Acute toxicity of individual polycyclic aromatic hydrocarbons (PAH) to early life stages of commercial marine organisms, zooplankton and phytoplankton

R. Beiras, J. Bellas, L. Saco-Álvarez, J.C. Mariño-Balsa, P. Pérez, N. Fernández

Universidade de Vigo, Laboratorio de Ecoloxía Mariña (LEM), Facultade de Ciencias do Mar , E-36310, Galicia.

(VEM2003-20068-C05-02)

Ecotoxicological evaluation

1. Reference toxicants: medium MW PAH

Phenathrene Fluoranthene Pyrene14 C 16 C 16 C178 MW 202 202435 µg/L solub. 260 133

-. Degradation products

1-OH Pyr

2. Water-accommodated fraction of the fuel (WAF)

3. Seawater from affected coastline

OH

R. Beiras et al. VERTIMAR Vigo

simplecontrolledartificial

unreproducibleenvironmentally relevant

still on progress

1. Ecotoxicological evaluation of medium MW (1) PAHs

(1) Low mollecular weight aromatics are too volatile and very high MW aromatics too insoluble (Neff & Stubblefield, 1995)

[fluoranthene] µg/l

control control acet 1 4 16 64 256

% b

iolo

gica

l re

spo

nse

0

20

40

60

80

100normal hatched larvaenon developed embryosabnormal larvae (alive)abnormal larvae (dead)

One week old Gadus morhua embryos exposed to fluoranthene for 7 days

[fluoranthene] µg/l

control control acet 1 4 16 64 256

% s

urv

iva

l

0

20

40

60

80

100

48 h 72 h

One day old Gadus morhua larvae exposed to fluoranthene for 72 h.

Fish embryos and larvae (cod)Dr. J. Bellas, Kristineberg Marine Station, Suecia

LOEC: 1 µg/L

LOEC: 64 µg/L

R. Beiras et al. VERTIMAR Vigo

? 64 µg/L

0

20

40

60

80

100

0 50 100 150 200 250 300

Fenantreno (µg l-1)

% S

uper

vive

ncia

48 h-OSC

48 h-LUZ

0

20

40

60

80

100

0 50 100 150 200 250 300

Fluoranteno [µg l-1]

% S

up

ervi

ven

cia

24 h- LUZ

24 h-OSC

Graf. 1 Graf. 2

Crustacean larvae (Palaemon serratus)Dr. J. C. Mariño-Balsa. Centro de Repoboación de Especies Mariñas, Muxía

R. Beiras et al. VERTIMAR Vigo

LIG

HT

DARK64 µg/L 32 µg/L

250 µg/L

Larvas de erizo - PAHsL. Saco-Álvarez, Universidade de Vigo.

-8.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.50.00.10.20.30.40.50.60.70.80.91.01.11.2

LIGHT

DARK

EC50LIGHT 2.393e-007 EC50

DARK 1.445e-006

LOG [fluoranthene] (M)

larv

al g

row

th(%

con

tro

l) ***

R. Beiras et al. VERTIMAR Vigo

Flu

-7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.00.00

0.25

0.50

0.75

1.00

1.25

LIGHT

DARK

EC50LIGHT 6.619e-006 EC50

DARK 8.515e-006

LOG [phe] (M)

larv

al g

row

th(%

con

tro

l)Larvas de erizo - PAHsL. Saco-Álvarez, Universidade de Vigo.

n.s.

R. Beiras et al. VERTIMAR Vigo

Phe

Larvas de erizo - PAHsL. Saco-Álvarez, Universidade de Vigo.

