Dissertation Thesis in Environmental Chemistry

Effects of selected environmental pollutants

on soil microbial community in laboratory
and field studies

Jitka Černohlávková

2009

Masaryk University, Faculty of Science

RECETOX - Research Centre for Environmental Chemistry and Ecotoxicology

Brno, Czech Republic

Supervisor:
prof. RNDr. Ivan Holoubek, Ph.D.

Consultant:
RNDr. Jakub Hofman, Ph.D.
BIBLIOGRAPHIC IDENTIFICATION

Author: Jitka Černohlávková

Title of dissertation: Effects of selected environmental pollutants on

soil microbial community in laboratory and field
studies

Title of dissertation (in Czech): Efekty vybraných polutantů životního prostředí na

půdní mikrobiální společenstva v laboratorních a
terénních studiích

Ph.D. study program: Chemistry

Specialization: Environmental chemistry

Supervisor: Prof. RNDr. Ivan Holoubek, Ph.D.

Year of defense: 2009

Keywords: soil ecotoxicology, microorganisms, variability of

microbial properties, laboratory tests, persistent
organic pollutants, pesticides, soil contamination,
bioindication

Keywords (in Czech): půdní ekotoxikologie, mikroorganismy, variabilita

mikrobiálních paramterů, laboratorní testy,
perzistentní organické polutanty, pesticidy,
kontaminace půd, bioindikace
© Jitka Černohlávková, Masaryk University, 2009
Aknowledgements

I would like to thank

ƒ my supervisor prof. RNDr. Ivan Holoubek, CSc. for theoretical backgrounds

and working conditions;

ƒ my supervisor RNDr. Jakub Hofman, Ph.D. for numerous advices and ideas,
motivation and support during all my postgraduate study;

ƒ RNDr. Jitka Bezchlebová, Ph.D. and for her invaluable help and friendly
cooperation;

ƒ all colleagues and friends from RECETOX, especially from the laboratory of
soil ecotoxicology and my office-mates, for many helps, support and pleasant
study atmosphere.

Special thanks belong to my family for support and encouragement during my studies.
Contents

TABLE OF CONTENTS

List of abbreviations.................................................................................................................. 7
Abstract ...................................................................................................................................... 8
Abstrakt (abstract in czech)...................................................................................................... 9
List of original papers ............................................................................................................. 10
The author's contribution in the papers ................................................................................ 10
Aims of the study..................................................................................................................... 11
1 Introduction to ecotoxicology of soil microorganisms............................................... 12
1.1 Microbial parameters .............................................................................................. 12
1.1.1 Microbial biomass .............................................................................................. 13
1.1.2 Parameters of carbon cycling............................................................................. 13
1.1.3 Parameters of nitrogen cycle ............................................................................. 16
1.1.4 Enzymatic activity .............................................................................................. 17
1.1.5 Microbial diversity .............................................................................................. 17
2 Variability of soil microbial parameters ....................................................................... 19
2.1 Spatial variability..................................................................................................... 19
2.2 Temporal variability................................................................................................. 22
2.3 Variability of soil sampling and manipulation .......................................................... 24
2.3.1 Soil sampling...................................................................................................... 24
2.3.2 Sample handling and manipulation .................................................................... 25
2.3.3 Storage of soil samples...................................................................................... 26
2.4 Study of soil variability (paper I).............................................................................. 28
2.5 Effect of soil storage on sensitivity of microorganisms to toxicants......................... 29
3 Laboratory tests with soil microoganisms .................................................................. 31
3.1 Procedures and standard methods of laboratory tests ........................................... 31
3.2 Laboratory tests at RECETOX................................................................................ 32
3.2.1 Evaluated microbial parameters......................................................................... 33
3.3 Persistent organic pollutants................................................................................... 34
3.3.1 Effects of POPs on soil microorganisms ............................................................ 36
3.4 Laboratory tests with selected POPs ...................................................................... 39
3.4.1 Effects of chlorinated paraffins (paper II) ........................................................... 39
3.4.2 Effects of toxaphene (paper III).......................................................................... 41
3.5 Effects of pesticides on soil microorganisms .......................................................... 44
3.5.1 Effects of selected pesticides (paper IV) ............................................................ 47
4 Soil microorganisms as bioindicators ......................................................................... 50
4.1 Soil quality .............................................................................................................. 50
4.1.1 Microorganisms as indicators of soil quality ....................................................... 51
4.1.2 Soil quality assessment...................................................................................... 53
4.2 Microorganisms as indicators of soil contamination................................................ 56
4.3 Case study in Zlín region ........................................................................................ 57
4.3.1 Effects of floods on soil microbial properties (poster 1)...................................... 58
4.3.2 Relationship between soil microbial properties and contamination (poster 2)... 59
4.4 Effect of road de-icing salt on soil microorganisms (paper V) ................................. 62
5 Overall conclusions ....................................................................................................... 63
6 References...................................................................................................................... 64

Appendices 74
1 – Paper I 74
2 – Paper II 92
3 – Paper III 99
4 – Paper IV 108
5 – Paper V 115
6 – Poster 1 123
7 – Poster 2 125
8 – Curriculum vitae 127

6
 List of abbreviations

LIST OF ABBREVIATIONS

ATP Adenosine triphosphate

BBA Biologische Bundesanstalt für Land- und Forstwirstschaft
BDE Brominated diphhenyl ethers
BOD Biochemical oxygen demand
BR Basal respiration
CEC Cation exchange capacity
CFE Chloroform fumigation extraction
CPPs Crop protection products
DGGE Denaturing Gradient Gel Electrophoresis
EPPO European and Mediterranean Plant Protection Organisation
FA Fulvic acids
FAO Food and agriculture Organisation of the United Nations
HA Humic acids
HCB Hexachlorobenzene
HCH Hexachlorocyclohexane
ISO International Organization for Standardization
LOEC Lowest observed effect concentration
MDS Minimum data set
NOEC No observable effect concentration
OECD Organisation for Economic Co-operation and Development
PAHs Polycyclic aromatic hydrocarbons
PAMO Potential ammonification
PAO Potential ammonium oxidation
PBTs Persistent bioaccumulative and toxic chemicals
PCBs Polychlorinated biphenyls
PCDDs Polychlorinated dibenzo-dioxins
PCDFs Polychlorinated dibenzo-furans
PCFs Perfluorochemicals
PCP Pentachlorophenol
PLFAs Phospholipid fatty acids
PNEC Predicted no effect concentration
POPs Persistent organic pollutants
PTS Persistent toxic substances
SCCPs Short chain chlorinated paraffins
SETAC Society for Environmental Toxicology and Chemistry
SIR Substrate-induced respiration
TCCD 2,3,7,8 - tetrachlorodibenzo-p-dioxin
TCDF 2,3,7,8 - tetrachlorodibenzofuran
TEQ Toxic Equivaletns
UN-ECE United Nations Economic commission for Europe
UNEP United Nation Environment Programme
WHC Water holding capacity

7
Abstract

ABSTRACT

Soil microoranisms play significant role in transformation and decomposition of organic

matter, food, nutrient and energy cycling in terrestrial ecosystems. They are important
in global cycle of carbon and nitrogen. Fertility, stability and production potential of soil
are also very dependent on microbial activities. These processes and whole microbial
communities could be negatively affected by environmental pollution, application of
pesticides and contamination of soil by other chemicals. This can lead to decrease of
soil quality and loss of their functions. The object of this work was to study effects of
environmental pollutants on soil microorganisms and relationship between soil
contamination and microbial activities under laboratory conditions as well as in field
studies. The interest was focused on assessing soil microbial biomass, respiration
activities and nitrogen mineralization.
Soil is very heterogeneous and complex system and neither microorganisms nor other
soil components are regularly distributed. This spatial variability often complicates soil
research. Moreover fluctuation of temperature, soil properties and other abiotic factors
influence microbial activities. Soil sampling, manipulation and storage are essential
steps that affect results of the analysis. In our study, we assessed spatial variability of
microbial properties at sampling sites and effects of various storage conditions on soil
microorganisms. According to the results, included in this work, variability and effects
differ according to soil type. The most changes in microbial communities were induced
by storage conditions (drying and freezing of soil). Storage at 4°C appeared most
appropriate with only minor changes up to 8 weeks of storage.
The impact of selected chemicals on microbial properties was evaluated in laboratory
tests according to standard procedures that consist of laboratory incubation of a
natural soil spiked with the tested chemical. In the tests performed during this work,
effects of two persistent organic pollutants (POPs) – chlorinated paraffins and
toxaphene – and two fungicides were assessed. Results showed that toxicity of both
POPs to microbial parameters assessed was very low even at the highest tested
concentration (thousand of ppm), probably due to their sorption on soil particles and
low bioavailability. The fungicides revealed significant adverse effect on nitrogen
transformation, however, this was rather short-term. In general, the laboratory
experiments showed that parameters of nitrogen transformation were more negatively
affected than overall microbial biomass or respiration.
Next concern was engaged in field monitoring of soil microbial communities. We were
interested mainly in evaluating relationship between microbial parameters, soil
properties and anthropogenic contamination. The bioindication potential of soil
microorganisms was assessed in fluvisols in the small river basin with high
environmental load in Zlín region. Despite significant soil contamination at some sites
results indicated that microbial parameters were more influenced by physical-chemical
soil properties, which masked the possible effects of contamination. Application of road
de-icing salt and its impact on soil properties near the road was the object of the next
study. The research showed on significant change in physical-chemical properties,
such us pH and base saturation, due to sodium ions accumulation. Microbial
parameters also indicated stress environmental condition and higher energy
requirement of soil microorganisms.

8
 Abstrakt (Abstract in Czech)

ABSTRAKT (ABSTRACT IN CZECH)

Půdní mikroorganismy hrají důležitou roli v procesech přeměny a rozkladu organické

hmoty v půdě, toku živin a energie v terestrických ekosystémech. Znečištění životního
prostředí, aplikace pesticidů a kontaminace půd dalšími polutanty může negativně
ovlivnit půdní mikroorganismy, což může vést k celkovému poklesu půdní kvality i
ztrátě samotných funkcí půdy. Předmětem této disertační práce bylo studovat vliv
vybraných polutantů životního prostředí na půdní mikroorganismy a vztahy mezi půdní
kontaminací a mikrobiálními aktivitami jak v laboratorních tak terénních studiích.
Pozornost byla zaměřena hlavně na posouzení celkové půdní mikrobiální biomasy,
respirační aktivity a parametrů mineralizace dusíku (amonifikace, nitrifikace).
Půda je velmi heterogenní a komplexní matrice, mikroorganismy ani ostatní složky
nejsou v půdě rovnoměrně rozloženy. Mikrobiální aktivity jsou navíc ovlivněny teplotou,
vlastnostmi půdy a dalšími abiotickými faktory, které se mění v čase. Vysoká
prostorová variabilita půdního prostředí často komplikuje jeho výzkum a správnou
interpretaci výsledků. Stěžejním krokem je přitom odběr, ale také manipulace a
skladování půdy před samotnou analýzou. V rámci této disertační práce byla
hodnocena prostorová variabilita mikrobiálních parametrů na lokalitě a vliv dalšího
zpracování, podmínek a délky uchování vzorků. Výsledky se do jisté míry lišily podle
typu půdy. Největší variabilita byla způsobena různými podmínkami skladování půdy
(zmrazením a vysušením vzorků). Jako nejvhodnější způsob skladování, který
způsoboval pouze malé změny v mikrobiálním společenstvu i po 8 týdnech, se ukázalo
skladování půdy ve 4°C.
Vliv vybraných polutantů na půdní mikrobiální společenstvo byl hodnocen
v laboratorních testech toxicity podle standardních postupů. Testy mají charakter
laboratorní inkubace přirozené půdy po aplikaci testovanou látkou. Během disertační
práce byl otestován vliv dvou perzistentních organických polutantů (POPs) -
chlorovaného parafínu a toxafenu - a dvou fungicidů. Výsledky ukázaly, že testované
POPs byly pro hodnocené mikrobiální parametry velmi málo toxické i v nejvyšších
testovaných koncentracích (tisíce ppm). Tento efekt byl způsoben pravděpodobně
jejich fyzikálně chemickými vlastnostmi – sorpcí na pevné částice a nízkou
biodostupností v půdním prostředí. Fungicidy naopak způsobily významnou, i když
pouze krátkodobou, inhibici amonifikace a nitrifikace. Vliv testovaných látek
v provedených laboratorních experimentech byl celkově více patrný u parametrů
mineralizace dusíku než celkové mikrobiální biomasy a respirace.
Další část disertační práce byla zaměřena na hodnocení stavu půdních
mikroorganismů v reálných ekosystémech. Zájem byl soustředěn na posouzení vztahu
mezi stavem mikrobiálních společenstev, reálnými přírodními podmínkami a
antropogenní kontaminací půd. Bioindikační potenciál půdních mikroorganismů byl
zkoumán na souboru nivních půd Zlínského kraje, které se vyznačovaly gradientem
antropogenního znečištění. I přes značnou kontaminaci některých lokalit byla
mikrobiální společenstva spíše ovlivněna variabilitou fyzikálně-chemických půdních
vlastností, která zastínila možný negativní vliv polutantů. Další práce se týkala
hodnocení vlivu aplikace chemického posypu silnic v zimním období na vlastnosti a
mikrobiální společenstva půd v blízkosti silnic. Zde byly zjištěny významné změny ve
fyzikálně-chemických vlastnostech půdy (nárůst pH), nárůst koncentrace sodíku
v blízkosti půd. Hodnocené mikrobiální parametry naznačovaly, pravděpodobně také
díky těmto změnám, zvýšené energetické nároky společenstva a stresující podmínky.

9
List of original paper

LIST OF ORIGINAL PAPERS

I. Černohlávková, J., Jarkovský, J., Nešporová, M., Hofman, J.:Variability of soil

microbial properties: effects of sampling, handling and storage; 2008 (submitted to
Ecotoxicology and Environmental Safety)

II. Bezchlebová, J., Černohlávková, J., Kobetičová, K., Lána, J., Sochová, I. and
Hofman, J. (2007): Effects of short chain chlorinated paraffins on soil organisms.
Ecotoxicology and environmental safety 67, p. 206-211

III. Bezchlebová J., Černohlávková, J., Lána, J., Sochová, I., Kobetičová, K. and
Hofman, J. (2007): Effects of toxaphene on soil organisms. Ecotoxicology and
environmental safety 68, p. 326-334

IV. Černohlávková, J., Jarkovský, J., Hofman, J. (2009): Effects of fungicides

mancozeb and dinocap on carbon and nitrogen mineralization in soils. Ecotoxicology
and environmental safety 72, p. 80-85

V. Černohlávková, J., Hofman, J., Bartoš, T., Sáňka, M., Anděl, P. (2008): Effects of
road de-icing salts on soil microorganisms, Plant Soil and Environment 54 (11), p. 479
– 489

THE AUTHOR'S CONTRIBUTION IN THE PAPERS

Paper I
Jitka Černohlávková participated in experimental design and analysed part of soil
samples for microbial parameters. She also participated in data evaluation and wrote
the manuscript.

Paper II
Jitka Černohlávková performed the toxicity test of short chain chlorinated paraffins on
soil microorganisms. She evaluated the data of microbial parameters and participated
in manuscript preparation.

Paper III
Jitka Černohlávková performed the toxicity test of toxaphene on soil microorganisms
and evaluated the results on microbial parameters. She participated in manuscript
preparation.

Paper IV
Jitka Černohlávková participated in experimental design, analysis of microbial
parameters and results evaluation. She wrote the manuscript and finalized the entire
article.

Paper V
Jitka Černohlávková performed the microbial analysis of soil and participated in data
evaluation. She wrote the manuscript and finalized the entire article.

10
 Aims of the study

AIMS OF THE STUDY

The topic of this work followed a diploma thesis of the author, which was engaged in
application of laboratory incubation of soil for ecotoxicity testing. During the diploma
work the laboratory test with indigenous soil microorganisms was successfully
introduced in laboratories of RECETOX. The dissertation was focused on the use of
soil microorganisms to evaluation the risks of several pollutants in laboratory tests.
Moreover the biondication potential of soil microorganisms in two field studies was
investigated.

The aims of this work were mainly:

ƒ to improve the method of laboratory testing and introduce further microbial

parameters that would enable to assess condition of microbial community in
more detail or to distinguish effects on different microbial groups and
processes. Special attention was pointed to nitrogen mineralization
(ammonification, nitrification) and long-term soil respiration,

ƒ to evaluate variability in results of microbial analyses with the emphasis on

effects of soil sampling, handling and storage,

ƒ to test effects of selected pollutants on microbial parameters in the laboratory

incubation of soil,

ƒ to evaluate relationship between soil contamination, microbial parameters and

other soil characteristics in the field studies.

11
Introduction

1 INTRODUCTION TO ECOTOXICOLOGY OF SOIL

MICROORGANISMS

Soil is very complex, heterogeneous and dynamic part of environment that consists of
living organisms and dead organic and inorganic matter. The soil represents a
favourable habitat for microorganisms and is colonized by a wide range of
microorganisms, including bacteria, fungi, algae, viruses and protozoa.
Microorganisms are found in large numbers in soil with bacteria and fungi being the
most prevalent. It is stated that usually more than 109 microorganisms are present per
gram of soil representing 4000 to 7000 different genomes and biomass of 300
to 3000 kg per ha (Ranjard and Richaume, 2001). The availability of nutrients is often
limiting for microbial growth in soil and thus most soil microorganisms is not
physiologically active in a given time. Regardless that, they present 80 – 90 % of whole
soil biota biomass and activity and are very important part of a soil ecosystem. They
form a robust community capable of surviving and functioning under extremes of
temperature, water availability, pH, energy resources, nutrient availability and salt
concentration. Microorganisms play a key role in transformation and decomposition of
organic matter in soil and are also involved in food, nutrient and energy cycles in
terrestrial ecosystems. They are essential for maintenance of soil stability and fertility.
Some soil microorganisms have been found to produce compounds (such as vitamins
and plant hormones) that can improve plant health and contribute to higher crop yield.
Due to huge genetic and function diversity soil microorganisms are subject of great
concern of scientists searching for new antibiotics, enzymes and other chemicals
useable in medicine, biotechnology or industry.

Form the ecological point of view soil is the main input of chemicals to the food chains.
Thanks to the position of microorganisms in soil structure elements and large active
surface they are strongly exposed to any soil contamination. A lot of chemicals and
pollutants can cause negative changes in their activities, quantity or diversity, which
consequently lead to decrease of soil quality. Parameters describing quantity, activity
or any other characteristic of soil microorganisms can be the first warning about
pollutants effects on soil and were included into minimal data sets of soil quality and
health indicators.

1.1 Microbial parameters

In comparison to other biotic systems, assessment of soil microorganisms does not

mostly deal with single species (their presence or abundance) but focuses on whole
microbial community and processes mediated by microorganisms. Several microbial
parameters are used to describe their state and function ability. Description of the most
important and used parameters was summarized already in my diploma and rigorous
thesis (Černohlávková, 2004, Černohlávková 2007). Because the microbial parameters
are discussed in relation with the presented studies, a brief overview is given at this
place. Moreover, some of them were used in the experimental part and basic
information is important for further understanding.

12
 Chapter 1

1.1.1 Microbial biomass

Determination of total microbial biomass is important parameter in soil microbiology.

Microbial biomass is fundamental for soil processes and the information about its total
amount serves as a basis to which other parameters could be related. In soil
microbiology, all living organisms smaller than 10 μm are taken into account, including
bacteria, fungi, algae, viruses and protozoa. The most important parts are, however,
bacteria and soil fungi. Total biomass is complex of active as well as dormant
microorganisms.

Several methods have been used for the estimation of microbial biomass in soil. The
methods can be divided into direct (e.g. microscopy) and indirect (quantifying
components of microbial cells). Direct counting and microscope technique gives
results that very closely represent the in situ soil conditions. Selective growth media
allow separating and quantifying of specific microbial populations. On the other hand
soil colloid particles could interfere in the microscopic analysis and only 0.1 – 10 % of
soil microorganisms grow in laboratory condition and thus the total numbers, based on
laboratory cultivation, are underestimated. These methods are also relatively time-
consuming. Despite the mentioned disadvantages, the direct methods are currently
used for soil monitoring purposes (Nielsen and Winding, 2002).

Indirect methods are generally cheaper, faster and easier to use than the direct
methods. They are based on quantifying the components, which are present in all
microbial cells in sufficient amount. For this purpose determination of microbial carbon
is the most suitable. Determination of microbial biomass enables chloroform
fumigation of soil. It covers formerly used the chloroform fumigation incubation
method (CFI) and the chloroform fumigation extraction method (CFE), which is also
standardized in ISO 14240-2 (1997b). The CFE method is used for soil microbial
biomass estimation also in laboratories at RECETOX. The CFE method is considered
to measure most of the soil microbial biomass, though some microorganisms (e.g.
spores) are insensitive to the fumigation process. Microbial biomass is expressed as
Cbio amount determined in soil extract. Besides carbon, other elements could be
analysed is extracts. We can get information about total microbial nitrogen or
phosphorus amount. This chemical analysis of microbial biomass provides information
about all soil living cells, without distinction between active and dormant stages (Van
Beelen and Doelman, 1997). Against carbon, adenosine triphosphate (ATP) is ideal
component for assessment of living microbial biomass (Contin et al., 2001). Another
possibility provides analysis of phospholipid fatty acids (PLFAs) or ergosterol in
fungal cells (Stahl and Parkin, 1996).

1.1.2 Parameters of carbon cycling

A major activity of soil microorganisms is decomposition of organic matter. Organic

matter in soil is largely derived from higher plants consisting of cellulose (15 - 60%),
hemicelluloses (10 - 30%) and lignin (5 - 30%). Indicators of carbon cycling represent
measurements at the ecosystem level.

Soil respiration (exchanges of gasses, particularly oxygen and carbon dioxide) is one
of the oldest, but still the most frequently used parameter for quantification of microbial
activities in soil. Aerobic decomposition results in complete oxidation of a substrate and
the release of large amounts of energy, gas, and heat with carbon dioxide and water

13
Introduction

as the end products. Soil respiration is strongly affected by physiological state of

microorganisms and nutrient availability. It depends very on the physical and chemical
properties like temperature, soil moisture, density or pH, as well. Nonetheless, it is
taken as one of the basal parameters thanks to its ecological relevance as indicator of
soil (and microbial) mineralization ability (Anderson and Domsch, 1985). Soil
respiration can be determined by either CO2 production or O2 consumption.
Measurement of CO2 concentration is more sensitive, because the atmospheric
concentration of CO2 is only about 0.033% versus 20% for O2.

