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Pergamon Chemosphere, Vol. 37, Nos 14 15, pp. 2935-2947, 1998 © 1998 Elsevier Science Ltd. All rights reserved
0045-6535/98/$ - see front matter
PII: S0045-6535(98)00334-8
COMPARATIVE SENSITIVITY OF 20 BIOASSAYS FOR SOIL QUALITY.
J. Bierkens, G Klein, P. Corbisier, R. Van Den Heuvel, L. Verschaeve, R. Weltens, G Schoeters.
Flemish Institute for Technological research - VITO, Boeretang 200, 2400 Mol, Belgium.
Increasing evidence suggests that the use of a single bioassay will never provide a full picture t?f the quality
of the environment. Only a test battery, composed of bioassays of different animal and plant species from
different trophic levels will reduce uncertainty, allowing an accurate assessment o f the quality of the
environment. In the present study, a test battery composed of 20 bioassays of varying biological endpoints
has" been compared. Apart from lethality and reproductive failure in earthworms, springtails, nematoda,
algae and vascular plants, these endpoints also included bioavailibility of metals (bacteria), heat-shock
induction (nematodes, algae), DNA damage (bacteria, earthworm, vascular plants), fl-galactosidase
(Daphnia) and esterase activity (algae) and a range of immunological parameters (earthworm). Four
chemicals (cadmium, phenol, pentachlorophenol and trifluralin) - each representing a different toxic mode
o f action - were applied in a dilution series (from 1 mg/kg up to 1000 mg/kg) onto OECD standard soil. The
tests have been performed both on these artificially contaminated soil samples and on aqueous extracts
subsequently obtained from these soils. The results show that the immunological parameters and the loss of
weight in the earthworms were among the most sensitive solid-phase assays. Esterase inhibition and heat-
shock induction in algae were shown to be extremely sensitive when applied to soil extracts. As previously
shown at the species level, no single biological endpomt was' shown to be the most sensitive for all four
modes o f toxic action.
Key words: Bioassays, Biomonitoring, Ecotoxicology, Environmental Quality, Soil, Test battery.
Introduction.
At present, risk assessment of contaminated soils is mainly based on chemical analyses of a priority list of
toxic substances, corrected for the total amount of organic and inorganic matter in the soil. This analytical
approach does not allow for mixture toxicity, nor does it take into account the bioavailibility of the
pollutants present. In this respect bioassays provide an alternative because they constitute a measure for
environmentally relevant toxicity, i.e. the effects of a bioavailable fraction of interacting pollutants set in a
complex environmental matrix. However, increasing evidence suggests that the use of a single bioassay will
never provide a full picture of the quality of the environment, i.e. that only a test battery, composed of
bioassays on different species belonging to different trophic levels will contain sufficient discriminatory 2935
2936
power and reduced uncertainty to allow for an accurate assessment of the quality of the environment [ 1,2].
Ideally, a multitude of biological endpoints (i.e. biological variables such as survival, reproduction, enzyme
activity, gene induction, ... ) should be included in this test battery.
In the present study, a test battery composed of 20 bioassays measuring different endpoints in different
animal and plant species has been compared. Four chemicals (cadmium, phenol, pentachlorophenol and
trifluralin) were applied in a dilution series (from 1 mg/kg up to 1000 mg/kg) onto OECD standard soil. The
chemicals were chosen in order to be able to make a comparison between four different toxic modes of
action [3]. The tests have been performed both on the artificially contaminated soil samples and on aqueous
extracts (eluates) subsequently obtained from these soils. The ultimate goal is to derive a representative,
cost-effective and quantitative test battery to assess soil quality. As such this study is part of the continuous
efforts of the Flemish Authorities to compose a test battery that can be used to generate biological effect
information for deriving ecological effects-based environmental quality criteria, evaluate hazards to
organisms on a site specific basis, and evaluate the effectiveness of cleanup operations.
