Dibutyltin oxide

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 September 2014 to 30 January 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Soil samples were removed from each dose group for analyses of nitrate concentrations immediately after dosing, and on day 28.

- Sampling method: A single sample of approximately 5.6 grams (5.0 grams dry soil) was removed from each test chamber at each sampling interval. Samples were weighed into plastic centrifuge tubes, and 25 mL of NANOpure® water were added. Tubes were capped, shaken by hand, and placed in an ultrasonic bath for at least 5 minutes. Tubes were placed on a gyratory shaker table set at 250 rpm for at least 30 minutes, and then centrifuged at 2000 rpm for 10 minutes. Portions of the supernatants were transferred to micro-centrifuge tubes, and further centrifuged at 12000 rpm for 5 minutes. Aliquots of the supernatants were transferred to auto-sampler vials and diluted with NANOpure® water, if needed. The vials were submitted for HPLC analysis.

Vehicle:

no

Details on preparation and application of test substrate:

APPLICATION OF TEST MATERIAL TO SOIL
- Method: Quartz sand (Q-Rok crushed quartzite supplied by U.S. Silica) was used as a carrier. Five dose mixes of the test material coated on quartz sand were prepared as specified below. The appropriate amount of the test material and quartz sand were added to a scintillation vial which was then capped and inverted to thoroughly mix. The dose mixes were used to dose the test chambers on the same day.
Just prior to test material application, the moisture contents of the soils were adjusted to approximately 50 % WHC. Each test chamber was amended with 0.50 grams of dried, ground alfalfa. Three test chambers served as controls, and were treated with 1.0 g of quartz sand. Three test chambers were treated with 1.0 g of each of the dose mixes. After dosing, soils were thoroughly homogenised by hand using stainless steel spatulas.

DOSE MIXES
- Dose mix 1: 2.0000 g test material, 18.0013 g quartz sand (nominal concentration 100 mg/g)
- Dose mix 2: 5.0010 g dose mix 1, 4.9995 g quartz sand (nominal concentration 50 mg/g)
- Dose mix 3: 2.5006 g dose mix 1, 7.5002 g quartz sand (nominal concentration 25 mg/g)
- Dose mix 4: 1.2505 g dose mix 1, 8.7506 g quartz sand (nominal concentration 12.5 mg/g)
- Dose mix 5: 0.6251 g dose mix 1, 9.3755 g quartz sand (nominal concentration 6.25 mg/g)

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20 ± 2 °C

Moisture:

Water was added to adjust the moisture content to approximately 50 % of the maximum water holding capacity (11.33 %). The moisture content of the soils in the test chambers was adjusted just prior to dosing on day 0 and at approximately weekly intervals thereafter.

Details on test conditions:

TEST SYSTEM
- Test container: Test chambers were 8-ounce French-square glass bottles with aluminium foil used as lids. Lids were perforated to allow circulation of air.
- Amount of soil: Approximately 110 grams of moist soil (equivalent to 100 grams of dry soil). Each test chamber was amended with 0.50 grams of dried, ground alfalfa.
- Conditions: Test chambers were kept in a temperature controlled room and incubated under aerobic conditions in the dark.
- No. of replicates per concentration: 3
- No. of replicates per control: 3

SOIL INCUBATION
- Method: series of individual subsamples

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site: The soil used was a Greensboro soil that was collected in Caroline County, Maryland, USA, at coordinates N 38° 56.3697’ - W 75° 46.3741’.
- History of site: The site was an open field between the road and lightly wooded area.
- Vegetation cover: The vegetation was grasses.
- Treatments with pesticides or fertilisers: No known treatments with pesticides or fertilisers occurred in the past five years.
- Other: Soil collection was not conducted during a drought or immediately following heavy rainfall.
- Depth of sampling: Soil was collected from the top 0 to 10 cm.
- Soil texture: % sand: 73; % silt: 22; % clay: 5
- Soil taxonomic classification: Sandy Loam
- Soil classification system: USDA
- pH (in water): 5.6
- Initial nitrate concentration for nitrogen transformation test: 11.1 mg/kg
- Maximum water holding capacity: 22.67 %. The moisture content of the soil was determined to be 10.0 %. NANOpure® water was added to each sample to adjust the moisture content to 11.33 %, or 50 % of the maximum water holding capacity.
- Cation exchange capacity: 5.5 meq/100 g
- Pre-treatment of soil: The soil was sieved through a 2-mm screen prior to characterisation and use in the study.
- Storage: The soil was maintained in an outdoor raised plot upon receipt.
- Initial microbial biomass: 114.9 µg/g, > 1 % of the organic carbon content

DETAILS OF PRE-INCUBATION OF SOIL (if any):
All test chambers were incubated under aerobic conditions in the dark in a temperature-controlled room set at approximately 20 °C for three days prior to application of the test material.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Soil samples were removed for analyses of nitrate concentrations immediately after dosing, and on day 28. Soil samples were removed for pH measurements immediately after dosing and on day 28.