Soil respiration could be assessed in two basic approaches, with and without any
substrate additions. The soil respiration without addition of any substrate or nutrients,
basal respiration (BR), is elementary parameter of soil microbial assessment and
monitoring. The ISO guideline (ISO 16072, 2002) describes methods for soil
respiration determination. The value of BR is than used as a base information and
control value to which other parameters could be referred. Basal respiration is limited
by available organic matter. Also its sensitivity to pollutants or as soil quality indicator is
discussed. Respiration represents activity of whole microbial community including
toxicants sensitive and resistant species. Moreover, some organic chemicals caused
increasing of basal respiration (Tu, 1993, Eisentraeger et al., 2000). Therefore this
parameter is regarded as low sensitive endpoints to pollutants and inhibition effects are
observed at high, often non-realistic concentration of toxicants (Fließbach and Mäder,
2004, Chen et al., 2001).

Respiration of soil after addition of available substrate enables to assess activity of soil
microorganisms that are not limited by amount of substrate in the soil. The substrate-
induced respiration was firstly designed for soil microbial biomass estimation
(Anderson and Domsch, 1978) but nowadays is widely used for microbial activity
evaluation. Elevated respiration rate after addition of small concentration of easily
available substrate allows determination of microbial community state and also
pollutants effects on soil microorganisms. Glucose-induced respiration is the most
often and used method, which was also standardized (ISO 14240-1, 1997a). Glucose
is easily utilized by wide spectrum of microorganisms and the respiration activity gives
non-specific response. On the other hand some oligotrophic microorganisms do not
utilize it. Another substrate like acetate, cellulose, chitin, aminoacids, sterilized soil or
complex organic substrate (plant residues) could provide more specific and additional
information about respiration activity (Panikov and Sizova, 1996, Hopkins et al., 1994).
Amount of substrate and duration of incubation is important factor.

Increase of respiration in the first hours after substrate addition reflects induced activity
of native microbial biomass. According to Stenström et al. 1998, the initial substrate-
induces respiration include respiration of two group of microorganisms with different life
strategy - growing and non-growing. In presence of further available nutrients, after a
definite time period (lag phase) a substrate-induced growth of whole microbial
community (growers followed by non-growing microbial groups) is initiated. This results
in further rapid increase of respiration (Stenström et al, 2001, Malkomes, 1999). Time
to reach the maximum respiration rate depends on glucose amount. Exponential
growth lasts till utilization of all available substrate, regularly within couple of days, after
that respiration decrease. Growth kinetic of soil microbial community is similar to
growth of single species bacterial culture and is characterized by a sigmoid curve with
initial lag phase, exponential (growth) and maximal phase (Zwietering et al., 1990).

14
 Chapter 1

Slope of the exponential growth (μ), calculated from log-transformed respiration rate, is
also important characteristic of growth kinetic.

Several studies were engaged in modelling of soil microbial growth kinetic

(Zwietering et al., 1990, Panikov a Sizova, 1996, Colores et al. 1996, Blagodatsky et
al., 2000). Moreover, modern automatic respiration measuring devices (e.g. Respicond
III, Nordgren, 1998) enable continual detection of microbial respiration and growth
kinetic. Application of mathematic models together with respiration kinetic
measurement enables to determine microbial biomass or activity of specific microbial
groups, biodegradation of xenobiotics or to evaluate effects of pollutants on microbial
community and different group of microorganisms (Johansson et al., 1998).

There are also some disadvantages and limitation of the kinetic approach, such as
sorption of substrate to soil, presence of extracellular enzymes that degrade the
substrate, degradation of substrate not associated with microbial growth. In
biodegradation studies can occur inhibition of microorganisms and co-metabolism of
xenobiotics.

Respiration curves of soil microbial community could be also used to determine

bioavailability and pool of nutrient in soil ecosystems. Demetz and Insam (1999)
showed that this approach is good and fast alternative to chemical analysis of
bioavailable phosphorus in forest soil. Soil microorganisms demand mainly source of
carbon, nitrogen and phosphor. Respiration kinetic after addition of these nutrients, in
different combination and concentration, enable determination of limiting elements for
given microbial community (Cleveland et al. 2002, Ilstedt et al., 2005, Teklay et al.,
2006). Results displayed that in soil microorganisms are mainly limited by supply of
carbon and afterward nitrogen. Limitation by phosphor was not so important, indicating
its sufficient bioavailable amount in soil (Ilstedt et al., 2006). Phosphorus may also
facilitate utilization of some carbon substrates (Ilstedt et al., 2005). An example of a
respiration kinetic of soil microbial community limited by phosphorus is shown in fig. 1.

with P-substrate addition

without P-substrate addition

Fig. 1: Hypothetical example of P-limited respiration kinetic (Ilstedt et al. 2006)

15
Introduction

Teklay et al. (2006) observed double peak of maximal respiration rate. They
hypothesized that the two phases might represent two nutrient pools: the first peak
could show an easily available small pool of N, while the second peak could show a
slowly available but larger pool of N (NH4+ fixed in the interstices).

Analyses of soil respiration kinetic have been applied for research of contamination
effects on nutrients availability (Hollender et al., 2003, Giesler et al., 2004),
ecotoxicological assessment of contaminated soil (Eisentraeger et al., 2000), for
monitoring of bioremediation processes (Riffaldi et al., 2006) or changes in chemical
and microbial properties after wildfire Ilstedt et al. (2003).

Evaluation of specific respiration curves parameters was also included to ISO

standards as method for determination abundance and activity of soil microflora or
assessment of the ecotoxic potential of soils and soil materials (ISO/DIS
17155, 2001).

1.1.3 Parameters of nitrogen cycle

Fundamental processes of nitrogen transformation are associated with microbial

activities. Soil microorganisms are also responsible for atmospheric nitrogen fixation
and release (Norton, 2000). The main microbial nitrogen transformation processes are
N-mineralization, nitrification, denitrification and N2-fixation and are shown in figure 2.

Fig. 2: Schematic illustration of nitrogen cycle in soil environment.

All fixed forms of nitrogen, NH4+, NO3-, and organic N, come from atmospheric N2.
Nitrogen fixation, when N2 is fixed to ammonia (NH3+), is carried out by over 100
aerobic and anaerobic free-living bacteria (Azotobacter, Clostridium, Beijerinckia) as
well as by some cyanobacteria (Anabeana, Nostoc), actinomycetes and symbiotic
bacteria (Rhizobacteriaceae) due to their nitrogenase enzyme complex. Nitrogenase is

16
 Chapter 1

extremely sensitive to oxygen, requiring low O2 tension. Nitrogen fixation is sensitive

parameter to chemicals and also other stress environmental condition.

The ammonium is assimilated by cells into amino acids and proteins and is
immobilized in microbial biomass. The reverse process, N-mineralization or
ammonification, when organic N is mineralised to ammonium is provided by a wide
variety of soil microorganisms and reflects the turnover of organic material in soil and
the available indigenous N-pools to plants. Ammonification depends on C/N ration in
organic matter.

Ammonium is subsequently either immobilised by soil microorganisms as assimilated

into new biomass or oxidised to nitrite (NO2-) and subsequently to nitrate (NO3-) by
aerobic nitrification. This two-step process is carried out different microbial
population. Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosovibria a Nitrosolobus
are main genera capable of oxidizing ammonia, whereas Nitrobacter, Nitrospira of
nitrite oxidization. Both steps are aerobic and energy yielding. Nitrifying organisms are
chemoautotrophs, and use carbon dioxide as their carbon source for growth. The
second step of nitrification is usually the more sensitive one, and under stress
condition nitrite accumulation may occur (Atlas and Barther, 1997). Nitrification rates
are slowed at pH < 6 and seem to be completely inhibited below pH 4.5 (Maier and
Pepper, 1999). Nitrates are assimilated by plants or could leach to groundwater. In
anaerobic conditions nitrate is subsequently reduced to gaseous nitrogen (N2) via
nitrous oxide (N2O) by anaerobic denitrification (Atlas and Barther, 1997).

1.1.4 Enzymatic activity

Soil enzymatic activity serves as an indicator of function diversity and biochemical

potential of soil microorganisms. Changes in enzymatic functions provide early
indications of changes in soil health, and involve simple analytical procedures (Taylor
et al, 2002, Nielsen and Winding, 2002). Enzymes in soil occur inside microbial cells or
extracellular, free or bound on soil particles (Ljunghal and Eriksson, 1985).
Interpretation of these endpoints should respect the fact that extracellular enzymes are
active independently on microbial vitality or also after cells death. There exists number
of enzymes involving in macro-element cycling. Relative small number of them is,
however, applicable in the laboratory assessment. Dehydrogenase, glucosidase,
urease, sulphatase, phosphatase and cellulase are the most often used ones.
Dehydrogenase activity has been decided as appropriate sensitive parameter
(Rossel and Tarradellas, 1991) and the assessment is standardized in ISO 23753-1
(2005). They are intracellular enzymes that are bound in cell membrane and are
associated with the electron transport chain and ATP formation. Their activity therefore
reflects metabolic activity of whole community and changes in environmental
conditions.

1.1.5 Microbial diversity

Besides microbial processes and activity assessment, microbial diversity is next

important parameter that can be used in soil microbial monitoring and toxicity tests.
Information about microbial community structure and diversity has been noted as
important for understanding the relationship between environmental factors and
ecosystem functions and thus recommended in soil health monitoring programmes
(Nielsen and Winding, 2002). The number of species has traditionally been determined

17
Introduction

by taxonomic classification studies. These methods are very laborious, time-

consuming, of small ecological relevancy and not optimal for investigation of soil
environment. With development of molecular and biochemical techniques, analysis of
genetic, functional and structural diversity has been applied. The genetic diversity of
soil microorganisms is an indicator of the genetic resource and all actual and potential
activity. Genetic diversity of bacteria is most commonly studied by diversity of the 16S
rDNA genes (18S rDNA for fungi and eucaryotes) which occur in all bacteria and
which show variation in base composition among species. 16S rDNA genes are thus
used for phylogenetic affiliation of Eubacteria and Archaea and large databases exist
on sequences of 16S rDNA. Functional diversity of microbial populations in soil may
be determined by either expression of different enzymes (carbon utilisation patterns,
extra-cellular enzyme patterns) or diversity of nucleic acids (mRNA, rRNA) within cells,
which also reflects the specific enzymatic processes operating in the cells. Indicators of
functional diversity are also indicators of microbial activity and thereby integrate
TM
diversity and function. The BIOLOG system can be used to analyse carbon utilization
patterns; it is carried out in microplates containing different carbon sources (Garland
and Mills, 1991). The content and diversity of mRNA molecules, that reflex the type of
enzymes synthesised, can further give very accurate pictures of the in situ function and
activity of the microbial community. Structural diversity of microbial community can
be determined by identification of phospholipid fatty acids PLFAs. These polar lipids
are specific for subgroups of microorganisms (gram-negative or gram-positive bacteria,
methanotrophic bacteria, fungi, mycorrhiza, and actinomycetes) and are related to
microbial community structure (Zelles, 1999). Beside diversity, PLFAs can give
information about information on fungal:bacterial biomass and serves as marker of
stress (Frostegård et al., 1996).

Besides above described parameters, many others could be used. They provide rather
additional information about specific microbial activities (anaerobic processes,
mycorrhiza, iron reduction, etc.).

18
 Chapter 2

2 VARIABILITY OF SOIL MICROBIAL PARAMETERS

Soil matrix is very heterogeneous complex consisting of mineral and organic

component that is inhabited by live organisms. Factors that influence soil characters
can be divided to relatively stable factors (like soil profile, structure and mineral
composition) that change during long time period and dynamic factors (temperature,
water content, pH, activity of soil microorganisms) that are daily and seasonally
fluctuated. Despite dynamic changes and seasonal variation soil characteristics are
relatively more stable in time in comparison to other environmental matrices. On the
other hand, soil environment very differ in space. Soil microorganisms are very
affected by abiotic factors and vice versa. This high spatial variability often complicated
soil monitoring, research of soil microorganisms and finding causality between
microbial activities and soil pollution.

2.1 Spatial variability

The distribution of soil microorganisms are not uniform, but changes along
environmental gradients. Spatial patterning can occur both vertically, through soil
profile, and horizontally and depends on physical and chemical soil parameters,
available substrate amounts or competitions of organisms. Vertical distribution of
microorganisms is given by character of soil profile and horizons. It is obvious and well
documented that amount of microorganisms decrease in depth with decline of nutrient
amount, oxygen saturation and other life support factors. The greatest portion of
microbial biomass and activity is located to upper soil layer to 10 – 20 cm (according to
soil horizons) that is also often sampled in microbiological studies. From this point of
view the vertical distribution is therefore not so important as horizontal variability in
space. Spatial heterogeneity can occur simultaneously at a macroscopic level,
corresponding to terrain and landscape, and microscopic level taking into account
microbial habitat in aggregates. Soil organisms are usually not randomly distributed,
but exhibit spatially predictable, aggregated patterns over scale ranging form square
millimetres to hectares. Factors affected spatial heterogeneity of soil organisms over
scales are illustrated in figure 3 (according to Ettema and Wardle, 2003).

19
Variability of soil microbial parameters

Fig. 3: Determinants of spatial heterogeneity of soil organisms. Spatial heterogeneity in soil

organism distributions occurs on nested scales, and is shaped by a spatial hierarchy of
environmental factors, intrinsic population processes and disturbance. Disturbance operates at
all spatial scales and can be a key driver of spatial heterogeneity, for example through biomass
reduction of dominant organisms or alteration of the physical structure of the soil substrate.
Feedbacks between spatial patterns of soil biotic activity and heterogeneity of environmental
factors add further complexity (dotted arrows). The scales at which population processes
operate are dependent on soil organism body size, dispersal mechanisms and life-history
characteristics (Ettema and Wardle, 2003).

Soil mineral and organic components are assemblage into aggregates. This structural
organization creates a mosaic of microenvironment differing in substrates, nutrients,
water and oxygen concentrations, size of pores and other physical and chemical
properties that provide many different habitats for the biotic components. Defining
population and diversity of fine soil structures is important to understanding soil
ecological processes and impacts of human activities (e.g. management practices) on
soil ecosystem. Aggregates can be classified according to their size and relative
stability. Microaggregates (diameter < 250 μm) are formed by fine particles (silt and
clay) fixed by microbial exopolysacharides. They are generally the most stable soil
secondary structure. Microaggregates, coarse minerals particles, organic matter and
fungal hyphae and plant roots are temporary associated to macroaggregate (diameter
> 250 μm) that are less stable. Microbial life occurs in soil pores that comprise up to
50% of total soil volume. Fractionation studies showed that microbial community differ
in the interior versus exterior of soil aggregates (Mummey and Stahl, 2004). Inner part
of soil microaggregates is characterized by relatively stable conditions - low predation,
stable water availability, but low nutrients and oxygen availability.

Microorganisms inhabiting interior of microaggregates (principally bacteria and

archaea) are smaller size, slow turnover and contribute little to overall measurable
microbial activity. Yet they may play important roles in anaerobic processes and soil
structure stabilization. Quantification of microorganisms showed that more than 80% of
bacteria are preferentially located in the inner part of aggregates, in the fraction
< 50 μm (mostly in the 2 – 20 μm), independent on soil type. Location of
microorganisms within the soil structure depends on their size, nutrients demands and

20
 Chapter 2

resistance to environmental conditions (water amount, gasses diffusion, predation).

Van Gestel et al. (1996) reported that microbial biomass is associated with aggregate
clay fraction (< 20 mm) that ensures microbial growth and survival by their capacity to
buffer nutrients supply. Together with organic matter allocation, it determined the
spatial distribution of microorganisms. Similarly Kandeler et al. (2000) revealed, using
molecular techniques (PLFA, molecular techniques, DNA, DGGE), that living and
death soil bacteria were mainly associated with silt and clay fraction, whereas fungi
and their exoenzymes were found associated with particulate organic matter in the
coarse sand fraction. Ranjard and Richaume (2001) reviewed that gram-positive
bacteria prefer outer part because are higher resistant to fluctuating conditions,
oppositely gram-negative bacteria are more abundant in the inner part. Actinomycetes
are too large to colonize inner part of aggregates. On the other hand higher salt
concentration, nutrients and water availability in the inner part are sufficient for
presence of halotolerant, nitrifying, denitrifying or free N2-fixing bacteria.

Fine-scale heterogeneity of soil populations and activities correlated to substrate

hotspots and exhibit patchy distribution. Besides substrate amount, presence of
pollutants also affects microbial distribution. Becker at al. (2006) reported that soil
contaminated by heavy metal (thousands of mg Pb, Cr kg-1) showed that heterogeneity
of microbial metabolic potential was apparent in samples located less then 1 cm apart.
The activity in uncontaminated soils was more homogenous. Surprisingly in this study,
metal content and activity was not significantly correlated, which indicated either less
metal bioavailability or selection of resistant microbial communities in some microsites
with high metal contamination. Kandeler et al. (2000) also found that metal
contamination lead to selection of resistant fungi population. They showed decrease of
microbial biomass in silt and clay fraction in polluted soil and discussed that adsorption
of microorganisms onto organic-mineral microaggregates did not protect them against
the heavy metal pollution.

Nested spatial distribution is exhibited also in large-scale, in meter distances, even

where topography and soil texture are relatively uniform. At this scale microbial
variability is related primary to plant growth, vegetation cover or landscape gradient.
There are also greater distinguish between soil types with obviously lower spatial
heterogeneity in arable soil, whereas higher in forest soils. The distribution is
influenced by individual plant growth, land use, soil carbon and moisture content, trees
size and spacing (Ettema and Wardle, 2002, Monokrousos et al., 2004). For example
Cornstanje et al. (2007) found that microbial urease activity varied from short-scale
(< 1 m) in the pasture soil to longer-scale variation in forest and arable soil (≥ 15 m)
and the variation was dependent on soil organic carbon. Soil moisture and water
content is further important factor affected microbial spatial variability. Görres et al.
(1998) showed that carbon mineralization was connected to spatial variability of soil
moisture and the relationship was also more apparent in old-field soil than in forest soil
with more stable conditions and ecosystem. Saetre and Baath (1999 and 2000)
reported that spatial pattern of microbial community structure (PLFA profile) in forest
soil may be on one order to magnitude higher than in agricultural field. The pattern
induced in their forest soil varied at scale 1 – 10 m and was depends on tree species
and position. They suggested that soil microbial community was driven by organic
matter quality associated to the tree species. Modern geostatistical analyses enable to
model, predict and display the distribution of measured parameters in space. An
example is given in figure 4 (Saetre et al., 1999).

21
Variability of soil microbial parameters

Fig 4: Spatial patterns of soil microbial biomass and activity in a mixed spruce-birch stand; A)
microbial biomass (μmol PLFAs g-1 OM), B) microbial respiration (μg CO2-C h-1g-1 OM). Grey
and white circles represent spruce and birch trees, respectively. Spatial scale in metres. (Saetre,
1999).

Spatial variability of soil properties is natural attribute of soil matrix that, however,
unfortunately complicates its research. In usual practice, when comparing microbial
parameters at different sites, randomly collected samples are pooled together to
reduce spatial variation. The sampling design depends on the study purpose and is
discussed below. Soil spatial variability can, however, be so high that exceed other
changes or factors studied. These inconveniences were many times reported by
various soil microbial studies (e.g. Ross et al., 1995, Laverman et al., 2000, Lorenz
and Kandeler 2006). Chen et al. (2003) found high spatial variability of microbial
biomass carbon and nitrogen that was moreover increased by soil compaction and
cultivation treatment in a hoop pine plantation. Field variability is often inadequately
considered, resulting in data that are unlikely to detect significant changes in microbial
parameters across studied sites. This problem was well discussed by Broos et al.
(2007) who questioned the value of routinely measurement of soil microbial biomass
as an ecotoxicological endpoint. They reported that to identify significant decrease
(20% effect) of microbial biomass C between metal contaminated and clean sites a
very large number of soil replicates (up to 93) would be required to sample. This also
emphasizes the question of suitable microbiological indicator for ecotoxicological
studies that would be sensitive enough but less variable in space. Since this is very
difficult task, great importance should be paid to representative sampling of soil with
respects to the aim of the study.

2.2 Temporal variability

Temporal dynamics of soil microbial biomass and activities are an important part of its
turnover and determine the extent of soil nutrient release, mineralization and
availability of these nutrients for other component of the ecosystem. Fluctuations of

22
 Chapter 2

microbial properties are results of changes in environmental conditions. The variability

is influenced by both climatic and soil quality factors. The effect of macroclimatic
conditions on temporal variability is relatively predictable and is driven to a large
extent by seasonal temperature, rainfall or soil moisture fluctuation (Chen et al., 2003).
Wardle (1998) summarized several published studies focused on temporal changes of
soil microorganisms. He pointed out that seasonal fluctuations are affected by latitude.
Generally it seemed that higher seasonal variability of microbial biomass appeared at
higher latitude where winter freezing reduced biomass and spring thaw induced
microbial growth. Oppositely, warmer and tropical habitats showed no consistent
temporal trends. Moisture dynamic is further factor controlling temporal variability,
however, the relationship is not unequivocal. Long drought causes stress and
reduction of microbial activities whereas increase of soil moisture favours microbial
growth (Chen et al., 2003). On the other hand negative relationship was also reported
(Šimek et al., 1998). This might be caused by enhanced plant competition of nutrients
under superior moisture conditions or root growth (Wardle, 1998). In Mediterranean
soils temporal variability was affected by dry and wet seasons (Monokrousos et al.,
2004). Higher amount of fungal biomass, but lowest values of microbial respiration and
mineralization rate were found during a dry period of year.

