United States
Environmental Protection
Agency
Research and Development
Hazardous Waste Engineering
Research Laboratory
Cincinnati OH 45268
EPA/600/S2-85/048 June 1985
'/I
4>EPA Project Summary
Enzyme-Based Detection of
Chlorinated Hydrocarbons in
Water
Barbara H. Offenhartz and Janet L. Lefko
This study explores a new approach
for detecting hazardous levels of high
molecular weight chlorinated hydrocar-
bons based on enzyme-catalyzed reac-
tions. The lactate dehydrogenase (LDH)
catalyzed reaction
pyruvate + NADH* +
H* LDH Jactate + NAD+
[inhibitor]
is used to detect the presence of chlori-
nated hydrocarbons, which reduce the
rate of the reaction by reversibly inhib-
iting the enzyme. Detection is straight-
forward, since the reaction produces a
significant pH change. An analysis takes
5 minutes. Inhibition screening experi-
ments show that the LDH method can
detect aldrin, toxaphene, DDT, poly-
chlorinated biphenyls (PCBs), and poly-
chlorinated phenols at comparable
levels of toxicity at parts per million
(ppm) levels. A preliminary study of
potential interferences is reported, as
well as the results of an extensive
literature search for suitable enzymes.
In addition to LDH, four other commer-
cially available enzymes were found to
be potentially suitable: carbonic anhy-
drase, hexokinase, phosphorylase b,
and an ATPase. LDH was chosen on the
basis of a trade-off between sensitivity
to inhibition and suitability for com-
mercial method development.
The bench-top method studied in the
laboratory appears potentially adaptable
to water-quality monitoring systems
and pocketable field sensors. The
"NAD* is the oxidized form of the cofactor nicotma-
mide adenine dinucleotide.
NADH is the reduced form of NAD*.
development is patterned on the cho-
linesterase antagonist monitors (C AMs),
developed under the sponsorship of the
U.S. Environmental Protection Agency
(EPA), which use the cholinesterase
enzyme to detect organophosphate and
carbamate pesticides. Work has begun
on developing immobilized enzyme
preparations suitable for use in this type
of device.
Since the LDH enzyme is stable in a
variety of water/organic solvent mix-
tures, including methanol, ethanol, and
acetone, the method has potential for
processing extracts of soils and organic
wastes. For water samples, in which the
solubility of chlorinated hydrocarbons
is often below the ppm level, improve-
ment of detection levels or use of an
extraction step will be required to
achieve sub-ppm detection levels. In
the LDH method reported, the inhibi-
tors were solubilized in a 20 percent
methanol solution.
This Project Summary was developed
by EPA's Hazardous Waste Engineering
Research Laboratory, Cincinnati. OH,
to announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction
Simple and robust field methods for the
detection of chlorinated hydrocarbons at
hazardous levels are needed to determine
the boundaries of an area targeted for
decontamination, waste site manage-
ment, and to rapidly detect spills. Recent
advances in the development of analyti-
cal methods based on immobilized en-
zymes have proved useful in the detection
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of pesticides. Specifically, enzyme-based
CAM water quality monitors, developed
under EPA sponsorship, detect hazard-
ous levels of organophosphate and car-
bamate pesticides by their effect on the
rate of a cholinesterase-catalyzed reac-
tion. Design features common to CAM
monitors are: an immobilized enzyme
preparation, electrometric detection of
reaction products, and an automated
sampling cycle with automated sample
handling.
In the present study, an enzyme-based
detection method has been developed for
the detection of high molecular weight
chlorinated hydrocarbons. Based on an
extensive literature review, lactate dehy-
drogenase was chosen to replace choli-
nesterase. The reaction
pyruvate + NADH +
H+ LDH ^lactate + NAD*
[inhibitor]
was found to be useful in detecting chlor-
inated hydrocarbons, which reduce the
rate of the reaction. The inhibition of rab-
bit muscle MA isoenzyme by high molecu-
lar weight hydrocarbons is reversible and
is competitive with respect to pyruvate
and the coenzyme NADH. The experimen-
tal work described below shows that a
wide variety of chlorinated hydrocarbons
can be detected at hazardous levels by
this reaction. Nonchlorinated hydrocar-
bon analogs do not interfere. Either pH or
electrometric detection of the coenzyme
can be used. Enzyme activity is minimally
affected by organic solvents, so the
method appears adaptable to soil as well
as water samples. Since pollutant inhibi-
tion is reversible, the LDH enzyme can
probably be immobilized on a suitable
support medium to enhance the cost-
effectiveness of the method. Enzyme
immobilization is essential in instrumen-
tation designed for field use, reducing
both complexity and cost per test.
