PRIMARY RESEARCH PAPER

Terrestrial runoff may reduce microbenthic net communityproductivity by increasing turbidity: a Mediterraneancoastal lagoon mesocosm experiment

A. Liess • C. Faithfull • B. Reichstein •

O. Rowe • J. Guo • R. Pete • G. Thomsson •

W. Uszko • S. N. Francoeur

Received: 21 October 2014 / Revised: 31 January 2015 / Accepted: 3 February 2015

� Springer International Publishing Switzerland 2015

Abstract Terrestrial runoff into aquatic ecosystems

may have both stimulatory and inhibitory effects, due

to nutrient subsidies and increased light attenuation. To

disentangle the effects of runoff on microbenthos, we

added soil to coastal mesocosms and manipulated

substrate depth. To test if fish interacted with runoff

effects, we manipulated fish presence. Soil decreased

microphytobenthic chlorophyll-a per area and per

carbon (C) unit, increased microbenthic phospho-

rous (P), and reduced microbenthic nitrogen (N) con-

tent. Depth had a strong effect on the microbenthos,

with shallow substrates exhibiting greater microben-

thic net ecosystem production, gross primary produc-

tion, and community respiration than deep substrates.

Over time, micobenthic algae compensated for deeper

substrate depth through increased chlorophyll-a

synthesis, but despite algal shade compensation, the

soil treatment still appeared to reduce the depth where

microbenthos switched from net autotrophy to net

heterotrophy. Fish interacted with soil in affecting

microbenthic nutrient composition. Fish presence

reduced microbenthic C/P ratios only in the no soil

treatment, probably since soil nutrients masked the

positive effects of fish excreta on microbenthos. Soil

reduced microbenthic N/P ratios only in the absence of

fish. Our study demonstrates the importance of light for

the composition and productivity of microbenthos but

finds little evidence for positive runoff subsidy effects.

Keywords Bacteria � Dissolved organic carbon

(DOC) � Enclosure experiment � Microbenthos �Nutrient subsidy � Terrestrial subsidy

Introduction

Current climate change scenarios predict an increase

in frequency and intensity of extreme rainfall events,

even in regions such as the Mediterranean, where

overall precipitation is predicted to decrease (Sanchez

et al., 2004). It may seem contradictory that on one

hand overall precipitation decreases whereas on the

other hand storm events increase. Translated into

weather patterns, this means that the rain that falls

does not fall as a drizzle but in short bursts of heavy

rain fall interspaced by droughts. Heavy rainfall after a

Handling editor: Sonja Stendera

A. Liess (&) � C. Faithfull � B. Reichstein �O. Rowe � J. Guo � G. Thomsson � W. Uszko

Department of Ecology and Environmental Sciences,

Umea universitet, 901 87 Umea, Sweden

e-mail: antonia.liess@emg.umu.se

R. Pete

Laboratoire Ecosystemes Marins Cotiers, UMR5119

CNRS, IRD, IFREMER, Universite Montpellier 2,

Montpellier Cedex, France

S. N. Francoeur

Department of Biology, Eastern Michigan University,

Ypsilanti, MI 48197, USA

123

Hydrobiologia

DOI 10.1007/s10750-015-2207-3

dry period may erode the soil from the surrounding

catchment and increase the pulsed export of terrestrial

nutrients, sediment, and dissolved organic matter into

lakes, lagoons, and coastal ecosystems; this phe-

nomenon has already been observed in the NW

Mediterranean (Alpert et al., 2002). Modeling soil

erosion and sediment yield indicates that increased

rainfall intensity could lead to a significant increase of

sediment yield in Mediterranean watersheds (Nunes

et al., 2009). Since lagoons and estuaries are among

the most altered and vulnerable aquatic ecosystems

and seriously threatened in their ecological and

societal services (Canuel et al., 2012), the conse-

quences of possible future increases in soil erosion and

sediment transport to these systems need clarification.

In general, soils and sediments flushed into aquatic

habitats augment nutrient availability and lead to

greater phytoplankton production (Guadayol et al.,

2009; Pecqueur et al., 2011). But if terrestrial runoff

contains terrestrial dissolved organic carbon (DOC),

nitrogen (N), or phosphorous (P), these resources may

increase bacterioplankton production, boost the mi-

crobial loop (Azam et al., 1983), and intensify nutrient

competition between bacteria and phytoplankton

(Pengerud et al., 1987; Thingstad et al., 2008;

Barrera-Alba et al., 2009). Terrestrial runoff may

therefore shift coastal, phytoplankton-dominated

ecosystems towards bacterial dominance as has been

found in the Baltic Sea (Wikner & Andersson, 2012).

In addition, terrestrial runoff and suspended sediment

inflow to previously non-turbid systems may reduce

light transmission through the water column, thus

further limiting autotrophic production (Cloern,

1987), whereas in already very turbid systems, a

further reduction of light penetration may have

minimal effects. The relative importance of light

reduction, terrestrial DOC subsidies, and mineral

nutrient inputs compared to ambient conditions should

thus determine the relative importance of algal versus

bacterial production (Sandberg et al., 2004).

In shallow coastal zones, both benthic and pelagic

production contribute to net ecosystem productivity.

Whereas pelagic productivity is often more limited by

nutrients, benthic productivity along a depth gradient

is very dependent on light availability (Jager & Diehl,

2014), since nutrients, especially N, are often suffi-

ciently supplied by diffusion from anoxic benthic

sediments (Engelsen et al., 2008). Increased pelagic

production as well as increased turbidity of coastal

waters due to suspended soils and sediments can lead

to light limitation of benthic autotrophic production,

even at shallow depth (as shown for lakes: Ask et al.,

2009). Reduced light availably for algae reduces algal

carbon (C)-fixation rates and therefore algal C:

nutrient ratios (Sterner et al., 1997; Frost & Elser,

2002) but only in the absence of other nutrient

subsidies (Liess & Kahlert, 2007). Thus, terrestrial

runoff may shift the benthic balance from net

autotrophy to net heterotrophy and decrease mi-

crobenthic C: nutrient ratios, as algae and bacteria

growing under low light conditions tend to be low in C

and high in nutrient content (Sterner et al., 1997).

