Behavioural Endpoints in Earthworm Ecotoxicology

Transcription

Research Articles
Avoidance Response Test
Research Articles: Bioassays
Behavioural Endpoints in Earthworm Ecotoxicology
Evaluation of Different Test Systems in Soil Toxicity Assessment
Maike Schaefer
Department of Ecology, Institute for Environmental Research and Technology (UFT), University of Bremen, Leobener Strasse,
D-28359 Bremen, Germany ([email protected])
DOI: http://dx.doi.org/10.1065/jss2003.02.066
Abstract. Background, Aims and Scope. Endpoints in earthworm
ecotoxicology scheduled in guidelines are mortality and reproduction rates. However, not only the direct influence of pollutants on population parameters but also changes in behaviour
such as substrate avoidance can have an important impact on
soil ecosystems. In practice two different avoidance response
tests are applied in earthworm ecotoxicology: (i) a six-chamber
test system and (ii) a two-chamber test system. Both avoidance
response-test systems were compared to establish their respective advantages and disadvantages in order to advance the standardisation of behavioural tests. The earthworm avoidance-response tests were applied in addition to the standard acute and
chronic earthworm toxicity tests (ISO 11268) in order to compare the sensitivity of the test endpoints.
Methods. Test substrates were contaminated with crude oil and
2,4,6-Trinitrotoluene (TNT), respectively. The test species was
Eisenia fetida. The earthworms were exposed to the contaminated substrates and their mortality (14 d), reproduction rates
(number of cocoons after 28 d, juvenile hatching after 56 d),
and substrate preference (48 h) determined.
Results and Discussion. Whereas 1000 mg/kg TPH (Total Petroleum Hydrocarbons) did not show any lethal effects, 100%
mortality occurred in soil with comparable TNT concentration.
The acute tests consistently produced the highest effect concentrations whereas reproduction and substrate avoidance were the
more sensitive test parameters. Both behavioural test systems,
when compared, showed similar substrate avoidance after an
incubation time of 48 h. The six-chamber test system provides
the potential to test six different substrates/concentrations at
one time. It was observed, however, that earthworms did not
migrate among all test chambers within a test unit in order to
select the most appropriate substrate. Orientation was observed
only between directly neighbouring test compartments, which
complicates the interpretation of the test results.
Conclusion. Substrate avoidance and reproduction variables were
clearly more sensitive test endpoints than mortality. Therefore
avoidance-response tests proved to be useful test methods in
detecting effects of sublethal concentrations of pollutants on
earthworms. The test duration of the avoidance tests is much
shorter compared to the standard acute and chronic earthworm
toxicity tests, which makes them a quick screening tool for identifying potential soil toxicity. Both avoidance-response test systems showed comparable results regarding the test sensitivity.
Nonetheless, the incomplete substrate use in the six-chamber
avoidance test due to the reduced migration possibilities (orientation only to neighbouring chambers) might reduce the distinctness of test results as it allows only reliable information on
the most avoided and therefore most toxic substrate but not on
a clear dose-response pattern. Thus, to gain valid results, the
number of replicates and the arrangement of the different
substrates must be adopted. The two-chamber test system is
less time-consuming due to easy handling and test results can be
quantified more easily.
Recommendations and Outlook. In consequence of the better
validity of test results, lower expenses for test containers and
less time for handling, the use of the two-chamber system is
preferred over the six-chamber test system to assess the toxicity
of polluted soil. Because of the ecosystem consequences of behavioural effects and the fact that avoidance response tests can
reveal the toxic potential of pollutants in low concentrations,
such tests should be included into ecotoxicological test protocols.
