Presentation - National Water Research Institute

Why is peroxide important?
Typical levels in seawater
Production mechanism in seawater
Southern California surf-zone waters
› Lab and mesocosm experiments
› Temporal and spatial measurements
› In situ diel studies
Potential additional sources
Conclusions/future work

Hydrogen peroxide (H 2 O 2 ) occurs naturally
in fresh and marine waters
Important intermediate in redox processes
in chemical and biological aquatic systems
Acts as a strong oxidizing agent that reacts
with trace metals and pollutants
Elevated levels of H 2 O 2 have been shown to
cause damage and cell lysis in
microorganisms
Potential impacts on microbial water
quality

Microbial water quality standards for marine
recreational bathing waters.
Public beaches with >50,000 visitors/year mandated
to participate in water quality monitoring programs.
Microbial water quality assessed from concentration
of fecal indicator bacteria (FIB; US EPA).
Beach closures and postings when levels are
exceeded.
Observed diel cycles in surf zone
› lower bacteria levels during the day
Diel cycling attributed to:
› UV radiation-induced mortality
› biological predation
› oxidative stress from natural photochemically-produced
oxidizing agents like H 2 O 2

Field measurements in surface seawater over last two
decades (review Clark et al., 2008).
Prior measurements in mid-ocean and near-shore waters
Concentrations from

Endogenous conc.of 100 nM and 10 000 nM for exogenous
H 2 O 2 increased catalase hydroperoxidase in E.coli
(Gonzalez-Flecha& Demple, 1997).
Cells exposed to sunlight may be sensitized to lower
concentrations.
Some evidence for bacterial mortality from oxidative stress
at lower exogenous conc. of 10 2 nM eg.
› Angel et al. (1999) ~100 nM H 2 O 2 caused oxidative stress to
bacteria in coastal waters as indicated by increasing catalase
enzyme concentration
› Xenopoulos & Bird (1997) spiked lake waters incubated in situ
with 100 nM H 2 O 2 and observed inhibition of bacteria by ~40%
› Kohn & Nelson (2007) concluded that indirect photoinactivation
by photochemically produced reactive oxygen species was
more important than direct damage by UVB light in sunlightmediated
inactivation of MS2 coliphage in waste stabilization
ponds.

What are H 2 O 2 production and
destruction mechanisms and
concentrations in the dynamic surf
zone environment where FIB levels
are monitored and regulated?

Photochemical production from CDOM
considered primary source in natural waters
› higher H 2 O 2 concentrations and production
rates observed in waters with higher levels of
CDOM as measured by absorbance and
fluorescence
Other sources to surface seawater include
› atmospheric input via dry and wet deposition
› biological production by algae
› largely insignificant compared to in situ abiotic
photochemical production
› wet deposition an intermittent significant source
over localized areas during rain events

Net oceanic decomposition rates show
considerable spatial variability; generally
first order with a lifetime

CDOM refers to a spectrum of highly complex
macromolecular colored compounds that include
humic substances.
Most CDOM in coastal waters comes from riverine or
wetland inputs of terrestrially derived materials from
plant degradation
May also be produced from grazing phytoplankton
and viral-induced lysis in the ocean
Example of 3D Excitation Emission
Matrix(3D EEM) fluorescence
for CDOM in surf zone waters

CDOM absorbs sunlight.
Rxn of O 2 with photoexcited CDOM gives superoxide O 2- (1) and its
conjugate acid HO 2 (2) which dismutate to form H 2 O 2 and oxygen (3-5)
(Cabelli, 1997):
CDOM* + O 2 → CDOM + + O 2
-
(1)
O 2- + H 2 O → HO 2 + OH - (2)
HO 2 + HO 2 H 2 O 2 + O 2 k =8.6 x 10 5 M -1 s -1 (3)
2 O 2 O 2
2-
+ O 2 k =

