Dibutyltin oxide

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:

08 October 2014 to 22 January 2015

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))

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): Test soil was prepared by mixing the appropriate amount of test material with dry artificial soil before adding reverse osmosis water to hydrate the soil. Test soil components were mixed for up to a total of 20 minutes in order to achieve a homogeneous mixture.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworms
- Age at test initiation: Adult (mixed age). Earthworms were from synchronous cultures with individuals not differing in age by more than four weeks.
- Weight at test initiation (mean ± SD): The body weights of the adult worms available for use in the test ranged from 375 to 668 mg with a mean of 488.1 ± 86.90 mg based on 20 representative worms that were individually weighed.

ACCLIMATION
- Acclimation period: Adult earthworms were transferred to the test room and held in a glass container for acclimation to test conditions for 18 days prior to test initiation. One day prior to the test the worms (340 with clitellum) were removed from the chamber and divided into ten one-litre glass beakers containing prepared artificial soil substrate adjusted to a moisture content of approximately 34 % by weight, for the soil acclimation period.
- Acclimation conditions: Earthworms were fed cow manure throughout the acclimation period.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

28 d

Post exposure observation period:

Following the 28 day exposure period for the adults, the test soil and cocoons were returned to test chambers for an additional 28 days in order to determine the numbers of juveniles produced in each replicate by the end of the test on Day 56.

Test temperature:

20 ± 2 °C

pH:

Soil pH ranged from 6.5 to 6.7 at test initiation and from 6.1 to 6.5 at test termination.

Moisture:

Soil moisture content measured during the test ranged from 33 to 34 % at test initiation and from 37 to 38 % at test termination.

Details on test conditions:

TEST SYSTEM
- Test container: The test chambers were one-litre glass beakers covered with plastic wrap that was perforated for air exchange.
- Amount of soil or substrate: 750 g per test chamber
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): Artificial soil was prepared in bulk by mixing the following constituents in a soil mixer for approximately 30 minutes: quartz sand 63.0 kg; kaolin clay 18.0 kg; sphagnum peat 9.0 kg and calcium carbonate 0.9 kg.
- % sand: 70
- % sphagnum peat: 10
- % clay: 20
- Treatment of soil: Test chambers were weighed periodically to monitor soil moisture loss. Lost soil moisture was replaced by adding water to the soil surface until weights approximated those at the start of the test (Day 1) or those weights collected on Day 28 of the test (after adult earthworms were removed).

OTHER TEST CONDITIONS
- Photoperiod: The photoperiod during acclimation and testing was 16 hours of light and 8 hours of dark per day and was provided by overhead fluorescent bulbs.
- Light intensity: The target light intensity during the test was approximately 400 to 800 lux, and was verified on Day 7 of the test at the top of each test chamber using a SPER Scientific Inc model 840006C light meter.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Observations
At test initiation, the earthworms were placed on the surface of the soil in each replicate test chamber and were observed for burrowing behaviour. On Day 28 of test, the contents of each replicate chamber were removed to determine the number of surviving earthworms. All surviving earthworms were observed for gross physical and behavioural abnormalities.
- Body Weights
Group weights for all earthworms in each replicate were collected prior to being placed in the test chambers on Day 0. On Day 28, all surviving earthworms were removed from each replicate test chamber, rinsed with deionised water and blotted on dry paper. Group body weights were measured for worms in each test chamber, and average individual body weights were calculated.
- Collection and Enumeration of Juveniles
On Day 56, juveniles were removed from the test soil and counted and examined for physical abnormalities.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: No

Nominal and measured concentrations:

- Nominal concentrations: 0, 62.5, 125, 250, 500 and 1000 mg/kg dry soil

Reference substance (positive control):

yes

Remarks:

carbendazim

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality, clinical signs and body weight

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

MORTALITY AND CLINICAL SIGNS
There were no mortalities in the control and treatment groups during the 28 day adult exposure period, with the exception of one dead earthworm in the 62.5 mg/kg dry soil group which was considered to be an incidental mortality and not a result of treatment. All surviving earthworms in the control and the treatment groups were normal in appearance and behaviour.
Earthworms showed no aversion to soils treated with the test material. Since there was no treatment-related mortality of adult earthworms in the study, an LC50 value for mortality was not calculated.

BODY WEIGHTS
Average adult body weights at initiation and termination of the adult exposure period, and the change in body weight from initiation to termination, were calculated from the Day 0 and Day 28 measurements. When compared to control group, there were no apparent effects upon earthworm weight in the treatment groups during the 28-day adult exposure period. There were no statistically significant differences between mean final body weight, and change in body weight, for the treatment groups when compared to control group means. Therefore, based on body weight data, the NOEC was determined to be 1000 mg/kg dry soil, the highest concentration tested.