Turbidity further interacts with depth, since light

limitation through terrestrial runoff should be inten-

sified especially for deeper benthos and thus reduce

the depth where microbenthos switches from net

autotrophy to net heterotrophy.

Since fish are a natural part of lake, lagoon, and

coastal communities, it is important to understand

how fish presence interacts with terrestrial runoff and

how fish presence can change water column light

penetration. First, fish may resuspend sediments and

increase turbidity. Second, fish may change water

clarity and thus light availability for benthic algae by

affecting lower food web levels (Carpenter et al.,

2001). For example, the presence of zooplank-

tivorous fish can reduce herbivorous zooplankton

abundance, thereby leading to reduced grazing pres-

sure on phytoplankton (Carpenter et al., 2001). Freed

from grazing pressure, phytoplankton density and

light attenuation in the overlying water column can

increase, thereby increasing light limitation for

benthic algae (Liess et al., 2006). Finally, fish

recycle nutrients, which may increase nutrient avail-

ability for phytoplankton, and thus increase pelagic

primary producer biomass, in lakes (Attayde &

Hansson, 2001; Liess et al., 2006) as well as in

coastal lagoons. This may further increase light

limitation for benthic algae. Thus, fish may intensify

turbidity effects of terrestrial runoff and possibly

reduce nutrient-mediated runoff effects.

Coastal zones with enough light for benthic macro-

and microalgal growth are very productive, but their

role in the global C cycle still remains poorly

understood (Bauer et al., 2013). It is thus important

and timely to determine the potential consequences of

terrestrial runoff on benthic autotrophic and

heterotrophic production. In this study, we focus on

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the microbenthos, which is an assemblage of benthic

algae (the microphytobenthos), bacteria, detritus,

fungi, and meiofauna (ciliates, amoeba, and flagel-

lates). To test the effects of terrestrial runoff on

microbenthic community parameters on shallow and

deep substrates, we added soil to marine mesocosms

containing a natural food web and crossed the soil

treatment (soil/no soil) with a fish treatment (fish/no

fish) to test the following hypotheses:

I. Soil addition increases turbidity as well as

nutrient and DOC availability.

II. Soil addition decreases microphytobenthic

algal biomass and microphytobenthic au-

totrophic production due to light limitation

but increases microbenthic bacterial biomass

due to increased DOC availability.

III. Soil treatment effects are more pronounced at

deeper substrate depth; therefore, soil reduces the

depth where the microbenthic community

switches from net autotrophy to net heterotrophy.

IV. Fish intensify turbidity effects of terrestrial

runoff and reduce nutrient-mediated runoff

effects.

Materials and methods

Study site and ambient environmental conditions

The experiment was conducted in collaboration with

the Mediterranean Platform for Marine Ecosystem

Experimental Research (MEDIMEER) inside the

Thau Lagoon in Sete, France (Latitude: 43�2405200,Longitude: 3�4101700). This marine coastal lagoon on

Northwestern Mediterranean coast has an area of

75 km2, is shallow, with a mean depth of 4 m and a

maximum depth of 10 m, and connected to the sea by

three narrow channels. The Vene River is the main

inlet into the Thau Lagoon. Due to its shallowness, its

large area, and its tidal range (mostly less than half a

meter) in combination with its currents, principally

driven by dominant winds regularly blowing at

10 ms-1, even in summer, stratification in the Thau

lagoon is not observed (\1�C temperature difference

between surface water and 9 m depth, Metzger et al.

2007). Episodic river flash floods occur in the Vene

River and transport terrestrially derived nutrients, such

as N and silicate, into the Thau lagoon. These nutrients

have major impacts on the pelagic community, leading

to blooms of bacteria, picophytoplankton, and di-

atoms, since basal production in the Thau Lagoon is

typically N-limited (Picot et al., 1990; Pecqueur et al.,

2011). During the time of our experiment, nutrient

concentrations in the Thau Lagoon were low

(Table 1), and light levels were high, with daily

maximum rates of photosynthetically active radiation

(PAR) between 1,800 and 2,500 lmol m-2 s-1.

Experimental design

Study design

Experimental manipulations were conducted in twelve

cylindrical mesocosms (transparent 200-lm-thick

vinylacetate mixed-polyethylene bags, Insinoori-

toimisto Haikonen Ky, Finland), 3 m high (thereof

1 m above the water surface) 9 1.2 m diameter.

Mesocosms were closed at the bottom and supported

by a floating pontoon at circa 2.5 m depth. Fish

treatment (fish/no fish) was crossed with soil addition

(soil/no soil) in a 2 9 2 fully factorial design with 3

replicates. Benthic substrates for microbenthic com-

munity (algal, bacteria, fungi, and meiofauna)

colonization were suspended inside each mesocosm,

in a block design, at two depths (shallow: 0.5 m and

deep: 1.5 m). Benthic substrates consisted of stainless

steel metal plates (15 9 30 cm) suspended by string

from a metal bar placed across the mesocosms, with

unglazed ceramic tiles (5 9 5 and 2.5 9 2.5 cm)

attached. Metal plates were arranged on opposite sides

of the mesocosms in order to avoid shading of the deep

by the shallow tiles. The experiment was conducted

from the 8th of June (day 0) until the 20th of June 2011

(day 12). Two days before the start of the experiment,

lagoon water was filtered (\1,000 lm) and pooled in a

tank (nutrient concentrations, see Table 1). From this

tank, mesocosms were simultaneously filled with

water, including a natural pelagic phytoplankton and

zooplankton community. Soil and fish (see below)

were added to the mesocosms on day 0 of the

experiment. At night and during rain events, remov-

able polyethylene hoods covered the mesocosms to

avoid dilution by rainwater and contamination by

waterfowl. To mimic the natural water column mixing

in the Thau Lagoon (turnover rate = 1 day-1), pumps

(Iwaki, MD30MX) circulated the mesocosm water

column at a pumping rate of ca. 2,000 l per day.

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Natural soil

We removed organic matter-rich top soil from the

banks of river the Vene River flowing into the lagoon.

