Presentation - National Water Research Institute

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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

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