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Alkaline phosphatase activity in the western English Channel:Elevations induced by high summertime rainfall

Andrew P. Rees a,*, Sam B. Hope a,b,1,2, Claire E. Widdicombe a,1, Joanna L. Dixon a,1,E. Malcolm S. Woodward a,1, Mark F. Fitzsimons b,2

a Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth PL1 3DH, UKb Centre for Chemical Sciences, SEOES, University of Plymouth, Plymouth, Devon PL4 8AA, UK

a r t i c l e i n f o

Article history:Received 31 July 2008Accepted 5 December 2008Available online 14 December 2008

Keywords:alkaline phosphatasephytoplanktonphosphorusnitrogenwestern English ChannelL4River Tamar

a b s t r a c t

Alkaline phosphatase activity (APA) was determined in bulk particulate material and in a single-cell (ELF)assay at station L4 in the western English Channel during the summer of 2007. Throughout this period,the UK experienced its heaviest summertime rainfall since records began in 1914; with the result thatriverine run-off into coastal waters was also elevated relative to long-term averages. Between May andAugust 2007, three distinct periods of elevated river run-off were observed which resulted in salinityminima at L4 on days 141, 190 and 232. An extended period of high river run-off between days 170 and210 was responsible for decreases in near-surface salinity at L4 from 35.2068 to a minimum on day 190 of34.7422. This contributed to the development of haline stratification which supported the developmentof an intense bloom of the centric diatom Chaetoceros debelis, with maximum observed chlorophylla concentration of 8.69 mg l�1. Minima in salinity, and maxima in chlorophyll concentration on day 190were coincident with a peak in river-derived dissolved inorganic nitrogen (DIN) of 1.9 mmol l�1 whichwas >5 times greater than the summertime mean and 24 times the concentrations experienced at L4 onweeks immediately before and after. There was no accompanying increase in dissolved inorganicphosphorus (DIP), and the DIN:DIP ratio increased to 49. With the inherent phosphorus stress that thiscaused, rates of APA increased from <4 to 42.4 nmolP l�1 h�1. ELF analysis on day 197 identified two taxaactively expressing alkaline phosphatase: the dinoflagellate Prorocentrum micans and ciliate Tiarana sp.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Phosphorus is an essential macronutrient required by allorganisms; as a component of nucleic acids, for intracellulartransfer of energy and for the production of phospholipidmembranes, the building blocks for all organic material (Munn,2004; Ruttenberg and Dyhrman, 2005; Nicholson et al., 2006). Inmarine waters, the supply of P to phytoplankton is largely as theinorganic orthophosphate – DIP (Cembella et al., 1984; Nicholsonet al., 2006), however during times when DIP is depleted relative toother nutrients, microbial activity, phytoplankton growth andtherefore primary production is often considered to be P-limited(Karl et al., 1995; Shaked et al., 2006). Under such conditions ofphosphorus stress a number of bacteria and phytoplankton are able

to derive their P requirement from the dissolved organic phos-phorus (DOP) seawater component (Monaghan and Ruttenberg,1999; Ruttenberg and Dyhrman, 2005). This is achieved via theexpression of the enzyme, alkaline phosphatase – AP (see forexample, Perry, 1972; Cembella et al., 1984) which hydrolyzesorthophosphate from the organic moiety of the DOP compound.The use of DOP as a source of P by phytoplankton occurs either atlow concentrations of DIP, or at elevated ratios of DIN:DIP, as suchalkaline phosphatase activity (APA) can be used to determine thephytoplankton community P status because AP is highly regulatedand dependent on ambient DIP or intracellular concentrations (e.g.Nicholson et al., 2006).

Phosphorus limitation of microbial activity is generally associatedwith oligotrophy or nitrogen enriched coastal waters, but phos-phorus stress due to low levels of DIP may also occur seasonallyfollowing spring bloom removal of dissolved nutrients. This studywas based within the western English Channel (WEC), which islocated at the boundary of oceanic (Atlantic Ocean) and neritic(eastern English Channel) waters (Southward et al., 2005). Theseasonal pattern of nutrient succession which takes place in waters of

* Corresponding author.E-mail address: apre@pml.ac.uk (A.P. Rees).

