REFERENCE TO COPENDING APPLICATION

The present application is related to and claims benefit of priority to U.S. Provisional

Patent Application 61/484,327; filed May 10, 201 1 entitled "USE OF ALGAE IN RECOVERY", the entire subject matter of whichjs incorporated herein by reference.

FIELD OF THE DISCLOUSRE

The present disclosure relates to use of algae, for example, Euglena in recovery and isolation of nanosilver, other nano value metals, nano particles and other materials as described hereinbelow, capable of being absorbed or otherwise consumed thereby.

BACKGROUND

Nanosilver is a compound used in commercial and industrial processes to inhibit the growth of bacteria and fungi in such diverse applications as acne treatments, dressings, feminine hygiene products, babies' bottles, socks, undergarments, food preparation equipment, air-filtration devices, surface disinfection, and food containers. The poultry, livestock, and aquatics industries use it to prevent disease and enhance weight gain. Catheters and dialysis devices with nanosilver coatings are now available, and it is anticipated that artificial joints, pacemakers, and artificial heart valves will soon be manufactured using these coatings as well. Consequently, humans may be exposed to high levels of nanosilver due to its widespread use but its potentially detrimental effects on living organisms make its use controversial.

Negative results have been shown regarding the environmental effects of elemental and ionic silver and government regulations, in response, have established limits for silver in water. However, the study of nanosilver is relatively new and there is limited data regarding toxicity from exposure to nanosilver and in determining its short- term and long-term effects on the environment. As well, extensive background research indicates that there are no documented methods for separating nanosilver from other forms of silver in solution in order to establish levels of nanosilver in freshwater samples. This is necessary for establishing current and future levels of nanosilver in freshwater samples.

A major concern regarding the use of nanosilver is its permanence in water systems. Nanosilver particles are encapsulated in gluconic acid, do not break down, and may be released into the water system in wastewater effluent. These particles range between 1 and 100 nm in diameter, which may allow them to pass through the cell membrane of living organisms. If nanosilver is harmful to living organisms, then plants and animals in aquatic ecosystems are at risk. If nanosilver does not break down, there must be an accumulation of its levels in the environment and there are serious implications as to whether wastewater treatment facilities are capable of removing nanosilver from drinking water. While some initial studies have maintained that treatment facilities are, indeed, capable of removing the necessary silver from effluent water, more recent studies indicate that this may not be accurate. If treatment centres are unable to ensure the absence of nanosilver in effluent water, there is a serious health risk, as nanosilver particles represent a source of silver ions, which are reported to be toxic. As well, there is concern over the nanosilver levels in biosolids exported from wastewater treatment facilities for use as fertilizer on agricultural lands. There is evidence that nanosilver has a toxic effect on the denitrification process that is essential in the breakdown of organic matter to form soil. In fact, one study showed nanosilver to be even more toxic to denitrifying bacteria than ionic silver.

Algae are often used as indicators to evaluate the existence of water pollution in bodies of water. Euglena is a single-celled organism that is a primary producer in freshwater ecosystems. This form of algae has both plant and animal characteristics and is, therefore, representative of most organisms in the water system.

SUMMARY OF THE DISCLOSURE

The following presents a simplified summary of the general inventive concept herein to provide a basic understanding of some aspects of the disclosure. This summary is not an extensive overview of the disclosure. It is not intended to restrict key or critical elements of the disclosure or to delineate the scope of the disclosure beyond that explicitly or implicitly described by the following description and claims.

In an exemplary embodiment, the Euglena is operable as an indicator of the impact of this chemical on living organisms in freshwater.

The present disclosure provides a method of recovering pollutants from a water sample, comprising adding to the sample a quantity of algae, isolating the algae from the sample and determining a level of pollution in the sample according to an identified amount of the pollutant absorbed by the algae.

In exemplary embodiments, the algae includes Euglena.

In exemplary embodiments, the pollutant includes metals. For example, the metals may include value metals, such as compounded silver.

In exemplary embodiments, the pollutants include capped nanoparticles.

In an exemplary embodiment, there is provided a filter element comprising a substrate carrying Euglena, in sufficient quantity to absorb pollutants from freshwater passing therethrough.

