Master’s Thesis 2017 60 ECTS Faculty of Environmental Sciences and Natural Resource Management Characterization of pesticide hazard and risk to freshwater algae under ecologically relevant exposure conditions in Norway Linn Anette Haug Master of Science in Environment and Natural Resources – Specialisation Environmental pollutants and ecotoxicology 1 Acknowledgements This is the final project at the Master of Science in Environment and natural resources – Specialisation in Environmental Pollutants and Ecotoxicology at the Norwegian University of Life Sciences. This project was conducted in collaboration with and funded by The Norwegian Institute for Water Research (NIVA). The water sampling and chemical analyses was conducted by Geir Tveiti, Hans Ragnar Norli and Sven Roar Odenmarck (NIBIO), and the solid phase extractions (SPE) was performed by Kine Bæk (NIVA). First and foremost, I would like to thank my main supervisor; Senior researcher Knut Erik Tollefsen, and my co-supervisor; Research scientist Tânia Gomes, for their excellent guidance and support during this process. I also wish to thank Lene Fredriksen, Li Xie, You Song and Ana Catarina Almeida for valuable support in the laboratory, and everyone I got to know at NIVA, especially Natasa, Stefanie and David. You made every day a wonderful day. I would also like to thank my friends and family for all the valuable help with reading and discussing this thesis. Finally, a huge thank you to Bjørn-Erik, for all the motivation and great company during these stressful times, I couldn’t have done it without you <3 2 Sammendrag Balansen i det akvatiske økosystemet er under konstant press fra menneskelig påvirkning, og store mengder forurensende stoffer slippes ut i vannforekomster i hele verden. Blant disse er pesticider en stor kilde til utslipp, og dette skyldes i stor grad avrenning fra jordbruk og skogbruk. Et pesticid er ethvert stoff eller blandinger av stoffer produsert med det formål å forhindre, ødelegge eller begrense en skade eller sykdom på levende organismer. I Norge er bruken av sprøytemidler hovedsakelig nedadgående, bortsett fra når det gjelder herbicider, som har hatt en svak økning de siste årene. Herbicider utgjør den største andelen av sprøytemiddelbruk i Norge, og utgjorde 69,5% av bruken i 2014. Individuell risikovurdering er gjort for stoffene som er godkjent for bruk i Norge, men det er en økende bekymring omkring blandinger av disse stoffene og de mulige negative effektene disse blandingene kan ha på det akvatiske miljøet rundt spesielt jordbruksområdene. I løpet av sommeren 2015 (juni-august) ble det tatt vannprøver fra Heiabekken, Råde kommune, i forbindelse med overvåkningsprogrammet JOVA. I et samarbeid mellom Norsk institutt for vannforskning (NIVA) og NIBIO, ble det gjennomført ekstraksjon og kjemiske analyser av disse vannprøvene for blant annet å undersøke tilstedeværelsen av pesticider. Ved hjelp av en kumulativ toksisitetsvurdering basert på innsamlet litteratur, ble de største bidragsyterne for toksisitet i blandingen identifisert. Det ble gjennomført toksisitetstesting (72-timers veksthemming og 5-timers PSII-aktivitet) av en miljørelevant syntetisk pesticidblanding og de to viktigste herbicidene i blandingen, metribuzin og metamitron, på ferskvannsalgen Chlamydomonas reinhardtii. Eksperimentene viste at den syntetiske blandingen måtte oppkonsentreres ~100 ganger i forhold til de målte miljøkonsentrasjonene for å gi en 50% veksthemming, men at både blandingen og enkeltstoffene hadde en tydelig redusert dose-responskurve ved økende konsentrasjoner. PSII-aktiviteten ble også tydelig påvirket, spesielt av metribuzin som er regnet som den mest potente risikodriveren for alger i blandingen. Den kumulative toksisitetsvurderingen basert på CA-prediksjonsmodellen ble sammenliknet med de eksperimentelle toksisitetsstudiene gjort på C. reinhardtii. Giftigheten av blandingen ble relativt godt estimert, innenfor en faktor på 2, i forhold til de eksperimentelle studiene. 3 Abstract The aquatic ecosystem balance is under constant pressure from human influence, and substantial amounts of pollutants are released to water bodies all over the world. Among these, pesticides are an important group, mainly due to runoff from agriculture and forestry. A pesticide is any compound or combinations of compounds produced with the aim to prevent, destroy or mitigate a pest. In Norway, the use of pesticides is in general decrease, except for herbicide use, which has had a weak increase in recent years. Herbicides make up the largest proportion of pesticide use in Norway, constituting 69.5% of the use in 2014. Individual risk assessment is performed for the compounds approved for use in Norway, but there is an increasing concern regarding mixtures of these compounds and the possible adverse effects these mixtures may have on the aquatic environment around agricultural areas. During the summer 2015 (June- August) water samples were collected from Heiabekken, Råde municipality, in conjunction with the monitoring programme JOVA. In a collaboration between NIVA and NIBIO, extractions and chemical analyses of these water samples were performed to investigate the presence of pesticides. By a cumulative toxicity assessment based on collected