Journal of Hazardous Materials 366 (2019) 615–623

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Journal of Hazardous Materials

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Heavy metals distribution and their bioavailability in earthworm assistant T

sludge treatment wetland
Zhongbing Chen , Shanshan Hu
⁎

Department of Applied Ecology, Faculty of Environmental Sciences, Czech University of Life Sciences Prague, Kamýcká 129, 16521 Prague, Czech Republic

GRAPHICAL ABSTRACT

ARTICLE INFO ABSTRACT

Keywords: Sludge treatment wetlands (STWs) have been used for sludge treatment in recent years, however, heavy metals
Earthworms (HMs) are one of the limiting factors for the final sludge application, and the fate of HMs in STWs are still not
Heavy metals bioavailability well studied. Therefore, six STWs with two plant species and earthworm addition were investigated to evaluate
Heavy metals distribution their effects on HMs distribution and bioavailability. The results showed that plant uptake of HMs was insig-
Sludge treatment
nificant with earthworm addition. Earthworm had the highest enrichment for Cd with bioaccumulation factors
Sludge treatment wetlands
of 6.9–7.3. Moreover, earthworm had a positive effect to remove HMs in accumulated sludge. Meanwhile, the
bioavailability of Cd in accumulated sludge was decreased by earthworm addition, with the acid-soluble fraction
decreased from range 16.5–22.7% to range 7.2–10.1%. Furthermore, HMs mass balance in the STWs revealed
that HMs were widely distributed in the accumulated sludge, leachate and others, while their (except Cd) ac-
cumulation in the plants and earthworm are less than 1%. The Cd toxicity in the accumulated sludge can be
reduced by the addition of earthworm with enrichment of 3.6–8.2%. Overall, earthworm addition have positive
effects on distribution and bioavailability of HMs in STWs.

1. Introduction
Sewage sludge is mainly produced from the primary and secondary
sedimentation tanks in wastewater treatment process. Numerous heavy
metals (70–90%) such as Cu, Mn, Ni, Zn, Pb, Cr, Cd and As in
wastewater are transferred into the sewage sludge by precipitation and
adsorption [1]. Therefore, sewage sludge need to be further treated.
Traditional sludge treatment technologies include sludge concentration,
drying, composting, and anaerobic digestion. In China, less than 10% of
sewage treatment plants have sludge stabilization and disposal facilities

⁎
Corresponding author at: Faculty of Environmental Sciences, Czech University of Life Sciences Prague, Kamýcká 129, 16521 Prague, Czech Republic.
E-mail addresses: zhongbing.chen@gmail.com, chenz@fzp.czu.cz (Z. Chen).

https://doi.org/10.1016/j.jhazmat.2018.12.039
Received 15 August 2018; Received in revised form 17 November 2018; Accepted 12 December 2018
Available online 13 December 2018
0304-3894/ © 2018 Elsevier B.V. All rights reserved.
Z. Chen, S. Hu Journal of Hazardous Materials 366 (2019) 615–623

