extfieldsofstudy: Biology Medicine

text:
Effects of heat stress on body temperature, milk production, and reproduction in dairy cows: a novel idea for monitoring and evaluation of heat stress — A review

Heat stress exerts a substantial effect on dairy production. The temperature and humidity index (THI) is widely used to assess heat stress in dairy operations. Herein, we review the effects of high temperature and humidity on body temperature, feed intake, milk production, follicle development, estrous behavior, and pregnancy in dairy cows. Analyses of the effects of THI on dairy production have shown that body temperature is an important physiological parameter in the evaluation of the health state of dairy cows. Although THI is an important environmental index and can help to infer the degree of heat stress, it does not reflect the physiological changes experienced by dairy cows undergoing heat stress. However, the simultaneous measurement of THI and physiological indexes (e.g., body temperature) would be very useful for improving dairy production. The successful development of automatic detection techniques makes it possible to combine THI with other physiological indexes (i.e., body temperature and activity), which could help us to comprehensively evaluate heat stress in dairy cows and provide important technical support to effectively prevent heat stress.

INTRODUCTION
Heat stress is a non-specific physiological response of animal to thermal environment when animal produces more heat than it can dissipate [1]. The Holstein is the most popular dairy cattle breed in the world. In summer, the ability of this breed to dissipate heat via skin evaporation is limited by its relatively low surface area to body weight ratio, underdeveloped sweat glands, and short, dense body surface hair, all of which burden milk production [2]. Moreover, considerable metabolic heat is produced by the fermentation of the consumed roughage in the rumen, which also results in an additional heat load on the cow's body [3]. In addition, with the steadily warming global climate and the intensification of hot periods, dairy cows have been subjected to even more severe heat stress [4,5], which threatens to severely reduce production performance, especially milk yield [6,7]. Therefore, dairy cows used to mainly be raised in cool areas to reduce heat stress. To help mitigate the negative effects of ambient temperature and humidity on body temperature and milk production, studies have focused on the development of scientific techniques to manage heat stress and increase cooling [4,8]. These techniques can reduce the negative impact of hot and humid environments on dairy cows, and promote increases in the quality and efficiency of milk production in the dairy industry [9].
Body temperature is an important physiological parameter and is indicative of the health of dairy cows [10]. Studies have shown that high temperature conditions can lead to sig-nificant increases in body temperature and respiratory rate [11,12], and a significant decrease in feed intake [9], milk production [13,14], and reproductive performance [15]. The annual economic losses of livestock industries as a result of heat stress have been reported to be 1.69 to 2.36 billion USD in the United States, of these losses, up to 1,500 million USD occur in the dairy industry [16]. These data show that the most serious economic impact of heat stress has been on the dairy industry. Thus, if real-time changes in record body temperature, ambient temperature, and humidity can be monitored and recorded, it would be possible to evaluate whether cows are experiencing heat stress, as well as the degree of heat stress they are experiencing, based on these changes [17,18].
The temperature-humidity index (THI) is often used as an important environmental assessment index to evaluate heat stress in dairy production [19,20]. Studies that focus on the relationship between THI and body temperature will help us to understand the physiological status of cows exposed to different THI conditions, which is of great significance to the feeding and management of cows. Herein, we review the effects of ambient temperature and humidity on the body temperature of cows, as well as the effects of heat stress on feed intake, milk production, follicular development, estrus, and pregnancy. We hope that this review will provide theoretical guidance for the feeding and management of dairy cows.

EFFECTS OF HEAT STRESS ON THE BODY TEMPERATURE OF DAIRY COWS
Formulas for calculating temperature-humidity index One study indicated that animals show different sensitivities to ambient temperature and humidity [15]. Under the same ambient humidity, swine cannot tolerate higher temperatures because they have few sweat glands [21], while the impact of heat stress on cattle can be relieved by dissipating excessive heat through sweating [20]. Thus, THI can be used as an important environmental index to ascertain whether animals are in a state of heat stress, as well as the degree of this heat stress. THI-a comprehensive index composed of the effects of ambient temperature and humidity-has been developed into a meteorological safety index to monitor the possible effects of ambient temperature and humidity on animals [22]. It has been shown the degree of heat stress varies with climate characteristics, as humidity plays a larger role in physiological stress in areas with high humidity, while the ambient temperature is more influential in areas with semi-arid climates [20]. Thus, different formulas for calculating THI have been developed for different climates. Temperature-humidity indices (THIs) were calculated using eight different formulas, while eight coefficients of determination (r 2 ) were determined based on both THI values and the rectal temperature (RT) of 1,280 Holstein cows [23]. The r 2 values ranged from 0.42 to 0.43 for seven of the eight THIs, while the other r 2 was 0.39, indicating that the THIs vary depending on the formula used, and that the formula for calculating THI should be selected according to the environmental conditions [23].
At present, two formulas for calculating THI are commonly used in dairy production. One equation uses dry and wet bulb temperatures to calculate THI: THI = 0.72(Tdb+Twb)+40.6, where Tdb is the dry bulb temperature and Twb is the wet bulb temperature in °C [24]. The other equation is THI = (1.8×T+32)-(0.55-0.0055×RH9×[1.8×T-269]), where T is dry bulb temperature (°C) and RH is the relative humidity (%) [25]. According to the local climate characteristics, we can select a suitable equation for THI to ascertain potential heat stress in dairy cows, and to provide a basis for the effective prevention of heat stress on a given farm. Armstrong [26] showed that cows do not experience heat stress when the THI value is less than 72, they experience mild heat stress when 73<THI<79, they suffer moderate heat stress when 80<THI <89, and they exhibit a severe heat stress response when THI >90. Therefore, if THI can be divided into different stages according to the possible state of heat stress, this heat stressand the degree of stress that heat and humidity may causecan be inferred by the change in ambient temperature and humidity. Thus, heat stress in dairy cows can be subsequently reduced or alleviated via corresponding measures (e.g., such as equipping facilities with a sprinkler system and/or a fan to control environmental conditions) [6,9].

Effects on the body temperature of dairy cows
The body temperature of cows can be measured in terms of RT, vaginal temperature (VT), rumen temperature, subcutaneous tissue temperature, and temperature measured via the ear canal [27][28][29]. In addition, milk temperature has also been measured to infer the body temperature of a cow [9]. In production practice, RT is often used as a sensitive index to determine whether a cow has reached thermal balance [30] and to determine the influence of the thermal environment on growth, lactation, and reproduction in dairy cows [31]. A study by Xue et al [32] showed that RT rose from 37.8°C to 38.5°C when the THI value rose from 45 to 72, representing increases in THI and RT of 60% and 1.8%, respectively. However, when THI rose from 72 to 79, RT increased from 38.5°C to 39.35°C, representing increases in THI and RT of 9.7% and 2.2%, respectively. In other words, the percent increase in RT was greater when THI was greater than 72, indicating that the correlation between RT and THI is complex. Therefore, THI less than 72 is considered to be thermoneutral zone for dairy cows [33,34]. Wen [6] showed that RT was significantly higher during heat stress than during non-heat stress periods (p<0.01), and that for each unit increase in the THI value, the RT of dairy cows increased by 0.1062°C, 0.0739°C, and 0.0847°C during early, mid-, and late lactation, respectively. In addition, the p values of correlations between THI and RT in early, mid-, and late lactation were 0.0069, 0.0292, and 0.0093, respectively, indicating that THI significantly affects the RT of dairy cows in early, mid-, and late lac tation (p<0.05) [6]. Rejeb et al [35] also showed that the correlation coefficient (r) between THI and the RT of thirteen multiparous cows was as high as 0.87 when the THI value ranged from 65.62 to 83.27.
Because milk temperature is easier to monitor than RT, the relationship between milk temperature and THI has also been studied. West et al [9] showed that the milk temperature fluctuated from 39.3°C to 39.6°C (in Holsteins) and 39.1°C to 39.2°C (in Jerseys) when THI ranged between 72.1 and 83.6. Furthermore, the r value of the THI and milk temperature of Holstein and Jersey cows were 0.88 and 0.60, respectively, indicating that the milk temperature of the Holstein cows was affected more than that of the Jersey cows by THI. In addition, these results showed that milk temperature gradually increased with ambient temperature, reaching 39°C when the THI reached 72, which is the point at which the feed intake and milk production of cows declined and other heat-stress symptoms appeared, indicating that milk temperature can be effectively used to assess heat stress [9].
The relationship between VT and ambient temperature and humidity has also drawn attention, as a strong correlation between VT and RT has already been established [36,37]. The VT of 11 Holstein-Friesian cows (including six in a hot period and five in a cool period) were continuously monitored using data-recording thermal sensors for 25 days, while the ambient temperature and humidity were also continuously recorded. Nabenishi et al [29] study showed that the average VT (39.1°C) during the hot period was significantly higher than that (38.7°C) during the cool period (p<0.01). The average THI during the hot period (78.0) was also higher than that of the cool period (56.6; p<0.01). The r value between VT and maximum THI (mTHI) was 0.30 when THI ranged from 72.0 to 81.1 during the hot period, but was -0.08 when THI ranged from 48.0 to 68.8 in the cool period, indicating that VT is not substantially affected by temperature and humidity during cool periods [29]. With the development of sensing technology, the ability to continuously measure the VT of cows has been developed. As a result, the importance of VT has become increasingly prominent, where correlations between VT and RT have garnered more and more attention. A recent study on the VT and RT of 75 primiparous and 142 multiparous Holstein cows showed that, under the same THI conditions, the mean VT and RT of primiparous cows were 39.7°C and 39.4°C, respectively, while the mean VT and RT of multiparous cows were 39.5°C and 39.2°C, respectively. However, the correlations between THI and VT and RT were not explored further [38].
At present, many reports have focused on the effects of THI on the body temperature of dairy cows, where the relationships between THI and body temperature vary depending on the method used to measure body temperature [35,39]. Although RT [30], rumen temperature [40], milk temperature [9], and VT [29] can all be used to assess heat stress, VT is affected by external factors to a lesser extent than other measurements and may be more suitable for the evaluation of heat stress in dairy cows [41]. In addition to improving the heat stress assessment index [42], it is still not entirely clear if other temperature indices can be used to assess heat stress, or if the combination of body temperature and environmental indices could provide an accurate evaluation of heat stress. Therefore, further research is necessary to elucidate these possibilities.