Soil was homogenized, dried, and passed through a

1-mm mesh. Six kg of dried soil was mixed with 40 l

distilled water and 1.4 kg of chelating ion exchange

resin (Amberlite�, IRC748I) to remove potential

metal contamination. The solution was left in the dark

for 48 h, with regular mixing. The solution was then

screened through a 100-lm mesh (to remove all large

particles and the ion exchange resin), and one-sixth of

the volume, containing circa 1 kg of dried soil, was

added to each of the 2,300 l ‘‘soil’’ mesocosms,

leading to a suspended matter concentration of

435 mg l-1, which corresponds well to the concen-

tration of suspended matter observed following a

natural runoff event in the Thau Lagoon (336 mg l-1,

Pecqueur et al. 2011). Nutrient enrichment by the soil

solution (see Table 1) also corresponded well with

observed nutrient (N and Si) additions from natural

flood events (Pecqueur et al. 2011). The soil suspen-

sion was light brown due to the suspended fine

(\100 lm) soil particles and absorbed light equally

across the whole spectrum of PAR.

Fish

Five young-of-the-year European sea bass (Dicentrar-

chus labrax) were introduced to each ‘‘fish’’ treatment.

These pelagic-feeding fish were picked explicitly in

order to test only indirect effects of fish via light

climate change and fish-mediated nutrient cycling but

not through direct grazing on benthic communities.

Sea bass were provided by the aquaculture facility at

MEDIMEER. For 1 week prior to the experiment, fish

were fed with zooplankton captured daily in the Thau

Lagoon. Average fish length and weight (±SE) were

27.4 (±0.4) mm and 0.294 (±0.034) g at the beginning

of the experiment. After fish were introduced to the

mesocosms, they fed on the zooplankton community

inside the mesocosms.

Sampling procedure

Temperature, salinity, oxygen concentration, and pH

were measured on day 1, 2, 4, 9, and 12 for each

mesocosm. Light attenuation (Kd) was measured on

day 1, 2, 7, and 12. Dissolved [dissolved inorganic

nitrogen (DIN), soluble reactive phosphorous (SRP)],

and total nutrient concentrations [total nitrogen (Tot-

N) and total phosphorous (Tot-P)] were measured on

day 1, 2, 4, 6, 9, and 12 inside each mesocosm.

Additional nutrient samples for dissolved [DIN, SRP,

total dissolved nitrogen (TDN), and total dissolved

phosphorous (TDP) silicate (SiO2-Si)] and total nutri-

ents were taken on day 0 from pre-filtered

(\1,000 lm) lagoon water and from the initial soil

solution (see Table 1). Phytoplankton samples

(n = 4) were also taken on day 0 from the pre-filtered

(\1,000 lm) lagoon water, immediately after it was

pumped into the mesocosms. Samples for all benthic

Table 1 Initial nutrient concentrations of seawater and nutrients added with soil to the ‘‘soil’’ mesocosms are presented

Variable Explanation Sea water

nutrients

(mg l-1)

Nutrients

in 1 kg

soil (mg)

Soil nutrients

added per l

mesocosm water

(mg l-1)

DIN Dissolved inorganic N: NH4-N and nitrite/nitrate-N \0.001 90 0.039

SRP Soluble reactive P: dissolved P measured as PO4-P \0.001 32 0.014

TDN Total dissolved N: DIN plus dissolved organic N 0.17 280 0.12

TDP Total dissolved P: SRP plus dissolved organic P 0.007 53 0.023

Total-N Total dissolved plus particulate N 0.3 900 0.39

Total-P Total dissolved plus particulate P 0.02 140 0.061

DOC Dissolved organic C 1.9 3,900 1.7

TOC Total organic C: DOC plus particulate organic C 2.0 4,400 1.9

Silicate Dissolved SiO2-Si 0.04 320 0.14

The initial concentration of the seawater pumped into the mesocosms is reported in the column ‘‘sea water nutrients.’’ To each soil

addition mesocosm, we then added the nutrients and small particulates (\100 lm) contained in 1 kg of soil. Mesocosms contained

ca. 2,300 l of sea water, including the nutrients contained in sea water

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community parameters were taken on day 6 and 12,

and samples for phytoplankton chlorophyll-a (Chl-

a) and seston particulate carbon (POC) were taken on

day 2, 6, and 12 from all mesocosms.

Analyses of response parameters

Physical and chemical parameters

Kd (m-1), the vertical light attenuation coefficient for

downwelling PAR, was estimated. Incident solar

radiation was measured at 12.00 h (noon) using a

profiling radiometer PUV-2500 (Biospherical Instru-

ments Inc., CA, USA). Vertical profiles of irradiance

for photosynthetically available radiation

(PAR = 400–700 nm) were acquired in each meso-

cosm, and the attenuation coefficient was calculated as

Kd = ln (Ed(0)/Ed(z))/Dz, where Ed is the irradiance at

depth 0 and z for PAR (lE m-2 s-1) (Kirk, 1994).

Optical depth n for deep and shallow substrates was

calculated as n = Kdz, where z is the depth of the

substrate (Falkowski & Raven, 2007). We used -n as

a measure of light availability to microbenthos.

Temperature, salinity, oxygen concentration, and

pH were measured at 10.00 a.m. in the middle of the

mesocosms at 1 m depth, using a handheld probe

(WTW Multi 350i meter). Depth profiles at 20 cm

intervals conducted on day 3 and 9 revealed no change

in temperature, salinity, oxygen concentration, and pH

with depth. We also collected GF/F-filtered and

unfiltered water samples in plastic bottles from ca.

1 m depth from all mesocosms (frozen until analyzed).

Filtered samples were analyzed for DIN, SRP, and

DOC, as well as TDN, TDP, and TOC (for initial

lagoon water nutrient concentration and soil solution)

using standard methods (APHA, 1998). Unfiltered

water samples were analyzed for Tot-N and Tot-P

using standard methods (APHA, 1998). Seston par-

ticulate organic C (POC) samples were filtered

through 35 lm filters to obtain the edible seston C

fraction, before filtration onto precombusted, acid-

washed GF/F filters. POC was analyzed using a Carlo-

Erba elemental analyzer. To determine phytoplankton

Chl-a concentrations, we used high performance

liquid chromatography (HPLC) according to Vidussi

et al. (2011). Mesocosm water was filtered through

GF/F filters at low pressure (200 bar). Filters were

stored at -80�C until extraction. Pigments were

extracted with 2 ml of 95% methanol at -20�C for

1 h. Then, the samples were sonicated for several

seconds using a sonication probe and extracted at

-20�C for an additional 1 h. Finally, extracts were

filtered through GF/F filters and analyzed with an

HPLC system (Waters, Milford Massachusetts, USA).