due to the high cost and high energy consumption required [2]. In are below the EU, US and Chinese agricultural reuse standards
particular, small sewage treatment plants in some rural areas with [18–20,30], the detailed effects of earthworms on heavy metals re-
limited funds and weak technology are lack of supporting sludge moval processes are still not known.
treatment facilities. Therefore, a low investment, low operation and Therefore, the aim of this study were to: (1) clarify the distribution
maintenance cost, low energy consumption sludge treatment tech- of heavy metals in STWs with earthworm addition; (2) evaluate the
nology is highly in demand. effect of earthworms on heavy metals bioavailability in accumulated
Sludge treatment wetlands (STWs) results in positive sludge dewa- sludge of STWs.
tering and stabilization with low cost and low energy consumption can
be considered as an effective, safe, environmental friendly and eco- 2. Materials and methods
nomical alternative technology [3,4]. It has been widely used in some
countries in recent years, for example in Denmark [5], France [6], Span 2.1. Experimental setup
[7] and Poland [8]. Sludge dewatering in STWs is relied on the leachate
removal. The leachate is discharged from the bottom drainage system Six microcosms sludge treatment wetlands (STWs) were established
by the filtration of the sludge layer and gravel layer after the sewage in a greenhouse with temperature and humidity controlled at 25 °C and
sludge with high water content (more than 90%) injected into the 65%, respectively. The STW was constructed by plexiglass with size of
STWs, meanwhile, the sludge particles are intercepted in the sludge 50 × 20 × 50 cm (Length × Width × Height). Each STW was divided
layer [5]. In addition, the leachate in the accumulated sludge is further into a sludge treatment section and a leachate treatment section by a
removed by transpiration and evaporation, thereby reducing the water baffle with some small holes in the bottom (Fig. S1). The size of the
content in sludge [9]. Magri et al. [10] reported that total solids (TS) in sludge treatment section was 30 × 20 × 50 cm (Length × Width ×
the accumulated sludge was increased to 45%, and the highest water Height). All the STWs contained three layers with substrate size in-
loses reached to 78% by the STWs under the temperature of 15–25 °C. creasing from bottom to top. The layers consisted of 10 cm of 25–40 mm
The sludge stabilization is mainly achieved through the processes of coarse gravel, 10 cm of 8–16 mm medium gravel and 10 cm 4–8 cm of
organic matter mineralization and humification, which needs the fine gravel. The porosity of each STW was 0.44. Two common wetland
combination action of plants, organic matters and microorganisms [4]. plants Phragmites australis (average height of 70 cm) and Typha angu-
Previous studies showed that the ratio of volatile solids (VS) to TS (VS/ stifolia (average height of 120 cm) were planted (10 strains) in each
TS) in accumulated sludge can be reduced by 25–30% in STWs [11,12]. STW system. Eisenia foetida was selected based on previous study and
Sludge treated by STWs is often used for land application as a fer- 400 g E. foetida (about 850 bodies) was added into some STWs.
tilizer. More than 50% of the accumulated sludge has been used for land Therefore, the six treatments were T. angustifolia + E. foetida (T + E),
application in some European countries (e.g. Germany, France and T. angustifolia (T), P. australis + E. foetida (P + E), P. australis (P),
Poland) and the United States [11]. However, pollutants (e.g. heavy Unplanted + E. foetida (U + E) and Unplanted (U), respectively. The
metals, pathogens and toxic organic compounds) in sludge are still feeding period and rest period last 5 months and 2 months, respectively.
limiting their land application (less than 10%) in China [1]. Heavy In the feeding period, the sludge loading rate and feeding frequency
metals (e.g. Cu, Mn, Ni, Zn, Pb, Cr and Cd) are one of the important was 3 L/2d, with the average sludge loading rate of 45.6 kg TS/m2/yr.
factors affecting the land application. In STWs, heavy metals are gen- Raw surplus sludge was collected from a local domestic wastewater
erally distributed in plants, gravel layer, accumulated sludge and lea- treatment plant, and their main characteristics were summarized in
chate. Plants can not only uptake nutrients (e.g. nitrogen (N), phos- Table 1.
phorus (P)) in sludge to meet their own growth needs, but also can
enrich and transform heavy metals through the action of rhizosphere 2.2. Bio-concentration factor in plants
microorganisms and direct uptake [13–15]. Gravel layer play the most
important role on heavy metals removal for accumulating sludge in Bio-concentration factor (BCF) of heavy metals in plants were cal-
STWs. The main processes to remove heavy metals from sludge via culated by Eq. (1), which adopt from Cheng et al. [31].
gravel layer including filtration, adsorption and sedimentation [16].
Stefanakis and Tsihrintzis [16] reported that more than 47% of heavy Heavy metal content in plants (mg/kg)
BCF =
metals were removed by the gravel layer in STWs, and more than 35% Heavy metal content in the raw sludge (mg/kg) (1)
of them were accumulated in the sludge layer, but less than 3% were
discharged from effluent. Although previous studies reported that
2.3. Bioaccumulation factor in earthworms
heavy metals contents in sludge are all below the EU, US and Chinese
agricultural reuse standards [7,17–22], the heavy metals contents still
Bioaccumulation factor (BAF) of heavy metals in earthworms were
exist risks to land application, especially for Cd. Our previous study
calculated by Eq. (2), which adopt from Liu et al. [32].
reported that Cd concentration is above the Chinese agricultural reuse
standards and its bioavailability is also relatively high [12]. Therefore,
Table 1
it is very necessary to improve the STWs technology to decrease the
Characteristics of the raw surplus sludge, n = 48.
heavy metals contents in accumulated sludge.
Invertebrates, such as earthworms, play an important role on pur- Parameter Unit Mean Standard
ifying wastewater in STWs [23]. Previous studies reported that N and P
pH – 6.5–7.3 –
concentrations in effluent can be decreased by earthworm addition ORP mV −116 - 93 –
[24,25]. Moreover, some researches revealed that earthworms play an DO mg/L 0.01–0.95 –
important role in enriching heavy metals in vermicomposting process, TS % 0.5 0.2
VS % 0.3 0.1
thereby reducing the content of heavy metals in sludge [26,27]. In
Cu mg/kg 6.3 4.1
addition, earthworms are also used in STWs to improve the sludge Mn mg/kg 129.7 58.9
treatment efficiency in recent years. Chen et al. [28], Hu and Chen [29] Ni mg/kg 9.1 3.9
proved a hint that TS, VS/TS, N and P in accumulated sludge are im- Zn mg/kg 177.8 87.5
proved by STWs treatment with earthworms addition. However, no Cr mg/kg 19.2 11.2
Fe mg/kg 8865 3415
study focus on the effect of earthworms on the removal of heavy metals
Cd mg/kg 0.8 0.4
in STWs. Although Hu and Chen [29] reported that the heavy metals Pb mg/kg 65.7 16.5
contents in the accumulated sludge by STWs with earthworms addition

