Shallow sublittoral meiofauna communities and sediment polycyclic aromatic hydrocarbons (PAHs) content on the Galician coast (NW Spain), six months after the Prestige oil spill

Marine Pollution Bulletin 58 (2009) 581–588

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Shallow sublittoral meiofauna communities and sediment polycyclic aromatic hydrocarbons (PAHs) content on the Galician coast (NW Spain), six months after the Prestige oil spill P. Veiga a, M. Rubal a,*, C. Besteiro a,b a b

Departamento de Zooloxía e Antropoloxía Física, Universidade de Santiago de Compostela, Facultade de Veterinaria, Avd. Carballo Calero s/n, 27002 Lugo, Spain Estación de Bioloxía Mariña da Graña (EBMG), Casa do Hórreo, Rúa da Ribeira, 1. 15590 A Graña, Ferrol, Spain

a r t i c l e

i n f o

Keywords: Prestige Meiobenthos Taxonomic sufficiency PAHs Chrysene Triphenylene

a b s t r a c t The aim of this work was to detect the impact of Prestige oil spill on meiobenthic community structure at higher levels of taxonomic aggregation. In addition, the relationship between sediment individual polycyclic aromatic hydrocarbon (PAH) concentration and meiofauna community structure was investigated. Six months after the Prestige oil spill, meiobenthos community and sediment PAHs content from seven shallow subtidal localities along the Galician coast were studied. Two sites presented differences in community structure, characterized by high densities of nematodes, gastrotrichs and turbellarians, and low densities of copepods. Chrysene and triphenylene were only found at these two disturbed sites and could be responsible for differences of meiobenthos community structure. However, differences in community structure of sites could be linked with sedimentary parameters, and discrimination between the effect of PAHs and sedimentary parameters was impossible due to the lack of baseline studies on meiobenthos and PAHs contents in this area. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Biogenic and petrogenic hydrocarbons are natural substances resulting from the decay of planktonic animals and algae, and consequently must be considered as part of the marine environment (Kingston, 2002). However, as a result of hydrocarbon extraction, transport and transformation, large amounts of oil and its derivates are spilled on the coastal environment, having negative impacts on benthic communities (Mc Lusky and Martins, 1998; Kingston, 1992, 2002). Even where oil spills are not the main source of hydrocarbons on coastal areas, their effects on ecosystems have been the object of many studies (e.g., Decker and Fleeger, 1984; Danovaro et al., 1995; Ansari and Ingole, 2002) as they tend to attract high-profile coverage in the news media. Polycyclic aromatic hydrocarbons (PAHs) have proved to be the main components responsible for effects on animals, due to their carcinogenic, mutagenic and toxic effects (Lotufo and Fleeger, 1997). While many laboratory works have studied the effect of a single PAHs on meiobentic taxa (e.g. Lotufo, 1998), few field studies have studied the relationship between the environmental PAHs concentrations

* Corresponding author. Present address: CIIMAR/CIMAR-LA – Centro Interdisciplinar de Investigação Marinha e Ambiental, Universidade do Porto, Laboratório de Biodiversidade Costeira, Rua dos Bragas, 289, 4050-123 Porto, Portugal. Fax: +351 223 390 608. E-mail addresses: [email protected], [email protected] (M. Rubal). 0025-326X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2008.11.002

and the observed community disturbance. Meiofauna has proved to be more efficient than macrofauna as indicator of pollution (Boucher, 1980; Fleeger and Chandler, 1983), but most of the pollution impact studies have focused so far on macrobenthic community. The effect on meiobenthos has been partly disregarded because of difficulties in its taxonomic determination. In order to solve this problem the Taxonomic Sufficiency (TS) principle was proposed by Ellis (1985). The TS principle states that the identification of community components should be made to the level that provides the required information for the purpose of the work. This principle has been applied to the meiofauna in many pollution studies with success (e.g. Heip et al., 1988; Herman and Heip, 1988). The Galician Atlantic coast (NW, Spain) has suffered three major oil spills in the last 30 years, due to the wreck of the Monte Urquiola, Aegean Sea and Prestige. In November 2002, the Prestige tanker spilled 50,000 tons of heavy fuel-oil (M-100), in successive black tides. The Monte Urquiola and the Aegean Sea affected a smaller area of the Galician coast, the Prestige spill affected the whole area, and reached the Bay of Biscay up to the Atlantic French coast. The persistence of the negative effects of spills can be very variable, and depends on many factors (Kingston, 2002). Nevertheless, it has been proved that long term negative effects are common in benthic communities after a spill (Peterson et al., 2003). Considering the amount of Prestige fuel that settled in shallow sublittoral areas of the Galician coast, the persistence of the fuel one year after the

