Seagrass beds are among the most valuable ecosystems in the world (Costanza et al. 2014), but they are also among those most affected by human activities (Pitanga et al. 2012) and have decreased significantly in recent decades (Short and Wyllie-Echeverria 2000), now occupying a much lower area than in the past (Short et al. 2011). This reduction in coverage has been related to direct and indirect human activities (Hastings et al. 1995, Baden et al. 2003), along with global climate change (Short et al. 2006). Seagrasses provide valuable goods and services (Cullen-Unsworth and Unsworth 2013), and their habitat is specifically targeted for conservation and restoration (Green and Short 2003, Cunha et al. 2012).

Seagrass meadows can be found in the intertidal and subtidal zones along temperate and tropical coastal areas and require specific conditions to grow and develop (Green and Short 2003). Zostera is the most widespread seagrass genus throughout the world, with 14 species currently recognized (Short et al. 2011). The species Zostera noltei is the dominant intertidal seagrass along the Atlantic coast of Europe and northern Africa (Moore and Short 2006), and its meadows are associated with flat areas within soft bottoms (sand mud mixtures) sheltered from high water current (Valle et al. 2015).

Among the several pressures and impacts affecting seagrasses, those occurring in the intertidal zone are mainly caused by human presence in general (Eckrich and Holmquist 2000, Pitanga et al. 2012), and by shellfish activity for human consumption and for live bait in particular (Boese 2002, Dolch and Reise 2010, Nordlund and Gullström 2013).

Although it is evident that shellfish harvesting exerts a high pressure on seagrass meadows (Pitanga et al. 2012), there is only limited research that demonstrates the magnitude of the impact: e.g. Alexandre et al. (2005) and Cabaço et al. (2005) in Portugal; Feigné et al. (2007, in Auby et al. 2011) in France; and Park et al. (2011) in Korea. We therefore aimed to assess the effect of shellfishing activity on Z. noltei beds in an estuary with extensive intertidal flats, where we hypothesized that recreational and shellfishing activities (in terms of digging and trampling) are negatively affecting the development (i.e. shoot density) of the seagrass. This objective was addressed by undertaking a field-based experiment in which we tested the effect of trampling and digging on Z. noltei seagrass beds within sandy environments.

Mean values (and standard deviation) for plant shoot density in both pressure experiments (trampling and digging) are shown in Table 2. In the trampling experiment, while at site A densities increased by 27% from time 0 to time 7 months, at site B no clear differences were found during the experiment, and at site C the densities had decreased by 23% at time 4 months. On the other hand, in the digging experiment, after 7 months the densities had increased in all the treatments. No clear differences were found between treatments, except for time 2 months, when a slight decrease in densities was found in treatments B, C and D (10%, 26% and 8%, respectively), whereas an increase of 35% was observed in treatment A.

Results from the GLMM applied to the trampling experiment (Table 3) showed significant negative differences in Z. noltei shoot density in the interactions between lanes B and C and time (p<0.0001, Fig. 5A) in comparison with the control (lane A). This can be observed in changes in slopes over time on both plots between trampling treatments in Figure 5A: Zostera shoot densities increased throughout the growing season in the control lane (treatment A), appeared to be stable following moderate trampling (treatment B), and clearly declined under the impact of intense trampling (treatment C). This result indicates that applied pressure in both lanes (B, low trampling pressure; and specially C, high trampling pressure) affected negatively the shoot density in comparison with the control lane (A).

Results from the GLMM applied to the digging experiment (Table 3) showed significant positive differences in Z. noltei shoot density in the interactions between lanes B, C and D and time (p<0.0001, Fig. 5B). As in the case of trampling, Zostera shoot densities also increased throughout the growing season in the control lane (treatment A). However, the slopes in the B, C and D lanes were slightly higher than in the control (Fig. 5B). This result indicates that applied pressures (B, low intermittent pressure, C, high intermittent pressure, and D, constant pressure) positively affected the shoot density in comparison with the control (A). Given that in the digging experiment pressure was only applied between time 0 and time 2, we interpreted this result as recovery of shoot density in times 4 and 7 (Table 3).

Most human activities in estuaries, such as pressure from trampling exerted by simply walking on seagrass, can casue direct physical damage to estuarine plants. At first, this impact might not seem very high because walkers move continuously (without stops) and do not usually produce major footprints and sediment movements except in very fine and soft sediment. Furthermore, the walk normally occurs in areas with relatively compact sediment. Similar to the findings of Eckrich and Holmquist (2000) and Travaille et al. (2015), our trampling experiment reduced the shoot density in seagrass beds in the Oka estuary. Trampling impact was in the Oka estuary negatively was shown to affect the survival and growth rate of Z. noltei planting units in a restoration project carried out by Valle et al. (2015). The impact of trampling increases with standing (which facilitates the sinking of the walker and the physical alteration of the substratum), slower movement and intensified trampling on a small area, which usually occurs when somebody is looking for something in the sediment, as in the case of shellfishing.

