Effects of Pollutants on Hior-obinl
Activities in Estuarine Surface Filas
State Univ.
Atlanta
Prepared for
Environmental Research Lib,
Gulf Breeze, FL
Mar 81
[
i.V
Li
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EPA-GOO/4~61~00'>
March ISGi
EFFECTS OF POLLUTANTS ON M1CROBIAL
ACTIVITIES IN ESTUARINE SURFACE FILMS
by
D. G. Ahcarn
W. L. Cook
aad
S. A. Crow
Department of Biology
Georgia State Uaiversity
Atlanta, Georgia 30303
Grant No. R-304477
Project Officer
Al W. Bourquin
Gulf Breeze Environmental Research Laboratory
Gulf Breeze, Florida 32561
U. S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPIiOT
ENVIRO^OTNTAL FJISEAKCH LABORATORY
GULF bPEEZE, FLORIDA 32561
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ji
I
1
;'
r> CHfMrAL FT IIVDATA
//'/.CM fuJ I" til, '1: '.'V • II /.'.. /'..' I'l fl'lf ll'l.l'
-J7
EPA-600/4-81--009
1. T I ' I r Afjf) ^ijm , 7 , |
Effects of Pollutants on Microbial Activities
In Estutrine Surface Films
7. AUTHOHiS)
D.G. Ahearn, W.L. Cook, and S.A. Crow
9. Pfc RFOHMING ORGANIZATION NAME AND ADDRESS
12. SPONSORING AOENCV 'JAME AND ADDRESS
U.S. Environmental Protection Agency
Environjj32fvj^3\l Research Laboratory
Office'of Research and Development
Gulf Breeze,vFL 32561
j. racir
b. HEP", ir.' L '. •!
March 1981 Icsuinp. D«te_.
6. PErtrrnviNij OP.GAMZATION couc
PER, -.:>•.• MO ORGArjI^ATIC M
13 PHOGRA.V F LEMfcT.'T NO
_A87E1A _
11. co7jTrTAcT~GR~AN"r~N7.
R-804477
13. TYFE OF ftPOUT AND PERIOD COVt RCD
14. SPONSORING AGtMCY COOt
EPA/600/4
IB. SU^Pl RVIENTAPY NOTFS
Samples of inshore surface films from Escambia Bay, Florida and from sites in
thp North Sea-yielded populations of aerobic, heterotrophic microorganisms up to
lO^-fiil"1 or 106 cm~2. Hydrocarbonoclastic organisms were in relativc-ly low pop-
ulations. A comparison of species of yeasts prevalent in North Sea v/aters before
and a?ter oil production activities indicated a shift to a more widespread distrib-
ution of hydrocarbonoclastic forms with possible inhibition of a non-hydrocarbon
utilizing species. Examination of various hydrocarbons and chlorinated compounds
with the potential of being sequestered in natural films indicated that 66» could
potentially alter microbial metabolic processes in the slick. In microcosm studies.
of estuarine systems representative compounds demonstrated a selective effect for
microfungi.
This report was submitted by Georgia State University in fulfillment of
Grant No. R-304477 under partial sponsorship of the U.S. Environmental Protection
Agency. This report covers the period from May 9, 1976 to Oct. 9, 1979 and was
completed November 9, 1979.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Microorganisms
Oil slick
Pollutants
Microcosms
1) OISTHIBLMION STAIf." NT
Release to public
b.IDENTIFIERS/OPEN ENDED TtRMS
Hydrocarbons
Escambia Bay
Hydrocarbonoclastic
organisms
Microbial Metabolic
processes
19 SI CURITY CLf :>S /rill!
Unclassified
20 SECURITY CLASS /Tliu
Unclassified
i. COS AT I I
06/F
06/M
06/T
21 --O Of ('AGtS
EPA Fo.n J2JO-1 («••• t-T
. x. •-'-;-'*L 7"C!-"•-'!<"A'
'
'inr-rt'-*-' •'~-*-'i-;-'
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DISCLAIMER
This report has been reviewed by the Gulf Breeze Environmental
Research Laboratory, U. S. Enviromaental Protection Agency, end approved
for publication. Approval does not signify that the contents necessarily
reflect the views and policies of the U. S. Environmental Protection
Agency, nor doea mention of trade names or commercial products constitute
endorsement or recommendation for use.
