ANALYSIS OF UNKNOWN WATER SAMPLES
TEAM #1: CATIE BARTONE & BRENNAN MIDDLETON
NOVEMBER 9, 2015
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TABLE OF CONTENTS​:
APPENDICES​:
APPENDIX A​: SUMMARY/ABSTRACT
APPENDIX B​: INTRODUCTION
APPENDIX C​: EXPERIMENTAL PROTOCOL
APPENDIX D​: RESULTS/DISCUSSION
APPENDIX E​: CONCLUSION/RECOMMENDATIONS
APPENDIX F​: TABLES/FIGURES
APPENDIX G​: REFERENCES CITED
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APPENDIX A: SUMMARY/ABSTRACT
In a series of four laboratory experiments, the analysis of four unknown water samples
was conducted. In these experiments, water quality parameters including pH, conductivity,
turbidity, dissolved oxygen, biochemical oxygen demand, alkalinity, hardness, as well as the
microscopic examination of microorganisms were analyzed. Many different methods were used
to analyze and measure these associated values for each respective unknown water sample. In the
first experiment, all four basic water quality parameters were measured using different meters to
quantify the differing amounts of dissolved ions or molecular oxygen within each sample. In the
second experiment, hardness and alkalinity levels were measured using various different meters
to test different levels of each associated parameter. In the third experiment, a week­long
experiment was performed to test differing levels of biochemical oxygen demand. In the final
experiment, there were many submethods in order to find total solids and total volatile solids for
each respective sample. It was deduced that each unknown water sample originated from various
different natural environments. In the first unknown water sample, it was concluded that this
sample had originated from a natural river or stream. In the second unknown water sample, it
was concluded that this sample also originated from a nearby river or stream. In the third
unknown water sample, it was concluded that this sample originated from a pond or lake
exhibiting high photosynthetic activity. In the fourth unknown water sample, it was deduced that
this sample was in fact taken from drinking water, most likely derived from a groundwater
source. Culminations of all analyses created educated conclusions for the origins of unknown
water samples.
APPENDIX B: INTRODUCTION
In a series of four laboratory experiments, the analysis of four unknown water samples
(UWSs) was conducted by APSE Engineering (CE 254) in Votey Lab 225 from September 8,
2015 to October 6, 2015. In this series of experiments, these four different UWSs, all collected
from water sources within a fifty­mile radius of the University of Vermont’s campus, were
analyzed for a series of different water quality parameters. Resulting concentrations for each
parameter were then compared to accepted values from a variety of different water sources so
educated conclusions could be made as to the origin of each UWS.
In the first laboratory experiment, four basic water quality parameters were tested,
including pH, conductivity, turbidity, and dissolved oxygen. pH, or measurements of
acidity/basicity, are one of the most important and frequently used measurements in water
quality analysis, as well as in water supply and wastewater treatment facilities for processes
including acid­base neutralization, water softening, precipitation, coagulation, disinfection, and
corrosion control (Standard Methods, 4­90). Values of pH can range from 6.5­8.0 in natural
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water systems​—​anything outside of this range can stress the physiological systems of most
organisms (EPA 2012). Conductivity measures of the ability of an aqueous solution to carry an
electric current, directly indicating the presence of inorganic dissolved ions in a sample. It can be
a good indication of the geology of the area through which the water flows, as well as whether a
discharge or some other source of pollution has entered a stream. Conductivity of rivers in the
United States typically range from 50­1500 µmhos/cm ​(micromhos per centimeter), to 10,000
µmhos/cm in industrial waters (EPA 2012). Turbidity is a representation of the clarity of water,
or amount of suspended and colloidal matter in it. It is important in manufacturing products
destined for human consumption and in many other manufacturing processes, and can greatly
affect natural water systems by increasing temperatures and decreasing dissolved oxygen (DO)
concentrations (EPA 2012). Turbidity can range from less than 1 NTU, such as a pristine
mountain stream, to 1000s of NTU in murky natural and industrial waters. DO is a measure of
the gaseous oxygen concentrations present in a water sample, and typically range from 5­15
mg/L with changes in temperature. DO is important for the respiration of aquatic animals,
decomposition, and various chemical reactions that require oxygen (EPA 2012). In addition to
these four general water quality parameters, the microscopic examination of water samples was
also performed. Microorganisms ranging from algae to bacteria can be a good indication of the
sample’s source, as well as the presence of pollutants.
