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.     

Role of waste management with regard to climate protection: a case study.

According to the Kyoto Protocol and the burden-sharing agreement of the European Union, Austria is required to cut greenhouse gas (GHG) emissions during the years 2008 to 2012 in order to achieve an average reduction of 13%, based on the level of emissions for the year 1990. The present contribution gives an overview of the history of GHG emission regulation in Austria and identifies the progress made towards the realization of the national climate strategy to attain the GHG emission targets. The contribution uses Austria as an example of the way in which proper waste management can help to reduce GHG emissions. The GHG inventories show that everything must be done to minimize the carbon input due to waste deposition at landfill sites. The incineration of waste is particularly helpful in reducing GHG emissions. The waste-to-energy by incineration plants and recovery of energy yield an ecologically proper treatment of waste using state-of-the-art techniques of a very high standard. The potential for GHG reduction of conventional waste treatment technologies has been estimated by the authors. A growing number of waste incinerators and intensified co-incineration of waste in Austrian industry will both help to reduce national GHG emissions substantially. By increasing the number and capacity of plants for thermal treatment of waste the contribution of proper waste management to the national target for reduction of GHG emissions will be in the range of 8 to 14%. The GHG inventories also indicate that a potential CO2 reduction of about 500 000 t year(-1) is achievable by co-incineration of waste in Austrian industry.     

Heading for the hills: climate-driven community relocations in the Solomon Islands and Alaska provide insight for a 1.5 °C future

Whilst future air temperature thresholds have become the centrepiece of international climate negotiations, even the most ambitious target of 1.5 °C will result in significant sea-level rise and associated impacts on human populations globally. Of additional concern in Arctic regions is declining sea ice and warming permafrost which can increasingly expose coastal areas to erosion particularly through exposure to wave action due to storm activity. Regional variability over the past two decades provides insight into the coastal and human responses to anticipated future rates of sea-level rise under 1.5 °C scenarios. Exceeding 1.5 °C will generate sea-level rise scenarios beyond that currently experienced and substantially increase the proportion of the global population impacted. Despite these dire challenges, there has been limited analysis of how, where and why communities will relocate inland in response. Here, we present case studies of local responses to coastal erosion driven by sea-level rise and warming in remote indigenous communities of the Solomon Islands and Alaska, USA, respectively. In both the Solomon Islands and the USA, there is no national government agency that has the organisational and technical capacity and resources to facilitate a community-wide relocation. In the Solomon Islands, communities have been able to draw on flexible land tenure regimes to rapidly adapt to coastal erosion through relocations. These relocations have led to ad hoc fragmentation of communities into smaller hamlets. Government-supported relocation initiatives in both countries have been less successful in the short term due to limitations of land tenure, lacking relocation governance framework, financial support and complex planning processes. These experiences from the Solomon Islands and USA demonstrate the urgent need to create a relocation governance framework that protects people’s human rights.     

Tales of island tails: biogeomorphic development and management of barrier islands

The Frisian islands (Southern North Sea) have extensive island tails, i.e. the entire downdrift side of an island consisting of salt marshes, dunes, beaches and beach plains, and green beaches. Currently, large parts of these tails are ageing and losing dynamics, partly due to human influence. This may mean a loss of young stages on the long term, and current management is not enough to counteract this. To aid the development of new interventions aiming at (re)introducing natural dynamics, a conceptual model of island-tail development under natural and disturbed conditions was developed, based on existing data, field visits and literature. The development of an island tail follows the general pattern of biogeomorphic succession. The first phase consists of a bare beach plain. In the second phase, embryonic dunes form. In the third phase, green beaches, dunes and salt marshes form, including drainage by creeks and washovers. In the fourth phase, vegetation succession continues and the morphology stabilises. Human interference (such as sand dikes and embankments) reduces natural dynamics and increases succession speed, leading to a reduction in the diversity in landforms and vegetation types. Both for natural and human-influenced island tails, succession is the dominant process and large-scale rejuvenation only occurs spontaneously when large-scale processes cause erosion or sedimentation. Island tails cannot be kept permanently in a young successional stage by reintroducing natural dynamics through management interventions, as biogeomorphic succession is dominant. However, such interventions may result in local and temporal rejuvenation when tailored to the specific situation.     

