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II. Methodology Applied in the Study of the Effects of Pesticides on the Soil Microflora

II. Methodology Applied in the Study of the Effects of Pesticides on the Soil Microflora

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4



GINETTE SIMON-SYLVESTRE AND J.-C. FOURNIER



microflora and some special microorganisms. This is the direct measurement of

the qualitative change appearing after pesticide treatments. ( b ) Indirect techniques for estimating the activity of the total microflora or of some microorganisms that play an important part in the biological cycles. We should also

mention some particular methods such as the measurement of the biomass in the

soils.



I . Composition of the Soil Microjlora: Counts of the

Microorganisms

Very rarely are counts of the microorganisms made directly with a microscope, in soil suspensions from experiments with pesticide treatments. Such

counts have the advantage of being carried out under ecological conditions, but it

is difficult to distinguish between living microorganisms and dead ones, for

evidently the dead organisms are not counted in the characterization of the

biological level of a soil. The use of fluorescence and of coloration techniques,

however, makes it possible to reduce such errors to a certain extent.

In most cases, the count of the microorganisms is carried out after they have

been grown on media favorable to their development. These are usually synthetic

organic media, either liquid or solid (with gelose) or mineral (silicogel). The

seeding is effected from inoculum suspensions, more or less diluted with water,

into the medium or on the surface. After a period of incubation, the number of

the microorganisms is estimated from the count of the colonies or from the

estimation of the highest dilution that permits the growth of the microorganisms.

In the latter case, probability tables, prepared by MacCrady (1915) and Swaroop

(1951), indicate the number of microorganisms per gram of soil on the basis of

the observed growths. This method of determination may be used on soil samples

taken directly in the field from treated experimental plots, or in trials carried out

in vitro in a laboratory, or in a greenhouse.

We have listed here some of the culture media used most often for counting the

total microflora and the main groups of microorganisms.

For counting the total microflora, the soil aqueous extract seems to be preferred, whether it is liquid or solidified by gelose (Huge, 1970; Kaiser and Reber,

1970; Kaszubiak, 1970; Lozano-Calle, 1970; Catroux and Fournier, 197 1; Karki

et al., 1973; Simon et al., 1973; Simon-Sylvestre, 1974; Oleinikov et al.,

1975). Some workers add yeast and mineral salts to this medium (for example,

Bunt and Rovira, 1955; Voets and Vandamme, 1970; Voets et al., 1974). Other

media used for the determination of the total microflora include that of Kaunat

(1965) (mineral gelose medium with casein, peptone, and glucose) used by

Kaiser and Reber, (1970); and Thoronton’s medium, used by Sharma and Saxena

(1974).



EFFECTS OF PESTICIDES ON SOIL MICROFLORA



5



For fungi, Martin's medium (1950) with rose bengal is often used (Huge,

1970a; Kaszubiak, 1970; Voets and Vandamme, 1970; Tu, 1972; Camper et al.,

1973; Karki et al., 1973; Simon et al., 1973; Sharma and Saxena, 1974;

Simon-Sylvestre, 1974). It contains streptomycin, which impedes the growth of

bacteria without having the drawbacks of the acidified media (Houseworth and

Tweedy, 1973; Focht and Josseph, 1974), in which certain fungi do not grow

because of the excessive acidity, whereas other quickly growing species overgrow all the preparations. However, some fungi (Pythiaceae, in particular) are

sensitive to streptomycin and cannot grow on media containing this antibiotic.

Finally, we should mention the work of Kaiser and Reber (1970), who use a

gelose medium with Maltea Moser and chloramphenicol.

Bacteria alone are often counted on nutritive gelose (Houseworth and Tweedy,

1973; Focht and Josseph, 1974) or on Czapek's gelose medium (Wainwright and

Pugh, 1974). Spore-forming bacteria may be separated from the others by heating the soil for 10 minutes at 90 "C (Voets et al., 19740 or by heating a soil

suspension for 15 minutes at 75°C (Oleinikov et a / . , 1975).

