Seaweeds and plastic debris can influence the survival of faecal indicator organisms in beach environments

Marine Pollution Bulletin xxx (2014) xxx–xxx

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Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul

Seaweeds and plastic debris can influence the survival of faecal indicator organisms in beach environments Richard S. Quilliam ⇑, Julie Jamieson, David M. Oliver Biological and Environmental Sciences, School of Natural Sciences, University of Stirling, Stirling FK9 4LA, UK

a r t i c l e

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Keywords: Beach grooming FIO revised Bathing Water Directive (rBWD) Waterborne pathogens Wrack

a b s t r a c t The revised Bathing Water Directive (rBWD) introduces more stringent standards for microbial water quality and promotes more pro-active management of the beach environment through the production of a bathing water profile (BWP). The aim of this study was to determine whether living seaweeds in the littoral zone are colonised by faecal indicator organisms (FIOs), and to quantify the survival dynamics of waterborne Escherichia coli in microcosms containing senescing seaweeds. Living seaweed (Fucus spiralis) was not associated with FIO colonisation, although could be providing a protected environment in the underlying sand. Senescing seaweeds enhanced waterborne E. coli survival compared to plastic debris, with the brown seaweed Laminaria saccharina facilitating greater E. coli persistence than either Chondrus crispus or Ulva lactuca. This has important implications for FIO survival on bathing beaches as the majority of beach-cast biomass is composed of brown seaweeds, which could support significant levels of FIOs. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Escherichia coli and enterococci are faecal indicator organisms (FIOs) commonly used by environmental regulators around the world to provide a measure of microbial pollution in bathing waters (Mansilha et al., 2009). Although FIOs are not pathogenic, compared with quantifying individual waterborne pathogens, their enumeration is relatively straightforward (Quilliam et al., 2011). The primary habitat of E. coli and enterococci is the mammalian gut; however, it is becoming clear that FIOs can persist in the environment for significant periods of time (Ferguson and Signoretto, 2011; Byappanahalli et al., 2012; Hernandez et al., 2014). The environmental pathways for contamination of bathing waters by FIOs can include both diffuse and point-source inputs, e.g. pasture grazed by livestock and sewage discharges. Diffuse source inputs are driven largely by high rainfall, and the subsequent generation of run-off within agricultural catchments is known to contribute to rapid incidental losses of FIOs from land to water via a number of hydrological pathways (Oliver et al., 2005). High flow conditions in streams and rivers may also remobilise stores of FIOs from streambed sediments, to the further detriment of downstream microbial water quality (Muirhead et al., 2004; Yakirevich et al., 2013). While diffuse source contributions ⇑ Corresponding author. Tel.: +44 1786 467769. E-mail address: [email protected] (R.S. Quilliam).

of FIOs to bathing waters tend to dominate during wet weather, the risk of point-source microbial inputs via spills from combined sewer overflows (CSOs) can also increase if sewerage infrastructure struggles to cope with potentially high volumes of urban run-off during intense rainfall and flood conditions (Kay et al., 2008). Maintaining and improving the microbial quality of EU bathing waters is regulated by the Bathing Water Directive (76/160/EEC) and the revised Bathing Water Directive (rBWD; 2006/7/EC). From 2015, the number of designated bathing waters falling below the legally enforceable ‘sufficient’ standard is likely to rise with the first reporting of classifications linked to the rBWD (Chawla et al., 2005; Oliver et al., 2014). The rBWD introduces more stringent standards for microbial water quality and also promotes pro-active management of the beach environment through the production of a bathing water profile (BWP) for all designated bathing waters. The BWP is intended to provide a qualitative appraisal of potential pollutant sources linked to physical, geographical and hydrological characteristics of the beach environment. Compliance with the (r)BWD also drives designations such as the Blue Flag award, the loss of which has the potential to impact on tourism and local coastal economies (Wyer et al., 2010). The rBWD thus provides significant impetus for EU regulators to identify and manage potential environmental reservoirs of FIOs, in addition to agricultural and sewage related inputs, which could contribute to decreased water quality at bathing beaches.

http://dx.doi.org/10.1016/j.marpolbul.2014.05.011 0025-326X/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Quilliam, R.S., et al. Seaweeds and plastic debris can influence the survival of faecal indicator organisms in beach environments. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.05.011

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R.S. Quilliam et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx

