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WP1 Adsorption and equilibrium partition of persistent pollutants to microplastics WP2 Uptake and accumulation of microplastics and associated persistent pollutants WP5 Trophic transfer @EPHEMARE_ FACEBOOK.COM/EPHEMARE WWW.JPI-OCEANS.EU/EPHEMARE Fibres, after 2h Fibres, after 48h PVC, after 48h PE, after 48h MPs Diffusion in digestive tract? 0 0.002 0.004 0.006 0.008 0.01 average k efflux, delayed (min -1 ) MP, low [C] MP, high [C] MP + CPF, low [C] MP + CPF, high [C] * Developing an uptake/excretion model in the Mediterranean mussel (M. galloprovincialis) WP2 WP5 WP1 Nile Red dyeing as a novel approach to monitor microplastic ingestion in vivo Any microplastic particle, of any shape, can be dyed with Nile Red Transparent larvae (such as medaka or zebrafish) are exposed to the dyed particles Their ingestion can be monitored in vivo using a stereoscope with a fluorescent filter (such as the Texas Red): the particles will fluoresce even after ingestion + Zebrafish and medaka larvae (~0-2 days post hatching) were incubated with MPs (PE, PVC, and polyester fibres), for 2 to 48 hours Monitoring of ingested MPs was performed after incubation and 2 days after. After exposure larvae were transferred in clean water and left undisturbed for 4 additional days PVC: 2 out of 14 larvae were still positive PE: 17 out of 18 larvae were still positive Dye seemed to diffuse out of the polyester fibres, although this hypothesis was invalidated during successive tests. Mussels were exposed to microalgae (MA), polyethylene microplastic (MP), and pollutant-spiked MP (MP+CPF) diets, to study the amount of MPs ingested and excreted and develop a mathematical model. Initially, just two parameters were considered: k uptake and k efflux, delayed . Uptake Low [C] 0 0.005 0.01 0.015 0.02 0.025 0.03 average k uptake (min -1 ) MA MP MP + CPF 0 0.01 0.02 0.03 0.04 0.05 0.06 average k uptake (min -1 ) MA MP MP + CPF Nile Red dyeing protocol Zebrafish (D. rerio) and Medaka (O. latipes) exposed to Nile Red- dyed microplastics Delayed efflux Average k uptake for the three treatments, at two different concentrations Average k efflux, delayed for MP and MP + CPF treatments High [C] It appears exposure to MPs in mussels decreases uptake of food particles, and increases excretion, particularly with contaminated MPs. Adsorption and equilibrium partition of persistent pollutants to microplastics Model target contaminants were selected from a group of candidates initially proposed by all partners. The selection of the two target concentrations (high and low) for each compound was based on data found in the literature. Chemical pollutants selected for the EPHEMARE project are: perfluorooctane sulfonate (PFOS), benzo(a)pyrene (BaP), and oxybenzone (Benzophenone 3, BP3). Initially, cadmium was selected as representative heavy metal; however, pre-tests showed it would not be possible to adsorb cadmium on LDPE particles within a reasonable time. LDPE and PVC microplastic particles of different sizes have been prepared with the three different compounds in two concentrations and distributed to all partners in the project for ecotoxicological testing. perfluorooctane sulfonate benzo(a)pyrene oxybenzone high low high low high low LDPE 500-125 μm 27.1 - 56.5 μg/g 180.9 ng/g 14.1 μg/g 378.17 pg/g 115 ng/g 1.1 ng/g 25-20 μm 46.1 μg/g 61.2 ng/g 15.1 μg/g 384.19 pg/g 62 ng/g 0.15 ng/g 13-11 μm 70.2 μg/g 9.2 ng/g 16.9 μg/g 364.34 pg/g 82 ng/g < 0.05 ng/g 6-4 μm 55.7 μg/g 118.2 ng/g 16.6 μg/g 4990.84 pg/g 24 ng/g 0.14 ng/g PVC 250-100 μm 159.5 μg/g 1603.2 ng/g 11.5 μg/g 102.2 pg/g 66 ng/g 0.13 ng/g Longterm sorption experiment PFOS (water) = 600 mg/L, LDPE (water) = 10 g/L 7 30 90 180 1 10 100 1000 10000 100000 time [days] PFOS on LDPE [ g/g] The preparation of the MP particles showed that may be longer exposure time would change the sorption rate of the compounds to the particles. Therefore, 10 g/L LDPE MP particles (500-125 μm) has been exposed to either PFOS (600 mg/L) or BaP (5000 μg/L) for 180 days. Exposure vessels has been constantly shaken and samples has been taken at 7, 30, 90 and 180 days. The results for PFOS shows that until 90 days there is no change of the sorption rate; however, after 180 days the sorption rate of PFOS was more than 200 times higher than before. For BaP during the first 7 days the sorption was increasing but for the subsequent time points the BaP concentration adsorbed on the MP particles was constantly declining. Longterm sorption experiment BaP (water) = 5000 g/L, LDPE (water) = 10 g/L 0 7 30 90 180 0 100 200 300 time [days] BaP on LDPE [ g/g] MP uptake and transfer in simplified food chains: Establishment of food chains Analyses of uptake rates, transfer and accumulation pattern in all trophic organisms Histological analyses and fluorescence microscopy Transfer kinetics After successful establishment of food chain, feeding of POP-spiked MPs (self-spiked or from WP1) Aurelia aurita Adult zebrafish Medaka larvae Artemia salina spec. Fluorescent Microplastics (MPs) Neomysis integer Nereis diversicolor Seabass Persistent organic pollutant (POP) transfer via MP transfer in food chains Paramecium spec. Zebrafish larvae CYP1A induction in adult zebrafish liver (EROD assay) and zebrafish/medaka larvae (qPCR) Fluorescence tracking of benzo(a)pyrene POP transfer via MPs and trophic food chains shown successfully in different species! *** CYP1A induction in medaka larvae after trophic transfer of spiked MPs CYP1A induction in adult zebrafish liver after trophic transfer of spiked MPs Fluorescence tracking of BaP signal in Artemia spec. nauplii Fluorescence tracking of BaP in intestinal tract and liver of adult zebrafish * uptake Gill Gut Transfer across lumen? Tissues k uptake k release k storage k efflux, rapid k efflux, delayed Steffen Keiter (1), Bettie Cormier (1), Ronny Blust (2), Raewyn M. Town (2), Camilla C. Carteny (2), Xavier Cousin (3), Thomas Braunbeck (4), Annika Batel (4) (1) Örebro University (2) University of Antwerp (3) Ifremer (4) University of Heidelberg

