Laboratory Investigation: Assessment of Silver Nanoparticles on Freshwater
Ecosystems.
Curricular Links: Biology 20
20–B1.2k explain how terrestrial and aquatic ecosystems support a diversity of
organisms through a variety of habitats and niches; e.g.,
• aquatic: littoral, limnetic, profundal and benthic zones30–C3.2sts explain that
scientific research and technological development help achieve a sustainable society,
economy and environment (SEC4a) [ICT F2–4.2, F2–4.8]
20–B1.3k identify biotic and abiotic characteristics and explain their influence in an
aquatic and a terrestrial ecosystem in the local region; e.g., stream, or lake.
Initiating and Planning
Students will:
20–A1.1s formulate questions about observed relationships and plan investigations
of questions, ideas, problems and issues • propose a relationship between
producers and available energy of a system (IP–SEC1)
Performing and Recording
Students will:
20–A1.2s conduct investigations into relationships among observable variables and
use a broad range of tools and techniques to gather and record data and information
• perform an experiment to demonstrate solar energy storage by plants (PR–NS3,
PR–NS4, PR–NS5)
Performing and Recording
Students will:
20–A2.2s conduct investigations into relationships between and among observable
variables and use a broad range of tools and techniques to gather and record data
and information
• measure and record the pH and the amount of nitrates, phosphates, iron or sulfites
in water samples (PR–NS2, PR–NS3, PR–NS4) [ICT P2–4.1].
Analyzing and Interpreting
Students will:
20–A3.3s analyze data and apply mathematical and conceptual models to develop
and assess possible solutions
• design and evaluate a model of a closed biological system in equilibrium with
respect to carbon dioxide, water and oxygen exchange (PR–ST2, AI–ST1)
Class time: Minimum of 3 classes.
Allow at least one class for:
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Research
Experimental design
Experimentation
Teacher Background information
Researchers in Switzerland have examined what happens to the silver nanoparticletreated textiles after washing. The scientists studied release of nanoparticles in
laundry water from nine different textiles, including different brands of
commercially available anti-odor socks. The nanoparticle release varied during
washing from less than 1 to 45 percent of the total nanosilver in the fabric and that
most came out during the first wash.
See the study by Bernard Nowak in the reference section below.
Read more:
http://www.nanowerk.com/spotlight/spotid=13362.php#ixzz2j4VEGIO7
Read more:
http://www.nanowerk.com/spotlight/spotid=13362.php#ixzz2j4UpEqCk
The study shows that the release of the nanoparticles depends on the way the silver
is incorporated into the textile fiber. Nowack (2009) explains that his team's results
demonstrate that little dissolution of silver nanoparticles occurs under conditions
relevant to washing (pH 10) with dissolved concentrations 10 times lower than at
pH 7. However, bleaching agents such as hydrogen peroxide can greatly accelerate
the dissolution of silver.
Purpose
You will design a study to assess the impact of silver nanoparticles on aquatics
ecosystems. Ideally, your study will examine the impact of the silver nanoparticles
on both plants and a representative aquatic invertebrate. (You cannot conduct your
study on fish or other vertebrates).
Hypothesis
State a hypothesis for your investigation.
Sample: If silver nanoparticles find their way into aquatic ecosystem, then
nanoparticles will have an adverse effect on food chains.
Prediction
Make a prediction about how silver nanoparticles will affect Elodea and
Daphnia, before beginning the project.
Sample: Silver nanoparticles decrease the growth rate of elodea and decrease
the rate of photosynthesis by interfering with biochemical processes. Silver
nanoparticles cause the death of Daphnia. As the concentration of silver
nanoparticles increases, so does the mortality rate of Daphnia.
1.
Present a bibliography of the articles that you have read prior to
beginning the project.
Sample below:
Taken from Learn Alberta Reference library (Science in Context)
Science in Context:
Czyzewski, By Andrew. "Silver surfer." The Engineer 20 Feb. 2012: 31. Science In
Context. Web. 28 Oct. 2013.
http://ic.galegroup.com/ic/scic/MagazinesDetailsPage/MagazinesDetailsWindo
w?failOverType=&query=&prodId=SCIC&windowstate=normal&contentModule
s=&mode=view&displayGroupName=Magazines&limiter=&currPage=&disableH
ighlighting=false&displayGroups=&sortBy=&source=&search_within_results=&a
ction=e&catId=&activityType=&scanId=&documentId=GALE%7CA280930394&
userGroupName=albertak12&jsid=46a3aa2350fcf03f4dd384c73985b53a
summary: Nanoparticles aid the clean-up of mercury in water A process for creating
extremely small silver nanoparticles could usher in more effective, cheaper ways of
cleaning mercury-contaminated water.