Keywords: Avoidance response test; crude oil; earthworm ecotoxicology; Eisenia fetida; six-chamber avoidance test; 2,4,6Trinitrotoluene (TNT); two-chamber avoidance test
Introduction
Earthworms are common test organisms in terrestrial ecotoxicology. Both an acute earthworm toxicity test and a reproduction test with the endpoints mortality, reproduction (cocoon production and juvenile hatching) and adult
body weight change are standardised and well described in
guidelines (ISO 11268-1/2). Other endpoints such as behavioural, morphological and physiological changes are reported
occasionally, but they have not been evaluated in a standardised way (Kula 1998). Endpoints of the standardised
earthworm toxicity tests reflect direct effects (lethal and sublethal) of chemicals, whereas behavioural tests focus on indirect effects. The reduction of population size due to mortality or reduced reproduction is an ecological consequence
of exposure to chemicals in soil. However, behavioural
changes such as substrate avoidance can be also ecological
relevant. Emigration of earthworms and the subsequent loss
of their beneficial functions in soil (aeration, drainage, enrichment of organic material, etc.) can lead to a degradation
of soil qualities. Additionally, loss of earthworms from an
area might also affect the numbers and distribution of their
vertebrate predators. Thus, migration of earthworms can
impact an ecosystem. Consequently, tests with behavioural
endpoints should be included in ecotoxicological test batteries in order to assess the toxic impacts of chemicals on
soil ecosystems. While Darwin (1881) showed that earth-
JSS – J Soils & Sediments 3 (2) 79 – 84 (2003)
© ecomed publishers, D-86899 Landsberg, Germany and Ft. Worth/TX • Tokyo • Mumbai • Seoul • Victoria • Paris
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worms select between different foods, recent tests proved
that earthworms respond to chemical stimuli (Slimak 1997,
Mather and Christensen 1998). High numbers of chemoreceptors, concentrated in the prostomium and anterior segments and the distribution of epidermal tubercles and nerve
endings in and around singular body segments contribute to
the capacity of earthworms to react to chemicals in their
environment (Wallwork 1983). The epithelium in the mouth
region accommodates groups of sensory cells, which can be
stimulated by chemical substances associated with taste.
These cells are associated with the selection of food, the detection of unfavourable environmental conditions (e.g. soil
acidity) (Mangold 1953), and the detection of mucus secretions of other earthworms (Edwards and Lofty 1972). This
sensitivity towards chemicals, coupled with their locomotory abilities enable earthworms to avoid adverse habitats
(Stephenson et al. 1998). Avoidance-response tests reflect
these behavioural properties of the earthworms. Principle
of avoidance response tests is the preference or avoidance of
substrates after a specific exposure period. The scope of this
study included (i) a comparison of the results of the behavioural tests (six-chamber avoidance response test) with those
from the standardised earthworm toxicity tests using soils
contaminated with crude oil and 2,4,6-Trinitrotoluene (TNT)
and (ii) a comparison of two different earthworm avoidance response tests. The first avoidance test system was developed by Stephenson et al. (1998) and consists of six compartments per test container. The second test system,
described by Yeardly et al. (1996) and more precisely by
Hund-Rinke and Wiechering (2001), uses test units that are
divided into two equal sections. Both avoidance-response
test systems were compared to identify the advantages and
limitations of the two systems to make a further step into
the direction of a standardisation of behavioural tests.
1
1.1
Material and Methods
Test substrates
Two test substrates with different contaminants were used
in the test systems. To match the standard earthworm toxicity tests (ISO 11268-1/2) soils were sieved (≤5 mm), calcium
carbonate was added to adjust pH to 6.0 ± 0.5 and distilled
water was added to approximate 60% of the maximum
water holding capacity. All tests were performed in climate
chambers at a temperature of 20 ± 2°C. The lights in the
climate chambers were switched off for the entire test duration. Previous tests had shown, that evaporation was always
significantly higher in test containers located near the light
source due to the heat build-up of the lamps.
2,4,6-Trinitrotoluene (TNT) contaminated soil. The soil was
collected from a former ordnance plant 'Werk Tanne' in
Clausthal-Zellerfeld, Germany (Warrelmann et al. 2000). It
was a silty loam soil with a C:N ratio of 18 and a pH of 3.5.
