1. Technical Field:

This invention is related to field of biology. Control of the prevalence of Bilharziasis using novel models of photothermal and photosensitizing drugs. It includes some sort of sterility to Biomphilaria alexandrina snails - intermediate hosts of Schistosoma mansoni- that aims at production of lab sterile strains.

2. Background Art: 2.1 Bilharziasis:

This disease, which is known as bilharziasis, is a chronic, debilitating infection that damages the liver and other vital organs in humans. It is the major public health problem in Egypt and many other tropical areas. The infection is called snail fever, because certain aquatic, fresh water snails serve as intermediate hosts in the parasites' life cycle (WHO, 1993). In spite of the considerable efforts that are made worldwide to prevent infection and morbidity (Pereira et at, 2006), it is estimated to infect 200 million people around the world, while more than 600 million people are at risk, causing high levels of morbidity and mortality in 74 countries in tropical and subtropical areas (WHO, 2002 and Steinmann et at., 2006).

There are many varieties of schistosomes, but only four, which are particularly important to man:

S. haematobium occurs throughout Africa and in Arabia, South West Asia, and around the Mediterranean. The urinary tract and the portal system are mainly affected, but the lungs and colon do not escape, and the central nervous system may occasionally be involved.

S. mansoni is also prevalent throughout Africa, particularly in the north, in Arabia, and in the north of South America. It mainly affects the colon, the portal system and the lungs, very rarely the central nervous system. S. japonicum is found mostly in Asia; in China and Japan, Philippines, and other Pacific islands. It primarily affects the colon and small intestine, the portal system and the lungs, rarely the central nervous system.

The fourth variety, S. intercalatum, is much less common and occurs only in equatorial Africa, particularly Zaire, and affects the digestive tract and the portal system. There is one other, S. mekongi, clinically similar to S, japonicum, but found only in the Mekong river basin (Pasvol and Hoffman, 2001).

In rural Egypt, Schistosomiasis is the major public problem with almost six million Egyptians infected as at mid - 1996 (El khoby et a , 1998). In 1983, the prevalence of Schistosomiasis in rural Egypt was greater than 50%, but a ten-year campaign of diagnosis and treatment has reduced the prevalence and intensity of infection (Cline et aL, 1989). Egyptians have a long history of symptoms caused by Schistosomiasis, notably haematuria (blood in the urine), which appeared classically in young boys and was once deemed to be a sign of puberty. In Egypt, it was reported at 1851 that Theoder Bilharz discovered, in autopsy material, the causative agent of haematuria: Schistosoma (El khoby et aL, 1998). And Leiper at 1914 had discovered the two genera of snails {Bulinus and Biomphalaria) that transmit the two species Schistosoma haematobium and S. mansoni, respectively (Leiper, 1915).

During the past four decades, numerous efforts have been made to overcome Schistosomiasis throughout the world. In 1 985, the World Health Organization (WHO) reported that reducing or curtailing the transmission of the parasite by snail control strategies could be a rapid and efficient method (WHO, 1980). Thus, priority was given to the extensive use of molluscicides in the field. Five years later, as a result of advances in parasitological diagnostic techniques and chemotherapy, the WHO modified the strategy, giving priority to the control of morbidity rather than to the control of transmission, and preferentially developed chemotherapy (Lardans and Dissous, 1998). In 1993, the WHO raised the alarm with a necessary return to snail control strategies in association with chemotherapy (WHO, 1993). Existing control methods are aimed principally at the management of snail population that inhibit endemic areas , including elimination of natural water bodies such as marshes and ponds , and regulation of human settlement in areas with significant risk (Lardans and Dissous ,1998). In areas concerned with irrigation development, proper drainage and environmental engineering have been effective in reducing S. haematobium and S. japonicum transmission (WHO, 1993). The use of molluscicides has always been considered to be a major supportive procedure in integrated Schistosomiasis control. Perret and Whitfield (1996) provided an overview of currently available synthetic and natural molluscicides and their respective efficiencies. Among synthetic compounds, niclosamide is still the molluscicide of choice, being highly active against all stages of the snail life cycle and the free-living larval stages of Schistosome. Although niclosamide is not toxic to humans, domestic animals and crops, the molluscicide is costly toxic to fish (Ayad, 1974). Moreover, the application of niclosamide did not prevent recolonization of some water bodies by surviving snails, which could lead to the selection of molluscicide - resistant populations. Natural molluscicideal compounds are present in a large number of plants (Kloss and McCullough, 1982). For example, saponins (from phtolacca dodencandra) and isoflavonoids (from Millettia thonningii) have been characterized for their molloscicidal activity (Kloss et ai, 1987). The distribution of molluscicdal plants in infected areas has been investigated extensively, but, their toxicity and the problems encountered with their large - scale production has considerably limited their exploitation (Belot, 1993).

