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

Bioaccumulation: terrestrial

Link to relevant study record(s)

Reference

Endpoint:

bioaccumulation: terrestrial

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Oct 2003 to 16 Dec 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Draft OECD Guideline 317 (Bioaccumulation in Terrestrial Oligochaetes), 2001

Version / remarks:

First Draft, May 2001

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Details on sampling:

- Sampling intervals: During the uptake phase, measurements of test item concentrations (total radioactivity) were taken on Day 1, 2, 4, 7, 14, and 21. After the 21-day uptake phase, the elimination phase was started by transferring exposed earthworms to artificial soil without test item. During the elimination phase, the measurements of the total radioactivity was performed in samples of Day 22, 23, 25, 28, 35, and 42 of the study (i.e. Day 1, 2, 4, 7, 14, and 21 of the elimination phase).
- Soil Samples: After the soil application four days before the start of the study, 12 samples of the artificial soil of each test concentration were taken for verification of homogeneity of the test item in the artificial soil. From each test concentration, two samples were taken from the top, two from the middle, and two from the bottom of the first and last test vessel applied. The samples were analysed by liquid scintillation counting (LSC). At the start of the study (Day 0), two samples were taken from the designated test vessel of each test concentration for determination of the exposure concentrations. At each sampling date during the uptake and the elimination phase, two soil samples per test concentration were taken from the test vessels assigned for sampling of worms and soil at the respective sampling date. The samples were analysed by LSC. For the determination of the LSC background, two samples were taken from the artificial soil of the control at the beginning and of the end of the uptake phase and of the elimination phase. At the start of the uptake phase (Day 0) and at Day 7, 14, and 21 of the uptake phase, two samples were taken from the artificial soils of both test concentrations for determination of the concentration of parent test item by HPLC. The samples for total radioactivity determinations were analysed directly after sampling. The soil samples taken for determination of parent and metabolites were stored deep-frozen at -20 °C until analysis.
- Worm Samples: For sampling, the worms were removed from the artificial soil at the sampling dates. They were quickly rinsed with tap water. The excess water was removed from the worms by carefully blotting them dry by using a dry paper towel. Then, for each worm sample, the worms were weighed and were put in the sampling vessel. The worms were killed immediately thereafter by freezing at -20 °C. The worms were not allowed to purge their gut content before being killed. After killing by deep-freezing, the sampled worms were stored deep-frozen at -20 °C until analysis. Four samples of one worm each were taken from the acclimated worm batch for determination of LSC background values just before the start of the uptake phase (Day 0). Additionally, four samples of one worm each were taken at Day 21 (end of uptake phase, start of elimination phase) and at the end of the experiment at Day 42 for LSC background determination from the worms of the control. For measurement of total radioactivity by LSC, four samples of one worm each were taken from both test concentrations at six occasions during the uptake phase (Day 1, 2, 4, 7, 14, and 21) and at six occasions during the elimination phase (Day 22, 23, 25, 28, 35, and 42). Additional four worm samples were taken at Day 21 (at the end of the uptake phase) for residue analysis by LSC as additional information. For residue analysis, the worms were allowed to purge their gut content before sampling. The worms were washed and weighed as described above. Then, the worms were incubated in glass dishes on wetted filter paper for eight hours for purge. Then, the worms were washed and weighed again. The incubation period of eight hours was chosen based on the results of a pre-experiment (without GLP) to determine the optimal incubation period and the incubation method to be used. At Day 7, 14, and 21 of the exposure phase, one additional sample of ten pooled worms was taken from both test concentrations for measurement of the concentration of parent test item and metabolites by HPLC. At the start and the end of the uptake phase, a sample of ten worms (pooled) were taken from the control for the determination of the worm lipid/wet weight ratio by means of a chloroform/methanol extraction.

