PRIMARY RESEARCH PAPER

Effects of sewage water on bio-optical propertiesand primary production of coastal systems in West Australia

P. A. Staehr Æ A. M. Waite Æ S. Markager

Received: 31 August 2007 / Revised: 23 September 2008 / Accepted: 6 October 2008 / Published online: 11 November 2008

� Springer Science+Business Media B.V. 2008

Abstract Relationships between key phytoplankton

attributes including Chl a-specific light absorption,

pigment composition and concentration, photosynthe-

sis, primary production and community structure were

studied in two open shallow nutrient-poor coastal

systems receiving similar amounts of sewage water.

Both systems were significantly nitrogen limited.

However, differences in wastewater treatment (pri-

mary vs secondary) and sewage dilution (50%)

between the two systems caused a greater difference

between systems than locally around the outflows. For

both systems, water at the outlet had significantly

lower water transparency caused by a 20% higher

absorption by coloured dissolved organic matter.

Nutrient concentrations were also elevated, gradually

decreasing with distance north (governing current) of

the outflows, causing higher abundance of nano-sized

phytoplankton, higher content of carotenoid pigments,

20–50% higher Chl a-specific absorption coefficients

and higher photosynthetic capacity. Although maxi-

mum rates of Chl a-normalised photosynthesis were

strongly related to nitrate availability, no effects were

found on the derived areal primary production or algal

biomass suggesting that photosynthetic and optical

parameters are more sensitive indicators of nutrient

enrichment than biomass or productivity.

Keywords Light absorption � Nutrients �Pigmentation � Photosynthesis � Primary production �Sewage water

Introduction

It is widely accepted that anthropogenic activities have

increased the load of nutrients from land to coastal

ecosystems (Conley, 1999) and that this may signif-

icantly enhance phytoplankton productivity (Borum &

Sand-Jensen, 1996). This increase in production may

cause a number of negative effects, e.g. enhanced

consumption of oxygen required to mineralise the

increasing amounts of organic matter produced within

the ecosystem, which consequently may result in

mass mortality of benthic invertebrates and fish.

Other effects may include a loss of benthic vegetation

from shading and harmful algal blooms. Inputs of

freshwater from land also carry dissolved organic

Handling editor: Tasman Peter Crowe

P. A. Staehr (&)

University of Copenhagen, Freshwater Biological

Laboratory, Helsingørsgade 51, Hillerød 3400, Denmark

e-mail: pastahr@bio.ku.dk

A. M. Waite

School of Environmental Systems Engineering,

University of Western Australia, Crawley, WA 6009,

Australia

S. Markager

Department of Marine Ecology, National Environmental

Research Institute, Frederiksborgvej 399, 358, DK-4000,

Denmark

123

Hydrobiologia (2009) 620:191–205

DOI 10.1007/s10750-008-9628-1

material (DOM), which may have a negative impact on

phytoplankton productivity in shallow well-mixed

waters, through elevated attenuation of light (Arrigo

& Brown, 1996). Although nutrients and DOM

entering coastal ecosystems from freshwater sources

may cause ecological changes in relatively sheltered

coastal ecosystems, this may not necessarily occur in

more exposed ecosystems where nutrients and DOM

become rapidly diluted.

Light and nutrient availability affect phytoplankton

productivity by limiting either phytoplankton abun-

dance (Dugdale & Wilkerson, 1992) or growth rate

(Dugdale & Goering, 1967). The absence of biomass

accumulation following nutrient enrichment, how-

ever, does not necessarily imply limitation of growth

rate, due to complications arising from grazing (Banse,

1992) and nutrient recycling (Dugdale & Wilkerson,

1992). Therefore, although changes in phytoplankton

abundance are important for understanding the eco-

logical functioning of a system, measurement of bulk

parameters such as chlorophyll a and cell numbers

may give little information on physiological responses

of a population to changes in prevailing growth

conditions (Graziano et al., 1996). On the other hand,

acclimation of the optical properties of phytoplankton

is known to represent an important set of physiological

responses to changes in availability of light and

nutrients (Stæhr et al., 2002). Significant changes in

the composition and intracellular concentration of

photosynthetic pigments, known as pigment packag-

ing, have been found to occur very rapidly (Berner

et al., 1989). This will affect the ability of the algae to

absorb the photosynthetically available radiation

(PAR) and transfer the absorbed energy into photo-

synthates. Studies of primary production in the ocean

have shown that variations in parameters of the

photosynthesis–light curve (e.g. initial slope, aB, and

maximum photosynthesis, PmaxB ), used to describe

primary production of phytoplankton, were positively

related to changes in nitrate availability (Babin et al.,

1996; Stuart et al., 2000). Therefore, changes in the

photosynthetic or optical parameters are likely to be

useful indicators of nutrient enrichment.

In the present study, we investigated the effects of

local inputs of sewage water on phytoplankton light

absorption, pigmentation, photosynthesis, primary

production, biomass, and size distribution at local

scales (c. 5–60 km). The study was conducted during

late summer in a subtropical coastal ecosystem

generally characterised by high irradiance and low

nutrient concentrations. Under these conditions, it was

expected that phytoplankton would have a high

potential for growth and thus be strongly influenced

by the anthropogenically derived nutrients and dis-

solved organic matter. In particular, we had the

opportunity to contrast a system having ammonium

as the main nitrogen (N) source (S sites) with a system

experiencing primarily nitrate inputs (N sites). The

relative importance of acclimation and changes in

phytoplankton community structure on primary pro-

duction and bio-optical properties of phytoplankton are

discussed.