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wreck of the ship and the very low incidence of natural weathering (Díez et al., 2007), biological effects on the benthic fauna could be expected. The aims of this work were to test the ability of meiobenthos, studied at higher taxa levels of aggregation, to detect the Prestige oil spill effects and establish the relative fuel sensitiveness or tolerance of the main meiobenthic taxa. Relationships between individual PAHs concentration, community structure and main meiobenthic taxa abundance were studied in order to identify the responsible PAHs for negative impacts on meiobenthic community. Finally the spatial distribution pattern of the affected sites was studied by examining the meiofaunal community, environmental variables and PAHs content at seven shallow subtidal locations along the Galician coast.

2. Material and methods 2.1. Sampling and samples processing From 6 to 12 May 2003, six months after the Prestige oil spill, seven localities were studied along the Galician coast (Fig. 1). At each, two sites with different depths were sampled, one shallower (7–11 m) and another one deeper (14–20 m). Samples were collected using a Van Veen grab (sampling surface of 0.1 m2); this grab has proved to be a reliable sampler for this kind of work at the depth and weather conditions of this cruise (Somerfield and Clarke, 1997). Four 10 cm long corers with an inner diameter of 3.6 cm (sampling surface 10 cm2) were taken from the undisturbed inner part of the grab. One of these corers was immediately frozen for granulometry analysis, determination of organic matter and

Fig. 1. Study area and sampling locations. Each location is indicated by a full dot and consecutive numbers (1–7).

sediment carbonate content. The remaining three corers were used for the meiobenthos study. A second grab was collected at each site to study the environmental variables. Sediment temperature (T), pH and redox potential (Eh) were measured in situ, and samples to study sediment PAHs content were taken and stored in the dark at 20 °C. Meiofauna samples were processed immediately after collection. They were treated with a 7% MgCl2 solution for 10 min for narcotization of the fauna, and preserved in 10% neutralized formaldehyde solution with Rose Bengal. The meiofauna was extracted by decanting through a 30 lm mesh size sieve (Pfannkuche and Thiel, 1988). This procedure was repeated six times for each sample. All meiobenthic organisms were counted and identified to higher taxa under a stereomicroscope. The median particle size (Md), and quartile deviation (QDu), were calculated following the procedures described by Buchanan (1984). The organic matter content (OM) was calculated by measuring the loss of weight on ignition in a furnace at 450 °C for 4 h. The determination of the sediment carbonate content (%) was realized by hydrochloric acid treatment of the sample. Analyses of PAHs were carried out by the General Services to Research (SXAIN) at the A Coruña University (Spain). Samples for PAHs analyses were dried, in previously decontaminated glass containers, in darkness. PAHs were extracted from the dried samples and analysed by gas chromatography and mass spectrometry. Forty parent PAHs, including the 14 parentals PAHs, were considered until a detection level of 0.1 ppb. 2.2. Data analyses Densities of the most abundant groups are presented as mean (±SD). Significant differences in the abundance of these groups between sampled sites were studied by a one-way analysis of variance (one-way ANOVA), followed by a Tukey honestly significant difference (HSD) multiple-comparison test, whenever applicable (Zar, 1999). Site was the fixed factor and density of the main meiobenthic taxa the variable. Prior to the analysis, the normality of the data was checked by Kolgomorov–Smirnov normality test and variance homogeneity by a Cochran test. These analyses were performed with the STATISTICA 6.0. package. Multivariate analyses were performed using PRIMER 5.0. Mean meiofauna abundance data were square root transformed for the calculation of the Bray–Curtis similarity matrix. The relationship between meiobenthic communities of the sampled localities was displayed using hierarchical agglomerate clustering technique (CLUSTER) and a non-metric multidimensional scaling (MDS). SIMPER analysis based on all replicated samples was performed to identify the contribution percentage of each taxa to the similarity and dissimilarity within and between groups identified from the CLUSTER and MDS analyses (Clarke, 1993). To assess the relationship between the principal meiobenthic taxa with environmental parameters and sediment PAHs content, correlation analysis was performed, using Spearman rank (Zar, 1999). When PAHs concentrations were below the detection limit zero values were considered for correlation calculation. Only the environmental variables and sediment PAHs well correlated with the main meiofaunal groups were used in posterior analyses. The relationship between meiobenthic assemblage structure and environmental variables were examined using the BIOENV procedure as an exploratory method (Clarke and Ainsworth, 1993). As a second stage approach, correlation-based principal components analysis (PCA) was performed, using the environmental variables and sediment PAHs which better match with the meiobenthic community structure in the BIOENV analysis. The concordance of graphical distribution of the studied sites between PCA and MDS plots was then tested (Warwick and Clarke, 1991).

