Chemosphere, VoI.II, No.9, pm 909 - 914, 1982 0045-6535/82/090909-06503.

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Printed in Great Britain © 1 9 8 2 Pergamon Press Ltd.

INFLUENCE OF PESTICIDES ON ACTIVITIES OF INVERTASE, AMYLASE AND LEVEL OF

ADENOSIHE TRIPHOSPHATS IN ORGANIC SOIL

C. M. Tu
Research Centre, Agriculture Canada
London, Ontario H6A 5B7, Canada
ABSTRACT

Laboratory tests showed that none of the pesticide treatments inhibited

invertase and amylase activities. With the exception of nitrapyrin at the low
level, all treatments inhibited ATP in the soil for i day and then showed a rapid
recovery.

INTRODUCTION

Soil microorganisms, chiefly bacteria, actinomycetes and fungi, occupy a

unique position in biological cycles in terrestrial habitat and are essential for
plant growth. Nany studies I'2'3 have shown that some pesticides had little effect
on microbial activities related to soil fertility. Pesticide effects on soil
enzymatic activities, a factor that has so far received little attention, may be
important. Adenosine 5'-triphosphate (ATP) is a useful indicator of life in soil.
It occurs in all living cells and can be measured readily and accurately by the
luciferin-luciferase system 4. No information is available on changes of the
content of ATP in organic soils due to the treatments.
In this study, experiments were conducted under laboratory conditions~to
elucidate effects of 39 pesticides on activities of invertase, amylase and ATP
contents in an organic soil.

MATERIALS AND ~ETHODS

An organic soil was collected in early spring and sifted through 2-mm mesh.
The soil contained 49% organic matter, 1.88% Kjeldahl nitrogen, 186% moisture
holding capacity and had a pH in water of 6.7. These properties were determined
by the methods reported previously 5'6. The pesticides (listed in Table I) were
of analytical grade or of ~ 94% purity. Roundup was a 36% emulsifiable concen-
trate of glyphosate. With exception of the fumigants, required amounts of pesti-
cide were applied to the soil using a carrier sand as described elsewhere 7. Five
non-persistent fumigants, D-D, Telone, Telone C-17, Telone II and Vorlex, were
injected directly into the soil. Soils treated with streptomycin, p-benzoquinone,
HgCI 2 and autoclaving were also prepared to compare the effects of these treat-
ments on soil enzyme activities with those of the pesticides.
Activities of soil enzymes were determined at 1 and 2 days for invertase
and 1 and } days for amylase. Triplicate samples of 2-g soil were allowed to
stand with 0.6 ml toluene for 15 min before incubating with 4 ml acetate-phosphate

909
910

buffer (0.5 M acetic acid-0.5 ~ Na2HP04) at pH 5.5 and 5 ml of 5% sucrose

solution or 2% starch. After shaking, they were placed in the incubator at 28°C.
Controls with or without added substrate were included. Enzyme activities were
determined using the Prussian blue method for the reducing sugar of Folin and
Malmros ~. Values for the hydrolysis of sucrose or starch by soil were corrected
for the reducing sugars produced on incubation of soil with toluene and buffer
without added substrate. Reducing sugars produced were estimated as glucose.
Twenty-three pesticides (Table 3) were selected to study their effects on
ATP contents in the soil. One gram of treated and/or non-treated soil was
incubated at 2~°C and ATP content was analyzed after 24 and 48 h. Soil moisture
was maintained at 60% of the moisture holding capacity. Soil was extracted with
5 ml Tris-EDTA buffer (50 mM Tris-4 mM EDTA) at pH 7.75. Soil microbial ATP
in 100 ul suspension was extracted using 100 ul nucleotide releasing reagent
(NRB, catalogue No. 4015, Lumac Systems Inc., P. O. Box 2805, Titusville, Florida
32750, U. S. A. or Handel Scientific Co., Rockwood, Ontario, Canada) and mixed
for 60 sec. ATP was added to other sample suspensions as an internal standard to
correct for extraction losses. The cuvette was placed in the counting chamber of
a Luminometer model 1070. ATP was then determined by adding 100 ul biolumines-
cence reagent (Lumit-HS, catalogue No. 4101, a highly purified luciferin-lucife-
rase) using a repeatable dispenser.
All analytical results were calculated on the basis of oven-dry (105°C)
weight of soil. Results were expressed as means of triplicate samples.

