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Comparative abiotic or biotic degradation of carboxinby two Entisols with different surface properties orPseudomonas aeruginosa strain: A toxicity study usingthe crustacean Thamnocephalus platyurusMarina Isidori a , Elio Coppola b , Maria Rosaria Iesce c , Flavio Cermola c , Giovanni Papa a &Alfredo Parrella aa Dipartimento di Scienze della Vita, Seconda Università di Napoli, Caserta, Italyb Dipartimento di Scienze Ambientali, Seconda Università di Napoli, Caserta, Italyc Dipartimento di Scienze Chimiche, Università Federico II, Napoli, Italy

Version of record first published: 02 Jul 2012

To cite this article: Marina Isidori, Elio Coppola, Maria Rosaria Iesce, Flavio Cermola, Giovanni Papa & Alfredo Parrella (2012):Comparative abiotic or biotic degradation of carboxin by two Entisols with different surface properties or Pseudomonasaeruginosa strain: A toxicity study using the crustacean Thamnocephalus platyurus , Journal of Environmental Science andHealth, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 47:9, 891-900

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Journal of Environmental Science and Health, Part B (2012) 47, 891–900Copyright C© Taylor & Francis Group, LLCISSN: 0360-1234 (Print); 1532-4109 (Online)DOI: 10.1080/03601234.2012.693879

Comparative abiotic or biotic degradation of carboxin bytwo Entisols with different surface properties orPseudomonas aeruginosa strain: A toxicity study using thecrustacean Thamnocephalus platyurus

MARINA ISIDORI1, ELIO COPPOLA2, MARIA ROSARIA IESCE3, FLAVIO CERMOLA3, GIOVANNIPAPA1 and ALFREDO PARRELLA1

1Dipartimento di Scienze della Vita, Seconda Universita di Napoli, Caserta, Italy2Dipartimento di Scienze Ambientali, Seconda Universita di Napoli, Caserta, Italy3Dipartimento di Scienze Chimiche, Universita Federico II, Napoli, Italy

The microcrustacean Thamnocephalus platyurus was used to detect the toxicity reduction of carboxin in abiotic degradation comparedto biotic degradation. The abiotic degradation was obtained using two sterilized Entisols with different surface properties while thebiotic degradation by Pseudomonas aeruginosa was obtained using the fungicide as the only C source. The results showed that thehighest toxicity reduction rates for the abiotic degradation were achieved in 20 days with 49.2% for the coarser soil, 60.7% for the soilwith a finer texture, whereas for the biotic degradation, 60.6%. Analysis (1H NMR) showed that the soils transformed carboxinto produce sulfoxide and enol in different concentrations depending on the soil properties, while P. aeruginosa metabolized thefungicide to produce inorganic compounds such as ammonium and nitrite, minor degradation pathways were oxidized to sulfoxideand hydrolytic ring-opening to 2-[(2-hydroxyethyl)thio]acetoacetanilide enol. These results indicated that the degradation of carboxinoccurred via abiotic catalytic processes as well as via biotic transformation leading to less toxic derivatives and such phenomena arecaused by exchange/surface features of soils, rather than by the mere content of clay or organic matter fractions.

Keywords: Carboxin, toxicity testing, abiotic degradation, Pseudomonas aeruginosa, Entisols.

Introduction

Carboxin (5,6-dihydro-2-methyl-1,4-oxathiine-t3-carboxa-nilide; CAS name: 5,6-dihydro-2-methyl-N-phenyl-1,4-oxathiine-3-carboxamide; CAS number: 5234-68-4), alsoknown as Vitavax, is a systemic anilide fungicide inhibitingthe mitochondrial function, used to control plant diseasescaused by Basidiomycetes.[1]

Carboxin can be oxidized in the environment by abioticand biotic transformation as shown by Balasubramanyaand Patil.[2, 3] Oxidation to sulfoxide is favoured by naturalsunlight in water,[4] by UV light in organic solvents[5] aswell as by the presence of humic substances and soil.[6]

