Estrogenic activity in Finnish municipal wastewater effluents.

Effluents from wastewater treatment plants (WWTPs) are a major source of estrogenic compounds to the aquatic environment. In the present work, estrogenic activities of effluents from eight municipal WWTPs in Finland were studied. The main objectives of the study were to quantify the concentrations of selected estrogenic compounds, to evaluate their contribution to estrogenic potency and to test the feasibility of the commercial bioassays for wastewater analysis. The effluent samples were analyzed by two in vitro tests, i.e. ERα-CALUX(®) and ELISA-E2, and by liquid chromatography mass spectrometry for six estrogenic compounds: estrone (E1), 17ß-estradiol (E2), estriol (E3), 17α-ethinylestradiol (EE2), 17α-estradiol and bisphenol A (BPA). Estrogenic effects were found in all of the effluent samples with both of the bioassays. The concentrations measured with ELISA-E2 (8.6-61.6 ng/L) were clearly higher but exhibited a similar pattern than those with chemical analysis (E2

Weight-of-evidence approach in assessment of ecotoxicological risks of acid sulphate soils in the Baltic Sea river estuaries.

Acidity and leaching of metals from acid sulphate soils (ASSs) impair the water quality of receiving surface waters. The largest ASS areas in Europe are found in the coasts of the northern Baltic Sea. We used weight-of-evidence (WoE) approach to assess potential risks in 14 estuary sites affected by ASS in the Gulf of Finland, northern Baltic Sea. The assessment was based on exposure and effect profiles utilizing sediment and water metal concentrations and concurrent pH variation, sediment toxicity tests using the luminescent bacterium Vibrio fischeri and the midge Chironomus riparius, and the ecological status of benthic macroinvertebrate communities. Sediment metal concentrations were compared to national sediment quality criteria/guidelines, and water metal concentrations to environmental quality standards (EQSs). Hazard quotients (HQs) were established for maximum aluminium, cadmium and zinc concentrations at low pH based on applicable US EPA toxicity database. Sediment metal concentrations were clearly elevated in most of the studied estuaries. The EQS of cadmium (0.1 µg/l) was exceeded in 3 estuaries out of 14. The pH-minima were below the national threshold value (5.5) between good and satisfactory water quality in 10 estuaries. V. fischeri bioluminescence indicated toxicity of the sediments but toxic response was not observed in the C. riparius emergence test. Benthic invertebrate communities were deteriorated in 6 out of 14 sites based on the benthic invertebrate quality index. The overall ecotoxicological risk was assessed as low in five, moderate in three and high in five of the estuary sites. The risk assessment utilizing the WoE approach indicated that harmful effects of ASSs are likely to occur in the Baltic Sea river estuaries located at the ASS hotspot area.

Assessment of ecological risks at former landfill site using TRIAD procedure and multicriteria analysis.

Old industrial landfills are important sources of environmental contamination in Europe, including Finland. In this study, we demonstrated the combination of TRIAD procedure, multicriteria decision analysis (MCDA), and statistical Monte Carlo analysis for assessing the risks to terrestrial biota in a former landfill site contaminated by petroleum hydrocarbons (PHCs) and metals. First, we generated hazard quotients by dividing the concentrations of metals and PHCs in soil by the corresponding risk-based ecological benchmarks. Then we conducted ecotoxicity tests using five plant species, earthworms, and potworms, and determined the abundance and diversity of soil invertebrates from additional samples. We aggregated the results in accordance to the methods used in the TRIAD procedure, conducted rating of the assessment methods based on their performance in terms of specific criteria, and weighted the criteria using two alternative weighting techniques to produce performance scores for each method. We faced problems in using the TRIAD procedure, for example, the results from the animal counts had to be excluded from the calculation of integrated risk estimates (IREs) because our reference soil sample showed the lowest biodiversity and abundance of soil animals. In addition, hormesis hampered the use of the results from the ecotoxicity tests. The final probabilistic IREs imply significant risks at all sampling locations. Although linking MCDA with TRIAD provided a useful means to study and consider the performance of the alternative methods in predicting ecological risks, some uncertainties involved still remained outside the quantitative analysis.

Behavior of titanium dioxide nanoparticles in Lemna minor growth test conditions.

