2-chloro-N-[[[4-(trifluoromethoxy)phenyl]amino]carbonyl]benzamide

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiated/completed: 14 November 2008 - 14 January 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The study consisted of 2 parts. Adult earthworms were exposed to the test item for a period of 4 weeks (first part): after this period, the adults were removed from the test vessels and the cocoons and juvenile earthworms remained in the test vessels for additional 4 weeks (second part). The total duration of each run of the study was 8 weeks.

Details on preparation and application of test substrate:

According to OECD 222 and EPPO (2003), 5% peat was used in the artificial soil
considering the influence of the properties of the test item (Log POW = 2) on
bioavailability. The artificial soil will be prepared with the following composition (percentage distribution on dry weight basis):
• Sphagnum peat (shredded): 5%
• Kaolinite clay: 20% (content of Kaolinite = 30.2%)
• Industrial quartz sand (Sort: F 36): 73.8% (Particle size: 0.20 mm – 0.05 mm = 75.09%)
• Calcium carbonate (CaCO3): 0.2 % (for the adjustment to pH 6.0 ± 0.5)
• Food (dried ground cow manure): 1%

The artificial soil was prepared by mixing the dry components intensely in a
laboratory mixer. Then, the soil was moistened with deionised water to reach a water content of 58 % of the maximum water holding capacity.

Directly after application the individually weighed adult earthworms were exposed to the treated artificial soil. Before weighing, the earthworms were quickly washed with water and surplus water was absorbed on filter paper.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Eisenia fetida was used as test species for the study (Eisenia fetida andrei; strain of Prof. Graff, Forschungsanstalt für Landwirtschaft, 38104 Braunschweig, Germany).
This strain has been held in the laboratory of the testing facility for several years at 22 ± 2°C. The substrate consisted of about 54.6 % by weight of natural soil, 33.8 % peat and 4.2 % straw, 0.7 % calcium carbonate and 6.8 % animal manure (dry weight in each case). During cultivation, they were fed with animal manure at 14 day intervals. At the same time, the substrate was also replenished with water. In order to obtain a synchronised culture of earthworms, 100 adult earthworms were kept in a rearing vessel with substrate and food for a period of 4 weeks. After this time, the adult earthworms were picked out of the substrate again and the offspring hatched from the cocoons.
For 4 days prior to the test start, the test organisms were acclimated to the artificial soil and test temperature. The mean body wet weight of the test organisms at the start of the test ranged from 0.25 to 0.44 g per worm. The worms were adult with a well developed clitellum and approximately 8 months old. The age of the worms from the synchronised culture differed by not more than 4 weeks.

Study type:

laboratory study

Substrate type:

artificial soil

Total exposure duration:

8 wk

Test temperature:

20 ± 2°C

pH:

The mean pH value of the artificial soil was pH 6.00 (initial). At the end of the test the pH values ranged from 6.80-6.90.

Moisture:

The moisture of the artificial soil was adjusted to nominal 26.2 g water / 100 g dry
weight artificial soil (¿ nominal 20.8% soil moisture) corresponding to approximately
58 % of the maximum water holding capacity of the artificial soil.
Prior to the start of the test the mean soil moisture was 18.4%. At day 0 the mean soil
moisture was 19.7% and at day 56 it was 18.0%.

Details on test conditions:

Non-re-usable plastic boxes (length x width x height ca. 16.5 cm x 12 cm x 6 cm, area approximately 200 cm2) were used as test vessels. Each test vessel contained an amount of approximately 500 g artificial soil (dry weight) to obtain a depth of approximately 5 cm soil in the test vessels. The test vessels were covered with transparent lids to prevent earthworms from escaping and to reduce evaporation during the test period. The lids were perforated to allow air exchange. The test vessels were labelled with the study number and all necessary additional information to assure unmistakable identification. The vessels were kept in a temperature-controlled room at 20 ± 2°C under a 16-hour light to 8-hour darkness photoperiod and a light intensity at light period between approximately 400 - 800 Lux. For each test concentration of the test item group, 4 replicates of 10 earthworms (4x10) and for the control group 8 replicates of 10 earthworms (8x10) were tested.


Evaluations:

Mortality and Adverse Effects:
After 4 weeks of exposure, the content of each test vessel was emptied and the living adult earthworms were counted and checked for any abnormal behaviour or other adverse effects (e.g. lack of movement, rigidity etc.). Those earthworms, which did not move after gentle mechanical stimulus were considered to be dead. Also missing earthworms (compared to the number of initially placed test organisms) were considered to have died, since dead earthworms completely decompose in the soil in short time and hence cannot always be found.