RESULTS AND DISCUSSION

The availability of methods for determination of soil enzyme activities and

ATP contents made possible a detailed analyses of the biomass activities in a
laboratory soil system. Invertase activity, as indicated by the formation of
reducing sugars is an index of the activity of microflora involved in hydrolysis
of sucrose, which would appear to be predominantly 8-fructofuranosidase 9. Acti-
vities and contents of enzymes in soil were based on the formation of glucose.
None of the pesticide treatments inhibited invertase activity in the organic soil
(Table 1), while some had a stimulatory effect. The activities of invertase
ranged from 1 to 50 mg glucose/g soil in the 1-day samples and 4 to 89 mg/g in
the 2-day samples.
Soil amylase probably consists of different enzymes or enzyme systems which
have a role in the hydrolysis of starch. Hofmann and Hoffman l0 revealed that
soil contained cosiderably higher concentration of ~-amylase than a-amylase. The
effect of different treatments on the activities of amylase is summarized in
Table 2. No inhibitory effect was observed with treatments of pesticides.
However, a stimulatory effect was evident with a number of chemicals. The appa-
rent inhibition by autoclaving on sucrose- and starch-hydrolyzing enzymes in
samples could be due mainly to inactivation of enzymes in the soil.
 911

1ABLE i
Activity of invertase a s r e l a t e d t o t r e a t m e n t o f an o r g a n i c s o i l with different chemicals
and a u t o c l a v i n g {mg r e d u c i n g s u g a r / g s o i l ) .

Treatment application ........ Inc),hat)gn ~,c~ od ~ay) ..........

(*,~I~] l 2 1 2 '
Control 0 0 b 81 b 81
Autoclaving 0 NI)** 1° 4* ND ND
Streptomycin 100 200 8 69 17 78
o-Benzoquinone 50 ND 8 87 ND ND
HgCI 2 70 ND 18 66 ND ND
Chlorfenvinpbos S 10 3 8b 28" 77
Chlorpyrifos 5 10 16 89 39 ° 78
Diazinon 5 lO 44* 88 3b* 79
Ethion S lO 44* 88 4l* 77
Ethoprop S lO 44' 88 SO° 77
Fensulfothion 5 lO 39* 89 27* 72
Fonofos S 10 26* 88 17 67
Leptophos S 10 d]* 89 42* 74
Malathion S 10 19 82 27 ° 77
Parathion S lO IS 88 21 75
Phorate 5 10 38* 89 45 ° 75
Terbufos S l0 33" 88 29* 76
Thionazin 5 10 28" 89 38" 71
Triazophos S IO 42" 89 38 ° 71
Trichloronat S 10 3S* 88 32* 73
Chlordane S 10 22 88 25 78
Dieldrin S 10 13 87 31 ° 71
Lindane S 10 23 87 31" 74
Carbofuran S lO 22 87 36" 70
Metalkamate S I0 18 80 41" 73
Oxamyl S lO 19 86 27* 7S
Permethrin 5 10 31" 87 37* 75
2,4-D S lO 14 74 8 75
Atrazine 5 10 8 76 28" 73
Dalapon S 10 b 83 16 76
Dicomba S lO 5 70 20 76
Nitrofen 5 lO S 77 34" 76
Picloram S i0 12 84 19 75
Roundup a S 10 ll 80 24 7b
Simazine S 10 10 85 21 74
Captan S l0 23 85 18 68
blaneb S lO 17 77 28* 65
Thiram S 10 iS 72 18 75
D-Db ISO 3(~ 2b* 89 40* 76
TeloneC 30 bO 19 77 35" 79
T e l o n e C-17 d 30 60 8 82 14 b4
Telone lie 30 o0 4 83 20 63
Vorlex f 40 80 8 79 21 60
Nitrapyrin 30 60 18 79 24 bl

