s2fieldsofstudy:
Agricultural And Food Sciences
Biology

provenance: AgriculturalAndFoodSciences,1970-2015-Agricultural And Food Sciences-2010.jsonl.gz.gz

text:
New Record of Red Palm Weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae) on Arecanut (Areca catechu) from Meghalaya, India

The Red Palm Weevil (RPW), Rhynchophorus ferrugineus (Oliver) (Cole?ptera: Curculionidae), locally called Asian Palm Weevil, is a native of Southern Asia and Melanesia (Malumphy & Mo ran 2007). The RPW is a devastating pest of palms having a wide geographical distribution in Europe and Asia (Wattanapongsiri 1966; Zhang et al. 2003; Al-Ayedh 2008). It attacks various palm species such as Phoenix sylvestris, Cocos nuc?fera, and Metroxylon sago in India, Pakistan, Sri Lanka, Myanmar, Indonesia, The Philippines, and the Gulf states (Rahalkar et al. 1985; Murphy & Biscoe 1999). In India it is one of the most-de structive pests of coconut causing infestation of as many as 12% of 5-10-years-old coconut palms (Sekhar 2000). RPW has been reported as the most-destructive pest of coconut (Cocos nuc?fera) from major Indian coconut growing states includ ing Karnataka, Kerala, Goa (Venkitasubban 1936; Lever 1979; Faleiro et al. 2003; Prabhu & Patii 2009). It is a serious pest of date palm (Phoe nix dactylifera) in Punjab state of India (Lai 1917) causing considerable damage to the crop during 1916-17 (Meckanna 1918) and a pest on sugar

The Red Palm Weevil (RPW), Rhynchophorus ferrugineus (Oliver) (Coleoptera: Curculionidae), locally called Asian Palm Weevil, is a native of Southern Asia and Melanesia (Malumphy & Moran 2007). The RPW is a devastating pest of palms having a wide geographical distribution in Europe and Asia (Wattanapongsiri 1966;Zhang et al. 2003;Al-Ayedh 2008). It attacks various palm species such as Phoenix sylvestris, Cocos nucifera, and Metroxylon sago in India, Pakistan, Sri Lanka, Myanmar, Indonesia, The Philippines, and the Gulf states (Rahalkar et al. 1985;Murphy & Biscoe 1999). In India it is one of the most-destructive pests of coconut causing infestation of as many as 12% of 5-10-years-old coconut palms (Sekhar 2000). RPW has been reported as the most-destructive pest of coconut (Cocos nucifera) from major Indian coconut growing states including Karnataka, Kerala, Goa (Venkitasubban 1936;Lever 1979;Faleiro et al. 2003;Prabhu & Patil 2009). It is a serious pest of date palm (Phoenix dactylifera) in Punjab state of India (Lal 1917) causing considerable damage to the crop during 1916-17 (Meckanna 1918) and a pest on sugar- cane (Rahalkar et al. 1972). RPW has not been reported previously on Arecanut (Areca catechu) in India. Herein, we report new records of RPW on a new host, Arecanut, from Meghalaya state (20.1°-26.5°N and 85.49°-92.52°E) in the North Eastern Hill Region of India.
In May 2008, severely infested Arecanut plants were observed in Arphewmer village of Umling block under Ri-Bhoi (90°55'15-91°16'N and 25°40' to 25°21'E) district of Meghalaya, but no larvae or adults were found in the infested plants. In Nov 2009, adults and larvae of RPW were first found in nearly wilted and dead Arecanut plants in Tura Municipality areas (25°30'N, 90°16'E) under West Garo hills district of Meghalaya province located near the north eastern border of Bangladesh in the north eastern part of India. RPW adults and larvae were collected after splitting the symptomatic stems/shoots from severely infested plants and the specimens were identified following the taxonomic key of Wattanapongsiri (1966) and Zhang et al. (2003). The infestation of RPW ranged between 1-3% of the plants (n = 511) in the rural and semi urban areas of Tura sub-division of Meghalaya.
In Dec 2009, the survey was extended to Sarin, Mawpud, Assainguitin village, and State Government Horticultural Farm at Phudkhylla village under Ranikor block of the West Khasi Hills (25°10'-25°51'N, 90°44'-91°49'E) district of Meghalaya, where we found infested Arecanut plants showing entry holes and wavy tunnels without larvae/adults in all the locations, except in Assainguitin village. Altogether, the RPW infestation in these areas was 1.5% considering all the locations.
At the end of Dec 2009 and first week of Jan 2010, again intensive survey was conducted in bordering (India-Bangladesh) areas, and RPW was collected from Arecanut plants in Pyrdwah and Lynkhat villages and State Government Horticultural Farm at Pomshutia village under East Khasi Hills (25°07'-25°41'N, 91°21'-92°09'E) district of Meghalaya. In these locations, the RPW infestation ranged from 2.5 to 10%. In the second week of Jan 2010, similar survey was conducted in Umdoh, Umlympiang, Muktapur and Umtyrngai villages of Jaintia Hills (20°58'-26° 03'N, 91°5 9'-92° 51'E) district of the state but no infested Arecanut plants were observed.
The infested plants showed signs of wilting, drooping of dried leaves (Fig. 1a), and dropping of leaves. The topless stem remained standing, showing symptoms similar to Phytophthora rot of Arecanut. About 1 ft. below the defoliated top, black sunken holes (Fig. 1b) could be seen, occasionally with dried frass. When the stem was split from the top, wavy tunnel could be seen with full of frass and insect larvae (Figs. 1c and 2A). Adults (Figs. 2B and 2C) were recovered sporadically depending on the time of survey/ location, and severity of infestation. The larvae collected from the infested Arecanut plants were reared in the laboratory artificially on finely ground Arecanut frass.
Adults measured 33.2-34.05 mm (average 33.62 mm) in length, 11.9-13.1 mm (average 12.5 mm) in width, and weighed 0.99-1.28 g (average 1.13 g). Adults are reddish brown to black with 6 black spots of variable size on the pronotum. The snout in males appeared smooth and slender with a hairy pad at the apex; in females the snout was slightly longer and shiny black without a hairy pad. Larvae are creamy white with a dark head. Vidyasagar (2007) has indicated that coconut, date palm, sago palm, and wild date or toddy palm (Phoenix sylvestris), palmyrah (Borassus flabellifer), oil palm (Elaeis guineenis), the talipot palm (Corypha umbraculifer), the sugar palm (Arenga sacharifera, and the sedang palm (Livistonia cochinchinensis) are potential hosts of RPW in India. RPW was not previously reported on palms including coconut in this state and this is the first record of RPW in Meghalaya state on Arecanut, and the first report of RPW on Arecanut from India. An early action is needed to prevent further spread of the pest in other states of the region and to protect the Arecanut industry in the state of Meghalaya and India.

SUMMARY
The red palm weevil (RPW) Rhynchophorus ferrugineus was newly recorded in 2008-2010, from 5 districts of Meghalaya, India and from a total of 7 districts surveyed in all bordering (India-Bangladesh) areas. There are isolated distributions of RPW indicating rapid spread in the state. The pest may migrate to other Indian states in the near future causing threat to Arecanut plantations. Our finding appears to be the first published report of RPW on Arecanut in this country. The programme was funded by ICAR (Indian Council of Agricultural Research) Research Complex for Northeastern Hill Region, Barapani, Meghalaya.


text:
THE INFLUENCE OF THI VALUES AT DIFFERENT PERIODS OF LACTATION ON MILK QUALITY AND CHARACTERISTICS OF LACTATION CURVE

: The experiment included 90 cows. Cows were chosen according to the time of calving, so that the first third of lactation occurred during the summer in 30 cows (G1), the second third of lactation occurred during the summer in 30 cows (G2), and in the last 30 cows the last third of lactation was in summer period (G3). The value of THI was between 72 and 82, which indicates the existence of the moderate intensity of heat stress. Heat stress does not damage the milk yield, milk fat and protein percentage on the level of the whole lactation, regardless of the lactation period in which the cows were exposed to stress. There was no correlation between THI and milk yield and quality at the level of the whole lactation. Heat stress did not show a significant effect on the parameters of lactation curve, except the peak of yield, which occurred later in cows exposed to heat stress. Increased value of THI showed nonsignificant effect on yield and quality of milk in the first third of lactation. In the middle and at the end of lactation THI was in a significant negative correlation with the yield and quality of milk. Our study showed a significantly lower heat-induced milk yield, milk fat and protein percent in the middle and at the end of lactation.