1 Tel.: þ44 1752 633100; fax: þ44 1752 633101.2 Tel.: þ44 1752 584555; fax: þ44 1752 233035.

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Estuarine, Coastal and Shelf Science 81 (2009) 569–574

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the western English Channel (WEC) is described by Butler et al.(1979). The nature of these waters is defined by complete mixing ofthe water column during the winter period and thermal stratificationover the summer months. The annual variance in the dissolvedinorganic nutrient (DIN & DIP) distribution is characterised bydistinct minima in concentrations during summertime followingremoval by phytoplankton and a corresponding, but not necessarilyequal increase in dissolved organic nitrogen (DON) and DOP. Thismeans that there are always significant amounts of dissolved nutri-ents present and the ratio between total N and total P is relativelyconstant through the year (Butler et al., 1979). Although inorganicnutrients become relatively depleted (DIP< 0.05 mmol l�1;DIN< 0.5 mmol l�1), these shelf waters remain productive andsupport regular algal blooms (Holligan and Harbour, 1977) fromspring through to autumn. Davies and Smith (1988) found duringa summertime survey of the WEC that all phytoplankton communi-ties, which varied in terms of chlorophyll a concentration from 0.3 to>10 mg l�1, displayed a level of APA suggesting that at that time, allpopulations were showing evidence of phosphorus stress.

Productivity in the WEC is considered to be limited by nitrogen(e.g. Jordan and Joint, 1998), but it would seem from the work ofDavies and Smith (1988) that due to the low concentrations of DIPexperienced during summer the microbial communities are alsolikely to be phosphorus-stressed, inferring an important role forDOP in supporting primary and bacterial productivity. This studywas conducted to perform an analysis of the factors which mayinfluence phosphorus stress of microbial communities undersummertime conditions in coastal waters of the WEC and toqualitatively assess which component of the phytoplanktoncommunity were responsible for APA.

2. Methods

2.1. Study area

Seawater samples were collected weekly at station L4 (Fig. 1),which is a long-term monitoring station in the Western ChannelObservatory (WCO) (www.westernchannelobservatory.org.uk)during June and July of 2007 (Days 169–197). The L4 samplingstation is located approximately 16 km south west of Plymouth in55 m of water, and is the most frequently visited of the WCO long-term sampling stations (see for example Southward et al., 2005).

Seawater samples were collected from the near surface andsecchi depth (operationally defined as the depth at which thesecchi disk was no longer visible) into acid-cleaned containerswhich were maintained in the dark at ambient seawater temper-atures. Samples were returned to the shore-based laboratory whereexperimental procedures were initiated within 3 h of collection.

2.2. Bulk alkaline phosphatase acitivity

At each depth (surface and secchi), seawater samples werestored in two 5 l acid-cleaned containers, the contents of one wasfiltered through a 0.2 mm polycarbonate filter in order to determinerates of dissolved APA, the other was unfiltered to allow total APA tobe determined. The fraction of APA associated with microbial cellsis referred to hereafter as particulate APA (pAPA), being thedifference between the total and dissolved components, and it isthis fraction to which we restrict this study. APA was assayed usinga sensitive fluorometric protocol similar to Perry (1972). APA inseawater samples was assayed as an increase in fluorescence, as anartificial P substrate, 4-methylumbelliferyl phosphate, was enzy-matically hydrolyzed to release the fluorescent product methyl-umbelliferone. 8 ml of seawater sample was distributed intoa series (8 time-points� 3 replicates) of 12 ml acid-cleaned glass

tubes, to each of which, 1 ml of substrate (0.5 mg ml�1

4-methylumbelliferyl phosphate in 10 nmol l�1 Tris buffer) wasadded to give a final concentration of 0.25 mmol l�1 (cf. Perry, 1972 –0.1 mmol l�1; Davies and Smith, 1988 – 2.0 mmol l�1). These werethen incubated at 25 �C in the dark and fluorescence determined(excitation wavelength of 365 nm, emission wavelength of 455 nm)initially and at 30 min intervals. The procedure was calibrated oneach occasion against six triplicate methylumbelliferone standards,with a concentration range of 5–35 nmol l�1, which was sufficientto account for all analyses made.

