Single and multigenerational studies of silver nanoparticle toxicity and adaptive mechanisms in the nematode Caenorhabditis elegans

Philosophiae Doctor (PhD) Thesis 2019:37

Lisa Magdalena Rossbach

Single and multigenerational studies of silver nanoparticle toxicity and adaptive

mechanisms in the nematode Caenorhabditis elegans

Enkelt og multigenerasjonsstudier av toksisitet og adaptive mekanismer i nematoden

Caenorhabditis elegans

Norwegian University of Life Sciences Faculty of Environmental Sciences and Natural Resource Management

Single and multigenerational studies of silver nanoparticle toxicity and adaptive mechanisms in the

nematode Caenorhabditis elegans

Enkelt og multigenerasjonsstudier av toksisitet og adaptive mekanismer i nematoden Caenorhabditis elegans

Philosophiae Doctor (PhD) Thesis Lisa Magdalena Rossbach

Norwegian University of Life Sciences

Faculty of Environmental Sciences and Natural Resource Management

Ås (2019)

Thesis number 2019:37 ISSN 1894-6402 ISBN 978-82-575-1596-6

PhD supervisors

”‘ˆ‡••‘”‡„‘”ƒŠǤ—‰Š–‘

‡’ƒ”–‡–‘ˆ˜‹”‘‡–ƒŽ…‹‡…‡•ǡ

ƒ…—Ž–›‘ˆ˜‹”‘‡–ƒŽ…‹‡…‡•ƒ†ƒ–—”ƒŽ‡•‘—”…‡ƒƒ‰‡‡–ȋȌǡ

‘”™‡‰‹ƒ‹˜‡”•‹–›‘ˆ‹ˆ‡…‹‡…‡•ȋȌ Ǧƒ‹Žǣ†‡„‘”ƒŠǤ‘—‰Š–‘̷„—Ǥ‘

‡•‡ƒ”…Š”‘ˆ‡••‘”ƒ‰Ǥ”‡†‡ǡ

‡’ƒ”–‡–‘ˆ˜‹”‘‡–ƒŽ…‹‡…‡•ǡ

ƒ…—Ž–›‘ˆ˜‹”‘‡–ƒŽ…‹‡…‡•ƒ†ƒ–—”ƒŽ‡•‘—”…‡ƒƒ‰‡‡–ȋȌǡ

‘”™‡‰‹ƒ‹˜‡”•‹–›‘ˆ‹ˆ‡…‹‡…‡•ȋȌ Ǧƒ‹Žǣ†ƒ‰Ǥƒ†‡”•Ǥ„”‡†‡̷„—Ǥ‘

‡•‡ƒ”…Š‡”Žƒ‹”‡‘—–”‹•

‹˜‹•‹‘‘ˆ˜‹”‘‡–ƒŽƒ†ƒ–—”ƒŽ‡•‘—”…‡•

‡’ƒ”–‡–‘ˆ‘‹Ž—ƒŽ‹–›ƒ†Ž‹ƒ–‡Šƒ‰‡

‘”™‡‰‹ƒ•–‹–—–‡‘ˆ‹‘‡…‘‘›‡•‡ƒ”…ŠȋȌ Ǧƒ‹ŽǣŽƒ‹”‡Ǥ…‘—–”‹•̷‹„‹‘Ǥ‘

Evaluation Committee

”‘ˆ‡••‘”‡”‡•ƒ ‡”ƒ†‡•

•–‹–—–‡‘ˆ‹ˆ‡ƒ†ƒ”–Š…‹‡…‡•

‡”‹‘–Ǧƒ––‹˜‡”•‹–›

†‹„—”‰ŠǡͳͶͶǡ

‹–‡†‹‰†‘

Š‘‡ǣΪͶͶͳ͵ͳͶͷͳͶͷͻͻ Ǧƒ‹ŽǣǤ ‡”ƒ†‡•̷Š™Ǥƒ…Ǥ—

”Ǥ•’‡ƒ”‹—••‡

‘”™‡‰‹ƒ•–‹–—–‡‘ˆ‹”‡•‡ƒ”…ŠȋȌ

•–‹–—––˜‡‹‡ͳͺ ʹͲͲ͹Œ‡ŽŽ‡”

‘”™ƒ›

Š‘‡ǣΪͶ͹͸͵ͺͻͺʹʹͻ

Ǧƒ‹Žǣ•’‡Ǥƒ”‹—••‡̷‹Ž—Ǥ‘

”Ǥƒ•ǦŠ”‹•–‹ƒ‡‹‡

‡’ƒ”–‡–‘ˆ˜‹”‘‡–ƒŽ…‹‡…‡•ǡ

ƒ…—Ž–›‘ˆ˜‹”‘‡–ƒŽ…‹‡…‡•ƒ†ƒ–—”ƒŽ‡•‘—”…‡ƒƒ‰‡‡–ȋȌǡ

‘”™‡‰‹ƒ‹˜‡”•‹–›‘ˆ‹ˆ‡…‹‡…‡•ȋȌ Ǧƒ‹ŽǣŠƒ•Ǧ…Š”‹•–‹ƒǤ–‡‹‡̷„—Ǥ‘

ͳ

Contents

…‘™Ž‡†‰‡‡–•ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͶ ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͷ ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͺ

‹•–‘ˆƒ’‡”•ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳʹ

„„”‡˜‹ƒ–‹‘•ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳ͵

ͳǤ –”‘†—…–‹‘ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳͷ ͳǤͳǤ ƒ…‰”‘—†ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳͷ ͳǤʹǤ ‹Ž˜‡”ƒ‘’ƒ”–‹…Ž‡•ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳ͹

ͳǤ͵Ǥ ‹Ž˜‡”ƒ‘’ƒ”–‹…Ž‡–‘š‹…‹–›–‘„‹‘–ƒǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳͺ ͳǤͶǤ ‡ƒ…–‹˜‡‘š›‰‡•’‡…‹‡•ƒ†‘š‹†ƒ–‹˜‡•–”‡••ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳͻ ͳǤͷǤ ‹Ž˜‡”ƒ‘’ƒ”–‹…Ž‡•ƒ†”‡ƒ…–‹˜‡‘š›‰‡•’‡…‹‡•ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤʹͳ ͳǤ͸Ǥ †ƒ’–ƒ–‹‘–‘™ƒ”†••–”‡••‘”•ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤʹʹ ͳǤ͹Ǥ ‰—Ž–‹‰‡‡”ƒ–‹‘ƒŽ•–—†‹‡•ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤʹͷ ͳǤͺǤ ‹•ƒ†„Œ‡…–‹˜‡•ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤʹ͹

ʹǤ ‡–Š‘†‘Ž‘‰›ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤʹͺ ʹǤͳǤ ‡‡”ƒŽ‘—–Ž‹‡ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤʹͺ ʹǤʹǤ ͵ͲͲ•–‘…’”‡’ƒ”ƒ–‹‘ƒ†…Šƒ”ƒ…–‡”‹œƒ–‹‘ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵Ͳ ʹǤʹǤͳǤ –‘…’”‡’ƒ”ƒ–‹‘‘ˆ–Š‡͵ͲͲ•–‘…•—•’‡•‹‘•ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵Ͳ ʹǤʹǤʹǤ ƒ‘’ƒ”–‹…Ž‡…Šƒ”ƒ…–‡”‹œƒ–‹‘ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵Ͳ ʹǤʹǤ͵Ǥ ”ƒ•‹••‹‘Ž‡…–”‘‹…”‘•…‘’›ƒƒŽ›•‹•ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵ͳ ʹǤʹǤͶǤ ›†”‘†›ƒ‹…†‹ƒ‡–‡”ƒ†œ‡–ƒ’‘–‡–‹ƒŽǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵ʹ ʹǤʹǤͷǤ ‡–”‹ˆ—‰ƒ–‹‘ǡ—Ž–”ƒˆ‹Ž–”ƒ–‹‘ƒ†•‹œ‡ˆ”ƒ…–‹‘ƒ–‹‘ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵ʹ ʹǤ͵Ǥ Š‘‹…‡‘ˆ‘”‰ƒ‹•ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵͵

ʹǤͶǤ ‘š‹…‹–›–‡•–ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵Ͷ

ʹ

ʹǤͷǤ š’‘•—”‡ƒ†…‘…‡–”ƒ–‹‘•ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵Ͷ ʹǤ͸Ǥ †’‘‹–•ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵ͷ ʹǤ͹Ǥ Š”‘‹…—Ž–‹‰‡‡”ƒ–‹‘ƒŽ‡š’‘•—”‡ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵ͷ ʹǤ͹ǤͳǤ ”‘••–‘š‹…‹–›–‡•–‡š’‘•—”‡ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵ͺ ʹǤ͹ǤʹǤ ƒ’Ž‹‰ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵ͺ ʹǤͺǤ ͸‰‡‡”ƒ–‹‘–‘š‹…‹–›–‡•––‘™ƒ”†•‘–Š‡”–‘š‹…ƒ–•ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵ͺ ʹǤͻǤ ’”‘†—…–‹‘ƒ†ƒ–‹‘š‹†ƒ–†‡ˆ‡•‡•‡…Šƒ‹••ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵ͺ ʹǤͻǤͳǤ ‡ƒ–‘†‡•–”ƒ‹•ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵ͺ ʹǤͻǤʹǤ ǦͳǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵ͻ ʹǤͻǤ͵Ǥ ›‡”ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵ͻ ʹǤͻǤͶǤ ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͶͲ ʹǤͻǤͷǤ —Ž–‹‰‡‡”ƒ–‹‘ƒŽ‡š’‘•—”‡ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͶͳ ʹǤͻǤ͸Ǥ ‘š‹…‹–›–‡•–ƒ†•ƒ’Ž‹‰ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͶʹ ʹǤͳͲǤ ƒ–ƒƒƒŽ›•‹•ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͶʹ

͵Ǥ ‡•—Ž–•ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͶ͵

͵ǤͳǤ ƒ’‡”ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͶ͵

͵ǤʹǤ ƒ’‡”ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͶ͸

͵Ǥ͵Ǥ ƒ’‡”ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͶͺ

͵ǤͶǤ ƒ’‡”ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͷͲ ͶǤ ‹•…—••‹‘ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͷʹ ͶǤͳǤ Šƒ‰‡•‹•‹œ‡†‹•–”‹„—–‹‘‹–Š‡•–‘…ƒ†‡†‹ƒǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͷͷ ͶǤͳǤͳǤ ‡…‘˜‡”›‘ˆ‰…‘…‡–”ƒ–‹‘•‹•–‘…•ƒ†‡š’‘•—”‡‡†‹ƒǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͷͷ ͶǤͳǤʹǤ ‹œ‡†‹•–”‹„—–‹‘‘ˆ–Š‡‰‹–Š‡‡š’‘•—”‡‡†‹ƒǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͷ͸

ͶǤʹǤ ‘š‹…‹–›ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͷͺ ͶǤʹǤͳǤ Ͳ–‘š‹…‹–›ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͷͺ ͶǤ͵Ǥ —Ž–‹‰‡‡”ƒ–‹‘ƒŽ‡š’‘•—”‡ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͸ͳ ͶǤ͵ǤͳǤ ”ƒ•ˆ‡”‘ˆ‡ˆˆ‡…–•‘ˆ’ƒ”‡–ƒŽ‡š’‘•—”‡–‘—‡š’‘•‡†‘ˆˆ•’”‹‰ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͸ͳ

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ͶǤ͵ǤʹǤ ‘š‹…”‡•’‘•‡ƒ…”‘••‰‡‡”ƒ–‹‘•ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͸͵

ͶǤ͵Ǥ͵Ǥ –‹‘š‹†ƒ–†‡ˆ‡•‡•ƒ†’”‘†—…–‹‘„›–Š‡‰•ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͸Ͷ ͷǤ ‹‹–ƒ–‹‘•‘ˆ–Š‡™‘”ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͸͸

͸Ǥ ‘…Ž—•‹‘ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͸ͺ

͹Ǥ ‡ˆ‡”‡…‡•ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͹Ͳ

””ƒ–ƒǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǥǤǤǤǤǤǤǤǤǤͺʹ

Ͷ

Acknowledgements

Š‡…—””‡–Š™‘”™ƒ•’”‹ƒ”‹Ž›ˆ—†‡†„›–Š‡‘ȋ‰”ƒ–—„‡”ʹͻͳͻͻȌƒ†

ȋ‰”ƒ–—„‡”ʹʹͳ͵ͻͳȌ’”‘Œ‡…–•ǡƒ•™‡ŽŽƒ•–Š‡‡–”‡‘ˆ˜‹”‘‡–ƒŽ

ƒ†‹‘ƒ…–‹˜‹–›ȋȌǡˆ—†‡†„›–Š‡‘”™‡‰‹ƒ‡•‡ƒ”…Š‘—…‹ŽȋʹͲͳ͵ǦʹͲʹ͵Ȍ–Š”‘—‰Š

‹–•‡–”‡•‘ˆš…‡ŽŽ‡…‡ȋ‘Ȍˆ—†‹‰•…Š‡‡ȋʹʹ͵ʹ͸ͺȀ ͷͲȌǤŽŽ‘ˆ–Š‡‡š’‡”‹‡–ƒŽ

™‘” ƒ† •ƒ’Ž‡ ƒƒŽ›•‹• ™ƒ• …‘†—…–‡† ƒ– –Š‡ •‘–‘’‡ Žƒ„‘”ƒ–‘”› ƒ– –Š‡ ‘”™‡‰‹ƒ

‹˜‡”•‹–›‘ˆ‹ˆ‡…‹‡…‡•Ǥ

™‘—Ž† Ž‹‡ –‘ ‰‹˜‡ •’‡…‹ƒŽ –Šƒ• –‘ › •—’‡”˜‹•‘”•ǡ ”‘ˆǤ ‡„‘”ƒŠ —‰Š–‘ ȋǡ

ƒ‹•—’‡”˜‹•‘”Ȍǡ”Ǥƒ‰†‡”•”‡†‡ȋǡ…‘Ǧ•—’‡”˜‹•‘”Ȍǡƒ†”ǤŽƒ‹”‡‘—–”‹•

ȋǡ …‘Ǧ•—’‡”˜‹•‘”Ȍǡ ˆ‘” ƒŽŽ –Š‡ •…‹‡–‹ˆ‹… ƒ†˜‹•‡‡–ǡ •—’’‘”–ǡ ƒ† ‡…‘—”ƒ‰‡‡–

†—”‹‰–Š‡Žƒ•–Ͷ›‡ƒ”•Ǥƒ‰ǡ™‹–Š‘—–›‘—ǡ‘‡‘ˆ–Š‹•™‘—Ž†Šƒ˜‡„‡‡’‘••‹„Ž‡Ǥ–”—Ž›

ƒ’’”‡…‹ƒ–‡ –Šƒ– ›‘— ƒŽ™ƒ›• –‘‘ –Š‡ –‹‡ –‘ †‹•…—•• ȋƒ† ”‡Ǧ†‹•…—••Ȍ › ”‡•‡ƒ”…ŠǤ ‘

Žƒ‹”‡ǡ–Šƒ›‘—ˆ‘”–Š‡ˆ”‹‡†Ž›ƒ••‹•–ƒ…‡ƒŽŽ–Š‡•‡›‡ƒ”•ǡƒ†•‡‡‹‰›™‘”–Š”‘—‰Š

–‘–Š‡‡†Ǥ†ǡŽƒ•–Ž›ǡƒŠ…ƒ‘–„‡†‘‡™‹–Š‘—–ƒƒ‹•—’‡”˜‹•‘”Ǥ‡„‘”ƒŠǡƒŽŽ–Š‡

‹†™‘”†•–Š”‘—‰Š‘—––Š‡Š™‡”‡ƒŽ™ƒ›•Š‹‰ŠŽ›ƒ’’”‡…‹ƒ–‡†ȋƒ†‘ˆ–‡‡‡†‡†ȌǨ …‘—Ž†‘–Šƒ˜‡ƒ•‡†ˆ‘”ƒ‘”‡…ƒ”‹‰ǡ’ƒ–‹‡–ǡƒ†•—’’‘”–‹˜‡ƒ‹•—’‡”˜‹•‘”Ǥ

