Limnol. Oceanogr., 55(5), 2010, 1835–1850
2010, by the American Society of Limnology and Oceanography, Inc.
doi:10.4319/lo.2010.55.5.1835
E
High concentrations of mycosporine-like amino acids and colored dissolved organic
matter in the sea surface microlayer off the Iberian Peninsula
Gavin H. Tilstone, Ruth L. Airs,* Victor Martinez-Vicente, Claire Widdicombe, and Carole Llewellyn
Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth, Devon, United Kingdom
Abstract
Mycosporine-like amino acids (MAAs), phytoplankton pigments, and inherent optical properties were
analyzed in sea surface microlayer (SSM), near-surface (0–2 m), and subsurface (0–110 m) samples from stations
off the Iberian Peninsula in June–July 2005. During a visible surface slick, MAA concentrations reached
290 mg L21 in the SSM, which correlated with an increase in abundance of the dinoflagellate, Prorocentrum
micans (. 4000 cells mL21) and covaried with low Ekman transport. High levels of colored dissolved organic
material (CDOM) were also found in the SSM, and a prominent absorption shoulder between 300 and 340 nm
was evident in the CDOM absorption spectra, indicative of MAAs in the dissolved fraction. Low CDOM slope
ratios (SCDOM(275–295) : (350–400)) during the development of the surface slick suggest microbial production of
CDOM, possibly exuded from the sudden proliferation in P. micans. In the absence of a well-defined SSM, higher
SCDOM(275–295) : (350–400) ratios were found, suggesting photobleaching of CDOM at the surface. The
particulate absorption spectra of MAAs (aPMAA(l)) were effective at absorbing ultraviolet (UV) radiation in a
narrow spectral band between 310 and 350 nm. Since aCDOM(l) exhibits an exponential rise in absorption in the
UV and was high in the SSM and the subsurface compared with aPMAA(l), CDOM would be the principal
mechanism of UVA and B attenuation in the water column, providing an additional sunscreen to phytoplankton.
The sea surface microlayer (SSM), commonly defined as
the upper 0–1 mm of the surface ocean (Liss and Duce
1997), represents a physically stable environment due to
surface tension and the presence of organic films. It is a
unique environment where physical, chemical, and biological processes are distinctly different compared with
underlying waters (Hardy 1982), particularly under calm
conditions. A slick, which can be defined as a visibly
surfactant-influenced surface (Liss and Duce 1997), occurs
where surface films become more concentrated. Data on
physical, biological, or chemical differences between slick
and nonslick SSM conditions are scarce because of a low
frequency of sampling opportunities. Optical imagery of
the sea surface indicates that surface films cover large areas
of the coast and open ocean, with 75% of sea surface
photographs showing slicks when the wind speed is
between 2 and 3 m s21 (Romano 1996).
The SSM has been found to be enriched in bacteria
(Bezdek and Calucci 1972), amino acids (Kuznetsova et al.
2004), anthropogenic pollutants (Wurl and Obbard 2004),
flagellates (Joux et al. 2006), and carbohydrates (Momzikoff et al. 2004) in the dissolved and particulate fractions
compared with near-surface waters. Mechanisms of enrichment include upward transport of particles clinging to
bubbles, and atmospheric deposition and diffusion processes, and may be unique to the sources and characteristics
of a particular component. Bubble transport or upward
transport of components attached to buoyant particles has
been proposed to be a major vector for SSM enrichment of
microorganisms (Joux et al. 2006). Enrichment or depletion
of autotrophic phytoplankton in the microlayer can be
highly variable and depends on weather conditions and
vertical stratification (Cullen et al. 1989). There is a
* Corresponding author: ruai@pml.ac.uk
growing body of evidence of enhanced concentrations of
biological material in the SSM. This suggests that field
sampling needs to be adjusted for the presence of
microlayers and that modeling studies on light attenuation,
air–sea gas exchange, or even export of organic material
from the photic zone may need to be modified to account
for this.
Organisms in the SSM can be exposed to high levels of
ultraviolet radiation (UVR), both UVA (320–400 nm) and
UVB (280–320 nm). Several studies report degrees of
photoinhibition of phytoneuston productivity in high
summer light intensities (Williams et al. 1986), indicating
photoprotection may be an essential requirement for
survival. One photoprotective mechanism that some
phytoplankton species use to counteract the deleterious
effects of UVR is the production of UV-absorbing
compounds called mycosporine-like amino acids (MAAs)
(Shick and Dunlap 2002). MAAs exhibit broad UV and
visible (VIS) spectra with absorption maxima between 310
and 366 nm (Shick and Dunlap 2002). Studies of MAAs in
the marine environment are mainly limited to particulate
material from coastal waters and corals. No determination
of these compounds in the SSM has been reported, despite
studies revealing high SSM UV absorption compared with
subsurface water (Carlson 1982). Colored dissolved organic
matter (CDOM) is a major contributor to the attenuation
of UVR in both coastal and oceanic waters (Tedetti and
Sempere 2006), but few studies have been conducted on
CDOM levels in the SSM and its role in UVR attenuation
(Carlson and Mayer 1980). MAA type absorption signatures have been detected in CDOM samples (Whitehead
and Vernet 2000); the presence of such compounds in SSM
samples, however, has yet to be verified.
The objective of this research was to assess whether the
phytoplankton community, pigment composition, MAAs,
1835
1836
Tilstone et al.
Deployment schedule of MLSD, GS, and NSSD sampling devices. GMT, Greenwich Mean Time.
Table 1.
