-Correction Experiment Report – ICESS – April, 2003




-Correction Experiment Report
Submitted by: Nathalie Guillocheau
Institute: ICESS – UCSB
Date of experiments: June 12, 2002 (Experiment 1)
August 14, 2002 (Experiment 2)
January 15, 2003 (Experiment 3)
Date of submission: April, 2003
1) Background and objectives
Absorption coefficients of algal and non-algal particles are measured on a regular
basis since 1996 in the Santa Barbara Channel during Plumes and Blooms cruises. The
specific absorption of chlorophyll a at 676 nm [a*ph(676)] should not exceed the
theoretical maximum of 0.0206 m2.mg-1 (Bricaud and Stramski, 1990). Our light
absorption spectra show abnormally high values of a*ph(676). About 43% of our values
exceed the theoretical maximum of 0.0206 m2.mg-1. Among the possible reasons of this
overestimate, the pathlength amplification factor () is likely to have a major influence.
Our measurements are corrected for pathlength amplification using the Mitchell (1990)
coefficients obtained from mixed cultures.
The objective of these experiments is thus to examine the relationship between the
optical density of natural samples in suspension (ODs) and the optical density of the same
sample on a GF/F filter (ODf) in order to determine our own  factor and to compare it to
other values.
2) Sampling procedures
Seawater samples volumes of 6, 12 and 19 liters were collected at surface during
three Plumes and Blooms cruises on June 11, 2002 at station 2 (experiment 1), on
August 13, 2002 at station 6 (experiment 2) and on January 15, 2003 at station 6. The
chlorophyll concentrations were respectively 1.67, 5.29 and 0.73 g.l-1. The samples
1
-Correction Experiment Report – ICESS – April, 2003
were stored in the dark until we came back to the lab and then placed overnight in a
refrigerator.
HPLC pigment data were available for the first experiment. The CHEMTAX
program (Mackey et al. 1996) was used to estimate the relative chlorophyll a biomass
due to different phytoplankton groups. The sampling information is summarized in
Table 1.
Table 1. Sampling information. The phytoplankton composition is estimated from HPLC pigment
concentrations processed with CHEMTAX. The percentages represent the relative contribution
of each algal group to chlorophyll a.
Experiment #
Date
PnB station
Depth
Chla (mg.l-1) Phytoplankton composition
Experiment 1
11-Jun-02
2
surface
1.67
Diatoms: 34%
Cryptophytes: 20%
Chlorophytes: 15%
Prasinophytes: 11%
Synechococcus: 10%
Haptophytes-T4: 5%
Haptophytes-T3: 3%
Dinoflagellates: 2%
Experiment 2
13-Aug-02
6
surface
5.29
HPLC results pending
Experiment 3
15-Jan-03
6
surface
0.73
HPLC results pending
3) Optical density measurements and calculations
The first step consisted on concentrating the water samples to get OD values close to
the ones we typically get (OD436 around 0.5 or less). Seawater samples were filtered
on 47mm 0.6m Nuclepore membranes. The material retained on those filters was
gently scraped off with a lab spoon and resuspended in 0.2 m filtered seawater.
The concentrate was poured into a 1 cm quartz cuvette and the absorbance relative to
0.2 m filtered seawater was measured on a dual-beam Perkin-Elmer Lambda 2
spectrophtometer equipped with a RSA-PE-20 Labsphere integrating sphere. The
cuvette content was then gently filtered on a 25 mm GF/F filter. The absorbance of
the total particulate material relative to the same blank filter saturated with seawater
was measured with the same equipment. We did three replicates of each spectrum in
the range 400-750nm.
2
-Correction Experiment Report – ICESS – April, 2003
Respectively 6, 12 and 19 liters have been concentrated and resuspended for
experiments 1, 2 and 3. The OD obtained on experiment 1 were low but still in the
range of our typical values (OD436 around 0.26). On the contrary, on experiment 2,
the values were too high and the concentrate has been ¼ diluted in order to get OD436
close to 0.5. The OD values ranged from 0.05 to 0.39 for experiment 3.
The baseline was automatically corrected and the blank reference was substracted
from the raw data. All the spectra were set to zero at 750 nm and the numbers from
the three replicates were then averaged. The absorbances of the sample filter and of
the suspension have thus been computed as:
 [OD
ODf or ODs =
raw
 ODbl  OD 750]
( 3 replicates)
3
The cuvette and the GF/F filter pathlength were compared to apply a correction
factor:
Cuvette pathlength = 0.01m
GF/F filter pathlength =
volume filtered
3.5 x 10 -6 (m 3 )
=
= 0.0092 m
clearance area
3.801 x 10 -4 (m 2 )
A pathlength correction factor of (0.01/0.0092) was thus applied to ODf.
4) Results and discussion
Experiments 1 and 2 samples were both collected during the summer 2002. The
results of these two experiments were close, so we decided to pool the data together.
