Spectrophotometry and nutrient analysis Nutrients: the “bread and butter of phytoplankton” Edited : Dr. Anastasios Anestis

Water pollution Industrial waste Marine – Lakes - Rivers  Every physical, chemical or biological alteration that renders water unsuitable for the organisms that live in it and use it UrbanwasteFertilisers

Eutrophication  Increase of phytplankton pigment concentration due to continuous flow of nutrients to an aquatic system  Increase of zooplankton populations  Accumulation of dead organic matter for decomposition  Increase of decomposers  Increase of biological and chemical oxygen demand (BOD and COD respectively)  Asphyxia of macrofauna (e.g. fish) and macroalgae

Nutrients  Mainly inorganic compounds (ΝΟ 3 -, ΝΟ 2 - PΟ 4 3-, ΝH 4 + και SiΟ 2 )  Organic compounds (urea, amino acids etc.)  They play a major role in aquatic ecosystems – essential “food” for phytoplankton

Ammonium (ΝH 4 + )  Easily assimilated by all planktonic organisms.  Toxic in high concentrations (e.g. the Bay of Elefsina 4x higher than the rest of the Aegean Sea).  It represents an indication of urban wastes and leaching of fertilisers containing ammonium.  In the water: ΝH 3 + Η 2 Ο  ΝH ΟΗ -  As temperature or pH increase the reaction shifts to the right.

Nitrites (ΝΟ 2 - )  Indicative of urban and industrial wastes  They give a general sense of the level of pollution  They create organic salts and as a result they produce an intense azo-dye (purple) measured spectrophotometrically  The more intense the colour the higher the concentration of ΝΟ 2 -.

Nitrates (ΝΟ 3 - )  The final stage of oxidation of nitrogen compounds  In untreated waste (for e.g. Elefsina’s Bay) nitrogen is bound in organic compounds and therefore concentration of nitrates is relatively small  In treated waste (for e.g. near Psitalia’s sewage treatment plant) they represent the most abundant source of nitrogen due to nitrification  They are measured after they are reduced (Cu-Cd reduction) to nitrites

Phosphates (PΟ 4 3- )  Indicative of urban waste and leaching of fertilisers containing phosphates.

Dissolved Oxygen (D.O.)  Dissolved oxygen in the water is a crucial environmental variable for the well-being of the ecosystem  Seaweeds and all forms of organic matter when they are decomposed DO is used  DO concentration is so low that life in the water cannot be supported, resulting to death by asphyxia

Methodology (Spectrophotometry)  Samples are filtered through a 0,45μm millipore filter to remove the particulate matter  Reagents are added to react with the salts we want to measure to produce a colour product  The produced coloured products can absorb at a specific wavelength and this absorption is measured against the readings of a standard solution

How absorbance is measured (Spectrophotometry) A spectrophotometer is a relatively simple piece of equipment composed of: A spectrophotometer is a relatively simple piece of equipment composed of:  A source of light usually Xe or Ar, that transmits light in the visible region ( nm)  A prism that separates light radiation and allows the passage of monochromatic radiation that can be absorbed by the salts in the sample  A quvette of known diameter  A photosensitive cells that produces an electical charge in proportion to the intensity of light that hits it after it has pass through the sample  An amplifier and a counter for the measurement of light intensity i.e. the concentration of the salt in the sample c in mg/L (milligrams per litre)

In a few words Spectrophotometry is a relatively simple measurement i.e. Light source Prism Quvette Photo cell Signal amplifier- Concentration counter Monochromatic radiation

Be aware that…  When we measure absorbance we must be careful to avoid anything that has the potential to affect the initial sample concentration; for e.g. inappropriate storage, improper filtering, contamination etc.  Do not leave any fingerprints on the quvette-absorbance will be affected

Absorbance of radiation Generally absorbance depends on 3 main factors: 1.The ion (salt) that we want to determine its concentration 2.The distance that light covers in the quvette 3.The initial concentration of the compound (nutrient) we want to measure So the absorbance of radiation is proportionate of the concentration of the compound we want to measure (for the specified compound) and the distance that the light has to travel

The greater the absorbance the greater the concentration of the compound we want to measure

The passage of light through the sample Sample in the quvette Initial light intensity Transmitted light intensity (measurable) NO 3 - NO 2 - PO 4 -3

Finally… Nutrient analysis and other environmental variables follows detailed analytical protocols Accuracy in the analysis is achieved through careful planning and experience Processing and results of data can lead to safe conclusions for the sustainable management of coastal ecosystems In the Mediterranean there is an immediate relationship between environmental variables with the economy; e.g. fisheries, tourism, waste management and freshwater management