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The categorisation system was developed through an iterative procedure including a careful evaluation at each stage. This was necessary because each of three rounds yielded substantial feedback from the expert taggers, identifying issues to be resolved and proposing improvements to the system. This process led to a much clearer understanding of the structure of sensitive data resources and a wider agreement on definitions to be applied in the tagging process. In addition, the allocation of exactly one tag per category improved during the development for many categories, indicating that the selection process was straightforward for most resources and categories. As a result, the categorisation system could be simplified and the structure improved, appropriately representing a trans-disciplinary effort. This may also be important from the user perspective. At the end of the day, the system should be so intuitive that the users searching for terms would have the same logic as the experts entered the tags.To be beneficial for the domain of LS, the categorisation system and the toolbox requires broad community approval38,39. In the project, we began the approval process with nominated experts from 6 LS RIs, embedded in a larger working group of the H2020-funded project EOSC-Life, covering 13 LS RIs. Though this can be seen as a useful starting point, the toolbox obviously needs community approval at a much larger scale. As the categorisation system is specifying a part of essential metadata for resources about sensitive data, it will be relevant to the FAIR Digital Objects (FDO) Forum for a « resources in the life sciences » FDO. The categorisation system can be used to derive FDO attributes and values for such FDOs. FDOs for the sensitive data itself, when levels of sensitivity and specific access protocols need to be specified is an interesting possible extension, and the categorisation system could support as a backbone information for access governance and technical choices. FDOs are to be “machine actionable”, so desirable mappings between different categorisation systems will be operationalisable. New European projects such as FAIRCORE4EOSC (https://faircore4eosc.eu/), FAIR-IMPACT (https://fair-impact.eu/) and other projects working on pragmatic semantic improvements for FAIR appliance will provide possibilities for registering metadata schemas and mappings that should reuse interdisciplinary approaches in the heterogeneous field of life sciences.The RDA has established and is maintaining a Metadata Standards Catalogue (MSC) (https://rdamsc.bath.ac.uk/mapping-index,5). An appropriate goal for the categorisation system would be to be included in this catalogue, after further refinement and alignment with other vocabularies addressing sensitive data in the life sciences. In any case, the work on the categorisation system can contribute to discussions on methodologies for aligning metadata schemas across scientific domains, while the categorisation system itself can be seen as an important contribution to the process of developing the most useful and appropriate cross-disciplinary terms and categories for describing sensitive data. We keep in mind that similar approaches have been applied via long and iterative processes in other scientific domains, such as understanding and predicting the evolution of climate (essential climate variables, https://public.wmo.int/en/programmes/global-climate-observing-system/essential-climate-variables) and essential biodiversity variables for mapping and monitoring species populations40. There are biases and gaps in the existing system that need to be tackled in the future. The initial content of the toolbox demonstrator, consisting of 110 resources related to sensitive data, has been primarily selected by four RIs with a focus on clinical and biomedical research (BBMRI, EATRIS, ECRIN, Euro-Bioimaging). Other areas and sensitive data types, such as environmental, classified, and proprietary data are under-represented, as are some disciplines, such as zoology, ecology, plant and mycological sciences, and microbiology. This indicates a need for a broader coverage of resources linked to sensitive data in the future work. Another question that needs to be investigated is how interoperable the categorisation system is with other domains outside the LS that systematically deal with sensitive data, for example, the Social Science and Humanities41). In addition, systematic data on the usability/user-friendliness of the toolbox from a broad sample of potential users from the life sciences are needed. Initial and informal evaluation of these aspects by the experts involved so far has been very positive but is clearly limited in scale and needs to be supplemented by more evidence.There are major challenges to the sharing of sensitive data, including interoperability, accessibility, and governance. The primary objective of the toolbox is to improve discoverability of resources and digital objects linked to the sharing and re-use of sensitive data (F in FAIR)4. The systematic application of a standardised typology for resources about sensitive data, as defined by the categorisation system, helps to better structure, and organise the issues and results in metadata enrichment (F4, R1.3 of the FAIR principles in Supplementary, Table S1). The toolbox alone will not be enough for the ‘I’ of the FAIR principles, but it may become a useful backbone for building more interoperable classification systems for sensitive data resources.It is perhaps more common to base findability on a tagging system using keywords (plus title text). That is, for example, how PubMed works—it does not categorise resources, it adds MESH terms to them (https://pubmed.ncbi.nlm.nih.gov/). Another option would have been to try to derive keywords from text or title. In our case, a categorisation system with pre-defined dimensions and pre-listed tags was preferred by the expert group. Keywords, in isolation, suffer from several disadvantages:

(a)

A range of equivalent terms may be used to mean the same thing – making searching for that concept difficult, requiring multiple ‘Or’ statements.

