Instituto de Investigaciones Químicas y Ambientales de Barcelona

Torres-Martínez J.A., Mora A., Mahlknecht J., Kaown D., Barceló D.

Over the past few decades, the La Paz aquifer system in Baja California Sur, Mexico, has been under severe pressure due to overexploitation for urban water supply and agriculture; this has caused seawater intrusion and deterioration in groundwater quality. Previous studies on the La Paz aquifer have focused mainly on seawater intrusion, resulting in limited information on nitrate and sulfate pollution. Therefore, pollution sources have not yet been identified sufficiently. In this study, an approach combining hydrochemical tools, multi-isotopes (δ 2 H H2O , δ 18 O H2O , δ 15 N NO3 , δ 18 O NO3 , δ 34 S SO4 , δ 18 O SO4 ), and a Bayesian isotope mixing model was used to estimate the contribution of different nitrate and sulfate sources to groundwater. Results from the MixSIAR model revealed that seawater intrusion and soil-derived sulfates were the predominant sources of groundwater sulfate, with contributions of ~43.0% ( UI 90 = 0.29) and ~42.0% ( UI 90 = 0.38), respectively. Similarly, soil organic nitrogen (~81.5%, UI 90 = 0.41) and urban sewage (~12.1%, UI 90 = 0.25) were the primary contributors of nitrate pollution in groundwater. The dominant biogeochemical transformation for NO 3 - was nitrification. Denitrification and sulfate reduction were discarded due to the aerobic conditions in the study area. These results indicate that dual-isotope sulfate analysis combined with MixSIAR models is a powerful tool for estimating the contributions of sulfate sources (including seawater-derived sulfate) in the groundwater of coastal aquifer systems affected by seawater intrusion. • Multiple isotopes and Bayesian mixing model traced nitrate and sulfate sources. • First-time use of Bayesian mixing model to assess sulfate from seawater intrusion. • Fertilizer - ẟ15 N is affected by the mineralization–immobilization–turnover process. • Groundwater overuse promotes nitrification, seawater intrusion and sulfate increase.

Garcia-Arellano H., Gonzalez-Alfonso J.L., Ubilla C., Comelles F., Alcalde M., Bernabé M., Parra J., Ballesteros A.O., Plou F.J.

While testing the ability of cyclodextrin glucanotransferases (CGTases) to glucosylate a series of flavonoids in the presence of organic cosolvents, we found out that this enzyme was able to glycosylate a tertiary alcohol (tert-butyl alcohol). In particular, CGTases from Thermoanaerobacter sp. and Thermoanaerobacterium thermosulfurigenes EM1 gave rise to the appearance of at least two glycosylation products, which were characterized by mass spectrometry (MS) and nuclear magnetic resonance (NMR) as tert-butyl-α-D-glucoside (major product) and tert-butyl-α-D-maltoside (minor product). Using partially hydrolyzed starch as glucose donor, the yield of transglucosylation was approximately 44% (13 g/L of tert-butyl-α-D-glucoside and 4 g/L of tert-butyl-α-D-maltoside). The synthesized tert-butyl-α-D-glucoside exhibited the typical surfactant behavior (critical micellar concentration, 4.0–4.5 mM) and its properties compared well with those of the related octyl-α-D-glucoside. To the best of our knowledge, this is the first description of an enzymatic α-glucosylation of a tertiary alcohol.

Janesick A.S., Dimastrogiovanni G., Chamorro-Garcia R., Blumberg B.

