Benzenesulfonamide, N-[2-[[(phenylamino)carbonyl]amino]phenyl]-

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:

2021-05-05 to 2021-10-18

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:

adopted July 29th 2016

Deviations:

no

Remarks:

no critical deviations

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) 2016/266 amending Regulation (EC) No. 440/2008, EU Method C.33 “EARTHWORM REPRODUCTION TEST (EISENIA FETIDA / EISENIA ANDREI)

Version / remarks:

adopted 07. Dec. 2015

Deviations:

no

Remarks:

no critical deviations

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Chemical name: N-{2-[(phenylcarbamoyl)amino]phenyl}benzenesulfonamide
- CAS no.: 215917-77-4
- EC no.: 806-543-7
- Molecular formula: C19H17N3O3S

Analytical monitoring:

no

Details on sampling:

not applicable

Vehicle:

yes

Remarks:

Acetone

Details on preparation and application of test substrate:

- Method of mixing into soil: Acetone was used to spike quartz sand which was mixed with the soil after evaporation of the solvent. A Stock solution containing 56.102 g/L in acetone was prepared (corresponds to 1000 mg/kg nominal). The whole artificial soil was divided into 3 portions of an appropriate amount. The required amount of test item was given to each portion using 88.0 g with test item spiked quartz sand per concentration. Afterwards it was well mixed.
- Controls: 500 g artificial soil was only mixed with quartz sand without test item for the blank control (88.0 g) and quartz sand spiked only with acetone (88.0 g) for the solvent control
- Chemical name of vehicle: Acetone
- Evaporation of vehicle before use: yes

Test organisms (species):

Eisenia andrei

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Source: bred in the laboratory
- Age at test initiation: 2 - 12 months with clitellum (adult animals)
- Weight at test initiation: 268 – 600 mg (range in this study)

ACCLIMATION
- Acclimation period: 24 hours
- Acclimation conditions: A small part of the final soil mixture mixed with horse manure (0.5 g per worm) was used for the acclimatisation of the animals used. For this, it was adjusted with demineralised water to a water content of 40 % to 60 % of the maximum water holding capacity calculated in relation to dry matter.
- Health during acclimation: no mortality reported

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

28 d

Test temperature:

19.0 – 22.1 °C

pH:

7.3 on day 0 and 6.5 on day 56 (for the treatment)

Moisture:

58.72 % of water holding capacity (WHC )on day 0 and 53.70% of WHC on day 56

Details on test conditions:

TEST SYSTEM
- Test container: glass preserving jars, nominal volume 1 L
- Amount of soil or substrate: 500 g artificial soil plus 88.0 g quartz sand
- No. of organisms per container: 10
- No. of replicates per treatment group: 8
- No. of replicates per control: 8
- No. of replicates per vehicle control: 8

COMPOSITION OF ARTIFICIAL SOIL
Sphagnum Peat: 10 % of dry matter of artificial soil; air dried before use
Kaolin Clay: 20 % of dry matter of artificial soil
- Bolus alba (white clay), pharmaceutical quality
- Quartz Sand: round grains, 69.5 % of dry matter of artificial soil
- Calcium Carbonate: 0.5 % used for pH adjustment
- Horse manure: air dried, finely ground and pasteurised.
The horse manure used during this test was taken from animals which are not subject to medication or treatment with substances, such as growth promoters, nematicides or similar veterinary products that could adversely affect the worms during the test.
Additionally, the horse manure was checked in a separate 2-week-test for its influence on worm growth and cocoon production before usage in the test.


