Long-term selection for 8-week body weight in chickens — direct and correlated responses

Summary A bidirectional selection experiment for 8-week body weight in chickens was conducted. In addition to 27 generations of selection, random samples were chosen from each selected line in generations 6, 13, 19 and 26 to initiate lines in which selection was relaxed. Genetic change was evident in the selected high-weight line through the first 75% of the study after which response in the direction opposing selection occurred. Selection for low body weight resulted in considerable reduction in body size, particularly in the last quarter of the study. Correlated responses evaluated were body weight at several ages, conformation, age at onset of lay, various reproductive and egg quality traits and ratio (female: male) of some traits. Data from lines where selection was relaxed indicated that natural selection opposed artificial selection with the effects greater in later generations.     

Error propagation methods for LCA—a comparison

Purpose

The analysis of uncertainty in life cycle assessment (LCA) studies has been a topic for more than 10 years, and many commercial LCA programs now feature a sampling approach called Monte Carlo analysis. Yet, a full Monte Carlo analysis of a large LCA system, for instance containing the 4,000 unit processes of ecoinvent v2.2, is rarely carried out by LCA practitioners. One reason for this is computation time. An alternative faster than Monte Carlo method is analytical error propagation by means of a Taylor series expansion; however, this approach suffers from being explained in the literature in conflicting ways, hampering implementation in most software packages for LCA. The purpose of this paper is to compare the two different approaches from a theoretical and practical perspective.

Methods

In this paper, we compare the analytical and sampling approaches in terms of their theoretical background and their mathematical formulation. Using three case studies—one stylized, one real-sized, and one input–output (IO)-based—we approach these techniques from a practical perspective and compare them in terms of speed and results.

Results

Depending on the precise question, a sampling or an analytical approach provides more useful information. Whenever they provide the same indicators, an analytical approach is much faster but less reliable when the uncertainties are large.

Conclusions

For a good analysis, analytical and sampling approaches are equally important, and we recommend practitioners to use both whenever available, and we recommend software suppliers to implement both.     

Heat shock proteins in animal neoplasms and human tumours—a comparison

Heat shock proteins (HSPs) are implicated in all phases of cancer from proliferation, impaired apoptosis and sustained angiogenesis to invasion and metastasis. The presence of abnormal HSP levels in several human tumours suggests that these proteins could be used as diagnostic and/or prognostic markers, whilst the direct correlation between HSP expression and drug resistance in neoplastic tissues means they could also be used to predict cancer response to specific treatment. HSPs have also been successfully targeted in clinical trials modifying their expression or chaperone activity. Preliminary studies in veterinary medicine have also demonstrated the presence of altered HSP expression in neoplasms, and the study of carcinogenesis and the role of HSPs in animal models will surely be an additional source of information for clinical cancer research.     

Selection for fertility in mice — the selection plateau and how to overcome it

Summary A long-term experiment for increasing the traits first day litter size (LS1) and litter weight (LW1) was conducted with two populations for 33 generations. The selection plateau was reached in population DU-C (selection and estrus synchronization (h² = 0.02±0.01); in population DU-K (selection) the plateau (h² = 0.05±0.2) was nearly reached. Selection progress per generation was in LS1 b = 0.11±0.02; b = 0.12±0.04 (1st to 18th generation DU-K, DU-C) and b = 0.10±0.03; b = 0.07±0.05 (19th to 33rd generation, DU-K, DU-C) in LW1 b = 0.16±0.04 g; 0.19±0.07 g (DU-K, DU-C) b = 0.20±0.09 g; 0.001±0.09 g (DU-K, DU-C). Reverse and relaxe selection as well as systematic inbreeding was applied for 10 generations. Reverse selection yielded h² = 0.28±0.11 (R-DU-K) and h² = 0.17±0.05 (R-DU-C) and showed that there was still additive genetic variance. Relaxe selection did not cause alterations in the selection parameters, whereas inbreeding lead to inbred depressions (b = LS1 = –0.42±0.15; –0.45±0.12; b = LW1 = –1.13±0.20; –0.82±0.18 I-DU-K, I-DU-C). The plateau was based upon the heterozygote advantage. Several methods for overcoming the plateau were applied. A new selective useful variance could be created by crossing the plateau populations (h² = 0.14±0.04). A short-term progress in overcoming the plateau (1st to 3rd generation) could be obtained by litter size standardization (LS = 388). Tandem selection (selection for body weight — BW42) as well as crossing of inbred strains were not suitable for overcoming the selection plateau. Altering the environmental conditions as a possibility for overcoming the plateau has been discussed.     

