Environmental metabolites of fluoroquinolones: synthesis, fractionation and toxicological assessment of some biologically active metabolites of ciprofloxacin

Background, aim, and scope

Biowastes produced by humans and animals are routinely disposed of on land, and concern is now growing that such practices provide a pathway for fluoroquinolone (FQs) antibacterial agents and their environmental metabolites (FQEMs) to contaminate the terrestrial environment. The focus of concern is that FQs and FQEMs may accumulate in amended soils to then adversely impact on the terrestrial environment. One postulated impact is the development of a selective environment in which FQ-resistant bacteria may grow. To find evidence in support of an accumulation of antibacterial-like activity, it was first necessary to establish whether any biologically active FQEMs could be synthesized by physicochemical factors that are normally present in the environment. However, many FQEMs are not commercially available to be used as standards in such studies. FQEMs were therefore synthesized using well-defined processes. They were subsequently analyzed using spectroscopy (UV-vis) and high performance liquid chromatography with mass spectral detection. The antibacterial-like activities of fractionated FQEMs were then assessed in novel bacterial growth inhibition bioassays, and results were compared to those obtained from instrumental analyses.

Materials and Methods

Parent FQs were either exposed to sunlight or were synthesized using defined aerobic microbial (Mycobacterium gilvum or a mixed culture derived from an agricultural soil) fermentation processes. Mixtures of FQEMs derived from photo- and (intracellular) microbial processes were isolated by preparative chromatography and centrifugation techniques, respectively. Mixtures were subsequently fractionated using analytical high-performance thin layer chromatography (HPTLC), and excised analytes were tested in bioautography assays for their antibacterial-like activities. Two bacteria, Escherichia coli (E. coli) and Azospirillum brasilense (A. brasilense) were used as reporter organisms in testing FQ standards and any subtle differences between biologically active FQEMs of ciprofloxacin (CF).

Results and discussion

FQEMs produced in the photo-synthetic process had UV-vis profiles that were indistinguishable from the parent FQs, and yet mass spectral data revealed the presence of N-formylciprofloxacin (FCF). In contrast, the UV-vis profiles of FQEMs synthesized by M. gilvum and a mixed culture of microorganisms had UV-vis profiles that were similar to one another and markedly different to the parent fluoroquinolones. Mass spectral studies confirmed the presence of FCF and N-acetylciprofloxacin in both microbial ferments. In addition, a photo-FQEM (Cp 6), three M. gilvum FQEMs (Cm 5, Cm 8, and Cm 10) and a mixed culture FQEM (Cs 6) of CF and many other FQEMs of CF, norfloxacin (NF), and enrofloxacin (EF) were fractionated using HPTLC, although their identities have yet to be confirmed. Differences between bioautography results were obtained when E. coli or A. brasilense were used as reporter organisms. Parent FQs (CF and EF) and the FQEMs of CF (Cp 6, Cm 8, and Cs 6) displayed antibacterial-like activity when using E. coli as the reporter organism. In contrast, A. brasilense was insensitive to parent CF and sensitive to EF and all tested FQEMs of CF. Results are consistent with photo- and microbial processes modifying CF in different ways, with the latter changing the UV-vis chromophores. It can be inferred that a lack of detection of analytes (especially photo-FQEMs) when using UV-vis does not necessarily indicate an absence of analyte. Additionally, similarities between the UV-vis profiles of FQEMs extracted from the (monoculture) M. gilvum and the mixed culture microbial aerobic ferments are consistent with similar processes operating in both ferments. Results of HPTLC and bioautography studies revealed that mixtures of (photo- and microbial) FQEMs could be fractionated into individual components.

Conclusions

Bioactive FQEMs of ciprofloxacin, as a representative FQ, can be synthesized by photo- and microbial processes, and their detection required the use of both instrumental and bioautography analytical techniques. It is likely that such FQEMs will also be present on agricultural land that has been repeatedly amended with FQ-contaminated biosolids.

Recommendations and perspectives

The use of instrumental analytical techniques alone and especially photometric detection techniques will underestimate antibacterial-like activities of FQEMs. Moreover, the extraction technique(s) and the selected toxicological endpoint(s) require careful consideration when assessing bioactivity. It is therefore recommended that instrumental analytical techniques and several bioautography assays be performed concurrently, and bioautography assays should use a variety of reporter organisms. Two types of bacterial growth bioassays are recommended in any assessment of antibacterial-like activity derived from CF (and possibly from other FQs). A standardized E. coli bioassay should be used as a general screening procedure to facilitate intra- and inter-laboratory exchange of data. Additionally, soil-specific (region-specific) growth inhibition bioassays should be undertaken using several species of endemic soil bacteria. It is likely that the two sets of data will be useful in future risk assessment processes.     

