Journal of Integrative Agriculture 2017, 16(8): 1819–1831

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RESEARCH ARTICLE

Impacts of chemical fertilizer reduction and organic amendments

supplementation on soil nutrient, enzyme activity and heavy metal
content

NING Chuan-chuan, GAO Peng-dong, WANG Bing-qing, LIN Wei-peng, JIANG Ni-hao, CAI Kun-zheng

Key Laboratory of Tropical Agro-environment, Ministry of Agriculture/South China Agricultural University, Guangzhou 510642, P.R.China

Abstract
Excessive use of agro-chemicals (such as mineral fertilizers) poses potential risks to soil quality. Application of organic
amendments and reduction of inorganic fertilizer are economically feasible and environmentally sound approaches to de-
velop sustainable agriculture. This study investigated and evaluated the effects of mineral fertilizer reduction and partial
substitution of organic amendment on soil fertility and heavy metal content in a 10-season continually planted vegetable
field during 2009–2012. The experiment included four treatments: 100% chemical fertilizer (CF100), 80% chemical fertilizer
(CF80), 60% chemical fertilizer and 20% organic fertilizer (CF60+OM20), and 40% chemical fertilizer and 40% organic
fertilizer (CF40+OM40). Soil nutrients, enzyme activity and heavy metal content were determined. The results showed that
single chemical fertilizer reduction (CF80) had no significant effect on soil organic matter content, soil catalase activity and
soil heavy metal content, but slightly reduced soil available N, P, K, and soil urease activity, and significantly reduced soil
acid phosphatase activity. Compared with CF100, 40 or 60% reduction of chemical fertilizer supplemented with organic
fertilizer (CF60+OM20, CF40+OM40) significantly increased soil organic matter, soil catalase activity and urease activity
especially in last several seasons, but reduced soil available P, K, and soil acid phosphatase activity. In addition, continu-
ous application of organic fertilizer resulted in higher accumulation of Zn, Cd, and Cr in soil in the late stage of experiment,
which may induce adverse effects on soil health and food safety.

Keywords: chemical fertilizer, organic amendments, soil fertility, vegetable, soil health, heavy metal

