ANALISIS AIR DANAU Oleh Sri Sunarsih Jurusan Teknik Lingkungan, Fak Sains Terapan, IST AKPRIND Yogyakarta
Alkalinitas dan kesadahan • Alkalinitas: ukuran kemampuan air untuk menetralkan asam kuat (menerima dan dinetralkan oleh proton) – Dinyatakan dalam mg CaCO3 per liter mikroekivalen – Alkalinitas air alamiah berkisar 20 - 200 mg/L • Dalam air alamiah yang dominan bikarbonat dan karbonat • Kesadahan : Ukuran total konsentrasi ion Ca dan Mg – Dinyatakan dalam mg CaCO3 per liter • Sebenarnya ion Ca dan Mg diperlukan untuk pertumbuhan normal dan daya tahan tumbuhan dan binatang. • Kesadahan dapat mempengaruhi toleransi ikan terhadap logam toksik
Analisis Alkalinitas • • • •
pH meter Buret* Termometer Pengaduk Magnetik dan stirer • Top loading balance
Analisis Alkalinitas • Reagen – 0.04 N H2SO4
• Analisis total alkalinitas dengan titrasi sampai sampel mencapai pH tertentu (disebut titik akhir titrasi) • Pada pH tersebut, semua senyawa basa dalam sampel sudah habis • Jumlah asam yang digunakan sesuai dengan alkalinitas total sampel • Hasilnya dinyatakan dalam milligram per liter kalsium karbonat (mg/L CaCO3) • Juga dapat dinyatakan dalam milliekuivalen dengan dibagi 50
Analisis Alkalinitas ( 2 B C ) N 50000 total alkalinity , mg CaCO 3 / L mL sample or
( 2 B C ) N 999100 total alkalinity , eq / L mL sample Where: B = mL titrant first recorded pH (i.e., to pH = 4.5) C = total mL titrant to reach pH 0.3 unit lower, and N = normality of acid (titrant)
Analisis kesadahan • Idealnya kesadahan ditentukan dengan perhitungan secara terpisah antara kalsium dan magnesium.
Satuan kesadahan adalah mg CaCO3/L
2 .497 [Ca ] 4 .118[ Mg ] Dengan Ca dan Mg dalam mg/L
Analisis alkalinitas dan kesadahan Ada kit tes titrasi yang tersedia untuk alkalinitas dan kesadahan www.lamotte.com
www.hach.com
ANALISIS NUTRIEN • Kolorimetri & spektrofotometri • nitrogen and phosphorus using spectrophotometry • Specific techniques for students to review in or out of class included: – developing calibration curves – QA/QC : standards, spikes, etc
Kolorimetri & spektrofotometri 1.Makin tinggi kosentrasi warna = absorbansi makin tinggi,
2. Menyiapkan larutan kalibrasi standar (konsentrasi sampel harus berada dlm kisaran kalibrasi
• add a dye that binds specifically to nutrient of interest • measure the increase in “color” as an estimate of analyte concentration
3. Membandingkn absorbansi sampel dengan absoebansi standard dan mengestimasi konsentrasi sampel
Prinsip:
Kolorimetri & spektrofotometri 4. Menambahkan reagen untuk membentuk warna 5. Membandingkan • rendah….…. ke ……. tinggi
•
Konsentrasi fosfat
•
Menggunakan peta warna Mengguakan kolorimeter Menentuan absorbansinya dengan spektrofotometer
Komparator warna dan kolorimetri Test Kits – Ada banyak merek
Tabung warna Images from www.hach.com
Cakram warna
Kolorimeter saku
Instrumen pengukur warna
Hach DR2400 portable spectrophotometer
Bausch & Lomb spectrophotometer 20
Standard Kalibrasi Standard dibuat dari lrutn stndr yang lebih pekat, diencerkan dengan presisi tinggi
Ortho-P: NH4-N dan NO3-N:
Menggunakan NH4NO Use dried KH2PO4, K2HPO4, 3 kering sebagai standar (masing-masing NaH 50%)2PO4 or Na2HPO4
Water chemistry “101” Procedure: • See specific analyses • Reagents are added to each sample and standard identically • Mix after each step • Incubate at room temp or in water bath for 20 min to ~ 2 hrs, depending on the analyte
Kurva kalibrasi Standar NH4-N standards
Garis lurus: A = a + b*c
Estimasi konsentrasi
maka, jika sampel memiliki absorbansi 0.290…
Konsentrasinya kira-kira ~ 0.33 ppm N … N
Standard curves – troubleshooting Example #1 – Live with it or re-run the batch
#1
Errors in preparing the 0.25 and 0.50 ppm standards perhaps ?