*" N D = not d e t e r m i n e d
° S i g n i f i c a n t l y different from control at P=O.OS.
a G I y p h o s a t e 36% p r e s e n t a s i s o p r o p y l a m i n e s a l t .
b 1,3-dichloropropene, 1 , 2 - d i c h l o r o p r o p a n e and r e l a t e d C3 h y d r o c a r b o n s m i x t u r e .
c 1 , 3 - d i c h l o r o p r o p c n e and r e l a t e d C3 h y d r o c a r b o n s .
d 1 , 3 - d i c h l o r o p r o p e n e and r e l a t e d C3 h y d r o c a r b o n s 83% and c h l o r o p i c r i n 17%.
e 1 , 3 - d i c h l o r o p r o p e n e 92% and r e l a t e d C3 h y d r o c a r b o n s .
f 1 , 3 - d i c h l o r o p r o p e n e and r e l a t e d C3 h y d r o c a r b o n s 80% and m e t h y l i s o c y a n a t e 20%.

The invertase activity was consistantly greater than that of amylase in the
organic soil. Drobnik ll showed that both invertase and amylase are produced
adaptively in soil and amylase is extracellularly accumulated enzymes 12.
ATP which is a common molecule for energy packaging and transport has been
suggested both as a direct index of biomass 13 and as a criterion for evaluation
of microbial activity 14" ATP is an extremely labile cell constituent. A good
extraction procedure is required to disrupt the cell quickly and completely so
that neither further synthesis nor degradation of ATP occurs.
912

TABLE 2
Effects of pesticides and a u t o c l a v i n g on a c t i v i t y o f a m y l a s e i n an o r g a n i c s o i l
(mg r e d u c i n g s u g a r / g s o i l )

Rates o f Low t r e a t m e n t r a t e High t r e a t m e n t rate

Treatment application Inc~.. period~
(vg/g) 1 3 1 3
Control 0 0 2.3 4.4 2.3 4.4
Autoclaving 0 ND** 0.4* 0.7* ND ND
Streptomycin 100 200 i,9 lO.l 5.6 13.1
0-8enzoquinone SO ND 9.3 8.5 NO ND
HgC12 70 ND 14.1" 15.7 ND ND
Chlorfenvinphos 5 10 15. 0 " 21.9" 12.9 12.2
Chlorpyrifos 5 10 9.0 28.6* 19.6" 23.2*
Diazinon S IO 11.2 18.2" 15.5" 20.6*
Ethion 5 10 8.5 29.6* 12.5 31.7"
Ethoprop 5 10 11.4 26.8* 11.5 18.4"
Fensulfothion 5 10 17, 6 " 21.5" 12.4 17.4
Fonofos B 10 6.7 17.5" 6.6 9.2
Leptophos 5 10 9,1 16.0 11.0 20.2*
Malathion 5 10 7.2 19.5" 11.5 11.0
Parathion S 10 4.9 20.5* 8.7 11.0
Phorate S 10 9.2 28.4" 18.3" 31.3"
Terbufos 5 10 9,2 14.9 17.5" 17.9"
Thionazin 5 10 8.8 15.6 14.1" 14.7
Triazophos 5 10 23,4" 54.5* 10.1 14.0
Trichloronat $ 10 9.7 11.0 13.9" 17.8"
Chlordane 5 10 6.6 22.6* 10.7 12.P
Dieldrin S 10 4.8 19.5" 3.9 9.7
L indan e S 10 6,0 14.6 11.2 13.2
Carbofuran 5 10 8.1 18.0" 8.4 11.7
Metalkamate 5 10 9.1 13.3 10.9 13.6
Oxamyl S lO 5.b 19.7" 14.2" 14,6
Permethrin 5 10 7.4 21.4" 12.9 13.4
2,4-D S 10 5.3 11.9 5.0 7.8
Atrazine 5 10 5.1 10.4 4.3 7,2
Dalapon 5 10 7.8 11.6 3.8 9.1
Dicomba S 10 6.8 6.9 4.2 7.8
Nitrofen 5 10 8.0 7.6 4.9 7.0
Picloram 5 10 9,3 8.2 3.9 4.9
Roundup S IO 5.3 8.8 4.9 7.2
Simazine 5 lO 7.0 8.4 4,3 7.2
Captan 5 |0 7.4 9.1 4.6 14.1
Maneb S 10 2.3 5.8 4.4 16.1
Thiram 5 10 3,9 9.0 4.9 15.3
D-D IS0 300 6,4 29.5" 10.2 12.2
Telone 30 60 7,8 15.6 9.8 23.7"
T e l o n e C-17 30 60 5,2 6.0 4.9 9.0
Telone II 30 60 5.8 7.2 3.9 16.6
Vorlex 40 gO 7,6 9.8 4.1 13.8
Nitrapyrin 30 60 2.9 9.6 S.1 16.2

