Application of Bioassays in Studies on Phytotoxic Herbicide Residues in the Soil Environment

Full text

Chapter Number
Application of Bioassays in
Studies on Phytotoxic Herbicide
Residues in the Soil Environment
Tomasz Sekutowski
Institute of Soil Science and Plant Cultivation – National Research Institute,
Pulawy Department of Weed Science and Tillage Systems, Wroclaw
Poland
Science is food for the mind
War and Peace, L. Tolstoy 1869
1. Introduction
The primary aim of the application of herbicides is to protect plantations against the
competitive action of many weed species found in the field of a given crop. Herbicides may
be used both directly to the soil and in foliar applications. In relation to the type and method
of application (single vs. split dose) a portion (in foliar applications) or the entire amount (in
soil-applied agents) of herbicide reaches the soil (Praczyk & Skrzypczak, 2004; Woźnica,
2008). Each active ingredient in a herbicide which penetrates the soil medium, undergoes
certain biophysical and biochemical processes. At the time the herbicide active ingredient
enters the soil it is separated between the solid phase (soil particles) and the aqueous phase
(soil solution). In the soil medium only this portion of the active ingredient is available to
plants, which is found in the liquid phase. However, herbicide molecules adsorbed or
chemically bound with the solid phase are not absorbed by plants. Under field conditions
this balance is constantly disturbed as a result of the action of the edaphone and through
changes in temperature and moisture content of soil, which affects the availability of
herbicide to weeds and crops (Vicari et al., 1994; Sadowski, 2001).
Depending on the applied cultivation regime and climatic and soil conditions observed in a
given vegetation season only a portion of herbicide active ingredient residue found in the
soil is available to plants and under advantageous conditions may exhibit phytotoxic action.
Thus the determination of the level of residue, degradation rate and translocation of
herbicide active ingredients in the soil is so significant both for the agricultural practice and
for the protection of the agricultural environment (Sadowski et al., 2002; Sadowski &
Kucharski, 2004).
At the selection of a detection technique the most important criterion in the evaluation is the
concentration, at which a given analyte may be found in the tested sample. Instrumental
methods, such as gas chromatography (GC) or liquid high performance chromatography
(HPLC), make it possible to determine the total content of active ingredients in the soil at the
time of the application or several weeks after the application of herbicides (Ahmad &
Crawford, 1990; Sadowski, 2001; Sadowski et al., 2001a; Kucharski & Sadowski, 2006). This

Herbicides, Theory and Applications
2
problem appears when the herbicide is used once or several times in the vegetation season
in small doses of <50 g/ha, since already at the moment of application the level of herbicide
active ingredients is slight and does not exceed 10
-2
mg/kg (Sadowski et al., 2002). The
evaluation of risk resulting from the occurrence of herbicide residue in the soil medium
until recently was based only on the results of chemical analyses, which supplied
information on the presence, content and type of the chemical substance, preventing an
evaluation of harmful ecological effects of the herbicide residue. Thus the traditional,
chemical approach to the assessment of the level of herbicide residue in the
agrophytocenosis for well over a decade has been supplemented by ecotoxicological
analyses. In such analyses the level of herbicide residue is evaluated on the basis of a
specific, comprehensive response of standard indicator organisms to the active ingredient
varying both chemically and in terms of its concentration, contained in the tested soil
sample. In such analyses the biotest methods is used with the application of e.g. a plant
biodetector. This method is to determine a biologically effective level of the herbicide active
ingredient residue immediately after application, as well as to follow the dynamics of
decline for this substance in the soil environment in the course of several months or even for
more than a year. Biotests also facilitate an objective evaluation of the level of residue, due
to the fact that all higher plants have a certain sensitivity to different xenobiotics (e.g.
herbicides) found in the soil environment. The phytotoxic effect of active ingredients
originating from herbicides may be observed on the basis of the reduction of dry or fresh
weight of roots or aboveground parts (stems, leaves) of test plants (Günther et al., 1993;
Stork & Hannah, 1996; Sarmah et al., 1999; Sadowski et al., 2002; Demczuk et al., 2004;
Sekutowski & Sadowski, 2005; 2006; 2009). Thanks to the wide-scale application of the
bioindication method using plants it is possible to evaluate the degree of contamination not
only for the soil, but also for the entire agrophytocenosis (Dećkowska et al., 2008).
2. The behavior of herbicides in the soil environment
Active ingredients of herbicides, after penetrating to the soil, are separated between the
solid phase (soil particles) and the liquid phase (soil solution). In the soil only this portion of
the active ingredient is available to plants, which is found in the soil solution within the
rhizosphere. In turn, herbicide molecules adsorbed or chemically bound with the solid
phase are not available to plants. They may constitute a certain reserve, which under
advantageous climatic conditions may become available to plants. Availability of these
active ingredients in the soil fluctuates constantly, since they are removed from the soil
solution as a result of immobilization, elution or diffusion. Bounding of these chemical
substances by the soil sorption complex is a factor determining their occurrence and through
accumulation may significantly alter their deposition time in the soil environment. Under
field conditions this equilibrium is constantly disturbed by changes in temperature,
moisture content, cultivation measures and the soil entomofauna, which has a crucial effect
on the amount of the herbicide active ingredient which is available to weeds or crops within
a specified period of time. On the one hand, the process of binding reduces mobility of these
residues in the soil profile and their penetration to aquatic zones, while on the other hand, it
reduces the possibility of their removal from soil using plants themselves and soil
microorganisms (Sadowski et al., 2001a, 2002; Praczyk & Skrzypczak, 2004; Woźnica, 2008).
The process of degradation and transfer of herbicide active ingredients depends on many
environmental and soil factors, which determine their adsorption and absorption in the soil.