In an exemplary embodiment, there is provided a method of filtering fresh water, comprising the step of providing the filter element of claim 7, passing a sample of freshwater therethrough, isolating the Euglena from the substrate and recovering pollutants from the Euglena. In an exemplary embodiment, there is provided a method for remediation of a sample of fresh water, comprising depositing in the sample a sufficient amount of Euglena to absorb waterborne pollutant materials capable of passing the membrane of Euglena and removing the Euglena from the sample.

In exemplary embodiments, the method further comprises recovering the pollutant materials from the Euglena.

In exemplary embodiments, the pollutant includes capped nano metals or nano non- metals.

In exemplary embodiments, the capped nano metals includes gold, silver, platinum, titanium and lead.

In an exemplary embodiment, there is provided a method of sequestering a constituent from a water sample, comprising adding to the sample a quantity of Euglena so as to sequester the constituent therein.

In exemplary embodiments, the constituent is a transition metal component.

In an exemplary embodiment, there is provided a method of indicating water quality comprising providing a non-polluted water sample, providing a first quantity of Euglena, adding the first quantity to the non-polluted water sample, providing a polluted water sample, providing a second quantity of Euglena, adding the second quantity of Euglena to the polluted water sample, culturing the first quantity of Euglena and the second quantity of Euglena for a given time period, and comparing the growth or an attribute of the first quantity of Euglena to the second quantity of euglena, so as to determine in the respective growth rates. In exemplary embodiments, the attribute is the shape.

In exemplary embodiments, the Euglena forms a ball shaped configuration in the polluted water sample.

In exemplary embodiments, the second quantity forms a ball shaped configuration in the polluted water sample, while the first quantity adopts an oval shaped configuration in the non-polluted sample.

In an exemplary embodiment, there is provided a method of indicating impact of a contaminant on organisms in a water sample comprising providing an uncontaminated water sample, providing a first quantity of Euglena, adding the first quantity of Euglena to the uncontaminated water sample, providing a contaminated water sample, providing a second quantity of Euglena, adding the second quantity of Euglena to the contaminated water sample, culturing the first quantity of Euglena and the second quantity of Euglena for a given time period, and comparing differences in population or attribute of the first quantity of Euglena to the second quantity of euglena, so as to determine respective growth rates thereof.

In an exemplary embodiment, there is provided a method of recovering a constituent from a water sample, comprising providing a water sample with the constituent, adding a quantity of Euglena to the water sample, and after a given time period, recovering the Euglena from the water sample, and isolating the constituent from the Euglena.

In exemplary embodiments, the isolating includes isolating the constituent from a vacual cell structure of the Euglena.

In exemplary embodiments, the constituent is a quantity of capped nanoparticles. In an exemplary embodiment, there is provided a method for isolating capped silver nanoparticles from a silver solution containing a capped silver nanoparticle constituent and one or more of an elemental silver constituent and an ionic silver constituent in the silver solution, comprising filtering substantially all of the elemental silver constituent from the silver solution, and/or adding to the silver solution an ionic constituent in sufficient concentration to react with the ionic silver constituent to remove substantially all of the ionic silver constituent from solution.

In exemplary embodiments, the ionic constituent includes bromide, carbonate, chloride, oxide, phosphate, sulphate and/or dichromate.

BRIEF DESCRIPTION OF THE DRAWINGS Figure A is a schematic representation of a filter; and

Figures 1 to 16 are representations of certain observations related to the exemplary embodiments.

DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

It should be understood that the invention is not limited in its application to the details of construction and the arrangement of the step set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including,"

"comprising," or "having" and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. There are limited studies demonstrating specific effects of nanosilver on living organisms. However, studies which have been done with ionic silver indicate that ingestion of elemental and silver compounds by rats results in the bioaccumulation of silver in the brain tissue of offspring and that ionic silver prohibits the growth and differentiation of nerve tissue. Chronic exposure to elemental and ionic silver is known to cause permanent damage to the subcutaneous and mucous membranes which can lead to discolouration of the skin. It has also been known to cause irritation of the eyes, skin, respiratory, and intestinal tracts, as well as blood cell changes and damage to the liver and kidneys. While some serious consequences of exposure to silver have been documented, the effects of nanosilver might be especially great, since nanosilver particles provide a greater relative surface area, allowing for maximal exposure of silver within the body. This means that greater absorption of nanosilver could occur at lower

concentrations than that of other forms of silver. Environmentally, this physical property of high surface area to volume ratio has raised concerns that nanoparticles themselves may combine with pollutants and amplify their effects. It has been hypothesized that nanosilver can diffuse through the cell membrane and release silver ions directly inside the cell; that nanoparticles can be absorbed through the skin, eyes, and nose; and that they can cross the blood-brain barrier. In terms of human health consequences, it has been hypothesized that nanosilver could cause drug to drug interactions within the liver.

