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Destination of urban pruning organic waste by means of biochar applied to the soil : Analysis of the compactation curve

Urban pruning waste can have multiple uses from direct burning wood as energy to its use as organic fertilizers in the composting process. However, the lack of suitable places for the disposal of this type Brazilian Journal of Development Braz. J. of Develop., Curitiba, v. 6, n. 11, p. 92665-92679, nov. 2020. ISSN 2525-8761 92666 of waste is a problem for municipalities with large urbanization. Thus, “biochar” presents itself as an alternative proposal for the disposal of urban pruning organic waste. In this context, the present work aimed to verify the behavior of soil compaction curves through the application of biochar as a form of conditioning and waste disposal. Soil collection was carried out at the Institute of Science and Technology of Sorocaba (ICTS –UNESP Sorocaba) followed by drying and sieving for soil characterization through particle size analysis. The tests were performed with application of biochar in the soil using the Proctor Normal Compaction Test (NBR 7182/1986) at 35 t/ha and 70 t/ha in different textures. The data of the compaction curves and comparative statistical analysis presented satisfactory results. The results expose a maximum specific weight reduction and optimum humidity increase in all samples with application of biochar. Thus, the proposal for the redestination of urban pruning organic waste by means of biochar applied to the soil can be considered an alternative for urban management of pruning organic waste and may help in integrated solid waste management.

of waste is a problem for municipalities with large urbanization. Thus, "biochar" presents itself as an alternative proposal for the disposal of urban pruning organic waste. In this context, the present work aimed to verify the behavior of soil compaction curves through the application of biochar as a form of conditioning and waste disposal. Soil collection was carried out at the Institute of Science and Technology of Sorocaba (ICTS -UNESP Sorocaba) followed by drying and sieving for soil characterization through particle size analysis. The tests were performed with application of biochar in the soil using the Proctor Normal Compaction Test (NBR 7182/1986) at 35 t/ha and 70 t/ha in different textures. The data of the compaction curves and comparative statistical analysis presented satisfactory results. The results expose a maximum specific weight reduction and optimum humidity increase in all samples with application of biochar. Thus, the proposal for the redestination of urban pruning organic waste by means of biochar applied to the soil can be considered an alternative for urban management of pruning organic waste and may help in integrated solid waste management.

INTRODUCTION
In the same trend of urban population growth, there is also an increase in the volume of solid waste generated in cities. The problem related to solid waste is part of an economic, political, social and ecological, whose complexity involves a wide network of interrelations that demand comprehensive approaches on various aspects MEDEIROS et al, 2020). In this context, urban pruning waste, even with multiple uses ranging from burning as firewood for power generation to use as organic fertilizers, presents a major management and disposal challenge within the municipalities. In this scenario, it highlights the volume of material and the lack of adequate places for the disposal of this type of waste.
Considering these factors, there is a shortage of efficient models for the management of pruning residues from urban afforestation and also the high cost of some techniques for their treatment, resulting in numerous environmental problems, due to the inadequate disposal of these materials (CHALUPE, 2016). According to NBR 10. 004/2004004/ (ABNT, 2004, pruning waste is classified as solid class II waste, that is, non-hazardous. If this type of waste is disposed of in open places, such as landfills, it can cause a series of problems, because in addition to reducing the capacity of the landfill, it can mix with other pre-existing waste, causing environmental impacts, such as increased risk fire in landfills and vacant lots, landscape degradation and air and water pollution. In this way, the search for alternatives for the use and redestination of organic waste, especially tree pruning, has become a challenge for sustainable urbanization (ANDREOLI et al, 2014).
In this context, the placement of biochar in soil conditioning can have good results in the environmental management of organic waste in cities. Studies carried out in the last decade prove the capacity of "black earth" or "biochar" to accumulate organic matter, allowing for increased fertility and carbon storage in tropical soils. Among the benefits of applying charcoal as a soil conditioner, in addition to high fertility, are high content of stable carbon in its organic fraction and increased biological activity, contributing to the formation and growth promotion characteristics of these soils (SCHOENHOLTZ et al, 2014;KIM et al, 2019).
Currently, the intensification of the urbanization process can be considered one of the major inducers of environmental problems due to the use and occupation of land, leading to the removal of vegetation and soil degradation (KIM et al, 2017, STEFANOSK et al, 2013STANGANINI and LOLLO, 2018). In that context, knowledge of the dynamics of land use and land cover is increasingly important for understanding space, planning and management of natural resources, which contributes to the conservation and environmental sustainability (Maidjelele et al, 2020).Thus, the biochar incorporated into the soil presents itself as an alternative management practice with great potential for soil improvement, contributing to soil recovery and conservation (TIECHER, 2016;GUILHEN, 2018;Kumar et al, 2019).
The term "biochar" results from "biomass + charcoal" (biomass + coal). This idea arose from the observation of the existence of our "black land of the Indians" in soils of the Brazilian Amazon.
The "biochar" is produced basically by burning organic matter in environments with little or no oxygen.
The process is known as pyrolysis or gasification, the same process that is used to create wood charcoal (Kumar et al, 2019;MANGRICH et al, 2011). The main function of Biochar is based on the following benefits: (1) soil improvement, (2) carbon sequestration, (3) carbon neutral energy production, (4) waste management and (5) water quality protection through greater efficiency in the use of nutrients (BRICK and MADISON, 2010).
Although several studies deal with changes in soil properties resulting from the use of biochar, few assess the effects of applying biochar to the soil's response to compaction and its potential for use in sustainable management (VERHEIJEN et al, 2010). In this scope, the present work aimed to verify the behavior of soil compaction curves through the application of biochar, as a way of conditioning and disposal of waste.

MATERIAL AND METHODS
The soil used in the present study was collected at the Campus of UNESP Sorocaba/SP.
Approximately 40 kg of sub-surface soil was removed, disregarding the organic horizon, as organic matter can influence the results of specific weight. The soil remained on drying for 15 days before test.
Then, the soil was sieved through a 4.75 mm sieve (ASTM 4 / TYLER 4) and soil characterization through granulometric analysis.
The biochar used in the tests was eucalyptus charcoal (Eucalyptus grandis, first the charcoal was broken until it reached a finer texture. Then the charcoal grains were separated into four different diameters, using 2 mm sieves (ASTM 10), 1.18 mm (ASTM 16), 600m (ASTM 30), 300m (ASMT 50), 150m (ASTM 100) and 75m (ASTM 200). The biochar plots with a diameter between 75m e 600m "fine texture", and between 600m and 2 mm "thick texture." As a descriptive element, the letter Ø was used as a symbol of the diameter retained in the sieving, for example: 75 µm <Ø <600 µm.
The rates of application of biochar in the soil were 35 t/ha and 70 t/ha. In this way, it was sought through these dosages to identify the contrast between applications, that is, quantities [tons] and particle sizes [size of coal particles]. The quantity and texture were adjusted according to the surface area of the 0.79 cm 2 cylinder and its depth 12.75 cm with the Proctor Normal Compaction Test cylinder following NBR 7182/1986(ABNT, 1986 and the calculation of the proportion of Biochar applied to the soil in each test he followed the equation below: Where: Mbio = Biochar mass to be applied (g); Mbiocil = Biochar mass for the cylinder surface (g); Msoil = Mass of soil (g); M1000cm³ = Mass of dry and sieved soil in the 1000 cm³ (1288g).

