Journal of Cleaner Production 232 (2019) 1452e1464

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Journal of Cleaner Production

journal homepage: www.elsevier.com/locate/jclepro

Environmental risk of chlorine-controlled clogging in drip irrigation

system using reclaimed water: the perspective of soil health
Peng Song a, b, Gary Feng b, John Brooks b, Bo Zhou a, c, Hongxu Zhou a, Zhirui Zhao d,
Yunkai Li a, *
a
College of Water Resources and Civil Engineering, China Agricultural University, Beijing, 100083, China
b
Genetics and Sustainable Agricultural Research Unit, United States Department of Agriculture, Starkville, MS, 39762, USA
c
College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, WI, 53706, USA
d
Research Center for Eco-environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China

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

Article history: Chemical chlorination is an effective method to control the emitter bio-clogging in drip irrigation using
Received 1 November 2018 reclaimed water, but the broad spectrum of strong oxidative bactericidal action of chlorine causes certain
Received in revised form risks to soil microbial communities and even soil health. Therefore, in this study, spring maize was
10 May 2019
selected as the research object, field chlorine experiments with drip irrigation using reclaimed water
Accepted 4 June 2019
were carried out in two years, high-throughput sequencing technology combined with phospholipid
Available online 6 June 2019
fatty acids (PLFAs) technology was used, the biological bio-indicator of soil health (microbial community
structure of soil) in the root zone of the spring maize were systematically studied on the effects of
Keywords:
Reclaimed water
different chlorination modes under the drip irrigation using reclaimed water. The effects of microbial
Drip irrigation community structure on soil enzyme activity and spring maize yield and quality were analyzed. The
Chlorination results showed that the total amount of PLFAs and bacteria content significantly decreased by 17.7
Soil microbial community %e44.7 % and 7.0 %e47.3 %, which reduced the microbial community diversity. The relative abundance of
Nitrospirae, Actinobacteria and Firmicutes decreased at the phylum level. The changes of microbial
community structure reduced urease, catalase and phosphatase activities, inhibited the conversion and
absorption of nutrients in the soil, which led to a decrease in crude fat and protein by 2.2 %e16.6 % and
2.2 %e14.1 %, respectively. But the yield of spring maize didn't significantly reduce. In comparison, long-
term use of high concentration short duration chlorination mode was more likely to have adverse effects
on soil health than low concentration long duration chlorination mode. The research results can provide
a reference for the management of reclaimed water network systems and soil health.
© 2019 Elsevier Ltd. All rights reserved.

1. Introduction
With the acceleration of urbanization, population growth, and
industrial development, water shortages have become one of the
main bottlenecks restricting the sustainable development of soci-
etal economics. The shortage of agricultural water can effectively be
solved by using reclaimed water for farmland irrigation (Capra and
Scicolone, 2007; Adrover et al., 2012; Chen et al., 2013; Becerra-
Castro et al., 2015; Alon, 2016; Guo et al., 2017). The use of
reclaimed water for drip irrigation can minimize health and envi-
ronmental risks (Tripathi et al., 2016), improve water use efficiency
* Corresponding author.
E-mail address: liyunkai@126.com (Y. Li).
and crop yield (Lu et al., 2016). However, it still contains a large
number of solid particulates, nitrogen, phosphorus, organic matter
and microbial flora, which greatly increases the risk of clogging in
drip irrigation systems (Capra and Scicolone, 2005). The primary
focus of drip irrigation systems is on the potential for emitters to
clog. According to the survey, one third of the drip irrigation pro-
jects, due to improper handling of the clogged emitter, have been
abandoned in China (Li et al., 2015). The application areas of sub-
surface drip irrigation in Xinjiang Construction Corps was forced to
decrease from 8000 ha in 2005 to 67 ha in 2014 due to the emitter
clogging problem (Li et al., 2018). Since the first International Drip
Irrigation Conference was held in Israel in 1971, many have tried to
solve the emitter clogging problems in drip irrigation by means of
properly installing filter equipment, periodic acid and chlorine
treatment, and optimization of emitter channel design,

