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Applied Microbiology and Biotechnology

, Volume 101, Issue 13, pp 5235–5245 | Cite as

Groundwater contamination with 2,6-dichlorobenzamide (BAM) and perspectives for its microbial removal

  • Lea Ellegaard-JensenEmail author
  • Benjamin Horemans
  • Bart Raes
  • Jens Aamand
  • Lars Hestbjerg Hansen
Mini-Review

Abstract

The pesticide metabolite 2,6-dichlorobenzamide (BAM) is very persistent in both soil and groundwater and has become one of the most frequently detected groundwater micropollutants. BAM is not removed by the physico-chemical treatment techniques currently used in drinking water treatment plants (DWTP); therefore, if concentrations exceed the legal threshold limit, it represents a sizeable problem for the stability and quality of drinking water production, especially in places that depend on groundwater for drinking water. Bioremediation is suggested as a valuable strategy for removing BAM from groundwater by deploying dedicated BAM-degrading bacteria in DWTP sand filters. Only a few bacterial strains with the capability to degrade BAM have been isolated, and of these, only three isolates belonging to the Aminobacter genus are able to mineralise BAM. Considerable effort has been made to elucidate degradation pathways, kinetics and degrader genes, and research has recently been presented on the application of strain Aminobacter sp. MSH1 for the purification of BAM-contaminated water. The aim of the present review was to provide insight into the issue of BAM contamination and to report on the current status and knowledge with regard to the application of microorganisms for purification of BAM-contaminated water resources. This paper discusses the prospects and challenges for bioaugmentation of DWTP sand filters with specific BAM-degrading bacteria and identifies relevant perspectives for future research.

Keywords

Water treatment Drinking water Pesticide contamination BAM Biodegradation Aminobacter sp. MSH1 

Notes

Acknowledgements

Lea Ellegaard-Jensen, Jens Aamand and Lars Hestbjerg Hansen were supported by the research project MEM2BIO (Innovation Fund Denmark, contract number 5157-00004B).

Benjamin Horemans was supported by the FWO postdoctoral fellowship grant (12Q0215N) and the BELSPO IAP-project μ-manager no. P7/25. Bart Raes was supported by the C1 project no. C14/15/043 of KU Leuven.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