Š”‘—‰Š‘—– –Š‹• Šǡ Šƒ† –Š‡ ’Ž‡ƒ•—”‡ –‘ ™‘” ™‹–Š ƒŽŽ ‘ˆ –Š‡ Ž‘˜‡Ž› •–ƒˆˆ ˆ”‘ –Š‡

•‘–‘’‡Žƒ„‘”ƒ–‘”›ǢŠƒ›‘—–‘›‘—ƒŽŽǨ††‹–‹‘ƒŽŽ›ǡƒŽŽ›‰”ƒ–‹–—†‡–‘ƒǡ‡˜‰‡‹›ƒǡ

”‹…ƒǡ•–‡Žƒǡ ”Þ›†‹•ǡŠƒ‡ǡ‡„ƒ†—‡–‹ǡƒƒDzŠ‡ƒ‰dzǡˆ‘”ƒŽŽ–Š‡Žƒ—‰Š–‡”ǡ•—’’‘”–

ƒ† —ˆ‘”‰‡––ƒ„Ž‡ –‹‡•Ǥ ’‡…‹ƒŽ –Šƒ• –‘ ‡”‡–Š‡ǡ Ž˜ƒ ƒ† ”‹…ƒǡ ˆ‘” Š‡Ž’‹‰ ‡

–Š”‘—‰Š•‘‡‘ˆ–Š‡Šƒ”†‡•–„‹–•Ǥ’‡…‹ƒŽ–Šƒ•–‘ƒˆ‘”–Š‡‡˜‡”Ǧ‡†‹‰’ƒ–‹‡…‡ǡŠ‡Ž’

™‹–Š‰Ž‹•Šǡƒ†ƒŽ™ƒ›•ˆ‹†‹‰ƒ™ƒ›–‘ƒ‡‡•‹Ž‡Ǥ

Ž•‘ǡ ƒ› –Šƒ• –‘ › …‘Ǧƒ—–Š‘”•ǡ ƒ† ‘Ž† ƒ† ‡™ …‘ŽŽ‡ƒ‰—‡•ǡ ‹ ’ƒ”–‹…—Žƒ” ƒ„Ž‘ǡ

‡‡ǡ‡–ǡ‹‡ǡ‡‡ǡ—Ž‹ƒǡ‹”‹ƒǡƒ”‹–ǡƒ”Ž†”‡ƒ•ǡ‹‰‡ǡ‡‡ƒ†‹Ž†‡Ǥ’‡…‹ƒŽ

–Šƒ• –‘ ƒ‰ ƒ”—•ǡ ˆ‘” ƒŽŽ –Š‡ ’ƒ–‹‡…‡ ƒ† –Š‡ Ž‘‰ǡ •‘‡–‹‡• •‡‡‹‰Ž› ‡˜‡”Ǧ

‡†‹‰ǡ†ƒ›•ǡ™‘”‹‰‘›†ƒ–ƒǤ

Šƒ ›‘— –‘ › ˆƒ‹Ž› ˆ‘” „‡Ž‹‡˜‹‰ ‹ ‡ ƒ† ƒŽ™ƒ›• ’”‘˜‹†‹‰ ‡ ™‹–Š ƒŽŽ –Š‡

‘’’‘”–—‹–‹‡•–‘ˆ‘ŽŽ‘™›†”‡ƒ•Ǥ

‹•ƒ‘••„ƒ…Šǡ%•ǡʹͲͳͻ

ͷ

SUMMARY

•—„•–ƒ–‹ƒŽ‹…”‡ƒ•‡‹ƒ‘ƒ–‡”‹ƒŽ’”‘†—…–‹‘ƒ†ƒ”‹•‡‹‘˜‡Žƒ’’Ž‹…ƒ–‹‘•ƒ”‡

’”‡†‹…–‡†‹–Š‡…‘‹‰›‡ƒ”•ǤŠ‡”‡ƒ”‡…Ž‡ƒ”Ž›ƒ—„‡”‘ˆ’‘•‹–‹˜‡ƒ•’‡…–•‘ˆƒ”ƒ‰‡

‘ˆ†‹ˆˆ‡”‡–Dzƒ‘Ǧƒ’’Ž‹…ƒ–‹‘•dzǤ‘™‡˜‡”ǡ–Š‡‹‡˜‹–ƒ„Ž‡”‡Ž‡ƒ•‡‹–‘–Š‡‡˜‹”‘‡–‘ˆ

ƒ‘ƒ–‡”‹ƒŽ•ˆ”‘•—…Šƒ’’Ž‹…ƒ–‹‘•ƒŽ•‘…ƒ””‹‡•ƒ’‘–‡–‹ƒŽ”‹•Ǥ‹Ž˜‡”ƒ‘’ƒ”–‹…Ž‡•

ȋ‰•Ȍƒ”‡–Š‡‘•–…‘‘Ž›—•‡†ƒ‘’ƒ”–‹…Ž‡•–‘†ƒ–‡ǡ™Š‡”‡–Š‡‹”ƒ–‹„ƒ…–‡”‹ƒŽ

’”‘’‡”–‹‡• ƒ”‡ —•‡† –‘ ‡Šƒ…‡ ƒ —„‡” ‘ˆ …‘‡”…‹ƒŽ ’”‘†—…–•ǡ •—…Š ƒ• ‹ ™‘—†

†”‡••‹‰•ǡ…‘•‡–‹…•ǡ–‡š–‹Ž‡•ǡƒ†ˆ‘‘†’ƒ…ƒ‰‹‰ǤŠ‡Š‹‰Š†‡‰”‡‡‘ˆŽ‡ƒ…Š‹‰‘ˆ–Š‡‰

• ˆ”‘ …‘•—‡” ’”‘†—…–• ”‡•—Ž–• ‹ ƒ……——Žƒ–‹‘• ‹ Žƒ†ˆ‹ŽŽ• ƒ† –Š‡ –‡””‡•–”‹ƒŽ

‡˜‹”‘‡–Ǥ‡•’‹–‡„‡‹‰ƒ‘‰•––Š‡‘•–‡š–‡•‹˜‡Ž›•–—†‹‡†ƒ‘ƒ–‡”‹ƒŽ–‘†ƒ–‡ǡ

–Š‡”‡‹••–‹ŽŽƒ…‡”–ƒ‹ƒ‘—–‘ˆ…‘–”‘˜‡”•›ƒ„‘—––Š‡‹”–‘š‹…‹–›Ǥ—Ž–‹–—†‡‘ˆ•–—†‹‡•

ƒ•…”‹„‡†–‘š‹…‡ˆˆ‡…–•–‘‹‘‹…”‡Ž‡ƒ•‡•ǡ™Š‹Ž‡‘–Š‡”•–—†‹‡•Šƒ˜‡‹†‡–‹ˆ‹‡†’ƒ”–‹…Ž‡•’‡…‹ˆ‹…

‡ˆˆ‡…–•Ǥ —”–Š‡”‘”‡ǡ ”‡•—Ž–• ˆ”‘ ‰ –‘š‹…‹–› –‡•–• ƒ”‡ Šƒ”† –‘ …‘’ƒ”‡ǡ †—‡ –‘

†‹ˆˆ‡”‡…‡• ‹ ‡š’‘•—”‡ ‡†‹ƒǡ ‘”‰ƒ‹••ǡ ’ƒ”–‹…Ž‡ …Šƒ”ƒ…–‡”‹•–‹…• ƒ† –Š‡ ‡†’‘‹–•

•–—†‹‡†Ǥƒ•–Ž›ǡ–‘š‹…‹–›•–—†‹‡•’”‹ƒ”‹Ž›ˆ‘…—•‘–Š‡‡š’‘•—”‡‘ˆƒ•’‡…‹ˆ‹…Ž‹ˆ‡•–ƒ‰‡‘”

ƒ•‹‰Ž‡‰‡‡”ƒ–‹‘‘ˆƒ‘”‰ƒ‹•ǤŠ‡Žƒ…‘ˆ—Ž–‹‰‡‡”ƒ–‹‘ƒŽ•–—†‹‡•…‘—Ž†‡ƒƒ

Žƒ”‰‡—…‡”–ƒ‹–›ƒ„‘—–Ž‘‰Ǧ–‡”‡ˆˆ‡…–•Ǥ

Š‹•Š”‡•‡ƒ”…Š’”‘Œ‡…–™ƒ•ƒ‹‡†ƒ–—†‡”•–ƒ†‹‰†‹ˆˆ‡”‡–ƒ•’‡…–•‘ˆ–Š‡–‘š‹…‹–›‘ˆ

–Š‡ ”‡ˆ‡”‡…‡ ‰ ͵ͲͲ –‘™ƒ”†• –Š‡ ‡ƒ–‘†‡ Caenorhabditis elegansǡ ‹

…‘’ƒ”‹•‘–‘‰͵Ǥ‘ƒ††”‡••–Š‹•ƒ‹ǡƒ”ƒ‰‡‘ˆ•‹‰Ž‡ƒ†—Ž–‹‰‡‡”ƒ–‹‘ƒŽ•–—†‹‡•

™‡”‡•‡–—’—•‹‰C. elegans ƒ•ƒ‘†‡Ž‘”‰ƒ‹•Ǥ–—†‹‡•™‡”‡†‡•‹‰‡†–‘–‡•––Š”‡‡

Š›’‘–Š‡•‡•ǣ

1. NM300K Ag NPs would be toxic to C. elegans, but the relatively low dissolution of these NPs would make these less toxic than AgNO3.

2. Multigenerational exposure will lead to an adaptation towards Ag NPs at lower concentrations, but an increase in sensitization at higher concentrations, across generations.

3. The production of reactive oxygen species (ROS) involved in the toxic mechanisms of Ag NPs, would trigger antioxidant defenses following exposure, and changes in toxic responses over generations could be related to these defense mechanisms.

͸

–ƒ†ƒ”† –‘š‹…‹–› –‡•–• ™‡”‡ …ƒ””‹‡† ‘—– –‘ –‡•– ˆ‘” –‘š‹… ”‡•’‘•‡ǡ ™Š‹Ž‡ –Š‡ …Š”‘‹…

—Ž–‹‰‡‡”ƒ–‹‘ƒŽ‡š’‘•—”‡•™‡”‡…ƒ””‹‡†‘—–‘ƒ‰ƒ”’Žƒ–‡•Ǥ ‘”ƒŽŽ‡š’‘•—”‡•‰͵

™ƒ•—•‡†ƒ•ƒ’‘•‹–‹˜‡…‘–”‘ŽǤ

‘”‰͵ƒ†‰•‡š’‘•—”‡•ǡ•‹œ‡ˆ”ƒ…–‹‘ƒ–‹‘‡ƒ•—”‡‡–••Š‘™‡†•—„•–ƒ–‹ƒŽ

…Šƒ‰‡•‹‰•’‡…‹ƒ–‹‘‘˜‡”–‹‡ǡ…Šƒ”ƒ…–‡”‹œ‡†„›ƒ‹…”‡ƒ•‡‹Žƒ”‰‡”ƒ‰‰”‡‰ƒ–‡•ǡ …‘—’Ž‡†™‹–Šƒ†‡…”‡ƒ•‡‹–Š‡Ž‘™‘Ž‡…—Žƒ”ƒ••ȋȌ‰ˆ”ƒ…–‹‘ȋδ͵ƒȌǤ–Š‡