Materials and methods

Study sites

The study sites included two areas off the West Coast

of Southern Australia, close to Perth. The areas are

referred to as Ocean Reef (N) and Sepia Depression

(S) located c. 40 km apart (Fig. 1). Both areas have

protecting reefs that restrict exchange of water with

the Indian Ocean and reduce wave energy on this

largely open coastline. The tidal range is low, but

both water bodies tend to be vertically mixed at least

once per day (Wallace, 2000) due to strong diurnal

Fig. 1 Map of the study area and sampling stations located in

the coastal zone of Perth, SW Australia

192 Hydrobiologia (2009) 620:191–205

123

(sea breeze) winds (Masselink & Pattiaratchi, 2001)

and overnight cooling. Along-shore transport is

primarily wind-driven in a northerly direction during

summer (Thompson & Waite, 2003). Approximately

equal amounts of sewage water run into the Sepia

Depression area (c. 101 9 106 l d-1) and the Ocean

Reef area (c. 80 9 106 l d-1) (Lord & Hillman,

1995). The sewage water is discharged into relatively

shallow zones (Ocean Reef, c. 10 m, Sepia Depres-

sion, c. 20 m) with predominantly sandy sediments,

and sewage was not tertiary treated in either of the

areas during the period of our study, such that

nitrogen and phosphorus were not effectively

removed from the effluent. However, at Ocean Reef

(a closed reef system with a depth of c. 10 m) the

effluent was subject to secondary treatment, such that

nitrate is the primary nitrogen source, while at Sepia

Depression the effluent was primary treated at the

time of this study, such that ammonium was the

primary nitrogen source (Thompson & Waite, 2003).

The study sites were part of a long-term monitoring

project (Thompson & Waite, 2000) that investigated

the temporal and spatial impact of sewage water on

primary production of the ecosystems. This study

presents data from intensive sampling on 25 and 28

January 2000. Sampling was conducted before noon

(8–12 am) to avoid the influence of the afternoon sea

breeze. The weather was warm (25–30�C) but partly

overcast at Ocean Reef while skies were clear at Sepia

Depression. Waters were high saline (35 ppt.) and

warm (22�C) at both study sites.

Four sampling stations were located within each

study site c. 5 km apart thus covering a transect of c.

15 km. Stations S2 and N2 were located at the sewage

outlets and the other stations were located north and

south of these. Previous work has shown that surface

Chl a biomass declines with distance from the coast,

suggesting the importance of bottom-derived or

terrestrial sources of nutrients for the productivity of

the nearshore zone (Pearce et al., 1999). Sample

stations were therefore placed at similar distances

from shore to minimise this effect. However, differ-

ences in coastline features (Fig. 1) and possible

mixing regime and current meant that those effects

were probably still important in generating some

differences we saw between the sampling areas.

Hydrodynamic modelling of these waters suggests

that the net water movement is northwards in both

areas, and on average 50% faster in the southern region

(Pattiaratchi et al., 1995). Based on previous studies

by Thompson & Waite (2003), stations S3 and N3, just

north of the outlets, were selected as the most likely

place for an impact of the sewage water under ‘‘low’’ to

‘‘normal’’ northward flow. Under conditions of low

physical mixing and ‘‘normal’’ to ‘‘high’’ northward

flow it was hypothesised that there could be an impact

of the sewage water extending to stations S4 and N4.

Stations S1 and N1 were selected as control sites which

generally have been found to receive nutrient-poorer

water from the south (Thompson & Waite, 2003).

Field measurements

Sampling locations were established by GPS within

±50 m. Depth-integrated samples of the upper 10 m

were pumped from the water column to fill a 20 l

carboy while raising the pump intake at a constant

rate. Subsamples were taken from the carboy for

analysis of inorganic nutrients, pigments, Chl a size

fractions, primary production and spectral light

absorption by particles and coloured dissolved

organic material (CDOM). At each station we also

measured the irradiance spectrum using a LI-COR

LI-1800 underwater spectroradiometer. Salinity and

temperature profiles, measured with a CSIRO/YEO-

KAL model 606 logger, showed no signs of strati-

fication in any of the stations. Horizontal changes in

Chl a were determined by pumping water from 1 m

depth using a WetStar fluorometer interfaced with a

Trimble GPS system (Sunnyvale, CA, USA). Data

were logged to a computer every 2 s and interpolated

by the SigmaPlot (SPSS) software producing con-

tours of surface Chl a in the vicinity of the

wastewater outlets. Sample treatment and analysis

are further described in the following sections.

Laboratory measurements

Water samples were analysed for nitrate (NO3-)

according to the guidelines of Wood et al. (1967),

ammonia (NH4?) according to Solorzano (1969) and

Phosphate (PO4-) according to Murphy & Riley

(1962).

High-performance liquid chromatography (HPLC)

analysis provided estimates of the concentration of

chlorophylls, carotenoids and other accessory pig-

ments. Immediately after water sampling, 2,000 ml

of water was filtered onto 25 mm Advantec GF 75

Hydrobiologia (2009) 620:191–205 193

123

glass fibre filters and samples were stored in liquid

nitrogen for c. 6 months until further analysis. In the

laboratory, filters were transferred to 2.5 ml metha-

nol, sonicated on ice for 30 s, and filtered (0.2 lm).

