Toxicokinetics of perfluorooctane sulfonate in rabbits under environmentally realistic exposure conditions and comparative assessment between mammals and birds.

This article describes the toxicokinetics of perfluorooctane sulfonate (PFOS) in rabbits under low repeated dosing, equivalent to 0.085µg/kg per day, and the observed differences between rabbits and chickens. The best fitting for both species was provided by a simple pseudo monocompartmental first-order kinetics model, regulated by two rates, and accounting for real elimination as well as binding of PFOS to non-exchangeable structures. Elimination was more rapid in rabbits, with a pseudo first-order dissipation half-life of 88 days compared to the 230 days observed for chickens. By contrast, the calculated assimilation efficiency for rabbits was almost 1, very close to full absorption, significantly higher than the 0.66 with confidence intervals of 0.64 and 0.68 observed for chickens. The results confirm a very different kinetics than that observed in single-dose experiments confirming clear dose-related differences in apparent elimination rates in rabbits, as previously described for humans and other mammals; suggesting the role of a capacity-limited saturable process resulting in different kinetic behaviours for PFOS in high dose versus environmentally relevant low dose exposure conditions. The model calculations confirmed that the measured maximum concentrations were still far from the steady state situation, and that the different kinetics between birds and mammals should may play a significant role in the biomagnifications assessment and potential exposure for humans and predators. For the same dose regime, the steady state concentration was estimated at about 36µg PFOS/L serum for rabbits, slightly above one-half of the 65µg PFOS/L serum estimated for chickens. The toxicokinetic parameters presented here can be used for higher-tier bioaccumulation estimations of PFOS in rabbits and chickens as starting point for human health exposure assessments and as surrogate values for modeling PFOS kinetics in wild mammals and bird in exposure assessment of predatory species.

Toxicokinetics of perfluorooctane sulfonate in birds under environmentally realistic exposure conditions and development of a kinetic predictive model.

This article describes the toxicokinetics of perfluorooctane sulfonate (PFOS) in birds under low repeated dosing, equivalent to 0.085 µg/kg per day, representing environmentally realistic exposure conditions. The best fitting was provided by a simple pseudo monocompartmental first-order kinetics model, regulated by two rates, with a pseudo first-order dissipation half-life of 230 days, accounting for real elimination as well as binding of PFOS to non-exchangeable structures. The calculated assimilation efficiency was 0.66 with confidence intervals of 0.64 and 0.68. The model calculations confirmed that the measured maximum concentrations were still far from the steady state situation, which for this dose regime, was estimated at a value of about 65 µg PFOS/L serum achieved after a theoretical 210 weeks continuous exposure. The results confirm a very different kinetics than that observed in single-dose experiments confirming clear dose-related differences in apparent elimination rates in birds, as described for humans and monkeys; suggesting that a capacity-limited saturable process should also be considered in the kinetic behavior of PFOS in birds. Pseudo first-order kinetic models are highly convenient and frequently used for predicting bioaccumulation of chemicals in livestock and wildlife; the study suggests that previous bioaccumulation models using half-lives obtained at high doses are expected to underestimate the biomagnification potential of PFOS. The toxicokinetic parameters presented here can be used for higher-tier bioaccumulation estimations of PFOS in chickens and as surrogate values for modeling PFOS kinetics in wild bird species.

Correlation between physicochemical and ecotoxicological approaches to estimate landfill leachates toxicity.

Leachates from municipal solid waste (MSW) landfills may contain a huge diversity of contaminants; these wastewaters should be considered as potentially hazardous complex mixtures, representing a potential environmental risk for surface and groundwater. Current MSW landfill wastes regulatory approaches deem exclusively on the physicochemical characterization and does not contemplate the ecotoxicological assessment of landfill leachates. However, the presence of highly toxic substances in consumer products requires reconsideration on the need of more specific ecotoxicological assessments. The main aim of this study was to evaluate the toxicity of different MSW landfill leachates using a battery of toxicity tests including acute toxicity tests with Daphnia magna and the anuran Xenopus laevis and the in vitro toxicity test with the fish cell line RTG-2. The additional objective was to study the possible correlation between physicochemical properties and the toxicity results obtained for untreated landfill leachates. The results showed that the proposed test battery was effective for the ecotoxicological characterization of MSW landfill leachates. A moderate to strong correlation between the measured physicochemical parameters and the calculated toxicity units was detected for all toxicity assays. Correlation factors of 0.85, 0.86 and 0.55 for Daphnia, Xenopus and RTG-2 tests, respectively, were found. The discriminant analysis showed that certain physicochemical parameters could be used for an initial categorization of the potential aquatic acute toxicity of leachates; this finding may facilitate leachates management as the physicochemical characterization is currently the most common or even only monitoring method employed in a large majority of landfills. Ammonia, alkalinity and chemical oxygen demand (COD), together with chloride, allowed a proper categorization of leachates toxicity for up to 75% of tested samples, with a small percentage of false negatives.