EFFECTS OF HEAT STRESS ON PRODUCTION AND REPRODUCTION IN DAIRY COWS
Under normal conditions, fluctuations in the body temperature of cows are very small. The normal body temperature ranges from 38.5°C to 39.5°C in calves, from 38.0°C to 39.5°C in heifers, and from 38.0°C to 39.0°C in adult cows [43]. While an ambient temperature between 0°C and 25°C has been showed to be beneficial to production performance [44,45], ambient temperatures in excess of 25°C have been shown to cause an increase in the body temperature of dairy cows, thereby increasing heat stress [45,46]. A study by Robinson et al [47] showed that heat production was 124.5 kcal/kg of BW 0.75 when beef cattle were exposed to an ambient temperature of 20°C, but was 141.8 kcal/kg of BW 0.75 when they were exposed to hotter conditions (35°C). This indicates that heat production increases with an increase in ambient temperature, resulting in a concomitant increase in the body temperature of the cow. However, body temperatures that exceed normal values are not ideal, and cows have been shown to decrease their feed intake and heat exchange capacity accordingly [48,49]. As a result, this leads to lower milk production and reproductive indices [24], as well as increased costs for the dairy industry in the summer months [34].

Effects on feed intake
A large amount of metabolic heat is produced by digesting roughage, which increases the body temperature of cows. As ambient temperature increases in the summer months, and as body temperature concomitantly increases, cows decrease their feed intake to mitigate heat stress [19], thereby leading to a gradual decline in milk production and a change in milk content. It has been reported that the feed intake of dairy cows begins to decrease when the ambient temperature reaches 25°C, and sharply decreases when the ambient temperature exceeds 40°C, after which feed intake is approxi-mately 20% to 40% lower than the normal intake [50,51]. Thus, an observed decrease in feed intake is an important index of heat stress in dairy cows, where the combination of decreased feed intake and increased ambient temperature gradually result in lower milk yield [32]. If environmental factors can be actively monitored and management practices altered accordingly to alleviate heat stress in dairy cows, milk yield and quality can be improved by boosting feed intake [4].
A study of 22 Holstein and eight Jersey cows found that THI negatively affected feed intake, where a THI>72.1 resulted in a decrease in the feed intake of Holstein and Jersey cows by 0.51 kg and 0.47 kg, respectively, for each unit of increase in THI [9]. In addition, Xue et al [32] found that the dry matter intake (DMI) increased from 18.5 to 19.8 kg/d when THI increased from 42 to 68, but decreased from 19.8 to 15.8 kg/d when the THI increased from 68 to 80. This indicates that DMI responds to THI in a biphasic manner, where DMI increases slowly with an increase in THI until a critical point, after which it decreases sharply with an increase in THI when the THI value is higher. Therefore, cows gradually enter a state of heat stress when THI exceeds 68, and DMI decreases gradually with increasing heat stress.

Effects on milk yield
As previously mentioned, heat stress leads to a decrease in feed intake of dairy cows [19], thereby leading to a reduction in milk production [13]. Gantner et al [34] showed that the THI values in spring and summer in the three regions (east, Mediterranean, and central) of Croatia easily exceeded 72, during which the cows were prone to develop heat stress. However, in autumn and winter, when THI values were usually less than 72, the cows rarely experiencing heat stress. In addition, the milk yields were significantly different between the heat-stress and non-heat-stress periods (p<0.01), and indicating that the milk yield of dairy cows in regions with different microclimates can be significantly affected when THI reaches heat-stress levels. A study by West et al [9] that investigated the effects of heat stress on different breeds of cows, showed that the milk yield of Holstein cows (n = 22) and Jersey cows (n = 8) decreased by 0.69 kg and 0.45 kg, respectively, for every unit increase in THI, indicating that a higher degree of breed selection led to a greater impact of heat stress. Moreover, a study that investigated seasonal effects on milk yield showed that the milk yield of Holstein cows decreased by 10% to 40% in summer in comparison to the milk yield in winter [52], further highlighting the influence of heat stress on milk production.
Heat stress not only decreases milk yield, but also affects milk content and somatic cell count [53,54]. An analysis of milk produced by 1,675,686 cows in Croatia from January 2005 to April 2010 showed that the fat and protein content of the milk decreased significantly as a result of increased THI (p< 0.01) [34]. A separate analysis-based on 119,337 milk-production values from 15,012 dairy cows in Georgia, USAshowed that the fat and protein content of milk decreased by 0.012 kg and 0.009 kg, respectively, for each unit of increase in THI above 72 [13]. Yet another study regarding milk components in the Mediterranean showed that, when the average THI increased from 68 to 78 (from spring to summer), the protein and fat contents of milk decreased from 2.96% and 3.58% to 2.88% and 3.24%, respectively. In addition, feed intake and milk yield decreased by 9.6% and 21%, respectively [33]. These studies illustrate that the performance of dairy cows in summer is greatly affected by heat stress, which is indirectly reflected in changes in feed intake, milk yield, and milk components [33,55].

Effects on follicular development and estrus
Heat stress can affect the function of tissues and organs in dairy cows and hinder the synthesis of some proteins and hormones, which can in turn lead to low fertility by affecting the synthesis of proteins and hormones associated with the reproductive organs [56]. First, heat stress has been shown to exert a direct effect on follicular development in dairy cows [57]. Wolfenson et al [58] showed that the number of dominant ovarian follicles in a heat-stressed group of dairy cows began to decline earlier than in the cooled group during the first follicular wave. The number of large follicles (diameter ≥10 mm) was significantly higher in the heat-stressed group than in the cooled group (p<0.01), resulting in 53% more large follicles in the heat-stressed group during the first follicular wave. Moreover, during the second follicular wave, the numbers of small and medium follicles were higher in the cooled group than in the heat-stressed group, and the number of medium follicles was significantly lower than that in the first follicular wave (p<0.02). In addition, the dominant ovarian follicles appeared earlier and were fewer in number than in the cooled group, indicating that heat stress can affect the development of follicles in dairy cows [58]. However, Wilson et al [59] showed that the dominant ovarian follicles in the heat-stressed group were equal in size to or smaller than those of the thermoneutral group during the second follicular wave, and that the percentage of cows having two follicular waves in the heat-stressed group and thermoneutral group was 18% and 91%, respectively. The serum concentrations of progesterone were similar for heat-stressed and thermonutral group until d 16. After d 16, serum progesterone content was significantly higher in the heat-stressed group than that in the thermoneutral group (p< 0.01) [59]. In addition, the day of functional luteolysis (defined as serum progesterone <1 ng/mL) was delayed in four out of 11 heat-stressed cows (day 29.1±2.4) in comparison to that of the thermoneutral cows (day 20.4± 2.4; p<0.05) [59]. Collectively, these studies all indicate that follicular development is adversely affected by heat stress.
In addition to affecting follicular development, heat stress also affects estrus [60]. A study on non-lactating Japanese Black cows indicated that their estrus cycle was significantly longer in summer than in winter (p<0.05), and that walking activity was also significantly lower in summer than in winter (p<0.001). However, there was no significant difference in the duration of estrous between the two seasons [41]. In addition, the estrus and conception rates decreased when THI exceeded 72 [22], indicating that heat stress increased feeding costs by reducing reproduction efficiency.

Effects on pregnancy
Heat stress also has a serious impact on pregnancy in dairy cows [61]. A statistical analysis of pregnancies in 20,606 cows from seven farms showed that the pregnancy rates were 39.4% (THI<72.0), 38.5% (72.0 to 73.9), 36.9% (74.0 to 75.9), 32.5% (76.0 to 77.9), and 31.6% (>78.0) [15]. This indicates that the pregnancy rate of dairy cows decreases in a THI-dependent way, when THI was great than 72.0, with a decrease in pregnancy rate of 1.03% per unit increase in THI [15]. The effects of heat stress on pregnancy rate were explored in another study involving 1,199 crossbred dairy cows for 30 days before and after insemination. In this study, according to whether the ambient THI values less than 72 or not, the cows were divided into four groups: thermoneutral-thermoneutral (TN-TN), thermoneutral-heat stress (TN-HS), heat stressthermoneutral (HS-TN), and heat stress-heat stress (HS-HS). The results showed that the pregnancy rate in the HS-HS group (20.5%) was significantly lower than those of the TN-TN (32.6%), TN-HS (30.3%), and HS-TN (31.8%) groups (p<0.05), while there were no significant differences between the latter three groups (p>0.05) [62]. These studies show that the pregnancy rate of dairy cows was decreased in response to heat stress, and that the influence of heat stress was enhanced by increased duration and intensity. However, another study showed that pregnancy rate was significantly higher after embryo transfer than that after artificial insemination (day 21, 47.6% vs 18.0%; days 45 to 60, 29.2% vs 13.5%; p<0.001) [63]. Related studies reported that heat stress negatively affects the development of bovine oviduct [64] and oocyte quality [65]. Therefore, the decline of pregnancy rate after artificial insemination may be due to the decreases in oocyte quality and oviductal function in response to heat stress.

