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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:
weight of evidence
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
equivalent or similar to guideline
OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)
no glucose added
Principles of method if other than guideline:
The soil, sampled 14 June 2004 beneath pasture grazed by dairy cattle near Lincoln, New Zealand (43°38.70′S, 172°28.62′E), was a poorly-drained Temuka silt loam (Fluvaquentic Endoaquept; Soil Survey Staff 1998). Samples were collected at 10 locations, 20 m apart along a 200 m long transect, from a 200 × 200 mm area to a depth of 50 mm. The roots were removed, the soil passed through a 2 mm mesh sieve, and returned to a refrigerator. At each location, a bulk density sample (75 mm diameter) was taken from the soil to a depth of 50 mm, dried for 48 h in an oven at 105°C, and weighed.
Composite samples of soil and ryegrass were biochemically analysed. Soil pH in water, total C and N, and microbial C and N concentrations were determined as described earlier by Ross et al. (1999). To calculate soil microbial C from the 0.5 M K2SO4 extractable-C flush value in the fumigation-extraction method, a kEC-factor of 0.41 was used, based on the value in Sparling et al. (1990) and adjusted for determination of extracted C using a total C analyser (TOC-5000A, Shimadzu Oceania Pty Ltd, NSW, Australia) rather than dichromate oxidation. The soil phosphorus status was estimated following Olsen et al. (1954). Cellulose, lignin and ash contents of the ryegrass were determined by isolation using an acid-detergent fibre pre-extraction and cellulose hydrolysis with sulphuric acid (Rowland & Roberts 1994).
Fourteen days after sampling, the processed soils were removed from the refrigerator. From each location sampled, three subsamples, 0.35 kg each, were placed in plastic bags and the soil pre-incubated at 20°C for 1 day. Ground ryegrass (10 g) was then gently mixed into a randomly chosen soil subsample from each location. The ryegrass total C and N contents were 416 and 43 g kg–1, respectively, so the C and N application rates (see soil container description below) were 5300 kg C ha–1 (12 g C kg–1) and 550 kg N ha–1 (1.2 g N kg–1).
Each soil subsample, including 0.190 kg of water, was packed to a depth of 50 mm into a 60 mm deep by 100 mm diameter plastic container (bulk density 890 kg m–3). One subsample from each location received a urea application containing 500 kg N ha–1 (1.1 g N kg–1) in a 10 ml aqueous solution (1.4 mol litre–1) equivalent to a 1 mm fall of rain. The other subsamples received 10 ml of water. During the 24- day incubation, 10 ml of water was sprayed daily on the surface of each subsample to maintain the water content, although we report an exception in the Results. During incubation, the soil temperature averaged 19.2 ± 1.2°C.
Soil carbon dioxide efflux rate (Fco2), indicating microbial respiration and carbonate hydrolysis if urea was applied, was measured using a portable chamber with an infrared gas analyser (SRC-1 and EGM-3, PP Systems, Hitchin, UK). The chamber connected directly to the soil container. To ensure fresh air circulation, the contained soils were briefly shifted close to an exterior door and each measurement took 2 min. The Fco2 was expressed as a mass (μg CO2) per unit mass (dry weight) of contained soil (kg) per unit time (s). On the first day, Fco2 was measured 1.5 and 4 h after the subsamples were packed into containers. Thereafter, measurements were made once daily for 9 days and then at 2- to 4-day intervals for another 15 days.
GLP compliance:
Analytical monitoring:
Details on preparation and application of test substrate:
- Method: One subsample from each location received a urea application containing 500 kg N ha–1 (1.1 g N kg–1) in a 10 ml aqueous solution (1.4 mol litre–1) equivalent to a 1 mm fall of rain.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Water
- Concentration of vehicle in test medium (stock solution and final test solution): 1.4 mol
- Evaporation of vehicle before use: no
Test organisms (inoculum):
Total exposure duration:
24 d
Test temperature:
19.2 ± 1.2°C
Organic carbon content (% dry weight):
Nitrogen content (% dry weight):
Nominal and measured concentrations:
Control and 1.1 g N kg–1 equivalent to 2358 mg/kg
Reference substance (positive control):
24 d
Dose descriptor:
Effect conc.:
>= 2 358 mg/kg soil dw
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
respiration rate
Remarks on result:
other: The respiration rate was increased for the first 6 days. Thereafter, the respiration rate was very similar if not identical to the control.
Reported statistics and error estimates:
Analysis of variance was performed on log10 transformed Fco2 values (GenStat Release 7.2, Lawes Agricultural Trust, Rothamsted, UK). The transformation was necessary to stabilise the withinday variance. Significant differences between averages were calculated, and averages (treatment by day) were back transformed for expression in the original units. Standard errors of averages were estimated by statistical differentials (Kempthorne & Folks 1971). For each replicate, Fco2 data were integrated over time by the trapezoidal rule, and treatment responses expressed as differences with respect to the control.
Validity criteria fulfilled:
not applicable
respiration inhibition test (without glucose addition) NOEC >= 2358 mg/kg
Executive summary:

The study from Kelliher et al. 2007 investigated the respiration of natural soil from grassland after urea application with a rate of 2358 mg/kg. The test was performed similar to the OECD 217 with the deviation that no glucose was added and that the test duration was 24 instead of 28 days. Within the first 6 days after urea addition, the respiration was significantly increased when compared to the untreated control. After 6 days, the respiration rate of the samples which received urea were not statistically significant to the control.

Based on the results of this study, the NOAEC is >= 2358 mg urea/kg.

Description of key information

NOEC as determined in the study similar to the OECD 217 was > 2358 mg urea/kg dw

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:
2 358 mg/kg soil dw

Additional information

The most relevant study for this endpoint is from (Kelliher et al, 2007). The NOEC as determined in the study similar to the OECD 217 was > 2358 mg urea/kg dw.


This information demonstrate that terrestrial microorganisms are less sensitive to urea than earthworms and therefore the terrestrial plants are less relevant for the risk assessment of urea than earthworms.


Further long-term data are available from Zhang et al. (2008) and Zhang et al. (2019). These studies were performed without adjusting the pH. The studies resulted in a NOAEC of 457 mg urea/kg dw Zhang et al. (2008) and > 33 mg/kg dw. Both studies have limited relevance for the risk assessment of the effect of urea on microorganisms since it deviated from good agricultural practice in regard to the pH adjustment which resulted in a drop of the pH-value with increasing application amount.