Pergamon

PII: S0045-6535(97)00003-9

Chemosphere, Vol. 34, No. 4, pp. 759-770, 1997 Copyright © 1997 Elsevier Science Ltd

Printed in Great Britain. All rights reserved 0045-6535/97 $17.00+0.00

F I N G E R P R I N T O F P O L Y C Y C L I C A R O M A T I C H Y D R O C A R B O N S IN T W O

P O P U L A T I O N S O F S O U T H E R N SEA L I O N S (Otariaflavescens)

L. MARSILI **, M.C. FOSSI ' , , S. CASINI*, C. SAVELLI*, °B. JIMENEZ,

*M. JUNIN, *H. CASTELLO

*Dipartimento di Biologia Ambientale, University of Siena, Via delle Cerchia 3, 53100 Siena, ITALY.

°Departamento de Anb.lisis Instrumental y Quimica Ambiental, Institute of Organic Chemistry, CS.I.C,

Juan de la Cierva 3, 28006 Madrid, SPAIN.

~'Museo Argentino de Cencias Naturales "Bernardino Rivadavia", Av. Angel Gallardo 470, 1405

Buenos Aires, ARGENTINA.

(Received in Germany 7 October 1996; accepted 15 November 1996)

ABSTRACT

The fingerprint of 14 polycyclic aromatic hydrocarbons (PAHs) was investigated in biopsy, fur, blood,

liver and faeces of live and dead specimens of two Argentinian population of southern sea lion (Otaria

f/ave,sz:ens). One colony lives in Mar del Plata harbour which is particularly polluted with petroleum, the

second (control) colony lives at Punta Bermeja (Patagonia). The highest concentrations of the five

carcinogenic PAHs were found in the Mar del Plata sea lions. © 1997 Elsevier Science Ltd. All rights reserved

INTRODUCTION

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants in all parts of the world [1], formed

by incomplete/inefficient combustion of organic materials, diagenesis and biosynthesis. Because they are

strongly hydrophobic, they are taken up readily by animal tissues The scientific interest in these compounds

principally concerns their carcinogenic, mutagenic and teratogenic effects 121. Some PAHs have been shown

to cause tumours in marine mammals [3] but there are few examples of cause and effect relationships. Here

Corresponding Author

759

760

we determined total levels of PAHs and their fingerprint in different biological material from live and dead

specimens cff southern sea fions (Otariaflavescens) living in two Argentinian colonies at Mar del Plata and

Punta Bermeja (Fig. t). The principal aim of the study was to determine wheter PAHs are implicated in the

health status of Mar del Plata sea lions. In this colony a high percentage of old sea lions have diseases of the

skin and mucous membranes with fur loss and baldness. Young and old sea lions often have tumours. A

preliminary study on organochlorine levels in organs and tissues of the Mar del Plata sea lions showed

significantly lower levels than in other marine mammals of the northern hemisphere and Mediterranean and

confirmed the existence of a metabolic imbalance between high toxifying and low detoxifying potential in

pinnipeds, as reported in other marine mammals [4-6]. There is little information on concentration of PAHs in

pinnipeds [7, 8] and marine mammals in general from the different oceans of the world.

Figure I - Study areas

MATERIALS AND METHODS

761

STUDY AREAS (Fig. 1)

The Mar del Plata harbour accommodates about 400 vessels, including coastal fishing boats, large

cargo ships, naval war ships and small yachts operating in the harbour on a daily basis. The water is heavily

polluted with oil, organic material and fish scraps from fish processing factories. The sea lion colony consists

of 650-750 males [9]. Faecal analysis showed that this colony feeds mainly on cod (Merluccius hubbsi),

mackerel (Trachurtts picturatus) and squid (llex illecebrosus) [10]. Punta Bermeja, in the San Matias Gulf,

Rio Negro Province, is not a polluted environment, but may be affected by pollution from the Rio Negro

river, which has intensive agriculture in its catchment area.