Besides climatic changes, microorganisms are affected by fluctuation of organic

matter input as well. Increased supply of organic carbon enhances microbial biomass.
On the other hand it has stabilizing effects on the microbial biomass and thus reduces
temporal variability. This effect has been shown also in aquatic ecosystem and might
explain resistance of soil food webs to disturbance in general (Wardle et al, 1995).
To the contrary soil C:N ratios, which indicates resource quality and helps to regulate
microbial mineralization processes, showed no relationship with temporal variability as
reviewed by Wardle (1998). Seasonal fluctuations of microbial parameters differ
according to soil type and are to some extent dependent on land use and soil
disturbance. Görres et al. (1998) reported that organic matter and soil moisture had
significant effect on carbon mineralization during year, whereas not so provable in the
forest soil. To the contrary, microbial biomass at hill pasture were unaffected by large
differences input of plant material over a period of six month in the study form Ross et
al. (1995). The pools of microbial biomass were relatively constant, whereas other
labile organic pools and some enzymatic activities were more fluctuate during season.

The impact of soil disturbance on microbial temporal variability is more complicated.

Populations subjected to significant disturbance may be expected to be less resistant
to environmental changes and have large tendency to temporal fluctuation. However,
Wardle (1998) not proved this hypothesis after analysing a lot of studies. Although
more persistent ecosystems like forest or grassland soils showed higher microbial
biomass values than more disturbed arable soils, the temporal variability of all soils
was similar. This means that temporal variability is independent on differences in
vegetation and land use type and little depends on above- and bellow-ground stability.
A possible explanation of this fact is that the soil microflora is regulated by disturbance
regimes at the microscopic spatial scale and the differences are not apparent between
ecosystem types at macroscopic level. Other factors may, however, be more important
in regulating temporal variability.

23
Variability of soil microbial parameters

2.3 Variability of soil sampling and manipulation

2.3.1 Soil sampling

The result of soil microbial analysis is the final point of long process consisted of soil
sampling design, sampling procedure, sample handling, its storage, laboratory analysis
and statistical evaluation. A lot of attention is often focused on the laboratory analysis.
This part of process presents, however, only minor part of final variability. The majority
of overall variability originates in the phase of soil sampling (up to 92%) and only about
8% is given by further soil manipulation (Sáňka, 1998). As mentioned above, soil is
very heterogeneous matrix with high variability. Therefore, soil sampling strategy must
be carefully considered with concern for the study purpose. Prior to every sampling, it
is important to determine major steps and factors like the position and density of
sampling points, time of sampling, sampling procedures, transport and sample
manipulation, subsequent treatment and analytical requirements. The details of a
sampling program depend on the information needed; wherever we are focused on the
average value or the distribution of given soil parameters. Data quality demands
enable to set design of sampling to achieve the desired aims of study with sufficient
accuracy but minimal cost. Statistical tools for experimental design and plan are very
helpful in this matter.

Sampling patterns are based on the estimation of the distribution of soil parameters in
an area and on on-site conditions. In general, several sampling patterns could be
characterized like irregular sampling, sampling in circular or other regular grids,
random and stratified sampling, systematic sampling or sampling along a linear source.
The basic patterns could be further combined according to certain events. More
detailed information about soil sampling design and procedures are given e.g. in Paetz
and Wilke (2005) or Alef and Nannipieri (1995).

Methods of soil sampling are also standardized and integrated to ISO guidelines in
documents:

ƒ ISO 10381-1/2002: Soil quality -- Sampling -- Part 1: Guidance on the design

of sampling programmes.
ƒ ISO 10381-2/2002: Soil quality -- Sampling -- Part 2: Guidance on sampling
techniques
ƒ ISO 10381-3/2001: Soil quality -- Sampling -- Part 3: Guidance on safety
ƒ ISO 10381-4/2003: Soil quality -- Sampling -- Part 4: Guidance on the
procedure for investigation of natural, near-natural and cultivated sites
ƒ ISO 10381-5/2005: Soil quality -- Sampling -- Part 5: Guidance on the
procedure for the investigation of urban and industrial sites with regard to
soil contamination
ƒ ISO 10381-6/1993: Soil quality -- Sampling -- Part 6: Guidance on the
collection, handling and storage of soil for the assessment of
aerobic microbial processes in the laboratory
ƒ ISO 10381-7/2005: Soil quality -- Sampling -- Part 7: Guidance on sampling of
soil gas
ƒ ISO 10381-8/2006: Soil quality -- Sampling -- Part 8: Guidance on sampling of
stockpiles

24
 Chapter 2

This thesis is focused on soil microorganisms and therefore further interest is

concerned to the methods of soil sampling and handling for microbial analyses. These
are integrated in the ISO 10381-6 (1993). In most cases a mixed sample composed of
several subsamples is taken to be a representative of microbial community at the
investigated site. Numbers of subsampes are usually from 10-30 subsamples, but they
depend on the site properties and size. Maximal area for one mixed sample should not
exceed 4-5 ha (Krištůfek and Šimek, 1998). For example, in the scope of soil microbial
monitoring conducted by RECETOX usually 8-10 subsamples are taken at the area of
about 50 m2. If the aim of the study is to investigate spatial variability, samples are not
homogenized but analysed independently. This was also one of the aims of our study
(paper I, see chapter 2.4). Sampling techniques differs, if disturbed or undisturbed
samples are required. With the exception of soil cores, sampling of soil means
disruption of its natural structure. If natural soils show distinct horizons, samples should
be taken from these horizons. Sampling depth depends on soil type. Mostly, upper soil
layer (0-10 cm) is taken for microbial monitoring; for agricultural soil maximum to
20 cm. Any surface vegetation cover, moss-covered litter layer, visible roots, large
pieces of plant or wood and soil fauna should be removed to minimize the addition of
fresh organic carbon to the soil. Sampling should be avoided during or immediately
followings long periods (> 30 days) of drought, freezing or flooding.

2.3.2 Sample handling and manipulation

ISO guideline recommends methods for soil processing and storage conditions and
durations. Soil should be processed as soon as possible after sampling. After
homogenisation, sieving through 2 mm mesh size is recommended. This facilitates
gaseous exchange between particles and therefore maintaining the aerobic nature of
the soil. More organic material such as mor layer or peat that will not pass through
a 2 mm sieve should be sieved using 5 mm mesh size. Drying of soil before sieving
should be performed at ambient temperature and not more than is necessary than is
necessary to facilitate sieving. Sample for microbiological analysis should be stored in
the dark at 4±2°C with free access to air. It is preferable to analyse soils as soon as
possible after sampling. If storage is unavoidable, this should not exceed 3 months
unless evidence showing continued microbial activity is provided. The active soil
microflora decreases with storage time, even at low temperatures, and the rate of
decrease depends on the composition of the soil and the microflora involved. In the
ISO 10381-6 (1993) freezing and drying of samples is not allowed during storage.
Before the specific microbial tests soils should be pre-incubated to allow re-establish
equilibrium of microbial metabolism. Pre-incubation conditions will depend on the study
purpose but should be similar to test conditions. The period of pre-incubation from 2 to
28 day is generally adequate.

Despite these recommendations, the optimal handling and storage conditions and their
effects on various microbial parameters are still discussed. The preferred conditions
also differ among published studies. Firstly, there is a question of soil sieving. Pre-
treatment of soil samples is essential to limit interferences of plant roots and to unify
soil particles size before microbial parameters analysis. Various mesh sizes are,
however, used in soil microbial studies. Although standard recommendations exist, it is
understandable that sieving procedure may differ according to type and character of
sieved material. Moreover, although certified meshes are available at the market, the
sieving procedure itself is quite subjective. Different people use different routine (force,
time, recovery effort etc.) when sieving. Some labs use grinding the soil before sieving,

25
Variability of soil microbial parameters

the others use shredder or various kinds of automated equipments. Sieving through
smaller mesh requires more force and therefore more fine roots with rhizosphere
microbial biomass can be added to the sample (Joergensen and Emmerling, 2006).
On the other hand, grinding the stones means increase of mineral fraction in the
sample. In general, fine sieving caused rather increased yield of microbial parameters
probably by release of available substrate. Hassink (1992) observed that C and N
mineralization was increased after fine sieving (1 mm mesh size). This effect was more
marked in loam and clay than in sandy soil, but was only temporary and disappeared
after 1-2 weeks. These results suggest that fine sieving removed part of the physically
protected organic matter presents in small pores. In loam and clay soils small pores
constituted a higher percentage of the total pore space than in sandy soil and therefore
the sieving disruption had larger effects on mineralization. Breaking up of soil
aggregates may release a pool of available substrate. Degens et al. (1998) also
reported that 0.5 mm sieving caused increase of microbial biomass and functional
diversity that was influenced by increasing the exposure of organic matter to
microorganisms. 2 mm sieving had only little effect on the soil functional diversity.
Sieving through larger mesh size (> 4 mm) resulted in more course structure and
therefore in lower results of e.g. microbial biomass C (Ross et al., 1985) or CO2
evolution rate (Petersen and Klug, 1994). The differences in the mentioned studies
were, however, only small, indicating that substrates were not released by sieving to
any large extent.

2.3.3 Storage of soil samples

The effect of soil storage on microbial properties is more complicated. Despite

immediate analysis of soil is recommended, this is not mostly possible. The storage
conditions (temperature, duration) may, however, influence analysed parameters to
different extent. Generally, drying of soil is considered to be unsuitable due to great
changes induced in microbial community. Drying of soil causes a decrease in water
potential and an increase of osmotic potential (osmotic stress) that decreases microbial
metabolism. Lee at al. (2007) reported significant reduction of microbial biomass,
respiration, several enzyme activities and DNA content after two weeks incubation of
dry stored soil. Rewetting of dried soil leads to burst of C and N mineralization by
mobilization of soil organic matter and by killing many of soil microorganisms (Fierer
and Schimel, 2002). The history of stress plays important role in determining
magnitude of the rewetting CO2 pulse. The more rewetting events previously
experienced, the less CO2 released after a rapid rewetting due to less organic matter
available. It has also been shown that soils frequently exposed to drying rewetting
cycles (agriculture, grassland soil; soils from Mediterranean climate) are more adapted
to moisture stress and less affected by drying treatment than forest soil. Moisture
stress results also in microbial community structure changes in favour to fungi and
G+ bacteria that can withstand osmotic shock (Lee et al., 2007, Fierer and Schimel,
2002, Fierer et al., 2003).

Temperature of storage also affected microbial community and wet cold storage
at 4 °C is usually appropriate for majority of microbial parameters. Effects could,
however, be related to soil type and characteristics. Brohon et al. (1999) found that
microbial parameters were differently influenced during storage according to the soil
physical chemical characteristics. They reported that presence of clay and neutral soil
pH might improve resistance of microorganisms to temperature changes. Lee at al.
(2007) observed increased biomass in forest soil with high organic matter content after

26
 Chapter 2

2 weeks of storage at 4°C, but no change in sandy and clayey agricultural soils. In the
same study cold storage was also appropriate for soil enzymatic activities. Only minor
changes were found in soil microbial community structure (PLFA profile) during storage
at 4°C against a significant shift at 25°C (Petersen and Klug, 1994). On the other hand
cold storage may not be suitable for tropical soils, where microbial communities are not
adapted to low temperatures. For example Verchot (1999) observed significant
reduction of nitrification potential in these soils and recommended storage at room
temperature.

Freezing of soil samples is further discussed topic. Freezing, in general, causes

increased concentration of salts (similar to drying) in the liquid phase during ice
formation and consequently osmotic stress for cells. Cells are killed as well by
intracellularly formed ice crystals, which could damage cell structures. To the contrary,
if organisms survive freezing they can profit from fresh pools of bioavailable substrate,
which is released from soil aggregates disintegrated by freezing. Organic molecules
are another pool of available, easily degradable substrates revealed from biomass
killed by freezing (Stenberg et al., 1998). Induction of carbon and nitrogen
mineralization by freeze-thaw cycles has also been reported in the literature (Feng at
al., 2007, Sternberg et al., 1998) and was attributed to increased level of labile
substrate from killed microorganisms. On the other hand in some instances, freezing
(-20°C) has been shown to be preferable to cold storage (2°C) for longer time
(Sternberg at al., 1998). They found that only nitrogen mineralization capacity was
greatly influenced by freezing, microbial biomass, respiration and denitrification
showed only small deviation from fresh soil up to one year of storage. Lee et al. (2007)
also found that freezing (-20 or 80 °C) did not affect microbial biomass and respiration.
Results were, however, to some extent dependent again on soil type. Soils with high
organic matters content, such as forest soil, may be more susceptible to the effects of
storage. Very low temperature of storage up to -80°C is, however, most appropriate
and recommended for analysis of soil DNA.

Based on this new research, the International Organization for Standardization revised
the guideline ISO 10381-6 from the year 1993. The new guideline (so far as a final
draft ISO/FDIS 10381-6, 2008) specifies storage conditions and periods for different
microbial parameters in more detail (table 1).

27
Variability of soil microbial parameters

Tab. 1: Storage conditions and periods for different microbial parameters according to ISO/FDIS
10381-6 (2008)

2.4 Study of soil variability (paper I)

Soil sampling and monitoring of microbial as well as chemical and other soil properties
have a long tradition at RECETOX. Sampling, handling and storage procedure were
based on standard recommendation and practical experiences. However, any
validation studies about influence of these processes on soil microbial community and
study results had not been done before. Hence, the main goal of this study was to
determine sources and extent of variability of soil microbial properties by
sampling, handling and storage of soil.

Variability was assessed for three soil types, arable, grassland and forest soil.
Parameters of microbial biomass, respiration, ammonification and nitrification were
evaluated. The soils used for this study were sampled at sites that have been

28
 Chapter 2

monitored for long time. They are located approximately 20 km east from Brno around
the cement works. Due to their properties, these soils were selected as suitable
representatives of different soil types. Moreover, properties of the arable soil fulfil the
OECD recommended criteria for laboratory toxicity testing of chemicals. These soils
have been used in many experiments and laboratory tests performed at RECETOX.
Therefore the purpose of this study was to investigate the effects of soil spatial
variability within sampling site, sample fractionation (sieving through 1, 2 or 4 mm
mesh), and different way (cold, frozen, air dried) and duration (1 – 32 weeks) of
storage.

The results of this study are summarized and discussed in paper I (submitted
manuscript). Generally, effects were greatly dependent on soil type. Variability was
most appeared in grassland soil, followed by forest and arable soil. Despite
variability of microbial properties were found within the sampling sites, the common
procedure of sampling at least eight soil points to prepared mixed sample seemed to
be optimal with small variability. Sieving method had only minor effects, indicating
higher yield when smaller mesh size was used. The largest span of variability was
induced by storage conditions. Drying and freezing of soil caused significant
changes in microbial biomass carbon and respiration. Storage at 4°C appeared most
appropriate for evaluated parameters with only minor changes up to 8 weeks of
storing.

In general, this study proved that current sampling, handling and storage procedures
were sufficient for microbial parameters assessed in RECETOX laboratories.
Nevertheless, it was shown that soil microorganisms are very sensitive and for
next experimental works we should keep in mind that all aspects of soil
sampling, manipulation and storage condition could affect our results.

2.5 Effect of soil storage on sensitivity of microorganisms to toxicants

The effects of chemicals (crop protection products) on soil microorganisms are

evaluated under controlled laboratory conditions using defined natural soil to minimize
differences in the results. As discussed above, handling and storage of soils influenced
measured intrinsic microbial parameters. It is also assumed that stressful conditions
such as temperature or aeration changes during storage can affect microbial sensitivity
to additional chemical stressors. For this reason, the recommendations for chemical
testing (OECD, 1995) prefer the use of freshly collected soil. If storage is necessary, it
has been recommended to store soil at 4°C for up to 3 months or at -20°C not more
than for 1 year. However the fate and impact of chemicals on soils with different
characteristics has been relatively well studied, there exist only few studies dealing
with their influences on soil microbial community after different storage conditions.
Pesaro et al. (2003) studied the effects of freeze-thaw stress during storage on
microbial communities and methidathion degradation. Although they observed
significant permanent changes in the microbial community induced by temperature

29
Variability of soil microbial parameters

stress, the mineralization of this crop protection product (CPPs) appeared unaffected
by frozen storage. Next study by Pesaro et al. (2004) revealed that soil drying and
rewetting resulted in decrease of degradation of other CPPs metalaxyl-M and
lufenuron. Similarly, Parkin and Shelton (1992) reported reduction of insecticide
carbofuran mineralization by drying of soil. More recent research by Trabue et al.
(2006) showed that the storage of soil resulted in lower metsulfuron-methyl
degradation compared to fresh soil. They also pointed out that the microbial biomass
was significantly reduced after 3 month of storage at 4°C.

Influence of soil storage condition and toxicity of model toxicant (cadmium, 5000 ppm)
on soil microorganisms was the objective of study conducted at RECETOX
(Nešporová, Diploma Thesis, 2008). The results showed that sensitivity of
microorganisms to cadmium increased after 3-months storage (in 4°C, -20°C and dry)
against the fresh state. The inhibition effect of cadmium was higher in case of microbial
biomass, substrate-induced respiration, ammonification and nitrification. On the other
hand, basal respiration showed lower sensitivity after all type of storage.

Recommendations for soil sampling, manipulation, storage and toxicity testing

according to experiences from the RECETOX laboratories:

ƒ Differences in values of microbial parameters exist within the sampling sites

(about 15 m2). To minimize spatial variability at least 8 subsamples of soil at
the site should be taken and homogenized together.

ƒ When soil sieving, mesh size has only minor impact on microbial parameters.
Soil should be sieved through 2 mm mesh.

ƒ Soil should be stored in plastic boxes or bags at 4°C in dark. Under these
conditions, values of microbial parameters most approximate levels in fresh
sampled soils.

ƒ Duration of storage should be as short as possible. In our study, microbial

parameters did not significantly change up to eight weeks of storage.

ƒ Storage of soil affects its sensitivity in toxicity tests. After 3-months of

storage, basal respiration was lower sensitive; parameters of microbial
biomass, substrate-induced respiration ammonification and nitrification
were higher sensitive. For toxicity tests, soil should be store for minimal
period.

ƒ Although our results were mostly common for arable, grassland and forest soil,
there could be larger differences among other soils and soil types.

30
 Chapter 3

3 LABORATORY TESTS WITH SOIL MICROOGANISMS

Due to essential role of microorganisms for soil structure formation and soil quality
maintenance it is important to assess the effects of chemicals on soil microorganisms
as a part of routine risk evaluation scheme for new and existing chemicals. Laboratory
tests with soil microorganisms were developed to prospective assessment of
pesticides and later adopted for all produced chemicals. In comparison with another
single-species ecotoxicological tests, in this case, effects on whole soil indigenous
microbial community is evaluated. Laboratory tests use exposure of natural soil
containing native microflora to added chemicals under defined laboratory conditions.
Introduction and optimisation of soil laboratory incubation tests for evaluation the
effects of persistent organic pollutants (POPs) were a topic of my diploma thesis.
Within my postgraduate study, the work was focused on more specific endpoints in
these laboratory tests. Effects of POPs – toxaphene and chlorinated paraffin – and
selected pesticides were assessed.

3.1 Procedures and standard methods of laboratory tests

Demands for soil microbial community assessment arose with intensive agriculture
development. Increase pesticides application led to worries about soil quality and
production preservation. Following, several international symposia and workshops
were held in 70´s and 80´s last century; “Recommended Laboratory Tests for
Assessing the Side Effects of Pesticides on Soil Microflora” has been published
(Somerville and Greaves, 1987), including proposal concerning interpretation of
results. This reference should be considered as a key source document. On the basis
of this, Food and Agriculture Organisation of the United Nations (FAO), Society for
Environmental Toxicology and Chemistry (SETAC), European and Mediterranean
Plant Protection Organisation (EPPO) introduced their procedures and guidelines for
assessing fate and ecotoxicity of pesticides included effects on non-target soil
microorganisms (FAO, 1989, Lynch, 1995, EPPO, 2003). In the similar way, American
organisation US EPA also published Ecological Effects Test Guidelines with soil
microbial community toxicity test (US EPA, 1996). In Germany registration of products
used in agriculture, forestry and horticulture has been under strict regulation since
1990 by “Biologische Bundesanstalt für Land- und Forstwirstschaft” (BBA, 1990).

The OECD designed laboratory test method to investigate the long-term effects of
chemicals, after single exposure, on nitrogen and carbon transformation activity of soil
microorganims (OECD 2001a,b). These tests are principally based on the
recommendation of the previous mentioned organisations. Although the standard
laboratory tests were firstly introduced to plant protection products assessment, they
are also required when the potential side effect or exposure of other chemicals are
expected. Potential adverse effects of tested substances are evaluated during aerobic
incubation of surface soil treated with tested chemicals. The test runs for at least 28
days and parameters of nitrogen and carbon mineralization are weekly determined.
Detailed characteristic of condition, sample preparation, chemicals application and
results interpretation are described within these guidelines.

The guidelines recommended characteristic of soil that should be used for testing. The
soil should represent a worst-case situation, since adsorption of the test chemical is

31
Laboratory tests with soil microorganisms

minimal and its availability to the microflora is maximal. The recommended soil
characteristics are:

ƒ sand content between 50% and 75%

ƒ pH 5.5 – 7.5
ƒ organic carbon content: 0.5 – 1.5%
ƒ microbial biomass carbon content should be at least 1% of the total soil
organic carbon

When major use of tested chemicals is expected in particular soil with different
conditions (e.g. acidic forest soils), the effects should be tested in soil with similar
character

Application of tested substance to soil is very important step of testing procedure.

Water soluble chemicals could be applied as water solution. Other non-toxic solvents
should be used, however, organic solvents such as acetone or chloroform should be
avoided due to they can damage soil microflora. Test chemical should be also applied
using an inert solid carrier such as fine quartz. In such cases, carrier is coated with the
test substance dissolved in an appropriate solvent that is evaporated before mixing
with the soil. To avoid increasing of result variability it is necessary to homogenously
mix the chemical with the soil. If sand is used as carrier, a ration of 10 g of sand per
kilogram of soil dry weight is recommended for optimum distribution of the test
substance in soil. Control samples are treated with the equivalent amount of water,
quartz sand or another carrier.

Concentration doses of tested substances should reflect expected environmental

concentrations. OECD guidelines (2001a,b) recommend at least two concentrations
should be tested for agrochemicals. The lower concentration should be derived from
minimum amount that could reach the soil under real conditions. The higher dose
should be a multiple of the lower dose, but should not exceed ten times the maximum
of single application rate. A geometric series of at least five concentrations are used
when non-agrochemicals are tested.