Station
1.1
1.2
1.3
2.1
2.2
2.3
3.1
4.1
4.2 am
4.2 pm
4.3
4.4
4.5
5.1
6.1
6.2
6.3
21
22
23
24
25
26
27
28
29
29
30
01
02
03
04
05
06
Date
Latitude
(uN)
Jun 05
Jun 05
Jun 05
Jun 05
Jun 05
Jun 05
Jun 05
Jun 05
Jun 05
Jun 05
Jun 05
Jul 05
Jul 05
Jul 05
Jul 05
Jul 05
Jul 05
42
42
41
41
41
41
41
42
42
42
42
42
42
41
41
41
41
02.20
00.29
59.30
44.65
44.60
43.60
51.99
12.48
12.82
12.49
12.51
12.40
12.37
46.73
46.93
47.00
46.92
Average
Equipment
Average
Time of
Ekman transport* incident surface
deployment
Qx (Qy)
irradiance
Longitude sampling (h) wind speed
(m s21)
(GMT)
(W m22)
(m3 s21 km21)
(uE)
MLSD GS NSSD
10
10
10
09
09
09
08
09
09
09
09
09
09
09
09
09
09
30.70
29.67
30.37
19.82
19.60
19.60
57.78
00.52
00.32
00.68
00.53
00.52
00.84
01.01
06.58
06.80
06.80
13:15
13:20
12:50
06:00
05:50
13:40
13:20
06:20
9{
6{
3{
3{
1{
1{
51
8{
31
3{
5{
8{
5{
11{
12{
21
12{
2568(261)
2525(324)
2403(235)
2233(143)
2146(21)
2170(226)
296(246)
85(2207)
154(2300)
113(2301)
45(2350)
2161(2280)
2448(243)
2699(121)
21190(323)
21271(318)
21610(427)
898{
937{
640{
650{
833{
838{
611
625{
911
840{
936{
911{
904{
916{
895{
311
937{
no
no
no
Y
Y
Y
Y
no
Y
Y
no
N
N
no
no
Y
no
deployment{
deployment
deployment
Y
Y
N
Y
Y
Y
Y
Y
deployment
Y
Y
Y
Y
deployment
N
Y
Y
Y
deployment
deployment
Y
Y
deployment
* Qx is Ekman transport perpendicular to the coast, Qy is parallel to the coast.
{
Average taken between 12:00 and 14:00 h.
{
No deployment means sea conditions were too rough to launch the rigid inflatable boat and to operate the sampling devices.
1
Average taken between 05:00 and 07:00 h.
and inherent optical properties (IOPs) were enhanced in the
microlayer compared with near-surface and subsurface
layers during a cruise off the Iberian Peninsula in June–July
2005. We assess the factors that lead to the accumulation of
MAAs in the SSM and analyze their relationship with the
IOPs, particularly CDOM. The results are discussed in the
context of the potential effects of MAAs and CDOM on
the attenuation of UVR.
Methods
Sample collection—Cruise 172 aboard the RRS Charles
Darwin took place off the Iberian Peninsula, NE Atlantic in
June–July 2005. Six stations in total were occupied, four of
which were sampled using SSM and near-surface equipment (Table 1). The station sampling started in oligotrophic waters and progressed to more productive waters
nearer to the coast (Fig. 1). The upwelling indices and
alongshore (Qy) and cross-shore (Qx) Ekman transport
(Table 1) were downloaded from the National Oceanographic and Atmospheric Administration (NOAA) National Marine Fisheries Service web site (www.pfeg.noaa.
gov/products/las.html). In the following sections, station
(Sta.) and days are indicated by, for example, ‘‘2.3,’’
meaning the third day at Sta. 2. The SSM was sampled
using a Garrett screen (GS; Garrett 1965) and a surface
microlayer sampling device (MLSD). The GS, a handheld
device consisting of a 50-cm2 frame enclosing a stainless
steel mesh, was deployed from a rigid inflatable boat. The
screen was slid gently into the water at an angle and then
lifted horizontally back through the surface. Small rectangular cells of water from the sea surface are captured in the
interstitial spaces of a wire mesh by means of surface
tension, which are released when the screen is tipped at an
angle into a prerinsed, amber glass collection bottle.
Thickness of the SSM sample collected by the screen is
typically 300–400 mm (Zuev et al. 2001).
The MLSD is a rotating drum device mounted between
the hulls of a small (1.5 m long) catamaran platform
(Fig. 2). The rotating glass drum picks up water from the
SSM by surface tension and is reported to sample a layer
typically of 20–100 mm (Zuev et al. 2001). The sample
picked up by the glass drum is then removed by a Teflon
wiper blade and collected in a small sample pot. The
contents of the pot were continuously removed by a
peristaltic pump to a larger (2.7 liter) storage vessel (amber
glass Winchester, prerinsed with milliQ water) on the upper
hull. The MLSD was deployed tethered to a near-surface
sampling device (NSSD; see below).
The two devices (GS and MLSD) were used to sample
the SSM to provide a reasonable chance of sampling
success over the range of physical conditions likely to be
experienced during a research cruise. Rather than taking an
average concentration measurement from the two devices,
we took the pragmatic approach of assuming the maximum
concentration observed was closest to the actual maximum
concentration present in the SSM (Momzikoff et al. 2004).
Enrichment factors (EFs) were calculated as follows:
EF~microlayer sample concentration :
average NSSD sample concentration (0:25 to 2:17 m)
A floating NSSD (Fig. 2) was used to sample the
uppermost 2 m of the water column at approximately 20
cm intervals. This device consisted of a flotation ring (1.2 m
in diameter) supporting a central vertical spar. The spar
Sea surface microlayer MAAs and CDOM
Fig. 1.
1837
Map showing location of stations during cruise CD172 off the Iberian Peninsula.
carried a series of eight 4-liter sampling bottles spaced at
20–30-cm intervals, with the uppermost bottle at a depth of
25 cm. The NSSD was deployed from the ship and allowed
to drift away on a conductor core tether. A 24 Niskin bottle
stainless steel conductivity-temperature-density sampling
device (CTD) rosette was used to profile the water column
from the near surface to below the 1% light level. The
maximum time from sample collection to filtering was
25 min for all samplers.
