Evaluation of potential inhibitors of methanogenesis and methane
oxidation in a land®ll cover soil
A.S.K. Chan
a, T.B. Parkin
b,*
a
Department of Microbiology, Iowa State University, Ames, IA 50011, USA b
USDA, Agricultural Research Service, National Soil Tilth Laboratory, 2150 Pammel Drive, Ames, IA 50011-4420, USA
Accepted 1 March 2000
Abstract
Biological methane (CH4) production is an anaerobic process, while CH4 consumption occurs predominantly under aerobic
conditions; however, both processes can occur simultaneously in soil. Thus, ®eld measurements of CH4¯ux re¯ect the net result
of both consumption and production reactions. Speci®c inhibitors of either CH4consumption or production processes oer the
opportunity for assessing the rates of these two processes independently. The objective of this work was to identify potential gaseous inhibitors of either CH4oxidation or methanogenesis, and to evaluate the eect of inhibitor concentration on these two
processes. For these studies, sieved cover soil from a municipal land®ll was treated with a variety of compounds including acetylene (C2H2), ethylene (C2H4), ethane (C2H6), methyl chloride (CH3Cl) and methyl ¯uoride (CH3F). Each experiment
consisted of six dierent treatments which included sterile soil, untreated soil and soil with test compound concentrations of 0.00%, 0.01%, 0.1% and 1%. Incubations were conducted under both aerobic and anaerobic conditions. Several compounds completely inhibited CH4 oxidation while not signi®cantly in¯uencing CH4 production; including C2H2 at 0.001%, C2H4 at
0.1%, and CH3F at 0.1%. One compound (CH3Cl) was unique; in that a concentration of 0.1% inhibited methanogenesis by
88.9% but CH4 oxidation was not signi®cantly aected. We recommend the use of C2H2 or C2H4 for inhibition of CH4
oxidation, and CH3Cl for inhibition of methanogenesis.72000 Elsevier Science Ltd. All rights reserved.
Keywords:Methanogenesis; Methane oxidation; Methanotrophy; Inhibitors; Land®ll
1. Introduction
Recent concern about global warming has intensi®ed interest in the role of terrestrial systems in controlling atmospheric CH4 levels. Terrestrial systems can
func-tion as net sources or sinks for atmospheric CH4.
Methane ¯ux measured at the soil/atmosphere inter-face is the net eect of two processes Ð CH4oxidation
and methanogenesis (Knowles, 1993). A negative CH4
¯ux, i.e., consumption of CH4by soil, occurs when the
magnitude of the CH4 uptake process is larger than
methanogenesis. This is commonly observed in arable
soils, when conditions are predominately aerobic (Schutz et al., 1990; Mosier et al., 1991; Bronson and Mosier, 1993; Hansen et al., 1993). On the other hand a positive CH4¯ux indicates net CH4production, and
is observed when the magnitude of the methanogenic process is larger than CH4 uptake. This is the case in
rice paddies and wetlands (¯ooded or water saturated areas) which are predominately anaerobic (Schutz et al., 1990; Lauren and Duxbury, 1993). Both soil and wetland systems often support a mixture of anaerobic and aerobic sites, and it is under these conditions that the use of inhibitors are of value in distinguishing CH4
consumption and production activities.
Inhibitors have been applied to a variety of ecosys-tems to quantify the role of CH4 oxidizers in
mitigat-ing atmospheric CH4 increases. Boeckx and Van
Cleemput (1997), using CH3F in ®eld chambers,
deter-0038-0717/00/$ - see front matter72000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 7 1 - 7
www.elsevier.com/locate/soilbio
* Corresponding author. Tel.: +1-515-294-6888; fax: +1-515-264-8125.
mined that CH4 oxidation was responsible for
con-sumption of 34±67% of the CH4 produced in a
wet-land system. Using CH3F in investigations of CH4
cycling in the rhizosphere of Pontederia cordata, Sagit-taria lancifolia and Typha latifolia, Lombardi et al. (1997) determined that CH4 oxidation consumed
22.9% of the CH4 produced in ®eld experiments and
64.9% of the CH4 produced in greenhouse
exper-iments. King (1996) used C2H2as an inhibitor of CH4
oxidation and determined that 1±58% of the potential CH4 produced was consumed by CH4 oxidation
as-sociated with burweed (Sparganium eurycarpum) roots. Data from Oremland and Culbertson (1992a) show that CH4 oxidation in a seasonally exposed sandbar
accounted for 75±96% of the total CH4 production.