During laboratory incubation several microbial parameters should be assessed (see

chapter 1). The International Organisation for Standardisation (ISO) published an array
of standards that describes methods of laboratory determination of microbial
endpoints.

3.2 Laboratory tests at RECETOX

From the wide spectrum of sites monitored by RECETOX, it was selected soil that
more or less fulfils the OECD recommended criteria for laboratory testing. It is arable
soil, a loamy sand cambisol. Grassland soil, sandy cambisol, was the second soil
used for laboratory tests. Both soils were sampled at long-term monitored sites about
20 km from Brno. Contaminations of the soils (organic pollutants and heavy metals
contents) were comparable to the background levels in the Czech Republic. More
detailed information of actual soil characteristics is given in the individual papers
attached to this thesis. For all tests, soil from upper layer (0-10 cm) was collected.
Mixed samples were taken as 6 - 10 subsamples (about 1 kg each) of each sampling
site from the sampling area approximately 15 × 15 m. Soils were handled and stored

32
 Chapter 3

according to ISO 10381-6 (1993). Samples were sieved through 2 mm and stored in
dark at 4°C before experiments.

Prior to a test start and chemicals application, soils were preincubated for 4-6 days to
restore microbial activities. Moisture content was adjusted to 40% of WHC and
samples incubated in dark at 20±2°C. Further incubation, during test duration, was also
carried out in dark at 20±2°C.

3.2.1 Evaluated microbial parameters

Microbial biomass was determined as microbial carbon content by the fumigation–

extraction method (ISO 14240-2, 1997b). Soil at 60% WHC (10 g) was exposed to
ethanol-free chloroform vapours in dessicator for 22 hrs and after that both, exposed
and unexposed, soil was extracted with 0.5M K2SO4 (1:4 w:v) and C in extracts was
determined after dichromate-sulphuric acid digestion by back titration using Mohr salt
and diphenylamine as indicator.

Basal respiration was measured according to (ISO 16072, 2002). Sub-samples (10 g)
were weighed into small glass bottles with rubber caps and incubated at 22°C. Gas
(1 ml) was sampled from the bottles after 24 h with a syringe, and CO2 was quantified
by GC with H2 mobile phase, Porapack Q stationary phase and thermal conductivity
detector.

Substrate-induced respiration was measured as CO2 production in the first 6 h after

the addition of glucose. Glucose (5 mg.g-1 dry soil) was added to samples as water
solution, used at the same time for the WHC adjustment to 80% (ISO 14240-1, 1997a).

Ammonification rate was determined according to Alef (1995a) as the release of

NH4+-N during the incubation of water-flooded soil (1:8 w:v) at 36-40°C for 1 week.

Potential ammonification (PAMO) was measured as arginine ammonification assay

according to Alef and Kleiner (1987). Production of NH4+-N ions was measured after
3 hours incubation of soil samples (2 g) with 0.5 ml of L-arginine (11.5 mM solution)
at 30 °C. Simultaneously, soil samples with the 0.5 ml of distilled water were stored
at -20 °C.

Basal nitrification rate was measured as the release of NO3--N during aerobic
incubation of soil sub-samples (2 g) at 22°C for 1 week (Alef, 1995b). Short-term
potential nitrification activity of soil microorganisms was determined as potential
ammonium oxidation (PAO) according ISO 15685 (2004). Samples of soil (20 g) were
mixed with 80 ml of potassium phosphate buffer solution (pH 7.2) containing 3.8 mM
(NH4)2SO4 and 15 mM NaClO3. Soil slurries were incubated on a rotary shaker
at 25 °C for 5 hours. The potential nitrification rates were calculated by linear
regression of accumulated nitrite over time.

Inorganic form of nitrogen were extracted from soil by 1 hour shaking with 1 M KCl
solution in ration 1:10 w:v, following centrifugation at 2000 rpm for 5 min. Ammonium
(NH4+-N) content in extracts was measured calorimetrically by the method according to
Forster (1995). The assay based on Berthelot reaction (the reaction with salicylate and
hypochlorite in alkaline solution) was modified to microvolume assay and carried out in
microplates. Absorbance of samples was measured after 60 min at a wavelength of

33
Laboratory tests with soil microorganisms

660 nm. The nitrate (NO3--N) was determined after reduction with Devarda’s alloy
(20 hours) as NH4+-N. Nitrite (NO2--N) concentration in soil extracts was analysed by
Griess-Ilosvay colorimetric determination (ISO/TS 14256-1, 2003), where nitrite forms
after addition of sulphanilamide and N-(naphtyl)ethylendiamine dihydrochloride
a diazo-compound. Analysis was again modified to microvolume assay and carried out
in microplates. Absorbance of samples was measured after 15 min at a wavelength of
540 nm.

Substrate-induced growth kinetic was evaluated using the OxiTop® system. This
device was originally developed to biological oxygen BOD analysis of wastewater. It
can be applied to determine of respiration of solid matrices such as compost, waste or
soil. OxiTop® is manometric system detection pressure changes in a close bottle. By
microbial respiration O2 in bottle is consumed and released CO2 is captured into
absorber (NaOH solution). Pressure drop is detected by sensor in a measure head.
Respiration rate is related to decreasing of pressure in the bottle. Schematic illustration
of the device is in figure 5.

infrared sensor
pressure sensor

CO2 trap (1M NaOH)

incubaiton flask

soil sample

Fig. 5: Schematic illustration of the OxiTop® respirometric system

3.3 Persistent organic pollutants

The first group of chemicals that were tested in our studies with microorganisms
belongs to persistent organic pollutants. Persistent Organic Pollutants (POPs) are
man-made carbon-based chemical substances that have been released into the
environment on a large scale since the 1950’. Due to their chemical properties POPs
pose a global threat to human health and the environment. Common properties of
these chemicals are: toxic characteristics, persistence (lasting for years or even
decades before degrading into less dangerous forms), bioaccumulation (mainly in fatty
tissues), disposition to long-range transboundary atmospheric transport and finally
potential of POPs to cause significant adverse human health or environmental effects.

The groups of compounds that make up POPs are also classed as PBTs (Persistent,
Bioaccumulative and Toxic) and belong to the wide group of persistent toxic
substances (PTS). National, regional and international bodies are developing ways to
manage PBTs and POPs to better protection of human health and the environment.

34
 Chapter 3

There exist two main POPs initiatives: the United Nations Economic commission
for Europe (UN-ECE) Protocol on POPs (the Aarhus Protocol) and the United
Nation Environment Programme (UNEP) POPs Convention (the Stockholm
Convention). The initiatives aim to control, reduce or eliminate discharges, emissions
and losses of specific POPs to the environment. Chemicals listed on these protocols
are in table 2.

Tab. 2: List of prior persistent organic pollutants

Prior POPs according to the UNEP and the UN-ECE

organo- aldrin
chlorine DDT and metabolites
pesticides dieldrin
endrin
Stockholm con. /UN-ECE
heptachlor
chlordane
mirex
toxaphene
chlordecone
UN-ECE
lindane (gama HCH)
industrial hexachlorobenzene (HCB)
Stockholm con. /UN-ECE
products polychlorinated biphenyls (PCBs)
hexabromobiphenyl (HBB) UN-ECE
industrial polychlorinated dibenzo-dioxins (PCDDs)
Stockholm con. /UN-ECE
by- polychlorinated dibenzo-furans (PCDFs)
products polycyclic aromatic hydrocarbons (PAHs) UN-ECE

The UN-ECE executive body adopted the Protocol on Persistent Organic Pollutants
in June 1998 in Aarhus (Denmark) (entered into force in October 2003). It focuses on
a list of 16 substances (eleven pesticides, two industrial chemicals and three by-
products/contaminants). The ultimate objective is to eliminate any discharges,
emissions and losses of POPs. The Stockholm Convention is a global treaty to
protect human health and the environment from persistent organic pollutants (POPs). It
was adopted in May 2001 and entered into force in May 2004. It focuses on eliminating
or reducing releases of 12 POPs, the so-called "Dirty Dozen". In implementing the
Convention, Governments will take measures to eliminate or reduce the release of
POPs into the environment.

There are many chemicals with POP-like characteristics that are currently not listed in
the Stockholm Convention or UN-ECE POPs protocol. The Persistent Organic
Pollutants Review Committee reviews the “new“ POPs. Candidate’s POPs have to fulfil
the criteria of persistence, bioaccumulation, potential for long-range environmental
transport and evidence of adverse effects to human health or to the environment.
Chemicals that have been proposed for adding to the Stockholm Convention are e.g.
pesticides endosulfan, hexachlorocyclohexane chlordecone, methoxychlor,
pentachlorophenols; brominated flame retardants, pefluorochemicals, chlorinated
chemicals as chlorobenzene, short-chain chlorinated paraffins; polycyclic aromatic
hydrocarbons, brominated dioxins and others. Other chemicals, which are also of
concern, are alkylphenols used in industrial detergents or phthalate esters used as
softeners in PVC.

35
Laboratory tests with soil microorganisms

3.3.1 Effects of POPs on soil microorganisms

Despite the majority of POPs were banned and are not more used, they persist in the
environment. Therefore the risk potential for the non-target soil organisms
(microorganisms) and main microbial processes should be assessed. Nowadays, the
evaluation is not important only for the “old” POPs, but mainly for the “new” PTS
chemicals that are still used. The main criteria for the lists of POPs were their toxicity
and significant adverse effects especially on human health or environment. From this
point of view the POPs toxicity were evaluated. The effects on soil organisms (soil
earthworms) were also assessed; however the data about influence on soil dwelling
microorganisms are limited. If there exist studies about effects of POPs on soil
microorganisms, they are focused primarily on organochloride pesticides. The studies
were done mainly from 1950´s to 1980´s, however more recent studies also exist. The
second approach comprises studies, in which soil microorganisms are used as
potential tools for POPs biodegradation. This chapter summarized available data about
POPs toxicity on soil microbial community.

Toxicity of persistent organochlorine pesticides

Pesticides listed on the Stockholm convention are mainly insecticides that were used
against termites and other insect diseases on a range of agricultural crops.
Organochlorine insecticides are more toxic to insects and less toxic to non-target
organisms, but unfortunately they can damage a wide variety of beneficial as well as
harmful organisms due to their persistence in the environment. The effect of an
insecticide on microorganisms depends upon the nature of the insecticide, the
concentration of the insecticide, the types of microorganisms, and the environment in
which the microorganisms are growing. Substances that are degraded
microbiologically would increase CO2 production (carbon mineralization), and the
magnitude of the increase would be a function of the extent to which the carbon of the
pesticide molecule was oxidized completely. Toxic substances would depress CO2
production and the effect would be temporary or persistent, depending on the stability
of the compound. Moreover, the sensitivity of microorganisms to toxicants is under
nutritional and environmental control and may be influenced by an exogenous supply
of carbohydrate (Bartha et al., 1967). Microorganisms are also able to accumulate very
high concentrations of organochlorine insecticides like DDT or dieldrin (Lal and
Saxena, 1982).

Bartha et al. (1967) reported that chlorinated pesticides (DDT, DDD, and
methoxychlor) had no influence on carbon mineralization (no degradation) and also
nitrification at concentration 1500 ppm. They caused, however, a significant increase in
the amount of nitrate produced by the nitrifying population of soil. Ahmend et al. (1998)
observed significant depression of heterotrophic bacteria and fungi and also a sharp
decrease in nitrifying bacteria in Egyptian soil contaminated by residues of chlorine
pesticides (up to 9.5 μg/kg).

Results of studies dealing with persistent organochlorine pesticides are often

antagonistic. Reduction or changes in microbial diversity are, however, common
features. These were reported for HCH (Feidieker at al., 1994, Bhatt et al., 2007) or
DDT contamination (Kantachote et al., 2001, Edvantoro et al., 2003). Some studies
indicated that pesticides did not affect overall microbial community functions, but at
high concentration could change microbial community structure either in favour of

36
 Chapter 3

bacteria - by HCH (Das et al., 1995), aldrin (Fletcher and Bollen, 1954) - or fungi - by
DDT and dieldrin (Stojanvic et al., 1972), chlordane (Tu et al., 1991). According to
Audus (1960), aldrin, dieldrin, chlordane, endrin and heptachlor at 10 000 ppm had a
selective effect on bacterial growth. They inhibited a wide range of gram-positive
bacteria without affecting gram-negative bacteria. Nitrifying bacteria were found
sensitive and were reduced in presence of chlorinated pesticides as lindane (Martinez-
Toledo, 1993, Ogunseitan and Odeyemi, 1985). Lal and Saxena (1982) reviewed that
soil bacteria were not affected at field-applied concentration of aldrin, dieldrin, endrin
and DDT. Mirex, considered as the most stable and persistent pesticide, did not affect
total soil fungi and bacteria even at high concentration (20 g/kg), however, reduction of
actinomycetes population at 10 g/kg was reported in the study from Jones and Hodges
(1974). The unchanged microbial activities at high doses of organochlorine pesticides
are obviously due to their very low water solubility and availability. Higher toxicity was
detected for pentachlorophenol due to its higher water solubility and ionic character
(Welp and Brümmer, 1998; Zelles et al., 1986a, b).

Toxicity of PCBs, PAHs and other POPs

The number and position of the chlorine atoms affect the toxicity of polychlorinated
biphenyls. Dušek and Tesařová (1996) observed a significantly lower microbial
biomass (decreased by 23%), lower specific respiration rate (decreased by 14%) and
lower ability of C utilization by microorganism in the PCBs polluted sites (around
municipal waste incineration the Czech Republic, 14.0 ng/g soil) in comparison with the
control plot (4.4 ng PCBs/g soil). High PCB concentrations in soils decreased the
microbial biomass in the study of Anan'eva et al. (2005).

Toxicity of PAHs was studied by Sverdrup et al. (2002a). Their results showed that
tested compounds (eight PAHs and their O-, S- and N- derivates) affected nitrate
production, typically at soil concentrations above 20 mg/kg. No effect of the selected
substances was found on bacterial diversity. Yang et al. (2007) used the microbial
phospholipid-fatty-acid (PLFA) patterns as biomarker in soil contaminated by PAHs.
They found that the Gram-negative bacteria were more sensitive to PAHs than Gram-
positive bacteria and fungi. It was observed that organic matter addition (rice straw,
sewage sludge) reduced negative effect of PAHs and allows their mineralization
(Hamdi et al., 2007). Scelza et al. (2007), reported that even the soil, with no history of
PAH contamination, very likely had an indigenous microbial Phe-degradative capacity.

Polychlorinated dibenzo-para-dioxins (dioxins) and polychlorinated dibenzofurans

(furans) are by-products resulting from the production of other chemicals like
pesticides (phenoxy herbicides) and other chlorinated substances. Chemical residues
in soil are increased especially at herbicide storage sites and chemical industrial sites.
Young (2006) reported enhanced degradation of TCDD in the presence of high
amounts of phenoxy herbicides. The fungal species Penicillium, Mucor, and Fusarium
were, however, the main microorganisms associated to dioxin metabolism. A long-term
effect of dioxins on soil microbial diversity was studied by Mitsevich et al. (2000) in the
areas of South Vietnam treated with dioxin-containing defoliants (Agent Orange). In
this study fungi and actinomycetes were the most sensitive microorganisms and were
significantly reduced at heavy contaminated sites (dioxin concentrations up to
thousands ng TEQ/ kg soil).

37
Laboratory tests with soil microorganisms

Soil microorganisms have very large enzymatic facilities. Due to their metabolic and
genetic flexibility, short reproduction cycle and high adaptive potential, the attention of
scientists is often focused on their application for biodegradation and bioremediation of
organic compounds and POPs. Microbial degradation is important process influencing
POPs fate in soil and in the environment. Interaction of POPs with microbial cells
could, on the other hand, protect substances against degradation and enhance the
persistence of chemicals in soil.

38
 Chapter 3

3.4 Laboratory tests with selected POPs

Two persistent organic pollutants – chlorinated paraffin and toxaphene - were tested
within my doctoral studies. The results of these laboratory studies were published by
Bezchlebova et al. in 2007 (attached paper II and paper III). Detailed information about
effects on microorganisms was also presented in my rigorous thesis (Černohlávková,
2004). Because laboratory testing took up an important part of my doctoral study, basic
information about selected POPs, experimental designs and result are summarized at
this place.

3.4.1 Effects of chlorinated paraffins (paper II)

Chlorinated paraffins are industrial products with wide use as metal working fluids,
sealants, flame retardants in rubbers and textiles and others. Due to its persistence,
high potential to bioconcentration and bioaccumulation they have been proposed for
listing in the Stockholm convention on POPs and are currently under review (POPRC,
2007). The toxicity to aquatic organisms has been well studied, however, only limited
information exist about effects of SCCPs on soil organisms. Hence, the aim of this
study was to determine the toxic effects of short chain chlorinated paraffin (C12,
64% chlorine content) on soil organisms (Folsomia candida, Eisenia fetida,
Enchytraeus albidus, Enchytraeus crypticus, Caenorhabditis elegans) as well as on
indigenous soil microbial community. The parameters of microbial biomass content,
basal and substrate-induced respiration were assessed in the 28 days laboratory
incubation test.

Tab. 2: Chlorinated paraffins characteristics:

Chlorinated paraffin properties

Molecular weight 320 – 500
Melting point -30,5 – +20 oC
alkanes(C10-13)
chloro(50-70%)
Boiling point > 200 oC
Water solubility 0.15-0.47 mg/l
Log KOW 5.47 - 7.30 (63%Cl)
Chemical formula CxH(2x-y+2)Cly (x=10-13, y=1-13) Vapor pressure 0.021 Pa at 40°C
CAS number 85535-84-8

The paraffin (technical mixture included all short-chain paraffin fractions C10–13) was
tested as cyclohexane solution at concentrations 500, 1000, 5000 and
10 000 mg/kg dry soil. Preincubated soils (natural arable soil) adjusted to 40% of
WHCmax were weight to glass bottles (per 10 g) and treated with the relevant amount of
paraffins solution (1 ml). Control samples were spiked with the same amount of clear
solvent (cyclohexane). Lucerne meal (0.5%) was added to each sample as organic
carbon source. Three replicates were designed for all treated levels. Samples were
kept overnight to evaporate cyclohexane. Next day, the total water content was
adjusted to 60% of WHCmax. Bottles with soil were covered by rubber caps and

39
Laboratory tests with soil microorganisms

incubated at 20 ± 2°C in dark, periodically aerated. Microbial parameters (microbial

biomass, basal respiration, substrate-induced respiration) were measured after 14
and 28 days. Results are illustrated in fig. 6.

Microbial biomass Basal respiration

(μ gC.g dry soil-1) (µg CO2-C.g dry soil-1.h -1)
400 2.5

2
300

1.5
200
1

100 14 d 0.5 14 d
28 d
28 d
0 0
control
10 100 1000 1010000
000 100 000
100000 control
10 100 1000 10 000
10000 100 000
100000
s hort-chain chlorinated paraffins (m g/kg) s hort-chain chlorinated paraffins (m g/kg)

Substrate induced respiration

(µg CO2-C.g dry soil-1.h -1)
12

3 14 d
28 d
0
control
10 100 1000 10 000
10000 100 000
100000
s hort-chain chlorinated paraffins (m g/kg)

Fig. 6: Effects of short-chain chlorinated paraffins on biomass, basal and substrate-induced

respiration of indigenous soil microorganisms. The boxes show mean ± standard error (n = 3).

Results indicated significant increased of microbial biomass content at the highest

tested concentration after 28 days of incubation. Basal respiration was not
inhibited with increasing SCCPs concentrations. Substrate-induced respiration was
the most sensitive parameter. Despite this endpoint was affected at 14 day of the
incubation at any exposure concentration, after 28 days a significant decrease of
microbial respiration (ANOVA; Dunnett’s test; p<0.05) was found at the highest
exposure concentration (72% of solvent control). In general, the toxicity of the selected
SCCPs on soil microorganisms was very low. High hydrophobic character of SCCPs
could explain their low toxicity. They are bound to solid particle and organic matter and
therefore little bioavailable for soil organisms. The risk of SCCPs for soil
microorganisms is, according to presented results and predictive soil concentration,
negligible.

40
 Chapter 3

3.4.2 Effects of toxaphene (paper III)

Toxaphene is non-specific contact insecticide, technical mixture contain more than

670 congeners, mainly polychlorinated bornanes containing 67 - 69% chlorine. Its use
was banned in 80's and toxaphene was included to the list of prior persistent organic
pollutants according to Stockholm convection (UN/ECE, 1998, UNEP, 2001).
Toxaphene is hydrophobic and has tendency to bioaccumulation and bioconcentration.
Toxaphene is very toxic for aquatic organisms and fish. There was, however, little
information about ecotoxicological effects on soil organisms. The aim of our study was
to investigate the effects of toxaphene on soil invertebrates (Folsomia candida, Eisenia
fetida, Enchytraeus crypticus, Enchytraeus albidus, and Caenorhabditis elegans) and
impact on soil microorganisms.

With the experiences from the previous study, that revealed low bioindicative potential
of carbon cycle parameters to effect of persistent organic compounds, in this study
beside endpoints of carbon transformation also parameters of nitrogen cycle were
evaluated. To reduce variability and undesirable effect of spiking procedure the design
of toxaphene application was modified against the paraffin test and was following:
Toxaphene standard (ampoule of 1 g neat) was dissolved in acetone and serial
dilutions were prepared to get soil concentrations of 1, 10, 100, and 1000 mg/kg dry
soil. Two kilograms of soil (natural arable soil) were adjusted to 60% water holding
capacity (WHCmax) and pre-incubated in closed jar at 22 °C to allow microbial activity to
stabilize. After 1 week, 250 grams of preincubated soil was spiked with the toxaphene
solutions. Spiking was the same for all concentrations considered. Firstly, 25 g of soil
was thoroughly mixed with 2ml of toxaphene solution, and this soil was kept overnight
to evaporate acetone. After that, the rest of the soil (225 g), lucerne meal (0.5%) and
water (amount evaporated overnight) were added and thoroughly mixed. Five hundred
grams of soil was prepared in the same way for acetone controls. Spiked soils were
incubated in 1l tightly closed glass jars at 22°C in darkness and periodically aerated.
After 28 days of incubation, following microbial parameters were measured:
microbial biomass, basal respiration, substrate-induced respiration,
ammonification, arginine ammonification, and nitrification. Three sub-samples
(replicates) with appropriate weight of soil were prepared for each concentration and
the control.