The survival of FIOs in beach sand is well documented and certain species of freshwater macroalgae washed up on bathing beaches have been shown to harbour FIOs, including human pathogenic bacteria (Ishii et al., 2006; Skalbeck et al., 2010). The nuisance filamentous green alga Cladophora growing in eutrophic regions of the Great Lakes in the US is often associated with significant levels of Shigella, Salmonella, Campylobacter, and E. coli O157, with evidence for enhanced survival of Salmonella and Shigella in association with Cladophora in freshwater microcosms (Ishii et al., 2006; Byappanahalli et al., 2009). There is also some evidence that marine beach-cast wrack can play a role in enhancing the persistence of FIOs (Imamura et al., 2011). The seaweed surface is an ideal environment for the formation of biofilm, which provides a nutrient-rich habitat for bacterial communities and a niche for protection from harmful UV light and predation (Egan et al., 2013). Living seaweeds can harbour relatively high concentrations of pathogenic species of bacteria, such as Vibrio, e.g. Vibrio vulnificus and Vibrio parahaemolyticus (Mahmud et al., 2007, 2008). However, there remains a significant lack of understanding about the role of living and senescing seaweeds in facilitating the persistence of FIOs in the marine environment, and the effect this has on bathing water quality for the microbial compliance parameters currently used by EU regulators (Anderson et al., 1997; Hannah and Cowey, 2009). In catchments containing areas of intensive agriculture, increased inputs of FIOs to bathing waters could be coupled with excessive growth of seaweeds due to concomitant eutrophic conditions. Understanding the role seaweeds play in facilitating the persistence of FIOs in bathing waters therefore, is crucial for informing environmental management decisions (and improving BWPs) designed to decrease the risk of exposure to potential human pathogens. Consequently, the aim of this study was to, (i) determine the spatial distribution of FIOs and Vibrio spp. colonising living seaweed fronds across the intertidal zone of a beach; and (ii) to quantify the survival dynamics of waterborne E. coli in microcosms containing senescing brown, red or green seaweeds, in the presence or absence of an autochthonous bacterial community.

2. Materials and methods 2.1. Field site and transect sampling Bracken Bay in Ayr, Scotland, consists of a 0.84 km stretch of sand and shingle beach with a bedrock foreshore. This site is adjacent to a popular tourist holiday park and is situated approximately 800 m south–west of a designated bathing water site (Heads of Ayr beach). The rural catchment draining into this part of the coast is just over 4 km2 with intensive dairy farming being the main agricultural activity. Average summer rainfall for the region is 392 mm compared to 331 mm across the rest of Scotland. Transect sampling was carried out in a single day in May 2013 during a low tide. Two transects were used to provide environmental and spatial data in relation to coverage by the tide. The first transect ran from the strandline at the top of the beach down to the water’s edge; the second transect equidistantly cut across the middle of the first transect at the 40 m point and remained parallel to the sea. Samples of living and attached Fucus spiralis together with the sand directly beneath this brown seaweed were collected at 10 m intervals along each of the transects, transferred to zip-lock plastic bags, stored in a cool box for return to the laboratory and processed within 12 h. In addition, samples of seawater were collected in sterile plastic bottles at approximately 1 m below the surface and a selection of plastic rubbish debris was collected from the beach for further microbiological analysis.

