N-phenyl-N-[(trichloromethyl)thio]benzenesulphonamide

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

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:

2016

Deviations:

no

Principles of method if other than guideline:

The first experiment was conducted as a limit study with one test concentration only. As the No Observed Effect Concentration (NOEC) could not be determined for reproduction in the first experiment, a second experiment was conducted with lower test concentrations in a dose response design to achieve a NOEC.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Vehicle:

no

Details on preparation and application of test substrate:

The test item was weighed separately for each concentration using an analytical balance and 10 g of fine quartz sand was added to each weighing. After establishing a homogeneous distribution of the test item within the sand by using mortar and pestle and mixing with a spoon the mixture was added to artificial soil equivalent to 2090 g dry weight.
The control was treated with the same amount of untreated sand per g substrate as the test item groups. While mixing the artificial soil in a laboratory mixer for approximately 5 minutes the soil of each treatment group was moistened with deionised water. In the 1st experiment the treatment groups were treated in two batches, which were then split into the replicates. In the 2nd experiment each group was treated in one batch (two in the control) which was then split into the replicates.

Test organisms (species):

Eisenia andrei

Animal group:

annelids

Details on test organisms:

Earthworm (Eisenia andrei), adult earthworms, approximately 8 months (1st experiment) and approximately 10 months (2nd experiment) months, with well-developed clitellum, age range between test individuals not differing by more than 4 weeks

Body weight:
300 to 583 mg (1st experiment)
335 to 600 mg (2nd experiment)

source: Bred under standardised conditions at ibacon laboratories in a breeding medium of cattle manure, peat, sand, calcium carbonate and straw, fed with cattle manure, stored at room temperature.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Test temperature:

18 - 22 °C

pH:

5.7 - 6.8

Moisture:

51.4 - 60.2 % of maximum water holding capacity (WHC)

Details on test conditions:

Test conditions
- Test Environment: Controlled environment room, in a ventilated area
- Temperature: Within the range of 18 °C to 22 °C
- Light Intensity: Within the range of 400 lux to 800 lux
- Light Regime: 16 h light : 8 h dark
- Water Content Maintenance: Once a week the water content of the soil was checked by weighing each container and evaporated water was replenished. In this way, it was ensured that the difference in water content between experimental start and end was less than 10%.
- pH: pH was determined at the start and end of the test for each treatment group, according to ISO 10390 (CaCl2).
1st experiment:
pH range at experimental start: 5.9 to 6.0, and thus did not deviate from the values given by the guidelines for initial pH (6.0 ± 0.5)
pH range at experimental end: 6.8
2nd experiment:
pH range at experimental start: 5.7 to 5.9, and thus did not deviate from the values given by the guidelines for initial pH (6.0 ± 0.5)
pH range at experimental end: 6.5
- Water Content: Water content was determined at the start and end of the experiment for each test concentration, according to ISO 11465. Therefore, one soil sample was taken from one replicate of each test item concentration. For the control soil samples from two replicates were taken and the mean of the two samples is reported.
At experimental start: 19.5% to 19.6% (51.4% to 51.6% of the maximum water holding capacity, i.e. within the recommended range 40 60 % of the total water holding capacity).
At experimental end: 21.3% to 21.4% (56.0% to 56.4% of the maximum water holding capacity)
2nd experiment:
At experimental start: 20.2% to 20.6% (53.2% to 54.2% of the maximum water holding capacity, i.e. within the recommended range 40 60 % of the total water holding capacity).
At experimental end: 19.9% to 22.9% (52.3% to 60.2% of the maximum water holding capacity)
- Test Substrate: Artificial Soil: Based on OECD 222 but with reduced organic matter content:
• 5% Sphagnum-peat, air-dried and finely ground (<2 mm, with no visible plant remains); (Floragard, Vertriebs GmbH für Gartenbau, 26138 Oldenburg, Germany)
• 20% Kaolin clay (Kaolinite content >30%) (Erbslöh, 65558 Lohrheim, Germany)
• 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)
• 0.2% Calcium carbonate (CaCO3) was added to adjust pH to 6.0 ± 0.5 (Merck, 64293 Darmstadt, Germany).
According to OECD 222, 5% peat was used in the artificial soil considering a potential influence 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.

Food
- Feeding: Air dried and finely ground cattle manure was used as food. 5 g/container was scattered on the soil surface at day 1 after application and was moistened with 5 g deionised water; 5 g/container (moistened with 2 g deionised water) was added each week for the first 4 weeks of the experiment, when the food of the previous week had almost been consumed. If the food was not quite fully consumed, the added amount of food was adjusted to replace the visually estimated consumption. Four weeks after application, the food was mixed into the substrate following removal of the adult earthworms.
- Food Source: Cattle manure is collected at an organic farm, the cows are not treated with antibiotics or worm treatments. Cattle manure is dried and grinded at ibacon.

