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Toxicity to soil macroorganisms except arthropods

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toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
56 days
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline test with GLP certificate

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
GLP compliance:
yes (incl. certificate)

Test material

Test material form:
other: Liquid
Details on test material:
Active Ingredient(s) / Purity: 84.69%
Certificate of Analysis Date: March 31, 2011
Certificate of Analysis Reference Code: R0004
Manufacturing Date: March 30, 2011
Aggregate State at Room Temperature: Liquid
Colour: Colourless to light yellow (according to MSDS)
Density: 1.25 g/cm³ (at 25°C) (according to MSDS)
Solubility: In water: 0.415 mg/L (according to MSDS)
Expiry Date: March 29, 2012
Storage: At room temperature (20 ± 5 °C), in the dark

Sampling and analysis

Analytical monitoring:
Details on sampling:
not sampled

Test substrate

Details on preparation and application of test substrate:
Artificial Soil: Based on OECD 222 but with reduced organic matter content:
• 5% Sphagnum-peat, air-dried and finely ground (2 mm); (Floragard, Vertriebs GmbH für Gartenbau, 26138 Oldenburg, Germany)
• 20% Kaolin clay (Erbslöh, 65558 Lohrheim, Germany)
• approximately 0.2% calcium carbonate (CaCO3) added to adjust pH to 6.0 ± 0.5 (Merck, 64293 Darmstadt, Germany)
• approximately 74.8% fine quartz-sand (F34) containing more than 50% by mass of particle size 0.05 mm to 0.2 mm; (Quarzwerke Frechen, Postfach 1780, 50207 Frechen, Germany)
According to OECD 222 and EPPO (2003), 5 % of peat was used in the artificial soil considering the influence of the properties of the test item on bioavailability.
The artificial soil was moistened to approximately half of the final water content 1 day before the application. The additional water required to achieve the final water content was added when applying the test item.
Maximum Water Holding Capacity of the Artificial Soil: 42% of the dry weight

Application: A stock solution was prepared by dissolving 8120 mg of BDP in 20 mL acetone. Then two batches were prepared by adding 5 mL of the stock solution to 20 g fine quartz sand. The treated sand was left for at least one hour in a fume hood until the solvent had evaporated. Then artificial soil equivalent to 2030 g dry weight (total amount of substrate 2050 g) was added to test item blended sand, resulting in the nominal concentration of 1000 mg test item/kg soil.
There were no significant deviations to the nominal target concentration (< 5%). The control was treated with the same amount of acetone and sand as the test item groups. While mixing the artificial soil in a laboratory mixer for approximately 5 min the soil of each treatment group was moistened with deionised water. Each group was treated in two batches which were then split into the replicates.

Test organisms

Test organisms (species):
Eisenia fetida
Animal group:
Details on test organisms:
Age: 11 to 12 months, with well-developed clitellum, age range between test individuals not differing by more than 4 weeks
Body Weight: 340 mg to 600 mg
Origin: Bred under standardised conditions (see OECD 207, in a breeding medium of cattle manure, peat, sand, calcium carbonate and straw, fed with cattle manure, stored at room temperature) by IBACON
Acclimatisation: 1 day, in artificial soil, under test conditions.

Study design

Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
Total exposure duration:
56 d
Post exposure observation period:
no data

Test conditions

Test temperature:
Within the range of 18 °C to 22 °C
pH was checked at the start and end of the test for each test treatment group, according to ISO 10390 (CaCl2).
pH at experimental start: 5.7 and thus did not deviate from the values given by the guidelines (6.0 ± 0.5)
pH at experimental end: 6.4
Water content was checked at the start and end of the experiment for each test concentration, according to ISO 11465.
At experimental start: 20.7% to 22.0% (49.2% to 52.4% of the maximum water holding capacity)
At experimental end: 24.2% to 24.8% (57.5% to 59.0% of the maximum water holding capacity)
Details on test conditions:
No. of Replicates: 8 per control and per test item treated group
No. of Individuals: 10 per replicate, i.e. 80 individuals per control and per test item treated group
Introduction of Individuals: All worms were washed with tap water, dried with dry paper towels, weighed individually and randomly assigned to batches of 10 worms. The different batches were sorted into four classes on the basis of the total weight and two batches of each weight class was assigned to each treatment group (two batches for the control) to ensure weights were homogeneous. The earthworms were placed on the surface of the artificial soil after application.