By utilizing the CH3F technique on plants (S. lancifo-lia), Schipper and Reddy (1996) determined that 65% of the total CH4produced was oxidized.
Several gaseous compounds have been examined as potential inhibitors of CH4oxidation and
methanogen-esis. Ethylene was observed to inhibit methanogenesis in marine sediments at concentrations of 20% and 5%, but not at 1.25%; however, C2H6 had no eect
on CH4production (Oremland and Taylor, 1975). The
eects of C2H4 or C2H6 on CH4 oxidation have not
been evaluated. Historically, C2H2has been used as an
inhibitor of both CH4 oxidation and methanogenesis,
depending upon the concentration. Inhibition of methanogenesis has been reported at C2H2
concen-trations of 0.5% (King, 1996) and at 1.25% (Oremland and Taylor, 1975), while CH4 oxidation is much more
sensitive to C2H2 and inhibition has been reported to
occur at a C2H2 concentration as low as 0.003%
(Watanabe et al., 1995).
Methyl ¯uoride has a similar dierential eect on CH4 oxidation and production. Oremland and
Cul-bertson (1992b) recommended that in soil, a CH3F
concentration of 0.4% would selectively block CH4
oxidation without aecting methanogenesis. Schipper and Reddy (1996) found no signi®cant eect of 1% CH3F on methanogenesis, but CH4 oxidation was
completely inhibited in soil slurries. Also, Epp and Chanton (1993) observed that while CH4 oxidation in
the rhizosphere of aquatic macrophytes was inhibited by 1.5% of CH3F, methanogenesis was not
signi®-cantly aected. However, there have been reports of methanogenesis inhibition at lower CH3F
concen-trations. Frenzel and Bosse (1996) observed that CH4
production in soil slurries was reduced by 75% in the presence of 0.1% CH3F and 90% inhibition of
metha-nogenesis occurred at 1% CH3F. Also, >99%
inhi-bition of methanogenesis has been observed at CH3F
concentrations as low as 0.01% in anaerobic sediments (King, 1996).
Whereas inhibitors have been evaluated in materials collected from a wide variety of habitats, none have
been performed with land®ll cover soil. Land®lls rep-resent major contributors to atmospheric CH4 (Topp
and Pattey, 1977) and the cover soils of land®lls have been reported to have high CH4 oxidation activity
(Jones and Nedwell, 1993; Bogner, 1997; Borjesson and Svensson, 1997). Keeping in view the importance of land®lls to the global CH4 budget, along with the
lack of information about inhibitor ecacy for land®ll cover soils, our investigations were designed to evalu-ate ®ve gaseous compounds over a wide concentration range for their inhibitory eects on methanogenesis and CH4 oxidation associated with a land®ll cover
soil. This study includes compounds which have been previously investigated (C2H2, C2H4, C2H6 and
CH3F), as well as an evaluation of the inhibitory
eects of methyl chloride (CH3Cl) and low
concen-trations of C2H4 and C2H6, which have not been
pre-viously determined.
2. Materials and methods
2.1. Soil properties
The soil used in our experiments was topsoil col-lected from a capped city land®ll located on Dayton Road, Ames, Iowa. Initial testing indicated that this soil had high CH4 oxidizing activity under aerobic
conditions and high methanogenic activity under an-aerobic conditions.