Example #2 – Fit a straight line from 01000 and a 2nd line from 1200-2000 ugN/L
Use non-linear quadratic instead of a line for 0-2000 ugN/L
Re-read in smaller cuvette or dilute and re-run
#2
The line becomes nonlinear after ABS ~ 1.0 (~ 1000 ugN/L)
Absorbance @ 880 nm
Some data from northern Minnesota lakes 0.600 0.500
ABS = (-0.0010) + (0.00254)* P
Calibration curve
R2 = 0.9997 n=12
0.400 0.300 0.200
= std
Sample #1 = 11.2 ugP/L
0.100 0.000 0
50
100
150
ortho-P (ug/L)
200
250
Sample #1 - Replicate = 12.6 ugP/L Sample #1 + 50 Spike = 59.4 ugP/L
Conclusion:
% RPD = 100* (1.4)/ 11.9 = 12%
The data are valid
% R = 100* (59.4-11.9)/50 = 95%
ANALISIS TSS DAN KEKERUHAN
Analisis total padatan tersuspensi 1. Air yang volumenya tertentu disaring dengan kertas saring yang sudah dicuci, dikeringkan (pada 103-105oC), dan ditimbang (~ + 0.5 mg) 2. dibilas, dikeringkan dalam oven, ditimbang dengan neraca analitis untuk mengukur berat TSS mg/L (ppm) 3. Kertas saring disimpan untuk analisis lain misal padatan tersuspensi volatil (VSS) yang memprediksi senyawa organik
Analisis total padatan tersuspensi Penyaring jenis apa yang digunakan?
Analisis total padatan tersuspensi • Jenis penyaring: – Filter membran menahan partikulat dan organisme sub-mikron – Filter gelas mikrofiber 100% terbuat dari gelas borosilikat. – Polikarbonat – memiliki ukuran pori yang tepat tetapi alirannya lambat www.whatman.com
Total padatan tersuspensi • Ada beberapa set peralatan – Corong yang terikat klem, disekrup, atau dgn magnetic – Peralatan plstik yg bermanfaat unt di lapangan
multiple towers
Total padtn tersuspensi • Peralatan yg diperlukan
Neraca Analitik
oven pengering
Penyaring dan petri dish
Total padatan tersuspensi • Menghitung TSS: TSS (mg/L) = ([A-B]*1000)/C dengan A = berat akhir kertas saring (mg) B = berat awal kertas saring (mg) C = Volume air yang disaring (Liter)
Hubungan kekeruhan denganTSS
Aturan umum : 1 mg TSS/L ~ 1.0 - 1.5 NTU kekeruhan BUT – hamburan oleh kekeruhan bergantung pada ukuran partikel sehingga perkiraan tersebut hanya merupakan pendekatan kasar
Turbidity - meter • Kebanyakan menggunakan nefelometrik optik dan daibaca dlm satuan NTU (nephelometric turbidity units) • Pengukuran kekeruhan dilakukan dengan: – Turbidimeter (untuk sampel diskrit) – Sensor kekeruhan yg dapat dibenamkan (USGS menganggap sbg metoda qualitative)
• Instrumen Laboratorium : – Turbidimeter
Kekeruhan Turbidimeter Nefelometrik optik • Kekeruhan nefelometrik ditaksir menggunakan efek hamburan sinar oleh partikel tersuspensi • detektor terletak pada sudut 90o terhadap sumber sinar
http://www.bradwoods.org/eagles/turbidity.htm
Satuan Kekeruhan • Nephelometric Turbidity Units (NTU) • Standardnya adalah formazin atau material lain yg tersertifikasi • Satuan JTU berasal dari teknologi yang lebih awal menggunakan nyala lilin yg dilihat melalui tabung air • 1 NTU = 1 JTU (Jackson Turbidity Unit)
Kekeruhan – standard formazin • Contoh 1 set standard formazin
Kekeruhan • Here is a range of NTUs using clay
Turbidi – meters dan probes • Bench and portable instruments and kits vs.
Submersible Turbidimeters YSI 6820 with unwiped turbidity
YSI wiping turbidity
Hydrolab
Turbidity - methods • Comparability of different methods: – With the proliferation of automated in situ turbidity sensors there is concern about the comparability of measurements taken using very different optical geometries, light sources and light sensors. – The US Geological Survey and US Environmental Protection Agency are currently (August 2002) developing testing procedures for a field comparison of a number of instruments produced by different manufacturers.
