RESUMO
The use of a multi-biomarker approach with three-spined sticklebacks (Gasterosteus aculeatus) through an active biomonitoring strategy appears to be a promising tool in water quality assessment. The present work proposes to assess the efficiency of these tools in the discrimination of some sites in a large scale on the Meuse basin in Europe. The study was part of an EU program which aims to assess water quality in the Meuse across the French-Belgian border. Sticklebacks were caged 21 days upstream and downstream from the wastewater treatment plants (WWTPs) of Namur (Belgium), Charleville-Mézières (France), Bouillon (Belgium) and Avesnes-sur-Helpe (France). First, the state of a variety of physiological functions was assessed using a battery of biomarkers that represented innate immunity (leucocyte mortality and distribution, phagocytosis activity, respiratory burst), antioxidant system (GPx, CAT, SOD and total GSH content), oxidative damages to the membrane lipids (TBARS), biotransformation enzymes (EROD, GST), synaptic transmission (AChE) and reproduction system (spiggin and vitellogenin concentration). The impacts of the effluents were first analysed for each biomarker using a mixed model ANOVA followed by post-hoc analyses. Secondly, the global river contamination was assessed using a principal component analysis (PCA) followed by a hierarchical agglomerative clustering (HAC). The results highlighted a small number of effects of WWTP effluents on the physiological parameters in caged sticklebacks. Despite a significant effect of the "localisation" factor (upstream/downstream) in the mixed ANOVA for several biomarkers, post-hoc analyses revealed few differences between upstream and downstream of the WWTPs. Only a significant decrease of innate immune responses was observed downstream from the WWTPs of Avesnes-sur-Helpe and Namur. Other biomarker responses were not impacted by WWTP effluents. However, the multivariate analyses (PCA and HAC) of the biomarker responses helped to clearly discriminate the different study sites from the reference but also amongst themselves. Thus, a reduction of general condition (condition index and HSI) was observed in all groups of caged sticklebacks, associated with a weaker AChE activity in comparison with the reference population. A strong oxidative stress was highlighted in fish caged in the Meuse river at Charleville-Mézières whereas sticklebacks caged in the Meuse river at Namur exhibited weaker innate immune responses than others. Conversely, sticklebacks caged in the Helpe-Majeure river at Avesnes-sur-Helpe exhibited higher immune responses. Furthermore, weak defence capacities were recorded in fish caged in the Semois river at Bouillon. This experiment was the first to propose an active biomonitoring approach using three-spined stickleback to assess such varied environments. Low mortality and encouraging results in site discrimination support the use of this tool to assess the quality of a large number of water bodies.
Assuntos
Smegmamorpha/fisiologia , Poluentes Químicos da Água/análise , Qualidade da Água , Animais , Antioxidantes/metabolismo , Biomarcadores/metabolismo , Monitoramento Ambiental , Europa (Continente) , Proteínas de Peixes , França , Estresse Oxidativo , Rios , Smegmamorpha/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Vitelogeninas/metabolismoRESUMO
The potential health risks associated with the pharmaceuticals released into the environment through effluents from sewage treatment plants have become a major cause for concern. Owing to the lack of effective indicators, monitoring the concentration of these pollutants in the aquatic environment is challenging. The aim of this study was to assess the toxicity of a mixture of five pharmaceutical drugs (paracetamol, carbamazepine, diclofenac, irbesartan, and naproxen) using the aquatic moss Fontinalis antipyretica as a bioindicator and bioaccumulator. We examined the effects of the drug mixture on the cellular antioxidant system, chlorophyll content, and morphological traits of F. antipyretica. The plant was exposed for 5 months to three concentrations of the mixture, including the environmental concentration (MX1), and 10- (MX10) and 100-times (MX100) the environmental concentration. The results showed that only carbamazepine and irbesartan were accumulated by the species. The bioconcentration level increased with exposure time, with the maximum uptake at the 4th month of exposure. The increase in bioaccumulation with exposure time was more evident in plants exposed to MX100. Analysis of the activity of antioxidant enzymes showed that superoxide dismutase (SOD, EC 1.15.1.1.) and catalase (EC 1.11.1.6.) were highly sensitive to the drug mixture. The activity of the enzymes was significantly higher in plants exposed to MX100; however, the activity of guaiacol peroxidase (GPX, EC 1.11.1.7.) was not significantly affected. Plants exposed to MX10 and MX100 had significantly lower total chlorophyll content and chlorophyll a/b ratio compared with those of plants in the control group; however, photosynthetic activity was restored after 5 months of exposure. The morphological characteristics of F. antipyretica were less sensitive to the treatment conditions.
