Chlorothalonil Degradation under Anaerobic Conditions in an Agricultural Tropical Soil

CHLOROTHALONIL DEGRADATION UNDER ANAEROBICCONDITIONS IN AN AGRICULTURAL TROPICAL SOIL

ZARHELIA CARLO-ROJAS1, RICARDO BELLO-MENDOZA1∗,MIGUEL SALVADOR FIGUEROA2 and MIKHAIL Y. SOKOLOV1

1 Departamento de Biotecnología Ambiental, El Colegio de la Frontera Sur, Carretera AntiguoAeropuerto km 2.5, Tapachula, Chiapas, Mexico; 2 Área de Biotecnología, Facultad de CienciasQuímicas, Universidad Autónoma de Chiapas, Carretera a Puerto Madero km 2.0, Tapachula,

Chiapas, Mexico(∗ author for correspondence, e-mail: [email protected], Fax: +52 (962) 628 98 06)

(Received 6 December 2002; accepted 11 August 2003)

Abstract. Chlorothalonil, a halogenated benzonitrile compound, is one of the most widely usedfungicides in the world. Anaerobic microcosm assays were established to evaluate the combinedeffect of the initial content of carbon (6.3, 9.45 and 12.6 mg g−1), nitrogen (0.6, 1.8 and 3 mgg−1) and chlorothalonil (432, 865 and 1298 ηg g−1) on the biodegradation of this fungicide bymicrobiota from an agricultural tropical soil. A Box-Behnken experimental design was used andchlorothalonil depletion was followed by HPLC with UV detection. The initial carbon content andfungicide dose were found to have a significant effect on removal efficiency. After 25 days of incub-ation, a high chlorothalonil depletion was observed in all biologically active microcosms (56–95%)although abiotic loss in a sterile blank was also notable (37%). The results suggest a high potentialfor chlorothalonil biodegradation under anaerobic conditions by indigenous microbial communitiesfrom soil that has been continuously exposed to high doses of the fungicide.

Keywords: bioremediation, Box-Behnken, C/N ratio, fungicide, microcosms, soil microbial com-munity

1. Introduction

Chlorothalonil (CTN) or 2,4,5,6-tetrachloroisophtalonitrile, is one of the mostwidely used fungicides in the world. This synthetic compound belongs to the groupof halogenated benzonitriles. Although the exact mechanism is not well understood(EPA, 1999), it is believed to act by contact, inhibiting cell respiration enzymesrelated to glutathion (Arvanites et al., 2001).

In the Soconusco region of Chiapas, Mexico, CTN is used as a protectant fun-gicide in foliage and soil of banana and other crops. Banana, in particular, ischaracterised by a high demand for agrochemicals all year long and has beenreported to be the recipient of 47% of the total load of active pesticide ingredientsin the zone (SERNyP, 1998). Moreover, CTN is the most used pesticide in theregion with 14.3% of the total active ingredient load (SERNyP, 1998).

CTN might cause severe pollution problems in tropical ecosystems as indicatedby a recent study in which this compound was found to be one of the two pesticides

Water, Air, and Soil Pollution 151: 397–409, 2004.© 2004 Kluwer Academic Publishers. Printed in the Netherlands.

398 Z. CARLO-ROJAS ET AL.

with the highest persistence in water and sediments in the Brazilian environment(Oubiña et al., 1998).

When analysing the pollution and toxicity risks associated with CTN, its simil-arity with other dangerous molecules should be considered (Table I). Also, one ofits principal metabolites, 4-hydroxychlorotalonil, is 30 times more toxic in acutetoxicity and more mobile in soil and water than CTN itself (UNEP-ILO-WHO,1996). On the other hand, during CTN synthesis, hexachlorobenzene (HCB) isgenerated as a by-product. This highly toxic and recalcitrant compound remains asan impurity in the commercial product. The use of CTN and other 3 organochlorinepesticides is the greatest source of human exposure to HCB (UNEP-ILO-WHO,1996; EPA 1999).

Reported data on CTN degradability (Table II) are very divergent, a fact whichmight be attributable to the variability on test environmental conditions and alsoto the functional groups of its structure. A quick removal of CTN from water hasbeen reported under rigorous oxidative conditions but these results could not becompared to those obtained by natural processes. Nonetheless, this type of datais the basis for the current classification of CTN (EPA, 1999). The degradationpattern of CTN in natural ecosystems is not well understood. It is therefore ne-cessary to study this under laboratory conditions similar to those prevailing in theenvironmental compartment with the highest inputs.