Vehicle:

yes

Remarks:

acetone

Details on preparation and application of test substrate:

- The test substrate: The test substrate was prepared six days prior to the start of the study. For the study, 57 portions of test substrate were prepared. For each portion, 580 g of the prepared artificial soil (corresponding to 570 g dry weight) was mixed with 2.8 g calcium carbonate and 20 g food in a laboratory mixer. Dried horse manure was used as food for the test organisms. The test substrate was pre-moistened by adding 100 mL of purified water to each portion of the test substrate. After mixing, the portions of test substrate were filled into the test vessels. The test vessels were incubated under test conditions for 2 days until application of the test item. Four days prior to the start of the study, the test item was applied to a portion of sand. Aliquots of 10 g of the treated sand of the corresponding concentration or control were incorporated into each portion of the pre-moistened test substrate. During mixing, the residual volume of 130 mL of purified water was added to each portion to obtain a nominal moisture content of 40% in the soil. Then, each portion of the test substrates was filled into the test vessel. The test vessels (with the exception of the test vessels designated for determination of homogeneity) were incubated for four days under test conditions until the start of the test.
- The test item: No aqueous application solution could be prepared due to the low water solubility of the test item. However, the test item is well soluble in the organic solvent acetone. Thus, the test item was dissolved in acetone and mixed with an adequate amount of quartz sand. After gently evaporating the solvent, the remaining dry sand/test item mixtures were intensively mixed and divided into portions for the application of each soil replicate. The weighed portions were incorporated directly into the artificial soil of each replicate by means of a laboratory mixer. The application solutions in acetone were prepared one day before application and were stored at about 4 °C. The nominal concentration of the test item in the application solution for the test concentration of 0.26 mg/kg dry soil was 0.8 mg/10 mL acetone (1.6 MBq/10 mL acetone). For the test concentration of 0.026 mg/kg dry soil, the nominal concentration of the test item in the acetone application solution was 0.08 mg/10 mL acetone (0.16 MBq/10 mL acetone). For both concentrations, the amount of 300 g of sand was treated with the respective application solution. The application was performed in six batches of 50 g sand each to ensure a homogeneous application of the test item on the sand. For each batch, the volume of 10 mL of the respective application solution was applied to the 50 g sand in a mortar. The solvent was completely evaporated at room temperature under a hood for 2 hours. The remaining sand/test item mixtures were intensively mixed by means of a pestle. Then, the different batches of the sand/test item mixtures of each test concentration were combined and mixed on a roller mixer for 30 minutes. Then, the sand/test item mixture of each test concentration was divided into aliquots of 10 g and filled into separate vials. The aliquots of 10 g sand/test item mixture were quantitatively incorporated into each replicate of pre-moistened test substrate (of 590 g dry weight: 570 g dry substrate plus 20 g food) of the respective test concentration by intense mixing in a laboratory mixer. In total, each replicate consisted of 600 g test substrate (dry weight). The moisture content was brought to 40% by adding 30 ml of purified water to each replicate of premoistened soil.
- Control: Sand treated with acetone as at the two test concentrations was added to the replicates of the control, but no test item was added. The control substrate was prepared analogous to the test substrates of the two test concentrations. Two portions of 50 g sand each were treated with 10 ml acetone (but without test item) identical to the application in the treatments. From the treated sand, aliquots of 10 g were incorporated into each control replicate and the moisture content was brought to 40% by adding 130 mL of purified water to each replicate of pre-moistened soil.

Test organisms (species):

Eisenia fetida

Details on test organisms:

TEST ORGANISM
- Common name: Earthworms
- Source: from a synchronized culture maintained at the test facility
- Feeding: During breeding and keeping, they were fed with suitable food (e.g. horse manure and potatoes).
- Age at test initiation: Adult with a clitellum (around 4½ to 5½ months old); The age of the worms from the synchronized culture did not differ by not more than 4 weeks.
- Weight at test initiation (mean and range, SD): 356 ± 23 mg (Prior to weighing, they were quickly rinsed with tap water and dried carefully by means of dry paper towel. Worms of individual weight in the range of 300 to 500 mg were selected for the study.)

ACCLIMATION
- Acclimation period: Six days prior to the test start (i.e. to the start of the uptake phase)
- Acclimation conditions: The test organisms were acclimated to the artificial soil and test temperature.

Total exposure / uptake duration:

21 d

Total depuration duration:

21 d

Test temperature:

19 - 22 °C

pH:

- Control: 6.1
- 0.026 mg/kg treatment: 6.0 - 6.2
- 0.26 mg/Kg treatment: 6.0 - 6.2

TOC:

Not measured

Moisture:

40%

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Cylindrical glass vessels (diameter 10 cm, height 14 cm) with a volume of about 1 L; The test vessels were covered by transparent lids to prevent worms from escaping and to reduce evaporation during the test period. However, the lids were sufficiently loose fitting to allow air exchange.
- No. of organisms per container: 5
- No. of replicates per treatment group: 25
- No. of replicates per control / vehicle control: 7
- Loading rate: 2.1 mg of worm tissue (wet weight) per gram of wet soil

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
The artificial soil (without calcium carbonate and food) was prepared in a batch of 20 kg by intensely mixing of 2 kg sphagnum peat, 4 kg kaolinite clay, and 14 kg sand (dry weight basis) in a cement mixer.