STATISTICAL ANALYSIS OF THE DATA
The statistical analysis of the data was performed with the aid of the ASSISTAT Version 7.6 beta software (2012) in a completely randomized design (DIC). Therefore, the analysis of variance (ANOVA) was carried out, ending with the application of an average test (Tukey, 5%). The variables analyzed were maximum specific weight and optimum humidity for each trial, treatments were (S, SBt, SBT, Sbt, SbT), and 4 repetitions were performed.
With the values of humidity and the specific weight of the dry soil, the graphs were constructed referring to the compaction curves for each test. Figure 1 represents the compaction curves for Tests 1 to 4, referring to the soil without Biochar (Fig. 1). The Compaction Curves of the same soil tests were similar to each other as expected due to the control of the test. The greatest variation between maximum specific weight was 0.033 g/cm³ between tests 1 and 2. The optimum humidity content also had little variation in tests 1 to 4, with 0.58% being the difference between tests (2 and 4) with greater divergence of optimum humidity. Figure 2 presents the graph referring to tests 5,6,7 and 8 represent the soil incorporated with thick biochar at the application rate of 35 t/ha, the curves shown show the maximum specific weights and the optimum humidity content for the same samples. The compaction curves for the soil incorporated with coarse biochar at the rate of 35 t/ha also showed few variations from one test to another, except for test 7, which showed a certain specific weight reduction in the first humidity content. However, the biggest difference between the maximum values of specific weight was 0.031 g/cm³ obtained in tests 6 and 7. The biggest difference between the values of optimum humidity was 2.09%. Figure 3 shows the compaction curves for Proctor Normal Tests 9 to 12, performed with coarse biochar in the amount of 70 t/ha. The compaction curves obtained from the tests with soil and coarse Biocarbon in greater quantity converge more for the wet branch (after reaching the maximum specific weight) than for the dry branch, even though the greatest difference in maximum dry specific weight was 0.026 g/cm between tests 10 and 11. The greatest variation in optimum humidity between tests 10 and 11 was 1.44%.  Figure 4 shows the compaction curves for Tests 13 to 16, performed with fine biochar in the amount of 35 t/ha. For the soil with thinner Biochar at the rate of 35 t/ha, the maximum variation in dry specific weight was even smaller than the previous ones, showing a difference of 0.020 g/cm³ between tests 13 and 15. The biggest difference between the variation of optimum humidity was 1.55%.  In addition to the reduction in specific weight and increase in optimum humidity in the soil with the application of biochar, there is also a lower slope in the wet branch (right side) of the curves of the soil with biochar, demonstrating a smaller variation in specific weight with the variation of moisture.
This property may be associated with a lower susceptibility of these soils to compaction according to the increase in humidity. The retention of water by fine clay particles showed similar characteristics described by VERHEIJEN et al. 201).
To understand the variations of the proposed treatments, a univariate statistic was performed with the assistance of the ASSISTAT software version 7.6 betas (2011) in order to identify the differences between the means in the interpretation of the data obtained from optimum humidity and maximum specific weight in the 5 treatments with 4 repetitions each. The comparison of the averages selected in the program was based on the Tukey test.
The results generated by the program for the statistical analysis of the optimum moisture content in different samples are shown in Table 3. The results generated by the program for the statistical analysis of the maximum specific weight in different samples are shown below in table 4. 0.04632 * significant at the level of 5% probability (.01 = <p <.05) ** significant at the level of 1% probability (p <.01) ns not significant (p> = .05) Comparative analysis for the verification between changes in soil compaction in relation to the application of different amounts and textures of biochar can be seen in figures 6 and 7 that elucidate bar graphs for the treatments. Differences can be observed between the average optimum humidity [%] and also in the average maximum specific weight [g/cm3] for the different treatments. Thus, according to table 6, a Tukey test was applied at a significance level of 5%. Considering the average moisture content, a significant increase in optimum moisture was observed in almost all applications of biochar to the soil. Sample 5 of Soil + fine biochar 70 t/ha was the one that most increased the optimum humidity. This means greater water retention in the soil made possible by coal fines, which for the same amount of water in a soil without biochar and another with biochar, the former will be more likely to reach greater densities when it undergoes some application of energy. Table 6 shows that the means followed by the same letter do not differ statistically from each other by means of the Tukey test at the 5% significance level.  The averages between the maximum specific weights of the different soil treatments with Biochar were reduced considerably when compared to the soil without Biochar, mainly for the applications of Biochar of 70 t/ha. The "SBT" sample (Soil+Biochar coarse 70 t/ha) was the one that most reduced the maximum specific weight of the soil-Biocarbon system, promoting a mass reduction greater than 6%.
Although the specific weight of the Biochar is much less than that of the soil, it can increase the specific weight when incorporated, since the maximum compaction capacity depends on the particle size of the system. As important as disposal and appropriate treatments, it is necessary to produce less and less waste and reuse more and more waste generated, reducing the high rate of waste, thus contributing to a more balanced and responsible society .The explanation for the thicker Biocarbon grains being better in reducing specific weight may be in the arrangement between the Biocarbon coarse particles with the soil particles and the friction between them. In a study on erodibility and compaction (GUILHEN, 2014), observed similar effects in analysis and highlighted that the addition of biochar to the soil resulted in decreased erosion throughout the dry state, while the opposite was observed in the wet state.

CONCLUSION
The results represented by the compaction curves and the statistical analysis of the data show a reduction in specific weight in all soil samples with incorporation of biochar, with the application of thick biochar (600 µm <Ø <2 mm) in the concentration of 70 t/ha more efficient in reducing soil compaction under the same energy as the rest of the tests.
It is also noted that the optimum moisture content, for the maximum specific weight, increases in all additions of biochar in the soil, and samples containing fine biochar (75 µm <Ø <600 µm) reached optimum humidity levels, more higher than other tests. This represents greater water retention in soils with biochar fines, which can be very interesting in very permeable soils. In addition, the increase in optimum humidity content can minimize the risk of maximum compaction in agricultural soil preparation in very rainy regions.
Thus, biochar has the potential to reduce soil compaction and increase water retention. These properties allow the biochar to be used for the disposal of urban organic waste and soil conditioning.
As an indication for the production of future works, it is suggested that tests be carried out in the field, based on biochar from different plant residues and applications on larger scales, as the proposal for the destination of organic urban residues originating from pruning applied to the soil can assist in urban waste management in an integrated manner and with more sustainability.

ACKNOWLEDGMENT
We thank the Laboratory for Research in Land Management and Recovery at the Institute of Science and Technology -ICTS / UNESP for the help and technical support, and the research group for their support in writing and editing the work.


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

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Assessment of Physicochemical, Microbiological and Sensory Attributes of Maldive Fish with the Incorporation of Garcinia cambogia (Goraka)

This study was conducted to evaluate the potential use of Garcinia cambogia for the improvement of quality attributes of Maldive fish. Maldive fish were prepared using three different incorporation levels of G. cambogia as 0%, 1.25%, 1.67% and 2.5%. Sensory results revealed that sample incorporated with 2.5% of G. cambogia had the best qualities. G. cambogia treated samples showed significant reduction (P0.05) of histamine content compared to commercial Maldive fish samples. The highest level of 2-thiobarbituric acid-reactive substances (TBARS) was identified in the commercial sample, while the lowest level found in Maldive fish incorporated with 1.25% of G. cambogia. Maldive fish incorporated with the highest level (2.5%) of G. cambogia showed greater suppression of bacterial count. Further, significantly lower (P0.05) fungal counts and bacterial counts were observed in all treatments compared to commercial samples and the control. In conclusions, Maldive fish incorporated with 2.5% of G. cambogia results enhanced sensory and quality attributes and safe product for consumers.

INTRODUCTION
Fish is a nutritious food source which comprises of quality proteins, vitamins, essential fatty acids and minerals. Fish consumption has been gradually increased over the years mainly due to the proven health benefits such as being a rich source of omega 3 fatty acids that reduces cardiovascular diseases and its use as a lean alternative to meat (Das et al., 2009;Siscovick et al., 2000). In many of the developing countries, smoked and dried fish are traditionally accepted food items and act as the major source of protein.
Maldive fish is a hard dried product obtained by salting, hot smoking and drying the flesh of fresh or frozen wholesome fish of the tuna species (Mohamed, 2013). It is widely used as an ingredient for flavoring and thickening of many dishes in Maldive islands and as well as in neighboring countries like Sri Lanka. Maldive fish is a favorite addition to curries and "sambals" (chilly chutneys) to impart a salty and fishy taste and it is a traditional fish product equivalent to the fish sauce in Vietnam and Thailand. The excess harvest of fresh or frozen tuna species such as Skipjack tuna (Katsuwonus pelamis), Frigate Mackerel (Auxis thazard), Yellowfin tuna (Thunnus albacores) and Mackerel tuna (Euthynnus affinis) are commonly used to produce Maldive fish (Mohamed, 2013).
Production of Maldive fish is gradually open out for countries like Sri Lanka, India, Japan and other south Asian countries. In Sri Lankan context, Maldive fish production is a seasonal industry which gives the highest profit during the glut season of fish and when the costs of raw materials are low. However, the products failed to satisfy the quality and quantity of local market requirement. Histamine poisoning due to decarboxylation of histidine into histamine, lipid oxidations which decrease the shelf life of the product are the major quality defective reactions. When consider about the microbial quality, the association between smoked fish and fungal growth is remarkable. Salted food can be affected by the halophilic organisms which has a preference for high salty conditions. In order to overcome these quality defects, manufactures tend to use some natural ingredients in the Maldive fish production process. G. cambogia is one of the natural ingredients used in the production process of Maldive fish in Sri Lanka (Gunaratne, 1999). It is also called as Malabar tamarind and famous for its antimicrobial, cytotoxic, antioxidant and anticancer properties of the fruit rind due to the activity of different secondary metabolites such as benzophenones, flavonoids and xanthones (Iinuma et al., 1998;Koshy et al., 2001;Masullo et al., 2008). As per the current trends, consumers highly prefer food products with natural preservatives such as spices than the chemical preservatives (Jakhar et al., 2018). Thus, the main objective Assessment of Physicochemical, Microbiological and Sensory Attributes of Maldive Fish with the Incorporation.... of this study was to ascertain the effect of G. cambogia on the improvement of physicochemical, microbiological and sensory attributes of Maldive fish.