Interactions between soil minerals and ionic and non-ionicorganic molecules which catalyze transformation anddegradation reactions are well known, especially in the

Address correspondence to Alfredo Parrella, Dipartimento diScienze della Vita, Seconda Universita di Napoli, Via A. Vivaldi,43, I-81100, Caserta, Italy; E-mail: alfredo.parrella@unina2.itReceived February 26, 2012.

presence of clay minerals with a high specific surface, suchas smectites.[7] On the other hand, the reaction betweenpesticides and experimentally redox-modified smectitescan produce genotoxins as abiotic degradation products.[8]

Carboxin is biodegraded by the blue-green alga Anabaenasp., the green alga Chlorella vulgaris, cyanobacteria Nostocsp. and Tolypothrix.[2] In addition, protozoan species(Colpoda spp) can degrade the fungicide while amongmicroorganisms Pseudomonas aeruginosa was shown tobe able to utilize carboxin as its exclusive source of C andN, causing its transformation to sulfoxide, and then tosulfone, further degraded to inorganic compounds.[9]

No data exists on the influence of soils with differentpedogenic features and physical-chemical properties in theabiotic degradation of carboxin. Studies in the field onthe performance of pesticide degradation in natural soilsmay be masked by solar light and the presence of soil mi-croorganisms. In this framework, the present study aimsto investigate the effectiveness of soil in carboxin degra-dation without photochemical side-reactions, consideringthe soil abiotic catalytic properties, with special referenceto surface/exchange features.

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To this purpose, poorly differentiated soils such as En-tisols were chosen for their similar pedogenetic and chem-ical characteristics as well as a comparably low organicmatter content. Furthermore, the Entisols had a widelycontrasting clay content, thus with dramatically differentcation exchange capacity as well as specific surface area.The soils were sterilized to check for abiotic degradationand a standard OECD artificial soil[10] was used as referencetest substratum. Because carboxin is biologically degradedby soil microorganisms producing the more stable, less hy-drolysable and without fungitoxic activity sulfoxide,[4] in thepresent study we used Pseudomonas aeruginosa to degradecarboxin when it was the only source of carbon and nitrogenfor its metabolism to compare biotic and abiotic degrada-tion. The derivatives obtained were chemically identifiedand the acute toxic effects of pure carboxin as well as ofthe biotic and abiotic transformations were determined onthe crustacean Thamnocephalus platyurus. This crustaceanwas chosen for its high sensitivity to pure chemicals and forthe ease of preparation for screening tests, being stored incysts.

Materials and methods

Chemicals

Carboxin analytical standard grade (99% purity), dimethyl-sulfoxide, potassium dichromate were purchased fromSigma-Aldrich Chemicals (St.Louis, MO, USA).

Soils and reference artificial soil

The two soils selected for the present investigation were rep-resentative of the Lower Volturno River Basin, situated inthe Campanian Plain (Campania Region). The area, cov-ering about 600 km2, represents one of the most valuableagricultural lands of southern Italy for the buffalo breed-ing and for the production of fruits, tomatoes, vegetablesand cereals. Both soils belong to the Entisols order.[11, 12] AVertic Xerofluvent (hereafter CSV) was sampled in CastelVolturno municipal territory, and a Typic Xeropsamment(hereafter MND) was sampled in Mondragone municipalterritory.

A reference artificial soil (RAS) was used as control. TheRAS utilized was the OECD reference soil,[10] a dry weightmixture of 750 g kg−1 silica fine sand, including < 10 gkg−1 CaCO3, 200 g kg−1 clay as kaolin, and 50 g kg−1

sphagnum peat moss. The kaolin clay contained silicateclay minerals prevailingly represented by kaolinite (300 gkg−1), with mica and smectites as accessory minerals.

Soil analysis

The soil properties were determined on the fine-earth frac-tion (Ø < 2 mm). Both soils were air-dried, crushed,screened to pass a 2 mm sieve and then sterilized at 121◦Cfor 3 h, repeating the same procedure three times with 24 h

intervals between each treatment.[13] The analyses were car-ried out according to the Italian standard methods for soilanalysis.[14] Both the soils were proved to be pesticide free.