Materials and methods
Preparation of soil and soil extracts
The OECD standard soil [4] that was used throughout the study consisted of 10 % sphagnum peat, 20 %
kaolinite clay, 69 % industrial quartz sand and 1 % CaCOs at pH 7.0 +05 and 60 % of maximal retention
capacity. The control soil and the dilution series of contaminated soil samples were prepared by mechanical
mixing. Contamination was performed by adding the contaminant dissolved in a predetermined volume of
28 ml distilled water to 100 g soil while mixing for 1 hour. The dilution series for all four contaminants was
four logl0 dilutions (1000 mg/kg to 1 mg/kg). Hydrophobic products (PCP and trifluralin) were dissolved
using dimethylsulfoxide (DMSO) in a final concentration of 1% of dry mass. Following contamination, the
soils were allowed to stabilise ( 'age') in a sealed container at 6°C for 14 days. The pH of the soil samples
was checked and, if required, re-adjusted prior to onset of the different experiments to pH 7 [5].
In order to obtain the soil extracts required for some tests, the appropriate amount of soil was contaminated
and aged as described above. Consecutively, these spiked soils were stirred for 4 hours in EPA elution
medium [6] in a ratio 1A (weight/volume) and left untouched overnight. Following a final stir for 1 hour next
morning, the eluates were filtered over a 0.22 ~tm Millipore filter.
Bioassays
An overview of the bioassays used in this study is given in Table 1. Essential features of the different assays
are included in the legend of Table 1. Details on the experimental procedures are given in the respective
references. A brief protocol for each assay is given below. Experiments were run in triplicate. For PCP and
trifluralin solvent controls were included.
2937
Comet assay vascular plants: Beans of Vich7 faba (var. 3x white) were imbibed overnight in deionized
water and then grown for 4 days (12 h light-dark) on the soils. Leaves were chopped in 0.5 ml MBS (MES
buffered saline: 80 g NaCI, 2 g KCI and 0.5 g MES for 1 litre, pH 6) with 10 mM EDTA by razor blade.
The suspension containing liberated nuclei was filtrated through a 50 Ixm nylon cloth [7]. 15 Ixi of nuclei
suspension was spreaded in 300 gl of molten 0.8% LMP Agarose (BRL) on a microscopic slide precoated
with 0.5% Agarose (BRL) with the help of 26 x 50 mm cover slip. Embedded nuclei were lysed in 2.5 %
SDS in 0.Sx TBE buffer [8] for 20 minutes at RT followed by a wash in 05xTBE for 10 min Then DNA
was unwinded in 0.3 M NaOH and 1 mM EDTA at 27°C for 20 rain. After rinsing and equilibration in
0.5x TBE, the slides were electrophoresed at 1 V/cm for 10 minutes either in 0.5x TBE, pH-8 or 0.03 M
NaOH, 1 mM EDTA, pH-12.5. After electrophoresis the slides were rinsed in distilled water and allowed
to air dry [9]. Dried slides were stained with 10 I.tg/ml ethidium bromide for 5 minutes. Then comets were
viewed in Zeiss Axioplan Fluorescence microscope (200x) and analysed with equipped image analysis
system Comet 3.1 (Kinetic Imaging). 50 comets were analysed per point and % of DNA in tail was taken as
a measure of DNA damage.
Comet assay Eiseniafetida: Adult earthworms were exposed for 1 day. After exposure of the earthworms,
their coelomocytes were obtained according to the non-invasive extrusion method described by Eyambe et
al. [10]. Individual earthworms were rinsed in saline (4°C), and the content from the lower gut was expelled
by massage to reduce faecal contamination of the extrusion fluid in which the worms were placed for three
minutes. The extrusion medium into which the coelomocytes are "spontaneously" secreted consisted of 5%
ethanol + 95% saline + 2.5 mg/ml guaiacol glycerol ether (pH 7.3). The comet assay was performed as
described above.
fl-Galactosidase activitv in adult daphnids: The ASTM E-47 Proposal P235 [11] describes an
ecotoxicological bioassay based upon the B-galactosidase activity in Daphnids. Diminishing I]-galactosidase
activity is taken as a direct measure for interference of the toxicant with normal metabolism in Daphnia
magna. To measure this gut enzyme's activity the living animals are fed with a non-fluorescent substrate
methyl umbelliferyl galactosidase (MUG) for 15 minutes. MUG is cleaved by the 13-galactosidase enzyme
thereby liberating galactose and forming a fluorescent product, umbelliferol. The bioassay was originally
described for new-born Daphnids, evaluating the fluorescence by counting the number of organisms that
highlight under UV after a one hour exposure period [10, 11]. To make this technique suitable for our
purposes the following adaptations were made: (a) adult Daphnids were used and (b) fluorescence was
measured with a cytofluorometer in 96 wells (5 animals per well, 4 replicates) after (c) 2 hours of exposure.
