Wetlands are currently degraded by both natural and anthropogenic activities, which deteriorate their quality, and push them to the brink of extinction in the process of unplanned development, giving rise to the need for suitable conservation strategies. Unfortunately, over the years, less attention has been given to wetland losses world over, including Bangalore. The degradation of the wetlands has altered their functions, affecting the ecological balance. The present study of water bodies in Bangalore attempts to: 

1.     Assess the water quality of selected water bodies through physico-chemical parametric studies. Generally, wetland functions directly relate to their physical, chemical and biological integrity. Water quality evaluation for wetlands leads to information about their misuse by indicating the pollution status. Gale et al. (1993) define water quality objectives as the overall direction and purpose of the project, and furthermore define goals as milestones to be met during the course of a project. Since the quality of aquatic life depends on water quality, a thorough assessment of the same becomes an integral part of wetland evaluation. The assessment of the chemical criteria of the water body helps in:

·         Evaluating the chemicals that cause toxicity to aquatic life.

·         Studying the long-term effects on the ecosystem.

·         Conducting the status and monitoring of wetland resources by studying their physico-chemical and biological parameters.

2.        Designate uses that protect the structure and function of wetlands for protection of fish, birds, wildlife, and recreation.

The baseline values attached to wetlands in terms of designating the viable usage of these water bodies based on established standards protecting their functions is also attempted.

3.        Analyse the qualitative and quantitative aspects of plankton population of the water bodies.

The biological integrity of the wetlands is the driving force for their sound ecological functioning. Wetlands that support a vast diversity of fish, birds, mammals, etc., depend directly on it by supporting vast and diverse forms of plankton providing a nutrient base and a complex food-web. In many cases, planktons also act as the biological indicators of pollution being very sensitive to changes in water quality.

 Water is a dynamic medium and its quality varies spatially and temporally. In order to characterise any water body, studies on the major components, hydrology, physico-chemical and biological characteristics, should be carried out.

 Hydrological  features:

A thorough knowledge of the hydrological properties of the water body must be acquired before an effective water quality monitoring system is established. Each of the inland water body is characterised by the following unique hydrological features.

Large variations in the water residence time occur in different types of inland water bodies. The hydrodynamic characteristics of each type of water body are highly dependent on the size of the water body, climatic conditions and the drainage pattern associated with it. Ground water greatly depends on the recharge regime i.e. infiltration through unsaturated aquifer zone, that allows renewal of the groundwater.

 Physical, chemical and biological properties:

The physical and chemical properties of a freshwater body are characteristic of the climatic, geochemical, geomorphological and pollution conditions (largely) prevailing in the drainage basin and the underlying aquifer. The biota in the surface water is governed entirely by various environmental conditions that determine the selection of species as the physiological performance of the individual organisms. The primary production of organic matter, in the form of phytoplankton and macrophytes is more intense in lakes and reservoirs than in rivers. In contrast to the chemical quality of waterbodies, which can be measured by suitable analytical methods, biological quality is a combination of both qualitative and quantitative characterisation. This can be carried out in two levels:

 SAMPLING

The sample collected should be small in volume, enough to accurately represent the whole water body. The water sample tends to modify itself to the new environment. It is necessary to ensure that no significant changes occur in the sample and preserve its integrity till analysed (by retaining the same concentration of all the components as in the sample). The essential objectives of water quality assessment are to:

 Sampling strategies

The sampling strategies may be as follows:

 Stratified random sampling: Here, the sample sites are selected randomly within the areas related to the more homogenous components of an otherwise heterogeneous variable. For example, sampling can be done within the epilimnion, thermocline and hypolimnion of a stratified lake or reservoir as mere sampling of the surface water will not give a true picture of the water quality. Instead sampling can be done at deep water and littoral zones of an aquatic habitat.

 Systematic sampling: This sampling method is considered most satisfactory as it gives a more representative sample for water quality assessment. Sample sites are chosen at appropriate locations so as to cover the entire water body. Sampling is carried out at regular depths so as to get various depth profiles. The sampling locations are also decided on the basis of pollution loads entering the river and other important events in the water body like organised bathing, sewage entry points and tributary entry points.

 Rapid sampling: This sampling is carried out when there is constraint of time for detailed sampling. A rapid assessment of the water quality can be done by mixing equal volumes of water from different locations of water body. However, this method cannot give detailed assessment of various habitats of the water body.

 Site selection

Sampling sites for the water body/lake are selected to represent the water quality at different points and depths. Generally sampling test sites selected for monitoring include:

Generally three types of water samples are collected.

 Sampling frequency

The quality of water varies with time in a water body due to various natural and human induced factors. The monitoring has to be done in a way that records all the changes in the quality. The sampling frequencies generally adopted in monitoring are:

Variations in water quality are mainly due to changes in the concentrations of the components of the water flowing into the water body. These variations can be man-made or natural and can either be cyclic or random.

In lakes, the mass of water and good lateral mixing provide inertia against any rapid modifications due to inputs and outputs.

 Sampling container

The sampling container should not react with the sample, be of adequate capacity to store the sample and be free from contamination.

 Sampling method

To characterise the water quality of wetlands in Bangalore, seven tanks were selected based on their location and the source of pollution. They were Bannergatta, Hebbal, Kamakshipalya, Madivala, Sankey, Ulsoor and Yediur. Grab sampling was done at the inlet, centre and outlet in most of the waterbodies studied to assess their physical and chemical qualities at monthly intervals, except during some seasons when the centre of the lake was not accessible due to excessive growth of water hyacinth. The samples were collected in thoroughly cleaned 2.5-litre inert plastic containers, which were rinsed with distilled and lake/tank water before collection.

 The sampling time for respective tanks was as follows:

Note:

Water samples were collected in a sampling bottle avoiding floating materials. The stoppers of the sample containers were closed properly to prevent outside contamination. The container was labelled describing the name of the water body, date, time, sampling-point, and conditions under which it was sampled.

 Preservation of the sample

Between the time a sample is collected and analysed in the laboratory, physical, chemical and biochemical reactions may take place in the sample container leading to changes in the intrinsic quality of the sample, making it necessary to prevent or minimize these changes with suitable preservatives such as alcohol and mercuric chloride. Highly unstable parameters such as pH, temperature, transparency, free carbon-di-oxide, dissolved oxygen, etc., are measured at the sampling site.

 The preservation procedure includes keeping the samples in the dark, adding chemical preservative (Table 14), lowering the temperature to retard reactions or combinations of these.

 Table 14: Preservation Methods

(Source: Analytical Methods Manual, Water quality branch, Environment Canada, 1981)

 PARAMETRIC ANALYSES

The parametric analyses carried out to assess the water quality are broadly divided into:

Field measurement: The field parameters measured include pH, conductivity, dissolved oxygen, temperature and transparency.

 Physical parameters

 COLOUR

In natural water, colour is due to the presence of humic acids, fulvic acids, metallic ions, suspended matter, plankton, weeds and industrial effluents. Colour is removed to make water suitable for general and industrial applications and is determined by visual comparison of the sample with distilled water.

 Visual comparison: About 20ml of the sample and 20ml of distilled water are taken in two separate wide mouthed test tubes. The results are tabulated (as clear, greenish, greyish, brownish, blackish, etc) by comparing the colour of the sample with distilled water.

 TEMPERATURE

Impinging solar radiation and atmospheric temperature brings about spatial and temporal changes in temperature, setting up convection currents and thermal stratification. Temperature plays a very important role in wetland dynamism affecting the various parameters such as alkalinity, salinity, dissolved oxygen, electrical conductivity etc. In an aquatic system, these parameters affect the chemical and biological reactions such as solubility of oxygen, carbon-di-oxide, carbonate-bicarbonate equilibrium, increase in metabolic rate and affects the physiological reactions of organisms, etc. Water temperature is important in relation to fish life. The temperature of drinking water has an influence on its taste. 

 Apparatus required: Thermometer- 0.1^o C division.

 Procedure: Temperature measurement is made by taking a portion of the water sample (about 1litre) and immersing the thermometer into it for a sufficient period of time (till the reading stabilizes) and the reading is taken, expressed as °C.

 TRANSPARENCY (LIGHT PENETRATION)

Solar radiation is the major source of light energy in an aquatic system, governing the primary productivity. Transparency is a characteristic of water that varies with the combined effect of colour and turbidity. It measures the light penetrating through the water body and is determined using Secchi disc.

 Apparatus required: Secchi disc, a metallic disc of 20cm diameter with four quadrats of alternate black and white on the upper surface. The disc with centrally placed weight at the lower surface is suspended with a graduated cord at the centre.

 Procedure: Transparency is measured by gradually lowering the Secchi disc at respective sampling points. The depths at which it disappears  (X₁) and reappears (X₂) is noted. The transparency of the water body is computed as follows                  

   Transparency (Secchi Disc Transparency)     =              (X₁  + X₂ )/2                            

Where, X₁ = Depth at which Secchi disc disappears

             X₂ = Depth at which Secchi disc reappears

 TURBIDITY

Turbidity is an expression of optical property, wherein light is scattered by suspended particles present in water (Tyndall effect) and is measured using a nephelometer. Suspended and colloidal matter such as clay, silt, finely divided organic and inorganic matter, plankton and other microscopic organisms cause turbidity in water. Turbidity affects light-scattering, absorption properties and aesthetic appearance in a water body. Increase in the intensity of scattered light results in higher values of turbidity.

 Apparatus required: Nephelometer (it detects scattered light at 90^o to the incident beam of light. It consists of a light source for illuminating the sample. One or more photoelectric detectors with a display unit indicate the intensity of light scattered at 90^o to the path of incident light), sample cells, lab glassware and Monopan balance.

 Principle: Nephelometric measurement is based on comparison of the intensity of scattered light of the sample with the intensity of light scattered by a standard reference suspension (Formazin polymer) under similar conditions.

 Reagents:

·         Solution 1:1.0 g Hydrazine sulphate is dissolved in 100ml of distilled water.

·         Solution 2: 10.0g of Hexamethylenetetramine is dissolved in distilled water and made up to 100ml in a volumetric flask.

 Procedure: The nephelometer is calibrated using distilled water (Zero NTU) and a standard turbidity suspension of 40NTU. The thoroughly shaken sample is taken in the nephelometric tube and the value is recorded.

                Turbidity (NTU) = (Nephelometer readings) (Dilution factor*)

* If the turbidity of the sample is more than 40 NTU, then the sample is diluted and the dilution factor is accounted in final calculations.

 Chemical parameters

 pH:

The effect of pH on the chemical and biological properties of liquids makes its determination very important. It is one of the most important parameters in water chemistry and is defined as -log [H⁺], and measured as intensity of acidity or alkalinity on a scale ranging from 0-14. If free H⁺ are more it is expressed as acidic (i.e pH<7) and if OH^- ions are more then it is expressed as alkaline (i.e pH> 7).

 In natural waters, pH is governed by the equilibrium between carbon dioxide/bicarbonate/carbonate ions and ranges between 4.5 and 8.5 although mostly basic. It tends to increase during day largely due to the photosynthetic activity (consumption of carbon-di-oxide) and decreases during night due to respiratory activity. Wastewater and polluted natural waters have pH values lower or higher than 7 based on the nature of the pollutant.

 (* The colorimetric indicator method can be used only for approximate pH values). 

 Apparatus required:

 Procedure:

 ELECTRICAL CONDUCTIVITY

Conductivity (specific conductance) is the numerical expression of the water's ability to conduct an electric current. It is measured in microSiemens per cm and depends on the total concentration, mobility, valence and the temperature of the solution of ions. Electrolytes in a solution disassociate into positive (cations) and negative (anions) ions and impart conductivity. Most dissolved inorganic substances are in the ionised form in water and contribute to conductance. The conductance of the samples gives rapid and practical estimate of the variation in dissolved mineral content of the water supply. Conductance is defined as the reciprocal of the resistance involved and expressed as mho or Siemen (s).

   G=(1/R)

G – Conductance (mho or Siemens) and R - Resistance

 Apparatus required: Conductivity meter

 Procedure: The electrode of the conductivity meter is dipped into the sample, and the readings are noted for stable value shown as mS/cm.