fertility and crop productivity (Hera 1996). However, long-

1. Introduction
Chemical fertilizer plays an important role in increasing soil
Received 9 March, 2016 Accepted 29 August, 2016
Correspondence CAI Kun-zheng, E-mail: kzcai@scau.edu.cn
© 2017 CAAS. Publishing services by Elsevier B.V. All rights
reserved.
doi: 10.1016/S2095-3119(16)61476-4
term excessive use of chemical fertilizers has contributed to
reduce soil organic matter (SOM) content, with a consequent
decline in the agricultural soil quality, and even an increase
in soil acidification and environment pollution (Roelcke et al.
2004; Dinesh et al. 2010; Guo et al. 2010), which has be-
come a major concern (Chaudhry et al. 2009).
At present, there is an increasing requirement for ag-
ricultural production not only to meet high standards of
quality but also to be produced by environmentally sound
practices. Previous studies have indicated that application
of organic fertilizer fulfills the requirements of sustainable
1820 NING Chuan-chuan et al. Journal of Integrative Agriculture 2017, 16(8): 1819–1831
agriculture, and organic fertilizer has apparent advantages
over chemical fertilizer in many aspects. For example, or-
ganic fertilizer has higher organic matter content and richer
nutrient elements; it can enhance soil physical properties
mainly by improving aggregate stability and decreasing soil
bulk density; it can also improve soil biological and bio-
chemical properties and optimize soil microbial community
structure (Zhang et al. 2009b; Diacono and Montemurro
2010). However, exclusive use of organic fertilizer may not
meet the nutrient requirement for crops as it releases nutri-
ents slowly, and only a fraction of the N and other nutrients
become available for plants in the first year after application
(Hartl et al. 2003).
Taking into account the advantages and disadvantages
of organic and inorganic fertilizer, combined application of
organic amendments and mineral fertilizers has become
an effective approach of nutrient management. Numerous
studies have shown that balanced application of inorganic
and organic amendments can increase SOM and maintain
soil productivity (Arancon et al. 2004; Blair et al. 2006;
Gong et al. 2009). A short-term field experiment by Lazcano
et al. (2013) showed that partial replacement of inorganic
fertilizer by vermicompost or manure had a significant
positive impact on soil microbial activity, and maintained
nutrient supply and crop yield at similar levels with inorganic
fertilizer. Herencia et al. (2007) found that use of straw
compost could reserve soil nutrient stability and maintain
competitive yield. Through a 15-year field experiment,
Zhang et al. (2009a) also showed that a suitable combined
application of chemical fertilizer and straw or cow manure
could increase crop yield, as well as maintain soil fertility
and soil buffering capacity. Furthermore, incorporation of
organic fertilizer can improve chemical fertilizer utilization
efficiency. For example, incorporating straw compost can
reduce adsorption of phosphate through humus wrap Fe,
Al, and Ca oxides, which greatly improves the effectiveness
of inorganic phosphate (Lee et al. 2004).
Conversely, organic fertilizer can also be a source of
environmental pollution and has a risk for human health,
due to the presence of heavy metals, residual additives,
and even microbial pathogens, particularly when they are
improperly used (Hao and Chang 2003; Kumpiene et al.
2008). Several studies have reported that the concentra-
tion of heavy metals in organic fertilizers is generally higher
than the normal concentration in soil; thus, heavy metal
accumulation in soil may exist when organic fertilizer is re-
peatedly applied (Zhao et al. 2006; Kumpiene et al. 2008).
More specifically, excess organic fertilizer may lead to soil
salinization (Hao and Chang 2003) and increase the risk of
heavy metal accumulation by crops. In addition, organic
fertilizers may also produce more greenhouse gases (e.g.,
CO2, CH4) when applied into soil, accelerating greenhouse
effects (Iqbal et al. 2009).
Many effects, such as nutrient release, carbon seques-
tration, and possible build-up of toxic elements in the soil,
evolve slowly and thus need long-term investigation (Di-
acono and Montemurro 2010). A number of studies have
shown that long-term effects of organic fertilizer are usually
more evident than its short-term effects (Udom et al. 2004;
Herencia et al. 2008; Liu et al. 2013). However, responses
of soil biological and biochemical properties to changes of
soil environment and fertilizer management are sensitive
and rapid enough (Gil-Sotres et al. 2005). For example,
short-term increases in microbial biomass have been
reported previously and attributed to the supply of organic
C substrates (Arancon et al. 2006; Dinesh et al. 2010).
Whether it is feasible or not, sustainable application of or-
ganic fertilizer in agriculture depends on the characteristics
of organic fertilizer (nutrient and toxic element contents,
availability, transportation costs, and environmental regu-
lations), as well as application rates and amount, which is
well reviewed in detail by Westerman and Bicudo (2005).
In the present study, field trials of inorganic fertilizer re-
duction and incorporation of partial organic fertilizer were
conducted in continuous 10-season vegetable cultivation
to investigate the effects of mineral fertilizer reduction and
supplementation with organic amendments on soil fertility
and microbial activity, and assess the risk of heavy metals
in soil under different fertilization regimes.
2. Materials and methods
2.1. Study site
The site of field trial was an intensive vegetable farm
located in Yisha Village (22°44´40´´N, 113°29´51´´E),
Nansha District, Guangzhou, Guangdong Province, Chi-
na. This area has a subtropical monsoon climate, and the
annual average temperature and rainfall are 21.9°C and 1
647.5 mm, respectively. The rainfall and average tem-
perature per month during experimental years are shown
in Fig. 1. The basic physical and chemical properties of the
soil are shown in Table 1.
2.2. Experimental fertilizers
Chemical fertilizer in this experiment was a kind of chemical
compound fertilizer (N:P:K, 15%:15%:15%; YARA Interna-
tional Company, Norway). Organic fertilizer was produced
by Guangzhou Liangtian Fertilizer Company, its main raw
material was pigeon droppings. Its basic physical and
chemical properties are shown in Table 1.
 NING Chuan-chuan et al. Journal of Integrative Agriculture 2017, 16(8): 1819–1831 1821

2.3. Experimental design
The field trial was conducted from June 2009 to October
2012 including 10-growing seasons of vegetable crops; the
crop growing sequence of each season is shown in Table 2.
There were four treatments with four replicates: (1) CF100,
100% chemical fertilizer, the local custom chemical fertiliz-
ers application amount; (2) CF80, 80% chemical fertilizer
of CF100; (3) CF60+OM20, 60% chemical fertilizer and
20% organic fertilizer; and (4) CF40+OM40, 40% chemical
fertilizer and 40% organic fertilizer. Organic fertilizer and
chemical fertilizer application amounts were all calculated
with pure N. According to the content of P and K in organic
and chemical fertilizer, the total amount of P and K from
CF60+OM20 were 1147.9 and 1227.2 kg ha–1, respectively;
and that from CF40+OM40 were 891.4 and 1 049.6 kg ha–1,
respectively. Treatments were arranged in a randomized
complete block design. The area of each trial plot was 80 m2.
In this experiment, organic fertilizer was used as basal
fertilizer; meanwhile, partial chemical fertilizer was applied
as basal fertilizer, and then the other partial chemical
fertilizer was applied as top dressing. Top dressing was
scattered between crops based on fertility characteristic of
vegetables. Basal fertilizer was applied five times during
the experiment. The soil was no-till except the date of basal
fertilizer applied. It was divided into five periods according to

700 Rainfall Temperature 35

600 30

500 25

Temperature (°C)
Rainfall (mm)

400 20

300 15

200 10

100 5

0 0
1 2 3 4 5 6 7 8 9 101112 1 2 3 4 5 6 7 8 9 101112 1 2 3 4 5 6 7 8 9 101112 1 2 3 4 5 6 7 8 9 101112
2009 2010 2011 2012

Fig. 1 The rainfall and average temperature per month (2009–2012) in study area. 1–12 in the X-axis represent the 12 months
in a year.