Passive drainage and biofiltration of landfill gas: results of Australian field trial

A field scale trial was undertaken at a landfill site in Sydney, Australia (2004-2008), to investigate passive drainage and biofiltration of landfill gas as a means of managing landfill gas emissions from low to moderate gas generation landfill sites. The objective of the trial was to evaluate the effectiveness of a passive landfill gas drainage and biofiltration system at treating landfill gas under field conditions, and to identify and evaluate the factors that affect the behaviour and performance of the system.The trial results showed that passively aerated biofilters operating in a temperate climate can effectively oxidise methane in landfill gas, and demonstrated that maximum methane oxidation efficiencies greater than 90% and average oxidation efficiencies greater than 50% were achieved over the 4 years of operation. The trial results also showed that landfill gas loading was the primary factor that determined the behaviour and performance of the passively aerated biofilters. The landfill gas loading rate was found to control the diffusion of atmospheric oxygen into the biofilter media, limiting the microbial methane oxidation process. The temperature and moisture conditions within the biofilter were found to be affected by local climatic conditions and were also found to affect the behaviour and performance of the biofilter, but to a lesser degree than the landfill gas loading.     

Modelling of post-fragmentation waste stream processing within UK shredder facilities

With the introduction of producer responsibility legislation within the UK (i.e., waste electrical and electronic equipment directive and end-of-life vehicles directive), specific recycling and recovery targets have been imposed to improve the sustainability of end-of-life products. With the introduction of these targets, and the increased investment in post-fragmentation facilities, automated material separation technologies are playing an integral role within the UK's end-of-life waste management strategy. Post-fragmentation facilities utilise a range of purification technologies that target certain material attributes (e.g., density, magnetism, volume) to isolate materials from the shredded waste stream. High ferrous prices have historically meant that UK facilities have been primarily interested in recovering iron and steel, establishing processing routes that are very effective at removing these material types, but as a consequence are extremely rigid and inflexible. With the proliferation of more exotic materials within end-of-life products, combined with more stringent recycling targets, there is therefore a need to optimise the current waste reclamation processes to better realise effort-to-value returns. This paper provides a background as to the current post-fragmentation processing adopted within the UK, and describes the development of a post-fragmentation modelling approach, capable of simulating the value-added processing that a piece of automated separation equipment can have on a fragmented waste stream. These include the modelling of the inefficiencies of the technology, the effects of material entanglement on separation, determination of typical material sizing and an appreciation for compositional value. The implementation of this approach within a software decision-support system is described, before the limitations, calibration and further validation of the approach are discussed.     

Validation of a passive atmospheric deposition sampler for polybrominated diphenyl ethers

An atmospheric deposition sampler was validated with respect to polybrominated diphenyl ethers (PBDEs), a compound group that is widely used as flame retardants in many types of consumer products. The deposition sampler consists of an adsorption cartridge that is connected to a glass funnel. Extraction tests with spiked cartridges using soxhlet extraction with acetone revealed recoveries of >80% for all of the investigated PBDEs. Once adsorbed, PBDEs are stable, as proven by extractions of spiked cartridges that were stored outdoors and collected after different periods of time, up to 84 days. High recoveries indicate that degradation of adsorbed PBDEs does not play a role under realistic field conditions. Bulk deposition rates of PBDEs were determined in a field test with 3 replicates, and a possible breakthrough of target compounds was assessed in the field using a second adsorption cartridge in series. No breakthrough of target compounds could be observed within a sampling period of 61 days, and a bulk deposition rate of approximately 1 ng m(-2) day(-1) for the sum of all analysed PBDEs was measured. The highest deposition rates were measured for octa-brominated congeners, followed by BDEs 99, 183, 153, and 47. Overall, the sampler was successfully validated regarding the atmospheric deposition of PBDEs.     