Growth:
At the beginning (prior to exposure) and at the end of the 4 weeks of exposure, the adult test organisms of each vessel were weighed (at the start each individually, at the end together of each test vessel). Before weighing, the earthworms were quickly washed with water; surplus water was adsorbed on filter paper. Changes in body weight values of the surviving test organisms of the treatment groups during the test period were compared to the values of the control group. The homogeneity of variances of the data was checked by Cochran’s test. The homogeneity hypothesis was rejected.

The homogeneity of variances of the transformed data was checked by Cochran’s test. The homogeneity hypothesis was rejected in any case.
The data were statistically evaluated by means of a multiple sequentially rejective U-test after Bonferroni-Holm.

Reproduction:
At the end of the test after 8 weeks, the number of surviving juveniles per test vessel was determined. The test vessels were placed in a water bath at 50-60 °C. By this treatment, alive juvenile earthworms rose to the soil surface. The emerging earthworms were removed and counted. Afterwards the content of each test vessel was checked additionally by carefully stirring up the artificial soil with the help of tweezers. The reproduction of the surviving test organisms per test vessel at the end of the study was compared to the control values.
The homogeneity of variances of the data was checked by Cochran’s test.
The homogeneity hypothesis was accepted. The normal distribution of the data was tested by Kolmogorov-Smirnov test. The normality hypothesis of the data was accepted. The data were statistically evaluated by means of a Williams multiple sequential t-test.

Assessment of Food Consumption:
At each feeding date, the amount of food consumed by the adult earthworms was visually estimated for each test vessel.

Soil Moisture:
For all moisture determinations a defined weight of 10 g of either ground peat or of artificial soil was dried up to a constant weight in a microwave ("Bosch", Inventory- No.: 695293) at appropriate capacity. Moisture content was calculated from the weight difference before and after drying. Peat moisture was determined prior to preparation of artificial soil. Artificial soil moisture was determined prior to test start, at day 0 and day 56 of the test. At day 0 and day 56 the artificial soil moisture was determined from well mixed samples of the control replicates.

The maximum water holding capacity (WHC max.) of the artificial soil was determined in 2 samples of the artificial soil: the soil was filled into a cylindrical vessel made of 2A-steel (diameter 6 cm, height 7.8 cm). The bottom of the steel vessel was perforated by 19 holes with diameters of 4 mm. The perforated bottom was covered by gauze or filter paper to avoid loss of soil. The vessel was put into a water-filled bucket for at least 3 hours with the water level lying above the soil surface. After this time, the cylinder was removed and was suspended or was hung up to allow the water to drip off for 2 hours. The maximum water holding capacity of the artificial soil was determined by drying the soil and weighing it afterwards. The test vessels were covered with transparent lids to reduce evaporation. Water evaporation losses will be compensated at least once a week by adding deionised water up to the initial weight of the test vessels.

The moisture of the artificial soil was adjusted to nominal 26.2 g water / 100 g dry weight artificial soil corresponding to approximately 58 % of the maximum water holding capacity of the artificial soil. Prior to the start of the test the mean soil moisture was 18.4%. At day 0 the mean soil moisture was 19.7% and at day 56 it was 18.0%.

Prior to the test, the mean pH value of the artificial soil was pH 6.00. The pH was measured in a sample of the wetted artificial soil. At the end of the test, the pH was determined in mixed samples from the control replicates and all treatment groups.

Test Environment:
The desired test temperature (room temperature) of 20°C ± 2 was recorded with suitable instruments, documented in the raw data. During the test period, the temperature was in the range of 18 to 22°C. The desired light meter ("TK02 – R0001") at the beginning, the middle and the end of the test, documented in the raw data. The measured mean light intensity was 587 Lux at day 0, 488 Lux at day 28 and 505 Lux at day 56 of the study.

Nominal and measured concentrations:

Nominal concentrations: 100, 178, 316 and 1000 mg test item/kg dry
weight artificial soil.