BTEX exposures in an area impacted by industrial and mobile sources: Source attribution and impact of averaging time

The impacts of emissions plumes from major industrial sources on black carbon (BC) and BTEX (benzene, toluene, ethyl benzene, xylene isomers) exposures in communities located >10 km from the industrial source areas were identified with a combination of stationary measurements, source identification using positive matrix factorization (PMF), and dispersion modeling. The industrial emissions create multihour plume events of BC and BTEX at the measurement sites. PMF source apportionment, along with wind patterns, indicates that the observed pollutant plumes are the result of transport of industrial emissions under conditions of low boundary layer height. PMF indicates that industrial emissions contribute >50% of outdoor exposures of BC and BTEX species at the receptor sites. Dispersion modeling of BTEX emissions from known industrial sources predicts numerous overnight plumes and overall qualitative agreement with PMF analysis, but predicts industrial impacts at the measurement sites a factor of 10 lower than PMF. Nonetheless, exposures associated with pollutant plumes occur mostly at night, when residents are expected to be home but are perhaps unaware of the elevated exposure. Averaging data samples over long times typical of public health interventions (e.g., weekly or biweekly passive sampling) misapportions the exposure, reducing the impact of industrial plumes at the expense of traffic emissions, because the longer samples cannot resolve subdaily plumes. Suggestions are made for ways for future distributed pollutant mapping or intervention studies to incorporate high time resolution tools to better understand the potential impacts of industrial plumes.

Implications: Emissions from industrial or other stationary sources can dominate air toxics exposures in communities both near the source and in downwind areas in the form of multihour plume events. Common measurement strategies that use highly aggregated samples, such as weekly or biweekly averages, are insensitive to such plume events and can lead to significant under apportionment of exposures from these sources.     

Socio-economic Impacts on Flooding: A 4000-Year History of the Yellow River, China

We analyze 4000-year flood history of the lower Yellow River and the history of agricultural development in the middle river by investigating historical writings and quantitative time series data of environmental changes in the river basin. Flood dynamics are characterized by positive feedback loops, critical thresholds of natural processes, and abrupt transitions caused by socio-economic factors. Technological and organizational innovations were dominant driving forces of the flood history. The popularization of iron plows and embankment of the lower river in the 4th century BC initiated a positive feedback loop on levee breaches. The strength of the feedback loop was enhanced by farming of coarse-sediment producing areas, steep hillslope cultivation, and a new river management paradigm, and finally pushed the flood frequency to its climax in the seventeenth century. The co-evolution of river dynamics and Chinese society is remarkable, especially farming and soil erosion in the middle river, and central authority and river management in the lower river.     

Evaluation of simplified mass transfer models to simulate the impacts of source zone architecture on nonaqueous phase liquid dissolution in heterogeneous porous media

Nonaqueous phase liquid (NAPL) dissolution was studied in three-dimensional (3D) heterogeneous experimental aquifers (25.5 cm x 9 cm x 8.5 cm) with two different longitudinal correlation lengths (2.1 cm and 1.1 cm) and initial spill volumes (22.5 ml and 10.5 ml). Spatial and temporal distributions of NAPL during dissolution were measured using magnetic resonance imaging (MRI). At high NAPL spill volume, average effluent concentrations initially increased during dissolution, as NAPL pools transitioned to NAPL ganglia, and then decreased as the total NAPL-water interfacial area decreased over time. Experimental results were used to test six dissolution models: (i and ii) a one-dimensional (1D) model using either specific NAPL-water interfacial area values estimated from MR images at each time step (i.e., 1D quasi-steady state model), or an empirical mass transfer (Sh') correlation (i.e., 1D transient model), (iii and iv) a multiple analytical source superposition technique (MASST) using either the NAPL distribution determined from MR images at each time step (i.e., MASST steady state model), or the NAPL distribution determined from mass balance calculations (i.e., MASST transient model), (v) an equilibrium streamtube model, and (vi) a 3D grid-scale pool dissolution model (PDM) with a dispersive mass flux term. The 1D quasi-steady state model and 3D PDM captured effluent concentration values most closely, including some concentration fluctuations due to changes in the extent of flow reduction. The 1D transient, MASST steady state and transient, and streamtube models all showed a monotonic decrease in effluent concentration values over time, and the streamtube model was the most computationally efficient. Changes during dissolution of the effective NAPL-water interfacial area estimated from imaging data are similar to changes in effluent concentration values. The 1D steady state model incorporates estimates of the effective NAPL-water interfacial area directly at each time point; the 3D PDM does so indirectly through mass balance and a relative permeability function, which causes reduced water flow through high saturation NAPL regions. Hence, when model accuracy is required, the results indicate that a surrogate of this effective interfacial area is required. Approaches to include this surrogate in the MASST and streamtube models are recommended.     

