bis(2,2,6,6-tetramethyl-1-(phenylthio)piperidin-4-yl)carbonate

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:

2019-01-16 to 2019-01-30

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:

OGYÉI- National Institute of Pharmacy and Nutrition, 21.04.2016

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): A mixture comprised of 10 g fine quartz sand with an amount of test item appropriate for the test concentration in the soil (1000 mg/kg soil dry weight) was first prepared. The test item - quartz sand mixture was then added to the pre-moistened artificial soil and mixed thoroughly to obtain homogeneous test substrate. Final moisture was adjusted to about 60 % of the maximum water holding capacity (WHC) by mixing an appropriate amount of deionised water.
For the untreated control, 10 g fine quartz sand without addition of the test item was mixed into the pre-moistened artificial soil.
The test substrate was prepared by mixing the amount of soil needed for four replicates in one step. The amount of air-dried soil used for preparing the test substrate (sufficient for four replicates) was 2617 g (containing the quartz sand - test item mixture) which corresponded to 2593 g dry weight (moisture of air-dried soil was determined to be 0.92 %). The amount of water needed to obtain approx. 60 % of the maximum water holding capacity of the soil was 583 g (sufficient for four replicates).
- Controls:
Negative control: Untreated control group ran parallel to the test item treated group. An appropriate amount of deionised water was mixed into the artificial soil.
Positive control: At least once a year a study with the reference item 2-Chloroacetamide is performed to verify the sensitivity of the earthworms and the precision of the test procedure.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworms (Eisenia fetida)
- Source: Bias Labs Ltd., Kirkcaldy, UK.
- Age at test initiation (mean and range, SD): about 11 months (not differing in age by more than 4 weeks)
- Weight at test initiation (mean and range, SD): 300-600 mg, including gut contents

ACCLIMATION
- Acclimation period:1 day, in artificial soil (day [–1] – day 0)
- Acclimation conditions (same as test or not): same
- Health during acclimation (any mortality observed): no mortality observed

Study type:

laboratory study

Substrate type:

artificial soil

Remarks:

5 % peat, 20 % kaolin clay; 75 % quartz sand

Limit test:

yes

Total exposure duration:

14 d

Post exposure observation period:

none

Test temperature:

18.1 – 21.9 °C

pH:

6.16 - 6.45

Moisture:

60 % of the maximum WHC

Details on test conditions:

TEST SYSTEM
- Test container (material, size): glass containers of approximately 1 litre capacity, covered with a transparent and punctured lid to allow gaseous exchange
- Amount of soil or substrate: Each test vessel contained 750 g wet weight of the artificial soil
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): Artificial soil was prepared, as follows: 5 % sphagnum peat (as close to pH 5.5-6.0 as possible, no visible plant remains, finely ground, dried to measured moisture content), 20 % kaolin clay (kaolinite content above 30 %), 75 % industrial sand (fine sand is dominant with more than 50 % of the particles between 50 and 200 μm)
- Maximum water holding capacity (in % dry weigth): 39.03 g water / 100 g soil dry weight
- Pretreatment of soil: The pH of the soil was adjusted to 6.0 ± 0.5 by addition of approx. 0.2 % calcium carbonate. The air-dry constituents were mixed thoroughly with a large scale mixer. The moisture content of the test medium was adjusted to approximately 60 % of the maximum water holding capacity (WHC) at the start of the test by adding an appropriate amount of deionised water (containing the test item amount appropriate for test concentration). The moistened soil was then divided in the test containers.

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity: 400-800 lux (measured mean value: 628 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Mortality and Clinical Symptoms: assessed after 7 and 14 days of exposure
- Biomass determinted at the end of treatment

VEHICLE CONTROL PERFORMED: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: single concentration (limit test)
- Range finding study: No toxic effects were observed in the preliminary range-finding test (1, 10, 100, 1000 mg/kg soil dry weight) wherefore a limit test was performed using a single test concentration of 1000 mg/kg soil dry weight.
- Test concentrations: 1000 mg/kg soil dry weight

Nominal and measured concentrations:

1000 mg/kg soil dry weight (nominal)

Reference substance (positive control):

yes

Duration:

14 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:

14 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:

14 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

biomass

Duration:

14 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

biomass

Details on results:

-Mortality
After 7 and 14 days of exposure no mortality was observed in the treatment or the control group.
Accordingly, the NOEC based on mortality was determined to be ≥ 1000 mg / kg soil dry weight.
The 14-day LC50 value was determined to be higher than 1000 mg / kg soil dry weight.

-Biomass
The biomass of the control group was reduced with 11.89 % after 14 days of exposure, which is considered to be within the normal range of this test system. The biomass reduction of the test item treated group (8.62 %) showed no statistically significant difference compared to the control (T-Test; α = 0.05; 2-sided). Accordingly, the NOEC based on biomass was determined to be ≥ 1000 mg / kg soil dry weight.