References

  1. Agriculture & Environment Research Unit (2016) PPDB: Pesticide Properties DataBase. University of Hertfordshire, http://sitem.herts.ac.uk/aeru/footprint/index2.htm.
  2. Albers CN, Jacobsen OS, Aamand J (2014) Using 2,6-dichlorobenzamide (BAM) degrading Aminobacter sp. MSH1 in flow through biofilters—initial adhesion and BAM degradation potentials. Appl Microbiol Biotechnol 98:957–967. doi: 10.1007/s00253-013-4942-6 CrossRefPubMedGoogle Scholar
  3. Albers CN, Ellegaard-Jensen L, Harder CB, Rosendahl S, Knudsen BE, Ekelund F, Aamand J (2015a) Groundwater chemistry determines the prokaryotic community structure of waterworks sand filters. Environ Sci Technol 49:839–846. doi: 10.1021/es5046452 CrossRefPubMedGoogle Scholar
  4. Albers CN, Feld L, Ellegaard-Jensen L, Aamand J (2015b) Degradation of trace concentrations of the persistent groundwater pollutant 2,6-dichlorobenzamide (BAM) in bioaugmented rapid sand filters. Water Res 83:61–70. doi: 10.1016/j.watres.2015.06.023 CrossRefPubMedGoogle Scholar
  5. Benner J, Helbling DE, Kohler HPE, Wittebol J, Kaiser E, Prasse C, Ternes TA, Albers CN, Aamand J, Horemans B, Springael D, Walravens E, Boon N (2013) Is biological treatment a viable alternative for micropollutant removal in drinking water treatment processes? Water Res 47:5955–5976. doi: 10.1016/j.watres.2013.07.015 CrossRefPubMedGoogle Scholar
  6. Beynon KI, Wright AN (1968) Persistence, penetration, and breakdown of chlorthiamid and dichlobenil herbicides in field soils of different types. J Sci Food Agric 19:718–722. doi: 10.1002/jsfa.2740191208 CrossRefGoogle Scholar
  7. Beynon KI, Wright AN (1972) The fates of the herbicides chlorthiamid and dichlobenil in relation to residues in crops, soils, and animals. In: Gunther FA, Gunther JD (eds) Residue reviews: residues of pesticides and other contaminants in the Total environment. Springer New York, New York, pp 23–53. doi: 10.1007/978-1-4615-8485-8_2 CrossRefGoogle Scholar
  8. Björklund E, Anskjær GG, Hansen M, Styrishave B, Halling-Sørensen B (2011) Analysis and environmental concentrations of the herbicide dichlobenil and its main metabolite 2,6-dichlorobenzamide (BAM): a review. Sci Total Environ 409:2343–2356. doi: 10.1016/j.scitotenv.2011.02.008 CrossRefPubMedGoogle Scholar
  9. Clausen L, Larsen F, Albrechtsen HJ (2004) Sorption of the herbicide dichlobenil and the metabolite 2,6-dichlorobenzamide on soils and aquifer sediments. Environ Sci Technol 38:4510–4518. doi: 10.1021/es035263i CrossRefPubMedGoogle Scholar
  10. Clausen L, Arildskov NP, Larsen F, Aamand J, Albrechtsen H-J (2007) Degradation of the herbicide dichlobenil and its metabolite BAM in soils and subsurface sediments. J Contam Hydrol 89:157–173. doi: 10.1016/j.jconhyd.2006.04.004 CrossRefPubMedGoogle Scholar
  11. Dealtry S, Ding G-C, Weichelt V, Dunon V, Schlüter A, Martini MC, Papa MFD, Lagares A, Amos GCA, Wellington EMH, Gaze WH, Sipkema D, Sjöling S, Springael D, Heuer H, van Elsas JD, Thomas C, Smalla K (2014) Cultivation-independent screening revealed hot spots of IncP-1, IncP-7 and IncP-9 plasmid occurrence in different environmental habitats. PLoS One 9:e89922. doi: 10.1371/journal.pone.0089922 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Desaint S, Hartmann A, Parekh NR, Fournier JC (2000) Genetic diversity of carbofuran-degrading soil bacteria. FEMS Microbiol Ecol 34:173–180. doi: 10.1016/S0168-6496(00)00092-1 CrossRefPubMedGoogle Scholar
  13. Dunon V, Sniegowski K, Bers K, Lavigne R, Smalla K, Springael D (2013) High prevalence of IncP-1 plasmids and IS1071 insertion sequences in on-farm biopurification systems and other pesticide-polluted environments. FEMS Microbiol Ecol 86:415–431. doi: 10.1111/1574-6941.12173 CrossRefPubMedGoogle Scholar