Microbenthic community measurements

Two large (25 cm2) and four small (6.25 m2) benthic

tiles were collected by lifting each tray out of the

mesocosms and removing two tiles (ensuring colo-

nized surfaces were not disturbed). Large tiles were

first used for respiration and production estimates and

then scraped and pooled to measure microbenthic

nutrient content and algal Chl-a. Small tiles were

retained for benthic bacterial analyses. Tiles were kept

submerged in mesocosm water until analyses were

completed.

Microbenthic community production and respiration

We measured benthic and pelagic community respira-

tion (RE), net ecosystem production (NEP), and

estimated gross ecosystem production (GPP =

NEP ? RE) inside the mesocosms using the oxygen

light–dark bottle method (Wetzel & Likens, 2000, as

modified by Francoeur et al., 2013). These assays

measured the integrated respiration (including small

animals and protozoa) and primary production of the

pelagic and benthic mesocosm communities. Oxygen

measurements were corrected for temperature and

salinity (Pijanowski, 1973). Equations (modified from

Wetzel & Likens, 2000) were used to calculate RE,

NEP, and GPP and to extrapolate the incubation data to

daily values using insolation data. Benthic respiration,

NEP, and GPP were corrected for pelagic activity in the

benthic incubation chambers (see below) and set to 0 in

those few cases where benthic activity was not

detectable over the pelagic background.

To measure benthic RE and NEP, two tiles from

each benthic tray were sealed in a clear plastic

chamber (*700 ml) filled with water from the

corresponding mesocosm containing a magnetic stir

bar. Initial oxygen concentrations were measured in

each chamber by inserting a needle-type optical

microprobe (WPI OxyMicro TX3) through a septum

in the chamber wall. Chambers were then submerged

in a flow-through water bath filled with lagoon water

maintained at in situ temperature and covered to

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exclude light. After 2–4 h, the light-excluding cover

was removed, water in the chambers was mixed using

the magnetic bar, and oxygen concentrations were

measured. Uncovered chambers were then suspended

at the corresponding depth in their respective meso-

cosm and incubated for an additional 1–3 h, before

mixing and measuring final oxygen concentrations. To

measure pelagic RE and NEP, opaque and clear glass

BOD bottles (300 ml) were filled with mesocosm

water. Nitrogen gas was bubbled in the clear bottles to

reduce dissolved oxygen to subsaturating concentra-

tions. Initial oxygen concentrations were measured in

each bottle by inserting the optical microprobe into the

neck of open bottles, and then all bottles were

carefully sealed to exclude any bubbles and incubated

for 4–6 h at a depth of 1 m (all mesocosms). After

incubation, bottles were opened and final oxygen

concentrations were measured.

Microbenthic Chl-a and nutrient content

The microbenthic community was divided into three

subsamples, filtered onto three replicate precombusted

GF/C filters, and stored frozen for analysis of

microbenthic community CN, P, and microphytoben-

thic Chl-a. Chl-a was measured spectrofluorometri-

cally (APHA, 1998) within 2 days of sampling. CN

was measured with a CHN-Analyzer (Carlo-Erba

elemental analyzer) and P (PO4, after hydrolysis by

heating and potassium persulfate treatment) with a

spectrophotometer (Grasshoff et al., 1983) within

3 month of sampling.

Benthic bacteria cell counts

The surface of a small tile was scraped, and the

dislodged material was preserved (4% final concen-

tration formaldehyde) in 1 ml of filter-sterilized sea

water. Cells were dispersed in synthetic sea water by

gentle sonication (1 min) and attached to 0.22 lm

black polycarbonate membrane filters (GE Water and

Process Technologies). Cells were stained with DAPI

(40,6-diamidino-2-phenylindole, dihydrochloride) and

visualized on a fluorescent microscope using UV-

excitation, blue emission light. A minimum of 20

randomly selected fields of view or 200 cells were

counted per slide, and cell number per surface area

was calculated.

Statistical analyses

For mesocosm nutrient and light conditions, repeated-

measures analyses of variances (RM-ANOVAs) were

used to test the effects of treatment factors (fish and

soil) and time. Additional 2-way ANOVAs tested the

effects of soil and fish on light attenuation for each

single measured day. Blocked design ANOVAs or

linear mixed effect (Lme) models (where one value

was missing in the data set due to loss of an incubation

chamber) were used for benthic response variables

with mesocosm as a block factor and depth, soil and

fish as treatment factors, for day 6 and day 12

separately (Table 2). We used a multiple linear

regression model to test the effects of the factors

‘sampling day’ (categorical predictor) and ‘optical

depth’ (continuous predictor) on GPP. The sig-

nificance level for all analyses was set at a = 0.05.

All statistical analyses were computed using the

program R (R 2.14.0 2011). When necessary, data

were log-transformed to achieve normality and ho-

mogeneity of variances. All reported results are

statistically significant.

Results

Physical and chemical factors and pelagic Chl-a

The addition of soil increased light attenuation, Tot-N,

and Tot-P, especially in the beginning of the

experiment (Fig. 1). Soil addition substantially re-

duced light penetration over the course of the ex-

periment, as shown by greater light attenuation (Kd) in

soil treatments (Fig. 1A; RM-ANOVA, soil effect:

P = 0.007). Kd was highest at the start of the

experiment and declined with time (RM-ANOVA,

time effect: P \ 0.001) but less so in soil treatments

with fish, which still had high Kd at the end of the

experiment (Fig. 1A; RM-ANOVA, time 9 soil 9

fish interaction P = 0.021). Water temperature

(mean ± SD: 20.3�C ± 0.9), salinity (mean ± SD:

36.6 ± 0.1), oxygen concentration (mean ± SD:

9.1 mg l-1 ± 0.7), and pH (mean ± SD: 8.21 ±

0.04) remained stable among treatments and over time

(2-way RM-ANOVA, all effects and interactions:

