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Diss Factsheets

Ecotoxicological information

Toxicity to soil microorganisms

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Administrative data

Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Study conducted in accordance with scientific principles but not following the standard methodology for testing this endpoint. The test was conducted without GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: von Mersi, W. and Schinner, F. 1991. An improved and accurate method for determining the dehydrogenase activity of soils with iodonitrotetrazolium chloride. Biology and Fertility of Soils (1991) 11:216-220)
Principles of method if other than guideline:
Abstract: Conditions for a rapid, precise [100 µg iodonitrotetrazolium chloride (INT)-formazan/ml assay mixture] and easily reproducible assay of potential soil dehydrogenase activity are described, using 2(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (iodonitrotetrazolium chloride, INT) as the substrate. Reduced iodonitrotetrazolium formazan (INTF) was measured by spectrophotometry (464 nm) after extraction with N,N-dimethylformamide and ethanol. With this method, the coloured complex formed is highly stable. The effects of pH, buffr concentration, temperature, substrate concentration, amount of soil weight, and reaction time on dehydrogenase activity were investigated. The rate of substrate hydrolysis was proportional to soil weight; the optimal INT reduction was achieved with 1 M TRIS buffer (pH 7.0) at 40ºC. It was possible to determine the biotic and abiotic substrate reduction by comparing assays of autoclaved and unsterile soil samples. Different investigations have confirmed that the intracellular enzyme is highly correlated with the microbial biomass, and indicate that this activity is suitable as an indirect parameter of microbial biomass, measurement.
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetradecanol
EC Number:
204-000-3
EC Name:
Tetradecanol
Cas Number:
112-72-1
Molecular formula:
C14H30O
IUPAC Name:
tetradecan-1-ol
Test material form:
other: white, semi-solid

Sampling and analysis

Analytical monitoring:
no

Test substrate

Vehicle:
yes
Details on preparation and application of test substrate:
- Method of mixing into soil: sand spike, using 10% of the total dry weight of soil. This involved adding an appropriate amount of compound to analar grade acetone to form a stock solution for the 1000 mg/kg dose. Aliquots of the stock solution were then diluted with acetone to prepare lower doses. Acetone solutions were added to silica sand in 500 ml amber glass jars. Mixtures were mixed thoroughly with a stainless steel spatula. The acetone was left to evaporate in a fume cupboard, leaving the test item coated onto the sand. Appropriate amount of moist soil was added to each of the glass jars. The dosed or undosed sand was incorporated into the bulk of the soil using a palette knife. Jars were then sealed with screw top lids containing aluminium foil inserts and tumbled overnight (ca. 16 h) on a rotary soil tumbler 50 rpm. Following mixing on the tumbler, de-ionised water was added to the jars and mixed using a metal spatula.
- Controls: One aliquot of sand was dosed with acetone only to serve as solvent control. A jar with an aliquot of sand only served as untreated control - both with the appropriate amount of moist soil added thereafter.
- Chemical name of vehicle: acetone
- Concentration of vehicle in test medium: acetone solutions was added between 1-7 ml to 5-35g of silica sand. The sand was left to evaporate the acetone completely before initial exposure, so that the concentration of vehicle would be negligible.
- Evaporation of vehicle before use: yes

Test organisms

Test organisms (inoculum):
soil

Study design

Total exposure duration:
2 h

Test conditions

Test temperature:
40ºC
Moisture:
15-20%
Details on test conditions:
TEST SYSTEM
- Testing facility:
- Test container (type, material, size):
- Amount of soil:
- No. of replicates per concentration:
- No. of replicates per control:
- No. of replicates per vehicle control:

SOIL INCUBATION
- Method: bulk / series of individual subsamples

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
The soil used in toxicity tests was purchased from LandLook (Midlands) c/o G.R. Beard, Leicester Lane, Cubbington Heath, Leamington Spa. The soil is described as soil series, Bromsgrove, standard soil number 33, variant. It was collected from a field that had been put to set-aside for the previous 3 - 4 years and used for cereal crops prior to 1998. The collection site is on Heath Farm, Leicester Lane, Leamington Spa, situated at OS map reference 32896926. Soil was collected from a depth of 5 - 20 cm to avoid the surface root mat. All soil was sieved to 2 mm by LandLook prior to dispatch.

The pH of the soil in the first series of tests (rangefinder tests) ranged from 5.8 to 6.8 at the start of the exposure periods and from 6.1 to 6.7 at the end. These ranges are close the range of pH 6.0 ± 0.5 recommended in test guidelines (OECD, 1984 and ISO, 1999) The pH of soil used in the second test (definitive tests) tended to be slightly lower, with a measured range of 5.4 - 6.1. Again, these are close to the recommended range of pH 6.0 ± 0.5.

- Initial microbial biomass as % of total organic C: Microbial biomass 296.6 µgC/g (range finder soil); 237.4 µgC/g (definitive test soil), equivalent to 1.8% of total OC in the definitive test soil (based on 1.3% OC in test soil).

DETAILS OF PREINCUBATION OF SOIL (if any): none

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : dehydrogenase activity, determined spectrophotometrically using INTF as indirect indicator.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
not conducted
Nominal and measured concentrations:
Nominal concentrations: 10, 30, 100, 300, 1000 mg/kg (dry weight)
Reference substance (positive control):
yes
Remarks:
Dimethoate

Results and discussion

Effect concentrations
Duration:
2 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
other: microbial biomass
Remarks on result:
other: (based on results seen in definitive test, conclusion not reported in study)
Results with reference substance (positive control):
No significant inhibition seen.

Carbendazim (used as reference substance in range-finder): stimulation of nitrification activity reported at all concentrations; insignificant stimulation or inhibition of dehydrogenase activity.

Dimethoate (used as reference substance in definitive test): stimulation of dehydrogenase activity at all loading levels.

Any other information on results incl. tables

Table 1. Results of definitive study

Tetradecanol concentration (mg/kg dw)

% inhibition (+ve figures) or stimulation (-ve figures) of biological activity compared to solvent controls

30

-17

100

-29

300

+18

1000

+26

Dimethoate (reference compound)

concentration (mg/kg dw)

 
 1  -11
 10  -7
 100  -11
 1000  -7

Applicant's summary and conclusion

Validity criteria fulfilled:
not applicable
Conclusions:
An EC50 >1000 mg/kg dwt in soil based on effects on dehydrogenase activity (as an indirect indicator of microbial biomass) was observed in a non-standard study. The definitive results appear to show the microbial biomass increasing with increasing amounts of the test substance, at lower test loadings.