A cost/effective screening method for assessing the toxicity of nutrient rich effluents to algae.

Screening whole effluent toxicity tests are cost/effective methods for detecting the presence of toxic concentrations of unknown pollutants, but the application must solve the problem associated with the effect of high and variable concentrations of nutrients in the effluent on the results of algal toxicity tests. This work proposes a cost/effective test, based on three dilution levels measured at a single point time and a discriminant model for establishing if this kind of complex samples, with difficult interpretation of dilution-response curves, should be considered toxic to algae. This procedure identified properly around 85% of the samples considered toxic by expert judgement.

Occurrence of pharmaceutically active compounds in surface waters of the Henares-Jarama-Tajo River system (Madrid, Spain) and a potential risk characterization.

The Henares-Jarama-Tajo river system is the largest drainage basin in the Province of Madrid, Spain. This area is characterized by the presence of intensive urban and industrial activities influenced by a continental Mediterranean climate with rainfalls presenting substantial fluctuations along the different seasons. This research aimed to monitor seasonal variations in concentrations of 22 pharmaceutically active compounds (PhACs) in this river system and to establish the potential risk of sublethal effects on aquatic organisms. A total of 10 sampling sites were selected along the river system with samples collected in each of the four seasons during a year-round schedule. Most of the PhACs detected were present in sampling sites downstream in the vicinity of the most populated cities (i.e. Madrid, Guadalajara and Alcalá de Henares). Only two PhACs, fluoxetine and paraxantine, were detected in all sites regardless of the season, and showed median (+/- interquartile range) concentrations of 21.4 (+/-31.2) ng L(-1) and 8.5 (+/-5.3) ng L(-1), respectively. Other PhACs were detected with a frequency >80% and included, caffeine, diphenylhydantoin, hydrochlorotiazide, ibuprofen, ketoprofen, diclofenac, sulfamethoxazole, atenolol, naproxen, carbamazepine and propanolol. Seasonal variations were observed with the highest concentrations in December and the lowest in September. By combining measured environmental concentrations with toxicity data (either publicly available or obtained experimentally in our laboratory), and by calculating an Maximum Risk Index (MaxRI) that each combination of PhACs should have for non exceeding the risk threshold, a high potential for long-term risk (MaxRI<10) was estimated for most of the sampling sites and sampling dates. This research allowed the characterization of the potential risk for each of the PhACs to exert sublethal effects on aquatic organisms using acute screening methods, justifying the need for chronic data in order to refine the risk of these compounds to aquatic organisms.

A new method for total mercury and methyl mercury analysis in muscle of seawater fish.

In this work we have developed a cost-effective method for the analysis of methyl mercury (MeHg) in seawater fish muscle. The novelty of this method lies in the use of microwave-assisted extraction with acidic solution (HCl), addition of toluene, and subsequent extraction with cysteine acetate solution where only MeHg is present because of its affinity for cysteine groups. MeHg in cysteine phase and total mercury in the homogenate muscle tissue were determined using a direct Hg analyzer (DMA-80). Validation, precision, and accuracy of the method were evaluated and monitored with a tuna fish certified reference material (CRM 463) containing MeHg.

Sewage sludge applied to agricultural soil: Ecotoxicological effects on representative soil organisms.