Table 1 The basic properties of soil in study site and organic fertilizer
Organic matter Total N Total P Total K Total Cu Total Zn Total Cd Total Cr Total Pb
pH
(%) (%) (%) (%) (mg kg−1) (mg kg−1) (mg kg−1) (mg kg−1) (mg kg−1)
Soil 6.28 2.90 0.18 0.20 1.37 56.38 123.30 0.64 59.00 41.61
Organic fertilizer 8.73 16.60 3.50 0.94 1.73 42.18 262.80 0.85 67.87 28.15

According to GB 15618-2008 (2008), the concentration of Cu, Zn, Cd, Cr, Pb in soil in this region (pH<6.5) is 50, 200, 0.3, 150,
250 mg kg−1 for the second level environment quality standard, respectively.

Table 2 The planting sequence of vegetable crops and essential date (yr-mon-d)
Season Latin name English name Sowing date Harvest date Soil sampling date
1st Cucumis sativus L. Cucumber 2009-6-15 2009-8-10 2009-8-7
2nd Vigna unguiculata Carob 2009-8-18 2009-10-26 2009-10-24
3rd Cucumis sativus L. Cucumber 2010-3-28 2010-6-11 2010-6-5
4th Momordica charantia L. Bitter 2010-6-23 2010-9-23 2010-9-20
5th Brassica oleracea L. Cabbage 2010-10-18 2011-1-16 2011-1-13
6th Zea mays L. Sweet corn 2011-3-15 2011-6-18 2011-6-17
7th Momordica charantia L. Bitter 2011-7-2 2011-9-22 2011-9-20
8th Brassica chinensis L. Bok choy 2012-2-16 2012-3-28 2012-3-25
9th Momordica charantia L. Bitter 2012-4-13 2012-7-25 2012-7-23
10th Lagenaria siceraria S. Gourd 2012-8-3 2012-10-16 2012-10-15
1822 NING Chuan-chuan et al. Journal of Integrative Agriculture 2017, 16(8): 1819–1831
the date of basal fertilizer applied, and each period included
two crops planting seasons. The total and each period fer-
tilizer applied amount during different treatments are shown
in Table 3, where the first day of each period was the date
of basal fertilizer applied.
2.4. Soil sampling
Soil samples (0–20 cm depth) were collected in each plot
prior to vegetable harvest in each season; soil sampling
and harvest dates are shown in Table 2. Five soil samples
were collected from each plot and mixed to give a composite
sample. The samples were air-dried at room temperature
and sieved through a broad-mesh screen (15 mm) to remove
stones and plant debris and to disrupt large soil aggregates.
Soil was then ground and sieved through 1-, 2-, and 0.15-mm
meshes for measurement of soil nutrients, enzyme activity,
and heavy metal content, respectively.
2.5. Soil chemical analysis
SOM was determined using the Walkley-Black method
(Walkley and Black 1934). Available N (alkaline hydrolysis
nitrogen) was measured using alkaline hydrolysis diffusion
method (Bao 2000). Available P was determined with a
spectrophotometer (TU-1901 UV-VIS; Beijing Puxi Instru-
ment Company, China) by extracting from 5.0 g of soil with
100 mL of 0.5 mol L−1 NaHCO 3 (Page et al. 1982). Available
K was determined by a flame atomic absorption spectrometry
(FAAS; Zennit 700P; Analytik Jena AG, Germany) by ex-
tracting from 5.0 g of soil with 50 mL of 1.0 mol L−1 NH4 OAc
(Page et al. 1982).
The soil total Cu, Zn, Cd, Cr, and Pb contents were an-
alyzed by FAAS after digestion with HClO4, HNO3 and HF
(Agemian and Chau 1976; Tessier et al. 1979).
2.6. Soil enzyme activity analysis
Soil urease activity was measured by incubating 10 g of
soil with 10 mL of 10% urea solution at 37°C for 24 h (Yao
and Huang 2006). The activity was colorimetrically de-
Table 3 The total and each period fertilizer application amount (kg N ha –1) of different treatments
Total fertilizer
Treatments1) 2009.6.11–
application amount
2009.10.26
CF100 1 756 273
CF80 1 404.8 218.4
CF60+OM20 1 404.8 218.4
CF40+OM40 1 404.8 218.4
1)
CF100, 100% chemical fertilizer; CF80, 80% chemical fertilizer; CF60+OM20, 60% chemical fertilizer and 20% organic fertilizer;
CF40+OM40, 40% chemical fertilizer and 40% organic fertilizer.
termined with a spectrophotometer (TU-1901 UV-VIS) at
578 nm within 1 h. The activities of soil acid phosphomon-
oesterase were assayed on 1 g of oven-dry equivalents of
buffered soil solutions incubated at 37°C for 1 h after
adding enzyme-specific substrate solution. The product of
all reactions, p-nitrophenyl phosphate, was colorimetrically
measured using a spectrophotometer at 412 nm (Tabatabai
1994). Soil catalase was measured by incubating 5 g of soil
with 5 mL of 0.3% H2O2 at 30°C for 30 min. The suspen-
sion was titrated with 0.1 mol L−1 KMnO 4solution. Activity
was expressed as mL 0.1 mol L–1 KMnO4 g−1 soil 30 min−1
(Johnson and Temple 1964).
2.7. Statistical analysis
Data of the results are presented as mean value and stan-
dard error. Data were statistically analyzed by one-way
ANOVA with repeated measures and post-hoc method
(Duncan’s method) for multiple comparisons at a 5% signifi-
cance level. The homogeneity of variances and normality of
distribution were tested with the Levene’s and Kolmogorov-
Smirnov tests, respectively. All statistical analyses were
carried out using SPSS (ver. 17.0).
3. Results
3.1. Soil fertility
In the 1st–6th season, similar content of SOM was de-
tected in different treatments during the corresponding
season (Fig. 2). SOM in the CF40+OM40 treatment was
significantly higher than that in the CF100 treatment in two
seasons (the 7th and 10th seasons). In the 9th season, the
content of SOM from CF80 was similar to that from CF100
and CF60+OM20 treatments, which was significantly lower
than that from CF40+OM40 treatment.
The effect of planting season on the content of soil
available N was statistically significant (P<0.05) (Table 4,
Fig. 3-A). Except the 1st season, soil available N from CF80
treatments was similar to that from CF100 during the
corresponding season. Soil available N from CF40+OM40
Each period fertilizer application amount
2010.3.25– 2010.10.16– 2011.6.30– 2012.4.11–
2010.9.23 2011.6.18 2012.3.28 2012.10.16
414 326 394 349
331.2 260.8 315.2 279.2
331.2 260.8 315.2 279.2
331.2 260.8 315.2 279.2
 NING Chuan-chuan et al. Journal of Integrative Agriculture 2017, 16(8): 1819–1831 1823