Investigation of Building - Influenced Atmospheric Dispersion Using a Dual Source Technique

Dispersion of atmospheric contaminants in the vicinity of an isolated cubical model building was investigated in the field. A dual source/receptor technique was used in the experiments, which was proved to be very useful for the investigation of pollution dispersion. This experimental technique involved the simultaneous release of two different tracer gases from two different point sources, and the deployment of a FID (Flame Ionization Detector) co-located with a UVIC®(Ultra-Violet Ion Collector) detector. Both mean concentrations and concentration fluctuation statistics were examined. In this paper concentration fluctuation statistics are presented. The effect of the upwind source location on intermittency values and on the cumulative density function (cdf) is examined. The exact location of a source placed upwind of an obstacle has a very significant and complex effect on both mean concentrations and concentration fluctuations. As the lateral or vertical displacement between the two sources is increased, cross-correlation values between data taken simultaneously by two co-located detectors decrease rapidly.     

Remediation of Leaking USTs: A System for Accessing Case Histories and Related Documents

The U.S. Environmental Protection Agency's regulations require corrective action to be taken in response to a leaking underground storage tank (UST). Increasingly active state-run programs and the introduction of new cleanup technologies make UST corrective actions a multidisciplinary effort. The diversity of experience among personnel involved with this type of work has increased.

The EPA's Risk Reduction Engineering Laboratory has made its databases available to the public to help support this diverse audience. Its Computerized On-Line Information System (COLIS) allows for the quick and simple retrieval of data relating to UST incidents, as well as other hazardous-waste-related information. This paper describes the development and features of two important COLIS subsystems: the Case History File and the Library Search System. Sample search sessions are used to illustrate features of particular interest to personnel involved with UST sites.     

Distributing power? Community energy projects' experiences of planning,policy and incumbents in the devolved nations of Scotland and Wales

Community-owned energy projects are viewed as compelling contributors towards renewable energy targets. They contribute to curtailing the use of carbon intensive energy sources, consequently aiding mitigation of climate change, and can contribute towards a sustainable, localised economy. The success and expansion of the sector varies. However, there is a lack of knowledge regarding the development of the sector between sub-state nations. This qualitative, comparative study looks at the sector in the sub-state, devolved nations of Wales and Scotland within the UK. Through a series of in-depth interviews with community energy practitioners in four case study sites in Scotland and Wales, this study shows how policy and governance practices can influence the sector and those working at grassroots level. The study shows a disparity in confidence and outlook for the sector, based on the perceived (in)effective governance in each devolved nation.     

Land use change in Southern Romania after the collapse of socialism

The drastic socio-economic and political changes that occurred after the breakdown of socialism in Eastern Europe triggered widespread land use change, including cropland abandonment and forest cover changes. Yet the rates and spatial patterns of post-socialist land use change remain largely unclear. We used Landsat TM/ETM+ images to classify land cover maps and assess landscape pattern changes from 1990 to 2005 in Argeş County, Southern Romania. Cropland abandonment was the most widespread change (21.1% abandonment rate), likely due to declining returns from farming, tenure insecurity, and demographic developments during transition. Forest cover and forest fragmentation remained remarkably stable during transition, despite widespread ownership transfers. Cropland abandonment provides opportunities for increased carbon sequestration, but threatens cultural landscapes and biodiversity. Continued monitoring is important for assessing whether abandoned croplands will eventually reforest or be put back into production and to better understand the consequences of post-socialist land use change for ecosystems and biodiversity.     

Base cation depletion, eutrophication and acidification of species-rich grasslands in response to long-term simulated nitrogen deposition

Pollutant nitrogen deposition effects on soil and foliar element concentrations were investigated in acidic and limestone grasslands, located in one of the most nitrogen and acid rain polluted regions of the UK, using plots treated for 8-10years with 35-140kg Nha(-2)y(-1) as NH(4)NO(3). Historic data suggests both grasslands have acidified over the past 50years. Nitrogen deposition treatments caused the grassland soils to lose 23-35% of their total available bases (Ca, Mg, K, and Na) and they became acidified by 0.2-0.4 pH units. Aluminium, iron and manganese were mobilised and taken up by limestone grassland forbs and were translocated down the acid grassland soil. Mineral nitrogen availability increased in both grasslands and many species showed foliar N enrichment. This study provides the first definitive evidence that nitrogen deposition depletes base cations from grassland soils. The resulting acidification, metal mobilisation and eutrophication are implicated in driving floristic changes.     

Accounting for the Impact of Management Scenarios on Typha Domingensis (Cattail) in an Everglades Wetland

Environmental Management - The coupled regional simulation model, and the transport and reaction simulation engine were recently adapted to simulate ecology, specifically Typha domingensis...