Environmental Pollution 255 (2019) 113184

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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

Ecological risk of combined pollution on soil ecosystem functions:

Insight from the functional sensitivity and stability*
Rong Jiang a, b, Meie Wang a, *, Weiping Chen a, Xuzhi Li a, e, María Balseiro-Romero c, d,
Philippe C. Baveye c
a
State Key Laboratory of Urban and Regional Ecology, Research Centre for Eco-environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China
b
University of Chinese Academy of Sciences, Beijing, 100049, China
c
UMR ECOSYS, AgroParisTech, Universit
e Paris-Saclay, Avenue Lucien Br
etigni
eres, Thiverval-Grignon, 78850, France
d
Department of Soil Science and Agricultural Chemistry, Universidade de Santiago de Compostela, Santiago de Compostela, 15782, Spain
e
State Environmental Protection Key Laboratory of Soil Environmental Management and Pollution Control, Nanjing Institute of Environmental Sciences,
Ministry of Ecology and Environment, Nanjing, 210042, China

a r t i c l e i n f o a b s t r a c t

Article history: Assessing the ecological risk of combined pollution, especially from a holistic perspective with the
Received 16 June 2019 consideration of the overarching functions of soil ecosystem, is crucial and beneficial to the improvement
Received in revised form of ecological risk assessment (ERA) framework. In this study, four soils with similar physicochemical
23 August 2019
properties but contrasting heavy metals contamination levels were selected to explore changes in the
Accepted 4 September 2019
Available online 10 September 2019
integrated functional sensitivity (MSI), resistance (MRS) and resilience (MRL) of soil microbial commu-
nities subjected to herbicide siduron, based on which the ecological risk of the accumulation of siduron
in the four studied soils were evaluated. The results suggested that the microbial biomass carbon, activity
Keywords:
Combined pollution
of denitrification enzyme and nitrogenase were indicative of MSI and MRS, and the same three pa-
Functional sensitivity rameters plus soil basal respiration were indicative of MRL. Significant dose-effect relationships between
Functional stability siduron residues in soils and MSI, MRS and MRL under combined pollution were observed. Heavy metal
Soil microbial communities polluted soils showed higher sensitivity and lower resistance to the additional disturbance of herbicide
Ecological risk assessment siduron due to the lower microbial biomass, while the resilience of heavy metal polluted soils was much
higher due to the pre-adaption to the chemical stresses. The quantifiable indicator microbial functional
stability was incorporated in the framework of ERA and the results showed that the accumulation of
siduron in the studied soils could exhibit potential harm to the integrated functional stability of soil
microbial community. Thus, this work provides insights into the application of integrated function of soil
microbial community into the framework of ERA.
© 2019 Elsevier Ltd. All rights reserved.

1. Introduction
Functional process performed by soil ecosystem contributes to a
wide range of ecosystem services that are essential to the sus-
tainability of human society, including element cycling and
Abbreviations: ERA, Ecological risk assessment; SI, stress index; RS, resistance
index; Rl, resilience index; MSI, integrated functional sensitivity of soil microbial
community; MRS, integrated functional resistance of soil microbial community;
MRL, integrated functional resilience of soil microbial communities; PEC, predicted
environment concentration; PNEC, predicted no effect concentration.
*
This paper has been recommended for acceptance by Dr. Yong Sik Ok.
* Corresponding author. 18 Shuangqing Road, Haidian District, Beijing, 100085,
China.
E-mail address: mewang@rcees.ac.cn (M. Wang).
nutrient supply, energy supply and natural attenuation capacity for
the pollutants. etc (Haines-Young and Potschin, 2010; Baveye et al.,
2016; Adhikari and Hartemink, 2016). With the intensively increase
of anthropogenic activity, soil ecosystems are susceptible to
contamination by mixtures of heavy metals and organic chemicals
(Yang et al., 2012; Szolnoki et al., 2013; Gulan et al., 2017), which are
expected to affect the ecological functions, services and sustain-
ability of soil ecosystem (Naveed et al., 2014). Evaluating and pre-
dicting the potential ecological risk caused by the pollutant
mixtures in soil ecosystem is critically important for environmental
managers and decision-makers. However, at present, the most
widely used endpoints or protection goals in ERA procedure are the
acute toxicity of single species, such as terrestrial plants or soil
dwelling invertebrates (EFSA Panel on Plant Protection Products