2.2. Microbiological quantification and physico-chemical analysis Following EU guidelines, all seawater samples were stored at 4 °C and processed within 6 h of collection. Each water sample was briefly shaken and 100 mL was vacuum-filtrated through a 0.45 lm cellulose acetate membrane (Sartorius Stedim Biotech., Gottingen, Germany). The membrane was aseptically transferred to the surface of a plate containing membrane lactose glucuronide agar (MLGA) (CM1031, Oxoid, Basingstoke, UK); the plate was inverted, incubated at 37 °C and enumerated 24 h later. Sand samples (5 g) were added to 10 ml of sterile seawater and shaken for 15 min at 225 rev min 1, vortexed four times in 30 s bursts, and allowed to settle for 5 min. Seaweed samples (25 g) were added to 100 ml sterile seawater (sterilised by autoclaving), and plastic debris (4 g) added to 10 ml sterile seawater, and shaken for 15 min at 225 rev min 1 and vortexed four times in 30 s bursts. The seaweed wash solution, the plastic debris wash solution, and the sand supernatant were subsequently serially diluted with sterile seawater and used to simultaneously culture loosely attached epiphytic bacteria. Selective media were used to quantify E. coli (MLGA; Oxoid), total Vibrio spp. (TCBS cholera agar; CM0330, Oxoid, UK), enterococci (Slanetz & Bartley agar; CM0377, Oxoid), and heterotrophic bacterial cells (R2A agar; CM0906, Oxoid). All plates were inverted and incubated at 37 °C (E. coli, Vibrio spp. and enterococci) for 24 h, or 25 °C (heterotrophic bacteria) for 48 h, and bacterial Colony Forming Units (CFU) enumerated. Electrical conductivity (EC) and pH were measured directly using standard electrodes, and the seawater turbidity measured with an LP2000 turbidity meter (Hanna Instruments, Bedfordshire, UK) and expressed as nephelometric turbidity units (NTU). An isolate of E. coli colonising the seaweed was further cultured for use in subsequent microcosm experiments. 2.3. Senescing seaweed microcosms Microcosms were created by adding either 100 ml of seawater, or 100 ml of sterile seawater, to a sterile 180 ml polypropylene container with a polyethylene screw cap (Fisher, UK). Seawater microcosms included the native microbial community (including bacterial grazers), which would be competing with E. coli for both resources and space. Comparable sterile seawater microcosms were included to assess how well E. coli competed with this autochthonous community. Representative red (Chodrus crispus), brown (Laminaria saccharina) and green (Ulva lactuca) seaweeds, that were freshly detached and with no obvious signs of disease or damage, were collected from the surf zone at the field site described above. Fronds from each species were cut into strips (2.0 cm by 7.8 cm) and vigorously rinsed in sterile seawater three times to remove loosely attached epiphytic E. coli cells. A single strip of seaweed was added to each replicate (n = 4) microcosm. Control microcosms contained a piece of polythene freezer bag cut to the same size as the seaweed and rinsed in sterile seawater as described above. Each microcosm was inoculated with 1 ml of 3.4  109 E. coli CFU generated from a fresh overnight liquid culture (18 h, 37 °C, 80 rev min 1, grown in LB broth, washed three times in sterile seawater and re-suspended in sterile seawater) of an environmental isolate of E. coli capable of colonising seaweed (isolated from the beach sampling survey), and all microcosms were incubated at 10 °C in the dark. At 1, 2, 3, 4, 5, 6, 7, 9, 13, 21, 27 and 38 days, 1 ml of water was removed from each microcosm, and the concentration of E. coli and total heterotrophic bacteria quantified as described above. On day 38 the seaweed and plastic samples were removed from the microcosms and following vigorous washing in sterile seawater, loosely attached epiphytic E. coli were quantified. Finally, the seaweed tissue was rinsed several times in sterile seawater and then ground in a pestle and

Please cite this article in press as: Quilliam, R.S., et al. Seaweeds and plastic debris can influence the survival of faecal indicator organisms in beach environments. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.05.011

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R.S. Quilliam et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx

mortar, and the resulting homogenate used to enumerate tightly bound and endophytic E. coli cells.

Table 2 Seawater characteristics at the field site, and microbial colonisation of plastic debris. Plastic debris

2.4. Statistical analysis

Vibrio spp. E. coli Enterococci THB pH EC (mS/m) Turbidity (NTU)

Spatial relationships of bacterial concentrations, and pH and EC, on the beach were analysed by regression analysis, and CFU microcosm measurements analysed by analysis of variance (ANOVA) and Tukey multiple comparison tests (Minitab 12.0 software, Minitab Inc., PA, USA).

Seawater

2.64 ± 2.14 log10 CFU g 0.0 0.0 4.62 ± 3.55 log10 CFU g – – –

1

1

0.0 2.15 ± 0.96 log10 CFU 100 ml 0.0 4.98 ± 3.84 log10 CFU 100 ml 7.4 ± 0.2 63.0 ± 0.23 1.62 ± 0.56