Particulate material for MAAs, pigment analysis, and
absorption coefficients was obtained by filtering water
collected from the GS, MLSD, or NSSD (typically 1–2 liter
volumes) through 25-mm glass fiber filters (GFFs), which
were flash frozen in liquid nitrogen and stored at 280uC for
transport to the U.K. They were then stored in liquid
nitrogen for a maximum of 1 month prior to analysis.
MAA analysis—MAAs were extracted from particulate
material on filters by sonication with an ultrasonic probe
(35 s, 50 W) using 1 or 2 mL 75% acetonitrile. During
extraction solvent tests, 75% acetonitrile was found to
Fig. 2.
outperform 25% methanol for extracting MAAs by
extracting 9% more shinorine and 36% more mycosporine-glycine from phytoplankton samples. Extracts were
incubated at 45uC for 2 h (Tartarotti and Sommaruga
2002) and then centrifuged (5 min at 20 3 g) to remove
cellular debris.
MAAs were analyzed by high performance liquid
chromatography (HPLC). The extracts were injected
(100 mL) onto an Agilent 1100 system comprising an
autosampler, quaternary pumping system, and diode array
detector. Instrument control and data acquisition were
performed using ChemStation software. MAAs were
separated using a Luna (Phenomenex) 5-mm NH2 column
(250 3 4.6 mm) with gradient elution. The mobile phase
comprised acetonitrile and ammonium carbonate solution
(1 mol L21, buffered to pH 10), in the following
proportions: mobile phase A 5 85/15 (v/v) acetonitrile : ammonium carbonate solution; mobile phase B 5
25/75 (v/v) acetonitrile : ammonium carbonate solution.
Mobile phases A and B were premixed and degassed by
gently bubbling with N2 gas for 10 min. The gradient
(A) NSSD and (B) catamaran microlayer sampler (MLSD).
1838
Tilstone et al.
program was as follows: 100% A for 10 min; linear gradient
to 25% A, 75% B over 35 min at a flow rate of 1 mL min21.
MAAs were monitored at 330 and 310 nm using a
photodiode array detector, scanning between 250 and
500 nm.
For quantification, pure samples of palythene, shinorine,
mycosporine-glycine, palythine, and palythinol, previously
isolated from culture extracts by preparative HPLC, were
verified by liquid chromatography mass spectrometry and
quantified by UV–VIS spectrophotometry using published
extinction coefficients (Gröniger et al. 2000), and then
analyzed by HPLC to establish response factors. These
were then used to quantify the MAAs in sample extracts.
HPLC grade solvents (Fisher) and ammonium carbonate
(Sigma-Aldrich) were used.
Carreto et al. (2005) report that extraction of MAAs at
45uC could cause hydrolysis of shinorine methyl ester to
produce shinorine. Both of these products have the same
absorption maxima and very similar extinction coefficients,
and the detection of shinorine methyl ester would not affect
the interpretation of our data. It should be noted, however,
that we cannot preclude the possibility that a proportion of
the shinorine detected in our samples did not originate
from shinorine methyl ester.
Particulate MAA concentrations were converted to
absorption coefficients using extinction coefficients from
Gröniger et al. (2000). Photodiode array spectra were
normalized to the correct extinction coefficients at peak
wavelengths and then converted to absorption (m21). The
coefficients were multiplied by MAA concentration to
provide an estimate of unpackaged total MAA absorption
in the UV spectral region from 250 to 500 nm. A MAA
packaging index of 0.80 was then used to convert
unpackaged MAA absorption to packaged absorption in
phytoplankton cells (aPMAA). The choice of packaging
index is based on observations by Laurion et al. (2004) that
dinoflagellates, which have high MAA : Chl a ratios under
high UVR, exhibit an index of 0.8. This may be simplistic,
since dinoflagellates can respond differently to varying light
treatments due to differences in photoadaptation and
genetic characteristics, which may cause the packaging
index to vary. The packaging index is only relevant,
however, when MAA concentrations are high, as recorded
at Sta. 2.
Pigments analysis—Pigments were extracted into 2 mL
100% acetone containing an internal standard (apocarotenoate; Sigma) using an ultrasonic probe (35 s, 50 W).
Extracts were centrifuged to remove filter and cell debris
(3 min at 20 3 g) and analyzed by HPLC using a reversedphase C8 column and gradient elution (Barlow et al. 1997)
on an Agilent 1100 Series system with chilled autosampler
(4uC) and photodiode array detection (Agilent Technologies). The HPLC was calibrated using a suite of standards
(DHI) and pigments in samples identified from retention
time and spectral match using photodiode array spectroscopy (Jeffrey et al. 1997).
Microscopy—Sea water samples of 250 mL were fixed in
acid Lugol’s solution at a final concentration of 2% and
stored in cool dark conditions until analysis in the
laboratory by settlement microscopy (Utermöhl 1958).
Subsamples of 25 mL were concentrated by sedimentation
for . 36 h, and all cells between 10 and 200 mm were
enumerated at 3200 and 3400 magnification and, where
possible, identified to species level. Linear dimensions of
individual cells were measured using an ocular micrometer,
and cell volumes were calculated using appropriate
geometric shapes. Mean cell volumes for each species were
converted to carbon using the equations of Menden-Deuer
and Lessard (2000).
Inherent optical properties—The absorption coefficients
of CDOM (aCDOM), phytoplankton (aph), and nonalgal
particles (aNAP) were determined in water samples collected
using the GS, MLSD, NSSD (at two to four depths), and
CTD (at two to four depths) sampling devices deployed at
the stations shown in Table 1. aph-aNAP samples were
frozen on board the ship in liquid nitrogen, and absorption
coefficients were determined in the laboratory directly after
the cruise. The parameters were measured spectrophotometrically as follows: the absorption coefficients of total
particulate (ap 5 aph + aNAP) and aNAP retained on GFFs
were measured before and after pigment extraction
following the transmission-reflectance method of Tassan
and Ferrari (1995). Scans were performed from 350–750 nm
at a 1-nm bandwidth using a dual beam Perkin Elmer
Lambda 800 spectrophotometer (L800) retrofitted with a
spectralon coated 60-mm Labsphere integrating sphere.
