{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T01:56:58Z","timestamp":1760234218559,"version":"build-2065373602"},"reference-count":59,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2021,4,20]],"date-time":"2021-04-20T00:00:00Z","timestamp":1618876800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100008398","name":"Villum Fonden","doi-asserted-by":"publisher","award":["15471"],"award-info":[{"award-number":["15471"]}],"id":[{"id":"10.13039\/100008398","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100006129","name":"FCT","doi-asserted-by":"publisher","award":["UIDB\/00329\/2020"],"award-info":[{"award-number":["UIDB\/00329\/2020"]}],"id":[{"id":"10.13039\/100006129","id-type":"DOI","asserted-by":"publisher"}]},{"name":"French National Research Agency and EUR H2O\u2019Lyon","award":["ANR-17-EURE-0018"],"award-info":[{"award-number":["ANR-17-EURE-0018"]}]},{"name":"Slovenian Research Agency","award":["P2-0191"],"award-info":[{"award-number":["P2-0191"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Water"],"abstract":"<jats:p>Current standardized laboratory test protocols use model species that have limitations to accurately assess native species responses to stressors. We developed and tested a novel acute in situ protocol for testing field-collected organisms. We used Asellus aquaticus and NaCl as a reference toxicant to test for the effects of location (laboratory vs. in situ), medium (synthetic vs. field water), substrate (presence vs. absence), and protocol replicability. We further tested the protocol using groundwater-adapted isopods: Proasellus assaforensis for the effect of location, P. cavaticus of medium and P.lusitanicus of substrate. Our results showed that A.aquaticus\u2019 lethality obtained with the novel acute in situ protocol did not significantly differ from those from laboratory testing. However, laboratory tested P.assaforensis showed a higher sensitivity, suggesting that its acclimation to laboratory conditions might have pernicious effects. A. aquaticus and P. cavaticus showed a higher mortality using synthetic medium in situ and under laboratory conditions, which overestimated the stressor\u2019s effect. Besides, substrate use had no significant effect. The novel acute in situ protocol allows the use of native species under realistic scenarios. It is particularly well adapted for assessing the risk of groundwater ecosystems but it can be applied to a wide range of ecosystems.<\/jats:p>","DOI":"10.3390\/w13081132","type":"journal-article","created":{"date-parts":[[2021,4,21]],"date-time":"2021-04-21T00:16:02Z","timestamp":1618964162000},"page":"1132","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Novel Protocol for Acute In Situ Ecotoxicity Test Using Native Crustaceans Applied to Groundwater Ecosystems"],"prefix":"10.3390","volume":"13","author":[{"given":"Andrea","family":"Casta\u00f1o-S\u00e1nchez","sequence":"first","affiliation":[{"name":"Natural History Museum of Denmark, University of Copenhagen, 2100 Copenhagen, Denmark"}]},{"given":"Florian","family":"Malard","sequence":"additional","affiliation":[{"name":"Univ Lyon, Universit\u00e9 Claude Bernard Lyon 1, CNRS, ENTPE, UMR 5023 LEHNA, F-69622 Villeurbanne, France"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8254-2975","authenticated-orcid":false,"given":"Gabriela","family":"Kal\u010d\u00edkov\u00e1","sequence":"additional","affiliation":[{"name":"Faculty of Chemistry and Chemical Technology, University of Ljubljana, SI-1000 Ljubljana, Slovenia"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4756-7034","authenticated-orcid":false,"given":"Ana Sofia P. S.","family":"Reboleira","sequence":"additional","affiliation":[{"name":"Natural History Museum of Denmark, University of Copenhagen, 2100 Copenhagen, Denmark"},{"name":"Centre for Ecology, Evolution and Environmental Changes (cE3c) and Departamento de Biologia Animal, Faculdade de Ci\u00eancias, Universidade de Lisboa, 1749-016 Lisbon, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2021,4,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"983","DOI":"10.1016\/j.envint.2006.06.005","article-title":"An examination of ecological risk assessment and management practices","volume":"32","author":"Hope","year":"2006","journal-title":"Environ. Int."