 TOTAL SOLIDS:

Total solids is the term applied to the residue left in the vessel after evaporation of the sample and its subsequent drying in an oven at a temperature of 103-105 ^oC. Total solids include Total Suspended Solids (TSS) and Total Dissolved Solids (TDS).

 Principle: A known volume (50 ml) of well-mixed sample is evaporated in a pre-weighed dish and dried to constant weight in an oven at 103-105 ^o C. The increase in weight over that of the empty dish gives the total solids.

 Apparatus: Evaporating dishes - 100ml porcelain dish, steam bath, drying oven, desiccator, Monopan balance and measuring jars.

 Procedure: A known volume of the well-mixed sample (50ml) is measured into a pre-weighed dish and evaporated to dryness at 103 ^o C on a steam bath. The evaporated sample is dried in an oven for about an hour at 103-105 ^o C and cooled in a desiccator and recorded for constant weight.

 Calculation:

                                Total solids  (mg/L)   =       (W₁-W₂) (1000) / Sample volume (ml)

W₁    = Weight of dried residue + dish

W₂  = Weight of empty dish

 TOTAL DISSOLVED SOLIDS

Dissolved solids are solids that are in dissolved state in solution. Waters with high dissolved solids generally are of inferior palatability and may induce an unfavourable physiological reaction in the transient consumer.

 Principle: The difference in the weight of total solids and the total suspended solids expressed in the same units gives the total dissolved solids.

 Apparatus: Glass-fiber filter disks, membrane filter funnel, filtration apparatus, suction flask and pump, drying oven and Grooch crucible.

 Procedure: The difference in the weights of Total Solids (W₁) and Total Suspended Solids (W₂) expressed in the same units gives Total Dissolved Solids (TDS).

 Calculation:    

                                Total Dissolved Solids  (mg/L)  =   (W₁-W₂) (1000) / Sample volume (ml)

W₁    = Weight of total solids + dish

W₂  = Weight of total suspended solids         

 TOTAL SUSPENDED SOLIDS

Suspended solids are the portions of solids that are retained on a filter of standard specified size (generally 2.0 µ) under specific conditions. Water with high-suspended solids is unsatisfactory for bathing, industrial and other purposes.

 Principle: A well – mixed sample is filtered through a weighed standard glass fibre filter and the residue that is retained on the filter is dried to a constant weight at 103-105 ^o C. The increase in the weight of the filter determines the total suspended solids.

 Apparatus: Porcelain dish (100ml capacity), glass fiber filter disk, suction pump and flask, measuring jar, membrane filter funnel, oven and filtration apparatus.

 Procedure: The known volume of vigorously shaken sample (50ml) is filtered into a pre-weighed glass fibre filter disk fitted to suction pump, and washed successively with distilled water. The filter is carefully removed from the filtration apparatus and dried for an hour at 103-105 ^o C in an oven, cooled in dessicator and weighed for constant weight.

 Calculation:

                                Total Suspended Solids  (mg/L)  =  (W₁-W₂) (1000) /  Sample volume (ml) 

W₁    = Weight of dried glass fibre filter + residue

W₂  = Weight of glass fibre filter disk before filtering  

 TOTAL HARDNESS

Hardness is predominantly caused by divalent cations such as calcium, magnesium, alkaline earth metal such as iron, manganese, strontium, etc. The total hardness is defined as the sum of calcium and magnesium concentrations, both expressed as CaCO₃ in mg/L. Carbonates and bicarbonates of calcium and magnesium cause temporary hardness. Sulphates and chlorides cause permanent hardness. 

Table 15: Hardness Chart (for drinking water)

Principle: In alkaline conditions, EDTA (Ethylene-diamine tetra acetic acid) and its sodium salts react with cations forming a soluble chelated complex when added to a solution. If a small amount of dye such as Eriochrome black-T is added to an aqueous solution containing calcium and magnesium ions at alkaline pH of 10.0 ± 0.1, it forms wine red colour. When EDTA is added as a titrant, all the calcium and magnesium ions in the solution gets complexed resulting in a sharp colour change from wine red to blue, marking the end point of the titration. Hardness of water prevents lather formation with soap rendering the water unsuitable for bathing and washing. It forms scales in boilers, making it unsuitable for industrial usage. At higher pH>12.0, Mg⁺⁺ ion precipitates with only Ca⁺⁺ in solution. At this pH, murexide indicator forms a pink color with Ca⁺⁺ ion. When EDTA is added, Ca⁺⁺ gets complexed resulting in a change from pink to purple indicating end point of the reaction.

  Apparatus required: Lab glassware - burette, pipette, conical flask, beakers, etc.

 Reagents:

 Procedure: Exactly 50ml of the well-mixed sample is pipetted into a conical flask, to which 1ml of ammonium buffer and 2-3 drops of Eriochrome black -T indicator is added. The mixture is titrated against standard 0.01M EDTA until the wine red colour of the solution turns pale blue at the end point.

 Calculation:

                                Total hardness (mg/L) =           (T) (1000) /  V

Where, T = Volume of titrant

V = Volume of sample

 CALCIUM HARDNESS

The presence of calcium (fifth most abundant) in water results from passage through or over deposits of lime stone, dolomite, gypsum and other calcium bearing rocks. Calcium contributes to the total hardness of water and is an important micro-nutrient in aquatic environment and is especially needed in large quantities by molluscs and vertebrates. It is measured by EDTA titrimetric method. Small concentration of calcium carbonate prevents corrosion of metal pipes by laying down a protective coating. But increased amount of calcium precipitates on heating to form harmful scales in boilers, pipes and utensils.

 Principle: When EDTA (Ethylene-diamine tetra acetic acid) is added to the water containing calcium and magnesium, it combines first with calcium. Calcium can be determined directly with EDTA when pH is made sufficiently high such that the magnesium is largely precipitated as hydroxyl compound (by adding NaOH and iso-propyl alcohol). When murexide indicator is added to the solution containing calcium, all the calcium gets complexed by the EDTA at pH 12-13. The end point is indicated from a colour change from pink to purple. 

Apparatus required: Burettes, pipette, conical flask, beakers and droppers.

  Reagents:

 Procedure: A known volume (50ml) of the sample is pipetted into a clean conical flask, to which 1ml of sodium hydroxide and 1ml of iso-propyl alcohol is added. A pinch of murexide indicator is added to this mixture and titrated against EDTA until the pink color turns purple.

 Calculation:

                                Calcium as Ca   (mg/L)  =  T  (400.5) (1.05) / Sample taken, ml

Where, T= volume of titrant, ml

                                  Calcium hardness   (mg/L as CaCO₃)  =   T  (1000) (1.05)  / Sample taken, ml                               

 MAGNESIUM HARDNESS

Magnesium is a relatively abundant element in the earth's crust, ranking eighth in abundance among the elements. It is found in all natural waters and its source lies in rocks, generally present in lower concentration than calcium. It is also an important element contributing to hardness and a necessary constituent of chlorophyll. Its concentration greater than 125 mg/L can influence cathartic and diuretic actions.

 Principle: Magnesium hardness can be calculated from the determined total hardness and calcium hardness.

 Calculation:

                Magnesium (as Mg, mg/L)  = (T - C) x 0.243

 where, T = Total hardness mg\L as CaCO₃

            C = Calcium hardness mg\L as CaCO₃

 High concentration of magnesium proves to be diuretic and laxative, and reduces the utility of water for domestic use while a concentration above 500 mg/L imparts an unpleasant taste to water and renders it unfit for drinking. Chemical softening, reverse osmosis and electro dialysis or ion exchange reduces the magnesium hardness to acceptable levels.

 NITRATES

Nitrates are the most oxidized forms of nitrogen and the end product of the aerobic decomposition of organic nitrogenous matter. The significant sources of nitrates are chemical fertilizers from cultivated lands, drainage from livestock feeds, as well as domestic and industrial sources. Natural waters in their unpolluted state contain only minute quantities of nitrates. The stimulation of plant growth by nitrates may result in eutrophication, especially due to algae. The subsequent death and decay of plants produces secondary pollution. Nitrates are most important for biological oxidation of nitrogenous organic matter. Certain nitrogen fixing bacteria and algae have the capacity to fix molecular nitrogen in nitrates. The main source of polluting nitrates is the domestic sewage let into water bodies. Nitrates may find their way into ground water through leaching from soil and at times by contamination. They can be measured by the phenoldisulphonic method.

 Principle: Nitrates react with phenoldisulphonic acid and produce a nitrate derivative, which in alkaline solution develops yellow colour due to rearrangement of its structure. The colour produced is directly proportional to the concentration of nitrates present in the sample.

 Apparatus required:  Nessler's tube, pipettes, beakers, spectrophotometer, cuvettes, measuring jar and hot water bath.

 Reagents:

·         Stock nitrate solution: 721.8 mg (0.722g) of AR potassium nitrate is dissolved in distilled water and made up to 100ml for stock solution.

·         Standard nitrate solution: Standard nitrate solution is prepared by evaporating 50ml of the stock solution to dryness in the water bath. The obtained residue is dissolved in 2ml of phenol disulfonic acid and diluted to 500ml, to give 1ml = 10 mg. The solution of various strengths ranging from 0.0 (blank) to 1.0 mg/L at intervals of 0.2 mg/L is prepared by diluting stock solution with distilled water.                           

Procedure: A known volume (50ml) of the sample is pipetted into a porcelain dish and evaporated to dryness on a hot water bath. 2ml of phenol disulphonic acid is added to dissolve the residue by constant stirring with a glass rod. Concentrated solution of sodium hydroxide or conc. ammonium hydroxide and distilled water is added with constant stirring to make it alkaline. This is filtered into a Nessler's tube and made upto 50ml with distilled water. The absorbance is read at 410nm using a spectrophotometer after the development of colour. The standard graph is plotted by taking concentration along X-axis and the spectrophotometric readings (absorbance) along Y-axis. The value of nitrate is found by comparing absorbance of sample with the standard curve and expressed in mg/L.

 Calculation:

 Nitrates (as NO₃ mg/L)  =   Absorbance of sample  X  Conc. of std  X 1000 / Absorbance of Std. X Sample taken

 The high concentration of nitrate in water is indicative of pollution.

 PHOSPHATES

Phosphates occur in natural or wastewaters as orthophosphates, condensed phosphates and naturally found phosphates. Their presence in water is due to detergents, used boiler waters, fertilizers and biological processes. They occur as detritus in the bodies of aquatic organisms. They are essential for the growth of organisms and a nutrient that limits the primary productivity of the water body. Inorganic phosphorus plays a dynamic role in aquatic ecosystems and is one of the most important nutrients when present in low concentration, but in excess along with nitrates and potassium, causes algal blooms. It is calculated by the stannous chloride method.

 Principle: In acidic conditions orthophosphate reacts with ammonium molybdate forming Molybdophosphoric acid, reduced further to molybdenum blue by stannous chloride. The intensity of the blue colour is directly proportional to the concentration of phosphate. The absorbance is noted at 690nm using spectrophotometer.

 Apparatus required: Spectrophotometer, lab glassware, hot plate and Nessler's tube.

 Reagents:

 Procedure: To 50ml of the filtered sample, 4ml of ammonium molybdate reagent and about 4-5 drops of stannous chloride reagent is added. After about 10 min but before 12 min, the colour developed is measured photometrically at 690nm and calibration curve is prepared. A reagent blank is always run with same treatment with distilled water as sample. The value of phosphate is obtained by comparing absorbance of sample with the standard curve and expressed as mg/L.

 Calculation:

 Phosphates  (as P mg/L)     =      Absorbance of sample X  Conc. of std  X 1000 / Absorbance of Std. X Sample taken

 High phosphorus content causes increased algal growth till nitrogen becomes limiting, although blue green algae continues to dominate because of its ability to utilize molecular nitrogen. Besides sedimentation, high uptake by phytoplankton is one of the reasons for fast depletion of phosphorus in water.

 SULPHATES

Sulphates are found appreciably in all natural waters, particularly those with high salt content. Besides industrial pollution and domestic sewage, biological oxidation of reduced sulphur species also add to sulphate content. Soluble in water, it imparts hardness with other cations. Sulphate causes scaling in industrial water supplies, and odour and corrosion problems due to its reduction to hydrogen sulphide. It can be calculated by turbidometric method.