The aerobic metabolic pathway has been considered the most suitable for CTNmicrobiological degradation (Katayama et al., 1997; Mori et al., 1998; Regitanoet al., 2001), although very few studies have been conducted on the possible oc-currence of degradation by anaerobic routes. Nevertheless, some metabolites havebeen reported which might be associated with the mechanisms of sulphate-reductionand reductive dechlorination that occur in anoxic environments (UNEP-ILO-WHO,1996; Regitano et al., 2001). Van der Meer et al. (1992) showed that the degrad-ation of halogenated benzenes occurred at a higher degree by reductive dechlor-ination than by hydrogenation (aerobic route). This could extend to the chlorin-ated benzenic structures of CTN and its metabolites. Wackett and Hershberger(2001) suggest benzenic ring reduction via carboxilation in anaerobic conditionslike one of the general rules of biodegradation, thus it is necessary to gather moreinformation about CTN anaerobic metabolism.

The sandy, acidic soils in the banana plantations of the Soconusco region sporad-ically present anoxic conditions by flooding and elevation of the phreatic mantle.Under these conditions, it would be possible to find facultative and anaerobic bac-terial populations, metabolically adapted to degrade CTN due to their continuousexposition to this compound. This work studies the degradation potential of CTNby indigenous microorganisms from a tropical soil under anaerobic conditions andtheir response to stimulation by different carbon/nitrogen ratios.

CHLOROTHALONIL DEGRADATION IN ANAEROBIC SOIL MICROCOSMS 399

TABLE I

Chlorothalonil and related compounds


TABLE II

Chlorothalonil half-life

Matrix Conditions Half-life Metabolic Source

route

Soil More degradation to more 1–3 months Aerobic UNEP-ILO-WHO,

temperature and humidity 1996

Groundwater Water basic pH 9.0, 65%to mayor metabolite

2.5 months –

Soil 10–37 days Aerobic EPA, 1999

Water 9 days Anaerobic

Water Fresh water 1.4 hr Aerobic

Water Experimental conditionsaerated wastewater

2–8 hr Aerobic

2. Materials and Methods

2.1. CHEMICALS

Chlorothalonil (PESTANAL grade, 99.2% purity) was obtained from Riedel-deHaën. Acetonitrile, methanol and hexane (85% n-hexane) were HPLC grade where-as acetone was analytical reagent grade. All solvents were provided by J. T. Baker.Other chemicals used (dextrose, ammonium chloride, sodium sulphate anhydrousand sodium chloride) were analytical reagent grade. Tri-distilled water was usedfor the preparation of aqueous solutions and as an eluent for HPLC analysis. Thepesticide utilised was Daconil 2775 (75% CTN) produced by Zeneca agrochemic-als.

2.2. SOIL

Soil samples were obtained from a banana plantation located in the Soconuscoregion of Chiapas (southern Mexico, 14◦36′7′′N and 92◦11′10′′W) at 28 m a.s.l.The climate is rainy tropical, with a precipitation of 1400 mm per year and rain-falls during the summer months. The average temperature is 27 ◦C. The plantationhas been cultivated for over 35 yr and is characterised by a high utilisation ofagrochemicals, including CTN.

The physical-chemical characteristics of the samples correspond to a sandyloam, slightly acidic (pH = 6.2) soil and are shown in Table III.

Single soil samples were taken at a 30 cm depth according to EPA (Mason,1992). Sampling sites were selected along water run-off areas (e.g. damp patches).Soil slides were obtained and only the core was used to minimise air contact. Allmaterials and tools used were sterilised. Samples were put into black polyethylene


TABLE III

Physical-chemical characteristics of the soil

Parameter Value Method

Texture Sandy loam Bouyucos

Sandy 55.32%

Silt 36.52%

Clay 8.16%

Electric conductivity 0.07 mS cm−1 Whiston’s bridge

pH 6.2 pH meter

Ion exchange capacity 6.82 meq 100 gr−1 Addition

Organic matter 0.67% Walkley y Black

Organic carbon 0.63% Combustion

Total nitrogen 0.06% Macro-Kjeldahl

Phosphorus 68.2 mg kg−1 Olsen-Bray

Potassium 400 mg kg−1 Flame photometer

Calcium 850 mg kg−1 Atomic absortion

Manganese 1.8 mg kg−1 Atomic absortion

Cooper 1.22 mg kg−1 Atomic absortion

Zinc 1.36 mg kg−1 Atomic absortion

Iron 76.2 mg kg−1 Atomic absortion

Magnesium 157.5 mg kg−1 Atomic absortion

Sodium 55 mg kg−1 Flame photometer

Sulphate 10.3 mg kg−1 Colorimetric

bags taking care that no air was introduced. The samples were transported to thelab on ice and stored there in a fridge at <4 ◦C. A composite sample was preparedin the lab with the single subsamples obtained in the field (Mason, 1992).