Application of sewage sludge to agricultural lands is a current practice in EU. European legislation permits its use when concentrations of metals in soil do not increase above the maximum permissible limits. In order to assess the fate and the effects on representative soil organisms of sewage sludge amendments on agricultural lands, a soil microcosm (multi-species soil system-MS3) experiment was performed. The MS3 columns were filled with spiked soil at three different doses: 30, 60 and 120tha(-1) fresh wt. Seed plants (Triticum aestivum, Vicia sativa and Brassica rapa) and earthworms (Eisenia fetida) were introduced into the systems. After a 21-d exposure period, a statistically significant increase for Cd, Cu, Zn and Hg concentrations was found for the soils treated with the highest application rate. Dose-related increase was observed for nickel concentrations in leachates. Plants and earthworm metal body burden offer much more information than metal concentrations and help to understand the potential for metal accumulation. Bioaccumulation factor (BAF(plant-soil)) presented a different behavior among species and large differences for BAF(earthworm-soil), from control or sewage-amended soil, for Cd and Hg were found. B. rapa seed germination was reduced. Statistically significant decrease in fresh biomass was observed for T. aestivum and V. sativa at the highest application rate, whereas B. rapa biomass decreased at any application rate. Enzymatic activities (dehydrogenase and phosphatase) as well as respiration rate on soil microorganisms were enlarged.

A Daphnia magna feeding bioassay as a cost effective and ecological relevant sublethal toxicity test for Environmental Risk Assessment of toxic effluents.

Environmental Risk Assessment of chemical products and effluents within EC countries require the use of cost effective standardized toxicity tests that in most cases are restricted to acute responses to high doses. Thus, subtle ecological effects are underestimated. Here we propose a short-term one day Daphnia magna feeding inhibition test as a cost effective and ecological relevant sublethal bioassay. The sensitivity and reliability of the proposed bioassay was tested in the laboratory against standardized bacteria, algae growth, D. magna and fish acute toxicity test by using 16 chemical mixture x water type combinations that included four different water types fortified with four complex chemical mixtures. Water types included ASTM hard water and three selected effluents diluted 1/10 in water to mimic worse field situations that many overexploited arid river ecosystems suffer during summer months when effluents are discharged into them with little dilution. The results obtained denoted a greater sensitivity of the proposed feeding bioassay in 51 out of 65 tests performed with an average sensitivity 50 fold greater than that of the standardized tests. The greater differences were obtained for mixtures that included narcotic chemicals and the lowest differences for those containing pesticides. Furthermore, feeding responses to the studied contaminant mixtures behaved differently to increasing TOC content than those based on bioluminescent bacteria and algae. Increasing TOC coming from sewage treated effluents decrease toxicity to the latter bioassays but increased those of D. magna feeding bioassays. These results empathize the need to include additional bioassays to monitor more accurately and realistically the toxicity of effluents or surface waters dominated by effluent discharges, a quite common situation in America and Mediterranean arid regions.

Cytochrome P4501A induction caused by the imidazole derivative Prochloraz in a rainbow trout cell line.

A variety of aquatic pollutants are able to induce cytochrome P4501A (CYP1A) in fish by ligand binding to the aryl hydrocarbon receptor (AhR). High-affinity AhR ligands are planar aromatic polycyclic molecules such as the prototypical ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The present work investigates the ability of the imidazole derivative, Prochloraz (PRO), to induce CYP1A. Computational studies on the molecular structure of PRO indicated that it is highly unlikely for PRO to have both aromatic rings of the molecule, i.e. the imidazole and the benzene ring, in the same plane. Thus, the possible conformers do not take planar structures, in contrast to the typically planar AhR ligands. Experimentally, the capability of PRO to induce CYP1A was assessed using the rainbow trout liver cell line, RTL-W1, as in vitro model. PRO increased in a concentration-dependent way the catalytic activity of CYP1A (determined as 7-ethoxyresorufin-O-deethylase, EROD, activity) in RTL-W1 cells. The potency of PRO was lower than that of a reference AhR-ligand, beta-naphthoflavone (betaNF). In addition to the catalytic level, PRO activated CYP1A also at the transcriptional level as determined by RT-PCR analysis of CYP1A mRNA. These results indicate that PRO, although its structure is not corresponding to the typical features of CYP1A-inducing AhR ligands, still is able to activate CYP1A expression.

In vitro toxicity of selected pesticides on RTG-2 and RTL-W1 fish cell lines.