The soils for the toxicity assessment with the acute, reproduction and avoidance tests were taken from five experimental plots
that differed in their TNT concentrations (referring to soil dry
wt.): TNT1 (2 mg/kg), TNT2 (7 mg/kg), TNT3 (29 mg/kg) and
TNT4 (1142 mg/kg). Soil from an uncontaminated plot (TNT0)
at the same site served as control substrate. The standard soil
Lufa 2.2. (Agricultural Research Centre, Speyer, Germany)
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was additionally used as an uncontaminated reference soil. As
the soil was acutely lethal to earthworms at concentrations
>1000 mg/kg, soils with lower test concentrations were used to
compare the two avoidance-response test systems [500 mg/kg
(TNT500) and 280 mg/kg (TNT280)].
Crude oil contaminated soil. The crude oil contaminated soil
came from a former refinery at the harbour of Bremen, Germany. The initial total petroleum hydrocarbon (TPH) concentration (referring to soil dry wt.) was 1074 mg/kg (Oil3).
The standard soil Lufa 2.2. was used as an uncontaminated
control substrate, as no uncontaminated reference soil from
the experimental site was available. In order to assess the toxic
effects of different TPH-concentrations, the contaminated soil
was mixed with Lufa 2.2. in volume concentrations 1:1 (Oil2
= 316 mg/kg) and 1:2 (Oil1 = 200 mg/kg). The Lufa 2.2. soil
was a loamy sand with a C:N ratio of 11 and a pH of 5.4. The
oil-contaminated soil was a silty sand with a pH of 7.05. The
test substrate used to compare the two avoidance-response
tests came from the same site but it had a higher TPH concentration (9900 mg/kg). The uncontaminated control substrate
used for the test unit comparison was Lufa 2.2.
1.2
Test organisms
Ten adult individuals of Eisenia fetida were placed into each
test container. Before and at the end of a test, earthworms
were incubated for 24 h on wet filter paper to empty their
guts. They were then washed, dried superficially and, weighed.
1.3
Avoidance tests
1.3.1 Six-chamber avoidance test
Round plastic containers (Ø 28 cm, 10 cm height) with six
different chambers connected to a central chamber (Ø 6.5 cm)
were used as test containers (see Fig. 1 below). Originally
(Stephenson et al. 1998), each of the six compartments was
connected to adjacent chambers and to the central cylinder by
three arches (1.0 cm wide, 0.5 cm height). A preliminary test
had shown that these few passages, did not enable a satisfying
migration. Earthworms did not seem to sense the neighbouring substrates. To improve migration, the test system was
modified by increasing the number of passage holes (Ø 5 mm)
up to 14, allocated equally alongside the separator. Ten worms
were placed into the soil-free central chamber at test start.
Because of their negative phototactical reaction, the earthworms moved quickly (approximately 5 min) into the soil filled
chambers [400 g moist (60% maximal water holding capacity) soil each]. The compartment entered by each earthworm
was recorded (t0). The arrangement of the different substrates
in test chambers was alternated between replicates to ensure
an optimal distribution. Only one compartment per test unit
contained the uncontaminated reference soil, whereas all other
test chambers were filled with the contaminated soils differing in their TNT respectively TPH contamination. As only
three different oil contaminated substrates were tested, test
units were modified by dividing each test container into four
compartments instead of six compartments by removing two
dividing slides. After all of the test organisms had migrated
into the soil filled chambers, the central chamber was closed
with a wooden buckler. Thus, movement of earthworms was
JSS – J Soils & Sediments 3 (2) 2003
Research Articles
only possible among the soil-filled compartments within a test
unit. To prevent worms from escaping, test containers were
covered with a plastic lid. The air-filled interspace between the
soil and the container-lid enabled a sufficient oxygen supply
during incubation. At the end of the exposure period of 48 h
(t48), each test compartment within a test unit was separated
from its neighbouring chambers by thin plastic dividers (without passages) that prevented further movement of worms among
compartments. The location of the worms in the test units was
determined by removing the soil from each compartment and
recording the number of worms present. Five replicates per test
were applied. Whereas for the toxicity assessment, six different
substrates/concentrations (TNT) respectively four substrates
(Oil) were applied in this test system, only two substrates (contaminated/uncontaminated control) were used for the comparison of the two avoidance response tests.