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Table 1 Environmental parameters: water depth in metres (Depth); sediment pH, redox potential in mV (Eh), sediment temperature in Celsius degrees (T); percentage of organic matter (OM); percentage of carbonate (CaCO3); percentage of each grain size class: Gravel (G), very coarse sand (VCS), coarse sand (CS), medium sand (MS), fine sand (FS), very fine sand (VFS), silt and clay (S/C), median particle diameter in millimetres (Md) and quartile deviation (QDu). Station St.1A St.1B St.2A St.2B St.3A St.3B St.4A St.4B St.5A St.5B St.6A St.6B St.7A St.7B

Date 12/05/2003 12/05/2003 11/05/2003 11/05/2003 11/05/2003 11/05/2003 10/05/2003 11/05/2003 09/05/2003 09/05/2003 08/05/2003 08/05/2003 06/05/2003 06/05/2003

Location 0

00

43°45 14 43°450 1900 43°290 5100 43°300 0100 43°180 5800 43°190 1200 43°110 4400 32°120 0800 42°490 4900 42°490 1200 42°330 1100 42°330 0400 42°130 1700 42°130 1400

0

00

07°43 48 07°430 5600 08°190 3800 08°190 4800 08°320 5300 08°320 5000 09°030 0400 09°030 0000 09°060 5600 09°070 2300 09°020 1200 09°020 3800 08°530 5500 08°530 4500

Depth

pH

Eh

T

OM

CO 3

G

VCS

CS

MS

FS

VFS

S/C

Md

QDu

8.0 16.5 9.2 18.7 8.3 17.1 7.7 16.1 11.0 16.0 7.3 14.1 7.0 20.0

8.11 8.24 8.13 8.16 8.11 8.16 8.17 8.00 7.72 8.55 8.30 8.79 8.19 7.80

319.0 380.7 170.6 303.9 131.9 151.6 315.1 151.3 84.2 188.8 127.4 138.8 61.2 4.8

16.4 15.1 15.0 15.1 15.8 16.0 16.2 16.1 15.4 16.6 15.8 15.7 14.3 14.2

0.62 0.93 1.25 1.62 1.57 2.43 1.47 2.25 2.00 0.68 2.60 1.52 0.98 2.25

44.04 49.12 55.73 59.53 61.98 64.42 60.51 73.16 66.36 47.11 65.11 58.98 44.70 59.96

0.02 0.04 0.55 0.02 0.18 0.01 0.06 0.15 0.11 0.30 0.05 0.68 2.33 3.14

0.29 0.17 4.18 0.17 2.00 0.23 0.30 0.74 0.16 4.50 0.93 2.41 2.10 2.06

3.58 1.51 28.47 3.60 12.83 2.36 6.12 9.49 0.83 41.60 6.98 4.07 20.11 8.54

67.75 45.61 52.83 39.34 42.61 32.47 56.47 60.44 5.17 42.87 37.38 12.04 61.76 31.52

