Marisa R Almeida
email: calmeida@fc.up.pt

Ana P Mucha

Marta F C Delgado

M Isabel Caçador

A A Bordalo

M Teresa

S D Vasconcelos

Can PAHs influence Cu accumulation by salt marsh plants?

Keywords: Mixture of pollutants, Salt marsh plant, Phytoextraction, Cu, PAHs, Halimione portulacoides

come

Introduction

Estuaries are often considered sinks for pollutants, receiving important anthropogenic inputs from upstream catchments as well as from metropolitan areas and industries located at the vicinity of those areas. Many temperate estuaries possess salt marshes that are able to perform important ecological roles not only because they are among the most productive ecosystems on Earth [START_REF] Mclusky | The estuarine ecosystem ecology, threats and management[END_REF], but also due to the ability to improve water and sediment quality by removing nutrients [START_REF] Magalhães | Temporal and spatial patterns of intertidal sedimentwater nutrient and oxygen fluxes in Douro River estuary, Portugal[END_REF] and pollutants [START_REF] Vernberg | Salt-marsh processes: A review[END_REF], including metals [START_REF] Almeida | Influence of the sea rush Juncus maritimus on metal concentration and speciation in estuarine sediment colonized by the plant[END_REF]Reboreda and Caçador, 2007;[START_REF] Williams | Metal accumulation within salt marsh environments: a review[END_REF] and petroleum compounds [START_REF] Daane | Isolation and Characterization of Polycyclic Aromatic Hydrocarbon-Degrading Bacteria Associated with the Rhizosphere of Salt Marsh Plants[END_REF][START_REF] Jackson | Potential for Enhancement of Biodegradation of Crude Oil in Louisiana Salt Marshes using Nutrient Amendments[END_REF]. Due to the importance of salt marsh ecosystems and their capability for degrading pollutants, it is imperative to design remediation strategies to favour recovery of coastal sediments.

Plants can take up chemical species from the environment, but the uptake is dependent not only on the plant species [START_REF] Burke | Release of Metals by the Leaves of the Salt Marsh Grasses Spartina alterniflora and Phragmites australis[END_REF][START_REF] Fitzgerald | Copper and lead concentrations in salt marsh plants on the Suir Estuary, Ireland[END_REF][START_REF] Weis | Release into the environment of metals by two vascular salt marsh plants[END_REF][START_REF] Windham | Uptake and distribution of metals in two dominant salt marsh macrophytes, Spartina alterniflora (cordgrass) and Phragmites australis (common reed)[END_REF], but also on the sediment characteristics [START_REF] Almeida | Influence of the sea rush Juncus maritimus on metal concentration and speciation in estuarine sediment colonized by the plant[END_REF]. Metal uptake by plants depends on the form in which metal is present in the sediment, particularly in the rhizosphere (the region of soil surrounding a plant root). The metal form is determined by the sediment constituents, the overlying and interstitial water chemistry, and the behaviour of micro-organisms [START_REF] Chapman | Assessing sediment contamination in estuaries[END_REF].

The capability of salt marsh plants to accumulate trace metals is, in principle, advantageous, as it reduces the levels of those metals in sediments and, therefore, the potential toxicity to other benthic organisms is also reduced. Therefore, salt marsh plants can be used in phytoremediation processes. These remediation techniques are considered an effective, low cost, preferred cleanup option for moderately polluted areas [START_REF] Weis | Metal uptake, transport and release by wetland plants: implications for phytoremediation and restoration[END_REF], namely by Cu. This metal is an essential element for plant growth, which can be toxic when present in higher levels.

Sites polluted with trace metals are also frequently affected by organic pollutants of different types. Petroleum compounds, including polycyclic aromatic hydrocarbons (PAHs), are among the most frequently found pollutants in coastal environment including estuaries.

PAHs are introduced from a number of sources including run-off and erosion, effluent discharges, atmospheric deposition, naturally occurring biological processes, as well as from shipping and oil spills [START_REF] Zhang | Distribution and sources of polynuclear aromatic hydrocarbons in Mangrove superficial sediments of Deep Bay, China[END_REF].

The combined presence of different pollutants might influence remediation processes (either in bioremediation or in phytoremediation), because different compounds may interact among themselves and/or with plants and their rhizosphere biota. The interactions might cause antagonistic or synergetic effects on metal uptake. Some evidence of such phenomena already exists, despite only a few studies having been carried out in medium contaminated with a mixture of inorganic and organic pollutants, most of them on bioremediation [START_REF] Amor | Kinetics of inhibition in the biodegradation of monoaromatic hydrocarbons in presence of heavy metals[END_REF][START_REF] Hoffman | Medium composition affects the degree and pattern of cadmium inhibition of naphthalene biodegradation[END_REF][START_REF] Baldrian | Influence of Cadmium and Mercury on Activities of Ligninolytic Enzymes and Degradation of Polycyclic Aromatic Hydrocarbons by Pleurotus ostreatus in Soil[END_REF][START_REF] Riis | Influence of heavy metals on the microbial degradation of diesel fuel[END_REF]. For instance, studies of the influence of trace metals on microbial degradation of aromatic and aliphatic hydrocarbons, either in solution [START_REF] Amor | Kinetics of inhibition in the biodegradation of monoaromatic hydrocarbons in presence of heavy metals[END_REF][START_REF] Hoffman | Medium composition affects the degree and pattern of cadmium inhibition of naphthalene biodegradation[END_REF] or in soil [START_REF] Baldrian | Influence of Cadmium and Mercury on Activities of Ligninolytic Enzymes and Degradation of Polycyclic Aromatic Hydrocarbons by Pleurotus ostreatus in Soil[END_REF][START_REF] Riis | Influence of heavy metals on the microbial degradation of diesel fuel[END_REF] have demonstrated biodegradation inhibition caused by inhibition of the microbial activity as a result of metal toxicity (namely of Cd, Cr, Cu, Hg, Ni, Pb and Zn) [START_REF] Amor | Kinetics of inhibition in the biodegradation of monoaromatic hydrocarbons in presence of heavy metals[END_REF][START_REF] Riis | Influence of heavy metals on the microbial degradation of diesel fuel[END_REF]. In addition, [START_REF] Singer | Phytoremediation of mixed-contaminated soil using the hyperaccumulator plant Alyssum lesbiacum: Evidence of histidine as a measure of phytoextractable nickel[END_REF] have examined the effects of PAHs on the ability of the hyperaccumulator plant Alyssum lesbiacum to phytoextract Ni from co-contaminated soil. They observed that plant growth was negatively affected by PAHs, notwithstanding the lack of significant effect on the phytoextraction of Ni per unit of biomass of shoot.

A very recent study has examined the depth profiles of PAHs in salt marsh sediments and observed higher concentration of PAHs in sediments colonised by the salt marsh plant Sarcocornia fruticosa than in sediments non-colonised [START_REF] Martins | The influence of Sarcocornia fruticosa on retention of PAHs in salt marsh sediments[END_REF]. Thus, authors have hypothesised that the elevated concentrations of PAHs in colonised sediments might be attributed to the transfer of dissolved PAH compounds towards the roots, and subsequent sequestration onto organically rich particles, as the plant takes up water. Relatively high concentrations of non-polar organic compounds (with hydrophobic characteristics) around roots might influence the extent of interactions between ionic and polar metal ions compounds with roots and, consequently, metal uptake. The literature on this topic is scarce, not only in situ and ex situ but also in vitro. However, it has been shown that,the pH buffer HEPES (N-hydroxyethylpiperazine-N'-ethanesulfonic acid), often used in biological and biochemical studies, increased Cu toxicity for the marine dinoflagellate Amphidinium carterae, yet HEPES was not toxic (Lage et al., 1996). Surfactant activity of HEPES has been demonstrated [START_REF] Vasconcelos | Electrochemical evidence of surfactant activity of the HEPES pH buffer which may have implications on trace metal availability to cultures in vitro[END_REF] and considered as a possible explanation for the influence of the pH buffer on Cu toxicity to A. carterae. In addition, several studies have reported the benefits of chelate-assisted phytoextraction, i.e., the use of metal chelators, such as EDTA or histidine, to enhance metal phytoextraction capacity of plants (e.g., [START_REF] Vassil | The role of EDTA in lead transport and accumulation by Indian mustard[END_REF][START_REF] Kramer | Free histidine as a metal chelator in plants that accumulate nickel[END_REF]. For instance, [START_REF] Vassil | The role of EDTA in lead transport and accumulation by Indian mustard[END_REF] observed a significant increase in the accumulation of Pb by the plant Brassica juncea due to the direct uptake of the metal complex Pb-EDTA.

Therefore, in principle, the presence of organic pollutants may change the mechanisms of metal uptake by plants, thus, changing metal phytoextraction potential.

So far, few studies have investigated the efficiency of metal phytoextraction in soils contaminated with mixture of inorganic and organic pollutants [START_REF] Singer | Phytoremediation of mixed-contaminated soil using the hyperaccumulator plant Alyssum lesbiacum: Evidence of histidine as a measure of phytoextractable nickel[END_REF] and studies concerning salt marsh plants are, to our knowledge, non-existent.

