CMUJ nat-sci 15-1.indb - Chiang Mai University Journal

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Doi: 10.12982/cmujns.2016.0001
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
1
Variation of Zinc Concentration in Rice Caryopsis
and Husk among Southern Rice Varieties Grown
in Southern and Northern Thailand
Nantiya Panomjan1,2*, Sansanee Jamjod1,5, Benjavan Rerkasem4,
Bernard Dell3 and Chanakan Prom-u-thai1,5*
1
Department of Plant Science and Natural Resources, Faculty of Agriculture,
2
Department of Plant Science, Faculty of Technology and Community Development, Thaksin University, Phatthalung 93100, Thailand
3
School of Veterinary and Life Sciences, Murdoch University, Perth 6150, Australia
4
Plant Genetic Resource and Nutrition Laboratory, Faculty of Agriculture, Chiang
Mai University, Chiang Mai 50200, Thailand
5
Lanna Rice Research Center, Chiang Mai University, Chiang Mai 50200, Thailand
*Corresponding authors. E-mail: [email protected], [email protected]
ABSTRACT
This study evaluated the concentration of Zn in rice caryopsis (intact
with pericarp and embryo) and husk (palea and lemma) among rice varieties
from southern Thailand and whether the pattern in the Zn concentration of
selected varieties was altered by production in a different genetic resource
area in northern Thailand. Forty-eight southern rice varieties were grown in
a paddy field at Phatthalung Rice Research Center, Thailand, designated as
the southern location. Seeds of each variety were harvested and analyzed for
Zn concentration in the caryopsis and husk and compared with the standard
check varieties with low (RD21) and high (Namroo) Zn concentrations. Four
varieties with different Zn concentrations in the caryopsis were selected from
among the 48 above varieties and grown in a demonstration field at Chiang
Mai University, Thailand, designated as the northern location, for examining the effect of planting location on Zn concentration in the caryopsis. Zn
concentrations ranged widely, from 19.2 to 33.3 mg Zn/kg in the caryopsis
and from 6.2 to 20.0 mg Zn/kg in the husk among the 48 southern varieties
planted in the southern location. Zn concentrations in the caryopsis and husk
planted at the southern location correlated significantly (r = 0.55*). The rice
varieties Chaw Gam Preud, Leuang Hawm, Mai Yah, and Nahn Hak had the
highest caryopsis Zn concentrations; all were higher than the high standard
Zn check variety (29.0 mg Zn/kg). The wide range of Zn concentrations in the
caryopsis among rice varieties from southern Thailand provides an opportunity
for selective inclusion in breeding strategies to enhance dietary Zn uptake by
rice consumers. The caryopsis Zn concentrations were reduced by 20-42% in
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➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
three out of four southern cultivars grown in northern Thailand; this indicates
the importance of considering the environment and agronomic practices when
selecting cultivars with high Zn concentrations in pre-breeding programs.
Keywords: Rice caryopsis, Grain zinc, Local rice, Southern Thailand, Northern
Thailand, Planting location
INTRODUCTION
Zinc (Zn) deficiency is a well-documented problem, occurring in both
crops and humans. Soil condition is the major cause of Zn deficiency in cereal
crops (Alloway, 2008); an estimated 50% of the world’s cereal production areas
are affected by constraints in soil Zn supply, leading to decreased crop yields
and nutritional quality (Cakmak, 2002). Diets low in available Zn cause severe
health complications, including impairing physical growth, the immune system,
and learning (Gibson, 2006). In Southeast Asia, most of the population derives
the majority of their nutritional and caloric needs from rice (Hotz and Brown,
2004). Therefore, strategies to decrease the incidence of Zn deficiency among the
population by increasing the Zn concentration in rice grain are worth considering. Previous studies have reported a wide variation in Zn concentrations among
varieties of rice caryopsis (with intact pericarp and embryo), e.g., 14 to 58 mg
Zn/kg in a germplasm collection at the International Rice Research Institute (Gregorio, 2002) and 17 to 59 mg Zn/kg among Thai rice varieties (Saenchai et al.,
2012). Breeding and selection of Zn-rich rice varieties have been confirmed as a
means to increase the intake of this nutrient in rice eaters (Welch and Graham,
2004; Bouis and Welch, 2010).
Thai rice varieties are genetically diverse (Chang, 1976; Dennis, 1987), with
many traditional varieties collected for conservation. This resource is invaluable
for future rice breeding programs in Thailand. In particular, genetic diversity
allows local farmers to select for specific traits, such as starch quality (Pusadee et
al., 2014). Research has been undertaken on variation in grain quality for fragrant
(aromatic) rice landraces (Prathepha and Srisa-Ard, 2014), colored rice (red and
black rice varieties) (Sompong et al., 2011a; Sompong et al., 2011b) and waxy
(glutinous) rice (Chakhonkaen et al., 2012), with a focus on special properties, such
as nutritional and cooking qualities for the international market. Interest has also
been expressed in developing rice products for other benefits, such as cosmetics.
For Thai rice, variation in Zn concentration of local upland rice from the
north (ranging from 17 to 26 mg Zn/kg) and improved and modern rice varieties in
the central plain (ranging from 39 to 59 mg Zn/kg) have been reported previously
(Saenchai et al., 2012; Jaksomsak et al., 2014; Jaksomsak et al., 2015). However,
the southern rice germplasm has not yet been evaluated for Zn concentration
and other grain qualities. Even though the rice growing area in southern Thailand is only about 10% of Thailand’s total rice growing area, some southern rice
varieties are well known for their premium quality, such as Sang Yod and Leb Nok.
Southern Thailand, sitting between the Indian and Pacific Oceans, experiences a
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
3
different climate regime than rice production areas elsewhere in Thailand and this
may influence grain quality, including grain Zn concentration. For example, in
northern Thailand, where climate and soil differ between upland and lowland rice
production areas, the Zn concentration in rice caryopsis was lower (21 to 38 mg
Zn/kg) for upland than paddy rice (28 to 41 mg Zn/kg) (Jaksomsak et al., 2015).
To explore this further, this study evaluated the variation in Zn concentration in
non-glutinous local rice varieties from southern Thailand, and whether the pattern
in the grain Zn concentration of selected varieties was altered by production in a
different genetic resource area in northern Thailand.
MATERIALS AND METHODS
Plant culture
One southern – Phatthalung Rice Research Center (PRRC; latitude 7° 57’
N, longitude 100° 13’ E and altitude 27 m AMSL) – and one northern – Chiang
Mai University (CMU; latitude 18° 47’ N, longitude 98° 59’ E and altitude 330
m AMSL) – location were selected for field trials in Thailand. The southern
experiment was undertaken in 2011-2012 with 48 local varieties from southern
Thailand. The northern experiment was undertaken in 2012-2013 with four varieties selected among the 48 southern varieties. The experiments were conducted
in randomized complete block design with three independent replications. Seed
for the southern experiment was obtained from the conservation field at PRRC.
From the results of Zn concentration in the rice caryopsis of the 48 southern
varieties, 4 varieties representative of low (Look Paw), medium (Sang Yod and
Look Krahd), and high (Chaw Gam Preud) Zn in the caryopsis were selected for
use at the northern location.
Plants were grown as lowland rice, i.e. on wetland soil. Briefly, the seeds
were soaked in water overnight and incubated moist until germination and raised
as seedlings for 30 days in the field. Single seedlings of each genotype were
transplanted into hills at 0.25x0.25 m spacing in 2x5 m2 plots in three replicated
blocks. Fertilizer, as 15N-15P2O5-15K2O, was applied twice, at the rate of 93.75
kg/ha, at 25 and 45 days after transplanting. Weeds were removed by hand. Seed
was harvested at maturity by hand, sun-dried to 14% moisture content, dehusked
manually, and the Zn concentration determined.
Chemical analysis
Sub-samples of rice caryopsis (0.5 g) and husk (0.2 g) were oven-dried at
70°C for 72 hours and dry-ashed in a muffle furnace at 535°C for 8 hours. The
ash was dissolved in HCL (1:1; HCl:deionized water) and the Zn concentration
determined using an atomic absorption spectrophotometer (Z-8230 Polarized
Zeeman AAS, Hitachi, Japan) (Allan, 1961). Soybean leaf reference material
and seed samples of the standard low (RD21) and high (Namroo) Zn varieties
were included in each analysis batch as a certified reference standard to check
the quality of plant analysis (“check varieties”). The standard low and high Zn
check varieties were previously used in grain Zn analysis (Saenchai et al., 2012;
Jaksomsak et al., 2015).
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Data analysis
The data were subjected to analysis of variance (ANOVA) and means
that were significantly different were separated at P<0.05 by the least significant
difference (LSD) test. The Pearson correlation model was used to evaluate the
relationship between variety and Zn concentration in the husk and caryopsis.
RESULTS
The Zn concentration in rice caryopsis varied widely among the 48 rice
varieties from southern Thailand grown at the southern location, ranging from
19.2 to 33.3 mg Zn/kg (Figure 1). Forty-four varieties had a medium to high Zn
concentration in the caryopsis (20 to 29 mg Zn/kg) compared with the low (RD
21) and high (Namroo) Zn concentration, the standard check varieties, which had
19 and 29 mg Zn/kg, respectively. However, four varieties, Chaw Gam Preud,
Leuang Hawm, Mai Yah, and Nahn Hak, had higher grain Zn concentrations than
the high-Zn standard check variety. Furthermore, a wide variation in Zn concentration was also observed in the husk among the 48 varieties (Figure 2) – from
6.2 to 20.0 mg Zn/kg, with 4 mg Zn/kg and 13 mg Zn/kg in the low and high
Zn standard check varieties, respectively. Thirty-four varieties had a medium
Zn concentration (4-13 mg Zn/kg) – between the Zn concentration in the husk
of the low and high standard check varieties – and 14 varieties had a high Zn
concentration in the husk (>13 mg Zn/kg) – higher than the Zn concentration in
the husk of the high standard check variety.
Figure 1. Variation in Zn concentration in rice caryopsis among 48 varieties from
southern Thailand, grown at the southern location. Mean averaged from
3 replications.
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
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Figure 2.Variation in Zn concentration in the husk of 48 rice varieties from
southern Thailand, grown at the southern location. Mean averaged from
3 replications.
The Zn concentration in the caryopsis and Zn concentration in the husk for
the 48 southern rice varieties grown at the southern location correlated significantly
(r= 0.55, p< 0.05) (Figure 3).
Figure 3.Relationship between Zn concentration in the caryopsis and husk of 48
varieties from southern Thailand grown at the southern location.
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Seed of the four selected rice varieties (Look Paw, Sang Yod, Look Krahd,
and Chaw Gam Preud) from southern Thailand were grown in the north at Chiang
Mai University to compare the Zn concentration in the caryopsis between the two
locations. The soil series at the two locations differed; the north contained more
clay and was more acidic than the south. In the south, soil N concentration was
3 times higher than the north, and the P, K, and Zn concentrations were 48, 4,
and 2 times lower than the north, respectively (Table 1). In contrast to the lower
soil Zn in the south than the north, three of the selected varieties had higher Zn
concentrations in the caryopsis when grown in the south compared to the north.
(Figure 4). The caryopsis Zn concentrations of Sang Yod (medium Zn), Chaw Gam
Preud (high Zn) and Look Krahd (medium Zn) were 20-42% higher when grown
in the south, but there was no effect of location on the grain Zn concentration in
Look Paw (low Zn).
Table 1.Soil characteristics of the rice cultivation sites at the southern (Phatthalung Rice Research Center, Phatthalung) and northern (Chiang Mai
University, Chiang Mai) Thai locations.
Soil characteristic
Southern Thailand
Northern Thailand
Klaeng
San Sai
Texture
Sandy clay
Sandy loam
Soil pH
5.8
5.1
% Organic matter (Walkley and Black)
2.0
1.8
% Total N (Kjeldahl)
1.8
0.7
Soil series
P (mg/kg) (Bray II)
0.7
33.9
K (mg/kg) (Sodium acetate extraction)
17.8
70.9
Zn (mg/kg) (DPTA extraction)
0.9
1.5
Note: All values are means of 10 replications for soil sampled from 0 to 15 cm depth.
Figure 4.Zinc concentration in rice caryopsis of four rice varieties from southern
Thailand grown in wetland rice fields at Phatthalung Rice Research
Center (south) and Chiang Mai University (north), Thailand.
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
7
DISCUSSION
The variation in Zn concentration in rice caryopsis among the 48 southern
varieties grown in southern Thailand (19 to 33 mg Zn/kg) was within the range
of 17 to 59 mg Zn/kg observed by Saenchai et al. (2012) for the northern Thai
rice varieties. The correlation between Zn concentration in the caryopsis and husk
among 48 rice varieties grown in the south suggests that the husk is an important
source of Zn for the caryopsis; this agrees with the findings of other researchers
(Gao et al., 2012; Impa et al., 2013). The amount of Zn in the husk is also a
major source of Zn transport to the grain during early germination (Prom-u-thai
et al., 2012).
Varieties with high Zn concentration provide a useful source of genetic
material for future breeding programs, for example, Chaw Gam Preud, Leuang
Hawm, Mai Yah, and Nahn Hak, with high Zn concentrations in the caryopsis, and
Ekoo3, with a high Zn concentration in the husk. The positive correlation between
Zn concentration in the caryopsis and husk is not recommended for screening, as
the correlation is quite weak. Further investigation of specific varieties is required
to understand the relative importance of reproductive tissues in Zn supply and
transport for the caryopsis.
In comparing the southern and northern growing locations, preliminary
findings on the four cultivars grown in common at the two, but in different years,
showed that grain Zn concentration was higher in the south than the north, despite
the lower levels of Zn in the southern soil. It is unclear to what extent climate and
soil might have affected supply of Zn to the grain. Studies elsewhere have shown
interactions between genotype and the environment (Gregorio, 2002; Chandel et
al., 2010) and that the caryopsis Zn content was affected by soil texture, pH, and
organic matter content (Chandel et al., 2010). Although the soil Zn concentration in
the south was about two times lower than the north, the south had higher organic
matter and soil N than the north. In particular, the N-nutritional status is critical
in root uptake and root-to-shoot translocation of Zn, as well as Zn remobilization
during both vegetative and generative stages of development (Erenoglu et al.,
2011). Specific studies where N fertilization have enhanced seed Zn are described
by Hao et al. (2007) and Dash et al. (2010). Further work is required to ascertain
the influence of environment on Zn uptake by rice caryopsis in southern Thailand.
This study showed a wide variation in grain Zn in the caryopsis and husk among
48 southern rice varieties grown in southern Thailand. Grain Zn concentration
was higher when grown in the south than the north, despite the lower levels of
Zn in the southern soil. This provides a useful source of genetic material for
potentially improving the Zn content of rice caryopsis in southern Thailand, as
several varieties were found to have higher grain Zn concentration than the standard high Zn check variety.
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ACKNOWLEDGEMENTS
This research was financially supported by a Thai Government Science and
Technology Scholarship from the Ministry of Science and Technology, Thailand,
The Graduate School and Lanna Rice Research Center, Chiang Mai University,
and Thailand Research Fund (RSA5580056). The Phatthalung Rice Research
Center, Thailand is also acknowledged for providing rice seed for this experiment.
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The Effects of Nitrogen as NO3- and NH4+ on the Growth
and Symbiont (Anabaena azollae) of Azolla pinnata R. Brown
Arunothai Jampeetong1,2, Thunyachol Sripakdee1, Tanaporn Khamphaya1
and Sutthathorn Chairuangsri1,2*
1
Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai
50200, Thailand
2
Environmental Science Program, Faculty of Science, Chiang Mai University,
*Corresponding author. E-mail: [email protected]
ABSTRACT
The growth, morphology, and symbiont (Anabaena azollae) of Azolla
pinnata R. Brown were investigated under different external N-supply regimes to
inform the plant’s potential in wastewater treatment. Azolla pinnata plants were
supplied with nitrogen as NO3- or NH4+ at four different concentrations (0, 0.5,
1, and 5 mM) and incubated in a greenhouse for 14 days. The relative growth
rates of NO3--fed plants were not significantly different between treatments,
but decreased significantly at the highest NH4+ concentration. Moreover, the
NO3- concentration did not affect root number. The highest NH4+ concentration
(5 mM) decreased both the root length and number of symbionts (Anabaena
azollae) in the mature leaves of Azolla pinnata. Because Azolla pinnata continued to grow well with supplied NO3- and NH4+, and retained their ability
to absorb nitrogen, they offer potential for treating wastewater, except at the
highest NH4+ concentration, which led to toxicity.
Keywords: Azolla pinnata, Anabaena azollae, Heterocyst, NH4+ toxicity, Symbiont
INTRODUCTION
Azolla pinnata R. Brown is a free-floating aquatic fern belonging to the
family Azollaceae. It is widely distributed in Asia and along the coast of tropical
Africa (Wagner, 1997). The plant consists of alternately arranged leaves on a
prostrate, floating rhizome, with one or two roots hanging in the water column.
The leaf is bilobed, consisting of a chlorophyllous floating dorsal lobe and a
colorless and partially submerged ventral lobe. A cavity in the ventral leaves houses
symbiotic cyanobacteria, Anabaena azollae (Pabby et al., 2003). This symbiont
fixes N2 from the atmosphere and produces a high N level in the plant tissue of
Azolla pinnata, making the plant useful as green manure for rice fields, where it
has been used for several centuries (Shi and Hall, 1988; Peters and Meeks, 1989;
Forni et al., 2001; de Macale and Vlek, 2004).
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More recently, Azolla spp. have been used in water treatment (Kitoh et al.,
1993; Forni et al., 2001; Nahlik and Mitsch, 2006; Costa et al., 2009). Several
studies have shown that Azolla spp. can both grow in and remove nutrients from
wastewater (Reddy and DeBusk, 1985; Kitoh et al., 1993; Vermaat and Hanif,
1998; Costa et al., 2009). In general, two forms of inorganic nitrogen – NH4+
and NO3- – are commonly found in wastewater at concentrations of 1 to 5 mM
(Kadlec and Wallace, 2009). Both the form and concentration of N may affect
plant growth, morphology, and the symbiont. A previous study by Ito and Watanabe
(1983) showed that NO3- and NH4+ at concentrations of 1 mM did not inhibit
the acetylene reduction activity of the symbiont Anabaena azollae in the leaves
of Azolla pinnata; however, they did not determine its effects on the growth and
morphology of the plants.