https://doi.org/10.1016/j.jclepro.2019.06.050
0959-6526/© 2019 Elsevier Ltd. All rights reserved.
 P. Song et al. / Journal of Cleaner Production 232 (2019) 1452e1464
electrochemical method, the microbial antagonism, the magneti-
zation treatment (Taylor et al., 1995; Li et al., 2008; Liu and Huang,
2009; Zhou et al., 2019).
Chlorination is a common method for preventing and treating
bio-clogging in drip irrigation systems using reclaimed water
(Rav-Acha et al., 1995; Dehganisanij et al., 2005; Elif et al., 2006; Li
et al., 2010a). In fact, chlorination, with the advantages of economy
and high efficiency, is often used for disinfection of sewage and
drinking water (Sharma et al., 2016). The commonly used hypo-
chlorous acid is a small neutral molecule that will quickly adsorb
on the surface of bacteria or viruses, and enters the interior of cells
by penetrating the cell wall or altering cell wall permeability.
Proteins and nucleic acid inside the bacteria or viruses are dena-
tured under strong oxidative forces (Cloete, 2003). Therefore, its
strong oxidative sterilization has broad spectrum targeting. Pre-
vious studies have focused on the disinfection of water pipelines.
It have shown that the use of chlorine disinfection can reduce the
total number of bacteria or coliforms by more than 95 % (Li et al.,
2010b; Wang et al., 2017). Norton (2000) found that Gram-
negative bacteria accounted for 97 % of the cultivated bacterial
population before chlorination; whereas, following chlorination,
98 % of the cultivated total bacteria were Gram-positive. Thus, the
community composition was altered due to chlorination and leads
us to the next question, “how chlorine affect the soil microbial
composition and community structure after chlorinated irrigation
water is applied to the soil?”.
Soil microbes are significantly associated with soil enzyme ac-
tivity (Acosta-Martínez, 2008) and are directly involved in soil ni-
trogen cycling, promoting uptake, transformation, and
bioavailability (Schimel and Bennett, 2004). Soil organic phos-
phorus mineralization is also dependent on soil microbial processes
(Richardson et al., 2009), relying on soil enzymes to hydrolyze
organic phosphorus to phosphate and other small molecular
phosphorus compounds for plant absorption and utilization.
Studies have shown that the accumulation of chlorine may
damage the plant roots to some extent (Coelho and Resende,
2001); likewise, it also exacerbates the accumulation of salt ions,
resulting in uneven nutrient distribution. Chlorination can reduce
the yields and quality of chlorine-sensitive tobacco, potato and
other crops. Li et al. (2012a) found that chlorination inhibited the
conversion process and accumulation of soil nitrogen, which
decreased nutrient uptake, and led to a decrease in tomato yield.
Drip irrigation using reclaimed water significantly increased to-
mato soluble sugar and water-soluble total acid, but the ascorbi-
cacid (Vc) content and soluble solids in fruits were significantly
reduced (Li et al., 2012a). Li et al. (2014) preliminary investigated
the effect of chlorination practice on soil chemical properties and
found that the residual chlorine in the soil increased after chlo-
rination, especially for the frequent chlorination at a high injection
concentration. Since there is still uncertainty risk about chlorina-
tion application, it is urgent to comprehensive examine the effects
of chlorination in drip irrigation systems on crop system soil
health.
Considering the planting area of the spring maize continuously
increased in North China and is still increasing, and it required
supplementary irrigation during the growing stage, which always
utilized the reclaimed water due to the extreme underground
water shortage on the North China plain, a field experiment was
carried out with a drip irrigation system using reclaimed water and
the potential risk of chlorination to control clogged emitters from a
soil health perspective. Two objectives will be addressed: (1) the
effect of chlorination on soil microbial richness and diversity (po-
tential biological indicators of soil health); and (2) the effect on crop
yield and quality as a result of changes to soil enzyme activity.
1453
2. Materials and methods
2.1. Experiment sites and design
The experiment was carried out in the Beijing Tongzhou
Experimental Station of China Agricultural University (N 39
420 , E
116
410 ), which is in a temperate continental semi-humid monsoon
climate, with an average annual rainfall of 550e600 mm and an
average temperature of 11.4e12.4
C. Spring maize “Agricultural
University 86” was cultivated in 2013 and 2014 into clay loam soil.
The spring maize (2013) was planted on May 5 and harvested on
September 20. Spring maize (2014) was planted on May 15, due to
water supply pipeline repairs, and was harvested on September 26.
The spring maize was planted in a wide and narrow ridge, with a
ridge shoulder width of 0.6 m, height of 0.3 m, length of 30 m,
groove center distance of 1.4 m, and an experimental plot of 3
ridges, with a plot area of 126 m2. Two rows of maize were planted
per ridge. The row spacing of maize on the ridge was 0.5 m, the
plant spacing was 0.3 m, and the planting density was about 47601
plants/ha. Three plots were set for each treatment and were
randomly arranged.
The dripline emitters with a 0.3 m spacing in between were
purchased from Metzerplas (http://www.metzerplas.com.au/). The
rated discharge of drip emitters was 1.2 L/h and the operating
pressure was kept at 0.1 MPa during the experiment period. The
emitter characteristics are shown in Table 1. The spacing of the
dripline emitter is equal to the spacing of the maize planting, which
ensures that the emitter coincides with the maize root zone. The
drip irrigation system layout is shown in Fig. 1.
The chlorination treatment was conducted for three time in
each year. The day was at May 23, June 14, August 16 in 2013 and
June 24, July 25, August 27 in 2014. So, the total amount of chloride
injected in the field was 428.7 g/hm2 for each year.
Residual chlorine concentration and chlorination duration were
experimentally varied. The emitter controlling effects on soil health
and crop growth were explored under different chlorination
modes. Three chlorination modes were set in this experiment
(C1.25T2, C2.50T1, and C5T0.5), as shown in Table 2. The drip irri-
gation system would stop 12 h after each chlorination, followed by
the adjusting flushing valve at the end of the drip irrigation system
was opened for lateral flushing treatment for 5 min. A proportional
fertilization pump (MixRite 2504, Tefen, Israel) was installed for
chlorination in the first part of each experiment plot. When chlo-
rination was required, the chlorination valve was opened, and the
sodium hypochlorite solution with a residual chlorine concentra-
tion greater than 9 % entered into the system. The pH of the
reclaimed water was about 7.14, to enhance the bactericidal effect
of chlorination, an appropriate amount of hydrochloric acid (HCl)
was added to the reclaimed water before the start of chlorination so
that the pH approximated 6.0. The dose of HCl added to the inflow
was adjusted in real-time by monitoring the reclaimed water pH
(SevenGo Pro, Mettler Toledo, Switzerland). Two hundred and 40
mL of HCl were typically added to 1 m3 reclaimed water for chlo-
rination practices each time. When chlorine added, the front and
rear valves of the proportional fertilization pump were opened, the
main pipeline valve was closed, and the suction part of the pro-
portional pump was placed into the chlorination barrel containing
a certain concentration of sodium hypochlorite solution.
The experimental site was equipped with an automatic weather
station (HOBO, U30), continuously observing meteorological con-
ditions during the experiment, including temperature, humidity,
rainfall, wind speed, sunshine, radiation, and pressure. The total
amount of rainfall during the growth period of spring maize was
203.74 mm in 2013, mainly concentrated in July and August. It was
348.6 mm in 2014, and the rainfall distribution was relatively
1454 P. Song et al. / Journal of Cleaner Production 232 (2019) 1452e1464

Table 1
Emitter characteristic parameters.

Channel type Geometry parameters of water flow channel length  width  depth/mm Emitter flow q (L/h) Emitter structure Manufacturers

Serrated Angle 48.20  0.50  0.64 1.20 Metzerplas

1- water pump, 2-value, 3- pressure gauge, 4- sand filter, 5- water meter; 6- adjustable proportional pump, 7-120-mesh screen filter, 8-

main valve, 9- chlorination valve, 10- flush valve

Fig. 1. Layout of experimental settings.

Table 2
The different chlorine treatment in field experiment.

Number Chlorination concentration (mg/L) Chlorination duration/h Chlorination content (g/hm2)

C0T0 0.00 0 0
C1.25T2 1.25 2 428.7
C2.50T1 2.50 1 428.7
C5T0.5 5.00 0.5 428.7

scattered. Rhizosphere soil (NRS), then the fine-grained soil within 5 mm of

The content of soil water was measured by a TRIME (TRIME-IPH-
PICO, IMKO, German) moisture meter, and the soil moisture mea-
surement position of each treatment was located at the center of
the plot (as shown in Fig. 2). The frequency of measuring was 3 days
after the three-leaf stage of maize. The measurement layer was
0e10, 10e20, 20e40, 40e60, and 60e80 cm, respectively. Mean-
while, at each treatment, the irrigation time were also recorded and
the single irrigation volume, total irrigation volumes were
measured by water meters.
2.2. Sampling and analysis
After the maize was harvested, the root soil was collected by
shaking root separation method. The sampling points are shown in
Fig. 3. The maize root and surrounding soil (0e20 cm) were dug.
First, the large root-free soil was gently shaken off as non-
the root surface was gently brushed off as Rhizosphere soil (RS). A
circular wetting body is formed on the surface of the ground
directly below the drip emitter, which had a radius of 30 cm. Soil
(0e20 cm) was collected from this wetting body marginal soil
(WMS) at a distance 30 cm from the vertical dripline. Bare soil was
sampled from the area unplanted with spring maize and no drip
irrigation. The soil samples from different locations were placed in
a portable cooler and stored on ice. After being brought back to the
laboratory, they were passed through a 2 mm sieve as soon as
possible and placed in a 4
C refrigerator for temporary storage.
Soil urease, catalase and phosphatase activities were measured
in rhizosphere samples. Urease activity was measured by phenol-
sodium hypochlorite colorimetry and was represented by the
mass of NH3eN generated from each gram of soil after 24 h [in units
of mg g1 (24 h)1] (Huang, 2012). The titration method (0.1
mol L1standard KMnO4 solution titration) was used to determine
 P. Song et al. / Journal of Cleaner Production 232 (2019) 1452e1464 1455

45 0
10
36

Irrigation/mm
20
27

Rainfall/mm
30
18 40
Rainfall (204.14mm)
Irrigation (270.00mm) 50
9
60
0 70
14 29 44 59 74 89 104 119 134
Days after planting/d

a 2013

45 0
10
36
Rainfall(316.90mm) 20

Irrigation/mm
Irrigation(241.50mm)
Rainfall/mm

27 30
18 40
50
9
60
0 70
15 30 45 60 75 90 105 120 135
Days after planting/d

b 2014

Fig. 2. Rainfall and irrigation during the experiment.