Soil texture (artificial soil)
- % sand:67
- % peat (shredded, sieved through 2 mm sieve): 10
- % clay (content of Al2O3: 36.4%): 19
- CaCO3: 0.5
- Food (dried horse manure): 3.3%
- Moisture (%): a nominal soil moisture content of 40% was chosen for the test substrate, corresponding to approximately 59% of the total water holding capacity (WHC) of the artificial soil.
- Maximum water holding capacity (in % dry weight): 68 g water per 100 g dry soil (including food)

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light to 8-hour darkness
- Light intensity: Within 520-680 Lux


VEHICLE CONTROL PERFORMED: Yes

Nominal and measured concentrations:

Nominal concentrations: 0 (vehicle control), 0.026 and 0.26 mg/kg dry soil
Measured concentrations: 0.00012 (vehicle control), 0.027 and 0.2474 mg/kg dry soil (accumulation phase) (See Table 1 - Table 3 in 'Any other information on results incl. tables')

Lipid content:

19 700 mg/kg bw w.w.

Time point:

start of exposure

Lipid content:

15 400 mg/kg bw w.w.

Time point:

end of exposure

Key result

Type:

BSAF

Value:

0.12 dimensionless

Basis:

whole body w.w.

Time of plateau:

21 d

Calculation basis:

other: mean concentrations (soil and fish) during the uptake phase

Remarks on result:

other: low dose

Key result

Type:

BSAF

Value:

0.13 dimensionless

Basis:

whole body w.w.

Time of plateau:

21 d

Calculation basis:

other: mean concentrations (soil and fish) during the uptake phase

Remarks on result:

other: high dose

Type:

BSAF

Value:

6.86 dimensionless

Basis:

whole body w.w.

Time of plateau:

21 d

Calculation basis:

other: mean concentrations (soil and fish) during the uptake phase

Remarks on result:

other: low dose; corrected for lipid content

Type:

BSAF

Value:

7.39 dimensionless

Basis:

whole body w.w.

Time of plateau:

21 d

Calculation basis:

other: mean concentrations (soil and fish) during the uptake phase

Remarks on result:

other: high dose; corrected for lipid content

Remarks on result:

other: Not determined

Metabolites:

An overview of the results is provided in Table 9 in 'Any other information on results incl. tables'.
At the low dose concentration on day 7, 14 and 21 one radioactive fraction (S1; 93.8 - 99.2% of the radioactivity recovered in the soil) was detected in the organic extract. On day 0, besides the radioactive fraction S 1 (91.1% of the radioactivity recovered in the soil), the radioactive fraction S3 amounting to 2.3% of the radioactivity recovered in the soil was found. The radioactive fraction S3 was not detected at subsequent sampling dates. At the high dose concentration on day O one radioactive fraction (S 1) amounting to 94,0% of the radioactivity recovered in the soil was found in the organic extract. On day 7, besides the radioactive fraction S1 (99.0% of the radioactivity recovered in the soil), the radioactive fraction S2 amounting to 0.7% of the radioactivity recovered in the soil was detected. The radioactive fraction S2 was already found as impurity of 0.4% in the stock solution. On day 14 and 21 two radioactive fractions (S1 and S4) amounting to 93.9 - 99.0% and 0.6 -1.0% of the radioactivity recovered in the soil for radioactive fraction S1 and S4 were detected, respectively. The radioactive fraction S1 showed a retention time ranging from 17.2 - 17.6 min. The unlabelled parent item eluted at an identical retention time (17.2-17.4 min). In conclusion based on HPLC analyses, radioactive fraction S1 was identical to the parent item. The radioactive fraction S3 was detected in a low amount on day 0 at the low dose concentration. S3 showed a retention time of 16.6 min. The reference item M6 eluted at an identical retention time. In conclusion based on HPLC analyses, radioactive fraction S3 was identical to M6. The radioactive fraction S4 was detected in negligible amounts on day 14 and 21 at the high dose concentration. S4 showed a retention time of 15.5 min. The reference item M4 eluted at an identical retention time (15.2 min). In conclusion based on HPLC analyses, radioactive fraction S4 was identical to M4. The radioactive fraction S2 was detected in negligible amount on day 7 at the high dose Concentration. S2 was already present as impurity in the stock solution.
Overall, during the whole exposure period at both test concentrations almost exclusively parent item was detected in the extract of exposure soil. Besides the parent item the reference items M4 and M6 were found in very low amounts.