Several studies have shown that many aquatic or wetland plants, such as
Phragmites australis (Cav.) Trin. ex Steudel and Salvinia natans (L.) All., prefer
NH4+ over NO3-, but that NH4+ is toxic at high concentrations (Kitoh et al., 1993;
Britto and Kronzucker, 2002; Tylova et al., 2005; Cao et al., 2009; Jampeetong and
Brix, 2009a, 2009b; Jampeetong et al., 2012). Because symbionts furnish Azolla
pinnata with nitrogen, external nitrogen in wastewater may affect the symbiotic
relationship and subsequent plant growth. To study this, we examined the growth,
morphology, and symbiotic response of Azolla pinnata R. Brown to two different
forms – NH4+ and NO3- – and concentrations of inorganic nitrogen; the results
can be applied to developing better water treatment systems.
Plant material and experimental set up
Azolla pinnata was obtained from a natural pond at Chiang Mai University,
Chiang Mai, Thailand. The plants were cleaned and grown on a standard N- and
P-free growth medium prepared according to Smart and Barko (1985), to which
0.5 mM of NO3- or NH4+, 100 µM KH2PO4, and a commercial plant micronutrient
solution (Tropica, Egaa, Denmark) (1 mL: 10 L growth solution) were added. The
pH was adjusted to 6.6±0.1.
After the plants had acclimated for 14 days, approximately 2 grams of the
ramet from the stock culture was placed in a plastic pot (4 pots per treatment)
containing 2 liters of a standard N- and P- free growth medium prepared according
to Smart and Barko (1985), to which 100 µM KH2PO4, and a commercial plant
micronutrient solution (Tropica, Egaa, Denmark) were added. The pH was adjusted
to 6.6±0.1 using HCl and NaOH. The treatments consisted of two N forms: NH4+
or NO3- prepared from (NH4)2SO4 or KNO3, respectively, at different concentrations (0, 0.5, 1, and 5 mM). The plants were incubated in the greenhouse at the
Department of Biology, Faculty of Science, Chiang Mai University, Thailand. The
growth medium was changed every 3 days and the plants were cleaned gently by
hand. At the beginning of the experiment, plant ramets similar to experimental
plants (n=10) were selected to estimate the fresh weight and dry weight ratio.
The fresh weight of all plants was measured, and then they were dried until they
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
13
reached a constant weight. The fresh to dry weight ratio was calculated and used
for the relative growth rate calculation.
Growth and morphological study
After 14 days, root number and root length of the plants in each treatment
were measured. The plants were then harvested and freeze dried. The relative
growth rate (g g-1d-1) was calculated by the formula:
where W1 and W2 are the initial and final dry weights (g) of plant material from
each pot, and t1 and t2 are the initial and final time (days).
The shoot area (cm2) was estimated from digital photos taken of each pot at
the same angle and distance. The relative shoot area growth rate (RSGR, cm2cm-2
day-1) was calculated in a similar way to the relative growth rate.
Counting Anabaena and heterocysts
Anabaena azollae in both young (1st in position) and mature (6th in position)
leaves was determined. The leaves were broken using a needle, and then wet
mount slides were made. Anabaena azollae filaments in each leaf were counted.
The heterocyst frequency was measured by randomly counting 200 cells of A.
azollae and recording the number of heterocysts found.
Inorganic nitrogen in the whole plant
Five milligrams of freeze-dried plant materials from each replicate were
extracted with 15 mL of distilled water at 98°C in a water bath for 20 minutes.
Then the NH4+ and NO3- in the extracts were analysed by a modified salicylate
method (Quikchem Method no. 10-107-06-3-B; Lachat Instruments, Milwaukee,
WI, USA). The absorbance of the extracts was measured at 690 and 220 nm using
a UV-VIS spectrophotometer (Lambda 25 version 2.85.04, USA) to determine
NH4+ and NO3-, respectively.
Statistics
The data were analyzed by one-way and two-way analysis of variance
(ANOVA) using Statgraphics Plus ver. 4.1 software (Manugistics, Inc., MD, USA).
The normality of the distribution and homogeneity of variance were tested using
Cochran’s C-test. If necessary, data was log-transformed to ensure homogeneity
of variance. Multiple comparisons of means were identified by Scheffe’s test
(p<0.05).
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RESULTS
Growth and morphology
Both the form and concentration of N affected relative growth rates (RGRs)
of Azolla pinnata, with significant interactive effects between these two factors
observed (Table 1, Figure 1a). The plants grown on the three lowest concentrations of NH4+ had higher relative growth rates than that grown on NO3-. However,
increasing NO3- concentrations did not affect plant growth, whereas the highest
NH4+ concentration (5 mM) decreased growth significantly. Varying the concentrations of either NH4+ or NO3- did not significantly affect relative shoot area
growth rates (RSGRs) (Fig.1b).
Neither the N form nor concentration significantly affected root number
(Figure 2a). However, the higher NH4+ concentrations negatively affected root
length (Figure 2b), with significant interaction between N form and N concentration (Table 1).
Table 1.Degrees of freedom (d.f.), F-ratios, and significance of a two-way
ANOVA of relative growth rate (RGR), relative shoot area growth rates
(RSGR), root number, root length, number of Anabaena azollae, number
of heterocyst, and NH4+ in the plant tissue of Azolla pinnata grown on
NO3- or NH4+ at four different concentrations (0, 0.5, 1, and 5 mM) for
14 days.
Main effects
Interaction
d.f.
N form (A)
(NO3- / NH4+)
N concentration
(B)
AxB
3
1.96
2.34
4.13*
RSGR (cm cm d )
3
8.33
0.05
0.18
Root number
4
3.50
0.37
1.20
Root length (mm)
4
3.16
3.10*
5.17**
2.87
12.38***
5.40**
24.33***
12.4***
5.29**
RGR (g g-1d-1)
2
-2
-1
Number of Anabaena azollae
(filament)
- young leaves
7
- mature leaves
7
Number of heterocyst (cell)
- young leaves
7
6.42*
10.72***
2.07
- mature leaves
7
12.13**
9.16*
4.04*
NH4+ in the plant tissue
(mg g-1 dw)
7
1360.58***
15.45***
15.87***
Note: *(in column) indicate significant differences between factors, * (P<0.05), ** (P<0.01), and
*** (P<0.001).
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15
Figure 1.The relative growth rate (RGR) (a) and the relative shoot area growth
rate (RSGR) (b) of Azolla pinnata (mean±SD) grown with NO3- (grey
column) or NH4+ (dark column) as the nitrogen source at four different
concentrations (0, 0.5, 1, 5 mM) for 14 days. Different letters above
columns indicate significant differences between treatments.
Anabaena and heterocyst
Both the form and concentration of N affected the amount of Anabaena
azollae; the interaction between these two factors was observed (Table 1). Fewer
Anabaena azollae were found in young leaves than mature leaves. In young leaves,
the amount of Anabaena azollae was mildly affected by the form and concentration
of N compared to mature leaves, in which high concentrations of NH4+ reduced
the number of Anabaena azollae (Figure 3a, b). Similarly, heterocyst counts per
200 vegetative cells of Anabaena significantly decreased in plants fed with 5 mM
NH4+; NO3- had no affect on either the young or mature leaves (Figure 3b, d). The
effects of N concentration depended on the N form, as shown by the significant
interaction term in the ANOVA results (Table 1).
Figure 2.Root number (a) and root length (b) of Azolla pinnata (mean±SD)
grown with either NO3- (grey column) or NH4+ (dark column) as the
nitrogen source at four different N concentrations (0, 0.5, 1, 5 mM).
Different letters above columns indicate significant differences between
treatments.
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Figure 3.Number of Anabaena azollae (a, b) and heterocyst (c, d) in young
leaves (dark column) and mature leaves (grey column) of Azolla pinnata
(mean±SD) grown with either NH4+ or NO3- as the nitrogen source at
four different N concentrations (0, 0.5, 1, 5 mM) for 14 days. Different
letters above columns indicate significant differences between treatments.
Inorganic nitrogen in the whole plant
The NH4+ in the plant tissue of Azolla pinnata treated with NO3- at different
concentrations did not significantly differ. In contrast, in the NH4+-fed plants,
the concentration of NH4+ in the plant tissue increased when NH4+ was supplied
at high concentration (Figure 4a). NO3- in the plant tissue was unaffected in the
NO3--fed plants, even as the external concentration of NO3- increased. The NO3in the plant tissue of the NH4+-fed plants was not determined (Figure 4b).
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17
Figure 4.Water extractable NH4+ -N (a) and NO3- -N (b) in the tissue of Azolla
pinnata (mean±SD) grown with either NH4+ (dark column) or NO3(grey column) as the nitrogen source at four different N concentrations
(0, 0.5, 1, 5 mM) for 14 days. Different letters above columns indicate
significant differences between treatments. n.d. = not determined.
DISCUSSION
The form (NO3 or NH4 ) and concentration (0, 0.5, 1 and 5 mM) of
externally supplied nitrogen affected the growth and morphology of Azolla
pinnata R. Brown. The effects were lower in the NO3--fed plants. Most aquatic
plants prefer inorganic nitrogen in the form of ammonium (NH4+), because of
the lower energy needed for uptake and assimilation (Cedergreen and Madsen,
2002; Tylova-Munzarova et al., 2005; Jampeetong and Brix, 2009a). However,
in our study, Azolla pinnata showed a negative response – low growth rate, short
roots, and leaf chlorosis, especially in mature leaves – to the highest concentration
(5 mM) of NH4+. Others have found similar effects of high NH4+ concentrations
in other free-floating plants, such as Azolla filiculoides (Kitoh et al., 1993) and
Salvinia natans (Jampeetong and Brix, 2009b). Ito and Watanabe (1983) reported
that biomass decreased after Azolla pinnata was exposed to 10 mM NH4+ for 4
days, but growth and morphology data were not precisely determined. They also
reported that both the form and concentration of nitrogen affected the symbiont
Anabaena azollae in the leaves of Azolla pinnata; a high NH4+ concentration
(10 mM) inhibited acetylene reduction activity, indicating decreased nitrogenase
enzyme activity. However, they did not determine the amounts of Anabaena and
heterocyst. In our results, we found that Anabaena azollae and its heterocyst
decreased with increasing external NH4+ concentration, particularly in mature
leaves. Maejima et al. (2001) found a similar result; both Anabaena azollae
and heterocyst decreased more than 50% in mature leaves, while young leaves
were not affected. In contrast, externally supplied NO3- did not affect Anabaena
azollae and its heterocyst. Therefore, the plants can obtain NO3- from both externally
supplied NO3- and from the atmosphere by fixing N2. Costa et al. (2009) found
similar results with Azolla filiculoides grown on combined nitrogen wastewater.
-
+
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Even though we did not determine the NH4+ uptake of Azolla pinnata, the
+
NH4 concentration in the plant tissue increased when the plants were supplied
with high concentrations of external NH4+. This indicated that the ability of the
plants to take up external NH4+ was not suppressed. Jampeetong et al. (unpublished data) found that Azolla pinnata grown on 1 mM NH4NO3 showed that the
uptake of NH4+ was 27 times higher than NO3-. Moreover, Cary and Weerts (1992)
showed that Azolla pinnata and Azolla filiculoides can obtain nitrogen from an
external supply, even though both species benefit from a symbiotic association.
However, in our study, Azolla pinnata appeared not to have a mechanism to
prevent over-accumulation of NH4+ in its cells. This over-accumulation has been
shown to be toxic (Britto and Kronzucker, 2002). Similar results were recorded
in several NH4+ intolerant species, including Thalassia hemprichii and Zostera
marina (van Katwijk et al., 1997; Christianen et al., 2011).
Many aquatic plants have been used for wastewater treatment. Most species
had high growth rates and high acquisition of nitrogen (Koerselman and Meuleman,
1996; Abe and Ozaki, 1998). According to our results, Azolla pinnata had high
growth rates and biomass production. Hence, this species offers potential for
treating various types of wastewater in constructed wetland systems.
In conclusion, both the form and concentration of N affected the growth
and morphology of Azolla pinnata. The plants grown with NH4+ up to 1 mM had
higher growth rates than NO3--fed plants, but the growth rate and root length of
the plants decreased at the highest concentration (5 mM). Anabaena azollae and
its heterocyst also decreased in the mature leaves of the plants fed with a high
NH4+ concentration, whereas the youngest leaves were not affected. Azolla pinnata
was able to take up external NO3- or NH4+, but high NH4+ concentrations may
cause NH4+ toxicity and lead to plant destruction. Azolla pinnata offers potential
for removing nutrients from wastewater, but exposure to NH4+ contamination
must be less than 5 mM in order to maintain plant growth and the potential of N
uptake from the polluted water.
ACKNOWLEDGEMENTS
This research was supported by the Institute for the Promotion of Teaching
Science and Technology (IPST), Thailand. The authors would like to thank Mr.
Alvin Yashinaga for improving the English of the manuscript.
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Identification of Antioxidants in Lamiaceae Vegetables by
HPLC-ABTS and HPLC-MS
Trakul Prommajak1, Sang Moo Kim2, Cheol-Ho Pan3, Sang Min Kim3,
Suthat Surawang1 and Nithiya Rattanapanone1,4*
1
Faculty of Agro-Industry, Chiang Mai University, Chiang Mai 50100, Thailand
Department of Marine Food Science and Technology, Gangneung-Wonju National
University, Gangneung 210702, Republic of Korea
3
Functional Food Center, Korea Institute of Science and Technology, Gangneung
210340, Republic of Korea
4
Posthavest Technology Research Institute, Chiang Mai University, Chiang Mai
50200, Thailand
2
ABSTRACT
Antioxidants in five Lamiaceae vegetables, namely lemon balm, sweet
basil, clove basil, holy basil, and lemon basil, were identified by HPLC-ABTS
and HPLC-MS. The major antioxidants in the samples were hydroxycinnamic
acids. Rosmarinic acid exhibited the highest antioxidant activity in each sample,
except clove basil, in which chicoric acid contributed the highest antioxidant
activity. Lemon balm had the highest concentration of rosmarinic acid. Chicoric
acid (highest in clove basil) and dihydroxy-dimethoxyflavone (highest in basil)
were also important antioxidants in many Ocimum spp. Other minor antioxidants were caffeic acid, caftaric acid, syringic acid, syringic acid, rutin, and
acacetin-acetylglucoside.
Keywords: Lamiaceae, Antioxidant, HPLC-ABTS, Rosmarinic acid, Chicoric acid
INTRODUCTION
Free radicals in food cause lipid peroxidation, which adversely affects its
nutritional and organoleptic properties (Yen and Duh, 1994). Free radicals in the
human body are associated with many degenerative diseases, e.g. cardiovascular
disease, diabetes, and cancer (Boeing et al., 2012). Consumption of fruits, vegetables, and antioxidants was associated with a lower risk of cancers and cardiovascular disease (Genkinger et al., 2004).
Lamiaceae is a family of annual or perennial aromatic herbs. It contains
236 genera that can be grown under a variety of soil and climatic conditions
worldwide, with the exception of the cold regions of high latitude (Harley et al.,
2004). Lavender, thyme, peppermint, patchouli, rosemary, and sage are used to
extract volatile aromatic oil or prepare perfumes. Some plants are used for medicinal purposes. For example, the leaves of Ocimum sanctum are used to treat
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➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
the common cold. Flowers of O. basillicum are used to treat digestive and renal
diseases. Leaves and tubers of some Lamiaceae plants are used as food (Reddy
et al., 2004).
Lamiaceae plants also contain antioxidative phenolic compounds that effectively scavenge free radicals. The major antioxidants in the plants of this family
are hydroxycinnamic acids and flavonoids. Rosmarinic acid has also been found,
notably in many Lamiaceae plants, e.g. basil, sage, lemon balm, peppermint, thyme,
lavender, rosemary, marjoram, hyssop, savory, and oregano (Zgórka and Głowniak,
2001; Dorman et al., 2003). The other hydroxycinnamic acids responsible for the
antioxidant capacity of Lamiaceae plants include caffeic acid, chlorogenic acid, and
ferulic acid (Chen and Ho, 1997). Chicoric acid, another hydroxycinnamic acid,
was recently found in basil and had not yet been identified in other Lamiaceae
plants (Lee and Scagel, 2009). Lamiaceae plants also contain flavonoids. Apigenin
and luteolin derivatives have been identified in germander, sage, thyme, savory,
marjoram, and oregano (Dorman et al., 2003; Valant-Vetschera et al., 2003).
Online HPLC-antioxidant assay is a technique developed for simultaneous
determination of compounds and their antioxidant capacity. The compounds eluted
from a column react with an oxidant probe, e.g. 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) radical cation (ABTS•+), and decrease the absorbance
of the chromophore. This method avoids the decomposition of the compounds
that occurs in a conventional process (He et al., 2010).
Lemon balm, basil, clove basil, holy basil, and lemon basil are common culinary herbs in Thailand. To the best of our knowledge, the profiles of the phenolic
compounds with their antioxidant capacity of these plants cultivated in Thailand
have not been reported. Therefore, the objective of this study was to identify and
quantify the bioactive compounds that are responsible for antioxidant activity in
Lamiaceae vegetables from Thailand using LC-ABTS and LC-MS assays.
MATERIAL AND METHODS
Materials
Lemon balm (Melissa officinalis L.), sweet basil (Ocimum basilicum L.),
clove basil (Ocimum gratissimum L.), holy basil (Ocimum sanctum L.), and lemon
basil (Ocimum × citriodorum) were purchased from a local market in Chiang Mai
Province, Thailand during August-September 2012. Methanol and water for HPLC
analysis were purchased from Fisher (Seoul, Korea). 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and potassium persulfate
were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Preparation of vegetable extracts
The edible parts of vegetables, without bruises or visual defects, were
selected and washed in water for about 1 min. The vegetables (10 g) were extracted
in 60% ethanol (50 ml) using a blender (Model MR 4050 CA, Braun, Spain) for
30 sec. Then, the extracts were filtered through a double layer of muslin cloth
and centrifuged at 2,500xg for 20 min. The supernatant was kept at -80°C until
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
23
analysis. Extraction was performed in duplicates.