H 2 O 2 levels had never been directly measured in the surf zone
where microbial water quality is monitored.
To assess the importance of H 2 O 2 in monitored marine
recreational waters, we conducted a spatial and temporal study
of surf zone concentrations at popular bathing beaches in
Southern California on short (24 h in situ field studies; 30 to 60 min
intervals) and long (weekly/monthly/annual) time scales.
Focused much of this study on Huntington State Beach as part of
a larger multi-investigator study aimed at elucidating sources
and cycling of surf-zone FIB (Boehm et al., 2002; Grant et al.,
2005).
Discuss trends observed between H 2 O 2 levels and tide, time of
day and season.
Compare concentrations, production and loss rates obtained in
the surf zone to previous near and off-shore seawater studies.

Enzyme-mediated fluorescence peroxidase technique by
Zika and Saltzman (1982) used for most previous surface
seawater measurements (Clark et al., 2008).
Filter field fluorometer (Turner 10-AU-0) with excitation ~354
nm and emission ~496 nm.
Detection limit ~5 nM in seawater.
2-4 replicate measurements averaged, with an average

Change in %Fluorescence
Refridgerated concentrated 1
mM stock solution from a
commercial 30% H 2 O 2 solution
standardized with sodium
thiosulfate used to prepare diluted
1x10 -5 M standard H 2 O 2 solution
prior to measurements.
To calibrate fluorometer, 100 μL
phosphate buffer and 25 μL horse
radish peroxidase (HRP) added to
20 mL seawater in cuvette, 40 μL
scopoletin added and initial
fluorescence recorded (%Flu 0 )
after 1 min. 50 μL aliquots of
diluted H 2 O 2 sequentially added
and fluorescence change ( %Flu)
recorded after 2 min delay.
To measure H 2 O 2 , 20 mL seawater
sample, 100 μL phosphate buffer
and 40 μL scopoletin added to
cuvette. After 2 min, %Flu 0
recorded, 25 μL HRP added, and
final %Flu recorded after 1 min. Δ
%Flu converted to H 2 O 2
concentration with calibration
plot of %Flu vs. [H 2 O 2 ]
40
35
30
25
20
15
10
5
y=0.46 + 0.102x
0
0 50 100 150 200 250 300 350 400
[Hydrogen Peroxide], nM

Huntington State Beach
13 beaches covering 40-mile stretch of
coastline in Orange County, Southern
California.
› selected for accessibility, popularity for
bathing/surfing, historically sporadic poor
water quality, wide spatial coverage.
North to south: Seal Beach Pier;
Huntington State Beach (HSB) Pier; HSB
main beach; HSB at Talbert Marsh outlet;
Newport Beach 17 th Street; Newport
Beach Pier; Big Corona; Little Corona;
Crystal Cove State Park Beach Tower;
Crystal Cove State Park Beach tide pools;
Laguna Beach Pier; Laguna Beach cliffs;
Doheny State Beach.
Diel studies at HSB and Crystal Cove
Lab studies included salt marshes/river
mouths source waters
Talbert Marsh
Santa Ana River
Newport Back Bay

Seal Beach
Doheny State Beach

unfiltered water from HSB
anti-correlation between FIB and H 2 O 2 diel cycles
Panel B.
Mesocosm
Solid symbols
– dark controls
Open symbols
- sunlight
Panel A. In situ beach

[H 2
O 2
] (nM)
Measured in salt
marsh/river mouth
source waters and
adjacent surf zone
waters
Irradiation 300 W ozonefree
Xenon lamp
Average initial hydrogen
peroxide production
rates (HPPR) were higher
in bulk source waters
(11±7.0 nM s -1 ) than the
surf zone (2.5±1nM s -1 ).
10000
8000
6000
4000
2000
Salt marsh/river mouth
(SJC, UNBB)
Beach (HSBP, SCP)
0
0 10 20 30 40 50 60
Time (min)
Clark et al., Chemosphere, 2009

To examine production as a function
of CDOM size, water samples were
size fractionated by tangential flow
ultrafiltration on custom built Soluble
Organic Concentrator (SOC;
Separation Engineering Inc.,
Escondido Ca) with a 1000 Dalton
polyethersulfone membrane.
› Concentrate made back up to original
volume with artificial seawater adjusted
to sample’s initial salinity to minimize
potential dilution and solution medium
effects.