Introduction
Temperature humidity index (THI) is an important indicator of heat stress loading. Heat stress in dairy cows occurs when THI index is higher than 72 (Dikmen and Hansen, 2009). In our geographical area, it happens from the second half of May to the end of September.
Lactation curve is a graphical representation of the milk yield during standard lactation (305 days). Lactation curve includes next parameters: the initial level of and peak of milk yield, period to reach peak of yield and the persistence of lactation (Ferris et al., 1985).
Heat stress in dairy cows is characterised by reduced milk yield and decrease of the quality of milk (Cincović and Belić, 2009). Decline in productivity occurs because of reduced food intake, as a result of endocrine and metabolic adaptation to a high body and environmental temperature (Hristov et al., 2007;Bernabucci et al., 2010). Stage of lactation is an important factor that affects the damage of milk production during heat stress. It was found that mid-lactating dairy cows were the most heat sensitive compared to early and late lactating cows (Johnson, 1987).
The aim of our study was to determine the influence of heat stress (higher THI value) on the lactation curve.

Materials and Methods
The experiment included 90 cows. Cows were chosen according to the time of calving, so that in 30 cows the first third of lactation occurred during the summer (G1), the second third of lactation occurred during the summer in 30 cows (G2), and in the last 30 cows the last third of lactation occurred in summer period (G3). Only healthy Holstein-Friesian cow breeds in the second and third lactation were selected for the experiment. Average milk production in previous lactation was about 20 litres of milk a day. Cows were grown under the same conditions of nutrition and care.
Milk production was monitored daily during 44 weeks (305 days of standard lactation). Data on the percentage of milk fat and protein in milk were measured weekly using standard spectrophotometric methods (Milcoscan device). The following parameters of lactation curves were observed: a-milk yield at the beginning of lactation, b-daily increase in milk yield until a peak (linear), c-moment of peak in milk yield (days), d-peak in milk yield (L), e-day drop in milk yield after reaching peak (linear).
THI-index was calculated on the basis of meteorological data and measurements on the farm according to the formula Shehab-El-Deen et al. (2010), mTHI = 0.8 x mAT + (mRH x (mAT -14.4)) + 46.4; mTHI-mean values THI, mAT-mean atmospheric temperature, mRH-mean relative humidity in decimal.
By means of statistical analyses the correlation coefficient between THI and lactation curve parameters and quality of milk was examined. Similarly, the significance of the difference between the average milk production, milk fat and protein in heat stressed cows and cows in thermoneutral zone was examined for the entire lactation and particularly for each third of lactation (Z-and t-test).

Results and Discussion
In our geographical climate during the late spring, summer and early autumn conditions for the development of heat stress are created (Figure 1). Heat shock did not lead to a significant drop in milk production (G1: G2: G3; 21.95: 21.95: 21.84 L/day), milk fat percentage (G1: G2: G3; 3.86: 3.89: 3.93) and percentage protein (G1: G2: G3; 3.22: 3.26: 3.22) at the level of the whole lactation (Figures 2, 3 and 4). A significant correlation between THI and milk production and quality at the level of the whole lactation was not obtained. Heat stress had no significant effect on the parameters of lactation curve (a, b, d, e), except for the moment of achieving the peak in milk yield (c) ( Table 1).   Increased value of THI did not show a significant impact on production and quality of milk in the first third of lactation. In the middle and at the end of lactation THI was in significant negative correlation with production and quality of milk ( Table  2). The average amount of milk, milk fat and protein were statistically significantly lower in the middle and at the end of lactation during the action of heat stress (Table 3).  Previous studies have shown that the cows are more sensitive to heat stress when they are in the middle or at the end of lactation (Calamari et al., 1997;Spiers et al., 2004;Cincović and Belić, 2009). This phenomenon can be explained by the fact that at the beginning of lactation the productivity depends on the possibility of cows to use their own sources of energy, while the later productivity depends on the energy used from food (Bernabucci et al., 2010). It is known that during heat stress, reduction of food intake and negative energy balance occur (West, 2003). Negative energy balance is the result of consumption of cold thermoregulation, because the thermal stress increases body temperature . Negative energy balance during heat stress is characterised by a reduced concentration of glucose, whose decline is significantly correlated with the decrease of milk quantity and quality . Previous research on a large number of animals showed that season could have a significant impact on the parameters of lactation curve (Strandberg and Lundberg, 1991;Fadlelmoula et al., 2007), but the experiments were carried out in geographic regions that are hotter than Serbia.

Conclusion
Heat stress did not change the level of milk production throughout whole lactation in our environment, and lactation curve did not show more important deviations. Increased value of THI showed a nonsignificant effect on yield and quality of milk in the first third of lactation. In the middle and at the end of lactation THI was significantly negatively correlated with yield and quality of milk. The average amount of milk yield, milk fat and protein percent is statistically significantly lower in the middle and at the end of the lactation during the exposure to heat stress.


extfieldsofstudy: Biology Medicine

text:
Homeopathic Preparations to Control the Rosy Apple Aphid (Dysaphis plantaginea Pass.)

A laboratory model system with the rosy apple aphid (Dysaphis plantaginea Pass.) on apple seedlings was developed to study the effects of homeopathic preparations on this apple pest. The assessment included the substance Lycopodium clavatum and a nosode of the rosy apple aphid. Each preparation was applied on the substrate surface as aqueous solution of granules (6c, 15c, or 30c). Controls were aqueous solutions of placebo granules or pure water. In eight independent, randomized, and blinded experiments under standardized conditions in growth chambers, the development of aphids on treated and untreated apple seedlings was observed over 17 days, each. Six experiments were determined to assess the effects of a strict therapeutic treatment; two experiments were designed to determine the effects of a combined preventative and therapeutic treatment. After application of the preparations, the number of juvenile offspring and the damage on apple seedlings were assessed after 7 and 17 days, respectively. In addition, after 17 days, the seedling weight was measured. In the final evaluation of the six strictly therapeutic trials after 17 days, the number of juvenile offspring was reduced after application of L. clavatum 15c (-17%, p = 0.002) and nosode 6c (-14%, p = 0.02) compared to the pure water control. No significant effects were observed for leaf damage or fresh weight for any application. In the two experiments with combined preventative and therapeutic treatment, no significant effects were observed in any measured parameter. Homeopathic remedies may be effective in plant-pest systems. The magnitude of observed effects seems to be larger than in models with healthy plants, which renders plant-pest systems promising candidates for homeopathic basic research. For successful application in agriculture, however, the effect is not yet sufficient. This calls for further optimization concerning homeopathic remedy selection, potency level, dosage, and application routes.

INTRODUCTION
Pests limit yields and quality in agricultural plant production. One example of a yield-and qualitylimiting pest is the rosy apple aphid (Dysaphis plantaginea Pass.), considered to be a major pest in apple orchards [1]. This aphid species causes irreversible damage to leaves, branches, and fruits.
In recent years, increasing levels of resistance to insecticides and concerns about pesticide residues in agricultural products have stimulated an increased interest of fruit growers in new control strategies. While considering new strategies, the question arises if homeopathic preparations could be of use to control this aphid or other pest species.
Generally, plants are able to react to potentized substances [2,3,4,5]. This capability of plants to react to homeopathy has mostly been proven by using healthy plants, but the observed differences to untreated plants were generally small. The reaction of diseased plants to homeopathic treatment should theoretically be more pronounced. However, only limited scientific data on the effects of homeopathic preparations on diseased plants are known [2,3,4,5,6,7,8,9] and some of these assessments lack statistical evidence. Therefore, scientifically sound model systems have to be developed in order to assess the potential of homeopathic treatments for plant pests and diseases. In a preliminary study, a plant-disease model with apple and apple scab (Venturia inaequalis Cke.) was developed for controlled laboratory trials [10]. It consisted of apple seedlings that were artificially inoculated with apple scab and treated with homeopathic preparations. This plant-disease model allowed assessments under standardized conditions and data complying with statistical requirements [11].
The objective of this study was to develop a similar plant-pest model system for the assessment of homeopathic preparations based on the experience with the plant-disease model. The apple seedlings were inoculated with one fundatrix of D. plantaginea. The offspring and the damage to leaves could easily be assessed. In repeated laboratory experiments, homeopathically prepared Lycopodium clavatum and a nosode of the rosy apple aphid were tested for possible impact on aphid offspring and leaf damage.