Application of Bioassays in Studies on Phytotoxic Herbicide Residues in the Soil Environment
3
Such factors include the type of soil, granulometric composition (particularly the content of
clays and zeolites), temperature, moisture content, content of organic matter (humus), pH of
soil as well as the content of soil entomofauna biomass (Walker & Welch, 1989; Vicari et al.,
1994; James et al., 1999; Sarmah et al., 1999; Sadowski & Kucharski, 2004).
The sorption capacity of herbicides is defined by the index of soil sorption of the herbicide
(K
d
) and sorption of the herbicide to organic carbon (K
oc
). Active ingredients of herbicides
characterized by very high mobility in the soil environment have K
oc
< 100 ml/g (clopyralid,
nicosulfuron, sulfosulfuron, sulcotrione, dicamba), while herbicides with K
oc
>2000 ml/g
exhibit poor mobility (trifluralin, diquat, pendimethalin, diclofop, fenoxaprop-P) (Praczyk &
Skrzypczak, 2004; Woźnica, 2008).
The translocation of herbicides in the soil profile is frequently disturbed by crops themselves
or by weeds, absorbing water from the soil solution. Since a portion of the root system of
plants frequently reaches a depth of 50 - 60 cm and has a very big suction power we may
often observe the process of leaching or even the movement of residue of certain herbicides
(e.g. chlorsulfuron) from deeper soil layers towards the rhizosphere (Walker et al., 1989;
Sadowski et al., 2001b). In the opinion of Sadowski & Kucharski (2004), elution of herbicide
active ingredients being derivatives of sulfonylurea (chlorsulfuron, sulfosulfuron) and
phenoxyacetic acid (2.4 D, MCPA) from the soil profiles is strongly dependent on the initial
moisture content and the absorbing capacity of soil. They showed in their studies that with
an increase in the initial moisture content of soil, the degree of leaching for these substances
increased markedly, reaching a certain maximum. When soil reached the maximum water
capacity (under field conditions this process is observed during heavy rains), then the
percentage of leached active ingredient of a herbicide is markedly reduced. Also Beckie &
McKercher (1990), Oppong & Sagar (1992) and Günter et al. (1993) were of an opinion that
apart from moisture content, also absorbing capacity of soil has a decisive effect on
herbicide mobility. In their studies Günter et al. (1993) showed that in soil with poor
absorbing capacity metsulfuron and triasulfuron were subjected to elution much faster than
in soil with a high absorbing capacity. Mobility was also dependent on the active ingredient
itself, with metsulfuron being much more active than triasulfuron.
Also the depth to which herbicide active ingredients penetrate under field conditions is not
specifically defined, since it depends on many factors (e.g. absorbing capacity,
granulometric composition, cultivation measures). On the basis of studies concerning the
translocation of herbicide active ingredients Helling & Turner (1968) determined the relative
mobility index of herbicides (R
f
), dividing them into five classes. In another study Walker &
Welch (1989) showed that chlorsulfuron (ALS group) was capable of penetrating to a depth
of 50 cm 63 days after application, despite the fact that a bigger part of its residue was
detected in a layer up to 25 cm deep. In turn, another active ingredient from the same
chemical group, i.e. triasulfuron, did not penetrate deeper than 10 cm, and its residue
remained at that depth throughout the entire period of the experiment (125 days).
Stability of active ingredients of herbicides in the soil is also dependent on its physico-
chemical properties and on the course of degradation dynamics. A very important indicator,
which defines potential persistence of the herbicide active ingredient in the soil
environment, is the half-life period (DT
50
). It is a time period required for the degradation of
the active ingredient to half its initial concentration in soil. The value of DT
50
is a
characteristic feature of individual active ingredients of herbicides and it may range from
several days (e.g. quizalofop-P, mesotrione, MCPA) to as long as several months (e.g.
trifluralin, ethofumesate, pendimethalin). Most active ingredients of herbicides used in