The surface area of the individual particles is an impor-tant factor in pesticide adsorption. The surface area of asoil has contributions from primary particles, amorphousmineral coatings and organic matter. Surface area is anextensive quantity (i.e., it depends on how much soil ispresent). A more satisfying alternative is the introductionof an intensive quantity, the specific surface, which is eitherthe surface area per mass (Sm) or per volume (Sv).

With reference to soil surface area (SSA), the typicalranging values are 15–20 and 280–500 m2g−1 for kaoliniteand smectite, respectively.[15] As a comparison, the SSA ofa sandy soil is estimated at less than 10 m2g−1.[15]

Carboxin was initially dissolved in dimethylsulfoxide(DMSO) and then diluted in reconstituted freshwater(moderately hard EPA medium) indicated as water dilu-tion for toxicity testing of chemicals in standard proce-dure.[16] The hardness of the freshwater was 80–100 mg L−1

CaCO3. The DMSO concentration never exceeded 0.01%(v/v). Then, 100 mL of carboxin solution (280 mg L−1) wereadded to 15 g of each soil previously autoclaved then vac-uum dried at 60◦C and −50 KPa up to a constant weight.The high concentration of carboxin used was justified byits low acute toxicity found for terrestrial as well as foraquatic organisms (in the order of dozen of mg L−1) asreported by Montague.[17] Furthermore, the high concen-tration of carboxin did not make its possible adsorption tothe soils significant. On the other hand, both carboxin andits degradation products are very mobile compounds.[18]

The suspensions of soils with carboxin solution wereplaced at 20 ± 0.5◦C in the dark on a rotating agitator inorder to continuously support soil/carboxin contact. Darkconditions were applied because serious modifications ofcarboxin are reported in literature for the photo-oxidationprocess and the formation of carboxin sulfoxide or sulfonewith different toxic activity (Fig. 1). In these conditions,every 5 days starting from 0 to 20 days, aliquots of thesamples were taken and centrifuged at 2,000 g at 4◦C for10 min. The clarified supernatant was kept to be subjectedto the acute toxicity testing.

Preparation of Pseudomonas aeruginosa suspension withcarboxin

To evaluate the capability of Pseudomonas aeruginosa(ATCC 9027) to degrade carboxin and compare its ac-tivity with those of the soils, the bacteria were grown inNutrient Broth (Oxoid, Milan, Italy) at 36 ± 1◦C for 48 h,then the bacterial suspension centrifuged at 2,000 g at 4◦Cfor 10 min and the pellet re-suspended in freshwater andre-centrifuged to remove any trace of the nutrient broth.A bacterial suspension (1 mL) was added to the carboxinsolution prepared as reported above to obtain a microbial

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Abiotic and biotic degradation of carboxin 893

S

O

C

HN

CH3

O

S

O

C

HN

CH3

OO

NC

CH3

H

O

NH2 OH

CONH

COOHS

O

C

HN

CH3

O

O O

NH+4 NO2

-NH+4 NO2

-

carboxin (vitavax)

carboxin sulfoxide

oxanilic acidacetoanilide

4-aminophenol

sulfone (oxicarboxin -plantavax)

95%

5%reducing environment

slow oxidation

rapid oxidation

Fig. 1. Staples by carboxin degradation in environment.[1, 3, 4, 6, 8, 9, 26]

concentration of about 200·104 CFU/mL. These condi-tions were not toxic for bacteria as shown by an evidentmicrobial growth curve (plate inclusion method in Cetrim-ide Agar Oxoid). The toxicity tests of the bacterial suspen-sion with carboxin were performed after inoculation, at 5,10, 15 and 20 days of exposure at 20 ± 0.5◦C on a rotatingagitator in the dark in order to avoid the phototransforma-tion of carboxin.

