Fig. 2 plots the coefficients of interactions between the intensity measure in mother;s birth-province and a dummy for mother;s age at the time of the policy from estimating equation (1).36 The coefficients of the interactions (δl for each l) are jointly significant for l = 7a10 and insignificant for l = 11a17 (p-values are 0.000 and 0.211, respectively). Imposing the restriction that Hygromycin B mothers aged 11a17 in 1997 were not affected by the CSL (imposing the restriction of zero for the coefficients of the interactions between the intensity measure and year-of-birth dummies for unexposed mothers) in order to gain precision, the first-stage takes the form:
equation(2)Sijk=α+βk+γj+l=710(IntensityjYOBil)δl+Xijkθ+uijkwhere the omitted control group is now mothers aged 11a18 in 1997. A discrete treatment dummy indicating whether mothers aged 7a10 in 1997 (post-reform cohort) interacted with the intensity measure is also used as an instrumental variable (in the above equation rather than year of birth dummies).37
Fig. 2.uEffect of the CSL on primary school completion. Notes: The sample includes all mothers between the ages of 18 and 29 at the time of the survey. Females aged 11 in 1997 is the youngest unaffected cohort (solid vertical line). Each point on the solid line represents the coefficient of the interaction between the intensity measure in mother;s birth-province and a dummy for mother;s age at the time of the policy. Dashed lines are 95% confidence intervals.Figure optionsDownload full-size imageDownload as PowerPoint slide
The second-stage model is:
equation(3)Yijk=φ+βk+γj+Sijkμ+Xijkθ+vijkwhere Sijk is instrumented using the interaction between the intensity measure and mother;s year-of-birth dummies.38 In all specifications, the standard errors are adjusted for clustering on the province of mother;s birth.39 Alternative levels of clustering, including, clustering at the regional and sub-regional levels (12 and 20 clusters, respectively), clustering at the year of birth*region of birth and year of birth*province of birth levels, and two-way non-nested clustering (following Cameron, Gelbach, Miller, 2006uanduThompson, 2011), yield similar results.40 Specifications are estimated for a sample of mothers ages 18a29, where mothers ages 22a29 are the unexposed cohorts.41 Furthermore, I use a tighter age window as a robustness check (mothers ages 18a25, where mothers ages 22a25 are the unexposed cohorts). It should be noted that a few husbands (1.4% of the sample of analysis; 24 observations) were exposed to the policy; however, the results are robust to excluding these observations.
Recall the exclusion restriction requires that the intensity of the reform should not directly affect the outcomes of interest, besides via changing schooling. In other words, the excluded instruments should be uncorrelated with the error term in (3). One particular concern is that the intensity of the reform is not random as the government likely devoted greater resources to provinces with a greater number of students taking up enrollment as a consequence of the reform. Specifically, the number of primary school aged children not enrolled in primary school in the year prior to reform influence the number of additional classrooms allocated by the government. The estimations therefore control for province-level (i) primary school aged children and (ii) enrollment rates in 1995, both interacted with mother;s year of birth dummies. Variation in the excluded instrument is therefore after controlling for Transformation non-random component for resource allocations. This would include, for example, variation in additional classrooms associated with government officials favoring certain provinces or arbitrary allocation of resources.

Recent HCA biomarker studies have focused on detection and characterization of HCA-DNA adducts (Tang et?al., 2013, Gu et?al., 2012 and Brown et?al., 2001) which reflect the actual detrimental impact of these carcinogens at the genomic level rather than the indicative harm represented by protein adducts. Other studies are aiming to detect HCA-peptide adducts following proteolysis by stabilising the adduct\’s arylsulfinamide bond via oxidation to an arylsulfonamide bond which is resistant to acid, heat and reducing agents commonly used in proteolytic MDL 12330A hydrochloride (Peng et?al., 2012, Peng and Turesky, 2013 and Peng and Turesky, 2014). While characterization of such stabilised adducts is ongoing (Peng and Turesky, 2014), their existence has yet to be demonstrated in human blood in vivo. However, an alternative approach is also warranted. Markers of HCA status, or more general dietary factors such as red meat intake, may be revealed via untargeted proteomic, lipidomic or metabolomic analyses. Preliminary metabolomic studies of the cohort of blood samples collected in this study are currently underway at the Institute for Global Food Security at Queen\’s University Belfast.
