Influence of pH and heavy metals in the determination of yellow substance in estuarine areas

REMOTE SENS. ENVIRON. 37:89-100 (1991)

Influence of pH and Heavy Metals in the Determination of Yellow Substance in Estuarine Areas G. M. Ferrari Institute for Remote Sensing Applications, Commission of the European Communities, Joint Research Centre, Ispra, Italy

A

Z'Jkbsorption and fluorescence changes of yellow substance in the presence of pH and heavy metal excursions were examined for water samples collected in the deltaic area of the Po river (Sacca di Goro) and for solutions of natural humic and fulvic acids isolated from lake waters and podsol soils, respectively. An important effect on the light absorption and induced fluorescence was found for the pH variations that occur during the mixing of fresh and saline waters. For this reason, correction factors are required to normalize the absorption and fluorescence values for use in remote-sensing water quality of estuarine /coastal areas. If the factor is chosen to equal i at pH 7, the percentage error in the uncorrected estimate of the yellow substance absorption varies from 0% to 18% with a mean of 5% relative to a set of samples collected in Sacca di Goro. Among the heavy metals Cu 2÷, Pb 2+, and Cd 2+ present as pollutants in trace levels in estuarine environments, only Cu e ÷ and Pb 2+ in high concentration (> 15 t z g / L for Pb and > 10 Izg / L for Cu) have been found to depress the induced fluorescence response of water

Address correspondence to G. M. Ferrari, Institute for Remote Sensing Applications, Commission of the European Communities, Joint Research Centre--Ispra Site, 21020 lspra (Va), Italy. Received 27 September 1990; revised 14 April 1991.

samples containing yellow substance and humic acid solutions. 1.

INTRODUCTION

The varying mixture of dissolved organic compounds known as yellow substance (YS) is an important parameter for studying the environment, especially in reference to the estuarine/ deltaic areas. River organic matter discharges, mixing of bottom releases for organic matter, and the organic decay products of plankton decomposition are some of many possible sources of YS in the water. YS is characterized by radiation absorption with exponential dependence on wavelength (Jerlov, 1976): Ay( A) = Ay( Ao)e - kta- x°~, where Ay(A) is the absorption coefficient at a fixed wavelength A. YS interferes with the absorption of chlorophyll and suspended matter in the visible domain (400-700 nm). The relatively low spectral resolution of the present satellite remote sensors prevents discrimination between chlorophyll, suspended matter, and yellow substance contents. Current algorithms for chlorophyll and suspended

ooa4-4257/91/sa.5o ©Elsevier Science Publishing Co. Inc., 1991 655 Avenue of the Americas, New York, NY 10010

89

90

Ferrari

matter retrieval from remote measurements of sea color (Bricaud and Morel, 1987) operate on the assumption that the optical signature of YS is either negligible, constant, or well correlated with chlorophyll or total seston. The spectral discrimination of YS from chlorophyll and suspended sediment will be possible in the 1990s when platforms with high spectral resolution sensors will be launched. Moreover, the use of airborne multispectral scanners, which make use of the recent development of CCD sensor technology, has opened new possibilities in spectroscopic characterization of the components of the water body which determine the water color (Vane and Goetz, 1988). Another remote sensing application for YS assessment is the laser-induced fluorescence technique employed for the water quality studies with good results by Bristow et al. (1985). These techniques require more and more accurate ground truth observations among them is the YS role to define the algorithms to interpret those complex spectral signatures. However, for all the reasons described above, YS must be carefully assessed as it can become an important source of error in remote sensing of water quality. This is especially so when attempting to retry parameters such as chlorophyll-like pigment and total suspended matter. Therefore, in situ measurements of YS are an essential support to biooptical assessments, especially in estuarine/deltaic waters where dissolved organic matter can be present as a variable independent from other parameters (see below). An estimate of YS can be obtained through direct measurement of absorbance using a standard beam transmission method (Austin and Petzold, 1977) on the dissolved fraction filtered through a 0.22 ~m pore size membrane. This avoids the laborious corrections required for scattering due to residual particles remaining in solution when using filters > 0.22 /.~rn pore size (Bricaud et al., 1981). Although the beam transmission method is the most correct measurement technique for satellite remote sensing requirements, the induced fluorescence method offers significant advantages: It can be rapidly performed in situ avoiding elaborate filtrations, and can quantify v e ~ low YS contents because of its better information content in the fluorescence configuration response. However, since fluorescence data are expected to be converted into equivalent ab-