literature, the main contributors to toxicity in the mixture was identified. Toxicological testing (72 h growth inhibition and 5 h PSII-efficiency) was conducted on an environmentally relevant synthetic mixture of pesticides, and the two main herbicides in the mixture, metribuzin and metamitron, to the freshwater alga Chlamydomonas reinhardtii. The experiments showed that the synthetic mixture had to be concentrated up to a factor of ~100 compared to the measured environmental concentrations, before a 50% growth inhibition could be seen. Still, both the synthetic mixture and the single compounds had a clear dose-response reduction with increasing concentrations. PSII-efficiency was also clearly affected, especially by metribuzin, which is regarded as the most potent risk driver for algae in the mixture. The cumulative toxicity assessment with the CA prediction model was compared to the experimental toxicity studies conducted on C. reinhardtii. The toxicity of the mixture was well estimated, within a factor of 2, compared to the experimental studies. 4 Contents Introduction ................................................................................................................................ 8 Background ............................................................................................................................. 8 Theory ..................................................................................................................................... 9 Pesticides ............................................................................................................................ 9 Monitoring of exposure in agricultural environments globally and in Norway ............... 10 Adversity and Mode of Action (MoA) ............................................................................... 12 Combined toxicity assessment (CTA) ................................................................................ 19 Cumulative risk assessment (CRA) .................................................................................... 20 Test organisms .................................................................................................................. 23 Effects of pesticides in algae ............................................................................................. 24 Objectives ............................................................................................................................. 25 Materials and methods ............................................................................................................ 26 Field sampling ....................................................................................................................... 26 Surface water sample collection ....................................................................................... 26 Extraction and analysis of water samples ......................................................................... 27 Cumulative risk assessment (CRA) ........................................................................................ 29 Experimental testing ............................................................................................................. 30 Chemicals .......................................................................................................................... 30 Synthetic mixture .............................................................................................................. 32 Single compounds ............................................................................................................. 33 Algae culturing .................................................................................................................. 34 Growth inhibition assay .................................................................................................... 34 Photosystem II-efficiency .................................................................................................. 35 Calculations, statistics and graphical treatments ............................................................. 36 Results ...................................................................................................................................... 37 JOVA summary ...................................................................................................................... 37 Experimental effort............................................................................................................... 37 Growth inhibition studies ................................................................................................. 38 PSII-efficiency studies ....................................................................................................... 42 Cumulative risk assessment.................................................................................................. 51 5 Discussion ................................................................................................................................. 52 Experimental studies ............................................................................................................ 