Selection of Homeopathic Preparations
Based on previous experiments [10], two types of preparations were included in the assessments with the new plant-pest model system: the specially selected homeopathic remedy L. clavatum and a nosode of the rosy apple aphid.
As there is no systematically compiled Materia medica for plants, it is not possible to approach diseased plants by the classical homeopathic remedy selection procedure. Especially, a thorough anamnesis, a crucial tool to extract the characteristics of a case, is not possible. Therefore, we tried to extrapolate from the observed plant disease patterns to similar human symptoms, and to select corresponding remedies with the aid of the Complete Millenium Repertory (MacRepertory Pro V. 7.5.6.1, Kent Homeopathic Associates Inc., San Rafael, CA). The repertorization ended up with four potential remedies for the treatment of the rosy apple aphid: L. clavatum, sulfur, Mercurius solubilis, and Natrium muriaticum. All are used for constitutional therapies, but are also important acute remedies for a great number of diseases. For feasibility reasons, only one remedy -L. clavatum, being the most evidentwas chosen for the present study.
To complement this classical homeopathic remedy, a nosode of the rosy apple aphid was chosen as another therapeutic approach for the apple seedlings.
Both preparations were applied in three potency levels only (6c, 15c, and 30c) due to restrictions in laboratory, manpower, and financial resources. These potency levels were chosen based on experiences in human therapy and plant experimentation [4,5].

Homeopathic Preparations and Controls
All homeopathic preparations were prepared as centesimal potencies (with a 1:100 dilution ratio, supplied as granules) by the company Spagyros AG (Gümligen, Switzerland). The homeopathic preparations were first potentized in the solid phase (triturated) for three or six potency levels, then potentized in the liquid phase, and finally impregnated on sugar granules. Solid potentization (trituration) was performed by hand using a porcelain mortar and pestle (Haldenwanger, Berlin, Germany). To obtain a potency level z, 62 mg of potency level z-1 were triturated in 2 g lactose (Hänseler, Herisau, Switzerland) for 20 min (6 min grinding, 4 min scraping, 6 min grinding, 4 min scraping). To this mixture, 2 g lactose were added and triturated analogously for 20 min. This procedure was repeated with another 2 g lactose to finally obtain 6 g of potency level z. Liquid potentization was done by hand in brown glass bottles of hydrolytical class 3 (Müller & Krempel, Bülach, Switzerland). To obtain a potency level z, 1 part of potency level z-1 was added to 99 parts of dilution medium and succussed by 60 firm vertical beats against a leather cushion. Liquid potentization medium was either water (aqua purificata, TKA-Pacific, TKA Wasseraufbereitungssysteme GmbH, Niederelbert, Germany) or mixtures of water with ethanol 94% (Eidgenössische Alkoholverwaltung, Bern, Switzerland). Water was used for the first potency step after trituration, 30% ethanol for the second potency step after trituration, 43% ethanol for all other potency levels, except for the last potency level (for granule impregnation) where 73% ethanol was used.
For the nosode preparations, leaves of apple seedlings with typical symptoms and aphids were triturated in lactose up to 3c, and further potentized in liquid potentization medium as described above. L. clavatum (Dixa, St. Gallen, Switzerland) was triturated up to 3c or 6c (for the 6 or 15/30c granules, respectively), and further potentized in liquid potentization medium as described above.
Granules were prepared in the potency levels 6c, 15c, and 30c; 100 g of granules size 2 (made of glucose and invert sugar; Werner, Tornesch, Germany) were impregnated with 1 ml of the corresponding liquid potency level in a rotating drum for 15 min. Placebo granules were of identical origin, but not impregnated. Granules were stored in separate brown glass vials.
A person not involved in the experiments (S.B.) coded all homeopathic preparations, including placebo granules, by assigning a random letter code to the brown glass vials used for storage of the granules. Codes were revealed only after all experiments and statistical analyses were accomplished.

Choice of Controls
Treatment was applied by dissolving 10 homeopathic sugar granules in 250 ml tap water and watering the plants with these solutions. We used two sorts of controls. The first control consisted of remedy-untreated seedlings (treatment with tap water only), since this type of control is the official standard in plant-pest experimentation according to the European and Mediterranean Plant Protection Organization (EPPO) standards [11]. As a second control (carrier control), we used tap water with dissolved untreated granules (not impregnated with succussed or potentized ethanol or lactose, respectively). This type of control was chosen to account for any effects of the carrier substance (sugar). Specificity of any homeopathic treatment effects could be controlled by comparing the effects of both potentized substances at the potency levels used.

Aphids and Apple Seedlings
In Spring 2008, fundatrices of D. plantaginea were sampled in organic apple orchards in northwestern Switzerland. They were transferred to apple seedlings and reared in climatic chambers at constant conditions (temperature 12ºC; photoperiod 13.5L:10.5D; photosynthetic active radiation 250 mol photons/m 2 sec). For each experiment new offspring of the same age were used.
Apple seedlings of the variety Golden Delicious were planted in pots (diameter 0.1 m) with standard substrate (Einheitserde Typ 0; Einheitserde, Germany). For each experiment, new seedlings at the stage of five to six leaves were used.

Plant-Pest Model System
The model system consisted of a potted apple seedling that was transferred in a transparent plastic tube (diameter 0.1 m) and put on a saucer to prevent contact of differently treated seedlings. On each apple seedling, a young fundatrix was inoculated (with the exception of a mock-inoculated quality check). For the treatments, 10 granules of each homeopathic preparation (or untreated placebo granules) were diluted in 250 ml tap water and pivoted until granules were dissolved. Twenty (20) ml of these solutions were pipetted (20 ml single-serving syringe for each treatment) on the substrate surface of the potted seedlings. Control seedlings got 20 ml tap water.

Experiments with Therapeutic Treatments
The objective of the six identical, blinded experiments was to assess the effects of therapeutic treatments with L. clavatum (6c, 15c, 30c, or placebo granules) and the nosode (6c, 15c, 30c, or placebo granules) on the offspring of aphids, on leaf damage, and on seedling weight. Water-treated seedlings served as main control. Three days after the transfer of the fundatrices, the seedlings were treated for the first time; three additional treatments followed in a 3-to 4-day interval.
Each experiment consisted of a completely randomized block design with 10 replicates (seedlings). Thus, each experiment included 100 plants in total (10 seedlings  10 treatments: three potency levels of L. clavatum, three potency levels of the nosode, two placebo granules treatments, one control treated with water only, and one uninfected [mock-inoculated] negative control).
The six experiments were repeated in a 14-day interval between April 21 and June 19 in 2008. To show any treatment effects, the number of juvenile offspring and the damage on apple leaves (percentage of total leaf surface) were assessed after 7 and 17 days. In addition, the fresh weight of apple seedlings (without roots) was measured after 17 days.

Experiments with Combined Preventative and Therapeutic Treatment
In contrast to the first experimental setup, the objective of the two identical, blinded, and repeated experiments was to assess the effects of a combined preventative and therapeutic treatment with L. clavatum (6c, 15c, 30c, placebo) and the nosode (6c, 15c, 30c, placebo) on the offspring of aphids, on leaf damage, and on seedling weight. Water-treated seedlings served as control. Three days before the transfer of the fundatrices, the seedlings got one preventative treatment; three therapeutic treatments followed in a 3-to 4-day interval.
The experimental design and assessments were identical as in the first experimental setup. The two experiments were carried out in a 14-day interval between May 13 and June 16 in 2008.

Statistical Analysis
Data of the final assessments (after 17 days) were evaluated for statistical significance based on two-way analysis of variance (ANOVA) F tests with  = 5%. Independent ANOVA factors were (1) treatment (n = 9: one control, two placebos, and six homeopathic preparations; the mock-inoculated quality check was excluded before data analysis), and (2) experimental number (n = 6 or 2). Dependent variables were the number of juvenile offspring, leaf damage, and fresh weight. Data on juvenile offspring were square root transformed prior to statistical analysis to achieve a normal data distribution. The LSD test was used for planned comparison of treatment means with the untreated water control (p = 0.05); according to EPPO Standards (2004), the untreated control has to be the main reference [11]. Planned comparisons were evaluated with the LSD test only if the preceding F test was significant (p < 0.05). This procedure (protected Fisher's LSD) gives a good safeguard against type I error without being too conservative, i.e., it also gives good security against type II error [12].
Leaf damage data of the preliminary assessments after 7 days were square root transformed prior to statistical analysis to achieve a normal data distribution. Data were evaluated for statistical significance based on two-way ANOVA F tests with  = 5% analogously as for the final assessment after 17 days as outlined above. The number of juvenile offspring after 7 days was evaluated with the Chi-square test.
All analyses were made using the software STATISTICA Version 6 (Stat Soft, Inc., Tulsa, OK).

Plant-Pest Model
The eight experiments with therapeutic and combined preventative/therapeutic treatments of apple seedlings to control the offspring of rosy apple aphids could be realized under standardized conditions. Less than 1% of the seedlings had to be excluded from data analysis, in all cases due to wrong manipulations during treatments. However, a technical problem might have enhanced the variability of results. The age of apple seedlings and fundatrices used in the experiments varied between the experiments: 4 days for apple seedlings, 2 days for the rosy apple aphids. Otherwise, no additional technical problem occurred with this new plant-pest model system. None of the mock-inoculated plants bore any rosy apple aphids. Thus, there was no cross-contamination between the plants regarding aphids within each experiment.

