Kfctoblologicsl Decor, taciinafcion of
Pentachloropbanol-ConLaminated Rafcural
Minnesota Univ., Minneapolis
for
Research Lab.-DutuV.h,
Sep 84
•3w«Ru».w&ijiaaKaH^^j*%s^^
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EPA-600/D-84-225
September 1984
MICROBIOLOGICAL DECONTAMINATION OF PENTACHLOROPHENOL-CONTAMINATED
NATURAL WATERS
M. M. MARTINSON, J.G. STEIERT, D.L. SABER, W.W. MOHN.
and R.L. CRAWFORD*. University o-f Minnesota, Gray
Freshwater Biological Institute, Navarre, MN 55392 and
Department of Microbiology,, Minneapolis, MN
55455.
EPA Project Officer
J. W. Arthur
E.PA Grant 810016
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE-Or RESEARCH AED'-DEVEI.OPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
DUltmi, rQi 53804
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-
TECHNICAL REPORT 1)ATA
(F e 2e r .d !nztt c? u on the , veree b efoir comp!ethig)
rR’o T3
EPA—600/D- 84-225
3. RtCIPIE .NTn ACCSItNJ Q.
2 6d63
4. T$TLt AP O S%JBTITLE
M crobioiogical Decontanination of Pentachiorophenol—
Contaminated Natu a1 Waters
—
5. REPORT DATE
epternber 1984
L PE -RFOAVING ORGANIZATION CODE
7. AUTIIOR(SF — .
N.H. Nartinson, J.G. Stelert, ILL. Saber, LW. Mobn,
LPERFORM NG D GANIZATION RgPORT N C ).
.
and R.L C:awford
I. PEOt G ORGANIZATION NAME AN
0 ADOPESS
10. C C ,MAU TNo. —
University of Minnesota
Minneapolis, Minnesota 55455
.
it bu1RAcT,cR p4T pip.
810016
2. SP 7NSORING AciENCY liAME AN A)DRISS —
Environnental esearch Laboratory
Off ice of Research and Development
U.S. En’ironmental Protection Agency
3.TYOFRCPo -’T AND PERIOD COVERED
14 .SPO NSOR G AGENCY
uluth _Mir.nesata 5580 ’
EPA6001 03
• SUPPLEMENtARY IiOTES
1&AFST RACT
inoculation of entach1oropherto1—contarntnated natural waters wIth e11s of a
pentachiorophenol—degrading F]avobncteri was shov to be an effective netiod for
decontamination oE PCB—polluted aquatic environments. Nt erous types of i. ater were
deccnta a nated, including: river ter, lake water, and grcuridwater. Decz ntaninat1on
vas nost effective between 15 C and 30 C, and between pi 7.5 and pH 9.0. Inoculation
of waters with as few as 10 4 ceIls/mi resulted in effective PCI ’ removal. PCB concac-
trations betwe n 10 ppb and 100 ppm vere reduced to undetectable levels, usually ithin
48 hc rs. Rtc obioL gica1 decontamination of PCP—p iluted waters appears to be a
promising waste treatment altarmitiva when compared to traditional treatment
techniques. -
I
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NOT ICE
This document ha beer. reviewed in accordance with
U.S. Environzental Protection Agency policy and
approved for publication. ention of trade na as
cr co ercial products does not onstitutc tndorse-
sent or recoiamendati n for use.
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M rtinson, et al. 2
MICROBIOLOGICAL DECONTAMIN. TION OF PENTACHLOROPHENOL—
CONTAMINATED NATURAL WATERS
M.M. MARTI SON, 3.6. STEIERT, C.L. SABER, W.W. MOHW, and
R.L. CRAWFORD*. university of Minnesota, Gray Freshwater
Biological Institute, Navarre, MN 5392 and Department i 4
Microbiology, tiinneapolis,MN 5453.
Summary.
Inoculation of pentach1oropheno t—contaminted natural waters with
cells of a pentachlorophenoi—degrading Flavobacteriurn was shown
to be an effective method for d contaminaticn of PCP—polluted
aquatic environments. Nut erous types of waters were
decontaminated, includina river water, lake water, and
groundwater. Decontamination was m t e ective between 15 C and
50 C and be 4 t een pH 7.5 and pH 9.0. nc culation of waters with
as few as 10 celig/mI resulted ir, effective PCP emoval. PCP
concertrations bet een lupph and I00p were reduced to
undetectable levels, usually within 48 h ,urs. Miorobiolonical
decoritc,mination of PCP—pcl luted waters appa ars to be a prot i sing
waste troatment lternativa when compared to t,aditic,nal
treatment technologi ’s.
