Antimicrobial activity of pharmaceutical cocktails in sewage treatment plant effluent - An experimental and predictive approach to mixture risk assessment.

Municipal wastewater contains multi-component mixtures of active pharmaceutical ingredients (APIs). This could shape microbial communities in sewage treatment plants (STPs) and the effluent-receiving ecosystems. In this paper we assess the risk of antimicrobial effects in STPs and the aquatic environment for a mixture of 18 APIs that was previously detected in the effluent of a European municipal STP. Effects on microbial consortia (collected from a separate STP) were determined using respirometry, enumeration of culturable microorganisms and community-level physiological profiling. The mixture toxicity against selected bacteria was assessed using assays with Pseudomonas putida and Vibrio fischeri. Additional data on the toxicity to environmental bacteria were compiled from literature in order to assess the individual and expected joint bacterial toxicity of the pharmaceuticals in the mixture. The reported effluent concentration of the mixture was 15.4 nmol/l and the lowest experimentally obtained effect concentrations (EC10) were 242 nmol/l for microbial consortia in STPs, 225 nmol/l for P. putida and 73 nmol/l for V. fischeri. The lowest published effect concentrations (EC50) of the individual antibiotics in the mixture range between 15 and 150 nmol/l, whereas 0.9-190 µmol/l was the range of bacterial EC50 values found for the non-antibiotic mixture components. Pharmaceutical cocktails could shape microbial communities at concentrations relevant to STPs and the effluent receiving aquatic environment. The risk of antimicrobial mixture effects was completely dominated by the presence of antibiotics, whereas other pharmaceutical classes contributed only negligibly to the mixture toxicity. The joint bacterial toxicity can be accurately predicted from the individual toxicity of the mixture components, provided that standardized data on representative bacterial strains becomes available for all relevant compounds. These findings argue for a more sophisticated bacterial toxicity assessment of environmentally relevant pharmaceuticals, especially for those with a mode of action that is known to specifically affect prokaryotic microorganisms.

Assessment of toxicity and biodegradability on activated sludge of priority and emerging pollutants.

Several methods for evaluating the toxicity and biodegradability of hazardous pollutants (chlorinated compounds, chemical additives and pharmaceuticals) have been studied in this work. Different bioassays using representative bacteria of marine and terrestrial ecosystems such as Vibrio fischeri and Pseudomonas putida have been used to assess the ecotoxicity. Activated sludge was used to analyse the effect of those pollutants in a biological reactor of a sewage treatment plant (STP). The results demonstrate that none of the compounds is toxic to activated sludge, except ofloxacin to P. putida. The additives tested can be considered moderately toxic according to the more sensitive V. fischeri assays, whereas the EC50 values of the pharmaceuticals depend on the specific microorganism used in each test. Regarding the biodegradability, respirometric measurements were carried out for fast biodegradability assessment and the Zahn-Wellens test for inherent biodegradability. The evolution of the specific oxygen uptake rate (SOUR) showed that only diethyl phthalate was easily biodegradable and acetylsalicylic acid was partially biodegradable (98% and 65% degradation, respectively). The persistence of dichloromethane, ofloxacin and hidrochlorothiazide was confirmed along the 28 days of the Zahn-Wellens test whereas 1,1,1-trichloroethane showed inherent biodegradability (74% removal). Most of the chlorinated compounds, pharmaceuticals, bisphenol A and ethylenediaminetetraacetic acid were partially degraded in 28 d with total organic carbon (TOC) reduction ranging from 21% to 51%. Sulphamethoxazole showed certain biodegradation (50% removal) with TOC decrease around 31%, which indicates the formation of non-biodegradable by-products.

Genetic Lineages and Antimicrobial Resistance in Pseudomonas spp. Isolates Recovered from Food Samples.

