CULTURAL STUDIES AND EFFECTS OF ‘
PESTICIDES IN NITRIFYTNG SYSTEMS.

Thesis for theDegree. of Ph. D.
MICHIGAN STATE UNIVERSITY
GEORGE GREGORY mos
1969 ,

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This is to certify that the
thesis entitled
CULTURAL STUDIES AND EFFECTS OF
PESTICIDES IN NITRIFYING SYSTEMS

presented by

George Gregory Nakos

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Soil Science

(KM/M

lVlajor professor

 

Date September CLO2 1%2

 

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JUL 1 9 2000

ABSTRACT

CUETURAL STUDIES AND EFFECTS OF PESTICIDES
IN NITRIFYING SYSTEMS

By

George Gregory Nakoa

Physiological characteristics of nitrosomonas eurogaea and gitro-
bacter agilis in mixed liquid culture and effects of agricultural

 

chemicals on their oxidation of ammonium and nitrite were examined
under stationary flask and perfusion conditions.

Persistent heterotrophic contaminants in the mixed culture did
not interfere with stoichiometric conversions of ammonium or nitrite
unless an organic energy source was addedo In the presence of glucose9
trichloro=bisa(#achloropheqyl) ethane [EDT19 N, N-dimethylpz, 2-
diphenylacetamide [diphenamid] 9 and Zachloro-alt, 6abis (ethylamino )-s.
triazine [simazine] completely suppressed growth of these contaminants
at concentrations which had no effect on ammonium or nitrite oxidationo
This suggests a new approach for purifying cultures of uitrosomonas
and Nitrobacter.

Optimum hydrogen ion concentration for nitrification in liquid
culture was found to be at pH about 800 to 900 for flitrosomonas and
600 to 900 for Nitrobactego Disappearance of nitrite at pH below 300
was observed and was likely due to chemical reactions of nitrite.

In stationary flask culture high ammonium levels had little or no
effect on the rate of ammonium oxidation but nitrate production was
restricted to.a low, linear rate characteristic for a static or resting

populationo

George Gregory Nakos

This indicates that growth of flitgggggtgg,rather than nitrite oxidation
‘was suppressed. ‘When ammonium was reduced.below about 100 ppm.NH¢-N,
there was an abrupt increase in rate of nitrate production consistent
with resumption of growth. Nitrite accumulated intermediately in amounts
and over periods of time which were proportional to the initial concen-
trations of ammonium. In enriched perfusion systems, no intermediate
accumulations of nitrite occurred with concentrations as high as 750 ppm
Nah-N. These observations suggest a bacteriostatic effect of either
ammonium or free ammonia on Nitrobacter.

Twenty-seven chemicals were tested for inhibitory effects on
nitrification in mixed culture. Nine were highly specific in their
action against Nitrosomonas over the entire range of the test concentra-
tions (2.5 to 50 ppm). Two were equally specific in their action
against Nitrobacter at 2.5 ppm and five more over the range 10 to 50
ppm. The rest of the chemicals affected both organisms. The concentra-
tions required for complete inhibition of ammonium and/or nitrite
oxidation were increased for ten of the chemicals when soil was added
to the incubation mixture. ‘Water soluble materials in extracts from
the same soil were ineffective in reducing inhibitory effects. This
indicates that insoluble organic or inorganic constituents were
responsible for inactivation by soil.

At low but biologically active concentrations, in mixed culture
of the nitrifying bacteria, 3-amino-1,2,h-triazole [amitrolé] combined
‘with 2-chloro-6-(trichloromethyl) pyridine [N-Serven synergistically
inhibited the oxidation of ammonium byquitggggmgngg. The inhibition

of Nitrobacter by potassium azide and sodium aside in combination was

George Gregory Nakos

also synergistic. Is opropyl-N-phenylcarbamate [IPCJ and is apropyl-N-
(trichlorophenyl) carbamate [GIPC] at low concentrations were antagonistic
in their inhibition of both Nitrosomonas and W. Both synergistic
and antagonistic responses approached additivity as the concentration

of one or both chemicals in a cosbination increased.

In perfusion systems, the inhibition of ammonium and/or nitrite
oxidation by amitrole, CIPC, IPC, potassium aside and sodium aside was
increased on increasing perfusate volumes and was affected by the nature
of column material. The pattern of Nitrosgonas inhibition by andtrole
and its residual effects appeared to depend mostly upon mechanisms. of
its sorption and desorption by column materials. CIPC and IPC inhibited
nitrite oxidation by Nitrobacter. Living or dead organic materials
rather than inorganic column constituents seemed to be responsible for
sorption of these two chemicals, and for inhibition carried over into a
second perfusate. Potassium and sodium azide prevented nitrite oxidation
and none of the chemicals was sorbed by column constituents, which is in
agreement with the absence of any residual effect on re-perfusion with
untreated medium.

Amitrole concentrations in perfusate were determined chemically. A
Nitrosomonas and Nitrobacter bioassay was developed for estimating concen-

trations of the other chemicals.

CULTURAL STUDIES AND EFFECTS OF PESTICIDES
IN NITRIFYING SYSTEMS

By

George Gregory Nakos

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF HIILOSOPHY

Department of Soil Science

1969

TACKNONLEDGMENTS

The author wishes to express his sincere gratitude to Dr.TA.R.
Wolcott for his interest and guidance throughout the course of this
study; The freedom he gave in the selection and execution of research
and his help in the preparation of the manuscript are appreciated.

Special thanks are due to Dr. K. Sommer of the university of Bonn,
Germany, for his teaching and orientation during the initial steps of
the project.

as deeply appreciates the financial support of‘the.George~Bouyoucos
Fellowship and the encouragement of the University of Thessaloniki,
Greece, in awarding him the fellowship to undertake graduate studies
at Michigan State University.

The partial support of the 0.3. Public Health Service under Grant
CC 002h6 is gratefully acknowledged.

ii

'TABLE OF CONTENTS

mmLmeDUcrION000.000.0.00000000000.

pm: I: PHYSIOLOGICAL STUDIES wrrn W AND W

BART

Introduction 0 e e e e 0
Materials and Methods . .
Biological material .
Perfusion apparatus .

Suppression of heterotrophic c

pH effects

Effects of ammonium levels .
Liquid cultur. e e e e e
Perfusion: conditions . .

Rflsults 0nd Discussion 0 e e e e

Heterotrophic contaminants .

pH effects 0 e e e e e e e 0

Effects of ammonium levels .

Summary 0 e e e e e e e e e e e
RbferOHCOS e e e e o e e e e e 0

II:

Introduction

Materials and Methods 0 0
Biological material .
PbStiCidOs e e e e e
3011 CXtr06t e e e 0
3°11 e e e e e e e 0
Interpretation of dat

Results and Discussion .
Fungicides e e e e e
IflSOCtiOid.’ e e e e
Harbicidos e e e e o
Aniline derivatives .
Significance e e e 0

Summary 0 e e e e e e e

RDtOrOHOOS e e e e e e e

0 0 0 0

0 0 0 0 0 0 0 0

0.......0.0.0..

111

O o e e e

e e e e e e e o o e (to o e e

0 0 0 0 . 0 0 0 0 .

n

000......000000

e e o e e e e e o e B's o o e

...000....0.0.0

EFFECTS OF.AGRICULTURALICHEMICALS 0N
LIQUID CULTURES CONTAINING

0 O
0 O
0 0
0 0
nant
0 0
0 0
0 0
0 0
0 0
0 O
0 0
0 0
0 0
0 0

. 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0.00....0000000

0 0 0 0 0 0 0 0 0 0 0 . . 0 0

00.000.00.00...

.0....0.0......

NITRIFICATION

0 0 . 0 0 0 0 0 0 0 0 0 0 0 0

00......0000000

SOIL.OR.SOIL EXTRACT

0.00.00.00.0000

00.00.00.000...

9

0

Page

2;:32;CD~Q~J‘O~O(h(h\n\d\9\dl0 IV I“

TABLE OF CONTENTS (continued)

Page

PART III: SINERGISTIC AND ANTAGONISTIC INHIBITION OF
NITRIFICATION BY HERBICIDES . . . . . . . .

e
e
e
e
e
e
\9
fi

Introduction . . . . .

Materials and MflthOds e e e e e e e e e e e e e e e o e e e e 37
BiOlOfiiCCl M‘t0r101 e e e e e e e e e e e e e o e e e e e 37
ChemicAIS e e o e e e e e e e e e e e e e e e e e e e e e 38
Ass‘y'e e e e e e e o e e e e e e e e e e e e e e e e e e 38
Interpretation 0 e e e e e e e e e e e e e e e e e e e e 38

Results and D13¢u3310n e e e e e e e e e e e e e e e e e e e 39

Summary 0 e e e e e e e e o e e e e e e e e e e e e e e e e o “4

References 0 e e e e e e e e e e e e e e e e e e e e e e e e “5

PART IV: EFFECTS OF PESTICIDES 0N NITRIFICATION IN PERFUSION
SYSTEMS 0 0 0 0 0 0 0 0 0 . 0 0 0 0 0 0 0 0 0 . 0 0

31

Introduction . . . . .

Materials 0nd MflthOds e e e e e e e e e e e e e e e e e e e e 47
3101081081 material 0 e e e e e e e e e e e e e e e e e e “7
Chemicals 0 e e e e e e e e e e e e o e e e e e e o e e e u?
Perfu310n studies 0 o e e e e e e e e e e e e e e e e e e 47
Sorption StUdies e e e e e e e e e e e e e e e e e e e e “9
BiOOSSQy e e e e e e e e e o e e e e e e e e e o e e e e “9

Rbsults ‘nd Discussion 0 e e e e e e e o e e e e e e o e e 51
Nitrification in control systems e e e e e e e e e e e c 51
Stud165'31th amitrole e e e e e e e e e e e e e e e e e e 53
Studies "1th cabeMCtOB e e e e e e e e e e e e o e e e e 58
Studies with asides e e e e e e e e e e e e e e e e e e e 65

Summary 0 e e e e e e e e e e e e e e e e e e e e e e e e e 72

References 0 e e e e e e e e e e e e e e e e e e e e e e e e 73

APPENDIX A - A listing of the trade or cannon and chemical names
and formula. or compounds 0 e e e e e e e e e o e 0
APPENDIX B' - Chemical names and formlae of aniline derivatives
TAPPENDIX C - PerfuBion apparatus e e e o e e e e e o e e e e o e
‘PPENDIX D" Culture fQPIBntor e e e e s o e e o o o e o e e e e

74
75
77

iv

LIST OF TABLES
Page
PART I:
TABLE

I Cation exchange and water holding capacities of
perfusionunits.................... Ll‘

II Effects of chemicals on W (Nm), m
bacter (Nb) and heterotrophic contaminants (Kt)
inmixedculture................... 9

III Effects of ammonium levels on the rates of ammonium
oxidation and nitrate production under perfusion
conditions eeeeeeeeeeeeeeeeeeeeee 16

PART II:
TABLE

I Effects of various fungicides on a mixed culture of

Nitrosogonas (Nm) and Nitrobgcter (Nb) in a liquid
medium with added soil extract or soil . . . . . . . . 25

II Effects of various insecticides on a mixed culture

of Nitrosgmnas (Nm) and Nitrgacter (Nb) in liquid
medium with added soil extract or soil .. . . . . . . . 27

III Effects of various herbicides on a mixed culture of

flitrosomonas (Nm) and Nitrobacter (Nb) in a liquid
medium with added soil extract or soil . . . . . . . . 28

IV Effects of aniline derivatives on a mixed culture

of Nitrosomog (Nm) and Nitrobacter (Nb) in a
liquid medium With add“ 301]- erct e e e e e e e e e 30

V Effects of aniline derivatives on a mixed culture

of Nitrosogonas (Nm) and Nitrobagtgg (Nb) in a liquid
modiumwithadded8011 0000000000000... 32

LIST OF TABLES (continued)
Page
BART III:
TABLE

I Combined effects of amitrole and N-Serve on
amonium oxidation by Nitrosomopg . e e e . . . . e . ’40