26.41 47.68 12.07 55.24 38.57 57.90 35.18 26.93 61.62 8.33 52.18 71.48 10.89 25.67

0.13 3.02 0.06 0.85 2.01 5.08 0.29 0.18 29.93 0.15 1.56 7.36 1.07 17.24

1.82 1.98 1.85 0.78 1.79 1.93 1.57 2.07 2.19 2.25 0.91 1.96 1.73 11.83

0.31 0.24 0.40 0.23 0.28 0.21 0.29 0.32 0.17 0.47 0.23 0.19 0.37 0.22

0.40 0.55 0.55 0.51 0.60 0.50 0.51 0.34 0.47 0.59 0.56 0.35 0.40 0.94

3. Results 3.1. Environmental parameters Benthic environmental and physicochemical characteristics are reported in Table 1. Sediment pH and temperature were quite similar at all the studied sites, with values ranging from 7.72 to 8.79, and from 14.2 °C to 16.6 °C, respectively. The Eh showed positive values at all sites except at St7A and St7B, where sediment was reduced showing negative Eh values of 61.2 mv and 4.8 mv, respectively. Sediment organic matter content was relatively low, ranging between 0.62% and 2.60% while the sediment content in carbonate was high, ranging between 44.04% and 73.16%. The Md varied from 0.17 mm up to 0.47 mm which suggests that sediments were principally of fine to median sands. The percentage of silt and clay was always below 4%, except at the site St7B (11.83%), which was the only muddy sand site. The QDu ranged between 0.34 (very well sorted) and 0.94 (moderately sorted), but most of the stations were moderately well sorted. The concentration of PAHs that showed significant correlation with meiobenthos or values above effects range low (ERL) proposed by Long et al. (1995) are listed in Table 2. The R PAHs varied from 131.3 ppb to 948.1 ppb, being always below the ERL, but when individual PAHs were considered at each studied site, the 2-Methylnaphthalene in St1B, Acenaphthylene at St7B, Fluorene at St1A, St1B, St3A, St5A and St6A presented concentrations over ERL. 3.2. Meiofauna A total of 24 major taxa, composed of 37,791 individuals were identified in this study. The collected meiofauna was largely composed of nematodes and copepods; nematodes were the dominant taxa at 9 of the 14 studied sites and copepods at five. Turbellarians,

gastrotrichs and polychaetes were present at all studied sites, while ostracods, were absent at St 6A. The rest of the meiofaunal taxa occurred in small numbers. The mean values ± SD of the total number of taxa, total meiofauna density and main taxa density at each studied site are represented in Fig. 2. The number of taxa ranged from 7 at St 6A to 12 at St 2B and St 7A. One-way ANOVA analysis revealed significant differences (F13,28 = 4.29, p < 0.001) in the number of taxa between sites (Fig. 2A). Even when significant differences were detected no clear groups of sites could be identified as function of number of taxa. Mean values of total meiofauna density ranged between 240.00 ± 89.60 individuals 10 cm2 at St 1A and 3474.00 ± 1286.85 individuals 10 cm2 at St 5A. One-way ANOVA analysis revealed significant differences in the total meiofauna density between sites (F13,28 = 15.82, p < 0.001) (Fig. 2B). St5A was significantly different from all the other sites, except St7A and St7B due to its high density. Nematode density ranged from 33.67 ± 12.90 individuals 10 cm2 at St 2A to 2823.33 ± 1061.88 individuals 10 cm2, at St 5A. One-way ANOVA analysis revealed significant differences in the nematode density between sites (F13,28 = 34.97, p < 0.001) (Fig. 2C). The most remarkable difference between sites was the high density at St5A with significant differences compared to the other sites except St7B. Copepod density (including nauplii) ranged between 21.00 ± 7.21 individuals 10 cm2 at St5A and 366.33 ± 275.14 individuals 10 cm2 at St7A. One-way ANOVA analysis revealed significant differences in copepod density between sites (F13,28 = 8.08, p < 0.001) (Fig. 2D). St5A and St6B were significantly different from the rest due to their low values. Turbellarian density ranged from 3.00 ± 2.64 individuals 10 cm2 at St 1A and 3.00 ± 2.65 individuals 10 cm2 at St5B up to 173.67 ± 152.94 individuals 10 cm2 at St 6B. One-way ANOVA analysis revealed significant differences in the turbellarian density between sites (F13,28 = 7.38, p < 0.001) (Fig. 2E). The most notable differences were detected at sites St5A and St6B, due to their high densities. Gastrotrich density varied from 2.00 ± 2.65 individuals