This study aimed to investigate the effect of PAHs on the uptake of Cu by the salt marsh plant Halimione portulacoides. Laboratory experiments were carried out using media prepared with sediment and water retrieved from a salt marsh. Controlled conditions were chosen to facilitate the interpretation of the results. H. portulacoides was the plant selected because it is commonly found in Portuguese temperate salt marshes and is capable of accumulating several metals, including Cu, which can be useful for metal phytoextraction [START_REF] Caçador | Seasonal variation of Zn, Pb, Cu and Cd concentrations in the root-sediment system of Spartina maritima and Halimione portulacoides from Tagus estuary salt marshes[END_REF].

Experimental Section

Materials and Reagents

To prevent contamination, all sampling and labware materials were soaked in 20 % (v/v) HNO 3 solution for at least 24 h, rinsed several times with bi-deionised water (conductivity < 0.1 µS cm -1 ) and dried in a Class 100 laminar flow hood. Sample manipulation was carried out in a clean room with Class 100 filtered air. Suprapure concentrated HNO 3 and concentrated HCl, both from Merck, were used without further purification. Methanol used was of chromatographic grade from Panreac. All other reagents used were pro analysis grade or equivalent. The stock standard solution containing a mixture of the sixteen EPA priority PAHs (each PAHs at 2 g L -1 ) in dichloromethane-benzene (1:1 (v:v)) and the stock standard mixture solution of deuterated PAHs containing acenaphthene-d10, chrysene-d12, naphthalene-d8, perylene-d12 and phenanthrene-d10 (each at 2 g L -1 ) in dichloromethane were obtained from Supelco. The stock Cu standard solution was obtained from BDH (Spectrosol)).

Samples

H. portulacoides used in the experiments was grown in a greenhouse. For that, the plant was collected at the salt marsh, then the roots and a small part of the stem were cut and the remaining part was placed in dark-walled vases filled with Light Expanded Clay Aggregate (LECA, supplied by MaxitGroup Portugal) and supplied with one quarter strength modified Hoagland nutrient solution [START_REF] Hoagland | The Water-Culture Method for Growing Plants Without Soil[END_REF]

Experiments

Experimental design is shown in Fig. 1. Two sets of experiments were carried in January 2007: one with sediment elutriate (set I), and another with sediment soaked in the respective elutriate (set II).

Sediment elutriate was obtained by mixing 50 g of wet sediment with 250 ml estuarine water, being vigorously stirred for 30 min with a magnetic stirrer (complemented by manual agitation every 10 min to ensure complete mixing), according to EPA protocols [START_REF] Usepa | Evaluation of dredged material proposed for ocean disposal[END_REF]. Afterwards, the mixture was left to settle for 12 hours. Then, for set I, elutriate was separated from the sediment by decantating and filtering through 0.45 µm pore size filters (cellulose nitrate membrane, Millipore), to remove particulate suspended matter (except colloids) and to reduce the presence of micro-organisms. For set II the medium consisted of sediment soaked in the elutriate, as obtained after settling, without further manipulation. In the preparation of elutriates, sediment was used as collected, i.e. with below-ground biomass incorporated, because the sediment was collected in a vegetated area. The separation/elimination of below-ground biomass, mostly plant roots, from the sediment was only possibly through sediment washing which could alter metal fractionation in the sediment. A change in the forms of the metals in the sediment may alter the exchange between water and sediment and influence the obtained results.

In each set, media were amended with 0, 10 2 and 10 4 µg l -1 Cu 2+ (as Cu(NO 3 ) 2 ), as well as with 0 and 0.1 µg l -1 of each of the sixteen EPA priority PAHs (naphthalene,

Samples treatment and chemical analysis

Cu and PAHs concentrations were measured in solutions at the beginning and at the end of the experiments, as well as the pH. The initial pH was ca. 6.5 in all media before addition of any of the pollutants. In the media that were amended with 10 4 µg l -1 Cu (and only in these media), after the addition of Cu, the pH decrease and it was adjusted (with 400 µL of a saturated solution of CaCO 3 ) to ca. 6.5 at the beginning of the experiments. As the initial Ca concentration in the media was already relatively high (water salinity 2.3) the addition of CaCO 3 did not have significance for nutrient availability. Cu concentrations were also measured in the different tissues (root, stems and leaves) of plants not exposed (initial conditions) and exposed to the different media (final conditions). Cu concentrations in sediment used for the elutriate preparation (initial conditions) and in sediments of the media at the end of the experiments were also measured.

For determination of pH, Cu and PAHs in the soluble phase, all solutions were previously filtered through 0.45 µm pore size filters of cellulose nitrate and glass fibre filters, respectively. In set II, solutions were firstly separated from sediments by decantation.

For determination of Cu in sediments and in the different parts of the plant (which were thoroughly rinsed with bi-deionised water), samples were dried at 30 ºC to a constant weight in trays covered with filter paper. Sediments were fractionated and homogenized by sieving through nylon nets of 2 mm. Plants were separated into stems, leaves and roots, which were separately crushed and homogenized. Portions of each dry sample (for plants it was a combination of several plants per experiment) were, afterwards, digested with suitable amounts of concentrated HNO 3 (and 30 % H 2 O 2 , only for plant tissues) in closed PTFE vessels using a high-pressure microwave system (MLS-1200 Mega, Millestone) as described before [START_REF] Almeida | Influence of the sea rush Juncus maritimus on metal concentration and speciation in estuarine sediment colonized by the plant[END_REF]. The procedure for sediments only provides total-recoverable metal contents, because it does not allow a complete dissolution of the sample, particularly of silicates. Nevertheless, it has the advantage of being a strong acid digestion that dissolves all elements that could become "environmentally available".

Atomic absorption spectrometry with flame atomisation (AAnalyst 200, Perkin Elmer)

was used for determination of total-recoverable levels of Cu in sediment and plant samples using the procedures described elsewhere [START_REF] Almeida | Influence of the sea rush Juncus maritimus on metal concentration and speciation in estuarine sediment colonized by the plant[END_REF].

Cu concentration in solutions was determined by anodic stripping voltammetry using procedures and conditions described elsewhere [START_REF] Vasconcelos | Antagonistic interactions of Pb and Cd on Cu uptake, growth inhibition and chelator release in the marine algae Emiliania huxleyi[END_REF]. Voltammetric equipment consisted of an Autolab voltammeter (Ecochemie) connected to a Metrohm 663-VA electrode stand provided with a hanging mercury drop electrode.

PAHs in solutions were determined by using a method developed by Evtyugina et al. Blank solutions were prepared for each type of sample following the respective sample treatment. Three independent replicates of each sample were prepared and analysed and, after blank subtraction, the mean and respective standard deviations were calculated.

Statistically significantly differences among samples were evaluated through ANOVA tests using SPSS software and Tukey pairwise comparisons were used.

Biological parameters

At the end of the experiments, the photosynthetic efficiency and levels of chlorophylls in plants were also determined as plant stress indicators. Photosynthetic parameters were measured fluorometrically using a Diving-PAM (Pulse Amplitude Modulation) device (Walz, Germany). Each pulse was given immediately after putting the leaf under the fibre optic light guide of the Diving-PAM. Photochemical efficiency (as Genty's parameter) was determined as in [START_REF] Genty | The relationship between the quantum yield of photosynthetic electron transport and photochemical quenching of chlorophyll fluorescence[END_REF]. The chlorophyll a and b and carotenoid contents were determined according to [START_REF] Lichtenthaler | Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents[END_REF]. For that, leafs of each sample were taken and placed in covered tubes with 100% methanol over-night at -4ºC, macerated in low luminosity conditions, and supernatant samples were subjected to spectrophotometrical absorbance determination at 470, 644.8 and 661.6 nm [START_REF] Lichtenthaler | Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents[END_REF].

Due to the relatively low biomass available (chemical analysis required most of the available material), biological parameters were only determined in one plant per experiment.

Results and Discussion

Biological parameters

Although plants have the capacity to degrade or sequester many toxic compounds, they are sensitive to many of them [START_REF] Davis | Benefits of vegetation for soils with organic contaminants[END_REF]. The stress response of plants to the presence of pollutants can alter the plants capacity to control the uptake of those pollutants, increasing the uptake and sometimes causing serious problems to the viability of the plant.

However, these effects depend on the pollutant, on the plant species and on the concentration and time of exposure to the pollutant [START_REF] Davis | Benefits of vegetation for soils with organic contaminants[END_REF].

In a previous study carried out in vitro with seawater amended with 5x10 3 µg l -1 Cu and H. portulacoides, during an eight weeks trial, no apparent damage (with no decrease in the chlorophyll a and b content) in the plant was observed [START_REF] Reboredo | Cu and Zn uptake by Halimione portulacoides (L.) aellen. A long-term accumulation experiment[END_REF].