Note: 1- dripline, 2- Wet body, 3- Sampling point, blue line represents the sampling position

Fig. 3. Soil sampling location.

1456 P. Song et al. / Journal of Cleaner Production 232 (2019) 1452e1464
catalase activity, which was expressed by the number of milliliters
of KMnO4 solution added per gram of dry soil (in units of mL g1)
(Guan, 1986). Soil phosphatase activity was measured using a
disodium phosphateebenzene colorimetric assay, and the mass of
phenol released from each gram of soil after 24 h was used to
represent phosphatase activity in units of mg g1 (24 h)1(Guan,
1986).
The rhizosphere soil, the non-rhizosphere soil and wetting body
marginal soil were subjected to PLFAs (phospholipid fatty acid
assay) testing according to Pennanen et al. (1999). The naming of
phospholipid fatty acids is based on the method proposed by
Frostegard et al. (1993). Different strains have different PLFA bio-
markers. Specific biomarkers characterize specific microorganisms
and represents single genus or species (Green and Scow, 2000). The
changes in phospholipid composition can explain the changes in
microbial community structure in environmental samples and can
identify and quantitatively describe microbial communities.
Branched chain fatty acids a14:0, 15:0, a15:0, 16:0, a16:0, i16:0, total
amount indicated Gram-positive bacteria content; 16:1u7, 18:1u7,
18:1u5, cy15:0, cy17:0 were used to estimate Gram-negative bac-
teria content; fatty acid 10Me16:0, 10Me17:0, 10Me18:0 means
actinomycete content; fungal biomass estimated by 18:2u6
(Frostegard et al., 1993; Baath, 2003; Medeiros et al., 2006).
The Shannon-Wiener diversity index reflects the richness, pro-
portion, and uniformity of individuals in the community:
X
s
H¼ ðNi=NÞlnðNi=NÞ
i¼1
where H is the diversity index; S is the number of species of mi-
croorganisms; and Ni represents the content of the i fatty acid
methyl ester detected in the same sample, and N represents the
total content of various fatty acid methyl esters detected in the
same sample. Ni/N is the content ratio of each microorganism.
The Simpson dominance index indicates the degree of com-
munity measurement in which the various classes within the
community are in an advantageous or inferior state. The greater the
value of the dominance index, the more prominent the dominant
species status:
X
s tration (|measured concentration - design concentration |/design
D¼1  ðNi=NÞ2
i¼1
where D is the dominance index; the other parameters are the
same as in equation (1).
The Pielou evenness index indicates that the closer each indi-
vidual number is, the higher the uniformity of distribution of
various individuals, and vice versa:
H
J¼
ln S
where H is the Shannon-Wiener index; S is the number of species of
microorganisms.
Soil bacterial community structure has been measured using
phospholipid fatty acid (PLFA) profiling, which gives a broad-scale,
quantitative overview of the living bacterial community (Willers
et al., 2015). However, the development of next-generation
sequencing technologies of the microbial 16S rRNA gene have
allowed information about soil bacterial communities to be ob-
tained at a much finer taxonomic resolution (Smets et al., 2016) and
focus of the study is on the details of bacterial ecology. Moreover,
the PLFAs composition of microorganism was specific to species
(Saetre and Baath, 2000). The combination of the two technologies
will answer a broader range of microbiological and ecological
questions.
Soil sample aliquots representing the three chlorination treat-
ments were composited and labeled as DI.C, while soil with no
chlorination treatment under drip irrigation were composited and
labelled as DI. Bare soil was labelled as CK. The three composited
soil samples were analyzed using the Roche Genome Sequencer
FLX Sequencing Platform (Herlemann et al., 2013) utilizing the 454
high-throughput paradigms for analyses of the eubacterial 16S
rRNA gene. Sequences were subjected to decontamination, prun-
ing, chimeric sequence and other filtering treatments to obtain
optimized sequences, and they were binned to operational taxo-
nomic units (OTUs) through binning at an OTU cutoff value of 0.03.
Each cell is arranged at three equal intervals along the direction
of the drip tape. 10 corns (2 rows, 5 spikes per row) were selected
for each test site, and a total of 30 corns were air-dried and
threshed. Then, after drying, the maize yield was measured and the
grain weight was converted into yield per hectare.
The test methods for crop quality indicators were as follows: the
fat content was determined by Soxhlet extraction, the starch con-
tent was determined by near-infrared method, the protein content
was determined by Kjeldahl method, and the ash content was
determined by 550
C firing. The cellulose content was determined
by medium filtration method, the ascorbic acid (Vc) content was
determined by 2,6-dichloroindophenol titration.
2.3. Quality assurance and quality control
(1)
The quality assurance and quality control of the field chlorina-
tion test consisted of three sets of replicates for no chlorination
filed blank and chlorination treatment. Chlorine concentration
control: The residual chlorine concentration of the drip irrigation
pipes was tested with a portable residual chlorine meter (EXTECH-
CL200 Extech Instruments Corporation, United States). The pro-
portional pump was adjusted according to the measured concen-
tration of the residual chlorine, which was tested every 10 min
using water samples from the end of the driplines. In addition, the
residual chlorine concentration at the end of the drip irrigation
system was kept consistent with the design value. This chlorination
method makes the relative error of the residual chlorine concen-
(2) concentration) 0e8 %. Soil microbial PLFA content, soil enzyme is
taken from three repeated test analyses. According to the analysis
and treatment methods specified by the China national material
standard network, the quality test is carried out for each sample.
Each sample has parallel and blank samples. A comparative analysis
was carried out, and the test results reflected that the measured
value and the error value were within the standard range, and the
measured parallel sample error was within 8 %.
(3) 2.4. Statistical analysis
Basic calculations were performed using Excel, while statistical
tests were performed using SPSS 17.0 software, the difference was
considered significant at a probability level of p < 0.05.
3. Results and analysis
3.1. Effects of chlorination on soil microbial content and
composition in root zone
The soil microbial content and community structure of PLFAs-
labeled in 0e20 cm soil cores under different chlorination modes
is shown in Fig. 4. A total of 19 PLFAs biomarkers were detected in
 P. Song et al. / Journal of Cleaner Production 232 (2019) 1452e1464
(a) 2013
Note: RS represents Rhizosphere soil; NRS represents Non-Rhizosphere soil; WMS represents Wet body marginal soil
Fig. 4. Soil microbial PLFA content and composition at different sampling points.
2013, with approximately 14, 4, and 2 bacterial, fungal, and acti-
nomycete biomarkers. The PLFAs biomarkers detected in 2014 were
almost the same as 2013, containing 18 PLFAs biomarkers; however,
the fungal 18:1u9t was not detected in 2014. Chlorination treat-
ment reduced the number of microbial groupings, but microbial
biomarkers were inconsistent for the different sample locations
(Fig. 4). Among them, the rhizosphere soil had the greatest rich-
ness, including 15 species, which reduced to 9e12 species after
chlorination. As the concentration of chlorination increased, the
species richness gradually decreased. Non-rhizosphere soil was the
next most rich sample location, which included 12 species, and
subsequently decreased to 8e9 species after chlorine treatment.
Overall, the representative biomarkers were similar under the
three chlorination concentrations.
The soil at the wetting body marginal was only partially affected
by chlorine, and moisture did not increase richness; on the con-
trary, richness was the lowest of the three soil sample locations
(Fig. 4). Wetting body marginal soil may not be affected by the
reclaimed water irrigation and may be beyond the effect of root
substrates, making the soil PLFAs biomarkers richness poorer than
the rhizosphere and non-rhizosphere soil. There were eight species
in the control group (C0), and 6e7 species in the different chlori-
nation treatment groups. The different treatment groups were
basically similar in PLFAs biomarkers. Under the different locations
and treatments, the dominant bacterial PLFAs (genera) were a15:0
(Bacillus), 16:0 (Pseudomonas), 18:0 (Hydrogenobacter), the former
two of which represent typical soil aerobic or facultative-anaerobic
heterotrophic bacteria, while the latter represents a genus
comprised of aerobic, thermophilic, chemolithotrophic bacteria
(Pitulle et al., 1994). The proportion of the three to all microbes
tested in 2013 and 2014 was 29.0 %e67.8 %, 39.3 %e72.8 %,
respectively.
For the different chlorination modes, the total amount of PLFAs
biomarkers in the rhizosphere soil of was the greatest with 2013
and 2014 values of 69.9 and 65.3 nmol/g, respectively. The chlori-
nation treatment significantly reduced, compared with the control
group, the soil microbial content of rhizosphere, non-rhizosphere,
and wetting body marginal soil decreased by 26.2 %e44.4 %, 19.9
%e44.7 %, 17.7 %e44.2 %, respectively. Under different chlorination
modes, (increased chlorination and decreased duration) the total
1457
(b) 2014
amount of microorganisms gradually decreased, showing the trend
of PLFAC0T0>PLFAC1.25T2>PLFAC2.50T1>PLFAC5T0.5. Sample location
also demonstrated the same trend, with increasing distance from
the plant inversely associated with PLFA signature amounts,
PLFARS > PLFANRS > PLFAWMS.
Table 3 shows the different microbial classification of PLFAs
biomarkers. All three microbial groups decreased to a different
extent following chlorination treatment. Bacteria accounted for
72.7 % and 83.9 % of the total PLFA signature. Compared with the
control group, the bacterial content in the chlorination treatment
group decreased by 16.9 %e42.5 %. The low concentration and short
duration chlorination mode (C1.25T2) was different from the con-
trol group but not significant; however, bacterial content of chlo-
rination treatments C2.50T1 and C5.0T0.5 were significantly
different (p < 0.05) compared with control group, which showed
relatively higher concentrations of chlorine over medium and short
durations significantly reduced populations. Fungi and actinomy-
cetes were less abundant in the soil and gradually decreased with
increased chlorination concentration. Fungi significantly decreased
(p < 0.05) in high concentration and short duration chlorination
mode, while chlorination treatment did not cause a significant drop
for the actinomycete content. Both dominant bacteria 16:0 and
a15:0 significantly decreased significantly (p < 0.05) with the in-
crease in chlorination concentration (Fig. 5, Table 4), with re-
ductions of 7.0 %e47.3 % and 3.1 %e44.4 %, respectively. Based on
the monitoring results, the ratio of bacteria: fungi were lowest in
the control treatment group, indicating a more stable soil
ecosystem (Wardle et al., 2004; Orwin et al., 2018), but gradually
increased with increasing chlorination. The results show that
chlorination changed the structure of soil microbial community.
The ecological index of PLFAs biomarkers was analyzed in
Table 5. It was found that the chlorination treatment changed the
microbial community structure, and Shannon index decreased
significantly (p < 0.05). With the increase of chlorine concentration,
the diversity index decreased gradually. The decrease ranged from
5.3 % to 14.2 %, and the microbial diversity index was the lowest in
the high concentration and short duration chlorination mode. The
Pielou index was basically unchanged before and after adding
chlorine. The Simpson index did not change significantly, indicating
that the dominant bacteria were not affected by chlorination.
1458 P. Song et al. / Journal of Cleaner Production 232 (2019) 1452e1464

