The use of new approach methodologies for the environmental risk assessment of food and feed chemicals.

New Approach Methodologies (NAMs) provide tools for supporting both human and environmental risk assessment (HRA and ERA). This short review provides recent insights regarding the use of NAMs in ERA of food and feed chemicals. We highlight the usefulness of tiered methods supporting weight-of-evidence approaches in relation to problem formulation (i.e., data availability, time, and resource availability). In silico models, including quantitative structure activity relationship models, support filling data gaps when no chemical property or ecotoxicological data are available, and biologically-based models (e.g., toxicokinetic-toxicodynamic models, dynamic energy models, physiologically-based models and species sensitivity distributions) are applicable in more data rich situations, including landscape-based modelling approaches. Particular attention is given to provide practical examples to apply the approaches described in real-world settings. We conclude with future perspectives, with regards to the need for addressing complex challenges such as chemical mixtures and multiple stressors in a wide range of organisms and ecosystems.

Terrestrial microcosms in a feasibility study on the remediation of diesel-contaminated soils.

Phytoremediation and bioremediation are site-specific processes, and feasibility studies should be performed as an initial step in scaling-up these processes. Soil microcosms can be a useful tool for investigating the efficiency of remediation alternatives under realistic conditions. In the present study, the degradation of diesel fuel in soil was studied in artificially assembled microcosms during a 180-day experiment. Microcosms consisted of soil columns seeded with a mix of Festuca arundinacea and Trifolium pratense into which earthworms were introduced and native soil microflora was used. Remediation assays were performed with different combinations of soil organisms' assemblages (micro-organisms, earthworms and plants) to determine the effects of these organisms and their interaction on diesel degradation. The microcosm system allowed for the study of the efficiency of remediation under field-relevant conditions. This system provided information about the dissipation of the pollutants as well as chemical leaching and possible toxic effects on the organisms during the remediation process. The decrease in soil hydrocarbon levels depended on the organisms' assemblage. Plants were not effective in the remediation of these soils despite their stimulation of microbial biomass. Conversely, earthworms had a beneficial impact on the dissipation of hydrocarbons that did not appear to be related to a generic improvement of plant or microbial activity. The grass species was more tolerant than the legume species to diesel fuel-contaminated soils. Leaching of hydrocarbons was negligible and independent of the organisms' assemblage.

Dynamics of BNF-induced in vivo ethoxyresorufin-O-deethylase (EROD) activity during embryonic development of medaka (Oryzias latipes).

This study aimed to characterize quantitatively the temporal basal and induced ethoxyresorufin-O-deethylase (EROD) activity as indicator of cytochrome P4501A (CYP1A) function during embryonic development of medaka (Oryzias latipes). For this purpose, non-invasive methods over fluorescence images of the whole embryo (non-organ-specific [NOS] EROD activity) or specifically of the gallbladder (organ-specific [OS] EROD activity) were used. To induce this EROD activity, embryos were continuously exposed to ß-naphthoflavone (BNF; 0.005, 0.05, 0.5, 5 µg/L). Analytical chemistry suggested no signs of BNF dissipation. Mean fluorescence intensity values for EROD induction increased with BNF concentration throughout embryonic development. Significant increments in the NOS activity were seen from exposures to ≥ 0.5 µg BNF/L as early as 2 days post-fertilization (dpf), and in the OS EROD activity as soon as the gallbladder was conspicuous (i.e. 4 dpf). Morphometric in vivo analysis of the gallbladder during embryonic development did not indicate significant dilation after BNF treatment suggesting normal hepatic bile formation. The conditions optimized in this study using intact embryos should allow the quantitation of EROD activity induced by specific chemicals, mixtures and environmental samples in terms of BNF-equivalents, offering a proper estimation of their potency. These results demonstrate the utility of medaka in a fish embryo test for a non-invasive CYP1A analysis expressed as EROD activity, fitting in the three R principles for the minimization of animal use in ecotoxicology evaluations and that are among the objectives of the European Community regulation for the Registration, Evaluation, Authorization and Restriction of Chemical substances (REACH).

Semifield assessment of the runoff potential and environmental risk of the parasiticide drug ivermectin under Mediterranean conditions.