Preliminary Assessment: Effects of Therapeutic Treatments after 7 Days
Data of all six experiments were analyzed jointly. In 34.4% of all cases, no juvenile offspring were observed. Thus, the number of juvenile offspring was strongly non-Gaussian; we therefore did not apply parametric analysis of variance to the data. The proportion of seedlings without and with juvenile offspring did not differ between the treatments (p = 0.78, Chi-square test). No significant differences were observed between treated and untreated apple seedlings for the percentage of damaged leaves (two-way ANOVA, p = 0.57 for the factor treatment, F test, square root transformed data). Furthermore, we did not observe any fundatrix that left an apple seedling.

Final Assessment: Effects of Therapeutic Treatments after 17 Days
To investigate the efficacy of therapeutic treatments with homeopathic preparations, data of the six experiments were analyzed jointly in a two-way ANOVA with the independent factors "treatment" (nine parameters) and "experiment number" (six experiments). The dependent variables (percentage of damaged leaves, juvenile number, and fresh weight) were analyzed separately. In all three cases, the factor "experiment number" yielded highly significant effects (p < 0.001). This reflects the differences in absolute values between single experiments, due to differences in age of apple seedlings and fundatrices between the experiments as discussed above. The results regarding the factor "treatment" (six homeopathic preparations and three controls) are discussed in the following paragraphs.
No significant differences were observed between treated and untreated apple seedlings for the percentage of damaged leaves (Fig. 1A, p = 0.84, F test for factor "treatment"). However, there were some treatment effects concerning the number of juvenile offspring (Fig. 1B, p = 0.005, F test for factor "treatment", square root transformed data). The two placebo controls were not significantly different from the untreated water control. Therefore, the effect of the granule carrier substance (glucose and invert sugar) seems to be negligible. However, the two treatments -L. clavatum 15c (p = 0.002, LSD test of square root transformed data) and nosode 6c (p = 0.02, LSD test, square root transformed data)significantly differed from the untreated water control (Fig. 1B). Compared to the water control, juvenile offspring were reduced by 17% under L. clavatum 15c treatment and by 14% under nosode 6c. Numerically, L. clavatum 15c reduced the number of juvenile offspring in all six experiments, while the nosode 6c reduced them in five of six experiments. The absolute number of juvenile offspring varied between experiments. This effect allowed us to observe that the treatment effects of L. clavatum 15c and nosode 6c were stronger (up to 30% offspring reduction) with less absolute number of juvenile offspring on the apple seedlings (Fig. 2).
No significant differences in fresh weight were found between treated and untreated apple seedlings (Fig. 1C, p = 0.062, F test for factor "treatment"). However, there was a tendency for reduced fresh weight for all treated apple seedlings.

Effects of Combined Preventative and Therapeutic Treatments
Data of the two experiments were analyzed jointly in two-way ANOVA. No significant differences were observed between treated and untreated apple seedlings for the percentage of damaged leaves, number of juvenile offspring, and fresh weight of apple seedlings for any assessment time.

DISCUSSION
Based on the hypothesis that diseased organisms should show a more evident reaction to treatments with homeopathic preparations than healthy organisms, the first objective of this study was to develop an easyto-handle plant-pest model. It should serve to screen homeopathic preparations under constant climatic conditions in the laboratory. Data gained with this model system should serve as the basis for further experiments under field and farm conditions. Several considerations led to the tested model system: (1) even if leaf disks are easy to handle and allow a greater number of replicates [13], a total plant model would be closer to natural conditions; (2) effects of homeopathic preparations on diseased plants might be more easily detected in perennial plants due to their longer life; (3) pests should be used as model organisms that cause important, but not lethal, damage; (4) the model system should allow statistically reproducible datasets. Apples are a perennial crop with several important pests that cause severe damage. Apple seedlings are juvenile, total plants that can easily be produced and are used as a model for pesticide screening [14]. On apple seedlings, the rosy apple aphid rapidly causes visible, drastic damage on leaves. Therefore, the rosy apple aphid on apple seedlings was chosen as a potential plant-pest model to test homeopathic therapies.
We were very satisfied with the handling of this plant-pest model and could not identify any major disadvantage associated with this experimental setup. In addition, we observed no cross-contamination between plants regarding aphids, which reduced the probability of erroneous results. The mean coefficient of variation was 34% for leaf damage, 25% for the number of juvenile offspring (square root transformed), and 31% for fresh weight at the final assessment after 17 days. In future experiments, this rather high variability might be reduced by an even more homogenous plant and pest age selection. Nevertheless, a model system with interacting organisms will always show a certain range of variability. Due to this fact, the statistical power of this type of model system will fail to show small differences in efficacy (less than 10%) of tested homeopathic remedies. The second objective of this study was to assess the efficacy of two specifically selected homeopathic preparations to control the rosy apple aphid. By repeated therapeutic treatments with L. clavatum 15c and the nosode 6c, the juvenile offspring could significantly be reduced compared to the pure water control. The reduction of juvenile offspring by 17 and 14%, respectively, is not relevant for agricultural practice, however. But, compared to studies with healthy plants [15,16,17] that document effects of homeopathic preparations in the order of 1-3% on growth [4,18], the resulting treatment effect of the homeopathic preparations was more pronounced in our pest-plant system. Thus, the latter might be an interesting tool for homeopathic basic research. This general result is in line with other toxicological and phytopathological investigations, which all showed homeopathic treatment effects in the order of 10-20% [13,19,20,21].
Due to lack of resources, we were not able to implement systematic negative control experiments [22] to document system stability continuously and to ensure appropriateness of statistical evaluation empirically. Although significance levels were quite low and although effects of L. clavatum 15c were numerically reproducible over all six single trials with curative treatment, one might argue that the effects found were artifacts (false-positive results). For future use in homeopathic basic research, systematic negative control experiments are therefore highly recommended to control system variability.
The homeopathic remedies were prepared according to standard procedures: after trituration in the solid phase, further potency levels were prepared by manual succussion in water/ethanol mixtures. Finally, granules of the potency levels 6c, 15c, and 30c were prepared by impregnation with the corresponding liquid potency level. Granules were stored light-protected in standard brown glass vials until use. For plant application, 10 granules of each homeopathic preparation (or untreated placebo granules) were diluted in 250 ml tap water and pivoted until granules were dissolved. 20 ml of these solutions were pipetted on the substrate surface of the potted seedlings. We have no idea whether this application route is optimal for the treatment of plants. Other ways to prepare the homeopathic potencies (e.g., direct potentization in water without using ethanol or granules) or to apply them to the plants (e.g., by foliar spray instead of root application, by another dosage [amount and frequency]) should be evaluated in future experiments. Furthermore, other remedies should be tested. In addition to L. clavatum, sulfur, M. solubilis, and N. muriaticum were also promising candidates, but could not be tested due to lack of resources. Analogously, apart from 6c, 15c, and 30c, other potency levels could be screened for larger efficiency.
Based on our main interest to determine the potential of homeopathic preparations for plant protection, we used two sorts of control groups. The first control consisted of remedy-untreated seedlings (treatment with tap water only), since this type of control is the official standard in plant-pest experimentation according to the EPPO standards [11]. As a second control (carrier control), we used tap water with dissolved untreated granules (not impregnated with succussed or potentized ethanol or lactose, respectively). This type of control was chosen to account for any effects of the carrier substance (sugar) present in traces in the watering solution. From the point of view of homeopathic basic research, the "perfect" control would be triturated lactose, potentized analogously to the verum preparations and impregnated on granules. Our main interest in this experiment was to determine the potential of homeopathic preparations for plant protection, however, and therefore we chose the controls mentioned above. When conducting further experiments with this system for basic research questions, we propose to use potentized lactose as the most appropriate control.
After these six experiments on the therapeutic effect of the two preparations on the control of the rosy apple aphid, the mode of action is not clear. Only the repellent effect on aphids can be excluded, as none left the treated apple seedlings. If the lower number of offspring is due to effects on fertility or on slowing down the development of aphids, it is not yet known.
All experiments have been done under identical, standardized climatic conditions. However, the timing of the rearing of apple seedlings and rosy apple aphids brought some difficulties: the age of the apple seedlings and the fundatrices used varied between (not within) the experiments. What first looks like a drawback can be easily dealt with through application of two-way ANOVA. It furthermore revealed the important information that the effect of the homeopathic preparations was stronger (up to 30% juvenile inhibition) for experiments with a lower absolute number of juvenile offspring (n ≈ 30 on the control plants), compared to about 10% offspring inhibition for experiments with a larger juvenile offspring (n > 40 on the control plants, cf. Fig. 2). This means that the capacity of the system to react to homeopathic preparations was at its upper limit. Consequently, the model system might be improved by using bigger, 1-year-old apple plants that might be hypothesized to have a stronger self-regulation to cope with the seemingly rather severe aphid attack.
Contrary to the number of juvenile offspring, no differences between treatments were found for leaf damage. Usually, the number of rosy apple aphids and the damage are positively correlated [23]. A possible reason for the contradiction in these experiments might be that the homeopathic preparations have no immediate effect on the rosy apple aphid. Therefore, the damage of the one fundatrix and her offspring might have surpassed the damage threshold for these apple seedlings within the 17-day test period. This model system is perfectly adapted to show effects of immediately active agents against aphids [24], but it might fail to show differences when treatments have a retarded effect, e.g., induced by the stimulation of self-defense mechanisms of plants. As mentioned above, the model system should be adapted by using older apple plants that would be able to recover from a severe aphid attack. It then might be possible to observe also a retarded effect of treatments with homeopathic preparations on leaf damage.
No statistically significant effect of combined preventative and therapeutic treatments could be observed within the two serial experiments. This may be due to the fact that the statistical power of these two experiments is lower than the power of the six experiments with therapeutic treatment only. However, the trends seen in juvenile number in the experimental groups of L. clavatum 15c (2% decrease compared to the water control) and of the nosode 6c (7% increase) are not in line with the results of the strictly therapeutic trials. Despite the lower statistical power of the two experiments, one can at least raise the hypothesis that an additional prophylactic treatment does not seem to increase the response of the system considerably; the additional prophylactic treatment rather seems to counteract the curative treatments. It seems premature to us to speculate about a possible correspondence of our results to the widespread view that the main strength of homeopathy is rather disease treatment than specific disease prophylaxis.

