Toxic and endocrine disrupting effects of wastewater treatment plant influents and effluents on a freshwater isopod Asellus aquaticus (Isopoda, Crustacea).

In the present study a biological "in vivo" assay, with freshwater isopod Asellus aquaticus, was used to define and evaluate the potential impact of the wastewater treatment plant (WWTP) wastewaters on local wildlife. Samples of both untreated and mechanically and biologically treated WWTP wastewater, were tested in the presence and absence of the formulated sediment for their lethal and sublethal effects. Chronic exposures to wastewater samples caused concentration dependent reduced locomotion, body pigmentation and molting frequency in exposed organisms. The observed effects indicate the overall toxicity and endocrine disruption of the wastewater samples. In contrast stimulations of the feeding rate and growth rate of the test organisms during the chronic exposure to sublethal levels of wastewater samples were observed, indicating an improvement in nutritional quality of the wastewater. The most sensitive exposure endpoint was molting frequency of tested organisms, which indicated the presence of estrogenically active endocrine disrupting compounds (EDCs). Raw wastewater caused up to 42% molting frequency reduction of exposed A. aquaticus when exposed to five times diluted untreated wastewater sample, while undiluted treated wastewater caused a 61% molting frequency reduction. The presence of estrogenically active compounds in the wastewater was confirmed with the yeast estrogen screen assay (YES test), which assigned the highest estrogenic activity to a mechanically and biologically treated wastewater sample, and lower estrogenic activity to all other tested samples. The importance of presence of the formulated sediment was determined, as it lessened the effects of all WWTP wastewater samples in all observed exposures.

Hazard identification and risk characterization of bisphenols A, F and AF to aquatic organisms.

Production of bisphenol A (BPA) analogues such as bisphenol F (BPF) and bisphenol AF (BPAF) has recently increased, due to clear evidence of adverse effects of BPA on humans and wildlife. Bisphenols (BPs) have already been released into aquatic environment without previous available information about potential adverse effects of BPs and their potential risk to aquatic ecosystems. In this study, lethal and sublethal effects of BPF and BPAF to bacteria, algae, crustacea and fish embryos were investigated and the results were compared to the adverse effects obtained for BPA. We found that BPAF was the most toxic compound to Daphnia magna, Danio rerio and Desmodesmus subspicatus; the lowest 72 h EC50 (median effective concentration) and 21 d NOEC (no observed effect concentration) values were determined at 2.2 mg/L regarding zebrafish hatching success and 0.23 mg/L of BPAF obtained for growth and reproduction of water fleas, respectively. In most cases, BPA was more toxic to D. magna, D. rerio and D. subspicatus in comparison to BPF, but pigmentation of zebrafish embryos after 48 h of exposure and reproduction of water fleas after 21-day D. magna reproductive test exposure to BPF were much more impaired. Risk quotients (measured environmental concentration/21 d NOEC) showed that BPA, BPF and BPAF are recently not chronically hazardous to the survival, reproduction and growth of water fleas in surface waters. On the other hand, we importantly show that currently present BPAF concentrations in surface waters could cause a potential ecological risk to aquatic organisms. In the near future, higher concentrations of BPF and BPAF in surface waters are anticipated and for this reason further testing using test systems with various aquatic species and endpoints are needed to provide additional information about toxic impacts of BPF and BPAF on aquatic biota.

Unexpected toxicity to aquatic organisms of some aqueous bisphenol A samples treated by advanced oxidation processes.