(b)

They may have multiple meanings (polysemy) especially if “drawn from”, or “applied to”, a wide range of scientific disciplines.

(c)

The different aspects of the resource covered by keywords, i.e., the types or dimensions of keyword applied, may be inconsistent and / or incomplete.

The categorisation system, on the other hand, guarantees that all 7 validated dimensions required are used in the tagging process and that the tags selected are standardised and defined. The toolbox categories also aid browsing of results by enabling sequential filtering using the categories and tags.In addition, there is a useful link between developing community approved categories for metadata, in this case for characterising resources dealing with sensitive data, and community understood (but implicit) ontologies used in the same area. Categories and ontologies can complement each other—without a common underlying ontology, metadata terms can be interpreted inconsistently, and without defining metadata categories, ontologies may remain implicit and inconsistent. We found, for example, that discussions on the best categorisation to use for scientific disciplines, or data types, exposed the implicit (and different) ontologies being used by different people and is based on the personal views of those in the group. Those would have been obviously rooted in and / or influenced by the language and working assumptions of their discipline(s), and their roles and experiences, (current and previous). That will be more and more the case with interdisciplinary research development and development in research careers. Developing categories in metadata can therefore play an important role in describing, understanding and, ultimately, harmonising the implicit ontologies scientists use in thinking about the area of sensitive data.In the development of the categorisation system, existing ontologies, classifications, and terminologies were taken into consideration (Table 2). However, many more have relationships to the categorisation system. An example is the Subject Resource Application Ontology (SRAO), an application ontology describing subject areas/academic disciplines used within FAIRsharing records by curators and the user community42. A first crosswalk has demonstrated considerable agreement between the toolbox category “research field” and subsections of SRAO42 and EDAM15. The toolbox has been registered as a resource (database) at FAIRsharing, a curated, informative, and educational resource on data and metadata standards, inter-related to databases and data policies (https://fairsharing.org/3577). It is planned to create a collection group of resources (standards, databases, policies) in FAIRsharing linked to the toolbox and the underlying categorisation system. This will also cover relationships to ontologies and classifications.There is a need to explore the applicability of the toolbox to specific domains. One example could be the European Joint Programme on Rare Diseases (EJP RD), where resources are made progressively FAIR at the record level to support innovative basic, translational and clinical research (https://www.ejprarediseases.org/coordinated-access-data-services/fairification-support/). The goal is to identify, refine and expose core standards for dataset interoperability, asset (data, sample, subject) discovery, and responsible data sharing, concentrating on data level rather than resource level information. Knowledge exchange between EJP RD and the toolbox could be of benefit in exploring the complementary of both approaches in adequately characterising resources linked to sensitive data and thus improving data discoverability.The first pilot study demonstrated major variation in tagging of resources if independent taggers are assessing the same resource (inter-observer variation). The example of BBMRI has shown that this variation can be considerably reduced if adequate training is performed; which in return is resource intense. Thus, to arrive at a valid and reliable tagging process, there is a necessity for adequate training and support to reduce inter-observer variation. Specific training sets and training programs as well as intercalibration tools need to be developed and implemented and approved by the community.Another option to be explored should be AI—or ML-algorithms to support automatic (or at least semi-automatic) tagging of resources. It is not easy to use AI/ML in this field due to the multilingualism and the misinterpretation of terms. Often there are different meanings between scientific disciplines and a common backbone for the application of AI/ML is difficult to achieve. It is necessary to come to a common understanding between people involved in the assessment of resources related to sensitive data in all life sciences. Nevertheless, the toolbox can become of major importance for research and application of AI/ML techniques in this field. It may serve as a resource