1.3.2 Two-chamber avoidance test
In this system square plastic containers (20 x 20 x 10 cm)
were filled with test substrates up to a height of 7 cm [1600 g
moist (60% max. water content) substrate in total]. One
section of the test vessel was filled with the uncontaminated
reference soil, separated by a plastic separator from the contaminated test substrate as shown in Fig. 1. After the separator was removed, ten worms were placed on the centre
line on the soil surface. After the worms had entered the soil
Six chamber test unit
(t0), the substrate choice was noted and containers were covered with a plastic lid allowing sufficient aeration. Unhindered migration was possible between the two test substrates.
After the incubation time of 48 h (t48), the two soils within a
test unit were separated with an inserted separator and the
number of worms in each test substrate were sorted, counted
and recorded. Five replicates were run for each test.
1.3.3 Evaluation
Substrate preference of <20% of the test substrate compared
to the uncontaminated control at t48 was assessed as a repellent (toxic) effect in both avoidance test systems. Twenty
percent substrate preference was chosen as the threshold for
toxicity, based on the effect concentration identified from
the standard acute/reproduction tests. Hund-Rinke and Wiechering (2001) also regarded a soil as toxic at an avoidance
behaviour of >80%.
1.4
Acute and reproduction tests
The acute test was performed according to ISO guideline
11268-1, the reproduction test according to ISO 11268-2.
Effects were assessed to be toxic when mortality in the test
soil was >20% according to Kreysa and Wiesner (1995),
respectively when the reproduction rate (cocoon production
and juvenile hatching) in the test soil was <20% compared
to the uncontaminated control treatment.
Dual test unit
1.5
10 worms
Chemical analyses and soil parameters
10 worms
10 cm
10 cm
TNT concentrations in soil samples were determined according to EPA-Method 8330: Nitroaromatics and Nitroamines by HPLC.
28 cm
Contaminated
Control
soil
soil
20 cm
Incubation 48 h
Incubation 48 h
free migration possible
Total petroleum hydrocarbon (TPH) measured by gas chromatography (GC) appears promising as an analytical indicator of acute toxic effects to earthworms in soils containing petroleum hydrocarbons (Saterbak et al. 1999). Soil was
extracted with hexane/acetone (1:1) and TPH concentration
analysed by GC.
Soil parameters were determined according to the following
guidelines: maximum water content: ISO 11274, and soil
pH (CaCl2): DIN 19684. The C:N ratio was determined in
a Leco® C-N analyser.
free migration possible
1.6
6 divider to
stop worms
from further
migration
Extraction of earthworms by handsorting
divider stops further migration
Extraction of earthwoms by hand sorting
Fig. 1: Principles of the two different avoidance response tests [six chamber test unit according to Stephenson et al. (1998), slightly modified, and
dual test unit according to Hund-Rinke and Wiechering (2001)]. Further
explanation see text
Statistical methods
Results in percent (%) substrate preference were shown to
give an impression of the effect level compared to the uncontaminated control substrates. Results were tested for their
normal distribution (Kolmogorov-Smirnov-Test) and homogeneity of variances (Levene-Test). One-way ANOVA procedures were used to assess the effects of TNT and oil contamination on the test endpoints mortality, reproduction
variables and substrate preference. An adverse effect was
significant at p = 0.05. For post-hoc comparison of means,
Scheffés test was applied. Statistical analyses were performed
with SPSS software (SPSS 9.0 for Windows; SPSS Inc, Chicago, Illinois USA.).
81
Results
2.1
Avoidance-response test (six-/ four-chamber test)
versus standard tests (ISO 11268-1/2)
Oil-contaminated soil with a total petroleum hydrocarbon
(TPH) concentration up to 1074 mg/kg was not acutely toxic
(lethal) to E. fetida, whereas the reproduction test and the
avoidance-response test revealed significant toxic effects at
this concentration. Oil3 was clearly avoided as only 16% of
all individuals were found in this substrate at t48. Oil had
also a significant effect on reproduction (ANOVA p <0.01,
F = 8.489). Significant adverse effects (p <0.05) occurred in
earthworms exposed to 200 mg/kg TPH (Oil1). A concentration-responding decrease of reproduction in the oil polluted substrates was observed (Table 1).
