LAB: WATER QUALITY TESTING
INTRODUCTION
More and more people are realizing that we need to find better ways to share our world’s natural
resources with each other and with other species to ensure that the planet can support life in the
future. By allowing poison in our waters, we are slowly drinking it ourselves. Everything we do
affects our water. We are beginning to see how important each of us is to making the world a better
place to live, no matter what kind of work we do or how intelligent society says we are.
There are two general methods of monitoring water quality: chemical monitoring and biological
monitoring. Chemical monitoring provides good data on what was happening at the instant that it is
measured. In this lab, we will be testing for pH, temperature, turbidity, nitrates, phosphates, and dissolved
oxygen. Biological monitoring can provide a longer term picture. Indicator organisms respond to certain
conditions consistently and the structure of the macroinvertebrate community is a reliable indicator of
water quality.
MATERIALS
pH test kit
thermometer
turbidity test kit
dissolved oxygen test kit
Nitrates test kit
Phosphates test kit
Waders
Kick net
Dip net
White sorting pans
Forceps
70% alcohol
Vials
PROCEDURE
pH, Temperature, and Turbity
pH:
1. Fill the test tube (0230) to the 5.0 ml line with sample water.
2. Add 10 drops of reagent (WR Ind).
3. Cap and mix.
4. Insert test tube into Octet Comparator. Match the sample color to a color standard and record the pH. For
optimum color comparison, the comparator should be positioned between the operator and a light source,
so that the light enters through the special light-diffusing screen in the back of the comparator. Avoid
viewing the comparator against direct sunlight or an irregularly lighted background.
Temperature:
1. Submerge the thermometer beneath the water for 60 seconds before reading.
2. Record your answer in degrees Fahrenheit.
Turbidity:
This test is made by comparing the turbidity of a measured amount of the sample with an identical amount
of turbidity-free water containing a measured amount of standardized turbidity reagent. The readings are
made by looking down through the column of liquid at a black pie shape. It turbidity is present, it will
interfere with the passage of light through the column of liquid. Small amounts of turbidity will cause a
“blurring” of the black pie shape in the bottom of the tube. Large amounts of turbidity may provide
sufficient “cloudiness” so that it is not possible to see the black pie shape when looking down through the
column. Any color that may be present in the sample should be disregarded. This determination is
concerned only with the haziness or cloudy nature of the sample.
1. Fill one Turbidity Column (0835) to the 50 ml line with the sample water. If the black shape on the
bottom of the tube is not visible when looking down through the column of liquid, pour out a sufficient
amount of the test sample so that the tube is filled to the 25 ml line.
2. Fill the second Turbidity Column with an amount of turbidity-free water that is equal to the amount of
sample being measured. Distilled water is preferred; however, clear tap water may be used. This is the
“clear water” tube.
3. Place the two tubes side by side and not the difference in clarity. If the black shape is equally clear in
both tubes, the turbidity is zero. If the black dot in the sample tube is less clear, proceed to step 4.
4. Shake the Standard Turbidity Reagent (7520) vigorously. Add 0.5 ml to the “clear water” tube. Use the
stirring rod to stir contents of both tubes to equally distributed turbid particles. Check for the amount of
turbidity by looking down through the solution at the black shape. If the turbidity of the sample water is
greater that that of the “clear water”, continue to add Standard Turbidity Reagent in 0.5 ml increments to
the “clear water” tube, mixing after each addition until the turbidity equals that of the sample. Record the
total amount of Turbidity Reagent added.
5. Each 0.5 ml addition to the 50 ml size sample is equal to 5 Jackson Turbidity Units (JTUs). If a 25 ml
sample size is used, each 0.5 ml addition of the Standard Turbidity Reagent is equal to 10 JTUs. Rinse both
tubes carefully after each determination.
Nitrates and Phosphates
Nitrate:
1. Fill sample bottle (0688) with sample water.
2. Fill one test tube (0820) to the 2.5 ml line with water from the sample bottle.
3. Dilute to the 5 ml line with Mixed Acid Reagent (V-6278). Cap and mix. Wait 2 minutes.
4. Use the 0.1 g spoon (0699) to add one level measure (avoid any excess) of Nitrate Reducing Reagent (V6279). Cap and invert gently 50-60 times in one minute. Wait 10 minutes.