One ml filtrate was then transferred into HPLC vials

containing 250 ll water. HPLC analyses were per-

formed on a Shimadzu LC 10A system with a

Supercosil C18 column (250 9 4.6 mm, 5 lm) using

a slight modification of the Wright et al. (1991)

method as described in Schluter & Havskum (1997).

This method does not provide full baseline separation

of lutein and zeaxanthin, so these pigments were

manually separated based on their absorption spectra

recorded by photodiode array detection. Pigments

were identified by retention times and absorption

spectra identical to those of authentic standards,

and quantified against standards purchased from

the International Agency for 14C Determination,

Hørsholm, Denmark.

In accordance with Babin et al. (1996) we com-

puted a ‘‘non-photosynthetic pigment index’’ (NPP

index) from the HPLC analyses. The NPP index

was determined as the weight-to-weight ratio of

non-photosynthetic pigments (i.e. zeaxanthin plus

diadinoxanthin and O-carotene) to total pigments. Con-

centrations of Chl a were analysed in three size

fractions: 0.7–5 lm * picoplanktonic algae, 5–20 lm

* nanoplanktonic algae and [20 lm * microalgae.

About 2,000 to 3,000 ml of each water sample was

gently filtered through a 25 mm Advantec GF 75 glass

fibre (pore size of 0.7 lm), 25 mm poretics filter (pore

size of 5 lm) and a 25 mm nylon mesh (pore size of

20 lm). The Chl a retained by the different filter types

was then determined using the HPLC method previ-

ously described. The three Chl a size fractions were

finally determined by subtraction.

Spectral light absorption (300–800 nm by 0.5 nm)

of particulate material was determined using the

quantitative filtering technique described by Kishino

et al. (1985). Immediately after water sampling,

2,000 ml of water was filtered onto 25 mm Advantec

GF 75 glass fibre filters and samples were stored in

liquid nitrogen for c. 1 month until further analysis.

Absorption measurements were made with a Shimadzu

UV-2401PC UV-Vis recording spectrophotometer

equipped with an integrating sphere. CDOM samples

were filtered through a 0.2 lm Minisart syringe filter

that had been pre-washed with Milli-Q water

and 10 ml of sample before use. Samples where

then stored under cold and dark conditions until

spectrophotometric analysis using a GBC UV/VIS

920 spectrophotometer. Measurements were per-

formed using a 10 cm cuvette over the 300–800 nm

range with 0.5 nm increments and referenced to Milli-

Q water according to Stedmon et al. (2000).

Photosynthetic rates (P) were measured as a function

of irradiance (E) on depth-integrated samples by the14C technique (Steemann-Nielsen, 1952) according to

the guidelines by Lewis & Smith (1983). Water

samples were incubated at irradiances of 20, 40, 80,

160, 320, 640 and 1,280 lmol photons m-2 s-1.

Calculations

Subsurface irradiance measured at different depths

was used to calculate the attenuation coefficient for

downwelling irradiance (Kd) over the PAR region

(400–700 nm). Kd was determined from 10 PAR

integrated irradiance vs depth measurements by

estimating the parameters in the exponential equation

Ez ¼ E0 e�zKd with a non-linear least squares regres-

sion technique. Ez is PAR irradiance at depth (z) and

E0 is irradiance at the surface.

To evaluate the importance of growth irradiance

on different photosynthetic parameters, we estimated

the mean irradiance level experienced by the plank-

ton community (Emean). Emean was calculated for all

samples collected in the surface waters according to

the equation Emean ¼ E0ð1� e�KdzÞ=Kdz (Riley,

1957), in which E0 is the incident light and z is the

mixing depth assumed to be the total water depth at

the sampling stations. E0 was calculated as the mean

surface irradiance over the duration of each sampling

period * 4 h. Assuming the absorption of bottom

reflected light to be negligible we estimated the

fraction of surface irradiance reaching the bottom

(Ebot) as Ebot ¼ expð�KdzÞ 9 100%, where z is the

maximum depth.

Optical density spectra of the particulate matter

were smoothed using a 10 nm running average, and

corrected for background absorption by subtracting the

average optical density measured between 750 and

800 nm. The corrected optical densities were con-

verted into particulate absorption coefficients (ap)

using the b-correction factor given by Bricaud &

Stramski (1990): ap(k) = 2.3 * OD(k) * AF/V *

(1.63 * OD(k)-0.22), where V is the filter volume, AF

194 Hydrobiologia (2009) 620:191–205

123

is the effective filter area, OD is the optical density of

the particulate material and k is the wavelength.

Samples were measured before and after methanol

extraction in order to correct for absorption by detritus

(adet). Absorption spectra were studied per unit con-

centration of Chl a by calculating the Chl a-specific

absorption aph* (k) as aph

* (k) = aph(k) m-1/mg Chl

a m-3, where the absorption by phytoplankton pig-

ments (aph) were determined as aph(k) = ap(k)

- adet(k). Spectrally averaged (400–700 nm) Chl

a-specific absorption (aph* ) was calculated by summing

aph* over all measured wavelengths (400–700 nm by

0.5 nm) and dividing by the number of wavelengths

measured (n = 601), (Markager & Vincent, 2001).

The absorption coefficients of CDOM was calcu-

lated by the equation Ak = 2.303Ak/L, where Ak is

the optical density at wavelength k and L is the path

length. Before calculations, optical density spectra

were smoothed using a 10 nm running average.