sorption values for remote sensing applications, this study was performed with the aim of evaluating the most important variability factors which can affect the estimate of YS in the estuarine (or deltaic) area with emphasis on pH and heavy metals (Cu, Pb, Cd) changes, pH variations occurring in the fresh/saline mixing waters are normally within the range 6.5-9.1. Fluorescence changes with changing pH have been extensively studied by several authors (Smart et al., 1976; Saar and Weber, 1980; Willey, 1984; Vodacek and Philpot, 1987), while absorption changes at varying pH have been observed in the Ems-Dollart estuary and the Dutch Wadden Sea (Laane, 1982). According to Carder et al. (1989), the effects of pH on specific absorption for the humic acids are very small whereas Zepp and Schlotzhauer (1981) found values for fulvic acid at pH 11 which are as much as 50% higher than are those at pH 6. The same authors have found this effect less marked for the humie acid. The interaction of heavy metals (Cu, Pb, Cd) with dissolved organic matter (YS) has been studied for the more or less marked metals quenching effect interfering in the fluorescence process of YS (Levesque, 1972; Cline and Holland, 1977; Saar and Weber, 1980; Willey, 1984). In particular, Willey (1984) studied the effect of the salinity change on natural fluorescence in estuarine mixing. He observed that at low salinity (0-5%o) a slight fluorescence increase due to the presence of magnesium. The magnesium addition to fluorescent matter in solution could enhance the fluorescence by: 1. Replacement of hydrogen by magnesium and adding crosslinking to the structure, 2. Replacement of a fluorescence depressing metal like copper or iron by magnesium thus subdues the quenehing aspect of the metal. The problem is: How can we quantitatively separate the effects due to change in composition/concentration of YS (mainly humie and fulvie acids) from those due to heavy metal and pH variations on the spectral response, and, moreover, how can we quantify those changes and provide appropriate corrections to reduce errors in the remote sensing estimate of pigments and suspended matter concentrations? This work is an attempt to introduce correction (normalization) factors to enhance the correlation between absorption and fluorescence, and to improve the estimate

Absorption and Fluorescence of Yellow Substance 91

via remote sensing of the chlorophyll-like pigment, taking into account the difficulties that occur in making a YS distinction among the presently investigated factors and other chemical factors affecting YS fluorescence in natural waters (Vodacek, 1985; Vodacek and Philpot, 1987). Induced fluorescence measurements have been indicated as a satisfactory procedure to obtain YS absorption as a support of remote sensing of sea surface color. That conclusion was justified by the good correlation between fluorescence and absorption obtained by including YS data of different origin and presumably different composition. This leads us to the convinction that the correlation (Ferrari and Tassan, 1991) expressed by Ay(375) = (.052 ___.055) + (.973 __+.045)FL, where Ay is the absorption coefficient at 375 nm in m-1 and FL is the fluorescence peak height at 420 nm, can be applied, for practical purposes, to a wide range of coastal water types. However, using a group of data relative to a single environment such as that of the Sacca di Goro (the deltaic area of the Po River), some corrections can be introduced in order to reduce the variability due to the pH changes and heavy metals.

2.

MATERIALS AND METHODS

To test some of the main parameter changes which affect the fluorescence/absorbance of YS in estuarine waters, a series of laboratory measurements has been performed using single solutions of humic and fulvic acids as well as natural samples collected in measurement campaigns (May 1989 and April 1990) in Sacca di Goro (Maracci et al., 1989; 1991). The choice of these substances, of very different origins, was suggested by the need to verify whether the absorption and fluorescence variations under pH and heavy metal changes might be influenced by the chemical behavior of the substances. Humic and Fulvic Acids (HA and FA) Solutions of humic and fulvic acids (Table 1) were prepared by dissolving natural humic acids extracted (Kearny, 1976) from lake waters of southern Germany: B~irnsee and Hohlohsee. Solutions