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"756","DOI":"10.1080\/10408444.2016.1190685","article-title":"The pros and cons of ecological risk assessment based on data from different levels of biological organization","volume":"46","author":"Rohr","year":"2016","journal-title":"Crit. Rev. Toxicol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1099","DOI":"10.1890\/1051-0761(1997)007[1099:TROMSI]2.0.CO;2","article-title":"The Role of Mesocosm Studies in Ecological Risk Analysis","volume":"7","author":"Boyle","year":"1997","journal-title":"Ecol. Appl."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"379","DOI":"10.1897\/04-025R.1","article-title":"Insecticide species sensitivity distributions: Importance of test species selection and relevance to aquatic ecosystems","volume":"24","author":"Maltby","year":"2005","journal-title":"Environ. Toxicol. Chem."},{"key":"ref_5","unstructured":"(2020, December 10). ECOTOX-Database, Available online: https:\/\/cfpub.epa.gov\/ecotox."},{"key":"ref_6","unstructured":"EC (2003). Technical Guidance Document in Support of Commission Directive 93\/67\/EEC on Risk Assessment for New Notified Substances and Commission Regulation (EC) No 1488\/94 on Risk Assessment for Exiting Substances, European Commission."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"951","DOI":"10.1080\/10807030500257788","article-title":"Assessing the Need for Groundwater Quality Guidelines for Pesticides Using the Species Sensitivity Distribution Approach","volume":"11","author":"Hose","year":"2005","journal-title":"Hum. Ecol. Risk Assess."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1007","DOI":"10.1016\/S0025-326X(01)00135-7","article-title":"Can Saltwater Toxicity be Predicted from Freshwater Data?","volume":"42","author":"Leung","year":"2001","journal-title":"Mar. Pollut. Bull."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1016\/j.scitotenv.2015.04.064","article-title":"Aquatic acute species sensitivity distributions of ZnO and CuO nanoparticles","volume":"526","author":"Adam","year":"2015","journal-title":"Sci. Total Environ."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"12328","DOI":"10.1038\/s41598-020-69050-7","article-title":"Salinity and temperature increase impacts groundwater crustaceans","volume":"10","author":"Hose","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1007\/s11356-012-1117-4","article-title":"A plea for the use of copepods in freshwater ecotoxicology","volume":"20","author":"Kulkarni","year":"2013","journal-title":"Environ. Sci. Pollut. Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"234","DOI":"10.1897\/IEAM_2006-027.1","article-title":"Review of aquatic in situ approaches for stressor and effect diagnosis","volume":"3","author":"Crane","year":"2007","journal-title":"Integr. Environ. Asses."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1897\/IEAM_2006-031.1","article-title":"In situ-based effects measures: Determining the ecological relevance of measured responses","volume":"3","author":"Baird","year":"2007","journal-title":"Integr. Environ. Asses."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1016\/j.envpol.2004.07.008","article-title":"In situ exposures using caged organisms: A multi-compartment approach to detect aquatic toxicity and bioaccumulation","volume":"134","author":"Burton","year":"2005","journal-title":"Environ. Pollut."},{"key":"ref_15","first-page":"13","article-title":"In situ online biomonitoring of groundwater quality using freshwater amphipods exposed to organic fertilizer and rainfall events","volume":"16","author":"Gerhardt","year":"2020","journal-title":"Curr. Top. Toxicol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"292","DOI":"10.1016\/j.scitotenv.2019.05.030","article-title":"Recommendations for ecotoxicity testing with stygobiotic species in the framework of groundwater environmental risk assessment","volume":"681","author":"Fiasca","year":"2019","journal-title":"Sci. Total Environ."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"122132","DOI":"10.1016\/j.jhazmat.2020.122132","article-title":"Measuring the biological impact of drilling waste on the deep seafloor: An experimental challenge","volume":"389","author":"Lelchat","year":"2020","journal-title":"J. Hazard. Mater."