In this study, photocatalytic and catalytic wet-air oxidation (CWAO) processes were used to examine removal efficiency of bisphenol A from aqueous samples over several titanate nanotube-based catalysts. Unexpected toxicity of bisphenol A (BPA) samples treated by means of the CWAO process to some tested species was determined. In addition, the CWAO effluent was recycled five- or 10-fold in order to increase the number of interactions between the liquid phase and catalyst. Consequently, the inductively coupled plasma mass spectrometry (ICP-MS) analysis indicated higher concentrations of some toxic metals like chromium, nickel, molybdenum, silver, and zinc in the recycled samples in comparison to both the single-pass sample and the photocatalytically treated solution. The highest toxicity of five- and 10-fold recycled solutions in the CWAO process was observed in water fleas, which could be correlated to high concentrations of chromium, nickel, and silver detected in tested samples. The obtained results clearly demonstrated that aqueous samples treated by means of advanced oxidation processes should always be analyzed using (i) chemical analyses to assess removal of BPA and total organic carbon from treated aqueous samples, as well as (ii) a battery of aquatic organisms from different taxonomic groups to determine possible toxicity.

Adverse effects of bisphenol A on water louse (Asellus aquaticus).

Experiments were performed to study the effects of short and long-term exposure to bisphenol A (BPA) on a freshwater crustacean isopod Asellus aquaticus (L.). Two life stages of isopods were exposed to a range of BPA concentrations, from aqueous and two dietary sources, in the form of with BPA spiked conditioned alder leaf (Alnus glutinosa) discs, or spiked formulated sediment, to determine the relative importance of each source of exposure on the uptake of this contaminant. Several lethal and sublethal endpoints were evaluated in this study to measure the potential effects of BPA on A. aquaticus, including mortality, growth and feeding rate inhibition, mobility inhibition, de-pigmentation and molting disturbances. They signify a correlation to BPA levels and a difference in BPA uptake efficiency from different uptake sources. Results of acute exposure to BPA show a greater sensitivity of test systems using juvenile specimens with a 96 h LC50 of 8.6 mg L(-1) BPA in water medium and a 96 h LC50 of 13.5 mg L(-1) BPA in sediment. In comparison, adult isopods show a 96 h LC50 of 25.1 mg L(-1) BPA in water medium and a 96 h LC50 of 65.1 mg L(-1) BPA in sediment. Observed endpoints of chronic exposures suggest the alder leave discs to be the most efficient uptake source of BPA, in contrast to uptake from water or heterogeneous sediment. Significant (p<0.05) growth inhibition, with a 21d NOEC of 0.5/2.5 mg L(-1) (for juvenile/adult organisms), and feeding rate inhibition, with a 21d NOEC of 0.5/1.0 mg L(-1) (for juvenile/adult organisms), were proven to be the most sensitive toxicity endpoints. An even more sensitive effect turned out to be molting frequency, which was significantly reduced; a 21d NOEC was 1.0 mg L(-1) of BPA for adult organisms and an even lower 21d NOEC of 0.05 mg L(-1) of BPA for juveniles. The observed endpoints are recorded at very low, non-toxic exposure concentrations, indicating that BPA acts as an endocrine disrupting compound, as well as a toxic substance. We also determined the importance of the direct dietary uptake of the pollutants, significant for juveniles as well as adult animals.

Efficiency of advanced oxidation processes in lowering bisphenol A toxicity and oestrogenic activity in aqueous samples.

Bisphenol A (BPA) is a well-known endocrine disruptor with adverse oestrogen-like effects eliciting adverse effects in humans and wildlife. For this reason it is necessary to set up an efficient removal of BPA from wastewaters, before they are discharged into surface waters. The aim of this study was to compare the efficiency of BPA removal from aqueous samples with photolytic, photocatalytic, and UV/H2O2 oxidation. BPA solutions were illuminated with different bulbs (halogen; 17 W UV, 254 nm; and 150 W UV, 365 nm) with or without the TiO2 P-25 catalyst or H2O2 (to accelerate degradation). Acute toxicity and oestrogenic activity of treated samples were determined using luminescent bacteria (Vibrio fischeri), water fleas (Daphnia magna), zebrafish embryos (Danio rerio), and Yeast Estrogen Screen (YES) assay with genetically modified yeast Saccharomyces cerevisiae. The results confirmed that BPA is toxic and oestrogenically active. Chemical analysis showed a reduction of BPA levels after photolytic treatment and 100 % conversion of BPA by photocatalytic and UV/H2O2 oxidation. The toxicity and oestrogenic activity of BPA were largely reduced in photolytically treated samples. Photocatalytic oxidation, however, either did not reduce BPA toxic and oestrogenic effects or even increased them in comparison with the baseline, untreated BPA solution. Our findings suggest that chemical analysis is not sufficient to determine the efficiency of advanced oxidation processes in removing pollutants from water and needs to be complemented with biological tests.