In the second laboratory experiment, alkalinity and hardness levels were tested.
Alkalinity, or the acid­neutralizing capacity of a water sample, is a direct indication of carbonate,
bicarbonate, and hydroxide concentrations in a water sample (although other bases contribute to
alkalinity if present). According to Figure 1 (Appendix F), alkalinity in surface waters can range
from <50 to >400 µeq/L (where 20 µeq/L = 1 mg/L as CaCO3 (Water Quality Terms)).
Alkalinity values are important to the interpretation and control of water and wastewater
treatment processes, and can be in a much higher range of 2000­4000 mg CaCO3/L in an
operating anaerobic digester (Standard Methods, 2­26). Water hardness is understood to be a
measure of the capacity of water to precipitate soap, or more accurately the sum of calcium and
magnesium concentrations expressed as mg CaCO3/L. Hard water requires more soap for home
laundry and washing, and can contribute to scaling in boilers and industrial equipment. Values
from 0­60 mg/L are classified as “soft,” while anything greater than 180 mg/L is seen as “very
hard” (USGS 2013). Patterns of hardness in the US can be seen in Figure 2. Water alkalinity and
hardness are primarily a function of the geology of the area where the water is located. Ions
typically responsible for alkalinity and hardness originate from the dissolution of geological
minerals into rainwater and groundwater. Surface water and groundwater sources are especially
likely to have high hardness and alkalinity due to the dissolution from limestone into
bicarbonates and carbonates, which contribute to total hardness for each UWS.
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In the third laboratory experiment, the biochemical oxygen demand (BOD) for each
water sample was measured. BOD measures the amount of molecular oxygen utilized during a
specified incubation period for the biochemical degradation of organic material (Standard
Methods, 5­2). BOD is an indirect measure of organic matter, thus sources of it include leaves
and woody debris, dead plants and animals, animal manure, wastewater treatment and
food­processing plants, and urban stormwater runoff. Higher concentrations of BOD typically
represent a more polluted sample, with domestic waste values ranging from 150­400 mg/L and
industrial waste from 100­3000 mg/L (Washington State Department of Ecology 1998). Not only
do high BOD values indicate pollution, but they are typically accompanied with low DO which
is necessary for healthy aquatic ecosystems. Lower values such as 1­5 mg/L indicate clean or
unpolluted water.
In the final laboratory experiment, a solids analysis was conducted for each UWS. Solids
refer to matter suspended or dissolved in a water sample, which can have a variety of effects on
water quality. Waters with high dissolved solids typically have inferior palatability, thus 500
mg/L or less is desired for drinking water (Standard Methods, 2­55). Higher concentrations of
suspended solids can even serve as carriers for toxic constituents, thus samples are more likely to
be polluted (EPA 2012). Solids concentrations are also important in the control of wastewater
treatment processes and the assurance of effluent water quality compliance. For this experiment,
total solids (TS), or the sum of total suspended solids (TSS) and total dissolved solids (TDS),
was determined gravimetrically. Volatile solids were also determined, which helps to quantify
organic matter concentrations.
Using these different yet interconnected water quality parameters, important conclusions
can be made about the possible origins and environments in which these water samples were
collected. Experimental procedures were conducted in compliance with ​Standard Methods for
the Examination of Water and Wastewater​, as well as laboratory handouts. Appendix C gives an
in­depth look at the experimental protocol undertaken for each experiment.
APPENDIX C: EXPERIMENTAL PROTOCOL
For the determination of pH in laboratory 1, a Beckman Φ pH/ISE Meter pH meter
consisting of a potentiometer, a glass electrode, a reference electrode, and a
temperature­compensating device was used. Using the pH meter provided, a calibration was
performed. Calibration of the pH meter included using a two­standard operation, using pH 4.0
and pH 10.0 buffers as a standard for all other pH readings and observations. After calibrating
the meter to these two standards, a pH 7.0 buffer was used to verify the calibration performed.
Prior to experimentation, the electrodes were rinsed and dried. Subsequently, the glass bulb at
the end of the electrode was fully immersed in solution, making sure the stir bar did not come
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into contact with the glass bulb. Upon completion of immersion within the solution, the pH
reading was then recorded and analyzed.