616
Z. Chen, S. Hu
Heavy metal content in earthworms (mg/kg)
BAF =
Heavy metal content in the raw sludge (mg/kg) (2)
2.4. Sample analysis
Accumulated sludge samples in the six STWs were collected every 6
days during the feeding period, and were analyzed for heavy metals
(Cu, Mn, Ni, Zn, Cr, Fe, Cd and Pb) contents and their fractions. Outflow
leachates in the six STWs were taken every 6 days during the feeding
period. Samples of earthworms were collected in the end of the ex-
periment. Plants were harvested in the end of the experiment and the
total dry biomass of aboveground and belowground were weighted. The
aboveground and belowground biomass were analyzed to determine
the heavy metals contents. All heavy metals analysis in plants and
sludge were carried out by acid digestion with HF-HClO4-HNO3, fol-
lowed by a flame atomic absorption spectrometer (Agilent 240/280FS
AA) [33]. The heavy metals analysis in leachate according to the
monitoring approaches described by Environmental quality standard
for surface water (GB 3838-2002) [34].The heavy metals fractions were
extracted by Community Bureau of Reference (BCR), then analyzed by
atomic absorption spectroscopy (Agilent 240/280FS AA).
2.5. Data analysis
Paired t-test was used to compare the difference of heavy metals
contents in the accumulated sludge among the six STWs regarding to
the effects of plant species and earthworms addition. Two-way Analysis
of variance (ANOVA) was used to assess the effects of plants and
earthworms in STWs. The level of significance was set to P < 0.05.
Principal component analysis (PCA) was completed to display data
groupings and correlations for general water environmental parameters
and heavy metals in the six STWs. The statistic software OriginPro 8.5
for windows was used to perform the test and to plot the graphs. The
two-way ANOVA and PCA were conducted using the SPSS 21.0 statis-
tical software.
3. Results and discussion
3.1. Heavy metals in plants
Plant uptake is one of the removal pathway for heavy metals re-
moval in STWs. Heavy metals concentrations in both aboveground and
underground parts of the plants were significantly higher than those of
the plants in the beginning of the experiment (p < 0.05) (Fig. 1).
Meanwhile, the heavy metals concentrations in the underground parts
were 1.0–35.9 times higher than those in the aboveground parts
(Table 2). Moreover, the earthworm had insignificant effect on the
heavy metals uptake by plants except Zn and Fe in the P. australis STWs
(p > 0.05). This result was in agreement with previous studies, which
revealed that roots were primary sites for heavy metals uptake [35,36].
Plant roots delivered oxygen to provide rhizosphere microbial activ-
ities, thus speeding up the removal of heavy metals in the sludge and
leachate [5,12,16]. In addition, Shanker et al. [37] reported that highly
accumulation of heavy metals in the underground parts could be a re-
sults of heavy metals binding on cell walls or heavy metals precipitation
within vacuoles. Meanwhile, the concentrations of Cr and Cd (20 and
1.4 mg/kg on average, respectively) in the plants were higher than
those in the wastewater study of Vymazal [14], which showed that the
Cr and Cd concentrations in the plants were below 6 and 0.2 mg/kg,
respectively. This indicated that heavy metals uptake by plants were
not inhibited under the high heavy metals conditions.
BCFs for Mn, Ni, Zn, Cr, Fe, Cd and Pb in the plants were 3.9–4.5,
5.4–8.2, 0.8–1.1, 1.1–1.2, 0.1-0.3, 1.5–1.8 and 0.6–0.7, respectively.
The BCF in the P. australis had insignificant difference between those in
the T. angustifolia (p > 0.05) (Table 2). Moreover, the BCF in our study
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Journal of Hazardous Materials 366 (2019) 615–623
was lower than the previous studies, which reported that the BCF in the
plants roots for Cu, Zn, Cr, Fe, Cd and Pb in CWs were 375–1140,