REPRODUCTIVE OUTPUT
The mean number of juveniles in the negative control group on Day 56 was 144.5 ± 28.46 (mean ± standard deviation), with a coefficient of variation of 19.7 %. In the 62.5, 125, 250, 500 and 1000 mg/kg dry soil treatment groups, the mean number of juveniles was 129.5, 153.8, 135.3, 127.0 and 84.0, respectively (Table 1). The juveniles collected from the control and treatment groups were normal in appearance and behaviour.
The mean number of juveniles produced in the 1000 mg/kg dry soil group was statistically significantly different from the mean number of juveniles in the control group with Dunnett’s Test (p<0.05). Therefore, the NOEC for reproduction was determined to be 500 mg/kg dry soil, and the LOEC was 1000 mg/kg dry soil.

ENVIRONMENTAL CONDITIONS AND PHYSICAL PROPERTIES OF TEST SOIL
Soil temperature ranged from 20.8 to 21.1 °C in each of the groups at test initiation and from 20.4 to 21.4 °C at test termination. Average light intensity during the 16-hour light period was 524 ± 50.7 lux, with a range of 420 to 591 lux.

VALIDITY CRITERIA
The test was considered to be acceptable based on the validity criteria. Adult mortality of control earthworms was less than 10 %, with no mortalities occurring in the control group. There were 109 or more juveniles produced in each of the eight replicates for the control group, thereby meeting the criterion of 30 or more juveniles per container. The coefficient of variation of reproduction in the control group was 19.7, meeting the less than the 30 % criterion.

Results with reference substance (positive control):

The testing facility conducted a reference toxicity test with carbendazim in 2006 to document that the earthworms being cultured were sensitive to a known toxicant. The LC50 value for the mortality of the adult earthworms exposed to carbendazim for 28 days was 7.149 mg a.i./kg dry soil, with a 95 % confidence interval of 6.338 and 8.273 mg a.i./kg dry soil. There were effects upon adult earthworm weight at concentrations of 0.3 mg a.i./kg and in the 2, 4 and 8 mg a.i./kg groups.
The EC50 value for reproduction was 0.8914 mg a.i./kg dry soil, with a 95 % confidence interval of 0.8416 and 0.9718 mg a.i./kg dry soil. The NOEC was 0.5 mg a.i./kg dry soil, and the LOEC was 1 mg a.i./kg dry soil, based on the numbers of juveniles produced.

Reported statistics and error estimates:

Statistical calculations were made using a personal computer and commercially available software (SAS Institute, Inc. 1999, SAS/STAT User’s Guide, Version 8.2.). The EC/LC50 is defined as the concentration of test substance that caused a 50 % reduction relative to the control group for a variable of concern. No EC/LC50 calculations were warranted for body weights, adult mortality or reproduction due to the lack of a 50 % effect at any test level.

Table 1: Number of Juveniles Produced

Nominal Concentration (mg/kg dry soil)	Replicate	Number of Juveniles¹ (Day 56)	Mean Number of Juveniles (Mean ±SD)
Control	A	164	144.5 ± 28.46²
	B	114
	C	124
	D	129
	E	164
	F	109
	G	170
	H	182
62.5	A	105	129.5 ± 17.52
	B	129
	C	144
	D	140
125	A	170	153.8 ± 19.97
	B	135
	C	172
	D	138
250	A	133	135.3 ± 14.24
	B	132
	C	155
	D	121
500	A	118	127.0 ± 6.68
	B	126
	C	133
	D	131
1000	A	70	84.0 ± 17.76*
	B	107
	C	70
	D	89

¹All juveniles were normal in appearance and behaviour.

SD = Standard deviation

²Coefficient of Variation = (SD/mean) x 100 = 19.7 %.

*Treatment group mean was statistically significantly different (p<0.05) from the control group mean when compared using Dunnett’s 2-Tailed Test of Means.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, based on body weight and survival data of adult earthworms, the no observed effect concentration (NOEC) was determined to be 1000 mg/kg dry soil, the highest concentration tested. Based on reproduction, the NOEC was determined to be 500 mg/kg dry soil and the lowest observed effect concentration (LOEC) was 1000 mg/kg dry soil.

Executive summary:

The effects of the test material on earthworm reproduction were investigated in a study conducted in accordance with the standardised guideline OECD 222 under GLP conditions.

Adult earthworms (Eisenia fetida) were exposed to a geometric series of five concentrations of the test material in artificial soil. Nominal concentrations of 62.5, 125, 250, 500 and 1000 mg/kg dry soil were selected. A negative control group was maintained concurrently in soil prepared without the addition of the test material.

