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Estimation of the cancer risk to humans resulting from the presence of cyclophosphamide and ifosfamide in surface water Full text
2010
Kümmerer, Klaus | Al-Ahmad, Ali
Background, aim, and scope Anti-tumour agents and their metabolites are largely excreted into effluent, along with other pharmaceuticals. In the past, investigations have focused on the input and analysis of pharmaceuticals in surface and ground water. The two oxazaphosphorine compounds, cyclophosphamide and ifosfamide are important cytostatic drugs used in the chemotherapy of cancer and in the treatment of autoimmune diseases. Their mechanism of action, involving metabolic activation and unspecific alkylation of nucleophilic compounds, accounts for genotoxic and carcinogenic effects described in the literature and is reason for environmental concern. The anti-tumour agents cyclophosphamide (CP) and ifosfamide (IF) were not biodegraded in biodegradation tests. They were not eliminated in municipal sewage treatment plants. Degradation by photochemically formed HO radicals may be of some relevance only in shallow, clear, and nitrate-rich water bodies but could be further exploited for elimination of these compounds by advanced oxidation processes, i.e. in a treatment of hospital waste water. Therefore, CP and IF are assumed to persist in the aquatic environment and to enter drinking water via surface water. The risk to humans from input of CP and IF into surface water is not known. Materials and methods The local and regional, i.e. nationwide predicted environmental concentration (PEClocal, PECregional) of CP and IF was calculated for German surface water. Both compounds were measured in hospital effluents, and in the influent and effluent of a municipal treatment plant. Additionally, published concentrations in the effluent of sewage treatment plants and surface water were used for risk assessment. Excretion rates were taken into account. For a worst-case scenario, maximum possible ingestion of CP or IF by drinking 2 L a day of unprocessed surface water over a life span of 70 years was calculated for adults. Elimination in drinking water processing was neglected, as no data is available. This intake was compared with intake during anti-cancer treatment. Results and discussion Intake of CP and IF for anti-cancer treatment is typically 10 g within a few months. Under such conditions, a relative risk of 1.5 for the carcinogenic compounds CP and IF is reported in the literature. In the worst case, the maximum possible intake by drinking water is less than 10⁻³ (IF) and 10⁻⁵ (CP) of this amount, based on highest measured local concentrations. On a nationwide average, the factor is approx. 10⁻⁶ or less. Conclusions The additional intake of CP and IF due to their emission into surface water and its use without further treatment as drinking water is low compared to intake within a therapy. This approach has shortcomings. It illustrates the current lack of methodology and knowledge for the specific risk assessment of carcinogenic pharmaceuticals in the aquatic environment. IF and CP are directly reacting with the DNA. Therefore, with respect to health effects a safe threshold concentration for these compounds cannot be given. The resulting risk is higher for newborns and children than for adults. Due to the lack of data the risk for newborns and children cannot be assessed fully. The data presented here show that according to present knowledge the additional risk of cancer cannot be fully excluded, especially with respect to children. Due to the shortage of data for effects of CP and IF in low doses during a whole lifespan, possible effects were assessed using data of high doses of CP and IF within short-term ingestion, i.e. therapy. This remains an unresolved issue. Anyway, the risk assessment performed here could give a rough measure of the risks on the one hand and the methodological shortcomings on the other hand which are connected to the assessment of the input of genotoxic and carcinogenic pharmaceuticals such as CP and IF into the aquatic environment. Therefore, we recommend to take measures to reduce the input of CP and IF and other carcinogenic pharmaceuticals. We hope that our manuscript further stimulates the discussion about the human risk assessment for carcinogenic pharmaceuticals in the aquatic environment. Recommendations and perspectives CP and IF are carcinogens. With respect to newborn and children, reduction of the emission of CP and IF into effluent and surface water is recommended at least as a precautionary measure. The collection of unused and outdated drugs is a suitable measure. Collection of patients' excreta as a measure of input reduction is not recommended. Data suitable for the assessment of the risk for newborn and children should be collected in order to perform a risk assessment for these groups. This can stimulate discussion and give new insights into risk assessment for pharmaceuticals in the environment. Our study showed that in the long term, effective risk management for the reduction of the input of CP and IF are recommendable.