Assuntos
Bryopsida , Preparações Farmacêuticas , Poluentes Químicos da Água , Antioxidantes , Bryopsida/metabolismo , Catalase/metabolismo , Clorofila A , Estresse Oxidativo , Superóxido Dismutase/metabolismo , Poluentes Químicos da Água/toxicidadeRESUMO
Pharmaceuticals are emerging pollutants of concern for aquatic ecosystems where they are occurring in complex mixtures. In the present study, the chronic toxicity of an environmentally relevant pharmaceutical mixture on juvenile rainbow trout (Oncorhynchus mykiss) was investigated. Five pharmaceuticals (paracetamol, carbamazepine, diclofenac, naproxen and irbesartan) were selected based on their detection frequency and concentration levels in the Meuse river (Belgium). Fish were exposed for 42 days to three different concentrations of the mixture, the median one detected in the Meuse river, 10-times and 100-times this concentration. Effects on the nervous, immune, antioxidant, and detoxification systems were evaluated throughout the exposure period and their response standardized using the Integrated Biomarker Response (IBRv2) index. IBRv2 scores increased over time in the fish exposed to the highest concentration. After 42 days, fish exposed to the highest concentration displayed significantly higher levels in lysozyme activity (p < 0.01). The mixture also caused significant changes in brain serotonin turnover (p < 0.05). In short, our results indicate that the subchronic waterborne exposure to a pharmaceutical mixture commonly occurring in freshwater ecosystems may affect the neuroendocrine and immune systems of juvenile rainbow trout.
Assuntos
Oncorhynchus mykiss , Poluentes Químicos da Água/toxicidade , Acetaminofen/toxicidade , Animais , Bélgica , Biomarcadores/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Carbamazepina/toxicidade , Diclofenaco/toxicidade , Irbesartana/toxicidade , Fígado/efeitos dos fármacos , Fígado/metabolismo , Lisossomos/metabolismo , Naproxeno/toxicidade , Síndromes Neurotóxicas , Rios , Serotonina/metabolismoRESUMO
The presence of natural estrogens estrone (E1), 17ß-estradiol (E2), estriol (E3) and synthetic estrogen 17α-ethynylestradiol (EE2) in the aquatic environment has raised concerns because of their high potency as endocrine disrupting chemicals. The European Commission (EC) established a Watch List of contaminants of emerging concerns including E1, E2 and EE2. The proposed environmental quality standards (EQSs) are 3.6, 0.4, 0.035 ng/L, for E1, E2, EE2, respectively. A thorough evaluation of analytical procedures developed by several studies aiming to perform sampling campaigns in different European countries highlighted that the required limits of quantification in surface water were not reached, especially for EE2 and to a lesser extent for E2. Moreover, data regarding the occurrence of these contaminants in Belgian surface water are very limited. A sampling campaign was therefore performed on a wide range of rivers in Belgium (accounting for a total of 63 samples). The detection frequencies of E1, E2, E3 and EE2 were 100, 98, 86 and 48%, respectively. E1 showed the highest mean concentration (= 4.433 ng/L). In contrast, the mean concentration of EE2 was 0.042 ng/L. The risk quotients (RQs) were calculated based on the respective EQS of each analyte. The frequency of exceedance of the EQS was 31.7% for E1, EE2, while it increased to 44.4% for E2. The extent of exceedance of the EQS, represented by the 95th percentile of the RQ dataset, was higher than 1 for E1, E2, EE2. The use of a confusion matrix was investigated to try to predict the risk posed by E2, EE2, based on the concentration of E1.