 Principle: Sulphate ions are precipitated in acetic acid medium with barium chloride to form barium sulphate crystals of uniform size. The scattering of light by the precipitated suspension (barium sulphate) is measured by a Nephelometer and the concentration is recorded.

 Apparatus required: Nephelometer, magnetic stirrer, Nessler's tubes and lab glassware.

 Reagents:

·         Conditioning reagent: 50 ml of glycerol was mixed in a solution containing 30 ml of conc. hydro chloric acid, in 300ml distilled water (10% HCl), 100 ml of 95% ethyl alcohol or isopropyl alcohol and 75g NaCl.

·         Barium Chloride.

·         Standard sulphate solution: 147.9mg of AR grade sodium sulphate was dissolved in distilled water and made up to 1000ml, to give 1ml = 100mg sulphate.

 Procedure: 100ml of the sample is filtered into a Nessler's tube containing 5ml of conditioning reagent. About 0.2g of barium chloride crystals is added with continued stirring. A working standard is prepared by taking 1ml of the standard, 5ml of conditioning reagent and made up to 100ml, to give 100 NTU. The turbidity developed by the sample and the standards are measured using a Nephelometer and the results are tabulated.

 Calculation:

Sulphate (as SO_4, mg/L)   =   (Nephlometric reading) (0.4) (Dilution Factor)

 CHLORIDES

The presence of chlorides in natural waters can mainly be attributed to dissolution of salt deposits in the form of ions (Cl^-). Otherwise, high concentrations may indicate pollution by sewage or some industrial wastes or intrusion of sea water or other saline water. It is the major form of inorganic anions in water for aquatic life. A high chloride content has a deleterious effect on metallic pipes and structures, as well as agricultural plants. They are calculated by Argentometric method.               

Principle: In alkaline or neutral solution, potassium chromate indicates the endpoint of the silver nitrate titration of chlorides. Silver chloride is quantitatively precipitated before the red silver chromate is formed.

 Apparatus required:  Lab glassware.

 Reagents:

·         Potassium chromate indicator solution: 50g of potassium chromate is dissolved in minimum amount of distilled water and silver nitrate is added drop wise till a red precipitate is formed. The mixture is allowed to stand for about 12 hours and diluted to 1000ml with distilled water.

·         Silver nitrate solution (0.014N): 2.395g of silver nitrate is dissolved in distilled water and made up to 1000ml.

 Procedure: A known volume of filtered sample (50ml) is taken in a conical flask, to which about 0.5ml of potassium chromate indicator is added and titrated against standard silver nitrate till silver dichromate (AgCrO₄) starts precipitating.

 Calculation:

  Chlorides (Cl^-) =   (A-B) (N) (35.45) / Sample taken in ml

Where,

A - Volume of silver nitrate consumed by the sample

B - Volume of silver nitrate consumed by the blank

N - Normality of silver nitrate  (Standard methods, APHA, 16th edn, pp 286-88) 

DISSOLVED OXYGEN

Oxygen dissolved in water is a very important parameter in water analysis as it serves as an indicator of the physical, chemical and biological activities of the water body. The two main sources of dissolved oxygen are diffusion of oxygen from the air and photosynthetic activity. Diffusion of oxygen from the air into  water depends on the solubility of oxygen, and is influenced by many other factors like water movement, temperature, salinity, etc. Photosynthesis, a biological phenomenon carried out by the autotrophs, depends on the plankton population, light condition, gases, etc. Oxygen is considered to be the major limiting factor in waterbodies with organic materials. Dissolved oxygen is calculated by many methods.

 Method 1: Membrane electrode method

 Principle: The membrane electrode has a sensing element protected by an oxygen-permeable plastic membrane that serves as a diffusion barrier against impurities. Under steady conditions, the electric current read is directly proportional to the D.O concentrations (electric current is directly proportional to the activity of molecular oxygen).

 Apparatus required: Oxygen-sensitive membrane electrode and lab glassware.

 Procedure: The calibrations are carried out following the manufacturer’s calibration procedure. The electrode is dipped into the sample, and the reading noted.

 Method 2: Winkler’s method

 Principle: Oxygen present in the sample oxidizes the dispersed divalent manganous hydroxide to precipitate as a brown hydrated oxide after addition of potassium iodide and sodium hydroxide. Upon acidification, manganese reverts to its divalent state and liberates iodine from potassium iodide, equivalent to the original dissolved oxygen content of the sample. The liberated iodine is titrated against N/80 sodium thiosulphate using fresh iodine as an indicator.

 Apparatus required: BOD bottles- 300ml capacity, sampling devices, lab glassware - measuring cylinder, conical flasks, etc., and Bunsen burner.

 Reagents:

·         Manganese sulphate: 480g of manganous sulphate tetrahydrate was dissolved and made up to 1000ml with distilled water (Discard if it changes colour with starch).

·         Alkaline iodide-azide reagent: 500g of sodium hydroxide and 150g of potassium iodide along with 10g of sodium azide (NaN₃) was dissolved and made up to 1000ml with distilled water.

·         Conc. sulphuric acid

·         Starch indicator: 0.5g of starch was dissolved in distilled water and was boiled for few minutes.

·         Stock sodium thiosulphate: 24.82g of sodium thiosulphate pentahydrate (Na₂S₂0₂. 5H₂O) was dissolved in distilled water and made upto 1000ml.

·         Standard sodium thiosulphate (0.025N): 250ml of the stock sodium thiosulphate pentahydrate was made upto 1000ml with distilled water to give 0.025N.

 Procedure: The samples were collected in BOD bottles, to which 2ml of manganous sulphate and 2ml of potassium iodide were added and sealed. This was mixed well and the precipitate was allowed to settle down. At this stage 2ml of conc. sulphuric acid was added, and mixed well until all the precipitate dissolved. 203ml of the sample was measured into the conical flask and titrated against 0.025N sodium thiosulphate using starch as an indicator. The end point is the change of colour from blue to colourless.

 Calculations:

                203ml because (200) (300)/ (200-4) = 203ml.

                1ml of 0.025N Sodium Thiosulphate  =  0.2mg of Oxygen

                 Dissolved    Oxygen (as mg/L)   =       (0.2) (1000 ml of Sodium Thiosulphate) / 200

 (Water analysis, APHA, 16th edn, pp 423-17)      

 BIOLOGICAL OXYGEN DEMAND

Biological Oxygen Demand (BOD) is the amount of oxygen required by microorganisms for stabilizing biologically decomposable organic matter (carbonaceous) in water under aerobic conditions. The test is used to determine the pollution load of wastewater, the degree of pollution and the efficiency of wastewater treatment methods. 5-Day BOD test being a bioassay procedure (involving measurement of oxygen consumed by bacteria for degrading the organic matter under aerobic conditions) requires the addition of nutrients and maintaining the standard conditions of pH and temperature and absence of microbial growth inhibiting substances.

 Principle: The method consists of filling the samples in airtight bottles of specified size and incubating them at specified temperature (20^o C) for 5 days. The difference in the dissolved oxygen measured initially and after incubation gives the BOD of the sample.

 Apparatus required: BOD bottles - 300ml capacity, air incubator - to be controlled at 20^oC -\+ 1^o C, oximeter and magnetic stirrer.

 Reagents:

·         Preparation of dilution water: To 1000ml of water, 1ml each of phosphate buffer, magnesium sulphate, calcium chloride and ferric chloride solution is added, before bringing it to 20^o C and aerating it thoroughly. 

 Procedure: The sample having a pH of 7 is determined for first day DO. Various dilutions (at least 3) are prepared to obtain about 50% depletion of D.O. using sample and dilution water. The samples are incubated at 20^o C for 5 days and the 5^th day D.O is noted using the oximeter. A reagent blank is also prepared in a similar manner.

 Calculation:                            

                BOD, mg/L  =                (D₁ - D₂) - (B₁ - B₂) X f  / p

D₁  - 1^st day D.O of diluted sample, mg/L

D₂  - 5^th day D.O of diluted sample, mg/L

P - decimal volumetric fraction of sample used.

B₁  - 1^st day D.O of control, mg/L

B₂  - 5^th day D.O of control, mg/L

 (Water analysis, APHA, 16th edn)

 CHEMICAL OXYGEN DEMAND

Chemical oxygen demand (COD) is the measure of oxygen equivalent to the organic content of the sample that is susceptible to oxidation by a strong chemical oxidant. The intrinsic limitation of the test lies in its ability to differentiate between the biologically oxidisable and inert material. It is measured by the open reflux method.

 Principle: The organic matter in the sample gets oxidized completely by strong oxidizing agents such as potassium dichromate in the presence of conc. sulphuric acid to produce carbon-di-oxide and water. The excess potassium dichromate remaining after the reaction is titrated with Ferrous Ammonium Sulphate (FAS) using ferroin indicator to determine the COD. The dichromate consumed gives the oxygen required for the oxidation of the organic matter.

 Apparatus required: Reflux apparatus, Nessler’s tube, Erlenmeyer flasks, hot plate and lab glassware.

 Reagents:

·         Standard potassium dichromate solution (0.250M): 12.25g of potassium dichromate dried at 103^o C for about 2 hours was dissolved in distilled water and made upto 1000ml.

·         Standard ferrous ammonium sulphate (FAS) 0.25N: 98g of FAS was dissolved in minimum distilled water to which 20ml of conc. sulphuric acid was added and made upto 1000ml using distilled water to give 0.25N of ferrous ammonium sulphate.

·         Ferroin indicator: 1.485g of 1,10-Phenanthroline monohydrate and 695mg of ferrous sulphate were dissolved in 100ml of distilled water.

·         Conc. sulphuric acid

·         Silver sulphate crystals

·         Mercuric sulphate crystals                                                                       

the cooled mixture (to make up to 50ml) and titrated against 0.25M FAS using ferroin indicator, till the colour turns from blue green to wine red indicating the end point. A reagent blank is also carried out using 10ml of distilled water.

 Calculation:

                                COD (mg/L)   =     (Blank reading - Sample reading) X N X F X 1000 / Sample taken, ml

 To calculate F,                              

                F     =    10000 /  Titrant value of blank.

 FLUORIDE

Fluorides have dual significance in water supplies. High concentration causes dental fluorosis and lower concentration (<0.8 mg/L) causes dental caries. A fluoride concentration of approximately 1mg/L in drinking water is recommended. They are frequently found in certain industrial processes resulting in fluoride rich wastewaters. Significant sources of fluoride are found in coke, glass and ceramic, electronics, pesticide and fertiliser manufacturing, steel and aluminium processing and electroplating industries. It is calculated by SPADNS method.

 Principle: The colorimetric method of estimating fluoride is based on the reaction of fluorides (HF) with zirconium SPADNS solution and the 'lake' (colour of SPADNS reagent), which is greatly influenced by the acidity of the reaction mixture. Fluoride reacts with the dye ‘lake’, dissociating (bleaching) the dye into a colourless complex anion (ZrF₆ ^2-). As the amount of fluoride increases, the colour produced becomes progressively higher or of different hue.

 Apparatus required: Spectrophotometer and lab glassware.

 Reagents:

·         Standard fluoride solution.

^·          Stock: 221.0mg of AR grade sodium fluoride was dissolved in distilled water and made up to 1000ml to give 1ml = 100 m g of F^-

·         Working Standard: 100ml of the stock fluoride was diluted to 1000ml to give 1ml = 10 mg of flouride.

·         SPADNS Solution: 958mg of SPADNS is dissolved in 500ml of distilled water.[1]

·         Zirconyl-acid reagent: 133mg zirconyl chloride octahydrate (ZrOCl₂ .8H₂O) was dissolved in about 25ml of distilled water. 350ml of conc. HCl was added and diluted to 500ml with distilled water.

·         Acid zirconyl-SPADNS reagent: Equal volume of SPADNS and zirconyl acid reagent was mixed.

 Procedure: A standard graph was prepared by using fluoride concentrations ranging from 0.005mg/L to 0.150mg/L at 570nm. A reference solution was prepared by adding 4ml of acid zirconyl-SPADNS reagent to 21ml of distilled water. A known volume of filtered sample (21ml) was taken in a test tube, 4ml of acid zirconyl-SPADNS reagent was added to the sample along with a reference solution. The mixture was left for about 30 min for complete colour development and the optical density was read at 570nm.