Production Rate (nM/s)
HPPR increased with
increasing absorbance
coefficient.
HPPR were higher in
the permeates (1kDa), suggesting
greater photoreactivity
in the smaller size
fraction of dissolved
material and/or
quenching of photoreactivity
by the larger
material.
20
18
16
14
12
10
8
6
4
2
0
0 2 4 6 8 10 12 14 16
Absorption coefficient (300 nm), m -1
Clark et al., Chemosphere, 2009

DOC (ppm C)
Absorbance coefficient -
measure of how much CDOM is
present or how photoactive it is
› abs decreases as the CDOM is
diluted or photochemically
bleached on exposure to sunlight
Higher abs and dissolved organic
matter content (as DOC) for
source waters vs. surf zone.
Linear relationship for DOC vs.
abs for all bulk/size-fractionated
samples suggests dilution
dominates variability in abs, with
minor aging/bleaching effects.
No significant change in linear
relationship for DOC vs. abs
observed for permeate/
concentrate for source/surf zone
waters - even distribution of
optically active DOM across size
range
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0 2 4 6 8 10 12
Absorbance coefficient (m -1 )
Clark et al., Chemosphere, 2009

HPPR varied significantly (5x) for surf zone samples
with the same absorbance coefficients.
To normalize production rates for samples with
different absorption coefficients (and hence CDOM
levels), apparent quantum yields calculated for H 2 O 2
photochemical production
Quantum yield would ideally be given by #
molecules H 2 O 2 produced/# photons absorbed.
However, since H 2 O 2 is the product of secondary
reactions and CDOM is not well characterized, a true
quantum yield cannot be calculated.
Instead, we calculated an apparent quantum yield
by normalizing production rate to absorption
coefficient and lamp flux

Apparent quantum yield
0.24
0.20
0.16
surf
surf
0.12
0.08
surf
0.04
0.00 source
source
source
Bulk Concentrate Permeate
Clark et al., Chemosphere, 2009
Source waters showed no
significant difference in
between bulk, large (>1
kDa)) and small (

concentration (nM)
200
180
160
140
120
100
80
60
40
20
0
1 2 3 4 5 6 7 8 9 10 11 12 13
beach sites
Clark et al., Water Research, 2010
13 beaches; flood tide at
noon
Summer dry season
concentrations averaged
122 ± 38 nM
Beaches with tide pools had
lower levels (50-90 nM).
Little annual variation.
Average surf zone
concentrations measured
here comparable to 183 nM
for near-shore So. Cal. waters
(Avalon, Boehm et al., 2009) and
within range previously
obtained in estuarine (10 -
350 nM), coastal (14-240 nM),
and some ocean waters (8-
150 nM).

Measured H 2 O 2 levels at
HB as a function of time of
day for 5 wks in summer
for 3 local time periods:
morning (08:00-09:30),
noon (11:00-13:00) and
evening (20:00-21:00)
Morning levels after ~2 h
of sunshine averaged 124
± 24 nM, statistically lower
than at noon (160 ± 34
nM).
Evening samples at 94 ±
18 nM were statistically
lower than the noon
samples.
No significant tidal effects
Clark et al., Water Research, 2010

24 hour diel study at HSB, 30
min intervals
Conc from 5 to 370 nM
Exhibited diel variability
After sunrise, conc. increased
from 5 to ~200 nM.
During the day, conc. ranged
from 200 to 370 nM, with ave.
daytime concentration of 238
± 79 nM.
Concentrations decreased to
44 nM at 21:00 and below
detection limit before sunrise.
Average night-time conc. of
23 ± 24 nM.
Longer-time scale points
overlay shorter-time resolution
points - robust diurnal cycling
over range of time scales.
Clark et al., Water Research, 2010