Assuntos
Estrogênios , Poluentes Químicos da Água , Estrogênios/análise , Bélgica , Água , Estradiol/análise , Medição de Risco , Poluentes Químicos da Água/análise , Monitoramento Ambiental/métodosRESUMO
The complex mixtures of contaminants released in wastewater treatment plant (WWTP) effluents are a major source of pollution for aquatic ecosystems. The present work aimed to assess the environmental risk posed by WWTP effluents by applying a multi-biomarker approach on caged rainbow trout (Oncorhynchus mykiss) juveniles. Fish were caged upstream and downstream of a WWTP for 21â¯days. To evaluate fish health, biomarkers representing immune, reproductive, nervous, detoxification, and antioxidant functions were assayed. Biomarker responses were then synthesized using an Integrated Biomarker Response (IBR) index. The IBR highlighted similar response patterns for the upstream and downstream sites. Caged juvenile females showed increased activities of innate immune parameters (lysozyme and complement), histological lesions and reduced glycogen content in the hepatic tissue, and higher muscle cholinergic metabolism. However, the intensity of the observed effects was more severe downstream of the WWTP. The present results suggest that the constitutive pollution level of the Meuse River measured upstream from the studied WWTP can have deleterious effects on fish health condition, which are exacerbated by the exposure to WWTP effluents. Our results infer that the application of IBR index is a promising tool to apply with active biomonitoring approaches as it provides comprehensive information about the biological effects caused by point source pollution such as WWTP, but also by the constitutive pollutions levels encountered in the receiving environment.
Assuntos
Oncorhynchus mykiss , Poluentes Químicos da Água , Purificação da Água , Animais , Monitoramento Biológico , Biomarcadores/metabolismo , Ecossistema , Monitoramento Ambiental/métodos , Feminino , Oncorhynchus mykiss/metabolismo , Águas Residuárias/análise , Águas Residuárias/toxicidade , Poluentes Químicos da Água/análiseRESUMO
A biomonitoring approach based on a single model species cannot be representative of the contaminations impacts on the ecosystem overall. As part of the Interreg DIADeM program ("Development of an integrated approach for the diagnosis of the water quality of the River Meuse"), a study was conducted to establish the proof of concept that the use of a multispecies active biomonitoring approach improves diagnostic of aquatic systems. The complementarity of the biomarker responses was tested in four model species belonging to various ecological compartments: the bryophyte Fontinalis antipyretica, the bivalve Dreissena polymorpha, the amphipod Gammarus fossarum and the fish Gasterosteus aculeatus. The species have been caged upstream and downstream from five wastewater treatment plants (WWTPs) in the Meuse watershed. After the exposure, a battery of biomarkers was measured and results were compiled in an Integrated Biomarker Response (IBR) for each species. A multispecies IBR value was then proposed to assess the quality of the receiving environment upstream the WWTPs. The effluent toxicity was variable according to the caged species and the WWTP. However, the calculated IBR were high for all species and upstream sites, suggesting that the water quality was already downgraded upstream the WWTP. This contamination of the receiving environment was confirmed by the multispecies IBR which has allowed to rank the rivers from the less to the most contaminated. This study has demonstrated the interest of the IBR in the assessment of biological impacts of a point-source contamination (WWTP effluent) but also of the receiving environment, thanks to the use of independent references. Moreover, this study has highlighted the complementarity between the different species and has emphasized the interest of this multispecies approach to consider the variability of the species exposition pathway and sensibility as well as the mechanism of contaminants toxicity in the final diagnosis.
Assuntos
Monitoramento Ambiental , Poluentes Químicos da Água , Animais , Monitoramento Biológico , Ecossistema , Rios , Águas Residuárias , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/toxicidadeRESUMO
Biomonitoring of aquatic environments requires new tools to characterize the effects of pollutants on living organisms. Zebra mussels (Dreissena polymorpha) from the same site in north-eastern France were caged for two months, upstream and downstream of three wastewater treatment plants (WWTPs) in the international watershed of the Meuse (Charleville-Mézières "CM" in France, Namur "Nam" and Charleroi "Cr" in Belgium). The aim was to test 1H-NMR metabolomics for the assessment of water bodies' quality. The metabolomic approach was combined with a more "classical" one, i.e., the measurement of a range of energy biomarkers: lactate dehydrogenase (LDH), lipase, acid phosphatase (ACP) and amylase activities, condition index (CI), total reserves, electron transport system (ETS) activity and cellular energy allocation (CEA). Five of the eight energy biomarkers were significantly impacted (LDH, ACP, lipase, total reserves and ETS), without a clear pattern between sites (Up and Down) and stations (CM, Nam and Cr). The metabolomic approach revealed variations among the three stations, and also between the upstream and downstream of Nam and CM WWTPs. A total of 28 known metabolites was detected, among which four (lactate, glycine, maltose and glutamate) explained the observed metabolome variations between sites and stations, in accordance with chemical exposure levels. Metabolome changes suggest that zebra mussel exposure to field contamination could alter their osmoregulation and anaerobic metabolism capacities. This study reveals that lactate is a potential biomarker of interest, and 1H-NMR metabolomics can be an efficient approach to assess the health status of zebra mussels in the biomonitoring of aquatic environments.