Course of the Test
- No. of Replicates: 1st experiment: 8 per control and test item treated group
2nd experiment: 8 per control, 4 per test item treated group
- No. of Individuals: 1st experiment: 10 per replicate, i.e. 80 individuals per treatment group
2nd experiment: 10 per replicate, i.e. 80 individuals per control, 40 individuals per test item treated group
- Introduction of Individuals: All earthworms were rinsed with tap water, dried with dry paper towels, weighed individually and randomly assigned to batches of 10 earthworms. The different batches were sorted into four classes on the basis of the total weight. In the 1st experiment two batches of each weight class were assigned to each treatment group, in the 2nd experiment one batch 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.
- Test Duration: 8 weeks
- Exposure Time of Adult Earthworms: After 4 weeks, the artificial soil was transferred to a tray and adult earthworms were counted, removed and weighed per replicate after being rinsed under tap water and dried on paper towels. The remaining soil (without the adult earthworms) 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 earthworms. In addition, the soil of each container was emptied out onto a tray and checked visually for any remaining juvenile earthworms.

Test Parameters
- Mortality: Number of dead adult earthworms at day 28 after application (including any missing ones)
- Behavioural and Morphological Abnormalities: Number of affected adult earthworms (e.g. lack of movement, rigidity) at day 28 after application
- Food Given: Cumulative amount of food added to each test container during the test period
- Mean Body Weight: Body weights were determined at start (day 0) and 28 days after application
- Reproduction: Number of juveniles 56 days after application

Nominal and measured concentrations:

Nominal concentrations: 1000, 833, 694, 579, 482, 402 mg test item/kg soil dw + control

Reference substance (positive control):

yes

Remarks:

Carbendazim; the effects of the reference item were investigated in a separate GLP study

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

482 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Results with reference substance (positive control):

In the most recent test with the reference item Carbendazim (performed under ibacon Study No. 105687022 from January to March 2021), there were statistically significant effects on reproduction at a concentration of 0.694 mg a.i./kg soil dry weight and above, which is in line with the guideline OECD 222 (effects should be observed between 1 and 5 mg a.i./kg soil dry weight). The EC50 for reproduction was calculated as 0.81 mg a.i./kg soil dry weight.

Reported statistics and error estimates:

In both experiments, mortality data were analysed for significance by using the Fisher’s Exact Binomial Test (two-sample comparison in the 1st experiment, multiple comparison, with Bonferroni Correction in the 2nd experiment, α = 0.05, one-sided greater). The LC50 at day 28 was not determined by statistical analysis as no mortality above 50% was observed.
The body weight change and reproduction data were tested for normal distribution and homogeneity of variance (α = 0.01) using the Shapiro-Wilk´s test and the Levene´s test in both experiments. As the data for body weight changes and reproduction were normally distributed and homogeneous in all cases of both experiments, Student t-test was used to compare treatment and control values in the 1st experiment (two-sample comparison, two-sided for weight and one-sided smaller for reproduction, α = 0.05). In the 2nd experiment, Dunnett’s t-test was used to compare treatment and control values for body weight changes (multiple comparison, two-sided, α = 0.05) and Williams t-test was used to compare treatment and control values for reproduction (multiple comparison, one-sided smaller, α = 0.05) since the reproduction values did follow a monotonicity trend. The EC50 was not determined by statistical analysis as no mortality above 50% was observed.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ToxRat® Solutions GmbH.

Validity criteria fulfilled:

yes

Remarks:

Control Mortality: Should not exceed 10% over initial 4-week test period; Reproduction of Control: Should be ≥ 30 earthworms per replicate container; Coefficient of Variation of Reproduction in Control: Should not exceed 30%

Conclusions:

In an earthworm reproduction and growth study with the test item the No Observed Effect Concentration (NOEC) for mortality and growth of the earthworm Eisenia andrei was determined to be ≥1000 mg test item/kg soil dry weight, i.e. the highest concentration tested. The Lowest Observed Effect Concentration (LOEC) for mortality and growth was determined to be >1000 mg test item/kg soil dry weight. The NOEC for reproduction was determined to be 482 mg test item/kg soil dry weight and the LOEC for reproduction was determined to be 579 mg test item/kg soil dry weight. The LC50 and the EC50 values were estimated to be >1000 mg test item/kg soil dry weight.

Executive summary:

A study was conducted to assess the toxicity of the test item towards the reproduction and growth of earthworms according to OECD 222 for a 56-day period in treated artificial soil. The first experiment was conducted as a limit study with one test concentration only (1000 mg/kg soil dw). As the No Observed Effect Concentration (NOEC) could not be determined for reproduction in the first experiment, a second experiment was conducted with lower test concentrations in a dose response design to achieve a NOEC.