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Page 1: time [days] - Home | JPI OCEANSjpi-oceans.eu/sites/jpi-oceans.eu/files/public... · transferred in clean water and left undisturbed for 4 additional days • PVC: 2 out of 14 larvae

WP1 Adsorption and equilibrium partition of persistent pollutants to microplastics

WP2 Uptake and accumulation of microplastics and associated persistent pollutants

WP5 Trophic transfer

@EPHEMARE_ FACEBOOK.COM/EPHEMAREWWW.JPI-OCEANS.EU/EPHEMARE

Fibres, after 2h

Fibres, after 48h

PVC, after 48h

PE, after 48h

MPs

Diffusion in digestive tract?

0

0.002

0.004

0.006

0.008

0.01

aver

age k e

fflu

x, d

elay

ed(m

in-1)

MP, low [C] MP, high [C]MP + CPF, low [C] MP + CPF, high [C]

*

Developing an uptake/excretion model in the Mediterranean mussel (M. galloprovincialis)

WP2

WP5

WP1

Nile Red dyeing as a novel approach to monitor microplastic ingestion in vivo

Any microplastic particle, of any shape, can be dyed

with Nile Red

Transparent larvae (such as medaka or zebrafish) are

exposed to the dyed particles

Their ingestion can be monitored in vivo using a

stereoscope with a fluorescent filter (such as the

Texas Red): the particles will fluoresce even after

ingestion

+

• Zebrafish and medaka larvae

(~0-2 days post hatching)

were incubated with MPs

(PE, PVC, and polyester

fibres), for 2 to 48 hours

• Monitoring of ingested MPs

was performed after

incubation and 2 days after.

• After exposure larvae were

transferred in clean water

and left undisturbed for 4

additional days

• PVC: 2 out of 14 larvae were

still positive

• PE: 17 out of 18 larvae were

still positive

• Dye seemed to diffuse out of

the polyester fibres,

although this hypothesis was

invalidated during

successive tests.

Mussels were exposed to microalgae

(MA), polyethylene microplastic (MP), and

pollutant-spiked MP (MP+CPF) diets, to

study the amount of MPs ingested and

excreted and develop a mathematical

model. Initially, just two parameters were

considered: kuptake and kefflux, delayed.

Uptake

Low [C]

0

0.005

0.01

0.015

0.02

0.025

0.03

aver

age k u

pta

ke(m

in-1)

MA MP MP + CPF

0

0.01

0.02

0.03

0.04

0.05

0.06

aver

age k u

pta

ke(m

in-1)

MA MP MP + CPF

Nile Red dyeing protocolZebrafish (D. rerio) and Medaka (O. latipes) exposed to Nile Red-

dyed microplastics

Delayed efflux

Average kuptake for the three treatments, at two different concentrations Average kefflux, delayed for MP and MP + CPF

treatments

High [C]

→ It appears exposure to MPs in mussels decreases uptake of food particles, and increases excretion, particularly with contaminated MPs.

