Environmental Risk Assessment of Active Human Pharmaceutical Ingredients in Urban Rivers in Japan.

Active pharmaceutical ingredients (APIs) have become a public concern owing to their possible adverse effects on aquatic organisms. Ministry of Health, Labor and Welfare in Japan (MHLW) issued "Guidance on the Environmental Risk Assessment (ERA) in new pharmaceutical development" in 2016. To evaluate the validity of phase 1 in the MHLW's ERA guidance, we monitored the measured environmental concentrations (MECs) of approved APIs in urban rivers and sewage treatment plants (STPs) in Japan and compared these MECs with the predicted environmental concentration (PEC). We collected water samples from urban seven rivers and three STPs during each season. Fifty-one APIs for human and veterinary use and the artificial sweetener sucralose were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Forty-four APIs were observed in the rivers and 42 were found in the influent and effluent of STPs, with levels ranging from nanograms to micrograms per liter. The action limit in phase I of the MHLW's guidance was set to 10 ng/L, and there was no API except for ketoprofen, for which PEC of the MHLW's guidance (PECjapan) was lower than 10 ng/L and the maximum MEC (MECmax) was 10 ng/L or greater. Almost all APIs also had median MECs that were lower than those of the respective PECjapan. These results indicate that the PECjapan values in phase I of the MHLW's guidance were appropriate. However, some APIs had MECmax values that were greater than those of the respective PECjapan due to overestimation of the dilution factor of river water and/or underestimation of API production.

High-temperature calcined fullerene nanowhiskers as well as long needle-like multi-wall carbon nanotubes have abilities to induce NLRP3-mediated IL-1ß secretion.

Because multi-wall carbon nanotubes (MWCNTs) have asbestos-like shape and size, concerns about their pathogenicity have been raised. Contaminated metals of MWCNTs may also be responsible for their toxicity. In this study, we employed high-temperature calcined fullerene nanowhiskers (HTCFNWs), which are needle-like nanofibers composed of amorphous carbon having similar sizes to MWCNTs but neither metal impurities nor tubular structures, and investigated their ability to induce production a major proinflammatory cytokine IL-1ß via the Nod-like receptor pyrin domain containing 3 (NLRP3)-containing flammasome-mediated mechanism. When exposed to THP-1 macrophages, long-HTCFNW exhibited robust IL-1ß production as long and needle-like MWCNTs did, but short-HTCFNW caused very small effect. IL-1ß release induced by long-HTCFNW as well as by long, needle-like MWCNTs was abolished by a caspase-1 inhibitor or siRNA-knockdown of NLRP3, indicating that NLRP3-inflammasome-mediated IL-1ß production by these carbon nanofibers. Our findings indicate that the needle-like shape and length, but neither metal impurities nor tubular structures of MWCNTs were critical to robust NLRP3 activation.

[Steps to Regulatory Science].

The research achievements in the field of regulatory science from the beginning of my research are described in an overview. First, I was interested in the complexity of development and pursued my studies on the mechanisms of DNA replication and repair, the mutagenicity of air pollutants, and the oncogene. After researching new phenomena based on the discovery of basic research in molecular/biochemistry, my research interests turned to the field of regulatory science which applies scientific evidence to social systems. I was able to successfully contribute to the field of drinking water quality in Japan through the establishment of drinking water quality standards and standard values, primarily for organic and agricultural chemicals, the development of analysis techniques, and the creation of an organization for ensuring safety. Research on the water quality in public water areas, which are also the sources of drinking water, was another subject in which I was involved. I took part in developing the concept and evaluation methodology for the environmental impact assessment of active pharmaceutical ingredients as well as conducting environmental monitoring on urban rivers in Japan. I have also been engaged in studies on the security and safety of human health with an ecosystem conservation background. It has been a great pleasure to collaborate on research projects with so many people toward a common aim.

[Investigation and Improvement of Techniques That Affect the Amount of Dispersal during Preparation of Anticancer Drugs].