 Calculation:

                 F^- mg/L      =   (O.D sample) (Conc. of the Standard) (1000) / (O.D Standard)  (sample taken)

 FREE CARBON-DI-OXIDE

The important source of free carbon-di-oxide in surface water bodies is mainly respiration and decomposition by aquatic organisms. It reacts with water partly to form calcium bicarbonate and in the absence of bicarbonates gets converted to carbonates releasing carbon-di-oxide.

[1] · SPADNS - Sodium 2-(parasulfophenylazo)-1,8-dihydroxy-3, 6-naphthalene disulfonate

Principle: Free carbon-di-oxide reacts with sodium carbonate or sodium hydroxide to form sodium bicarbonate. The completion of the reaction is indicated by the development of pink colour, characteristic of phenolphthalein indicator at an equivalent pH of 8.3

 Apparatus required: Lab glassware - measuring jar, pipette, conical flask, etc.

 Reagents:

·         Sodium hydroxide solution (0.22N): 1g of sodium hydroxide was dissolved in 100ml of distilled water and made up to 1000ml to give 0.22N.

·         Phenolphthalein indicator

 Procedure: A known volume (50ml) of the sample was measured into a conical flask. 2-3 drops of phenolphthalein indicator was added and titrated against 0.22N sodium hydroxide till the pink colour persisted indicating the end point[1](*).

Calculation:

                                Free CO₂  (mg/L)  =    (V_t) (1000) / V_s

Where,  V_t - volume of titrant (ml)

             V_s - volume of the sample taken (ml)

 POTASSIUM

Potassium ranks seventh among the elements in order of abundance, behaves similar to sodium and remains low. Though found in small quantities (<20mg/L) it plays a vital role in the metabolism of fresh water environment.

 Principle: Trace amount of potassium can be determined by direct reading of flame photometer at a specific wavelength of 766.5nm by spraying the sample into the flame The desired spectral lines are then isolated by the use of interference filters or suitable slit arrangements. The intensity of light is measured by the phototube.

 Working principle of Flame photometer: The emission of characteristic radiations by alkali and alkaline earth metals and the correlation of the emission intensity with the concentration of the element form the basis of flame photometry. The principle of the flame photometer depends on the "Emission Spectroscopy" in which the electrons of the metals after absorbing energy get excited from ground state to higher energy level and return back to the ground state with emission of light. The sample under test is introduced into flame in solution by means of atomizer. The radiation from the flame enters a dispersing device and isolates it (radiation) from the flame to the desired region of the spectrum. The photo tube measures the intensity of isolated radiation, which is proportional to the concentration of the element present in the sample. 

 Apparatus required: Flame photometer, lab glassware and Whattman filter paper. 

Reagents:

Procedure: The filter of the flame photometer is set at 766.5nm (marked for Potassium, K) and the flame is adjusted for blue colour. The scale is set to zero and maximum using the highest standard value. A standard curve of different concentration is prepared by feeding the standard solutions. The sample is filtered through the filter paper and fed into the flame photometer. The concentration is found from the standard curve or as direct reading.

 SODIUM

Sodium is one of the most abundant elements and is a common constituent of natural waters. The sodium concentration of water is of concern primarily when considering their solubility for agricultural uses or boiler feed water. The concentration ranges from very low in the surface waters and relatively high in deep ground waters and highest in the marine waters. It is calculated by flame photometric method.

[1] (*) If the pink color appears on adding phenolphthalein it indicates the absence of free carbon-di-oxide.

Reagents:

 Procedure: The filter of the flame photometer is set to 589nm (marked for Sodium, Na). By feeding distilled water the scale is set to zero and maximum using the standard of highest value. A standard curve between concentration and emission is prepared by feeding the standard solutions. The sample is filtered through filter paper and fed into the flame photometer and the concentration is found from graph or by direct readings.

 Apparatus required: Flame photometer, lab glassware and Whattman filter paper.

 Heavy metals

 Heavy metals are elements (properties of metals satisfied) of high atomic numbers. They have high utilities in industrial applications from papers to automobiles, by their very characteristic properties. They are found in the deep bowels of the earth as ores (complexes of mixtures). The metals are segregated from these ores, leaving behind the tailings that find their way into the environment as toxic pollutants. They get into the water bodies directly from point sources as sewage, and non-point sources as runoff and more insidiously as atmospheric deposition pollutants transported from long distances. Heavy metals affect every level of the food web, from producers in the trophic levels to the highest order carnivore by residing in the system and magnifying at every trophic status.

 Atomic absorption spectrophotometer (AAS)

 Working principle: Atomic absorption spectrophotometer's working principle is based on the sample being aspirated into the flame and atomized, when the light beam from AAS’s is detected through the flame into the monochromator, and onto the detector that measures the amount of light absorbed by the atomized element of the flame. Since metals have their own characteristic absorption wavelength, a source lamp composed of that element is used, making the method relatively free from spectral or radiational interferences. The amount of energy of the characteristic wavelength absorbed in the flame is proportional to the concentration of the element in the sample.

Procedure: The sample is thoroughly mixed by shaking, and 100ml of it is transferred into a glass beaker of 250ml volume, to which 5ml of conc. nitric acid is added and heated to boil till the volume is reduced to about 15-20ml, by adding conc. nitric acid in increments of 5ml till all the residue is completely dissolved. The mixture is cooled, transferred and made upto 100ml using metal free distilled water.

 Operations for analysing heavy metals:

·         Lamp selection:

·         Lamp for the element to be detected is selected.

·         Operating current is suitably adjusted.

·         The lamp is aligned for the visible beam to fall on the slit of the monochromator

·         Wave length selection and slit adjustment:

·         Appropriate wavelength for the element to be detected is selected. The wavelength controller is moved clockwise or anti-clockwise slowly to get maximum percentage of transmittance.

·         The slits are adjusted to get closest to the required wavelength and avoid excess stray light.

·         Flame adjustment:

·         On selecting suitable wavelength, the acetylene-air mixture is lit at the recommended pressure.

·         The burner level is so adjusted that the beam from the cathode crosses 1cm from the top of the burner and the beam is stabilized.

·         Analysis:

·         A calibration graph is obtained by feeding the standard solutions of suitable concentration.

·         The samples are aspirated by feeding them through the capillary and the readings are noted. (Distilled water is aspirated between samples).

 Apparatus required: AAS and lab glassware.

 Reagents:

·         Air-Acetylene flame

·         Metal free water

·         Standard metal solutions

 LEAD

Lead is relatively a minor element in the earth's crust but is widely distributed in low concentrations in uncontaminated soils and rocks. Lead concentration in freshwater is generally much higher. High concentration of lead results from atmospheric input of lead originating from its use in leaded gasoline or from smelting processes. Industrial processes such as printing and dyeing, paint manufacturing, explosives, photography and mine or smelter operations may contain relatively high values of lead. Lead is toxic to aquatic organisms.

 Principle: Lead can be determined at a wavelength of 283.3 nm by AAS with aspiration of the sample into the oxidising air-acetylene flame. When the aqueous sample is aspirated, the sensitivity for 1% absorption is 0.5 mg/L and the detection limit is 0.05 mg/L.

 Standard lead solution: 1.598g of lead nitrate was dissolved in about 200ml of water containing 1.5ml of conc. nitric acid and diluted to 1000ml of metal free water to give 1ml = 1mg lead. A series of standards ranging from 1mg to 5mg were prepared from the stock and a standard graph was made.                 

 COPPER

Copper is a widely distributed trace element because most copper minerals are relatively insoluble and are sorbed to solid phases, hence only low concentrations are normally present in natural waters. Because of the presence of sulphide, copper would be expected to be even less soluble in anoxic systems. The presence of higher concentrations of copper can usually be attributed to corrosion of copper pipes, industrial wastes or particularly in reservoirs, where copper is used as algaecides. Copper is an essential trace element in the nutrition of plants and animals including man. It is required for the function of several enzymes and is necessary in the biosynthesis of chlorophyll. High levels are toxic to organisms but the response varies greatly with species.      

Principle:

Copper can be determined at a wavelength of 324.7 nm by AAS with aspiration of the sample into an oxidising air-acetylene flame. When the aqueous sample is aspirated, the sensitivity for 1% absorption is 0.1 mg/L and the detection limit is 0.01 mg/L.

 Standard copper solution: 1g of copper salt is dissolved in 15ml of 1+1 nitric acid and diluted to 1000ml to give 1ml = 1mg copper. A series of standards ranging from 1mg to 5mg were prepared from the stock and a standard graph was prepared.

NICKEL

Nickel is present in less than 1 mg/L in surface waters. Nickel is relatively a non-toxic element. It is essential for animal nutrition. Certain nickel compounds have shown to be carcinogenic in animal experiments. However, soluble nickel compounds are not currently regarded as either human or animal carcinogens, but higher concentrations of nickel can react with DNA (deoxy ribonucleic acid), resulting in the damage of DNA. 

Standard nickel solution: 1.273g of nickel oxide was dissolved in a minimum quantity of 10% HCl and diluted to 1000ml with distilled water to give 1ml = 1mg of nickel. A series of standards ranging from 1mg to 5mg were prepared from the stock and analysed.             

IRON

Iron is an abundant element in the earth's crust, but exists generally in minor concentrations in natural water systems. Iron is found in the +2 (ferrous) and +3 (ferric) states depending on the oxidation-reduction potentials of the water. The ferric state of iron imparts orange strain to any settling surface, including laundry articles, cooking and eating utensils, and plumbing fixtures.                 

 Principle:

Iron can be determined at a wavelength of 248.3 nm by AAS with aspiration of the sample into an oxidising air-acetylene flame. Under standard conditions, iron produces 1% absorption at 0.12 mg/L and a linear response up to about 5 mg/L.

  Standard iron solution: 1g of iron was dissolved in 50ml of 1+1 nitric acid and diluted to 1000ml with distilled water to give 1ml = 1mg of iron. A series of standards ranging from 1mg to 5mg were prepared from the stock and analysed.             

 CHROMIUM

The concentration of chromium in natural waters is usually very low. Elevated concentrations of chromium can result from mining and industrial processes. Chromate compounds are routinely used in cooling waters to control erosion. Chromium in water supplies is generally found in the hexavalent form.   

Principle:

Total chromium can be determined at a wavelength of 357.9 nm by atomic absorption with aspiration of sample into a reducing air-acetylene flame. Under standard concentrations, chromium produces 1 % absorption at 0.25 mg/L and is detectable down to 0.003 mg/L.

 Standard chromium solution: 2.828g of AR grade potassium dichromate was dissolved in about 200ml of distilled water, with 1.5ml of conc. nitric acid and made up to 1000ml with the same to give 1ml = 1mg of chromium. A series of standards ranging from 1mg to 5mg were prepared from the stock and analysed.

 CADMIUM

Cadmium is largely found in nature in the form of sulphide, and as an impurity of zinc - lead ores. The abundance of cadmium is much less than that of zinc. Cadmium may enter the surface waters as a consequence of mining, electroplating plants, pigment works, textile and chemical industries. Cadmium is toxic to man. There is evidence that cadmium affects reproductive organs in humans and is also a potential carcinogen. A specific disease called "itai-itai" has been observed in Japan due to excess cadmium. In addition, due to bioaccumulation, certain edible organisms may become hazardous to the ultimate consumer. 

  Principle: Cadmium can be determined at a wavelength of 228.8 nm by atomic absorption with aspiration of sample into an oxidising air-acetylene flame. When the aqueous sample is aspirated, the sensitivity for 1 % absorption is 25 mg/L and the detection limit is 2 mg/L.  

Standard cadmium solution: 1.000g of cadmium metal was dissolved in minimum volume of 1+1 HCl and made upto 1000ml with distilled water to give 1ml = 1mg of cadmium. A series of standards ranging from 1mg to 5mg were prepared from the stock and analysed.