As for PAHs, several plants have been shown to tolerate crude oil pollutants in soil.

However, the mechanisms of the PAH toxicity to plants are poorly understood, and the phytotoxicity appears to vary depending on the particular PAH and plant species and presently, it is not clear to what extent PAH exposure triggers stress signalling pathways common to other abiotic or biotic stresses in plants, and whether signalling components specific to PAH stress exist [START_REF] Alkio | Stress response to polycyclic aromatic hydrocarbons in Arabidopsis include growth inhibition and hypersensitive responselike symptoms[END_REF] and references therein).

In the present study, photosynthetic efficiency and levels of chlorophylls were measured (Fig. 2), to be used as plant stress/damage indicators, as plants were in the same mature state (see experimental section). In general, plants were not significantly affected by the addition of pollutants to the medium. The photochemical efficiency of the plants was identical either with or without addition of pollutants (either alone or combined) to the media. Genty's parameter was also identical for plants exposed to the different media. The only exception was for 10 4 µg l -1 Cu added together with PAHs to the medium with sediment, where photosynthetic efficiency was reduced to nil and photosynthetic pigments decreased significantly, apparently due to serious damages to the plant caused by the treatment. However, this result may be accidental because (a) Cu levels observed in plants of this experiment were similar to those found when Cu was added in the absence of PAHs; and (b) plants exposed to higher amounts of soluble Cu when PAHs were added to the medium (set I) did not revealed appreciable damage. Therefore, no significant stress/damage to plants, caused by the addition of pollutants to the medium, was observed after the 6 day exposure period. So, the effects observed in the present work (see below) may not be related to plant damage.

pH in the medium

The initial pH was ca. 6.5 in all media. At the end of the experiments pH ranged from 6.0 to 7.0, except in set I experiments carried out with plants in elutriates not amended with any pollutant and in elutriates amended only with PAHs, where the pH was ca. 4. Plants can exude a variety of compounds such as organic acid anions, phytosiderophores, sugars, vitamins, amino acids, purines, nucleosides and inorganic ions (e.g. HCO 3 -, OH -, H + ) [START_REF] Jones | Organic acids in the rhizosphere -a critical review[END_REF]. The lower pH values observed in the two mentioned cases were probably caused by root exudation. The fact that pH values did not vary significantly when media were amended with Cu suggests that, in these cases, chemical exchanges among media and roots were of a different nature. Root exudation may be a response to nutrients deficiency or to stress induced by pollutants, being highly variable and dependent on physiochemical environment besides other factors [START_REF] Jones | Organic acids in the rhizosphere -a critical review[END_REF].

Cu in medium vs. Cu in plants

Absence of added PAHs

Cu levels were measured at the beginning and after six days in sediments (when present), in solution (subsequently called soluble fraction, although it included also the colloid fraction)

and in plant tissues.

Levels of Cu in sediments (Fig. 3) did not vary significantly (P>0.05) with the addition to the medium of 10 2 µg l -1 of Cu, because the amount of Cu added to the medium (0.5 µg/g) did not significantly increase the Cu above the initial level in sediment (ca. 50 µg/g). However, when the medium was amended with 10 4 µg l -1 , Cu concentration in sediment increased significantly (Fig. 3; P<0.01). In this case, exposure of plants, which could accumulate Cu (see below), caused a significant decrease of Cu in the sediment.

Cu concentrations in solution increased significantly and markedly in all media amended with both 10 2 µg l -1 and 10 4 µg l -1 Cu (P<0.01). Fig. 4 shows that after the experiments carried out in the absence of plants the soluble Cu was much higher in elutriate (set I) than in solution in contact with the sediments (set II). This difference is explained by the fact that sediment metals are removed from solution through various processes, such as precipitation as hydroxides, adsorption to Fe and Mn oxides or complexed/adsorbed by a variety of organic (e.g. humic substances) or inorganic (e.g. carbonates) ligands from the sediment [START_REF] Almeida | Influence of the sea rush Juncus maritimus on metal concentration and speciation in estuarine sediment colonized by the plant[END_REF]. Cu sulphide probably did not played a role in the removal of Cu for the following reasons: (a) sediment was collected in a vegetated area (colonized by H. portulacoides) and previous studies have indicated that salt marsh plant roots (including H. portulacoides, unpublished data) could oxidize the medium, resulting in undetectable levels of acid volatile sulphide [START_REF] Almeida | Comparison of the role of the sea clubrush Scirpus maritimus and the sea rush Juncus maritimus in terms of concentration, speciation and bioaccumulation of metals in the estuarine sediment[END_REF][START_REF] Genty | The relationship between the quantum yield of photosynthetic electron transport and photochemical quenching of chlorophyll fluorescence[END_REF] in the laboratory at the onset of the experiment, sediment was intensively stirred with estuarine water under aerobic conditions for elutriate preparation, which promoted oxidation of the sulphide that might remain in the sediment. In elutriate experiments (set I) only the solubility product of Cu hydroxide and metal adsorption/absorption to colloid particles in suspension could control removal of Cu from solution. Nevertheless, it must be noted that the elutriate (set I) amended initially with 10 4 µg l -1 Cu had at the end of the experiments only 6 % of the initially added metal (only ca. 600 µg l -1 Cu was measured in the soluble form, see Fig. 4). This large decrease of Cu concentration in solution can be a disadvantage in hydroponics experiments investigating the effects caused by high concentrations.

Levels of Cu in the tissues of plants pre-exposed (controls) and after six days exposure are shown in Fig. 5. H. portulacoides could accumulate Cu in roots either when it was exposed to elutriate contaminated with Cu (set I) or when it was exposed to media of set II, in which the sediment contained a relatively high initial concentration of Cu (ca. 50 µg/g) before any Cu was added. In addition, the levels of Cu in roots increased with the degree of Cu contamination. Accumulation of Cu in stems was also observed (Fig. 5), with a pattern similar to that found for roots. These results indicate that at least a fraction of the Cu fixed by the plant was absorbed into the roots and translocated to stems in a short period of time (six days). The levels of Cu in leaves did not vary significantly during the experiments, indicating that translocation to the leaves would require a longer exposure period. These results are compatible with those obtained by [START_REF] Reboredo | Cu and Zn uptake by Halimione portulacoides (L.) aellen. A long-term accumulation experiment[END_REF] in hydroponics experiments, carried out in seawater, which indicated that H. portulacoides could accumulate Cu not only in its roots and stems but also in its leaves after a period of two weeks exposure. The amount of Cu accumulated per gram of plant tissue was much higher in roots that in stems (ratio root/stem up to 14), confirming that metal in halophyte plants are mainly accumulated in the roots with small quantities translocated to stems and leaves [START_REF] Almeida | Comparison of the role of the sea clubrush Scirpus maritimus and the sea rush Juncus maritimus in terms of concentration, speciation and bioaccumulation of metals in the estuarine sediment[END_REF][START_REF] Weis | Release into the environment of metals by two vascular salt marsh plants[END_REF][START_REF] Windham | Uptake and distribution of metals in two dominant salt marsh macrophytes, Spartina alterniflora (cordgrass) and Phragmites australis (common reed)[END_REF], except in the case of more mobile elements like Cd, Mn or Zn [START_REF] Almeida | Comparison of the role of the sea clubrush Scirpus maritimus and the sea rush Juncus maritimus in terms of concentration, speciation and bioaccumulation of metals in the estuarine sediment[END_REF][START_REF] Caçador | Seasonal variation of Zn, Pb, Cu and Cd concentrations in the root-sediment system of Spartina maritima and Halimione portulacoides from Tagus estuary salt marshes[END_REF]. The capability of H. portulacoides to accumulate Cu mostly in its roots has been also observed before [START_REF] Caçador | Seasonal variation of Zn, Pb, Cu and Cd concentrations in the root-sediment system of Spartina maritima and Halimione portulacoides from Tagus estuary salt marshes[END_REF].

PAH effects

PAHs (0.1 µg l -1 of each of the sixteen EPA priority PAHs) were added to the medium every day during the six days experiments. Levels of PAHs were measured in elutriate before any addition (control levels) and in all solutions after the experiments.

Levels of PAHs were only measurable in amended elutriates (set I) without plants (Table 1), the remaining media displaying values below the limits of detection (included in Table 1).

The marked reduction of PAHs in the solution in contact with sediment (set II) may result from either adsorption on organic matter present in the sediment or degradation by microorganisms. In set II experiments micro-organisms were present, whereas in set I experiments micro-organisms presence was markedly reduced (results not shown). Therefore, the elutriates with measurable PAHs levels had a much lower microbial population than the other conditions, but even in these cases PAH concentrations were much lower than the dosed values indicating either sorption to the container walls or compound degradation by the remaining micro-organisms. In the presence of plants, even in elutriate experiments (set I), extensive removal of PAHs from the solution also occurred. For instance, a faster degradation of PAHs has been reported in vegetated soils than in non vegetated soil, which has been mediated by root-associated micro-organisms [START_REF] Chaudhry | Utilising the synergy between plants and rhizosphere microorganisms to enhance breakdown of organic pollutants in the environment[END_REF]. In addition, PAHs could also be adsorbed on the root surface or absorbed into root tissues [START_REF] Lin | Uptake of polycyclic aromatic hydrocarbons by maize plants[END_REF] ).

