(Sindh), Pakistan Fehmida Firdous, Sohail Barkati and Solaha Rahman 29

Pakistan Journal of Scientific and Industrial Research Series B: Biological Sciences

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Contents

Floral Biology, Psychophily, Anemochory and Zoochory in Chromolaena odorata (L.) King and H. E. Robins (Asteraceae) Pakkurti Vara Lakshmi, Aluri Jacob Solomon Raju, Dandangi Jeevan Ram and Kunuku Venkata Ramana 1

Sustaining Soil Productivity by Integrated Plant Nutrient Management in Wheat Based Cropping System under Rainfed Conditions Mohiuddin Dilshad, Mohammad Iqbal Lone, Ghulam Jilani, Muhammad Azim Malik, Muhammad Yousaf, Rizwan Khalid and Fakhra Shamim 9

Combining Ability Study in a 6 × 6 Diallel Cross of Maize Amanullah, Shah Jehan Khan and Muhammad Mansoor 18

Reduction of Aflatoxin B1 Contamination in Pakistani Wheat Varieties by Physical Methods Arshad Hussain, Ghosia Lutfullah and Shafqatullah 23

Studies on Transplantation of Marine Turtle Nests at Karachi Coast (Sindh), Pakistan Fehmida Firdous, Sohail Barkati and Solaha Rahman 29

Effects of Cowdung and Poultry Manure on Growth Performance of Indian Major Carps (Catla catla, Labeo rohita) and Exotic Carp (Cyprinus carpio) in Thatta District (Sindh), Pakistan Mohammad Shoaib, Syed Anser Rizvi, Faisal Ameer and Mohammad Nasir 34

Morphological Variations, Patterns of Frontal Ambulacrum Pores and Paleoecology of Heteraster renngarteni Poretzkaja (Echinoidea: Spatangoida) from Aptian Sediments of Baghin Area, Kerman, Iran Mohammed Raza Vaziri and Ahmad Lotfabad Arab 41

Antibacterial Activity of Saponin and Alkaloidal Extracts of Whole Plant of Phyllanthus niruri L., (Syn. P. franternus Webster) Victor Adeyinka Ajibade and Oladiran Famurewa 47

Development of a Predictive Model for Preparation of Banana Modified Drink Daramola Bode 53 Pak. J. Sci. Ind. Res. Ser. B: Biol. Sci. 2011 54 (1) 1-8

Floral Biology, Psychophily, Anemochory and Zoochory in Chromolaena odorata (L.) King and H.E. Robins (Asteraceae)

Pakkurti Vara Lakshmi, Aluri Jacob Solomon Raju*, Dandangi Jeevan Ram and Kunuku Venkata Ramana Department of Environmental Sciences, Andhra University, Visakhapatnam 530 003, India

(received August 25, 2010; revised January 6, 2011; accepted January 18, 2011)

Abstract. The study investigates the pollination biology of Chromolaena odorata an exotic species of India, and also its importance as forage source for insects especially butterflies. The plant possesses floral characteristics typifying psychophily. It is an important nectar source for insects and attracts butterflies, hawk moths, bees, wasps, flies and other insects, which act as facultative pollinators while collecting the forage. Among the butterflies, Nymphalids play a prime role in the pollination. Seed set rate is very high in each head inflorescence in open pollination. Seed dispersal is by both anemochorous and zoochorous modes but former is the principal one. Keywords: Chromolaena odorata, nectar, psychophily, insects, anemochory, zoochory, pollination, butterflies

Introduction the importance of this plant as forage source for insects, especially butterflies. The notable finding in Plant-insect interactions are important to understand this work is that C. odorata is an important nectar the invasiveness of certain exotic weeds around the host plant for local butterflies and at the same time world. Invasive species threaten the native biological butterflies are contributing to the expansion of the diversity of any ecosystem (Dupont et al., 2002) by distribution range through fertilization of flowers and interacting with native species in several ways. Many subsequent seed set. introduced plant species compete with natives for resources (Daehler, 2003) or benefit from mutualistic Materials and Methods interactions with native resident species, often to Chromolaena odorata is a noxious perennial weed. the detriment of native mutualism (Traveset and It is a free standing shrub and grows up to 1 m. It Richardson, 2006; Richardson et al., 2000). Several is maintained by a system of abundant, yellowish, studies have described the disruption of plant-animal fine lateral roots. The opposite, three-nerved leaves interactions caused by competition for pollinators are ovate-lanceolate, usually with a dentate margin between invasive exotics and native plants (Moragues and a long pointed tip. Seed germination starts in and Traveset, 2005). On the contrary, Larson (2008) May-June and active growth occurs during June- reported that invasive plants often occur in monotypic October. Their shoots root when touching the stands and provide a dense source of sustenance for ground. The plant possesses underground organs insects that rely on floral resources. A dense stand of which ensure the plant survival during drought or flowering invasive plant may attract more pollinators mechanical damage or in case of fire. Flowering to the area, and in the process enhances pollination occurs once in a year. Flowers are very small, white of neighbouring natives also. It is in this context, the to light purple, bisexual and zygomorphic. Calyx study has been contemplated to understand how is reduced to pappus and represented by hairs. Chromolaena odorata, an exotic species in India, is Corolla is tubular with five teeth at the tip. Stamens able to thrive well and attract a guild of insect species are 5, epipetalous, arise from the base of the corolla, during its flowering season in order to achieve great with filaments free but anthers united, representing success in sexual reproduction and in seed dispersal syngenesious condition. The anthers are dithecous, to expand its distribution range in the world. This paper have their connective prolonged into hood and their describes the pollination biology of C. odorata and bases produce hairy outgrowths, which in turn form *Author for correspondence; E-mail: [email protected] a protective envelope for the nectary. The ovary is 1 2 Aluri Jacob Solomon Raju et al. bicarpellary and syncarpous, unilocular with a rate in open-pollination. Field studies were carried single basal ovule (Fig. 1i,j). The style is surrounded out also for seedling aspects. by the nectary at the base and it is forked into two parts. Results and Discussion The the present study, C. odorata was found at an Floral biology. Flowering occured during October- elevation of over 700 m in the Seshachalam Hills of December. The inflorescences consisted of corymbs of southern eastern ghats (lat. 17° 43′ N; long. 83° cylindrical heads, emerging at the terminal part of the 90′ E) of Andhra Pradesh, India in open, sunny areas branches (Fig. 1a). A head consisted of 21 to 28 tubular of forest margins and open gaps in the forest and was florets (Fig. 1b,c). The florets opened during 0600- used for the study during the summer season of 2009. 0800 h (Fig. 1d-g). The anthers dehisced by longitudinal C. odorata grows profusely during the active growth slits prior to anthesis. The pollen grains were 196 ± 14.6 period and suppresses the growth of native low per anther and the total pollen output per floret was ground herbaceous flora. Twenty five tagged mature 980 ± 11.8 (Fig. 1h). The pollen-ovule ratio was 980:1. buds, five each from five individuals were followed The narrow anthers form a hollow space and pollen is for recording the time of anthesis and anther de- liberated into this space. The receptive surfaces of the hiscence; the mode of anther dehiscence was also stylar branches stayed in closed state and as they noted by using a 10× hand lens. Pollen was collected grew through the hollow space, brushed out the pollen from ten mature but undehisced anthers of staminate liberated in the hollow space. Further, the style stretched flowers of five different plants. Each anther was out beyond the anthers, and spread out its branches dabbed with a needle in a drop of lactophenol- which then became receptive and ready to receive aniline-blue. The anther tissue was then observed pollen from the foragers. The receptivity remained so under compound microscope and the total number of until the noon of the 2nd day and the stylar branches pollen grains was counted. The mean pollen output withered away gradually. The nectar was secreted in per anther was multiplied by the number of anthers traces only. The florets remained in place for about of a flower for obtaining the mean number of 3-4 days and later fell off if not pollinated. pollen grains per flower. The pollen-ovule ratio was Flower visitors and pollination. The florets were determined as per Dafni et al. (2005). Twenty five visited during day time by butterflies for nectar flowers, five each from five individuals were used to test stigma receptivity with hydrogen peroxide from collection and by other insects for pollen and/or nectar mature bud stage to flower drop (Dafni et al., 2005). collection; and by diurnal hawk moths, during dawn Regular observations were made on the insect and dusk hours for nectar collection. The butterflies species visiting the flowers for forage. The insects included 23 species representing Papilionidae, Pieridae, were observed for their foraging behaviour such as Nymphalidae, Lycaenidae and Hesperiidae (Table 1; mode of approach, landing, probing behaviour, the Fig. 1l-p; Fig. 2a-i). Foraging activity of nymphalid type of forage they collect, contact with essential butterflies showed a general trend characterized by organs to result in pollination and inter-plant gradual increase from morning to noon and gradual foraging activity in terms of cross-pollination. The decrease towards the evening (Fig. 4). This foraging observation days for these aspects were thirty and trend was observed almost throughout the flowering the time spent each day was five to eight hours. season for butterflies of all families during this study. Twenty flowers each from fifty plants were selected The data collected on the foraging visits of butterflies for collecting information on insect activity. Three of each family showed that Nymphalids made 61%, to five specimens of each butterfly species collected Papilionids 17%, Pierids 11%, Lycaenids 8% and at different times of the day were brought to Hesperiids 3% of the total visits. The cylindrical heads laboratory for examining their proboscis under equipped with a cluster of florets provided convenient microscope for the presence of pollen grains in landing place for butterflies and this arrangement also order to assess their role in pollen transfer and enabled them to probe several flowers during each visit pollination. Fruit and seed aspects were recorded in succession for nectar before their departure. The based on fifty sample heads collected from ten flowering heads borne terminally stood out prominently plants. A sample of ten tagged cylindrical heads and the butterflies were found to be attracted to them consisting of 489 flowers was followed for fruit set even from a long distance. The butterfly proboscides Floral Biology of Chromolaena odorata 3

Fig. 1. Chromolaena odorata: a. Flowering inflorescences; b. Individual cylindrical head; c. Mature head; d. & e. Mature florets; f. Open floret; g. L.S. floret; h. Pollen grain; i. Ovary; j. Ovule; k. Mature seeds ready for dispersal; l. Pachliopta hector; m. Papilio polytes; n. Acraea violea; o. Ariadne ariadne; p. Junonia lemonia. 4 Aluri Jacob Solomon Raju et al.

Fig. 2. Chromolaena odorata: a. Euploea core; b. Phalanta phalantha; c. Danaus genutia; d. Danaus chrysippus; e. Tirumala limniace; f. Delias eucharis; g. Catopsilia pyranthe; h. Tarucus nara; i. Ypthima asterope; j. Macroglossum gyrans; k. Cephonodes hylas; l. Apis dorsata; m. Apis cerana; n. Xylocopa sp. Floral Biology of Chromolaena odorata 5

Table 1. List of insect foragers on Chromolaena odorata daytime. They hovered over the floret heads, inserted

Family Scientific name Common name the proboscis, and collected nectar quickly from as many florets as possible from several flowering heads Lepidoptera Papilionidae Pachliopta hector Crimson Rose in quick succession. Their proboscis gained contact Papilio polytes Common Mormon with the stylar surfaces of each floret they visited Papilio demoleus Lime Butterfly and this contact was considered to be resulting in Pieridae Catopsilia pyranthe Mottled Emigrant pollination. They collected nectar from a number of Anaphaeis aurota Pioneer plants within a patch and in different patches Delias eucharis Common Jezebel Nymphalidae Ariadne ariadne Angled Castor contributing to cross-pollination. Junonia lemonias Lemon Pansy Junonia hierta Yellow Pansy Other insect foragers included Hymenoptera and Precis iphita Chocolate Pansy Diptera (Table 1; Fig. 2 l-n, Fig. 3 a-d). Of these, bees Acraea violae Tawny Coster and wasps were frequent foragers during flowering Euploea core Common Indian Crow peak season in November; other foragers visited the Phalanta phalantha Common Leopard florets/heads occasionally. They approached the Danaus genutia Striped Tiger Danaus chrysippus Plain Tiger flowering plants in upright position and landed on the Ypthima asterope Common Three- Ring head inflorescences prior to probing the florets. Bees Melanitis leda Common Evening Brown were found to probe the florets for nectar as well as Tirumala limniace Blue Tiger pollen; it was not clear whether they were successful Parantica aglea Glassy Tiger Neptis hylas Common Sailer in getting the nectar with their short proboscides but Lycaenidae Everes lacturnus Indian Cupid they voraciously collected pollen. The wasps and other Tarucus nara Rounded Pierrot insects probed the flowers to collect nectar only; their Hesperiidae Borbo cinnara Rice Swift success depended on the force with which they Sphingidae Cephonodes hylas Coffee Hawk moth Macroglossum gyrans Diurnal Hawk moth stretched out their tongue into the tubular corolla for Hymenoptera nectar collection. Apidae Apis dorsata Rock Honey Bee Apis cerana Indian Honey Bee A. florea Dwarf Honey Bee Xylocopa sp. Large Carpenter Bee Crabronidae Stizus sp. Digger Wasp Scoliidae Scolia quadripustulata Blue-winged Wasp Scolia sp. Blue-winged Wasp Diptera Syrphidae Helophilus sp. Hover Fly

invariably contained pollen grains ranging from 15-35 in Papilionids, 14-23 in Pierids, 29-189 in Nymphalids, 8-17 in Lycaenids and 9-21 in Hesperiids. All these butterflies stretched out their proboscis to reach the floret base for accessing nectar; while doing so the proboscis invariably contacted the stylar surfaces and hence affected pollination. They frequently moved between individual plants of C. odorata which had Fig. 3. Chromolaea odorata: a. Stizus sp.; patchy distribution along the forest margins and in the b. Scolia guadripustulata; c. Scolia sp.; open gaps of the forest interior. d. Helophilus sp. Hawk moths (Fig. 2j,k) showed intense foraging activity during dawn hours, when the florets offered Fruiting ecology. Natural fruit set was 89%. The fruits fresh nectar, while reduced activity during dusk hours were small and one-seeded. The seeds were dry, light when the florets offered only the left-over nectar and 4 mm long, brownish grey to black achene with a which was not fresh; they rarely foraged during small hook and tipped with pale brown 5-6 mm long

Floral Biology of Chromolaena odorata 7

Asteraceae, Tridax procumbens Danaid and Pierid In C. odorata, seed production is prolific in each head butterflies have been shown to be the main pollinators inflorescence in open pollination. The fruit produces (Balasubramaniam, 1989). Therefore, psychophily is a single seed due to the presence of a single ovule in adaptive and advantageous for the plant to maximize each floret (Fig 1 i, j). Anemochory is the principal pollination with specialized florets and nectarivore of mode for seed dispersal; it is highly effective due to butterflies. seed dispersal during summer season when ambient temperature is high and relative humidity is very low. The diurnal hawk moths, Macroglossum gyrans and Further, zoochory is also functional due to the presence Cephonodes hylas, also visited C. odorata flowering of small hooks on the seeds; animals especially heads during dawn and dusk hours. The dawn foraging domestic animals as well as humans disperse seeds. activity is energetically profitable for them while the Therefore, the dual modes of seed dispersal are foraging at dusk period may not be profitable as the instrumental in spreading the plant in the tropical florets by that time are most likely to be devoid of latitudes rapidly (Kushwaha et al., 1981). nectar due to butterflies and other insects during day time. They collected nectar on clear sunny and rainy References days and utilized this floral source until exhausted. Baker, H.G., Baker, I. 1983. Floral nectar sugar consti- Balasubramaniam (1989) reported that hawk moths tuents in relation to pollinator type. In: Handbook visited the florets of Tridax procumbens during rainy of Experimental Pollination Biology, C.E. Jones weather when most butterflies took shelter among foliage in nearby bushes. and R.J. Little (eds.), pp. 117-141, Van Nostrand Rheinhold, New York, USA. Balasubramaniam (1989) reported that honey bees, Balasubramaniam, M.V. 1989. Studies on the ecology flies, ants and thrips acted as facultative pollinators of butterfly pollination in South India. Annals of of Tridax procumbens. In the present study, bees Entomolology, 7: 31-41. visited the flowering heads to probe both nectar and Daehler, C.C. 2003. Performance comparisons of pollen but it was not clear whether they accessed the co-occurring native and alien invasive plants: nectar; however, they collected pollen voraciously implications for conservation and restoration. and easily. The wasps and flies also visited the flow- Annual Review of Ecology, Evolution and System- ering heads; the former collected nectar easily with atics, 34: 183-211. their tongue lengths exceeding the corolla tube while Dafni, A., Kevan, P.G., Husband, B.C. 2005. Practical the latter attempted to collect nectar but it was not Pollination Biology, 590 pp., Enviroquest Ltd., clear whether they were successful in nectar collec- Cambridge, Canada. tion. Nevertheless, bees, wasps and flies affected Dupont, Y.L., Hansen, D.M., Valido, A., Olesen, J.M. pollination while collecting the forage; they may be 2004. 2002. Impact of introduced honey bees on designated as facultative pollinators since they the native pollination of the endemic. Echium visited the florets/heads during peak flowering wildprettii (Boraginaceae) on Tenerife Canary season or occasionally. Further, the pollen collecting activity of bees can contribute to the unavailability Islands. Biological Conservation, 118: 301-311. of pollen for pollination purpose and hence they Faegri, K., van der Pijl, L. 1979. The Principles of acted primarily as pollen thieves. The study shows Pollination Ecology, 244 pp., Pergamon Press, that C. odorata being an exotic plant is psychophilous Oxford, New York. and its prevalence in the forest areas is a potential Kushwaha, S.P.S., Ramakrishnan, P.S., Tripathii, R.S. source of nectar for butterflies for a period of three 1981. Population dynamics of Eupatorium months. Although it is a menace due to its prevalence odoratum in successional environments following and suppressive activity on the growth of certain slash and burn agriculture. Journal of Applied native low ground herbs, its flowers are most attrac- Ecology, 18: 529-535. tive to butterflies throughout its distribution range Larson, D.L. 2008. Invasive plants and pollinator and act as a provisioning post for the butterflies. interactions. Endangered Species Bulletin Fall 8 Aluri Jacob Solomon Raju et al.

2008, 33: pp. 46-49, USGS Northern Prairie 63-93. Wildlife Research Center, Minnesota, USA. Traveset, A., Richardson, D.M. 2006. Biological Moragues, E., Traveset, A. 2005. Effects of Carpobrotus invasions as diruptors of plant reproductive spp. on the pollination success of native plant mutualisms. Trends in Ecology and Evolution, 21: species of the Balearic Islands. Biological Con- 208-216. servation, 122: 611-619. Van Doorn, W.G. 2002. Does ethylene treatment Richardson, D.M., Allsopp, N., D’Antonio, C.M. mimic the effects of pollination on floral lifespan Milton, S.J., Rejmanek, M. 2000. Plant invasions and attractiveness? Annals of Botany, 89: 375- the role of mutualisms. Biological Reviews, 75: 383. Pak. J. Sci. Ind. Res. Ser. B: Biol. Sci. 2011 54 (1) 9-17

Sustaining Soil Productivity by Integrated Plant Nutrient Management in Wheat Based Cropping System Under Rainfed Conditions

Mohiuddin Dilshada*, Mohammad Iqbal Loneb, Ghulam Jilanib, Muhammad Azim Malikb, Muhammad Yousaf b, Rizwan Khalida and Fakhra Shamimb aSoil Fertility Survey and Soil Testing Institute, Rawalpindi, Pakistan bPMAS Arid Agriculture University, Rawalpindi, Pakistan

(received April 14, 2010; revised July 27, 2010; accepted August 18, 2010)

Abstract. The study of the use of organic (FYM) and inorganic (NPK) nutrient sources with biofertiliser on wheat-fallow and wheat-maize cropping system under rainfed environment revealed significant increase in biometric parameters of wheat during winter and summer seasons of two years. During both the seasons, application of ½ NPK + ½ FYM + Biopower (brand) produced the highest grain yield (3684 kg/ha) and (3781 kg/ha) of wheat with the maximum N uptake of 357 kg/ha, P uptake of 51 kg/ha and K uptake of 215 kg/ha. Wheat-maize cropping system was found to be profitable economically with integrated use of mineral and organic and/or Biopower under rainfed conditions of Pakistan. Keywords: integrated plant nutrient management, wheat-maize cropping, wheat-fallow cropping, rainfed area, biofertiliser, farm yard manure

Introduction tion had similar effects when compared with organic Growing concerns about the environmental conse- fertiliser and chemical fertiliser treatments. quences of use of mineral fertilizers and their future Hence, the present experiment was carried out to cost perspectives emphasize the need to develop new evaluate the integrated use of organic and mineral agricultural technologies that are sustainable both fertilisers with biofertiliser ‘Biopower’ on the yield of economically and ecologically. Use of chemical wheat. fertilizers alone does not sustain productivity under continuous intensive cropping, whereas inclusion of Materials and Methods organic materials improves physical soil properties A field experiment pertaining to wheat based cropping (Benbi et al., 1998), builds up soil fertility and increa- system was conducted at the research farm of Soil ses crop yield (Yaduvanshi, 2003). Organic materials Science and SWC Department of Arid Agriculture hold great promise due to their local availability as a University, Rawalpindi. Split-plot design was used for source of multiple nutrients and ability to improve soil two cropping system (CS) treatments in main plots and characteristics. According to several authors the nine integrated plant nutrient management (IPNM) improvement of fertility and quality of soil, especially practices in subplots. All treatments were applied with under low input agricultural systems, requires the three replications. The sub-plot size was 6 m x 4 m input of organic materials (Soumare et al., 2003; (24 m2). The experiment was conducted in winter Ouedraogo et al., 2001). The effect of organic nutri- 2004-05 (Rabi season) with Triticum aestivum cv. ents on crop yield is long term and not immediate. Chakwal 97 as test crop followed by Zea mays cv. A However, the use of biofertilisers along with organic/ gaiti 2002 in one main plot in summer of 2005 (Kharif inorganic materials effectively stimulates the supply season), while the second main plot remained fallow. and the release of nutrients from the nutrient sources. All treatments of subplots were applied at the same Combination of both N sources with effective micro- rate to wheat and maize. The same experiment was organisms (EM) increased the NPK concentrations repeated in winter 2005-06 and in summer 2006 on the in cotton crop (Khaliq et al., 2006). Wu et al. (2005) same crops with identical treatments. National Insti- observed that half the amount of biofertiliser applica- tute for Biotechnology and Genetic Engineering *Author for correspondence; E-mail: [email protected] (NIBGE), Faisalabad, Pakistan has developed a 9 10 Mohiuddin Dilshad et al. microbial inoculum ‘Biopower’. The biofertiliser longitude 71° 10 to 73° 55 E with 1.82 million hect- ‘Biopower’ was used for seed inoculation containing ares area. Fig. 1 and 2 present the data on mean monthly N-fixing bacteria, belonging to genus Azotobacter, rainfall and temperature during the experimental period.. Azospirillum, Azoarcus and Zoogloea. Fallow plots Chemical analysis. Composite soil sample were collec- were kept weed-free by repeated cultivation immedi- ted from the experimental field at two depths (0-15 cm ately followed by planking. There were two treatments and 15-30 cm) before sowing. One soil sample was of cropping systems: wheat-fallow (CS ) and wheat- 1 collected for each treatment from the subplot before maize (CS ). There were nine treatments of integrated 2 sowing and after harvest of wheat. Soil samples were plant nutrient management practices. These included: analyzed for various physical and chemical character- control (without NPK fertilizer, FYM or biofertiliser); istics. Soil texture was determined by hydrometer half of recommended NPK; full dose of recommended method as described by Koehler et al. (1984) and pH NPK (120-90-60); FYM @ 20 t/ha; FYM on N require- in soil water suspension (1:10) was determined with ment basis + make-up dose of P/K fertilizer; ½ NPK + pH meter (McLean, 1984). Organic carbon was deter- FYM @ 10 t/ha; ½ NPK + Biopower; ½ FYM + mined by the method given by Nelson and Sommers Biopower; and ½ NPK + ½ FYM + Biopower. The (1982). Total nitrogen was determined by Kjeldahl experimental site was located in the subtropical digestion method (AOAC, 1982). Available phos- subhumid Pothwar plateau, Pakistan at an altitude of phorus was determined by Spectronic 601 as described 513 m extending over latitude 32° 10 to 34° 9 N and by Soltanpur and Schwabe (1977). Ammonium acetate- extractable potassium was determined by flame photometer (PFP, Jenway). Data for yield components and 400.0 2003-04 2004-05 2005-06 yield was recorded at physiological maturity of wheat. 350.0 Plant and grain samples from individual treatments 300.0 were analyzed for total nitrogen by Kjeldahl digestion 250.0 method; phosphorus was determined by AB-DTPA 200.0 extractable-P method and total K concentration, by 150.0 flame photometer (Ryan et al., 2001). 100.0 Monthly rainfallMonthly (mm) 50.0 Statistical and economic analysis. Growth, yield and 0.0 soil parameters were recorded and then analyzed statistically according to standard statistical procedures described by Sokal and Rohlf (1997). Data Months showing significant difference at P=0.05 was put to comparison of treatment means by Duncan’s multiple Fig. 1. Monthly rainfall during the study (2004-2006). range test. All the data was processed using MSTAT software for statistical analysis. For economic analy-