CONCLUSIONS
According to our results, homeopathic remedies may be effective in plant-pest systems. The magnitude of observed effects is larger than in models with healthy plants, which renders plant-pest systems attractive for homeopathic basic research. The larger effect size has to be put into relation with the enhanced variability and the larger expenditures for handling and cultivation, however. In addition, the effect size is not yet sufficient for successful application in agriculture. This all calls for further optimization of this model system regarding homeopathic remedy selection, potency level, dosage (amount and frequency), application form (e.g., granules or dilution), and application routes (e.g., foliar spray vs. root treatment). Finally, the use of systematic negative control experiments is to be recommended in future investigations to control system variability.


extfieldsofstudy: Chemistry Medicine

text:
Biosorption of Lanthanides from Aqueous Solutions Using Pretreated Buccinum tenuissimum Shell Biomass

Biosorption experiment from aqueous solutions containing known amount of rare earth elements (REEs) using pre-treated Buccinum tenuissimum shell was explored to evaluate the efficiency of shell biomass as sorbent for REEs. In this work, four kinds of sieved shell samples: (a) “Ground original sample”, (b) “Heat-treatment (480°C, 6 hours) sample”, (c) “Heat-treatment (950°C, 6 hours) sample” and (d) “Heat-treatment (950°C, 6 hours) and water added sample” were used. Furthermore, to confirm the characteristics of the shell biomass, the crystal structure, the surface morphology, and the specific surface area of these shell samples were determined. Consequently, the following matters have been mainly clarified. (1) The crystal structure of the shell biomass was transformed from aragonite (CaCO3) into calcite (CaCO3) phase by heat-treatment (480°C, 6 hours); then mainly transformed into calcium oxide (CaO) by heat-treatment (950°C, 6 hours), and calcium hydroxide (Ca(OH)2) by heat-treatment (950°C, 6 hours) and adding water. (2) The shell biomass showed excellent sorption capacity for lanthanides. (3) Adsorption isotherms using the shell biomass can be described by Langmuir and Freundlich isotherms satisfactorily for lanthanides except “heat-treatment (950°C, 6 hours) sample”. (4) Shell biomass (usually treated as waste material) can be an efficient sorbent for lanthanides in future.

Introduction
Rare earth elements (REE) have gained considerable attention owing to their unique properties and a wide range of applications [1][2][3][4]. These elements and their compounds have found a variety of applications especially in metallurgy, ceramic industry, and nuclear fuel control [5]. For example, current applications of lanthanum as a pure element or in association with other compounds are in super alloys, catalysts, special ceramics, and in organic synthesis [6]. However, the shortage of trace metals including REEs (and the problem of stable supply for these metals) has been concerned in recent years. Therefore, the establishment of the removal or recovery method for trace metals is important from the viewpoint of resources recovery.
In our research, the objective elements are mainly rare earth elements (REEs) and uranium (U) from the viewpoint of resources recovery, although REEs do not represent a common toxic threat. In a previous paper [20], we carried out laboratory model experiments for biosorption of REEs and U using seaweed biomass. Moreover, preliminary experiment for biosorption of REEs, Th and U using Buccinum tenuissimum shell biomass has been performed recently [21]. Consequently, the following has been mainly obtained: (1) the shell biomass shows excellent sorption capacity for REEs; (2) Adsorption isotherms using the shell biomass can be described by Langmuir and Freundlich isotherms satisfactorily for REEs, but not for Th and U.
In addition to ground original sample and heattreatment (480 • C, 6 hours) sample used in our preliminary work [21], heat-treatment (950 • C, 6 hours) sample and heattreatment (950 • C, 6 hours) and water added sample of Buccinum tenuissimum were also used for comparison in present work. Furthermore, the effect of competitive ion on uptake potential of REEs was also studied using original sample to investigate the efficiency of seaweed biomass as sorbent for REEs for more practical use. Among REEs, lanthanides (i.e., La-Lu) were selected in this study, and sorption isotherms of lanthanides were analyzed using Langmuir and Freundlich equations. Moreover, to evaluate the characteristics of the shell biomass used in this work, the crystal structure, the surface morphology, and the specific surface area of the biomass (four kinds of sieved shell samples) were determined by XRD (X-ray powder diffraction), by SEM (Scanning Electron Microscope), and by BET (Brunaeur, Emmet, and Teller) and Langmuir method, respectively.

Reagents and Apparatus.
Lanthanides standard solutions used for making the calibration curve were prepared by diluting the multielement standard solutions (XSTC-1 for REEs; 10 mg·dm −3 5% HNO 3 solution) purchased from SPEX CertiPrep, Inc. (USA). All other chemical reagents were purchased from Kanto Chemical Co., Inc. (Japan). All reagents used were of analytical grade, and water (>18.2 MΩ in electrical resistance) which was treated by an ultrapure water system (Advantec Aquarius: RFU 424TA, Advantec Toyo, Japan) was employed throughout the work.
An ICP-MS instrument (Agilent HP4500, USA) was employed to determine the concentration of REEs. The operating condition of ICP-MS is shown in Table 1. The specific surface area of biomass sample was measured using Micrometritics TriStar3000 (USA). The BET method and Langmuir method were applied to determine the surface area. Nitrogen (N 2 ) gas was used to determine the adsorption isotherms. The crystal structure and the surface morphology of biomass were determined by X-ray powder diffraction (RIGAKU RINT2500HR, Japan) and by SEM (JEOL, JSM-5800, Japan), respectively. The measurement of pH in solution was carried out using a pH meter (HORIBA, F-21, Japan).

2.2.
Samples. Buccinum tenuissimum shellfish samples used for shell biomass in this work were collected at shermen's cooperative association on the coast of Niigata Prefecture, Japan in June, 2008. After being separated from the meat by boiling, organism shells were washed thoroughly with ultrapure water after being washed with tap water repeatedly. Afterwards, dried overnight in an electric drying oven (Advantec DRA 430DA, Advantec Toyo, Japan) at 60 • C, the biomass was ground and sieved through a sieve (SANPO Test Sieves) to remove particles having size more than 600 μm. Afterwards, a part of this sieved materials was heated for 6 hours at 480 • C or 950 • C in an electric furnace (ISUZU Muffle Furnace STR-14K, Japan). Moreover, adequate ultrapure water was added to a part of heat-treatment (950 • C, 6 hours) samples that were heated at 100 • C on a hotplate for evaporation to near dryness (removing water) and finally dried in an electric drying oven at 60 • C. Based on the above-mentioned procedure, four kinds of sieved samples: (a) ground original sample, (b) heattreatment (480 • C, 6 hours) sample, (c) heat-treatment (950 • C, 6 hours) sample, and (d) heat-treatment (950 • C, 6 hours) and water-added sample have been prepared for sorption experiments of lanthanides in this work.

