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DEDICATED
T 0
MY FAMILY
THE EFFECTS OF SELECTED PESTICIDES ON MICROORGANISMS
IN TERRESTRIAL AND AQUATIC ENVIRONMENTS
by
Norris C. Charles
A thesis submitted ta the Faculty of Graduate Studies and Research, McGill University, in partial fulf~lment cf the requirements for the
degree of Maste! of Science.
Department of Hicrobiology Macdonald·Campus of McGill University Montreal 800, Quebec, Canada.
@ Norris C. Charles 1973
July 1972 .
•
. : 1
A suggested short title:
PESTICIDES AND THE MICROBIAL ENVIRONMENT
Norris C. Charles
M.Sc.
A B S T R ACT
Norris C~ Charles Microbiology
THE EFFECTS OF SELECTED PESTICIDES ON MICROORGANISMS
IN TERRES TRIAL AND AQUATIC ENVIRONMENTS
The effects of Baygon, Carbaryl and its metabolite l-naphthol,
C~rboxin, Chlorfenvinphos, Dasanit and Elocron, on microorganisms
in terres trial and aquatic environments were investigated. No per
manent deleterious effects were observed on (1) the soil bacterial
and fungal populations at application rates from 5 lbs/acre (2.5
ppm) to 20 lbs/acre; (2) soil respiration testing soil treated at
25 and 250 ppm and (3) soil nitrification at 25 to 500 ppm applica
tion rates.
Enumeration of the constituent rhizosphere populations of the
aquatic angiosperm Lemna mir~r, revealed a disruption at aIl pesti
cide concentrations studied (0.01 to 50.0 ~g/ml). The disruption
observed was the reduction in numbers of several species of aquatic
invertebrates with their extinction at higher concentrations. With
the exception of Baygon, as the concentration of . pesticides increased
so did the bacterial populations.
Although only high pesticide concentrations upset soil micro
bial metaoolism, normal pest control practices will disrupt the
aquatic biosphere.
RESUME
H.Sc. Norris C. Charles Microbiologie
LES EFFETS DE CERTAINS PESTICIDES SUR LES MICROORGANISMES
DES HILIEUX TERRESTRE ET AQUATIQUE
Nous avons étudié l'effet des pesticides suivants, Baygon,
Carbaryl, Carboxin, Chlorfenvinphos, Dasanit et Elocron de même que
du l-Nap'hthol (metabolite du Carbaryl)· sur les microorganismes des
mil~e~x aquatique et terrestre •
. Nous n'avons observé aucun effet néfaste vis-a-vis (1) des
populations fongiques et bactériennes du sol aux taux d'application
de 5 lbs/acre (2.5 ppm) à 20 lbs/acre; (2) de la respiration des
sols traités a 25 et 250 ppm et (3) de la nitrification des sols
aux taux d'application de 25 a 250 ppm.
L'analyse des populations de la rhizosphère de l'angiospérme
Lemna minor a révélé un changement pour chacune des concentrations
utilisées (0.01 à 50~O pg/ml). Ce changement consistait en une
diminution de plusieurs espèces d'invertébrés aquatiques accompagnée
de leur disparition aux concentrations élevées. La population. des
bactêries aquatiqùes augmentait en nombre; parallèlement aux concen-
trations des pesticides, sauf pour Baygon.
La pratique courante de contrôle par les pesticides débalancera
la biosphère aquatique mais seules les concentrations· élevées de
pe'sticide affe'cteront le métabolisme inicrob"i~n du sol.
TABLE, OF CONTENTS
ACKNmrr.EDGEHENTS . . . GLOSSARY . . . . . . LIST OF TABLES
LIST OF FIGURES . . . . . . . . . . INTRODUCTION . . . . .. . LITERATURE REVIEW
PART 1. EFFECT OF SELECTED PESTICIDES ON MICROORGANISMS IN TERRESTRIAL ENVIRONMENTS • • •
INTRODUCTION AND LITERATURE REVIEW •
Or>garzoahZorine aompounds • • • • • •
Or>ganophosphate aompounds • • • • • • • •
MethyZaaT'bamate aompounds ••
MATERIALS AND METHODS
A. THEEFFECT OF SELECTED PESTICIDES ON'SOIL
Page
ix
x
xiii
xvi
1
3
24
25
25 26 '
28
30
BACTERIA AND FUNGI • • • • • • • • • • • •• 30
EACTERIAL AND FUNGAL COUNTS
}ŒDIA •• • • • • • • • • •
, (1) Soil Extract Agar • • •
(2) Rose-bega1 Streptomycin Agar
B. EFFECT OF SELECTED PESTICIDES ON SOIL
30
33
33
34
RESPIRATION • • • • • • • • • • • • • • 35
C. EFFECT OF SELECTED PESTICIDES ON SOIL NITRIFICATION • • 37
RESULTS AND DISCUSSION •• 38
A. THE EFFEeT OF SELECTED PESTICIDES ON SOIL BACTERIA AND FUNGI • • • • • • • • 38
vi
. r -, ,
TA BLE o F CON T E N T S
I;Jasanit
ChZol'fenvinphos
Baygon and CarbaryZ •
EZocl'on
Carboxin ' ••••••
B. THE EFFECTS OF SELECTED PESTICIDES ON SOIL
:;>age
38
38
39
39
40
RESPIRATION ••••••••• 48
Baygon and EZocl'on • • • • •
ChZol'fenvinphos and Dasanit •
Carboxin ••
Z-NaphthoZ
. .
. .
C. THE EFFECTS OF SELECTED PESTICIDES ON SOIL
48 50
52
52
NITRIFICATION 60
Baygon • • • • • 60
EZocl'on •
ChZol'fenvinphos •
Dasanit ••• • • • •
Carboxin ••
Z-NaphthoZ
DISCUSSION •
PART II. THE EFFECT OF SELECTED PESTICIDES ON MICROORGAN-
60
60
61
61
62
77.
ISMS IN -AQUATIC ENVIRm~!ENTS • • • • ~ •• 79
INTRODUCTION AND LITERATURE REVIEH •
MATERIALS AND METHODS •••
ANALYSIS OF POND WATER •
APPARAIUS • • • • • • •
SAMPLING AI.'ID ENmIERATION PROCEDURE
EXTRACTION k'ID DETERHINATION OF PESTICIDE
80
85
85
87
87-
LEVELS '...... • • • • • • • • • .' 88
vii
TA BLE o F CON T·E N T S
RESULTSAND DISCUSSION
Z-NaphthoZ
Baygon •
EZocron • • • • •
ChZorfenvinphos •
Dasanit
Carborin
DISCUSSION . . . . . . . . . . . . . . . . . . . GENERAL DISCUSSION AND CONCLUSIONS
BIBLIOGRAPHY • • • • • • • • • • • . . . . . . .
viii
.Page
91 ~91'
92
93
95
96
97
99
149
151
ACKNOWLEDGEMENTS
The author wishes to express his sincere appreciation for the'
helpful guidance, advice and encouragement of his director, Dr. A.
C. BlackïNood, Professor, Department of Microbiology; Dean, Faculty
of Agriculture; Vice-Principal, Macdonald Campus of McGill University;
through?ut the course of th~s research and in the preparation of
the manuscript. Thanks are due ta Dr. F. Ali Khan of the Department
of Entomology for her assistance in the identification of the fresh
water invertebrates of the aquatic ecosystem.
The author is obliged to Mrs. Edna Rowell for the typing of
the manuscript and to Mr. James Gilchrist for· the preparation of the
graphs appearing in the thesis.
Appreciation is aIs a extended ta Chemagro Corporation, Ciba
Geigy Canada Ltd., Shell Chemicals, Union Carbide Chemical Company
and UniRoyal Agticultural Chemicals Division, for supplying the
pesticides~
Last but not least the author is deeply indebted to Environment
Canada for financial support.
ix
COIl'.rnon Name
Aldrin
Amiben
Amitrole
Bayer 37289
Baygon
BHC
Cap tan
Carbaryl
Carbofuran
Carbophenothion
Carboxin
Chlorqane
Chlorfenvinphos
ClPC
2,4-D
Dalapon
GLOSSARY
Chemical Name
1,2,3,4,lO,lO-hexachloro-l,4,4a,S,8,8a-hexahydro-
1,4-endo,exo-S,8-dimethano-napthalene
3-amino-2,S-dichlorobenzoic acid
3-amino-l,2,4-triazole
O-ethyl 0-2,4,5-trichlorophenyl ethyl phosphono
thioate
O-isopropoxyphenyl methyl carbamate
l,2,3,4,S,6~hexachlorocyclohexane
N-[(trichloromethyl)thio]-4-cyclohexene-l,2-
di carb oximide
I-naphthyl N-methylcarbamate
2,3-dihydro-2,2-dimethyl-7-benzofuranyl
methylcarbamate
S-[(p-chlorophenyl)thiomethyl]O,O-diethyl
phosphorothioate
S,6-dihydro-2-methyl-l,4-oxathiin-3-carboxanilide
1,2,4,S,6,7,8,8-octachloro-2,3,3a,4,7,7a
hexahydrà-4,7-methanoindene
2-chloro-l-(2,4-dichlorophenyl) vinyl diethyl
phosphate
isopropyl N-(3-chlorophenyl) carbamate
2,4-dichlcirophenoxy acetic acid
2,2-dichloropr~pionic acid
x
i ,-
Common Name
Dasanit
DDD
DDT
Diazinon
Diazoxon
Dieldrin
Diuron
Dursban
Dyfonate
Elocron (C-8353)
Furadan
Heptachlor
Heptachlor epoxide
IPC
Isolan
Lindane
GLOSSARY
Chemical Name
O,O-diethyl O-[p-(methylsulfinyl)phenyl]
phosphorothioate
1,1-dichloro-2,2-bis (p-chlorophenyl) ethane
dichloro diphenyl trichloroethane
O,O-diethyl O-(2-isopropyl-6-methyl-4-pyrimidinyl)
phosphorothioate
O,O-diethyl~O-(2-isopropyl-4-methyl pyrimidinyl) phosphate
hexachloroepoxyoctahydro-endo, exo-dimethano-'
naphthalene 85%
3-(3,4-dichlorophenyl)-1,1-dimethylurea
O,O-diethyl 0-3,5,6-trichloro-2-pyridyl
phosphorothioate
O-ethyl S-phenyl-ethylphosphonodithioate
2-(1,3-dioxolane-2-yl)-phenyl-N-methyl carbamate
2,3-dihydro-2,2-dimethyl-7-benzofuranyl
methylcarb~mate
1,4,5,6,7,8,8-heptachloro-3a,4,7,7a-tetrahydro-
4,7-methanoindene
1,4,5,6,7,8,8~heptachloro-2,3-epoxy-2,
3,3a,7a-tetrahydro-4,7-methanoindene
isopropyl N-phenylcarbamate
l-isopropyl-~~ethyl-5-pyrazolyl di-methylcarbamate
Y-l,2,3,4,5,6-hexachlorocyclohexane
ri.
Common Name
Linuron
Malathion
Methyl Parathion
Monuron
Nabam
a .. Naphthol
Parathion
Phorate
Propanil
Sevin
Systox
Thimet
Temik
Trichlorofon
Vitavax
Zinophos
/1'-- .
GLOSSARY
Chemical Name
3-(3,4-dichlorophenyl)-1-methoxy-l-methylurea
O.O-dimethyl dithiophosphate of diethyl
mercaptosuccinate
O,O-dimethyl O-p-nitrophenyl phosphorothioate
3-(p-chlorophenyl)-1,1-dimethylurea
disodium ethylene-l,2-bisditiocarbamate
,l-Naphthol
O,O-diethyl O-p-nitrophenyl phosp4o~othioate
O,O-diethyl S-(ethylthio)methyl phosphorodithioate
3',4'-dichloroproprionanilide
(see CarbarYl)
O,O-diethyl O(and S)-2-(ethylthio)ethyl
phosphorothioates
(see Phorate)
2-(methyl-2-methylthio)propionaldehyde
O-(methylcarbamoyl) oxime
0,0-dimethyl(l-hydroxy-2,2,2-trichloroethyl)
phosphoromate
(see Carboxin)
O,O-diethyl O-Z pyrazinyl phosphorothioate
xii
LIS T o F TABLES
Table Page
1. Mode of application of pesticides to plots • • • • • 31
2. The effects of various concentrations of Baygon on soil nitrification • • 65
3. The effects of various concentrations of Elocron on soil nitrification • • 67
4. The effects of various concentrations of Chlorfenvin-phos on soil nitrification. • • • • 70
5. The effects of various concentrations of Dasanit on soil nitrification • • 72
6. The effects of various concentrations of Carboxin on soil nitrification. . . . . . . . . . . . . . . . . 74
7. The effects of various concentrations of l-naphthol on soil nitrification 76
8. Summary of conditions for extraction and determina-tion of pesticides in samples • • • • •• 90
9. Population levels of f!~mn.g minoT' rhizosphere before exposure to l-naphthol • • • • • • • • • • • • • •• 104
10. . Population. levels of Lemna minoT' rhizosphere after 2 days of exposure to l-naphthol • • • • • • • • • •• 105
Il. Population levels of Lemna minoT' rhizosphere after 10 days of exposure to l-naphthol . • • . • • • •• 106
12. Population levels of Lemna minoT' rhizosphere after 20 days of exposure to l-naphthol • • • • . • • •• 107
13. Population levels of Lemna minoT' rhizosphere after 30 days of exposure to l-naphthol • • • • • • • •• 108
xiii
-.-
Table Page
14. Population levels of Lemna minoT' rhizosphere before exposure toBaygon ••••••••••••••••• 112
15. Population levels of Lemna minoT' rhizosphere after 2 days of exposure to Baygon • • • • • • • • • • • • • 113
16. Population levels of Lemna minoT' rhizosphere after 10 days of exposure to Baygon •• • • • • • • • • • 114
17. Population levels of Lemna minoT' rhizosphere after 20 days of exposure to Baygon • • • • • • • • 115
18. Population levels of Lemna minoT' rhizosphere after 30 days of exposure to Baygon •• • • • • • • • • • 116
19. Population levels of Lemna minoT' rhizosphere before exposure to Elocron • • • • • • • • • • • • • • • • 120
20. Population levels of Lemna minoT' rhizosphere after 2 days exp os ure to Elocron • • • • • • • • • • • • • • 121
21. Population levels of Lemna minoT' rhizosphere after 10 days exposure to Elocron • • • • • • • 122
22. Population levels of Lemna minoT' rhizosphere after 20 days exposure to Elocron • • • • • • • 123
23. Population levels of Lemna minoT' rhizosphere after 30 days exposure to Elocron • • • • • • • • • • 124
24. Population levels of Lemna minoT' rhizosphere before exposure to Chlorfenvinphos • • • • • • • • • • 128
25. Population levels of Lemna minoT' rhizosphere affer-2 days exposure to Chlorfenvinphos • • • • • • • • • • 129
26. Population levels of Lemna minoT' rhizosphere after 10 days exposure to Chlorfenvinphos •••• • 130
xiv
, 'l ....
Table Page
27. Population 1eve1s of Lemna mino~ rhizosphere after 20 days of exposure to Ch1orfenvinphos · · · · · · · 131
28. Population levels of Lemna mino~ rhizosphere after 30 days of exposure to Chlorfenvinphos · · · · · · · 132
29. Population levels of Lemna mino~ rhizosphere béfore exposure to Dasanit . . . . . · · · · · · · · · · · 136
30. Population levels of Lemna mino~ rhizosphere after 2 days of exposure to Dasanit · · · · · · · · · · 137
31. Population levels of Lemna mino~ rhizosphere after 10 days of exposure to Dasanit · · · · · · · · 138
32. Population levels of Lemna mino~ rhizosphere after 20 days of exposure to Dasanit · · · · · · · · 139
33. Population 1evels of Lemna mino~ rhizosphere after 30 days of exposure to Dasanit · · · · · · · · · · · 140
34. Population 1evels of Lemna mino~ rhizosphere before exposure to CarboXin . . . . . · · · · · · · · · · · 144
35. Population levels of Lemna mino~ rhizosphere after 2 days exposure to Carboxin · · · · · · · · · · 145
36. Population leve1s of Lemna mino~ rhizosphere after 10 days of exposure to Carboxin · · · · · · · · · · 146
37. Population levels of Lemna mino~ rhizosphere after 20 days of exposure to Carboxin · · · · · · · .147
38. Population levels of Lemna mino~ rhizosphere after 30 days of exposure to Carboxin · · · · · · · · · · 148
xv
".-
...- ' ;
LIS T o F FIGURES
Figures Page
1. The chemical names and structural formulae of the pesticides • • 32
2. The effects of Dasanit on soil bacterial and fungal populations • • 42
3. The effects of Chlorfenvinphos on soil bacterial and fungal populations • • • • • • • • • •• 43
4. The effects of Baygon on soil bacterial and fungal populations • • • • • • • • • • • •. • • • • • 44
5. The effects of Carbaryl on soil bacterial and fungal popula.tions • • • • • • • • • • • • • • 45
6. The effects of Elocron on soil bacterial and
7.
8.
9.
fungal populations • •
The effects of Carboxin on soil bacterial and fungal populations • • • • • • • • • • • • • •
The effects of Baygon on soil respiration rate • •
The effects of Elocron on soil respiration rate
10. The effects of Chlorfenvinphos on soil respiration rate . . . . ._ . . . . . . . . . . . .
11. The effects of Dasanit on soil respiration rate
12. The effects of Carboxin on soil respiration rate •
13. The effects of l-naphthol on soil respiration rate
14. The effects of Baygon on soil nitrification
xvi
46
47
54
55
56
57
58
59
64
Figures
15.
16.
The effects of E10cronon soi1 nitrification • • •
The effects of the supernate of a pseudomonad grown on E10cron, on soi1 nitrification
17. The effects of Ch10rfenvinphos on soi1 nitrifica-tion • • • • •
18. The effects of Dasanit on soi1 nitrification •
19. The effects of Carboxin on soi1 nitrification
20. The effects of 1-naphtho1 on soi1 nitrification
21. Eco10gica1 Assay F1ask • •
22. Assemb1ed Assay Apparatus . . . . . . . 23. The effects of 0.1 ~g/m1 of 1-naphtho1 on the
numbers,of Protozoa, Rotifers and Gastrotrichs of
Page
66
68
69
71
73
75
86
86
the Lemna minop rhizosphere • • • • • • • 101
24. The effects of 50.0 ~g/m1 of 1-naphtho1 on the numbeISof Protozoa, Rotifers and Gastrotrichs of the Lemna minop rhizosphere • • • • • • • • • • • 102
25. The effects of 1-naphtho1 on the bacteria1 po.pu1ation of the Lemna minop rhizosphere • • • • • • • 103
26. The effects of 0.1 ~g/m1 of Baygon on the numbers of Protozoa, Rotifers and Gastrotrichs of the Lemna minop rhizosphere. • • • • • • • • • • • • • 109
27. The effects of 50.0 ~g/m1 of Baygon on the numbers of Protozoa, Rotifers and Gastrotrichs of the Lemna minop rhizosphere • • • • • • • • • • • • • 110
The e~fects of Baygon on the bacteria1 popu~ation of Lemna minop rhizosphere • • • • • • • • • • • • 111
Figures
29. The effects of 0.1 pg/ml of Elocron on the numbers of Protozoa, Rotifers and Gastrotrichs of the
Page
Lemna minor rhizosphere • • • • • • • • • • • • 117
30. The effects of 50.0 pg/ml of E1ocron on the numbers of Protozoa, Rotifers and Gastrotrichs of the Lemna minor rhizosphere • • • • • • • • • • • • 118
31. The effects of E1ocron on the bacteria1 population of the Lemna minor rhizosphere •••••••••• 119
32. The effects of 0.1 pg/ml of ct.lorfenvinphos on the numbers of Protozoa, Rotifers and Gastrotrichs of the Lemna minor rhizosphere • • • • • • • • •• 125
33. The effects of 50.0 pg/ml of Ch1orfenvinphos on the numbers of Protozoa, Rotifers and Gastrotrichs of the Lemrva minor rhizosphere • • • • • • • • •• 126
34. The effects of Ch1orfenvinphos on the bacteria1 population of the L~ minor rhizosphere 127
35. The effects of 0.1 pg/ml of Dasanit on the numbers of Protozoa, Rotifers and Gastrotrichs of the Lemna minor rhizosphere • • • • • • • • • • • • • • 133
36. The effects of 50.0 pg/ml of Dasanit on the'numbers of Protozoa, Rotifers and Gastrotrichs of the Lemna minor rhizosphere • • • • • • • • • • • • • • • • • 134
37. The effects of Dasanit on the bacteria1 population of the Lemna minor rhizosphere • • • • • • • • • • 135
38. The effects of 0.1 pg/ml of Carboxin on the numbers of Protozoa, Rotifers and Gastrotrichs of the Lemna
39.
minor rhizosphere • • • • • • • • • • • 141
The effects of 50.0 pg/ml of Carboxin on the numbers of Protozoa, Rotifers and Gastrotrichs of the Lemna minor rhizosphere • • • • • • • • • • . • 142
40. The effects of Carboxin on the bacteria1 population of the Lemna minor rhizosphère • • • • • • • • • • 143
xviii,
1
INTRODUCTION
The control of pests is basically an ecological problem - a
problem of the relations between living organisms and their environ-
ment. Pesticides are among the most effective weapons available to
control pests. In many instances, they are the only means available
for the control of certain pests. However, in the use of pesticides,
~Ye mus t be keenly aware of, and concemed and knowledgeable about
their effects on man's total environment. Our soils, water, and
plants are primary recipients of pesticides in a complex ecosystem
containing many other values.
There is the danger of potential accumulation of toxic levels
of pesticides in natural food chains, and dangerous residues in crops
and food. The possible inhibition of beneficial soil microorganisms
by application of pesticides and the potential danger of herbicide
residues accumulating in soils with subsequent injury to following
crops in a routine system should also be seriously considered.
In recent years there have been many reviews and symposia on the
persistence of pesticides in the environment. The reviews include
"Pesticides and the Living Landscape" (Rudd, 1964), which was a much
more balanced review of the influence of pesticides in nature than
"Silent Spring" (Carson, 1962) and "Pesticides in Soils and Water"
(U.S. Department of Health, Education, and Welfare, 1964) a comprehen-
sive annotated bibliography. Symposia include "Research in Pesti
cides" (C.O. Chichester, ed., 1965); "Pesticides and Their Effects
on Soils' and Water", (A. S.A. special publication No. 8, 1966);
"Pesticides in the Soil: Ecology Degradation and Movement", (Michigan
State University, 1970) and "International Symposium. on Identifica
tion and Measurement of Environmental Pollutants", (Ottawa, 1971).
For a better understanding of the pests and the merit of some
of these pesticides, six pesticides were studied for their effects
on the microbial population, respiration and nitrification in the
soil and on the rhizosphere of the aquatic angiosperm Lemna mino~
(duckweed). The six pesticides were, the insecticides Chlorfenvin
phos, Baygon, Elocron (C-8353), Carbaryl (Sevin) and its principal
metabolite, l-naphthol; a systemic fungicide, Carboxin (Vitavax),
and a nematocide, Dasanit, which is aIso an insecticide.