5. Conclusions
Carcinogenic and mutagenic heterocyclic aromatic amines are known to form adducts with blood proteins. The characterisation and use of such adducts as internal dosimeters in epidemiological studies of HCA exposure have been studied for many years without a clear candidate adduct being proven in vivo. The hydrolytic release from protein adducts of intact HCAs which distinguished between vegetarians and meat-eaters was demonstrated fifteen years ago (Magagnotti et al., 2000) but correlation with dietary HCA intake by this method has not been repeated since. The current study systematically assessed a range of hydrolysis conditions for release of HCAs from in vivo plasma adducts measured by validated mass spectrometry (limits of detection 1-5 pg/ml plasma). Food diaries from cross-sectional study participants demonstrated HCA intakes ranging from to 2.5 μg/day. Despite sufficient assay sensitivity and use of positive control HCA-protein adducts synthesised in vitro, a HCA was detected in only one sample (PhIP, 9.7 pg/ml) demonstrating that acid-labile HCA adducts do not reflect dietary HCA intake as previously postulated and are present at such low concentrations that they are not viable biomarkers MDL 12330A hydrochloride of HCA exposure. This conclusion is in keeping with the current direction of research in this area which aims to fully characterize the structure of HCA-protein adducts and to utilize stabilised HCA-peptide adducts as dosimeters following protein digestion.

During signal acquisition, unwanted image acquisition artifacts and physiological noise obscure the true underlying signal of interest. To improve the signal-to-noise ratio (SNR), various preprocessing operations, i.e. temporal frequency filtering or magnitude image smoothing, are incorporated in the processing and reconstruction pipeline, and physiologic noise sources are commonly regressed out from the signal [13], [14], [15] and [16]. It is well documented that the application of these operations induces local spatial and temporal correlations into neural regions that were previously uncorrelated [17], [18] and [19]. The linear framework developed in this Cyclophosphamide monohydrate manuscript also describes how signal processing alters the structure of the spatial covariance matrix, such that induced correlation is a result of increased overlapping frequency content between voxels after processing. Signal processing will alter the activated voxel\’s temporal frequency spectrums, by spreading voxel task activated peaks temporally and spatially. Correlation will be induced between voxels as a result of increased overlapping frequency content between the two voxel\’s Fourier frequency spectrums. This notation for spatial correlation is advantageous since various physiological signals are also confined to specific frequency ranges. Respiratory and cardiac cycle fluctuations are characterized around 0.2-0.3 Hz and 1 Hz in a voxel\’s temporal frequency spectrum, although they are often aliased to low frequencies in fMRI signal acquisition [13], [20] and [21]. The summation notation of spatial correlation that is described here, allows relative contributions to the correlation to be quantified by segregating the natural partitions in a voxel\’s temporal frequency spectrum. Compared to magnitude-only correlations, applying this framework with complex-valued data more accurately identifies regions of spatial correlation, and reduces the false positives in correlation maps. This result is most significant in low magnitude CNR data sets since including the phase in the complex-valued correlation results in increased sensitivity of identifying correlated regions.

The time series of winter mean sea to air heat flux and wind stress curl over the north-central Iceland Sea (Fig. 10) are consistent with this interpretation. Both show a long-term decline that would lead to a reduction in the buoyancy flux from the ocean to the CPI-169 as well as a reduction in the doming of the isopycnals of the Iceland Sea Gyre. The diminished occurrence frequency of high heat flux events over the region (Fig. 11) as well as a northward shift in the location of the heat flux maximum (Fig. 12) contribute to the reduction in the buoyancy flux. There is evidence of a step-like discontinuity in both the turbulent heat flux and wind stress curl time series around 1995 that is consistent with the observed long-term behavior of the depth of the σθ=28.03kg/m3 isopycnal (Fig. 7). The timing of these discontinuities is simultaneous with the transition from NAO positive to NAO neutral conditions (Flatau et al., 2003uanduCohen and Barlow, 2005) that resulted in a number of other changes in the oceanography of the region (e.g. H?kkinen and Rhines, 2004, Pálsson et al., 2012uanduStraneo and Heimbach, 2013). However, the muted dependence of the airasea forcing over semilunar valve region on the depth of the Icelandic Low as compared to the Lofoten Low (Table 1) suggests that further work is required in order to understand the relative importance of large-scale atmospheric circulation patterns like the NAO and ILD to the climate of the Nordic Seas.