Toxicity testing

Three independent bioassays on the anostracan crustaceanThamnocephalus platyurus were run following the manu-facturing procedure. Second and third instar fairy larvaehatched from cysts, provided by MicroBioTest (Nazareth,Belgium), were used. After 20–22 h of incubation at 25◦C insynthetic freshwater under continuous lighting (light source3000–4000 lux), the organisms were exposed to two-fold di-lutions of carboxin solution incubated with soils as well aswith P. aeruginosa, with three replicates for dilution, fivedilutions, and ten crustaceans per well (1.0 mL of test so-lution). After 24 h in a 25◦C incubator in the dark, thenumber of dead crustaceans was recorded and the concen-tration that promotes 50% mortality in the organisms in24 h was indicated as LC50. While toxicity testing, refer-ence tests were performed in freshwater medium (negativecontrol) and potassium dichromate as positive control. TheDMSO concentration in the exposure solutions, includingcontrols, was 0.01% (v/v), which is a non-toxic dose asestimated in preliminary tests.

Chemical identification

Nuclear magnetic resonance (NMR) spectra were recordedat 500 MHz for 1H on a Varian 500 Fourier transform NMRspectrometer using CDCl3 as solvent. GC/MS analyseswere conducted on an Agilent (AMDIS) gas chromato-graph with a Hewlett-Packard detector using a low bleedcolumn, 30 m × 0.25 mm ID × 0.25 µm film thickness.The analyses were carried out by a splitless injection mode(1 min) flow pulse of 5.0 mL min−1, with inlet set at 250◦C.The helium carrier flow was at 1 mL min−1 and the oventemperature program was set at 40◦C and increased to 70◦Cat a rate of 10◦C min−1; to 150◦C at a rate of 25◦C min−1;to 200◦C at a rate of 3◦C min−1; to 280◦C at a rate of 8◦Cmin−1. The HPLC apparatus consisted of an Agilent 1100HPLC system equipped with a UV detector set at 254 nm.The column was a Phenomenex HYDRO RP-18, 4 µm, 250× 4.5 mm. Analytical TLC was made on Kieselgel 60 F254or RP-18 F254 plates with 0.2 mm layer thickness (Merck).Preparative TLC was performed on Kieselgel 60 F254 plateswith 0.5 or 1 mm film thickness (Merck).

Soil supernatant analysis

The three clarified supernatants were obtained as abovefrom each soil, the bacterial suspension and the controlwere analyzed as follows:

• an aliquot of each sample at 0, 5, 10, 15 and 20 days wasdirectly injected in the HPLC using MeOH/H2O (2:3v/v) as eluent and a flow set to 0.5 mL min−1.

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• an aliquot from each of the samples at 20 days was evapo-rated under vacuum and the residue analyzed by GC-MSand 1H NMR.

• an aliquot of the sample from CSV at 0, 5, 10, 15 and20 days was evaporated under vacuum and each residueanalyzed by 1H NMR.

Nitrogen inorganic compounds were investigated(NH4

+, NO2−, NO3

−) using the procedures reported byAPHA, 2005.[19]

Authentic samples of carboxin sulfoxide and sulfonewere prepared by an oxidation procedure[20] while enol wasprepared by hydrolysis.[21, 22] They were used as standardcompounds. Since sulfone is reported to undergo glass-catalyzed decomposition,[23] pyrex or plastic containerswere used in all experiments. Toxicity tests on the purecompounds carboxin, its sulfoxide and sulfone were previ-ously performed on T. platyurus obtaining EC50 values of61.0, 56.6 and 26.8 mg L−1, respectively, with confidencelimits of 55.3–67.3; 40.9–78.2; 17.9–40.2 mg L−1 for p =0.05. Enol was shown to be non-toxic.

Data analysis

The mixture of carboxin with the soils and the bacterial in-oculum was tested in three independent assays. The resultswere analyzed using the statistic software ToxcalcTM.[24]

The LC50s were evaluated by concentration/response re-gression using the trimmed Spearman-Karber method withconfidence limits for P = 0.05. The LC50 values were sub-sequently converted to toxic units (TUs) where TU = 100/LC50 to better calculate the toxicity reductions. Toxicityvalues were also compared using Tukey’s HSD multiplecomparison for p ≤ 0.05 to verify the significance of dataat different time exposures. Furthermore, results from dif-ferent experiments were compared to those of the initialtoxicity and reported as a percentage of toxicity reductionefficiency: %TRE = (1–TUday/TU start) × 100 where day =5, 10, 15 and 20 exposure days.