MATERIALS AND METHODS
Maldive fish samples were prepared using three different incorporation levels of G. cambogia, which were Treatment 1, Treatment 2 and Treatment 3 as 1.25%, 1.67% and 2.5% of G. cambogia respectively. A control sample was prepared without adding G. cambogia. Commercially available Maldive fish samples were obtained from local markets in Sri Lanka for comparision.

Preparation of Maldive fish
Fresh ripened fruits of G. cambogia were cut into 4 pieces and seeds along with the seed covering were removed. After that, fruit rinds were allowed to boil for about 1.5-2 h without addition of water. During the boiling, stirring was done until it became a paste.
Fresh tuna fish (Katsuwonus pelamis) was cleaned by removing head, gills, gut and fins. Washed with clean water and 1.5 kg of fish was weighed accurately. Five liter of water was added to a container with G. cambogia paste and 250 g of salt was added. The mixture was heated up to about 55-60C and fish was dipped in the container and boiled for 45 minutes. It was allowed to cool and water was drained off. After that fish was cut into four longitudinal pieces and all the bones were removed. Smoking was done for 18 h and finally sun drying was done for 5-7 days.

Determination of histamine content
Fluorometric method was employed with modifications for the analysis of histamine content (Horwitz and GW, 2005). Histamine content was calculated by using the standard curve.

Determination of TBARS content
The content of TBARS was analyzed using Spectrophotometric method (Horwitz and GW , 2005). TBARS value was calculated using the standard curves prepared with 1,1,3,3tetramethoxy methane as a precursor of malonaldehyde (Horwitz and GW, 2005).

Determination of myoglobin content
Maldive fish samples were cleaned by removing exterior layer and cut into small pieces. Two grams of sample was accrately weighed and 20 mL of ice cold 40 mM phosphate buffer (pH 6.8) was added to each sample. Then the samples were blended for 30 seconds at high speed. These homogenates were centrifuged at 3000 g using centrifuge (CF 15D2, Himac, Hitachi, Japan). After that, the supernatant of each sample was filtered using a cheese cloth and followed by filtration with whatman No.1 filter paper. The volume of the filtrate was accurately measured. The absorbance values were measured at emmision wave length of 525 nm and 700 nm.

Sensory evaluation
A ranking test was done to identify the best level of treatments. The T1(1.25%), T2(1.67%) and T3(2.5%) were compared and all the treatments were presented as both sliced and whole maldive fish forms. Most preffered sample was obtained the rank one and the least prefered sample obtained the rank three.

Statistical analysis
Completely Randomized Design (CRD) was used for the analysis of quality parameters and mean separation was done by Duncan's Multiple Range (P<0.05) test. Data were analyzed using SAS software (SAS Inst. Inc., Cary, NC, USA). Sensory attributes were analyzed separately and ranking test was performed using Friedman test using Minitab 14 software.

Physicochemical analysis
Histamine is a protruding food born chemical hazard which is produced due to bacterial decarboxylation of histidine. Most of the fish species used in Maldive fish production is associated with histamine formation; especially fish belong to family Scombridae (Mustafa, 2018;Thadhani et al., 2002). Histamine determination results (Table 1) indicated that all the Maldive fish samples have relatively much lower levels compared to the maximum allowable histamine level of 200 mg/kg (Fletcher et al., 1998). According to the European Legislation, up to a maximum of 200 mg/kg in fresh fish and 400 mg/kg of histamine in fishery products subjected to enzyme maturation in brine are allowable (Visciano et al., 2014). Commercial Maldive fish sample gave relatively higher histamine content than other Maldive fish samples and it could be attributed to the poor quality of the fish used for Maldive fish production and improper processing conditions. Since, histamine forming bacteria are mesophilic, a rapid histamine decarboxylation can take place as a result of large proliferation of this bacteria at optimum temperature (26C) and pH conditions. According to the results, T2 and  (Thadhani et al., 2002). According to previous study findings, mean histamine concentration of inner and outer portions of Maldive fish vary from 190 to 90 ppm respectively and our study findings also in line with that values (Gunaratne, 1999). Fish is highly susceptible for lipid oxidation since it contains high level of polyunsaturated fatty acids. It promotes the rancidity and discoloration of fish and fish products (Tsaknis et al., 1999). Hydroperoxides are primary products of lipid oxidation and readily decomposed to produce various TBARS, mostly carbonyl compounds (Ke et al., 1984). The highest level of TBARS value was identified in the commercial sample, while the lowest level present in the Treatment 1 which contained 1.25% G. cambogia (Fig 1). All the Maldive fish samples exceed the recommended level of TBARS which is 8 mg MDA/kg except treatment 1, indicating high levels of lipid oxidation (Kocatepe et al., 2014;Özyurt et al., 2009). Higher lipid oxidation levels could be attributed to the bad handling and storage conditions of fish used for Maldive fish production. And also, lipid oxidation of a fish species vary with season and location within the tissue (Flick Jr et al., 1992). All the G. cambogia treated samples showed lower level of TBARS compared to both control and commercial sample and it reveals that G. cambogia have significant impact upon reduction of lipid oxidation. It has been reported that garcinol and guttiferone k isolated from fruit of G. cambogia has exhibit in vitro protective effects against lipid and protein oxidation. Furthermore these compounds cause to reduce the formation of carbonyl groups and thiobarbituric acid reactive substances (Semwal et al., 2015).
Significantly higher myoglobin content was observed in the treatment 3 which is treated with the highest level of G. cambogia and no significant difference was observed in between treatment 1 and 2 (Fig 2). Myoglobin is the sarcoplasmic hemeprotein that primarily responsible for the meat color and it is liable to oxidation during the heating and the storage (Suman and Joseph, 2013;Ishikawa et al., 2006). Bailey et al., (1990) have reported that, fish contain 65-23 nmol/g of myoglobin in tissue. According to the current study, all the Maldive fish samples comprise of much higher myoglobin content and it implies proper handling and storage temperature of fish used in Maldive fish production (Hui et al., 2004).

Microbiological assessment
Maldive fish samples yielded both bacteria and fungi from all the samples. Commercial sample had the highest total bacterial count and fungal count which could be attributed to unhygienic handling of fish, postharvest delay, improper salting and sun drying processes (Saritha et al., 2012).   Smoked and dried fish products are associated with several food borne bacterial pathogens, toxicogenic fungi and mycotoxins. Higher bacterial counts lead to elevated biogenic amine levels in dried fish products due to the action of histamine producing bacteria such as Listeria monocytogenes , Clostridium botulium, Clostridium perfringens, Staphylococcus aureus (Mohamed, 2013). Total bacterial count was significantly reduced in the G. cambogia treated samples compared to the control sample. Sample with the highest concentration of G. cambogia showed greater suppression of bacterial count than other G. cambogia treated samples (Fig 3). Previous studies have proved the in vitro antibacterial activity of the fruit rind extracts of G. cambogia using hexane, ethyl acetate and ethanol as the extraction solvents. According to the nutritional profile of the G. cambogia; availability of xanthones, isoxanthones and garcinol are the responsible compounds for the antibacterial property of G. cambogia (Cuesta-Rubio et al., 2005;Iinuma et al., 1998). Fungal count results also gave similar observations as in bacterial count and relatively higher fungal count was observed in all the samples (Fig 3). This is in agreement with the fungal count values reported by Abolagba et al. (2011) . Yeast and molds are associated with the spoilage of dry foods especially containing high salt or sugar content. They act as abundant food spoilage agents that can grow over wide range of pH, temperatures and especially at reduced water activity (a w ). Hence, Maldive fish is highly susceptible for fungal contamination as it is a seafood product with low water activity (W illiams et al., 2004). The lowest level of fungal count was associated with treatment 3 containing 2.50% G. cambogia concentration while the highest count was observed in the commercial sample (Fig 4). Even though the mean count was gradually reduced there was no significant difference in terms of microbial load between T1 , T2 and T3. Presence of garcinol, iso-garcinol, benzophenons in G.cambogia impart antifungal and antimicrobial effect and thereby reduce the yeast and mold count of Maldive fish with increased levels of G. cambogia (Cuesta-Rubio et al., 2005).