Perspectives | Correspondence A Section 508–conformant HTML version of this article is available at http://dx.doi.org/10.1289/EHP1122. The Correspondence section is not peer-reviewed. Personal opinions expressed herein are the sole responsibility of the authors. ­ EHP neither endorses nor disputes the content of the letters it publishes. Reply to “Comment on ‘On the Utility of ToxCast™ and ToxPi as Methods for Identifying New Obesogens’” http://dx.doi.org/10.1289/EHP1122 Refers to http://dx.doi.org/10.1289/EHP881 We thank Houck et al. for taking the time to critique our paper, which evaluated the utility of ToxCast™ bioactivity data and the ToxPi tool for predicting PPARγ acti- vation and induction of adipogenesis. Our science-based critique of the performance of certain ToxCast™ assays should have elicited a response that engaged with the substance of our paper. Instead, Houck et al. highlight irrelevant or marginally relevant points and criticize us for analyses performed by scien- tists at the U.S. Environmental Protection Agency (EPA) while ignoring what we consider to be fundamental problems with ToxCast™ assays. Here we respond to their major points. In their first point Houck et al. suggest that we failed to cite the correct publications for the NovaScreen®, GeneBLAzer®, Attagene, and Tox21 assays. We cited the original developers of these assays and Knudsen et al. (2011) for using them in ToxCast™ Phase I to profile 309 chemicals. We did not perform targeted testing of Tox21 assays. In their second point Houck et al. make multiple criticisms regarding the assays, our analysis of them, and possible reagent differ- ences. We want to clarify that we undertook this project as a collaboration with Dr. Kristina Thayer of the National Toxicology Program (NTP) in 2010 as an activity related to the January 2011 NTP workshop “Role of Environmental Chemicals in Diabetes and Obesity” (Thayer et al. 2012). Prior to the workshop, Dr. Thayer provided us with a list of PPARγ activators generated by EPA scientists during late 2010. We agreed to test the top 20 ranked chemicals if they were provided. In rechecking our material transfer agree- ments, we found that the top 20 PPARγ hits were indeed provided by the EPA at the request of the NTP; it was our under- standing that these were from the same stocks as were used in ToxCast™. We added an additional chemical, chloro­thalonil (purchased separately), because the ToxCast™ Novascreen® assay results suggested it bound avidly to PPARγ (AC 50 = 0.54 μM). Following the workshop, we were provided with a second group of compounds (the “ToxPi chemicals”) by the NTP (material transfer agreement dated 27 October 2011). A 12 ToxPi chemicals were generated by predic- tion models based on assays applicable to biological processes associated with diabetes and obesity, assays that were nominated by experts in obesity, diabetes, and metabolism. The adipocyte differentiation model, which my group helped construct, was based on assay results for PPARγ, PPRE, RXR, GR, LXR, LXRE, SREBP1, and C/EBP; see Table S2 in Auerbach et al. (2016) and Figure 3 in our paper. Prediction models were generated by Dr. David Reif, then of the EPA, and the results provided to the investigators by the NTP, together with test chemicals. These models reflected the data analysis pipeline used by the EPA in 2011. It was our understanding that these chemicals were from ToxCast™ stocks, but whether this was the case is unclear in our correspondence. We tested all of the chemi- cals provided to us in good faith and reported the results. Changes in the EPA data processing pipeline caused the lists of prioritized chemi- cals to change numerous times between 2011 and 2015. Auerbach et al. (2016) used a data processing pipeline that evolved during the period of 2014–2015. Their prediction models utilized ToxCast™ Phase II data on 1,860 compounds; the original 2011 ToxPi prioritized chemicals were based on 309 ToxCast™ Phase I chemicals. Therefore, the targeted testing analysis originally contem- plated and undertaken by multiple groups was no longer straightforward, and Auerbach et al. (2016) was published as a review article. Surprisingly, none of the chemicals on the 2011 list that we identified as active on PPARγ, on RXR, and in adipogenesis assays were included on the 2015 list of priori- tized chemicals (see Table 2 in Auerbach et al. 2016). Three chemicals appeared on both lists (tebufenpyrad, pyridaben, and fenpyroximate) but did not induce adipo- genesis. That none of the active chemicals from the 2011 ToxPi list appeared in the 2015 list, whereas 3 inactive chemicals were incorrectly predicted to be active on both lists, indicates that the prioritization process needs improving. It is instructive to consider where the chemicals we found to be active are ranked by Auerbach et al. (2016). The 1,890 ToxCast Phase II chemicals were ranked from 1 to 810 by EPA scientists using the 2011 ToxPi list of assays; position 810 comprised 1,050 chemicals with a score of 0 (See Table S3 in Auerbach et al. 2016). The chemicals active in our assays were ranked as follows: tebupirimfos, 48; t ­ riphenyltin hydroxide, 71; spirodiclofen, 96; triflumizole, 150; zoxamide, 223; bisphenol A, 290; quinoxyfen, 444; flusilazole, 490; fludioxonil, 525; forchlorfenuron, 663; and pymetrozine and acetamiprid, 810. Therefore, the source of the low ranking is the ToxCast™ assays themselves, not the source of the chemicals. Since both the 2011 and the 2015 lists were generated by EPA scientists, it is unclear how our analysis of which chemicals to test was faulty, as Houck et al. allege. Houck et al. state that we did not consider chemical source in our discussion of why the assay results in our study disagreed with ToxCast™ results. While it is possible that one batch of a chemical has a contami- nant that produces spurious activity, or has degraded such that the active material is no longer active, we minimized this possibility by receiving test chemicals from reputable sources (the NTP and the EPA). These chemicals were used exclusively for receptor activation assays and in most of the adipo- genesis assays. In some cases, the stock of chemicals provided to us was exhausted, and we repurchased them from commercial sources. We did not observe differences in the ability of the repurchased chemicals to induce adipogenesis from the originals provided by the NTP and the EPA. Houck et al. further state that we did not consider methodological and platform differences in our criticism of the results of ToxCast™ assays. We have been doing nuclear receptor activation assays since 1992 and have contributed to the development of these technologies and the interpretation of results. While it can be made to appear that there are differences in our techniques that would obviate comparison of receptor activa- tion assays across species or platforms, this is not the case for PPARγ and virtually all other nuclear receptors (with the exception of the xenobiotic receptors SXR/PXR and CAR, which exhibit strong species selectivity for a subset of compounds). Occasional differences arise in PPARγ activation across species, but these are exceptional. It is also possible that a chemical can act as a receptor agonist in one cell type and be inactive or a receptor antagonist in another. Such chemicals are uncommon, and one would not expect to find many, if any, among the 37 PPARγ or PPRE activators identified by the ToxCast™ assays we tested. Whether chemicals can be metabolized to active forms by the cells used in the Attagene assays is also unlikely to be a valid criti- cism. The ability of chemicals to cause hits in the NRF2 assays noted by Houck et al. (information that was not conveyed to us volume 125 | number 1 | January 2017 • Environmental Health Perspectives