P [ 0.1). Total water nutrient concentrations (Tot-N,

Tot-P) increased with soil addition (2-way RM-

ANOVA, soil effect: P \ 0.001) but decreased over

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123

time (Fig. 1B and C; time 9 soil interaction, 2-way

RM-ANOVA: P \ 0.001). Similarly, pelagic Chl-

a concentrations were increased by soil addition, but

these positive soil effects decreased over time

(Fig. 1D; 2-way RM-ANOVA, soil effect: P \0.001, time effect: P \ 0.001, interaction soil 9 time:

P \ 0.001). DOC was unaffected by experimental

manipulations but decreased over time (2-way

RM-ANOVA, time effect: P = 0.022; mean ± SD,

DOC on day 2: 14 ± 4 lg l-1; on day 12:

2.1 ± 0.06 lg l-1). However, particulate organic ses-

ton C (POC) was greater in the soil treatments

(mean ± SD, soil: 0.88 ± 0.20 mg l-1, no soil:

0.45 ± 0.08 mg l-1), peaking at day 2 and declining

over time in all mesocosms (2-way RM-ANOVA, soil

effect: P \ 0.001, time effect: P \ 0.001). DIN and

Table 2 ANOVA results for the effects of soil, fish, and depth on microbenthic parameters on day 6 and on day 12 of the experiment

Soil treatment (S) Fish treatment (F) Depth (D) S 9 F S 9 D F 9 D S 9 F 9 D

P HSD P HSD P HSD P P P P

Day 6

C (mg/m2)a 0.207 0.392 0.801 0.389 0.797 0.758 0.941

Chl-a (mg/m2)a 0.549 0.093 0.172 0.994 0.017 0.396 0.272

Bacteria cells 0.171 0.230 0.488 0.814 0.266 0.249 0.391

NEP 0.095 0.250 0.042 Sh [ D 0.446 0.615 0.966 0.239

GPP 0.350 0.038 NF [ F 0.004 Sh [ D 0.285 0.910 0.862 0.200

Respiration 0.823 0.111 0.006 Sh [ D 0.543 0.783 0.771 0.372

Chl-a/C (%)a 0.326 0.153 0.433 0.219 0.110 0.738 0.486

NEP/Chl-a 0.048 NS [ S 0.849 0.002 Sh [ D 0.089 0.438 0.067 0.663

Na 0.553 0.969 0.833 0.486 0.918 0.921 0.889

Pa 0.032 S [ NS 0.570 0.061 0.440 0.677 0.444 0.523

C/N (molar)a 0.319 0.173 0.212 0.992 0.647 0.540 0.564

C/P (molar)a 0.140 0.494 0.214 0.017 0.999 0.835 0.707

N/P (molar)a 0.062 0.516 0.079 0.047 0.838 0.624 0.515

Day 12

C (mg/m2)a 0.567 0.342 0.006 D [ Sh 0.290 0.478 0.268 0.470

Chl-a (mg/m2)a 0.033 NS [ S 0.074 <0.001 D [ Sh 0.679 0.316 0.137 0.836

Bacteria cells 0.518 0.228 0.113 0.006 0.762 0.927 0.209

NEPb 0.230 0.087 0.034 Sh [ D 0.070 0.125 0.985 0.821

GPPb 0.556 0.850 0.020 Sh [ D 0.306 0.648 0.147 0.659

Respiration 0.637 0.526 0.045 Sh [ D 0.505 0.257 0.205 0.798

Chl-a/C (%)a 0.022 NS [ S 0.015 F [ NF <0.001 D [ Sh 0.275 0.206 0.026 0.996

NEP/Chl-a 0.064 0.924 0.008 Sh [ D 0.346 0.086 0.919 0.487

Na 0.016 NS [ S 0.267 0.001 D [ Sh 0.123 0.030 0.750 0.342

Pa 0.215 0.395 0.961 0.275 0.841 0.949 0.498

C/N (molar)a 0.008 S [ NS 0.838 0.044 Sh [ D 0.450 0.039 0.141 0.049

C/P (molar)a 0.042 NS [ S 0.054 0.140 0.708 0.903 0.659 0.331

N/P (molar)a <0.001 NS [ S 0.029 NF [ F 0.066 0.858 0.378 0.856 0.113

S for soil, NS no soil, F fish, NF no fish, D deep, Sh shallow tiles

P is the probability and HSD describes the significant differences between treatments as found by the Tukey HSD test. Significant

results are in bolda Log10-transformedb Lme instead of ANOVA because of one missing value each

Hydrobiologia

123

SRP concentrations were low and constant throughout

the experiment (2-way RM-ANOVA: P [ 0.1,

mean ± SD, SRP: 4 ± 0.6 lg l-1, DIN: 13 ±

0.7 lg l-1), indicating that dissolved soil nutrients

(see Table 1) were taken up immediately.

Microbenthic community C, microphytobenthic

Chl-a, and benthic bacterial biomass

On day 6, C per area was not affected by experimental

treatments, whereas Chl-a per area was slightly greater

on shallow than on deep tiles but only in the soil

treatment (Fig. 2A, B; Table 2). By day 12, both C and

Chl-a per area were greater on deep than on shallow

tiles, whereas bacterial density was not affected by

depth (Fig. 2A–C; Table 2). Additionally, soil treat-

ments on day 12 had lower Chl-a per area than no soil

treatments (Fig. 2B; Table 2). Bacterial abundance

per area was not affected by experimental treatment on

day 6, but on day 12, bacterial abundance per area was

greater in soil than no soil treatments when fish were

absent and lower in soil than no soil treatments when

fish were present (Fig. 2C; Table 2).