Identification and quantification of antioxidants
Separation of the compounds in the crude vegetable extracts was performed
by Agilent 1200 series HPLC (Agilent Technologies, Santa Clara, CA, USA) using
the method described by He et al. (2010) with slight modification. Sample (10
μl) was eluted by 100% methanol (solvent A) and methanol/water/formic acid
(20/80/0.25, v/v/v) (solvent B) through a Kromasil 100-5C18 (250×46 mm) column
with a flow rate of 0.7 ml/min and column temperature of 30°C. Gradient elution
began with the initial condition of 100% B, then changed to 90% B at 6.25 min,
80% B at 12.5 min, 55% B at 50 min, and 20% B at 68.75 min, before changing
back to 100% B at 75 min. The compounds were detected by diode array detector
(DAD) at 210, 254, 280, 330, and 520 nm.
The compounds that escaped from DAD were subjected to online-ABTS
analysis. ABTS reagent (2 mM ABTS and 3.5 mM potassium persulphate) was
prepared and kept in a dark condition at room temperature for 16 h to stabilize the
radical before use. The reagent was pumped at a flow rate of 0.5 ml/min to react
with the eluate, before passing through a multiple wavelength detector (MWD) for
detection of ABTS at 734 nm. The presence of antioxidants in a sample resulted
in negative peaks in the chromatogram.
Antioxidant concentrations were quantified from calibration curves. Flavonoids were quantified as the molar equivalent of quercetin. Other phenolic
compounds were quantified as the molar equivalent of caffeic acid. Antioxidant
capacities of the compounds were calculated from the area of the negative peak
and expressed as Trolox equivalents.
For HPLC-MS analyses, compound separation and UV-Vis detection were
performed using the same conditions as HPLC-ABTS. Atmospheric pressure
ionization-electrospray (API-ES) ionization was performed by an Agilent 6120
quadrupole mass spectrometer in both positive and negative modes. Capillary
voltages were 4,000 V for positive and 3,500 V for negative ionization. The
drying gas flow rate was 9 L/min, the drying gas temperature was 350°C, and
the nebulizer pressure was 35 psig. Mass spectra were recorded in the range of
m/z 100-1,500. The compounds were identified by comparing molecular weights,
fragments, and UV absorption with that reported in the literature.
Statistical analysis was performed by R version 2.15.1.
RESULTS
Identification of antioxidants
Various compounds accounted for the antioxidant activity of the ethanol
extracts of the five Lamiaceae vegetables. HPLC-ABTS chromatograms revealed
that the vegetables had similar antioxidant profiles (Figure 1). Mass spectra of
identified compounds are shown in Figure 2. Compound 1 was identified as syringic
acid (Table 1). Compounds 2, 3, 4, and 7 were hydroxycinnamic acids, identified
as caftaric acid, caffeic acid, chicoric acid, and rosmarinic acid, respectively.
Compounds 5, 6, and 9 were flavonoids, identified as acacetin-acetyl glucoside,
24
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
rutin (quercetin rutinoside), and dihydroxy-dimethoxyflavone, respectively. For
general phenolic compounds, UV absorption at 280 nm was detected. But for
hydroxycinnamic acids and flavonoids, absorption maxima around 330 nm were
Figure 1.HPLC-ABTS chromatograms of the ethanolic extract of Lamiaceae
vegetables. Positive peaks: DAD signal at 280 nm for detection of
phenolic compounds. Negative peaks: MWD signal at 734 nm for
detection of ABTS radical scavenging activity.
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
25
Figure 2.Mass spectra of antioxidative compounds identified from Lamiaceae
vegetables. All mass spectra were from negative-ion mode, except
compound 8.
280
221
297sh
245
244
222
284
220
202
252
1
2
3
4
5
6
7
8
9
332
232
330
230
244
330
324
330
λmax (nm)
No.
280
330sh
330
313
359
609
487
473
179
311
197
[M–H]–
627
−
719
−
−
947
−
623
395
[2M-H]–
−
−
−
−
−
−
315
165
−
611
−
−
181
−
149 [tartaric acid–H]–
−
199
[M+H]+
–
Other
MS negative ions (m/z)
337
187
383
633
511
497
−
335
221
[M+Na]+
−
−
743
1243
−
971
−
647
−
[2M+Na]+
Other
610
303 [quercetin+H]+
449 [M–hexose]
−
−
314
164
360
488
163 [glucose+H]+
221 [acetyl glucoside]
285 [acacetin+H]+
163 [caffeoyl]
474
180
312
198
MW
163 [caffeoyl]
295 [M–caffeic
acid+H]+
135 [M–COOH]
163 [caffeoyl]
153 [M–COOH]
166 [M–OCH3]
MS positive ions (m/z)
Table 1. Identification of antioxidants in Lamiaceae vegetables by HPLC-MS.
Dihydroxydimethoxyflavone
Coumaric acid
Rosmarinic acid
Rutin
Acacetin-acetyl
glucoside
Chicoric acid
Caffeic acid
Caftaric acid
Syringic acid
Proposed name
Vieira et al.
(2003)
Sánchez-Rabaneda
et al. (2003)
Lee and Scagel
(2009)
Eyles et al.
(2007)
Lai et al.
(2007)
Lee and Scagel
(2009)
(Hossain et al.
(2010)
Lee and Scagel
(2009)
Sun et al.
(2007)
References
26
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
27
also detected (Tomas-Barberan and Ferreres, 2012).
Quantification of antioxidative compounds
The phenolic acid concentrations in five Lamiaceae vegetables are shown
in Table 2. Of the phenolic compounds, rosmarinic acid was the most abundant
in lemon balm, sweet basil, and holy basil, while chicoric acid was the most
abundant in clove basil and lemon basil.
Table 2.The phenolic acid concentrations in Lamiaceae vegetables.
Concentrationa (mg/g DW)
Compounds
Lemon
balm
Sweet
basil
Clove
basil
Holy basil
Lemon
basil
Syringic acid
0.30±0.01
0.07±0.01
0.07±0.00
0.12±0.03
0.08±0.00
Caftaric acid
0.52±0.12
2.60±0.35
1.26±0.02
4.13±0.12
0.71±0.02
Caffeic acid
1.03±0.03
2.44±0.53
0.35±0.07
0.22±0.03
0.63±0.09
Chicoric acid
NDb
5.67±0.25
17.12±0.41
3.32±0.43
8.46±0.23
Acacetin-acetyl glucoside
ND
0.58±0.01
2.30±0.02
3.19±0.04
0.75±0.03
Rutin
5.32±0.64
1.31±0.04
ND
ND
ND
Rosmarinic acid
32.31±2.33 13.32±1.12
1.21±0.08
17.19±1.19
7.65±0.07
Coumaric acid
Dihydroxy-dimethoxyflavone
ND
ND
14.08±0.16
ND
ND
0.14±0.00
3.35±0.44
ND
0.83±0.81
1.53±0.04
Note: aMeans of duplicate analyses. Flavonoids were quantified as molar equivalent of quercetin.
Other phenolic compounds were quantified as molar equivalent of caffeic acid. bND, not detected.
Antioxidant capacity of phytochemicals
ABTS radical cation (ABTS•+) is a blue/green chromophore produced by the
reaction between ABTS and potassium persulfate. It has an absorption maximum
at 734 nm. In the presence of hydrogen-donating antioxidants, e.g. polyphenols
and flavonoids, the radical cation becomes colorless. In an online HPLC-ABTS,
it produced a negative peak in the presence of antioxidants (Koleva et al., 2001).
Rosmarinic acid possessed the highest antioxidant capacity among the
compounds in the tested samples, except clove basil, in which chicoric acid
contributed the highest antioxidant capacity (Table 3). Chicoric acid and dihydroxy-dimethoxyflavone were also responsible for a moderately large portion of
antioxidant capacity in sweet basil and lemon basil. Caffeic acid, identified in all
samples, was highest in sweet basil.
28
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
Table 3.Antioxidant capacity of phenolic compounds in Lamiaceae vegetables.
Antioxidant capacitya (mmol TE/g DW)
Compounds
Lemon
balm
Sweet
basil
Clove
basil
Holy
basil
Lemon
basil
Syringic acid
0.37±0.04
0.12±0.03
0.06±0.01
0.13±0.02
0.10±0.01
Caftaric acid
0.19±0.03
0.83±0.08
0.39±0.01
1.21±0.06
0.19±0.00
Caffeic acid
0.78±0.06
1.77±0.16
0.28±0.03
0.14±0.03
0.50±0.02
Chicoric acid
NDb
2.10±0.04
4.18±0.11
1.18±0.13
2.65±0.01
ND
0.08±0.00
0.30±0.00
1.22±0.20
0.06±0.02
Rutin
0.44±0.05
0.20±0.00
ND
ND
ND
Rosmarinic acid
9.29±0.32
7.11±0.05
1.16±0.07
7.46±0.06
4.85±0.03
ND
ND
1.19±0.08
ND
ND
0.18±0.01
3.54±0.19
ND
0.24±0.10
1.86±0.06
Coumaric acid
Note: aMeans of duplicate analyses. bND, not detected.
Trolox equivalent antioxidant capacity (TEAC) of antioxidants
The TEAC value is the specific antioxidant capacity of a given substance
compared to the standard Trolox (Huang et al., 2005). It is calculated as the ratio
between the antioxidant capacity (molar equivalent of Trolox from area of negative
peak) and molarity of a compound. The antioxidant activity of the hydroxycinnamic acids decreased in the following order: rosmarinic acid > chicoric acid >
caffeic acid > caftaric acid (Table 4).
Table 4.Trolox equivalent antioxidant capacity (TEAC) of phenolic compounds
in Lamiaceae vegetables.
TEACa
Compounds
Lemon
balm
Sweet
basil
Clove
basil
Holy
basil
Lemon
basil
Mean±SD
Syringic acid
2.46
3.47
1.89
2.19
2.51
2.50±0.59
Caftaric acid
1.10
1.00
0.97
0.91
0.81
0.96±0.11
Caffeic acid
1.35
1.31
1.48
1.13
1.43
1.34±0.13
Chicoric acid
NDb
0.75
1.16
1.69
1.48
1.27±0.41
ND
0.71
0.63
1.87
0.41
0.91±0.66
Rutin
0.50
0.91
ND
ND
ND
0.71±0.29
Rosmarinic acid
1.04
1.93
3.45
1.56
2.28
2.05±0.91
Coumaric acid
ND
ND
0.14
ND
ND
0.14
4.17
3.33
ND
0.92
3.81
3.06±1.47
Note: aMolar of Trolox with equivalent antioxidant capacity of a 1 molar substance. bND, not detected.
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
29
DISCUSSION
Phenolic compounds are secondary metabolites in plants that play a role in
plant adaptation to the environment, for example, protection against ultraviolet
radiation, herbivores, pathogens, and abiotic stress (Bourgaud et al., 2001). The
identified phenolic compounds were synthesized through the phenylpropanoid
Figure 3.Phenylpropanoid pathway for synthesis of some phenolic compounds
in Lamiaceae plants. Each arrow represents one enzymatic reaction.
Adapted from: Petersen et al. (2009); Saltveit (2009); and Bel-Rhlid
et al. (2012).
30
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
pathway as shown in Figure 3.
Although the culinary herbs used in this study contained similar antioxidant
compounds, their concentrations varied; this affected the total antioxidant capacity of the extracts. The variation in phenolic compounds in plants is affected by
species, genotypes, climate, location, and growing conditions (Asami et al., 2003;
Anttonen and Karjalainen, 2005; Scalzo et al., 2005).
The caffeic acid concentrations in the Lamiaceae plants in this study (from
0.22 mg/g DW in holy basil to 2.44 mg/g DW in sweet basil) were similar to the
range found in 10 Lamiaceae plants (from 0.1 mg/g DW in lavender to 2.6 mg/g
DW in sweet basil) by Zgórka and Głowniak (2001).
The concentration of caftaric acid in sweet basil (2.82 mg/g DW) found
here was higher than those reported in 15 basil cultivars (from 0.09 to 0.49 mg/g
DW) by Kwee and Niemeyer (2011). Caftaric acid has also been found in other
plant families – 0.51-1.68 mg/g DW in manatee grass (Syringodium ﬁliforme)
(Nuissier et al., 2010) and 1.9-6.5 mg/g DW in purple coneflower (Echinacea
purpurea), depending on growth temperature and light adiation (Wu et al., 2007).
The concentration of chicoric acid in sweet basil (6.17 mg/100 g DW)
found here was higher than those reported in 15 sweet basil cultivars (0.03-2.78
mg/g DW) by Kwee and Niemeyer (2011) and those reported in commercial dry
basil (0.06-0.16 mg/g DW) by Lee and Scagel (2010). For other plant families,
the concentration of chicoric acid has been reported as 0.94-5.26 mg/g DW for
manatee grass (Nuissier et al., 2010) and 0.43-19.27 mg/g DW for Echinacea
spp. (Pellati et al., 2004).
Chicoric acid has been previously identified as a major phenolic compounds
in Echinacea spp. (Perry et al., 2001). In addition to its antioxidant activity, this
compound also has been shown to have antiviral properties by targeting human
immuno-deficiency virus type 1 (HIV-1) integrase (Robinson Jr, 1998). Echinacea,
indigenous to North America, has been commercialized worldwide for treating the
common cold (Barrett, 2003). This study found chicoric acid, a main bioactive
compound in Echinacea, in clove basil, which is widely cultivated in Southeast
Asia.
Rosmarinic acid was the most abundant of the hydroxycinnamic acids found
in basil (13.25 mg/g DW); others have also reported this (Lee and Scagel, 2010;
Bušić et al., 2013). The rosmarinic acid concentration in basil (13.25 mg/g DW)
was similar to that of sweet basil from New Zealand (10.86 mg/g DW) (Shan et
al., 2005). However, this is higher than Kwee and Niemeyer (2011) found in 15
different basil cultivars (0.10 to 6.09 mg/g DW). Basil variety and nitrogen fertilization affected the concentration of the phenolic compounds. Rosmarinic acid
concentrations ranged from 5.41 mg/g DW in basil cv. Sweet Thai irrigated with
5 mM nitrogen to 47.89 mg/g DW in basil cv. Dark Opal grown in the summer
and irrigated with 0.1 mM nitrogen (Nguyen and Niemeyer, 2008). Rosmarinic
acid content in sage (Salvia spp.), another Lamiaceae plant, has been shown to
vary from 13.3 to 47.3 mg/g DW (Bandoniene et al., 2005). In addition to its antioxidant activity, rosmarinic acid also has been shown to have anti-inflammatory
activity by inhibiting diesel exhaust particles-induced lung injury (Sanbongi et al.,
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
31
2003) and antiviral activity against herpes simplex virus type 1 and 2 (Mazzanti
et al., 2008; Astani et al., 2012).
Syringic acid has been identified in other Lamiaceae plants, e.g., basil,
oregano, rosemary, sage, spearmint, and thyme, in concentrations ranging from
0.05 to 0.12 mg/g DW (Kivilompolo and Hyötyläinen, 2007).
Aglycone of acacetin has been previously identified in many Ocimum spp.,
e.g., O. basillicum, O. × citriodorum, O. americanum, and O. minimum (Grayer
et al., 2004).
Rutin or quercetin-3-O-rutinoside has been previously identified in Lamiaceae plants, e.g., rosemary, oregano, basil, and thyme, with concentrations ranging
from 0.3 to 34.0 mg/g FW (Lee and Scagel, 2009; Hossain et al., 2010; Sofic
et al., 2010). Rutin has also been identified in clove basil (Grayer et al., 2000),
although it was not detected in this study.
The isomers of dihydroxy-dimethoxyflavone, including cirsimaritin
(5,4’diOH-6,7-diOMe flavone) and ladanein (5,6-diOH-7,4’-diOMe flavone),
have been identified in hoary basil (O. americanum), sweet basil, and lemon basil (Grayer et al., 2004). The isomers have also been found in other
Lamiaceae plants. For example, cirsimaritin and ladanein were identified in
catmint (Nepata spp.) (Jamzad et al., 2003). Cirsimaritin and 5,4’-diOH-7,3diOMe ﬂavone have been identified in Teucrium spp. Cirsimaritin, ladanein,
and velutin (5,4’-diOH-7,3’-diOMe flavone) have been identified in Salvia spp.
(Valant-Vetschera et al., 2003).
The number and position of hydroxyl groups has been shown to influence
the antioxidant activity of phenolic compounds (Arts et al., 2003). Rosmarinic
acid and chicoric acid have four hydroxyl groups attached to two aromatic rings,
while caffeic acid and caftaric acid have two hydroxyl groups attached to an
aromatic ring. While the number and position of the hydroxyl groups attached to
the aromatic rings is similar in rosmarinic acid and chicoric acid, rosmarinic acid
has a higher TEAC value. The difference might due to the presence of a tartaroyl
group in chicoric acid. A similar trend has also been found between caffeic acid
and caftaric acid, with the latter containing a tartaroyl group.
Flavonoid aglycone (dihydroxy-dimethoxyflavone) had a higher TEAC
value than flavonoid glycosides (rutin and acacetin-acetyl glucoside). Glycosylation
negatively affected the antioxidant activity of flavonoids, due to a steric effect
(Rice-Evans et al., 1996).
The TEAC values of the phenolic compounds found here (Table 4) were
similar to those reported previously in the literature – 1.31 and 1.26 for caffeic acid
(Cai et al., 2006; Rice-Evans et al., 1996) and 2.13 for rosmarinic acid (Nenadis
et al., 2004).
The TEAC value represents the specific antioxidant capacity of a compound.