2938
Growth inhibition measurements in Raphidocelis subcapitata: Growth inhibition experiments were
performed in 96 multiwell plates (Corning). Algae were inoculated at 2x105 cells/ml in a total volume of
100 I.d/well Jaworski [13], and kept at a 12/12 light/dark regimen and 24°C throughout the experiments. At
day 1 the different pollutants were added in serial dilutions for 6 remaining days. Enumeration was
performed by measuring the chlorophyll A content in the algae at 360 and 645 nm for the excitation and
emission filters, respectively (Cytofluor 2350, Millipore, Bedford, MA), Percent inhibition (It) in the
treatment groups was calculated by using the formula It = ((AUF~ - AUFt) / AUFc) x 100, where AUFc and
AUFt are the arbitrary units of fluorescence in the controls and the test population [14]. Ten wells were
counted per treatment group.
Enzyme Linked lmmunosorbent Assay for Hsp70: ELISA was performed as described by Anderson et al.
[15], with minor modifications. Briefly, samples diluted to 3 t~g/100 ~tl using PBS were loaded in
polycarbonate immunoassay plates (Maxisorp, Nunc). In order to construct a standard curve, recombinant
human Hsp70 at a stock concentration of 500 ng/100 I.tl was serially diluted in half so that the lowest
concentration was 0.49 ng. The final volume in each well was 100 ~tl. The plates were covered and
incubated overnight at 4°C. The next day the wells were washed thrice with 200 ~tl PBS prior to addition of
200 ~tl PBS-3% BSA for 8 hours at 4°C (blocking step). After a washing step (3 x PBS) the plate was
incubated with the anti-Hsp70 antibody (100 ~!) at a dilution 1/500 overnight (O/N) at 4°C. Subsequently
the wells were washed trice with 200 ~tl PBS containing 0 .1% Tween 20, and again incubated with goat
anti-mouse immunoglobulin (IgG at 1/1000) O/N at 4°C. Since no western blot has been carried out on
proteins obtained from R. subcapitata, cross-reactivity of the anti-Hsp70 antibody with other proteins
cannot be excluded. The plate was once again washed 3 times with PBS containing 0 .1% Tween 20, and
OPD (o-phenylenediamine dihydrochloride) added at a concentration of 500 ~tg/ml. The reaction was
stopped using 50 p.l 3M H2SO4 and the plate was read in a 96-well multiplate reader (Bio Rad, Richmond,
CA) using a 490 nm filter with reference 595 nm. Background activity was determined in wells containing
PBS alone. Non-specific binding was derived from wells containing only secondary antibody.
Bacterial biosensor assay (Biomet ~ assay): Freeze dried bacterial biosensors that respond to zinc,
cadmium, cobalt and lead (strain AE1433; [16]) were resuspended in 5 ml reconstitution medium (minimal
Tris medium supplemented with 0.2% acetate buffered with 20 mM 4, morpholinepropanesulfonic acid
(MOPS) at pH 7 and containing 20 ~tg/ml tetracycline) to obtain about 34 xl06 Colony Forming Units/ml.
Twenty microliters of the different soil extracts were added to a series of tubes (Lumacuvette-P#9200-0)
containing 180 ml of the reconstituted biosensor. Those tubes were kept at 23°C for 5 hours and the
bioluminescence was measured during 10 s in a Lumac M2010 luminometer.
