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Additional file 1: Table S1. LC-MS/MS parameters for selected alkaloids: retention time (tR), precursor ion, product ion, CVand CE. Table S2A. Measured concentrations of the alkaloids in the acute tests at the start of the experiment, and at the end of the 96 h experiments at a minimum of three selected concentrations. Recoveries related to the nominal concentrations are given in percent, as is the recovery of the start concentration at the end of exposure. Table S2B. Measured sparteine concentrations in the chronic tests at the start of each media renewal and after three days in selected treatments. Recoveries related to the nominal concentrations are given in percent, as is the recovery of the start concentration at the end of each three-day period between media renewal. Selected samples before and after the addition of food are included to investigate if sparteine sorbed to the food algae. Table S3. The negative Log-likelihood values (LL), and parameters derived from fitting mobility of daphnids exposed to individual alkaloids and an alkaloid plant extract daily over 96 h to a GUTS-RED-SD model are given together with derived EC50 values after 48 h (standard timeframe for toxicity on D. magna) and 96 h. All values are given with their 95% confidence intervals (CI). Figure S1. Concentration-response curves for the alkaloids after 96 h of exposure. N = 4 replicates per group; 5 daphnids per replicate. Curves obtained from R version 3.5.1.

Sewage sludge is widely used as an organic fertilizer, but also presents several risks to environmental and human health. This chapter presents a quantitative environmental risk assessment (ERA) and a qualitative human health risk assessment associated with farmland application of sewage sludge as compared to cattle and pig slurry. The analysis reflects conditions pertaining to Denmark, and neighboring European countries. The quantitative ERA was performed by estimating the cumulative risk of 138 (sludge) or 20 (slurry) identified contaminants 6 months after the 100th year of application. Environmental risk was quantified as the ratio of the predicted environmental concentration (PEC) divided by the predicted no effect concentration (PNEC). PEC/PNEC was 2–3 for sewage sludge and cattle slurry, whereas it was 8 for pig slurry. Metal compounds (especially Zn and Cu) accounted for the majority (>50%) of the environmental risk associated with farmland application of animal slurries, whereas phthalates and triclocarban collectively accounted for more than 50% of the corresponding risk associated with sewage sludge. Human health risks via dietary exposure to potentially toxic elements (PTEs) and pharmaceuticals in food crops were concluded not to cause any reason for concern. Based on reviewed literature, we further conclude that Danish sewage sludge does not represent a higher risk than animal manure for environmental dissemination of antibiotic resistance. Overall, it is concluded that sewage sludge from contemporary Danish society does not constitute a higher risk to soil organisms or human health than pig slurry.

This Guidance document describes harmonised risk assessment methodologies for combined exposure to multiple chemicals for all relevant areas within EFSA's remit, i.e. human health, animal health and ecological areas. First, a short review of the key terms, scientific basis for combined exposure risk assessment and approaches to assessing (eco)toxicology is given, including existing frameworks for these risk assessments. This background was evaluated, resulting in a harmonised framework for risk assessment of combined exposure to multiple chemicals. The framework is based on the risk assessment steps (problem formulation, exposure assessment, hazard identification and characterisation, and risk characterisation including uncertainty analysis), with tiered and stepwise approaches for both whole mixture approaches and component‐based approaches. Specific considerations are given to component‐based approaches including the grouping of chemicals into common assessment groups, the use of dose addition as a default assumption, approaches to integrate evidence of interactions and the refinement of assessment groups. Case studies are annexed in this guidance document to explore the feasibility and spectrum of applications of the proposed methods and approaches for human and animal health and ecological risk assessment. The Scientific Committee considers that this Guidance is fit for purpose for risk assessments of combined exposure to multiple chemicals and should be applied in all relevant areas of EFSA's work. Future work and research are recommended., This publication is linked to the following EFSA Supporting Publications article: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2019.EN-1589/full, http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2019.EN-1602/full