Many healthcare workers who handle anticancer drugs are at risk for occupational exposure. However, there are no established permissible limits for occupational exposure to anticancer drugs; thus, in this study, we aimed to search for and improve procedures that have a greater impact on the amount of spatter for handling anticancer drugs in vials, which are frequently used, based on the quantitative evaluation of the amount of exposure. We used sodium riboflavin phosphate (FMN) as a simulated anticancer drug and measured the amount of FMN dispersed to the handling area by the wiping method and the amount of FMN dispersed in both gloves using high-performance liquid chromatography with fluorescence detection (HPLC-FL). In this study, it was suggested that the overall amount of dispersal in the preparation process was affected by the different methods of injecting the drug solution into the infusion bottles and whether recapping. It was also found that the variation in the amount of dispersal differed depending on the selected preparation technique. It was suggested that the amount of dispersal could be reduced by selecting an appropriate dissolution method for multiple vials, recapping, an appropriate method for injecting the drug into the infusion bottle, and properly preparing the internal pressure of the infusion bottle. The results of this study suggest that there are some techniques and procedures in the preparation process of vials that have a significant effect on the amount of dispersal, and that proper implementation of these techniques can contribute to the reduction of dispersal.

Quantitative comparison of anticancer drug dispersal before and after introducing appropriate preparation procedures during anticancer drug preparation.

BACKGROUND: In Japan, engineering controls for preparing injectable anticancer drugs are inadequate and compliance with appropriate preparation procedures is vital. In this study, we evaluated the effects of adherence to appropriate anticancer drug formulation and packaging procedures on reducing anticancer drug dispersal in clinical practice, especially in Japan. METHODS: We quantitatively evaluated the effectiveness of implementing procedures that were experimentally verified to help reduce the amount of anticancer drug dispersed during preparation based on procedures described in the "Anticancer Drug Preparation Manual." The target facilities were four regional hub hospitals in the Kanto area. Contamination of sheets and gloves with 5-fluorouracil (5-FU) and gemcitabine (GEM) in a safety cabinet during formulation was evaluated using wipe tests. Subsequently, the proper preparation procedure was shown on a video, training was provided, and the wipe tests were repeated. RESULTS: Forty-one and 39 pharmacists were engaged in drug preparation before and after intervention, respectively. 5-FU had the highest dispersal per prepared vial on the sheet before intervention. The dispersal amount per prepared vial decreased significantly (P = 0.01) after intervention. The amount of GEM dispersed before and after intervention did not differ significantly. However, the percentage of sheets below the detection limit after intervention was 62%, increasing from 46% before intervention. The amount dispersed on gloves was not significantly reduced by proper preparation technique. Although not explicitly noticeable and quantifiable, pharmacists must consider that a significant amount of anticancer drug is dispersed on gloves despite following appropriate preparation procedures. CONCLUSIONS: Quantitative amounts of anticancer drugs dispersed in the preparations of 5-FU and GEM were found in our study. The difference in the amount of contamination before and after intervention was significantly reduced only for the contamination of sheets with 5-FU. There was no decrease in the amount of glove contamination. There was also no difference between medical facilities. Despite following appropriate preparation procedures, dispersed amounts cannot be maintained below the detection limit, indicating the need for a combination of education and engineering controls.

[Importance of researches on chronic effects by manufactured nanomaterials].

Manufactured nanomaterials are the most important substances for the nanotechnology. The nanomaterials possess different physico-chemical properties from bulk materials. The new properties may lead to biologically beneficial effects and/or adverse effects. However, there are no standardized evaluation methods at present. Some domestic research projects and international OECD programs are ongoing, in order to share the health impact information of nanomaterials or to standardize the evaluation methods. From 2005, our institutes have been conducting the research on the establishment of health risk assessment methodology of manufactured nanomaterials. In the course of the research project, we revealed that the nanomaterials were competent to cause chronic effects, by analyzing the intraperitoneal administration studies and carcinogenic promotion studies. These studies suggested that even aggregated nanomaterials were crumbled into nanosized particles inside the body during the long-term, and the particles were transferred to other organs. Also investigations of the toxicokinetic properties of nanomaterials after exposure are important to predict the chronically targeted tissues. The long lasting particles/fibers in the particular tissues may cause chronic adverse effects. Therefore, focusing on the toxicological characterization of chronic effects was considered to be most appropriate approach for establishing the risk assessment methods of nanomaterials.

[Analysis of volcanic-ash-based insoluble ingredients of facial cleansers].