In the conductivity measurement procedure, an OMEGA CDH 287 Meter
Resistivity Probe was used for experimentation. Calibration of this meter was performed by
inserting the probe into a solution of known conductivity, and adjusting the measurement to the
known value as a calibration point. Upon calibration with this standard solution, the probe was
then placed in the sample solution for an observed conductivity measurement. Subsequently, the
conductivity on the digital display was read while gently stirring the probe in the solution until a
stabilized value could be recorded.
A Cole Parmer turbidimeter was used for the experimental determination of turbidity. A
calibration was performed using two formazin standards provided, with respective turbidity
readings of 0.5 Nephelometric Turbidity Units (NTU) and 10 NTU. These two formazin
standards were set as known values for instrument calibration. Subsequently, the measurement of
turbidity for each respective water sample was performed. Each sample was poured into a glass
turbidity cell, with liquid level just above the mark on the glassware. Each cell was then gently
tapped to remove any air bubbles suspended within solution. Subsequently, the exterior of each
cell was then wiped carefully to avoid contributions from lint and fingerprints. Each cell was
aligned and inserted into the compartment cell of the turbidimeter, capped, and recorded as
values in NTU.
For DO determination, the Accumet Portable Waterproof Dissolved Oxygen Meter was
utilized. Calibration of this instrument was performed using the 100% saturated method in air, in
which the probe was placed in moist air, and the resulting temperature was read. Subsequently,
the first calibration table was referenced in order to determine the calibration value. The altitude
or atmospheric correction factor was determined using the second calibration table. The product
of the calibration value and the correction factor yielded the value inputted into the calibration.
Upon completion of this calibration procedure, the determination of DO was performed for each
UWS. Here, the probe was placed in each sample making sure not to touch the membrane in
order to avoid contamination of sensing element and the trapping of minute air bubbles under the
membrane. Sufficient flow across the membrane surface was provided in order to overcome
erratic response. The probe was stabilized in order to effectively sample temperature and
dissolved oxygen values in mg/L.
Finally, the microscopic examination of microorganisms was performed using a Leica
Galen III Microscope. “Wet mounts” were prepared by pipetting a few drops of each UWS onto
separate slides and covering with a coverslip. Objective lenses set at three different
magnifications (10x, 20x, and 40x) were used to examine each sample. The microscope was
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adjusted until a desired region was in focus and small details of microorganisms could be
observed. Key types of organisms present in the UWSs were identified in conjunction with
diagrams in the Standard Methods (SM) procedure, “Identification of Aquatic Organisms,” and
text from ​Wastewater Organisms: A Color Atlas​. ​Relative sizes, shapes, and differing mobilities
were noted in order to create accurate and useful depictions of aquatic organisms present.
In the second laboratory experiment, alkalinity was determined using two different
methods—the indicator color change method and the potentiometric titration method. The
sulfuric acid titrant was standardized by 0.05 N sodium carbonate solution, which was prepared
by adding exactly 2.4798 g of sodium carbonate to a 1.00 L volumetric flask and filling it to the
mark with distilled water (DI). From the known standard concentration and standard/titrant
volumes, exact normality of the titrant solution could be calculated and used in the analysis. In
the indicator method, Bromcresol Green indicator was titrated until a color change was observed.
In the potentiometric titration method, standard acid was added in increments of ½ milliliters or
less, such that a change of less than 0.2 pH units occurred per increment. pH was then observed
using the pH meter. Each titration was continued until a wide range of pH values were observed,
typically ranging from pH 10­11 to pH 4.5 or lower. The Gran Function method could then be
used to analytically determine the amount of alkalinity present in the sample in mg CaCO3/L.
Hardness was determined using a similar indicator method. However, a calcium
carbonate solution was used as a standard (prepared with 0.5014 g of calcium carbonate in 500
mL of solution) to determine the concentration of our Ethylenediaminetetraacetic acid (EDTA)
titrant. EDTA is an aminopolycarboxylic acid that latches onto metal ions such as calcium and
iron. After being bound by EDTA, metal ions remain in solution but exhibit diminished
reactivity (AVA Chemicals Private Limited). Calmagite was used as an indicator, which is a
complexometric indicator used in analytical chemistry to identify the presence of metal ions
within solution. As with other metal ion indicators, calmagite will change color when it is bound
to an ion. EDTA was added to the calmagite indicator in small increments until a color change
was observed, and an equivalence point volume recorded.