Show more [+] Less [-]Winter accumulation of acidic pharmaceuticals in a Swedish river Full text
2010
Daneshvar, Atlasi | Svanfelt, Jesper | Kronberg, Leif | Weyhenmeyer, Gesa A.
Purpose In this study, seasonal variations in the concentration profile of four analgesics and one lipid regulator were monitored on their way from a wastewater treatment plant (WWTP) effluent, along a river, and into a lake. Methods From December 2007 to December 2008, water samples were collected monthly (n = 12) from an upstream point, the effluent, four downstream points of the WWTP, and at the point where the river merges with the lake, and the concentrations of ibuprofen, naproxen, bezafibrate, diclofenac, and ketoprofen were determined. The analytical methodology involved solid-phase extraction of the target compounds from water samples followed by liquid chromatography coupled with tandem mass spectrometry for compound separation and detection. Results The studied pharmaceuticals were found in the effluent at concentrations ranging from 31 to 1,852 ng l⁻¹ depending on the season. In the river and lake, the concentrations were much lower (6-400 ng l⁻¹) mainly due to dilution but also to a season-dependent contribution from natural transformation processes. The mean mass flow of all analgesics was highest during winter while the highest mean mass flow of the lipid regulator bezafibrate was observed in spring. Conclusions The WWTP is the main source of the target compounds in the aquatic environment. The observed winter accumulation signifies the importance of natural transformation processes, which can only be estimated based on mass flow data, on the fate of pharmaceuticals in the environment.
Show more [+] Less [-]Partitioning of endocrine disrupting compounds in inland waters and wastewaters discharged into the coastal area of Thessaloniki, Northern Greece Full text
2010
Arditsoglou, Anastasia | Voutsa, Dimitra
Background, aim, and scope In the Water Framework Directive 2000/60/EC, environmental objectives for the proper quality of inland, surface, transitional, coastal, and ground waters have been set. Member states are required to identify chemical pollutants of significance in the water bodies, to establish emission control measures, and to achieve quality standards. A specific category of pollutants are the compounds that may possess endocrine-related functions known as endocrine disrupting compounds (EDCs). This means that member states have the obligation to take action in order to prevent human exposure to these compounds via aquatic environment. The objective of this research was to study the occurrence and distribution of phenolic and steroid EDCs in inland waters and wastewaters discharged in the area of Thermaikos Gulf, Thessaloniki, Northern Greece. Materials and methods Samples were collected from three rivers, four streams, and four municipal and industrial wastewaters from the area of Thessaloniki, Northern Greece, during the period 2005-2006. The samples were analyzed for 14 EDCs (nonylphenol, octylphenol, their mono- and di-ethoxylate oligomers, bisphenol A, estrone, 17α-estradiol, 17β-estradiol, estriol, mestranol, and 17α-ethynylestradiol). The compounds were recovered by solid phase extraction and ultrasonic extraction from the dissolved phase and particulate phase, respectively, and determined by employing gas chromatography-mass spectrometry. Results Results revealed the presence of phenolic EDCs (NP, NP1EO, NP2EO, tOP, OP1EO, OP2EO, and BPA) in all water and wastewater samples. Steroid EDCs were not found at detectable concentrations. The relationships between field partition coefficients of EDCs and concentration of total suspended solids, dissolved, and particulate organic carbon were investigated. Discussion Rivers exhibited concentrations of EDCs similar to minimally impacted surface waters worldwide. The concentrations of NP and OP occasionally exceeded the environmental quality criteria proposed for inland waters. The concentrations of EDCs in streams exhibited wide variations due to low flow rate in these systems and the impact of wastewaters from various pollution sources. Wastewater from tannery activities showed extremely high concentrations of NP, whereas relatively high concentrations of EDCs were determined in effluents from the industrial wastewater treatment plant. Field partition coefficients of EDCs are negatively correlated with concentrations of total suspended solids and dissolved organic carbon and positively correlated with particulate organic carbon. Conclusions The examined rivers (Aliakmon, Axios, and Loudias) exhibited concentrations of EDCs similar to minimally impacted surface waters worldwide. However, special attention should be paid to these systems since the concentrations for NP and OP occasionally were above the proposed quality standards, revealing the impact of urban, industrial, and agricultural activities. High concentrations of EDCs were determined in streams, urban, and industrial wastewater posing significant risk to the aquatic environment they discharged. Recommendations and perspectives The occurrence of EDCs in inland waters and wastewaters discharged to Thermaikos Gulf results in an increased risk to the marine environment. Thus, these systems should be regularly monitored, especially for NP, OP, and BPA that are considered as priority hazardous compounds in the Water Framework Directive.