1

1

3.2. Survival of waterborne E. coli in seaweed microcosms 3. Results 3.1. Spatial distribution of bacteria colonising living seaweed and sand across the intertidal zone The concentration of bacteria present in the sand beneath the seaweed at each point of both the vertical and horizontal transect was consistently greater than the number of cells recovered from the living seaweed fronds (Table 1). In general, the distribution of Vibrio spp., E. coli and enterococci was patchy across the two transects, and although there were ‘hotspots’ at the top of the beach near the strandline, these were not spatially consistent across the beach. The spatial distribution of culturable total heterotrophic bacteria colonising the seaweed and inhabiting the sand remained fairly constant, and there was no correlation with distance within the littoral zone (Table 1). The electrical conductivity of the sand significantly decreased with distance from the sea (P < 0.05), whilst localised increases in sand pH across the beach were patchy and were not significantly correlated with distance along either of the two transects. At the time of sampling, the concentration of E. coli in the seawater was equivalent to the ‘excellent’ classification (i.e. <250 CFU 100 ml 1) stipulated by the rBWD, whilst neither enterococci nor Vibrio spp. were detected in the seawater (Table 2). Loosely attached Vibrio spp. and autochthonous bacteria were isolated from a selection of plastic rubbish on the beach, which included plastic packaging, plastic bottles and discarded children’s toys, although no E. coli or enterococci cells were recovered (Table 2).

The presence of the brown seaweed, L. saccharina, significantly enhanced E. coli persistence compared with the other two types of seaweed or the plastic control (P < 0.001), and by day 38 the concentration of E. coli in the seawater of the non-sterile microcosm had only decreased by 0.92 log10 CFU cm 2, and by 1.24 log10 CFU cm 2 in the microcosm containing sterile seawater (Fig. 1). The red seaweed, Chondrus crispus, also facilitated the persistence of E. coli in the seawater, although by 21 d less than 50% of the initial inoculum remained detectable, and the concentration continued to decrease. E. coli persistence in the seawater of the microcosms containing the green seaweed U. lactuca had become undetectable after 13 d (Fig. 1). The rate of E. coli die-off was most rapid in the non-sterile microcosm containing the clear piece of plastic, with no E. coli being recovered in the water by 9 d (Fig. 1a); however, in the sterile microcosm containing the piece of plastic, E. coli cells remained detectable up to 27 d (Fig. 1b). The concentration of heterotrophic bacteria recovered from the non-sterile seawater was also significantly higher in the microcosms containing the brown seaweed; similarly, the bacterial die-off rate in the sterile seawater microcosms containing brown seaweed was significantly slower (Fig. 2). There was little difference in bacterial die-off rates in the red and green seaweed and the plastic control non-sterile microcosms (Fig. 2a), nor in the sterile microcosms containing the red seaweed or the plastic control (Fig. 2b). The bacterial die-off rate was the most rapid in the sterile microcosms containing the green seaweed (Fig. 2b), which had undetectable levels by 21 d.

Table 1 Microbial colonisation of living and attached brown seaweed Fucus spiralis and the sand directly beneath each sampled seaweed. Distance from Strandline (m)

0 10 20 30 40 50 60 70 80 90 Horizontal transectb 40 30 20 10 0 10 20 30 40 50 a b

Vibrio spp. (g

1

F. spiralis

Sand

F. spiralis

Sand

67220 1107 546 0 81 40 0 0 0 0

18548 48907 8762 131579 54198 30561 40354 100000 20833 60966

18548 4891 0 0 0 0 0 0 0 0

32056 0 190 188 191 0 0 3400 12500 402

938 2054 802 315 40 40 0 39 1024 0

6415 21792 17822 7421 7600 7443 16265 24675 12941 15637

0 0 120 0 0 0 0 0 0 0

189 1018 396 557 0 1145 402 0 196 0

) (CFU)

E. coli (g

1

) (CFU)

) (CFU)

THBa (g

F. spiralis

Sand

F. spiralis

751 0 0 0 0 440 955 80 0 0

3226 596 381 0 191 0 0 0 0 0

4.38 4.55 4.13 4.12 3.98 3.33 4.10 3.96 3.93 3.83

313 1422 0 0 0 0 0 158 0 0

0 0 0 186 0 573 0 0 0 193

4.23 3.69 4.11 4.09 4.00 3.23 4.50 4.07 4.00 4.13

Enterococci (g

1

1

pH

EC (lS cm

Sand

Sand

Sand

5.15 4.73 4.71 4.75 4.93 5.06 4.90 5.07 4.73 4.70

7.7 7.3 7.1 7.3 7.1 7.0 7.0 7.1 7.0 7.2

204 730 1345 657 1304 1511 1650 1415 1379 1088

4.53 4.50 4.59 4.15 4.36 4.59 4.67 4.96 4.96 4.56

7.4 7.3 7.6 7.5 7.5 8.2 8.2 7.7 7.5 7.2

650 1150 443 350 369 1274 863 1404 1303 2075

) (log10 CFU)

1

s

1

)

THB, total heterotrophic bacteria. The horizontal transect ran parallel to the sea and crossed the perpendicular transect at 40 m from the strandline point.