Assuntos
Dreissena , Poluentes Químicos da Água , Animais , Bélgica , Monitoramento Ambiental , França , Metabolômica , Espectroscopia de Prótons por Ressonância Magnética , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/toxicidadeRESUMO
Natural estrogens (estrone: E1, 17ß-estradiol: E2, estriol: E3) and the synthetic estrogen (17α-ethinylestradiol: EE2) are endocrine disruptors harmful to aquatic wildlife. The European Commission included these molecules in the surface water Watch Lists issued in 2015 and 2018 under the Water Framework Directive regarding emerging aquatic pollutants, proposing maximum detection limits (LOD) of 0.035 ng/L for EE2 and 0.4 ng/L for E1 and E2. Attaining these limits represents a challenge even with the most up-to-date analytical tools, in particular in surface water. A two-step sample preparation, involving a preliminary extraction of a whole water sample on a solid-phase extraction (SPE) disk and further purification on a Florisil SPE cartridge, was optimized. The purified extract was derivatized subsequently and quantified by LC-MS/MS. The main goal was to maximize the recoveries to achieve the very low LODs required by the European Watch Lists. The method was fully validated in seven surface water. The LODs calculated were below the maximum acceptable limits required by the European Commission.
Assuntos
Estrogênios/análise , Poluentes Químicos da Água/análise , Calibragem , Cromatografia Líquida , Disruptores Endócrinos/análise , Monitoramento Ambiental/métodos , União Europeia , Água Subterrânea/química , Limite de Detecção , Silicatos de Magnésio/isolamento & purificação , Minerais/química , Reprodutibilidade dos Testes , Extração em Fase Sólida , Espectrometria de Massas em TandemRESUMO
Pharmaceutical substances are ubiquitous in the aquatic environment and their concentration levels typically range from ng/L up to several µg/L. Furthermore, as those compounds are designed to be highly biologically active, assessing their impacts on non-target organisms is important. Here, we conducted a mesocosm experiment testing a mixture of five pharmaceuticals (diclofenac, carbamazepine, irbesartan, acetaminophen and naproxen) on fish, three-spined stickleback (Gasterosteus aculeatus). The mixture concentration levels were chosen on the basis of the contamination of the Meuse river in Belgium which had been measured previously during a monitoring campaign undertaken in 2015 and 2016. Three nominal mixture concentration levels were tested: the lowest concentration level mixture was composed by environmentally-relevant concentrations that approximate average realistic values for each pharmaceuticals (Mx1); the two other levels were 10 and 100 times these concentrations. Although no impact on stickleback prey was observed, the mixture significantly impaired the survival of female fish introduced in the mesocosms at the highest treatment level without causing other major differences on fish population structure. Impacts on condition factors of adults and juveniles were also observed at both individual and population levels. Using a modelling approach with an individual-based model coupled to a bioenergetic model (DEB-IBM), we concluded that chronic exposure to environmentally-relevant concentrations of five pharmaceuticals often detected in the rivers did not appear to strongly affect the three-spined stickleback populations. Mechanisms of population regulation may have counteracted the mixture impacts in the mesocosms.