Show more [+] Less [-]Ice phase as an important factor on the seasonal variation of polycyclic aromatic hydrocarbons in the Tumen River, Northeastern of China Full text
2010
Cong, Linlin | Fang, Yingyu | He, Miao | Wang, Xinshun | Kannan, Narayanan | Li, Donghao
Background, aim and scope The climatic characteristic is a major parameter affecting on the distribution variation of organic pollutants such as polycyclic aromatic hydrocarbons (PAHs). The Tumen River is located in Northeastern of China. The winter era lasts for more than 5 months in a year, and the river water was frozen and covered by ice phase. Coal combustion is an essential heating source in the Tumen River Basin. The objective of this research is to study ice phase effect on the seasonal variation of PAHs in the Tumen River environment. Materials and methods Samples were collected from 13 sites along the River in March, July, October, and December of 2008. In addition, the ice sample, under ice water and air particulate were also collected in winter. The samples were analyzed for 16 PAHs (naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, beazo[a]anthene, chrysene, beazo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indeno(1,2,3-cd)pyrene, dibenz(a,h)anthracene, and benzo(ghi)perylene). The compounds were extracted from the water samples and solid samples using LLE and Soxhlet extraction technique, respectively, and it is determined by gas chromatography-mass spectrometry. Results and discussion Among 16 PAHs, fluorene, phenanthrene, and pyrene were found to be present in high concentrations and at high detection frequencies. The total concentration of PAHs in the water, particulate, sediment and ice phase ranged from 35.1-1.05 × 10³ ng L⁻¹, 25.4-817 ng L⁻¹, 117-562 ng g⁻¹and 62.8-136 ng g⁻¹, respectively. The levels of PAHs were generally higher in spring than other seasons. The ice phase in winter acts like a major reservoir of the pollutants and it is major contributor on the seasonal variation of PAHs in Tumen River. The PAHs found in water, particulate, and sediment in the Tumen River were possibly derived from similar pollution sources a proposition based on the compositions and isomer ratios of PAHs. Conclusions The distribution of PAHs was showed clear seasonal variation in the Tumen River environment, the ice phase and air pollution look like an important factor affecting on the seasonal variation. Recommendations and perspectives The ice phase as an important factor affecting on the seasonal variation of PAHs in Tumen River environment. Further studies regarding the effects of air pollution on the river and the mechanisms of migration and transformation of them in the environment are currently being conducted in our laboratory.
Show more [+] Less [-]Source identification of sulphate forming salts on sandstones from monuments in Salamanca, Spain—a stable isotope approach Full text
2010
Schleicher, Nina | Recio Hernández, Clemente
Background, aim and scope Salt efflorescences markedly contribute to the alteration and deterioration of building material, in this case the Villamayor Sandstone of the facades in the Old Town of Salamanca, Spain (United Nations Educational, Scientific and Cultural Organization world cultural heritage site). A better understanding of the mechanisms of salt formation and the involved elements would allow more precise measures in monument conservation. The magnesium which is required for the salt precipitation originates from selective processes of hydrolysis. The source of sulphate, however, is presently not as clear. Identifying the source of the sulphur was the main goal of this research. Isotope ratio measurement of δ³⁴S and δ¹⁸O was used to clarify the origins of Mg sulphate salts. Materials and methods A total of 56 Mg sulphate samples were collected in two different seasons (July and November 2005) from monuments of the Old Town of Salamanca. These sampled salt efflorescences were analysed for δ³⁴S and δ¹⁸O by mass spectrometry. A ‘dual-inlet' type by VG Isotech was used for δ³⁴S and continuous flow type Isoprime by GV Instruments for δ¹⁸O. Samples were measured in triplicates and standard material was analysed for quality control. Results δ³⁴S values range between 3.6‰ and 15.4‰ with a median value of 10.2‰ for the July samples and of 10.1‰ for November samples. The results of the sulphur ratios hint towards a bimodal distribution (with modes at δ³⁴S = 6‰ and 12‰) for