Titanium dioxide nanoparticles (TiO(2) NPs) have raised concern of environmental risks due to their widespread applications, but little is known about the potential toxicity of TiO(2) NPs to aquatic plants. The aim of this work was to study the effects of TiO(2) NPs on Lemna minor and to study the behavior of TiO(2) NPs under modified ISO 20079 test conditions. TiO(2) NPs had a tendency to aggregate in ISO (Steinberg) growth medium, but modification of the standard growth medium enabled the exposure of L. minor to TiO(2) NPs. By dilution of the growth medium (1:10), and exposure under semi-static conditions with medium renewal every second or third day, the size of TiO(2) particles remained rather stable throughout the test period. TiO(2) NPs showed no adverse effect on the growth rate or chlorophyll a content of L. minor, even at a high exposure concentration of 5 mg L(-1) and extended exposure time of 14 days. TiO(2) NPs attached onto L. minor cell walls, but no cellular uptake was observed. Although TiO(2) NPs were not toxic to L. minor, the potential transfer of TiO(2) NPs in aquatic food chains, e.g. attached to the plant leaves and other biological surfaces may be of importance, causing exposure of other organisms and contributing to the environmental fate of nanoparticles.

More accuracy to the EROD measurements--the resorufin fluorescence differs between species and individuals.

Ethoxyresorufin-O-deethylase (EROD) activity is a biomarker of exposure to planar aromatic hydrocarbons, and it is often measured from the S9 fraction. The effect of the liver S9 fraction of seven boreal freshwater fish species on the fluorescence of resorufin was studied. The S9 fractions diminished resorufin fluorescence by 40-80%, and there were large differences between species. Thus, using a resorufin standard curve without the S9 fraction leads to a large underestimation of the EROD activity. Therefore a microwell plate EROD method was developed that takes into account the effect of each sample on resorufin fluorescence. At least two mechanisms were involved in the decrease of the fluorescence: opaqueness of the sample, and enzymes (DT-diaphorase and plausibly NADPH-CYP450 oxidoreductase) that reduce resorufin to a non-fluorescent form.

Size-exclusion chromatographic study of ECF and TCF softwood kraft pulp bleaching liquors.

BACKGROUND, AIMS, AND SCOPE: Currently, elemental chlorine-free (ECF) and totally chlorine-free (TCF) bleaching systems are widely used for pulp production. Low and medium molecular weight lignin break-down products are known to have harmful effects on the environment. According to some recent results, also high molecular weight (HMW) material consisting mainly of lignin and carbohydrates may cause toxic effects to the environment. For these reasons, toxicity and structure studies of HMW materials are of great importance. This investigation is a part of a larger project to obtain more structure information of HMW materials and toxicity of ECF and TCF bleaching effluents. Size-exclusion chromatography (SEC) has been commonly used for the characterization of organic macromolecules such as lignin, but to our knowledge, no reports have appeared dealing with the comparison of SEC of ECF and TCF bleaching liquors. The aim of the present study was to get more information about the molecular weight distribution (MWD) of HMW fractions of waste liquors from ECF and TCF bleaching sequences by SEC. METHODS: The MWDs of organic materials dissolved during different stages of ECF bleaching (O-D-EOP-D-ED) and TCF bleaching (O-Z-Q-P-Z-Q-P-P) of softwood (Pinus sylvestris) kraft pulp were determined and compared by SEC. All effluent samples from the above bleaching stages were ultrafiltrated using a membrane with a cutoff value of 1,000 Da. SEC was performed on high and also low molecular weight fractions and non-fractionated effluents. In the SEC experiments, a Superdex 75 column was used with 0.1 M NaOH solution as the eluent. Standards used for calibrating the SEC system were albumin, carboanhydrase, cytochrome C, tannic acid, dehydrodiacetovanillone, and vanillin. RESULTS AND DISCUSSION: The chromatograms of liquors from TCF bleaching stages vary more than those from ECF bleaching. Peroxide and chelating stages contained mostly high molecular weight (HMW) matter whereas chlorine dioxide and ozone stages had more low molecular weight compounds. The lignin content in HMW matter was higher than in stages that consisted of low molecular matter. Bleaching effluents contained the highest amounts of HMW material, mainly lignin, in the beginning of the sequences; the amounts decreased towards the end of the bleaching sequence. CONCLUSION: Determinations of MWD by the SEC method showed that effluents from the TCF sequence contained more HMW material than those from the ECF stage. This might be due to peroxide stages (P) that dissolve HMW lignin effectively. However, the molecular weights of ozone stages (Z) were very low compared to other stages. Chlorine dioxide stages also dissolved mostly low molecular weight lignin. Ultrafiltration of bleaching liquors showed that high molecular weight fraction also included some low molecular weight compounds and vice versa. High polydispersity and high lignin content correlated with the amount of HMW material in ECF and TCF bleaching stages. RECOMMENDATION AND OUTLOOK: Our liquor samples were studied by using a UV detector commonly used for lignin preparations; in upcoming investigations, it will be interesting to determine carbohydrates such as hemicelluloses. The results are applicable in papermaking in order to improve commonly used bleaching procedures, to test new potential bleaching systems, and to study chemical behavior of HMW materials in various bleaching liquors. The present results also form a good basis for toxicity measurements of ECF and TCF bleaching effluents and for more comprehensive spectroscopic and chromatographic experiments with samples taken from various bleaching stages. From the behavior of liquors studied, it appears that our other structure investigations by spectroscopic and chromatographic (NMR, Py-GC/MS, etc.) methods mostly correlate well with the present results.