CONCLUSION
The body temperature and feed intake of dairy cows are affected by rising ambient temperature and humidity, which in turn affect milk production and reproduction. Although THI integrates ambient temperature and humidity information to assess heat stress, there are still many shortcomings in the research performed to date. First, the precision of THI monitoring was not sufficient. Many studies only used seasonal averages, where only a few working days were monitored. Moreover, just a few time points (e.g., morning, noon, and night) were used when monitoring working days. As a result, the results of monitoring were intermittent, did not record circadian changes in THI, and could not timely and precisely reflect the heat stress status of the cows. Second, it is not adequate for all regions and farms to determine the state of heat stress according to whether THI exceeds 68 or 72. Instead, specific problems should be analyzed more concretely. Third, heat stress is the physiological reaction of a given cow to its thermal environment, and is not only affected by THI, but also varies between breeds, individuals, farms, and management practices. Thus, inferring heat stress using only THI is not sufficiently analytical. The magnitude of heat stress should be more accurately reflected using a comprehensive assessment of environmental index and physiological parameter. With the rapid development of mechanical and electrical sensing technology, automatic monitoring equipment for the body temperatures and activities of cows has been gradually improved [29,66]. Moving forward, these techniques will be important in the scientific evaluation of heat stress in cows by combining THI, body temperature, and other indices (e.g., activity) to make the management of dairy cows more refined, and even individualized, to effectively forecast heat stress.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2019.jsonl.gz.gz

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A New, Rapid and Efficient HPLC Method to Assay Resveratrol in Food Supplements

Resveratrol has numerous benefits for human health: antioxidant, anti-inflammatory and cytoprotective [1]. Resveratrol is very abundant in beverages such as red wine, fruits such as grape berry and particularly in dried roots of the plant Polygonum cuspidatum. Also, resveratrol can be found in other natural foods such as bilberries, blueberries, peanuts, pistachios and in valuable by-products like grape skin and grape pomace [2, 3]. Resveratrol has multiple benefits. Among these are: -facilitation of increased oxidation of fatty acids, mitochondrial biogenesis, mitochondrial respiration, and gluconeogenesis. -inhibits expression of HIF-1α and VEGF, explaining its effective action against cancer. -triggers apoptosis of activated T cells and suppresses proinflammatory molecules, and thus is of benefit in autoimmune diseases. -alters gut microbiota and influences stem cell proliferation and differentiation [4]. According to its chemical structure, resveratrol (3,42 ,5trihydroxystilbene, Fig.1.) is a polyphenolic compound. It is similar to diethylstilbestrol, a synthetic estrogen [4, 5]. Resveratrol has been the subject of intense interest in recent years due to a range of unique anti-aging properties.These include cardiovascular benefits via increased nitric oxide production, down-regulation of vasoactive peptides, lowered levels of oxidized low-density lipoprotein, and cyclooxygenase inhibition; possible benefits on Alzheimer disease by breakdown of betaamyloid and direct effects on neural tissues; phytohormonal actions; anticancer properties [6].

Resveratrol has numerous benefits for human health: antioxidant, anti-inflammatory and cytoprotective [1]. Resveratrol is very abundant in beverages such as red wine, fruits such as grape berry and particularly in dried roots of the plant Polygonum cuspidatum. Also, resveratrol can be found in other natural foods such as bilberries, blueberries, peanuts, pistachios and in valuable by-products like grape skin and grape pomace [2,3].
Resveratrol has multiple benefits. Among these are: -facilitation of increased oxidation of fatty acids, mitochondrial biogenesis, mitochondrial respiration, and gluconeogenesis.
-inhibits expression of HIF-1α and VEGF, explaining its effective action against cancer.
-triggers apoptosis of activated T cells and suppresses proinflammatory molecules, and thus is of benefit in autoimmune diseases.
Resveratrol has been the subject of intense interest in recent years due to a range of unique anti-aging properties.These include cardiovascular benefits via increased nitric oxide production, down-regulation of vasoactive peptides, lowered levels of oxidized low-density lipoprotein, and cyclooxygenase inhibition; possible benefits on Alzheimer disease by breakdown of betaamyloid and direct effects on neural tissues; phytohormonal actions; anticancer properties [6]. and on the other hand, due to its antioxidant effects, in the manufacturing of cosmetic products, industry which increasingly focuses on the development of active cosmetics [9,10].
In order to supply this molecule in high concentrations, the food supplements market offers various products. Also, innovative pharmaceutical forms containing resveratrol are being tested [11]. In most supplements, resveratrol (and other antioxidants) is combined with various plant extracts containing polyphenols in order to enhance healthpromoting effects [12], including antioxidative properties [13]. Also selling supplements can reduce costs and optimize the activity of pharmaceutical units [14]. The presence of the actual content stated by the producers is crucial for the therapeuticbenefits and an indicator of product quality [15].

Experimental part Materials, reagents and standards
Three food supplements and a standardized extract of Polygonium Cuspidatum were analyzed. Food supplements have been purchased from pharmacies and otherspecialized stores.
The supplements analyzed were coded as follows: -PB001 -is presented as hard gelatin capsules containing 50 mg resveratrol/cps. Antioxidant preservatives are able to inhibit reactions promoted by oxygen, thus avoiding the oxidation and rancidity of commonly used fats, oils, waxes, surfactants, perfumes, etc. [7,8]. Because of this, resveratrol can be used on one hand in manufacturing of food supplements -PB002 -is presented as hard gelatin capsules containing 50 mg resveratrol/cps.
-PB004 -is a Polygonium Cuspidatum extract with a minimum resveratrol content of 50%.
Resveratrol was extracted with analytical purity methanol from VWR.

Sample and standard preparation
From each product 20 capsules were taken and the average mass was performed. The sample solution was prepared using 50 mg of powder content from 20 capsules in 50 mL of methanol and sonicated for 15 min. From this solution 1 mLwas diluted ten times using the same solvent.
The standard solution was prepared in the same manner. The standard and sample solutions were previously filtered using 0.45µm filters before injection. The standard was injected six times and each sample was prepared in triplicate and injected once.

Equipment and chromatographic conditions
The identification and quantification of resveratrol was performed using a HPLC method developedon basis of the European Pharmacopoeia. The separation of resveratrol was performed by HPLC using an Agilent 1200 HPLC system consisting of a quaternary pump, degassing device, autosampler, diode array detector (PDA), and Agilent ChemStation software. A Zorbax SB-C18, 250x4.6 mm, 100 Å (S/No86996-11, B/No:5701-029) column was used. The mobile phase used for isocratic elution consisted of distilled water (MilliQ purity, 0.5µS/cm at 20 o C)-methanol HPLC grade (60: 40) (v/v). The acquisition parameters used were: flow rate 1.5 mL/min, injection volume 10 µL, column temperature 25 o C and detection was performed at 303 nm.

Results and discussions
Identification was made by comparison of retention times and by spectral data of PDA with those obtained for the standard as is shown in figure 2 and figure 3. The retention time of resveratrol was approximatively 8.8 min. The system suitability parameters of resveratrol peak are presented in Table 1.
Quantification was performed on the basis of the external standard method against average of 6 peak areas of standard solution chromatogram. The results are shown in figure 4.  The content of resveratrol in the analyzed samples ranged between 91 and 119 % from the claimed content on the product label. According to this data, samples PB001, PB002 and PB004 contained more resveratrol than was indicated on the label and the sample PB003 contained less resveratrol than claimed. The precision of resveratrol was determined as the variation coefficient (RSD %) for six successive injections of the standard solution which was 0.96.

Conclusions
Resveratrol was successfully separated using the used and described method. The method provided a fast and cost efficient way for assaying resveratrol in food supplements.


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Introduction performance of four excellent and new citrus varieties in Jintang, Sichuan

In order to further enrich the structure of citrus varieties in the Jintang area of Sichuan, and to solve the problems of old and single varieties and relatively concentrated listing periods. In this experiment, four excellent and new citrus varieties, ‘Kanpei’, ‘Niahinoka’, ‘Or’ and ‘Qingfeng’ is introduced, and the local cultivar ‘Shiranui’ is used as a control. The traits and fruit quality indicators are investigated, measured and analyzed. The results show: The ‘Kanpei’ and ‘Or’ trees are strong, the ‘Niahinoka’, ‘Qingfeng’ and ‘Shiranui’ are medium, ‘Or’ has the highest ratio of sugar to acid, ‘Niahinoka’ has the highest vitamin C and soluble solids. Compared with the control, the ratio of sugar to acid and the per mu yield in the ‘Niahinoka’ is 27.78% and 13.62% higher than ‘Shiranui’, ‘Qingfeng’ performance is poor. According to a comprehensive analysis, ‘Kanpei’, ‘Niahinoka’ and ‘Or’ are suitable for introduction, and ‘Qingfeng’ is not suitable for introduction.