ii
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FOREWORD
The protection of our estuarine and coastal areas from damage caused
by toxic organic pollutants requires that regulations restricting the
introduction of these compounds into the environment be formulated on a
sound scientific basis. Accurate information describing dose-response
relationships for organisms and ecosystems under varying conditions is
required. The Environmental Research Laboratory, Gulf Breeze, contributes
to this inforaation through research programs aimed at determining:
. the effects of toxic organic pollutants on individual species
and communities of organisms;
. the effects of toxic organlcs on ecosystem processes and
components ;
. the significance of chemical carcinogens in the estuarine
and marine environments.
Research described in this report examines the fate of pesticides in
estuarine surface layers . An understanding of the response of microblal
populations to pollutants should aid In attempts to determine where toxic
chemicals reside in the environment and to develop better methods to assess
effects of such chemicals on biological processes.
Heary F. Enos
Director
Environmental Research Laboratory
Gulf Breeze, Florida
111
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ABSTRACT
Samples of inshore surface films frora Escaobia Bay, Florida and from
sites in the North Sea yielded populations of aerobic, heterotrophic micro-
8 —1 6 —2
organisms up to 10 ml or 10 cm" . Hydrocarbonoclastic organisms occured
in relatively low populations. A comparison of species of yeasts prevalent
in North Sea waters before and after oil production activities indicated &
shift to a ciore widespread distribution of hydrocarbonoclastic forms with
possible inhibition of a non-hydrocarbon utilizing species. Examination of
various hydrocarbons and chlorinated compounds with the potential of being
sequestered in natural films indicated that 66% could potentially alter
microbial iretabolic processes in the slick. In microcosm studies of estuarine
systems, representative compounds demonstrated a selective effect for micro-
fungi.
This report was submitted by Georgia State University in fulfillment
of Grant No. R-8C4477 under partial sponsorship of the D. S. Environmental
Protection Agency. This report covers the period from May 9, 1976 to
Oct. 9, 1979 and was completed November 9, 1979.
iv
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CONTENTS
Page
Foreword ..... .......... Ill
Abstract iv
Tables vl
1. Introduction 1
2. Conclusions 4
3. RecoEsnendationa 5
4. Materials 6
5. Experimental
Environmental Sampling < 7
Laboratory Studies . 7
6. Results and Discussion 10
References 19
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TABLES
Page
1. Heterotrophic Microbial Populations In Surface Slicks
of Escambia Bay 10
2. Yeasts from the North Sea 12
3. Fungi Isolated from AEOCO Cadiz Oil 13
4. Conparison of S£dcpjGeJLl£/Hanmialian-Microsorja Mutagenicity Test
ReaultB with Publiolied Reports Using the Same Test 15
5. Prevalent Fungi Isolated ?roa Salt Marsh Microecosystema
After Addition of Selected Pesticides 16
6. Batch Culture of Selected Fungi With C-Labelled Pesticides .... 18
vi
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SECTION I
IPProduction
Surface files on natural waters have been ehown to contain high
concentration!} of organic carbon, nitrogen and phosphorous (Uilliam 1967),
alkones and chlorinated hydrocarbons (Seba and Corcoran 1969, Ledet and
Lasater 1974). This organically enriched raicrohabitat also has been ehown
to contain high densities of bacteria relative to underlying waters.
Sieburth (1965) reported bacterial populations up to 4 x 10 ml~ in surface
films. The predominant bacteria were poeudoisoaads which expressed lipolytic
activity. Harver (1966) found that bacteria, small algae, end colorless
flagellates were concentrated in the upper 60 um of surface water. In the
studies of Crow et al. (1975), samples of the upper 10 u» of Inshore surface
films obtained by adsorption to membranes yielded tnicroblal populations up
a _i e _o
to 10 ml or 10 cm . These populations were typically 10 to 100 times
greater than those in underlying waters at a depth of 10 en. Predominant
bacteria in the films were motile, nonpigmented, gram-negative rods. Colony-
4 -1
forming units of yeasts and Isolds were found in concentrations to 10 ml
-2
or 28 cm . The predominant species in the surface films were proteolytic
and amylolytic.but exhibited only weak to negligible hydrocarbonoclastic
and lipolytic activities.