Table 3
Different groups of PLFA content and their ratio.

(a) 2013

Treatment Bacteria/nmol▪g1 Fungi/nmol▪g1 Actinomyces/nmol▪g1 16:0/nmol▪g1 a15:0 nmol▪g1 B/F

C0T0 43.15a±7.19 6.98a±1.84 6.59a±3.06 10.51a±0.84 5.55a±0.64 6.28a±0.58

C1.25T2 33.95 ab ± 3.68 5.31 ab ± 1.13 5.03a±1.97 9.13 ab ± 0.97 5.04 ab ± 0.71 6.48a±0.64
C2.50T1 28.13b ± 6.14 4.31 ab ± 1.44 4.17a±1.86 7.90b ± 1.40 4.30b ± 0.54 6.71a±0.83
C5T0.5 28.17b ± 4.44 4.06b ± 0.99 4.10a±1.76 6.82b ± 1.78 3.89b ± 0.57 7.02a±0.57

(b)2014

Treatment Bacteria/nmol▪g1 Fungi/nmol▪g1 Actinomyces/nmol▪g1 16:0/nmol▪g1 a15:0 nmol▪g1 B/F

C0T0 41.07a±9.06 5.91a±2.01 6.09a±2.63 8.87a±1.01 5.85a±0.80 7.19a±1.06

C1.25T2 29.02 ab ± 7.54 3.83 ab ± 1.36 3.94a±1.85 7.63 ab ± 0.46 5.10b ± 0.76 7.81a±0.97
C2.50T1 26.34b ± 7.29 3.22 ab ± 1.12 3.18a±1.37 6.46bc±1.54 4.58b ± 0.86 8.34a±0.77
C5T0.5 24.02b ± 5.55 2.80b ± 0.86 2.67a±1.18 5.26c±1.36 3.52b ± 0.79 8.73a±0.74

Note: B/F represents the ratio of Bacteria: Fungus. The different small letters mean significant difference (p < 0.05) among the different treatment.

8 a15:0 12 16:0
2013RS
7
Content of PLFA/nmol·g -1

Content of PLFA/nmol·g -1
10 2013NRS
6 2013WMS
8
5 2014RS
2014NRS
4 6
2014WMS
3
4 fit curve 2013RS
2 fit curve 2013NRS
2 fit curve 2013WMS
1
fit curve 2014RS
0 0
fit curve 2014 NRS
0 1 2 3 4 5 0 1 2 3 4 5
fit curve 2014WMS
Chlorine concentration/mg·L -1 Chlorine concentration/mg·L -1

Fig. 5. Correlation between 16:0, a15:0 and chlorination concentration.