Procedure
The objectives of the effort reported
were to identify enzymes that could be
incorporated in automated water moni-
toring systems or other field detectors of
chlorinated hydrocarbons and to develop
an enzymatic detection method that
demonstrated potential for adaptation to
field use.
Selection criteria were developed to
facilitate the literature search and to
provide a scheme for ranking the enzymes
identified. Sensitivity to inhibition by a
broad spectrum of chlorinated hydrocar-
bons and insensitivity to interferants
were of primary importance. Practical
considerations dictated that enzymes
considered were commercially available
and that enzyme characteristics were
well-documented. Field method charac-
teristics of reliability, ease of use, and low
cost were addressed by requiring elec-
trometric detection of enzymatic reaction
products and a reusable immobilized
enzyme preparation. Adaptability of inhi-
bition detection schemes to the EPA-
developed CAM-1 field monitor was
desirable. Lactate dehydrogenase, car-
bonic anhydrase, hexokinase, and an
ATPase have potential for enzymatic
detectors. Lactate dehydrogenase, the
most promising enzyme identified, was
used for field method development.
The inhibition of lactate dehydrogenase
by the organochlorine insecticides (diel-
drin, aldrin, endrin, chlordane, mirex,
kepone, and DDT) has been documented
previously. Therefore, laboratory tests on
the inhibition of LDH by chlorinated
hydrocarbons and potential interferants
were carried out to supplement published
information. Si nee the three-dimensional
structure and reaction mechanism of LDH
Table 1. Inhibition of Lactate Dehydrogenase
are well established, testing focused on
compounds most likely to show inhibition.
The spectrophotometric inhibition as-
say procedure used for the inhibition
studies was adapted to a pH assay more
suitable for field use. The sensitivities of
the two methods are compared. Prelim-
inary results on immobilizing LDH in a
polyacrylamide gel are reported.
LDH Inhibition Studies
The results of the experimental inhibi-
tion studies are shown in Table 1, which
summarizes some 70 inhibition assays
on seven representative test compounds.
The inhibition results are consistent with
the published literature where overlap of
data exists, e.g., for aldrin. LDH sensitivity
to chlorinated hydrocarbons approximate-
ly parallels the relative toxicity of the
compounds. The 2,4,5-T inhibition, 19
percent at the highest concentration
tested, is marginally significant, and the
2,4,5-T should be retested at higher
concentrations.
The "100**" values (Table 1) require
an explanation. If an inhibitor that is
undetected and undissolved is present at
the lower concentrations tested, then the
% Inhibition" at Several Concentrations
Test Compound
Cyclodiene Insecticides
Aldrin 700**
Toxaphene 100**
Chlorinated Ethane Derivatives
DDT WO**
Polychlorinated Biphenyls
Aroclor 1242 100**
Polychlorinated Phenols
Pentachlorophenol 74 ±8
Chlorophenoxy Derivatives
2,4.5- Trichlorophenoxy A cetic A cid 19 ±7
Misc. Chlorinated Hydrocarbons
Lmdane 0
58±8
40±2
22 ±3
22±72
23±6
7±7
29±3
13±4
6±5
* Per cent inhibition was calculated by assigning 100 percent activity to a control containing no test
compound.
^Conversion factor applicable: 1 x 70~5 M = 2-4 mg/l
**Undissolved test compound present.
Experimental conditions: [NADH] = 0.220 mM; [pyruvate] =1 00 mM; [test compound] = 0 (control),
1 x W'eM, 1 x 10~5Mor 1 x 10~*M;0.1 unit of LDH in 0.11 M phosphate-methanol buffer (80:20
v/v%), pH 7.3, 23°C. The addition of enzyme, pre-equilibrated in phosphate-methanol buffer,
started the reaction. Enzyme activity was determined by the method of initial reaction rates. The
disappearance of NADH was followed spectrophotometrical/y with a Hitachi Model 100-80 at 340
nm. The 300-340 nm background absorbance was checked for the presence of undissolved
reaction components.