The assessment of the TNT-contaminated soils showed only a
significant acute (lethal) toxic effect at TNT4 (ANOVA: F =
27.15; p <0.001). All worms in this treatment were dead after
2 days and even avoided to migrate into the test substrate.
TNT4 was therefore classified as highly toxic. Cocoons and
hatched juveniles were only found in the uncontaminated controls Lufa 2.2, TNT0 and in TNT1 (2 mg/kg). Due to the general low reproduction rates (even in the uncontaminated reference soils) the absence of reproduction in the TNT2–4
treatments were interpreted as a toxic tendency. ANOVA revealed a significant effect of treatment at the six-chamber avoidance response test (F = 3.96; p <0.01). Comparing the data,
significant differences between TNT4 (p <0.01) and TNT3
(p <0.05) compared to the control TNT0 emerged. Only 10%
of all individuals entered the TNT2 treatment, which suggests
that the earthworms were also avoiding soils with this level of
TNT concentration. Lufa 2.2, TNT0 and TNT1 showed no
toxic effects (Table 1). To summarise: 1.) acute (lethal) toxic
effects occurred only in the soil with the highest TNT concentration, whereas the reproduction test and the avoidance
response test revealed effects at much lower concentrations.
2.) The reproduction test showed a clear dose response pattern for the oil contamination. 3.) The sensitivities of the
avoidance test and reproduction test were similar concerning the TNT contaminated soil; effect thresholds occurred
in both the reproduction and avoidance behaviour tests in
soils with concentrations greater than 7mg/kg TNT.
2.2
Six chamber versus two-chamber avoidance response tests
2.2.1 Crude oil contaminated soil
The distribution of earthworms in test soils was equal in
both test systems at test start (t0). Both avoidance tests revealed significant substrate avoidance (≥90%) of the contaminated soil at the end of incubation (t48). The oil-contaminated soil was therefore classified as clearly toxic. 96%
of all worms preferred the reference soil in the six-chamber
test system, 90% migrated into the Lufa at the two-chamber avoidance response test (Fig. 2). No significant difference was observed between the results of the two avoidance-response test systems.
00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00
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Lufa t0
Oil t0
Lufa t48
100
90
Substrate preference (%)
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10
0
00 00 00 00
Six Chamber Unit
00 0 0 0
0 00 00 00 Dual Chamber Unit
0 00 00 00 00 00 00 00 00 00
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Oil t48
Fig. 2: Substrate preference [% of total numbers (n = 50 worms) + SE] at
test start (t0) and test end (t48) of the six chamber and dual chamber avoidance tests. Oil substrate: 9900 mg/kg TPH, silty sand; Lufa 2.2: loamy sand
Table 1: Mortality [% of dead animals after 14d related to numbers of individuals at test start t0], reproduction [total numbers of cocoons (28d) and hatched
juveniles (56d)] and substrate preference [% worms found in each substrate after 48h] in dependence of Total Petroleum Hydrocarbon (TPH) and 2,4,6Trinitrotoluene (TNT) concentration. Results were obtained from five replicates (n = 50 worms) for the avoidance response test and three replicates (n = 30
worms) for acute and reproduction tests
Pollutant
Acute Test
TNT (mg/kg)
Mortality (%)
Cocoons
Juveniles
Preference (%)
Lufa 2.2.
0
7
2
4
20
TNT 0
0
0
5
12
38
TNT 1
2
16
1
3
30
TNT 2
7
20
0
0
10
TNT 3
29
23
0
0
2
TNT 4
1142
100
0
0
0
TPH (mg/kg)
Mortality (%)
Cocoons
Juveniles
Preference (%)
0
0
104
163
30
Oil 1
200
0
47
71
27
Oil 2
316
0
38
54
27
Oil 3
1074
6
20
35
16
Substrates
Substrates
Lufa 2.2.