Table 2 Concentrations of sediment PAHs (ppb). Only RPAHs, PAHs with values over ERL and PAHs that presented significant correlations with some meiobenthic taxa were shown. Values above the ERL were expressed in bold numbers. Values below the detection limit were expressed as non detected (n.d.). PAHs

St1A

St1B

St2A

St2B

St3A

St3B

St4A

St4B

St5A

St5B

St6A

St6B

St7A

St7B

2-Methylnaphthalene Acenaphthylene Fluorene 11 H-Benzofluorene Triphenylene Chrysene Benzo(b + j)fluoranthene RPAHs

57.5 11.6 31.3 2.6 n.d. n.d. n.d. 649.9

80.8 24.8 54.4 5.8 n.d. n.d. 10.5 948.1

5.0 0.6 7.5 0.5 n.d. n.d. n.d. 142.1

14.5 2.8 12.6 1.2 n.d. n.d. n.d. 209.9

41.2 10.0 22.9 2.0 n.d. n.d. n.d. 520.7

9.1 1.2 13.8 5.8 n.d. n.d. 1.5 315.8

6.1 1.9 8.6 0.4 n.d. n.d. n.d. 153.8

7.7 22.3 9.5 1.1 n.d. n.d. n.d. 420.9

36.6 9.6 22.4 1.8 0.1 0.4 6.6 576.1

10.9 3.4 12.9 1.1 n.d. n.d. n.d. 239.3

51.8 8.7 26.3 1.3 n.d. n.d. n.d. 436.0

3.5 1.2 6.5 0.6 0.1 0.2 n.d. 131.3

9.3 3.8 15.2 2.9 n.d. n.d. n.d. 223.5

39.2 56.7 10.6 7.8 n.d. n.d. 7.0 451.0

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Fig. 2. Mean ± SD values of: number of meiobenthic taxa (A), total meiobenthic density (B), nematode density (C), copepod density (D), turbellarian density (E), gastrotrich density (F), polychaete density (G) and ostracod density (H). All the densities are expressed in individuals 10 cm2. Different letters identify significant differences among sampling sites by Tukey honestly significant difference multiple-comparison test (p < 0.05).

10 cm2 at St4A to 534.67 ± 217.66 at St5A, being the second most important taxa at St5A and St6B. One-way ANOVA analysis revealed significant differences in the gastrotrich density between

sites (F13,28 = 14.09, p < 0.001) (Fig. 2F). Sites St5A and St6B were significantly different from the other sites due to their high density. Polychaete density was quite low, ranging between

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50

A

Similarity

60 70 80 90 S t 3A

S t 1B

S t 6A

S t 3B

S t 5B

S t 4B

S t 2B

S t 4A

S t 1A

S t 2A

S t 7B

S t 7A

S t 6B

S t 5A

100

II

I

Stres St s s :0,05

B

SSt t4A 1A

S t 2A t 3A SS t 1B

S t 6B

S t 6A S t 3B

S t 2B S t 4B S t 5B

S t 7A

S t 5A

S t 7B

C

2.0

S t 5B

PC2

1.5

S t 2A S t 7A

1.0 0.5

S t 5A

4B SStt1A 4A SStt3A

S t 6B

0

1B SStt7B 2B 6A SStt3B

-0.5 -5

-4

-3

-2

-1

0

1

2

PC1 Fig. 3. Cluster analysis dendogram (A), MDS plot (B), from the mean values of meiofaunal densities, and (C) PCA ordination from the main environmental parameters. Roman numbers indicate the different groups found in the cluster analysis.

