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

Cheng Jiemin1,Ming H Wong2
1. College of Population, Resources and Environment, Shandong Normal University, Jinan Shandong 21001, China;
2.Croucher Institute for Environmental Sciences, Department of Biology, Hong Kong Baptist University,
Hong Kong, China
: The objective of this study was to investigate the ef-
fects of earthworm density on the availability of nutrients and
heavy metals in metal contaminated soils. Pb/Zn mine tailings
were mixed throughly with a red yellow podzolic soil at the ratio
(w/w) of 75:25. Earthworms (Pheretima sp.) were introduced to
the mixture at four different densities, zero, three, six and nine
individuals per pot planted with ryegrass (Loliun multiflorum).
The results indicated that earthworm activity significantly en-
hanced ryegrass shoot biomass. However, as denser earthworm
population was introduced, shoot biomass tended to decrease.
Earthworm activity significantly increased soil pH and availability
of N, P and K in the tailings and soil mixture. There was a general
tendency that uptake of Zn by ryegrass increased after earthworm
inoculation, although the increase in extractable Zn in tailings and
soil mixture was not significant. On the contrary, there seemed to
be a lower uptake of Pb by ryegrass under earthworm inoclation,
despite the fact that higher extractable Pb concentrations were
observed. The present project indicated that the improved growth
of ryegrass was due to improved nutrient availability and other
soil conditions, by inoculation of earthworms at an appropriate
rate. Further studies are needed to illustrate the relationship be-
tween metal availability and earthworm activity in the field.
 : lead, zinc, nitrogen, phosphorus, phytostabilization,
Pb/Zn mine tailings
  
Earthworms play an important role in adding soil or-
ganic matter, enhancing nutrient cycling, and improving
physio-chemical conditions of soil, thus subsequently
promoting plant growth (Anderson, 1988; Parmelee et al,
1990; Zhang and Schrader, 1993). Earthworms ingest plant
litter and other organic detritus from the soil surface to-
gether with lager quantities of inorganic soil constituents.
The organic portions are decomposed once passed through
their digestive system and the casts produced are rich in
Corresponding author: Cheng Jeimin (mcheng2002@hotmail.com)
Chinese Journal
Chinese Journal of Population, Resources
nutrients for plant (Lavell et al, 1983; Parmelee et al, 1990).
The physical effects of earthworms on soils result from
burrowing and casting, which provide channel systems and
stabilized aggregates to allow easy penetration of water and
air, are extremely important for root growth (Anderson,
1988; Zhang and Schrader, 1993). The pH of earthworm
casts is generally closer to neutrality than that of the soils
from which they are derived (Edwards and Lofty, 1977).
The modulation of pH has been often explained by the
action of Ca-gland (Lee, 1985), which excretes calcium
compounds into the vicinity. The removal of dead roots,
leaves and other plant litter from the soil surface and the
improvement of soil fertility are two major contributions of
earthworm activity towards the increase in plant biomass
(Van Rhee, 1971).
Earthworms appear to be more sensitive to heavy metals
than other soil invertebrates, and have been used in assess-
ing metal toxicity in contaminated soils (Spurgeon and
Hopkin, 1999). Recent researches showed that some spe-
cies of earthworm can survive in heavy metals contami-
nated soils (Langdon et al, 2001; Ma et al, 2002; Caroline
et al, 2003) and even affect the chemistry activities of
heavy metals in soil-plant system. Earthworm may enhance
the bioavailability of nutrients and metals to plant in soil by
gut digestion and nutrients accumulation (Devliegher et al,
1996). Since earthworm is capable of improving plant
growth and uptake of heavy metals, it was suggested to be
used in enhancing phytoremediation in heavy metal con-
taminated soil to solve the two limiting factors of phytore-
mediation: the less biomass of hyper-accumulator plants
and less bioavailability of soil heavy metals (Gao et al,
2005).
Earthworms feed on dead plant roots, leaves and other
plant litter from the soil surface, but they might attack
growing roots. In fact, apart from seizing plant roots,
earthworms can also spread diseases and excrete casts to
of Population, Resources and
and Environment
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ruin the soil surface, make them potential pests of plants,
under crowded conditions (Lee, 1985). Dense earthworm
brings about a reduction of vegetable. The effect of dense
earthworm population on the availability of nutrients and
heavy metals in soil is still far from clearly defined and
depends mainly on circumstantial evidence.
High toxicity of heavy metal, the low contents of nu-
trient and the poor physical structure are major limits to
plant growth on the mine tailings (Ye et al, 2000; 2002).
Therefore, in this research, the farm soil was mixed sepa-
rately with Pb/Zn mine tailings at the ratio of 25:75 (w/w).
The aim is to investigate the possibility of inoculating
earthworms for revegetation of lead/zinc metal mine tail-
ings by planting ryegrass. More specifically the present
study aims to study (1) the effects of earthworm density
on growth of ryegrass and earthworms, and (2) the effects
of earthworm density on availability of nutrients and
heavy metals.
  
