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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