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data for Table 1 could overestimate
inhibition effects projected for field de-
vices. When undissolved inhibitor is
present, coprecipitation of enzyme and
inhibitor has been observed. This effect
mimics inhibition by reducing the active
enzyme concentration and has been
extensively documented. Undissolved in-
hibitor, if present in field samples, will
react differently with the immobilized
enzyme preparations of field devices than
with the free enzyme used in the inhibition
studies. For this reason, the distinction
between coprecipitation and inhibition is
of more than academic interest. The
possibility of coprecipitation was ruled
out by mechanistic studies of the type
illustrated in Figure 1. Linear double-
reciprocal plots of initial reaction velocity
versus substrate concentration are only
consistent with reversible inhibition and
are inconsistent with the occurrence of
coprecipitation.
Interferences
Nonhalogenated aromatic pollutants,
excepting phenols and amines, are even
less soluble than their chlorinated ana-
logs, so that interference is unlikely.
Furthermore, interference is not expected
on structural grounds. Phenol was tested
and exhibited no significant inhibition at
concentrations below 0.1 M.
The organophosphate pesticide Diazi-
non and the carbamate Sevin inhibited
LDH only at concentrations greater than
0.001 M. This indicates that cholinester-
ase (CAM) and LDH may have comple-
mentary detection capabilities toward
Category X hazardous substances.* The
LDH appears to selectively detect high
molecular weight chlorinated hydrocar-
bons, whereas the CAM method detects
organophosphate and carbamate pesti-
cides.
Cyanide tends to inhibit most enzymes.
Concentrations as high as 0.01 M were
required to exhibit 50 percent inhibition
of LDH. The LDH method is hence un-
usually insensitive to toxic levels of
cyanide. On the other hand, heavy metal
ions, such as free mercuric ion (Hg+2),
showed complete inhibition consistent
with the presence of sulfhydryl (-SH)
groups on the enzyme. Use of a suitable
chelating agent will be necessary to
suppress interference in environmental
applications when high levels of heavy
metal ions are present.
720 -
1
V,
90 -
10
[pyruvate]-1 mM
15
20
Figure 1. The effect of Aroclor 1242 on lactate dehydrogenase. Lineweaver-Burk double
reciprocal plot of the effect with respect to pyruvate. Experimental conditions.
[NADH] = 0.220 mM; [pyruvate] = 0.050 - 1 00 mM; [Aroclor 1242] = (Q) 0 mM.
( A;0.005mM, (D)0.010mM,(»J0.050mM; 0 J units LDH inO.1 J M phosphate -
methanolbuffer (80:20 v/v%J atpH 7.3. 23°C Spectrophotometric determination of
the initial rate of disappearance of NADH (Vj) with a Hitachi Model 100-80 at 340
nm
•Category X hazardous substance refers to the 21
organic compounds designated in 40 CFR 117 3 as
having a reportable quantity of one pound
Detection Methods
In the laboratory work, the rate of the
reaction was followed by spectrophoto-
metrically determining the absorbance of
the reaction mixture at the 340 nm peak
of NADH. However, this detection method
is not particularly suitable for field use.
Natural waters tend to have background
absorption and to foul optical surfaces.
Furthermore, optical methods of detection
are relatively expensive. Two cheaper
alternatives are available, electrometric
detection (monitoring of the [NADH]/
[NAD*] couple) and pH detection (the
reaction consumes hydrogen ion). Both
methods are relatively insensitive to
interference, although high concentra-
tions of oxidants (e.g., chlorine) can distort
electrometric detection, just as high
concentrations of acids and bases can
distort pH detection. Only the pH detection
method was examined in this study.
Sincethe reaction consumes hydrogen
ion, the pH of the reaction mixture should
increase with time. The extent of the pH
increase depends upon the buffering
capacity of the reaction mixture. When
the reaction mixture was titrated with
hydroxide, addition of 10"4 M OH" at a pH
initially equal to 6 produced a 2-unit pH
change rather than the 3-unit change
expected in the absence of a buffer. The
difference is traceable to the buffering
effect of the commercial enzyme prepara-
tion. As shown in Figure 2, when the
reaction mixture (primarily sodium ace-
tate) is titrated in the absence of enzyme,
a far sharper pH change is observed. (A
sodium acetate buffer limits pH decrease
but has no effect on pH increase.) Never-
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pH
Figure 2.