Modeling thermal sensation in a Mediterranean climate—a comparison of linear and ordinal models

A simple thermo-physiological model of outdoor thermal sensation adjusted with psychological factors is developed aiming to predict thermal sensation in Mediterranean climates. Microclimatic measurements simultaneously with interviews on personal and psychological conditions were carried out in a square, a street canyon and a coastal location of the greater urban area of Athens, Greece. Multiple linear and ordinal regression were applied in order to estimate thermal sensation making allowance for all the recorded parameters or specific, empirically selected, subsets producing so-called extensive and empirical models, respectively. Meteorological, thermo-physiological and overall models - considering psychological factors as well - were developed. Predictions were improved when personal and psychological factors were taken into account as compared to meteorological models. The model based on ordinal regression reproduced extreme values of thermal sensation vote more adequately than the linear regression one, while the empirical model produced satisfactory results in relation to the extensive model. The effects of adaptation and expectation on thermal sensation vote were introduced in the models by means of the exposure time, season and preference related to air temperature and irradiation. The assessment of thermal sensation could be a useful criterion in decision making regarding public health, outdoor spaces planning and tourism.     

Behavioural and body mass changes before egg laying in the Barn Owl: cues for clutch size determination?

To investigate laying decision and clutch size determination in indeterminate layers, we analysed in-nest activity (nest presence, and copulation, prey deliveries, and entrance frequencies) and female body mass change, as well as their relation to clutch size variation in five Barn Owl pairs (Tyto alba) nesting in eastern France. Body mass of the female and behaviour [copulation frequency, entrance frequency, and prey delivery to the nest by the male (in number and mass)] were monitored using an automated weighing system and a video camera. There was a consistent change of behaviour and foraging activity among pairs ca. 18 days before laying indicating that the females may be tied to the nest at this time. Barn Owls being indeterminate layers have their clutch size determined at the oviposition of the first egg of the clutch. Window correlation analyses between the clutch size and the female body mass gain indicate that the clutch size might be determined no later than a few days before the laying of the first egg. Our results suggest that female Barn Owls may use the pre-laying period to determine the clutch size using cues such as the male food deliveries (a proxy for male quality).     

Mycorrhizal symbiosis and Lygodium microphyllum Invasion in South Florida—a biogeographic comparison

Lygodium microphyllum (Old World Climbing Fern) is one of the most problematic weeds in south Florida, invading numerous habitats from mangroves to pine flatwoods natural ecosystems. Much of the research efforts on L. microphyllum has been focused on reproductive potential, spore release, growth under different environmental conditions, belowground rhizome dormancy and survival strategies that describes its invasiveness. However, the role of an important mutualistic association with arbuscular mycorrhizal fungi (AMF) in the competitive ability and successful invasion of L. microphyllum by enhancing nutrient uptake has not been previously considered. Analysis of field root and soil samples from the ferns introduced and native range as well as a 7-week growth chamber experiment were done to determine the level of mycorrhizal colonization in the roots of L. microphyllum and the dependency on mycorrhizal fungi for growth and phosphorus (P) uptake. The field root samples showed that L. microphyllum was heavily colonized by AMF in relatively drier conditions, which are commonly found on some Florida sites compared to more common wetter sites where the fern is found in its native Australia. The results from the growth chamber experiment showed that the mycorrhizal treatment plants had significantly higher relative growth rate and biomass compared to the non-mycorrhizal plants. Similarly, L. microphyllum was highly dependent on the mycorrhizal fungi for growth and P uptake. Our results suggest that AMF play a significant role in vegetative reproduction and likely enhance the invasiveness of L. microphyllum in south Florida natural areas.     