for AI/ML to better find resources in the field by serving as a kind of gold standard to compare with. Another promising approach would be to consider a knowledge graph as an intelligent representation. For the categorisation system the approach could be used to interlink categories to a resource (e.g., “source related to sensitive data” has “geographical scope”) and to link individual tags between categories if possible (e.g., “clinical research data” result from “clinical research”). This would give a richer representation of the knowledge behind the categorisation system and the option to be integrated in existing approaches (e.g., OpenAIRE, https://www.openaire.eu/). Therefore, we will consider knowledge graphs as an intelligent knowledge representation of the categorisation system in the future.A major challenge will be the transition of the toolbox demonstrator to a mature toolbox and ultimately its maintenance, extension, and sustainability. Development of the toolbox demonstrator has been financed by EOSC-Life, but this project will end in 2023. Discussion on sustainability has been initiated with several life-science infrastructures (e.g., BBMRI, EATRIS, ECRIN and ELIXIR, another European Life-Science Infrastructure). Key aspects of sustainability that need to be considered are maintenance of the toolbox portal and tagging tool and of the toolbox content including expert time for tagging as well as human resources to maintain the system. Different approaches are under evaluation: an organization considering the resource core to its operations and taking full responsibility, or a joint ownership across multiple organisations (e.g., multiple RIs) or a community taking responsibility, either funded by future grants or through in-kind contributions from motivated research parties/individuals. Further costs to be covered will include system maintenance, input from a toolbox manager, tagging of resources by experts, as well as advertisement to the envisioned user groups, hardware costs and costs for debugging and major extension of functionality if needed. More

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Effect of phosphorus starved cultures of Dunaliella tertiolecta on growth represented as optical density under stress of nickel ionsIn the case of normal culture, phosphorus starved control culture (without nickel stress), and phosphorus-starved treated cultures, data presented in Table 1 and graphed in figure (S1, Supplementary Data) clearly showed a progressive increase in optical density with increasing culturing period in case of normal culture, phosphorus-starved control culture, and phosphorus-starved treated cultures. Our findings are consistent with those of18 who found that in phosphorus starved cultures of three algae species, Microcystic aeruginosa, Chlorella pyrenoidesa, and Cyclotella sp., the biomass, specific growth rate, and Chl-a all declined significantly.The optical density achieved during the four periods of culturing was lower in phosphorus-depleted control cultures than in normal cultures (i.e., cultures contained phosphorus). When compared to a normal control (without nickel addition), the optical density was reduced by 9.1% after 4 days of culturing under phosphorus deprivation and by 10.0 percent after 8 days of culturing. In the case of 5 mg/L dissolved nickel, however, the obtained optical density values in phosphorus starved treatment cultures rose with the increase in culturing period during all culturing periods as compared to phosphorus-starved control (without nickel addition) cultures.At 10 mg/L dissolved nickel and after 4 days of culturing, the optical density although less than those in case of concentration 5 mg/L, yet it was higher than control (− P) but by increasing the culturing period more than 4 days, the optical density was less than control (− P). Our results are similar to those of19 who observed that the decrease in cell division rate signaled the onset of P-deficiency. The cultures that showed no significant increase in cell number for at least three consecutive days under the experimental conditions were considered P-depleted. In addition20, observed that the growth rate of Dunaliella prava was found to be dramatically lowered when phosphorus was limited. The content of chlorophyll fractions, total soluble carbohydrates, and proteins all fell considerably as a result of phosphorus restriction.The results concerning the effect of dissolved nickel on the growth of Dunaliella tertiolecta under conditions of phosphorus limitation show that phosphorus starved Dunaliella had lower growth as compared to the control (phosphorus-containing culture medium). These results are in agreement with those obtained by7 who reported that the optical density of Chlorella kessleri cell suspension decreased with phosphorus deficiency compared to control. Also21, found that Chlorella vulgaris cells grew 30–40% slower in phosphorus-starved