https://doi.org/10.1016/j.envpol.2019.113184
0269-7491/© 2019 Elsevier Ltd. All rights reserved.
2 R. Jiang et al. / Environmental Pollution 255 (2019) 113184
and their Residues (PPR) et al., 2017). There is a growing need to
develop methods for risk assessment of combined pollution from a
holistic perspective with consideration of overarching functions of
soil ecosystem (Faber and Van Wensem, 2012; Thomsen et al.,
2012; EFSA Scientific Committee, 2016; US Environmental
Protection Agency, 2016).
It is well known that microorganisms play a determinant role in
maintaining the sustainability of soil ecosystem functioning and
services (Nannipieri et al., 2003; Aislabie et al., 2013) and many
researches have carried out to investigate the response of soil mi-
croorganisms under the combined stress of heavy metals and
organic chemicals (Shen et al., 2005; Zhang et al., 2005; Wang et al.,
2007). However, most of the microbiological responses to the
exposure of contaminants (e.g. soil respiration, enzyme activity and
bacterial growth) are rather unstable in soils and usually appear as
wave-like and irregular fluctuations (Trasar-Cepeda et al., 2000;
Khan and Scullion, 2000; Labud et al., 2007; Zabaloy et al., 2008)
owing to the high degree of biological diversity and the inherent
spatial and temporal heterogeneity of soil microenvironment (Vos
et al., 2013; Baveye et al., 2018). Moreover, studies at lower bio-
logical levels (e.g. genetic, cellular, or individual) investigating the
short-term and high-dose exposure usually explicate mechanisms
or modes of toxic actions of xenobiotic pollutants. However, the
interpretation of toxicological events for risk management de-
cisions is typically associated with adverse responses observed at
higher biological levels (e.g. population, community, or ecosystem)
following long-term exposure to environmental concentrations
(Bradbury et al., 2004; Munns et al., 2009; De Laender et al., 2011;
De Laender and Janssen, 2013). Assessment of combined effects of
contaminant mixtures at the ecosystem function level could help to
address the ecological significance of toxic effects, as well as
improve the ecological risk assessment (ERA) framework for com-
bined pollution in soils.
At this point, robust methods are lacking to assess toxic effects
of contaminants at a community or ecosystem level because of
difficulties associated with distinguishing natural changes from
those caused by anthropogenic disturbances (Rohr and Crumrine,
2005). Moreover, it is particularly problematic to establish dose-
effect relationships between concentrations of contaminants and
changes in endpoints at the community or ecosystem level since
many ecosystem components are dynamic and interdependent,
and the contaminants may directly or indirectly affect the com-
munity structure/function through a number of ecological mecha-
nisms (Clements and Kiffney, 1994; Munns et al., 2009).
The integrated functional sensitivity and stability of soil mi-
crobial communities, may gauge changes of ecological functions,
characterize the stressed soil ecosystems and contribute essentially
to maintaining soil ecosystem functioning and services when the
additional environmental disturbances arose (Griffiths et al., 2001;
Allison and Martiny, 2008; Shade et al., 2012; Griffiths and
Philippot, 2013). Thus these two attributes appear to be particu-
larly reasonable and applicable assessment endpoints in evaluating
the realistic toxic effect of contaminants on the functioning process
of soil ecosystem (EFSA Panel on Plant Protection Products and
their Residues (PPR) et al., 2017; US Environmental Protection
Agency, 2016). The terms of soil resistance (the inherent capacity
of soil to withstand stress/disturbance) and resilience (the ability of
soil to recover after stress/disturbance) were introduced to quantify
the stability of soil microbial communities subjected to different
types of stress/disturbance (Griffiths et al., 2001). The stress index
(SI) is used to quantify the sensitivity of soil microbial communities
to stress (Wang et al., 2017). Further, the functional parameters in
the functioning process, such as carbon cycle (microbial biomass
carbon-MBC, substrate induced respiration-SIR, basal respiration-
SR) and nitrogen cycle (potential nitrification rate -PNR,
denitrification enzyme activity-DNR, and nitrogenase activity-NI)
can be used to reflect the functional sensitivity and stability of
soil microbial communities (Orwin et al., 2006; Griffiths et al.,
2008; Li et al., 2016).
The functional sensitivity and stability depend on the intrinsic
traits of the community itself and are also closely linked to the
characteristics of stress, such as the intensity, frequency, and
duration (Tobor-Kapłon et al., 2005; Bressan et al., 2008; Folke et al.,
2010). The evolution of stress or disturbance over time would affect
the stability of soil microbial communities (Philippot et al., 2008).
Many studies have revealed that the initial stress or disturbance
history could suppress the functional stability of soil microbial
ecosystems due to the consumption of energy resources to cope