Differential expression of secreted phosphoprotein 1 in response to estradiol-17β and in ovarian tumors in chickens

Roundabout 2 (Robo2) is a member of the membrane protein receptor family. The chemorepulsive effect of Slit2-Robo2 signaling plays vital roles in nervous system development and neuron migration. Slit2-Robo2 signaling is also important for maintaining the normal morphogenesis of the kidney and urinary collecting system, especially for the branching of the ureteric bud (UB) at the proper site. Slit2 or Robo2 mouse mutants exhibit multilobular kidneys, multiple ureters, and dilatation of the ureter, renal pelvis, and collecting duct system, which lead to vesicoureteral reflux. To understand the effect of Robo2 on kidney development, we used microinjection and electroporation to overexpress GFP-Robo2 in an in vitro embryonic kidney model. Our results show reduced UB branching and decreased glomerular number after in vitro Robo2 overexpression in the embryonic kidneys. We found fewer metanephric mesenchymal (MM) cells surrounding the UB but no abnormal morphology in the branching epithelial UB. Meanwhile, no significant change in MM proliferation or apoptosis was observed. These findings indicate that Robo2 is involved in the development of embryonic kidneys and that the normal expression of Robo2 can help maintain proper UB branching and glomerular morphogenesis. Overexpression of Robo2 leads to reduced UB branching caused by fewer surrounding MM cells, but MM cell apoptosis is not involved in this effect. Our study demonstrates that overexpression of Robo2 by microinjection in embryonic kidneys is an effective approach to study the function of Robo2.     

Spontaneous egg or chick abandonment in energy-depleted king penguins: a role for corticosterone and prolactin?

Various exogenous or endogenous factors may induce an emergency response in birds, redirecting current activity towards survival. In fasting, breeding penguins, the achievement of a critical energy depletion was suggested to induce egg abandonment and departure to sea for re-feeding. How such a behavioral shift is hormonally controlled remains unknown. The possible involvement of corticosterone and prolactin was examined by characterizing the nutritional and hormonal states of king penguins at egg abandonment. Further, we tested if these states differ according to whether an egg or a chick is abandoned, and according to the timing of breeding. In every case of abandonment, birds were in phase III fasting characterized by accelerated protein catabolism. However, body condition at egg abandonment was lower in early than in late breeders, suggesting that king penguins are willing to tolerate a larger energy depletion when their potential breeding success is high. At egg and chick abandonment, plasma corticosterone levels were, respectively, increased by 2- and 4-fold, whereas plasma prolactin levels were, respectively, depressed by 3- and 1.4-fold. The increase in plasma corticosterone and the decrease in plasma prolactin could be involved in the control of abandonment by, respectively, stimulating the drive to re-feed and diminishing the drive to incubate or brood. The smaller decrease in prolactin levels and the greater increase in corticosterone levels observed at chick vs egg abandonment suggest that, in addition to nutritionally-related stimuli, tactile or audible stimuli from the egg or chick could intervene in the endocrine control of abandonment.     

Maternal investment in relation to sex ratio and offspring number in a small mammal – a case for Trivers and Willard theory?

1.  Optimal parental sex allocation depends on the balance between the costs of investing into sons vs. daughters and the benefits calculated as fitness returns. The outcome of this equation varies with the life history of the species, as well as the state of the individual and the quality of the environment.
2.  We studied maternal allocation and subsequent fecundity costs of bank voles, Myodes glareolus , by manipulating both the postnatal sex ratio (all-male/all-female litters) and the quality of rearing environment (through manipulation of litter size by −2/+2 pups) of their offspring in a laboratory setting.
3.  We found that mothers clearly biased their allocation to female rather than male offspring regardless of their own body condition. Male pups had a significantly lower growth rate than female pups, so that at weaning, males from enlarged litters were the smallest. Mothers produced more milk for female litters and also defended them more intensively than male offspring.
4.  The results agree with the predictions based on the bank vole life history: there will be selection for greater investment in daughters rather than sons, as a larger size seems to be more influencial for female reproductive success in this species. Our finding could be a general rule in highly polygynous, but weakly dimorphic small mammals where females are territorial.
5.  The results disagree with the narrow sense Trivers & Willard hypothesis, which states that in polygynous mammals that show higher variation in male than in female reproductive success, high-quality mothers are expected to invest more in sons than in daughters.     

Focal chromosomal copy number aberrations in cancer—Needles in a genome haystack

The extent of focal chromosomal copy number aberrations (CNAs) in cancer has been uncovered through technical innovations, and this discovery has been critical for the identification of new cancer driver genes in genomics projects such as TCGA and ICGC. Unlike constitutive copy number variations (CNVs), focal CNAs are the result of many selection events during the evolution of cancer genomes. Therefore, it is possible that a single gene in a focal CNA gives the tumor a selective growth advantage. This concept has been instrumental in the discovery of new cancer driver genes. However, focal CNAs lack a consensus definition; therefore, we propose one based on pragmatic considerations. We also describe different strategies to identify focal CNAs and procedures to distinguish them from large CNAs and CNVs.     