CDOM’s spectral properties were modelled from

300 to 650 nm using the equation aCDOM(k) = a-

k0eS(k0-k) ? K, where k0 was equal to 400 nm and K

is a background constant that allows for any baseline

shift or attenuation not due to organic matter

(Markager & Vincent, 2000; Stedmon et al., 2000).

The parameters a400, S and K were estimated

simultaneously via non-linear regression, using the

secant iterative method in the SAS/STAT software

package (SAS Institute Inc., 1994).

The fraction of surface light absorbed by each of

the four components; water, CDOM, non-pigmented

particles and phytoplankton, was calculated as the

fraction absorbed by each component (n) relative to

the total absorption, an/atot, where atot = aw ? aCDO-

M ? aph ? adet. The fraction for each component

(Fn) between 400 and 700 nm was summed between

400 and 700 nm after weighting with the surface

spectrum I0(k) according to:

FnðPARÞ ¼

P700

400

FnðkÞ � E0ðkÞ

E0ðPARÞ

where E0(PAR) was calculated from measurements

of the surface irradiance spectrum E0(k) by addition

of E0(k) over the PAR range.

Photosynthetic parameters were obtained from a

non-linear regression fit of the P vs E data to a

saturating exponential model (Webb et al., 1974),

modified by the inclusion of an offset (c), PB

Pest(1 - exp(-aBE0/Pest)) ? c, where aB is the light-

limitation parameter (g C g-1 Chl a m2 mol-1), and

PmaxB , the light-saturated photosynthetic rate

(g C g-1 Chl a h-1), was calculated as Pest ? c.

The offset (c) was incorporated to avoid bias in the

estimation of aB, which occurs when the curve is

forced through the origin (Markager et al., 1999).

The saturation irradiance (Ek) was calculated as PmaxB /

aB. The maximum photon yield of carbon fixation

/Cmax was calculated as /C max ¼ ðaB=ð12 � �a�phÞ,where 12 is the molar weight of carbon (g) and �a�ph is

the mean Chl a-specific absorption coefficient of

algae, weighted by the spectral distribution of the

incubation lamp according to the method used by

Markager et al. (1999). Primary production

(mg C m-2 d-1) was finally calculated for the upper

10 m as PA ¼P10m

i ¼ 1 PBmax � ð1 � expð�aB �

�

Ez daily=PBmaxÞÞÞ � Chla½ �, where Ez_daily is the

daily (06:00–19:00 h * 13 h) average PAR intensity

at depth z, determined as Ez_daily = E0_dailye-Kdz.

Statistical analysis

Simple correlations between environmental variables,

photosynthesis parameters and bio-optical properties

of phytoplankton were tested by Pearson’s correlation

analysis. Normality of the data was tested using

Kolmogorov–Smirnov analysis. Student’s t-test were

applied to test for overall differences between the two

regions.

Results

Nutrients

In the southern study region, ammonium was the

primary form of nitrogen (N) released through the

outfall after primary treatment; phosphate and ammo-

nium concentrations were highest close to the outfall

(primary treated wastewater). In the northern region,

nitrate was the primary N source released from the

outfall after secondary treatment, and concentrations

peaked closest to the outfall (Fig. 2). In general,

concentrations of these key nutrients at the northern

and southern outfall, respectively increased from

control stations (N1 and S1) to stations located at the

sewage outlets after which concentrations decreased

Hydrobiologia (2009) 620:191–205 195

123

gradually or became undetectable with increasing

distance to the north in both areas. The molar ratio of

nitrate ? ammonium to phosphate (N:P) was below

the Redfield level of 16 for all stations, suggesting

that nitrogen was the main potentially limiting

nutrient. However, since concentrations of nitrate

and phosphate were cross-correlated (r = 0.67), any

correlations between N concentrations and phyto-

plankton physiological indicators around the outfall

could surface statistically as a parallel relationship

with phosphate concentrations. This confounding

factor is exacerbated by the fact that N concentrations

often reach non-detectable levels, lowering the sta-

tistical power to detect the effects of N alone, when

comparisons are made across a wide range of N

concentrations.

Phytoplankton distribution

Depth integrated (0–10 m) Chl a concentrations were

low (0.15–0.71 mg Chl a m-3) and the variation was

not significant except at station S4 where the Chl a

concentration was high (Table 1). However, the

horizontal mapping of the surface Chl a concentra-

tions did show a pattern (Fig. 3). At Sepia there was a

gradual increase from about 5 km north of the outlet

and northwards. The increase did not start before

station S3, providing some agreement with the depth-

integrated measurements. At Ocean Reef the surface

mapping showed a more complex pattern, with a

patch of elevated values south of the outlet and a

smaller patch to the north and with the lowest values

around the outlet. Chl a size fractionation indicated

that the phytoplankton at Sepia Depression was

dominated by pico- and microplankton, whereas the

nano-fraction remained low, between 4 and 17%

(Fig. 4A). At Ocean Reef, the nano-sized algae were

a larger fraction of the phytoplankton (27–69%), with

maximum abundance at the sewage outlet station

(N2). Size distributions of algae were significantly

different between sampling areas (P = 0.02) and the

relative abundance of the phytoplankton size groups

was highly correlated to nitrate (Table 2). Thus, the

nano-fraction increased with increasing nitrate while

pico- and micro-sized phytoplankton decreased, sug-

gesting a response in community structure to inputs

of nitrate. Since the systems were clearly nitrate

limited, effects of phosphate also indicated by

correlations with phytoplankton size groups, are most

likely to be a result of the significant cross-correlation

between the different nutrients (Table 2).