Nitrate (+nitrite) (NO3ÿ, NO2ÿ) and ammonium
(NH4+) were determined by colorimetric analyses of 2
Molar KCl soil extracts (KCl:soil, 4:1) on a Lachat autoanalyzer (Lachat Instruments, Mequon, WI) fol-lowing the procedure described by Keeney and Nelson (1982). The pH was determined on 1:1 (dH2O:soil)
extracts with a standard glass electrode as described in the study by McLean (1982). Soil water content was determined gravimetrically after overnight drying at 1058C (Gardner, 1982). Microbial biomass carbon measurements were determined by
fumigation-extrac-Table 1
Properties of land®ll soil used in inhibitor evaluation studiesa
pH 6.71 (0.79)
NO3ÿ(mg N gÿ1dry soil) 3.89 (1.34)
NH4+(mg N gÿ1dry soil) 0
Moisture (%) 54.32 (0.82)
Microbial biomass (mg C gÿ1dry soil) 595.8 (12.63)
Soluble C (mg C gÿ1dry soil) 31.12 (7.09)
Total N (%) 0.15 (3.94)
Total C (%) 1.92 (1.04)
Soil texture 57% sand 25% silt 18% clay
a
tion (Rice et al., 1996). Fifty grams of 5 mm sieved soil was extracted with 100 ml of 0.5 Molar potassium sulfate (K2SO4). Soluble organic carbon was measured
on a Dohrmann DC-180 carbon analyzer (Rosemount Analytical Services, Santa Clara, CA). Biomass carbon was calculated using the correction factor (K = 0.33) of Sparling and West (1988). Total N and C were determined by combustion on a Carlo Erba Carbon/ Nitrogen Analyzer (Fisons Instruments, Rodano, Milano, Italy). Soil texture analyses (sand, silt and clay) were performed by Midwest Laboratories (Omaha, NE). General properties of this soil are listed in Table 1.
2.2. Laboratory experiment
For these tests 20 g portions of sieved (0.5 cm mesh) soil were incubated in 250 ml erlenmeyer ¯asks. Flasks were sealed with a rubber stopper in which a glass tube accommodating a butyl rubber septum (20 mm)
(Bellco Glass, Vineland, NJ, USA) was mounted. Compounds were tested at concentrations of 0%, 0.001%, 0.01%, 0.1%, 1%, and in some cases 10% (v/ v). Autoclaved soil was included as a sterile control. Each treatment was replicated three times. The eects of these compounds on CH4 consumption and CH4
production were evaluated in two dierent incubation regimes. Aerobic conditions (headspace of approxi-mately 20% O2) with added CH4 (initial headspace
concentration of 1±3%) were used to evaluate com-pound in¯uences on CH4 oxidation, while anaerobic
conditions (He atmosphere) with a 10% H2 and 10%
CO2headspace were established to evaluate compound
eects on methanogenesis. Incubations were performed in the dark at room temperature (218C) and spanned up to 30 h for the evaluation of CH4consumption and
up to 7 days for the evaluation of CH4production.
2.3. Gas chromatography
Methane and the initial test compound concen-trations were measured by injecting 0.2 ml of gas sample obtained from the headspace of each ¯ask into a Tracor 540 gas chromatograph (Tracor Instruments Austin, Austin, TX) equipped with a ¯ame ionization detector running at 2008C, a Porapak Q column (All-tech Associates, Deer®eld, IL, USA) and He carrier gas ¯owing at the rate of 30 ml minÿ1. Certi®ed stan-dards of CH4, C2H2, C2H4, and C2H6 were obtained
from Scott Speciality Gases, Troy, MI, USA. Methyl chloride and CH3F were obtained from Matheson Gas
Products, Montgomeryville, PA.
2.4. Rate determinations
An example of the response of these processes to increasing concentrations of CH3F is shown in Fig. 1.
Methane consumption appeared to be a ®rst order process (Fig. 1(A)), but increased concentrations of CH3F reduced CH4consumption. Rate coecients for
CH4 consumption were estimated by applying linear
regression to the natural log of the concentration ver-sus time data (slope = ÿk, Paul and Clark, 1996). Methane production rate increased with time but was not well described by ®rst order kinetics (Fig. 1(B)). The impact of inhibitors on this process was evaluated by computing the maximum rate of CH4 production.
This analysis was done by ®tting the CH4
concen-tration versus time data with a curve and evaluating the ®rst derivative of the ®t equation using the Table Curve software package (SPSS, Chicago, IL).
2.5. Statistical analysis
Results for each inhibitor tested are presented in terms of a mean and a con®dence limit. Statistical Fig. 1. Eect of headspace methyl ¯uoride concentration on methane
dierences between treatments and the control were determined by Tukey's test (Sigma Stat software pack-age, SPSS, Chicago, IL). Eects within test concen-trations can be determined by observation of the overlap of 97.5% con®dence limits (Birnbaum, 1961; Natrella, 1963; Parkin, 1993).