Concern has even been suggested that overuse of nanosilver for its anti-microbial properties could result in increased antibiotic resistance. Given the results of these early studies, there is a strong basis for further study into the effects of nanosilver on living organisms.

The annual world investment in nanosilver technology has gone from billions of dollars to an estimated trillion in the last five years. In Europe, millions of tons of nanosilver are produced yearly and it has been estimated that, in Europe, up to 15% of the total silver added to the environment each year may come from textiles and biocidal plastics treated with nanosilver. It has been estimated that over 400 tons of silver are released into wash water in Europe alone annually from nanosilver embedded fabrics such as socks. In a world hungry for antimicrobial agents in the face of superbugs and the threat of pandemics, the demand for nanosilver is increasing dramatically. Every year, new applications are developed for nanosilver. However, since it has not been possible to establish accurate levels of nanosilver in freshwater environments, nanosilver levels in our freshwater ecosystems are not being monitored currently and may already be approaching a lethal threshold which could challenge the sustainability of these ecosystems. Water resources and ecosystem management concerns would dictate that further study needs to be done on this source of water pollution. In addition, the possible effect of nanosilver on water quality suggests that there could be potential for negative repercussions related to the health of all living organisms.

Several problems were investigated in this project including:

1. Does nanosilver affect the structure and function of Euglena?

2. Can a method be developed to determine nanosilver concentration in

freshwater samples?

3. Is nanosilver present as an environmental contaminant in Canada's Trent Severn Waterway near Peterborough, Ontario?

4. Does the current water filtration system in Peterborough, Ontario, Canada effectively remove nanosilver from water?

5. Does Euglena absorb nanosilver?

The following hypotheses guided this study:

1. Nanosilver will be shown to negatively affect structure and function of

Euglena.

2. A method of determining the amount of nanosilver in freshwater samples will be developed.

3. Canada's Trent Severn Waterway near Peterborough, Ontario will contain nanosilver as an environmental contaminant.

4. The current water filtration system in Peterborough, Ontario will be shown to be ineffective at removing nanosilver from water.

5. Euglena will absorb nanosilver. 1. Materials and Methods:

EXPERIMENT #1 : DETERMINATION OF THE LC50 FOR EUGLENA CULTURED IN NANOSILVER

A concentrated Euglena culture was grown in algae optimum growth media, and nanosilver solutions of: 0, 5, 10, 40, 75, and 100 μg/L concentrations were added.

Cultures were incubated for 48 hours in a controlled growth chamber at 20 C, in an 18- hour light cycle on a shaker table to ensure agitation. Using a fluorometer and microscope, chlorophyll concentrations and cell counts were determined in test samples and compared to a control group to determine the lethal concentration causing 50% mortality in the sample population (LC50).

EXPERIMENT #2: SHORT-TERM AND LONG-TERM EFFECTS OF NANOSILVER ON EUGLENA

Euglena cultures were grown in 10μg/L and 50 μg/L nanosilver solutions. Chlorophyll concentration and cell counts were determined at 0, 1 , 2, 3.5, 5.5, 24, 48, 72, and 96 hours.

EXPERIMENT #3: VARIABLE CONCENTRATIONS OF EUGLENA AND

NANOSILVER

A dilution series of Euglena cultures (0.25, 0.5, 1.0, 3.75, and lOmL of the stock culture) was grown in nanosilver solutions of: 0, 1 , and 10μg/L nanosilver. Chlorophyll concentration and cell counts were then determined.

EXPERIMENT #4: DETERMINATION OF NANOSILVER ABSORPTION BY

EUGLENA

For each experiment #1, 2, and 3, Euglena cultures were grown for 48 hours and 100 mL of each culture, (control and test samples) was filtered. The total silver content on the filter and in the filtrate was determined using inductively coupled plasma mass spectrometry.