winter samples, which is less obvious during summer. δ¹⁸O values range from 7.1‰ to 41.1‰. However, most values range from 7.1‰ to 20.8‰, whereas only few summer samples show outliers towards higher δ¹⁸O values. The median δ¹⁸O value for July samples is 15.5‰ and for November samples 14.6‰. Discussion The isotopic ratios of the analysed sulphate samples were compared with values of possible source materials. Sulphur sources in the case of Salamanca are barites from the Villamayor Sandstone itself, sea spray, sulphides from regional rocks, biogenic sulphur (soil, avian excreta), as well as sulphur from anthropogenic sources such as building materials (especially mortar) or traffic exhaust. Salamanca is a representative site for non-industrial cities with no heavy industry and thus, there are no significant SO₂ emissions from industry. Conclusions Based on the measured isotopic ratios, it was ascertained that more than one sole sulphur source is present. However, based on additional information about the source material and possible transport ways, some sources could be excluded whereas others only played a minor role. Finally, there is strong indication that the main sulphur source is atmospheric pollution and the exhaust emissions from vehicles in particular, while mortar as building material also contributes to a minor extent. The δ¹⁸O values support this hypothesis. Moreover, the reported δ¹⁸O values are a strong indicator of the secondary nature of the Mg sulphates. Isotope ratio measurement and especially the combined use of δ³⁴S and δ¹⁸O values have proven to be a good instrument in clarifying the origin of salt efflorescences on buildings. Recommendations and perspectives Further studies should investigate more closely the isotopic composition of atmospheric aerosols in Salamanca in order to get a more detailed knowledge about the main sulphur sources, as well as to quantify the relation between the isotopic values and the amount and mineralogical form of the salts.
Show more [+] Less [-]A universal method to assess the potential of phosphorus loss from soil to aquatic ecosystems Full text
2010
Pöthig, Rosemarie | Behrendt, Horst | Opitz, Dieter | Furrer, Gerhard
Background, aim, and scope Phosphorus loss from terrestrial to the aquatic ecosystems contributes to eutrophication of surface waters. To maintain the world's vital freshwater ecosystems, the reduction of eutrophication is crucial. This needs the prevention of overfertilization of agricultural soils with phosphorus. However, the methods of risk assessment for the P loss potential from soils lack uniformity and are difficult for routine analysis. Therefore, the efficient detection of areas with a high risk of P loss requires a simple and universal soil test method that is cost effective and applicable in both industrialized and developing countries. Materials and methods Soils from areas which varied highly in land use and soil type were investigated regarding the degree of P saturation (DPS) as well as the equilibrium P concentration (EPC₀) and water-soluble P (WSP) as indicators for the potential of P loss. The parameters DPS and EPC₀ were determined from P sorption isotherms. Results Our investigation of more than 400 soil samples revealed coherent relationships between DPS and EPC₀ as well as WSP. The complex parameter DPS, characterizing the actual P status of soil, is accessible from a simple standard measurement of WSP based on the equation [graphic removed] . Discussion The parameter WSP in this equation is a function of remaining phosphorous sorption capacity/total accumulated phosphorous (SP/TP). This quotient is independent of soil type due to the mutual compensation of the factors SP and TP. Thus, the relationship between DPS and WSP is also independent of soil type. Conclusions The degree of P saturation, which reflects the actual state of P fertilization of soil, can be calculated from the easily accessible parameter WSP. Due to the independence from soil type and land use, the relation is valid for all soils. Values of WSP, which exceed 5 mg P/kg soil, signalize a P saturation between 70% and 80% and thus a high risk of P loss from soil. Recommendations and perspectives These results reveal a new approach of risk assessment for P loss from soils to surface and ground waters. The consequent application of this method may globally help to save the vital resources of our terrestrial and aquatic ecosystems.