II Combined effects of potassium azide and sodium
aside on nitrite oxidation by Nitrobagter . . . . e . 41

III Combined effects of CIPC and IPC on ammonium and
nitrite oxidation by Nitrosomenas and Nitgobagter . . 43

BART IV:
TABLE

I Structural formulae and preperties of chemicals
used in the p0?f“810fl.8t“d105 e e e e e e e e e e e e “8

II Cation exchange and water holding capacities of
perfusion units 0 e e e e e e e e e e .~§ e e e e e e 50

III Effects of amitrole on ammonium oxidation as
influenced by concentration, Porfusate volume
and column material 0 e e e e e e e e e e e e e e e e 5“

IV Effects of CIPC on nitrate formation as influenced
by concentration, perfusate volume and column
material 0 e e e e e e e e e e e e e e e e e e e e e e 59

V Effects of.IPC on nitrate formation as influenced
by concentration, perfusate volume and column
material 0 e e e e e e e e e e e e e e e e e e e e e e 60

VI ‘Ambunts of IPC removed from the initial 9-hour
perfusate as estimated.by the Nitrosoggnas-Nitro-
bQCter biO‘ss‘y e e e e e e e e e e e e e e e e e e e 65

VII Effects of potassium.azide (KN ) on nitrate formation
as influenced by concentration, perfusate volume and
OOIUMD m3t0r181 e e o e e e e e e e e e e e e e e e e 67

PART IV:
TABLE
VIII

LIST OF TABLES (continued)
Page

Effects of sodium azide (NaN ) on nitrate formation
as influenced by concentration, perfusate volume
WCOIWMtOflCleeeeeeeeeeeeeeeeee Q

Amounts of potassium aside (KN ) removed from the
initial 9-hour perfusate as esgimated by the

Nitgosomonas-Nitrobacgr bioassay . . . . . . . . . . e 70

vii

PART I:
FIG.
1.

2.

PART IV:
FIG.
1.

2.

3.

’4.

5.

LISI‘ OF FIGURES

Effect of pH on nitrification in liquid mixed

culture of Nitgg enemas and Nitrobagter.
(Incubation for days . Initial nitrogen levels =

. Nah-N, 50 ppm; NOE-N, 50 ppm; No'5-N,‘ 10 ppm) . . . . .

Effects of ammonium levels on disappearance of
ammonium and appearance of nitrite and nitrate in
liquid mixed culture of Nitzos gonas and mm.

pH=8eO-8e3eeeeeeeeeeeeeeeeeeee

Nitrification in glass bead columns (left) and
vermiculite-parlite (right) inoculated with

Nitrosogqnas and Nitrobacter. Perfusate volume =

200 OOOCOOOOOOOOOOOOOOOOOOOO

Ammonium oxidized (-——-) and changes in sorbed
amitrole ( ) during perfusion with 50 ml
solution. containing, initially, 10 ppm amitrole . . .

 

 

Ammonium oxidized and changes in sorbed amitrole
after replacing the perfusate in Fig. 2 with 200 m1
untreated nutrient solution e e e e e e e e e e e e e

Effects of CIPC and IPC on ammonium oxidation during
perfusion through glass beads ( ) and
vermiculite-perIite ( -). (Perfusate
volume=200ml).....ooo..........o

 

 

 

Nitrite found during initial perfusion (A ) with
200 ml nutrient solution containing 80 ppm IPC or
1&0 ppm CIPC and during subsequent perfusion (0)
With 200 ml untreated nutrient solution . . e e e . .

viii

Page

13

56

56

62

63

 

PART IV:
FIG.
6.

7.

LIS]? OF FIGURES (continued)

Page
Effects of sodium (NaN ) and potassium (KN ) aside
on ammonium oxidation during perfusion thrgugh glass
beads (—————) and vermiculite-perIite
(— "— —)e (Perfusate V01“ 3 200 III) e e e e e e 69

Nitrite found during initial perfusion (A ) with

200 ml nutrient solution containing 1.0 ppm

M or 1.5 ppm KN and during subsequent perfusion

(03 with 200 .1 treated nutrient solution . . . . . . 71

GENERAL INTRODUCTION

The application of pesticides to soils and plants to control
insects, plant diseases and weeds is a virtual necessity for human
survival in our present economic system. EValuation of the effects of
these materials shows that in addition to reducing numbers of specific
parasites they may alter the ecological balance of beneficial microbial
populationS.

Nitrification in soil is considered an important biological process
with regard to effective nitrogen nutrition of plants and other organisms
living in soil. This process is mainly effected by two groups of
aerobic chemolithotrophic bacteria, Nitros omonas and Nitrobacter.
Ammonium is oxidized to nitrite by Nitrosogonas and nitrite to nitrate
by Nitrobacter.

The objectives of this research were (1) to study some physiological
characteristics of these two organisms in mixed culture, (2) to look for
synergistic or antagonistic interactions among chemicals which inhibit
one or both organisms and (3) to investigate the extent to which solid
surfaces in stationary flask or perfusion culture may alter the degree
to which these organisms are inhibited by several agricultural chemicals .

The research developed logically in four stages. These are the
basis for subdivision of the thesis into four parts. Each part has
been developed with an independent format to facilitate later publication.

 

PART I: PHYSIOLOGICAL STUDIES‘WITHZNITfiQfiQflQNAfigAND NIIBQBAQZEE
Introduction

Nitrification is traditionally considered to be the biological
conversion of ammonium.to nitrate. This is mostly effected in
nature in two steps by two highly specialized groups of aercbic
chemolithotrophic bacteria. In the first step, ammonium is oxidized
to nitrite by the Nitrosomonas group; in the second, nitrite is
oxidized to nitrate by Nitrobagter.

The isolation of nitrifying bacteria in pure culture is difficult.
However, heterotrOphic microorganisms (contaminants) present in liquid
culture of Nitrosgmpnas and NitrObacter seem to have no effect on the
Observed rates of ammonium and nitrite oxidation (9,11).

Nitrification under field and laboratory conditions is affected
by, among other factors, the hydrogen ion concentration (16,20) and
the concentration of ammonium present in the growth medium (5,6,8,19).

Working with mixed liquid cultures of W and Nitrobacter,
this study is concerned with: (a) the selectivity of certain chemicals
in preventing heterotrOphic growth without influencing nitrification,
(b) the effect of hydrogen ion concentration on ammonium and nitrite
oxidation and (c) the effect of ammonium levels on the rates of
ammonium oxidation and nitrate production under liquid.flask culture
and perfusion conditions of growth.

Materials and Methods

Wm- matures ofmsmmandmo-
93299.11 eggs were kindly supplied by Dr. C.L. San Clemente, Department
of Microbiology and Public Health, Michigan State University, East
Lansing, Michigan. A mixed stock culture of these two organisms has
been maintained in the dark for more than two years with no change
in the apparent rate of ammonium oxidation.

The mixed stock culture was maintained in a mineral medium
composed of (nagzsou, 0.300 g; 0.012, 0.136 g; MgS04’7H20, 0.175 g;
NaI-ICOB, 0.500 g; NaZHPOu, 13.500 g; mm“, 0.700 g; Fe304°7320, 5 mg;
Nanoou, 0.375 mg, and one liter of deionized water. The final pH of
the medium was adjusted to 8.0-8.3. All media preparations were
autoclaved (15 p.s .i., 1210). The culture was incubated with forced
aeration at 20-250 in four-liter fermentors and transferred every
10-15 days. An inoculum of l-5fi was used to initiate growth. No
turbidity developed in the stock culture .

Perfusion amratus. Twa1ve positive pressure soil perfusion
percolators (13,18) with cylindrical chambers (5.25 cm2 x 20.2 cm)
and conical reservoirs (250 ml capacity) were used. The chambers
(columns) were packed with three different materials (Table I). Four
units of each material were employed: (1) Glass beads (1.00-1.05 m,
B. Braun, Apparatebau, Melsungen). (ii) Vermiculite-perlite fixture
(commercial sources). (iii) Soil-perJite mixture (air-dried Sims clay
loam, 3-5 m). The cation exchange capacity of the column materials
was determined by the method of Chapman (4) .

 

 

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Ne 33 m 2 .ooflnom «m m .ooaosog

on 8.0 m one Jones .30
3.3{3 innocence Andamav biomass mangoes... he
mcgdonnaopss ownenouonsoSeo vases.» one bag

 

 

mods: measuring Mo moapdoemse made: hope: one owcmmoxe 5.3.8

H H.349

The glass beads were boiled first with concentrated H230,+ and
then with 10% New! for half an hour and washed 10 times with distilled
water. Glass wool was placed below each column to prevent movement
of fine materials into the reservoir. After packing, all units were
autoclaved for half an hour (15 p.s.i., 1210).

Each percolator was. connected by way of a manifold system to a
pressure pump. The air that entered the apparatus had been passed
through 10.20% sulfuric acid and a humidifying chamber. The perfusate
was percolated at an approximate rate of 5 ml per minute .

All units were inoculated with the above mixed culture of
fltrosomgnas and Nitrobactgr and maintained in the described medium
but with 0.l&7l g (NI-145804 and 0.3 g KNO2 to give initial concentration
of 100 ppm nan-n and 50 ppm NOE-N. ‘

In order to stabilize the organisms at a constant rate of substrate
oxidation under the described conditions , percolation was conducted for
l5o30 days with 200 ml of nutrient solution per unit, changed every 1-2‘,
days .

W 9; hgtgrotroggg contagnapts . The original Nitrobacgr
culture contained heterotrophic contaminants which had proven extremely
difficult to eliminate (9). HeterotrOphic contaminants were still
present in the mixed stock culture of Nitros omonas and Nitrobacter
after 18 months ' continuous culture in the inorganic salt medium. This
was readily demonstrated by the rapid development of turbidity when
transfers were made into the same medium amended with glucose.

A number of chemicals were tested for specific inhibition of
heterotrOphic growth in a 50 ml incubation mixture in 250 ml Erlenmeyer
flasks .

The mixture comprised: (i) the desired quantity (1‘ chemical,
transferred to the flask in acetone solution of appropriate concentration
and evaporated to dryness at 155-500, under vacuum in a water bath, (ii)
25 ml nutrient solution with ammonium and nitrite to give a final concen-
tration of 50 ppm in NEH-N and 50 ppm in NOE-N, (iii) 25 ml mixed stock
culture which had recently completed oxidation of nitrogen contained in
the medium and (iv) either none or 400 ppm glucose-carbon to give CsN—vli.

Duplicates were incubated for 6 days in the dark at 20-25C without
shaking. Flasks without glucose were analyzed for ammonia, nitrite and
nitrate. Those with glucose were visually examined for heterotrophic
growth on the basis of turbidity.

‘LH effects . The pH-values limiting the oxidation of ammonium and
nitrite were investigated experimentally. Test mixtures (50 ml in
250 ml flasks) contained: (1) 25 ml nutrient solution with 100 ppm
NHn-N and 100 ppm NOE-N and (ii) 25 ml mixed stock culture which had
recently oxidized all the nitrogen in the growth medium. The mixture
was adjusted with 0.1 N KOH or 0.1 N HCl to pH-values ranging from 3.0
to 11.0. The pH was determined daily and maintained at the initial
values by addition of the required amounts of base. After 6 days °
incubation in the dark, at 20-25C, without shaking, all flasks :ere
analyzed for ammonia, nitrite and nitrate.

Efgts 2; My; M22- The effect of ammonium levels on the
rates of ammonium oxidation and nitrate production was studied under

liquid culture and perfusion conditions 0

Liquid culture. The 50 m1 incubation mixture in a 250 ml flask
contained: (1) 25 ml nutrient solution with ammonium to five the
desired concentration and (ii) 25 ml mixed stock culture which had
recently oxidized all the nitrogen contained in the medium. Four
flasks for each concentration were incubated in the dark, at 20-2 50,
without shaking. The pH was adjusted to 8.0-8.3 daily.