Alternative methods of snail control have been developed using different biological control agents. Predators (mainly fish) have occasionally been applied with success in islands to limit snail populations (Pointier and Gayard, 1992). Snail pathogens, particularly dominant trematodes {Echinostomatidae) with the ability to sterilize snails, have been used in control trials, but their field application exhibited an efficacy lower than that of mollusciciding (Lardans and Dissous, 1998). The introduction of snail competitors has also been considered as a promising way to displace target snail populations (Madsen, 1990) or to compete for parasites by a possible decay effect (Combes and Mone, 1987). Since 1920, when the first antimony - based cure for Schistosomiasis was introduced, Egypt has taken the leading role in field testing new drugs against Schistosoma and new molluscicides to kill the snail hosts and reduce transmission. Control programs were a regular feature in rural Egypt, with treatment campaigns using whatever was the current drug of choice and snail control using a range of molluscicides (El khoby et al., 1998). Major program in the late 1960s aimed to eradicate Schistosomiasis from the oasis of Fayoum, using blanket mollusciciding with niclosamide, supplemented by chemotherapy using ambilhar (El khoby et al., 1998)

Although the program reduced the prevalence of the disease, by 1985 there was as much, if not more, Schistosomiasis in Fayoum as ever. Another large control program commenced in 1977 in middle Egypt, using nicolsamide to control the Bulinus snails, and Bilarcil (metrifonate) to treat cases of S. haematobium. The project was subsequently expanded southwards to cover an area of two million feddans and some 12 million people (El khoby et al., 1998). Investigations on the effects of radiation (gamma, x- ray and ultraviolet) on the Schistosome snail hosts have not been numerous. Amongst are Laviolette and Voulet (1961). It is worth to mention that radiation plays an important role on the life of snails through effects on their mortality, growth rate, and egg - laying capacity. Much effort has been directed towards snail control strategies as a mean of reducing the prevalence of Schistosomiasis. Essentially, these efforts have been based upon elimination of hosts by molluscicides and / or by the use of competitor species, but, further progress is required for a more efficient prevention of disease transmission in endemic areas (Lardans and Dissous, 1998).

2.2 Photosensitization:

Photosensitization is a treatment involving the administration of a photoactive compound that selectively accumulates in the target cells and is followed by irradiation with visible light. The combination of the two absolutely nontoxic elements, drug and light, in the presence of oxygen results in the selective destruction of the target cell (Luksiene Z., 2005)

It is important to note that truly major advances have been made in photosensitized antimicrobial chemotherapy, in particular disinfection of the blood and blood products, or treating local infections (Luksiene Z., 2005).

By any means, prevention of any disease by controlling its causative agents of prevalence, including the secondary - non human- hosts-, is of great importance. Thus, development of new antimolluscicidal methods is necessary. In this context, photosensitization has been shown to be really effective as different microorganisms such as drug-resistant bacteria, yeasts, viruses and parasites can be inactivated by this method. So far, a photosensitization phenomenon can open new and interesting avenues for the development of novel, effective and ecologically safe molluscicides' modality.