  14. Ekelund F, Harder CB, Knudsen BE, Aamand J (2015) Aminobacter MSH1-mineralisation of BAM in sand-filters depends on biological diversity. PLoS One 10:e0128838. doi: 10.1371/journal.pone.0128838 CrossRefPubMedPubMedCentralGoogle Scholar
  15. Ellegaard-Jensen L, Albers CN, Aamand J (2016) Protozoa graze on the 2,6-dichlorobenzamide (BAM)-degrading bacterium Aminobacter sp. MSH1 introduced into waterworks sand filters. Appl Microbiol Biotechnol 100:8965–8973. doi: 10.1007/s00253-016-7710-6 CrossRefPubMedGoogle Scholar
  16. European Commission EU (2011) COMMISSION IMPLEMENTING DECISION of 11 April 2011 concerning the non-inclusion of dichlobenil in Annex I to Council Directive 91/414/EEC. Official Journal of the European Union, Brussels, 11 April 2011.Google Scholar
  17. European Council EU (1998) Drinking Water Directive (COUNCIL DIRECTIVE 98/83/EC). Official Journal of the European Communities, Brussels, 3 November 1998.Google Scholar
  18. Harwood CS, Parales RE (1996) The beta-ketoadipate pathway and the biology of self-identity. Annu Rev Microbiol 50:553–590. doi: 10.1146/annurev.micro.50.1.553 CrossRefPubMedGoogle Scholar
  19. Hernández-Leal L, Temmink H, Zeeman G, Buisman CJ (2011) Removal of micropollutants from aerobically treated grey water via ozone and activated carbon. Water Res 45:2887–2896CrossRefPubMedGoogle Scholar
  20. Holtze MS, Sørensen J, Hansen HCB, Aamand J (2006) Transformation of the herbicide 2,6-dichlorobenzonitrile to the persistent metabolite 2,6-dichlorobenzamide (BAM) by soil bacteria known to harbour nitrile hydratase or nitrilase. Biodegradation 17:503–510. doi: 10.1007/s10532-005-9021-y CrossRefPubMedGoogle Scholar
  21. Holtze MS, Hansen HCB, Juhler RK, Sørensen J, Aamand J (2007) Microbial degradation pathways of the herbicide dichlobenil in soils with different history of dichlobenil-exposure. Environ Pollut 148:343–351CrossRefPubMedGoogle Scholar
  22. Holtze MS, Sørensen SR, Sørensen J, Aamand J (2008) Microbial degradation of the benzonitrile herbicides dichlobenil, bromoxynil and ioxynil in soil and subsurface environments—insights into degradation pathways, persistent metabolites and involved degrader organisms. Environ Pollut 154:155–168. doi: 10.1016/j.envpol.2007.09.020 CrossRefPubMedGoogle Scholar
  23. Horemans B, Raes B, Vandermaesen J, Simanjuntak Y, Brocatus H, T’Syen J, Degryse J, Boonen J, Wittebol J, Lapanje A, Sørensen SR, Springael D (2016) Biocarriers improve bioaugmentation efficiency of a rapid sand filter for the treatment of 2,6-dichlorobenzamide (BAM)-contaminated drinking water. Environ Sci Technol. doi: 10.1021/acs.est.6b05027
  24. Janniche GS, Clausen L, Albrechtsen H-J (2011) Inherent mineralization of 2,6-dichlorobenzamide (BAM) in unsaturated zone and aquifers—effect of initial concentrations and adaptation. Environ Pollut 159:2801–2807. doi: 10.1016/j.envpol.2011.05.010 CrossRefPubMedGoogle Scholar
  25. Johnsen AR (2015) Pesticider. In: Thorling L (ed) Grundvand. Status og udvikling 1989–2014. GEUS, Denmark, pp 105–129 (in Danish)Google Scholar
  26. Kämpfer P, Neef A, Salkinoja-Salonen MS, Busse HJ (2002) Chelatobacter heintzii (Auling et al. 1993) is a later subjective synonym of Aminobacter aminovorans (Urakami et al. 1992). Int J Syst Evol Microbiol 52:835–839. doi: 10.1099/ijs.0.01897-0 PubMedGoogle Scholar
  27. Kimura K, Amy G, Drewes JE, Heberer T, Kim T-U, Watanabe Y (2003) Rejection of organic micropollutants (disinfection by-products, endocrine disrupting compounds, and pharmaceutically active compounds) by NF/RO membranes. J Membr Sci 227:113–121CrossRefGoogle Scholar