0.67 ± 0.58 individuals 10 cm2 at St1A and 22.00 ± 12.12 individuals 10 cm2 at St5B. One-way ANOVA analysis revealed significant differences in the polychaete density between sites (F13,28 = 4.46, p < 0.001) (Fig. 2G). Only sites St5B and St7A showed significant differences with low density at St1A and St4A. Ostracod density ranged from 0.33 ± 0.58 at St3B to 40.67 ± 10.41 individuals 10 cm2 at St2A being the second most important taxa in this site. One-way ANOVA analysis revealed significant differences in the ostracod density between sites (F13,28 = 17.78, p < 0.001) (Fig. 2H). St2A and St5B showed the highest ostracod densities and were significantly different from all the other sites except the sites St4A, St4B and St7A. The CLUSTER ordination identified two main groups (Fig. 3A). One group (I) clustered sites St5A and St6B as the most distinctive sites and the second group (II) clustered the rest of the sites together. The multidimensional scaling (MDS) ordination deriving from the mean meiofaunal taxa density is presented in Fig. 3B. The low stress level (0.05) indicated that an acceptable representation of the similarities in community structure was achieved (Clarke, 1993). The ordination of the sites was identical to the one obtained in the CLUSTER dendogram. Results of the SIMPER

analysis are presented in Table 3. The responsible taxa of the similarity of group (I) were nematodes, gastrotrichs and turbellarians while in group (II) were copepods, nematodes and gastrotrichs. The two groups presented a high percentage of dissimilarity, and the responsible taxa were nematodes, gastrotrichs, copepods and turbellarians.

Table 3 SIMPER analysis results. Percentage of similarity within each group and percentage of dissimilarity between groups are presented in bold. Taxa responsible of similarity or dissimilarity and their individual contribution are shown in roman. % Similarity

% Dissimilarity

Group I

51.60

Group I–II

Nematodes Gastrotrichs Turbellarians

76.23 13.0 8.28

63.57 12.70 9.63

Group II Copepods Nematodes Gastrotrichs

53.92 45.93 43.71 3.98

11.50

69.02

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Table 4 Spearman rank correlations (r) between major meiobenthic taxa and environmental variables. Variables Eh OM Md MS FS VFS Chrysene Benzo(b + j) fluoranthene Triphenylene 11 H-Benzofluorene * **

Nematodes **

0.710 0.480** 0.619** 0.683** 0.322* 0.814** 0.457** 0.515** 0.443** 0.490**

Copepods 0.173 0.121 0.577** 0.510** 0.482** 0.502** 0.605** 0.266 0.606** 0.006

Turbellarians *

0.320 0.194 0.497** 0.408** 0.558** 0.513** 0.602** 0.109 0.607** 0.063

Gastrotrichs

Ostracods

0.231 0.308* 0.518** 0.617** 0.629** 0.512** 0.588** 0.260 0.584** 0.050

0.090 0.522** 0.782** 0.600** 0.717** 0.621** 0.212 0.390* 0.223 0.424**

Significant correlations p < 0.05. Significant correlations p < 0.001.

The results of the Spearman rank correlation analyses between the six most important taxa and environmental variables are shown in Table 4. Eh was negatively correlated with nematodes. There was a positive relationship between OM and nematodes and a negative relationship with ostracods. Md and percentage in median sand (MS) were negatively correlated with nematodes, turbellarians and gastrotrichs; being this relationship positive for copepods and ostracods. There was a positive relationship between content in fine sand (FS) and turbellarians and gastrotrichs and a negative relationship for copepods and ostracods. Finally, content in very fine sand (VFS) was positively correlated with nematodes, turbellarians and gastrotrichs, being this relationship negative for copepods and ostracods. On the other hand, there was a positive relationship between some PAHs content in sediment and nematodes, turbellarians and gastrotrichs and a negative relationship for copepods and ostracods. Polychaetes were not correlated with any environmental parameter or aromatic hydrocarbon. The BIOENV analysis performed with the environmental parameters, PAHs and meiobenthos community, indicated that Md, VFS, chrysene and triphenylene were the most important environmental factors in determining meiofauna assemblage structure (r = 0.566). These results showed that sedimentary characteristics, chrysene and triphenylene were the main factors structuring the meiobenthic community. As a second stage analyses, to verify the relationship between meiobenthic community structure and environmental parameters, a PCA based on the Md, VFS and content of chrysene and triphenylene was performed (Fig. 3C). A high degree of similarity was found between the spatial distribution of the sampled sites, at the PCA and MDS plots. This degree of concordance corroborated the results pointed out by the BIOENV analysis.