Actinomycetes are generally counted on media containing antibiotics: nystatine and actidione in the work of Camper et al. (1973), and albamycine and

streptomycin in the studies of Focht and Josseph (1974). Sharma and Saxena

(1974), on the other hand, use only a (nitrates + saccharose) medium.

The (gelose + sodium albuminate) medium is often chosen to evaluate bacteria and actinomycetes (Chandra, 1966; Tu, 1972; Camper, 1973). Van Faassen

(1974), however, carries out the separation of bacteria and actinomycetes on a

(soil extract + gelose) medium enriched in glycerol, asparagine, casein, glucose, and cycloheximide as antifungic.

In addition to providing estimates of the totals of the major groups of microorganisms, these methods may be used in research on the identification and evaluation of the percentages of various microorganisms. We must, however, place

restrictions on the use of Martin's medium, which often affects the morphology

of the colonies. This method of identification, which is long and dull, is suitable

only for certain microorganisms, such as the pathogenic ones.

Some criticisms may be expressed concerning methods of dilution counts:

(1) Regardless of the technique used, the number of counted microorganisms

is always much lower than that read by direct numeration, by a factor of 10-1000

on the average for the total numeration; very special organisms, such as

anaerobic bacteria, autotrophic bacteria, and strictly cellulolytic organisms, do

not grow because their feeding requirements are too specific.

(2) In spite of the standardization of the techniques of dilution, inoculation,

and incubation, the precision of the measurements remains relative, for in addition to technical errors, there are the difficulties of sampling due to the unavoid-



6



GINETTE SIMON-SYLVESTRE AND J.-C. FOURNIER



able heterogeneity of the analyzed soils (Meynel and Meynel, 1965; Ricci,

1974). Whatever the circumstances, differences lower than a half power are not

often significant.

(3) No distinction can be made between inactive microorganisms and those

really active in the soil. In fact, the dilution methods often lead to the reduction

or to the suppression of the phenomena of biostasis and synergism, which normally take place in the soil.

(4) The significance of the count of microorganisms sometimes has a restricted but far-reaching effect in the case of organisms with vegetative and

reproductive forms, such as fungi. In fact, the mycelia may be cut into several

pieces, the spores scattered and the count is not precise.

2 . Measurement of Microflora Activity

An interesting method of testing the effects of pesticides on the soil microflora

consists in measuring its biological activity. We may evaluate either the total

activity or the activity of a particular group of microflora.

Measurement of the activity of the soil microflora provides ordinary indexes of

the biological state of the soils and therefore of their fertility. We may determine

the real activity of the microflora or its potential activity-that is, its ability to

adapt to new ecological conditions, to the addition of various substances or

substrates, or to the modification of any of the environmental factors.

There are two types of techniques for determining total activity: ( a ) Classical

techniques, which allow the activity of the microflora proper to be characterized.

The treated soil may then be used just as it is, as the basis of the analysis-the

ecological conditions are then respected-or just for seeding of the media (grains

of soil, suspension dilutions). ( b )Other techniques, which determine the activity

of the microflora by measurement of enzymatic activity.

a . Classical Techniques. Measurement of biological activity. The measurement of biological activity, described by Pochon and Tardieux (1962), consists in evaluating the rapidity with which the soil microorganisms grow in the

more or less synthetic, liquid or solid media used for the counts of the microorganisms, and always in the presence of a specific substrate. The number of living

microorganisms (see the tables of MacCrady) increases as the incubation progresses. The speed of the reaction is thus known, and also its limits.

These simple methods give a first approximation of the activity of the microflora, but most of the criticisms expressed with regard to the count methods by

suspension dilutions apply here also.

These techniques are still used to study most of the major physiological groups

of the soil microflora, for example, the microorganisms active in the nitrogen and

carbon cycles: ammonifying bacteria (with tyrosine), nitrifying bacteria (with

ammonium sulfate and sodium nitrite), denitrifying bacteria (with potassium



EFFECTS OF PESTICIDES ON SOIL MICROFLORA



7



nitrate), proteolytic bacteria (with serum), and amylolytic bacteria (with starch).

