Chemosphere 86 (2012) 212–215

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Chemosphere

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Short Communication

Biotic transformation of anticoccidials in soil using a lab-scale bio-reactor as aprecursor-tool

Martin Hansen a,⇑, Erland Björklund a, Kristine A. Krogh a, Asbjørn Brandt b, Bent Halling-Sørensen a

a Section of Toxicology and Environmental Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmarkb Section of Veterinary Medicines, The Danish Medicines Agency, Axel Heides Gade 1, DK-2300 Copenhagen, Denmark

a r t i c l e i n f o a b s t r a c t

Article history:Received 26 July 2011Received in revised form 30 September2011Accepted 2 October 2011Available online 1 November 2011

Keywords:SalinomycinRobenidineAerobicAnaerobicDegradationSoil bacteria

0045-6535/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.chemosphere.2011.10.001

⇑ Corresponding author. Tel.: +45 35336455; fax: +E-mail address: mah@farma.ku.dk (M. Hansen).

Two anticoccidial agents, salinomycin and robenidine, heavily used in the worldwide veterinary meatproduction, were investigated for their potential biotic degradation by cultured soil bacteria. The degra-dation-study was performed in lab-scale bio-reactors under aerobic and anaerobic conditions incubatedfor 200 h with a mixed culture of soil bacteria. Samples were analyzed by LC-MS/MS and potential trans-formation products were tentatively identified. Salinomycin was degraded under aerobic conditions andtraces could be found after 200 h, however, seems more persistent under anaerobic conditions. Fourtransformation products of salinomycin were discovered. Robenidine was degraded under aerobic andanaerobic conditions, however, traces of robenidine were observed after 200 h. Five biotic transformationproducts of robenidine were discovered.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction robenidine (Hansen et al., 2009c), and the environmental fate of

Anticoccidial pharmaceuticals or coccidiostats are intensivelyand legally used in meat production industry as prophylactic vet-erinary feed additives and as growth promoters (European FoodSafety Authority, 2009; US Food and Drug Administration, 2009).These drugs possess antimicrobial and antiparasitic potency ofwhich the primarily aim is to reduce outbreaks of the parasitic dis-ease coccidiosis. As stated in recent reviews anticoccidials are nowreported in several environmental compartments (Hansen et al.,2009a) and have also been identified as potential environmentalcontaminants that will pose an eco-toxicological risk (Hansenet al., 2009b). Despite their heavy application and possible eco-tox-icological risk, the knowledge on environmental fate and effects ofthese emerging contaminants is limited. In many countries it is notrequired to monitor the usage of feed additives, thus no completepicture of the consumption data is available. However, the usage ismonitored in Denmark, where total consumption of anticoccidialsreflects that ionophores are used in the highest amounts, salino-mycin being the most heavily applied (Statens Serum Institutet al., 2004). The more potent synthetic anticoccidials are con-sumed in smaller quantities, where robenidine is the most used(Statens Serum Institut et al., 2004). Previous research investigatedthe abiotic stability and antibacterial potency of salinomycin and

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anticoccidials are emerging as a focus area, nevertheless, onlyscarce biodegradation data is available for salinomycin (Vertesyet al., 1987; Schlusener and Bester, 2006; Schlusener et al., 2006;Ramaswamy et al., 2010), while no data is available for robenidine.Likewise another ionophore, monensin, has initially been investi-gated and the soil dissipation was found to strongly depend on soilorganic matter and water content with half-lives up to 23 d (Yos-hida et al., 2010).

The aim of this initiative was to describe a fast methodology todiscover environmental realistic transformation products usinglab-scale batch bio-reactors combined with LC-MS2, which latercan be applied to investigate the 23 approved anticoccidials world-wide (Hansen et al., 2009a). Salinomycin and robenidine were se-lected among the coccidiostatics to simulate biotransformationunder aerobic and anaerobic conditions, providing initial soil deg-radation profiles and preliminary information on transformationproducts. The obtained data will improve environmental riskassessment and observed transformation products can be eco-tox-icologically evaluated (Hansen et al., 2009b).

2. Materials and methods

2.1. Chemicals and materials

Salinomycin SV sodium (purity 94%), was purchased from Sig-ma–Aldrich, Germany and robenidine hydrochloride (purity 96%)

M. Hansen et al. / Chemosphere 86 (2012) 212–215 213

was obtained from Qmx Laboratories, Thaxted, Essex, United King-dom. Stock solutions of 1000 ppm were prepared in dimethylsulf-oxide. Nutrition media was prepared from 5 g sodium chloride, 3 gyeast extract, 1 g glucose and 5 g peptone in 1 L MilliQ water. Allchemicals were of analytical grade. Bio-reactors (1 L in volume)were obtained from Skandinavisk Glasblæseri, Copenhagen, Den-mark. Nitrogen was supplied by a generator and air was obtainedfrom a compressed air system.