INTRODUCTION: The antiparasitic ivermectin is of particular concern to regulatory agencies. Ivermectin can reach the environment through the direct emission of dung from livestock on pasture and via manure application on agricultural lands. METHODS: A semifield study was conducted for assessing the ivermectin dynamic in runoff and drainage waters from dung-treated soils placed on experimental trays. The experiment was conducted under natural Mediterranean conditions. Realistic pasture and arable land applications were assessed using dung of treated animals and compared with a positive control (spraying the ivermective solution without dung). RESULTS: Similar concentrations were obtained in all three treatments for drainage waters, with values ranging from <5-10 to about 20 ng/l. However, strong treatment-related variation was observed in runoff waters, with the highest concentrations found in the spray treatment (9-188 ng/l), followed by the arable land (<5-88 ng/l) scenario, and concentrations not exceeding 6 ng/l in the pasture scenario. Ivermectin levels in runoff particles were up to 1,660 and 5,890 ng/kg dry weight for the pasture (I1) and arable land (I2) scenarios, respectively. Ivermectin was only detected in the drainage and runoff waters collected in the first rainfall events after treatment. CONCLUSIONS: The measured concentrations in water (0.006-0.118 ng/ml) and runoff particles (0.052-5.89 ng/mg dry suspended matter) are orders of magnitude higher than those provoking effects on aquatic and benthonic communities under experimental and mesocosm conditions, suggesting a clear risk for aquatic systems in the vicinity of pasture areas of treated animals or arable soil fertilized with its manure.

Screening risk assessment tools for assessing the environmental impact in an abandoned pyritic mine in Spain.

This paper describes a new methodology for assessing site-specific environmental impact of contaminants. The proposed method integrates traditional risk assessment approaches with real and variable environmental characteristics at a local scale. Environmental impact on selected receptors was classified for each environmental compartment into 5 categories derived from the whole (chronic and acute) risk assessment using 8 risk levels. Risk levels were established according to three hazard quotients (HQs) which represented the ratio of exposure to acute and chronic toxicity values. This tool allowed integrating in only one impact category all the elements involved in the standard risk assessment. The methodology was applied to an abandoned metal mine in Spain, where high levels of As, Cd, Zn and Cu were detected. Risk affecting potential receptors such as aquatic and soil organisms and terrestrial vertebrates were assessed. Whole results showed that impact to the ecosystem is likely high and further investigation or remedial actions are necessary. Some proposals to refine the risk assessment for a more realistic diagnostic are included.

In vitro cellular responses in the RTG-2 cell line to complex mixtures of dioxins and dioxin-like PCDDs, PCDFs and PCBs.

High-resolution gas chromatography/mass spectrometry (HRGC/MS) is the standard method for analysing dioxin, furan and polybrominated retardants in hazardous waste. Determination of dioxin-like compounds using in vitro bioassays such as ethoxyresorufin-O-deethylase (EROD) is an important tool to evaluate their Ah receptor-mediated toxic effects, because it detects all arylhydrocarbon receptor ligands in a variety of sample matrices. In the present work, we compared RTG-2 cell line EROD bioassay with HRGC/MS for assessing waste samples (liquid and solid) contaminated with polychlorinated dibenzo-p-dioxins and dibenzofurans, polychlorinated biphenyls (dioxin-like PCBs) and other xenobiotics. For liquid samples, HRGC/MS-toxic equivalent (HRGC/MS-TEQ) values ranged from 273.26 to 5.84 ng TEQ l(-1) and correlated well (correlation coefficient 0.99) with values obtained by EROD-TEQ, which ranged from 128 to 2.5 ng TEQ l(-1). For solid samples, HRGC/MS-TEQ values ranged from 3.44 to 0.49 ng TEQ g(-1) and correlated less well than liquid samples (correlation coefficient 0.64) with values obtained by EROD-TEQ ranging from 2.27 to 0.93 ng TEQ g(-1). The overestimation of RTG-2 EROD-TEQ (1.2 +/- 0.92 of values established by HRGC/MS) and the absence of false-negative results may limit analytical costs by eliminating the need for follow-up GC/MS analysis on the negative samples. We suggest that RTG-2 EROD bioassay is an inexpensive means for preliminary dioxin and furan positive screenings of waste samples.

Forecasting risk along a river basin using a probabilistic and deterministic model for environmental risk assessment of effluents through ecotoxicological evaluation and GIS.