 ZINC

Zinc is an abundant element in rocks and ores and is present in natural waters only as a minor constituent. The main industrial use of zinc is in galvanizing and may enter the drinking waters from galvanized pipes. Another important use is in the preparation of alloys, including brass and bronze. It is an essential element in human nutrition with food being the main source of zinc to the body. Zinc may be toxic to aquatic organisms but the degree of toxicity varies greatly depending on water quality characteristics as well as the species being considered.

 Principle: Zinc can be determined at a wavelength of 213.9 nm by AAS aspiration of the sample into an oxidizing air-acetylene flame. When the aqueous sample is aspirated, the sensitivity for 1% absorption is 20 mg/L and the detection limit is 5 mg/L. 

Standard zinc solution: 1.000g of zinc metal was dissolved in 20ml of 1+1 HCl and diluted to 1000ml in distilled water, to give 1ml = 1mg of zinc. A series of standards ranging from 1mg to 5mg were prepared from the stock and analysed.

 PLANKTON ANALYSIS:

The physical and chemical characteristics of water affect the abundance, species composition, stability and productivity of the indigenous populations of aquatic organisms. The biological methods used for assessing water quality include collection, counting and identification of aquatic organisms; biomass measurements; measurements

of metabolic activity rates; toxicity tests; bioaccumulation; bio magnification of pollutants; and processing and interpretation of biological data. The work involving plankton analysis would help in:

1.        Explaining the cause of colour and turbidity and the presence of objectionable odour, tastes and visible particles in waters;

2.        The interpretation of chemical analyses.

3.        Identifying the nature, extent and biological effects of pollution.

4.        Providing data on the status of an aquatic system on a regular basis.

 Plankton: A microscopic community of plants (phytoplankton) and animals (zooplankton), found usually free floating, swimming with little or no resistance to water currents, suspended in water, non-motile or insufficiently motile to overcome transport by currents, is called "Plankton".

Phytoplankton (microscopic algae) usually occurs as unicellular, colonial or filamentous forms and is mostly photosynthetic and grazed by zooplankton and other organisms occurring in the same environment. Zooplankton principally comprises of microscopic protozoans, rotifers, cladocerans and copepods. The species assemblage of zooplankton also may be useful in assessing water quality. 

The structure of photosynthetic populations in the aquatic ecosystems is dynamic and constantly changing in species composition and biomass distribution. An understanding of the community structure is dependent on the ability to understand the temporal distribution of the different species. Changes in species composition and biomass may affect photosynthetic rates, assimilation efficiencies, and rates of nutrient utilization, grazing, etc. 

Plankton, particularly phytoplankton, has long been used as an indicator of water quality. Because of their short life spans, planktons respond quickly to environmental changes. They flourish both in highly eutrophic waters while a few others are very sensitive to organic and/or chemical wastes. Some species have also been associated with noxious blooms causing toxic conditions apart from the tastes and odour problems (Figure 4-6).

Figure 4: Clean Water Algae

Figure 5: Polluted Water Algae

Figure 6: Taste and Odour Algae

Plankton net: The plankton net is a field equipment used to trap plankton. It has a polyethylene filter of a defined mesh size and a graduated measuring jar attached to the other end. A handle holds the net. The mesh size of the net determines the size range of the plankton trapped. The mesh number 30 of size 60 mm was used for collecting samples.

Sampling Procedure: The manner in which sampling is done should conform to the objectives of the study. The “surface samples” (samples collected from the surface) were collected as close to the water surface as possible, mostly towards the centre of the lake at regular monthly intervals. A known volume of the sample, 5L to 50 L, is filtered and planktons are filtered and preserved for further analysis.

Labels: The sample label had the date, time of sampling, study area-lake name and the volume measured pasted on the containers of 50ml capacity.

 Preservation: The samples collected into the 100ml polyethylene vials were preserved by adding suitable amounts of 1ml chloroform to act as the narcotizing agent and 2ml of 2% formalin for preservation and analyses.

Concentration technique: The plankton nets were used to collect samples for the qualitative and quantitative estimation of the plankton, by filtering a known volume of water (5-50 liters) through the net depending on the plankton density of the tanks. 

Qualitative and quantitative evaluation of plankton: Detailed analyses of phytoplanktonic populations were done by estimating the numbers in each species. The phytoplankton consisting of individual cells, filaments and colonies were counted as individual cells. When colonies of species were counted, the average number of cells per colony was counted, and in filamentous algae, the average length of the filament was determined.

Sedimentation and enumeration by microscope: Preserved samples in bottles were mixed uniformly by gentle inversion and then exactly 1ml of the sample was pipetted out into the S-C cell for analysis.

 Microscope:

 Compound microscope:

A monocular compound microscope was used in the counting of plankton with different eyepieces such as 10X, 15X and 20X. The microscope was calibrated using plankton counting squares.

Counting:

Counting cell: Sedgwick-Rafter (S-R) cell:

The Sedgwick-Rafter cell is a devise used for plankton counting and is about 50mm long by 20mm wide and 1mm deep. The cell is covered by a relatively thick cover-slip and is calibrated to contain exactly 1.0 ml.  

Method:

Filling the cell:

The cover slip is placed diagonally across the S-R cell and filled with the sample carefully without air bubbles with a large bore pipette. The sample is allowed to settle for about 5 minutes before the actual counting begins.

 Note: Since the configuration of the S-R cell does not allow the use of high power microscope objectives, the identification of organisms smaller than 10 – 15 mm is difficult or impossible, limiting the usage to only larger forms of relatively dense populations.

Strip counting:

A "strip" is the length of the cell that constitutes a volume approximately 50 mm long, 1-mm deep accounting to 25mm³ or 1/40 (2.5%) of the total cell volume. By moving the mechanical stage from left to right, the organisms can be examined in a systematic manner. By knowing the surface area of the portion counted in relation to that of the total, a factor was determined to expand the average counts of the plankton to the total area of the counting surface. This total area represents the number of organisms present per given volume of the sample. This volume expanded to an appropriate factor yields the organisms per litre of water for the lake.

 The total number of planktons in the S-R cell is obtained by multiplying actual count in the strip by the number (enumeration factor) representing the portion of the S-R cell counted. The number of the strips counted is a function of the precision desired and the number of units (cells, colonies) for the strips measured. In this study, 500 cells were counted for estimation. 

The plankton count in the S-R cell was got from the following,

Number/ml =      C X 1000 mm / L X D X W X                                  

Where:

                                C = number of organisms counted

                                L = Length of each strip (S-R cell length) in mm

                                D = Depth of a strip (S-R cell depth) in mm

                                W = Width of a strip in mm

                                S = number of strips counted 

V₁ = (50)(1)(W)

                                                     =  mm³   

The plankton counts per strip are then determined by multiplying the actual count by the factor representing the counted portion of the whole S-R cell volume.

                                                No./ml = (C) (1000 mm³) / (L) (D) (W) (S) 

Where

                C = Number of organisms counted.

                L = Length of each strip in mm (of S-R cell)

                D = Depth of the strip in mm (S-R cell)

              W = Width of the strip in mm (Whipple grid image width)

                S = Number of strips counted. 

Phytoplankton Counting Units: Some plankton are unicellular while others are multicellular (colonial), posing a problem for enumeration. For analysis, a colony of plankton was accounted as a single count. The large forms that cross two or more boundaries of the grid were counted separately at lower magnification and their number included in the total count.  

The quality of water affects species composition, abundance, productivity and physiological conditions of the aquatic community. The structure and composition of these aquatic communities is an indicator of the water quality. Bio-monitoring methods, which involve the use of plants and animals to assess periodic changes in environmental quality; can be used to assess the water quality. Some of the advantages of using bio-monitoring techniques are as follows: 

(i)                   Biological communities reflect overall ecological integrity (i.e., chemical, physical and biological integrity). Therefore bio-monitoring results directly assess the status of a water body.

(ii)                 Biological communities integrate the effects of different pollutant stressors and thus provide a holistic measure of their impact.

(iii)                Routine monitoring of biological communities can be relatively inexpensive, particularly when compared to the cost of assessing toxic pollutants either chemically or with toxicity studies.

(iv)               Where criteria for specific ambient impact do not exist (e.g., non-point source impacts that degrade habitat), macroinvertebrates may be the only practical means of evaluation.

The methods useful in bio-monitoring include the collection, identification and counting of bioindicator organisms, biomass measurements, measurements of metabolic activity rates, and investigations on the bioaccumulation of pollutants. The communities that are useful in bio-monitoring are plankton, periphytes, macrophytes, fishes, macroinvertebrates, amphibians, aquatic reptiles, birds and mammals. These organisms reflect a certain range of physical and chemical conditions. Some organisms can survive a wide range of conditions and are tolerant to pollution. Others are very sensitive to changes in conditions and are intolerant to pollution. These organisms are called bioindicators (EPA, 1989).

Macroinvertebrates are invertebrates visible to the naked eye. Adults of this group are larger than 0.5 mm. These are an ecologically important group in any aquatic system, more so in freshwater habitats. Some examples of organisms that are sensitive to pollution are mayflies, stoneflies and some caddis flies. Examples of pollution tolerant organisms are sludge worms, leeches and midge larva. 

Advantages of using benthic macroinvertebrates are:

·         Macroinvertebrate communities integrate the effects of short-term environmental variations. Most species have a complex life cycle of approximately one year or more. Sensitive life stages will respond quickly to stress; the overall community will respond more slowly.

 Ecological Importance:

Macroinvertebrates convert and transport nutrients from one part of the water body to another, influencing nutrient cycling. This process is important for keeping the general health of a water body. They convert organic matter such as leaf litter and detritus into food in their body. They in turn become the main source of food for higher aquatic organisms such as fish, forming a basic link between organic matter and higher aquatic animals in the food web. They are sensitive to changes in habitat and pollution, especially to organic pollution. Because of this, they are considered to be important indicators of water quality.

 Habitat:

Freshwater provides a wide range of micro-habitats for these organisms such as stones, organic debris, clay and plants. Based on the microhabitat, the organisms can be broadly classified into the following: 

Sediment living: These animals live on or in sediment such as gravel, cobbles, silt, clay, rocks and sand. Since most organisms are bottom dwellers they are called as benthic organisms.

Vegetation living: These organisms cling to the stems and leaves of plants, which give shelter, and a range of foods for many types of organisms. Water plants include algae and moss. 

Food Habits:

Based on their food and feeding habits, macroinvertebrates may be divided into shredders, collectors, scrapers (grazers) and predators. 

·         Shredders feed on coarse organic matter such as leaves and algae. They are omnivorous and feed on bacteria, fungi and protozoans present along with coarse organic matter. These animals break the coarse material into finer material in their body and then excrete them as faeces. Some examples of shredders are stonefly nymphs and caddisfly larvae.

·         Collectors feed on decomposing finer organic matter including the faecal matter released by shredders. They also feed on coarse organic matter along with bacteria and fungi. Animals, which strain minute particles transported by the flowing water, are called filter collectors and are generally found in high velocity flows, for example caddisfly larvae. Animals, which feed on organic matter deposited at the bottom, are called gathering collectors; for example, mayfly nymphs and beetles. 

·         Scrapers feed essentially on plants including algae.  They are also known as grazers. Some examples are mayfly nymphs and caddisfly larvae. 

·         Predators feed on other aquatic insects and their body parts are adapted to capture live prey, for example, scoop like jaws in damselfly and spear like mouth parts in water striders. 

Even though there is an overlap among the feeding habits of the organisms, it helps to understand the organisms in relation to organic resource availability. The composition of the macroinvertebrates changes based on the availability of food. As one moves downstream, the number of grazers declines and that of collectors increases. 

Sampling Sites:

The composition and population of macroinvertebrates are influenced by water flow velocity, volume of water and substrates including vegetation, food, and water temperature pattern. Therefore, the sample should be collected from different microhabitats to get a representative picture. The sampling points of macroinvertebrate collection should be close to water sampling points (collected for physico-chemical analyses). The sampling sites should be longitudinal to the flowing water systems. In case of any disturbance or pollution in the water body, the samples are taken from 3 sites – area before disturbance, area immediately after disturbance and area away from the disturbance. This is decided on site based on factors such as type of pollution, load of pollution, flow velocity and similarity in microhabitat. The samples are collected once every season or at least twice a year, in March-April and late October. For quantitative sampling, an area of 3feet x 3feet is generally recommended. 