The spectrophotometer was calibrated using Holmium
Oxide filters. To obtain the absorbance of nonalgal
particles the GFFs were scanned again after depigmentation with 15% NaClO (1% active Cl). Phytoplankton
absorption coefficients were derived from the difference
between ap and aNAP fractions. To convert the absorbance
measurements into absorption a beta correction (see
equation 18 in Tassan and Ferrari 1995) was used between
350 and 750 nm. The same beta factor was used for both ap
and aNAP following Tassan and Ferrari (1998). ap and aNAP
were not measured , 350 nm, since it is difficult to
determine pathlength wavelength amplification at UV
wavelengths for particles in suspension with the integrating
sphere configuration used.
For aCDOM(l), seawater samples were filtered through
0.2-mm Whatman Nuclepore membrane filters using acidcleaned glassware. The first two 250-mL aliquots of filtered
seawater were discarded. The third sample of 125 mL was
spiked with 0.5 mL solution of 10 g L21 of NaN3 per
100 mL of sample to prevent CDOM degradation (Ferrari
et al. 1996). Samples were measured from 250 to 850 nm on
immediate return to the laboratory in 10-cm quartz
cuvettes, relative to a bidistilled MilliQ reference blank
also spiked with the same concentration of NaN3, using the
L800. aCDOM(l) was calculated from the optical density of
the sample and the cuvette pathlength following Babin et
al. (2003). aCDOM slopes (SCDOM) were calculated using
linear regression on log-transformed spectra from 275 to
295 nm and 350 to 400 nm, and the ratio between the two
(SR) was calculated following Helms et al. (2008). In
addition, detectable peaks and shoulders in the aCDOM
Sea surface microlayer MAAs and CDOM
1839
Table 2. Microlayer enrichment factors (EFs) of predominant photosynthetic pigments and total MAAs, referenced to average
concentration in NSSD samples (25–217 cm). GS, Garrett screen; MLSD, microlayer sampling device. Values in parentheses are
microlayer concentration in mg L21. nd, no deployment.
EFs—unitless (microlayer concentration (mg L21))
Station
Peridinin
Hexfuco
Diadino
Fuco
Chl c2
Chl a
Total MAAs
MAAs : Chl a
GS
MLSD
GS
MLSD
GS
MLSD
GS
MLSD
GS
MLSD
GS
MLSD
GS
MLSD
GS
MLSD
2.1
2.2
2.3
3.1
4.4(0.03)
7.5(0.05)
1.3(0.09)
1.1(0.07)
1.9(0.06)
2.1(0.06)
1.4(0.06)
1.4(0.06)
1.5(0.06)
1.9(0.07)
1.7(0.43)
1.9(0.48)
8.9(4.42)
8.8(4.39)
10.3
9.1
nd
438.3(2.01)
nd
3.6(0.17)
nd
124.8(3.16)
nd
9.0(0.23)
nd
58.4(1.20)
nd
59.0(9.49)
nd
276(192.8)
nd
20.3
437.9(1.62)
170.7(0.63)
10.9(0.39)
6.4(0.23)
100.3(2.21)
34.4(0.76)
1.9(0.04)
0.0(0.0)
81.8(1.12)
31.8(0.44)
63.1(7.09)
33.2(3.74)
336.3(289.7)
119(102.1)
40.9
27.3
2.9(0.03)
1.4(0.01)
1.1(0.06)
0.9(0.05)
4.0(0.28)
0.8(0.06)
6.8(0.56)
1.2(0.10)
3.7(0.27)
0.7(0.05)
3.6(1.45)
0.9(0.36)
1.3(1.57)
1.0(1.16)
1.1
3.2
spectra between 290 and 350 nm were removed by refitting
aCDOM(l) with SCDOM calculated from 290 to 350 nm at 10nm intervals for Sta. 4.2 am, which had very low MAAs
(Table 2). The difference between the measured aCDOM(l)
and this refitted curve (aCDOM-DMAA) gave MAA absorption for the dissolved fraction (aDMAA), and MAA
concentration in this fraction was estimated from the
absorption coefficients at 310 nm. apCDOM is defined as the
sum of aph, aNAP, and aCDOM.
Results
MAAs, phytoplankton pigments, and microplankton
community composition—The water column at Sta. 2 was
highly stratified, with a mixed layer depth at approximately
10 m. This followed low Ekman transport velocities parallel
(Qy) and perpendicular (Qx) to the coast, and several days
of calm, sunny conditions (Table 1), which resulted in a
visible surface slick. The mean wind speed increased at
Stas. 3–6, which augmented Qx and Qy from Stas. 4.4 to 6.3
(Table 1), and there was no visible surface slick observed at
these stations.
The total concentration of MAAs in particulate material
in the SSM at Sta. 2 increased by . 65 times from Stas. 2.1
to 2.3 and far exceeded concentrations observed at Stas. 3–
6 (Table 2; Fig. 3A–C). EFs for MAAs in the SSM
increased by , 40 times from Stas. 2.1 to 2.3 (Table 2).
