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Ecotoxicological information

Toxicity to soil microorganisms

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toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
supporting study
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Study conducted in accordance with scientific principles but not following the standard methodology for testing this endpoint. The test was conducted without GLP.
according to 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:
Analytical monitoring:
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 (inoculum):
Total exposure duration:
2 h
Test temperature:
Details on test conditions:
- 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:

- Method: bulk / series of individual subsamples

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).


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


not conducted
Nominal and measured concentrations:
Nominal concentrations: 10, 30, 100, 300, 1000 mg/kg (dry weight)
Reference substance (positive control):
2 h
Dose descriptor:
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.

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









Dimethoate (reference compound)

concentration (mg/kg dw)

 1  -11
 10  -7
 100  -11
 1000  -7
Validity criteria fulfilled:
not applicable
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.

Description of key information

Key value for chemical safety assessment

Additional information

Short term terrestrial toxicity

A 2-h EC50 value of >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 (Shell, 2004).

Whilst fully consistent with expectations, this cannot be considered to be key data.

This data is considered to be indicative only (non assignable reliability) and not suitable for quantitative application as part of the chemical safety assessment.

Long term terrestrial toxicity

In accordance with Column 2 of REACH Annex IX toxicity testing with terrestrial microorganisms (required in Section 9.4.2) is not needed as the chemical safety assessment according to Annex I indicates that this is not necessary.

Moreover, considerable technical difficulties would be expected in the conduct of such a test, due to the very rapid biotic removal of the substance from the test system. Please refer to discussion of the long-term aquatic invertebrate and fish studies, and evidence of rapid removal in non-sterilised soils during method development for the adsorption/desorption study with natural soils, carried out with the close structural analogue decan-1-ol, in the environmental fate section.

The terrestrial chemical safety assessment has been conducted using the Equilibrium Partitioning method (EQPM). It is recognised that the aquatic PNEC used in the EQPM does not take into account any indicator for effects in aquatic microorganisms. However, analogous alcohols within the Category are very rapidly biodegradable and show no significant inhibitory effects on respiration of activated sludge or specific microbial strains relevant to WWTP, at or above the limit of solubility (based on inhibition tests and lack of toxicity in ready biodegradability test). A lack of toxicity to soil microbiota was suggested by a recent experimental finding associated with a recent study of adsorption/desorption (OECD 106, Wildlife International, 2015) using decan-1-ol. Significant technical difficulties were encountered during method development for this study using natural standard soils, in that it was not possible to detect sufficient substance and establish equilibrium in non-sterilised soil samples.Therefore it is unlikely that the PNECterrestrial based on aquatic ecotoxicity test results would not be protective for terrestrial microorganisms. The chemical safety assessment using EQPM does not suggest any unacceptable risks for the terrestrial compartment.

Therefore, further toxicity testing with terrestrial microorganisms does not need to be conducted.