  
The plough layer (0–20 cm) of soil (red-yellow podzolic
soil) was sampled from the Laitiao Village, the New Terri-
tories in Hong Kong. The Pb/Zn mine tailings were col-
lected from the Fankou Pb/Zn mine located at Shao- guang,
Guangdong Province, China. The earthworms (Pheretima
sp.) were collected from the same location where soil sam-
ples were taken. All test worms were selected as adult and
had a mean length of 7–8 cm and mean fresh weight of 5 g
per ten individuals.
  
Selected physico-chemical properties of soil and tailings
samples were tested: pH (CaC0l 2 -extraction, pH me-
ter), organic matter (H2SO4-K2Cr2O7 digestion), total and
available N (Kjeldahl digestion), total and available P
(HClO4-H2SO4 and Na2CO3 extraction respectively, fol-
lowed by molybdenum blue method), total and extractable
Zn, Cu, Pb, Cd (HNO3-HClO4-HF digestion and DTPA
extraction respectively, followed by atomic absorption
spectrophotometry (AAS)), according to Sparks et al.
(1996).
     

Samples of soil and mine tailings were air-dried, cru-
shed to pass through 2 mm sieve and mixed thoroughly.
The mixture of tailings and soil at the ratio of 25:75 (w/w)
was filled into 24 pots each containing 1 kg of the mixture;
and 0.35 g urea and 0.35 g K2HPO4 were mixed thoroughly
for each pot to facilitate the growth of ryegrass.
Different number of earthworms was introduced to the
pots: zero, three, six and nine individuals per pot respec-
tively. Distilled water was added into each pot to achieve
moisture content at about 70%. All the pots were incu-
bated in a temperature controlled greenhouse at 20°C for
three days. The moisture content was carefully controlled
and adjusted if necessary. Fifteen seeds of ryegrass (Lo-
lium multiflorum) were sowed into each pot, and the pots
were placed in the same greenhouse under a randomized
block design. After a week, seeds germinated well and
there were thinned to ten seedlings per pot, and water
daily.
        

At a growth period of six weeks, earthworms were
picked by hand, washed carefully with distilled water and
weighed. Mortality, if any, was recorded. The washed
earthworms were then placed on wet filter papers for 48 h

Table 1 Selected properties of the tailings and soil

Org. Extract. Extract.

Total N Total P Avail N Avail P Total Pb Total Zn
pH mat. Pb Zn
(%) (%) (mg/kg) (mg/kg) (mg/kg) (mg/kg)
(%) (mg/kg) (mg/kg)

Tailings 7.06 0.70 0.039 0.096 20.32 69.51 3559 6575 710.3 310.3

Soil 6.67 2.79 0.27 0.12 1223 3412 61.8 174.5 1.05 18.90

44
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to empty the ingested materials. The emptied earthworms