2.3. MICROCOSMS

Firstly, soil humidity was determined by drying at 105 ◦C for 48 hr. 200 g (dryweight) of moist soil were then deposited in each of 16 clean and sterilised, boro-silicate glass bottles (250 mL) with hole screw caps fitted with Teflon layerednitrile septa in their centre. Preliminary tests showed that the bottles closures werehermetic even after repeated punctures with a needle. Additionally, a sterilisedsoil sample was used as an abiotic control. For this, the soil was autoclaved for90 min and once again 24 hr later to ensure the elimination of thermoresistantendospores (Madsen, 1997; Schmidt and Scow, 1997). All bottles were then spikedwith different doses (86.5, 173.1 and 259.6 µg in 2 mL of methanol) of the pesti-cide according to the experimental design. Stock solutions of dextrose (0.4 M)


and ammonium chloride (0.686M) were prepared and used as carbon and nitrogensources, respectively. Different volumes of these solutions were placed in eachbottle so as to reach the carbon and nitrogen doses required for each treatment.The humidity of the mixture in each bottle was then adjusted to 60% by addingan isotonic solution (0.85% NaCl). This resulted in a medium volume of 214 cm3.Finally, the bottles were closed and refluxed with nitrogen gas of ultrahigh purity(99.99% N) in order to create anaerobic conditions. For this, two needles, one forthe injection and another for the evacuation of the gas, were used. The bottles werelabelled and covered with aluminium foil to protect them from light and preventphotodecomposition. Incubation was conducted at room temperature (29 ◦C) for a25-day period. Biogas produced during anaerobic degradation was regularly meas-ured by the displacement of a plunger in a calibrated syringe inserted into eachbottle. After measurement, the syringes were withdrawn and the accumulated gaswas vented. This was done in order to prevent any interference in the microbialactivity by overpressure.

2.4. PESTICIDE EXTRACTION

Pesticide was extracted from soil samples (60% humidity) by refluxing with hex-ane/acetone (1:1 v/v) for 7 hr in a Soxhlet apparatus (EPA, 1979). The extractswere further concentrated in a rotary evaporator and thereafter by flushing withhigh purity nitrogen gas until a volume of 2 mL was reached. 64% of CTN wasrecovered from a spiked soil sample (abiotic) 50 hr after its addition. An internalstandard was used to identify the compound.

2.5. CHROMATOGRAPHIC ANALYSIS

CTN separation was carried out on a HPLC equipment (Gynkoteck 300C) fittedwith a UV detector (Merck-Hitachi L7400). A 5 µL volume sample was injectedonto a 250 mm LiChroCART 250-4 (Merck) analytical column using acetoni-trile:water (80:20 v/v) as the eluent. This mixture was previously degassed bysonication (Branson 1510). CTN was detected at 232 nm. This wavelength showedthe highest absorbance when tested in an UV/Vis spectrophometer (Beckman DU-6) in scan mode. Chromatograph plotting and area calculation were conducted inan HP3396A integrator.

The linearity of the system was tested by running calibration standards in therange of 1–10 mg L−1. The correlation coefficient obtained was satisfactory (r2

= 0.9973). The reproducibility of the peak areas was measured by four 5 µLinjections of a 5 mg L−1 solution of CTN in a mixture of acetonitrile and water(80:20 v/v). The coefficients of variation measured for the area and the retentiontime were 2.1 and 0.7%, respectively.


TABLE IV

Factors and levels of the Box-Behnken experi-mental design

Factors X1 X2 X3

C N Chlorothalonil

(ηg g−1)

Low level 10 1 432

Medium level 15 3 865

High level 20 5 1298

2.6. EXPERIMENTAL DESIGN

A Box-Behnken experimental design (Box and Behnken, 1960) was used to eval-uate the effect of carbon, nitrogen and initial CTN concentration on the pesticideremoval efficiency. Thus, three variables were considered at three levels (Table IV).Carbon and nitrogen concentrations were varied in order to evaluate their stimulat-ory effects on biodegradation. The combination of the three doses of carbon andnitrogen resulted in a higher number of C/N ratios. The lowest level consideredfor the C/N ratio was that found in the soil (10/1). Microcosms were not amendedwith phosphorous since this element was found to be at an optimal level in the soil(Table III). The levels considered for the fungicide represent 1, 2 and 3 times thedose used by the banana producers.