Details on results:

An overview of the results is provided in Table 1 - Table 8 in 'Any other information on results incl. tables'.
- Behavior and body weights of the earthworms: At the start of the study, the earthworms were placed onto the test substrates of the test vessels. All worms retired from the surface immediately after introduction and no difference in the behavior was observed between control and the two treatments. During the study, no mortality of worms or other adverse effects were observed, neither in the control nor in the two treatments.
At the start of the study, the mean weight of the earthworms was 356 ± 23 mg (mean ± standard deviation (SD) of 20 worms taken from the acclimated worm batch). During the study, the individual weights of the worms sampled for LSC measurements were in the range of 262 to 552 mg. The mean weights of the worms sampled from the control and from the low and the high test concentration were 409 ± 35 mg (mean ± SD), 395 ± 54 mg, and 386 ± 55 mg, respectively. The mean weight of the worms of the pooled worm samples (taken at Day 0, 7, 14, and 21 for analysis of parent and for lipid content determinations) was 395 ± 20 mg (mean ± SD). Thus, the mean weights of the worms sampled during the study were slightly higher than the mean weight determined at the start of the study indicating that the worms have taken up food and soil.
During the uptake phase (at Day 2), one worm of each test concentration was dissected to investigate whether the worms ingest the soil. The whole gut of the worms of both test concentrations was filled with soil particles demonstrating that the worms ingested the substrate at both test concentrations.
- Test item concentrations in the soils during the uptake phase: After application of the test item to the artificial soil (Day -4), the homogeneity of the test item in the soil was investigated and confirmed. At the nominal test concentration of 0.026 mg/kg dry soil, the mean measured test item concentration was 0.026 ± 0.002 mg/kg dry soil (mean ± SD). At the nominal test concentration of 0.26 mg/kg dry soil, the mean measured test item concentration was 0.24 ± 0.01 mg/kg dry soil (mean ± SD).
At the sampling dates during the uptake phase, the measured concentrations of total radioactivity were in the range of 98 to 108% of the nominal value at the low test concentration. At the high test concentration, 91 to 102% of the nominal concentration was found. The mean measured soil concentrations of radioactivity of the treated soils during the uptake phase (arithmetic mean values of the measurements over the whole uptake phase) were 0.027 and 0.25 mg/kg dry soil. The concentration of parent test item was determined in extracts from soil samples taken at the start of the uptake phase and then weekly during the uptake phase. In the extracts, 91 to 99% of the recovered radioactivity was identified to be parent test item at the low test concentration. At the high test concentration, 94 to 99% was determined to be parent test item. Thus, the test item in the soil was stable during the uptake phase of 21 days at both test concentrations.
A fast uptake of radioactivity was observed within the first day of the uptake phase. Then, no further increase of the concentration of radioactivity was determined during the uptake phase. The concentrations of radioactivity were in the range of 2.4 to 3.8 μg-eq/kg worm for the low test concentration. For the high test concentration, concentrations of radioactivity in the range of 28.0 and 37.6 μg-eq/kg worm were measured during the uptake phase. Compared to the soil concentrations, the radioactivity levels in the earthworms were approximately eight fold lower than the soil concentrations. The mean measured concentrations in the worms during the uptake phase (arithmetic mean value of the measurements over the whole uptake phase) were 3.2 and 31.6 μg-eq/kg worm for the low and the high test concentration, respectively.
During the elimination phase, a fast decrease of the radioactivity concentration in the worms was observed. The measured concentrations were below the detection limit after 7 days elimination (Day 28) and after 14 days elimination (Day 35) for the low and the high test concentration, respectively (see Tables 4 and 5 of the attached analytical phase report). The fast increase of the radioactivity concentrations at the start of the uptake phase, the constant radioactivity levels in the worms during the uptake phase, and the fast decrease of the radioactivity in the elimination phase indicate that the measured radioactivity in the worms can be attributed, at least in part, to the treated soil ingested by the worms.
- Residual concentrations in the worms: At the end of the uptake phase, four worms of each test concentration were allowed to purge their gut content before sampling to determine the residual concentration in the worms. For depletion, the worms were incubated on moistened filter paper for eight hours. During incubation, the weight loss per worm was approximately 50 mg per worm. The mean concentration of radioactivity in these worms was 2.5 and 20.8 μg-eq/kg worm at the low and the high test concentration, respectively. These concentrations correspond to 78 and 66% of the mean total radioactivity measured in the worms during the uptake phase (where the worms were not allowed to purge their gut content before sampling). However, the mean concentration measured in worms incubated for one day in clean artificial soil without test item (at Day 1 of the elimination phase) were significantly lower (22 and 13% for the low and high test concentration, respectively). This indicates that the depletion of the worms gut contents for determination of residuals was not complete within eight hours. Therefore, the concentration measured in worms at Day 22 of the study (Day 1 of the elimination phase) can be regarded as residual concentrations which were 0.7 and 4.0 μg-eq/kg worm for the low and high test concentration, respectively.
- Bioaccumulation factor: Due to the lack of accumulation of the test item in worms (BAF < 1, see below), no half-life calculations and accumulation/elimination kinetics could be performed. Consequently, the average amount of radioactivity in worms during the whole uptake phase was used for calculations of bioaccumulation factors. Bioaccumulation factors (BAF) were calculated on the basis of mean measured concentrations in the soils and in the worms during the uptake phase for both test concentrations. The mean BAF was 0.12 ± 0.02 (mean ± SD) and 0.13 ± 0.01 for the low and the high test concentration, respectively. The lipid content in the worms was similar at day 0 (19. 7 mg/g) and day 21 (15.4 mg/g). Taking into account the average Cw levels, average lipid-BAF values of both days ranged from 6.0 - 7.7 and 6.5 - 8.3 as calculated for low and high dose, respectively. In conclusion, no accumulation of the test substance in the earthworm was detected. Consequently BAF values were negligible.