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"324","DOI":"10.1016\/j.ecoenv.2005.12.009","article-title":"Ten challenges for improved ecotoxicological testing in environmental risk assessment","volume":"63","author":"Breitholtz","year":"2006","journal-title":"Ecotoxicol. Environ. Saf."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"125422","DOI":"10.1016\/j.chemosphere.2019.125422","article-title":"Ecotoxicological effects of anthropogenic stressors in subterranean organisms: A review","volume":"244","author":"Hose","year":"2020","journal-title":"Chemosphere"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"641","DOI":"10.1093\/biosci\/biz064","article-title":"Scientists\u2019 Warning on the Conservation of Subterranean Ecosystems","volume":"69","author":"Mammola","year":"2019","journal-title":"BioScience"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"4643","DOI":"10.1007\/s11356-013-2390-6","article-title":"Sensitivity of hypogean and epigean freshwater copepods to agricultural pollutants","volume":"21","author":"Baratti","year":"2014","journal-title":"Environ. Sci. Pollut. Res."},{"key":"ref_22","unstructured":"EMA (European Medicines Agency) (2020, December 15). Guideline on Assessing the Environmental and Human Health Risks of Veterinary Medicinal Products in Groundwater. Committee for Medical Products for Veterinary Use. Available online: https:\/\/www.ema.europa.eu\/en\/documents\/scientific-guideline\/guideline-assessing-environmental-human-health-risks-veterinary-medicinal-products-groundwater_en.pdf."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1016\/S0025-326X(01)00253-3","article-title":"Integrating toxicology and ecology: Putting the \u201ceco\u201d into ecotoxicology","volume":"44","author":"Chapman","year":"2002","journal-title":"Mar. Pollut. Bull."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1002\/etc.5620170112","article-title":"A critical evaluation of safety (uncertainty) factors for ecological risk assessment","volume":"17","author":"Chapman","year":"1998","journal-title":"Environ. Toxicol. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1016\/j.envpol.2004.11.023","article-title":"Copper toxicity to different field-collected cladoceran species: Intra- and inter-species sensitivity","volume":"136","author":"Bossuyt","year":"2005","journal-title":"Environ. Pollut."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.aquatox.2012.12.023","article-title":"A new bioassay for the ecotoxicological testing of VOCs on groundwater invertebrates and the effects of toluene on Niphargus inopinatus","volume":"130\u2013131","author":"Avramov","year":"2013","journal-title":"Aquat. Toxicol."},{"key":"ref_27","first-page":"179","article-title":"Animal standardisation for mixed species ecotoxicological studies: Establishing a laboratory breeding programme for Gammarus pulex and Asellus aquaticus","volume":"21","author":"Bloor","year":"2010","journal-title":"Zool. Baetica"},{"key":"ref_28","unstructured":"US EPA (2016). Ecological Effects Test Guidelines OCSPP 850.1020: Gammarid Amphipod Acute Toxicity Test."},{"key":"ref_29","unstructured":"(2021, January 15). Danish Meteorological Institute. Available online: http:\/\/www.dmi.dk."},{"key":"ref_30","unstructured":"OECD (2004). Test No. 202: Daphnia sp., acute immobilization test, adopted: April 2004. OECD Guidelines for the Testing of Chemicals, OECD Publishing."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1550","DOI":"10.1007\/s11270-013-1550-0","article-title":"Acute toxicity of copper sulfate and potassium dichromate on stygobiont Proasellus: General aspects of groundwater ecotoxicology and future perspectives","volume":"224","author":"Reboleira","year":"2013","journal-title":"Water Air Soil Pollut."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Mermillod-Blondin, F., Lefour, C., Lalouette, L., Renault, D., Malard, F., Simon, L., and Douady, C.J. (2013). Thermal tolerance breadths among groundwater crustaceans living in a thermally constant environment. J. Exp. Biol., jeb.081232.","DOI":"10.1242\/jeb.081232"},{"key":"ref_33","unstructured":"Rice, E.W., Baird, R.B., and Eaton, A.D. (2017). Standard Methods for the Examination of Water and Wastewater, American Public Health Association, American Water Works Association and Water Environment Federation. [23rd ed.]