cultures than in control cultures. Furthermore22, showed that diatoms were unable to thrive when phosphorus levels were insufficient. Diatom dominances were reduced to 45 and 55% in enclosures where phosphate was not provided23 observed that, under salt stress, Chlorella’s metabolic rate was substantially lower than Dunaliella’s.It can be concluded that when microorganisms are deprived of phosphorus, dissolved nickel uptake decreases, resulting in an increase in algal metabolism24. Also25, examined the effects of phosphorus and nitrogen starvation on the life cycle of Emiliania huxleyi (Haptophyta) and proved that various biochemical pathways’ metabolic load increased under P-starvation while it decreased under N-starvation.Effect of phosphorus starved cultures of Dunaliella tertiolecta on chlorophylls content under stress of nickel ionsTable 2 and figure (S2, Supplementary Data) show the sequences of change in the amount of chlorophylls a and b in phosphorus-depleted cultures of Dunaliella tertiolecta in response to various dissolved nickel concentrations. The results show that total chlorophyll content rose steadily until the end of the experiment under normal conditions (a control containing phosphorus). These results are in harmony with those obtained by24. The ratio between chlorophylls “a” and “b” remained nearly constant till the end of the 12th day. At the 16th day of culturing, the ratio decreased from 2.9:1 to 2.4:1. On the contrary, the total chlorophylls under control (in the absence of nickel element) in case of phosphorus-starved cultures showed a progressive increase up to the 12th day. At the 12th day the total chlorophylls in case of phosphorus-starved cultures decreased by 10.7% compared to the normal control. At the 16th day, the total chlorophylls in case of untreated phosphorus starved culture decreased by 20.8% compared to those obtained at normal control26. Reported that the chlorophyll content of Chlorella sorokiniana was significantly reduced due to a lack of nitrogen and phosphorus in the medium.Table 2 Effect of different concentrations of dissolved nickel (mg/L) on chlorophylls content (µg/ml) of Dunaliella tertiolecta under the stress of phosphorus starvation.Full size tableThe total chlorophyll content of Dunaliella tertiolecta in the phosphorus-starved cultures treated with 5 mg/L of dissolved nickel increased gradually until the 12th day, when the content of the total chlorophylls reached 2.11 µg/ml, i.e., higher than the phosphorus-starved control (− P) by 15.3%. At the 16th day, the total chlorophylls, although lower than those obtained at the 12th day, were still higher than the control (− P). At a concentration of 10 mg/L of dissolved nickel, slight increase in the content of total chlorophylls was recorded from the beginning to the end of the culturing period, i.e., from the 4th to the 16th day. At the other concentrations of dissolved nickel (15, 20, and 25 mg/L), a pronounced decrease in the total chlorophylls could be observed from the 4th to the 16th day of culturing compared to control (− P). Our results are going with an agreement with those obtained by27 who found that chlorophylls were inhibited maximum at higher dissolved nickel concentrations but activated at lower values. The normal ratio between chlorophylls “a” and “b” (3:1) was upset after the 8th day of culturing under concentrations 5, 10, and 15 mg/L of dissolved nickel. At 20 and 25 mg/L of dissolved nickel, this ratio was unstable from the beginning to the end of the experiment. The fact that dissolved nickel is extremely mobile and hence only absorbed to a minimal level may explain the sensitivity of the tested alga to nickel in response to phosphorus deficiency, and an increase in phosphorus concentration favors its absorption by microorganisms28. It can be concluded that when microorganisms are deprived of phosphorus, dissolved nickel uptake decreases, resulting in an increase in algal metabolism.Effect of different concentrations of dissolved nickel on photosynthesis (O2-evolution) of phosphorus starved cells of Dunaliella tertiolecta
Data represented in Table 3 and graphed in figure (S3, Supplementary Data S3) showed that the effect of phosphorus limitation on the photosynthetic activity of Dunaliella tertiolecta in response to five different concentrations of dissolved nickel revealed that, under phosphorus limiting conditions, the amount of O2-evolution was lower than in untreated cultures (the control). The evolution of O2 after 4 days of culturing in case of phosphorus starved control decreased by 8.7% compared to normal control, while after 12 days it decreased by 30.4%. The rate of O2-evolution at different concentrations of dissolved nickel over 5 mg/L caused successive reductions in the O2-evolution of phosphorus starved cells. Application of 5 mg/L of dissolved nickel, the results cleared that the rate of O2-evolution increased under the effect of all tested