SAMPLING PROCEDURES

Samples of fur and faeces were obtained in a completely non invasive way from several members of

each colony. Fur samples were cut with scissors. Blood and biopsy samples were obtained from a few sea

lions in each colony after anaesthetising them with a mixture ofketamine and rompum Peripheral blood was

obtained from the hind-flipper capillary network, and biopsy samples from the hind-flipper surface Biopsy

samples include skin as well as subcutaneous fat. Liver was obtained from dead animals in a good state of

conservation. Three livers were obtained from Punta Bermeja sea lions and one from Mar del Plata sea lions.

The samples were sent to the Department of Environmental Biology in Siena for chemical analysis and other

studies.

ANAESTHETIC DRUGS

Anaesthetic darts were shot with a blow-gun with the help of a member of the Fundacion Fauna

Argentina (Mar del Plata). The mean dose was 3.17 mg/Kg rompum (range: 1.42-5.33 mg/Kg) and 2.78

mg/Kg ketamine (range: 1.42-5.60 mg/Kg). Apnea and thermoregulatory disturbance were the two most

common side effects of anaesthesia. An analeptic drug, doxapram, was injected ira. to improve the general

respiratory and cardiac condition of the animals.

PAH ANAL YSIS

PAHs were analysed by HPLC/Fluorescence system. Extraction was carried according to Griest &

Caton [11] and Holoubek et al. [12], with some modifications. Samples were extracted with a mixture of

KOH/methanol (1/4). Extraction with 200 ml of cyclohexane was performed to obtain the PAH fraction,

which was purified in a chromatographic column containing Florisil. The organic fraction was concentrated

to 1 ml in acetonitrile and analysed by HPLC with fluorescence detection. A reversed-phase column

(Supelcosil LC-18, 25 c m x 4.6 mm i.d., 0.5 lam particle size) was used with an acetonitrile/water gradient.

The initial concentration of the gradient was 60% acetonitrile, increasing over 20 rain to 100% acetonitrile,

and then remaining stable for 10 rain. The flow rate was 1 ml/min. Quantification was carried out using an

762

external standard consisting of 16 PAHs from Supelco (EPA 610 polynuclear aromatic hydrocarbons

mixture). Table 1 lists the 14 PAHs studied, with their molecular formulae, molecular weights and

carcinogenic properties

Table 1 - Polycyclic aromatic hydrocarbons investigated (0 = not carcinogenic; =1= = uncertain or weakly carcinogenic;

++, +++ = strongly carcinogenic [13]).

Naphthalene Naph

Ace Acenaphtheoe

Fluorene FI

Phcnanthrene Phen

Anthracene Ant

Fluoranthene Fit

Pyrene

Benzo(a)anthracene

Chrysene (93%)

Benzo(b)fluoranthene

Bcnzo(k)fluoranthene

Benzo(a) pyrene

Dibenzo(a,h)anthracene

Benzo(g,h,i)pervlene

Pyr

B[a]A

Chry

BIbIF

Blklr

BIaIP DlahlA

B[ghi]Per

CIoH8

C12Hs

Ct3HI0

CI4HI0

CI4HI0

C16H10

CI6HI9

CII~Ht2

CI~HI2

C29H12

C2oH12

CIoH12

C22H14

C22H12

128.2 0

152.2 0

166.2 0

178.2 0

178.2 0

202.2 0

202.2 0

228.3 +

228.3 +

252.3 ++

252.3 0

252.3 +++

279.2 +++

276.3 0

The results were expressed in ng/g dry weight (d.w) or fresh weight (£w). Recoveries ranged between

80 and 98% in all samples.

To calculate the water content of samples, about 1-5 g of fur, liver, biopsy, faeces or blood were

placed in an oven at 100°C for 24 h. The percentage of water was 1-3% in fur, 60-70% in liver, 8-13% in

biopsy, 75-85% in faeces and 80-90% in blood.