However, in the current study it is not possible to determine the exact causes contributing to the removal of PAHs from solution.

PAHs influenced the levels of Cu in solution, when plants were present (Fig 6).

Experiments carried out in the presence and absence of PAHs showed that the levels of soluble Cu (normalized for the mass of roots) were significantly lower in the PAH exposed compared with the unexposed when experiments were amended with 0 µg l -1 and 10 2 µg l -1 added Cu. However, soluble Cu levels were markedly higher (P<0.05) in those amended with 10 4 µg l -1 Cu. Variations in the pH of solutions, which can influence Cu solubility, were small (maximum difference in pH of solutions with and without addition of PAHs was 0.5, see Fig. 6) and, probably, did not influenced the observed differences in Cu concentrations. The effects caused in the soluble fraction by the addition of PAHs were much smaller in the experiments carried out in the presence of sediment (set II) than in set I, which might result from a higher removal of PAHs from solution, namely due to the adsorption or faster degradation of PAHs in the sediments.

In contrast, in the absence of plants, the Cu levels in solution were, in most cases, statistically identical in the media amended and not amended with PAHs (Fig. 4), which indicated that PAHs by themselves did not control Cu solubility. Thus, the observed effects were the result of unknown interactions among PAHs, plants and probably Cu (as the PAHs effects depended on the levels of Cu in the medium).

PAHs did not significantly influence the levels of Cu in sediment (Fig. 3), probably because the levels of Cu were already relatively high, masking possible small variations caused by PAHs in the presence of plants.

Influence of PAHs on Cu uptake. A comparison of Fig. 5 and 6 shows that accumulation of Cu by plants in elutriate experiments (set I) reflected the pattern of variation of Cu levels observed in solution. Particularly for 10 4 µg l -1 Cu added, the amounts of metal accumulated either in roots or in stems were significantly and markedly higher when the medium was amended with PAHs, that is, when the soluble Cu was much higher (P<0.01). However, the level of Cu in leaves did not increase in experiments using media with and without PAHs, possibly because of the short exposure time.

Together, these results suggest that PAHs can alter the Cu sorption by plants or modify the Cu solubility. For instance, [START_REF] Mucha | Exudation of organic acids by a marsh plant and implications on trace metal availability in the rhizosphere of estuarine sediments[END_REF] has shown that plants can exude organic compounds capable of complexing metals, which can change metal availability. It is possible that PAHs might control exudation of Cu ligands capable of forming soluble and bioavailable Cu complexes. Dissolved metals in waters and pore waters of soils and sediments are present as free ions (aqua-complexes), or as complexes with organic or inorganic ligands. Increases in metal availability, particularly an enhanced uptake by plants, in the presence of metal complexes have been found (Degryse et al., 2006a and references therein), including Cu (Degryse et al., 2006b). Therefore, a possible role of PAHs in changing exudation deserves investigation. Alternatively, PAHs, may passively penetrate the root cells membranes without any carrier [START_REF] Alkio | Stress response to polycyclic aromatic hydrocarbons in Arabidopsis include growth inhibition and hypersensitive responselike symptoms[END_REF], which can then facilitate metal (or a metal complex) penetration into the cell.

In the experiments carried out in the presence of sediments, the influence of PAHs in Cu accumulation by plants was not significant (Fig. 5), which is consistent with the small differences in soluble Cu observed among media amended and not amended with PAHs (Fig. 6).

The and photosynthetic efficiency (Genty's parameter) after plant exposure both to elutriate (set I)

and sediment soaked in elutriate (set II), amended with 0, 10 2 and 10 4 µg l -1 Cu, as well as with 0 and 1.6 µg l -1 of total PAHs concentration. Lighter bars: 0 µg l -1 PAHs; Darker bars:

1.6 µg l -1 total PAHs concentration. Mean and standard deviation are shown. Set II -Sediment soaked in elutriate 10 4 µg l -1 10 2 µg l -1 1.6 µg l -1 total concentration

  for approximately 2 months to allow new root biomass growth. All plants used in the experiments were of the same age and of similar size. Plants total weight (dry weight) ranged from 1.1 to 3.2 g. Plant roots, stems and leaves represented, respectively, 8-17 %, 52-67 % and 17-35 % of total plant weight. Sediment was collected in the salt marsh area of the Cavado river estuary (41.5228 N; 8.7846 W) during the winter season (January) 2007 at low tide in an area colonized by H. portulacoides. Sediments were retrieved by means of a plastic shovel, placed in individual sterile bags and immediately refrigerated in an ice chest. Simultaneously, estuarine water was also collected in plastic bottles and refrigerated.

  acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, chrysene, benz[b]fluoranthene, benz[k]fluoranthene, benz[a]pyrene, indeno[1,2,3-cd]pyrene, dibenz[a,h]anthracene and benz[ghi]perylene). As NO 3 is a nutrient of plants, the addition of NO 3 -to the media may influence plants activity. However, NO 3 concentration was the same with or without addition of PAHs to the media for each added Cu concentration. Therefore, the added NO 3 -is not expected to influence the results of the present study, which were obtained by comparison between media with the same Cu (and NO 3 -) concentration and with or without addition of PAHs. Cu was added to the medium solution only at the beginning of the experiments, whereas PAHs were added every day throughout the experiment to overcome a possible PAHs removal from solution (e.g. by adsorption and/or degradation). PAHs were added by pipetting a small volume (10 µL) of a working standard solution prepared by dilution of the stock standard solution. The total amount of PAHs added per day was 1.6 µg l -1 . After the addition of pollutants, the solution was manually stirred for homogenous distribution within the medium. Groups of H. portulacoides (five plants per experiment) were exposed to the media for six days. Control experiments without plants were also carried out in parallel. Experiments carried out with sediment soaked in the respective elutriate (set II) allowed simulation of nutrients and contaminants exchange among plants, solution and sediment, as in the natural environment. Experiments carried out with elutriate alone (set I) is a much simplified medium which facilitated the interpretation of results. Solutions are more homogenous media than sediments. In this case there is only exchange of nutrients and contaminants between solution and plant. Sediment elutriates are used in EPA protocols for elutriate sediment toxicity tests in sediment quality trials (USEPA, 1991) as they can give anestimation of the amount of metal that is exchanged between sediment and aqueous phase during re-suspension processes, such as floods, strong tides, regional sea level rise or dredging operations, which frequently occur in estuaries.The 10 2 µg l -1 Cu concentration corresponded to the higher Cu concentration observed in sediment interstitial water at a site contaminated with Cu(Mucha et al., 2007), i.e. the concentration that could be bioavailable in a site contaminated with Cu. The 10 4 µg l -1 Cu concentration was chosen because this would be the Cu concentration in solution if all Cu present in the mass of sediment used could be bioavailable during the experiments carried out in elutriates. Although this concentration may not be realistic in terms of environmental pollution, it would probably cause a more marked effect on plants facilitating the attainment of measurable results. In addition, Cu concentration in the sediment used was similar to the ERL (Effect Range Low, which is 35 µg/g dry sediment for Cu), the sediment quality guideline that indicates the concentration above which adverse biological effects may occasionally occur[START_REF] Long | Incidence of adverse biological effects within ranges of chemical concentrations in marine and estuarine sediments[END_REF]. So, the 10 4 µg l -1 Cu concentration may be of environmental concern. The PAHs concentration selected (each PAHs at 0.1 µg l -1 ) has been set as the Environmental Quality Standard for several organic compounds being of environmental and regulatory concern and of relevance in the Drinking Water Directive and for Water Treatment Plants (Lacorte et al., 2001). During the experiments, plants were subjected to natural day:night regime with natural sunlight. For each experiment each container was wrapped in aluminium foil to prevent light influencing the results, because in the natural medium plant roots are not exposed to direct light. Containers were in a randomized position and were randomly rotated each day. To maintain solution levels during experiments, estuarine water, identical to that used in the preparation of elutriates, was added to the media for compensation of losses, due to evaporation and absorption.

(

  2007), which was adapted from the literature (e.g.[START_REF] King | Determination of polycyclic aromatic hydrocarbons in water by solid-phase microextraction-gas chromatography-mass spectrometry[END_REF]. Determinations were carried out in a Varian CP-3800 GC provided with a split/splitless injector and a Saturn 2200 ion trap mass spectrometric (MS) detection system, after extraction with a solid-phase microextraction (SPME) device with fibres coated with a 100 µm thickness polydimethylsiloxane film. The GC-MS determination was carried out in selected ion monitoring mode. All samples were dosed with a known concentration of deuterated PAHs, before extraction. Quantification of PAHs was carried out through response factors obtained from PAHs standards with deuterated PAHs. At least one deuterated PAHs per class of aromaticity was used to determine accurate concentrations for all PAHs. The accuracy of the analytical procedure was checked by spiking aliquots of all filtered solutions from the different media with known amounts of PAHs and determining recovery percentages, which were between 80 and 120 %.

