Soil Biology
Series Editor: Ajit Varma 5
Volumes published in the series
Vo lume 1
A. Singh, O.P. Ward (Eds.)
Applied Bioremediation and Phytor emediation
2004
Vo lume 2
A. Singh, O.P. Ward (Eds.)
Biodegradation and Bioremediation
2004
Vo lume 3
F. Buscot, A. Varma (Eds.)
Microorganisms in Soils: Roles in Genesis and Functions
2005
Vo lume 4
S. Declerck, D G. Strullu, J.A. Fortin (Eds.)
In Vitro Culture of Mycor rhizas
2005
Rosa Margesin
Franz Schinner (Eds.)
Manual for Soil Analysis –
Monitoring and Assessing
Soil Bioremediation
With 31 Figures
123
Prof. Dr. Rosa Margesin
Leopold Franzens University
Institute of Microb iology
Technikerstr. 25

E
X Jelonek, Schmidt & Vöckler GbR, Leipzig, Germany
31/3150-YL - 5 4 3 2 1 0 - Printed on acid-free paper
Preface
The increasing use of soil bioremediation technologies requires new con-
cepts and methods to assess the feasibility of a remediation technology
and to monitor the success of the treatment. The knowledge of the reac-
tion of the soil microflora to contamination facilitates the optimization of
biodegradation. Manual of Soil Analysis – Monitoring and Assessing Soil
Bioremediation differs from other books on soil analysis in that the moni-
toring and assessing of soil bioremediation are the central themes.
In this comprehensive laboratory manual, sampling, pretreatment and
storage of soil, feasibility studies for soil bioremediation, and the most
important methods to analyze physical, chemical, and biological soil pa-
rameters are presented. Chapters written by experts for those involved in
resear ch, teaching, and routineanalyses outline molecular and immunolog-
ical techniques, the use of conserved internal markers, radiorespirometry,
bioreporter technology, the interpretation of fatty acid profiles, soil mi-
cro bial and enzyma tic methods, and the assessment of ecotoxicity using
bioassays. Particular emphasis has been placed on the comprehensible and
complete description of the experimental procedures. The broad spectrum
of modern soil biological methods provides an exc e llent complementation
of traditional soil investigation and characterization. Our book, however,
does not claim to present all modern methods available, it rather contains
a selection of the most suitable methods for investigating contaminated
soil. More biological methods can be found in our volume Methods in Soil
Biology (Schinner,
¨
Ohlinger, Kandeler and Margesin 1996, Springer).
We are most grateful to the authors for their excellent contributions and

1.6.2 Specific Considerations for Biological Parameters 42
1.6.3 Preparingthe Samples After Storage 44
References 44
2 Determination of Chemical and Physical Soil Properties 47
Berndt-Michael Wilke
2.1 Soil Dry Mass and Water Content 47
2.2 Water-Holding Capacity 50
VIII Contents
2.3 Bulk Density – Total Porosity 52
2.3.1 Core Method 52
2.3.2 Excavation Method 54
2.3.3 Clod Method 57
2.4 Water Retention Characteristics – Pore Size Distribution 59
2.4.1 Determination of Soil Water Characteristics
Using Sand, Kaolin, and Ceramic Suction Tables 62
2.4.2 Determination of Soil Water Characteristics
by Pressure Plate Extractor 65
2.5 Soil pH 68
2.6 Soil Organic Matter – Soil Organic Carbon 71
2.6.1 Dry Combustion Method 72
2.6.2 Loss On Ignition Method (LOI) 74
2.7 Soil Nutrients: Total Nitrogen 76
2.7.1 Dry Combustion Method (“Elemental Analysis”) 77
2.7.2 Modified Kjeldahl Method 79
2.8 Soil Nut rients: Inorganic Nitrogen 82
2.8.1 Extraction 83
2.8.2 Quantification of Nitrate Nitrogen 84
2.8.3 Quantification of Ammonium Nitrogen 86
2.9 Soil Nutrients: Phosphorus 87
2.9.1 Extraction of Total Phosphorus 88

5.2.2 Selective Microbial Enrichment 134
5.2.3 Controls 135
5.2.4 Soil Microcosms 136
5.2.5 Slurry Bioreactors 137
5.2.6 Land Treatment 139
5.2.7 Composting 140
5.2.8 Scale-Up 141
5.3 ProcessMonitoring and Evaluation 142
5.4 Bioaugmentation 143
5.5 Effect of Surfactants 144
5.5.1 Screening of Microbial Cultures
for Biosurfactant Production 145
5.5.2 Effect of Biosurfactants 146
5.5.3 Effect of Chemical Surfactants 146
5.6 Optimization of Environmental Conditions 147
5.7 Optimization of Nutritional Factors 148
5.8 Conclusions 150
References 151
6 Feasibility Studies for Microbial R emediation
of Metal-Contaminated Soil 155
Franz Schinner , Thomas Klauser
References 159
7 Feasibility Studies for Phytoremediation
of Metal-Contaminated Soil 161
Aleksandra Sas-Nowosielska, Raf al Kucharski, Eugeniusz Malkowski
7.1 Introduction 161
7.2 Phytoextraction 161
7.2.1 Treatability Study 162
7.2.2 Full-Scale Application 166
7.2.3 Conclusions 170