35.0 sis, after considering the cost of fertilizer N, P, K, farm- 2003-04 2004-052004 20052005-06 yard manure and biofertilizer ‘Biopower’ application, 30.0 income from seed yield was used for economic analy- 25.0 sis (CIMMYT, 1988) using the formula: 20.0 Value cost ratio = value of increased yield 15.0 (VCR) obtained/cost of mineral/

Temperature (°C)Temperature 10.0 organic sources. 5.0 Results and Discussion 0.0 Composite soil sample analysis before the start of the experiment showed that the soil was sandy loam in Months texture with reaction of 7.8 and ECe of 0.25 dS/m. It Fig. 2. Mean monthly temperature (°C) during the had low soil organic carbon of 0.32 g/100 g. The total study (2004-2006). soil nitrogen, available P and extractable K were Soil Productivity through IPNM for Wheat Crop 11 estimated as 152, 3.45 and 80 µg/g, respectively, produced maximum average plant height of 108.9 cm showing low fertility status of soil (Table 1). followed by 106.4 cm due to treatment of ½ NPK + ½

FYM(T6). The recommended NPK treatment (T3) produced 104.6 cm of plant height, which was statistically Table 1. Physical and chemical properties of composite soil samples before the start of experiment at par with the treatment (T6) and (T8). The results during 2005-06 showed that application of ½ NPK + Soil series : Rawal; Soil order : Inceptisol; Parent material : Loess ½ FYM + Biopower (T9) caused the highest plant

Soil depth height of 111.8 cm followed by 110.1 cm in T3, which Parameter 0-15 cm 15-30 cm were at par with the other, statistically. The plant height

Clay (%) 16 17 due to T3 and T6 were also statistically at par (Table 2). Silt (%) 39 40 Maximum plant height by T9 (½ NPK + ½ FYM + Sand (%) 45 43 Biopower) was due to the improved availability of Soil texture Sandy loam Sandy loam plant nutrients through Biopower. Rashid et al. (1998) Soil pH 7.80 7.91 and Ahmed et al. (1998) also reported that Integrated ECe (dS/m) 0.25 0.21 Bulk density (g/cc) 1.40 1.53 plant nutrient management (IPNM) produced higher Soil moisture (g/100 g) 8.82 9.2 plant height for wheat crop in rainfed areas. Chatha Total N (µg/g) 152 154 et al., (2006) also estimated that combined use of Organic C (g/100 g) 0.32 0.33 mineral and organic fertilizers resulted in higher plant Available P (µg/g) 3.45 3.55 Extractable K (µg/g) 80 85 height of wheat and maize in rainfed conditions. Many earlier researchers had also recorded that combined use of organic and chemical fertilizer to soil increased effi- Yield and yield components of wheat. Plant height. ciency of chemical fertilizer and crop yield significantly The height of a plant is an expression of inherited bio- (Bhatti, 2006; Jadoon et al., 2004; Nawaz et al., 2000). logical and environmental factors. During 2004-05, Biological yield. During the first year (2004-05), the application of ½ NPK + ½ FYM + Biopower (T9) application of ½ NPK + ½ FYM + Biopower (T9)

Table 2. Effect of integrated use of organic and mineral fertilisers and/or biopower on plant height of wheat during 2004-06 Treatments Plant height (cm) 2004-05 2005-06

CS1 CS2 Mean CS1 CS2 Mean

g h G F T1 Control 96.7 92.9 94.8 84.5 82.1 83.3 fg g F E T2 NPK (60-45-30) kg/ha 98.5 96.8 97.7 96.2 95.8 96.0 cde bc BC AB T3 NPK(120-90-60) kg/ha 103.3 105.9 104.6 111.6 108.6 110.1 ef def E CD T4 Full FYM @ 20 t/ha 100.2 101.2 100.7 105.0 102.3 103.6 cd cd CD CD T5 FYM* 103.6 103.5 103.5 107.3 104.4 106.1 cd bc B BC T6 ½ NPK + ½ FYM 106.4 106.5 106.4 109.6 105.0 107.3 def de DE D T7 ½ NPK + Biopower 101.3 102.7 102.0 101.5 103.7 102.6 cde cd CD CD T8 ½ FYM + Biopower 103.3 103.8 103.6 105.7 104.7 105.2 b a A A T9 ½ NPK + ½ FYM + BP 107 110.9 108.9 112.6 111.1 111.8 Mean 102.2Y 102.7X - 103.8 ns 101.9 ns - Analysis of variance P-value LSD SE P-value LSD SE Treatment (T) < 0.001 1.995 0.6941 < 0.001 3.467 1.206 Cropping system (CS) ns 0.0253 T x CS 0.02 2.821 0.9817 ns CV (+ %) 1.66 - - 2.87 - - CS = cropping system; biofertiliser biopower (seed inoculation at sowing); NPK @ 120-90-60 kg/ha; FYM (farm yard manure), @ 20 t/ha; FYM* = N equivalent + P make up dose; data are average of three replications; means followed by the same superscripts are not significantly different (P < 0.05; DMR test) from each other. 12 Mohiuddin Dilshad et al.

produced the highest biological yield of 9698 kg/ha yield was produced in CS2 than CS1 during 2004-05 followed by 8899 kg/ha by treatment of ½ NPK + ½ and 2005-06. However, yield of 2005-06 was lower FYM; difference between these treatments was signi- than that of 2004-05 with significant difference bet- ficant (Table 3). Mineral fertilizer NPK treatment (T3) ween the two cropping systems for the first year and yielded 8622 kg/ha, which was significantly lower to the second year of wheat. It may be due to the low both the T9 and the T6 treatments. Application of FYM rainfall received during the second crop year (Fig. 1). with P make-up dose produced biological yield of This is evident from the integrated use of organic and 7518 kg/ha, which was significantly higher than the T4 mineral fertilisers and/or Biopower showing better that gave 7140 kg/ha. The ½ FYM + Biopower treat- performance regarding the biological yield of wheat. ment (T8) gave biological yield of 7206 kg/ha, which Integrated plant nutrient management had advantage was statistically at par with the T4. Biopower with ½ over the sole application of mineral and organic and/or NPK produced a biological yield of 6969 kg/ha that biofertilisers. Ahmed et al. (1998) reported that IPNM was significantly lower than the T 8. produced higher biological yield of wheat in rainfed

The results during 2005-06 showed that the highest areas. Highest biological yield produced by T9 (½ NPK biological yield of 9381 kg/ha was produced by T9, + ½ FYM + Biopower) may be due to better avail- which was statistically at par with the biological yield ability of plant nutrients (Hafeez et al., 2002). Khaliq of 9335 kg/ha by T6 and 8753 kg/ha due to NPK et al. (2006) also reported higher growth and yield of cotton by integrated use of organic and inorganic fertilisers (T3). Application of FYM (T4 and T5) produced biological yield of 6725 kg/ha and 6308 kg/ha, nutrient sources with the effective microorganisms. respectively, which were statistically at par with one Grain yield. Grain yield in wheat Triticum aestivum other. L. is the result of a number of complex morphological Significant interaction between integrated nutrient and physiological processes affecting each other. management practices and cropping system (Table 3) During 2004-05, application of ½ NPK + ½ FYM + was observed during 2004-05, while this interaction Biopower (T9) produced the highest grain yield of was nonsignificant during 2005-06. Higher biological 3684 kg/ha followed by the grain yield of 3492 kg/ha

Table 3. Effect of integrated use of organic and mineral fertilisers and/or biopower on biological yield of wheat during 2004-06 Treatments Yield (kg/ha) 2004-05 2005-06

CS1 CS2 Mean CS1 CS2 Mean

i i H D T1 Control 4290 4255 4273 4200 ns 4106 ns 4153 h h G C T2 NPK (60-45-30) kg/ha 6149 6200 6175 6066 6160 6113 c c C A T3 NPK(120-90-60) kg/ha 8565 8679 8622 8580 8926 8753 e e E BC T4 Full FYM @ 20 t/ha 7199 7140 7170 6351 6264 6308 d d D BC T5 FYM* 7505 7531 7518 6637 6812 6725 b b B 5A T6 ½ NPK + ½ FYM 8789 9010 8899 9290 9380 933 g g F BC T7 ½ NPK + Biopower (BP) 6819 7119 6969 6575 6575 6575 f g E B T8 ½ FYM + BP 7176 7236 7206 7040 6776 6908 a a A A T9 ½ NPK + ½ FYM + BP 9641 9755 9698 9226 9535 9381 7348Y 7436X 7094 7151 Analysis of variances P-value LSD SE P-value LSD SE Treatment (T) < 0.001 109.0 37.92 < 0.001 588.8 186.9 Cropping system (CS) 0.0014 ns T x CS 0.0418 ns CV (+ %) 1.26 6.43 CS = cropping system; biofertiliser Biopower (seed inoculation at sowing); NPK @ 120-90-60 kg/ha; FYM (farm yard manure), @ 20 t/ha; FYM* = (N equivalent + P make up dose); data are average of three replications; means followed by the same superscripts are not significantly different (P < 0.05; DMR test) from each other. Soil Productivity through IPNM for Wheat Crop 13

by the treatment T6 (½ NPK + ½ FYM), while the use FYM + Biopower were applied in CS1 and CS2. How- of the recommended NPK dose produced 3292 kg/ha ever, during 2005-06, CS1 produced higher grain yield grain yield which were significant. The difference bet- than CS2. The reason might be that after the harvest of ween the highest and the lowest grain yield obtained wheat in 2004-05, the field was kept fallow (CS1), while was 1938 kg/ha. There was significant difference bet- in CS2, maize was grown which might have depleted ween CS1 and CS2 for the average of all the nine treat- the soil of nutrients and moisture. The results showed ments (Table 4). that integrated use of organic and mineral fertilisers and/or biofertilisers showed better performance re- The results during 2005-06 showed that application of garding grain yield of wheat. Both FYM treatments ½ NPK + ½ FYM + Biopower (T ) caused the highest 9 (T andT ) showed significant increase in grain yield. grain yield of 3781 kg/ha, which was statistically sig- 4 5 Results of previous studies indicated that use of nificant to all the nine treatments. The application of ½ organic sources as FYM produced equivalent or more NPK + ½ FYM produced significantly lower grain yield plant biomass and grain yield of wheat as the applica- of 3611 kg/ha than T , while T produced significantly 9 3 tion of inorganic fertilizers alone (Alam and Shah, lower grain yield of 3424 kg/ha than T . The grain yield 6 2003; Bakhtiar et al., 2002; Khanum et al., 2001). produced during 2005-06 was higher than that produced Chatha et al. (2006) also reported that the combined during 2004-05, depicting nonsignificant difference use of mineral and organic fertiliser produced higher between the two cropping systems for the first year grain yield of wheat crop in rainfed areas. Biofertilser and the second year of wheat. There was no effect of ‘Biopower’ treatments (T and T ) showed significant cropping system on grain yield of wheat during both 7 8 increase in grain yield over T depicting Biopower the years; however, lower values of grain yield were 3 response (Hafeez et al., 2002). The highest grain yield recorded in 2004-05. in IPNM treatment T9 may be due to better availability A nonsignificant interaction between the integrated of plant nutrients, while mineral fertilizers released nutrient management practices and the cropping nutrients readily to soil (Ahmad et al., 2002). Increase system (Table 4) was observed in both the years. The in grain yield due to IPNM can be attributed to impro- highest grain yield was observed when ½ NPK + ½ vement of organic matter content (Singh and Swarup,

Table 4. Effect of integrated use of organic and mineral fertilisers and/or biopower on grain yield of wheat during 2004-2006 Treatments Yield (kg/ha) 2004-05 2005-06

CS1 CS2 Mean CS1 CS2 Mean

H I T1 Control 1654 ns 1636 ns 1646 1580 ns 1466 ns 1523 G H T2 NPK (60-45-30) kg/ha 2388 2408 2399 2333 2314 2324 C C T3 NPK(120-90-60) kg/ha 3266 3317 3292 3429 3418 3424 E E T4 Full FYM @ 20 t/ha 2830 2804 2818 2930 3017 2974 D D T5 FYM* 2974 2936 2956 3173 3145 3159 B B T6 ½ NPK + ½ FYM 3450 3533 3492 3625 3597 3611 F G T7 ½ NPK + Biopower 2638 2771 2705 2695 2624 2660 E F T8 ½ FYM + Biopower 2826 2837 2832 2858 2765 2812 A A T9 ½ NPK + ½ FYM + BP 3675 3692 3684 3793 3768 3781 Mean 2856 2881 2935 2901 Analysis of variances P-value LSD SE P-value LSD SE Treatment (T) < 0.001 55 19 < 0.001 66 23 Cropping system (CS) 0.0519 0.0357 T x CS 0.068 ns 0.165 ns CV (+ %) 1.64 1.93 CS = cropping system; biofertilizer Biopower (seed inoculation at sowing); NPK @ 120-90-60 kg/ha; FYM (farm yard manure), @ 20 t/ha; FYM* = N equivalent + P make up dose; data are average of three replications; means followed by the same superscripts are not significantly different (P < 0.05; DMR test) from each other. 14 Mohiuddin Dilshad et al.

2001) and consequently improvement of physical performance regarding grain yield of wheat. Integrated properties particularly water holding capacity of soil plant nutrient management had advantage over the (Hati et al., 2007). Khaliq et al. (2006) also recorded sole application of mineral and organic and/or higher growth and yield of cotton by integrated use of biofertiliser. During 2004-05, application of ½ NPK + organic and inorganic nutrient sources with effective ½ FYM + Biopower (T9) recorded 14, 4 and 10% microorganisms. Ahmad et al. (2008); Bhatti (2006) increase in uptake of N, P and K, respectively, by and Wu et al. (2005) also documented that combined wheat over mineral fertiliser treatment (T3), while use of organic and mineral fertilisers produced better during 2005-06, application of ½ NPK + ½ FYM + grain yield. Biopower (T9) recorded 6.5, 3.8 and 1.5% increase in uptake of N, P and K, respectively by wheat over NPK uptake. The uptake of macronutrients like N, P mineral fertiliser treatment (T3). Integrated plant and K is the direct indication of the growth and the nutrient treatment, especially with Biopower, improved biomass production. During 2004-05, application of NPK uptake over mineral fertilisers (Hafeez et al., ½ NPK + ½ FYM + Biopower (T9) recorded maxi- 2002; Bakhsh et al., 2001). mum N uptake of 357 kg/ha followed by 313 kg/ha by The results of this experiment revealed that full dose recommended dose of NPK fertiliser treatment (T3). of mineral NPK fertilisers caused maximum uptake of Maximum P uptake of 51 kg/ha was recorded by T9 followed by P uptake of 49 kg/ha in mineral fertiliser N, P and K nutrients by both the wheat and the maize plants in both the growing seasons. Biofertiliser treat- treatment (T3). Maximum K uptake of 215 kg/ha was ments, especially those with Biopower, improved NPK recorded by T9 followed by K uptake of 195 kg/ha uptake over half the dose of NPK. This was due to the by mineral fertilisers treatment (T3) as depicted in Table 5. fact that Biopower contained four N-fixing bacteria, which resulted in increased availability of nutrients to During 2005-06, application of ½ NPK + ½ FYM + wheat plants. Microbial inoculums not only increased Biopower (T9) recorded maximum N uptake of the nutritional assimilation of wheat plants (total N, P 367 kg/ha followed by N uptake of 344 kg/ha by the and K) but also improved soil properties, which lead recommended dose of NPK fertiliser treatment (T3). to better vegetative growth and crop yield (Ahmad

Maximum P uptake of 55 kg/ha was recorded by T9 et al., 2008). followed by P uptake of 53 kg/ha by T . Maximum K 3 The economic analysis. The economic analysis of the uptake of 210 kg/ha was recorded by T followed by K 9 cropping systems revealed that wheat-maize cropping uptake of 207 kg/ha by mineral fertiliser treatment (T ). 3 system was profitable with the integrated use of The results showed that integrated use of organic and mineral, organic and/or biofertiliser under rainfed mineral fertilizers and/or biofertiliser rendered better conditions. The results indicated that organic sources

Table 5. Effect of integrated plant nutrient management on NPK uptake (kg/ha) of wheat Treatments NPK uptake (kg/ha) 2004-05 2005-06 NP KNPK

T1 Control771566671762

T2 NPK (60-45-30) kg/ha 134 25 112 131 27 110

T3 NPK(120-90-60) kg/ha 313 49 195 344 53 207

T4 Full FYM @ 20 t/ha 221 37 160 205 34 145

T5 FYM* 237 43 174 222 39 157

T6 ½ NPK + ½ FYM 299 45 194 329 49 206

T7 ½ NPK + Biopower 200 30 148 200 28 139

T8 ½ FYM + Biopower 209 31 153 217 30 146

T9 ½ NPK + ½ FYM + Biopower 357 51 215 367 55 210 CS = cropping system; biofertiliser biopower (seed inoculation at sowing); NPK @ 120-90-60 kg/ha; FYM (farm yard manure), @ 20 t/ha; FYM* = N equivalent + P make up dose; data are average of three replications (P < 0.05; DMR test). Soil Productivity through IPNM for Wheat Crop 15

Table 6. Value cost ratio (VCR) due to integrated plant nutrient management on seed yield of wheat Treatments Value cost ratio 2004-05 2005-06

CS1 CS2 CS1 CS2

T1 Control - - - - T2 NPK (60-45-30) kg/ha 1.84 1.94 1.89 2.13

T3 NPK(120-90-60) kg/ha 2.02 2.11 2.32 2.45

T4 Full FYM @ 20 t/ha 1.18 1.07 1.35 1.55

T5 FYM* 1.28 1.26 1.54 1.62

T6 ½ NPK + ½ FYM 2.00 2.11 2.28 2.37

T7 ½ NPK + Biopower (BP) 2.35 2.71 2.67 2.77

T8 ½ FYM + Biopower 2.25 2.31 2.46 2.50

T9 ½ NPK + ½ FYM + BP 2.20 2.24 2.41 2.51 Prices of mineral and organic/biofertilisers in 2004-06 for VCR Urea (50 kg bag) Rs.468.0 (US $ 7.8) Diammonium phosphate (50 kg bag) (US $13.35) Triple super phosphate (50 kg bag) Rs. 801.0 (US $13.35) Sulphate of potash Rs.996.0 (US $16.6) Farmyard maure Rs.1200.0 (US $ 20) Biofertilizer Biopower (1.0 kg packet) Rs.100.0 (US $1.6) (US $1.0 = Rupees 60 during 2004-2006) CS = cropping system; biofertiliser Biopower (seed inoculation at sowing); NPK @ 120-90-60 kg/ha; FYM (farm yard manure), @ 20 t/ha; FYM* = N equivalent + P make up dose. alone or their integrated use may improve yield but rainfed areas. The results confirm that besides incre- cannot compete mineral sources for sustaining crop asing the crop yield, IPNM saved mineral fertilisation productivity (Table 6). There is no scope for reducing which had potential effects on sustainable agricultural the consumption of chemical fertilisers but due to production in less fertile soils of rainfed Pothwar their increasing cost and environmental concerns, region of Pakistan. The high concentration of minerals chemical fertilisers may not be used since integrated in wheat plants demonstrated more efficient use of the use of fertilisers is showing a profitable VCR applied mineral nutrients by IPNM in both the crop- (CIMMYT, 1988). The VCR values of 2.20 and 2.41 ping systems. However, more intensive and systematic were calculated for CS1 while 2.24 and 2.51 for CS2 studies are required to provide better understanding were calculated in 2004-2005 and 2005-06, respect- of the usefulness of IPNM in making crop production ively, in T9. The VCR estimated for IPNM in T9 showed more profitable and income generating activity for that integrated use of organic and inorganic fertiliser small farmers of rainfed Pothwar region of Pakistan. sources had better net profit for wheat yield for the Integrated plant nutrient approach is going to be the farmer under rainfed conditions. Thus, IPNM may mainstay in the next millennium. prove more viable and sustainable for wheat based cropping systems in rainfed Pothowar area of Punjab References province of Pakistan. Ahmad, R., Naveed, M., Aslam, M., Zahir, Z.A., Arshad, M., Gilani, G. 2008. Economizing the use Conclusion of nitrogen fertilizer in wheat production through The investigations presented in this study indicate enriched compost. Renewable Agriculture and distinct benefits of IPNM over the use of mineral Food System, 23: 243-249. fertilisers. The results of this study showed that inte- Ahmad, S.I., Abbasi, M.K., Rasool, G. 2002. Integrated grated use of NPK (60-45-30) kg/ha and farmyard plant nutrition system (IPNS) in wheat under manure @ 10 t/ha with biofertiliser ‘Biopower’ as in rainfed conditions of Rawalakot, Azad Jammu and

T9, was most appropriate and economical for better Kashmir (Pakistan). Pakistan Journal of Soil yield of wheat in wheat based cropping systems of Science, 21: 79-86. 16 Mohiuddin Dilshad et al.

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Combining Ability Study in a 6 × 6 Diallel Cross of Maize

Amanullaha*, Shah Jehan Khana and Muhammad Mansoorb aFaculty of Agriculture, Gomal University, Dera Ismail Khan, Pakistan bArid Zone Research Institute, Ratta Kulachi, Dera Ismail Khan, Pakistan

(received March 29, 2010; revised December 23, 2010; accepted December 28, 2010)

Abstract. The F1 generation of 6 × 6 diallel cross in maize was evaluated for combining ability under irrigated conditions of Dera Ismial Khan, Pakistan. Based on the variance data, general combining ability (GCA) and specific combining ability (SCA) showed the importance of additive gene effect in some characters under study like kernel rows/ear, kernels/row, kernels/ear, 1000 grain weight and grain yield.