Pigment analysis revealed small differences in the

relative abundance of the accessory pigments within

and between water systems (Fig. 4B), suggesting that

phytoplankton species composition was not strongly

affected by the inflow of nutrient-enriched sewage

water. However, a noticeable difference was found at

station S4, which, compared to the other stations in

the Sepia Depression area, had a twofold higher

phytoplankton biomass (Table 1) and relatively

higher concentrations of Chl c and fucoxanthin,

indicating high abundance of diatoms. In general,

dominance of the accessory pigments fucoxanthin,

190-hexanoyloxyfucoxanthin and zeaxanthin sug-

gested that the phytoplankton communities in both

systems were dominated by diatoms, prymnesio-

phytes and cyanobacteria. Phytoplankton counts,

using an inverted light microscope, documented the

overall dominance of diatoms but not the importance

of prymnesiophytes and cyanobacteria (data not

shown). However, prymnesiophytes and cyanobacte-

ria are generally small (*2–10 lm) and therefore

difficult to recognise using an inverted microscope.

HPLC determined the pigment concentrations, and

a pigment ratio file (Henriksen et al., 2002) was

furthermore applied in the CHEMTAX program to

calculate the relative abundance of the different

phytoplankton classes according to the guidelines by

Fig. 2 Concentrations (lM) of nitrate (NO3-) and phosphate

(PO4-) and the corresponding N:P molar ratio at Sepia

Depression and Ocean Reef

196 Hydrobiologia (2009) 620:191–205

123

Mackey et al. (1996). These results (not shown)

suggested that more than 75% of the phytoplankton

community was composed of diatoms, cyanobacteria

and prymnesiophytes, in order of relative importance.

Furthermore, the CHEMTAX calculations confirmed

the increased dominance of diatoms at Ocean

Reef station N4 (78%) compared to stations N1–N3

(24–43%).

Light absorption

The diffuse attenuation coefficient for downwelling

irradiance (Kd) was slightly higher at Sepia Depres-

sion (0.20–0.24 m-1) than at Ocean Reef (0.19–

0.22 m-1), although not statistically significant

(P = 0.58). Furthermore, the Kd values were close

to the annual average of 0.19 ± 0.01 m-1

(mean ± 95 CL) observed for both areas (Thompson

& Waite, 2000).

Total light absorption in the PAR region (atot)

ranged from 0.18 to 0.22 m-1 at Sepia Depression

and from 0.20 to 0.24 m-1 at Ocean Reef (Fig. 5A).

Although total light absorption was almost equal

among sampling stations and areas, differences in

water depth caused very low light levels at the sea

floor at Sepia Depression (0.6–1.4%) whereas the

shallower Ocean Reef area received a significantly

larger fraction of the surface irradiance (9.1–16.4%)

(see Fig. 5B). Total light absorption was dominated

by water (55–75%) but the variation in atot was

primarily accounted for by variations in CDOM

absorption. At both Sepia Depression and Ocean

Reef, CDOM absorption increased from the control

stations to the sewage outlets. Also the relative

absorption of CDOM decreased gradually from south

to north (Fig. 5B). However, differences in CDOM

absorption were not significant among sampling areas

(P = 0.33). Correlation analysis showed a significant

positive relation among CDOM absorption and

nitrate and phosphorus concentrations (Table 2),

indicating that nitrate, phosphate and CDOM origi-

nated from the same source.

Light absorption by phytoplankton (aph) contrib-

uted little to the overall light absorption and varied

little among sampling stations and areas (Fig. 5).

Phytoplankton light absorption was however closely

related to variations in Chl a concentrations

(r = 0.95, P \ 0.001). Normalising aph(k) to Chl a

provided the Chl a-specific absorption coefficientTa

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13

05

0.6

1\

0.0

10

.87

0.0

39

0.2

10

.89

0.1

24

13

.33

46

.49

.31

2.9

20

12

31

75

N4

90

.20

31

60

.39

\0

.01

0.8

70

.02

40

.31

0.8

30

.11

32

2.3

32

9.0

9.4

13

.41

95

43

26

5

Un

its:

Dep

th,

m;

Em

ean

and

EK

,lm

ol

qu

anta

m-

2s-

1;

Kd,

m-

1;

ino

rgan

icn

utr

ien

ts,lM

;a

CD

OM

(400–700

nm

),m

-1;

Ch

la

,m

gm

-3;

Nan

o,

%o

fto

tal

Ch

la

bet

wee

n5

and

20

lm;

aph

*,

m2

g-

1C

hl

a;

Pm

ax

B,

gC

g-

1C

hl

ah

-1;aB

,g

Cg

-1

Ch

la

mo

l-1

ph

oto

ns

m-

2;/

Cm

ax,

mm

ol

Cm

ol-

1p

ho

ton

s;P

A,

mg

Cm

-2

d-

1

Hydrobiologia (2009) 620:191–205 197

123

(aph* ), from which the mean spectral coefficient (aph

* )

can be determined. This coefficient was slightly

higher at Ocean Reef (P = 0.05) and showed a strong

dependence on the size composition of the phyto-

plankton and decreased significantly with increasing

abundance of microalgae (r = 0.85, P \ 0.01). Fur-

thermore, aph* decreased with increasing Chl a

concentration, indicating increased pigment packag-

ing with increasing phytoplankton biomass (Table 2).