EXPERIMENT #5: EXAMINATION OF EUGLENA CELLS USING A COMPOUND MICROSCOPE

Euglena cells grown in nanosilver were examined under a compound microscope for changes in morphology, mobility, and behaviour. EXPERIMENT #6: EXAMINATION OF EUGLENA CELLS USING AN

ELECTRON MICROSCOPE

Euglena samples, grown in nanosilver solutions and solutions lacking nanosilver, were preserved and prepared, as described below, for microscopic examination.

1. 7.5mL of 2.5% gluteraldehyde (pH of 7.2) were added to 15 mL of each Euglena culture, (Euglena grown in nanosilver solutions and Euglena grown in solutions lacking nanosilver). After two hours, these samples of Euglena were centrifuged for ten minutes at 3000rpm.

2. A pipette was used to remove gluteraldehyde from centrifuged Euglena pellets.

5mL of sodium phosphate buffer was then added to each Euglena pellet. After twenty minutes, sample of Euglena were centrifuged for ten minutes at 3000rpm and the gluteraldehyde was removed from each sample. 5mL of sodium phosphate buffer was then added to each pellet. After 20 minutes, each mixture of Euglena and sodium phosphate buffer was centrifuged for 10 minutes at 3000rpm. This procedure (Euglena with sodium phosphate buffer) was repeated a second time.

3. A 1% tetra osmium tetra oxide was added to each Euglena pellet, allowed to stand for 1 hour in the dark, and then centrifuged for 10 minutes at 3000rpm. This procedure was repeated.

4. In preparing these treated samples of Euglena for microscopic examination, an agar solution was produced and then added to each Euglena pellet. After the agar had solidified, it was cut into blocks. The samples of Euglena in agar were next dehydrated using ethanol ranging from low to high concentration. The dehydrated samples were then put into increasing concentrations of epoxy until the 100% epoxy stage was reached. After being cooked over night to harden the epoxy, the cooled samples were sectioned, using a glass blade, into sections about one micron thick.

5. These sections were stained, placed on a copper grid and then loaded into the electron microscope for examination.

EXPERIMENT #7: SEPARATION OF NANOSILVER FROM OTHER FORMS SILVER IN SOLUTION Elemental silver, ionic silver, and nanosilver, each in known concentration, were added to de-ionized water to create a solution. A glass fibre membrane filter was then used to remove the elemental silver. Nitric acid was added to the remaining solution to form silver nitrate with the ionic silver. Hydrochloric acid was then added to this solution to react with the silver nitrate and form a silver chloride precipitate. This suspension was centrifuged to separate the silver chloride precipitate from solution. The supernatant was then analysed and compared with the original concentration of nanosilver added to the solution.

EXPERIMENT #8: DETERMINATION OF THE LEVEL OF NANOSILVER

CONCENTRATIONS IN WATER SAMPLES

1. Water samples upstream and downstream from Peterborough, Ontario were tested using the method developed in experiment #7 to evaluate nanosilver concentration.

2. Influent and effluent samples were collected at Peterborough's wastewater treatment facility. These samples were tested using the method developed in experiment #7 to measure nanosilver concentration.

3. Effluent water samples at an industrial site upstream from Peterborough, Ontario were collected. These samples were tested using the method outlined in experiment #7 to measure nanosilver levels.

EXPERIMENT #9: REMOVAL OF NANOSILVER FROM WATER SAMPLES BY EUGLENA

Cultures of Euglena were grown in solutions with varying concentration of nanosilver (l μg/L·, lC^g/L, and 50 μg L of nanosilver) and exposure time to nanosilver (48 hours, 72 hours, and 96 hours) in order to study the ability of Euglena to absorb of nanosilver. Each sample was then filtered using glass fibre filter paper. In each case, the silver

concentration in the filtrate and the concentration of silver in the filter were tested and compared with control solutions containing nanosilver alone.

. Results:

1. Nanosilver was shown to be harmful to Euglena with the LC50 for Euglena, grown in the presence of nanosilver, being calculated as 25.0 by determining the decrease in total chlorophyll concentration and then confirmed by cell count (see Figures 1 and 2). In experiments in which Euglena were grown in nanosilver solutions containing 5 g/L, 10 μg L, 4(^g/L, 75μg L, and 100 μg L respectively, an inverse relationship was found between nanosilver concentration and chlorophyll concentration. As nanosilver concentration increased, chlorophyll concentration decreased proportionally (see Figure 1). A similar pattern was found with Euglena cell count. As nanosilver concentration increased, the number of typical, fully formed Euglena cells decreased proportionally (see Figure 2).