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Plartinson, et al. 3
INTRODUCTION
Pentachlorophe 1 (PCP) is a widely used biocide that
contaminates soil r i water in many parts of the United
States (Edgehill and Finn, 1983; Pignatello, et al., 1993).
This compound. is acutely tnxic, so environmental
cofltamination is of concern both as an ological threat and
as a public health menace. It is possible to ,.solate pure
cultures of bacteria that grow on FCP a a sole source of
carbon and energy (Chu and Kirach, 1972; Edgehill and Finn,
1983 Pignatello, et al., 1983; Stanlaká and Finn, 1982
Suzuki, 1977; Trevors, 1982 and Watanabe, 1973). . Such pure
cultures have potential for use as PCP detoxilication
agents. For example, work by Edgehill arid Finn (1983)
suggests that:inoculation of PCP—contaminated soils with
PCP—degrading bacteria may be a feasible method for removing
PCP 4rom such environments. Here we show that
addition of cells o4 a PCP—degrading El st 1 to
P P—conta r iated natural waters is effective
decontamination teiJir ique.
2
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Martin on, et al. 4
IIATERIfILS AND METHODS
During previou5 work (Pigriatello, et al., 1953) we isolated
from PCP-contam nated stream sediments a pure culture of a
6r m—negative bacterium that gro s aerobically on PCP as a
sole source of carbon and energy. This bacterium will
tolerate PCP at concentrations of 300—400 mg/L, completely
degrading such quantities of PC? within 24—43 hours and
liberating all FCP chlorine as chloride ions. Extensive
characterization of this strain using the methods of Holmes,
et al. (1981), Weeks (1974), Smibert and Krie (1931)
Hendrie and Shewan (1979), Kodaka, et al. (1982), and
Skerman (1969), summarized in the RESULTS section, indicates
that it belongs in the genus E1t i . We have used
this strain for the detoxification studies reported here.
The Eiavo acte iurn was rown axenicelly to mid—logarithmic
stage in a define growth medium (Pignat. llo, et al., 1983)
containing L—glutamic acid (4.0 gIL) as the only source of
carbon arid energy. Pentachlc rophe c1 then was added (20
ppm ) t.o induce the PCP degradative enzymes. When
degradation of the added PCP was n early complete, as
determined by measuring ultravio et absorbance of the
culture fluid at 3 18—320nm tPi jnatello. et al., 1983), the
microbial cells were added directly (without removal of th
spent growth medium) to PCP—coritaminated waters to give 106
to 107 cells/mI of water. e1l densities in the initial
inoculac were determined by direct microscopic counts
(Hot bie, et al., 1977) or by using a standard curve of
culture turo oty I vs. call numbe-/mi.
Concentrations of PCP in natural waters were deter iined by a
gas—chrornatoQraphic procedure described previously
(Pignatello, et al., 19 3) or by a u ing the absorb nc
of water at 3lOnm (Piqnatello, et al.. 1983). The latter
procedure was used fo ’ most samples. The gas
chromato raphic procedure w s erpioyed when inc.reased
sensit v.ty was na”ded 10O ppb) ar per odicaiiy as
check or; ie spectrophotometric assay.
iis issippi River wot r was c loc.toc on y 7, 1
temperature, 2u CI o> yçen cercentrz tLcn, Orng/L; H, 5 2)
from o i coo- e> perimenta1 streams at the Nontic 1lc
Ecological Research St tion. a ieid station o th United
States Environ;nental Pr c.tect.ion r cy Eriv . ronriental
esearch Laborat r’, at &ul uth 1i nn scta ( rthur e
1982). River water (20—liter portion aa distributcd tnt :;
al—glass, 5—gallon aq rio in four t at nt qr . 1 roup
A 2 replicates) received o .ty water. t oun .. (2 rericatea)
received water nius cter ai c& C
replicates) received water plus O70 -l 40 pd
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Martinson, et al. 5
(average for the four aquari , 1110 ppb). Group D (4
replicates) receiv . d water, 10’ bacteria/mi, and PCP (1020—
1110 ppb, average 1080 ppb). Concentrations of PCP within
aU aquaria were mor itored by periodic sampling of the water
and determination ef (PCPJ qas—chromato raphically. All
aquaria were protected from exposure to direct light to
minimize photolytic decom e ’ ition of PCP. After 48 hours, 10
fathead minnows ( aL prornelas) were added to each
aquarium, and fish survival monitored up to 168 hours past
the beginning of the experirnent.