Please cite this article in press as: Quilliam, R.S., et al. Seaweeds and plastic debris can influence the survival of faecal indicator organisms in beach environments. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.05.011

R.S. Quilliam et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx

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Time (d) Fig. 1. Survival of waterborne E. coli in mesocosms containing green seaweed Ulva lactuca (closed circle), brown seaweed Laminaria saccharina (closed triangle), red seaweed Chondrus crispus (open circle) or plastic (open triangle) in non-sterile (a) or sterile (b) microcosms. Data points are the mean of four replicates ± the standard error.

At 38 d the senescing strips of seaweed and the pieces of plastic were removed from the microcosms and E. coli loosely attached to their surfaces were quantified. In the non-sterile microcosm, the concentration of attached E. coli was significantly higher on the brown seaweed (P < 0.05) than either the red or green seaweed (Fig. 3a); no E. coli cells were recovered from the piece of plastic. In contrast, in the non-sterile microcosms, E. coli was re-isolated from the surface of the strip of plastic, but was undetectable on the green seaweed (Fig. 3b). Brown seaweed was colonised by slightly higher, but not significantly different, concentrations of E. coli than the red seaweed (Fig. 3b). Following the quantification of loosely attached epiphytic E. coli, the seaweed tissue was ground in a pestle and mortar, and the resulting homogenate used to enumerate tightly bound and potentially endophytic bacterial cells. In both microcosms, the highest numbers of tightly bound E. coli cells were associated with the red and brown seaweed, with significantly fewer, or none, attached to the green seaweed. The piece of plastic control was also ground with a pestle and mortar but no E. coli cells were recovered from it (Fig. 3). 4. Discussion 4.1. FIO colonisation of living seaweed fronds and plastic rubbish Living seaweeds can by colonised by a wide range of bacteria, with some evidence that attachment by microbial communities can be host specific (Egan et al., 2013). There are several reports of FIOs being associated with living seaweed fronds, although these have come from warm coastal waters in areas with high pollution and nutrient input (Vairappan and Suzuki, 2000; Musa and Wei, 2008). Our survey of living F. spiralis fronds in the intertidal zone

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Time (d) Fig. 2. Survival of waterborne heterotrophic bacteria in mesocosms containing green seaweed Ulva lactuca (closed circle), brown seaweed Laminaria saccharina (closed triangle), red seaweed Chondrus crispus (open circle) or plastic (open triangle) in non-sterile (a) or sterile (b) microcosms. Data points are the mean of four replicates ± the standard error.

found little evidence of consistent colonisation by E. coli and enterococci. Seaweeds employ several methods to limit epiphytic growth on their surfaces, including the regular sloughing off of their surface layers and by producing secondary metabolites to prevent biofilm formation (Engal et al., 2006; Egan et al., 2014). However, despite these antifouling strategies, living seaweeds such as F. spiralis are still colonised by relatively high concentrations of autochthonous bacteria, including Vibrio spp., and the antagonism from these native microbes may be more important for regulating subsequent colonisation by FIOs (Egan et al., 2013). Antimicrobial compounds are commonly produced by epiphytic bacteria attached to living seaweeds, with a particularly high number of antibiotic-producing species colonising green seaweeds (Lemos et al., 1985). The most abundant form of litter in the marine environment is plastic, and the negative and detrimental consequences of plastic debris on fish, reptiles, birds and mammals, and the introduction of alien species, is well documented (Derraik, 2002; Gregory, 2009). The so-called ‘Plastisphere’, which is capable of supporting diverse microbial communities, could serve as a source of hydrocarbon and provide a novel hydrophobic marine ecological habitat (Zettler et al., 2013), and offers the potential for the wider dissemination of potentially pathogenic bacteria, e.g. Vibrio. The concentration of Vibrio spp. and culturable heterotrophic bacteria found colonising plastic litter in our beach survey was about the same as that colonising the living seaweed fronds, which demonstrates the potential for plastic litter to contribute to microbial loading and water quality. However, although we did not recover either E. coli or enterococci from this plastic debris, our method was only