4. Discussion Immediately after an oil spill an acute toxicity effect is found in benthic communities, resulting in a severe decrease of density and diversity. As the sediment hydrocarbon levels decrease, the benthic communities recover their normal density and diversity values (Wormald, 1976; Giere, 1979; Bodin, 1988). The amount of hydrocarbons that reaches shallow subtidal sediments is lower than the intertidal, but has shown low degradation rates, particularly in the case of the Prestige (Díez et al., 2007). However, the persistence of hydrocarbon residues in shallow sediments can have long term toxic consequences (Peterson et al., 2003). The study of meiobenthic community structure at higher taxa level of aggregation allowed us to discriminate two groups of localities. The low number of copepods and high density of nematodes found among group (I) sites has been previously identified as consequence of fuel or diesel spills (Wormald, 1976; Elmgren et al., 1983; Carman et al., 1997; Ansari and Ingole, 2002) and in laboratory experiments

(Grassle et al., 1981; Elmgren and Frithsen, 1982; Frithsen et al., 1985). However, this reduction in copepod density has not been found in other oil spill studies (Boucher, 1985; Fleeger and Chandler, 1983; Decker and Fleeger, 1984; Feder et al., 1990; Danovaro et al. 1995) where crude oil was spilled. Crude oil has shown lower toxicity than its derivates, such as fuel or diesel. Indeed after a short toxic phase, the crude effect is similar to an excessive amount of organic matter (Bodin, 1988; Giere, 1993). Consequently copepods seem to be more sensitive to fuel or diesel. However, high densities of opportunistic nematode species have been observed after oil spills (Heip et al., 1985), probably as a consequence of the increase in bacteria density in sediments (Danovaro et al., 1996). These bacteria have been found to be an important food source for nematodes after an oil spill (Heip et al., 1985) or in oil seepages (Montagna and Spies, 1985). High densities of turbellarians and gastrotrichs were found at group (I) sites too. Few data are available about these taxa sensibility to pollution. Hummon et al. (1990) and Evans et al. (1993) have studied the effect of pollution on gastrotrichs and noted that its diversity decreased but, like Raffaelli (1982) and Read et al. (1983), they found high densities of some species at polluted sites. In the case of turbellarians some degree of tolerance to oil pollution (Giere, 1979; Gooday, 1980; Bonsdorff, 1981) has been recorded. These data pointed out that gastrotrichs and turbellarians should have a similar response to pollution than nematodes. However information about the pollution tolerance of these soft meiofaunal taxa is still scarce and more research is needed. Sites clustered in group (II) presented similar Md than group (I) sites but a lower amount of fine and very fine sands. However, at group (II) sites meiobenthic community structure was similar to non-polluted areas (e.g. Heip et al., 1990; Vanaverbeke et al., 2000), and should therefore be considered as a reference group. Most work on oil pollution so far has not correlated the biological effects detected to PAHs concentrations at the study area, or P have only considered the PAHs rather than individual PAHs concentrations. However, disturbed sites at group (I) were the only ones that presented detectable concentrations of chrysene and triphenylene. These two PAHs showed significant correlations with the most important meiobenthic taxa and were two important factors to explain the meiobenthic community structure at the studied sites. Chrysene was considered as an indicator for the Exxon Valdez oil spill, and its concentration explained a small but significant proportion of the invertebrate eelgrass community structure (Dean and Jewett, 2001). One of the most abundant PAH components of the spilled oil by the Arrow cargo was chrysene, which showed long persistence in the environment and induced toxic responses in biota (Lee et al., 2003). Recent ecotoxicity studies performed with Prestige fuel contaminated sediments, which include chrysene, induced sublethal toxicity in fish larvae (Morales-Caselles et al., 2006). Therefore, chrysene could be the responsible of