40 CF100 CF80 CF60+OM20 CF40+OM40

35 a
a aa a
Soil organic matter (g kg–1)

b bab a aa ab a
30 aaaa aaaa aaaa ab b ab
a aa bb
a
aaa
25

20

15

10

0
1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th
Season

Fig. 2 Effects of chemical fertilizer reduction supplemented with organic fertilizer on soil organic matter. CF100, 100% chemical
fertilizer; CF80, 80% chemical fertilizer; CF60+OM20, 60% chemical fertilizer and 20% organic fertilizer; CF40+OM40, 40% chemical
fertilizer and 40% organic fertilizer. Data were statistically analyzed by one-way ANOVA with repeated measures and different
small letters indicate significant differences (P<0.05) among four treatments of each season by Duncan’s method for multiple
comparisons. All the data are the means of four replicates±standard error.

Table 4 Season variance analysis for all tested parameters of different treatments 1)
CF100 CF80 CF60+OM20 CF40+OM40
Parameters
F-value P-value F-value P-value F-value P-value F-value P-value
Organic matter 3.655 0.008 3.23 0.014 8.30 0.000 6.55 0.000
Available N 30.53 0.000 59.15 0.000 37.25 0.000 52.64 0.000
Available P 8.19 0.000 15.58 0.000 14.13 0.000 19.64 0.000
Available K 44.67 0.000 44.70 0.000 56.04 0.000 58.15 0.000
Catalase activity 29.47 0.000 27.90 0.000 23.59 0.000 22.53 0.000
Urease activity 38.32 0.000 30.43 0.000 44.26 0.000 51.10 0.000
Acid phosphomonoesterase activity 21.86 0.000 16.26 0.000 17.01 0.000 11.43 0.000
Total Cu 1.19 0.355 3.59 0.008 2.26 0.062 1.56 0.194
Total Pb 3.98 0.005 2.35 0.053 4.01 0.005 2.78 0.027
Total Zn 1.81 0.130 1.81 0.129 4.93 0.001 12.15 0.000
Total Cd 2.81 0.026 1.80 0.132 2.71 0.030 3.17 0.015
Total Cr 0.96 0.497 3.63 0.008 3.65 0.008 8.59 0.000
1)
CF100, 100% chemical fertilizer; CF80, 80% chemical fertilizer; CF60+OM20, 60% chemical fertilizer and 20% organic fertilizer;
CF40+OM40, 40% chemical fertilizer and 40% organic fertilizer.
The effect of planting season on the tested parameters were analyzed separated with respect to each different treatments by one-way
ANOVA with repeated measures.