2.3. Cell-specific alkaline phosphatase activity

A qualitative, cell-specific assay was conducted on Day 197 usingenzyme labelled fluorescence – ELF (Dyhrman and Palenik, 1999;Nicholson et al., 2006) to examine which microplankton specieswere responsible for APA. To determine APA on a cell-specific basisrequires the addition of a phosphomonoester substrate (ELF-97)which produces a yellow green fluorescent precipitate at the site ofester hydrolysis and thus labelling any cells that express AP. A singlesurface seawater sample of 100 ml was filtered onto a 0.2 mm 25 mmpolycarbonate filter at low pressure (< 40 cm Hg vacuum) until thefilter was just dry. 1 ml of 70% ethanol was added and left for 30 minin the dark at room temperature. 100 ml of ELF product (MolecularProbes, ELF97 Endogenous phosphatase detection kit) was addedand the sample was incubated for a further 30 min before cells wereexamined for ELF fluorescence. Microscopic analysis was performedin a Sedgewick Rafter chamber using a Leica epifluorescencemicroscope. Cells were identified using phase contrast illuminationand then examined with a 100-W mercury lamp and a long passDAPI filter set (excitation 350 nm, emission 520 nm) for ELF activity.

2.4. Inorganic nutrient analysis

Dissolved inorganic nitrogen (DIN) and DIP concentrations weredetermined on surface samples only using a 5 channel Bran and

4°5°6°Longitude (W)

Latitu

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E1

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Fig. 1. Location of L4 within the western English Channel (original map derived fromwww.aquarius.ifm-geomar.de).

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Luebbe AAIII, segmented flow autoanalyser within 3 h of samplecollection, using analytical protocols detailed in Woodward andRees (2001).

2.5. Chlorophyll a concentration

Chlorophyll a concentrations were measured on surface watersby fluorometric (Holm-Hansen et al., 1965) analysis of extractedpigments. 100 ml samples were filtered through glass fibre filters(GF/F – Whatman Ltd.). Pigments were extracted by addition of 90%acetone and stored in the dark at 4 �C for approximately 12 h priorto analysis.

2.6. Phytoplankton biomass

Samples for phytoplankton enumeration were fixed with acidLugol’s solution (2% final concentration) and stored in cool, darkconditions until analysis by inverted settlement microscopy(Utermohl, 1958). Where possible cells were identified to specieslevel and divided in groups comprising diatoms, dinoflagellates andciliated protozoa according to Widdicombe et al. (2002). Cellvolumes were calculated using appropriate geometric shapes andconverted to biomass using the equations of Menden-Deuer andLessard (2000).

2.7. Environmental variables

A Sea-Bird 19þ CTD was deployed at each station for thedetermination of seawater temperature and salinity, metereo-logical observations were logged at the PML meteorological station.Flow data for the Rivers Tamar, Tavy and Lynher were kindlyprovided by the UK Environment Agency.

3. Results

3.1. Environmental conditions

The UK Met Office reported that the rainfall for England andWales was double the monthly average during July 2007 (www.metoffice.gov.uk/climate), whilst the BBC weather centre reportedthat Met Office figures showed that the summer of 2007 was thewettest on record (since 1914), with a total of 358.5 mm falling onthe UK, which was marginally higher than the previous record from1956 of 358.4 mm. In the south west of England, rainfall for thesummer was 164% of the 1961–1990 average, which resulted inanomalously high run-off from local rivers. Between May and theend of August 2007, river run-off was characterised by 3 periods ofelevated flow, each of which depressed the observed surfacesalinity at station L4 from a pre and post rainfall mean of 35.2068 tominima of 35.0091, 34.7422 and 34.9998 on days 141, 190 and 232respectively (Fig. 2). In Fig. 3a, the flow at the Environment Agencygauging station at Gunnislake (30 km upstream from the mouth ofthe River Tamar) is shown for the study period (days 169–197) in2007, and for comparison, the same period in 2006. Between day170 and beyond day 200, river-derived freshwater at L4 contributedto the maintenance of a stratified water column (Fig. 3b), whichfavoured the development of an intense bloom of the centricdiatom Chaeotoceros debelis as indicated by the fluorescence andchlorophyll profiles (Figs. 3c and 4). Although diatoms dominatedthe community throughout this period, their biomass peaked onday 183 (174 mgC l�1), while dinoflagellate biomass increased overthe subsequent two weeks (reaching 10.2 mgC l�1 on day 197).Penetration of incident irradiation through the water column wasassociated with changes in phytoplankton biomass so that the

Secchi depth varied from 6.5 m, 5 m, 4 m and 8 m on days 176, 183,190 and 197 respectively.