2939
Genotoxicity testing using a SOS bioluminescence Salmonella typhimurium test (VITOTOX ~ assay): The
VITOTOX ~ test has been previously described [17]. Briefly, to prepare the test cultures, 5 ml 869 medium
was inoculated with TA104recN2-4 or T104pri bacteria (see legend Table I) and grown overnight (ON) at
37°C on a rotative shaker. Next morning, 20 txi of the ON culture was diluted in 2.5 ml 869 medium and
incubated for 1 h at 37°C with shaking. Subsequently, the culture was diluted 10 times in 869 medium and
90 Ixl of the dilution was added per well ofa 96-weU microtiter plate that already contained 10 Ixl of the test
product solution. The 96-well plate was placed into the Microlumat LB96P luminometer (EG and G
Berthold) and measuring was performed with the following parameters: 1 s/well; cycle time 5 min; 60
cycles; incubation temperature 30°C. After completing the measurements the signal-to-noise ratio, being the
light production of induced cells divided by the light production of non-induced cells, was calculated for
each measurement. A compound was considered genotoxic when the signal-to noise ratio was equal or
higher than 2 for at least two concentrations and when a clear dose-effect response was observed.
Esterase inhibition in Raphidocelis subcapitata: Algae (100 IA at a density of 5.106 algae/ml) were
distributed over the wells of a 96 well microtiter plate that contained alreadyl00 p.l of the different test
solutions. Following an incubation period of 4 h (23°C; 4000 Lux) 10 p.l fluorescein-diacetate (25
mg/100ml) was added for a period of 15 min Fluorescence was measured using a Cytofluor 2350
Fluorometer (Millipore) at 485 nm and 530 nm for the excitation and emission filters, respectively. Percent
inhibition in the treatment groups was calculated by using the formula It = ((AUFc -AUFt)/AUFc)) x 100,
where AUFc and AUFt are arbitrary units of fluorescence in the controls and the test population,
respectively [14]. Per treatment group ten wells were counted.
Immunological parameters in Eisenia foetida: A series of parameters on the coelomocytes of E. foetida
have been determined. The coelomocytes were obtained using an extrusion buffer as described above. After
extrusion the harvested cells were mixed gently with 4°C PBS, centrifuged at 150 g and resuspended at a
density of 106 Cells/ml in PBS. Viability, Phagocytosis and agglutination were assayed using a fuorescent
activated cell sorter (FACS; Star plus, Becton Dickinson). For this purpose 105 ceils/0.5 ml were incubated
with 25 ktg/ml propidium iodide (PI), 10 ktl of a 3.8 10 TM latex FLUO particles solution (Boehringer) and 20
p.l rabbit anti rat-FITC (Prosan/DAKO), respectively. Viability was analysed immediately after the addition
of PI. Following an incubation period for 1 hour at 4°C, non-ingested latex particles were removed by
centrifuging the cell suspension over a Foetal Calf Serum (FCS) cushion. Coelomocytes intended to
determine agglutination were incubated for 1 hour at RT in the dark and theroughly washed before analysis.
Viability was expressed as the percentage of total cell number at counting. Phagocytosis and agglutination
in the different treatment groups were expressed as a percentage of control coelomocytes.