Table 4
Correlation between a15:0 and 16:0 and chlorination concentration.

a15:0 16:0

Sample time Fit curve Sample time Fit curve

2
2013 R ¼ 0.38 x þ 6.17 R ¼ 0.92* 2013 R ¼ 0.47 x þ 11.00 R2 ¼ 0.91*
NR ¼ 0.42 x þ 5.17 R2 ¼ 0.90* NR ¼ 0.95 x þ 9.95 R2 ¼ 0.93*
WF ¼ 0.20 x þ 4.93 R2 ¼ 0.91* WF ¼ 0.76 x þ 9.58 R2 ¼ 0.92*
2014 R ¼ 0.44 x þ 6.47 R2 ¼ 0.93* 2014 R ¼ 0.67 x þ 9.51 R2 ¼ 0.91*
NR ¼ 0.52 x þ 5.87 R2 ¼ 0.94* NR ¼ 0.58 x þ 8.50 R2 ¼ 0.95**
WF ¼ 0.42 x þ 4.94 R2 ¼ 0.92* WF ¼ 0.88 x þ 7.84 R2 ¼ 0.92*

Table 5
Changes in ecological index marked by PLFAs.

Treatment 2013 2014

Shannon Pielou Simpson Shannon Pielou Simpson

C0T0 2.31a±0.22 0.95a±0.01 0.89a±0.02 2.19a±0.27 0.91a±0.05 0.87a±0.03

C1.25T2 2.15b ± 0.16 0.94a±0.01 0.87a±0.02 2.07b ± 0.29 0.94a±0.02 0.86a±0.04
C2.50T1 2.05b ± 0.36 0.95a±0.03 0.85a±0.04 1.90c±0.21 0.92a±0.03 0.83a±0.04
C5T0.5 2.02b ± 0.27 0.96a±0.01 0.85a±0.04 1.88c±0.26 0.93a±0.03 0.83a±0.04

Note: The different small letters mean significant difference (p < 0.05) among the different treatment.

3.2. Effect of chlorination on soil bacterial community structure in dominant phylum, with relative abundance in the three treatment
root zone composites (CK, DI, DI.C) were 24.5 %, 26.6 %, 27.7 % in 2013, and
20.0 %, 21.2 %, 23.0 % in 2014. The relative abundance of Actino-
The high-throughput sequencing analysis was conducted to bacteria, Firmicutes and Nitrospirae in the soil decreased from
explore changes to the soil bacterial community because of chlo- 14.3 % to 11.3 % (2013), from 18.1 % to 12.0 % (2014); from 3.9 % to
rination. Fig. 6 shows the phylum level distributions of the 2.3 % (2013), from 6.6 % to 2.3 % (2014); from 2.1 % to 1.8 % (2013),
composited treatment samples. A total of 35 groups were obtained from 3.5 % to 1.6 % (2014) respectively. Bacteroidetes and Gemma-
from the phylum taxonomic level. Overall, Proteobacteria was the timonadetes were more abundant in the soil irrigated with
 P. Song et al. / Journal of Cleaner Production 232 (2019) 1452e1464 1459

100% Proteobacteria reclaimed water and chlorination treatment. The relative abun-
90% dance of increasing from 7.3 % to 9.0 % (2013), 3.2 %e9.2 % (2014);
Chloroflexi and from 4.4 % to 4.9 %. (2013), 4.2 % rose to 4.8 % (2014) respec-
80% tively. The relative abundance of Chloroflexi and Planctomycetes
Actinobacteria
70% had increased or decreased in the two-year experiment in the soil
Relative abundance

Acidobacteria irrigated with reclaimed water and chlorination treatment, which

60%
Bacteroidetes demonstrated some inconsistency year on year and indicates other
50% environmental factors affecting these phyla. Principal component
Planctomycetes
40% analysis of b diversity indices between CK, DI and DI.C is shown in
Firmicutes Fig. 7. The libraries sequestered in three different quadrants, which
30%
means significant differences were obtained among these three
Gemmatimonadetes
20% treatments, indicating that chlorine in the drip irrigation system
10% Nitrospirae has a relatively large impact on the soil microbial community
diversity.
0% Cyanobacteria
As it can be seen from Table 6, the number of OTU, ace, and chao
CK DI DI.C CK DI DI.C Verrucomicrobia indices increased in the sample with the drip irrigation treatment,
2013 2014 but the three indices decreased after the chlorine added. This
Others
Sample name indicated that the drip irrigation using reclaimed water can in-
crease species richness, but the strong oxidative bactericidal effect
Fig. 6. Composition of bacteria in the sample at the level of phylum.

Note: The abscissa represents the first principal component, the percentage represents the contribution of the first principal component to

the sample difference; the ordinate represents the second principal component, and the percentage represents the contribution of the

second principal component to the sample difference.

Fig. 7. Bacterial 16S rRNA PCA analysis.

1460 P. Song et al. / Journal of Cleaner Production 232 (2019) 1452e1464

Table 6
Alpha Diversity index diversity analysis.

Time Sample name Reads OTU ACE chao shannon simpson

2013 CK 7991 1675 2284 (2159, 2352) 2194 (2095, 2317) 6.72 (6.69, 6.75) 0.0024 (0.0022, 0.0025)
DI 8298 1783 2362 (2275, 2465) 2299 (2203, 2418) 6.79 (6.76, 6.82) 0.0024 (0.0022, 0.0025)
DI.C 7239 1659 2326 (2213, 2459) 2227 (2107, 2374) 6.75 (6.72, 6.79) 0.0023 (0.0021, 0.0025)
2014 CK 10246 2151 2899 (2796, 3018) 2930 (2798, 3089) 6.92 (6.89, 6.95) 0.0021 (0.002, 0.0022)
DI 10143 2394 3330 (3211, 3467) 3272 (3134, 3436) 7.05 (7.02, 7.07) 0.0024 (0.0022, 0.0026)
DI.C 10631 2374 3188 (3082, 3311) 3131 (3009, 3277) 7.02 (6.99, 7.05) 0.0019 (0.0018, 0.0021)

Table 7
Changes in soil enzyme activity.

(a) Urease activity

Treatment 2013 2014

RS NRS WMS RS NRS WMS

C0T0 13.09a±1.11 12.76a±0.95 11.80a±0.90 13.08a±0.63 11.71a±0.79 11.15a±0.90

C1.25T2 12.12a±0.61 11.05b ± 0.53 9.76b ± 0.74 11.83b ± 0.62 9.49b ± 0.59 8.86b ± 0.42
C2.50T1 12.09a±0.33 10.48bc±0.45 9.25b ± 0.30 11.84b ± 0.66 9.39b ± 0.51 8.26b ± 0.36
C5T0.5 11.81a±0.39 9.53b ± 0.37 8.58b ± 0.47 11.68b ± 0.55 9.07b ± 0.62 8.05b ± 0.41

(b) Catalase activity

Treatment 2013 2014

RS NRS WMS RS NRS WMS

C0T0 1.01a±0.03 0.94a±0.03 0.85a±0.04 0.91a±0.05 0.76a±0.03 0.72a±0.10

C1.25T2 0.89b ± 0.03 0.87a±0.02 0.71b ± 0.05 0.88a±0.03 0.74a±0.03 0.67 ab ± 0.03
C2.50T1 0.78c±0.04 0.76b ± 0.03 0.64c±0.04 0.84 ab ± 0.03 0.71b ± 0.04 0.63 ab ± 0.03
C5T0.5 0.74c±0.04 0.70b ± 0.05 0.62c±0.03 0.78b ± 0.02 0.66b ± 0.03 0.59b ± 0.05