Sorption Experiment for Lanthanides Using Seaweed
Biomass. The following sorption experiments were performed using Buccinum tenuissimum shell biomass. Each sample of 0.2 g was contacted with 100 cm 3 of multielement standard solution (prepared by XSTC-1) including known initial lanthanide concentration (10 to 500 μg · dm −3 ) in a 200 ml conical flask. Afterwards, the suspensions were shaken for 30 minutes in a water bath at room temperature (25 • C) at pH 5. Experimental conditions (i.e., pH, contact time, and biosorbent dose rate) in this work were determined based on our preliminary experiments [21]. The pH of each solution was adjusted by using 0.1 mol·dm -3 HNO 3 /0.1 mol·dm -3 NH 3 aq. Following with each sorption experiment, the suspension containing biomass and lanthanides standard solution was filtered through a 0.10 μm membrane filter (Advantec Mixed Cellulose Ester, 47 mm) to remove lanthanides that have been adsorbed into the shell, and the concentration of these metals in the filtrate was determined with an ICP-MS.

Effect of Competitive Ions on the Sorption of Lanthanides.
The effect of competitive ion on the sorption of lanthanides was studied as the following experiment. In this experiment, the initial lanthanides concentration was taken as 100 μg·dm −3 based on preliminary experiments [21]. In a 200 ml conical flask, each biomass sample (0.2 g) was contacted with 100 cm 3 of lanthanide solution under the presence of calcium (Ca), magnesium (Mg), sodium (Na), and potassium (K) ion at different concentrations 50, 100, and 200 mg·dm -3 . Other experimental conditions and methods were basically the same as that mentioned above in Section 2.3. After filtration through a 0.10 μm membrane filter, the matrix in the filtration was removed using chelate disk (47 ϕmm) (Empore Sumitomo 3M Co.) according to the procedure of Takaku et al. [22], and the concentration of these metals was determined with an ICP-MS.

Adsorption Isotherms
The uptake of lanthanides by each sample was calculated using the following mass balance equation [23]: where q = metal uptake (mg · g -1 ); C o = initial metal concentration (μg·dm -3 ); C e = final (after sorption at equilibrium) metal concentration (μg·dm -3 ); V = volume of the solution (cm 3 ); and W= dry weight of each sample (g). Adsorption isotherms of sorption data were studied at varying initial concentration from 10 to 500 μg·dm -3 under optimized condition of pH, contact time, and biosorbent dosage in this work. The adsorption data obtained for lanthanides were analyzed using Freundlich and Langmuir equations.

Langmuir and Freundlich Isotherm Model.
Two common adsorption model, Langmuir and Freundlich isotherm model was applied based on Dahiya et al. [13,14] to evaluate the adsorption data obtained in this study.
Langmuir model assumes monolayer sorption onto a surface and is given by where C e (mg·dm −3 ) is the concentration of metal ion at equilibrium in the aqueous solution, q e (mg · g −1 ) is the amount of adsorption at equilibrium, a (mg · g −1 ) is the maximum adsorption capacity, and b (dm 3 · mg −1 ) is the equilibrium adsorption constant (Langmuir constant) [24,25]. A plot of C e /q e versus C e gives a straight line with slope of 1/a, and intercept is 1/(ab); b can be related to the adsorption free energy G ads (J · mol −1 ) by where R is the gas constant (8.314 J · K −1 · mol −1 ), T is the absolute temperature (K) at equilibrium. The Langmuir constant (b) can be used to determine the suitability of the adsorbent to adsorbate by using dimensionless parameter, Hall separation factor (R L ) [26], which is defined as where C 0 (mg·dm −3 ) is the initial concentration. The slope of the linearized Langmuir isotherm can be used to interpret the type of sorption using the value of R L as follows: On the other hand, Freundlich isotherm can also be used to explain adsorption phenomenon as given below: where K F and 1/n indicate the adsorption capacity and the adsorption intensity of the system, respectively. It is shown that 1/n values between 0.1 and 1.0 correspond to beneficial adsorption. The linearized Freundlich model isotherm is represented by log 10 q e = log 10 K F + 1 n log 10 C e .
A plot of log 10 q e versus log 10 C e gives a straight line with slope of 1/n and intercept is log 10 K F . . It is suggested that ground original sample contains a lot of organic materials such as protein, and most of organic matter seem to disappear by heat treatment (480 • C, 6 hours). Moreover, fine crystal particle was not observed in (c) heat-treatment (950 • C, 6 hours) sample. This may be attributable to the phenomena that many crystals were connected largely with each other due to hightemperature sintering. Meanwhile, relative clear crystal (sizes are mostly 1.0-4.0 μm) was observed in (d) heat-treatment (950 • C, 6 hours) and water-added sample.

Characteristics of Buccinum tenuissimum Shell
Furthermore, the measurement of specific surface area of the four kinds of sieved samples was performed in this study; and the results are shown in Table 2 along with the main crystal structure of these samples. Remarkably,  decrease of specific surface area (i.e., from 3.32 m 2 /g to 0.390 m 2 /g for BET, or from 5.35 m 2 /g to 0.612 m 2 /g for Langmuir) was found after heat treatment (480 • C, 6 hours). It is suggested that the crystal structure transformation (i.e., from aragonite (CaCO 3 ) into calcite (CaCO 3 ) phase) and also the difference of the surface morphology can be closely related to the remarkable decrease of specific surface area of the shell biomass. On the other hand, the surface area of heattreatment (950 • C, 6 hours) sample was 1.88 m 2 /g for BET or 3.10 m 2 /g for Langmuir, respectively, and that of "heattreatment (950 • C, 6 hours) and water-added sample" was 6.37 m 2 /g for BET or 9.91 m 2 /g for Langmuir, respectively.

Comparison for Sorption Capacity of Lanthanides by
Four Kinds of Sieved Biomass. The comparison for sorption capacity of lanthanides by four kinds of sieved Buccinum tenuissimum shell samples is shown in Figure 3. In this experiment, the initial lanthanides concentration was taken as 100 μg · dm -3 . From this figure, it is found that all kinds of sieved samples showed excellent sorption capacity under this experimental condition. However, the sorption capacity in sample (b) (i.e., the main phase is calcite) decreases slightly relative to that of the original material (i.e., (a) the main phase is aragonite) and others. The decrease of sorption capacity in sample (b) may be attributable to the remarkable decrease (i.e., by a factor of less than one eighth) of specific surface area of the biomass.
Prieto et al. [27] pointed that the sorption capacity of calcite is considerably lower than that of aragonite for Cd. In case of lanthanides, similar tendency of sorption capacity were suggested from our work.

Effect of Competitive Ions on the Sorption of Lanthanides.
The percentage removal of REEs under the presence of common ions (Ca 2+ , Mg 2+ , Na + , and K + ) at different concentrations 50, 100, and 200 mg · dm −3 is shown in Figure 4. From this figure, the remarkable decrease of sorption capacity of lanthanides was not observed. Even when the concentrations of common ions are 200 mg · dm −3 , the percentage removal of light REE (LREE) such as La or Ce decreased slightly (2-3%) whereas the removal decreased about 5% for heavy REE (HREE) such as Yb or Lu. This implies that the shell biomass can be an efficient adsorbent for lanthanides in aqueous environment such as   seawater, although it requires further investigations to apply the shell biomass to use as an adsorbent for lanthanides more practically.

Characteristics of Buccinum tenuissimum Shell Biomass
After Adsorption of Metals. X-ray diffraction (XRD) patterns of four kinds of sieved samples after adsorption of metals are shown in Figure 5. Similar to the XRD patterns before adsorption of metals (Figure 1), aragonite and calcite were found as the main crystal structure in (a) ground original sample and (b) heat-treatment (480 • C, 6 hours) sample, respectively. However, the decrease of peak and increase of noise were also observed in both patterns, particularly in the ground original material as shown in Figure 5(a). Bottcher [28] pointed out that the natural powdered aragonite was transformed to mixed rhombohedral carbonates by the reaction with (Ca, Mg)-chloride solutions. Therefore, there is the possibility that the transformation of aragonite occurred by the reaction with lanthanides in our experiment. Moreover, according to XRD analysis, the main crystal structure of (c) heat-treatment (950 • C) sample was transformed from calcium oxide (CaO) to the mixture of calcium hydroxide (Ca(OH) 2 ) and calcite (CaCO 3 ) after exposing metals and that of (d) heat-treatment (950 • C) and water added sample was transformed from calcium hydroxide(Ca(OH) 2 ) to calcite (CaCO 3 ) after adsorption of metals. These changes may be due to the reaction with water or carbon dioxide in atmosphere.
SEM pictures of the shell biomass after adsorption of metals are shown in Figure 6 ((a) ground original sample, (b) heat-treatment sample (480 • C), (c) heat-treatment sample (950 • C), and (d) heat-treatment and water-added sample (950 • C)). By comparing SEM pictures in Figure 2 with that in Figure 6, it is found that the morphology of sample (a) and (b) has hardly changed even after exposing metals. From this observation, these sieved samples should be predicted to withstand the repeated use, and hence it can be a good adsorbent.
In contrast to sample (a), clear crystal structure (sizes are mostly 0.25-2.0 μm) was observed in sample (b) even after adsorption of metal. In case of Cd conducted by Kohler et al. [29], the difference of procedure for reaction with metals between aragonite and calcite was suggested. According to their work, the precipitation of several distinct types of crystals was observed after exposing metals in the case of aragonite. Then, it is anticipated that similar phenomenon were occurred by adsorption of lanthanides in case of our samples.
On the other hand, the surfaces of samples (c) and (d) after exposing metals have changed largely compared to that before adsorption of metals ( Figure 2). This is in good accord with the results of XRD patterns. Particularly, remarkable transformation was observed in the morphology of sample (d). The reaction of sample (d) with metal solution is supposed to proceed rapidly.