Please cite this article in press as: Quilliam, R.S., et al. Seaweeds and plastic debris can influence the survival of faecal indicator organisms in beach environments. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.05.011

R.S. Quilliam et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx 12

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Ulva lactuca

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Fig. 3. E. coli cells colonising the seaweed after 38 days, either weakly (open bars) or firmly attached (closed bars) in non-sterile (a) or sterile (b) microcosms. Data points are the mean of four replicates ± the standard error.

designed to remove very loosely attached bacterial cells, i.e. to mimic the tide washing over them several times a day, and the potential for this source of beach litter to act as a reservoir for FIOs clearly needs further investigation. 4.2. Survival of FIOs in sand beneath living seaweed fronds The contamination of beach sand with FIOs and the subsequent effect this can have on bathing water quality has been investigated in both fresh water and marine environments (e.g. Kinzelman et al., 2003; Shibata et al., 2004; Yamahara et al., 2007). Living seaweeds may play a role in facilitating the persistence of FIOs in sand, by providing shelter from harmful UV radiation during low tide and by creating a favourable microclimate and a potential nutrient source. Although the sand directly below each living clump of F. spiralis in our beach survey often harboured relatively high numbers of E. coli, this was patchy and did not correlate with distance from the sea. This probably reflects the heterogeneous delivery of FIOs to the beach, but will also be affected by a range of other environmental variables such as the uniformity of sand grain size (Skalbeck et al., 2010). Clearly, the dynamics of seaweed-sandFIO interactions are underexplored, particularly with regard to possible impacts on bathing water quality. Understanding these interactions from a regulatory perspective is important given the potential for physical mobilisation and resuspension of ‘hotspots’ of FIOs during rising tides or increased wave activity. If living seaweeds can influence the survival of FIOs in the underlying sand, this could impact microbial compliance records associated with EU beaches once the more stringent rBWD standards come into full effect. Similarly, sand beneath piles of beach-cast wrack can support high concentrations of FIOs (Imamura et al., 2011); however, decaying seaweed often attracts birds that continually add fresh

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faecal matter directly onto the sand, which will significantly effect FIO concentrations over time (Kinzelman et al., 2008). A greater understanding of this interaction would be important to underpin any potential revision of FIO source risks identified in BWPs at EU designated bathing waters, as currently seaweeds are only recognised in BWPs as a potential nuisance at times of overproduction and for generating offensive odours when decaying.

4.3. The influence of senescing seaweeds on the persistence of E. coli in seawater The enhanced survival of FIOs in the presence of seaweed may in part be due to the protection provided from harmful UV radiation and desiccation. Additionally, seaweeds could also be providing a source of nutrients in the form exudates, such as dissolved organic carbon (DOC), being released into the surrounding water. The absence of the autochthonous seawater microbial community only made a difference to the rate of E. coli survival in our microcosms containing the piece of plastic, although this was probably due to the plastic being used for biofilm formation. However, our data suggests that E. coli was poor at competing with the native marine microbes in producing biofilms and we found no evidence of colonisation of the plastic in non-sterile seawater. The concentrations of total waterborne culturable heterotrophic bacteria in the senescing seaweed microcosms were greater in the presence of the brown seaweed, supporting reports that these three different groups of seaweed produce varying levels of antimicrobial compounds (Egan et al., 2014). The presence of seaweed in our microcosms significantly increased the survival rate of E. coli compared to the inert piece of plastic, therefore, the seaweed is providing something more than just physical protection or a surface on which to develop biofilm. Damaged and decaying seaweeds release a number of organic compounds including amide- and free amino-N, and osmoprotectants such as glycine betaine. Therefore, the differential persistence of waterborne E. coli cells in the microcosms containing the three different seaweed groups is likely to result from the release of host-specific compounds (together with any antimicrobial compounds) and their associated native microbial communities together with the antagonistic strategies they may employ. The presence of the brown seaweed L. saccharina maintained a greater rate of E. coli survival than the other two seaweed species, with the presence or absence of the native microbial community making little difference to the die-off rate. This could have important implications for FIO survival on bathing beaches as the majority of beach-cast biomass is composed of brown seaweeds, which are able to support significant levels of FIOs (Imamura et al., 2011). Whether this biomass could mask a reduction in FIO loading to bathing waters driven by catchment and wastewater treatment improvements is an important question. The potential for seaweed to impact, either directly or indirectly, on bathing water quality is currently unaccounted for in traditional catchment-based microbial risk assessments and not considered in source apportionment studies (e.g. Kay et al., 2010). During high-flow storm conditions, the role of seaweed-sourced FIOs contributing to microbial impairment of bathing waters could be hypothesized as being less important relative to the considerable FIO load delivered to the bathing zone from diffuse catchment sources. However, their contribution during relatively calm non-storm days may be more significant and difficult to foresee. The removal of beach-cast wrack, which often contains litter and plastic debris, through mechanical beach cleaning is a common management practice at beaches where large quantities of seaweed are deposited (Defeo et al., 2009). Whilst this practice reduces the risk of human exposure to this potential health hazard, so-called beach grooming can actually increase