From the factorial design (3 × 3), the Box-Behnken matrix selects 13 differentlevel combinations plus two replicates of the mid-level treatment for a total of 15microcosms. Experimental error is calculated from the three mid-level replicates(i.e., microcosms 11, 12 and 13). A sterilised control was also run (Table V).

The surface response analysis was performed with JMP® Statistic Softwareversion 4.0 (1989–2000) by SAS, Inc.

3. Results and Discussion

The removal efficiency achieved by the microcosms is shown in Table V. A reduc-tion in CTN concentration above 56% was observed in all treatments after 25 daysof incubation. The sterile control also showed an important decline in CTN concen-tration (37%) indicating that abiotic processes might play an important role in thedepletion of the fungicide. However, this reduction was lower than that attained byall other biologically active microcosms. Regitano et al. (2001) conducted a seriesof experiments on the degradation of CTN involving the incubation of tropical,acidic soil samples for 90 days. Their results showed that although most of the


TABLE V

Removal efficiency of chlorothalonil and total biogas production

Treatments Factor Removal Cumulative pH

C N chlorothalonil efficiency biogas

(× 0.063 g (× 0.006 g (ηg g−1) (%) production (mL)

g−1 soil) g−1 soil)

1 10 3 432 81 22 6.4

2 20 3 432 56 292 5.0

3 15 1 432 ND 128 5.5

4 15 5 432 ND 203 5.3

5 10 3 1298 75 2 6.1

6 15 1 1298 77 103 5.7

7 15 5 1298 66 248 5.5

8 20 3 1298 95 528 5.3

9 20 1 865 89 111 5.4

10 20 5 865 82 228 5.3

11 15 3 865 77 34 5.5

12 15 3 865 71 13 5.1

13 15 3 865 74 138 5.5

14 10 5 865 72 0 6.2

15a 10 1 865 83 0 6.5

16a,b 10 1 865 37 0 6.1

a No nutrient added.a Sterile control.

compound was lost within the first 7 days, this was attributed to abiotic factors,whilst effective mineralisation was found to occur at a rather slow rate, as evaluatedby 14C assays.

The high degree of CTN removal achieved in this work (up to 95%) is in agree-ment with the results reported for different soil types and incubation conditions(Katayama et al., 1997; Regitano et al., 2000). Nonetheless, it should be noted thatsoil binding might have contributed to these high removal efficiencies. A prelimin-ary test showed a 64% recovery after spiking a soil sample with CTN and extractingthe fungicide with solvents. Also, it has been reported that a CTN reduction of up to80% could be due to abiotic causes (Katayama et al., 1995). Regitano et al. (2001)reported a high formation of soil-bound residues in the first day of microcosmsincubation. This binding process varies widely due to the diversity of the mineralcomponents, the nature and content of organic matter, the proportion and size ofthe particles and, particularly, the clay content of the soil (Scow and Fan, 1995;Fushiwaki and Urano, 2001; Gamble et al., 2001). Soil aggregates do not onlyhave a physical-chemical influence but are also related to biological activity since


Figure 1. Surface response of chlorothalonil removal efficiency as a function of C and CTN doses,showing a saddle point.

they provide micro spaces allowing microbial diversity (Van Eaden et al., 2000;Regitano et al., 2001).

As expected, biogas production was related to the amount of dextrose addedto the microcosms due to the fast fermentation of this carbon source by anaerobicmicroorganisms (Table V). The highest gas production corresponded to the greatestcarbon dose and, conversely, treatments with the lowest carbon dose showed no, orvery low, gas production. It is however interesting to note that the highest CTNremoval was attained by the treatment (20/3 C/N, 1298 ηg g−1 CTN) with thehighest biogas production (528 cm3). This might be indicative of co-metabolicprocesses taking place, as has been reported elsewhere (Katayama et al., 1995;Motonaga et al., 1995; Regitano et al., 2001). Katayama et al. (1995) have alsopointed out that there is no direct relationship between microbial growth and CTNdegradation.

Dextrose fermentation was also responsible for the acidification of the me-dium (Table V). One of the treatments with the most elevated carbon dose (20/3C/N, 432 ηg g−1 CTN) showed the sharpest drop in pH (6.2 to 5.0), whereastreatments with no addition of dextrose did not show any change in pH (6.2–6.4). The acidification of the medium did not seem to affect CTN depletion. CTNdegradation in acidic soils (pH = 4.5) has been reported under aerobic condi-tions (Regitano, 2001). Moreover, the hydrolytic nature of CTN degradation hasbeen demonstrated (Rouchaud et al., 1998) and the microorganisms responsiblefor dextrose fermentation might have produced the enzymes required for CTNtransformation.