Numerous other water samp&es were collected from the
vacinity of the L ray Freshwater Bioloqical Institute during
May and June of 9B4. Groundwaters were collected from a
well in Deephaven, 1N (water pH, 6.9) and a well in Spring
Park, MN (pH, 6.4). Oligotrophic lake water (ph, 7.2) was
collected from Christmas Lake, near Excelcior, MN.
Eutrophic lake water (pH, 6.9) came from Lake Minnetonka,
Minnetonka Beach, 1N . A second river water sample (pH,
7.1) came froo the Crow River near Delano, MN. Port or;s
(100 ml) of each sample were placed in seperate 2 0a l
flasks, and FCP was added to a conc ntraticn of 100 ppm.
Each flask then received 10 F vobacteriun cells/mi.
Control flasks received only PCP. All +1as were thcubate
with shaking, in the dark, at 2 C. Pentachiorophenol
concentrations in the various waters were determir
periodically by measuring the or mnce of small al±quots
of water t 3lBrim. 1+ i- ecessarv. aliquots were centrifuged
to remov microbial cells prior to analysis for PCP.
Periodic .hecks of PC concentration :n no-centrifu c
aliquots con4irmed that ecv-eases in A were due to
sappeararsre of i P, not adsorption of PCP to microbial
cells.
For e aminat on of ef4ects of ph, temperature, PCP
concentration, and Fievcb cteriuo cell density on rates o
PCP removal from naturo l waters we tzed water collected
fram Lake Mir netonka, M . Freshly—collected wat r (pH 6.9—
7.0’ was di ;tr but into fi sks (1( ml/flask) nd
supplelz1ented th PEP. For er3ture e pt rjmar ts all
flasks received lOOppi! of PCP plus apprcximate y 10’
bacteri i reil / 1. TN ’ we’-r- ir’u ted n th€ th rk. with u
ita c)n at ter,r tur—es e n i C ‘n 40 C.. To ne
effects of ctor,.u cell density, .‘ater samples
cant r r j 1C 0prn o{ PC& e -o ccL’iat. d with 1 - ferent
Co., ntrat’ons of h r teri l r?lls ‘aryir CZ ‘t ec i
celi / . i aa d . ‘ J ce I . ‘m1 . For :H tud s , ‘sater
e cor t i -a r! l( 0ppr CP were d.te: to v rt r3
ph’s by eddit o: c f+ r-ring acent (T - l ph’s
1Eg: p!-rs • )-7. 0, erh -L- i’rid; oh’ 7 ( --C3 ,0, and
borate: p ’ ; . (i rt a c:cr. r r . io .’ of At
tr ;uut or, pH. pH 7. .) ‘u:J) c s werc run wit
different bu+ rr r.cj ‘n i . TP1 p o r ,ori
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Martinson, et al. 6
to confirm that effects on PCP removal rates were due to pH
and not particular types of bufferring agents, After
adjustment of pH. water samples were inoculated with
approximately 10’ E Q jy cells/mi; ph’s were
monitored throughout the experiment, and maintained within
0.1 unit of the initial pH. To examine the effects of PCP
concentration on removal rates, two approaches were used. In
tne first, a moderately low PCP concentration of 1.Oppm was
usti, with variable densities of the In the
second, all flasks received the same density of bacteria
(10’s cells/mi), but different conceitrations of FCP (0.1,
1.0, 10.0, and 100.Oppm). Flasks for pH, inoculas size, and
PCP concentration studies were incubated in the dark,
without agitation, at 25 C. Aliquots of water were removed
periodically for determination of EPCP).