26.7–33.9, 0.9–53.8, 41–82, 9.8–32.9 and 2.4–600, respectively
[38,39]. This might due to the abundant sludge particles with high
heavy metals contents was intercepted in the sludge layer. Stefanakis
and Tsihrintzis [16] and Peruzzi et al. [40] also detected that the heavy
metals concentrations in plants were significantly lower than those in
the accumulated sludge.
3.2. Heavy metals in earthworms
Earthworm accumulation is another important factor for heavy
metals translocation in STWs. The heavy metals concentrations in
earthworms of the three earthworm STWs were significantly higher
than those of the blank earthworms (p < 0.05) (Table 3). The con-
centrations of Cr and Pb in the earthworm under the U + E STWs were
one time higher than those under the T + E STWs. However, the op-
posite result was obtained by Fe. In addition, the concentrations of Mn
and Pb in the earthworm under the P + E STWs was significantly higher
than those under the T + E STWs (p < 0.05). The possible reason for
the heavy metals enrichment by earthworm in the accumulated sludge
was due to the activities of enzymes in earthworms [41]. Moreover, the
different enrichment ability to heavy metals by earthworms was influ-
enced by various affinity of transporters on their enamel cell mem-
branes [42].
BAFs were arranged as follows: Cd > Mn > Ni > Zn > Pb > 1
> Cr > Fe (Table 3). Similar results were observed by Suleiman et al.
[43], which revealed that the BAF in earthworms (E. fetida, Eisenia
andrei and Dendrobaena veneta) ranked as: Cd > Co > Cu > 0 > Zn
> Ni > Pb > Cr. Liu et al. [32] and Rorat et al. [44] also found that
the earthworms had high tendency to accumulate Cd and Zn. The
bioaccumulation of heavy metals by earthworms including absorption,
internal distribution, storage and excretion [32]. Cd is influenced by
pH, electrical conductivity and organic matter content in the sludge,
especially for pH, which is easily affect the Cd absorption and trans-
formation [12,45]. Hu et al. [12] reported that the pH values in the
accumulated sludge without earthworm addition were below 6.5.
However, the pH values in accumulated sludge was increased to 6.7–7.5
with earthworm addition in our study. The reason might be the
earthworms secrete intestinal Ca and NH4+-N during the treatment that
lead to the pH values maintain neutral in the sludge, which create an
advantage condition for heavy metals accumulation by earthworms
[43]. In addition, Rorat et al. [44] showed that earthworms can accu-
mulate essential metals (e.g. Cu, Zn) by a fast enrichment with low
dose, while the non-essential metals (e.g. Cd) can be accumulated by
earthworms with long-term absorption and slow excretion. This might
be another reason that Cd had the high bio-accumulation in earth-
worms.
3.3. Heavy metals in effluent
Generally, the effluent concentrations of all measured heavy metals
in the six STWs were lower than inflow, significant decrease were ob-
served between the Cu, Fe, Ni, Mn, Zn and the inflow (p < 0.05)
(Fig. 2). Moreover, the effluent concentrations of heavy metals were
below the Chinese integrated wastewater discharge standard (GB8978-
1996) [46]. The mass removal efficiencies of Fe, Mn and Zn in the six
STWs were more than 90%, which were similar to the results of Hu
et al. [12] (Table 4). In addition, the removal efficiencies of Cu, Mn, Ni,
Zn, Cr, Cd and Pb in the planted or earthworms STWs (T, T + E, P,
P + E and U + E) had insignificant difference between the unplanted
STWs (U) (p > 0.05). Šíma et al. [47] reported that the highest re-
moval efficiencies were achieved for Zn and Cu (89.8 and 81.5%) in the
CWs due to precipitation of insoluble sulfides (ZnS and CuS). Mustapha
et al. [39] proved that sedimentation and plant uptake were the main
processes of heavy metal removal from wastewater by CWs. Vymazal
Z. Chen, S. Hu Journal of Hazardous Materials 366 (2019) 615–623