Various researchers have reported the accumulation of various pesti-
cides and polychlorinated aroaatics in surface filras. The bacterial
bioconcentration of chlorinated hydrocarbon insecticides from
aqueous systems appears to be a commonly occurring phenotaenon (Crimea and
Morrieon 1975). Such binding of pesticides to cells suggests that the
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presence of chlorinated hydrocarbons in surface films (Seba and Corcoran
1969) may be related in part to their microbial densities.
In other studies (Smith et nl. 1975), heptachlor was shown to enhance
or inhibit hexadecane utilization by Candida maltosa (from a freshwater oil
slick) dependent upon aeration and pasticide concentration. The heptachlor
in these culture systems appeared to be bound to the cells, but not meta-
bolized. Walker and Cooney (1975) found stimulation of oxidation by hexa-
decane by Cladosporium resinae in the presence of aon-utilizable substrates.
The alteration of taicrobial ecosystems in eetuarine habitats by crude
oil has been reported (Crow et al. 1975, Hood et al. 1975) and inhibition of
eetuarlne bacteria by PCS formulations is known f.o occur (Bourquin and
Cassidy 1975, Bourquin et al. 1975). Potential alteration of nutrient
cycling in coastal areas mediated through hydrocarbon pollution will be of
greater concern with the advent of the superports, development of offshore
drilling along tha eastern coast, and production from the Coaipeche Bay area
in the Gulf of Mexico. Since PCB's, chlorinated pesticides, and detergent
molecules are preferentatlly soluble in or bmnd to hydrocarbons, the
potential of an altered surface-film microflo-a with chronic oil pollution
can be expected to increase.
The induction of bio-alteration by recalcitrant molecules is fre-
quently unobserved in studies of macroscopic organisms. Unfortunately, long-
term detrimental effects of pollutants usually are seen too late to prevent
environmental damage. The rapid generation of bacteria and their metabolic
responsiveness obviates come of theaa difficulties. Microorganisms, primary
decomposers in the food web, can reflect potential deleterious environmental
effects within a time span of days. Knowledge of the basic microecology of
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estuarine surface files, particularly aa affected by recalcitrant pollutants,
may permit predictions of impending ecological stress of higher living forms.
The objective of thia research was to examine the effects of hydro-
carbons, pesticides and chlorinated biphenyls on the species composition and
physiology of predominant groups of micororganisms in ettuarine and marine
surface films. Three basic ap roaches were taken to achieve the objective:
(1) heterotrophic microorganisms were isolated from natural and man-mediated
estuarine and oceanic surface films; representative isolates were examined
for their interactions with select compounds; (2) selected pesticides,
chlorinated biphenyls and polynuclear aromatic hydrocarbons were screened
for their potential mutagenic and inhibitory capacity for Tlcroorganipms.
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SECTION 2
Conclusions
Various hydrocarbons from polluting crude oil have the potential to
niter the microbial composition of es'.uarine and oceanic surface slicks.
Chlorinated aromatic compounds and various pesticides which have been shown
to be sequestered in surface films may further affect microbial activities.
Microcosm laboratory studies may be pmploved to show >'he selective effect
of pesticides on microbial populations in estuarine habitats.
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SECTION 3
Laboratory microcosms should be established with ssaterials from
selected eatuarine habitats of known niicroccology. These taicrocosscs should
be enriched with traces of natural nutrients (glucose, cellulose, amino
adds, etc.) and the response (metabolism and populations) oonitored (short-
term) in the absence and presence of xenobictic molecules. The results
should be compared with findings from field studieo of environoents ex-pooed
to the same xenobiotic. A major purpose of these studies is to deft-mine
the shortest exposure time for significant results from laboratory nicro-
coEms.
