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faster photobleaching at longer wavelengths. For example, synchronous fluorescence at ex
= 375 nm (SF375) decreases more rapidly than synchronous fluorescence at ex = 350 nm
(SF350) (Pullin and Cabaniss,
1997
). Similar results were found by Tzortziou et al. (2007)
for the Rhode River estuary and by Osburn et al. (2009b) for the Mackenzie River in which
the longer excitation wavelength fluorescence bleached faster than did the shorter exci-
tation wavelength fluorescence. Few EEM measurements have been made of DOM after
photobleaching. Mayer et al. (
1999
) showed that tryptophan protein fluorescence is more
photo-labile than was tyrosine fluorescence. Using estuarine DOM, Moran et al. (2000)
was one of the first reports exploring both photochemical and biological DOM degrada-
tion via fluorescence. They reported greater bleaching at humic (A, C, M) peaks than at
protein peaks, though bleaching of the latter did occur. Coupled with this was blue shifting
of the DOM fluorescence ex/em maxima due to the preferential loss of emission at longer
wavelengths. Typically, DOM photobleaching is quite rapid initially, followed by a slower
degradation stage (Koussai and Zika,
1990
; Pullin and Cabaniss,
1997
; Moran et al.,
2000
;
Del Vecchio and Blough,
2002
; Osburn et al.,
2009b
).
On monochromatic light exposure, the fluorescence signal is lost (bleached) as a func-
tion of the irradiation wavelengths. Along the excitation axis, monochromatic light expos-
ure induces the greatest fluorescence loss nearest the irradiation wavelength (as observed
for absorption) (Patsayeva et al.,
1991
; Boehme and Coble,
2000
; Del Vecchio and Blough,
2002
,
2004
). However, losses also occur above and below the irradiation wavelength.
A possible explanation for the loss at the excitation wavelength is direct photochemical
destruction of fluorophores all absorbing at this wavelength. However the broad emis-
sion loss (extended across the entire range investigated) cannot arise from destruction of
non interacting DOM components all excited at the irradiation wavelength and showing
a continuum of red-shifted emission (very unlikely) but could instead arise from a more
complex model of interacting species (Del Vecchio and Blough,
2004
; Goldstone et al.,
2004
). The secondary loss (away from the irradiation wavelength) could be due (a) to the
destruction (via indirect photochemistry) of a different class of fluorophores only excited
at long wavelengths; or (b) to the direct photochemical destruction of a subset class of
fluorophores excited at both wavelengths (or somehow coupled with fluorophores excited
at short wavelengths). The continuous red shift of secondary loss with increasing irradi-
ation wavelengths does not favor the superposition of a large number of fluorophores all
overlapping the lowest excited state band and showing a continuous red shift of the highest
excited band. Instead, Del Vecchio and Blough (
2004
) argue for a more complex model of
interacting species originating the long wavelength emission.
The net effect on DOM fluorescence from this complex photochemistry is a blue shift
in excitation after irradiation, but red shifting may also occur within emission spectra. An
example of DOM fluorescence photobleaching showing a subtle red shift in emission is
presented in
Figure 7.7
for a Sphagnum-dominated bog (
Figure 7.7A
) and for DOM gener-
ated from an algae culture (
Figure 7.7B
) (cf. Osburn et al.,
2001
). For each type of DOM,
fluorescence emission spectra (400-600 nm, ex370) decreased significantly. After photo-
exposure, the bog DOM peak emission shifted slightly from 462 to 469 nm; the algae
