1. Too much of a good thing…
natural in
water ^
nutrients
waters in
We all know that nutrients can be found in the foods and beverages that we consume (even if they were artificially added – Vitamin Water)…
But did you know that nutrients can also be found in natural waters? And did you know that monitoring these nutrients is an important aspect of maintaining good water quality in our rivers, lakes, and streams?
Today we are going to discuss the role of nutrients in the aquatic environment & how sometimes too much of a good thing can be very bad
We’ll also discuss how state and federal agencies measure particular nutrients to get a snapshot of a water body’s health.
Too much of a good thing…
Image Source: vitaminwater.com

2. What Are Nutrients?
Nutrients: chemical elements that provide nourishment essential for growth and the maintenance of life; often called “minerals”
Primary Producers responsible for nutrient uptake
Nutrients
Producer
Consumer
So, what are nutrients?
Nutrients are substances that plants and animals utilize to live and grow; sometimes you will hear them referred to as “minerals”
There are several important nutrients that we will discuss on the next slide; but you can see some of them in the sample Nutrition Facts Label
Primary producers form the base of the food chain and thereby play an important role in carrying nutrients to higher organisms
Plants use sunlight as their source of energy for photosynthesis, and mineral nutrients as the building blocks for new growth
Images mined from Google Image search

3. 15 Important Plant Nutrients in Natural Waters
Non-minerals
Minerals
Macro-
Micro- H
HYDROGEN
1.0079 1 C
CARBON 6 O
OXYGEN 8
Limiting Nutrients N
NITROGEN 7 P
PHOSPHOROUS 15 K
POTASSIUM 19 Ca
CALCIUM
40.078 20 Mg
MAGNESIUM 12 S
SULFUR
32.066 16
In natural water systems, there are several important nutrients that are essential for plant growth – 15 of them are listed here
Non-mineral: Hydrogen, Carbon, & Oxygen
Readily available in air and water
In a process called photosynthesis, plants use energy from the sun to change carbon dioxide (CO2 - carbon and oxygen) and water (H2O- hydrogen and oxygen) into starches and sugars. These starches and sugars are the plant's food. 
Considered “non-mineral” b/c they are biogenic (resulting from the activity of living organisms)
Mineral:
Macro-nutrients: Nitrogen, Phosphorous, Potassium, Calcium, Magnesium, and Sulfur
Mineral nutrients that are in the greatest demand; plants use large amounts for their growth and survival. 
Micro-nutrients: Copper, Iron, Zinc, Silica, Chlorine, and Manganese
Mineral nutrients essential for growth, but only needed in small quantities
Nitrogen & Phosphorous tend to be the limiting nutrients in standing water bodies
Phosphorous compounds are usually abundant in the ocean – Nitrogen is the limiting nutrient
Nitrogen compounds are more abundant in lakes, rivers, and streams – Phosphorous is the limiting nutrient
Nitrogen and phosphorus are frequently called “limiting nutrients.” This means that at any given time, one or the other of these nutrients may be the hardest nutrient for a plant to acquire and therefore be the only nutrient that is limiting the plant's growth. So, the plant's growth is often directly related to the amount of whichever limiting nutrient (nitrogen or phosphorus) that it is are able to get.
Generally speaking, phosphorus is a limiting nutrient in freshwater systems and nitrogen is a limiting nutrient in saltwater systems.
So, back to our fundamental question, “Why do nutrients cause pollution?” Limiting nutrients cause pollution when they enter a waterbody in relatively high amounts. If the conditions are right, where there is a lot of sunlight, the water is clear, and the temperature is high, nutrients cause algae to grow. When this happens, the algae use the sunlight to make food for themselves and increase their numbers. This is called an algae bloom. Algae blooms can result in large floating “mats” of algae. When the algae die, however, they sink as dead organic matter. Organic matter on the bottom of the water column is used by bacteria for food. In order for the bacteria to break down the organic matter they use dissolved oxygen. This can lead to hypoxia and fish kills. Cu
COPPER
63.545 29 Fe
IRON
55.845 26 Zn
ZINC
65.39 30 Si
SILICA
28.086 14 Cl
CHLORINE
35.453 17 Mn
MANGANESE
54.938 25