with the pre-disturbances (Bruins et al., 2000; Loreau, 2000) or
promote the functional stability because of the pre-adapting and
evolving in response to the pre-disturbances (Díaz-Ravin ~ a et al.,
1994; D. Vinebrooke et al., 2004). However, this is still under
debate (e.g. Müller et al., 2002; Tobor-Kapłon et al., 2005; 2006;
Arthur et al., 2012; Li et al., 2014; 2016).
Elevated concentrations of heavy metals such as Cu, Pb, Zn and
Cd in urban soils have been broadly noticed (Wang et al., 2012).
Meanwhile, as one of the most widely used herbicide in urban
lawn, the detection of siduron in urban and suburban surface water
(Jiang et al., 2018a; 2018b) as well as its adverse effect on soil biotas
(Jiang et al., 2017; Uwizeyimana et al., 2018; Li et al., 2019),
including soil microorganisms (Wang et al., 2017) have been
extensively reported. Thus, the co-existence of heavy metals and
siduron can be found commonly in urban soil and imposes adverse
impact on the microbial functional process in soil. Thus, in this
general context, four natural field soils with contrasting heavy
metal pollution level were chosen to investigate the functional
sensitivity and stability of microbial community in response to
siduron, based on which the ecological risk of siduron was evalu-
ated. Thus this work can provide insight into the application of
functional sensitivity and stability of microbial community into the
framework of ecological risk assessment.
2. Materials and method
2.1. Study sites and soil sampling
Soils were collected from two public parks located in Beijing,
China. Soil from Chaoyang park (C) was selected as the reference
soil. The other three (L, M and H) were collected from Nanguan Park
and contaminated with multiple heavy metals at different levels
(Wang et al., 2017). The two public parks were about 5 km apart,
comprise a soil that has been classified as fluvo-aquic and has never
received siduron prior to our experiments. Topsoil (0e20 cm) was
sampled by mixing five soil cores obtained in a 10 m
10 m square,
after removing covering plants and roots. Fresh soil samples were
sieved to pass through a 2 mm screen, and then stored at 4  C until
microbiological tests. The storage time is no more than two weeks
and soil samples were pre-incubated for 24 h to activate soil
microorganism before incubation experiment. The general physi-
cochemical characteristics of the four soils were reported before in
our previous study (Jiang et al., 2018a) and summarized in Table S1.
Those four soils had similar physicochemical properties; however,
the comprehensive pollution level (Pn) of heavy metals ranged from
1.64 for soil C to 60.1 for the heaviest contaminated soil (H) based
on the background value of Beijing (Chen et al., 2005).
2.2. Microcosm incubation
Fresh soil samples from the four studied sites were mixed with
 R. Jiang et al. / Environmental Pollution 255 (2019) 113184
different levels of siduron (0, 6, 30 and 60 mg kg1DW based on
0
, 1
, 5
and 10
of the recommended application dose of
siduron in the field) (US Environmental Protection Agency, 2008).
For each soil, there were a total of 15 vials (3 replicates
5 time
points) for each siduron treatment and control treatment. A 100 g
of three replicated soil samples were weighted into 300 ml vials.
The vials were loosely covered to allow air exchange and incubated
at a constant temperature of 25  C in dark for 120 days based on the
half-life time of siduron in soils (90e120 day) (Domine et al., 1993).
Soil moisture was replenished regularly to maintain a constant
level (equivalent to 70% of the water content obtained after free
drainage for 2 days) and the designated containers were withdrawn
for analyses at the 0-, 30-, 60-, 90- and 120-day incubation
intervals.
2.3. Microbiological and siduron residue analyses
Basal respiration (SR) and substrate-induced respiration (SIR)
were determined according to Elberling and Brandt (2003) and Li
et al. (2014). Microbial biomass carbon (MBC) was estimated by
the fumigation-extraction method (Wu et al., 1990). Soil potential
nitrification rate (PNR) was determined as described by Kurola et al.
(2005). Soil denitrification enzyme activity (DNR) was determined
referring to Barton et al. (2000). Nitrogenase activity (NI) was
determined using the acetylene (C2H2) reduction technique (Patra
et al., 2006). The residual of siduron in soil samples were extrac-
ted by methanol to measure using a high-performance liquid
chromatograph (Agilent 1260 HPLC). The details of microbiological
and siduron residue analyses were available in Supporting
Information.
2.4. Data analysis
2.4.1. Calculation of stress index (SI), resistance index (RI) and
resilience index (RL)
The stress index (SI) (Wang et al., 2017) was used to assess the
sensitivity of the soil microbial community under combined
pollution by heavy metals and siduron. The SI (Eq. (1)) was evalu-
ated by comparing the relative stress response (RSR) (Eq. (2)) with
the two threshold values, Th 1 and Th 2, following Dagnino et al.
(2008).
RSR < Th1; SI ¼ 0Th1 < RSR  Th2; SI
¼ ðRSR  Th1Þ=ðTh2  Th1Þ; RSR > Th2; SI ¼ 1