Perfusion conditions . 150 m1 portions of nutrient solution were

 

used for. each ammonium level studied. Between changes in ammonium
treatments , all units were maintained under standard conditions for
3-1} days , with daily changes of 200 ml nutrient solution containing
100 ppm NHu-N and 50 ppm NOE-N.

The pH of the perfusate was adjusted to 8.0-8.3 every ll-5 hours
with 0.1 KOH solution.

Five-ml perfusate portions were withdrawn through a sampling
port kept sterile by immersion in alcohol, and analyzed for ammonia,
nitrite and nitrate. Ammonia was determined by Nesslerization (3),
nitrite by the modified Griess-Ilosvay method (2) and nitrate by
the phenol-disulfonic-acid method (17) . All colorimetric measurements
were made in a Bausch and Lamb Spectronic 20.

Results and Discussion

fietgrotrophic contagnants e From a total of 85 chemicals tested,
ll! prevented heterotrophic growth as judged by lack of visually
observed turbidity in the growth medium (Table II). Their effective

dosages ranged, on the average, between 150 and 250 ppm.

DDT, diphenamid , and simazine, having no effect on ammonium
and nitrite oxidation (Table II), were the most suitable for use as
inhibitors of heterotrophic microorganisms present in the culture of
Nitros omonas and Nitrobacteg.

All the others which prevented heterotrophic growth also inhibited
ammonium and nitrite oxidation within their effective ranges (Table II).
A green color in addition to turbidity appeared in most flasks
with glucose after the second day of incubation, suggesting the presence
of Pseudomom species . Isolation of heterotrophic species which

developed in the cultures was not attempted.

Gram-negative rods isolated from cultures of Nitgos omonas and/or
Nitrobacter are presumed to be Pseudmonas (9) but it has not as yet
been established whether Psgugomonas species are the only persistent
contaminants or merely the dominant ones (9,11).

DDT, diphenam‘d and simazine are Specific for the particular case

examined here. They may or may not be in isolates of Nitros omonas or

Nitrobacter from other sources .

2E 9313932. The effect of hydrogen ion concentration on nitri-
fication in liquid mixed culture is shown in Fig. 1. At the 5 percent
level of probability, differences from one pH to another which are
greater than n.7, 8.0 and 7.!» ppm for NHu-N, Hog-N and NOS-N are
significant . The optimum pH values for ammonium oxidation lay between
pH 8.0 and 9.0 and those for nitrite between pH 6.0 and 9.0. Ammonium
was not oxidized at pH 6.0 or below, and its oxidation was sharply

TABLE II

Effects of chemicals on Nitros omnas (Nm), Nitrobacter (Nt) and
heterotrOphic contaminants (Ht) in mixed culture

 

 

 

 

 

 

 

Chemical 250 ppm 150 ppm 25 ppm

ET N m Nb lit Nm Nb Ht Nm Nb
DDT NT - - NT - - T - -
Diphenamid NT - .. NT - - T - -
2,4-Dichloroaniline NT +++ 4+0- NT +++ 4-H- T +++ -
2,5-Dich10roaniline NT +++ «H-t» NT +++ + T +~H~ -
3,14-Dichloroaniline NT +++ +++ NT +-H- +++ T +++ -
3, 5-Dich10roaniline NT +++ +++ NT +++ +++ T +++ -
8 «- quinolinol NT +++ ++ NT +++ ++ T 4-H -H-
IPC NT +++ +++ NT +++ +++ T - ~H-
m—chloroaniline NT +++ + T +++ - T +++ -
N-Ser've NT +++ +44- T +++ o T 4+:- -
Potassium Azide NT +++ 4-H- T +44- +++ T +++ +H-
Sevin NT +++ «H- NT «Hr-+- + T 4—H- +
Simazine NT - - NT - - T - -
Sodium Azide NT +++ +++ NT +++ +04- T -H+ +4-0-
NT=Not Turbid, T=Turbid

- 90-10% of ammonium or nitrite was oxidized at the end of incubation
+ 50- 90$ of ammonium or nitrite was oxidized at the end of incubation
++ 10- 50$ of amonium or nitrite was oxidized at the end of incubation
+~H 0- 10% of ammonium or nitrite was oxidized at the end of incubation

10

linited above pH 9.0. At pH 9.0, 100% of the nitr0gen initially
present could be accounted for after 6 days. Recoveries at pH 10.0
and 11.0 were 87$ and 71%, respectively. Thus volatilization of NH3,
rather than ammonium oxidation, was mainly reaponsible for decreasing
ammonium concentrations in this very alkaline pH range.

The absence of ammonium oxidation in liquid cultures below pH
6.0 has been reported (12,20). In monoculture, pI-I optima for ammonium
oxidation by Nitros omonas (7) and for nitrite oxidation by Nitrobacter
(1) are in the alkaline range and the activity of both organisms drops
off sharply below pH 7.0 and above pH 9.0. The data for nixed cultures
in Fig. l are consistent with these observations made in liquid
culture by others. In soil systems, Morril and Dawson (16) observed
that the most favorable pH for ammonium oxidation was above pH 7.6 and
that for nitrite oxidation was in the range of 6.2 to 7.0.

Chemical instability of nitrite gives rise to gaseous losses of
nitrogen from acid media. Gerretson and de Hoop (10) observed such
losses below about pH 5.5 and maxinmm losses occurred at pH 3.0-3.5.
In the present study, nitrite disappeared completely at pH 3.0 (Fig. 1).
Only 45% of the added N was recovered after 6 days.

fleets g ago-gum 1931;. In liquid mixed culture of W
and Nitrobactgg, at pH 8.0—8.3, amonium was oxidized with average rates
of 27, 35, 41, 41 ppm NI-Iu-N per day for the 400, 500, 700 and 1,000 ppm
levels , respectively (Fig. 2A). Within each level of ammonium, the
oxidation rate remained approximately constant until depletion levels

 

 

 

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12

were approached (Fig. 2A). There was some evidence in the data for
ammonium (Fig. 2A) and for nitrite (Fig. ZB) that ammonium oxidation may
have been more rapid after about 18 days when the 1,000 ppm concentration
had been reduced to 460 ppm NHu-N.

Nitrite accumulated initially at all levels at an average rate
of 23 ppm NOE-N per day. Nitrite continued to accumulate at each
level until the ammonium concentration was reduced below about 100 ppm N.
The periods of nitrite accumulation and the maximum nitrite concentrations
attained (1&0, 21m, 360 and 675 ppm NOE-N) were directly related to the
initial ammonium concentration.

Nitrate was produced initially at all levels at an average rate
of about 10 ppm NOS-N per day (Fig. 20). This low initial rate of
nitrate production was maintained. at each level until ammonium
concentration was reduced into the range of 100 to 200 ppm min-N.
At that point, nitrate production increased to 30, 31+ and 65 ppm
NOE-N per day for the first three ammonium levels, respectively.
These rates were roughly preportional to the levels of nitrite which
had accumulated by this time. These enhanced rates in the first two
levels declined later as substrate was depleted.

No enhancement of nitrate production rate occurred at the highest
ammonium level but could have been expected to appear if incubation
had been continued beyond 23 days .

Levels of ammonium (Fig. 2A) higher than about 100 ppm NHu-N had
negligible effects on ammonium oxidation but did suppress the rate of
nitrate production with a resultant accumulation of nitrite. Similar

 

13

 

 

l,000 - A A 0400 ppm AMMONIUM
E \A 0500 .. ..
3 300 _ 0700 .. ..
2
{‘3‘ \A Al,000 .. ..
D .
E 600 ' \ \
f n
’5 e [i
o O D
g o \
< 200 ~ \0 e
\ :0 A
0 i o i fix

NITRITE - NITROGEN (ppm)
to A on
o o o
o o o
\
8h
/7\
/w
0 \

 

 

 

C
/

o fli L 1 01§L
E C
3600 ~ :1
2
§ e
c: O

400 ~

'2' 8
£1
s 200- /g A
g ; Z /

O l l L

 

4 a |12 IS 20 24
mcuetnou TIME (DAYS)

FIG. 2. Effects of ammonium levels on
disappearance of ammonium and appearance of
nitrite and nitrate in liquid mixed culture

of Nitrosoaggg and Nitrobactgr. pH = 8.0 -
8.3

lb

accumulations of nitrite have been reported frequently when anhydrous
ammonia (8) or ammonium forming fertilizers (6) are introduced into
neutral or alkaline soils at levels higher than 300 ppm. .Ammonium
concentration and pH are both involved. It appears that respiratory
processes in,fli§g§bgg§gg,are interfered with by either ammonium.or free
r153 formed as the equilibrium 1012142,th + 3* is shifted to the right at
alkaline pH (1).

Ammonium or free N83 apparently do not Specifically inhibit the
nitrate oxidizing enzyme itself, since extracts of’Nitggbggtgg,oxidized
nitrite in the presence of ammonium concentrations which drastically
reduced oxygen uptake by whole cells (1). This is indicated also by
the data in Fig. 20 which shows that nitrate was produced at a low but
steady rate even in the presence of high concentrations of ammonium.

There was little evidence of lag when these high ammonium
concentrations were removed, indicating a fully active Nitrobacter
population (Fig. 20). Thus, the effect of excess ammonium or free
ammonia in these systems suppressed growth of Nitrobacter rather than
nitrite oxidation with a resultant reduction in the observed rate of
nitrate formation.

Under perfusion conditions, at pH 8.0-8.3, increasing levels of
ammonium.had no effect on rates of ammonium oxidation and nitrate
production, and nitrite never accumulated during percolation (Table III).

Rates of ammonium oxidation and nitrate production were approximately
equal and almost the same for the two perfusion systems. This indicates

that the very great difference in cation exchange capacity between glass

15

heads and the vermiculite-parlitesystem (Table I) had no effect on
the oxidizing capacities of these two systems. It would appear that
the level of activity in these two systems were determined by other
factors involved in their maintenance. The most likely factors are
the accessible or colonizable surface area of the column materials,
rate of percolation and the maintenance levels of substrate (100 ppm
NHu-N plus 50 ppm NOE-N ) .

In perfusion studies, using soil, Stojanovic and Alexander (19)
found that nitrite accumulated when ammonium concentrations in the
perfusate exceeded 200 ppm. Their initial pepulation was the indigenous
soil inoculum that had survived air-drying. Graphs presented show a
slow linear increase in nitrate over periods up to 20 days in the presence
of high ammonium concentrations, similar to that observed in the liquid
cultures used in this study (Fig. 2C). The linearity of nitrate
accumulation indicates a non-proliferating population of Nitrobacter.
This restricted Nitrobacter population was inadequate to cope with
nitrite produced by the associated Nitros gmonas population which was
not suppressed by high ammonium concentrations .

In the enriched perfusion systems used here (Table III) a large
and constant Nitrobacter population was maintained by inclusion of
50 ppm NOE-N in the maintenance perfusate. Ammonium oxidation and
nitrate production rates at all ammonium levels showed the linearity
characteristic of a constant population. The nitrifying capacity of
these systems was deternined by the constant size of Nitrosomonas and
Nitrobactgr population and was unaffected by ammonium concentrations

16

which could certainly have suppressed growth of Nitrobacter. These
observations suggest a bacteriostatic effect of either ammonium or free

ammonia on Nitrobacter similar to that observed with chlorate (14).
TABLE III

Effects of ammonium levels on the rates of ammonium oxidation and

nitrate production under perfusion conditions

 

 

Column Ammonium levels (Pg/ ml)
material

 

100 250 420 750

 

Ammonium oxidized

 

ppm Nita-N per hour
Glass beads 21 20 19 20

Vermiculite— Perlite 22 24 23 24

 

Nitrate oxidized

 

ppm NOS-N per hour
Glass beads 23 20 21 20

Vermiculite-Perlite 24 21 25 24

 

17
Summary

Trichloro-bis -(4-chlorophenyl) ethane [DDT] , N ,N-dimethyl-2, 2-
diphenylacetamide [diphenamid] and Zechloro-l-r, 6-bis (ethylamino )-
Sotriazine [simazine] , prevented growth, of heterotrophic microorganisms
(contaminants) in culture with the nitrifying bacteria with no effect
on ammonium or nitrite oxidation, thus providing a new approach for
purifying cultures of Nitros omonas and flitrobacter.