In the third laboratory experiment, a typical five­day BOD experiment was performed. It
is important to note, however, that this experiment was performed over seven days. In this
experiment, traditional methods of estimating the five­day BOD of a sample involved filling an
airtight bottle with a water sample, along with a specified volume of SEED and dilution water,
and incubating it at a specified temperature for “five” days. Dissolved oxygen values were
measured prior to the five­day test on day “zero.” After complete incubation, final dissolved
oxygen values were measured so that BOD could be calculated from the difference in oxygen
consumption between day zero and day five. In compliance with the Standard Methods
procedure, all samples that did not have a minimum DO depletion of 2.0 mg/L and a residual DO
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of at least 1.0 mg/L were not included in analysis. SEED and dilution water controls were also
tested for BOD so that their DO contributions could be factored in to final calculations for the
UWSs. Azide Modification (Winkler Titrations) was used to experimentally determine
concentrations of DO, following Standard Methods 4500­0 C. Azide modifications are used for
most wastewater, effluent, and stream samples, especially if samples contain more than 50 pg
NO2—NL and not more than 1 mg ferrous iron/L. Sodium thiosulfate was used as a titrant, and
potassium bi­iodate as a standard which helped to determine the exact concentration of our
titrant.
In the fourth laboratory experiment, gravimetric methods were utilized in order to
determine various solids concentrations. These describe a set of methods in analytical chemistry
for the quantitative determination of an analyte based on the mass of a solid (ECS UMASS). TS
was the first parameter identified. Here, 25­mL of the UWS was dried at 103°C for one
hour—just enough time for all the water to evaporate off and leave dried residue representative
of the samples TS concentration (difference in dish weight before and after drying). TS is the
sum of TDS and TSS concentrations, which could also be determined. The suspended or
particulate fraction of a water sample was separated from the dissolved components through
filtration. Filtration is the separation of solids from liquids by passing a water sample through a
porous medium. The solids fraction that passed through a filter with nominal pore diameter less
than two micrometers was obtained, dried at 103°C and 108°C for an hour each and weighed in
order to find TDS content. TSS, or the solids fraction that remains on said filter, was determined
by weighing the residue remaining after drying at 103°C for one hour. Through combustion,
volatile solids could be found. Combustion is used to differentiate between organic and inorganic
matter within a water or wastewater sample, which is useful in the control of wastewater
treatment plant operation. However, loss on ignition is not restricted to organic material, as
losses of some mineral salts due to decomposition and volatilization may occur throughout the
process. TS, TSS and TDS residues were ignited at 550°C for 30 minutes to produce total
volatile (TVS), volatile suspended (VSS) and volatile dissolved solids (VDS). Here, the
difference in weights before and after ignition were calculated so that the total weights loss could
be presented as the various volatile solids parameters.
APPENDIX D: RESULTS/DISCUSSION
APPENDIX E: CONCLUSIONS/RECOMMENDATIONS
In each subsequent experiment, many water quality parameters were observed, measured,
and analyzed to form important deductions as to where these unknown water samples may have
originated.
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In the analysis of the first unknown water sample, it was deduced that this sample had
originated from a natural river or stream. In this sample, the average dissolved oxygen value was
approximately 8.88 milligrams per liter, the highest of all four unknown water samples analyzed.
Water sources with relatively high concentrations of dissolved oxygen are typically indicative of
water bodies containing waterfalls or otherwise high, turbulent currents. Rapidly moving water,
such as in a mountain stream or large river, tends to contain an increased amount of dissolved
oxygen, whereas stagnant water tends to contains far less (Water Properties: Dissolved Oxygen).
While looking under the microscope at this water sample, microorganisms similar in shape,
color, and size to Anacystis were observed. Anacystis is a blue­green algae typically found in
water, on rocks and soils that are usually associated with minor to severe water pollution
(Wastewater Organisms: A Color Atlas). Because microorganisms similar to Anacystis were
observed, it can also be deduced that this unknown water sample was most likely exposed to
some type of effluent or pollution. It can be concluded that the first unknown water sample, with
a mean hardness value of 146 milligrams calcium carbonate per liter, is moderately hard based
off of the hardness classifications used by the United States Environmental Protection Agency
(USEPA National Recommended Water Quality Criteria for Freshwater and Human
Consumption of Water and Organism). Hardwater streams generally indicate higher levels of
animal and plant productivity. Hardness also helps limit the damaging effects of toxic or
dangerous metals. Hardwater streams are often noted for high levels of productivity because
magnesium and calcium are usually associated with other important nutrients and minerals.