treatment was significantly lower than that from CF100
treatments during the 1st, 4th, and 7th seasons. For the
8th season, the lowest soil available N was detected in
CF60+OM20 treatment, which was significantly lower than
that in CF100 treatment. However, for the 3rd season, the
highest soil available N was detected in CF40+OM40 treat-
ment, which was significantly higher than that in CF100 and
CF80 treatments. In addition, significant difference among
the four treatments was not found in the 2nd, 5th, 6th, 9th,
and 10th seasons.
Generally, soil available P decreased after chemical fer-
tilizer reduction in most seasons (Fig. 3-B). For example,
soil available P in CF60+OM20 treatment during the 3rd,
9th and 10th seasons was significantly lower than that in
CF100 treatment. Except the 5th, 7th, and 8th seasons, soil
available P from CF40+OM40 treatment was signifi- cantly
lower than that from CF100 treatment, where the contents
in the 1st, 2nd, 3rd, 4th, and 6th seasons were only 76.7,
70.4, 62.1, 67.5 and 75.2% of CF100 treatment,
respectively.
Similarly, soil available K decreased significantly with
the chemical fertilizer reduction during the corresponding
season, except the 1st season (Fig. 3-C). For the CF80
treatment, soil available K during the 3rd, 7th, and 9th sea-
sons was significantly lower than that of CF100 treatment.
Except the 1st season, soil available K from CF40+OM40
treatment was significantly lower than that from CF100 treat-
ment. For example, compared with CF100, CF60+OM20
1824 NING Chuan-chuan et al. Journal of Integrative Agriculture 2017, 16(8): 1819–1831

CF100 CF80 CF60+OM20 CF40+OM40

A 500
Soil available N (mg kg–1)
400 a aaa a
a aa ab ab
b b
300 aab
a
ab aaa
aba b
200 a
ab bb a a a aa
a a
aa
a
a
a
bb
100

0
1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th
B 250
a
Soil available P (mg kg–1)

200
a aaa bb aab
aaa
150 a a b bb
aa a ab a aa aaa
a b ab b a
100 b c b a
ababa
b
50

0
1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th
C 700
a
Soil available K (mg kg–1)

600
bb
500

400 aa c a
ab a ab b
b

300 b aa bc
ab b ca
aa aab ab
200 a aa cc b bb
c ab bb
a b
100
0

1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th
Season

Fig. 3 Effects of chemical fertilizer reduction supplemented with organic fertilizer on soil available N (A), available P (B) and
available K (C). CF100, 100% chemical fertilizer; CF80, 80% chemical fertilizer; CF60+OM20, 60% chemical fertilizer and 20%
organic fertilizer; CF40+OM40, 40% chemical fertilizer and 40% organic fertilizer. Data were statistically analyzed by one-way
ANOVA with repeated measures and different small letters indicate significant differences (P<0.05) among four treatments of each
season by Duncan’s method for multiple comparisons. All the data are the means of four replicates±standard error.

and CF40+OM40 treatments reduced soil available K by activity from CF40+OM40 treatment was significantly higher
34.8 and 25.5% in the 2nd season, 52.3 and 57.4% in the 3rd than that from CF100 treatment during the corresponding
season, and 31.6 and 54.5% in the 4th season, respectively. season. Moreover, compared to CF60+OM20, soil catalase
activity of CF40+OM40 treatment was higher from the 5th
3.2. Soil enzyme activity to 7th seasons.
The effect of planting season on soil urease activity was
Soil catalase activity of four treatments maintained at a rel- statistically significant (P<0.05) (Table 4, Fig. 4-B). Com-
atively high level in the first six seasons (1st–6th season), pared with CF100, significant lower soil urease activity was
and then it dropped to a lower level in the last four seasons detected in CF80 treatment during the 4th and 6th seasons.
(7th–10th season) (Fig. 4-A). Compared with CF100, Conversely, CF60+OM20 and CF40+OM40 treatment
CF80 did not influence soil catalase activity. Higher soil enhanced soil urease activity during the 5th, 6th and 10th
catalase activity was found in CF60+OM20 treatment than seasons, especially CF40+OM40 treatment. For example,
that in CF100 treatment during the 3rd, 4th, 8th, and 10th in the 10th season, soil urease activities from CF60+OM20
seasons. Except for the 1st and 2nd seasons, soil catalase and CF40+OM40 treatments were increased by 19.28 and
 NING Chuan-chuan et al. Journal of Integrative Agriculture 2017, 16(8): 1819–1831 1825

A CF100 CF80 CF60+OM20 CF40+OM40

g–1 soil 30 min–1)
5.0

a aa
Soil catalase activity (mL

aa aba
4.0 a aa a ab ab ab
a aa b
b b ababa
3.0 b aa aa
0.1 mol L–1 KMnO

a
a
4

b ab b
2.0
b bb b
bbb
1.0

0.0
1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th

B 800
a
700
Soil urease activity (mg
NH4+-N kg–1 soil 24 h–1)

600 bb
aaa
500 a ab aba c
400 ab ab
a b a ab a a a
b a aa a
aa
a
b bcab
300 c
a aa a
200 b bbb
100
0

1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th
C
500 a
Soil acid phosphomonoesterase

a a ab
a
activity (mg P 2O 5 kg–1 soil h–1)

400 ab a ab abb
a ab
ab a
b a b ab
300 b a a bbb b bb
a a
aa a bbbaaa a
200 a

100

0
1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th

Season

Fig. 4 Effects of chemical fertilizer reduction supplemented with organic fertilizer on soil catalase activity (A), urease activity (B)
and acid phosphomonoesterase activity (C). CF100, 100% chemical fertilizer; CF80, 80% chemical fertilizer; CF60+OM20, 60%
chemical fertilizer and 20% organic fertilizer; CF40+OM40, 40% chemical fertilizer and 40% organic fertilizer. Data were statistically
analyzed by one-way ANOVA with repeated measures and different small letters indicate significant differences (P<0.05) among four
treatments of each season by Duncan’s method for multiple comparisons. All the data are the means of four replicates±standard error.