This thesis is organized in two parts. The first part examines
the effects of these selected pesticides on microorganisms in the
soil while in the second part the effects of the pesticides on micro
organisms in an aquatic environment are considered.
2
3
LITERATURE REVIEW
Because of their extended persistence, slow rate of decomposi-
tion and the fa ct that the bulk of any pesticide application almost
invariably reach the soil, lakes and rivers, it is desirable that
the actual or potential effects upon the soil and water microorgan-
isms be investigated.
The most significant of the interactions of pesticides with
soil microorganisms appears to be the influence of the soil microbial
population (or some segment of it) in decomposing the chemicals, a
process commonly termed 'microbial detoxication'.
Pesticides may be destroyed or removed by several different
methanisms which operate in natural environments. These involve
such processes as photodecomposition, volatilization, leaching,
adsorption, chemical (non-biologica1) decomposition, microbial decom-
position and plant or organism uptake.
The physica1 factors influencing pesticide behaviour in the
soi1 have been considered in severa1 review articles (Hartley, 1964;
Upchurch, 1966; Bailey and White, 1970; Mortland, 1970 and White
and Mortland, 1970).
PHOTODECOMPOSITION
Certain organic compounds undergo molecular changes when exposed
to electromagnetic radiation. These changes have been demonstrated
by noting altered u.v. absorption spectra, detecting, isolating and
identifying new products or modified properties of specifie pesti
cides following their exposure to light. Actually only the wavelength
region between 285 - 450 m~ appears to be of wide importance to
pesticide photolysis in sunlight (Crosby, 1969). Light within this
wavelength range energizes quite a variety of common reactions
including oxidation, reduction, elimination, hydrolysis, substitution
'nd isomerization. Usually, air, water and light combine to produce
a multiplicity of reactions.
4
Crosby and Tutass (1966) demonstrated that the widely used herbi
cide, 2,4-dichlorophenoxyacetic acid (2,4-D) undergoes photodecomposi
tion in aqueous solution in which the key reaction was the photochemical
replacement of the chlorine atoms of the aromatic ring by hydroxyl
groups. The decomposition products were 2,4-dichlorophenol, 4-chloro
catechol, 2-hydroxy-4-chlorophenoxyacetic acid, 1,2,4-benzenetriol, and
finally polymerie humic acids.
The photodecomposition of chlorobenzoic acids (Crosby and Leitis,
1969) has also been reported. Ultraviolet irradiations of 2-, 3- and
4-chlorobenzoic acids as aqueous solutions of their sodium salts led
to replacement of the chlorine by hydroxyl and hydrogen to produce the
5
corresponding hydroxy-benzoic acids and benzoic acid itself. In sun-
light the monochlorobenzoic acid remained unaffected, while Amiben
(3-a~ino-2,5-dichlorobenzoic acid) decomposed rapidly.
Photo-oxidations are among the most general and im"portant envir-
onmental reactions. Crosby and Tang (1969) showed that the principal
pathways for the substituted urea herbicide, 3-(p~chlorophenyl)-1,1-
dimethy~ urea (Monuron), involved the stepwise photo-oxidation and
demethylation of the N-methyl groups, hydroxylation of the aromatic
nucleus and pol}~erization.
Photochemical decomposition has been observed for Elocron (C-8353)
(Pape et aZ~ 1970). The compound undergoes a solution-phas"e photo-
chemical reaction in methyl alcohol and in water. A benzoxazine d~
rivative is formed in greater than 85% yield in methanol and multiple
products are formed in water - two that areinsolublebeing the benzal-
dehyde derivative and the substituted phenol. Studies indicate that
these reactions may be of significance in determining the persistence
of this compound under field conditions.
Elimination reactions are also represented among photochemical
reactions. For example, Carbaryl undergoes alkaline hydrolysfs to
provide l-naphthol~methylamine and carbon dioxide. Aly and El-Dib
(1971)"showed that Carbaryl produced five degradation products, one
of which was l-naphthol,two were degradation products of the latter
and two others were unidentified. Further examination revealed that
6
under neutral conditions or in non-aqueous solvents the decomposition
led to I-naphthol and methyl isocyanate by elimination (Crosby et aZ.~
(1965).
Crosby et aZ.~ (1965), also investigated the photolysis of Baygon
in ethanol and hexane but no products were found when sunlight was
the light source. However, there were slight products under strong
ultraviolet light. Aly and El-Dib (1971) showed that aqueous solu-
tions of Baygon resulted in the formation of several degradation
products. This supports the view that photodecomposition of carba-
mate esters is affected by the nature of the solvent. The pH of the
aqueous medium was found to be an imporant factor in determining the
rate of photolysis of Baygon, being slow at low pH values and tending
to increase with a high pH value.
Lamherton et aZ.~ (1970), reported that the stability of l-naphthol
in sea water is considerably affected by light. It is relatively
stable in the absence of oxygen and the presence of light. Its degrad-
ation in water may be attributed more to photo-oxidation than photo-
decomposition. The products formed included 1,4-naphthoquinone and
2-, or 3-hydroxy-l,4~aphthoquinone. A reddish-blue precipitate, which
was not identified, was also formed.
So far there have been no reports in the literature of decomposi-
tion of Chlorfenvinphos, Carboxin and Dasanit attributed to the effects
of light.
VOLAIILITY
Pesticides may evaporate and be 10st to the atmosphere as vola
tile gases. Vo1ati1ity may be an important avenue of 10ss from soi1
for some pesticides especia11y those with high vapour pressures
(Edwards, 1965). Ga11aher and Evans (1961) found that even non-vola
tile DDT disappeared more rapidly in the first three months than in
the subsequent nine months.
Laboratory and field studies by Harris and Lichtenstein (1961)
showed that vapors toxic to both vinegar f1ies (DrosophiZa meLanogas
ter Meigen) and housef1ies (MUsaa domestiaa L.) were given off from
Aldrin-, Heptach10r-, Phorate-, Lindane-, Heptach10r epoxide- and
Die1drin-treated soi1s. There was no ev~dence of vo1ati1ization from
soi1s treated with DDT, Carbary1, Ma1athion and Parathion. An increase
in the rate of Aldrin vo1ati1ization from soi1 resulted from increase
in: (1) insecticide concentration in the soi1 (2) soi1 moisture
(3) relative humidity of air passing over the soi1 (4) soi1 tempera
ture (5) rate of air movement over the soi1 surface. A decrease in
the rate of Aldrin vo1ati1ization was noted in dry soi1s containing
increasing amounts of clay and organic matter and in wet soi1s con
taining increasing amounts of organic matter. The amount of soi1 in
a given volume (bu1k density) had no effect on the rate of Aldrin
vo1ati1ization.
Parochetti and Warren (1966) observed that 10sses of the pheny1-
7
carbamate herbicides, IPC and CIPC from moist soil decreased as the
per cent clay, the organic matter, or both increased. Volatilization
was negligible from dry soil but increased with increasing moisture
and temperatqre. They concluded that the adsorptive capacity of the
soil was the major factor influencing volatility of the herbicides.
The volatilization of many pesticides,e.g. phenylureas) has been
considered to be almost negligible under field conditions yet it is
conceivable that chemicals classed as being qui te non-volatile by
classical measurements may be lost in appreciable amounts under certain
environmental conditions (Upchurch, 1966).
The volatilization of the s-Triazines is known to be influenced
by molecular structure and the degree to which they are adsorbed to
the soil (Kearney et aZ~ 1964).
Chisholm and Koblitsky (1959) suggested that a large proportion
of chlorinated hydrocarbons may be lost by codistillation of insecti
cide into the atmosphere with the water vapour escaping from the soil.
Upchurch (1966) has also postulated the same explanation for some
losses of herbicides.
LEACHING
The extent to ~hich a pesticide is leached is determined princi
pally by: (1) solubility of the pesticide in water (2) amount of
8
9
water passing downward through the soil profile and (3) adsorptive
relationships between the pesticide and the soil.
The strength of adsorption bonds between the pesticide molecules
and soil colloidal surfaces is more important than water solubility
in determining the leaching of pesticides (Klingman, 1963).
Edwards et al, (1971) concluded that more Chlorfenvinphos leached
vertically into drainage water than laterally over the surface into
natural waters. The amounts of Chlorfenvinphos appearing in the
leachates were about nine times greater than in a similar experiment
using Dieldrin following the application of similar doses. Dieldrin
is more insoluble than Chlorfenvinphos.
CONCENTRATION
In a review, Edwards (1966) stated that not aIl workers agreed
on the influence of concentration on persistence. Edwards (1964)
plotted published results together with some unpublished data as
graphs of percentage of original dose against time. Regression showed
that more proportionally disappeared each year from small rather than
large doses. Larger quantities of insecticide disappeared from soil
treated with a large than small dose, i.e. rate of breakdown does not
appear to remain constant irrespective of dose but falls off logarith-
mically. Except for the rapid losses that occur immediately after an
/--
( insecticide is applied, the loss of a particular dosage seems to be
r
at a constant rate whether the insecticide is a residue of a much
larger dose or a new application.
ADSORPTION
Comprehensive reviews of the factors which influence adsorption,
desorption, and movement of pesticides in soils has been made recent
ly by Bailey and White (1964); Kunze (1966); Bailey and White (1970),
and White and Mortland (1970).
In the review by Bailey and White (1970) the adsorption theories
of Freundlich, Longmuir, Gibbs and Brunauer, Emmet and Teller (BET)
are briefly discussed. Mathematical expressions were derived to
explain theories involved in adsorption models. Lambert (1967) has
mathematically derived a fundamental relationship between adsorption
by soils and chemical structure of certain classes of chemicals.
According to Bailey and White (1970) the factors influencing
adsorption and desorption are the physico-chemical nature of the ad
sorbent, the physico-chemical nature of the pesticide, soil reaction,
surface acidity, nature of the saturating cation on the colloid ex
change site, temperature and nature of formulation.
The physical adsorption of pesticides by soils involves the con
centration of the molecules of the compound, either from the vapour
phase or from solution, at the solid surfaces of the soil particles.
10
Il
\ ~-
Idea11y, this process is reversib1e, the position of equilibrium
being a function of the concent~ations of the herbicide in the two
phases (Furmidge and Osgerby, 1967). Adsorption invo1ves an inter-
action between electrical charges on the pesticide molecu1e and the
e1ectrica1 charges on the soi1 component and is regulated by the
characteristics of the soi1 solutions (Bai1ey and White, 1964).
Many attempts have been made to corre1ate the extent of adsorp-
tion with individua1 soi1 properties, such as clay content (Sheets,
1958; Hilton and Yuen, 1963; Audus, 1964; Ta1bert and F1etcha11,
1965; Sheets and Harris, 1965; Edwards, 1966; and Weber, 1970), or-
ganic matter content (Sheets, 1958; Upchurch, 1958; Hilton and Yuen,
1963; Hance, 1965 (a and b); and Ta1bert and F1etcha11, 1965), cation
exchange capacity (Sheets, 1958; Upchurch, 1958) and pH (Harris and
Warren, 1964; and Edwards, 1966).
Clay content and organic matter tend to be corre1ated with each
other and with important soi1 properties such as structure, water-
holding capacity, pH buffering and specific surfaces. However, in a
review·by Wo1cott (1970), he stated that organic matter content has
been judged to be the most usefu1 (Edwards, 1966; Kay and E1rick,
1967; Yaron et aZ~ 1967; Day et aZ~ 1968; Lambert, 1968; Doherty and
Warren, 1969). Wo1cott (1970) a1so stated that high organic soi1s
(peats and mucks) adsorb or inactivate pesticides to a greater extent
than kaolinite or montmori11onite and re1ease them incomp1ete1y under
treatments which results in complete desorption from the clays
(Harris and Warren, 1964; Talbert and F1etchal1, 1965; Scott and
Weber, 1967; Coffey and Warren, 1969; Doherty and Warren, 1969; and
Shin et aZ~ 1970).
CHEMICAL DECOMPOSITION
The ready sus ceptib i1it y of organophosphate insecticide to de
gradation has contributed recent1y to their increasing agricu1tura1
use. This increase is based part1y on the implication that rapid
degradation of these compounds minimizes residue prob1ems. Certain
organophosphates are known to be degraded by both chemica1 and micro
bia1 pathways. The degradation of Diazinon is principa11y by chemica1
hydro1ysis (Konrad et aZ~ 1967) whi1e Ma1athion has been observed to
degrade by both chemica1 and microbia1 means (Matsumura and Boush,
1966).
Factors which affect the rate of hydro1ysis and types of hydro-
1ytic products formed from organophosphate insecticides are tempera
ture, pH and the ionic strength of the system. The most important is
perhaps pH (Faust and Suffet, 1966).
Gomaa et aZ~ (1969) showed that Diazinon and Diazoxon are hydro-
1ysed by first order kinetics in the pH range 3.1 to 10.4 at an ionic
strength of 0.02 M. Under natura1 water conditions they are character
ised by long residua1 1ife. Under specifie conditions (pH lower than
12
,"'.
13
5.0 or higher than 9.0, and temperature higher than 20°C) hydrolysis
proceeds within a relatively short period of time. The major hydrolysis
p~oduct·· is 2-isopropyl-4-methyl-6-hydroxypyrimidine.
Various soil fractions may participate in facilitating the
initial catalytic hydrolysis of the phosphate insecticides. In soils
(Konrad et aZ3 1967), hydrolysis is apparently catalysed by adsorp
tion on some soil component rather than by acid hydrôlysis. Mort land
and Raman (1967) showed that the catalytic hydrolysis of several
organophosphates including Diazinon and Dursban occurred in cupric
chloride solutions and in copper-montmorillorite clay suspensions.
Thus, clay minerals which bond metallic cations more strongly than
does montmorillorite and organic soils allow little or no hydrolysis.
The breakdown of Chlorfenvinphos in soils and in crops grown in
soils was examined by Beynon and Wright (1967). Soils of four differ
ent types were treated with a relatively high dosage level (15 ppm)
of 14C-Chlorfenvinphos and eight breakdown products were formed. A
breakdown pathway for Chlorfenvinphos was prepared and this involved
the acid hydrolysis product 2,4-dichlorophenacyl chloride. This com
pound is the in vitro hydrolysis product of Chlorfenvinphos in crops,
soils (Beynon et aZ3 1966), in tissue of sheep (Robinson et aZ3 1966)
and cows (Ivey et aZ3 1966).
When Chlorfenvinphos was applied to potato foliage there was
some evidence for the conversion of the tra:ns (S)isomer to the cis
(
Ca) isomer. Both isomers were degraded rapidly and the initial half
life of Chlorfenvinphos (ais and trans) was about three days (Beynon
et aZ, 1968). The initial half-1ife of Chlorfenvinphos in soils
varied from two weeks to more than twenty-three weeks depending on
the soil type (being most persistent in peat and least in sandy loam),
the formulation and the dosage level (Beynon et aZ, 1966).
Beynon et aZ, (1971) showed that Chlorfenvinphos disappeared
rapidly from natural waters and it did 50 in two distinct phases, a
rapid initial phase and a much slower second phase. Possibly the
first p4ase represented the initial precipitation of the heavier
particles and plankton containing the adsorbed pesticide. Later the
Ch10rfenvinphos was gradually adsorbed by other suspended matter,
which then precipitated more slowly or the second phase could repres
ent the slower removal of residues by metabolism. Chlorfenvinphos
decomposes slowly in water (half-life - 170 days at pH 6 and 80 days
at pH 8) at 20 - 30 Oc hydrolyses probably did not contribute much
to the initial disappearance.
Dasanit, an organophosphorus insecticide con tains an alkylthio
ether linkage which is oxidized to a sulfoxide in the parent compotmd.
Further oxidation results in the Dasanit oxygen analogue, Dasanit
sulfone and the Dasanit oxygen analogue sulfone. Katague and Anderson
(1967) detected these three metabolites when studying the metabolism
of Dasanit in cotton plants.
14
15
Bowman and Hill (1971) determined Dasanit and the above three
metabolites in corn grass and milk. The determination from plant
mate rial of the same compound using gas chromatography with flame
photometric detection was studied by Williams et al~ (1971).
Carbamates have already gained an important place among pesti-
cides. They do not have the persistence so detrimental to the use of
most of the o~gandchiorine compounds but they are degraded less
rapidly than the organophosphate compounds. Like the latter, they
are cholineesterase inhibitors.
The carbamate insecticides are exemplified by Carbaryl (S;evin).
The ester groups present in insecticides of this class indicates that
they should be subject to hydrolysis and they actually are. An anal-
ytical method for Carbaryl is based on the alkaline hydrolysis of the
compound tonaph.thol (Johnson et al'~ 1963) and this is indicative of
the instability of this type of compound in alkaline media.
Johnson and Stansbury (1965) reported that the primary factors
influencing disappearance rate from various foliage surfaces seem to
be weathering and dilution by plant growth rather than chemical (or
photochemical) degradation or systemic effects. Accumulated analyti-
cal data showed that the normal half-life of Carbaryl on growing
crops to be three days in spinach, two days in berries, four days in
apples and in soil approximately eight days under normal conditions.
16
Karinen et aZ., (1967) determined the persistence of Carbaryl in
marine estuarine environment in laboratory aquaria. At low tempera-
ture (BOe) and under conditions whe~e adsorption by mud was prevented,
Carbaryl degraded slowly, persisting for severa! weeks. One of the
products of decomposition under these conditions, I-naphthol, subs~
quent1ytphotodecomposed. The above products were acce1erated by a
higher temperature (20°C). ~~en Carbary1 was applied to sha110w mud
f1ats, the pesticide was rapid1y removed from water by adsorption on
bottom mud. Degradation proceeded in the medium, ultimately to the
rupture of the naphtho1 ring to produce C02 and possib1y methane.
Intermediate products in the degradation process are polar compounds
arising from modification of the naphthy1 portion of the Carbary1.
Even with such processes, however, Carbary1 and I-naphtho1 persisted
in the mud for two to six weeks.
Eiche1berger and Lichtenberg (1971) studied the persistence of
carbamates in river water. The concentration of Carbary1 (10 ~g/l)
diminished in one week and comp1etely disappeared in two. However,
I-naphtho1, the suspected decomposition product of Carbary1 was not
detected in the water immediately after the parent compound disappeared.
Either 1-naphtho1 decomposed as rapidly as the Carbaryl or the Carbaryl
did not follow the suspected path of decomposition.
After one week, Baygon was 50% hydrolysed to its phenol, after
two weeks 70%, after four weeks 90%, and after eight weeks 95%. The
i . ~-
17
phenol was aIso degraded during this time and none was found in water
after eight weeks.
Nothing was found in the literature concerning the chemical de-
gradation of Elocron, however, work is now being undertaken in this
laboratory to determine its chemical decomposition products.
Carboxin (Vitavax) is a highly effective systemic fungicide.
Edgington and Corke (1967) studied its breakdown in normal and sterile
soil using the soil perfusion technique. Decomposition occurred
only in normal soil and the rate of breakdown was logarithmic. They
concluded that it was biologically degraded. Complete decomposition
took ten to thirty days.
Chin et aZ~ (1970) studied its degradation in water and soil.
The experiment indicated that the main change in water was oxidation
to its sulfoxide. The oxidation rate was found to be retarded by
higher pH. At pH's of 2 and 4, further but slower oxidation to the
sulfone was detected. Hydolysis was not detected under any of these
conditions. The rapid decrease in activity of Carboxin in soil was
due to its quick oxidation to the less active sulfoxide. In this
experiment, neither hydrolysis nor further oxidation to sulfone were
detected by thin layer chromatography. In a separate experiment,
sterilized soil was extracted seven days after fortification and
standing at room temperature. About 20% of Carboxin was oxidized to
18
the sulfoxide. This would indicate that microbial activity is not an
essential factor for its degradation.
MICROBIAL DEGRADATION
Microbial breakdown of pesticides have had several reviews
(Ahmed and Casida, 1958; Audus, 1960; Alexander, .1966; Kearney, 1966;
Alexander, 1967; Kearney et aZ~ 1967; Kearney and Helling, 1969;
Kaufman, 1970; and Kaufman and Kearney 1970). The mechanisms proposed
. by which microorganisms develop the capability for pesticide degrada-
tion involve mutation, adaptation and constitutive enzyme systems. Most
pesticide degrading soil microorganisms have been isolated from soil
by enrichment culture technique. Free of soil environment, microbial
reaètions can be studied in detail at cellular and enzyme levels.
Kaufman (1970), Kearney and Helling (1969) discussed the princi-
pal microbial reactions associated with pesticide decomposition by
soil microorganisms. These reactions include dehalogenation, dealkyl-
ation, amide or ester hydrolysis, oxidation, reduction, ether fission,
aromatic ring hydroxylation and ring cleavage. Many of these reactions
have been demonstrated as occurring in soils but most of the informa-
tion has been obtained through pure culture technique.
In his review, Kaufman (1970) also discussed the biodegradation
of pesticide combinations. He stated that several interactions of
pesticide combinations affected pesticide metabolism in the soil by
19
microorganisms. For instance, the microbial degradation of the her-
bicide Dalapon was found to be inhibited in the presence of a second
herbicide Amitrole.
Microbial degradation of the ph~nylcarbamate herbicide ClPC is
inhibited by residues of certain methyl carbamates. Kaufman et aZ~
(1970) stated that Carbaryl and Baygon were among the methyl carbam-
ates that increased the persistence of ClPC. Soil pH, soil type,
time of treatment and methylcarbamate concentrations did not signifi-
cantly affect the interactions. Kinetic studies reveâled that methyl-
carbamates were competitive inhibitors of thephenylcarbamate hydrol-
ysing enzyme. Kaufman et aZ~ (1971) also showed that PCMC, a methyl-
carbamate, retarded the degradation of Propanil in soil and greatly
reduced the formation of 3,3',4,4'-tetrachloroazobenzene (TCAB).
A synergistic relationship between two organisms in the degrada-
tion of a pesticide which neither can achieve by itself was reported
by Gunner and Zuckerman (1968). When Arthrobaater and Streptomyaes
were incribated separately, the pyrymidyl ring of Diazinon was not
attacked. When, however, Arthrobaater and Streptomyaes were incribated
together radical changes in the Diazinon molecule were evident. The
pyrimidyl ring was cleaved and two metabolites were observed.
The very slow degradation of some compounds might result from a
Change in penneability of the organism to the substrate or might be
( attributable to steric hindrance by the sribstrate molecule affecting 1
20
the active enzyme. A pesticide may not be capable of inducing form-
ation of the enzyme(s) appropriate for detoxification if the chemical
is of very low solubility, or low concentration or impermeable to the
cell (Alexander, 1968).
The resistance of certain organic mole cules to microbia1 attack
may be related to their specifie structural characteristics. Many
compounds resistant to microbia1 degradation are derivatives of sub-
strates which are decomposed rapidly by numerous bacteria e.g.
benzoic acid is readi1y degraded, while the herbicida1 ch10rinated
derivative 2,3,6-trich10robenzoic acid is very resistant to attack
(Alexander and Lustigman, 1966). The type of substitution and the
number and position of the substituents on the molecule are important
factors in determining the resistance of the mo1ecule to degradation.
The relation of structure to pesticide decomposition is discussed
by Kearney and Plimmer (1970).