-8.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.50.0

0.2

0.4

0.6

0.8

1.0

1.2

LIGHT

DARK

EC50LIGHT 4.275e-007 EC50

DARK 1.285e-006

LOG [pyr] (M)

larv

al g

row

th(%

con

tro

l) ***

R. Beiras et al. VERTIMAR Vigo

Pyr

Phenanthrene

[Phe] nM

0 395 791 1582 3164

Gro

wth

rat

e (d

ay-1

)

0.04

0.06

0.08

0.10

0.12

0.14

ANOVA: p<0.05

Dunnett test *

EC10=1169nM, 208 µg/L

EC50=4320nM, 768 µg/L

**

R. Beiras et al. VERTIMAR Vigo

Phytoplankton (Isochrysis galbana)P. Pérez, Univ. de Vigo

70 140 280 560 µg/L

Fluoranthene

[Phe] nM

0 173 346 692 1730

Gro

wth

rat

e (d

ay-1

)

0.04

0.06

0.08

0.10

0.12

0.14

*

ANOVA: p<0.05

Dunnett test *

R. Beiras et al. VERTIMAR Vigo

Phytoplankton (Isochrysis galbana)P. Pérez, Univ. de Vigo

35 70 140 280 µg/L

[Flu]

Pyrene

[Pyr] nM

0 74 148 297 593

Gro

wth

rat

e (d

ay-1

)

0.04

0.06

0.08

0.10

0.12

0.14

0.16 ANOVA: p>0.05

R. Beiras et al. VERTIMAR Vigo Phytoplankton (Isochrysis galbana)

P. Pérez, Univ. de Vigo

15 30 60 120 µg/L

Phenanthrene Fluoranthene Pyrene sp endpoint incubation parameter µg/L µM % sat µg/L µM %sat µg/L µM %sat

cod embryogenesis 7d, dark LOEC n.t. 64 0.32 24.6% n.t.urchin larval length 2d, dark EC10 90 0.51 20.7% 40 0.2 15.4% n.t.

larval growth 2d, dark EC10 n.c. 27 0.13 10.4% 70 0.34 52.6%2d, light EC10 113 0.63 26.0% 21 0.1 8.1% 23 0.12 17.3%

copep. egg production 2d, dark EC50 231 1.3 53.1% 87 0.43 33.5% 51 0.25 38.3%shrimp larval survival 3d, dark LC50 132 0.74 30.3% 94 0.47 36.2% >128

3d, light LC50 91 0.51 20.9% <32 <0.16 <12% >128phytop. growth rate 3d, light EC10 208 1.17 23.0% 99 0.49 38.1% >120

Summary

Conclusions on individual PAHs

-Medium MW PAHs cause toxicity on early life stages of marine organisms at concentrations in average 0,8 µM for Phe, 0,3 µM for Flu and 0,2 µM for Pyr, which corresponds to approx. 30% saturation for the three PAHs

-Light enhances the toxicity of Flu and Pyr but not Phe

2. Ecotoxicological evaluation of WAF

2. magnetic stirring (24 h) 3. GF filtration

1. mixing Prestige fuel oil with filtered seawater (40 g/L)

Preparation of water-accommodated fraction (WAF)

4. dilutions with FSW

Fish larvae (Cyprinodon variegatus)Dr. J. Bellas, I. Fernández-Méijome, Univ. de Vigo

WAF

control 12.5 25 50 100

% s

urvi

val

0

20

40

60

80

100

24 h 48 h 72 h 96 h

WAF

control 12.5 25 50 100

% s

urvi

val

0

20

40

60

80

100

24 h48 h72 h96 h

Luz Osc

WAF

control 25 37.5 50 75

% s

urvi

val

0

20

40

60

80

100

24 h48 h72 h96 h

WAF

control 25 37.5 50 75

% s

urvi

val

0

20

40

60

80

100

24 h48 h72 h96 h

Luz Osc

R. Beiras et al. VERTIMAR Vigo

Sea-urchin larvae -WAFI. Fernández Méijome, A. Méndez, Universidade de Vigo.