Table 1. Homogeneity of radioactivity in the soil as determined by LSC in the first and the last vessel of both test concentrations at day -4 (values given as μg parent equivalents per kg dry soil based on a specific radioactivity of 120'000 dpm/μg).

Day-4
Vessel	Sampling level	low dose (µg-eq/kg dry soil)	high dose (µg-eq/kg dry soil)
first	top	20.3	227.0
		27.1	250.6
	middle	18.6	259.2
		28.8	249.0
	bottom	25.2	224.2
		23.8	240.1
second	top	23.9	229.3
		36.1	254.1
	middle	27.3	221.5
		27.0	221.3
	bottom	26.4	228.0
		22.7	248.0
Mean		25.6	237.7
± SD (n=12)		2.4 (9.4%)	13.3 (5.6%)

Table 2. Actual concentration of total radioactivity in the soil during the uptake phase at target concentrations of 0.026 mg/g for the low dose and 0.260 mg/g for the high dose (mean values of duplicates given as μg parent equivalents per kg dry soil based on a specific radioactivity of 120'000 dpm/μg).

Time interval (days)	Accumulation Phase
	low dose		high dose
	(µg-eq/kg dry soil) Cs low	% of nominal concentration	(µg-eq/kg dry soil) Cs high	% of nominal concentration
0	27.3	105	235.3	91
1	26.7	103	243.4	94
2	27.5	106	236.7	91
4	27.6	106	252.4	97
7	28.0	108	250.1	96
14	25.6	98	249.3	96
21	26.2	101	264.7	102
Mean	27.0	104	247.4	95
± SD (n=7)	0.9 (3.2%)		10.1 (4.1%)

 

Table 3. Concentration of total radioactivity in the soil during elimination (A) and in the control during the whole test (B). Values (mean of duplicates) are given as μg parent equivalents per kg dry soil based on a specific radioactivity of 120'000 dpm/μg.