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Martinscn, et al. 7
RESULTS AND Y ISCUSS1ON
We have isolated froni a variety of PCP—contaminated
environrnertts about 40 pure cultur , s of bacteria that are
able to use PCP as a sole source of caron and energy for
growth. All of these strains are Grain—negative and exhibit
a yellow insoluble pigment. They grow optimally between 2
and 30 C, and tolerate FCP at concentrations up to 300—
400pprn. A single strain w s used in the detoxification
studies reported here. This bacterium is rod shsped; about
2 micometers long. It is not motile., It possesses
cytoplasmic inclusions when grown on rich media, and
exhibits positive tests for oxidase, catalase.. and
phosphatase. It gave negative tests with respect to the
following: casein hydrolysis, gelatin hydrolysis, ipase,
amylase, arginine dihydrolase, N production from nitrate,
indole production, urea hydrolysis, MRVP test, modi$ic ations
of litmus milk, deoxyrihonuclease production, grouth on
rutrogen—free media, growth on MacConkey Agar, csiiulase
production, agar- degradation, and growth at 40 C.. The
bacterium will grow in the prese 1 -ce of 27. Ned, and produces
ammonia when grown on peptcne. It grows weil
microaerophilically, and in the presence of low oxygen
concentrations produces acid from glucose, maltose,
trehalose, salicin, and cellobiose. Under strictly
anaerobic conditions it will not ferment ç lucose. No acid
is produced under aerobic conditions froa zylcsa, manriose,
fructose, maltose, sorbose, galactose , salicirs, inuli i,
dextrin, pyruvate, acetate, succinate, aspartate q1uta ate,
or butyrate. The bacterium will not grow at the ei pense of
sucrose, lactoss’, rhamnose, mannitol, raffi c,se erabinase,
dulcitol, inositol, glucosamine, gluconate tartarate , —
alanine, propionate, salicylate, valerate, oxalate, eth riol,
propanol, citrate, trehalose, berizoate glycine, furnarate,
arginine, starch, 2—chlorobenzaate 3--chioro5er,zoate, 4—
chloroben oate, 2,4—dichlor-obeno te 4 6 —dichloro—
resorcinol. —chioropn ,nol. —chlorop ’eno , —chlor-c phenol,
2,4—dichiorophenol, or 2,4—dichiorophonoxy etic acid whur%
these compour d are supplied as th only rce c f c rbcn
arid energy a concentration of O ppir in e - ncr r i ral
aalts medium. A light to hea’ y pellicle devr .Iopei upon
stationary growth in liquid media. 1h ’ bacteriw is
resistant to Novcb acin at a disc on ertr tion of CHt cg.
The microorq nism grows t the expense of F P acetate ,
succi nate, qI ur.o e. mel tose, ce l Oiose
ga actose,s-a1icin. inu 1 ir , de trin, pyru -’at ’ , p 3rtat4!-’ ,
butyrate, glutceate, and )3—hyc r vbuty’-at ner these i re.
supplied as sole u -co of carton ood g o tN ccurs
between pH 6.9 and pH 6. , and at t p ra r bet n C
and 7 C. 4o 4iuor ecen1- p entz re r ducnd. . The
guanine + cytosir.o content of the bact s DN i 4 .
Ł
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Martinson, et al. 8
The bacterium harbors one or more plasmids. The above
characteristics indicate that this bacterium should be
classified in the genus Flavobacteriurn (Holrnes, et al.,
1984; Smibert and Krieg. 1981; Hendrie and ShEwan, 1979; and
Kodaka, et al., 1982).
Mississippi River water was distribL eb into aquaria as
described under MATERIALS ND METHODS.. All aquaria, except
treatment group A, received approximately lppm of PCP. Some
aquaria also received bacteria, wh 2e others did not.
Addition of approximately 10 ELg cells/mi to
PCP—contarninated river water resulted in removal a-f >90% of
the biocide within 48 hours! as summarized in Table 1.
Removal of the PCP detoxified the water, as shown by
survival of PCP—sensitive minnows irs treated water, but not
in untreated water (Table 2).
The ability of the EL to remo’ ’e PCP -from natural
waters other than that from the Mississippi River was
examined. The bacterium readily decontaminated -five
additional waters, incluthng oligotrophic (low procuctivity)
lake water eutrophic (highly productive) lake water,
groundwater, and another river water. Four of the water
samples were decontaminated of 1O( ppm o4 PCP within about 48
hours, at which time PCP no longer was detectable. One well
water sample (Spring Park, MN) was decontaminated
completely, but only after a 72—hour lag period. We
suspect this was due to the lower initial pH cf this water
as conpared to the others. Other e> pes-is ’ents (see below)
indicated that pH’s oF 7.O inhibited FCP removal by the
Fl avobacteri urn.
Temperature was an important variable affecting PCP
removal rates. The Flavcbacter urn removed PCP within 50
hours at between 20 C and 30 C. Removal rates slowed
somewhat at 15 C ( 110 hours for complete remov l), hut
still were significant. In most experiments, alow removal
of FCP was oh ved 35 C, with no removal at 40 C.