The profiles of individual MAAs with depth demonstrate
both the enrichment of individual MAAs in the SSM
compared with the near surface, and the dominance of
mycosporine-glycine and shinorine (Fig. 3A–C). SSM
enrichment of MAAs (taken here as EF . 2) was also
observed at Stas. 4.2 afternoon, 4.5, and 6.2. The low
absolute concentrations of MAAs in the SSM at Sta. 6.2,
however, concurrent with a relatively high fucoxanthin
(fuco) concentration, may indicate MAAs were present in
these fuco-containing autotrophs at very low concentra-
4.2 (morning) 4.2 (afternoon)
1.3(0.02)
1.0(0.02)
1.0(0.06)
0.8(0.04)
1.1(0.03)
0.6(0.02)
1.4(0.05)
0.7(0.03)
1.2(0.04)
0.7(0.02)
1.2(0.29)
0.9(0.20)
1.3(0.86)
0.6(0.38)
2.3
1.9
8.8(0.03)
3.8(0.01)
1.1(0.05)
1.2(0.05)
1.6(0.05)
1.2(0.03)
1.1(0.3)
1.0(0.03)
1.6(0.04)
1.3(0.03)
1.6(0.28)
1.3(0.23)
2.9(2.92)
1.2(1.22)
10.4
5.3
4.5
6.2
1.3(0.02)
nd
0.8(0.03)
nd
5.2(0.35)
nd
4.8(0.61)
nd
4.5(0.22)
nd
3.6(1.26)
nd
2.2(2.16)
nd
1.7
nd
2.6(0.01)
2.8(0.01)
1.0(0.07)
1.1(0.08)
5.1(0.31)
2.3(0.14)
6.7(2.32)
2.6(0.88)
5.4(0.67)
2.5(0.32)
2.4(2.18)
5.2(4.84)
2.7(0.31)
3.8(0.44)
0.1
0.1
tions or may suggest a different source. The ratios of
MAAs : Chl a were high at Sta. 2, increasing from 10 at Sta.
2.1 to over 40 at Sta. 2.3. The MAAs : Chl a ratios were
generally lower at Stas. 3–6, and the lowest ratio was
observed at Sta. 6, where high Chl a values were assigned to
a diatom pigment signature, which coincided with the
beginning of an upwelling event (Tables 1, 2).
The EF for Chl a at Sta. 2 increased by . 30 times from
Sta. 2.1 to 2.3 (Table 2). Values were much higher than
those at Stas. 3–6 (Table 2) where surface slicks were not
observed. The dominant phytoplankton pigments changed
during the development of the slick at Sta. 2. At Sta. 2.1,
19-hexanoyloxyfucoxanthin (hexfuco), fuco, chlorophyll c2
(Chl c2) and diadinoxanthin (diadino) were the most
dominant pigments (Table 2; Fig. 3D). Peridinin, however,
gave the highest EF (Table 2). At Stas. 2.2, 2.3, concentrations of diadino, peridinin, and Chl c2 increased in the
SSM (Table 2; Fig. 3E,F), indicative of dinoflagellates, and
were high relative to the underlying near-surface water
(Table 2). The dominant pigments at Stas. 3–6 were fuco,
diadino, and Chl c2, indicative of diatoms. Notably
peridinin gave a high EF at Sta. 4.2 pm but with a
relatively low absolute concentration. Compared with the
other major pigments present, hexfuco showed little SSM
enrichment at any sampling station (Table 2).
The phytoplankton community in the SSM was dominated by autotrophic dinoflagellates, whereas in nearsurface waters, flagellates dominated the total biomass
(Fig. 4A,B). At Sta. 2.2 the autotrophic dinoflagellate
biomass increased (Fig. 4C), comprising almost entirely
Prorocentrum species. In the near surface, autotrophic
dinoflagellates, heterotrophic dinoflagellates, and ciliated
protozoa dominated the biomass; the autotrophic dinoflagellate biomass, however, was two orders of magnitude
lower than in the SSM (Fig. 4C,D). At Sta. 2.3, the SSM
continued to be dominated by autotrophic dinoflagellates,
whereas the near-surface water was dominated by ciliated
1840
Tilstone et al.
Fig. 3. Microlayer and near-surface profiles of predominant MAAs at (A) Sta. 2.1, (B) Sta.
2.2 (inset: near-surface profiles from 25 to 217 cm), and (C) Sta. 2.3 (inset: near-surface profiles 25
to 217 cm), and microlayer and near-surface profiles of predominant phytoplankton pigments at
(D) Sta. 2.1, (E) Sta. 2.2 (inset: near-surface profiles 25 to 217 cm), and (F) Sta. 2.3 (inset: nearsurface profiles 25 to 217 cm).
Sea surface microlayer MAAs and CDOM
1841
Fig. 4. Phytoplankton biomass (mgC L21) at Sta. 2.1 in (A) the SSM and (B) at 217 cm; Sta.
2.2 in (C) the SSM and (D) at 217 cm; and Sta. 2.3 in (E) the SSM and (F) at 217 cm. Numbers
above the bars are percentage biomass of the total for each group.
protozoa and flagellates (Fig. 4E,F). In the SSM sample
from Sta. 2.3, the maximum Prorocentrum micans cell
abundance observed was 4681 cells mL21. The increase in
MAA concentration at Sta. 2 closely tracked the increase in
Prorocentrum cell numbers observed by microscopy
(Fig. 5A). We also found a significant relationship between
Qy and MAAs (F1,6 5 21.28, p 5 0.0058), which explained
69% of the variance in MAAs (Fig. 5B).
Inherent optical properties—aCDOM(l) in the SSM at Sta.
2 was also high compared with near-surface and subsurface
values (Fig. 6) and always higher than aph(443). The ratio
aCDOM(443) : aph(443) increased from 26 at Sta. 2.1 to 40 at
Sta. 2.3. At Sta. 2.2, aCDOM(l) spectra had a prominent
shoulder between 270 and 325 nm, which increased at Sta.
2.3 (Fig. 6C,E). The corresponding SR was comparatively
high in the SSM at Stas. 2.1, 4.2, and far lower at Stas. 2.2,
2.3. At the latter stations, SR rose sharply with depth
(Fig. 6).
aph(443) in the SSM was nearly an order of magnitude
higher at Sta. 2.2 than at Stas. 2.1 and 4.2 (Fig. 7A,C,G). In
the surface CTD bottle aph(443) was between 6 and 10
times lower than in the SSM at Stas. 2.2, 2.3 (Fig. 7C–F).