2. In examining the short-term and long-term effects of nanosilver on Euglena, it was determined that, after one hour, Euglena cells grown in 10μg/L and 50μg/L solutions of nanosilver showed a significant decrease in typical, fully formed Euglena cells as well as a significant increase in the number of atypical round Euglena cells when compared with control cultures (0 μg/L nanosilver) (see Figures 1 1 and 12). The trend continued until no fully formed Euglena cells were observed. This occurred at 2 hours exposure in a 50μg L nanosilver solution and at 48 hours exposure in a 10μg L nanosilver solution (see Figure 4).

3. A very small decrease in chlorophyll concentration was observed almost immediately (0 and 5.5 hours) in solutions containing 10.0 μg L and 50.0 μg L of nanosilver when compared to the control (0 μg L). However, at 24 hours, there was a statistically significant decrease in chlorophyll in the 50.0 μg/L nanosilver cultures when compared with control cultures. In addition, at 48 hours, there was a further dramatic and statistically significant decline in chlorophyll concentration in both the 10.0 μg/L and 50^g/L nanosilver solutions compared to the control. At 72 hours, a significant recovery of Euglena, as registered by chlorophyll concentration, was found when compared to the control. Finally, at 96 hours, a significant decrease in chlorophyll concentration was again seen. Lastly, it should be noted that between 72 hours and 96 hours an exponential increase was seen with respect to both cell count and chlorophyll concentration in all control cultures (see Figure 3).

4. When a dilution series of Euglena (0.25mL, 0.5mL, lmL, 3.75mL, and lOmL of stock culture) were grown in 1.0 μg/L and 10.0 μg L nanosilver solutions, it was determined that: a) As the concentrations of both Euglena and nanosilver increased, the concentration of chlorophyll decreased proportionally (Figure 5).

b) As the concentration of Euglena culture decreased, the number of typical, fully formed Euglena cells exposed to both l.C^g/L and 10.C^g/L of nanosilver also decreased significantly until there were few or no remaining typically structured Euglena cells (see Figure 6)

c) In passing nanosilver-treated water through a filter, it was determined that no nanosilver was caught in the filter. However, when Euglena was cultured in nanosilver-treated water and then passed through the same type of filter, it was observed that the concentration of nanosilver in the filtered water was dramatically decreased.

d) The difference between nanosilver in the unfiltered water and filtered water increased proportionately with the amount of time the Euglena was allowed to grow before filtration occurred. The most dramatic results were observed after 96 hours (see Figure 7).

e) Although increased time was observed to increase nanosilver absorption, it was also observed that the absorption of nanosilver did begin immediately upon its introduction to a given sample (see Figure 12).

5. When Euglena was grown in nanosilver solutions of increasing concentration (1.0 μg/L, 10.0 g/L, and 50 μg L) and then filtered, it was determined that Euglena had removed: a) all nanosilver present in the 1 μg/L solution at 48 hours; b) all nanosilver present in the 10 μg/L at 72 hours; and c) 99% of the nanosilver present in the 50 μg/L solution at 96 hours (see Figure 7 and 8). Generally, it was found that Euglena were able to remove higher concentrations of nanosilver from solution as time increased (see Figures 8).

6. A combination of filtration techniques and ion exchange reactions described earlier (in experiment #7) were successfully used to isolate nanosilver from solutions containing known concentrations of elemental silver, ionic silver, and nanosilver (see Figure 10). The combination of filtration techniques and ion exchange reactions, which were shown to successfully isolate nanosilver in solutions containing ionic silver and elemental silver, were applied to determine the level of nanosilver contamination present in water samples collected upstream and downstream from Peterborough, Ontario along the TSW (see Table 1). The level of nanosilver contamination upstream from Peterborough was determined to be 0.1 13 μg/L nanosilver. Water samples taken downstream from

Peterborough showed a significant increase in nanosilver testing at 0.488 μg L.

7. Identified filtration techniques and ion exchange reactions were also used to determine nanosilver concentration in influent and effluent water collected from a waste water facility (see Table 1). The nanosilver concentration in influent water was determined to be 7^g/L while the nanosilver concentration in effluent water was determined to be 67.0 μξ/L.