Alkali-catalyzed transesterification is extensively used in biodiesel production from biomass such as soybeans and corn because it provides fast reaction time and convenient separation, and does not produce AMG 487 corrosion. Alkaline-catalyzed reactions are inhibited by high concentrations of free fatty acids (FFAs) (>1% w/w) in the feedstock, which will form soap and cause difficulty during washing, as shown in Reaction 3. Water can also be a problem (>0.1% w/w) because it hydrolyzes esters to form FFAs, as shown in Reaction 4, which will consequently form soap (Atadashi et al., 2013). The literature indicates that the FFA level of sewage sludge can reach up to 65%, which is considerably beyond the limit of 1% (Suresh Kumar et al., 2008). Hence, acid catalysts are selected for sewage sludge in the present research.Figure optionsDownload full-size imageDownload as PowerPoint slide
Figure optionsDownload full-size imageDownload as PowerPoint slide
Meanwhile, previous studies have indicated that sewage sludge is a potential feedstock for biodiesel (Mondala et al., 2009, Kargbo, 2010, Revellame et al., 2010a and Wang et al., 2016). The yield of FAMEs from sludge is significantly affected by the combined effects of methanol-to-sludge mass ratio, acid catalyst concentration, and temperature. For biodiesel production, primary, secondary, and blended sludge have been used. Dufreche et al. (2007) demonstrated that biodiesel could be produced from secondary sludge by using different polarities, through extraction using supercritical CO2, or via in situ transesterification. The highest biodiesel yield was obtained via in situ transesterification (6.23% of dried sludge). Mondala et al. (2009) reported the acid-catalyzed in situ transesterification of primary and secondary sludge. The maximum yield of FAMEs obtained from primary sludge (14.5%) was higher than that obtained from secondary sludge (2.5%) when in situ transesterification was conducted at 75 °C using an acid concentration of 5% (v/v) and a methanol-to-sludge mass ratio of 12:1. Revellame et al. (2010a) demonstrated that the optimum yield of biodiesel from activated sludge via in situ transesterification (4.88%) was obtained at 55 °C at a methanol-to-sludge ratio of 25 and using a H2SO4 volume of 4%. They hypothesized that a temperature above 60 °C significantly decreased biodiesel yield because of the acid-catalyzed polymerization of unsaturated fatty acids or their esters. Revellame et al. (2010b) also investigated the in situ transesterification of wet activated sludge (84.5% weight moisture) for biodiesel production; they demonstrated that this process was less economical than that for dried sludge (5% weight moisture). Pastore et al. (2013) reported the production of FAMEs via a two-step process: the preliminary extraction of dried sludge using hexane in acidic ambient followed by methanolysis. However, this process required multiple reactions, as well as washing and separation steps.
The aforementioned studies show that in situ transesterification conditions can be varied to obtain optimum biodiesel yield. However, only a few studies have reported on the production of biodiesel from heat-dried blended sludge samples obtained from different main wastewater treatment technologies, e.g., normal anaerobic-anoxic-oxic (A2/O) and A2/O by membrane bioreactor (MBR), as sources (Mondala et al., 2009, Revellame et al., 2010a and Wang et al., 2016). Complex sewage sources and several complicated wastewater treatment technologies will lead to different results with respect to the composition of non-polar and polar lipids in sewage sludge, which will further result in biodiesel yield. At present, several treatment processes are used in WWTPs in China, such as conventional activated sludge treatment, A2/O, sequencing batch reactor (SBR), anaerobic-oxic, oxidation ditch, and MBR. Among these, A2/O is the most widely used technology in large WWTPs (capacity treatment > 104 t/d) in China. In terms of design capacity, the A2/O process accounts for 33.2%, followed by the oxidation ditch process (28.2%), and the SBR process (10.3%) (Qiu et al., 2010 and Jin et al., 2014). Meanwhile, the MBR process has become an increasingly attractive option for wastewater treatment and water reclamation in which excellent, stable, and effluent quality is required (Sun et al., 2011). Although MBR is currently a minor process in China, its use is rapidly increasing, with an estimated annual growth rate of 20-30% in the next 5 years (Wang et al., 2014). Therefore, two types of drying blended sludge from the A2/O process and the MBR process were used as promising lipid feedstock to produce biodiesel in this study.