As a rule, Photosensitizers are usually aromatic molecules that can form long-lived triplet excited states. Despite the possible synthetic Photosensitizers, there are, however, many examples of natural Photosensitizers that have evolved over the years either in plants or in fungi (Wood and Bruhn, 2000).

Chlorophyll is the pigment that gives plants and algae their green color. Plants use chlorophyll to trap light needed for photosynthesis. The basic structure of chlorophyll is a Porphyrin ring similar to that of heme in hemoglobin, although the central atom in chlorophyll is magnesium instead of iron. The long hydrocarbon (phytol) tail attached to the Porphyrin ring makes chlorophyll fat-soluble and insoluble in water. Two different types of chlorophyll (chlorophyll a and chlorophyll b) are found in plants .The small difference in one of the side chains allows each type of chlorophyll to absorb light at slightly different wavelengths.

Chlorophyllin is a semi-synthetic mixture of water soluble sodium copper salts derived from chlorophyll. During the synthesis of Chlorophyllin, the magnesium atom at the center of the ring is replaced with copper and the phytol tail is lost. Unlike natural chlorophyll, Chlorophyllin is water-soluble. Chlorophyllin has been used orally as an internal deodorant and topically in the treatment of slow-healing wounds for more than 50 years without any serious side effects. Chlorophylls and Chlorophyllin form molecular complexes with some chemicals known or suspected to cause cancer, and in doing so, may block carcinogenic effects. Scientists are hopeful that Chlorophyllin supplementation will be helpful in decreasing the risk of liver cancer in high-risk populations with unavoidable, dietary aflatoxin exposure.

Little is known about the bioavailability and metabolism of chlorophyll or Chlorophyllin. The lack of toxicity attributed to Chlorophyllin led to the belief that it was poorly absorbed .However; significant amounts of copper chlorine were measured in the plasma of humans taking Chlorophyllin tablets in a controlled clinical trial. More research is needed to understand the bioavailability and metabolism of natural chlorophylls and chlorine compounds in synthetic Chlorophyllin.

2.3 Nanoscience:

Over the past few decades, inorganic nanoparticles, whose structures exhibit significantly novel and improved physical, chemical, and biological properties, phenomena, and functionality due to their Nano scale size, have elicited much interest. Nano phasic and Nano structured materials are attracting a great deal of attention because of their potential for achieving specific processes and selectivity, especially in biological applications (Sukdeb Pal, et al)

NanoScience deals with those processes that take place on the nanometer scale, that is, from approximately 1 to 100 nm. Nanoparticles of metals have been investigated extensively in recent years due to their novel material properties which differ greatly from the bulk substances.

Discoveries in the past decade have demonstrated that the electromagnetic, optical, and catalytic properties of noble-metal Nano crystals are strongly influenced by shape and size (Burda et al., 2005 and Mulvaney, P. 1996). This has motivated an upsurge in research on the synthesis routes that allow better control of shape and size (Sun, Y. G., et al 2003, Zhou, Y.S. et al ,1999, and Jana et al 2001), with projected applications in Nano electronics and spectroscopy (Hranisavljevic, J., N., et al 2002,. Hermanson, K. D.,etal 2001 and E. W. Kaler. 2000). Recent studies have demonstrated that specially formulated metal oxide nanoparticles have good antibacterial activity and antimicrobial formulations comprising nanoparticles could be effective bactericidal materials (Sukdeb Pal, et al).

Among inorganic antibacterial agents, silver has been employed most extensively since ancient times to fight infections and control spoilage. The antibacterial and antiviral actions of silver, silver ion, and silver compounds have been thoroughly investigated (Sun, Y. G., et al 2003, Oka, M„ T, et al,l994 and Oloffs, A., C. et al, 1994). However, in minute concentrations, silver is nontoxic to human cells. The epidemiological history of silver has established its non toxicity in normal use. Catalytic oxidation by metallic silver and reaction with dissolved mono valent silver ion probably contribute to its bactericidal effect (James, G. V. 1971).