This work presents a computer model for Risk Assessment of Basins by Ecotoxicological Evaluation (RABETOX). The model is based on whole effluent toxicity testing and water flows along a specific river basin. It is capable of estimating the risk along a river segment using deterministic and probabilistic approaches. The Henares River Basin was selected as a case study to demonstrate the importance of seasonal hydrological variations in Mediterranean regions. As model inputs, two different ecotoxicity tests (the miniaturized Daphnia magna acute test and the D.magna feeding test) were performed on grab samples from 5 waste water treatment plant effluents. Also used as model inputs were flow data from the past 25 years, water velocity measurements and precise distance measurements using Geographical Information Systems (GIS). The model was implemented into a spreadsheet and the results were interpreted and represented using GIS in order to facilitate risk communication. To better understand the bioassays results, the effluents were screened through SPME-GC/MS analysis. The deterministic model, performed each month during one calendar year, showed a significant seasonal variation of risk while revealing that September represents the worst-case scenario with values up to 950 Risk Units. This classifies the entire area of study for the month of September as "sublethal significant risk for standard species". The probabilistic approach using Monte Carlo analysis was performed on 7 different forecast points distributed along the Henares River. A 0% probability of finding "low risk" was found at all forecast points with a more than 50% probability of finding "potential risk for sensitive species". The values obtained through both the deterministic and probabilistic approximations reveal the presence of certain substances, which might be causing sublethal effects in the aquatic species present in the Henares River.

Toxic effects of an oil spill on fish early life stages may not be exclusively associated to PAHs: studies with Prestige oil and medaka (Oryzias latipes).

Polycyclic aromatic hydrocarbons (PAHs) are assumed to be the primary determinant of oil petroleum toxicity. Since the PAH content in Prestige oil was relatively high, we investigated the effects of different oil fractions (crude or weathered oil -0.05 to 50 g/L, and shaken or sonicated water accommodated fractions, WAFs, 25-100%, v/v) on the embryo-larval development of medaka (Oryzias latipes). Concentrations of summation operator16PAHs analyzed in the incubation medium were highest in the shaken WAF followed by the crude oil, the sonicated WAF and the weathered oil. Both oils (> or =0.25 g/L) induced developmental abnormalities whereas no significant effects were seen in the WAF exposures. In vivo morphometric analysis of the surface of the gallbladder during advanced embryo organogenesis (192 h post-fertilization, hpf) revealed significant dilation in both WAF exposures (>3 x 10(4) microm(2) at > or =25%, v/v, compared to <1.7 x 10(4) microm(2) at 0%, v/v) followed by the crude oil (>2.2 x 10(4) microm(2) at > or =0.05 g/L). Fluorescent aromatic compounds were observed in the gallbladder and the yolk sac of 168-hpf embryos exposed to all oil fractions. Results suggest the presence of components in both oils capable of penetrating the chorion and inducing a toxicity not observed in the WAFs. Hence, the hazard and risk assessment of Prestige oil should not be based solely on the presence of PAHs since proximity or direct contact may induce toxicity not associated exclusively to these compounds. This research offers a new hypothesis for explaining the reported biological observations, which could be correlated to direct oil exposure rather than the traditional mechanism of waterborne PAH exposure. Further research is needed to identify those oil components responsible for toxicity.

A new hazard index of complex mixtures integrates bioconcentration and toxicity to refine the environmental risk assessment of effluents.

A new methodology to evaluate the overall environmental hazard of unknown mixtures, based on bioconcentration potential and toxicity, was developed using a combination of two methodologies: 1) the estimation of the octanol-water partition coefficient (K(ow)) using reverse-phase high performance liquid chromatography (RP-HPLC) and 2) the toxicity identification evaluation (TIE). Forty seven compounds with known K(ow) and different molecular structures where used for the calibration of the log K(ow) in relation to the retention time in reverse-phase high performance liquid chromatography (RP-HPLC). A linear regression with an R(2)=0.81 and an sd=0.69 was established between log K(ow) and RP-HPLC retention time. This K(ow) estimation method was furthermore validated using seven additional compounds, showing acceptable estimations of the log K(ow) of unknown substances. Two different mixtures were tested, one containing 3,4 Dichloroaniline, Diazinon and 4-Nonylphenol and another one containing a mixture of 16 pesticides. Both mixtures were first tested as a whole effluent and then fractionated and tested, using a miniaturized Daphnia magna test. An equation is presented, that combines both methodologies and establishes a relative hazard index RHI, ranging from 1 to 10 for any particular mixture of chemicals. The results show how the method presented can refine the security factors that could be included in the environmental risk assessment of effluents in the future.

Risk-based ecological soil quality criteria for the characterization of contaminated soils. Combination of chemical and biological tools.