(ii). Optimum hydrogen ion concentration for nitrification in
liquid culture was found to be at pH about 8.0 to 9.0 and 6.0 to 9.0
for ammonium and nitrite oxidation, respectively. No nitrificatiOn
was observed at pH . below about 6.0 nor above about 9.0. Disappearance
of nitrite at pH below 3.0, was observed and was likely due to chemical
reactions of nitrite.

(iii). In liquid culture, ammonium levels above about 100 ppm
NHVN had little or no effect on the rate of ammonium oxidation.
Nitrate production was restricted to a low, linear rate characteristic
for a static or resting population, indicating that growth of mm
gag; rather than nitrite oxidationwas suppressed. When ammonium
was reduced below about 100 ppm NHIfN, there was an abrupt increase
in rate of nitrate production consistent with resumption of growth.
Nitrite accumulated intermediately in amounts and over periods of
time which were proportional to the initial concentrations of
ammonium. In enriched perfusion systems , no intermediate accumulations
of nitrite occurred with concentrations as high as 750 ppm NHuaN.

These observations suggest a bacteriostatic effect of either

ammonium or free ammon‘? a on Nitrobacter.

1.

2.

3.

4.

5.

7.

8.

9.

10.

11.

12.

13.

18

References

ALEEM, M.I.H. and ALEXANDER, M. 1960. Nutrition and phySiolog' of
Nitrobacter agilis. Appl. Microbiol. 8: 80-84.

BARNES, H. and FOLKARD, A.R. 1951. The determination of nitrite.
Analyst, 76: 599-603.

98:23:, T., ALTHER, R. and sown. 1953. Die photometrische
N -bestimmung mit Nessler°s Reagenz. Pharmaceutica Acta
He vetica, 28: 273.

CHAPMAN, H.D. 1965. Cation exchange capacity. ”In Methods of
Soil Analysis“, Part 2, pp. 891-901, American Society of
Agronom, Madison, Wisconsin.

CHAPMN, H.D. and LIEIG, G.F. 1952. Field and laboratory
studies on nitrite accumulation in soils. Soil Sci. Soc.
Amer. Proc. 16:. 276-»282.

DUISBERG, P.C. and BUEHRER, T.F. 1954. Effect of amonia and its
oxidation products on rate of nitrification and plant growth.
8011 3010 78: 37-180

ENGEL, 14.3. and ALEXANDER, M. 1958. Growth and autotrophic
metabolism of Nitrosomonas europgea. J. Bacteriol. 76: 217-222.

ENO, C.F., BLUE, W.G., and GOOD, J.M. 1955. The effect of
anhydrous ammonia on nematodes , fungi, bacteria, and
nitrification in some Florida soils. Soil Sci. Soc. Amer.

Proc. 19: 55.580

GARRETSON, A.L. and SAN CLEMEITE, 0.1.. 1967. Characterization of
heterotrophic bacteria in mixed culture with itrobacter ggili .
Qo Bullo Mich. Ste UMV. Agrico ups Stno 503 15 1 o

GERRETSEN, F.C. and DE HOOP, H. 1957. Nitrogen losses during
nitrification in solutions and in acid sandy soils. Can. J.
Microbiol. 3: 359-380.

GUDERSEI , K . 1956. Observations on mixed cultures of Eitros omomg
and heterotrophic soil bacteria. Plant Soil, 7: 2 34.

KAJA, L. 1963. The oxidation of nitrites by Nitrobacter in
aqueous solutions, of different pH. Soils and Fertilizers
273 2198 (AbStPe)o

LEES, H. 1949. The soil percolation technique. Plant Soil,
1: 221-239.

14.

15.

16.

17.

18.

19 o

20.

19

LEES, H. and QUASTEL, J.H. 1945. Bacteriostatic effect of potassium
chlorate on soil nitrification. Nature, London, 15: 276-278.

McLAREN, A.D. and SKUJINS, J.J. 1963. Nitrification by' itrobacter
agilig,on surfaces and in soil with respect to hydrogen ion
concentrations. Can. J. NucrObiol. 9: 729-731.

mRRIL’ LOGO and DAme, JeEe 1962. Grath rates or nitflfying
chemolithotrophs in soil. J. Bacteriol. 83: 205-206.

ROLLER, E.M. and MCKAIG, N. 1939. Some critical studies of
phenoldisulfonic acid method for the determination of nitrates.
Soil Sci. 47: 397-407.

SPERBER, JoIe and SYKES, 801’. 19640 A perquion apparatus with
variable aeration. Plant Soil, 20: 127:130.

STOJANOVIC, B.J. and AMBER, M. 1958. The effect of inorganic
nitrogen on nitrification. Soil Sci. 86: 208-215.

‘WEBER, D.F. and GAINEI, P.L. 1962. Relative sensitivity of
nitrifying organisms to hydrogen ions in soils and in solutions.
3011 3010 9“: 138.1450

RKRT II: EFFECTS OFHAGRICUDTURAL CHEMICALS ON NITRIFICATION IN LIQUID
CUDTURES CONTAINING SOILnOR.SOIL.EXTRACT

Introduction

Chemicals used for pest control in agricultural practice are incorporated
into the soil directly or indirectly. Knowledge of their effects on
biological processes in soil may help in their proper usage in the future.

Nitrification insoil is affected in varying degrees by different
insecticides (3,6), fungicides (12,18) and herbicides (14).

Present evidence indicates that the effect of a pesticide on
nitrification and the magnitude of that effect depend upon its chemical
properties and the physico-chemical and biological characteristics
of the environment. For example, in different studies, chlordane was
found to decrease nitrification in soil at 50 ppm (4), to have no effect
up to 200 ppm (10,11,16), and to increase nitrification when applied at
10-100 ppm (7). DDD prevented nitrification in liquid culture at 10
ppm (8) but 50 to 100 ppm (4,7) were required under field conditions
and it had no effect when used at normal field rates (11). Parathion
inhibited nitrate production by Nitrobactgr at 10 ppm in liquid culture
(8) but high field rates increased numbers of nitrifying bacteria (13).
Sevin retarded nitrate formation in soil at about lSO-ppm(l) and
potassium and sodium azide strongly inhibited nitrification at very
low concentrations in soil (15). CIPC at normal field rates had
little effect on nitrification (17) but inhibited nitrate production
at concentrations of 150 ppm (1,17). N-Serve inhibited ammonium
oxidation in the range of 0.05 to 20 ppm (9) depending on the nature
of the soil.

20

21

Most of the above and other chemicals have been found by K. Sommer
and A .R. Wolcott (unpublished data) to prevent ammonium and/ or nitrite
oxidation in liquid culture of Nitros omonas and Nitrobacter at concen-
trations ranging from about 2.5 to 50 ppm. Thir' study examines the relative
importance of soluble vs . insoluble soil materials in modifying the

influence of these chemicals on nitrification.

Materials and Methods

giological material. The mixed stock culture of Nitrosomonas m9;
and Nitrobacter agilis and the maintenance medium used in these studies

have been described.1

Pesticides. Twenty-seven chemicals were selected which earlier
studies had shown to inhibit Nitros omonas and/ or Nitrobacter in liquid
culture at concentrations of 50 ppm or less (K. Sommer and A.R. Wolcott,
unpublished data). The chemicals were dissolved in acetone. Appropriate
concentrations of each chemical were transferred into 250 m1 Erlenmeyer
flasks and the solvent was evaporated to dryness, at 45=5OC under
vacuum in a water bath. Duplicate flasks of each concentration were
used to prepare test cultures containing either soil extract or soil
as described below.

$41, M. Air-dry Sims clay loam was used for soil extract
preparations (1,000 g of soil plus one liter of tap water, with
autoclaving for half an hour). The 50 m1 incubation mixture in the
250 m1 flask contained (1) the chemical to be tested, (ii) 25 m1 of

 

1
Part I, p. 3.

22

soil extract containing ammonium and nitrite to give a final concentration
of 50 ppm in ammonium-nitrogen and 50 ppm in nitrite-nitrogen, and (iii)
25 m1 of mixed stock culture which had recently completed the oxidation
of all nitrogen contained in the maintenance culture medium.

After 6 days0 incubation at 20-250 in the dark without shaking, the
material in all flasks was analyzed for ammonia, nitrite andnitrate by
methods cited in Part I, p. 7 A 6-day incubation was used since this
was the time required for complete oxidation to nitrate in control

flasks to which no test chemical was added.

Soil. The soil (Sims clay loam) was incubated before use for 10

 

days at 20-250 at 75 percent of its waterbholding capacity. The
incubation mixture in a 250 m1 flask contained (1) the chemical to be
tested, (ii) one gram of moist 8011, (iii) 25 ml nutrient solution

with ammonium and nitrite to give a final concentration of 50 ppm
ammonium-nitrogen and 50 ppm nitrite-nitrogen and (iv) 25 ml of mixed
stock culture which had recently completed the oxidation of the nitrogen
contained in the maintenance medium.

All flasks were incubated at 20-250 in the dark, without shaking.
Nitrification was slower in the presence of soil than when only 8011
extract was added, and 10 days was required for complete conversion in
control flasks . Ammonia , nitrite and nitrate were determined as before
at this time.

Qtegpretation 2; gm. The use of a mixed culture made it possible
to compare inhibitory effects on Nitros omonas and Nitrobacter directly
in the same environment at each concentration of test chemical. The

degree of depression of Nitros omonas activity was estimated from the

23

percentage dis appearance of, added ammonium-nitrogen (disappearance
assumed due to oxidation). Depression of Nitrobacter activity was

estimated from percentage conversion of nitrite calculated as follows:

Final '-N - tial O.- x100
95 nitrite oxidation = Added N 2-N + (NHu-N oxi zed

The notation in Tables I-V identifies arbitrary ranges of depression
based upon the extent to which available substrate was oxidized:
Toxic, O-lOfi; strongly depressive, 10-50$; depressive, 50—90%; no
effect, 90-10%.

Results and Discussion

In Tables I-V, the degree of inhibition shown for cultures to which
soil extract was added is the same as that observed by Sommer and
Wolcott in the absence of soil extract, in the same medium, using .
inocula from the same stock source (unpublished data). Thus for
these chemicals , soluble organic and inorganic compounds in soil
extract did not change the concentration needed to depress or prevent
ammonium or nitrite oxidation by W or itrobacter.

The addition of 1 gram of pro-incubated soil to the liquid medium,
however, reduced the activity of a number of the chemicals, thereby

increasing the concentration required for a given degree of inhibition.

Fungicides. From the chemicals in Table I, 8-quinolinol and
terrazole were highly specific in their action against Nitrosgggnag.
Higher concentrations of both were required in the presence of soil to
depress ammonium oxidation to the same degree as in the presence of

soil extract. Thiram was also more active against Nitrosomonas than

24

mtrobacter. The addition of soil materials had no effect either on
the degree of specificity or the concentration required for a given
degree of inhibition of either organism.

Over this range of concentrations , Dowicide- 7 and sodium azide
inhibited both organisms about equally in liquid culture. The addition
of soil materials had no effect on the activity of sodium azide but
the activity of Dowicide-7 was markedly decreased.