Sensory quality analysis
Maldive fish possess unique bright red colour which affect quality and palatability (Becker et al., 2012). T2 and T3 samples obtained the highest colour preference since the bright red color of the Maldive fish samples enhanced with increased G. cambogia concentration. Most preferred sample (α=0.05) for the tenderness was T3 (2.5%) which having the highest G. cambogia concentration (Fig 4). As the surface appearance, consumers consider combination of various factors such as the characteristic spindle shape, absence of molds and yeast. According to the results the most preferred sample (α=0.05) for the surface appearance was T2 (1.67%) (Fig 4). T3 is selected as the sample which is having the best flavor and it suggests that the increment of the concentration of G. cambogia has significantly improved the flavor of Maldive fish. Since G. cambogia has a potential to suppress the microbial growth it can be used as natural antimicrobial compound in Maldive fish production and it improves the quality characteristics. According to the sensory and microbiological property analysis, 2.5% of G. cambogia is the best level to be incorporated in Maldive fish production.

CONCLUSION
Since G. cambogia has a potential to suppress the microbial growth it can be used as natural antimicrobial compound in Maldive fish production and it improves the quality characteristics. According to the sensory and microbiological property analysis, 2.5% of G. cambogia is the best level to be incorporated in Maldive fish production.


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Evaluation of the anti-diabetic effect of the methanol leaf extract and fractions of Dennettia tripetala G. Bak (Annonaceae) in alloxan- induced diabetic mice

This study evaluated the anti-diabetic properties of the methanol extract and fractions of Dennettia tripetala leaves in alloxan diabetic rats. The crude methanol extract (ME), n-hexane (NF), ethyl acetate (EAF), butanol (BF) and water (WF) fractions, were tested for anti-diabetic activity in alloxan-induced diabetic mice. The extracts and its fractions were screened for phytochemical constituents. Glibenclamide was used as the standard anti-diabetic treatment Fasting blood glucose (FBG) levels were determined using a glucometer. Lipid profile parameters were also assessed. Histopathological examination of the pancreas was performed. The presence of glycosides, saponins, reducing sugars, fats and oil, alkaloids, carbohydrates, flavonoids, proteins and tannins were detected in ME, and the fractions. Treatment of the rats with the extract and its fractions reduced FBG levels significantly (p < 0.05) within 10 hours of acute treatment and 14 days short term treatment. The highest reduction was by the crude extract,( ME 500 mg/kg) with percentage reduction of 62.89 %. The study revealed significant (p < 0.05) reductions in serum triglyceride, serum cholesterol and LDL levels by the extracts and its fractions while a significant (p < 0.05) increase in HDL levels was evident . The effect of the extract and fractions on body weight indicated a moderate weight gain. Treatment with high dose of the extract and fractions (500 mg/kg) resulted in marked rejuvenation of the pancreatic β cells. The results of this study suggest that the leaves of Dennetia tripetala are endowed with potent anti-diabetic and antilipidemic properties.

INTRODUCTION
Diabetes mellitus (DM) is a common disorder of the endocrine system which leads to impairment of glucose homeostasis resulting in severe complications such as retinopathy, angiopathy, nephropathy, neuropathy and neurological disorders [1]. It is a serious global health challenge characterized by hyperglycemia resulting from absolute or relative deficiency of insulin or by insulin resistance at the cellular level [2]. Features of DM may include lipoprotein abnormalities [3], elevated basal metabolic rate [4], reactive oxygen species scavenging enzymes deficiency [5] and damage to pancreatic beta cells induced by high oxidative stress [6]. Trivedi [7] reported that diabetes mellitus ranks seventh among the leading causes of death and is considered third when it's lethal and debilitating complications are taken into consideration. Despite the tremendous achievements by pharmaceutical companies to produce different types of oral hypoglycaemic agents alongside insulin for the treatment of diabetes, there is still an increase in the demand of natural products with antidiabetic activities [8]. The use of medicinal plants as agents to control diabetes mellitus have been reported [9]. Metformin, a very effective hypoglycemic drug was discovered by the traditional approach, through the use of Galega officinialis [10]. The continuous efforts by man to exploit medicinal plants for the treatment of several diseases including diabetes is essentially because the side effects associated with herbal medicine are few compared to that associated with chemically synthesized drugs. Dennettia tripetala (pepper fruit) is of Annonaceae family. It is a tropical plant that is mostly dominant in West African region especially Cameroon, Ivory Coast and Nigeria [11,12]. The plant is characterized by its pungent like spicy and pepperish taste, which makes it suitable for spices and condiments [13]. Its mature fruit is masticated as a fresh green, freshly ripened red, black dry fruit and dry de-hulled seed [14]. In Nigeria, it is locally called "Nkarika" ( Calabar), "Ako" (Edo), "Opipi" (Idoma), "Mmimi" (Igbo) and "Ata igbere" (Yoruba). The medicinal properties of the plant has been associated to the presence of saponins, flavonoids, tannins, alkaloids, terpenoids, and cyanogenic glycosides [15,16]. Its activities against microbial related and coronary diseases are linked to the presence of these phytochemicals [17]. The leaves and roots have been used locally to treat various ailment including fever, infantile convulsion, typhoid, worm infestation, vomiting and stomach upset [18]. The leaf extract has been demonstrated to possess antimicrobial activity against Staphylococcus aureus. [18], antinociceptive, antiinflammatory, antioxidant and antimicrobial activities [19]. In the present study, the antidiabetic properties of Dennettia tripetala was assessed by evaluating the antihyperglycemic and hypolipidemic activities of the extract and fractions in alloxan-induced diabetic mice.

Plant Materials
Fresh leaves of Denettia tripetala were collected from Irri Town, Isoko South Local Government Area, Delta State, Nigeria. The plant was authenticated by Mr. Patrick Ugwuozo of the Department of Botany, Nnamdi Azikiwe University, Awka, with voucher number PCG/UNIZIK/0631. The fresh leaves were washed in a running tap to remove dust and other debris, and air dried for two weeks. The dried leaves were pulverized with electrical blender and kept in clean air tight amber bottle. About, 750 g of the powdered material was cold macerated in 80% ethanol. The mixture was agitated continually for two days (48 hours), and then filtered using Whatman filter paper No 1. The filtrate was concentrated to dryness using water bath at 40 0 C. The extract was stored in a refrigerator before use. The percentage yield of the extract was calculated.

Fractionation of Crude Extract
Fractionation was carried out using n-hexane, ethyl acetate and butanol following the method described by Ihekwereme et al [19]. Methanol crude extract (100 g) was dispersed in 500 ml of distill water and then poured into a separating funnel. N-hexane 500 ml was then added to the funnel and was shaken thoroughly to mix. The mixture was allowed to separate into two distinct layers. The n-hexane portion, (upper layer) was separated and the other portion was subjected to fresh n-hexane until the n-hexane solvent became clear. After the n-hexane phase, the other portion was subjected to ethyl acetate and butanol successively. The various fractions were concentrated to dryness using water bath at 40 0 C.

Experimental Animals
A total of 100 male albino mice, (18-35 g) were purchased from the Laboratory Animal Facility of the Department of Veterinary Physiology and Pharmacology, University of Nigeria, Nsukka and transferred to the animal House of the Department of Pharmacology and Toxicology, Nnamdi Azikiwe University. They were housed in clean metal cages, with free access to water and feed (Guniea Feed®, Nigeria). Animals were handled in compliance with the National Institute of Health Guidelines for care and use of laboratory animals (Pub No. 85-23, revised 1985).

Experimental Design
A total of 65 mice consisting of 60 diabetic mice randomized into 12 groups of 5 mice each and 5 non-diabetic mice and were treated as follows: Group 1 (Normal control) received 5 % Tween 80 (5 ml), Group 2 (Diabetic control) received 5 % Tween 80 (5 ml), Group 3 received glibenclamide (5 mg/kg), Groups 4 and 5 received 250 and 500 mg/kg of methanol crude extract respectively, Groups 6 and 7 received 250 and 500 mg/kg of ethyl acetate fraction respectively, Groups 8 and 9 received 250 and 500 mg/kg of n-hexane fraction respectively, Groups 10 and 11 received butanol fraction 250 and 500 mg/kg respectively while Groups 12 and 13 received aqueous fraction 250 and 500 mg/kg respectively.

Blood Glucose determination
Baseline pretreatment blood glucose level (zero hour) of the mice was taken. Then diabetic animals were then treated as stated. Blood glucose level was measured at 2, 4, 6, 8 and 10 hours post treatment using One Touch Ultramini Glucometer (LifeScan GmbH Switserland) [20]. Daily administration of glibenclamide, crude extract and fractions was also carried out for a period of 14 days (2 weeks) on the same animals. Blood glucose level was measured on day 2, 4, 6, 8, 10, 12 and 14. Blood samples were obtained from the tail vein of the mice.