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SECTI02I 4
Materials
Areas Satcoled
Gulf Breeze, Florida
Range Point salt marsh
Escambia Bay
North Sea
35 stations between
54'H, 8'E and 66° 40'N, 10CE
Materials and Sources
8
23 aacples
70 samples
Polycarbonate oeobranes, Nucleopore Corporation, Pleaoariton, California
Standard Media (prepared with 50 per cent seawater)
Marine Agar 2216
Mycological Agar
Spirit Blue Agar
Tryptic Soy Agar
MOP Madlua
bushnell liuas Broth
Specialized media (listed below) were prepared according to the nethodi
of Colvell and Wiebe (1970) and Hankin and Anagnostakis (1975).
Proteolytic Enuoeration Media
Amylolytic Enumeration Media
Lipolytic Enumeration Kedia
Hydrocarbon Enumeration Media (1 per cent hexadecana in
Buohneli-Haas broth)
Phosphatase Media
Basal Broth
Yeast: YKB (Difco)
Bacteria: Buohnell-Haas Broth
Clicmicala Studied
Aldrin
Aroclor 1221
Aroclor 12A2
Aroclor 1260
BHC
Bux
Captafor
Captan
Carbaryl
Chlordane
Chlordene
ODD mixed ieotners
DDT mixed isotaers
DDT-O.P1
DOT-P1,?1
Diazinon
Dichlone
Dicofol
Dieldrin
Elndosulfan
Endrin
Halovax 1000
Halowax 1051
Halowax 1099
Heptachlor
Heptachlor epoxide
Hexachlorohvenzcne
1-Hydrorychlordene
Malathion
Methyl Oxychlor
Methyl Parathion
Mirex
PentacMorophenol (PCP)
Trans Nonachlor
Tetrachlorophenol
Toxaphene
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SECTION 5
Experimental Procedures
Envlrouneutal Sampling
The surface slick sanples froia Escambia Bay and the North Sea ware
collected with sterile polycarbonate membranes according to previously des-
cribed procedures (Crow et al. 1975; 1976; 1977). In brief, sterile poly-
carbonate nensbranes were floated on the water. The raccabrane and adhering
surface fila were retrieved with either a sterile plastic dish or bucket
which was submerged under the membrane and underlying waters or, in calm
waters, by directly retrieving the membrane with a sterile forceps frota the
water surface. The membranes were placed into bottles containing sterile
seawater or placed directly onto a solid nutrient mediua.
Samples from the AIBOCO Cadiz spill included: a viscous, brown-black
crude with low water of eoulsion, a browa Ecuese. and surface film collected
from tidal pools. Samples of each were collected in sterile 10 el vials from
various sites within 10 loa north of the Portsal harbor.
Laboratory Studies
Bottles containing stcmbraaes were returned to the laboratory under
refrigeration and processed within an hour of collection. The bottles were
agitated for 3 nin on a wrist-action shaker. Aliquots were serially diluted
and 0.1 ml of dilutions plated onto appropriate media. Bacteria were char-
acterized physiologically with media prepared according to the fonsula of
Colvell and Wiebe (1970). Proteolysls was determined with 2.OX skim oilk
and 0.1Z yeast extract in 1.7Z agar and with Thioglycollatc gelatin nediua
(Difco) prepared with artificial seawater. Oxidative or ferraentative carbo-
hydrate roetaboliBia wao determined with Hof raediua (Difco). Llpaae and ureaoe
7
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activities were determined en Spirit Blue agar (Difco) and Orea agar (Difco),
respectively. Both were prepared with artificial seswater at 2O°/00 salinity
Yeaste and filamentous fungi were identified and examined for their capacity
to grow on various hydrocarbons according to described procedures (Ahearn
et al. 1971; Crow and Ahearn, 1979).
The Aacco Cadiz samples vrere held at room temperature and periodically
over a 6-Gonth period, 0.1 ml was cultured in 20 ml of enrichment broths and
0.1 ml was diluted 1:10 in a .1% Tveen 80 sea water solution and inoculated
onto Buschnell-Eaaa agar with .01% hexadecane, mycological agar prepared with
sea water and marine &gar. The enrichment broths were: filtered sea water
with a 0.01% yeast extract, and sea water with .07% (KH,), SO, and sea water
with both the (NH.K SO, and yeast extract. The enrichment broths were
incubated et 20*0 for up to 14 days and 0.01 al samples inoculated onto the
isolation agars by spread plate procedure every 3-4 days. Representative
bacteria and fungi froa the various selective media were characterized for
their interactions with various pesticides and oil constituents. The muta-
genic and inhibitory effect of selected pesticides, chlorinated biphenyls
and polynuclear arenatic hydrocarbons waa established using the tester
strains developed by Ames et al. (1975).