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:

2-chloro-6(trichloromethyl)pyridine

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Details on results:

SOIL NITRATE ANALYSES
A summary of the results from analyses of soil nitrate concentrations is presented in Table 1. The mean nitrate concentrations in the control soil were 38.8 and 350.6 mg NO₃/kg on days 0 and 28, respectively. The nitrate concentrations increased in the controls throughout the test period. The relative standard deviation (RSD) among control replicates was <15 % at each sampling interval; therefore, the test was considered valid.
The measured concentrations for all nitrate standards were within 15 % of the nominal concentrations throughout the study. All linear calibration curves had r² values >0.999 for each set of analyses. There were no interferences observed in the extraction solvent. Recoveries from fortified samples ranged from 96.6 to 101.0 %; therefore, the extraction method was acceptable.
On day 28, there were statistically significant differences (Dunnett’s two-tailed test p<0.05) in nitrate concentrations of the soils treated at 125, 250, 500, and 1000 mg/kg dry soil when compared to the control (untreated) soil. The nitrate concentrations of the soils treated at 125, 250, 500, and 1000 mg/kg dry soil were higher than the nitrate concentrations of the control (untreated) soil, therefore not an adverse effect.
The calculated nitrate formation rates for individual replicate samples are presented in Table 1. The mean nitrate formation rates and percent inhibition for each treatment are presented in Table 2. The mean nitrate formation rate in the control soil was 11.14 mg NO₃/kg/day after 28 days. On day 28, there were statistically significant differences (Dunnett’s two-tailed test p<0.05) in nitrate formation rates of the soils treated at 125, 250, 500 and 1000 mg/kg dry soil when compared to the control soil. The calculated nitrate formation rates of the soils treated at 125, 250, 500, and 1000 mg/kg dry soil were higher than the calculated rates of the control (untreated) soil.
On day 28, all soils treated with the test material had higher nitrate concentrations than the control soil. The EC50 value for inhibition of nitrate formation was greater than 1000 mg/kg dry soil (the highest concentration tested).

TEST CONDITIONS
During the 3-day acclimation period, temperatures ranged from 20.3 to 21.2 °C. During the 28-day test period, temperatures ranged from 19.5 to 22.2 °C.
The measured moisture contents of the soil samples ranged from 11.2 to 12.8 %, and were equivalent to 49.2 to 56.3 % of the maximum water holding capacity (WHC) during the test. These values were measured after water had been added to the soils at each interval to adjust the moisture contents. The adjusted soil moisture contents were within the required range.
During the 28-day test period, the calculated soil moisture contents before and after adjustments ranged from 9.8 to 13.0 %, and were equivalent to 43.2 to 57.5 % WHC. Throughout the study, the soil moisture contents were maintained within the required range.

SOIL PH MEASUREMENTS
Immediately after application of the test material, there were no pH differences attributed to the test material. On day 28, the pH measurements of the treated soils were lower than the pH measurement of the control soil.

Results with reference substance (positive control):

500 mg/kg of a known inhibitor of nitrogen transformation was applied to the same soil and showed 84 % inhibition of the nitrate formation rate after 28 days.

Reported statistics and error estimates:

Statistical comparisons of measured nitrate concentrations were made at each sample interval using the ANOVA function in Microsoft Excel. Where differences among treatments were significant, a Dunnett’s test comparison was made to the control group using SAS (release 8.02).

Table 1: Measured Nitrate Concentrations and Calculated Nitrate Formation Rates in Soil

Treatment (mg/kg)	Replicate	Measured Nitrate Concentrations (mg NO₃/kg dry soil)		Calculated Nitrate Formation Rates
		Day 0	Day 28	Days 0 to 28
Control	A	37.8	339.5	10.78
	B	38.4	356.0	11.34
	C	40.1	356.4	11.29
	Mean	38.8	350.6	11.14
	RSD (%)	3.1	2.8	2.8
62.5	A	38.6	361.5	11.53
	B	37.1	381.3	12.29
	C	38.8	378.7	12.14
	Mean	38.2	373.8	11.99
	RSD (%)	2.5	2.9	3.4
125	A	37.1	393.0	12.71
	B	36.5	392.7	12.72
	C	38.4	396.6	12.79
	Mean	37.3	394.1*	12.74*
	RSD (%)	2.6	0.6	0.4
250	A	39.6	374.3	11.95
	B	37.6	426.1	13.88
	C	37.1	404.9	13.14
	Mean	38.1	401.8*	12.99*
	RSD (%)	3.5	6.5	7.5
500	A	38.9	400.1	12.90
	B	37.8	367.0	11.76
	C	35.4	412.7	13.48
	Mean	37.4	393.3*	12.71*
	RSD (%)	4.8	6.0	6.9
1000	A	40.7	421.3	13.59
	B	40.3	395.9	12.70
	C	39.0	424.6	13.77
	Mean	40.0	413.9*	13.35*
	RSD (%)	2.2	3.8	4.3