Allen, Terry J. "Nanotech: teeny tiny particles, big risks: nanotechnology, one of the
fastest growing industries today, may be a major threat to human health and the
environment. Or not. The fact is, when it comes to nanotech, we don't have any idea
what the facts are." Mother Earth News June-July 2008: 24. Science In Context. Web.
28 Oct. 2013.
Document URL
http://ic.galegroup.com/ic/scic/MagazinesDetailsPage/MagazinesDetailsWindow?f
ailOverType=&query=&prodId=SCIC&windowstate=normal&contentModules=&mo
de=view&displayGroupName=Magazines&limiter=&currPage=&disableHighlighting
=false&displayGroups=&sortBy=&source=&search_within_results=&action=e&catId
=&activityType=&scanId=&documentId=GALE%7CA179815449&userGroupName=
albertak12&jsid=592cefdaf13a8e589ce31e3c6bb72901
summary: manufacturers are rushing to add tiny, inadequately tested particles, 1
nanometer to 100 nanometers in size, to a wide, growing array of consumer
products. A nanometer is one-billionth of a meter, or about the length a fingernail
grows per second. A new Samsung washing machine that disperses 100 quadrillion
antibacterial nanosilver particles per load illustrates the regulatory morass. Under
the latest EPA shift, products like the washing machine don't need to prove they are
environmentally safe as long as they don't claim to kill germs.
Fecht, Sarah. "Killer paper." Popular Mechanics Apr. 2011: 15. Science In Context.
Web. 28 Oct. 2013.
Document URL
http://ic.galegroup.com/ic/scic/MagazinesDetailsPage/MagazinesDetailsWindo
w?failOverType=&query=&prodId=SCIC&windowstate=normal&contentModule
s=&mode=view&displayGroupName=Magazines&limiter=&currPage=&disableH
ighlighting=false&displayGroups=&sortBy=&source=&search_within_results=&a
ction=e&catId=&activityType=&scanId=&documentId=GALE%7CA252741233&
userGroupName=albertak12&jsid=d8b3955818dcdde4f67ea2d381392de0
summary: Russian and Israeli scientists have invented a new potential weapon
in the fight against food poisoning--a paper wrapper that can kill bacteria.
Lu, Cathy. "Dirtproof clothing." National Geographic Kids Apr. 2008: 7. Science In
Context. Web. 28 Oct. 2013.
Document URL
http://ic.galegroup.com/ic/scic/MagazinesDetailsPage/MagazinesDetailsWindo
w?failOverType=&query=&prodId=SCIC&windowstate=normal&contentModule
s=&mode=view&displayGroupName=Magazines&limiter=&currPage=&disableH
ighlighting=false&displayGroups=&sortBy=&source=&search_within_results=&a
ction=e&catId=&activityType=&scanId=&documentId=GALE%7CA178348328&
userGroupName=albertak12&jsid=dc094a0766ba4222b1ce22d9507e798d
summary: Go ahead, sneeze into your sleeve or splash in that mud puddle. A
team at Cornell University developed the ultimate garment: one that fights
germs and never needs to be washed.
Source Citation
"Antimicrobial, anti-odor fabric." R & D Feb. 2008: 36. Science In Context. Web. 28
Oct. 2013.
Document URL
http://ic.galegroup.com/ic/scic/MagazinesDetailsPage/MagazinesDetailsWindo
w?failOverType=&query=&prodId=SCIC&windowstate=normal&contentModule
s=&mode=view&displayGroupName=Magazines&limiter=&currPage=&disableH
ighlighting=false&displayGroups=&sortBy=&source=&search_within_results=&a
ction=e&catId=&activityType=&scanId=&documentId=GALE%7CA175630506&
userGroupName=albertak12&jsid=e35324ee20d9a74ae4996d93f8a6c384
summary: In the battle against stinky bacteria, one needs a smart and powerful ally.
As a person sweats, odor-causing microbes thrive in damp fibers, Sweat makes
SmartSilver nanoparticles generate millions of silver ions
Full Text: COPYRIGHT 2005 Centaur Communications Limited.
http://www.centaur.co.uk/
Source Citation
"Ioning the dirty laundry." The Engineer 28 Nov. 2005: 7. Science In Context. Web. 28
Oct. 2013.