Fig. 1. Heavy metals concentrations in the underground and aboveground parts of plants (a, b show the significant difference (p < 0.05); BT: blank T. angustifolia;
BP: blank P. australis).

and Březinová [15] also revealed that the emergent plants, which can
greatly enhance the removal of heavy metals in wastewater.
Two-way ANOVA analysis showed that the heavy metals removal
efficiencies were insignificant affected by plants and earthworms
(p > 0.05) (Table 5). Stefanakis and Tsihrintzis [16] reported that
more than 35% of heavy metals were still remained in the accumulated
sludge due to the substantial by the gravel and sludge layers, and less
than 3% of heavy metals was discharged with the outflow. Results of
PCA indicated that the variation in water parameters (pH, ORP, DO)
and heavy metals removal were captured by two main axes, together
explaining 51.4 and 48.4% of the variation in planted and unplanted
STWs, respectively (Fig. 3). In the planted STWs, PCA axis 1 was largely
determined by ORP, removal efficiencies of Ni, Cd, Mn, Fe, Zn and Cr,
among them, removal efficiencies of Ni and Cr had the highest positive
correlation with the ORP (r = 0.7–0.8). In the unplanted STWs, varia-
tion along PCA axis 1 was determined by all these parameters, mean-
while, pH, ORP and DO were largely determined onto PCA axis 2. The
results of scores of four STWs on AX 1 and AX 2 showed that earthworm
had significant effect on the water parameters and the heavy metals
removal in the unplanted STWs. However, the earthworm effect in the
planted STWs is weak.
3.4. Heavy metals in accumulated sludge
3.4.1. Total heavy metals
All heavy metals concentrations in the accumulated sludge were
below the EU, US and Chinese agricultural reuse standards [18–20,30],
and also below the standards of woodland reuse and land reuse in China
[48,49] (Table 6). Similar studies reported that heavy metals in accu-
mulated sludge after STWs treatment can meet the EU and US standards
for agricultural reuse [12,21,35,50]. Matamoros et al. [51] also re-
ported that six heavy metals (Pb, Ni, Cu, Zn, Cd and Cr) in the accu-
mulated sludge after a 20-year old STWs treatment in Denmark were all
below the EU legal limits for agricultural reuse [19].
The concentrations in the accumulated sludge by the planted or
earthworm STWs were 1.7–6.5, 8.9–40.2, 4.7–7.1, 14.4–39.5, 5.2–10.2,
669.7–4011.3, 0.3–0.4, 1.9–68.8 mg/kg for Cu, Mn, Ni, Zn, Cr, Fe, Cd
and Pb, respectively, which were lower than in the unplanted STWs.
Meanwhile, the concentration of Cu, Mn, Ni, Zn, Cr, Fe, Cd and Pb in
the accumulated sludge under the STWs without earthworm addition
were 0.5–1.7, 8.9–31.6, 0.5–4.7, 7.4–18.0, 3.1–5.2, 128.5–3419.0,
0–0.3, 1.9–4.0 mg/kg, which were higher than those in the earthworm
assistance STWs, respectively. The results revealed that heavy metals

Table 2
Heavy metals bio-concentration factor (BCF) and the ratio of the underground to aboveground parts in plants.
Plant Mn (mg/kg) Ni (mg/kg) Zn (mg/kg) Cr (mg/kg) Fe (mg/kg) Cd (mg/kg) Pb (mg/kg)

Underground/Aboveground T+E 2.3 ± 1.6 4.9 ± 0.9 4.9 ± 0.8 1.5 ± 1.2 35.9 ± 7.8 6.0 ± 1.5 1.5 ± 0.8
T 2.0 ± 0.8 3.3 ± 1.0 4.6 ± 0.3 2.0 ± 1.0 17.3 ± 4.9 6.0 ± 2.2 1.6 ± 1.2
P+E 3.4 ± 1.5 1.7 ± 1.0 3.1 ± 1.2 1.6 ± 1.0 28.0 ± 6.5 11.0 ± 2.8 1.5 ± 1.0
P 4.7 ± 1.4 2.0 ± 1.3 4.4 ± 1.7 1.7 ± 1.0 7.1 ± 6.9 6.0 ± 2.0 1.0 ± 0.9
BCF T+E 3.9 ± 1.7 5.4 ± 0.6 0.8 ± 0.3 1.2 ± 0.3 0.2 ± 0.01 1.8 ± 0.4 0.7 ± 0.4
T 4.5 ± 1.3 6.4 ± 0.7 0.9 ± 0.4 1.1 ± 0.2 0.3 ± 0.08 1.8 ± 0.6 0.6 ± 0.4
P+E 4.5 ± 1.3 7.9 ± 0.7 0.8 ± 0.4 1.2 ± 0.2 0.3 ± 0.06 1.5 ± 0.4 0.7 ± 0.3
P 4.4 ± 1.9 8.2 ± 0.8 1.1 ± 0.6 1.2 ± 0.5 0.1 ± 0.09 1.8 ± 0.5 0.7 ± 0.5