Determination of serum total cholesterol
Serum total cholesterol (TC) was evaluated using Randox commercial assay kits following the methods described by Ezeigbo [21]. One millilitre (1 ml) of the working cholesterol reagent was added into tubes labeled blank, standard and test groups. Ten microlitres of standard cholesterol reagent, and samples were added into their respective tubes. They were mixed and allowed to stand for 10 minutes at room temperature. Absorbance of samples and standard were read with the aid of a spectrophotometer at 500 nm. Total cholesterol level in sample was calculated using the formula below; ISSN: 2250-1177 [131] CODEN (USA): JDDTAO

Determination of serum triglyceride
Serum triglyceride was evaluated using Randox commercial assay kits following the methods described by Tietz [22]. One millilitre (1 ml) of the working triglyceride reagent was added into tubes labeled blank, standard and test groups. Ten microlitres of standard triglyceride reagent, and samples were added into their respective tubes. They were mixed and allowed to stand for 10 minutes at room temperature. Absorbance of samples and standard were read with the aid of a spectrophotometer at 500 nm. Total cholesterol level in sample was calculated using the formula below;

Determination of serum high density Lipoprotein cholesterol (HDL-cholesterol)
Serum HDL-cholesterol was evaluated using Randox commercial assay kits following the methods developed by National Institute of Health Consensus Development Conference Statement [23]. One hundred microlitres (100 ul) of samples and standard cholesterol reagent were dispensed into test tubes containing 250 ul of HDL cholesterol precipitate (R1). The mixture was centrifuged at 4000 rpm for 10 minutes. Thereafter, 100 uL of samples and standard supernatants were added to another set of test tubes labeled samples and standard containing cholesterol reagent. The mixture was incubated for 10 minutes at room temperature and absorbance of standard and samples were measured against reagent blank at 500 nm within 60 minutes using Spectrophotometer. HDL-cholesterol level in sample was calculated using the formula below;

Determination of serum low density Lipoprotein cholesterol (LDL-cholesterol)
Low density lipoproteins (LDL) cholesterol in serum was calculated using the equation described by Friedewald et al. [24]. The Friedewald's equation estimates the value of HDL-C using the values of total cholesterol, triglyceride and HDLcholesterol.

Histopathological studies
The pancreases harvested from the animals were preserved using 10% neutral buffered formalin placed in pre labeled universal containers. The pancreases were dissected and placed in labeled tissue cassettes and the thickness maintained between 3-5mm [25]. The pancreases were subjected to automatic tissue processing using the Leica TP2010 automatic tissue processor for 18 hours passing them through the four stages of tissue processing namely: fixation (using 10% neutral buffered formalin), dehydration (using ascending grades of isopropyl alcohol), clearing or dealcoholisation (using xylene) and finally impregnation or infiltration (using molten paraffin wax) [26]. The tissues were then embedded in paraffin wax using the Leica automated tissue embedder and sectioned to get ultra thin sections at five (5) microns, using the thermo scientific semi automated rotary microtome. The tissues were floated out from the thermo scientific digital floating bath on frosted end pre labeled slides and dried on the thermo scientific digital slimline hot plate. The tissues were further dried in the hot air oven overnight and subjected to hematoxylin and eosin staining to demonstrate the general tissue structure. Stained slides were mounted in DPX and allowed to dry before viewing under the microscope using X10 and X 40 magnification.

Statistical Analysis
The data obtained were expressed as mean ± SD and were analysed using Statistical Product and Service Solutions (SPSS), version 20. Tests of statistical significance were carried out using one way Analysis of Variance (ANOVA) followed by post hoc turkeys test. P values < 0.05 were considered statistically significant.

Phytochemical analysis.
The extract and fractions contained saponins, carbohydrates, resins, alkaloids, flavonoids, tannins, cardiac glycosides and reducing sugars. Flavonoids, saponins, alkaloids, and tannins were present in large amount in the crude extract. The ethyl acetate fraction contained saponins, flavonoids and tannins in large amount while steroids, alkaloids and cardiac glycosides were in moderate amount. The n hexane fraction had saponins, flavonoids and alkaloids in large quantity but is devoid of other phytoconstituents. The butanol fraction had terpenoids, steroids and tannins in large amounts but is devoid of other constituents. Water fraction contained more of saponins Table 1).

Effect of crude extract and fractions on fasting blood glucose (FBG) level .
The crude methanol extract and fractions reduced fasting blood glucose levels in alloxan-induced diabetic mice from 2 -10 hr (p < 0.05). The highest reduction (62.58 %) in FBS was by the BF (250 mg/kg), at the 6 th hour. By the 10 th hour, a percentage reduction of 61.76 and 61.09 % was evoked by ME, 250 and 500 mg/kg respectively (

Effect of two weeks treatment on blood glucose level
Daily treatment with the crude methanol extract and fractions for 14 days reduced fasting blood glucose levels from the 2 nd to the 14 th day with the exception of ME (500 mg/kg). Significant (p < 0.05) reduction in FBG levels was evident at 500 mg/kg of the extract and fractions. The highest reduction in FBG levels was by ME (500 mg/kg 62.89 %), followed by BF (250 mg/kg 59.40 %) at day 14 (Table  3).

Effect on serum lipid profile
The levels of triglyceride, total cholesterol and LDL-C were significantly (p < 0.05) elevated and the level of serum HDL-C was decreased in the diabetic control group as compared to normal control.
After supplementation with methanol extract and fractions the alteration in lipid metabolism was significantly (p < 0.05) affected as evidenced by decreased serum triglyceride (TG), total cholesterol (TC) and LDL-cholesterol levels and increased HDL cholesterol concentration (Table 5)

Effect on body weight
There was reduction in the body weight of the animals after induction of diabetes with alloxan monohydrate. A significant (p < 0.05) increase in body weight was noticed after treatment with the extract and fractions (Table 6).

Histopathology
The pancreas of the non diabetic normal control mice (Group 1) showed sheets of normal sized glands lined by columnar epithelium with basally located nuclei surrounded by vesicular cytoplasm. Interspersed within these glands are islands of normal sized Islet of Langerhans. There was normal pancreatic histoarchitecture of islet β cells ( Figures  1A & B). The pancreas of the diabetic mice untreated (Group 2) were severely degenerated, and showed sheets of small sized glands lined by columnar epithelium with basally located nucleus and vesicular cytoplasm. No Islet of Langerhan was seen (Figures 1C & D). Photomicrograph of a pancreatic section of Group 3, (glibenclamide treated diabetic mice) (Figures 1E & F) revealed normal histoarchitecture with increased number of islet cells when compared to the untreated Group 2 that were severely degenerated. Histological examinations revealed a mildly distorted architecture of the pancreatic β cells islet of diabetic mice in group 4, 6, 8, 10 and 12 treated with low doses (250 mg/kg) of methanol extract and different fractions (Figures 2C & D, 3C & D, 4C & D, 5C & D and 6C &  D). Diabetic Groups 5 and 7 that were treated with high dose (500 mg/kg) of the crude extract and EF also had mildly despaired architecture of the pancreatic β cells islet ( Figures  2E & F, and 3E & F). Histological examinations showed a marked rejuvenation of the pancreatic β cells islet of diabetic mice in Groups 9, 11 and 13 treated with high doses (500 mg/kg) of HF, BF and WF (Figures 4E & F, 5E & F and 6E &  F) . The pancreas of all the diabetic mice (Groups 4 -13), treated with the two doses of the methanol extract and fractions, showed clusters of variably sized regenerated Islet of Langerhans within sheets of normal exocrine pancreas.      surrounded by a fine capsule (long arrow). The photomicrograph shows the regenerative effect of butanol fraction (500 mg/kg).

Figure 6
Group 12 (Plates C and D) compared with Group 2 (Plates A and B). The pancreas of this group (WF 250 mg/kg) revealed secretory acini with bulky pancreatic islet (long arrow). The histology reveals intralobular duct at low power (short arrow). The photomicrograph indicates the regenerative effect of water fraction (250 mg/kg) Group 13 (Plates E and F) compared with Group 2 (Plates A and B). The pancreas of this group (WF 500 mg/kg) revealed secretory acini with bulky pancreatic islet (long arrow). The nucleus appears pyknotic. The photomicrograph indicates the regenerative effect of water fraction (500 mg/kg)