Additionally, Silver Nano crystallites have widely been paid attention due to their application potential in medicine (Jain P. and Pradeept., 2005 and

Elechiguerraj.L., et al 2005,) catalysis electrical conductivity , optical properties , and as the substrates for surface-enhanced Raman spectroscopy ( Mariam Jokar, et al , 2010) .

Recent literature reports encouraging results about the bactericidal activity of silver nanoparticles of either a simple or composite nature. Consequently, this study was designed to detect the capability of silver Nano particles to act as a moUuscicidal tool.

3. Disclosure of invention:

Th is invention introduces novel modal ity for control and producing lab steri le strains of Biomphilaria alexandrina snails (the intermediate host of S. mansoni) It was the pioneer that was concerned with the effect of silver nanoparticles (SNPs) along with two other photosensitizers on the Biomphilaria alexandrina snails (the intermediate host of S. mansoni). The main aim of this work was to investigate the efficiency of these chromophores on the biological behavior change as a consequence of the change in the initial tissue structure of Biomphilaria alexandrina snails to confirm the control of the viability of the snails using LC90 and damage the genital organs using LC50 and LC25.

4. Methodology: Effect of the examined chromophore(s) on the survival of Biomphilaria alexandrina snails as a function of the examined chromophore(s) concentration:

In this experiment d ifferent proposed concentrations of the examined chromophore(s) were proposed to be tested 10 ^"3 , 10 ^'4 , 1 0 ^"5 and 10 ^"6 M/L. Snails were incubated in dark for 24 hours in SNPs (three replicates, each of ten snai ls IL for each concentration) , for 12 hours in copper Chlorophyllin and 3 hours for magnesium Chlorophyllin . then they were exposed to the sun light for nearly the day time then they were allowed to recover for another 24 hours and their viabil ity was tested as previously mentioned. Control samples were exposed to sun light without incubation in any chromophores and recovered for 24 hours.

Prel iminary observations implied that the lethal dose and the sub lethal concentration for each chromophore independently.

Effect of the examined chromophore(s) on the survival of Biomphilaria alexandrina snails as a function of incubation period:

In this experiment, different incubation periods were proposed to be tested 3, 6, 9, 12, 24 and 48 hours. As mentioned, snails were incubated in lethal concentration (Lc90) and the sub lethal concentrations (Lc50) or the incubation periods proposed for each chromophore independently, then they were transferred to dechlorinated water and exposed to sun light, then they were allowed to recover and their viabi l ity was tested together with the light and dark control samples.

This experiment was repeated with different other concentrations under the same conditions. Three repl icates were prepared for each incubation period.

Prel iminary observations impl ied that 24 hours of incubation was the optimum one for si lver nanoparticles, 12 hours for copper nanoparticles, and 3 hours for magnesium nanoparticles

Histological studies:

To examine the photo thermal effect of the tested chromophore on the tissues of the Biomphilaria alexandrina snails histological studies were performed, after the snails were incubated in the examined concentration of the tested chromophore and exposed to the light source for the proposed period of time, they were dissected to remove the shel ls and then sections (5-8μιτι) were fixed using BOUlN's fixative then they were processed, embedded and stained with Delafield's haematoxyl ine and eosin accord ing to Mohamed and Saad (1990). Sections of control snails' hermaphrodite gland were prepared simultaneously.

Biochemical studies:

After incubation of the snails they were d issected to remove the shel ls and homogenized using UP 200H ultrasonic processor in 2ml dec lorinated water. The suspension obtained was centrifuged for 40 minutes at room temperature at 4000 rpm , The pellet was d iscarded while the aliquots of supernatants were involved in different biochemical runs to estimate the levels of SGOT, SGPT, alkal ine phosphatase, total protein, albumin, globulin, glucose, cho lesterol and triglycerides using the relevant kit accessories for each test and the relevant device recommended for each investigation.