Adsorption and equilibrium partition of persistent pollutants to microplastics

Model target contaminants were selected from a group of candidates initially proposed by all partners. The selection of the two

target concentrations (high and low) for each compound was based on data found in the literature.

Chemical pollutants selected for the EPHEMARE project are: perfluorooctane sulfonate (PFOS), benzo(a)pyrene (BaP), and

oxybenzone (Benzophenone 3, BP3). Initially, cadmium was selected as representative heavy metal; however, pre-tests showed it

would not be possible to adsorb cadmium on LDPE particles within a reasonable time.

LDPE and PVC microplastic particles of different sizes have been prepared with the three different compounds in two concentrations

and distributed to all partners in the project for ecotoxicological testing.

perfluorooctane sulfonate benzo(a)pyrene oxybenzone

high low high low high low

LDPE

500-125 µm 27.1 - 56.5 µg/g 180.9 ng/g 14.1 µg/g 378.17 pg/g 115 ng/g 1.1 ng/g

25-20 µm 46.1 µg/g 61.2 ng/g 15.1 µg/g 384.19 pg/g 62 ng/g 0.15 ng/g

13-11 µm 70.2 µg/g 9.2 ng/g 16.9 µg/g 364.34 pg/g 82 ng/g < 0.05 ng/g

6-4 µm 55.7 µg/g 118.2 ng/g 16.6 µg/g 4990.84 pg/g 24 ng/g 0.14 ng/g

PVC 250-100 µm 159.5 µg/g 1603.2 ng/g 11.5 µg/g 102.2 pg/g 66 ng/g 0.13 ng/g

Longterm sorption experimentPFOS(w ater) = 600 mg/L, LDPE(w ater) = 10 g/L

7 30 90 180

1

10

100

1000

10000

100000

time [days]

PF

OS

on

LD

PE

[

g/g

]

The preparation of the MP particles showed that may be longer exposure time would change the sorption rate

of the compounds to the particles. Therefore, 10 g/L LDPE MP particles (500-125 µm) has been exposed to

either PFOS (600 mg/L) or BaP (5000 µg/L) for 180 days. Exposure vessels has been constantly shaken and

samples has been taken at 7, 30, 90 and 180 days. The results for PFOS shows that until 90 days there is no

change of the sorption rate; however, after 180 days the sorption rate of PFOS was more than 200 times

higher than before. For BaP during the first 7 days the sorption was increasing but for the subsequent timepoints the BaP concentration adsorbed on the MP particles was constantly declining.

Longterm sorption experiment

BaP(w ater) = 5000 g/L, LDPE(w ater) = 10 g/L

0 7 30 90 180

0

100

200

300

time [days]

BaP

on

LD

PE

[

g/g

]

MP uptake and transfer in simplified food chains: Establishment of food chains

• Analyses of uptake rates, transfer and

accumulation pattern in all trophic

organisms

• Histological analyses and fluorescence

microscopy

• Transfer kinetics

• After successful establishment of

food chain, feeding of POP-spiked MPs

(self-spiked or from WP1)

Aurelia aurita

Adult zebrafish

Medaka larvae

Artemia salina spec.Fluorescent

Microplastics

(MPs)

Neomysis integer

Nereis diversicolor

Seabass

Persistent organic pollutant (POP) transfer via MP transfer in food chains

Paramecium spec.

Zebrafish larvae

• CYP1A induction in adult zebrafish liver (EROD assay) and zebrafish/medaka larvae (qPCR)

• Fluorescence tracking of benzo(a)pyrene

• POP transfer via MPs and trophic food chains shown successfully in different species!

*** CYP1A induction in medaka larvae

after trophic transfer of spiked MPs

CYP1A induction in adult zebrafish liver after

trophic transfer of spiked MPs

Fluorescence

tracking of BaP

signal in Artemiaspec. nauplii

Fluorescence

tracking of BaP in

intestinal tract

and liver of adult

zebrafish

*

uptake

Gill

Gut

Transfer across lumen?

Tissues

kuptake

kreleasekstorage

kefflux, rapid

kefflux, delayed

Steffen Keiter (1), Bettie Cormier (1), Ronny Blust (2), Raewyn M. Town (2), Camilla C. Carteny (2), Xavier Cousin (3), Thomas Braunbeck (4), Annika Batel (4)

(1) – Örebro University (2) – University of Antwerp (3) – Ifremer (4) – University of Heidelberg