3. Results
Methane oxidation was inhibited by all of the test compounds, but inhibitor eectiveness diered (Fig. 2). Acetylene had a strong inhibitory eect on CH4
oxi-dation over the entire concentration range (Fig. 2(A)).
At C2H2 concentration of 0.001% the ®rst order rate
constant dropped 4.210ÿ2 minÿ1, corresponding to an average of 93.5% inhibition and at higher C2H2
concentrations CH4 oxidation was 100% inhibited.
Methane oxidation was less sensitive to C2H4
(Fig. 2(B)). At 0.001% C2H4, CH4oxidation was
unaf-fected, but partial inhibition (33%) was observed at 0.01% C2H4, with near complete inhibition (96%)
occurring at concentrations of 1%. Neither C2H6 nor
CH3Cl (Fig. 2(C) and (D)) were eective inhibitors of
CH4 oxidation at concentrations less than 0.1%;
how-ever, at a concentration of 1%, a 53% and 78% inhi-bition was observed for C2H6and CH3Cl, respectively.
Methyl chloride was completely inhibitory at a concen-tration of 10%. Increased concenconcen-trations of CH3F
progressively inhibited CH4 oxidation (Fig. 2(E)), and
concentrations at and exceeding 0.1% complete inhi-bition was observed.
The eects of the test compounds on methanogen-esis are presented in Fig. 3. Acetylene only slightly inhibited CH4production at 0.001%, but with
increas-ing concentration greater inhibition was observed (Fig. 3(A)). Ethylene was ineective at inhibiting CH4
production at low concentrations, but at 1%
signi®-cant inhibition was observed. In contrast, C2H6
showed essentially no eect on CH4 production
ac-tivity (Fig. 3(C)). Methyl chloride, on the other hand, was completely inhibitory at concentrations of 1% and 10%, and partial inhibition (89%) was observed at 0.1% (Fig. 3(D)). Methyl ¯uoride exhibited slight inhi-bition (39%) at 1% and inhiinhi-bition was nearly com-plete (93%) at 10% (Fig. 3(E)).
A comparison of the eects of the inhibitors on both processes is presented in Fig. 4, where CH4
con-sumption and production activity are expressed as a percentage of the respective rates with no inhibitor added. Acetylene, at a concentration of 0.001% did not signi®cantly reduce CH4 production activity, but
did inhibit CH4 oxidation (Fig. 4(A)). Methane
oxi-dation at 0.001% C2H2 was reduced to 6.6% of the
control. At higher C2H2concentrations, both CH4
oxi-dation and methanogenesis were signi®cantly lower than the control (no inhibitor). There was no signi®-cant eect of C2H4 on CH4 production at
concen-trations less than 1%, but 89% inhibition of methanogenesis was observed at 1% C2H4(Fig. 4(B)).
Methane oxidation was more sensitive to C2H4 than
methanogenesis and a C2H4 concentration of 0.1%
and greater resulted in 90% inhibition. Methanogen-esis was unaected by C2H6over the range of
concen-trations tested (Fig. 4(C)). Methane oxidation was only partially inhibited (35% and greater) by C2H6 at
higher concentrations (>0.1%).
The pattern of inhibition produced by CH3Cl was
unlike those of the other inhibitors tested, in that methanogenesis was more sensitive than CH4oxidation
(Fig. 1(D)). Methanogenesis was impacted by CH3Cl
at concentrations exceeding 0.01%, and at a CH3Cl
concentration of 0.1% methanogenesis was inhibited by 89%. Methane oxidation was not signi®cantly aected by CH3Cl less than 0.1%, but concentrations
of CH3Cl greater than 1% did inhibit CH4 oxidation.
Methanogenesis was unaected by CH3F
concen-trations at and below 0.1% (Fig. 4(E)). Partial inhi-bition of methanogenesis (38.8%) was observed at 1% CH3F and 92.6% inhibition at 10% CH3F. Methane
oxidation was partially inhibited (39.5%) by CH3F at
0.01% and completely inhibited by CH3F
concen-trations >0.1%.
4. Discussion
Inhibitors present potentially powerful tools in the study of microbial process; however, they must be used judiciously due to potential nonspeci®c or unin-tended eects. As pointed out by Oremland and Capone (1988), ``...employing inhibitors in ecological/ biogeochemical studies must be tempered by a knowl-edge of their limitations.'' This caution is underscored by the variability in the literature concerning inhibitory eects of C2H2and CH3F on CH4 oxidation and
pro-duction in the wide range of systems they have been applied.