Raw food is a reservoir of Pseudomonas isolates that could be disseminated to consumers. The presence of Pseudomonas spp. was studied in food samples, and the phenotypic and genotypic characterizations of the recovered isolates were analyzed. Two samples of meat (3%, turkey and beef) and 13 of vegetables (22%, 7 green peppers and 6 tomatoes) contained Pseudomonas spp. A total of 20 isolates were identified, and were classified as follows (number of isolates): P. aeruginosa (5), P. putida (5), P. nitroreducens (4), P. fulva (2), P. mosselli (1), P. mendocina (1), P. monteilii (1), and Pseudomonas sp. (1). These 20 Pseudomonas isolates were clonally different by pulsed-field-gel-electrophoresis, and were resistant to the following antibiotics: ticarcillin (85%), aztreonam (30%), cefepime (10%), imipenem (10%), and meropenem (5%), but were susceptible to ceftazidime, piperacillin, piperacillin-tazobactam, doripenem, gentamicin, tobramycin, amikacin, ciprofloxacin, norfloxacin, and colistin. Only one strain (Ps158) presented a class 1 integron lacking the 3' conserved segment. The five P. aeruginosa strains were typed by multilocus sequence typing in five different sequence-types (ST17, ST270, ST800, ST1455, and ST1456), and different mutations were detected in protein OprD that were classified in three groups. One strain (Ps159) showed a new insertion sequence (ISPa47) truncating the oprD gene, and conferring resistance to imipenem.

Assessment of planctomycetes cell viability after pollutants exposure.

In this study, the growth of six different planctomycetes, a particular ubiquitous bacterial phylum, was assessed after exposure to pollutants. In addition and for comparative purposes, Pseudomonas putida, Escherichia coli and Vibrio anguillarum were tested. Each microorganism was exposed to several concentrations of 21 different pollutants. After exposure, bacteria were cultivated using the drop plate method. In general, the strains exhibited a great variation of sensitivity to pollutants in the order: V. anguillarum > planctomycetes > P. putida > E. coli. E. coli showed resistance to all pollutants tested, with the exception of phenol and sodium azide. Copper, Ridomil® (fungicide), hydrazine and phenol were the most toxic pollutants. Planctomycetes were resistant to extremely high concentrations of nitrate, nitrite and ammonium but they were the only bacteria sensitive to Previcur N® (fungicide). Sodium azide affected the growth on plates of E. coli, P. putida and V. anguillarum, but not of planctomycetes. However, this compound affected planctomycetes cell respiration but with less impact than in the aforementioned bacteria. Our results provide evidence for a diverse response of bacteria towards pollutants, which may influence the structuring of microbial communities in ecosystems under stress, and provide new insights on the ecophysiology of planctomycetes.

Toxicity assessment of SiC nanofibers and nanorods against bacteria.

In the present study, evidence of the antibacterial effects of silicon carbide (SiC) nanofibers (NFSiC) and nanorods (NRSiC) obtained by combustion synthesis has been presented. It has been shown that the examined bacteria, Pseudomonas putida, could bind to the surface of the investigated SiC nanostructures. The results of respiration measurements, dehydrogenase activity measurements, and evaluation of viable bacteria after incubation with NFSiC and NRSiC demonstrated that the nanostructures of SiC affect the growth and activity of the bacteria examined. The direct count of bacteria stained with propidium iodide after incubation with SiC nanostructures revealed that the loss of cell membrane integrity could be one of the main effects leading to the death of the bacteria.

Use of microcalorimetry to determine the costs and benefits to Pseudomonas putida strain KT2440 of harboring cadmium efflux genes.