Types of Sampling:

Sampling can be either qualitative or quantitative. In quantitative sampling, the number of individuals of each species is counted and the area sampled is measured. This is expressed as numbers per unit area of the water body.  In qualitative sampling, different types (species) of macroinvertebrates are identified. Here, the area sampled is not measured. This sampling is useful for pollution monitoring. Comparative studies can also be done in the sampling site over a period of time (Sunil Kumar M and Shailaja Ravindranath, 2001). 

Substrata to be sampled are boulders, large stones, gravel, gravel-sand, sand or mud and various parts of water plants including patches of algae. Various devices are available for sampling invertebrates. Most of the devices use nets, with the mesh size varying from 0.125 to 0.25 mm. This is because some of the insect stages (nymph/larva) are smaller than 500 micrometers and do not meet the conventional definition of macroinvertebrates. However, they are considered important water quality indicators. 

Types of samplers:

Qualitative samplers:

D frame net: This is mainly used to sample gravel or cobble substrata and soft sediments. The net is dragged on the substrata to collect samples.

Kick screens: They are used to sample riffle areas and a large area can be sampled then with a D frame net. The kick screen is held upright with the base touching the river bottom and remaining open upstream. After disturbing the streambed i.e., brushing the surface of the stones to dislodge any organisms present, the net is held so as to catch the dislodged organisms. The net is removed to prevent the organisms from escaping. 

Quantitative samplers:

Square foot sampler: This has a square (1ftx1ft) frame hinged to another frame of the same size. The cloth sides on the frames help the water to flow into the net. During sampling, the frame is pushed into the substrate and the substrate area is disturbed within by rubbing the rocks. The dislodged organisms are carried into the cloth net by the flowing water.

Grab sampler: They are so called as they are used to grab a sediment sample. There are various grab samplers, out of which the most commonly used is Ekman grab sampler. The Ekman grab sampler is useful in sampling silt and sludge in deep waters with little current. The grab is made of stainless steel and has two jaws, which help in collecting the samples. The jaws of the device are kept open before the sampling.

Scoop: This device is shaped as a dust pan, with a wire screen at the bottom and back. 

Artificial substrate samplers:

They provide for the colonisation and collection of organisms along the rivers, lakes, canals and tanks where it is difficult to collect benthic animals due to strong currents and deep waters. Various types of artificial samplers are available, the most common being Hester Dendy and Basket samplers. 

Data Analysis:

The level of pollution can be found by using any one of the following indices.

Diversity index

Biotic index

Biological water quality evaluation system

Diversity Index:

Diversity here refers to the number of different species found in the community. Greater the number more is the diversity. Diverse communities tend to be more stable than less diverse ones. Due to pollution and habitat degradation, the number of species in the community is reduced by the elimination of the species sensitive to water and habitat changes. The community then has a reduced number of species but a great proportion of pollution tolerant species. 

Two main types of Diversity indices are commonly used. They are: 

(i)                   Sequential Comparison Index (SCI): The SCI is a measure of the distribution of individuals among groups of organisms. It is based on the theory of runs. A run is the count of similar organisms in successive picks. A run ends when a dissimilar organism is encountered and a new run begins when an organism picked up from a sample looks different from the previous one. This test does not require the knowledge of identification of macroinvertebrates. 

PROCEDURE:

On a white metal or plastic tray, straight parallel lines are drawn with a distance of 1 or 1.5 inches between the lines. The container with benthic organisms is shaken and the contents poured over the tray. Pouring water over the tray separates the clumps of organisms. Now, each organism is moved using forceps to the nearest drawn line. If the organisms are between two lines, it is moved to either one of the lines. In the end, all the organisms should be associated with any one of the lines. Now the organisms are carefully observed. If similar organisms occur successively, it is considered as one run. If an organism is not similar to the previous one, it is considered as a new run. The number of runs and the total number of organisms after going through the entire sample is recorded. 

Calculation 

SCI =  number of runs / total number of organisms picked 

Interpretation: The SCI has a range of 0-1 with the following water quality ratings.

(ii)                        Shannon Weiner index: The index is widely used for several biological communities. It can also be used for the evaluation of stresses to benthic macroinvertebrate populations due to changes in water quality. Weiner and Shannon independently developed the index and hence the name.

The Shannon Weiner diversity index (H) is given by 

              H =  - ₁/N  log₂ n₁/N 

Where

s = number of species in a sample,

N = total number of individuals in a sample,

n₁ = number of individuals in each species. 

The relationship between the Shannon-Weiner index (H) and the quality of water is as follows.

Biotic index:

Biotic index is a measure to find the level of pollution using bioindicators present in water. This concept is based on the fact that each species has varying degree of tolerance to organic pollution.  Some species are sensitive to pollution and may not survive in such conditions. Others are tolerant and hence survive in a wide range of situations. Biotic index using macro invertebrates is calculated based on animal scores, which range from 1 to 10. Sensitive taxa are given higher scores while tolerant ones are given lower scores. Lower scores therefore indicate poor water quality. Animal scores for the major macroinvertebrates found in a waterbody are given in table 16

Table 16: Animal scores for the major macroinvertebrates found in a waterbody

 This is based on the Biological Monitoring Working Party (BMWP) score system. If there are different scores for the same animal group, the score that has maximum number of families is considered. 

The level of pollution can be ascertained from the above table as follows:

The scores for each animal in the list are found out. The scores are added and the total score is divided by total number of species. This gives the biotic index, which indicates the average pollution tolerance of the taxa. Higher the index, less polluted is the water. Comparison of the biotic index can be made between micro habitats / sampling area in the same water body. 

Biological water quality evaluation system:

This is based on the classification of indicator macroinvertebrates based on their sensitivity and tolerance to organic pollution. The indicator organisms are divided into 5 classes representing different degrees of water quality. Table 17 can be used to evaluate the water quality.

Table 17: Indicator organisms and water quality

Table 18: Classification based on abundance of the individual macroinvertebrates

Indicator Information (Sunil Kumar M and Shailaja Ravindranath, 2001)

1.         Flat worms (Phylum Platyhelminthes)

Flat ribbon like worms with elongated body; segments are absent or no true segments; carnivorous; found in ponds and flowing water, for example, Planaria.

 2.        Segmented worms (Phylum Annelida)

Segmented worms; elongated body with a definite head.

*              Leeches (Class Hirudinea) - Flattened body with suckers at both ends, found in shallow waters under stones/rocks where there is a lot of vegetation; parasites on aquatic birds, fish and molluscs; active during night (nocturnal).  They can live without oxygen for several days.

*              Sludge worms (Class Oligocheata) - Round body with many segments; live in mud or in high organic debris; feed on detritus.

 3.             Arthopods (Phylum Arthropoda)

Animals with jointed legs; largest phylum in the animal kingdom consisting of about 75% of animal species.

*              Crustaceans (Class Crustacea)

Its body is protected by a crust (hard chitinous skin); many are carnivores, but some are herbivores.  They also feed on detritus.  Most species are less tolerant to pollution.  They are mainly filter feeders.

 Freshwater Prawn (Order Decapoda)

Front pair of legs have pincers, long, thread - like pair of antennae and a laterally compressed body. 

Freshwater Lobster (Order Decapoda)

Front pair of pincers is huge. 

Freshwater Shrimp (Order Decapoda)

Long, thin, laterally compressed body with slender legs, depressed crust and small extension of head (rostrum).

*              Water mites (Class Arachnida)

Animals with 8 legs; fairly pollution tolerant; resemble spiders; small animals with nearly round shaped body; carnivores; easy to identify. 

*              Insects (Class Insecta)

Animals with 6 legs; largest class in animal kingdom.  Adult insects are rare in water bodies.  Land living (terrestrial) insects pass through their earlier life cycle stages (metamorphosis) in freshwater.  Some insects undergo incomplete metamorphosis passing through 3 stages namely, egg, nymph and adult.  The nymph looks almost like the adult except that its wings are not completely developed.  Some examples are: Mayfly and Dragonfly.  Others undergo complete metamorphosis, passing through 4 stages: egg, larva, pupa and adult.  The larva looks entirely different from the adult.  In the pupa stage it becomes inactive.  The nymph and the larva stages are active and live longer than the adult form.  Hence they are good water - quality indicators. 

Mayfly nymph (Order Ephemeroptera)

They are grazers and collectors; gills present on either side of the abdomen; found in highly oxygenated waters; about 2.5 cms in length. 

Stonefly nymph (Order Plecoptera)

Similar to Mayfly nymphs; gills are rarely found around the base of their legs; live in well - oxygenated water; herbivores or detritivores, some are even carnivores; size 2.5 cms. 

Dragonfly nymph (Order Odonata)

Robust nymphs with pointed abdomen; predator, easily eat up a tadpole or small fish; found generally in ponds and slow moving water bodies; often covered with algae and organic debris; indicate low organic pollution. 

Damselfly nymph (Order Odonata)

Nymphs slender with 3 plate like fill filaments; predators; found mainly in ponds and slow moving waters; covered with algae or organic debris; indicate low organic pollution. 

Water Bug nymph (Order Hemiptera)

Surface dwellers; found in ponds and flowing waters; head is prolonged with a beak-like structure which helps in sucking fluids from plants/animals; largely predators. 

Caddisfly larva (Order Trichoptera)

They form a large benthic community.  Some live in hollow cases made up of small stones, gravel, leaves, twigs, sand, while others are free-living.  They have a cylindrical body and hard-shelled head capsules; the last segment bears 3 small hooks; grow up to 4 centimetres, generally carnivores. 

Beetle (Order Coleoptera)

Many species of beetles are found in water.  Some are surface dwellers, while others are submerged species.  Wings form a rigid cover on the abdomen; many are carnivores; some eat detritus and algae.

Truefly larva (Order Diptera)

One of the largest order of aquatic insects; includes many families.  They are the most common indicators of highly polluted waters.  They have pseudopodia in one or more segments.  They have less than 15 segments; resemble worms; many are detritivores; some are carnivores.  Some examples are, rat tailed maggot, mosquito larva and species of midge larva such as red worm.

4.             Molluscs (Phylum Mollusca)

Benthic animals, having a soft body covered with a hard calcareous shell secreted by the body.  Some are tolerant to pollution while others are sensitive.  They generally feed on algae, higher plants and detritus.

 Freshwater Snails (Class Gastropoda)

They have one spiral shell.  They are usually found everywhere.  Some snails are tolerant to conditions of oxygen depletion.  In these snails, the shell spirals to the right.  Others are sensitive to oxygen depletion; in these animals the shell spirals to both right and left.

 Freshwater limpets (Class Gastropoda)

The shell is a small cone.  They are susceptible to pollution and are found in well-oxygenated waters.

Freshwater mussels (Class Bivalva)

They have a shell with 2 halves connected by a hinge.  They are less tolerant to pollution.

Indicators of Good Water Quality (http://mason.gmu.edu/)

I) Insects

 (A)   Stonefly nymphs (Order Plecoptera)

  Roach-like stonefly nymphs (Family Peltoperlidae) 

Common stonefly nymph  (Family   Perlidae)

Slender Winter Stonefly nymph (Family Capniidae) 

(B)   Mayfly nymphs  (Order Ephemeroptera) 

Brush Legged Mayfly nymph (Family Oligoneuridae) 

 Flat headed Mayfly nymph (Family Heptageniidae)   

Burrowing Mayfly nymph (Family Ephemeridae) 

 (C)     Caddisfly Larvae (Order Trichoptera) 

Netspinning Caddisfly larva (Family Hydropsychidae)  

 Fingernet Caddis larva (Family Philopotamidae) 

Case-making Caddis larva (various families)  

 Free living Caddis larva (Family Ryacophilidae)  

(D) Dobsonfly (Order Megaloptera, Family Corydalidae)    

(E)     Water Penny (Order Coleoptera, Family Psephenidae)

 (F) Riffle Beetle (Order Coleoptera, Family Elmidae)           

II) Others:

Gilled Snail (Order Gastropoda, Family Viviparidae)     

Shell usually opens on right. Shell opening covered by a thin plate (operculum).  