The results showed that F1 mean squares due to GCA were highly significant for all the parameters under observation. Higher magnitude of GCA components of variance (%) for kernels/ear, 1000 grain weight and grain yield, indicated predominance of additive and additive type of gene action while for kernel rows/ear, and kernels/row, nearly equal importance of both additive and non-additive gene effects were observed. The estimates of GCA effects revealed that parents Jalal and Sarhad White were good general combiners for most of the traits under discussion. The crosses, Kisan × Azam and Azam × Sadaf showed good SCA

effects with excellent mean performance for most of the traits studied in F1. Keywords: combining ability, diallel cross, Zea mays L.

Introduction General combining ability (GCA) and specific com- Maize is grown over a diverse environment ranging bining ability (SCA) effects were more important from tropical to north latitude. However, in Pakistan it for kernel rows and additive genetic variance was is mainly grown in provinces of Punjab and NWFP predominant for all the grain characters (Bagheri and during spring and summer seasons. Its global average Jelodar, 2010). is much higher than average yield of Pakistan. The The present investigation was undertaken to charac- global area under maize crop is 147.56 million terize six varieties and their 30 F1 cross combinations hectares producing 701.273 million metric tons with for their general and specific combining ability effects, average yield of 4752 kg/ha. In Pakistan, it is respectively, and identify the potential hybrids for cultivated on 1017 thousand hectares with annual cultivation under agro-climatic conditions of Dera production of 3088 thousand tons and average yield Ismail Khan, Pakistan. The reason for selection of of 3036 kg/ha (Federal Bureau of Statistic, 2006- these six maize varieties was that these varieties were 2007). promising and acclimatized to the climatic conditions Utilization of hybrid vigour has proved successful for of the area. Unfortunately, their performance varied increased productivity of maize crop in almost all the from character to character i.e., one variety was better countries of the world. Combining ability analysis in one character and the other was better in other assumes considerable importance as it is a established character, hence, keeping in view their GCA and fact that all the lines cannot produce good hybrids as SCA, we were trying to explore their exploitation in compared to other lines producing superior hybrids. breeding programme. The frequencies of desirable inbred lines, in maize, Materials and Methods have been placed between 0.1 and 0.01 percent (Wang et al., 2007). Six maize varieties namely, Kisan, Azam, Sarhad White, Sarhad Yellow, Jalal and Sadaf were evaluated in a full The conventional crop breeding methodology mainly diallel mating design to generate 30 F crosses. Five depends upon the development of inbred lines of maize 1 plants were involved in each cross. The parents and from open pollinated varieties or other heterogeneous their crosses were evaluated at Agricultural Research sources and their evaluation for commercial use. Institute, Ratta Kulachi, Dera Ismail Khan during Kharif *Author for correspondence; E-mail: [email protected] (rainy) season of 2005.The experimental material was

18 Combining Ability of a Diallel Cross of Maize 19 arranged in a randomized complete block design of mean squares due to GCA, SCA and reciprocal (RCBD) with four replications. Each entry was sown effects for all the characters studied, indicating there in four rows of 5 m length having row to row distance by the differences among parental lines for GCA and of 75 cm and plant to plant spacing of 20 cm. Planting among crosses for SCA effects. The ratio of estimated was done on ridges using 2-3 seeds per hill with the GCA to SCA variance indicated the preponderance of help of dibbler. Plants were thinned to one plant per the latter component in controlling the expression of hill when the plants reached to four-leaf stage. all the traits. The SCA variance involves both Fertilizer was applied @ 150-75 NP kg/ha; nitrogen dominance and epistasis which together constitute and phosphorus were applied in the form of urea and the non-additive type of gene action, whereas the DAP, respectively. All the phosphorus and half dose GCA variance is a reflection of additive and additive × of nitrogen were applied at seedbed preparation and additive type of gene action. The non-additive compo- the remaining dose of urea was applied when the nent of gene action could be optimized in upgrading plants attained knee height. Other cultural practices the genetic potential of the crop by adopting and protection measures were carried out according to reciprocal recurrent selection and hybrid technology, the recommended procedure for D. I. Khan. The data which has already become a field reality in maize were recorded for six traits i.e., kernels/ear, kernel breeding. rows/ear, kernels/row, 1000 grain weight, ear length The perusal of Table 2 enunciated that the GCA and grain yield. components of variance (%) were greater in magnitude Numbers of kernels/row were counted from 10 ears and more important for kernel rows/ear (28.21), randomly selected from each plot, and then average kernels/row (19.07), kernels/ear (26.61), 1000 grain was calculated. Number of kernels/ear was counted in weight (24.04 g) and grain yield (39.02 kg/ha), thereby 10 randomly selected plants in each plot. Total number showing the predominance of additive type of gene of kernels divided by 10 gave the average number of action for inheritance of the above characters. These kernels/ear. Ten ears randomly selected from each plot findings for additive type of gene action for different were measured and the average ear length was parameters corroborate the conclusion drawn by calculated. 1000 grain weight was calculated with the earlier workers (Haq et al., 2010; Akbar et al., 2009; help of electronic balance, up to two decimal points, Ojo et al., 2007). However, some researchers had from the bulk produce of 10 randomly selected plants observed non-additive type of gene action for most of of each plot. Grain yield was calculated using fresh the traits and such deviations may be due to different ear weight at harvest, assuming 80% shelling and genetic background of the cultivars used and different adjusted to 15% moisture content. Combining ability environmental conditions encountered. analysis was performed using Griffing (1956) method-2 In case of reciprocal variance (Table 2), some traits model-II. exhibited smaller values as compared to GCA and SCA except kernels/row (57.73) and 1000 grain weight Results and Discussion (60.23 g). It is relevant that the maternal effect for the Pooled analysis of variance for combining ability above traits were due to plasma gene action which is (Table 1) indicated the presence of high significance found rarely in such diallel studies. These results are

Table 1. Pooled analysis of variances for different traits in a 6 × 6 variety diallel of maize Source of DF Kernels/ear Kernel rows/ear Kernels/row 1000 Grain Ear length (cm) Grain yield variation weight (g) (kg/ha)

GCA 5 2580.81* 3.04* 4.80* 1625.80* 4.364* 1737399* SCA 15 475.59* 0.45* 0.35* 58.70* 0.40* 164628.7* Reciprocal 15 466.60* 0.64* 2.84* 314.77* 1.20* 228674.9* Error 105 304.36 0.22 0.60 58.70 0.25 57687.33

* = significant @ 0.05 probability. 20 Amanullah et al. closely supported by Kenga et al. (2005) and Long Estimates of SCA effects of the crosses are presented et al. (2004) who noticed some reciprocal variance in Table 4. The F1 hybrid (Kisan x Sadaf) was the best showing positive maternal effects for different specific combination for exploitation of positive SCA parameters. effects for the characters under study. The hybrid (Azam x Sadaf) produced the highest SCA values for The perusal of GCA effects (Table 3) revealed that Azam was an ideal general combiner for three charac- the traits like kernels/ear (33.77) and kernel rows/ear ters i.e. kernel rows/ear (0.82), kernels/row (0.97) and (0.93). For kernels/row cross combination, Azam x 1000 grain weight (19.14 g) while the same cultivar Sarhad Yellow with value of 0.81, for 1000 grain weight exhibited negative GCA effect for one character under Sarhad White x Sarhad Yellow with value of 7.63 g, discussion i.e. kernels/ear (-26.69). The Kisan variety for ear length Kisan x Sadaf with value of 0.49 cm and surpassed and produced maximum GCA effects for for grain yield Sarhad White x Jalal remained the kernels/ear (14.29) and grain yield (241.99 kg/ha). The topper with SCA value of 449.36 kg/ha. In the above same variety performed as the second best general crosses it is obvious that when Azam variety was used combiner for 2 traits i.e. kernel rows/ear (0.42) and as female parent, the cross showed maximum SCA kernels/row (0.34) under study. The variance due to effect; moreover the GCA estimates depict that Azam GCA showed the importance of both additive and had positive and prominent GCA effect for three traits. non-additive gene effects in the inheritance of almost As depicted through the values given against each all the characters studied. These results are in line with parent, it can be concluded that comparatively low x the findings of Bello and Olaoye (2009) and Viana high parents performed well in SCA determination. (2007). These results are in line with the findings of Mufti

Table 2. Estimates of components of variance and their percentages in parenthesis due to general, specific combining ability and reciprocal effects for various traits in 6 × 6 variety diallel maize Components of Kernel rows/ear Kernels/row Kernels/ear 1000 Grain Ear length (cm) Grain yield variation weight (g) (kg/ha) GCA 0.22 0.37 175.89 125.38 0.33 131351.7 (28.21) (19.07) (26.61) (24.04) (28.95) (39.02) SCA 0.13 -0.15 99.42 58.70 0.09 62095.0 (16.66) (-7.73) (15.04) (11.25) (7.89) (18.45) Reciprocal 0.21 1.12 81.12 314.20 0.47 85493.8 (26.92) (57.73) (12.28) (60.23) (41.23) (25.39) Error 0.22 0.60 304.36 23.37 0.25 57687.3 (28.21) (30.93) (46.06) (4.48) (21.93) (17.14) Total 0.78 1.94 660.79 521.65 1.14 336627.8 100 100 100 100 100 100

Table 3. Estimates of GCA effects for different traits of 6 × 6 variety diallel of maize Varieties Kernel rows/ear Kernels/row Kernels/ear 1000 Grain weight (g) Ear length (cm) Grain yield (kg/ha) Kisan 0.42 0.34 14.29 -5.81 -0.16 241.99 Azam 0.82 0.97 -26.69 19.14 0.17 223.79 Sarhad W -0.14 -0.08 -4.04 2.73 0.57 123.43 Sarhad Y -0.39 -0.64 0.31 -13.21 -1.04 -101.40 Jalal -0.45 -0.72 10.56 -8.19 -0.14 -729.07 Sadaf -0.24 0.13 5.56 5.33 0.60 241.26 CD(gi-gi)* 0.38 0.62 13.96 3.87 0.40 192.19

*gi = GCA effects for line i. Combining Ability of a Diallel Cross of Maize 21

Table 4. Estimates of SCA effects of F1 crosses from 6 × 6 variety diallel of maize F1 Hybrids Kernels/ear Kernels rows/ear Kernels/row 1000 Grain Ear length (cm) Seed yield weight (g) (kg/ha) Kisan × Azam -31.58 -0.09 0.33 4.05 -0.22 69.69 Kisan × Sarhad White 7.52 -0.13 -0.26 2.30 0.39 27.67 Kisan × Sarhad Yellow 0.54 -0.01 -0.44 -1.56 -0.01 -338.87 Kisan × Jalal 14.92 -0.44 0.14 -1.66 -0.28 -15.70 Kisan × Sadaf 2.04 0.60 0.16 -3.44 0.49 260.72 Azam × Sarhad White 13.25 -0.67 -0.38 3.35 0.10 18.13 Azam × Sarhad Yellow 13.52 -0.05 0.81 -22.13 -0.22 -150.04 Azam × Jalal -0.98 -0.36 0.01 2.94 -0.11 218.01 Azam × Sadaf 33.77 0.93 -0.59 -5.24 -0.01 -79.70 Sarhad W × Sarhad Y -3.75 0.16 0.22 7.63 -0.49 -2.18 Sarhad White × Jalal -0.13 0.35 0.31 -6.80 -0.38 449.36 Sarhad White × Sadaf -13.63 0.14 0.20 -3.53 -0.86 -440.35 Sarhad Yellow × Jalal -3.85 0.22 -0.13 2.09 -0.48 243.69 Sarhad Yellow × Sadaf -5.48 -0.74 -0.49 7.60 -0.37 360.11 Jalal × Sadaf -9.98 -0.05 0.47 2.48 0.22 -463.47 CD(sij*-slk) 31.21 0.84 1.39 8..65 0.89 429.74 CD(sij*-skl) 27.92 0.75 1.24 7.74 0.80 384.37 *sij = specific combining ability between ith and jth line.

et al. (2002) who reported that parent with the best Bello, O.B., Olaoye, G. 2009. Combining ability for GCA, used as pollen parent, always produced better maize grain yield and other agronomic characters combinations. in a typical southern guinea savanna ecology of Nigeria. African Journal of Biotechnology, 8: Conclusion 2518-2522. Variety Jalal followed by Sarhad White proved to be Federal Bureau of Statistic, 2006-07. Agricultural good general combiners for most of the traits under Statistics of Pakistan, pp. 23-24, Ministry of Food, discussion. The cross Kisan × Sadaf showed positive Agriculture and Livestock, Economic Wing, Islamabad. Government of Pakistan. SCA effects for all the traits under study in F1 generation. Though cultivar Sadaf remained on top in only Griffing, B. 1956. Concept of general and specific one trait i.e. ear length but its combination with the combining ability in relation to diallel crossing other cultivars produced the best specific combina- systems. Australian Journal of Biological tions. Hence, it is concluded that comparatively low × Sciences, 9: 463-493. high parents performed well in SCA determination and Haq, M.I, Ajmal, S., Munir, M., Gulfaraz, M. 2010. resulted in superior combinations. Gene action studies of different quantitative traits in maize. Pakistan Journal of Botany, 42: 1021- References 1030. Akbar, M., Saleem, M., Ashraf, M.Y., Husain, A., Kenga, R., Alabi, S.O., Gupta, S.C. 2005. Heterosis Azhar, F.M., Ahmad, R. 2009. Combining ability and combining ability for grain yield and its studies for physiological and grain yield traits in components in induced sorghum mutants. maize at two temperature regimes. Pakistan African Crop Science Journal, 13: 143-152. Journal of Botany, 41: 1817-1829. Long, J.K., Banziger, M., Smith, M.E. 2004. Diallel Bagheri, N., Jelodar, N.B. 2010. Heterosis and com- analysis of grain iron and zinc density in Southern bining ability analysis for yield and related-yield African-adapted maize inbreds. Crop Science, traits in hybrid rice. International Journal of 44: 2019-2026. Biology, 2: 222-231. Mufti, M.U., Saleem, M., Hussain, A. 2002. Diallel 22 Amanullah et al.

analysis of yield and yield components in maize Viana, J.M. 2007. Heterosis and combining ability (Zea mays L.). Pakistan Journal of Agriculture analyses from the partial diallel. Bragantia: Revista Research, 17: 22-26. de Ciencias Agronomicas, 66: 51-60. Ojo, G.O.S., Adedzwa, D.K., Bello, L.L. 2007. Com- Wang, Y., Fu, S., Wen, Y., Zhang, Z., Xia, Y., Liu, Y., bining ability estimates and heterosis for grain Rong, T., Pan, G. 2007. Selection of maize inbred yield and yield components in maize (Zea mays lines with high regeneration and susceptibility to L.). Journal of Sustainable Development in Agrobacterium tumifacien. Journal of Genetics Agriculture and Environmental, 3: 49-57. and Genomics, 34: 749-755. Pak. J. Sci. Ind. Res. Ser. B: Biol. Sci. 2011 54 (1) 23-28

Reduction of Aflatoxin B1 Contamination in Pakistani Wheat Varieties by Physical Methods

Arshad Hussain*a, Ghosia Lutfullahb and Shafqatullaha aFood Technology Centre, PCSIR Laboratories Complex, Jamrud Road, Peshawar-25120, Pakistan bCentre of Biotechnology and Microbiology, University of Peshawar, Peshawar, Pakistan

(received April 29, 2010; revised August 30, 2010; accepted November 16, 2010)

Abstract. In the study of effect of physical treatments, such as washing and heating, on the AFB1 contami-

nated wheat varieties, it was observed that the reduction of AFB1 was directly proportional to washing time

in all the varieties. The concentration of AFB1 was reduced more by heating than washing. The level of AFB1 in dried wheat decreased to more than 50% and 90% by heating in oven at 150 and 200 °C, respectively.

However, the reduction of AFB1 in wet wheat in which water (10%) was intentionally added was higher on heating at 100 °C for 30 min than that in the dried wheat.

Keywords: wheat, aflatoxin B1, toxicity reduction, washing, heating

Introduction of mycotoxin contaminated raw material, as well as Aflatoxins (AFs) are toxic secondary metabolites foods. produced by species of Aspergilli, especially Aspergillus Many countries regulate aflatoxin levels in their foods. flavus and Aspergillus parasiticus. These fungi can grow USA and EU (Europe Union) permit level lower than on certain foods and feeds under favourable condi- 20 ppb and Korea and Japan 10 ppb (Chiavaro et al., tions of temperature and humidity and generate AFs 2001). It is generally accepted that mycotoxin levels before and/or during harvest, handling, shipment and in food need to be reduced as low as technologically storage (Peraica et al., 1999; Bushby and Wogan, 1984). possible. Several physical, chemical and biological AFs are produced under optimum temperature and methods have been applied for the removal and inhibi- moisture conditions on Aspergillus contaminated tion of aflatoxigenic mould growth and subsequent carbohydrate-rich grains such as peanut, corn, cotton, aflatoxin biosynthesis. However, few of these have and wheat (Jaimez et al., 2000). AFs have the highest practical applications (Abrunhosa et al., 2002). toxicity among mycotoxins and possess carcinogenic Ammoniation, one of the chemical methods, was activity as well. They affect not only the health of reported to detoxify AFs in various raw materials with humans and animals but also the economics of agri- high efficiency (Buser and Abbas, 2002). The level of culture and food (Hwang et al., 2004). Epidemiological studies have shown that AF exposure is associated AFs was reduced to over 40% by roasting and heating peanuts (Rustom, 1997). Buser and Abbas (2002) with increased risk of hepatocellular carcinoma, parti- reported that an extrusion process is able to decrease cularly in combination with hepatitis B virus (IARC, the level of AFs to 33%. As a biological treatment, 2002). The aflatoxin form has 17 members including B , 1 antagonistic microorganisms were used to reduce B , G , G , M and M , among whom aflatoxin B is con- 2 1 2 1 2 1 toxicity of AFB (Cho and Kang, 2000). Studies on sidered so far the strongest hepatocarcinogen agent. 1 AFB , AFG , and AFM have higher carcinogenic the reduction of aflatoxin by treatment with naturally 1 1 1 occurring compounds such as, antioxidants have been activity than AFB , AFG , and AFM , respectively. 2 2 2 investigated worldwide. These studies are usually Recently, contamination of mycotoxins in food carried out through the elucidation of metabolic path- including imported ones has received much atten- ways in aflatoxin forming microorganisms (Huwig et al., tion because of the increase in international food 2001). A mushroom extract has been reported to inhibit trade under new trade treaties. Therefore, it is highly AFB1- 8, 9-epoxide formation (Lee et al., 2003). The use necessary to investigate means of toxicity reduction of heat treatment has been investigated with the double *Author for correspondence; E-mail: [email protected] aims of processing the food and partially or totally

23 24 Arshad Hussain et al.

reducing mycotoxin contamination. Predominantly, in homogenized and analyzed for AFB1 analysis accord- the case of peanuts and corn, the conventional practice ing to standard method, by thin layer chromatography

of roasting has proved to reduce the contamination level (AOAC, 2000). The analysis of AFB1 in wet wheat by more than 40 % (Taha et al., 2001). samples was performed in the same way as for the dried ones. The present study was undertaken for developing a

process feasible for decontamination of AFB1 by Chemicals analyses. All the chemicals of analytical physical treatments with minimum possible detrimental grade used in the present study were procured from effect on the nutritive value of wheat and thus convert BDH (Poole, England), Merck (Darmstadt, Germany) and the unusable contaminated commodity into usable form. Sigma Chemicals (St. Louis, USA). In the present study, the reduction of aflatoxin B con- 1 Standard of aflatoxin B . Standard of aflatoxin B (2.02 tamination in wheat varieties was monitored employing 1 1 g/mL) was purchased from Biopure (Tecknopark Tullin, physical treatment methods of washing and heating, μ Austria). Standard stock solution of AFB of concentra- which are generally undertaken in the home cooking, 1 with a view to providing basic and valuable information 1 μg/mL was prepared by diluting in benzene/ tion about reduction of aflatoxin in wheat. acetonitrile (98:2; v/v). This stock solution was then stored at 4 °C in refrigerator. Materials and Methods Determination of aflatoxin B1. Aflatoxin B1 was Collection of samples. Total 30 samples of different determined according to the standard method of local wheat varieties such as Auqab, Ghaznavi-98, AOAC (2000) by thin layer chromatography. Briefly, Saleem- 2000, Kyber-87, Pirsabak-2005 were acquired 50 g test sample was extracted with 250 mL acetone/ from NIFA Peshawar and Agricultural University water (85:15 v/v) using blender for 3 min and filtered Peshawar, Khyber Pukhtoonkhwa. through Whatman filter paper No. 4 and 150 mL of filtrate was collected in 400 mL beaker. Then 170 mL Processing of samples. Wheat varieties were artifi- of 0.02 N sodium hydroxide and 30 mL ferric chloride cially contaminated with aflatoxin B (AFB ). For wash- 1 1 along with about 3 g basic copper carbonate were ing, the contamination levels employed on wheat varieties [(Auqab, Ghaznavi-98) and (Saleem-2000, added to the filtrate in 400 mL beaker, mixed well and added to the mixture in 600 mL beaker. 150 mL of this Kyber-87, Pirsabak-2005)] were 43 μg/kg and 46 μg/kg, solution mixture was filtered and transferred to a respectively, for AFB . For heating treatment, the con- 1 500 mL separating funnel. To this 150 mL of 0.03% tamination levels employed on dried wheat samples sulphuric acid was added and extracted twice with 10 [(Auqab, Saleem-2000) and (Ghaznavi-98, Kyber-87, mL of chloroform. Lower chloroform extract layer was Pirsabak-2005)] were 100 μg/kg and 55 μg/kg, respectively, for AFB , while on the wet wheat samples transferred to another separating funnel and 100 mL 1 of 0.02 M potassium hydroxide was added, swirled [(Auqab), (Saleem-2000) and (Pirsabak-2005)] were gently for 30 sec and left for layer separation. The 28.6 μg/kg, 14.86 μg/kg and 18.56 μg/kg, respectively. chloroform extract layer was collected in a vial and Adequate amount of the standard dissolved in ben- 8 mL of it was evaporated to dryness at 45 C under a zene were dripped on to 100 g portions of samples ° gentle stream of nitrogen on a heating block. The held in 600 mL beakers, taking precautions so as to avoid contaminating the wall. After 24 h the contami- residue was redissolved in 200 μL benzene/ acetonitrile (98:2 v/v) and subjected to thin-layer chroma- nated wheat was used for the aflatoxin reduction by tography. Final identification and quantification of physical methods of washing and heating. AFB1 was performed by one-dimensional TLC on Washing and heating. Wheat (20 g) contaminated with precoated silica gel plates (Merck, Germany). The

AFB1 was put in 200 mL water and agitated to 155 rpm plates were developed in a saturated chamber with for 10, 20, 30 min using stirrer. Contaminated wheat was chloroform/xylene/acetone (60: 30: 10; v/v/v). The heated in an oven at various temperatures for 30, 60 and samples spots were observed under long wave ultra-

90 min. The AFB1 level of heated wheat was analyzed by violet light (λ=365 nm) and determined by visual com- TLC. Wet wheat samples were prepared by soaking parison with AFB1 standard spots. Confirmation of wheat in water up to 10% moisture levels. Wet samples the identity of AFB1 was carried out with the spray of were heated at 100 °C for 30 min. Then, wheat was 50% sulphuric acid and using the trifluoroacetic acid Reduction of Aflatoxin in Wheat 25

(TFA) reaction (Scott, 1984) and its amount was AFB1 by washing. However, the value did vary accord- calculated as follows: ing to the wheat variety. Due to low solubility of AFs in water, it is generally hard to remove AFs by washing. AFB (μg/kg) = S × Y × V / X × W 1 However, in this study about 40% of AFB , usually Where: 1 attached to the surface of wheat, could be removed by S = Standard which matches the unknown (μL) washing. The reduction degree of 10 and 30 min wash- Y = Concentration of standard (μL/mL) ing did not show a significant difference, which means V = Final dilution of sample extract (μL) it is very difficult to remove AFB bonded or attached to X = Sample extract spotted giving florescent 1 wheat strongly. Washing of Saleem 2000 for only 20 min intensity equivalent to standard (μL) showed a significant difference (P< 0.05) compared to W = Weight of the sample contained in final washing for 10 min. This result was in agreement with a extract (g) previous report (Yeo and Kim, 2002). To investigate the Statistical analysis. The statistical significance of the effect of heating treatment on the destruction of AFB1, data was analyzed using Student’s t-test. Each sample all the contaminated wheat samples were heated in oven was analysed thrice and standard deviation was calcu- at various temperatures (50, 100, 150, 200 °C) for differ- lated (Steel et al., 1997). The data was expressed as ent periods (30, 60, 90 min). The effect of heating on the average mean ± standard deviation (SD). Method of level of AFB1 is shown in Table 2. Dried wheat samples recovery varied from 94 to 104 % and the quantification were selected for heat treatment. Heating at 100 °C or limits was 1 g/kg for AFB (Simionato et al., 2003). μ 1 lower did not lead to any marked reduction in AFB1 levels, whereas at temperatures higher than 150 °C, the Results and Discussion reduction of AFB1 was much higher. Wheat samples (Auqab, Ghaznavi-98) and (Saleem- Heating the variety Auqab at 150 °C, caused AFB 2000, Kyber-87, Pirsabak-2005) were artificially 1 destruction of about 60%, 75%, and 82% for 30 min, contaminated with AFB 43 μg/kg and 46 μg/kg, 1 60 min, and 90 min, respectively. In case of heating at respectively. The effect of washing on the reduction 200 °C, over 90% of AFB were destroyed. Compared to of AFB in wheat varieties is shown in Table 1. The 1 1 the heating at 50 °C, the heating at 100 °C for 60 min reduction of AFB in all the wheat samples was 1 displayed a significant difference in AFB reduction proportional to washing time. 1 (P<0.05). However, heating of Auqab over 150 °C,

The level of AFB1 in wheat samples such as Saleem- showed a significant difference against heating at 2000, Kyber-87and Pirsabak-2005 decreased by 62%, 50 °C, regardless of the heating time (P < 0.01 or P < 56%, and 58%, respectively, after 30 min washing. When 0.05). The effect of heating on Ghaznavi was almost wheat samples Auqab and Ghaznavi-98 were washed similar to that on other wheat varieties. Greater decrease for 30 min, about 60% and 56% reduction in AFB1 was in AFB1 was apparent in Saleem-2000 by heating at obtained, respectively. There was no difference 150 °C. However, it is impossible to heat foods over between wheat samples in terms of the reduction (%) of 100 °C for reducing AFs level.