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Table 3
Heavy metals concentrations and bioaccumulation factors in the earthworms (a, b show the significant difference (p < 0.05)).
Earthworms Concentrations (mg/kg) BAFs

Blank E T+E P+E U+E T+E P+E U+E

a b c b
Mn 234 ± 15 506 ± 35 789 ± 33 412 ± 32 3.9 6.1 3.2
Ni 19.5 ± 4.6a 31.4 ± 11.2b 42.9 ± 12.0b 29.8 ± 10.7b 3.5 4.7 3.3
Zn 96 ± 27a 177 ± 24b 239 ± 22b 185 ± 25b 1.0 1.4 1.0
Cr 0.7 ± 0.5a 7.7 ± 4.6b 6.0 ± 3.8b 17.4 ± 3.7c 0.4 0.9 0.9
Fe 1124 ± 97a 3146 ± 96b 4426 ± 83b 2200 ± 45c 0.4 0.3 0.2
Cd 0.03 ± 0.02a 5.8 ± 0.2b 5.5 ± 0.1b 5.7 ± 0.1b 7.3 6.9 7.1
Pb 8.4 ± 1.9a 48.4 ± 11.7b 83.0 ± 16.4c 89.1 ± 17.7c 0.7 1.3 1.4

contents in the accumulated sludge may be influenced by the plants and
earthworms. Plants can uptake heavy metals through their roots and
stems [16,35], the earthworms also can enrich heavy metals in the
accumulated sludge partially due to the action of enzyme in their bodies
[52]. Liu et al. [32] reported that the concertation of Cu, Ni, Cd, Pb and
Zn in the sludge were lower than initial sludge due to the adsorption by
earthworms. Xu et al. [53] also revealed that the heavy metals con-
certation of Cr, Zn, Pb, Cd, Cu and Ni in sludge were decreased by 27%,
31%, 14%, 24% and 23% after the earthworm treatment, respectively.
3.4.2. Heavy metals fractions
Generally, Fe, Cr, Cu and Pb mainly existed in the form of residual
and oxidation fractions, and the sum of these two fractions reached over
50% during the whole experiment (Fig. 4). Moreover, both plants and
earthworms showed insignificant effect on the fractions of the Cu, Fe
and Pb based on two-way ANOVA analysis (p > 0.05) (Table S1). This
was in agreement with the previous studies [12,54,55]. However, both
plants and earthworms had a significant effect on the acid-soluble state
of Cr. This indicated that the bioavailability of Cr can be influenced by
plants and earthworm. In addition, plants and earthworms had a sig-
nificant effect on the oxidation state and residual state for Mn and Ni
(p < 0.05). Moreover, plants had an extremely significant effect on the
acid-soluble state and reduction state for Zn (p < 0.001).The total
residual and oxidation fractions of Ni in the accumulated sludge of the
six STWs were between 37.6–43%. Additionally, the residual fraction of
Ni in the accumulated sludge under the U + E STWs was 10% higher
than the U STWs during the rest period. This clearly indicated that the
bioavailability of Ni was declined by earthworm addition, which is si-
milar to the result of Xia et al. [56]. Meanwhile, Mn and Zn mainly
existed in the form of acid-soluble and reduction fractions (the sum of
two fractions reached to 90.0%). Hu et al. [12] reported that it is
beneficial for use the treated sludge with high bioavailability of Zn and
Mn as fertilizer to improve the physical and chemical properties for
calcareous soil. During the rest period, the acid-soluble and oxidation
fractions of Cd in the accumulated sludge of the six STWs was decreased
to 40.1%, especially the acid-soluble fraction of Cd in the three earth-
worm STWs, which decreased from range 16.5–22.7% to range
7.2–10.1%. Furthermore, the acid-soluble state of Cd was obtained
significant difference by the plants and earthworms (p < 0.05). It re-
vealed that the bioavailability of Cd can be decreased by the earth-
worm’s activity during the rest period [57]. Wu et al. [58] also reported
that carbonate precipitates fraction of Cd in the soil was decreased by
52–85% and residual fraction of Cd increased by 59–123% with
earthworm addition.