Malki M., Choukr-Allah R., Bouchaou L., Hirich A., Ait Brahim Y., Krimissa S., Hssaisoune M., Nghira A., Barceló D.

Groundwater quality in Souss-Massa Basin is influenced by natural and anthropogenic contaminations. In fact, the geological formations are the main sources of mineralization in Souss-Massa aquifer. The impact of marine intrusion is noticed in the coastal area. However, human activities are also responsible for the deterioration of groundwater quality. Indeed, agriculture is the main activity in the region with the presence of some industrial food unities which contribute to the degradation of water quality due to different uses of water resources as well as the reject of industrial and urban wastewater. The evaluation of urban pollution shows that it is directly linked to demographic changes, the rate of connection to the sanitation system, and wastewater treatment. However, wastewater treatment activity is quietly growing in the area. In fact, more than six treatment plants are operational in different cities. Furthermore, the Hydraulic Basin Agency and its partners have initiated several studies for new treatment plant implementation in other locations. Despite this progress, sanitation sector is still in remarkable delay. In fact, the total liquid discharges through the Souss-Massa Basin are equal to 28.8 million m3 of which 54.7% are only treated, and 45% are rejected directly in the environment and consequently generating a pollution load of more than 7.1 T/year of BOD, for example. The Agadir City is generating more than 50% of this pollution load. Regarding the production of solid waste, the total amount of waste produced in the area is evaluated at nearly 565.000 T/year. The distribution of waste between provinces shows that for the urban area of Agadir and Inezgane Ait Melloul, agglomeration contributes with over 80% of waste, while Taroudant and Ait Baha contribute with 68%. The assessment of industrial pollution shows that 36% of potential polluting industries are located at the province of Chtouka Ait Baha, 35% at the province of Inezgane Ait Melloul, 24% at Taroudant, 4% at Tiznit, and 1% at the province of Agadir Ida Ou Tanane. Agriculture is the dominant activity in the basin where the agricultural pollution in the Souss-Massa Basin is mainly connected to the agricultural production and the livestock. Citrus and vegetables are considered as a main source of nitrate pollution because of the important quantities of fertilizers used for these crops. The pollution load based on amounts of nitrogen leached is valued at 1,511 tons of nitrates per year.