Identification and characterization of a QTL on chromosome 2 for cytosolic glutamine synthetase content and panicle number in rice

A quantitative trait locus (QTL) associated with the protein content of cytosolic glutamine synthetase (GS1; EC 6.3.1.2) in senescing leaves, panicle number, and panicle weight was characterized in rice (Oryza sativa L.). A near-isogenic line (NIL), C-22, developed by marker-assisted selection was grown under different nitrogen levels in the greenhouse and in a paddy field. Chromosome 2 of C-22 had an approximately 50-cM segment substituted from the Kasalath (indica) chromosome in a Koshihikari (japonica) genetic background. C-22 showed a 12–37% lower content of GS1 protein in leaf blades than Koshihikari, which was in good agreement with a QTL region positively affected by the japonica chromosome. At an early vegetative stage, C-22 had more active tillers than Koshihikari in the greenhouse. At the reproductive stage, both panicle number and total panicle weight of C-22 were significantly higher than those of Koshihikari, particularly when the plants were grown under a low-nitrogen condition. These traits of C-22 were further confirmed in a paddy field. Thus, tiller development was positively affected by the Kasalath chromosome at an early vegetative stage, which resulted in an increased panicle number and panicle weight at the mature stage in C-22. These data indicate that the target QTL (Pnn1; panicle number 1) is important in the development of tillers and panicles in rice. Linkage analyses for panicle number and ratio of developing tiller formation in the second axil (RDT) revealed that Pnn1 was delimited at the 6.7-cM region.     

Three‐dimensional reconstruction and comparison of vacuolar membranes in response to viral infection

The vacuole is a unique plant organelle that plays an important role in maintaining cellular homeostasis under various environmental stress conditions. However, the effects of biotic stress on vacuole structure has not been examined using three‐dimensional (3D) visualization. Here, we performed 3D electron tomography to compare the ultrastructural changes in the vacuole during infection with different viruses. The 3D models revealed that vacuoles are remodeled in cells infected with cucumber mosaic virus (CMV) or tobacco necrosis virus A Chinese isolate (TNV‐A^C), resulting in the formation of spherules at the periphery of the vacuole. These spherules contain neck‐like channels that connect their interior with the cytosol. Confocal microscopy of CMV replication proteins 1a and 2a and TNV‐A^C auxiliary replication protein p23 showed that all of these proteins localize to the tonoplast. Electron microscopy revealed that the expression of these replication proteins alone is sufficient to induce spherule formation on the tonoplast, suggesting that these proteins play prominent roles in inducing vacuolar membrane remodeling. This is the first report of the 3D structures of viral replication factories built on the tonoplasts. These findings contribute to our understanding of vacuole biogenesis under normal conditions and during assembly of plant (+) RNA virus replication complexes.     

IPHEN—a real-time network for phenological monitoring and modelling in Italy

This paper aims to describe the Italian PHEnology Network (IPHEN), a cooperative project started in 2006 with the aim of producing nationwide maps of analysis and forecast of plants phenological stages mainly used to satisfy the needs of agriculture, health and environmental care. Iphen is a data processing system composed of the following main segments (a) collection of atmospheric and phenological data, (b) processing of data with suitable phenological and geo-statistical models and (c) phenological maps of analysis and forecast. In more detail, IPHEN maps of analysis (featuring phenological stages reached at the date of processing) are produced with models based on a Normal Heat Hours approach which weighs hourly air temperature effectiveness for plant phenological progression applied to national grids of hourly temperature derived from the operational agro-meteorological network of CRA-CMA. A correction scheme based on phenological surveys provided by volunteer observers is applied to the first guess maps of analysis to obtain final maps. Forecast maps (prediction of the days of occurrence of relevant phenological stages) are produced on the basis of GFS model medium range forecasts and climatic data. Freeware IPHEN maps for grapevine, common and Arizona cypress, black elder, olive and locust trees are broadcasted weekly on the CRA-CMA website. The positive operational results of IPHEN are testified by 150 maps broadcasted during the 2011 season for the above-mentioned species. The system performances and reliability have been analysed focusing on the analysis of phenological simulation errors and on the sensitivity of phenological maps to anomalous atmospheric circulation patterns. The error analysis shows that phenological models are characterized by advances/delays that justify the adoption of an observation based correction scheme. The sensitivity analysis highlights that the system is responsive to the effects of circulation blocking systems leading to phenological advances and delays.     