Both aph* and aph436

* /aph676* (the ratio between Chl a-

specific absorption in the blue to the red part of the

spectrum) increased with increasing carotenoid: Chl a

ratio as expected (Table 2).

Photosynthesis

None of the samples experienced photoinhibition at high

incubation irradiances (1,200 lmol photons m-2 s-1),

indicating that the phytoplankton were adapted to high

irradiances (Fig. 6). This was further supported by the

high irradiances experienced by the algae (Emean, see

Table 1), which on average constituted 104 ± 34%

(mean ± 1SD) of the irradiance needed to start

saturation of photosynthesis (Ek). The maximum photo-

synthetic rate normalised to Chl a (PmaxB ) ranged from 5.5

to 12.4 g C g-1 Chl a h-1 and was positively related to

nitrate (Fig. 6) and the fraction of nano-sized phyto-

plankton (Table 2). Although mean PmaxB was higher in

Sepia Depression (12.2 ± 3.5 g C g-1 Chl a h-1;

mean ± SD) than Ocean Reef (8.5 ± 2.6 g C g-1 Chl

a h-1), differences were not significant (P = 0.14) due

to a large within-area variability in photosynthesis at

saturating light (Fig. 5). The initial slope of the P–E

curve normalised to Chl a (aB) ranged from 4.8 to

13.4 g C g-1 Chl a m-2 mol-1 photons (Table 1) and

decreased with increasing Emean (Table 2). Higher

concentrations of non-photosynthetic pigments (NPP)

resulted in a reduction in aB and photon yield (/Cmax)

A

Longitude (°E)

115.37

Latit

ude

(°S

)

32.08

32.21

0.750.70

0.650.60

0.80

0.85

0.55

0.55

0.90

S1

S4

S3

S2Outlet

115.39 115.43

31.41

31.50

1.0

1.0

1.0

0.9

0.9

0.9

0.8

0.80.8

0.8 0.8

1.2

1.1

1.1

0.7

0.7

1.31.31.2

0.7

1.1

0.9

0.9

B

N1

N2Outlet

N3

N4

115.41

Fig. 3 Map of the surface

(1 m) horizontal

distribution of Chl a at (A)

Sepia Depression and (B)

Ocean Reef, estimated from

high frequency fluorometry

data. Dotted grey line is the

GPS track of the boat

198 Hydrobiologia (2009) 620:191–205

123

(Table 2). /Cmax values ranged from 14 to 43 mmol

C mol-1 photons with no obvious enhancement at outlet

stations. Photon yield was, however, higher at Ocean

Reef (P = 0.02), suggesting an effect of higher concen-

trations of nitrate, compared to Sepia Depression

(Table 1). Ek values ranged from 142 to 645 lmol

quanta m-2 s-1 (Table 1) and increased significantly

with Emean. Daily area primary production (PA) calcu-

lated for the upper 10 m ranged from 98 to

378 mg C m-2 d-1 and was strongly related to phyto-

plankton biomass [Chl a] but unrelated to nutrient

concentrations and light availability (Table 2). Since

[Chl a] varied more within the sampling areas

than between them, large differences in area produc-

tion were only observed between sampling stations

(Table 1). Samples with a high content of non-photo-

synthetic pigments, furthermore, had a low area

production.

Discussion

Phytoplankton size structure, productivity and bio-

optics were significantly affected by inputs of sewage

water, enriched in N (either nitrate or ammonium)

and phosphate among other nutrients. The general

response of the phytoplankton community was an

increase in nano-sized (5–20 lm) phytoplankton cou-

pled with an increase in the photosynthetic capacity

per unit chlorophyll, reflected in PmaxB (Fig. 7). Inter-

estingly, there was also a significant impact of sewage

water on the absorption by coloured dissolved organic

matter (CDOM). Even away from the outfalls, the

impact of CDOM on light absorption was unusually

important for oligotrophic waters ([20% light absorp-

tion). The effects were not always seen at the outlets

but rather some distance ‘‘downstream’’ (this would be

either north or south of the outlets, depending on wind

Fig. 4 Changes in

phytoplankton composition

at Sepia Depression and

Ocean Reef. (A) Changes in

the relative abundance of

three size classes

determined by Chl a size

fractionation and (B)

changes in the relative

abundance of

phytoplankton classes

determined from pigment

ratios. All data are average

of two replicates

Hydrobiologia (2009) 620:191–205 199

123

history) where the phytoplankton community has

acclimated or adapted to the conditions. Instantaneous

changes in primary production or algal abundance near

the outfalls were not directly observed, but the clear

relationship between maximum photosynthetic rate

and nitrate concentrations, for example, indicated that

maximum achievable primary production rates were

stimulated by the local availability of inorganic

nitrogen (nitrate ? ammonia), which here included

inputs of nutrient-rich sewage water. It was particu-

larly of note that nitrate inputs generated the greatest

nanoplankton response, which may be a model for

regional upwelling systems off the Western Australian

coast subject to sporadic nitrate inputs (Twomey et al.,

2007).