Associated with peaks in river run-off and salinity minima wereelevated concentrations of surface DIN at L4 (2.26, 1.90 and2.23 mmol l�1 relative to 2007 summertime mean of 0.35 mmol l�1),which were not paralleled with changes in DIP (mean� 1s.d.¼ 0.034� 0.004 mmol l�1).

3.2. Alkaline phosphatase activity

pAPA was measured on five occasions at the surface (days 169,176, 183, 190 and 197) and on four occasions at the secchi depth(days 176, 183, 190 and 197). Prior to the peaks in fluoresence andDIN which were observed on day 190, orthophosphate release frompAPA was less than 0.4 nmolP l�1 h�1 (c¼ 0.26 nmolP l�1 h�1) insurface waters and less than 4.0 nmolP l�1 h�1 (c¼ 2.3 nmolPl�1 h�1) at the secchi depth (Fig. 4). On day 190, pAPA rates of 42.4and 40.3 nmolP l�1 h�1 at surface and secchi depth respectivelywere observed, returning to 1.0 and 3.0 nmolP l�1 h�1 by day 197.

Unfortunately ELF analysis was not performed during the peakof APA on day 190, however on day 197 a single investigation wasperformed to assess which component of the phytoplanktoncommunity was expressing the AP enzyme by labelling individualcells with ELF. Two separate species, the dinoflagellate Pro-rocentrum micans and ciliate Tiarana sp. were observed to displayindications of phosphorus stress, as shown by the green fluores-cence of the ELF substrate (Fig. 5) which is precipitated at the site ofenzyme activity.

4. Discussion

The expression of AP by phytoplankton during times of phos-phorus stress is to be expected (e.g. Perry, 1972; Dyhrman andPalenik, 1999). Davies and Smith (1988) performed a study of APA at27 sites through the WEC in July 1987, and found a measurable rateof APA at each station, indicating a degree of phosphorus stress forall of the communities encountered. Davies and Smith (1988) foundin their study that for a chlorophyll range of approximately0.3 mg l�1 to >10 mg l�1, that APA was inversely related to chloro-phyll concentration, implying that phytoplankton populations arelargest when DIP is greatest. This is in contrast to the present study,where maximum rates of pAPA were coincident with maxima inphytoplankton chlorophyll (Fig. 4), although the two studies aregeographically distinct, this one being close to shore and theinfluence of local rivers, whilst the study of Davies was further

0

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lo

w (m

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34.6

34.8

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190 232141

Fig. 2. Flow rates for River Tamar (A) and its subsidiaries, Tavy (6) and Lynher (B)from Environment Agency gauging stations for January to December 2007, and themarked changes in surface salinity observed at station L4 (,).

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offshore in waters that were more typical of oceanic than coastal. Inrecent history the coastal waters close to Plymouth have beencharacterised by summertime nitrogen depletion with respect toDIP, and whilst concentrations of all inorganic nutrients remain lowthroughout the summer, it would appear that N rather than P is thenutrient which limits phytoplankton productivity. During most of

this study period, ratios of DIN:DIP were significantly lower thanthe canonical Redfield value of w16 (Redfield et al., 1963), indeedfor four of the five weeks that measurements of APA were taken, theratio of DIN:DIP was less than 5, and it was only on three occasionsover the whole summer, when minima in salinity coincided withelevated DIN concentrations did DIN:DIP ratio increase significantlyto 50, 62 and 19 on days 141, 190 and 232 respectively. Despite thelow ratios of DIN:DIP observed, pAPA was taking place at ratescomparable to those observed by Davies et al. (1988), whichsuggests that under these conditions that either the Redfield ratio isnot indicative of nutrient limitation, that is the N:P requirement isdifferent to 16, which may be so (e.g. Geider and LaRoche, 2002), orthat the phytoplankton community were accessing nitrogen sour-ces other than NO3

�, NO2� and NH4

þ to balance the available P (sum ofDIP and AP-liberated P). It is well known that urea is often used byphytoplankton (e.g. Wafar et al., 1995; Rees et al., 2002), and it isquite likely that other components of the dissolved organicnitrogen pool are also contributing to the algal nitrogen require-ments (Bronk et al., 2007).