(c) Phosphatase activity

Treatment 2013 2014

RS NRS WMS RS NRS WMS

C0T0 17.03a±0.51 16.38a±0.46 14.57a±0.27 16.63a±0.36 15.02a±0.76 13.40a±0.24

C1.25T2 16.28a±0.34 14.32b ± 0.31 13.35b ± 0.39 15.48b ± 0.25 13.67b ± 0.43 12.88a±0.61
C2.50T1 15.18b ± 0.66 13.48c±0.36 12.22c±0.40 13.79c±0.58 12.97c±0.70 12.02b ± 0.22
C5T0.5 14.63b ± 0.65 13.36c±0.33 11.31d ± 0.73 12.69d ± 0.31 12.97c±0.46 10.38c±0.32

Note: The different small letters mean significant difference (p < 0.05) among the different treatment.

of chlorine deleteriously affected species richness. Similar patterns
were noted for Shannon diversity index values.
3.3. Effect of chlorination on soil enzyme activity
concentration; the three modes decreased by 4.4 %e12.6 %, 10.3 %e
17.7 %, and 13.6 %e22.3 %, respectively. The high concentration and
short duration chlorination mode had the largest decrease when
compared to control soil (p < 0.05).

The effects of different chlorination modes on soil enzyme ac-
tivities was shown in Table 7. The effects of chlorination treatment
on the activity of the three enzymes significantly reduced (p < 0.05)
activity except soil urease activity of the rhizosphere in 2013.
Overall, this trend was consistent over the study period. Urease
activity decreased gradually as chlorine concentration increased,
which decreased by 7.5 %e20.6 % compared with the control group.
Urease activity of the rhizosphere soil (C0T0) was the highest,
reaching 13.09 mg NH3eN/g. Compared with the non-chlorination
treatment group, the soil urease activity of the rhizosphere soil,
non-rhizosphere soil and wetting front soil decreased by 7.5 %e10.7
%; 13.3 %e22.5 %; 17.3 %e27.8 % respectively. Catalase activity fol-
lowed an overall similar trajectory to urease activity. The different
chlorination modes were reduced by 2.8 %e17.4 %, 6.6 %e25.3 %,
13.3 %e27.2 %, respectively, all of which were significant in 2013.
Interestingly, catalase activity has the ability of reducing soil toxic
oxidative effects (Trasar-Cepeda et al., 1999). Overall, catalase ac-
tivity decreased in the presence of increasing chlorine concentra-
tion but was only significant reduced (p < 0.05) during the high
chlorine concentration. Alkaline phosphatase activity was between
10.4 and 17.0 mg/g. It decreased with the increase of chlorine
3.4. Effect of chlorination on quality and yield of spring maize
It can be seen from Table 8 that the different chlorination modes
had significant effects on the quality index of spring maize, spe-
cifically fat, protein, Vc and cellulose content (p < 0.05). The chlo-
rination treatment resulted in a significant decrease in fat and
protein content (p < 0.05). Compared with the control group, the
chlorination treatment decreased by 2.2 % e16.6 %, 2.2 %e14.1 %,
respectively. The content gradually decreased with the increasing
of chlorination concentration. The high chlorination, short duration
had the most significant effects on the decline of fat and protein
content. The Vc content reached the lowest content under the low
chlorine concentration, long duration mode. The Vc content
increased gradually with the increase of chlorination concentra-
tion, which may be related to adversity stress. Vc is an important
antioxidant in plants, it can remove active oxygen and reduce
peroxidation damage under the adversity stress. Studies have
shown that Vc content in fruits can be increased during moderate
deficit irrigation (Zushi and Matsuzoe, 1998; Tezara et al., 1999).
Drip irrigation using reclaimed water significantly reduced the Vc
content in tomato fruit; however, Li et al. (2012a) demonstrated
 P. Song et al. / Journal of Cleaner Production 232 (2019) 1452e1464 1461

Table 8
Changes in spring maize quality and yield.

(a)2013

Treatment Fat g/100 g Protein g/100 g Starch g/100 g Ash g/100 g Vc g/100 g Cellulose % Yield t/hm2

C0T0 4.00a±0.06 9.62a±0.48 68.77a±1.11 1.42a±0.04 0.32b ± 0.007 17.08a±0.87 10.83a±0.28

C1.25T2 3.87b ± 0.03 8.71b ± 0.45 65.80a±2.41 1.35a±0.01 0.28a±0.003 19.14b ± 0.29 10.56a±0.41
C2.50T1 3.39c±0.04 8.49b ± 0.43 66.22a±1.79 1.34a±0.03 0.34c±0.009 19.98b ± 1.16 10.50a±0.57
C5T0.5 3.33c±0.05 8.26b ± 0.33 65.27a±2.07 1.33a±0.03 0.36d ± 0.012 19.63b ± 0.47 10.13a±0.38

(b)2014

Treatment Fat g/100 g Protein g/100 g Starch g/100 g Ash g/100 g Vc g/100 g Cellulose % Yield t/hm2

C0T0 3.69a±0.05 7.03a±0.02 66.46a±0.23 1.27a±0.02 0.37b ± 0.004 20.71a±0.69 10.44a±1.04

C1.25T2 3.61b ± 0.02 6.75c±0.03 65.27a±1.31 1.15a±0.04 0.29d ± 0.006 25.20c±0.26 10.25a±0.51
C2.50T1 3.45c±0.04 6.87b ± 0.04 66.30a±0.82 1.15a±0.02 0.32c±0.004 23.83b ± 0.35 9.84a±0.53
C5T0.5 3.38d ± 0.05 6.58d ± 0.04 64.22a±0.69 1.16a±0.02 0.39a±0.004 24.77bc±0.90 9.66a±0.33

Note: The different small letters mean significant difference (p < 0.05) among the different treatment.