Stability and toxicity of selected chlorinated benzophenone-type UV filters in waters.

In our study, the transformation of two most widely used UV filters, benzophenone-3 (BP3) and benzophenone-4 (BP4), in chlorinated water with disinfection reagents sodium hypochlorite (NaClO) and trichloroisocyanuric acid (TCCA) was studied. Based on the HPLC/MS and UV-Vis analysis the formation of two different chlorinated products (5-chloro-2-hydroxy-4-methoxybenzophenone and 3,5-dichloro-2-hydroxy-4-methoxybenzophenone) was established. Identity of chlorinated products was confirmed by means of comparison of retention times with independently synthesized standards. Photostability study showed that dichloro-derivative in water is less stable then parent compounds, which is not the case for monochloro-derivatives. Toxicity of chlorinated compounds tested by Vibrio fischeri was found to be in the same range as that of the starting compounds. Preliminary testing of real water samples from swimming pools and sea swimming areas confirmed the presence of BP3 and its 3,5-dichloro derivative.

Hindered disulfide bonds to regulate release rate of model drug from mesoporous silica.

With the advancement of drug delivery systems based on mesoporous silica nanoparticles (MSNs), a simple and efficient method regulating the drug release kinetics is needed. We developed redox-responsive release systems with three levels of hindrance around the disulfide bond. A model drug (rhodamine B dye) was loaded into MSNs' mesoporous voids. The pore opening was capped with ß-cyclodextrin in order to prevent leakage of drug. Indeed, in absence of a reducing agent the systems exhibited little leakage, while the addition of dithiothreitol cleaved the disulfide bonds and enabled the release of cargo. The release rate and the amount of released dye were tuned by the level of hindrance around disulfide bonds, with the increased hindrance causing a decrease in the release rate as well as in the amount of released drug. Thus, we demonstrated the ability of the present mesoporous systems to intrinsically control the release rate and the amount of the released cargo by only minor structural variations. Furthermore, an in vivo experiment on zebrafish confirmed that the present model delivery system is nonteratogenic.

Antioxidant responses and whole-organism changes in Daphnia magna acutely and chronically exposed to endocrine disruptor bisphenol A.

Bisphenol A (BPA) is a well known endocrine disruptor. Significantly less is known about its ability to induce antioxidant defense and oxidative stress in aquatic organisms. This is an interesting subject because BPA can act both as a prooxidant (induces the formation of reactive oxygen species) and an antioxidant. The aim of the present study was to elucidate if BPA induces changes in the activities of antioxidant enzymes catalase (CAT) and glutathione S-transferases (GSTs) and the level of lipid peroxidation in crustacean Daphnia magna. These biomarkers were followed after acute (48h) and chronic (21d) exposure period. Interestingly, CAT and GSTs were changed in the same manner after both exposure durations. The most observable change in daphnids was the increase of GSTs activities, measured with 1,2-chloro-4-dinitrobenzene as a substrate. No GSTs activities with ethacrynic acid and 1,2-dichloro-4-nitrobenzene as substrates were detected. The activity of CAT and the level of lipid peroxidation remained unchanged in daphnids exposed to sublethal concentrations of BPA. The reproduction rate was considerably affected already at 1.73mg/L while the growth was only affected at the highest concentration (13.8mg/L), where also significant mortality was observed.

Effects of four CeO2 nanocrystalline catalysts on early-life stages of zebrafish Danio rerio and crustacean Daphnia magna.