5. Insert test tube into Nitrate-N Comparator (3109). Match sample color to a color standard. Record as
ppm Nitrate as Nitrate Nitrogen.
6. Convert this number to ppm Nitrate by multiplying it by 4.4.
Total Phosphate:
1. Fill test tube (0843) to the 10 ml mark with sample water.
2. Use 1.0 ml pipet with (0354) to add 1.0 ml of Phosphate Acid Reagent (V-6282).
3. Cap and mix.
4. Use the 0.1 g spoon (0699) to add one level measure of Phosphate Reducing Agent (V-6283).
5. Cap and mix until dissolved.
6. Wait 5 minutes.
7. Remove stopper from test tube. Place tube in Phosphate Comparator (3122) with Axial Reader (2071).
Match sample color to a color standard. Record as ppm Orthophosphate.
Dissolved Oxygen
Steps 1-7 must be completed in the field. The remainder of the procedure can be completed the following
day in the lab.
1. Fill the water sampling bottle (0688-DO) by submerging it and allowing it to fill completely. Tap the
sides of the bottle to dislodge any air bubbles and replace the cap while the bottle is still submerged. Make
sure that there are not air bubbles trapped inside the bottle (this will affect your measurement).
2. Add 8 drops of Manganous Sulfate Solution (4167).
3. Add 8 drops of Alkaline Potassium Iodide Azide (7166).
4. Cap and mix.
5. Allow precipitate to settle.
6. Use the 1.0 g spoon to add Sulfamic Acid Powder (6286) or add 8 drops of sulfuric acid, 1:1 (6141WT).
7. Cap and mix until the reagent and precipitate dissolve. At this point the sample has been “fixed” and
contact between the sample and the atmosphere will not affect the test result.
8. Fill titration tube (0299) to the 20 ml line with the fixed sample.
9. Fill titrator with Sodium Thiosulfate, 0.025N (4169). To accomplish this, first depress the plunger of the
titrator to expel any air. Then, insert the titrator into the plug in the top of the 0.025N Sodium Thiosulfate
titrating solution. Invert the bottle and slowly withdraw the plunger until the bottom of the plunger is
opposite the zero mark on the scale. If small air bubbles appear in the titrator barrel, expel them by partially
filling the barrel and pumping the titration solution back into the reagent container. Repeat until the bubble
disappears.
10. Insert the tip of the titrator into the opening of the titration tube cap. Slowly depress the plunger to
dispense the titrating solution until the yellow-brown color changes to a very pale yellow. Gently swirl the
tube during the titration to mix the contents. Titrate until sample color is pale yellow.
11. Add 8 drops of Starch Indicator (4170WT). The sample should turn blue.
12. Continue titration until blue color just disappears and solution is colorless.
13. Record the test result where the titrator tip meets the scale. Read the result in ppm Dissolved Oxygen.
Each minor division on the titrator scale equals 0.2 ppm.
Macroinvertebrates
Sampling:
1. Place the kick screen in the middle of the stream so that both poles are pushed into the substrate and the
bottom of the net is resting on the bottom. Allow the net to bow slightly so that the current will carry
dislodged macroinvertebrates into the net.
2. Have one person vigorously kick the sediment one square meter upstream of the net for 60 seconds.
3. Gently lift the net moving upstream slightly as you lift being careful not to lose any of the organisms
caught in the net.
4. Bring the net to a flat area and dump the sample into a sorting tray.
5. Remove organisms with forceps and store them in 70% alcohol. You will be identifying these organisms
to Order when we return to the lab.
Richness:
Richness is a measure of the number of different types of organisms you collect. For this lab, record
richness as the number of different orders identified in each sample.
Shannon’s Diversity Index:
Sample 1
May flies
Stone flies
Caddis flies
True flies
Sample 2
5
1
5
1
5
1
5
17
Sample 3
2
2
2
34
Pollution Tolerance Index (PTI):
The Pollution Tolerance Index is based on the concept of indicator organisms and tolerance levels.