‰͵‡š’‘•—”‡•–Š‡‰ˆ”ƒ…–‹‘™ƒ•ͲǦͷͶΨ‘ˆ–‘–ƒŽ‰…‘…‡–”ƒ–‹‘ƒ––‹‡œ‡”‘

ȋǦͲȌǡ„—–‘Ž›ͲǦͲǤͲ͸Ψƒ–ͻ͸ŠǤ‘’ƒ”ƒ–‹˜‡Ž›ǡ‹–Š‡‰‡š’‘•—”‡ǡƒ”‡†—…–‹‘ˆ”‘

̱ͳ͸Ψƒ–ǦͲǡ–‘ͲǤͲͷΨƒ–ͻ͸Š™ƒ•‘„•‡”˜‡†ǤŠ‹•…‘ˆ‹”‡†ƒŽ‘™†‹••‘Ž—–‹‘‘ˆ‰•

ƒ† ƒ Š‹‰Š‡” ‹‹–‹ƒŽ …‘…‡–”ƒ–‹‘ ‹ ‡š’‘•—”‡ ‡†‹ƒ ˆ‘” ‰͵Ǥ —”–Š‡”‘”‡ǡ

”‡•—Ž–••—‰‰‡•–ƒŠ‹‰Š‹–‡”ƒ…–‹‘‘ˆ‰™‹–ŠEscherichia coliǡ–Š‡‡ƒ–‘†‡•ǯˆ‘‘†•‘—”…‡ǡ

™Š‹…Šǡ‹–—”ǡˆƒ…‹Ž‹–ƒ–‡–Š‡†‹‡–ƒ”›‰—’–ƒ‡„›C. elegansǤ

 –Š‡ •‹‰Ž‡ ‰‡‡”ƒ–‹‘ ‡š’‘•—”‡•ǡ ƒ …‘…‡–”ƒ–‹‘Ǧ†‡’‡†‡– †‡…”‡ƒ•‡ ‹ ‡ƒ–‘†‡

”‡’”‘†—…–‹‘ǡˆ‡”–‹Ž‹–›ǡƒ†‰”‘™–Š™ƒ•‡ƒ•—”‡†ˆ‘”„‘–Š‰ƒ†‰͵Ǥ‘™‡˜‡”ǡ

–Š‡…‘…‡–”ƒ–‹‘•‡…‡••ƒ”›–‘ƒ…Š‹‡˜‡ƒ…‘’ƒ”ƒ„Ž‡†‘•‡Ǧ”‡•’‘•‡™‡”‡͹ȂͳͲ–‹‡•

Š‹‰Š‡”ˆ‘”–Š‡•…‘’ƒ”‡†–‘‰͵ǤŠ‹•‹•ˆ—”–Š‡”•—’’‘”–‡†„›ͷͲ‡•–‹ƒ–‹‘•ǡˆ‘”

™Š‹…Š‰•Š‘™‡†͵Ǧ͹–‹‡•Š‹‰Š‡”Ž‡˜‡Ž•ˆ‘”‰”‘™–Šǡͺˆ‘Ž†Š‹‰Š‡”ˆ‘”ˆ‡”–‹Ž‹–›ǡƒ†

ʹȂͻˆ‘Ž†Š‹‰Š‡”ˆ‘””‡’”‘†—…–‹‘ǡƒ•…‘’ƒ”‡†–‘‰͵Ǥ‰—’–ƒ‡„›–Š‡‡ƒ–‘†‡•

™ƒ• …‘’ƒ”ƒ„Ž‡ „‡–™‡‡ –Š‡ –™‘ ˆ‘”• ‘ˆ ‰Ǥ ‘™‡˜‡”ǡ ˆ‘ŽŽ‘™‹‰ †‡’—”ƒ–‹‘

ƒ’’”‘š‹ƒ–‡Ž›ʹˆ‘Ž†Š‹‰Š‡”…‘…‡–”ƒ–‹‘•‘ˆ‰͵™‡”‡”‡–ƒ‹‡†„›‡ƒ–‘†‡•Ǥ

 –Š‡ —Ž–‹‰‡‡”ƒ–‹‘ƒŽǡ …Š”‘‹… ‡š’‘•—”‡ǡ ‡ƒ–‘†‡• ™‡”‡ …‘–‹—‘—•Ž› ‡š’‘•‡† –‘

–Š”‡‡…‘…‡–”ƒ–‹‘•‘ˆ‡‹–Š‡”‰͵ȋͲǤͲͳǡͲǤͲͷ‘”ͲǤͳ‰‰^ǦͳȌ‘”‰•ȋͲǤͳǡͲǤͷ‘”ͳ

‰‰^ǦͳȌǡ’”‹‘”–‘‡š’‘•‹‰†‹ˆˆ‡”‡–‰‡‡”ƒ–‹‘•–‘Š‹‰Š‡”…‘…‡–”ƒ–‹‘•ȋ‘ˆ‰͵‘”

‰•Ȍ‹•–ƒ†ƒ”†–‘š‹…‹–›–‡•–•ǤŠ‡…‘–‹—‘—•…Š”‘‹…‡š’‘•—”‡–‘ͳ‰‰^Ǧͳ‰•

”‡•—Ž–‡†‹ƒƒ†ƒ’–‹˜‡”‡•’‘•‡ǡƒ•‡ƒ•—”‡†„›ƒ‹…”‡ƒ•‡‹”‡’”‘†—…–‹‘…‘’ƒ”‡†

–‘…‘–”‘Ž•ǡ–‘‰•Ǥ‘™‡˜‡”ǡ–Š‡ƒ†ƒ’–ƒ–‹‘…ƒ‡ƒ–ƒ…‘•–‘ˆ”‡†—…‡†‰”‘™–ŠǤŠ‡•‡

‡ƒ–‘†‡•™‡”‡’”‘†—…‹‰‘ˆˆ•’”‹‰ƒ–ƒ–‘–ƒŽ„‘†›Ž‡‰–Š‘ˆͲǤͺȂͳǡ™Š‹Ž‡…‘–”‘Ž

ƒ† ‰͵ ‡š’‘•‡† ‡ƒ–‘†‡• ‘Ž› ’”‘†—…‡† ‘ˆˆ•’”‹‰ ƒ– εͳǤ —”–Š‡”‘”‡ǡ –Š‡

…‘–‹—‘—• ‡š’‘•—”‡ –‘™ƒ”†• ‰ • Ž‡† –‘ ƒ ‹…”‡ƒ•‡† •‡•‹–‹˜‹–› –‘™ƒ”†• ‰͵Ǥ

‘’ƒ”ƒ–‹˜‡Ž›ǡ –Š‡ —Ž–‹‰‡‡”ƒ–‹‘ƒŽ ‰͵ ‡š’‘•—”‡ ”‡•—Ž–‡† ‹ ‘ …Šƒ‰‡ ‹ –‘š‹…

”‡•’‘•‡–‘™ƒ”†•‰͵ǡ„—–ƒ†‡…”‡ƒ•‡†•‡•‹–‹˜‹–›–‘™ƒ”†•‰•Ǥƒ•–Ž›ǡƒ†‡…”‡ƒ•‡†

•‡•‹–‹˜‹–› –‘™ƒ”†• –Š‡ ‘™ ‹†—…‡” ’ƒ”ƒ“—ƒ– ™ƒ• ‡ƒ•—”‡† ˆ‘ŽŽ‘™‹‰ –Š‡ •‹š

͹

‰‡‡”ƒ–‹‘ƒŽ‡š’‘•—”‡–‘‰•ǡŠ‹–‹‰ƒ–…Šƒ‰‡•‹–Š‡‹˜‘Ž˜‡‡–‘ˆ–Š‡•—’‡”‘š‹†‡

ȋȌƒ–‹‘š‹†ƒ–†‡ˆ‡•‡•›•–‡‹–Š‡‘„•‡”˜‡†ƒ†ƒ’–‹˜‡”‡•’‘•‡Ǥ

–—†‹‡•—•‹‰ˆŽ—‘”‡•…‡–Ž›Žƒ„‡ŽŽ‡†C. elegans ”‡’‘”–‡”ƒ†„‹‘•‡•‘”•–”ƒ‹••Š‘™‡†–Šƒ–

–Š‡‡š’‘•—”‡–‘„‘–Šˆ‘”•‘ˆ‰”‡•—Ž–‡†‹ƒ‰…‘…‡–”ƒ–‹‘Ǧ†‡’‡†‡–‹…”‡ƒ•‡‹

ƒ–‹‘š‹†ƒ–†‡ˆ‡•‡•ǡƒ•™‡ŽŽƒ•ƒ‹…”‡ƒ•‡‹’‡”‘š‹†‡Ž‡˜‡Ž•ƒ†…Šƒ‰‡•‹–Š‡…‡ŽŽ—Žƒ”

”‡†‘š•–ƒ–—•‹–Š‡‡ƒ–‘†‡•Ǥ —”–Š‡”‘”‡ǡˆ‹†‹‰••Š‘™‡††‹•–‹…–†‹ˆˆ‡”‡…‡•‹–Š‡

„‹‘†‹•–”‹„—–‹‘‘ˆ‰ǤŠƒ‰‡•‹…‡ŽŽ—Žƒ””‡†‘š•–ƒ–—•‹–Š‡Ž—‹ƒŽ…‡ŽŽ•ǡ•—‰‰‡•–‰•

™‡”‡’”‹ƒ”‹Ž›…‘–ƒ‹‡†™‹–Š‹–Š‡‹–‡•–‹‡Ǥ…‘’ƒ”‹•‘ǡ‰͵ƒ…–‡†‘”‡‡˜‡Ž›

ƒ…”‘••–Š‡™Š‘Ž‡„‘†›Ǥ…”‘••‰‡‡”ƒ–‹‘•ǡŠ‘™‡˜‡”ǡƒ‹…”‡ƒ•‡‹sod-1 ‡š’”‡••‹‘‹

–Š‡ ͵ ‰‡‡”ƒ–‹‘• •—‰‰‡•–• –Š‡ ‹˜‘Ž˜‡‡– ‘ˆ ƒ–‹‘š‹†ƒ– †‡ˆ‡•‡• ‹ –Š‡ ƒ†ƒ’–‹˜‡

”‡•’‘•‡Ǥ‡˜‡”–Š‡Ž‡••ǡƒ†‡…”‡ƒ•‡‹sod-1ǡ…‘„‹‡†™‹–Šƒ•‹—Ž–ƒ‡‘—•‹…”‡ƒ•‡‹

‘š‹†ƒ–‹˜‡ •–”‡•• †‡˜‡Ž‘’‡– ‹ –Š‡ ͸ ‰‡‡”ƒ–‹‘•ǡ •—‰‰‡•– –Šƒ– –Š‡ ƒ‹–‡ƒ…‡ ‘ˆ

”‡’”‘†—…–‹˜‡…ƒ’ƒ…‹–›‹•–Š‡‘”‡„‡‡ˆ‹…‹ƒŽƒ†ƒ’–‹˜‡”‡•’‘•‡‘ˆ‡ƒ–‘†‡•ǡ…‘’ƒ”‡†

–‘‘š‹†ƒ–‹˜‡•–”‡••”‡•’‘•‡•Ǥ

‘…‘…Ž—†‡ǡ„‘–Šˆ‘”•‘ˆ‰•Š‘™‡†–”ƒ•ˆ‘”ƒ–‹‘•‘˜‡”–‹‡‹–Š‡‡š’‘•—”‡‡†‹ƒ

™‹–Šƒ†‡…”‡ƒ•‡‹–Š‡‰ˆ”ƒ…–‹‘ǡ‹…‘Œ—…–‹‘™‹–Šƒ‹…”‡ƒ•‡‹–Š‡ƒ‰‰”‡‰ƒ–‡†

ˆ”ƒ…–‹‘ǤŠ‹•Š‹‰ŠŽ‹‰Š–•–Š‡‹’‘”–ƒ…‡‘ˆ‘‹–‘”‹‰–Š‡‰•’‡…‹ƒ–‹‘–Š”‘—‰Š‘—––Š‡

‡š’‘•—”‡’‡”‹‘†Ǥ‹ˆˆ‡”‡…‡•‹–‘š‹…‹–›„‡–™‡‡‰͵ƒ†‰•…‘—Ž†„‡”‡Žƒ–‡†–‘

†‹ˆˆ‡”‡…‡• ‹ –Š‡ ‹‹–‹ƒŽ ˆ”ƒ…–‹‘• ƒ• ™‡ŽŽ ƒ• …Ž‡ƒ” †‹ˆˆ‡”‡…‡• ‹ „‹‘†‹•–”‹„—–‹‘Ǥ

‘”‡‘˜‡”ǡŽ‘™†‹••‘Ž—–‹‘‘ˆ–Š‡‰•ƒ›’”‡˜‡––Š‡‹…‘”’‘”ƒ–‹‘‘ˆ‰ˆ”‘–Š‡‰

•‹–‘…‡ŽŽ—Žƒ”…‘’‘‡–•Ǥ‘’ƒ”‹‰–Š‡–™‘ˆ‘”•‘ˆ‰•—‰‰‡•–•–Šƒ–‰•Ž‡ƒ†

–‘‹…”‡ƒ•‡•‹’”‘†—…–‹‘–Šƒ–ǡ‹–—”ǡ…‘—Ž†Ž‡ƒ†–‘–Š‡‘„•‡”˜‡†–‘š‹…‡ˆˆ‡…–•ǡƒ•

‡ƒ•—”‡† „› †‡…”‡ƒ•‡• ‹ ”‡’”‘†—…–‹‘ǡ ˆ‡”–‹Ž‹–› ƒ† ‰”‘™–ŠǤ ††‹–‹‘ƒŽŽ›ǡ ‹– ™ƒ•

…‘…Ž—†‡†–Šƒ–‰ˆ”‘‰͵‡š’‘•—”‡‹•‘”‡”‡ƒ†‹Ž›‹…‘”’‘”ƒ–‡†‹–‘…‡ŽŽ•ǡŽ‡ƒ†‹‰

–‘–Š‡‹–”ƒ…‡ŽŽ—Žƒ”’”‘†—…–‹‘‘ˆƒ…”‘••–Š‡™Š‘Ž‡„‘†›‘ˆ–Š‡‡ƒ–‘†‡•Ǥ˜‡”ƒŽŽǡ

–Š‡”‡•—Ž–••—‰‰‡•–‡†–Šƒ–‰͵Šƒ†ƒ†‹ˆˆ‡”‡––‘š‹…‘†‡‘ˆƒ…–‹‘–‘–Šƒ–‘ˆ‰•Ǥ —”–Š‡”‘”‡ǡ†ƒ–ƒ•—‰‰‡•–‡†–Šƒ–C. elegans ™ƒ•ƒ„Ž‡–‘†‡˜‡Ž‘’ƒƒ†ƒ’–‹˜‡”‡•’‘•‡

