Degradation of organophosphorus pesticides inaqueous extracts of young green barley leaves(Hordeum vulgare L)Janis J Durham, Joji Ogata, Sadatoshi Nakajima, Yoshihide Hagiwara andTakayuki Shibamoto*Department of Environmental Toxicology, University of California, Davis, CA 95616, USA

Abstract: The degradation of the organophosphorus pesticides malathion, chlorpyrifos, guthion,

diazinon, methidathion and parathion in an aqueous extract of young green barley leaves (Hordeum

vulgare L) was monitored by gas chromatography. Aqueous solutions of various amounts of freeze-

dried young barley leaves containing 5.75mglÿ1 of malathion were incubated at 37°C and pH 7.4 over

prolonged time periods. Over 95% of the malathion degraded in 4h in a 3% (30g lÿ1) solution of young

green barley leaves. When the barley solution was autoclaved at 120°C for 25min prior to the addition

of malathion, no degradation of malathion was observed. When 10mglÿ1 each of the above six

pesticides was incubated in a 15% (150g lÿ1) solution of young green barley leaves for 3h at 37°C and pH

7.4, malathion and chlorpyrifos degraded 100%, whereas parathion (75%), diazinon (54%), guthion

(41%) and methidathion (23%) showed lesser degrees of degradation.

# 1999 Society of Chemical Industry

Keywords: barley leaves; malathion; organophosphorus pesticides; pesticide degradation

INTRODUCTIONAlthough pesticides have made a very important

contribution towards increasing both the quality and

quantity of crop production in the world over the last

half century, concern about the impact of pesticide

residues in foods on human health has grown,

especially in developing countries.1

In order to prevent any possible adverse effects

caused by pesticide residues in foods, such residues

should be reduced as much as possible. However, it is

impossible to completely remove all pesticide residues

from foods. Therefore it is ideal if pesticide residues

can be degraded into non-toxic materials in foods.

Monitoring pesticide degradation in a natural plant

extract is one avenue to investigate the possible

reduction of pesticide residue levels in foods. There

are some reports on the role of food plants in pesticide

degradation. For example, some food plants have been

shown to be capable of metabolically degrading

pesticides;2 also, carrot cell suspension cultures

degraded a pesticide.3

In the present study the degradation of organophos-

phorus pesticides in an aqueous extract of young green

barley leaves was investigated. Aqueous extracts from

young barley leaves were selected for this study

because they are known to contain many enzymes

which may degrade pesticides.4 For example, more

than 20 enzymesÐincluding cytochrome oxidase,

peroxidase, catalase, fatty acid oxidase and trans-

hydrogenaseÐin an aqueous green barley extract were

reported at the Japanese Society of Pharmaceutical

Science.5 Organophosphorus pesticides were chosen

because they are among the most commonly used

insecticides.6 Of the organophosphorus pesticides,

malathion has been the most widely used on various

crops. For example, in 1992, 360tons of malathion

was applied to over 70 crops in California alone.7

MATERIALS AND METHODSReagentsAll reagents were analytical grade (Fisher Scienti®c,

Santa Clara, CA, USA). Malathion, guthion, chlor-

pyrifos, diazinon, methidathion and parathion stan-

dards were purchased from Chem Service Co (West

Chester, PA, USA). Structures of pesticides used in

the present study are shown in Fig 1 along with their

CAS name. A gas chromatographic internal standard

(IS) was prepared by the addition of 60mg of

parathion to 50ml of ethyl acetate. Trizma1 buffer

(pH 7.4) was prepared by combining 13.2g Trizma1

HCl with 1.94g Trizma1 base (both purchased from

Sigma Chemical Co, St Louis, MO, USA) in a 2l

volumetric ¯ask and adding deionised water to

volume. C18 solid phase extraction (SPE) cartridges

were bought from Varian Corp (Harbor City, CA,

USA). Bovine serum albumin (BSA) was purchased

from Bio-Rad Laboratories (Hercules, CA, USA).

Journal of the Science of Food and Agriculture J Sci Food Agric 79:1311±1314 (1999)

* Correspondence to: Takayuki Shibamoto, Department of Environmental Toxicology, University of California, Davis, CA 95616, USA(Received 18 August 1998; revised version received 12 January 1999; accepted 29 March 1999)

# 1999 Society of Chemical Industry. J Sci Food Agric 0022±5142/99/$17.50 1311

Preparation of freeze-dried young green barleyleavesYoung green barley leaves (Hordium vulgare L, var

nudum Hook) were harvested 2 weeks after germina-

tion.8 The young green barley leaves were freeze-dried

for 3 days in a freeze-dryer, Model 50-SRC-5 (VIRTIS

Co, Gardner, NY, USA). The freeze-dried leaves were

subsequently ground with a Wiley Mill Standard

Model No 3 (Arthur H Thomas Co, Philadelphia,

PA, USA) equipped with a 2mm mesh size sieve to

form a ®ne and uniform powder.