 

j
j ref

RSRi ¼ j
MRref
i  MRi =MRi

where, RSRji is the relative stress response for the ith microbial
parameter from the siduron treatments; MRji and MRref i are the ith
microbial response from the siduron treatments and the control
treatment, respectively.
The resistance RS (Eq. (3)) of soil microbial parameters at day 0,
30, 60, 90, 120 were calculated based on the change in microbial
parameters with or without siduron treatments, and the resilience
RL (Eq. (4)) at day 30, 60, 90, 120 were further calculated based on
the different relative changes between day x and day 0, according
to Orwin and Wardle (2004):
2jDx j
RSðtx Þ ¼ 1 
ðCx þ jDx jÞ
3
2jD0 j
RLðtx Þ ¼ 1 (4)
ðjD0 j þ jDx jÞ
D0 ¼ C0  P0 (5)
Dx ¼ Cx  Px (6)
where, C0 and Cx are the values of the tested parameter in control
on the first and the xth day of treatment, respectively; P0 and Px are
the values of the tested parameter in siduron treatments on the first
and the xth day of treatment, respectively.
The SI is a standardized value that ranges from 0 to 1 as the
stress increases from none to the highest, while RL(tx) and RS(tx) are
bounded between 1 and þ1. A RL (tx) or RS (tx) value of þ1 in-
dicates no disturbance (maximal resistance) and full recovery
(maximal resilience), respectively. While values closer to 1 indi-
cate stronger disturbance and slower recovery respectively.
2.4.2. Calculation of the integrated microbiological stress index
(MSI), resistance index (MRS) and resilience index (MRL)
The computations outlined in the previous section would result
in a set of stress (SI), resistance (RI) and resilience (RL) indices in
terms of each microbial parameters. Significances of the indices
were consolidated and prioritized through the principle compo-
nent analysis (PCA) that would sort out factors most closely related
to the stress of siduron. The dissimilarities in sensitivity and sta-
bility between various soils were further tested by similarities
analysis (ANOSIM) and permutational multivariate analysis of
variance (PERMANOVA). Then, the integrated microbiological
stress index (MSI) (Eq. (7)), resistance index (MRS) (Eq. (8)) and
resilience index (MRL) (Eq. (9)) for each siduron treatment of each
soil were calculated by combining the representative parameters to
quantify the integrated sensitivity and stability of the entire soil
microbial community to the stress caused by siduron.
X
n
MSI ¼ SIi =n (7)
i¼1
X
n
MRS ¼ RSi =n (8)
i¼1
X
n
MRL ¼ RLi =n (9)
i¼1
(1)
where, SIi, RSi and RLi refer to the stress index, resistance index and
resilience index, respectively, for the ith representative microbial
(2)
parameter and n refers to the number of representative parameters
considered.
2.4.3. Estimation of dose-effect relationship between MSI, MRS,
MRL and the residues of siduron in soil
The dose-effect relationship between soil residual concentra-
tions of siduron and individual integrated indices were plotted for
each soil based on the data from the siduron treatments for the
entire time intervals. To linearize the dose-effect relationship be-
tween functional sensitivity and stability of soil microbial com-
munity and residual concentration of siduron, a ratio method was
introduced referring to the pharmacological statistics method (Sun,
1987), where the X-axis was the residual concentrations of siduron
(3) in soil and the Y-axis was the ratio of residues of siduron in soil to
each integrated index.
According to the calculation equations of SI, RS, and RL (Eqs. (1),
4 R. Jiang et al. / Environmental Pollution 255 (2019) 113184
(3) and (4)), when the response of the microbial community in the
siduron treatment was equal to a half of that in the control, e.g.,
RSR ¼ 0.5 for SI, Dx ¼ 1/2 Cx for RS, and Dx ¼ 1/2 D0 for RL, the cor-
responding half effects of SI, RS and RL values were 1/2, 1/3 and 1/3,
respectively. The 50% effect for those integrated indices (MSI, MRS,
and MRL) were 1/2, 1/3 and 1/3, respectively, according to Eqs.
(7)e(9). The half effect concentrations of siduron (EC50) for the
sensitivity, resistance, and resilience of the soil microbial commu-
nity functioning were thus calculated.
2.4.4. Ecological risk assessment of the accumulation of siduron in
the four studied soils based on the endpoint of functional sensitivity
and stability
In our previous reported work, the accumulation level of
siduron in the top layer of the four studied soils (PECsiduron) under
two application pattern, including park & residential turf and golf
course greens (US Environmental Protection Agency, 2008), after
continuous application of ten years were simulated using HYDRUS-
1D model (Jiang et al., 2018a). In this work, based on the EC50 of the
functional sensitivity and stability, the predicted no effect con-
centration of siduron (PNECsiduron) can be calculated by dividing the
EC50 by an assessment factor of 10 (European Chemicals Agency
(ECHA), 2008). The risk characterization ratio (RCR), a ratio be-
tween PECsiduron and PNECsiduron, was used to characterize the
ecological risk of siduron.
2.4.5. Statistical analysis
Multi-way ANOVA of microbial parameters between soils, in-
cubation intervals and siduron treatments were conducted using