–‘™ƒ”†•–Š‡‡š’‘•—”‡‘ˆ‰•ǤŠ‹•”‡•’‘•‡…ƒ‡™‹–Š–Š‡ƒ••‘…‹ƒ–‡†…‘•–‘ˆ”‡†—…‡†

‰”‘™–Šǡ ‹…”‡ƒ•‡† •‡•‹–‹˜‹–› –‘™ƒ”†• ‰͵ ƒ† ‡^͵Ϊǡ ƒ† ”‡†—…‡† ‘š‹†ƒ–‹˜‡ •–”‡••

†‡ˆ‡•‡Ǥ

ͺ

SAMMENDRAG

‡§”‡•–‡¤”‡‡ˆ‘”˜‡–‡•‡„‡–›†‡Ž‹‰Þ‹‰‹’”‘†—•Œ‘ƒ˜ƒ‘’ƒ”–‹Ž‡”ǡ‘‰‡

”‡‡›‡„”—•‘”¤†‡”ˆ‘”†‹••‡Ǥ‡–‡”ƒ‰‡’‘•‹–‹˜‡‡ˆˆ‡–‡”›––‡––‹Ž„”—ƒ˜—Ž‹‡

ƒ‘ƒ–‡”‹ƒŽ‡”Ǥ ‡– ‡” ‹‹†Ž‡”–‹† ——‰¤‡Ž‹‰ ƒ– „”—‡ ˜‹Ž ‡†ˆÞ”‡ —–•Ž‹’’ ƒ˜

ƒ‘ƒ–‡”‹ƒŽ‡”‘‰†‡”‡†—–‰ŒÞ”‡‡”‹•‹‘ˆ‘”‹ŽŒÞ‡–Ǥގ˜ƒ‘’ƒ”–‹Ž‡”ȋ‰Ȍ‡”’‡”

¤ „Žƒ– †‡ ‡•– ‘‡”•‹‡Ž– ƒ˜‡†–‡ ƒ‘’ƒ”–‹Ž‡‡Ǥ ‡”‡• ƒ–‹„ƒ–‡”‹‡ŽŽ‡

‡‰‡•ƒ’‡”„”—‡•–‹Ž¤ˆ‘”„‡†”‡’”‘†—–‡‰‡•ƒ’‡”‹•¤”„ƒ†ƒ•Œ‡”ǡ‘•‡–‹ǡ–‡•–‹Ž‡”

‘‰ƒ–‡„ƒŽŽƒ•Œ‡Ǥ–Ž‡‹‰ƒ˜‰ˆ”ƒ•Ž‹‡’”‘†—–‡”Šƒ”ˆÞ”––‹Žƒ——Ž‡”‹‰ƒ˜•ÞŽ˜

‹ ƒ˜ˆƒŽŽ•†‡’‘‹‡” ‘‰ –‡””‡•–”‹•‡ ‹ŽŒÞ‡”Ǥ ‡Ž˜ ‘ •ÞŽ˜ƒ‘’ƒ”–‹Ž‡” ‡” „Žƒ– †‡ „‡•–

˜‹–‡•ƒ’‡Ž‹‰•–—†‡”–‡ƒ‘ƒ–‡”‹ƒŽ‡”•‘ˆ‹‡•ǡ‡”†‡–Ž‹‡˜‡Ž›––‡–•–‘”—•‹‡”Š‡–

‘‰ƒ‰‡Ž’¤‘•‡•—•˜‡†”Þ”‡†‡–‘•‹•‡‡ˆˆ‡–‡”ƒ˜‰Ǥ”‡‡•–—†‹‡”›––‡”

–‘•‹•‹–‡–‡–‹ŽŽ‡ƒ•Œ‡ƒ˜‹‘‡”ǡ‡•ƒ†”‡ˆ‘”•ÞŠƒ”˜‹•–’ƒ”–‹‡Ž•’‡•‹ˆ‹‡‡ˆˆ‡–‡”Ǥ

‡– Šƒ” ‘‰•¤ ˜‹•– •‡‰ ˜ƒ•‡Ž‹‰ ¤ •ƒ‡Ž‹‰‡ •Ž‹‡ •–—†‹‡”ǡ ’¤ ‰”— ƒ˜ —Ž‹Š‡–‡” ‹

ˆ‘”•Þ•‘’’•‡––‡‡ ”‡Žƒ–‡”– –‹Ž ‡•’‘‡”‹‰•‡†‹—ǡ –‡•–‘”‰ƒ‹•‡”ǡ

’ƒ”–‹‡Ž‡‰‡•ƒ’‡” ‘‰ Š˜‹Ž‡ ‡ˆˆ‡–’ƒ”ƒ‡–‡”‡ •‘ ‡” ¤Ž–Ǥ ƒŒ‘”‹–‡–‡ ƒ˜

‡•’‘‡”‹‰••–—†‹‡‡ Šƒ” ˜§”– ˆ‘—•‡”– ’¤ ‡ ‡‡Ž– ‰‡‡”ƒ•Œ‘ ‡ŽŽ‡” ‡– •’‡•‹ˆ‹–

Ž‹˜••–ƒ†‹— Š‘• –‡•–‘”‰ƒ‹•‡Ǥ ‡– ‡” †‡”ˆ‘” •–‘” ƒ‰‡Ž ’¤ †ƒ–ƒ ˆ”ƒ

—Ž–‹‰‡‡”ƒ•Œ‘••–—†‹‡”ǡ ‘‰ †‡”‡† Ž‹–‡ —•ƒ’ ‘ ’‘–‡•‹‡ŽŽ‡ ‡ˆˆ‡–‡” ƒ˜

Žƒ‰–‹†•‡•’‘‡”‹‰Ǥ

¤Ž•‡––‹‰‡ ˆ‘” †‡––‡ †‘–‘”‰”ƒ†•ƒ”„‡‹†‡– Šƒ” ˜§”– ¤ —†‡”•Þ‡ ‘‰ ˆ‘”•–¤ •‡–”ƒŽ‡

ƒ•’‡–‡”˜‡†–‘•‹•‹–‡–ƒ˜”‡ˆ‡”ƒ•‡ƒ‘’ƒ”–‹‡Žƒ–‡”‹ƒŽ‡‰͵ͲͲ•ƒ‡Ž‹‰‡–

‡†•ÞŽ˜‹–”ƒ–ȋ‰͵Ȍ˜‡†„”—ƒ˜Caenorhabditis elegans•‘–‡•–‘”‰ƒ‹•‡Ǥ–—†‹‡

Šƒ” „‡•–¤–– ƒ˜ ‡ ”‡‡ ‡‡Ž–‰‡‡”ƒ•Œ‘•Ǧ ‘‰ —Ž–‹‰‡‡”ƒ•Œ‘•‡•’‘‡”‹‰‡” ‡†C.

elegansǤ

•’‡”‹‡–‡‡„Ž‡†‡•‹‰‡––‹Ž¤–‡•–‡–”‡Š›’‘–‡•‡”ǣ

1. NM300K Ag NP er toksiske for C. elegans, men toksisiteten er lavere enn for AgNO3 fordi ioner det i relativt liten grad frigjøres ioner fra partiklene.

2. Eksponering over flere generasjoner vil føre til en adapsjon til lave konsentrasjoner av Ag NP, mens høye konsentrasjoner vil medføre økt sensitivitet.

ͻ

3. Reaktive oksygenforbindelser (ROS) generert som resultat av Ag NP eksponering, vil aktivere antioksidantsystemer, og vil over generasjoner medføre endringer i toksiske responser relatert til forsvarsmekanismer mot oksidativt stress.

C. elegans „Ž‡ ‡•’‘‡”– ˜‡† „”— ƒ˜ •–ƒ†ƒ”† –‘•‹•‹–‡–•–‡•–‡”‡ŽŽ‡”’¤ ƒ‰ƒ”’Žƒ–‡”ˆ‘”

”‘‹•—Ž–‹‰‡‡”ƒ•Œ‘•‡•’‘‡”‹‰Ǥގ˜‹–”ƒ–ȋ‰͵Ȍ„Ž‡„”—–•‘’‘•‹–‹˜‘–”‘ŽŽ

˜‡†ƒŽŽ‡‡•’‘‡”‹‰•ˆ‘”•Þ‡‡Ǥ–Þ””‡Ž•‡•ˆ”ƒ•Œ‘‡”‹‰‡‡†”‡–•‡‰‘˜‡”–‹†ˆ‘”„¤†‡

‰͵‘‰‰Ǥ‡•—Ž–ƒ–‡‡˜‹•–‡„‡–›†‡Ž‹‰†ƒ‡Ž•‡ƒ˜•–Þ””‡ƒ‰‰”‡‰ƒ–‡”ǡ‘„Ž‡–‡†

‡ ”‡†—•Œ‘ ‹ Žƒ˜‘Ž‡›Ž§”– ȋȌ ‰ ȋδ͵ ƒȌǤ ‡† •–ƒ”– ȋǦͲȌ ƒ˜ ‰͵

‡•’‘‡”‹‰‡‡—–‰Œ‘”†‡ͲǦͷͶΨƒ˜–‘–ƒŽ‡‰†‡‰ǡ‡•˜‡†‡•’‘‡”‹‰••Ž—––

ȋǦͻ͸Ȍ ˜ƒ” ƒ†‡Ž‡ ”‡†—•‡”– –‹Ž Ͳ Ȃ ͲǤͲ͸ ΨǤ ‹Ž •ƒ‡Ž‹‰‹‰ ˜‹•–‡ ‰

‡•’‘‡”‹‰‡ ‡ ”‡†—•Œ‘ ˆ”ƒ ͳ͸ Ψ ˜‡† ǦͲǡ –‹Ž ͲǤͲͷ Ψ ȋǦͻ͸ȌǤ ‡––‡ „‡”‡ˆ–‡– Žƒ˜

ˆ”‹‰ŒÞ”‹‰ ƒ˜ •ÞŽ˜‹‘‡” ˆ”ƒ ͵ͲͲ ’ƒ”–‹Ž‡‡ ‘‰ •‘ ˆ‘”˜‡–‡– ‡ „‡–›†‡Ž‹‰ ŠÞ›‡”‡

‹‹–‹ƒŽˆ”ƒ•Œ‘‹‡•’‘‡”‹‰•‡†‹‡–ˆ”ƒ‰͵‡•’‘‡”‹‰Ǥ‡•—Ž–ƒ–‡‡˜‹•‡”‡

ŠÞ›‹–‡”ƒ•Œ‘‡ŽŽ‘•ÞŽ˜‘‰‡ƒ–‘†‡‡•§”‹‰•‹Ž†‡ȋEscherichia coliȌǤ‡––‡ƒ

ˆ‘”Žƒ”‡Š˜‘”ˆ‘”‰‹‰͵‡•’‘‡”‹‰‡‡‹•¤’ƒ••„‡‰”‡•‡–‰”ƒ†ˆ‘”‡Ž‹‰‰‡”•‘ˆ”‹‡

‹‘‡”ǡ‘‰„‹†”ƒ”–‹ŽÞ–‘’’–ƒ˜‹ƒ†‹‡––Ǥ

•’‘‡”‹‰‘˜‡”‡‡‡Ž–‰‡‡”ƒ•Œ‘˜‹•–‡†‘•‡ƒ˜Š‡‰‹‰‡”‡†—•Œ‘‡”‹‡ƒ–‘†‡‡•

”‡’”‘†—•Œ‘ǡˆ‡”–‹Ž‹–‡–‘‰˜‡•–‹”‡•’‘•–‹Ž„¤†‡‰‘‰‰͵Ǥ‡–˜ƒ”‡•‹‰‹ˆ‹ƒ–

ˆ‘”•Œ‡ŽŽ‹–‘•‹•‹–‡–ǡ‡––‡”•‘†‡–”‡˜†‡•͹ǦͳͲ‰ƒ‰‡”ŠÞ›‡”‡†‘•‡ƒ˜‰ˆ‘”¤ˆ¤

•ƒ‡Ž‹‰„ƒ”‡ˆˆ‡–‡†‰͵Ǥ‡––‡—†‡”•–Þ––‡•ƒ˜‡•–‹‡”–‡ͷͲ‘•‡–”ƒ•Œ‘‡”

•‘ ˜‹•‡” ͵Ǧ͹ ‰ƒ‰‡”ˆ‘”•Œ‡ŽŽ ˆ‘” ˜‡•–ǡ ͺ ‰ƒ‰‡” ˆ‘”•Œ‡ŽŽ ˆ‘” ˆ‡”–‹Ž‹–‡–ǡ ‘‰ ʹǦͻ ‰ƒ‰‡”

ˆ‘”•Œ‡ŽŽ‹”‡’”‘†—•Œ‘‡ŽŽ‘‰‘‰‰͵ǤƒŽ›•‡”ƒ˜‡•’‘‡”–‡‡ƒ–‘†‡”˜‹•–‡

”‡Žƒ–‹˜–Ž‹–‘’’–ƒˆ”ƒ„‡‰‰‡–›’‡”•ÞŽ˜ǡ‡•ƒƒŽ›•‡”‡––‡”†‡’—”‡”‹‰˜‹•–‡…ƒǤʹ‰ƒ‰‡”