In c eneral it appears that tempe atures in ess of about
35 C re detric iental to PCP re cv l. Shifting o a water
sample het -,eer 23 C arc i 33 C t l0- l4 hour intorvals. as
min -t occur in on outroor tr tmer,t system due to diurnal
v -i . tions of t po - : l y 51 i ;htiy si owed CE romoval
os coopared to incu5at one at 2 C or 31) C y ithcut
varzaticn. Thus, exposur-e o-f the eacterium to 33 C for
periods of 1O--4 hours did not in t v tu the lls. This
observation is ancouragin -i as r 1rcis outdoor pplicatior.s
in war n clirate-.
Inoculurs c nsitv also a-Ffect.pd CF re - oval rates.
r a inq the densitie o’ F1a’ b cteriu:n cells
signi+icant)y irrease C - recn val rntes. s ohown
Figure 1. Hc. -.:ever ovun rather low dersi ti s of the
7
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Martinson, et al. 9
bacterium (e.g., 5.5x10 4 cells/mi) were reasonably effective
at removal of PCP.
The pH optimum for r oval of PCP from lake water
was about ph 8.5. Rapid removal of PCfr occurred between pH
7.5 and pH 9.0. The still was active at pH’5
as low as pH 6.5, but remnval rates slo.aed considerably at
these pH’s. No removal was observed at pH 6.0. Th . , the
bacterium performs best as a PCP decontamination agent at
somewhat alkaline pH ’s. It per+o m i poorly at pH 7.0 ar
lower, or pH 9.5 and higher. Rates of PCP removal vari
slightly depending on the type of water beirg
decontaminated and in water samples collected st different
times of the year; however, the oierall response to ph
(optimum, pH 8.5) was consistent.
The FlavobacteriLim was able to ren ve PCP from natural
waters over a wide range of PCP concentrations.
PCP concentrations ranging between lOppb and lOOppm were
decontamir.ated equally w ll. Ratea o-f PCP removal (Z PCP
removed/hour) were similar at all concentrations, but total
time required for complete re noval increased with increasing
PCP concentraUon because of somewhat longer initial lag
phases preceding degradation at the nigher PCP
concentrations. PCP concentraticns usually were reduced to
levels below cur detection limit of about 0. 1 ppb.
Decontamination of natural waters tnat ha’ve b en
polluted with F’CP clearly may be accomplished o ing
microbiological techniques. As with any bio ogcal syatem,
many variables may influence t e outcome of decontamination
attempts. Some particularly important variables includ€ pH,
temperature, pollutant concentration, type of natura! water
to be treated, density of added microorganisms, and the
abilities of the added microbes to compete with the natural
microclora already in the water. Data reported he—e s .ow
that our PCP—degrading EL2 t is a very ‘,ersatile
PCP decontamination aaent. It removes PCP from i ost types
of natural water. It accomplishes this quite rapidly and
over a range of ph and temperature that ecompass S
conditions encountered in most natural systems. Either high
or low concentrations o PCP are treatable, and
concentrations u -i treated water are reduced to very a+e
levels, usually below detection by qas—chrornatoç,raphiL
measere nent techniques As 4e as 10’ —l0
oells/r l are sufficient to detoxify most waters. Cn oir g
experiments indicate that the costs of growing the
concentrating the c ils , and transport ng
the i to a pollution site will be competitive with existing
clean—up technologies such as the use of activated carbun
filtration. Results of several laree field tridis of cur
rnicrcb:ological clean—up process will be- reported later.
8
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Martinson, et al. 10
REFERENCES
Arthur, 3. W., Z schke, J. A., and Erickson, (3. L. (1982).
Effects of elevated water temperature on macroinvertebrats
communities in outdoor experimental char;nels. Water Res.,
16, 1465—1477.
Chu, J. p., and Kirsch, E. 3. (1972). Metabolism of
pentachiorophenol by an axenic bacterial culture. A 22 .
Microbiol., 23, 1033—1035.
Edgehill, R. U., and Finn, R. K. (1982). Isolation,
characterization and growth kinetics of ba..terja
metabolizing pentachiorophenol. Ac 1. Environs. Nicrobiol.,
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Hendrie, M. S., arid Shewan, 3. M. <1979). The identification
of pse idomc,nads. in, Id nti+jcati Methods far
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Hobbie, 3. E., Daley, R. 3., and Jasper. S. (1977 ) Use of
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microscopy. A i. rons. icrobio1., 33, 1225—1228.