Herbicides, Theory and Applications
4
agricultural plantations has DT
50
of less than 60 days (e.g. florasulam, clomazone,
clopyralid, bentazone), while in vegetable growing it is below 20 days (e.g. clethodim,
cycloxdim, metazachlor, pyridate) (Praczyk, 2004; Praczyk & Skrzypczak, 2004; Woźnica,
2008). Half-life (DT
50
) is only a rough indication of the potential persistence of herbicide
active ingredients in soil. Under field conditions degradation of a herbicide and its
translocation may occur faster or much slower, since it is a result of interactions between
chemical properties of the active ingredient itself and moisture content, temperature,
absorbing capacity of soil, pH and soil microorganisms. Thus the risk of persistence and
translocation of herbicide active ingredients in soil may not be considered only on the basis
of one of the above mentioned parameters (e.g. DT
50
, K
oc
, R
f
), as under field conditions the
interactions of all these factors affect the rate of chemical and biological processes, which in
turn determine the behavior of active ingredients of herbicides in the soil environment.
Table 1 presents characteristics of selected active ingredients of herbicides, which have a
decisive effect on their behavior in the soil environment.
Active
ingredient
Group
HRAC
Solubility in
water [mg/l]
DT
50
[days]
K
OC
[ml/g]
R
f
movement index
in soil environment
quizalofop-P A 0.4 <1 1024
florasulam B 6360 (pH 7) 2-18 4-54
trifluralina K1 0.22 60-132 2500-13700
diquat D 700000 1000 >32000
pendimethalin K1 0.3 30-150 6700-29400
small
(R
f
= 0.0-0.34)
amidosulfuron B 9 (pH 5.8) 3-29 33.7
clomazone F3 1100 15-45 104-608
ethofumesate N 50 15-250 97-245
alachlor K3 242 15-30 170-200
MCPA O 734 5-6 25-157
medium
(R
f
= 0.35-0.64)
sulfosulfuron B 1627 (pH 7) 11-47 5-89
metamitron C1 1700 7-70 91-392
bentazone C3 570 12-45 13-176
mesotrione F2 2200 3-7 19-390
clopyralid O 143 14-56 4.6
large
(R
f
= 0.65-1.0)
Source: Helling & Turner, (1968); Praczyk & Skrzypczak, (2004); Woźnica, (2008); modified
Table 1. Examples of active ingredient of herbicides and selected physico-chemical
properties affecting their behavior in soil
Annually repeated application of herbicides in the same field may affect the dynamics of
degradation and translocation, as well as the level of residue of their active ingredients.
After penetrating into the soil the action of a herbicide on a crop or weeds is determined
within the rhizosphere by the degree of availability and the sensitivity of the plant to the
active ingredient. Strong vertical translocation of certain active ingredients of herbicides
several days after application, particularly in lessive soils may be dangerous for the soil

Application of Bioassays in Studies on Phytotoxic Herbicide Residues in the Soil Environment
5
environment due to the possible penetration into the ground waters causing their
contamination (Beckie & McKercher, 1990; Sadowski & Kucharski, 2003). Thus studies are
necessary which would facilitate an evaluation of a threat posed by the application of
herbicides in relation to agrophytocenosis. In ecotoxicology the adopted methods for the
determination of the levels of bioavailable phytotoxic residue of herbicide active ingredients
in soil include biotests, due to their high efficiency, relatively very high sensitivity and
limited testing costs in comparison to instrumental methods (Fahl et al., 1995; Hollaway et
al., 1999; James et al., 1999; Sadowski et al., 2002; Sadowski & Kucharski, 2004; Sekutowski &
Sadowski, 2006). Plant species exhibiting high sensitivity to the action of selected active
ingredients of herbicide, such as Sinapis alba, Fagopyrum esculentum, Sorghum saccharatum,
Lepidium sativum, Helianthus annuus, Zea mays or Cucumis sativus are used as detectors. In
particular cases biotests may also provide information on transport and on the situation of
applied active ingredients (Günther et al., 1993; Sadowski & Kucharski, 2004). We may find
numerous examples in literature concerning applications of plant biodetectors in studies on
herbicide active ingredient residue (Günther et al., 1993; Stork & Hannah, 1996; Sarmah et
al., 1999; Sekutowski & Sadowski 2005; 2006; 2009).
3. Division of biological methods used in studies on the soil environment
Analytical methods using biological material are becoming promising alternatives for
conventional analytical methods and in certain cases they may even replace them (Hollaway
et al., 1999). They are commonly applied mainly due to their specificity and low unit costs.
In toxicological analyses we may distinguish two groups of applications for biological
methods in the assessment of the effect of xenobiotics (e.g. herbicides) on the soil
environment:
a. bioanalytical tests, which are connected with the use of biological organisms as
receptors of specific chemical substances, e.g. herbicides. Due to the method of the
utilization of the biological component we distinguish:
- biosensors, in which the biological component is the active element (e.g. an enzyme,
antibodies – ELISA test),
- biotests, in which a whole plant organism or its part (e.g. seeds, roots) are the control
and measuring element (Hollaway et al., 1999; van Wyk & Reinhardt, 2001).
b. biomonitoring, which may be conducted in two ways:
- through the formation of passive accumulation samplers based on typical analytical
tests of biological samples,
- through observation of plant or animal bioindicators (Fahl et al., 1995; Alonso-Prados et
al., 2002).
4. Bioassay
Bioassay or biotest (Greek bios – life + Latin testari - indicate) may be defined as an
experimental biological sample (the whole organism or its part), which aim is to detect a
toxic substance found in the environment or to identify its harmful action, by quantitative
determination of the effect of the tested substance in relation to the control object.
In studies conducted using biotests three methods are typically applied, with the first two
being conducted under controlled (laboratory) conditions, while the third being run using a
population of organisms living under natural conditions (in situ).