41
Laboratory tests with soil microorganisms

Tab. 3: Toxaphene characteristics:

Toxaphene properties
Chemical class and structure Melting point 65-90 °C
Boiling point >120°C
polychlorinated
Water solubility 3 mg/l
bornanes
(67 - 69% chlorine) Log KOW: 3,3
Log KOC: 2.47 - 5.00
CAS number 8001-35-2 Vapor pressure 0.2 - 0.4 mm Hg at 20°C
Chemical formula C10H10Cl8 (approx) Henry's law const. 0.063 atm m3/mol
Molecular weight 414 (average)

Results of this study are shown in fig. 7. and are discussed in paper III. Generally,
toxaphene did not have strong negative effects on soil microorganisms in
measured parameters. The highest tested concentration of 1000 mg/kg caused
inhibition only of microbial biomass and ammonification. Parameters of nitrogen
transformation, basal and arginine ammonification, was negatively affected also even
at the highest tested concentration. Basal respiration was not negatively affected at
any of the tested concentrations and also not clear relationship was found on
nitrification activity. Stimulation of microbial activities was observed at low toxaphene
concentration for substrate-induced respiration.

In soil, toxaphene is strongly bound to solid particles and almost non-bioavailable

for microorganisms. Negative influence on soil environment could appear only at
heavy pesticide contaminated sites. Microorganisms are also able to biodegraded
toxaphene under anaerobic conditions (e.g. Lacayo-Romero et al., 2006). Because of
low environmental concentrations in the Czech Republic and high effective
concentration the risk of toxaphene for soil microorganisms can be neglected.

42
 Chapter 3

S ubstrate-induced
Microbial biomass C Basal respiration respiration
(μg . g-1) (μg CO 2-C . g-1 . h-1) (μg CO 2-C . g-1 . h-1)
600 1.6 16

450 1.2 12

300 0.8 8

150 0.4 4

0 0.0 0
0 1 10 100 1000 0 1 10 100 1000 0 1 10 100 1000
Toxap hene concentration (mg/kg) Toxap hene concentration (mg/kg) Toxap hene concentration (mg/kg)

Ammonification Nitrification Arginine ammonification

(μg NH 4-N . g-1 . d-1) (μg NO 3-N . g-1 . d-1) (μg NH 4-N . g-1 . h -1)
5 2.5 4

4 2.0
3
3 1.5
2
2 1.0
1
1 0.5

0 0.0 0
0 1 10 100 1000 0 1 10 100 1000 0 1 10 100 1000
Toxap hene concentration (mg/kg) Toxap hene concentration (mg/kg) Toxap hene concentration (mg/kg)

Fig. 7: Effects of toxaphene on biomass, respiration (basal and substrate-induced),

ammonification and nitrification of indigenous microorganisms after 28 days. The boxes show
means ± standard errors (n = 3).

43
Laboratory tests with soil microorganisms

3.5 Effects of pesticides on soil microorganisms

Other chemicals, which were in focus of my doctoral studies, were selected pesticides.
Pesticides present a group of chemicals that are purposely applied to environment with
aim to suppress plant and animal pests and to protect agricultural and industrial
products. The majority of pesticides are not specific and during their application they
can reach soil and affect non-target organisms such as soil microorganisms. The
International Organization has assigned more than 1100 of official pesticide names for
Standardization (ISO) (The Compendium of Pesticide Common Names Internet
database, http://www.alanwood.net/pesticides). Pesticides can be grouped related to
biological effects, chemical structure, application form or origin. The main groups of
pesticides according to target organisms are herbicides (against plant), fungicides
(against fungi), bactericides, insecticides, algicides, acaricides, rodenticides,
molluscicides, nematocides and many further (e.g. against viruses, birds, plant growth
regulators). Chemical structure of pesticides determines mode of action and also
distribution and fate in environment. The most important properties are molecular
weight, polarity and charge, solubility, lipofility or volatility. The majority of pesticides
are chemicals related to chlorinated hydrocarbons, organophosphates, dinitrophenols,
carbamate and thiocarbamate, derivates of urea, esters, anilines and many others.

Registration of pesticides comes under strict rules and includes assessment of impact
on soil microorganisms. Design of laboratory testing, concentration set-up and
properties of soil used simulate the worst-case scenario. Therefore, the results from
the laboratory testing show higher toxicities than the observations from the field
situations. In the field, pesticides are applied to various soil types, often with higher
sorption capacity or clay amount. Adverse effects of pesticides are often reported at
high (non-realistic) concentrations (Ahtiainen et al., 2003). On the other hand
environmental condition could increase the exposition of microorganisms to pesticides
impact.

Processes of carbon and nitrogen mineralization are the most important microbial
endpoints evaluated in the risk assessment of pesticides. However, specific microbial
groups and functions must be considered as well because they might be crucial for
some soil processes and functions (nitrification, N2 fixation, denitrification, contaminant
degradation etc.). Impacts of pesticides could also be different on different microbial
group (fungi vs. bacteria, between bacterial species) that complicates statistical
evaluation of test results (Giller, 1998).

Effects of pesticides on carbon mineralization

There exist a lot of studies dealing with pesticide effects on soil microorganisms.
However, they strongly differ in design, used methods of measurement, application or
concentration of tested pesticides as well as in observed results. The parameters
evaluated within carbon mineralization are in particular soil basal and substrate-
induced respiration, respiration kinetic, microbial biomass carbon content or
determination of mineralization with radiolabeled C14. Application doses of pesticides
vary according to specific substance and application form, but range mainly from units
to tens mg per kilo of soil. Many studies reported that microorganisms are able to
tolerate low concentrations of pesticides (Gigliotti and Allievi, 2001, Strickland et al,
2004, Katayama et al., 2001, Downing et al., 2004). To the contrary, stimulation of
carbon mineralization (Das and Mukherjee, 2000, Elmholt, 1992, Chen et al., 2001,

44
 Chapter 3

Malkomes, 1999, Malkomes 2005, Harden et al., 1993) as well as soil enzymatic
activities (Tu 1990, Tu 1993) was often observed at application doses. These results
indicate that microorganisms can utilize some pesticides at lower concentration
as source of carbon.

Negative effects such as decrease of respiration activity and biomass amount

appeared only at concentration several times higher than application doses and seem
to be rather short-term. Recovery to original values, similar as before application, was
obtained often in 3-4 weeks (Fließbach and Moder, 2004, Chen et al, 2001,
Munier-Lamy et al. 2000, Perruci et al., 2000). This negative effect should be caused
by higher toxicity to sensitive microbial species followed by growth of resistant species
or utilization of the tested chemical. More significant adverse effect were observed by
fungicides (thiram, captan, dithianon), which are toxic also to soil fungi and
actinomycetes and cause changes in microbial community structure in favour of
bacteria (Anderson et al., 1981, Liebich et al., 2003, Pal et al., 2005). It was further
reported that amendments of organic substrates to soil (alfalfa, straw, compost)
increase mineralization potential and could decrease and soften inhibition effects of
pesticides (Liebich et al., 2003, Malkomes, 1999, Chen et al., 2001, Perruci et al.,
2000).

Effects of pesticide on nitrogen mineralization

When nitrogen transformation processes are assessed, ammonification, nitrification,

denitrification, N2 fixation or microbial biomass nitrogen content are the main evaluated
endpoints. The majority of studies did not show negative impacts of pesticides on
ammonification process. However, this endpoint is related to majority of
microorganisms and it is not very sensitive in toxicity testing (Martens, 1997, Vonk,
1991). Martens and Bremner (1997) tested effects of 17 fungicides on urea hydrolysis
(endpoints of ammonification) and a decrease was observed even at multiple
application concentrations. On the other hand nitrification bacteria are considered to
be very sensitive (Anderson, 2003). Moreover, nitrification assay is relatively easy and
fast. Therefore nitrification and potential nitrification activity are more frequently
used endpoints when pesticides are tested. Inhibition of nitrification was proved by
sulphonylurea herbicides (Gigliotti and Allievi, 2001). Dithiocarbamate fungicides
(mancozeb, maneb, zineb) were again very toxic to potential nitrification and
denitrification (Pell et al., 1998, Martens and Bremner, 1997, Kinney et al., 2005). Chen
et al. (2001) reported that some pesticides caused increasing amount of ammonium
ions and lower nitrate production due to inhibition of nitrification bacteria. However, this
effect was only short-term.

Denitrification seems to be less affected, moreover rather stimulated in many cases,

probably by release of organic matter from death bacterial cells (Pell et al., 1998).
Stimulation of nitrogen mineralization was observed by fungicides metalaxyl,
mefenoxam (Monkiedje et al., 2002), benomyl and captan (Chen et al., 2001) as well
as by insecticides (Das and Mukherjee, 2000). Again, increased mineralization activity
was related to available organic matter released from bacterial cells, applied chemicals
or from unavailable forms in soil. Similarly as for carbon mineralization, amendments of
organic matter to soil caused increasing nitrogen mineralization. On the other hand,
amendments of inorganic fertilizer could lead to higher nitrate formation, which can
mask the negative effects of pesticides.

45
Laboratory tests with soil microorganisms

Overall, pesticides certainly impact soil microorganisms. Depending on the

particular substance, microbial community composition and situation in the soil, they
may have both stimulation and inhibition effects on soil microorganisms. Various
soil microbial groups are differently affected and composition and balance of the
microbial community is very often changed after pesticide application. Negative
effects were proved to be rather short-term and appeared even at higher
concentration than recommended as application dose. Repeated application and
combination of several pesticides seemed to be more adverse (Kinney et al., 2005).
Evaluation of carbon, nitrogen cycles endpoints as well as specific group of
microorganisms is necessary for objective pesticide assessment. Moreover,
assessment under conditions close to real application and agriculture practise should
be evaluated.

46
 Chapter 3

3.5.1 Effects of selected pesticides (paper IV)

In the last laboratory study presented within this thesis we focused on evaluation the
risk of pesticides that are frequently used in the Czech Republic. The main
criterions for pesticide selection were wide usage and high consumption in agricultural
praxis and potential of toxic effects on soil microorganisms together with the lack of
information about that. After consultations with experts from State Phytosanitary
Administration, two fungicides - dinocap and mancozeb - were selected. In this
study, effects of fungicides on soil nitrogen transformation (potential
ammonificiation and potential nitrification) and carbon transformation (basal
respiration, substrate-induced respiration with the evaluation of growth kinetic)
were measured. Substrate-induces growth kinetic was analysed using the OxiTop®
measurment system. This study was aimed as well to validation of this device for
routine application for toxicity testing of chemicals.

Results and discussion of this study were published in scientific paper that is attached
to this thesis as paper III. Here, some additional information about selected fungicides,
experimental design and main results are presented.

Test chemicals and soil treatment

Dinocap – dinitrophenol fungicide and acaricide – is used as a contact fungicide for

control of powdery mildew on fruit, vegetable, vine and ornamental crops. Dinocap is of
low persistence in the soil environment, with reported field half-lives of 4 to 6 days. It is
highly photosensitive and readily broken down by sunlight. It is also subject to
microbial degradation. More than 1000 l of dinocap was consumed in the Czech
Republic for protect vine, vegetables and fruit orchard in 2005 (State Phytosanitary
Administration). Detailed information is in table 4.

Tab. 4: Dinocap characteristics

Dinocap properties
Chemical class and structure
Chemical formula C18H24N2O6
Molecular weight 364.4 g/mol
2,6-dinitro- Water solubility <0.1 mg/l
4(or 6)-octylphenyl practically insoluble
crotonates Commercial product Novozir MN 80
Active ingredients mancozeb 80 %
Formulation liquid concentrate
CAS number 39300-45-3 Application dose 0.2 % solution,
spray fungicide

47
Laboratory tests with soil microorganisms

Mancozeb – ethylene(bis)dithiocarbamate polymeric fungicide – is used to protect

many fruit, vegetable, nut and field crops against a wide spectrum of fungal diseases.
Mancozeb is of low soil persistence, with a reported field half-life of 1 to 7 days.
However, it rapidly and spontaneously degrades to ethylenethiourea that may persist
for longer, on the order of 5 to 10 weeks. In the Czech Republic, mancozeb is one of
the most used fungicides. In 2005, more than 130 tons were applied (State
Phytosanitary Administration). Detailed information is in table 5.

Tab. 5: Mancozeb characteristics

Mancozeb properties
Chemical class and structure CAS number 8018-01-7
Molecular weight 266.3 g/mol
manganese
Water solubility 6 mg/l, insoluble
ethylenebis
Commercial product Karathane LC®
(dithiocarbamate)
polymeric Active ingredients dinocap 350 g/l
complex with zinc Formulation spray fungicide,
salt wettable powder
Application dose 0.05 % solution per ha

Experimental design

This study was carried out with two soils – arable and grassland. Detailed properties
are given in the paper IV. Fungicides were tested in their commercial formulation
Karathane LC (containing 35% of active dinocap) and Novozir MN 80 (containing 80%
of active ingredients mancozeb). Both fungicides were tested at recommended
application rate and 10 times lower rates. Application solutions were prepared
according to label instruction as water emulsion (Karathane LC) or water dispersion
(Novozir MN 80). Final fungicides concentrations were 2.8 and 28 mg.kg-1dw for
dinocap and 25.6 and 256 mg.kg-1dw for mancozeb. A large amount of soil (about
250 g) was spiked with appropriate amount of fungicides solution to obtained 60% of
WHC. Control samples were spiked with the same amount of distilled water. Endpoints
of nitrogen transformation (potential ammonificiation and potential nitrification) were
measured the 1st day and after 14 days of incubation; basal respiration substrate-
induced growth kinetic after 1 week.

Results and discussion

All results are presented and discussed in the attached paper IV. In general, both
fungicides showed significant adverse effect on nitrogen transformation, but
mancozeb was more toxic. This effect was rather short-term, however, at the higher
tested concentration lasting even after 14 days. Moreover, accumulation of ammonium
ions in soil indicated disruption of nitrogen cycle, specifically nitrification process,
by mancozeb. To the contrary, parameters of carbon mineralization were mostly

48
 Chapter 3

stimulated. Overall, parameters of basal respiration did not show great differences
tested variants. However, specific parameters of growth kinetic revealed enhanced
growth activity (specific growth rate -μ) and shorter time to the maximal respiration
rate at lower concentration of mancozeb. The Oxitop® system seemed suitable for soil
respiration kinetic determination, yet some disadvantages (e.g. high sensitivity to
temperature oscillation) occurred during measurement.

Results of this study emphasized that focusing on specific microbial parameters such
as nitrification gives better understanding the complex effects of chemicals on
microbial activities.

49
Soil microorganisms as bioindicators

4 SOIL MICROORGANISMS AS BIOINDICATORS

As pointed out frequently in the previous text, soil microbial community has a
fundamental role in terrestrial ecosystems. Besides laboratory tests, where microbial
communities are exposed to chemicals under controlled conditions, monitoring of soil
microbial parameters is important with respect to general evaluation of soil state and
quality.

Intensification of soil use during the last decades, particularly in agriculture, has
caused fear that soil quality could be changed for long time or irreversibly. In the last
50 years, about 2 billion of the 8.7 billion ha of agricultural land, permanent pastures,
and forests and woodlands have been degraded. Negative changes are reduction of
soil fertility and one of the key reasons of this is inadequate soil and water
management (Arshad and Martin, 2002). On the other hand, subsequent proposals for
sustainable land use have shown that there is a lack of suitable parameters for the
assessment of “soil quality” from the viewpoint of soil microbiology. Therefore need of
methods and concepts for soil character and quality assessment arose.

4.1 Soil quality

The interpretation of the term “soil quality” is not unequivocal, but differs in accordance
to viewpoint of management praxis such as soil fertility in agriculture, stability of
ecosystems, society's priorities, etc. Larson and Pierce (1991) defined soil quality as
the capacity of soil to function within the ecosystem boundaries and to interact
positively with surrounding ecosystems. The Soil Science Society of America (SSSA)
contributed to development of the soil quality concept. This led initially to a simple
definition for soil quality: “the capacity (of soil) to function”. An expanded version
defines soil quality as ”the capacity of a specific kind of soil to function, within natural or
managed ecosystem boundaries, to sustain plant and animal productivity, maintain or
enhance water and air quality, and support human health and habitation” (Karlen et al.,
1997). Doran and Safley (1997) define soil quality in similar way. The interpretation of
“soil quality” is sometime used in context or as a synonym to “soil health”. The term
“soil health“, however, focuses more on the biotic components of a soil, reflecting, i.e.,
the maintenance of soil organisms and their proper functioning as regulators of nutrient
cycling and therewith of soil fertility. In the pertaining literature both terms are
intermingled (Anderson, 2003).

Assessment of soil quality is complicated process, which has not been appropriately
standardized so far. The ideal soil microbiological and biochemical indicator to
determine soil quality should be simple to measure, should work well in all
environments, be sensitive to long-term changes in soil management, but at the same
time should be resistant to short time fluctuation (e.g. weather) and also should reliably
reveal which problems existed where (Scholter et al., 2003). It is unlikely that some
single ideal indicator can be defined because of the multitude of microbiological
components and biochemical pathways. Thus, the basic indicators and the number of
estimated measures are still under discussion. Depending upon the nature of the
function under consideration the actual properties selected will vary.

50
 Chapter 4

Nortcliff et al. (2002) grouped attributes that might be used as indicators of soil
quality:

ƒ physical attributes (e.g. soil texture, dry bulk density, water holding capacity
porosity, aggregate strength and stability, soil crusting, soil compaction, top
soil depth, etc....),
ƒ chemical attributes (pH, salinity, aeration status, organic matter content,
cation exchange capacity, status of plant nutrients - NH4, NO3, P, K,
concentrations of potentially toxic elements, and possibly the most important
attribute, the capacity of the soil to buffer against change),
ƒ biological and biochemical attributes (biodiversity, populations of micro-,
meso- and macroorganisms, respiration rate or other indicators of microbial
activity, phytotoxicity and so on),
ƒ visible attribute (visible evidence of the decline in soil quality like evidence of
erosion in the form of rills and exposure of subsoil, surface ponding of water,
surface run-off and poor plant growth).

4.1.1 Microorganisms as indicators of soil quality

Biomass, respiration, mineralization potential, microbial diversity or enzymatic activities

are the major microbial parameters determined within the scope of nationals and
international monitoring programmes of soil quality. Common question remain, which
microbial parameters are the most suitable to indicate soil quality.

Microbial biomass is a fundamental parameter to which all others are closely related.
It has been suggested that the microbial biomass content is an integrative signal of the
microbial significance in soils because it is one of the few fractions of soil organic
matter that is biologically meaningful, sensitive to management or pollution and finally
measurable. Microbial biomass reflects the dynamics of soil organic matter as its sink
and source joined with the pivotal functions of organic matter transformations and
cycling. Despite pollutants (e.g. heavy metals) may reduce soil microbial biomass, it
must be realized that differences may occur without direct correlation to soil quality
(Dilly and Munch, 1998). Another problem is that the presently widespread biomass
estimates (fumigation-extraction, substrate-induced respiration, fumigation- incubation,
ATP content), either direct or indirect (biochemical) techniques, were not properly valid
and checked for separating active and dormant microorganisms or different microbial
groups. Microbial biomass amount gives therefore information about whole community
without definition of its heterogeneity. Although the soil microbial biomass is the eye of
the needle through which all organic matter needs to pass through, it is a susceptible
soil component and may be therefore include into soil quality indicators (Scholter et al.,
2003).

The observed loss of aboveground biodiversity of animal and plant species as a result
of the expansion of farm production and intensification of land management has also
initiated questions if also below-ground (microbial) biodiversity is affected and if so, to
which degree? And second if soil microbial diversity could be applied to assess
environmental stress and work as and indicator of environmental changes (Anderson,
2003). Some studies (mentioned in Scholter et al., 2003) proved relationship between
microbial diversity changes and type of land use. On the other hand huge species
diversity and adaptation to altered condition hinder the usage of microbial diversity as
sensitive soil quality indicator. The species spectrum of any soil is never a constant

51
Soil microorganisms as bioindicators

entity, but shifting permanently, even under “normal” agricultural management as well
as by natural stresses (freezing, draught) and it would be very difficult to determine
man-made effects by taxonomic species diversity, since they could be masked by
natural impacts. With the rise of molecular genetic tools in microbial ecology it became
apparent that we know only a very small part of the diversity in the microbial world.
Most of this unexplored microbial diversity seems to be hiding apparently in the high
amount of yet uncultured bacteria. Assessment of microbial community diversity using
this molecular approach has, however, still a lot of obstacles such as the lack of
quantitative recovery of nucleic acids. Beside genetic, functional diversity should be
also assessed. The basic question here is, if microbial controlled soil functions, i.e.
decomposition of organic material (a guarantee of soil fertility) depend on species
diversity. Application of the BIOLOG technique for example showed only little decrease
in the functional diversity of these two communities, although in the acidic soils was
fewer bacteria. There was, however, a difference in the utilization of substrate types
between the two communities (Kreitz and Anderson, 1997).

Due to the mentioned complexity of the whole microbial community it might be useful
to look at “keystone” species or “keystone microbial controlled processes” as
indicators of environmental change or disturbances. Sensitive indicator could be e.g.
nitrogen fixing bacteria, arbuscular mycorrhiza or actinomycetes (Anderson, 2003).

Assessment of microbial activities, both specific for individual species and total
community, gives further valuable information thanks to direct relationship to soil
properties (amount and availability of nutrients, organic matter) and metabolic
processes. Short-term laboratory tests designed from 2 to 5 h best enabled to assess
microbial activities because they minimize changes in biomass structure during the
experiments. Laboratory assays have also the advantage of standardizing
environmental factors, and thus allow the comparison of soils from different
geographical locations and environmental conditions and also results from different
laboratories. Such microbial activity measurements include enzymatic assays that
catalyse substrate-specific transformations and may be helpful to ascertain effects of
soil management, land use and specific environmental conditions. The question, which
enzymes from the wide range are most suitable as soil quality indicators, remains.
Enzymes with a critical role in the N and C cycling (processes of nitrification, N2 fixation
or denitrification) should be good and sensitive indicators. Several “keystone enzymes“
(e.g. proteases) are, however, regulated by extracellular enzymes with no microbial
cell control (Schloter et al. 2003).

