

Remarks:

See validity criteria in 'Any other information on results incl. tables'

Conclusions:

In Collembolan reproduction study with Folsomia candida, performed in accordance with ISO 11267, the 4-week NOEC was determined to be 0.2 mg/kg soil dw. for reproduction.

Executive summary:

The toxicity of the test substance to springtails (Folsomia candida) was tested according to ISO 11267, in compliance with GLP criteria. Five replicates of 10 juvenile Collembola (10 – 12 days old) per treated group were exposed in artificial soil to Lufenuron 050 EC at nominal concentrations of 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 mg a.s./kg dry weight soil and a deionised water control. The sensitivity of the test system was checked, as done routinely, using a toxic standard and was within the range as quoted in the guideline ISO 11267 (1999). During the test, the temperature ranged between 18 – 21 ˚C, the lighting intensity was 600 lux and the photoperiod was 16 hours light and 8 hours darkness. After 28 days, the mortality of parental Collembola and the number of juvenile Collembola were determined.

In the control group, the mean mortality of the parental Collembola was 8%. The mean mortality levels were 4%, 6%, 8%, 6%, 4%, 14% and 44% in 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 mg/kg dry weight of soil treatment groups, respectively. No statistically significant effects on parental mortality were found for concentrations up to and including 3.2 mg/kg dry weight of artificial soil substrate. The test item concentrations including and above 0.4 mg/kg d.w. resulted in a statistically significant decrease in the number of juvenile Collembola compared to control (46% - 73% decreased in reproduction). According to these results, the NOEC on basis of reproductive effects was determined to be 0.2 mg/kg dry weight of artificial soil substrate. The EC50 for number of juvenile Collembola was calculated to be 0.59 mg/kg dry artificial soil weight of substrate.

Description of key information

All available data was assessed. One study on soil dwelling arthropods is included here and its effect values were used as key value. Another study on honeybees is included as supporting study.

4-wk, NOEC = 0.2 mg/kg soil dw, Folsomia candida, reporduction, ISO 11267, Friedrich 2003

Key value for chemical safety assessment

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

0.2 mg/kg soil dw

Additional information

Three standard guidelines followed, and GLP-compliant studies are available for this endpoint. In addition, there is a field study that did not follow the standard guidelines but followed EPPO Bulletin 22 and is similar to the OECD Guidance document on the honey bee (Apis Mellifera L.) brood test under semi-field conditions (OECD Series on Testing and Assessment No. 75) and is in compliance with GLP criteria. One study (Friedrich 2003; ISO 11267; Reliability 1) was selected as the key study because it concerns a reproduction test carried out on the soil-dwelling arthropod, springtail (Folsomia candida), while the other study was an acute oral and contact toxicity study in honeybees. Five replicates of 10 juvenile Collembola (10 – 12 days old) per treated group were exposed in artificial soil to Lufenuron 050 EC at nominal concentrations corresponding to 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 mg a.s./kg dry weight soil and deionised water control. During the test, the temperature ranged between 18 – 21 ˚C, the lighting intensity was 600 lux and the photoperiod was 16 hours of light and 8 hours of darkness. After 28 days, the mortality of parental Collembola and the number of juvenile Collembola were determined. No statistically significant effects on parental mortality were found for concentrations up to and including 3.2 mg a.s./kg dw. The results show that the NOEC on basis of reproductive effects was determined to be 0.2 mg/kg dry weight of artificial soil substrate. The EC50 for the number of juvenile Collembola was calculated to be 0.59 mg/kg dry artificial soil weight of the substrate.

In one of the supporting studies, toxicity to honeybee (Apis mellifera) was determined in acute oral (48-h) and contact (48-h) toxicity tests with the test substance (Kleiner 1997; EPPO170; Reliability 1). For both oral and contact toxicity tests, 10 bees/ replication and 3 replicates/concentration were used. The nominal doses were 100 and 197 µg/bee for oral exposure and 100 and 200 µg/bee for contact exposure. An untreated solvent control was involved in both tests. The mortality and behaviour of the honeybees were compared to the control to study the effects. The test substance caused negligible mortality (7 %) up to a dose of 197 µg/bee in the oral toxicity test and up to a dose of 200 µg/bee in the contact toxicity test. No behavioural anomalies were observed. Based on this information, the 48-hour oral LD50 was determined to be > 197 µg/bee, and the contact LD50 was > 200 µg/bee. In the other supporting study on honeybees (Apis mellifera), the test organisms were exposed to nominal dose rates of 0.0063, 0.013, 0.025, 0.050, and 0.10 μg/larva, equivalent to 0.18, 0.38, 0.73, 1.5, and 3.0 mg/kg diet, respectively. Negative and solvent controls were also included in the exposure (Shaw 2022; OECD TG 237; Reliability 1). Mean measured diet concentrations were 0.19, 0.34, 0.65, 1.3, and 2.8 mg/kg diet. Dimethoate was used as a positive control in the study. Each treatment contained 12 larvae from three different hives for a total of 36 larvae per test group evenly divided between two plates (i.e., 18 larvae per plate). The test substance was added to the royal jelly diet and provided to the honey bee larvae for consumption on test day 4. The 72-hour LD50 was determined to be 0.054 μg/larva based on nominal dose rates. Based on nominal diet concentrations, the 72-hour LC50 was determined to be 1.6 mg/kg diet.

In the field study (supporting study; Petto 1994; Reliability 1), the effect of the test substance on the bee brood was investigated. Three free-flying honeybee (Apis mellifera) colonies were fed with the 1 L of ready-to-use syrup containing 6.6 ml/L of Lufenuron EC 050 (equivalent to 0.33 g a.s./L), pure ready-to-use syrup (negative controls) or ready to use syrup contaminated with Dimilin (25% diflubenzuron;10g/L; positive controls). Two colonies were exposed for 48 hours, and the third one was exposed for 72 hours. One brood-comb per colony was investigated ultimately before application and one, two and three weeks after application. All brood stages found on one side of the respective combs were recorded for each inspection. The individual brood stages found on the first inspection were tracked until the end of the experiment (three weeks) to check individual developmental success. In this manner, the normal development time of the honeybee brood of 21 days (workers) was covered. The experiment was performed in a later summer season (August to September). The results show that after exposure to the test formulation, egg-laying of the queens was reduced for about one week after application. Afterwards, egg-laying was comparable to the time before application. The development of the larvae was strongly reduced for about two weeks. As a consequence, fewer pupae were built.

Metabolites – available information

One toxicity study on springtail (Folsomia candida) is available with the major degradation product, M6. As the dossier has been prepared to address the test substance itself, this study is not summarised as endpoint study record but is briefly discussed here. The toxicity of M6 on the reproduction of juvenile Collembola (10-12 days old) was studied according to ISO 11267. The study complied with GLP. The 28-day NOEC was determined to be 6.4 mg/kg soil dw. The EC50 for the number of juvenile Collembola was calculated to be 21.8 mg/kg (Friedrich 2003).