The effects of selected pesticides on fungal development were
examined using nicrocomas (Pritchard et al. 1979). A sediment-water test
system contained 50 g of sand and detritus with 250 ml water (10-17 ppt
salinity) frca the Range Point salt oarsh. This system was maintained et
rooia temperature (23-25eC) with the water fraction aerated with a bubbler
tube. The other microcosm, a continuous flow system, coateined 144 g of
sand, 250 ml detritus and 250 ml of vater from the Range Point salt march
8
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layered Into 1000 ml growth vessels. The temperature was maintained at 23°C;
salinity at 12-14 ppt and air was provided at & rate of 30 cc/min.
1 A
Peutachlorophanol (UL. C, Pathfinder Lab, Inc.) was introduced into
14
the sediment-vrater test system at 140 ug/1. Carbaryl (Haphthyl-1- C,
California Bionuclear) and methylparathion (2,6- C ring-lrbeled, Amersham-
Serala Corporation) were introduced into the continuous-flow microcosm from
the reservoir at a rate of 14 ml/hr,giving a final concentration in the
growth vessel of 214 ug/1 for the foraer aiid 75 pg/1 for the latter.
At approxinately weekly intervalst1.0 ml of water and detritus were
removed from each growth vessel and from the headbox water. Detritus and
water from the Range Point salt marsh also were examined weekly for fungi.
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SECTION 6
Results and Discussion
Environmental Studies
Surface slick materials from Escambia Bay were plated on selective
media to determine the prevalence of physiologic types (Table 1). For these
Banples.lipolytic and amyloiytlc*T>rgani8i!i8 were more prevalent than proteo-
lytic and hydrocarbonoclastic types.
Table 1. Heterotrophic Kicrobial Populations in Surface Slicks of
Escambia Bay.
t CM"2
Total Aerobic
Heterotrophs
Amylolytic
Proteolytic
Hydrocarbonoclastic
Lipolytic
Yeauts
No. Sanples
23*
21
15
8
13
14
Range
1-2.8
1-5.8
1-4.6
1-2.7
1-1.8
1-2.4
xlO7
xlO7
x 105
x 104
x 107
xlO3
Mean
1.24
2.8
3.2
3.7
1.4
1.3
x 106
xlO6
xlO4
x 103
xlO6
xlO2
*No. sampl£8 positive of 23 total samples.
No yeasts with significant hydrocaroonoclestic activities were
obtained. None of the samples, however, was from sites influenced by not-
able hydrocarbon pollution. The influence of oil production activities
appeared to affect the composition of the surface film flora of the North
Sea.
Species of yeasts from surface waters of the North Sea were compared
with thooe isolated in an esvlier study prior to the development of oil
10
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production (Table 2). Candida gaillierpondii, a hydrocarbonoclastic yeast,
was obtained frequently in 1976, whereas only several atypical isolates
initially identified as Candida sp. were obtained in 1964-66. In 1976, the
incidence of Aureoobasidium palluIana appeared reduced. Meyers et al. (1968)
found yeasts at all stations in 992 of 84 samples at cell densities between
35-50 cella/L with the maximum density of >3,000 cells/L. In 1976, yeasts
were isolated from 100% of the surface samples collected at the 35 stations
and from 28 of the 35 samples collected at 10 m. least densities at the
surface averaged 76 cells/L and 35 cells/L at 10 m.
Relatively few fungi were isolated from the Amoco Cadiz oil (Table 3).
