Chapter 8

TABLE OF CONTENTS Total Phosphorus

Page Section 1: Introduction to Total Phosphorus....................................................................................... Section 2: Glossary............................................................................................................................. Section 3: Safety and Hygiene............................................................................................................ Section 4: Sampling............................................................................................................................ Section : Sample Preser!ation......................................................................................................... Section ": Sample #ontainers............................................................................................................ Section $: Interpretations.................................................................................................................... %ui& '.1................................................................................................................................................ Section ': Total Phosphorus............................................................................................................... Section (: )*uipment and +eagents................................................................................................... Section 1,: -a.oratory Procedure...................................................................................................... Section 11: Total Phosphorus............................................................................................................. Section 12: )*uipment and +eagents................................................................................................. Section 13: -a.oratory Procedure...................................................................................................... Section 14: #ali.ration #ur!e............................................................................................................. Section 1 : #alculations..................................................................................................................... Section 1": Interferences.................................................................................................................... Section 1$: %01%#............................................................................................................................. %ui& '.2................................................................................................................................................ 0ns2ers to %ui&&es.............................................................................................................................. 0ppendi3 0: +eferences 0ppendi3 4: Phosphorus +eagents 0ppendi3 #: Sample 4ench Sheet 0ppendi3 5: Preparation of a #ali.ration #ur!e 0ppendi3 ): 6ethods #hec7list 0ppendi3 8: 6ethod #hec7list / +eagent 0ppendi3 G: 6ethod #hec7list 5igester 0ppendi3 H: Phosphorus 0scor.ic 0cid 9Single +eagent: /" " "/$ $ $/' ' '/( 2 2 2 3 3 3 3 4 4 4 4/

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Chapter 8
TOTAL PHOSPHORUS
Section 1 !NTRO"UCT!ON TO TOTAL PHOSPHORUS ;aste2ater is relati!ely rich in phosphorus compounds. Phosphorus is a nutrient used .y organisms for gro2th. It occurs in natural 2ater and 2aste2ater .ound to o3ygen to form phosphates. Phosphates come from a !ariety of sources including agricultural fertili&ers< domestic 2aste2ater< detergents< industrial process 2astes and geological formations. The discharge of 2aste2ater containing phosphorus may cause algae gro2th in *uantities sufficient to cause taste and odor pro.lems in drin7ing 2ater supplies. 5ead and decaying algae can cause o3ygen depletion pro.lems 2hich in turn can 7ill fish and other a*uatic organisms in streams. 8or this reason< phosphorus remo!al is an essential role of 2aste2ater treatment plants and testing for phosphorus in the plant effluent is critical. Phosphates are classified as orthophosphates< polyphosphates and organic phosphates. In this procedure< orthophosphates can .e determined directly .y colorimetric analysis. =ther types re*uire a digestion step to con!ert the >com.ined? phosphate to the ortho form for analysis. This gi!es the Total Phosphorus result. Section # $LOSSAR% Blan& 0 preliminary analysis omitting only the sample to pro!ide an un.iased reference point or .aseline for comparison. The 4lan7 is usually run on distilled 2ater. Nutrient 0ny su.stance used .y li!ing things that promotes gro2th. The term is generally applied to nitrogen and phosphorus in 2ater< .ut is also applied to other essential and trace elements. Phosphate cur'e 0 cali.ration cur!e that plots concentrations of 7no2n standards against the instrument@s response 9color intensity< a.sor.ance< milli!olts< etc.:. Section ( SAFET% AN" H%$!ENE ;hene!er samples of 2aste2ater are handled< it is !ery important that operators 2ash his or her hands .efore eating or smo7ing. ;hile some la.oratory chemicals are not dangerous< many of them are poisonous or harmful to s7in and clothing. +u..er glo!es and safety glasses should .e used. It is important to 2ash thoroughly 2ith soap and 2ater after handling la.oratory chemicals< especially if chemicals come into contact 2ith the s7in. Aeep .ench areas free of clutter and clean .ench surfaces 2ith disinfectant after testing. +ead the la.els carefully and 7no2 2hat to do in case of an accidental spill. 0l2ays clean up spills *uic7ly and in the safest possi.le manner using disposa.le rags or to2els. 0cids and .ases can .e corrosi!e. #are should .e ta7en 2hen handling them. Be!er add 2ater to acid. 0l2ays add acid to 2ater !ery slo2ly .ecause acids and .ases can generate heat 2hen mi3ed 2ith 2ater. The Total Phosphorus test in!ol!es heating samples in the digestion step. To a!oid inCuries caused .y heat< use glo!es or tongs to handle hot or potentially hot glass2are. Be!er assume that a .ea7er or flas7 is cool.