Herbicides, Theory and Applications
6
a. phytotoxicity tests conducted in a laboratory, during which the substance exhibiting
phytotoxic action is artificially introduced to the tested object (e.g. soil). Next the test is
performed with an appropriately selected indicator organism e.g. a plant (a phytotest).
Thus collected results are a source of information on toxicity of a given substance under
controlled conditions. The main aim of such a test is to calibrate the biotest, which will
next be used to estimate phytotoxicity of tested samples (e.g. collected from
contaminated areas).
b. phytotoxicity tests conducted at a laboratory on the basis of respective samples (e.g.
soil) collected from contaminated areas. Phytotoxicity of such samples is compared
with the phytotoxicity of reference samples (biotests). On this basis the interval is
determined, within which residue e.g. of herbicides may have an adverse effect on
crops (e.g. residual effect).
c. phytotoxicity tests conducted on the site in which a population of sensitive organisms is
living (conditions of their natural occurrence) (Kuczyńska et al., 2005, Namieśnik &
Szefer 2009).
Moreover, biotests may be classified in terms of the used organism (e.g. bacteria, plants,
animals), which constitute the active element of the test. In ecotoxicology in studies on the
residue of different xenobiotics (e.g. herbicides) the most frequently applied include plants
and their seeds, due to the specific action of the tested preparations and in view of the
humane, economic and practical aspects.
On the basis of the dose ↔ final effect dependence, which may be expressed e.g. by the
reduction of fresh or dry weight of the test plant in comparison to the control object, we may
determine values of indicators being a quantitative measure of phytotoxicity of the tested
substance. Phytotoxic action of active ingredients contained in herbicides may be
determined using such indicators as ED
10
, ED
50
or ED
90
(effective dose), i.e. determining the
concentration of the active ingredient causing a specific biological effect at 10%, 50% or 90%
its maximum value. Another applied indicator is index IC
50
or IC
90
(inhibition
concentration), i.e. the concentration of e.g. herbicide in the soil environment, which causes
a reduction of fresh or dry weight of the test plant (roots, stems, leaves) by 50% or 90% in
comparison to the control (not treated with this herbicide).
The dose ↔ final effect dependence may also be used to predict risk, i.e. to determine the
dose and persistence of herbicide residue, at which the probability of phytotoxic effects is
high or small. An example exhibiting this dependence may be here a study conducted by
Sadowski et al. (2007) or Sadowski & Sekutowski (2008), referring to the phytotoxic action of
herbicide active ingredient residue on successive crops. Those authors using biotests
showed that herbicide residue in soil may be hazardous for successive crops at two critical
moments. The first refers to resowing, i.e. situations when for different reasons, most
frequently independent of the farmer, the plantation is eliminated. In turn, the other
moment refers to residue persisting in soil and exhibiting phytotoxic action immediately
after the crop is harvested or even for the next several months. A similar phenomenon on
fields in which chlorsulfuron and metsulfuron were applied, observed in the form of
extensive damage to sugar beet or rape plantations found in the period of 2 successive years,
was reported by Walker & Welch (1989) and Walker et al. (1989). The above mentioned
effect is manifested only because crops (e.g. beet, rape) exhibit very high sensitivity to
herbicides from the ALS group. Plant species with a narrow range of tolerance (stenobionts)
characterized by high sensitivity to specific chemical groups or active ingredients of
herbicides are referred to as indicator species or bioindicators. Thus biotests are very often

Application of Bioassays in Studies on Phytotoxic Herbicide Residues in the Soil Environment
7
used in biomonitoring, to evaluate the consequences potentially caused by herbicides on
individual elements of agrophytocenosis (e.g. crops, soil or water).
4.1 Criteria for the selection of a bioindicator
Species of indicator plants should be characterized by a narrow range of responses and
exhibit high sensitivity to specific chemical substances, with their response being specific
and adequate to the concentration of the chemical substance and easily observable (e.g.
strong inhibition of root growth).
Bioindicators should meet the following requirements:
- common occurrence,
- a wide range of distribution,
- a long life cycle or several generations within a year,
- being easily recognizable,
- genetic homogeneity,
- high sensitivity to specific chemical substances,
- stability and repeatability of responses,
- low unit costs and easy laboratory culture.
In turn, plant bioindicators used in phytotests should have the following characteristics:
- small and even seeds,
- uniform germination power and energy of seeds,
- a short emergence period (1-2 days),
- a short vegetation period,
- high biomass of stems, leaves or roots,
- high sensitivity in relation to one chemical group (e.g. phenoxy acids, sulfonylurea).
When selecting a bioindicator for a test it is also necessary to take into consideration the age
and sensitivity of individual tissues to the tested herbicide. A similar opinion was also
expressed by Shim et al. (2003) and Demczuk et al. (2004), who in their studies conducted
using different weed species and Cucumis sativus plants observed a diverse response of
individual plant tissues to tested active ingredients of herbicides. They showed that
sensitivity to residue of sulfonylurea herbicide depended to a considerable degree on the
age of tissues and their location. The youngest roots and leaves of test plants turned out to
be most sensitive.
Thus one of the basic guarantees of an appropriately conducted biotest is the selection of an
appropriate test plant. An example of a dependence between the phytoindicator and the
response to the herbicide active ingredient is presented in Fig. 1-2. In the analyses 3 test
plants were used, i.e. Sinapis alba, Fagopyrum esculentum and Cucumis, as well as 2 active
ingredients of herbicides belonging to different chemical groups (phenoxy acids – 2.4 D and
sulfonylurea – nicosulfuron). For the detection of 2.4 D residue Cucumis sativus proved to be
most suitable, since root growth inhibition by 50% (IC
50
) occurred already at a concentration
of
0.18 mg/kg. For the two other species, i.e. Sinapis alba and Fagopyrum esculentum, IC
50
ranged from 0.4 to 0.5 mg/kg (Fig. 1).
In turn, in the detection of nicosulfuron residue the highest sensitivity was found for the test
with the use of Sinapis alba. Root length reduction by 50% (IC
50
) occurred already at a
concentration of 0.125 mg/kg. Sensitivity of the test (IC
50
) with the use of Fagopyrum
esculentum and Cucumis sativus was markedly lower and amounted to 0.25 mg/kg for
Fagopyrum esculentum and 0.55 mg/kg for Cucumis sativus, respectively (Fig. 2).