Table 1. Humic and Fulvic Acid Matter Solutions HA HA FA

Origin

Conc. (mg / L)

InitialpH

B~irnsee(September 1986) Hohlohsee (July 1986) Podsol (1986)

22.5 24.4 12.3

5.6 5.2 5.2

of fulvic acids were prepared using fulvic acid isolated from Podsol soil. Humic acids were almost completely soluble because of the possible previous transformation into humate and with successive arrangement until pH 5. A chemical characterization of H A / F A / Y S was not attempted since this has not relevant to the scope of the work. The mother solutions were diluted with distilled water to obtain different concentrations; they were preserved in amber-colored bottles stored in the refrigerator. Natural humic and fulvic acids of terrestrial origin were chosen as being the main contributors to the composition and to fluorescence/absorbance properties of YS in the estuarine deltaic waters. This can be considered as an empirical approach since not all authors agree about the origin of humic substances in the sea. According to Hama and Handa (1980), Miinster (1985), and Satoh and Abe (1987), humic substance usually represents more than half of the dissolved organic matter (DOM) in natural waters (lakes). Hfjerslev (1979, 1988) concluded that fluorescent matter does not form prevalently in situ in the sea, which is in contradiction with Duursma (1974) and Postma et al. (1976), who suggested that degradation products could be a source of fluorescence matter. Nevertheless, it is generally recognized that most of the fluorescence observed in waters is due to humic substances (Laane and Koole, 1982), the aromatic rings and functional groups of which are the most important fluorescence centres. The estuarine/deltaic areas represent a complex aquatic environment, where dissolved organic matter may be ascribed to various sources such as river run-off and in situ production or decay of planktonic matter. Its formation and diffusion can be influenced by local environmental factors: bottom topography, circulation, temperature, and degree of interrelation with the open sea. Thus, the empirical approach concerning the use of humic and fulvic acids can be justified because the final product which constitutes YS is classified into two broad categories: humic and fulvic acid, or other

92 Ferrari

products with the same reactivity and fluorescence/adsorption characteristics.

Table 3. Humic and Fulvic Acid Solutions as Well as Natural Water Samples Used for the Heavy Metal Quenching Tests

Origin

Natural Estuarine Samples

Humic acid

Some natural water samples were selected from those collected during the remote sensing calibration campaigns (Maracci et al., 1989; 1991) in Sacca di Goro (deltaic area of the Po River) (Table 2) with the aim of submitting them to the same treatments to compare the results with those of humic and fulvic acids. The water samples were prepared by filtration at 0.22 /zm pore size Millipore membrane; they were preserved in ambercolored bottles after adding 2 mL of NaN 2 (1%) as an inhibitor of bacterial degradation according to the procedures described by Ferrari and Tassan (1991).

pH Measurements The pH was measured with a WTW pH 530 meter (Weilhein, Germany) using an Ingold combination electrode provided with a temperature compensation system and calibrated by a standard buffer solution at pH 4, 5, and 9. Each pH adjustment was obtained by the addition of HC1 3% a n d / o r Na2CO 3 5% with Eppendorf pipettes. The reversibility of pH excursion was verified for each sample using solutions with 1% MgC12 in order to check the effect due to Mg 2+ ions present in sea water (Willey, 1984).

Heavy Metals The presence of metals, even in low concentrations, may affect the fluorescence in natural wa-

Table 2. River and Estuarine Samples after Filtration through a 0.22/~m Pore Size Millipore Membrane Sample No.