#hapter ' / 2

Section ) SA*PL!N$ Samples used for the determination of phosphorus can .e either gra. or composite. The type of sample used 2ill depend on the monitoring re*uirements< plant operating procedures and the testing and sample storage capa.ilities of indi!idual plants. Samples should .e collected from 2ell/mi3ed areas in the process flo2 so that they are representati!e of the total flo2. Section + SA*PLE PRESER,AT!ON If Total Phosphorus is the only determination to .e made< acidify the sample to less than pH 2 2ith sulfuric acid and cool to 4D#. 0cidified and cooled samples may .e held for up to 2' days. If the test is run immediately< no preser!ati!e is re*uired. Samples to .e tested for dissol!ed reacti!e phosphorus< dissol!ed acid/hydroly&a.le phosphorus< or total dissol!ed phosphorus must .e filtered. See the 1'thth )dition of >Standard 6ethods for the )3amination of ;ater and ;aste2ater? for information on these tests. Section - SA*PLE CONTA!NERS Special sampling de!ices and storage containers are not necessary for phosphorus testing. Sampling de!ices should dra2 from 2ell/mi3ed areas of tan7s or pipes< .e made of resistant materials that 2ill not rust or corrode< .e capa.le of ta7ing samples that are proportional to the plant@s flo2< and .e easily and thoroughly cleaned. Storage containers should .e made of corrosion resistant material 2hich can stand repeated refrigeration. These containers should ha!e lea7/proof tops. 5o not store lo2 concentration samples in plastic .ottles since phosphorus may .e adsor.ed on the container 2alls. +inse all sampling containers 2ith diluted 91 E 1: hydrochloric acid. Then rinse 3 times 2ith distilled 2ater. Be!er use commercial detergents for cleansing any glass2are used in the storage or analysis of samples for phosphorus determination. Section . !NTERPRETAT!ONS 4ecause of its effect on recei!ing streams< testing for phosphorus is critical to 2aste2ater treatment plants. +anges for phosphorus concentrations are gi!en .elo2. Sample type Influent Primary )ffluent 0cti!ated Sludge )ffluent Tric7ling 8ilter )ffluent 0d!anced Treatment )ffluent range / 4 / 3 / 4 / ,.1 / 1, ( ' ' 2., mg1mg1mg1mg1mg1-

;hat is normal for one plant is not necessarily normal for another. )ach operator must determine 2hat range reflects normal conditions for his or her plant. Falues outside the esta.lished normal range indicate that a condition e3ists 2hich could yield poor effluent *uality.

#hapter ' / 3

/ui0 811 1. 2. 3. ;hy is phosphorus remo!ed from 2aste2aterG ;hat are the three .asic forms of phosphorus found in 2aste2atersG ;hat must .e done .efore polyphosphates can .e measuredG