The rainbow trout fish cell lines RTG-2 and RTL-W1 were used to determine the cytotoxic effects of the pesticides bifenthrin, cypermethrin, cyhalothrin, lambda-cyhalothrin, quinalphos and chlorpyrifos. Cytotoxicity was measured by EROD and beta-Gal enzymatic activities, the neutral red (NR) uptake assay, and the FRAME KB protein (KBP) assay. The beta-Gal activity was unaffected by the pesticide exposure. The EROD activity was induced by cyhalothrin and lambda-cyhalothrin (RTG-2 and RTL-W1) and by bifenthrin (RTL-W1). Dose dependent inhibition responses were observed for EROD activity in cells exposed to quinalphos (RTL-W1) and chlorpyrifos (RTG-2 and RTL-W1). RTL-W1 offered a better response for EROD induction. The EC50 values on EROD endpoint were more sensitive than NR and KBP. The acute fish toxicity of chlorpyrifos and quinalphos depends highly on the species; the species sensitivity distributions cover several orders of magnitude and the values obtained for EROS were within the lowest part of the reported ranges.

Stage-specific toxicity of cypermethrin to medaka (Oryzias latipes) eggs and embryos using a refined methodology for an in vitro fertilization bioassay.

Using original artificial fertilization methods with medaka (Oryzias latipes), the effects of exposure to cypermethrin on gametes, fertilization, and embryonic development were investigated. The relative sensitivity was studied with 96-hour duration, 24-hour renewal exposures to six nominal concentrations of cypermethrin ranging from 3.1 100.0 microg cypermethrin/L. Tests were initiated at different developmental stages: unfertilized egg (stage 0), late morula (stage 9), eminence of swim bladder (stage 29), and maximum flexion of the atrioventricular region (stage 34). Cypermethrin did not affect the fertilization process. Predominant sublethal effects in embryos included transient visceral edemas intimately associated to the gall bladder with subsequent pericardial edemas. Other sublethal effects were observed in surviving larvae and included spastic movements with or without ability to respond to stimulus (>/=6.3 microg cypermethrin/L), spinal curvatures, and delayed or absence of swim bladder inflation (>/=12.5 microg cypermethrin/L). The exposure of the gametes (stage 0) or animals during earlier embryonic development (stages 9 or 29) was not a critical window for cypermethrin exposure. Although the incidence of edemas in embryos occurred mainly during exposure of these early developmental stages, embryo and larva lethality and the incidence of transient sublethal effects in hatchlings showed that the later exposure window (stage 34) was the most sensitive. The stage 34 group involved advanced organogenetic stages in which the chorion partially degraded before hatching. Our studies reinforced the idea that a combination of morphologic and functional impairment evaluation is a more sensitive response to developmental toxicants than morphologic defects alone.

Ecotoxicological assessment of doxycycline in aged pig manure using multispecies soil systems.

This paper assesses the ecotoxicity of the antibiotic doxycycline in aged spiked pig manure using a multispecies soil system (MS 3) covering plants, earthworms and soil microorganisms. The study reproduced realistic exposure conditions, as well as higher exposure doses covering the uncertainty factors typically employed for covering interspecies variability. MS 3, consisting of columns of natural sieved soil assembled with earthworms and seeds from three plant species, were employed. Pig manure was spiked with doxycycline (75 or 7500 microg/ml), aged for 15 days under aerobic/anaerobic conditions and added on top of the soil columns (120 ml/column, equivalent to 220 kgN/ha). Water and doxycycline free manure were used as negative controls. Doxycycline (7500 microg/ml) solution was used as a positive control. No effects on plants or earthworms were observed. Significant effects on soil phosphatase activity, indicating effects on soil microorganisms, were observed at the highest exposure dose, affecting all soil layers in the doxycycline-solution-treated MS 3 (positive control) but only the top layer in the spiked pig manure system. Chemical analysis confirmed the different behavior of doxycycline in both systems (with and without manure) and those effects were observed in soil with measured concentrations over 1 mg/kg soil. The detection of doxycycline in leachates revealed a potential mobility. Leachate concentrations were similar for doxycycline solution and spiked manure treatments.

Modelling the absorption and desorption of cadmium on paper pulp using kinetic approaches.