The same compounds, even though analyzed from different samples, should have
similar TEAC value. However, some of the same compounds extracted from
different Lamiaceae plants had different TEAC values. A possible explanation
for this variation is the low reaction kinetics of an ABTS assay, which takes a
long time to reach the end point (Karadag et al., 2009). A normal reaction time
32
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
for an ABTS assay has been reported as 6 min (Re et al., 1999). In contrast, the
reaction in an online HPLC-ABTS assay takes only a few minutes before reaching
a detector. Different compounds require different reaction times with the ABTS
radical to reach the end point. Walker and Everette (2009) have reported end points
for Trolox and ascorbic acid of within 2 s; gallic acid of more than 15 s; rutin,
quercetin, and curcumin of about 1 min; and chlorogenic acid, caffeic acid, and
ferulic acid of more than 1 min. Higher antioxidant concentrations prolonged the
reaction rates. Gallic acid at the concentration of 50 μM required upwards of 5 min
to reach steady state, while 250 μM gallic acid required 15 min (Pérez-Jiménez
and Saura-Calixto, 2008). Although the reaction time between the ABTS radical
and different substances may differ, each substance had the same reaction time
from mixing point to detector (as determined by flow rate of HPLC). Therefore,
the same substance from different samples had the same reaction time, and should
respond according to their concentration.
In this study, the TEAC value of rosmarinic acid (derivative of caffeic
acid) varied from 1.04 to 3.45. A plot between concentration and TEAC value of
rosmarinic acid showed a negative relationship (Figure 4). The higher the concentration of rosmarinic acid in the samples, the lower the TEAC values. These
results indicated that ABTS might not be appropriate for quantifying the antioxidant
capacity of antioxidants with slow reaction kinetics by an online HPLC-ABTS,
unless the antioxidant concentrations were low enough to reach steady state before
reaching a detector.
Figure 4.Relation between rosmarinic acid concentration and TEAC value obtained by HPLC-ABTS.
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
33
CONCLUSION
The Lamiaceae vegetables studied here contained similar compounds that
were responsible for their antioxidant activity. However, these compounds were
present at different concentrations. The major antioxidants were hydroxycinnamic
acids and flavonoids. Rosmarinic acid was responsible for the highest antioxidant
activity in all samples, except clove basil. The rosmarinic acid content was highest
in lemon balm. Other major antioxidants were chicoric acid and dihydroxy-dimethoxyflavone. Chicoric acid content was highest in clove basil. Dihydroxy-dimethoxyflavone content was highest in sweet basil. The minor antioxidants were
caffeic acid, caftaric acid, syringic acid, rutin, and acacetin-acetylglucoside. In
addition to antioxidant activity, Lamiaceae plants also possessed other medicinal
properties. Ocimum spp., especially clove basil, contained a significant amount
of antiviral chicoric acid, making it an alternative source to Echinacea.
ACKNOWLEDGEMENTS
The Thailand Research Fund, through the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/0260/2551), supported this research. A KIST Gangneung
Institute Intramural Grant (2Z04220) supported C. Pan and S. Kim.
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Doi: 10.12982/cmujns.2016.0004
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39
A Simple Device for Collecting Exhaled Breath Condensate
(EBC) to Study Inflammatory Biomarkers of PM10 Exposure
in Thai Schoolchildren
Waraphan Phornwisetsirikun1,2, Tippawan Prapamontol2*,
Somporn Chantara3, Prasak Thavornyutikarn3,
and Somrak Rangkakulnuwat4,5*
1
Environmental Science Program and Center of Excellence on Environmental
Health and Toxicology, Faculty of Science, Chiang Mai University, Chiang Mai
50200, Thailand
2
Environment and Health Research Unit, Research Institute for Health Sciences,
3
Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang
Mai 50200, Thailand
4
Department of Pediatrics, Faculty of Medicine, Chiang Mai University, Chiang
Mai 50200, Thailand
5
Pediatrics Service, Chiangmai Ram Hospital, Chiang Mai 50200, Thailand
*Corresponding authors. E-mail: [email protected], [email protected]
ABSTRACT
This study developed a portable device to collect exhaled breath condensate (EBC) and used it to collect EBC samples from schoolchildren exposed to
ambient PM10. The developed device was validated, including investigating the
effect of collecting duration and breathing patterns on EBC volume, with five
healthy volunteers. All five volunteers tolerated the device well, completing the
EBC collection procedure without difficulty. Collecting normal tidal breathing
for 10 minutes yielded the required EBC volume. We conducted a follow-up study
with 104 healthy schoolchildren from two different primary schools in Chiang
Mai, Thailand. We measured exhaled H2O2 concentrations in both the rainy
and dry season; ambient PM10 was significantly higher in the dry season. In
the dry season, the mean exhaled H2O2 concentration was significantly higher
in both groups (p<0.05). This study showed that the developed EBC collector
device was cost effective, safe, rapid, and simple to use and exhaled H2O2 could
be used as a biomarker for elevated PM10 exposure before clinical symptoms
appeared.
Keywords: Exhaled breath condensate collector device, Inflammatory marker,
Schoolchildren, Chiang Mai, PM10
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INTRODUCTION
Previous studies have shown that airborne particulate matter less than 10
µm in aerodynamic diameter (PM10) is a complex mixture of many pollutants –
chemicals and transition metals, that are capable of redox cycling and that can
stimulate inflammatory responses, especially in children (Kelly, 2003; Schwartz,
2004). Evidence has suggested that organic components deposited on the particle
surface play an important role in mediating the toxic effect and inducing oxidative
stress in the lungs, especially when antioxidant defenses have been overwhelmed.
Airway inflammation plays an important role in the pathophysiology of various
respiratory diseases. Respiratory tract health is traditionally assessed using airway
biopsies, bronchoalveolar larvage (BAL) fluid, and bronchoscopy. Although these
techniques provide direct information about the degree of airway inflammation,
they are invasive, difficult, and not suitable for repeated use in children (Montushi
and Barnes, 2003).
However, the precise mechanism of the relationship between ambient PM10
and respiratory health remains unclear, partly because of the lack of a non-invasive, biological sample, collection procedure for assessing lung inflammation.
Recently, research has focused on analyzing biomarkers in exhaled breath. Exhaled
breath condensate (EBC) is a new matrix for monitoring airway inflammation and
oxidative stress markers of various respiratory conditions (Montushi and Barnes,
2003). EBC contains a number of volatile and non-volatile compounds derived
from the respiratory surface, such as hydrogen peroxides, lipid peroxidation-derived
products, and protein carbonyl groups (Taylor, 2011). In addition, EBC collection
is non-invasive compared to bronchoscopy or induced sputum; it also facilitates
repeated measurements in the same individual, a significant advantage over some
of the other methodologies available for measuring inflammatory responses. Hydrogen peroxide (H2O2) is an oxidant produced by the alveolar membrane and
can be measured in exhaled air. H2O2, a marker of oxidative stress, is pathologically indicative of lung inflammation (van Beurden et al., 2002; Murata et al.,
2014). Recently, numerous researchers have explored the utility of studying airway
inflammation and oxidative stress in the context of pollution exposure (Jansen et
al., 2005), as well as clinical monitoring (Antus and Kardos, 2015; Corradi et al.,
2015; Garcia-de-la-Asuncion et al., 2015). EBC has been proposed as a simple,
non-invasive tool for measuring airway inflammation. The biomarkers in EBC
include H2O2, which has been proposed as a method for assessing the health effects
of air pollution in exposed populations (Doniec et al., 2005; Epton et al., 2008).
Although a variety of devices to collect EBC samples are commercially
available, they are expensive and not suitable for field study. The developed EBC
collecting device was based on the similar principle as the system of Dean et al.
(2007), but with some modifications. The condensation chamber was constructed
of polypropylene, following the recommendation of Horvath (2005) for EBC
collection devices.
This study aimed to develop a portable device to collect EBC samples and,
using these samples, assess airway inflammation in urban and highland schoolchildren exposed to ambient PM10 in northern Thailand.
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
41
MATERIAL AND METHODS
Development and validation of a portable EBC collecting device
The portable EBC collecting device (Figure 1) consists of a mouthpiece
with a one-way valve in which inspiratory and expiratory air are separated. The
mouthpiece is connected to a flexible plastic tube (30 cm in length and 2 cm in
internal diameter) that allows subjects to find a comfortable position. The tube
connects to a 50 mL polypropylene collecting tube that acts as a sampling container. Polypropylene is a thermoplastic polymer used in variety of healthcare
and laboratory applications, including syringes, tubing, hospital disposables, test
tubes, beakers, and pipettes (Sastri, 2014). The collecting tube is placed inside
a stainless steel chamber and designed to connect with a second one-way valve
that allows the excess air in an expired breath to flow toward the top. A rubber
ring between the flexible plastic tube and the hole in the stainless steel chamber
creates an airtight connection. The stainless steel chamber contains liquid nitrogen
in order to cool down the collecting tube.
Figure 1.Schematic diagram of the developed portable EBC collecting device.
The developed device was validated for: (1) technical problems, such as
any discomfort while wearing the device, (2) length of time required to collect
adequate EBC volume, and (3) overall acceptance of the device by the volunteer
subjects. Five healthy volunteers were recruited (12, 17, 25, 40, and 44 years old).
Each volunteer was asked to breathe normally into the developed device for 10
minutes to collect EBC samples. After that, they were given a 30-minute break
before collecting EBC samples a second time, for 20 minutes. Validation criteria
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(1) and (3) were assessed by verbal communication between volunteer users and
a trained research technician. For criteria (2), two EBC samples were collected
and the condensate EBC volume of each sample was measured using a calibrated
1000-µL pipette.
Application of the developed portable EBC device to collect EBC samples
Study site. Two primary schools, one urban and one rural/highland, were
selected as study sites. Chiang Mai Rajabhat University Demonstration School (or
urban school) in Chiang Mai city represented the urban location. A recent study
reported on the EBC malondialdehyde (MDA) as a biomarker of effect during
elevated ambient PM10 levels at this study site (Phornwisetsirikun et al., 2014).
Srinaeroo School (or highland school) represented the rural highland location.
The urban school is located in the northern part of Chiang Mai city, in the
valley at about 300 m above mean sea level. This study site is adjacent to a street
on one side and surrounded by workplaces and commercial areas on the other
sides. The school is located within 2.5 km of the air quality monitoring station
at the Yupparaj Wittayalai School in downtown Chiang Mai city. The PM10 data
from this station was assumed representative of the urban participants’ exposure
to PM10.
The highland school is located on Doi Suthep Mountain, about 35 km
northwest of Chiang Mai city and about 1300 m above mean sea level. With no
air quality monitoring station nearby, a portable airborne dust monitor (E-sampler,
Met One Instruments Inc., USA) was used to collect PM10 data at the location.
This portable monitor was calibrated with the air quality monitoring station at
the Yupparaj Wittayalai School, before using at the highland school. The PM10
data from the portable airborne dust monitor was assumed representative of the
highland participants’ exposure to PM10.
Criteria of study subjects. To be included in the study, the schoolchildren
had to have attended the selected primary school for at least one year and live
within 2.5 km of the air quality monitoring station for the urban participants or
the portable airborne dust monitor location for the highland participants. The
children had to be 10-12 years old, not diagnosed with asthma or other chronic
respiratory diseases, not on long-term medication, and willing to participate in
the study.
The Human Experimentation Committee of the Research Institute for Health
Sciences, Chiang Mai University, Thailand, approved the study protocol (Certificate HEC approval No. 1/2010). Children and parents signed written informed
consent before participation.
Study period. The study was conducted during July 2011 (rainy season)
and March 2012 (dry season).
Exhaled H2O2 analysis
EBC samples were collected from the schoolchildren using the developed
device, as shown in Figure 2, during both the rainy season (low PM10 level) and
dry season (high PM10 level). After rinsing their mouths, subjects were instructed
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
43
to form a complete seal around the mouthpiece and maintain a dry mouth during
collection by periodically swallowing excess saliva. The subjects sat comfortably
and wore nose clips. They were instructed to breathe normally and expire very
slowly through the mouthpiece; this continued for 10 minutes to collect condensate of about 1.2 mL per subject. The collected EBC samples were immediately
stored at -70°C until analysis.
Figure 2.Collection of EBC samples using the developed device.
The concentration of H2O2 in EBC was measured using a spectrophotometric
assay by means of horseradish peroxidase-catalyzed oxidation of tetramethylbenzidine, according to the method previously described by Gallatin and Pratch
(1985). The detection limit was approximately 0.1 µM.
Data analysis
The statistical analysis was performed using the Statistical Package for
the Social Sciences for Windows (SPSS, Thailand) Version 17. The differences
between exhaled H2O2 levels and pulmonary function indices in the rainy and
dry seasons were determined using paired t-test. A p-value of less than 0.05 was
considered statistically significant.
RESULTS
Development and validation of a portable EBC collecting device
The developed EBC collecting device was validated with five healthy
volunteers; they reported no technical problems or complaints while wearing the
device. EBC samples from five healthy volunteers were collected and the mean
volumes by normally breathing over a period of 10 and 20 minutes were 1.24±0.07
(n=5) and 1.30±0.05 (n=5) mL, respectively (p=0.017). As 10 minutes yielded
adequate volume for the biomarker test in the present study, a collection time of
10 minutes was used throughout the study. The procedure of EBC collection using
the developed device was found safe, rapid, and simple to use and operate.
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Application of the EBC device to collect EBC samples for investigating the
airway inflammation of schoolchildren
General characteristics of the study participants. Table 1 shows the general
characteristics of the participants. The participants, all between the ages of 10-12
years, had a median height of 129-144 cm and weight of 27-34 kg. However, the
urban children were significantly taller and heavier than their highland counterparts, partly skewed by one overweight subject of 59 kg.
Table 1. General characteristics of the participants.
Participant
characteristics
Male : Female
Urban school
(n = 54)
Highland school
(n = 50)
p-value
27 : 27
33 : 17
0.101
Age, year **
11 (10-12)
11 (10-12)
0.213
Height, cm **
144 (30-164)
129 (113-155)
0.000*
Weight, kg **
34 (25-59)
27 (20-50)
0.000*
Note: *significant difference between schools (p < 0.05), ** median (range).
PM10 and exhaled H2O2 concentrations. In both schools, the 24-hour
mean PM10 in the dry season was significantly higher than in the rainy season.
However, it was five times higher at the urban school, while only twice as high
at the highland school; this difference between these two locations was also statistically significant. Both levels of PM10 during the study period did not exceed
Thailand’s 24-hour mean PM10 limit of 120 µg/m3 (Thailand Air Quality and Noise
Standards, 2004). This phenomenon has been explained by the westerly wind that
blows into Chiang Mai City (Wiriya et al., 2013). Also, Doi Suthep Mountain, to
the west, is a National Park and open biomass burning is strictly prohibited.
At the urban school, the mean concentration of exhaled H2O2 was 0.17 µM
in the rainy season and 0.21 µM in the dry season (p = 0.003). At the highland
school, the mean concentration of exhaled H2O2 was 0.16 µM in the rainy season
and 0.18 µM in the dry season (p=0.001). The concentrations of exhaled H2O2 in
both schools increased significantly in the dry season.
Table 2.Comparison of PM10 and exhaled H2O2 concentrations of the schoolchildren from urban and highland schools between the rainy and dry
seasons.
Variables
Mean ± SD
p-value
Rainy season
Dry season
Mean 24 h PM10 (n=5), µg/m3
16.7 ± 1.3
90.7 ± 27.0
0.003*
Exhaled H2O2 conc. (n=54), µM
0.17 ± 0.08
0.21 ± 0.08
0.003*
Mean 24 h PM10 (n=5), µg/m3
22.6 ± 1.0
50.6 ± 8.8
0.002*
Exhaled H2O2 conc. (n=50), µM
0.16 ± 0.04
0.18 ± 0.05
0.001*
Urban School
Highland School
Note: *significant difference between schools (p < 0.05).
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45
DISCUSSION
We developed a cost-effective EBC collecting device for in-house and field
use; each device costs about THB 600 (about USD 20) (Figure 2) and consumes
liquid nitrogen for cooling of about THB 5 per sample. The device was employed
in a field study of 104 primary schoolchildren in 2011-2012 in Chiang Mai. The
collection procedure, which took only 20 minutes per person, was simple and did
not require skilled medical staff. A trained operator was sufficient to collect the
EBC samples from the subjects. Our collection procedure compared favorably with
Mutlu et al. (2001), who reported that collection usually takes 5-10 minutes in
adults and up to 15-20 minutes in children to obtain 1-3 mL of EBC. Furthermore,
our developed device, which can be used in the field, offered similar performance
to commercially available devices used in research and clinical studies, including children with respiratory diseases, which collect 1.5-2 mL of EBC in 10-15
minutes (Romieu et al., 2008; De Prins et al., 2014; Rosa et al., 2014).
Exhaled H2O2, the inflammatory biomarker used in our study, has been
shown to be a significant biomarker from ambient elevated PM10 exposure. Our
results here also confirm our previous findings, which reported that the EBC
malondialdehyde (MDA) biomarker of oxidative stress was raised in children
exposed to PM10 air pollution (Phornwisetsirikun et al., 2014). These results are
also consistent with other studies of EBC biomarkers of pulmonary inflammation
(Ralph et al., 2006; Gergelova et al., 2008; Chow et al., 2009).
Although possible confounding factors may exist in measuring exhaled
H2O2 concentration, the present study design was a follow-up study of the same
person, in order to minimize individual variability. Therefore, the present study
results demonstrated that the exhaled H2O2 concentrations, as well as the MDA
concentrations from our previous report (Phornwisetsirikun et al., 2014), are suitable biomarkers of elevated PM10 exposure. In conclusion, our study developed
an economical device for collecting EBC that was non-invasive, safe, rapid, and
simple to use. In addition, the results indicated that exhaled H2O2 concentration
provided good information about inflammation in the respiratory system of children (i.e., healthy schoolchildren in the present study) before clinical symptoms
appear.
ACKNOWLEDGEMENTS
The authors thank the schoolchildren for their cooperation throughout the
study period, and the Research Institute for Health Sciences (RIHES), Chiang
Mai University for laboratory support. This study was funded by a grant from
the Center of Excellence on Environmental Health and Toxicology, Faculty of
Science, Chiang Mai University and the Commission for Higher Education through
the National Research University Program, Chiang Mai University, Chiang Mai,
Thailand.