Document URL
http://ic.galegroup.com/ic/scic/MagazinesDetailsPage/MagazinesDetailsWindo
w?failOverType=&query=&prodId=SCIC&windowstate=normal&contentModule
s=&mode=view&displayGroupName=Magazines&limiter=&currPage=&disableH
ighlighting=false&displayGroups=&sortBy=&source=&search_within_results=&a
ction=e&catId=&activityType=&scanId=&documentId=GALE%7CA139237699&
userGroupName=albertak12&jsid=ebf8ab09e2feed6cbf1b55eb961035e8
summary: A waterless washing machine that uses negative ions, compressed air and
deodorants to clean clothes has won the 2005 Electrolux Design Lab Award.
Source Citation
Pierce, Julia. "Going for gold: creation of low-cost nanoparticles for food packaging
could prolong shelf life." The Engineer 8 Oct. 2004: 16. Science In Context. Web. 28
Oct. 2013.
Document URL
http://ic.galegroup.com/ic/scic/MagazinesDetailsPage/MagazinesDetailsWindo
w?failOverType=&query=&prodId=SCIC&windowstate=normal&contentModule
s=&mode=view&displayGroupName=Magazines&limiter=&currPage=&disableH
ighlighting=false&displayGroups=&sortBy=&source=&search_within_results=&a
ction=e&catId=&activityType=&scanId=&documentId=GALE%7CA123927285&
userGroupName=albertak12&jsid=9a76e57cb20a814e9205276748b44556
summary: PLASTIC STORAGE containers and food packaging could soon be
manufactured with built-in anti-microbial properties, following the creation of
low-cost, polymer-compatible gold and silver nanoparticles.
"Nanoprobe will find explosives and drugs." Machine Design 2 Sept. 2004: 44.
Science In Context. Web. 28 Oct. 2013.
Document URL
http://ic.galegroup.com/ic/scic/MagazinesDetailsPage/MagazinesDetailsWindo
w?failOverType=&query=&prodId=SCIC&windowstate=normal&contentModule
s=&mode=view&displayGroupName=Magazines&limiter=&currPage=&disableH
ighlighting=false&displayGroups=&sortBy=&source=&search_within_results=&a
ction=e&catId=&activityType=&scanId=&documentId=GALE%7CA122262166&
userGroupName=albertak12&jsid=b1e8092856db2f21037fb13224d8e0ec
Summary: Oak Ridge National Laboratory (ORNL) researchers have developed a
tiny probe which could make it easier to detect and analyze illicit chemicals,
explosives, and drugs in minute amounts.
Design
Some classes may assign groups to do either the impact of silver nanoparticles on
aquatic plant or aquatic animal communities.
2.
Identify your manipulated and responding variables.
Elodea: Manipulated variable (concentration of silver nanoparticles
added or exposure to silver particles.)
Elodea: Responding variables – the amount of oxygen produced –
represents the rate of photosynthesis and/or changes on mass or
length to measure growth rate of elodea.
If a photometer is used, the amount of chlorophyll can be measured.
Daphnia: Manipulated variable (concentration of silver
nanoparticles added)
If growth rate of elodea is measured as a responding variable, the
manipulated variable is exposure to silver.
Daphnia: Responding variable – LD 50. Manipulated variable
(concentration of silver nanoparticles added or exposure to silver
particles.)
3.
Identify controls for your experiments.
 Daphnia or elodea placed in a test tube without silver
nanoparticles, but in the same carrier solvent used in
dissolving nanoparticles.
 Exposure time to potential toxic chemical (silver), should
multiple trials be run.

4.
5.
Light, water temperature, water pH, etc must also be
controlled.
Identify the concentrations of solutions used. Why did you choose these
concentrations?
Students should choose something less that a 0.01 mol/L solution
because even with the washing of a great many clothes, silver
nanoparticle concentrations would be very low.
Identify the duration of exposure for both plants and animals. Why did
you choose the duration of exposure?
We recommend 48 –h for Daphnia and 72 h for elodea. The standard
bioassays of Daphina in labs is 48-h. Plants take longer to show
effects of the silver.

Present your study for approval prior to beginning any experimental
work.
Materials
We recommend using small test tube or beakers for the study. Students should use
at least three different dilutions.
Evidence
Present your data using a table format. Some groups may use spreadsheets for data
tables. Make sure you list the concentration of the silver solution and the duration of
exposure for the test plant or animal.
Elodea
Expect little impact of silver particles on elodea after 72 –h when compared
with the control. The actual data is dependent upon the size of the elodea
sprig, the strength of the light source, temperature of water etc. In studies that
we conducted we did not find a significant variation in the rate of
photosynthesis between the sample containing silver particles and the control.
