We detect several heavy metals with a single test strip.
Heavy metals have been part in a lot of iGEM projects over the last years, so why work with them again?
Heavy metals are still a major problem. Therefore, they have been part in a lot of iGEM projects. There are many concepts to create heavy metal sensors. Some of them work extraordinary well. But most of these sensors never made it to real world applications. We aim to make a use of well characterized sensors as well as concepts and new ideas. All this sensor systems shell work on the same principle, so that we can use them to create a modular easy to handle paper based cell free test strip for detection of more substances, heavy metals in this case, in parallel. "In my opinion the test stripe system has great potential in the field of monitoring contamination in industrial wastewater. It`s a fast and easy available system for qualitative control of several heavy metals.” (Dr.rer.nat. Andreas Bermpohl, manager of Biotec GmbH)
Why heavy metals? Heavy metals are part of Earth’s crust. Therefore, they do occur naturally in our environment. (Heavy Metals - Lenntech) In low doses some of them as copper or nickel are even essential trace elements for animals and humans (Rashmi Verma and Pratima Dwivedi 2013). A major problem is their bioaccumulation, which leads to toxicity and long term effects which include fatal diseases like cancer (Martin et al. 2009), Parkinson`s or Alzheimer’s disease (Gaggelli et al. 2006) (figure 1).
Which heavy metals? The heavy metal sensors we chose for detection are specific to arsenic, copper, chromium, lead, mercury and nickel. Their concentrations in drinking water are regulated by the WHO, because of their immediate and long term health effects (figure 2).
Our biosensors We decided to work with already existing, well-characterized sensors as well as with established but not well-characterized concepts of other teams and moreover create new sensor systems. Therefore, we established a basic construction plan for our sensor systems, which is based on a promoter with a specific operator region in front of a super folder GFP (sfGFP), which was used for detection trough fluorescence analysis. In addition we used fitting activators or repressors for our inducible promoters under the control of BBa_K608002, which consists of a constitutive promoter with a strong ribosomal binding site (RBS) (figure 3). We combined these into a device consisting of constitutive promoter and RBS reverse and the promoter and operator region in upstream of the sfGFP. So we have repressor or activator constitutively expressed in reverse orientation. This was done to minimize the background transcription of the inducible system in upstream of our heavy metal promoter operator system. In addition, these devices are optimized for the usage in a cell free protein synthesis(CFPS). This is the basis for the development of cell free biosensors on a test strip, which can be used to detect several heavy metals at once in the open field.
Click on the test strip for more information about the heavy metals and how they can be detected:
Arsenic is found in nature in both organic and inorganic forms, typically as arsenite (AsIII) or arsenate (AsV). The average arsenic concentration in sea water is about 1-2 µg/L (Kaur et al. 2015). Inorganic arsenic is naturally present at high levels in the groundwater of a number of countries, including Argentina, Bangladesh, China, India, Mexico, and the USA (World Health Organization 2012). Arsenic contamination is most dramatic in Bangladesh, where over one million people suffer from arsenic poisoning. Strong local and seasonal fluctuations in arsenic concentrations make it necessary to test each well regularly (van der Meer 2003).
Inorganic arsenic compounds are highly toxic. Acute effects of arsenic intake can range from gastrointestinal distress to death. Chronic exposure can result in skin lesions, vascular diseases and cancer. These chronic effects are referred to as arsenicosis, and there is no effective therapy for them. Due to its toxicity and frequency, arsenic ranks first on the Priority List of Hazardous Substances prepared by the US Environmental Protection Agency (EPA) and the Agency for Toxic Substances and Disease Registry (ATSDR). The World Health Organization recommends a limit of 10 µg/L in drinking water, but some countries have adopted a national standard of 50 µg/L (World Health Organization 2012; Chen, Rosen 2014).
Arsenic can be accurately detected by means of techniques such as atomic absorption spectroscopy (AAS), atomic fluorescence spectrometry or high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS). However, they are expensive and not suitable for field testing (Chen, Rosen 2014). Chemical test kits are available, which mostly rely on the Gutzeit method. This method is based on the generation of arsine gas from a sample solution. Arsine then reacts with a mercuric bromide impregnated test strip, which results in a color change (Kearns 2010). The accuracy and reliability of this method has been called into question (Rahman et al. 2002). The need for an inexpensive and reliable detection method has led to the development of various arsenic biosensors. Among them are both whole-cell-based and cell-free biosensors. For a recent review, refer to Kaur et al. 2015.