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49
Effects of a Theory-based Breastfeeding Promotion
Intervention on Exclusive Breastfeeding in China
Hongwei Wan1, Sujitra Tiansawad2*, Susanha Yimyam2
and Punpilai Sriarporn2
1
Shanghai Proton and Heavy Ion Hospital, Fudan University, Pudong, Shanghai
201321, the People’s Republic of China
2
Faculty of Nursing, Chiang Mai University, Chiang Mai 50200, Thailand
ABSTRACT
The benefits of exclusive breastfeeding (EBF) are well known; EBF is
recommended for the first six months of an infant’s life. However, EBF rates
remain low and unsatisfactory in China. This study aimed to investigate the
effectiveness of a breastfeeding promotion intervention based on the theory of
planned behavior (TPB) to promote long-term exclusive breastfeeding rates
among first-time Chinese mothers. A longitudinal, randomized, controlled trial
was conducted at Shanghai First Maternity and Infant Hospital in Shanghai,
China. The participants were 285 first-time Chinese mothers. The intervention
group (n=157) was offered a specially designed nursing intervention program
based on TPB, whereas the control group (n=128) received routine nursing
care. Data of exclusive breastfeeding practices were collected at 3 days, 6
weeks, and 4 and 6 months postpartum. Data pertaining to breastfeeding mediating factors were collected three times at the first day after birth, 3 days, and
6 weeks postpartum. A Chi-square test and repeated measures ANOVA were
used for the data analysis. The results showed that the intervention group had
significantly higher scores than the control group in breastfeeding knowledge,
attitude, subjective norm, and perceived control (at least p< 0.01). The rates of
exclusive breastfeeding at the different time points of the intervention group
(40.1%, 57.3%, 56.7%, and 42.0%) were consistently and significantly higher
than those of the control group (10.9%, 29.7%, 15.6%, and 10.2%) (at least
p< 0.01). It could be concluded that this theory-based breastfeeding promotion
intervention was effective for improving long-term exclusive breastfeeding rates
in China.
Keywords: Exclusive breastfeeding, Breastfeeding promotion intervention, Theory
of Planned Behavior, Randomized controlled trial
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INTRODUCTION
Breastfeeding is a relatively basic means of nourishing a baby that exerts
a significant effect on his or her optimal health with the establishment of a lifetime foundation (Oweis et al., 2009). The benefits of breastfeeding, particularly
exclusive breastfeeding (EBF), to mothers, children, and societies have been
well established (World Health Organization [WHO], 2002). EBF for the first
six months has been recommended for the health of infants, because breast milk
provides the best nutrients for infants’ optimal growth (WHO, 2002). The WHO
and the United Nations Children’s Fund (UNICEF) have put forth an objective to
increase breastfeeding rates to 75% in the early periods of an infant’s life, to 50%
at 6 months, and to 25% at 1 year of age (WHO, 2002). Despite evidence of the
benefits of EBF for at least 6 months, only a limited number of mothers comply
with such recommendations (Zhu et al., 2013; McIsaac et al., 2014). Many studies
have revealed a growing number of risk factors that may impede a mother’s ability
to sustain longer periods of EBF. For instance, demographic factors such as age,
education, marital status, and annual income were reported to be significantly
associated with breastfeeding duration (McIsaac et al., 2014). In general, married
older women with higher education and income were reported to breastfeed for
longer (McIsaac, et al., 2014). Other factors, including breastfeeding knowledge
and access to related information sources (Tang et al., 2013), attitude (Zhang, et
al., 2009; Jessri et al., 2013), subjective norms (Lynn and Rempel, 2004), and
self-efficacy or control (Meedya et al., 2014), were positively related to EBF,
whereas mothers’ working status or employment was negatively associated with
EBF (Yimyam, 2011).
In China, the National Program of Action for Children Development has
set a target rate of 80% for EBF at 4 to 6 months in different provinces since
the 1990s (Shen, 2008). However, a recent report by the Consumer Association
indicated that in 30 Chinese cities, only 28.6% of mothers exclusively breastfed
their newborns, 40% fed their babies a mixture of breast milk and formula within the first 6 months, and almost 31.4% did not breastfeed at all (Zhao, 2010).
Another survey conducted in three cities in China in 2012 revealed that the EBF
rate at 6 months was only 25.2% (Zhu et al., 2014). Recently, a study conducted
in Shanghai found that the EBF rate among first-time Chinese mothers at 4 months
was 34.1%, and the rate sharply dropped to 3.3% at 6 months (Wan et al., 2015).
The findings of these studies have suggested that few cities and provinces in China
could meet the WHO goal or the national target rate for EBF. Chinese women
cited working as the most common reason to stop breastfeeding. Perceived insufficient breast milk and worrying about the insufficient nutrition of breast milk
were also reported as reasons for ceasing breastfeeding (Zhu et al., 2014; Wan
et al., 2015). A recent predictive study among first time Chinese mothers supported
that breastfeeding knowledge, attitudes, subjective norms, and perceived control
could predict their EBF practice at 4 months postpartum in a positive direction,
whereas working status could negatively predict EBF practice (Wan et al., 2015).
This means that employed women were more likely to discontinue EBF at 4
months, when their maternity leave ended. Three of these predictors – attitudes,
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51
subjective norms, and perceived control – are major components of the theory of
planned behavior (TPB) (Ajzen, 1991) that is one of the most widely employed
theories in behavior change studies. According to TPB (Ajzen, 1991), a person’s
favorable or positive attitude towards a behavior and beliefs that significant others
will approve the behavior (subjective norms) will drive the person’s intention to
perform the behavior. The person’s perception of control over behavioral performance together with the behavioral intention will have a direct effect on the
behavior. Employment, a factor outside the person’s control, may function as a
barrier to behavior performance. Thus, the evidence supports that this theory can
be applied in breastfeeding research.
To promote breastfeeding, several interventions using various approaches
have been studied in both developed and developing countries. The existing
promotion interventions have included prenatal education, professional support,
and/or counseling; interventions during postnatal hospitalization; and postnatal
follow-up home support (Hannula et al., 2008; Imdad et al., 2011). With regard
to timing of interventions, a systematic review of 53 studies from developed and
developing countries revealed that all prenatal, postnatal, or combined interventions had significant effects on improving EBF rate at 4-6 weeks while combined
prenatal and postnatal interventions had the highest effects on the rate at 6 months
(Imdad et al., 2011). In terms of components of the interventions, it was found
that education alone, professional support alone, or combined approaches could
significantly promote EBF at 4-6 weeks, but only combined approaches could be
effective at 6 months (Imdad et al., 2011). Findings of this review suggest that to
achieve great impact on long-term EBF practice, components of the interventions
should include combined approaches of prenatal and postnatal interventions. Interestingly, greater increase in EBF rate was found in developing countries compared
to developed countries. However, relatively few studies have been reported from
developing countries. Therefore, further studies in developing countries are still
needed.
In China, studies examining effectiveness of the interventions for promoting long-term exclusive breastfeeding are scarce. One community-based home
visit intervention was conducted for 200 postpartum women in Shanghai, China,
with community nurses providing home visits twice during the two weeks after
delivery (Yang, 2009). The EBF rate at 6 months reached 24% in the home visit
group, which was significantly higher than the control group, of which the EBF
rate was 5%. Nonetheless, these low rates of EBF in both groups did not meet
the national target goal for breastfeeding. Therefore, the objective of this study
was to assess the effectiveness of a theory-based comprehensive breastfeeding
promotion intervention in improving long-term EBF practice in China. The TPB
was chosen for guiding the intervention development, because its usefulness has
been shown in breastfeeding research (Bai et al., 2011). The expected main outcome of the study was a high rate of EBF practice at 6 months, especially in the
intervention group. A specific question of “Are the EBF rates at 3 days, 6 weeks,
4 months, and 6 months postpartum of the Chinese mothers in the intervention
group higher than those of the control group?” was examined.
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Research design
This was a longitudinal, randomized, controlled-trial study. The intervention
group was offered the specially designed breastfeeding promotion intervention,
plus routine nursing care. The control group received only routine nursing care
that included one prenatal breastfeeding education class, rooming-in, breastfeeding
initiation within half an hour after delivery, postnatal lactation consulting support
by primary nurses, and pamphlets on breastfeeding presented in the ward during
their hospitalization. The prenatal education class generally aims to promote
pregnant women’s intention to breastfeed by providing information regarding its
benefits. A positive attitude towards breastfeeding may be enhanced, but it is not
emphasized. The above-mentioned postnatal nursing care is the routine approach
for the Baby-Friendly Hospital designed to promote and support breastfeeding
initiation. They are beneficial for most postpartum mothers; however, information
provided and teaching/learning strategies are not specifically planned to improve
the individual mothers’ attitude, subjective norm, and perceived control.
Participants
The study was conducted from October 2013 to June 2014 in the Shanghai
First Maternity and Infant Hospital, Tongji University, which is a teaching hospital
accredited as an “AAA”, tertiary care, specialty hospital with an average of 15,00017,000 new births per annum. The participants were first-time Chinese mothers
who met the following inclusion criteria: (1) physically and mentally capable of
communicating, reading, and writing in Mandarin; (2) able to be interviewed by
telephone at home until 6 months postpartum; (3) not having illnesses or problems that prohibit breastfeeding for both mother and baby; (4) having attended
at least one prenatal education class; and (5) having either a husband, mother,
or mother-in-law who met the following four inclusion criteria as a significant
other: (a) able to communicate and read in Mandarin; (b) having regular contact
with the participant; and (c) able to attend the intervention activities twice.
A permuted block random sampling method was used to assign the participants into the intervention or control group. Through randomizing participants
within blocks, an equal number was assigned to the treatment group (T) and
control group (C). Given a block size of four, there were six different possible
arrangements (1=TCTC, 2=CTCT, 3=TTCC, 4=CCTT, 5=TCCT, 6=CTTC) in a
block to assign participants equally to two groups. A random number sequence
was used to choose a particular block, which set the allocation order for the first
four subjects. The process was then repeated. The allocation proceeded by randomly selecting one of the orderings and assigning the participants to two groups
according to the specified sequence. As each participant was admitted to a private
room in the hospital, there was little cross contamination between the two groups.
The sample size was estimated in two ways. Firstly, the proportion of the
participants with EBF for 6 months was estimated as 50%, the WHO target rate,
in the intervention group (p1 = 0.5) and 30%, an average rate from previous
studies, in the control group (p2 = 0.3). Then, the sample size was estimated as
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53
n= [(1.96+0.842)2 × (0.5 × 0.5+ 0.3 × 0.7)] / 0.202 = 90 subjects per group. When
considering an attrition rate of nearly 20%, the sample size was estimated as
216. Secondly, because the study examined five breastfeeding factors, including
breastfeeding knowledge, attitude, subjective norms, breastfeeding control, and
working status or employment between two groups, repeated measures ANOVA
were used to estimate the sample size. According to the table of sample size
estimation by ANOVA, with a power of 0.8 and a medium effect size, the sample
size was estimated as 280*(1+20%) = 336 participants (considering the attrition
rate of 20%). The researchers enrolled participants and assigned them to their
groups. At the beginning of the study, 352 participants were recruited, including
180 in the intervention group and 172 in the control group. However, during the
intervention period, 67 (19%) participants dropped out of the study at different
stages of the intervention, as shown in Figure 1. Incomplete participation was
only one criterion for termination. No other participant was terminated from the
study. Finally, 285 participants, including 157 in the intervention group and 128
in the control group, completely participated in the study. The final number was
almost equal to the expectant sample size that was adequate for analysis.
Figure 1.Flow Diagram of Participants through Each Stage of Interventions.
Theory-based comprehensive nursing intervention program
The comprehensive nursing intervention program was developed, by three
members of the research team (the first three co-authors), mainly based on the
TPB (Ajzen, 1991) and the findings of the previous study (Wan et al., 2015).
The TPB assumes that behavioral intention is the most important determinant of
behavior. Behavioral intention is influenced by three factors, including a person’s
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attitude toward a given behavior, subjective norms, and perceived behavioral control (Ajzen, 1991). The attitude refers to a person’s overall positive or negative
feeling with respect to performing the behavior. It is determined by a person’s
beliefs or expectations about the outcome of the behavior and evaluation of that
outcome. The subjective norm is defined as a person’s subjective belief about
the approval or disapproval of the behavior from people important to them. It is
determined by two factors: beliefs about what significant others think the person
should do and the person’s motivation to comply with those beliefs. The perceived
behavioral control is a person’s belief in her own ability to perform the behavior.
This is influenced by control beliefs and control power. Control beliefs are beliefs
about resources available for performing the behavior, and control power is the
perceived effect of resources on the difficulty of performance (Ajzen, 1991).
Therefore, the breastfeeding promotion interventions were designed to
change women’s breastfeeding attitudes, subjective norms, and perceived breastfeeding control by focusing on their underlying beliefs and values. Interventions
to improve women’s breastfeeding knowledge and attitudes were planned by
providing education with adequate essential information on EBF. The significant
others of the women, including their husbands, mothers, or mother-in-laws, were
invited to be involved in the activities to increase the women’s subjective norm.
Finally, because the women’s working status was also a predictor of EBF at 6
months postpartum, the experiential learning activities attempting to enhance the
women’s perceived breastfeeding control were designed to prepare employed
women for EBF during their working hours. The protocol of this intervention
targeted five different stages of breastfeeding, starting at the first and second
days postpartum and completed at six months, as presented in Table 1. The first
intervention did not begin during pregnancy, because all women of both groups
had already attended prenatal education class.
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55
Table 1.Intervention protocol.
Time & Place
Intervention steps (activities and contents)
Objectives
First and second Step 1: Promoting BF initiation at hospital.
days postpartum - Individual advice and support: practicing
at hospital
correct BF techniques at day 1 postpartum
(even though not enough breast milk came
out) and solving any BF problems every day
during the 2 or 3 days of hospitalization.
- Group education: at day 2 postpartum,
a 60-min session was provided, including
30 minutes of lecture and video watching
for information about BF advantages and
attitudes, 15 minutes of practice for BF
position and latching-on, and 15 minutes of
discussion on BF planning and sharing with
other experienced mothers or nursing staff
for successful BF initiation. The participants’
significant others were invited to the session.
-To reinforce important information on BF knowledge and
practical skills;
-To change BF attitudes and
subjective norms to support the
participant;
-To improve BF control by
learning practice skills;
-To solve any problems, such
as breast pain and breast engorgement.
During postpar- Step 2: Promoting BF initiation at home.
tum before 6 weeks - Telephone counseling twice a week within
at home
the first 2 weeks and once a week during
weeks 3 to 6 postpartum.
- Telephone counseling to follow up BF
practice and solve any BF problem.
- If there are major problems, provide a
home visit or ask the mother to come to the
hospital for further help.
-To enforce important BF
knowledge and practical skills;
-To address problems;
-To provide information on
nutrition for sufficient breast
milk to breastfeed successfully.
At 6 weeks post- Step 3: Promoting continuation of BF.
partum at hospital - A 30-min group session of discussion on
BF during the regular check-up.
- Mothers and their significant others were
re-informed about the long-term benefits and
importance of EBF.
-To improve EBF knowledge,
positive attitudes, and subjective norms;
-To balance continued BF with
work.
During 6 weeks to Step 4: Promoting continuation of BF.
-To encourage longer BF con3 months at home -Individual BF advice by telephone support tinuation.
once every two weeks from 6 weeks until
3 months.
D u r i n g 3 t o 6 Step 5: Promoting continuation of BF.
months postpar- A series of individual advice via telephone,
tum at home
focusing on techniques for preparing for
work and management of breast milk, such
as breast milk expression, storing breast milk
in ice box, giving breast milk to babies by
cup, and dealing with various BF problems
at the workplace.
-Once a week within 2 weeks before working;
-Once during the first 2 days of work;
-Once a week within a month after working;
-Once every 2 weeks during the 4th and the
6th months.
-To comprehend BF techniques
when working;
-To learn how to manage breast
milk at work;
-To address various problems
associated with BF at the workplace;
-To encourage successful longer BF continuation.
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Measurement
The research team developed the measures of demographic characteristics
and breastfeeding practice. This included mother’s age, marital status, educational level, family yearly income, mother’s working status, and three questions
on breastfeeding practice. The first question, “Do you currently breastfeed?”
asked about the participant’s current infant feeding. If the response was “yes”,
she was asked two additional questions to obtain detailed information regarding
how she gave breast milk to her infant (e.g., with or without formula, water, and
other foods). Then, the type of breastfeeding practice was identified. Practice of
EBF was identified when an infant was fed only breast milk. No other liquids
or solids, with the exception of drops or syrups consisting of vitamins, mineral
supplements, or medicines, were given (WHO, 2002). If the answer was “no”,
she was categorized in a group of no current EBF.
The modified Breastfeeding Attrition Prediction Tool (BAPT) and the Breastfeeding Knowledge Scale (BKS) were used to monitor changes in breastfeeding
mediating factors. The original BAPT, consisting of three subscales (subjective
norm, attitude, and control), was developed and revised by Janke (1994). It was
modified and translated into Chinese by the first author and back translated into
English by three bilingual professors, two in medicine and one in nursing. The
modifications included deleting five items of the breastfeeding subjective norm
subscale and changing the response scale from six to five levels. The deleted
items identified family doctor, baby doctor, midwife, educator, and Lee League,
which do not exist in the Chinese health care system as women’s significant
others. A 5-point Likert scale was used because this scale is easy for participants
to understand and make judgments. The subscales of breastfeeding attitude, the
breastfeeding subjective norm, and the perceived control consisted of 29, 8, and 10
items, respectively. The Cronbach’s alpha coefficient of the total scale was 0.88,
and the coefficients of the subscales were acceptable, ranging between 0.81 and
0.92. The BKS was developed and modified by Zhu et al. (2013). The modified
BKS is a 5-point Likert scale with 25 items. Its content validity index (CVI) was
1.00 as reviewed by five experts in breastfeeding. The Cronbach’s alpha coefficient
was 0.80.