This study mainly investigates the difference between two types of heat-dried sludge obtained from the A2/O process and the MBR process in Beijing to produce biodiesel via acid-catalyzed in situ transesterification. The optimized biodiesel yield obtained from these sludge samples was investigated using a three-factor and four-level orthogonal test and single-factor tests. The effects of methanol-to-sludge mass ratio, acid concentration, and temperature on biodiesel yield were examined by range analysis. In addition, the compositions and purity of FAMEs were analyzed via gas chromatography-mass spectrometry (GC-MS). This study also aimed to determine which sludge would be a better biodiesel resource, i.e., A2/O sludge or MBR sludge.

1. Introduction
With rapid advances in technology, the use of engineered nanoparticles has been steadily increasing in the recent past. In spite of the best recycling efforts, these nanoparticles very often end up as inclusions of waste materials which are thermally broken down in waste incineration plants. Waste incineration is also proving to be an attractive alternative as it results in reduction of the waste volume and at the same time providing the possibility of energy recovery (Buonanno and Morawska, 2015). Recent legislations are aiming to reduce waste disposal by landfilling and this has kindled more interest in waste incineration. Volatilization and recombination of the metal inclusions in waste materials, during their incineration often lead to nanoparticle formation. Without proper filtration measures the nanoparticles could end up being released to the atmosphere, which creates environmental and health hazards. The risk of nanoparticle release from the combustion of nanowaste has been addressed by several studies in the last years (Bouillard et al., 2013, Cernuschi et al., 2010, Gottschalk and Nowack, 2011, Kumar et al., 2013 and Walser et al., 2012). Bouillard et al. (2013) could show that the combustion of a polymeric LY335979 mixed with carbon nanotubes results in a transfer of carbon nanotubes to the gas phase requiring a proper filtration to prevent release. Cernuschi et al. (2010) investigated the nanoparticle concentration in the exhaust of four differently configured waste incineration plants. Walser et al. (2012) balanced the mass flow of the nanoparticle input to the different waste fractions (slag, quench water, fly ash, clean gas) of a waste incineration plant. Furthermore, they concluded that wet scrubbers and electrostatic precipitators are effective in separating the ultrafine particles. However, the underlying mechanisms of nanoparticle separation and how these processes could be improved by adjusting the operational conditions have not been addressed. Therefore this study focuses on a detailed investigation of the separation processes during the transport of the nanoparticles to the filtration unit as well as the filtration process itself based on pulse-jet cleanable fibrous filter media covered by a filter cake.
In this sense the separation of nanoparticles in waste incineration plants can be described as a twofold process. Nanoparticles formed during waste incineration are carried to the bag house filters by the flue gas along with fly ash particles. These products are cooled down at different stages before reaching the bag house filters, and consistently undergo Brownian collisions resulting in adherence to fly ash particles as well as self-aggregation of the nanoparticles. Collisions and subsequent adherence to fly ash particles is termed as scavenging of nanoparticles and results in easier nanoparticle removal at the bag house filters as the fly ash particles have sizes in the micrometre range (Friedlander et al., 1991 and Lee and Wu, 2005). However, self-aggregation and diffusion scavenging are competing processes as self-aggregation of the nanoparticles increases the particle size thereby reducing the effectiveness of nanoparticle scavenging. The effectiveness of scavenging depends not only on the size and concentration of the nanoparticles and the fly ash particles but also on the experimental conditions. The extent of scavenging depends on the differences in size of the colliding entities and the mass transfer regime in which collisions are happening (Gopalakrishnan and Hogan, 2011 and Gopalakrishnan et al., 2011). An accurate population balance modelling is necessary to study the effectiveness of nanoparticle scavenging for various parameters that are typical for a waste incineration plant.