Adsorption Isotherms.
The adsorption data obtained for lanthanides using Buccinum tenuissimum shell biomass were analyzed using Langmuir and Freundlich equations.
The correlation coefficient (R 2 ) of Langmuir and Freundlich isotherms for lanthanides using ground original shell biomass is shown in Table 3 along with other relevant parameters. From this table, it is found that R 2 value for lanthanides is comparatively high. It indicates the applicability of these adsorption isotherms satisfactorily for lanthanides   in this sample. The dimensionless parameter Hall separation factor (R L ) for lanthanides is in the range of 0 < R L < 1, which means that the sorption for lanthanides by this shell biomass is favorable. Furthermore, the negative value of G indicates that the sorption is spontaneous. The higher R 2 value for Freundlich model rather than for Langmuir isotherm (0.638-0.886 for Langmuir isotherm and 0.844-0.932 for Freundlich one) suggests that the adsorption on this sample is due to multilayer coverage of the adsorbate rather than monolayer coverage on the surface. It is noted that the value of 1/n less than unity indicates better adsorption mechanism and formation of relatively stronger bonds between adsorbent and adsorbate [13]. That is to say, favorable adsorption for lanthanides by this shell biomass is presented.
On the other hand, R 2 and other parameters of Langmuir and Freundlich isotherms for lanthanides using heattreatment (480 • C) sample is shown in Table 4. It is noteworthy that R 2 value for REEs in this sample is still more large (0.947-0.982 for Langmuir isotherm and 0.948-0.975 for Freundlich one), compared with the original ground sample (Table 3). Futhermore, this result indicates the stronger the monolayer adsorption (the surface adsorption) on the heattreatment sample relative to on the original sample (before heat treatment). Judging from the value of R L or 1/n in Table 4, the heat-treatment (480 • C) sample also exhibits the favorable property for lanthanides adsorption.
The correlation coefficient (R 2 ) and other parameters of Langmuir and Freundlich isotherms for lanthanides using heat-treatment (950 • C) sample is shown in Table 5. It is found that R 2 value for lanthanides in this sample is fairly small compared with the values of ground original sample or heat-treatment (480 • C) sample (in case of La, Ce, Yb and Lu, R 2 cannot be estimated due to the lack of sorption data at low initial concentration). The low correlation coefficient (R2) in this heat-treatment (950 • C) sample may indicate that the removal of lanthanides occurred not by adsorption mechanism, Particularly R 2 value is remarkably small for Langmuir isotherm, and then other relevant parameters cannot be estimated. As for Freundlich one, not only R 2 8 Bioinorganic Chemistry and Applications  value is relatively small (0.157-0.625), but the value of 1/n for most lanthanide is more than unity. That is to say, the almost perfect removal of lanthanides for this sample (as shown in Figure 3) may be due to other mechanism rather than the adsorption on the biomass. However, the cause or mechanism of lanthanides removal on this sample has yet to be sufficiently clarified in our work, and further investigation to survey the mechanism is needed.
Finally, R 2 and other parameters of Langmuir and Freundlich isotherms for lanthanides using heat-treatment (950 • C) and water-added sample is shown in Table 6. It is Table 6: Coefficient of Langmuir and Freundlich isotherms for lanthanides using Buccinum tenuissimum shell biomass after heattreatment (950 • C, 6 hours) and adding water.

Langmuir
Freundlich found that R 2 value for lanthanides in this sample is fairly large particularly for Langmuir isotherm (0.992-0.999 for Langmuir isotherm and 0.885-0.951 for Freundlich one). This result is similar to that for heat-treatment (480 • C) sample and indicates the stronger the monolayer adsorption on this sample. Judging from the value of R L or 1/n in Table 6, this sample also exhibits the favorable conditions for lanthanides adsorption.
As mentioned above, biosorption studies have been mainly focused on toxic metals elements such as Cd, Pb, As, and Cr so far, and a few reports are focused on lanthanides. Hence, the comparison of maximum adsorption capacity (q m ) of the biomass for lanthanides in the present study with that of different biosorbents (for lanthanides and other elements) in the literatures [5,6,8,[15][16][17][18][19] are presented in Table 7. As clearly seen in Table 7, the biosorption capacity of Buccinum tenuissimum shell biomass for lanthanides in this work is smaller than that of the presented biomasses. However, the sorption experiments in this work were carried out under low concentration of lanthanide (i.e., 100 cm 3 of multielement standard solution including known initial lanthanide concentration (10 to 500 μg · dm −3 )) instead of that in single component system.
Then, sorption experiment for three lanthanides (La, Eu and Yb) in single component system is being planned using the solution individually prepared by each nitrate salt: La(NO 3 ) 3 · 6H 2 O, Eu(NO 3 ) 3 · 6H 2 O, or Yb(NO 3 ) 3 · 3H 2 O (supplied by Kishida Kagaku Company, Japan) and this Buccinum tenuissimum shell biomass.

Conclusion
Biosorption characteristic of Buccinum tenuissimum shell biomass was studied for lanthanides. Sorption isotherms of lanthanides were analyzed using Langmuir and Freundlich equations to confirm the efficiency of shell biomass as sorbent.
The shell biomass samples showed excellent sorption capacity for lanthanides under this experimental condition, and the effect of all the common ions (Ca 2+ , Mg 2+ , Na + , and K + ) up to the concentration of 200 mg·dm −3 on the sorption capacity of lanthanides is considerably small. From these results, it was quantitatively clarified to some extent that shell biomass can be an efficient sorbent for lanthanides. It is very significant information from the viewpoint of environmental protection that the shell (usually treated as waste material) can be converted into a biosorbent for lanthanides.


text:
Effect of Aqueous Extract of Embelica officinalis on Selenite Induced Cataract in Rats.

Cataract is clouding of the eye lens that reduces the amount of incoming light and results in deteriorating vision. Blindness is thought to reach 75 million by 2020. Of these, unoperated cataract may be expected to account for at least 35 million. Thus, the burden of cataract is increasing remorselessly. Embelica officinalis is reported to have a very good antioxidant property and thus we hypothesized that it could be a good candidate in treatment of cataract. Hence, the aim of this study was to investigate the effect of aqueous extract of Embelica officinalis on selenite induced cataract in rats. For the purpose of this study, cataract was induced in young suckling (on the 10th day of life) albino wistar rats using sodium selenite (a single dose of sodium selenite; 20μM/kg; subcutaneously). After induction of cataract, the test drug (Embelica Officinalis) and the reference standard (ascorbic acid) were administered orally for 18 days. The progression or disappearance of cataract was observed with the help of an ophthalmoscope (OM-18, Takagi resolution 1.6). At the end of this study the alterations in the levels of total protein, soluble protein, reduced glutathione and malondialdehyde were estimated in the lens homogenate. Results showed that treatment with Embelica officinalis, as well as ascorbic acid, produced a significant decrease (p < 0.05) in malondialdehyde and a simultaneous increase in lens glutathione levels (p < 0.05). The malondialdehyde content was decreased by 48% in animals treated with Embelica officinalis. Similarly, lens glutathione was increased by 82.5% in animals treated with Embelica officinalis. There was also a significant (p < 0.05) increase in protein content (total protein = 59.36% and soluble protein = 105.78%) in animals treated with Embelica officinalis, indicating improvement in cataractogenic condition in the selenite induced cataract model. At the end of the treatment, disappearance of cataract was observed in test and standard treated animals. In conclusion, it could be said that aqueous extract of Embelica officinalis delayed the progression of cataract in sodium selenite induced cataractogenic rats.