SPSS 18. The fit of dose-response relationship between the micro-
bial index and siduron concentration was conducted using Ori-
ginPro. Principal Component Analysis (PCA), similarities analysis
(ANOSIM) and permutational multivariate analysis of variance
Fig. 1. The residue rate (%) of siduron in the four studied soils treated with siduron at 6 ppm (A), 30 ppm (B) and 60 ppm (C), respectively.
(PERMANOVA) of the stress index, resistance index, and resilience
index of the four studied soils were conducted using Past 3.
3. Results and discussion
3.1. Changes in siduron residue in soils
The amount of siduron residue at different incubation intervals
over a 120 days period (Fig. 1) was the lowest in clean soil C and the
highest in the soil M that has the highest soil organic matter (SOM)
content. The soil-borne siduron were all reduced more or less to
95% during a 120-day incubation when the input was at the 6 mg
siduron kg1 of soil. By contrast, the reductions of added siduron
were 93, 83, 66, and 82% and 72, 52, 47, and 66% in soils C, L, M, and
H and for 30 and 60 mg siduron kg1 soil, respectively. The results
indicated that the ability of the microbial community to attenuate
siduron was adversely impacted in heavy metal polluted soils, as
heavy metals can affect the physiology of microorganisms involved
in the degradation of organic chemicals (Sandrin and Maier, 2003)
due to the formation of unspecific complex compounds in the cell
(Roane et al., 2001; Olaniran et al., 2009; 2013). While, the atten-
uation of siduron in soil M with the highest content of organic
matter was the lowest, due to the strong adsorption ability of
organic matter to siduron, which made the siduron less bioavail-
able (Jiang et al., 2018b).
3.2. Microbial activity of siduron treated soils
As shown in Table S2, the responses of the soil microbial com-
munities as characterized by changes of MBC, SR, SIR, PNR, DNR and
NI were all significantly affected (p < 0.05) by the siduron input
levels, extents of heavy metal pollution, durations of incubation,
and their two factors and three factors interactions, with the
 R. Jiang et al. / Environmental Pollution 255 (2019) 113184
exception of the binary effect of siduron and time for SR and ternary
effects of soil, siduron, and time for SR and PNR. The results sug-
gested the sensitivity and instability of the microbial community
when exposed to the stresses caused by heavy metals and siduron.
Microbial Carbon Biomass, Basal Respiration and Substrate
Induced Respiration. The MBC of the four studied soils undergoing
different treatments (Fig. S1 in the Supplement Information)
ranged from 152 to 469 mg kg1, and decreased by 29.6%, 67.6% and
53.1% in the heavy metal contaminated soils L, M and H, respec-
tively compared with the clean soil C at day 0 without the addition
of siduron. Without the siduron input, the MBC of all soils,
regardless of the extent of heavy metal pollution, increased at the
end of 120-day incubation period. However, the MBC decreased
when the soils were treated with siduron in comparison to those
not treated with siduron.
As with the MBC, the SIR of the four studied soils ranged from
15.7 to 25.5 mL CO2 g1 h1 and decreased in the order of
C > L > H > M before treatment with siduron at day 0 (Fig. S2 in
Supplement Information). However, the SR in heavy metal
contaminated soils L, M and H were 1.5, 1.01, and 2.01 times higher
than those in reference soil C before treatment with siduron at day
0. Changes in the SIR in the four soils exhibit similar trends in both
treatments with or without the addition of siduron. However, the
differences in SIR among siduron treatments were not significant
(p > 0.05). As shown in Fig. S3 in the Supplement Information, the
SR in heavy metal contaminated soils decreased when subjected to
the stress caused by siduron, and were significantly inhibited at day
30 in soil L and at day 90 in soil M and H (p < 0.05). In contrast, the
addition of siduron activated SR in clean soil C.
Potential Nitrification Rate, Activity of Denitrification
Enzyme, and Nitrogenase Activity. The PNR, DNR and NI were
associated with key processes of the nitrogen cycle (Figs. S4, S5, and
S6 in the SI). The PNR of the four studied soils ranged from 1.43 to
2.27 mg N g1 h1 and were 21%, 59% and 17% higher than that of the
reference in the heavy metal contaminated soils L, M, and H,
respectively. The PNR in soil C increased significantly with the in-
crease of siduron concentration at day 90 and 120 (p < 0.05) (Fig. S4
in the SI). In contrast, PNR in heavy metal contaminated soil L and
M decreased with the increasing siduron concentration throughout
the experiment, while there were no differences in PNR among
treatments in soil H.
Before addition of siduron, the DNR in the heavy metal
contaminated soils L, M, and H were 40%, 75%, and 16% lower than
that in the reference soil C, respectively (Fig. S5 in SI). The DNR
increased significantly in the clean soil C, while it decreased in
heavy metal contaminated soils in all the treatments (p < 0.05)
after a 120 day incubation period. The DNR in the reference soil C
spiked with siduron was significantly inhibited throughout the