Herbicides, Theory and Applications
8
0
10
20
30
40
50
60
70
80
90
100
0 0.025 0.05 0.125 0.25 0.5 0.75 1 1.2
2.4 D concentration in the soil [mg/kg]
Roots lenght reduction [%]
Sinapis alba
Fagopyrum esculenthum
Cucumis sativus
Fig. 1. 2.4 D effect on the tested plant in terms of roots lenght reduction
0
10
20
30
40
50
60
70
80
90
100
0 0.025 0.05 0.125 0.25 0.5 0.75 1 1.2
Nicosulfuron concentration in the soil [mg/kg]
Roots lenght reduction [%]
Sinapis alba
Fagopyrum esculenthum
Cucumis sativus
Fig. 2. Nicosulfuron effect on the tested plant in terms of roots lenght reduction

Application of Bioassays in Studies on Phytotoxic Herbicide Residues in the Soil Environment
9
4.2. Conventional bioassays
A conventional bioassay, used in the detection of herbicide active ingredients in soil,
consists in the sowing of seeds of a test plant (adequately sensitive to the tested substance or
chemical group) into the soil sample containing the residue. Examples of procedures
required for the establishment of such a bioassay are presented in a diagram in Fig. 3.
1
Drying of soil moisture to
the presumed
2
Add water to get the
assumed humidit
y
3
Alignment moisture
throughout the batch of
soil (24 h in a closed
container
)
4
Spraying the soil layer
(
0.5-1.0 cm
)
5
Thorou
g
hl
y
mix
6
Filling containers with
soil sprayed
7
Sowin
g
the test plants
8
Set of test
p
lants
9
Determination of dry weight
of test plants
Source: Sadowski et al., (2002); modified
Fig. 3. Example diagram of a conventional bioassay setting
4.2.1 Availability of herbicide active ingredients to plants
The bioassay method is also used in the determination of values of ED
50
, ED
90
or IC
50
, IC
90
.
The duration of a conventional bioassay depends to a considerable degree on the test plant,
or rather on the tested part of the bioindicator (roots, leaves) and the active ingredient of a
given herbicide, and it may range from 7 days (roots) to 14 days (leaves, stems). After a
period of 7 or 14 days from the establishment of the test fresh and then dry weight of roots
or leaves and stems is determined (by cutting and drying at a temperature of 105
0
C, and
weighing on an analytical scale). Next the percentage loss of fresh and dry weight is
calculated in relation to the control plants (sown into the soil containing no herbicide), while
thus collected results for the dependence between weight loss in the phytotest and the
concentration of the herbicide active ingredient in the soil are used in the graphic
presentation of this dependence (Fig. 4).
Figure 5 presents an example of a phytotest using Cucumis sativus established in soil
containing different concentrations of chlorsulfuron. Results recorded from the bioassay
constitute a source of information on the toxicity of chlorsulfuron under controlled
conditions. The theoretical objective of such a test is to determine IC
50
for chlorsulfuron and
to calibrate the phytotest, which will next be used in the estimation of phytotoxicity of

Herbicides, Theory and Applications
10
R
2
=
0
.
7
5
5
9
R
2
=
0
.9
01
8
0
10
20
30
40
50
60
70
80
90
100
0 0.2 0.4 0.6 0.8 1 1.2
Sulfosulfuron concentration in soil [mg/kg]
Weight reduction in test plants [%]
Fresh weight
Dry weight
trendline (dry weight)
trendline (fresh weight)
Fig. 4. Changes n fresh and dry weight of Sinapis alba under the influence of different
sulfosulfuron concentrations in soil
Control
0.020
0.050
0.100
0.150
0.250
0.500
0.750
chlorsulfuron [m
g
/k
g
]
0.100
1.500
Fig. 5. Te effect of chlorsulfuron on fresh weight reduction of Cucumis sativus (determination
of IC
50
)
samples of soil collected from a field containing residue of chlorsulfuron (the practical
objective). Thanks to this test it will al be possible to determine whether in that field plants
from family Cucurbitaceae will be exposed to the phytotoxic action of chlorsulfuron residue.
4.2.2 Distribution of herbicide active ingredients in the soil profile
Knowledge on the translocation and distribution of active ingredients in the soil profile and
factors affecting this process is required both for the protection of the soil environment and
a more efficient use of herbicides. Most studies in this field have been conducted mainly