Another point that should be taken into consideration is that the
environment may render the substrate inaccessible to the microbia1
decomposers, thereby effectively preventing decomposition. The com-
pound may be deposited in a micro-environment or adsorbed to soil
colloids where it would be protected from microbia1 attack (Alexander,
1965) •
The bio1ogica1 decomposition of Carbaryl can occur by hydrolysis
... ' ", >
21
î '<.. •.. 1
of the carbamate ester· group fo11owed by degradation of the hydro-
1ysed fragments (Casida,1963), hydroxy1ation of the insecticide
mo1ecu1e (Baron et aZ.~ 1969; Dorough et aZ.~ 1963; and Oonnithan and
Casida, 1968) or conjugation of the original or transf6rmed mo1ecu1e
(Kuhr and Casida, 1967; Knaak et aZ.~ 1968; Paulson et aZ.~ 1970).
These studies were carried out in vivo with plants, insects, mamma1s,
or with in vitro studies of enzyme systems iso1ated from insects and
mamma1s. Bo11ag and Liu (1971) described the decomposition of Carbary1
with severa1 microbia1 iso1ates from the soi1. It was estab1ished
that aIl iso1ated microorganisms hydro1ysed Carbary1 to 1-naphtho1. A
fungus identified as Fusarium soZani a1tered 1-naphtho1 rapid1y, where-
as one bacterium, a gram-negative coccus degraded the hydrolysis product
gradua11y and a third iso1ate accumu1ated it under certain conditions.
Mixed cultures of the iso1ates were very effective in degrading Carbary1
as weIl as 1-naphtho1 and this suggests that complete biodegradation is
performed by combined growth.
Liu and Bol1ag (1971) iso1ated a fungus from soi1, GZioeZadium
roseum, which metabo1ized Carbary1, through a non-hydrolytic mechanism
to three metabo1ites, 1-naphtho1 N-hydroxymethy1carbamate, 4-hydroxy-1-
naphthy1 methylcarbamate, and 5-hydroxy-1-naphthy1 methy1carbamate.
This shows that there was hydroxy1ation of the side chain and a1so
hydroxy1ation of the aromatic ring. These are important detoxication
reactions. Bo11ag and Liu (1972) described the capacity of severa1
other soil fungi to hydroxy1ate Carbary1 (side chain and ring hydroxy-
lation)in the manner described above. The detoxication mechanism
seems to be widespread among fungi. Carbaryl metabolism in plant
insects and animaIs also involve the formation of these hydroxyl-
ated metabolites (Dorough; 1970; Kuhr, 1970).
Pelletier (1972) isolated a pseudomonad organism which attacked
the side chain of Carbaryl. He also isolated another pseudomonad 14C-l-
organism which metabolized}-naphthol,60 - 70% of the radioactive
carbon 14C going to 14C02 through salicylic acid and 30 - 40% of 14C
formed three metabolites, one of which ,vas 1,4-dihydroxynaphthalene.
When the two cultures were mixed and the substrate was Carbaryl,
there was evidence of complete degradation of the compound to C02 and
cellular constituents.
The microbial degradation of Elocron has been accomplished 'vith
a pseudomonad organism. It is believed that the aromatic ring is
broken and there is evolution of C02 (Levac, 1972).
So far in the literature there have been no reports of microbial
degradation of Baygon and Dasanit. As reported before in this review,
Edginton and Corke (1967) stated that the breakdown of Vitavax is
microbial but Chin et aZ~ (1970), believe that microorganisms are not
essential for its degradation.
Severa1 attempts have been made in th~s study, using enrichment
culture tecllniques, to microbia11y degrade Ch10rfenvinphos. Un for-
22
(
l \
.23
tunately, they have aIl proven futile. In the literature there are
no reports of microbial degradation.
The primary requirement for a modern pesticide is effective con-
trol of the target organism. The second requirement is that a pesti-
cide should degrade to ecologically acceptable products in a reasonable
period of time. Of a1l the systems operating in the natural environ-
ment, the greatest potential for detoxification appears to exist in
the soils. However, further studies are required on:
(1) Elucidation of metabolic pathways;
(2) Studies at the biochemical and enzyme level to determine which
specific linkages are being attacked;
(3) The resultant fate and reactions in soil and water of metabolites;
(4) The actual degradative agent and the influential environmental
factors;
(5) The relation of molecular structure to pesticide decomposition.
Broduce,compounès that are more susceptible to degrâdation;
(6) The effects of·pesticides and their metabolites on microbial
activities in soil and water (the main area of research in
this thesis).
24
PAR T 1.
THE EFFECT OF SELECTED PESTICIDES ON MICROORGANISMS IN
TERRESTRIAL ENVIRONMENTS
,r-
(
25
l N T R 0 DUC T ION
and
LITERATURE REVIEW
Normally, the quantities of the common herbicides, fungicides,
nematicides and insecticides applied to soils or which reach soil
from above-ground spraying operations are not sufficient to have per-
manent deleterious effects on activities of soil microorganisms
(4udus, 1964; EolIen, 1961). If there is any selective inhibition
or elimination of species, this is compensated by the survival of
other more resistant and equally potent species. This is part of the
'biological buffering capacity' of the soils and therefore the meta-
bolic integrity of the soil is maintained (Domsch, 1964; Domsch, 1970).
However, this does not preclude the potential danger of toxic
pesticide degrad2'tion products accumulating in the environment.
Higher application rates of some pesticides have been shawn to be toxic
to some forms of soil microorganisms (Hale et aZ.~ 1~57; Bollen, 1961;
and Breazeale and Camper, 1970).
OrganoahZorine aompounds
For many years, the organochlorine compotmds were used exten-
sively for insect control. Severa! studies (Smith and Wenzel, 1948;
(
26
Eno and Everett, 1958; Martin et aZ.~ 1959; and Bollen, 1961),
indicated that these materials had little effect on microbial acti-
vities in the soil. In an excellent review on the persistence and
behaviour of soil insecticides, Barris (1970) reported that Tu
conducted studies on the influence of Dieldrin (2,000 ppm) on soil
microorganisms and little effect was apparent on either fungi or
bacteria. Similar results were obtained with other cyclodiene
insecticides.
~ganophosphate compounds
Tu (1970 and 1972) examined the effects of five organophosphor-
ous pesticides on microbial activities in the soil: Bayer 37289,
Diazinon, Dursban, Zinophos and Dasanit. The first four pesticides
were applied at rates of 10 and· 100 ~g/g and Dasanit at 1 and 5 ~g/g
of sandy loam. Bacterial and fungal counts were temporarily decreased
for 1 - 2 weeks but population levels recovered rapidly to levels
similar to that of the control. Ammonium production from added pep-
tone-N increased in treated soils. Nitrate production was about equal
to the control after two weeks of incubation in the tceated soils.
Sulfur oxidation was equal to or better than that obtained with un-
treated soil in most cases. The higher 02 consumption from the decom-
position of native organic matter was greater in treated soils than
in the controls. In addition, 02 consumption increased with increasing
concentration of pesticide both in soils with and without supplemental
glucose, suggesting the possibilities of microbial degradation of the
i i...
insecticiges or their degradation products and of uncoupli~g oxida-
tive phosphorylation.
Sideropoulos et aZ.~ (l963) also reported on work done with some
organophosphate insecticides, Systox, Thimet, Malathion and Diazinon,
at 5 - 25 lbs/acre in the field. With the exception of Diazinon,
whichincreased bacterial numbers, there was no stimulatory or inhib-
itory effect of the other pesticides on fungal, Streptomyces and
bacterial counts. Laboratory studies were also performed using soil
from the field plots. Insecticide concentrations of 5 - 1,500 ppm
were used. Fungi and Streptomyces counts were not affected by pesti-
cide concentration. Bacterial counts increased with increasing
concentrations above 40 ppm, Diazinon giving much greater stimulation.
Diazinon also stimulated C02 4production and the C02 evolution propor-
tional to the amount of insecticide used.
Bartha, et aZ.~ (1967) found that the organophosphates, Malathion,
Parathion and Thimet caused an initial increase and subsequent decrease
on C02 production in the soil; the magnitude of the effect was related
to the concentration applied (150 and 1,500 ppm). At 150 ppm nitri
fication was depressed on the 6t~, 12th and lSth day. Organophosphates
are not chemically or biologically stable. They undergo degradation
in.soil possibly increasing respiration (Sideropoulos, 1963; Tu, 1972)
and bacterial numbe~s.
27
28
Funke et aZ.~ (1970) studied the effect of some organophosphates
on nitrification. Some inhibition at 500 ppm occurred with Dyfonate
Bayer 68138, Bayer 37289 and Dursban. At 500 ppm Dursban was phyto-
toxic and inhibited the growth of a1fa1fa and sweet c10ver in plastic
growth pouches. Bayer 37289, Trich1orofon, Bayer 68138 and Dasanit
were most toxic to severa1 pure cultures of Rhizobium. Dursban,
Carbophenothion and Diazinon were 1ess toxic to the Rhizobium spp.
MethyZeaPbamate eompounds
There was strong inhibition of nitrification with temporary
accumulation of nitrites with the methy1carbamates Temik and Baygon
at 500 ppm (Funke et aZ.~ 1970). The nitrifier population essentia11y
recovered by 30 days. At·5 and 50 ppm, there was on1y slight inhibi-
tion. Furadan, on the other hand did not inhibit nitrification
strong1y at any concentration studied. The three carbamates, Baygon,
Temik and Furadan strong1y inhibited growth of a1fa1fa and sweet
c10ver in plastic growth pouches at 50 and 500 ppm. Baygon and Temik
were the most toxic te Rhizobium spp.
Funke et aZ.~ (1972) showed that pure cultures of Nitrosomonas
euzoopea were strong1y inhibited by Baygon and Temik. Niti>obaeter>
agiZis was moderate1y inhibited at high concentrations but on1y
slight1y and for a few days at 10w concentrations. 02 uptake for 12
hours as affected by 500 ppm Baygon showed high activity, apparent1y
due to the metabolism of the "dry carrier': Temik experimenta1 resu1ts
29
differed 1itt1e from controIs. Pesticide residues remaining after
30 days were approximate1y 95% of Baygon and 50% of Temik.
l'wo methy1carbamates, Iso1an and Carbary1 at 150 ppm and 1,500
ppm, depressed C02 production and nitrification. These effects per-
sisted for at least 18 days with nitrate production and 30 days with
C02 production (Bartha et aZ. 3 1967).
Oxygen consumption from decomposition of native organic matter
was greater in soils treated with Carbofuran, a methylcarbamate, at 1
and 5 ~g/m1 of a sandy loam (Tu, 1972). Oxygen consumption increased
with increasing amount of pesticide. Ammonium production from added
peptone was slightly increased, nitrate production was better after
2 weeks of incubation and there was a slight but significant decrease
in su1fur oxidation. Bacteria1 and funga1 counts were depressed for
1 and 2 weeks respectively and recovered.
.30
MATERIALS ~d METHODS
A. THE EFFECT OF SELECTED PESTICIDES ON SOIL BACTERIA &~D FUNGI
The pesticides Baygon ~d Dasanit, obtained·from Chemagro Cor-. .
poration; Carbaryl from Union Carbide Chemical Company; Carboxin
from UniRoyal Agricultural Chemicals Division; Chlorfenvinphos from
Shell Chemicals and Elocron from Ciba-Geigy Canada Ltd., were applied
at recornmended rate, 2 x recommended rate ~d 4 x recommended rate,·
to the soil in field plots (8' x 8'). The plots were located at
Macdonald College and the soil classified as a Chateauguay Clay loam:
Organic matter 5.2%
pH 7.3
Water holding capacity 56.7%
Table 1 summarizes the mode of application of the pesticides to
the plots ~d Figure 1 gives the chemical name and structural formula.
Plots treated with 2 x ~d 4·x recommended dose were applied in
the manner described for the re~ommended dose while three control
plots were sprayed with 4 litres of water.
BACTERIAL AND FUNGAL COUNTS
Samplings were carried out ·on. the 2nd , 4th , 8th and 16th days.
Eight samples were taken· with a core sampler (1 1/2" cIiameter) to a
'fABLE 1.
MPDE OF APPLICATION OF PESTICIDES TO PLOTS
Recommended.dose Pesticide Formation Mode of application
per acre per plot
Baygon 50% wettable powder 10 lbs 6.6 g mixed in 4 litres water
Carbaryl 85% wettable powder 5.9 lbs 3.9 g " Il " Il "
Carboxin 75% wettable powder 6.7 lbs 4.4 g Il Il Il Il Il
Chlorfenvinphos 25% wettable powder 20 lbs 13.2 g Il with soil*
Dasanit 15% granular 33.3 lbs 22 Il Il Il * g
Elocron technical 5 lbs 3.2 g Il in 4 litres water
* 4 litres of water were sprayed on the plot after application of the pesticide.
"
lA> 1-'
32
FIGURE 1.
Figure 1. Chemical names and structural formulae of Dasanit,
Elocron, I-Naphthol, Carbaryl, Chlorfenvinphos,
Carboxin and Baygon.
1 /
/
DASANIT
. O,O-Diethyl O-[p-(methylsulfinyl)phenyl]
phosphorothioate
ELOCRON
2-(1,3-dioxolane-2-yl)-phenyl-N-methylcarbamate
OCONHCHa
~J
I-NAPHTHOL
OH
· CARBARYL
I-naphthyl methylcarbamate
CHLORFENVINPHOS
2-chloro-l-(2,4-dichlorophenyl)vinyl diethyl phosphate
CARBOXIN
5,6-dihydro-2-methyl-l,4-oxatniin-3-carboxanilide
,-
!.
SAYGON
O-Isopropoxyphenyl methylcarbamate
o H Il 1
O-C-N-CH / 3
<0> /CH3 o -O-CH
'CH 3
depth of 6" from each plot. The samples from each plot were mixed
together and homogenized. Ten grams of the homogenized soil were
added to 90 ml of sterile water and glass beads. After vigorous
shaking of the soil suspension for 2 - 3 minutes, suitable dilutions
were made for plating in quintuplicate. For total counts, soil ex
tract agar (Lochhead, 1940) was used; the fungal count was made in
Rose-bengal streptomycin agar (Martin, 1950).
From the final dilution of the soil suspension as prepared for
the total bacterial count, 20 ml of the freshly agitated suspension
were transferred to a sterile test-tube which was then immersed in
a water bath maintained at 85°C for 10 minutes (Clark, 1965). One
millilitre of the suspension was plated in soil extract agar to
determine the sporeforming bacterial counts.
The incubation time and temperature for the total bacterial,spore
forming bacterial and fungal counts were 3 days and 25°C.
l'wo 10 g portions of each--plot sample were dried in an oven at
110°C and weighed to obtain the dry weight.
}IEDIA
(1) Soil-extract agar (Lochhead, 1940)
20 g
0.5 g
Dextrose 0.1 g
Soil extract 1000 ml
33
( \
The pH was adjusted to 7.0 with either dilute HCl or dilute NaOH.
The medium was then autoclaved at 15 pounds pressure for 15 minutes.
The soil extract was prepared by mixing 1 kg of fertile soil
with 1.5 litre of water, and the mixture autoclaved for 30 minutes at
l5 pounds per square inch. After partial cooling and settling, the
supernatant suspension was filtered in a Buchner funnel through medium-
grade filter paper.
(2) Rose-bengal streptomycin agar (Martin 1950)
Thirty-six grams of Bacto Cooke Rose-bengal agar (Difco) were
dissolved in 1000 ml of water and autoclaved (15 pounds pressure for
15 minutes). Streptomycin at a concentration of 0.3 g in 100 ml of
water was sterilized by passing it through a bacteriological-grade,
sinterea-glass filter at room temperature. Prior to use, the agar
medium was melted and cooled to a temperature of 45°C and 1 ml of
sterile streptomycin solution was added to 100 ml of agar medium to
provide 30 ~g of streptomycin per ml of solution.
34
B. EFFECT OF SELECTED PESTICIDES ON SOIL RESPIRATION
The Gilson respirometer was used in the experiments to de termine
the effects of Baygon, Carboxin, Ch10rfenvinphos, Dasanit, E10cron
and 1-Naphtho1 on soi1 respiration. The formulations of the pesti
cides used were as fo110ws:
Baygon
Carboxin
Ch10rfenvinphos
Dasanit
E10cron
1-Naphtho1
Ana1ytica1 grade 99.2%
75 Wrecrysta11ized from ethano1 and water
Technica1 grade 92%
Ana1ytica1 grade 94.5%
Technica1 grade recrysta11ized from 20%
a1coho1 in water
Recrysta11ized grade III Sigma
The theory and detai1s of respirometry techniques are described by
Umbreit et al.~ (1972).
Fresh Chateauguay clay 10am from Macdonald Co11ege was air-dried
for 2 days and sieved so as to co11ect the 0.8 - 1 mm fraction. Four
grams of the 0.8 - 1.0 mm fraction were added to the main chamber of
each Gilson respirometer f1ask. The appropriate dilution of each
pesticide solution or suspension was prepared so that the final desired
pesticide concentration inthe soi1 cou1d be achieved by adding 1.2 ml
of f1uid to bring the moisture content of each samp1e to 60% water
holding capacity. In the control f1asks on1y water was added to bring
35
the soil to water holding capacity. The rates of application of
pesticides were 25 and 250 ppm. Water was also added to the center
weIl of each flask to help maintain the relative humidity of the at
mosphere in the vessel and 1 ml of a 5% KOH solution placed in the
side arm of each flask for C02 absorption.
Each flask containing a particular treatment was randomly attached
to a manometer and lowered in a water bath set at 25°C. The stopcocks
were left open and the flasks allowed to equilibrate for about 1/2
hour. The stopcocks were then closed and readings, taken at the be
ginning and end of a 6-hour period for each day, we~e used to.calculate
the rate of oxygen uptake expressed in micro-litres oxygen per gram of
air-dried soil per hour. After the final reading for the day was
taken, the stopcocks were le ft open until the beginning of the next
initial reading. Readings were taken for 8 days. Each pesticide
treatment and the control were in triplicate.
36
37
C. EFFECT OF SELECTED PESTICIDES ON SaIL NITRIFICATION
The soil perfusion apparatus as described by Lees and Quastel
(1946) and Chase (1948) was employed to see the effects of Baygon,
Carboxin, Chlorfenvinphos, Dasanit, Elocron, and l-Naphthol on
nitrification in the soil. The formulations of the pesticides used
were the same as described in Section B, Materials and Methods, Part I.
Each unit contained 50 g of air-dried, 2 - 4 mm fraction, Chateauguay
clay loam in the column and 200 ml solution in the reservoir. The
100 pg/ml ammonium-N added in the form of (NH4)2S04 to each unit was
based on the 200 ml solution in the reservoir. The various concen-
trations of pesticides (25, 125, 250 and 500 ppm) added to the units
were based on the weight of 50 g air-dried soil in the column. Each
treatment was applied to duplicate perfusion units.
Soilcolumns were percolated continuously with the solutions.
Perfusates were analyzed periodically for nitrite-N and nitrate-No
Nitrite-N was determined colorimetrically by the sulfanilic acid-a-
naphthylamine method (Standard Methods, 1965) and nitrate-N by the
phenoldisulphonic acid method (Chase, 1948).
( -
RESULTS and DIS C U S S ION
A. THE EFFECT OF SELECTED PESTICIDES ON SOIL BACTERIA AND FUNGI
Dasanit
The two organophosphate pesticides showed no permanent deleter
ious effects on the total numbers of bacteria, sporeforming bacteria
and fungi. Tu (1972) showed that Dasanit at concentrations of 1 and
5 ppm significantly decreased the bacterial population which even
tually recovered. Figure 2 shows that Dasanit slightly depressed
the numbers of bacteria two to three times less than the control on
the 8th day after spraying. Recovery occurred by the l6th day.
The highest concentration of Dasanit stimulated the numbers of spore
formers. This probably means that Dasanit decreases the numbers of
non-sporeformers and therefore allows the sporeformers to grow at an
increased rate. This is a phenomenon that often follows partial
sterilization of the soil and the increase could be attributed to
increased metabolic activity of the surviving population. With the
fungi, Dasanit showed some indication of depressing the numbers with
a maximum on the 4th day and recovery by the 8th day. Tu (1972)
also observed a reduction of the number of fungi by Dasanit.
ChZol'fenvinphos
Chlorfenvinphos (Figure· 3) aIso depressed the numbers of
bacteria two to three times less than the control on the 8th day.
38
Recovery occurred by the l6th day. The highest concentration of
Chlorfenvinphos increased the numbers of bacterial sporeformers.
Again it seems as though the high concentrations reduced the numbers
of non-sporeformers allowing the sporeformers to increase in numbers.
The high concentration of Chlorfenvinphos slightly depressed the
numbers of fungi at the 4th and 8th days but recovery was made by
the l6th day.
Baygon and CaPbaPuZ
The methyl carbamates, Baygon and Carbaryl, tend to reduce
39
total bacteria, sporeformers and fungi (Figures 4.and5).,All,poplllations
subsequently recovered to the levels of the control by the l6th day.
Bartha et aZ.~ (1967) found that Carbaryl decreased the C02 produced
from the soil, thus supporting the data obtained in these experi-
ments.
EZoa!'on
Elocron (Figure 6) increased the numbers of total bacteria and
sporeformers for the first 4 days after which there was a reduction
in the populations which subsequently recovered to levels of the
control. Elocron stimulated the bacteria andsporeformers by acting
as a metabolizable substrate. The subsequent decrease might be due
to toxic metabolites accumulating. As shown later, a pseudomonad
isolated from this soil in pure culture produced metabolites from
Elocron that were toxic to the nitrifying organisms while Elocron
40
itself was not. Mold counts were slightly increased by the second
highest concentration of Elocron (10 lbs/acre) but the results
were not consistent.
Ca1'boxin
The data presented (Figure 7 ) shows that there was a trend
towards a reduction of the total numbers of bacteria, the inhibition
was more marked with increasing concentrations of Carboxin. The
trend obtained with the sporeformers indicates a stimulatïon of
sporeformers. This could mean that Carboxin is toxic to non-spore-
formers allowing the sporeformers to increase in numbers. The effects
disappeared by the l6th day when the populations of the control and
treatments were about the same. With the fungi, there was a slight
tendency to decrease the numbers. One would expect this since Car-
boxin is a fungicide, however, it is effective against certain patho-
genic fungi (UniRoyal Technical Data Sheet No. 3).
Reported resu1ts of experiments in which growth and metabolic
activity of soi1 bacteria in situ has been investigated following
organochlorine insecticides app1ication,range, from description of
increased activity (Fletcher and Bollen, 1954) or of a total lack of
effect (Eno and Everett, 1958) to inhibition of growth and metabo1ic
activity (Pramer and Bartha, 1968). The data presented above for
other types of pesticides a1so show inconsistencies as increasing
concentrations did not al ways correlate with increasing or decreasing
population of microorganisms.