** ND = not d e t e r m i n e d .
Significantly different from c o n t r o l at P=0.05.

The method employed enabled measurement of ATP directly in soil extracts.

In preliminary trials, water and cold Tris-EDTA buffer were used and the Tris-
ZDTA buffer proved to be suitable for extraction of soil. A greater reproduci-
bility and uniform counts from a Luminometer on ATP values were obtained with the
addition of the microbial cell nucleotide releasing reagent prior to the addition
of luciferin-luciferase. Many methods such as boiling Tris-EDTA buffer at pH
7.75, mineral acids (perchloric acid, trichloroacetic acid, H2SO 4, HIq03), bases
(KOH, NaOH), organic solvents (butanol, ethanol, chloroform) or Tris-EDTA-
arsenate-butanol, have been applied for extraction of ATP from microbial cells 15.
 913

TABLE 3
ATP c ha nge s i n an o r g a n i c s o i l a f t e r different treatments.
(vg ATP/g s o i l )

Rates of Low t r e a t m e n t r a t e High t r e a t m e n t r a t e

Treatment treatment Incubation period (day)
(ug/g) ~ 2 1 2

Control 0 0 15 . 4 2 24.36 15.42 24.36

Autoclaving 0 ND** 0.06" 0.19" ND ND
llgCl2 70 IS0 2.99* 14.34" 0.55* 10.43"
Chlorfenvinphos 5 lO 4.70" 2].63 9.09" 16.07"
Chlorpyrifos S l0 6.72* 14.09" 6.94* 14.56"
Diazinon S 10 6.72* 21.45 6.88" 18.91
Ethion S 10 6.63* 19.$6 7.79* 20.65
Fensulfothion S 10 7.41" 17.30 8.83" 16.59"
F onofos S 10 6.6]" 19.27 6.77* 18.38
Phorate S 10 5.44" 17.75 6.85 ° 14.60"
Terbufos 5 10 6.64" 17.39 11.37" 16.35"
Chlordane 5 lO 4.59" 14.55" 12.48" 11.79"
Lindane 5 10 7.75" 21.12 9.77* 25.36
Carhofuran S 10 4.82" 18.61 6.66" 22.97
Oxamyl 5 l0 5.05" 26.23 3.97" 15.64"
Permethrin 5 10 9.15" 23.46 8.57 ° 13.SI*
2,4-D 5 l0 6.01" 21.34 5.89" 17.15"
Atrazine 5 l0 4.74" 17.06 6.51" 17.06"
Roundup S 10 7.02" 23.02 7.23" 9.98*
Captan 5 10 5.70" 21.68 6.05" 14.14"
Maneb 5 10 3.54* 27.28 4.08 t 10.43"
Thiram 5 l0 8.43* 17.43 6.17" 10.16"
D-D 180 300 S.I0* 23.78 6.83* 11.51"
Telone C-17 30 60 3.56" 15.26 9.80* 8.79*
Vorlex 40 80 9.57" 25.58 5.89" 13.56"
Nitrapyrin 30 60 12.10 26.72 9.77" 18.98