Generally, there is consistency in reports regarding the inhibitory concentrations of CH3F required to
inhibit CH4 oxidation. Data from Oremland and
Cul-bertson (1992b) indicate complete inhibition of CH4
oxidation by soil at CH3F concentrations of 0.3% and
1.7% over a 150 h period. Decreasing CH3F
concen-trations which resulted in a decrease in the time inhi-bition was sustained, with CH3F concentrations of
0.16 and 0.06% resulting in nearly complete inhibition of CH4 oxidation for 100 and 30 h, respectively. In
short term incubations (130 h) of S. eurycarpum roots, King (1996) observed nearly complete inhibition of CH4 oxidation (86±96%) over a CH3F
concen-tration range of 0.01±1.0%. Our aerobic incubations of land®ll topsoil yielded results similar to these past studies. Over a 30 h incubation, we observed a 39% inhibition of CH4 oxidation at a CH3F concentration
of 0.01%, and complete inhibition at 0.1% CH3F.
The eects of CH3F on methanogenesis are less
con-sistent. Oremland and Culbertson (1992a) reported a 36% inhibition of methanogenesis in anoxic salt marsh sediments incubated with 1.25% CH3F, but observed
no inhibition of methanogenesis at 0.4% CH3F in
an-aerobic incubations of composted paper waste. Epp and Chanton (1993) found that in the rhizosphere of aquatic macrophytes, CH3F concentration of 1.5%
was sucient to inhibit CH4 oxidation, but did not
aect methanogenesis. However, King (1996) observed that methanogenesis was aected by much lower CH3F concentrations. He reported that methane
pro-duction in anaerobic incubations of peat material was inhibited by 89% and 96% at CH3F concentrations of
0.01% and 0.1%, respectively and suggested that a possible reason for the higher sensitivity of methano-genesis to CH3F may be due to dierences in sediment
sources (marine vs. freshwater). Recently, Frenzel and Bosse (1996) observed that methanogenesis in dierent systems exhibited a dierential sensitivity to CH3F. In
the rhizosphere of cat-tail (T. latifolia), these workers found that methanogenesis was unaected by CH3F
up to concentrations of 1.0%. Methane production in a hypersaline microbial mat was also found to be insensitive to CH3F concentrations up to 4%, but in
anoxic rice ®eld soil low CH3F concentrations (0.1%)
inhibited methanogenesis by 75%. The dierential eects of CH3F on methanogenesis were thought to be
related to the characteristics of the methanogenic populations, with acetoclastic methanogens showing a greater sensitivity to CH3F than organisms using
methylamine or formate (Frenzel and Bosse, 1996). Evidence supporting this eect is provided by Janssen and Frenzel (1997) who observed that the growth of acetoclastic methanogens was inhibited by lower CH3F
concentrations than growth of methanogens using H2
or formate. Our results are consistent with past obser-vations in that, in anaerobic incubations of soil with added H2 and CO2, methanogenesis was not impacted
by CH3F concentrations of <0.1%, but at high CH3F
levels (>1.0% ) CH4production was inhibited.
litera-ture concerning the inhibitory eects of C2H2on CH4
oxidation and production. Early work indicated that C2H2 at high concentrations (>1.25%) strongly
inhi-bits methanogenesis (Oremland and Taylor, 1975; Sprott et al., 1982). Recently, CH4production has also
been reported to be sensitive to much lower C2H2
con-centrations. King (1996) observed that 0.01% C2H2
inhibited methanogenesis associated with peat by >60%. Also, data of Watanabe et al. (1997) indicate that in a 7-day incubation of soil amended with glu-cose, C2H2 at a concentration of 0.001% only had a
slight eect on methanogenesis, but that 0.01% C2H2
resulted in 60% inhibition. Our observations of 80% inhibition of methanogenesis at a 0.01% C2H2, but no
signi®cant eect at 0.001% C2H2 are consistent with
these recent studies.