Sample preparations for pesticide degradationexperimentsTrizma1 buffer solutions (400g, pH 7.4) containing

powdered barley leaves (4 and 12g) were prepared at

25°C. The resulting mixtures, 1% and 3% concentra-

tions (w/w) of barley samples in the buffer, were stirred

for 10min using a magnetic stirrer. After two table-

spoonfuls of talc or celite were added, the sample

solutions were stirred for an additional 5min. The

sample solutions were subsequently ®ltered and each

®ltrate was divided into three 100ml aliquots to

conduct three replications. A buffer solution (100ml)

without barley was treated as the control preparation.

One of the 3% barley buffer solutions was autoclaved

for 25min at 120°C to deactivate the enzymes.

Each sample was spiked with a 0.5ml acetone

solution of malathion (4.6mgmlÿ1) in a 200ml ¯ask.

After spiking, the ¯asks were sealed and mixed by

inverting each ¯ask several times by hand. These

sample ¯asks were then covered with aluminium foil to

prevent possible photodegradation of the malathion.

The samples were allowed to stand at 25 or 37°C for

the duration of the experiment. Conditions used to

prepare each malathion sample are summarised in

Table 1.

At 10, 30, 60, 120 and 240min after spiking with

malathion, 10ml subsamples were transferred from

each sample into preconditioned C18 SPE cartridges.

Each cartridge was preconditioned with two column

volumes of ethyl acetate, followed by drying atÿ10psi

for 3.5min and then the addition of two column

volumes each of methanol and distilled water. After

Figure 1. Organophosphorus pesticides used in the experiment.

Table 1. Conditions for sample preparations

Sample Barley conc. Solvent Temperature

I 1% Trizma 25°CControl a 0 Trizma 25°C

II 3% Trizma 25°CControl a 0 Trizma 25°C

III 1% Trizma 37°CControl b 0 Trizma 37°C

IV 3% Trizma 37°CControl b 0 Trizma 37°C

V 1% water 37°CControl c 0 water 37°C

VI 3% water 37°CControl c 0 water 37°C

VIIa 3% water 25°CControl d 0 water 25°Ca Barley solutiion was autoclaved at 120°C for 25 min.

1312 J Sci Food Agric 79:1311±1314 (1999)

JJ Durham et al

eluting each subsample, the cartridges were rinsed

with a small amount of distilled water. Subsequently

the cartridges were dried at ÿ10psi for 5min.

Malathion trapped in the cartridges was eluted with

ethyl acetate using a centrifuge. Each cartridge was

placed in a 15ml centrifuge tube and then 1ml of ethyl

acetate was added. The samples were centrifuged for

1min and the process was repeated with 0.5ml of ethyl

acetate. The eluate organic layer and at least three

ethyl acetate rinses were pipette transferred from each

tube to a corresponding 2ml volumetric ¯ask. Excess

solvent was removed under a nitrogen gas stream. Gas

chromatographic IS (0.2ml) was added to each 2ml

sample prior to analysis.

In another experiment, Trizma1 buffer solutions

(400g, pH 7.4) containing various amounts of barley

leaf powder (0, 2.5, 5, 10, 15, 20%) were prepared at

25°C. Solutions were spiked with 10mglÿ1 of chlor-

pyrifos and incubated at 37°C for 3h. The sample

solutions were treated exactly as in the experiment

with malathion. The amount of chlorpyrifos remaining

in the sample solutions was analysed by GC.

Also, Trizma1 buffer solutions (400g, pH 7.4)

containing 15% of barley leaf powder were prepared at

25°C. A solution was spiked with 10mglÿ1 each of

malathion, guthion, chlorpyrifos, diazinon, methi-

dathion and parathion and incubated at 37°C for

3h. The sample solutions were treated exactly as in the

experiment with malathion. The amounts of pesticides

remaining in the sample solutions were analysed by

GC.

Protein measurement in barley solutionsThe Lowry protein determination method used in this

experiment was a simple colorimetric method that has

been commonly used in many laboratories throughout

the world.9 A stock solution of BSA standard was

prepared by adding 10mg of BSA to 10ml of distilled

water. Standard protein solutions (50, 100, 200, 300

and 500mgmlÿ1) to prepare a calibration curve were

prepared in distilled water using the above stock

solution. After Lowry's reagent was added to each

solution, the absorbance at 660nm was measured

using a Hewlett-Packard 8452A diode array spectro-

photometer.