References 228
11 Bioreporter Technology for Monitoring Soil Bioremediation 233
Steven Ripp
11.1 General Introduction 233
11.2 A n Overview of Reporter Systems
for Soil Bioremediation Application 235
11.3 Single Point Measurements of Soil Contaminants 241
11.4 Continuous On-Line Vapor Phase Sensing
of Soil Contaminants 244
11.5 Quantification of Soil-Borne lux-Tagged Microbial Popula-
tions Using Most-Probable-N umber (MPN) Analysis 247
References 249
Contents XI
12 Interpretation of Fatty Acid Profiles of Soil Microorganisms 251
David B. Hedrick, Aaron Peacock, David C. White
12.1 ObtainingFatty Acid Profiles from Soil Samples 251
12.2 Transforming Fatty Acid Peak Areas
to Total Microbial Biomass 252
12.3 Calculation and Interpretation of Community Structure 254
12.3.1 Standard Community Structure Method 254
12.3.2 Custom Community Structure Methods 255
12.3.3 Factor Analysis 255
12.4 Calculation and Interpretation
of Metabolic Stress Biomarkers 256
12.5 Naming of Fatty Acids 257
References 258
13 Enumeration of Soil Microorganisms 261
Julia Foght, Jackie A islabie
13.1 Sample Preparation and Dilution 261
13.2 Direct (Microscopic) Enumeration 264

17.4 Dehydrogenase Activity 316
References 319
18 Assessment of Ecotoxicity of Contaminated Soil Using Bioassays 321
Adolf Eisentraeger, Kerstin Hund-Rinke, Joerg Roembke
18.1 General Introduction: Strategy 321
18.2 Sample Preparation 323
18.3 Water-Extractable Ecotoxicity 330
18.3.1 Vibrio fischeri Luminescence-InhibitionAssay 330
18.3.2 Desmodesmus subspicatus Growth-Inhibition Assay 331
18.4 Water-Extractable Genotoxicity 332
18.4.1 The umu Test 332
18.4.2 Salmonella/Microsome Assa y (Ames Test) 333
18.5 Habitat Function:
Soil/Microorganisms, Soil/Soil Fauna, Soil/Higher Plants 334
18.5.1 Respiration Curve Test 334
18.5.2 Ammonium Oxidation Test 337
18.5.3 Combined Earthworm Mortality/Reproduction Test 340
18.5.4 Collembola Reproduction Test 342
18.5.5 Plant Growth Test 344
18.5.6 Test Performance for the Derivation
of Threshold Values 346
18.6 Combined Performance of B ioassays and Assessment of the
Results 348
18.6.1 Water-Extractable Ecotoxic Potential 348
18.6.2 Water-Extractable Genotoxicity 349
18.6.3 Assessment of the Habitat Function 350
18.6.4 Overall Assessment – Combined Strategy 353
References 355
Subject Index 361
Contributors

Department of Soil Biology and Plant Nutrition, University of Kassel, Nord-
bahnhofstr. 1a, 37213 Witzenhausen, Germany
Jørgensen, Kirsten S.
Finnish Environment Institute, P.O. Box 140, 00251 Helsinki, Finland
Klauser, Thomas
Institute of Microbiology, Leopold Franzens University, Technikerstrasse
25, 6020 Innsbruck, Austria
Kucharski, Rafal
Land Management Department, Institute for Ecology of Industrial Areas,
Kossutha 6 St, 40–833 Kato wice, Poland
Kuhad, Ramesh C.
Department of Biotechnology, Kurukshetra University, Kurukshetra –
136119, Haryana, India
Lindstrom, Jon E.
Shannon & Wilson, Inc., 2355 Hill Road, Fairbanks, Alaska 99709; Institute
of Arctic Biology, University of Alaska Fairbanks, Fairbanks, Alaska 99775,
USA
Malkowski, Eugeniusz
DepartmentofPlantPhysiology,FacultyofBiology and EnvironmentalPro-
tection, University of Silesia, Jagiello
˜
nsk a 28 St, 40–032 Katowice, Poland
Margesin, Rosa
Institute of Microbiology, Leopold Franzens University, Technikerstrasse
25, 6020 Innsbruck, Austria
Paetz, Andreas
Deutsches Institut für Normung (DIN), Normenausschuss Wasserwesen
(NAW), 10772 Berlin, Germany
Peacock, Aaron
Center for Biomarker Analysis, U niversity of Tennessee, 10515 Research