The substance termed "Shirasu balloons", produced by the heat treatment of volcanic silicates, is in the form of hollow glass microspheres. Recently, this substance has gained popularity as an ingredient of facial cleansers currently available in the market, because it lends a refreshing and smooth feeling after use. However, reports of eye injury after use of a facial cleanser containing a substance made from volcanic ashes are on the rise. We presumed that the shape and size of these volcanic-ash-based ingredients would be the cause of such injuries. Therefore, in this study, we first developed a method for extracting water-insoluble ingredients such as "Shirasu balloons" from the facial cleansers, and then, we examined their shapes and sizes. The insoluble ingredients extracted from the cleansers were mainly those derived from volcanic silicates. A part of the ingredients remained in the form of glass microspheres, but for the most part, the ingredients were present in various forms, such as fragments of broken glass. Some of the fragments were larger than 75 microm in length. Foreign objects having a certain hardness, shape, and size (e.g., size greater than 75 microm) can possibly cause eye injury. We further examined insoluble ingredients of facial scrubs, such as artificial mineral complexes, mud, charcoal, and polymers, except for volcanic-silicate-based ingredients. The amounts of insoluble ingredients extracted from these scrubs were small and did not have a sharp edge. Some scrubs had ingredients with particles larger than 75 microm in size, but their specific gravities were small and their hardness values were much lower than those of glass microspheres of ingredients such as "Shirasu balloons". Because the fragments of glass microspheres can possibly cause eye injury, the facial cleansers containing large insoluble ingredients derived from volcanic ashes should be avoided to use around eyes.

[Volatile organic compounds (VOCs) emitted from large furniture].

Indoor air pollution by volatile organic compounds (VOCs), which may cause a hazardous influence on human being such as sick building (sick house) syndrome, has become a serious problem. In this study, VOCs emitted from nine pieces of home furniture, three sets of dining tables, three sets of chest of drawers and three sofas, were analyzed as potential sources of indoor air pollution by large chamber test method (JIS A 1911). Based on the emission rates of total VOC (TVOC), the impacts on the indoor TVOC was estimated by the sample model with a volume of 20 m3 and ventilation frequency of 0.5 times/h. The estimated TVOC increment values were exceeded the provisional target value for indoor air (400 microg/m3) in three sets of dining tables, one set of chest of drawer and one sofa. The estimated increment of formaldehyde were exceeded the guideline value (100 microg/m3) in one set of dining table, two sets of chest of drawers and one sofa. These results revealed that VOC emissions from furniture may influence significantly indoor air quality. Also, in this study, to establish the alternative method for large chamber test methods, emission rates from representative three parts of furniture unit were evaluated using the small chamber and emission rate from full-sized furniture was predicted. Emission rates of TVOC and formaldehyde predicted by small chamber test were 3-46% and 6-252% of the data obtained using large chamber test, respectively.

[Volatile organic compounds (VOCs) emitted from wood furniture--estimation of emission rate by passive flux sampler].

The aim of this study was to evaluate aldehydes and other volatile organic compounds (VOCs) emission from furniture, which may cause hazardous influence on human being such as sick building/sick house syndrome. In this study, VOCs emitted from six kinds of wood furniture, including three set of dining tables and three beds, were analyzed by large chamber test method (JIS A 1911). Based on the emission rates of total VOCs (TVOC), the impacts on the indoor TVOC was estimated by the simulation model with volume of 20 m3 and ventilation frequency of 0.5 times/h. The estimated increment of formaldehyde were exceeded the guideline value (100 microg/m3) in one set of dining table and one bed. The estimated TVOC increment values were exceeded the provisional target value for indoor air (400 microg/m3) in two sets of dining tables and two beds. These results revealed that VOC emissions from wood furniture may influence significantly indoor air quality. Also, in this study, to establish the alternative method for large chamber test methods, emission rates from representative five areas of furniture unit were evaluated by passive sampling method using flux sampler and emission rate from full-sized furniture was predicted. Emission rates predicted by flux passive sampler were 10-106% (formaldehyde) and 8-141% (TVOC) of the data measured using large chamber test, respectively.

Involvement of macrophage inflammatory protein 1alpha (MIP1alpha) in promotion of rat lung and mammary carcinogenic activity of nanoscale titanium dioxide particles administered by intra-pulmonary spraying.

