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Journal of Analytical and Applied Pyrolysis 110 (2014) 470–475

Contents lists available at ScienceDirect

Journal of Analytical and Applied Pyrolysis

journa l h om epage: ww w.elsev ier .com/ locate / jaap

hort communication

hermally assisted hydrolysis and methylation (THM) analysis: A newerspective for biochemical investigation of fatty acid composition innchytraeid tissues

abricio Augusto Hansela,∗, Cintia Carla Nivaa,b, Tamires Oliveira de Meloc,alimécio G. Guerrero Jr c, George Gardner Browna

Embrapa Florestas, Estrada da Ribeira, km 111, C.P. 319, Guaraituba, Colombo 88411 000, PR, BrazilUniversidade Positivo, Pós Graduac ão em Gestão Ambiental, Rua Prof. Pedro Viriato Parigot de Souza, 5300, Curitiba 81280 330, PR, BrazilDepartamento de Química e Biologia, Universidade Tecnológica Federal do Paraná, Curitiba 81280 340, PR, Brazil

r t i c l e i n f o

rticle history:eceived 15 May 2014ccepted 30 August 2014vailable online 8 September 2014

eywords:nchytraeidaeligochaeta

a b s t r a c t

Fatty acids are ubiquitous components in all organisms, and their applications in taxonomic and ecolog-ical studies of enchytraeids are scarce, and their small size (ca. 133 �g fresh weight) may be the mainreason. Thermally assisted hydrolysis and methylation (THM) reaction with pyrolysis–gas chromatogra-phy (Py–GC) allow determination of fatty acid composition for small size samples. Thus, our objectivewas to test this methodology with soil enchytraeids cultured in laboratory. We used THM on-line Py–GCusing trimethylsulfonium hydroxide (TMSH) reagent to investigate fatty acid composition in tissues oftwo enchytraeid species: Enchytraeus crypticus and Enchytraeus n. sp. cultured on soil and agar. A total

ipidshospholipidsatsass spectrometry

of 12 fatty acids were consistently identified, ranging from C10 to C16. The major fatty acids were C14:0

(myristic acid) and an unsaturated C14:1. Fatty acid distribution was dependent on species and culturingmethod, suggesting the need of standardization of the culturing substrate and diet in chemosystematicsstudies. THM using TMSH provided insight on the fatty acid composition of Enchytraeus tissues and maybe promising for application in taxonomic and ecological studies of this group of neglected soil animals.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Fatty acids are ubiquitous components in all organisms, and theyccur as constituents of epicuticular tissues and waxes (esters),ats/oils (triacylglicerols) and cell membranes (phospholipids),cting as energy stores, cell wall components, and performing pro-ective functions in some cases [1–4]. Since fatty acid compositionsre influenced by environmental condition and vary significantly

ith life stage, species, and diet, they are used in ecological

tudies involving trophic interaction, organic matter decomposi-ion, environmental quality and microbial symbioses, as well ashemosystematics [4–8]. One prominent application is the use of

∗ Corresponding author. Tel.: +55 41 3675 5792.E-mail addresses: fabricio.hansel@embrapa.br, f.a.hansel@hotmail.com

F.A. Hansel), cintiacn@gmail.com (C.C. Niva), melo.tamireso@gmail.comT.O.d. Melo), paligimenes1@gmail.com (P.G. Guerrero Jr),eorge.brown@embrapa.br (G.G. Brown).

ttp://dx.doi.org/10.1016/j.jaap.2014.08.019165-2370/© 2014 Elsevier B.V. All rights reserved.

iso anteiso C15 and C17 phospholipid fatty acids to characterizegrampositive bacteria populations in soil [9].

Only a few annelids have had their fatty acids studied thus far,including the earthworm Lumbricus terrestris [7,10–12], and threeenchytraeid species in the Enchytraeus genus (Enchytraeus albidus,Enchytraeus fragmentosus and Enchytraeus bigeminus) [13]. The pur-pose of these studies was to evaluate the fatty acid composition, thepresence of odd fatty acid components [10], obtain the distributionof fatty acids in the digestive tract, show microbial symbioses, andverify their usefulness in chemosystematics. However, the studyof fatty acid patterns in enchytraeids, a neglected group of soil ani-mals, has not been carried out since the work by Jacob et al. [13]. Thesmall size of enchytraeids may be the main problem in the inves-tigation (ca. 133 �g fresh weight), since conventional “wet” fattyacid analysis requires extensive time consuming sample handling,increasing the risk of contamination especially when small samplesizes are used [14].