Ten earthworms were housed in each of the replicate chambers. On the day of test initiation, the earthworms were rinsed briefly with well water, blotted on paper, weighed in groups of 10, and then placed on the soil surface of a test chamber. The adult earthworms were fed cow manure weekly during the first 28 days of the test, removed from the test chambers on Day 28, and observed for mortality and signs of toxicity. Test soil and cocoons were returned to test chambers for an additional 28 days in order to determine the numbers of juveniles produced in each replicate by the end of the test on Day 56. Cow manure was also added to the soil once at the beginning of the cocoon exposure period to serve as a source of food for juvenile worms emerging from cocoons. The number of juveniles present in each replicate at test termination was used to determine the no- observed-effect-concentration (NOEC) for reproduction.

There were no mortalities in the control and treatment groups during the 28 day adult exposure period, with the exception of one dead earthworm in the 62.5 mg/kg dry soil group which was considered to be an incidental mortality and not a result of treatment. All surviving earthworms in the control and the treatment groups were normal in appearance and behaviour.

When compared to control group, there were no apparent effects upon earthworm weight in the treatment groups during the 28-day adult exposure period.

The mean number of juveniles in the negative control group on Day 56 was 144.5 ± 28.46 (mean ± standard deviation), with a coefficient of variation of 19.7 %. In the 62.5, 125, 250, 500 and 1000 mg/kg dry soil treatment groups, the mean number of juveniles was 129.5, 153.8, 135.3, 127.0 and 84.0, respectively. The juveniles collected from the control and treatment groups were normal in appearance and behaviour.

The mean number of juveniles produced in the 1000 mg/kg dry soil group was statistically significantly different from the mean number of juveniles in the control group with Dunnett’s Test (p<0.05).

Under the conditions of this study, based on body weight and survival data of adult earthworms, the no observed effect concentration (NOEC) was determined to be 1000 mg/kg dry soil, the highest concentration tested. Based on reproduction, the NOEC was determined to be 500 mg/kg dry soil and the lowest observed effect concentration (LOEC) was 1000 mg/kg dry soil.

Description of key information

Eisenia fetida 28 d NOEC (toxicity) = 1000 mg/kg dry soil, NOEC (reproduction) = 500 mg/kg dry soil and LOEC (reproduction) = 1000 mg/kg dry soil.

Key value for chemical safety assessment

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

500 mg/kg soil dw

Additional information

The effects of the test material on earthworm reproduction were investigated in a study conducted in accordance with the standardised guideline OECD 222 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Adult earthworms (Eisenia fetida) were exposed to a geometric series of five concentrations of the test material in artificial soil. Nominal concentrations of 62.5, 125, 250, 500 and 1000 mg/kg dry soil were selected. A negative control group was maintained concurrently in soil prepared without the addition of the test material.

Ten earthworms were housed in each of the replicate chambers. On the day of test initiation, the earthworms were rinsed briefly with well water, blotted on paper, weighed in groups of 10, and then placed on the soil surface of a test chamber. The adult earthworms were fed cow manure weekly during the first 28 days of the test, removed from the test chambers on Day 28, and observed for mortality and signs of toxicity. Test soil and cocoons were returned to test chambers for an additional 28 days in order to determine the numbers of juveniles produced in each replicate by the end of the test on Day 56. Cow manure was also added to the soil once at the beginning of the cocoon exposure period to serve as a source of food for juvenile worms emerging from cocoons. The number of juveniles present in each replicate at test termination was used to determine the no- observed-effect-concentration (NOEC) for reproduction.

There were no mortalities in the control and treatment groups during the 28 day adult exposure period, with the exception of one dead earthworm in the 62.5 mg/kg dry soil group which was considered to be an incidental mortality and not a result of treatment. All surviving earthworms in the control and the treatment groups were normal in appearance and behaviour.

When compared to control group, there were no apparent effects upon earthworm weight in the treatment groups during the 28-day adult exposure period.

The mean number of juveniles in the negative control group on Day 56 was 144.5 ± 28.46 (mean ± standard deviation), with a coefficient of variation of 19.7 %. In the 62.5, 125, 250, 500 and 1000 mg/kg dry soil treatment groups, the mean number of juveniles was 129.5, 153.8, 135.3, 127.0 and 84.0, respectively. The juveniles collected from the control and treatment groups were normal in appearance and behaviour.

The mean number of juveniles produced in the 1000 mg/kg dry soil group was statistically significantly different from the mean number of juveniles in the control group with Dunnett’s Test (p<0.05).

Under the conditions of this study, based on body weight and survival data of adult earthworms, the no observed effect concentration (NOEC) was determined to be 1000 mg/kg dry soil, the highest concentration tested. Based on reproduction, the NOEC was determined to be 500 mg/kg dry soil and the lowest observed effect concentration (LOEC) was 1000 mg/kg dry soil.