Besides basic microbial parameters itself, derived eco-physiological indicators

should be use to evaluate the soil quality. Eco-physiological approach was introduced
already in 1980' based on ecological principles of ecosystem functioning stated by
Odum (Anderson and Domsch, 1985). Its application in the soil quality assessment
was presented in Anderson (2003). The term eco-physiology already implies an
interlinkage between cell-physiological functioning and environmental factors.
Relationships between microbial biomass and its activity under various environmental
conditions could be useful indicator of whole microbial community state, energy flows
through ecosystem or possible stress. Such indicators are often used the qCO2 -
„metabolic coefficient“(community respiration per biomass unit) and a ration Cbio:Corg
(amount of biomass per total organic matter content). With the link to soil quality, it was
shown that monocultural soil system lead to higher energy requirements and lower
biomass (thus higher qCO2, lower Cbio:Corg) in comparison to soil with long-term crop

52
 Chapter 4

rotation systems. Many other eco-physiological coefficients should be used like e.g.
SIR/BR, specific growth (μ), death (qD), nutrient uptake (Vmax) and so on (Anderson et
al., 1994). Malý et al. (2002) used similar coefficient for nitrogen - Nbio/Norg, Cbio/Nbio, qN
as bioindicators in routine monitoring of arable and grassland soils. They concluded
that eco-physiological coefficients are potential sensitive indicators of soil quality.
These derived eco-physiological coefficients are, however, dependent on
environmental changes. This presents a certain disadvantage when soil quality
assessed. Experiments should be therefore carefully designed and properly
interpreted.

4.1.2 Soil quality assessment

Great attention to develop and standardize methods for soil quality assessment and
protection is paid under nationals and international levels. The international afford are,
however, complicated by different national approach to soil quality conception
(Nortcliff, 2002). In Europe, an international project including states from Central
Europe (Czech Republic, Slovakia, Hungary, Germany and Russia) was set up
between 1995 and 1997. The aim of the project was to estimate whether some
microbiologically-based soil characteristics would be capable of indicating
anthropogenically-caused alterations in soil quality. There examined almost 50
regularly sampled differently anthropogenically-affected sites and over 20 individual,
both microbial and abiotic, parameters evaluated. The project results and conclusions
were summarized by Filip (2002). It was concluded that N2-fixing bacteria, total
microbial biomass, soil respiration, dehydrogenase activity, and perhaps also the
humification activity of soil microorganisms could serve as sensitive indicators of soil
quality. A detail on sensitivity of microbial endpoints is given in table 6.

53
Soil microorganisms as bioindicators

Tab. 6: Sensitivity of microbial parameters in soil quality assessment according to Filip (2002).

Soil quality assessment is complicated due to soil complexness and multiple functions.
Heterogeneity, seasonal changes and other problem of soil variability were discussed
in chapter 2 and should be kept in mind during monitoring of soil quality indicators.
Moreover decreasing quality could appear after accumulation of more adverse effects.
The aim of the soil quality assessment is therefore to set up easy method or index
system to quantify and predict soil quality. Karlen et al. (2003) suggested to score
indicators includes in minimum data set according to relationship to the soil’s inherent
capability. Indicator scoring can be accomplished in a variety of ways (e.g. linear or
nonlinear, optimum, more is better, more is worse) depending upon the function. The
unitless values are then combined into an overall index of soil quality and can be used
to compare effects of different practices on similar soils or temporal trends on the same
soil. Doran and Parkin (1994) presented another way and propose soil quality index
(SQ) that consist of six elements: food and fibre production, erosivity, the ground water
quality, the surface water quality, the air quality and the food quality. According to
these scientists, advantage of this approach is that soil functions can be assessed
based on specific performance criteria established for each element, for a given
ecosystem. Data sets can be compared by designing sunray plots (or stars plots,
orientors). An example is given in the figure 8. This approach enables to display
aggregated “microbial pattern“ and compare sites between each other or changes in
time. The shape of the sunray plot indicates the state of overall microbial community.

54
 Chapter 4

On the other hand, this approach has also limitations such as abiotic factors are
omitted from the analysis (Scholter et al., 2003).

A B C

Fig. 8: Examples of sunray plots as was proposed in (A) Schouten et al. (2000) and used by (B)
Dilly and Blume (1998) and (C) Hofman et al. (2003).

The important question during soil quality assessment is determination of threshold

values or critical limits for the proposed soil indicators. Critical limit is the desirable
range of values for a selected soil indicator that must be maintained for normal
functioning of the soil ecosystem health. Within this critical range, the soil performs its
specific functions in natural ecosystems. Determination of these ranges is very difficult,
because they differ between indicators and also soil environment. Variation of 10%
should be critical for one case, but not affect another. Assessment should also take
note to certain land use (field, forest, pasture, recreation area) and specific crop. For
example, a pH below 6.5 reduces the yield of alfalfa but not affect other farming
products (Arshad and Martin, 2002).

In many cases, no reference values are available. Measuring soil parameters in a

specific soil system over time rather than in comparison with other systems is
recommended as a dynamic assessment approach. Monitoring activities and
programs that are in progress in many countries have to define these reference and
threshold values for repeated monitoring activities. Detailed information about
monitoring program e.g. in Denmark is available in the report by Nielsen and Winding
(2002). In the Czech Republic, Central Institute for Supervising and Testing in
Agriculture (www.ukzuz.cz) has realized monitoring of agriculture soils. Its results
showed that for example microbial biomass in monitored agricultural soil ranged from
-1
92 – 670 μg C.g (Sáňka and Materna, 2004). Long-term monitoring of soil microbial
parameters has been conducted also at RECETOX. Some results were summarized
e.g. in Hofman et al. (2003 and 2004). Research by Hofman et al. (2004) indicated
ranges of microbial biomass in forest (300 – 8000 μg C.g-1, median 1078 μg C.g-1) and
grassland soils (150 - 3000 μg C.g-1, median 880 μg C.g-1). Another summary of
microbial, chemical and textural parameters form long-term monitoring of over
160 arable and grassland soil in the Czech Republic is given in study from
Růžek et al. (2006). Their results showed that whereas microbial biomass ranged from
350 – 550 μg C.g-1 across various soil taxonomical units, in urban soils microbial
biomass were much lower (about 180 μg C.g-1).

Mathematical modelling can be a useful tool, when data are missing. Models could
help to describing relationships of several indicators, evaluating obtained data, predict

55
Soil microorganisms as bioindicators

soil health and up-coming changes. Furthermore, modelling will aid in reducing the
number of sampling locations, decisions of sampling frequency and of indicators within
a MDS (Nielsen and Winding, 2002).

4.2 Microorganisms as indicators of soil contamination

Microbial properties have been shown to serve as an early warning of decreasing soil
quality by agricultural practise like tillage (Powlson et al., 1987, Kandeler et al., 1999).
Besides that, evaluation of microbial activities has appeared as sensitive indicator of
the pollution impact on soil ecosystem. Relationship between microbial properties and
contamination was more studied and proved in case of metal polluted sites (Aceves
et al., 1999, Wang et al., 2007, Dai et al., 2004). Metal speciation is also important to
determine, because it affects the bioavailabitity and toxicity to soil microorganisms
(Kunito et al., 1999, Welp and Brummer, 1997). It was often observed that use of more
and specific parameters had better bioindication potential. For example Barajas-
Aceves (2005) reported that linked parameters (biomass specific respiration, biomass
as a percentage of soil organic C) may well serve as useful indicators of environmental
stress and Dumestre et al. (1999) showed that the lag period determined from the
substrate-induced mineralization tests was a sensitive indicator of copper pollution.
Avidano et al. (2005) monitored several microbial (metabolic profile, enzymatic activity
pattern, bacterial density and species diversity) in industrial highly polluted soils with
both metals and organic pollutants. They found that used parameters were affected by
pollutants even if their low concentration and observed synergistic effects of different
kind of pollution. They concluded that use of different microbiological indicators seem
to be more efficient and informative than the analysis of single parameter. Various
parameters differ in sensitivity to critical contamination as showed Simona et al. (2004)
for different microbial enzymatic activities in metal contaminated sites. Therefore they
used an integrative index (AME) that expressed the average microbial response from
assessed endpoints. This method was, according to the authors, more suitable than
single microbial parameters for bioindication of stress induced by heavy metals.

Bioindication potential of microorganisms in case of soil organic pollution has not

been so often studied, but it was proved e.g. for petroleum and hydrocarbons
contaminated sites. Megharaj et al. (2000) showed that microbial biomass, soil enzyme
-1
activity decreased in correlation to hydrocarbons pollution (5 200 – 21 430 mg kg
soil). Maila et al. (2006) evaluated soil microbial diversity (community level
physiological profile and PCR- DGGE) of different geographic locations contaminated
by hydrocarbons. Their results indicated that different locations contaminated by
different hydrocarbons had different microbial communities and that hydrocarbons
rather than the geographical origin of the sample were more important in determining
the functional or species diversity within the bacterial communities. The bioindication
potential is, however, often demeaned by mentioned high spatial and temporal
variability (Broos et al., 2007, Wardle 1997, Gelsomino et al., 2006).

56
 Chapter 4

4.3 Case study in Zlín region

One part of my doctoral studies was concerned on the monitoring of fluvisols at Zlín
region. This region was selected as model area of small river basin with a gradient of
environmental load affected by agriculture, industrial and urban activities. The
monitoring in Zlín region was performed earlier, during 1997 – 2002. Since 2005, this
area has been monitored within the scope of project INCHEMBIOL and besides
fluvisols also sediments and air have been sampled. In 2005 and 2006 I was
participating monitoring of fluvisols with the aim to study interaction between microbial
parameters, soil contamination and physical-chemical characteristics. In this thesis I
want to present results of fluvisols monitoring that were focused on the role of soil
microorganisms as bioindicators of soil contamination. The data were presented at two
conferences as posters (appendices 6 and 7).

Fluvisols are young soils that are developed in river floodplains and characterized by
periodical inundation. High temporal and spatial heterogeneity and variability are
typical for flooded soils. Permanent sedimentation, denudation and ground water
fluctuation are the main processes participation on fluvisols genesis. Condition and
properties of these soils depend on presence, intensity and duration of floods.
Deposition of river sediments is a source of organic matter but also contaminants
presented in river water. Accumulation of heavy metals (Krüger at al., 2005,
Middlekoop, 2000, Brinkmann et al., 2000, Tighe et al., 2005) as well as persistent
organic pollutants (Witter et al.2003, Umlauf et al., 2005, Elhottova et al., 2006,
Schwartz et al., 2006) was well documented in floodplain soils. Fluvisols have been
therefore understood as important indicator ecosystems, which reflect the
contamination level of the whole river basin. That is why fluvisols were selected as
model soil type for understanding the interactions between soil contamination,
microbial parameters and soil properties.

Study area and monitoring activities

The study area at Zlín region is the flooding area round rivers Morava and its tributary
Dřevnice in the southeastern part of the Czech Republic. This area, with 10 cities and
72 villages with largest industrial city Zlín, has high economic and cultural significance
and is noted for its industrial (chemical, plastics-and-rubber, boot-and-shoe, food-stuff)
and agricultural activities. The Zlín region goes along Dřevnice River from rural
countryside at east to town agglomeration at west, which makes a gradient for different
pollution sources. The soil monitoring in this region has long tradition. Over 37 fluvisols
were monitored by RECETOX during 1996 - 2002 under the research project
concerned to ecological risk assessment. The sampling sites were located in close
proximity to the river flow or up to 10 m from riverbanks. Soils were sampled
repeatedly during a year; on the whole 9 sampling campaigns were done (in May,
June, September, November 1997, May and September 1998 and 1999, April 2000).
In July 1997, the enormous floods (100-year floods) affected the region and caused
several ecological damages. On the other hand the floods allowed evaluating fluvisols
changes, long-term development and comparison with the initial state. Since 2005
these fluvisols has been monitored within the scope of the project INCHEMBIOL
Several original sampling sites got lost or changed and it was necessary selected new
similar sites.

57
Soil microorganisms as bioindicators

4.3.1 Effects of floods on soil microbial properties (poster 1)

The first work was focused on evolution of fluvisol sites in time and comparison of
soil properties before and after the floods in 1997 and also after several years.
Thanks to repeated monitoring of fluvisols, it was possible to assess the impact of the
floods immediately after the disaster. Despite several sites were totally destroyed,
some interesting results were observed and reported in the project document
(Holoubek et al., 2000). My work was focused on soil microbial properties assessment.
Results that summarized monitoring activities and effects of the floods on soil microbial
properties were presented as a poster presentation at SETAC 2006 (appendix 6).
The comparison of microbial properties was performed using sunray plots as was
described in Hofman et al. (2003). Many visible changes also were observed in 2005.
Despite not all sites were preserved due to reconstruction of riverbanks after the flood,
in some cases it was possible to sample the same sites. For the analysis, campaigns
before the floods (July 1997), after the floods (September 1997) and 8 years after (May
2005) were selected. Eight parameters – microbial biomass Cbio, BR (basal
respiration), PR (potential respiration, Corg (organic carbon), qCO2, PR/Cbio, PR/BR,
Cbio/Corg – were standardized (centered by average subtraction and normalized by
dividing by the standard deviation) in the framework of the soil set evaluated. The
standardized values were plotted into sunray plots with eight axes. In this way, one plot
for each soil sample was obtained, characterizing the status of the microbial biomass.
Different shapes of the plots enabled comparing sites as well as developing of
microbial profiles and temporal changes at each site. Moreover the shape can indicate
biological quality of soil, where the “best“ shows maximal values of Cbio, BR, BR, Corg
and other parameters not exceeding the average.

Results showed that soils texture shifted from medium to coarse structure, pH, sorption
capacity and microbial biomass increased. To the contrary, there was found decrease
of organic matter and available carbon content and soil respiration activity.
Concentrations of pollutants indicated increase of heavy metal and PAHs amount and
oppositely decrease of PCBs and chlorinated pesticides content at the majority of sites.
Results of sunray plots are shown in figure 7. Assessment of soil microbial parameters
indicated changes in microbial profile after the flood. However, it was interesting, that
background sites (fig. 9A) at upper part of Dřevnice river basin showed only little
changes during whole monitoring period that indicated relatively stable soil ecosystem.
To the contrary at some sites microbial biomass decreased and metabolic coefficients
increased that could show on stress situation after the floods. At some sites, where
contamination decreased, the microbial profile indicated improvement of microbial
community state after the floods. On the other hand, sites next to the Morava River,
those were more polluted, revealed less stable microbial ecosystem with very
different shape of the sunray plot in time (fig. 9B). Surprisingly after 8 years form the
floods, the sunray plots showed very similar pattern as before the floods in some cases
(fig. 9C). This phenomenon was, however, only rare.

58
 Chapter 4

A B C
Cbio Cbio Cbio
3 3 3

Cbio/Corg BR Cbio/Corg BR Cbio/Corg BR

0 0 0

PR/Cbio -3 PR PR/Cbio -3 PR PR/Cbio -3 PR

PR/BR Corg PR/BR Corg PR/BR Corg

qCO2 qCO2 qCO2

before the flood 2 month after the flood in 2005

Fig. 9: Results of sunray plots: (A) background site at Dřevnice River, (B) site next to the Morava
River, more contaminated, (C) example of restore of microbial community near to origin state,
site at Morava River

Microbial communities are very changeable and therefore microbial pattern illustrated
for one selected sampling event (and compared with another) do not necessary mean
significant shift to better or worse soil quality. Moreover due to many methodological
problems of sunrays plot assessment, this approach in maybe more relevant for
comparing sites among themselves within one sampling campaign than comparing
evolution of sites in time.

4.3.2 Relationship between soil microbial properties and contamination

(poster 2)

The second work related to fluvisols monitoring was more concerned to assess the
relationship between microbiological properties and soil contamination. The main
objectives of the study were to describe the distribution of pollutants and soil
microbial properties in the river-basin, to determine levels of contaminant and
microbial properties and explore relationship among soil contamination, microbial
parameters and physical-chemical properties. Results were presented at SETAC
2008 as poster presentation (appendix 7).

In this study data from more recent sampling campaigns – in May 2005 and May 2006
– were used. Descriptive statistic revealed that fluvisols were relatively rich in organic
matter content, had high amount of microbial biomass and respiration activity.
Moreover, concentration of environmental pollutants – PAHs, PCBs and heavy
metals – exceeded soil permitted limits in most cases. For easier interpretation the
monitored area was divided to three parts according to environmental load: 1)
background agriculture area at Dřevnice river basin above big agglomeration, 2) sites
at Morava River and below Zlín city, load by agriculture, traffic and industry, and 3)
sites after rivers confluence, load by industry and traffic. The result after this separation
showed increasing of contamination level from background area to downstream sites.
Microbial parameters were, however, not significantly correlated with organic

59
Soil microorganisms as bioindicators

pollution (except PAHs) and heavy metal. Levels of contaminants and microbial
properties of fluvisols are summarized in table 7.

Tab. 7: Summary statistics - levels of fluvisols contamination and microbial properties

-1 -1
Organic pollutants (µg.kg ) Heavy metals (mg.kg )
Σ PAHs Σ PCB Σ HCH Σ DDT Cd Ni Zn
1997
median 8381.0 30.85 1.24 9.42 0.28 38.0 86.1
quantile 10% 670.0 3.74 0.45 1.76 0.20 27.0 64.3
quantile 90% 15364.0 182.01 5.08 133.24 0.55 52.8 169.0
2005
median 6902.4 8.86 0.35 3.95 0.31 37.4 93.7
quantile 10% 681.3 1.63 0.17 0.90 0.16 24.9 70.9
quantile 90% 12845.2 23.71 0.82 9.77 0.90 50.2 146.0
2006
median 3313.9 4.81 1.16 2.91 0.33 40.2 89.3
quantile 10% 246.8 0.63 0.60 0.76 0.23 29.6 70.6
quantile 90% 7493.9 12.31 1.59 7.68 0.86 59.0 133.5

Soil microbial Corg Cbio BR PR

properties (%)
-1
(µg C.gdw )
-1 -1
(µg CO2-C.gdw. .h )
1997
median 1.79 1415.4 0.96 6.33
quantile 10% 0.89 859.8 0.32 1.80
quantile 90% 2.31 2251.0 2.85 16.67
2005
median 2.23 462.8 1.46 15.84
quantile 10% 1.40 209.5 0.84 8.25
quantile 90% 3.77 863.6 2.36 24.12
2006
median 3.64 600.0 0.91 15.44
quantile 10% 2.28 406.0 0.67 10.33
quantile 90% 5.09 761.2 1.22 20.21

More detailed analysis of mutual relationships among parameters was done using PCA
and factorial analysis (fig. 10). Soil physicochemical properties were used as the
driving factors, which are able to differentiate the localities. Pearson correlations of
microbial parameters and contaminants with the factorial axes illustrated the mutual
dependence of the parameters. Result showed that the most variability among sites
(39%) was explained by soil organic matter (Corg, HCs, Ntot, HA, FA), parameters
related to soil reaction (pH), and cation exchange capacity (CEC). The second
most important part of the variability (22%) was driven by the soil particles. The
clearest relationship was found between basal microbial parameters (Cbio, BR and SIR)
and soil organic matter. From contaminants, surprisingly nickel and lead were
correlated too. All other parameters were less correlated to soil physicochemical
properties.

60
 Chapter 4

1
0.01-0.05 m m <0.01 m m

0.05-0.1 m m
0.5
BR
HA:FA Ni SIR
CaCO3 qCO C bio/C org C bio HA
Factor 2 (22 %)

2
SIR/C bio CEC
DDE C ext SIR/BR C org Ntot
0 γ HCH
V
pH (KCl) C ext/C bio DDTs DDD HCs
Zn HCB Pb
pH(H 2O) PCB 153 HCHs Hg
PA Hs αHCH Cr FA
PCBs DDT Cd
Q4/6
-0.5 soil properties βHCH

heavy metals
organic polutnats
biological parameters 0.05-2 m m
-1
-1 -0.5 0 0.5 1
Factor 1 (39 %)

Fig. 10: Correlation of soil contamination and microbial parameters with the main factors of
physicochemical properties

Our results suggest that microbial parameters were fundamentally influenced by

measured physicochemical soil properties, which masked the possible effects of
contamination. However, full “picture” is not available because also other factors such
as periodical inundation or larger floods may play role. Despite microbial parameters
were strongly affected by the floods due to redistribution of material and distortion of
the upper soil layers, the clear relationship with contamination was not found.

61
Soil microorganisms as bioindicators

4.4 Effect of road de-icing salt on soil microorganisms (paper V)

In this study, we investigated the impact of road salt application on soil chemical,
physical-chemical properties and microbial community state. The study was
performed in the Krkonoše Mountains in cooperation with Evernia s.r.o. company
and was designed on request of The Krkonoše National Park management in April
2004. The study area, mixed and spruce forest, was located along the main road,
regularly treated with de-icing salt during winter seasons. Soils were sampled in three
distances from the road assuming gradient of salt impact. Microbial community
state (microbial biomass, basal and substrate-induced respiration, eco-physiological
coefficients qCO2, Cbio/Corg) was used as bioindicator of soil quality. Beside that,
standard bacterial toxicity tests (Vibrio fischeri bioluminescence inhibition test –
Microtox and Pseudomonas putida growth inhibition test) were used to evaluate toxicity
of soil extract. Results of these two approaches were compared with chemical and
physical-chemical analysis of soils. Results of this study were published as paper V,
attached to the thesis.

In general, a negative impact of the road de-icing on soils was evident. Changes in soil
properties were dependent on the relative distance from the road with the most
adverse effect at 1m distances from the road. The physical-chemical parameters
showed mainly sodium ions accumulation, increased base saturation and pH
values. Soil microorganisms showed to be stressed by environmental conditions
and indicated higher energy requirement. Despite the clear relation to the amount of
organic carbon, microbial biomass and respiration were reduced thanks to the higher
salinization. On the other hand, increasing soil salinity caused rather a stimulation
effect in the case of water extracts bacterial toxicity tests.