OTHER TEST CONDITIONS
- Photoperiod: 16 hours light / 8 hours dark
- Light intensity: 673 lux

EFFECT PARAMETERS MEASURED:
- Mortality: after 28 days
- Number of juveniles: after 56 days
- Weight change: after 28 days

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable (limit test)
- Justification for using less concentrations than requested by guideline:
- Range finding study: The concentration to be tested was based on the result of a
non-GLP pre-test
- Results used to determine the conditions for the definitive study: yes

Nominal and measured concentrations:

Nominal: 1000 mg/kg; measured values not available

Reference substance (positive control):

yes

Remarks:

Benomyl (CAS-No. 17804-35-2)

Key result

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:

reproduction

Key result

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

28 d

Dose descriptor:

LC50

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:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Mortality at end of exposure period: no
- Changes in body weight of live adults (% of initial weight) at end of exposure period: no significant differences between treatment and negative control
- No. of offspring produced: 132 (mean for treatment after 56 days)
- No. of unhatched cocoons: not reported
- Morphological abnormalities: not reported
- Behavioural abnormalities: not reported
- Other biological observations: not reported

Results with reference substance (positive control):

The 56d-EC50 of Benomyl (CAS-No. 17804-35-2) was tested in a current reference test. The value was determined as 1.86 mg/kg dry soil, lying within the required range of the Guidelines of 1 – 5 mg/kg.

Table1: Number of juveniles produced after 56 days

Concentration in mg/kg	Vessel No.	Number of Juvenile Worms after 56 Days	Mean Number of Juvenile Worms after 56 Days
Blank control	1	98	104
	2	69
	3	97
	4	105
	5	174
	6	107
	7	88
	8	95
Solvent control	9	111	118
	10	121
	11	112
	12	98
	13	91
	14	135
	15	130
	16	144
1000	17	151	132
	18	108
	19	92
	20	90
	21	146
	22	159
	23	148
	24	161

 

 

Table 2: Weight change adult worms in 28 days

Concentration in mg/kg	Vessel No.	Total Weight Day 0 in g	Total Weight Day 28 in g	Weight Change after 28 Days in %	Mean Weight Day 28 in mg
Blank control	1	4.289	4.609	7	4.391
	2	4.055	4.383	8
	3	4.139	4.401	6
	4	4.211	4.410	5
	5	4.041	4.424	9
	6	4.159	4.460	7
	7	3.891	4.324	11
	8	3.935	4.118	5
Solvent control	9	4.193	4.529	8	4.714
	10	4.214	4.534	8
	11	4.814	4.771	-1
	12	4.216	4.571	8
	13	4.374	4.720	8
	14	4.163	4.785	15
	15	4.573	4.851	6
	16	4.647	4.953	7
1000	17	4.336	4.893	13	5.147
	18	4.384	5.367	22
	19	4.178	5.118	23
	20	4.138	4.817	16
	21	4.243	5.037	19
	22	4.162	5.393	30
	23	4.174	5.195	24
	24	4.435	5.353	21

 

 

Table 3: Detailed Data Water Content Day 0

Concentration in mg/kg	Weight [g] empty glass vessel	Weight [g] wet soil with glass vessel	Weight [g] dry soil with glass vessel
Blank control	40.6957	88.7781	74.7132
Solvent control	54.5462	110.4884	93.8994
1000	52.0131	100.6807	85.0768

 

Table 4: Detailed Data Water Content Day 56

Concentration in mg/kg	Weight [g] empty glass vessel	Weight [g] wet soil with glass vessel	Weight [g] dry soil with glass vessel
Blank control	40.6961	52.3280	48.7536
Solvent control	50.5873	60.1473	57.1694
1000	47.7385	58.4918	55.2498

 

Table 5: pH values and water content

Concentration in mg/kg	pH Value Day 0	Water Content in % of WHC Day 0	pH Value Day 56	Water Content in % of WHC Day 56
Blank control	7.3	51.44	6.2	55.20
Solvent control	7.4	52.45	6.4	56.29
1000	7.3	58.72	6.5	53.70

 

Table 6: Validity data for the pooled concentrations

Parameter	Demand	Found	Assessment
Offspring in each control replicate	≥ 30 juveniles	at least 69	valid
Coefficient of variation of reproduction in the controls	≤ 30 %	22.8 %	valid
Adult mortality in the controls over the initial 28 days	≤ 10 %	0 %	valid

 

Validity criteria fulfilled:

yes

Conclusions:

In an Earthworm Reproduction Test according to OECD TG 222 with Eisenia andrei, the 56d-EC10 and the 56d-EC50 based on reproduction were determined to be > 1000 mg/kg soil dw (nominal).