Inhibition of prostaglandin synthesis in eggshell gland mucosa as a mechanism for P,P'-DDE-induced eggshell thinning in birds—a comparison of ducks and domestic fowls

1. A recent report from the present laboratory indicated that the DDT-metabolite p,p'-DDE, which produces eggshell thinning in sensitive species of birds, inhibits prostaglandin synthesis in the eggshell gland.2. This was found to be true both after in vitro addition to a homogenate of eggshell gland mucosa from ducks, and after in vivo administration to ducks at a dose regimen that produces eggshell thinning.3. The purpose of the present study was to compare the p,p'-DDE-sensitive duck and the insensitive domestic fowl regarding the effects of p,p'-DDE administration on calcium and prostaglandin metabolism of the eggshell gland mucosa.4. Administration of p,p'-DDE caused significant eggshell thinning in ducks (−18.5%) but not in the domestic fowl.5. The shell thinning in ducks was accompanied by significant reductions in ⁴⁵Ca uptake and prostaglandin synthesis by a homogenate of the eggshell gland mucosa, as well as significant reductions of the prostaglandin E₂ content in the shell gland and the content of calcium and bicarbonate present in the shell gland lumen at the time of slaughter; the content of calcium in the eggshell gland mucosa was significantly increased.6. None of the changes was observed in the domestic fowl.7. Based on these findings, inhibition of a prostaglandin-stimulated bicarbonate transport across the eggshell gland mucosa is discussed as a possible mechanism for p,p '-DDE-induced eggshell thinning in birds.     

Selection of efficient vesicular-arbuscular mycorrhizal fungi for wetland rice — a preliminary screen

Eighteen different VA mycorrhizal fungi were screened for their symbiotic responses with wet land rice cultivar Prakash (IET 2254) under pot culture. Of the 18 fungi, Acaulospora sp. (ICRISAT), Glomus fasciculatum (Riverside) and G. mosseae (Invermay) were found to be the best in improving plant height, tiller number, total biomass, panicle number, grain weight and plant P and Zn concentrations. Increases in grain yield caused by inoculation with Acaulospora sp. (ICRISAT), G. fasciculatum (Riverside) and G. mosseae (Invermay) were 62%, 59% and 35%, respectively.     

In vitro response of leaf tissues from Lolium multiflorum — a comparison with leaf segment position,leaf age and in vivo mitotic activity

Summary Immature gramineous leaves provide a convenient system for comparing the response of cells in culture with their state of differentiation. Callusing frequency is compared with leaf segment position, leaf age and in vivo mitotic activity in Lolium multiflorum. (1) In a succession of one millimeter sections from the immature leaf base, callus was formed from the first and second sections but not the third or subsequent sections. The frequency of those explants callusing decreased with distance from the base of the leaf and with leaf age (or leaf extension growth). (2) In vivo, the proportion of cells in mitosis declined from around 10–14% at the base of young leaves to zero at 5 mm from the base and beyond. Mitotic activity also declined in leaves as they aged, and dividing cells were not observed in leaves 30 days from initiation or older. (3) A high frequency of callus formation was associated with a high mitotic index in the explant. But for corresponding mitotic indices, cells further away from the leaf base were less responsive in culture. (4) It is proposed that cells are becoming differentiated even in highly meristematically active regions of the leaf and concomitantly losing their ability to respond in culture.     

Mean surface temperature prediction models for broiler chickens—a study of sensible heat flow

Body surface temperature can be used to evaluate thermal equilibrium in animals. The bodies of broiler chickens, like those of all birds, are partially covered by feathers. Thus, the heat flow at the boundary layer between broilers’ bodies and the environment differs between feathered and featherless areas. The aim of this investigation was to use linear regression models incorporating environmental parameters and age to predict the surface temperatures of the feathered and featherless areas of broiler chickens. The trial was conducted in a climate chamber, and 576 broilers were distributed in two groups. In the first trial, 288 broilers were monitored after exposure to comfortable or stressful conditions during a 6-week rearing period. Another 288 broilers were measured under the same conditions to test the predictive power of the models. Sensible heat flow was calculated, and for the regions covered by feathers, sensible heat flow was predicted based on the estimated surface temperatures. The surface temperatures of the feathered and featherless areas can be predicted based on air, black globe or operative temperatures. According to the sensible heat flow model, the broilers’ ability to maintain thermal equilibrium by convection and radiation decreased during the rearing period. Sensible heat flow estimated based on estimated surface temperatures can be used to predict animal responses to comfortable and stressful conditions.     