that chlorination treatment could possibly alleviate the decrease of
Vc content in fruits, which corroborates our results. The cellulose
index increased significantly in the chlorination treatment group
by 12.1 %e21.7 % compared with the non-chlorination treatment
group, but results weren't consistent across study years. In 2013,
the highest cellulose content was 19.98 % at medium concentration
medium duration chlorination mode, and in 2014, the low con-
centration long duration chlorination mode reached the highest
value of 25.20 %. The average values of starch and ash indicators
were 66.51 g/100 g, 1.36 g/100 g in 2013 and 65.56 g/100 g and
1.18 g/100 g in 2014, showing no significant difference. The yield of
spring maize with different chlorination modes was slightly lower
than that of the control group. The yield of spring maize gradually
decreased with the increase of chlorination concentration, which
was consistent across the study period. The yield of spring maize
was about 10,504.69 kg/hm2 in 2013, but decreased by 2.5 %e6.5 %
after adding chlorine. The yield was around 10,050.56 kg/hm2 in
2014, but decreased after chlorine treatment between 1.9 % and 7.5
%. The chlorination treatment had a certain inhibitory effect on the
yield, but the effect was not obvious and did not reach a significant
level.
4. Discussion
Emitter clogging has become a bottleneck problem that restricts
the application and promotion of drip irrigation technology using
reclaimed water (Liu and Huang, 2009; Li et al., 2012b; Pei et al.,
2014). The solution to this problem determines the irrigation uni-
formity, engineering service life, and application benefit of drip
irrigation systems (Adin and Sacks, 1991; Taylor et al., 1995).
Chlorination treatment is one of the most economical and effective
methods to reduce emitter clogging (Taylor et al., 1995; Coelho and
Resende, 2001; Song et al., 2017). The input of chlorine ensured the
system stability and high irrigation uniformity, but the effects of
strong oxidation and chlorine ions on the soil environment and
health have not been determined. Soil health could be improved by
rational irrigation schemes (Martinez et al., 2011; Chen et al., 2013;
Robert et al., 2014; Nicolas et al., 2016). Soil microbes and enzymes
are important components of soil ecosystems, they participate in
the cycling of soil C, N, P and other mineral processes and complex
biochemical processes, affecting crop growth, quality, and the
evolution of soil organic matter and fertility (Sun et al., 2007).
However, the risk on soil health biological indicators such as mi-
crobial and enzyme activity, as well as yield quality associated with
chlorination in drip irrigation systems remains unknown.
4.1. The risk of chlorination to soil health
It was not surprising that the rhizosphere microbial population
had the greatest microbial content, as rhizosphere microbes are
expected to obtain abundant nutrients and energy from the root
system, which continuously secretes a large amount of organic
matter into the growth medium (Rani and Juwarkar, 2012). How-
ever, the chlorine had a more deleterious effect on the non-
rhizosphere soil (decrease by 44.7 %), possibly due to a more
stressed condition or physiological state (Dimkpa et al., 2009).
Overall, this trend was noted in both experimental years; however,
the decline in 2014 was lower than 2013, which may indicate soil
microbiome adaptation to the effect of chlorine (Binet et al., 2001;
Rentz et al., 2005). Or the rainfall during the growth period in 2014
was greater than that in 2013, and the leaching effect on chloride
ions reduced the harmful effect on soil microorganisms. It was
determined that chemical chlorination reduced the microbial
population and changed diversity, resulting in a significant
decrease in microbial diversity, which led to significant decreases of
the total amount of soil microbes, including bacteria, which
accounted for the largest proportion of soil microorganisms. A
gradual increase in bacteria/fungi ratio indicates a decrease in soil
stability (Orwin et al., 2018). Fungi and actinomycetes decreased as
well. The dominant flora didn't change, but with the increase of
chlorine concentration, the beneficial microbial genera a15:0 (Ba-
cillus), 16:0 (Pseudomonas) decreased significantly. Bacillus spp.
(PLFA a15:0) can play an important role in solubilizing phosphorus-
containing potassium compounds in the soil, while Pseudomonas
(PLFA 16:0) is a well-known soil saprophyte.
Additionally, the application of high-throughput sequencing
was briefly applied, which found that Proteobacteria was the
dominant phylum, while the relative abundance of Bacteroidetes,
Gemmatimonadetes, Cyanobacteria, and Verrucomicrobia was
greater in the drip irrigation using reclaimed water and chlorina-
tion than that of control soil. Guo et al. (2017) found that the
relative abundances of Proteobacteria, Gemmatimonadetes and
Bacteroidetes in the soil irrigated with reclaimed water were
significantly higher than those irrigated with clear water, which
was similar to our findings.
Chlorination treatment did not result in a decrease in the rela-
tive abundance of these dominant flora. The ratio of Proteobacter-
ia:Acidobacteria may reflect the nutrient level of the soil with
higher ratios indicative of greater nutrient status (Smit et al., 2001).
Upon analysis, in 2013 the ratio was 1.85, 2.12, 2.02 in the control
soil, soil irrigated with reclaimed water, and soil added chlorine,
respectively, while in 2014, ratios were 1.64, 1.80, 1.77 indicating
that overall ratios remained similar. At the same time, the
1462 P. Song et al. / Journal of Cleaner Production 232 (2019) 1452e1464

chlorination treatment significantly reduced the relative abun-
dance of Nitrospirae, which decreased from 2.1 % to 1.8 % (2013),
from 3.5 % to 1.6 % (2014), which may have an effect on nitrogen
cycling. The relative abundance of Firmicutes had a decrease of 30.7
% and 31.2 %, respectively for two years compared with the non-
chlorinated treatment group irrigated with reclaimed water. The
a15:0 (Bacillus) belongs to the Firmicutes phylum, so the results
obtained by the two test methods were consistent. Therefore, the
effect of chlorination on the ratio of Proteobacteria:Acidobacteria,
Nitrospirae, Actinobacteria and Firmicutes suggests reduced soil
nutrient cycling and potential soil biological health problems.
4.2. Effects of soil microbial community structure changes on soil
enzyme activities
The formation and accumulation of soil enzymes is mainly
derived from microbial activities in the soil. In soil ecosystems, soil
microbial diversity is significantly associated with soil microbial
biomass and soil enzyme activity (Acosta-Martínez et al., 2008).
The increase of soil microbial content can stimulate soil enzymes,
which can increase organic matter decomposition and nutrient
release. According to the correlation analysis (as shown in Table 9),
soil urease activity was significantly positively correlated with the
total amount of microorganisms, bacteria contents, fungi contents,
actinomycetes contents and 16:0 contents.
Soil catalase activity was also significant and positively corre-
lated with the total amount of microorganisms’ bacteria (PLFA) and
16:0 contents, while phosphatase activity showed a significant
positive correlation with total amount of microorganisms, bacteria
contents, 16:0 contents and a15:0 contents. Yang et al. (2016)
studied the long-term application of chlorinated fertilizer in a
rice-wheat rotation system, which caused a change in soil microbial
community structure and led to a decrease of soil biological activity
such as urease, phosphatase and catalase activity. This indicated
that chlorination could directly lead to changes in soil microbial
contents and community structure, which leads to a decline in soil
enzymes, thus causing a decline in soil quality.
4.3. Effects of soil microbial community structure changes on spring
maize yield and quality
As early as 1952, Hofmann proposed the use of soil enzyme
activity as an indicator of soil biological activity and productivity.
Enzyme activity was directly related to soil fertility. Under normal
conditions, soil urease and phosphatase activities were closely
related to soil organic C and total N content (Yang et al., 2012).
Studies have shown that soil enzyme activity was generally
significantly related to some quality indicators of rice (Fu et al.,
2009). The change of soil enzyme activity affected the growth of
rice plants, which in turn affected rice yield and rice quality. In this
experiment, the correlation analysis between soil enzymes, soil
microbial flora and the relative indicators of spring maize quality,
yield indicators are shown in Table 10. The results showed that
there was a significant positive correlation between fat and protein
indexes, indicating that the changes in fat and protein content may
have been affected by the decrease of total amount of microor-
ganisms. Further, the reduction of soil urease and phosphatase
activity suggests potential reduction in urea hydrolysis, thus ni-
trogen availability, as well as reduced use of organic phosphorus.
This may explain the reduction in fat and protein content. There
was a significant positive correlation between starch index and soil
microbial flora and soil enzyme activity, but chlorination did not
have a significant effect on the index, indicating that the change in
starch content may be caused by the interaction of different bac-
teria groups. The ash and Vc indicators did not show a significant
correlation with microbial content and soil enzyme activity, which
may be related to other environmental factors. The cellulose index
showed a significant negative correlation with urease activity and
total amount of microorganisms, indicating that chlorination
treatment indirectly promoted the increase of cellulose content.
Soil enzyme activity and microbial flora (except fungi) showed
significant correlation with corn yield, indicating that chlorination
treatment may change microbial community structure, soil enzyme
activity and change the maize quality at the same time. Long-term
chlorination may have adverse effects on spring maize yield.