85
80
7.5
7.0
65
O Enzyme Reaction
1 0
2.0
30
4.0
Equivalents OH added or AY+ consumed x w~4 M
Tit ration of the LDH reaction mixture. In titration A the components NAOH and
pyruvate in buffer are titrated with 0.1 N NaOH In titration B the •enzyme in buffer is
titrated In titration C the enzyme reaction consumes H* (initial pH-7.0). The change
in [H*] is calculated from the change in[NADH], determinedspectrophotometrically
at 340 nm; A///*./ 5 &[NADH] Experimental conditions: [NADH] = 0220
mM; [pyruvate] = 1 00 mM; 0.4 units LDH in 1 mM acetate buffer-methanol 180 20)
v/v%; 23°C.
theless, as shown in Figure 3, the buffer-
ing effect of the enzyme preparation is not
so great as to eliminate pH detection as a
viable method. In the presence of high
concentrations of a chlorinated hydro-
carbon, the pH change would be zero; the
pH change actually observed, in excess of
1 unit, clearly indicates the absence of
inhibitors.
We conclude from these results that a
0.001 M acetate buffer in 20 percent
methanol solution supports a viable de-
tection scheme. At this pH and methanol
concentration, the enzyme activity is
about 25 percent greater than the stan-
dard LDH assay, and the solution could be
adapted readily to acidic water samples.
The only potential problems would occur
in waters that are more basic than pH 7,
such as sea water or brackish water
samples. Electrometric detection of
[NADH]/[NAD+] could probably be used in
such circumstances.
LDH Immobilization
Immobilization of the LDH enzyme is a
necessary step toward developing reli-
able, cost-effective field methods. Many
different types of immobilization tech-
niques have been successfully applied to
LDH. Several look quite promising for
developing continuous flow applications
and field detector designs. However, each
immobilization method must be examined
with care, since immobilizing an enzyme
can also reduce its activity and/or reduce
its sensitivity to inhibition. Among the
various LDH immobilization techniques,
covalent bonding methods, employing a
spacer molecule between the enzyme
and its support, are best suited to repro-
ducing the free enzyme environment in
an immobilized preparation. As of this
writing, this technique has not been
attempted, although it is clearly recom-
mended for future method development.
Conclusions
An LDH enzyme-based detector has
been developed that can detect at least
five chlorinated hydrocarbons at concen-
trations of 10~4 to 10~6 M in a 20 v/v%
methanol/water solution. Based on liter-
ature reviews, it is thought that the
detector will also be sensitive to other
high molecular weight chlorinated hydro-
carbons. Phenol test data and analysis of
the literature indicate that interference
by nonchlorinated analogs will not occur.
The simplicity and rapidity of the method
indicates promise for field applications.
The inhibition of LDH by chlorinated
hydrocarbons can be monitored either by
pH or electrometric changes in the reac-
tion mixture. This flexibility also enhances
the potential of the detection method for
field use.
Although the LDH enzyme-based de-
tector has potential for field application,
more work is required in several areas
including: (1) development of an extrac-
tion/concentration procedure (with an
enzyme-compatible solvent) to precede
the use of the LDH detection method for
chlorinated hydrocarbons that have very
low water solubility; (2) immobilization of
the LDH enzyme on a suitable support to
make re-use of the enzyme possible and
to simplify the detection procedure; and
(3) evaluation of the sensitivity of the
detection method to potential interferants
in environmental samples.
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pH
80
75
7.0
65
^ Addpyruvate
time, mm
Figure 3. pH detection of the LDH catalyzed reaction. Experimental conditions [NADH] -
0.220 mM; [pyruvate] -1 00 mM; 0 4 units LDH in 1 mM acetate buffer-methanol
(80:20 v/v%); 23°C. The pH was measured with a Beckman Model 76 pH meter
Barbara H. Offenhartz and Janet L. Lefko are with B & M Technological Services.
Inc.. Cambridge, MA 02142.
Michael D. Royer is the EPA Project Officer (see below).
The complete report, entitled "Enzyme-Based Detection of Chlorinated Hydro-
carbons in Water." (Order No. PB 85-191 176/AS; Cost: $10.00, subject to
change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Re/eases Control Branch
Hazardous Waste Engineering Research Laboratory—Cincinnati
U.S. Environmental Protection Agency
Edison. NJ 08837
.Government Printing Office: 1985 — 559-111/10861
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