The extracted organic material (EOM%) from freeze-dried samples was 38.5% + 3.0 for liver (n=4),

10.4% + 12.1 for faeces (n=17), 1.4% + 0.9 for blood (n=6) and 48.1% + 18.9 for biopsy (n=6).

STATISTICAL ANAL YSIS

The data was processed by summary statistics and ANOVA using Statistica and Excel (Microsoft)

programs for a significance level (p) of 0.05.

763

RESULTS AND DISCUSSION

Total PAHs found in both colonies studied are summarized in Table 2. Total PAHs content was

calculated as the sum of the 14 single PAHs. The main components of these residues were low molecular

weight PAHs.

Table 2 - PAH concentrations (ng/g d.w.) in fur, biopsy, blood, l iver and faeces of sea lions from the Mar del Plata

and Punta Bermeja colonies ( ~ = arithmetic mean; S.D. = standard deviation).

FUR 1

BIOPSY 3

B L O O D 3

LIVER M.dP. n=l;

P.B. n=3

FAECES 4

521 / 379 /

2785 1676 578 263

1393 422 2294 1295

527 / 274 182

408 82 424 94

In the Mar del Plata colony, the PAH concentrations decreased in the following order: biopsy > blood

> liver = fur > faeces. In the Punta Bermeja colony, the pattern was rather different: blood > biopsy > faeces

> fur > liver. It is worth noting that the higher concentration of PAHs was not always in the Mar del Plata

samples. In fact total PAHs in the blood of the Punta Bermeja sea lions were about twice those of the Mar

del Plata animals. PAHs were about the same in faeces and fur of the two colonies. PAH levels in livers were

low in relation to fat content which was high (EOM% = 38.5). This may be because most of the PAHs

detected, especially those found in high concentrations, were the 6asiest to metabolise. PAHs are metabolised

by the microsomal mixed-function oxidase system (MFO), often termed aryl hydrocarbon hydroxylase

(AHH), which is most abundant in the liver. Hence the liver operates as a demolisher of these contaminants

rather than an accumulator. This is why liver concentrations of PAHs were not correlated with total lipids. It

is therefore better to give PAH concentrations in terms of dry weight of tissue

There were interesting differences in PAH fingerprint between the various biological materials and

between the two colonies. In fur of the Mar del Plata sea lion (Table 3), acenaphthene (32.7%) accounted for

the largest percentage of total PAHs, followed by pyrene (128%), chrysene (11.9%) and anthracene

(11.8%). The five most carcinogenic PAHs (see Table 1) accounted for 20.49% of the total (Fig. 2). In fur of

the sea lion from Punta Bermeja, acenaphthene (33.4%) again accounted for the largest percentage of total

PAHs, but was followed by naphthalene (22.4%), fluoranthene (157%), phenanthrene (15.7%) and

anthracene (13.9%). The five most carcinogenic PAHs accounted for only 0.97% of the total (Fig. 2).

764

¢,f) "1-

"6

!

20 '

15

10

. F u r IB iopsy r-qBIood

I L i v e r 1 Faeces

0 ' / I I Mar del Plata Punta Bermeja

Figure 2 - Percentage of carcinogenic PAHs (Benzo(a)anthvacene, ChtTesene, Benzo(b)fluoranthene, Benzo(a)pvrene and Dibenzo(a,h)anthracene) in sea lions fi'om the colonies of Mat" del Plata and Punta Bel'meja.