Our arsenic biosensor
We choose to work with the chromosomal arsenic operon of E. coli, which was used by the team from Edinburgh in 2006. This operon encodes an efflux pump, which confers resistance against arsenic. The expression is controlled by the repressor ArsR, which negatively autoregulates its own expression. AsIII can bind to three cysteine residues in ArsR. The resulting conformational change deactivates the repressor (Chen, Rosen 2014).
Chen, Jian; Rosen, Barry P. (2014): Biosensors for inorganic and organic arsenicals. In Biosensors 4 (4), pp. 494–512. DOI: 10.3390/bios4040494.
Kaur, Hardeep; Kumar, Rabindra; Babu, J. Nagendra; Mittal, Sunil (2015): Advances in arsenic biosensor development--a comprehensive review. In Biosensors & bioelectronics 63, pp. 533–545. DOI: 10.1016/j.bios.2014.08.003.
Kearns, James Kalman (2010): Field Portable Methods for the Determination of Arsenic in Environmental Samples. Dissertation.
Rahman, Mohammad Mahmudur; Mukherjee, Debapriyo; Sengupta, Mrinal Kumar; Chowdhury, Uttam Kumar; Lodh, Dilip; Chanda, Chitta Ranjan et al. (2002): Effectiveness and Reliability of Arsenic Field Testing Kits: Are the Million Dollar Screening Projects Effective or Not? In Environ. Sci. Technol. 36 (24), pp. 5385–5394. DOI: 10.1021/es020591o.
van der Meer, Jan Roelof (2003): EAWAG news 56e: Bacterial Biosensors to Measure Arsenic in Potable Water.
World Health Organization (2012): Arsenic fact sheet. Available online at http://www.who.int/mediacentre/factsheets/fs372/en/, checked on 8/12/2015.
Chromium is an essential part of the earth´s crust. It is the sixth most abundant one and used in metallurgical, chemical and refractory form. The three most important oxidative forms of chromium are the elemental metal (Cr), the trivalent (CrIII) and the hexavalent (CrVI) (Mitchell D. Cohen et al.).
While the trivalent form is an essential dietary mineral and the most common natural form, it is of interest to detect the hexavalent form because of its potential toxicity and carcinogenic effects. Most of it is produced trough industrial uses (Paustenbach et al. 2003). Chromium intoxication can result in damage to the nervous system, fatigue and mental instability (Singh et al. 2011). Its potential cancerogenity is the result of chromium VI being , able to enter cells , while this is not possible for chromium III compounds. Inside of the cells, chromium IV is reduced to chromium III and can not leave the cells anymore, where it results in oxidative stress reactions (Mitchell D. Cohen et al.). Because of its toxicity the World Health Organization (WHO) recommends a limit of 50 µg/L chromium in drinking water. In contrast to this guideline concentrations of 120 µg/l chromium were detected in drinking water in the USA (Guidelines for drinking-water quality 2011, WHO 2003).
Chromium in drinking water is detected trough atomic absorption spectroscopy (AAS) or ion chromatography with post column derivatization and UV visible spectroscopic detection (U.S. EPA, OW, OGWDW, SRMD, Technical Support Center). Moreover, chromium detection at home can be detected by a basic titrimetric method using an iodide reaction for measurement (GIORGIA).
Our chromium biosensor
We work with the chromate inducible operon of Ochrobactrum triti ci5bvl1, which enables a tolerance for chromium VI and superoxide. The expression of the operon depends on the bondage of the repressor chrB to the operator sequence (Branco et al. 2008). The chrBACF operon (BBa_K1058007) and the chrB repressor were introduced by the team BIT 2013.
Branco, Rita; Chung, Ana Paula; Johnston, Tatiana; Gurel, Volkan; Morais, Paula; Zhitkovich, Anatoly (2008): The chromate-inducible chrBACF operon from the transposable element TnOtChr confers resistance to chromium(VI) and superoxide. In: Journal of bacteriology 190 (21), S. 6996–7003. DOI: 10.1128/JB.00289-08
Background document for development of WHO Guidelines for Drinking-water Quality, checked on 9/9/2015. Copper excess, zinc deficiency, and cognition loss in Alzheimer's disease - Brewer - 2012 - BioFactors - Wiley Online Library. Available online at http://onlinelibrary.wiley.com/doi/10.1002/biof.1005/abstract.