Table 1. Reduction of Aflatoxin B1 in wheat varieties through washing for different time periods Variety Initial After 10 min After 20 min After 30 min level of Final level Reduction Final level Reduction Final level Reduction

AFB1 (μg/kg) (%) (μg/kg) (%) (μg/kg) (%) (μg/kg) Auqab 43 21.3 ± 1.7 50 21.2 ± 0.5 50 17.2 ± 2.6 60 Ghaznavi-98 43 25.2 ± 2.5 41 19.4 ± 0.4 55 18.9 ± 1.1 56 Saleem-2000 46 22.8 ± 1.2 50 18.2 ± 1.5* 60 17.1 ± 3.0 62 Kyber-87 46 25.0 ± 3.1 45 23.6 ± 2.4 48 20.0 ± 8.9 56 Pirsabak-2005 46 24.4 ± 3.0 46 21.9 ± 4.4 52 18.8 ± 3.0 58

The values are mean ± standard deviation (n = 3); * P<0.05; ** P<0.01. 26 Arshad Hussain et al.

Regarding the type of heating used to destroy AFs, it 100 °C (Table 3). The initial levels of AFB1 in Auqab, was reported that the use of oven for heating was Saleem-2000 and Pirsabak-2005 were 28.6 and 18.5 μg/L, more effective in reducing AFs than the use of other respectively, and after heating the reduction of AFB1 means of heating such as microwave (Soliman, 2002; in these varieties was 40%, 47% and 43%, respectively. Soliman et al., 2001). For investigating the effect of In comparison with the dried wheat samples, reduction moisture content on the reduction of AFB level, the 1 of AFB1 in wet samples of Auqab, Saleem-2000 and varieties, Auqab, Saleem-2000 and Pirsabak-2005 Pirsabak-2005 varieties was higher by 20%, 24% and (10 % moisture content) were heated for 30 min at 18%, respectively.

Table 2. Effect of heating for different time periods on reduction of AFB1 in dried wheat Temperature Initial After 30 min After 60 min After 90 min

(°C) level of AFB1 level Reduction AFB1 level Reduction AFB1 level Reduction AFB1 (μg/kg) (%) (μg/kg) (%) (μg/kg) (%) (μg/kg) Auqab 100 50 98.0 ± 3.0 2 95.0 ± 3.3 5 82.0 ± 4.0 18 100 80.0 ± 1.9 20 69.2 ± 2.4* 31 57.3 ± 1.7* 42 150 40.0 ± 2.5** 60 25.0 ± 2.0** 75 17.6 ± 0.7* 82 200 4.0 ± 0.2** 96 11.0 ± 1.0** 90 7.0 ± 0.3** 93 Ghaznavi-98 55 50 51.6 ± 3.6 6 52.7 ± 2.0 4 49.0 ± 0.4 11 100 45.6 ± 3.2 17 36.0 ± 1.6* 34 38.4 ± 2.8* 30 150 25.9 ± 2.3** 53 18.0 ± 1.8** 67 12.0 ± 2.9** 78 200 5.0 ± 0.7** 91 6.4 ± 0.3** 88 4.7 ± 0.8** 91 Saleem-2000 100 50 95.0 ± 5.0 5 93.0 ± 3.3 7 82 ± 15.5 18 100 77.0 ± 1.9 23 70.0 ± 2.4* 30 62 ± 4.7* 38 150 38.6 ± 3.5** 61 24.0 ± 2.0** 76 12.0 ± 0.7* 88 200 5.0 ± 0.2** 95 9.0 ± 1.0** 91 6.0 ± 4.9** 94 Kyber-87 55 50 52.0 ± 3.6 5 52.0 ± 2.0 5 47.2 ± 0.4 14 100 44.0 ± 3.2 20 45.0 ± 1.6* 18 35.0 ± 2.8* 36 150 24.2 ± 2.3** 56 18.9 ± 1.8** 66 12.2 ± 2.9** 77 200 6.4 ± 0.7** 88 4.2 ± 0.3** 92 3.7 ± 0.8** 93 Pirsabak-2005 55 50 51.5 ± 3.6 6 50.0 ± 2.0 9 49.3 ± 0.4 10 100 46.5 ± 3.2 15 42.3 ± 1.6* 23 33.5 ± 2.8* 39 150 24.5 ± 2.3** 55 14.6 ± 1.8** 73 11.5 ± 2.9** 79 200 2.8 ± 0.7** 95 3.6 ± 0.3** 93 2.0 ± 0.8** 96 The values are mean ± standard deviation (n = 3); * = P<0.05; ** = P<0.01.

Table 3. Effect of heating at 100 °C for 30 min on reduction of AFB1 in wet wheat Auqab Saleem-200 Pirsabak-2005 Moisture Initial level Final level Reduction Initial level Final level Reduction Initial level Final level Reduction

of AFB1 (μg/kg) (%) of AFB1 (μg/kg) (%) of AFB1 (μg/kg) (%) (μg/kg) (μg/kg) (μg/kg)

10% 28.6 17.0 40 14.8 7.8 47** 18.5 10.5 43* ± 3.0 ± 2.3 ± 2.0 The values are mean ± standard deviation (n=3); * = P<0.05; ** = P<0.01. Reduction of Aflatoxin in Wheat 27

Heating of wet Saleem-2000 varieties showed a signifi- method to detect aflatoxins in food and feed with cant difference compared to the heating of the dried cyclodextrins as fluorescence enhancers added variety under the same conditions (P < 0.05). The rela- to the eluent. Journal of Chromatography A, 937: tionship between moisture content in foods and 31-40. destruction of AFs has been already reported by many Cho, J.I., Kang, G.J. 2000. Control of aflatoxin B1 researchers (Mendez-Albores et al., 2004; Torres et al., production of Aspergillus parasiticus using 2001). According to these reports, the increased mois- antagonistic microorganisms and its application in ture content enhances the destruction of AFs during Meju. Food Science and Biotechnology, 9: 151- cooking or baking. In addition, AFs are stable up to 156. their melting points when heated without moisture Huwig, A., Freimund, S., Kappeli, O., Dutler, H. 2001. (Mann et al., 1967). Moisture is required to hydrolyze Mycotoxin detoxification of animal feed by the lactone ring of the AFs when cooking at home at different adsorbents. Toxicology Letters, 122: temperature of 85-95 °C (Samarajewa, et al., 1990). For 179-188. detoxifying of AFs, the most important step involves Hwang, J.H., Chun, H.S., Lee, K.G. 2004. Aflatoxins in opening of the lactone ring of the AFs (Buser and Abbas, foods: analytical methods and reduction of toxicity 2002). by physicochemical processes. Journal of the Korean Society for Applied Biological Chemistry, Conclusion 47: 1-16. In the present study of the effects of physical treat- IARC 2002. IARC Monographs on the Evaluation of ments such as washing and heating in oven on the Carcinogenic Risks to Humans: Some Traditional Herbal Medicines, Some Mycotoxins, Naphthalene reduction of aflatoxin B1, reduction of AFB1 toxicity was found to be directly proportional to washing time in all and Styrene, vol. 82, International Agency for Research on Cancer, Lyon, France. the studied wheat varieties. The level of AFB1 decreased more by heating than washing. The reduction of afla- Jaimez, J., Fente, C.A., Vazquez, B.I., Franco, C.M., toxin by heating was more in case of wet wheat than Cepeda, A., Mahuzier, G., Prognon, P. 2000. Review: in the dried wheat. The study provides a base for deve- Application of the assay of aflatoxins by liquid loping a method feasible for converting unusable con- chromatography with fluorescence detection in taminated wheat commodity in to usable form, through food analysis. Journal of Chromatography A, 882: 1-10. decomposition of aflatoxin B by physical treatments 1 Lee, S.E., Jeong, J.H., Kim, D.G., Choi, W.S. 2003. Inhibi- and its reduction in wheat. tory effect on aflatoxin B1-8, 9-epoxide formation References and anti-complementary activity of methanol extract from Hericium erinaceus cultivated with Abrunhosa, L., Serra, R., Venancio, A. 2002. Bio- Artemisia iwayomogi. Food Science and Biotech- degradation of ochractoxin A by fungi isolated nology, 12: 183-186. from grapes. Journal of Agricultural and Food Mann, G.E., Codifer, L.P., Dolear, F.G. 1967. Effect of Chemistry, 50: 7493-7496. heat on aflatoxins in oilseed meals. Journal of AOAC 2000. Official Methods of Analysis, 17th edition, Agricultural and Food Chemistry, 15: 1090- Association of Official Analytical Chemists. 1092. Washington DC., USA. Mendez-Albores, J.A., Arambula-Villa, G., Loarca-Pina, Buser, M.D., Abbas, H.K. 2002. Effects of extrusion M.G., Gonzalez-Hernandez, J., Castano-Tostado, E., temperature and dwell time on aflatoxin levels in Moreno-Martinez, E. 2004. Aflatoxins fate during cottonseed. Journal of Agricultural and Food the nixtamalization of contaminated maize by two Chemistry, 50: 2556-2559. tortilla-making processes. Journal of Stored Bushby, W.F., Wogan, G.N. 1984. Aflatoxins. In: Chemi- Products Research, 40: 87-94. cal Carcinogens, F. Edwards (ed.), pp. 945-1135, Peraica, M., Radic, B., Lucic, A., Pavlovic, M. 1999. Toxic Mapple Press, New York, USA. effects of mycotoxins in humans. Bulletin of the Chiavaro, E., Dall, Asta, C., Galaverna, G., Biancardi, World Health Organization, 77: 754-766. A., Gambarelli, E., Dossena, A., Marchelli, R. 2001. Rustom, I.Y.S. 1997. Aflatoxin in food and feed: occu- New reversed-phase liquid chromatographic rrence legislation and inactivation by physical 28 Arshad Hussain et al.

methods. Food Chemistry, 59: 57-67. cooking on aflatoxin residues. Journal of Agricul- Samarajewa, U., Sen, A.C., Cohen, M.D., Wei, C.I. 1990. tural and Food Chemistry, 49: 3291-3295. Detoxification of aflatoxins in foods and feeds by Steel, R.D., Torrie, J.H., Dickey, D. 1997. Principles and physical and chemical methods. Journal of Food Procedures of Statistic: A Biometrical Approach, Protection, 53: 489-501. 3rd edition, McGraw Hills Book Co. Inc, New York, Scott, P.M. 1984. Effects of food processing on mycotoxins. USA. Journal of Food Protection, 47: 489-499. Taha, O.G., Fonseca, T.T., Sylos, C.M. 2001. Efeito da Simionato, E.M.R.S., Astray, R.M., Sylos, C.M. 2003. radiação microondas na redução dos teores de Ocorrência de ocratoxina A e aflatoxinas em arroz. aflatoxinas em amendoin. Alimentos e Nutrição, Revista do Instituto Adolfo Lutz, 62: 123 -130. Araraquara, 12: 163-170. Soliman, K.M. 2002. Incidence, level, and behavior of Torres, P., Guzman-Ortiz, M., Ramirez -Wong, B. 2001. aflatoxins during coffee bean roasting and Revising the role of pH and thermal treatments in decaffeination. Journal of Agricultural and Food aflatoxin content reduction during the tortilla and Chemistry, 50: 7477-7481. deep frying processes. Journal of Agricultural and Soliman, K.M., El-Faramawy, A.A., Zakaria, S.M., Food Chemistry, 49: 2825-2829. Mekkawy, S.H. 2001. Monitoring the preventive Yeo, H.J., Kim, J.G. 2002. Reduction of aflatoxin during effect of hydrogen peroxide and y-radiation of the cooking and processing of rice. Journal of Food aflatoxicosis in growing rabbits and the effect of Hygiene and Safety, 17: 79-86. Pak. J. Sci. Ind. Res. Ser. B: Biol. Sci. 2011 54 (1) 29-33

Studies on Transplantation of Marine Turtle Nests at Karachi Coast (Sindh), Pakistan

Fehmida Firdousa, Sohail Barkatib* and Solaha Rahmanb aSindh Wildlife Department, Karachi, Pakistan bDepartment of Zoology, University of Karachi, Karachi-75270, Pakistan

(received April 29, 2010; revised August 2, 2010; accepted September 16, 2010)

Abstract. Egg clutches of two species of marine turtles, namely Chelonia mydas and Lepidochelys olivacea, were collected during 1974 to 1997 and transplanted to the protected enclosures. The emerging hatchlings were released to the natural environment. The experiment helped to produce an average of 19495.5 hatchlings per year of green and 1174.5 per year of olive ridley turtles. Keywords: transplantation, turtles, Chelonia mydas, Lepidochelys olivacea, green turtles

Introduction protected enclosures and the emerging hatchlings were Overexploitation including indiscriminate slaughter- released to the natural environment. ing and destruction of nesting sites are considered main reasons of inclusion of the marine turtles of Materials and Methods Pakistan in the list of threatened species in Red The present study was carried out on the sandy beaches Data Book of IUCN (Groombridge, 1982). Intensive of Sandspit (24° 50´ 49"N-66° 53´ 34"E) and programmes for conservation of marine turtles were Hawkesbay (24° 51´ 37"N-66° 51´ 11"E) along Karachi launched in various parts of the world. Rules and coast, Sindh, Pakistan. This is about 5 km long area ordinances formulated and implemented strictly in located south west of Karachi. many countries viz., Australia, USA, Durban, Surinam, Individual eggs of two species of turtles (C. mydas (green State of Sabah, Oman, India etc. turtle) and L. olivacea (olive ridley) were collected from Reacting to the alarming situation of their depletion from wild just after being laid during the period 1974 to 1997 Pakistani waters, the Government of Sindh (Pakistan) (green turtle) and 1980 to 1997 (olive ridley); these were declared all marine turtles as “Protected” under Sindh then relocated to protected enclosures built just above Wildlife Protection Ordinance (Salm, 1975) and Sindh the high tidal zone at Sandspit and Hawkesbay to Wildlife Protection Act 1993. Eggs of turtles are provide secure and safe environment. usually removed from the nests and buried at a safest Three enclosures measuring 80 × 80 ft each (6400 sq. place (Stancyk et al., 1980). Among others some recent ft), one on Hawkesbay and two on Sandspit were built studies on relocation of nests and hatching success are on high tidal zone at a distance of 50 to 3000 m from those of da Silva et al. (2007), Trullas and Paladino the nesting sites. The enclosures were made secured (2007), Kornaraki et al. (2006), Vanzolini (2003), to through a metal fence. About 1.0 m 1.0 m area was mention a few. × reserved for each nest. Eggs were collected and An intensive project on study of ecology of sea turtles transferred within a maximum of 6 h of deposition with of Karachi among other aspects, included establishing minimum disturbance to eggs. After removal, eggs were enclosures (hatcheries) for hatching and rearing the counted, placed in a bucket, carried to the enclosures hatchlings for safety return to the natural environment (hatchery) and buried in the nest prepared for this (Firdous et al., 2009). purpose. Eggs were usually handled manually. Baldwin In the present study, egg clutches of Chelonia mydas and Lofton (1959) also reported manual handling of and Lepidochelys olivacea were transplanted to eggs of loggerhead turtles at Little Cumberland Island. The hatchlings were released to the sea soon after *Author for correspondence; E-mail: [email protected] their emergence. The nests were excavated after the

29 30 Fehmida Firdous et al.

hatching process was completed. Hatching success The average number of eggs per clutch per year var- was calculated as the ratio of total number of hatchlings ied from 62.2 (1994) to 104.7 (1980). More nests produced to the number of eggs present in the nest. (8375) were available for transplantation during the first 6 years (1981-1986) compared to the last 13 years A thermistor thermometer (C-8150-60) was used to (8673) of study. measure the temperature of the nest containing eggs. The probes of the instrument are epoxy-tipped and are The eggs of green turtle transplanted to the enclosures equipped with 15-ft. PVC leads. A probe was placed ranged from 4610 in 1979 to 127337 in 1985 with an within the centre of the clutch which was connected average of 76524.5 eggs per year. More eggs (117810 through a wire to a meter over the earth’s surface. The eggs per year) were transplanted during the first six years nest was then filled with sand and left for incubation. (1981-86) compared to the last 11 years (1987-97) of The probes were waterproof and could withstand the the study period (60410.7 eggs per year). burial in sand without affecting their sensitivity. Tem- Olive ridley. Only 654 nests of olive ridley (L. olivacea) perature was recorded at least twice a week till were found and transplanted over a period of 18 years hatchlings emerged. Soon after emergence hatchlings (1980 to 1997) with an average of 43.6 nests per year. were released to the sea. The number of nests fluctuated considerably from year to year varying from a minimum of 2 (1996, 1997) to a Results and Discussion maximum of 120 (1984). The nests reduced to just few Temperature changes. The average temperature ranged (2-8) during the last few years of the study (1993-1997). between 16.06 and 29.03 °C. Seasonal changes of temperature in different years followed almost similar 35 Ambient temp. pattern from 1984 to 1997 (Figs. 1 and 2). Generally Nesting( % ) Hatching( % ) June and July were the months of high temperature, 30

whereas relatively low temperature was observed 25 during December and February. 20

Nesting and hatching. Details regarding number of 15 egg clutches (nests) transplanted, number of eggs per clutch, incubation period, number of hatchlings per 10 clutch and hatching percentage (hatching success) of 5

green and olive ridley during the period 1979 to 1997 0 is summarized in Tables 1 and 2. The values are given Jan Feb Mar Apr May Jun Jul Aug Sept Oct Nov Dec as total, minimum, maximum and average of respec- Fig. 1. Relationship between temperature changes tive parameters. and percent nesting and percent hatching The incubation period of green turtles varied from 22 of C. mydas. to 160 days whereas the yearly averages ranged between

56 and 70 days. The incubation period of olive ridley 60 Ambient Temp.temp. varied from 42 to 69 days, whereas the yearly averages Nesting (% ) 50 ranged between 46 and 52. Hatching (% ) 40 Transfer of nests to protected enclosures. A total of 1531980 eggs of both the species were transplanted to 30 the enclosures, of which 1453966 (94.9%) eggs were of 20 green turtle and 78014 (5.09%) eggs were of olive ridley. 10 Green turtles. A total of 17048 nests of green turtle were transplanted during the 19 years investigation 0 Jan Feb Mar Apr May Jun Jul Aug Sept Oct Nov Dec period (1979 to 1997) with an average of 894.4 nests per year. Maximum number of nests (1549) was trans- Fig. 2. Relationship between temperature changes planted during the year 1985 and minimum (44) in and percent nesting and percent hatching 1979 (Table 1). of L. olivacea. Transplantation of Marine Turtle Nests 31

Table 1. Yearly information about the number of egg clutches, eggs transplanted, incubation period, number of hatchings and percentage of hatching for green turtle (C. mydas) from Karachi Coast (Sandspit and Hawkes Bay) between 1979 and 1997, values of standard error (SE) are also given

Year No. of egg clutches No. of eggs per clutch Incubation period (days) No. of hatchlings / clutch Hatching transplanted Min Max. Av. SE Min Max. Av. SE Total Min Max. Av. SE (%)

1979 44 81 135 104.7 2.44 40 86 62 9.62 1196 3 80 28.4 3.7 26 1980 799 45 159 97.5 0.7 34 134 58 0.73 33907 1 125 60.7 1.02 43 1981 1168 27 136 84.5 0.46 40 136 70 0.74 21916 1 88 27.0 0.60 22 1982 1405 7 122 89.8 0.51 31 136 65 0.73 22587 1 109 23.2 0.57 18 1983 1457 9 151 84.3 0.26 22 151 66 0.79 18645 1 120 25.4 0.82 15 1984 1339 9 65 83.2 0.61 43 127 62 0.61 39994 1 132 36.6 0.77 36 1985 1549 13 168 82.2 0.57 41 132 58 0.57 48095 1 125 40.5 0.74 38 1986 1457 20 120 82.4 0.56 47 120 60 0.56 44037 1 135 40.2 0.77 37 1987 704 20 160 87.6 0.84 39 160 59 0.84 13693 1 121 26.1 0.96 22 1988 866 11 150 83.6 0.71 43 128 66 0.71 14719 1 106 24.1 0.89 20 1889 717 28 121 88.7 0.71 43 121 64 0.71 20379 1 105 33.5 0.93 32 1990 839 25 132 83.7 0.68 28 132 60 0.68 21297 1 117 32.6 0.90 30 1991 1031 13 145 85.3 0.64 41 145 65 0.84 14119 1 104 24.3 0.86 16 1992 785 7 163 85.9 0.71 44 132 64 0.97 12514 1 85 25.2 0.87 19 1993 802 10 150 83.2 0.65 45 120 62 0.92 7385 1 83 16.8 0.76 11 1994 519 27 131 82.2 0.69 43 146 63 1.15 6889 1 101 27.8 1.25 16 1995 464 19 194 84.8 0.86 39 120 56 0.98 4962 1 87 22.9 1.51 13 1996 475 32 181 84.3 0.92 36 122 61 0.87 9489 1 105 32.1 1.43 24 1997 628 30 135 83.1 0.69 46 156 64 0.69 10895 1 104 28.0 1.05 21

Table 2. Yearly information about the number of egg clutches, eggs transplanted, incubation period, number of hatchlings and hatching percentage for Olive Ridley (L. olivacea) from Karachi Coast (Sandspit and Hawkes Bay) between 1980 and 1997; values of standard error (SE) are also given

Year No. of egg clutches No. of eggs per clutch Incubation period (days) No. of hatchlings / clutch Hatching transplanted Min Max. Av. SE Min Max. Av. SE Total Min Max. Av. SE (%)

1980 16 84 132 99.2 5.33 43 66 50 1.72 967 25 92 60.4 4.20 61 1981 11 83 146 120.9 5.93 49 52 50 0.33 403 1 93 44.7 7.72 30 1982 95 75 160 112.8 1.95 42 69 50 0.54 2386 2 116 27.7 1.98 22 1983 25 90 170 128.6 3.58 48 65 50 0.35 472 5 98 33.7 7.01 15 1984 120 10 160 119.1 2.12 43 65 51 0.32 5424 1 126 50.6 2.29 38 1985 57 94 163 120.0 1.99 47 57 50 0.29 2448 1 110 49.9 4.60 36 1986 61 78 164 121.8 2.45 46 56 51 0.25 3860 1 135 64.3 4.72 52 1987 113 64 185 126.4 1.76 44 53 48 0.18 2799 1 101 25.6 2.11 20 1988 31 86 186 122.4 3.70 46 50 48 0.23 230 1 40 11.5 2.63 6 1889 43 50 150 115.5 3.48 45 54 48 0.36 1175 2 105 29.3 3.84 24 1990 29 50 146 112.2 4.46 46 56 49 0.39 963 2 70 37.0 3.57 30 1991 13 45 136 107.5 6.62 47 68 52 1.95 304 2 60 30.4 6.86 22 1992 20 85 155 121.2 3.82 46 54 47 0.30 670 1 78 39.4 5.56 28 1993 4 105 126 118.5 4.62 45 48 46 0.88 54 2 45 18.0 13.57 11 1994 4 75 122 118.2 8.74 48 51 49 0.75 97 11 54 24.2 10.11 21 1995 8 107 152 126.7 4.97 45 55 50 2.05 76 5 58 19.0 13.0 7 1996 2 94 140 117.0 47 51 49 2.00 173 81 92 86.5 5.50 74 1997 2 127 135 131.0 4.00 ------32 Fehmida Firdous et al.