Time (min)
5
10
15
Daphnia
Amount of gas (oxygen) collected – mL of oxygen
Control (no silver
Experimental (silver
nanoparticles)
nanoparticles)
0.5
0.5
1.1
1
1.6
1.7
The effects on Daphnia are dependent upon the type of silver used. When
using AgNO3 (aq), we found a great variation in LD50 depending upon water
temperature. Most LD 50 studies recommend using a 48-h exposure for
Daphnia.
Sample data for Daphnia LD50
Concentration Initial number
ppm
of Daphnia
1000
500
100
50
10
10
10
10
Total number of dead Daphnia (hours)
24
5
3
2
1
48
8
6
3
2
72
10
8
6
4
Analysis
6.
Why must a control be used in the experiment?
Some other contaminant in the water other than silver could kill
Daphnia or have a negative effect on the growth of elodea or the rate
of photosynthesis.
7.
Why is Daphnia an excellent animal to model toxicity. (Give 3 reasons)
Daphnia is commonly found in both ponds and lakes.
Daphnia are principal grazers of algae, bacteria, protozoa, and the
primary forage of fish. Because of their pivotal position in food
webs, they are widely utilized as an indicator species to assess the
response of ecosystems to environmental change.
The reproductive cycle of Daphnia is ideal for experimental
genetics. Generation time in the lab ranges from 5 - 10 days, making
it possible to examine genome regulatory changes throughout its
development.
Daphnia are reasonably complex invertebrates with a nervous
system, circulatory system and digestive system. The toxic material
can be studied at a systems level.
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8.
Identify two possible sources of error in measuring the growth of a sprig
of elodea.
 Water remains attached to the sprig of elodea. Some of the
large droplets need to be removed.
 Measurement could be taken for a given leaf or the entire
sprig.

In addition, some groups may even consider measuring
changes in mass to represent growth.
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Evaluation
9.
If a student finds the LD 50 for a 1.0 mol/L silver nitrate after 48-h for
Daphnia in the laboratory, can you conclude that silver nanoparticles are
harmful for Daphnia? Explain why or why not.
Death of 50% of the population may have been caused because the
silver nanoparticles killed the microorganisms that provided food
for the Daphnia. Scientists have already shown that various
microbes are destroyed by the silver nanoparticles. It is possible
that Daphnia had a reduced food supply.
10.
On the basis of your project, would you want to ban the use of silver
nanoparticles in clothing? Explain why or why not.
The evidence does not provide conclusive proof. For most
experiments. The concentration of silver nanoparticles used in the
experiment would be greater than what would be collected in a lake
or river. We found that any test with elodea was inconclusive.
However, the LD 50 test of Daphnia, assuming a difference between
control and experimental groups, might suggest further studies.
Students will begin to understand that a single set of results is not
conclusive and gain a better understanding of why testing take a
very long time. Notable results for Daphnia can not be extended to
all aquatic invertebrates. Daphnia may not have been affected
directly by silver as a toxin, but rather the food source of Daphnia
might have been depleted by the silver.
Synthesis
11.
12.
13.
What additional studies would you want to perform to draw any
conclusion?
A few examples are provided.
 Multiple concentrations of silver nanoparticles.
 Work on other representative organisms as indicator species.
 Use water samples of differing temperature and pH.
 Increase the length of time of the study. Short term versus
long term effects.
What things would you change if you were to repeat your study?
 Students might measure the number of bubbles for elodea,
rather than working with volume.
 Students might suggest a better way to count Daphnia.

Students might use other indicator end point– for example
they might measure heart rate of Daphnia.
Teacher Resources
†
‡
L. Geranio , M. Heuberger and B. Nowack * The Behavior of Silver
Nanotextiles during Washing, Environ. Sci. Technol., 2009, 43 (21), pp 8113–8118
Technology and Society Laboratory, and Laboratory of Advanced Fibers, Empa Swiss Federal Laboratories for Materials Testing and Research, CH 9014 St. Gallen,
Switzerland’
http://pubs.acs.org/doi/abs/10.1021/es9018332
Enrique Navarro, Toxicity of Silver Nanoparticles to Chlamydomonas
reinhardtii, Environ. Sci. Technol., 2008, 42 (23), pp 8959–8964
DOI: 10.1021/es801785m
Publication Date (Web): October 1, 2008
Copyright © 2008 American Chemical Society
http://pubs.acs.org/doi/abs/10.1021/es801785m
Readings for Students
1. Nanoparticles.org
http://www.nanoparticles.org
Silver as an Antimicrobial Agent
http://microbewiki.kenyon.edu/index.php/Silver_as_an_Antimicrobial_Agen
t#Current_uses
General information:
http://www.nano.gov
http://www.education.nnin.org