Indicator organisms are those organisms that are sensitive to water quality changes and respond in
predictable ways to changes in their environment. Therefore, their presence or absence indicates something
about water quality.
The PTI is calculated by multiplying the number of types of organisms in each tolerance level by the index
value for that level (1 = pollution tolerant, 2 = moderately tolerant, 3 = sensitive) and adding the resulting
three numbers. The pollution tolerance taxa groups for calculating this index can be found at:
http://www.epa.gov/bioindicators/html/benthosclean.html
Water quality is then assessed according to the following scale:
excellent 23 and above
good 17-22
fair 11-16
poor 10 or less
Data Sheet
A. WATER QUALITY MEASUREMENTS
pH
Temperature (ºC)
Dissolved
Oxygen (ppm)
Nitrate (ppm)
Phosphate (ppm)
Turbidity (FTU)
How Should We Interpret Water Quality Measurements?
Water quality measurements from various sections of the river under study will be
compared to determine whether or not land use practices in the watershed have an impact
on water quality. The parameters that have been selected for measurement are
representative of some of the best indicators of water quality. The following descriptions
provide an indication of why these parameters are important and how the values you
obtain might be interpreted.
A.
pH
pH is a measure of the hydrogen ion (H+) concentration in a solution. Acid strength is
based on how readily it releases hydrogen ions in water - strong acids release lots of H+,
weak acids release smaller amounts. A pH reading of 7 indicates neutrality (neither
acidic nor basic); numbers less than 7 are acids, greater than 7 are bases (alkaline). Since
the pH scale is logarithmic, a change of one pH unit represents a ten-fold change in the
acidity of the solution. Most species can tolerate pH values from 6 to 8; optimal levels
for most fish are between 7.1 and 7.8 (Table 1). Values above or below these values may
affect some species. Amphibians and some aquatic insect larvae are particularly sensitive
to acidic conditions.
Table 1. Lethal pH Limits for Some Aquatic Organisms
(Adapted from Murdoch and Cheo, 1996)
pH Value
Impacts
4.0 to 4.5
all fish, amphibians and many invertebrates dead
4.5 to 5.0
caddisflies and mayflies dead
5.0 to 5.5
salmonid eggs and alevin dead, decomposing bacteria decline
5.5 to 6.0
most fish and amphibians decline
6.0 to 6.5
snails and tadpoles decline
6.5 to 8.2
most species can tolerate
8.5 to 9.0
salmonids begin to decline with prolonged exposures
> 11.0
salmonids dead
> 11.5
most fish dead
Most biochemical reactions that occur in living organisms are sensitive to pH. Therefore,
pH values that lie outside of a species range of tolerance can have direct effects on
survivability and overall health of the organism. In addition to these direct effects of pH
changes, there are indirect effects as well. Acidic conditions (low pH) can increase the
release of metals such as aluminum or copper from sediments and increase their
concentration in the water. These metals can disrupt gill function or cause deformities in
fish. Another indirect effect is illustrated by the relationship between pH and ammonia.
Ammonia in water may occur either as ammonium ion (NH4+) or ammonia (NH3). NH3
is highly toxic to fish and other aquatic organisms. As pH levels increase, a greater
portion of the ammonia exists in this toxic form.
High acidity (low pH) in waterways can be caused by carbon dioxide dissolved in water,
tannic acid from the decomposition of conifer needles and bark, acid rain, coal mining
operations and industrial pollutants.
B.
Water temperature
Water temperature in rivers and streams is influenced by a number of factors, several of
which are related to land use. Heated effluent from industry or runoff from urban streets,
for example, may increase stream temperatures directly. Also, the loss of shading
provided by trees in the riparian zone or increased turbidity due to agricultural runoff
may result indirectly in increased stream temperature. Stream temperatures are also
influenced by ambient air temperature and the water source (e.g., springs, snow melt, and
runoff). Temperatures influence those organisms that can occur in streams (Table 2).
Since most aquatic organisms are ectothermic ("cold-blooded"), water temperature
controls metabolic rate and, often, the timing of reproductive activities. In most areas,
thermal pollution (high temperatures) is generally more limiting than low temperatures.
