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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

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 dont 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 its essential
for a good taxonomist to keep current (not
easy by any means)
Its 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
B

Rod

Sphere

Ellipsoid

A / 6

AB2 / 6

AB / 4
2

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