4. Nutrient Sources : Natural
Atmosphere
Terrestrial weathering
Water erosion
Groundwater
Decomposition of organic matter
Upwelling
So, how do these nutrients get into the water?
Atmosphere:
Atmospheric gases absorbed by plants or dissolved in water
Deposition of dust/soil/ash from wind erosion or volcanic emissions
Terrestrial weathering/water erosion:
Rocks & Soil
Groundwater
Dissolved lithogenic compounds
Decomposition of organic matter
Upwelling
Image source: clean-water.uwex.edu

5. Nutrient Sources: Anthropogenic
Atmosphere
Terrestrial runoff
Groundwater
Discharge outfalls
Spills
So, how do these nutrients get into the water?
Atmosphere:
Deposition of substances from industrial or automotive emissions
Terrestrial runoff:
Lawn and agricultural fertilizers
Livestock/Animal waste
Landfills
Automotive pollutants on impervious road surfaces
Groundwater
Fertilizers, animal waste, and pollutants leached from surface soils
Leaking underground storage tanks
Discharge outfalls
Waste water treatment facilities
Industrial facilities
Stormwater
Spills
Image source: clean-water.uwex.edu

6. Nutrient Pollution? Human Concerns Environmental Concerns
Drinking water related disease or illness
Drinking water treatment costs
Decrease in perceived aesthetic value of water bodies
Environmental Concerns
Increased plant & algae growth
Decreased water transparency
Decrease of SAV
Depletion of dissolved oxygen (DO)
Increased incidence of fish kills
as waters become hypoxic or anoxic
Eutrophication
In the past 50 years, the EPA has coined the term “Nutrient Pollution”
But, if nutrients support growth of aquatic plants which form the base of the food web, how can they be pollutants & why would we be concerned about them in our waters?
When nutrients exceed levels required for normal plant growth, detrimental effects are often seen:
Human Concerns
Left untreated, high levels of certain nutrients in drinking water can lead to health problems – Example: Consuming high nitrates and nitrites in drinking water can lead to adverse reproductive/developmental effects and organ toxicity (esp. breathing problems & liver failure); also, infantile Methemoglobinemia (“blue baby syndrome”), caused by excessive nitrates in drinking water
Many of us rely on water treatment plants to remove these nutrients from our drinking water; higher levels of nutrients in water sources drives up the cost of this removal
Water bodies become murky and may even smell bad due to increased plant and algal growth
Environmental Concerns
Excessive plant and algae growth
Not immediately a problem; in fact, increased plant growth may initially increase dissolved oxygen levels in the water which can be beneficial to other living organisms
However, as plant growth continues to increase, water transparency is decreased – also known as increased turbidity
The turbid water retains heat longer & increases water temps which may not be desirable for organisms living in the water  more importantly, the increased temperatures decrease the waters capability to retain dissolved oxygen (oxygen dissolves better in cold water – think of a cold soda vs a warm soda)  decreases in oxygen may stress aquatic organisms
Decreased water transparency also decreases the ability for sunlight to penetrate the water  decreased sunlight means reduced photosynthesis for Submerged Aquatic Vegetation (SAV)  and decreased photosynthesis results in plant death
Additionally, if the plants/algae continue to grow, they eventually use up the nutrients and begin to die as well
Dead plants sink to the bottom
On the bottom, decomposing bacteria feed on the dead plant material  as the decomposers respire, they deplete more oxygen from the water (hypoxic and/or anoxic waters)
In highly eutrophic (or hypereutrophic) conditions, aquatic organisms are highly stressed and overall biomass is usually impacted:
Many benthic, sessile species may die
Fish may leave the area (if they can)
In areas where fish can not escape the eutrophic conditions, there will be an increased incidence of fish kills
Eutrophication (Greek: eutrophos meaning well-nourished, from eu meaning good and trephein meaning to nourish) – defined as an increase in the rate of supply of nutrients or organic matter to an ecosystem; Increased plant growth is an ecosystem’s response to increased levels of nutrients
Hypereutrophication
Image Source: oceanservice.noaa.gov