Show more [+] Less [-]Assessment of metal contaminations leaching out from recycling plastic bottles upon treatments Full text
2010
Cheng, Xiaoliang | Shi, Honglan | Adams, C. D. (Craig D) | Ma, Yinfa
Background, aims, and scope Heavy metal contaminants in environment, especially in drinking water, are always of great concern due to their health impact. Due to the use of heavy metals as catalysts during plastic syntheses, particularly antimony, human exposure to metal release from plastic bottles has been a serious concern in recent years. The aim and scope of this study were to assess metal contaminations leaching out from a series of recycling plastic bottles upon treatments. Methodology In this study, leaching concentrations of 16 metal elements were determined in 21 different types of plastic bottles from five commercial brands, which were made of recycling materials ranging from no. 1 to no. 7. Several sets of experiments were conducted to study the factors that could potentially affect the metal elements leaching from plastic bottles, which include cooling with frozen water, heating with boiling water, microwave, incubating with low-pH water, outdoor sunlight irradiation, and in-car storage. Results Heating and microwave can lead to a noticeable increase of antimony leaching relative to the controls in bottle samples A to G, and some even reached to a higher level than the maximum contamination level (MCL) of the US Environmental Protection Agency (USEPA) regulations. Incubation with low-pH water, outdoor sunlight irradiation, and in-car storage had no significant effect on antimony leaching relative to controls in bottle samples A to G, and the levels of antimony leaching detected were below 6 ppb which is the MCL of USEPA regulations. Cooling had almost no effect on antimony leaching based on our results. For the other interested 15 metal elements (Al, V, Cr, Mn, Co, Ni, Cu, As, Se, Mo, Ag, Cd, Ba, Tl, Pb), no significant leaching was detected or the level was far below the MCL of USEPA regulations in all bottle samples in this study. In addition, washing procedure did contribute to the antimony leaching concentration for polyethylene terephthalate (PET) bottles. The difference of antimony leaching concentration between washing procedure involved and no washing procedure involved (AC) was larger than zero for samples A to G. This interesting result showed that higher antimony concentration was detected in experiments with no washing procedures compared with those experiments with washing procedures. Our study results indicate that partial antimony leaching from PET bottles comes from contaminations on the surface of plastic during manufacturing process, while major antimony leaching comes from conditional changes. Conclusion The results revealed that heating and microwaving enhance antimony leaching significantly in PET plastic bottles. Plastic bottle manufacturers should consider the contaminations during manufacturing process and washing bottles before first use was strongly recommended to remove those contaminants.
Show more [+] Less [-]Characteristics of exhaust gas, liquid products, and residues of printed circuit boards using the pyrolysis process Full text
2010
Chiang, Hung-Lung | Lo, Cho-Ching | Ma, Sen-Yi
Introduction The pyrolytic method was employed to recycle metals and brominated compounds blended into printed circuit boards (PCBs). Methods PCBs were crushed into pieces 4.0-4.8 mm in size, and the crushed pieces were pyrolyzed at temperatures ranging from 200 to 500°C. The compositions of pyrolytic residues, liquid products, and exhaust were analyzed by inductively coupled plasma atomic emission spectrometer, inductively coupled plasma mass spectrometry, and gas chromatography-mass spectrometry. Pyrolytic exhaust was collected by an impinger system in an ice bath cooler to analyze the composition fraction of the liquid product, and uncondensable exhaust was collected for gas constituent analysis. Results Phenol, methyl-phenol, and bromo-phenol were attributed mainly to the liquid product. Metal content was low in the liquid product. In addition, CO, CO₂, CH₄, and H₂ were the major components of pyrolytic exhaust. Conclusions Brominated and chlorinated compounds—i.e., dichloromethane, trans-1,2 dichloroethylene, cis-1,2 dichloroethylene, 1,1,1-trichloroethane, tetrachloromethane, bromophenol, and bromoform—could be high, up to the several parts per million (ppm) level. Low molecular weight volatile organic compounds (VOCs)—i.e., methanol, acetone, ethyl acetate, acrylonitrile, 1-butene, propene, propane, and n-butane—contributed a large fraction of VOCs. The concentrations of toluene, benzene, xylene, ethylbenzene, and styrene were in the ppm range.