They are mentioned in the reports of Huge (1970a), Kaiser and Reber (1970),

Lozano-Calle (1970), Ritter et al. (1970), Catroux and Fournier (1971), and

Simon et al. (1973).

Kinetic tneasurement of degradation of the substrates. All these kinetic

measurements are made directly on the soil itself. In incubation, a substrate is

added to the soil, which is incubated under favorable conditions of temperature

and moisture. The products formed from the added substrate are chemically

determined in the course of time. The activity curve of the microorganisms

studied can be plotted from the data. This method is particularly suitable for

studying ammonification and nitrification. The nitrogenous substrate added varies, depending on the workers. For nitrification, it is most often ammonium

sulfate (Chandra and Bollen, 1961; Balicka and Sobieszczanski, 1969; Bardiya

and Gaur, 1970; Dubey, 1969; Dubey and Rodriguez, 1970; Szernber et al.,

1973; Wainwright and h g h , 1973; Campbell and Mears, 1974; Focht and Josseph, 1974; Horowitz et al., 1974a; Voets et al., 1974; Abueva and Bagaev,

1975). Correa Salazar (1976) uses a variant of this method, in which soil suspensions take the place of soils. Sometimes the following substrates are used:

monoammonium phosphate (Shaw and Robinson, 1960; Bartha et af., 1967),

ammonia (Chandra and Bollen, 1961), urea (Vlassak and Livens, 1975), asparagine (Szember et al., 1973), cotton meal, (Eno, 1962), peanut oil cake

(Akotkar and Deshmukh, 1974), or plant remains (Bliev, 1973).

In the case of organic substrates, the breakdown of the nitrogenous compounds

allows measurement of the activity of the ammonifying as well as the nitrifying

bacteria.

A variant of this technique, percolation, is also used for nitrification studies.

Through a column filled with soil a solution of ammonium sulfate circulates in a

closed circuit; subsequent chemical analyses give information about the biological evolution of this ammonium salt (Jaques, et al., 1959; Torstensson, 1974).

Urea may also be used (Namdeo and Dube, 1973). Technical difficulties make

this method a difficult one.

Finally, measurement of the mineralization of the organic matter in the soil,

under well-controlled conditions, can give good information on biological activity (Drouineau and Lefevre, 1949; Dommergues, 1960). However, not many

papers on this technique mention research on the effects of pesticides.

Measurement of total soil respiration. Measurement of the total soil respiration (oxygen uptake and evolution of carbon dioxide) is often carried out according to Warburg’s technique, directly on the soil, treated or not-just as it is

(endogenous respiration) (Giardina et al., 1970; Tu, 1975; Weeks and Hedrick,

1971), or enriched with nutrients to measure their effects on the metabolism

(glucose: Bartha et al., 1967; Tu, 1972; mannitol: Johnson and Colmer, 1958).

This technique is suitable for short-term measurements and generally for small



8



GINETTE SIMON-SYLVESTRE AND I.-C. FOURNIER



soil samples. For long-term measurements the technique of sweeping the soil

samples with COz-free air is often used. In its passage through the soil, this air

becomes heavy with COBfrom the catabolic activities; it is then possible to trap

and to titrate the COOformed. Some workers use titrated solutions of baryta

(Elkan and Moore, 1960; Agnihotri, 1971; Smith and Weeraratna, 1975) or of

soda (Eno, 1962; Grossbard, 1971; Van Faassen, 1974).

Measurement of oxygen uptake is often carried out with electrolytic respirometers. The general principle of the operation of these apparatuses is the

incubation of soil samples in closed cells and the periodic introduction of oxygen, by electrolysis, to compensate for the soil uptake (MacGarity et a f . , 1958;

MacFayden, 1961).

The measurement of soil respiration is sometimes used in the study of particular groups of microflora. Thus, Anderson and Domsch (1973), using antifungic

or antibiotic substances, were able to distinguish the relative importance of fungi

and of bacteria in soil respiration. However, there seem to be no studies on the

combined use of pesticides.

