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)

Version / remarks:

1984

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, 107, 192, 346, 623 and 1121 mg enzyme concentrate dry matter/kg dry weight of artificial soil.

Reference substance (positive control):

yes

Remarks:

chloroacetamide

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

1 121 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mortality

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

> 1 121 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mortality

Details on results:

- Mortality at end of exposure period: 0
- Changes in body weight of live adults (% of initial weight) at end of exposure period: control: +16.4%, 107 ppm: +15.2%, 192 ppm: +21.1%, 346 ppm: +17.1%, 623 ppm: +17.6%, 1121 ppm: +22.8%

Results with reference substance (positive control):

- Results with reference substance valid: yes

Validity criteria fulfilled:

yes

Conclusions:

Acute exposure of earthworms to Subtilisin, batch PBI48037 showed no toxicity up to 1121 mg enzyme concentrate dry matter/kg soil for an exposure of 14 days. Therefore, the LC50 is greater than 1121 mg enzyme concentrate dry matter/kg dry weight of artificial soil.
The overall no observed effect concentration (NOEC) was considered to be 1121 mg enzyme concentrate dry matter/kg soil dry weight of artificial soil corresponding to 576 mg active enzyme protein/kg soil dw.

Executive summary:

A study was performed to determine the acute toxicity (LC50) of Subtilisin, batch PBI48037 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 107, 192, 346, 623 and 1121 mg enzyme concentrate dry matter/kg soil dry wt. 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 1121 mg enzyme concentrate dry matter/kg soil dry wt., the highest concentration tested.

The overall no observed effect concentration (NOEC) was considered to be 1121 mg enzyme concentrate dry matter/kg soil dry wt.

Description of key information

Acute exposure of earthworms to Subtilisin, batch PBI48037 showed no toxicity up to 1121 mg enzyme concentrate dry matter/kg soil for an exposure of 14 days. Therefore, the LC50 is greater than 1121 mg enzyme concentrate dry matter/kg dry weight of artificial soil.
The overall no observed effect concentration (NOEC) was considered to be 1121 mg enzyme concentrate dry matter/kg soil dry weight of artificial soil corresponding to 576 mg active enzyme protein/kg soil dw.

Key value for chemical safety assessment

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

1 121 mg/kg soil dw