experiment, whereas the effects of siduron on DNR in heavy metal
contaminated soils were not as consistent as those in soil C. The
nitrogenase activity (NI) in the four studied soils ranged from 6.45
to 252 ng N g1 h1 before siduron treatment at day 0 and was
significantly up regulated in the heavy metal contaminated soils
and the NI in soil L, M and H increased by 22.8, 39.1, and 5.16 times
respectively compared with the reference soil C. Exposure to
siduron strongly increased the nitrogenase activity in soil C and L
(Fig. S6 in SI); however, the addition of siduron did not affect NI
significantly in soil M and H.
Responses of soil microbial communities to disturbances or
stress can provide an effective ‘early warning’ of the deterioration
of soil quality and ecological function (Winding et al., 2005).
However, soil function is difficult to recover within a short-term
period as degradation and aging of pollutants, as well as develop-
ment of resistant microorganisms, will take a long time (Deng,
2012). Thus, in this work, the integrated functional stability was
5
calculated following a 120-day stress of herbicide siduron under
environment concentration in in-situ heavy metal polluted soils
with the incubation interval of 30 days. In this study, long-term
heavy metals exposure exerted a detrimental effect on MBC SIR
and DEA, while SR, PNR and NI were facilitated. The sensitive
response of the aforementioned parameters to the stress of heavy
metals was also reported in studies by Smolders et al. (2001),
Karaca et al. (2002) and Oliveira et al. (2009). Different microbio-
logical responses to the subsequent herbicide siduorn were
observed among the four studied soils, while the responses were
rather unstable at different exposure concentrations and time
points. Thus, it is difficult to determine the explicit effects of
siduron on soil microbial community function using actual exper-
imental data. The same applications to the establishment of dose-
response relationships between siduron concentration and micro-
bial parameters are also rather difficult.
3.3. Stress, resistance and resilience index of soil microbial
community under combined pollution of heavy metals and siduron
The index method was introduced to quantitatively evaluate the
long-term joint toxic effects of siduron on soil microbial commu-
nity functioning instead of qualitatively describing the microbial
response to the stress of siduron with actual experimental data. The
configuration of the SI, RS, and RL values of the treatments in the
four soils in the PCA graph (Fig. 2) graphically illustrates the func-
tional sensitivity, resistance, and resilience of the soil microbial
community at 30, 60, 90 and 120 day after the addition of different
levels of siduron. The length of vectors indicates the strength of the
different variables.
As shown in Fig. 2, the first two principal components account
for about 80%, 68% and 72% of the total variance for SIs, RSs and RLs,
respectively. The vectors corresponding to SI and RS of NI, DNR and
MBC represent the integrated functional sensitivity and resistance
of the entire soil microbial community subject to siduron treat-
ments (Fig. 2A and B). A significant influence of phenylurea herbi-
cides including diuron, linuron, and chlortoluron on MBC, NI and
DNR was also reported in studies by El Fantroussi et al. (1999),
Yeomans and Bremner (1985) and Jena et al. (1987). The same three
parameters plus SR were representative indicators of the resilience
of soil microbial function (Fig. 2C). The SR representing the overall
rate of total metabolic processes can indicate metabolic changes in
response to stresses (Nannipieri et al., 2003) and is one of the most
common indicators in studies of soil microbial community stability
(Orwin et al., 2006; Griffiths et al., 2008). The parameters SIR and
PNR were not screened as representative indicators in this study,
although many studies have reported that they are sensitive to the
presence of pollutants and could reflect different levels of pollution
(Smolders et al., 2001; Broos et al., 2007).
The SIs, RSs and the RLs of the four studied soils were distinc-
tively separated (Fig. 2), indicating that the change trend in
response to the stresses became more clear and visible after the
introduce of the integrated index, because the variations in soil
microbial parameters were normalized based on the control
treatment and beginning status (Asensio et al., 2013; Wang et al.,
2017). The application of the dissimilarity tests ANOSIM and PER-
AVOVA further reveals significant differences in SIs, RSs and RLs
between reference soil C and heavy metal contaminated soils L, M,
H (Table S3), suggesting that the functional sensitivity, resistance,
and resilience of the soil microbial community to the addition of
siduron depended strongly on the levels of pre-contamination of
heavy metals in soils. However, the difference in SIs and RSs be-
tween soil M and H is insignificant (p > 0.05) (Table S3), indicating
that the functional sensitivity and resistance of microbial commu-
nity might also be affected by some physicochemical properties of
6 R. Jiang et al. / Environmental Pollution 255 (2019) 113184