  28. Knudsen BE, Ellegaard-Jensen L, Albers CN, Rosendahl S, Aamand J (2013) Fungal hyphae stimulate bacterial degradation of 2,6-dichlorobenzamide (BAM). Environ Pollut 181:122–127. doi: 10.1016/j.envpol.2013.06.013 CrossRefPubMedGoogle Scholar
  29. van der Kooij D (1992) Assimilable organic carbon as an indicator of bacterial regrowth. J Am Water Works Assoc 84:57–65Google Scholar
  30. Król JE, Penrod JT, McCaslin H, Rogers LM, Yano H, Stancik AD, Dejonghe W, Brown CJ, Parales RE, Wuertz S, Top EM (2012) Role of IncP-1β plasmids pWDL7::rfp and pNB8c in chloroaniline catabolism as determined by genomic and functional analyses. Appl Environ Microbiol 78:828–838. doi: 10.1128/aem.07480-11 CrossRefPubMedPubMedCentralGoogle Scholar
  31. Layh N, Hirrlinger B, Stolz A, Knackmuss H-J (1997) Enrichment strategies for nitrile-hydrolysing bacteria. Appl Microbiol Biotechnol 47:668–674. doi: 10.1007/s002530050993 CrossRefGoogle Scholar
  32. Madsen HT, Ammi-said A, Van der Bruggen B, Søgaard EG (2015) Addition of adsorbents to nanofiltration membrane to obtain complete pesticide removal. Water Air Soil Pollut 226:160. doi: 10.1007/s11270-015-2419-1 CrossRefGoogle Scholar
  33. Martini MC, Albicoro FJ, Nour E, Schlüter A, van Elsas JD, Springael D, Smalla K, Pistorio M, Lagares A, Del Papa MF (2015) Characterization of a collection of plasmid-containing bacteria isolated from an on-farm biopurification system used for pesticide removal. Plasmid 80:16–23. doi: 10.1016/j.plasmid.2015.05.001 CrossRefPubMedGoogle Scholar
  34. McDonald IR, Kämpfer P, Topp E, Warner KL, Cox MJ, Connell Hancock TL, Miller LG, Larkin MJ, Ducrocq V, Coulter C, Harper DB, Murrell JC, Oremland RS (2005) Aminobacter ciceronei sp. nov. and Aminobacter lissarensis sp. nov., isolated from various terrestrial environments. Int J Syst Evol Microbiol 55:1827–1832. doi: 10.1099/ijs.0.63716-0 CrossRefPubMedGoogle Scholar
  35. Meth-Cohn O, Wang M-X (1997) An in-depth study of the biotransformation of nitriles into amides and/or acids using Rhodococcus rhodochrous AJ2701. J Chem Soc Perkin 1:1099–1104. doi: 10.1039/a607977f CrossRefGoogle Scholar
  36. Ministry of Agricultural Food and Rural affairs (2015) Guide to weed control 2016–2017. Ministry of Agriculture, Food and Rural Affairs, Ontario, Canada http://www.omafra.gov.on.ca/english/crops/pub75/pub75.pdf.Google Scholar
  37. Miyazaki S, Sikka HC, Lynch RS (1975) Metabolism of dichlobenil by microorganisms in the aquatic environment. J Agric Food Chem 23:365–368. doi: 10.1021/jf60199a026 CrossRefPubMedGoogle Scholar
  38. Montgomery M, Yu TC, Freed VH (1972) Kinetics of dichlobenil degradation in soil. Weed Res 12:31–36. doi: 10.1111/j.1365-3180.1972.tb01184.x CrossRefGoogle Scholar
  39. Porazzi E, Pardo Martinez M, Fanelli R, Benfenati E (2005) GC–MS analysis of dichlobenil and its metabolites in groundwater. Talanta 68:146–154. doi: 10.1016/j.talanta.2005.04.044 CrossRefPubMedGoogle Scholar
  40. Pukkila V, Kontro MH (2014) Dichlobenil and 2,6-dichlorobenzamide (BAM) dissipation in topsoil and deposits from groundwater environment within the boreal region in southern Finland. Environ Sci Pollut Res Int 21:2289–2297. doi: 10.1007/s11356-013-2164-1 CrossRefPubMedGoogle Scholar
  41. Pukkila V, Gustafsson J, Tuominen J, Aallonen A, Kontro MH (2009) The most-probable-number enumeration of dichlobenil and 2,6-dichlorobenzamide (BAM) degrading microbes in Finnish aquifers. Biodegradation 20:679–686. doi: 10.1007/s10532-009-9255-1 CrossRefPubMedGoogle Scholar
  42. Reemtsma T, Alder L, Banasiak U (2013) Emerging pesticide metabolites in groundwater and surface water as determined by the application of a multimethod for 150 pesticide metabolites. Water Res 47:5535–5545. doi: 10.1016/j.watres.2013.06.031 CrossRefPubMedGoogle Scholar