Fig. 1 .Fig. 2 .

 12 Fig. 1. Experimental design of the work with each experimental treatment carried out in

Fig. 3 .

 3 Fig. 3. Cu concentration (mean and standard deviation) observed in sediments used for

Fig. 4 .

 4 Fig. 4. Cu concentration (mean and standard deviation) observed in solution after the

Fig. 5 .

 5 Fig. 5. Cu concentrations (mean and standard deviation) observed in the different tissues of

Fig. 6 .

 6 Fig. 6. Cu concentration (mean and standard deviation (error propagation)) observed in

Fig

  Fig. 1.

Fig

  Fig. 2.

Table 1

 1 PAHs concentrations (ng l -1 , mean and standard deviation (in parenthesis, n=3)) measured in elutriate without plants and without sediments (set I, Fig.1). After all the experiments with plants and after the experiments with sediments all sixteen EPA priority PAHs were below limits of detection (LD, included in the Table)

	.2	4.0	5.8	5.9	7.3	6.8

l -1 (dark bars) total PAHs concentration. Determinations were carried out in pre-exposed plants (white bars) and after six days exposure (grey and black bars).

Acknowledgements

This work was partially funded by Fundação para a Ciência e Tecnologia (FCT), Portugal, through project POCTI/CTA/48386/2002. Authors acknowledge Margarita Evtyugina for PAHs analysis.


Ronald A Kastelein

Willem C Verboom

Nancy Jennings

Dick De Haan

Sander Van Der Heul

The influence of 70 and 120 kHz tonal signals on the behavior of harbor porpoises (Phocoena phocoena) in a floating pen

Keywords: Acoustic alarm, Avoidance threshold level, Bycatch, Dolphin, Fisheries, Fishing, Nets, Odontocete, Pinger, Ultrasound

Two harbor porpoises in a floating pen were subjected to five pure tone underwater signals of 70 or 120 kHz with different signal durations, amplitudes and duty cycles (% of time sound is produced). Some signals were continuous, others were intermittent (duty cycles varied between 8-100 %). The effect of each signal was judged by comparing the animals' surfacing locations and number of surfacings (i.e. number of respirations) during test periods with those during baseline periods. In all cases, both porpoises moved away from the sound source, but the effect of the signals on respiration rates was negligible. Pulsed 70 kHz signals with a Source Level (SL) of 137 dB had a similar effect to a continuous 70 kHz signal with a SL of 148 dB (re 1 Pa, rms). However, a pulsed 70 kHz signal with a SL of 147 dB had a much stronger deterring effect than a continuous 70 kHz signal with a similar SL. For pulsed 70 kHz signals (2 s pulse duration, 4 s pulse interval, SL 147 dB re 1 Pa, rms), the avoidance threshold Sound Pressure Level (SPL), in the context of the present study, was estimated to be around 130 dB (re 1µPa, rms) for porpoise 064 and around 124 dB (re 1µPa, rms) for porpoise 047. This study shows that ultrasonic pingers ( 70 kHz) can deter harbor porpoises.

Such ultrasonic pingers have the advantage that they do not have a "dinner bell" effect on pinnipeds, and probably have no, or less, effect on other marine fauna, which are often sensitive to low frequency sounds.

Introduction

Many harbor porpoises (Phocoena phocoena) are accidentally caught in gillnets and drown [START_REF] Trippel | Incidental mortality of harbour porpoise (Phocoena phocoena) by the gill-net fishery in the lower Bay of Fundy[END_REF][START_REF] Read | Bycatch of marine mammals in U.S. and global fisheries[END_REF]. One potential alternative to reducing bycatch of small odontocetes by time and area closures of fisheries or by fish gear modifications [START_REF] Read | Bycatch of marine mammals in U.S. and global fisheries[END_REF], is to deter the animals from the nets acoustically. Harbor porpoises are deterred by certain anthropogenic underwater noises (Amundin and Amundin, 1973;[START_REF] Polacheck | The Swimming Direction of Harbor Porpoises in Relationship to a Survey Vessel[END_REF][START_REF] Johnston | The effect of acoustic harassment devices on harbour porpoises (Phocoena phocoena) in the Bay of Fundy, Canada[END_REF][START_REF] Olesiuk | Effect of sound generated by an acoustic harassment device on the relative abundance of harbor porpoises (Phocoena phocoena) in Retreat Passage, British Columbia[END_REF][START_REF] Koschinski | Behavioural reactions of free-ranging porpoises and seals to noise of a simulated 2 MW windpower generator[END_REF]Kastelein et al., 2005a;[START_REF] Carstensen | Impacts of offshore wind farm construction on harbour porpoises: acoustic monitoring of echolocation activity using porpoise detectors (T-POSs)[END_REF][START_REF] Madsen | Wind turbine noise and marine mammals: implications of current knowledge and data needs[END_REF].

Field studies with acoustic alarms on set gillnets have led to reduced harbor porpoise bycatch [START_REF] Lien | Field tests of acoustic devices on groundfish gillnets: assessment of effectiveness in reducing harbour porpoise by-catch[END_REF][START_REF] Kraus | Effect ultrasonic sounds on harbor porpoises Laake[END_REF]Laake et al., 1998;[START_REF] Trippel | Mitigation of harbour porpoise (Phocoena phocoena) by-catch in the gillnet fishery in the lower Bay of Fundy[END_REF][START_REF] Gearin | Experimental testing of acoustic alarms (pingers) to reduce bycatch of harbour porpoise, Phocoena phocoena, in the state of Washington[END_REF][START_REF] Anonymous | Annex I, Report of the Sub-Committee on Small Cetaceans[END_REF][START_REF] Barlow | Field experiments show that acoustic pingers reduce marine mammal bycatch in the California drift gill net fishery[END_REF]. Behavioral studies in the field show that porpoises avoid acoustic alarms (Laake et al., 1998;[START_REF] Culik | Reactions of harbour porpoises Phocoena phocoena and herring Clupea harengus to acoustic alarms[END_REF][START_REF] Johnston | The effect of acoustic harassment devices on harbour porpoises (Phocoena phocoena) in the Bay of Fundy, Canada[END_REF][START_REF] Olesiuk | Effect of sound generated by an acoustic harassment device on the relative abundance of harbor porpoises (Phocoena phocoena) in Retreat Passage, British Columbia[END_REF].

Acoustic alarms work by eliciting avoidance behavior and in some cases perhaps by alerting animals. One undesirable side-effect of alarms is noise pollution, which is increasingly believed to be damaging to the marine environment. Knowledge of the effects of alarm noises and of the hearing systems and behavior of many marine animals is very limited, but marine animals are likely to be disturbed by noise in their environment, and intense sounds may have negative physiological, auditory, and behavioral effects. For marine mammals, sound is particularly important as a means of communication, and echolocating mammals also use sound for orientation, and to locate prey, conspecifics and predators.

-4 -Kastelein et al., Effect ultrasonic sounds on harbor porpoises Therefore, sounds used in acoustic alarms should have characteristics such that they reduce odontocete bycatch, but also cause minimal noise pollution.

So far, the fundamental frequencies of signals of most commercially available acoustic alarms to reduce small cetacean bycatch have been below 20 kHz, although some recently developed pingers produce ultrasonic signals [START_REF] Kastelein | Effects of acoustic alarms, designed to reduce small cetacean bycatch, on the behaviour of North Sea fish species in a large tank[END_REF]. However, when not masked by background noise, harmonics produced by conventional alarms may be more audible to harbor porpoises than the fundamental frequency, as the hearing sensitivity of harbor porpoises increases up to around 120 kHz [START_REF] Kastelein | The influence of acoustic emissions for underwater data transmission on the behaviour of harbour porpoises (Phocoena phocoena) in a floating pen[END_REF].