Although effects of sewage water on photosyn-

thesis and bio-optics were comparable between the

two outfalls (Ocean Reef vs Sepia Depression),

differences between the sampling areas were overall

larger than the variability within the areas (Table 1),

reflecting differences in nutrient type (nitrate result-

ing from nitrification during secondary treatment vs

ammonium due to lack of nitrification during primary

Nitrate (µM)

PB m

ax (

g C

g-1

Chl

a h

-1)

00.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

5

10

15

Sepia DepressionOcean Reef

r2 = 0.75p < 0.05

Fig. 7 Light-saturated photosynthetic rate (PmaxB ) as a function

of nitrate (NO3-)

Fig. 5 Light absorption by CDOM, phytoplankton, detritus,

and water at Sepia Depression and Ocean Reef. Data are based

on spectrophotometric measurements. (A) Absolute absorption

values (m-1) and (B) relative absorption values (%) and %

surface irradiance reaching the sea floor (white dot)

Fig. 6 Photosynthesis normalised to chlorophyll as a function

of irradiance at Sepia Depression and Ocean Reef. Curves were

fitted using a saturating exponential model by Webb et al.

(1974). Sewage outlets are located at S2 and N2

200 Hydrobiologia (2009) 620:191–205

123

treatment) and in water depth (10 vs 20 m) diluting

the sewage water approximately twice as much at

Sepia Depression (Pattiaratchi et al., 1995). Using

water depth as a surrogate for water dilution, we

found decreasing availability of nitrate and phosphate

at the deeper and more diluted sites, but increasing

light availability per unit depth due to lower atten-

uation by CDOM (Table 2). The importance of

CDOM in light attenuation is novel for this coastal

region and has not previously been documented at

these outfalls. Differences in sewage treatment

between the two areas suggest (as indicated above)

that the outlet at Ocean Reefs would be a more direct

source of nitrogen than the outlet at Sepia Depres-

sion, because of lower dilution rates.

Another parameter that showed an unexpected

behaviour was the Chl a-specific absorption coeffi-

cient (aph* ). From previous studies we would expect an

increase in nutrient availability to cause an increase in

cell size and, due to chloroplast packaging, a decrease

in aph* (Stæhr et al., 2002). In this study, however,

aph* increased with increasing concentrations of inor-

ganic nitrogen and phosphoros. The most likely

explanation for this contradiction is that the phyto-

plankton responding to nutrient enrichment were the

nano-sized algae, which are in the middle of the size

spectrum. Studies of phytoplankton composition tradi-

tionally predict an increased dominance of large

phytoplankton with increasing nutrient availability

(Stolte & Riegman, 1995; Philippart et al., 2000)

because large cells are thought to have a greater storage

capacity for nutrients and to be better competitors under

high and fluctuating nutrient regimes (Sommer, 1984;

Kiørboe, 1993). The biomass of smaller algae is

believed to be controlled by microzooplankton (Things-

tad & Sakshaug, 1990; Kiørboe, 1993). The success of

nano- over pico- and micro-phytoplankton found in this

study therefore suggests that although concentrations of

the limiting nutrient, nitrate, was elevated, concentra-

tions were not high enough to support growth of large

diatoms as is often seen in eutrophic coastal systems

(Kiørboe, 1993; Stæhr et al., 2004). Low silicate

availability (\2 lM year round) may also have reduced

diatom growth (Lourey et al., 2006).

Changes in photosynthetic performance

The high light level needed to start saturation of

photosynthesis (Ek), combined with the absence of

photoinhibition at high irradiance, indicated that the

phytoplankton assemblages were well acclimated to

high light conditions. Higher mean irradiance (Emean)

was accompanied by lower values of aB, which

caused a positive correlation between Emean and Ek

(Table 2). PmaxB and aB both increased with increasing

nitrate concentrations, showing that the photosyn-

thetic performance was strongly dependent on the

availability of nitrogen and therefore responded to

enrichment from sewage water.

The photosynthetic yield (/Cmax) values reported in

this study (14–43 mmol C mol-1 photons) is in the

lower range of values found in temperate and tropical

studies of marine phytoplankton between 10 and

100 mmol C mol-1 photons (Tyler, 1975; Kishino

et al., 1986; Lewis et al., 1988; Cleveland et al., 1989;

Babin et al., 1996). Low /Cmax values have previously

been suggested to result from light absorption by

partially degraded or inactive chlorophyll (Lewis

et al., 1988; Cleveland et al., 1989). The suggestion

by Cleveland et al. (1989) was based on a significant

negative relationship between the ratio aph436/aph676

and /Cmax, plus the fact that the aph436/aph676 ratio

often is higher in field samples than in healthy cultures,

where values are usually below 2 (Cleveland & Perry,

1987; Cleveland et al., 1989). The aph436/aph676 ratios

of this study were all above 2 (2.18 to 3.48) and were

significantly negatively correlated to /Cmax. Further-

more, /Cmax values decreased with increasing

concentration of non-photosynthetic pigments. The

dependency of /Cmax on inorganic nitrogen, in natural

phytoplankton assemblages, has previously been

described by a hyperbolic function of nitrate (Kolber

et al., 1988; Falkowski et al., 1991; Geider et al.,

1993; Babin et al., 1996). The apparent lack of

response of /Cmax to nutrient enrichment found in

this study therefore seems to be related to differences

in nitrogen source (ammonia at Sepia D. and nitrate at

Ocean R.), and high concentrations of partially

degraded or inactive pigments, possibly related to

high rates of microheterotrophy.