Aquatic organisms are generally more susceptible to the influences of toxic chemicals,
parasites and diseases at temperatures at the upper end of their range of tolerance. Water
temperature also influences dissolved oxygen levels (see discussion below).
C.
Dissolved oxygen (D.O.)
Dissolved oxygen is measured in parts per million (ppm) with a dissolved oxygen meter
or chemically with a Winkler Titration. Dissolved oxygen levels of 7-10 ppm are typical
in unpolluted water and generally considered adequate for most aquatic life. In salmonid
streams, dissolved oxygen requirements are higher. Salmon embryo and larval stages can
show some impairment at DO levels as high as 8 or 9 ppm. In other aquatic habitats,
levels below 4.5 ppm can cause acute mortality of fish and invertebrates. The primary
sources of oxygen in streams are photosynthetic production by algae and higher plants
and diffusion from the air above the water surface. Diffusion rates increase in turbulent
waters such as those created by wind action, rapids, and waterfalls. Dissolved oxygen
levels may fluctuate significantly throughout the day especially in bodies of water with
extensive plant growth. For this reason, if dissolved oxygen levels are to be compared
through time, samples should be collected at approximately the same time of day, at the
same water depth, and under similar conditions of light intensity. Levels rise from
morning to afternoon as a result of photosynthesis, reaching a peak in late afternoon.
Photosynthesis then begins to shut down as light intensity decreases. At night
photosynthesis stops but plants and animals continue to respire thus consuming oxygen.
Dissolved oxygen levels typically decline at night.
Low dissolved oxygen levels generally indicate polluted water and high Biological
Oxygen Demand (BOD). Low dissolved oxygen readings can be expected in stagnant
water with large amounts of organic material. As the organic material decomposes,
oxygen is consumed in the process. Dissolved oxygen can influence the species that
occur in a body of water. When dissolved oxygen levels drop below a critical level, fish,
amphibians, aquatic invertebrates and aerobic bacteria which rely on this oxygen for
aerobic metabolism will decline and eventually perish. Additionally, at low dissolved
oxygen levels, anaerobic bacteria proliferate and break down the remaining organic
material producing toxic gases such as hydrogen sulfide and methane.
The amount of dissolved oxygen is also a function of water temperature - cold water is
capable of retaining high amounts of dissolved oxygen, warmer water is less capable.
This relationship is illustrated for temperatures from 10 - 41°C in Table 3. This
information can be used to determine the percent saturation for dissolved oxygen in a
water sample. Percent saturation is a measure of the amount of dissolved oxygen in a
water sample relative to the maximum amount that could be in that sample. For
example, suppose we obtained a dissolved oxygen reading of 8.0 ppm for a water sample
and the temperature of the sample was 20°C. The percent saturation could be calculated
by dividing your reading with the maximum dissolved oxygen concentration at 20°C
("9.07 ppm" from the Table 3) and multiplying by 100:
Percent Saturation = (8.0/9.07) X 100 = 88.2 %
Table 3. Maximum Dissolved Oxygen Concentration
Temperature (°C)
Dissolved Oxygen
(ppm)
Temperature (°C)
Dissolved Oxygen
(ppm)
10
11.27
26
8.09
11
11.01
27
7.95
12
10.76
28
7.81
13
10.52
29
7.67
14
10.29
30
7.54
15
10.07
31
7.41
16
9.85
32
7.28
17
9.65
33
7.16
18
9.45
34
7.05
19
9.26
35
6.93
20
9.07
36
6.82
21
8.9
37
6.71
22
8.72
38
6.61
23
8.56
39
6.51
24
8.4
40
6.41
25
8.24
41
6.31
Stream habitats are considered healthy at 90 - 100% saturation; levels for ponds are
generally lower.
D.
Turbidity
Turbidity is a measure of the "cloudiness" of the water. Sediment, algae, bacteria and
zooplankton all contribute to what is technically known as the Total Suspended Solids
(TSS) that increase the turbidity. As turbidity increases, the degree to which sunlight
penetrates the water column declines. This obviously has an impact on photosynthetic
rates in algae and submerged vegetation. High turbidity can also raise surface water
temperature as suspended particles near the surface absorb more heat from sunlight.