N2L 3G1
Suortti, Anna-Mari
SGS Inspection Services, Syväsatamantie 24, 49460 Hamina, Finland
Tien, Albert J.
Holcim Group S up port Ltd Corporate Social Responsibility Occupational
Health and Safety, Im Schachen, 5113 Holderbank, Switzerland
XVI Contributors
Ulfig, Krzysztof
Institute for Ecology of Industrial Areas, 40–844 Katowice, 6 Kossutha,
Poland
Ward, Owen P.
Department of Biology, University of Waterloo, Waterloo, Ontario, Canada
N2L 3G1
Whit e, David C.
Center for Biomarker Analysis, U niversity of Tennessee, 10515 Research
Drive, Suite 300, Knoxville, Tennessee 37932–2575, USA
Whyte, Lyle G.
Dept. of Natural Resource Sciences, McGill University, Macdonald Cam pus
21, 111 Lakeshore Road, St. Anne de Bellevue, Quebec, Canada H9X 3V9
Wilke, Berndt-Michael
Institute of Ecology, Berlin University of Technology, Franklinstrasse 29,
10587 Berlin, Germany
1
Soil Sampling and Storage
Andreas Paetz, Berndt-Michael Wilke
1.1
Objective of Soil Sampling
1.1.1
Principal Objectives
General

c
 Springer-Verlag Berlin Heidelberg 2005
2A.Paetz,B M.Wilke
Some consideration should be given to the degree of detail and precision
that will be required, and also to the manner in which the results are
to be expressed and presented, for example, concentrations of chemical
substances, maximum and minimum val ues, arithmetic means, median
values, etc. Additionally, a list of parameters of interest should be compiled
and the relevant analytical procedures consulted; these will usually give
guidance on precautions to be observed during sampling and subsequent
handling of soil samples.
It may often be necessary to carry out an exploratory sampling-and-
analysis program before the final objectives can be defined. It is important
to take into account all relevant data from previous programs at the same
or similar locations and other information on local conditions. Previous
personal experience can also be very valuable. Time and money allocated
to the design of a proper sampling program are usually well justified be-
cause they ensure that the required information is obtained efficiently and
economically.
It is emphasized that complete achievement of objectives of soil inves-
tigations depends mainly on the design and execution of an appropriate
sampling program. The four principal objectives of soil sampling may be
distinguished as follows and are discussed below:
• Sampling for determination of general soil quality
• Sampling for characterization purposes in preparation of soil maps
• Sampling to sup port legal or regulatory action
• Sampling as part of a hazard or risk assessmenthack
Theutilizationofthesoilandsiteisofvaryingimportancedependingon
the primary objective of an investigation. For example, while consideration
of past, present, and future site use is particularly relevant to sampling for

Sampling may be required to establish base-line conditions prior to an
activity that might affect the composition or quality of soil, or it may be re-
quiredfollowing an anthropogenic effect such as the input of an undesirable
material that may be from a point or a diffuse source. Sampling strategies
need to be developed on a site-specific basis. To adequately support legal
or regulatory action particular attention should be paid to all aspects of
quality assurance including, for example, “chain-of-custody procedures.”
The guidance given in ISO 10381-5 (1995) is particularly relevant; that in
ISO 10381-4 (2003) may also be relevant.
Sampling for Hazard and Risk Assessment
When land is contaminated with chemicals and other substances poten-
tially harmful to human health and safety or to the environment, it may
be necessary to carry out an investigation as a part of a hazard and/or risk
assessmen t i.e., to determine the nature and exten t of contamination, to
identify hazards associated with the contamination, to identify potential
targets and routes of exposure, and to evaluate the risks relating to current
and future use of the site and neighboring land. A sampling program for
risk assessment (in this context: phase I, phase II, phase III, and phase IV
investigations) may have to comply with legal or regulatory requirements,
and careful attention to sample integrity is recommended. Sampling strate-
gies should be developed on a site-specific basis. The guidance given in
ISO 10381-5 (1995) is particularly relevant, and that in ISO 10381-4 (2003)
may also be relevant.
4A.Paetz,B M.Wilke
1.1.2
Specific Objectives
General
Depending on the principal objective(s) it will usually be necessary to
determineforthebodyofsoilorpartthereof:
• The nature, concentrations, and distribution of naturally occurring sub-