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community structure differences at sites in group (I). No available information about toxicity of triphenylene or PAHs mixtures including it was found, consequently the negative effect of triphenylene at sites in group (I) is not clear. On the other hand, considering the ERL values proposed by Long et al. (1995) as reference, both chrysene and triphenylene presented low concentrations, clearly below the ERL proposed for chrysene or other PAHs. The only group (I) site with PAHs values above ERL was St5A, where fluorene showed concentrations above this limit. Even if fluorene is considered one of the most harmful PAHs, in this study it was present in important concentrations at sites in group (II), but did not show any correlation with major meiobenthic taxa density. Even ERL values are helpful to predict negative effects in meiobenthos, ERL values were calculated using macrobenthic invertebrates. Detected differences at sites of group (I) cannot be related with certainty to the Prestige oil spill. It must be noted that Md and VFS content were important factors explaining community structure. On the other hand the intense maritime traffic off the Galician coasts or the activities of A Coruña oil refinery could be diffuse sources of chronic PAHs pollution. No information on the baseline levels of PAHs of this area was found, but it is supposed that these diffuse sources of PAHs should affect all the studied sites in a similar way. Moreover, the non affected sites St2A, St2B, St3A and St3B were located near A Coruña refinery, while the affected St5A and St6B were far from this hydrocarbon source but located in an area strongly affected by Prestige fuel (Junoy et al., 2005). A recent study in this area by Morales-Caselles et al. (2008), found sediment PAHs levels with potential sublethal effects on benthic fauna four years after the Prestige oil spill. The St5A and St6B area was heavily affected by Prestige spill and meiofauna community showed a disturbed structure six months after the spill, but the lack of differences on meiofauna community structure at sites St5B and St6A, located at the same area than St5A and St6B, prove that Prestige effect follows a patched distribution. This distribution makes very difficult to evaluate the actual area impacted by fuel and its effects on benthos communities. Acknowledgements The authors would like to thank Francisco Arenas and Matías H. Medina for comments and criticism on the manuscript and Agnès Marhadour for reviewing the final version of the manuscript. Juan Freire (Universidade da Coruña) provided the data of sediment PAHs. Jesus Troncoso (Universidade de Vigo) provided environmental data of the studied sites. This research was supported by the Ministerio de Ciencia y Tecnología (Programa Nacional: Acción urgente Prestige-Recursos Naturales). References Ansari, Z.A., Ingole, B., 2002. Effect of an oil spill from M V Sea Transporter on intertidal meiofauna at Goa, India. Marine Pollution Bulletin 44, 396–402. Bodin, P., 1988. Results of ecological monitoring of three beaches polluted by the ‘‘Amoco Cadiz” oil spill: development of meiofauna from 1978 to 1984. Marine Ecology Progress Series 42, 105–123. Bonsdorff, E., 1981. The Antonio Gramsci oil spill impact on the littoral and benthic ecosystems. Marine Pollution Bulletin 12, 301–305. Boucher, G., 1980. Impact of Amoco Cadiz oil spill on intertidal and sublittoral meiofauna. Marine Pollution Bulletin 11, 95–101. Boucher, G., 1985. Long term monitoring of meiofauna densities after the Amoco Cadiz oil spill. Marine Pollution Bulletin 16, 228–333. Buchanan, J.B., 1984. Sediment analysis. In: Holme, N.A., McIntyre, A.D. (Eds.), Methods for the Study of Marine Benthos. Blackwell, Oxford, pp. 41–65. Carman, K.R., Fleeger, J.W., Pomarico, S.M., 1997. Response of a benthic food web to hydrocarbon contamination. Limnology and Oceanography 42, 561–571. Clarke, K.R., 1993. Non parametric multivariate analyses of changes in community structure. Australian Journal of Ecology 18, 117–143. Clarke, K.R., Ainsworth, M., 1993. A method of linking multivariate community structure to environmental variables. Marine Ecology Progress Series 92, 205– 219.

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