Key result

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Key result

Duration:

8 wk

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Key result

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

8 wk

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

EFFECTS OF TREATMENTS ON SURVIVAL OF ADULTS:

A mortality rate of 1.25% of adult earthworms was observed after 28 days of exposure at the control group. No mortality of adult earthworms was observed at any test concentration of triflumuron SC 480 G in this study (see Table 1 and 2).
EFFECTS OF TREATMENTS ON BIOMASS OF ADULTS:

The mean body wet weight of the ten earthworms in each replicate from the control and from all treatment groups at test start The mean body weight of the adult earthworms in the control group had increased during the 4 weeks of exposure. The mean body weight was 0.48 g per worm (+ 54.5% of the mean initial weight). The mean changes in body weight of the test concentrations of 100, 178, 316, 562 and 1000 mg test item/kg dry weight artificial soil were + 61.8%, + 60.8%, + 58.9%, + 68.8% and + 66.5%, respectively. No statistically significant different values for the growth relative to the control were observed at the test concentrations of 100, 178, 316 and 1000 mg test item/kg dry weight artificial soil. A statistically significant increase of the growth relative to the control was observed at the test concentration of 562 mg test item/kg dry weight artificial soil. This slight increase in growth compared to the control is considered not to be treatment related, but rather caused by biological variability of the test system. However, no statistically significant different value for the growth relative to the control was observed at the highest test concentration of 1000 mg test item/kg dry weight artificial soil. (Results of a multiple sequentially rejective U-test after Bonferroni-Holm, two-sided, a = 0.05.).

EFFECTS OF TREATMENTS ON THE REPRODUCTION
Table below shows the total number of living offspring, produced by all adults within 28 days of continuous exposure expressed per test vessel and per surviving adult (reproduction rate).
In the control group, on average 206.0 juvenile earthworms per test vessel were found (corresponding to a mean reproduction rate of 20.8 juveniles per surviving adult).
At the test item treatment groups exposed to triflumuron SC 480 G up to and including the highest test concentration of 1000 mg test item/kg dry weight artificial soil, the mean reproduction rate was in the range of 87.4% to 111.0% of the control value. No statistically significant different values for the number of juveniles per test vessel relative to the control were observed at any test concentration, including the highest concentration of 1000 mg test item/kg dry weight artificial soil. (Results of a Williams multiple sequential t-test, one-sided smaller, a = 0.05).

Results with reference substance (positive control):

The most recent toxic standard reference test, with the reference test item mixed into the artificial soil, was performed from January to April, 2008 (Study No.: Rg 02/08; Report No. LRT-Rg-R-Ref-10/08; NON-GLP). Effects of the reference test item on mortality and changes in body weight of the adults after an exposure period of 28 days and the number of offspring after 56 days

Number of surviving adult earthworms and % mortality at day 28

Test concentration (mg test item/kg dws*)	Box No.	Number of earthworms exposed Day 0	Number of earthworms survived Day 28	Sum of dead earthworms	% Mortality per replicate	% Mortality per test concentration
Control	1	10	10	0	0	1.25
	2	10	10	0	0
	3	10	10	0	0
	4	10	9	1	10
	5	10	10	0	0
	6	10	10	0	0
	7	10	10	0	0
	8	10	10	0	0
100	1	10	10	0	0	0
	2	10	10	0	0
	3	10	10	0	0
	4	10	10	0	0
178	1	10	10	0	0	0
	2	10	10	0	0
	3	10	10	0	0
	4	10	10	0	0
316	1	10	10	0	0	0
	2	10	10	0	0
	3	10	10	0	0
	4	10	10	0	0
562	1	10	10	0	0	0
	2	10	10	0	0
	3	10	10	0	0
	4	10	10	0	0
1000	1	10	10	0	0	0
	2	10	10	0	0
	3	10	10	0	0
	4	10	10	0	0

* dws = Dry weight artificial soil

 

 

Reproduction of the earthworms
(Values in this table are rounded values)

Test concentration (mg test item/kg dws*)	Box No.	Reproduction rate (per surviving adult)		Juvenile earthworms per test box				Statistical evaluation **
			Mean ± SD		Mean ± SD	CV (%)	% of control
	1	16.3		163
	2	16.9		169
	3	18.2		182
Control	4	18.1***	20.8 ± 3.9	163***	206.0 ± 41.4	20.1	100.0	---
	5	24.9		249
	6	23.1		231
	7	22.6		226
	8	26.5		265
	1	22.6		226
100	2	13.4	18.1 ± 4.2	134	180.5 ± 42.4	23.5	87.6	n.s.
	3	20.5		205
	4	15.7		157
	1	23.0		230
178	2	17.1	18.6 ± 3.5	171	185.8 ± 34.5	18.6	90.2	n.s.
	3	14.9		149
	4	19.3		193
	1	15.1		151
316	2	17.5	18.9 ± 3.2	175	188.8 ± 31.9	16.9	91.6	n.s.
	3	20.7		207
	4	22.2		222
	1	15.1		151
562	2	16.4	18.0 ± 3.3	164	180.0 ± 33.2	18.5	87.4	n.s.
	3	17.8		178
	4	22.7		227
	1	24.5		245
1000	2	25.0	22.9 ± 2.9	250	228.8 ± 28.7	12.6	111.0	n.s.
	3	23.3		233
	4	18.7		187