Results and discussion

Soils

The soil survey and classification showed that the featuresof the sterilized soils selected for the experiment fit in theEntisols order.[11, 12] Such pedotypes were chosen simplybecause they have little or no evidence of pedogenesisprogress,[11] i.e. the clear development and differentiationof pedogenic horizons. Indeed, the only features commonto all soils of the order are the virtual absence of diagnostichorizons and the mineral nature of the soils. This suffi-ciently guarantees the absence of peculiar processes linkedto intense differentiation of soil horizons, and producesbetter and more widely generalized results. On many land-scapes, the soil material is not in place long enough forpedogenic processes to form distinctive horizons; this typi-

Table 1. Selected physical and chemical parameters of the Entisolsutilized (samples from Ap horizon).

Soil

Parameter RAS CSV MND

pH 6.0 7.7 7.7Electrical conductivity dS m−1 0.153 0.925 0.338Organic carbon g kg−1 — 4.4 5.5Peat g kg−1 50 — —Total nitrogen g kg−1 <0.1 0.6 0.7Cation Exchange Capacity cmol[+] kg−1 2.9 25.1 8.6Carbonate contents as CaCO3 g kg−1 <1 144 42Sand g kg−1 750 123 521Silt g kg−1 0 622 387Clay g kg−1 200 255 91SSA∗ m2 g−1 (estimated) 4 100 35

∗SSA = Specific Surface Area [15]

cally occurs on flood plains, like the alluvial Low VolturnoBasin where the investigated soils developed, which receivesnew deposits of alluvium at relatively frequent intervals. Be-cause of the similar geo-pedoclimatic origin, both soils be-long to the mixed, calcareous, mesic classes of mineralogy,reaction and temperature, respectively. The soils also ex-hibit (Table 1) analogous sub-alkaline pH (7.7), due to thepresence of carbonates, and, according to the aim of the in-vestigation, a comparably poor organic matter content (4.4and 5.5 g kg−1 for CSV and MND, respectively). However,their texture is widely different, being finer for CSV, as silty-loam, and coarser for MND, as loamy-sand. Furthermore,the larger clay content of CSV vs. MND, 254.8 vs. 90.9 gkg−1, is mainly made up of non redox smectitic, swellingclay mineral, typically present in the studied area.[25] Con-sequently, at a subgroup taxonomic hierarchical level, CSVsoil is classified as Vertic Xerofluvent. The Xerofluvents aresoils on flood plains along rivers or streams or on alluvialfans, mostly in areas with Mediterranean climates.[11] Ad-ditionally, despite the relatively moderate content of clayfraction, CSV soil exhibits vertic properties due to theabundance of smectitic, swelling clay minerals which resultin cyclical soil swelling/shrinking with deep downwardscracking from the soil surface.[11] On the contrary, MNDsoil is a Typic Xeropsamment, i.e. the characteristic, coarse-textured Entisols on dunal zones of Mediterranean climates(USDA, 1999)[11]. The presence of smectitic, swelling clayminerals in CSV soil also accounts for the Cation ExchangeCapacity (CEC), which is noticeably high (25.1 cmol[+]kg−1) despite the poor content of organic matter and themoderate amount of the clay fraction itself. Conversely, theCEC of MND soil is as low as 8.6 cmol[+] kg−1, coherentwith the low content of both the organic carbon and clayfraction.

The Cation Exchange Capacity of the reference artificialsoil (RAS) was determined (2.9 cmol[+] kg−1).

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Abiotic and biotic degradation of carboxin 895

Table 2. Carboxin toxicity values at intervals of 5 days in TUs with confidence limits for P = 0.05.