The eggs of olive ridley transplanted to the enclosures in June-July. Moderate temperatures were favourable ranged from 234 in 1996 to 14299 in 1984 with an for nesting and hatching. average of 43341 eggs per year. Period between 1982 In case of green turtles nesting and hatching was very and 1987 may be regarded as years of maximum num- low during the colder months (December to March) ber of eggs transplantation. A decline in number of eggs and at minimum or negligible during the warmest part in the last five years is due to a simultaneous decline in of the year (April to July). On the contrary, in case of the number of nests of olive ridley. olive ridley, nesting and hatching mainly occurred in Release of hatchlings. A total of 391556 hatchlings July to October period and was totally absent in the (green and olive ridley combined) emerged from the months of low temperature (November to April). eggs with an average of 20608.2 hatchlings per year, The hatching rates of green turtle reported from the number varying from 922 (in 1979) to 49436 (in various places are: 58 and 61% at Florida (Witham 1986). Maximum hatchlings were produced during the and Futch, 1977), 60% natural hatching rate (Hirth period 1984-1986. The share of olive ridley was 5.39% and Carr, 1970), 50.7% in 1959 and 50.8% during compared to 94.6% of green turtle hatchlings. Details 1960 in Costa Rica and 54.4% from Ascension of hatchings and related aspects are given by Firdous et Island (Carr and Hirth, 1962), 67% in hatchery al. (2009). (Bustard and Greenham, 1968), 33.9% and 21.4 at Green turtle. A total of 370414 hatchlings (×19495.5 Nancite in 1983 and 1982, respectively. Low hatching per year) were produced during the study period (1979- rates were assigned to monsoon (Hendrickson, 1958) 1997) varying from 1196 (in 1979) to 48095 (in 1985). and prolonged wet weather (Bustard, 1972). The number of hatching egg clutches hatched varied In case of olive ridley hatching rate of 90-94% was from 47 (in 1995) to 85% (in 1989). reported from two transplanted nests in Sri Lanka The number of hatchlings produced per year varied (Zwinenberg, 1976; AGRW, 1968). Pritchard (1969) from 1 to 135. The yearly averages ranged between observed a higher hatching rate (98-100%) from the 16.8 and 60.7. The annual hatching success varied from same area. da Silva et al. (2007) reported almost same 11% (1993) to 43% (1980). Maximum hatchlings were average in situ hatching success (80.0%) compared produced between 1980 and 1986. It dropped down to relocated nests (78.7%) of olive ridley in Brazil. to below 10,000 (7563 per year) between 1993 and Similarly, Kornaraki et al. (2006) did not find any 1996 before rising again to 11077 in 1997. significant difference in hatching success rates between hatchery and natural nesting. Olive ridley. A total of 21142 olive ridley hatchlings (1174.5 per year) were produced during the period In the present study, the hatching rate of green (25%) 1980 - 1997 varying from 11 (1994) to 4766 (1984). and olive ridley (27%) turtles are low as compared to The egg clutches hatched varied from 50 (1995) to the rates reported in the literature from other parts of 100% (1980, 1994 and 1996). The total number of the world. Minimum rate of hatching during the months olive ridley hatchlings produced per year ranged of June and July may be correlated to monsoon sea- between 54 (1993) and 5424 (1984). The number of son and prolonged wet weather as also described by hatchlings produced per year varied from 1 to 135. Hendrickson (1958) and Bustard (1972). Hatching The period between 1982 and 1987 (excluding 1983) rates in terms of number of clutches hatched during may be recognized as years of high production, the present study period varied from 47-85% and 50- whereas hatchlings were produced in significantly low 100% for green and olive ridley turtles, respectively number (11- 54) during 1993 - 1995. No hatchling (Tables 1 and 2). was produced in 1997 although 262 eggs were trans- The only published reference on hatching intensity of planted to the enclosures (Table 2). turtles from Karachi coast is that of Minton (1966) Nesting and hatching of green and olive ridley turtles who worked on herpetology of West Pakistan during were at their peak in August - October, when tempera- the period 1958 to 1966. He mentioned without quan- ture started decreasing from its maximum in June-July tifying the rates that maximum hatching in green turtle (Figs. 1-2). The nesting generally started in the post and olive ridley at Hawkesbay occurred during late monsoon season (August- onwards) which coincided September and early October. This is in consistence with decreasing temperature after reaching to its peak with the present results. Transplantation of Marine Turtle Nests 33

References Kornaraki, E., Matossian, D.A., Mazaris, A.D., AGRW 1968. Olive-backed loggerhead turtles. Loris, Matsinos, Y.G., Margaritoulis, D. 2006. Effec- 11: 203-204. tiveness of different conservation measures for Baldwin, W.P., Lofton, J.P. 1959. The loggerhead turtles loggerhead sea turtle (Caretta caretta) nests at of Cape Romain, South Carolina. Abridged and Zakynthos Island, Greece. Biological Conserva- annotated by D. K. Caldwell. Bulletin of Florida tion, 130: 324-330. State Museum, 4: 309-348. Minton, S.A. 1966. A contribution to the Herpetology Bustard, H.R. 1972. Sea Turtles: Their Natural History of West Pakistan. Bulletin of American Museum of and Conservation, 220 pp., Collins, London, UK. Natural History, 134: 29-184. Bustard, H.R., Greenham, P. 1968. Physical and Pritchard, P.C.H. 1969. Studies of the Systematics and chemical factors affecting hatching in the green sea Reproductive Cycles of the Genus Lepidochelys, turtle (Chelonia mydas L). Ecology, 49: 269-276. Ph.D. Dissertation, University of Florida, USA. Carr, A.F., Hirth, H. 1962. The ecology and migration Salm, R. V. 1975. Summary report on existing and of sea turtles. 5. Comparative features of isolated potential marine parks and reserves around Sri green turtle colonies. American Museum Novitates, Lanka, Southeast and Western India and Pakistan. 2091: 1-42. In: Regional Meeting on Marine Parks and Reserves, da Silva, A.C.C.D., de Castillos, J.C., Lopez, G.G., Tehran, Iran, 6-10 March 1975, IUCN, Morges, Barata, P.C.R. 2007. Nesting biology and conser- Switzerland. vation of the olive ridley sea turtle (Lepidochelys Stancyk, S. E., Talbert, O. R., Dean, J. M. 1980. olivacea) in Brazil, 1991/1992 to 2002/2003. Jour- Nesting activity of loggerhead turtle (Caretta nal of Marine Biological Association of the caretta) in South Carolina, II. Protection of nests United Kingdom, 87: 1047-1056. from raccoon predation by transplantation. Firdous, F., Barkati, S., Rahman, S. 2009. Hatching and Biological Conservation, 18: 289-98. rearing of two species of marine turtles from Trullas, S.C., Paladino, F.V. 2007. Microenvironment Karachi, Pakistan. Records of Zoological Survey of olive ridley turtle nests deposited during an of Pakistan, 19: 1-7. aggregated nesting event. Journal of Zoology, 272: Groombridge, B. 1982. The IUCN Amphibia-Reptilia 367-376. Red Data Book, Part I: Testudines, Crocodylia, Vanzolini, P.E. 2003. On clutch size and hatching Rhynchocephalia, 426 pp., IUCN, Conservation success of the South American turtles Podocnemis Monitoring Centre, Gland, Switzerland. expansa (Schweigger, 1812) and P. unifilis Troschel, Hendrickson, J.R. 1958. The green sea turtle Chelonia 1848 (Testudines, Podocnemididae). Anais da mydas (Linn.), in Malaya and Sarawak. Proce- Acadmic Brasileira de Ciencias, 75: 415-430. edings of Zoological Society of London, 130: 455- Witham, R., Futch, C.R. 1977. Early growth and oceanic 535. survival of pen-reared sea turtles. Herpetologica Hirth, H., Carr, A. 1970. The green turtle in the Gulf of 33: 404-409. Aden and the Seychelles Islands.Verhandelingen Zwinenberg, A.J. 1976. The Olive ridley, Lepidochelys der Koninklijke Nederlandes Akademie Van olivacea (Eschscholtz, 1829), probably the most Wetnshchappen Afdeling Naturkunde Tweede numerous marine turtle today. Bulletin of the Reeks -Deel, 58: 1-44. Maryland Herpetological Society, 12: 75-95. Pak. J. Sci. Ind. Res. Ser. B: Biol. Sci. 2011 54 (1) 34-40

Effects of Cowdung and Poultry Manure on Growth Performance of Indian Major Carps (Catla catla, Labeo rohita) and Exotic Carp (Cyprinus carpio) in Thatta District (Sindh), Pakistan

Mohammad Shoaiba*, Syed Anser Rizvia, Faisal Ameerb and Mohammad Nasirb aDepartment of Zoology, University of Karachi, Karachi - 75270, Pakistan bFMRRC, PCSIR Laboratories Complex, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi - 75280, Pakistan

(received April 20, 2010; revised August 24, 2010; accepted September 24, 2010)

Abstract. In the manuring trials conducted in earthen ponds of 120 m2 for one year, the juveniles of Indian major carps (Catla catla and Labeo rohita) and exotic carp (Cyprinus carpio) responded positively during warmer months i.e. March to August. The mean weight increase and mean specific growth rate (SGR) were better in the ponds fertilised with cowdung. Total growth rate per day calculated for Catla catla was 32.96 g, for Labeo rohita 95.02 g and for Cyprinus carpio 14.37 g under the influence of cowdung. Moreover individual growth rates/day were also high i.e. 2.06, 2.50 and 1.43 g, respectively. Percent contribution in fish production was 23.15%., 66.74% and 10.09%, respectively. Total growth rate of the three was significantly higher in cowdung (142.36 g) as compared to that in poultry manure i.e. 106.64 g. Both the fertilisers significantly differed in terms of monthly and treatment variations (P<0.05) except for treatment variation for Cyprinus carpio. Water temperature, light penetration, electrical conductivity, total dissolved solids, total suspended solids and nitrates were highly significant (P<0.05) both for treatment and monthly variation except pH and dissolved oxygen. Keywords: poly-culture, carp farming, organic manure, Catla catla, Labeo rohita, Cyprinus carpio

Introduction (iii) physicochemical parameters of fish pond of Use of animal wastes like poultry manure and cowdung carps (Indian major and exotic carps) in poly-culture has been practiced for long for generation of algal system. blooms which play a vital role in developing ecofriendly, Conventionally, three species of Indian major carps economical and socially viable cultural system. namely Catla catla (suface feeder), Labeo rohita According to Dhawan and Kaur (2002), cowdung and (column feeder) and Cirrhina mrigala (bottom feeder) poultry manure proved to be the best organic fertilisers were stocked in a fish pond (Javed et al., 1992). when used adequately. Presently instead of C. mrigala, Cyprinus carpio Conversely, indiscriminate use of these fertilisers in fish has been stocked as bottom feeder considering the ponds, instead of improving the primary productivity, findings of Milstein et al. (2002). may also lead to pollution. Therefore, developing standards for doses is necessary which would control Materials and Methods the ecological parameters of pond water for favourable Experiments were conducted in three replications for growth of fish like major and common carps (BARC, both manuring trials in earthen ponds (size ~120 m2, 1997). The cowdung and poultry manure, when water depth 1.5 m) in Thatta district (Sindh), Pakistan. recycled, produce soluble phosphorous, nitrogen and The experimental neonates of Indian major carp and carbon for algal growth and natural food production exotic carp were obtained by means of induced (Knud-Hansen et al., 1991). breeding technique. The ponds were stocked with Work has been on record on the utilization of cowdung stocking density of 16:38:10 (Catla catla: Labeo and poultry manure for growth of conventional Indian rohita: Cyprinus carpio) according to Sheri et al. major carps. The present study was conducted to work (1986). out the effects of these manures on (i) growth perfor- Pond preparation. The existing fish ponds were mance, (ii) production of planktonic biomass and emptied and sun dried. To avoid any infection, liming *Author for correspondence; E-mail: [email protected] was done after fifteen days interval. The inlets and

34 Effect of Organic Fertilizers on Carp Growth 35

outlets of all ponds were sealed with fine mesh gauze to increase right from the beginning (16.47 g in for blocking the intrusion of any predator into the pond September) and continued upto 92.93 g in July but or the exit of experimental fish from the pond. lowered slightly in the last month of experiment (August, 86.80 g) (Table 1). Better specific growth Manuring of fish ponds. Nitrogen content of the test rate (SGR) was noted during the whole experimental manures was estimated by means of Kjeldahl method periods. following the analytical procedure of AOAC (1990). The amount of manure used in the ponds was calculated on the basis of nitrogen availability. Poultry Table 1. Growth performance of Catla catla under the manure at the rate of 0.17 g N/100 g and cowdung at influence of poultry manure and cowdung the rate of 0.2 g N/100 g of stocked fish were intro- Months Poultry manure Cowdung duced daily. Mean Mean wt. Mean Mean Mean wt. Mean weight increase SGR weight increase SGR Parameters studied. During the study, following September 78.15 58.92 0.813 68.53 16.47 0.306 parameters were recorded: October 137.32 59.0 0.323 85.00 22.57 0.336 1. Monthly increase in weight = Final mean November 172.27 16.44 0.126 107.57 30.97 0.360 December 188.7 13.12 0.093 138.5 48.0 0.416 weight (g) - Initial mean weight (g) January 201.83 12.64 0.083 186.5 68.1 0.400 2. Specific growth rate February 214.47 14.78 0.093 247.6 67.43 0.343 (%/day) = Log Wtt-Log Wti March 229.3 39.63 0.226 315.0 82.33 0.340 × 100 Time April 269.2 49.68 0.240 397.3 84.93 0.276 3. Individual performance of experimental Indian May 318.8 55.5 0.223 482.3 87.83 0.240 major carps and common carp. June 374.3 67.5 0.223 570.1 88.90 0.210 4. Physical parameters of pond water included July 441.9 84.7 0.236 659.0 92.93 0.186 August 526.5 112.9 0.336 751.9 86.80 0.153 temperature, light penetration, pH, electrical conductivity, total dissolved solids (TDS) and total suspended solids (TSS). Table 2 exhibits growth performance of Labeo rohita 5. Chemical parameters included nitrate nitrogen, in ponds receiving poultry manure and cowdung. dissolved oxygen and total hardness of water. L. rohita responded similar to C. catla in poultry Prior to chemical analysis, water samples were manured ponds i.e. the attainment of weight was collected and brought in the laboratory. For observed during warmer months up to 130.79 g in dissolved oxygen, water samples were fixed August. In cowdung treated ponds, the weight gain immediately after sampling as described by increased in the beginning and continuously showed Welch (1952). Nitrate nitrogen and total hard- addition upto August (95.13 g). With respect to ness were determined by Merck chemical test kits for testing water and wastewaters. Table 2. Growth performance of Labeo rohita under Statistical analysis. The obtained data were subjected enrichment of the poultry manure and cowdung to statistical analysis using MINITAB Release 14. Months Poultry manure Cowdung Mean values for various parameters and significance Mean Mean wt. Mean Mean Mean wt. Mean levels were determined using two way analysis of weight increase SGR weight increase SGR variance (ANOVA). September 58.77 47.49 0.847 88.27 19.66 0.283 October 106.25 41.07 0.463 107.93 32.06 0.373 Results and Discussion November 147.3 39.47 0.333 140.00 42.03 0.376 Growth of fish. When fertilised with poultry manure, December 186.8 14.24 0.100 181.97 70.90 0.470 January 201.0 10.36 0.066 252.87 90.10 0.436 Catla catla mean weight (g) was observed to lower February 211.4 23.31 0.150 342.97 93.96 0.346 considerably during the four months of November to March 234.7 39.29 0.220 437.00 93.06 0.276 February (16.44, 13.12, 12.64 and 14.78 g) because of April 274.0 58.14 0.283 530.07 94.33 0.233 lowering of water temperature (Table 1). It started to May 332.1 76.3 0.296 624.40 95.93 0.203 improve after winter; from March to August, mean June 408.5 114.4 0.343 720.33 97.00 0.176 July 522.9 130.4 0.313 817.33 95.40 0.153 weight increased upto 112.9 g. While in cowdung August 653.2 130.7 0.253 912.73 95.13 0.136 treated ponds the mean weight of C. catla started 36 Mohammad Shoaib et al. growth per day it was noticed that SGR was healthy Table 4. Individual performance and percent contri- (0.305%/day) with poultry manure treatment as com- bution of experimental fishes under enrichment of the pared to 0.288%/day in the case of cowdung. poultry manure and cowdung Fishes Growth Individual growth Contribution Approximately same size of juvenile Cyprinus carpio rate/day rate/day (%) were stocked in poultry manure and cowdung treated Catla catla 23.07 1.44 21.64 ponds. The minimum weight increase was recorded Labeo rohita 68.00 1.78 63.76 during December to March (12.129 g in January), the Cyprinus carpio 15.55 1.55 14.58 winter months, while weight increased by 100.8 g Total growth rate of three species in poultry manure 106.64 g/day (maximum) in July when ponds were enriched with Catla catla 32.96 2.06 23.15 poultry manure. C. carpio did not show good response Labeo rohita 95.02 2.50 66.74 earlier in cowdung enriched ponds as generally they Cyprinus carpio 14.37 1.43 10.09 prefer food items in bottom soil; the organic materials Total growth rate of three species in cowdung 142.36 g/day sometimes took about two months to settle in the bottom soil or pond embarkment. Later on, their weight increased gradually in the range of 33.70 g in January rate per day of C. catla was 1.44 g, of L. rohita 1.78 g upto 57.10 g in August. SGR followed, the trend of and that of C. carpio 1.55 g. Total growth rate at the L. rohita, being 0.243%/day with poultry manure time of harvest was recorded to be 106.64 g/day. treatment and 0.203%/day with cowdung treatment Percent contribution of all fishes was recorded as (Table 3). 21.64%, 63.76% and 14.58% (Table 4). Statistically it was also noticed that C. catla mean weight signifi- Table 3. Growth performance of Cyprinus carpio cantly differed (P<0.05) both in case of the treatment under enrichment of the poultry manure and cowdung and the monthly variations (Table 5). Months Poultry manure Cowdung Mean Mean wt. Mean Mean Mean wt. Mean Table 5. Analysis of variance (GLM) of experimental weight increase SGR weight increase SGR fishes for monthly and treatment variations September 84.25 35.05 0.496 88.20 23.00 0.326 Fishes Months Treatments October 119.3 41.08 0.433 111.20 25.93 0.296 November 161.53 35.52 0.296 137.20 39.07 0.290 F-value P-value F-value P-value December 198.80 22.6 0.076 168.00 34.87 0.290 Catla catla 27.34 0.000* 12.82 0.000* January 209.75 12.12 0.076 206.20 33.70 0.216 Labeo rohita 12.33 0.000* 16.09 0.002** February 221.84 13.54 0.086 239.90 44.20 0.236 Cyprinus carpio 66.04 0.000* 0.90 0.362* March 236.8 22.13 0.166 284.1 42.77 0.196 * = non-significant; ** = significant. April 267.5 37.67 0.213 326.9 45.40 0.180 May 311.9 58.19 0.283 372.3 47.50 0.170 June 381.0 77.80 0.306 419.8 50.57 0.156 Physicochemical parameters of water. Poultry July 472.4 100.8 0.246 470.3 54.53 0.153 August 567.9 62.3 0.243 524.8 57.10 0.146 manure and cowdung were introduced to supply adequate levels of nutrients necessary for production of primary products and ultimately for the fish. During this process, the physicochemical parameters of water Individual performance. In cowdung treated ponds, considerably fluctuated affecting the growth rate of the growth rate per day of C. catla was 32.96 g, of experimental fish. Tables 6 and 7 show mean annual L. rohita, 95.02 g and that of C. carpio, 14.37 g. values along with their values for analysis of variance Individual growth rate per day was 2.06, 2.50 and (F-values and P-values) for both the treatments and the 1.43 g, respectively. Percent contribution of these monthly variations. Significant differences (P<0.05) experimental fishes was recorded as 23.15%, 66.74% were observed in light penetration, electrical conduct- and 10.09%. Total growth rate at the harvest of all the ivity, nitrates, total dissolved solids and total suspended three species was 142.36 g per day (Table 4). solids of both the treatment and the monthly variations. The growth rates per day of C. catla 23.07 g, L. rohita However, non-significand difference was noticed only 68.00 g and C. carpio 15.55 g were recorded in in temperature in case of treatment variation while the poultry manure treated ponds. The individual growth monthly variation remained significantly different. Effect of Organic Fertilizers on Carp Growth 37