Holmes, ., Owen, R. 1 , and M :Menkin, 1. A. (1984). in,
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N. R. (ed. . Williams ans Wilkins. altimore, IT). pp. 353—
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Kod ka, H., Armfieid,. A. V., Larrbard, 3. L., and Dowell,
Jr. V. R. (1992). Practical prc dur-e for demonstrating
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Carlsc n, P. E., and Crawtord, S. L. (19B3).Biodec r cjatjon
and phatolysis of Pentachlor-ophenol in rtific .a1 freshwater
trea ns. Anpi. Er viron n. icrobiol. 4 . 1024- 1031.
Skeriran, V. . D. (ed .). (19 9). Abstracts of
Microb olo j:al M’ thc,d . Jchn Wi19y and Sons, New York.
pp. 27-32.
Smibcrt, P. h., anrJ Kr e , N. P. (1 B1). in, Cf.
±C 1f.c io)ci . 6 rhardt, P., Mw-ray, P.
G. E.. Co t:i1cw, R. N., Naster, E. ., Woods, W. A., rieg,
N. P... and 6. E’. (ans.). Aiior c.an Scciety for
Micro jol ogy. Washi r oi i, ;. ap. 40c—443.
St r.1 k , 6. J . and F n, P. K. (1 B2). Isolation and
char ..icterizatj of a pe tachiorophenoi —J r j,n bacterium.
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Martinson, et al. 11
44, 1421--1427.
Suzuki, 1. (1977). Metabolism of pentathiorophenol by a
oi1 microbe. . B12(2), 113—127..
Trevors, 3. 1. (1982). Effect of temperature on the
degradation of pentachiorophenol by Pse dcrnona5 species.
11, 471—475.
Watanabe, I. (1973). Isolation of pentachloropher o
decomposing bacteria from soil. fl a Nutr.
iI ! 19, 109—116.
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Cowan, S. T.., Halt, 3. S., Liston, 3., Murray, R. S., Niven,
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ACKNOWLEDGMENTS
This work was supported in part by the U. S. Environmental
Protection Agency under a cooperative agree nent (CR— 1001o—
01—1) with the E.P.A., St. Olaf college (Northfieid, MN),
and the University of Minnesota. This paper has not been
Ettbiected to the E..P.A.’s peer and policy review procec ;ros.
Additional support came frřm the National institute o’f
Environmental Health Sciences under grant number 1 F
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Martinson, et al. 12
Table 1. Removal of Pentachiorophenol from River Water by a
TIME
Z OR
IGZNAL PCP
REM INING
(hours)
Aquaria with
(Treatment
Bacteria
Group D)
Uninotu lated
Aquaria
(Treatment Group C)
-0 100.0 4.0 100.0 +2.5
4 93.8 ±4.0 103.2 ±1.2
8 87.0 ±2.9 102.8 +0.5
12 79.2 ±7.9 98.3 ±0.1
20 64.2 ± .7 100.5 ±7.0
34 40.3 ±14.9 102.0 ±9.2
45 24.5 ±15.6 105.5 15 3
48 (fish added) ND ND
51 17.4 ±l2 6 92.1 ±0.5
57 11.7 ± - 94. ±4.8
69 9.3 ±8.9 89.7 ±3.8
ND, not determined
Results are + standard deviation (Young, 1962)
11
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N rtin on rt al. 13
Table 2. Fish Survival in Cor tatninated nd Decontai tthated
River Water
TREAThENT
$ROUP
0.0
2.0
CHour* .
2 .S 3.0
Z FISH SURVIVA
1Ol1owin’ a4d ti
.5 4.0 6.0
L
Ofl 04
9.0
igh)
10
48
168
A
100
100
100
100
100 100 100
100
100
100
100
B
100
100
100
100
100 100 100
100
200
100
100
C
100
90
48
12
8 0 0
0
0
0
0
D
100
00
100
100
100 95 90
85
80
78
78
A, r bacteri ! o PCP 4, bact ’rio PC?; C, PCPIno
bacteriai 0, ECE a v bacteria. The dif4erence bet 4e n
tre t nent gr ’p C 0 after 16 3 hours is significant at
the 1 v Yn ig, 1962). 04 the ish that d± not
s viv n trr t t eroup D, 1l &ierc in single, atypicak
aau riu no fish were lost i 3 of tne 4 replicate aquar .
-------
P1 rtinv rn. t a l *4
Fz jure I. E4f ct nf Inoculum Size an R mava1 of PentachiorophenQi
from Natural Water by a vc,bacteri m
1 3
-------
‘e.
.1
H
N
P1..
H.
N
i L
1 cPiL
f4
I H
i
H+f
ft
-‘4...
II
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