Table 3 - PAH fingerprint expressed as concentration (ng/g f.w.) and percentage of total PAHs in the fur

of a sea lion each of the hvo cohmies (% = i)ercentage of Total PAHs).

i

I Iiiiiiiiiii i i ii i i !iiiiiiiiiiiiiiiiiiii iii i / i i !! i ii iiiiii iiiiiiiiiiiiiiiiiiiiiiiii I, iiiiiiiiiiiiii lliiiiiiiiiiiiiii! i!iiiiiiiiiill iiiiiiiiiiiiiiii Naphthalene 8.120

Acenaphthene 165.2

Fluorenc 4.100

Phenanthrenc 28.47

An[hracenc

Fluoranthcne

b, rene

Benzo(a)anlhracene

Chn~sene (93%)

59.81

47.57

64,91

5.890

60.15

31.49 Benzo(b) fluoranthene

Benzo(k)fluoranthene 6.660

Benzo(a)p.vrene

Dibenzo(a.h)anthracene

2.97[)

3.070

1.58

32.7

0.81

5.63

11.8

9.40

12.8

1,16

11.9

6.23

1.32

0.59

0.61

3.44

82.33 22.4

122.8 33.4

11.34 3.08

55.18 15.0

14.29 13.9

57.91 15.7

17.18 4.67

1.170 0.32

0.760 0.21

0.940 0.26

0.810 0.22

0.230 0.06

0.90O 0.24

2.310 0.63 Benzo(8,h,i)pe~lene 17.40

765

Fur is the first compartment to come into contact with these contaminants which may be in the water or

on the shore. PAHs are not metabolized in the fur Hence the percentage composition of the various PAHs in

this compartment could reflect the kind of contamination of the area studied. We found that total PAHs in fur

did not differ significantly between the two stations, but their fingerprints were quite different. Low

molecular weight PAHs which are more readily metabolized and are not carcinogenic, accounted for about

95% of the total in the fur of the Punta Bermeja sea lion, and the five most toxic were less than 1% of the

total. In the Mar del Plata sea lion, 20% of the total consisted of these carcinogenic PAHs. It therefore seems

that the two colonies come into contact with two different PAH mixtures, one consisting only of non toxic

PAHs and the other containing the five most carcinogenic ones up to 20%.

The skin biopsy specimens also included blubber; these tissues are the first area of accumulation and

metabolisation of contaminants absorbed by this route. Many enzymes, including benzo(a)pyrene

monooxygenase, metabolize these substances in the skin [14]. The greatest difference in total PAHs between

the two colonies was found in this compartment, with the Mar del Plata sea lions having levels five-fold those

of the other colony. Table 4 shows that naphthalene, acenaphthene and phenanthrene were the three most

abundant in both colonies, accounting for 68.0% in the Mar del Plata sea lions and 74.8% in the controls.

The five most toxic PAHs accounted for 5.40% and 1.79% of the total, respectively (Fig. 2).

Talfle 4 - Fingerprint of PAHs expressed as concentration (ng/g f.w.) and percentage of total PAHs in biopsy of sea

lions from the two colonies (n = number of samples; "~ = arithmetic mean; S.D. = stamlard deviation; % = percentage of

total PAHs).

l liiiiii!i!ii!i!!!iii!ii!!i!iiiiiiiiiiiiiiiiiiiiiiiiiiiiill ill,ill iii i i i l ! i ~iiiii~i~iiii!iii!i ii!ii!ilil ii!iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii!iill i i i i i i!iii i i i i i i i i i i i I

, :i::~i ~i~ i~?:/~:~/~:~i:~iii~i i::::~ii:~:~::~:~::::i::::iii::i~i~:i:i:i:~: i :::::::::::::::::::::::::::::::::::::::::::::: ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::::::::::::::::::::::::::::::::::::::::::::::::: ::~:j:~:::~:~::~::~:~:~:~:~::~:~:M:~:~:~: ==================================================================== ~:::~:i:iii:ii:~!~:~:~::::~:~!:~ ~?~??::~:: •