ŠÞ›‡”‡”‡–‡•Œ‘ƒ˜•ÞŽ˜Š‘•‰͵‡•’‘‡”–‡‡ƒ–‘†‡”Ǥ

‘” —Ž–‹‰‡‡”ƒ•Œ‘•–—†‹‡ „Ž‡ ‡ƒ–‘†‡‡ ‘–‹—‡”Ž‹‰ ȋ”‘‹•Ȍ ‡•’‘‡”– ˆ‘” –”‡

—Ž‹‡‘•‡–”ƒ•Œ‘‡”ƒ˜‡–‡‰͵ȋͲǤͲͳǡͲǤͲͷ‡ŽŽ‡”ͲǤͳ‰^ǦͳȌ‡ŽŽ‡”‰ȋͲǤͳǡͲǤͷ

‡ŽŽ‡”ͳǤͲ‰^ǦͳȌǤˆˆ‡–‡”ƒ˜‡•’‘‡”‹‰‡„Ž‡¤Ž–ˆ‘”Š˜‡”‰‡‡”ƒ•Œ‘˜‡†„”—ƒ˜‡

•–ƒ†ƒ”†–‘•‹•‹–‡–•–‡•–Ǥ‘–‹—‡”Ž‹‰ȋ”‘‹•Ȍ‡•’‘‡”‹‰˜‡†ͳǤͲ‰^Ǧͳ”‡•—Ž–‡”–‡‹‡

ƒ†ƒ’–‹˜–‘Ž‡”ƒ•‡”‡•’‘•ǡ˜‹•–˜‡†Þ–”‡’”‘†—•Œ‘‹§”˜§”ƒ˜‰•ƒ‡Ž‹‰‡–

‡† ‘–”‘ŽŽȀƒ†”‡ „‡Šƒ†Ž‹‰‡”Ǥ ‡ ލ–‡ ”‡’”‘†—•Œ•ƒ’ƒ•‹–‡–‡ ‡†ˆÞ”–‡ ‡

‘•–ƒ† ‹ ˆ‘” ƒ˜ ”‡†—•‡”– ˜‡•–Ǥ ‹••‡ ‡ƒ–‘†‡‡ ’”‘†—•‡”–‡ ƒ˜‘ ˜‡† ‡ –‘–ƒŽ

ͳͲ

”‘’’•Ž‡‰†‡’¤ͲǤͺǦͳǡ‡•‘–”‘ŽŽ‘‰‰͵Ǧ‡•’‘‡”–‡‡ƒ–‘†‡””‡’”‘†—•‡”–‡

ƒ˜‘ — ¤” ”‘’’•Ž‡‰†‡ ˜ƒ”εͳ Ǥ –‹ŽŽ‡‰‰ ˜‹•–‡ †‡– •‡‰ ƒ–‰ ‡•’‘‡”–‡

‡ƒ–‘†‡”„Ž‡‡”•‡•‹–‹˜‡ˆ‘”‰͵Ǥ‹Ž•ƒ‡Ž‹‰‹‰ˆÞ”–‡—Ž–‹‰‡‡”ƒ•Œ‘‰͵Ǧ

‡•’‘‡”‹‰ ‹‡ –‹Ž ‘‡ ‡†”‹‰ ‹ ”‡•’‘• ‘– ‰͵ǡ ‡ ‡ ”‡†—•‡”– •‡•‹–‹˜‹–‡–

‘˜‡ˆ‘” ‰ Ǥ ‡ƒ–‘†‡” ‡•’‘‡”– ˆ‘” ‰ ‘˜‡” •‡• ‰‡‡”ƒ•Œ‘‡” ˜‹•–‡ ‘‰•¤

”‡†—•‡”–•‡•‹–‹˜‹–‡–ˆ‘”ƒ”ƒ“—ƒ–•‘‘ˆ–‡„”—‡•ˆ‘”¤‹†—•‡”‡Ǥ‡––‡ƒ–›†‡”‡

ƒ†ƒ’•Œ‘”‡Žƒ–‡”––‹Žƒ–‹‘•‹†‡”‹‰•ˆ‘”•˜ƒ”•‡ƒ‹•‡”‹Ž—†‡”–•—’‡”‘•‹††‹•—–ƒ•‡

ȋȌǤ

‡†„”—ƒ˜ˆŽ—‘”‡•…‡•‡”‡†‡C. elegans”‡’‘”–‡”Ǧ‘‰„‹‘•‡•‘”•–ƒ‡”ǡ„Ž‡†‡–˜‹•–

ƒ–„‡‰‰‡–›’‡”•ÞŽ˜‹†—•‡”–‡‘•‡–”ƒ•Œ‘•ƒ˜Š‡‰‹‰Þ‹‰‹—––”›ƒ˜sod-1‰‡‡–ǡލ–

‹–”ƒ…‡ŽŽ—Ž§” ’‡”‘•‹†‘•‡–”ƒ•Œ‘ ‘‰ ‡†”‹‰‡”‹ …‡ŽŽ‡‡• ”‡†‘••–ƒ–—•Ǥ ‡•—Ž–ƒ–‡‡

˜‹•‡”†‹•–‹–‡ˆ‘”•Œ‡ŽŽ‡””‡Žƒ–‡”––‹Ž„‹‘†‹•–”‹„—•Œ‘ƒ˜‰Ǥ†”‹‰‡‡‹”‡†‘•Ǧ•–ƒ–—•‹

‹–‡•–‹ƒŽ‡ …‡ŽŽ‡” –›†‡” ’¤ ƒ– ‰ ’”‹§”– ‹–‡”ƒ‰‡”‡” ‡† –ƒ”‡’‹–‡Ž‡–Ǥ

•ƒ‡Ž‹‰‹‰ ’¤˜‹”‡– ‰͵ …‡ŽŽ‡” ‘‰ ˜‡˜ ‹ Š‡Ž‡ ”‘’’‡Ǥ —Ž–‹‰‡‡”ƒ•Œ‘•–—†‹‡

˜‹•–‡Þ–sod-1‰‡—––”›‹ ͵‰‡‡”ƒ•Œ‘‡ƒ–›†‡”‡’‘–‡•‹‡ŽŽ”‘ŽŽ‡ˆ‘”ƒ–‹‘•‹†ƒ–

ˆ‘”•˜ƒ”•‡ƒ‹•‡”‹ƒ†ƒ’•Œ‘’¤†‡––‡•–ƒ†‹‡–‹‡•’‘‡”‹‰‡Ǥ‡‡‡†”‹‰‡˜ƒ”

‹‡˜ƒ”‹‰ǡ‘‰‹ ͸‰‡‡”ƒ•Œ‘‡˜ƒ”sod-1‰‡—––”›‡–‡†”‡‰—Ž‡”–—†‡”‘–”‘ŽŽ‹˜¤ǡ

•ƒ–‹†‹‰•‘‡ƒ–‘†‡‡˜‹•–‡Þ–‘•‹†ƒ–‹˜–•–”‡••Ǥ‡––‡–›†‡”’¤ƒ–‘’’”‡––Š‘Ž†‡Ž•‡

ƒ˜”‡’”‘†—•Œ‘••—•‡••‡”‡˜‹–‹‰‡”‡ƒ†ƒ’–‹˜”‡•’‘•ˆ‘”‡ƒ–‘†‡‡‡Þ–ˆ‘”•˜ƒ”

‘–‘•‹†ƒ–‹˜–•–”‡••Ǥ

‡–‘Ž—†‡”‡•‡†ƒ–„‡‰‰‡–›’‡”‰–”ƒ•ˆ‘”‡”‡•‹ŽÞ’‡–ƒ˜‡•’‘‡”‹‰•–‹†‡ǡ˜‡†

ƒ–†‡Žƒ˜‘Ž‡›Ž§”‡ˆ”ƒ•Œ‘‡”‡†—•‡”‡•‹–ƒ–‡††ƒ‡Ž•‡ƒ˜ƒ‰‰”‡‰ƒ–‡”ǡŠ˜‹Ž‡–

—†‡”•–”‡‡” ˜‹–‹‰Š‡–‡ ƒ˜ ‰ •’‡•‹‡”‹‰ ‰Œ‡‘ Š‡Ž‡ ‡•’‘‡”‹‰‡Ǥ ‘”•Œ‡ŽŽ‡ ‹

–‘•‹•‹–‡–‡ŽŽ‘‰͵‘‰‰ƒ˜§”‡›––‡––‹Ž†‡‹‹–‹ƒŽ‡ˆ”ƒ•Œ‘‡ǡ•ƒ–

ˆ‘”•Œ‡ŽŽ‡” ‹ „‹‘†‹•–”‹„—•Œ‘‡Ǥ ‡ Žƒ˜‡ ˆ”‹‰ŒÞ”‹‰‡ ƒ˜ ‹‘‡” ˆ”ƒ ‰ ˆ‘”Š‹†”‡”

–”ƒ•Ž‘ƒ•Œ‘ƒ˜•ÞŽ˜‹–”ƒ…‡ŽŽ—Ž§”–Ǥƒ‡Ž‹‰‹‰ƒ˜†‡–‘–›’‡‡•ÞŽ˜–›†‡”’¤ƒ–‰

‹†—•‡”‡” •‘ ˆÞ”‡” –‹Ž ‘„•‡”˜‡”–‡ –‘•‹•‡ ‡ˆˆ‡–‡” ‹Ž—†‡”– ”‡†—•‡”–

”‡’”‘†—•Œ‘ǡˆ‡”–‹Ž‹–‡–‘‰˜‡•–ǡ‡•‰͵‹‘”’‘”‡”‡•Ž‡––‡”‡‹…‡ŽŽ‡‡‘‰†‡”‡†

ƒ’¤˜‹”‡‡œ›‡”‘‰’”‘–‡‹‡”†‹”‡–‡ǡ‡†’¤ˆÞŽ‰‡†‡’”‘†—•Œ‘ǤƒŽ‡–•‡––

˜‹•‡””‡•—Ž–ƒ–‡‡ƒ–‰͵‘‰‰‹†—•‡”‡”–‘•‹•‹–‡–˜‡†—Ž‹‡˜‹”‹‰•‡ƒ‹•‡”Ǥ

–—†‹‡˜‹•‡”‘‰•¤ƒ–C. elegans‡”‹•–ƒ†–‹Ž¤—–˜‹Ž‡Þ––‘Ž‡”ƒ•‡‘–‰‡•’‘‡”‹‰

˜‹ƒ ƒ†ƒ’–‹˜‡ ’”‘•‡••‡”Ǥ ‡‡ ƒ†ƒ’–‹˜‡ ”‡•’‘•‡ ‡†ˆÞ”‡” ‡ ‘•–ƒ† ‹ ˆ‘” ƒ˜

ͳͳ

”‡†—•‡”–˜‡•–ǡލ–•‡•‹–‹˜‹–‡–‘–‰͵‘‰‡^͵Ϊ‘‰”‡†—•‡”–ƒ’ƒ•‹–‡–‘–‘•‹†ƒ–‹˜–

•–”‡••Ǥ

ͳʹ

List of Papers

Paper I. Characterizing the behavior, uptake and toxicity of NM300K silver nanoparticles in Caenorhabditis elegansǤȋŽ‡‹˜‡ǡǤǡ‘••„ƒ…ŠǡǤǤǡƒŽŽ‡‰‘Ǧ””‡ƒǡ ǤǤǡ ”‡†‡ǡ ǤǤǡ —‰Š–‘ǡ ǤǤǡ Ƭ ‘—–”‹•ǡ Ǥǡ ʹͲͳͺǤEnvironmental Toxicology and ChemistryǤ͵͹ȋ͹Ȍǣͳ͹ͻͻȂͳͺͳͲǡǣͳͲǤͳͲͲʹȀ‡–…ǤͶͳͶͶȌ

Paper II. In vivo assessment of silver nanoparticle induced reactive oxygen species reveals tissue specific effects on cellular redox status in the nematode Caenorhabditis elegans. ȋ—„‹––‡† –‘Environmental Science: NanoǤ ‘••„ƒ…Šǡ ǤǤǡ

—‰Š–‘ǡǤǤǡ‘—–”‹•ǡǤǡƬ”‡†‡ǡǤǤȌ

Paper III. Adaptive tolerance to a multigenerational silver nanoparticle (NM300K) exposure by the nematode Caenorhabditis elegans is associated with increased sensitivity to AgNO3.ȋ‘••„ƒ…ŠǡǤǤǡƒ”‡‘–‹ǡǤǡ‹†‡ǡǤǤǡ—‰Š–‘ǡǤǤƬ”‡†‡ǡ ǤǤǡʹͲͳͻǤNanotoxicologyͳͺǣͳȂͳ͸ǡǣͳͲǤͳͲͺͲȀͳ͹Ͷ͵ͷ͵ͻͲǤʹͲͳͺǤͳͷͷ͹ʹ͹ʹȌ

Paper IV. Effects on Caenorhabditis elegans antioxidant defense and reactive oxygen species (ROS) metabolism following multigenerational exposure to AgNO3

or NM300K Ag NPsȋƒ—•…”‹’–Ǧ‘••„ƒ…ŠǡǤǤǡ—‰Š–‘ǡǤǤǡƬ”‡†‡ǡǤǤȌ

ͳ͵

Abbreviations

‰ ‹Ž˜‡”

‰ ‹Ž˜‡”ƒ‘’ƒ”–‹…Ž‡•

ƒ ƒŽ–‘ǡƒ–‘‹…ƒ••—‹–

›ƒ‹…Ž‹‰Š–•…ƒ––‡”‹‰

š ˆˆ‡…–‹˜‡…‘…‡–”ƒ–‹‘š

”‡‡ˆŽ—‘”‡•…‡–’”‘–‡‹

Ž—–ƒ–Š‹‘‡

Ž—–ƒ–Š‹‘‡”‡†—…–ƒ•‡

Ž—–ƒ–Š‹‘‡†‹•—Žˆ‹†‡

Ž—–ƒ–Š‹‘‡•Ǧ–”ƒ•ˆ‡”ƒ•‡

Ž—–ƒ–Š‹‘‡’‡”‘š‹†ƒ•‡

Ǧ †—…–‹˜‡Ž›…‘—’Ž‡†’Žƒ•ƒƒ•••’‡…–”‘‡–”›

Ǧ †—…–‹˜‡Ž›…‘—’Ž‡†’Žƒ•ƒ‘’–‹…ƒŽ‡‹••‹‘•’‡…–”‘•…‘’›

‘™‘Ž‡…—Žƒ”ƒ••

‘™‡•–‘„•‡”˜‡†‡ˆˆ‡…–…‘…‡–”ƒ–‹‘

‘†‡”ƒ–‡Ž›Šƒ”†”‡…‘•–‹–—–‡†™ƒ–‡”

‘”™‡‰‹ƒ‹˜‡”•‹–›‘ˆ‹ˆ‡…‹‡…‡•

‘‘„•‡”˜‡†‡ˆˆ‡…–…‘…‡–”ƒ–‹‘

ƒ–—”ƒŽ‘”‰ƒ‹…ƒ––‡”

‘Ž›˜‹›Ž’›””‘Ž‹†‘‡

‡ƒ…–‹˜‡‘š›‰‡•’‡…‹‡•

—’‡”‘š‹†‡†‹•—–ƒ•‡

•’Ǧ ‹‰Ž‡’ƒ”–‹…Ž‡Ǧ

ͳͶ

”ƒ•‹••‹‘‡Ž‡…–”‘‹…”‘•…‘’›

‡ŽŽ‘™ˆŽ—‘”‡•…‡–’”‘–‡‹

ͳͷ

1. Introduction 1.1. Background

‘š‹…‘Ž‘‰›†‡ƒŽ•™‹–Š‡‰ƒ–‹˜‡‹’ƒ…–•‘ˆ…Š‡‹…ƒŽ•ƒ†–‘š‹…ƒ–•‘Š—ƒ•ǡ‘”‰ƒ‹••

ƒ†–Š‡‡˜‹”‘‡–ǡƒ†‹•‘ˆ–‡–”ƒ…‡†„ƒ…–‘ƒ”ƒ…‡Ž•—•Ǥƒ”ƒ…‡Ž•—•…‘‹‡†–Š‡’Š”ƒ•‡

DzSolely the dose determines that a thing is not a poisondz ȋ‘Žƒ †‘•‹• ˆƒ…‹– ˜‡‡—Ȍ