were killed at 100°C–110°C for 30 min, and further dried
at 60°C overnight, and weighed. Ryegrass was harvested
and divided into shoot and root portions. They were
washed, dried at 60°C for a week and weighed. The
growth substrates (mixtures of soil and tailings) were
air-dried, and grounded to pass 1 mm sieve. The Pb and
Zn concentrations in the shoot portion of ryegrass
(HNO3-HClO4-HF digestion, followed by AAS) were
tested. The pH (pH meter), and available N (Kjeldahl
Fig. 1 Effect of earthworm population density on biomass of
digestion), P (molybdenum blue method) and K (extracted
ryegrass
by DTPA, followed by AAS) and extractable concentra-
tions of Pb and Zn (extracted by DTPA, followed by AAS)
Note: Data of ryegrass fresh weight shoot or roots fol-
were determined (Shi, 1996).
lowed by the same letter are not significantly (p<0.05) dif-
   ferent (n=3) according to the ANOVA test (Tukey HSD) in
SPSS 11.0.
Data were analyzed using one-way ANOVS and
Fig. 2 indicates the changes of fresh weight as well as
Tukey’s HSD tests as available in the SPSS statistical
growth rate of earthworms under different treatments. It
package (Armitage and Berry, 1994).
was obvious that the growth of earthworms was adversely
affected by the increasing number of individuals added to
   the pot. The fresh weight of earthworms which receiving
the lowest number of individuals (three) increased, but the
   fresh weights of both treatments receiving higher numbers
According to Table 1, the soil sample was slightly of earthworms (six, nine) decreased, when compared with
acidic and possessed moderate contents of organic matter, their original weights (both P < 0.05). The growth rates in
total and available of N and P, and relatively low levels of treatments of three, six and nine earthworms were 4.4%,
total and DTPA-extractable metals. On the contrary, the -26.4% and -31.6% respectively.
tailings sample was near neutral, and contained very low
levels of organic matter and essential nutrients, but very
high total and extractable concentrations of the Pb and
Zn.

         


The shoot biomass of treatments receiving three and six
individuals per pot was significantly higher (P < 0.05) than
Fig. 2 Effect of earthworm population density on its growth rate
the control (without any earthworm) and the one receiving
nine individuals per pot. As to root biomass, all treatments
  
with earthworms had significantly lower (P < 0.05) values

than the control, and there were no significant differences
among all the treatments (Fig. 1). Table 2 indicates that earthworm activity significantly

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Table 2 Effects of earthworm population density on pH and   

concentrations of available N, P and K in growth substrates

Earthworm Available (mg/kg) The major objective of the present project is to test
number pH whether it is possible to inoculate earthworms for remedia-
N P K
tion Pb/Zn mine tailings, by planting ryegrass. The data
d d c
0 6.87 12.24 50.64 24.43c presented here suggest that earthworm addition, at an ap-
3 7.01c 37.01c 53.45b 29.11b propriate rate, increased shoot biomass of ryegrass (Fig. 1).
The present results also indicated that earthworm activity
6 7.07b 44.11b 54.83b 30.45b
enhanced availability of nutrients. Beneficial effects of
9 7.12a 81.51a 59.39a 32.42a earthworms on plant growth have been well-documented.
Note: Different letters in the same column indicate significant differ- Their contributions included the addition of soil organic
ence at P < 0.05. matter, the enhancement of nutrient cycling, and the im-
provement of physio-chemical soil conditions (Parmelee et
increased all parameters as compared with the control al, 1990; Zhang and Schrader, 1993). It is obvious that
(without any earthworms). There was also a general ten- earthworm activity enhances the transformations of soil
dency that the higher the number of earthworms added to nutrients and improves soil nutrient status, which is subse-
each pot, the higher the values of all parameters were ob- quently utilized by vegetable (Subler et al, 1997). The pre-
tained. Table 3 shows that earthworm activity increased the sent study shows that earthworm activity significantly in-
available portions of Pb and Zn extracted by DTPA in tail- creased the concentrations of available N, P and K in soil,
ings and soil mixture. There were significant differences compared with control without any earthworm, and the
(P < 0.05) in terms of extractable Pb concentrations be- values increased according to the increase in individuals
tween control and those added with earthworms, but not added to each pot. Nitrogen mineralization is in fact one of
extractable Zn (P > 0.05). When adding more earthworms the key indicators of soil fertility (Hauser, 1993).
into each pot, a general trend that higher uptake of Zn, and The basic requirements for earthworms are adequate
lower uptake of Pb by ryegrass was observed (Table 3). food supply and suitable growth conditions such as mois-
ture, temperature, soil texture, and pH (Waters, 1955). The
Table 3 Effects of earthworm density on the extractable concen- inadequate food supply and crowded living space observed
trations of heavy metalsin growth substrates and metal uptake by in treatments receiving a higher number of individuals per
ryegrass pot (six, nine) did not seem to fulfill the basic requirements
and hence the poor growth as indicated by the lower mean
DTPA-extractable
Concentrations in fresh weight and growth rate observed (Fig. 2). Earth-
concentrations in growth
ryegrass (mg/kg) worms feed on dead plant roots, leaves and other plant
substrates (mg/kg)
litter from the soil surface, but they might attack growing
Earthworm roots as observed under the crowded conditions, especially
Zn Pb Zn Pb
number no food was added during the pot trial which lasted for six
0 73.39a 180.68b 138.97b 40.06a weeks. Dense earthworm population brings about a reduc-
3 84.67a 209.62ab 152.06a 15.10b
tion of ryegrass. In fact, apart from seizing plant roots,
earthworms can also spread diseases and excrete casts to
6 78.51a 194.22a 165.32a 16.64b
ruin the soil surface, make them potential pests of plants
9 80.26a 205.40a 168.52a 37.27ab under crowded conditions (Lee, 1985).
Note: Different letters in the same column indicate significant differ- Soil pH is an important parameter affecting heavy metal
ence at P < 0.05. solubility in soils and uptake by plants. The present results

