Biol Fertil Soils (2002) 36:72–78
DOI 10.1007/s00374-002-0507-z
O R I G I N A L PA P E R
Jiemin Cheng · Ming H. Wong
Effects of earthworms on Zn fractionation in soils
Received: 9 November 2001 / Accepted: 16 May 2002 / Published online: 4 July 2002
© Springer-Verlag 2002
Abstract Laboratory incubation experiments were con-
ducted to examine the effect of earthworm (Pheretima
sp.) activity on soil pH, zinc (Zn) fractionation and N
mineralization in three soils. No Zn uptake by earth-
worms was observed. Zinc addition decreased pH of red
soil (soil 1) and hydragric paddy soil (soil 3) by 0.5 and
0.2 unit, respectively, but had no effect on alluvial soil
(soil 2). The effect of Zn on soil pH was possibly due to
a specific adsorption mechanism between Zn and oxides.
Earthworm activity significantly decreased the pH of the
red soil, a key factor affecting Zn solubility, but not of
the other two soils. Earthworm activity significantly in-
creased DTPA-Zn (DTPA-extractable) and OxFe-Zn
(NH2OH-HCl-extractable) in the red soil, but had little
effect on other fractions. In the alluvial soil, earthworm
activity significantly increased OxFe-Zn but decreased
organic-Zn (organic-associated Zn). In the hydragric
paddy soil, earthworm activity significantly increased
MgCl2-Zn (MgCl2-extractable) and organic-Zn. The lev-
el of CaCl2-extractable Zn in all three soils was not af-
fected by earthworm activity. Nitrogen mineralized as a
result of earthworm activity was equivalent to 110, 120
and 30 kg N ha–1 in soils 1, 2 and 3, respectively. Zinc
added at rates less than 400 mg Zn kg–1 did not seem to
affect the activity of N-mineralizing microorganisms.
The present results indicated the possibility of increasing
the metal bioavailability of relatively low level metal-
contaminated soils, with a higher organic matter content,
by earthworm inoculation.
Keywords Earthworm · Zinc fractionation · Nitrogen
mineralization
J. Cheng
College of Resources and Environmental Science,
Nanjing Agricultural University, Nanjing 210095, China
M.H. Wong (✉)
Institute for Natural Resources and Environmental Management,
and Department of Biology, Hong Kong Baptist University,
Kowloon Tong, Hong Kong, China
e-mail: mhwong@hkbu.edu.hk
Tel.: +86-852-34117050, Fax: +86-852-34115995
Introduction
Beneficial roles of earthworms on soil fertility, nutrient
cycling and plant growth have been commonly observed.
Earthworm activity improves soil physical conditions by
burrowing and casting, with the formation of a channel
system and stabilized aggregates that allow easy penetra-
tion of water and air (Anderson 1988; Zhang and
Schrader 1993). Earthworms ingest organic debris with
various C:N ratios, convert it into earthworm tissue with
a low C:N ratio (Moody et al. 1995), excrete N-rich ma-
terials (Lee 1985) and consequently enhance nutrient
(particularly C and N) cycling. They indirectly shape soil
C and N cycling through earthworm-microorganism in-
teractions (Winding et al. 1997). Earthworm activity
modifies soil pH (Lee 1985), a key chemical factor af-
fecting bioavailability of nutrient elements and heavy
metals in soils. Plants growing in soils containing earth-
worms showed better translocation of nutrients (e.g. N
and P) from root to shoot than soils without earthworms
(Hu et al. 1998). Consequently better growth and in-
creases in the phytoremediation efficiency of plants, es-
pecially hyper-accumulators, could be achieved by en-
hanced earthworm activity in soils contaminated by
heavy metals.
The response of earthworms to heavy metals is re-
ported to be species-dependent. For most species, in gen-
eral, heavy metals are toxic, and negatively affect their
population density (Pizl and Josens 1995) and reproduc-
tion (Spurgeon et al. 1994). Certain species such as
Eisenia fetida are sensitive to Cd and other chemicals
and therefore can be used as bioindicators of soil toxicity
(van Gestel and van Dis 1988). Other species, however,
may tolerate and even preferentially accumulate certain
heavy metals in their tissues (Morgan et al. 1992; Niu
and Cui 1997).
Plants such as Brassica juncea and Thlaspi rotundifo-
lium, capable of accumulating large amounts of heavy
metals, are called hyper-accumulators. Continued culti-
vation and harvest of these plants could eventually de-
contaminate metal-contaminated soils. (Blaylock et al.

1997; Reeves and Brooks 1983). Restricted heavy metal
bioavailability in metal-contaminated soils is often a ma-
jor limitation when using these plants for phytoremedia-
tion. Heavy metals, once in the soil, may become non-
soluble or unavailable to plants through precipitation,
fixation and other reactions with organic and inorganic
soil components. The proportion of soluble to non-solu-
ble metals may depend on the origins of the metal con-
taminant, its residence time, total metal contents and soil
conditions such as pH (Young et al. 2000). Enhanced
availability of metals to plants may be important to the
success of phytoremediation.
Chelating agents have been used to increase heavy
metal solubility in soils and hence increase metal accu-
mulation in plants (Blaylock et al. 1997; Huang et al.
1997). Large scale application of this technology, how-
ever, might be limited by the high cost.