Fig. 2. Concentrations of heavy metals in inflow and outflow under the six STWs.

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Table 4
Heavy metals removal efficiencies in the six STWs.
Heavy metal T + E (%) T (%) P + E (%) P (%) U + E (%) U (%)

Cu 70.5±24.6 67.7±26.8 71.1±22.8 73.6±20.0 71.1±24.3 62.4±29.2

Mn 93.6±3.4 93.5±4.3 95.5±2.2 95.6±3.1 94.0±5.3 92.7±4.2
Ni 73.0±9.9 72.8±10.7 74.5±8.3 77.8±8.9 75.3±10.2 66.7±6.5
Zn 96.3±2.7 95.9±2.2 95.8±4.0 95.8±12.0 96.0±3.1 95.7±2.3
Cr 48.1±20.5 48.9±23.6 51.4±23.3 54.8±21.3 46.7±28.8 45.9±16.9
Fe 97.8±1.9 98.6±1.4 98.0±2.8 97.1±6.4 98.2±2.5 98.7±1.4
Cd 38.4±25.2 39.9±22.8 42.6±16.4 47.1±17.4 43.0±24.1 24.0±16.2
Pb 49.6±28.7 42.8±32.1 54.8±28.7 61.8±28.5 59.3±27.1 42.6±27.3

Table 5 plants and earthworms except Cd (Fig. 5). Earthworms had a strong
Two-way ANOVA analysis for the effects of plants and earthworms in STWs on enrichment for Cd, which showed the mean enrichment proportion of
heavy metals removal efficiencies and water parameters (the values in bold 3.6, 8.2 and 7.3% in the T + E, P + E and U + E STWs, respectively. Xu
show the significant difference (p < 0.05)). et al. [53] reported that the order of enrichment in earthworm was
Parameters Plants Earthworms Plants x earthworms Cd > Hg > As > Zn > Cu > Pb. The average proportions of Fe, Zn,
Pb and Mn for all STWs in leachate were lower than those of the Ni, Cd
pH < 0.001 < 0.001 0.01 and Cr. Stefanakis and Tsihrintzis [16] showed that the lower propor-
ORP 0.17 0.001 0.06
DO 0.44 0.001 0.64
tions of Fe, Zn and Mn were owing to the precipitation of respective
Water loss 0.01 0.02 0.01 oxides within the well-aerated STW system. Compared with the planted
Cu 0.71 0.60 0.72 STWs, the heavy metals accumulation in the sludge under the un-
Mn 0.08 0.64 0.78 planted STWs was higher, especially the concentration of Fe and Pb in
Ni 0.13 0.38 0.06
the U STW, the proportion reached to 98.9% and 79.7%, respectively.
Zn 0.60 0.56 0.86
Cr 0.55 0.83 0.94 This can be explained by the presence of plants and earthworms that
Fe 0.58 0.83 0.54 increase the system porosity, resulting in the entrapment of sludge
Cd 0.15 0.36 0.09 particles in the gravel layer and discharge with the leachate. Mean-
Pb 0.32 0.39 0.33 while, more than 47.6% of Mn, Ni, Zn and Pb were accumulated in the
substrate, Fe, Cd and Cr were also reached more than 20%. Stefanakis
and Tsihrintzis [16] found that more than 47% of heavy metals (Fe, Zn,
3.5. Heavy metals mass balance
Mn, Cd, Pb, Cu, Cr and Ni) was removed by the substrate, which play an
important role in heavy metals removal. The heavy metals removal in
Heavy metals in STWs were existed in accumulated sludge, plants,
CWs was mainly relied on the gravel layer due to precipitation trans-
leachate, earthworm and other places (e.g. substrate). On the average,
formation and adsorption [59]. The heavy metals accumulated in the
less than 1% of the heavy metals mass contents was accumulated in the

Fig. 3. Principal component analysis (PCA) of heavy metals removal efficiencies and water parameters in the planted and unplanted STWs (C and D); scores of four
STWs treatments on AX1 and AX2 (A and B).