Hormonal studies:

After incu bation of the snai ls with the Lc25 and Lc l O of the tested chromophore, they were dissected to remove the shells and homogenized using U P 200H u ltrasonic processor in 2m l dechlorinated water. The suspension obtained was centrifuged for 40 m inutes at room temperature at 4000 rpm, The pellet was d iscarded while the aliquots of supernatants were involved in different ELISA runs to estimate the levels of testosterone, progesterone and estradiol using the relevant kit accessories for each test Where, the absorbance of the calibrators, controls and the test samples were measured using ELISA reader, stat fax, device.

The accumulation rate of the examined chromophore(s) in the snails' tissues as a function of incubation period:

In this experiment light, dark controls and the test groups were incubated with the proposed concentrations for 3, 6, 9, 12, 24 and 48 hours each accord i ng to the type of the examined chromophore (5 snails for each aquaria in 50 m l) then, the snai ls were rinsed with dechlorinated water, dissected to remove their shel ls then each category of samples were put in2 ml dechlorinated water in 5 ml plastic test tubes and homogenized using the UP200H Ultra Sonic Processor. Then the suspension was centrifuged for 45 m inutes at 4000rpm as mentioned in the general methods and the aliquots of supernatants were measured by reading the absorbance of the dechlorinated water using PERKIN ELMER lambda 40 spectrophotometer. The examined chromophore(s) was determined from Beer's Lambert law.

The accumulation rate of the examined chromophore(s) in the snails' tissues as a function of the examined chromophore(s) concentrations: In this experiment, light & dark controls along with the test samples incubated with different concentrations of the examined chromophore(s) for periods that were estimated to be the optimum according to the type of the exam ined chromophore . Then as mentioned in the genera] methods they were rinsed with dechlorinated water ,dissected to remove their shells, then they were put in 2ml dechlorinated water in 5 m l plastic test tu be to be homogenized and centrifuged .The aliquots of supernatants were measured by reading the absorbance of the dechlorinated water using PER IN ELMER lambda 40 spectrophotometer. The examined chromophore(s) was determined from Beer's Lambert law.

The accumulation rate of the examined chromophore(s) in the snails' tissues as a function of the chromophore(s) released by the snails:

In this experiment, light and dark controls along with the test samples were incubated with different concentrations of the examined chromophore for periods that were identified to be the optimum accord ing to the type of exam ined chromophore(s), then they were transferred with dark and light control to dechlorinated water med ia and after 6,9, 12, 24 and 48 hours of recovery the snails were dissected respectively each in a time to remove their shells , homogenized , centrifuged and the al iquots of supematants were measured by read ing the absorbance of the dechlorinated water using PERKIN ELMER lambda 40 spectrophotometer. The examined chromophore(s) concentration was determined from Beer's Lambert law.

Estimating the effect of nanoparticles and / or photosensitizers on some biological parameters of B. alexandrina snails at the optimum treatment conditions via Snails' fecundity

In this experiment, 1000 adu lt snails of sizes ranges from 7-9mm that were bred in TBRI were involved. First of all, the snails were allowed to be tested for their reproductive capacity for one week. As the number of laid eggs was obtained and the number of the survived snails were determined and the reproductive index was calculated. For three separate experiments, the remained survived snails were d istri buted even ly to be tested for each chromophore. Where, for each experiment the snai ls were incubated with two concentrations (Lc25 and Lc l O) for each chromophore and the dark and light controls run along with the test samples for the results' accuracy. In all experiments the incubation period was determined to be the optimum for each chromophore where it was 24 hours in case of silver nanoparticles, 12 hours for copper Chlorophyl lin and 3 hours for magnesi um Chlorophyl l in. While the exposure ti me was the day l ight (aprox. 8 hours) for al l the experiments. Thereafter, the snails were tested for the percent of survivorship, put under observation to detect the rate of reproduction where the number of survived snails and the num ber of laid eggs were recorded weekly for six successive weeks. From which the reproductive rate (Ro) was recorded for each chromophore. Worthy to mention that defined number of each test and controls groups was taken to be re-incubated and re- exposed to sun light to detect the effect of repeated stimulations.

