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

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 June 2002 - 22 July 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

AMENDMENT OF SOIL
- Type of organic substrate: Soil samples (100 g dry weight equivalent) were amended with glucose to stimulate respiration.
The soils for nitrogen transformation were amended with a source of nitrogen consisting of 1 mm sieved ground lucerne at a rate of 0.5 % w/w. The lucerne had a total carbon content of 42.73 % w/w and a total nitrogen content of 2.96 % w/w giving a C:N ratio of 14.4:1. The soils for the respiration test were not amended with lucerne.

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: One day prior to the start of the test, three 50.00 g amounts of quartz sand were weighed out into separate 500 mL dark glass bottles. Two stock solutions were then prepared by weighing 6.63 and 33.21 mg of the test material for the 1x and 5x PEC treatments, respectively. These amounts were then made up separately to 100 mL using HPLC grade acetone. A single 1 mL aliquot of each stock was then taken and made up to 20 mL using HPLC grade acetone. One 20 mL solution of acetone only was prepared for the control sand. These 20 mL solutions were then evenly poured over the designated sand and mixed. Once mixed, the unopened bottles were placed under a sample concentrator for between 60 and 160 minutes in order to evaporate the acetone. The bottles were then sealed and left overnight.
Prior to starting the test the sealed bottles were placed on a roller at approximately 60 rpm for 30 minutes in order to homogenise the sand.
Nominally 10 g amounts of each treated sand, per 1000 g dry weight of soil, were then weighed out for each treatment: 0 (control), 1x PEC and 5x PEC, evenly distributed over the soil and well mixed. The quantity of water required to bring the soil to 50 % of its maximum water holding capacity (MWHC) was then added and again well mixed.

VEHICLE:
- Chemical name of vehicle: Acetone (HPLC Grade)
- Evaporation of vehicle before use: Yes

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20 ± 2 °C

Moisture:

47.5 to 49.7 % MWHC

Details on test conditions:

TEST SYSTEM
- Test container (type, material, size): The storage vessels used for both the soil nitrogen transformation and soil respiration tests were 2.5 L square plastic containers and perforated with a single 3 mm hole to allow gaseous exchange.
- Amount of soil: 500 and 1000 g (dry weight equivalent) amounts of soils per treatment were weighed out for nitrogen transformation and soil respiration, respectively.
- No. of replicates per concentration: 3
- No. of replicates per control: 3

SOIL INCUBATION
- Method: The soil samples were incubated for 28 days at 25 ± 2 °C in the dark

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site: The soil used in this study was collected on 25 March 2002 from a set-aside area of an organic arable field (Club Field) at Waltham Place Estate, Church Hill, White Waltham, Berkshire, SL6 3JH, UK. OS Map reference (UK): Landranger Sheet 175 (854 771)
- History of site: Club Field had not been cropped for one year (2002), one year previously was field beans (2001) following two years of red clover and rye grass ley (1999 and 2000).
- Treatments with pesticides or fertilizers: No pesticide, crop protection products, organic or inorganic fertilizers and biological material have been applied to the site of soil collection (Club Field) for at least five years.
- Depth of sampling: 5 to 20 cm
- Soil texture: 73 % sand
- Soil taxonomic classification: Sandy loam
- Soil classification system: UK textural classification
- pH (in water): 6.0
- Total nitrogen: 0.0119 % w/w
- Maximum water holding capacity (at 0.001 bar): 39.5 % w/w
- Cation exchange capacity (meq/100 g): 7.6
- Storage (condition, duration): Upon arrival the soil was spread out in lined plastic trays and left to gently air dry in the dark. The soil was periodically mixed to ensure an even drying. The soil was then passed through a 5 mm sieve to remove large debris. Final sieving through a 2 mm sieve was carried out between the 28 March 2002 and 04 April 2002. The soil was transferred to black plastic bags, loosely tied and stored in a Cold Room at ca. 4 °C. The soil was stored for 74 days before being acclimated for testing. Soils were acclimated at 20± 2 °C in the dark.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Extractions for nitrogen transformation and soil respiration measurements were made within the first six hours of dosing and again after 7, 14 and 28 days.

VEHICLE CONTROL PERFORMED: Yes

RANGE-FINDING STUDY: No

Nominal and measured concentrations:

- Nominal concentrations: 0, 1 x PEC (equivalent to 0.013 mg/kg dry soil), and 5 x PEC (equivalent to 0.065 mg/kg dry soil).