41
More consistent resu1ts have been obtained with pure cultures
of microorganisms growing on synthe tic media using other pesticides
than those under investigation. Organoch10rine insecticides are
inhibitory to the growth of gram-positive bacteria (Gray and Rogers,
1955; Duda, 1958; Trudgi11 et al., 1971). A range of sensitivity
has been observed; Bacillus subtilis was inhibited by 10wer Ch10r-
dane concentrations than were required to produce simi1ar resu1ts
with Sareina lutea and Streptococcus faecalis. Effects observed
with Ch10rdane-treated Bacillus subtilis inc1ude cessation of growth,
inhibition and eventua1 cessation of respiration, 10ss of viabi1ity
coup1ed with a de1ayed re1ease of po1ymeric mole cules from within
the bacteria (Trudgi11 et al., 1971). Inhibition of who1e ce Il res-
piration is para11e1ed by inhibition of e1ectron transport capabi1ity
of subcellular membrane fragments. Disruption of this and other mem-
brane and mesosome associated phenomena has been postu1ated as being
the primary cause of cessation of growth and loss of viabi1ity (Widdus
et al., 1971).
Future research a10ng the 1ines discussed in the above paragraph
with Ch10rdane should be considered with the pesticides under dis-
cussion but it must be remembered that pure culture studies and use
of artificia1 media yie1d desirable information but the results may
not always be projected with certainty to the field where the soi1 as
a medium, the environment, and the population as a who1e, are exceed-
ing1y comp1ex and variable.
42
\j
F, l G URE 2.
( \
,J
Figure 2. The effects of Dasanit on bacteria1 and funga1 popu1a-
tions in the soi1 over a period of 16 days.
Applications were at Field Rate (F.R.) of 5 lbs/acre
(2.5 ppm) , 2 x Field Rate and 4 x Field Rate.
\ 1
------.-----
8 10
DASANIT
16
- -. ~
• CONTROL o lx F.R. a 2x F.-R. A 4xF.R.
Figure 3. The effects of Ch1orfenvinphos on bacteria1 and funga1
populations in the soi1 over a period of 16 days.
Applications were at Field Rate (F.R.) of 5 lbs/acre
(2.5 ppm) , 2 x Field Rate and 4 x Field Rate.
)
i '- :
1 2 4 8
DAYS
CHLORFENVINPHOS
12 16
.. -- _. __ ._-_ .. _ .. ,._ ....• __ ... _. __ .. --."
, e CONTROL, o IxF.R. Il 2x F.R . ..,4xF.R.
, ~1
Figure 4. The effects of Baygon on bacterial and fungal popula
tions in the soil over a period of 16 days.
Applications were at Field Rate (F.R.) of 5 lbs/acre
(2.5 ppm), 2 x Field Rate and 4 x Field Rate.
SAYGON
DAYS
• CONTROL o 1 x F.R. Il 2 x F.R. ~ 4x F.R.
Figure 5. The effects of Carbaryl on bacterial and fungal popula
tions in the. soil over a period of 16 days.
Applications were at Field Rate (F.R.) of 5 lbs/acre.
(2.5 ppm), 2 x Field Rate and 4 x Field Rate.
12 4 8
DAYS
CARBARYL
12 16·
• CONTROL o IxF.R. -.2xF.R. Â 4xl~R.
Figure 6. The effects of Elocron on bacterial and fungal popula
tions in the soil over a period of 16 days.
Applications were at Field Rate (F.R.) of 5 lbs/acre
(2.5 ppm) , 2 x Field Rate and 4 x Field Rate.
.. '
ELOCRON
--,---~~~ 20 "s
, . DAYS
• CONTROL o IxF.R . • 2xF.R. Â 4xF.R.
Figure 7. The effects of Carboxin on bacteria1 and funga1 popula
tions in the sail over a period of 16 days.
Applications were at Field Rate (F.R.) of 5 lbs/acre
(2.5 ppm), 2 x Field Rate and 4 x Field Rate.
)
CARBOXIN
<t ; \/ ffi 101
-CONTROL .... o IxF.R.
0 <t
.. 2x F.R. a)
l.I.. 106
A 4xF.R. 0 ci Z
105
1 2 4'
DAYS
48
B. THE EFFECTS OF SELECTED PESTICIDES ON SOIL RESPIRATION
Dai1y rates of 02 uptake were determined and recorded as micro-
litres of 02/g air-dried soi1/hour. The percentage change in the rate
of soi1 respiration in the treated soi1 compared to the control was
then p10tted against time. A11 six pesticides at both concentrations
of 25 and 250 ppm showed an increase in the rate of oxygen uptake in
the treated soils over the control (Figures 8 - 13).
Baygon and EZoapon
The rate of 02 uptake of the soi1 treated with the two methy1car-
bamates, Baygon and E1ocron showed the most marked increasé with
increasing concentrations of pesticide (Figures 8. and 9.) .Possib1ëexplana-
tions for such behaviour are:
(1) Bio1ogica1 degradation of E1ocron and Baygon by indigenous soi1
microorganisms. From this soi1 severa1 bacteria which utilized E1ocron
as sole carbon source were iso1ated. A pseudomonad grew in pure culture
in minimal medium with E1ocron (as low as 100 llg/m1) as the sole carbon
source (Levac, 1972). No attempt was made to iso1ate any organisms
which uti1ized Baygon.
(2) Another exp1anation for the increase in the rate of 02 consumption
is the uncoup1ing of oxidative phosphorY1ation by the compounds or the
metabolites. With Baygon this seems to be a possibi1ity, for the
results obtained resemb1ed that of the effect on soi1 of 2,4-dinitro-
, '-
49
phenol which is known to uncouple oxidative phosphorylation (Barth a
et aZ.~ 1967). Unfortunately these experiments could not be contin-
ued for 30 days as Bartha et aZ.~ (1967) did (because of growth of
seedlings in the flask) to see if a depression in respiration would
have resulted after the 8th and 9th day but the results indicate a
peak of 02 uptake followed by a decrease towards the contrCillevel.
2,4-Dinitrophenol is considered to function as an uncoupler of oxi-
dative phosphorylation. Stimulation of oxygen utilization and inhib-
ition of 32p incorporation into ATP has been observed in plants.
Plant tissues treated with 2,4-dinitrophenol are rapidly depleted of
their carbohydrate reserves, apparently by the increased rate of
respiration induced by the herbicide (Moreland, 1967).
The dinitrophenols also un coup le oxidative phosphorylation in
aerobic soi1 microorganisms (Butt and Lees, 1960), and the observed
rate of 02 uptake in dinitrophenol-treated soil might be explained
on this basis. Stevenson and Katznelson (1958) observed oxygen up-
take in soi1 containing acetate was enhanced by the addition of dinitro-
phenol.
The graph obtained with Elocron-treated soil does not entirely
resemble that for 2,4-dinitrophenol-treated soi1 since at 8 days the
rate of 02 consumption is still increasing while at 8 or 9 days un-
coupling of oxidative phosphorylation would cause a decrease in soi1
respiration due to the lack of ATP in the microorganisms and their
eventual death. This observation encourages the conclusion that 02
uptake increased as a result of the microbial degradation of Elo-
cron.
(3) A third explanationis that the pesticides or their metabolites
are being used as electron acceptors, passing the electrons to 02
and thereby by-passing the respiratory chain and increasing the rate
of 02 consumption, thus acting in a similar way to menadione.
(4) The fourth possibility could be one of oxidation of the chemical
itself, or of its breakdown products. With methylcarbamates it is
more likely that there is first hydrolysis of the original compound
followed by oxidation of the metabolites. For instance with Baygon,
o-isopropoxyphenol (Aly and El-Dib, 1971) is formed which would then
be oxidized. From Elocron also is produced a substituted phenol (Pape
et aZ.~ 1970) which may also be-subject to oxidation.
ChZorfenvinphos and Dasanit
The results for the organophosphates, Chlorfenvinphos and Dasanit
treated soils are illustrated in Figures 10 and Il. Explanations
for such increases could be explained along the same lin es as with
the methylcarbamate-treated soils.
50
The chances that 02 consumption increased due to microbial attack
on Chlorfenvinphos are slight for several attempts were made to iso
late organisms which could degrade Chlorfenvinphos but all proved futile.
51
, t .....
Oxidation of Chlorfenvinphos is not part of the breakdown path-
way proposed by Beynon and Wright (1967) and such an explanation for
the rate of 02 increaSe is probably not valide
The other possibility involves uncoupling of oxidation phosphory-
lation as was discussed with the methylcarbamates. The curves obtained
with Chlorfenvinphos (Figure la) do not show any difference between
concentrations. The reason for this could be that 25 ppm were ade-
quate to uncouple oxidative phosphorylation to maximum effect.
The results obtained with Dasanit-treated soils showed increasing
O2 uptake with increasing concentrations (Figure rI) as found by Tu
(1972). These results can be explained considering the same four
possibilities as discussed wove for the methylcarbamates. The
shapes of the curves for the Dasanit-treated soils are somewhat similar
to the curves obtained for 2,4-dinitrophenol and so the compound or
its metabolites may act as uncouplers of oxidative phosphorylation or
as electron acceptors passing the electron on to 02.
There is also the possibility of the oxidation of Dasanit to the
Dasanit oxygen analogue, Dasanit sulfone and the Dasanit oxygen anal-
ogue sulfone. These metabolites were detected in cotton plants by
Katague and Anderson (1967), corn, grass and milk by Bowman and Hill
(1971) •
52
CCIl.'boxin
Carboxin-treated soi1s consumed 02 in a simi1ar manner to Ch10r-
fenvinphos-treated soi1s (Figure 12) i.e. there was an increased rate
of 02 consumption over the control but litt1e difference between the
two concentrations. One can again specu1ate on the uncoupling of
oxidative phosphorylation being responsible for the increased;'rate.'of
soi1 respiration and that 25 ppm of Carboxin is enough to cause the
maximum uncoup1ing. The structure of Carboxin involves a benzene ring
to which a nitrogen atom is attached and bears a resemb1ance to the
structure of dinitropheno1.
No attempt was made to iso1ate organisms that degraded earboxin
in this experiment but Edgington and Corke (1967) studied its break-
down in normal and sterile soi1 and conc1uded that it was bio10gica11y
degraded. Also oxidation of the Carboxin cannot be~overlooked for
the rapid decrease in activity of Carboxin in soi1 was due to its
quick oxidation to a less active sulfoxide (Chin et aZ.~ 1970). Both
of these possibilities may cause an increase in the rate of 02 con-
sumption.
Z-NaphthoZ
1-Naphthol did not increase the rate of soil respiration as much
as the other compounds (Figure 13). Pseudomonads were iso1ated from
this soil which degraded l-Naphthol and so biological degradation of
1-Naphtho1 could exp1ain the increase in rate of 02 consumption of
53
the treated soi1. 1-Naphtho1 has a1so been shown to undergo photooxi-
dation, some of the products being 1,4-naphtholquinone and 2-, (or 3)-
hydroxy-l,4-naphtho1quinone (Lamberton et aZ.~ 1970). This would-also
increase the rate of 02 consumption. Other possibilities include
uncoupling of oxidative phosphorylation and l-Naphthol being used as
an electron acceptor.
The sensitivity of microorganisms to toxicants is under nutri-
tional and environmental con troIs which are influenced by an exogenous
supp~y of organic matter. The data indicate that indigenous soil
microorganisms can tolerate the chemicals but it must be noted that
the results are the sum total. of 02 exch~nge in the flask and there-
fore definite conclusions cannot be made. For example, the pesticides
may inhibit the growth and activity of one segment of the soil population,
therebY· enabling those microbes not affected by the pesticide to multi-
ply and reach a high population, supplanting the inhibited microflora.
The increased proliferation and activity of the non-inhibited microbes
coincide with the increased rate of 02 uptake.
, .'
Figure 8. The effects at 25 and 250 ppm of Baygon on soil respira-
tion rates over a period of 8 days.
z 0
. ,_.~ .. C)
~ CD
E ~ c.Q. Q.o 1010 {\lN
• 0
o V
. 0 •
01 1 0
/ / •
\ 1 0 •
\ \ 0 •
\ \ 0 •
\ \ 0 •
\ \ 0 .'
31\1~ NOI1\1~ldS3~ 110S NI V %
CI)
~ 0
Figure 9. The effects at 25 and 250 ppm of Elocron on soil
respiration rates over a period of 8 days.
z 0 0= 0 0 -1 w
e a 0.0. 0.0 lOlO (\IN
• 0
0 •
\ 1 0 •
\ 1 o~
•
0 •
1 \ 0 •
\ \ •
1 1 o~~
. 0
31"~ NOI1"~ldS3~ 110S NI V 0/0
·0 N J
<0
tO
CI)
~ ~o
ft)
N
Figure 10. The effects at 25 and 250 ppm of Chlorfenvinphos on
soil respiration rates over a period of 8 days.
Cf)
0 J: 0-Z 5= z w IL. a:: 0 .J J: 0
E 5. 0.0. 0. 0 lOlO C\JN o 0
o 10
0 0
\ / o •
X • 0
1 \ • 0
\/ • 0
\/ 00
\\ ., •
o 000 0 V ro C\J -
3!\1~ NOI!\1~ldS3~ liaS NI v 0/0
o -1 o C\J
1
Cf)
~ O·
ro
C\J
-
Figure Il. The effects at 25 and 250 ppm of Dasanit on soil
respiration rates over a period of 8 days.
"\,..,
l--2: <t Cf)
<t 0
E&. Q.o. Q.o
1010 (\IN
• 0
o Il)
G>
~ GO
f fi)
/ 0 •
I 1 o •
-/ 1 o •
\ \ O~\
o e.
o 0 o· 00 V ,.., C\I
3.L'O'~ NOI.L"~ldS·3~ 110S NI V 0/0
o -1
o .C\I
1
10
Cf)
V ~ 0
If)
Figure 12. The effects at 25 and 250 ppm of Carboxin on soi1
respiration rates over a period of 8 days.
)
2 -x 0 al a: <t ()
E a Q.Q. Q.o 1010 C\IC\I
• 0
o 10
ft 0
1 01 •
\/ o 0
y J.
1\ o e
\\ 0 •
\/ oe
\ ce
o 0 O· 0 0 q- ft) C\I
31V~ NOI1\f~ldS3~ 110S NI V °/0
. 0 . -
1 o C\I
1
Q)
1'-
(0
10
en V ?i
0
Figure 13. The effects at 25 and 250 ppm of I-naphtho1 on soi1
respiration rates over a period of 8 days.
/
-1 0 l: l-l: 0-<t Z
1 -
E E 0. 0.Q. 0.0 1010 NN • 0
o 10
o •
1 10
Il • 0
~ 00
~ CI>
A 00
1\ 0 •
\\ • o 0 0 0 0 v ft) (\J
31'1H NOI1'1~ldS3~ 110S NI V 0/0
o -• o N
1
.__ ._ •. 1~.-
C/)
~ 0
\
C. THE EFFECTS OF SELECTED PESTICIDES ON SOIL NITRIFICATION
Baygan
Figure 14 shows that Baygon has an inhibiting effect on soil
nitrification. For example, on the 5t h day there was 75% inhibition
at 500 ppm and 40% inhibition at 125 ppm. At 25 ppm there was only
a slight difference from the control (Table 2). By the l4th day,
recovery·.was evident at aIl concentrations of pesticide. Figure 14
and Table 2 also show a temporary accumulation of nitrite at aIl .con-
centrations whiCh is evidence of toxicity towards the Nitrobaater
group. The data presented above are supported by Funke et aZ.~ (1970).
EZoaron
Elocron applied at a concentration of 500 ppm did not affect soil and Table 3
nitrification (Figure 152. However, when 500 ppm of Elocron as the
sole carbon source in a basal medium was inoculated with a pseudomonad,
which fully utilized the Elocron, and the supernate used to treat the
soil in the perfuSion column, there was inhibition of nitrification
(Figure 16). The supernate therefore contained some metabolite that
was toxic to the nitrifying organisms.
ChZorfenvinphos . -",-.
Figure 17 shows that Chlorfenvinphos at a concentration of 500 ppm
did not have a marked effect on nitrification. On the 5th day there
was 35% inhibition at 500 ppm and virtually no inhibition at 25 ppm
60
(Table 4). There was a slight delay and temporary accumulation of
nitrite at 250 and 500 ppm of Chlorfenvinphos. This demonstrates that
Nitrobaater was only slightly inhibited at the higher concentrations.
By the l2 th and l3th day (Figure 17) no inhibition of nitrification
was observed.
Concentrations of 1,000 and 1,500 ppm were aIso used to treat
the soil and in addition to greater inhibition of nitrification there
was also greater production of N03-N (150 ppm).
Dasanit
At 500 ppm Dasanit inhibited nitrifi"cation (Figure 18 and Table
5) e.g. on the 7th day about 60% inhibition of nitrate production was
noted. At 25 ppm only slight inhibition occurred which increased
with increasing concentrations of Dasanit. A temporary accumulation
of nitrite and a delay in nitrite utilization was found at aIl pesti
cide levels.
Carboxin
Carboxin has an inhibitory effect on soil nitrification (Figure19
and Table 6). Inhibition was observed at 125, 250 and 500 ppm
ranging from 20% (125 ppm) to 60% (500 ppm). Table 6 displays a tem
porary accumulation of nitrite and a delay of nitrite utilization at
all levels of pesticide indicating an upset of the metabolic activity
of Nitrobaater.
61
i
'-'
62
Z-NaphthoZ
1-Naphtho1 was the most toxic of the chemica1s to the nitrifying
organisms. Figure 20 and Table 7 show that for the first 3 days no
nitrate was formed. On the 5th , 6th , 7th and 8th days, there was
inhibition ranging from 65 - 75% at 500ppm.and as much as 10% inhibi-
tion at 25 ppm. A temporary accumulation of nitrite and a delay in
nitrite utilization was found at aIl concentrations of the chemical
indicating an upset of Nitrobaatel' metabo!1sm. The deleteriou"~· effect
on the nitrifying organisms did not last for more than 11 to 12 days.
The data presented indicate that with the exception of Elocron
the pesticides had the ability to inhibit nitrification and this in-
hibition increased with increasing concentrations of the chemical. Also,
the ability to retard nitrification decreased with time suggesting that
the substances either underwent transformation and detoxification in
soil or there developed in soil a nitrifying population that was resis-
tant to their action.
Elocron was exceptional in that there was no affect on nitrifica-
tion even at 500 ppm, but unidentified metabolites produced when
Elocron was utilized by a pseudomonad did cause inhibition (Figure 16).
Previously Elocron was found to first stimulate the bacterial population
and then inhibit it. This phenomenon that toxic substances are produced
when a pseudomonad organism degrades Elocron is worth further investi-
gation.
Future research with these pesticides should include a study ta
determine the mode of action and on bath growth of cultures of Nitro
somonas and Nitrobacter and on the respiration of ce Il suspensions
and cell-free extracts. An investigation should also be made ta de
termine the mechanism of inhibition at the molecular level by examin
ing the· enzymes which are inhibited in these nitrifying organisms by
the pes ticides •
.63
Figure 14. The effects at 125 and 500 ppm of Baygon on
soil nitrification.
110
100
90
l1J 80 ~ en
::) l1. a: l1J 70 a. l1. 0 -E 60 a: w a. z 50 'IC) 0
~ ON 40 2 l1. 0 0»
30 ~
20
la
a 2·
SAYGON
--N03-N"
---- N02 -N
4
• CONTROL Â 125 PPM o500PPM
6 8 DAYS
la 12 14
65
t -.,
TA BLE 2.
The effect of various concentrations of Baygon on soil nitrification
N02-N ~g/ml of perfusate N03-N ~g/ml of perfusate
Day
Control 25 125 250 500 Control 25 125 250 500 ppm ppm ppm ppm ppm ppm ppm ppm
1 0.3 0.3 0.3 0.6 0.7 0.0 0.0 0.0 0.0 0.0
2 0.7 0.8 1.0 2.0 1.7 3.0 1.6 1.1 0.6 0.2
3 1.2 1.4 1.9 3.1 3.2 4.2 3.2 1.9 1.4 0.6
4 2.0 3.1 3.2 5.0 5.6 9.0 6.8 3.9 2.9 1.8
5 , 2.7 4.9 6.1 8.2 9.6 19.8 17.4 12.8 8.5 5.8
6 2.1 4.8 6.3 6.3 6.2 31.2 28.1 21.8 16.1 12.1
7 1.3 1.8 1.8 2.2 2.6 51.6 45.7 35.2 32.1 28.2
8 0.5 0.7 0.6 1.4 1.5 64.4 61.5 51.2 48.3 44.1
9 0.3 0.0 0.3 0.5 0.6 77 .1 69.8 67.7 61.4 58.2
10 0.0 0.0 0.0 0.0 82.9 77.3 76.1 74.8 72 .5
11 88.4 84.2 82.2 79.8 76.9
12 93.1 92.6 88.1 85.2 86.5
13 98.4 99.5 93.0 96.0 92.1
14 102.0 105.2 103.2 99.3 106.6
----------_.
66
FIG URE 15.
Figure 15. The effects at 500 ppm of Elocron on soil
nitrification.
-\..,
/ -1
, "
110
90
LU t( 80 (J) :J LL 0:: LIJ 0- 70 LL 0 -E 0:: 60 laJ 0-
z 1 50 ort)
~ ON Z 40 LL 0 CP =\ 30
20
10
o - 2 4
ELOCRON
NO -N 3
---- NO ~N 2 • CONTROL o TREATED
li 1/ 1 •
6 8 DAYS
- 0_0
~.-.
~'(/ ·f~~/ •
;,! •. : ...... ---. ,.
10 12 14
67
L,
TA BLE 3.
The effect of various concentrations of E10cron on soi1 nitrification
NQ2-N J.lg/ml of perfusate NOg-N J.lg/ml of perfusate
Day
Control 25 125 250 500 Control 25 125 250 500 ppm ppm ppm ppm ppm ppm ppm ppm
1 0.5 0.5 0.7 0.7 1.0 0.0 0.0 0.0 0.0 O'~O
2 0.9 0.8 1.0 1.3 1.8 2.5 2.1 2.0 1.8 1.5
3 2.0 2.2 2.2 2.3 2.5 3.2 2.8 2.6 2.6 2 .5
4 2.7 2.8 3.0 3.2 3.5 7.5 7.6 7.7 7.3 6.5
5 3.5 3.3 3.0 3.3 4.0 22.5 19.8 20.1 20.5 18.5
6 2.1 2.3 2.5 2.7 2.9 32.1 33.2 31.5 34.1 33.0
7 1.3 1.6 1.9 2.0 2.0 42.5 42.1 45.9 46.5 45.8
8 0.5 0.5 0.9 1.0 1.3 55.0 56.1 57.2 58.8 58.5
9 0.0 0.0 0.3 0.5 0.5 71.5 71.0 72.1 70.8 74.2
10 0.0 0.0 0.0 85.1 84.2 86.5 84.2 88.3
11 95.0 96.2 98.0 98.1 97.3
12 99.8 98.9 99.3 99.2 102.5
13 104.8 105.3 105.8 106.3 106.7
14 105.5 103.5 105.4 106.7 107.3
Figure 16. The effects of the supernate of a pseudomonad grown
on 500 ppm of Elocron, as the sole carbon source,
on soil nitrification.
1
\
(
lLJ
ti Cf) :::::> lL 0::
110
1LJ 70 a.. LI.. o E 0:: lLJ a..