Engrained experience—a comparison of microclimate perception schemata and microclimate measurements in Dutch urban squares

Acceptance of public spaces is often guided by perceptual schemata. Such schemata also seem to play a role in thermal comfort and microclimate experience. For climate-responsive design with a focus on thermal comfort it is important to acquire knowledge about these schemata. For this purpose, perceived and “real” microclimate situations were compared for three Dutch urban squares. People were asked about their long-term microclimate perceptions, which resulted in “cognitive microclimate maps”. These were compared with mapped microclimate data from measurements representing the common microclimate when people stay outdoors. The comparison revealed some unexpected low matches; people clearly overestimated the influence of the wind. Therefore, a second assumption was developed: that it is the more salient wind situations that become engrained in people’s memory. A comparison using measurement data from windy days shows better matches. This suggests that these more salient situations play a role in the microclimate schemata that people develop about urban places. The consequences from this study for urban design are twofold. Firstly, urban design should address not only the “real” problems, but, more prominently, the “perceived” problems. Secondly, microclimate simulations addressing thermal comfort issues in urban spaces should focus on these perceived, salient situations.     

Eco-efficiency indicator framework implemented in the metallurgical industry: part 1—a comprehensive view and benchmark

Purpose

The purpose of this work was to develop an indicator framework for the environmental sustainability benchmarking of products produced by the metallurgical industry. Sustainability differentiation has become an important issue for companies throughout the value chain. Differentiation is sometimes not attainable, due to the use of average data, lack of comparative data, certain issues being overshadowed by others, and a very narrow palette of indicators dominating the current sustainability assessments. There is a need for detailed and credible analyses, which show the current status and point out where improvements can be made. The indicator framework is developed to give a comprehensive picture of eco-efficiency, to provide methods that enable relevant comparisons as well as the tools for communicating the results. In this way, the methodology presented in this study aims to make differentiation easier and thus aid companies in driving the development toward more sustainable solutions.

Methods

The framework is based on the existing indicator framework Gaia Biorefiner, which is primarily intended for bio-based products. In this work, the framework was further developed for application in the metallurgical industry. The indicator framework is built by first looking at the issues, which are critical to the environment and global challenges seen today and which the activities of the metallurgical industry may have an impact on. Based on these issues, suitable indicators are chosen if they exist and built if they do not. The idea is that all indicators in a group form a whole, showing areas of innovation while refraining from aggregating and weighting, which often compromise a comprehensive and objective view. Both qualitative and quantitative indicators are included. The indicators are constructed following the criteria set by the EU and OECD for building indicators. Each indicator further has a benchmark. The rules for building the benchmark are connected to the indicators. Suitable data sources and criteria for the benchmark and the indicators are gathered from literature, publicly available databases, and commercial LCA software. The use of simulation tools for attaining more reliable data is also studied.

Results and discussion

The result is a visual framework consisting of ten indicator groups with one to five indicators each, totaling up to 31 indicators. These are visualized in a sustainability indicator “flower.” The flower can be further opened up to study each indicator and the reasons behind the results. The sustainability benchmark follows a methodology that is based on utilization of baseline data and sustainability criteria or limits. A simulation approach was included in the methodology to address the problem with data scarcity and data reliability. The status of the environment, current production technologies, location-specific issues, and process-specific issues all affect the result, and the aim of finding relevant comparisons that will support sustainability differentiation is answered by a scalable scoping system.

Conclusions

A new framework and its concise visualization has been built for assessing the eco-efficiency of products from the metallurgical industry, in a way that aims to answer the needs of the industry. Since there is a baseline, against which each indicator can be benchmarked, a sustainability indicator “flower” can be derived, one of the key innovations of this methodology. This approach goes beyond the usual quantification, as it is also scalable and linked to technology and its fundamental parameters. In part 2, a case study “A case study from the copper industry” tests and illustrates the methodology.