Application of Bioassays in Studies on Phytotoxic Herbicide Residues in the Soil Environment
11
using lysimeters. Unfortunately, the primary drawback of the lysimeter model is connected
with the high cost of one assay and limitations related with the collection of soil samples,
resulting from the disruption of the soil profile in the lysimeter column. After several
samplings the lysimeter column has to be refilled with a new undisturbed soil profile. In
turn, analyses conducted under laboratory conditions using bioassays do not have such
limitations. Moreover, they are more efficient and provide the experimenter with more
flexibility and control over a much bigger number of parameters observed during the
process of herbicide translocation. Soil collected from such a model is used as a substrate for
bioassays and the filtrate may be used in chemical analyses. This method makes it possible
to determine in a very precise way the distribution of phytotoxic residue of herbicide active
ingredients in the soil profile. Another advantage of this model is the possibility of arbitrary
modeling of irrigation in the soil profile, which facilitates a comprehensive evaluation of the
residue balance in the soil – water system. Figure 6 presents an example diagram of such a
model in action, in which the bioassay method was used to determine the distribution of
herbicide residue.
In the opinion of Sadowski & Kucharski (2004) the degree of leaching and as a consequence
the distribution of a portion of herbicide active ingredients depends on the initial soil
moisture content. Figure 7-8 presents the distribution of certain active ingredients of
1
Herbicide
a
pp
licatio
n
Leachate of
instrumental
analysis
Sowin
g
the test plants
0
2
4
6
8
10
Drying of soil moisture
to the presumed
Add water to get the
assumed humidity
3
Water
Phytotoxic
residues of
the herbicide
active
ingredient
4
5
7
6
8
9
2
Alignment moisture
throughout the batch
of soil (24 h in a closed
container)
Source: Günther et al., (1993); Sadowski & Kucharski, (2004); modified
Fig. 6. The application of the phytotest method to determine residue of herbicide active
ingredients in the soil profile

Herbicides, Theory and Applications
12
0
2
4
6
8
10
12
14
2.4-D MCPA
dichlorprop-P
isoproturo
n
pendimethali
n
chlorsulfuro
n
sulfosulfuro
n
Depth of soil profile [cm]
Initial humidit
y
of soil = 0%
- visible ph
y
totoxic effects
Source: Sadowski & Kucharski, (2004); Sekutowski & Sadowski, (2006); modified
Fig. 7. Rate of translocation of active ingredients depending on initial soil moisture content
0
2
4
6
8
10
12
14
2.4-D MCPA
dichlorprop-P
isoproturo
n
pendimethali
n
chlorsulfuro
n
sulfosulfuro
n
Depth of soil profile [cm]
Initial humidit
y
of soil = 2%
- visible ph
y
totoxic effects
Source: Sadowski & Kucharski, (2004); Sekutowski & Sadowski, (2006); modified
Fig. 8. Rate of translocation of active ingredients depending on initial soil moisture content

Application of Bioassays in Studies on Phytotoxic Herbicide Residues in the Soil Environment
13
herbicides depending on changes in the initial soil moisture content. Most active
ingredients, which were transferred on air dry soil (0% moisture content) were detected by
test plants (Sinapis alba) mainly in the surface soil layer (Fig. 7). The highest leaching level
was found for isoproturon (0-11 cm), while the least leached substance turned out to be
pendimethalin (0-3 cm). An increase in the initial soil moisture content by 2% caused a
marked shift of residue deeper within the soil profile practically for all the tested active
ingredients. Only pendimethalin residue remained at the same level (Fig. 8).
From the practical point of view the distribution of the main portion of herbicide active
ingredients, as well as the degree of their leaching to deeper soil layers are highly
significant, since they determine the effectiveness of herbicides (particularly those soil-
applied). Moreover, they also determine the degree of herbicide translocation outside the
root zone, which may increase the risk of their being transferred to ground waters
(Sadowski & Kucharski, 2004).
4.2.3 Dynamics of degradation of herbicide active ingredients in soil
Dynamics of degradation occurs most intensively in the surface soil layer (0-20 cm) and it is
closely related with the processes of degradation and translocation of herbicide active
ingredients. In this layer the intensity of biological and chemical processes is dependent to a
high degree on the temperature and soil moisture content, as well as the cultivation regime
(Sadowski, 2001; Sadowski & Kucharski, 2004; Rola & Sekutowski, 2005). In order to
determine the dynamics of degradation and translocation of herbicide active ingredients in
the soil, at specified time intervals samples are collected, onto which test plants
(phytoindicators) are sown. An example of a phytotest for different herbicide active
ingredients is presented in Fig. 9.
An example given here presents an experiment conducted using the bioassay method (with
Sinapis alba as a phytodetector) under field conditions referring to the dynamics of
translocation and degradation of rimsulfuron depending on the tillage method applied. In
the first 6 weeks after application no marked differences were observed in the course of the
dynamics of rimsulfuron degradation depending on the tillage method used. Accelerated
1 = Control
2 = foramsulfuron + iodsulfuron (45 + 1.5 g/ha)
3 = nicosulfuron (60 g/ha)
4 = rimsulfuron (12 g/ha)
5 = rimsulfuron (15 g/ha)
6 = tifensulfuron (11.5 g/ha)
7 = dicamba + rimsulfuron (240 g/ha + 7.5 g/ha)
1 2 3 4 5 6 7
Test plant – Sinap
i
s a
lb
a
Fig. 9. Rimsulfuron degradation rates in the 0-20 cm soil layer