Please cite this article in press as: Quilliam, R.S., et al. Seaweeds and plastic debris can influence the survival of faecal indicator organisms in beach environments. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.05.011

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concentrations of FIOs in the underlying sand and in the associated surf zone water (Kinzelman et al., 2003, 2004; Russell et al., 2014). It is unclear whether FIOs are able to actively colonise seaweed, or whether the bacterial cells simply become passively absorbed onto the seaweed surface (Anderson et al., 1997). At the end of the microcosm experiment we recovered E. coli cells that were either loosely or strongly associated with the senescing seaweed tissue. Overall, there was little difference in E. coli associated with the brown and the red seaweeds, regardless of presence of the native microbial community, which suggests that E. coli is able to become firmly attached to these two types of seaweed. Taken together, when compared to the concentration of the original inoculum, the concentrations of waterborne E. coli and those cells recovered from the red and brown seaweed surfaces indicate that E. coli survival can be greatly enhanced; although, determining whether it was capable of reproducing in the presence of these senescing seaweeds was beyond the scope of this study. Interestingly, E. coli cells were only recovered from the green seaweed that was in the non-sterile seawater microcosm; whether this is because native marine microorganisms play an important role in facilitating E. coli colonisation through biofilm formation remains unclear. 4.4. Conclusions and environmental management considerations There are several reports of piles of decaying seaweeds contributing to a reduction in bathing water quality (Weiskel et al., 1996; Anderson et al., 1997), and due to its offensive smell local authorities often remove piles of beach-cast seaweed before it becomes a public nuisance. However, removal of these wrack beds can have significant consequences for the provisioning of a number of ecosystem services (Defeo et al., 2009). The removal of wrack from sandy beaches by mechanical grooming for the purpose of gaining tourism awards such as the Blue Flag can have negative impacts on invertebrate biodiversity, feeding shorebirds and nutrient cycling, which is crucial for an area with such little primary production (Dugan et al., 2003, 2011; Gilburn, 2012). Beach grooming can also increase the concentration of FIOs remaining in the sand (Kinzelman et al., 2004) and create hotspots of faecally-derived bacteria across a beach, resulting in the sand acting as both a source and a sink for FIOs and potentially pathogenic bacteria such as Vibrio spp. In this study we have shown that FIOs were not associated with the living attached brown seaweed F. spiralis. Whether this is a universal phenomenon, whereby living seaweeds can inhibit colonisation by FIOs, or whether this is specific to this particular seaweed host requires further work. What is clear from this study is that senescing seaweeds can facilitate the survival and persistence of E. coli for a significant period of time, and could be contributing to reduced bathing water quality. With the imminent arrival of the rBWD, beach managers will be looking at all potential environmental reservoirs of FIOs in order to meet the new microbial compliance standards. Consideration of the extent and volume of senescing seaweed as a potential FIO source at designated bathing waters should be included within updated BWPs. Seaweeds have the potential to significantly facilitate the survival of FIOs, but where there is conflict on bathing water beaches the management of seaweeds will need a sustainable and sensitive approach in order to fully deliver the valuable ecosystem services that decaying seaweeds provide. References Anderson, S.A., Turner, S.J., Lewis, G.D., 1997. Enterococci in the New Zealand environment: implications for water quality monitoring. Water Sci. Technol. 35, 325–331.

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Please cite this article in press as: Quilliam, R.S., et al. Seaweeds and plastic debris can influence the survival of faecal indicator organisms in beach environments. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.05.011