Table 6. Physicochemical parameters of water enriched with poultry manure and cowdung Variables Poultry manure Cowdung Mean±SE Min Max Mean±SE Min Max Temperature (°C) 21.65 ± 2.16 11.98 31.15 22.18 ± 2.07 12.52 30.85 Light penetration (cm) 12.55 ± 1.34 7.93 23.59 18.492 ± 0.856 14.800 22.700 pH 8.017 ± 0.101 7.40 8.70 8.5325 ± 0.0580 8.250 8.950 Electrical conductivity 1.904 ±0.0423 1.700 2.09 1.780 ± 0.0193 1.660 1.870 (m.mhos.cm) Total dissolved solids (mg/L) 266.47 ± 6.59 222.33 308.66 244.8 ± 22.3 150.0 370.0 Total solids (mg/L) 1689 ± 102 1186 2238 1946 ± 130 450 1203 Dissolved oxygen (mg/L) 5.741 ± 0.584 2.90 9.93 13.225 ± 0.219 11.80 14.30 Nitrates (mg/L) 2.785 ± 0.379 0.220 5.220 5.252 ± 0.0665 4.880 5.650 Total hardness (mg/L) 266.47 ± 6.59 222.33 308.66 244.8 ± 22.3 150.0 370.0

pH, dissolved oxygen and total hardness also differed Table 7. Analysis of variance for various physico- non-significantly only for monthly variation and was chemical parameters of water enriched with different highly significant for poultry manure and cowdung fertilizers (Table 7). Variables Treatment Months F-values P-values F-values P-values Indian major carps, including Catla catla (surface ** feeder) and Labeo rohita (column feeder) along with Temperature (°C) 2.72 0.060 211.89 0.000 Light penetration (cm) 36.46 0.000 4.36 0.000** Cyprinus carpio (bottom feeder) were stocked to pH 10.97 0.000 1.33 0.254* evaluate the response of pond enrichment. These fishes Electrical conductivity 70.11 0.000 18.14 0.000** are excellent respondent to different pond niches. The (m.mhos.cm) present study revealed that these fishes fully utilized Total dissolved solids 38.82 0.000 2.41 0.025** the available food, generated by pond enrichment. (mg/L) During the study, the stocking of C.carpio with C. catla Total solids (mg/L) 31.43 0.000 2.95 0.008** and L. rohita produced positive environmental results Dissolved oxygen 89.87 0.000 1.48 0.188* because the C.carpio damaged the embarkment with- (mg/L) out creating any harmful effect on the rest of two fishes. Nitrates (mg/L) 49.26 0.000 2.34 0.029** Besides this, the nutrients released by the soil were Total hardness (mg/L) 8.34 0.000 1.90 0.075* evenly dispersed in the experimental ponds to promote ** = significant; * = non significant. the planktonic biomass. The present results regarding the stocking of major carps and common carp also conform to those of Wahab et al. (2002) who found (1979) also favoured the use of cowdung to rear carp that the feeding habit of common carp was certainly fingerlings instead of inorganic fertilisers. However, helpful for nutrient mixing from soil compared to Banergee et al. (1979) reported that poultry manure conventional bottom feeder fish. There were no effects can maximize fish production as it can provide on the performance of C. catla and L. rohita. Con- nitrogen adequately for development of phyto- sidering present results of manuring trials, the best plankton and zooplankton which serve as food items growth (142.36 g/day) was observed with the use of for fish. Kang’ombe et al. (2006) suggested that cowdung (Table 5) followed by the poultry manure chicken manure treatment produced large amount of (106.64 g/day) which concluded that cowdung has chlorophyll along with large number of zooplanktons an edge over poultry manure (organic fertiliser). indicating high levels of primary producers than cattle Rappaport and Sarig (1978) claimed that the cowdung and pig manure. feed for fish culture produced highly positive results as compared to that of the poultry droppings, which As mentioned in Tables 1-3, growth rates of all the confirm present findings. Machaodo and Costagnolli experimental fishes were slow due to low feeding rate 38 Mohammad Shoaib et al. up to the month of February. The available primary and Fitzpatrick (1979) who suggested total optimum producers gradually flourished with the increase in the growth of fish may coincide with the temperature temperature and thus in the decomposition rate of because the latter maximizes the metabolic efficiency poultry manure and cowdung. of fishes. Jinghran (1982) stated that major carps thrived well in temperature range of 18.3-37.8 °C, while Goolish and Adelman (1984) found that a unit change the grass carp, silver carp and common carp preferred in temperature directly affects the growth of fish as the temperatures below 30 °C. Annual variations in tem- two are proportional to one another. Javed (1988) perature have a great impact on primary productivity reported that the stocked fish (major carps) responded and also speed up the chemical change in water and significantly when the temperature was between 30.00 soil. Singh (1964) reported that temperature and light to 33.73 °C. Conversely to this, during the present penetration were the two important factors responsible study, major carps showed the best growth within for regulating the maximum and minimum production the temperature range of 22.89 °C to 31.15, 26.42 to of phytoplankton. However, the report of Parveen 30.85 °C, 23.40 to 30.80 °C and 27.77 to 30.68 °C (1986) contradicts the present findings as the correla- when the pond received poultry manure or the tion coefficient between the dry weight of planktonic cowdung. The findings of Barthelmes and Bramick biomass and physical factors like temperature, light (2003) seemed to deviate. They reported silver carp penetration, electrical conductivity etc. was non- grew better in the temperature range of 18.00 °C to siginificant. Brezonic et al. (1984) suggested that high 22.00 °C. pH values prompted the growth of phytoplankton and Individual performance of weight gain of L. rohita resulted in blooms, while on the contrary, Nazneen remained better with cowdung (250 g/day) followed (1980) argued that high values of pH during phyto- by poultry manure (1.78 g/day). The maximum weight plankton blooming period were the result of and not gained by L. rohita may be due to its efficiency in the cause of phytoplanktonic blooms. According to conversion of available food (phytoplankton) to Hawkins and Griffith (1986), the pattern of light varies flesh (Jhingran and Pullin, 1985). C. catla attained at different times of the year which exclusively depends maximum weight gain (2.06 g/day) under the enrich- on the stratification conditions and dominance of ment of cowdung followed by poultry manure phytoplankton species. (1.44 g/day). The C. carpio, as bottom feeder fish, Dissolved oxygen contents, when both the fertilizers gained 1.55 g/day of weight in pond receiving poultry were used, showed irregularity in variation related to manure while in cowdung treated pond, 1.43 g/day temperature and abundance of phytoplanktons. The weight gain was observed (Table 5). The above- results of present experiments were similar to the ones mentioned results are very much similar to the reported by Chiba (1971). Sharma et al. (1979) reported findings of some other workers including Javed and that the concentration of dissolved oxygen depended Sial (1991) who reported that Indian major carp yield on both the physical and the biological factors. of 3360.40 and 3215.60 kg/ha/year can be obtained Depletion of dissolved oxygen was due to respiration through the use of broiler and layer droppings, res- of bacteria and phytoplankton and it was compensated pectively, when applied at the rate of 0.10 g N/100 g of by zooplankton and algae during daytime through body weight on daily basis. Natarjan and Varghese photosynthesis. (1980) concluded that poultry manure was better fertilizer for maximizing the pond productivity The availability of nitrogen in the form of nitrate was (2728.0 kg/ha/year) than digested sewage sludge highly noticed during the present study. Nitrates in (2156.00 kg/ha/year) and cowdung (1811.00 kg/ha/ both the fertilisers significantly differed in months and year). Garg and Bhatnagar (1999) optimized the rate treatments. It is an important factor in the planktonic of cowdung for increasing the pond productivity and biomass production and is accordingly related to the fish biomass; 4.45 kg fish biomass and 2.36% (SGR) findings of Mahboob and Sheri (1993), who mentioned weight per day were observed when the 15000 kg/ha/ significant contribution of nitrates towards the growth year cowdung was applied. of fish. The present findings regarding the effect of tempera- Total hardness was also highly significant during the ture on fish yield are in line with the results of Beitinger whole experimental period but non-significant for both Effect of Organic Fertilizers on Carp Growth 39

the types of applied fertilizers. These results are in line Goolish, E.M., Adelman, I.R. 1984. Effect of ration with the reports of Vasisht and Jindal (1980) that size and temperature on the growth of juvenile phytoplankton showed a direct relationship with the common carp (Cyprinus carpio L.). Aquaculture, light penetration, pH, dissolved oxygen and hardness 36: 27-35. of water. Parveen (1986) reported that correlation Hawkins, P.R., Griffiths, D.J. 1986. Light attenuation coefficient for nitrates, total hardness and dry weight in a small tropical reservoir (Solomon Dam, North of planktonic biomass was non-significant. Queensland): Seasonal changes and effect of artificial aeration. Australian Journal of Marine The present study concluded that for ecological and and Freshwater Research, 37: 199-208. economical sustainability, optimum dose of fertilisers Javed, M., Hassan, M., Sial, M.B. 1992. Fish pond for fish were evidently necessary for maximum net revenue. fertilization. IV. Effect of cowdung on the growth performance of major carps. Pakistan Journal of References Agricultural Sciences, 29: 111-115. Javed, M., Sial, M.B. 1991. Fish pond fertilization (iii) AOAC 1990. Official Methods of Analysis of the Effect of layer manure fertilization on the growth AOAC, 15th edition, Association of Official performance of fish viz., Catla catla, Labeo rohita Analytical Chemists, Arlington, VA, USA. and Cirrihinus mrigala. Pakistan Journal of Banergee, R.K., Ray, P., Singil, G.S., Dutta, B.R. 1979. Agricultural Sciences, 28: 115-120. Poultry droppings- its manurial potentiality in Javed, M. 1988. Growth Performance and Meat aquaculture. Journal of the Inland Fisheries Quality of Major Carps as Influenced by Pond Society of India, 2: 94-108. BARC 1997. Fertilizer Recommendation Guide, Fertilization and Feed Supplementation. Ph.D. Publication No. 41, 196 pp., Bangladesh Agricul- Thesis, 281 pp., University of Agriculture, tural Research Council, Dhaka, Bangladesh. Faisalabad, Pakistan. Barthelmes, D., Bramick, U. 2003. Variability of a Jhingran, V.G., Pullin, R.S.V. 1985. A hatchery manual cyprinid lake ecosystem with special emphasis on for the common, Chinese and Indian major carp. the native fish fauna under intensive fisheries ICLARM Studies and Reviews 11, 191 pp., Asian management including common carp (Cyprinus Development Bank, Manila, Phillipines and carpio) and silver carp (Hypophthalmichthys International Center for Living Aquatic Resources molitrix). Limnologica, 72: 331-342. Management, Manila, Phillipines. Beitinger, T.L., Fitzpatric, L.C. 1979. Physiological and Jhingran, V.G. 1982. Fish and Fisheries of India, ecological correlates of preferred temperature in 666 pp., 2nd revised edition, Hindustan Publishing fish. American Zoologist, 19: 319-329. Corporation, New Delhi, India. Brezonic, P.L., Crisman, T.L., Schluze, R.L. 1984. Kang’ombe, J., Brown, J.A., Halfyard, L.C. 2006. Planktonic communities in Florida (USA) Effect of using different types of organic animal softwater lakes of varying pH. Canadian Journal manure on plankton abundance and on growth of Fisheries and Aquatic Sciences, 41: 46-56. and survival of Tilapia rendalli (Boulenger) in Chiba, K. 1971. Studies on the carp culture in running ponds. Aquaculture Research, 37: 1360-1371. water pond. (iii) On the relation between fish Knud-Hansen, C.F., McNabb, C.D., Batterson, T.R. growth or harvest and environmental conditions 1991. Application of limnology for efficient in fish ponds. Bulletin of Freshwater Fisheries nutrient utilization in tropical pond aquaculture. Research Laboratory Tokyo, 20: 111-215. Verhandlungen der Internationalen Vereinigung Dhawan, A., Kaur, S. 2002. Pig dung as pond manure. fur Theoretische und Angewandte Limnologie, Effect on water quality, pond productivity and 24: 2541-2543. growth of carps in poly-culture system. Naga: Machado, C.R., Costagnolli, N. 1976. Preliminary The ICLARM Quarterly, 25: 11-14. observation related to the culture of Rhamdia Garg, S.K., Bhatnagar, A. 1999. Effect of different hilarii, a Brazillian cat fish. FAO Technical doses of organic fertilizer (cowdug) on pond Conference on Aquaculture, Kyoto, Japan 26th productivity and fish biomass in still water May, 1976, Fishery Resources and Environment ponds. Journal of Applied Ichthyology, 15: 10-18. Division, 12 pp., FAO, Rome, Italy. 40 Mohammad Shoaib et al.

Mahboob, S., Sheri, A.N. 1993. Regression studies on on intensive growth fish farming at the Ginosar the physicochemical characteristics and planktonic Station ponds. Bamidgeh, 29: 57-70. life of fishpond. Pakistan Journal of Zoology, 25: Sharma, B.K., Kumar, D., Das, S.R., Chakraborty, D.P. 19-21. 1979. Observations on swine dung recycling in Milstein, A., Wahab, M.A., Rahman, M.M. 2002. composite fish culture. In: Proceedings of Envoirnmental effect of common carp Cyprinus Symposium on Inland Aquaculture (Abstract), carpio (L.) and mrigal. Cirrhinus mrigala (Hamilton) Central Inland Capture Fisheries Research as bottom feeders in major Indian carp polyculture. Institute, Barrackpore, India. Aquaculture Research, 33: 1103-1117. Sheri, A.N., Sial, M.B., Javed, M. 1986. Nutrient Natarajan, M., Varghese, T.J. 1980. Studies on the requirements of the fish. I. Pond fertilization with effects of poultry manure, digested sewage study N.P.K. (20:20:5). Pakistan Journal of Agricultural cake and cowdung on the growth of Catla catla Sciences, 23: 266-277. (Hamilton) and Cyprinus carpio var. Communis Singh, C.G. 1964. Seasonal fluctuations in diatoms (Linneaus). Agricultural Waste, 2: 261-271. populations. Tropical Ecology, 5: 42-51. Nazneen, S. 1980. Influence of hydrological factors Vasisht, H.S., Jindal, R. 1980. Rheological survey of a on the seasonal abundance of phytoplankton in Pucka stream of Patiala (Punjab, India). Limnologica, Kinjhar Lake, Pakistan. Hydrobiologia, 65: 262- 12: 77-83. 282. Wahab, M.A, Rahman, M.M., Milstein, A. 2002. The Parveen, N. 1986. Study on the Relationship Between effect of common carp, Cyprinus carpio (L) and Physicochemical and Biological Aspects of mrigal, Cirrhinus mrigala (Hamilton) as bottom Fisheries and Storage Water Tank. M. Phil. Thesis, feeders in Major Indian carp polycultures. 56 pp., University of Agriculture, Faisalabad, Aquaculture Research, 33: 547-556. Pakistan. Welch, P.S. 1952. Limnological Methods, 538 pp., Rappaport, V., Sarig, S. 1978. The results of manuring Mc Graw Hill Book Co. Inc., New York, USA. Pak. J. Sci. Ind. Res. Ser. B: Biol. Sci. 2011 54 (1) 41-46

Morphological Variations, Patterns of Frontal Ambulacrum Pores and Paleoecology of Heteraster renngarteni Poretzkaja (Echinoidea: Spatangoida) from Aptian Sediments of Baghin Area, Kerman, Iran

Mohammed Raza Vaziri* and Ahmad Lotfabad Arab Geology Department, Faculty of Sciences, Shahid Bahonar University, Kerman, Islamic Republic of Iran

(received July 30, 2009; revised July 25, 2010; accepted August 30, 2010)

Abstract. Detailed macro- and microscopic analysis of spatangoid echinoid, Heteraster renngarteni Poretzkaja showed a remarkable variation in morphology and alternation of short and long pores in the frontal ambulacrum. The differentiation of pores in the frontal ambulacrum has been interpreted as an adaptive strategy for survival in a shallow shelf environment whereas, variation in morphology appears to be influenced mainly by grain size of the substrate. Keywords: echinoids, Iran, ambulacrum pores, Heteraster renngarteni, Cretaceous, Kerman

Introduction of spatangoids (Villier et al., 2001), there is no revised Phylogenetic schemes for spatangoid relationships have monograph of the genus. Description of Iranian mate- been proposed by Smith (1984) and more recently by rial will improve knowledge about the genus and help Neumann (1999). The order Spatangoida appeared first compare it with the specimens reported from the other in the lowermost Cretaceous (Berriasian). Within the parts of the world. spatangoids, the family Toxasteridae originated probably H. renngarteni, on which this study focused, is a rather from a disasteroid ancestor. During the Berriasian to the large toxasterid echinoid and occurs abundantly in Aptian, diversification of the family led to appearance Aptian marls of Baghin area, west of Kerman, Iran. of three genera (Toxaster, Aphelaster, and Heteraster). Apparently, this primitive spatangoid is a highly endemic These genera were a rootstock for all the later Spatan- goida (Villier et al., 2004). taxon, described previously from Azerbaijan (Ali-zadeh, 1988) and Turkmenistan (Lobacheva, 1968) but is The genus Heteraster appeared first during the reported for the first time from Iran herein. Hauterivian. From this time to the Barremian, diversity of the genus is very low (fewer than 5 species). The acme Geological setting and stratigraphy. The species of diversity occurs in the Aptian and Albian with about Heteraster renngarteni, described here, was collected 20 species (Villier et al., 2001). Afterward, diversity from Aptian strata that crop out in Baghin area, west decreases again and finally the genus disappears during of Kerman city (Fig. 1). The Aptian sediments of the Early Cenomanian. The genus Heteraster is unique Baghin consists mainly of alternation of green marls among spatangoids, because its frontal ambulacrum and thin layers of yellow shaly limestone (Fig. 2) with consists of an alternation of short and long pores. The an abundant benthic fauna of Orbitolina, brachio- patterns of pore distribution in Heteraster were docu- pods, oysters, bivalves, gastropods and corals. Echi- mented by Devries (1955) and Villier et al. (2001). noids are also among the most conspicuous and diverse constituents of the Aptian fauna of Baghin Cotteau (1890) proposed the name Heteraster as a area, represented by Stomechinidae, Emiratidae, synonym of Enallaster. Adkins (1928) Lambert and Pygaulidae, Holectypidae, Cidaroida, Stirodonta, Thiery (1924) and Lambert (1920), regarded Enallaster Hemiasteridae, Toxasteridae and Acropeltidae, as a junior synonym of Heteraster, while Cooke (1946) among which the last two are the dominant groups of considered Enallaster at least as a subgenus of Heteraster. the assemblage. The Aptian marls are covered by a Although the genus Heteraster comprises of 34 species gray, bioturbated and thick-bedded limestone, *Author for correspondence; E-mail: vazirimohammadreza @yahoo.com relatively barren of macro-invertebrates.

Morphology of Heteraster renngarteni from Iran 43

Fig.3. Camera lucida drawings of apical: (a, d, f, h, i, k, l, n, p, q), oral: (c) and apical disc (b,e,g,j,m,o) views in different specimens of Fig. 4. Heteraster renngarteni Poretzkaja; a-c: (SBM H. renngarteni Poretzkaja, from the Aptian 22011): apical, oral and posterior views; d: of Baghin area. Scale bars represent 1 cm. (SBM 22013); e: (SBM 22018); f: (SBM 22012); g: (SBM 22022) and h: (SBM 22031) abnormal development and not as a distinct variety. Fron- apical views. All from the Aptian marls of tal sulcus was deep and well developed. Paired petals Baghin area. Scale bar represents 1 cm. were broad and unequal in length. The anterior petals reached the ambitus, slightly curved backward, opened The alternation pattern of short and long pores in the distally. Posterior petals were about 80% of the length frontal ambulacrum was also variable (Fig. 5). In the of the anteriors ones and straighter (Figs. 3 and 4). Ante- apical part of the ambulacrum, all the specimens rior ambulacrum was as wide as petals, sunken from apex exhibited an identical pore pattern. In this part all the to peristome. Interambulacral areas were about twice as pores were small and showed same size, while along wide as the ambulacral areas. No fasciole was present. the medial part, differentiation began and arrangement Periproct was relatively large and situated longitudinally was quite irregular with one short isopore alternating towards top of posterior truncate face (Fig.4c). Oral sur- with one, two, three or four elongate anisopores, but face was flat, peristome relatively large, pentagonal in usually one short to two or three long. outline, one-fourth the length from the front (Fig. 4b). In Results and Discussion some specimens, tuberculation was highly dense (Fig. 3d). H. renngarteni specimens found in Baghin area differed Morphology and arrangement of pore pairs in the from H. oblongus and H. peroni in having a nearly cen- frontal ambulacrum. The specimens of H. renngarteni tral position of the apical disc and less flexuous petals. found in the Baghin area showed considerable variation It is closest to the H. couloni (from Western Europe) in morphology and also pore arrangement in the frontal and H. heckeri (from Azerbaijan). It can be differenti- ambulacrum. The specimen outline can be cordiform, ated from H. couloni by the structure of ambulacrum ovate, circular, subquadrate or even hexagonal (Figs. 3 III and the relative width of the petals. Some specimens and 4), but usually cordiform. were very similar to H. heckeri Melikov, 1989 as the 44 Mohammed Raza Vaziri and Ahmad Lotfabad Arab

Table 1. Alternation of two types of pores in the frontal ambulacrum, from apex to ambitus in seven specimens of H. renngarteni

Sample Total number Number of Elongated no. of pores elongated pores pores (%)

1201 35 25 71.4 1202 33 23 69.6 1203 32 22 68.7 1204 47 34 72.3 1205 42 30 71.4 1206 25 17 68 1207 27 19 70.3

The warm environment is also confirmed by conspicuous presence of reef corals (shallow water corals) among faunal assemblage of Baghin area and indicates Fig. 5. Heterogeneity of pores in the frontal ambu- that the Aptian marls were deposited where the water lacrum and architectural types of the apical depth was shallower than 50 m and temperature, warmer disc in some specimens of H. renngarteni than 26 °C (Tsaparas and Diacantoni, 2005). Poretzkaja, from the Aptian of Baghin area; × 2.5 (appro.). Detailed macroscopic analysis also shows that there is wide morphological variation in the test shape of H. renngarteni. Spatangoid irregular sea urchins are detritivorous benthic pores showed a sieve-like feature (Fig. 5h). It seems organisms (Saucède et al., 2006), and almost all of them that H. renngarteni is synonymous with H. heckeri. live buried in sandy to muddy sediment from superficial Ecological interpretation. Echinoid requirement for layers to more than 20 cm deep. This mode of life enables oxygen depends on many factors including: size, the spatangoids to gain access to new food resources and deve-lopmental stage, temperature and animal activities escape from predators (Smith, 1984). Variation in test shape (Farmantarmanian, 1966). In warm waters of shallow of echinoids appears to be related to sediment granulometry, environment the metabolic rate is high, while gas exchange to the depth of burrowing, and to water depth (Saucède, decreases. Therefore, echinoids dwelling in shallow warm et al., 2007; Kanazawa, 1992; Néraudeau and Moreau, waters have more trouble in obtaining oxygen. 1989; Zaghbib-Turki, 1989; Nichols, 1959; Smith and Paul, 1985). This study shows that there is no marked The relation between functional anatomy of tube feet diffe-rence in percentage of respiratory tube feet among and morphology of the related pores has been described different specimens of H. renngarteni (Table 1). Those by Nichols (1959) and Smith (1980, 1978). Long pores tube feet represent about 70% of the pores in frontal are related to respiratory function, while short ones are ambulacrum in nearly all the studied samples. This obser- characteristic of tube feet since their main function is vation suggests that considerable variation in the test burrowing or food transport. In H. renngarteni, the long outline of Heteraster renngarteni, collected from Baghin pores are frequent and show about 70% of the alterna- area, is not dependent upon spatial distribution (i.e. occu- tion of the frontal ambulacrum (Table 1). pation of different levels within the sediments to minimize This percentage is similar to that of H. ucetiae, which competition between species or water depth), but may inhabits shallow environment (Villier et al., 2001). This results from granulometric parameters. In other words, fact suggests that the high percentage of long pores in although H. renngarteni population was located in marly H. renngarteni can be considered as an adaptive strat- facies, this highly sensitive community was more influ- egy for living in shallow environment, because, as stated enced by the grain size than the other factors (Fig. 6). above, in shallow- water species, the rate of respiration Closely spaced tubercles in some specimens, that supported increases and demand for oxygen is high, so respira- a dense cover of spines, might be a device for stabilization tory tube feet are frequent (Table 1). of the walls of burrows in more loose sediments. Morphology of Heteraster renngarteni from Iran 45

Fig. 6. Schematic diagram of the habitat of the different specimens of H. renngarteni from the Aptian marls of Baghin area. Grain symbols do not imply to any particular grain size, but just show that the difference in the test shape is significantly influenced by granulometry of the sediments.