Herbicides, Theory and Applications
14
0
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4 5 6 7 8 9 1011121314151617181920
Weeks after rimsulfuron application
Initial rimsulfuron concentration in the soil [%]
Reduced tillage
Conventional tillage
Source: Sekutowski, (2009)
Fig. 10. Rimsulfuron degradation rates in the 0-20 cm soil layer (mean in the years 2005-
2007)
translocation and dynamics of degradation was found as late as 7 weeks after application
and it was markedly diversified depending on the tillage method (Fig. 10).
The presented examples of the application of plants as phytodetectors in the bioassay
method more precisely illustrate the phytotoxic action of herbicide active ingredients (even
those found in trace amounts) for agrophytocenosis than their concentration in soil
determined using chemical analyses. A similar opinion was presented by Hollaway et al.
(1999), who in their studies concerning the detection of sulfonylurea herbicide residue in soil
using three methods, i.e. bioassay, ELISA and HPLC, stated that a bioassay using Pisum
sativum and Lens culinaris plants as bioindicators was most sensitive. Biotests detected
residue of sulfonylurea herbicides at 0.1 – 1.0 µg/kg soil, ELISA at 0.1 – 10 µg/kg, while
HPLC at 3 – 10 µg/kg, respectively.
Depending on soil and climatic conditions only a portion of residue contained in soil is
available to plants. Biotests used in biomonitoring make it possible to evaluate whether this
part of residue may exhibit phytotoxicity towards agrophytocenosis.
The presented examples of conventional phytotests using different plants and their seeds as
phytodetectors, conducted according to standardized national procedures, frequently
happen to be complicated, they require considerable laboratory space and are time-
consuming (BN-83 9180-25, 1983; BN-83 9180-27, 1983; BN-84 9180-30, 1984; PN-ISO 17616,
2010). For several years now ready-to-use tests (toxkits) have been commercially available,
sold in the form of packages, allowing the evaluation of phytotoxicity of tested samples
within a short time (1-3 days). They contain cryptobiotic forms of bioindicators (e.g. seeds of
plants – Phytotoxkit
TM
), coming from standard breeding, which may be stored for 6 months
and when needed prepared for the test within a very brief time (Phytotoxkit, 2004).

Application of Bioassays in Studies on Phytotoxic Herbicide Residues in the Soil Environment
15
4.3 Phytotoxkit microbiotest
The necessity to conduct analyses of many soil samples within a relatively short time has led
to the introduction of miniature phytotoxicity tests, called microbiotests or second
generation tests, as alternatives for conventional phytotests. An example of such a
microbiotest is a rapid (72 h) test - Phytotoxkit
TM
(Phytotoxkit, 2004). Professor Guido
Persoone (with a team of co-workers) from the University of Ghent in Belgium was the
creator of the toxkit tests (Persoone, 2005). The principle of such a phytotest is based on
germinating seeds of Sorghum saccharatum, Lepidium sativum and Sinapis alba, which as a
result of contact with the tested herbicide active ingredient found in soil exhibit a specific
reaction (a lack of germination or reduced root length). The use of standard seeds facilitates
test standardization and maintenance of reproducible results irrespective of the laboratory,
at which analyses are being conducted. The specific nature of Phytotoxkit
TM
results in the
omission of all labor-consuming activities connected with conventional biotests, thus
considerably reducing the time required to obtain the reading (from 14 to 3 days). Moreover,
this test makes it possible to obtain a direct measurement of root length using image tools,
thanks to which a graphic presentation of the dependence between root length reduction in
phytodetectors and the phytotoxic concentration of tested herbicide active ingredients is
faster and much easier in comparison to a conventional biotest. This test makes it also
possible to more comprehensively estimate the phytotoxic effect of herbicide residue not
only on the soil environment, but also on the entire agrophytocenosis. An example of a
Phytotoxkit
TM
tst conducted using a standard set of plants is presented in Fig. 11.
The diverse chemical character of herbicides prevents the use of only one type of a
Phytotoxkit
TM
containing standard phytodetectors supplied in the kit. Due to the specific
response of different plant species to the presence of herbicide active ingredients belonging
to different chemical groups it is necessary to supplement knowledge on the applicability of
other plants. Thus the test is very often modified, which consists in the replacement of
standard test plants with other plant species, such as e.g. Helianthus annuus, Cucumis sativus
or Fagopyrum esculentum. Thanks to the modification of Phytotoxkit
TM
it was possible to
extend the collection of plants potentially applicable in the determination of herbicide
residue, e.g. derivatives of benzoic acid, phenoxy acids and sulfonylourea (Fig. 12).
Similarly as in case of conventional biotests, Phytotoxkit
TM
may be used in the
determination of values of ED
50
and IC
50
and the determination of the level of residue, rates
of degradation and translocation of herbicide active ingredients in soil. An example in this
respect may be an experiment conducted using a modified Phytotoxkit
TM
under laboratory
conditions, consisting in the determination of ED
10
and ED
50
for dicamba. The run biotest
Test containers -
Phytotoxkit
TM
Sorghum saccharatum Sinapis alba
Fig. 11. Phytotoxkit
TM
with standard test plants.