Measurements of radioactivity. Labeled elements are helpful in making

these determinations. Thus, the radiorespirometric technique represents a neat

and practical method of measuring the rapidity of the mineralization of a radioactive substrate (labeled with 14C). Mayaudon (1973) offers a true index of the

biological activity of the soil, expressed in relation to the pmoles (lo-’’ A41

of 14C02evolved, in the course of the mineralization of dl-glutamic acid.

The tests may be repeated on treated and control plots. Mayaudon (1973)

studied the effects of the different pesticides used alone or in mixtures under

sugar beet. Domsch et a1. (1973), with a similar technique, followed the influence of benomyl on the breakdown of 14C-labeledglucose.

Other radioactive substrates may be used for biological studies, such as those

of Lauss and Danneberg (1975) on the decomposition of plant residues labeled

with 15N, in the presence of pyramin. Sometimes the pesticide itself is labeled,

but its microbial degradation is studied more often than its effects on the soil

microflora (Grossbard, 1970b, 1973).

Measurements in situ. In order to work under more ecological conditions,

some workers make their activity measurements directly in situ, either on the soil

treated with pesticides under field conditions, or on the plants. Respiration of the

soil, according to Dommergues’s technique (1968), may be measured in situ, but

not many papers have been published on pesticide research. The situation is

similar with Billes’ method (197 l), which includes titration by electrolysis of

COPdissolved in a sodium chloride solution colored by phenol-phthalein.

Cellulolysis, in contrast, has attracted several workers: its study involves

periodic checks on the behavior of strips of calico and of cellulose powder, both

buried in the treated soil. The decrease in the strength of the calico strips,

measured mechanically, and the loss of weight of the cellulose powder, enclosed



EFFECTS OF PESTICIDES ON SOIL MICROFLORA



9



in nylon bags, are dependent on the activity of cellulolytic organisms

(Klyuchnikov et a l . , 1964; Grossbard, 1974; Grossbard and Wingfield, 1975;

Grossbard and Long, 1975, oral communication).

The method of “mold soil” also gives direct measurements of activity, but the

data obtained are only qualitative. The soil serves as culture medium; it is

increased with elective substrates chosen according to the research objectives.

Microbial readings are carried out on lamellae, set on the soil. This method is

described in an earlier work on DDT and hexachlorocyclohexane (Drouineau et

a l . , 1947).

In our final example, relating to symbiotic nitrogen fixation, plants are used

for the determination of biological activity. Immediately after the rooting up of

the leguminous plants, the root nodules are counted and examined under the

microscope, and the leghemoglobin is determined. Goss and Shipton (1965)

follow this technique on plants the seeds of which have been disinfected with an

insecticide. Garcia and Jordan (1969) use the same method after spraying herbicides on a leguminous plant field.

b. Measurement of the Enzymatic Activity of Soils. We must also consider

this technique, which is based on soil enzymology. In fact, a large portion of the

enzymes present in the soil is of biological origin, both extra- and intracellular.

Several workers have endeavored to evaluate this fraction after a pesticide treatment, hoping to find a correlation between enzymatic activity and soil fertility.

Thus, some research workers have determined the presence of several enzymes

such as tryptophanase, urease, phytase, invertase, and saccharase (Voets and

Vandamme, 1970; Bliev, 1973; Zubets, 1973; Verstraete and Voets, 1974; Voets

et a l . , 1974; Karanth et a l . , 1975; Lethbridge and Bums, 1976). Others have

limited their studies to one determination only-for example, the determination

of nitrogenase in studies on atmospheric nitrogen fixation (Eisenhardt, 1975;

Neven et a l . , 1975; Peeters et al., 1975, or of dehydrogenase, which gives an

estimate of the soil’s respiratory activity (Ulasevich and Drach, 1971; Karki et

a l . , 1973; Karki and Kaiser, 1974; Van Faassen, 1974).

These enzymatic methods are attractive because of their simplicity and their

good reproducibility, as compared with the classical techniques used in soil

microbiology; yet interpretation of the data requires some prudence, in view of

the fact that at present we have limited knowledge of the enzymes-their origins,

the relationships between them, and the role they play in soil fertility.