Data collection
Before data collection, the Research Ethics Review Committee of the Faculty
of Nursing, Chiang Mai University, Thailand and Shanghai First Maternity and
Infant Hospital, Shanghai, China granted ethical approval. Each participant was
approached on the first day after delivery and informed about the purpose of the
study, procedures, confidentiality and anonymity preserved, and potential risks and
benefits. Then, written informed consent was obtained. Demographic information
was collected at the first day after birth and before starting the intervention. Data
pertaining to breastfeeding knowledge, attitude, subjective norms, and perceived
breastfeeding control were collected three times for monitoring: on the first day
after birth as baseline data, 3 days postpartum before discharge from the hospital,
and 6 weeks postpartum during a follow-up visit. All the questionnaires were
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
57
self-completed by the participants. The data pertaining to breastfeeding practice
were collected four times – at 3 days and 6 weeks postpartum using face-to-face
interview at the hospital, and at 4 and 6 months postpartum using telephone interviews. The same three questions on breastfeeding practices were asked.
Data analysis
The licensed software SPSS 17.0 was employed to analyze all data. The level
of significance was set at an alpha of 0.05. Data were tested and found normal
distribution. The data were analyzed using descriptive statistics, t-test, Chi-square
test, and repeated measures ANOVA. T-test and Chi-square tests were used for
comparison of participants’ characteristics between the intervention and control
groups. Because the breastfeeding rates were compared by percentages between
two groups, a Chi-square test was used. Repeated measures ANOVA was used to
analyze changes of breastfeeding mediating variables of the two groups at three
different times (baseline, 3-days, and 6-week postpartum).
RESULTS
Characteristics of the participants
In this study, 285 first-time mothers participated, with 157 in the intervention
group and 128 in the control group. The mother’s age ranged from 21 to 40 years,
with average ages of 29.6 and 29.0 years in the intervention and control groups,
respectively. The majority of the mothers were married, lived in the city, and
held associate or bachelor’s degrees (75.8% of the intervention group and 66.4%
of the control group). Nearly half (47.8% and 44.5% for the two groups) of the
mothers had an annual family income of RMB 100-199 thousand (USD ~6,30013,000), and more than one-third of the mothers in each group had an annual
family income of RMB 200-300 thousand (USD ~13,000-20,000). The majority
of the mothers were employed (94.9% of the intervention group and 95.3% of
the control group). There were no significant differences in these demographic
characteristics between the two groups, as shown in Table 2.
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➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
Table 2. Demographic characteristics of the participants in two groups.
Characteristics
Age (years)
Marital status
Married
Unmarried
Experimental
group (n = 157)
Control group
(n = 128)
Frequency (%)
Frequency (%)
Range 21-40
Mean = 29.61
SD = 3.39
Range 23-41
Mean = 29.02
SD = 3.76
Statistics
P
t = 1.396
.164
155 (98.73)
2 (1.27)
126 (98.44)
2 (1.56)
χ
2
=0.09
>.05
7 (4.46)
119 (75.80)
12 (9.38)
85 (66.41)
χ
2
=4.07
>.05
31 (19.74)
31 (24.22)
11 (7.00)
75 (47.77)
58 (36.94)
17 (10.83)
8 (6.25)
57 (44.53)
50 (39.06)
13 (10.16)
χ
2
=0.59
>.05
Working status
Employed
Unemployed
149 (94.90)
8 (5.10)
122 (95.31)
6 (4.69)
χ
2
=0.03
>.05
Residence
City
Town/village
146 (93.00)
11 (7.00)
115 (89.84)
13 (10.16)
χ
2
=0.91
>.05
Education level
≤ Secondary school Associate & Bachelor
degree
≥ Master degree Family annual income (thousand RMB)
< 100
100-199
200-300
> 300
Comparison of breastfeeding mediating factors
Four breastfeeding mediating factors, including knowledge, attitude, subjective norm, and perceived control, were examined for the interaction effects of
time and group. Baseline mean scores of the four factors of two groups were not
different at the significance level of 0.05. The findings revealed that there were
statistically significant differences in mean scores of breastfeeding knowledge,
attitude, subjective norm, and perceived control between two groups at different
time points (F=6.95, p <0.001; F=13.58, p <0.001; F=9.90, p <0.001; and F=7.17,
p <0.01, respectively). Further analysis of the differences between mean scores
of knowledge and attitude of two groups showed that the intervention group had
significantly higher scores than the control group at 6 weeks (p <0.001). The
subjective norm scores of the intervention group were significantly higher than
those of the control group at both 3 days and 6 weeks (p <0.001) whereas the
perceived control scores of the two groups were significantly different at 3 days
only (p <0.01), as shown in Table 3.
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
59
Table 3.Comparison of breastfeeding mediating factors between two groups at
different time points.
Breastfeeding monitoring
factors
Experimental
group (n = 157)
Mean (SD)
Control group
(n = 128)
Mean (SD)
t
p
Breastfeeding knowledge
Baseline
At 3 days
At 6 weeks
99.24 (11.36)
106.54 (19.62)
110.79 (15.71)
98.15 (10.19)
101.73 (18.19)
102.04 (18.91)
0.710
4.528
18.073
.400
.034
<0.001
Breastfeeding attitude
Baseline
At 3 days
At 6 weeks
103.80 (19.51)
111.42 (29.24)
119.02 (24.48)
101.95 (24.19)
103.46 (25.90)
103.66 (27.55)
0.50
5.69
24.31
.481
.018
<0.001
Breastfeeding subjective norm
Baseline
At 3 days
At 6 weeks
31.43 (4.31)
33.27 (3.85)
32.95 (4.48)
31.16 (5.48)
31.14 (5.66)
30.97 (5.64)
0.23
13.97
10.79
.635
<0.001
<0.001
Breastfeeding perceived control
Baseline
At 3 days
At 6 weeks
36.87 (8.51)
41.67 (9.64)
42.25 (10.48)
37.28 (9.16)
37.99 (10.65)
40.78 (10.18)
0.15
9.36
1.43
.696
<0.01
.233
Exclusive breastfeeding rates at different time points
As illustrated in Figure 2, the EBF rates at 3 days, 6 weeks, 4 months, and
6 months postpartum in the intervention group (40.1%, 57.3%, 56.7%, and 42.0%,
respectively) were consistently higher than those in the control group (10.9%,
29.7%, 15.6%, and 10.2%, respectively). The chi-square test showed that the EBF
rates at all four time points in the intervention group were significantly higher
than those in the control group (χ2= 30.47, p <0.001; 21.77, p <0.001; 50.34,
p <0.001; and 35.77, p <0.01; respectively), as shown in Table 4.
Figure 2. Exclusive breastfeeding rates of two groups at different time points.
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➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
Table 4. Comparison of exclusive breastfeeding rates at different time points.
Time
Group
N
v
Frequency
Percentage
157
3
63
40.13
3-day
Experiment
control
128
3
14
10.93
6-week
Experiment
157
3
90
57.32
control
128
3
38
29.69
Experiment
157
3
89
56.69
control
128
3
20
15.63
Experiment
157
3
66
42.04
control
128
3
13
10.16
4-month
6-month
χ
2
P
30.47
<0.001
21.77
<0.001
50.34
<0.001
35.77
<0.01
DISCUSSION
The purpose of this longitudinal study was to evaluate the effectiveness
of a specially designed, theory-based, breastfeeding promotion intervention on
improving long-term EBF for first-time Chinese mothers. Overall, the findings
suggest that the intervention was effective in increasing the EBF rates of the intervention group within 6 months postpartum. The EBF rates at 4 and 6 months
among the intervention group were significantly higher than the control group in
this study and were higher than the Chinese mothers in a recent study conducted
by these researchers (Wan et al., 2015).
With regard to the breastfeeding initiation rate, it is interesting that although
breastfeeding initiation was usually motivated in the study hospital, the EBF
rates at 3 days postpartum of both the intervention (40.1%) and, in particular,
the control groups (10.9%) were not high. The plausible explanations of these
low EBF rates may be related to the mothers’ perception of insufficient breast
milk, poor breastfeeding technique by either the new mothers or the infants, and
no hospital policy to restrict formula or other foods. A study conducted in three
cities of China revealed that the most difficult time for mothers to maintain EBF
was 0-3 days (Zhu et al., 2014). Mothers’ worrying about insufficient nutrition
for their babies and improper breastfeeding technique were the main problems.
However, after that difficult time, the EBF rates in the current study increased in
both groups. The increased rates of EBF at 6 weeks may be a result of the full
adjustment to breast milk production and the improved breastfeeding skills of the
mothers and the infants.
When EBF rates were compared between the two groups, the intervention
group revealed significantly higher rates than the control group at all four time
points. The findings demonstrated the effectiveness of the intervention program
in promoting breastfeeding initiation and continuation. To promote breastfeeding
initiation, in addition to the routine prenatal education class, the program included a
series of interventions after delivery at the hospital and at home. Individual advice
and support and group education were provided at days 1 and 2 after delivery,
and the participants were provided counseling by telephone at least seven times
during the first six weeks at home. These combined strategies led to an almost
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
61
93% increase in the EBF rate at 6 weeks (the rates were 57.3% in the intervention
group compared to 29.7% in the control group). A review conducted by Imdad et
al. (2011) revealed that the educational interventions significantly increased the
EBF rate at 4-6 weeks by 43% (or 1.43 times). A previous study in Italy reported
that postnatal telephone support during the first 6 weeks also led to an increased
EBF rate in first time mothers (Simonetti et al., 2012).
To promote breastfeeding continuation, this study also employed various
methods for the intervention. The invitation of the mothers’ significant others –
including the husband or partner and own mother or mother-in-law – to participate
in a postnatal group discussion that mainly focused on practical aspects to deal
with breastfeeding problems was one among several approaches. Their involvement in the group discussion at 6 weeks postpartum was expected to improve the
mother’s perception of social pressures for breastfeeding from referent persons
or subjective norms (Ajzen, 1991). A previous non-experimental study based on
TPB found that the mothers who continued breastfeeding at 6 weeks received
more positive support from their partners and own mothers than the mothers
who did not continue breastfeeding; likewise, subjective norms were important
determinants of breastfeeding initiation and continuation (Swanson and Power,
2005). Another study evaluating the comprehensive peer support program in the
United Kingdom found that significant others who were breastfeeding supporters
could provide the mothers praise and reassurance and could help them overcome
their obstacles to lead to an improved EBF rate (Thomson et al., 2012).
After 6 weeks until 6 months postpartum, telephone counseling and support
were regularly provided. This intensified telephone support focused on preparing
the mothers for breastfeeding when they returned to work. This intervention aimed
to enhance the mothers’ perceived breastfeeding control or confidence in managing
breast milk or their infant feeding and appeared to be effective because the EBF rate
at 4 months remained high (56.7%). However, the EBF rate at 6 months slightly
decreased to 42.0%. This decreased rate may be attributed to the declined effect
of perceived breastfeeding control because of no or less workplace support. Since
the perceived control is determined by control beliefs concerning the presence
or absence of facilitators and barriers to behavioral performance and perceived
power or effect of each factors to facilitate or inhibit the behavior (Ajzen, 1991),
its effect can decline in situations in which prior facilitators are absent or new
barriers to behavioral performance are present. Most of the mothers in this study
were employed, and those who discontinued breastfeeding after 4 months cited
returning to work as their main reason. This reason is similar to one of the major
reasons given by most employed mothers who had completely stopped breastfeeding in previous studies (Tahir and Al-Sadat, 2013; Yimyam, 2013a; 2013b). It was
also reported that mothers who were unable to nurse their infants during work
shifts had 4.98 times higher odds of weaning before 4 months of age (Brasileiro
et al., 2010; Yimyam, 2013a; 2013b). It was evident that employed women face
various barriers in the workplace, such as not having a private room for breast milk
expression and still requiring lactation information and support (Zinn, 2000). In
Cohen and Mrtek’s (1994) study, 75% of the women who had access to a worksite
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➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
breast pump room and lactation professional counseling continued to breastfeed
their children to 6 months. In another study, 58% of the women with access to
a similar physical facility and lactation professional services with an additional
education class continued to breastfeed their children to 6 months (Ortiz et al.,
2004). Therefore, the workplace policy and implementation of the breastfeeding
promotion program for employed women must be considered.
Although the EBF rate at 6 months postpartum of the intervention group in
this study did not achieve the Chinese national target rate, this rate was four times
higher than the control group. In addition, the monitored scores of breastfeeding
knowledge, attitude, subjective norm, and perceived control of the intervention
group were higher than the control group. These findings indicate that the program
was relatively effective for enhancing the breastfeeding contributing factors and
consequently improving long-term EBF rate in China. They support the findings
of recent systemic reviews (Haroon et al., 2013; Skouteris et al., 2014). Haroon
and colleagues concluded that combined individual and group counseling appeared
to be more effective than individual or group interventions alone. Another review
by Skouteris et al. (2014) summarized other aspects of the effective interventions,
and stated that the most successful interventions tended to include various forms
of education and support and commenced in the postnatal period and continued
for a relatively long period (from 3 weeks to 6 months) to provide ongoing assistance to mothers.
In conclusion, this intervention program was effective in improving EBF
rates at 4 and 6 months postpartum among first-time Chinese mothers. The program involves a series of strategies during both hospitalization and the postnatal
period at home. These include intensifying breastfeeding knowledge, improving
positive breastfeeding attitudes, enforcing breastfeeding subjective norms, and
strengthening perceived breastfeeding control. Therefore, the strategies included
in this theory-based intervention program should be incorporated into the regular
services of nurses in hospitals or in community healthcare for first time Chinese
mothers to improve EBF. For example, breastfeeding education should be conducted by primary nurses as a part of routine care in the hospital, and follow-up
telephone counseling should be performed to encourage new mothers to breastfeed
exclusively and to help them address various problems associated with breastfeeding during their stay at home. Finally, a session of counseling and discussion
is recommended as an intervention for preparing new mothers to return to work.
Implementation of the breastfeeding promotion intervention in a healthcare setting
and community, however, may not be sufficient, because problems remain in the
workplace. Thus, policymakers are recommended to seriously consider issuing
workplace policy for providing a private breastfeeding room and breastfeeding
time at the workplace. This policy will significantly facilitate the breastfeeding
practice among working mothers.
Since participants of this study were recruited from one setting in Shanghai,
a large city in China, generalization of our findings to mothers in other areas should
be considered with caution. Therefore, it is recommended that further studies be
conducted in other settings or other areas in China.
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
63
ACKNOWLEDGEMENTS
This study was supported by funding from the Shanghai Science and Technology Committee under Grants 134119a1000, 14495810900, and 13ZR1432800.
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67
Importance of Tien Yen Estuary (Northern Vietnam) for EarlyStage Nuchequula nuchalis (Temminck & Schlegel, 1845)
Hau Duc Tran1*, Thuy Thi Ta2 and Thanh Trung Tran1
1
Hanoi National University of Education, 136 Xuan Thuy, Cau Giay, Hanoi,
Vietnam
2
Hanoi Metropolitan University, 98 Duong Quang Ham, Cau Giay, Hanoi, Vietnam
ABSTRACT
To understand the importance of an estuary for the early stage of Nuchequula nuchalis (Spotnape Ponyfish), we collected samples (5,430 individuals)
from the shallow areas of the Tien Yen estuary, northern Vietnam, from March
2013 to February 2014. The water temperature, salinity and turbidity at the collection sites varied from 18 to 29°C, 0 to 19 psu, and 4 to 96 NTU, respectively.
The fish caught were mainly postflexion larvae, with a few juveniles. While the
fish occurred from April 2013 to January 2014, they were heavily concentrated
during two months only – April and May 2013, when more than 99% of the
postflexion larvae were collected. The fish were distributed mostly in the middle
part of the estuary, implying that this area is its main habitat during the early
stages of its lifecycle.
Keywords: Nuchequula nuchalis, Larvae and juveniles, Occurrence, Estuarine
habitat, Northern Vietnam
INTRODUCTION
Nuchequula nuchalis (Spotnape Ponyfish) is distributed in East Asia (Kimura
et al., 2008b), including Taiwan and from south of Fujian to the Gulf of Tonkin
(Woodland et al., 2001); this probably includes northern Vietnamese coastal waters.
This species, with others in the Leiognathidae family, are commercially important
in Asian wild fisheries and aquaculture, and inhabit coastal and estuarine waters
(Woodland et al., 2001). Occurrence of the early stage of N. nuchalis has only been
recorded in northern Kyushu, Japan, and its spawning season is from the middle
of May to the end of July (Fujita, 1960); little is known about the distributional
pattern of the early stages of this fish in an estuary.
Tran and Ta (2014a) recorded 193 fish species, belonging to 142 genera
and 83 families, in the Tien Yen estuary. Some research has been conducted on
the early life stages of fish in this estuary, such as descriptions of Sillago sihama
(Tran et al., 2014) and Nuchequula nuchalis (Tran et al., 2014). The distribution
patterns of some species have also been elucidated, namely Oryzias curvinotus
(Ta et al., 2014) and species of Lateolabrax (Tran and Ta, 2014b); it is presumed
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➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
that the Tien Yen estuary plays a crucial role as their nursery ground. It has also
been shown that fish populations in the Tien Yen River have been dramatically
reduced recently, partly due to anthropogenic pressures (Tran and Ta, 2014a).
Thus, understanding early-stage fish recruitment in this estuary is a priority to
prevent further reduction and/or promote replenishment.
Recently, a number of larvae and juveniles belonging to the Nuchequula
genus were collected along the shore of the Tien Yen estuary, northern Vietnam;
based on morphology, they were identified as (Tran et al., 2014). This study reports
occurrence data of the larvae and juveniles of N. nuchalis in this northern Vietnam
estuary, probably the southernmost distribution area that has been recorded.
Spotnape ponyfish larvae and juveniles were sampled monthly from the Tien
Yen estuary, northern Vietnam, from March 2013 to February 2014 (Figure 1).
The river, 82 km long, flows into the northern Vietnam coast on the Gulf of
Tonkin; the headwaters are partly located in China. The maximum width of
the estuary is 2 km and its maximum depth is approximately 8 m. The estuary
is characterized by a large tidal flat, with average tidal fluxions of 3-4 m (Vu,
2009). The salt wedge can reach approximately 15 km upriver from the river
mouth. Sampling stations (TS2-TS9) were chosen to represent the entire estuary,
and the uppermost station was located at the end of the tidal basin (Figure 1).