Elevated levels of metals can clog the gills of aquatic animals. In theory, scientists believe that if
there is a higher concentration of calcium and magnesium ions in a water body, these harmless
atoms end up in the gills of fish and other aquatic life, and not the damaging metals, essentially
displacing these metals (USEPA National Recommended Water Quality Criteria for Freshwater
and Human Consumption of Water and Organism).
Alkalinity of natural water sources is determined by the soil and bedrock through which it
passes, and the main sources for natural alkalinity are rocks which contain carbonate,
bicarbonate, and hydroxide compounds. Because the alkalinity values are low in this UWS, this
sample was most likely taken from a source with not many rocks or minerals present (Water
Hardness and Alkalinity). It can be inferred that the first unknown water sample, given a BOD
value of 1.979 milligrams per liter, was most likely derived from a nearby, relatively clean,
natural water source. Because this value is on the cusp of the two to eight milligrams per liter
range as seen as typical values for moderately polluted river BOD ranges, this river may have
recently been polluted by an incoming waste stream (Biochemical Oxygen Demand and
Carbonaceous Biochemical Oxygen Demand in Water and Wastewater). In addition to these
water quality parameters, the approximate value for total solids was 0.14 milligrams per liter
solution, which can also indicate some minor pollution due to the correlation between total solids
and turbidity. In addition to this parameter, this water sample also observed average values for
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biochemical oxygen demand at about 2 milligrams per liter, which means this sample was most
likely derived from a nearby, relatively clean, natural water body.
In the analysis of the second unknown water sample, it was deduced that this was most
likely derived from a moderately polluted river or stream. It appeared to have a precipitate of
decomposing organic material, with some embedded, natural sediments indicative of these types
of water bodies. Rivers and streams that have rapid currents can create a phenomena of
dislodging sediment and organic material and turning up this material into the current of the river
or stream, where the water sample is being collected for analysis. Because of the high turbidity
readings from this sample, it is indicative that this may have been the case. It can be concluded
that the second unknown water sample, with a mean hardness value of 351.5 milligrams calcium
carbonate per liter, is very hard based off the hardness classifications used by the United States
Environmental Protection Agency (USEPA National Recommended Water Quality Criteria for
Freshwater and Human Consumption of Water and Organism). Because this water sample also
has the highest alkalinity values, it was most likely taken from a stream laden with rocks
containing carbonate, bicarbonate, and other compounds. Limestone is also rich in carbonates, so
waters flowing through limestone regions or bedrock containing carbonates typically generate
higher alkalinity and hardness values. In addition to high alkalinity values, this sample had an
extremely high BOD value of 2195.667 milligrams per liter. Based on typical values stated
above, this water source may have generally come from some sort of wastewater stream,
possibly a river or stream recently polluted by some sort of industrial or chemical process.
Initially, our results have led us to believe that this sample was also most likely derived
from a clean, nearby natural water body similar to that of the first unknown water sample, but
because of these high BOD levels, we are no longer led to believe that this is the case. In the
second unknown water sample, it also appears to have a precipitate of decomposing organic
material, with some embedded, natural sediments typically indicative of rivers and streams.
Rivers and streams with rapid currents can also create conditions of high levels of turbidity,
which was also observed. In the continued analysis of this unknown water sample, it was also
observed that there were relatively high values of alkalinity and hardness; also indicative of
streams or rivers laden with rocks containing carbonate, bicarbonate, and other compounds
associated with these three (Alkalinity and Stream Water Quality). In the continued analysis of
the second unknown water sample, the mean value for total solids was approximately 3.05
milligrams per liter. In addition to this parameter, this sample also has very high experimental
values for biochemical oxygen demand, averaging at approximately 2,200 milligrams per liter. In
this continued analysis, this unknown water sample also appeared to have a precipitate of
decomposing organic material, with some embedded, natural sediments indicative of a river or
stream environment, which may have contributed to total solids within the sample, creating a
slightly higher value.