Introduction
Jintang is affiliated to Chengdu City, Sichuan Province, and is located in the northeastern part of the Chengdu Plain. It is a key development county within the "Chengdu Plain Economic Circle" and a "characteristic industrial development zone" in Chengdu. With its unique location, Jintang' climate that meets the needs of most citrus, it's citrus has also been awarded the title of famous product for many times. In recent years, there are problems such as aging, singleness and weak brand awareness in citrus cultivation in Jintang area and most citrus products can only be sold in the low-end market, which further leads to a gradual decrease in the share of citrus sales, which restricts the development of local citrus industry. In order to improve this situation, adapting the modern high-grade and diversified consumption characteristics of citrus [1] , and improving the economic benefits of local citrus. In this experiment, four kinds of excellent and new citrus varieties are introduced in Jintang. Through the analysis of their biological characteristics and fruit quality, we intend to select suitable varieties of orange for planting in jintang area with excellent performance for promotion. It has a subtropical monsoon climate with an average elevation of 468m, an average annual temperature of 16.4℃, an average temperature of 6.0℃ in January, and an average temperature of 26.8℃ in July. The soil type is mainly red-purple soil; the annual average rainfall is 845.4mm, the annual frost-free period is about 296d, the average annual sunshine hours is 1210.4h, the effective accumulated temperature (≥10℃) is 5450℃, and the annual average relative humidity is 84%.

Test materials and design.
Test materials: the experiment is conducted from January 2017 to March 2018. In January 2017, the three-year-old citrus 'Qingfeng', 'Or', 'Kanpei', 'Niahinoka' and the local main variety 'Shiranui', which are robust and have the same level of cultivation management, are selected as test materials. Test design: the experiment used 'Shiranui' as the control, 'Qingfeng', 'Or', 'Kanpei', 'Niahinoka' as the test subjects, and each variety selected 9 plants with the same age, tree potential and tree shape and no pests and diseases. The plant spacing is 3m×4m, and the 3 plants are one plot, and 3 replicates is set.

Measurement of Test Indicators.
Observation of phenological period and tree status: The phenological period mainly observes the germination stage, the bud stage, the flowering stage, the fruit color change stage and the fruit ripening stage. Each phenological period is observed 3-5 times, the method is based on Lifeng Liang's standard [2] .

Analysis of Test Indicators.
The data is collated using Excel 2013, and the results are statistically analyzed using SPSS 20.0.

Phenophase and tree status analysis
It can be seen from Table 1 that in the Jintang area, 'Qingfeng', 'Kanpei' and 'Niahinoka' are characterized by medium maturity, and 'Or' and 'Shiranuhi' are late maturity. In the germination, bud and flowering period, 'Niahinoka' is the earliest, and 'Shiranuhi' is the latest. In the color change period and maturity period, 'Kanpei' is the earliest, and 'Shiranuhi' is the latest. It can be seen that the phenological periods of the tested varieties are not nearly overlapping, and the maturity period is staggered, which is conducive to the market supply of citrus in Jintang area.  Table 2 shows that: The tree height and crown diameter of 'Kanpei' are the largest, significantly larger than 'Shiranuhi', 11.74% and 23.60% higher respectively; Compared with the control 'Shiranuhi', the tree height and crown diameter of 'Niahinoka' and 'Or' are significantly higher than 'Shiranuhi' by 5.68%, 18.03% and 6.47%, 17.58% respectively; The tree height of 'Qingfeng' is significantly lower than 'Shiranuhi', 9.57% lower, and the difference of crown diameter is not significant; Taken together, 'Qingfeng', 'Niahinoka' and 'Shiranuhi' have medium tree potential, while 'Or' and 'Kanpei' have strong tree potential.   Table 3 shows that: The number of vesicles in 'Niahinoka' is the largest, with an average of 12, while that in 'Shiranuhi' is 11. There is no significant difference between the two; 'Qingfeng' is significantly less than 'Shiranuhi', 18.18%. There is no significant difference between 'Or' and 'Kanpei' and 'Shiranuhi'. The number of seeds of 'Shiranuhi' is higher than that of 'Niahinoka', 'Kanpei', 'Or' and 'Qingfeng', which is 5.80%, 11.82%, 29.45% and 58.91% higher than that of 'Niahinoka', 'Kanpei', 'Or' and 'Qingfeng', respectively. There are significant differences in the number of petals and seeds among different varieties, which can meet the different needs of consumers and processing industry.  Table 4 shows that: There are significant differences in vertical and horizontal diameter and fruit shape index among the five varieties. The fruit shape index of 'Shiranuhi' is the largest, with an average value of 1.268, which is significantly higher than that of the other four varieties, 15.87%, 26.19%, 28.57% and 32.54% higher than 'Qingfeng', 'Or', 'Niahinoka' and 'Kanpei', respectively. The fruit shape of 'Shiranuhi' is oblong or pear-shaped, 'Qingfeng' is elliptic, 'Kanpei' is near circular, 'Or' and 'Niahinoka' are flat circular.  Table 5 shows that: The V C content of 'Niahinoka' is the highest, 43.526+0.002 mg · 100m L -1 , which is significantly higher than that of 'Shiranuhi' (18.93%). Compared with the control group, the V C content of 'Or', 'Kanpei' and 'Qingfeng' is significantly higher than it, 11.95%, 9.28% and 4.36% higher respectively. The soluble solid content of 'Niahinoka' is the highest, 12.835±0.145%, which is significantly higher than that of 'Shiranuhi', 10.37% higher. The soluble solids of 'Or' and 'Qingfeng' are significantly lower than those of 'Shiranuhi', 9.57% and 15.04% respectively. There is no significant difference between 'Kanpei' and 'Shiranuhi'. The sugar-acid ratio of 'Or' is the highest, 11.687±0.774, which is significantly higher than that of 'Shiranuhi', 51.03% higher. Compared with the control group, 'Niahinoka' is significantly higher than 'Shiranuhi' by 27.78%, and 'Qingfeng' is  Table 6 shows that: The average fruit weight of the control 'Shiranuhi' is the largest, 332.332+2.603g, which is significantly higher than that of 'Niahinoka', 'Or', 'Kanpei' and 'Qingfeng', which are 18.15%, 24.58%, 39.52% and 51.75% respectively. The highest yield per mu is 'Niahinoka', with an average yield of 1144.008+20.185 kg, which is significantly higher than that of 'Or' (13.62%); 'Or' is significantly higher than that of 'Shiranuhi' (8.71%); 'Kanpei' and 'Qingfeng' are not significantly different from that of 'Shiranuhi'.

Comprehensive evaluation and analysis of fruit quality
As can be seen from

Conclusion and discussion
(1) Among the four varieties introduced in Jintang, 'Or' showed late maturity. Compared with 'Shiranuhi', the germination period of 'Or' is earlier than one month, and the fruit ripening period is February of the next year. It also has the characteristics of vigorous tree vigor, early fruiting and high yield, excellent flavor quality, high sugar, residue and aroma. Thus, it can be seen that 'Or' has excellent comprehensive properties and can be marketed at the same time as 'Shiranuhi'. It can meet people's needs for different flavors and is suitable for planting in Jintang area. (2) The ripening period of 'Kanpei' fruit is earlier, in mid-December. Compared with 'Shiranuhi', 'Kanpei' have strong potential, high yield, sweet and sour taste and high nutritional value. It can make up for the shortage of market supply in the off-season without 'Shiranuhi' by two months earlier.
(3) 'Qingfeng' is medium ripening, the germination stage is mid-February, and the fruit ripening stage is late December. Tree vigor is moderate, susceptible to brown spot. Compared with the control, the content of V C is significantly higher than that of 'Shiranuhi', which is 4.36%, but the content of soluble solids is 15.04%, the ratio of sugar to acid is 27.45%, the average weight of single fruit is 51.76%, and the yield per mu is 8.02%. The comprehensive performance is poor, probably due to the inadaptability to the climate in Jintang area.
(4) The earliest germination period of 'Niahinoka' is in mid-January and the fruit maturity is in late December. Among the tested varieties, 'Niahinoka' has the highest V C content and soluble solids content, which are significantly higher than 'Shiranuhi', 18.93% and 18.93% respectively. The sugaracid ratio and yield per mu are significantly higher than 'Shiranuhi', 27.78% and 13.62% respectively. The market date is about 1.5 months ahead of schedule, the sweetness is high and the comprehensive adaptability is strong. It is suggested that introduction be made.
In the study of citrus varieties, high sugar, low acid and high V C content are ideal fruit quality traits [8] . Through this experiment, 'Or', 'Kanpei' and 'Niahinoka' are more suitable for the environmental conditions of Jintang area, and their comprehensive cultivation performance is excellent, and they have the characteristics of making up for the current off-season market of Citrus in Jintang area and regulating the concentration of citrus fruit on the market. It is suggested to introduce them. Because of the poor comprehensive performance of 'Qingfeng', it is not recommended to introduce them.