SoilExposure Control (no soil; no P. aeruginosa(days) P. aeruginosa) RAS CSV MND inoculum

0 4.67 a (4.36–4.98) 4.06 a (3.79–4.34) 3.82 a (3.54–4.10) 3.86 a (3.61–4.13) 3.25 a (3.07–3.44)5 4.53 a (4.17–4.80) 3.75 ab (3.46–4.04) 3.55 a (3.32–3.80) 3.55 a (3.22–3.80) 2.72 b (2.60–2.83)

10 4.42 a (4.13–4.74) 3.40 bc (3.07–3.64) 2.28 b (1.95–2.34) 2.17 b (2.03–2.32) 2.12 c (1.95–2.22)15 4.32 a (4.20–4.43) 3.20 c (2.98–3.40) 1.60 c (1.43–1.83) 2.07 b (1.95–2.23) 1.71 d (1.50–1.88)20 4.30 a (4.17–4.50) 3.10 c (2.84–3.26) 1.50 c (1.32–1.63) 1.96 b (1.83–2.10) 1.28 e (1.12–1.36)

Mean followed by the same letter are not significantly different using Tukey’s HSD multiple comparison test for P ≤ 0.05.

The specific surface area (SSA) of samples RAS, MND,CSV has been estimated to 4, 35 and 100 m2g−1 (Table 1).

Toxicity

The results of the tested soils, the negative control (car-boxin in freshwater), the reference soil and carboxin solu-tion with bacteria inoculum, at different days of exposureare reported in Table 2 and expressed as Toxic Units. Com-paring toxicity data with Tukey’s HSD statistic method, nosignificant differences were observed for the negative con-trol. Differences were found for the reference soil startingfrom the fifteenth day. Both Entisols showed significant dif-ferences starting from the tenth day. The initial TUs of soilswere practically the same, but at the end of the experimentsthe Vertic Xerofluvent (CSV) showed the lowest TU val-ues, 1.50 vs. 1.96 of the Typic Xeropsamment (MND). TheTUs of the two soils were significantly different (P < 0.05),starting from the fifteenth day.

As reported in Figure 2, the reference soil showed a sig-nificant influence on toxicity reduction (24%). The Entisolsdemonstrated, after 10 days, that the toxicity of the super-natant was reduced up to 42% (CSV) and 44% (MND); af-terwards, the trend of reduction seemed to reach a plateaufor MND (46% and 49% after 15 and 20 days), whereasCSV reduction continued to increase up to 58% and 61%after 15 and 20 days, respectively.

The degradation of carboxin was particularly empha-sized by microbial flora. In fact, although in the absenceof nutrient broth (see above), P. aeruginosa concentrationmoves from 2·106 to 5·106 CFU/mL, indicating the exclu-sive use of carboxin as a nutrient, the only organic com-pound in the synthetic medium. It is to underline thatTU values reported at day 0 have to be considered thehighest toxicity of pesticide compatible with the micro-bial growth. In fact, in previous range finding tests it wasshown that higher concentrations of the pesticide were toxicto the microorganism. In the experimental plan, toxicity

0

25

50

75

0 5 10 15 20 25days

% r

edu

ctio

control RAS CSV MND P. aeruginosa

Fig. 2. Toxicity reduction of carboxin solution in the different experimental procedures after contact time reported.

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Table 3. Results in mgL−1 of compounds identified by 1H NMR and HPLC apparatus in biotic and abiotic degradation of carboxinafter 20 days contact time. Derivatives from negative control (carboxin solution at 280 mgL−1) or in co-presence of P. aeruginosa,were absent (abs) or less than LOD (Limit of Detection).

Compound Control RAS CSV MND P. aeruginosa inoculum

Carboxin 258.0 ± 9.8 83.0 ± 5.6 77.0 ± 8.0 70.0 ± 4.5 72.0 ± 6.7Sulfoxide abs 33.0 ± 2.5 2.7 ± 0.4 24.0 ± 1.5 <LODEnol abs 64.0 ± 4.0 11.0 ± 5.9 21.0 ± 1.5 <LODOther (inorganic) <LOD abs abs abs <LOD

0

250

500

750

1000

RAS CSV MND

sand

+ c

lay

cont

ent

sand + clay content TRE %

0

25

50

75

100

125

RAS CSV MND

CE

C

0

25

50

75

SS

A

CEC SSA TRE %

Fig. 3. Influence of cation exchange capacity (CEC in cmol[+] kg−1), specific surface area (SSA in m2 g−1) and sand + clay content(in g kg−1) of soils on toxicity reduction efficiency of carboxin.