‹–”‘†—…‹‰–Š‡…‘…‡’–‘ˆ†‘•‡ƒ††‘•‡”‡•’‘•‡Ǥ‘…‡–”ƒ–‹‘ƒ†‡š’‘•—”‡–‹‡ƒ”‡

–™‘ƒŒ‘”ƒ•’‡…–•‘ˆ–‘š‹…‘Ž‘‰›ǡ„‘–Š‘ˆ™Š‹…Š†‡–‡”‹‡–Š‡‡ˆˆ‡…–•‘ˆ•‹‰Ž‡…Š‡‹…ƒŽ•ǡ

ƒ†–Š‡”‡„›…”‹–‡”‹ƒˆ‘”ƒ••‡••‹‰–Š‡”‹•‘ˆ•’‡…‹ˆ‹……Š‡‹…ƒŽ•ȋƒŽ‡”ǡʹͲͲ͸ȌǤ”‡…‡–

›‡ƒ”•ǡƒ‘–‘š‹…‘Ž‘‰›Šƒ•‡‡”‰‡†ƒ•ƒ•—„Ǧˆ‹‡Ž†‘ˆ–‘š‹…‘Ž‘‰›ǡ™Š‹…Šƒ‹•–‘‹†‡–‹ˆ›–Š‡

Ž‹ „‡–™‡‡ –Š‡ ƒ‘’ƒ”–‹…Ž‡ ’Š›•‹…‘…Š‡‹…ƒŽ …Šƒ”ƒ…–‡”‹•–‹…•ǡ •—…Š ƒ• •‹œ‡ǡ •—”ˆƒ…‡

’”‘’‡”–‹‡•ƒ†…Šƒ”‰‡ǡƒ†–Š‡‹”–‘š‹…‹–›ȋ‘ƒŽ†•‘ et al.ǡʹͲͲͶǡ‹ƒ‰ et al.ǡʹͲͲͻȌǤ

Š‡ –‡” Dzƒ‘dz ‘”‹‰‹ƒ–‡• ˆ”‘ –Š‡ ”‡‡ ™‘”† ˆ‘” Dz†™ƒ”ˆdzǤ ‹…Šƒ”† ‡›ƒ ‹• „›

ƒ›ƒ……Žƒ‹‡†ˆ‘”’”‡†‹…–‹‰–Š‡”‹•‡‘ˆ–Š‡‹‘˜ƒ–‹˜‡ˆ‹‡Ž†‘ˆƒ‘–‡…Š‘Ž‘‰›‹ƒ–ƒŽ

‰‹˜‡„ƒ…‹ͳͻͷͻDzThere is plenty of room at the bottom: An invitation to enter a new field of Physicsdz ȋŠƒ et al.ǡ ʹͲͳ͹Ȍǡ ”‡…‘‰‹œ‹‰ –Š‡ —‹“—‡ ’”‘’‡”–‹‡• ‘ˆ ƒ–‡”‹ƒŽ•

ƒ‹’—Žƒ–‡†ƒ––Š‡ƒ–‘‹…•…ƒŽ‡Ǥ‘†ƒ›ǡ‘†‡”ƒ‘–‡…Š‘Ž‘‰›‹•†‡ˆ‹‡†ƒ•Dzcreating products and applications based primarily upon the synthesis of molecules in the nanoscale (10^-9 m) size rangedzȋƒ”Š‡‹–ǡʹͲͳͺȌǤŠ‡…Šƒ‰‹‰’”‘’‡”–‹‡•‘ˆƒƒ–‡”‹ƒŽ™Š‡‹–••‹œ‡

”ƒ‰‡ ˆƒŽŽ• „‡Ž‘™ ͳͲͲ ǡ ƒ† Š‡…‡ –Š‡ ƒ‹’—Žƒ–‹‘ ‘ˆ ƒ–‡”‹ƒŽ ’”‘’‡”–‹‡•ǡ ƒ‡•

ƒ‘–‡…Š‘Ž‘‰›ƒ‹†—•–”‹ƒŽŽ›‹’‘”–ƒ–ˆ‹‡Ž†‘ˆ”‡•‡ƒ”…Šǡ™‹–Šƒ‹–‡”‡•–‹‰”ƒ‰‡‘ˆ

ƒ’’Ž‹…ƒ–‹‘•ƒ†—•‡•Ǥ

Ž–Š‘—‰Š ƒ‘ƒ–‡”‹ƒŽ• ‘……—” ƒ–—”ƒŽŽ›ǡ ‹…‹†‡–ƒŽ ƒ† ƒ—ˆƒ…–—”‡† ƒ‘ƒ–‡”‹ƒŽ•

ƒ––”ƒ…––Š‡ƒ‹ˆ‘…—•‹–‡”•‘ˆŠ‡ƒŽ–Šƒ†•ƒˆ‡–›‘ˆŠ—ƒ•ǡˆ‘”‹•–ƒ…‡‘……—’ƒ–‹‘ƒŽ

™‘”‡”•ƒ†…‘•—‡”•ǡƒ•™‡ŽŽƒ•ƒ†˜‡”•‡‡ˆˆ‡…–•‘–Š‡‡˜‹”‘‡–ȋ—– et al.ǡʹͲͳ͵ǡ

ƒœƒ”‡˜ƒƒ†‡ŽŽ‡”ǡʹͲͳͶǡƒ”Š‡‹–ǡʹͲͳͺȌǤƒ–—”ƒŽŽ›‘……—””‹‰ƒ‘ƒ–‡”‹ƒŽ•ǡ•—…Šƒ•

ˆ”‘˜‘Ž…ƒ‹…‡”—’–‹‘•ǡƒ†‹…‹†‡–ƒŽŽ›’”‘†—…‡†ǡ•—…Šƒ•„›Ǧ’”‘†—…–•‘ˆ…‘„—•–‹‘

’”‘…‡••‡•ǡƒ”‡‘ˆ–‡–‡”‡†—Ž–”ƒˆ‹‡’ƒ”–‹…Ž‡•ǡƒ†Šƒ˜‡ƒ–‡†‡…›–‘„‡Š‡–‡”‘‰‡‡‘—•

‹ –‡”• ‘ˆ –Š‡‹” ’Š›•‹…ƒŽ ƒ† …Š‡‹…ƒŽ ’”‘’‡”–‹‡•Ǥ  –Š‡ ‘–Š‡” Šƒ†ǡ ‡‰‹‡‡”‡†

ƒ‘ƒ–‡”‹ƒŽ•ƒ”‡†‡•‹‰‡†™‹–Š’Š›•‹…‘…Š‡‹…ƒŽ’”‘’‡”–‹‡•ˆ‘”ƒ•’‡…‹ˆ‹…ˆ—…–‹‘Ǥ

–Š‡‡ƒ”Ž›ʹͲͲͲǯ•ǡ‘”‡–Šƒ͵ͷ…‘—–”‹‡•‹‹–‹ƒ–‡†”‡•‡ƒ”…Š‹–‘ƒ‘•…ƒŽ‡’”‘†—…–‹‘

ȋ‘…‘ǡʹͲͲ͵ȌǤŠ‹•™ƒ•ˆ‘ŽŽ‘™‡†„›ƒ•–‡ƒ†›‹…”‡ƒ•‡‹ƒ‘ƒ–‡”‹ƒŽ’”‘†—…–‹‘ǡ™‹–Š

ͳ͸

ƒ”‘—† ͶͲͲͲ ”‡‰‹•–‡”‡† ƒ‘ƒ–‡”‹ƒŽ• Ž‹•–‡† ‹ –Š‡ ƒ‘™‡” †ƒ–ƒ„ƒ•‡ –‘ †ƒ–‡

ȋƒ‘™‡”ǡ ʹͲͳͻȌǤ  ʹͲͲͷ –Š‡ –‘–ƒŽ ‰Ž‘„ƒŽ ‹˜‡•–‡– ‹ ƒ‘–‡…Š‘Ž‘‰‹‡• ™ƒ•

ƒ’’”‘š‹ƒ–‡Ž›̈́ͳͲ„‹ŽŽ‹‘‰Ž‘„ƒŽŽ›ǡƒ†‡š’‡…–‡†–‘‹…”‡ƒ•‡–‘ͳ–”‹ŽŽ‹‘„›ʹͲͳͳǦʹͲͳͷǤ

ʹͲͲͶǡƒ”‘—†ͳͲ^͵–‘•™ƒ•’”‘†—…‡†ƒ—ƒŽŽ›ǡ™‹–Šƒˆ—”–Š‡”’”‡†‹…–‡†‹…”‡ƒ•‡–‘

ͳͲ^ͶȂͳͲ^ͷ–‘•ƒ—ƒŽŽ›„›ʹͲͳͲȋ…‹‡…‡‘Ž‹…›‡…–‹‘ǡʹͲͲͶǡƒ””‹•‘ǡʹͲͲ͹ǡƒ˜ƒ””‘ et al.ǡʹͲͲͺƒȌǤ”‘†—…–‹‘‘ˆƒ‘ƒ–‡”‹ƒŽ•‹•‡•–‹ƒ–‡†–‘–”‹’Ž‡„›ʹͲʹͲǡ™Š‡”‡‡–ƒŽ‘š‹†‡

ƒ‘’ƒ”–‹…Ž‡’”‘†—…–‹‘ƒŽ‘‡‹•‡•–‹ƒ–‡†–‘‹…”‡ƒ•‡–‘—’–‘ͳǤ͸‹ŽŽ‹‘–‘•ƒ—ƒŽŽ›

„›ʹͲʹͲȋ ‘”•–‡” et al.ǡʹͲͳͳǡ‹……‹‘ et al.ǡʹͲͳʹǡ —–—”‡ƒ”‡–•ǡʹͲͳ͹ǡ— et al.ǡʹͲͳ͹ǡ

‡ƒ† et al.ǡ ʹͲͳͺȌǤ —‡ –‘ ˜ƒ•– †‹ˆˆ‡”‡…‡• ‹ –Š‡ ™ƒ› ƒ”‡– ”‡•‡ƒ”…Š “—ƒ–‹ˆ‹‡•

ƒ‘ƒ–‡”‹ƒŽ• ƒ’’Ž‹‡† „› ’”‘†—…‡”•ǡ —…‡”–ƒ‹–› „› ’”‘†—…‡”• ‘ –Š‡ ‡šƒ…– ƒ‘—–•

—•‡†ǡƒ•™‡ŽŽƒ•ƒŽƒ…‘ˆ…‘’ƒ”ƒ–‹˜‡Š‹•–‘”‹…ƒŽ†ƒ–ƒǡ–Š‡‡•–‹ƒ–‹‘‘ˆ‡šƒ…–“—ƒ–‹–‹‡•‘ˆ

ƒ‘ƒ–‡”‹ƒŽ•…—””‡–Ž›—•‡†‹’”‘†—…–‹‘‹•†‹ˆˆ‹…—Ž–ȋ‹……‹‘ et al.ǡʹͲͳʹǡ‹‡•‡ et al.ǡ ʹͲͳͺȌǤ

Š‡ ‹…”‡ƒ•‹‰ —•‡ ƒ† ƒ’’Ž‹…ƒ–‹‘ ƒ‡• ‹– ‡…‡••ƒ”› –‘ †‡ˆ‹‡ ƒ† …ƒ–‡‰‘”‹œ‡

ƒ‘ƒ–‡”‹ƒŽ•Ǥ‘˜‡”‡–ƒŽ‘”‰ƒ‹œƒ–‹‘•ƒ•™‡ŽŽƒ•‹†—•–”‹‡•Šƒ˜‡ƒ†‡‡ˆˆ‘”–•–‘

†‡ˆ‹‡ƒ‘ƒ–‡”‹ƒŽ•ǡ”‡•—Ž–‹‰‹ƒ™‹†‡”ƒ‰‡‘ˆƒ˜ƒ‹Žƒ„Ž‡†‡ˆ‹‹–‹‘•–‘†ƒ–‡ȋ‘˜‡”Š‘ˆ

et al.ǡ ʹͲͳͷȌǤ Š‡ –‡”ƒ–‹‘ƒŽ ”‰ƒ‹œƒ–‹‘ ˆ‘” –ƒ†ƒ”†‹œƒ–‹‘ ȋȌ †‡ˆ‹‡•

ƒ‘ƒ–‡”‹ƒŽ• ƒ• ƒ DzMaterial with any external dimension in the nanoscale or having internal structure or surface structure in the nanoscale”ǡ™‹–Šƒƒ††‹–‹‘ƒŽ†‡ˆ‹‹–‹‘‘ˆ

Dzƒ‘•…ƒŽ‡dzŽ›‹‰‹–Š‡•‹œ‡”ƒ‰‡‘ˆͳȂͳͲͲȋǡʹͲͳͷȌǤŠ‡—”‘’‡ƒ‘‹••‹‘

‘–Š‡‘–Š‡”Šƒ†ǡŠƒ•‹…Ž—†‡†ƒ††‹–‹‘ƒŽˆƒ…–‘”•–‘–Š‡•‹œ‡Ž‹‹–ǡ‹–Š‡‹”†‡ˆ‹‹–‹‘ǣdzA natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50 % or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm – 100 nm. In specific cases and where warranted by concerns for the environment, health, safety or competitiveness the number size distribution threshold of 50 % may be replaced by a threshold between 1 % and 50 %” (European Commission, 2011). ‘‘–‘