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Mobility Behavior and Environmental Implications of Trace Elements Associated With Suburban Soils from the Steel Industry

Our work was aimed on the study of mobility of steel trace elements (STEs), using single-step extraction procedure (Na2EDTA) and ecotoxicity in suburban soils from the Košice, Slovakia. The total element contents were determined by X-ray fluorescence (XRF) analysis, Zn, Pb, Cd, Ni, and As in extracts and elements contents in earthworms tissues by the atomic absorption spectrometry (AAS) after 7 days bioassays (chronic tests of mortality). An ecotoxicological study was performed with earthworms (Dendrobaena veneta) for the screening evaluation of element mobility of urban grass and agricultural soils samples. Elements concentrations (Zn, Pb, Cd, Ni and As) in the suburban soils of Košice, indicating the significant contamination with Cd, and As were measured at sites located south of the ironworks. From the results of Pearson correlation analysis of yields, it can be seen that Pb - Zn, Cd, As; Cd - Zn, As; and As - Zn have obvious correlation, and the correlation between chemical parameters and nickel is not relevant.

Introduction
Steel trace elements constitute a significant part of soil contaminants, which, at some concentrations, may be toxic to the urban ecosystem. Total concentration of elements might could be suitable as useful indicators for assessment of soil contamination. However, the total concentration of elements is not a suitable indicator of mobility, bioavailability, or toxicity. Element characteristics are mainly depending on the different chemical forms of binding between trace elements and solid phases of the samples [1]. The study of investigate the trace element availability and mobility in soils, a number of extraction methods have been extensively used [2,3,4]. The application of the single-step extraction procedure (with Na2EDTA) is very interesting from a time-saving point of view, and as the first screening information about the most mobile portions (trace elements) in the soil samples. The singlestep extractions into the strong chelating agents (e.g. Na2EDTA) are suitable for the evaluation of mobile and potentially mobile element contents.
Košice has about 239 000 inhabitants and is located in the Košice basin, in the river valley Hornád in the eastern part of Slovakia (Europe). The technology of steel production has a significant impact on the urban environment. Annual raw steel production capability is 4.5 million metric tons. Also, the agricultural and industrial activities contribute to contamination of the soil in this region [5,6].
Many authors have shown that bioassays provide a general indication of metal toxicity in soils [7][8]. Earthworms play important role in soil ecotoxicological risk assessment [9] as the metal content in earthworm tissues is partly dependent on the metal content of soils. The activity of earthworms can be affected by exposure to contaminants. These tests can be successfully used to assess the trace element mobility on soil pollution, and to understand the ecological risk, transport processes of elements and the correlations with the soil characteristics. The aim of this paper was the investigated of element mobility in soil collected from around the plant U. S. Steel Košice by using a single-step extraction; to used bioassays of earthworms (Dendrobaena veneta) to evaluate toxicity of soil, and to analyze a possible relationship between observed toxicity and mobility. The obtained information may provide better understanding of environmental risks of steel trace elements in urban soils.

Material 2.1.1. Study area
Urban grass soils (UGS) and agricultural soils (AS) were collected in the Košice suburban, in surrounding of U. S. Steel Košice, which is located about 10km south-westwards from the centre of Košice. Soils were taken in 2016 -2017. In these samples the pH was measured. Then the representative samples marked as Ke-01-12 (Table 1)

Bioassays with Dendrobaena veneta
The earthworms were purchased from a local supplier. Three replicates were performed for each test (100 g of dry weight sediment) with 10 earthworms. The distilled water was added to each box for the purpose of obtaining 30% moisture of sediment. After that, the boxes with soils were kept for 7 days at room temperature in dark room. The similar results with earthworms bioassay we obtained in our previous works [10,11].

Methods 2.2.1. Analysis of single-step extraction
A single-step extraction with 0.05 mol L-1 Na2EDTA [10] was used for the soils from the studied area. The single-step extractions into the strong chelating agents (e.g. EDTA) are suitable for the evaluation of mobile and potentially mobile element contents [12]. The solid soil/solution extraction ratio (weight/volume ratio) of 1:150 (0.05 mol L-1 Na2EDTA (75 mL) was added to the soil sample (0.5 g) in a 100 mL polyethylene vessel). Tightly closed vessels were shaken for 6 h. The extract was separated by filtration and stored in a polyethylene vessel. This solution was used for the determination of the extractable portions of Zn, Pb, Cd, Ni and As in the soil samples. The total concentrations of the steel trace elements (Zn, Pb, Cd, Ni and As) and the element determinations of soil residues in soil samples were measured using SPECTRO XEPOS X-ray fluorescence spectrometer (XEPOS 3). The concentrations of trace metals after single-step extraction (Na2EDTA) and after 7 days bioassay earthworms in the studied soils were measured by XRF method.

Analysis of bioassays and soils
The experiments were carried out in the OECD Guidelines 317 for the testing of chemicals relating to environmental fate, chronic tests. The earthworms tissue were lyophilized (at -50°C and 50Pa), then wet acid digestion (HNO3/HF/H3BO3 (5:2:20)) to total destruction of samples was used to prepare samples for measurement by the atomic absorption spectrometry (AAS-Variant, Australia). The physico-chemical parameters of soils were determined (pH value in H2O and KCL solutions (ISO/DIS 10390)), organic matter (OM) (STN EN 12879). The used control reference soil (CRM -TM52, MicroBioTests, Belgium) contained: 85% (w/w) quartz, 10% (w/w) kaolin, 5% (w/w) peat and calcium carbonate (pH of 6±0.5), (OECD soil).

Statistical analysis
The certified river sediment LGC6187 was used to validation of data. All data analysed using SPSS v.9.0 statistical software. The statistical dependence between chemical parameters (and elements yield of single-step extraction (Na2EDTA)) and total trace element concentrations were evaluated by Pearson matrix correlation. Principal correlation analysis was used comparison of the potential relationships among the data. As control, earthworms feeded in non-contaminated soil were used CRM.

Results and discussions
The recent years, (STEs) pollution has become a serious ecological problem. Industrial activities related to ore processing cause the main source of trace element pollution. The pollutants entering the soil gradually accumulate and form a layer (i.e. the industrialized soils) [12]. The soil types examined here were of silty-clay texture from various areas to keep representativeness and their quality was established with reference to law (220/2004, No.2, Slovak Republic, about the quality of the soil).
The steel trace element total concentration of the studied soils along with their TS, pH and OM are listed in Table 1. All soils have weakly alkaline pH value (5.67-7.95) and low OM range (4.1-10.3 %). The highest concentrations of studied elements were measured at sites located south of the ironworks in their vicinity. At the main gate of the U.S. Steel-plant (locality Ke-04) high concentrations Zn, Pb, Cd and As (exceed of 5% for Zn; 3% for Pb; 21% for Cd; 3% for As) were detected for UGV soils.
The highest values of elements from the areas were determined in AS-soils: U.S.Steel-slag heap (Ke-09) for Cd exceeds of 9% and Pereš-south (Ke-12) exceeds of 6.9% (Table 1). Effect of elements on mortality D. veneta after 7-days of exposure at the end of the tests showed that the earthworms' mortality was influenced by industrialized soils after exposure (average of mortality for the UGV soils range 1-4%, and for the AS soils range 1-3%). Mortality was not statistically significantly influenced. On the other hand, earthworms in some cases caused decrease of metals concentration in the soils, between 2-40% for Zn, between 1-17% for Pb, between 1-5% for Cd, between 5-30% for Ni, and between 2-20% for As for all studied soils (Table 2.). The concentrations of trace metals after single-step extraction (Na2EDTA), after 7 days bioassay earthworms and the elements value of residual in the studied soils are shown in Table 3. In this study, the single-step extraction was applied to define toxicity and mobility (bioavailability) of five soil trace elements (Zn, Pb, Cd, Ni, and As) in surrounding of U. S. Steel Košice suburban soils. The comparison of the elements after extraction and after bioassays with residual showed the lower results of elements after extraction of 7-days bioassays (Table 3.). The yields of the single-step extraction, after 7-days bioassay are between 5-73% for Zn, between 2.1-47% for Pb, between 1-34% for Cd, between 3-17% for Ni, and between 6.8-67% for As in studied industrialized soils, see Figure 1.-3. The maximum yields are ranged 67-73% for Zn and As. The potentially mobility of the elements in the soils was in followed sequence: Zn >As >Pb >Cd >Ni. Relative high mobility of Zn, As, moderate mobility of Pb, Cd, and minimal mobility of Ni were observed in our experimental and local geological conditions. It was confirmed that relative content of Ni and Cd are not present in mobile form which is bio-accessible to plants, but elements are bound in crystal lattices and mineral matrices.  Table 3. Relative bioassays, distribution of elements obtained by the single-step extraction, comparison of elements after extraction and after 7-days bioassays of soils extraction, and elements value of residual.    The correlation between elements can reflect whether they are from the same source, and the highly correlated elements may have the same source [5]. From the results of Pearson correlation analysis (Table 4), it can be seen that Cd and Zn, Pb, As have highest obvious correlation (r = 0.89 to 0.90). The results show that Zn, Pb, Cd and OM have obvious correlation (Tables 4.). The results show (r = 0.78 to 0.79 for Pb and Cd, As) the relatively highest positive correlation between yield (CMY) and risk steel trace (  Table 4-5. According to the correlation coefficients, the trace element contamination level in the studied soils was followed: As> Cd> Pb> Zn> Ni. The elements show strong correlation coefficients and it indicates the degree of urban contamination (i.e. As, Cd, Zn and Pb). It shows on the anthropogenic pollution of industrialized soils in surrounding of U. S. Steel Košice.