Mitchell D. Cohen; Biserka Kargacin; Catherine B. Klein; and Max Costa: Mechanisms of Chromium Carcinogenicity and Toxicity, zuletzt geprüft am 19.08.2015.
Paustenbach, Dennis J.; Finley, Brent L.; Mowat, Fionna S.; Kerger, Brent D. (2003): Human health risk and exposure assessment of chromium (VI) in tap water. In: Journal of toxicology and environmental health. Part A 66 (14), S. 1295–1339. DOI: 10.1080/15287390306388.
Singh, Reena; Gautam, Neetu; Mishra, Anurag; Gupta, Rajiv (2011): Heavy metals and living sys-tems: An overview. In: Indian journal of pharmacology 43 (3), S. 246–253. DOI: 10.4103/0253-7613.81505.
U.S. EPA, OW, OGWDW, SRMD, Technical Support Center: Method 218.7: Determination of Hexavalent Chromium in Drinking Water by Ion Chromatography with Post-Column Derivatization and UV-Visible Spectroscopic Detection.
Copper is an essential trace element for humans, animals and plants. The human body contains concentrations of 1.4 to 2.1 mg/kg body mass. Copper is ingested through the gut and transferred to most tissues through the liver. It is used in coating alloys and used to make pipes, valves and fittings. Moreover coppersulfate pentahydrate is used in algae control by adding it to surface water. Therefore copper concentrations in drinking water vary widely and range from 0,005 mg/L to 30 mg/L. After the guidelines for drinking water the maximal concentration of copper is 2 mg/L (Guidelines for Drinking-water Quality, Fourth Edition ).
Copper is essential for human health, but in to high doses it can cause anemia, kidney and liver damage as well as stomach and intestinal irritation and immunotoxicity (ATSDR). A with copper associated disease is Wilsons disease which manifests in a misfunction, so that copper can not be excreted by the liver into bile. If not treated this can lead to brain and liver damage (US EPA ORD NCEA Integrated Risk Information System (IRIS) 2014). Some studies associate high copper levels with aging diseases such as atherosclerosis and Alzheimer’s disease (Brewer 2012).
The most important analytical method for the detection of copper in water is the inductively coupled plasma mass spectrometry (ICP-MS),which has the lowest detection limit (0.02 μg/L). Other methods often used for detection are the atomic absorption spectrometry (AAS) with flame detection, which has the highest (20 μg/L) as well as graphite furnace atomic absorption spectroscopy, inductively coupled plasma atomic emission spectroscopy and stabilized temperature platform graphite furnace atomic absorption (cavillona 2004).
Our copper Biosensor
We used the native operator of copper homeostasis from E. coli K12. This includes the promoter (CopAP) and its regulator CueR. CueR is a MerR like regulator, which stimulates the transcription of CopA, a P-type ATPase pump (Outten et al. 2000). CopA is the central component in obtaining copper homeostasis, it exports free copper from cytoplasm to the periplasm. This is enabled by copper induced activation of the operon transcription via CueR. The CueR-Cu+ is the DNA-binding transcriptional dual regulator which activates transcription (Yamamoto, Ishihama 2005). We combined CueR (BBa_K1758320) under the control of a constitutive promoter with the operator site of CopA Promoter and sfGFP (BBa_K1758321) for measuring output signals.
ATSDR: TOXICOLOGICAL PROFILE FOR COPPER, checked on 8/27/2015.
cavillona (2004): Copper in Drinking-water.
Background document for development of WHO Guidelines for Drinking-water Quality, checked on 9/9/2015. Copper excess, zinc deficiency, and cognition loss in Alzheimer's disease - Brewer - 2012 - BioFactors - Wiley Online Library. Available online at http://onlinelibrary.wiley.com/doi/10.1002/biof.1005/abstract, checked on 8/28/2015.
Grass, Gregor; Rensing, Christopher (2001): Genes Involved in Copper Homeostasis in Escherichia coli, checked on 8/26/2015. Guidelines for Drinking-water Quality, Fourth Edition, checked on 9/9/2015.