25
TABLE I

Effects of various fungicides on a mixed culture of Nitros omonas (Nm)

 

and Nitgobactgr (Nb) in a liquid medium with added soil extract or soil

 

 

Fungicide concentration (ppm)

 

 

 

 

 

Common
or 2,5 10,0 20,9 50,0
Trade Name Nm Nb Nm Nb Nm Nb Nm Nb
Soil extract
Dad Gide-7 +++ ++ +++ +++ +44» +++ +++ +=H~
8-vquinolinol ++ - 4-H» - +44- - +++ «-
Sodium azide ++ +++ +++ -H-+ +-H- +++ ~H-+ +++
Terrazole +++ - +++ - +++ - +++ «-
Thiram +++ - +++ + +++ ++ +++ ++
Soil

Dowicide-7 - - - +++ - -H-+ +++ 4+0»
8c-quinolinol - - + - ++ - +++ ..
Sodium azide 4+ +++ +++ +++ +++ +44 +++ 44+
Terrazole - - 4.4+ . +4... a. +++ -
Thiram +~H> - +++ + +++ ++ +++ ++

 

Toxic, +++g strong depression, ++; depression, +; no effect, -

26

W. Among the insecticides in Table II, captan and
sevin were highly specific for Nitrosomgnas. Nitrite oxidation was
unaffected even at 50 ppm. At the lowest concentration, the degree
of interference with ammonium oxidation was reduced in the presence
of soil materials.

All of the other insecticides in Table II were specifically
depressive only on Nitrobacter. Higher concentrations were required
for a given level of depression in the presence of soil. The strong
depression by Linda. at 20 gnd 50 ppm was completely eliminated
when soil materials were added.

Herbicides . N-Serve, a chemical which is being develOped commercially
as a nitrification inhibitor, is included. with herbicides in Table III.
At concentrations up to 50 ppm, its action against W was
highly specific. The same was true for amitrole. The addition of
soil did not interfere with the action of either chemical even at the
lowest concentration.

The other herbicides were more inhibitory to Nitrobacter than
Nitggsomnas. In the case of CIPC, this specificity at 50 ppm was
enhanced by the addition of soil. Soil materials had no effect on
the activity of IPC but they completely eliminated the toxicity of
zytron to Nitrobacter at 10 ppm. Higher concentrations of. zytron
(20 and 50 ppm) were still toxic after addition of soil.

2?

TABLE II

Effects of various insecticides on a mixed culture of Nitrosomonas (Nm)

and Nitrobactgr (Nb) in liquid (medium with added soil extract or soil

 

 

Insecticide concentration (ppm)

 

 

 

 

 

606:0!) 2,: 10,0 20,0 50,0
Trade Name Nm Nb , Nm Nm Nm Nb
Soil extract
Captan 4-H- - 4-H- - ~H-+ - +++ -
Chlordane - - - ++ 9 ++ .. +4....
DDD - +4- - +++ .- +4+ .. +++
Kelthane - «H- .- +++ - +4.... .. +4.4,
Lindane - - - + - +4» «- «H»
Parathion - «- - «1+ a He.» .- +=H~
Sevin H - +++ .. =H-+ .. +++ 6
Soil

Captan ++ - +++ - +++ =- i-H- «-
Chlordane - .. - + - ++ .. +4....
DDD - .. «- +++ . +4—9- - 44+
Kelthane - .. .. 4—H. - +4.... ., +4....
Lindane - .- - a .. .. e. a
Parathion - .. .. - - +4. .. .H.
Sevin - - +++ - +4-4- - +++ -

 

Toxic, 4-H»; strong depression, +0»; depression, +; no effect, -

28

new III

Effects of various herbicides on a mixed culture of Nitrosomonas (Na)

and W (Nb) in a liquid medium with added soil extract or soil

 

 

Herbicide concentration (ppm)

 

 

 

 

 

Common
or 2,: 10,0 20,0 50,0
Trade Name N m Nb Nm Nb Nm Nb Nm Nb
Soil extract
Amitrole +++ - +++ - +-l-+ - +++ -
CIPC - - - ++ - ++ ++ 4-H-
IPC - - - - - ++ + +++
MPG?! - - - +++ - +++ - +++
N-Serve" +++ - +++ - 4+!- - +H~ -
Soil

Amitrole 4-H- - +'H- - +++ - 4-H» -
CIPC - - - ++ .. ++ - +44»
IPC - «- .. - .. ++ + 44+
mron - - - - «- +++ - +++
N-nServe * +++ - +++ - +++ - +++ ..

 

Toxic, -H-+; strong depression, +-3-; depression, +; no effect, -

*Not a herbicide

29

M We Aniline compounds have been isolated as
breakdownproducts of diuron (5) and other pesticides used in
agriculture (2). Such breakdown products may be inhibitory to soil
nitrification even though the initial compounds have no such effect .

Host aniline derivatives were toxic to Nitrosgmonas in the range
from 2.5 to 20 ppm; 2,3, 5,6-tetrachloroaniline strongly inhibited
Nitrobacter at 10 ppm and only 2,4-dichloroaniline, 2,3,4-, and 2,4,5-
trichloroaniline affected both organisms (Table IV).

In the presence of soil, 10 ppm or more were required for the
same inhibitory effect on. Nitros omega with practically no change in
the amounts needed for Nitrobacter (Table V).

W. Nine of the chemicals in this experiment were highly
specific in their inhibitory effect on W. They interfered
with ammonium oxidation, with no effects on Nitrobactgg in the same
liquid culture , over the entire range of concentrations employed
(2.5 to 50 ppm).

Seven chemicals were equally specific in their action against
Nitrobacter at concentrations above 10 ppm. Only two of these (DD!)
and Kelthane) interfered with nitrite oxidation at 2. 5 ppm, but none
had any effect on ammonium oxidation at concentrations up to 50 ppm.

Other chemicals such as thiram, CIPC, 2,4,6-trichloroaniline,
which inhibited both organisms at the higher concentrations , showed
marked specificity at lower concentrations . Sodium azide was toxic
to both organisms at concentrations of 10 ppm or more, but there was
evidence that its specificity against Nitrobacter might be expected to

increase at lower concentrations .

30

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32

These specific interferences occurred at concentrations which
may be expected in the soil solution following normal field applications.
The addition of solid soil materials in these experiments increased the
concentrations required for interference by a number of chemica13.

Such protective action would be expected to increase with increasing
soil-to-solution ratio. However, the toxicity to nitgggggggag,of
NeServe, amitrole, and thiram at 2.5 ppm was unaffected and captan
at this concentration was still strongly depressive in the presence
of soil.

In the field, effects of soil on these chemical interferences
with nitrification will vary with the nature of the soil itself and
its microbial population, with vegetation, management and climatic
conditions. Very different effects of a chemical may actually be
observed in different experimental situations.

Thus it has been observed (1,7,13) that nitrification was increased
with high field rates of DDD and parathion. In the present study,
nitrobacter was inhibited by both chemicals, even in the presence of
soil9 after a 10-day incubation period. This result cannot be compared
directly with the cited studies where a longer period of time intervened
between treatment and observation.

Inhibition of nitrification has been reported when chlordane and
lindane were applied at a rate of 50 ppm soil weight bases (4) and
when CIPC'was applied at a rate of 150 ppm (1,17). The importance of
soil chemical and physical properties in modifying effects of exotic
chemicals on nitrification can be inferred from the early work on N-Serve
(9) where it was found that concentrations effective in reducing nitrifi»

cation rates varied with soil type from .05 to 20 ppm, increasing generally
with organic matter content.

33

In this study, soil reduced inhibitory effects of test chemicals
on nitrification, whereas soil extract was without effect, which suggests
that insoluble organic.or inorganic soil materials were responsible
for inactivation. Since pre-incubated soil was used, the possibility
that activities or cell materials of the microflora may have influenced
the result cannot be ignored.

From a practical standpoint, if a given pesticide inhibits
nitrosomonas specifically, it may exert a useful side-effect'by,
minimizing losses of nitrate or nitrite through leaching or denitri-
fication, thus conserving soil and fertilizer nitrogen. By contrast,
if’flitrobacter is specifically inhibited,_losses of nitrogen through
leaching or chemical reactions of nitrite may be enhanced or toxic

effects of nitrite on crops may result.

 

 

 

 

34
Summary

Tuentyeseven chemicals Were tested for inhibitory effects on
Eitresomones and yitrebacter in mixed liquid culture. Nine Were
highly specific in their action against Nitrosomonas over the entire
range of test concentrations (2.5 to 50 ppm). Two were equally
specific in their action against flitrobacter at 2.5 ppm and five

more over the range 10 to 50 ppm. The rest of the chemicals affected
both organisms.

The concentratiOns required for complete inhibition of ammonium
and/or nitrite oxidation in liquid culture were increaSed for ten of
the chemicals when soil was added to the incubation mixture. Water
soluble materials in extracts from the same soil were ineffective in
reducing inhibitory effects. This suggests that insoluble organnc or

inorganic constituents were responsible for inactivation by soil.

1.

2.

3.

7.

8.

9.

10.

11.

12.

13.

1h.

35

References

BARTHA, R., LANZILOTTA, R.P., and PRAMER, D. 1967. Stability and
effects of some pesticides in soil. Appl. Microbiol. 15: 67-75.

BARTHA, R. and PRAMER, D. 1967. Pesticide transformation to aniline
and azo compounds in soil. Science, 156: 1617.

BOLLEN, W.B. 1961. Interactions between pesticides and soil
microorganisms. Ann. Rev. Microbiol. 15: 69-92.

BROWN, A.L. 1954. Effects of several insecticides on ammonification
and nitrification in two neutral alluvial soils. Soil. Sci.
Soc. Amer. Proc. 18: 417-4120.

DALTON, RoLe’ EVANS, AeWe and RHODES, ReCe 1966c Disappearance
of diuron from cotton field soils. Needs, 1%: 31-33.

ENO, C.F. 1958. Insecticides and the soil. J. Agr. Food Chem.
6: 3u8-351.

ENO, C.F. and EVERETT, P.H. 1958. Effects of soil applications
of 10 chlorinated hydrocarbon insecticides on soil microorganisms
‘and the growth of stringless black valentine beans. Soil Sci.
5°09 Amara PrOCe 22: 235-2380

GARRETSON, A.L. and SAN CLEMENTE, C.L. 1968. Inhibition of nitrifying
chemolithotrophic bacteria by several insecticides. J. Econ.
Rte 61: 285-2880

GORING, C.A.I. 1962a. Control of nitrification by 2-chloro~6-
(trichloromethyl) pyridine. Soil Sci. 93: 211-218.

MARTH, E.H. 1965. Residues and some effects of chlorinated
hydrocarbon insecticides in biological material. Res. Rev.
9 3 1‘89 0

MARTIN, J.P., HARDING, R.B., CANNELL, G.H., and ANDERSON, L.D.
1959. Influence of five annual field applications of organic
insecticides on soil biological and physical properties. Soil
Sci. 87: 33h—338.

MARTIN, J.P. and PRATT, P.F. 1958. Fumigants, fungicides, and soil.
J. Agr. Food Chem. 6: 345-348.

NAUMANN, K. 1960. The effect of pesticides on soil microflora.
Soils and Fertilizers, 23, 1356 (Abstr.).

NWMN’ A030 1958. HerbiCides and the 30110 Jo Agr. FOOd Chem.
6: 352-353.

15.

16,

17.

18.

36

NISHIHARA, T. 1963. The search for chemical agents which efficiently
inhibit nitrification in soil and studies of its utilization
in agricultural'practice. Soils and Fertilizers 26: 3072 (Abstr.).

SHAW, W.M. and ROBINSON, B. 1960. Pesticide effects in soils on
nitrification and plant growth. Soil Sci. 90: 320-323.

TEATER, R.H., mR'TENSEN, J.L, and PRATT, P.F. 1958. Effect of
certain herbicides on rate of nitrification and carbon dioxide
evolution in soil. J. Agr. Food Chem. 6: 2114-216.

WOLGOTT, A.R., MACIAK, F., SHEPHERD, L.N. and LUCAS, R.E. 1960.
Effects of telone on nitrogen transformations and on growth of
celery in organic soils: Down to Earth, 16: lO-llt.

PART III: SINERGISTIC AND ANTAGONISTIC INHIBITIONS 0F NITRIFICATION
BY HERBICIDES

Introduction

Ammonium is oxidized to nitrite mainly by Nitggggggng§.and nitrite
to nitrate by Nitrobacter. One or both steps may be Specifically
inhibited by various chemicals.