Effects of four different nanocrystalline CeO(2)-based catalysts on crustaceans Daphnia magna and early-life stages of zebrafish Danio rerio were studied. Pure CeO(2) and CuO-CeO(2) mixed oxides with a nominal 10, 15 and 20 mol.% CuO content were tested. Pure CeO(2) provoked no effects, but CuO-CeO(2) mixed oxides induced some sublethal effects on fish and affected daphnids' survival. The most pronounced effects were found on fish body growth, which was reduced at 10 mg/L in case of CuCe20 and 50 mg/L in cases of CuCe10 and CuCe15. Daphnids' survival was affected above 80 mg/L of CuCe20, while CuCe10 and CuCe15 did not affect daphnids. None of the materials was highly toxic to daphnids and fish in comparison to some other environmental pollutants. Differences in effects between the materials could not be explained by their specific physicochemical properties. This work indicates that more attention should be placed at potential toxicity of nanostructured materials, such as nanocrystalline mixed-oxides.

Assessment of landfill leachate toxicity reduction after biological treatment.

In the present article, the efficiency of biological treatment of landfill leachates was evaluated by implementation of physicochemical characterisation and a complex toxicity assessment. An array of toxicity tests using bacterium Vibrio fischeri, alga Desmodesmus subspicatus, crustacean Daphnia magna, and embryo of fish Danio rerio, as well as unconventional methods using biochemical biomarkers (protein content, enzymes cholinesterase, and glutathione-S-transferase), were employed. Toxicity of leachates varied depending on the season of collection in relation to their different physicochemical characteristics. Uncommon effects of leachates on organisms, such as hormetic-like increases of algal growth and reproduction of daphnids, were identified. New approaches using the activities of enzymes were found unsuitable for routine hazard assessment of leachates. Although physicochemical parameters and toxicity decreased significantly after biological treatment, the effluents did not meet the demands of the current Slovenian legislation; thus, the existing biological treatment was found inappropriate. The development of advanced treatment techniques for landfill leachates is thus encouraged.

Conversion and Estrogenicity of 17ß-estradiol During Photolytic/Photocatalytic Oxidation and Catalytic Wet-air Oxidation.

Estrogen 17ß-estradiol (E2), produced by human body and excreted into municipal wastewaters, belongs to the group of endocrine disrupting compounds that are resistant to biological degradation. The aim of this study was to assess the efficiency of E2 removal from aqueous solutions by means of catalytic wet-air oxidation (CWAO) and photolytic/photocatalytic oxidation. CWAO experiments were conducted in a trickle-bed reactor at temperatures up to 230 °C and oxygen partial pressure of 10 bar over TiO2 and Ru/TiO2 solids. Photolytic/photocatalytic oxidation was carried out in a batch slurry reactor employing a TiO2 P-25 (Degussa) catalyst under visible or UV light. HPLC analysis and yeast estrogen screen assay were used to evaluate the removal of E2 and estrogenicity of treated samples. The latter was completely removed during photolytic/photocatalytic oxidation under UV (365 nm) light and photocatalytic oxidation under visible light. In CWAO experiments, complete removal of both E2 and estrogenicity from the feed solution were noticed in the presence of TiO2 and Ru/TiO2 catalysts.

Comparison of Fenton's oxidation and ozonation for removal of estrogens.

This study compares efficiency of Fenton's oxidation and ozonation of 17beta-estradiol (E2) and 17alpha-ethinylestradiol (EE2) as two possible processes for removal of estrogens from aqueous solutions. The effectiveness of Fenton's oxidative removal was studied at different ratios of reagents Fe2+:H2O2 (1:0.5; 1:10; 1:20; 1:33), where with some molar ratios up to 100% removal of E2 and EE2 was achieved in the first few minutes of reaction. The best molar ratio for E2 (17beta-estradiol) removal was 1:33, while in the case of EE2 the most efficient one was 1:20 ratio. Ozonation was much faster, because complete removal of estrogens was achieved in 30 seconds (pH approximately eaqual 6), but the time of ozonation was extended up to 60 minutes trying to decompose formed by-products, expressing estrogenic activity, detected by YES (Yeast Estrogen Screening) assay. The obtained results showed that the removal efficiency of estrogens from waters should be assessed by a combination of chemical analyses and bioassay.