El Mahdad E., Ouhajou L., El Fasskaoui M., Aslikh A., Nghira A., Fdil F., Baroud A., Barceló D.

By its transitional location between the Mediterranean and Saharan environment and its opening on the Atlantic, the Souss basin represents a specific hydraulic space across Morocco and the Maghreb countries. Its limited water resources formed through the phases of the long history of Morocco a real support to help the building of the foundations of the Moroccan hydraulic civilization. The authors of this chapter of varied specialties and belonging to different institutions relevant to water present from their experiences a synthesis of good practice in water planning and management in Souss basin. The surveyed subjects concern the contributions of the new water law and the need for its renewal for a better adaptation to space and society developments, the implementation and evaluation of the operation of the hydraulic basin Agency, the ingrained hydraulic prospective in the basin level, and its transition from a sector planning to integrated planning. Other current issues are discussed. It concerns participatory management of water that presents a promising approach at the local and national levels, the advanced state of resources development which has just reached the desalination of seawater for population supply and irrigation, and attempts of widespread access to water between state intervention and local initiative. Also, the aspects of the conservation of natural sites and actions to reduce water excess as the risk of flooding were analyzed. At the end of this chapter, the international dimension of planning, development, and management of water is discussed. Souss basin has benefited from many water projects in the framework of cooperation between Morocco and foreign partners.

Janesick A.S., Dimastrogiovanni G., Vanek L., Boulos C., Chamorro-García R., Tang W., Blumberg B.

In ToxCast™ Phase I, the U.S. EPA commissioned screening of 320 pesticides, herbicides, fungicides, and other chemicals in a series of high-throughput assays. The agency also developed a toxicological prioritization tool, ToxPi, to facilitate using ToxCast™ assays to predict biological function.We asked whether top-scoring PPARγ activators identified in ToxCast™ Phase I were genuine PPARγ activators and inducers of adipogenesis. Next, we identified ToxCast™ assays that should predict adipogenesis, developed an adipogenesis ToxPi, and asked how well the ToxPi predicted adipogenic activity.We used transient transfection to test the ability of ToxCast™ chemicals to modulate PPARγ and RXRα, and differentiation assays employing 3T3-L1 preadipocytes and mouse bone marrow-derived mesenchymal stem cells (mBMSCs) to evaluate the adipogenic capacity of ToxCast™ chemicals.Only 5/21 of the top scoring ToxCast™ PPARγ activators were activators in our assays, 3 were PPARγ antagonists, the remainder were inactive. The bona fide PPARγ activators we identified induced adipogenesis in 3T3-L1 cells and mBMSCs. Only 7 of the 17 chemicals predicted to be active by the ToxPi promoted adipogenesis, 1 inhibited adipogenesis, and 2 of the 7 predicted negatives were also adipogenic. Of these 9 adipogenic chemicals, 3 activated PPARγ, and 1 activated RXRα.ToxCast™ PPARγ and RXRα assays do not correlate well with laboratory measurements of PPARγ and RXRα activity. The adipogenesis ToxPi performed poorly, perhaps due to the performance of ToxCast™ assays. We observed a modest predictive value of ToxCast™ for PPARγ and RXRα activation and adipogenesis and it is likely that many obesogenic chemicals remain to be identified.Janesick AS, Dimastrogiovanni G, Vanek L, Boulos C, Chamorro-García R, Tang W, Blumberg B. 2016. On the utility of ToxCast™ and ToxPi as methods for identifying new obesogens. Environ Health Perspect 124:1214-1226; http://dx.doi.org/10.1289/ehp.1510352.

García-Villoria J., Navarro-Sastre A., Fons C., Pérez–Cerdá C., Baldellou A., Fuentes-Castelló M.Á., González I., Hernández-Gonzalez A., Fernández C., Campistol J., Delpiccolo C., Cortés N., Messeguer A., Briones P., Ribes A.

Acuña V., Aristi I., Aymerich I., Barceló D., Corominas L., Petrovic M., Poch M., Rodríguez-Mozaz S., von Schiller D., Sabater S., Elosegi A.