Section 8 TOTAL PHOSPHORUS "ESCR!PT!ON OF "!$EST!ON STEP Since phosphorus e3ists in se!eral distinct forms in 2aste2ater samples and the appro!ed test method measures only the orthophosphate form< pretreatment methods ha!e .een de!eloped to con!ert the !arious forms of phosphate/phosphorus to the orthophosphate form. If the only determination to .e made is Total Phosphate/Phosphorus< the sample is digested to con!ert .oth the polyphosphate and the organic phosphate to the ortho form at the same time. If the analyst must test for the !arious types of phosphate< an acid/hydrolysis must .e performed. Since this is not re*uired for most facilities< it is not included in this te3t. Section 2 E/U!P*ENT AN" REA$ENTS E/U!P*ENT 1. 2. 3. 4. . Hot plate Tongs or glo!es Scoop 9,.4 g capacity: 12 m- )rlenmeyer flas7s 9acid 2ashed: , m- graduated cylinders 9acid 2ashed:

REA$ENTS 1. 2. 3. 4. Phenolphthalein indicator Sulfuric acid solution 0mmonium persulfate< crystal Sodium hydro3ide< 1 B See 0ppendi3 4 for the procedures for preparation of the reagents used in this method. Section 13 LABORATOR% PROCE"URE 1. 2. 3. 6easure , m- or an appropriate amount of sample diluted to , m- 2ith distilled 2ater. 0dd 1 drop phenolphthalein indicator. If a red color de!elops< add sulfuric acid solution until color Cust disappears. 0dd 1 m- of sulfuric acid solution and ,.4 g of ammonium persulfate.

#hapter ' / 4

4. . ".

4oil gently for 3, to 4, minutes or until the total !olume is 1, m-. #ool< add 1 drop of phenolphthalein and neutrali&e to a faint pin7 color 2ith 1 B sodium hydro3ide. 6a7e up to , m- 2ith distilled 2ater. The digested sample is then tested for total phosphate as outlined in Section 12.

NOTE If precipitate forms< do not filter. Section 11 TOTAL PHOSPHORUS ASCORB!C AC!" PROCE"URE 0n accepted method for determining phosphate/phosphorus is the 0scor.ic 0cid Procedure. The procedure is suita.le for concentrations of ,.,1 to " mg1- P= 4/P. 0mmonium moly.date and potassium antimonyl tartrate react in acidic solution 2ith orthophosphate to form a heteropolyic acid 9phosphomopyd.ic acid: 2hich can .e reduced .y ascor.ic acid to form an intense .lue color. Section 1# E/U!P*ENT AN" REA$ENTS E/U!P*ENT 1. 2. 3. Spectrophotometer 2ith an infrared phototu.e for use at '', nm 2ith a light path of at least 2. cm 91 in: 0cid 2ashed glass2are 0n analytical .alance capa.le of 2eighing to ,.1 mg accuracy

REA$ENTS 1. 2. 3. 4. . ". Sulfuric acid solution B

Potassium antimonyl tartrate solution 0mmonium moly.date Solution 0scor.ic acid solution #om.ined reagent Standard phosphate solution

See 0ppendi3 4 for the procedures for preparation of the reagents used in this method. Section 1( LABORATOR% PROCE"URE 1. Pipette ,., m- or an appropriate amount diluted to , m- of digested sample into an acid cleaned< dry 12 m- )rlenmeyer flas7.

NOTE If only orthophosphate is to .e determined< an undigested sample is used. 2. 0dd 1 drop of phenolphthalein indicator. If a red color de!elops< add disappears. B sulfuric acid until the color

#hapter ' /

3. 4. .

0dd '., m- of com.ined reagent and mi3 thoroughly. 0llo2 at least 1, minutes 9.ut not more than 3, minutes: for color de!elopment. 6easure a.sor.ance at '', nm using a reagent .lan7 to &ero the spectrophotometer. The reagent .lan7 is made using , m- of distilled 2ater carried through the digestion step and ascor.ic acid procedure. In highly colored or tur.id samples< prepare a sample .lan7 .y adding all the reagents to the sample e3cept the ascor.ic acid and potassium antimonyl tartrate. Su.tract the a.sor.ance of this .lan7 from the a.sor.ance of the sample.

".

#hec7 the sample@s a.sor.ance against the cali.ration cur!e and determine the concentration. #orrect for dilution.