Feasibility of Fenton's Oxidation for Removal of Estrogens from Aqueous Solutions.

Presented research has been focused on the effectiveness of degradation of natural (17-estradiol - E2) and synthetic (17α-ethinylestradiol - EE2) hormones with Fenton's oxidation. Numerous studies have demonstrated, that the impact of hormones on organisms was shown already at environmental concentrations close to 1 pg L-1, so their effective removal from wastewaters is important. Fenton's oxidation is very efficient in terms of oxidation of organics, while its main withdrawal is the formation of waste sludge, leading to adsorption of organics, which sometimes makes it impossible to reliably value the extent of actual degradation. The ratio among degradation and adsorption of estrogens on sludge during oxidative treatment was determined as a part of our study. It depends upon the molar ratio of reagents Fe2+ and H2O2 (1:0.5, 1:10; 1:20; 1:33), reaction time and initial concentrations of the investigated estrogens E2 (0.279/27.9 mg L-1) and EE2 (0.296/29.6 mg L-1). The most efficient removal of E2 was achieved at 1:33 molar ratio of reagents (100%, 26-50% adsorption) while EE2 removed efficiently at 1:10 and 1:33 ratios (99-100%, 20-76% adsorption). It was confirmed, that adsorption plays an important role in Fenton's oxidative treatment of estrogens. It is prevailing process at higher pHs, while at lower ones (pH = 3.0-5.0) oxidation is dominant, because it is not limited by mass transport.

Biochemical biomarkers in environmental studies--lessons learnt from enzymes catalase, glutathione S-transferase and cholinesterase in two crustacean species.

BACKGROUND, AIM AND SCOPE: For reliable environmental risk assessment of pollutants, knowledge on the effects at different levels of biological organisation is needed. During the early days of biomarker research in environmental studies approximately two decades ago, biochemical biomarkers were considered as the most promising tool for such purposes. Among these, three enzymes have often been studied: catalase (CAT), glutathione S-transferase (GST) and cholinesterase (ChE). However, despite their intensive research, their measurements in invertebrates have not been commonly applied in environmental risk assessment (ERA) or for regulatory purposes. MAIN FEATURES: In the present review, we summarise our past experiences in biochemical biomarker research in two crustacean species: water flea Daphnia magna and terrestrial isopod Porcellio scaber. This is to orientate their use and to provide recommendations for the use of novel biomarkers in environmental studies, such as proteomic or genomic responses. RESULTS AND DISCUSSION: We assessed the intrinsic properties of biochemical biomarkers CAT, GST and ChE in the D. magna and the isopod P. scaber. It was found that they are not in agreement with the expectations that were previously given for their use in environmental studies. To advance their use in environmental risk assessment, we suggest that based on their properties, their role should be more specifically defined. ERA includes several distinct steps, among them hazard identification, effect assessment and finally risk characterisation, each of which requires a different type of toxicity data. We recommend that the use of biochemical markers is most appropriate for hazard identification because this is a procedure whose purpose is to characterise the potential hazard of the substance in question and is more flexible in terms of using different tools. Furthermore, our results imply that biochemical markers are not always more sensitive than whole-organism responses, as was anticipated. Their sensitivity depends on the mode of action, duration of exposure and test species. Therefore, we suggest that combining both a battery of biomarkers from different levels of biological organisation and an array of biomarkers within a single level could identify hazard adequately. CONCLUSIONS: The lesson learnt from biochemical biomarkers in environmental studies utilizing crustacean model species is that, for successful application of each group of biomarkers, their intrinsic properties are needed to be known before an (eco)toxicity study is designed. We suggest that a substantial body of experience obtained with biochemical biomarkers should be exploited to new emerging biomarkers in environmental studies in order to facilitate their application. RECOMMENDATIONS AND PERSPECTIVES: The future of biomarkers lies in a combination of traditional biochemical and new-generation biomarkers. The latter are not only a potential replacement for existing biomarkers but will also provide new knowledge which might encourage renewed research and development of traditional biomarkers. For research purposes, complete ecotoxicity information should include contributions from molecular fingerprint of an organism, as well as whole organism, population and ecosystem responses. Still, the type of biomarkers used for routine purposes will depend on their reproducibility, their ease of use, robustness, affordability of the methodology and the type of chemicals, organisms and ecosystem of interest.