Titanium dioxide (TiO(2)) is evaluated by World Health Organization/International Agency for Research on Cancer as a Group 2B carcinogen. The present study was conducted to detect carcinogenic activity of nanoscale TiO(2) administered by a novel intrapulmonary spraying (IPS)-initiation-promotion protocol in the rat lung. Female human c-Ha-ras proto-oncogene transgenic rat (Hras128) transgenic rats were treated first with N-nitrosobis(2-hydroxypropyl)amine (DHPN) in the drinking water and then with TiO(2) (rutile type, mean diameter 20 nm, without coating) by IPS. TiO(2) treatment significantly increased the multiplicity of DHPN-induced alveolar cell hyperplasias and adenomas in the lung, and the multiplicity of mammary adenocarcinomas, confirming the effectiveness of the IPS-initiation-promotion protocol. TiO(2) aggregates were localized exclusively in alveolar macrophages and had a mean diameter of 107.4 nm. To investigate the underlying mechanism of its carcinogenic effects, TiO(2) was administered to wild-type rats by IPS five times over 9 days. TiO(2) treatment significantly increased 8-hydroxydeoxy guanosine level, superoxide dismutase activity and macrophage inflammatory protein 1alpha (MIP1alpha) expression in the lung. MIP1alpha, detected in the cytoplasm of TiO(2)-laden alveolar macrophages in vivo and in the media of rat primary alveolar macrophages treated with TiO(2) in vitro, enhanced proliferation of human lung cancer cells. Furthermore, MIP1alpha, also detected in the sera and mammary adenocarcinomas of TiO(2)-treated Hras128 rats, enhanced proliferation of rat mammary carcinoma cells. These data indicate that secreted MIP1alpha from TiO(2)-laden alveolar macrophages can cause cell proliferation in the alveoli and mammary gland and suggest that TiO(2) tumor promotion is mediated by MIP1alpha acting locally in the alveoli and distantly in the mammary gland after transport via the circulation.

Activation of the human transient receptor potential vanilloid subtype 1 by essential oils.

Transient receptor potential vanilloid subtype 1 (TRPV1) is a non-selective cation channel activated by capsaicin. TRPV1 is expressed not only on human sensory neurons but also on human epidermal and hair follicle keratinocytes. Therefore, TRPV1 could have the potential to be a therapeutic target for skin disorders. To search for novel TRPV1 agonists, we screened 31 essential oils by using human TRPV1-expressing HEK293 cells. TRPV1 was activated by 4 essential oils: rose, thyme geraniol, palmarosa, and tolu balsam. The dose-response curves for TRPV1 activation by the essential oils revealed a rank order potency [the half-maximal effective concentration (EC(50))] of rose>palmarosa>thyme geraniol>tolu balsam, and rank order efficiency (% activity in response to 1 microM capsaicin) of tolu balsam>rose>palmarosa>thyme geraniol. Moreover, the dose-response curves for TRPV1 activation by citronellol (main constituent of rose oil) and geraniol (main constituent of thyme geraniol and palmarosa oils) were consistent with the potency and efficiency of each essential oil. In contrast, benzyl cinnamate and benzyl benzoate (main constituent of tolu balsam oil) and geranyl acetate (main constituent of thyme geraniol oil) did not show TRPV1 activity. In this first-of-its-kind study, we successfully investigated the role of some essential oils in promoting human TRPV1 activation, and also identified two monoterpenes, citronellol and geraniol, as new human TRPV1 agonists.

[Analysis of preservatives used in cosmetic products: salicylic acid, sodium benzoate, sodium dehydroacetate, potassium sorbate, phenoxyethanol, and parabens].

Preservatives are used to inhibit the growth of microorganisms in cosmetic products. The Japanese standards for cosmetics set restrictions on the maximum amount of each preservative added to cosmetics as per the purpose of use of cosmetics. For the investigation into the actual conditions of commonly used preservatives in commercial cosmetics, we analyzed parabens, phenoxyethanol, sodium benzoate, sodium dehydroacetate, salicylic acid, and potassium sorbate by high-performance liquid chromatography (HPLC). Twenty-one samples were obtained from cosmetic product manufacturers located in 14 prefectures in Japan. Among different acid- and salt-based preservatives, sodium benzoate was observed to have been used in many products. These acid- and salt-based preservatives were used with parabens in personal washing products, such as shampoo and soap. The labels of two of the cosmetic product samples displayed inaccurate ingredient information, that is, a preservative other than the one used in the corresponding product was listed on them. The amount of preservatives used did not exceed regulatory limits in any of the analyzed samples.

[Volatile organic compounds (VOCs) emitted from furniture and electrical appliances].