Acknowledgement burrowing and locomotion in spatangoid echinoids. Palaeontology, 35: 733-750. The authors are deeply indebted to professor Loïc Lambert, J. 1920. Sur quelques genres nouveaux d, Villier, Université de Provence, France for his guidance e´chinides. Memoires de la Societe Academique and constructive discussion during the course of study. d’Agriculture des Sciences, Arts et Belleslettres du Special thanks to individuals who helped to collect De´partement de Aube; Serie 3, 55: 145-174. Heteraster renngarteni from Baghin area, including J. Lambert, J., Thiery, P. 1924. Essai de Nomenclature Hasani, M. Derisi, S. Jaber and M. Rami. Raisonnée des Echinides.Chaumont, Ferrière, Fasc. 6 and 7, 1909-1925. References Lobacheva, S.B. 1968. On the early cretaceous sea urchins of genus Epiaster, Turkmenistan. Annual Adkins, W.S. 1928. Handbook of Texas Cretaceous Review of the Soviet Paleontological Society, 18: Fossils, University of Texas Bulletin (Austin), 268-281 (in Russian). 2838: 1-385. Néraudeau, D., Moreau, P. 1989. Paléoécologie et Ali-zadeh, Ak.A. 1988. Cretaceous fauna of Azerbaijan. paléobio-géographie des faunes d’échinides du Baku. Elm., 648 pp., (in Russian) available on Cénomanien nord-aquitain (Charente-Maritime, http://www.jurassic.ru/cretaceous.eng.htm. France). Geobios, 22: 293-324. Cooke, C.W. 1946. Comanche Echinoids. Journal of Neumann, C. 1999. New spatangoid echinoids (Echi- Paleontology, 20: 193-237. nodermata) from the Upper Cretaceous of Jordan: Cotteau, G. 1890. Echinides nouveaux ou peu connus. their taxonomy and phylogenetic importance. th 9 part. Memoires Societe Zoologique de France, Berliner Geowissenschaftliche Abhandlung, E, 3: 537-550. 30: 175-189. Devries, A. 1955. Note sur le ge nre Heteraster. Bulletin de Nichols, D. 1959. Mode of life and taxonomy in la Société Géologique de France, 6: 315-323. irregular sea-urchins. Systematics Association Farmantarmaian, A. 1966. The respiratory physiology Publication, 3: 61-80. of echinoderms. In: Physiology of Echinodermata, Saucède, T., Mooi, R., David, B. 2007. Phylogeny and R. A. Boolootian (ed.), pp. 245-266, Wiley Inter- origin of Jurassic irregular echinoids (Echinoder- science, New York, USA. mata: Echinoidea). Geological Magazine, 144: Kanazawa, K. 1992. Adaptation of test shape for 333-359. 46 Mohammed Raza Vaziri and Ahmad Lotfabad Arab

Saucède, T., Alibert, P., Laurin, B., David, B. 2006. Tortonian Scleractinian corals from the island of Environmental and ontogenetic constraints on Gavdos (South Greece). Revue de Paléobiologie, developmental stability in the spatangoid sea 24: 629-637. urchin Echinocardium (Echinoidea). Biological Villier, L., Neraudeau, D., Clavel, B., Neumann, C., Journal of the Linnean Society, 88: 165-177. David, B. 2004. Phylogeny of early Cretaceous Smith, A.B., Paul, C.R.C. 1985. Variation in the irregular spatangoids (Echinodermata: Echinoidea) and echinoid Discoides during the Early Cenomanian. taxonomic implications. Palaeontology, 47: Special Papers in Palaeontology, 33: 29-37. 265-292. Smith, A.B. 1984. Echinoid Palaeobiology, 190 pp., Villier, L., David, B., Neraudeau, D. 2001. Ontogenetic Allen and Unwin, London, UK. and morphological evolution of the ambulacral Smith, A.B. 1980. The structure, function, and evolu- pores in Heteraster (early spatangoids). In: tion of tube feet and ambulacral pores in irregular Echinoderm 2000, M Barker (ed.), pp. 563-567, echinoids. Palaeontology, 23: 39-83. Balkema, Rotterdam, The Netherlands. Smith, A.B. 1978. A functional classification of the Zaghbib-Turki, D. 1989. Les Échinides indicateurs des coronal pores of regular echinoids. Palaeontology, paléoenvironments: un exemple dans le Céno- 21: 759-789. manien de Tunisie. Annales de Paléontologie Tsaparas, N., Marcopoulou-Diacantoni, A. 2005. (Invertébrés), 75: 63-81. Pak. J. Sci. Ind. Res. Ser. B: Biol. Sci. 2011 54 (1) 47-52

Antibacterial Activity of Saponin and Alkaloidal Extracts of Whole Plant of Phyllanthus niruri L., (Syn. P. franternus Webster)

Victor Adeyinka Ajibadea* and Oladiran Famurewab aMicrobiology Unit, Department of Science Technology, Federal Polytechnic, P.M.B 5351, Ado-Ekiti, Nigeria bDepartment of Biological Sciences, Faculty of Science, Osun State University, P.M.B. 4494, Oke-Baale, Osogbo, Nigeria

(received April 2, 2010; revised June 7, 2010; accepted August 23, 2010)

Abstract. Saponins identified as phylagenin-13-O-α-D-glucopyranoside and phylangenin-25-O-β-D- glucopyranoside and alkaloid, extracted from the whole plant of Phyllanthus niruri, were tested for minimum inhibitory concentration (MICs) against Staphylococus aureus, Staphylococus pyrogenes, Escherichia coli, Salmonella typhi and Klebsiella pneumoniae. MIC of saponin against S. aureus SSH22 and SSH23 ranged from 5-15 μg/mL, and against E. coli OAUTH71 and K. pneumoniae OAUTH 54, from 15-60 μg/mL. MICs increased with the increase in concentration of cells used in the inoculum. S. aureus SSH22 exhibited a paradoxical biphasic response to saponin in nutrient broth, whereas bacterial activity against E. coli SSH31 increased with concentration up to the highest concentration of saponin tested. Activity against E. coli OAUTH71 was more pronounced in the phosphate-buffered saline than in the nutrient broth. The other active compound extracted (alkaloid) gave MIC values between 200 and 600 μg/mL. Keywords: Phyllanthus niruri, saponin, alkaloid, antibacterial activity

Introduction Tiny amounts of some of them can immobilise even an Extracts of many plants are known to elicit certain elephant; others can be clinically used as analgesic, reactions in human body when applied in a prescribed antimalarial, and antispasmodic agents, for pupil manner including Phyllanthus niruri L., (Syn. dilation, and treatment of hypertension, mental dis- P. franternus Webster) belonging to the family orders and tumours. Morphine, codeine, atropine and Euphorbiaceae. It has been claimed to be an excellent ephedrine are just a few of the plant alkaloids currently remedy for jaundice and hepatitis (Tabasum et al., used in medicine (Naik and Juvekar, 2003). Other 2005; Qudhia and Tripathi, 2002). The plant is consi- alkaloids, including cocaine, nicotine and caffeine, dered analgesic, aperitif, digestive, emmanagogue, enjoy a widespread non-medical use as stimulant or laxative and stomachic tonic (Khanna et al., 2002) and sedative (Naik and Juvekar, 2003). Some alkaloids are is helpful in treating oedema, anorexia and diabetes medically useful for the cure of human diseases e.g. (George and Pamplona-Roger, 2002.). Its roots, leaves, atropine in treatment of bronchial asthma (Natarraj, fruits, milky juice as well as the whole plant are used as 2000); intestinal and biliary colic, and to dilate pupil medicine. Recently, lignansniranthin, nirtetralin and of the eye (Naik and Juvekar, 2003). Saponins are phyltetralin have been isolated from the leaves glycosides with a distinctive foaming characteristic. (Tabasum et al., 2005). Many of the active constituents They are found in various parts of the plant leaves, found in the plant are biologically active lignans, stems, roots, bulbs, flowers and fruits. The name glycosides, flavonoids, saponins, alkaloids, ellagitannins “saponin” originated from soapwort plant (saponaria), and phenylpropanoids (Dhar et al., 1968); common the root of which was used historically as a soap. lipids sterols and flavonoids also occur in the plant Saponins are believed to be useful in the human diet for controlling cholesterol, but some (including those (Barros et al., 2003). Alkaloids are organic nitrogen- produced by the soapberry) are poisonous if swallowed containing compounds found in 20-30% of vascular and can cause urticaria (skin rash) in many people plants and at lower doses, are useful pharmacologically. (Marston et al., 2000). Digitalis type saponins These compounds are renowned for their potent strengthen heart muscle contractions, causing the pharmacological activities (Sharma and Gupta, 1994). heart to work more efficiently (Haridas et al., 2001). *Author for correspondence; E-mail: [email protected] Saponins inhibit some kind of cancerous cell growth in

47 48 Victor Adeyinka Ajibade and Oladiran Famurewa animals particularly in cases of lung and blood Marston et al. (2000). The milled plant (170 g) was cancers, without killing normal cells (Ray, 2007). defatted using 700 mL of petroleum ether for 72 h using soxhlet for about 24 h. Methanol (700 mL) was Materials and Methods used to extract saponin from the defatted sample and Collection of plant material. Phyllanthus niruri was the residue was left overnight under reflux at 70 °C. It collected from shrubs around the Federal Polytechnic was then filtered and the filtrate was evaporated to Compound, Ado-Ekiti, Nigeria during the months of dryness. The yield was dissolved in distilled water July to September 2008 and was identified at the (300 mL) and in the ratio of 1:1 with methanol placed Department of Plant Science, University of Ado-Ekiti, in a separating funnel. The set up was left for three Nigeria. A voucher specimen was deposited at the days when two layers were formed. The bottom layer herbarium of the Department of Science Technology, was removed and the upper layer, which is the crude Federal Polytechnic, Ado-Ekiti. The sample used for saponin, was poured into an evaporating dish and the analysis was air-dried at room temperature of ±28 dried by evaporation for 2 weeks. °C and pulverized. Bacteriological assay. The isolated crude extracts of Test organisms. The following bacteria were used the plant were investigated biologically and the active in the study: Bacillus subtilis NCTC 8263, Staphy- compounds were detected and purified. The bioassay lococcus aureus SSH22, S. aureus SSH23, Strepto- monitoring of fractions was undertaken using agar coccus pyogenes SSH43, Escherichia coli SSH31, disc diffusion technique (Denni and Hussain, 1991). Klebsiella pneumoniae OAUCH54 and Salmonella Aliquots (20 μL) of the extracts were placed on typhi SSH45, which were obtained from clinical susceptibility test discs. These were then applied to the surface of overdried nutrient agar plates, which isolates at the University Teaching Hospital (SSH), had been seeded with an overnight culture of test Ado-Ekiti, and Specialist Laboratory of the Centre for organism. Culture plates were incubated overnight Disease Control (SLT), Ile-Ife and Obafemi Awolowo at 37 °C and zones of inhibition were estimated University Teaching Hospital (OAUTH), Ile-Ife semi-quantitatively. Gentamicin sulphate (Nicholas Annex, Wesley, Ilesa, Nigeria. They were authenticated Laboratories, UK) and Ampicillin (Beecham) were used using conventional methods. The organisms were as positive controls. stored on the nutrient agar (LAB) slant at 4 °C until required for use. Characterization of saponin. The crude saponin was characterized using 13C NMR spectra described by The media used were: oxoid nutrient broth Olugbade et al. (2000). Optical rotations at 20 °C were (Basingstoke, UK) which was solidified with 1.5% taken on a Perkin-Elmer 241 polarimeter. Spectra were Lab M agar No. 1 (Amersham Basingstoke, UK) to recorded as follows: pulse of 92.9°, total time of 9 h make nutrient agar, oxoid iso-sensitest agar and 20 min 51 sec; WALTZ-16 modulated and 19237 minimal salts (DM) Davis Mingiolis medium which repetitions. Infra-red spectra were obtained as nujol was solidified with 1.5% Lab M agar No. 1 to make mull on Pye Unicam SP 300 Infrared Spectrophoto- solid media. meter. The H1-NMR was taken in deuterochloroform Extraction of crude alkaloid. The method of Naik with tetramethylsaline (TMS) as internal standard and Juvekar (2003) was employed for the extraction. using a Bruker WH 300 Mhz spectrophotometer The dried, coarsely powdered whole plant of P. niruri employing Fourier transform techniques. The MS (200 g) was moistened with 25% ammonium hydroxide, using the electron impart technique was obtained by allowed to stand overnight and then Soxhlet extracted direct insertion at an ionization voltage of 70 eV using with 95% ethanol. After concentraction under vacuum, AE-MS 902 (V.G. Micromass Ltd., Manchester, UK). the syrup residue (30 g) was treated with conc. HCl. The mass spectrophotometer was fitted with a DS-55 The acidic filtrate was washed with benzene, made computer data output at inlet temperature of 170- basic (pH 10) with 25% ammonium hydroxide and 240 °C. extracted with chloroform to collect the alkaloidal fraction (2 g). Results and Discussion Extraction of crude saponin. The saponin was Four bacterial strains were used for determination of extracted according to the method described by the antibacterial activities (Table 1). S. aureus SSH22, Antibacterial Activity of Phyllanthus niruri L. 49

S. aureus SSH23, E. coli SSH31 and K. pneumoniae determined using three different concentrations of the OAUCH 54. The activities of the initial solvent extracts inoculum (undiluted, over-night culture, and diluted and the active compounds were compared with the 10-2 and 10-4). The first two concentrations of the cells standard antibiotics, gentamicin and ampicillin. gave confluent growth on nutrient agar, whereas application of 20 μL of the 10-4 dilution gave about 20 isolated colonies per application. MICs of gram Table 1. Bacteriological monitoring of active compound positive organisms ranged between 3 μg/mL for the isolates 10-4 dilution of S. aureus SSH22 and 30 μg/mL against Test Concen- Zone of inhibition (mm) undiluted S. pyogenes SSH43. For gram-negative extract tration S.aureus S. aureus E. coli K. pneumo- organisms, the range of MICs was between 15 μg/mL (fractions) SSH23 SSH22 SSH31 niae OAUTH54 for E. coli OAUTH71 (10-4 dilution) to over 60 μg/mL PEE 20% v/v 10.0 10.0 10.0 5.0 CH 20% v/v 15.0 15.0 10.0 10.0 for S. typhi SSH45 (Table 2). From these results, it was SAP 5 μg 10.0 5.0 5.0 - concluded that saponin had activity on these tests 20 μg 26.0 26.0 15.0 5.0 bacteria within the range of standard antibiotics used ALK 200 g 15.0 15.0 - - μ as control. The effect of saponin on these bacterial 600 μg NT NT 10.0 - GEN 2 μg 10.0 10.0 10.0 5.0 strains was, therefore, investigated further. AMP 5 μg 5.0 5.0 5.0 5.0 MET 50% v/v - - - - PEE = petroleum ether extract; CH = chloroform; SAP = Table 2. Minimum inhibitory concentration (MIC) of saponin; ALK = alkaloid; GEN = gentamicin; AMP = ampi- saponin in nutrient agar at various concentrations of cillin; MET = methanol; NT = not traced. inoculum* Test organisms Inoculum density (cfu) -4 -2 It was observed that saponin at 5 μg/mL and 20 μg/mL 2 x 10 2 x 10 20 MIC ( g/mL) had activity against S. aureus SSH22, S. aureus SSH23 μ and E. coli SSH31 within the range of the standard S. aureus SSH23 5 3 3 S. aureus SSH22 15 5 5 antibiotics used. However, K. pneumoniae OAUTH54 B. subtilis 15 15 5 showed resistance to the saponin at a concentration of S. pyogenes SSH33 30 15 15 5 μg/mL. Five compounds were extracted from P. niruri K. pneumoniae 60 45 30 using column chromatography together with moni- S. typhi SSH45 >60 60 45 toring of the solvent fractions using TLC and an anti- * = MICs based on triplicate results. microbial assay. The compounds, which were found in the petroleum ether and chloroform fractions, were identified by MS, UV, IR and 13C-NMR as phylagenin- Bactericidal activity of saponin. Data obtained from 13-O-α-D-glucopyranoside and phylangenin-25-O-β- viable counts of S. aureus SSH22 and E. coli SSH31, D-glucopyranoside saponin, which showed the appear- suspended in nutrient broth containing saponin, ance of the anomeric C-1 signal at 105 ppm and C-7, at confirmed the MICs of the extract in nutrient agar 98 ppm, alkaloid and another compound that is yet to (Table 2). The bactericidal activity against S. aureus be identified. SSH22 commenced at concentrations above MICs (5 μg/mL) and increased with the increase in the con- All the fractions investigated were active in the centration of the extract (Fig. 1). However, the extract antimicrobial assays (Table 1). Saponin isolated from became less active towards S. aureus SSH22 at con- P. niruri had previously been shown to be bactericidal centration above 60 μg/mL, giving a biphasic response to E. coli (Calbo et al., 2006). However, antibacterial (Fig. 2). activities against E. coli and S. typhi have been The bactericidal activity of the saponin extract against previously reported for alkaloid (Naik and Juvekar, E. coli SSH31 in nutrient broth increased with the 2003). concentration of the extract (Fig. 3). No biphasic The effect of saponin on some bacterial strains was, response was seen. The extract was more active in DM therefore, investigated further (Table 2). The MICs of basal salt solution than in nutrient broth; 30 μg/mL the saponin extract against the test bacteria were was highly bactericidal in DM salt solution (Fig. 4), 50 Victor Adeyinka Ajibade and Oladiran Famurewa whereas it was only bacteriostatic in nutrient broth 1200 (Fig. 3), corresponding to the MIC determined in the 1000 nutrient agar (Table 1). 800

600 120 400 100 200 80 0 60 12345 40 Saponin concentration (μg/mL) 20 0 Fig. 3. Absence of biphasic response of E. coli 12345 Time (min) SSH31 when exposed to increasing concentration of saponin in nutrient broth 5 μg/mL 10 μg/mL 15 μg/mL 30 μg/mL 60 μg/mL (data as in Fig. 2).