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Abiotic and biotic degradation of carboxin 897

S

O

C

HN

CH3

O

S

O

C

HN

CH3

OOcarboxin (vitavax)

carboxin sulfoxide

anilineenol

hydrolysis

oxidation

H3C O H

SNHOH

O

NH2

NH+4 NO2

-

Fig. 4. Abiotic degradation products from carboxin by soils and P. aeruginosa without solar light.

reduction increased with the incubation time, from 16.3%(after 5 days) to 34.8, 47.4 and 60.6% at 10, 15 and 20 days,respectively. The results of toxicity reduction were signifi-cant for P = 0.05.

Degradation products

The test solutions were carefully examined by 1H NMRand HPLC and, those after 20 days also by GC-MS. Thelatter mixtures were separated by chromatographic meth-ods to isolate different compounds which were identifiedspectroscopically (1H NMR, MS) and by comparison ofthe 1H NMR spectra with those of specifically preparedauthentic samples.

No change in the chromatogram was observed in thecontrol solution (without soil and without inoculum). Inaccordance with previous reports,[26] carboxin was stablein this condition and very small amounts of unidentifiedproduct(s), if any, were detected after 20 days by 1H NMR.

In contrast, chemical analysis of samples from soils(RAS, CSV and MND) showed the presence, after 20 daysof the time test, of sulfoxide and enol together with carboxinwhile only carboxin was present in negative control (car-boxin in freshwater) and inorganic nitrogen compounds.

Both spectral and chromatographic analyses of the mix-tures from the treatment with Pseudomonas showed the

presence of carboxin and only after 15–20 days, of sulfox-ide and enol but at concentration levels less than LOD.Results from P. aeruginosa inoculum showed that carboxindecreased at 72 mg L−1 with a reduction of 64% from startand equivalent to 61% toxicity reduction of pesticide. Table3 reports the product distribution after 20 days.

The other sole product which was revealed in trace byGC-MS in all the mixtures was aniline. Sulfone was care-fully checked but was never detected, neither chromato-graphically (HPLC, TLC) nor spectroscopically (1H NMR,GC-MS).

It is noteworthy that sulfoxide present in RAS and MNDsamples represented about 20% of total organic compoundswhile the respective content in CSV samples was one orderof magnitude lower. Besides, enol concentration decreasedas RAS > MND > CSV.

Our results demonstrate that both the investigated Enti-sols were able to reduce the toxicity of carboxin. Further-more, CSV and MND exhibited the same toxicity reductionefficiency during the first 10 days experiment, with a sig-moidal trend which suggests an activation process (Fig. 2).CSV reached a significantly higher value of the plateaucompared to MND. The reduction of RAS showed noevidence of an activation process, and reached a final valueless than 50% and 40% when compared to CSV and MND,

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Fig. 5. Influence of cation exchange capacity (CEC in cmol[+] kg−1) and specific surface area (SSA in m2 g−1) of soils on degradationof carboxin to sulfoxide and enol.

respectively. Since both carboxin and its transformationproducts are not adsorbed to the soil because its adsorp-tion coefficient is very low,[18] the reduction efficiency ofsoils is probably due to their different characteristics in therespective content of coarser and/or finer mineral particles.RAS contains more sand than MND (750 vs. 521 g kg−1,respectively), but approximately the same amount of CSVclay (200 vs. 255 g kg−1, respectively) (Table 1). Figure 3shows the content of sand or clay of the investigated soilsagainst the respective TRE values. As it would be expected,the TRE decreases as the sand content increases, and theTRE is not linearly proportional to the clay content. The

former could be explained taking into account that thesurface activity of sand is known to be very poor, the lat-ter by considering that both Entisols contain smectites asclay mineral, whereas RAS contains kaolinite. This impliesthat the clay fraction of the investigated Entisols is charac-terized by similar surface properties, as Cation ExchangeCapacity (CEC) and Specific Surface Area (SSA), whichare dramatically lower than those of kaolin clay present inRAS.