,60
~ 50 o~
-- NO-N 3 ------- NO - N 2
.. -
.t  CONTROL 0,. TREATED
•
.".
o
/ o
/ o
~ C\I .. ' ~ M
~ "40" "~, "' "" ","".- . 1 LI.. o g: 30
20
10
o
• / 1
//1 " /:/
A--./-!: ..... -'----~ .- __II~~;:..-- • -.----.-~ ...... _I: .. -. ~. -- ~ --- -.. -
2 4 6 8 DAYS
10
o
12 14
Figure 17. The effects at 500 ppm of Ch10rfenvinphos on soi1
nitrification.
)
Î
\ 110
100
90
lLJ
!i ~ 80 li.. 0:: IJJ Cl.. Lt.. 70 o Ë 0:: 60 lLJ Cl..
ON
..z50 le)
o Z
IL 40 o -E . ~30 ~
20
10
CHLORFENVINPHOS
-N03-N ---N02-N
• CONTROL o 500ppm
•
2 4 6 8 10 .. 12 14 .
DAYS
70
(
T AB L E 4.
The effect of various concentrations of Ch1orfenvinphos on soi1 nitrification
N02-N ~g/m1 of perfusate NOg-N ~g/m1 of perfusate
Day
Control 25 125 250 500 Control 25 125 250 500 ppm ppm ppm ppm ppm ppm ppm ppm
1 1.8 0.9 0.6 0.5 0.0 0.0 0.0 0.0 0.0 0.0
2 4.7 2.1 1.5 1.0 0.4 2.5 2.4 3.0 2.2 1.5
3 5.6 3.8 3.8 3.2 1.8 3.5 3.0 3.0 2.2 1.5
4 4.6 4.6 5.5 7.5 4.6 8.5 8.0 6.8 5.5 5.0
5 3.1 3.0 4.5 4.9 8.6 20.0 18.9 16.3 15.1 13.0
6 2.0 1.8 2.8 3.0 5.5 36.0 37.0 30.1 24.0 20.0
7 1.1 0.5 1.5 1.9 2.5 46.0 46.3 41.2 36.1 30.1
8 0.0 0.0 0.9 1.1 1.3 60.0 61.0 56.2 48.5 41.8
9 0.0 0.2 0.5 74.3 72.3 69.3 60.0 52.3
10 + 0.0 82.3 79.0 74.2 73.2 70.2
11 0.0 0.0 92.1 93.0 94.2 90.2 81.3
12 96.1 98.0 100.1 93.0 92.1
13 100.6 101.3 102.3 100.0 102.5
14 99.8 100.2 104.0 103.1 106.5
Figure 18. The effects at 500 ppm of Dasanit on soil
nitrification.
1 .1
, 110 \
100
90
lLJ 80 !i en ::J LL-
ffi 70 a. LL-O - 60 E œ: lLJ
0.",50 0
~ If)
~ 40 LL-O -~30 0' ~
20
10
o 2 . 4
DASANIT
- N03-N ---.N02 -N
• CONTROL 0 o 500 ppm 0/··
L./ ,/;'tl
/0/ Il
/ 0
/ •
0
6 8 DAYS
0
10 . 12 .14
72
L TABLE 5.
The effect of various concentrations of Dasanit on soi1 nitrification
N02-N ~g/ml of perfusate NOg-N ~g/ml of perfusate
Day
Control 25 125 250 500 Control 25 125 250 500 ppm ppm ppm ppm ppm ppm ~pm ppm
1 0.3 0.3 0.3 0.2 0.0 0.0 0.0 0.0 0.0 0.0
2 0.6 0.6 0.6 0.5 0.3 1.4 1.3 0.9 0.9 0.5
3 0.8 1.0 1.0 1.6 2.3 3.9 3.5 2.5 1.5 1.0
4 1.2 1.5 2.1 3.8 5.5 9.0 7.5 4.6 3.6 2.5
5 3.5 4.2 5.9 7.6 9.2 19.8 15.6 10.3 7.8 5 .0
6 1.7 1.5 4.4 6.6 11.6 31.2 27.2 20.3 16.3 9':8
7 0.5 0.6 3.1 5.6 10.5 53.5 41.3 30.5 24.1 20.9
8 0.0 0.8 1.8 1.8 2.5 63.0 59.2 50.3 46.3 39.2
9 0.0 0.0 0.5 0.5 1.0 75.1 73.1 70.5 63.2 56.0
10 0.0 0.0 0.0 84.1 82.1 80.1 75.2 73.9
11 89.6 89.8 87.1 83.1 84.1
12 93.1 94.6 91.5 90.1 92.0
13 94.0 97.2 95.5 95.2 97.3
14 98.9 102.0 98.0 101.1 103.2
Figure 19. The effects at 500 ppm of Carboxin on sail
nitrification.
tOO
90
ILl tt 80 en ::::> IJ.. Cl::
·W 70 Q.
IJ.. o - 60 E Cl:: W Q.
N 50 o ~
If)
~ 40 IJ.. o
E 30 ~ ~
20
10
o 2 4
CARBOXIN
-NOaooN --- N02- N
. • CONTROL 0500 ppm
6 8 DAYS
.0
10 12 . 14
74
! \
T A BLE 6.
The effect of various concentrations of Carboxin on soil nitrification
N02-N ~g/ml of perfusate NOg-N ~g/m1 of perfusate
Day
Control 25 125 250 500 Control 25 125 250 500 ppm ppm ppm ppm ppm ppm ppm ppm
1 0.3 0.3 0.3 0.4 0.5 0.0 0.0 0.0 0.0 0.0
2 0.6 0.8 0.9 0.9 0.9 1.4 1.3 1.0 0.9 0.0
3 0.8 4.0 5.3 3.2 1.2 3.9 3.5 2.5 1.5 1.0
4 1.2 6.3 7.6 5.8 3.8 9.2 8.6 6.1 3.5 3.1
5 3.5 3.6 8.0 6.9 6.1 19.8 18.8 12.4 7.8 5.4
6 1.7 2.5 7.5 8.1 8.3 31.2 30.1 21.1 17.1 14.2
7 0.5 1.2 3.0 6.5 9.4 53.5 48.2 40.1 29.2 ,. 19.1
8 0.0 0.3 1.5 4.8 6.1 63.0 60.1 58.2 38.5 26.4
9 0.0 0.5 1.8 4.5 75.1 74.3 71.1 55.5 42.6
10 0.0 0.3 2.1 84.1 85.3 80.6 67.6 57.8
11 0.0 0.0 89.6 89.9 88.5 81.6 77.1
12 93.1 95.0 92.9 91.6 89.8
13 94.0 96.3 97.6 97.2 95.6
14 98.9 99.2 101.6 101.3 99.8
Figure 20. The effects at 500 ppm of l-naphthol on soil
nitrification.
I-NAPHTHOL
- NO-N 0 110 3
/ i --- N02 -N
\._> • CONTROL
J.-. 100 o 500ppm
90 /7
./ w / !:i 80 Cf) • :::> LL 0::. 0 W Q.. 70· LL 0 -E 60 ct: LLJ Q.. 0
ON50 e
~ o~ z 40 LL 0
~30 Cl ~
20
10
o 2· 4 . 6 . 8 10 f2 . 14
DAYS
. ,
L
76
TABLE 7.
The effect of various concentrations of~-Naphtholon sail nitrification
N02-N pg/m1 of perfusate NOg-N pg/ml of perfusate
Day
Control 25 125 250 500 Control 25 125 250 500 ppm ppm ppm ppm ppm ppm ppm ppm
1 0.3 0.5 0.3 0.1 0.0 0.0 0.0 0.0 0.0 0.0
2 0.9 1.5 1.0 0.5 0.2 1.5 1.4 0.2 0.0 0.0
3 2.9 3.4 2.4 1.8 1.5 3.8 2.3 0.5 0.2 0.0
4 3.9 6.1 3.9 3.4 3.0 7.7 6.1 2.31 1.6 1.2
5 5.5 7.9 6.3 6.1 6.0 14.5 11.3 6.8 4.7 2.6
6 0.7 2.6 3.1 7.5 8.5 23.4 19.6 14.3 9.5 5.2
7 0.0 0.0 1.0 6.4 11.3 47.9 44.8 33.0 23.3 11.0
8 0.0 5.2 12.1 77.2 67.2 53.3 37.1 24.0
9 0.9 1.5 87.4 77.0 73.1 60.4 52.3
10 0.0 0.0 91.2 84.7 91.2 80.7 73.5
11 95.1 91.0 96.5 93.2 90.1
12 97.6 94.5 101.5 103.4 105.0
13 97.0 97.2 105.4 106.8 111.2
DIS eus S ION
In considering the effect of pesticides and other agricultural
chemicals applied to the soil, excess rates of application must be
distinguished from ranges that result in pra~tice. The highest
treatment used in these experiments(500 ppm) corresponds to about
200 x field rate. ~10st methods of application, however result in
concentrations zones so that lateral and vertical mosaics of varying
concentration result. Probably variations occur from zero concentra
tion to 500 ppm in laboratory studies and in field plots even though
treatments are usually made in a manntar to obtain a dis'tribution as
uniform as possible throughout the soil sample. This is one reason
why application higher than field rates were included in laboratory
studies. Another factor is the possible build up of concentration of
some pesticides over a peribd of years.
Laboratory studies on soils under controlled conditions are appro
priate but may give results that not necessarily will follow in the
field, where changes in environmental factors, drainage, and plant
roots introduce variables, thus field studies are necessary for prac
tical conclusions and recommendations. Again, because of low ra~es
of treatments, adequate distribution and sampling are serious problems.
Despite these limitations, the research data presented here show that
only when the pesticides are applied to the soil at many times the
recommended field rates do they appreciably reduce beneficial activities
77
78
of soi1 microorganisms and even these effects are temporary.
Some pesticides exert a rather marked effect on soi1 chemica1
properties. For example, they may increase the supply of soluble
plant nutrients. The elements are released through the decomposition
of dead bodies of organisms killed by the initial treatment, from the
decomposition of pesticide residues from the soil organic matter as
a result of stimulated microbial activity or fram inorganic soil con-
stituents. There is some evidence of this phenomenon occurring. On
examining the data from the effects of l-Naphtho1, Chlorfenvinphos
and Dasanit on nitrification, more than 100 ~g/m1 of N03-N were pro-
duced at the end of the experiment. Theoretica1ly this should not be
found sinèe the total nitrogen content in initial CNH4>2S04 perfusate
was 100 ~g/ml. At the end of 14 days the quantity of N03-N was still
increasing (Figure 13). When 1,500 ppm of Chlorfenvinphos were used,
N03-N produced was more than 150 ~g/m1 of perfusate at the end of 20
days and still increasing whereas the untreated ~oU produced a maximum
of apprvximately 100 ppm, the theoretical value. It seems 1ikely that
the ammonium-nitrogen accumulated until the·nitrifiers became re-es-
tablished, and a higher concentration of N03-N was formed.
79
PAR T II
THE EFFECT OF SELECTED PESTICIDES ON MICROORGANISMS
IN AQUATIC ENVIRONMENTS
l N T RaD U C T ION
~d
LITERATURE R E VIE W
A large part of our present day pesticide problem is ultimately
tied to the fresh water ecosystem. Pesticides are used in so m~y
types of terrain to control so m~y types of pests that almost aIl
lakes ~d streams are contaminated. In addition to accidentaI con
tamination, ~y compounds are deliberately applied directly to fresh
water for suppression of aquatic ~imals and pl~ts. The problem is
intensified because of the extreme susceptibility of fresh water
org~isms. The complexity of fresh water environments ~d their var
iety makes it difficult to comprehend the total effect of pesticides.
This diversity of organisms in fresh water has several import~t
consequences giving rise to a multiplicity of experimental systems
~d techniques for evaluation of the toxicity of environmental con
tamination. Another problem is the extrapolation from the effects of
certain chemicals or classes of chemicals on individual target organ
isms to include ~ entire ecosystem. AIs 0 , investigators have synthe
sized communities of 2 or 3 members ~d have extrapolated from this
to include ~ entire ecosystem. The problem is obvious since an
80
ecosystem is a natural assemblage of organisms which are in dynamic
equilibrium.
Co 1er and Gunner (1969) showed that there is an enhancement of
microbial activity by the exudation of organic substances from the
floating aquatic angiosperm Lemna minor (duckweed). Lemna minor,
therefore, can support a rhizosphere of microorganisms and is said to
have a carrying capacity which is a function of photosynthetic capa
bility. Further support for Lemna minor's ecologically catalytic role
as an enrichment source is found in the isolation of amine acids
solely from the top half inch of water.
A technique was de".:::'l.oped by Co!l:er and Gunner (1970) which allowed
the advantages of controlled laboratory conditions and field study,
using the rhizosphere of Lemna minor for a microbiotic ecoassay for
environmental pollutants. The stress was imposed with Diazinon and
changes were charted in the rhizosphere composition of ~ëmna minor.
The rhizosphere under the stress of 0.01 ~g/m1 Diazinon were less pro
ductive than the control, while the ecosystem inhibited by pesticide
residue in excess of 0.01 ~g/m1 became, with the exception of bacteria,
virtually barren.
Other studies comparing pesticides and aquatic microorganisms
include the work of Wurster (1968) who found that concentrations of
DDT as low as a few parts per billion in water reduced photosynthesis
in laboratory cultures of four species of coastal and oceanic phyto-
81
plankton representing four major classes of algae, and in a natural
phytoplankton community from Woods Hole, Mass. The cultures of the
test phytoplankton were exposed to different cOncentrations of DDT
for 20 to 24 hours with 14 hours of ligbt and 10 hours of dark. Aft~r
this exposure period 14C-bicarbonate was added and the algae were
illuminated for an additional 4 or 5 hours. Radio-assay of the fil
tered cultures was employed to estimate the amount of carbon fixed by
photosynthesis.
The effects of 17 toxicants on the growth of 5 species of algae
in pure culture over a period of 10 days under continuous illumination
were studied (Ukeles, 1962). In contrast to WUrster (1968) the toxi
cants were incorporated in the sterile basal medium at different con
centrations. The substituted urea compounds and a mercuric compound
were the most effective in inhibiting growth of all algal species at
the lowest concentrations. Carbaryl was very toxic to two of the
species.
Another evaluation of the use of algae in biological assays for
pesticide pollution was studied by Lazaroff (1967). Algal development
in enrichment cultures showed delayed growth in the ~esence of 1.0 ppm
of the fungicides Captan and Nabam and the insecticides DDT and Lindane.
Thiocarbamate pesticides were found to interfere vith photoinduced
development in the blue-green alga Nostoc muscorum. Lazaroff (1967)
aIso tested an assay system based on the inhibition of motility but
82
not growth, in EugZena ~aaiZis which can detect Parathion at concen
trations of 10 ppb. Malathion is without effect.
Investigations of the effects of pesticides on bacteria in aqua
tic environments are less frequent. Lichtenstein et aZ.~ (1966) showed
that DDT, Dieldrin and Methyl parathion do not affect growth while
Parathion and BHC show inhibition of total bacterial numbers stored
in lake water for three months. In soil percolated water, only DDT
did not have an adverse effect.
Another problem is biological magnification of pesticides. Hunt
and Bischoff (1960) illustrated this by the death of fish-eating birds
following DDD application to the waters at Clear Lake, California, to
control gnats. Waters containing 0.02 ppm or less of DDD produced
plankton containing 5 ppm and fish containing hundreds to thousands
of ppm of DDD. Western Grebes that fed on the fish died, containing
somewhat smaller residues than the fish (80,000 times concentration of
DDD in the water).
A modelecosystem for evaluation of pesticide biodegradability
and ecological magnification was devised by Metcalf et aZ~~ (1971).
Thè model ecosystem had a terrestrial-aquatic interface and seven-ele
ment food chain. It simulates the application of pesticides to crop
plants and the eventual contamination of the aquatic environment. The
results obtained with DDT after one month in the food chains of the
model system show remarkable approximations to that observed after
.83
many years under natural conditions.
Thi:di1.teractl:on of pesticides with aquatic microorganisms may be
observed by:
(1) Lethal effects upon a particular organism~
(2) Changes in growth rate.
(3) Changes in specific metabolic rates, e.g. in photosynthesis.
(4) Indirect actions that result from stress on one or more organisms
that permit previously suppressed competitors to flourish,
further stressing dominant populations.
(5) Biological magnification which is the ability of microorganisms,
plants and animaIs to concentrate many types of pesticides in
their body tissues. The Chl6rinated insecticides have the great
est affinity for this type of process.
84
85
MATERIALS and METHODS
A technique was developed (Coler and Gunner, 1969; 1970; Gunner
and Coler, 1971) for observing the response of an intact aquatic eco-
system under a range of controlled laboratory conditions. In these
studies changes were noted in the rhizosphere composition of Lemna
minor, a floating pollution tolerant angiosperm, when exposed to var-
ious stresses. Lemna minor obtained from a pond at Macdonald College
was cultured at room tempe rature and an illumination of 100 ft-c in
pond water in an aquarium. The pond water was filtered through filter
paper and then passed through a millipore filter to make it as clear
as possible.
ANALYSIS OF POND WATER
pH 7.6 Zinc < 0.02 mg/l
Specifie Lead < 0.5 mg/l conductance 200 micromhos/cm
Alkalinity: Total 66.15 mg/l Soluble P 20 llg/l
Hardness: Total 65.83 mg/l Cadmium < 0.05 mg/l
N02 + N03 (N) 850 llg/l Calcium 14 mg/l
Iron < 0.05 mg/l Magnesium 7.5 mg/l
Copper < 0.1 mg/l C.O.D. 20 mg/!
Figure 21 Ecological assay flask:
A. Exhaus_t port;
B. Sampling port;
C. Aerator.
•
Figure 22. View of assembled assay apparatus.
·6M-:::a .-- 1 .rw't;:;;«.';;:.,L! .. 5! ..... ~ __ . ,--, ~'r 'iClJ _
~ ~~,...,.. ;:T~ - y "! " .
. . \; -•. .... . ~ . .~ t~"~ . "-- ":_,----- .
, " ;;
MPARATUS
Disposable tissue culture flasks (30 ml) were modified as des
cribed by Coler and Gunner (1970). These flasks proved to be effec
tive housing for rhizosphere studies. Figure 21 gives a view of the
assay flask while Figure 22 shows the assembled apparatus.
Twelve flasks were used in each experiment with two controls
and duplicates for each concentration of pesticide. Millipore fil
tered pond water and 20 - 25 duckweed plants at the same stage of
development were placed in each flask and allowed to equilibrate at
room temperature and an illumination of 100 ft-c for 2 - 3 weeks;
population counts of the organisms were taken at the end of each week
then introduced so that the pesticide concentrations were 0, 0.01,
0.1, 1.0, 10.0 and 50.0 ~g/ml with a total.volume of medium of 25 ml.
Population inventories were taken on the 2nd, 10th, 20th and 30th
days. The pesticides studied were Baygon, Carboxin, Chlorfenvinphos,
Dasanit, Elocron and l-naphthol and their formulations were the same
as given in Materials and Methods, Part-l,Section B.
SAMPLlNG AND ENUMERATION PROCEDURE
The sampling procedure involved withdrawing 0.25 ml of medium
from the area adjacent to the undersurface of the plant and its roots
with the use of a syringe and needle. Suitable dilutions were made
and total bacterial counts were estimated by plating on plate count
87
agar, each dilution ~n triplicate. The nurnbers of sporeforming bact
eria were also determined by the method described in Materials and
Methods, Part I, Section A, and then plating on plate count agar. The
protozoa, rotifers, gastrotrichs, oligochaetes and nematodes were
enumerated byrernoving two plants at a time and placing them in a few
drops of sterile pond water on a microscopie slide and observing dir-
ectly the root system and undersurface of the plant with a microscope.
This procedure reduces some of the ecological disruption which arises
in handling and desiccation while still provides the stability and
visibility of a microscopie slide. The population inventory per flask
was made from an average of 10.plants which l .. ere replaced in the flask
after population inventories were taken.
The pH of the medium and the pesticide levels in each flask were
determined at each sampling.
EXTRACTION AND DETE~lINATIO~ OF PESTICIDE LEVELS
Ten millilitres of medium were withdrawn at each·sampling for
pesticide determination. To accomplish this, each treatment had a
second set of duplicates, making 4 flasks per treatment. This allowed
10 ml portions to be extracted in duplicate for e~ch pesticide deter-
mination on the 2nd , 10 th , 20th and 30th day.
The pesticide in the 10 ml of medium was extracted by shaking in
a separatory funp.el wi.th . three equal volumes of the extraction solvent
·88
which was then evaporated to dryness on a rotary evaporator at 35°C
and redissolved in 1 ml of acetone. One millilitre of the solution
was injected into a GLC Varian Aerograph, Series 1700, Flame Ioniza
tion Detector. The following conditions in the GLe were the same for
aIl compounds ~
Helium flow,
Hydrogen flow,
Air flow,
40 p.s.i.;
40 ml/min;
400 ml/min.
The two columns used in the analyses had the following charac
teristics:
(1) 10% (Silicone) S.E. - 52 on Chromosorb W
(60 to 80 mesh), hexamethyldisilazane treated in a stainless
steel column, 6 ft. x 0.25 in. (O.D.).
(2) 3% (Silicone) S.E. - 30 on Varaport No. 30,
(100 to 120 mesh) , distilled water treated in a stainless
steel column, 6 ft. x 0.25 in. (O.D.).
The conditions used for extracting and determining the various
pesticides are summarized in Table 8.
Standards were prepared by dissolving known quantities of the
pesticide in pond water and using a similar procedure for extrac
tion and determination.
89
or A BLE 8.
SUMMARY OF CONDITIONS FOR EXTRACTION AND DETERMINATION OF PESTICIDE IN SAMPLES
Pesticide Extraction Co1umn Co1umn 'Detector Injector solvent temperature temperature temperature
Baygon Ch1oroform 3% S.E. 30 i3SoC 225°C 215°C
Carboxin Ch1oroform 3% S.E. 30 190°C 230°C 220°C
Ch1orfenvinphos Hexane 3% S.E. 30 190°C 230°C 220°C
Dasanit Ch1oroform 3% S.E. 30 200°C 210°C 22"O°C
E1ocron* Ether 10% S.E. 52 150°C 225°C 225°C
1-Naphtho1 Ch1oroform 3% S.E. 30 120°C 225°C 210°C
* The procedure used for extracting and determining E1ocron (Levac, 1972) was slight1y different from the other pesticides, 10 ml of medium was first acidified ta pH 2, then the pesticide was extracted with three equa1 volumes of ether which was 1ater evaporated ta dryness at 35°C on a rotary evaporator. The residue was redisso1ved in 0.7 ml of pyridine and 0.3 ml of Tri-sil ta produce a trimethy1si1yl derivative, then shaken for 30 seconds and a110wed ta stand for 15 minutes and centrifuged for 10 minutes at 4000 rpm. 1 ~1 of the supernatant was then injected into the GLC.
\0 o
RESULTS and DISCUSSION
After introducing the Lemna minol' plants and the medium into
the flasks, about 2 to 3 weeks elapsed before the population of
the rhizosphere became stable. Pesticides were introduced then.
The Tables 9, 14, 19, 24, 29 and 34 IIbefore exposure to pesticidell
illustrated.how reproducible are the counts of the rhizosphere con
stituents. Note that when the number of organisms per plant were
greater than thirty they became too numerous to count and were
listed as > 30. Note also that there were no sporeformers in the
rhizosphere.