30.71% compared to CF100, respectively. 3.3. Soil heavy metal content

During the 2nd, 8th, 9th, and 10th seasons, soil acid
phosphomonoesterase activity decreased significantly The level of soil total Cu and Pb from different treatments
with reduction in chemical fertilizer (Fig. 4-C). In general, was relatively stable through the entire trial period. Further-
significant lower soil acid phosphomonoesterase activity in more, significant difference among four treatments was not
CF80 treatment was found than that in CF100 except the noted in the total soil concentrations of Cu and Pb during
5th and 7th seasons. During the 2nd, 3rd, and 10th the corresponding season (Fig. 5-A and B).
season, CF60+OM20 treatment significantly reduced soil Through the entire trial period, no significant difference of
acid phosphomonoesterase activity compared with CF100. soil total Zn content was found between CF80 and CF100
Furthermore, lower soil acid phosphomonoesterase activity treatments (Fig. 6-A). However, organic fertilizer treatments
in CF40+OM40 treatment was detected than that in CF100 especially CF40+OM40 significantly enhanced soil total Zn
treatment during the 2nd, 3rd, 6th, 8th, 9th and 10th content compared with CF100 in the last several seasons
seasons. (6th–10th). Compared with CF100, significant higher soil
1826 NING Chuan-chuan et al. Journal of Integrative Agriculture 2017, 16(8): 1819–1831

A CF100 CF80 CF60+OM20 CF40+OM40

Soil total Cu content (mg kg–1)

70
a a aa
60 a a a a a a aa a a a a aa a a aa aa a aaa
a aa a a aaa a
50 a aa
40
30
20
10
0
1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th
B
60
Soil total Pb content (mg kg–1)

50 a aa a a a aa aa a
aaa a a aa a a
a aaa aa aa a aa a aa a aa
40 aa a

30
20
10
0
1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th

Season

Fig. 5 Effects of chemical fertilizer reduction supplemented with organic fertilizer on soil total Cu (A) and Pb (B) contents. CF100,
100% chemical fertilizer; CF80, 80% chemical fertilizer; CF60+OM20, 60% chemical fertilizer and 20% organic fertilizer; CF40+OM40,
40% chemical fertilizer and 40% organic fertilizer. Data were statistically analyzed by one-way ANOVA with repeated measures
and different small letters indicate significant differences (P<0.05) among four treatments of each season by Duncan’s method for
multiple comparisons. All the data are the means of four replicates±standard error.

total Zn content in CF60+OM20 treatment was detected
during the 1st, 6th and 8th season. Moreover, significant
higher soil total Zn content was detected in CF40+OM40
treatment during the 6th, 7th, 8th, and 10th seasons.
Similarly, CF100 treatment did not influence soil total Cd
and Cr content compared with CF100 through the entire
trial period (Fig. 6-B and C). However, in the last several
seasons (6th–10th), organic fertilizer treatments especially
CF40+OM40 significantly enhanced soil total Cd and Cr
content compared with CF100.
4. Discussion
4.1. Fertilization regimes and soil nutrient
Organic fertilizer generally contains more organic matter
than inorganic fertilizer. Previous studies showed that
application of organic fertilizer can increase SOM (Edme-
ades 2003; Monaco et al. 2008). Based on a continuous
10-season vegetable cultivation trial, we found that chemical
fertilizer reduction and incorporation of organic fertilizer
(CF60+OM20 and CF40+OM40) could increase SOM,
especially in the last several seasons (7th, 9th, and 10th
seasons; Fig. 2), which was mainly resulted from accu-
mulation of organic matter due to repeated application of
organic fertilizer.
Long-term application of organic fertilizer can accelerate
soil nutrients activation, improve soil nutrients content,
maintain available nutrients balance, and then improve soil
fertility (Garcia-Gil et al. 2004; Huang et al. 2010). Organic
fertilizer can also increase the activity of microorganisms
and enzymes with regard to nutrient transformation, thus
increasing soil available nutrients (Lazcano et al. 2013).
Diacono and Montemurro (2010) analyzed overall litera-
tures and pointed out that long-term application of organic
amendments enhances soil available K, extractable P, and
organic carbon content, but results in deferred N availability.
Conversely, our study found that combined application of
inorganic and organic fertilizers didn’t have significant effect
on soil available N content throughout the trial period, but
it reduced soil available P and K content compared with
CF100 in several planting seasons (Fig. 3). The main rea-
son was attributed to the lower P and K contents of organic
fertilizer compared with chemical fertilizer (Table 3). Another
explanation was that the control (CF100) in our study is
“100% chemical fertilizer application”, whereas the control
in other references is “no fertilization”. In fact, increases in
soil microorganisms can also reduce short-term nutrients
 NING Chuan-chuan et al. Journal of Integrative Agriculture 2017, 16(8): 1819–1831 1827