The presence of the larger fly ash particles creates a continuously growing dust cake on the fibrous filter medium of the bag house filters thereby increasing the filtration efficiency of the filter system. The dust cake also results in an increase of the pressure drop across the filter making it necessary to periodically remove the dust cake by a pulsed gas jet. The filtration behavior of fibrous filter media has been studied extensively for the separation of coarse fly ash particles (L?ffler et al., 1988 and Binnig et al., 2009) as well as the separation of nanoparticles (Kim et al., 2007, Wang and Tronville, 2014, Liu et al., 2011, Buha et al., 2013 and Wang and Otani, 2013). However, to the best of our knowledge detailed investigations dealing with the effect of a dust cake on a filter medium for the filtration of particles in the lower nanometre regime are not available so far. As suggested before (Buonanno and Morawska, 2015), very few studies on measuring ultrafine emissions from incineration plants are available. Schiller and Schmid (2014) have briefly discussed the filtration efficiency of a precoated filter medium for three precoating thicknesses, but not in the context of waste incineration but for pellet heaters in private households representing a totally different operational scale. This study investigates three of the main operational parameters of dust cake covered filter media namely the filter cake thickness, the face velocity and the flue gas temperature for particles larger than 2 μm. Buonanno et al. (2012) have studied the ultrafine emissions from incineration plants and the role of fiber filters on the filtration efficiencies. However, a detailed analysis on the effect of different operation parameters on the filtration efficiencies and possible emissions from incineration plants has not been done before.

2.3. Analytical methods
The volume of gas produced was measured once a day using a Ritter meter (MGC-10 PMMA, Germany), and the gas composition once or twice a week (% of CH4, CO2 and O2, ppm(v) H2S) by passing 24-h samples through a Biogas Check Analyser (Geotechnical Instruments, United Kingdom). All gas volumes were normalized to 1 atm, 273 K and are expressed as NmL. To avoid intake of air during withdrawal of reactor sludge, the gas space of the reactors was connected to an external gas balloon filled with nitrogen. The dilution of the produced gas with nitrogen from the external gas balloon together with any contamination by air during the collection of the gas was compensated for by adjusting the methane content according to the formula CH4/(1-(O2 + Balance)), where Balance = 1-CH4-CO2-O2-H2S. In addition, the produced biogas was adjusted by a factor 0.96 to account for the water vapour in the gas at 37 °C. The number 0.96 was calculated taking the theoretical value for water vapour pressure at 37 °C multiplied by the air moisture content of the gas (estimated by Testo 605-H1, Nordtec Instrument AB, Sweden). To adjust for the variation in feeding time, the gas production was divided by the amount of hours since the last feeding and then multiplied by 24.
TS and VS were measured in duplicate twice a week for the reactor sludge and all substrate batches according to the Swedish Standard method (SS 028113), with the modification of using 10-15 ml of digested sludge for the analysis. The Benzoquinonium of the reactor sludge and substrate was determined with InfoLab pH 7310 (WTW Germany) according to European Standard EN 12176. For a summary of pH, TS and VS of the substrates, see Table S2.
Concentrations of volatile fatty acids (VFA) were measured twice a week for the reactor sludges, and once per substrate batch of the fibre sludge and activated sludge, respectively, as described by Jonsson and Borén (2002). Total metal concentrations were determined once a month for the reactor sludge and centrifuge reject and once per substrate batch of fibre sludge and activated sludge (Eurofins Environment Testing Sweden AB).
3. Results and discussion
Reactor performance in terms of methane production for the different experimental Phases (I-IV) is depicted in Fig. 1. The process was initially unstable and both reactors accumulated VFA as soon as the OLR was increased above 0.6 g VS L?1 day?1 (days 12, 17 and 27). Elemental analyses showed that the Mg content of the feed was low (10 mg/L) compared to values reported for optimal growth of methanogens (730 mg/L; Ahring et al. (1991)). Accordingly, Mg was supplied to R1. Elemental analyses showed that the nutrient levels in the activated sludge were much higher than in the fibre sludge (cf. Table S3). For that reason, in the case of R2, activated sludge was mixed in with the fibre sludge to investigate whether the Mg content therein (40 mg/L) was enough to stabilize the reactor. However, when the OLR was increased to 0.75 g VS L?1 day?1 on day 47, VFA started to accumulate in R2 (from 0.6 mmol/L to 1.8 mmol/L) but not in R1. To avoid a decrease in pH by further accumulation of VFA, R2 was also supplied with MgO from day 49. The adjusted Mg and Ca additions enabled a stable process at loadings up to 2.9 g VS L?1 day?1 in both reactors (Fig. 1).