3 . Measurement of the Biomass



This kind of measurement, which has not received much attention, provides an

estimate of the effects of pesticides on the soil microflora.

Several methods have been suggested for estimating the microbial biomass,

including measurements of ATP (MacLeod et a ! . , 1969; Lee et a!., 1971;



10



GINETTE SIMON-SYLVESTRE AND J.-C. FOURNIER



Ausmus, 1973) and measurement of muramic acid (Millar and Casida, 1970).

The biomass may also be calculated from the biovolume by multiplying the

number of organisms in a soil sample by the volume of an organism of medium

volume (Russell, 1973).

A particularly interesting method is proposed by Jenkinson and PowIson

(1976). These authors deduce the biomass of the microorganisms from the measurement of the flush of C02 evolved after fumigation of the soil with C H Q .

They note that the CO, evolved comes from the carbon mineralization in the

bodies of the microorganisms killed by the treatment; 50% of this carbon is

mineralized during the ten days after the treatment by the surviving or reinoculated microorganisms recolonizing the medium.

4 . Complementary Techniques



The preceding techniques for measuring activity are, in general, applied in

research concerning the major biological cycles, the carbon and nitrogen cycles.

They constitute the classical techniques, as opposed to less familiar methods,

which are, nevertheless, also interesting, owing to the additional information

they provide, especially with respect to the effects of pesticides on the soil

microflora.

a . Phosphorus and Sulfur Cycles. Research relating specifically to the

phosphorus and sulfur cycles includes studies on the mineralization of organic

phosphorus (Tyunyaeva et a f . , 1974, according to the technique of Menkina);

studies on the oxidation of elementary sulfur (Tu, 1970, 1972, 1973) (laboratory

experiments with incubation of powdered sulfur in the presence of pesticides);

and studies on the mineralization of organic sulfuf (Simon-Sylvestre and

Chabannes, 1975) (monthly determinations of sulfates on soil samples from

experimental plots treated under field conditions).

6 . Pure Cultures. Assays have also been carried out with pure cultures of

soil microorganisms. It is easy to follow the effects of a pesticide on these varied

organisms, but the responses are individual. Furthermore, we cannot assume that

these results would be the same for soil, where the interactions and competition

between the microorganisms are numerous.

This kind of experiment requires pure cultures of different organisms, such as

symbiotic nitrogen-fixing organisms (Brakel, 1963; Makawai and Ghaffar, 1970;

Pajewska, 1972; Mendoza, 1973; Pantera, 1974; Suriawiria, 1974; Eisenhardt,

1975; nonsymbiotic nitrogen-fixing organisms (Pajewska, 1972; Szegi et al.,

1974; Peeters et a l . , 1975); fungi (Lorinczi, 1974); Cellulolytic organisms

(Lembeck and Colmer, 1967; Szegi, 1970); Cellulolytic fungi (Grossbard,

1974); nitrifying bacteria (Garretson and San Clemente, 1968); and bacteria

(Kulinska and Romanov, 1970; Ujevic and Kovacikova, 1975). Moreover, with

this type of experiment, we must determine the action of pesticides on anti-



EFFECTS OF PESTICIDES ON SOIL MICROFLORA



11



biotic production by actinomycetes (Krezel and Leszczynska, 1970) and

bacteria (Kosinkiewicz, 1970; giberellic substance production by bacteria

(Sobieszczanski, 1970); free amino acid secretion by bacteria and actinomycetes

(Balicka et a l . , 1970); and pigment formation (Kulinska and Romanov, 1970).

We should also consider the influence of pesticides on some antagonisms and on

pathogenic microorganisms.

Finally, pure strains of microorganisms may be used as “indicators” of soil

phenomena. Most of the tests that evaluate the toxicity of substances may be

applied to the study of the effect of pesticides on the microflora. Thus, Breazeale

and Camper (1972) studied the action of a line of herbicides on the growth rate of

different microorganisms.