(A) 

Time interval		Elimination Phase
I	II	low dose		high dose
		dpm/g sample*	(µg-eq/kg dry soil)	dpm/g sample*	(µg-eq/kg dry soil)
22	1	5	0.1 (< BG)	36	0.4
23	2	10	0.1 (< BG)	16	0.2
25	4	9	0.1 (< BG)	65	0.8
28	7	10	0.1 (< BG)	24	0.3
35	14	8	0.1 (< BG)	9	0.1 (< BG)
42	21	17	0.2	25	0.3

< BG: Below background (i.e. 0.12 μg-eq/kg dry soil)

(B)

Time interval		Control (BG)
I	II	dpm/g sample*	µg-eq/kg dry soil
0		3	0.04
21		6	0.07
21	0	16	0.19
42	21	17	0.20
Mean		11	0.12
± SD (n=4)		7	0.09

*wet soil

I: Days after the onset of accumulation

II:Days after the onset of depuration

Table 4.Concentration of radioactivity (mean values of four worms per time point; A) and bioaccumulation factors (BAF; B) in the worm at an average soil concentration (Cs) of 27.0 and 247.4 μg-eq/kg for the low and high dose, respectively.

(A)

Phase	Time interval		µg-eq/ka worm
	I	II	low dose	high dose
A	1	--- --- --- --- --- ---	3.8	28.0
A	2		2.4	37.6
A	4		3.1	31.6
A	7		3.1	32.3
A	14		3.4	29.8
A	21		3.7	30.4
Mean			3.2	31.6
± SD (n=6)			0.5 (15.5%)	3.3 (10.4%)
*depleted	21	---	2.5	20.8
E	22	1	0.7	4.0
E	23	2	0.4	2.6
E	25	4	0.4	1.3
E	28	7	0.3 L	1.1
E	35	14	< 0.05 L	0.3 L
E	42	21	0.2 L	0.5

L: below or at limit of detection

(B)

Phase	Time interval	BAF**
	I	low dose	high dose
A	1	0.14	0.11
A	2	0.09	0.15
A	4	0.11	0.13
A	7	0.11	0.13
A	14	0.13	0.12
A	21	0.14	0.12
Mean		0.12	0.13
± SD (n=6)		0.02 (15.5%)	0.01 (10.4%)
*depleted	21	0.09	0.08

*worms were allowed to purge their gut content before sampling

I: Days after the onset of accumulation

II: Days after the onset of elimination

Table 5. Background levels in control worms during incubation. Values (mean of four worms per time point) are given as dpm per g worm and are expressed in parent equivalents on a fresh weight basis (i.e. μg/kg) taking into account the specific radioactivity of 120'000 dpm/μg.

Time interval (days)	dpm per g worm	µg-eq/kg worm
0	23	0.19
21	18	0.15
42	9	0.07
Mean	17	0.14
SD (n=3)	--	0.06
Limit of detection*	--	0.32

* Mean+ 3x SD

 

Table 6. Bioaccumulation factors calculated for the low dose soil concentration of 27.0 μg/kg (A) and the high dose soil concentration of 247.4 μg/kg (B) based on lipid content in control worms as determined in a pool of 10 worms on day 0 and 21.

(A)

day	Low dose soil concentration
	Lipid (mg/g worm)	Cw (mean) (µg-eq/kg worm)	Cw (µg-eq/kg worm)	Cs (mean) (µg/kg)	BAF* (Lipid)
0	19.7	3.2	163	27.0	6.02
21	15.4	3.2	208	27.0	7.70
Mean BAF lipid					6.86

(B)

day	Low dose soil concentration
	Lipid (mg/g worm)	Cw (mean) (µg-eq/kg worm)	Cw (µg-eq/kg worm)	Cs (mean) (µg/kg)	BAF* (Lipid)
0	19.7	31.6	1'606	247.4	6.49
21	15.4	31.6	2'052	247.4	8.29
Mean BAF lipid					7.39

*Cw/Cs

Table 7.Extraction of total radioactivity in soil treated with 14C-labelled test substance at an average low dose level of 27.0μg/kg (values in percentage of the radioactivity recovered in the soil before (A) and after normalization (B)).