  43. Ren D, Madsen JS, Sorensen SJ, Burmolle M (2015) High prevalence of biofilm synergy among bacterial soil isolates in cocultures indicates bacterial interspecific cooperation. ISME J 9:81–89. doi: 10.1038/ismej.2014.96 CrossRefPubMedGoogle Scholar
  44. Reungoat J, Macova M, Escher BI, Carswell S, Mueller JF, Keller J (2009) Removal of micropollutants and reduction of biological activity in a full scale reclamation plant using ozonation and activated carbon filtration. Water Res 44:625–637CrossRefPubMedGoogle Scholar
  45. Sahar E, David I, Gelman Y, Chikurel H, Aharoni A, Messalem R, Brenner A (2011) The use of RO to remove emerging micropollutants following CAS/UF or MBR treatment of municipal wastewater. Desalination 273:142–147CrossRefGoogle Scholar
  46. Schipper PNM, Vissers MJM, van der Linden AMA (2008) Pesticides in groundwater and drinking water wells: overview of the situation in the Netherlands. Water Sci Technol 57:1277–1286. doi: 10.2166/wst.2008.255 CrossRefPubMedGoogle Scholar
  47. Schlömann M (1994) Evolution of chlorocatechol catabolic pathways—conclusions to be drawn from comparisons of lactone hydrolases. Biodegradation 5:301–321. doi: 10.1007/BF00696467 CrossRefPubMedGoogle Scholar
  48. Schultz-Jensen N, Knudsen B, Frkova Z, Aamand J, Johansen T, Thykaer J, Sørensen S (2014) Large-scale bioreactor production of the herbicide-degrading Aminobacter sp. strain MSH1. Appl Microbiol Biotechnol 98:2335–2344. doi: 10.1007/s00253-013-5202-5 CrossRefPubMedGoogle Scholar
  49. Schultz-Jensen N, Aamand J, Sørensen SR (2016) Bioaugmentation potential of free and formulated 2,6-dichlorobenzamide (BAM) degrading Aminobacter sp. MSH1 in soil, sand and water. AMB Express 6:33. doi: 10.1186/s13568-016-0204-1 CrossRefPubMedPubMedCentralGoogle Scholar
  50. Sekhar A, Horemans B, Aamand J, Sørensen SR, Vanhaecke L, Vanden Bussche J, Hofkens J, Springael D (2016) Surface colonization and activity of the 2,6-dichlorobenzamide (BAM) degrading Aminobacter sp. strain MSH1 at macro- and micropollutant BAM concentrations. Environ Sci Technol 50:10123–10133. doi: 10.1021/acs.est.6b01978 CrossRefPubMedGoogle Scholar
  51. Simonsen A, Holtze MS, Sørensen SR, Sørensen SJ, Aamand J (2006) Mineralisation of 2,6-dichlorobenzamide (BAM) in dichlobenil-exposed soils and isolation of a BAM-mineralising Aminobacter sp. Environ Pollut 144:289–295CrossRefPubMedGoogle Scholar
  52. Simonsen A, Badawi N, Anskjaer GG, Albers CN, Sorensen SR, Sorensen J, Aamand J (2012) Intermediate accumulation of metabolites results in a bottleneck for mineralisation of the herbicide metabolite 2,6-dichlorobenzamide (BAM) by Aminobacter spp. Appl Microbiol Biotechnol 94:237–245. doi: 10.1007/s00253-011-3591-x CrossRefPubMedGoogle Scholar
  53. Sjøholm OR, Aamand J, Sørensen J, Nybroe O (2010a) Degrader density determines spatial variability of 2,6-dichlorobenzamide mineralisation in soil. Environ Pollut 158:292–298CrossRefPubMedGoogle Scholar
  54. Sjøholm OR, Nybroe O, Aamand J, Sørensen J (2010b) 2,6-Dichlorobenzamide (BAM) herbicide mineralisation by Aminobacter sp. MSH1 during starvation depends on a subpopulation of intact cells maintaining vital membrane functions. Environ Pollut 158:3618–3625. doi: 10.1016/j.envpol.2010.08.006 CrossRefPubMedGoogle Scholar
  55. Sørensen SR, Holtze MS, Simonsen A, Aamand J (2007) Degradation and mineralization of nanomolar concentrations of the herbicide dichlobenil and its persistent metabolite 2,6-dichlorobenzamide by Aminobacter spp. isolated from dichlobenil-treated soils. Appl Environ Microbiol 73:399–406. doi: 10.1128/aem.01498-06 CrossRefPubMedGoogle Scholar