An underlying cause of the process disturbance could be that high Ca levels affected the ion balance between Ca and Mg. Analyses of the fibre sludge showed a high calcium content (290 mg/L), which could be explained by its natural occurrence in wood together with release from the chemical recovery and from paper production steps (fillers, coating) in the kraft process (Y Borgström 2015, pers. comm., 9 September). Together with the CaOH2 addition of 100-1100 mg/L, this could have resulted in inhibitory levels of Ca, even though much higher concentrations have been reported in order to reach inhibitory levels (cf. Chen et al. (2008)). There are several reports on the antagonistic relationship between cations, for example between Na+ and Mg2+ (Ahring et al., 1991), Na+ to Mg2+ or Ca2+ (McCarty and McKinney, 1961), or the antagonistic effects of combinations of cations (Kugelman and McCarty, 1965). In a study by Suárez et al. (2014), microcrystalline cellulose was digested in the presence of four different mineral mixtures. The mineral mixture with a high Ca content and a high Ca:Mg ratio showed a reduced methane production, while the mineral mixture with a high Ca content but lower Ca:Mg ratio improved the methane production. This confirms that the ratio of Ca to Mg is important for AD of cellulose-based materials. Thus, in the case of pulp and paper mill wastewaters, Mg supplements may be needed to reduce the risk of process instabilities caused by high Ca concentrations. In this study, a ratio of Ca to Mg of 42:1 gave an unstable system, while a change to 16:1 made it possible to start increasing the OLR. On day 107, both reactors were running at an OLR of 2.6 g VS L?1 day?1 without VFA accumulation at Ca:Mg ratios of 8:1 and 13:1 for R1 and R2, respectively.

A second area of research addresses the relation between waste production and GDP growth. These studies aim at analyzing the effectiveness of waste policies through the environmental Kuznets Curve (Grossman and Krueger, 1991 and Kuznets, 1955) as a baseline, and testing hypotheses with econometric modeling. According to these scholars scarce evidence of delinking as an effect of waste management regulations and policy tools exists (Mazzanti and Zoboli, 2005). Only a relative decoupling4 has been reported by some studies investigating the broad spectrum of households, municipal or industrial waste (Mazzanti, 2008, Mazzanti and Zoboli, 2008 and Sj?str?m and ?stblom, 2010). Considering the issue of packaging waste, except for Rouw and Worrell, 2011 and van Sluisveld and Worrell, 2013, and Worrell and van Sluisveld (2013) focused on the packaging policies in the Netherlands, no specific paper has been found in our review. In the Netherlands case, even if different voluntary agreements have been implemented to reduce the environmental impact of packaging, packaging waste figures have been increasing annually. This calls for clearer and more robust policy measures.
The last group consists of papers based on technical approaches. These articles adopt the perspectives of the Life Cycle Assessment (LCA), design for the environment or the cradle to grave frameworks to analyze packaging waste prevention with a special focus on the environmental performance of packaging in relation to other critical features such as marketing and communication (Ferreira et al., 2015, Grant et al., 2015, Lee and Xu, 2005, Madival et al., 2009, Rossi et al., 2015, Wikstr?m et al., 2013 and Williams et al., 2008).
So, as a result of the review carried out, we found that a broad analysis of packaging waste prevention policies, comparing the specific measures issued at country level, the organizational solutions developed to face the regulatory targets, and the results obtained, is currently missing. This paper aims at addressing this DMOG lack of investigation, responding to the following research questions: What is the state of the art of the packaging and packaging waste prevention policies? What are the main features and tendencies of the prevention policies emerging at the global level?
Therefore, our investigation has an exploratory purpose because it aims at understanding what is happening in a substantially new field in order to identify useful insights and generate new ideas for future policies and further research (Robson, 2002 and Yin, 2009).
3. Methodology
In order to carry out our study we adopted a theoretical sampling technique (Eisenhardt, 1989 and Yin, 2009). This non-probability sampling approach, where the elements or subjects of the study are purposefully selected, is particularly suitable for investigating emerging phenomena and developing novel insights (Eisenhardt and Graebner, 2007).
In particular, we selected multiples cases (Yin, 2009), chosen because they are “extreme exemplars” (Eisenhardt and Graebner, 2007: 27), that is, leading countries/states in the prevention field. In fact, according to Siggelkow (2007: 20), “it is often desirable to choose a particular organization precisely because it is very special in the sense of allowing one to gain certain insights that other organizations would not be able to provide”.