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46 Chinese Journal of Population, Resources and Environment 2008 Vol. 6 No.2
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show a significant increase in soil pH by earthworm inocu-
lation (Table 2), which goes along with the well- docu-
mented fact that earthworm activity tends to raise soil pH
to neutrality (Edwards, 1977; Hu et al, 1998), which
mainly due to the action of Ca-gland excreting calcium
compounds into soil environment (Lee, 1985).
Chelate agents have been commonly used in both agro-
nomic and environmental studies to evaluate the availabil-
ity of metals to plants (McGrath and Cegarra, 1992; Young
et al, 2000). There is generally a good correlation between
the amount of metals extracted from soils by Ethylene
Diamine Tetraacetic Acid (EDTA) or DTPA, and the
amount of uptake of the same metals by plants (Chen,
1996). It has also been indicated that the available contents
of heavy metals in soils are inversely related to soil pH,
with a higher metal availability under lower pH (McBride,
1989). However, as the pH values of all the treatments in
the present study were near neutral, the effects of pH on
metal availability were minimal. In this study, the Zn con-
centration in soil extracted by DTPA was slightly increased
(P > 0.05) by earthworm activity, but the Zn uptake by
ryegrass (shoots) was significantly increased (P < 0.05)
(Table 3). Although the extractable Pb concentrations in
soil decreased according to earthworm inoculation, there
was a decrease in Pb uptake by ryegrass. The reason for
this was not clear. It has been observed that Pb uptake by
corns grown on sludge-treated soils is insignificant and
also poorly translocated into the grain, suggesting that Pb
appears to be one of the least mobile and bioavailable
metal in the sludge-soil-plant system (Juste and Mench,
1992). In the study of Abdul et al. (1996), earthworm had
no significant effect on metal bioavailability in contami-
nated soil with three different levels of Cd, Cu, Fe, Pb and
Zn concentrations. It indicated that the effects of earth-
worm on soil heavy metals depend on species of heavy
metals and soil characters.
According to the above discussion, it can be concluded
that earthworm inoculation at an appropriate density for the
revegetation of metal mine tailings, could increase nutrient
cycling and promote plant growth. To satisfy different
remediation targets in contaminated soils with distinct bi-
ology conditions, pollution extent, and species of heavy
metals, more earthworm species tolerant of heavy metals
should be studied, especially those can be artificial cultured
ChineseJournal
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and be easily reproduced.
Acknowledgements: This research was funded by the Natural Science
Fund of China (Grant No.40271068) and Natural Science Fund of
Shandong Province of China (Grant No.Y2002E02).
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