7. Eutrophication Example
Farmer Brown fertilizes his crops…
Farmer Brown goes home & it rains…
Run-off from Farmer Brown’s cropland enters the nearby rivers, lakes, streams, etc.
If enough nutrient-rich run-off enters these environments, eutrophic conditions may lead to:
Increased plant/algae growth
Decreased water transparency
Decreased submerged aquatic vegetation (SAV)
Depletion of dissolved oxygen (DO)  hypoxic or anoxic conditions
Increased fish kills
Images mined from Google Image search

8. P N “Limiting Nutrients” Nitrogen & Phosphorous 15 N
NITROGEN 7
Nitrogen & Phosphorous
Major chemical forms of Nitrogen & Phosphorous:
Nitrogen Gas (N2)
Nitric Oxide (NO)
Nitrogen Dioxide (NO2)
Ammonia (NH3)
Nitrite (NO2-)
Nitrate (NO3-)
Phosphate (PO43-)
Hydrogen Phosphate (HPO42-)
Dihydrogen Phosphate (H2PO4-)
Phosphoric Acid (H3PO4)
Organic Phosphates
Polyphosphates
Nitrogen and Phosphorous are rarely found in their pure elemental forms in the environment
Instead, they are typically found as chemical compounds such as these listed on the screen…
When people discuss water quality and nutrients that cause pollution, they are usually talking about nitrogen and phosphorus. Most other nutrients, like calcium, magnesium, sodium, and potassium are naturally in high abundance in nature. Nitrogen and phosphorus are frequently called “limiting nutrients.” This means that at any given time, one or the other of these nutrients may be the hardest nutrient for a plant to acquire and therefore be the only nutrient that is limiting the plant's growth. So, the plant's growth is often directly related to the amount of whichever limiting nutrient (nitrogen or phosphorus) that it is are able to get.
Generally speaking, phosphorus is a limiting nutrient in freshwater systems and nitrogen is a limiting nutrient in saltwater systems.
So, back to our fundamental question, “Why do nutrients cause pollution?” Limiting nutrients cause pollution when they enter a waterbody in relatively high amounts. If the conditions are right, where there is a lot of sunlight, the water is clear, and the temperature is high, nutrients cause algae to grow. When this happens, the algae use the sunlight to make food for themselves and increase their numbers. This is called an algae bloom. Algae blooms can result in large floating “mats” of algae. When the algae die, however, they sink as dead organic matter. Organic matter on the bottom of the water column is used by bacteria for food. In order for the bacteria to break down the organic matter they use dissolved oxygen. This can lead to hypoxia and fish kills.

9. Measuring Nutrients
Nitrogen & Phosphorous Levels For Drinking & Surface Waters

10. National Water Quality: Nutrient Criteria
Numeric Nutrient Strategy: 1998
U.S. Environmental Protection Agency
Nitrate-Nitrogen
Nitrite-Nitrogen
Total Phosphorous
In 1998, EPA outlined a Numeric Nutrient Strategy to describe the approach that EPA would follow to develop nutrient information and work with the states and tribes to adopt numeric nutrient criteria.
Numeric nutrient criteria are a critical tool for protecting and restoring a waterbody's designated uses related to nitrogen and phosphorus pollution. These criteria enable effective monitoring of a waterbody for attaining its designated uses; facilitate formulation of NPDES discharge permits; and simplify development of total maximum daily loads (TMDLs) for restoring waters not attaining their designated uses (i.e., impaired waters).
The only national water quality criteria in existence are for nitrate-nitrogen, nitrite-nitrogen, and total phosphorus (EPA standards).

11. National Water Quality: Nutrient Criteria
Ongoing process
North Carolina?
The map shows a national summary of current numeric total nitrogen (TN) and total phosphorus (TP) EPA-approved criteria.
Notice how many states do not yet have state-specific criteria set.
North Carolina isn’t even projected to be onboard in the next three years.
Click on the map to go to the interactive map which allows you to view the progress from It also shows the planned progress in the next three years. (Just tab through the years at the top.)
This does not mean that there are not National Criteria…if a state has not set its own “EPA accepted” criteria, then it defaults to the EPA’s National Criteria (see next two slides).