Neutral Red (NR) testing and Rhodamine 123 (Rh123) determinations were performed using a Cytofiuor
2350 Fluorometer (Miilipore). For the NR assay 105 coelomocytes/well were incubated overnight at 4°C
Tab
le 1
: Ove
rvie
w o
f the
bio
assa
ys u
sed
in t
his
stud
y. (
Solid
-pha
se t
ests
are
giv
en in
bol
d)
.gL
Bio
logi
cal
endp
oint
Bio
avai
libi
lity
Det
oxif
icat
ion
&
cell
ular
def
ence
Subl
etha
l da
mag
e
Let
hali
ty &
repr
oduc
tive
eff
ects
Tes
t
Met
al s
enso
rs c°
Gen
otox
icit
y se
nsor
s (:)
Tox
icit
y
Hsp
70 °
)
Est
eras
e in
hibi
tion
13-g
alac
tosi
dase
inhi
biti
on
Phy
toto
xici
ty ~
)
Gen
otox
icit
y (6
)
Imm
unot
oxic
ity
(s)
Mor
tali
ty
Rep
rodu
ctio
n
Rep
rodu
ctio
n
Org
anis
m
dlca
lige
nes
eutr
ophu
s i1
)
Salm
onel
la ty
phim
uriu
m c
2)
Pho
toba
cter
ium
pho
spho
reum
~3)
Rap
hMoc
eles
sub
capi
tatc
t c4)
Cae
norh
abdi
tis
eleg
ans
Rap
hido
cele
s su
bcap
itat
a c4
)
Dap
hnia
mag
na
Lepi
dium
sat
ivum
15)
Eis
enia
foet
ida
16)
Vic
ia fa
ba 1
6;
Eis
enia
foet
ida
Eis
enia
foet
ida
Fol
som
ia c
andi
da
Lep
idiu
m s
ativ
um r
T;
Eis
enia
foet
ida
Cae
norh
abdi
tis
eleg
ans
Rap
hido
cele
s su
bcap
itat
a f4
)
Tro
phic
lev
el
Bac
teri
a
Bac
teri
a
Bac
teri
a
Uni
cell
ular
alg
ae
Nem
atod
es
Uni
cell
ular
alg
ae
Cru
stac
ea
Vas
cula
r pl
ants
Ann
elid
a
Vas
cula
r pl
ants
Ann
elid
a
Ann
elid
a
Spr
ingt
ails
Vas
cula
r pl
ants
Ann
elid
a
Nem
atod
a
Uni
cell
ular
alg
ae
Ref
eren
ce
[18,
161
[171
[171
[19,
201
[19,
20l
[21]
[22]
[23]
I241
[251
[26]
[4]
[271
[28l
[291
[30]
(1)
Spec
ific
met
al s
enso
rs (
Bio
met
®-a
ssay
) ar
e av
aila
ble
for
Cd~
Zn, C
u, C
r, N
i and
TI.
The
bac
teri
al s
trai
n A
E143
3 us
ed m
thi
s st
udy
resp
onds
to th
e pr
esen
ce o
f Cd,
Zn
, C
o an
d Pb
; (2
) Th
e V
itoto
x ® a
ssay
mak
es u
se o
f tw
o ge
netic
ally
mod
ifie
d st
ratu
s of
S.
typh
imur
ium
Whe
reas
TA
t04r
ecN
2-4
reac
ts w
ith a
n m
crea
se o
f bi
olum
mes
cenc
e in
res
pons
e to
gen
otox
ic p
rodu
cts,
the
biol
unun
esce
nce
m T
104p
ri d
ecre
ases
m a
dos
e de
pend
ent w
ay i
n th
e pr
esen
ce o
f to
xic
com
poun
ds;
(3)
Mic
roto
x as
say;
(4)
pre
viou
sly
know
n as
Sel
enas
trum
capr
icor
nutu
m; (
5) R
oot e
long
atio
n an
d lo
ss o
f wei
ght
m g
arde
n cr
ess;
(6)
In
the
com
et a
ssay
on
eart
hwor
m
and
gard
en b
ean
mig
ratio
n of
sin
gle
and
doub
le s
tran
d br
eaks
is
take
n as
a m
easu
re f
or g
enot
oxic
ity (
DN
A d
amag
e);
(7)
Ger
mm
atio
n ga
rden
cre
ss;
(8)
Imm
unot
oxic
ity in
clud
es a
set
dif
fere
nt a
ssay
s, in
clud
ing
the
num
ber
of c
oelo
moc
ytes
, in
clus
ion
of p
ropi
dium
iod
ide
(PI)
, la
tex
inge
stio
n, n
eutr
al r
ed u
ptak
e:
rhod
amin
e123
sta
inin
g, a
gglu
tinat
ion;
(9)
EL
ISA
sys
tem
mea
suri
ng to
tal H
sp70
(he
at-s
hock
pro
tein
s-70
) lev
els.
2941
with a 100 Ixl of a 6.6 mg/l NR (Sigma) solution. In order to determine Rh123 105 cells/well were ON
incubated at room temperature in the dark with 100txl 20~tg/ml Rh123 in PBS. Before measurements the
cells were washed and centrifuged (150 g) and subsequently resuspended in 200 Ixl of a 90% ethanol 10%
glacial acetic acid mixture for 10 min while being shaken. The excitation and emission filters for the
respective assays were 530 nm (NR) or 485 nm (Rh123) and 645 nm (NR) or 530 nm (Rh123).