Adult earthworms (Eisenia andrei), approximately 8 and 10 months old, were exposed to 6 different concentrations of the test item incorporated into the soil (control, 402, 482, 579, 694, 833 and 1000 mg test item/kg soil dw). 4 replicates were prepared for the test item treatments (402, 482, 579, 694 and 833 test item/kg soil dw) and 8 replicates for the control and 1000 mg test item/kg soil dw with 10 earthworms each. The earthworms were fed wit cattle manure from an organic farm.

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

A slight mortality of 2.5% and was found at the concentration of 833 mg test item/kg soil dry weight, which was not statistically significantly different compared to the control, where no earthworm died (Fisher’s Exact Test, a = 0.05, one-sided greater).

The body weight changes of the earthworms after 4 weeks exposure to the tets item were not statistically significantly different compared to the control up to and including the highest test concentration of 1000 mg test item/kg soil dw (Student t-test, α = 0.05, two-sided in the 1^st experiment and Dunnett’s t-test, α = 0.05, two-sided in the 2^nd experiment).

The reproduction rates were not statistically significantly different compared to the control up to and including the test concentration of 482 mg test item/kg soil dry weight (Williams t-test, a = 0.05, one-sided smaller). At the concentration of 579 mg test item/kg soil dry weight and above, reproduction was statistically significantly different compared to the control group (Student t-test, α = 0.05, one-sided smaller in the 1^st experiment and Williams t-test, α = 0.05, one-sided smaller in the 2^nd experiment). No behavioural abnormalities were observed in any of the treatment groups. The feeding activity in all the treated groups was comparable to the control.

The No Observed Effect Concentration (NOEC) for mortality and growth of the earthworm Eisenia andrei was determined to be ≥1000 mg test item/kg soil dw, i.e. the highest concentration tested.

The NOEC for reproduction was determined to be 482 mg test item/kg soil dry weight and the LOEC for reproduction was determined to be 579 mg test item/kg soil dry weight.

The LC₅₀ and the EC₅₀ values were estimated to be >1000 mg test item/kg soil dry weight.

Description of key information

A study was conducted to assess the toxicity of the test item towards the reproduction and growth of earthworms according to OECD 222 for a 56-day period in treated artificial soil. The first experiment was conducted as a limit study with one test concentration only (1000 mg/kg soil dw). As the No Observed Effect Concentration (NOEC) could not be determined for reproduction in the first experiment, a second experiment was conducted with lower test concentrations in a dose response design to achieve a NOEC.

Adult earthworms (Eisenia andrei), approximately 8 and 10 months old, were exposed to 6 different concentrations of the test item incorporated into the soil (control, 402, 482, 579, 694, 833 and 1000 mg test item/kg soil dw). 4 replicates were prepared for the test item treatments (402, 482, 579, 694 and 833 test item/kg soil dw) and 8 replicates for the control and 1000 mg test item/kg soil dw with 10 earthworms each. The earthworms were fed wit cattle manure from an organic farm.

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

A slight mortality of 2.5% and was found at the concentration of 833 mg test item/kg soil dry weight, which was not statistically significantly different compared to the control, where no earthworm died (Fisher’s Exact Test, a = 0.05, one-sided greater).

The body weight changes of the earthworms after 4 weeks exposure to the tets item were not statistically significantly different compared to the control up to and including the highest test concentration of 1000 mg test item/kg soil dw (Student t-test, α = 0.05, two-sided in the 1^st experiment and Dunnett’s t-test, α = 0.05, two-sided in the 2^nd experiment).

The reproduction rates were not statistically significantly different compared to the control up to and including the test concentration of 482 mg test item/kg soil dry weight (Williams t-test, a = 0.05, one-sided smaller). At the concentration of 579 mg test item/kg soil dry weight and above, reproduction was statistically significantly different compared to the control group (Student t-test, α = 0.05, one-sided smaller in the 1^st experiment and Williams t-test, α = 0.05, one-sided smaller in the 2^nd experiment). No behavioural abnormalities were observed in any of the treatment groups. The feeding activity in all the treated groups was comparable to the control.

The No Observed Effect Concentration (NOEC) for mortality and growth of the earthworm Eisenia andrei was determined to be ≥1000 mg test item/kg soil dw, i.e. the highest concentration tested.

The NOEC for reproduction was determined to be 482 mg test item/kg soil dry weight and the LOEC for reproduction was determined to be 579 mg test item/kg soil dry weight.

The LC₅₀ and the EC₅₀ values were estimated to be >1000 mg test item/kg soil dry weight.

Key value for chemical safety assessment

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

482 mg/kg soil dw