Piotr Stepnowski

Application of Spectroscopic Methods for Structural Analysis of Chitin and Chitosan

Chitin, the second most important natural polymer in the world, and its N-deacetylated derivative chitosan, have been identified as versatile biopolymers for a broad range of applications in medicine, agriculture and the food industry. Two of the main reasons for this are firstly the unique chemical, physicochemical and biological properties of chitin and chitosan, and secondly the unlimited supply of raw materials for their production. These polymers exhibit widely differing physicochemical properties depending on the chitin source and the conditions of chitosan production. The presence of reactive functional groups as well as the polysaccharide nature of these biopolymers enables them to undergo diverse chemical modifications. A complete chemical and physicochemical characterization of chitin, chitosan and their derivatives is not possible without using spectroscopic techniques. This review focuses on the application of spectroscopic methods for the structural analysis of these compounds.

Progress in evaluation of risk potential of ionic liquids—basis for an eco-design of sustainable products

Motivated by the prevailing need for a sustainable development and taking the principles of Green Chemistry as a starting point, the present paper describes new and updated findings regarding a sustainable product design for ionic liquids. The focus is on environmental risk. Nevertheless, cytotoxicity testing and first indicative results from a genotoxicity study extend present knowledge also with regard to possible effects on humans. The structural variability of commercially available ionic liquids as well as the abundance of theoretically accessible ionic liquids is illustrated and the consequences for an integrated risk assessment accompanying the development process are discussed. The side chain effect on toxicity for imidazolium type ionic liquids was confounded by more complex biological testing. Also, an influence of an anion on cytotoxicity is shown for the first time. Testing of presumed metabolites of the imidazolium type cations showed a significantly lower biological activity in cytotoxicity studies than their parent compounds. The importance of a purity assessment for ionic liquids is pointed out and a collection of methods that is believed to be adequate is presented. In addition to risk analysis, the use of life cycle analysis for the multi-objective problem of designing ionic liquids is sketched and an eco-design scheme for ionic liquids is proposed. In conclusion, the paper illustrates the complex nature of the development processes ionic liquids are currently undergoing and provides guidance on which aspects have to be kept in mind.

Reversed-phase liquid chromatographic method for the determination of selected room-temperature ionic liquid cations

The separation of selected 1-alkyl- and 1-aryl-3-methylimidazolium-based room temperature ionic liquid cations has been performed using reversed-phase high-performance liquid chromatography with electrospray ionization mass detection. The RP-HPLC method development started with the selection of a column taking into account especially the resolution of low molecular congeners of the selected group. Mobile phase composition was optimized for peak resolution, sensitivity and high reproducibility of retention values. The results of the method development were applied to the determination of exemplary ionic liquid species present in the medium used in cytotoxicity studies.

Toxicity of imidazolium and pyridinium based ionic liquids towards algae. Chlorella vulgaris, Oocystis submarina (green algae) and Cyclotella meneghiniana, Skeletonema marinoi (diatoms)

This paper reports on an investigation into the toxicity of 1-alkyl-3-methylimidazolium ionic liquids (ILs) towards two green algae (Chlorella vulgaris and Oocystis submarina) characteristic of freshwater and brackish environments, as well as two brackish and marine diatoms (Cyclotella meneghiniana and Skeletonema marinoi). The test kit of IL compounds consisted of five 1-alkyl-3-methylimidazolium chlorides (from -ethyl to -decyl) for evaluating the expected alkyl chain length effect, together with 1-butyl-3-methylimidazolium tetrafluoroborate, dicyanamide, trifluoromethanesulfonate, methyl sulfate an α-methyl[poly(oxy-1,2-ethanediyl)]sulfate for investigating the influence of the anion on IL toxicity towards various algal species. A pronounced alkyl chain effect was confirmed with all the organisms studied. EC50 values were linearly very well correlated with the number of carbon atoms in the IL alkyl chains. The results indicate that diatoms are far more sensitive than green algae to ILs. Cell size also plays an important part in the intoxication process: a tenfold difference in cell size results in a 100% more sensitive reaction to ionic liquids in both the green algae and diatoms. No significant differences were observed between alkylimidazolium salts and an alkylpyridinium compound of similar lipophilicity. It was also found that the use of tetrafluoroborate and trifluoromethanesulfonate as counteranions in the IL structure gave rise to the most pronounced toxic effects in comparison with the other anions tested. In the case of tetrafluoroborate this is probably caused by the potential hydrolysis of this entity, which leads to the formation of fluoride and a further increase in toxicity. Whereas in the case of trifluoromethanesulfonate it is likely caused by relatively high lipophilicity of this anion, additionally known to be strongly associated with alkylimidazolium cations, that in turn may enhance cell wall penetration.