The major objective of the present study is to evaluate
the feasibility of using earthworms to modify the metal
availability of Zn-contaminated soils. Zinc is a common
soil contaminant and its removal is needed in the biore-
mediation of metal-contaminated soils.
Materials and methods
Collection of soil samples and preparation
Plough layers (0–20 cm) of a red soil, alluvial soil and a hydragric
paddy soil were sampled from Yintan (Jiangxi Province), Rugao
(Jiangsu Province) and Wuxian (Jiangsu Province; soils 1, 2, 3 re-
spectively). The soil samples were air-dried and sieved (2 mm
mesh) and stored at 20°C before use.
Experimental design and procedures
Each of the three air-dried soils was divided into five equal parts
of 3.0 kg each and amended to contain 0, 100, 200, 300 and
400 mg Zn kg–1 respectively by adding appropriate concentrations
of ZnCl2. Distilled water was added to the soils to achieve a mois-
ture content of 70%. To ensure even distribution of Zn, soils were
frequently mixed while adding ZnCl2 and water. The soils were in-
cubated at room temperature (at about 20 C) for 2 months allow-
ing Zn to distribute into various fractions and to be stabilized.
During this period, soil moisture content was carefully monitored.
The concentrations of Zn (extracted by CaCl2) were tested on day
40, 50 and 60. It was assumed that Zn differentiation was complet-
ed when CaCl2-extractable Zn remained unchanged.
At the end of the incubation period, Zn-treated samples were
separated into six sub-samples (0.5 kg soil each placed in a pot of
height 15 cm and diameter 10 cm). Three pots each received three
earthworms of Pheretima sp. (two species of earthworms were
collected from a vegetable farm in Fang Ling, Hong Kong and
they were sent to Prof. J.Y. Xu for identification; one was identi-
fied as Pheretima guillelmi, but positive identification of the sec-
ond one, a more hardy species, used in this experiment has not yet
been achieved; in the present paper it is referred to as Pheretima
sp.), 0.6 g fresh weight, and 6 cm in length each, collected from an
uncontaminated soil from the Nanjing Agricultural University,
while three pots did not contain any earthworms. The pots con-
taining earthworms were covered with a polyethylene net. They
were placed in an air-conditioned room at 20 C for 40 days. Soil
moisture content was carefully controlled.
After 40 days, earthworms were picked by hand, washed, then
blotted with filter papers to remove adhering water, and their fresh
73
weight determined. Mortality, if any, was recorded. The washed
earthworms were put on wet filter papers for 2 days to empty in-
gested materials. The earthworms were then heated at 100–110°C
for 5 min and further dried at 60°C for 4 h, weighed, and analyzed
for Zn. Soil sub-samples were air-dried and ground to pass
through a 1-mm sieve.
Analytical methods
The air-dried soil samples were tested for pH (using a pH meter,
10 g soil in 10 ml 0.01 M CaCl2), organic matter (wet combustion
method, using H2SO4-K2Cr2O7), total N (Kjeldahl digestion meth-
od), total and available phosphate P (colorimetric method, using
HClO4-H2SO4 and CoNa2CO3 extraction respectively), available
K (ammonium acetate method), CaCl2-extractable Zn [extracted
with 0.01 M CaCl2 with a solution:soil ratio of 5:1, followed by
atomic absorption spectrophotometry (AAS)] and total Zn (digest-
ed with HCl-HNO3-HClO4, followed by AAS) according to the
methods described in Sparks et al. (1996). Zinc contents of earth-
worms were also determined using AAS after digestion with
HNO3-HClO4.
The distribution of Zn in various fractions in the soils at the
end of the incubation period was also analyzed. These fractions in-
cluded CaCl2-extractable Zn (same method described above), and
DTPA-extractable Zn (extracted with DTPA, pH 7.3, with a solu-
tion:soil ratio of 2:1). In addition, other fractions were also tested
using the sequential extraction procedures described by Tessier et
al. (1979), with modifications: (1) exchangeable Zn–MgCl2-Zn:
extracted with 1 M MgCl2 (pH 7.0); (2) Zn bound to carbon-
ates–NaOAc-Zn; extracted with 1 M sodium acetate (adjusted to
pH 5.0 using acetic acid); (3) Zn bound to iron and manganese ox-
ides–OxFe-Zn: extracted with 0.04 M NH2OH-HCl in 25% (v/v)
HOAc (pH 2.0); (4) Zn bound to organic matter – Organic-Zn: by
oxidizing the organic matter with 30% H2O2 (pH 2.0) followed by
MgCl2 extraction. Zn brought into solution was measured by
AAS.
Nitrogen mineralization was evaluated by comparing mineral-
N contents in soils before and after earthworm introduction. Min-
eral-N was extracted with 1 M KCl. Ammonium in the extracts
was measured by the indophenol blue method (Sparks et al. 1996).
Statistical analyses
Duncan's multiple range test was used to determine differences in
earthworm fresh weight among different Zn treatments, and the
ANOVA test was used to determine differences in mineralized N
contents after 40 days' incubation between treatments with and
without earthworms.
Results
General properties of soils
Table 1 lists the general properties of the three soils. Soil
3 had the highest contents of organic matter, total N, and
total and extractable Zn. Soil 1 was the most acidic, and
had the lowest contents of organic matter, total N, total
and available P, and extractable Zn. Soil 2 had a near
neutral pH, moderate levels of most of the components
tested, but contained the highest levels of total and avail-
able P.