Exposure Time of Adult Worms: After 4 weeks, the artificial soil was transferred to a tray and adult worms were counted, removed and weighed per replicate after they had been washed under tap water and dried on paper towels. Missing earthworms and earthworms that failed to respond to gentle stimulation were considered to be dead. The remaining soil (without the adult worms) was then returned to the respective test containers.

Exposure Time of Offspring: Additional 28 days

Removal of Juveniles: Juveniles were removed by placing the test units in a water bath at 50 - 60 °C and counting all emerging worms. In addition the soil of each container was emptied out onto a tray and checked visually for any remaining young worms.
Nominal and measured concentrations:
Nominal: 1000 mg/kg dw
BDP was determined to be stable during the test, so only nominal concentration was used in the test.

Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
4 wk
Dose descriptor:
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
4 wk
Dose descriptor:
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
8 wk
Dose descriptor:
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Details on results:
No mortality was observed in the treatment group or the control for adult worms. The NOEC for mortality was determined to be above 1000 mg test item/kg soil. The body weight changes in the test item treated group was not significantly different compared to the control (Student t-test, a = 0.05, two-sided). The NOEC for body weight changes was determined to be above 1000 mg test item/kg soil.
The reproduction rates were not significantly different compared to the control in the test item treated group (Student t-test, a = 0.05, one-sided smaller). The NOEC for reproduction was determined to be above 1000 mg test item/kg soil.
Results with reference substance (positive control):
In the most recent test with the reference item Luxan Carbendazim 500 FC (performed under IBACON Study Number 46643022 from October 2010 to December 2010), there were statistically significant effects on reproduction at a concentration of 1.0 mg carbendazim/kg soil and higher; the EC50 for reproduction was calculated as 1.21 mg carbendazim/kg soil. The results indicated that the sensitivity of the worms was consistent with the level proposed by the OECD 222 guideline (significant effects between 1 and 5 mg carbendazim/kg soil).
Reported statistics and error estimates:
The body weight change and reproduction data were tested for normal distribution and homogeneity of variance (a = 0.05) using the Shapiro-Wilk´s test and the Levene´s test, respectively).
As the data for body weight changes and reproduction were normally distributed and homogeneous in both cases, Stundent t-test was used to compare treatment and control values (multiple comparison, two-sided for weight and one-sided smaller for reproduction, a = 0.05).
The software used to perform the statistical analysis was ToxRat Professional, Version 2.10.05, ® ToxRat Solutions GmbH.

Applicant's summary and conclusion

Validity criteria fulfilled:
Fulfilled all validity criteria
In an earthworm reproduction and growth study with BDP the No Observed Effect Concentration (NOEC) for mortality, growth, reproduction and feeding activity of the earthworm Eisenia fetida was determined to be 1000 mg test item/kg soil, i.e. the highest concentration tested.
Executive summary:

Test Item:

BDP; batch no.: 20110330; purity: 84.69%

Test Species:

Earthworm (Eisenia fetida), adult worms (with clitellum and weight range 340 to 600 mg), 11 to 12 months old, source: from an in-house culture.

Test Design:

56-day test in treated artificial soil prepared according to OECD 222 (5% peat only); one concentration of the test itemwas incorporated into the soil;2 treatment groups (one test item concentration, control); 8 replicates for the test item treatment and 8 replicates for the control with 10 worms each.

Assessment of adult worm mortality, behavioural effects and biomass development was carried out after 28 days exposure of adult worms in treated artificial soil. Reproduction rate (number of offspring) was assessed after additional 28 days (assessed 56 days after application).

BDP did not show effects on mortality and body weight of the earthworms at the concentration of 1000 mg test item/kg soil (Student t-test,a= 0.05).No statistically significant effects on reproduction were observed at 1000 mg test item/kg soil (Student t-test,a =0.05). No behavioural abnormalities were observed and the feeding activity in all the treated groups was comparable to the control.