Organic chemicals are widely used as ingredients in household products. Therefore, furniture and other household products as well as building products may influence the indoor air quality. This study was performed to estimate quantitatively influence of household products on indoor air quality. Volatile organic compound (VOC) emissions were investigated for 10 products including furniture (chest, desk, dining table, sofa, cupboard) and electrical appliances (refrigerator, electric heater, desktop personal computer, liquid crystal display television and audio) by the large chamber test method (JIS A 1912) under the standard conditions of 28 degrees C, 50% relative humidity and 0.5 times/h ventilation. Emission rate of total VOC (TVOC) from the sofa showed the highest; over 7900 microg toluene-equivalent/unit/h. Relatively high TVOC emissions were observed also from desk and chest. Based on the emission rates, the impacts on the indoor TVOC were estimated by the simple model with a volume of 17.4 m3 and ventilation frequency of 0.5 times/h. The estimated TVOC increment for the sofa was 911 microg/m3, accounting for almost 230% of the provisional target value, 400 microg/m3. The values of estimated increment of toluene emitted from cupboard and styrene emitted from refrigerator were 10% and 16% of guideline values, respectively. These results revealed that VOC emissions from household products may influence significantly indoor air quality.

[Research strategy for evaluation methods of the manufactured nanomaterials in NIHS and importance of the chronic health effects studies].

Manufactured nanomaterials are one of the most important substances for the nanotechnology. The nanomaterials possess different physicochemical properties from bulk materials. The new properties may lead to novel biological effects and also may or may not cause unknown adverse effects. However, the toxicological evidences are very limited, and there are no standardized evaluation methods at present. Some domestic and international activities are ongoing, in order to share the information or to standardize the methods. In 2005, our institute launched the research on the establishment of health risk assessment methodology of manufactured nanomaterials by funding from the research grants of the Japanese Ministry of Health, Labour and Welfare. The project contains four themes. The first is development of measurement methods of nanomaterials from biological samples. The second is development of dispersion methods in in vitro systems. The third is development of inhalation exposure systems. And the last is development of in vivo systems for evaluating long-term health effects. As evaluation materials, fullerene, titanium oxide and multi-walled carbon nanotubes were chosen because of their high production volumes. In the course of the research project, we revealed that the nanomaterials were competent to cause chronic effects, by analyzing intraperitoneal administration studies and carcinogenic promotion studies. These studies suggested that even aggregated nanomaterials were crumbled into nano-sized particles inside the body during the long-term, and the particles were transferred to other organs. Additionally, long lasting particles/fibers in the particular tissues may cause chronic adverse effects. The phsyco-chemical properties or toxicity mechanism related with these chronic effects were considered to be different from those properties or mechanism related to acute toxicity. Therefore, we suggested that the toxicological characterization of chronic effects by nanomaterials would be important for the future research. Also, investigations of the toxicokinetic properties and biological interaction with nanomaterials are important to predict the chronically targeted tissues after exposure.

[Studies on an analytic method for detection of prohibited ingredient, strontium dichloride in cosmetics].

Strontium dichloride is one of the prohibited ingredients in cosmetics due to the Japanese Pharmaceutical Affairs Act. We established the analytical method for strontium dichloride in cosmetics by capillary electrophoresis (CE). The toothpaste was dispersed into water. After ultrasonication for 10 min, the solution was centrifuged at 3000 rpm for 10 min. The supernatant was filtrated through a membrane (0.45 microm), diluted 100-times with water, and injected into CE. The calibration curve showed linear between the concentrations of strontium dichloride (from 2 to 50 microg/ml) and the peak area of strontium ion. Detection limit of strontium dichloride is 2 microg/ml. There was no interference of the ingredients in the toothpaste.

[Detection of clobetasol propionate in a cream advertised to be effective against atopic dermatitis].

Addition of medical ingredients to cosmetics is prohibited. However, last year some cases of illegal cosmetics containing steroids were successfully identified. We have already reported an analytical method to detect steroids in cosmetics [Bull. Natl. Inst. Health Sci, 126, 51-56 (2008)]. In this study, we initially examined whether this method could be applied for the detection of some new steroids as target chemicals. We then used this developed method to detect steroids in cosmetics obtained from manufacturers by spot checks. These manufacturers have been advertising the effectiveness of a steroid-free cream against atopic dermatitis. The results revealed that clobetasol propionate (CP) was present in this facial moisturizing cream, which was available in the market. The steroid was extracted with methanol. After ultrasonication and centrifugation, the resulting supernatant was injected into the high-performance liquid chromatography system equipped with an ODS column. The separation was achieved using a mixture of acetonitrile and water as the mobile phase. The retention times of the observed peaks were in accordance with those of some preservatives and CP. The presence of CP was also confirmed by thin-layer chromatography. The concentration of CP in the cream was approximately 0.039%. CP is a steroid that has the strongest effect as compared to those of other steroids. The cream was therefore recalled for safety reasons.