Outten, F. W.; Outten, C. E.; Hale, J.; O'Halloran, T. V. (2000): Transcriptional activation of an Escherichia coli copper efflux regulon by the chromosomal MerR homologue, cueR. In The Journal of biological chemistry 275 (40), pp. 31024–31029. DOI: 10.1074/jbc.M006508200.
US EPA ORD NCEA Integrated Risk Information System (IRIS) (2014): Copper (CASRN 7440-50-8) | IRIS | US EPA. Available online at http://www.epa.gov/iris/subst/0368.htm, updated on 10/31/2014, checked on 9/2/2015.
Yamamoto, Kaneyoshi; Ishihama, Akira (2005): Transcriptional response of Escherichia coli to external copper. In Molecular microbiology 56 (1), pp. 215–227. DOI: 10.1111/j.1365-2958.2005.04532.x.
Lead is a heavy metal with widespread occurrence. The relatively simple extraction methods and several desirable properties have made it useful to humans. Lead and lead compounds are used in a high variety of products, such as pipes and plumbing materials, solders, gasoline, batteries, ammunition and cosmetics. Therefore, lead plays a major role in the industry and is one of the most used metals. Compared to other metals its occurrence is relative low. Lead can be detected in different parts of the environment, like air, soil and water. A high concentration of lead in drinking water is often induced by obstruct pipes that consist of lead or that has a part of lead, respectively. This allow water to be easily contaminated. That is often a problem in houses, where lead is used in household plumbing. Due to the fact that lead is also occurring in water, it could result in adverse health effects (WHO: Fact sheet number 379, Lead poisoning and health).
Lead has no biological role in the body, but is a highly poisonous metal. The ingestion of lead could affect almost every organ and system in the body (EPA Health Effects: How Lead Affects the body). The main target for lead toxicity is the nervous system. It can have acute or chronic health effects. The acute health effects are occurring immediately after contact with lead. This can be irritation of the eyes or can cause headache, irritability, disturbed sleep, and mood as well as personality changes. Exposure to higher lead concentrations over a long-term could cause serious damage to the brain and to the kidneys. And finally the damage can cause death (Golub, M. S., 2005). The poisoning is mostly resulting of ingestion of water or food, which is contaminated with lead or lead compounds (Ferner, D. J., 2001). It is quickly taken up into the bloodstream and spread in the body (Bergeson, L., 2008). The World Health Organization recommends a limit of 10 µg/L in drinking water, concentrations in drinking water are generally below 5 μg/L. Nevertheless, there are much higher concentrations that have been measured if lead fittings are existing (WHO: Guidelines for Drinking-water Quality, fourth edition).
Due to the effects on health, the detection of lead in drinking water is of importance in all parts of the world. Therefore, a simple system for a fast review of the water quality is a worthwhile aim. The method for detection is currently based on the principle of flame atomic absorption spectrometry (FAAS) (Abdallah, A. T. and Moustafa, M. A., 2002). This detection method is difficult to implement in developing countries, and time consuming thus there are hardly any quality standards.
Our lead biosensor
For our biosensor we use parts of the chromosomal lead operon of Cupriavidus metallidurans. Naturally the operon for lead resistance contains pbrT, which encodes a Pb(II) uptake protein, pbrA, which encodes a P-type Pb(II) efflux ATPase, pbrB, which encodes a predicted integral membrane protein and pbrC, which encodes a predicted prolipoprotein signal peptidase. The expression of the operon is regulated by the repressor pbrR. As a MerR like regulator, its stimulates the transcription of the operon in the presence of Pb2+ Borremans et al., 2001). Our sensor system combines pbrRunder the control of a constitutive promoter and the pbrA promoter for the lead depending expression of sfGFP.
Abdallah, A. T. and Moustafa, M. A. (2002): Accumulation of lead and cadmium in the marine prosobranchNeritisaxtilis, chemicall analysis, light and electron microscopy. Environmental Pollution, 116, 185-191.
Bergeson, L. L. (2008). "The proposed lead NAAQS: Is consideration of cost in the clean air act's future?". Environmental Quality Management 18: 79–84. :10.1002/tqem.20197 .
Borremans B., Hobman J. L., Provoost A., Brown N. L., van der Lelie D. (2001), Cloning and Functional Analysis of the pbr Lead Resistance Determinant of Ralstoniametallidurans CH34, J. Bacteriol., 183, 5651 – 5658
EPA Health Effects: How Lead Affects the body, checked on 2015-09-17.