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Avoidance Test
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2.2.2 TNT-contaminated soil
Both avoidance-response test systems showed also comparable results when assessing the TNT contaminated soil. TNT
concentrations of 500 mg/kg (TNT500) and 280 mg/kg
(TNT280) resulted in substrate avoidance >90% in both test
systems (Fig. 3 and 4). Whereas 54 – 62% of all individuals
migrated into the contaminated soils at test start (t0),
substrate avoidance had increased at t48 to 90% (six-chamber unit) and 92% (two-chamber unit) for TNT500 and
ranged between 92% (six-chamber unit) and 96% (twochamber unit) for TNT280.
100
90
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0
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t0
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TNT500
t0
TNT0
t48
TNT500
t48
00 00 00 00 Six Chamber Unit
00 00 00 00
Dual Chamber Unit
test start (t0) and test end (t48) of the six chamber and dual chamber avoidance test. TNT500 = concentration of 500 mg/kg TNT
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t0
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TNT280
t0
TNT0
t48
TNT280
t48
00 00 00 00
Six Chamber Unit
00 00 00 00
Dual Chamber Unit
test start (t0) and test end (t48) of the six chamber and dual chamber avoidance test. TNT280 = concentration of 280 mg/kg TNT
3
Discussion
The ability of the various earthworm toxicity tests (acute,
chronic, avoidance response) used in this study to detect an
adverse or toxic response differed for the types of pollutants
(TNT versus crude oil). The highest concentration (TPH:
1074 mg/kg) of the oil-contaminated soil resulted in no
mortality, but it clearly reduced reproduction and substrate
preference. Significant lethal effects (100% mortality) occurred in earthworms exposed to the highest TNT concentration of 1142 mg/kg. The results of the reproduction test
were limited to indications of a toxic effect of TNT (because
all adults had died in TNT4). Renoux et al. (2000) noted that
earthworms stayed on the soil surface instead of burrowing
into the TNT contaminated soil. This phenomenon was also
observed in this study in the acute test treatment with the highest TNT concentration and was interpreted as a clear behavioural response. The results of the avoidance response test
showed significant toxic effects at 29 mg/kg TNT, which was
lower than the NOEC of 55 mg/kg identified from the reproduction test (Robidoux et al. 2000). Comparison of all tests
showed that the endpoint mortality required the highest effect
concentration. This endpoint indicates maximum damage of
an organism, and consequently a relatively high concentration of the pollutant is necessary to cause an effect (HundRinke and Wiechering 2001). Therefore acute toxicity provides relatively little information on actual effects on a
population in the natural environment (Slimak 1997). Acute
bioassays may be very useful for a first rapid screening of highly
polluted soils, but bioassays using sublethal endpoints are required for a more accurate assessment of the long-term ecological risks of polluted soils (Van Gestel et al. 2001). Endpoints
of chronic (reproduction) or behavioural (substrate avoidance)
tests are thus more likely to detect an adverse effect in response
to lower exposure concentrations. Saterbak et al. (1999) observed concentration response patterns more often for the reproduction endpoints than for survival. This was clearly confirmed by the reproduction data of the oil contaminated soil in
this study. Organisms can exhibit behavioural responses at levels of chemical stress lower than those that can be identified
from the acute test, and possibly also from sublethal toxicity
tests (Yeardly et al. 1996). This was observed in the avoidance
response test assessing the TNT contaminated soil. Substrate
avoidance and reproduction are clearly more sensitive than mortality (Table 2). Another advantage of the avoidance response
test is that results can be obtained already after 48 h, compared
to the time-consuming acute (14 d) and reproduction tests (cocoon production after 28 d, juvenile hatching after 56 d) with
their long incubation periods. Yeardly et al. (1996) proved that
48 h are sufficient to measure an avoidance response.