Herbicides, Theory and Applications
16
Helianthus annuus Cucumis sativus Fagopyrum esculentum
Fig. 12. Phytotoxkit
TM
with alternative test plants.
showed that significant differences in the reduction of root length in Fagopyrum esculentum
and Cucumis sativus were obtained for concentrations ranging from 0.025 mg/kg to 0.25
mg/kg. The strongest response to the tested substance was recorded for Fagopyrum
esculentum, while it was weakest in case of Sinapis alba. The detoxication capacity in relation
to dicamba in Fagopyrum esculentum (ED
50
)
was eliminated already at a concentration of
0.125 mg/kg, while a further increase in the concentration of the tested substance in soil (1.2
mg/kg)
resulted in root length reduction by 99%. In turn, ED
50
for the
other two species, i.e.
Cucumis sativus and Sinapis alba fell within the range of 0.25 – 0.5 mg/kg soil (Fig. 13).
0
10
20
30
40
50
60
70
80
90
100
1.2 1 0.75 0.5 0.25 0.125 0.05 0.025 0
Dicamba concentration in the soil [mg/kg]
Root lenght reduction [%]
Sinapis alba
Fagopyrum esculentum
Cucumis sativus
ED
50
ED
10
Source: Sekutowski & Sadowski, (2009)
Fig. 13. The effect of dicamba on root length reduction in tested plant
The above example very well shows the response (sensitivity) of the phytodetector to the
tested active ingredient of the herbicide. In analyses using plants as detectors, it is crucial to
select an appropriate plant for the tested herbicide active ingredient. A sufficiently sensitive
plant detector makes it possible to conduct tests on microresidue of 0.01 mg/kg soil.

Application of Bioassays in Studies on Phytotoxic Herbicide Residues in the Soil Environment
17
4.4 Sets of biotests (batteries)
The selection of an appropriate biotest in studies on agrophytocenosis depends on the type
of required information, the concentration of herbicide active ingredient residue in the
analyzed sample of soil (water), as well as the species-specific sensitivity of the tested plant.
In case of the use of only one phytodetector species the estimated phytotoxicity reflects the
sensitivity of only this one tested species. Such a procedure may result in an error connected
with an underestimation of phytotoxicity of the analyzed herbicide active ingredient in
relation to the entire agrophytocenosis. This risk may be minimized thanks to the
application of a battery of biotests, which action is based on the use of plant species of
different sensitivities to active ingredients of herbicides belonging to one chemical group.
Batteries of tests may be formed within one test (e.g. Phytotoxkit
TM
), which may include
several species of test plants exhibiting different sensitivity to a given chemical group.
Moreover, sets of batteries may be established within several tests using different
biodetectors of varying sensitivity to the same chemical group, e.g. Phytotoxkit
TM
→ ELISA
→ HPLC (Hollaway et al., 1999).
5. Conclusion
Bioassays are methods commonly applied in ecotoxicology in the determination of the levels
of bioavailable phytotoxic residue of herbicide active ingredients in soil. Tests with the use
of rapidly germinating seeds have several very important advantages, as they are cheap and
easy to perform, they do not require expensive laboratory equipment and they yield
reproducible results. The phytotoxic effect of herbicide active ingredient may be stated on
the basis of the dynamics of germination, seedling growth, reduction of dry or fresh weight
of roots or aboveground parts (stems, leaves) of test plants. On the basis of selected
parameters, such as the reduction of root length, the toxic effect of herbicide active
ingredients may be determined already after approx. 24 h, while the dynamics of root
growth - after 3-5 days from the onset of the test (Phytotoxkit
TM
). In turn, the reduction in
fresh or dry weight of aboveground parts of plants may be established after approx. 10-14
days (a conventional biotest).
Unfortunately, drawbacks of such a method include first of all the fact that it is impossible
to identify the tested active ingredient. This problem may be solved by using different
biological factors forming a set of biotests (Phytotoxkit
TM
→ ELISA → HPLC), which will
make it possible to precisely determine the herbicide active ingredient. It also needs to be
stressed that biotests with the application of rapidly germinating seeds of selected plant
species may be a good supplementation or even an alternative to classical instrumental
measurements, used in the detection of phytotoxic residue of herbicide active ingredients in
soil.
Probably the scope of bioassay application within the next few years will be increasing and
thus collected information will constitute the basis for the initiation of analyses using
classical analytical methods.
6. Acknowledgement
I would like to express my gratitude to Prof. J. Sadowski for providing me with literature
and for his valuable remarks and suggestions in the course of preparation of this
manuscript.

Herbicides, Theory and Applications
18
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