R. Beiras et al. VERTIMAR Vigo

20

40

60

80

100

-3,5 -3 -2,5 -2 -1,5 -1

Log dilution

Le

ng

th (

% C

on

tro

l)

Light Dark

WAF

Dilution

0 1/32 1/16 1/8

Gro

wth

rat

e (d

ay-1

)

0.040

0.045

0.050

0.055

0.060

0.065

**

***

***

ANOVA: p=0.000

Dunnett test *

Thalassiosira rotula

Phytoplankton (Thalassiosira rotula)P. Pérez, Univ. de Vigo

R. Beiras et al. VERTIMAR Vigo

dilution WAF

0.0 0.1 0.2 0.3 0.4 0.5 0.6

grow

th r

ate

%

0

20

40

60

80

100

120

p<0.001

R2=0.69

Thalassiosira rotula

R. Beiras et al. VERTIMAR Vigo

Findings on WAF

-Whilst the sensitivity of marine organisms to PAHs was very similar among phyla, large differences in sensitivity to WAF are appreciated, with urchins the most and fish the less sensitive

-Light moderately enhances the toxicity of WAF

-If we multiply the WAF dilutions shown above by the concentrations of medium MW PAHs measured in WAF from Prestige fuel oil (J.J. González et al.) the PAH concentrations obtained are orders of magnitude below the toxicity thresholds. Thus medium MW PAHs alone are not responsible for the toxicity of the WAF on marine organisms.

Summary

sp end point incubation parameter WAF dilution TU C. variegatus (fish) larval survival 4d, dark LD10 0.35 2.9

4d, light LD10 0.34 2.9P. lividus (urchin) larval growth 2d, dark ED10 0.0076 249

2d, light ED10 0.0042 131T. rotula (phytop.) growth rate 3d, light ED10 0.04 25

3. Ecotoxicological evaluation of environmental samples

Nov Dec Jan Feb Mar Apr May

larv

al le

ngth

(%

cont

rol)

0.2

0.4

0.6

0.8

1.0

1.2

M1 M2M3 ***

Nov Dec Jan Feb Mar Apr May

La

rval

leng

th (

%co

ntro

l)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

M1 M2 M3

******

* **

**

Natural seawater from coastline affected by the oil-spillL. Saco Álvarez, Univ. de Vigo

Tox. in Nov.

Tox. in Nov. & Dec.

Dark Light

R. Beiras et al. VERTIMAR Vigo

M1: Furnas42º 38’ 38” N, 9º 02’ 15” W

Light-enhanced toxicity of M1 seawater sampled in Nov and Dec 2002 on Paracentrotus lividus sea-urchin embryos.

0%

20%

40%

60%

80%

100%

Site 21 Site 17 Site 13 Site C Site 5 Control

D-l

arva

e

R. Beiras et al. VERTIMAR Vigo

Sediment elutriates from coastline affected by the oil-spillDr. R. Beiras, Univ. de Vigo

0%

20%

40%

60%

80%

100%

0.00 0.50 1.00

Dilution

no

rma

l D-l

arv

ae

.

M1,1

M1,2

M1,3

Control

0%

20%

40%

60%

80%

100%

0,00 0,50 1,00

Dilution

no

rmal

D-l

arva

e.M2,1

M2,2

M2,3

Control

No toxicity of sediments from the Galician shelf (9 to 13th Feb 2003, IEO cruise) on V. pullastra clam embryos

No toxicity of M1 and M2 sand elutriates sampled Dec 17th on Crassostrea gigas oyster embryos.

Findings on natural samples

-We present direct evidence of toxicity of seawater taken the first few weeks from impacted sites on water-column organisms.

-In contrast the fraction of weathered fuel accumulated in the sand as solid particles and tar balls, visually much more conspicuous, lacked toxicity to water column organisms

Final Conclusions

-Except for phytoplankton (less sensitive), toxicity of Phe, Flu and Pyr is very similar for very different marine organisms, and it appears at concentrations around 30% saturation in seawater.

-The toxicity of the Prestige fuel oil WAF is much more selective (organism-specific) and it cannot be explained on the basis of its content in medium MW PAHs alone.

-Our findings stress the impact to water column organisms of the less conspicuous and frequently overlooked water-accommodated fraction, rather than the more visible oil slick