Turbidity - calibration • Turbidity free water = zero (0 NTU) standard – USGS recommends filtering either sample water or deionized water through a 0.2 um or smaller filter to remove particles – WOW uses deionized water that is degassed by sparging (bubbling) with helium, to minimize air bubbles that may give false turbidity readings
Turbidity - standards • Standards range depends on anticipated sample values – Lakes - typically 0-20 NTU – Streams and wetlands - 0-20, 0-50 or 0-100 NTU – 2 non-zero standards typically adequate (response is linear) • Types of standards – Formazin particles (either from a “recipe” or purchase a certified, concentrated stock solution usually 4000 NTU) – Other commercially available materials, e.g., polystyrene
Source
Turbidity – standards Concentrations
Table of standards 2 to 20 NTU
Hach Company
Suggested holding times
Prepare daily
20 to 40 NTU
Prepare monthly
Standard Methods (APHA 1995)
All dilutions
Prepare daily
EPA Region 5
All dilutions
Prepare weekly
ANALISIS BOD
BOD • BOD mengukur jumlah oksigen yang diperlukn oleh mikroorganisme untuk menguraikan senyawa organik, termasuk untuk mengoksidasi senyawa anorganik • Uji BOD mengukur jumlah oksigen yg diperlukan selama waktu tertentu (biasanya 5 days at 20o C)
BOD 5 • DO is measured initially and again after a 5-day incubation at 20o C – BOD is computed from the difference between initial and final DO
• The rate of oxygen consumption is affected by a number of variables: – temperature – pH – the presence of certain kinds of microorganisms – the type of organic and inorganic material in the water
BOD - analysis • Equipment needed: – Incubation bottles – Air incubator or water bath thermostatically controlled at 20 +/- 1o C – DO meter and probe
BOD • Reagents: – Dilution water – provides nutrients necessary for microorganism growth – Seed – a population of microorganisms capable of oxidizing the organic matter in the sample – Commercially available or freeze-dried culture – A “conditioned” bacteria source (effluent from a biological treatment source such as a wastewater treatment plant).
– Glucose-glutamic acid standard
BOD – QA/QC • Assure quality with: – Seed control – determine the BOD of the seeding source – Dilution water blank – used to check for quality of unseeded dilution water and incubation bottle cleanliness • Steps to Include: – Read and record temperature of incubator – Prepare replicate bottles for dilution water blanks and seed controls – Include at least one set of replicate samples per analysis
BOD - procedure • Blanks – Prepare dilution water, bring to 20o C and aerate – Add sufficient seeding material to produce a DO uptake of 0.05 to 0.1 mg/L in 5 d (dilution water)
• Samples – Add sample to bottle and dilute. – Dilutions should result in a residual DO of at least 1 mg/L and DO uptake of at least 2 mg/L after 5 day incubation
BOD – procedure • Steps in procedure: – Fill bottles with enough dilution water so the stopper displaces all of the air, leaving NO air bubbles – Read initial DO – Incubate for 5 days at 20o C – Read final DO – Calculate BOD5 correcting for the exact duration
Phytoplankton/Algae – counting methods
BOD Calculations – When dilution water is not seeded:
D1 D 2 BOD5day (mg / L) P – When dilution water is seeded:
( D1 D 2) ( B1 B 2) f BOD5day (mg / L) P
Algae – chlorophyll instrumentation • Spectrophotometer: – Visible with 1-2 nm bandwidth – Matched cuvettes, 1-5 cm
• Fluorometer: – Requires excitation and emission filters specifically for chlorophyll measurement
Algae – chlorophyll filtration Apparatus - extraction – Prewashed 47 mm glass fiber filters (GF/C, GF/F, AE, or equivalent) – Gelman polycarbonate filtration tower or equivalent – Vacuum pump (5 to 7.5 psi) – Centrifuge (clinical) – DIW/acetone (90%) washed 15 mL Corex centrifuge tubes with caps
Algae – chlorophyll filtration (cont.)