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Table 6
Heavy metals contents in the accumulated sludge as well as national and international standards for land use.
STWs Cu Mn Ni Zn Cr Fe Cd Pb
(mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg)

T+E 2.5 ± 0.7 117.1 ± 6.4 4.2 ± 0.3 140.7 ± 7.3 13.6 ± 4.5 10378 ± 116 1.2 ± 0.2 32.4 ± 1.7
T 3.0 ± 1.4 128.7 ± 38.3 5.6 ± 0.2 162.3 ± 16.4 16.7 ± 3.2 11648 ± 1773 1.2 ± 0.1 41.3 ± 2.4
P+E 2.1 ± 0.4 102.8 ± 23.6 4.1 ± 0.2 150.1 ± 29.8 12.2 ± 0.8 8306 ± 1182 1.1 ± 0.1 56.6 ± 6.4
P 2.7 ± 0.05 129.7 ± 4.2 4.6 ± 0.2 157.5 ± 14.9 16.7 ± 3.0 11752 ± 425 1.2 ± 0.3 60.6 ± 5.7
U+E 6.9 ± 1.6 134.1 ± 2.8 6.5 ± 0.2 175.1 ± 15.0 17.2 ± 3.7 11189 ± 425 1.2 ± 0.1 108.2 ± 7.1
U 8.6 ± 0.5 143.0 ± 1.9 11.2 ± 0.4 189.5 ± 29.9 22.4 ± 4.3 12317 ± 319 1.5 ± 0.3 110.1 ± 3.9
EU 1000-1750 – 300-400 2500-4000 – – 20-40 750-1200
(86/278/EEC)
Proposed in 3rd Draft 1000 300 2500 1000 750
US 4300 – 420 7500 – – 85 840
(Part503,1994)
Agricultural reuse 250 – 100 500 600 – 5 300
(GB-18918-2002)
Woodland reuse 1500 – 200 3000 1000 – 20 1000
(CJT-362-2011)
Land reuse 800 – 100 2000 600 – 5 300
(CJT-291-2008)

gravel layer in the P + E STW was the highest. This indicates that
earthworms and P. australis can promote the heavy metals removal
from the input sludge simultaneously. Xu et al. [60] reported that the
enzyme activity (catalase, urease, and invertase) around the plants
rhizosphere were increased by earthworm addition, meanwhile, plants
roots create an aerobic condition which can promote heavy metals
precipitation (e.g. co-precipitated in ferric oxides, hydroxides) in the
substrate. Therefore, the combination of earthworm and plant increased
the heavy metals removal by substrate.
4. Conclusion
Heavy metals uptake by plants were mainly occurred in the un-
derground parts, which were 1.0–35.9 times higher than in the
aboveground parts. Moreover, earthworm had insignificant effect on
heavy metals accumulation in the plants. Earthworm had the highest
enrichment for Cd, meanwhile, the BAF in the earthworm was
in the following order: Cd > Mn > Ni > Zn > Pb > Cr > Fe.
Earthworm had significant effect on heavy metals removal from ef-
fluent in the unplanted STWs. Meanwhile, the heavy metals contents in
the accumulated sludge were decreased by the earthworm addition.
Furthermore, earthworm can decrease the Cd bioavailability, with the
acid-soluble fraction decreased to 7.2–10.1% in the six STWs during the
rest period. In addition, the heavy metals mass balance showed that the
major distribution of heavy metals (except Cd) in STWs was in the
gravel layers, accumulated sludge and leachate, which sum up to 99%,
while the heavy metals in the plants and earthworms were negligible.
Notably, 3.6–8.2% of Cd was distributed in the earthworms under the

Fig. 4. Heavy metal fractions in the accumulated sludge of the STWs (P: P. australis system; T: T. angustifolia system; U: Unplanted system; E+: Earthworm addition;
E-: Without earthworm addition).

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Z. Chen, S. Hu
Fig. 5. Heavy metals mass balance in the six STWs.
earthworm STWs. Overall, earthworm addition can reduce the toxicity
of heavy metals in the accumulated sludge, thus, STWs treatment
technology can be improved by earthworm addition.
Acknowledgements
Shanshan Hu would like to thank the China Scholarship Council for
PhD scholarship (CSC, No. 201706760061). This work was financially
supported by the Fundamental Research Funds for the Natural Science
Foundation of Hubei Province (Grant No. 2014CFB928).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jhazmat.2018.12.039.
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