NaServe at 10 ppm and amitrole at normal field rates inhibited
soil nitrification for four to eight weeks, respectively (1,2).
Potassium azide decreased soil nitrification at 50 ppm, and sodium
azide is known to be a potent inhibitor of certain microbial oxidases
(6). CIPC and IPC retarded nitrate production in soil (7).

The above chemicals have been.tested alone for their effects on
nitrification. Use of herbicides in various combinations for more
efficient weed control is an increasingly important practice (#).
Frequently, insecticides, fungicides and herbicides are applied in
varying combinations on the same crap. ‘We examine here the effects
of combinations of several chemicals on ammonium and/or nitrite

oxidation by Nitrosomoggs and Nitrobacter in a liquid mixed culture.

materials and methods

fiiological mategia . The mixed culture of Nitrosggpnas europgga
and Nitrobacter agilis and the maintenance medium used in these studies
have been described.1

 

lPart I, 13.3.

37

38

ghchals. 3»amino-1,2,l+-triazole [amitrole] , isopropyl-N-
(3-chlorophenyl) carbamate [CIPS], isopropyl-N-phenyl carbamate [IPC],
2-chloro-6-(trichloromethyl) pyridine [NoServe] , potassium azide, and
sodium azide were used. Amitrole, potassium.aaide and sodium azide
‘were dissolved in sterile medium at 220. CIPC, IPC, and.N-Serve were

dissolved in sterile medium at 40—500 in a water bath.

will. The 50 ml incubation mixture in a 250 ml flask contained:
(i) 25 m1 mineral medium with ammonium (100 ppm NEE-N) and nitrite
(100 ppm NOE-N) plus the appropriate concentrations of chemicals to
be tested and (ii) 25 ml mixed stock culture which had recently
completed the oxidation of nitrogen contained in the growth medium.
Duplicate flasks were incubated for it days, at 20-250, in the dark
without shaking.

Amonia was determined by Nesslerization, nitrite by. the modified
Griesscllosvay method, and nitrate by the phenol-disulfonic-acid
method.2 All colorimetric measurements were made in a Bausch and

Lomb Spectronic 20.

Interggtation. According to Colby (3) and Gowing (4) the expected
additive response to a given combination of two herbicides would be:

Igloo-g)
x + 100 [1]

E
where: E 8 expected response

X = the percent inhibition of ammonium or nitrite oxidation by
herbicide A at m ppm.

Y = the percent inhibition of ammonium or nitrite oxidation by
herbicide B at n ppm.

 

2PM I, p. 7

 

39
Equation [1] simplifies to the form used by Limpel et a1. (5):
11
E = x + Y - 100 [2]

When the observed response is greater than expected, the response

to the combined chemicals is synergistic; when less than expected,

it is antagonistic. If the observed and expected responses are equal,
the response to the combinations is additive (3).

Results and Discussion

Effects of aldtrole and N—Serve, separately and in combination, on
ammonium oxidation by Nitros omonas are presented in Table I. Amitrole
by itself produced inhibitions of 32 and 60 percent at concentrations
of 0.5 and 1 ppm. Concentrations of 0.2 and 0.3 ppm NuServe were
less than the 0.4 ppm. threshold above which depression of ammonium
oxidation by this chemical was detected in this assay system.

By the adopted criteria [eq. 2], the reaponse of Him—m
to these two cheudcals in combination would be interpreted as synergistic.
Neither had any effect, separately or in combination, on Nitrobacter
in the same culture. I

In the case of the two azide salts (Table II), concentrations
which resulted in less than complete inhibition of, Nitrobacter
had no effect on ammonium oxidation by itros omonas, regardless of
whether the salts were added singly or in combination. Sodium
azide by itself was somewhat more inhibitory to Nitrobacter than the
potassium salt at equivalent concentrations. The response to the two

salts in comination would be interpreted as synergistic.

40

TABLE I

Combined effects of amitrole and N~Serve on ammonium oxidation by

Nitrosomonas

 

 

Inhibition (i)

 

 

Chemical Dosage a b
ppm Observed Expected Difference
Anitr016 0 e 5 32
1.0 60
NaServe 0.2 0
0.3 0
0 .240 e 5 5"" 32 +22
NeServe
0.2+1.0 72 60 +12
.9.
0.3+0.5 54 32 +22
Amitrole
0.3+l.0 72 60 +12

 

aCalculated according to equation [2]

bPositive difference denotes synergism

The synergisms expressed in Tables I and II have in common the
feature that it appears to be the activity of the more potent inhibitor
that is enhanced in the presence of the less active chemical. This
may be inferred from the fact that, in Table I, the largest synergism
occurred when N-Serve at either concentration was combined with the
lower concentration of amitrole. In Table II, the largest synergism

occurred when potassium azide at either concentration was combined

41

TABLE II

Coubined effect of potassium azide and sodium azide on nitrite oxidation

by Nitrobacter

 

 

Inhibition (4)

 

 

Chemical Dosage a b
ppm Observed Expected Difference
Potassium 0.2 31
azide
0.3 42
Sodium 0.2 34
azide
0.3 56
Potassium 0.2+0.2 74 54 +20
azide
0e2+003 75 70 + 5
+
0.3+0.2 81 62 +19
Sodium
£21d9 0 03+0 03 78 74 + 1"

 

aCalculatedaccording to equation [2]

bPositive difference denotes synergism

with the lower concentration of sodium azide. This behavior is analogous
to that Observed by Limpel et a1. (5). They found that the herbicidal
effectiveness of dimethyletetrachloroterephthalate was enhanced to a
greater degree by a number of chemicals when these were combined

with the lower dosages of the phthalate.

42

Data for IPC. and CIPC are presented in Table III. At the
concentration employed, both chemicals depressed the activity of both
Nitrosomonas and Nitggbggtgge Both chemicals were more inhibitory to
Nitrobacter than to Nitrosomonas. At equivalent concentrations, IPC
was more inhibitory to Nitrosomenas than was CIPC.

At low concentrations, the response to these chemicals in combination
was antagonistic. In other words, the observed inhibition was less
than‘wculd have been expected if.they had acted independently of
each other to produce an additive response. The antagonism decreased
as the concentration of either chemical was increased.

Synergistic, antagonistic or additive reopenses can‘be expected
when chemicals are applied in combination (3,4,5). However, it is
apparent that the extent to which such interactions are expressed

is dependent upon concentration and specific activity of each chemical.

 

43

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Summary

At low but biologically active concentrations in liquid culture,
3~amino-1,2,4-triazole [amitrole] combined with 2-chloro-6- (trichloro—
methyl) pyridine [u- Serve] synergistically inhibited the oxidation of
ammonium by Nitros omoms . The inhibition of Nitrobacter by potassium
azide and sodium aside in combination was also synergistic.

Is opropyl-Naphenyl carbamate [IPC] and is apropyl-N- (trichlorophemrl)
carbamate [CIR] at low concentrations were antagonistic in their
inhibition of both Nitros omonas and nitrebegtgr.

Both synergistic and antagonistic responses approached additivity

as the concentration of one or both chemicals in a combination increased.

l.

2.

3.

4.

5.

7.

45
lbferences

ANSORGE, 11., MARKERI‘, 3., and JAUERI', R. 1967. Studies on inhibition
of nitrification by s ome chemical preparations . Soils and
Fertilizers, 31: 318 (Abstr.).

CHANDRA, P. 1964. Herbicidal effects on certain soil microbial '
activities in some brown soils of Saskatchewan. Weed Res . 4: 54-63.

COLBY, S.R. 1967. Calculating synergistic and antagonistic responses
of herbicide combinations. Needs 15: 20-22.

GOWING, D.P. 1960. Comments on tests of herbicide mixtures. ' Weeds
8: 379-3910 .

LIMPEL, L.E., SCHULDT, P.H., and LAMONT, D. 1962. Weed control
by dimethyl tetrachloroterephthalate alone and in certain
combinations. Proc. N.E. Weed Control Conf. 16: 48-53.

REUSZER, HM. 1965. Effects of potassium azide on soil micro-
organisms. Agron. Abstr. p. 87.

QUASIEL, J.H. and SCHOLEFIEID, 39.0. 1953. Urethanes and 8011
nitrification. Appl. Microbiol. 1: 282-287.

 

PART IV: EFFECTS OF PESTICIDES 0N NITRIFICATION IN PERFUSION SYSTEMS
Introduction

Nitrification refers traditionally to the biological. oxidation of
ammonium to nitrate. In soil this conversion is effected mainly

by the aerobic chemolithotrophic bacteria Nitros genes and Nitrobacter,

 

in two steps. In the first step, ammonium is oxidized to nitrite by the
W group: in the second nitrite is oxidized to nitrate by
Nitrobactgr.

Pesticides used in agriculture have been reported to inhibit one
or both steps in nitrification. Amitrole at 4 ppm, inhibited soil
nitrification for eight weeks and residual. depressive. effects were
apparent for an additional eight weeks (2,). Potassium azide at about
30 ppm inhibited soil nitrification for four weeks (5). In perfusion
studies cm: at 160 ppm almost completely inhibited nitrification (4).

K. Sommer and A.R. Wolcott (unpublished data), using a liquid
mixed culture of fltros omonas and itrobacter, found that amitrole
completely inhibited ammonium oxidation at 5 ppm with no effect on
nitrite oxidation up to 250 ppm. CIPC inhibited ammonium oxidation
at 150 ppm or less and nitrite oxidation at 23 ppm. IPC completely
inhibited ammonium oxidation at 23 ppm and slightly depressed nitrite
oxidation at 25 ppm. Potassium azide and sodium azide completely
inhibited ammonium oxidation at 23 and 5‘ ppm and nitrite oxidation at
5 and 1 ppm, respectively. This paper investigates the influence of
perfusate volumes and solid matrix materials on the inhibitory effects
of these chemicals on a mixed culture of Nitros omonas and Nimbagter

in perfusion systems.
46

47

Materials and Methods

W Ml. The mixed culture of Nitros omonas europggg
and Nitgobactg; M and the maintenance medium have been des cribed.1

 

thgcals. Some pertinent properties of the chemicals used are
given in Table I. Amitrole, potassium azide and sodium azide were
dissolved in nutrient solution at 25C. CIPC and IPC were dissolved
in nutrient solution at no.45c in a water bath.

Perfusion studies. The perfusion apparatus and the conditions
of growth of Nitros omonas and Nitrobagter on the columns have been
described in Part I, p.2. The column materials and their total

cation exchange and water-holding capacities are given in Table II.
The experimental medium was the same as the equilibration medium,

except for the addition of the desired concentration of test chemical.
In a given experimental run, duplicate columns of each matrix material
were perfused with 50, 100, 150 or 200 ml of medium containing a
given concentration of a test chemical. Fifty and 100 ml were tested
in one run followed by 200 and 150 ml in the next, so that after a Zarun
cycle each unit had been perfused with a total of 250 m1. A 3 or haday
equilibration period then followed before the next concentration was
tested.

Five-ml aliquots were withdrawn aseptically after 0, 3 and 5 hours
irl'the 50 and 100 ml runs and after 0, 5 and 9 hours in the 200 and

150 m1 runs. Ammonia, nitrite and nitrate were determined by methods
cited in Part I, p. 3.

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49

§ogption studies. In several experiments, sorption and desorption
of chemicals by the solid matrix was followed by observing changes in
concentration or activity of the chemical in the perfusate.
Amitrole in the perfusate was determined according to meig (10).
An aliquot was treated with nitrous acid coupled with N-(t-naphthyl)
etl'grlenediamine, and the resultant color intensity was measured at 455 mp.
CIPC, IPC and KN3 in the perfusate were estimated by the fiitrosomonas

and flitrobacter bioassay described below.

fiioassay. Bioassays have been preposed as useful tools in residue \1
Studies with a number of pesticides (1,3).