ƒŽŽ†‡ˆ‹‹–‹‘•‹•–Šƒ–ǡˆ‘”ƒ’ƒ”–‹…Ž‡–‘„‡…‘•‹†‡”‡†Dzƒ‘dzǡ‘‡‘ˆ‹–•†‹‡•‹‘•—•–

„‡ „‡Ž‘™ ͳͲͲ ȋ‘˜‡”Š‘ˆ et al.ǡ ʹͲͳͷȌǤ ‘™‡˜‡”ǡ …”‹–‹…• ’‘‹– –‘ ‹…‘•‹•–‡…‹‡•

„‡–™‡‡ †‹ˆˆ‡”‡– †‡ˆ‹‹–‹‘•ǡ ƒ• ™‡ŽŽ ƒ• –Š‡ •–”‘‰ ˆ‘…—• ‘ –Š‡ •‹œ‡ Ž‹‹– ‘ˆ ͳͲͲǡ

™Š‹…ŠŠƒ•Ž‹––Ž‡–Š‡‘”‡–‹…ƒŽ‘”‡˜‹”‘‡–ƒŽ”‡Ž‡˜ƒ…‡ȋ‘˜‡”Š‘ˆ et al.ǡʹͲͳͷȌǤ–Šƒ•„‡‡

’”‘’‘•‡†–Šƒ–ǡˆ‘”†‡ˆ‹‹–‹‘•–‘„‡”‡Ž‡˜ƒ–ˆ‘””‹•ƒ••‡••‡–ǡˆƒ…–‘”•Ž‹‡†‹•–”‹„—–‹‘

ͳ͹

–Š”‡•Š‘Ž†ǡ•‹œ‡Ǧ†‡’‡†‡–’”‘’‡”–‹‡•ǡ–Š‡•–ƒ–‡‘ˆƒ‰‰Ž‘‡”ƒ–‹‘Ȁ†‡Ǧƒ‰‰Ž‘‡”ƒ–‹‘ƒ†

ƒ‰‰”‡‰ƒ–‹‘Ȁ†‹•ƒ‰‰”‡‰ƒ–‹‘•Š‘—Ž†„‡‹…Ž—†‡†ȋ‘˜‡”Š‘ˆ‡–ƒŽǤǡʹͲͳͷȌǤ

—‡–‘–Š‡‹”•ƒŽŽ•‹œ‡ǡ‡ƒ‹‰ƒŠ‹‰Š‡”•—”ˆƒ…‡–‘˜‘Ž—‡”ƒ–‹‘ǡƒ‘ƒ–‡”‹ƒŽ•Šƒ˜‡

‹…”‡ƒ•‡†”‡ƒ…–‹˜‹–›ƒ††‹ˆˆ‡”‡–•—”ˆƒ…‡…Š‡‹•–”›…‘’ƒ”‡†–‘–Š‡‹”Žƒ”‰‡”’ƒ”–‹…Ž‡•‘ˆ

–Š‡•ƒ‡ƒ–‡”‹ƒŽǡ’‘••‹„Ž›Ž‡ƒ†‹‰–‘ƒ†˜‡”•‡‡ˆˆ‡…–•‘Š—ƒ•ƒ†‘”‰ƒ‹••ȋ”‘™

et al.ǡ ʹͲͲͲȌǤ Š‡”‡ˆ‘”‡ǡ ”‡‰—Žƒ–‘”› ƒ‰‡…‹‡• ƒ”‡ …‘ˆ”‘–‡† ™‹–Š –Š‡ “—‡•–‹‘ ƒ„‘—–

™Š‡–Š‡”–Š‡ƒ‘’ƒ”–‹…—Žƒ–‡ˆ‘”‘ˆƒ…‘’‘—†‡‡†•–‘„‡ƒƒ‰‡††‹ˆˆ‡”‡–Ž›–Šƒ

‹‹–•„—Ž‘”†‹••‘Ž˜‡†’Šƒ•‡•ȋ—†Ǧ‹‡ et al.ǡʹͲͳ͸ȌǤ

1.2. Silver nanoparticles

‡…ƒ—•‡ ‘ˆ –Š‡‹” ƒ–‹„ƒ…–‡”‹ƒŽ ’”‘’‡”–‹‡•ǡ •‹Ž˜‡”ƒ‘’ƒ”–‹…Ž‡• ȋ‰ •Ȍ ƒ”‡ ƒ ˜‡”•ƒ–‹Ž‡

ƒ–‡”‹ƒŽǡ —•‡† ‹ ƒ ™‹†‡ ”ƒ‰‡ ‘ˆ ’”‘†—…–•ǡ ˆ‘” ‹•–ƒ…‡ ƒ• ™‘—† †”‡••‹‰ǡ ‹ •’‘”–•

‡“—‹’‡–ǡƒ•™‡ŽŽƒ•„ƒ„›’”‘†—…–•ȋ —‰ƒ†‘™‡ǡͳͻͻ͸ǡƒ” et al.ǡʹͲͲͻǡ‘™ƒ…ǡ ʹͲͳͲȌǤ‘•–‘ˆ–Š‡ƒ‘•‹Ž˜‡””‡Ž‡ƒ•‡†‹–‘–Š‡‡˜‹”‘‡–‘”‹‰‹ƒ–‡•’”‹ƒ”‹Ž›ˆ”‘

–‡š–‹Ž‡•ǡ™‹–Šƒ††‹–‹‘ƒŽ•‘—”…‡•ˆ”‘…Ž‡ƒ‹‰ƒ‰‡–•ǡ…‘•‡–‹…•ƒ†‡†‹…ƒŽ’”‘†—…–•

ȋ‹‡•‡ et al.ǡʹͲͳͺȌǤ‡˜‡”–Š‡Ž‡••ǡƒŽ–Š‘—‰Š‡˜‹”‘‡–ƒŽ”‡Ž‡ƒ•‡•‘ˆ‰•ǡˆ”‘ˆ‘”

‹•–ƒ…‡ –‡š–‹Ž‡• ȋڎ‡” et al.ǡ ʹͲͳͷȌǡ Šƒ• „‡‡ •Š‘™ ‡š’‡”‹‡–ƒŽŽ›ǡ †ƒ–ƒ ‘ ƒ…–—ƒŽ

‡˜‹”‘‡–ƒŽ”‡Ž‡ƒ•‡•…‡ƒ”‹‘•‹••…ƒ”…‡ȋ‘––•…ŠƒŽ et al.ǡʹͲͳ͵ȌǤ‹ˆˆ‹…—Ž–‹‡•ƒ”‹•‡ˆ”‘

ƒ Žƒ… ‘ˆ ƒ’’”‘’”‹ƒ–‡ ƒƒŽ›–‹…ƒŽ –‡…Š‹“—‡• ƒ„Ž‡ –‘ †‹•–‹‰—‹•Š ‰ • ˆ”‘ ƒ–—”ƒŽŽ›

‘……—””‹‰ ’ƒ”–‹…Ž‡• ƒ† …‘ŽŽ‘‹†•ǡ ƒ• ™‡ŽŽ ƒ• –Š‡ Ž‘™ …‘…‡–”ƒ–‹‘• ‹ ‡˜‹”‘‡–ƒŽ

‡†‹ƒǡŠ‹†‡”‹‰ƒ……—”ƒ–‡“—ƒ–‹ˆ‹…ƒ–‹‘ȋ— et al.ǡʹͲͳ͸ȌǤ

––‡’–•Šƒ˜‡„‡‡ƒ†‡–‘‡•–‹ƒ–‡ˆŽ—š‡•—•‹‰‘†‡ŽŽ‹‰–‘‘Ž•Ǥ‹‡•‡ et al.ȋʹͲͳͺȌ …ƒŽ…—Žƒ–‡† –Š‡ ƒ—ƒŽ ’”‘†—…–‹‘ ‘ˆ ‰ • –‘ „‡ δ ͳͲͲͲ –‘•Ǥ ‘†‡ŽŽ‡† ”‡Ž‡ƒ•‡ †ƒ–ƒ

’”‡†‹…–‡†‡˜‹”‘‡–ƒŽ…‘…‡–”ƒ–‹‘•‹ˆ”‡•Š™ƒ–‡”•›•–‡•‹–Š‡’‰–‘‰^Ǧͳ”ƒ‰‡ǡ

™‹–Šƒ”‘—‰ŠŽ›ʹǦ͸ˆ‘Ž†‡•–‹ƒ–‡†‹…”‡ƒ•‡„›ʹͲͷͲȋ‹‡•‡ et al.ǡʹͲͳͺȌǤ ‘”ƒ‰”‹…—Ž–—”ƒŽ

•‘‹Ž•ǡŠ‘™‡˜‡”ǡ‘†‡Ž‡†…‘…‡–”ƒ–‹‘•”ƒ‰‡„‡–™‡‡͵Ͳ’‰Ȁ‰ȋ‹‹—‹ʹͲͳ͹Ȍ—’

–‘ͳͲρ‰Ȁ‰•‘‹Žȋƒš‹—‹ʹͲͳͷȌȋ‹‡•‡ et al.ǡʹͲͳͺȌǤ‘™‡˜‡”ǡƒŽƒ”‰‡—…‡”–ƒ‹–›

™‹–Š‹–Š‡•‡‘†‡Ž‡†‡•–‹ƒ–‹‘•‹•ƒ…‘™Ž‡†‰‡†Ǥ— et al.ȋʹͲͳ͸Ȍ…ƒŽ…—Žƒ–‡†Š‹‰Š‡•–

”‡Ž‡ƒ•‡• ‘ˆ ‰ • ˆ”‘ ‡Ž‡…–”‘‹…• ƒ† ƒ’’Ž‹ƒ…‡• ™‹–Š ̱͵ͺΨ ‘ˆ –Š‡ –‘–ƒŽ ƒ‘ ‰

ƒ’’Ž‹…ƒ–‹‘ ƒ† ̱ʹͷΨ ˆ”‘ –‡š–‹Ž‡•ǡ …‘ŽŽ‡…–‹‰ ‹ Žƒ†ˆ‹ŽŽ• ƒ† •‡†‹‡–Ǥ ‘™‡˜‡”ǡ

‡•–‹ƒ–‡• ’”‡•‡–‡† •Š‘—Ž† „‡ …‘•‹†‡”‡† ™‹–Š …ƒ”‡ǡ ƒ• ‘†‡Ž• —•‡† †‘ ‘– …‘•‹†‡”

…Š‡‹…ƒŽ–”ƒ•ˆ‘”ƒ–‹‘•ƒ††‹••‘Ž—–‹‘‘ˆ–Š‡•ǡ™Š‹…Šƒ”‡‘ˆ’ƒ”–‹…—Žƒ”‹’‘”–ƒ…‡

‹–Š‡…ƒ•‡‘ˆ‰•ȋ— et al.ǡʹͲͳ͸ȌǤ

ͳͺ

1.3. Silver nanoparticle toxicity to biota

‡•’‹–‡ Š‹‰Š —…‡”–ƒ‹–‹‡• ƒ„‘—– ƒ…–—ƒŽ ‡˜‹”‘‡–ƒŽ …‘…‡–”ƒ–‹‘•ǡ ’”‡˜‹‘—•

‘™Ž‡†‰‡ ƒ„‘—– –Š‡ –‘š‹… ’”‘’‡”–‹‡• ‘ˆ –Š‡ ‹‘‹… ˆ‘”• ‘ˆ †‹ˆˆ‡”‡– ƒ–‡”‹ƒŽ•ǡ Ž‡† –‘

‹…”‡ƒ•‹‰…‘…‡”ƒ„‘—––Š‡’‘–‡–‹ƒŽƒ†˜‡”•‡‡ˆˆ‡…–•‘ˆƒ‘ƒ–‡”‹ƒŽ•ȋ„‡”†Ú”•–‡” et al.ǡ ʹͲͲͷǡ ‹……‹‘ et al.ǡ ʹͲͳʹȌǤ  ”‡•’‘•‡ǡ –Š‡ —„‡” ‘ˆ ’—„Ž‹•Š‡† •–—†‹‡• ‘

ƒ‘’ƒ”–‹…Ž‡–‘š‹…‹–›Šƒ•‹…”‡ƒ•‡†„›͸ͲͲΨ‘˜‡”–Š‡Žƒ•–†‡…ƒ†‡ȋƒœ“—‡œǦ—Ó‘œ et al.ǡ ʹͲͳ͹ȌǤŠ‡”‡ˆ‘”‡ǡ‘„–ƒ‹‹‰†‡–ƒ‹Ž‡†‘™Ž‡†‰‡ƒ„‘—––Š‡’‘–‡–‹ƒŽ–‘š‹…‡ˆˆ‡…–•ƒ†–‘š‹…

‡…Šƒ‹•• ‘ˆ •—…Š ‡š’‘•—”‡• ‹• ‘ˆ …Ž‡ƒ” ‹–‡”‡•–Ǥ ‡•’‹–‡ „‡‹‰ ‘‡ ‘ˆ –Š‡ ‘•–

‹–‡•‹˜‡Ž› •–—†‹‡† ƒ‘ƒ–‡”‹ƒŽ• –‘ †ƒ–‡ǡ …‘–”‘˜‡”•› •–‹ŽŽ ‡š‹•–• ƒ„‘—– –Š‡ –‘š‹…

’”‘’‡”–‹‡•‘ˆ‰•ǡ™‹–Š”‡•—Ž–•†‹ˆˆ‡”‹‰‘”‡˜‡…‘–”ƒ†‹…–‹‰‡ƒ…Š‘–Š‡”ȋƒœ“—‡œǦ

—Ó‘œ et al.ǡʹͲͳ͹ȌǤ‘™‡˜‡”ǡ–Š‡’‘–‡–‹ƒŽ‡˜‹”‘‡–ƒŽŠƒœƒ”†‘ˆ‰•‹•ƒ––”‹„—–‡†

–‘–Š‡™‡ŽŽǦ‘™ƒ–‹„ƒ…–‡”‹ƒŽ’”‘’‡”–‹‡•‘ˆ‰ǡ–‘‰‡–Š‡”™‹–Š–Š‡‹”ƒ„‹Ž‹–›–‘’”‘†—…‡

”‡ƒ…–‹˜‡ ‘š›‰‡ •’‡…‹‡• ȋȌ ‘ –Š‡ •—”ˆƒ…‡ ‘ˆ –Š‡ ’ƒ”–‹…Ž‡• ȋƒœ“—‡œǦ—Ó‘œ et al.ǡ ʹͲͳ͹ȌǤŠ‡ƒ–‹„ƒ…–‡”‹ƒŽƒ…–‹‘‘ˆ‰•Šƒ•„‡‡ƒ––”‹„—–‡†ǡƒŽ‘•–‡–‹”‡Ž›ǡ–‘‰^Ϊ