concentrations till the end of the experiment. It is clear from our data that the rate of O2-evolution depended mainly on the concentration of the nickel element and the length of culturing period. The lower the rate of O2-evolution, the higher the element’s concentration, and the longer the culturing period. This coincided with the findings of7 who found that low phosphorus treatment causes Chlorella kessleri to lose its photosynthetic activity. In this regard, it was discovered that phosphorus deficiency resulted in a decrease in photosynthetic electron transport activity29 found that the O2-evolution of Chlamydomon reinhardtii declined by 75%. This decrease reflects damage of PSII and the generation of PSII QB-non reducing centers.Table 3 Effect of different concentrations of dissolved nickel (mg/L) on photosynthetic activity (O2-evolution calculated as µ mol O2 mg chl-1 h-1) on phosphorus supplemented and starved cells of Dunaliella tertiolecta.Full size tableAlso30 found that P- deficiency has been correlated with lower photosynthetic rates. In the case of the treated phosphorus-starved cultures with lower concentrations (5 mg/L) of dissolved nickel, the rate of photosynthesis increased when compared to the phosphorus-starved control, but was less than that of the normal control (without nickel treatment). On the contrary, it was found that, in the treated phosphorus-starved cultures at concentrations of 10, 15, 20 and 25 mg/L of the tested element, the rate of photosynthesis decreased from the beginning to the end of the experiment. With increasing concentration, duration of the culturing period, and kind of element, the condition of decrease in O2-evolution became more pronounced; the same results were also recorded by24. The stimulation of growth and photosynthesis in the presence of some concentrations of dissolved nickel under phosphorus-limiting conditions is observed by31 they report that in Cu2+ sensitive Scenedesmus acutus, intracellular polyphosphate plays a key role in shielding photosynthesis from Cu2+ toxicity but not in copper resistant species.Effect of different concentrations of dissolved nickel on respiration (O2-uptake) of phosphorus starved cells of Dunaliella tertiolectaData obtained in Table 4 and graphed in figure (S4, Supplementary Data S4) concerning the rate of respiration of Dunaliella tertiolecta under phosphorus-limiting conditions was higher than that of untreated phosphorus-starved (control) for a short period of time only, i.e., after 4 days, at concentrations 5, 10 and 15 mg/L of dissolved nickel, After 8 days of culturing, the rate of O2- uptake increased only at 5 mg/L of dissolved nickel, while at the other concentrations it decreased gradually with increasing the concentration of the element. This finding is consistent with the findings of23, who discovered that Dunaliella cells increased their O2 absorption and evolution rates in the presence of 2 M salt NaCl in the media. In terms of oxygen uptake rate, Dunaliella cells demonstrated an increase in salt concentrations. In 1.5 M NaCl, it increased significantly by 60–80%.Table 4 Effect of different concentrations of dissolved nickel (mg/L) on respiration activity (O2-uptake calculated as µ mol O2 h-1) on phosphorus supplemented and starved cells of Dunaliella tertiolecta.Full size tableConcerning the increase in respiration in P-depleted green alga species cultures5 suggested that Scenedesmus, for example, can utilize the energy stored in starch and lipids for active phosphorus uptake from lake sediments. This process is aided by an increase in phosphatase production32 and these cells’ ability to operate anaerobically33. When unicellular green algae or higher plants are exposed to P deficiency, the majority of newly fixed carbon appears to be allocated to the synthesis of non-phosphorylated storage polyglucans (i.e., starch) or sucrose, with less photosynthetic activity directed to respiratory metabolism and other biosynthesis pathways34. It can be concluded from the obtained results that, when the alga was cultivated under phosphorus deficiency and treated with varied amounts of dissolved nickel, the growth was the most sensitive characteristic, followed by photosynthesis, and then dark respiration. In the few comparative studies with several species of green algae, growth was more sensitive than the other physiological processes examined. Out of them35, reported that growth was more susceptible to phosphorus deficiency in Chlorella pyrenoidosa and Asterionella gracilis than photosynthesis and respiration (the least sensitive processes). Growth was also more sensitive than photosynthesis in Nitzschia closterium 36 . Another important fact reported by37 is that under low phosphorus conditions, Dunaliella parva accumulates lipids rather than carbohydrates. These findings imply that phosphorus stress may prevent starch and/or protein production, leading to an increase in carbon flux to lipids. More