Net photochemical production rate of H 2 O 2 between
low morning levels at 08:30 and maximum at 16:00,
estimated from initial and final concentrations to be
49 nM h -1 , is on the high end of 2-10 nM h -1 reported
for surface seawater and 17-56 nM h -1 in estuarine
and coastal waters
Substantial dynamic short-term variability in H 2 O 2
concentrations on the time-scale of min
› attribute this to variability in surf-zone waters as water
parcels are transported into and out of the sampling site
by long-shore and rip currents. Physical properties showed
the same short-term variability
Note that ave a(300) was 1.4 ± 0.5 m -1 , relatively low
value consistent with smaller CDOM pool relative to
the previous studies in estuarine/near-shore waters
with large terrestrial riverine inputs.

4 diel studies in surf zone waters in
July/August 2008 at Crystal Cove State
Beach
Diel cycles in H 2 O 2 obtained for all 4 field
experiments
Overall, studies 1-3 consistent with respect
to overall trends, measured max and min
conc. and relationship between sunlight
and H 2 O 2
Field study 4 different; attributed to
occurring after a major storm with
significant increases in plant wrack and
surf zone turbulence
Maximum conc. of 160-200 nM measured
within 1h of solar noon
Levels dropped at night to 20-40 nM,
consistent with photochemical
production from sunlight

Field
study 1
Clark et al., Marine Pollution Bulletin, 2010

Field
study 2
Clark et al., Marine Pollution Bulletin, 2010

Field
study 3
Clark et al., Marine Pollution Bulletin, 2010

Field
study 4
Clark et al., Marine Pollution Bulletin, 2010

Day-time production and (initial) night-time dark loss rates
averaged 16±3 nMh -1 and 12±4 nMh -1 respectively.
Apparent quantum yields ranged from 0.04 to 0.09, with
average of 0.07 ± 0.02, similar to quantum yields of 0.09 ±
0.04 from lab studies
Production largely dominated by sunlight, with some
dependence CDOM levels in waters with highest abs.
Evidence for dark biological production?
› Between midnight and sunrise, net peroxide decay rate appears
to slow and peroxide levels remain constant well above the
detection limits of the instrument
› If observed initial night-time decay rate remains constant and
constant levels measured during midnight to sunrise period are
due to balance between measured night-time decay and dark
production process, estimated maximum dark production rates
range from 10.0 to 21.3 nM h -1 .

Absorption coefficients used as a proxy for amount of
CDOM in natural waters. Average absorption coefficients
for each field study ranged from 1.3 to 1.9 m -1 at 300 nm,
with overall average of 1.7 ± 0.2 m -1 .
Cycling in absorption coefficients and hence CDOM levels
was observed on the order of 2 to 4 h, suggesting longshore
transport of parcels water with different optical
characteristics
Absorption coefficients varied with tide; higher values at
ebb vs. flood tides.
Increases in absorption coefficients at ebb tides suggest
inputs from intertidal beach zone.
Increased absorption coefficient pulses on flood tides,
suggests near-shore CDOM source, possibly due to kelp
beds

Significant correlation between sunlight and H 2 O 2 but
no significant correlation between absorption
coefficient (measure of CDOM levels) and H 2 O 2
Enhanced HPPR (for low abs., low CDOM waters)
Variability in HPPR for the same absorbance
Enhanced for surf zone waters vs. source waters
Increases in H 2 O 2 at ebb and flood tides during night
suggest additional non-CDOM production
mechanisms are operating in surf zone
Samples were filtered so enhanced production rates
cannot be due to large particles, algae or plankton,
but must be due to dissolved species