Microbenthic community production

and respiration

Depth had a negative effect on area-specific benthic

NEP, GPP, and RE, both on day 6 and 12 (Fig. 3A–C;

Table 2). In addition, fish presence reduced area-

1 2 3 4 5 6 7 8 9 10 11 12Day

0

20

40

60

80

100

120

Tot-P

(μg

L-1)

1 2 3 4 5 6 7 8 9 10 11 12Day

200

300

400

500

600

700

800

Tot-N

(μg

L-1)

1 2 3 4 5 6 7 8 9 10 11 12Day

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Ligh

t atte

nuat

ion

Kd (

m-1

) No soil/ no fish No soil/ fish Soil/ no fish Soil/ fish

A

C

B

1 2 3 4 5 6 7 8 9 10 11 12Sampling day

0

2

4

6

8

10

12

14

Pel

agic

Chl

-a (μ

g L-1

) D

Fig. 1 Mean (±SE) abiotic conditions and pelagic Chl-a of the

mesocosms over time. A Light penetration (Kd,m-1), B total

nitrogen (Tot-N, lg l-1), C total phosphorous (Tot-P, lg l-1),

and (D) pelagic Chl-a (lg l-1) in the different soil and fish

treatments

02E54E56E58E51E6

1.2E61.4E61.6E6

0.0

0.2

0.4

0.6

0.8

1.00

20406080

100120140160

Deep Shallow

Day 6No soil Soil

Day 12No soil Soil

020406080

100120140160

C (μ

g cm

-2)

0.0

0.2

0.4

0.6

0.8

1.0

Chl

-a(μ

g cm

-2) B

A

02E54E56E58E51E6

1.2E61.4E61.6E6

Bac

teria

(cel

ls c

m-2

) C

No fish Fish No fish Fish No fish Fish No fish Fish

Fig. 2 A Mean (±SE) microbenthic C per area, B microbenthic

Chl-a per area, and C benthic bacteria per area on day 6 and day

12 in the different soil, fish, and depth treatments

Hydrobiologia

123

specific benthic GPP on day 6 (Fig. 3B; Table 2). The

positive effect of light availability on GPP is illustrat-

ed in the positive connection between benthic GPP and

light availability at benthic substrate depth (Fig. 4).

Together with sampling day, which affected benthic

algal light adaptation, light availability at substrate

depth explained 44% of the variation in GPP (Fig. 4).

Soil treatment or its interaction with other factors did

not affect GPP on any of the sampling days (Table 2),

possibly since soil effects on light attenuation were

already greatly reduced by day 6 (Fig. 1A).

Microbenthic Chl-a/C and NEP/Chl-a

Treatment effects were different between day 6 and

day 12. On day 6, Chl-a/C ratios were not affected by

experimental treatment, but by day 12, Chl-a/C ratios

were greater on deep than on shallow tiles and in no

soil than in soil treatments (Fig. 5A; Table 2). Further,

fish presence had positive effects on microbenthic

Chl-a/C ratios but only on the deep tiles (Fig. 5A;

Table 2). NEP/Chl-a ratios illustrate the productivity

of benthic algal Chl-a. Chl-a-specific NEP was greater

on shallow tiles both on day 6 and 12. On day 6, Chl-a-

specific NEP was greater in no soil than in soil

treatments (Fig. 5B; Table 2), suggesting that Chl-

a produced more oxygen per mass unit at greater light

availability.

D12

-200

-100

0

100

200

300

0

200

400

600

800

Day 12

0100200300400500600700800

Com

mun

ity re

spira

tion

(mg

O2

upta

kem

-2 d

-1)

Net

eco

syst

em

prod

uctio

n (m

g O

2 m

- 2 d

-1)

Gro

ss p

rimar

y pr

oduc

tion

(mg

O2

m-2

d-1

)

Day 6

0

200

400

600

8000

200

400

600

800

D6

-200

-100

0

100

200

300

Deep Shallow

No soil Soil No soil SoilDay 6 Day 12

0

B

C

A

No fish Fish No fish Fish No fish Fish No fish Fish

Fig. 3 A Mean (±SE) microbenthic net ecosystem production,

B microbenthic gross primary O2 productivity, and C microben-

thic community respiration on day 6 and day 12 in the different

soil, fish, and depth treatments

-4.0 -3.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.00

200

400

600

800

1000

Ben

thic

GP

P (m

g O

2 m

-2 d-1

) r2 = 0.44F(2, 44) = 17P < 0.0001

Day 6Day 12

Substrate light availability (-ξ = -Kdz)

Fig. 4 Microbenthic area-specific GPP for day 6 (black

symbols) and day 12 (gray symbols) plotted against substrate

light availability: -n = (-Kd)z, where z is substrate depth and

Kd is mesocosm light attenuation. Whole model R2, F, and

P values from multiple regression analyses with the factors

sampling day and substrate light availability -n are presented in

figure panel. The predictor variables, sampling day, and light

availability were significant (P \ 0.05) in explaining benthic

GPP. Lines indicate linear regressions for benthic GPP against

light availability for day 6 (black) and day 12 (gray)

-4

-2

0

2

4

6

8

Day 6No soil Soil

Day 12No soil Soil

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Deep Shallow

Chl

-a/ C

(%) A

-4

-2

0

2

4

6

8

No fish Fish No fish Fish No fish Fish No fish Fish

B

NE

P/ C

hl-a

(mg

O2

prod

uctio

nμg

- 1 d

-1)

Fig. 5 A Mean (±SE) microbenthic Chl-a/C and B microben-

thic net primary production/Chl-a on day 6 and day 12 in the

different soil, fish, and depth treatments

Hydrobiologia

123

Microbenthic community nutrient content

Soil addition strongly affected microbenthic commu-

nity nutrient content through interactions both with

depth and fish treatment (Fig. 6; Table 2). N per area

was affected only by experimental treatments on day

12, when N per area was greater on deeper tiles,

especially in no soil treatments (Fig. 6A; Table 2). P

per area on day 6 was greater in soil than in no soil

treatments, but these effects were gone by day 12

(Fig. 6B; Table 2). C/N ratios were not affected by

experimental treatments on day 6, but by day 12, a

positive soil effect on microbenthic C/N ratios was

visible on the deeper tiles, whereas greater depth (less

light) led to lower C/N ratios only in the no soil/no fish

treatment (Fig. 6C; Table 2). Microbenthic C/P ratios

on day 6 were reduced by fish presence in no soil

treatments but increased with fish presence in soil

treatments (Fig. 6D; Table 2). By day 12, C/P ratios

were lower in soil than in no soil treatments (Fig. 6D;

Table 2). On day 6, soil addition reduced microben-

thic N/P ratios in the absence of fish but had no effect

on N/P ratios when fish were present (Fig. 6E;

Table 2). On day 12, N/P ratios were lower in soil

than in no soil treatments and in fish than in no fish

treatments (Fig. 6E; Table 2).