A simultaneous collection (TS1) was made at the outer estuary as a control site.
Figure 1.Location and sampling stations in the Tien Yen estuary. Stations where
Spotnape Ponyfish were sampled are indicated by solid circles (stations
TS1–TS9).
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
69
Collections were made in shallow areas of the Tien Yen estuary by a small
seine net (1 × 4 m, 1 mm mesh-aperture) (Kinoshita et al., 1988). Two persons
kept the net stretched, and waded backward in the waters, from ankle- to neckdepth along the shoreline for a distance of ca. 50 m (2 min.). A day’s collection
usually consisted of one to four hauls at each bank water station. Catch Per Unit
of Effort (CPUE) is the number of individuals in each haul (ca. 2 minutes or
50 m). All specimens were fixed in 10% formalin, and sorted specimens were
transferred to 80% ethanol. Water temperatures (°C), salinities (psu) and turbidity
(NTU) were measured at each station during the sampling periods using a Water
Quality Checker (WQC-22A, TOA DKK – temperature: 0-50°C ± 0.1°C; salinity:
0-4psu ± 2.5%; turbidity: 0-800NTU ± 0.1%).
The N. nuchalis specimens were divided into developmental stages, in accordance with Kendall et al. (1984). Standard length was measured to the nearest
0.1 mm using an ocular micrometer attached to a stereo microscope. In this study,
standard length indicates postflexion larvae and juveniles.
The linear relationships between paired quantitative variables, namely CPUE
and temperature, CPUE and salinity, or CPUE and turbidity, were ascertained using
the Spearman’s Rho rank correlation, because these data, tested by Shapiro-Wilk
(n < 50), were non-normal curve distributions. The value of Spearman rank (r)
is from -1 to + 1. A value closer to 1 indicates strong agreement, while r closer
to -1 indicates strong agreement in the reverse direction. In Figures 2 and 3, the
CPUE was lg (x+1)-transformed.
RESULTS
Seasonal changes of N. nuchalis along the shallow waters of the Tien Yen
estuary from March 2013 to February 2014 are described in Table 1. Nearly all
(99.5%) of the specimens were collected in April and May. No fish were collected
in March, then the average CPUE increased remarkably to 115.8 individuals per
haul in April, reaching a peak in May of 459.3 individuals per haul. Thereafter,
the value fluctuation from July to January varied from 0.1 to 2.1 individuals per
haul (Table 1); with no specimens collected in June, November, and February. The
average fish size increased from April to July, then decreased in August, before
remaining roughly constant until January (Table 1). The size range varied the
most in May. The fish collected in April were the smallest, with a body length of
around 7.0 mm SL (Table 1).
Average water temperature was 23.8°C in March 2013, then rose to 29.4°C
in September, before declining to 17.3°C in January 2014; eventually, increasing to
20.6°C in February (Table 1). The water temperature was higher than 24°C from
March to November 2013, and lower than 21°C from December 2013 to February
2014 (Table 1). On the other hand, the average salinity fell from around 16.8
psu in March, to approximately 3 psu in May to August, after which, the salinity
increased to 11.5 psu by November, and subsequently stabilized around 12.7 psu
until February (Table 1). The monthly relationship between the temperature and
salinity showed a reciprocal pattern (r = -0.75, p < 0.05) (Table 1). The turbidity
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➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
showed a different pattern, with extremely turbid water (150 NTU) in May (Table
1). In some other months, turbidity was less than 20 NTU (Table 1). The monthly
relationship between salinity and turbidity also showed a negative pattern, but
the difference was not significant (r = -0.68, p > 0.05) (Table 1). Additionally,
tidal changes did not show a connection with the fish collection during the study
period (Table 1).
Table 1.Environmental parameters, CPUE and body length of N. nuchalis larvae
and juveniles collected in Tien Yen estuary during March 2013 to
February 2014.
Mar
Tide
CPUE
(individuals
per haul)
Max
Min
SD
Average
Body length
(mm)
Max
Min
SD
Average
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Jan
Feb
E
E, L
S
L
S
S, H
E, H
E, L
E
S, L
E, L
E, L
0
0
0
0
809.3 3168.0
0
0
305.8 1194.5
115.8 459.3
0
0
0
0
4
0
1.5
0.6
1.3
0
0.5
0.2
12.0
0
4.9
2.1
2.2
0
0.8
0.3
0
0
0
0
1.0
0
0.4
0.1
4.0
0
1.5
0.6
0
0
0
0
37.1
36.6
0.4
36.9
23.7
23.7
0
23.7
29.1
23.1
2.0
26.1
24.9
22.3
2.0
26.1
25.4
25.4
0
25.4
27.8
21.2
2.6
24.2
8.7
5.9
0.6
7.1
19.1
6.2
1.9
9.0
Temperature
(°C)
Max
Min
SD
Average
24.8
23.1
0.6
23.8
25.2
21.7
1.3
22.9
28.6
26.3
0.8
27.2
33.7
26.4
2.5
29.0
29.8
28.0
0.7
29.0
30.2
27.7
1.0
28.9
29.9
28.3
0.6
29.4
25.9
24.7
0.4
25.5
27.4
25.3
0.7
25.9
18.8
17.7
0.4
18.1
17.8
16.6
0.4
17.3
21.0
20.2
0.3
20.6
Salinity
(‰)
Max
Min
SD
Average
27.3
4.5
8.7
16.8
21.0
0
7.3
9.1
18.6
0
6.8
3.3
11.5
0
4.2
2.9
10.9
0
4.0
2.0
13.7
0
5.4
3.1
17.5
0
7.9
6.7
20.3
0
7.5
7.0
24.8
0.3
9.6
11.5
24.4
0.1
9.5
10.8
25.2
0.9
8.0
12.5
26.0
0.2
8.6
12.7
Turbidity
(NTU)
Max
Min
SD
Average
14.0
1.0
4.8
4.7
29.0 150.0 37.0
0
2.0
2.0
10.2 46.2 13.1
11.3 96.3 19.1
55.0 105.0 22.0
12.0 2.0
3.0
15.1 34.4 7.0
26.3 40.9 10.2
70.0
2.0
28.7
32.6
41.0
1.0
17.0
14.8
6.0
0
2.4
4.1
17.0
0
6.8
4.6
10.0
0
3.6
3.6
Note: E. ebb tide, L. low tide, S. spring tide, H. high tide.
Because none of the larvae or juveniles were caught in stations TS8 and
TS9, where the salinity was 0 psu during the study period, this paper omits the
above two stations in the next parts (Figure 2). Among the seven stations from
TS1 to TS7, fish occurred abundantly at stations TS4 and TS5 – the middle part
of the estuary – the average CPUE was 264.4 and 77.8 individuals per haul,
respectively. In contrast, the average CPUE ranged from 0.06 to 0.9 individuals
per haul at stations TS1, TS2, TS3, TS6, and TS7 (Figure 2). The average fish size
reduced slightly from TS1 to TS5 (Figure 2). Only one specimen was collected at
station TS6 (37.1 mm SL). The size of specimens collected at stations TS4 and
TS5 was larger than those collected at the other stations (Figure 2).
A few differences in the average water temperature were found among
the seven stations (TS1–TS7), while the average salinity showed a significantly
different pattern from the temperature (Figure 2). Salinity was highest in the outer
stations (TS1, TS2), where the tidal effects were largest, and decreased moving
upstream; the salinity was less than 10 psu from stations TS3 to TS7, with TS5-
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
71
TS7 at ca. 1 to 5 psu. The average turbidity fluctuated among the seven stations;
it was lowest at station TS1 (16 NTU) and highest at station TS5 (29.5 NTU)
(Figure 2).
Figure 2.Spatial variation of N. nuchalis average CPUE collected in the bank
waters of the Tien Yen estuary, during March 2013 to February 2014.
Average water temperature, salinity, and turbidity are shown by diamond, triangle, and square, respectively. Vertical lines and thick bars
denote ranges of and average ±SE of standard length, respectively.
DISCUSSION
Nuchequula nuchalis occurred in the Tien Yen estuary during two periods
– from April to July 2013 and from August 2013 to January 2014, with a peak
in May 2013. The spawning season of this fish in northern Vietnam may take
place earlier, and last longer than in northern Kyushu, Japan, where the breeding
season is from middle May to the end of July (Fujita, 1960). In Japan, Equulites
rivulatus larvae appear in the summer, showing a greater deviation in spawning
months at lower latitudes (Haque and Ozawa, 1995b), unlike that in sparid fish.
At higher latitudes, various species of sparids spawn year round, in contrast to
lower latitudes, where spawning correlates with the months of lowest sea surface
temperature (Sheaves, 2006). The spawning period of N. nuchalis reported in this
study showed a similar tendency as that of Equulites rivulatus. Smaller collections
in April and May were from around 5.0 mm SL (Table 1), and in the laboratory,
hatching larvae were 1.4 mm in total length (Fujita, 1960). Based on this, the
peak of the spawning season of the fish in this study was estimated in March or
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April, during which the average water temperatures at the river bank were lower
than 25°C (Table 1). As can be seen from Table 1 and Figure 2, the average
water temperature and CPUE did not show a positive relationship by month (r
= 0.07) or by station (r = -0.28). Therefore, the assemblages of this fish larvae
and juveniles in the bank waters of the estuary seem to be unrelated to changes
in water temperature.
The peak of occurrence of the N. nuchalis larvae and juveniles was in
May, when average turbidity was highest (Table 1). However, although the average turbidity and fish caught were positively related by month, the difference
was not significant (r = 0.41, p > 0.05) (Table 1), and there was no relationship
by stations (r = 0) (Figure 2). There is no evidence that turbidity influenced the
distribution of Leiognathus equulus fish in northeastern Queensland, Australia
(Johnston et al., 2007), although turbidity provided early stages with cover from
predators and could be beneficial for increasing benthic production of fish (Cyrus
and Blaber, 1987). While May, the month with the highest turbidity, accounted for
more than 99% of the fish collected over the study period, station TS4, at which
the turbidity was only 2 NTU (the lowest of the seven stations), yielded 99% of
May’s total specimens (Figure 3). Hence, factors other than water temperature
and turbidity may contribute to the distribution of this fish along the bank waters
of the estuary.
While the occurrence of N. nuchalis larvae and juveniles might be related
to the stations along the estuary, the Spearman’s correlation coefficient between
salinity and CPUE at the seven stations is -0.14 (Figures 2, 3). Most of specimens
were collected in the middle part of the estuary (stations TS4 and TS5), at which
the average salinity ranged from 3 to 8 psu (Figure 2). At station TS4, the peak
catch occurred in May, when the salinity reached only 0.3 psu, while another
peak appeared in April at station TS5, when the salinity was just 2.1 psu (Figure
3). At stations TS6 and TS7, only larger specimens were collected; this implies
that larger juveniles were more tolerant of freshwater (Figures 2, 3). In contrast,
at station TS1 at the mouth of the estuary, where average salinity was around
20 psu, few larvae and juveniles were collected (Figures 2 and 3). This finding
indicates that the variation in salinity along the Tien Yen estuary could be one of
the factors that influence the occurrence of N. nuchalis larvae and juveniles. This
phenomenon was also observed with Liza richardsonii, L. dumerili, and Mugil
cephalus (Whitfield, 1994). However, despite some other months having lower
salinity than April and May, few specimens were collected (Figure 3). Therefore,
occurrence of the early stage of N. nuchalis in the estuary was partly driven by
its reproductive cycle, concentrated in March or April (Table 1).
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
73
Figure 3.Comparison of the CPUE of Spotnape Ponyfish and water conditions
among seven stations in the Tien Yen estuary, during March 2013 to
February 2014. Otherwise, same as in Figure 2.
This study confirmed that the Tien Yen River plays a crucial role for in the
early stages of N. nuchalis’s lifecycle (Ta et al., 2014; Tran and Ta, 2014b). Recently,
overfishing, destructive fishing methods, water pollution, and poor management
have placed considerable pressure on the Tien Yen estuary fish resources (Tran and
Ta, 2014a), possibly affecting the early life stages as well. Hence, more attention
needs to be paid to preserving the estuary and its fishery resources. This study yielded a number of larval and juvenile along the bank waters of
the Tien Yen estuary in northern Vietnam during March 2013 to February 2014,
with the vast majority found in May in the middle part of the estuary. Salinity is
a possible factor influencing the occurrence of the larval and juvenile stages of
N. nuchalis in an estuarine environment.
74
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
ACKNOWLEDGEMENTS
The authors are grateful to Ken Maeda for his critical reading of the initial
manuscript and two anonymous reviewers for their helpful comments. The Nagao
Natural Environment Foundation (Japan), financially supported this study, through
the second author. All the surveys in the present study comply with the current
laws of Vietnam.
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Myoung, J.G. and Y.U. Kim. 1984. Morphology of larvae and juveniles of Leiognathus nuchalis (Temminck et Schlegel). Bulletin of the National Fisheries
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Doi: 10.12982/cmujns.2016.0007
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77
Bismuth Coated Screen-printed Electrode Platform
for Greener Anodic Stripping Voltammetric Determination
of Cadmium and Lead
Autchara Paukpol1 and Jaroon Jakmunee1,2*
1
Department of Chemistry, and Research Laboratory for Analytical Instrument
and Electrochemistry Innovation, Faculty of Science, Chiang Mai University,
2
Center of Excellence for Innovation in Chemistry, Faculty of Science, Chiang
Mai University, Chiang Mai 50200, Thailand
ABSTRACT
A greener electrochemical platform was developed to determine trace
amounts of cadmium and lead. It is based on a disposable screen-printed carbon ink electrode modified with an in situ plated bismuth film employed as a
more environmentally-friendly working electrode alternative to the widely used
mercury electrode. The bismuth coated screen-printed electrode (Bi-SPE) was
used for the simultaneous determination of trace Cd(II) and Pb(II) by squarewave anodic stripping voltammetry (SWASV). Operational parameters such as
Bi(III) concentration, deposition potential, deposition time and rotation speed
during preconcentration of the metals were optimized. The Bi-SPE presented
well-defined, reproducible, and sharp stripping voltammograms. Peak current
increased linearly with the metal concentration in a range of 5-40 µg L-1 for
Cd(II) and 2-40 µg L-1 for Pb(II). The limits of detection were 1.7 µg L-1 for
Cd(II) and 0.7 µg L-1 for Pb(II), which are better than those of the flame
atomic absorption method. The proposed method was successfully applied to
determine trace cadmium and lead in river water samples. Accuracy of the
developed method was examined by spiking Cd(II) and Pb(II) standard solutions
into river water samples, and percentage recoveries were obtained in the range
of 86.4-110.6%. The SWASV with the new Bi-SPCE electrode provided advantages, including high sensitivity, low detection limits, low background current,
portability, fast and cost-effective determinations, and, importantly, the use of
relatively non-toxic chemicals.
Keywords: Bismuth coated screen-printed electrode, Square-wave anodic stripping
voltammetry, Cd(II), Pb(II), Water samples
78
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
INTRODUCTION
The growing concerns about Cd(II) and Pb(II) poisoning have led to increasing demands for highly sensitive, reliable, reproducible, and low cost methods
for quantitation in water. Electrochemical stripping analysis has been recognized
historically as a powerful technique for Cd(II) and Pb(II) detection. In stripping
voltammetry, the mercury film or hanging mercury drop electrode (MFE/HMDE)
has been widely applied as a working electrode owing to its remarkable sensitivity.
However, the toxicity of mercury has stimulated the search for environmentally-friendly material to produce a greener, mercury-free electrode. The bismuth
film electrode (BiFE) is considered the best alternative to MFE due to its ability
to form fusing alloys with other metals (Wang et al., 2000; Locatelli and Torsi,
2001; Arduini et al., 2010). Different materials have been used as substrates for
BiFEs, including glassy carbon (Wang et al., 2001a; Kefala et al., 2003), carbon
paste (Krolicka et al., 2002; Svancara et al., 2003), wax-impregnated graphite
(Kefala et al., 2003), pencil-lead (Demetriadis et al., 2004), and screen-printed
carbon ink (Wang et al., 2001b). Screen-printing technology is well established
for the production of low-cost, reproducible, and sensitive electrochemical sensors.
Screen-printed sensors have been widely used for environmental, biomedical, and
industrial monitoring (Goldberg et al., 1994; Laschi et al., 2006).
In this study, bismuth film was deposited in situ on carbon working electrode
and used in square wave anodic stripping voltammetry (SWASV) to determine
Cd(II) and Pb(II) in water samples; the optimization, characterization and attractive analytical performance of bismuth coated screen-printed electrode was also
reported.
Chemicals and reagents
All the chemicals were of analytical reagent grade. Ultrapure water (Milli-Q
water, resistivity of 18.2 MΩ cm-1) obtained from a Millipore water purification
system (Millipore, Sweden) was used throughout. The solutions of Bi(III) (Carlo
Erba, Italy), Cd(II) (Fluka, Switzerland) and Pb(II) (Merck, Germany) were prepared from 1000 mg L-1 standard solutions by appropriate diluting with Milli-Q
water. An acetate buffer solution (0.1 M, pH 4.5) was prepared by mixing the
acetic acid (Lab Scan, Thailand) and sodium acetate (Carlo Erba, Italy) and served
as a supporting electrolyte.
Apparatus
Voltammetric measurements were performed with a Metrohm model 757
VA computrace with GPES software for control of the voltammograph linked with
a personal computer. A bismuth coated screen-printed carbon electrode served as
the working electrode, with the Ag/AgCl (3 M KCl) and platinum wire as the
reference and counter electrodes, respectively. Plasma cleaner (PDC-32G, Harrick
Plasma, USA) was used to pretreat the working electrode.
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
79
Bismuth coated screen-printed carbon electrode (Bi-SPCE) preparation
SPCEs were produced with a homemade manually screen-printing apparatus (Upan et al., 2015). Carbon ink (Henkel, Germany) was used to print the
working electrode. The substrate was a flexible polyester film. The diameter of
the working electrode was 3 mm. SPCEs were modified with bismuth using the
in situ plating approach. Firstly, SPCEs were cleaned by subjecting them to air
plasma in a chamber of the plasma cleaner. The chamber was evacuated to 0.15
torr and the plasma was generated at low frequency RF level. Then, the chamber
was backfilled with air and operated at pressure 0.4 torr for 60 s. The plasma
treated SPCEs were electrochemically treated in a 0.1 M acetate buffer solution at
pH 4.5 by applying a positive potential of +1.6 V vs Ag/AgCl for 120 s, followed
by +1.8 V vs Ag/AgCl for 60 s, and then thoroughly rinsed with water.
