DEPARTMENT OF BIOLOGICAL AND ENVIRONMENTAL SCIENCES The ecological cost to tolerance against the herbicide diflufenican in the green algae Raphidocelis subcapitata ventuell bild ska placeras här Johanna Bengtson Degree of Bachelor of Science with a major in Environmental Science ES1510, Examination course in Environmental Science, 15 HP Basic level Semester/year: Spring 2021 Supervisor: Natàlia Corcoll Cornet, Department of Biological and Environmental Sciences Examiner: Thomas Backhaus, Department of Biological and Environmental Sciences Abstract Chemical pollution is one of the drivers of global change. Although some organisms can develop tolerance to toxic chemicals, little is known about how this can affect the organism and make them more susceptible to other environmental stress factors (so-called ecological cost of adaptation). The main goal of this project is to understand how the freshwater algae Raphidocelis subcapitata may change its sensitivity to two general environmental stressors (i.e., light and temperature) and a chemical contaminant (i.e., copper) when adapted to the herbicide diflufenican. The algae were exposed to two levels of diflufenican during a 2,5-month period: 0,1 nM – which is ecologically relevant for European surface waters and to 3,7 nM – that corresponds to the 72h-EC50 value for diflufenican. Algae exposed for the same period of time to 0 nM diflufenican was used as controls. Adaptation was assessed as growth rate and the tolerance of the algae to diflufenican as 72h-EC50value. Additionally, short-term tests were performed to assess the sensitivity of the algae when exposed to: i) copper via growth inhibition assays (72h- EC50value), ii) increased light via PI-curves and iii) increased temperature (4°C) via growth rate assays (72h-growth rate). Obtained results showed that after 2,5 months, algal cultures adapted to 0,1 and 3,7 nM had similar growth rates compared to the control culture (1,02± 0,03). Diflufenican tolerance increased from 4,84 nM in the controls, to 8,15 nM in the algal culture adapted to 0,1 nM and to 17,25 nM in the algal culture adapted to 3.7 nM. These results indicate that R. subcapitata has a high phenotypic plasticity. Adaptation to diflufenican did not lead to increasing sensitivity to copper. But, adapted algae had a lower capacity to cope with light stress, as observed with, lower ETRmax and Ik values. Additionally, adaptation to diflufenican leads to a lower capacity to cope with increased temperature. Overall, this study shows that R. subcapitata can adapt to the toxic levels of the herbicide diflufenican, this adaptation has negative ecological consequences since algae became more vulnerable to general environmental factors such as light and temperature which may have negative effects under the scenario of climate change. Key words: freshwater algae, adaptation, tolerance, ecological cost, diflufenican, climate change 2 Sammanfattning Kemiska föroreningar är en av drivkrafterna bakom en föränderlig miljö. Även om vissa organismer kan utveckla tolerans mot giftiga kemikalier, så saknas kunskap om hur detta kan påverka organismer och huruvida det kan göra dom mer mottagliga för andra stressfaktorer i miljön (så kallad ekologisk kostnad vid adaptation.) Fokus i denna studie är att förstå hur motståndskraften i sötvattensalgen Raphidocelis subcapitata påverkas av två stressfaktorer i miljön (ljus och temperatur), samt vid kemisk exponering av koppar när dom adapterats till herbiciden diflufenican. Algerna exponerades för två olika koncentrationer av diflufenican under en 2,5-månadsperiod: 0,1 nM – vilket representerar en ekologisk relevant koncentration för europeiskt ytvatten och för 3,7 nM – vilket motsvarar 72h-EC50 värde för diflufenican. Under samma tidsperiod har alger som exponerats för 0 nM diflufenican använts som kontroll. Adaptationen bedömdes utifrån algernas tillväxthastighet och deras tolerans mot diflufenican genom 72h-EC50 värden. Dessutom undersöktes algernas känslighet genom exponering av: i) koppar (inhibering av tillväxt under 72 h), ii) ökad ljusstrålning via PI-kurvor samt iii) ökad temperatur (tillväxthastighet under 72 h). Resultaten visade att algerna i behandlingarna 0,1 nM och 3,7 nM efter 2,5 månad hade liknande tillväxthastighet som kontrollen (1,02 ± 0,03). Tolerans mot diflufenican ökade från 4,84 nM i kontrollerna till 8,15 nM hos algerna i behandlingen med 0,1 nM och till 17,25 nM i kulturen som exponerats för 3,7 nM. Resultaten indikerar att R. subcapitata har en hög fenotypisk plasticitet. Adaptation mot diflufenican ledde inte till ökad känslighet mot koppar men adapterade alger hade sämre kapacitet att hantera ökad ljusstress, vilket observerades genom, lägre ETRmax- och Ik – värden. Dessutom leder tolerans till en sämre förmåga att klara av ökad temperatur. Sammantaget så visar denna studie att R. subcapitata kan adaptera sig till toxiska nivåer av herbiciden diflufenican, men det har negativa ekologiska konsekvenser eftersom algerna blir mer sårbara mot miljöfaktorer så som ljus och temperatur, vilket i sin tur kan ha en negativ påverkan under rådande klimatförändringar. 3 Abbreviations DFF = diflufenican FU = Fluorescence units EC50 = Effective concentration 50 % effect PI-curve= Photosynthesis vs. irradiance curve rETR-I curve = relative electron transport vs. irradiance curve PAM = Pulse Amplitude Modulated ETRmax =maximum electron transport rate α = the efficiency of light used Ik = half of the light intensity when photoinhibition occurs µ = growth rate 4 Table of contents Background .............................................................................................................................................. 6 The importance of algae...................................................................................................................... 6 Global pollution ................................................................................................................................... 6 Diflufenican ......................................................................................................................................... 6 Multiple stressors and plasticity ......................................................................................................... 7 Ecological cost of tolerance ................................................................................................................. 8 Aim and/or hypothesis ...................................................................................................................... 10 Methods and materials ......................................................................................................................... 11 Algae cultivation and adaptation phase ............................................................................................ 11 Cell growth and calibration curve ................................................................................................. 12 Short-term stress ............................................................................................................................... 13 Chemical growth inhibition assays in microplates ........................................................................ 13 Increased temperature experiment .............................................................................................. 14 Electron transport rate-irradiance curves ..................................................................................... 14 Statistical evaluation ......................................................................................................................... 15 Results ................................................................................................................................................... 16 Growth rate (µ) after adaptation ...................................................................................................... 16 Tolerance to diflufenican .................................................................................................................. 17 Dose-response tests: Copper ............................................................................................................ 18 Algal growth rate under increased temperature at 25°C .................................................................. 19 Increased light intensities.................................................................................................................. 19 Discussion .............................................................................................................................................. 21 Physiological cost of adaptation ........................................................................................................ 21 Ecological cost of developing tolerance ............................................................................................ 21 Ecosystem effects .............................................................................................................................. 22 General remarks ................................................................................................................................ 22 Conclusions ............................................................................................................................................ 23 Acknowledgements ............................................................................................................................... 23 References