Show more [+] Less [-]Biomonitoring perfluorinated compounds in Catalonia, Spain: concentrations and trends in human liver and milk samples Full text
2010
Kärrman, Anna | Domingo, José L. | Llebaria, Xavier | Nadal, Martí | Bigas, Esther | van Bavel, Bert | Lindström, Gunilla
Background, aim and scope Perfluorinated compounds (PFCs) are global environmental pollutants that bioaccumulate in wildlife and humans. Laboratory experiments have revealed toxic effects such as delayed development, humoral suppression, and hepatotoxicity. Although numerous human blood levels have been reported, little is known about distribution in the human body. Knowledge about PFC distribution and accumulation in the human body is crucial to understanding uptake and subsequent effects as well as to conduct risk assessments. The present study reports PFC levels in human liver and breast milk from a general population living in Catalonia, Spain. Liver and milk levels are compared to previously reported levels in blood from the same geographic area as well as to other existing reports on human liver and milk levels in other countries. Materials and methods Human liver (n = 12) and milk (n = 10) samples were collected in 2007 and 2008 in Catalonia, Spain. Liver samples were taken postmortem from six males and six females aged 27-79 years. Milk samples were from healthy primipara women (30-39 years old). Both liver and milk were analyzed by solid-phase extraction and ultra-performance liquid chromatography tandem mass spectrometry. Results Six PFCs were detected in liver, with perfluorooctanesulfonate (PFOS, 26.6 ng/g wet weight) being the chemical with the highest mean concentration. Other PFCs such as perfluorohexanesulfonate (PFHxS), perfluorooctanoic acid (PFOA), and acids with chain lengths up to C11 were also detected, with mean levels ranging between 0.50 and 1.45 ng/g wet weight. On the other hand, PFOS and PFHxS were the only PFCs detected in human milk, with mean concentrations of 0.12 and 0.04 ng/mL, respectively. Discussion While milk concentrations were similar to reported levels from other countries, liver samples contained more PFCs above quantification limits and higher PFOS concentrations compared to the only two other reports found in the literature. Differences between the results of the present study and those concerning previous investigations can be due to declining levels of some PFCs, which have been reported for the USA. The relationship between PFC concentrations in human liver, milk, and blood was assessed using blood concentrations previously determined in Catalonia. Those levels resulted in liver/serum ratios of 1.7:1, 1.4:1, and 2.1:1 for PFOS, perfluorodecanoic acid, and perfluoroundecanoic acid, respectively. Accumulation in liver is suggested for PFOS and the perfluorocarboxylic acids with carbon chain lengths C9, C10, and C11. For PFOA and PFHxS, fivefold and 14-fold higher concentrations, respectively, were seen in serum as compared to liver. The mean concentration of PFOS and PFHxS in milk was only 0.8% and 0.6% of the reported mean serum level, respectively. Conclusions The results of the present study show that several PFCs could be detected in human liver samples of subjects living in Tarragona. Concerning human milk, the mechanism by which PFCs are transferred from mother's blood to breast milk is still unclear. Considering that PFCs are strongly bound to the protein fraction in blood, the possibility of PFCs entering the milk and accumulating to levels observed in maternal plasma is limited. Recommendations and perspectives Interestingly, the potential accumulation difference for PFCs with different chain lengths might be of great importance for risk assessment. Continuing studies on the distribution of different PFCs in human tissue are therefore justified.