Method of comparison of the different bioassays
For each of the different bioassays used in this study, the LOEC and NOEC were calculated for each
contaminant separately. In order to allow a comparison of different test systems and endpoint units of
measure, a w-value has been calculated:
W ~ NOECx / NOECaverag¢
where NOECx = NOEC of each individual test and NOECav,,age the NOEC of all responding tests in the
battery. As a result, tests that are less sensitive than average will be characterised by w>l, whereas the more
sensitive tests will obtain w<l. NOEC rather than LOEC values were used to calculate the w-value because
a LOEC was not obtained for all tests.
Table 2: Comparison of the sensitivity of the solid phase assays used in this study for cadmium, PCP,
phenol and trifluralin. (Most sensitive test for each pollutant is given in bold)
Organism Biological endpoint w-values
Cd PCP phenol trifluralin
Eiseniafoetida Acute toxicity 3.225 0.204 1 ~449 1.792
Eiseniafoetida Reproduction 0.323 n.d. (1) n.d. n,d.
Eiseniafoetida Weight loss 0.323 0.204 0.145 0.002
Eiseniafoetida Genotoxicity 3.225 2.041 0.015 1.792
Eiseniafoetida Immunopathology 0.032 0.204 1.449 0.002
Eiseniafoetida Cellular immunity 0.0003 0.204 0.145 0.018
Eiseniafoetida Humoral immunity 0.0003 2.041 1.449 1.792
Folsomia candida Reproduction 0.0032 rid. n,d n.d.
Lipidium sativum Germination 0.323 2.041 1,449 1,792
Lipidium sativum Weight loss 0.323 0.021 1.449 1.792
Viciafaba Genotoxicity 3.225 2.041 1.449 0.018
(1) n.d.; not done
2942
Results
The sensitivity of the different test systems was calculated for each pollutant separately (Table 2 and 3) and
for the 4 pollutants together (Figure 1 and 2). A distinction was made between solid and liquid phase tests,
because the concentrations in the soil samples and the eluates were assumed different. Chemical analyses
were only performed for Cd showing recoverable concentrations of 976 mg/kg and 5.125 mg/l for the soil
and the eluate samples of the 1000 mg/kg spiked soils, respectively. Similar ratios between the
concentrations in the soil and eluate samples were obtained for the soils spiked with 100, 10 and 1 mg/kg,
respectively.
As can be seen from tables and figures, the immunological parameters and the loss of weight in the
earthworms were among the most sensitive solid-phase assays. Esterase inhibition and heat-shock induction
in algae were shown to be extremely sensitive when applied on the soil extracts. In calculating the relative
sensitivity for all products together, no reproduction tests on Eiseniafoetida and Viciafaba were included,
because too few data were available. Since cellular immunity, immunopathology and humoral immunity
consist in each case of different endpoints, always the most sensitive of these endpoints has been withheld
for comparison. Because of its low solubility, trifluralin could only be detected by one eluate test, i.e. the
Table 3: Comparison of sensitivity of liquid phase bioassays for cadmium, PCP, phenol and
trifluralin. (Most sensitive test for each pollutant is given in bold)
Organism Biological endpoint w-values
Cd PCP phenol trifluralin
Photobacterium Microtox 1.843 0.103 0.63 1.091
phosphoreum
Alcaligenes eutrophus Metal sensors 0.184 0.103 0.63 1.091
Salmonella typhimurium Vitotox TA104recN2-4 1 . 8 4 3 0.103 0.63 1.091
Salmonellatyphimurium Vitotox TAl04pri 0.184 0.103 6.31 1.091
Caenorhabditis elegans Lethality 1.843 0.103 0.63 1.091
Caenorhabditis elegans Reproduction 1.843 10.31 0.63 1.091
Caenorhabditis elegans Stress protein induction 1 843 0.01 0.006 1 091
Daphnia m a g n a 13-galactosidase 1.843 0.01 0.631 1.091
inhibition
Lipidium sativum Root elongation 0.184 0.01 0.631 1.091
Raohidoceles subcapitata Reproduction 0.018 0.10 0.631 1.091
Raphidoceles subcapitata Stress protein induction 0.184 0.01 0.631 1.091
Raphidoceles subcapitata Esterase inhibition 0.184 1.031 0.006 0.001
Figure 1: Relative sensitivity of solid-phase bioassays for all four pollutants.