Conclusions
In this study, the single-step extraction was applied to define toxicity and mobility (bioavailability) of five soil trace elements (Zn, Pb, Cd, Ni, and As) in surrounding of U. S. Steel Košice suburban soils. The present investigation reported that the soil of surrounding of U. S. Steel Košice is highly contaminated by trace elements Cd and As. Relative high mobility of Zn, As, moderate mobility of Pb, Cd, and minimal mobility of Ni were observed. The better understand the mobility and environmental risk of these elements, it would be useful to determine their geochemical speciation in soils.


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Study on the Optimization of Mercury Ion (II) Adsorption with Activated Carbon from a Biomass Combination of Palm Bunches and Rice Husk

– This research deals with the adsorption of mercury ions [Hg (II)] using a combination of natural biomass between oil palm bunches and rice husks (PB-RH). After drying at 40 o C, PB-RH biomass was carbonized by using a tube furnace for 2 hours at 500 °C and followed chemical activation with citric acid. Activated carbon formed was contacted with an aqueous solution containing Hg (II) ion and stirred at a constant rate of 150 rpm. The concentration of Hg (II) ions in the aqueous phase were analyzed with Atomic Absorption Spectrophotometer (AAS) and the Central Composite Design (CCD) method with Design-Expert software version 6.0.8 was conducted to calculate the optimization of adsorption capacity for mercury ion. The Hg ion adsorption capacity was estimated to observe the performance of the PB-RH biomass combination on the adsorption of Hg (II) ions with some variables, such as contact time, biomass weight, and activator concentration. Results showed that all variable response, contact time, biomass weight, and activator concentration influenced the mercury (II) ion adsorption capacity. The optimum condition of Hg (II) ion adsorption occurred at 30 g of activated PB-RH biomass with 0.6 mole/L concentration of activator during 99.88 minutes with 99.42 mg/L initial concentration of Hg (II). The adsorption capacity occurred at 10.1 mg/g as the optimum condition for the adsorption of Hg (II) ions by PB-RH. These results indicate that optimal conditions were achieved at 30 g of activated PB-RH biomass with 0.6 mole/L concentration of activator during 99.88 minutes with 99.42 mg/L initial concentration of Hg (II).

Introduction
Mercury (Hg) is a form of a natural heavy metal element that often pollutes the environment. Most of the mercury comes from nature in the form of other elements combined. In other words, not much mercury is found in a way separate from other elements. In the environment, mercury components are widely distributed in corals, soil, air, water, and living organisms through complex physical, chemical, and biological processes. This element is toxic to one in a billion total parts and is one of the most toxic elements. Nowadays, Mercury poisoning is now one of the most severe health hazards in the world (Sharma, Sharma, & Arya, 2015).
An extensive study has been conducted by many researchers to remove and to minimize the effects of mercury poisoning because it is responsible indirectly for influencing the environment and often causes health hazards. Several researchers continue to develop new alternatives with low-cost mercury removal techniques at a laboratory scale that might be able to be designed for commercial applications, such as the use of biomass as an adsorbent (Salam, Reiad, & ElShafei, 2011). Adsorption is the best conventional method and universal for the processing of organic and inorganic waste (Sumanjit, Seema, Mahajan, & Gupta, 2015) also very easy in reconstruction, operation, and even maintenance (Gupta, Ali, Saleh, Nayak, & Agarwal, 2012) (Yu et al., 2015) with relatively low operating costs. Activated carbon is the most popular material for this adsorption; however, high costs limit their use on a large scale (El-Said, Badawy, & Garamon, 2012). The application of activated carbon in adsorption will increase the absorption capacity but requires a high cost in the process to prepare a surface area of activated carbon with high uptake and optimal adsorption capacity (Karaoǧlu, Doǧan, & Alkan, 2010).
Several alternatives using cheap raw materials intended that can lower the total cost required to obtain the optimal results are using natural materials, such as agricultural wastes and useless plants. Natural biomass materials, such as wastes from agriculture, that abundance may have the potential to be cheap absorbents. Because of their low costs, when the lifetime of these materials runs out, they may be removed with low-cost regeneration. The large quantities and availability of agricultural by-products make it a challenge as raw materials for natural adsorbents. Some agricultural wastes can be used as an adsorbent for mercury treatment, including Bambusa vulgaris var. striata. It has an adsorption capacity of 218.08-248.05 mg/g (González & Pliego-Cuervo, 2014;Mistar et al., 2019), rice husk (Aprianti, Sheilla, & Machdar, 2018), coconut shell 3.02 mg/g, and rice straw 0.11 mmole/g (Goel, Kadirvelu, & Rajagopal, 2004b). Furthermore, water hyacinth (Zhang et al., 2015) and bamboo (Mistar et al., 2019;Tan, Qiu, Zeng, Liu, & Xiang, 2011) as useless plants, have a potential to be adsorbent to reduce heavy metals. In this study, we modified palm bunches (PB) and rice husk (RH) as an adsorbent to adsorb Hg (II) ion from the water phase.We combined PB-RH biomass as an adsorbent. The purpose of this study was to find the optimal conditions of the Hg (II) adsorption process with activated carbon from a combination of PB-RH biomass using a surface response methodology (RSM).

Adsorbent Preparation
The preparation method was carried out based on our previous work (Suhendrayatna, Zaki, Delima Habdani Harahap, & Verantika, 2018;Zaki, Hadi, & Adha, 2018), consist of adsorbent preparation, activation, and adsorption. PB and RH were carbonized at 500°C in a furnace for 2 hours. The charcoal produced was cooled and follow with the activation process using citric acid with various concentration of 0.2M; 0.4M; 0.6M; and 0.8M, respectively. After this preparation, activated carbon was applied in the adsorption treatment as an adsorbent.

Mercury ion Adsorption and Analysis
A batch experiment was designed in this research. An aqueous solution (100 ml) containing Hg(II) ion was contacted with a suitable weight of activated carbon PB-RH in a shaker at 150 rpm for 100 min at room temperature. Neutral pH was maintained by dripping an appropriate volume of the base (sodium hydroxide) or acid (hydrochloric acid) solution under measuring with a digital pH meter. The adsorbent was removed from the aqueous phase by centrifugation, and Hg ion concentration in the supernatant was analyzed by atomic absorption spectrophotometer. Design-Expert software version 6.0.8 has been conducted to calculate Optimum adsorption of Hg (II) ion based on the Central Composite Design (CCD) method. This method was occurred by applying four factors and three levels to study the effects of contact time, biomass weight, citric acid concentration, and initial concentration on the Hg (II) adsorption capacity.

Statistical analysis
RSM is a combination of statistical and mathematical techniques that are used to model and analyze responses that are influenced by some independent variables or factors x to optimize responses. This methodology is more profitable than traditional single-parameter optimization because it saves time, raw Aceh Int. J. Sci. Technol., 8(3) : 161-168 December. 2019 10.13170/aijst.8.3.15160 materials, and space. In this study, there were a total of 30 runs to optimize the four parameters in the current CCD method. Table 1 shows the experimental and prediction results of the Hg (II) ion adsorption capacity based on factorial design. Maximum Hg (II) ion adsorption capacity (7.7 mg/g) occurred at run 16 under the experimental condition of contact time 40 min, biomass weight 25 gram, citric acid concentration 0.2 mole/L, and initial concentration 80 mg/L. Furthermore, the lowest Hg (II) ion adsorption capacity was recorded 1.0 mg/g at the run of 29 under the experimental condition of contact time 60 min, 20-gram biomass weight, 0.4 mole/L citric acid concentration, and 40 mg/L initial concentration.
The statistical model summarizes the general description of the selection of the right model and matches the experimental data. CCD method describes the actual mathematical model applied to predict the Hg (II) ion adsorption capacity based on the suggested model. Based on experimental data being substituted in the quadratic model, the equation of mathematical model that can be applied to the Hg (II) adsorption capacity is as follows. Y = 3,59356 -0,14802 X1 -1,03973 X2 -0,072948 X3 + 0,014965 X4 + 1,35666E-003 X1 2 + 2,78541 X2 2 + 4,87860E-004 X3 2 + 7,66041E-004X4 2 + 0,021333 X1X2 + 9,47316E-005 X1X3 + 1,45083E-003 X1X4 + 0,016431 X2X3 -0,047792 X2X4 -5,13171E-005 X3X4 … (1) Equation model (equation 1) consists of each variable that influenced to increase the Hg (II) ion adsorption capacity. These four variables were biomass weight (X1), citric acid concentration (X2), contact time (X3), and initial concentration (X4). Regarding the most significant coefficient in equation (X1), biomass weight was the most influential variable among these four variables. Base on the equation 1, the actual values for the Hg (II) ion capacity adsorption were tabulated in Table 1 within the prediction column. The prediction value shows the relationship between the predicted values based on the equation model proposed by the response surface. In contrast, the actual amount was provided based on the experimental value. This condition determined the accuracy of the model obtained. The analysis results of variance and adequacy of the design model were tabulated in Table 2. ANOVA is a CCD factorial that useful for generating the interactions between variables of process and response variables . F-ratio, which can determine the effectiveness of a model, was calculated based on components in ANOVA. Table 2 shows that all variables have a P-value higher than 0.05 and only variable D values below 0.05. These results suggested that the variable D (concentration variable) has a significant influence on the adsorption capacity of Hg (II) ions, which has a probability value (probability > F) <0.05, and that value was less than 0.0001.