In preliminary studies CIPC and IPC at 10-20 ppm and potassium
azide at 0.3 to 0.5 ppm depressed nitrite oxidation by Nitrobacter
in liquid culture. At higher concentrations they were also inhibitory
t° Litresomonas. By utilizing responses of these organisms in mixed
culture, it was possible to develop a standardized bioassay for each
of t'1'1ese chemicals. Reproducible standard curves were obtained covering
the following ranges of concentration: CIPC, 10 to 70 ppm; IPC, 20 to
60 ppm; and m3, 0.2 to 0.5 ppm.

A 50 percent inoculum was used from a mixed stock culture which
had recently completed the oxidation of all the nitrogen in the
Mintenance medium. The assay mixture in a 250 ml Erlenmeyer flask
cglltprised (i) 25 ml inoculum, (ii) an aliquot of the perfusate to be
tag ted, (iii) (NHQ)2SOu and KNOZ to give final concentrations of 50 ppm
NHL‘fN and 50 ppm NOE-N and (iv) nutrient solution as needed to give a
ritual volume of 50 ml. The resulting mixtures were incubated, without

shaking, for 6 days in the dark at 20-256.

‘ ___

 

50

 

 

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51

Concentration of the tested chemical was estimated graphically,
using standard curves with concentration plotted against the ordinate

and percent inhibition of nitrite oxidation against the abscissa.
Results and Discussion

flitrification in_control sygtems. Standard nitrification patterns
during control runs are shown in Fig. l for the 200 ml perfusate.

Some nitrate was always present at the beginning of percolation.
Analyses at zero time for nag-N and NOE-N were usually quantitative
for the concentration desired after dilution by perfusate solution
carried over in the column from the previous percolation.

During the first 3 or U hours of percolation, ammonium oxidation
and nitrate production were essentially linear and occurred at equal
rates. The rate of ammonium oxidation declined after 4 hours, but
nitrate was produced with little change in rate until both substrates
were exhausted at 6 hourse

Nitrification rates during control runs ranged from 20 to 25 ppm
N per hour. This is 20 to 25=fold greater than in the stationary
flask cultures used in Parts I, II and III. There were no consistent
differences in rate between column materials, except as structural
deterioration resulted in a gradual decline in the activity of the
soil-perlite systems to the point that they were discarded.

In Tables II-V and VII, VIII data taken after 5 hours are presented.
Normally at this time, 80 to 90 percent of both substrates had.been
oxidized in control runs with 200 ml perfusate containing no chemical

(Fig 11). In control runs with 150 ml of perfusate, the percentage

52

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53

conversion was usually closer to 90 than 80 percent. With 50 and lOOuml
perfusate volumes, both substrates in control runs were normally
oxidized completely prior to the fifth hour.

Control runs were made at each perfusate volume after equilibration
preceding each change in concentration. Since there were no consistent
differences for column materials, data for all control runs at a
given volume were averaged over all perfusion units for the period
during which a given chemical was under study. The average total
conversion is given in column 2 of Tables III-V and VII, VIII. This
value was taken as 100 percent in calculating conversions in the

presence of test chemicals to "percent of the control”.

Studigs with M. In Part II, amitrole had no effect on
nitrobacter at concentrations up to 50 ppm in liquid culture but it
caused 90 to 100% inhibition of fiitrosomonas at 2.5 ppm.

A similarly toxic degree of inhibition occurred when glass beads
were perfused with 150 or 200 ml of nutrient solution containing 1.7
ppm of amitrole (Table III). The inhibition was substantially reduced
when smaller volumes of perfusate containing up to 12 ppm were used.
The protection afforded by vermiculite was greater than by glass beads,
and that afforded by soil at each concentration and volume was still
greater.

Amitrole inhibited Nitrosomonas at extremely low concentrations
but its activity was very greatly reduced in the presence of solid
matrix materials. It is known that amitrole is strongly adsorbed by
soil materials (2,8). Accordingly, an experiment was conducted in which
perfusate concentrations of both ammonium and amitrole were followed

through two successive percolations. The results are presented in Figs,

2 and 30

5“

TABLE III
Effects of amitrole on ammonium oxidation as influenced by concentration,

perfusate volume and column material

 

 

 

 

Perfus ate NH -N Percent of the controla
volume 03d. zed in
(ml) the control Amitrole concentration (ppm)
Pg l.’/ 4.0 6.0 12.0

 

Glass beads

 

 

 

 

 

50 5.500 83 77 58 36
100 10,300 81 51 33 28
150 13,000 2 5 5 ND
200 17,100 i 7 4 2 ND

Vermiculite-Perlite

50 5,500 92 84 80 6a
100 10,300 75 59 50 43
150 13,000 25 22 11 ND
zoo 17,100 22 11 13 ND

Soil-aPerlite

50 5,500 100 95 94 82
100 10,300 96 93 92 69
150 13,000 56 56 49 ND
200 17,100 51 oz 42 ND

 

aF‘ive-ehour percolation; ND=not determined

55

During the first 2 hours of the first percolation, 75 percent of
the added amitrole had disappeared from the perfusate in the soil-perlite
systems and 85 to 90 percent from the perfusates in the other two
systems (Fig. 2). All this sorbed amitrole was later released and
recovered in the perfusate. In the glass bead systems, all of the
added amitrole was accounted for in the perfusate by the fifth hour of
the second percolation (Fig. 3). In the other two systems, complete
release of sorbed amitrole was not detected until the last sampling
at 18 hours in the second percolation.

Patterns of amitrole sorption and release were clearly related to
fi1trosomonas activity.. The rate of ammonium oxidation in treated
systems decreased abruptly as release of sorbed amitrole was observed
in the perfusate. The degree of inhibition was much less during the
first 2 or 3 hours of percolation when amitrole was most extensively
sorbed.

None of the 0.26 mg of amitrole retained on soil or vermiculite
at the end of the first percolation had reappeared five hours later
in the second perfusate. After 18 hours, however, 1.2 and 1.3 ppm
were found for soil and vermiculite, respectively. The calculated
concentration, assuming complete release of 0.26 mg, would have been
1.3 ppm. The calculated 0.25 ppm for glass beads was found in the
second perfusate at 5 hours. The perfusate concentrations for soil
and vermiculite after 18 hours were well within the inhibitory range

indicated in Table III for a 200~ml percolation volume.

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57

These amitrole concentrations in the second perfusate were
related to the observed inhibition in ammonium oxidation after
about 3 hours (Fig. 3). Thus, the glass bead system which had suffered
the greatest inhibition during the first percolation oxidised more
ammonium during the second percolation (even though less than the
control) because negligible amounts of amitrole had been left on the
columns (Fig. 2).

The data in Fig. 2 and 3 and in Table III show that amitrole
inhibits nitrosomonas at extremely low solution concentrations, and
that the protective effect of solid matrix materials involves sorption
of amitrole from solution. In the case of the glass beads, the sorption
must be due to living cells and cellular debris, since clean glass beads
did not ad=orb any amitrole. In the soil and vermiculite systemS,
mineral surfaces as well as dead and living organic materials must be
involved.

Infra-red studies (8) have shown that sorption of amitrole by
NHu-montmorillmiie involves the formation of the aminotriazolium

cation by the following reaction:

R—NHZ‘rNI-Qf-clay .9 anrgtclay + NH3 [1]

In the experiments of Fig. 2 and 3, this reaction at exchange
sites on clay or organic materials would have been shifted to the
right by the high Han-N concentration of the fresh nutrient solution.
This would account for the extensive sorption of amitrole observed in
the first hours of percolation (Fig. 2). H

Subsequent desorption and increasing inhibition by amitrole in

Figs, 2 and 3 appears to be related to activities of the nitrifiers

58

themselves. The mechanisms for displacement of sorbed aminotriazolium
are not clear.

The data suggests that interference with nitrite formation by
flitggggmgg§§,may involve competition between the aminotriazolium cation
and NthN or NH3 for uptake sites or for sites on the oxidizing enzyme.

The extensive sorption and later release in an active form which
is suggested by these data would explain the residual inhibition

observed by Chandra (2).

Studies gith,g§:§§g§§g§. Both CIPC and IPC reduced the amounts of
nitrate formed in 5 hours (Tables IV, V). At 80 ppm, CIPC was somewhat
more inhibitory than IPC. 'With both chemicals, inhibition increased
with increasing concentration and perfusate volume. Nitrate production
was somewhat retarded in 50 ml volumes at 2 hours but this was no
longer apparent at 5 hours.

The inhibition increased with increasing concentration or volume
to a greater extent with glass beads than with vermiculite-perlite.
Hewever, even at the highest concentration and volume, complete
inhibition was never attained. This is in contrast with the complete
inhibition of nitrobacter observed in Part II of this thesis in
stationary flask cultures with 50 ppm of GIPC and IPC, respectively.

On the other hand Hale et al. (4) reported a 90 percent inhibition of
nitrification in enriched soil perfusion systems with 160 ppm.CIPC.
This compares favorably with the results obtained here using the 200 ml
perfusate volume. It is apparent that inhibitory concentrations will
vary with size and activity of the population and the experimental

conditions under which the chemicals are tested.

59

TmBLE IV
Effects of CIPC on nitrate formation as influenced by concentration,

perfusate volume and column material

 

 

 

 

 

 

 

 

Perfusate N0'-N Percent of the control‘
volume produ ed in
7 (ml) the control CIPC concentration (ppm)
(pg)
40 80 140
Glass beads
50 6,180 100 100 100
100 12,350 84 82 65
150 18,500 93 29 31
200 23,800 85 20 22
Vermiculite-Perlite

50 6,180 100 100 100
100 12,350 82 97 80
150 18,500 100 51 38
200 23 .800 97 52 29

 

aFive-hour percolation

At the highest concentration of CIPC used here (140 ppm), the rate
of ammonium cxidation was somewhat reduced (Fig. 4). However, both

chemicals were otherwise specific in their action against Nitrobacter,

 

'with the result that nitrite accumulated intermediately (Fig. 5).

60

TABDE'V

Effects of IPC on nitrate formation as influenced by concentration,

perfusate volume and column material

 

 

 

 

 

 

 

 

Perfusate N0.-N Percent of the control.
volume produ ed in '
(ml) the control IPC concentration (ppm)
(pg)
40 60 80
Glass beads
50 9.770 100 100 100
100 16,410 100 100 92
150 20,250 93 70 62
200 23,800 78 61 62
Vermiculite-Perlite

50 9,770 100 100 100
100 16,410 100 100 100
150 20,250 92 89 80
200 23,800 96 85 78

 

aFive-hour percolation

High nitrite concentrations were maintained over a longer period of

time with CIPC than with IPC because ammonium oxidation*was retarded

and continued over a longer period of time.

The more rapid oxidation

of ammonium in the vermiculite system than in the glass heads when

61

perfused with CIPC (Fig. 4) served to maintain a higher level of
nitrite in the CIPC-vermiculite system (Fig. 5).

After 9 hours, the initial perfusates in Fig. 5 were replaced
'with 200 ml fresh nutrient solution containing no test chemical.

Some residual delay in nitrite oxidation was apparent, notably with
CIPC and in the glass bead systems. However, the retardation occurred
during the first 3 hours of percolation, after which nitrite disappeared
as rapidly as in the controls.

The nitrosomonas-Nitrobactgr bioassay was used to estimate the
extent to which the chemicals had.been inactivated or removed from the
initial perfusate at 9 hours. ipproximately'BO percent (24 ppm) IPC
(Table VI) and 50 percent (80 ppm) of CIPC (data not given) had dis»
appeared according to this assay;

‘With both chemicals, the indicated removals were the same for
glass bead columns as for vermiculiteeperlite columns. Thus there was
no evidence that the very great difterence in cation exchange capacity
of inorganic surfaces in the two matrix materials had influenced the
activity of these chemicaIS. Neithertwas adsorbed by clean glass beads,
so inactivation in the nitrifying glass'bead systems must be ascribed
to effects of living cells or cellular debris adhering to the surfaces
of the beads.