Ferner D.J., (2001)Toxicity, heavy metals.eMed J.;2(5):1
Golub, M. S., (2005). "Summary".Metals, fertility, and reproductive toxicity.ISBN 978-0-415-70040-5.
WHO Guidelines for Drinking-water Quality fourth edition, checked on 2015-09-09.
WHO lead poisoning and health, fact sheet number 379, reviewed August 2015, checked on 2015-09-17.
Mercury is found in water, typically as methylmercury, which is build out of inorganic mercury by different marine bacteria Pseudomonas spp. under aerobic conditions. Additional mercury(II)chloride with a high solubility, and mercury sulfide are found in water. The main natural source of Mercury exposure is through volcanic activity (WHO 2005). Additional, there are many kinds of emission caused by humans. For example mercury contamination can be caused by medical waste (damaged measurement instruments), fluorescent-lamps, chloralkali plants and thermal power plants (Verma et al., 2013). The natural occurring concentration of mercury in groundwater and surface water are in general less than 0.5 µg/L but can rise to higher concentrations by local mineral deposit. Due to volcanic activity, the mercury concentration in water can rise frequently up to 5.5 µg/L (Izu Oshima Island in Japan) (WHO, 2005).
In the environment, mercury is one of the most toxic elements (L.A. Rojas, 2011). The most toxic compounds are organometallic mercury molecules like methylmercury and dimethylmercury, because they can easily permeate the cellular membrane. These organometallic compounds are better soluble in lipids. Because of this fact, it is easier to permeate the cellular membrane. Acute effects of a mercury intoxication can range from diseases of the liver, kidney, gastrointestinal tract, neuromuscular and neurological problems. Inorganic mercury accumulates in the kidneys and has a long biological half-life, before it is not detectable anymore. In contrast to organic mercury, inorganic mercury is not able to cross the blood-brain barrier or blood-placenta barrier, so it accumulate in the organs (Park et al., 2012). A chronic intoxication of mercury results in kidney changes, changes in the central nervous system and other effects like cancer (Holmes et al., 2009, WHO 2005). Additional studies show that mercury generates chromosomal aberrances (WHO, 2005). In addition, a relation between an early exposure of mercury and late initial of Alzheimer`s and other neurodegenerative diseases are discussed (Park et al., 2012).
Mercury can be detected by atomic absorption spectrometry with a detection limit of 5 µg/L and the Inductively Coupled Plasma Method with a detection level of 0.6 µg/L (WHO 2005). In addition, there are different chemical and biological test systems. One of these systems is the detection by ELISA with mercury specific antibodies (Wylie et al., 1991).
Our mercury biosensor
For our sensor, we use the mer operator from Shigella flexneri R100 plasmid Tn21 ( BBa_K346002 ) and its regulator merR ( BBa_K346001 ) constructed by the iGEM team Peking 2010. The MerR functions as an activator and regulates its own transcription (N.L. Brown et al., 2003). Our sensor system combines merR under the control of a constitutive promoter and the merT promoter for the mercury depending expression of sfGFP.
WHO (2005): Mercury in Drinking-water Background document for development of WHO Guidelines for Drinking-water Quality, checked 20.08.15
Holmes, P. ; James K.A.F.; Levy, L.S. (2009): Is low-level environmental mercury exposure of concern to human health? In SCIENCE OF THE TOTAL ENVIRONMENT 408 ( 2) pp. 171-182.
Brown, Nigel L.; Stoyanov, , Jivko V.;Kidd,Stephen P.;Hobman; Jon L. (2003): The MerR family of transcriptional regulators. In FEMS Microbiology Reviews, 27 ( 2) pp.145-163.
Rojas, LA.; Yanez, Carolina; Gonzalez, Myriam; Lobos, Soledad; Smalla, Kornelia; Seeger, Michael (2011). Characterization of the Metabolically Modified Heavy Metal-Resistant Cupriavidus metallidurans Strain MSR33 Generated for Mercury Bioremediation. PLOS ONE, 6 (3).
Verma, Rashmi; Dwivedi, Pratima (2013): Heavy metal water pollution- A case study. Recent Research in Science and Technology 2013, 5(5) pp.98-99. Park, JD.; Zheng, Wei (2012). Human exposure and health effects of inorganic and elemental mercury. In Journal Of Preventive Medicine And Public Health, 45 (6) pp. 344-352.
