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M.
Schaefer,
Department of Ecology, Centre for Environmental Research and
Technology (UFT), University of Bremen, Germany.
Soil ecotoxicological tests record the effect
of chemicals on soil. To evaluate exemplarily the toxic
potential of petroleum contaminations for soil fauna,
different earthworm toxicity tests were performed. Earthworms
are often used as test organisms because of their important
function e.g. as decomposer (Edwards & Bohlen, 1989), their
high biomass in soil [10-200 g fresh weight m-2 (Dunger
& Fiedler, 1989)] and their sensitive reactions towards
environmental influences. Dorn & Salanitro (2000) confirmed
that earthworms are sensitive indicators for oily soil. The
“OECD-Earthworm Toxicity Test” (ISO
11268-1/2)
is a widely used terrestrial ecotoxicological test which is
applied both in prospective as well as increasingly in
retrospective ecotoxicological research. Test organism of this
standardised test is the “compost worm” Eisenia fetida.
Tests with different sensitive test endpoints were performed:
the acute (ISO 11268-1) and reproduction test (ISO 11268-2) as
well as an avoidance response test. Test parameters were
mortality, reproduction, biomass and substrate preference.
Test substrate was a crude oil contaminated soil, which was
taken from a former oil refinery site at the harbour of
Bremen/Germany, where currently a soil remediation project is
taking place.
Aim of this study was to assess the toxicity of
oil contaminations with earthworm toxicity tests, operating on
different sensitive scales and to analyse the significance of
earthworms for the degradation of oil contaminations in soil.
Material and
Methods
Test species was the compost worm Eisenia
fetida. The adult worms, which came from a synchronised
culture, were at least 2 months old and weighed between 300 –
600 mg. Before their biomass was determined at test start and
test end, they were washed with tap water and incubated on wet
paper for 24h to empty their guts.
Test substrate was a crude oil contaminated
soil with total hydrocarbon concentration of 1074 mg/kg (®
“Oil.3”). Uncontaminated control substrate was the standard
soil Lufa 2.2. from the Agrarian Research Centre; Speyer/
Germany. In order to attenuate the hydrocarbon concentration,
the contaminated soil was mixed with Lufa 2.2. in volume
concentrations 1:1 (316 mg/kg ® “Oil.2”) and 1:2 (200 mg/kg ®
“Oil.1”).
To ensure constant test conditions, test
substrates were sieved (≤ 5 mm), calcium carbonate was added
to adjust pH to 6,0 ± 0,5 and distilled water was added to
reach 60% of the maximum water holding capacity. Water content
was readjusted weekly. All test were performed in climate
chambers at a temperature of 20 °C.
1l glass cylinders serving as test containers
in the acute and reproduction test were covered with plastic
foil to prevent worms from escaping. An integrated gauze (Ø
1mm) ensured optimal ventilation. Round plastic containers (Ø
28 cm, 10 cm height) with six different chambers connected to
a central chamber were used as test containers in the
avoidance response test. Slides with 10 holes (Ø 5 mm), which
enabled worms to migrate through chambers, divided each
chamber from neighbouring units. Test containers were covered
with a plastic lid, a wooden buckler closed the central
chamber during incubation time to prevent worms to migrate
through it.
Acute Test (ISO 11268-1)
10 adult worms were placed in each test container filled with
test substrate. After 14 days of incubation surviving worms
were sorted out by hand. Test endpoint was mortality. Control
variants without worms were applied in order to compare the
influence of earthworms on the degradation of hydrocarbons.
Reproduction Test (ISO 11268-2)
The reproduction test was set up like the acute test, but
because of longer incubation times additional food (5g ground
cattle dung/ per container) was given. Test endpoints were
cocoon production after 28 d and numbers of hatched juveniles
after 56 d. Therefore surviving worms were sorted out by hand
after 28 d, and produced cocoons further incubated for another
28d (total incubation time: 56d).
Avoidance Response Test
(according to Stephenson et al. 1998)
10 adult worms were placed into the soil free central chamber
of each container. After they had migrated into the
neighbouring soil filled chambers, the central chamber was
closed. Therefore free migration was only possible between the
chambers. After an incubation of 48 h, slides (without holes)
placed between the different chambers, stopped worms from
further migration. Worms were sorted out by hand from each
chamber. Test endpoint was substrate preference.
Three replicates were applied for each variant in each test.
Chemical analyses
Total hydrocarbon content was analysed by gas
chromatography (GC). Soil was extracted with Hexane- Acetone
(1:1).
Statistical analyses were performed with SPSS.
Results
Acute Test
No significant acute effect could be observed after an
incubation of 14 d. Highest mortality was found in Oil.3
(highest contamination), but did not exceed more than 6% and
therefore the substrates could be assessed as non toxic for
earthworms. In Oil.1; Oil.2 and Lufa 2.2. all individuals had
survived.
Reproduction Test
Compared to the acute test, the
reproduction test showed a concentration-responding decrease
of reproduction in the oil polluted substrates. Whereas worms
in Lufa 2.2. produced a total number of 104 cocoons after 28
d incubation, only 20 cocoons were found in Oil.3. As the
hatching rate of juveniles is related to the number of
cocoons, the same effect was observed after an incubation of
56 d. 163 juveniles had hatched in Lufa 2.2. whereas only 35
juveniles could be counted in Oil.3. Oil.1 and Oil.2 showed
also decreased numbers of cocoons and juveniles (Fig. 1). As a
substrate is assessed to be toxic at a decrease of
reproduction
³
20 % compared to the uncontaminated control, the heavily
contaminated Oil.3 has to be assessed as toxic.