Optimization of Hg (II) Adsorption
The quadratic equation was a model obtained from the Design-Expert software version 6.0.8 to predict the adsorption capacity of Hg (II) in this study. The selection of the model was based on various variables, such as the value of R 2 . Based on the surface response analysis of this model, two types of graphs can be used to describe the adsorption capacity; they were contour plots and 3D plots.
This plot is applied to facilitate the description of the effects of variables on the response. The contour plot was a 2D plot, which was a cross-section of a 3D plot that was useful to illustrate the impact of interactions between factors on the response. On the other hand, the 3D plot shows the effect of two variables on the response where the variable was created in a fixed condition. The model equation suggested in this study for the analysis of Hg (II) ion adsorption was the quadratic equation model. This model can be analyzed through contour plots, and 3D plots and Figures 1 to 3 show the relationship between three independent variables to the response variable. The optimization process in this study was carried out by adding the amount of biomass weight, contact time, and citric acid concentration. Optimization condition for Hg (II) ion adsorption capacity by PB-RH resulting from the calculation of Design-Expert software version 6.0.8 was tabulated in Table 3. These results indicate that optimal conditions were achieved at 30 g of activated PB-RH biomass with 0.6 mole/L concentration of activator during 99.88 minutes with 99.42 mg/L initial concentration of Hg (II).

Discussions
Activated carbon has been widely known and used as an absorbent of pollutants in waste treatment processes. Activated carbon consists of porous solids containing 85-95% carbon produced from materials including carbon, which is dehydrated, carbonated, and activated physically and chemically to obtain a large surface and has a high affinity. Activated carbon has a surface area in the range of 600 to 2000 m 2 /g, which is related to the pore structure that causes has an absorbent property (Bhatnagar & Sillanpää, 2010;Bhatnagar, Vilar, Botelho, & Boaventura, 2010). Carbonation is a process of combining indoors without oxygen and other chemicals. At the same time, activation is a treatment of charcoal to enlarge pores by breaking hydrocarbon bonds or oxidizing surface molecules to changes in properties, both physical and chemical. Activated carbon has been found as a versatile adsorbent, which can reduce various types of pollutants such as metal ions, pesticides, detergents, phenols, and other chemicals (Sud, Mahajan, & Kaur, 2008). However, its application is sometimes limited due to higher costs. Therefore, researchers continue to look for low-cost adsorbents for wastewater treatment, where cost factors play a significant role (Al-Mulla, Jabbar, & AL-Hamadani, 2018). One of the raw materials derived from abundant and inexpensive agricultural materials is rice husk, which has high organic (carbon) content with low inorganic content and can be easily activated. This rice husk has been tested and succeeded in removing heavy metal ions such as arsenic in water (Khalid, Ahmad, Kiani, & Ahmed, 1999), Sb (III), Pb (II) (Khalid et al., 1999;Wang & Lin, 2008), Hg (II) (Khalid et al., 1999), and Cd (II) (Kumar & Bandyopadhyay, 2006).
This study uses natural rice husk combined with palm bunches, followed by citric acid activator to remove Hg (II). The use of activator will affect the ability of activated carbon. Other types of activators that have been studied include KOH, ZnCl2, H2SO4, (Yalçın & Sevinc, 2000), depending on the type of pollutant to be sorbed. The citric acid activator used has also been tested inactivation for the absorption of Mn (II) ions (Suhendrayatna et al., 2018). After experimenting with several variables (time, citric acid concentration, and biomass weight), rice husk combined with palm bunches can remove Hg (II) from the water phase with its adsorption capacity of 10.01 mg/g. The optimal conditions were achieved at 30 g of activated PB-RH biomass with 0.6 mole/L concentration of activator during 99.88 minutes with 99.42 mg/L initial concentration of Hg (II). The adsorption capacity was one of the main parameters in the adsorption system. It describes the performance of adsorbents used in the absorption process. A comparison of adsorption capacity values of various types of natural adsorbents is presented in Table 4. Activated Carbon from PB-RH resulted in this study has a relatively low adsorption capacity (10.01 mg/g) in contrast to other Activated Carbon based natural materials reported in the literature. The difference in adsorption capacity is due to each characteristic of natural ingredients having different elemental contents, such as preparation methods and types of functional groups

Conclusion
This research leads to the conclusion that the concentration variable has a significant influence on the adsorption capacity of Hg (II) ions from the aqueous phase. By using the quadratic regression model in the optimization analysis, the optimal conditions occurred at 30 g of activated PB-RH biomass with 0.6 mole/L concentration of activator during 99.88 minutes with 99.42 mg/L initial concentration of Hg (II). The adsorption capacity occurred at 10.1 mg/g as the optimum condition for the adsorption of Hg (II) ions by PB-RH.


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Embryogenesis Callus Induction of Carica pubescens Using Divine Smoke Particulates Containing Amino Acids

Carica pubescens is one of plants that can live in the highlands which has a potential as medicinal plant to treat many diseases. The plant propagation is still done conventionally. Embryogenic callus culture is the alternative. The success of callus culture depends on the role of auxin growth regulator (2,4-D) and Amino Acids. The aim of this study is to determine the effet of amino acids through Divine smoke particulation to the induction of embryogenesis callus carica pubescens. This research is an experimental study with Complete Randomized Design (CRD) method. It has 7 treatments consist of control and the Amino Acids (Threonine, Tryptophan, Serine, Histidine, Asparagine, and Methionine). The result of this study showed a significant effect of amino acids through Divine smoke particulation to the induction of embryogenesis callus Carica pubescens which is grown on ½ MS media + 3 mg/L of 2,4-D. Tryptophan is the fastest treatment to induce callus which is grown 24 days after planting (DAP). Serine influences to the percentage of callus area in explants is 98,8% and the fresh weight of callus 0,6733 gr. The observation of morphology and anatomy embryonic callus color is yellowish white, friable texture and has a large cell nucleus

Introduction
Indonesia is one of the country that has high Biodiversity. One of them is the richness of plant species. One of plants that has the benefits is Carica pubescens Lenne & K. Koch. For the people of Indonesia, some of us are not so familiar with this one fruit. Carica fruit is hard to find because Carica fruit only grows in areas high and has a cold temperature or in the mountains. Carica fruit which be found in Bromo, Cangar Batu East Java, and Dieng Plateu Central Java. Carica fruit has many functions as a food ingredient because it can be processed into many kinds, like jam, sweets, syrup, chips. his fruit is used as a non-alcoholic soft drink and made into jam. Young fruit is usually dried to be used as a powder material for making skin diseases or cosmetics. Wonosobo residents themselves are making a home industry that process Carica fruit and sell it. C. pubescens became one of the typical hand of the area, and also export abroad [1]. Besides that, C. pubescens has potential as medicinal plant, contain antioxidants, The seed extract of C. pubescens can be potentially as larvacide of Aedes aegypti mosquitoes [2]. The seed of C. pubescens can use as an extract for antibacterial to Enteropathogenic Escherichia coli (EPEC) that it causes diarrhea to male mice [3].
The multiplication of C. pubescens in Indonesia is still done conventionally. If it grown conventionally, the germination of C. pubescens seed needs a long time. It is difficult to germinate because of the type of seeds, genetic factor, and the condition of the environment. Besides, a This hormone plays a role as inhibitor to the germination of the seeds. So, if the cultivation of C. pubescens is not developed optimally, it is concerned that this germplasm will be increasingly rare and extinct. Therefore it is necessary to do some conservation efforts because it is not cultivated specifically. So, it needs a tissue culture plant. In vitro culture has been proven to produce seedling in large number, in a short time, pathogen free, and does not need a lot of mother plant. Embryogenic callus culture has the potential become a plant through organogenesis and embryogenesis [4]. The embryogenic cells show characteristics common to meristematic cells: high division rates, cells are isodiametric and small with a dense cytoplasm with several starch grains, large nucleus and prominent nucleolus, small vacuoles, thin cell walls, and a higher metabolic activity. The embryogenic callus morphology show characteristics callus color is yellowish white with a friable texture Growth regulator 2,4-Dichlorophenoxyacetic acid is a type of strong Auxin and can be broken down into the plant's body [5]. Increasing formation and growth callus can be done by giving Amino Acids. Amino Acids are protein constituent that has a function to plant as transporting other substances, coordinating the activities of organisms, responding cells to stimuli, movement, protection against diseases, and catalyzing chemical reactions selective [6].
Amino Acids can be obtained from Divine smoke processed from tobacco and other syntheses that contain nicotine and tar with Amino Acids additives. The Amino Acids that added into explant plants can be through Divine smoke particulation which are able to penetrate the cells of the desired plant organs, because Divine smoke particulation works in nano molecules [7]. Divine smoke containing complex nanostructures can supply electrons in millivolts. In nano size, there is an electric field force. Divine smoke can function to supply energy while capturing mercury free radicals. even divine smoke supplies energy more efficiently than nutrients. Divine smoke can also be used to improve the quality of plant growth [8].
Tryptophan plays a role in protein synthesis that make the auxin higher. Methionine is one of an amino acids that contains sulfur, the plants need sulfur to form disulfides bond between protein chain, help to protein synthesis and growth plants [8] Therefore, The aim of this study is to determine the effect of amino acids through Divine smoke particulation to the induction of embryogenesis callus C. pubescens

Materials And Methods
This research is an experimental study with Complete Randomized Design (CRD). This study has 7 treatments consist of control and 6 Amino Acids (Threonine, Tryptophan, Serine, Histidine, Asparagine, and Methionine) through Divine smoke particulation with 3 replications, in order to obtain 21 experimental units. The study was done in April until August 2018. The research was done at Tissue Culture Laboratorium and Plant Physiology, Laboratorium Biology Department, Science and Technology Faculty, Islamic State University Maulana Malik Ibrahim Malang.