Overall, we found evidence that changes in key

optical parameters of primary production, light

absorption, pigmentation and phytoplankton size

composition were strongly related to variations in

availability in nitrate, ammonium and phosphoros but

less to the average light level, supporting the idea of

an oligotrophic ecosystem being strongly affected

primarily by the input of nutrients and less by inputs

Hydrobiologia (2009) 620:191–205 201

123

Ta

ble

2P

ears

on

corr

elat

ion

coef

fici

ents

for

the

corr

elat

ion

sam

on

gp

aram

eter

sfo

rp

ho

tosy

nth

esis

,p

rim

ary

pro

du

ctio

n,

lig

ht

abso

rpti

on

,p

igm

enta

tio

n,

nu

trie

nts

,E

mean,

dep

th,

CD

OM

,an

dre

lati

ve

abu

nd

ance

of

the

nan

o-s

ized

ph

yto

pla

nk

ton

EK

PmB

PA

aph

*C

hl

aC

aro

/Ch

la

NP

Pin

dex

aph436

*/a

ph676

*N

O3-

NH

4?

PO

4-

Em

ean

Dep

tha

CD

OM

Nan

o

aB-

0.6

7( *

)0

.49

0.1

80

.58

-0

.28

0.2

3-

0.6

10

.27

0.6

4*

-0

.57

0.8

5*

*-

0.6

8( *

)-

0.9

9*

*0

.37

0.7

7*

/C

max

-0

.69

( *)

0.1

00

.59

-0

.11

0.1

8-

0.2

2-

0.7

4*

-0

.75

*0

.23

-0

.50

0.4

50

.45

-0

.76

*-

0.0

20

.35

EK

0.2

50

.38

-0

.10

-0

.06

0.2

40

.59

0.4

6-

0.1

00

.26

-0

.44

0.8

0*

0.6

3( *

)-

0.0

1-

0.2

3

PmB

-0

.20

0.5

9-

0.3

90

.49

-0

.07

0.1

00

.87

**

-0

.37

0.5

7-

0.1

4-

0.5

40

.60

0.7

8*

PA

-0

.46

0.8

9*

*0

.75

*-

0.8

3*

*0

.88

**

-0

.01

-0

.38

-0

.13

0.0

30

.24

0.0

2-

0.0

2

aph

*-

0.6

4( *

)0

.67

( *)

0.0

10

.55

0.6

2( *

)-

0.1

30

.72

*-

0.3

6-

0.5

40

.57

0.7

0( *

)

Ch

la

-0

.81

*0

.54

-0

.70

( *)

-0

.26

-0

.09

-0

.47

0.3

20

.21

-0

.05

-0

.32

Car

o/C

hl

a0

.45

0.7

2*

0.4

60

.20

0.5

7-

0.1

9-

0.2

00

.46

0.5

6

NP

Pin

dex

0.7

1*

-0

.36

0.7

0( *

)-

0.2

50

.29

0.6

5( *

)-

0.1

8-

0.3

8

aph436

*/a

ph676

*-

0.0

10

.37

0.0

4-

0.4

9-

0.6

9( *

)0

.80

( *)

0.9

5*

*

NO

3-

-0

.46

0.6

7( *

)0

.60

-0

.84

*0

.62

( *)

0.8

3*

NH

4?

-0

.07

0.2

30

.56

0.0

5-

0.3

8

PO

4-

-0

.36

-0

.63

( *)

0.8

6*

*0

.92

**

Em

ean

0.6

4( *

)-

0.3

2-

0.5

3

Dep

th-

0.4

2-

0.8

0*

aC

DO

M0

.81

*

**

P\

0.0

1,

*P

\0

.05

,( *

)P

\0

.1

Par

amet

ers

and

un

its

are

exp

lain

edin

Tab

le1

202 Hydrobiologia (2009) 620:191–205

123

of coloured dissolved organic matter in the sewage

water. Obviously, sewage contains large quantities of

phosphoros and both N and P levels are enhanced in

areas of heavy sewage influence. However, given the

low N:P molar ratios (Fig. 2) both systems clearly

appear to be N limited and the overall response to

increasing P is only to be explained by the fact that N

and P co-vary.

Conclusions

Sewage outlets into two shallow coastal systems were

found to strongly influence a range of parameters

related to phytoplankton composition, productivity

and optical properties. Nanoplankton concentrations

were augmented by N input. Nitrate seemed to induce

a greater response than ammonium in the nanoplank-

ton—this seems to be an appropriate biogeochemical

model for upwelling-driven new production off the

Western Australian coast. The actual effects were

local to the outfalls and seem to disappear about

15 km from the outlet in the prevailing direction of

the current. Large variability was observed in the Chl

a-specific absorption coefficient, which varied pri-

marily as a function of phytoplankton size, while

changes in pigment content to nutrients and light had

minor influence. We have shown that significant

changes in photosynthetic and bio-optical properties

(light absorption and pigmentation) may occur within

short distances. Differences in these response vari-

ables were related to differences in the availability of

nitrate and ammonia. Differences in sewage treat-

ment and thus nitrogen source between the areas,

however, caused nitrogen to be overall less important

for the changes in photosynthetic and bio-optical

properties than would be expected in this nitrogen-

poor environment. Response variables were also

strongly related to water depth, suggesting that

sewage dilution was important for the observed

differences in photosynthetic and bio-optical proper-

ties of the studied areas.

Acknowledgements This article was prepared as a part of the

DECO (Danish Environmental monitoring of Coastal waters)

project. The DECO project was financed by the Danish Earth

Observation Program grant no. 9600667. The Carlsberg

Foundation has contributed with support for instrumentation.

We are grateful to Bridget Alexander for technical support and

Winnie Martinsen and for laboratory assistance. We thank

Peter A. Thompson for providing access to the extended

PLOOM dataset.

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