Estimating the effect of nanoparticles and / or photo sensitizers on some biological parameters of B. alexandrina snails at the optimum treatment conditions via Snails' growth.

In these experiments, 300 snails of sizes ranges 3-5mm were involved. Where, they were distributed evenly among the three experiments for the three examined chromophores. The snails were incubated with two concentrations for each chromophore regarding the light and dark control snails in each experiment. Thereafter, the survived snai ls were kept in the lab to recover where their sizes were measured weekly for eight successive weeks and their capability to lay eggs was also examined by recording the number of the survived snails and the number of laid eggs for each experiment.

Estimating the effect of nanoparticles and / or photo sensitizers on some biological parameters of B. alexandrina snails at the optimum treatment conditions via infection of snails with S. mansoni miracidia.

In these experiments, thousand adu lt snai ls of sizes ranges 7-9mm were involved. The snai ls were kept in the lab for a defined time to assure their voidedness of any sort of infection. Thereafter, they were distributed evenly between the three examined chromophores, taking two replicates for two concentrations (LC25 & LC10) with the light and dark controls runs within each experiment. The examined snai ls were allowed to be infected with S. mansoni miracidia. And they were d ivided in to two groups; one was to be incubated and exposed to l ight after three days of infection and the other was to be incubated and exposed to light after 21 days of infection. After incubation with the examined chromophore and exposure to sun light, the snails were kept to recover where they were examined for the production of cercariae after 21 days of infection. Worthy to mention is that, the period of incubation was defined according to the type of chromophore meanwh i le, the exposure period was the day l ight (8hours) for al l. 5. Brief description of tables and drawings:

Figure 1 shows light control sample (sectioned from the hermaphrodite system) when exposed to sun light. Intact ova cells and sperm threads are obvious in this section, as the sectioned snails were not incubated with SNPs but, they were exposed to sun light thus, the functional hermaphrodite system was preserved as implied in the figure. ((H. &E. stain, x400) G = Spermatogonia, I = Oogonia, O =Oocyte, T ^Spermatid (Sperm threads)

Figure 2 reveals a dark control sample where, sections were taken from the hermaphrodite system of snails that were incubated with 10 ^"5 M/L SNPs without being exposed to light irradiance so, as the section reveals no cellular destruction was observed.

Figure 3 show test sample (sectioned from the hermaphrodite system) incubated withl O ^"4 M/L SNPs (Lc90) for 24 hours and exposed to sun light. Degeneration of ova cells is observed when compared with the control samples as it implies hollow centers in the hermaphrodite system with degenerative ova cells and sperm threads which, was observed obviously when the snails were incubated with 10 ^"4 M/L SNPs for 24 hours and exposed to sun light.

Figure 4 shows test sample (sectioned from the hermaphrodite system) incubated withlO ^"5 M/L SNPs (Lc50)for 24 hours and exposed to sun light. It reveals a degenerative effect of ova cells and sperm threads for snails incubated with 10 ^"5 M/L SNPs for 24 hours and exposed to sun light. However, this degenerative effect observed was less than that observed when the snails were incubated with 10 ^"4 M/L at the same conditions.

Figure 5 reveals a dark control sample where, sections were taken from the hermaphrodite system of snails that were incubated with 10 ^~5 M/L copper Chlorophyllin without being exposed to light irradiance so, as the section reveals no cellular destruction was observed.

Figure 6 show test sample (sectioned from the hermaphrodite system) incubated withl O ^"4 M/L copper Chlorophyllin (Lc90) for 12 hours and exposed to sun light. Degeneration of ova cells is observed when compared with the control samples as it implies hollow centers in the hermaphrodite system with degenerative ova cells and sperm threads which, was observed obviously when the snails were incubated with 1 (T ⁴ M/L copper Chlorophyllin for 12 hours and exposed to sun light.