Fig. 2. Stress index (A), resistance index (B), and resilience index (C) of microbiological parameters to contamination with siduron in the four studied soils based on PCA. The vectors
represent the primary variable. Cases 1e5, 6e10, and 11e15 indicate the index under siduron concentrations of 6, 30, and 60 mg kg-1 at 0, 30, 60, 90 and 120 days, respectively.

soil (Griffiths and Philippot, 2013).
3.4. Dose-response relationship between siduron residual
concentration and the integrated sensitivity and stability of soil
microbial community functioning
The indices MSI, MRS, and MRL, denoting the integrated func-
tioning sensitivity, resistance, and resilience of soil microbial
communities exposed to siduron, were derived by averaging the
respective indices (i.e. stress, resistance, and resilience) due to the
impacted microbial activity factors (see Eqs. (7)e(9)). The signifi-
cant dose-response relationships between residual concentrations
of siduron and the corresponding MSI, MRS, MRL were all estab-
lished (p < 0.01) (Fig. 3, Table 1). The results indicated that the in-
tegrated index method is valid to quantify the integrated response
of soil microbial community function to the combined pollution of
heavy metals and agrichemicals.
The EC50MSI of siduron in the four soils ranges from 0.115 to
3.14 mg kg1 and decreases in the order of C > L > M > H (Table 1),
indicating that heavy metal contamination significantly increases
the sensitivity of microbial community functioning to the addition
of siduron. It is consistent with the results reported by Wang et al.
(2017), who used the soil from the same sites and observed higher
stress indexes in heavy metal contaminated sites relative to the
reference site under siduron stress. Similar to the EC50MSI, the
EC50MRS of the four soils ranges from 0.045 to 2.29 mg kg1, and
decreases as heavy metal pre-contamination levels increased,
which suggests that heavy metal contamination would severely
damage the resistance ability of microbial communities. The higher
sensitivity and lower resistance ability of heavy metal polluted soils
might be explained by the lower biomass of the microbial com-
munity (Figs. S1 and S2 in SI). The presence of a large amount of
biomass can minimize the likelihood of large changes in ecosystem
functioning after disturbances because of the high level of func-
tional redundancy (Ives et al., 2000; McCann, 2000; Balvanera et al.,
2006). The decreased resistance in a heavy metal contaminated soil
compared with the control soil due to the reduced microbial
biomass and diversity was also reported by Müller et al. (2002).
However, the EC50MRL in reference clean soil C is the lowest
(0.998 mg kg1), whereas the EC50MRL values in heavy metal
polluted soil L, M and H are about 1.01, 2.45, and 22.8 times higher
than in the reference clean soil C, respectively. It indicates that the
 R. Jiang et al. / Environmental Pollution 255 (2019) 113184 7

Fig. 3. Dose-response relationship between the residual concentration of siduron and MSI (A), MRS (B), and MRL (C) in the four studied soils based on the ratio method.

Table 1
Relationships between the residual concentration of siduron (C) and MSI, MRS, and MRL in the four studied soils.

Soil Regression equation R2 p Half effect concentration (mg kg1)

Sensitivity C C/MSI ¼ 7.59C-17.6 0.871 <0.01 3.14

L C/MSI ¼ 5.37C-9.37 0.918 <0.01 2.78
M C/MSI ¼ 10.8C-8.25 0.803 <0.01 0.934
H C/MSI ¼ 11.4C-1.08 0.672 <0.01 0.115
Resistance C C/MRS ¼ 1.59C þ 3.25 0.851 <0.01 2.29
L C/MRS ¼ 1.76C þ 0.479 0.983 <0.01 0.386
M C/MRS ¼ 1.47C þ 0.379 0.994 <0.01 0.249
H C/MRS ¼ 1.55C þ 0.066 0.974 <0.01 0.045
Resilience C C/MRL ¼ 2.94C þ 5.93 0.785 <0.01 0.998
L C/MRL ¼ 4.35C þ 7.45 0.912 <0.01 1.01
M C/MRL ¼ 10.2C-17.7 0.647 0.023 2.45
H C/MRL ¼ 3.13C-3.03 0.956 <0.01 22.8