In April 2017, the Organic Business Development Team released a report with 25 recommendations for the Minister of Environment and Food (Det økologiske erhvervsteam 2017). Among these was a recommendation that organic farmers should have opportunities for utilizing nutrients from treated domestic wastewater for nutrient recycling.A prerequisite for future use of nutrients from treated wastewater is, that quality requirements are met and that application can be explained to (and accepted by) consumers. In partial fulfilment of this, the business team identified a need for a scientific overview of the risks of using nutrients from treated municipal wastewater in relation to other authorized fertilizer sources – e.g. conventional animal manures. Thus, it was assumed that a comparative approach to assess potential risk of using sewage sludge and conventional manures, could usefully inform decision makers in the future regulation of organic farming systems.Dependent on the result of the scientific investigation, the Organic Business Development Team foresaw that Denmark could chose to work to expand Annex 1 of the EU Ecology Regulation, to allow the organic farmers to use nutrients from municipal wastewater or other acceptable derived sludge products. Mobilization of support for this should be done by the Ministry of Environment and Food in collaboration with the Organic Farming Industry.Thus, based on available literature, this report aims at creating an overview of the environmental and human risks associated with application of pig and cattle slurry as well as sewage sludge to agricultural soils. The risk evaluation was performed for the following compound groups: Metals, Chlorophenyls, Dioxins, Furans, Halogenated aliphatic and aromatic hydrocarbons (HAH), Linear alkylbenzenesulfonates (LAS), Polyaromatic hydrocarbons (PAH), Polybrominated diphenyl ethers (PBDE), Polychlorinated biphenyls (PCB), Poly- and perfluorinated alkylated substances (PFAS), Phenols, Phosphate-triestersVII, Phthalates, Polychlorinated naphtalenes (PCN), Polychlorinated alkanes (PCA), Triclosan, Triclocarban, Medicines, Estrogens, Antibiotic resistance genes.Additionally the fertilizer potential of the two nutrient sources was characterized and compared.

International audience; Numerous micropollutants have been detected concurrently in aquatic systems, but little is known about the mixture effects of micropollutants in aquatic organisms and the underlying mechanisms. Azole fungicides are known to act synergistically with other chemicals by inhibiting cytochrome P450 (CYP) catalyzed detoxification and thereby enhance the effect of already toxic substances in different organisms. Two widely used fungicide classes co-occur in surface water are strobilurin and azole fungicides. This study aimed to investigate the species’ sensitivity to both fungicide classes and to gain mechanistic insights on potential synergistic effects of azoles on strobilurin fungicides in non-target organisms. Therefore, we determined the toxicokinetics of a strobilurin fungicide azoxystrobin and an azole fungicide prochloraz in two aquatic invertebrate species Gammarus pulex and Hyalella azteca. Furthermore, we explored median lethal concentrations (LC50s) of azoxystrobin in the presence and absence of prochloraz, the inhibition strength (IC50s) of prochloraz, and its effect on the locomotory behavior of the two species. Bioaccumulation of azoxystrobin weresimilar in both species with bioaccumulation factors (BAFs) approximately 5 L kgww-1, while bioaccumulation of prochloraz was different in two species, with BAFs 57 and 110 L kgww-1in G. pulex and H. azteca, respectively.Many biotransformation products were found for both fungicides in both species, of which taurine and malonyl conjugates were only identified in H.azteca. Most BTPs result from oxidation and conjugation reactions, which occurred at the (E)-methyl β-methoxyacrylate group of azoxystrobin and imidazole ring of prochloraz. Prochloraz inhibited the CYP-catalyzed biotransformation of azoxystrobin in both species, leading to higher internal azoxystrobin concentrations and suggesting synergistic effects. The half maximal inhibition concentration of prochloraz IC50, PRZ, AZwas 0.1 and 0.02 μM in G. pulex and H. azteca, respectively. The LC50sof azoxystrobin alone were 157 and 200 μg L-1in G. pulexand H. Azteca, respectively. Prochloraz significantly decreased the LC50s of azoxystrobin in both species. Video-tracking of the locomotory behavior suggested that prochloraz induced hyperactivity inG. pulex, but not in H. azteca. Overall, results suggests H. aztecacomprise more diverse biotransformation reactions and G. pulextended to be more sensitive than H. aztecatoward prochloraz effects.

Temperature is an important factor affecting toxicity, determining chemical toxicokinetics and toxicodynamics in poikilothermic organisms. As also metabolic rates are affected by temperature, interactions between the emergence of toxic effects and time are very likely. The aim of this study was to investigate how temperature affected the toxicity of copper towards the nematode Caenorhabditis elegans when measured during short fixed timeframes compared to during full life-cycles. Copper toxicity was tested in two experiments at four or six temperatures in the range of 11 to 24°C, with copper concentrations spanning from one to 40 mg Cu/L agar, respectively. Reproduction and mortality were determined across the entire life-cycle, and the time to production of first egg and the population growth rate were calculated. The results showed that the 50% effect concentrations (EC50) of copper increased 1.5 to 2.5-fold with increasing temperature within the tested range, depending on endpoint. When calculating EC50 on reproduction after 24 or 96 hours, as is the typical setup for temperature/chemical interaction studies, results ranged from no temperature effect to effects much larger than for the full life cycle. Studies of temperature effects on toxicity must therefore be carefully designed in relation to the research question investigated.