A CF100 CF80 CF60+OM20 CF40+OM40

160
Soil total Zn content (mg kg–1)
a a a aba a
140 a a aa a a aa a b b b bb aba b bab
bb b aaaa aa aa cbc abb
120 a
100
80
60
40
20
0

1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th
B
1.2
Soil total Cd content (mg kg–1)

1.0 aa

a a
0.8 a a a a aa a a a a a
a a aa a aa a a aa b bab b ab ab bc ab
b
b b bc c
0.6 c
0.4

0.2

0.0

1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th
C 100
Soil total Cr content (mg kg–1)

80 a
aa
aaa a bbb a aa a
60 a a a aa a aa
a aa a aa bb ab a ab ab
aa b bb
b
40

20

0
1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th

Season

Fig. 6 Effects of chemical fertilizer reduction supplemented with organic fertilizer on soil total Zn (A), Cd (B) and Cr (C) contents.
CF100, 100% chemical fertilizer; CF80, 80% chemical fertilizer; CF60+OM20, 60% chemical fertilizer and 20% organic fertilizer;
CF40+OM40, 40% chemical fertilizer and 40% organic fertilizer. Data were statistically analyzed by one-way ANOVA with repeated
measures and different small letters indicate significant differences (P<0.05) among four treatments of each season by Duncan’s
method for multiple comparisons. All the data are the means of four replicates±standard error.