functional resilience of microbial community increases greatly as
the levels of pre-contaminated heavy metals increased. This might
be attributed to the pollution induced community tolerance, as soil
microbial communities in long-term field heavy metal contami-
nated soil had the opportunity to adapt to the toxicant (Mertens
et al., 2006; Brandt et al., 2010; Ferna
ndez-Calvin~ o et al., 2011).
An increasing body of evidence also suggests that long-term metal
exposure can positively affect the long-term resilience of the mi-
crobial community if additional stresses arose (Mertens et al., 2007;
Arthur et al., 2012; Li et al., 2014). It has also been reported that the
stability of a stressed microbial community to a subsequent
disturbance might depend on the nature of the disturbance (Tobor-
Kapłon et al., 2005; 2006). If the primary and subsequent distur-
bances were similar, the resilience of the soil microbial community
might be stronger (Philippot et al., 2008). Siduron and heavy metals
have a similar type of dose-response cure for soil biotas (Jiang et al.,
2018a). Meanwhile both chemicals were reported to have toxicity
mechanisms to soil microorganisms involved in the disruption of
essential biological molecules in functional processes, e.g. meta-
bolism, detoxification, photosynthesis (De Lorenzo et al., 2001;
Tchounwou et al., 2012; Hussain et al., 2015). Thus, it could be
concluded that those two chemicals were similar in toxicology to a
certain extent. The similarity of the primary and subsequent stress
might also contribute to the development of tolerance of microbial
communities (Rusk et al., 2004; Philippot et al., 2008; Li et al.,
2016).
3.5. Ecological risk assessment of the accumulation of siduron in
the studied soils based on the endpoint of integrated functional
sensitivity and stability
Our previous study showed that the accumulation of siduron in
the top 0e20 cm layer of the four studied soils ranged from 1.13 to
3.49 mg kg1, and from 43.8 to 107 mg kg1 under the application
8 R. Jiang et al. / Environmental Pollution 255 (2019) 113184
Fig. 4. Risk characterization ratio (RCR) of siduron in four soils after 10 years' application under patterns of park & residential turf (A) and golf course greens (B) based on the
PNECsoil derived from functional sensitivity and stability of soil microbial communities.
pattern of park & residential turf and golf course greens, respec-
tively after continuous application of ten years (Jiang et al., 2018a).
It needs to note that the functional resistance of the microbial
community in heavy metals polluted soils is more susceptible to
siduron stress than the functional sensitivity and resilience. Thus,
for heavy metal contaminated soil, the functional resistance of the
microbial community is used to determine the predicted no effect
concentration (PNEC). However, for the clean soil C, EC50MRL is
lowest and the derivation of PNEC is based on the endpoint of
functional resilience of the soil microbial community. Thus, the
lowest PNECsoil of siduron derived from functional sensitivity and
stability were 99.8, 38.6, 24.9, 4.50 mg kg1 in soil C, L, M, H,
respectively, based on which the potential ecological risks of the
accumulation of siduron in the top 0e20 cm soil layer after 10
years’ application were calculated.
As shown in Fig. 4, the risk characterization ratio (RCR) in four
studied soils ranged from 11.3 to 354, and from 439 to 12507, under
two application respectively after 10 years. All the RCR values are
far greater than 1 and increased in the order of C < L < M < H under
both application patterns. The RCR in heavy metal contaminated
soil L, M, H increased by 5.04, 12.3, 31.1 and 4.18, 9.82, 28.5 times,
respectively under application patterns of park & residential turf
and golf course greens compared with soil C. The results indicate
that the accumulation of siduron would exhibit potential harm to
the integrated functions of soil ecosystem and especially in heavy
metal polluted soils. The chronic bioassays in microcosm testing in
this study could provide more realistic and refined effect assess-
ments by evaluating the integrated functional sensitivity, resis-
tance, and resilience of the field soil microbial communities under
the exposure of environment concentration level. This work pro-
vided the insight into the application of integrated functions in the
ecological risk assessment of soil pollution.
4. Conclusion
This study demonstrated that the stress, resistance, and resil-
ience of microbial communities of heavy metal polluted soils when
exposed to herbicide siduron might be deduced from the mea-
surements of microbial activity factors such as the basal respiration
(SR), substrate-induced respiration (SIR), denitrification enzyme
activity (DNR), nitrogen activity (NI), potential nitrification rate
(PNR), and microbial biomass (MBC) of impacted soil microbial
communities. The stress index, resistance and resilience of each
scenario could be calculated. The overall stress (MSI), resistance
(MRS) and residence (MRL) indices of the soil microbial
communities were obtained by averaging each representative mi-
crobial activity. The MSIs, MSRs, and MRLs would be the ultimate
quantitative measurements on the wellbeing of the impacted soil
ecosystems. The residual dose Csiduron (mg siduron kg1 soil) versus
MSI (or MRS, MRL) relationships were established using ratio
method to obtain the 50% effect concentration EC50MSI, EC50MRS and
EC50MRL, from which the predicted no effect concentration of
siduron (PNECsiduron) was derived with an assessment factor. The
ecological risks of the accumulation of siduron on the integrated
functional stability of microbial communities in the studied soils
were further calculated using the risk ratio between the predicted
environment concentration of siduron (PECsiduron) and the corre-
sponding PNECsiduron. In this work, we propose that the use of in-
tegrated functional stability of soil microbial community as a
quantifiable endpoint would enhance the reality and acceptance of
results and further improve the potential for actual utility in
environmental decision-making.
Acknowledgements
We gratefully acknowledge the financial support provided by
the National Key R&D Program of China (No.2018YFC1800505) and
the Special Foundation of the State Key Lab of Urban and Regional
Ecology.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.envpol.2019.113184.
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