Hazard identification of imidacloprid to aquatic environment.

The use of a very effective insecticide against sucking pests, neonicotinoid imidacloprid, has been increasing extensively. For this reason elevated concentrations are expected in aquatic environment. Despite this fact, there is still a lack of data available on its possible risk for the environment. In this study, the potential hazards of imidacloprid and its commercial product Confidor SL 200 to aquatic environment were identified by the acute and chronic toxicity assessment using bacteria Vibrio fischeri, algae Desmodesmus subspicatus, crustacean Daphnia magna, fish Danio rerio and the ready biodegradability determination. We found out, that imidacloprid was not highly toxic to tested organisms in comparison to some other environmental pollutants tested in the same experimental set-up. Among the organisms tested, water flea D. magna proved to be the most sensitive species after a short-term (48 h EC50=56.6 mg L(-1)) and long-term exposure (21 d NOEC=1.25 mg L(-1)). On the contrary, the intensified toxicity of Confidor SL 200 in comparison to analytical grade imidacloprid was observed in the case of algae and slight increase of its toxicity was detected testing daphnids and fish. The activities of cholinesterase, catalase and glutathione S-transferase of daphnids were not early biomarkers of exposure to imidacloprid and its commercial product. Imidacloprid was found persistent in water samples and not readily biodegradable in aquatic environment. Due to increased future predicted use of commercial products containing imidacloprid and the findings of this work, we recommend additional toxicity and biodegradability studies of other commercial products with imidacloprid as an active constituent.

Effects of ingested nano-sized titanium dioxide on terrestrial isopods (Porcellio scaber).

The effects of ingested nano-sized titanium dioxide (TiO2; anatase, 15 nm) on the terrestrial isopod Porcellio scaber (Isopoda, Crustacea) after short-term (3-d) dietary exposure were studied. Activities of antioxidant enzymes, such as catalase (CAT) and glutathione-S-transferase (GST), in digestive glands were affected in a dose-independent manner, but higher-level isopod endpoints, including weight change, feeding rate, food assimilation efficiency, and survival, were not affected up to the highest tested concentration of TiO2 in food (3,000 microg/g). Exposure concentrations of 0.5, 2,000, and 3,000 microg nonsonicated TiO2/g food decreased CAT and GST activities, but intermediate concentrations (1, 10, 100, and 1,000 microg/g food) did not result in significant changes of enzyme activities. When the dispersion of TiO2 was sonicated, no effects on enzyme activities or higher-level biomarkers were observed. The experimental setup with terrestrial isopods designed for dissolved chemicals also is suitable for testing the effects of ingested nanoparticles, but the presentation of toxicity data needs to be adapted according to the mode of action of the nanoparticles and their specific characteristics.

Biochemical biomarkers in chronically metal-stressed daphnids.

Biochemical biomarkers are a popular measure of toxic effects on organisms due to their assumed fast response, and are usually assessed after acute exposure of the organism to the stressor. However, increasing interest in the use of biochemical biomarkers in environmental pollution monitoring calls for more laboratory long-term studies of contaminants' effects on biochemical endpoints. In this study, four biochemical biomarkers (protein content, activity of cholinesterase (ChE), catalase (CAT) and glutathione S-transferase (GST), were correlated with standardised reproductive and survival endpoints of water fleas (Daphnia magna) after chronic exposure to Cr (VI) and Cd. No effect on the reproduction and survival was noticed up to the highest tested concentration of Cr (VI) (52.5 microg/L), while the protein content, and the ChE and CAT activity decreased, and GST activity increased. Cd affected reproduction of daphnids above 0.656 microg/L, but the protein content and ChE activity were changed at 0.328 microg/L and 0.082 microg/L of Cd, respectively. Biochemical biomarkers in some cases proved to be equally or more sensitive than reproduction and mortality. We recommend more frequent use of a battery of biochemical biomarkers in combination with other higher-level biomarkers also in chronic studies and not only in the acute ones.