RSD = relative standard deviation or coefficient of variation

*Statistically significant (p<0.05) with Dunnett’s Two-Tailed Test

 

Table 2: Mean Nitrate Formation rates and Inhibition

Treatment (mg/kg)	Days 0 to 28
	Rate (mg NO₃/kg dry soil per day)	Inhibition (%)
0	11.14	-
62.5	11.99	-7.6
125	12.74	-14.4
250	12.99	-16.6
500	12.71	-14.1
1000	13.35	-19.9

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the EC50 value for inhibition of nitrate formation after 28 days was greater than 1000 mg/kg dry soil, the highest concentration tested. The test material did not have an adverse effect on nitrogen transformation in soil.

Executive summary:

A study was conducted to assess the effects of the test material on the nitrogen transformation activity of soil microorganisms in aerobic surface soils. The study was conducted in accordance with the standardised guideline OECD 216 under GLP conditions.

Test chambers were each filled with ~110.0 grams of moist soil (equivalent to 100 grams of dry soil). Water was added to adjust the moisture content to approximately 50 % of the maximum water holding capacity and test chambers were acclimated to test conditions for three days. Test chambers were amended with dried, ground alfalfa at 5.0 g/kg of dry soil. Quartz sand was added to the three test chambers that served as controls. Three test chambers each were treated with the test material at nominal concentrations of 62.5, 125, 250, 500 and 1000 mg/kg of dry soil. All test chambers were incubated in the dark under aerobic conditions at 20 ± 2 °C for the 28-day test period. Soil samples were removed for analyses of nitrate concentrations on days 0 and 28.

The nitrate concentrations increased in the controls throughout the test period. The relative standard deviation (RSD) among control replicates was <15 % at each sampling interval; therefore, the test was considered valid. Mean nitrate formation rates were calculated as mg NO₃/kg dry soil per day. The percent inhibition of nitrate formation, compared to untreated controls, was calculated for each treatment at each interval.

On day 28, there were statistically significant differences (Dunnett’s two-tailed test p<0.05) in nitrate concentrations of the soils treated at 125, 250, 500, and 1000 mg/kg dry soil when compared to the control (untreated) soil. The nitrate concentrations of the soils treated at 125, 250, 500, and 1000 mg/kg dry soil were higher than the nitrate concentrations of the control (untreated) soil, therefore not an adverse effect.

Under the conditions of this study, the EC50 value for inhibition of nitrate formation after 28 days was greater than 1000 mg/kg dry soil, the highest concentration tested. The test material did not have an adverse effect on nitrogen transformation in soil.

Description of key information

28 d EC50 for inhibition of nitrate formation >1000 mg/kg dry soil. The test material did not have an adverse effect on nitrogen transformation in soil.

Key value for chemical safety assessment

Short-term EC50 for soil microorganisms:

1 000 mg/kg soil dw

Additional information

A study was conducted to assess the effects of the test material on the nitrogen transformation activity of soil microorganisms in aerobic surface soils. The study was conducted in accordance with the standardised guideline OECD 216 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria of Klimisch et al. (1997).

The study was conducted using a single soil type (sandy loam). Test systems were dosed with nominal concentrations of 0, 62.5, 125, 250, 500 and 1000 mg/kg dry soil, and incubated at approximately 20 °C for 28 days. Soil samples were collected on days 0 and 28 and analysed to determine nitrate concentrations.

The nitrate concentrations increased in the controls throughout the test period. The relative standard deviation (RSD) among control replicates was <15 % at each sampling interval; therefore, the test was considered valid. Mean nitrate formation rates were calculated as mg NO₃/kg dry soil per day. The percent inhibition of nitrate formation, compared to untreated controls, was calculated for each treatment at each interval.

On day 28, there were statistically significant differences (Dunnett’s two-tailed test p<0.05) in nitrate concentrations of the soils treated at 125, 250, 500, and 1000 mg/kg dry soil when compared to the control (untreated) soil. The nitrate concentrations of the soils treated at 125, 250, 500, and 1000 mg/kg dry soil were higher than the nitrate concentrations of the control (untreated) soil, therefore not an adverse effect.

Under the conditions of this study, the EC50 value for inhibition of nitrate formation after 28 days was greater than 1000 mg/kg dry soil, the highest concentration tested. The test material did not have an adverse effect on nitrogen transformation in soil.