Section 1) CAL!BRAT!ON CUR,E Since the phosphate concentration is measured as a function of a.sor.ance< a standard cur!e of a.sor.ance !ersus 7no2n phosphate concentrations must .e prepared. Si3 standard phosphorus concentrations and a distilled 2ater .lan7 are treated 2ith the same digestion procedures as the samples. These " !alues are used to plot a.sor.ance !ersus phosphate concentration to gi!e a straight line passing through the origin. Prepare " dilutions of the ., mg1- Phosphate standard to result in the follo2ing final concentrations: final conc. ,.1 mg1,.2 mg1,.4 mg1,." mg1,.' mg11., mg1!olume of std. 1., m2., m4., m"., m'., m1,., mfinal !ol. in flas7 ,., m,., m,., m,., m,., m,., m-

Perform the Total Phosphorus procedure including the digestion step and color de!elopment. +ecord the a.sor.ances and plot the cur!e. 0t least one standard phosphate concentration must .e included 2ith each .atch of samples as a chec7 on the cali.ration cur!e. Prepare a ne2 cur!e e!ery si3 months. See 0ppendi3 5 for more information on the preparation of cali.ration cur!es. Section 1+ CALCULAT!ONS 5etermine the final concentration of the sample using the follo2ing e*uation: mg1- P H mg1- from the cur!e 3 , mdi!ided .y initial !olume used 9m-: 8or e3ample< if m- of Influent gi!es an a.sor.ance of ,.3 2hich represents ,. cali.ration cur!e< the corrected concentration of phosphate/phosphorus is: mg1- P H ,. ' 3 , m- di!ided .y m- H . ' mg1' mg1- on the

Section 1- !NTERFERENCES

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0rsenates react 2ith the moly.date reagent to produce a .lue color similar to that formed in the phosphate determination. #oncentrations as lo2 as ,.1 mg 0s1- interfere 2ith the analysis. He3a!alent chromium and nitrite interfere to gi!e lo2 results. Chapter 1. /A4/C 0 %uality 0ssurance1%uality #ontrol program is re*uired .y the BP5)S permit. %uality 0ssurance 9%0: is a set of operating principles that must .e follo2ed during sample collection and analysis. -a. .ench sheets must .e maintained that document 2hen the sample 2as collected< ho2 it 2as preser!ed< and 2hat results 2ere o.tained. %uality #ontrol 9%#: includes any testing 2hich is done to pro!e that the results are relia.le. =ne of e!ery ten samples analy&ed should .e a %# chec7. This may include duplicate samples< spi7e samples< reagent .lan7 analyses and 7no2n %# samples o.tained from outside sources. 5uplicate sample analysis in!ol!es analy&ing the same sample t2ice and comparing the results. The closer the results< the more accurate the analysis. +esults should not differ .y more than 1,I. Spi7e sample analysis in!ol!es adding 7no2n amounts of analyte to a sample and calculating the percent reco!ery. These are discussed further in #hapter 1,. In Phosphorus analysis< a distilled 2ater .lan7 must .e run 2ith e!ery .atch of samples tested. It is used .oth to &ero the spectrophotometer and to sho2 that the glass2are and reagents are not contaminated 2ith phosphates. 0t least one chec7 standard must also .e run 2ith each .atch. If a !ariety of samples are .eing tested 2ith results e3pected to co!er a 2ide range of concentrations< test t2o or more standards. The a.sor.encies of the standards must agree closely 2ith the true !alue or a ne2 cur!e must .e dra2n. Spi7e samples are run .y adding the ., mg1- Phosphate standard to a sample. To simplify the calculation< use the same amount of standard in the spi7e sample as you are using in your chec7 standard. 8or e3ample< an )ffluent sample is spi7ed 2ith 2 m- of the ., mg1- Phosphate standard yielding the follo2ing results: )ffluent sample result: ,.11" mg1Spi7ed )ffluent sample result: ,.31, mg1#hec7 standard 92 m-: result: ,.1($ mg1E5pecte6 spi&e result 7 sa8ple result 9 stan6ar6 result H ,.11" E ,.1($ H ,.313 Percent reco!ery H spi7e result di!ided .y e3pected result 3 1,,I H ,.31, di!ided .y ,.313 3 1,,I H ((I %# samples 2ith 7no2n concentrations of Phosphate can .e purchased from chemical supply companies. The results from duplicate< spi7e and outside %# samples should .e recorded in a %# note.oo7. Sample .ench sheets are included in 0ppendi3 #. /ui0 81#