* dws = Dry weight artificial soil

** Statistical comparison of mean reproduction per test vessel:

Result of a Williams Multiple Sequential t-test, one-sided smaller, α = 0.05 n.s.: mean value not statistically significantly different compared to the control

(p ≥ 0.05)

*** One dead adult earthworm

Validity criteria fulfilled:

yes

Conclusions:

The exposure of adult earthworms to the test item up to and including the highest test
concentration of 1000 mg test item/kg dry weight artificial soil did not affect mortality
of Eisenia fetida.
No statistically significant different values for the growth relative to the control were
observed at the test concentrations of 100, 178, 316 and 1000 mg test item/kg dry
weight artificial soil. A statistically significant increase of the growth relative to the
control was observed at the test concentration of 562 mg test item/kg dry weight
artificial soil. This slight increase in growth compared to the control is considered not
to be treatment related, but rather caused by biological variability of the test system.
However, no statistically significant different value for the growth relative to the
control was observed at the highest test concentration of 1000 mg test item/kg dry
weight artificial soil.
No statistically significant different values for the number of juveniles per test vessel
relative to the control were observed at the test concentration of 100, 178, 316, 562
and 1000 mg test item/kg dry weight artificial soil.
Overall, based on the biological and statistical significance of the effects observed on
reproduction, it is concluded, that the NOEC for this study is = 1000 mg test
item/kg dry weight artificial soil. Thus, the overall LOEC is determined to be
> 1000 mg test item/kg dry weight artificial soil.

Executive summary:

Adult Eisenia fetida (approx. 8 months old, 8 x 10 animals for the control group and 4 x 10 animals per test concentration of the treatment group) were exposed in an artificial soil (with 5% peat content) to the nominal test concentrations of 100 – 178 – 316 – 562 – 1000 mg test item/kg dry weight artificial soil. The test item was mixed into the soil. After 28 days the number of surviving animals and their weight alteration was determined. They were then removed from the artificial soil. After further 28 days, the number of offspring was determined.

A mortality rate of 1.25% of adult earthworms was observed after 28 days of exposure at the control group. No mortality of adult earthworms were observed at any test concentration of the test item in this study. No statistically significant different values for the growth relative to the control were observed at the test concentrations of 100, 178, 316 and 1000 mg test item/kg dry weight artificial soil. A statistically significant increase of the growth relative to the control was observed at the test concentration of 562 mg test item/kg dry weight artificial soil. This slight increase in growth compared to the control is considered not to be treatment related, but rather caused by biological variability of the test system.However, no statistically significant different value for the growth relative to the control was observed at the highest test concentration of 1000 mg test item/kg dry weight artificial soil.
Therefore, based on biological and statistical significance:
NOEC related to growth: ≥ 1000 mg test item/kg dry weight artificial soil LOEC related to growth: > 1000 mg test item/kg dry weight artificial soil

In regard to effects on reproduction, no statistically significant different values for the number of juveniles per test vessel relative to the control were observed at the test concentrations of 100, 178, 316, 562 and 1000 mg test item/kg dry weight artificial soil. Therefore, based on statistical significance: NOEC related to reproduction: ≥ 1000 mg test item/kg dry weight artificial soil, LOEC related to reproduction: > 1000 mg test item/kg dry weight artificial soil.

Overall, based on the biological and statistical significance of the effects observed on growth and reproduction, it is concluded, that the NOEC for this study is ≥ 1000 mg test item/kg dry weight artificial soil. Thus, the overall LOEC is determined to be > 1000 mg test item/kg dry weight artificial soil.

Description of key information

The exposure of adult earthworms to the test item up to and including the highest test concentration of 1000 mg test item/kg dry weight artificial soil did not affect mortality of Eisenia fetida.
No statistically significant different values for the number of juveniles per test vessel relative to the control were observed at the test concentration of 100, 178, 316, 562 and 1000 mg test item/kg dry weight artificial soil.
Overall, based on the biological and statistical significance of the effects observed on reproduction, it is concluded, that the NOEC for this study is = 1000 mg test
item/kg dry weight artificial soil. Thus, the overall LOEC is determined to be > 1000 mg test item/kg dry weight artificial soil.

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil macroorganisms:

1 000 mg/kg soil dw