A novel microcalorimetric approach was used to analyze the responses of a metal-tolerant soil bacterium (Pseudomonas putida strain KT2440) to metal resistance gene deletions in cadmium-amended media. As hypothesized, under cadmium stress, the wild-type strain benefited from the resistance genes by entering the exponential growth phase earlier than two knockout strains. In the absence of cadmium, strain KT1, carrying a deletion in the main component (czcA1) of a Cd/Zn chemiosmotic efflux transporter (CzcCBA1), grew more efficiently than the wild type and released ∼700 kJ (per mole of biomass carbon) less heat than the wild-type strain, showing the energetic cost of maintaining CzcCBA1 in the absence of cadmium. A second mutant strain (KT4) carrying a different gene deletion, ΔcadA2, which encodes the main Cd/Pb efflux transporter (a P-type ATPase), did not survive beyond moderate cadmium concentrations and exhibited a decreased growth yield in the absence of cadmium. Therefore, CadA2 plays an essential role in cadmium resistance and perhaps serves an additional function. The results of this study provide direct evidence that heavy metal cation efflux mechanisms facilitate shorter lag phases in the presence of metals and that the maintenance and expression of tolerance genes carry quantifiable energetic costs and benefits.

Environmental assessment of ester-based lubricants after application.

GOAL, SCOPE AND BACKGROUND: Lubricants based on renewable resources are an environmentally friendly alternative to petrochemical products due to their better ecotoxicological performance and their excellent biodegradability. To improve the technical performance of lubricants, and to reduce friction and wear, the use of additives is nowadays obligatory. The collaborative research center SFB 442 aims at developing environmentally acceptable lubricants that facilitate the avoidance of these additives by transferring their function to suitable coatings. For a complete assessment of the ecological performance of these newly developed lubricants, the whole life cycle including production, application as well as disposal and fate in the environment is studied. In the following study the focus was on the application and its influence on the environmental behavior of the lubricant. The application of lubricants leads, among other things, to the intake of metals due to abrasion from tools, work pieces or mechanical components. Previous examinations indicated a possible influence of metals on the toxicity of eluates prepared from used lubricants (Erlenkaemper et al. 2005). To clarify if the apparent toxicity of used lubricants solely results from the intake of metals, the extractability of these metals from the oil matrix is determined. By combining chemical analyses with bioassays, the bioavailability of metals that are present in the extract is estimated. To further investigate the relevance of metals on toxicity, toxic units (TU) were calculated and related to the results of the bioassays. Interactions between the metals were investigated with aqueous mixtures of metal chlorides and calculations based on the concept of concentration addition and independent action. METHODS: A lubricant mixture was applied to a tribological test bench that simulates real conditions of use and extremely short time load, respectively. Samples were taken at particular times, water soluble fractions (WSF) of these fluids were prepared and dilution series were investigated in several bioassays. Concentration of metals and total organic carbon (TOC) were determined in the eluates. TUs were calculated according to Sprague (1970) and mixture toxicity was calculated according to the concept of concentration addition (Loewe and Muischnek 1926) and independent action (Bliss 1939). RESULTS: Analyses of the metal content of the lubricant and the eluates clearly revealed the availability of the metals in the aqueous extracts. Especially copper, zinc, nickel and chromium were found and their concentrations increased during the time of use. The water extractable fraction, e.g., of copper, rose from 8.8% to 45.3% of the total content in the lubricant after 33.5 hours of use. Tests performed with the algal growth inhibition assay and the luminescence inhibition assay revealed the uptake or absorption by the organisms and, thus, the bioavailability of the metals. The calculation of TUs partly indicated a possible influence of the metals on ecotoxicity of the eluates. Copper reached concentrations equal to or higher than the EC50 value of copper chloride in the growth inhibition assays with algae and Ps. putida as well in the immobilization assay with daphnids. TUs for copper are also larger than 1 for the algal growth inhibition assay. The EL50 values indicated that the luminescence inhibition assay was the most sensitive test system, with values between 4.7% and 9.6%. While the toxicity towards algae and V. fischeri in the growth inhibition assay decreased until both organisms were no longer influenced by the exposure, the EL50 values for the D. magna immobilization assay and the Ps. putida growth inhibition assay decreased with the progressing use of the lubricant. The tested metal salt mixtures showed that Ps. putida, algae and daphnids are the most sensitive organisms with EC50 values below 1 mg/l. DISCUSSION: Although the intake of metals mainly occurred via abrasion of particles, the results revealed the availability of these metals in water. The availability varied for each of the four metals. For both the algal growth inhibition assay and the luminescence inhibition assay, an uptake or absorption of the metals could be demonstrated. The calculated TUs indicated an effect in some bioassays that was not verified in the test itself. The influence of copper on V. fischeri, for example, was not confirmed while the EL50 values for the daphnid bioassay decreased, meaning that the eluates became more toxic with progressing use of the lubricant. The calculations of mixture toxicity based on the concept of concentration addition demonstrated good correlations with the tested metal mixtures, but also a different sensitivity of the organisms. CONCLUSIONS: The results presented here reveal the availability of those metals in water that were taken in during the use of the lubricant in a tribological test bench and, thus, have the possibility of interacting with the organisms. The availability of the metals in the bioassays was proven by chemical analyses. The calculation of TUs and the corresponding EL50 values, however, indicate different availabilities of the metals. The results of the metal salt mixtures show good correlations with calculations of mixture toxicity based on concentration addition. Moreover, the varying sensitivity of the organisms when exposed to eluates or metal mixtures indicates a different bioavailability of the metals and/or the presence of other compounds that exert toxic action. RECOMMENDATIONS AND PERSPECTIVES: For further investigations, the organic oil matrix and its influence on the toxicity have to be taken into account. The toxicity of the eluates may not only be due to metals; additional effects could arise from changes in the lubricant itself.