The ideal alarm would be audible to harbor porpoises, but not to fish [START_REF] Popper | Effect ultrasonic sounds on harbor porpoises capabilities of teleost fishes[END_REF] and pinnipeds [START_REF] Southall | Marine Mammal Noise Exposure Criteria[END_REF]. The range of best hearing of harbor porpoises is 32-140 kHz [START_REF] Kastelein | The influence of acoustic emissions for underwater data transmission on the behaviour of harbour porpoises (Phocoena phocoena) in a floating pen[END_REF] -range highest sensitivity + 10 dB). Therefore, alarms designed to deter harbor porpoises should have their fundamental frequency between 32 and 140 kHz. If alarms are to be inaudible to pinnipeds, their frequency should be > 64 kHz [START_REF] Southall | Marine Mammal Noise Exposure Criteria[END_REF]. Such ultrasonic signals have the advantages over audible signals that less energy is needed to produce them, and that they are somewhat more subject to attenuation over distance. [START_REF] Kastelein | The effects of various sounds on a harbour porpoise (Phocoena phocoena)[END_REF] showed that harbor porpoises can be deterred by ultrasound, but the extent of the exclusion zone caused by the signals used in that study is not clear. [START_REF] Culik | Reactions of harbour porpoises Phocoena phocoena and herring Clupea harengus to acoustic alarms[END_REF] showed that harbor porpoises in the wild avoided an ultrasonic alarm producing sweeps between 20 and 160 kHz. [START_REF] Teilmann | Reactions of captive harbor porpoises, (Phocoena phocoena), to pinger-like sounds[END_REF]

Materials and methods

Study animals

The study was carried out on two male harbor porpoises that had been stranded and rehabilitated in the Netherlands. One animal (047) had stranded on the Dutch coast at the age of seven months, the other (PpSH064) on the German coast at the age of nine months (ages estimated from their body length and assuming that they were born in June). At the time of the study they were five and two years old respectively, healthy, weighed approximately 35 and 25 kg and were about 135 and 118 cm long respectively. The animals were fed fish five times a day: at 0845, 1030, 1230, 1430, and 1630 h.

Study area

The harbor porpoises were housed in a floating pen (34 m x 20 m; 2.4 m deep at the sides and 2.8 m deep in the center, containing two smaller pens; Fig. 1). The lower 10 cm of the surrounding pontoons (plywood boxes filled with Styrofoam and coated with fiberglass) were submerged. The bottom panel of the net was made of nylon and the four side panels were made of polypropylene; both types of net had a stretched mesh size of 9 cm and a twine diameter of 3 mm. The net was covered with algae. Seawater could flow through the pen, as could small fish and invertebrates, such as jellyfish.

-6 -Kastelein et al., Effect ultrasonic sounds on harbor porpoises

The pen was in a harbor in the southwest Netherlands, at Neeltje Jans (51° 37' N, 03° Therefore the tidal amplitude inside the harbor was similar to that in the nearby North Sea (2.5 m). No shipping occurred within 2 km of the study area during the study. The depth of the harbor around the pen depended on the tide (Fig. 1). The sea floor below the pen was smooth and covered with sandy silt; salinity was around 34 ‰. During the study the underwater visibility (determined by Secchi disc reading) was between 1.0 and 3.1 m, and the average monthly water temperatures were 18 °C (July), 21 °C (August), 16 °C (September) and 13°C (October). During the experiments both animals were kept in the main pen (Fig. 1).

Underwater background noise

Background noise in the pen, mostly from wind and tide, was measured several times under conditions similar to those during which actual sessions were carried out. At high tide the swell caused squeaking, impulsive noises from the steel joints of pontoon elements of the floating pen, especially in north to north-east winds. Drainage noise occurred when water fell from the quayside into the harbor at falling tide. Background noise levels were determined in the range 100 Hz -80 kHz and were converted to 'spectrum level' (dB re 1 µ Pa/√Hz; Fig. 2).

Equipment used to measure the background noise consisted of a broadband hydrophone (Brüel & Kjaer 8101, 0-100 kHz), a voltage amplifier system (TNO TPD, 0-300 kHz) and an analyzer system (Hewlett Packard 3565, controlled by a notebook computer; sample frequency 260 kHz, df = 31 Hz; the result was presented in 1/3-octave bandwidths). The system was calibrated with a pistonphone (Brüel & Kjaer 4223) and a white noise 'insert voltage' signal into the hydrophone pre-amplifier. Measurements were corrected for the frequency sensitivity of the hydrophone and the frequency response of the measurement equipment.

Stimuli

The porpoises were exposed to five ultrasonic signals (Table 1) The 70 kHz signal frequency was chosen to be above the hearing frequency range of pinnipeds, and the 120 kHz signal was chosen to coincide with the peak frequency of harbor porpoises' echolocation clicks [START_REF] Møhl | Echolocation: high-frequency component in the click of the harbour porpoise (Phocoena ph. L.)[END_REF][START_REF] Verboom | Acoustic signals by Harbour porpoises (Phocoena phocoena)[END_REF], 1997, 2003) and their most sensitive hearing [START_REF] Kastelein | The influence of acoustic emissions for underwater data transmission on the behaviour of harbour porpoises (Phocoena phocoena) in a floating pen[END_REF]. For each signal (frequency -pulse duration -pulse interval combination) the Source Level (SL) used in the experiment was determined during a pre-test by increasing the amplitude until the porpoises swam away from the sound source.

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-8 -Kastelein et al., Effect ultrasonic sounds on harbor porpoises

Acoustic recording and analysis

The SPL distribution of the signals in the pen was measured by using a hydrophone All SPLs reported are corrected for the response curves of the hydrophone, the amplifier, and analogue-to-digital conversion. The SPL distribution was determined at 13 measurement locations in the pen with the hydrophone 1.5 m below the water surface, the same depth as the sound sources (Fig. 3).

The SL of each signal was calculated from the measured SPL at a distance of 2 m from the sound source by adding 6 dB (Fig. 3). Because it is not clear which sound parameter(s) influenced the porpoises' behavior, the SLs of the five signals are presented in three ways: 1) the SL based on the root-mean-square (rms) value of the signal amplitude; 2) the rms SL averaged over the pulse interval (thus including the influence of duty cycle; 3) the source energy level (signal level x signal duration; Table 1).

Experimental procedures

The transducer was put into position (Fig. 1) several hours before a session started. A session was composed of a 15-minute baseline period (no acoustic stimulus produced), followed immediately by a 15-minute test period (stimulus produced). Daily, one session was conducted between 15.30 and 16.00 h. At this time of day the sun was behind the observers, so that their view of the animals was optimal. The observers were trained to recognize the individual porpoises. During the tests, people were not allowed on the pontoons.

Response variables and behavioral data recording

To quantify and compare the effects of the signals, we measured the displacement of the porpoises and changes in their breathing rate (as an indicator of their level of agitation). In order to do this, during the baseline and test periods, the locations where each animal surfaced were plotted on a map of the pen by two observers. Depending on the tide, the observers were 2 to 4.5 m above water level, on land to the west of the pen (Fig. 1). To facilitate the mapping of the positions, two ropes were strung across the pen (80 cm above the water level) with markers in the middle. On most days, the animals could be seen up to 1 m below the water surface, but on some days the water was so clear that the observers could see the bottom of the pen. Because on those days the porpoises were never seen swimming far away from their surfacing locations, it was assumed that these locations indicated their general swimming area. From the maps, the following response variables were derived: the number of respirations (or surfacings), and the distances between the transducer and the surfacings, during baseline and test periods.

Statistical analysis

Statistical analysis was done on Minitab release 14 [START_REF] Ryan | Minitab handbook[END_REF]) with a significance level of 0.05.

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-11 -Kastelein et al., Effect ultrasonic sounds on harbor porpoises

In preliminary analysis to investigate the effect of each signal on the distance of surfacings from the transducer, a separate analysis of covariance (ANCOVA) was used for each animal and for each signal, so that a total of ten ANCOVAs were carried out. In all, the session number was introduced as a covariate, so that any effect of session number could be both quantified and corrected for before the analysis of the only factor in the ANCOVA, which was the period (baseline or test). The assumptions of ANCOVA (normality of residuals and homogeneity of variances, see [START_REF] Zar | Biostatistical analysis[END_REF] were not always conformed to exactly, but ANCOVA is robust to small departures from these assumptions [START_REF] Zar | Biostatistical analysis[END_REF].

To evaluate potential differences between the responses to the signals, further ANCOVAs were carried out in which the dependent variable was the difference between the distance from the transducer of surfacings in baseline and test (calculated for each session as the mean distance from the transducer in the baseline period, minus the mean in the test period). As above, the session number was included as a covariate; the only factor was the signal. One ANCOVA was carried out for each animal.

To investigate the effects of the signals on the number of surfacings in each session, a paired t-test was carried out for each signal and for each animal. When assumptions of the ttest were not met, a Wilcoxon matched pairs test was used as a substitute. In both cases the difference between the number of surfacings in baseline and test sessions were compared to zero, thus controlling for any effects of session number.

Results

Both animals moved away from the transducer when each of the five signals was emitted (Tables 2 and3 Table 1. For Signal 4 (70 kHz, 2 s pulse duration, 4 s pulse interval, SL 147 dB re 1 Pa, rms), the avoidance threshold SPL (the SPL at the boundary between the areas that animals generally occupied during the transmission of sounds and areas that they generally did not enter during transmission) was estimated to be around 130 dB (re 1µPa, rms) for porpoise 064 and around 124 dB (re 1µPa, rms) for animal 047.

In most cases, the number of surfacings was similar in baseline and test sessions (Table 5 and Fig. 5).