2.2. Extraction of bacteria from soil

A well-described agricultural Danish soil (Jyndevad) was used(pH 6.9, content of calcium, magnesium, sodium and potassiumions were 5.19, 0.71, 0.09 and 0.19 cmol(+) kg�1, respectively,orthophosphate was 56.5 mg kg�1 soil, nitrogen and carbon con-tent was 0.12% and 2.43%, respectively, the distribution of clay, siltand sand was 5%, 1% and 94%, respectively). The soil has not beenexposed to antibiotics for the last 8 years. Separation of bacteriafrom soil was done using a Nycodenz density gradient as describedelsewhere (Lindahl and Bakken, 1995).

2.3. Batch bio-reactors

Six sterile bio-reactors were added 400 mL autoclaved (121 �Cfor 20 min) nutrition media. Three reactors were kept at aerobicconditions by constant bubbling of compressed air and three reac-tors were kept at anaerobic conditions by constant bubbling ofnitrogen. Room temperature was at all times kept at 19 ± 1 �C. Fur-thermore the reactors were shielded from light and incubated for3 d before spiking with 200 lL of the Nycodenz bacteria extractto all reactors and incubated another 24 h before spiking to10.0 ppm of salinomycin and robenidine in separate reactors andone matrix-matched blank (i.e., all reactors were spiked to 1%dimethylsulfoxide by volume) at both conditions. At the end ofthe study (200 h) the 400 mL media remaining in each of the sixbio-reactors was liquid–liquid extracted with dichloromethane(3 � 10 mL), and further evaporated (nitrogen) and reconstitutedin 500 lL mobile phase A.

Fig. 1. Degradation profiles of salinomycin (A) and robenidine (B) under aerobic and anaeproduct 1 (sTP1) from salinomycin. The chemical structures of salinomycin, sTP1 and ro

2.4. Chemical analysis

Bio-reactor samples of 1000 lL were centrifuged at 15,000 g for15 min and 200 lL supernatant was transferred to a HPLC vial andanalyzed within 1 h. Sampling was done at 0, 22, 68 and 200 h. Agi-lent 1100 series HPLC coupled to a Sciex API3000 ESI-MS/MS systemwas used with 5 lL injections (autosampler temperature controlledto 4 �C) onto a Phenomenex MAX-RP C12 2.0 � 150 mm, 4 lm col-umn with a 4.0 mm guard column kept at 40 �C. An isocratic300 lL min�1 flow of 10/90 or 40/60 mobile phase A/B was usedfor salinomycin and robenidine analysis, respectively. Mobile phaseA consisted of 95/5 water/acetonitrile (v/v) and 5 mM ammonia ace-tate, while mobile phase B consisted of 5/95 water/acetonitrile (v/v)also with 5 mM ammonia acetate. The MS was operated in positiveMRM mode monitoring 334.1 > 137.9 (40 V) and 336.1 > 139.1(40 V) ion transitions for robenidine, and 773.5 > 431.5 (75 V) and773.5 > 531.5 (50 V) for salinomycin. Other MS parameters; declus-tering, focusing, entrance and collision cell exit potential was set to20, 200, 10 and 15 V, respectively. Additionally, full scan spectrumsof each sample were acquired. Nitrogen was used as curtain, nebul-iser, auxiliary and collision cell gases with flow rates of 8, 6, 6 and4 L min�1, respectively. Source temperature and spray voltage was400 �C and 4500 V. A Valco valve was used as a diverter betweenthe HPLC and the MS, and was set to infuse into the MS from 5.0 to9.0 min. Retention times were 6.2 and 7.2 min for salinomycin androbenidine, respectively.

3. Results and discussion

3.1. Salinomycin

Degradation of salinomycin is substantial under aerobic condi-tions compared to anaerobic as seen in Fig. 1A. Already after 68 h ataerobic conditions no traces of salinomycin could be found. Theaerobic data complies with previous findings by Vértesy et al.,where salinomycin degraded in 20 h, even though the experimentswere done with enzymes extracted from a pure bacteria cultureand not by direct exposure to living bacteria (Vertesy et al.,

robic conditions. Dashed line with crosses displays the generation of transformationbenidine are shown to the right.

214 M. Hansen et al. / Chemosphere 86 (2012) 212–215

1987). The results indicate that uptake of salinomycin by soil bac-teria is the rate limiting process. The anaerobic experimentsshowed limited biodegradation of salinomycin during the 200 hthe experiment was conducted (Fig. 1A). Schlüsener found a lagtime of around 200 h in anaerobic pig manure experiments (Sch-lusener et al., 2006), while for soil experiments presented by thesame research group such a conclusion could not be drawn (Sch-lusener and Bester, 2006).