 Indicators of Moderate Water Quality:

 I)                    Insects

 (A)   Dragonfly nymph (Order Odonata, suborder Anisoptera)  

(B)   Damselfly nymph (Order Odonata, Suborder Zygoptera)                     

(C)   Watersnipe fly larva (Order Diptera, family Athericidae)

(D) Alderfly larva (Order Megaloptera, Family Sialidae)

 (E)   Cranefly larvae (Order Diptera, Family Tipulidae)

(F) Beetle larvae (Order Coleoptera)

 Whirligig Beetle larva (Family Gyrinidae)

Predaceous diving beetle larva (Family Dysticidae)

Crawling water beetle larva (Family Haliplidae)

(II) Others

               Scuds (Order Amphipoda, Family Gammaridae)

Sow bugs (Order Isopoda, Family Asellidae)

Crayfish (Order Decapoda, Family Cambaridae)          

Indicators of Poor Water Quality

(I)      Insects

(i) Midge larva (Order Diptera, Family Chironomidae)

(ii) Black fly larva (Order Diptera, Family Simulida

(II) Others:

(i)                   Pouch snail (Order Gastropoda, Family Physidae)

(i)                   Planorbid snail (Order Gastropoda, Family Planorbidae) 

(i)                   Leech (Class Hirudinea) 

(i)                   Aquatic Worm (Class Oligochaeta) 

Fish census techniques

 Introduction:

 Fish are the most abundant, widespread and diverse group of vertebrates, comprising 22, 000 species with various forms, size and habits. Sampling fish requires high level of resources (e.g., time, labour, and cost of equipment), and this increases with the size of the habitat (e.g., pond versus the sea). To census fish in the largest aquatic systems, data can be gathered from commercial catches, where available, or by visiting markets where fish are landed.

 Methods of capturing fish fall into two categories namely: passive methods, which rely on the fish swimming into a net or a trap, and active methods where fish are pursued. The fact that fish are poikilothermic influences the choice of method and timing of sampling. There are very few truly quantitative techniques, so statistics such as catch per unit effort are used commonly to generate indices of abundance. Variability in swimming speed, seasonality as a result of migration, diurnal and patchiness of distribution all influence how catchable a species is. It is vital to have at least some knowledge of the ecology and behaviour of fish in the habitat to be sampled. Factors such as depth, clarity, presence of vegetation or the speed of the current have to be considered. A hydrographical survey prior to sampling may be required (William Sutherland, 1996).

 Seasonality for Fish Collections:

 Seasonal changes in the abundance of fish community primarily occur during reproductive periods and (for some species) summer and winter migratory periods. Generally, the preferred sampling period is from mid to late summer. Although some fish species are capable of extensive migration, fish populations and individual fish tend to remain in the same area during summer. The stream fish assemblages are stable and persistent for 10 years, recovering rapidly from droughts and floods indicating that substantial population fluctuations are not likely to occur in response to purely natural environmental phenomena. However, comparison of data collected during different seasons is discouraged, as are data collected during or immediately after major flow changes (EPA, 1989).

 Fish Sampling Methodology

 Although various gear types are routinely used to sample fish, electrofishing equipment and seines are the most commonly used collection methods in fresh water habitats. Each method has advantages and disadvantages. However, electrofishing is recommended for most fish field surveys because of its greater applicability and efficiency. Local conditions may require consideration of seining as an optional collection method. Advantages and disadvantages of each gear type are presented below.

Electrofishing:

It involves passing an electric current through water via electrodes which stuns nearby fish, leading to their disorientation and easy capture. Power is supplied by a generator and is converted to the required form by an electrofishing unit or box. Several types of current maybe used, each producing slightly different effects. The most commonly used is DC, because it attracts fish to the anode and causes fewer harmful effects to the fish than AC. 

 Advantages of Electrofishing:

Electrofishing allows greater standardization of catch per unit of effort. Electrofishing requires less time and a reduced level of effort than some sampling methods (e.g., use of ichthyocides). Electrofishing is less selective than seining (although it is selective towards size and species). If properly used, adverse effects on fish are minimized. Electrofishing is appropriate in a variety of habitats. 

Disadvantages of Electrofishing:

Sampling efficiency is affected by turbidity and conductivity. Although less selective than seining, electrofishing is size and species selective. Effects of electrofishing increase with body size. Species specific behavioral and anatomical differences also determine vulnerability to electro shocking. Electrofishing is a hazardous operation that can injure field personnel if proper safety procedures are ignored.

 Seine netting:

A seine net is a wall of net fitted with floats at the top (float line) and a weighted line (lead line) on the bottom, and generally with a bulging section at the back of the net to hold the catch. The first step in seine netting is to encircle a known area of water. To do this, the net is generally fixed at one end, which can be a shore, a boat or buoy or by walking when in very shallow water, in an arc or semi circular fashion and returning to the fixed point. In shallow waters, they can be dragged through the water by two people, one at each end towards a fixed point.

Advantages of Seining:

Seines are relatively inexpensive. They are lightweight and are easily transported and stored. Seine repair and maintenance are minimal and can be accomplished onsite. Seine use is not restricted by water quality parameters. Effects on the fish population are minimal because fish are collected alive and are generally unharmed.

 Disadvantages of Seining:

Previous experience and skill, knowledge of fish habitats and behaviour, and sampling effort are probably more important in seining than in the use of any other gear. Sample effort and results for seining are more variable than sampling with electrofishing. Use of seines is generally restricted to slower water with smooth bottoms, and is most effective in small streams or pools with little cover. Standardization of unit of effort to ensure data comparability is difficult.

 Bankside counts:

It is possible to census fish without catching them. Bankside count is a good technique in shallow, slow-moving and clear waters with minimal vegetation, such as streams or even lakeshores. The stretch of water is divided into continuous and non-overlapping sections. The sections should be small enough such that all fish can be counted from a single vantage point Fish density can be calculated by measuring the area surveyed. Shore based visual counts are easy, fast and inexpensive, and appropriate when the water is shallow. But any disturbance from humans will reduce the accuracy of visual counts. Fish in deep waters, turbid waters or dense habits maybe overlooked.

 Trawling:

Trawling involves towing a cone-shaped net along the bottom or through the water at a specific depth. For mid water or surface trawls, the mouth of the net is to be fitted with floats at the top (head rope) and lead at the bottom (ground rope) to keep it open. For the trawls, the net is deployed from the stern or side of the boat. The net must be towed at a faster rate than the fish can swim. Trawling is a semi-quantitative method for estimating numbers and biomass. Trawling helps in censusing large water bodies where large areas can be covered in a short time. Trawling is limited to waters free of obstructions. It needs expensive equipment and is time consuming (William Sutherland, 1996).

 Lift, throw and push netting:

Lift nets fall into two types namely hand held scoop nets and buoyant nets. Hand held scoop nets are inserted below the water surface and brought up sharply whereas the buoyant nets are allowed to lie at the bottom for a fixed period before taking it up abruptly to the surface. Cast nets are circle nets that are deployed from a boat or bank. They consist of a central line, which is retained, in the hand for hauling the net after casting. Push nets have a pocket-shaped net attached to a triangular or D shaped frame, which keeps the mouth of the net open. These techniques are appropriate for small fishes in shallow waters or near the surface for deeper waters. This type of netting is fairly cheap and used extensively in developing countries, but a major disadvantage is that little is known about their selectivity and efficiency.

 Hook and Lining:

This technique relies on catching fish with a baited hook attached to a line. The bait maybe worms or pieces of meat, fish etc. The line maybe short and held by hand or maybe longer and attached to a pole. The line is then cast into the water either from the boat or the shore. When a fish bites the hook, the line becomes tensed and is pulled up. It is a cheap and good method to census large and predatory fish, which occur at low density. It is inappropriate to monitor entire fish communities as a result of its high selectivity. The fish is inevitably damaged and subjected to considerable stress.

 Trapping:

Traps for censusing fish fall into three categories namely: pot gear, fyke nets and trapping barriers (weirs). The fish pass through an entrance hole, but are confused by the blind trappings within the net and are trapped. Fyke nets have a succession of fish, which concentrates the fish in the final section. Pot traps and fyke nets are usually set temporarily whereas the weirs are more permanent, mostly used along rivers to catch migratory species.

Trapping is one of the most versatile fish censusing methods. It is used in a wide range of habitats  from fast flowing rivers to lakes and wetlands dominated by vegetation to estuaries. It is effective to catch species that are active by night. But trapping may be labour intensive. In highly vegetated areas the traps may be damaged (William Sutherland, 1996).

 Gill netting:

It is a passive technique and relies on the fish trying to swim through diamond shaped apertures in a net set vertically in the water column. The apertures are large enough to allow the fish’s head but not the body and fish become trapped. Gill nets usually have a head line along the bottom edge and a float line along the top. By varying the length of the head line, gill nets can be made to sink to the bottom or stay at the surface of water, allowing the capture of fishes at various depths.

 Gill netting is a low cost method for censusing fish. Gill nets are relatively cheap and long lasting. They are most effective in lakes and rivers with little current when the target species is highly mobile. The major disadvantage is that the fish in gill nets often die, especially if the net was set for too long or too many fishes are caught.

 Sampling Representative Habitat:

Fish sampling Processing and Enumeration:

Effort should be made when sampling to avoid regionally unique natural habitat. Samples from such situations, when compared to those from sites lacking the unique habitat, will appear different, i.e., assess as in either better or worse condition, than those not having the unique habitat. This is due to the usually high habitat specificity that different taxa have to their range of habitat conditions; unique habitat will have unique taxa. Thus, all sampling is focused on sampling of representative habitat (EPA, 1989).

Composite sampling is the norm for investigations to characterize the reach, rather individual small replicates. However, a major source of variance can result from taking too few samples for a composite. Therefore, each of the protocols (i.e., for periphyton, benthos, fish) advocate compositing several samples or efforts throughout the stream reach. Replication is strongly encouraged for precision evaluation of the methods.

When sampling wadeable streams, rivers, or waterbodies with complex habitats, a complete inventory of the entire reach is not necessary for bioassessment. However, the sampling area should be representative of the reach, incorporating riffles, runs, and pools if these habitats are typical of the stream in question. Midchannel and wetland areas of large rivers, which are difficult to sample effectively, may be avoided. Sampling effort may be concentrated in near-shore habitats where most species will be collected. Although some deep water or wetland species may be undersampled, the data should be adequate for the objective of bioassessment.

Processing of the fish biosurvey sample includes identification of all individuals to species, weighing and recording incidence of external anomalies. It is recommended that each fish be identified and counted. The data from the counted and weighed sub sample is extrapolated for the total.

Fish Environmental Tolerance characterisations:

Responses of individual species to pollution vary regionally and according to the type of pollutant. Tolerance characterisation of fishes is presented in a Table in the annexure (EPA, 1989). 

 IDENTIFICATION OF FISHES

 Scientific identification of fishes is based mainly on external characters such as body shape, length, depth, mouth and nature of fish spines, scales, etc. The best way to collect fish for a scientific or taxonomic study is to catch them alive through a fishing net, trap or any other device locally adopted except poisoning with toxic chemicals (Jayaram K.C., 1996).

Outline lateral view diagrams of A) a non-scaled catfish and B) a scaled fish showing body parts

Fish have different ecological preferences and inhabit waters best suited to them. Environmental factors influence the predominance of certain species of fish. For instance, river fishes prefer riffle or quiet areas; a hill stream with fast flowing water over rocky bed may not have large sized carps, while dimly lit, shallow swampy pools may have cat fishes, mussels, eels and may not have fishes like rohu, mrigal etc.

The fishes caught are segregated mainly based on the presence or absence of scales on the body. When scales are present, they are further separated based on body shape, number and length of fins. In the case of fishes without fins, they are separated according to the total number of barbels. After the segregation, they are identified according to the keys (Jayaram K.C., 1996).

Classification of fishes for scientific study is done through taxonomy or systematics. Under this, each fish is given a name of two words; the first one is generic name and the second specific name, followed by the name of the author who described it first. There may be many fishes under the first word, which is called Genus. This indicates the affinity of the fish grouped under the same genus due to common features. Similarly, a number of Genera (plural of genus) are grouped under the term Family, while a number of families are put under an Order. Many orders come under a Class. The characters differentiating orders and families are distinct, but down the hierarchy, they become insignificant. For identification, the fishes are first grouped under orders, then families, genera and species. Identification keys are available for all orders, families and genera.