This paper describes the development of soil quality criteria for the characterization of soils focused on the potential risk to the ecosystem. The approach combines both Generic Soil Quality standards (GSQs) for individual chemicals and direct ecotoxicity assays on soil samples taken from the site. Criteria establish three main risk levels with their corresponding trigger values. The trigger values to determine high risk or "polluted" soils are exclusively based on direct toxicity assessments. The trigger values for the other categories are established by a combination of the application of GSQs and the results of bioassays. Low-risk is assumed when no toxicity is observed and GSQs based on precautionary ecotoxicity thresholds are not exceeded; high-risk must be considered if acute toxicity above the proposed trigger value is observed in soil or leachate samples. In between these levels, the risk cannot be elucidated and a site-specific assessment is required. The GSQs take into account the current or future land use, thus defining three categories: industrial soils, urban/residential soils and natural/agricultural/forest soils, each of them with different ecological requirements. The GSQ values are established following an inverse risk assessment methodology, integrating ecotoxicity and exposure models and setting the soil levels associated to pre-established criteria for the assumption of low risk. The proposed methodology covers all relevant ecological receptors and processes, soil organisms, potential contamination of ground and surface waters, and exposure of terrestrial vertebrates due to bioaccumulation and biomagnification. Exposure routes and protection criteria are defined in each protection goal. The relevance of each receptor and route is established according to the land use.

Ecological risk assessment of contaminated soils through direct toxicity assessment.

A microcosm (MS-3) with a multispecies soil system is introduced as an experimental tool for direct toxicity assessment of contaminated soils. The capacity of MS-3 to determine soil ecotoxicity potential was evaluated using samples from three sites contaminated with organic and/or inorganic compounds. Soils were toxic to soil-dwelling organisms (earthworm, plants, and microorganisms) and to aquatic organisms (algae and RTG-2 cell fish). As expected, responses varied substantially among different soils and organisms. The application of this evaluation system provided complementary information to the chemical characterization. For soils containing metals the toxic response was lower than predicted from total metal concentrations. For hydrocarbons, the toxicity response agreed with estimated values. The induction of EROD activity suggested the presence of dioxin-like compounds, which had not been addressed in the chemical characterization. The proposed multispecies system affords the measurement of 11 endpoints covering three soil and three aquatic taxonomic groups, reproduces soil conditions and gradients, and appears as an excellent complementary tool to chemical analysis for characterization of contaminated sites.

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

This study proposed a battery of endpoints based on in vitro fish cell lines. Two fish cell lines and four toxicity endpoints are considered. The tetracycline (TC), oxytetracycline (OTC), sulfachlorpyridazine (SCP), and septrin(®) (ST) were selected as model antimicrobials and chlorpyrifos as positive control. EROD was induced by septrin(®) (formulation containing sulfamethoxazole and trimethoprim) at concentrations higher than 20mg/L, but inhibited by tetracycline, oxytetracycline and chlorpyrifos. Dose dependent inhibition responses were observed for ß-galactosidase (ß-Gal) activity in cells exposed to septrin(®), tetracycline or oxytetracycline. The EROD EC50s ranged between 2.29×10(-2)mg/L (chlorpyrifos) and 167.63mg/L (tetracycline). The ß-Gal EC50s ranged between 22.1 (septrin(®)) and 84.59mg/L (tetracycline). Data suggest that in vitro testing using a battery of endpoints can be a cost-effective solution for screening the toxicity of antimicrobials on fish. The absence of in vitro effects at concentrations well above those expected/measured in the environment may replace the need for conducting acute lethality tests on fish.

Teschoviruses as indicators of porcine fecal contamination of surface water.

Teschoviruses specifically infect pigs and are shed in pig feces. Hence, their presence in water should indicate contamination with pig fecal residues. To assess this hypothesis, we have developed a real-time reverse transcriptase PCR (RT-PCR) method that allows the quantitative detection of pig teschovirus (PTV) RNA. The method is able to detect 92 fg of PTV RNA per ml of sample. Using this method, we have detected the presence of PTV RNA in water and fecal samples from all pig farms examined (n = 5). Feces from other animal species (cattle, sheep, and goats) were negative in this test. To compare the PTV RNA detection method with conventional chemical determinations currently in use for evaluation of water contamination, we analyzed water samples collected downstream from a pig slurry spillage site. We have found a positive correlation within both types of determinations. The sensitivity of the PTV detection assay was similar to that achieved by unspecific organic matter determination and superior to all other conventional chemical analyses performed. Furthermore, the new method is highly specific, revealing the porcine origin of the contamination, a feature that is lacking in currently available methods for the assessment of water contamination.