Shellfishing combines both trampling and digging (Cochón and Sánchez 2005). Feigné et al. (2007, in Auby et al. 2011) concluded that collection of clams carried out on foot and by hand in Arcachon (France) has a strong impact on Z. noltei seagrass. Similarly, Cabaço et al. (2005) concluded that shellfishing negatively affects Z. noltei populations, despite the high recovery capacity of this species. In the Formosa estuary, Portugal, Cabaço et al. (2005) found that the high frequency and intensity of disturbance caused by shellfishing (especially in summer) prevents the total recovery of the seagrass meadow. These authors pointed out that seagrass can withstand the disturbance of shellfishing provided that, after an isolated disturbance, it is left to recover for one month. However, survival and recovery of the plant will depend on the type of physical damage (cutting or breakage), its magnitude and, if it is only a burial disturbance, on the burying magnitude (deep level and dwell time), as Z. noltei can only tolerate some degree of burial due to its high sensitivity to this disturbance (Cabaço et al. 2008). Furthermore, several authors have found a direct effect of shellfishing on seagrass density (Short and Wyllie-Echeverria 1996, Cabaço et al. 2005, Park et al. 2011), even with low-intensity pressure (Alexandre et al. 2005), but they refer to the activity of harvesting in general and do not specify the shellfishing method as we do here. In contrast, our digging experiment, where pressure was applied between time 0 and time 2, did increase the shoot density, which we associated with the recovery of seagrass in times 4 and 7.

In addition to the direct impact of shellfishing in seagrass abundances, the disturbances produced by gatherers can also affect benthic communities in different ways (García-García et al. 2015): by altering nutrient availability, by altering abundance and diversity of infaunal and epifaunal invertebrates, and by fostering a higher growth of green algae such as Ulva sp. It seems that Ulva sp. grows faster in the holes or puddles caused by shellfishing than on muddy platforms and in tidal ponds (van Alstyne et al. 2011), becoming an extra pressure for seagrass (Brun et al. 2003).

However, Boese (2002) does not consider recreational shellfishing as an important threat for intertidal seagrass, at least in the case of Z. marina. This author points out that seagrass remains in areas that have been heavily disturbed for decades by recreational gatherers, suggesting that long-term effects are minor. Nevertheless, the fact that seagrass does not disappear should not be considered as a minor alteration, since this seagrass would probably have reached larger surface areas if there had been no such shellfishing or if it were of a lower intensity. This might be happening in the Oka estuary, where the number of professional gatherers has decreased in the past decade, but the pressure on seagrass beds is not decreasing, probably due to an increase in the number of non-professional gatherers (especially illegal gatherers). According to Cabaço et al. (2008), the impact of an alteration will depend on the magnitude and frequency of disturbance, so recovery will also vary depending on the damage caused (Short and Wyllie-Echeverria 1996).

As the number of professional clam gatherers has decreased in the last decade, new licences, which are compulsory for shellfishing in the Oka estuary, have not been issued in recent years. This is supposed to be positive for seagrass conservation since the shellfishing pressure is apparently decreasing. However, the increase in recreational and, especially, illegal gatherers is alarming, mainly due to their lack of awareness of the magnitude of the impact they cause and because they are an unidentified (and therefore inaccessible) audience for addressing environmental awareness campaigns.

In these areas, in which different human interests coexist, it is crucial to develop management and conservation plans according to the natural habitats and the activities carried out, especially when the survival of a species or habitat of great interest is at risk. Z. noltei is the only seagrass occurring in the Basque coastal estuaries, and is presently restricted to 3 (Oka, Lea and Bidasoa) out of the 12 estuarine ecosystems of the coast (Valle et al. 2015). It has therefore been listed as an endangered species within the Basque Catalogue of Threatened Species of Wild and Marine Fauna and Flora (23 February 2011). In addition, the Oka estuary includes a wide variety of ecosystems of international importance and is the best preserved estuary in the region. It is the main part of Urdaibai designated as a Biosphere Reserve by UNESCO in 1984. It was also included as a Ramsar site under the Ramsar List of Wetlands of International Importance (year 1992), and in the Natura 2000 Network (Specially Protected Bird Area, SPBA ES0000144, Birds Directive 79/409/EEC) (1994). The Urdaibai shoreline and marshes have recently been chosen among the Atlantic biogeographic region Sites of Community Importance to be designated a Special Area of Conservation (SAC) (ES2130007, Habitats Directive 92/43/EEC) (2013). In such an important natural area, the management strategy must be (re)defined, recognizing the importance of communication, exchanging information, sharing responsibilities and managing the participation of all users (Guimarães et al. 2012).

The high frequency and intensity of current shellfishing disturbance in the Oka estuary will negatively affect the natural development of Z. noltei. To stop this trend of seagrass regression, shellfishing impact must be monitored and managed. The goods and services provided by seagrasses must be highlighted in order to promote less aggressive shellfishing techniques (Cochón and Sánchez 2005). Knowledge of the importance of a resource is the first step towards its management, protection and sustainable use (de la Torre and Rönnbäck 2004, Guimarães et al. 2012).

From our experiments we can conclude that Zostera noltei was affected by the alteration produced by trampling. Further experimentation and monitoring is needed for final confirmation of the potential impacts, especially in the case of digging. It should also assess, for example, the interaction between digging and trampling and enlarge the experimental sample size. All this will help to provide a more robust assessment of direct impacts on seagrasses in this area.

Solving the lack of knowledge of shellfish gatherers about seagrass meadows and their importance could be a key element for estuarine habitat conservation. Lack of information is a threat when activities are carried out in vulnerable natural environments: users are unaware of the severity of the impact that their activity produces. It is therefore necessary to increase understanding (especially among shellfish gatherers) of the importance of seagrass in marine ecosystems and how it can be conserved.