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.065 mg/kg soil dw

Basis for effect:

nitrate formation rate

Details on results:

SOIL MICROBIAL RESPIRATION
Soil microbial respiration in soil treated at the rates of 1x maximum predicted environmental concentration (PEC) and 5x PEC showed no deviation greater than 6.0 % when compared to the controls over the duration of the study, as shown in Table 1. This is below the 25 % limit as stated in OECD 217 (2000).
The total range of between-replicate variation for the controls for soil microbial respiration was -2.6 to +2.8 % during the study. The total range of between-replicate variation within respiration measurements was -4.3 to +8.4 % for the 1x PEC samples and -1.3 to +1.9 % for the 5x PEC samples.

SOIL NITROGEN TRANSFORMATION (concentrations and rates)
Soil nitrate-nitrogen transformation rates in soil treated at the rates of 1x maximum PEC and 5x PEC showed a deviation of 13 and 22 %, respectively, when compared to the controls between days 7 and 14. However, this deviation was reduced to a maximum of 2.3 % between days 14 and 28. By day 28 the greatest deviation was 2.3 % as shown in Table 2. This is below the 25 % limit as stated in OECD 217 (2000).
The total range of between-replicate variation for the controls for soil nitrate-nitrogen concentrations was -11 to +10 % during the study. The total range of between-replicate variation within soil nitrate-nitrogen concentrations was -8.1 to +10 % for the 1x PEC samples and -8.2 to +6.9 % for the 5x PEC samples.

VALIDITY OF THE STUDY
Evaluations of test results are based on relatively small differences between nitrogen and CO₂ concentrations in control and treated soil samples, so that variations in control measurements could lead to false results. OECD 216 (2000) and 217 (2000) state that the deviation between control replicate samples for the CO₂ and nitrate-nitrogen concentrations should be no greater than ± 15 %.
The range of percentage variation within the control samples for CO₂ concentrations was from -2.6 to +2.8 % and for nitrate-nitrogen concentrations was from -11 to +10 %. Therefore, control variation during the study for both soil CO₂ and nitrate-nitrogen concentrations waswithin the validity criteria of ± 15 %.

Reported statistics and error estimates:

Analysis of the test material treatments for soil respiration measurements (expressed as mean respiration rates as mg CO₂/kg/hour) and values for nitrate transformation rates (expressed as mg of N per kilogram dry weight equivalent of soil per day) was carried out using the Dunnett’s two-tailed test (at P = 0.05), to determine the location of statistically significant differences between treatments and controls using the computer program TOXSTAT 3.4 (1994).

Table 1: Percentage Variation in Mean CO₂ Relative to the Mean Control Value (%)

Sample time (days)	Concentration
	1x PEC	5x PEC
Day 0 (start)	+6.0	+2.0
Day 7	+1.3	+1.2
Day 14	+2.6	+2.3
Day 28 (end)	+2.2*	+1.2

* Statistically significant when compared with the controls (P = 0.05), tested using the two-tailed Dunnett’s test.

 

Table 2: Percentage Variation of Mean Nitrate-Nitrogen Transformation Rates Relative to the Mean Control Value (%)

Sample time (days)	Concentration
	1x PEC	5x PEC
Day 0 (Start) to 7	-5.5	+8.8
Day 7 to 14	-13	-22*
Day 14 to 28 (End)	+2.0	+2.3

* Statistically significant differences were found when compared with the controls (P = 0.05), tested using the two-tailed Dunnett’s test.

Validity criteria fulfilled:

yes

Conclusions:

No adverse effect of greater than 6.0 % compared to the untreated control soil was seen in the treatments up to and including the highest treatment tested (5x maximum predicted environmental concentration, equivalent to 0.065 mg/kg dry soil) for soil microbial respiration. By day 28 of the study the greatest deviation was 2.2 % from the controls. This is below the 25 % limit as stated in OECD 217 (2000).

Executive summary:

A study was conducted to determine the effects of the test material on soil microbial activity by the measurement of nitrogen transformations according to the standardised guideline OECD 216 and soil respiration according to the standardised guideline OECD 217 under GLP conditions.

