TOXICITY ASSESSMENT WP3 Organism Level WP4 Cellular and Mollecular levels

BIVALVES

Microplastics loaded with BaP

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Scrobicularia plana Mytilus galloprovincialis

Test Organisms

4-cm adults

Microplastics loaded with Chlorpyrifos

CPF

1. Incubation

2. Partitioning 3.CPF quantification

particles

water

GC-MS

Microplastics really act as vectors of pollutants but in a

similar way as other particles of the seston

Scope for Growth Responses

-5

0

5

10

15

20

25

30

MA MP CPF MA+CPF MP+CPF

SFG

(J/in

d st

/h)

SFG t1 (7 days)

cc

ab

a

bc

-5

0

5

10

15

20

25

30

MA MP CPF MA+CPF MP+CPF

SFG

(J/in

d st

/h)

SFG t2 (21 days)

bb

a a a

MP increased biochemical biomarkers. CPF, independently of the via of exposition,

affected growth and biochemical biomarkers.

02468

10121416

T0 T1 T1 T1 T1 T1 T2 T2 T2 T2 T2

IBR

Integrated Biochemical Biomarker Response

algae

microplastics

CPF Bioaccumulation

0

50

100

150

200

250

[CPF

] ng/

g dw

t= 7 days b

b

a

0

50

100

150

200

250

[CPF

] ng/

g dw

t= 21 days

b

a a

Higher CPF accumulation when pollutant is offered through particles, especially through

microalgae

Bivalves experiments under the WP3 & WP4 frameworks were directed to evaluate the effect of MPs and adsorbed PPs using a variety of biological responses, at cellular, biochemical, molecular and physiological levels in two species: the mussel Mytilus galloprovincialis ,the worldwide sentinel species used for monitoring pollution and the clam Scrobicularia plana, a benthic bivalve used in sediment pollution studies.

14 days 28 days

Exposure

7 days

LDPE dose: 10mg/L BaP adsorbed to PE: 15 µg/g

BaP dose: 150 ng/L

Mytilus galloprovincialis Filter feeder

Benzo(a)pyrene Organic pollutant

LD-PE Heterogeneous shape

20-25 µm in size

QUALITATIVE ANALYSIS OF MICROPLASTICS UPTAKE AND BaP BIOACCUMULATION

PRINCIPAL COMPONENT ANALYSIS (PCA) AND ELABORATION WITHIN THE WOE MODEL

Results of the PCA analysis on the whole set of biomarker responses indicated that separation of groups at the initial stage of the exposure (7 days) is related to the polymer itself. Over the time the separation is more evident for both BaP groups (14 days). After 28 days separation is clearly caused by exposure to the chemical alone. Results on the WOE model indicated that contaminated MPs cause a "MAJOR" risk in respect to the virgin ones mostly related to BaP bioaccumulation the risk associated to treatments is not time-dependent.

Hemolymph Gill Digestive Gland

0 10 20 30 40 50 60

Digestive Gland (ng/gr d.w. tissue)

0

5

10

15

20

25

30

Gills (ng/gr d.w. tissue)

a a a

b

a a ab

b

14 days 7 days 28 days 14 days 7 days 28 days

a a a a a a

b b b

b

b

b

a a

b

b

After 7, 14 and 28 days, organisms were collected; analyses included: Histological Examination (occurrence and localization of microplastics), Chemical Analyses (BaP accumulation), Biomarkers (immunological parameters, antioxidant defences, oxidative stress, DNA damages, neurotoxicity, peroxisomal proliferation).

Individuals factor map (PCA)

The uptake of PE particles was revealed by polarized-light microscopy, showing microplastics in the hemolymph, gills and digestive gland. Chemical analyses highlighted a marked accumulation of BaP both in mussels exposed to contaminated microplastics and to pollutant alone. In the digestive gland the maximum of bioaccumulation was observed already after 7 days of exposure, while in the gills the same condition was obtained after 28 days. Levels of BaP measured in the water of contaminated MPs group is below detection limit, thus excluding a significant desorption of BaP from plastics to seawater and confirming that bioaccumulation occurred after microplastics’ ingestion.

Scrobicularia plana exposure to LDPE microplastics with adsorbed

BaP, BP3 and PFOS*

7 days 0 3 7 14

Acclimation Exposure (days)

GST

Control LDPE (11-13 µm) LDPE (11-13 µm) +

BP3 (82 ng g-1)

LDPE (11-13 µm) +

BaP (17 µg g-1)

LDPE (11-13 µm) +

PFOS (70 µg g-1)

Treatments:

Biomarkers with elevated levels

when compared to controls, with time:

AChE

LPO

DNA damage

SOD

AChE

LPO

SOD

LPO

CAT

GPx

GST

Gills, Digestive gland, Hemolymph

Adsorbed contaminants present in microplastics affect clams;

Synergy between LDPE and BaP after 14 days for GST;

Tissue specific response

*For further details check poster by Mestre et al., entitled: Ecotoxicological effects of microplastics with adsorbed contaminants in the clam Scrobicularia plana

Partners involved in the bivalves framework: IEO: J.A. Campillo, L.Vidal-Liñán, B. Fernández, M. Albentosa; UAlg: S. O´Donovan, N. Mestre,M.J. Bebianno; UAnt: C. Catarci Carteny, R. Town, R.Blust; UPM: F. Regoli, S. Gorbi, C.G. Avio, L. Pittura

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