Statistical analysis of data (Table VI) showed that interacting carbon and initialCTN doses had a significant effect on CTN degradation (p = 0.0194). It should be


TABLE VI

Regression analysis of removal eficiency of chlorothalonil (%),according to the Box-Behnken experimental design

Term Coefficient T value Significance level

(p-value)

Intercept 74.01 22.41 <0.0001

X1 carbon 1.441125 0.71 0.4724

X2 nitrogen –6.585813 –2.66 0.1555

X3 chlorothalonil 9.9160625 4.00 0.0485a

(X1)2 12.114562 3.67 0.0471a

(X2)2 –4.833562 –1.46 0.4153

(X3)2 –9.332312 –2.83 0.0981

X1X2 0.9265 0.32 0.7353

X2X3 2.676625 0.66 0.8214

X1X3 11.46075 4.01 0.0194a

R2 = 0.937234 (R2 Adj = 0.748937). Coefficient of variation = 6.5%.a Variables with significant effect.

pointed out that although treatment 9 (20/3 C/N, 1298 ηg g−1 CTN) showed thehighest CTN removal efficiency (95%) this is not a maximum, according to thestatistical model. In fact, a saddle point was found, which means that there are tworegions where feasible maxima could be found (Figure 1).

The diversity of factors that interact in biological soil systems causes a highvariability of results wherever there has been an attempt to assess the effect of theC/N ratio on degradation behaviour due to their non-linear nature. The results ofthis study have not shown, in the evaluated range, an optimal value for the C/Nratio during the anaerobic degradation of CTN. However, two regions of strongbiological activity have been outlined. The occurrence of these two regions couldbe explained by the interaction of several microbial communities with differentenvironmental conditions that favour CTN metabolism. Regitano et al. (2001) dis-cussed CTN degradation by different metabolic pathways. Katayama et al. (1997)also suggest that pure bacterial cultures are not able to mineralise CTN, thus imply-ing the need for microbial consortia. Degradation can also be expected to diminishwhen the more toxic metabolites begin to accumulate in the system. These toxiceffects could be more severe to some microbial groups. The proportional particip-ation of fungi and bacteria on CTN degradation in soil has been found to undergoample variations depending on the amount and origin of the carbon source, thetoxicity of the compound and pH level (Mori et al., 1998).

Although CTN depletion under anaerobic conditions has been observed in thisstudy, the produced metabolites were not identified. However, Katayama et al.


(1997) suggest that the capacity of bacteria to make the chlorine substitution onCTN’s aromatic ring by metylthio, hydroxyl or hydrogen groups, is ubiquitous, be-ing metylthioisopthalonitrile and 4-hydroxychlorothalonil the major metabolites.During the degradation of CTN in acidic soils, the metabolites 3-carbamyl-2,4,5trichlorobenzoic acid and 4-hydroxychlorothalonil were found to predominate atpH values of 4.5–4.8 and 5.5, respectively (Regitano et al., 2001). Table I showsthe main metabolites reported for CTN degradation.

The dissipation of CTN in the environment involves losses by volatilisation,photodegradation, biodegradation, soil sorption and binding, and residues bindingto humic substances (Katayama et al., 1995; Mori et al., 1996; Winkler et al., 1996;Regitano et al., 2001). The presence of CTN in the environment might have con-sequences which are difficult to detect for non-target organisms in agroecosystemssuch as mycorrhizal fungi, worms, birds and aquatic organisms (Aziz et al., 1991;UNEP-ILO-WHO, 1996; EPA, 1999). It is necessary to revise the impacts of the in-tensive usage of CTN on plantations in the tropics. Although CTN dissipation fromthe application sites occurs, it is difficult to assess to what degree CTN remainsin the surrounding areas or is in fact transported far away (Beyer and Matthies,2001). Also the impact of this fungicide on soil fertility, given the magnitude andrecurrence of its application, is still unknown and requires evaluation.

Although the aerobic pathway is considered the most important in CTN degrad-ation, this study has shown that CTN degradation by indigenous microbial com-munities from a tropical soil also occurs under anaerobic conditions. The amountof abiotic CTN loss from the soil is also important. The interaction of the initialcontent of carbon and CTN had a significant effect on the degradation of the fungi-cide. The addition of organic matter should be contemplated as an endorsement tothe conservation of the soil physical-chemical properties that contributes to CTNdegradation.

Acknowledgements

The authors wish to thank M. S. Javier Valle Mora for his assistance in statisticsand Dr. Rolf M. Wittich for helpful discussion. Z. Carlo-Rojas also expresses hergratitude to CONACyT-Mexico for the scholarship granted.

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Chlorothalonil Degradation under Anaerobic Conditions in an Agricultural Tropical Soil