Glossary of terms for identification of fish:

 Adipose fin: A short fleshy fin, without rays behind the dorsal fin mainly on the back of catfishes.

Antrorse: Pointing forward or towards anterior direction especially in pectoral spine.

Axilla: Space behind base of a fin.

Axillary: Pertaining to the axilla.

Barbel/s: Slender, tactile whisker-like projection extending from the head of some fishes; functioning primarily as a sensory organ for locating food and locations.

Base: The part where a fin joins body, as in length of dorsal/anal fin base.

Branchial: As referred to gills.

Branchiostegeal rays: Numerous tiny thin bones arranged fanwise from the lower edge of the opercle to the ventral surface of the head and covered by the branchiostegeal membrane.

Breast: Ventral part of the body situated between head and pectoral fins.

Caudal peduncle: The narrow posterior part of the fish’s body between anal and caudal fin.

Ctenoid scale: Scales with rough, comb-like or toothed margin.

Cycloid scale: Scales that are smooth-edged, more or less circular with concentric striations.

Depth of body: The greatest vertical height of fish.

Dorsal: The back or upper part of the body.

Fin rays:

All paired and median fins in teleosts have long, mobile filament like prolongations called rays. The movements of the fins are due to the action of muscles, the movements possible due to the articulations and often flexibility of these rays. The term “ray” also applies to spines, whether they are included within the membrane of a fin or not.

The chief types of fin rays encountered are:

Branched ray: It is branched either from the base or middle or tip of the ray.

Different caudal fin shapes

(A-slightly emarginated or furcate, B-rounded with wavy margins, C-forked, D-wedge or paddle shaped, E-notched, F-truncate or cut square, G-rounded, H-lanceolate and J-ovate)

BR-branchiostegeal ray, C-chin, F-forehead, HL-head length, OP-interopercule, MB-maxillary barbell, MN-mandible, MO-mouth, MX-maxillary, NA-nape, NO-nostrils, OP-opercule, PMX-premaxillary, POP-preopercule, SOP-subopercule and SOR-suborbital

Gills: The respiratory apparatus of fishes, found within the gill openings.

Gill archers: The bony supports to which the gill rakers are attached.

Gill opening: The opening situated generally on either side of the head; the water used for breathing enters by the mouth and is expelled through gill-openings.

Gill rakers: These are thin needle like prolongations on the gill arches.

Gill slit: Each of the narrow spaces between the gill arches.

Gular plate: A hard plate covering the under part of the throat, often present in some fishes.

Isthmus: The fleshy interspace below the head and between the gill openings.

Nare, Naris, Nostril: On the snout of fishes the opening of the olfactory or organ of smell; in fishes these are usually a pair of nostrils on either side of head.

Opercule or operculum: The gill cover.

Opercular flap: A fleshy extension of the rear edge of opercule.

Origin of fin: The point where the first ray is inserted into the body of the fish.

Pectoral fins: The paired fins attached to the shoulder girdle.

Pelvic fins: The paired fins placed behind or below the pectoral fins.

Scale: One of the thin, bony or horny plates covering the whole or part of the body of most fishes. Scales can be macroscopic as on eel, small as on Chela and large as on Tor. A fish may have no scales as the catfishes.

 Different types of fins and rays

A-long dorsal fin, B-short dorsal fin, C-high dorsal fin, D-low dorsal fin, E-simple unbranched ray, F-thick ray, G-antrorsely serrated dorsal ray and H-branched ray

 Waterfowl Census Techniques:

 Birds are the easiest of all animals to census as they are often brightly coloured, relatively easy to see and highly vocal. The bird census and monitoring is an extremely cost-effective way of monitoring the overall health of the ecosystem. The censusing methods are broadly of two types: those for censusing species that are evenly distributed across the landscape and those for species that are highly clumped in distribution.

 Water birds are broadly defined as: “birds ecologically dependent on wetlands” and include traditionally recognised groups popularly known as wildfowl, waterfowl and shorebirds and waders. In addition to these groups there are other species that are dependant on wetlands such as kingfishers, passerines, etc.

 Wetland and water birds make use of a variety of conditions, from dry zones and meadows bordering lakes to open water zones. On the basis of their size, the availability of food and suitable foraging conditions, different birds can occupy different parts of the lake. Generally there are five major groups of water birds found based on the wetland zones they frequent. They are: (i) Open water birds, (ii) Waders and shoreline birds, (iii) Meadow and grassland birds, (iv) Birds of reed bed and other vegetation, and (v) Birds of open air space above wetlands (Krishna M.B., 1996).

Ducks, geese, grebes, cormorants, kingfishers, terns, gulls and pelicans represent the open water birds. Stilt, greenshank, sandpipers, storks, ibises, spoonbill, herons and egrets tend to frequent shallow waters. Rails, bitterns, coots, jacanas, moorhens, snipe, painted snipe etc, represent the vegetated portions of the wetland. The following section is adapted from “Shorebird Study Manual” (Howes and Blackwell, 1989)

Counting water birds:

 Counts should either be written immediately into a field notebook or dictated into a hand-held tape recorder.  All counts should be transcribed onto the count forms as soon as possible.  Water bird numbers can either be counted accurately or estimated, this decision depends on several factors:

·         The time period available;

·         The site conditions i.e., are the birds a long distance from the observer? Are they difficult to see amongst vegetation?

·         The size of the site. Should the count area be divided into smaller sub-sites?

·         The behaviour of the birds i.e., are they flying? Are there disturbances which may disrupt a count?

·         Weather conditions i.e., is there a heat haze, strong winds or rain?

·         The overall approximate numbers of birds present.  When counting, maximum accuracy should be aimed for, sometimes this is best achieved by counting each bird individually, sometimes by estimating numbers.

 Bird by bird counts should be made when:

-               small numbers (less than 3000)  birds are present.  This decision, based on total numbers present, will depend on      the observer’s experience;

-               there is limited movement of the birds, i.e., they are stationary, feeding or roosting;

-               there is little or no disturbance, e.g. by people or birds of prey, which may force the birds to fly often; or

-               the birds are scattered and are in an open area (e.g. resting in open water, or foraging on a wide intertidal mudflat).

 Numbers are usually estimated when:

-               there is a large number of birds present (3000+);

-               birds are continually in flight, i.e. large flocks moving to a roost site;

-               there is much disturbance, and therefore the birds are continually moving, taking prolonged observation on the ground difficult;

-               when birds are tightly packed together at a roost site and not all birds are wholly visible; or

-               when the identification of species is not possible due to poor light conditions (e.g. viewing into the sun) or the distance between the observer and the birds is large.

 Methods for accurate counting

 To count a water bird flock accurately, the counter can either write counts directly in a notebook, dictate numbers and species into a hand-held recorder or to another person to write down or use a tally counter.  A tally counter is a small, hand-held instrument which records and adds numbers at the press of a button.  This facilities fast counting and if the person counting is distracted, ensures that the number counted will not be forgotten.  Tally counters can also be used for estimating numbers of birds using blocks (see below) where for each block size the counter is pressed once.

 Accurate counts can be made using the following techniques:

-               Closely viewing individual birds, either with binoculars or telescope, and counting 1,2,3,4,5,6,7,....... etc.

-               Counting small groups of birds within a scattered flock and noting down each total, e.g. 3,7,4,2,11,17,3, etc.  Totals for each group are added to form the final total.

-               Counting flocks in multiples i.e., 2,4,6,8,10,12, ...... etc.  This method is faster than counting individually.

 Methods for estimating numbers

An easy and accurate method for estimating numbers of birds present in the 'Block Method'.  This can be used for large flocks, densely packed flocks or distant flocks, either in flight or on the ground.  This method involves counting or estimating a "block" of birds within a flock.  Depending on the overall flock size a "block" can be 10, 100 or 1,000 birds. The "block" is then used as a model to measure the remainder of the flock.  Some examples are given below.

It is very important to gain as much experience as possible by practicing counting and estimating large flocks of birds.  In time it will become easier to estimate large flocks of water birds accurately and using the block method will become the obvious choice.

 Counts can be made by a single observer or by a two-men team.  A single observer may find it difficult to count and record large numbers of birds without the aid of a hand-held tape recorder or tally counter.  However, it can be done by using gaps in the flock or markers such as rocks, bushes, fishing stakes or posts as breaks in counting when numbers can be written down in a notebook.

 A two-men team can divide activities, one observing (using binoculars and telescope), identifying, counting (or estimating), and dictating data to the second who records this in a field note book or on a prepared count form.  A two men team allows discussion on numbers estimated and species identification.

 Counts by either a single observer or a two-men team can be made in two ways:

-               a species by species count i.e., count all of one species then another etc, starting with the most abundant and finishing with the least abundant.  This ensures that even if the flock flies away during counting an estimate of the least abundant species can be made using the completed counts, e.g. there were 613 Dunlin and there were roughly half as many Terek Sandpipers, therefore the Terek Sandpiper count can be recorded as ca. 300.  This method is fairly slow but is best when birds seem settled and unlikely to fly.

 -               an all species count, i.e., observing a flock and counting Coot, 2; Mallard, 1; Pintail, 3; Wigeon, 4; etc..... until all birds have been counted.  This method is fast to use and is best for widely spaced flocks or in areas where birds are often moving.

 Techniques are outlined below for counting at high tide and low tide.

 Counting high tide roosts

High tide roosts are usually formed in areas adjacent to intertidal areas.  Birds in non-tidal areas may also gather at roosts during the evening.  Counting birds at a roost is a very effective method of identifying the species composition and overall abundance and fluctuations of species within an area, provided it is used by the majority of birds present within the area.  In many cases a high tide roost will contain a larger number of waterbirds from the adjacent intertidal area.

 The following method can be used to count waterbirds at a high tide roost.

-               scan the area with binoculars to see where the main concentrations of birds are.

-               count or estimate the overall flock size using the binoculars, e.g. 2,000 birds.

-               mount telescope on a tripod.

-               scan flock in one direction, i.e., left to right, with the telescope and make a note of all species seen.

-               make an assessment of the dominant species present e.g. Lesser Sand plover 40%, Dunlin 10% etc.

Completing these steps ensures that even if the roost is now disturbed and birds leave, some data have been collected.

-               where possible, accurate counts of individual species can now be made.  Using the telescope, move slowly through the flock counting each species.

 Counting birds flying into a roost

Once a roost site is known, a simple method of counting the birds using it is to count the birds as they fly. Flying flocks (such as large numbers which are packed closely together and change direction often) may be difficult to count accurately, so estimates need to be made and the most abundant species identified.  In some cases, flocks can be counted and species identified relatively easily (small flocks or flocks which fly in lines or small groups).  When counting a flying flock either begin at the front of the flock and move towards the back (in this way birds can be counted as they fly 'through' the field of view of the binoculars) or begin at the back and move towards the front (in this way it is possible to regulate the speed of the birds passing through the field of view).  Try both methods to see which suits you best.

 An observer must position himself suitably before high tide and wait for the arrival of the birds.  This can also be carried out by two men, each in a different position.  Each man independently counts the flocks of incoming birds and identifies as many species as possible.  When all birds have arrived at the roost the two observers can compare their counts and agree on a count figure.  Both men can then observe the roost and identify and count the different species present.  They will, therefore, have totals of three different counts and an accurate picture of the roost numbers and species composition.  A single observer should count the roost several times to get comparative counts and ultimately a maximum count.

 Counting foraging birds

Counts of this nature will either take place in an intertidal area or freshwater marsh/ rice field/lake shore, etc.  In most cases the distances between the observer and the feeding birds will be great and the landscape flat.

 A simple method, as given for counting at high tide roosts can be used to count birds widely scattered on the feeding area.

 Counting foraging birds can be made easier if they can be divided using markers.  Good markers are fishing stakes or traps, which are often left permanently in the mud flats and also physical features such as small bays, headlands or channels, etc.  Count the birds between the markers and add to get the total.

 In some cases the feeding area used by waterbirds will be too large to accurately survey (on the ground) on a regular basis.  If this is the case it will be necessary to select a small area (i.e. less than 25 ha), which can be marked.