Assessment of baseline toxicity of mono-cyclic aromatic compounds by pseudomonas initial oxygen uptake assay.

The objective of this study was to develop quantitative structure-activity relationships (QSARs) for the toxicity of mono-cyclic aromatic compounds in the Pseudomonas putida initial oxygen uptake assay. The QSARs were developed using response-surface based on descriptors for chemical hydrophobicity (logP) and electrophilicity (LUMO). The model log (Ki(-1)=0.434 (+/-0.011) log P-0.389 (+/-0.013) LUMO - 2.13(+/-0.031); n=155, r2=0.941, r2(adj)=0.940, s=0.119, F=1206 led us to conclude that the polar and non-polar narcotics were statistically indistinguishable. Pentafluorophenol, pentachlorophenol and most dinitrophenols classified as weak acid respiratory uncouplers in literature fit well into this model when they were treated as their corresponding phenoxides. This latter result suggests that the action mechanism of these phenols should be reevaluated.

Environmental exposure and risk assessment of fluoroquinolone antibacterial agents in wastewater and river water of the Glatt Valley Watershed, Switzerland.

The mass flows of fluoroquinolone antibacterial agents (FQs) were investigated in the aqueous compartments of the Glatt Valley Watershed, a densely populated region in Switzerland. The major human-use FQs consumed in Switzerland, ciprofloxacin (CIP) and norfloxacin (NOR), were determined in municipal wastewater effluents and in the receiving surface water, the Glatt River. Individual concentrations in raw sewage and in final wastewater effluents ranged from 255 to 568 ng/L and from 36 to 106 ng/L, respectively. In the Glatt River, the FQs were present at concentrations below 19 ng/L. The removal of FQs from the water stream during wastewater treatment was between 79 and 87%. During the studied summer period, FQs in the dissolved fraction were significantly reduced downstream in the Glatt River (15-20 h residence time) (66% for CIP and 48% for NOR). Thus, after wastewater treatment, transport in rivers causes an additional decrease of residual levels of FQs in the aquatic environment. Refined predicted environmental concentrations for the study area compare favorably with the measured environmental concentrations (MEC) obtained in the monitoring study. Total measured FQ concentrations occurring in the examined aquatic compartments of the Glatt Valley Watershed were related to acute ecotoxicity data from the literature. The risk quotients obtained (MEC/PNEC < 1) following the recommendations of the European guidelines or draft documents suggest a low probability for adverse effects of the occurring FQs, either on microbial activity in WWTPs or on algae, daphnia, and fish in surface waters.