With regard to this point, in order to strengthen robustness and reliability of our study we decided to involve field experts from the very beginning. A continuous dialogue with six officers of the national compliance scheme and other corporate representatives was realized through meetings, telephone conversations, and email exchanges for the whole period of the project.
This engagement process led to the selection of eleven countries/states that have demonstrated an advanced attitude with regard to packaging waste prevention. Seven are EU Member States (Belgium, France, Germany, Ireland, Italy, Spain, and Sweden), whereas the remaining four are extra-European countries/states (Australia, California, Canada, and Japan). The sample constituents have three elements in common:?The attention paid to environmental issues (considering, for example, the high targets established for waste recycling and recovery).?Similar social-economic structures, characterized by advanced consumption patterns.?Vast diffusion of modern distribution systems.

Fig. 8. Effect of curcumin on the expressions of NF-kB, iNOS, Nrf2 and HO-1 mRNA levels in L02 cells. Curcumin pretreatment markedly inhibited the expression levels of NF-kB (A) and iNOS (B) mRNAs, but further increased the expression levels of Nrf2 (C) and HO-1 (D) mRNAs. Values were presented as mean ± SD, from three independent experiments (n = 3). **p < 0.01, compared to the control group; #p < 0.05, ##p < 0.01, compared to the QCT alone group.Figure optionsDownload full-size imageDownload as PowerPoint slide
3.8. Inhibition of HO-1 reduces the protective effect of curcumin on QCT induced cell death
Compared to the QCT alone group, HO-1 inhibition by ZnPP IX significantly increased QCT induced caspase-9 and -3activations (Fig. 9A and B) and further activated the expression of NF- kB mRNA (Fig. 9C), finally aggravated QCT induced cell death (Fig. 9D). Meanwhile, ZnPP IX treatment partly abolished the inhibition of curcumin on the activation caspase-9, -3 and the expression of NF-kB mRNA, reduced the protection of curcumin on QCT induced cell death, compared to that in QCT plus curcumin (i.e., QCT + Cur) group (Fig. 9).
Fig. 9. Inhibition of HO-1 reduces the protective role of curcumin on QCT-induced caspase activation), inflammation and cytotoxicity in L02 cells. To determine the relative role of HO-1 activity in the protective role of curcumin on QCT induced cytotoxicity, GNF 2 were pre-treated wth curcumin (at 5 μM) for 2 h, then replaced and incubated with ZnPP IX (at 10 μM) plus with or without QCT(at 5 μg/mL) for additional 24 h, caspase-9 (A), caspase -3 (B), NF-kB mRNA expression (C) and cell viability (D) were measured. Values were presented as mean ± SD, from three independent experiments (n = 3). The values sharing the same superscript are not significantly different at p < 0.05 by Duncan’;s multiple range test.Figure optionsDownload full-size imageDownload as PowerPoint slide
4. Discussion
Series of animal studies showed that QdNOs including olaquindox (OLA), mequindox (MEQ), CBX, QCT and their metabolites could induce oxidative damage in rat or mouse tissues including liver, kidney, spleen and adrenal gland, however, the potential molecular mechanism is far from clear (Wang et?al., GNF 2 2011a, Wang et?al., 2015a, Wang et?al., 2010, Yu et?al., 2014a and Yu et?al., 2013a). It was suggested that QCT exhibited stronger toxicity and higher risk by using in vitro models than any other members of QdNOs, such as OLA, CBX, and MEQ, and this has drawn widely attention in China (Wang et al., 2015b). In present study, we investigated the protective role of curcumin on QCT induced toxicity and the potential mechanism in human normal hepatocyte L02 cells. The results of current study revealed that QCT exposure could cause mitochondrial dysfunction, apoptosis and inflammatory response in L02 cells, and curcumin could effectively attenuate these unwanted toxicity effect.