Prior studies:
› H 2 O 2 from decomposing seaweed (Collen and
Pedersen, 1996)
› Increased atmospheric H 2 O 2 at ebb tide attributed
to decaying seaweed in the intertidal zone (Morgan
and Jackson, 2002).
› Kupper et al. (2001) observed activated oxygen
species including H 2 O 2 from brown algal kelp
Kelp is likely an important intermittent source in
coastal waters with kelp beds.
Measured photochemical production of H 2 O 2
from senescent kelp and seagrass immersed in
seawater irradiated by sunlight (8 hrs) in
mesocosm experiments.

Irradiated kelp produced H 2 O 2 (75
nM) with substantial increase in
solution color, suggesting
photochemical production from
kelp-derived CDOM.
Dark control for senescent kelp was
30 nM, consistent with a direct
contribution (~40% of
photochemical production) from
decaying tissues.
H 2 O 2 photochemical production
rate from kelp ~5 nM h -1 , on the
order of CDOM production rates
previously measured in coastal and
oceanic waters.
No significant production from
seagrass

Measured photochemical production of H 2 O 2
from HSB sand immersed in seawater irradiated
by sunlight (8 hrs) in mesocosm experiments.
Double amount of H 2 O 2 produced vs. seawater
only, indicating photochemical production from
a beach sand component.
Dark control 20 nM H 2 O 2 over same time period,
indicating limited amount of direct H 2 O 2
contribution from leaching (

Seawater over sand
Seawater only
Solar simulator
Filtered seawater and
intertidal beach sand
from Crystal Cove
State Beach
With sand: 210 nM
Without sand: 180 nM
Net increase of 20%
when irradiated over
sand

Beach sand contained dark magnetic grains (iron
mineral?)
Preliminary study:
› Acid digestion of 2 sand samples with AA analysis
› Leachate from sand sample with more visible dark grains
had higher (x2) Fe concentrations
Production of H 2 O 2 from iron (Fe(II)/Fe(III)) in fresh
and coastal seawater (Miller et al., 1995; Southworth and
Voelker, 2003).
Hypothesize that iron in beach sand/dissolved in
seawater is a significant photochemical source of
peroxide compared to CDOM in study area

Explore contributions to hydrogen peroxide
production from kelp, intertidal beach
sand, dissolved metals and other potential
coastal sources
› Role of dissolved iron in production in seawater
• Photochemical production as a function of iron
and CDOM levels in a range of seawater and
beach sand samples
› Effect of microorganisms in filtered vs. unfiltered
seawater
› In situ diel sampling of kelp beds for CDOM and
photochemical and biological production of
peroxide
› Oil seeps?

The oxidant H 2 O 2 is photochemically generated in situ
in surf zone waters at concentrations on the order of
100 to 300 nM, at production rates higher than
expected from CDOM levels.
Diel cycling in H 2 O 2 in beach waters occurs, with
maxima in the afternoon after solar noon with lower
concentrations during the night at 10 to 20% of
daytime maxima.
Production appeared to be dominated by solar
intensity, with no evidence for tidal effects.
Higher dark loss rates suggest larger sinks are
associated with the turbulent surf zone.
Evidence for dark and photochemical production
from non-CDOM sources (kelp and intertidal beach
sand).
Concentrations are likely sufficient to cause indirect
photoinactivation of fecal indicator bacteria in
marine recreational waters.

Collaborators: Stanley Grant, Warren De
Bruyn
Site access:
› Dept. of Fish and Game
› California State Parks
› Harry Helling, Crystal Cove Alliance
Funding from:
› Office of Naval Research
› National Science Foundation
› American Chemical Society Petroleum Research
Fund
› Chapman University faculty grants

Scott Jakubowski
Liannea Litz
Charlotte Hirsch
Paige Aiona
Lauren Pagel
Benjamin Brahm
Jeanette Pineda
Lillian Burns