When comparing these two approaches, bacterial toxicity tests with single species
seemed to be not suitable for assessment in this kind of soil pollution. Only the
evaluation of the real microbial community state can provide the true information about
the effects on soil environment. The application of bacterial toxicity tests can be used
as an additional method, which can yield interesting information but only if it is
interpreted correctly.

62
 Overall conclusion

5 OVERALL CONCLUSIONS

This work showed several possibilities of using soil microorganisms in the evaluation of
pollutants effects on soil environment. It was also demonstrated that soil matrix is very
complex and complicated system and assessment of potential effects of chemicals is
not always simple. Main role of soil microorganisms is in processes of decomposition
and transformation of organic matter and nutrient cycling. The attention of my work
was therefore focused on evaluation of parameters connected with the carbon
mineralization (respiration activity, microbial biomass carbon content) and nitrogen
mineralization (ammonification, nitrification).

The first study explored variability of mentioned microbial parameters with attention
especially on soil sampling and handling. It was showed that microbial parameters
could significantly differ within the sampling sites. The main source of variability in
results was, however, made by different way and condition of soil storage. Drying and
freezing of soil samples disrupted microbial community and resulted in important
changes in microbial parameters. Storage at 4°C seemed to be most appropriate with
minor changes of microbial parameters till 8 weeks.

The effects of chemicals, especially side effects of pesticides, on soil microorganisms

have been widely studied in the laboratory tests. In this work the concern was focused
on selected persistent organic pollutants and two fungicides. Results of the laboratory
experiments were published in three scientific papers attached to this thesis. Tested
POPs – toxaphene and chlorinated paraffin – were relatively low toxic even at high
tested concentration (thousand of ppm). To the contrary, selected fungicides showed
significant adverse effect on nitrogen transformation, especially nitrification. In general,
parameters of potential microbial activities - substrate-induced respiration and newly
set up parameters of potential ammonification and nitrification - were more sensitive to
chemicals effects than overall biomass and respiration.

Monitoring and evaluation of effects of soil contamination on microbial properties in the

field often collide with complexity and heterogeneity of soil environment. Our research
of microbial parameters in fluvisols at Zlín region also demonstrated that relationship
between physical-chemical parameters and microbial activity was more important than
presence and effects of pollutant in soil. On the other hand, as was shown in the study
of impact of road de-icing salt, soil microorganisms could serve as bioindicators of
stress environmental condition caused by adverse physical-chemical properties.

The results and conclusions of studies with indigenous soil microbial communities are
often controversial. Microorganisms are affected by many factors, natural as well as
anthropogenic. In laboratory tests, it is important to assess effects of chemicals on
wide spectrum of endpoints. Different metabolic pathways (carbon and nitrogen
mineralization) could be affected. Besides parameters of overall microbial biomass or
respiration, specific endpoints such as nitrification and microbial growth should be
analyzed. Evaluation of relationships between soil contamination and microbial
parameters in the field studies is often complicated by low load of contaminants and
heterogeneity of soil physical chemical characteristics that masks the adverse effects
of pollutants.

63
References

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 Appendix 1

Paper I

Černohlávková, J., Jarkovský, J., Nešporová, M., Hofman, J.: Variability of soil
microbial properties: effects of sampling, handling and storage; 2008 (submitted to
Ecotoxicology and Environmental Safety)

74
 Variability of soil microbial properties: effects of sampling, handling and storage

Jitka Černohlávková, Jiří Jarkovský, Michala Nešporová, Jakub Hofman*

RECETOX - Research Centre for Environmental Chemistry and Ecotoxicology

Masaryk University, Faculty of Science
Kamenice 126/3
CZ-62500, Brno
Czech Republic

*corresponding author
Phone: +420 549 494 267
Mobile: +420 775 140 071
Fax: +420 549 492 840
E-mail: [email protected]

75
Abstract

We investigated the effect of soil spatial variability within the sampling site scale, the
effects of sample sieving (1, 2 and 4 mm), and storage conditions up to 32 weeks (wet
at 4 °C, -20 °C and air dried) on microbial biomass C, respiration, ammonification and
nitrification activities in arable, grassland and forest soil. In general, all results were
dependent on soil type. Arable soil showed the highest spatial variability, followed by
grassland and forest soil. Sieving did not cause large differences, however higher
biomass C and respiration activity were observed in 1 mm than in 4 mm fraction.
Storage at 4 °C seemed to be the most appropriate up to 8 weeks showing only minor
changes of microbial parameters. Freezing of soils resulted in large increase of
respiration. Dried storage indicated disruption of microbial communities even after 2
weeks.

Keywords: soil biomonitoring, microbial biomass, respiration, spatial variability,

sample storage

76
Introduction

Analysis of microbial properties has a fundamental role in soil monitoring as well as

for research the effects of pollutants on soil microorganisms. One of the great
inconveniences of soil microbiology is high variability of microbial parameters that is
caused by large heterogeneity of soil matrix or by way of sampling, handling or storage
of soil samples. All these aspects are potential source of variability in measured data
and complicate interpretation of results. Moreover, high variability of soil could mask
the effects on contamination and other researched issues. Therefore, great importance
should be paid to representative sampling of soil with respects to the aim of the study.

Microbial properties and activities very depend on distribution of organic matter,

nutrients, water content, texture, vegetation cover and other physical chemical
properties of soil. Microorganisms in soil are distributed in microaggregates and
heterogeneity of soil resources results in microhabitat diversity (Ettema and Wardle,
2002). Spatial heterogeneity of soil parameters were reported many times (Wei et al.,
2008, Berg and Bengston, 2007). Distribution of microbial biomass and their activities
also vary between soil types as well as within one site (Görres at al., 1997). The level
of variability of microbial parameters in a scale of one site could be so high, that the
data are unlikely to determine significant differences between environmental and
anthropogenic effects. This was e.g. reported by Broos et al. (2007). They documented
that due to spatial heterogeneity of soil physicochemical environment the microbial
biomass carbon was not sensitive indicator of metal contaminated sites.

Besides spatial variability, environmental conditions and sampling strategy, handling

and storage of soils may also affect microbial community and change the results.
Minimal duration of storage and analysis of fresh soil immediately after sampling is
preferred. However, some kind of soil storage is mostly necessary. Although ISO
10381-6 (1993) recommends the sampling technique, transport conditions, sieving (2
mm mesh size), and storage of soil (max 3 months at 4±2°C) the optimal conditions
and effects on microbial parameters are still discussed. These recommendations were
based on experiments with temperate soil assuming that low temperature of storage
slows microbial growth and activities. Freezing of sample should be rather avoided,
due to ice effects on microbial cells and soil structure (Stenberg et al., 1997). On the
other hand, long-term frozen soil in cold climatic zone could be more resistant to the
storage at low temperature and oppositely cold storage may adverse affected microbial
community of tropical soil, which is not adapted to low temperature (Verchot, 1999).
Microbial processes are also variously sensitive to the temperature of storage. While
total microbial biomass carbon and respiration seemed to be less affected by longer
storage and freezing, DNA concentration and community structure indicate bigger
changes (Lee et al., 2007, Feng et al., 2007). Long term freezing greatly influenced
mainly nitrogen mineralization capacity and available nitrogen forms (Sternberg at al.,
1997). Changes are, however, influenced by soil properties. For example, soils with
high organic matter may be more susceptible to the effects of storage (Lee et al., 2007).

The aim of this study was to determine sources and extent of variability of soil
microbial properties in sampling, handling and storage of soils. We investigated the
77
spatial variability of sampling, the effect of sample fractionation (sieving through 1, 2
or 4 mm mesh), the effects of different way (cold, frozen, air dried) and duration (1 –
32 weeks) of storage. Evaluated parameters were biomass carbon content, basal and
substrate-induced respiration, ammonification, and nitrification. To see the soil type
dependency, arable, grassland, and forest soil were studied.

Material and methods

Soils, sampling and handling

Three different soils were selected for this study: arable sandy loam soil, grassland
orchard soil and mixed forest soil with high organic carbon content. Soils were
collected at long-term monitored, uncontaminated sites approximately 20 km east from
Brno, the Czech Republic. Arable soil was sampled from plough layer (0-15 cm, A
horizon), grassland soil was sampled from the root layer (0-15 cm, A horizon) after
removal of vegetation, and forest soil was sampled from top organic horizon under
litter (5-10 cm, Oh/Of horizon). Mixed samples were taken as 6 - 10 subsamples (about
1 kg each) of each sampling site from the sampling area approximately 15 × 15 m.
Generally, soils were sampled and handled according to ISO 10381-6. All soils were
classified as cambisols with loamy sand texture. More detailed physical-chemical
characteristics are summarized in Table 1.

To elucidate spatial variability of soils, 8 individual subsamples (3-4 kg each) in

geometric net were sampled at each plot and not mixed together but analysed
separately. The distance between subsamples was about 1.5 m. Each subsample was
well homogenized, freshly sieved through 2 mm mesh and stored at 4 °C before
microbiological analysis.

To analyze the effect of sieving, mixed soil samples (see above) were freshly sieved
parallelly through 1 mm, 2 mm and 4 mm mesh size. Samples were stored at 4°C prior
to analysis.

To study the effects of storage conditions, mixed soil samples (see above) were freshly
sieved through 2 mm mesh. These were immediately analysed for microbial parameters
(fresh soil). Subsequently, soils were divided into two portions, which were stored at
field moisture cooled (at 4°C) or frozen (at -20°C). The third portion was air dried and
stored at 4°C. Microbial parameters were analyzed after 2, 4, 8, 16 and 32 weeks of
storage.

Microbial parameters

Microbial biomass carbon content (Biomass C), basal respiration (BR), substrate-
induced respiration (SIR), ammonification (AMO) and nitrification (PAO) were
analyzed in the spatial variability experiment. For the effects of sieving and storage
conditions, parameters of Biomass C, BR and SIR were measured. All analyses were
done in triplicates.

78
Dry matter of soil and maximal water holding capacity according to Forster (1995) of
samples were measured before analysis. Microbial biomass carbon content (Biomass
C) was measured by the chloroform fumigation extraction method according to ISO
14240-2 (1997). Simply, soil at 60% WHC was exposed to ethanol-free chloroform
vapours in dessicator for 22 hrs and after that both exposed and unexposed soil was
extracted with 0.5M K2SO4 (1:4 w:v) and C in extracts was determined after
dichromate-sulphuric acid digestion by back titration using Mohr salt and
diphenylamine as indicator.

Basal and substrate-induced respirations were analyzed after 3 days of soil pre-
incubation at 40% WHC at 20±2 °C. Basal respiration (BR) was quantified according
to ISO 16072 (2002) Soil (60% WHC) was incubated for 24 hrs at 20±2 °C in glass
jars closed with rubber lids without any addition of substrate. Substrate induced
respiration (SIR) was measured as the respiration response to the glucose addition in
the first 6 hours of incubation at 20±2 °C (ISO 14240-1, 1997). Glucose (5 mg.g-1 dry
soil) was added to samples as water solution, used at the same time for the WHC
adjustment to 80%. Respiration was determined as CO2 production. CO2 concentration
in the air sample withdrawn from the jars with syringe was measured at GC with H2
mobile phase, Porapack Q stationary phase and thermal conductivity detector.

Ammonification (AMO) was measured under anaerobic condition. Concentration of

ammonium ions was analyzed at beginning and after one week incubation of water-
flooded soil at 40 °C. The NH4+-N concentration was analyzed colorimetrically
according to Alef (1995). The assay based on Berthelot reaction (the reaction with
salicylate and hypochlorite in alkaline solution) was modified to microvolume assay
and carried out in microplates (Cernohlavkova et al., 2009).

Potential ammonification (PAMO) was measured as arginine ammonification assay

according to Alef and Kleiner (1987). Production of NH4+-N ions was measured after
3 hours incubation of soil samples with arginine (11.5 mM solution) at 30 °C.

Short-term nitrification activity of soil microorganisms was determined as potential

ammonium oxidation (PAO) according ISO 15685 (2004). Samples of soil (20 g) were
mixed with 80 ml of potassium phosphate buffer solution (pH 7.2) containing 3.8 mM
(NH4)2SO4 and 15 mM NaClO3. Soil slurries were incubated on a rotary shaker at 25
°C for 5 hours. Production of nitrite was analyzed by Griess-Ilosvay colorimetric
determination (ISO/TS 14256-1, 2003), where nitrite forms after addition of
sulphanilamide and N-(naphtyl)ethylendiamine dihydrochloride a diazo-compound.
Analysis was modified to microvolume assay and carried out in microplates. The
potential nitrification rates were calculated by linear regression of accumulated nitrite
over time

Data evaluation

For analysis of spatial variability of microbial parameters, mean, minimal and maximal
values, standard deviation (SD), and relative standard deviation (RSD) were calculated
from the 8 individual subsamples analyzed at each site. The effect of the number of
79
subsamples on the variability of the pooled sample was evaluated using simulation.
Results for the simulated pooled samples were computed as the means from all
possible combinations of originally measured values for defined number (2 – 8) of
subsamples. The relative standard deviation of means for the simulated pooled samples
was plotted against number of subsamples used for combination.

Effects of sieving and storage conditions and duration were statistically analyzed with
STATISTICA 8.0 software (StatSoft, Inc., 2008). Differences among sieving through
1, 2 and 4 mm mesh size were analyzed by means of ANOVA followed by Newman-
Keuls test. The statistical significance of differences between fresh, non-stored, soil
and other storage treatments was tested with Dunnett’s test; post-hoc comparison
among storage treatments was performed with Newman-Keuls test. Statistical
significance was evaluated using α=0.05 in all test.

Results

In general, forest soil showed the highest amount of microbial biomass carbon content
and also the highest respiration activities. It was followed by lower values of grassland
soil and the lowest values were revealed for arable soils. Variability of routine methods
for microbial parameters in our laboratory (relative standard deviation) was: 2-6% for
microbial biomass C, 1-7% for substrate-induced respiration, 6-15% for basal
respiration, 9-15% for ammonification, 9-13% for potential ammonification and 4-15%
for potential nitrification. All analysis were done in triplicates (n = 3).

Spatial variability

For spatial variability, 8 independent samples were separately handled and analyzed at
each site. Results of descriptive statistics are summarized in Table 2. Arable soil
showed the lowest amount of microbial biomass and the lowest level of microbial
activities, followed by grassland and forest soil. Relative standard deviation (RSD)
showed that the highest variability of carbon parameters (biomass C, BR and SIR) was
in the arable soil, followed by forest and grassland soil. Different results were obtained
for nitrogen transformation parameters. The highest variability was observed in
grassland soil for ammonification, in forest soil for potential ammonification and in
arable soil for potential nitrification. Simulation of the relationship between variability
of the results and the number of subsamples showed similar results for all soils and
parameters. Only exception was ammonification in grassland soil, which showed
dramatic increase of variability if lower number of subsamples was taken.

Effect of soil sieving

The results of microbial parameters of soils sieved through different mesh sizes are
summarized in Table 3. Generally, the results in 1 mm and 4 mm fractions did not
differ from 2 mm fraction more than 22% in arable and grassland soils and more than
8% in forest soil. The differences of microbial parameters between three fractions were
rarely significant in the forest and arable soils, i.e. mostly did not exceed the intrinsic
variability of parameters. Lower biomass C content for 4 mm fraction was observed in
80
arable soil when compared to 2 mm fraction. In forest soil, significantly different
biomass C was found in 1 and 4 mm fractions. Fraction size was found to have
significant effect on the results of microbial parameters in the grassland soil.

Effects of storage conditions and duration

Results of microbial parameters in soils under different storage conditions and times
are given in Table 4. The effects of storage were different for the three soil types and
for different parameters. There was often no clear pattern in the data and temporal
fluctuation of the results; both increase and decrease of the respective parameters were
recorded. Generally, microbial biomass was not affected significantly by storage until
the 4 weeks of storage. At -20 °C there was frequent significant decrease of biomass C
after 8 – 16 weeks. Significant decrease was observed after 8 weeks of dried storage
and was most pronounced in forest and grassland soil (57% and 88% of fresh values,
respectively). An increase was, however, found for arable soil. Soils stored at 4 °C
revealed the least changes when compared to the results of the fresh soil over whole
period.

Similarly to the biomass C, storage at 4 °C showed the least significant changes of

original basal respiration. Controversially, drastic changes were found for other ways
of storage. Significant increases were found especially for arable and grassland soils
stored at -20 °C. The respiration reached in these soils more than 140% of fresh soil
values during whole storage period. Storing dry soils induces dynamic fluctuation in
the results composed from decreases and increases of basal respiration.

Effect of storage conditions on substrate induced respiration was not consistent and
there were significant changes also for storage at 4 °C. Dried storage caused
significantly higher respiration in arable soil (140 -170%), whereas decrease of activity
in grassland soil in comparison to fresh value (up to 65%). Higher SIR was found in
grassland soil stored frozen whereas decrease was induced by this way of storage in
arable and forest soils. In all soils, substrate induced respiration significantly increased
after 8 weeks of storage under all type conditions.

Discussion

Spatial variability

The questions of soil heterogeneity and representative sampling have been often
discussed. High variability of soil parameters diminishes the potential of soil
microorganism as bioindicator of soil pollution. Broos et al. (2007) reported that field
variability of microbial biomass C limited its use as an ecotoxicological indicator.
They reported that very large number of replicates (up to 93) would be required to
identify significant decrease of microbial biomass C between metal contaminated and
clean sites. In our study the model was used to predict the variability of sampling and
determine optimal number of subsamples taken. RSD for simulated mean assessment
was bellow 5 % for 6 and more subsamples, except the ammonification in the
grassland soil. The results showed that pooled samples of six to eight subsamples is
81
acceptable because it gives representative assessment of really measured mean value
for these soils.

The microbial parameters are influenced by soil physical-chemical properties. Field

topology together with vegetation cover, root distribution crop residues leads to local
differences in water content and temperature and are major source of variability.
Spatial heterogeneity of soil resources results in microhabitat diversity and separation
of microbial population even at fine-scale (Ettema and Wardle, 2002). The distance
between individual pits, in our case, was about 1.5 m. Tessier et al (1998) discussed the
spatial correlation among total and soluble carbon content and microbial biomass in
manure treated soil. They showed that microbial biomass was similar to the spatial
variability of total C and that the adequate sampling distance to obtained non-
correlated representative samples at the plot was 1m.

Higher variability of the arable and grassland soil in comparison to forest soil could be
explained by soil land use and presence of small crop residues. Görres et al (1998)
reported that carbon mineralization was connected to spatial variability of soil moisture
and the relationship was more also apparent in old field soil than in forest soil with
more stable conditions and ecosystem. However, our data did not show so marked
differences between soils types, results are in agreement with their study.

The microbial parameters also fluctuate during the year. Our results were linked to the
one sampling event. Therefore different spatial pattern would be obtained in repeated
sampling. As reviewed in Joergensen and Emmerling (2006) information about spatial
variability are necessary prior analyzing seasonal changes or temporal variability.

Effects of sieving

Pretreatment of soil samples is essential to limit interferences of plant roots and to

unify soil particles size before microbial parameters analysis. Despite the ISO 10381-6
(1993) standard prescribes sieving through 2 mm mesh, the mesh size varies among
soil microbial studies. Majority of studies use fraction of 2 mm (e.g. Broos et al., 2007,
Feng at al., 2007, Görres et al., 1997, Lee et al, 2007), however, studies with 4 mm or
large fraction are not rare (e.g. Stenberg et al., 1998, Koponen et al., 2006, Fierer and
Schimel, 2002, Fierer et al., 2003). Our results showed no large differences in
microbial biomass and respiration activities between 1, 2 and 4 mm fractions. The
major variability was found in grassland soil, which might be related to heterogeneous
character of rhizosphere under permanent grass vegetation.

Although certified meshes are available at the market, the sieving procedure itself is
quite subjective. Different people use different routine (force, recovery effort etc.)
when sieving. Some labs use grinding the soil before sieving, the others use shredder
or various kinds of automated equipments. Sieving through smaller mesh requires
more force and therefore more fine roots with rhizosphere microbial biomass can be
added to the sample (Joergensen and Emmerling, 2006). On the other hand, grinding
the stones means increase of mineral fraction in the sample. Breaking up of soil
aggregates may, however, also release a pool of available substrate. Probably for that
82
reasons the microbial biomass content and respiration activities were higher at 1 mm
mesh size. In agreement with our results, Degens et al. (1998) observed that 0.5 mm
sieving caused increase of microbial biomass and functional diversity that was
influenced by increasing the exposure of organic matter to microorganisms.

In general our results partly support the hypotheses that fine sieving caused a
temporary increase in organic matter mineralization (Hassink, 1992). This could be
explained so that organic matter is presented in small pores and is physically protected
against decomposition. During fine sieving this part of organic matter is exposed to
mineralization. The grassland soil in our study contained more than 6% of total organic
carbon and therefore the effect of sieving procedure could be more apparent in
comparison to the arable soil with only 1.4% of Corg.

Sieving through larger mesh size did not break small soil particles and resulted in more
course structure. In our study, lower amount of available substrate could consequence
lower respiration activity of grassland soil sieved through 4 mm mesh. Similar results
was reported by Petersen an Klug (1994), who found higher CO2 evolution rate from
soil sieved at 2 mm than from soil sieved at 4 mm. Ross et al. (1985) also previously
reported that lower biomass C was found in 6 mm than 2 mm fraction of pasture soil.
However, differences in the mentioned studies, as well as in our one, were only small,
indicating that substrates were not released by sieving to any large extent.

Effects of storage

Storage of soils influenced measured microbial parameters. This might be expected,

because soil is living system, which is sensitive to temperature changes, aeration status
and moisture. Changes were observed even after 2 weeks, but diverged during time of
storage. Effects were not common for the three soil types and might be related to
difference in physical chemical and microbial characteristics of soils. Brohon et al.
(1999) also found that microbial parameters were differently influenced during storage
according to the soil physical chemical characteristics. They reported that presence of
clay and neutral soil pH might improve resistance of microorganisms to temperature
changes.