• Filter a known volume of water through a GF/C filter • Volume filtered depends upon algal density
• Add a few drops of saturated MgCO3 solution near the end • When all the water has been pulled through, fold the filter into quarters and wrap in foil
Algae – chlorophyll storage • Wrap the folded filter in a square of foil, label, then freeze • Record the volume filtered, date, site, depth, replicate # all with permanent marker • Store the filter in the freezer at < 20o C • EPA holding time for a frozen chlorophyll filter is 2 weeks
Algae – chlorophyll extraction & analysis • Chlorophyll extraction:
– Tear filter into several pieces – Place in a test tube – Add 10 mLs of 90% acetone – Extract overnight at 4oC
• Chlorophyll analysis: – After 18-24 hr extraction, centrifuge to settle filter debris – Read absorbance or fluorescence of the supernatant
Algae – chlorophyll measurement • Measure absorbance of a 90% acetone solution blank at 750 nm and at 664 nm to correct for primary pigment absorbance • Record sample absorbance at 750 nm and 664 nm • Estimate phaeophytin by acidifying the sample. Record the absorbance at 665 nm and again at 750 nm • Run working standard solutions of purified chlorophyll-a (Sigma Chemical Co. Anacystis nidulans by the procedure used for the blank)
Algae – chlorophyll and phaeophytin • What is phaeophytin? – Degradation product of chlorophyll – Absorbance wavelength (665 nm) is very close to that of chlorophyll (664 nm)
acid
H
Algae –spectrophotometry calculations
26 .7[ E E ] V chlorophyl l a ( g / L ) V L 26.7[1.7 E E ] V phaeophytin( g / L) V L 664 b
665 a
ext
sample
665 b
664 a
sample
Where: b = before acidification a = after acidification E664b - [{Abs664b(sample)–Abs664b(blank)}-{Abs750b(sample)–Abs750b(blank)}] E665a - [{A665a(sample)-Abs665a(blank)}-{Abs750a(sample)-Abs750a(blank)}] Vext = Volume of 90% Acetone used in the extraction (mL) Vsample = Volume of water filtered (L) L = Cuvette path length (cm)
ext
Algae – chlorophyll QA • Quality assurance – There are no commercial QA check standards – Lab replicates are usually not done – Essentially, the analysis is a one-shot deal, you don’t get a second chance, so be careful – Field replicates should be done every 10 samples – Cut filters in half and save one half if nervous
Algae- counting methods • • • •
Wet mounts Filter Counting chambers Utermohl – requires an inverted microscope (light from above)
• Sedgewick rafter chamber • Hemocytometer
Algae – counting methods Microscopes capable of magnifications of 100X to 1000X
Compound microscope
Inverted microscope
Less expensive inverted microscope
Algae- taxonomy • Use an algal taxonomic key that shows species from your geographical area • Phytoplankton are continually being described and re-classified so it’s essential for a good taxonomist to keep current (not easy by any means) • It’s a good idea to take photographs of slides for cataloging
Algae – determining biomass • Algal biomass (standing crop): – A quantitative estimate of the total mass of living organisms within a given area or volume
• Biovolume estimates: – Identification to genus and species level – Calculate cell volume by approximation to nearest geometrical shape – Count cells over a known area of the slide so cells per unit volume can be determined
• Chlorophyll
Algae – determining biovolume • Taxonomic keys often include questions about size • Determining size is basically like using a ruler. – The standard ruler for a microscope is called an "ocular micrometer," which is fitted into the eyepiece of your microscope
Algae – determining biovolume Some formulas to estimate biovolume from cell dimensions (Wetzel & Likens 2000) B
A
A B
A
Rod
Sphere
Ellipsoid
A / 6
AB2 / 6
AB / 4 2
3
Algae – chlorophyll determination
Algae – chlorophyll determination • Measuring chlorophyll-a concentration remains the most common method for estimating algal biomass • Chlorophyll-a concentration has also been shown generally, when comparing lakes, to relate to primary productivity (Wetzel 1983) • Can be used to assess the physiological health of algae by examining its degradation product, phaeophytin
Algae – chlorophyll basics • Algal biomass is most commonly estimated by chlorophyll-a. • Units are ug/L or mg/L (ppb and ppm) • Detection limit depends upon method used
Algae – chlorophyll methodology • Spectrophotometry and fluorometry, utilizing 90% acetone extraction, remain the most commonly used methods • Spectrophotometry is most widely used but fluorometry is more sensitive and may be used when low levels of chlorophyll are anticipated or when handling large volumes of water is logistically difficult
Algae – counting methods Microscopes capable of magnifications of 100X to 1000X
Compound microscope
Inverted microscope
Less expensive inverted microscope
Bacteria – 2 indicator methods Two basic methods: 1. membrane filtration 2. multiple-tube fermentation
http://picturethis.pnl.gov/picturet. nsf/f/uv?open&SMAA-3V9T37
http://www.intelligence.gov/2community_examples.shtml
Bacteria – membrane filter technique • The fecal coliform MF procedure uses an enriched lactose medium and incubation temperature of 44.5 ± 0.2o C for selectivity. • Results in 93% accuracy (APHA 1995) in differentiating between coliforms found in the feces of warm-blooded animals and those from other environmental sources. • Fecal Coliform is reported as colony forming units per 100 mL (CFU/100 mL).