The level of enhanced phytoplankton biomass (chlorophylla¼ 8.69 mg l�1) recorded on day 190, is not wholly unprecedented,but equally is not frequently observed. In a fifteen year record ofregular sampling at L4 between 1992 and 2007, chlorophyllconcentrations have only been observed to be greater than8 mg m�3 on six other occasions. These were associated with the

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Fig. 3. a) River flow at Gunnislake gauging station on the River Tamar for years 2007 (B) and 2006 (C), data courtesy of the Environment Agency. Panels b) Salinity and c)Fluorescence show contoured profiles of the whole water column, derived from CTD profiles on days 162, 176, 183, 190, and 197. The CTD was not deployed on day 169 due to seaconditions, contours between days 162 and 176, should be treated with caution, and are highlighted by the shaded area in panels b) and c).

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Fig. 4. Rates of particulate Alkaline Phosphatase Activity at surface (A) and secchi (>)depths, chlorophyll (C) and DIN (B) concentrations at Station L4 during June and July2007.

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peak of the spring bloom of 1999 and 2003 and during the summerof 1993 and 1997. During this study phytoplankton biomassappeared to increase from day 175 onwards (Fig. 3d), as thefreshwater stratification due to enhanced river run-off developed(Fig. 3c). The fact that APA is measureable, though low, suggeststhat the low concentrations of DIP were imparting a degree ofphosphorus stress on the microbial populations. It is interesting tonote that there is little or no change in inorganic nutrientconcentrations during this time (Fig. 4), which implies thatincreases in phytoplankton growth rates were balanced by rates ofnutrient supply. It is only on day 190, when DIN concentrations(coincident with the distinct minima in salinity) increased bya factor of 24, relative to day 183, to 1.9 mmol l�1, and the ratio ofDIN:DIP reached a maximum of 62 did an apparent condition ofsevere phosphorus stress occur, so that the rate of pAPA increasedfrom< 4.0 nmolP l�1 h�1 to w40 nmolP l�1 h�1. Whilst acknowl-edging our earlier arguments regarding the reconciliation of N:Pwith Redfield ratios, and at the same time wishing to determinewhether these rates are sufficient to match the phytoplankton andbacterial P requirements we find that this rate of pAPA is enough toameliorate phosphorus stress (at 16 N:1P) within 3 h.

Whilst the phytoplankton community was dominated by dia-toms, our single ELF analysis (Fig. 5) found examples of dinofla-gellates and ciliates which suggested P stress. It is possible thoughthat this ELF activity was a result of intracellular (as opposed to cell-surface) alkaline phosphatases which can be constitutivelyexpressed and not necessarily as a response to P stress (e.g. Dyhr-man and Palenik, 1999). There was no evidence of diatoms showinga similar condition. This is not unprecedented as other authors havereported similar findings. Huang et al. (2007) observed few diatomslabelled with ELF in the East China Sea in contrast to most of thedominant dinoflagellates, whilst Nicholson et al. (2006) reportedthat less than 12% of diatoms in Monteray Bay, USA were labelled

with ELF compared to, between 34 and 100% of dinoflagellateswhich were labelled. It would appear then that whilst the dino-flagellates may maintain their P requirements through APA on thedissolved organic P component, diatoms have either a high affinityfor phosphate uptake or storage mechanisms which are accessedduring periods of relative depletion. The ciliate Tiarina sp. may beexpressing AP in its own right, however as it is an active grazer ofnanoflagellates, cryptophytes and small dinoflagellates we believethat the fluorescence detected here may result from phytoplanktonthat the ciliate has consumed.