Though Lemna minol' exhibited no apparent distress when the
pesticides were introduced, a distinct reduction in the carrying
çapacity was noted with aIl of the pesticides levels tested. The
pH remained fairly constant throughout.
Z-NccphthoZ
The general pattern obtained for aIl the pesticides were basic
cally the same (Figures 23 and 24) i.e. a distinct and abrupt reduc
tion in the carrying capacity was observed at all levels tested. Even
at an initial concentration of 0.01 pg/m!, a few individual species were
reduced in numbers drastically. For example, the holotrichous ciliate
population was >30 per plant but when l-naphthol was introduced the
91
population was reduëed to 5 per plant after 2 days (Table 10). Many
more species were reduced in numbers by more than 50% with an initial
concentration of 0.01 ~g/ml of l-naphthol. At higher concentrations
more species were kil1ed until eventually the rhizosphere was barren
(with the" exception of bacteria) by the tenth clay (Table Il). By
this time no more l-naphthol was detected at the 0.02 ~g/ml level.
The loss may have beendue to volatilization, chemical or biological
degradation, photo-oxidation, adsorption to the biomass, or uptake by
a the roots (the roots turned purple colour).
A
At 20 and 30 days (Tables 12 and 13) there were ~ome signs of
recovery particularly by VoptiaeZLa and the holotrichous ciliate.
The bacteria showed a general increase in numbers with increasing con-
cent,rations of l-naphthol ove"r the period of 30 days (Figure 25). This
increase in "bacteria may be due to:
(1) Reduction of the predator pressure, e.g. "protozoas decreased
and the bacteria increased in numbers.
(2)" Utilization of l-naphthol as a carbon source by bacteria.
Baygon
Baygon (Figures 26 and 27) showed the same general disruption
patterns at low and high concentrations as found for"1-naphtho1. At
an initial concentration of 0.01 ~g/m1 of Baygon there was the extinc-
tion of at least one protozoan Oikomonas as early as 2 days (Table is).
92
Other organisms were a1so reduced in riumbers, some reductions were as
high as 80%. At the initial concentration of 10 pg/ml, apart from
bacteria, Bodo and a ho1otrichous ci1iate, the rhizosphere was almost
barren. Bodo was the most resistant protozoan. At 50 pg/ml, initial
concentration, there was complete ki11ing of a11 the aquatic inverte
brates except LepadeZZa after 2 days and the residue of Baygon was
43.8 pg/ml.
The pattem remained the same tmti1 on the 30th day there were
signs of some of the protozoa retuming at the 50.0 pg/ml initial
treatment, which now had a residue of 4.1 Pg of Baygon per ml (Table
18). This slight indication of a recovery of some organisms may be
due to adaptation of the organisms to the chemica1. The rate of disa
ppearance of Baygon was simi1ar to that obtained by Eiche1berger and
Lichtenberg (1971). The disappearance of Baygon cou1d be due to
chemica1 degradation, microbia1 degradation, photodecomposition, adsorp
tion by the biomass, uptake by the plant roots and vo1ati1ization. The
lower 1imit of det~ction of Baygon was 0.02 pg/ml.
Figure 28 shows a trend towards a decrease in the number of bac
teria in the Baygon-treated f1asks, about four times 1ess than the
control after a 30 day periode This reduction of the number of bacteria
may be due to the toxicity of the Baygon and its metabolites.
EZoC!1'on
E1ocron was not detected in any of the treatments by the 2nd day
. l
93
94
(Table 20) as apparently it undergoes decomposition in water (Levac
1972). However, the effect was quite apparent for even at 0.01 ~g/m1
initial treatment Euplotes became extinct (Table 20). Figures 29 and 30
illustrate the typica1 general pattern obtained with a low and
high concentration of pesticide introduced as an external stress.
At 50 ~g/ml, initial treatment, and at 2 days the rhizosphere was
completely barren except for the bacteria. This killing could be due
to toxicity of the initial Elocron or its metabolites.
At concentrations as low as 0.1 ~g/m1, several protozoa, roti
fers, oligocheates and nematodes were killed and there was complete
extinction of 5 organisms (Table 20). Over the 30 day period, the
rotifers made a slight recovery even at the 50 ~g/m1 initial treatment.
VOI'tiaella, the holotrichous ciliate, and to a lesser extent, Oikom
onas and Aeolosoma were the most resistant organisms besides the
bacteria (Table 23).
Figure 31 shows that the bacterial population increased with in
creasing concentrations of Elocron. This can be explained by:
(1) Loss of predators, e.g. protozoa were killed due to the
toxicity of Elocron and therefore there is an increase in
the number of bacteria.
(2) Utilization of the Elocron as a carbon sOurce.
ChZo!'fenvinphos
Chlorfenvinphos was very toxie to a number of organisms (Table
25) for even at 0.01 llg/ml, initial treatment, the Astasiidae and
the nematodes were completely kil1ed as ear1y as 2 days. At an ini
tial concentration of 10.0 llg/ml the on1y surviving organisms were
Vo!'tiaeUa and at 50.0 llg/ml initial treatment, the only organisms
present were the bacteria. However, at the 10-day mark, there was
an increase in the numbers of the protozoa particularly Oikomonas
(Table 26). This trend continued for the rest of the 30-day period
(Figures 32 and 33) •. The resistanee of the surviving organisms may
be one of adaptation for on the 30th day, there was a residue of 8.5
llg/ml of Chlorfenvinphos from the initial treatment of 50.0 llg/ml
and yet there was survival of Oikomonas and Bodo. Figure 54 shows that
the bacteria increased in numhers with inereasing concentration and
for the same reasons as discussed with Elocron.
Results by Beynon et aZ.~ (1968) showed that the half-life on
potato foliage was 3 days and Suett (1971) showed that carrots took
up Chlorfenvinphos from the soi1. Beynon et aZ.~ (1971) also showed
that Chlorfenvinphos disappeared rapidly from natural waters and it
did so in two distinct phases, a rapid initial phase and a much slower
second phase. Possibly the first phase represented the initial preci
pitation of the heavier particles and the plankton. Later the Chlor
fenvinphos was gradually adsorbed on to other suspended matter, which
then precipitated more slowly or the second phase could represent the
95
slower removal of residues by metabolism. Chlorfenvinphos decomposes
slowly in water (half-life 170 days at pH 6 and 80 days at pH 8) at
20 - 30°C and hydrolysis probably did not contribute much to the ini
tial disappearance.
From the data presented here (Tables 24 - 28) a similar 2-stage
disappearance of Chlorfenvinphos took place, also uptake by the roots
of the plants, similar to that shawn by carrot roots (Suett, 1971)
may have taken place.
Beynon et aZ.~ (1971) also suggested that contamination of ponds
by aerial spraying at commercial dosages (up to 5 kg/ha) or from run
off and leaching of Chlorfenvinphos from treated soil would not be
a serious hazard to free swimming aquatic animaIs. From the data
presented in Figure 32 and Tables 24 - -28, this conclusion can be
questioned since the levels of concentration used in these experiments
are weIl within the range of surface run-off residue levels and dras
tically upset and killed many aquatic invertebrates in this rhizosphere
of Lerrma minore
Dasanit
Dasanit displayed a similar general upset of the ecosystem pattern
(Figure 35). Table 30 shows that as early as 2 days and at an initial
concentration of 0.01 ~g/ml, certain species of protozoa and rotifers
were reduced in numhers. At 10.0 and 50.0 ~g/ml (except for the
bacteria), the rhizosphere was almost completely barren, Oikomonas and
VortiaeZZa were the most resistant genera (Table 30).
96
Over the 30-day period, no free swimming aquatic invertebrates
were seen in the rhizosphere at the 50.0 ~g/ml initial treatment and
only a few rotifers and holotrichous ciliates at the 10.0 ~g/ml ini
tial treatment, a1though Dasanit was found (lower 1evel of detection
is 0.02 ~g/ml)(Tab1e 33). During the course of the 30-day period
several peaks appeared in the gas chromatogram; these peaks were not
identified but possibly represent metabolites of Dasanit which are
toxic and maintained a barren condition of the rhizosphere except for
the bacteria.
Figure 37 shows an increase in the bacterial population per plant,
with increased numbers associated with increasing concentrations of
Dasanit. The reasons for this phenomenon have already been discussed
for E1ocron.
C~boxin
Carboxin-treated flasks (Figures 38 and 39) showed the same gen
eral trend in the changes in the rhizosphere as for the other pesti
cides. Disruption was noticed as early as the 2nd day with the reduc
tion of certain species at the 0.01 ~g/ml initial treatment leve1
(Table 35). At high concentrations the rhizosphere was virtually
barren except for the presence of bacteria. The most resistant aquatic
invertebrates were VortiaeZZa, a ho1otrichous ciliate and the rotifers.
Figure 40, which illustrates the response of the bacterial popu-
l,:
97
lation to different concentrations through time, shows that an
increase in bacterial numberswas related to increasing initial con
centrations of Carboxin.
The analyses for the Carboxin residues were not determined in
this experiment. According to Chin ét al., (1970), after 4 weeks
and at pH 7, there was no change or loss of Carboxin. However, in
trying to draw any parallel between the work of Chin et al., (1970)
and these experiments other factors such as uptake by roots and
adsorption of the biomass, must be considered. A possibility is that
oxidation to the sulfoxide may have occurred (Chin et al., (1970).
98
DIS C U S S ION
These data give relative numbers of various organisms in the
rhizosphere of this aquatic plant in the presence of varying concen
trations of pesticides. They support Coler and Gunner's view (1970)
that the significance of the results lieir. the novel use of an
entire biota as a bioassay system rather than the classical resort
to "universal indicator species" as a measure of the broad spectrum
of community responses to the various pesticides. The originators
of this technique, Coler and Gunner (1970) have already tested Dia
zinon as the exogenous stress and there was profound disruption in
the microbiocoenosis.
From the response of the populations to the various chemicals
in these experiments, the microecosystem sustained by the Duckweed
host proved to be an exceptionally sensitive instrument capable of
responding to normally unsuspected and undetectablé ·levels·of~stress.
The patterns of the disruption were basically the same for aIl pesti
cides, however, different constituents of the rhizosphere are more
sensitive or resistant to different pesticides •.
Reduction ~f the populations of the rhizosphere was attributed
to the reduction of the carrying capacity of the duckweed by Coler and
Gunner (1970). The toxicity of the Chemicals to the constituents of
the rhizosphere are responsible also for the reduction of populations
99
both qualitatively and quantitatively.
The extreme sensitivity of this assay system and the concentra
tions of pesticides used in experiments make difficult the decision
as to which of the pesticides used were the most orleast toxic.
Future research should be based on improving the techniques to pro
vide gradations of effect according to the concentration of the
pesticide.
The data presented on aIl these pesticides indicate that at very
low concentrations, undesirable effects on this aquatic ecosystem
occur.
100
\
;
Figure 23. The effects of 0.1 pg/ml of l-naphthol on the numhers
of Protozoa, Rotifers and Gastrotrichs per plant of
the Lemna minop rhizosphere over a period of 30 days.
fZ :5 100
~ en ~ 50 -:2: <t C) 0::: o U. o d z 8
4
o 2
I-NAPHTHOL O.l}J9/ml
-----0
5 10 15
DAYS
A PROTOZOA
o ROTIFERS o GASTROTRICHS
~""------.A.
20 25 30
Figure 24. The effects of 50~O lJg/ml of I-naphthol on the mnnbers
of Protozoa, Rotifers and Gastrotrichs per plant of
the Lemna minop rhizosphere over a period of 30 days.
1-Z ~ 100 a.
~ ~ 50 -Z <t (!) 0:: o L.L. o é :2
4
o 2 5
I-NAPHTHOL 50.0J,l9/ml
10 . 15 DAY 5
20
 PROTOZOA e ROTIFERS o GASTROTRICHS
25 30
Figure 25. The effects of 1-naphtho1 at 3 concentrations, 1.0,
10.0 and 50.0 ~g/m1 on the bacteria1 population per
plant of the Lemna minor rhizosphere over a period
of 30 days.
)
10 0 )(
~ 20 :2 <[ ..J a. ~ 16 a: LIJ t-0 <t al
li. 0
d z
o
•
5 10
I-NAPHTHOL
15 DAYS
20
o CONTROL o 50J,lg/ml . m 10J,J9/ml /). 1 J,l9/ml
25
-0
30
104
J
TA BLE ~.
Population levels of Lemna minor rhizosphere before exposure to
l-Naphthoi*
pH 7.60 7.55 7.50 7.45 7.50 7.50
Data reported as numbers per 0.25 ml a1iquot
Bacterial x 105 8.9 12.0 9.2 11.0 13.0 9.5
Data reported as numbers per plant by direct observation Protozoa
Astasiidae 4.5 3.5 3.0 4.0 3.5 3.25
Bodo >30 >30 >30 >30 >30 >30
EupZotes 5.0 5.5 4.0 4.5 4.25 6.5
Lacrymal'ia 4.0 4.5 3.5 4.0 4.5 3.5
Oikomonas >30 >30 >30 >30 >30 >30
VorticeZZa >30 >30 >30 >30 >30 >30
Holotrichous Ciliate >30 >30 >30 >30 >30 >30
Rotifers
LepadeZZa 5.5 6.5 3.5. 5.5 5.0 6.0
PhiZodina 4.0 3.0 4.0 4.5 3.5 4.0
Gastrotrichs
Lepidoderme Z Za 2.0 2.5 3.5 3.5 2.5 2.0
Oligochaetes
AeoZosoma 0.3 0.25 0.2 0.1 0.25 0.25
Nematodes 0.25 0.20 0.15 0.15 0.20 0.25
* Data reported as the average of 2 f1asks, 10 plants per f1ask samp1ed.
105
T A BLE 10.
Population levels of Lemna minor rhizosphere after 2 days of exposure to
l-Naphthoi*
. : l.:.NaRlitlié.l i:bncentration (llg/ml)
Initial nïl 0.01 0.1 1.0 10.0 50.0
After 2 days N.D.** N.D. N.D. .48 5.:!- 27.4
pH 7.55 7.45 7.40 7.45 7.50 7.55
Data reported as numbers pr 0.25 ml aliquot·
Bacterial x 105 11.0 9.0 9.7 12.0 .13.0 14.0
Data reported as nurnbers per plant by direct observation Protozoa
Astasiidae 3.5 2.5 3.0 1.0 2.3 nil
Bodo >30 >30 >30 >30 -20 nil
Eup7,otes 5.5 4.0 2.5 2.5 nil nil
La.crymaria 4.5 3.0 2.5 nïl 0.2 nil
Oi7wmonas >30 >30 >30 >30 -15 nïl
VorticeUa >30 >30 >30 >30 nil nïl
Holotrichous Ciliate >·30 5.0 5.5 nïl 1.5 nil
Rotifers
Lepade 7,],a 6:è 4.0 4.0 1.0 1.0 ni1
Phi7,odina 4.0 2.0 ni! 0.25 0.25 nil
Gastrotrichs
Lepidoderme 7, 7,a 2.0 1.5 1.5 0.25 nil nil
Oligochaetes
Aeo7,osoma 0.25 0.25 0.10 nïl nil nil
Nematodes 0.25 0.25 0.30 0.30 nil nil
* Data reported as the average of 2 flasks, 10 plants per flask sampled. ** Not detected
. ,
106
T A' BLE 11.
Population levels of Lemna minor rhizosphere after 10 days of exposure to
I-N'aphthoï*
, J,:.NâPhthd~ concentration (pg/ml)
Initial nil .01
** N.D. N.D. After 10 days
pH 7.50 7.45
0.1
N.D.
7.40
1.0 10.0 50.0
N.D. N.D. N.D.
7.45 7.50 7.55
Data reported as numbers per 0.25 ml a1iquot
Bacteria1 x 105 12.0 11.0 12.0 14.0 15.0 21.0
Data reported as numbers per plant by direct observation Protozoa
Astasiidae 4.5
Bodo >30
EupZotes 5.5
Laarymaria 6.5
Oikomoll(ls >30
VortiaeZZa >30
Ho1otrichous Ci1iate >30
Rotifers
LepadeZZa
PhiZodina
Gastrotrichs
LepidodermeZZa
Oligochaetes
AeoZosoma
Nematodes
*
6.0
6.75
1.75
0.25
0.35
4.0
>30
3,.2
3.5
5.0
>30
-10
3.0
3.5
1.5
0.1
0.15
2.5
>30
ni!
1.5
ni!
>30
-10
3.0
3.0
1.5
0.20
0.20
0.5
-10
ni!
ni!
ni!
5.0
ni!
0.25
1.0
ni!
0.20
0.20
0.1
-10
ni!
ni!
ni!
ni!
ni!
0.25
ni!
ni!
ni!
0.1
0.1
ni!
ni!
ni!
ni!
ni!
ni!
ni!
ni!
ni!
ni!
ni!
Data reported as the average of 2 f1asks, 10 plants per f1ask samp1ed.
** Not detected
107
T A :B L E 12.
Population levels of Lemna minop rhizosphere after 20 days of exposure to
i-Naphthof*
l~NaplithOlconcentration (pg/ml)
Initial
After 20 days
pH
ni! 0.01
N.D.** N.D.
7.60 7.30
0.10
N.D.
7.55
1.0 10.0 50.0
N.D. N.D. N.D.
7.55 7.55 7.60
Data reported as numbers per 0.25 ml a1iquot
:Bacteri~ll x 105 14.0 18.0 20.0 15.0 17.0 21.0
Data reported as numbers per plant by direct observation Protozoa
Astasiidae 3.0
Bodo >30
EupZotes 3.0
Laczoymazaia. 3.5
Oikomonas >30
VopticeZZa >30
Holotrichous Ci1iate >30
Rotifers
LepadeZZa
Phi Zodina
Gastrotrichs
LepidodemeZZa
Oligochaetes
Aeo Zosoma~-
Nematodes
*
6.0
4.5
2.75
0.3
0.2
1.0
>30
2.5
1.0
5.0
>30
>30
1.5
1.5
2.5
0.1
0.1
0.5
>30
1.5
0.5
ni!
-25
>30
2.0
2.5
6.0
0.2
0.1
0.5
5.0
0.5
0.2
ni!
-15
>30
1.5
2.0
2.0
0.1
0.2
1.0
3.5
ni!
0.1
ni!
-20
2.0
1.0
2.0
1.0
ni!
ni!
0.5
3.0
ni!
ni!
ni!
ni!
ni!
ni!
ni!
ni!
ni!
ni!
Data reported as the average of 2 f1asks, 10 plants per f1ask samp1ed.
** Not detected
108
T A BLE 13.
Population leve1s of Lemna minop rhizosphere after 30 days of exposure to
l-Naphth6i*
.. ]..i.~âpli.tliOJ. concentration (Ug/ml)
Initial
After 30 days
pH
nil
nil
7.60
0.01 0.1
N.D.** N.D.
7.40 7.60
1.0
N.D.
7.70
10.0 50.0
N.D. N.D.
7.60 7.65
Data reported as numbers per 0.25 ml a1iquot
Bacterial x 105 13.0 23.0 23":0 ... 17.0 17.0 25.0
Data reported as numbers per plant by direct observation Protozoa
Astasiidae 3.0
Bodo >30
EupZotes 3.0
Laaryma:Pia 3.0
Oikomonas >30
VortiaeZZa >30
Ho1otrichous Ciliate >30
Rotifers
LepadeZZa 6.5
PhiZodina 4.0
Gastrotrichs
Lepiàoder.meZZa 2.5
01igochaetes
AeoZosoma 0.25
Nematodes 0.3
*
0~25
>30
nil
nil
5.5
>30
>30
1.5
2.0
4.5
0.25
0.1
0.5
3.0
nil
0.25
nil
--20
>30
2.5
5.5
5.0
0.5
0.1
0.5
3.0
0.5
0.25
nil
-10
>30
1.5
4.25
2.0
0.2
0.2
0.1
1.0
0.25
0.1
nil
-10
>30
6.0
2.0
0.2
0.1
0.15
0.5
1.0
nil
nil
nil
nil
nil
nil
nil
nil
nil
nil
Data reported as the average of 2 flasks, 10 plants per flask sampled.
** Not detected
Figure 26. The effects of 0.1 pg/ml of Baygon on the numbers of
Protozoa, Rotifers and Gastrotrichs per plant of the
Lemna minor rhizosphere over a period of 30 days.
)
tZ « ..J
~ (/) 0
~ 0 50 z <[ (!) 0:: o Lt.. o d z
4
o
SAYGON O.t.ug/ml
~. •
-0
"--01-
0
----0_ -
5 10 15 20 0
o DAYS
 PROTOZOA e ROTIFERS o GASTROTRICHS
_A
•
25 30
Figure 27. The effects of 50.0 pg/ml of Baygon on the numbers of
Protozoa, Rotifers and Gastrotrichs per plant of the
Lemna minor rhizosphere over a period of 30 days.
1 /
.... 2 <t -1
~ ~ 50 !2 z « C) a: o IL o o 2:
0
_e_ . 1>:
·5 10
 PROTOZOA o ROTIFERS o GASTROTRICHS
-==-15 20 25 30
DAYS
Figure 28. The effects of Baygon at 3 concentrations, 1.0, 10.0
and 50.0 g/ml on the bacteria1 population per plant
of the Lemna minor rhizosphere over a period of 30
days.
\ /
'.
28
11)0
";( 20 1-:2 « .J
~ <t il: lLJ 1-U « en t.L o d z
4
o
._- --
BAYGON
o CONTROL o 50.ug/ml ra 10 JJg/ ml 6 1 JJ9/ml
- -0
a CJ
l----.-----~--~~----~----~2~5----~30 (5 20 10 .-5
DAYS
112
~
TA BLE 14.
Population levels of Lemna minop rhizosphere before exposure Baygon*
to
pH 7.60 7.60 7.65 7.55 7.65 7.60
Data reported as numbers per 0.25 ml aliquot
Bacterial x 105 10.0 9.0 8.6 9.6 9.7 8.4
Data reported as numbers per plant by direct observation Protozoa
Astasiidae 3.0 2.5 3.0 2.0 3.5 3.0
730(10 >30 >30 >39 >30 >30 >30
EupZotes 4.0 3.0 4.5 5.0 3.5 4.0
Lacrymapia 4.0 3.5 3.0 3.5 2.5 2.5
Oikomonas >30 >30 >30 >30 >30 >30
VopticeZZa >30 >30 >30 >30 >30 >30
Holotrichous Ciliate >30 >30 >30 >30 >30 >30
. Rotifers
LepadeZZa 6.0 6.0 5.75 5.5 6.0 4.5
PhiZodina 4.0 3.0 3.5 4.0 3.25 3.75
Gastrotrichs
LepidodemeZZa 2.0 2.0 2.0 2.0 2.5 2.0
Oligochaetes
AeoZosoma .25 0.3 0.2 0.2 0.25 2.0
Nematodes 0.1 0.1 0.2 0.2 ni! 0.20
* average of 2 flasks, 10 plants per flask sampled. Data reported as the
113
tj
TA BLE 15.