availability to the plants due to microbial competition (Geis- treatments showed an increasing trend (Fig. 3-B), indicating
seler et al. 2010). that excess phosphorus was applied into soil, which waste
In the present study, although the available P and K of resources and pollute groundwater (Sharpley et al. 2001).
organic fertilizer treatments (CF60+OM20 and CF40+OM40) In addition, soil available N and K of four treatments fluc-
were lower than complete chemical fertilizer treatments tuated dramatically in different seasons (Table 4, Fig. 3-A
(CF100), the absolute values of available nutrients were and C), because the fertilization time and the amount of
high enough for crop requirement. For example, avail- able fertilizer before each soil sampling was different; the other
P and K in the 10th season were (142.09±12.75) and reasons may be related to the planting of different crop spe-
(151.74±38.67) mg kg−1 for CF60+OF20, and (142.36±20.43) cies and climate conditions change (Fig. 1) in each season.
−1
and (149.31±48.47) mg kg for CF40+OF40, respectively.
Our previous study also showed that 20% chemical fertilizer 4.2. Fertilization regimes and soil enzyme activity
reduction did not influence vegetable crop yield (Wang et al.
2012). In particular, we noticed that soil available P of the four Soil enzymes are a kind of special protein owning biochem-
1828 NING Chuan-chuan et al. Journal of Integrative Agriculture 2017, 16(8): 1819–1831
ical and catalytic properties; they are involved in many
important biochemical processes in soil and have a close
relationship with soil fertility (Monreal and Bergstrom 2000;
Bohme et al. 2005). Several studies found that soil amend-
ments (municipal solid waste, farm yard manure, compost,
etc.) significantly increase the enzymatic activities of urease,
β-glucosidase, alkaline phosphatase, and o-diphenyloxidase
compared with the chemical fertilizer-treated soil, thus im-
proving soil biochemical quality (Chang et al. 2007; Lazcano
et al. 2013; Liang et al. 2014). In the present study, single
chemical fertilizer reduction (CF80) did not affect soil cat-
alase activity, but slightly reduced soil urease activity and
significantly reduced soil acid phosphatase activity (Fig. 4).
This finding was consistent with the results by Wang S Q
et al. (2008). The reason was speculated that soil available
nutrients and soil fertility decline because of the reduction
of chemical fertilizer.
More importantly, our results showed that combined
application of inorganic and organic fertilizers (CF60+OF20
and CF40+OF40) significantly improved soil catalase and
urease activities, and high ratio of organic fertilizer sup-
plementation resulted in higher enzyme activity (Fig. 4-A
and B). Similar results were also obtained from the study
by Pascual et al. (1999) and Marcote et al. (2001). It was
attributed to the increased inputs of organic substrates that
stimulate microbial growth and enzyme synthesis.
Increases in the enzyme activities involved in the release
of the main plant macronutrients with organic fertilizers
have been verified in several studies (Marinari et al. 2000;
Dinesh et al. 2010). However, our results found that
combined application of inorganic and organic fertilizers
(CF60+OF20 and CF40+OF40) resulted in reduced soil
acid phosphatase activity (Fig. 4-C), which was opposite
with the other studies (Chang et al. 2007; Saha et al. 2008).
Soil acid phosphatase is a kind of hydrolase that catalyzes
organic P transform into inorganic P, which is sensitive to
soil pH, and it usually can be detected in acid and neutral
soil, but can not be detected in alkaline soil (Beissner and
Roemer 1999). The soil in our experimental site is acidic
(pH 6.28), and organic fertilizer for this experiment is alka-
line (pH 8.73), along with the proportion of organic manure
increasing, soil pH was escalated and not conducive to
acid phosphatase activity.
Our results indicated that soil enzyme activities varied
under different fertilization regimes which depended on
enzyme types, the characteristics of organic amendments
and soil itself. Moreover, different crops planting and climate
conditions in each season (Table 4) were important factors
to soil enzymes activity, and it may influence soil enzymes
activity by soil microbes and root exudate changes and their
interactions, which need to be further studied.
4.3. Fertilization regimes and soil heavy metal content
Compared with chemical fertilizer, organic fertilizer or
manure often contains a higher content of heavy metals,
which are introduced into soil via fertilization (Diacono and
Montemurro 2010). Then long-term application of organic
fertilizer with high heavy metals content will lead to heavy
metals accumulation in soil, because most of the heavy
metals are relatively stable and only a small portion will
be removed (Rajkumar et al. 2009). Our present study
found that chemical fertilizer reduction and combined ap-
plication of organic fertilizer had no effect on soil total Cu
and Pb contents (Fig. 5), but significantly increased total
Zn, Cd, and Cr contents in soil in the late several seasons
(Fig. 6). Moreover, high ratio of organic fertilizer application
(CF40+OF40) resulted in more accumulation of Zn, Cd, and
Cr in soil. Similarly, Wang K F et al. (2008) found that long-
term application of organic manure significantly increased
the total content, available content, and activation rates
of Zn, Cu, Cd, and Pb, which increased the risk of heavy
metals contamination in paddy soils. A trial of 5 years by
Bartl et al. (2002) demonstrated that application of 32 t ha−1
biowaste compost didn’t influence the total contents of Cd,
Mn, Mo, and Ni in soil, but significantly increased the total
contents of Zn and Pb compared with unfertilized treatment.
As mentioned in Table 1, organic fertilizer for this study
contained less Cu and Pb, which would not affect Cu and
Pb concentration in soil, but contained more Zn, Cd, and
Cr, which may lead to Zn, Cd, and Cr accumulation in soil.
Throughout the trial period, organic fertilizer was added for
five times, thus heavy metals, including Zn, Cd, and Cr,
were slightly accumulated in the late seasons as a result.
Therefore, the effect of organic fertilizer on heavy metals in
soil is primarily attributable to the amount of heavy metals
contained by itself.
The toxicity of heavy metals in soil depends mainly on its
forms, rather than its total amount (Allace and Berry 1989).
Organic fertilizer is a class of most important organic sub-
stance that influences the chemical behavior of heavy metals
in soil. On the one hand, heavy metals carried by organic
manure possess strong bioavailability, and organic acid from
organic matter decomposition can activate the heavy metals
of strong soil-bound (Mclsaac et al. 1991); On the other
hand, humus can fix heavy metals through complexation or
chelation reaction, reducing heavy metals availability and
mitigating its toxicity to crops (Naidu and Harter 1998;
Udovic and McBride 2012). This study only examined the
effect of organic fertilizer application on total heavy metals
content in soil, and the available heavy metals need further
study. Anyway, the increasing total Zn, Cd, and Cr contents
in soil pose potential risks to soil health, especially in the
 NING Chuan-chuan et al. Journal of Integrative Agriculture 2017, 16(8): 1819–1831
case that the Cd content of original soil in this region has
far exceeded the safety standards of 0.3 mg kg−1.
5. Conclusion
of Plant Nutrition and Soil Science, 165, 161–165. Beissner L,
Based on 3-year and 4-month uninterrupted field experi-
ment, dynamic effect was understood well about chemical
fertilizer reduction and organic amendments supplemen-
tation on soil quality, which provides scientific basis for
agriculture practice. Our 10-season field experiments
showed that single chemical fertilizer reduction (CF80) had
no significant effect on SOM, soil catalase activity and soil
heavy metal content, but slightly reduced soil available N,
P, K, and soil urease activity, and significantly reduced soil
acid phosphatase activity. Compared with complete
chemical fertilizer application (CF100), chemical fertilizer
reduction and supplementation of partial organic fertilizer
(CF60+OM20 and CF40+OM40) significantly increased
SOM, soil catalase activity and urease activity, particularly
in the last several seasons, but reduced soil available P, K,
and soil acid phosphatase activity. In addition, our results
found that continuous application of organic fertilizer resulted
in slightly higher accumulation of Zn, Cd, and Cr in soil in
the late stage of experiment, which may induce adverse
effects on the soil quality and even human health. Finally,
it needs to be pointed out that planting season, crop type
and soil itself may also influence soil properties.
Acknowledgements
This study was financially supported by grants of the Key
Projects in the Key Technologies R&D Program of China
during the 12th Five-Year Plan period (2012BAD14B00) and
the Guangdong Provincial Science and Technology Plan
Key Project , China (2012A020100003, 2015A050502043).
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