Explant Material and Media
Explant material preparation was done by taking young embryos from the fruit of C. pubescens. Young embryos of C. pubescens were soaked in fungicides solution (benlate 0,5 mg/L), Then, it shaked for an hour. After that, it moved to Laminar Air Flow (LAF) to soak the young embryos using 30% and 20% chlorox solution for each 10 minutes. Then, continued using 70% alcohol for 10 minutes. The young embryos rinsed three times using sterile aquades for each 10 minutes. The Murashige and Skoog (MS) media that it used by measuring ½ MS media composition, 30 grams of sugar, and 7 grams of agar. The ½ media and the sugar are put into Erlenmeyer that it contains 1000 ml of aquades. Then it homogenized with stirrer. It added by 3 mg/L of growth regulator 2,4-Dichlorophenoxyacetic acid (2,4-D). The media measured by pH media until 5,7-5,8. It heated and stirred until boiling. After that, it inserted into a culture bottles about 10 ml for each bottle. The culture bottles which containing media are covered with plastic and tied with the rubber. Then, it sterilized with the autoclave.

Initiation Phase 2.2.1 Amino Acids through Divine smoke particulation
Amino Acids was given to young embryos of C. pubescens through Divine smoke particulation for 30 minutes. Then, these treatments can be sterilized and planted in growth media.

Explant Initiation
The young embryos of C. pubescens are grown in embryogenic callus induction media. The young embryos were grown on ½ MS + 3 mg/L of 2,4-D. After that, the bottle cultures that containing explant were incubated at storage room on 23-25ºC and it observed daily.

Observation Phase
The observations are about 60 Days After Planting (DAP). Parameters observed were: Time Appeared Callus (TAC), callus fresh weight, callus color and texture, and the percentage of callus area in explants.

Data analysis
Observation data is qualitative and quantitative data. qualitative data are the visual observations, include: callus color, callus texture, and callus anatomy. While quantitative data are time appeared callus (TAC), the percentage of callus area in explants, and callus fresh weight. Quantitative data were analyzed using One Way ANOVA statistical test. If there is a mean difference then it proceeds with the Duncan's Multiple Range Test (DMRT) test at 5% significant level.

Time Appeared Callus (TAC)
Swelling in explants is the initial stage of callus formation which indicates cell activity in explants. The appearance of callus begins with the existence of explant parts that grow enlarged or swollen. Then, in that part there are tissues which grow actively and undifferentiated. In this process will initiating callus [9]. This is one of the success propagation in tissue culture. Based on the average of Time Appeared Callus (Figure 2) shows that the giving of Amino Acids through Divine smoke particulation can accelerate the growth of callus.   Figure 2 shows that Tryptophan through the Divine smoke particulation gave the fastest average of time appeared callus at 24,44 DAP, and than serin 25,55 DAP. The amino acid tryptophan synthesizes the endogenous hormone, thus affecting callus growth faster. Tryptophan is one of the Amino Acids that can be synthesized into Indole-3-Acetic acid (IAA). Tryptophan works in protein synthesis and makes Auxin levels higher [10]. So, Tryptophan can induce callus to the explants of C. pubescens [11]. Tryptophan plays a role in biosynthetic pathways as precursors to Auxin synthesis [12]. Synthesis of IAA is doing by eliminating the carboxyl group (decarboxylation) from the side ring of Tryptophan to Tryptamine which is oxidized to form of IAA [13]. The other pathway of IAA synthesis is from Tryptophan that combines with alpha keto acid through the transaminase reaction to indolepyruvic acid. Then it was induced by indolepyruvic decarboxylation to indoleacetaldehyde which was subsequently oxidized to form IAA [14].

Percent of Callus Area in Explants
The result of C. pubescens growth callus are influenced by the type of Amino Acids and 2,4-D. Amino Acids are important for callus growth because they play a role as precursors. Amino Acids have long polymers as a basic unit form in the molecular field. Amino Acids consist of 20 types that play a role in the basic structure and function of the body. There are 20 types of Amino Acids in proteins that become the basic structure and functional in the plant. Carboxyl (-COOH) and (-NH3) are contained in an Amino Acid that has the same bond on the carbon atom [15]. In plants, D-amino acids can synthesized, contained, and absorbed by plants. Plants have ability to metabolise a chemical compound like new nitrogen source [16]. Based on the result of 7 treatments of Amino Acids through Divine smoke particulation which is grown on ½ MS media + 3 mg/L of 2,4-D had a significant effect on the induction of C. pubescens embryogenic callus.

Callus Fresh Weight
The 6 types of Amino Acids through Divine smoke particulation which is grown on ½ MS media + 3 mg/L of 2,4-D had an effect on callus fresh weight variables. Chart 3 shows that Serine through Divine smoke particulation produces the highest average of callus fresh weight compared to other Amino Acids.  Serin is needed as an essential role in cellular processes. Serine is the main source in one carbon unit for the methylation reaction that occurs through the generation of S-Adenosilmethionine. In the metabolic process, serine plays a role as a major contributor and works together with glycine, taurine, cysteine, D-serine, and phospholipid in a form. Serine can induce callus with the highest percentage of callus area in explants and fresh weight callus [17]. Methionine is one of an amino acids that contains sulfur, the plants need sulfur to form disulfides bond between protein chain, help to protein synthesis and growth plants [8].
The amount of particulate, performance and function of smoke as a source of energy for the metabolic system can be added to amino acids. Molecular nano complex structures like these are similar to molecular complexes naturally found in cell physiological systems such as chlorophyll which play a role in energy transfer systems. The addition of amino acids to Divine cigarettes, particle size and complex structures of non-chemical compounds resulting from Divine combustion processes are smaller than the process of burning cigarettes without amino acids. It was suspected that the embryogenic callus induction C. pubescens using Divine smoke particulation that contains amino acids was able to move the metabolic system which was added with amino acids [18].

Colour, Textur and anatomy of callus
The observation result that have been done to knowing the effect of the types of Amino Acids through Divine smoke particulation to the callus color and texture can produce different callus color and texture in each treatment. Callus has a varied color due to the presence of light pigmentation and the origin of explants [19]. Callus pigmentation can be equal on all callus surfaces or it can be distributed only partially on the surface. Callus color can change from white, green, brown, yellowish white, and greenish white [20].  Observation of callus C. pubescens color showed a yellowish white. It shows that the yellowish white callus is meristematic ( Figure 5). The callus color that produced is affected by the speed of the regeneration or the multiplication cells. The color of meristematic callus is yellowish white as usual because the cells still forming young tissue and regenerate continuously [21]. The texture of C. pubescens embryogenic callus is friable. Compared to compact callus, callus which has a friable texture can multiply or regenerate rapidly. One of the friable callus characteristics is the callus cells can separate each other easily. Visually, friable callus has a tenuous bond between cells and when the callus is taken with pinset, the callus will break easily and stick on the pinset. The structure of friable callus shows that the callus is active dividing (meristematic) [12].
Anatomy observations showed a large cell nucleus and cytoplasm with several starch grains ( Figure 6). The embryogenic cells show characteristics common to meristematic cells: high division rates, cells are isodiametric and small with a dense cytoplasm with several starch grains, large nucleus and prominent nucleolus, small vacuoles, thin cell walls and a higher metabolic activity [22].

Conclusion
The conclusion of this research showed a significant effect of 6 types of amino acids through Divine smoke particulation to the induction of embryogenesis callus Carica pubescens, which is grown on ½ MS media + 3 mg/L of 2,4-D. Tryptophan is the fastest treatment to induce callus which is growed 24 days after planting (DAP). Serine influences to the percentage of callus area in explants is 98,8% and the fresh weight of callus 0,6733 gr. The observation of morphology and anatomy embryonic callus color is yellowish white with a friable texture and has a large cell nucleus and cytoplasm with several starch grains

Acknowledgments
We would like to express our gratitude to Biology department, Science and Technology Faculty, Islamic State University of Malang. We would like to express our gratitude to our teams who help this study.