12. Monitoring & Assessment: Nitrogen7 N
NITROGEN
Common forms of Nitrogen that are evaluated in water:
Nitrate-nitrogen:
Drinking Water  EPA MCL = 10 mg/L
Surface Water  No standard; typically ≤ 1 mg/L
Nitrite-nitrogen:
Drinking Water  EPA MCL = 1 mg/L
Surface Water  No standard; typically ≤ 0.1 mg/L
Maximum Contaminant Level (MCL)
Total Nitrate  MCL = 44.3 mg/L
Wastewater Treatment Plant Effluents:
Nitrates may be in excess of 40 mg/L
Nitrites may be in excess of 5 mg/L

13. Monitoring & Assessment: Nitrogen7 N
NITROGEN
Nitrite-nitrogen:
Drinking Water  EPA MCL = 1 mg/L
Surface Water  No standard; typically ≤ 0.1 mg/L
To convert µg-at NO2/L to mg/L:
[µg-at NO2/L] × [(Molecular Weight of Nitrogen) ÷ (Molecular Weight of NO2)] × [0.001] = mg-N/L
To convert µg-at NO2/L to mg/L:
[µg-at NO2/L] × = mg-N/L
*** You need this conversion for the lab calculations this week!! ***

14. Monitoring & Assessment: Phosphorous
Common forms of Phosphorous that are evaluated in water:
Total Phosphorous:
Drinking Water  No standard
Surface Water – Lakes & Reservoirs  20 µg/L
Surface Water – Rivers & Streams  µg/L
Orthophosphate:
Surface Water – Lakes, Reservoirs, Streams, & Rivers  0.05 mg/L
Surface Water – Estuaries  mg/L
Surface Water – Marine and Coastal  0.05 mg/L
The listed criteria are for EPA aggregate ecoregion IX (that’s us here in the Southeastern plains, hills, and coastal region)
The recommended water quality criteria are set to reduce problems associated with excess nutrients in waterbodies in specific areas of the country
There are no standards set for drinking water because phosphorous is not a known toxicant to humans – unless consumed in unnatural quantities not expected to be seen in any environment.
Some drinking water facilities (such as Sweeney) even add phosphorous to the water to coat/protect the water pipes. And check out your sodas and many proteins (milk, nuts, beans, wheat, etc.)…some contain them because they were added to keep the product “fresh”…other foods simply have them from natural uptake in the environment (plants love phosphorous!).
However, increases in nutrients can lead to algal blooms which can in turn be toxic in drinking water!  an excellent example of this happened in Toledo, Ohio in August of 2014 when the algae toxins in Lake Eerie were so high that residents were in a water emergency. (Click the EPA image to watch a 10 minute PBSnews video about this story…you may not want to watch the entire video, kind of long!)

15. Methods for Measuring Nutrients
Quick, cheap (less accurate)
Test strips
Test kits
Color discs
More Expensive (more accurate)
Nitrite ion-specific electrodes
Phosphorous-specific electrodes
Spectrophotometric methods
-- Click thru images of each method –
Quick, cheap, less accurate:
Test strips
Test kit
Color disc
More Expensive, more accurate:
Nitrite ion-specific electrode
Phosphorous-specific electrodes
Spetrophotometer
Images source: jenway.com

16. Spectrophotometric Analysis: Nitrite
Naphthyl*
Water Sample
Sulfanilamide
Diazonation Rxn
543 nm
Abbreviated explanation of spectrophotometric methods for the measurement of Nitrite in water samples:
Reagents Sulfanilamide & N-(1-naphthy) ethylenediamine dihydrochloride are added and allowed to react with any nitrite present in a water sample
If nitrite is present, a diazonation reaction occurs in which sulfanilamide reacts with the nitrite to create a diazonium salt.
When the Naphthyl is added, a coupling reaction occurs that creates an azo dye compound (if nitrite is present)
The more nitrite that is present, the more azo dye compound that is created  resulting in a pink/red color
The more azo dye that is created, the higher the absorbance of light will be in the spectrophotometer (remember Beer’s Law = the concentration is directly proportional to the absorbance)
We will measure absorbance at 543 nm as this is the optimal wavelength for the azo dye compound
*** Similar analysis for phosphate analysis…just different reagents and different wavelength used ****
Images mined from Google Image search
* Naphthyl = N-(1-naphthyl) ethylenediamine dihydrochloride

17. Let’s Review & See a Quick Experiment:
5 minute YouTube video: University of Connecticut – Marine Science Dept: Nutrient Pollution Video