There is concern about the environmental effects of pharmaceuticals, since these substances have strong biological impacts and are found in an increasing number of sites, especially downstream from wastewater treatment plants (WWTP). Most information existing on the effects of pharmaceutical products is based on simple laboratory assays with single compounds, whereas pharmaceuticals in the environment typically appear in complex mixtures that include secondary metabolites as well as other pollutants. Therefore, real-world situations may contribute to the understanding of the fate and effects of pharmaceuticals in freshwaters. Here we report the effects of pharmaceuticals in the river Segre (Pyrenees, Iberian Peninsula) in a river segment affected by the effluent of a WWTP. The removal efficiencies of pharmaceuticals and their metabolites in both the WWTP and the river were analyzed by comparing the inflow and outflow concentrations at the WWTP and along the studied river segment, and their transformations and interactions were modeled. The WWTP had a higher removal efficiency (45%) than the river segment (20%), but the latter was also important. In general, the compounds most efficiently removed in the WWTP were also those more efficiently removed in the river. The removal efficiency in the river was higher during the day than during the night, suggesting that attenuation was driven by either photodegradation or biological transformation by primary producers. The effects of pharmaceuticals were analyzed across different scales, from those on biofilms to functional impairment of the river ecosystem. Laboratory toxicity tests showed that stream biofilms at the most polluted site developed community tolerance to anti-inflammatory drugs. Biofilms in the field also showed altered metabolic profiles and reduced algal diversity. WWTP effluents were able to alter the balance between autotrophic and heterotrophic processes: while ecosystem respiration was subsidized, gross primary production showed some stress effects.

Aristi I., Schiller D., Arroita M., Barceló D., Ponsatí L., García‐Galán M.J., Sabater S., Elosegi A., Acuña V.

The effluents of wastewater treatment plants (WWTPs) include a complex mixture of nutrients and pollutants. Nutrients can subsidise autotrophic and heterotrophic organisms, while toxic pollutants can act as stressors, depending, for instance, on their concentration and interactions in the environment. Hence, it is difficult to predict the overall effect of WWTP effluents on river ecosystem functioning. We assessed the effects of WWTP effluents on river biofilms and ecosystem metabolism in one river segment upstream from a WWTP and three segments downstream from the WWTP and following a pollution gradient. The photosynthetic capacity and enzymatic activity of biofilms showed no change, with the exception of leucine aminopeptidase, which followed the pollution gradient most likely driven by changes in organic matter availability. The effluent produced mixed effects on ecosystem-scale metabolism. It promoted respiration (subsidy effect), probably as a consequence of enhanced availability of organic matter. On the other hand, and despite enhanced nutrient concentrations, photosynthesis-irradiance relationships showed that the effluent partly decoupled primary production from light availability, thus suggesting a stress effect. Overall, WWTP effluents can alter the balance between autotrophic and heterotrophic processes and produce spatial discontinuities in ecosystem functioning along rivers as a consequence of the mixed contribution of stressors and subsidisers

Corcoll N., Acuña V., Barceló D., Casellas M., Guasch H., Huerta B., Petrovic M., Ponsatí L., Rodríguez-Mozaz S., Sabater S.

We assessed the tolerance acquired by stream biofilms to two non-steroidal anti-inflammatory-drugs (NSAIDs), ibuprofen and diclofenac. Biofilms came from a stream system receiving the effluent of a wastewater treatment plant (WWTP). The response of biofilms from a non-polluted site (upstream the WWTP) was compared to that of others downstream with relevant and decreasing levels of NSAIDs. Experiments performed in the laboratory following the pollution-induced community tolerance (PICT) approach determined that both algae and microbial communities from biofilms of the sites exposed at the highest concentrations of ibuprofen and diclofenac acquired tolerance to the mixture of these NSAIDs occurring at the sites. It was also observed that the chronic pollution by the WWTP effluent affected the microbial metabolic profile, as well as the structure of the algal community. The low (at ng L(-1) level) but chronic inputs of pharmaceuticals to the river ecosystem result in tolerant communities of lower diversity and altered microbial metabolism.

Grundmann V., Bilitewski B., Zehm A., Darbra R.M., Barceló D.