DISCUSSION
In this study, the crude methanol extract and fractions of D. tripetala reduced fasting blood glucose levels in alloxan induced diabetic mice when compared to the untreated diabetic control. The glucose lowering effect of the extract and fractions may be due to the stimulation of β -cells of pancreatic islets [27] akin to the action of glibenclamide, a sulphonylurea known to produce hypoglycemic effect primarily through release of insulin from the pancreatic β cells [28]. The results of these studies are in line with previous report on some plants with similar phytoconstituents, which have been confirmed to have both, in-vivo and in-vitro antidiabetic activity [29,30,31].
The presence of flavonoids and saponins has previously been reported in ethanolic fruit extracts of L. camara which demonstrated hypoglycemic activity in streptozotocin induced diabetic male Wistar rats [32]. Flavonoids act on various molecular targets and regulate different signaling pathways in pancreatic β-cells, hepatocytes, adipocytes and skeletal myofibers [33]. Saponin fraction isolated from Momordica charantia reduced blood glucose levels and increased insulin secretion and glycogen synthesis in alloxan induced diabetic mice [34]. Diabetic dyslipidaemia is marked in alloxan induced diabetic mice by elevated triglycerides, total cholesterol, LDL-C and decreased HDL-C, and these constitute important cardiovascular risk factors [29]. The extract and fractions supplementation resulted in significant attenuation in the level of serum HDL-C. After supplementation with the extract and fractions, the alteration in lipid metabolism was significantly attenuated as evidenced by decreased serum triglyceride (TG), total cholesterol (TC), and LDL-cholesterol levels and increased HDL-cholesterol concentration in diabetic mice. These findings are in agreement with that of Kaleem et al. [35], who reported that the ethanolic extract of Nigella sativa seeds produced significant decrease in serum lipid profile. The reduction in serum total cholesterol concentration by the extract is beneficial and may reduce the risk of cardiovascular disease. Elevated HDL-C is very significant in human health because it improves the transportation of cholesterol from plasma to the liver for biotransformation and excretion, thereby preventing atheroma formation and blood vessel occlusion (Nwajo, 2015).
Aja et al. [37], reported a significant (P < 0.05) decrease in the body weight of alloxan diabetic rats and a significant increase following the administration of ethanol extract of Cajanus cajan leaf. This is consistent with the observation of the World Health Organization, that diabetes mellitus is often characterized by rapid and significant weight loss leading to fatigue which is not easily reversed. Photomicrograph of the pancreas showed atrophic pancreatic islet with vacuolations which indicates significant damaged Islets of Langerhans in the beta cells. The regenerative effect of the extract and fractions on the pancreatic cells indicate possible secretagoge effects on the production of insulin. The regeneration of the Islets of Langerhans may be due to the effect of the extract and fractions on the pancreatic cells, which resulted in the production of insulin.


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CHANGES OF SOIL PHOSPHORUS FRACTIONS IN PARENT MATERIAL OF A MOLLISOL AT THE EARLY PEDOGENIC STAGE IN NORTHEAST CHINA

Fertility degradation of Mollisols affects world food security. Understanding the degradation mechanisms of Mollisols is essential in order to restore the soils’ productivity. A long-term field experiment was conducted to assess soil phosphorus (P) concentrations and fractions in a Mollisol at its early development stage under different cropping systems in Northeast China. Parent material (PM) and mature Mollisol (OM) were used for comparison. Organic P (Po) concentrations under natural fallow (Pnat), alfalfa (Medicago sativa L.) (Palf) and soybean-maize (S-M) rotation without fertilizer application (PCS-F) increased by 68-74% after 10 years. S-M rotation with mineral fertilizer (PCS + F), organic matter amendment (PCS + F + OM), or crop straw return (PCS + F + BM) increased Po concentrations by 74.1-108 mg kg, which is equivalent to 33% of total Po in the OM; and increased Ca2-P, Ca8-P, Al-P and Fe-P concentrations by 0.9-1.3, 7.1-8.8, 2.1-2.7 and 1.6-1.7 times, respectively. 67% of the applied fertilizer P was transformed to Pi for the treatments with chemical fertilizer. The results indicate that return of crop residue combined with chemical fertilizers enhances the accumulation of P in soils at their early development stage, which has important implications for the fertility restoration of a Mollisol in Northeast China and elsewhere in the world.

Introduction
Phosphorus (P) is one of the essential elements for crop growth and development. Over 80% of P absorbed by crops comes from a soil reserve (Fageria et al., 2008) and applied fertilizer P reverts to less available forms in the soil (Han et al., 2005a, b). Therefore, the fractions and availability of soil P, and the transformation and availability of applied fertilizer P have received much attention in agronomic research (Milic et al., 2019;Sun et al., 2019). Soil P fractions include inorganic P (Ca2-P, Ca8-P, Al-P, Fe-P, O-P, Ca10-P) and organic P (labile organic P, moderately labile organic P, moderately stable organic P and highly stable organic P). Generally, organic P (Po) accounts for 30-65% of the total P in soils (Pätzold et al., 2013;Jin et al., 2013), while organic P in Mollisol accounts for 42-69% of total P , which can be attributed to the relatively high organic matter contents (Hou et al., 2014). Crops directly utilize inorganic P (Pi) in soil solution (generally orthophosphates), which is in dynamic equilibrium with phosphate minerals and surface adsorbed P in soil (Xiang et al., 2004). With the advancement of soil development, Po fractions become dominant and P availability is more closely related to the activity of soil microorganisms (van der Bom et al., 2019; Richardson et al., 2011). Generally, plant available P comes from the pools of Fe-P, Al-P, moderately labile organic P (MLOP) and labile organic P (LOP) in acid soil; while from the pools of Ca2-P, Ca8-P, LOP and MLOP in alkaline soil (Li et al., 2005). Therefore, Po fractions play an important role in P supply, especially in soils with relatively high organic matter content (Vanden Nest et al., 2016;Nobile et al., 2020).
The development and utilization of a Mollisol in Northeast China underwent both natural virgin stages through the stages of cultivation without fertilizer application, cultivation with organic matter application, and cultivation with the combination of organic matter and inorganic fertilizers. The impacts of different cropping systems and agricultural practices on the content and fractions of P in Mollisol have been reported . Mollisol was fertile in nature and could sustain P supply under both natural and agricultural production systems. Excessive applications of chemical P fertilizers increased both available and unavailable P fractions . However, the soils in Northeast China suffered from severe erosion as a result of intensive agricultural practices, which has caused 60% loss of soil organic matter content (Miao et al., 2019). In some extreme cases, soil erosion has removed the surface layer, with exposed parent material (C horizon). It is important to understand P transformations in soil during the period from parent material to current stage.
The restoration of degraded soil has been undertaken at a large-scale and proven to be successful in terms of above-ground ecosystem properties. However, it still lacks understanding regarding soil development in relation to vegetation changes (Griffiths et al., 2008;Harris, 2009), which could be attributed to the fact that it takes a long time to build a high level of soil fertility under natural conditions (Harrison et al., 2008). Anthropogenic activities can accelerate the pedogenic process as indicated by a significant increase in soil organic matter content with all the treatments including no fertilizer, chemical fertilizer and chemical fertilizer plus organic residue amendments during the first eight years of soil development from parent material (You et al., 2017). The inputs of crop residue and manure changed the distribution of carbon (C) density fraction and humic substances. High organic matter input first caused an increase in labile C pool, followed by stable C fractions . Soil microbial activity increased with tillage and organic amendment at the early pedogenic stage of parent material, as evidenced by an increase in soil microbial biomass-C and total PLFAs, as compared to the parent material . The soil food web was quickly developed with fertility restoration practices (Li et al., 2016). However, information was limited about P transformations at the initial stage of soil development from the parent material.
Prior studies have focused on the impact of different vegetation types and agricultural practices on P fractionation in mature Mollisol, with much less attention being paid to young soils. Lu et al. (2015) reported that fertilizer P applied in Mollisol was transformed to Al-P and Fe-P, with less than 20% being accumulated as Po. Few reports are available about the effects of cropping systems (or vegetation types) and related agricultural practices on the changes of P fractions in the initial stages of soil development. Accordingly, a field experiment was established in 2004 to examine soil development from parent material (C horizon) of a Mollisol. To simulate the effects of cropping systems and agricultural practices on P fractions at the early development stage of soil, it was hypothesized that application of exogenous P including chemical fertilizer P and P in organic amendment can enhance the accumulation of P in parent material, which can reach relatively high levels within a short term. The objectives of this study were to: (1) identify P fractions in parent material and; (2) determine the effect of cropping systems and agricultural practices on P fraction in the initial stage of soil development.