Thermally assisted hydrolysis and methylation (THM) reactionsusing organic alkalis such as tetramethylammonium hydroxide(TMAH) and trimethylsulfonium hydroxide (TMSH) are a well-established derivatization process in fatty acid investigations

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15–18]. Nevertheless, TMSH is more effective, since TMAH causesppreciable isomerization/degradation of polyunsaturated fattycids. Its use has been applied in the investigation of a wide range ofynthetic and natural products [15,16,18–21]. Furthermore, TMSHan be used in combination with pyrolysis and gas chromatographyPy–GC) providing the following advantages for analysis of individ-al organisms: (i) THM occurs in situ, (ii) only small sample sizeequired, (iii) speed of analysis, (iv) no use of solvents, (v) smallolume of reagent, and (vi) reduced sample contamination [14,15].

Hence, in the following study, we investigated the use of THMsing a heated-filament pyrolyser to obtain the fatty acid composi-ion of enchytraeid tissues of cultured specimens. The usefulness,dvantages and disadvantages of this method to enchytraeids areerein discussed.

. Materials and methods

.1. Enchytraeids

Enchytraeus crypticus and Enchytraeus n. sp. were obtained fromingle strain cultures maintained in laboratory for more than 2ears in moist defaunated natural soil as substrate (22% sand,2.5% clay, 2.8% organic carbon and pH 5.2) at 22 ± 1 ◦C and fedeekly with oatmeal flakes ad libitum. E. crypticus is one of theost used species in soil ecotoxicological tests, while Enchytraeus

. sp. is a new species originally sampled from Piraquara City,razil (25◦23′2.03′′S, 49◦7′2.42′′W). The taxonomical description ofnchytraeus n. sp is in preparation as a separate paper. E. crypticusnd Enchytraeus n. sp. have a short life cycle of about 20 days, andeproduce by cocoon [22,23].

.2. Preparation of the enchytraeids

Five groups of 10 to 15 adult individuals (5–7 mm length for E.ypticus and 3–4 mm length for Enchytraeus n. sp.) were separatedrom the natural soil cultures and transferred to five culture boxesontaining 1% agar substrate and supplied weekly with oatmealakes (ca. 20 mg). After a period of 0, 7, 14 and 21 d, a group of fiveealthy adult individuals of each species were selected from the dif-

erent culture boxes and placed in petri dishes (Ø90 mm × 15 mmeight) containing deionized water (15 mL) for 24 h to remove

ntestinal contents (verified by stereomicroscope). Each enchy-raeid was inserted individually into a calcined pyrolysis quartzube (1000 ◦C, 4 h) filled with quartz wool at both ends, dried inn oven overnight (40 ◦C) and stored at −20 ◦C until required fornalysis.

.3. Thermally assisted hydrolysis and methylation (THM) withrimethylsulfonium hydroxide (TMSH)

Approximately 2 �L of TMSH (0.25 mol L−1 in methanol, Flukaigma-Aldrich Co. Ltd) was added to the quartz tube contain-ng each individual enchytraeid. The methanol was evaporatedefore insertion into the pyroprobe inlet. The on-line THM reac-ions were performed by fast pyrolysis to 350 ◦C and held for 10 ssing a pyropobe 5000 (CDS analytical, Oxford, USA), with theoil filament heater set at 10 ◦C ms−1. The additional pyroprobeonditions were: single step interface programme for transfer-ing volatiles were used from 40 ◦C to 280 ◦C (held for 60 s) at00 ◦C min−1, oven 290 ◦C, and transfer line 290 ◦C. The on-lineerivative fatty acid methyl ester (FAME) products were introducedn-line via pyroprobe transfer line into a split/splitless injector

t 290 ◦C using a splitless mode for 1 min. They were separatedsing a Focus GC gas chromatography and analysed in a Polaris

ion trap mass spectrometer (Thermo, Waltham, USA). The GCas equipped with a capillary column DB5-ms (60 m × 0.25 mm,

pplied Pyrolysis 110 (2014) 470–475 471

0.25 �m film thickness). The GC oven was programmed from 40◦

(held for 2 min) to 140 ◦C at 10 ◦C min−1 then from 140◦ to 290 ◦Cat 7 ◦C min−1 and held at 290 ◦C for 1 min. Helium, at a constantflow of 1.0 mL min−1, was the carrier gas. The GC–MS interface andion source temperatures were 290 ◦C and 200 ◦C, respectively. Theion trap mass spectrometer was operated in the positive electronionization mode at 70 eV scanning the range m/z 50–650 in a 0.58total scan time and emission current 250 mA.