Biomass
Worms in Oil.2 and Oil.3 had lost
up to 10% of their biomass at test end, whereas individuals in
Oil.1 and the control substrate Lufa 2.2. increased their
biomass up to 20%.
Avoidance Response
Test
Only Oil.3 showed a negative
effect, because only 16% of total numbers of applied worms
were extracted at test end in this substrate. For the
assessment, a toxicity criterion of 80% avoidance is proposed
(Hund-Rinke & Wiechering, 2001). Lufa 2.2., Oil.1 and Oil.2
showed no significant effects.
Changes of
hydrocarbon concentration by influence of earthworms
Earthworms influenced the
degradation of total hydrocarbon content in the polluted soil
positively. The highest concentration at test start of 1074
mg/kg (Oil.3) was reduced to 96 mg/kg after an incubation of
56 d, whereas the contaminated control without worms with a
start concentration of 812 mg/kg showed no reduction at all at
test end (Fig. 2). Hydrocarbon concentrations of the different
test substrates in variants with worms were reduced between
62,5% up to 91% compared to initial start concentration.
Linear regressions were calculated for the different test
variants. No significant degradation of hydrocarbons were
found in Lufa 2.2. and the two control variants without worms
(r2 < 0,4). The contaminated variants with worms
showed linear regressions which indicate a continuos decrease
of hydrocarbons in time (Oil.1: r2= 0,73; Oil.2: r2=
0,92; Oil.3: r2=0,90). The decontamination rate was
related to start concentration, degradation was fastest at
Oil.3 with the highest hydrocarbon concentration (B= -17,8)
compared to Oil.2 (B= -6,3) and Oil.1 (B= -4,3).
Discussion
The German
Soil Protection Law (1998) instructs a remediation of oil
contaminated soil at 1g/kg. The results of this study showed
that crude oil in this concentration did not have any lethal
effect on earthworms. The toxic potential of hydrocarbons is
based on the ability to bind at the apolar regions in
biogeneous membranes and to disorganise these (Kraß et al.
2000). Aromatic fractions in light oils may be responsible for
the acute toxicity in soils. Dorn et al. (2000) confirmed that
only low to no acute effects of heavy oil in high organic
carbon soils indicate that these soils pose little impact to
populations of soil-dwelling organisms, e.g. earthworms. As
crude oil is characterised as a heavy oil, the low mortality
in this test can be explained. Saterbak et al. (1999) observed
concentration response patterns more often for the
reproduction endpoints than for survival. These observations
are confirmed in this study. Whereas no acute effect could be
registered, a clear dose response pattern could be seen in the
reproduction test. The test organisms responded to the oil
contamination by a decrease of cocoon production and therefore
reduced numbers of hatched juveniles. The avoidance response
test showed also a toxic effect, but only at the highest
concentration. Earthworms are able to avoid unfavourable
surroundings because of their high numbers of chemo-receptors
located in the prostomium, which let them react sensitively
towards chemical influences (Wallwork 1983). The advantage of
the avoidance response test is to reveal results already after
48h, compared to the long test duration of the acute and
reproduction test.
Chemical
analyses showed the reduction of the total hydrocarbon
concentration during the test duration in variants with
earthworms, whereas in controls without worms no significant
reduction was observed. The positive influence of earthworms
on the reduction of oil pollution in soil can be explained by
a better aeration of the soil due to digging activities [as
according to Hellmann (1995) the degradation of hydrocarbons
is always bond to bacterial available oxygen] and the
enhancement of microbial activity. Earthworms have a complex
interrelationship with micro-organisms and promote microbial
biomass and activity in soil in decaying organic matter by
fragmenting it and inoculating it with micro-organisms, and
they disperse micro-organisms widely through soils (Edwards &
Bohlen, 1996). Ubiquist micro-organisms are able to oxidise
alkanes in three steps into carboxylic acid. Acids produced in
the last oxidising steps are identical to naturally occurring
fatty acids. Therefore earthworms can be seen as promotors in
terms of bioremediation of oil contamination.
Conclusions
Earthworms
are useful test organisms to assess the toxicity of oil
contaminated soils, because of their sensitive reactions
towards the pollutant.
The
application of earthworm toxicity tests differing in their
sensitivity revealed different answers depending on the
method. The different test answers lead once more to the
conclusion that the application of methods operating on
different scales is inevitable. While high hydrocarbon
concentrations are not necessarily lethal, they can cause a
decrease of reproduction rates (numbers of produced cocoons
and hatched juveniles) and induce substrate avoidance.
As Earthworms are known to increase the
metabolic activity of soil micro-organisms, this stimulation
may lead to an enhanced degradation of the mineral oil
compounds. Consequently earthworms should be considered as
additional organisms to support bioremediation.
Acknowledgements
Peter
Behrend (Dept. Bioorganic chemistry, UFT, Bremen) carried out
the chemical analysis.
References
Dorn P.B.,
J.P. Salanitro (2000): Temporal ecological assessment of oil
contaminated soils before and after bioremediation;
Chemosphere 40; pp. 419-426.
Dunger W.;
H.J. Fiedler (1989): Methoden der Bodenbiologie. Gustav
Fischer Verlag, Stuttgart, New York.
Edwards C.
A., P. J. Bohlen (1996): Biology and Ecology of Earthworms,
Chapman & Hall Ltd., London, New York.
Hellmann H.
(1995): Umweltanalytik von Kohlenwasserstoffen; VCH
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Hund-Rinke K.,
H. Wiechering (2001): Earthworm Avoidance Test for Soil
Assessment; Journal of Soils and Sediments, Vol. 1, No. 1, pp.
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International Standard ISO 11268-2
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