Assuntos
Preparações Farmacêuticas/análise , Rios/química , Smegmamorpha/crescimento & desenvolvimento , Poluentes Químicos da Água/toxicidade , Acetaminofen/análise , Acetaminofen/toxicidade , Animais , Bélgica , Carbamazepina/análise , Carbamazepina/toxicidade , Diclofenaco/análise , Diclofenaco/toxicidade , Feminino , Modelos Teóricos , Naproxeno/análise , Naproxeno/toxicidade , Dinâmica Populacional , Smegmamorpha/fisiologia , Poluentes Químicos da Água/análiseRESUMO
The effects of the ripeness stage of banana (Musa AAA) and plantain (Musa AAB) peels on neutral detergent fibre, acid detergent fibre, cellulose, hemicelluloses, lignin, pectin contents, and pectin chemical features were studied. Plantain peels contained a higher amount of lignin but had a lower hemicellulose content than banana peels. A sequential extraction of pectins showed that acid extraction was the most efficient to isolate banana peel pectins, whereas an ammonium oxalate extraction was more appropriate for plantain peels. In all the stages of maturation, the pectin content in banana peels was higher compared to plantain peels. Moreover, the galacturonic acid and methoxy group contents in banana peels were higher than in plantain peels. The average molecular weights of the extracted pectins were in the range of 132.6-573.8 kDa and were not dependant on peel variety, while the stage of maturation did not affect the dietary fibre yields and the composition in pectic polysaccharides in a consistent manner. This study has showed that banana peels are a potential source of dietary fibres and pectins.
Assuntos
Biotecnologia/métodos , Fibras na Dieta , Musa/metabolismo , Pectinas/análise , Plantago/metabolismo , Carboidratos , Celulose/química , Ácidos Hexurônicos/análise , Íons , Metilação , Peso Molecular , Oxalatos/análise , Polissacarídeos/análise , Especificidade da Espécie , TemperaturaRESUMO
An experimental design was used to study the influence of pH (1.5 and 2.0), temperature (80 and 90°C) and time (1 and 4h) on extraction of pectin from banana peels (Musa AAA). Yield of extracted pectins, their composition (neutral sugars, galacturonic acid, and degree of esterification) and some macromolecular characteristics (average molecular weight, intrinsic viscosity) were determined. It was found that extraction pH was the most important parameter influencing yield and pectin chemical composition. Lower pH values negatively affected the galacturonic acid content of pectin, but increased the pectin yield. The values of degree of methylation decreased significantly with increasing temperature and time of extraction. The average molecular weight ranged widely from 87 to 248kDa and was mainly influenced by pH and extraction time.
RESUMO
Natural estrogens (estrone: E1, 17ß-estradiol: E2, estriol: E3) and synthetic 17α-ethynylestradiol (EE2) are reported as strong endocrine disruptors even at extremely low concentrations. Therefore, the watch list from the European Commission regarding emerging aquatic pollutants recommended maximum detection limits of 0.035â¯ng/L for EE2 and 0.4â¯ng/L for E1 and E2. In this study, a UHPLC-ESI-MS/MS method allowing quantification of E1, E2, E3 and EE2 in aqueous matrices was developed. The analytes were derivatized using pyridine-3-sulfonyl chloride and a broad range of product ions were generated and their specificity was assessed by analyzing both surface and groundwater. At least two product ions for each estrogenic compound were proved to be specific and hence suitable for quantification and confirmation. In complex aqueous matrices, analyte responses were particularly affected by ion suppression. This phenomenon was reduced by optimizing the clean-up and selecting a suitable stationary phase for the chromatographic separation. The limits of quantification assessed in surface water with the optimized method ranged from 0.098â¯ng/L (EE2) to 2.73â¯ng/L (E3).