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In the analysis of the third unknown water sample, it was deduced that this sample was
taken from a pond or lake with high photosynthetic activity. It had a green color, a pungent odor,
and contained floating particulates including small, winding chains of green circles, plankton,
and other blue­green algae. In this analysis, it was also observed the sample was highly turbid,
with a mean turbidity value of 2050 NTU, meaning there seemed to be a higher magnitude of
particulate matter suspended within the sample. Microorganisms observed best matched that of
Anabaena, a particular type of cyanobacteria typically used to treat industrial wastewater.
Because of this, it was at first concluded that this sample was taken from a wastewater sample
because of the amount of variation and matter in the sample, as well as these specific
microorganisms present in the sample (Cyanobacteria and Cyanotoxins: Information for
Drinking Water Systems).
However, although it was first deduced this water sample had been taken from a
wastewater sample, this conclusion was then discarded due to other observations. In the
continued analysis of the third unknown water sample, a mean hardness value of 161 milligrams
calcium carbonate per liter, which is labelled hard from the hardness classifications used by the
United States Environmental Protection Agency (USEPA National Recommended Water Quality
Criteria for Freshwater and Human Consumption of Water and Organism), was measured. In the
continued analysis of this third unknown water sample, a mean value of approximately 1.54
milligrams per liter for total solids was measured. In addition to this parameter, this sample also
observed very low experimental values for biochemical oxygen demand, with values averaging
at approximately 6 milligrams per liter. High turbidity values, averaging at approximately 2050
Formazin Nephelometric Units (FNU), were also observed within this sample, which seem to
contribute to total solids concentrations as well.
In the continued analysis of this water sample, a mean BOD value of 6.512 milligrams
per liter was observed. Initially, we had expected this sample to have the highest BOD value out
of all four samples due to the decomposition of organic matter indicative of wastewater
treatment, but soon realized that this sample was most definitely not derived from a wastewater
source, and was in fact taken from a pond or lake with high levels of photosynthetic activity
instead. Chlorophyll is a photosynthetic pigment that causes this green color in algae and plants.
Concentrations of chlorophyll present in the water are directly related to to the amount of algae
living in the water, and excessive concentrations of algae tend to give ponds and lakes an
undesirable green appearance that was observed in this water sample in particular (Ecosystem
and Lake Productivity Chlorophyll Analysis).
In the analysis of the fourth unknown water sample, it was deemed practically clear with
no odor, with barely any microorganisms present, if at all. It can be concluded that this sample
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was most likely taken from a sample used for drinking water. It can be concluded that in the
analysis of this fourth unknown water sample, with a mean hardness value of 156 milligrams
calcium carbonate per liter, is hard based off of the hardness classifications used by the United
States Environmental Protection Agency (USEPA National Recommended Water Quality
Criteria for Freshwater and Human Consumption of Water and Organism). In the continued
analysis of the fourth unknown water sample, very low BOD values were observed, averaging at
about 0.902 milligrams per liter. Because this sample is relatively clear with no odor,
microorganisms, or particles present, it can be inferred that this water sample was likely taken
from a sample used for drinking water. Because of its relatively hard nature, with a hardness
value of 156 milligrams calcium carbonate per liter, this water sample was likely taken from a
drinking water sample exposed to dissolved ions like fluoride and calcium. In the continued
analysis of this unknown water sample, a mean value of 0.18 milligrams per liter for total solids
was observed. In addition to this parameter, this sample also observed very low values for
biochemical oxygen demand, averaging at 0.902 milligrams per liter. Because this sample is
relatively clear with no odor, microorganisms, or particles present, it can be further inferred that
this water sample is likely taken from a drinking water sample. In addition to these parameters,
furthermore, high values for hardness were also observed. Because of this sample’s relatively
hard nature, with hardness values averaging at approximately 156 milligrams calcium carbonate
per liter, this water sample was likely taken from a drinking water sample exposed to dissolved
ions like fluoride and calcium. In addition to these observations, this water sample was most
likely derived from a groundwater sample because of the amount of dissolved ions concentrated
within the sample (Water Hardness and Alkalinity).
APPENDIX F: FIGURES AND TABLES
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Figure 1. Total Alkalinity of Surface Waters in the United States
Figure 2. Distribution of Hardness Concentrations in the United States
APPENDIX G: REFERENCES CITED
13
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