Executive summary:

In an Earthworm Reproduction Test according to OECD TG 222 Eisenia andrei was exposed for 28 days to a nominal limit concentration of 1000 mg/kg artificial soil dw in 8 replicates each containing ten adult animals. Nominal concentrations were not analytically verified. Biological results are hence based on nominal test item concentrations. Acetone was used to spike quartz sand which was mixed with the soil after evaporation of the solvent. A stock solution containing 56.102 g test item/L in acetone was prepared (corresponds to 1000 mg/kg nominal). The whole artificial soil was divided into 3 portions of an appropriate amount. The required amount of test item was given to each portion using 88.0 g with test item spiked quartz sand per concentration. Afterwards it was well mixed. In addition, a negative control and a solvent control ran in parallel (8 replicates per control, each with 10 adult animals). For the negative control 500 g artificial soil was only mixed with quartz sand without test item (88.0 g) and quartz sand spiked only with acetone (88.0 g) was mixed with soil for the solvent control. The solvent was evaporated before the substrate was used in the test. After 28 days of exposure, the living adult animals were counted and removed 28 days later, the number of juveniles produced was determined. As a result, no mortality of adult animals after 28 days occurred in any replicate of the controls and the treatment. No toxicity after 28 d and no inhibition of reproduction after 56 d occurred in the test up to the limit dose of 1000 mg/kg. The 56d-EC₅₀ of Benomyl (CAS-No. 17804-35-2) was tested in a current reference test. The value was determined as 1.86 mg/kg, lying within the required range of OECD Guideline 222 of 1 – 5 mg/kg. For the test item the 28d-LC₅₀, 56d-EC₁₀ and the 56d-EC₅₀, respectively, were determined to be > 1000 mg/kg. No observations were made which might cause doubts concerning the validity of the study outcome. All validity criteria were met. The result of the test can be considered valid.

Description of key information

In an Earthworm Reproduction Test according to OECD TG 222 with Eisenia andrei, the 56d-EC₁₀ and the 56d-EC₅₀ based on reproduction were determined to be > 1000 mg/kg soil dw (nominal).

Additional information

In an Earthworm Reproduction Test according to OECD TG 222 Eisenia andrei was exposed for 28 days to a nominal limit concentration of 1000 mg/kg artificial soil dw in 8 replicates, each containing ten adult animals. Nominal concentrations were not analytically verified. Biological results are hence based on nominal test item concentrations. Acetone was used to spike quartz sand which was mixed with the soil after evaporation of the solvent. A stock solution containing 56.102 g test item/L in acetone was prepared (corresponds to 1000 mg/kg nominal). The whole artificial soil was divided into 3 portions of an appropriate amount. The required amount of test item was given to each portion using 88.0 g with test item spiked quartz sand per concentration. Afterwards it was well mixed. In addition, a negative control and a solvent control ran in parallel (8 replicates per control, each with 10 adult animals). For the negative control 500 g artificial soil was only mixed with quartz sand without test item (88.0 g) and quartz sand spiked only with acetone (88.0 g) was mixed with soil for the solvent control. The solvent was evaporated before the substrate was used in the test. After 28 days of exposure, the living adult animals were counted and removed 28 days later, the number of juveniles produced was determined. As a result, no mortality of adult animals after 28 days occurred in any replicate of the controls and the treatment. No toxicity after 28 d and no inhibition of reproduction after 56 d occurred in the test up to the limit dose of 1000 mg/kg. The 56d-EC₅₀ of Benomyl (CAS-No. 17804-35-2) was tested in a current reference test. The value was determined as 1.86 mg/kg, lying within the required range of OECD Guideline 222 of 1 – 5 mg/kg. For the test item the 28d-LC₅₀, 56d-EC₁₀ and the 56d-EC₅₀, respectively, were determined to be > 1000 mg/kg. No observations were made which might cause doubts concerning the validity of the study outcome. All validity criteria were met. The result of the test can be considered valid.