-please refer also to "any other information on results"

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: please refer to table 4 in "any other information on results"

Reported statistics and error estimates:

Statistical comparison of biomass change in control and in treated group was carried out using Independent Samples T-Test (2-tailed; α = 0.05) by SPSS software.
For determination of LC50 and NOEC for mortality no statistical procedure was necessary.

Table 1: Mortalities of Eisenia fetida after 7 and 14 days

Test Group	Number of dead worms/replicate										Mean [%]
	1			2		3			4
	day 7	day 14		day 7	day 14	day 7		day 14	day 7	day 14	day 7	day 14
Control	0	0		0	0	0		0	0	0	0.0	0.0
1000 mg/kg soil dry weight	0	0		0	0	0		0	0	0	0.0	0.0

Table 2: Summary of biomass changes (%) of Eisenia fetida

Test Group	Weight change [%] /replicate								Mean [%]		Standard Deviation
	1		2		3		4
Control	-15.31		-11.80		-11.29		-9.14		-11.89		2.56
1000 mg/kg soil dry weight	-7.14		-9.42		-10.48		-7.42		-8.62		1.60

Table 3: Water content, water holding capacity (WHC) and pH of the artificial soil samples

Test Group	Water content [g/100 g soil dry weight]		% water content of WHC*		pH
	day 0	day 14	day 0	day 14	day 0	day 14
Control	24.14	21.44	61.9	54.9	6.41	6.40
1000mg/kg soil dry weight	23.87	19.45	61.2	49.8	6.16	6.45

* WHC of the test substrate = 39.03 g / 100 g soil dry weight

Table 4: Summary of acute toxicity test with reference substance

Concentration [mg/kg soil dry weight]	Control	3.1		6.3	12.5	25	50
Mortality [%] 14-day	0.0	0.0		0.0	0.0	7.5	100
Weight change [%]	-3.34	-18.72		-10.39	-14.43	-19.05	NA
Endpoints [mg/kg soil dry weight]
14-day LC50(95 %confidence limits)			32.1(28.4 - 36.4)
14-day NOEC			25
14-day LOEC			50

NA: Not applicable, due to the 100 % mortality

Validity criteria fulfilled:

yes

Conclusions:

The test item had no effect on the mortality and biomass change of earthworms (Eisenia fetida) at the examined limit concentration of 1000 mg/kg soil dry weight under the test conditions used in this study.
The 14-day NOEC based on mortality and biomass was determined to be at least 1000 mg/kg soil dry weight. The LC50 (based on mortality) was determined to be higher than 1000 mg/kg soil dry weight.

Executive summary:

The study on the acute toxicity of the registered substance on earthworms (Eisenia fetida) in artificial soil was conducted as a limit test including a single concentration of 1000 mg/kg soil dry weight following OECD Guideline 207 under GLP compliance. At the start of the test the test item was mixed into artificial soil (5 % peat, 20 % kaolin clay; 75 % quartz sand). The worms were placed on the surface of the soil. Four replicates were prepared for the treatment group as well as for the control, each containing 10 worms. Assessment of mortality and behaviour was carried out 7 and 14 days after treatment. Assessment of weight change as sub-lethal parameter was carried out after 14 days. All validity criteria were met and therefore the study can be considered as valid. In conclusion, the test item had no effect on the mortality and biomass change of earthworms (Eisenia fetida) at the examined limit concentration of 1000 mg/kg soil dry weight under the test conditions used in this study.

The 14-day NOEC based on mortality and biomass was determined to be at least 1000 mg/kg soil dry weight. The LC₅₀ (based on mortality) was determined to be higher than 1000 mg/kg soil dry weight.

Description of key information

Key, acute toxicity on earthworms (Eisenia fetida): LC₅₀ > 1000 mg/kg soil dw (based on mortality), 14 -d NOEC >= 1000 mg/kg soil dw (based on mortality and biomass)

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

1 000 mg/kg soil dw

Additional information

The study on the acute toxicity of the test item on earthworms (Eisenia fetida) in artificial soil was conducted as a limit test including a single concentration of 1000 mg/kg soil dry weight following OECD Guideline 207 under GLP compliance. At the start of the test the test item was mixed into artificial soil (5 % peat, 20 % kaolin clay; 75 % quartz sand). The worms were placed on the surface of the soil. Four replicates were prepared for the treatment group as well as for the control, each containing 10 worms. Assessment of mortality and behaviour was carried out 7 and 14 days after treatment. Assessment of weight change as sub-lethal parameter was carried out after 14 days. All validity criteria were met and therefore the study can be considered as valid. In conclusion, the test item had no effect on the mortality and biomass change of earthworms (Eisenia fetida) at the examined limit concentration of 1000 mg/kg soil dry weight under the test conditions used in this study.

The 14-day NOEC based on mortality and biomass was determined to be at least 1000 mg/kg soil dry weight. The LC₅₀ (based on mortality) was determined to be higher than 1000 mg/kg soil dry weight.