Assuntos
Cromatografia Líquida de Alta Pressão , Monitoramento Ambiental/métodos , Estrogênios/análise , Água Doce/química , Espectrometria de Massas em Tandem , Poluentes Químicos da Água/análise , Água Subterrânea/química , Limite de DetecçãoRESUMO
Because of their intrinsic biological activity and ubiquitous environmental occurrence, human pharmaceutical compounds have received increasing attention from health and environmental agencies. In the present study, all-female juvenile rainbow trout (Oncorhynchus mykiss) were exposed to environmentally-realistic concentrations of a mixture of nonsteroidal pharmaceuticals for 42 days, and the effects on plasma levels of sex-steroids and the expression of genes encoding key proteins involved in ovarian development were assessed. Paracetamol, carbamazepine, diclofenac, irbesartan and naproxen were selected, as these have been detected in the Meuse River in Belgium. Fish were exposed to three concentrations of the mixture including the environmental concentration, 10- and 100-times the environmental concentration. Plasma levels of sex-steroid hormones, particularly 11-ketotestosterone, increased in a concentration-dependent way in exposed females. In addition, some key genes involved in ovarian steroidogenesis were significantly overexpressed after 7 days of exposure, such as key genes involved in the maintenance of the ovary. The steady-state mRNA level of genes implicated in germ cell fate were especially affected, such as that of foxl3 which increased by 5 fold at the highest concentration of the mixture. In conclusion, this study highlights that combined occurrence of common pharmaceutical drugs at concentrations present in surface water environments may act as endocrine-disrupting compounds in rainbow trout.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Oncorhynchus mykiss/genética , Ovário/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Acetaminofen/toxicidade , Animais , Bélgica , Carbamazepina/toxicidade , Diclofenaco/toxicidade , Disruptores Endócrinos/toxicidade , Feminino , Hormônios Esteroides Gonadais/sangue , Irbesartana/toxicidade , Naproxeno/toxicidade , Oncorhynchus mykiss/sangue , Oncorhynchus mykiss/metabolismo , Testosterona/análogos & derivados , Testosterona/sangueRESUMO
A method using Ion Chromatography hyphenated to an Inductively Coupled Plasma-Mass Spectrometer has been developed to accurately determine arsenite (As(III)), arsenate (As(V)), mono-methylarsonic acid (MMAA(V)), dimethylarsinic acid (DMAA(V)) and arsenobetaine (AsBet) in different water matrices. The developed method showed a high sensitivity with detection limits for each arsenic species close to 0.4pg injected. Arsenite and arsenate were the major species found in surface and well waters, but AsBet and DMAA(V) were found in some surface waters, which has never been reported before, while in some natural mineral waters located in volcanic region, the arsenic content exceeded the maximal admissible arsenic content by European legislation standards and the predominant form was As(V).
Assuntos
Arsenicais/análise , Poluentes Químicos da Água/análise , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica/métodos , Espectrometria de Massas/métodosRESUMO
Mercury (II) measurements were performed thanks to a newly developed electrochemical method using a disposable gold modified screen printed carbon electrode. The method has a wide dynamic range (1-100 µg/L), a good accuracy and a limit of detection in compliance with WHO standards. The application of the method to several groundwater samples made it possible to identify, for the first time, mercury content higher than the recommended WHO standard value in a gold mining activity area in the northern part of Burkina Faso. The accuracy of the assay was checked by ICP/MS.
RESUMO
In this study, inulooligosaccharides (F(n)-type inulin) resulting from the endo-inulinase hydrolysis of globe artichoke inulin were purified and characterized. The aim was to produce F(n) oligomer standards with the intention of identifying them in the complex inulin chromatogram. Inulin was extracted from globe artichoke and presented a high average degree of polymerization (DP) of about 80 as determined by high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). This inulin was hydrolyzed by a commercial endo-inulinase yielding a product with a very high F(n)/GF(n) molecule ratio, thus limiting the interference of GF(n) during the purification process. High-performance size exclusion chromatography was used to individually isolate and collect each retention peak corresponding to a specific oligomer. The purity of these fractions was checked by HPAEC-PAD and showed that relatively pure molecules were produced. Electrospray ionization mass spectrometry allowed the molecular weight determination of these purified oligomers and ascertained their DP as F2, F3 and F4. These F(2-4) standards were used with glucose, fructose, sucrose and GF(2-4) (commercially available) to spike commercial oligofructose products in order to determine the elution profile in the HPAEC-PAD chromatogram.
Assuntos
Inulina/química , Oligossacarídeos/química , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Eletroquímica , Hidrólise , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
A new sample preparation method for the water content determination of inulin by volumetric Karl Fischer (KF) titration was developed and compared to the usual method of introducing the sample directly in the methanol-based working medium, modified or not by formamide (1:3, v/v) in order to increase sample solubility. In the proposed method, inulins were externally prepared by dissolving them in pure formamide (2.5:7.5, w/w). The time of analysis of the liquid/liquid reaction of the new method between the dissolved sample and the reaction medium is about 1-2min, while the usual KF method is stopped after the 10min delay time. The developed method permits the determination of water included in the crystals of the sample, confirmed by analysing both crystalline and amorphous inulin samples. Another advantage of this new method is its applicability for the water content determination of other polysaccharides that are not readily soluble in the working medium. Moreover, water content determination can be done by any type of volumetric KF titrator, as this proposed technique is not dependent on any additional tools such as a built-in homogeniser or a heatable titration beaker.