."},{"key":"ref_34","unstructured":"R Team (2013). R Development Core Team. R. A. Lang. Environ. Stat. Comput., 55, 275\u2013286."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Markussen, B. (2020, December 14). LabApplStat: Miscellaneous Scripts Developed at the Data Science Laboratory, University of Copenhagen. Available online: https:\/\/github.com\/bomarkussen\/LabApplStat.","DOI":"10.32614\/CRAN.package.LabApplStat"},{"key":"ref_36","unstructured":"Russell, L. (2020, December 11). Emmeans: Estimated Marginal Means, Aka Least-Squares Means, Available online: https:\/\/cran.r-project.org\/web\/packages\/emmeans\/index.html."},{"key":"ref_37","unstructured":"Wickham, H., Chang, W., Henry, L., Pedersen, T.L., Takahashi, K., Wilke, C., Yutani, H., and Dunnington, D. (2020, December 11). ggplot2: Create Elegant Data Visualisations Using the Grammar of Graphics, Available online: https:\/\/cloud.r-project.org\/package=ggplot2."},{"key":"ref_38","first-page":"98","article-title":"Climate change going deep: The effects of global climatic alterations on cave ecosystems","volume":"6","author":"Mammola","year":"2019","journal-title":"Anthr. Rev."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1638","DOI":"10.1111\/1365-2435.13382","article-title":"Extending Janzen\u2019s hypothesis to temperate regions: A test using subterranean ecosystems","volume":"33","author":"Mammola","year":"2019","journal-title":"Funct. Ecol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"498","DOI":"10.1016\/j.chemosphere.2019.06.217","article-title":"The freshwater isopod Asellus aquaticus as a model biomonitor of environmental pollution: A review","volume":"235","author":"Harrison","year":"2019","journal-title":"Chemosphere"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"2849","DOI":"10.1002\/etc.3847","article-title":"How lethal concentration changes over time: Toxicity of cadmium, copper, and lead to the freshwater isopod Asellus aquaticus","volume":"36","author":"Ginneken","year":"2017","journal-title":"Environ. Toxicol. Chem."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"421","DOI":"10.1016\/S0306-4565(03)00027-5","article-title":"Thermal acclimation without heat shock, and motor responses to a sudden temperature change in Asellus aquaticus","volume":"28","author":"Lagerspetz","year":"2003","journal-title":"J. Therm. Biol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"196","DOI":"10.1016\/j.envpol.2014.05.020","article-title":"Population responses of Daphnia magna, Chydorus sphaericus and Asellus aquaticus in pesticide contaminated ditches around bulb fields","volume":"192","author":"Ieromina","year":"2014","journal-title":"Environ. Pollut."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1700","DOI":"10.1016\/j.chemosphere.2005.03.083","article-title":"Contaminated sediments and bioassay responses of three macroinvertebrates, the midge larva Chironomus riparius, the water louse Asellus aquaticus and the mayfly nymph Ephoron virgo","volume":"61","author":"Maas","year":"2005","journal-title":"Chemosphere"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1016\/j.ecoenv.2017.11.031","article-title":"The ecotoxicity of binary mixtures of Imazamox and ionized ammonia on freshwater copepods: Implications for environmental risk assessment in groundwater bodies","volume":"149","author":"Cifoni","year":"2018","journal-title":"Ecotoxicol. Environ. Saf."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"930","DOI":"10.1002\/etc.454","article-title":"Influence of water hardness and sulfate on the acute toxicity of chloride to sensitive freshwater invertebrates","volume":"30","author":"Soucek","year":"2011","journal-title":"Environ. Toxicol. Chem."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"521","DOI":"10.1002\/tox.20214","article-title":"Effects of copper pre-exposure routes on the energy reserves and subsequent copper toxicity in Daphnia magna","volume":"21","author":"Canli","year":"2006","journal-title":"Environ. Toxicol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"3746","DOI":"10.1016\/j.scitotenv.2009.10.067","article-title":"Interactions between effects of environmental chemicals and natural stressors: A review","volume":"408","author":"Holmstrup","year":"2010","journal-title":"Sci. Total Environ."