Show more [+] Less [-]Influence of linear alkylbenzene sulfonate (LAS) on the structure of Alphaproteobacteria, Actinobacteria, and Acidobacteria communities in a soil microcosm Full text
2010
Sánchez-Peinado, Mª del Mar | Gonzalez-López, Jesús | Martínez-Toledo, Mª Victoria | Pozo, Clementina | Rodelas, Belén
Background, aim, and scope Linear alkylbenzene sulfonate (LAS) is the most used anionic surfactant in a worldwide scale and is considered a high-priority pollutant. LAS is regarded as a readily biodegradable product under aerobic conditions in aqueous media and is mostly removed in wastewater treatment plants, but an important fraction (20-25%) is immobilized in sewage sludge and persists under anoxic conditions. Due to the application of the sludge as a fertilizer, LAS reaches agricultural soil, and therefore, microbial toxicity tests have been widely used to evaluate the influence of LAS on soil microbial ecology. However, molecular-based community-level analyses have been seldom applied in studies regarding the effects of LAS on natural or engineered systems, and, to our knowledge, there are no reports of their use for such appraisals in agricultural soil. In this study, a microcosm system is used to evaluate the effects of a commercial mixture of LAS on the community structure of Alphaproteobacteria, Actinobacteria, and Acidobacteria in an agricultural soil. Material and methods The microcosms consisted of agricultural soil columns (800 g) fed with sterile water (8 ml h⁻¹) added of different concentration of LAS (10 or 50 mg l⁻¹) for periods of time up to 21 days. Sterile water was added to control columns for comparison. The structures of Alphaproteobacteria, Actinobacteria, and Acidobacteria communities were analyzed by a cultivation independent method (temperature gradient gel electrophoresis (TGGE) separation of polymerase chain reaction (PCR)-amplified partial 16S rRNA genes). Relevant populations were identified by subsequent reamplification, DNA sequencing, and database comparisons. Results Cluster analysis of the TGGE fingerprints taking into consideration both the number of bands and their relative intensities revealed that the structure of the Alphaproteobacteria community was significantly changed in the presence of LAS, at both concentrations tested. The average number of bands was significantly lower in the microcosms receiving 50 mg l⁻¹ LAS and in the lower portion of soil cores. The clear differentiation of the samples of the upper portion of the soil columns amended with LAS was specifically related to the presence and intensity of a distinctive major band (named band class 7). There was a statistically significant positive correlation between the concentrations of LAS detected in soil portions taken from LAS 10 mg l⁻¹ and LAS 50 mg l⁻¹ microcosms and the relative intensity of band class 7 in the corresponding TGGE profiles. Prevalent Alphaproteobacteria populations in the soil microcosms had close similarity (>99%) to cultivated species affiliated to genera of the Rhizobiaceae, Methylocystaceae, Hyphomicrobiaceae, Rhodospirillaceae, Brucellaceae, Bradyrhizobiaceae, and Caulobacteraceae families. The population represented by band class 7 was found closely related to the genus Phenylobacterium (Caulobacteraceae). According to cluster analysis of TGGE profiles, the structure of both Actinobacteria and Acidobacteria communities in the soil microcosms was remarkably stable in the presence of LAS at the two concentrations tested, as most bands were universally present in all samples and displayed fairly similar relative intensities. Discussion Previous studies by others authors, based on biological and chemical tests, concluded that LAS toxicity was not an important microbial selection factor in sludge amended soil, while work based on the use of molecular fingerprinting to evaluate the impact of LAS in aqueous media and marine sediments showed that concentrations as low as 1 mg l⁻¹ significantly influence the development of the bacterial community structure. Although TGGE is not a strictly quantitative method due to the bias introduced by the PCR reaction, changes of band intensity through experiments are a consequence of a change in the relative abundance of the corresponding populations in the community and can be used as a semiquantitative measure of bacterial diversity. Our results evidence that the Phenylobacterium population represented by band class 7 was favored by the presence of increasing concentrations of LAS in the soil and turned into a dominant population, suggesting its possible ability to use LAS in soil as a source of nutrients. As studies with pure cultures are required to confirm the ability of this population to degrade LAS, isolation strategies are currently under development in our laboratory. The weak effect of LAS on the structure of Actinobacteria and Acidobacteria communities is particularly interesting, as to our knowledge, there are no previous reports regarding the effects of LAS on these bacterial groups in soil. Conclusions, recommendations, and perspectives The Phenylobacterium-related alphaproteobacterial population identified in this work was selectively enriched in LAS polluted soil and is a plausible candidate to play a relevant role in the biotransformation of the surfactant under the conditions tested. The surfactant had no remarkable effects on the Actinobacteria and Acidobacteria fingerprints in soil, even when present at concentrations widely exceeding those reached in soil immediately after sludge application. TGGE fingerprinting provides a reliable and low time-consuming method for the monitoring of the bacterial community structure and dynamics, and we recommend its integration with the biological and chemical analyses usually applied in risk assessment of LAS in the environment.
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