Discussion

Overview

Simulated terrestrial runoff (soil) increased turbidity

and Tot-N and Tot-P concentrations in the water

column, especially at the start of the experiment. Soil

added 3.9 g of terrestrial DOC, 90 mg of terrestrial

DIN, and 32 mg of terrestrial SRP per mesocosm.

Terrestrially derived DIN and SRP were already taken

up by the pelagic community by day 1 and thus only

increased pelagic Tot-N and Tot-P fractions, not

mesocosm DIN and SRP concentrations (supporting

hypothesis 1). Soil decreased benthic algal biomass

(Chl-a) and reduced Chl-a-specific productivity but

increased benthic bacterial biomass in the absence of

fish (supporting hypothesis 2). The positive effects of

soil on benthic bacteria, however, were only visible on

day 12, whereas soil had positive effects on pelagic

bacteria from day 1 (A. Liess, unpublished data). Since

pelagic bacteria are competitively superior to benthic

bacteria for water column nutrients, soil effects on

benthic bacterial abundance may have been due to

increased sedimentation of pelagic bacteria. This

sedimentation was probably relatively slow due to

water column mixing and thus not apparent on day 6.

Area-specific NEP, GPP, respiration, and Chl-a-

specific NEP were always lower on deeper tiles. Soil

did not intensify these depth effects but did appear to

reduce the depth where microbenthos switched from

net autotrophy to net heterotrophy (weak support for

hypothesis 3). Overall fish effects were reduced by

soil, since fish increased microbenthic nutrient content

8101214161820222426

Deep Shallow

0.000.020.040.060.080.100.120.140.160.180.20

0.00.20.40.60.81.01.21.4

8101214161820222426

C/ N

(mol

ar)

0100200300400500600700

C/ P

(mol

ar)

0

10

20

30

40

50

60

N/ P

(mol

ar)

No fish Fish No fish Fish No fish Fish No fish Fish

D

B

C

Day 6 Day 12No soil Soil

0.00.20.40.60.81.01.21.4

No soil Soil

0.00

0.04

0.08

0.12

0.16

0.20 Deep Shallow

P (μ

g cm

-2)

N (μ

g cm

-2)

A

E

Fig. 6 A Mean (±SE) microbenthic algal N and B P per area

(lg cm-2) and microbenthic nutrient ratios: C microbenthic

C/N molar ratios, D microbenthic C/P molar ratios, and

E microbenthic N/P molar ratios on deep and shallow tiles in

the different soil and fish treatments on day 6 and day 12 of the

experiment

Hydrobiologia

123

(day 6) and bacterial abundance (day 12) especially

without soil (supporting hypothesis 4).

Simulating terrestrial runoff with soil addition

in mesocoms

Simulating soil runoff with soil from the river bank of

the main tributary to the Thau Lagoon probably led to

a realistic simulation of a storm runoff event, as

described in previous field studies (Guadayol et al.,

2009; Pecqueur et al., 2011). The dissolved soil

nutrients were taken up equally quickly, i.e., in less

than one day, in our mesocosms, as recorded in the

oligotrophic environment of the Thau Lagoon (Pec-

queur et al., 2011). Both in the lagoon and in our

mesocosms, dissolved soil nutrients caused a brief

pelagic diatom bloom (Pecqueur et al., 2011; A. Liess,

unpublished data). Interestingly, increased nutrient

availability and turbidity after runoff or soil addition

were very transient effects. Thus, in order to estimate

nutrient inflow during storm runoff, the increase in

total nutrient concentrations in the water column and

in microbenthic nutrient content immediately after the

storm event appear to be good indicators of the

terrestrial nutrient subsidies. The rapid decrease in soil

induced turbidity may partly have been a mesocosm

artifact, since the mesocosm water was relatively

protected from wave action. Sediment resuspension

was thus potentially lower than in natural systems.

The importance of soil, light, and depth

for the microbenthic community

Soil reduced Chl-a-specific productivity on day 6.

This might have been due to increased turbidity with

soil at the start of the experiment or to sedimentation

of chlorophyll-containing but physiologically inactive

phytoplankton. Further, the pattern of reduced Chl-

a and increased microbenthic P content (lower

microbenthic C/P and N/P ratios) in soil treatments

on day 12 suggests microbenthos grazing. Pecqueur

et al. (2011) found that a flood in the Thau Lagoon

increased the abundance of some pelagic ciliate taxa.

Increased pelagic ciliate biomass with soil was even

found in our mesocosms (A. Liess, unpublished data).

Some of these ciliates probably settled, thus becoming

part of the microbenthos. Ciliate grazing on benthic

algae and bacteria may thus have reduced overall

microbenthic community biomass but increased

microbenthic community nutrient content (Liess &

Hillebrand, 2004).

In addition to microbenthic algae and other micro-

scopic organisms (flagellates, ciliates, amoeba), mi-

crobenthos includes bacteria. Positive effects of soil

addition on benthic bacteria were only visible on day

12 and may have been due to higher sedimentation

rates of pelagic bacteria, which were more abundant in

the soil treatments (A. Liess, unpublished data).

Declines in bacterial abundance on shallow, but not

deep, substrates from day 6 to 12 may also have been a

consequence of both settling of pelagic bacteria and

high initial bacterial growth rates in the absence of a

fully developed predator community. As the predator

community developed over time, bacteria declined on

shallow substrates, whereas higher rates of settling of

pelagic bacteria onto deep tiles, due to a greater

overlying water column, compensated for bacteria lost

to predation on deep tiles. Influence of bacterial

abundances by predation, specifically trophic cascad-

ing, also offers a potential explanation for the observed

fish effect on day 12. Differences in turbidity may have

affected the feeding selectivity of our visually hunting

fish and could be a possible explanation for the positive

effects of fish on bacterial abundances in no soil

treatments and the negative effects of fish on bacterial

abundances in the soil treatments. If visibility for fish

was reduced in the soil treatments, fish may have had to

prey on smaller, less rewarding but more abundant

prey, such as large bacterivorous ciliates. Whereas

with better visibility in the no soil treatment, fish may

have been able to see and select larger and more

lucrative prey (predatory meiofauna, e.g., nematodes).