Comparison of in vivo and in vitro methodologies for the assessment of arsenic bioavailability in contaminated soils

An in vivo swine assay was utilised for the determination of arsenic (As) bioavailability in contaminated soils. Arsenic bioavailability was assessed using pharmacokinetic analysis encompassing area under the blood plasma-As concentration time curve following zero correction and dose normalisation. In contaminated soil studies, As uptake into systemic circulation was compared to an arsenate oral dose and expressed as relative As bioavailability. Arsenic bioavailability ranged from 6.9+/-5.0% to 74.7+/-11.2% in 12 contaminated soils collected from former railway corridors, dip sites, mine sites and naturally elevated gossan soils. Arsenic bioavailability was generally low in the gossan soils and highest in the railway soils, ranging from 12.1+/-8.5% to 16.4+/-9.1% and 11.2+/-4.7% to 74.7+/-11.2%, respectively. Comparison of in vivo and in vitro (Simplified Bioaccessibility Extraction Test [SBET]) data from the 12 contaminated soils and bioavailability data collected from an As spiked soil study demonstrated that As bioavailability and As bioaccessibility were linearly correlated (in vivo As bioavailability (mgkg(-1))=14.19+0.93.SBET As bioaccessibility (mgkg(-1)); r(2)=0.92). The correlation between the two methods indicates that As bioavailability (in vivo) may be estimated using the less expensive, rapid in vitro chemical extraction method (SBET) to predict As exposure in human health risk assessment.     

A Thermal Desorption Procedure for Determining Residual Blowing Agent (CFC-11) in Rigid Foam

A method for measuring the cell gas content in rigid foams has been developed. A unique foam extraction device and a thermal desorption system were developed to expedite gas extraction and analysis. CFC-11 found by this method was in excellent agreement with the calculated value in a commercially formulated foam (7.1 versus 7.8 percent). Average method precision (i.e., relative standard deviation) from processing nine foam samples (in duplicate) was 1.9 percent. The method has also been successfully applied to foams containing non-GFC blowing agents.     

LC–ICP–MS analysis of arsenic compounds in dominant seaweeds from the Thermaikos Gulf (Northern Aegean Sea,Greece)

The content of total arsenic and arsenic compounds in the dominant seaweed species in the Thermaikos Gulf, Northern Aegean Sea was determined in samples collected in different seasons. Total arsenic was determined by acid digestion followed by ICP–MS. Arsenic speciation was analyzed by water extraction followed by LC–ICP–MS. Total arsenic concentrations in the seaweeds ranged from 1.39 to 55.0 mg kg⁻¹. Cystoseira species and Codium fragile showed the highest total As contents, while Ulva species (U. intestinalis, U. rigida,U. fasciata) had the lowest Arsenosugars, the most common arsenic species in seaweeds, were found in all samples, and glycerol-arsenosugar was the most common form; however, phosphate-arsenosugar and sulfate-arsenosugar were also present. Inorganic arsenic was measured in seven algae species and detected in another. Arsenate was the most abundant species in Cystoseira barbata (27.0 mg kg⁻¹). Arsenobetaine was measured in only one sample. Methylated arsenic species were measured at very low concentrations. The information should contribute to further understanding the presence of arsenic compounds in dominant seaweeds from the Thermaikos Gulf.