In the SSM there was a sharp decrease in the Chl a-specific
absorption coefficient of phytoplankton (aph*(443)), which
then rose to a maximum in the subsurface. aNAP(443) in the
SSM was similar to near-surface and subsurface waters at
Stas. 2.1, 4.2 and two times higher in the SSM at Stas. 2.2,
2.3 (Fig. 8). The ratio of aNAP(443) : apCDOM(443), however, was consistently lower in the SSM at Sta. 2 and increased at Sta. 4.2, where there was no visible slick, indicating that the accumulation of detrital material in the SSM
was not excessive. Subsurface aNAP(443) : apCDOM(443)
ratios were double those in the SSM, and at Sta. 4.2 there
was a sharp rise in the proportion of aNAP(443) relative to
aph(443) and aCDOM(443) at 5 m even though the absolute
aNAP(443) values decreased.
At all stations, the dissolved MAA absorption (aDMAA)
was always lower than aCDOM-DMAA and approximately a
quarter of aPMAA (Fig. 9). At Stas. 2.1, 4.2, aCDOM-DMAA
. aPMAA. At Stas. 2.2, 2.3, aCDOM increased relative to
aPMAA, except between 310 and 350 nm, where aPMAA was
higher. Based on the extinction coefficients of MAAs and
the absorption peak at 310 nm (Fig. 9B,C), we estimate the
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Tilstone et al.
Fig. 5. (A) Microlayer concentration of Prorocentrum cells (closed symbols) and MAAs
(open symbols) over three sampling days at Sta. 2. (B) Relationship between Ekman transport Qy
and MAAs during the cruise.
concentration of MAA in the dissolved fraction at Stas. 2.2,
2.3 to be 42 and 117 mg L21, respectively. There were
significant relationships between Chl a and MAAs for all
stations (F1,122 5 458.58, p , 0.0001) and for stations with
a surface slick (F1,5 5 16.25, p 5 0.015; Fig. 10A,B). There
was also a significant regression between aCDOM and MAA
concentration in the particulate fraction, when there was a
visible slick (F1,21 5 23.69, p , 0.0001; Fig. 10C), and
MAA concentration explained 79% of the variance in
aCDOM. Similarly, during slick conditions, MAAs explained
31% of the variance in a NA P (443) : a p C D O M (443)
(Fig. 10E,F), and this relationship disappeared during
nonslick conditions.
Discussion
Microlayer sampling using the GS and MLSD—Two
different microlayer samplers were used in this study to
provide a reasonable chance of sampling success over the
range of conditions likely to be encountered during a
research cruise. We also employed a near-surface sampler
to determine profiles over the depth range 0.2–2.2 m.
Sea surface microlayer MAAs and CDOM
Fig. 6. Microlayer and subsurface spectra of aCDOM(l) for (A) Sta. 2.1, (C) Sta. 2.2, (E) Sta.
2.3, and (G) Sta. 4.2 and profiles of aCDOM(320) and the ratio of SCDOM(275–295) : SCDOM(350–
400) at (B) Sta. 2.1, (D) Sta. 2.2, (F) Sta. 2.3, and (H) Sta. 4.2.
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Tilstone et al.
Fig. 7. Microlayer and subsurface spectra of aph(l) for (A) Sta. 2.1, (C) Sta. 2.2, (E) Sta. 2.3,
and (G) Sta. 4.2 and profiles of aph(443) and aph*(443) at (B) Sta. 2.1, (D) Sta. 2.2, (F) Sta. 2.3,
and (H) Sta. 4.2.
Sea surface microlayer MAAs and CDOM
Fig. 8. Microlayer and subsurface spectra of aNAP(l) for (A) Sta. 2.1, (C) Sta. 2.2, (E) Sta.
2.3, and (G) Sta. 4.2 and profiles aNAP(443) and the ratio aNAP(443) : aPCDOM(443) at (B) Sta. 2.1,
(D) Sta. 2.2, (F) Sta. 2.3, and (H) Sta. 4.2. Where aPCDOM(443) is the sum of aph(443), aNAP(443),
and aCDOM(443).
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Tilstone et al.
Fig. 9. Absorption coefficients of MAAs in the particulate (aPMAA(l)) and dissolved
fractions (aDMAA(l)), and aCDOM(l) with the dissolved MAAs removed (aCDOM-DMAA(l)) from
250 to 500 nm at (A) Sta. 2.1, (B) Sta. 2.2, (C) Sta. 2.3, and (D) Sta. 4.2.
Zhang et al. (2003) reported the thickness of the SSM using
a pH microelectrode to be 60 mm, which is the layer of
sudden change of physical and chemical properties. The
MLSD is reported to sample a thickness of 20–100 mm (Liss
and Duce 1997; Zuev et al. 2001) and was therefore
expected to sample the sudden change layer at 60 mm more
closely than the GS, which has a reported sampling
thickness of 300–400 mm (Zuev et al. 2001).
The microlayer model illustrated by Hardy (1982)
depicts phytoneuston at 10–100 mm, which we would
therefore also expect to be well sampled using the MLSD
and to be diluted by the additional volume sampled by the
GS. EFs determined using the GS in the presence of a welldefined slick at Sta. 2.3 were generally higher, however,
than those determined from the MLSD (Table 2). Daumas
et al. (1976) also used a GS and rotating drum device to
sample the microlayer, and from observations of Chl a
concluded that phytoplankton did not accumulate in the
uppermost layer sampled by the drum. In addition,
phytoplankton have been reported to be more efficiently
sampled by the GS than the glass plate sampler (Agogué et
al. 2004; Momzikoff et al. 2004), which samples in a similar
manner to the MLSD, using viscosity and cohesion forces
(Carlson 1982). The thicknesses sampled by the devices
may also be influenced by the presence of a surface slick
(Liss and Duce 1997). Carlson (1982) reported increased
sample thicknesses from a glass plate during slick
conditions and found that the thicknesses of plate samples
were influenced by sea surface conditions and bulk water
temperatures, whereas screen samples were not. The
different EFs observed in our study from the GS and
MLSD, particularly during a visible surface slick, are
therefore consistent with these previous studies. During
control tests, Agogué et al. (2004) demonstrated that even
the GS led to underestimated phytoplankton concentrations. We therefore used the maximum SSM concentration
sampled by either of the devices in the figures presented to
give a more comprehensive indication of the scale of the
potential SSM enrichment.