Comparative toxicity of imidacloprid, of its commercial liquid formulation and of diazinon to a non-target arthropod, the microcrustacean Daphnia magna.

Imidacloprid (IMI) is at the moment the insecticide with the world's fastest growing sales and is considered possible replacement for the widely used organophosphorus pesticide, diazinon, which is subject to phased revocation in many countries. In this study, biochemical, reproductive and survival parameters of the water flea (Daphnia magna) after chronic exposure to IMI, its commercial liquid formulation Confidor SL 200 and diazinon are presented and compared. According to the lowest observed effect concentrations, diazinon is more toxic to the reproduction of D. magna than IMI and Confidor SL 200, which exert similar toxicity. The same was observed for the survival, except that Confidor SL 200 is more toxic than IMI. In polluted aquatic environments, the actual levels of diazinon are potentially chronically hazardous to the reproduction of D. magna (risk quotient >1). According to very few measured environmental levels of IMI, the latter is not expected to be chronically hazardous, unless it is accidentally spilled in a small pond. In such case, the predicted concentrations of IMI would present a potential chronic risk to D. magna, and a potential acute risk to other aquatic invertebrates. In the future, higher environmental levels of IMI are expected due to its increasing use and physico-chemical properties. The literature survey summarized in this work suggests that further ecotoxicological studies with a broader spectrum of aquatic organisms are needed before IMI is classified as safer than currently applied pesticides.

Evaluation of point and diffuse sources of nutrients in a river basin on base of monitoring data.

The methodology of materials accounting is presented and applied to developing nutrient balance (nitrogen and phosphorus) in a river basin. The method is based on the balance principle: inputs and outputs of each nitrogen and phosphorus related sub-systems were balanced. The application of the methodology strategies was illustrated by means of a case study of the Krka river, Slovenia. Different pathways of emission to surface waters were taken into account: WWTP discharges, direct discharges, erosion/runoff and baseflow. Total annual emission into the river Krka was estimated to be 362 tonnes N/year and 73.3 tonnes P/year. The main sources of nitrogen are diffuse sources, emitted via baseflow (52%). Other important sources are effluents from WWTP, which account for 36% of total emissions. Other sources like erosion and direct discharges to surface water (animal manure, industry, households) are of lower magnitude. Erosion is main source of phosphorus emission (55% of total emission), WWTP effluents account for 37% of total emission, while other sources are less important. Besides reduction of point sources by means of wastewater collection and implementation of nutrient removal technology, managing agricultural nitrogen and phosphorus to protect water quality should become a major challenge in the Krka river basin.

The applicability of acetylcholinesterase and glutathione S-transferase in Daphnia magna toxicity test.

The most commonly used toxicity test worldwide is the acute Daphnia magna test. The relevance of acetylcholinesterase (AChE) and glutathione S-transferase (GST) activity in D. magna exposed to chromium, cadmium, and diazinon was evaluated in connection with this standard test. We found no link between enzyme activities and immobility. Concentrations of Cr(6+) up to 280 microg/L had no effect on AChE and GST activities, while 20% immobility was observed. At concentrations of 20-25 microg/L of Cd(2+) AChE activity was increased by about 50%. The effect of diazinon on both enzymes was insignificant up to concentrations that caused 27% immobility. Consequently, while the use of AChE and GST activities is recommended when the mode of action of chemicals is studied, the value of these biomarkers in routine acute toxicity tests is limited because the relationship between enzyme activities and immobility of D. magna exposed to different chemicals is unclear.