Discussion and conclusions

Reasons for the different effects of the five signals

Before and during the entire study period, the animals had a strong tendency to occupy the north-western corner of the pen. This was probably because the gangway to the pen was there (Fig. 1). From the gangway, the trainers arrived five times per day to feed the animals, and often started as soon as they stepped onto the pontoons around the pen. Therefore the transducer was placed at the northern end of the pen, so that the deterring effect of the test signals could be quantified. All five signals deterred the animals from the sound source, but only 70 kHz Signal 4 (2 s duration, 4 s interval, SL 147 dB) had an effect comparable to that of sonic pingers tested in previous studies with harbor porpoises in the same floating pen [START_REF] Kastelein | The effects of acoustic alarms on harbour porpoise (Phocoena phocoena) behaviour[END_REF]1997[START_REF] Anonymous | Annex I, Report of the Sub-Committee on Small Cetaceans[END_REF], 2001). During test periods with Signal 4, the porpoises hardly visited their preferred north-western corner of the pen (Fig. 1).

The animals did not swim to the areas with the lowest SPLs, but tolerated higher SPLs (Table 1). Each animal had a preferred individual general surfacing area, suggesting that the porpoises had little influence on each other's swimming behavior. No effect of the duty cycle, when comparing pulsed signals of 70 kHz and the same SL (137 dB re 1µ Pa, rms), was found (Signals 1 (0.3 s) and 2 (1 s)). For the 70 kHz pulsed signals (Signals 1, 2 and 4) the porpoises generally swam in an area with exposure SPLs of around 117 dB, while they accepted much higher exposure SPLs during continuous 70 kHz Signal 3 (129-131 dB; Table 1). More specifically, they swam further away from 70 kHz Signal 4 (2 s signal, 4 s interval, SL 147 dB) than from 70 kHz Signal 3 (continuous, SL 148 dB), although both were 70 kHz signals with similar source levels. Commercially available pingers are designed to produce pulsed signals mainly to reduce energy demands on the batteries, but the present study suggests that pulsation also increases the deterrent effect. Alarm devices for humans usually also consist of pulsed signals, as the perceived urgency is higher for pulsed signals than for continuous signals, to which habituation occurs also sooner [START_REF] Edworthy | Improving auditory warning design: Relationship between warning sound parameters and perceived urgency[END_REF][START_REF] Haas | Perceived urgency of and response time to multi-tone and frequency-modulated warning signals in broadband noise[END_REF][START_REF] Edworthy | Auditory warnings in noisy environments[END_REF].

The sensation level of a signal depends on its frequency, the porpoise's hearing threshold at that frequency (or frequency band), and the ambient noise level and spectrum.

The harbor porpoise has a U-shaped audiogram [START_REF] Kastelein | The influence of acoustic emissions for underwater data transmission on the behaviour of harbour porpoises (Phocoena phocoena) in a floating pen[END_REF], and is more sensitive (equivalent) spectrum level of the background noise (in dB re 1 µ Pa /√Hz), CR the critical ratio of the hearing system (in dB re √Hz), and DI the receiving directivity index (in dB). The DI of harbor porpoises for 70 kHz is approximately 8.5 dB and for 120 kHz approximately 11.2 dB (based on a regression line through three data points; Kastelein et al., 2005b). No information is available on the CR of harbor porpoises, but it can be estimated from human data and the measured CR of bottlenose dolphins, Tursiops truncatus [START_REF] Johnson | Masked tonal thresholds in the bottlenosed porpoise[END_REF]estimated CRs: 45.5 dB at 70 kHz and 49 dB for 120 kHz). Due to the spectrum and level of the ambient noise in the floating pen, the theoretical masked hearing thresholds for the two test frequencies in the present study were similar (76 and 74 dB; Table 1). This calculation shows that all five test signals were audible to the porpoises throughout the floating pen.

The porpoises tolerated lower exposure SPLs for 120 kHz signals than for 70 kHz signals. This is expected considering that they have higher hearing sensitivity for 120 kHz signals than for 70 kHz signals (Table 1). However, the theoretical masked detection thresholds (hearing thresholds determined by the background noise level) are similar for both test frequencies. It is possible that the 120 kHz continuous signal interfered with the porpoises' echolocation and was thus tolerated only at lower levels, or that harbor porpoise critical ratios around 120 kHz are lower than expected based on extrapolating those of bottlenose dolphins.

Effects on other marine fauna

Ultrasonic pingers > 70 kHz are not audible to pinnipeds, so have the advantage that they cannot attract pinnipeds to the catch in gill nets (by the so-called "dinner bell" effect).

The hearing frequency range of most fish is up to 2 kHz (higher for clupeid fish; [START_REF] Popper | Effect ultrasonic sounds on harbor porpoises capabilities of teleost fishes[END_REF]. The higher the frequency of a pinger, the less likely it is that fish species will react to it. Some commercially available pingers produce, in addition to the advertised signals, low frequency signals to which some fish species react behaviorally [START_REF] Kastelein | Effects of acoustic alarms, designed to reduce small cetacean bycatch, on the behaviour of North Sea fish species in a large tank[END_REF].

Such reactions may lead to reduced catch rates, which may encourage fishermen not to use the pingers.

Limitations

Some caution is needed when interpreting the data presented here. Only two animals were available for the present study, so it may be risky to extrapolate the findings to all members of the species. Age, sex, location, context and experience may influence the behavior of individuals, and differences in the reactions of two harbor porpoises to pingers were described by [START_REF] Kastelein | The influence of three acoustic alarms on the behaviour of harbour porpoises (Phocoena phocoena) in a floating pen[END_REF]. In addition, the animals in the present study may have influenced one another, although this is not likely because they occupied different areas in the pen, and did respond differently to the five test signals.

In the present study the alarm signals were switched on at the SLs reported. This sudden onset may have startled the porpoises. In the wild, a marine mammal can swim freely towards a sound source, and may be able to regulate the speed with which the exposure SPL increases.

However, in the present study the porpoises did not habituate to the sounds during the 15minute test sessions or over the course of the study (15 sessions over approximately 1 month per signal), and in a similar study porpoises returned to baseline behavior immediately after sounds ceased [START_REF] Kastelein | The effect of acoustic alarms on the behavior of harbor porpoises (Phocoena phocoena) in a floating pen[END_REF]. This suggests that little of the behavior of the animals observed during the 15-minute test sessions can be ascribed to a 'startle' effect.

The present study was limited by animal welfare considerations. The SL of the test signals was increased only to the point that the animals moved away from their preferred north-western corner of the pen, and not so high that they moved to the other side of the pen.

ACCEPTED MANUSCRIPT

-16 -Kastelein et al., Effect ultrasonic sounds on harbor porpoises Also, the animals were not exposed to the alarm signals for very long time periods (e.g. for several days) to test potential habituation to the sounds.

Each odontocete species probably reacts differently to particular sounds. In a study in which a striped dolphin (Stenella coeruleoalba) and a harbor porpoise were subjected to the same alarm, the porpoise swam as far away as possible from the sound source, whereas the striped dolphin showed no behavioral response [START_REF] Kastelein | Differences in the response of a striped dolphin (Stenella coeruleoalba) and a harbour porpoise (Phocoena phocoena) to an acoustic alarm[END_REF]. The results of the present study are probably only valid for harbor porpoises under the test conditions.

Nevertheless, the results are useful for evaluating which parameters are most likely to be effective in the field.

Suggestions for future research

The present study has shown that ultrasonic signals can deter harbor porpoises from a particular area for a certain period of time. However, the following research should be carried out to make acoustic alarms more efficient and less acoustically polluting: 1) Discover which parameters of sounds (e.g. pulse duration, duty cycle, SL, harmonic distortion, spectrum) are important in rendering them suitable for use as acoustic alarms.

For this research, the SL must be more tightly controlled experimentally than was possible in this study.

2) Study the effects of masking on sounds. During rainfall, part of an alarm's spectrum may be masked by ambient noise. Observations over seven years of pinger research suggest that porpoises are less deterred by pingers during strong winds (which cause waves) and rainfall than during calm, quiet weather conditions (Kastelein pers. obs.).

3) Test the effect of potential pinger sounds on other marine fauna, such as fish, and on nontarget marine mammal species.

- 5 -

 5 tested three 200 ms signals between 100 and 140 kHz, with a signal interval of 4 s, and noticed displacement in porpoises in a pool, but the animals' reaction decreased over time. Kastelein et al. (2008) subjected a harbor porpoise to a continuous 50 kHz pure tone signal. The animal swam away from the sound source, and the avoidance threshold Sound Pressure level (SPL) was estimated to be around 108 dB (re 1µPa, rms). The aim of the present study is to discover whether 70 ACCEPTED MANUSCRIPT Kastelein et al., Effect ultrasonic sounds on harbor porpoises and 120 kHz tonal signals are suitable for use in acoustic alarms to deter harbor porpoises, and if so, what sound parameters are important in causing the deterring effect.

  40' E). The harbor is horseshoe-shaped (500 m x 280 m) with the entrance towards the northeast and near the middle of the leeward side of the Oosterschelde surge barrier. The barrier is only closed during exceptionally high tides and storms, and was open at the time of the study.