8. Effluent water from an industrial site upstream from Peterborough, Ontario, tested using the same separation techniques, determined that the level of nanosilver

contamination in this water was determined to be 2.0 μg/L.

RESULTS OF ELECTRON MICROSCOPE EXPERIMENT/OBSERVATIONS (see Table 1).

9. Microscopic observation of Euglena cells cultured in nanosilver solutions using a compound microscope indicated that Euglena responded to exposure to nanosilver almost immediately, showing altered structure (see Figure 11). Rounded and lysed cells were seen dominating the field of view in each case of exposure (see Figure 11). The first abnormal type of cell appeared to be a dark green colour with chlorophyll densely packed within the cell (see Figure 12). The lysed cells were clear in colour and had the appearance of a ruptured cell, consisting only of a cell membrane with no organelles inside. In addition, as the concentration of nanosilver increased, the number of atypical, round Euglena cells and lysed cells increased while the number of typical, fully formed Euglena cells decreased proportionally (see Figures 13 and 14).

10. Examining Euglena cells grown in nanosilver with a transmission electron microscope revealed that nanosilver was being actively transported into the cell through the cell membrane and being deposited within the cell (see Figures 12, 15, 16).

Nanosilver particles were found to pass through the cell membrane and were identified in the cytoplasm and the vacuoles of these Euglena cells (see Figure 12). Table 1. Nanosilver Concentration at Different Locations Along the Trent Severn Waterway

4. Discussion:

Overall, the results of this investigation are important because they indicate serious reason for concern about the negative impact on aquatic organisms that could occur with the introduction of nanosilver into our freshwater ecosystems. In addition, our results also suggest that once introduced into a fresh water ecosystem, nanosilver would be damaging to living organisms at the base of many or all food chains in the ecosystem and be amplified through these food chains, thereby potentially threatening the entire fresh water ecosystem. Lastly, our investigation suggests that the negative impact of exposure to nanosilver could extend as far as the human population.

In this project, the LC50 was discovered to be 25μ ί of nanosilver for concentrated solutions of Euglena and 0^g/L of nanosilver for dilute solutions of Euglena. The

concentration of Euglena used as the dilute solution of Euglena was important as it

represents the total concentration of algae in the healthy freshwater ecosystem as reported for the TSW by researchers in the field. These results suggest that even at low

concentrations, nanosilver may have a lethal effect on primary producers in our fresh water ecosystems.

This is of concern considering 500μg of nanosilver is reported to be released in wastewater from socks alone when washed. In addition to socks silver nanoparticles are being found in an increasing numbers of consumer products such as food packaging, odour-resistant textiles, household appliances, and medical devices. Additionally, the total global investment in nano technology was around $10 Billion in 2005 and it is estimated that the annual value for all nano technology-related products will be $1 Trillion by 2014. With nanosilver also being used in the poultry, livestock, and aquatic industries to prevent disease and enhance weight gain in animals, we predict that the level of nanosilver contamination in fresh water ecosystems will continue to increase, creating an increased potential for bioaccumulation of nanosilver in fresh water ecosystems and we maintain that the potential for humans to be exposed to detrimental levels of nanosilver on a daily basis similarly will continue to increase.

A particularly exciting discovery in this investigation is that Euglena cells were found to absorb nanosilver particles immediately and that nanosilver' s toxic effects on Euglena are similarly immediate. Microscopy with living samples confirmed this.

Microscopy also confirmed that the number of typically oval, normal Euglena cells had dramatically decreased after 1 hour with no normally-structured Euglena cells visible at 2 hours. These results were supported in further testing with a fluorometer where a corresponding decrease in chlorophyll concentration and cell count was found at 24 and 48 hours respectively.

Furthermore, nanosilver' s toxic effect was determined to be dose-time dependent. Cultures with higher concentrations of Euglena absorbed proportionally more nanosilver than cultures with lower concentrations of Euglena. In addition, at 96 hours all the nanosilver had been absorbed in a 50μg L nanosilver solution. This result suggests that Euglena has the potential to be a new, previously unrecognized means of removing nanosilver from wastewater. Therefore, we propose that Euglena may serve as a bioremediation measure for nanosilver contamination in freshwater.