”‡Ž‡ƒ•‡• „› –Š‡ •ǡ ƒ• ™‡ŽŽ ƒ• –Š‡ ’”‘†—…–‹‘ ‘ˆ ”‡ƒ…–‹˜‡ ‘š›‰‡ •’‡…‹‡• „› –Š‡ •ǡ

ƒ‹‰ ˆƒ…–‘”• Ž‹‡ •Šƒ’‡ǡ …‘ƒ–‹‰ ‘” •‹œ‡ ‘ˆ –Š‡ • •‡…‘†ƒ”› ‹ –‡”• ‘ˆ –‘š‹…‹–›

–‘™ƒ”†•„ƒ…–‡”‹ƒȋ‹ et al.ǡʹͲͲ͹ǡ‹— et al.ǡʹͲͳͳǡ—”ž et al.ǡʹͲͳ͸ȌǤ‡˜‡”–Š‡Ž‡••ǡ™‹–Š

‹…”‡ƒ•‹‰…‘’Ž‡š‹–›‘ˆ‘”‰ƒ‹••ǡ–Š‡‡…Šƒ‹••‘ˆ–Š‡–‘š‹…‹–›‹•‘–ƒ•…Ž‡ƒ”…—–ǡ

™Š‡”‡‹‘–‘š‹…‹–›†‘‡•‘–•‘Ž‡Ž›‡š’Žƒ‹–‘š‹…”‡•’‘•‡ȋ‡ƒ† et al.ǡʹͲͳͺȌǤŠ‡”‡ˆ‘”‡ǡ

™Š‹Ž‡ ƒ› •–—†‹‡• …‘…Ž—†‡ –Šƒ– –‘š‹…‹–› …ƒ „‡ •‘Ž‡Ž› ƒ––”‹„—–‡† –‘ ‹‘‹… ”‡Ž‡ƒ•‡• ƒ

”ƒ‰‡‘ˆ•–—†‹‡•Šƒ˜‡ˆ‘—†’ƒ”–‹…Ž‡•’‡…‹ˆ‹…‡ˆˆ‡…–•ǡ‘”ƒ…‘„‹ƒ–‹‘‘ˆ–Š‡–™‘ȋƒ˜ƒ””‘

et al.ǡʹͲͲͺ„ǡ‹ et al.ǡʹͲͲͻǡ ƒ„”‡‰ƒ et al.ǡʹͲͳͳǡ‡‡” et al.ǡʹͲͳʹǡŠ‘‹ et al.ǡʹͲͳͺȌǤŠ‹•

ˆ—”–Š‡”Š‹‰ŠŽ‹‰Š–•–Š‡Š‹‰Š‹ˆŽ—‡…‡‘ˆ–Š‡’ƒ”–‹…Ž‡…Šƒ”ƒ…–‡”‹•–‹…•‘–‘š‹…‹–›Ǥ

˜ƒ‹Žƒ„Ž‡•–—†‹‡•ƒ”‡Šƒ”†–‘…‘’ƒ”‡†—‡–‘†‹ˆˆ‡”‡…‡•‹‡š’‘•—”‡‡†‹ƒǡ…Š‘‹…‡‘ˆ

‘”‰ƒ‹••ǡ –‡•– …‘†‹–‹‘•ǡ ƒ† †‹ˆˆ‡”‡– ‰ • ™‹–Š ˜ƒ”›‹‰ •Šƒ’‡ǡ •‹œ‡ǡ …Šƒ”‰‡ ‘”

…‘ƒ–‹‰ǡ ƒŽŽ …Šƒ”ƒ…–‡”‹•–‹…• –Šƒ– ‹’ƒ…– „‡Šƒ˜‹‘” ȋƒœ“—‡œǦ—Ó‘œ et al.ǡ ʹͲͳ͹ȌǤ

‡˜‡”–Š‡Ž‡••ǡ‰•ƒ’’‡ƒ”–‘„‡–‘š‹…–‘‘•–‘”‰ƒ‹••ǡ™‹–Šƒ‘ˆͷρ‰‰^Ǧͳ‹

–Š‡ ˆ”‡•Š™ƒ–‡” …ŽƒSphaerium corneumǡ ƒ ƒ…—–‡ ͷͲ ‘ˆ ͳʹͳρ‰‰^Ǧͳ ˆ‘”Daphnia magnaǡ ƒ† ͺǤͻͷ ƒ† ͳ͵Ǥͻρ‰‰^Ǧͳ ˆ‘”D. pulex and D. galeataǡ ”‡•’‡…–‹˜‡Ž› ȋ‹ et al.ǡ ʹͲͳͲƒǡڎ‡” et al.ǡʹͲͳ͵ǡŠ et al.ǡʹͲͳͶǡڎ‡” et al.ǡʹͲͳͷȌǤ‘™‡˜‡”ǡ‘•–•–—†‹‡•

”‡’‘”– Š‹‰Š‡” –‘š‹…‹–› ˆ”‘ ‰ ‹‘•ǡ ™‹–Š ƒ ͷͲ ˆ‘”D. pulex ‘ˆ ͲǤ͸ͺρ‰‰^Ǧͳǡ ƒ†

ʹǤͳ͵ρ‰‰^Ǧͳˆ‘”D. galeata ȋڎ‡”‡–ƒŽǤǡʹͲͳ͵)ǡƒ†–Š‡Ž‘™‡•–˜ƒŽ—‡•ˆ‘”ƒ’Š‹ƒ

ͳͻ

•’’‘ˆͲǤͲͲͳρ‰‰^Ǧͳȋ‹‡Ž›‡” et al.ǡʹͲͲʹȌǤڎ‡” et al.ȋʹͲͳ͵Ȍ”‡Žƒ–‡†‹ˆˆ‡”‡…‡•‹

–‘š‹…‹–› „ƒ… –‘ –Š‡ …‘ƒ–‹‰• ‘ˆ –Š‡ ’ƒ”–‹…Ž‡•ǡ ˆ‘” ‹•–ƒ…‡ ǡ ’”‡˜‡–‹‰ ’ƒ”–‹…Ž‡

†‹••‘Ž—–‹‘ǡ ƒ† Š‡…‡ ƒ Ž‘™‡” ‹‘‹… ˆ”ƒ…–‹‘ ‹ –Š‡ ‡š’‘•—”‡Ǥ ‘™‡˜‡”ǡ ˆ‘ŽŽ‘™‹‰ –Š‡

‹‰‡•–‹‘ǡ‰•ƒ›‡‹–Š‡”‡š‡”––Š‡‹”–‘š‹…‹–›†‹”‡…–Ž›‹–Š‡‹–‡•–‹‡ǡ‘”„‡–ƒ‡—’

‹–‘…‡ŽŽ•ƒ†–”ƒ•ˆ‡””‡†ƒ…”‘••†‹ˆˆ‡”‡––‹••—‡•Ǥ —”–Š‡”‘”‡ǡ‰•‘ˆ–‡•Š‘™Š‹‰Š

†‹••‘Ž—–‹‘ǡŽ‡ƒ†‹‰–‘Ž‘…ƒŽ‹œ‡†‹‘‹…”‡Ž‡ƒ•‡•ȋڎ‡” et al.ǡʹͲͳͷȌǤ

††‹–‹‘ƒŽ–‘š‹…‡…Šƒ‹••‘ˆ‰•…ƒ„‡”‡Žƒ–‡†–‘–Š‡‹”Š‹‰Šƒˆˆ‹‹–›–‘•—Žˆ—”Ǥ‰

ȋ‡‹–Š‡”‹‘‹…‘”’ƒ”–‹…—Žƒ–‡Ȍ…ƒŠƒ˜‡†‹”‡…–‹–‡”ƒ…–‹‘™‹–Šƒ…”‘‘Ž‡…—Ž‡•ǡ’‘••‹„Ž›

Ž‡ƒ†‹‰ –‘ …‘ˆ‘”ƒ–‹‘ƒŽ …Šƒ‰‡•ǡ ‹…Ž—†‹‰ ’”‘–‡‹ —ˆ‘Ž†‹‰ ‘” –Š‡ ƒ†•‘”’–‹‘ ‘ˆ

’”‘–‡‹•‘–‘–Š‡•—”ˆƒ…‡‘ˆ–Š‡’ƒ”–‹…Ž‡•ǡƒŽ•‘”‡ˆ‡””‡†–‘ƒ•–Š‡ˆ‘”ƒ–‹‘‘ˆƒ’”‘–‡‹

…‘”‘ƒȋŠ‘‹ et al.ǡʹͲͲͻǡ‹— et al.ǡʹͲͳͳǡƒ’–ƒ”•Š‹ et al.ǡʹͲͳ͵ȌǤŠƒ‰‡•‹–Š‡’”‘–‡‹

•–”—…–—”‡Šƒ˜‡–Š‡’‘–‡–‹ƒŽ–‘‹’ƒ…–†‘™•–”‡ƒ’”‘–‡‹Ǧ’”‘–‡‹‹–‡”ƒ…–‹‘•ǡ…‡ŽŽ—Žƒ”

•‹‰ƒŽ‹‰‘”–”ƒ•…”‹’–‹‘ȋƒ’–ƒ”•Š‹ et al.ǡʹͲͳ͵ȌǤ —”–Š‡”ǡ†‹”‡…–‹–‡”ƒ…–‹‘‘ˆ–Š‡

ƒ‘’ƒ”–‹…Ž‡• ™‹–Š ‡„”ƒ‡ ’”‘–‡‹• ƒ† ƒ…–‹˜‡ •‹‰ƒŽ‹‰ ’ƒ–Š™ƒ›• ƒ› Ž‡ƒ† –‘

‹Š‹„‹–‹‘ ‘ˆ …‡ŽŽ ’”‘Ž‹ˆ‡”ƒ–‹‘ ȋ•Šƒ”ƒ‹ et al.ǡ ʹͲͲͻǡ ‘’‹ƒ–Š et al.ǡ ʹͲͳͲǡ ‘Š et al.ǡ ʹͲͳʹȌǤ‰ƒ›ƒŽ•‘„‹†–‘‹”‘•—Žˆ—”…Ž—•–‡”•ƒ†‹Š‹„‹–‡œ›‡ƒ…–‹˜‹–‹‡•ǡ™‹–Š•‹Ž˜‡”

•—Žˆ‹†‡ ’”‡…‹’‹–ƒ–‹‘ǡ ”‡•—Ž–‹‰ ‹ •—……‹ƒ–‡ †‡Š›†”‘‰‡ƒ•‡ ‹Š‹„‹–‹‘ǡ …‘•‡“—‡–Ž›

Ž‡ƒ†‹‰–‘ˆ—”–Š‡”‡–ƒ„‘Ž‹…‹–‡”ˆ‡”‡…‡•ȋŠƒ†‘—” et al.ǡͳͻͺͺȌǤ‘”‡‘˜‡”ǡ–Š‡„‹†‹‰

‘ˆ‰–‘•—ŽˆŠ›†”›Žȋ–Š‹‘ŽȌ‰”‘—’•Šƒ•„‡‡•Š‘™–‘’”‘‘–‡‹”‘”‡Ž‡ƒ•‡•ǡŽ‡ƒ†‹‰–‘–Š‡

ˆ‘”ƒ–‹‘‘ˆŠ›†”‘š›Ž”ƒ†‹…ƒŽ•ȋ‘”†‘ et al.ǡʹͲͳͲȌǤ

1.4. Reactive oxygen species and oxidative stress

‘•–‘”‰ƒ‹••”‡“—‹”‡‘š›‰‡ȋʹȌ–‘Ž‹˜‡ǡ„—––Š‡’ƒ”–‹ƒŽ”‡†—…–‹‘‘ˆ–Š‡ʹƒ›Ž‡ƒ†

–‘ –Š‡ ˆ‘”ƒ–‹‘ ‘ˆ Ǥ  ‡—ƒ”›‘–‹… …‡ŽŽ•ǡ ƒ”‡ …‘–‹—‘—•Ž› ’”‘†—…‡† „› –Š‡

‹–‘…Š‘†”‹ƒȋ—”’Š›ǡʹͲͲͻǡ—•Š…ŠƒǡʹͲͳͳȌǤŠ‹•’”‘†—…–‹‘‘ˆ‹•…‘–‹—‘—•Ž›

…‘—–‡”„ƒŽƒ…‡†„›ƒˆ‹‡Ž›–—‡†ƒ–‹‘š‹†ƒ–†‡ˆ‡•‡•›•–‡ǡ‹‘”†‡”–‘ƒ˜‘‹†‘š‹†ƒ–‹˜‡

•–”‡••ȋ‹˜‹‰•–‘‡ǡʹͲͲ͵ǡ—”’Š›ǡʹͲͲͻȌǤš’‘•—”‡–‘ƒ™‹†‡”ƒ‰‡‘ˆ‡˜‹”‘‡–ƒŽ

•–”‡••‘”•ǡ ‹…Ž—†‹‰ ‰ • ȋ™ƒ‰ et al.ǡ ʹͲͲͺȌǡ ”ƒ†‹ƒ–‹‘ ‘” Š‡”„‹…‹†‡• ȋ•—…Š ƒ•

’ƒ”ƒ“—ƒ–Ȍȋ—–”‡•ǡʹͲͲʹȌǡƒ›’‘–‡–‹ƒŽŽ›‹†—…‡ƒƒ††‹–‹‘ƒŽ’”‘†—…–‹‘‘ˆǤ

‹„ƒŽƒ…‡„‡–™‡‡’”‘†—…–‹‘ƒ†‡—–”ƒŽ‹œƒ–‹‘”‡•—Ž–•‹ƒ‹„ƒŽƒ…‡‹–Š‡

”‡†‘š •–ƒ–—• ‘ˆ ƒ …‡ŽŽǤ ‡…‡ǡ ƒ †‹•–—”„ƒ…‡ ‹ –Š‡ ”‡†‘š Š‘‡‘•–ƒ•‹• ƒ› Ž‡ƒ† –‘ –Š‡

ˆ‘”ƒ–‹‘‘ˆ‘š‹†ƒ–‹˜‡†ƒƒ‰‡–‘„‹‘‘Ž‡…—Ž‡•ǡ‹…Ž—†‹‰’”‘–‡‹ǡŽ‹’‹†•ƒ†—…Ž‡‹…ƒ…‹†ǡ

ƒ•™‡ŽŽƒ•‹–‡”ˆ‡”‡…‡•‹…‡ŽŽ—Žƒ”•‹‰ƒŽ‹‰‡…Šƒ‹••ȋƒ› et al.ǡʹͲͳʹȌǤ