It is recognized that L4 is influenced by the River Tamar(Southward et al., 2005), though at 16 km separation, direct effectsare not always obvious. During the summer of 2007, three distinctminima in salinity were observed at L4 coincident with elevatedperiods of river run-off (Fig. 3). For most of the period there waslittle if any evidence of enhanced nutrient enrichment. This is inpart in respect of the modification of riverine nutrients that takesplace within the estuary (Uncles et al., 2002), so that under thegeneral condition there is only limited contrast between marineand end-of-estuary conditions. Inorganic nutrients in river waterentering the estuarine system are dominated by NO3

�, which duringsummertime is of the order of 200 mmol l�1 (A. Tappin, pers.comm.) Following the rapid removal of DIP at the freshwater end ofthe estuary and the conservative behaviour of nitrate (Uncles et al.,2002), DIN:DIP ratios in the estuary during summertime are foundin the range of 35–90 (Kitidis, PML – pers, comm.). Under condi-tions of enhanced flow such as those observed during this study,there is reduced time for nutrient modification and elevatedflushing can result in the observations of variable freshwaterstratification (Robinson et al., 1986; Southward et al., 2005) at L4with enhanced levels of DIN, as seen in Fig. 4.

Summer 2007 in the UK was the wettest since records began in1914. The UK Meteorological Office attributes this to the position of

Fig. 5. Photomicrographs of a) the ciliate Tiarina sp. and b) dinoflagellate Prorocentrum micans from Station L4 on day 197 following ELF treatment; under light microscopy (leftpanels) and under a long pass DAPI filter set (right panels). Scale bar¼ 20 mm.

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the jet stream; a ribbon of very strong winds which brings weathersystems across the UK, which from early June was further souththan normal with the result that rainfall systems were directedstraight over the UK. It is quite apparent that the elevated run-offfrom local rivers resulted in prolonged periods of freshwaterstratification and episodic pulses of high DIN, coincident withsalinity minima in coastal areas adjacent to Plymouth, or at least atStation L4. It is clear that during these periods of enhanced run-offthat the nutrient stoichiometry of local waters was altered so thatthe low DIN:DIP conditions usually experienced were reversed.This resulted in the microbial population becoming phosphorus-stressed and hence, the manner in which phytoplankton obtainedtheir nutrient requirements was necessarily modified to enable theorganic component of the phosphorus pool to be accessed. In otherregions elevated ratios of N:P as a result of riverine inputs or algalblooms have been associated with phosphorus-stressed commu-nities and the occurrence of HABs (e.g. Huang et al., 2007) and anychange in nutrient stoichiometry driven by environmental pertur-bations is expected to affect phytoplankton activity and diversityand, therefore, the whole ecosystem (e.g. Migon and Sandroni,1999). Our sampling programme did not resolve whether this wasto the detriment of existing populations so that communitieschanged to reflect environmental differences or whether theexisting population were able to adjust their functionalitysufficiently.

5. Conclusions

It is clear that the modified conditions of nutrient stoichiometryand the hydrodynamic regime resulted in clear modifications toecological function, if not the community composition of microbialpopulations at L4. Latest climate model projections indicate that bythe middle of the 21st century it is very likely that average winterrainfall will increase and that summer rainfall will decrease.However, although, it is likely that overall summers will becomedrier, it is also likely that summer rainfall events will becomeheavier (Pope et al., 2007). It is evident that these episodes of highrainfall result in the elevated flushing of river systems observedduring this study, and which have contributed to summertimeblooms of high phytoplankton biomass and enforced phosphorusstress on these phytoplankton and bacterial communities whichmust alter their ecological function in order to maintain rates ofproduction.

Acknowledgements

This work contributes to the NERC funded OCEANS2025 corescience programme of the Plymouth Marine Laboratory. We wouldlike to thank all of our colleagues that contribute to the WesternChannel Observatory, in particular to Tim Smyth for sourcing of dataand for several discussions which contributed to the completion ofthis manuscript and to James Fishwick who collected and processedCTD data. Nutrient analysis was performed by Lisa Al-Moosawi andCarolyn Harris, and chlorophyll analysis by Denise Cummings. Riverflow data was kindly provided by the Environment Agency.

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