#hapter ' / $

1. 2.

;hat four reagents are used to ma7e the com.ined reagent for the phosphorus testG ;hat is the storage time of this reagentG ;hat sample !olume 2ould you start 2ith if the estimated phosphorus content of the sample 2as 1 mg1-G 4 mg1-G 1, mg1-G

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Ans:ers to /ui00es /ui0 811 1. ;hy is phosphorus remo!ed from 2aste2aterG Phosphorus is re8o'e6 ;ro8 :aste:ater <ecause it pro'i6es a nutrient or ;oo6 source ;or algae1 "ea6 algae can cause serious o5=gen 6epletion pro<le8s in recei'ing strea8s :hich in turn can &ill ;ish an6 other a>uatic li;e1 Also? algae can cause taste an6 o6or pro<le8s in 6rin&ing :ater supplies1 2. ;hat are the three .asic forms of phosphorus found in 2aste2atersG The three <asic ;or8s o; phosphorus ;oun6 in :aste:aters are Orthophosphates Pol=phosphates? an6 Organic phosphates . 3. ;hat must .e done .efore polyphosphates can .e measuredG The sa8ples 8ust <e aci6 h=6rol=0e6 @or 6igeste6A to con'ert the pol=phosphates to the orthophosphate ;or8 ;or 8easure8ent1 /ui0 81# 1. ;hat four reagents are used to ma7e the com.ined reagent for the phosphorus testG ;hat is the storage time of this reagentG The ;our reagents that are use6 to 8a&e the co8<ine6 reagent ;or the phosphorus test are + N Sul;uric aci6 solutionB Potassiu8 anti8on=l tartrate solutionB A88oniu8 8ol=<6ate SolutionB an6? Ascor<ic aci6 solution1 The storage ti8e is ) hours1 2. ;hat sample !olume 2ould you start 2ith if the estimated phosphorus content of the sample 2as 1 mg1-G 4 mg1-G 1, mg1-G #3 to (3 8L ;or 1 8g4L + to 13 8L ;or ) 8g4L 1 to ) 8L ;or 13 8g4L

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APPEN"!C A Re;erences Standard 6ethods for the )3amination of ;ater and ;aste2ater< 1'thth )dition< 0;;0< 0PH0< ;P#8J ;ater Pollution #ontrol 8ederation< ;ashington< 5#< 1((2. 6ethods for #hemical 0nalysis of ;ater and ;astes< K.S. )P0 / ",,14/$(/,2,< 6arch 1($(. 0 8ield Study Program< =peration of ;aste2ater Treatment Plants< Aerri< Aenneth et al< Kni!ersity of #alifornia< Sacramento. NOTES