5 . Conclusions



All these methods of biological analysis of soil have flaws, yet they contribute

considerably to the analysis of the major biological phenomena of soil. However,

no numerical values are obtained. All the data are comparative, even for the

techniques that seem to be the most ecological; the conditions are often altered in

order to magnify the phenomena, and the optimum or potential activity is estimated rather than the actual activity. We must add to this criticism the fact that

the use of elective media allows us to study only the main groups of microorganisms; the interrelations between them in the soil are not considered, in order

to simplify the study of the problem. Therefore, it is difficult to interpret the data

obtained in the laboratory in terms of field conditions.

In short, we must emphasize comparative measurements and always work

under the same conditions, with the same techniques, on soil samples freshly

collected or stored at low temperatures. We should also prefer measurements of

activity, which give better information about the behavior of the phenomena, to

counts, which may lead to uncertain results.



B. EXPERIMENTAL PROTOCOLS FOR THE STUDY OF THE

INFLUENCE OF PESTICIDES ON THE SOIL MICROFLORA



This section will present a fairly extensive, but not exhaustive, discussion of

the characteristic conditions of experimentation described in recent studies of the

interactions between pesticides and the soil microflora. The different factors of

variation may be divided into three major groups, based on their association with

the environmental conditions, the pesticide, or the research worker.

Our purpose is to draw attention to the great diversity that exists among the

experimental protocols. As the studies concerned deal with biological processes,



12



GINETTE SIMON-SYLVESTRE AND J.-C. FOURNIER



which are very sensitive to environmental conditions, this diversity is reflected in

the variability of the effects studied.

Herbicides, which stimulated much of the research, have received the most

attention. To simplify the discussion and to give the reader a general overview of

the subject, the results are presented in tables that are very schematic and sometimes incomplete, for research workers often omit from their description of the

experimental conditions those factors affecting the results of pesticide treatment

that have already been proved.

1. Factors Associated with Environmental Conditions



Environmental conditions play an important part in the effect of pesticide

treatments on the soil microflora and therefore deserve particular attention.

a. Noncontrolled Physical Factors. In field studies-that is, in vivo-under

noncontrolled climatic conditions (Tables I and 11), where the scientist can regulate neither the rain nor the temperature, the data obtained the first year in a

specific location for a certain chemical are not necessarily repeated the next year

(Davidson and Clay, 1972; Simon-Sylvestre, 1974). Our limited knowledge of

the different climatic factors makes it difficult to demonstrate the part played by

TABLE I

Field Experiments



Pesticide



Soil type



Crop



Duration of

the trial



Authors



1 year



Mashtakov et a / . (1962)



1 year

16 weeks

2 years



Kozlova el al. (1964)

Kulinska (1967)

Bakalivanov and

Nikolova (1969)

Ulasevich and Drach

(1971)

Husarova (1972)

Kozyrev and Laptev

( 1972)

Karki er d . (1973)

Namdeo and Dube ( I 973)

Szember et ul. (1973)

Deshmukh and

Shrikhande (1974)



Simazine, atrazine,

2.4-D

Simazine, atrazine

Simazine

Simazine between

rows

Atrazine



Sod podzol, peat

podzol

Middle loam

Tchernozem

Tchernozem



Corn



Humus-bearing podzol



Corn



2 years



Alachlor, propachlor

Simazine, prometryn

2.4-D

Sodium chlorate

Dalapon, paraquat

Dinoseb

Simazine, atrazine,

cyanazine, 2,4-D,

TCA

Atrazine



Degraded tchernozem

Clayey illuvial podzol,

slightly loamy

Sandy loam

Grassland sward

Leached podzol

Clayey soil



Corn

Barley, flax,

winter rye



9 years

2 years



Grass

Rotation

Wheat



Loamy sand



Apple trees



Corn, lupine

Corn

Strawberry plants



150 days



3 years

I year



25 years



Voets et ul. (1974)



EFFECTS OF PESTICIDES ON SOIL MICROFLORA



13



each of them, and the research worker can determine the effects of their action

for a given year only.