2.4. Post analysis treatment

Individual fatty acid methyl esters were identified by mass spec-tra, and their structure confirmed using the molecular ion (M+.)and [M-32]+ for saturated and monoenoic/dienoic compounds,respectively. The superscript letters (a, b, c) in the monounsaturatedfatty acids through the text indicate different isomers. Integratedchromatograms were normalized and an individual fatty acidwas expressed as a relative amount (%) of the sum of all fattyacids detected (100%). Only fatty acids that occurred at >0.5% anddetected in all samples were reported and included in the statis-tical analyses. The results were reported using mean and standarddeviation of three samples, and the differences among fatty aciddistributions were evaluated by ANOVA at p < 0.05.

3. Results and discussion

A total of 12 fatty acids were consistently identified in thepyrograms, and accounted for 91–99% of total fatty acids detected(Table 1). The distribution of major fatty acids, ranging from C10 toC16, was characterized by a strong contribution of C14 fatty acids inboth species (Fig. 1 and Table 1). The major fatty acids were C14:0(myristic acid) and an unsaturated aC14:1 (Table 1), here identi-fied by elution order in non-polar column as 9-tetradecanoic acid(C14:1ω9) [24]. Other unsaturated C14 fatty acids (C14:2, bC14:1ω7,cC14:1ω5) were also identified by retention times and mass spec-tra [24], albeit in lower abundance (Fig. 1 and Table 1). In general,the presence/absence of fatty acids appeared to be constant afterculturing substrate change (soil to agar substrate), with only smalldifferences detected in fatty acids C12:1 and bC16:1, with the formerbeing absent in the initial stages of the experiment with E. crypticus(0 and 7 days), and the later in the Enchytraeus n. sp. tissues feedingon soil substrate (Table 1). This fatty acid range for enchytraeid tis-sues was similar to that observed by Jacob et al. [13] who also founddominance of C14 in the fatty acids of E. albidus, E. fragmentosus andE. bigeminus. However, differently from their observations, in ourdata no C18 fatty acids were detected in significant level (≥0.5%),and the relative abundance of C16 fatty acid was lower (Table 1).TMSH analysis is effective in the detection of the major classes ofacyl lipids present in soil mesofauna [14], and such differences maybe related to the Enchytraeus species, diet, and methodology, whichmay be less sensitive for detection of longer chain fatty acids. Inter-estingly, the remarkably high percentage of C14 fatty acid presentin enchytraeid tissues was not observed in L. terrestris in which thefatty acid C18:1ω7 was the most abundant compound in the bodyand gut walls [7,11].

Fatty acids are constituents of cell membranes and may beaffected by the diet and environmental conditions. So, consideringthat natural soil is richer in minerals, nutrients and microorgan-isms and thus a more complex substrate than agar, we expectedthe two enchytraeid species to show differences in their lipid pro-files when reared on soil versus agar, even though oatmeal was

offered as food source in both culturing methods. In fact, a distinctbehaviour was observed in the fatty acid distribution of the twospecies used in the present study. The mean values (%peak area)for monounsaturated and saturated C14 fatty acids of E. crypticus

472 F.A. Hansel et al. / Journal of Analytical and Applied Pyrolysis 110 (2014) 470–475

Table 1Relative fatty acid distribution (%) of E. crypticus and Enchytraeus n. sp. reared on natural soil or after 7 to 21 days of change to agar substrate. Fatty acid values were obtainedby TMSH thermal assisted hydrolysis and methylation using Py–GC–MS.