Table 2: Evaluation of the applied test systems towards the test endpoints
mortality (acute test), cocoon production/juvenile hatching (reproduction
test) and substrate avoidance (six chamber avoidance response test). Test
sensitivity: 1 = high, 2 = medium, 3 = low test sensitivity
Acute
Test
Reproduction
Test
Avoidance
Test
Crude Oil
3
1
2
TNT
3
2
1
Test substrates
Both avoidance response tests showed similar results, when
compared. The six-chamber avoidance response test allows
the testing of up to six different substrates/concentrations
simultaneously. This advantage compared to the two-chamber test system has to be put into perspective. It was observed that worms made preliminary selections on substrates,
as they often explored the different test units with their anterior end before they finally decided which substrate to enter.
Despite this pre-selection, the comparison between the distribution of individuals at test start and end of the six-chamber test system suggested that orientation of worms only
occurred to directly neighbouring compartments, but never
to all chambers of the test container. The closed central cylinder did not allow any shortcuts and therefore orientation
to opposite compartments could not occur. However, an
earlier experiment with an open central cylinder during incubation did not enhance the migration through it, as worms
avoided this soil-free passage. If the substrate of the initially
chosen test compartment was unfavourable, the worms might
83
then move to one of two neighbouring substrates, selecting
the most appropriate of these two soils. More suitable
substrates, which might be positioned in test chambers beyond the two neighbouring compartments might be ignored.
Results therefore give only reliable information about the most
avoided and therefore most toxic substrate, but they fail to
differentiate between the soils with lower levels of contamination. In consequence, if more than one contaminated substrate
(test concentration) is to be assessed, the number of replicates
and the arrangements of the different test substrates/ concentrations in each replicate has to be adjusted to ensure reliable
results. This would increase the effort expended to complete a
test. The two-chamber system enables only the assessment of
one soil compared to the control at one time. If more than one
substrate or concentration has to be tested, a high number of
test runs are necessary. Compared to the six-chamber test system the handling of this system is easier and time-saved. Both
TNT and crude oil contaminated soils showed no significant
differences between both test systems when two soils (test soil
+ control) were applied. The avoidance of the oil contaminated substrate was slightly lower in the two-chamber system. The lack of a separator between test substrates during
incubation in this system eases the movement of earthworms
from one soil into the other. Altogether, the two-chamber test
system is more useful in the assessment of one test substrate (+
control) due to its quick and uncomplicated handling.
4
Conclusions
Not only direct, but also indirect effects (expressed e.g. in
behavioural endpoints) of chemicals on earthworms can have
great consequences on soil ecosystems. Avoidance response
tests can produce results that indicate lower threshold effect
concentrations than either the standard acute or the reproduction test. The assessment of the oil contaminated soil
showed that the reproduction test results can be more sensitive than substrate avoidance (which was assessed by applying the six-chamber test system). The six-chamber avoidance response test showed similar results compared to the
two-chamber test unit, when only two different substrates
were tested in both test systems. As orientation of earthworms seemed to take place only between directly adjacent
chambers, the validity of test results becomes questionable
when applying six different soils at one time in the six-chamber test system. To overcome this shortcoming, an adopted
arrangement of soils in the test chambers is required, which
leads to an increase of the experimental expenditure. Compared to the six-chamber test system the two chamber test is
less expensive both to purchase and to operate (e.g. handling
time and effort) and can be more easily standardised.
5
Recommendations and Outlook
In consequence of the better reliability of test results, lower costs
of the test unit, and less time for handling, the use of the twochamber system is preferred to the six-chamber test system in
order to assess the toxicity of polluted soil. Because of the ecosystem consequences of behavioural effects and the fact that
avoidance-response tests can reveal the 'toxic potential' of pollutants in soil at low concentrations, this test should be included
in the available battery of ecotoxicological test protocols.
84
Research Articles
Acknowledgements. I like to thank Peter Behrend (Dept. Bioorganic
Chemistry, University of Bremen) for carrying out the chemical analyses, Juliane Filser, Andrea Ruf and Jürgen Warrelmann for their helpful
scientific advice and comments on the manuscript and Karen MorrowLitke for the linguistical review.
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Received: November 18th, 2002
Accepted: January 31st, 2003
OnlineFirst: February 1st, 2003

Behavioural Endpoints in Earthworm Ecotoxicology

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