#hapter ' 1 0ppendi3 0 1 Page 1

APPEN"!C B Phosphorus Reagents 5igestion reagents: 11 Sul;uric aci6 6igestion reagent #arefully add 3,, m- concentrated Sulfuric acid to appro3imately ",, m- distilled 2ater and dilute to 1 - 2ith distilled 2ater. #1 So6iu8 h=6ro5i6e 1 N 5issol!e 4, g of Sodium hydro3ide pellets in distilled 2ater. #ool and dilute to 1 -. 0scor.ic acid procedure: 11 Sul;uric aci6 solution + N 5ilute $, m- of concentrated Sulfuric acid to ,, m- 2ith distilled 2ater 9add acid to 2ater< #0+)8K--L:. #1 Potassiu8 anti8on=l tartrate solution 5issol!e 1.3$1 gm of Potassium antimonyl tartrate in 4,, m- of distilled 2ater in a ,, m!olumetric flas7 and dilute to ,, m-. Store in a glass stoppered .ottle. (1 A88oniu8 8ol=<6ate solution 5issol!e 2, g of 0mmonium moly.date in ,, m- of distilled 2ater. Store in a glass stoppered .ottle. )1 Ascor<ic aci6 solution 311 * 5issol!e 1.$" g of 0scor.ic acid in 1,, m- of distilled 2ater. Store at 4D# and discard after 1 2ee7. +1 Co8<ine6 reagent To prepare 1,, m- of com.ined reagent< com.ine the follo2ing reagents and mi3 thoroughly after each addition. , m- B Sulfuric acid m- Potassium antimonyl tartrate solution 1 m- 0mmonium moly.date solution 3, m- 0scor.ic acid solution +eagents must .e added in the order listed. 0ll reagents must .e at room temperature .efore mi3ing. If tur.idity occurs< sha7e the reagent and let stand until tur.idity clears. The com.ined reagent is sta.le for only 4 hours. -1 Stoc& Phosphate Solution 5issol!e 21(. mg of anhydrous potassium dihydrogen phosphate in distilled 2ater and dilute to 1 -. This yields a concentration of , mg1-. .1 Stan6ar6 Phosphate Solution

#hapter ' 1 0ppendi3 4 1 Page 1

5ilute 1, m- of the stoc7 solution to 1,, m- 2ith distilled 2ater. This yields a concentration of ., mg1-.

#hapter ' 1 0ppendi3 4 1 Page 2

APPEN"!C C Sa8ple Bench Sheet Phosphate Anal=sis Sample collection 5ate: MMMMMMM Sample preser!edG yes no 0nalysis run 5ate: MMMMMMM Sa8ple T=pe !nitial ,olu8e Time: MMMMMMM 4y: MMMMMMM Preser!ati!e: MMMMMMMMMMMM Time: MMMMMMM 4y: MMMMMMM Final ,olu8e A<sor<ance Conc1 Correcte6 Conc1

#hapter ' 1 0ppendi3 # 1 Page 1

APPEN"!C " Preparation o; a Cali<ration Cur'e In se!eral of the analyses presented in this manual< there is a need for the preparation of a standard cali.ration cur!e. The cur!e esta.lishes the relationship .et2een a.sor.ance or percent transmittance and the concentration of the desired parameter. Ksing this cur!e< samples can .e tested .y the measurement of their a.sor.ance or percent transmittance and the corresponding concentration can .e read off the graph. +ules for preparing cali.ration cur!es. 1. 2. 3. Prepare all standards in duplicate or triplicate. Kse the a!erage of a.sor.ance or transmittance for preparing the cur!e. Process the standards in the same manner as the samples 2ill .e analy&ed. If the samples 2ill undergo a digestion< the standards must .e digested as 2ell. 0 distilled 2ater sample 9.lan7: must .e processed in the same manner as the standards. This .lan7 2ill .e used as the reagent .lan7 for setting the spectrophotometer to read &ero a.sor.ance or 1,,I transmittance. 0 minimum of one standard should .e processed daily to chec7 the accuracy of the cali.ration cur!e and the *uality of the reagents. ;hen ne2 reagents are prepared< the cali.ration cur!e should .e chec7ed .y at least 3 standards. If the cur!e appears to differ from a straight line< the cur!e may .e straightened to appro3imate a straight line .y plotting the data on semi/logarithmic graph paper. Plot data 2ith the a.sor.ance or percent transmittance readings on the !ertical a3is and concentration on the hori&ontal a3is. Select graph paper 2ith appropriate graduations. 5o not use graduations 2hich re*uire constant estimation of readings.

4. . ". $. '.

#hapter ' 1 0ppendi3 5 1 Page 1