Moreover, these investigations in vivo are performed most often in the presence of a plant that may vary in size and age from a small plant sprung up some

weeks before, to a fruit tree several decades old. In experiments in plots, the size

of the plot often depends on the plant being tested; the area may vary from a few

square meters for vegetable crops (Ritter et a / . , 1970; Kolesnikov et al., 1973)

to several hundred square meters for arable crops, especially in Russia (Yurkevich and Tolkachev, 1972; Fisyunov et ul., 1973). Some “rhizosphere” effects modify the actions of pesticides and render the problem more complicated.

Thus, the species cultivated and the age of the plants are important variables that

must be considered. The soil tested is another variable-a factor that is often

underestimated by many microbiologists, in both in vivo and in vitro studies.

This factor will be discussed later in this section.

b. Controlled Physical Factors. In the greenhouse. This part of our study

is concerned with experiments in pots, which are intermediate between field

trials and laboratory assays. The most important characteristics are given in

Table 111. The fact that such experiments are usually performed under controlled

conditions of moisture and temperature brings them closer to experiments in

vitro, whereas the frequent presence of the plants reproduces in a rather artificial

manner the “rhizosphere” effects observed in the fields. The choice of the

plant tested, as well as the duration of the cultivation period, is limited by the

size of the pots. The quantity of soil may vary between 0.9 kg (Kaiser and

Reber, 1970) and 20 kg (Catroux and Fournier, 1971; Steenbjerg et ul., 1972).

The material used for the pots is sometimes specified, such as Mistcherlich pots

(Lozano-Calle, 1970; Wanic and Kavecki, 1972; Kozaczenko and Sobieraj,

1973), or Wagner pots (Kozlova et al. , 1964; Malichenko, 1971). Yet, considerable information on the scheme of these experiments may be missing (the

preservation of moisture, the problem of water flow, etc.).

Moreover, some microbiologists wait a longer period of time before beginning

their experiments in order that the balance of the microflora may be recovered

after the pots have been filled. Thus, Guillemat et a / . (1960) delay their experiments for 15 days at 20°C; Smith (1972) delays for 2 weeks to bring the pots

to field capacity; and Horowitz and Blumenfeld (1973) wait 4 months. This

procedure seems reasonable, since the favorable conditions of moisture and

temperature that are established often result in a waking of the microorganisms,

which is immediately indicated by a noticeable increase in their number and in

respiratory activity. On the other hand, Eno (1962) works immediately with

air-dried soil, as do Allott (1969) and Houseworth and Tweedy (1973).

The temperatures used in greenhouses vary, depending on the workers, and

even in the same room they are not always kept constant during the experiments.



TABLE II

Experiments in Vivo (Plots)



Pesticide



e



P



2.4-D, simazine, monuron,

dinoseb, petroleum oils

Simazine, atrazine

Methabenzthiazuron

Paraquat, simazine, diuron,

linuron, chlortiamid,

dichlobenil

Prometryn, carbamate,

2,4-MCPB, dinoseb

Methoprotryne

Vapam, DD, chloropicrin

MCPA, simazine, linuron,

triallate

2.4-D, 2,4-MCPA, SO,H2

2.4-D, simazine

TCA, EFTC, cyclurona

chlorbufarn



Soil type



Size of plot



Sod podzol (clayey

and loamy sand

Sandy soil

Loamy sand, sandy

loam



10 m 2

50-56 m 2

I m2



Crop



Spring barley,

winter barley



Serradella

Alluvial soil

Calcareous clay

Sandy loam



2x5111

4 x 22.5 m

1 x 10m



Tchernozem

(slightly humic)

Light chestnut soil



400 m 2

60 mz



wheat

Tomato

t Corn, barley,

carrot

Spring wheat



Duration of

the trial



Authors



2 years



Shklyar ef a / . (1961)



1 year



I year



Klyuchnikov e t a / . (1964)

Huge ( 1970a)



5 years



Huge (1970b)



70 days

1 year

2 years

7-8 years



4 months



Corn



1 year



Sugar beet



5 years



Kaszubiak (1970)

Micev (1970)

Ritter et a / . (1970)

Grossbard (1971)

Benrand and De Wolf

(1972)

Yurkevich and

Tolkachev (1972)

Zharasov er a / . (1972)



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