The direct sampling onto agar plates of all samples gave only a few coloniea,
indicating that fungal populations were <10 colony forming units per 100 ml that
represented only a few species, but when the samples were vigorously
agitated in a Tween 80 solution, densities in some samples ranged to nearly
50 cells/ml and yielded up to five different species. The greatest variety
of species was obtained from the surface films. In comparison to surface
films and water samples examined in earlier work, the fresh crude oil from
the Amoco Cadiz appeared selective and possibly inhibitory to normal marine
yeast flora. Certain volatile hydrocarbons, dependent upon concentration,
may be lethal £o yeasts (Ahearn et al. 1971). In preliminary tests, Iranian
crude (about 30% naphthenes) proved inhibitory to representative Isolates of
Debaryomyces hansenii, in spot tests. This species is the most cona&on yeast
in North Sea waters. The presence of odorous volatile oil fractions at the
shore adjacent to the wreck was quito noticeable, even 10 days after the
spill. The high concentrations of these volatile fractions may have markedly
reduced the densities and cpeciea of yesats brought into contact with the oil.
11
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Table 2. Yeasts from the North Sea
1964-66*
Incidence**
1976
Incidence
Debarcmrjces hansenii 38
Rhodotorula rubra 29
Aureobasidium pullulans 23
Candida diddensii 18
£. tropicalis 12
R. pilimanae 11
Hanseniaspora uvannn 10
C_. zeylanoides
£. obtusa
£. krusei
£. lipolytica
C_. sil--icola
£.* tei. is
Rhodosporidium
infinnio-niaiata
Rhodotorula gratr
Sporobolomycea roaeus
Hanceniacpora californica
£. guillierroondii <10
(Caadida ay.)
Total Sanples 84
I), hansenii
Candida guilliencondii
Cryptococcus laurentii
Rhodotorula rubra
Cr. albidua
Cr. gastricus
Torulopsis Candida
^. graminis
JR. lactosa
£. tropicalia
£. parapsilosia
Kloeckera apiculata
Rhodosporidium
capitatua
Sporoboloayces roseuo
S^. gracilis
£. albo-rusescens
Aureobaaidium pullulana
46
29
14
11
70
*See Meyers et al. 1967
**Per cent occurrence in total samples
12
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Table 3. Fungi Isolated from Amoco Cadiz Oil
Species
Rhodotorula rubra
Debaryotayces hansenii
Candida tropicalis
Brown-Black
Oil
+
+
Mousse Surface Film
+ +
+ +
+ +
£. lipolytica - +
£. guilliermondii + +
Aureobasidiun pullulans - -
Penicillium sp. + +
Cladosporium sp. - +
Mucor ap. - ~
Fusarium sp. + +
13
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Laboratory Studies—Hutaognlcity Teats
The Salaopella/aamaallan-nierQ&oiag mutagenicity test was used to
detect conrpouods with potential of altering surface-slick flora (Table 4).
More than 60% of the cocpounds increased the rates of nutagenicity of tha
salmonellae. Table 4 compares also the results of the current study with
publications using the Salmonella/Kanmalian-microsoaa rautagenicity test.
Shirasu et al. (1976) prescrcened all the pesticides with the rec-assay
system,using Bacillus subtilis and apparently some of the rautagena in our
test were eliminated by their prescreening. Marshall et al. (1976) used
Salmonella that were not as sensitive to mutagens as were the ones used in
our test; thua.we report more mutagens. Three compounds, methyl parathion,
carbaryl, and pentachlorophenol, were selected for further studies in micro-
cosms.
Microcosm Studies
Changes in the talcrofungal populations of microcosms established
from estuarine sediments and water of the Range Point salt marsh were
monitored upon the addition of selected pesticides (Table 5).
Addition of these pesticides to the microcosms altered the pet-
tern of species recovered. The prevalent fungi in the oediment of the
carbaryl microcosm changed from Trichoderma to an overgrowth of Fuaariina.
In the methylparathion growth vessel,Trichoderma, initially predominant
and was succeeded by a species of Penicilliurn. The Peoicilliua I, provision-
ally classified within the Penicillium chrysogenum series, was Isolated in
large numbers in the final six samplings. A different species, Penicilliun
II, provisionally classified within the Penicilliun canescens serieo, pre-
dominated during the sampling period in the sediment of the pentachlorophenol
sediment-vater test system.
14
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Table 4. Comparison of Sal.QonelU/maciEalian-7aicrosor« Muta^enlcity Test
Results with Published Reports Using the Same Test.