Naphthalene 536.8 257.2 23.3 138.8 139.4 24.4

Acenaphlhene 588.9 717.3 20.0 101.6 35.17 20.5

Fluorene 121.6 78.94 4.55 29.81 0.270 4.51

Phenanthrene 599.8 317.5 24.7 161.5 100.3 29.9

Anthracene 174.3 101.7 6.87 39.34 11.35 8.69

Fluoranthene 176.2 112.5 6.93 24.98 7.240 5.34

~,rene 156.6 76.60 6.49 23.06 4.330 5.13

Bcnzo(a)anlhracene 12.88 2.520 0.63 1.870 1.230 0.34

Ch~'sene (93%) 8.910 3.000 0.57 3.130 4.090 0.53

Benzo(b)fluoranthene 54.21 53.65 2.59 1.430 0.860 0.28

Benzo(k)fluoranthene 13.82 14.67 0.46 1.280 0.400 0.27

Benzo(a)pyrene 28.69 16.84 1.14 3.160 2.400 0.53

Dibenzo(a,h)anthracene 27.85 37.63 0.47 0.650 0.140 0.11

Benzo(~,h,i)perylene 45.43 51.91 1.43 4.310 3.(190 0.87

766

PAHs found in blood samples are presented in Table 5. In blood of controls, total PAHs were double

the levels found in blood of the Mar del Plata sea lions. The main components were again naphthalene,

acenaphthene and phenanthrene which accounted for 75.4% in Mar del Plata animals and 78.6% in Punta

Bermeja sea lions. No significant difference was found in the percentages of the five most carcinogenic PAHs

in the two colonies; these were 1.80% and 1.53% respectively (Fig. 2).

Table 5 - Fingerprint of PAHs expressed as concentration (ng/g d.w.) and percentage of total PAHs in blood of sea

lions from the two colonies (n = number of samples; ~ = arithmetic mean; S.D. = standard deviation; % = percentage of

total PAHs).

~iiiiiiiiiiiiiiiiiiiiiiiiiiiiiii~iiii~iiii!iii~ii~:i~ii~ii~:~:~i~iiiiii!i!!!~i~ii!!i~iiiiiiiiii!iiiiiiiiii i iiiiiiiiiiiiiiiiiiiii!!! i!!!ii!!i!ii!!!i!iiii!!~:!~i!~ ii5 ::iiiiiiiiiiiiiiiiiiii!!!i!ii!i!i!i!!i!ii

[ii iiiiii iiiiiiii iii ii ii ii iiiii iii iiiiii i N~iiiiiiiiii!iiiiiJiiiiiiiiiiii;Jiii:ii iiiii:iJ i:iii; i:ili!i!i! i;iiil i[i iii iii!!: ~i: i :iii~!iii !ii iiiii !ii iiiii !ili ii iil iiiiiiii iii i li~iNii }iii}iiiiiiiiiiiiiliii iiii}ill i i}~i i! i iii i i!i: [!ii i iiii ii i iii iill ~iii i iiiii; iliil i iiill ]ii ilii:i iii i:!ii iii i~iN i: ii i:ii ii ii!ii iiiiiiiiliiiiiiiiiiiii:i[iiiiiiiiiiii~Niii!iiiii:iii:iiiiiii:iii::!;:!iiil Naphthalene 383,8 363.0 24.4 827.8 931.7 29.4

Acenaphthene 391,4 99.40 30.1 559.0 110.5 27.5

Fluorene 62.53 22.35 4.44 102.1 43.00 4.66

Phenanthrene 289,7

81.32

86.70 20.9 460.5 149.7 21.7

Anthracene 13.92 6.03 100,3 19.74 4.96

Fluoranthene 64.35 5.620 4.96 92.88 26.82 4.46

Pyrene 82.93 3.48(I 6.25 1(17.8 31.28 5.14

Benzo(a)anthracene 4.640 1.(11(I 0.34 7.810 2.620 0.37

Ch~,sene (93%) 2.870 3.450 0.26 6.080 2.010 0.33

Benzo(b)fluoranthene 7.620 2.800 0,61 9.180 0.900 0.47

Benzo(k)fluoranthene 3.670 0.320 0.27 4.980 1.680 0.23

Benzo(a)pyrene 6.030 4.860 0.43 3.750 3.420 0.23

Dibenzo(a,h)anthracene 2.25(I 1.300 0.16 3.040 0.970 0.13

(I.86 Benzo(t~.h.i)per¢. lene 8.680 8.000 7.560 10.83 0.34

The PAH fingerprint in liver is shown in Table 6. In the Mar del Plata sample, the most abundant PAHs