Population leve1s of Lemna minop rhizosphere after 2 days of exposure to
Baygon *
Bazgon concentration (ug/ml)
Initial nil 0.01 0.1 1.0 10.0 50.0
After 2 days N.D.** N.D. 0.06 0.73 8.5 43.8
pH 7.65 7.60 7.70 7.60 7.60 7.60
Data reported as numbers per 0.25 ml aliquot
Bacterial x 105 8.5 13.0 6.5 8.4 11.0 10.0
Data reported as numbers per plant by direct observation Protozoa
Astasiidae 3.5 2.75 2.0 nil nil ni!
Bodo >30 >30 >30 >30 >30 ni!
EupZotes 5.0 1.0 O~ 75, 0.75 0.5 ni!
Laarymapia 4.5 2.0 1.0 1.5 0.5 nil
Oikomonas >30 ni! 3.0 ni! ni! nil
VoptiaeUa >30 >30 >30 >30 ni! nil
Holotrichous Ciliate >30 >30 >30 >30 -10 nH
Roterifers
LepadeZZa 6.0 5.0 2.0 2.5 1.0 0.5
PhiZodina 4.0 2.75 1.5 2.0 0.5 ni!
Gastrotrichs
LepidodemeZZa 2.0 1.0 1.5 1.0 0.5 nil
Oligochaetes
AeoZosoma 0.25 0.2 0.1 nil ni! nil
'NéIilatodes 0.2 0.45 0.1 0.1 nH nil
* Data reported as the average of 2 flasks, 10 plants per f1ask sampled.
** Not detected
114
l~
TA BLE 16.
Population levels of Lemna minop rhizosphere after 10 days of exposure to
Baygon *
Ba~8on concentration (ll·g/ml~
Initial ni! 0.01 0.1 1.0 10.0 50.0
After 10 days N.D.** N.D. 0.04 0.35 4.5 22.0
pH 7.55 7.45 7.55 7.35 7.4 7.45
Data reported as numbers per 0.25 ml aliquot
Bacterial x 105 11.0 16.0 8.6 6.9 10.0 9.6
Data reported as numbers per plant by direct observation Protozoa
Astasiidae 3.0 2.5 2.5 ni! ni! ni!
Bodo >30 >30 >30 8.0 ni! ni!
EupZotes 5.0 2.0 2.5 1.5 1.5 ni!
LacpyrnaPia 3.0 2.5 2.5 1.5 ni! ni!
Oikomonas >30 ni! ni! ni! ni! ni!
VopticeZZa >30 >30 >30 >30 ni! ni!
Holotrichous Ci!iate >30 >30 >30 5.0 2.5 ni!
Rotifers
LepadeZZa 6.75 6.5 6.0 3.0 2.0 1.5
PhiZodina 4.0 2.5 0.5 3.0 2.0 ni!
Gastrotrichs
Lepidodeme Z Za 1. 75 1.0 1.5 1.0 ni! ni!
Oligochaetes
AeoZosoma 0.25 0.1 0.1 0.1 ni! ni!
Nematodes 0.35 0.5 0.3 ni! ni! ni!
* Data reported as the average of 2 flasks, 10 plants per flask sampled.
** Not detected
115·
TABLE 17.
Population 1evels of Lemna minor rhizosphere after 20 days of exposure to
* Baygon
BaIgon concentration (llg/m1~
Initial nil 0.01 0.1 1.0 10.0 50.0
After 20 days N.D.** N.D. 0.02 0.23 2.20 10.1
pH 7.60 7.50 7.45 7.45 7.50 7.55
Data reported as numbers per 0.25 ml a1iquot
Bacterial x 105 13.0 13.0 15.0 8.4 9.0 6.0
Data reported as numbers per plant by direct observation Protozoa
Astasiidae 2.0 0.5 nil nil nil nil
Bodo >30 3.0 2.5 1.5 1.0 0.25
EupZotes 3.0 0.5 1.0 0.5 0.5 nil
Laarymaria 3.0 1.0 1.25 nil 0.1 nil
Oikomonas >30 nil nil nil nil nil
VortiaeZZa >30 >30 >30 >30 nil nil
Ho1otrichous Ciliate >30 >30 -10 3.0 0.5 0.25
Rotifers
LepadeZZa 10.0 3.5 2.5 1.5 0.5 nil
PhiZadiiza 4.0 3.0 3.0 2.0 0.5 nil
Gastrotrichs
Lepidodeme Z Za 2.0 0.75 0.25 0.25 nil nil
Oligochaetes
AeoZosoma 0.35 0.25 0.25 0.2 0.1 nil
Nematodes 0.25 0.75 0.25 nil nil nil
* Data reported as the average of 2 f1asks, 10 plants per fiask samp1ed.
** Not detected
L:
116
T A BLE 18.
Population levels of Lemna minor rhizosphere after 30 days of exposure to
* Baygon
Baygon concentration (~g/ml)
Initial
After 30 days
pH
ni! 0.01
N.D.** N.D.
7.60 7.45
0.1
N.D.
7.55
1.0
0.09
7.45
10.0 50.0
0.9 4.1
7.35 7.45
Data reported as numbers per 0.25 ml aliquot
Bacterial x 105 II.5 20.0 15.0 4.4 3.6 3.5
Data reported as numbers per plant by direct observation Protozoa
Astasiidae 3.0
Bodo ~30
Eup Zo tes 3.0
Laarymaria 1.5
Oikomonas > 30
VortiaeZZa >30
Holotrichous Ciliate >30
Rotifers
LepadeZZa
PhiZodina
Gastrotrichs
LepidoderrneUa
Oligochaetes
AeoZosoma
Nematodes
*
9.5
3.5
2.5
0.1
0.2
0.5
1.0
0.25
0.25
ni!
>30
>30
5.0
1.5
0.75
0.1
0.2
ni!
1.5
1.0
ni!
ni!
>30
4.0
1.0
2.0
ni!
0.2
0.35
ni!
1.5
0.5
ni!
ni!
>30
1.5
0.75
1.25
0.2
ni!
ni!
ni!
nil
0.1
0.1
nU
nU
0.5
1.5
0.2
ni!
nil
ni!
nil
0.2
0.1
0.1
ni!
nil
0.25
nil
ni!
nil
nil
ni!
Data reported as the average of' 2 flasks, 10 plants per flask sampled.
**Not detected
Figure 29. The effects of 0.1 ~g/ml of Elocron on the numbers of
Protozoa, Rotifers and Gastrotrichs per plant of the
Lemna mina!' rhizosphere over a period of 30 days.
)
)200
150
... z
100 <!: ..J
~ Cf)
:! 50 Cf) -Z <t (!) 0:: 0 u.. 0 0 z
o
ELOCRON OJ.ug/ml
_. •
 PROTOZOA • ROTIFERS . o GASTROTRICHS
_e __ --------0------____ __ -e-· 0 _0-------:--
5 10 15 20 ·25 30
DAYS
Figure 30. The effects of 5~0 pg/ml of Elocron on the numbers of
Protozoa, Rotifers and Gastrotrichs per plant of the
Lemna minor rhizosphere over a period of 30 days.
... z « .J
~ CI)
~ ~ z « C) a: 0 u.. 0 d z
100
50
8
4
o 5·
ELOCRON ·50JJg!ml
. 10· 15
DAYS
20
 PROTOZOA e ROTIFERS o GASTROTRfCHS
25 . 30
)
Figure 31. The effects of E1ocron at concentrations, 1.0, 10.0
and 50.0 pg/ml on the bacteria1 population per plant
of the Lemna minop rhizosphere over a period of 30
days.
-t .. j
32
24 11)0
)(
1- 20 Z <t ..J
~ <t 16 -a: lIJ 1-0 « m LI.. 0 . 0 :z
o 5
ELOCRON
e-------------e
·~B t._
0
10 15
DAYS
-t.
-0_
20
o CONTROL ., 50.0 JJg Iml • 10.0.ug 1 ml' t. 1.0 JJg/ml
•
t.
-0
25 30
120
\j
TABLE 19.
Population levels of Lemna minor rhizosphere before exposure to * Elocron
pH 7.60 7.65 7.60 7.55 7.65 7.60
Data reported as numbers per 0.25 ml aliquot
Bacterial x 105 8.9 9.1 8.1 10.1 9.7 8.4
Data reported as numbers per plant by direct observation
Protozoa
Astasiidae 3.5 2.5 2.0 3.0 2.25 2.0
Bodo >30 >30 >30 >30 >30 >30
EupZotes 4.0 6.0 6.5 2.5 3.5 4.0
Lac:X'ymaria 3.5 4.0 3.0 2.5 2.0 2.5
Oikomonas >30 >30 >30 >30 >30 >30
Vortic:eUa >30 >30 >30 >30 >30 >30
Holotrichous Ciliate >30 >30 >30 >30 >30 >30
Rotifers
LepadeZZa 6.0 6.5 4.5 3.5 4.0 3.5
PhiZodina 3.5 2.0 4.0 3.5 4.0 5,.0
Gastrotrichs
LepidodermeZZa 2.0 1.0 2.0 2.0 2.0 2.0
Oligochaetes
AeoZosoma 0.25 ni! 0.25 0.3 0.2 0.2
Nematodes 0.1 0.25 0.2 0.2 ni! 0.25
* Data reported as the average of 2 flasks, 10 plants per flask sampled.
'.
121
TA BLE 20.
Population 1eve1s of Lemna minor rhizosphere after 2 days of exposure to
E1ocron*
E1ocron concentration (llg!ml)
Initial nil 0.01 0.1 1.0 10.0 50.0
After 2 days N.D.** N.D. N.D. N.D. N.D. N.D.
pH 7.60 7.50 7.65 7.70 7.65 7.60
Data reported as numbers per 0.25 ml a1iquot
Bacteria1 x 105 8.5 10.0 5.9 14.0 16.0 18.0
Data reported as numbers per plant by direct observation Protozoa
Astasiidae 2.0 2.0 nil nil nU nil
Bodo >30 >30 ,,20 -10 -10 nU
EupZotes 6.5 nil nil nil nil nil
Lacrymaria 1.0 2.0 nil 0.5 nil nU
Oikomonas >30 >30 >30 >30 >30 nn
VorticeZZa >30 >30 >30 >30 -10 nU
Ho1otrichous Ciliate >30 >30 -10 -20 5.0 1.0
Rotifers
LepadeZZa 3.5 4.5 1.0 1.0 0.5 nil
PhiZodina 3.0 4.0 2.0 1.5 0.5 nil
Gastrotrichs
LepidodemeZZa 2.0 2.0 nil nil nU nil
01igochaetes
AeoZosoma 0.2 0.25 0.1 nil nU nil
Nematodes 0.1 0.2 nil 0.1 nU nil
* Data reported as the average of 2 f1asks, 10 plants per f1ask samp1ed.
** Not detected
122
T A BLE 21.
Population 1eve1s of Lemna minor rhizosphere after 10 days of exposure to
E1ocron*
E1ocron concentration (~g/m1)
Initial
After 10 days
pH
nil 0.01 ** N.D. N.D.
7.65 7.70
0.1
N.D.
7.60
0.1
N.D.
7.55
10.0
N.D.
7.65
Data reported as numbers per 0.25 ml a1iquot
50.0
N.D.
7.70
Bacteria1 x 105 12.0 13.0 16.0 18.0 20.0 27.0
Data reported as numbers per plant by direct observation Protozoa
Astasiidae
Bodo
Eupl,otes
Laarymaria.
Oikomonas
VortiaeUa
Ho1otrichous Ci1iate
Rotifers
Lepadel,l,a
Phil,odina
Gastrotrichs
Lepidode1'TTlel,l,a
Oligochaetes
Aeol,osoma
Nematodes
*
2.5
>30
4.0
1.5
>30
>30
>30
2.0
3.0
1.5
0.15
0.25
nil
nil
>30
>30
>30
2.5
2.0
2.0
0.1
0.1
0.5
-20
1.0
nil
>30
>30
-20
1. 75
2.0
0.25
0.2
nil
nil
-15
nil
0.2
>30
>30
-20
2.5
2.0
nil
nil
nil
0.1
ni!
nil
nil
5.0
>30
0.5
0.5
1.0
nil
nil
nil
nil
nil
nil
nil
nil
nil
3.5
0.5
0.75
nil
nil
Data reported as the average of 2 flasks, 10 plants per flask sampled.
** Not detected
123
T A BLE 22.
Population levels of Lemna minor rhizosphere after 20 days of exposure to
* Elocron
Elocron concentration (us/ml)
Initial
liter 20 days
pH
nil .01
** N.D. N.D.
7.70 7.60
0.1
N.D.
7.60
1.0
N.D.
7.65
10.0
N.D.
7.70
Data reported as numbers per 0.25 ml a1iquot
50.0
N.D.
7.70
Bacterial x 105 13.0 12.0 17.0 17.0 18.0 29.0
Data reported as numbers per plant by direct observation Protozoa
Astasiidae
Bodo
EupZotes
Laarymaria
Oikomonas
VortiaeUa
Holotrichous Ciliate
Rotifers
LepadeZZa
PhiZodia
Gastrotrichs
LepidodermeZZa
Oligochaetes
AeoZosoma
Nematodes
*
3.0
>30
4.0
2.5
-20
>30
>30
3.5
3.0
2.0
0.1
nil
2.0
-10
nil
1.5
-20
>30
>30
2.5
3.0
1.5
0.25
0.2
nil
-10
nil
nil
>30
>30
>30
1.5
1.0
2.0
0.1
0.1
0.5
nil
nil
nil
-20
-20
-20
1.2
1.0
nil
nil
nil
nil
nil
nil
0.5
nil
5.0
-10
1.5
0.75
nil
nil
nil
nil
nil
nil
nil
nil
nil
nil
1.0
0.75
nil
nil
nil
Data reported as the average of 2 flasks, 10 plants per flask samp1ed.
** Not detected
124
TA BLE 23.
Population 1evels of Lemna minor rhizosphere after 30 days of~·exposure to
E1ocron*
E1ocron concentration (J.Ig!ml)
Initial nil 0.01 0.1 1.0 10.0 50.0
After 30 days N.D.** N.D. N.D. N.D. N.D. N.D.
pH 7.75 7.65 7.60 7.50 7.55 7.55
Data reported as numbers per 0.25 ml a1iquot
Bacteria1 x 105 11.5 15.0 19.0 17.0 21.0 24.0
Protozoa Data reported as numbers per plant by direct observation
As tas iidae 2.75 3.0 nil nil nil nil
Bodo >30 -20 -10 -10 nil nil
E1A;"[)7,~i;e:~ 5.5 2.0 nil nil nil nil
IaCT'ymaria 3.0 2.5 1.5 nil nil nil
Oikomonas >30 >30 -20 -20 nil nil
Vortice Ua >30 >30 >30 >30 >30 nil
Ho1otrichous Ciliate >30 >30 >30 -20 -10 nil
Rotifers
Lepade 7, 7,a 3.75 2.5 2.0 1.5 1.5 1. 75
Phi7,odina 4.0 3.5 2.0 0.75 0.5 0.5
Gastrotrichs
Lepidoderme 7, 7,a 4.0 2.0 2.5 nil nil nil
Oligochaetes
Aeo7,osoma 0.2 0.2 0.1 0.2 0.25 nil
Nematodes 0.1 0.05 0.2 nil ni1 nil
* Data reported as the average of 2 f1asks, 10 plants per f1ask samp1ed.
** Not detected.
Figure 32. The effects of 0.1 ~g/m1 of Ch1orfenvinphos on the
numhers of Protoeoa, Rotifers and Gastrotrichs per
plant of the Lenma mirzor rhizosphere over a period
of 30 days.
)
)
i ... _"
... z :3 100
~ CI)
.~ 50 z « (!) 0:: o IL o ci z
4
0
CHLORFENVINPHOS 0.1 )Jg/ml
 PROTOZOA • ROTIFERS o GASTROTRICHS
__ -------A----__________ ~ ________ --4
_0_
e- -e ,.------0_ .
2 5 10 15 20 25 30
DAYS
Figure 33. The effects of 50.0 ~g/ml of Chlorfenvinphos on the
numbers of Protozoa, Rotifers and Gastrotrichs per
plant of the Lemna minop rhizosphere over a period
of 30 days.
)
)
t-~IOO ..J 0-
~ ~50 ~ z « (!) 0:: 0 I.L. 0 ci :2
o 2 5
CHLORFENVINPHOS 50.0 )Jg/ml
10 15 DAYS
...
20
 PROTOZOA • ROTIFERS o GASTROTRICHS
25 30
Figure 34. The effects of Ch1orfenvinphos at 3 concentrations,
1.0, 10.0 and 50.0 ~g/m1 on the bacterial popula-
tion per plant of the Lemna minor rhizosphere over
a period of 30 days.
\ 1
28
24 "b
)(
1-~20 .J Q.
" !$ 16 .0:
bJ 1-
~ al u.. o ci z
4
o
CHLORFENVINPHOS
o CONTROL .50ug/ml iii 10 JJ9/ml
.. ~·I }JO! ml
.--------.-----.
- _____ a
~
-----à
5
0_-----0 _0------
10 1.5
CAYS
20 25 30
128
-
TABLE 24.
Population levels of Lemna minor rhizosphere before exposure to
Chlorfenvinphos*
pH 7.50 7.40 7.50 7.55 7.60 7.55
Data reported as numbers per 0.25 ml aliquot
Bacterial x 105 3.7 5.2 4.4 3.0 5.3 3.8
Data reported as numbers per plant by direct observation Protozoa
Astasiidae 3.5 3.25 4.25 3.75 4.5 3.25
Boq.o >30 >30 >30 >30 >30 >30
EupZotes 4.0 3.0 4.25 4.0 3.5 3.75
Laarymaria 4.25 3.5 5.0 2.5 3.5 3.5
Oikomonas >30 >30 >30 >30 >30 >30
VortiaeZZa >30 >30 >30 >30 >30 >30
Holotrichous Ciliate >30 >30 >30 >30 >30 >30
Rotifers
LepadeZZa 5.0 5.5 4.75 5.25 5.0 5.5
PhiZodina 3.5 3.25 3.5 4.0 4.0 3.5
Gastrotrichs
Lepidoderme Z Za 1.5 1.75 2.0 3.0 1.5 2.0
Oligochaetes
AeoZosoma 0.1 0.2 0.25 0.2 0.1 0.2
Nematodes 0.1 0.1 0.15 0.20 0.2 0.2
* Data reported as the average of 2 flasks, 10 plants per flask sampled.
129
T A BLE 25.
Population 1eve1s of Lemna mino~ rhizosphere after 2 days of exposure to
Ch1orfenvinphos*
Chlorfenvinphos concentration (~g/m1)
Initial
After 2 days
pH
ni! 0.01
N.D.** N.D.
7.60 7.60
0.1
0.04
7.60
1.0 10.0 50.0
0.33" 3.5 '16.4 '
7.55 7.65 7.60
Data reported as numbers per 0.25 ml a1iquot Bacteria1 x 105 2.8 3.1 8.6 8.2 9.4 13.0
Data reported as numbers per plant by direct observation Protozoa
Astasiidae 3.5
Bodo >30
Eup lotes 4.5
Io.c7!ym~ia 3.0
Oikomonas > 30
Vo~ticella >30
Ho1otrichous Ci1iate >30
Rotifers
Lepadella
Philodina
Gastrotrichs
Lepidoderrnella
Oligochaetes
Aeolosoma
Nematodes
*
5.0
4.0
2.5
0.25
0.20
ni!
>30
3.0
0.25
>30
>30
>30
2.5
2.5
1.0
0.25
ni!
ni!
:..10
ni!
ni!
-20
>30
-10
1.0
0.75
1.0
0.1
ni!
ni!
5.5
ni!
ni!
5.5
>30
5.0
0.25
0.5
ni!
ni!
ni!
ni!
ni!
ni!
ni!
ni!
10
ni!
ni!
ni!
ni!
nil
nil
ni!
ni!
ni!
ni!
ni!
ni!
ni!
ni!
ni!
ni!
nil
ni!
Data reported as the average of 2 f1asks, 10 plants per f1ask samp1ed.
** Not detected
.130
.... ~
TA B LE 26.
Population levels of Lemna mtnop rhizosphere after 10 days of exposure to
Chlorfenvinphos *
Chlorfenvinphos concentration (Pg/ml)
Initial nil 0.01 0.1 1.0 10.0 50.0
After 10 days N.D.** N.D. 0.03 0.25 2.9 12.2
pH 7.60 7.60 7.60 7.55 7.65 7.60
Data reported as numbers per 0.25 ml aliquot
Bacterial x 105 4.2 6.6 7.0 8.0 16.0 26.0
Data reported as numbers per plant by direct observation Protozoa
Astasiidae 3.5 1.0 1.5 1.0 1.0 nil
Bodo >30 -20 5.0 -15 5.5 nil
EupZotes' 4.0 nil nil ni! ni1 nil
LaapymaPia 3.5 1.0 ni! ni! nil nil
Oikomonas >30 >30 >30 >30 >30 >30
VoptiaeUa >30 >30 -10 5.0 3.0 nil
Ho1otrichous Ciliate >30 >30 >30 5.0 5.5 nil
Rotifers
LepadeZZa 4.0 3.5 2.0 1.5 0.25 nil
PhiZodina 4.0 2.5 2.0 1.0 ni1 nil
Gastrotrichs
LepidodePIT/eUa 2.0 0.5 nil nil ni1 nil
Oligochaetes
AeoZosoma 0.2 0.25 0.2 0.1 ni1 ni1
Nematodes 0.1 0.1 ni! ni! nil nil
* Data reported as the average of 2 f1asks, 10 plants per flask sampled.
** Not detected
131
T A BLE 27.
Population levels of Lemna minop rhizosphere after 20 days of exposure to
Chlorfenvinphos*
Chlorfenvinphos concentration (~g/ml)
Initial
After 20 days
pH
ni! 0.01
N.D.** N.D.
7.60 7.60
0.1 1.0 10.0
0.02 0.2: 2.0
7.50 7.45 7.60
Data reported as numbers per 0.25 ml aliquot
Bacterial x 105 4.6 9.6 11.0 11.0 16.0
50.0
:.9.9
7.60
28.0
Data reported as numbers per plant by direct observation Protozoa
Astasiidae 4.0
Bodo >30
EupZotes 2.0
LaarymaPia 1.5
Oikomonas >30
VortiaeZZa >30
Holotrichous Ciliate >30
Rotifers
LepadeZZa
PhiZodina
Gastrotrichs
LepidodemeZZa
Oligochaetes
AeoZosoma
Nematodes
*
4.0
4.0
2.0
0.3
0.15
4.0
-20
ni!
1.0
>30
>30
>30
3.5
2.5
1.0
0.1
0.05
2.0
5.0
ni!
1.0
>30
-10
-10
3.0
2.0
ni!
0.1
ni!
1.0
5.5
nil
nil
>30
5.0
5.0
1.5
1.0
ni!
ni!
ni!
1.0
5.5
ni!
ni!
>30
3.0
2.0
0.5
ni!
ni!
ni!
ni!
ni!
ni!
ni!
ni!
>30
ni!
ni!
ni!
ni!
ni!
ni!
ni!
Data report~d as the average of 2 flasks~ 10 plants per flask sampled. ** Not detected
132
T A BLE 28.