The possibility that some degradation of the carbamates by hetero»
trophic contaminants might have occurred was not investigated. However,
the accumulation of neutral pesticide moleciles by sorption on living
and dead microbial tissues in soil has been reported by Re and Lockwood
(6). Mortland et a1. (7) found.that a more stable complex was formed

62

 

CD
CD

 

 

 

E
3 a I40 ppm CIPC
Lu \
*2 80 7 \ O 80 ppm IPC
(f) \
E \ e CONTROL
5 \
Q 60 " \ \
E \ a
E \ \
8 \ \
E 40 " \ \a
E \ \ \
' a
2 \ \
553 2()" \\ \\
CZ) \
\
5 \g \
2
< 1 1 1 1 \A 7,
2 4 6 8 l0

PERCOLATION TIME (HOURS)

FIG. 4. Effects of CIPC and IPC on ammonium oxidation during perfusion
through glass beads ( ) and vermiculite - perlite (_ — -—).
(Perfusate volume = 200 ml)

 

63

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64

between a carbamate and montmorillonite when the clay was first
saturated with an organic cation (pyridinium) rather than with metal
cations . Scott et al. (9) found that the phytotoxicity of CIPC was
reduced by addition of organic soil to the growth medium but not by
addition of Montmorillonite or Kaolinite clays.

These observations by others support the inference made here that
dis appearance of IPC and CIPC activity from the initial perfusate was
due largely to sorption by. living or dead organic matter. Both the
glass beads and the vermiculite-perlite were devoid of. organic matter
prior to enrichment with the nitrifying cultures. A similar high rate
of nitrifying activity had been maintained for months in both systems
before these experiments were conducted.

Similar quantities of organic materials with apparently equal
sorptive capacities would reasonably have accumulated. The reductions
in activity observed by bioassay (Table VI) are consistent with the
inference that sorption by organic materials was mainly responsible.

If all of the initially sorbed chemicals had been released again
into the second perfusate (Fig. 5)» the effective concentrations would
have been about 70 ppm (2ch and 20 ppm IPC. According to Tables IV and
V, 70 ppm CIPC in 200 m1 perfusate would be inhibitory, whereas negligible
inhibition would be expected with 20 ppm IPC. The residual retardations
observed during the second percolation in Fig. 5 were about what was
expected if the sorbed chemicals had equilibrated with the fresh nutrient
solution. No assays were made at this time, but release of sorbed CIPC
and IPC into the fresh solution was expected since water competes effect-w
tively with carbamates in the lie-bonding interactions involved in their

sorption by clays (7 ).

65

TABLE VI

Amounts of IPC removed from the initial 9-ahour perfusate as estimated
by the W-fltrobagtgr bioassay

 

 

 

Cblumn Perfusate IPC ooncent. in IPC
materials volume perfusate (ppm) removed
(m1) a (15)

 

at 0 hours after 9 hours

 

Glass 150 67" 44 34
beads 200 70 48 31
Vermiculite- 150 63 M 30
Perlite 200 66 “4 33

 

“corrected for water-holding capacity of each column material

Although the bioassay detected no difference between glass beads
and vermiculite-perlite, the residual protection afforded by the latter

system was distinctly greater.

Stages 213.3; m. Both the potassium and the sodium salts
reduced nitrate production in the same manner as the carbamates with
respect to effects of perfusate volume and column materials (Tables
VII, VIII). Sodium.azide appears to be more inhibitory to Nitrobacter

than potassium azide.

66

At higher concentrations both salts also inhibit Nitrosomenas
(Parts II and III of this thesis)e However, at the concentrations
used here, only KN3 at 1.5 ppm interfered with ammonium oxidation
(Fig. 6). Otherwise, both salts were highly specific in their action
against flitggbacter. As a result, nitrite accumulated intermediately
(Fige 7) as was the case with the carbamatese

The concentrations in both chemicals needed for complete inhibition
are in the range of those found effective in liquid culture in Parts II
and III of this thesise At the lower concentrations and larger volumes
in Tables VII and VIII inhibitory effects of both salts were less in
the vermiculite-perlite systems than in the glass beadse At the
highest concentrations, and volumes, however, the difference between
column materials were negligibleo This interaction with column materials
was of lesser magnitude than in the case of amitrole or the carbamates.

The nitrosomenas-gitrobacter‘bioassawaas used again to estimate
the degree of removal or inactivation of KN3 during the first 9~hour
percolation in Fige 7. The results of the bioassay in Table IX
indicate that negligible sorption or inactivation occurredo There is
a suggestion that the perfusate concentration in the 50 m1 perfusate
may have been increased by negative sorption of the anione However,
it must be recognized that the accuracy of the bioassay is qualitative
rather than quantitative.

The bioassay was not performed on the 200 m1 perfusates used in
the experiments of Fig. 7. Nevertheless, the evidence in Table IX

that azide was not sorbed by column materials to any extent is consistent

6?

TABLE VII

Effects of potassium azide (m3) on nitrate formation as influenced by

concentration, perfusate volume and column material

 

 

 

 

 

 

 

 

Perfus ate N0'-N Percent of the control‘
volume produged in
(ml) the control “3 concentration (ppm)
( z ' ' '
P 0. 5 1.5 2.5
Glass beads
50 7,830 100 100 100
100 15,120 68 45 no
150 20,250 42 19 16
200 23,800 27 10 9
Vermiculite—Perlite
50 7,830 100 100 100
100 15,120 80 69 64
150 20,250 70 i 36 17
200 23 ,800 52 24 10

 

aFivee-ahour percolation

with the immediate recovery in the rate of nitrite oxidation when the

perfus ates containing these salts were replaced with untreated nutrient

solution (Fig. 7).

Some interaction with solid matrix materials mst

68

TABLE VIII
Effects of sodium azide (Nana) on nitrate formation as influenced.hy

concentration, perfusate volume and column material

 

 

 

 

 

 

 

 

Perfusate N0'-N Percent of the controlg
volume produaed in
(ml) the control NaN3 concentration (ppm)
( s
r 0.1 0.5 1o0
Glass beads
50 7,600 100 100 100
100 16,400 91 58 1+2
150 20,250 38 2h 13
200 23,800 2? 16 ll
Vermiculite-Perlite
50 7,600 100 100 100
100 16,400 89 72 65
150 20,250 33 32 22
200 23,800 35 2h 18

aFiveuhour percolation

have occurred, however, to have given the larger intermediate accumulation
of nitrite in the glass beads during the initial percolation. The nature

of this interaction is not clearo

 

5
o

0 L0 ppm N0N3

    
  

a I.5 ppm KN3

(D
O

O CONTROL

60"
40-
A
‘\\A
20" \ \ \

 

 

AMMONIUM-NITROGEN IN PERFUSATE (ppm)

 

2 4 6 8 l0
PERCOLATION TIME (HOURS)

FIG. 6. Effects of sodium (NaNB) and potassium (KN3) azide on ammonium

oxidation during perfusion through glass beads (

 

) and vermiculite-
perlite (-— -~ -) (Perfusate volume = 200 ml)

70
TABLE IX

Amounts of potassium azide (m3) removed from the initial 9-hour

perfusate as estimated by the fiitrosgmonas-nitrobagter bioassay

 

 

Column Perfus ate KN3 concent . in 1013
materials volume perfus ate (ppm) removed
(m1) (%)

 

at 0 hours after 5 hours

 

Glass 50 1. 508L 1.90 (-21. 5)
heads 100 1.90 1.80 5.2
Vermiculite- 50 1.35 1.60 (45.5)
Perlite 100 1075 1050 805

 

a‘(2orrected for water-holding capacity of each column material

71

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72
Summary

The inhibition of ammonium and/ or nitrite oxidation by 3-amino-
l,2,4-triazole [amitrole , is opropyl-N(3-chlorophenyl) carbamate [C1130],
isopropyl-N-phenyl carbamate [IPC], potassium azide [1013] and sodium
azide [NaNBJ was increased on increasing perfusate volumes and was
affected by the nature of the column material.

The pattern of W inhibition by amitrole and its residual
effects appeared to depend mostly upon mechanisms of its sorption and
desorption by column materials .

CIPC and IPC inhibited nitrite oxidation by nitrobacter. Living
or dead organic materials rather than inorganic column constituents
seemed to be responsible for sorption of these chemicals, and for the
inhibition carried over into a second perfusate.

Potassium and sodium azide prevented nitrite oxidation and none
of the chemicals was sorbed by column constituents, which is in agree-
ment with the absence of any residual effect on re-perfusion with untreated
medium.

Amitrole concentrations in perfusate were deterdned chemically.

A flitros mom and nitrgagter bioassay was developed for estimating

concentrations of the other chemicals.

1o

2.

3.

1+.

5.

7.

8.

9.

10.

73

References

ATKINS, CA. and TCHAN, LT. 1967. Stuck of soil algae. VI.
Bioassay of atrazine and the prediction of its toxicity in
soil using an algal growth method. Plant Soil, 27: 432-1-042.

CHANDRA, P. 1964. Herbicidal effects on certain soil microbial
activities in some brown soils of Saskatchewan. Weed Res.

“3 SL630

DWI, J .E. 1958. Utility of bioassay in the determination of
pesticide residues. J. Agr. Food Chem. 6: 27th-281.

HALE, 11.0., HULGER, F.H. and CHAMPPEIL, W.E. 1957. The effects
of several herbicides on nitrification in field soil under
laboratory conditions . Weeds : 331-3141.

HUGHES, T.D. and WELCH, L.F. 1968. Nitrification inhibition with
potassium azide. Agron. Abstr. p. 91+.

K0, 111.11. and LOCKHOOD, J .L. 1968. Accumulation and concentration
of chlorinated hydrocarbon pesticides by microorganisms in soil.
Can. J. Microbiol. 14: 1075-1078.

MORTIAND, 14.11. and MEGGITT, VLF. 1966. Interaction of ethyl N,
N-di-n-propyl thiolcarbamate (EPIC) with montmorillonite.
Jo Age FOOd Chane 1n: 126.129e

RUSSELL, J.D., CRUZ, 14.1. and WHITE, J.L. 1968. The adsorption
cg 3-aminotriazole by montmorillonite. J. Agr. Food Chem.
1 3 21-214“

SCOTT, D.C. and WEBER, J .B. 1967. Herbicide phytotoxicity as
influenced by adsorption. Soil Sci. 1015: 151-158.

ZWEIG, G. 1961+. Analytical methods for pesticides plant growth
regulators and food additives. Volume IV, pp. 17-26, Academic
Press, New York, London.

APPENDICES

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76

APPENDIX B
Chemical names and formulae of aniline derivatives

 

 

 

Chemical name Formula

2,3odichloroaniline Csfisplzfl
2,h-dichloroaniline CéflSClzN
2,5-dichloroaniline 06H5012N
3,h-dichloroaniline c6HSCI2N
3,5-dichloroaniline C6H5C12N
m-chloroaniline C63601N

grchloroaniline céagcln

2,3,5,6-tetrachloroaniline C6H§CIhN
2,3,#-trichloroaniline Céhh013N
2,#,5—trichloroaniline CthClBN
2,4,6—trichloroaniline

 

77

APPENDIX C

PERFUSION APPARATUS

 

 

 

\I i PERFUSAIE
“Craig/(#275 i" ’ All?
/
A
All? ounn :3 I:
g, ’f . . / All? Ill/LET
”WI/5475 rr N mm?
Rams/om 1 co 0
a
' 1
:
i F 541m ma soar
! / will) sterilizing closure
E I

 

  

 

 

 

78

APPENDIX D

CULTURE FERMENTOR
007mm m m?

I
All? INLET g

4“ 4

 

 

 

 

 

AIR OUTLET

211/ [L

 

x—e_e —

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

srmuzma L

610503?“ -.
a mo FLU/0 mm
our; 57

 

SPA/P65}?

 

PLEASE NOTE; Not original copy.
Blurred print throughout. Filmed

as received.
UNIVERSITY MICROFILMS .

 

 

   

    

    
  

        

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