were naphthalene (62.5%) and benzo(g,h,i)perylene (13.3%); in the Punta Bermeja samples they were

naphthalene (44.9%), phenanthrene (19.2%) and acenaphthene (10.8%). The five most toxic PAHs

accounted for 8.22% and 1.57% of the total, respectively (Fig. 2), which is five times higher for sea lions

from Mar del Plata. This confirms the greater abundance of these components in Mar del Plata harbour.

Because of their complex structure, the liver cannot metabolize them completely and they accumulate.

767

Table 6 - Fingerprint of PAHs expressed as concentration (ng/g f.w.) and percentage of total PAHs in liver of sea lions

from the two colonies (n = number of samples; ~ = arithmetic mean; S.D. = standard deviation; % = percentage of total

PAHs; N.D. = not detected).

II :ii:: !iiiii!ili:iliiiiiiiiiiii:i::i;:!ii:i!i:il i [ ::i:iii~:i:ii:i:i:i ] ] ~i~iiiiii!iiiiii!i!i!:i!iliiiii!ilili:iiiiiiii:i:i ~i iii Naphthalene 194 62.5

2.62

76.23 54.07 44.9

Acenaphthene 8.12 17.41 14.52 1 (I. 8

Fluorene 9.14 2.95 6.9311 6.060 4.31

Phenanthrene 27.1 8.74 30.91 21.75 19.2

Anthracene 0.30 0.10 10.57 2.2711 6.57

Fluoranthene 0.27 0.119 9.270 1.100 5.76

[ Pvrene 14.2 4.58 11.33 9.840 7.05

Benzo(a)anthraccne 3.12 1.01 0.270 0.350 0.17

Cho,sene (93%) 18.1 5.84

Benzo(b)fluoranthene 0.13 0.04

! Benzo(k)fluoranthene 7.21 2.33

4.12 Benzo(a)p.vrene

Dibenzo(a,h)anthracene

1.33

13.3 Benzo(g,h.i)perylene

N . D .

0.89(I

0.670

13711

N.D.

0.990

ND.

/ /

0.120 0.55

1.150 11.42

1.270 11.85

/ /

1.100 0.62 41.2

Table 7 - F ingerpr in t of PAHs expressed as concentration (ng/g d.~v.) and percentage of total PAHs in faeces of ~ a

lions from the two colonies (n = number of samples; ~ = ar i thmetic mean; S.D. = s tandard deviation; % = percentage of

total PAHs).

I IIIIIIIIEIIIIII~II iliill i !~ ~ :iiiii ii~i ~!~ !3 ' !ill iiiii!!!!ii!!!ii!iiiiiiiiili ~iSil!?"':'!:'::!!" '!!i i i i iii! !ii iiilil iEiiiii i : !III!IIIIIIIII!!!IIIII~!~IIIIIE 151 ? ? ~ i! i!iiiiii iii :: i?

Naphthalene 179.0 79.97 42.4

Acenaphthene 39.71 15.18 9,78

Fluorene 15.95 1.070 3,98

P h e n a n t h r e n e 92.41 19.56 25.4

Anthracene 20.61 5.570 5.33

F luo ran thene 14.87 2.33(I 3.67

Pvrenc 17.35 4.790 451

Benzo(a)anth racene

Chrysene (93%)