  . The experimental electronics used to generate and project signals 1 and 2 were manufactured by Airmar Technology Corporation, Milford, U.S.A. Signals 3-5 were produced by a signal generator (Hewlett Packard, model 33120A). The high-frequency transducer, which was used for all five signals, was fixed in a standard Airmar gillnet pinger housing. To allow remote activation of the transducer, the internal battery (D-cell, 1.5 V) was replaced by an external power supply producing 1.5 V at the transducer end of the cable. The sound projection pattern of the transducer was omni-directional in the horizontal plane, and slightly flattened in the vertical direction due to an air cavity at the top of the transducer. The yellow polyurethane housing of the transducer had a diameter of 5 cm, was 15 cm long and weighed 0.3 kg. A 0.4 kg piece of lead, hanging from a string below the transducer, kept it vertically stable at 1.5 m below the water surface in the middle of the north side of the pen (Fig. 1).

(

  Brüel & Kjaer, model 8101) and a 30-m extension cable (Brüel & Kjaer) connected to a conditioning amplifier (Brüel & Kjaer, Nexus type 2690). The amplifier output was connected via a coaxial module (National Instruments, BNC-2090) to a computer (Joheco, PIV-1.6 GHz) with a data acquisition card (National Instruments, model PCI-MIO-16E-1), on which signals were digitized with 12 bits resolution. Sound signals were measured with a scan rate of 512 kHz. Monitoring, recording and analysis of signals were accomplished by the use of software modules developed by IMARES (National Instruments, Labview 6.0). A pistonphone (Brüel & Kjaer, model 4223) was used as a reference to calibrate the hydrophone, the conditioning amplifier and the analogue-to-digital conversion. Reference data were logged separately and used in the analysis module to calculate the SPL. The single frequency Fast Fourier Transform (FFT) analysis covered a bandwidth of 20 Hz to 256 kHz. The signals were subjected to FFT with Hanning window over the activated duration, and pure tones were analyzed in time blocks of 0.2 s (102564 samples).

  An underwater listening system was used to check that the signals were produced during the test sessions. The system was composed of a hydrophone (LabForce 1 BV, model 90.02.01) and a heterodyne bat detector (Batbox III, Stag Electronics Steyning, UK). The hydrophone was placed 1.5 m below the water surface, 1 m away from the transducer (Fig. 1). The five signals were tested between July and October 2002. A maximum of seven sessions per week were conducted. Tests were not carried out during rainfall or when wind speeds were above 4 Beaufort. Initially the aim was to investigate the effect of duty cycle, and only 70 kHz Signals 1 (0.3 s) and 2 (1s) were considered. These two signals were tested 15 times each, in random order, between 2 July and 9 August 2002. Because less effect was seen than expected with 70 kHz Signals 1 and 2, two additional signals (Signals 3 (70 kHz) and 5 (120 kHz)) were tested with higher amplitudes. These signals were tested in random order between 11 August and 25 September 2002. Each signal was tested 15 times, once a day. To study the effect of duty cycle on the behavior of the porpoises, 70 kHz Signal 4 (2 s duration, 4 s interval, SL 147 dB) -10 -Kastelein et al., Effect ultrasonic sounds on harbor porpoises was tested between 30 September and 23 October 2002. This signal was tested five times, instead of the intended 15 times, because due to poor weather conditions the number of days available was less than expected (the study period was limited by the end of the operational season for the floating pen).

  , Fig. 4). Animal 064 swam a similar distance away from the transducer in response to all five signals, but animal 047 responded significantly more ACCEPTED MANUSCRIPT -12 -Kastelein et al., Effect ultrasonic sounds on harbor porpoises strongly to Signal 4 (70 kHz, 2 s pulse duration, 4 s pulse interval, SL 147 dB re 1 Pa, rms) than to the other signals (Table 4 and Fig. 4). A rough indication of the SPL in the general area occupied by each porpoise during test sessions with each of the five signals is shown in

  to 120 kHz signals than to 70 kHz signals. This probably explains why Signal 5 (120 kHz) had a relatively strong deterring effect, despite it being continuous and of lower SL than the 70 kHz signals. During the present study, the signal detection level for part of the hearing frequency spectrum was influenced by the ambient noise at the study site. The theoretical masked detection thresholds (MDTs) for the 70 and 120 kHz test signals can be calculated as follows: MDT = L sp + CR -DI, in which the MDT is for pure tones -dB re 1 µ Pa, L sp is the -14 -Kastelein et al., Effect ultrasonic sounds on harbor porpoises

Fig. 1 .

 1 Fig. 1. Top view of the study area, showing the locations of the underwater transducer,

Fig. 2 .

 2 Fig. 2. Background noise limits in the floating pen between 100 Hz and 100 kHz. Each

Fig. 3 .

 3 Fig. 3. The Sound Pressure Level (SPL in dB re 1 µ Pa, rms) distribution in the floating pen for

Fig. 4 .

 4 Fig. 4. The adjusted mean distances between the porpoises' surfacings and the transducer

Fig. 5 .

 5 Fig. 5. The adjusted mean number of surfacings of the porpoises during baseline (white

Tables : Table 1

 :1 Parameters of the five test signals used, Source level (SL, expressed in three ways), Sound Pressure Level (SPL) range (dB re 1 µPa, rms) in the pen, approximate SPL in the general area occupied by each porpoise during test sessions, and the theoretical masked detection threshold (MDT) of the signals for porpoises in the ambient noise conditions of the study area.

	Sig-	n	Funda-	Pulse	Pulse	Duty	SL fund	SL av	SL E	SPL	SPL	MDT
	nal		mental	duration	interval	cycle	(dB)	(dB)	(dB)	range in	in area	(dB)
			frequency	(s)	(s)	(%)				pen	occupied
			(kHz)							(dB)	by
											porpoises
											(dB)
	1 15	70	0.3	4	8	137 126 132 124-105	~115	76
	2 15	70	1	4	25	137 129 135 124-105	~115	76
	3 15	70	Cont.	0	100 148 148 --137-118	~130	76
	4 5	70	2	4	50	147 144 150 137-115	~120	76
	5 15	120	Cont.	0	100 134 134 --119-102	~110	74
	n = number of sessions conducted
	SL fund =Source Level of fundamental frequency (rms pulse level) in dB re 1 µPa/1 m
	SL av = averaged Source Level per pulse cycle (including duty cycle influence) in dB re 1
	µPa/1 m
	SL

E = source energy level in dB re 1 µPa 2 s/1 m ACCEPTED MANUSCRIPT -30 -Kastelein et al., Effect ultrasonic sounds on harbor porpoises

Table 5

 5 Results of paired t-tests (where appropriate; otherwise Wilcoxon matched pair tests, marked with *) to compare the difference between numbers of surfacings in baseline and test (baseline minus test) to zero. Significant differences were found in only three cases. n = sample size (number of sessions), T/W = test statistic for paired t-test or Wilcoxon matched pair test, P = probability.

	Animal 047	n	T/W	P
	Signal 1	15	-0.29	0.778
	Signal 2	15	-2.38	0.032
	Signal 3	15	-1.29	0.217
	Signal 4	5	0.0*	1.000
	Signal 5	15	1.04	0.314
	Animal 064
	Signal 1	15	2.34	0.035
	Signal 2	15	1.25	0.233
	Signal 3	15	-2.85	0.013
	Signal 4	5	6.0*	0.787
	Signal 5	15	-0.30	0.765

ANCOVAs (one for each signal) for the effect of period (baseline and test) with session as the

Acknowledgements

We thank Rob Triesscheijn, Mariska Muijsers, Natascha Lor, Brigitte Slingerland, Marjolijn Essenburg, Famke de Hond, Betty Braaksma and Paul Wensveen. Steven Christensen (Airmar Technology Corporation, Milford, NH, U.S.A.) provided the experimental Airmar alarm signal generating system. We thank Lee Miller (Odense University, Denmark), Finn Larsen (Technical University of Denmark), Simon Northridge (Sea Mammal Research Unit, St.

Andrews, UK) and two anonymous reviewers for their comments on the manuscript. We thank Jan de Kam and Hans Jager (both of RWS, Topshuis, Neeltje Jans) for postponing dredging activities at Neeltje Jans for this study. The study was funded by SEAMARCO, Harderwijk, The Netherlands, and The Netherlands North Sea Directorate (contact: Christoph Reuther), and conducted under authorization of the Netherlands Ministry of Agriculture, Nature Management and Fisheries, Department of Nature Management, with endangered Species Permit FEF27 06/2/98/0184.

Table 2 ANCOVAs (one for each signal) for the effect of period (baseline and test) with session as the covariate on the distance between surfacings and the transducer for animal 047; Df = degrees of freedom, Adj. MS = adjusted mean squares, F = test statistic, P = probability. Both animals moved away from the transducer when each of the five signals was emitted. The bars indicate standard errors. An * indicates a significant difference between mean baseline and mean test values.

Source of variation

Figure 1