Biological effects of high molecular weight lignin derivatives.

A number of high molecular weight (HMW) lignin derivatives possessing varied chemical properties were screened for their biological effects in order to obtain more information on the possible structural features of HMW lignin-related effects. The studied compounds were both commercial and in-house extracted lignin derivatives. Bioassays used include reverse electron transport (RET), Vibrio fischeri, Daphnia magna, and juvenile rainbow trout (Oncorhynchus mykiss) hepatocytes. The studied lignin derivatives inhibited the in vitro systems and luminescence of V. fischeri bacteria to some extent-daphnids were not affected. It seems that, at least in the RET assay, certain pH-dependent functional groups in lignin may be of importance regarding the biological effects.

Multivariate statistics of the pyrolysis products of high molecular components in pulping wastewaters to explain their toxicity.

BACKGROUND, AIM AND SCOPE: At present, large-scale paper manufacture involves delignification and bleaching by elemental chlorine free (ECF), or totally chlorine free (TCF) processes. The wastewater is purified by secondary treatment (mechanical, chemical and biological) which removes most of the toxic substances from the discharge. However, we found residual toxicity in the high molecular (> 1000 D) matter (HMWM) of the discharge by test of the RET (reverse electronic transfer) inhibition. This fraction consists mainly of polydispersed lignin (LIG) and carbohydrate (CH) macromolecules. Structural units in these molecules are studied by pyrolysis gas chromatography/mass spectrometry (Pyr-GC/MS). In the present work, our aim was to find out those structural units which could explain the RET toxicity of LIG or CH molecules. We compared statistically RET toxicity values of the HMWM samples from treated wastewaters of pilot pulping experiments and intensity variation of the pyrolysis product gas chromatograms of these samples. This application is a novel study procedure. METHODS: Pyrolysis products (Py-GC/MS results) and inhibition of RET (reverse electronic transport toxicity) as TU50 and TU20 of HMWM (High Molecular Weight Material; Mw > 1000 D) were compared by multivariate statistics. The samples were from laboratory pilot stages of TCF (Totally Chlorine Free) and ECF (Elemental Chlorine Free) manufacture of softwood pulp. Py-GC/MS was done without and with the addition of TMAH (Tetra Methyl Ammonium Hydroxide). The name and structure of each abundant fragment compound was identified from its retention time and mass spectrum compared to authentic reference compounds or literature. Four sets of Toxicity Units (TUs) and GC peak areas of the pyrolysis fragments were obtained. The data were normalized by division with LIG (lignin content of each sample). TU values were dependent and the fragment values independent (explanatory) variables in statistical treatments by SPSS system. Separate analyses of correlations, principal components (PCA) and stepwise multiple linear regression (SMLR) were performed from the four sample sets TCF and ECF with and without TMAH. RESULTS AND DISCUSSION: From the CH fragments, 2-furfural in TCF, and from the LIG fragments, styrene in ECF showed the highest probabilities of originating from source structures of toxicity. Other possible compounds in concern were indicated to be CH fragment 2-methyl-2-cyclopenten-1-one in ECF and LIG fragments 2-methoxy-4-methylphenol, 4,5-dimethoxy-2-methylphenol and 2-methylphenol in TCF.

Evaluation of wastewater effluents by small-scale biotests and a fractionation procedure.

Municipal and industrial effluents were screened with a battery of biotests and with a modified toxicity identification evaluation Phase I procedure. The acute toxicities of the effluent samples were low and the submitochondrial reverse electron-transport (RET) test was the most sensitive toxicity test. Estrogenic effects were found in almost all effluent samples, and genotoxicity was detected in one concentrated effluent sample. The fractionation methods we used proved to be especially effective at tracking toxicity caused by metals and organic contaminants, with the RET test being particularly suited to evaluating pH-dependent toxicity. The used solid-phase extraction columns with both hydrophilic and hydrophobic binding properties turned out to be suitable for removing or reducing organic toxicity-causing substances from the effluent samples. The results of this study show that the use of only conventional acute toxicity tests for effluent assessment will not be sufficient-the genotoxic, hormonal, and even bioaccumulative potential of the effluents and effluent fractions should be evaluated as well.