A search for possible transformation products in the various ex-tracts were performed by full scan LC-MS. A known transformationproduct (sTP1) previously found in manure after 192 d kept at

Fig. 2. Full scan MS chromatograms (m/z 250–800) of bio-reactor samples (400 mL) contstudy (200 h). The 400 mL samples were liquid–liquid extracted using 3 � 10 mL dichlshown on the same scale with an off-set value of 2 � 106 cps-units. Corresponding masssTP1, sTP3 and sTP4 are shown to the right.

Fig. 3. Full scan MS chromatograms (m/z 230–360) of bio-reactor samples (400 mL) constudy (200 h). The 400 mL samples were liquid–liquid extracted using 3 � 10 mL dichlshown on the same scale with an off-set value of 2 � 106 cps-units. Corresponding mass s5 are shown to the right.

anaerobic conditions (Schlusener et al., 2006) was here easily de-tected under aerobic conditions (Fig. 2). The mass spectra of sTP1is provided in Fig. 2 and the tentatively elucidated structure is de-picted in Fig. 1. This is in line with previous findings where sTP1was identified as the sole transformation product (Vertesy et al.,1987). Schlüsener et al. performed soil experiments (Schlusenerand Bester, 2006) and observed degradation of salinomycin, how-ever they could not identify any transformation products.

A new transformation products of salinomycin (sTP2) was ten-tatively identified for the first time found under anaerobic condi-tions and peaked in the 68 h samples, and was not detected in

aining salinomycin under aerobic (A) and anaerobic (B) conditions at the end of theoromethane followed by evaporation prior to analysis. Matrix-matched blanks are

spectra for salinomycin (from the B chromatogram), and transformation products;

taining robenidine under aerobic (A) and anaerobic (B) conditions at the end of theoromethane followed by evaporation prior to analysis. Matrix-matched blanks arepectra for robenidine (from the A chromatogram) and transformation products rTP1-

M. Hansen et al. / Chemosphere 86 (2012) 212–215 215

the 200 h samples (data not shown). With the applied technologyit was not possible to elucidate the structure of sTP2. Upon termi-nation of the experiments (200 h) the remaining 400 mL mediawas liquid–liquid extracted. Under both aerobic conditions(Fig. 2A) and anaerobic conditions (Fig. 2B), two new possibletransformation products sTP3 and sTP4 were observed, thoughtheir structures are still to be elucidated. Furthermore, in the200 h-extract from aerobic conditions many unresolved chromato-graphic peaks were detected with similar retention time as salino-mycin (Fig. 2A).

3.2. Robenidine

The biotic degradation of robenidine is here reported for the firsttime. Degradation of robenidine is rapid under aerobic and anaero-bic conditions as seen in Fig. 1B. Consequently, robenidine was al-most completely transformed after 200 h under both aerobic andanaerobic conditions, the latter though at a somewhat slower rate(Fig. 1B). In none of the experiments could a lag phase be observedmeaning that the cultured soil bacteria required no adaptation.Only the 400 mL liquid–liquid extracted media with robenidineshowed traces of transformation products (Fig. 3). Under aerobicconditions three transformation products (rTP1-3) were observed(Fig. 3A), where rTP1 contained one chlorine while rTP2 and rTP3contained two chlorine atoms (mass spectra presented in Fig. 3).Two new transformation products (rTP4 and rTP5) were found inthe anaerobic reactor besides rTP2 and rTP3 (Fig. 3B). An abiotictransformation product with similar mass spectra as rTP2 was ob-served in a previous study (Hansen et al., 2009c), where an oxida-tion was suggested as transformation process.

4. Conclusions

Usage of small scale bio-reactors gives fast access to prelimin-ary new information on potential transformation of anticoccidialsin the soil environment. Future work will concentrate on valida-tion, as the transformation products generated from in bio-reactorsneeds verification from soil samples exposed to robenidine andsalinomycin. In addition, ecotoxicological studies of these transfor-mation products and mixtures hereof are highly needed. Degrada-tion profiles of salinomycin and robenidine are here shown for thefirst time when exposed to a cultured and mixed soil bacteria com-munity. It was also shown that soil bacteria degrade salinomycin

into a previously observed transformation product (sTP1) using en-zyme extracted from pure culture bacteria or bacteria present inmanure. Three new transformation products (sTP2-4) of salinomy-cin were tentatively identified. Under aerobic conditions sTP3 wasobserved, while sTP2 and sTP4 were seen under anaerobic condi-tions. Five new transformation products of robenidine were ob-served from the degradation by soil bacteria, where rTP1-3 werefound under aerobic conditions and rTP2-5 under anaerobicconditions.

Acknowledgements

This research was financially supported by Centre for Environ-mental and Agricultural Microbiology (CREAM), funded by the Vil-lum Kann Rasmussen Foundation.

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