In present study, QCT exposure at 5 μg/mL significantly decreased the cell viability of L02 cell (Fig. 1A), which was consistent with the previous study (Dai et al., 2015b). In addition, we found that QCT exposure markedly increased LDH activity (Fig. 1B), indicating that QCT could disturb the integrity of cell membrane. Curcumin pretreatment could markedly increase the cell viability and decrease the release of LDH (Fig. 1), then may protect L02 cell from QCT induced cytotoxicity. Apoptosis, one type of programmed cell death, is characterized by cell shrinkage, chromatin condensation, nucleus condenses and the formation of apoptotic bodies (Wang et al., 2011c). There are at least three major pathways participating in the apoptosis process, i.e., the mitochondrial pathway, death receptor pathway, and endoplasmic reticulum pathway (Wang et al., 2011c). Our previous study demonstrated QCT could induce the cell apoptosis in human HepG2 cell line (Zhang et al., 2013). In the present study, we found that QCT exposure also caused chromatin condensation, nucleus condenses and formation of apoptotic bodies in L02 cells, finally led to significant increase of the apoptotic rates. However, this trend could be significantly attenuated by pretreatment of curcumin in a dose-dependent manner (Fig. 2). These results indicated that curcumin attenuated QCT induced cytotoxicity involving the inhibition of cell apoptosis.

The reason for the low thiamine levels in the AIN-93-related diets in the present study is not clear, although irradiation seems to have been a factor (Table 3). It is known that heat and irradiation can reduce thiamine and vitamin A levels, and this is taken into account when formulating diets that will be autoclaved or irradiated. Irradiation has been reported to have relatively modest effects in a natural ingredient diet matrix (Caulfield et al., 2008), while thiamine was reported to be unstable on storage at room temperature in the purified AIN-76 diet relative to a natural ingredient diet (Fullerton et al., 1982). In the current study, the vitamin mix added to the AIN-93-based diets should have been sufficient to account for loss due to irradiation, and the diets were stored refrigerated to maintain nutrient levels. However, specifics of pellet preparation were likely a contributing factor as, in an attempt to normalize hardness across all purified diets, some had higher moisture content than others. Factors that contribute to thiamine loss in irradiated purified diets include a combination of moisture content, irradiation dose, and form of thiamine (personal communication, Dr. B. Mickelson, Envigo (Harlan) Teklad). The longer irradiation time and higher dose range used by the manufacturer of the purified diets (see Materials and Methods) may have contributed to the lower thiamine levels in these diets. In any case, although the thiamine levels fed to the animals in the study were below 5 ppm, they were clearly adequate in all cases to support growth, gestation, and lactation.
Endpoints to evaluate spermatogenesis (testes weight, testicular spermatid head count, caudal epididymal Nutlin3a count, motility, and morphology, and histopathology) were included based on the reports of soy/phytoestrogen detrimental effects on spermatogenesis in CD-1 mice (Cederroth et al., 2010). Although our data did not support such a link, abnormal spermatogenesis was noted in animals fed AIN-93, and its low thiamine may have contributed to this observed effect. Testicular degeneration had been reported decades ago in rats subjected to severe thiamine deficiency (Morris and Dubnik, 1947), and spermatogenesis defects have been reported in thiamine transporter knockout mice in the absence of the more severe thiamine deficiency manifestations (Fleming et?al., 2003 and Oishi et?al., 2004). An interesting aspect of the present study is that defects in spermatogenesis were observed only in the AIN-93 diet group, which had non-detectable levels of thiamine reported in both the G and M formulations, while the SPC and SPC-IF groups, which had detectable thiamine levels in the M, but not G, formulation, did not show effects on testicular weights, testicular spermatid head counts, or histology. This could indicate that an early developmental subclinical thiamine deficiency may be critical to the noted effect on spermatogenesis and/or that there is an interaction between the subclinical thiamine deficiency and the lack of soy protein. Furthermore, although consistent effects on testes were noted, there was no observed deficit in epididymal sperm in the AIN-93 diet group. This could indicate a delayed effect on testicular spermatogenesis, similar to the delayed effect on spermatogenesis reported in aromatase knockout mice (Robertson, 2002). Further targeted experiments in mice fed low thiamine diets would be needed to address these questions more definitively. The issue of thiamine deficiency affecting reproductive toxicity assessments in rodents is unlikely to be an issue when natural ingredient diets are used, due to their high thiamine content; however, this may be a potential issue for rodent studies when purified diets, and particularly sterilized purified diets, are used. It must also be noted that, while the low measured thiamine levels are possibly involved in the observed effect, other micronutrients that were lower in the AIN-93-related diets may have also played a role. For example, as noted previously, vitamin A levels were lower in these diets than in the natural ingredient diets and vitamin A also plays a critical role in the development of the testis and the maintenance of spermatogenesis (Chihara et?al., 2013 and Hogarth and Griswold, 2010).