Table 9
Correlation between soil microbial community groups and soil enzymes.

Urease activity Catalase activity Phosphatase activity

Total content of PLFAs 0.970** 0.862* 0.920**

Bacteria 0.938** 0.835* 0.881*
Fungi 0.859* 0.706 0.692
Actinomyces 0.847* 0.699 0.677
i16:0 0.769* 0.755* 0.791*
a15:0 0.706 0.685 0.717*

Note: In the table, * indicates significant (p < 0.05), **indicates extremely significant (p < 0.01).

Table 10
Relationship between soil microbial community, soil enzymes and crop quality, yield.

Fat Protein Starch Ash Vc Cellulose Yield

Urease activity 0.812* 0.799* 0.803* 0.695 0.123 0.737* 0.851*

Catalase activity 0.964** 0.823* 0.835* 0.589 0.477 0.618 0.823*
Phosphatase activity 0.892* 0.713* 0.787* 0.654 0.432 0.682 0.921**
Total content of PLFAs 0.838* 0.864* 0.762* 0.666 0.174 0.717* 0.838*
Bacteria 0.773* 0.711* 0.728* 0.419 0.064 0.483 0.886*
Fungi 0.691 0.310 0.720* 0.415 0.098 0.477 0.697
Actinomyces 0.608 0.310 0.716* 0.410 0.135 0.470 0.708*
i16:0 0.863* 0.295 0.688 0.347 0.411 0.394 0.758*
a15:0 0.830* 0.292 0.717* 0.329 0.449 0.379 0.755*

Note: In the table, * indicates significant (p < 0.05), **indicates extremely significant (p < 0.01).
 P. Song et al. / Journal of Cleaner Production 232 (2019) 1452e1464 1463

Table 11
Comparison of different chlorination references.

Chlorine raw material Chlorine mode Chlorine Water quality and quantity Effect of chlorine Reference
equipment of reclaimed water

Sodium hypochlorite 0.83 mg/Lþ3 h the CASS Chlorination had no adverse effect on soil health and had no this study
(NaCLO) proportional 226.8 mm effect on yield
pump
Sodium hypochlorite 1.3 mg/Lþ3 h the The secondary sewage Chlorination treatment is safe, weakens soil enzyme activity to a Hao et al.
(NaCLO) proportional effluent treated using certain extent, and has no significant effect on nitrogen (2019)
pump oxidation ponds absorption and yield
87.2 mm
Sodium hypochlorite 100 ppm þ five venturi Water from the canal and There was no effect on soil crops Chauhdary
(NaCLO) times/month apparatus. tubewell Obtain the application frequency under different chlorine et al.
150 ppm þ three e concentration (2015)
times/month
calcium hypochlorite 20 mg/L the The secondary sewage Chlorine accumulation, soil salinization and nitrogen absorption Li et al.
[Ca(OCl)2] sodium proportional effluent treated using were inhibited (2014)
hypochlorite (NaOCl) pump oxidation ponds
249.2 mm

4.4. Application of chlorine in drip irrigation using reclaimed water
Appropriate assessment of the responses of soil and crop is
essential for determining an optimal chlorination scheme. In the
past, the effects of chlorination on drip irrigation system control
mainly concentrated on soil salt transport, crop yield and quality
(Li et al., 2014; Hao et al., 2019). The dynamic of soil health after
chlorination is little concerned. Based on this, in order to fill this
gap, the feasibility of chlorination treatment was analyzed from
the relationship between soil microbial biomass, dynamic of
community structure, enzyme activity, crop yield and quality. A
suitable chlorination treatment mode was obtained and achieved
the effect of cleaner production. Therefore, we compared this
study with previous experimental studies on chlorination and the
practical application of chlorination in the field (Table 11), found
that low concentration þ long duration of chlorine can guarantee
the stability of the drip irrigation system, reduce the high con-
centration of chlorine on accumulation of soil salt, soil salinization
risk, it is a suitable application mode for chlorination in the field.
The 0.83 mg/Lþ3 h chlorination mode obtained in this study is
reasonable.
In China, sewage treatment volume increases year by year and
sewage treatment cost decreases gradually. At present, the average
cost of sewage treatment is $0.12/m3, which has been widely used
in farmland and garden irrigation (Adrover et al., 2012) and is of
great significance for alleviating the contradiction between supply
and demand of water resources and ensure social stability. Drip
irrigation using reclaimed water is extremely easy to cause emitter
bio-clogging, using liquid sodium hypochlorite solution as raw
material is to reduce the risk of soil salinization than other solid
calcium hypochlorite (Li et al., 2014), In the mode of chlorine,
although the proportion of fertilizer pump have a higher price than
venturi fertilizer, the concentration control accuracy and stability
have a clear advantage, which can significantly reduce the con-
centration of water consumption in the process of adjustment. It is
necessary to equip the pump house with a special fertilization tank
or fertilization tank with extending this research to large-area field
applications, and then input the chlorination raw materials into the
main pipeline of irrigation system according to the set proportion
in the form of fertilizer liquid. The irrigation was carried out in the
field using different rounds for chlorination to achieve the chlori-
nation mode in this study. At the same time, chlorination treatment
requires high corrosion resistance for the equipment, which further
promotes the technical research and development for the related
filtration and fertilization equipment, and promotes the utilization
of reclaimed water drip irrigation towards a cleaner and sustainable
development direction.
5. Conclusions
The following main conclusions can be drawn from this study:
(1) The total amount of microorganisms marked by PLFAs was
significantly reduced under the chlorination treatment in the
drip irrigation using reclaimed water. It also caused the
content of bacteria, fungi and actinomycetes to decrease,
resulting in decrease of microbial community diversity. The
dominant bacterial community members of a15:0, 16:0, and
18:0 did not change before and after chlorination. Among
them, the bacteria content was the most significantly
affected by chlorination.
(2) The dominant bacterial phylum was Proteobacteria, and
chlorination didn't change its dominance. However, it
reduced the relative abundance of Nitrospirae, Actino-
bacteria and Firmicutes, thereby potentially affecting
nutrient cycling.
(3) The change of microbial community structure caused by
chlorination directly led to a decrease of urease, catalase and
phosphatase activity by 7.5 %e27.8 %, 2.8 %e7.2 %, 3.9 %e23.7
%, respectively. This change possibly reduced fat and protein
content; however, long-term chlorination did not have
adverse effects on spring maize yield.
(4) High chlorine concentration and short duration mode is
more likely to produce greater negative soil effects, because
soil microbial and enzyme activities are significantly
reduced, which in turn may affect crop qualitative content.
Acknowledgements
We are grateful for the financial support from the National Key
Research Project of China (2017YFD0201504); National Natural
Science Fund of China (51621061 51339007).
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