Fattyacids#

Soil 7 days in agar 14 days in agar 21 days in agar

E. crypticus E. n. sp. E. crypticus E. n. sp. E. crypticus E. n. sp. E. crypticus E. n. sp.

C10:0 1.1 ± 0.3 2.5 ± 3.1 1.8 ± 0.2 4.4 ± 1.3 3.7 ± 0.5 7.2 ± 0.2 3.3 ± 0.6 3.0 ± 1.4C12:1 – – – – 1.5 ± 0.5 – 2.0 ±0.1 –C12:0 7.6 ± 0.9 6.6 ± 4.6 9.7 ± 1.5 8.8 ± 1.3 6.7 ± 1.3 9.9 ± 0.4 10.4 ± 1.9 12.9 ± 3.9C13:0 – 0.5 ± 0.2 0.5 ± 0.1 0.7 ± 0.1 – 0.9 ± 0.3 0.5 ± 0.1 1.5 ± 0.4C14:2 8.0 ± 2.8 0.7 ± 0.5 4.1 ± 1.7 1.1 ± 0.1 2.6 ± 0.7 1.1 ± 0.3 3.8 ± 0.5 –aC14:1 30.3 ± 2.3 24.7 ± 9.9 30. 6 ± 3.1 35.1 ± 0.7 25.6 ± 1.0 35.8 ± 1.7 25.9 ± 3.1 24.7 ± 2.9bC14:1 2.7 ± 0.5 5.5 ± 1.5 3.6 ± 0.6 5.5 ± 1.1 2.6 ± 0.2 5.5 ± 0.4 2.3 ± 0.7 5.5 ± 0.3cC14:1 10.6 ± 2.0 2.4 ± 0.5 9.5 ± 1.3 6.1 ± 0.4 9.9 ± 1.6 4.0 ± 0.4 11.6 ± 1.5 5.0 ± 0.1C14:0 35.8 ± 6.9 47.4 ± 0.6 35.2 ± 5.7 36.2 ± 2.3 37.8 ± 1.8 30.9 ± 2.0 33.0 ± 2.9 41.2 ± 3.5aC16:1 – 6.5 ± 3.9 – 0.6 ± 0.1 – 1.1 ± 0.3 – 2.2 ± 2.1bC16:1 1.3 ± 0.8 – 2.6 ± 1.6 0.9 ± 0.4 0.6 ± 0.2 0.7 ± 0.1 1.16 ± 0.2 2.0 ± 1.9C – 2.6 ± 1.1 0.8 ± 0.7 – – – – 0.9 ± 1,2

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# Cx refers to number of carbon atoms in the fatty acid chain, and numbers aonounsaturated fatty acids indicate different isomers.

ere not significantly different after the change in the culturingubstrate (p > 0.05) (data not shown), and their relative distribu-ion did not vary considerably during the 21 d (Fig. 2A). On the otherand, the mean values (% peak area) of aC14:1 and C14:0 fatty acids fornchytraeus n. sp. were significantly different (p < 0.001) after 14 d

f feeding on agar substrate (data not shown). These differences,epicted in Fig. 2B, show a clear shift in the relative distribu-ion in the aC14:1 and C14:0 fatty acids after 14 d. At the beginningf the experiment (soil substrate—day 0) the major fatty acid in

ig. 1. Partial total ion current (TIC) pyrograms showing the major fatty acids in E. cryptictoms in the fatty acid chain, and numbers after colons refer to number of double bondetters (a, b, c) in the monounsaturated fatty acids indicate different isomers.

he colon refer to the number of double bonds. Superscript letters (a, b, c) in the

Enchytraeus n. sp. tissues was C14:0;. After 7 d (agar diet) the rela-tive distribution of C14:0 fatty acid decreased and similar abundanceto the aC14:1 fatty acid was detected, while after 14 d the aC14:1 fattyacid component was dominant (Fig. 2B). After 21 d, no-significantdifferences were detected in the aC14:1 fatty acid (p > 0.05) com-

pared to day 0, and a return to the initial relative distribution wasobserved, being the major component C14:0 fatty acid (Fig. 2B). Thisindicates that Enchytraeus n. sp. was more sensitive than E. crypti-cus to substrate change from soil to agar. Unfortunately, no results

us (A) and Enchytraeus n. sp. (B) tissues. Cx above peaks refers to number of carbons.? and * Denote unidentified peaks and contamination, respectively. Superscript

F.A. Hansel et al. / Journal of Analytical and Applied Pyrolysis 110 (2014) 470–475 473

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ig. 2. Normalized relative abundance of C14 monounsaturated and saturated fatuperscript letters (a, b, c) in the monounsaturated fatty acids indicate different isom

ave been published using fatty acid composition of enchytraeidissues to study trophic interactions, but changes in the fatty acidomposition due to diet effects and trophic interaction have beeneported for other invertebrates such as collembolans and L. ter-estris [6,25,26]. Therefore, the present methodology opens a newvenue for application of the fatty acid distribution in feeding ecol-gy, and ecotoxicological studies [27], and to future studies on these of the present methodology in detection of differences in theattern of fatty acids in enchytraeids exposed to contaminants.