Concentrations of Chl a at 25 cm were 2% of the values
observed in the microlayer during the visible surface slick at
Sta. 2. The maximum EF observed for Chl a was 63.1,
significantly higher than values published during other SSM
studies, where EF values were typically 0.2 to 8 (Daumas et
al. 1976; Agogué et al. 2004; Joux et al. 2006), reaching 18
within a visible slick (Hardy and Apts 1989). As discussed
above, the EF is also dependent on the type of sampler
deployed based on the thickness of SSM that the instrument
samples. Agogué et al. (2004) reported an average EF for
Chl a of 1.76 (0.94–4.33, n 5 24, screen sampler) and 0.71
(0.26–1.13, n 5 11, glass plate), consistent with our
observations with the exception of Stas. 2.1, 6.2 (Table 2).
Phytoplankton enrichment in the SSM—The high Chl a EF
values observed in this study indicated a high concentration of
autotrophs in the SSM, attributed to the flat calm conditions,
Sea surface microlayer MAAs and CDOM
1847
Fig. 10. Relationship between MAA concentration and (A) Chl a during surface slick
conditions, (B) Chl a during nonslick conditions, (C) aCDOM(300) during surface slick conditions,
(D) aCDOM(300) during nonslick conditions, (E) aNAP(443) : aPCDOM(443) during surface slick
conditions, and (F) aNAP(443) : aPCDOM(443) during nonslick conditions. In A, C, E, open
diamonds are microlayer samples, filled shapes are CTD and near-surface samples.
low Qy, and moderately high incident irradiance (Table 1;
Fig. 5B). Similarly, Obernosterer et al. (2008) found particulate organic matter enrichment in the microlayer was
negatively correlated with wind speed. The SSM at Sta. 2
also showed a high concentration of diadino, reaching
3.16 mg L21 (Fig. 3E,F). Notably, diadino is a photoprotective carotenoid involved in the xanthophyll cycle. Under
nonslick conditions (Stas. 3–6) Chl a enrichment (taken here
as EF . 2) was observed in the SSM on three out of five
sampling occasions. EFs for Chl a between 1.6 and 5.2 were
observed, consistent with Chl a SSM enrichment in other
studies during nonslick conditions (Daumas et al. 1976;
Agogué et al. 2004; Joux et al. 2006). Chl a values alone,
however, are not indicative of the health of phytoplankton
communities. Reports on the health of phytoplankton in the
microlayer conflict with some reporting a highly light-
adapted, healthy community (Cullen et al. 1989), whereas
others report low primary production in the SSM compared
with subsurface waters (Ignatiades 1990). Daumas et al.
(1976) reported higher ratios of pheophytin to Chl a in the
SSM than in the underlying water, indicating damaged cells in
the surface. Chlorophyll allomers (oxidation products of Chl
a) were not detected in the SSM at Sta. 2. By contrast, we
detected chlorophyll oxidation products (3–12% of chlorophyll concentration) in the SSM at Stas. 3.1, 4.5, and 6.2,
possibly indicative of higher phytoplankton mortality at these
stations (data not shown). The aNAP(443) : apCDOM(443)
ratios that we report from the SSM (Fig. 8) also indicate
that the proportion of dead cells was low at Sta. 2, especially
compared with nonslick conditions. We also found that
aNAP(443) : apCDOM(443) ratios decreased with increasing
MAA concentration, suggesting that the phytoplankton
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Tilstone et al.
community in the SSM was actively synthesizing photoprotective pigments. We have no direct photophysiological
measurements, however, to ascertain the photophysiological
health of the phytoplankton community within the SSM.
High Chl a, diadino, peridinin, Chl c2 (Table 2; Fig. 3),
and Prorocentrum spp. biomass (Figs. 4, 5) occurred during
a visible surface slick; Qy was low and MAA concentrations
were high. Dinoflagellates, such as P. micans, are known to
bloom off the NW Iberian Peninsula after downwelling
cycles when Qy and Qx are low (Tilstone et al. 1994). P.
micans may have therefore produced the high MAA in the
SSM at Sta. 2. To date, MAA : Chl a ratios detected in
marine phytoplankton range from 0.4 to 2.5 mg (mg Chl a)21
(Villafañe et al. 1995; Llewellyn and Harbour 2003), with
values of mycosporine-glycine from a marine dinoflagellate
bloom off the Californian coast reaching 5.35 mg (mg Chl
a)21 (Whitehead and Vernet 2000) and total MAAs up to
16 mg (mg Chl a)21 in dinoflagellate cultures (Laurion and
Roy 2009). These values compare to maximum concentrations measured in the SSM during this study of 41 mg (mg Chl
a)21 for total MAAs (Table 2), 17.3 mg (mg Chl a)21 for
shinorine, and 15.6 mg (mg Chl a)21 for mycosporine-glycine.
The concentrations of MAAs measured in the SSM at Sta. 2
far exceed those measured previously in other marine
environments. The EFs were also some of the highest
reported. There were significant relationships between Chl a
and MAA for all stations (F1,122 5 458.58, p , 0.0001) and
for stations with a surface slick (F1,5 5 16.25, p 5 0.015;
Fig. 10). Previous studies of SSM EFs for other variables
have selected a single reference depth without having the
opportunity to measure values in the near surface. Our data
indicate that EFs for MAAs should be interpreted cautiously
if a single reference depth is used and that near-surface
samples are a requisite before MAA enrichment in the SSM
can be properly assessed.