Figure 7 show test sample (sectioned from the hermaphrodite system) incubated withl O ^"5 M/L copper Chlorophyllin (Lc50) for 12 hours and exposed to sun light. It reveals a degenerative effect of ova cells and sperm threads for snails incubated with 10 ^"5 M/L copper Chlorophyllin for 12 hours and exposed to sun light. However, this degenerative effect observed was less than that observed when the snails were incubated with 10 ^"4 M/L at the same conditions.

Figure 8 reveals a dark control sample where, sections were taken from the hermaphrodite system of snails that were incubated with 10 ^"5 M/L magnesium Chlorophyllin without being exposed to light irradiance so, as the section reveals no cellular destruction was observed.

Figure 9 show test sample (sectioned from the hermaphrodite system) incubated with 10 ^"4 M/L magnesium Chlorophyllin (Lc90) for 3 hours and exposed to sun light. Degeneration of ova cells is observed when compared with the control samples as it implies hollow centers in the hermaphrodite system with degenerative ova cells and sperm threads which, was observed obviously when the snails were incubated with 1(T M/L magnesium Chlorophyllin for 3 hours and exposed to sun light.

Figure 10 show test sample (sectioned from the hermaphrodite system) incubated withl O ^"5 M/L magnesium Chlorophyllin (Lc50) for 3 hours and exposed to sun light. It reveals a degenerative effect of ova cells and sperm threads for snails incubated with 10 ^"5 M/L magnesium Chlorophyllin for 3hours and exposed to sun light. However, this degenerative effect observed was less than that observed when the snails were incubated with 10 ^"4 M/L at the same conditions.

Figure 11 show the hormonal key markers for silver nanoparticles

Figure 12 hormonal key markers for copper Chlorophyllin

Figure 13 hormonal key markers for magnesium Chlorophyllin Table 1/4

The effect of SNPs on the fecundity of Biomphilaria alexandrina snails

Lx= Survivorship, Mx= Mean number of eggs / snail /week, Ro = net reproductive rate = Sum of LxMx

Mx at the start of the experiment was 1.41 eggs

Table 1- Survival rate (Lx) and fecundity (Mx) of B. alexandrina snails incubated with sub-lethal concentrations of SNPs (24 hrs) and exposed to light (9hrs).

(Lx= Survivorship, Mx= Mean number of eggs / snail /week, R ₀ = net reproductive rate = Sum of LxMx. The total number of eggs laid for the 1000 snails for whole experiment= 1406 eggs (1406egggs/1000 snails = 1.406) Table 2/4

The effect of copper Chlorophyllin on the fecundity of Biomphilaria alexandrina snails

Lx^ Survivorship, Mx= Mean number of eggs / snail /week, R ₀ = net reproductive rate = Sum of LxMx

Mx at the start of the experiment was 1.41 eggs

Table 2 - Survival rate (Lx) and fecundity (Mx) of B. alexandrina snails incubated with sub-lethal concentrations of Cu-chl (12 hrs) and exposed to light (9hrs).

(Lx= Survivorship, Mx= Mean number of eggs / snail /week, Ro = net reproductive rate = Sum of LxMx. The total number of eggs laid for the 1000 snails for whole experiment= 1406 eggs (1406egggs/1000 snails = 1 .406) Table 3/4

The effect of magnesium Chlorophyllin on the fecundity of Biomphilaria alexandrina snails

Lx= Survivorship, Mx= Mean number of eggs / snail /week, R ₀ = net reproductive rate = Sum of LxMx

Mx at the start of the experiment was 1.41 eggs

Table 3 - Survival rate (Lx) and fecundity (Mx) of B alexandrina snails incubated with sub-lethal concentrations of Mg-chl (3 hrs) and exposed to light (9hrs).

(Lx= Survivorship, Mx= Mean number of eggs / snail /week, o = net reproductive rate = Sum of LxMx. The total number of eggs laid for the 1000 snails for whole experiment^ 1406 eggs ( 1406egggs/l 000 snails = 1 .406) Table 4/4 biochemical markers for the examined chromophores References:

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