Instrumental analysis for pesticides in barleysolutionsA Hewlett-Packard (HP) 5890 Series II gas chroma-

tograph equipped with a 30m�0.25mm id DB-5

bonded phase fused silica capillary column (J&W

Scienti®c, Folsom, CA, USA) and a ¯ame photo-

metric detector (phosphorus mode) was used for

quantitative analysis of malathion according to the

GC internal standard method reported previously.10

The oven temperature was programmed from 160 to

220°C at 15°C minÿ1 and held for 12min. The

helium carrier gas ¯ow rate was 1.7ml minÿ1 with a

split ratio of 1:16. The injector and detector tempera-

tures were 220 and 225°C respectively.

RESULTS AND DISCUSSIONProtein contents of the samples were 2.4, 7.2, 11 and

31mgmlÿ1 in 1%, 3%, 5% and 15% barley solutions

respectively. The values were obtained using the

calibration curve prepared with BSA (y =0.012x�0.0116). The results of malathion degradation with

barley leaves are shown in Fig 2. The malathion

concentrations in samples I±VI decreased with time

except in the control samples, which remained fairly

constant. For example, the malathion concentration in

the test samples dropped to 63% in 4h, while the

control concentration remained between 87% and

95% (sample I).

More malathion was degraded in the 3% solutions

than in the 1% solutions. Also, malathion degradation

occurred more rapidly at 37°C than at room tempera-

ture. For sample III, malathion degradation in the 1%

barley leaf powder at 37°C (40%) was similar to that

of sample II in 3% barley leaf powder at 25°C (33%).

Thus the increase in temperature and the increase in

barley concentration had a similar effect on the

degradation of malathion. When both temperature

and barley concentration were increased, the greatest

decrease in malathion was observed. In sample IV the

malathion concentration dropped steadily to 5%,

while that of the control stayed between 90% and

96%.

There was no important difference resulting from

the use of water as opposed to buffer. The only change

in solution pH was noted when the temperature was

increased. The buffer pH, which was 7.4 at 25°C,

dropped to 7.2 in both samplesV and VI and control

solutions as the temperature was increased to 37°C,

and remained at that pH throughout the experiment.

The enzymatic degradation of malathion may be

found in the results of sample VII. The results show

that malathion degradation did not occur in the

autoclaved barley samples. This is likely due to loss

of enzyme activity. In the process of autoclaving the

barley solution, all the proteins were probably de-

natured and lost their activity. These results suggest

that the malathion degradation in barley leaves is an

Figure 2. Amounts of malathion remaining in barley solutions overprolonged time period. Refer to Table 1 for the description of samples.

J Sci Food Agric 79:1311±1314 (1999) 1313

Degradation of organophosphorus pesticides in barley leaves

enzymatic process. This experiment also provides

recovery ef®ciency of malathion from the system used.

Over 97% recovery was obtained from the samples

with deactivated proteins (refer to sample VII in Table

1).

Fig 3 shows the percentage of degradation of

chlorpyrifos (10mglÿ1) incubated at 37°C for 3h in

solutions containing various amounts of barley leaf

powder. Values are mean�standard deviation (n =3).

Signi®cant degradation occurred when the barley leaf

concentration increased over 5% (protein content

11mgmlÿ1), and 100% degradation was achieved in

the 15% barley leaf solution.

Fig 4 shows the degradation of six pesticides

(10mglÿ1 each) incubated at 37°C for 3h in a 15%

barley leaf solution. Values are mean�standard

deviation (n =3). Malathion and chlorpyrifos dis-

appeared completely and the remaining pesticides

showed varying degrees of degradation: parathion

75%, diazinon 54%, guthion 41% and methidathion

23%.

Comprehensive analysis of the degraded pesticide

samples was not performed. However, no additional

peak appeared on the GC-FPD of a test solution after

the degradation of malathion (eg sample IV), suggest-

ing that no volatile organic degradation products

containing sulphur or phosphorus were produced.

On the other hand, some highly polar compounds,

such as dimethyl phosphate which requires derivatisa-

tion before analysis by GC, may be present in the

samples. In order to better understand why an

aqueous extract of young green barley leaves degraded

the pesticides used, efforts will be made to determine

the degradation products to elucidate the pathway of

degradation. As mentioned above, it is impossible to

completely remove pesticide residues from food.

Therefore facilitating the degradation of pesticides

with natural plant materials may be one way to

decrease pesticide residues in foods. Investigation on

the actual application of barley extract is in order.

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Figure 3. Degradation of chlorpyrifols (10ppm) incubated at 37°C for 3 h insolutions containing barley leaf powder.

Figure 4. Degradation of organophosphorus (10ppm each) incubated at37°C for 3 h in a 15% barley leaf solution.

1314 J Sci Food Agric 79:1311±1314 (1999)

JJ Durham et al