Introduction
Cataract is clouding of the eye lens that reduces the amount of incoming light and results in deterioration of the vision. Cataract remains the leading cause of visual disability and blindness all over the globe (1, 2), which makes up at least 50% of blindness in most developing countries (3). Blindness is thought to reach 75 million by 2020. Of these, unoperated cataract may be expected to account for at least 35 million. This figure is equivalent to the combined present total populations of Australia, New Zealand, Sweden and Denmark. Thus, the burden of cataract is increasing remorselessly (3). Senile cataract, also called "age related cataract, " is the commonest type of cataract affecting equally persons of either sex usually above the age of 50 years. Increased oxidative stress is reported to be the cause for such a cataract. The only treatment presently available is surgery. Experimental cataract, correlating senile cataract, can be induced by subcutaneous injection of sodium selenite to suckling rats. Several biochemical processes occur during production of selenite cataract. These include altered epithelial metabolism, calcium accumulation, calpaininduced proteolysis, crystalline precipitation, phase transition, and cytoskeletal loss. A number of important changes in metabolism have been documented in lens epithelium during the formation of selenite cataract; usually well before any visible opacity. These include suppression of mitosis and entry of epithelial cells into prophase, nuclear fragmentation (4), decreased rate of epithelial cell differentiation (5), decreased synthesis and increased damage of DNA (6), and loss of calcium haemostasis (7). It has been hypothesized that these early changes in lens epithelium may result from oxidative damage caused by selenite, possibly due to critical sulfhydryl groups on molecules such as calcium ATPase or ion channels. Loss of calcium haemostasis could be prevented by antioxidants. Preparations containing Embelica officinalis are widely used for their preventive, curative, and health restorative properties. It possesses very good hypocholesterolemic (8), anticarcinogenic (9), antidiabetic (10), and antioxidant properties (10-13). Emblica officinalis is a rich source of vitamin C (ascorbic acid), and most of its biological actions may be due to vitamin C. Nevertheless, the reported vitamin C content in Embelica officinalis varies from negligible to 0.7% (14).
Presently, the only treatment available for cataract is surgery and thus the present study was aimed to investigate the effect of Embelica officinalis, a herbal drug having antioxidant activity, in treatment and prevention of maturation of cataract. A prevention or delay through such an applications in human will be a significant achievement.
Hence, the objective of the present study was a preliminary investigation into the effect of Embelica officinalis in selenite induced cataract.

Experimental
Identification and collection of agents (I) Embelica officinalis was collected from the field of nearby region and dried in shade. It was authentified by the Bioscience Department, Sardar Patel University and a specimen voucher was deposited. Powdered plant was prepared and stored in an air-tight container. An aqueous extract was prepared for the purpose of study, and administered orally (p.o.).

Estimation of ascorbic acid in Embelica officinalis
Aqueous extract of Emblica officinalis was estimated for the content of ascorbic acid, as per the method described in Indian Pharmacopoeia (15).

Selection of animals
All animals were housed at ambient temperature (25 ± 10°C) and relative humidity (55 ± 5%), and a 12h/12h light/dark cycle. Animals had free access to standard pellet diet and water given ad libitum. The experimental protocol was approved by the Institutional Animal Ethical Committee, as per the guidance of committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India (Protocol number project 5008, dated 10 th of January 2006).

Induction of cataract
Cataract was induced experimentally by selenite treatment. Thirty suckling wistar rats (9 days old pups) of either sex were divided into five groups, with 6 animals in each group. On the 10 th day of life, when they were in an absolute nutritive dependence from the mother, a single dose of sodium selenite (20 µM/kg) was administered s.c. to all the groups except for the normal control group (group l) (16).Groups I and group II (cataract control) animals received food and water ad libitum throughout the study period. Group III (test) and group lV (standard) received aqueous extract of Embelica officinalis (equivalent to 26.19 mg/kg ascorbic acid) and ascorbic acid (26.19 mg/kg), once daily orally, respectively.
The treatment was continued for 18 days after the initiation of cataract. The eyes of the animals were observed for the progression/ disappearance of the cataract. All the animals were sacrificed at the end of study, to isolate the lens and lens homogenate was prepared and used to estimate various biochemical parameters. Total protein and soluble protein contents were estimated, using the Lowry et al. (17) method, in the lens homogenate. Anti-oxidative properties were evaluated by estimating the glutathione (18) and malondialdehyde (19) levels.

Estimation of Malondialdehyde in lens (MDA)
MDA estimation was performed using the method described by Ohkawva et al (19). Briefly, to 0.2 mL of lens homogenate, 0.2 mL of 4% sodium dodecyl suphate, 1.5 mL of 20% acetic acid (prepared in 0.27 M HCl) and 1.5 mL of 0.5% thiobarbituric acid were added. The mixture was heated for 60 min at 95°C in a temperature controlled water bath to give a pink color. The mixture was then centrifuged at 3500 rpm for 10 min. Finally, absorbance of the supernatant layer was read spectrophotometrically at 532 nm.

Statistical analysis
Statistical analysis of various physical and biochemical parameters were carried out, using the one way analysis of variance (ANOVA),with p < 0.05 considered as a significant difference.

Effect of Embelica officinalis on catract progression
Embelica officinalis extract was found to have 7.5 mg of vitamin C (0.75%).
Presence of cataract in the sucklings was visible by the naked eye after the opening of their eyes, i.e. on the 15 th day of life in groups II, III and IV.on the 15 th day of their life cataract was seen to turn yellowish -white, localized in the center of the lens, termed as nuclear cataract (Figure 1a). The cataract progressed to mature catarct in the selenite control group on day 33 after birth ( Figure 1b). On the contrary, cataract was found to be disappeared in the ascorbic acid ( Figure 1c) and Embelica officinalis (Figure 1d) treated animals.

Effect of Embelica officinalis on lens protein content
Selenite injection in animals, caused a decrease in the amounts of total protein and soluble protein (Table 1). In the present study a significant decrease (p < 0.05) in the amount of soluble protein and total protein in selenite treated group was observed, as compared to the normal control. Treatment with Embelica officinalis (26.19 mg/kg; aqueous extract; p.o.) significantly increased (p < 0.05) the total protein content as well as the soluble protein levels. The increase in the total protein content in the ascorbic acid treated (26.19 mg/kg; p.o.) animals was also found to be more than that of the selenite control.

Antioxidant activity of Embelica officinalis (I) Effect of Embelica officinalis on lipid peroxidation (MDA content)
Rats administered with sodium selenite showed a significant increase (p < 0.05) in the levels of malondialdehyde (Table 1), compared to the normal control. The elevation produced in lipid peroxidation by selenite was decreased significantly (P < 0.05), as a result of Embelica officinalis (26.19 mg/kg, aqueous extract; p.o.) administration. The reversal offered by ascorbic acid (26.19 mg/kg) on malondialdehyde level was comparable with the respective normal control values.

Discussion
Cataract was produced in suckling rat pups by an overdose of the essential trace mineral selenium, when injected before completion of the critical maturation period of the lens (approximately 16 days of age).
It has been hypothesized that these early changes in lens epithelium may result from oxidative damage caused by selenite, possibly due to the critical sulfhydryl groups on molecules such as calcium ATPase or ion channels. Loss of calcium haemostasis could be prevented by antioxidants.
The significant increase in the protein content (both total and soluble proteins) indicates an improvement in the cataractogenic condition, with Embelica officinalis treatment.
L-Ascorbic acid (pure form) did not increase glutathione levels and nor it could decrease the malondialdehyde levels to a significant extent, as was observed with Embelica officinalis treatment. This suggests that cataract inhibition by Embelica officinalis is due to some other constituents other than vitamin C.
Some studies indicate that the biological actions, particularly antioxidant activities, of Embelica officinalis can not be attributed to ascorbic acid alone (11-13). The potent vitamin C-like anti-oxidative effect of Embelica officinalis fruit against reactive oxygen species, was also observed with low molecular weight hydrolysable tannins (14). Four such compounds, emblicanin A, emblicanin B, punigluconin, and pedunculagin have been isolated from Embelica officinalis pericarp and their structures have been established (14). Furthermore, Bhattacharya et al (12,13) have demonstrated the antioxidant property of tannoid rich fraction of Embelica officinalis in a stress induced oxidative damage model in rat brain. Therefore, it can be concluded that the antioxidant potential of Embelica officinalis, as evidenced by a significant decrease in malondialdehyde and a simultaneous increase in lens glutathione levels in the present study, may be attributed to tannoid principle and not fully because of ascorbic acid present in Embelica officinalis extract.
It is now well understood that oxidative stress is one of the important reasons for cataract formation, which in turn is associated with alteration in protein content of lens (i.e. decrease in both total and soluble proteins). The antioxidant property of Embelica officinalis was confirmed by an increase in lens glutathione content and a decrease in malondialdehyde content. However, at the same time the improvement in the cataractogenic condition was implicated by an increase in the protein content (both soluble and total protein) within Embelica officinalis.
This article is available online at http://www.ijpr-online.com