Hazardous properties of paint residues from the furniture industry.

The objective of this study was to screen nine excess paint residues for environmental hazard and to evaluate their disposability in a non-hazardous or hazardous-waste landfill. These residues were produced in the process of spray-painting furniture. Residues were classified according to their leaching and ecotoxicological properties. Leaching properties were determined with the European standard SFS-EN 12457-2 leaching-test. The toxicity of the leaching-test eluates was measured with plant-, bacteria- and enzyme-inhibition bioassays. Total organic carbon, formaldehyde and solvent concentrations in the solid wastes and in the leaching-test eluates were analysed. It seemed likely that leached formaldehyde caused very high acute toxicity in leaching-test eluates of the dry-booth residues. This hypothesis was based on the fact that the formaldehyde concentrations in the leaching-test eluates of the dry-booth residues were 62-75 times higher than the EC50 value reported in the literature for formaldehyde. The results of the water-curtain booth residues showed that the samples with the highest TOC and aromatic solvent concentrations were also the most toxic. The studied excess paint residues were complex organic mixtures and contained large amounts of compounds not identifiable from chemical data. Therefore, the evaluation of the hazard based solely on available chemical data is unlikely to be sufficient, as evidenced by our study. Our results show that harmful compounds remain in the solid waste and the toxicity results of their leaching-test eluates show that toxicity may leach from residues in contact with water at landfill sites. They also confirm the benefit of combining chemical and ecotoxicological assays in assessing the potential environmental hazard of complex organic mixtures found in wastes.

Extractability of metals and ecotoxicity of soils from two old wood impregnation sites in Finland.

Four metal-contaminated soil samples were classified using physical methods, extracted by selective extraction procedures and analyzed for chemical concentrations. De-ionized water, 0.01 mol/l barium chloride, 1 mol/l ammonium acetate and concentrated nitric acid were used as extraction solutions. Ecotoxicity of water extracts and soil samples was analyzed in order to describe the bioavailability of the contaminants. Samples from old wood impregnation plants contained high amounts of As, Cu, Cr and Zn, which originated from chromated copper arsenate, ammoniacal copper-zinc arsenate, and ammoniacal copper quaternary compound. Total As concentrations of the heavily contaminated samples varied from 752 to 4340 mg/kg, Cu concentrations from 339 to 2330 mg/kg, Cr concentrations from 367 to 2,140 mg/kg and Zn concentrations from 79 to 966 mg/kg. The extractabilities of metals differed according to soil type, extractant and element. Cu and Zn were proposed to cause the highest toxicity in the water extracts of the soils. Ecotoxicity tests displayed rather high differences in sensitivity both for water extracts and for solid soil samples. Reproduction of Enchytraeus sp. was the most sensitive and seed germination of Lactuca sativa the least sensitive and the other tests were in decreasing order of sensitivity: Folsomia candida>reverse electron transport>MetPLATE>Toxichromotest>Allium cepa root growth>Lemna sp. growth. As a conclusion, polluted soils rich in sand retain heavy metals with less firm bindings, particularly in the case of Cu and Zn, than soils rich in clay, indicating that chemical methods for measuring the bioavailability of metals need to be optimized taking into account the soil type, acidity, redox state and the individual contaminants.

Toxicity of industrial wastes and waste leaching test eluates containing organic compounds.

Leaching tests, CEN prEN 12457-2, CEN PrEN 12457-3, and NEN 7349, were conducted for varnish residue and urea resin waste, two industrial wastes containing organic chemicals. The leaching test eluates were analyzed for solvent concentrations and total organic carbon. Aqueous leaching tests were found to be suitable for both chemical and biological testing. Ecotoxicity was assessed by luminescent bacteria, plant root growth, reverse electron transport, and ToxiChromopad. The eluates were highly toxic but the toxicity decreased in later stages of the multistep leaching test (NEN 7349). Urea resin eluates were significantly less toxic than varnish residue eluates. The solid wastes markedly inhibited plant seed germination before and after the leaching test (CEN prEN 12457-3). The solid wastes were not biologically degradable in the standard test (ISO 14593), whereas the eluates from the CEN prEN 12457-3 test degraded slowly.