* Significantly different from control at P=O.OS

** ND = not d e t e r m i n e d .

Most of these methods introduced chemical quenchers that reduced the biolumines-
cent light reaction 16. Karl and La Rock 17, however, demonstrated that the EDTA
concentration was not high enough to depress light production determined by the
luciferin-luciferase system.
The firefly bioluminescence system has been employed for the determination
of ATP. The sensitivity, reproducibility and specificity of the system to ATP
depend greatly on the quality of the bioluminescent reagents. Crude extracts
contain endogenous ATP and enzymes competing with luciferase for ATP. The
luciferin-luciferase system utilized had a high specificity to ATP, and contained
neither contaminating enzymes nor endogenous ATP 18.
With the exception of nitrapyrin at the low rate, all treatments inhibited
ATP concentrations in the soils for 1 day with subsequent recovery being faster
with low level treatments (Table 3). It was demonstrated by Jenkinson and Oades 19
that ATP content was related to carbon content and more closely to nitrogen
content. A greater ATP values was observed in an organic soil than in a mineral
soil. In general, treatments that improved soil productivity increased ATP.
The pesticides selected for this study had slight effects similar to
those caused by the microbial and enzyme inhibitors on soil invertase, amylase
914

and ATP contents. The inhibitory effects were, however, short lived when
compared with those of reference compounds or treatments e.g. autoclaving. Appa-
rently, the soil indigenous microbes can tolerate the chemicals used for control
of soil pests.

ACKNOWLEDGEMENT

The technical assistance provided by Mr. G. Hietkamp is acknowledged.

REFERENCES

I. L. J. Audus, Herbicide behaviour in the soil. In: L. J. Audus (ed.) The

Physiology and Biochemistry of Herbicides, Academic Press, New York (1964).
2. W. B. Bollen, Annu. Rev. Microbiol. 15, 69 (1961).
3. C. M. Tu and J. R. W. Miles, Residue Rev. 64, 17 (1976).
4. B. L. Strehler and J. R. Totter, Arch. Biochem. Biophys. 40, 28 (1952).
5. C. ~. Tu and W. B. Bollen, Weed Res. 8, 28 (1968).
6. C. M. Tu, Appl. Microbiol. 19, 479 (1970).
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B. L. A. Kolmer, E. H. Spaulding and H. W. Robinson, Approved Laboratory Tech-
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lO. E. Hofmann and G. Hoffman, Z. Pflanzenernahr. Dung. Bodenkd. 70, 97 (1955).
ll. J. Drobnik, Folia Biol. l, 29 (1955).
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13. C. C. Lee, R. F. Harris, J. D. H. Williams, J. K. Syers and D. E. Armstrong,
Proc. Soil.Sci. Soc. Amer. 35, 86 (1971).
14. M. P. Greaves, R. E. Wheatley, H. Shepheard and A. H. Knight, Soil Biol.
Biochem. ~, 686 (1973).
15. A. Lundin and A. Thore, Appl. Microbiol. 30, 713 (1975).
16. Anonymous, Microbial applications. Luminescent Analytical Chemistry System
A. G. 40801 (1978).
17. D. M. Karl and P. A. La Rock, J. Fish. Res. Bd. Can. 32. 599 (1975).
18. Anonymous, Lumit-HS: Bioluminescence reagents for ATP measurements. Lumines-
cent Analytical Chemistry System A. G. 40601.
19. D. S. Jenkinson and J. M. Oades, Soil Biol. Biochem. Ii, 193 (1979).

(Received in USA 21 July 1982)