Ecotoxicity evaluation of selected sulfonamides

Sulfonamides (SAs) are a group of antibiotic drugs widely used in veterinary medicine. The contamination of the environment by these pharmaceuticals has raised concern in recent years. However, knowledge of their (eco)toxicity is still very basic and is restricted to just a few of these substances. Even though their toxicological analysis has been thoroughly performed and ecotoxicological data are available in the literature, a systematic analysis of their ecotoxicological potential has yet to be carried out. To fill this gap, 12 different SAs were chosen for detailed analysis with the focus on different bacteria as well as non-target organisms (algae and plants). A flexible (eco)toxicological test battery was used, including enzymes (acetylcholinesterase and glutathione reductase), luminescent marine bacteria (Vibrio fischeri), soil bacteria (Arthrobacter globiformis), limnic unicellular green algae (Scenedesmus vacuolatus) and duckweed (Lemna minor), in order to take into account both the aquatic and terrestrial compartments of the environment, as well as different trophic levels. It was found that SAs are not only toxic towards green algae (EC₅₀=1.54-32.25 mg L⁻¹) but have even stronger adverse effect on duckweed (EC₅₀=0.02-4.89 mg L⁻¹) than atrazine - herbicide (EC₅₀=2.59 mg L⁻¹).

Simultaneous analysis of non-steroidal anti-inflammatory drugs and estrogenic hormones in water and wastewater samples using gas chromatography-mass spectrometry and gas chromatography with electron capture detection

Non-steroidal anti-inflammatory drugs are the group of pharmaceuticals that is most often found in the environment, whereas estrogenic hormones are considered to be potent endocrine disruptors. However, the fate and persistence of these compounds in the environment are still unclear. In this study we propose two approaches for determining these compounds in environmental water samples: GC-MS using time windows and operating in selected ion-monitoring mode (SIM) and, for the first time, gas chromatography with electron capture detection (GC-ECD). The identification criteria of both methods fulfilled the requirements of Directive 2002/657/EC. The use of time windows improved the sensitivity of GC-MS measurements. In GC-MS analysis the pharmaceuticals were determined as trimethylsilyl, in GC-ECD as pentafluoropropionyl derivatives. The influence of such parameters as the type of reagent, type of solvent, reaction time, reaction temperature and microwave irradiation in a household microwave oven on the efficacy of silylation was investigated. Derivatization using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and 1% trimethylchlorosilane (TMCS) in pyridine (1:1, v/v) for 30 min in 60 °C was found to be optimal. Optimization of the solid phase extraction procedure (SPE) confirmed that the application of Oasis HLB cartridges, the acidification of loading samples to pH2 and the use of methanol as eluent gave the best absolute recoveries (ARs) of the target compounds. The following ARs of all the compounds were achieved: 58.2-106.8% in influent wastewater, 77.8-103.4% in effluent wastewater and 81.2-101.9% in surface water samples. Validation of the SPE-GC-MS method enables 13 pharmaceuticals to be determined with MDLs between 3.3 and 343.6 ng/L, depending on the analytes and matrices. GC-ECD analysis enables the determination of 6 pharmaceuticals in surface water samples with MDLs between 0.7 and 5.4 ng/L. The proposed methods were successfully used for analyzing selected pharmaceuticals in wastewaters and river waters in Poland.

Ionic liquids as lubricants or lubrication additives: An ecotoxicity and biodegradability assessment

This paper reports on the (eco)toxicity and biodegradability of ionic liquids considered for application as lubricants or lubrication additives. Ammonium- and pyrrolidinium-based cations combined with methylsulphate, methylsulphonate and/or (CF(3)SO(2))(2)N(-) anions were investigated in tests to determine their aquatic toxicity using water fleas Daphnia magna, green algae Selenastrum capricornutum and marine bacteria (Vibrio fischeri). Additional test systems with an isolated enzyme (acetylcholinesterase) and isolated leukaemia cells from rats (IPC-81) were used to assess the biological activity of the ionic liquids. These compounds generally exhibit low acute toxicity and biological activity. Their biodegradability was screened according to OECD test procedures 301 B and 301 F. For choline and methoxy-choline ionic liquids ready biodegradability was observed within 5 or 10 d, respectively. Some of the compounds selected have a considerable potential to contribute to the development of more sustainable products and processes.

Matrix effects and recovery calculations in analyses of pharmaceuticals based on the determination of β-blockers and β-agonists in environmental samples.