Acetylene also inhibits CH4oxidation, but most past
studies only have examined C2H2 concentrations
exceeding 0.1% (Yavitt et al., 1990; Bender and Con-rad, 1995; McDonald et al., 1996; Miller et al., 1998). For C2H2 to be useful in distinguishing between gross
and net rates of CH4 production, it must inhibit CH4
oxidation at a concentration of <0.01% because methanogenesis is also sensitive to 0.01% C2H2. King
(1996) reported an 89% inhibition of CH4 oxidation
associated with excised S. eurycarpum roots at 0.01% C2H2. An evaluation of lower C2H2concentrations on
CH4 oxidation have only been presented by
Wanta-nabe et al. (1995). In laboratory incubations of rice ®eld soils, these investigators observed 90% inhibition of CH4oxidation at a C2H2concentration of 0.003%.
The eects of C2H4 on CH4 cycling reactions are
largely unknown. Oremland and Taylor (1975) found nearly complete inhibition of methanogenesis at 5% C2H4, but there have been no reports of C2H4 eects
on CH4 oxidation. Our results not only con®rm that
high C2H4 concentrations inhibit methanogenesis, but
also that CH4 oxidation is nearly completely inhibited
(90%) at C2H4concentrations exceeding 0.1%. Despite
the fact that C2H4is produced in soils by
microorgan-ism (Arshad and Frankenberger, 1989, 1990) it is doubtful that this process signi®cantly impacts CH4
oxidation in nature. In anaerobic incubations of forest soils Sexstone and Mains (1990) measured C2H4
pro-duction rates of 205±71.8 nM C2H4±C kg soilÿ1 hÿ1.
Assuming a bulk density of 1 g cmÿ3 and an air ®lled porosity of 50%, we calculated that these rates of pro-duction would result in soil atmosphere C2H4
concen-trations of only 0.0005% and 0.00017% over an 1 h period (assuming no loss through diusion). Based on our results these concentrations are too low to impact either CH4consumption or production.
There is little information on the other compounds we examined. Our observations that methanogenesis is not signi®cantly aected by C2H6at concentrations up
to 1% are similar to results of Oremland and Taylor
(1975), who reported no inhibitory eect of C2H6 on
CH4 production at concentrations of 1.25% and 20%
using marine sediments. Methyl chloride, as far as we know, has never been used to study its eects on CH4
oxidation or methanogenesis; however, our results in-dicate that CH3Cl is distinctly dierent than the other
compounds of our study, in that methanogenesis was more sensitive than CH4 oxidation. Methanogenesis
was signi®cantly inhibited (89%) by 0.1% CH3Cl, but
CH4 oxidation was unaected by concentrations
<0.1%.
Conclusions of our study are summarized as rec-ommendations of concentrations of compounds which may be useful for distinguishing between CH4
oxi-dation and CH4 production (Table 2). Acetylene at a
concentration of 0.001% inhibited CH4 oxidation by
93.5% but did not signi®cantly impact CH4
pro-duction. Ethylene at a higher concentration (0.1%) had a similar eect. Methyl chloride is unique, in that at a concentration of 0.1% methanogenesis was inhib-ited by 89%, but CH4 oxidation was not signi®cantly
aected. Finally, CH3F at a concentration of 0.1%
showed complete inhibition of CH4 oxidation but did
not signi®cantly in¯uence CH4 production. Of the
compounds we investigated, C2H2, C2H4 and CH3Cl
show the greatest application for use in CH4 cycling
studies. Despite its widespread use, CH3F should be
used with caution, because of the reported dierential response of methanogenic bacteria to CH3F (Frenzel
and Bosse, 1996; Janssen and Frenzel, 1997).
Finally, it is realized that extrapolation of our results to other soils may be tenuous; however, for many of the compounds tested, similar ecacious con-centrations have been reported over a wide range of habitats, including mineral and organic soils, sediments and plant systems. Also, the land®ll soil used in our study had the highest CH4 oxidation and production
activity of any soil we have examined, and we specu-late that the inhibitor concentrations we report (with the exception of CH3F) may be at least as eective in
other soils. However, a prudent course of action would be to evaluate the precise inhibitor concentration Table 2
Recommended compound concentrations to selectively inhibit either methanogenesis or methane oxidation
Compound Concentration % (v/v)
% Average inhibition
CH4production CH4oxidation
Acetylene 0.001 8.8 93.5
Ethylene 0.1 17.7 90
Methyl chloride 0.1 88.9 11.2
required to achieve the desired degree of inhibition in each speci®c system of interest.
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