The presence of toxic metals on paper pulp and the migration of these metals to food from the food package is receiving significant attention. The final exposure levels for consumers depend on two main processes. First the potential of metals to bind paper pulp during manufacture. Second, the metal potential to migrate from paper to food during storage and use. Binding and unbinding processes are modelled for cadmium kinetics through kinetic approaches. The cadmium concentration in paper pulp is estimated from the cadmium concentration in the water-pulp liquor during manufacture, the temperature, and contact time. Two food simulants have been employed for desorption studies, water and acetic solution (3%, w/w). As expected, under acidic conditions desorption is total and rapid (close to 100% desorption reached in a few minutes). However, the desorption of cadmium into the neutral food simulant depends on the initial cadmium concentration in the paper pulp, temperature and contact time. Surface response curves for each combination are presented. Temperature did not affect cadmium binding, but played a significant role for the desorption processes into the neutral food simulant. The proposed equations offer a good fitting of the experimental values, with p < 0.001 and residuals within a factor of 3 for over 99% of the measured data. These models allow estimations of the expected exposure levels in consumers, on the basis of manufacture and use conditions. Linking the expected exposure with toxicity thresholds, such as the admissible daily intake (ADI), would allow a scientific assessment of the maximum acceptable cadmium levels in water-pulp liquors during manufacture and in the final paper pulp on the basic of the use patterns of each food packaging material.

Hazard and risk assessment of chemicals for terrestrial ecosystems.

Risk assessment for terrestrial ecosystems represents a great challenge due to their complexity. Pragmatic approaches, such as independent assessments for soil and 'above soil' organisms, are unrealistic. This communication presents a workable alternative, extending the role of the hazard identification. For each chemical, a set of selected ecological receptors and exposure routes is considered. Terrestrial vertebrates, soil-ground-foliar dwelling invertebrates, plants, and soil micro-organisms, are potential receptors subjected to direct and indirect exposures. Direct exposures cover those related to the emissions during the Life Cycle of the chemical. Indirect exposures focus on those occurring after the emission, related to the fate and behaviour of the molecule in the environment. Direct exposures are regulated by the production-use-disposal patterns. Indirect exposures are regulated by intrinsic (physicochemical, biological) properties. Hazard identification considers the toxicological profile and the exposure potential for each receptor, and selects the key receptors for the assessment. Risk analysis includes, for each receptor, all potential exposure routes and the corresponding timings. Food chain biomagnification is quantified on the basis of toxicokinetic data and a three vertebrate species model, suitable for top-predators and humans. A higher tier approach, considering the lack of homogeneity of the exposure and probabilistic assumptions, can also be conducted.

Binding of cadmium on raw paper pulp. Relationship between temperature and sorption kinetics.

Several assays have been carried out in order to study in detail the cadmium mobility from water to virgin pulp as previous step of cadmium mobility description from paper into food. Virgin pulp has been immersed in different concentration aqueous solutions at several temperatures and during short and long time. The absorbed cadmium amounts have been measured by atomic absorption spectroscopy. For a long time, the temperature of storage is not an important factor except during the first 3 h. In general, percentage of migrated cadmium into pulp is between 30% and 90% of initial charge. Kinetic equation of cadmium mobility has been described employing statistics methods with a very good correlation.

Toxicological characterisation of sludge from sewage treatment plants using toxicity identification evaluation protocols based on in vitro toxicity tests.

The ecotoxicological characterisation of complex mixtures, such as sludge from sewage treatment plants, is complex. Toxicity identification evaluation (TIE) protocols, developed by the United States Environmental Protection Agency (US EPA); to identify toxic pollutants in complex effluents, are useful tools in this context; to solve the difficulties in assessing unknown organic pollutants by analytical methods, the usefulness of bioassays to detect the relevant (toxic) organic compounds present in complex samples, and the possibilities of in vitro cytotoxicity tests as screening tools, offers a profitable combination. The sludge obtained from a sewage treatment plant was extracted by acetonitrile using a microwave extractor and fractionated in an HPLC system. The toxicity of every fraction was assayed using a RTG-2 cytotoxicty test, based on the fibroblastic RTG-2 fish cell line (ATCC, CCL N. 55). At exponential growth, three endpoints, beta-galactosidase activity, culture viability assayed by the neutral red assay (NR) and inhibition of growth rate using the FRAME KB protein assay (KBP), were used. By plotting the toxicity of each fraction vs elution time, the corresponding "toxicograms" were built. The UV and fluorescence chromatograms are compared to the three toxicograms (one for each toxicity endpoint).