[Development of a liquid chromatography-tandem mass spectrometry method for the determination of fullerenes C60 and C70 in biological samples].

Wide application of fullerenes in various areas would increase the risk of occupational and environmental exposure to human. However, information about toxicity and biological behavior of fullerenes is not sufficient for the risk assessment at present. For the determination of fullerene C60 in biological samples, an analytical method using high performance liquid chromatography--tandem mass spectrometry (LC-MS/MS) and extraction procedure from tissues of experimental animals was established in this study. Using LC-MS/MS with an atmospheric pressure chemical ionization in negative mode, C60 were identified and quantified. After optimization of mobile phase and separation column, good separation of peak of fullerene and sensitivity were obtained in case of using toluene and acetonitrile as the mobile phases and Develosil RPFULLERENE as the separation column. For method validation, rat brain, kidney, liver, lung, spleen tissues and blood were used for recovery tests. Good results were obtained and the recovery percentages were found to be between 98.1% and 106.5%.

Hepatotoxicity and Drug/Chemical Interaction Toxicity of Nanoclay Particles in Mice.

Nanomaterials are relatively new and unconventional materials with many useful properties, but their effects on biological systems are poorly understood. Nanoclay is a general term for layered mineral silicate nanoparticles that are ideally suited for use in clay-based nanocomposites. The potential biological hazards of nanoclays have not been addressed, however. Therefore, we investigated the in vivo effects and drug interactions of nanoclays. In mice, administration of nanoclay particles via the tail vein led to acute liver injury. Co-administration of nanoclay and carbon tetrachloride, paraquat, or cisplatin resulted in both liver and kidney injury. Our findings thus indicate that nanoclay particles are potentially hepato- and nephrotoxic.

Chronic toxicity of an environmentally relevant mixture of pharmaceuticals to three aquatic organisms (alga, daphnid, and fish).

Principles of concentration addition and independent action have been used as effective tools to predict mixture toxicity based on individual component toxicity. The authors investigated the toxicity of a pharmaceutical mixture composed of the top 10 detected active pharmaceutical ingredients (APIs) in the Tama River (Tokyo, Japan) in a relevant concentration ratio. Both individual and mixture toxicities of the 10 APIs were evaluated by 3 short-term chronic toxicity tests using the alga Pseudokirchneriella subcapitata, the daphnid Ceriodaphnia dubia, and the zebrafish Danio rerio. With the exception of clarithromycin toxicity to alga, the no-observed-effect concentration of individual APIs for each test species was dramatically higher than the highest concentration of APIs found in the environment. The mixture of 10 APIs resulted in toxicity to alga, daphnid, and fish at 6.25 times, 100 times, and 15,000 times higher concentrations, respectively, than the environmental concentrations of individual APIs. Predictions by concentration addition and independent action were nearly identical for alga, as clarithromycin was the predominant toxicant in the mixture. Both predictions described the observed mixture toxicity to alga fairly well, whereas they slightly underestimated the observed mixture toxicity in the daphnid test. In the fish embryo test, the observed toxicity fell between the predicted toxicity by concentration addition and independent action. These results suggested that the toxicity of environmentally relevant pharmaceutical mixtures could be predicted by individual toxicity using either concentration addition or independent action.

Analysis of herbicides in water using temperature-responsive chromatography and an aqueous mobile phase.

A simple and rapid method has been developed for herbicides in water using temperature-responsive liquid chromatography (LC) and a column packed with poly(N-isopropylacrylamide) (PNIPAAm), a polymer anchored on the stationary-phase surface of modified silica. PNIPAAm reversibly changes its hydrophilic/hydrophobic properties in water in response to temperature. The method was used to determine five sulfonylurea and three urea herbicides. Separation was achieved with a 10 mM ammonium acetate (pH 3.0) isocratic aqueous mobile phase, and by changing the column temperature. The analytes were extracted from water by off-line solid-phase extraction (SPE) with an N-vinyl-pyrrolidone polymer cartridge. The average recoveries of the eight herbicides from spiked pure water, tap water and river water were 70-130% with relative standard deviations (RSDs) of <10%. The limits of quantitation (LOQ) of the eight herbicides were between 1 and 4 microg l(-1).