(A)

Phase	Days of exposure (duplicates)
	0		7	14	21
	I*	II	I	I	I
Acetonitrile Acetonitrile Methanol Methanol/water (8:1; v/v) Methanol (reflux extraction)	54.9 26.5 8.7 -- --	60.2 26.5 9.2 -- --	55 25.8 11.0 3.0 2.8	60.6 25.8 10.3 2.9 2.9	69.7 26.8 8.6 3.7** 2.3**
Subtotal organic	90.1	95.9	97.6	102.5	111.1
Non-extractable*	6.0	6.7	1.8	0.8	0.9
TOTAL	96.1	102.6	99.4	103.3	112.0

 

(B)

Phase	Days of exposure (duplicates)
	0		7	14	21	Mean
	I*	II	I	I	I
Acetonitrile Acetonitrile Methanol Methanol/water (8:1; v/v) Methanol (reflux extraction)	57.1 27.6 9.1 -- --	58.7 25.8 9.0 -- --	55.3 26.0 11.1 3.0 2.8	58.7 25.0 10.0 2.8 2.8	62.2 23.9 7.7 3.3** 2.1**
Subtotal organic	93.8	93.5	98.2	99.2	99.2	96.8 ± 2.9
Non-extractable*	6.2	6.5	1.8	0.8	0.8	3.2 ± 2.9
TOTAL	100	100	100	100	100

-- Not performed

* Not further analysed

** Not pooled, consequently further analysed 93.8%

I: Sample 1

II: Sample 2

Table 8. Extraction of total radioactivity in soil treated with 14C-labelled test substance at an average high dose level of 247.4 μg/kg (values in percentage of the radioactivity recovered in the soil before (A) and after normalization (B)).

(A) 

Phase	Days of exposure (duplicates)
	0		7	14	21
	I	II*	I	I	I
Acetonitrile Acetonitrile Methanol Methanol/water (8:1; v/v) Methanol (reflux extraction)	58.4 32 12.2 -- --	58.7 30.9 9.8 -- --	53.6 26.6 10.5 2.4 3.4	58.2 29.2 9.6 2.3 3.4	63.1 24.7 8.1 2.8** 2.0**
Subtotal organic	102.6	99.4	96.5	102.7	100.7
Non-extractable*	6.6	5.0	0.3	0.4	0.4
TOTAL	109.2	104.4	96.8	103	101.1

(B)

Phase	Days of exposure (duplicates)
	0		7	14	21	Mean
	I	II*	I	I	I
Acetonitrile Acetonitrile Methanol Methanol/water (8:1; v/v) Methanol (reflux extraction)	53.5 29.3 11.2 -- --	56.2 29.6 9.4 -- --	55.4 27.5 10.8 2.5 3.5	56.5 28.3 9.3 2.2 3.3	62.4 24.4 8.0 2.8** 2.0**
Subtotal organic	94.0	95.2	99.7	99.6	99.6	97.6 ± 2.8
Non-extractable*	6.0	4.8	0.3	0.4	0.4	2.4 ± 2.8
TOTAL	100	100	100	100	100

-- Not performed

* Not further analysed

** Not pooled, consequently further analysed 93.8%

I: Sample 1

II: Sample 2

Table 9. HPLC analysis of soil extracts from the low dose (A) and high dose (B) exposure concentration (values in percentage of the radioactivity recovered in the soil).

(A) Low dose

Code radioactive fraction	Identity	HPLC Retention time (min.)	Days of exposure
			0	7	14	21
S1   S3	Parent   M6	17.2-17.6   16.6	91.1   2.3	98.2   n.d.	99.2   n.d.	93.8   n.d.
SUBTOTAL			93.5	98.2	99.2	93.8

 

(B) High dose

Code radioactive fraction	Identity	HPLC Retention time (min.)	Days of exposure
			0	7	14	21
S1	Parent	17.2-17.6	94.0	99.0	99.0	93.9
S2*	Unknown	17.0-17.1	n.d.	0.7	n.d.	n.d.
S4	M4	15.5	n.d.	n.d.	0.6	1.0
SUBTOTAL			94.0	99.7	99.6	94.9

* Already present in the stock solution in an amount of 0.4%.

n.d. not detected

Validity criteria fulfilled:

yes

Conclusions:

In a bioaccumulation of the substance in earthworm study, following the first draft of OECD TG 317, the mean BAF was calculated to be 0.12 ± 0.02 and 0.13 ± 0.01 for 0.026 and 0.26 mg/kg soil dw test concentrations, respectively. Thus, no bioaccumulation (BAF < 1.0) of the test item in the tested earthworm.