Plots of ODs versus ODf are presented in figure 1. The data were fitted by a second
order polynomial equation:
Experiments 1 and 2 (data pooled together):
ODs = 0.251 ODf + 0.283 (ODf)2 (in the following referred to as the PnB model)
r2 ≈ 1
The Mitchell (1990) quadratic equation fit is:
ODs = 0.392 ODf + 0.655 (ODf)2
3
-Correction Experiment Report – ICESS – April, 2003
Fig. 1. Relationship between ODs and ODf experiments 1 and 2 pooled together and the quadratic
equation fitting.
The experiment 3 sampling was conducted during the winter 2003. The results are
presented separately. The quadratic fit for this latter experiment is:
ODs = 0.332 ODf + 0.112 (ODf)2
r2 ≈ 1
Figure 2 compares the relationship between ODs and ODf for the PnB model and
several other published or unpublished expressions. The PnB model appears to
occupy an intermediate position among different published models whereas the
Mitchell (1990) model is situated above the others. Subsequently, the Mitchell (1990)
model generates lower  values than the PnB model(fig. 3). Indeed, the application of
the Mitchell (1990)  values lead to an overestimate of our absorption coefficients by
a factor 1.7.
4
-Correction Experiment Report – ICESS – April, 2003
Fig. 2. Comparison of the ODs and ODf relationship for different pathlength amplification
coefficients
4.5
4
3.5
3
2.5
PnB beta

Mitchell beta
Experiment 3 beta
2
1.5
1
0.5
0
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
ODf
Fig 3. Comparison of  values obtained with PnB, Mitchell (1990) and Experiment 6 models
5
-Correction Experiment Report – ICESS – April, 2003
.
.
Fig. 4. Spectral absorption curves normalized to unit [Chla + Phaeo a] concentration, measured on
Plumes and Blooms cruise PB138 (Aug 13, 2002) and computed using the Mitchell 1990 (doted lines)
and the PnB (continuous lines) beta correction coefficients
The chlorophyll specific coefficients (a*ph) determined with Mitchell (1990) and with
PnB pathlength correction coefficients are displayed on figure 4. The results of a*ph(676)
obtained with the PnB model are much more consistent with the theoretical values. This
suggests that the application of Mitchell (1990) quadratic equation coefficients to our
filter absorbances induce an overestimation of the spectral absorption coefficients. It
seems thus preferable to use bio-optical characteristics of local phytoplankton
assemblages for Plumes and Blooms spectral absorption coefficients calculation
The present experiments have been conducted over a limited period of time.
Considering that the estimates of  may vary with the phytoplankton community
composition, and more specifically with the particle size, it would be necessary to carry
out experiments over more diverse phytoplankton assemblages. Even though experiment
3 results are closer to PnB model than any literature models (Fig. 2), the experiment 3
coefficients result in about 20% higher estimates of a*ph() compared to the PnB model.
6
-Correction Experiment Report – ICESS – April, 2003
This observation suggests that the seasonal variability may need to be accounted for in
the data processing.
Our HPLC data analyzed with CHEMTAX software show marked variations in the
phytoplankton composition in relationship with the chlorophyll a concentration levels . A
progression from nanoplankton- to microplankton-dominated communities with
increasing chlorophyll a values is observed in the Santa Barbara Channel (unpublished
results). Work is in progress to deal with the seasonality of the correction factor in
relationship with the variability of the phytoplankton community composition.
Depending on the future results of the variability patterns, we should be able to
evaluate the need for multiple algorithms based on phytoplankton assemblages.
.
5) References
Bricaud, A. and Stramski, D. (1990) Spectral absorption coefficients of living
phytoplankton and non algal biogenous matter: a comparison between the Peru
upwelling area and Sargasso Sea. Limnol. Oceanogr., 35, 562-582.
Mackey, M.D., Mackey, D.J., Higgins, H.W. and Wright, S.W. (1996) CHEMTAX – a
program for estimating class abundances from chemical markers – application to
HPLC measurements of phytoplankton. Mar. Ecol. Prog. Ser. 144, 265-283.
Mitchell, B.G. (1990) Algorithms for determining the absorption coefficient for aquatic
particulate using quantitative filter technique. In Ocean Optics 10, Proceedings of
SPIE 1302. The International Society for Optical Engineering, Bellingham, WA, pp.
137-148.
Moore, L.R., Goericke, R. and Chisholm, S.W. (1995) Comparative physiology of
Synechococcus and Prochlorococcus: influence of light and temperature on growth,
pigments, fluorescence and absorption properties. Mar. Ecol. Prog. Ser., 116, 259275.
Nelson, N.B., Siegel, D.A. and Michaels, A.F. (1998) Seasonal dynamics of colored
dissolved material in the Sargasso Sea. Deep-Sea Res., 45, 931-957.
7