Microscopic examination of Euglena cells grown in nanosilver revealed rapid changes in the typical Euglena structure and function following exposure to nanosilver. A loss of motility was observed, along with a change of shape. These non-motile, abnormal cells appeared to be round in shape, compared to the typically ovoid shape of Euglena. Abnormal cells were dark green in colour and packed with condensed chlorophyll. Many lysed cells were also observed appearing colourless, lacking cellular content (protoplasm), and surrounded by a wrinkled and irregular cell membrane.

Thus, in one example, Euglena is an effective indicator species of water pollution at one or more predetermined sites, based on some knowledge of the sites, where the possibility of pollution may exist where Euglena does not grow effectively. Further, Euglena is believed to be useful in detecting, by changes in its growth rate, negative changes in a fresh water ecosystem, particularly to pollutants, such as nanosilver and other constituents that are capable of passing the membrane of the Euglena. In this case, microscopic examination may identify very small concentrations of nanoparticles, such as nonosilver particles.

Examination of Euglena cells with an electron microscope revealed that nanosilver was actively transported into the Euglena cells. This characteristic, which is generally common to animal cells, suggests that nanosilver could potentially be actively transported into all animal cells, including human cells, exposed to nanosilver.

Microscopic examination also provided some important information regarding the mechanism by which the Euglena cell is destroyed by nanosilver. This has important implications for future studies regarding the nature of the toxicity of nanosilver on living organisms.

One of the most significant discoveries of this investigation was the successful development of a method to identify nanosilver concentration in fresh water samples. Previously, it was reported to us by researchers in the field that a method had not been identified for differentiating between the various forms of silver (elemental, ionic and nanosilver) present in fresh water samples. Using filtration techniques and ionic displacement reactions, we were successful in isolating nanosilver in solution. Applying these methods, we were able to determine nanosilver concentration in water samples collected along the TSW near Peterborough, Ontario, Canada. In testing water samples collected upstream and downstream from Peterborough, we were able to show that nanosilver concentration was increased in downstream samples. In addition, we were able to show that sources of contamination exist along the water system between the two sampling sites and that the effect is cumulative in terms of nanosilver contamination. Additionally, our investigation of nanosilver levels in influent and effluent water at Peterborough's water treatment facility was important. We suggest that the level of nanosilver in the influent water was lower than that measured in the effluent water because the raw sewage solids were filtered out of the influent sample to allow testing of the water component. If this is the case, then the effluent water contained nanosilver that had been released from the solids during waste treatment. We determined that there were significant amounts of nanosilver in both influent and effluent water and, in doing so, disproved the claim that water treatment centres are able to successfully remove nanosilver from contaminated water samples.

The findings in this study confirm that serious attention needs to be paid to nanosilver contamination of our fresh water ecosystems.

5. Conclusions:

1. The LC50 for nanosilver and Euglena in vitro was determined to be 25μg/L.

2. An inverse relationship was discovered, for Euglena, between nanosilver

concentration and both chlorophyll concentration and cell count.

3. A more significant reduction in both cell count and chlorophyll concentration was found with longer-term exposure to nanosilver than with shorter-term exposure to nanosilver.

4. Euglena was found to immediately absorb 25% of the nanosilver present in solution.

5. Euglena was found to actively absorb and concentrate nanosilver.

6. After 48 hours of exposure to nanosilver, Euglena absorbed 100% of the

nanosilver present in the 1.0 g/L nanosilver solutions. At 72 hours, Euglena absorbed 100% of the nanosilver particles present in the 10^g/L nanosilver solutions. After 96 hours of exposure to nanosilver, Euglena was found to absorb 99% of the nanosilver present in the 50.( g/L nanosilver solutions.

Microscopy indicated that the number of atypical round, ^' non-motile Euglena cells increased a) as the length of time of exposure to nanosilver increased and b) as the concentration of nanosilver increased.

Electron microscopy confirmed that nanosilver particles were present inside Euglena cells.

A method was determined and successfully used to isolate nanosilver from solutions containing elemental silver, ionic silver, and nanosilver.

It was found that the concentration of nanosilver in water samples increased as you progressed downstream.

Testing of influent and effluent water from Peterborough Ontario's water treatment plant revealed that the level of nanosilver in effluent water was higher (67.0 μg L) than the level of nanosilver present in influent water (7.0 μg L).

When water samples from an industrial effluent site were measured for nanosilver it was found that 2.0 μ L of nanosilver was being added into the river.

Euglena was used to successfully remove nanosilver from contaminated water samples.