Suspended soil particles may also carry nutrients, pesticides and other pollutants and they
can bury benthic organisms. Turbid waters tend to be low in dissolved oxygen.
Turbidity can be measured with a turbidity meter in formazine turbidity units (FTU's).
Drinking water is generally very clear and would have a turbidity measurement less than
10 FTU. Very cloudy water would read about 1000 FTU. We will use a Portable
Microprocessor Turbidity Meter to measure turbidity in water samples. Alternatively,
relative measures of turbidity can be obtained by using a "spectrophotometer”. This
device passes a known quantity of light through a sample in a glass tube called a
"cuvette". A sensor on the opposite side of the cuvette detects the amount of light (%
Transmittance) that passes through and displays this amount on a scale. The wavelength
of light can be adjusted to measure the amount of various materials dissolved in the
sample. The accepted wavelength for the measure of turbidity is 550 nm.
E.
Phosphate
Phosphorus (P) usually occurs in natural systems as phosphate (PO4 -3 ). This phosphate
may be bound to organic compounds (organic phosphate) or inorganic compounds
(inorganic phosphate or orthophosphate). Inorganic phosphate is the form most
readily available to plants and therefore is generally of greater interest than organic
phosphate. Phosphorus tends to be less abundant than nitrates in freshwater ecosystems
and is, therefore, often a limiting factor for plant and algal growth. The addition of
phosphorus (in the form of phosphates) commonly results in algal blooms (cultural
eutrophication). Domestic sewage (particularly those containing significant amounts of
laundry detergents) and agricultural runoff are important sources of phosphates. Most
sewage treatment plants remove only about 50% of the nitrogen and 33% of the
phosphorus from domestic sewage. The remainder is dumped in the effluent into surface
water. In an effort to reduce the growth of nuisance aquatic plants and algae, the
Environmental Protection Agency has established a desired goal of 0.05 ppm total
phosphorus for streams entering lakes and 0.10 ppm for other flowing waters. At the
height of phosphate-based detergent use in 1970, effluent from U.S. sewage treatment
plants averaged 11 ppm. Reductions in the use of these detergents reduced this value to 5
ppm in 2002. Phosphorus levels as low as 0.01 ppm have been shown to have an impact
on algae growth in ponds. Some scientists use phosphorus levels as an indication of
whether a lake is oligotrophic (<0.01 ppm) or eutrophic (> 0.02 ppm). Phosphates
(inorganic phosphates only) can be measured with an ion-specific meter.
F. Nitrate
Nitrogen appears in several forms in water sources, including nitrate (NO3), nitrite
(NO2) and ammonia (NH3). Of these, nitrates are probably the most common
inorganic pollutant tested in water. Ammonia is a product of the decomposition of
plant and animal protein but tends to be taken up quickly by algae and plants. Nitrites
tend to occur at fairly low levels in most water samples because they are readily
converted to nitrates by bacteria. Nitrates are a common component of multi-nutrient
fertilizers whose nutrient content is indicated by three numbers called the "grade".
The first of these numbers indicates the nitrogen content of the fertilizer. A fertilizer
grade of 16-16-16, for example, contains 16% by weight nitrogen, 16% phosphate
(P2O5) and 16% potassium (K2O). Both nitrates and ammonia stimulate algal growth
and may be responsible for causing cultural eutrophication. If ingested they are
converted to nitrites in the intestines of humans where they combine with hemoglobin
in red blood cells causing the oxygen-carrying capacity to decline. In infants this
condition may be fatal. Contamination of groundwater by nitrates that are applied as
fertilizer or runoff from feedlots and dairies is a widespread problem in agricultural
regions of the country. Improperly treated sewage from sewage treatment plants and
septic systems that finds its way into waterways is also an important source. Nitrates
can be measured with an ion-specific meter.
The national drinking standard for nitrates in the U.S. is 10 ppm. Waters that have levels
as low as 1 ppm, however, can be sufficiently polluted to cause algal blooms.
Wynn W. Cudmore, Ph.D., Principal Investigator
Northwest Center for Sustainable Resources
Chemeketa Community College