Ref. No. [UMCES]CBL 2013-021
ACT VS12-05
following their internal SOP’s based on EPA SW846 Method 8015B,C. The Laboratory
provides reporting limits of 50 ppb for this hydrocarbon range. Reference samples were
collected in certified pre-cleaned amber glass bottles supplied by Test America. Bottles were
filled, stored and shipped according to their SOP’s. Reference samples, along with sampling
blanks, were shipped to the contract lab not more than three days after collection to meet their
holding time requirements.
Excitation Emission Matrix Spectroscopy (EEMS)
A SPEX ISA Fluoromax-2 scanning spectrofluorometer, operated in ratio mode, was
used to generate room temperature (22 ± 1°C) EEM fluorescence spectra for all reference
samples. To optimize sample throughput, fluorescence spectra were determined over an
excitation range of 230-500 nm at 5 nm intervals and an emission range of 300 – 600 nm at 3 nm
intervals. For each scan, an integration time of 1 second was used, and bandpass widths were set
to 5 nm for both excitation and emission spectrometers. Xenon lamp intensity as well as
emission monochrometer performance were verified and recalibrated once per day according to
the instrument manual.
For all generated EEM’s, dark counts were subtracted and spectra were subsequently
corrected for wavelength-dependent instrument effects using ISA-supplied and user-generated
correction files. Fluorescence spectra intensities were then normalized to the area under the
Raman peak, determined daily using MilliQ water (Murphy, 2011; Murphy et al. 2010). This
value exhibited less than 2% variation over the length of the study period. In addition to daily
Raman scans, daily EEM’s of MilliQ water were generated as background blanks and were
subtracted from all subsequent sample EEM’s. At the beginning and end of each analytical batch
a four-point calibration curve (0-50 ppb) of Quinine Sulfate (QS) in 50 mM H
2
SO
4
was run to
track drift in fluorometer response over time. The QS response factor was used to standardize
emission intensities across each analytical batch (Coble et al. 1993). Finally, all sample EEM’s
were corrected for Raman and Rayleigh scattering peaks, following Zepp et al. 2004.
Excitation and emission windows for each instrument (based on the reported FWHM for
the filter sets as provided by manufacturers) were mapped onto each reference sample EEM
space and corresponding integrated quinine sulfate normalized fluorescence intensities obtained
for direct comparison to instrument output under the various challenge concentrations.
Colored Dissolved Organic Matter (CDOM)
Approximately 50 ml of the CDOM designated subsample were filtered using 47 mm
GF/F filters with low vacuum pressure and poured into an acid-cleaned, combusted, 60 ml amber
glass bottle. All samples were stored in the dark at 4° C until analysis, within approximately one
month of collection. A dual-beam spectrophotometer was blanked with MilliQ water in cuvettes
in both the sample and reference positions. Matched 10 cm quartz or optical glass cells were
used for a dual-beam spectrophotometer. MilliQ samples were run intermittently during each
analytical batch to assess instrument baseline drift. Scans were run between 200 and 800 nm and
electronic files were saved for each sample.
MilliQ blank and turbidity (750 nm) corrected spectra were used to estimate CDOM
abundance by non-linear regression of the absorption spectra over 400 – 575 nm.