With reference to CEC, kaolinite exhibits a Cation Ex-change Capacity typically ranging 1–10 cmol[+] kg−1,about ten times lower than that of smectites, ranging 80–120

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Abiotic and biotic degradation of carboxin 899

cmol[+] kg−1,[27] whereas the CEC of sand is negligible. In-deed, RAS had a CEC as low as 2.9 cmol[+] kg−1, vs. 8.6cmol[+] kg−1 for MND and 25.1 cmol[+] kg−1 for CSV. Inagreement with the amount and the nature of clay mineralspresent in the RAS and in the Entisols, the ratio CEC/kgclay results in 14.5, 85.6 and 95.6 for RAS, MND and CSV,respectively.

On this basis, we can calculate that the SSA of RAS,MND, and CSV approximately extends 4, 35, and 100 m2

per g soil (Table 1). Figure 3 also shows the values of CECand SSA of the investigated soils against the respectiveTRE values. It is clearly evident that both parameters arepositively correlated with TRE. This data clearly suggeststhat the toxicity reduction in soil is an abiotic process whichis exponentially controlled by the Specific Surface Area ofa given soil.

Previous experimental data demonstrated that carboxinis degraded when applied in the field with 95% reductionin 7 days, the formation of carboxin sulfoxide (about 50%of parental compound) and a low concentration of car-boxin sulfone[28] (Fig. 1). Nevertheless, in our experimentswith soils we found, generally starting from 10 days, justtraces of sulfoxide and enol, and after 20 days, aniline invery low concentrations. It is interesting to note that enolhas been obtained only by a strong acid treatment or, ina small amount, by irradiation on acid medium. How-ever, recently it has been indicated as the only hydrolyticproduct of carboxin. It is then evident that in the pres-ence of soils the carboxin, also in conditions of darkness,is abiotically oxidized, even if this process is more activein the presence of solar radiation. The reduced toxicityfound in our experiments cannot be attributed to the trans-formation of carboxin into these products (sulfoxide wasfound toxic by DellaGreca et al., 2004)[4] but rather to themineralization of the pesticide, probably via enol (Fig. 4).Such a hypothesis is strongly supported by the observeddecrease of both sulfoxide and enol percentage yields inthe investigated samples. In fact, their relative concentra-tions are inversely proportional to the CEC and SSA ofthe respective samples (Fig. 5). The chromatographic andspectral analyses of Pseudomonas suspension indicate theexclusive presence of carboxin. The absence of other prod-ucts suggests that the action of microorganisms on thepesticide results in the accumulation of ammonium thatis partly oxidized to nitrite without further oxidation tonitrate.

Conclusions

Our study demonstrates that the degradation of car-boxin into less toxic and available degradation productsin the soils occurs significantly also in the dark via abi-otic catalytic processes. Such phenomena are caused byexchange/surface features of soils, rather than by the merecontent of clay or organic matter fractions.

As a consequence, the availability of carboxin sulfoxide,the first by-oxidation product, is decreased. Our resultsshow that a similar consequence is also partially verifiedfor soils that are relatively poor in clay (MND soil), butthe lowering of carboxin sulfoxide is in that case due to thepresence of organo-mineral complexes which make theiractive surface available for abiotic catalytic degradation.

It is also demonstrated that kinetic degradation is fasterand more complete when P. aeruginosa is present. In thiscase, during the time test, the biotic degradation of carboxingives as a result, as foreseen, inorganic nitrogen products,such as ammonia and nitrite, but, as shown in Figure 2,with a final toxicity level lower than the soils.

Finally, our study suggests that carboxin is a quicklyand naturally degraded pesticide, thus safeguarding, evenwithout solar light, the environment with a significant roleof soil and its microorganisms in the detoxification.

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