Benzo(b)fluoranthene

Benzo(k)fluoranthene

Benzo(a)pyrene

Dibenzo(a,h)anthracene

Benzo(g,h,i)perylene

1.2511

8.200

7.55(I

0.690

6.390

13 (18

(1.32

2.03

2.94

222.2

18.54

15.59

58.54

25.36

3/I.19

29.15

1.4311

5.150

11,15

85.51 51.1

17.611 4.75

1.380 3.78

11.02 14.5

20.74 5.75

13.57 7.14

17.41 7.10

1.040

7.950

8.060

039

1.10

2.56

0.530 0.240 0.12 1.35(I 1.4110 0.38

1.710 1070 0.42 2.540 1.560 11.69

0.770 0.200 11.22 2.080 1.5511 11.58

4.1170 1,0911 0.88 2.360 1.050 11.64

768

Table 7 shows PAHs in faeces. As reported in Table 2, total PAHs were almost the same in the two

colonies. Naphthalene and phenanthrene were the most abundant PAHs, accounting for 67.8% and 65.6% of

the total in the Mar del Plata and control colonies respectively (Table 7). The percentage of carcinogenic

PAHs in the two colonies was 6.29% and 5.07% respectively (Fig. 2). There were therefore no significant

differences in excretion of PAHs in the two colonies.

The results of the analysis of fur and biopsy suggest that sea lions from Punta Bermeja come into

contact with a less toxic mixture of PAHs than the sea lions of the other colony. An index of the metabolic

capacity of a sea lion can be obtained by calculating the ratio of PAH intake by contact (levels in biopsy) and

PAHs excreted in the faeces. In fact PAHs are mostly excreted through the faeces and the amount excreted

in the urine is about one-fourth to one-fifth that excreted in the faeces [15]. The higher this ratio, the lower

the metabolic capacity & t h e animal. For the sea lions ofPunta Bermeja we obtained a ratio of 1.36, versus

5.34 for the Mar del Plata colony (Fig. 3A). A ratio of one means that all PAHs absorbed are excreted; the

ratio of the harbour sea lions means that only about a fifth of the PAHs absorbed are excreted. If we consider

only the carcinogenic PAHs, the result is surprising. Mar del Plata sea lions had 145.89 ng/g d.w. of these

compounds in biopsy and 21.60 ng/g dw. in faeces. The ratio is therefore 6.75 (Fig. 3B) which means that

less than one sixth is excreted. In control sea lions, the same quantities were 11.26 ng/g d.w. in biopsy and

21.44 ng/g d.w. in faeces. Excretion was about double the quantity absorbed through the biopsy (ratio 0.53,

Fig. 3B). This suggests that PAHs are also absorbed by other routes, such as ingestion via food and water

and inhalation.

3A 5.34 1.36

B i o p s y ~ Faeces

Mar del Plata

Biopsy Pun~ Bermeja

Faeces

3B ~ F a e c e s

Biopsy 6.75

Mar del Plata

Biops!

0.53 Punta Bermeia

:aeces

Figure 3 - Ratio of Total PAHs in biopsies and faeces (3A) and ratio ofthe sum of the five carcinogenic PAHs in biopsies and faeces (3B) in the sea lions from the colonies of Mar del Plata and Punta Bermeja.

769

It can be concluded that the high levels of PAHs in the biological samples of sea lions at Mar del Plata,

especially the high percentage of carcinogenic PAHs, are related to the poor health of the animals in this

colony. As this study was only a preliminary one, it will be interesting to see whether the results are

confirmed by more detailed research involving a larger number of samples and the study of PAH fingerprints

in sea water and soil.

ACKNOWLEDGEMENTS

The authors would like to thank Mr. Juan Antonio Lorenzani and Mr. Julio Lorenzani of Fundacion

Fauna Argentina, Mar del Plata, Argentina, Mr. Edgardo Miguel Intrieri of the Reserva Faunistica de Punta

Bermeja "La Loberia", Viedma, Argentina, and Dr. Clorinda R. Costa, Viedma, Argentina, for providing

samples. This research was financed by the EC (Contract No. CI ! *-CT94-0018).

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