Microbial activity was assessed using a single sandy-loam soil (73 % w/w sand content, 1.4 % w/w organic carbon and a microbial biomass of 2.55 % of the total organic carbon). The soil samples for nitrogen analysis were amended with 0.5 % w/w ground lucerne. The soil samples for soil respiration were not amended. Both soils were then acclimated to the test conditions for three days. At the start of the test, replicate samples were treated with the test material, to achieve concentrations equal to the 1x maximum predicted environmental concentration (PEC) (equivalent to 0.013 mg/kg dry soil) and 5x PEC (equivalent to 0.065 mg/kg dry soil). One set of samples was left untreated to serve as controls.

The soil samples were incubated for a period of 28 days for soil respiration and soil nitrogen transformation at a temperature of 20 ± 2 °C in the dark. Soil respiration measurements and extractions for nitrogen were made within six hours of dosing the soil. Further measurements of soil respiration and extractions for nitrogen determinations were carried out 7, 14 and 28 days after dosing the soil. The soil respiration and nitrogen phases were considered complete once a deviation of <25 % from the control mean was seen at both treatments.

Soil nitrate-nitrogen transformation rates in soil treated with the test material at the rates of 1x PEC and 5x PEC showed a deviation of 13 and 22 %, respectively, when compared to the controls between days 7 and 14. However, this deviation was reduced to a maximum of 2.3 % between days 14 and 28. By day 28 the greatest deviation seen in the treatments up to and including the highest treatment tested (5x PEC, equivalent to 0.065 mg/kg dry soil) for soil nitrate-nitrogen transformation rates was 2.3 %. This is below the 25 % limit as stated in OECD 217.

No adverse effect of greater than 6.0 % compared to the untreated control soil was seen in the treatments up to and including the highest treatment tested (5x maximum predicted environmental concentration, equivalent to 0.065 mg/kg dry soil) for soil microbial respiration. By day 28 of the study the greatest deviation was 2.2 % from the controls. This is below the 25 % limit as stated in OECD 217.

Description of key information

No adverse effect of greater than 6.0 % compared to the untreated control soil was seen in the treatments up to and including the highest treatment tested (5x maximum PEC, equivalent to 0.065 mg/kg dry soil) for soil microbial respiration. By day 28 of the study the greatest deviation was 2.2 % from the controls.

Additional information

A study was conducted to determine the effects of the test material on soil microbial activity by the measurement of nitrogen transformations according to the standardised guideline OECD 216 and soil respiration according to the standardised guideline OECD 217 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Microbial activity was assessed using a single sandy-loam soil (73 % w/w sand content, 1.4 % w/w organic carbon and a microbial biomass of 2.55 % of the total organic carbon). The soil samples for nitrogen analysis were amended with 0.5 % w/w ground lucerne. The soil samples for soil respiration were not amended. Both soils were then acclimated to the test conditions for three days. At the start of the test, replicate samples were treated with the test material, to achieve concentrations equal to the 1x maximum predicted environmental concentration (PEC) (equivalent to 0.013 mg/kg dry soil) and 5x PEC (equivalent to 0.065 mg/kg dry soil). One set of samples was left untreated to serve as controls.

The soil samples were incubated for a period of 28 days for soil respiration and soil nitrogen transformation at a temperature of 20 ± 2 °C in the dark. Soil respiration measurements and extractions for nitrogen were made within six hours of dosing the soil. Further measurements of soil respiration and extractions for nitrogen determinations were carried out 7, 14 and 28 days after dosing the soil. The soil respiration and nitrogen phases were considered complete once a deviation of <25 % from the control mean was seen at both treatments.

Soil nitrate-nitrogen transformation rates in soil treated with the test material at the rates of 1x PEC and 5x PEC showed a deviation of 13 and 22 %, respectively, when compared to the controls between days 7 and 14. However, this deviation was reduced to a maximum of 2.3 % between days 14 and 28. By day 28 the greatest deviation seen in the treatments up to and including the highest treatment tested (5x PEC, equivalent to 0.065 mg/kg dry soil) for soil nitrate-nitrogen transformation rates was 2.3 %. This is below the 25 % limit as stated in OECD 217.

No adverse effect of greater than 6.0 % compared to the untreated control soil was seen in the treatments up to and including the highest treatment tested (5x maximum predicted environmental concentration, equivalent to 0.065 mg/kg dry soil) for soil microbial respiration. By day 28 of the study the greatest deviation was 2.2 % from the controls. This is below the 25 % limit as stated in OECD 217.