Use of bioassays for assessment of water-extractable ecotoxic potential of soils.

The characterization of contaminated soils is based on heterogeneous strategies considering chemical analyses or bioassays. In the report Bioassays for soils, test methods which are at an advanced state of development and standardization are recommended by the German DECHEMA (German Society for Chemical Apparatus, Chemical Engineering and Biotechnology e.V.). Following this report six soil samples contaminated with different organic and inorganic substances are applied to bioassays using the following organisms: Scenedesmus subspicatus, Daphnia magna, Vibrio fischeri, and Pseudomonas putida. Additionally, they are chemically analyzed. The test results demonstrate that toxic contaminants are present and bioavailable in water elutriates from two soils, indicating the necessity of bioassays and chemical analyses to predict potential risks from contaminated soils. Furthermore it is demonstrated that the described qualitative approach for the assessment of test results is not sufficient to quantify the risk potential of contaminated soils.

Environmental risk assessment for the widely used iodinated X-ray contrast agent iopromide (Ultravist).

Iodinated X-ray contrast media are diagnostic pharmaceuticals that are applied to enhance the contrast between organs or vessels examined and surrounding tissues during radiography. These substances are applied in doses up to ca. 200 g per person (corresponding to approx 100 g iodine) and are rapidly excreted. In the sewage system they contribute to the burden of adsorbable organic halogens (AOX). To assess the potential environmental impact of this release, studies on environmental fate and effects were conducted for a risk assessment of the frequently used X-ray contrast medium iopromide (brand name: Ultravist). A screening test for biological degradation (OECD Screening Test 301 E) led to iopromide being classified as not readily biodegradable. Therefore, the predicted environmental concentration (PEC) in surface water was calculated in a first step. The resulting concentration of 2 microgram/liter was then compared in a second step with the predicted no-effect concentration as derived from a battery of ecotoxicity tests. In short-term toxicity tests with bacteria (Vibrio fisheri, Pseudomonas putida), algae (Scenedesmus subspicatus), crustaceans (Daphnia magna), and fish (Danio rerio, Leuciscus idus) no toxic effects were detected at the highest tested concentration of 10 g/liter. In a chronic toxicity test with D. magna no effect was observed at the highest tested concentration of 1 g/liter. Using an assessment factor of 100 the ratio between the predicted environmental concentration (PEC) and the predicted no-effect concentration (PNEC) was calculated to be

Assessment of rapid bioassays for detecting cyanobacterial toxicity.

Simple and easy-to-use bioassays with Artemia salina (brine shrimp) larvae, luminescent bacteria and Pseudomonas putida were evaluated for the detection of toxicity due to cyanobacterial hepato- and neurotoxins. The hepatotoxins and a neurotoxin, anatoxin-a, were extracted from laboratory-grown cultures and natural bloom samples by the solid phase fractionation method and dissolved in diluent for different bioassays. The toxin concentration of cyanobacterial extracts was determined with HPLC. The Artemia biotest appeared to be quite sensitive to cyanobacterial hepatotoxins, with LC 50 values of 3-17 mg l-1. The Artemia test was also shown to be of value for the detection of toxicity caused by anatoxin-a. The fractionated extract of anatoxin-a was not lethal to Artemia but it disturbed the ability of the larvae to move forwards. Filtered cyanobacterial cultures with anatoxin-a, on the other hand, caused mortality of Artemia larvae at concentrations of 2-14 mg l-1. With the solid phase fractionation of cyanobacterial samples, no non-specific toxicity due to compounds other than hepato- and neurotoxins was observed. In the luminescent bacteria test, the inhibition of luminescence did not correlate with the abundance of hepatotoxins or anatoxin-a. The growth of Ps. putida was enhanced, rather than inhibited by cyanobacterial toxin fractions.