Generally, storage at 4 °C had the least effects on biomass C as well as on basal and
substrate-induced respirations in our study. The storage effects at these conditions
lasting for several weeks probably should not exceed intrinsic variability of the
microbiological method when the respective method is repeatedly measured. In our
study, microbial biomass C showed minor changes in forest and grassland soil. Slight
decrease in time was observed in arable soil but only after 32 weeks. Storage at 4 °C is
also recommended by ISO 10381-6 standard (1993) and was verified appropriate for
soil microbial analysis with no effect on microbial composition by further researchers
(Peterson and Klug, 1999, Lee et al., 2007). Moreover, Lee et al. (2007) found out that
either frozen storage (-20°C) did not affected soil enzymatic activities and biomass C
for 4 weeks.

83
To the contrary, storage at -20 °C did not seem to be much suitable in our case.
Increased level of basal respiration together with lower microbial biomass especially in
arable and grassland soil indicated changes of microbial communities. Freezing, in
general, causes increased concentration of salts in the liquid phase during ice formation
and consequently osmotic stress for cells. Cells are killed as well by intracellularly
formed ice crystals, which could damage cell structures. This might be the reasons of
microbial biomass decrease in our soils stored frozen. On the other hand, if organisms
survive freezing they can profit from fresh pools of bioavailable substrate, which is
released from soil aggregates disintegrated by freezing. Another pool of available,
easily degradable substrates is organic molecules revealed from biomass killed by
freezing (Stenberg et al., 1998). This might be an explanation for increases of basal
respiration activity in our soils when stored frozen. Freeze-thaw cycles induced carbon
and nitrogen mineralization has also been reported in the literature (Feng at al., 2007,
Sternberg et al., 1998) and was attributed to increased level of labile substrate from
damage microorganisms.

Drying of soil resulted in great changes of microbial biomass and respiration activities
in our study too. In literature, air drying and rewetting is not recommended for the
microbial analyses because it induces changes of microbial communities by
mobilization of soil organic matter and by killing many of soil microorganisms
(Joergensen and Emmerling, 2006, Lee at al., 2007). Lee at al. (2007) found out that
dry storage caused significant reduction of microbial activities after 2 weeks. They also
reported shift in microbial community structure in favour of G+ bacteria that are
generally more resistant to stress. Basal respiration in our case was also greatly reduced
after 2 weeks, but it increased after that. Similar explanation as in the case of freeze
storage might be used: Elevated basal respiration activity after longer dry storage might
be caused by utilization of easily available substrate which was released from dead
microorganisms or soil structure. According to Fierer and Schimel (2002), soils
frequently exposed to drying and rewetting (agriculture, grassland soils) may be more
adapted to moisture stress and less affected to drying treatment than forest soil. That is
in agreement with our results. Microbial biomass C was quite consistent up to 1 month,
after that difference occurred depending to soil type, first in forest soil followed by
grassland and arable soil. Drying of soil causes a decrease of water potential and
osmotic stress similar to effect of freezing. Repeated drying cycles result in selection of
microbes group such as fungi or G+ bacteria, which can withstand the osmotic shock
(Fierer and Schimel, 2002).

Conclusions

Our results proved that soil microbial properties were influenced by sampling,
handling and storage procedures. In general, effects were greatly dependent on soil
type. The microbial parameters showed spatial variability at the sampling sites. From
the soils used, the arable soil showed the highest variability of biomass C and
respiration activity. Grassland and forest soils appeared more variable in nitrogen
transformations. Despite the observed variability, it seems that the pooled samples
from 6 to 8 subsamples provided representative assessment of the mean value for these
soils. Sieving method was found to not affect the microbial results extensively.
84
Nevertheless, mesh size caused higher variability of results in grassland and forest soil.
Sieving through 1 mm mesh caused higher values of microbial parameters than 2 and 4
mm size. The largest span of variability of microbial parameters was induced by
storage conditions and durations. Storage at 4 °C seemed to be the most appropriate for
soil respiration and microbial biomass C analysis with only minor changes up to 8
weeks of storage.

This study shows that although microbial parameters are routinely used for soil
monitoring and standardized methods exist, the effects of sample handling and storage
are significant factors affecting the results. Spatial variability of microbial parameters
could also decrease the significance of causal links between ecological damage and
pollution. With the reference to other studies (Broos et al., 2007, Lee et al., 2007) we
would like to point out the importance of microbial parameters variability, which may
hamper the straightforward interpretation of the results and therefore must be seriously
considered.

Acknowledgements:
This research was supported by Ministry of Education of the Czech Republic, project
No. MSM 0021622412 INCHEMBIOL.

85
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M., McLaughlin, M.J., 2007. Limitations of soil microbial biomass carbon as an
indicator of soil pollution in the field. Soil Biol. Biochem. 39, 2693-2695.
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and dinocap on carbon and nitrogen mineralization in soils. Ecotoxicol. Environ.
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Fig. 1:

Dependence of the spatial variability on the number of subsamples for soil

microbial parameters: microbial biomass C, basal respiration - BR, substrate
induced respiration - SIR, ammonification - AMO, potential ammonification -
PAMO and potential nitrification - PAO. Plots represent relative standard
deviations of means calculated from simulated pooled samples combined from
2 - 8 subsamples.

30 30 30
Biom as s C SIR BR
15 15 15

m e an
0 m e an
0 m e an
0

-15 -15 -15

mean ± SD (% of mean)

-30 -30 -30

1.8 2.8 3.8 4.8 5.8 6.8 7.8 1.8 2.8 3.8 4.8 5.8 6.8 7.8 1.8 2.8 3.8 4.8 5.8 6.8 7.8
2 3 4 5 6 7 8 2 3 4 5 6 7 8 2 3 4 5 6 7 8

arable soil grassland soil forest soil

30 30 30
AM O PAM O PAO
15 15 15

m e an
0 m e an0 m e an
0

-15 -15 -15

-30 -30 -30

1.8 2.8 3.8 4.8 5.8 6.8 7.8 1.8 2.8 3.8 4.8 5.8 6.8 7.8 1.8 2.8 3.8 4.8 5.8 6.8 7.8
2 3 4 5 6 7 8 2 3 4 5 6 7 8 2 3 4 5 6 7 8

sample size

88
Table 1:
Selected characteristics of soils used in this study.

Arable soil Grassland soil Forest soil

Clay (<2 μm) (%) 19.0 19.1 47.6
Silt (2-50 μm) (%) 17.2 14.6 9.6
Sand (50-2, 000 μm) (%) 63.7 66.3 42.8
pH(H2O) / pH(KCl) 6.7 / 5.8 7.4 / 6.7 5.4 / 4.7
-1
CEC (meq kg ) 161.5 307.8 687.9
Corg (%) 1.38 6.54 11.00
Ntot (%) 0.21 0.44 0.62

Table 2:
Spatial variability in three different soils: summary statistics for 8 subsamples
taken at each site (N=8) for microbial biomass (Biomass C), basal respiration
(BR), substrate induced respiration (SIR), ammonification (AMO), potential
ammonification (PAMO) and potential nitrification (PAO). AMO in arable soil
and PAO in forest soil were not detected.

soil mean median min max SD RSD %

-1
Biomass C (µg C.gdw )
arable 351.3 342.1 278.1 545.2 87.0 24.8
grassland 1162.8 1207.1 945.6 1313.3 114.4 9.8
forest 1261.9 1229.3 989.8 1478.7 171.5 13.6
-1 -1
BR (µg CO2-C.gdw .h )
arable 0.87 0.88 0.62 1.17 0.17 19.7
grassland 1.09 1.10 0.93 1.26 0.13 12.0
forest 3.96 3.97 2.84 5.01 0.79 19.8
-1 -1
SIR (µg CO2-C.gdw .h )
arable 7.75 7.86 5.54 9.95 1.59 20.5
grassland 12.62 12.22 11.08 15.12 1.58 12.5
forest 22.90 22.98 16.64 31.03 4.16 18.2
-1 -1
AMO (µg NH4-N.gdw .d )
grassland 4.90 4.02 2.75 8.53 2.20 44.8
forest 16.79 16.54 14.11 19.94 2.12 12.6
-1 -1
PAMO (µg NH4-N.gdw .h )
arable 5.28 5.26 4.47 6.03 0.48 9.1
grassland 6.81 6.51 5.91 7.98 0.76 11.2
forest 5.47 5.42 4.30 6.82 0.72 13.3
-1 -1
PAO (µg NO2-N.gdw .h )
arable 0.122 0.129 0.082 0.146 0.021 17.0
grassland 0.459 0.442 0.358 0.654 0.097 21.2

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Table 3:
Microbial biomass (Biomass C), basal respiration (BR) and substrate induced
respiration (SIR) of soils sieved through 1, 2 and 4 mm mesh. The results are
expressed as mean ± SD (n = 3). In parentheses, results are shown as percentage
of 2 mm values. Asterisks mark statistically significant differences from 2 mm
values, crosses mark significant differences between 4 and 1 mm mesh.

Biomass C BR SIR
Soil -1 -1 -1 -1 -1
μg C gdw μg CO2-C gdw h μg CO2-C gdw h

arable soil
1 mm 387.64 ± 41.57 (91.3) 1.00 ± 0.10 (121.4) 10.35 ± 0.63 (95.5)
2 mm 424.66 ± 71.31 0.82 ± 0.07 10.84 ± 0.32
4 mm 349.85 ± 41.93 (82.2)* 0.90 ± 0.06 (109.3) 9.71 ± 0.45 (89.6)
grassland soil
1 mm 1201.03 ± 54.99 (109.3)* 1.36 ± 0.30 (122.0) 24.56 ± 0.83 (109.9)*
2 mm 1098.54 ± 83.59 1.11 ± 0.01 22.36 ± 0.74
† †
4 mm 1112.57 ± 16.37 (101.3) 1.03 ± 0.03 (92.1) 19.68 ± 0.62 (88.0)*
forest soil
1 mm 1479.17 ± 50.89 (97.4) 5.91 ± 0.20 (104.1) 29.67 ± 0.54 (103.0)
2 mm 1519.47 ± 14.99 5.68 ± 0.17 28.80 ± 2.82
†
4 mm 1578.66 ± 32.36 (104.0) * 5.90 ± 0.31 (104.0) 31.00 ± 0.86 (107.6)

Table 4:
Effects of storage conditions and duration on microbial biomass C, basal
respiration (BR) and substrate-induced respiration (SIR). Results are expressed
as percentage of values measured in fresh soils (few days after sampling).
Asterisks mark statistically significant differences (p < 0.05) from the fresh soil
values. Arrows show significant decrease or increase and ≈ symbol marks not
significant change from the measurement at previous time.

90
 2 weeks 4 weeks 8 weeks 16 weeks 32 weeks
-1
Biomass C - % of fresh values (µg C.gdw ± SD)
arable soil Fresh soil = 504.1 ± 74.5
stored at 4°C 106.4 ≈ 104.9 ≈ 90.2 ≈ 99.4 ≈ 85.1
stored at -20°C 81.5* ≈ 97.7 75.5* 93.6 ≈ 107.4
stored dried 103.2 ≈ 103.9 ≈ 106.8 133.9* 113.2
grassland soil Fresh soil = 1174.84 ± 106.43
stored at 4°C 106.5 120.3* 99.9 ≈ 94.1 107.1
stored at -20°C 97.9 ≈ 104.8 88.9* 99.5 90.3*
stored dried 100.7 ≈ 104.5 88.5* 111.6* 88.3*
forest soil Fresh soil = 1512.21 ± 24.36
stored at 4°C 106.9 114.8* 103.5 ≈ 100.8 108.8*
stored at -20°C 91.4* 100.3 92.8 ≈ 95.2 ≈ 100.6
stored dried 102.7 112.9* 57.4* 44.4* 72.7*
-1 -1
BR - % of fresh values (µg CO2-C.gdw .h ± SD)
arable soil Fresh soil = 0.77 ± 0.06
stored at 4°C 130.4 ≈ 130.1 ≈ 121.9 ≈ 133.9* 102.2
stored at -20°C 145.8* ≈ 144.8* ≈ 168.0* ≈ 176.7* 147.8*
stored dried 49.8* ≈ 63.6* 95.6 136.8* 67.5
grassland soil Fresh soil = 1.85 ± 0.21
stored at 4°C 119.2 ≈ 114.3 ≈ 117.9 86.7 ≈ 114.6
stored at -20°C 135.8* ≈ 142.7* 183.2* 145.1* ≈ 158.5*
stored dried 55.2* ≈ 90.9 121.5* 174.1* 106.2
forest soil Fresh soil = 5.75 ± 0.24
stored at 4°C 110.6 ≈ 96.2 ≈ 106.2 ≈ 90.9 ≈ 94.2
stored at -20°C 124.8* ≈ 115.6 130.5* 111.3 ≈ 115.4
stored dried 58.8* 88.0 ≈ 82.9 166.1* 96.0
-1 -1
SIR - % of fresh values (µg CO2-C.gdw .h ± SD)
arable soil Fresh soil = 14.32 ± 0.25
stored at 4°C 95.2 ≈ 89.4* 113.7* 87.0* 71.4*
stored at -20°C 87.5* ≈ 91.9 122.4* 90.3 ≈ 81.0*
stored dried 142.6* ≈ 156.2* 172.9* 141.0* ≈ 146.6
grassland soil Fresh soil = 25.23 ± 0.38
stored at 4°C 112.7* 104.4 135.5* 115.5* 102.6
stored at -20°C 116.5* ≈ 113.1* 144.9* 125.1* 103.7
stored dried 76.7* ≈ 94.7 ≈ 81.9 ≈ 70.4* ≈ 64.2*
forest soil Fresh soil = 37.19 ± 0.38
stored at 4°C 96.3 ≈ 85.7 114.1 88.0 72.1*
stored at -20°C 81.0* ≈ 98.1 131.3* 91.8 ≈ 81.9*
stored dried 119.6* 85.5* 119.7* 91.7 ≈ 89.1

91
 Appendix 2

Paper II

Bezchlebová, J., Černohlávková, J., Kobetičová, K., Lána, J., Sochová, I. and
Hofman, J. (2007): Effects of short chain chlorinated paraffins on soil organisms.
Ecotoxicology and environmental safety 67, p. 206-211

92
93
94
95
96
97
98
 Appendix 3

Paper III

Bezchlebová J., Černohlávková, J., Lána, J., Sochová, I., Kobetičová, K. and
Hofman, J. (2007): Effects of toxaphene on soil organisms. Ecotoxicology and
Environmental safety 68, p. 326-334

99
100
101
102
103
104
105
106
107
 Appendix 4

Paper IV

Černohlávková, J., Jarkovský, J., Hofman, J. (2009): Effects of fungicides mancozeb

and dinocap on carbon and nitrogen mineralization in soils. Ecotoxicology and
Environmental safety 72, p. 80-85

108
109
110
111
112
113
114
 Appendix 5

Paper V

Černohlávková, J., Hofman, J., Bartoš, T., Sáňka, M., Anděl, P. (2008): Effects of
road de-icing salts on soil microorganisms, Plant Soil and Environment 54 (11), p. 479
– 489.

115
116
117
118
119
120
121
122
 Appendix 6

Poster 1

Černohlávková, J., Hofman, J., Bezchlebová J., Dušek, L., Holoubek, I. (2006):
Monitoring of microbial parameters of fluvisols in the Czech Republic. SETAC 2006,
7-11 May. Haag, Holland. In Abstract Book of SETAC Europe the 16th Annual
Meeting, poster presentation

123
124
 Appendix 7

Poster 2

Černohlávková, J., Jarkovský J., Hofman J., Holoubek I. (2008): Soil contamination
and microbial biomass in fluvisols of the catchment area with high environmental load.
In Abstract Book of SETAC Europe the 18th Annual Meeting. p. 99. Warsaw, Poland,
25 -29/5/2008, poster presentation

125
126
 Appendix 8

Curriculum vitae

127
Curriculum vitae
Personal data

Name, surname, title: Jitka Černohlávková, M.Sc.

Date of Birth: 13.5.1981
Address: Kotlanova 2, 628 00 Brno
E-mail: [email protected]

Education

2004 – present Ph.D. candidate, branch environmental chemistry, RECETOX, Faculty of

Science, Masaryk University, Brno, Czech Republic.

1999 – 2004 M.Sc. degree, branch ecotoxicology, RECETOX, Faculty of Science,

Masaryk University, Brno, Czech Republic.

2007 State rigorous exam in biology – branch ecotoxicology

Rigorous thesis: Effects of Persistent Organic Pollutants (POPs) on Soil
Microorganisms

Employement history

2/2006 – 6/2006 laboratory technician, RECETOX, Masaryk University, Brno, Czech

Republic.
3/2005 – 8/2005 laboratory technician, RECETOX, Masaryk University, Brno, Czech
Republic.
8/2008 – 12/2008 laboratory technician, RECETOX, Masaryk University, Brno, Czech
Republic

Professional qualification
Ecotoxicology, environmental chemistry, environmental matrices sampling, environmental risk
assessment. Special focus on soil ecotoxicology (microbial and invertebrates), soil microbial
analysis, soils monitoring.

Languages
Czech – native, English

Research projects

Černohlávková, J., Hofman. J. (2006): Application of soil respiration curves for evaluation effects
of pesticides on soil microorganisms and organic substrate mineralization. Project of
Ministry of education and youth of the Czech Republic FRVS TO G4 2097/2006.

Scientific paper

Černohlávková, J., Jarkovský, J., Hofman, J (2009): Effects of fungicides mancozeb and dinocap
on carbon and nitrogen mineralization in soils. Ecotoxicology and environmental safety 72,
p. 80-85

Černohlávková, J., Hofman, J., Bartoš, T., Sáňka, M., Anděl, P. (2008): Effects of road de-icing
salts on soil microorganisms, Plant Soil and Environment 54 (11), p. 479 – 489.

128
Bezchlebova J., Cernohlavkova, J., Kobeticova, K., Lana, J., Sochova, I. and Hofman, J. (2007):
Effects of short chain chlorinated paraffins on soil organisms. Ecotoxicology and
environmental safety 67(2), p. 206-211.
Bezchlebova J., Cernohlavkova, J., Lana, J., Sochova, I., Kobeticova, K. and Hofman, J. (2007):
Effects of toxaphene on soil organisms. Ecotoxicology and environmental safety 68(3), p.
326-334.

International and national meetings

Černohlávková, J., Jarkovský J., Hofman J., Holoubek I. (2008): Soil contamination and
microbial biomass in fluvisols of the catchment area with high environmental load. In
Abstract Book of SETAC Europe the 18th Annual Meeting. p. 99. Warsaw, Poland, 25-
29/5/2008. poster presentation

Černohlávková, J., Jarkovský, J., Hofman, J.: Vliv vybraných pesticidů na nitrifikační a respirační
aktivitu půdních mikroorganismů, 24. kongres Československé společnosti mikrobiologické,
2.-5. říjen, 2007, Liberec, Abstract Book, ISSN 0009-0646, p-71.

Černohlávková, J., Hofman, J., Bezchlebová J., Dušek, L., Holoubek, I. (2006): Monitoring of
microbial parameters of fluvisols in the Czech Republic. SETAC 2006, 7.-11. May, 2006.
Haag, Holandsko. In Abstract Book of SETAC Europe the 16th Annual Meeting, poster
presentation

Černohlávková, J., Hofman, J., Holoubek, I. (2006): Effects of toxaphene on soil microorganisms
in laboratory incubation test. SETAC 2006, 7 -11 May, 2006. Haag, Holandsko. Abstract
Book of SETAC Europe the 16th Annual Meeting, poster presentation

Černohlávková, J., Anděl, P., Bartoš, T., Hofman, J. (2006): Vliv zimního solení na půdní
mikrobiální společenstva kolem komunikací. (Effects of Road Deicing Salts on Soil
Microorganisms). Studentská vědecká konference, 26 April 2006, Bratislava, Slovensko.
Sborník recenzovaných příspěvků, 1. svazek – biologická a environmentální sekce, ISBN
80-88870-58-5, p. 209 – 211, oral presentation

Černohlávková, J., Hofman, J. (2006): Effects of selected pesticides and organic substrates on
long-term respiration of soil microbial community. International conference on ecotoxicology
2006. Trends and Perspectives. September 17 – 20, 2006, Wisla, Poland. Abstracts Book,
p. 33 , oral presentation

Černohlávková, J. and Hofman, J. (2005): Laboratory testing of persistent organic pollutants

toxicity on soil microbial biomass - application limits, drawbacks and benefits. In Abstract
Book of SETAC Europe the 15th Annual Meeting. p. 155. Lille, France, 22-26 May, 2005.
poster presentation

Bezchlebová, J., Lána, J., Černohlávková, J., Sochová, I., and Hofman, J. (2005): Effects and
ecological risk assessment for short chain chlorinated paraffins in soil. In Abstract Book of
SETAC Europe the 15th Annual Meeting. p. 388. Lille, France, 22-26/5/2005. poster
presentation

Anděl, P., Černohlávková, J., Bartoš, T., Sáňka, M., and Hofman, J. (2005): Effects Of Road
Deicing Salts On Soil Microorganisms. SETAC Europe 15th Annual Meeting, In Abstract
Book of SETAC Europe the 15th Annual Meeting, 22 - 26 May 2005, Lille, France. poster
presentation

Bezchlebová, J., Černohlávková, J., Lána, J., Sochová, I., Hofman, J. (2005): Ecological risk
assessment of toxaphene residues in soils. In Book of Abstracts of Integrated sediment and
soil assessment, the 3rd International Workshop of RECETOX EU-Center of Excellence. 11
-12 February 2005, Brno, Czech Republic, 11-12/2/2005. poster presentation

129
Černohlávková, J., Hofman, J., Bezchlebová J., Dušek, L., Holoubek, I. (2005): Results of the
monitoring microbial biomass in different fluvisols from Zlín region in the Czech Republic. In
Book of Abstracts of ECOTOX 2005. ISBN 80-210-3799-7. p. 125. Brno, Czech Republic, 5-
7/9/2005, poster presentation

Bezchlebová, J., Černohlávková, J., Lána, J., Sochová, I., Hofman, J. (2004): Ecological risk of
toxaphene residues in soil. NICOLE Network Meeting, 15 – 16 November 2004, Sofia,
Bulgaria, poster presentation

Hofman, J., Černohlávková, J., Holubářová, B., Bezchlebová J., (2004): Filling the gaps in
ecotoxicological profile of toxaphene and short chain chlotinated paraffins, SETAC Europe
the 14th Annual Meeting, 18 – 21 April, 2004, Prague Czech Republic, oral presentation

130