Site description and soil sampling
The long-term restoration experiment was located in the center of the Mollisol' area in Northeast China at the State Key Experimental Station of Agroecology, Chinese Academy of Sciences, Hailun, Heilongjiang province (47°26N, 126°38S). The Mollisol was derived from the sedimentary material of loamy loess parent material (Xiong et al., 1987). Typical soil profiles consist of A-AB-BC-C horizons (A, 0-60 cm; AB, 60-115 cm; BC, 115-150 cm; C, > 170 cm depth). The depth to bedrock is > 20 m. Soil classification is Pachic Haploborolls (fine-silty, mixed, superactive, mesic Pachic Ultic Haploxeroll) according to the USDA Soil Taxonomy (Soil Survey Staff, 2014). The climate in the region is typical temperate continental monsoon, characterized by cold winters and hot summer; with the mean annual temperature of 2.2 ℃. The average total annual precipitation is 550 mm, with approximately 358 mm occurring from June to August.
The long-term restoration experiment started in June 2004 with 24 enclosed plots (each plot measured 1.4 m long, 1.0 m wide and 0.8 m deep). The enclosures consisted of cement strips (0.2 m wide and 0.8 m deep) that protruded 0.1 m above the ground. These enclosures were refilled with parent material taken from the 2 m depth (C horizon) below surface near the experimental station. The parent material had a semi-blocky structure. Nylon mesh with 75 µm openings was placed on the parent material at 0.8 m depth to prevent root penetration, but it allowed water and nutrient exchange. The parent material contained 4.79 g C and 0.41 g N kg -1 soil, had a pH of 6.88 (soil: DI water 1:2.5), bulk density of 1.35 g cm -3 , 420 g kg -1 clay (< 0.002 mm) and 356 g kg -1 silt (0.02-0.002 mm). The dominant clay minerals were vermiculite, chlorite and illite ( Fig. 1).

Figure 1. The experimental plots in the field
The following six treatments were assigned to the 24 plots with the parent materials as a randomized block design with four replications: natural fallow without weed control (vegetation consisted mainly of Leymus chinensi, Poa annua L. and Equisetum tataricus) (Pnat), N-fixing alfalfa (Medicago sativa L.) (Palf) and four Soybean (S)-Maize (M) rotation: (i) S-M without mineral fertilizer and biomass removed (PCS-F), (ii) S-M with mineral fertilizer and biomass removed (PCS + F), (ii) S-M with mineral fertilizer and partial return of residue as a soybean powder-maize straw mixture (PCS + F + OM) and (iv) S-M with mineral fertilizer and all biomass incorporated (PCS + F + BM) ( Table 1 and 2). The biomass in each plot of Pnat and Palf treatments was left in the fields as mulch. In P + F-OM, 2250 kg ha -1 ground soybean seeds that had been baked at 200 ℃ and 4500 kg ha -1 chopped (20-50 mm long pieces) maize straw were incorporated into the soil at the same time as the other residue return treatments. The C content in the soybean powder and maize straw was equivalent to the C content of a typical organic manure application and return of maize straw C in this region . In the PCS + F + BM treatment, the biomass of each year's crop was incorporated into the field, but the maize grain and soybeans were pulverized as above. The fertilizer sources were diammonium phosphate and potassium sulphate, applied at 54, 60, and 54 kg N, P and K ha -1 year -1 , respectively, for soybean and maize. An additional 140 kg N ha -1 year -1 was applied as urea at the V8 stage of maize growth. The parent material was tilled to 20 cm depth with a spade to incorporate the chopped crop residue, and ridges (70 cm wide and 25 cm high) were made after applying the organic amendments. The first crop of rotation was soybean. The crop densities were 70000 and 270000 plants ha -1 for maize and soybean, respectively. The varieties of soybean and maize were Dongsheng 7 and Haiyu 6, respectively. The crops were sown in rows in May and harvested in October. At harvest, the above-ground biomass in each plot was cut and weighed, and subsamples were oven-dried at 60 ℃ to determine the dry weight of grain and non-grain biomass.  Soil samples at the surface layer (0-20 cm) were collected from the six treatments in all four replications after harvest in October 2013. Composite soil samples, consisting of four random subsamples, were removed with a spade from the ridge and furrow locations and mixed thoroughly. The soil surface layer (0-20 cm) of each treatment was air-dried after removing all visible roots and plant fragments and sieved through a 2 mm or 0.25 mm sieve. The samples of parent material (PM) were taken below 2 m depth in 2004. Mature Mollisol (OM) was sampled from the A horizon (0-20 cm) from an adjacent experimental field, which had been cropped to a maize and soybean rotation since 1993 and received only mineral N (120 g kg -1 , urea) and P (51.8 g kg -1 , diammonium phosphate) fertilizers for maize and N (20.3 g kg -1 , urea) and P (51.8 g kg -1 diammonium phosphate) fertilizers for soybean. All aboveground biomass had been removed from field of the mature Mollisol and soil tillage had been manually conducted to the 20 cm depth using a spade.

Soil sample analysis
The soil samples were air-dried and sieved through a 2-mm sieve for the laboratory experiment. Total P (T-P) in soil samples was determined by digesting soil in a tri-acid mixture (HNO3, HClO4, and H2SO4 at a 3:1:1 ratio) and P concentration in the digested samples determined calorimetrically using the vanado-molybdate-yellow color method (Jackson, 1973). Soil total organic P was determined after the soil was combusted at 550 °C and extracted with 4 M H2SO4 (Anderson, 1960). Available P was analyzed with the colorimetric molybdenum method following extraction with 0.5 M NaHCO3 (Olsen et al., 1957).

Organic P fractions
Soil organic P (Po) was separated into four fractions using a modified sequential extraction procedure developed by Bowman et al. (1978) and modified by Fan et al. (1990). A flow diagram for the Po fraction procedure is given in Figure 2. Highly stable organic P (HSOP) was determined by subtracting the moderately stable organic P (MSOP) from the stable organic P.  (Yang et al., 2006)

Statistical analysis
The analysis of variance (ANOVA) was performed using the SPSS (version 16, IBM SPSS Statistics, Somer, NY) to determine the significant difference between the treatments. Standard errors were calculated for mean values of determinations. The multiple comparison test was performed using Fisher's (protected) Least Significant Difference (LSD) at the 0.05 significant level. A principal component analysis (PCA) of the soil phosphorus fractions for all the field treatments, mature Mollisol and parent material was generated using CANOCO 5.0 software (written by Cajo J. F. ter Braak and Peter Smilauer, http://www.canoco5.com).

Total P and Olsen P
Compared to parent material (PM), total P in unfertilized soils (Pnat, Palf and PCF-F) did not significantly increase (P > 0.05), but Olsen P did (P < 0.05) (Fig. 3). For the treatments with mineral fertilizers and organic matter amendment (PCS + F, PCS + F + OM and PCS + F + BM), total P concentrations increased by 282.1-330.6 mg kg -1 (Fig. 3), which was equivalent to the total P concentration in the OM. Olsen P concentrations increased by 93.3-114.9 mg kg -1 (Fig. 3), which was higher than that in the OM, indicating that application of chemical fertilizers and organic amendment enhanced P accumulation in soil after the initial10 years of treatments from parent material.

Total organic and inorganic P
Compared to PM, the treatments without chemical fertilizers (Pnat, Palf and PCS-F) increased organic P (Po) but decreased inorganic P (Pi) (Fig. 4). However, the treatments with chemical fertilizers and organic amendment (PCS + F, PCS + F + OM and PCS + F + BM) significantly increased Pi concentrations with the highest value of 586.8 mg kg -1 occurred to the PCS + F treatment, which was higher than that in OM (348.7 mg kg -1 ) (Fig. 4). The Po concentrations in all the field treatments increased by 68.2-138%, as compared to PM, and the highest value (186.2 mg kg -1 ) was observed in the PCS + BM treatment, which was 39.4% of Po in the OM. OM had a similar Pi concentration as the PM, but had more Po (Fig. 4). The percentages of Po in the total P were lower in PM than OM, which were 57.4%, 18.2% and 20.6 -31.9%, respectively for OM, PM and all the tested soil. The percentage of Po in total P within Pnat, Palf and PCS-F treatments was higher than those in the PCS + F, PCS + F + OM and PCS + F + BM treatments (Fig. 4). 320 mg P kg -1 was applied to the soil as diammonium phosphate every year during the 10 years of the experiment in the treatments with chemical fertilizers (PCS + F, PCS + F + OM and PCS + F + BM) ( Table 2). Compared to PM, total P concentrations in the treatments with chemical fertilizers increased by 282.1-330.6 mg kg -1 , which was comparable to the amount of P applied in fertilizers (Fig. 4). The different total P concentrations among treatments could be attributed to the removal of P in aboveground biomass, which possibly caused P loss in the PCS + F treatment. Total P concentrations in the PCS + BM treatment reached the highest value of 330.6 mg kg -1 (Fig. 3), which is higher than the total P input via fertilization. This can be attributed to biomass decomposition including aboveground and root biomass (Fig. 3). This indicates that most of the applied P accumulated in the top 0-20 cm of soil depth. This increased in total P was made up of the increase of 211.1-235.9 mg kg -1 of Pi and 74.1-108.0 mg kg -1 of Po over the 10-year period of the experiment (Fig. 4). Compared to PM, available P in the PCS + F, PCS + F + OM and PCS + F + BM treatments increased by 93.8-114.9 mg kg -1 , indicating that approximately 33% of the applied fertilizer P accumulated in the Olsen P pool (Fig. 4).