Measurement procedure
The electrochemical deposition on SPCEs coated by bismuth was carried
out according to the following in situ procedure in the presence of dissolved
oxygen. The SPCE was immersed in an electrochemical cell containing the specified
concentration of Bi(III) in an acetate buffer (0.1 M, pH 4.5) medium and Bi was
deposited on the metals on the surface of the electrode for a specified time while
the solution was stirred. The analytical measurement used the square wave anodic
stripping voltammetric mode (SWASV). ASV experiments consisted of three conventional steps: time controlled electrochemical deposition with solution stirring,
rest period, and a positive voltammetric stripping scan under the selected conditions. The optimum conditions for SWASV method were as follows: frequency
(f), 35 Hz; step potential (Estep) 4 mV; square wave amplitude 40 mV; deposition
potential (Edep) -0.9 V; deposition time (tdep) 180 s; the potential scanning range
from -0.9 to 0.3 V; equilibrium time (teq) 15 s; cleaning potential (Eclean) 0.3 V;
and cleaning time 30 s. During the electrodeposition and precondition steps, the
solution was stirred at 2000 rpm. In the analysis, an in situ plated Bi-SPE was
prepared by adding the Bi solution to a water sample diluted in 0.1 M acetate
buffer solution pH 4.5 to obtain 1 mg L-1 Bi(III) in the final solution. Calibration
graphs were used to quantify Cd(II) and Pb(II) in natural water samples.
Analysis of real samples
Water samples were collected from the Ping River, Chiang Mai, Thailand at
the sampling sites shown in Figure 1. Water samples were filtered through a 0.45
µm membrane. In the analysis, in situ plated Bi-SPCEs were prepared by spiking
the sample with 1 mg L-1 Bi(III) in 0.1 M acetate buffer at pH 4.5. Calibration
curves were used to quantify Cd(II) and Pb(II) in the samples.
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➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
Figure 1.The water sampling sites in the Ping River, Chiang Mai, Thailand.
RESULTS
Effect of experimental variables
Bi(III) concentration. Representative Cd(II) and Pb(II) peak currents for a
solution containing 100 µg L-1 each of Cd(II) and Pb(II) in 0.1 M acetate buffer
(pH 4.5) on in situ plated Bi-SPCEs with different Bi(III) concentrations in the
range 100-1400 µg L-1 are shown in Figure 2(a).
Deposition potential. The effects of deposition potentials in the range of
-0.75 to -1.60 V on the stripping currents of Cd(II) and Pb(II) are shown in Figure
2(b); the peak potentials for Cd(II) and Pb(II) were at -0.75 and -0.50 V vs Ag/
AgCl, respectively.
Deposition time. The effects of metal deposition times in the range of 60420 s for the solution containing 100 µg L-1 each of Cd(II) and Pb(II) in 0.1 M
acetate buffer (pH 4.5) are shown in Figure 2(c).
Rotation speed during preconcentration step. The effects of rotation
speeds of the stirrer in the range of 0-3000 rpm are shown in Figure 2(d).
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
81
(a)
(b)
(c)
(d)
Figure 2. Effects of the Bi(III) concentration (a), deposition potential (b), deposition time (c), and rotation speed during preconcentration (d) on the
stripping peak currents of Cd(II) ( ) and Pb(II)( ) in 100 µg L-1
Cd(II) and Pb(II) with 0.1 M acetate buffer pH 4.5 using in-situ prepared
Bi-SPCE, n = 3. SWASV parameter; step increment 4 mV, frequency
35 Hz, pulse height 40 mV, cleaning potential 0.3 V, and cleaning time
30 s.
Calibration data
Calibration was performed on Bi-SPCEs for the simultaneous determination of Cd(II) and Pb(II) by SWASV; the results are shown in Figure 3. A small
background current over a wide potential range was noticed. Calibration graphs
had different slopes in the ranges of 5-40 and 40-100 µg L-1 for Cd(II) and 2-40
and 40-100 µg L-1 for Pb(II), respectively. This may be a result of the limited
surface area of the electrode, leading to the saturation of metals deposited on
the electrode. At lower concentrations, the metals might disperse more on the
electrode surface; on the other hand, at higher metal concentrations, they might
deposit upon the previously deposited layer. The current obtained in the stripping
step was directly proportional to the rate of oxidation of the metals back into
the solution, which was related to the amounts of metals on the surface of the
electrode. The analytical sensitivities were Cd(II): 1.21, 0.17 µA per µg L-1 (R2
82
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
Peak curent (µA)
= 0.9995, 0.9911) and Pb(II): 1.21, 0.15 µA per µg L-1 (R2 = 0.9961, 0.9959).
On the same electrode, the relative standard deviations were 7.0% for Cd(II) and
5.8 % for Pb(II) at 20 µg L-1 level (n=15). The same SPCE electrode could be
used for at least 15-20 stripping cycles without loss of sensitivity. The limits of
detection were calculated, at a deposition time of 180 s, as 1.7 µg L-1 for Cd(II)
and 0.7 µg L-1 for Pb(II).
60
40
20
0
0
20
40
60
80
100
Peak curent (µA)
Cd(II) concentration (µg L-1)
60
40
20
0
0
20
40
60
80
100
Pd(II) concentration (µg L-1)
Figure 3. A series of voltammograms and calibration graphs for increasing concentrations of Cd(II) and Pb(II) in 0.1 M acetate buffer pH 4.5 using
in-situ prepared Bi-SPCE (1 mg L-1 Bi(III)), n = 3. SWASV parameter;
deposition potential -0.9V vs Ag/AgCl, deposition time 180 s, stirrer
speed 2000 rpm, step increment 4 mV, frequency 35 Hz, pulse height
40 mV, cleaning potential 0.3 V and cleaning time 30 s.
Accuracy of the method
The system accuracy was determined by spiking water samples with Cd(II)
and Pb(II) standard solutions of 5 µg L-1 and measuring the percent recoveries of
Cd(II) and Pb(II). The results are given in Table 1. Percent recoveries of Cd(II)
and Pb(II) are close to 100%, in the range of 86.4 to 110.6%, under the optimal
condition.
Application to river water
For the purpose of practical applicability, Bi-SPCEs were employed to determine Cd(II) and Pb(II) in river water samples. The anodic stripping peak current
was recorded with the calibration graph method under optimized conditions for
determining Cd(II) and Pb(II). The results obtained for nine water samples are
summarized in Tables 1 and 2.
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
83
Table 1.Determination of Cd(II) in water samples by the proposed SWASV with
Bi-SPCE.
Water
sample
Cd(II) found in
sample (µg L-1)
Spiked Cd(II)
(µg L-1)
Total conc. found
(µg L-1)
RSD
(%)
Recovery
(%)
1
n.d.
5
4.85 ± 0.13
2.7
96.9
2
n.d.
5
4.79 ± 0.11
2.3
95.7
3
n.d.
5
4.32 ± 0.11
2.5
86.4
4
n.d.
5
4.78 ± 0.07
1.5
95.6
5
n.d.
5
4.93 ± 0.14
2.8
98.6
6
n.d.
5
4.62 ± 0.08
1.7
92.4
7
n.d.
5
4.57 ± 0.09
2.0
91.4
8
n.d.
5
5.16 ± 0.07
1.4
103.2
5
5.18 ± 0.13
2.5
103.6
9
n.d.
Note: * n.d. = not detectable.
Table 2.Determination of Pb(II) in water samples by the proposed SWASV with
Bi-SPCE.
Water
samples
Pb(II) found in
sample (µg L-1)
Spiked Pb(II)
(µg L-1)
Found Pb(II)
(µg L-1)
RSD
(%)
Recovery
(%)
1
n.d.
5
4.91 ± 0.09
1.8
98.1
2
n.d.
5
4.97 ± 0.10
2.0
99.4
3
n.d.
5
5.15 ± 0.10
1.9
103.0
4
n.d.
5
5.31 ± 0.08
1.5
106.2
5
n.d.
5
4.52 ± 0.10
1.8
110.4
6
n.d.
5
5.53 ± 0.07
1.3
110.6
7
2.10 ± 0.01
5
7.34 ± 0.10
1.9
106.8
8
n.d.
9
n.d.
Note: * n.d. = not detectable.
5
5.22 ± 0.05
1.0
104.4
5
5.28 ± 0.12
2.3
105.6
DISCUSSION
The concentration of Bi(III) is a key parameter, as it controls the thickness
of the Bi film; the thickness of the film does not affect the peak potential of any
metals, but rather the peak height. The results showed a decrease of peak current
of the Cd(II) and Pb(II) with increasing thickness of the Bi film, especially the
Pb(II) peak, as shown in Figure 2(a). Since Pb(II) has a more positive reduction
potential than Cd(II), Pb(II) can be determined at lower Bi(III) concentrations.
At high Bi(III) concentrations, the peaks became wider and led to a substantial
decrease in sensitivity at Bi(III) concentrations over 1000 µg L-1, due to a saturation effect of the Bi film on the screen-printed carbon surface. This behavior is
attributed to the increased number of nucleation sites and increased alloy formation.
However, at Bi(III) concentrations greater than 1000 µg L-1, a reduction in the
84
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
peak intensity was observed, ascribed to the formation of a thick layer of Bi on
the electrode surface that partially blocks the conductive surface of the electrode,
reducing the number of electroactive sites (Kachoosangi et al., 2007). Thus, a
Bi(III) concentration of 1000 µg L-1 was selected for further experiments.
The deposition potential affects the quality of bismuth film to form fused
alloys with reduced metal ions. As shown in Figure 2(b), the stripping responses
of Cd(II) and Pb(II) were found to occur at potentials more negative than -0.75
V. As the deposition potential became more negative, the peak currents increased
up to -0.90 V for Cd(II) and -0.85 V for Pb(II). The Pb(II) peak current did not
increase significantly from -0.75 to -0.85 V, due to its more positive reduction
potential relative to Cd(II). The evolution of hydrogen gas from the SPCE surface that started to occur at -1.50 V could damage the bismuth film, and some
interfering species may be deposited on the electrode at more negative potential.
Therefore, a potential of -0.9 V was chosen as the optimal deposition potential.
The deposition time is the time required for Bi-SPCE to reduce Cd(II)
and Pb(II) to Cd(Bi) and Pb(Bi) alloys, respectively. It is generally accepted that
lower limits of detection (LODs) can be obtained with longer deposition times
(Castaneda et al., 2005). In Figure 2(c), Cd(II) and Pb(II) peak currents increased
rapidly with deposition times from 60-180 s. For deposition times longer than 180
s, the current responses increased negligibly, so this was considered the longest
practical time for a satisfactory compromise between high sensitivity and short
analytical times. Metal ions were preconcentrated for 180 s in further experiments.
The rotational speed of the stirrer controlled the mass transport behavior
during the preconcentration step, as shown in Figure 2(d). The stripping peak
currents for Cd(II) and Pb(II) continuously increased with each increment of
rotational speed up to 2000 rpm. Beyond 2000 rpm, peak currents decreased
because the bismuth film may have become mechanically damaged; therefore,
preconcentration was performed at 2000 rpm.
The SWASV with bismuth film working electrode can simultaneously determine trace amounts of Cd(II) and Pb(II) in real water samples using calibration
graphs. This method was highly sensitive, consumed low amounts of reagent and
sample, and was convenient to operate. It was also able to determine both metals
in water samples at concentrations lower than the detection limits of the standard
FAAS method. In comparison to the FAAS method, the developed voltammetric
method was more sensitive with lower detection limits, less costly, and consumed
fewer chemicals and energy (Roongjang et al., 2014).
The proposed method successfully analyzed real water samples, with recovery rates in the range of 86.4 to 110.6%. The results obtained for nine water
samples are summarized in Table 1 and 2. The concentrations of the investigated
metal ions in the Ping River water were very low and should have no environmental impact. According to the Thailand Pollution Control Department (PCD) Water
Quality Standards, the maximum acceptable concentrations for Cd(II) and Pb(II) in
drinking water are 10 µg L-1 and 50 µg L-1, respectively. The developed method
can be used for monitoring these trace metals in natural water sources. The
proposed Bi-SPCE was more environmentally friendly than the mercury-based
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
85
electrode. Moreover, the screen-printed carbon electrode is conveniently fabricated, less expensive, and can be mass produced, as compared to other carbon
electrodes, such as glassy carbon (Wang et al., 2001a; Kefala et al., 2003), carbon
paste (Krolicka et al., 2002; Svancara et al., 2003), and wax-impregnated graphite
(Kefala et al., 2003).
CONCLUSION
The developed anodic stripping voltammetric method with in situ coated
Bi film on screen-printed carbon working electrode offers a cost-effective and sensitive alternative analytical method for the simultaneous determination of trace
Cd(II) and Pb(II) in water samples. SPCE, which is an inexpensive, widely available
and disposable material, has been shown to offer comparable performance to more
expensive glassy carbon electrodes for the simultaneous determination of Cd(II)
and Pb(II). In addition, with the low toxicity of bismuth, low background current
and small size, Bi-SPCEs offer great scope for applications in which compact
instrumentation and low sample volumes are critical, such as field measurement
and on-site monitoring of heavy metals.
The developed method provides high sensitivity and low detection limits
compared to standard flame atomic absorption spectrometry (FAAS) (Roongjang
et al., 2014). The developed method was accurate in the range of 86.4 to 110.6%,
as determined from percent recovery of Cd(II) and Pb(II). Moreover, the SWASV
method involves inexpensive instrumentation with low operating costs.
ACKNOWLEDGEMENTS
This work was supported by the Thailand Research Fund (TRF), the
Commission on Higher Education (CHE) and Chiang Mai University (Grant
# RMU5380052). We thank the Science Achievement Scholarship of Thailand
(SAST) for providing a scholarship to AP. The Center of Excellence for Innovation
in Chemistry (PERCH-CIC) is acknowledged for partial support. We gratefully
thank Professor Richard L. Deming for his comments and editing of the manuscript.
86
➔ CMU J. Nat. Sci. (2016) Vol. 15(1)
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Kachoosangi, R.T., C.E. Banks, X. Ji, and R.G. Compton. 2007. Electroanalytical
determination of cadmium(II) and lead(II) using an in-situ bismuth film
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Kefala, G., A. Economou, A. Voulgaropoulos, and M. Sofoniou. 2003. A study
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Krolicka, A., R. Pauliukaite, I. Svancara, R. Metelka, A. Bobrowski, E. Norkus, K. Kalcher, and K. Vytras. 2002. Bismuth-film-plated carbon paste
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Laschi, S., I. Palchetti, and M. Mascini. 2006. Gold-based screen-printed sensor
for detection of trace lead. Sensors and Actuators B: Chemical 114: 460465. doi:10.1016/S1388-2481(01)00301-0
Locatelli, C., and G. Torsi. 2001. Voltammetric trace metal determinations by
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the presence of mutual interference. Journal of Electroanalytical Chemistry
509: 80-89. doi: 10.1016/S0022-0728(01)00422-3
Roongjang, S., U. Pusod, P. Phooviang, and K. Nantachit. 2014. Development
of Method for Determination of Lead, Iodine, and Protein Content in Fish
Sauce. Chiang Mai University Journal of Natural Sciences 13(3): 383-390.
doi: 10.12982/cmujns.2014.0043
Svancara, T., M. Fairouz, Kh. Ismail, R. Metelka, and K. Vytras. 2003. A contribution to the characterization of mercury and bismuth film carbon paste
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none
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89
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Chapter in book
Jackson, M.B. 1982. Ethylene as a growth promoting hormone under
flooded conditions. p.291-301. In P.F. Wareing (ed) Plant growth
substance. Academic Press, London.
Edited proceeding, symposia etc.
Pratt, A., R.J. Gilkes, S.C. Ward, and D.A. Jasper. 2000. Variations
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Proceeding of Remade Land 2000, the International Conference
on Remediation and Management of Degraded Lands. Fremantle,
30 Nov-2 Dec 2000. Promaco Conventions, Canning Bridge.
Dissertation
Senthong, C. 1979. Growth analysis in several peanut cultivars
and the effect of peanut root-knot nematode (Meloidogyne
arenaria) on peanut yields. Ph.D. Dissertation. University of Florida, Gainesville, Florida, USA.
➔ CMU J. Nat. Sci. (2015) Vol. 15(1)
INDEX TO VOLUME 15 NUMBER 1 (2016)
CHIANG MAI UNIVERSITY JOURNAL OF NATURAL SCIENCES
AUTHOR INDEX
Chairuangsri, S.
15 (1):11
Chantara, S.
15 (1):39
Dell, B.
15 (1):1
Jakmunee, J.
15 (1):77
Jamjod, S.
15 (1):1
Jampeetong, A.
15 (1): 11
Khamphaya, T.
15 (1):11
Kim, S.M.
15 (1):21
Pan, C.H.
15 (1):21
Panomjan, N.
15 (1):1
Paukpol, A.
15 (1): 77
Phornwisetsirikun, W.
15 (1):39
Prapamontol, T.
15 (1):39
Prommajak, T.
15 (1):21
Prom-u-thai, C.
15 (1):1
Rangkakulnuwat, S.
15 (1):39
Rattanapanone, N.
15 (1):21
Rerkasem, B.
15 (1):1
Sriarporn, P.
15 (1):49
93
94
➔ CMU J. Nat. Sci. (2015) Vol. 15(1)
Sripakdee, T.
15 (1):11
Surawang, S.
15 (1):21
Ta, T.T.
15 (1):67
Thavornyutikarn, P.
15 (1):39
Tiansawad, S.
15 (1):49
Tran, H.D.
15 (1):67
Tran, T.T.
15 (1):67
Wan, H.
15 (1):49
Yimyam, S.
15 (1):49
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CMUJ nat-sci 15-1.indb - Chiang Mai University Journal

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