Subtilisin

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 April 2010 - 14 May 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

certificate included in the study report

Analytical monitoring:

not required

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): The test substance was formulated with water in a series of concentrations and the same volume was measured from each dose formulation for mixing by hand in a bowl prior to the addition of distilled water. Distilled water was gradually incorporated into the treated soil and the soil further mixed until a moisture content equivalent to 35% of the dry weight was achieved. The soil in each test group was weighed at the start of the study to ensure that the correct moisture content had been achieved.
- Controls: distilled water

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm
- Source: Huntingdon Life Sciences
- Weight at test initiation (mean and range, SD): 300 - 600 mg

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

14 d

Test temperature:

19.7 - 20.4°C

pH:

6.0

Moisture:

34% at the start of the study, 31 to 32% at termination

Details on test conditions:

TEST SYSTEM
- Test container (material, size): one-litre glass container
- Amount of soil or substrate: 2200 g soil (dry weight) was divided approximately equally between four containers
- No. of organisms per container (treatment): ten earthworms per container
- No. of replicates per treatment group: 4
- No. of replicates per control: 4


SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
Artificial OECD 207 soil
- Composition (if artificial substrate): 7 % industrial quartz sand, 20% kaolin clay, 10% sphagnum peat
- Pretreatment of soil: Distilled water was gradually incorporated into the treated soil and the soil further mixed until a moisture content equivalent to 35% of the dry weight was achieved.


OTHER TEST CONDITIONS
- Photoperiod: continuous lighting
- Light intensity: 556 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Assessment of health: Any earthworms visible on the soil surface were observed daily for behavioural and pathological signs.
Assessment of mortality: On Days 7 and 14, the medium was emptied from the test containers and the earthworms removed for counting. Mortalities, live earthworms and those unaccounted for were recorded (earthworms which died beneath the soil surface may have decomposed and no longer be detectable at the counts on Days 7 and 14). Mortality was assessed, if necessary, by testing the reaction of each earthworm to a mechanical stimulus at either end. On Day 7, the medium was returned to the containers and surviving earthworms replaced on the surface.
Assessment of bodyweights: Bodyweights of earthworms were recorded in both treatment and control replicates prior to treatment and on Day 14.

VEHICLE CONTROL PERFORMED: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8

Nominal and measured concentrations:

nominal: 0, 95, 171, 309, 556 and 1000 mg TOS/kg dry weight of artificial soil

Reference substance (positive control):

yes

Remarks:

chloroacetamide

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: Total Organic Solids (TOS)

Basis for effect:

mortality

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: TOS

Basis for effect:

mortality

Key result

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

568 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

mortality

Details on results:

- Mortality at end of exposure period: 0
- Changes in body weigth of live adults (% of initial weight) at end of exposure period: control: +16.4%, 95 ppm: +15.2%, 171 ppm: +21.1%, 309 ppm: +17.1%, 556 ppm: +17.6%, 1000 ppm: +22.8%

Results with reference substance (positive control):

- Results with reference substance valid: yes

Validity criteria fulfilled:

yes

Conclusions:

The test substance is of low acute toxicity to earthworms. The LC50 after 14 days was greater than 1000 ppm (mg TOS/kg dry weight of artificial soil), the highest concentration tested.
The overall no observed effect concentration (NOEC) was considered to be 1000 ppm (mg TOS/kg dry weight of artificial soil) corresponding to 568 mg aep/kg dw.

Executive summary:

A study was performed to determine the acute toxicity (LC50) of the test substance to the earthworm, Eisenia foetida, in an artificial soil under laboratory conditions.The study was performed according to OECD Guideline No. 207 and EEC Directive 88/302/EEC, Part C, and in compliance with GLP.

Description:

Soil: Artificial medium, 70% quartz sand, 20% kaolin clay and 10% sphagnum peat and adjusted to pH 6.0 ± 0.5 with calcium carbonate. Moisture content was adjusted with distilled water to 35% soil dry weight. Methods: The test substance was added to the soil in water formulations and at nominal concentrations of 95, 171, 309, 556 and 1000 mg/kg soil dry wt. (ppm) adjusted for TOS. Four replicates of 10 earthworms (300-600 mg) were exposed to each treated soil. A further negative control group was exposed to soil treated with water only. Earthworms were counted and mortality assessed if necessary by testing the reaction of each worm to a mechanical stimulus 7 and 14 days after soil treatment. Bodyweights were recorded prior to soil treatment and at Day 14.

Results:

There were no mortalities. Bodyweights were similar in all groups with no evidence of a treatment related effect.

Conclusion:

The test substance is of low acute toxicity to earthworms. The LC50 after 14 days was greater than 1000 ppm, the highest concentration tested.

The overall no observed effect concentration (NOEC) was considered to be 1000 ppm or higher.

Description of key information

The test substance is of low acute toxicity to earthworms. The LC50 after 14 days was greater than 1000 ppm (mg TOS/kg dry weight of artificial soil), the highest concentration tested.
The overall no observed effect concentration (NOEC) was considered to be 1000 ppm (mg TOS/kg dry weight of artificial soil) corresponding to 568 mg active enzyme protein (aep)/kg dry weight of artificial soil.

Additional information

A study was performed to determine the acute toxicity (LC50) of the test substance to the earthworm, Eisenia foetida, in an artificial soil under laboratory conditions.The study was performed according to OECD Guideline No. 207 and EEC Directive 88/302/EEC, Part C, and in compliance with GLP.

After 14 days of expsoure to subtilisin no mortalities or other toxic effects were observed. In conclusion, subtilisin is of low acute toxicity to earthworms. The LC50 after 14 days was greater than 1000 ppm, the highest concentration tested. The overall no observed effect concentration (NOEC) was considered to be 1000 ppm.