Wylie, Dwane E.; Carlson, Larry D.; Carlson, Randy; Wagner, Fred W.; Schuster, Sheldon M. (1991). Detection of mercuric ions in water by ELISA with a mercury-specific antibody. Analytical Biochemistry, 194 (2) pp. 381-387.
The amount of natural occurring nickel in comparison to other heavy metals is quite low even if it is an element of the Earth’s crust. Therefore, small amounts of it are found in food, water, soil and air. Nickel concentration in drinking water is normally less than 0.02 mg/L, although through releases from taps and fittings the nickel may accumulate to concentrations up to 1 mg/L. In special cases of release from natural or industrial nickel deposits in the ground, there may be higher concentrations in drinking-water. Through unintended release the concentration can be higher than the guideline value of 0.07 mg/L (WHO: Guidelines for Drinking-water Quality, Fourth Edition)
Even though nickel is essential for mammals and a part of human nutrition, it may cause dermatitis as well as itching of fingers, hands and forearms by some people, who had long term skin contact. The main source of nickel exposure is food or water, but most people have contact to nickel trough everyday products as jewelry or stainless steel dishware or trough smoking tobacco(US; EPA et al., 2013). In Germany most drinking water pollutions by nickel happen in the last meters of the plumbing system. Wrong tapware is the main source of nickel contamination in drinking water.
The two most commonly used analytical methods for nickel in water are atomic absorption spectrometry and inductively coupled plasma atomic emission spectrometry. Inductively coupled plasma atomic emission spectroscopy is used for the determination of nickel detection limit of about 10 μg/L (ISO, 1996). Flame atomic absorption spectrometry is suitable in the range of 0.5–100 μg/L (ISO, 1986). A limit of detection of 0.1 μg/L can be achieved using inductively coupled plasma mass spectrometry. Alternatively, electrothermal atomic absorption spectrometry can be used. (cavillona 2005)
Our nickel biosensor
For our nickel sensor system we used the rcn-operon from E. coli which encodes a nickel- and cobalt-efflux system with a high sensitivity to nickel. In the absence of Nickel the respressor RcnR regonizes the operator site and blocks transcription of the operon, while in the presence of nickel it is abadoned due to a conformational change. Our sensor system combines rcnR under the control of a constitutive promoter and the prcnA promoter for the nickel depending expression of sfGFP. If Ni2+-ions bind to the repressor RcnR, it cannot attach to DNA and rcnA the nickel responsive promoter is activated. In the absence of nickel or cobalt, RcnR is bound to rcnR operator and blocks rcnA transcription. (EPA et al., 2013; Blaha et al., 2011; Iwig et al., 2006) Our output signal works through fluorescence.
Blaha, Didier; Arous, Safia; Blériot, Camille; Dorel, Corinne; Mandrand-Berthelot, Marie-Andrée; Rodrigue, Agnès (2011): The Escherichia coli metallo-regulator RcnR represses rcnA and rcnR transcription through binding on a shared operator site: Insights into regulatory specificity towards nickel and cobalt. In Biochimie 93 (3), pp. 434–439. DOI: 10.1016/j.biochi.2010.10.016.
cavillona (2005): Nickel in Drinking-water, checked on 9/9/2015.
EPA, U. S.; OAR; Office of Air Quality Planning and Standards (2013): Nickle Compounds | Technology Transfer Network Air Toxics Web site | US EPA. Available online at http://www.epa.gov/airtoxics/hlthef/nickel.html, updated on 10/18/2013, checked on 9/10/2015.
Guidelines for Drinking-water Quality, Fourth Edition, checked on 9/9/2015.
Iwig, Jeffrey S.; Rowe, Jessica L.; Chivers, Peter T. (2006): Nickel homeostasis in Escherichia coli - the rcnR-rcnA efflux pathway and its linkage to NikR function. In Molecular microbiology 62 (1), pp. 252–262. DOI: 10.1111/j.1365-2958.2006.05369.x. kreusche: Trinkwasser-Installation 27.6.07 Endfassung.qxd, checked on 9/10/2015.
ISO, 1996 US: Technical Factsheet on: Nickel, checked on 9/9/2015.