Inherent optical properties of the SSM and subsurface
waters—aCDOM(l) in the SSM at Sta. 2 was also high and
more than twice that in the near-surface waters, probably
derived as a by-product of Prorocentrum spp. We found a
significant relationship between MAA and aCDOM(300)
(Fig. 10C,D), and MAAs explained , 80% of the variance
in aCDOM(300). In the absence of a surface slick, MAAs
explained a lower percentage of the variance in aCDOM(300),
possibly suggesting that exudates from MAA-producing
organisms alone make a significant contribution to the
CDOM pool in this area. Other compounds resulting from
the breakdown of biological material such as low molecular
weight carbonyl compounds and dissolved phenols have been
found to accumulate in the SSM due to exposure of organisms
to increased sunlight (Carlson and Mayer 1980). At Stas. 2.2,
2.3, a prominent absorption shoulder was evident in the
aCDOM(l) spectra between 270 and 330 nm, indicative of
MAA in the dissolved fraction (Fig. 6B,C). Whitehead and
Vernet (2000) observed a similar spectral feature associated
with CDOM during a red tide off the upwelling coast of
California and found aCDOM(330) to be relatively high
(0.145 m21) and associated with 111.4 nmol L21 MAA in
the dissolved fraction. We estimate MAA concentrations in
the dissolved fraction, from the aDMAA spectra, to be
117 mg L21 at Sta. 2.3, corresponding to approximately
420 nmol L21, assuming the MAA distribution in the
dissolved phase matches that measured in the particulate
phase. The absorption peak of aPMAA was at 320 nm,
corresponding to a combination of the absorption maxima of
the most dominant MAAs, mycosporine-glycine (lmax
310 nm) and shinorine (lmax 334 nm), whereas the absorption
peak of aDMAA was at 300 nm. This shift in peak absorption
may indicate a different composition in the two fractions,
possibly due to chemical transformation in the dissolved
phase. It should be noted, however, that aPMAA(l) was
calculated from extinction coefficients in methanol (Gröniger
et al. 2000), and aDMAA(l) was measured from aCDOM(l)
spectra in aqueous solution, which could contribute to a shift
in the absorption maximum.
SR was generally higher in the SSM at Stas. 2.1, 4.2 caused
by higher SCDOM(275–295), indicative of photobleaching of
aCDOM(l) (Helms et al. 2008). The lower SR in the SSM at
Stas. 2.2, 2.3 due to high SCDOM(350–400) indicates a
microbial production of CDOM (Helms et al. 2008),
paralleled with the sudden proliferation in P. micans.
Bacteria and viral lysis of phytoplankton could be the cause
of the release of MAAs into the DOM pool, although the
absorbance of detrital material in the SSM was low and
would be expected to be higher under these conditions. This
may indicate that P. micans cells were actively secreting
MAAs into the surrounding waters. aph(l) in the SSM was
always higher than in the subsurface, and aph*(443) sharply
decreased in the SSM (Fig. 7). A decrease in aph*(443) is
often observed with increasing Chl a (Bricaud et al. 1995)
and reflects a general trend toward an increase in cell size
(Ciotti et al. 2002), pigment packaging, and concentrations
of accessory pigments (Bricaud et al. 1995), which occurred
in the SSM (Table 2). The sharp decrease in aph*(443) may
also be due to a relative increase in concentrations of MAAs.
aCDOM is responsible for much of the UV attenuation in
the open ocean (Bricaud et al. 1981) and absorbs the most
biologically harmful UVR. Carlson and Mayer (1980)
showed that material absorbing at 280 nm is enriched in
the microlayer compared with bulk seawater and that
dissolved phenolic material is the predominant lightabsorbing material. Although we have no measured values
of attenuation coefficient (Kd) in the UVR and we cannot
model them reliably because accurate in vitro measurements
of aph(l) and aNAP(l) were not available , 350 nm, Kd in the
UVA and B are driven largely by absorption. Although high
concentrations of MAA were measured in the SSM, they are
only effective at attenuating UVR in a narrow spectral band
between 310 and 350 nm (Fig. 9). aCDOM, on the other hand,
absorbs significantly more UVA and UVB at Stas. 2.2, 2.3
compared with aPMAA due to the exponential rise in CDOM
absorption in the UV (Fig. 9). MAAs were far lower in
subsurface waters than SSM levels (Fig. 3), but aCDOM
remained comparatively high in the subsurface (Fig. 6). This
suggests that aCDOM would attenuate UVA and UVB more
than aPMAA both in the SSM and lower in the water column.
Whitehead and Vernet (2000) suggested that the accumulation of DOM originating from exudates released by
phytoplankton may increase UV attenuation by the
dissolved pool, thus providing a UV shield during shallow
Sea surface microlayer MAAs and CDOM
blooms. Along the Iberian Peninsula in the SSM, the high
aCDOM and MAA in both the particulate and dissolved
fractions, associated with a bloom of P. micans, suggest that
in the UVA and UVB, Kd would be high. Further studies of
Kd in the UV associated with microlayer and nonmicrolayer
conditions are required to verify this.
Our study reports the first direct measurements of
MAAs, aCDOM, aph, and aNAP in the SSM and in
underlying near-surface water sampled at a finer resolution
than conventional CTD and rosette. We report the highest
known concentrations of MAAs in marine waters, which
were associated with a bloom of P. micans in the sea surface
microlayer. aCDOM(l) was also elevated, which may have
also served as a sunscreen to UV for phytoplankton both in
the SSM and subsurface layers.
Acknowledgments
This work was undertaken as part of the Plymouth Marine
Laboratory (PML) Core Research Program, 2001–2005. We are
grateful to the officers, crew, and scientists of the Royal Research
Ship Charles Darwin, Cruise 172 for their help and support, to
Tony Bale (PML) and Chris Gallienne (PML), who designed and
tested the near-surface sampling device. NEODAAS are acknowledged for providing satellite data images to the ship during the
cruise. We also thank Dariusz Stramski and two anonymous
referees, whose comments significantly improved this paper.
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Associate editor: Dariusz Stramski
Received: 27 August 2009
Accepted: 04 May 2010
Amended: 14 May 2010