However, since sea bass juveniles are pelagic-feeding

fish, the above predator–prey interactions would have

had to take place during periods of benthic predator

resuspension. Strong trophic interactions within the

microbenthic community between benthic bacteria,

microalgae, heterotrophic nanoflagellates, and ciliates

have been documented for lakes (Burgmer et al., 2010)

and marine systems (Epstein et al., 1992).

Depth had a very strong effect on the microbenthic

community. Reduced light availability with increasing

depth led to lower NEP, GPP, and community

respiration rates. Low respiration rates were probably

due to low algal respiration, which is autocorrelated to

algal production (Falkowski & Raven, 2007) but may

also indicate low bacterial or meiofaunal respiration.

High microbenthic algal production often co-occurs

Hydrobiologia

123

with high bacterial production (Neely & Wetzel, 1995;

Liess & Haglund, 2007; Kuehn et al., 2014), due to

higher food availability for heterotrophic organisms.

By day 12, deeper tiles had accrued significantly more

Chl-a. Thus, lower light availability at greater depth

probably led to compensatory algal Chl-a synthesis or

shade adaptation (Kirk, 1994; Greenwood & Rose-

mond, 2005), indicated by increasing Chl-a/C ratios

on deep tiles over time. As algae became shade-

adapted, GPP and respiration increased. Due to the

high ambient summer irradiance levels, deep algal

shade adaptation by day 12 was sufficient to compen-

sate for reduced light levels. Even so, deep benthic

algae initially displayed very low photosynthesis rates

and supported little respiration. This suggests that

deep benthic algae were not shade-adapted at first,

despite lower light levels, and that shade adaptation

occurred later or shade-adapted pelagic algae settled

out after the initial phytoplankton bloom.

Unexpectedly, no interactions between soil treat-

ment and substrate depth were found. This could either

have been due to the transient soil effect on light

climate or due to direct soil sedimentation effects on

microbenthic productivity. Even though, the deep

microbenthos in the soil/fish treatment exhibited

lowest area-specific NEP rates (net heterotrophy,

NEP \ 0), whereas the shallow communities in the

no soil treatments had highest area-specific NEP rates

(net autotrophy, NEP [ 0). Thus, a natural terrestrial

runoff event where soil remains suspended longer is

likely to reduce the depth where microbenthos

switches from net autotrophy to net heterotrophy.

Fish effects

Fish presence probably increased nutrient cycling in

the mesocosms, since fish affected microbenthic

nutrient stoichiometry. Fish reduced both C/P and

C/N ratios, especially under nutrient-poor and high

light conditions (no soil/shallow), and increased Chl-

a content. Fish can increase the availability of DIN and

SRP, as well as increase microbenthic Chl-a content

(Liess et al., 2006). Fish-mediated nutrient cycling in

general (Attayde & Hansson, 1999, 2001) and in

benthic systems in particular (McIntyre et al., 2008) is

very important for the productivity and biodiversity of

the system, especially under nutrient-limited condi-

tions. Thus, in a relatively nutrient-poor, shallow

system, such as the Thau Lagoon, nutrient recycling

by fish may be very important for microbenthos.

However, during terrestrial runoff events and associ-

ated nutrient inflow, fish-mediated nutrient recycling

should be less important. During these conditions, fish

probably affect the microbenthic community by

amplifying and prolonging the terrestrial runoff

turbidity effects.

Consequences of terrestrial runoff for plankton

and microbenthos

In order to gain a mechanistic understanding of how

terrestrial runoff will affect aquatic ecosystems, it is

important to examine terrestrial runoff effects on the

different aquatic food web components, both in

pelagic as well as in benthic systems. In recent years,

several pelagic studies have examined elevated

episodic runoff (e.g., storm) effects on coastal plank-

tonic communities (Guadayol et al., 2009; Pecqueur

et al., 2011). In general, terrestrial runoff increased

pelagic Chl-a concentrations relative to bacterial

biomass by providing nutrient subsidies to algae

(Guadayol et al., 2009). For example, in the Thau

Lagoon, phytoplankton is typically N-limited, and

storm-induced inputs of N from the watershed allevi-

ate this N-limitation (Picot et al., 1990). Pelagic

communities also shift towards an algal-dominated

state due to runoff (Guadayol et al., 2009; Pecqueur

et al., 2011). In contrast to these pelagic studies, we

found that terrestrial runoff had negatively affected

microphytobenthic biomass and productivity and

positively affected benthic bacterial biomass,

specifically: (1) decreasing microphytobenthic chloro-

phyll-a per area and per carbon unit, (2) reducing the

depth where microbenthos switched from net autotro-

phy to net heterotrophy, and (3) increasing bacterial

biomass in the absence of fish. Thus, our study

illustrates the importance of light for benthic systems

and indicates that terrestrial runoff nutrient subsidies

might be less important in light-limited benthic

systems, especially at deeper depths.

Conclusions

Shallow coastal marine ecosystems of up to one meter

depth are dominated by benthic production, but even

down to 13 m depth, benthic production can sig-

nificantly contribute to ecosystem productivity

Hydrobiologia

123

(Barranguet et al., 1994). Here, we demonstrated the

importance of light to these benthic ecosystems and

concluded that increased turbidity from terrestrial

runoff may have negative effects on costal ecosystem

productivity. Such negative effects are predicted to

increase in many areas of the world, including the

Mediterranean region, due to climate change.

Acknowledgements We thank A. Deininger, R. Lefebure, P.

Mathisen, K. Lange, T. Bayer, and A. Schroder for help in field

and laboratory. B. Mostajir, E. Le Floc’h, and F. Vidussi at the

MEDIMEER mesocom facility provided technical support.

J. Ask, S. Berger, and J. Nejstgaard provided helpful advice. The

manuscript profited from comments by Sebastian Diehl. This

research received funding from the European Union Seventh

Framework Program (FP7/2007–2013) under Grant Agreement

No. 228224, MESOAQUA and from the Oscar and Lili Lamms

Minnes Stiftelse.

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