  56. Springael D, Ryngaert A, Merlin C, Toussaint A, Mergeay M (2001) Occurrence of Tn4371-related mobile elements and sequences in (chloro)biphenyl-degrading bacteria. Appl Environ Microbiol 67:42–50. doi: 10.1128/AEM.67.1.42-50.2001 CrossRefPubMedPubMedCentralGoogle Scholar
  57. Top EM, Springael D (2003) The role of mobile genetic elements in bacterial adaptation to xenobiotic organic compounds. Curr Opin Biotechnol 14:262–269. doi: 10.1016/S0958-1669(03)00066-1 CrossRefPubMedGoogle Scholar
  58. Törnquist M, Kreuger J, Adielsson S (2007) Occuring of pesticides in Swedish water resources against a background of national risk-reduction programms - results from 20 years of monitoring. In: XIII Symposium Pesticide Chemistry - Environmental Fate and Human Health, Piacenza, Italy, 2007.Google Scholar
  59. T’Syen J, Tassoni R, Hansen L, Sørensen SJ, Leroy B, Sekhar A, Wattiez R, De Mot R, Springael D (2015) Identification of the amidase BbdA that initiates biodegradation of the groundwater micropollutant 2,6-dichlorobenzamide (BAM) in Aminobacter sp MSH1. Environ Sci Technol 49:11703–11713. doi: 10.1021/acs.est.5b02309 CrossRefPubMedGoogle Scholar
  60. Urakami T, Araki H, Oyanagi H, Suzuki KI, Komagata K (1992) Transfer of Pseudomonas aminovorans (den Dooren de Jong 1926) to Aminobacter gen. nov. as Aminobacter aminovorans comb. nov. and description of Aminobacter aganoensis sp. nov. and Aminobacter niigataensis sp. Nov. Int J Syst Bacteriol 42:84–92CrossRefGoogle Scholar
  61. Vandermaesen J, Horemans B, Degryse J, Boonen J, Walravens E, Springael D (2016) Mineralization of the common groundwater pollutant 2,6-dichlorobenzamide (BAM) and its metabolite 2,6-dichlorobenzoic acid (2,6-DCBA) in sand filter units of drinking water treatment plants. Environ Sci Technol 50:10114–10122. doi: 10.1021/acs.est.6b01352 CrossRefPubMedGoogle Scholar
  62. Verloop A, Nimmo WB (1970) Metabolism of dichlobenil in sandy soil. Weed Res 10:65–70. doi: 10.1111/j.1365-3180.1970.tb00924.x CrossRefGoogle Scholar
  63. Veselá AB, Pelantová H, Šulc M, Macková M, Lovecká P, Thimová M, Pasquarelli F, Pičmanová M, Pátek M, Bhalla TC, Martínková L (2012) Biotransformation of benzonitrile herbicides via the nitrile hydratase–amidase pathway in rhodococci. J Ind Microbiol Biot 39:1811–1819. doi: 10.1007/s10295-012-1184-z CrossRefGoogle Scholar
  64. Vlaamse Milieumaatschappij (VMM) (2012) Pesticiden in het grondwater in Vlaanderen 64 (in Dutch) Google Scholar
  65. Vosáhlová J, Pavlů L, Vosáhlo J, Brenner V (1997) Communication to the editor degradation of Bromoxynil, Ioxynil, Dichlobenil and their mixtures by Agrobacterium radiobacter 8/4. Pestic Sci 49:303–306. doi: 10.1002/(sici)1096-9063(199703)49:3<303::aid-ps519>3.0.co;2-t CrossRefGoogle Scholar
  66. Werlen C, Kahler HPE, Van Der Meer JR (1996) The broad substrate chlorobenzene dioxygenase and cis-chlorobenzene dihydrodiol dehydrogenase of Pseudomonas sp. strain P51 are linked evolutionarily to the enzymes for benzene and toluene degradation. J Biol Chem 271:4009–4016. doi: 10.1074/jbc.271.8.4009 CrossRefPubMedGoogle Scholar
  67. Yuan Z, VanBriesen JM (2008) Bacterial growth yields on EDTA, NTA, and their biodegradation intermediates. Biodegradation 19:41–52. doi: 10.1007/s10532-007-9113-y CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Department of Environmental ScienceAarhus UniversityRoskildeDenmark
  2. 2.KU Leuven, Department of Earth and Environmental SciencesHeverleeBelgium
  3. 3.Department of Geochemistry, The Geological Survey of Denmark and Greenland (GEUS)Copenhagen KDenmark

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