In recent years substantial progress has been made in analytical methods for determining pharmaceutical residues in environmental samples. Although much work has attempted to establish the influence of sample matrix complexity on results through the determination of matrix effects (ME), extraction efficiency (EE) and absolute recovery of analytes (AR), comparison of these parameters is very complicated because different authors use different methods to obtain them. Moreover, there are few literature data describing the influence of aqueous matrices (tap water and waste water) on results obtained with GC-MS methods. For these reasons, the main aims of the present study were: (1) to critically review the determination of matrix effects and recovery parameters using the two most common techniques for analyzing drugs in environmental samples: gas and liquid chromatography coupled with mass spectrometry or tandem mass spectrometry (GC-MS, GC-MS/MS and LC-MS, LC-MS/MS); (2) to postulate a uniform method for determining ME, EE and AR using GC techniques; (3) to investigate the influence of different aqueous matrices on the solid-phase extraction, derivatization and final determination of drugs using GC. β-Blockers and β-agonists, drugs commonly found in the environment, were chosen as model compounds for this investigation. The values of ME, EE and AR obtained were compared with analogous (or similar) data obtained by other researchers using LC-MS measurements. All the results confirmed that GC-MS analyses are much less sensitive to the complexity of sample matrices than LC-MS, so GC-MS measurements appear to be a very good alternative to LC-MS methods of determining pharmaceutical residues in environmental samples.

Toxicity of imidazolium and pyridinium based ionic liquids towards algae. Bacillaria paxillifer (a microphytobenthic diatom) and Geitlerinema amphibium (a microphytobenthic blue green alga).

Many studies done so far on the aquatic ecotoxicity of ionic liquids have dealt with phytoplankton organisms. Nonetheless, benthic organisms are just as important if we are to fully understand the prospective fate and effects of ionic liquids in the aquatic environment. An investigation was therefore undertaken into the toxicity of 1-alkyl-3-methylimidazolium ionic liquids towards the diatom Bacillaria paxillifer and the cyanobacteriumGeitlerinema amphibium, species characteristic of the benthic environment. As in the first part of this study (A. Latala, M. Nedzi and P. Stepnowski, Green Chem., 2009, 11, 580–588), five 1-alkyl-3-methylimidazolium chlorides (from ethyl to decyl) were chosen as the test kit compounds for examining the expected alkyl chain length effect, together with 1-butyl-3-methylimidazolium tetrafluoroborate, dicyanamide, trifluoromethanesulfonate and methyl sulfate and α-methyl[poly(oxy-1,2-ethanediyl)]sulfate for investigating the influence of anions on the toxicity of ionic liquids towards the algal species under investigation. A pronounced alkyl chain effect was found with both organisms. The results indicate that P. amphibium is more sensitive than B. paxillifer. It was also found that the use of trifluoromethanesulfonate and dicyanamide as counteranions in the IL structure gave rise to the most pronounced toxic effects in comparison with the other anions tested. It is assumed that the greater toxicities of long alkyl chain ILs will most probably not pose a greater threat to benthic organisms, since these compounds will undergo rapid and very strong adsorption to sedimentary matter. Short-chain compounds, which are known to be reversibly bound to sediments, may still pose a significant risk to benthic ecosystems, however.

The cuticular fatty acids of Calliphora vicina, Dendrolimus pini and Galleria mellonella larvae and their role in resistance to fungal infection.

Epicuticular lipids in many terrestrial arthropods consist of vast numbers of polar and non-polar aliphatic compounds, which are mainly responsible for the water balance in these animals but can also affect conidia germination of entomopathogenic fungi. In this work the qualitative and quantitative profiles of cuticular fatty acids from three insect species differing in their susceptibility to fungal infection were studied. In an innovative approach, laser light scattering detection was coupled with HPLC in order to identify the non-chromophoric chemicals usually present in cuticular extracts. The acids identified contained from 5 to 20 carbon atoms in the alkyl chain and included unsaturated entities such as C(16:1), C(18:1), C(18:2), C(18:3) and C(20:1). There was a marked dominance of acids containing 16-18 carbon atoms. The relative contents of fatty acids in the extracted waxes varied from trace amounts to 44%. Cuticular fatty acids profile of Calliphora vicina (species resistant to fungal infection) significantly differs from profiles of Dendrolimus pini and Galleria mellonella (both species highly susceptible to fungal infection). The major difference is the presence of C(14:0), C(16:1) and C(20:0) in the cuticle of C. vicina. These three fatty acids are absent in the cuticle of D. pini while G. mellonella cuticle contains their traces. The concentrations of four fatty acids dominating in the G. mellonella larval cuticle (C(16:0), C(18:0), C(18:1) and C(18:2)) were found to fluctuate during the final larval instar and correlate with fluctuations in the susceptibility of larvae to fungal infection. The possible role of cuticular fatty acids in preventing fungal infection is discussed.