Phosphorus fractions
The total P concentrations in soil increased after the initial 10 years of experiment regardless of treatments, whilst vegetation types and agricultural practices showed different effects on Pi fractions ( Table 3). There was an increase mainly in the concentrations of LOP and MLOP. Compared with the LOP of PM without P fertilizers, the highest concentration of LOP was found in the PCS + F + OM treatment, which accounted for approximately 25% of the amount in OM ( Table 3). MLOP concentrations in all the tested soils increased by 2.9-5.3 times, with the highest value in the PCS + BM treatment, which was similar to that in OM ( Table 3). Treatments with P fertilization increased MSOP by 15.3-62.2%, and this accounted for approximately 33% of the MSOP in the OM ( Table 3). The PCS-F and PCS + F treatments decreased the contents of HSOP, but the rest of treatments did not affect HSOP concentrations. The HSOP concentrations in all the tested soil were much lower than that in OM ( Table 3).  SOP, stable organic P; Ca2-P, NaHCO3-soluble P; Ca8-P, NH4Ac-soluble P, Al-P NH4F-soluble P; Fe-P, NaOH-NaCO3-soluble P; O-P, occluded P; Ca10-P, H2SO4-soluble P. Values followed by different letters at same column differ significantly at P < 0.05 In contrast with PM, the treatments without fertilization (Pnat, Palf and PCS-F) decreased the concentrations of Ca2-P, Al-P and Ca10-P and increased the concentrations of Ca8-P and Fe-P, whereas the treatments with fertilization (PCS + F, PCS + F + OM and PCS + F + BM) significantly increased the Pi fractions except for Ca10-P and O-P ( Table 3). The concentrations of Ca2-P, Ca8-P, Al-P, and Fe-P increased by 0.9-1.3, 7.1-8.8, 2.1-2.7 and 1.6-1.7 times ( Ca8-P, Al-P, and Fe-P in OM were much higher than those in PM, but the concentrations of O-P and Ca10-P in OM was lower than those in PM ( Table 3).

Pedogenic process of parent material under Mollisol
Principal component analysis demonstrated that a large difference was found between OM and PM (Fig. 4), parent material with the field treatments had developed into mature Mollisol over the 10-year period of the experiment. Compared with the treatments without fertilizers (Pnat, Palf and PCS-F), the soils receiving fertilized treatments were closer to mature Moillisol (Fig. 5). Inorganic P concentrations in the soils treated with chemical fertilizers (PCS + F + BM, PCS + F + OM and PCS + F) were similar to the Pi in the OM, but a large difference in Po concentration exists between PM and OM (Fig. 5).

Changes in different P fractions
Most P was present as calcium phosphates at the early pedogenic stage of rock, and vegetation occurrence converted Pi to Po in soil (Lu, 1980). In the parent material of Mollosil, the Pi concentration was 78.2 mg kg -1 , which accounted for 16.55% of that in mature Mollisol (Fig. 4). The unfertilized treatments (Pnat, Palf and PCS-F) did not increase total P, but increased Po to 131.5-136.1 mg kg -1 , accounting for 27.8-28.8% of Po in OM (Fig. 4), which is consistent with the organic carbon change measured by You et al. (2017), who reported that organic carbon increased by 7-60% in the same site. Soil organic carbon gradually increased in the parent material after vegetation occurrence, which resulted in an increase in Po, but a decrease in Pi concentration of a similar amount, indicating the transformation of Pi to Po during soil development from parent material. Specifically, Ca2-P, Al-P, O-P and Ca10-P were transformed to LOP and MLOP ( Table 3), indicating that plants absorbed Pi and returned Po in residues to soil.
The Pnat and Palf treatments demonstrated a similar effect on the transformation of P fractions, which differed from the PCS-F treatment ( Table 3) where SOC was only derived from root and litter. This resulted in a relatively low Po concentration with a decrease in HSOP in the PCS-F treatment. Perroni et al. (2014) has documented that Alfalfa growth resulted in an increase in Po in a desert scrub soil in Mexico. Parent material used in the present study had a low SOC (4.8 g kg -1 ) in this study, and the field treatments rapidly increased SOC (You et al., 2017) and subsequently Po in the soil. However, Alfalfa did not show any advantage in the accumulation of Po fractions in the parent material.
A mature Mollisol had been cultivated for hundreds of years, underwent no fertilization stage, farmyard manure stage and chemical fertilization stage. The accumulation of Po increased during soil development and utilization. However, the accumulation of Po concentration became steady at a certain level without the application of inorganic P fertilizers (Song et al., 2011). The transformation of Pi to Po occurred at a lower rate in the treatments without fertilizers where Po accumulation per year accounted for 1% of that in the mature Mollisol (Table 3). Apparently, external P inputs could accelerate the accumulation of Po in soil under field conditions.

Effects of fertilizer application on P fractions
In this study we attempted to accelerate P formation in the parent material by adding P fertilizers to contrast with the natural soil development process. P fertilizer applied to soil was mainly presented as Pi in the plant-available fractions in parent material under the imposed cropping systems and agricultural practices (Table. 3). The rate of Po accumulation in the treated parent material was relatively slow, but approximately 33% of the fertilizer P was transformed to Po. Total P concentration in the treated parent material was close to that in the mature Mollisol, which may be attributed to the application of P fertilizers over the 10-year of the experiment (Fig. 4). Soil development in the Mollisol parent material was accelerated by P fertilization based on the result of PCA (Fig. 5), whilst soil development in the vegetation restoration contexts may take hundreds of years to accomplish this level of fertility (Harrison et al., 2008). Po accumulation was as high as 40% in the mature Mollisol, which is an evidence of the acceleration of soil development in the parent material used in this study. Reddy et al. (2000) found that soil P fractionation was affected by soil type and fertilization practices, and the application of cattle manure increased the concentration of Po, activated fixed Pi in soil, and thus enhanced uptake of P by crops. Application of chemical fertilizers, straw and animal manure increased Pi concentration in the soils of Northwest China (Wei et al., 2011). Chemical fertilizers and organic manure also affected P sorption capacity in a Mollisol . The long-term experiment with a Mollisol showed that chemical fertilizers (N and P), and the combination of chemical fertilizers with pig manure increased both total P and Olsen P, dominantly in the forms of Ca2-P, Ca8-P and Al-P, but no significant increase in Po was noted (Song et al., 2011). Similar results were obtained in present study. P fertilization enhanced Pi accumulation in the treated parent material, which resulted in a significant increase in Pi with the treatments of PCS + F, PCS + F + OM and PCS + F + BM.
The concentrations of Pi showed a decreasing order of PCS + F + AM, PCS + F + OM > PCS + F > Pnat, Palf, PCS-F. The highest value reached 186.2 mg kg -1 , accounting for 39.4% of that in the mature Mollisol (Fig. 4). Accumulated Po in the parent material was mainly derived from crop residues, and a higher rate of organic amendment resulted in more accumulation of Po in soil.
Long-term field experiments have documented that organic amendment significantly increased the concentration of Olsen P and reduced soil adsorption capacity for P (Motavalli et al., 2002;Allen et al., 2006). Low molecular weight organic substances in organic amendment reduced the adsorption capacity of variable charge minerals for P by competition for adsorbing sites (Guppy et al., 2005). There was no significant difference in the total P and Olsen P concentrations between chemical fertilizers alone and the combination of chemical fertilizers and organic amendment. Applied fertilizer P was mainly presented in the Olsen P pools in the treated parent material. Compared with chemical fertilizer treatment alone (PCS + F), the treatments of chemical fertilizers with organic amendment (PCS + F + OM and PCS + F + BM) had no significant difference in Pi and Po concentrations or the Po : total P ratio.

Conclusions
The long-term field experiment was established in 2004 to determine the impact of cropping systems and agricultural practices on soil P concentration and fractionation during the first 10 years of soil development from parent material of a Mollisol. Po and Pi concentrations were 78.9 and 350.9 mg kg -1 in parent material, and 472.6 and 348.7 mg kg -1 in mature Mollisol. The treatments without chemical fertilizers (Pnat, Palf and PCS-F) enhanced P transformation from Ca2-P, Al-P, O-P, Ca10-P to Po in PM. The treatments with chemical fertilizers (PCS + F, PCS + F + OM and PCS + F + BM) increased total P concentration in PM to a level similar to that in OM, Po by 74.1-108 mg kg -1 , accounting for 20.6-31.9% of in the total P, lower than that in the Mollisol. The results from the present study indicate that agricultural practices with fertilizer application can enhance the accumulation of P during soil development from parent materials of a Mollisol in semi-arid and semi-humid climatic regions in a relatively short period. This information has important implications in the restoration of degraded Mollisols. The further studies are warranted to detect the mechanisms of P transformation during the process of fertility restoration.