Based on differences in fatty acid profiles of three Enchytraeuspecies, Jacob et al. [13] proposed the use of fatty acids in enchy-raeid chemosystematics. However, in the present investigation,nitially the fatty acid distributions of the two Enchytraeus specieshowed no statistically significant differences for the monoun-aturated fatty acids (0 day, Fig. 3A) and significant differencesere only detected in saturated C14 fatty acid (p < 0.05, Fig. 2A).

he standard deviation (�) plots, produced to check the precisionf fatty acid distribution during the experiment (Fig. 3C and D)howed fluctuations, particularly for the major fatty acids aC14:1nd C14:0, that had higher values in the soil substrate for bothpecies. After 14 d in agar substrate a better overall precision

or all C14 fatty acids was observed (Fig. 3C and D). A new plotor C14 monounsaturated and saturated fatty acids distributionas produced with data on enchytraeids after 14 d in agar sub-

trate (Fig. 3B). The histogram showed significant differences in the

ds along the 21 day experiment using E. crypticus (A) and Enchytraeus n. sp. (B).

distribution of 3 fatty acids (p < 0.001, Fig. 3B). Apparently, theless complex culturing substrate (agar) stabilized the enchytraeidfatty acid distributions yielding more precise measures, and signifi-cant differences in fatty acid distribution between the species weredetected. In practical terms, these results suggest that the presentanalytical method might not be recommendable for taxonomy ofenchytraeids coming directly from the field without a pretreatmentand, that standardized culture media such as agar are needed tobe able to perform adequate comparison in fatty acid profiles forchemosystematic studies of enchytraeid species.

The taxonomic identification of enchytraeids demands micro-scopical observation of some morphological traits of livespecimens, which is a difficult task for most people and, in somecases, morphological analysis is inconclusive to separate similargroups. Several techniques have been used to assess moleculardiversity and aid traditional taxonomy and phylogenetic studies,such as isozyme banding patterns [28,29], RAPD-PCR fingerprints[30] and DNA barcoding [31,32]. Using the fatty acid distribution weare able to distinguish both species, suggesting the method mightbe used in chemosystematic studies in combination with othertechniques for characterization and identification of Enchytraeus

populations. However, a special attention must be given particu-larly to the potential effects of diet and culturing substrate (e.g.natural soil vs. agar). Obviously, the effectiveness of this method-ology for taxonomical studies needs to be certified using a larger

474 F.A. Hansel et al. / Journal of Analytical and Applied Pyrolysis 110 (2014) 470–475

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ig. 3. C14 monounsaturated and saturated fatty acids comparative distribution inxperiment for E. crypticus (C) and Enchytraeus n. sp. (D). Significant levels in theonounsaturated fatty acids indicate different isomers.

umber of species, but doubtless this approach seems to be annteresting analytical tool for chemosystematics.

. Conclusions

THM using TMSH proved to be an interesting approach tonvestigate fatty acid distribution in Enchytraeus tissues. The Fattycid distribution was dependent on species and culturing method;or example Enchytraeus n. sp. was more sensitive to substratehange (soil to agar). Also a mixture of soil in diet, a more com-lex substrate, a lower precision in the determination of the majoratty acids was detected. In case of chemosystematic studies, theatty acid distributions may be used in combination with otherechniques for characterization and identification of Enchytraeusopulations, but their diet and rearing substrate must be standard-

zed. It is important to note that the proposed protocol is not auantitative method, so only the relative abundance of the fattycids is given, and apparently some limitations were encounteredn the detection of C16 and C18 fatty acids.

cknowledgments

Financial support for this study came from projects funded bymbrapa/Ministério da Agricultura and CNPq. Fellowships to CCNPNPD) was provided by CNPq and CAPES and to GGB, by CNPq.

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