Bacteria – membrane filter equipment • Materials needed for MF method: – Air incubator or water bath – Non-corrugated forceps – Heat sterilizer (BactiCinerator) – Filter flask and tower (Autoclavable) – Vacuum pump or water aspirator
Bacteria – membrane filter equipment • MF materials (continued): • Sterile 50 mm petri plates (with tight-fitting lids) • Sterile 0.45 um gridded membrane filters • Sterile absorbent pads • Autoclave (121o C at 15-17 psi)
http://www.nbtc.cornell.edu/biofacility/autoclave.html
Bacteria – membrane filter procedure Procedure: –Saturate the absorbent pad with M-FC broth –Select a sample volume that will yield 20-60 colonies/filter –Filter sample and dilution water through pad –Place pad into petri dish –Invert plates and place in incubator for 24 hrs
Bacteria – membrane filter counting • Fecal coliform colonies bacteria are various shades of blue. • Non-fecal colonies are gray to cream colored. – normally, few of these are present.
Bacteria – MF counting (cont.) http://water.usgs.gov/owq/FieldManual/Chapter7.1/images/Fig7.1-3.gif
image showing method of counting
Bacteria – multiple tube fermentation MTF image process
http://water.usgs.gov/owq/FieldManual/Chapter7.1/images/Fig7.1-3.gif
Bacteria – cleaning and sterilizing All equipment
Wash equipment thoroughly with dilute nonphosphate, laboratory-grade detergent. Rinse 3 X with hot tap water Rinse again 3-5 X with deionized or glass-distilled water.
Glass, polypropylene, or Teflon™ bottles
If sample will contain residual chlorine or other halogens, add Na2S2O3. If sample will contain > 10 ug/L trace elements, add EDTA. Autoclave at 121 C for 15 min or bake glass jars at 170 C for 2 hrs.
Stainless-steel field units
Flame sterilize with methanol (Millipore™ Hydrosol units only), or autoclave, or bake at 170 C for 2 hrs
Portable submersible pumps and pump tubing
Autoclavable equipment (preferred): autoclave at 121 C for 15 min. Non-autoclavable equipment: Submerge sampling system in a 200 mg/L laundry bleah solution and circulate solution through pump and tubing for 30 min; follow with thorough rinsing, inside and out, with sample water pumped from the well. **SEE NOTES
Bacteria – USGS summary
Test (media type)
Total coliform bacteria (m-Endo) Escherichia coli After primary culture as total coliform colonies on m-Endo (NA-MUG)
Ideal count range (colonies per filter) 20-80
None given but much fewer in number than total coliforms on the same filter
Typical colony color, size, and morphology
Colonies are round, raised and smooth; 1 to 4 mm di; and red with golden-green metallic sheen. Colonies are cultured on m-Endo media as total coliform colonies. After incubation on NA-MUG, colonies have blue florescent margins with a dark center. Count under a long wave ultra violet lamp in a completely dark room.
Fecal coliform bacteria (m-TEC)
20-60
Colonies are round, raised and smooth with even to lobate margins; 1 to 6 mm di; light to dark blue in whole or part. Some may have brown or cream colored centers.
Escherichia coli (m-TEC)
20-80
Colonies are round, raised and smooth; 1 to 4 mm di; yellow to yellow brown; many have darker raised centers.
Fecal streptococci (KF media)
20-100
Colonies are small, raised, and spherical; about 0.5 to 3 mm di; glossy pink or red in color.
Enterococci (m-E and EIA)
20-60
Colonies are round, smooth and raised; 1 to 6 mm di; pink to red with a black or red dish – brown precipitate on underside.
Fecal coliforms – troubleshooting
poor seal around the edges; poorly seated with air bubble
Dry spot from poor seating
Uneven; not mixed well; low volume
No matter which assay is used, after incubation there should be ~20-60 colonies evenly distributed across the Petri dish
Fecal coliforms – troubleshooting (cont.)
Too many – use less sample
Too few – use more sample
Looks good