Population levels of Lemna minor rhizosphere after 30 days of exposure to
Chlorfenvinphos*
Chlorfenvinphos concentration (pg/ml)
Initial ni! 0.01 0.1 1.0 10.0 50.0
After 30 days N.D.** N.D. 0.02 0.17 1.4 8.5
pH 7.50 7.55 7.65 7.60 7.60 7.50
Data reported as numbers per 0.25 ml a1iquot
Bacteria1 x 105 5.0 17.0 17.0 14.0 19+0. 26.0
Data reported as numbers per plant by direct observation Protozoa
As tas iidae 6.0 5.0 2.0 2.5 0.5 nil
Bodo >30 -10 5.0 5.5 5 •. 0- 2.0
EupZotes 2.0 ni! ni! ni! ni! nil
Ia.apyrna:r.'ia 2.5 1.5 0.5 ni! ni! ni!
Oikomonas >30 >30 >30 >30 >30 >30
VortiaeZZa >30 >30 -10 ni! 2.5 ni!
Holotrichous Ciliate >30 >30 -10 -10 5.5 nil
Rotifers
LepadeZZa 3:ê 3.75 2.0 2.0 1.5 ni!
PhiZodina 3.0 2.5 2.0 1.0 ni! ni!
Gastrotrichs
LepidodeZ'TTleZZa 1. 75 1.5 ni! ni! ni! nil
Oli8°chaetes
AeoZosoma 0.3 0.1 0.15 0.2 ni! ni!
Nematodes 0.2 0.1 ni! ni! ni! ni!
* Data reported as the average of 2 flasks, 10 plants per f1ask sampled.
** Not detected
Figure 35. The effects of 0.1 llg/ml of Dasanit on the mnnbers of
Protozoa, Rotifers and GastrotriChs per plant of the
Lemna mino~ rhizosphere over a period of 30 days.
)
:>200
t-~ 100 -1
~ Cf)
~ Cf) -z « (.!) 0: o IL o o 2
o 2 ·5
DASANIT O.lJJ9/mt
_Â
 PROTOZOA e ROTIFERS . ô GASTROTRICHS
-------- ~=======~
10 15
DAYS
20 25
Figure 36. The effects of 50.0 ~g/m1 of Dasanit on the numbers of
Protozoa, Rofiers and Gastrotrichs per plant of the
Lemna minop rhizosphere over a period of 30 days.
>200
fZ
150
:3 100
~ CI)
~ 50 -Z <t C) a:: o 12 la.. o o z
4
o 2 10
DASANIT 50.0.ug/ml
15
DAYS
20
 PROTOZOA o ROTIFERS o GASTROTRICHS
..
25 30
Figure 37. The effects of Dasanit at 3 concentrations, 1.0, 10.0
and 50.0 ~g/m1 on the bacterial population per plant
of the Lemna minop rhizosphere over a period of 30
days.
)
24 "b -)(
1-Z 20 <:t .J a.
""~ 16 ct: l1J t-~ al 12· LL. o o 2 8
4
o 5
DASANIT
o CONTROL .50JJg/ml Il 10 ug/ml
.~.
.-à-
------0-
10 15 DAYS
20
-Ii:
_1:::.
_0
25 30
136
T AB L E 29.
Population 1eve1s of Lemna mino!' rhizosphere before exposure to
Dasanit*
pH 7.70 7.60 7.55 7.60 7.60 7.65
Data reported as numbers per 0.25 ml 1iquot
Bacteria1 x 105 8.9 12.0 7.2 12.0 11.0 12.0
Data reported as numbers per plant by direct observation Protozoa
As tas iidae 3.0 4.5 5.5 4.0 3.5 4.0
Bodo >30 >30 >30 >30 >30 >30
EupZotes 5.0 5.5 6.5 4.75 5.5 6.75
Lacryma:t'ia 2.5 3.5 4.0 3.0 3.5 3.0
Oikomonas >30 >30 >30 >30 >30 >30
VorticeUa >30 >30 >30 >30 >30 >30
Ho1otrichous Ciliate >30 >30 >30 >30 >30 >30
. 'R6tifers
LepadeZ'la 6.5 5.5 3.5 5.5 4.0 4.5
PhiZodina 4.5 3.5 4.0 4.5 3.0 4.0
Gastrotrichs
Lepidoderme 'l 'la 2.5 2.0 2.5 2.5. 1. 75 ).·9
Oli8ochaetes
AeoZosoma 0.25 0.15 0.15 0.2 0.25 0.2
Nematodes 0.1 0.15 0.2 0.15 0.15 0.2
* Data reported as the average of 2 f1asks, 10 plants per f1ask samp1ed.
T A BLE 30.
Population 1eve1s of Lemna minop rhizosphere after 2 days exposure to
Dasanit*
Dasanit concentration (pg/ml)
Initial
After 2 days
pH
ni! 0.01
N.D.** N.D.
7.6 7.65
0.10
0.05
7.7
1.0
0.45
7.7
10.0 50.0
5.6 31. 7
7.65 7.7
Data reported as numbers per 0.25 ml a1iquot
Bacteria1 x 105 10.0 13.0 14.0 16.0 17.0 19.0
Data reported as numbers per plant by direct observation
Protozoa
Astasiidae 4.0
Bodo >30
EupZotes 3.0
Lactymapia 3.5
Oikomonas >30
VopticeZZa >30
Ho1otrichous Ciliate >30
Rotifers
UpadeZZa
PhiZodina
Gastrotrichs
Lepidode!'TTIe Z Za
Oligochaetes
Aeowsoma
Nematodes
5.0
4.5
3.0
0.25
0.15
4.0
>30
2.0
1.0
>30
>30
>30
2.25
1. 75
1.5
0.2
0.1
0.5
,..20
ni!
1.0
>30
>30
>30
0.5
0.75
0.5
0.2
0.1
ni!
-10
ni!
0.5
-20
-20
>30
0.25
0.5
ni!
0.1
ni!
ni!
ni!
ni!
ni!
-10
-10
ni!
0.2
0.5
ni!
ni!
ni!
ni!
ni!
ni!
ni!
ni!
ni!
ni!
ni!
ni!
ni!
ni!
ni!
* Data reported as the average of 2 f1asks, 10 plants per f1ask sampled. ** Not detected
137
138
,. II-
TABLE 31.
Population 1eve1s of Lemna mino~ rhizosphere after 10 days of exposure to D - * asan~t
Dasanit concentration (llg/ml)
Initial nil 0.01 0.1 1.0 10.0 50.0
After 10 days N.D.** N.D. N.D. 0.2- 3.9: . 20'-7-
pH 7.65 7.7 7.55 7.7 7.7 7.65
Data reported as numbers per 0.25 ml a1iquot
Bacteria1 x 105 8.6 12.0 12.0 14.0 19.0 22.0
Data reported as numbers per plant by direct observation Protozoa
Astasiidae 4.5 4.0 0.5 nil nil nil
Bodo >30 >30 >30 -10 nil nil
EupZotes 4.5 3.5 1.0 nil nil nil
Lac1Jyma1'ia 3.5 1.5 1.0 nil nil nil
Oikomonas >30 >30 >30 -10 nil nil
Vo~ticeUa >30 >30 >30 -10 nil nil
Ho1otrichous Ciliate >30 >30 >30 >30 5.0 nil
. RCitiférs
LepadeZZa 6.25 5.0 1.5 0.75 0.2 nil
PhiZodina 4.0 2.25 1.5 0.5 0.1 nil
Gastrotrichs
Lepidode1'Tl7eZZa 3.5 1. 75 0.5 0.2 nil nil
Oligochaetes
AeoZosoma 0.3 0.2 0.15 0.15 0.05 nil
Nematodes 0.1 0.1 0.2 0.05 0.05 nil
* Data reported as the average of 2 flasks, 10 plants per flask samp1ed. ** Not detected
.139
TA BLE 32.
Population levels of Lemna minor rhizosphere after 20 days of exposure to
Dasanit*
Dasanit concentration (llg/ml)
Initial ni! 0.01 0.1 1.0 10.0 50.0
After 20 days N.D.** N.D. N.D. N.D. a.8. 4.2.
pH 7.6 7.6 7.5 7.7 7.7 7.6
Data reported as numbers per 0.25 ml aliquot
Bacterial x 105 8.6 .15.0 16.0 18.0 23.0 26.0
Data reported as numbers per plant by direct observation Protozoa
As tas iidae 5.0 4.0 1.0 ni! ni! ni!
Bodo >30 >30 >30 -10 ni! ni!
EupZotes 4.5 3.0 3.5 ni! ni! ni!
Lacrymaria 3.0 2.0 2.0 ni! ni! ni!
Oikomonas >30 >30 >30 5.0 ni! ni!
VorticeZZa >30 >30 >30 -10 ni! ni!
Holotrichous Ci!iate >30 >30 >30 -10 ni! ni!
Rotifers
LepadeZZa 5.5 3.0 1.5 0.75 0.5 ni!
PhiZodina 4.5 2.0 1.0 0.5 0.5 ni!
Gastrotrichs
LepidoderrneZZa 3.5 2.5 2.0 0.1 ni! ni!
Oligochaetes
AeoZosoma 0.4 0.25 0.1 0.1 ni! ni!
Nematodes 0.2 0.15 0.15 ni! nil ni!
* Data reported as the average of 2 flasks, 10 plants per flask samp1ed. ** Not detected
140
T A BLE 33.
Population levels of Lemna mino~ rhizosphere after 30 days of exposure. to
Da.,anit*
Dasanit concentration (~g/ml)
Initial
After 30 days
pH
nïl 0.01
N.D.** N.D.
7.7 7.8
0.1
N.D.
7.6
1.0 10.0 50.0
N.D.· N.D. N.D.
7.65 7.65 7.7
Data reported as numbers per 0.25 ml aliquot
Bacterial x 105· 10.0 13.0 19.0 19.0 22.0 28.0
Data reported as numbers per plant by direct observation Protozoa
Astasiidae 5~5
Bodo >30
EupZotes 6.5
Lac~ym~ia 2.0
Oikomonas >30
Vo~ticeZZa >30
Holotrichous Ciliate >30
Rotifers
LepadetZa 3.75
PhiZodina 3.5
Gastrotrichs
Lepidode~eZZa
Oligochaetes
AeoZosoma
Nematodes
*
3.0
0.3
0.2
3.0
>30
5.0
1.5
>30
>30
>30
3.5
3.0
4.0
0.1
0.25
0.1
-10
3.0
3.0
-10
>30
>30
1.5
1.0
2.5
0.1
0.1
nïl
-10
nïl
1.0
5.0
-10
-10
2.0
0.5
1.0
0.2
0.05
nïl
nïl
nïl
nïl
nïl
nïl
nïl
1.0
0.5
0.1
0.5
0.1
ni1
ni!
nïl
nïl
ni!
ni!
ni!
ni!
ni!
ni!
ni!
ni!
Data reported as the average of 2 f1asks, 10 plants per flask samp1ed. ** Not detected
Figure 38. The effects of 0.1 ~g/ml of Carboxin on the numbers of
Protozoa, Rotifers and Gastrotrichs per plant of Lemna
minor rhizosphere over a period of 30 days.
1-'Z
:3 100·
~ en ~ 50 -z « C)
~ 12 IL o o z
4
o
CARBOXIN 0.1 pg/m 1
'-Â
-----~..-------. ,
Â. PROTOZOA • ROTIFERS o GASTROTRICHS
-e
"o---------o------------o------~ ____ o
5 10 15
DAYS
20 25 30
Figure 39. The effects of 50.0 ~g/ml of Carboxin on the numbers
of Protozoa, Rotifers and Gastrotrichs per plant of
Lemna minop rhizosphere over a period of 30 days.
1-z :j100
~ CI)
~ 50 2 ~ 0:: '0
li. o o ·z
4
5
CARBOXIN 50.0).lg Iml
10 15
DAYS
'.
20
 . PROTOZOA . • ROTIFERS o GASTROTRICHS
25 30
Figure 40. The effects of Carboxin at 3 concentrations, 1.0, 10.0
and 50.0 pg/ml on the bacterial population per plant
of Lemna minar rhizosphere over a period of 30 days.
"b -!z 20 <t -' ~
<t -0: lLI tU « CD LL o
~ 8
4
o
CARBOXIN o C.ONTROL .50JJg/ml .. 10 JJgI ml A IJJg/ml
e-_0
• • _________ m ____________ .~A
__ --------à----------__ _ -.A
5 10 15
DAYS
o
20 25 30
144
TABLE 34.
Population levels of Lemna minop rhizosphere before exposure to
Carboxin*
pH 7.7 7.6 7.65 7.7 7.6 7.6
Data reported as numbers per 0.25 ml aliquot
Bacterial x 105 12.0 9.0 12.0 13.0 11.0 12.0
Data reported as numbers per plant by direct observation Protozoa
Astasiidae 6.0 5.0 4.0 4.5 5.5 6.0
Bodo >30 >30 >30 >30 >30 >30
EupZotes 6.5 6.0 5.0 4.5 5.0 5.0
LacrymŒ1'ia 3.5 4.0 3.5 2.5 3.0 3.5
Oikomonas >30 >30 >30 >30 >30 >30
VorticeUa >30 >30 >30 >30 >30 >30
Ho1otrichous Ciliate >30 >30 >30 >30 >30 >30
Rotifers
LepaàeZZa 4.25 3.5 5.5 6.5 5.0 4.5
PhiZodina 3.5 3.25 3.5 4.0 3.5 3.5
Gastrotrichs
Lepidode!'TTleZZa 2.5 3.0 2.0 2.0 2.25 2.0
Oligotrichs
. . .Aeg·Z,ci8.Qi71a ...... .0.2 0.25 0.3 0.15 0.2 0.15
Nematodes 0.1 0.2 0.2 0.15 0.2 0.15
* Data reported as the average of 2 flasks, 10 plants per flask sampled.
145
T AB L E 35.
Population 1eve1s of Lemna minor rhizosphere after 2 days of exposure to
Carboxin*
Carboxin concentration (lIg/ml)
Initial ni! 0.01 0.1 1.0 10.0 50.0
After 2 days N.A.** N.A. N.~. N.A. N.A. N.A
pH 7.60 7.55 7.65 7.70 7.65 7.60
Data reported as numbers per 0.25 ml a1iquot
Bacterial x 105 9.6 13.0 12.0 16.0 18.0 20.0
Data reported as numbers per plant by direct observation Protozoa
Astasiidae 5.5 4.5 1.5 0.2 ni! ni!
Bodo >30 >30 -20 5.0 ni! ni!
Eup Zo tes 5.0 3.5 1.5 ni! ni! ni!
Laarymaria 2.5 1.0 0.5 ni! ni! ni!
Oikomonas >30 >30 >30 5.0 ni! ni!
VorticeUa >30 >30 >30 -20 5.0 ni!
Ho1otrichous Ci!iate >30 >30 -20 -20 5.0 ni!
Rotifers
Lepade 7, ],a 5.75 4.5 1.5 1.5 1.0 ni1
Phi7,odina 4.0 3.0 1. 75 0.5 0.25 ni1
Gastrotrichs
Lepidoderme7,],a 3.0 1.0 1.0 ni! ni! ni!
Oligochaetes
AeoZosoma 0.30 0.15 0.1 0.15 0.15 ni!
Nematodes 0.1 0.1 0.1 ni! ni! ni1
* Data reported as the average of 2 flasks, 10 plants per flask samp1ed. ** Not analyzed
146
-1 ""-
TABLE 36.
Population levels of Lemna minop rhizosphere after 10 days of exposure to
Carboxin*
Carboxin concentration (lIg/ml)
Initial nil 0.01 0.1 1.0 10.0 50.0
After 10 days N.A.** N.A. N.A. N • .A. N.A. N.A.
pH 7.55 7.60 7.65 7.65 7.60 7.70
Data reported as numbers per 0.25 ml a1iquot
Bacterial x 105 12.0 13.0 10.0 17.0 18.0 24.0
Data reported as numbers per plant by direct observation Protozoa
.1
As tas iidae 4.75- 3.5 1.25 0.05 ni! ni!
'"Bodb >30 >30 -10 5.0 ni! ni!
EupZ,otes 6.0 4.2 2.5 0.1 ni! ni!
Laapyma:1'ia 2.5 1.5 0.05 0.05 ni! ni!
Oikomonas >30 >30 -20 -10 ni! ni!
VoptiaeUa >30 >30 >30 -10 5.0 ni!
Holotrichous Ci!iate >30 >30 -20 -20 -10 0.15
Rotifers
LepadeZ,Z,a 5.5 3.0 2.5 1.25 1.0 ni!
PhiZ,odina 4.0 2.5 1.25 0.5 0.2 ni!
Gastrotrichs
Lepidode1'l7leZ,Z,a 3.0 2.2 1.5 1.0 0.05 ni!
Oligochaetes
AeoZ,osoma 0.2 0.15 0.1 0.15 0.1 ni!
Nematodes 0.3 0.15 0.1 0.1 nil ni!
* Data reported as the average of 2 f1asks, 10 plants per f1ask samp1ed. ** Not analyzed~
147
TABLE 37.
Population levels of Lemna minop rhizosphere after 20 days of exposure to
Carboxin*
Carboxin concentration (ll~/ml)
Initial nil 0.01 0.1 1.0 10.0 50.0
After 20 days N.A.** N .}~. N.A. N.A. N.A. N.A.
pH 7.60 7.70 7.70 7.80 7.65 7.70
Data reported as numbers per 0.25 ml aliquot
Bacterial x 105 8.6 10.0 15.0 16.0 18.0 27.0
Data reported as numbers per plant by direct observation Protozoa
Astasiidae 7.0 4.0 2.2 nil nil nil
Bodo >30 >30 >30 5.0 nil nil
Euplotes 4.5 2.5 1.0 0.15 nil nil
Lac!'Ymapia 3.5 1.0 0.5 0.5 nil nil
Oikomonas >30 >30 >30 5.0 nil nil
VopticeUa >30 >30 >30 -10 2.0 nil
Holotrichous Ciliate >30 >30 >30 -10 3.0 nïl
" "Rôtiférs
Lepadella 6.5 3.25 4.0 1.2 1.0 0.1
Philodina 3.75 2.2 2.0 0.5 nil 0.05
Gastrotrichs
Lepidodemella 2.5 2.0 1.0 1.2 nil nil
Oligochaetes
Aeolosoma 0.25 0.2 0.15 0.1 0.1 nil
Nematodes 0.15 0.15 0.05 0.05 nil nil
* Data reported as the average of 2 flasks, 10 plants per flask sampled. ** Not analyzed"
148
T A BLE 38.
Population 1evels of Lemna minop rhizosphere after 30 days of exposure to
Carboxin*
Carboxin concentration (pg/ml)
Initial
After 30 days
pH
ni! . 0.01
N • .A. ** N.A.
7.65 7.70
0.1 1.0 10.0 50.0
N.!:. N.A. N.A:.
7.65 7.80 7.65 7.70
Data reported as numbers per 0.25 ml a1iquot
Bacteria1 x 105 12.0 8.6 16.0 19.0 20.0 28.0
Data reported as numbers per plant by direct observation Protozoa
Astasiidae 5.5
Bodo >30
EupZotes 5.5
LaarymaPia 3.0
Oikomonas >30
VopticeZZa >30
Ho1otrichous Ciliate >30
Rotifers
Lepadella
PhiZodirr.a.
Gastrotrichs
LepidodeZ'TTleZla
01igochaetes
AeoZosoma
Nematodes
*
5;è 4.0
2.5
0.25
0.3
3.25
>30
3.5
2.0
>30
>30
>30
~;§
2.0
2.0
0.15
0.1
1. 75
>30
2.2
0.75
-20
>30
>30
§;ê 1.5
1.0
0.2
0.1
ni!
-10
ni!
0.2
-10
-20
-10
§;§
2.0
1.0
0.25
ni!
ni!
2.0
ni!
ni!
ni!
2.5
2.0
2:-13 0.1
ni!
0.05
ni!
ni!
ni!
ni!
ni!
ni!
ni!
ni!
0.2
0.2
ni!
ni!
ni!
Data reported as the average of 2 f1asks, 10 plants per f1ask samp1ed. ** . Not analyzed
GENERAL DIS eus S ION
and
CON C LUS ION S
In view of the comp1exity of terres trial ecosystems, a more
appropriate approach wou1d be a discussion of the side-effects of
pesticides on the total soi1 eco10gy rather than certain iso1ated
aspects. However, the actua1 accomp1ishments in this direction are
minimal :i.n these experiments or in 1iterature reports. Most of the
investigations have concentrated on numerica1 shifts in populations
or changes in certain physio1ogica1 activities, as nitrification,
ammonification or cellulose decomposition. Soi1 respiration experi
ments do give some indication of the overa11 changes in the terres
trial ecosystem. Attempts shou1d be made to quantify specifie bio
chemica1 activities of soi1 organisms in such a way that the compon
ents of the soi1 ecosystem can be weighed against each other. Exclu
sion of nematodes and protozoa in the terresttia1 ecosystem is a
serious shortcoming since manifold inter-re1ationships exist between
these groups and microorganisms. How and to what extent the regulating
function of nematodes and protozoa are affected by pesticides remain
to be investigated in the soi1.
Investigation of the effect of pesticides on a total aquatic
ecosystem was carried out in the ab ove research and the components of
149
the aquatic ecosystem were weighed against each other when external
stress was applied. The observed disruption of an intact microbio
coenosis, which included besides bacteria, protozoa, rotifers, nema
todes, gastrotrichs and oligochaetes is a better indication of the
response of an entire ecosystem to stress.
From these data very high concentrations of pesticide are nec
essary to drastically upset the microbial metabolism in the soil but
normal pest control practices will disrupt the aquatic biosphere, since
the pesticide levels in these experiments are weIl within the range
of surface runoff residue levels and they profoundly disrupted the
aquatic ecosystem.
150
\
BIBLIOGRAPHY
Ahmed, M.K., and J.E. Casida 1958 Metabo1ism of Some Organophosphorus Insecticides
by Microorganisms. J. Econ. Entomo1. 51:59-63.
Alexander, M. 1965 Biodegradation: Prob1ems of Mo1ecu1ar Reca1citrance
and Microbial Fa11ibi1ity. Adv. Appl. Microbiol. l= 35-80.
Alexander, M. 1966 Biodegradation of Pesticides.
In: Pesticides and their Effects on Soils and Water. ASA Special Publication No. 8:78-84.
Alexander, M. 1967 The Breakdown of Pesticides in Soils.
In: Agriculture and the Quality of Our Environment. N.C. Brady (Ed.) , Am. Assoc. Advan. Sei., Wash., D.C. pp 331-342.
Alexander, M. 1968 Degradation of Pesticides.
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Alexander, M., and B.K. Lustigman 1966 Effect of Chemica1 Structure on Microbia1 Degradation
of Substitute Benzenes. J. Agr. Food Chem. 14:410-413.
Aly, 0 .M., and M.A. El-Dib 1971 Photodecomposition of Some Carbamate Insecticides in
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151
ASA Special Publication 1966 Number 8
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Audus, L.J. 1960 Microbiologica1 Breakdown of Herbicides in the Soi1s.
In: Herbicides and the Soi1. E.K. Woodford and G.R. Sagar (Eds.) B1ackwe11, Oxford, pp 1-19.
Audus, L.J. 1964 Herbicide Behaviour in the Soi1. II. Interactions with
Soi1 Microorganisms.
Bailey, 1964
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