The project RISKCYCLE (“Risk-based management of chemicals and products in a circular economy at a global scale”) was funded by the European Union Framework Program 7 (“FP7”). It started in 2009 and its aims were successfully accomplished by the end of the project, three years later (2012). Within this coordination action a consortium of international experts investigated and defined the future research needs and gaps of for innovation in the field of risk-based management of chemicals and products. Various potential hazardous chemicals are used as additives in products worldwide. By developing new chemicals and product management approaches, the risks for human health and the environment should be minimized. As a first step, existing information about usage, risks, chemical properties and labelling chemicals and especially additives in consumer and industrial products were assembled and evaluated. RISKCYCLE focused on the fate and behaviour of these additives in six sectors: textile, electronics, plastics, leather, paper and lubricants. Additionally the project aimed at developing alternative testing strategies to minimize animal testing. The following article summarizes the background, the aims and issues and the most important outcomes of the EU funded project RISKCYCLE. It gives a brief overview about how the issues arise within a circular economy, how the project partners worked together in a coordination action and how results and conclusions were obtained.

Cayuela D., Montero L., Riva A., Prieto R., Marti M., Manich A.M.

Polylactide fibers (PLA), textured by false twist at different temperatures (135, 150 and 165℃) and draw ratios (1.30, 1.35 and 1.40) were stabilized by the application of a continuous thermal treatment at 100℃ and 50 m/min. The effect of thermal stabilization on the microstructure was determined by physicochemical tests such as iodine sorption or differential solubility. Iodine sorption and differential solubility are closely related to the crystallinity of the samples. Stabilization enhances crystallinity and orientation with the result that differences between the samples are diminished. Color value differences, albeit reduced, in some cases exceed the accepted limits. Color absorption is mainly dependent on the orientation and accessibility of the amorphous phase.

Cayuela D., Montero L., Riba M., Prieto R., Cano F., Manich A.M.

Polylactide fibers (PLA), false-twist textured at different temperatures (135, 150 and 165℃) and draw ratios (1.30, 1.35 and 1.40) were stabilized by a continuous setting treatment at 50 m/min and 100℃ using a specially designed chamber. The crystallinity and the orientation of the original textured filaments and those of the stabilized ones were measured. The effect of post setting on the dimensional stability, mechanical properties and thermomechanical behavior in accordance with the texturing conditions was studied. The relationships between crystallinity and the dimensional stability and between the orientation and both the relaxation shrinkage and the endset temperature of shrinkage were considered.

Antunes P., Viana P., Vinhas T., Rivera J., Gaspar E.M.

This paper reports, for the first time, a study of dioxin emissions from 10 siderurgies and metallurgies, secondary copper, aluminum and lead metallurgies, in Portugal. The study reports the emission factors and total emission amounts of PCDD/Fs, dioxin-like PCBs and hexachlorobenzene (HCB). The congener patterns were characterized and are discussed. The results showed that the total amount of PCDFs is higher than PCDDs in flue gas of each industrial unit. The toxic equivalent emission factors of pollutants emitted are 3098-3338 ngI-TEQt(-1) for PCDD/Fs and 597-659 ng I-TEQt(-1) for dioxin-like PCBs in siderurgies production (total estimated emission amounts released to atmosphere of 3.9-4.5 g I-TEQyr(-1)), 50-152 ng I-TEQt(-1) for PCDD/Fs and 24-121 ng I-TEQt(-1) for dioxin-like PCBs in ferrous foundries production (total estimated emission amounts released to atmosphere of 0.0010-0.0016 g I-TEQyr(-1)) and 5.8-5715 ng I-TEQt(-1) for PCDD/Fs and 0.49-259 ng I-TEQt(-1) for dioxin-like PCBs in non-ferrous foundries production (total estimated emission amounts released to atmosphere of 0.00014-0.12 g I-TEQyr(-1)). The HCB emission from siderurgies production is 0.94-3.2 mg t(-1) (total estimated emission amounts released 0.94-3.8 g yr(-1)), being much smaller, residual, in the emissions of the other types of plants (0.0012-0.026 mg t(-1) production and total estimated emission amounts released to atmosphere of 0.013-1.7 mg yr(-1)).

Marechal A., Fernandes D., Rotchell J., Porte-Visa C., Minier C.