Fig. 1. Bactericidal activities of various concentrations of saponin in nutrient broth against S. aureus SSH22. 100

80 90 μg/mL 1200 60 10 μg/mL 15 μg/mL 1000 Viability (%) Viability 40 30 μg/mL 50 μg/mL 800 20 90 μg/mL 600 0 123 4 56 400 Time (min) 200

0 Fig. 4. Activity of saponin in Davis Mingiolis basal 12345 salt solution against E. coli SSH31. Drug concentration (μg/mL)

The antibacterial activity of saponin is of some Fig. 2. Biphasic response of S. aureus to increasing interest since it was effective at concentrations as low concentrations of saponin in nutrient broth. Data plotted are survival levels after 270 min as 3 μg/mL against S. aureus SSH22 (Table 2). This exposure (1 μg/mL=10 μg/mL). concentration was of the same order of magnitude as the MIC determined for Gentamicin and Ampicillin (Table 2) and reported for other standard antibiotics Antibacterial activity of the extracts of saponin and such as chloramphenicol, penicillin, streptomycin or alkaloids has been previously reported (Naik and erythromycin (Kazakova et al., 2005). Increase in MICs Juvekar, 2003; Unander et al., 1990). The NMR and produced by increasing the cell density of the inocula IR characterization of the saponin extract gave a (Table 2) is similar to the observations of George and spectra which identified the extract as phylagenin-13- Pamplona-Roger (2002) of the antibacterial activity O-α-D-glucopyranoside and phylangenin-25-O-β-D- of 4-quinolone. Similarly, the suggestion that the glucopyranoside saponin. (Agrawal et al., 1985). In response of S. aureus SSH22 to the drug is biphasic view of the antimicrobial activity of this extract, (Fig. 3) could be due to inhibition of RNA and protein complete structural elucidation would be of interest. synthesis by high concentrations of the extract, which Antibacterial Activity of Phyllanthus niruri L. 51 is believed to be responsible for the biphasic response biological activity. Indian Journal of Experimental of antibacterial activity of quinolone (Marston et al., Biology, 4: 232-247. 2000). The bactericidal activity of saponin extract George, D., Pamplona-Roger, M.D. 2002. Encyclo- observed against E. coli SSH31 in phosphate buffered paedia of Medicinal Plants, Inter-American saline indicates that the extract is still bactericidal Publishing Association (IADPA), USA. towards cells in which cell division is inhibited. This is Haridas, V., Higuchi, M., Jayatilake, G.S., Bailey, D., similar to the observations of Naik and Juvekar (2003) Mujoo, K., Blake, M.E., Arntzen, C.J., Gutterman, regarding the bactericidal activities of the fluoroquino- J.U. 2001. Avicins: Triterpenoid saponins from lone, ciprofloxacin and norfloxacin and in contrast to Acacia victoriae (Bentham) induce apoptosis by the activity of the parent compound nalidixic acid, mitochondrial perturbation. Proceedings of the which requires cell division for activity. National Academy of Sciences USA, 98: 5821-5826. The data obtained in this study have led to the Kazakova, S.V., Hageman, J.C., Matava, M., Srinivasan, conclusion that the saponin content of P. niruri is A., Phelan, B., Garfinkel, T., Boo, S., McAllister, responsible for the significant antibacterial effect of S., Anderson, J., Jensen, D., Dodson, D., Lonsway, this plant on a wide range of organisms. This may D., McDougal, L.K., Arduino, M., Fraser, V.J., explain some of the ethnopharmacological claims for Killgore, G., Tenover, F.C., Cody, S., Jernigan, D.B. this plant, especially its application as poultice for 2005. A clone of Methicillin-resistant Staphy- the treatment of certain types of ulcers (Barros et al., lococcus aureus among professional football 2003), spleen disorders (Barros et al., 2003), super- players. New England Journal of Medicine, 352: ficial wounds (Khanna et al., 2002 ), chronic dysentery, 468-475. asthma, jaundice (George and Pamplona-Roger, 2002), Khanna, A.K., Rizvi, F., Chander, R. 2002. 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Development of a Predictive Model for Preparation of Banana Modified Drink

Daramola Bode Department of Food Technology, Federal Polytechnic, P.M.B 5351, Ado-Ekiti, Ekiti State, Nigeria

(received January 30, 2010; revised July 13, 2010; accepted July 28, 2010)

Abstract. Fresh ripe banana drink was modified to produce non-carbonated, non-alcoholic food drink comparable to selected (four) commercial fruits juices mimic. The effects of a hydrocolloid (0.01-0.1 g), tart enhancer (0.005-1.000 g) and onion extract (0.02-0.2 mL) were concurrently studied using the central composite rotatable design on some product characteristics namely, titratable acidity, pH, relative viscosity and total soluble solids as responses. Preparations were compared with food commercial drink substitutes with respect to evaluated characteristics. Equations for predicting the responses were developed and their adequacy confirmed using analysis of variance and residual assessment. The empirical model could be useful as a base data for preparation and manipulation capable of yielding a peculiar banana base drink brand. This process provides an additional means for utilization of ripe banana fruits thereby lending enhanced economic value to producers consequently contributing to reduction in post harvest losses of banana fruits. Keywords: banana drink, food grade additives, model for juice preparation

Introduction - Banana fruit has economic advantage in terms The physiological benefits of both nutrients and of availability and price, compared to other phytochemicals of banana fruit are profoundly fruits available in developing countries e.g., Nigeria. expressed in the literature (Englberger et al., 2003; Sharma and Caralli, 2002; Hills and Kirwood, 1989). - The product (juice) yield is high comparative In Nigeria, banana fruits are abundantly available and to the yield of other fruits like pawpaw (with could be found all the year round. However, the com- too much water content) or orange (with too parative short post-harvest shelf life of banana coupled much food fibre). with lack of sufficient quality transportation system as - Such preparation can be used to boost import well as storage facilities lead to spoilage of large substitution of non-carbonated, non-alcoholic proportion of this highly nutritious fruit before its reach- fruit juice mimic brands. ing the consumers (Yomeni et al., 2004). An indigenous challenge associated with production of Although, products such as clarified juice, banana banana juice, as with many other fruits and vegetables, powder, jam, candy and fermented products like etha- is its susceptibility to discolouration caused by nol brandy, could be obtained from ripe banana fruit enzymatic browning (Arslan and Dogan, 2005). Other constraints are flat taste and variable viscosity (depen- (Palmu et al., 1999), they are scarcely produced at ding on the brand of drink to be prepared). Attempt to commercial scale, probably due to the low techno- eliminate the limitations had singly been accomplished logical acquisition. Among the named products, using various methods. banana juice shall be focused here for modification. The investigation appears economically worthwhile Therefore, the objective of this investigation is to study for the following reasons: concurrently the effect of application of three food grade additives that can circumvent the shortcomings - World demand for fruit juices is increasing most by developing a predictive model using three addi- probably due to their health benefits and highly tives as process variables for modification of banana appreciated flavour and aroma (Palmu et al., juice with a view to preparing a non-carbonated, non- 1999). alcoholic drink comparable to the commercial fruit E-mail: daramola_ bode@yahoo. co.uk drinks mimic. 53 54 Daramola Bode

Materials and Methods Fresh ripe banana Material: Ripe fresh banana (Musa spp.) was obtained from commercial fruit pack shed in Ado-Ekiti, Nigeria. The banana fruits were ripe enough to be blended using domestic blending machine. Food plant gum (Khaya Washing gum) and onion (Allium cepa) white variety were obtained from a local processor and seller, respectively. Food grade citric acid was sourced from departmental Peeling laboratory stock. Four commercial fruit juice mimic brands were used as reference.

Preparation of banana modified drink. Fresh ripe Blending Onion extract banana (Musa spp.) fruit was washed, peeled and blended with onion (Allium cepa) extract. This was followed by extraction and filtration to obtain juice. The Extraction juice was then treated with citric acid and plant food gum to obtain banana modified drink. The schematic diagram for the modification process is shown in Fig. 1. Details of combination of additives in the ex- Filtration perimental design are presented in Table 1. Extraction and purification of plant food gum. The Khaya gum was hydrated in double strength chloroform Juice water for 5 days with intermittent stirring and extrane- ous materials were removed by straining through a Gum cheese cloth. The gum was precipitated from the solu- Formulation tion using absolute ethanol. The precipitated gum was filtered, washed with diethyl ether and then dried in hot Citric acid air oven at 40 °C (Tyler et al., 1981). Flash Pasteurization Preparation of onion extract. Onion (500 g) was homogenized with 500 mL of distilled water for 5 min. The homogenate was centrifuged at 12,000 rpm for 20 min at 4 °C. The supernatant was used as onion extract. Modified banana drink

Experimental design. A central composite rotatable Fig. 1. Flow sheet for processing of banana modi- design for K = 3 was used (Cochran and Cox, 1957). fied drink. The design generated 20 sample combinations com- prising of eight points peculiar to 23 factorial six star markers, namely, titratable acidity, appearance, appar- points and six central points for replication. The ent viscosity and total soluble solids were evaluated. effect of independent variables, amount of citric acid, Step-wise regression analyses were performed on the gum and onion extract on preparation quality indices/ data to yield equations for predicting selected physi-

Table 1. Process variables used in the central composite rotatable design (K = 3) Independent Code Levels variable* -1.682 -1 0 1 1.682

Onion extract (mL) X1 0.02 0.06 0.1 0.14 0.2

Citric acid (g) X2 0.005 0.025 0.125 0.5 1

Plant food gum (g) X3 0.01 0.03 0.05 0.07 0.1 * = 50 mL of banana juice was used for each treatment. Model for Banana Drink Preparation 55 cochemical indices of the product that have quality Y-column (response) to obtain corresponding sum of consequences. products that is 0 y to 13 y for Xo to X1 X3. Conse- quently, the coefficients b to b were calculated as: Analytical Methods. Total soluble solid determina- o 13 tion. Refractive index of sample was measured using bo = 0.166338 (0y) – 0.056791 ∑(iiy) ______(1) Abbe refractometer (ABBE 325 Zuzi). Corresponding b = 0.073224 (iy) ______(2) soluble solid was determined using appropriate proce- i dure and reference to appropriate designated table. bii = 0.062500 (iiy) + 0.006889 ∑(iiy) – 0.056791 Determination of pH. This was accomplished by dis- (0y)______(3) pensing 50 mL of the sample into 100 mL beaker and bij = 0.125000 (ijy) ______(4) reading the value directly on a pH meter (Omega H. HPx digital pH meter) which was previously standard- The quadratic model was fitted using the regression co- ized with buffer 4 and 9 solutions. Value was recorded efficients and the predicted response was calculated for when equilibrium pH was attained. each of the observed values. The model was assessed for adequacy by subjecting to analysis of variance and Titratable acidity determination. Titratable acidity residual analysis. was determined by titrating 100 mL of the sample against 0.1 M NaOH using phenophthalein as indicator Results and Discussion of end point and the results were expressed in g citric Four technological parameters namely, titratable acid, pH, acid equivalent /100 mL of the sample. total soluble solids and apparent viscosity were assessed Measurement of apparent viscosity. Each sample was in the modified drink. Also, the parameters were exam- placed in a Baroid division rheometer cup and the ined (Table 2) on unmodified drink (when necessary) as instrument head was lowered until the sleeve was well as the four commercial fruit drink mimic brands for immersed exactly to the scribed line. The lock screw on the purpose of reference and analytical discussion. the left leg of the instrument was tightened. The gear The central composite orthogonal design to fit the poly- shift was then set to 600 rpm before rotation of the crank nomial model for the modification was accomplished as for 15 sec. The dial reading was allowed to come to a elicited by Cochran and Cox (1957). The computed sums steady value before the value was measured. The gear of products and regression coefficients to fit the model shift was then set to 300 rpm and the reading was taken are shown in Table 3. like wise. The apparent viscosity in centipoise is given as 600 rpm reading-300 rpm reading (Osunsami, 1987). Titratable acidity (TA). A cursory look at Table 2 revealed that TA (0.1485 g citric acid equivalent/100 mL) Assessment of discolouration. Discolouration of of banana unmodified juice is less than TA (0.482-1.018 treated and untreated samples was determined subjec- g citric acid equivalent/100 mL) of any of the commer- tively following the procedure of Demooy and Demooy cial fruit juice mimic brands. The result obtained in this (1990). study did not compare with the acidity reported earlier Statistical analysis. The central composite orthogonal by Palmu et al (1999), because the acid equivalent used design was analysed as repored by Cochran and Cox was not stated. But the fact that the determined pH is (1957). Each of the X-matrix was multiplied by the the same suggests that the acidity could be similar if

Table 2. Physicochemical parameters of the unmodified juice and commercial drinks Parameter Control Berry blast Chivita 5-alive Dansa TA (citric acid equivalent/100 mL) 0.1485 0.482 0.749 1.018 0.4922 pH 4.65 3.36 3.00 2.90 3.21 App visc (Cp) 1.400 1.022 1.022 1.043 1.059 TSS (0Brix) 5.00 15.30 13.40 14.70 13.40

TA = titratable acidity; TSS = total soluble solids; App visc = apparent viscosity. 56 Daramola Bode

Table 3. Regression coefficients for the quadratic model equation for the banana modified drink Sum of Parameter Regression Parameter products TA pH TSS App visc coefficient TA pH TSS App visc

Oy 21.0894 68.4200 135.5000 27.3350 bo 0.8254 3.5112 6.1549 1.3615

Y 0.545 - 2.1092 2.5387 -0.5022 b1 0.0399 - 0.1544 0.1859 - 0.0368

2y 10.0092 - 10.8050 11.7801 - 0.1362 b2 0.7329 - 0.7912 0.8626 - 0.0010

3y 0.0332 - 0.8569 0.7341 - 0.4761 b3 0.0024 - 0.0627 0.0538 - 0.0349

11y 14.7245 45.1063 96.1646 18.9215 b11 0.0480 - 0.1119 0.3025 0.0166

22y 18.0699 48.2183 97.0134 18.8592 b22 0.2571 0.0826 0.3556 0.0127

33y 14.4416 45.2477 95.3159 18.3076 b33 0.0303 - 0.1030 0.2495 - 0.0218

12y - 0.0397 0.3000 0.75 - 0.0480 b12 - 0.0050 0.0370 0.0938 - 0.0060

13y - 0.1031 0.0000 1.35 - 0.0220 b13 - 0.0129 0.0000 0.1688 - 0.0028

23y - 0.3091 0.5000 - 2.15 - 0.010 b23 - 0.0386 0.0625 - 0.2688 - 0.0013 ∑(ii) y 47.2360 138.5723 288.4939 56.0883 R2 = 0.998 0.9858 0.9618 0.9122 TA = titratable acidity; TSS = total soluble solids; App visc = apparent viscosity.

same unit is used. Taking cognizance of the fact that ˆpH = 3.5112 – 0.1544 X1 – 0.7912 X2 – 0.0627 X3 - one of the major necessary modifications is suppres- 2 2 2 0.1119 X + 0.0826 X – 0.103 X + 0.037X X + sion of flat taste inherent to banana ordinary juice in 1 2 3 1 2 0X X + 0.0625 X X ______(6) order to impart tartness characteristic of all the selected 1 3 2 3 commercial fruit juice mimic brands, the quadratic The predicted pH (ˆpH) for each of the experimental model takes the form, run and their residuals are presented in Table 4. The residuals suggest that the fitted model is adequate. The Â = 0.8254 + 0.0399X + 0.7329 X + 0.00024 X + tt 1 2 3 view on adequacy of model fitness was verified by con- 2 2 2 0.048X1 + 0.2571X2 + 0.0303 X3 - 0.0050X1 X2 – ducting analysis of variance test. As the first and the second order terms were significant (Table 5) due to 0.0129X1 X3 – 0.0386 X2 X3 ______(5) the higher calculated F-ratio in comparison with the The predicted total titratable acidity, Â , for each of the tt tabulated values, and the calculated F-ratio for lack of experimental run and their respective residual are shown fit was lower than the tabulated value, attesting adequacy in Table 4. Examination of the residuals suggests that of the model. The pH model may even be advantageous the fitted model was reasonably adequate. The claim to predict acidity because it is a rapid objective test in was confirmed on model testing. In addition, the analy- comparison to titratable acidity measurements. sis of variance to test the fitness of the model is shown in Table 5. The first and the second order terms were Apparent viscosity. The apparent viscosity (Cp) of the significant as shown by the higher calculated F-ratio banana unmodified juice as determined in this study compared with the tabulated values. However, since the shows higher value (1.400) compared to the low values calculated F- ratio for the lack of fit was lower than the (1.022-1.059) of the tested commercial fruit mimic tabulated value, adequacy of the fitted model is affirmed. brands. This is not surprising since the constituents The coefficient of multiple determinations (R2) also (flavourant, colourant and sweetener) of the reference showed the fitness of the residuals. Since there are dif- drinks are monomers (citric acid, tetrazine yellow, ferences in TA of various commercial fruit juice mimic glucose for each of the constituents, respectively) of high brands used in this study, the model can be used to solubility index. simulate a particular brand with respect to TA with con- The apparent viscosity quadratic model (AˆvC) for the sequent sensorial tartness. modified drink takes the form:

pH. The pH value of the banana unmodified juice was 2 AˆvC = 1.3615–0.0368X1–0.010X2–0.035X3+ 0.0166 X1 4.65, similar to the earlier report by Palmu et al. (1999). 2 2 + 0.0127 X – 0.0218 X – 0.006X X – 0.0028 pH values of the commercial juice brands ranged 2 3 1 2 X X - 0.0013 X X ______(7) between 2.71 to 3.36 which revealed that all of them 1 3 2 3 were acidic comparative to the banana unmodified juice. Fitteness of the model (Table 4) was assessed and found adequate. Comparative study of the viscosity of each The pH quadratic model takes the form: Model for Banana Drink Preparation 57

Table 4. Residual analysis of assessed parameters

Run TA pH TSS App visc Observed Predicted Residuals Observed Predicted Residuals Observed Predicted Residuals Observed Predicted Residual

1 0.2571 0.3291 -0.072 4.45 4.4867 - 0.0367 5.65 5.9541 -0.3041 1.413 1.4407 -0.0277 2 0.5000 0.447 0.0553 4.10 4.1039 -0.0039 5.95 5.8007 0.1493 1.375 1.3847 -0.0277 3 1.9000 1.8821 0.0179 2.60 2.7053 -0.1053 8.00 8.0293 -0.0293 1.445 1.4353 0.0097 4 1.9600 1.9777 -0.0177 2.40 2.4705 -0.0705 8.00 8.2511 -0.2511 1.343 1.3553 -0.0123 5 0.4200 0.4369 -0.0169 4.30 4.2363 0.0637 6.35 6.2617 0.0883 1.374 1.3789 -0.0049 6 0.4483 0.5009 -0.0526 3.95 3.8535 0.0965 6.65 6.7835 -0.1335 1.285 1.3117 -0.0267 7 1.7483 1.8355 -0.0872 2.70 2.7049 -0.0049 6.95 7.2617 -0.3117 1.361 1.3683 -0.0073 8 1.9138 1.8795 0.0343 2.50 2.4701 0.0299 8.30 8.1587 0.1413 1.288 1.2771 0.0109 9 0.9713 0.8941 0.0772 3.50 3.4543 0.0457 6.95 6.981 0.2519 1.480 1.4704 0.0096 10 1.0000 1.0283 -0.0283 2.90 2.9349 -0.0349 7.30 7.3235 -0.0235 1.361 1.3466 0.0144 11 0.3550 0.3201 0.0349 5.00 5.0757 -0.0757 5.75 5.7101 0.0399 1.447 1.4142 0.0328 12 2.8000 2.7855 0.0145 2.50 2.4141 0.0859 8.80 8.6119 0.1881 1.372 1.3806 -0.0086 13 0.9000 0.9071 -0.0071 3.45 3.3253 0.1247 6.95 6.7704 0.1796 1.374 1.3588 0.0152 14 0.9713 0.9151 0.0562 3.00 3,1143 -0.1143 7.00 6.9514 0.0486 1.250 1.241 0.0090 15 0.8741 0.8254 0.0487 3.50 3.5112 -0.2112 6.00 6.1550 -0.155 1.360 1.3615 -0.0015 16 0.8322 0.8254 0.0068 3.55 3.5112 0.0388 6.30 6.1550 0.145 1.370 1.3615 -0.0085 17 0.7601 0.8254 -0.0653 3.55 3.5112 0.0388 6.30 6.1550 0.145 1.340 1.3615 -0.0215 18 0.8322 0.8254 0.0068 3.57 3.5112 0.0588 6.00 6.1550 -0.155 1.350 1.3615 -0.0115 19 0.8322 0.8254 0.0068 3.60 3.5112 0.0888 6.30 6.1550 0.145 1.377 1.3615 0.0155 20 0.8135 0.8254 -0.0119 3.50 3.5112 -0.0112 6.00 6.1550 -0.155 1.370 1.3615 0.0085 TA = titratable acidity (g citric acid equivalent /100 mL), TSS = total soluble solids (0Brix), App visc = apparent viscosity (Cp); residual = observed value – predicted value.

Table 5. Analysis of variance (ANOVA) for the Predictive model equations *Dependent Statistical term DF SS MS F-ratio calculated Tabulated variable 5% 1% TA First order 3 7.3577 2.4526 1751.86 5.41 12.06 Second order 6 0.9725 0.1621 115.79 4.95 10.67 Lack of fit 5 0.0330 0.0066 4.71 5.05 10.97 Error 5 0.0069 0.0014 Total 19 8.3701 pH First order 3 8.9283 2.9761 252.21 5.41 12.06 Second order 6 0.4857 0.0810 6.86 4.95 10.67 Lack of fit 5 0.0333 0.0067 0.57 5.05 10.97 Error 5 0.0591 0.0118 Total 19 9.5064 TSS First order 3 10.6727 3.5576 131.76 5.41 12.06 Second order 6 4.2301 0.7050 26.11 4.95 10.67 Lack of fit 5 0.4797 0.0959 3.55 5.05 10.97 Error 5 0.1350 0.027 Total 19 15.5175 App visc First order 3 0.0365 0.0122 61.00 5.41 12.06 Second order 6 0.0131 0.0022 11.00 4.95 10.67 Lack of fit 5 0.0054 0.0011 5.5 5.05 10.97 Error 5 0.0010 0.0002 Total 19 0.0560 * = see interpretation in Table 4; DF = degree of freedom; SS = sum of square; MS = mean square. 58 Daramola Bode run resulted in no acute increase in viscosity. This adequate to provide a means of predicting tested prepa- observation is presumably due to the span of quantity ration (drink) characteristics. The model can be useful of plant food gum used or/and solvation effect of tart for product development with respect to diversification enhancer (food grade acid) added to each run. How- of banana fruit utility with the view to enhancing ever, preparations are characterized by higher apparent agro-economic rewards. viscosity relative to that of the commercial fruit juice mimic brands used in this study. References Total soluble solids. The total soluble solids (oBrix) of Arslan, O., Dogan, S. 2005. Inhibition of polyphenol the banana unmodified juice was less (5.00) compared oxidase obtained from various sources by 2, 3- to high (13.40–15.30) total soluble solids of the diaminopropionic acid. Journal of the Science of commercial fruit juice mimic brands used in this study. Food and Agriculture, 85: 1499-1504. Much difference in the values is believed to be due to Cochran, W.G., Cox, G.M. 1957. Experimental Design, the method adopted in preparation of the banana juice 2nd edition, John Wiley and Sons, New York, (unmodified) in this study. For example, no enzyme or USA. heat was used to facilitate dissolution of pulp. Besides, Demooy, B.E., Demooy, C.J. 1990. Evaluation of cook- the TSS, figure could have been inflated by the syn- ing time and quality of seven diverse cowpea thetic colourant used in the commercial fruit juice mimic (Vigna unguiculata (L) walp.) varieties. Interna- brands. However, modification resulted in increase in tional Journal of Food Science and Technology, TSS and the quadratic model takes the form: 25: 202-212. Englberger, L., Darnton-Hill, I., Coyne, T., Fitzgerald, TSˆS = 6.1549 + 0.1859X + 0.8626X + 0.0538X 1 2 3 M.H., Marks, G.C. 2003. Carotenoid-rich bananas: + 0.3025X2 + 0.3556 X2 + 0.2495 X2 + 0.0938X X 1 2 3 1 2 a potential food source for alleviating vitamin A

+ 0.1688X1X3 – 0.2688X2X3 ______(8) deficiency. Food and Nutrition Bulletin, 24: One of the benefits of the model is that it can give insight 303-318. to response if one or combinations of the independent Hills, B.A., Kirwood, C.A. 1989. Surfactant approach variables are manipulated. Therefore, inspection should to the gastric mucosal barrier: Protection of rats by inform on direction to manipulate to affect the desired banana even when acidified. Gastroenterology, changes. Also, the fact that the assessed physicochemi- 97: 294-303. cal parameters of the commercial fruit juice mimic brands Osunsami, A.T. 1987. Investigation of Optimum vary suggests that it may not be mandatory to determine Conditions for the Isolation and Modification of optimum values by canonical means (RSM) and conse- Cassava Starch. MSc. Thesis, Department of Food quently, were not attempted in this study. Technology, University of Ibadan, Oyo State, Nigeria. Visual assessment showed that the banana unmodified Palmu, P.T., Jardinc, J., Matta, V. 1999. Production of a juice was discoloured (brown) due to enzymatic brown- banana (Musa cavendishii) extract containing no ing but all the samples treated with onion extract were polyphenol oxidase by ultra filtration. Journal of not characterized with discolouration. The mode of anti- the Science of Food and Agriculture, 79: 643-647. browning action of extract of onion is believed to be Sharma, J.L., Caralli, S. 2002. A Dictionary of Food and complexing of the bioactive components of onion with Nutrition, pp. 53-55, CBS Publishers and Distri- polyphenolases, thereby, making the phenolases not butors, New Delhi, India. available for oxidation of phenolic compounds to Tyler, V.E., Brady, L.R., Robbers, J.E. 1981. Pharma- quinone and other substrates that are responsible for cognosy, pp. 21-54, 8th edition, Lea and Febiger, brown pigments. Philadelphia, USA. Yomeni, M.O., Njoukam, J., Tchango, T.J. 2004. Influ- Conclusion ence of the stage of ripeness of plantains and Modification of banana drink using three classes of some cooking bananas on the sensory and physical additives concurrently yielded food preparations com- characteristics of processed products. Journal of parable to non- carbonated, non-alcoholic commercial the Science of Food and Agriculture, 84: 1069- fruit drink mimics. The model developed was found 1077.