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"3763","DOI":"10.1016\/j.scitotenv.2010.01.043","article-title":"Interactions between toxic chemicals and natural environmental factors\u2014A meta-analysis and case studies","volume":"408","author":"Laskowski","year":"2010","journal-title":"Sci. Total Environ."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"1859","DOI":"10.1111\/brv.12312","article-title":"Behavioural effects of temperature on ectothermic animals: Unifying thermal physiology and behavioural plasticity","volume":"92","author":"Abram","year":"2017","journal-title":"Biol. Rev."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"181","DOI":"10.3354\/cr00764","article-title":"Interactive effects of metal pollution and temperature on metabolism in aquatic ectotherms: Implications of global climate change","volume":"37","author":"Sokolova","year":"2008","journal-title":"Clim. Res."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"1856","DOI":"10.1242\/jeb.118851","article-title":"The effects of temperature on aerobic metabolism: Towards a mechanistic understanding of the responses of ectotherms to a changing environment","volume":"218","author":"Schulte","year":"2015","journal-title":"J. Exp. Biol."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"720","DOI":"10.1016\/j.scitotenv.2016.10.104","article-title":"Towards the review of the European Union Water Framework Directive: Recommendations for more efficient assessment and management of chemical contamination in European surface water resources","volume":"576","author":"Brack","year":"2017","journal-title":"Sci. Total Environ."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1016\/j.trac.2004.11.004","article-title":"Priority substances of the European Water Framework Directive: Analytical challenges in monitoring water quality","volume":"24","author":"Coquery","year":"2005","journal-title":"TrAC Trends Anal. Chem."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.scitotenv.2011.11.072","article-title":"Review of risk from potential emerging contaminants in UK groundwater","volume":"416","author":"Stuart","year":"2012","journal-title":"Sci. Total Environ."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"517","DOI":"10.1016\/j.marpolbul.2013.06.012","article-title":"Evaluating legacy contaminants and emerging chemicals in marine environments using adverse outcome pathways and biological effects-directed analysis","volume":"74","author":"Hutchinson","year":"2013","journal-title":"Mar. Pollut. Bull."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"287","DOI":"10.1016\/j.envpol.2011.12.034","article-title":"Emerging organic contaminants in groundwater: A review of sources, fate and occurrence","volume":"163","author":"Lapworth","year":"2012","journal-title":"Environ. Pollut."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1016\/j.ecolind.2014.07.024","article-title":"A review on the ecological quality status assessment in aquatic systems using community based indicators and ecotoxicological tools: What might be the added value of their combination?","volume":"48","author":"Beiras","year":"2015","journal-title":"Ecol. Indic."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"1135","DOI":"10.1111\/geb.12200","article-title":"Geographic variation in range size and beta diversity of groundwater crustaceans: Insights from habitats with low thermal seasonality","volume":"23","author":"Zagmajster","year":"2014","journal-title":"Glob. Ecol. Biogeogr."}],"container-title":["Water"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4441\/13\/8\/1132\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T05:50:22Z","timestamp":1760161822000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4441\/13\/8\/1132"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,4,20]]},"references-count":59,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2021,4]]}},"alternative-id":["w13081132"],"URL":"https:\/\/doi.org\/10.3390\/w13081132","relation":{},"ISSN":["2073-4441"],"issn-type":[{"type":"electronic","value":"2073-4441"}],"subject":[],"published":{"date-parts":[[2021,4,20]]}}}