Pesticide/Chemical
Aldrin
11HC
Bux
Captafol
Captan
Carbaryl
Chlordane
Chlordene
ODD mixed iaomcra
DDT mixed isomerc
DDT-0T PI1
DDT-P ,P
Diazinon
Dichlone
Dicofol
Dieldrin
Endosulfan
Endrin
Heptachlor
Heptachlor epo:cide
Hexachlorobensene
l-Hydroj:ychlordeae
Malathion
Methyl Oxychlor
Methyl paraChion
Mirex
Trans nonachlor
PCP
Tetrachlorophcnol
Toxaphene
Aroclor 1221
Aroclor 1242
Aroclor 1260
Halowax 1000
Halowax 1051
Halowax 1059
Kut£gen
+
+
+
+
+
+
+
-
•f
+
-
*
-
+
•t-
—
-
-
-
-
-
+
-
+
-
-
t
±
•f
±
*
±
-
+
±
—
Results of References
-(3)
-(3)
None
-(3)
-(2)i(3;'
None
None
None
-(2), (3)
None
None
-(2), (3)
-(3)
None
-(1),(2) (3)
None
None
-(2), (3)
-(2)
None
None
-(3)
Kone
None
None
Kone
-(3)
None
None
None
None
None
None
None
None
a
rautagen; - * nonmutagen
(1) McCann, J., E. Choi, E. Yrasisoki, end B. Aoen. 1975. Proc. Hat. Acad.
Sci.
(2) Marshall. T. C., W. H. Borough and H. E. Swim. 1976. J. Agric. iood
Chem. 2_4_:560-563.
(3) Shirasu, Y., M. Moriya, K. Kato, A. Furuhaski and T. Kada. 1S76. Muta-
tion Reo. 40:19-30.
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Table 5. Prevalent fungi isolated from salt marsh microecosystema after addition of selected
pesticides.
Pesticide
Carbaryl
Kethyl-
parathion
Penta-
chloropheaol
Microcosm
Environment
Water
Sediment
Water
Sediment
Water
SedLaent
Prevalent Fungi
Initially Isolated
Fungus Frequency Range
Fusarium
Trlchcderma
Cladosporiica
Trichodenoa
Cladosporlua
Penicilliira II
3/72
4/7
3/7
6/7
3/7
5/7
80-TNC3
30-TNC
20-80
60-TNC
20-120
40-950
Prevalent Fungi
Finally Isolated
Fungus Frequency Range
Fusarium
Fusarium
Penicilliuta I
Penicillium I
Penicillium II
Penicilliua II
6/8
6/8
3/8
6/8
2/8
8/8
30-340
20-350
10-20
30-290
10-120
70-460
Genus of most cossaonly isolated fungus.
Nuaber of times fungus isolated/nucber of weeks sampled.
Range of number of fungi isolated from 1 ml of sample.
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Labeled pesticide molecules were added to batch culture systems con-
tained otily sterile aeawater or dilute broth end inoculated with the fungi
(Table 6). The Fusarium isolated froa the carbaryl microcosm slowly released
14
traces of CO, at approximately the saae rate from both batch cultures.
Trichoderma sp. gave negligible release of C0_. The release of C0_ was
, taoafe.notable from PCP in the dilute broth culture. These low levels of
14
C02 release suggest low level contaminant molecules as their source, but
no such contamination was detected.
These preliminary studies indicate that pollutant pesticides may
select for fungi and possibly alter the normal recycling of nutrients in
microhabltats. Future studies should evaluate the use of such microeco-
systerns to predict rates of biodegradation and the fate of xenobiotic
molecules.
17
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Table 6. Batch culture of selected fungi with C labeled pesticides.
Fungus
Fusariua
Trichoderma
Penicillium I
PenicilliuTa II
Pesticide
Carbaryl
Carbaryl
Methyl-
parathion
Pentachioro-
phenol
Culture Medium
Dilute mycological broth
Seawater
Dilute mycological broth
Seawater
Dilute mycological broth
Seawater
Dilute nycological broth
Seawater
Oaya to Maximum
14
C0_ Evolution
10
13
20
20
17
13
20
20
Per cent of Pesticide
Molecules Degraded
0.45
0.51
0.11
0.01
0.25
0.72
0.17
1.74
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