2943
W'
2,500 JJ~l
2,000
1,500
1,000
0,500
0,000 A B C D E F G H I
A: Cellular immunity in Eiseniafoetida; B: Immunopathology in Eiseniafoe#da; C: Weight loss in Eisenia
foetida; D: Humoral immunity in Eiseniafoetida; E Weight loss in Lipidium sativum; F: Genotoxicity in
Viciafaba; G: Germination in Lipidium sativum; H: Genotoxicity in Eiseniafoetida; I: Acute toxicity test
on Eisenia foetida.
esterase inhibition test in Raphidoceles subcapttata, rendering it the most sensitive test when all four
products are considered.
Discussion
The ultimate goal of composing a test battery for soil quality is to create a quantitative tool to assess the
quality of soil samples. In this respect, the relative sensitivity of the bioassays is but one criterion that can be
looked at. Cost-effectiveness, accuracy, economy of the test system are as many others. Still, understanding
the relative sensitivity of the different bioassays is a prerequisite to arrive at a coherent, normalised test
battery.
As mentioned in the result section, the immunological parameters and the loss of weight in the earthworms
were among the most sensitive solid-phase assays. Esterase inhibition and heat-shock induction in algae
were shown to be extremely sensitive when applied on the soil extracts. However these data have to be
2944
interpreted with some caution since representatives of certain modes of toxic action (e.g. genotoxicity) have
as yet not been included in the comparison
Figure 2: Relative sensitivity of bioassays performed on soil extracts for all four pollutants.
W
1,800 -
1,600
1,400 -
1,200
1,000
0,800
0,600
0 ,400 -
. . . . . !i
0,200 -
0,000 - A B C D E F G H I J K L
A: Esterase inhibition in Raphidoceles subcapitata; B: Reproduction in Raphidoceles subcapitata; C: Stress
protein induction in Raphidoceles subcapitata; D: Root elongation in Lipidium sativum; E: Metal sensors
(Alcaligenes eutrophus); F: Stress protein induction in Caenorhabditis elegans; G: 13-galactosidase
inhibition in Daphnia magna; H: Vitotox TAI04pri (Salmonella typhimurium); I: Vitotox TA104recN2-4
(Salmonella typhimurium); J: Lethality in Caenorhabditis elegans; K Microtox (Photobacterium
phosphoreum); L: Reproduction in Caenorhabditis elegans.
This may explain the high w-values for the comet assays on both Eiseniafoetida and Viciafaba. In this
respect it is remarkable that two of the most frequently used acute toxicity assays for soil, the microtox
assay and the acute toxicity test on Eiseniafoetida, rank among the least sensitive tests in the battery.
As previously shown at the species level [2], in the present study no single biological endpoint was shown
to be the most sensitive test for all four modes of toxic action. This is not surprising since both a-specific
(Hsp70, esterase inhibition,...) and more specific (genotoxicity, immunotoxicity,...) bioassays have been
included in the test battery. A final conclusion on how the different bioassays compare will only be possible
after all different modes of toxic action (as defined by McCarthy and McKay [3]), have been included in the
comparison.
2945
Although sublethal effects are believed to occur early in the cascade of toxic events and at lower doses, this
is not necessarily reflected by our results. Stress protein induction in Caenorhabditis elegans is but one
sublethal endpoint that did not show high sensitivity for all the chemicals tested. This may reveal yet
another aspect involved in composing a test battery. Not only does one have to take into account the
biological endpoint as such, but also the test species under investigation. Caenorhabditis elegans is known
to be rather insensitive to heavy metals [31], which is reflected in a high w-value of 1.843. However,
omission of the stress protein assay in Caenorhabdias elegans on ground of its insensitivity would mean an
impoverishment of the test battery as nematodes represent the most dorntinant group of multicellular
organisms in soil.
Whereas in the present study only four toxic modes of action have been compared, in the near future this
study will be extended by comparing the sensitivity of the test battery with regard to more and different
toxic modes of action. Together with the present results, these data will be used to further optimise the
different assay systems, and to normalise the test battery. The ultimate goal is to derive a representative,
cost-effective and quantitative test battery to assess soil quality.
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