Executive summary:

The bioaccumulation potential of the 14C-dichlorophenyl ring-labelled test substance was investigated in earthworm, Eisenia fetida, in accordance with the draft OECD TG 317 and in compliance with GLP criteria. In this study, the earthworms were exposed to radiolabelled test substance (mixed with artificial soil which includes food for the earthworms) at concentrations of 0.026 and 0.26 mg/kg dry soil for 21-day (uptake phase, 125 earthworms per concentration) and 21 days depuration period. In addition, a solvent control (acetone) group (5 earthworms per replicate, 7 replicates in total) was included in the study as well and tested under the same test condition. The test condition was as following: 19 - 22 °C, pH 6.0 – 6.2, 40% moisture and 16 hours light/8 hours darkness cycle (light intensity within 520 – 680 Lux). During the uptake phase, measurements of test item concentrations (total radioactivity) were taken on Day 1, 2, 4, 7, 14, and 21. During the elimination phase, the measurements of the total radioactivity was performed in samples of Day 22, 23, 25, 28, 35, and 42 of the study (i.e. Day 1, 2, 4, 7, 14, and 21 of the elimination phase). LSC and HPLC were used for analysing the total radioactivity and identifying the degraded substances.

The results show that the mean measured concentrations of radioactivity in the treated soils during the uptake phase were 0.027 and 0.25 mg/kg dry soil at the low and high test concentration, respectively. Analyses of radioactivity in the treated soils during the uptake phase showed almost exclusively parent test item (91 - 99% of the radioactivity recovered) for both test concentrations. Thus, the test item in the soil was stable during the uptake phase of 21 days at both test concentrations. A fast increase of radioactivity was determined in the earthworms within the first day of the uptake phase. Then, no further increase of the concentration of radioactivity was determined during the uptake phase. The concentrations of radioactivity in the earthworms incubated during the uptake phase at the low test concentration were in the range of 2.4 to 3.8 μg/kg worm. At the high test concentration, concentrations of radioactivity in the range of 28.0 and 37.6 μg/kg worm were measured during the uptake phase. The mean measured concentrations in the worms during the uptake phase (arithmetic mean value of the measurements over the whole uptake phase) were 3.2 and 31.6 μg/kg worm for the low and the high test concentration, respectively. During the elimination phase, a fast decrease of the radioactivity concentration in the worms was observed. The measured concentrations were below the detection limit after 7 days elimination (Day 28) and after 14 days elimination (Day 35) for the low and the high test concentration, respectively. The mean BAF was calculated to be 0.12 ± 0.02 and 0.13 ± 0.01 for 0.026 and 0.26 mg/kg soil dw test concentrations, respectively. Thus, no bioaccumulation (BAF < 1.0) of the test item in the tested earthworm.

Description of key information

BAF = 0.12 and 0.13 (for 0.026 and 0.26 mg/kg soil dw treatments, respectively), earthworm, Eisenia fetida, artificial soil, draft OECD TG 317, Bätscher 2004

Key value for chemical safety assessment

BCF (terrestrial species):

0.13 dimensionless

Additional information

There is one OECD TG 317 (draft) followed and GLP complied study available for this endpoint. The 14C-Dichlorophenyl ring-labelled test substance was applied to earthworm, Eisenia fetida, at concentrations of 0.026 and 0.26 mg/kg dry soil for 21-day (uptake phase, 125 earthworms per concentration) and 21 days depuration period. In addition, a solvent control (acetone) group (5 earthworms per replicate, 7 replicates in total) was included in the study as well and tested under the same test condition. The test condition was as following: 19 - 22 °C, pH 6.0 – 6.2, 40% moisture and 16 hours light/8 hours darkness cycle (light intensity within 520 – 680 Lux). During the uptake phase, measurements of test item concentrations (total radioactivity) were taken on Day 1, 2, 4, 7, 14, and 21. During the elimination phase, the measurements of the total radioactivity was performed in samples of Day 22, 23, 25, 28, 35, and 42 of the study (i.e. Day 1, 2, 4, 7, 14, and 21 of the elimination phase). LSC and HPLC were used for analysing the total radioactivity and identifying the degraded substances. The results show that the mean measured concentrations of radioactivity in the treated soils during the uptake phase were 0.027 and 0.25 mg/kg dry soil at the low and high test concentration, respectively. The mean BAF was calculated to be 0.12 ± 0.02 and 0.13 ± 0.01 for 0.026 and 0.26 mg/kg soil dw test concentrations, respectively. Thus, no bioaccumulation (BAF < 1.0) of the test item was observed in the tested earthworm.