Registration Dossier

Diss Factsheets

Ecotoxicological information

Toxicity to soil microorganisms

Currently viewing:

Administrative data

Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Soil microbial toxicity of eight polycyclic aromatic compounds: Effects on nitrification, the genetic diversity of bacteria, and the total number of protozoans
Author:
Sverdrup LE, Ekelund F, Krogh PH, Nielsen N, Lohnson K
Year:
2002
Bibliographic source:
Environ Toxicol Chem 21, 1644-1650

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
ISO 14238
Deviations:
yes
Remarks:
drying/dehumidification, use of solvent, addition of fresh inoculum derived from soil
Principles of method if other than guideline:
Comparative study including four aromatic and four heteroaromatic compounds. In addition to nitrification, other parameters were analysed (the genetic diversity of bacteria, total number of protozoans).
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Phenanthrene
EC Number:
201-581-5
EC Name:
Phenanthrene
Cas Number:
85-01-8
Molecular formula:
C14H10
IUPAC Name:
phenanthrene
Test material form:
solid
Details on test material:
- Substance type: organic
- for additional information see study record
Specific details on test material used for the study:
TEST MATERIAL
- Name of test material (as cited in study report): phenanthrene
- Source of test material: Sigma-Aldrich, St. Louis, MO, USA
- Analytical purity: > 96 %

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 10 mg/kg soil dw (low conc.) and 1000 mg/kg soil dw (high conc.)
- Sampling time: start of the experiment
- Sample size: 1000 mg soil for low concentration and 100 mg soil for high concentration

Test substrate

Vehicle:
yes
Remarks:
acetone
Details on preparation and application of test substrate:
APPLICATION OF TEST SUBSTANCE TO SOIL
- Method of preparation of test solutions: a stock solution in acetone was prepared that corresponded to the highest test dosage. For each test concentration, dilutions from the stock solution with acetone were made such that 4 mL of the resulting acetone solution contained the amount of test material required to produce the final test concentration after mxing with soil.
- Method of mixing into soil: 4 mL acetone solution for each of the treatments (equal volumes) was added to a 20 g soil dw in a glass vessel and thoroughly mixed into the soil resulting in the desired test concentration. The solvent was evaporated for 24 h.
- Controls: without and with solvent (soil samples were treated the same way as the test samples but using acetone without test substance)

VEHICLE
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution): not specified
- Evaporation of vehicle before use: yes

AMENDMENT OF SOIL
- Nitrogen source: horn meal (Solsikken, Lina, Denmark) at 1 ± 0.05 g/kg soil dw was added to each glass vessel (horn meal nitrogen content: 9.1 %, C/N ratio. 3.48 (mol/mol))
- Inoculum: 3.5 mL inoculum prepared from fresh Askov soil was added to each soil sample (glass container with 20 g soil dw) and thoroughly mixed. The Askov soil samples used in the test were pretreated (dried at 80 °C). Therefore inoculum from fresh Askov soil (stored at 5 °C for a maximum of 3 days) was added to the test samples.
preparation of inoculum according to a procedure described by Lindahl and Bakken (1995, FEMS Microbiol Ecol 16:135-142). In general, a slurry of 20 g fresh soil in 180 mL of deionised water was prepared by repeated mixing with a kitrchen blender for one minute followed by cooling on ice. The amount of water within the 3.5 mL inoculum added to the microcosms corresponded to 57 % of the water holding capacity of the soil. The population density of microorganisms added was estimated to be 2 % of that in the fresh material (100 % extraction efficiency assumed).

Test organisms

Test organisms (inoculum):
soil

Study design

Total exposure duration:
4 wk

Test conditions

Test temperature:
20 °C
Moisture:
57 ± 5 % of water holding capacity; gravimetric determination of water loss;
twice a week during incubation period water was added corresponding to water loss
Organic carbon content (% dry weight):
1.6
Nitrogen content (% dry weight):
0.443
Details on test conditions:
TEST SYSTEM
- Test container: cylinders (5 cm height, 3.5 cm diameter); sealed with plastic lids; upper lid perforated with five holes (made by a needle); at the start of the test, the inoculated soil samples (see above) were tranferred from the glass vessels to the test containers
- Amount of soil: 20 g
- No. of replicates per concentration: 3
- No. of replicates per control: 3
- No. of replicates per vehicle control: 3

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Soil test substrate: Danish agricultural soil
- Geographic location: Askov, Jutland, Denmark
- Soil texture (if natural soil): sandy loam
- Composition: coarse sand (0.2 – 2 mm) 38.4 %,
fine sand (0.063 – 0.2 mm) 23.6 %,
coarse silt (0.02 – 0.063 mm) 10.0 %,
fine silt (0.002 – 0.02 mm) 12.3 %,
clay (<0.002 mm) 13 %,
humus 2.8 %
- Organic carbon content: 1.6 %
- Soil pH: 6.2, no adjustment
- Density: 1.135 g/cm³ dry soil
- Maximum water holding capacity (in % dry weight): no data
- CEC (Cation exchange capacity): 8.14 meq/100 g
- Pretreatment of soil prior to use: drying at 80 °C for 24 h, sieving (2 mm mesh)

ADDITION OF INOCULUM TO SOIL SAMPLES: yes; prepared acc. to Lindhahl and Bakken (FEMS Microbiol. Ecol.,1995) (see above under 'Details on preparation and application of test substrate')

EFFECT PARAMETERS MEASURED after incubation period: soil nitrification, bacterial diversity (bacterial DNA), number of total protozoans (amoebae and heterotrophic flagellates) and heterotrophic flagellates

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY: no data
Nominal and measured concentrations:
Nominal concentrations: 1, 3, 10, 30, 100, 300, 1000, 3000 mg/kg soil dw;
Measured levels prior to test: 10 and 1000 mg/kg soil dw, recovery: 9.6 and 880 mg/kg soil dw;
Mean value for measured/nominal concentrations: 0.88 (mean recovery rate)
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
4 wk
Dose descriptor:
NOEC
Effect conc.:
26 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Key result
Duration:
4 wk
Dose descriptor:
EC10
Effect conc.:
42 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Remarks on result:
other: 95% C.I.: 0 - 70 mg/kg soil dw
Key result
Duration:
4 wk
Dose descriptor:
EC50
Effect conc.:
250 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Remarks on result:
other: 95% C.I.: 220 - 380 mg/kg soil dw
Duration:
4 wk
Dose descriptor:
other: EC5
Effect conc.:
2 400 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: total number of protozoa
Duration:
4 wk
Dose descriptor:
other: EC5
Effect conc.:
250 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: number of heterotrophic flagellates
Details on results:
Nitrogen mineralisation was generally the most sensitive of the three endpoints measured.
[Note: All substances affected nitrate production from > 20 - < 100 mg/kg soil dw, except acridine, which showed no substantial toxciity at ≥ 1000 mg/kg soil dw (Report Table 2)].
Reported statistics and error estimates:
Nitrate production: NOEC: ANOVA and Dunnett's procedure; EC10 and EC50: Linear interpolation
Protozoans: EC5 instead of NOEC: Three parameter relationship (by means of SigmaStat)

Applicant's summary and conclusion