on other soil horizons or on other environmental compartments, e.g.,
the transfer of substances from a soil into a plant
6. To study the effect of waste disposal, including the disposal of sewage
sludge on a soil (which, apart from contributing to the pollution load,
may produceother chemical reactions such as the formation ofpersistent
compounds, metabolites, or the evolution of gases, such as methane)
7. To identify and quantify products released byindustrial processes and by
accident (usually done by in vestigation of suspect sites or contaminated
sites)
8. To evaluate soil derived from construction works with view to possible
or further utilization of such soils or disposal as waste
Commonly, sampling strategies are employed that require samples to
be taken either from identifiable soil horizons or from specified depths
(below ground surface). It is best to avoid mixing the two approaches,
particularly when sampling natural strata, as this can make it difficult to
compare results. However , a coherent combination of the two approaches
can sometimes be useful on old industrial sites where there is variation in
both the nature of fill and the depth of penetration of mobile contaminants
in to the gro und. i.e., where there are two independent reasons for changes
in soil/fill properties.
Knowledge of the way in which particular chemical substances tend to be
distributed between the different compartments (air, soil, water, sediment,
and living organisms) is advantageous f or the design of some sampling
programs. Similarly,knowledge ofthe behavior ofliving organisms affected
by chemical substances, or that influence the availability of substances due
to microbiological procedures, is also advantageous.
Sampling for the Determination of Physical Soil Parameters
The sampling of soil for the determination of some physical properties re-
quires special consideration since the accuracy and extrapolation of mea-
sured data rely on obtaining a sample that retains its in situ structural

including both fauna and flora in the micro and macro range. Ecotoxico-
logical questions are usually given first priority. For example, tests should
be made to verify the effects of chemicals added to the soil on life-forms
and also the possible effects of life-forms in the soil on plants (e.g., high-
valuecrops)andontheenvironment,especiallyonhumanhealth.Insome
cases, biological soil test procedures operate with fully artificial soils, but
normally the major task of s ampling i s to ch oose a reliable soil or s ite to
carry out the tests. Sampling for the assessment of aerobic microbial pro-
cesses is covered in ISO 10381-6 (1993). The sampling for the assessment
of anaerobic processes is described in ISO 15473 (2002).
1.2
Selection of Sampling Technique
The selection of appropriate sampling equipment depends on the objective
of sampling and should be done after consideration by the analyst or
scientist responsible for subsequent determination. ISO 10381-2 (2002)
gives guidance on commonly used equipment for sampling soil and related
material. Parts 4, 5, and 6 of ISO 10381 describe needs for specific purposes
within their scopes.
1 Soil Sampling and Storage 7
1.3
Sampling Strategy
1.3.1
General
The strategy for the site investigation (whether preliminary, exploratory,
or main) will be determined by the obj ectives. For example, the different
requirements of site investigations for the purpose of selling, determining
whether contaminationis present as suspected,or redevelopment will influ-
ence the spacing of sample locations and the number of samples analyzed,
and hence the cost of the investigation.
Before embarking on any phase or stage of investigation it is important

obtained, i.e., the amount of work required will vary with the age of the
site, the complexity of its historic usage, the complexity of the underlying
geology, etc.
It shall be remembered that the contamination on a site may be more
complex than initially indicated (for example by current usage) and ade-
quateinformationonsitehistoryshouldalwaysbeobtainedintheprelim-
inary investigation.
Desk Study
This includes collection of relevant information on the site, e.g., location,
infrastructure, utilization, history. Possible sources of this information are
publications, maps (check accuracy of map used), aerial photographs, and
satelliteimageryfrom,e.g.,landsurveyor’soffices,geologicalsurveys,water
managemen t boards, industrial inspection boards, mining boards, mining
companies, geotechnical institutions, regional and local (city) archives,
agricultural and forestry authorities, and building supervisory boards.
Particularly important is information on the physical and chemical prop-
erties and the possible spatial distribution of the soil parameter under
investigation; special attention must be paid to geological features such as
stratigraphy and hydrogeology.
Site Reconnaissance
A visit of the site should be part of the preliminary investigation, prefer-
ably in conjunction with the desk study, although it may be independent.
Depending on the local variability of the site and the technical difficulty
of the planned inv estigation, an experienced person should be chosen fo r
this task. Such a visit gives a first impression about the correlation of ex-
isting maps with reality, and it will provide much additional information
in a comparatively short time. In some cases, it may be necessary to draw
a first or additional map at this stage.
Samples are not often taken during preliminary investigation s; if they
are, they are usually needed to obtain an overview of the kind of soil in

remedial measures.
1.3.4
Main Site Investigation
The main site investigation quantitatively determines the amoun t and spa-
tial distribution of contaminants, their mobile and mobilizable fractions,
andthepossibilitiesofspreadintothe environment. Also included is the
possible future development of the contamination situa tion. This will in-
volve the collection and analysis of soil or fill, surface water , ground water ,
and soil gas samples in order to obtain the information necessary to enable
a full assessment of the hazards presented by the contamination to humans
and other potential receptors and also to enable appropriate containment
or remediation actions to be identified (sometimes) together with an initial
estimate of costs. The analysis of samples can be supported by model cal-
culations and investigation techniques that do not make use of sampling.