18. Lab Procedure Let’s get out our Lab Manuals…
Determination of Nitrite (pg. 71)

19. Pre-made Reagents: Sulfanilamide Solution
N-(1-naphthyl) ethylenediamine dihydrochloride
Primary Standard
0.345 g/L Sodium Nitrite Solution
Images mined from Google Image search

20. You will make… 1 Run Standard 3 Nitrite Standards 2 Reagent Blanks
A dilution of the pre-made Primary Standard
1:100
Used to make Nitrite Standards
3 Nitrite Standards
A dilution of Run Standard
1:25
Used to determine the Calibration (F) factor
F-factor should be around 2.00
2 Reagent Blanks
Used to “carry” the reagents in DIW – without nitrite present
These are blanks – their ABS will be mathematically blanked from final results
Run Standard (1): a “working” solution for each student group to use at lab station  used to create Nitrite Standards
Nitrite Standards (3): used to determine F-factor; essentially, we know the concentration of nitrite in these solutions, therefore, we can calibrate our unknown samples (e.g., Cape Fear River water sample) with this known amount
Reagent Blanks (2): Blanks; used to determine the Calibration Factor
Image sources:
aliexpress.com
amazon.com

21. Spectrophotometric Analysis: Standards & Blanks
1.0 mL Sulfanilamide
Mix & wait 2 minutes
1.0 mL Naphthyl*
Mix & wait 10 minutes
What will we use to blank the spectrophotometer?
543 nm
Nitrite Standards
Reagent Blanks
Diazonation Rxn
Nitrite Standards
Reagent Blanks
Images mined from Google Image search
* Naphthyl = N-(1-naphthyl) ethylenediamine dihydrochloride

22. Calculate F-factor Average absorbance of 3 Nitrite Standards (ABSstnd)
Average absorbance of 2 Reagent Blanks (ABSrb)
F1cm =
(ABSstnd ABSrb) × 10
This value should be around 2.00 which denotes that the
standards that you created are accurate & reliable.
The F-factor let’s us know that the standards that we created are accurate & reliable  which ultimately means that the reagents were made correctly & the techniques that you are using are precise enough to continue.

23. Spectrophotometric Analysis: Water Sample
1.0 mL Sulfanilamide
Mix & wait 2 minutes
1.0 mL Naphthyl*
Mix & wait 10 minutes
What will we use to blank the spectrophotometer?
543 nm
Cape Fear River Water Sample
Diazonation Rxn
Cape Fear River Water Sample
Images mined from Google Image search
* Naphthyl = N-(1-naphthyl) ethylenediamine dihydrochloride

24. Spectrophotometric Analysis: Turbidity Blank
The water sample was blanked in the spectrophotometer using DIW
However, the Cape Fear River water must also be blanked
We will accomplish this mathematically
543 nm
Cape Fear River Water
Must be calculated because the Spectrophotometer will not allow us to zero out more than one blank
= ABSturbidity
Images source: jenway.com

25. Calculate Corrected Sample Absorbance
Corrected Sample Absorbance = ABSsample ABSrb - ABSturbidity
This results in the actual absorbance of the nitrite in the sample
You have mathematically ‘blanked’ the equation
Removing the absorbance of the Reagent Blanks and the turbidity present in the Cape Fear River water

26. Calculate Amount of Nitrogen in Sample
g-at N/L = (Corrected Sample) × F-factor You can convert this value to mg/L to compare with EPA standards: mg/L of Nitrogen = (g-at N/L) × (atomic mass N) ÷ 1000

27. You could use the example data sheet provided in your lab manual to record your data in your lab notebook this week (looks like this one)…or…

28. Data: Nitrite Standards ABS Nitrite Standards ABS Water Sample Name
Nitrite Std #1
Nitrite Std #2
Nitrite Std #3
Avg. Nitrite Std. ABS
Nitrite Standards
ABS
Reagent Blnk #1
Reagent Blnk #2
Avg. Reagent Blnk ABS
Water Sample Name
Water Sample ABS
Avg. Reagent Blank ABS
Turbidity Blank ABS
Corrected Sample ABS
You could make individual tables in your lab notebook (like these) to record your data.
Just keep it neat and legible!