52 Growth inhibition studies ................................................................................................. 52 Photosystem II- efficiency ................................................................................................. 53 Adversity vs. Mode of Action ............................................................................................ 55 Predicted vs observed toxicity .............................................................................................. 56 Ecological relevance ............................................................................................................. 56 Conclusion ................................................................................................................................ 57 Future prospects ................................................................................................................... 57 References ................................................................................................................................ 58 Appendices ............................................................................................................................... 64 Appendix 1: Water sample collection................................................................................... 64 Appendix 2: Summary of the differences in properties of SPE and whole water samples .. 65 Appendix 3: Pesticide compound in the synthetic mixture ................................................. 66 Appendix 4: Stock solutions dilution of synthetic mixture, and final concentrations ......... 67 6 7 Introduction Background There are several anthropogenic activities that affect the balance of ecosystems, and emission of toxic metals and organic chemicals (POPs, PCBs, PPPs) is one of them. These emissions can be either deliberate, as in agricultural context, or by leaching, and accidental spills (Hartgers et al. 1999). Around 300 million tons of synthetic compounds are used in industry and consumer products every year. In addition, a lot of pollution comes from diffuse sources of agricultural activities, where 140 million tons of manure and several million tons of pesticides are added yearly (FAO statistical database 2006, according to Schwarzenbach et al. 2006). A pesticide is any substance or mixture of substances produced to prevent, destroy or mitigate a pest. Pesticides are divided into herbicides, insecticides, fungicides, depending on the target pest species. Pest species includes any living organism that may cause damage and economic loss, or transmits or produces disease. Examples of pest species are insects, birds, rodents, unwanted plants, or microorganisms. In Norway, the use of plant protection products is in general decrease each year, except for herbicide use, which has had a weak increase of 2,2 % in use from 2005 to 2014. Herbicides are the main type of pesticide used on crops in Norway, and constituted 69,5% of the pesticide use in Norway in 2014 (Statistics Norway) Several pesticides are used during the growth season in Norway. Individual risk assessment for these pesticides are, and has been, performed to identify potential environmental effects, but the possible effects caused by combinations of these compounds are often unknown. Therefore, focus has increased both nationally and internationally on developing methods to predict the combined effects of complex pollutant mixtures (Petersen et al. 2015; Cedergreen et al. 2008). The aim of this thesis is to characterize and evaluate hazard and risk of pesticides to algae as non-target organisms under Norwegian conditions with the use of prediction tools and experimental studies. 8 Theory Pesticides Surface waters are being subjected to increasing pressure because of contamination by various mineral and organic pollutants. Most of the organic pollutants are pesticides, which are not only used in agriculture and forestry, but also for weed clearance in road verges and railway tracks (Villeneuve et al. 2011). The term pesticides mainly include herbicides, fungicides and insecticides, all organic compounds designed to prevent attacks from, and/or kill, unwanted organisms. The Mode of Action (MoA) of main classes of pesticides are presented in table 1 below. Table 1. MoA of main classes of pesticides on target organisms (according to DeLorenzo et al. 2001) General mode of Main class Functional groups Site of action action Nervous system Organophosphates Carbamates Acetylcholinesterase inhibition Nervous system Organochlorides Cyclodienes GABA-receptor inhibition Acylanilides, ureas, Photosystem II cyclic ureas, Photosysnthesis (PSII), Hill reaction in phenylcarbamates, inhibition the electron triazines, triazinones transport Photosynthesis Photosystem I (PSI), Bipyridiniums inhibition light reaction Biosynthesis Carotene Pyridazinones Herbicides inhibition accumulation Biosynthesis Chloroacetamide Fatty acid synthesis inhibition Dinitroanilines, phosphoric amides, Biosynthesis Microtubule chlorthadimethyl, inhibition formation propyzamide, cholchicine, terbutol Phosphorylation, Multiple inhibiting Chlorophenols protein synthesis, Broad-spectrum actions lipid biosynthesis biocides Tributyl tins, trialkyl Respiratory system Mitochondrial tins inhibition ATPase 9