Period

13 1

May 1989 May 1989

3

May 1989

1 6

April 1990 April 1990

9

April 1990

Location River Po di Volano Embochure of the Sacca di Goro Sacca di Goro River Po di Volano Embochure of the Sacca di Goro Sacca di Goro

Salinity (%c)

In situ pH

0.46

7.42

5.31 11.71

6.48 8.75

1.86

6.95

7.75 20.89

6.95 7.49

Fulvic acid

Natural Water Samples Goro (April 1990)

Concentration (mg / L) pH

B~irnsee B~irnsee B[irnsee

0.5 2.7 8.5

5.45 5.57 6.49

Hohlohsee Hohlohsee Hohlohsee

0.6 3.1 12.2

5.65 6.03 .5"~. ,7

Podsol Podsol Podsol

2.5 6. l 12.3

5.45 5.60 5.20

Station

Ay375 (m 1)

pH

9 6 I

2.78 6.57 8.65

8.28

8.21 8.40

ters. In order to study their influence, aliquots of heavy metal solutions (Cu 2+, Pb z+, Cd 2+) were added to the fulvic and humic acids and to natural solutions, to reach the more or less realistic concentrations normally found in estuarine/deltaie areas subject to industrial and agricultural impact. As far as the Po River delta is concerned, i.e., the site where our investigation took place in 1985, the levels in the dissolved phase were found to be: for cadmium 0.05 ~ g / L max, for lead 2.26/xg/L max and for copper 2 / x g / L , both in the delta area and in the sea facing the river mouth (Ferrari and Ferrario, 1989). In this experiment, heavy metal was added both to humic and fulvic acids of three different concentrations and to natural samples collected in April 1990 in Sacca di Goro (Table 3). The final levels of the metal were reached for Cd (with CdSO 4) from 0.05 to 1.1 /xg/L, for Pb (with PbC12) from 1.74 to 34.7/~g/L, and for Cu (with CuSO 4) from 0.68 to 13.7/xg/L. The experiments were performed at constant pH varying the metal cation concentration and varying the pH keeping the metal concentration constant.

Beam Transmission and Induced Fluorescence Measurements

Absorption Measurements' A Perkin Elmer LA 3844 single-beam spectrophotometer and a MPF-66 fluorimeter, controlled by a PE 7500 professional computer, were adopted for

Absorption and Fluorescence of Yellow Substance

the absorbance and fluorescence measurements. The absorbance spectra usually closely follow an exponential decay between 300 nm and 500 nm, with a transition zone from 500 nm to 600 nm, which leads to a portion slowly decreasing to the end of the measured wavelength range. The water samples were contained in 4 cm long quartz cells. The pure water absorbance was measured using cells filled with standard Millipore distilled water and subtracted by the instrument software. The spectral interval investigated ranged from 250 nm to 800 nm. Despite the filtration with 0.22 /xm Millipore membranes, the samples can sometimes exhibit a residual disperse fraction of particulate matter, which scatter the light causing a false absorbance in the spectral region of high wavelength ( > 600 nm). The procedure to minimizing the error due to the spurious particle scattering affecting the absorbance is based on the extrapolation to lower wavelengths of the absorbance measured between 600 nm and 800 nm, where the contribution of the exponential fraction of dissolved organic matter is negligible.

93

samples was computed to be not negligible in our case. The fluorescence values were corrected for radiation absorption using the depression factor computed by the expression f ( A e, Ai, s) = e-AY(xe)s_ 1 e -y(~')s -- 1 AY(Ae)S

Ay(Ai)s

'

where Ay(A) is the yellow substance absorption coefficient at wavelength A in m-1, s is the sample dimension in m, and As and / ~ i a r e the wavelengths of exciting and induced radiations, respectively. The YS fluorescence at 420 nm was at least corrected for the minor water fluorescence background and normalized to the intensity of the Raman peak of distilled water to avoiding the effect of the instrumented drift. These procedures are extensively described in a paper in press (Ferrari and Tassan, 1991).

3.

RESULTS AND DISCUSSION

Fluorescence Measurements

pH Change Effect

The measurements were performed in the wavelength range 200-500 nm with bandpass 10 nm. The excitation wavelength was 308 nm, yielding a nonelastic Raman scattering peak at 345 nm and a broad fluorescence peak centered about 420 nm. Absorption of exciting radiation and self absorption of fluorescence and Rama quanta in the 1 cm 3

YS fluorescence spectra are altered under changing pH conditions. Ionization or protonization of functional groups with changing pH modifies the electronic state of the molecule and, thus, the observed fluorescence (Wehry, 1973). The impor-

8.08 z~ Hohlohsee (MA) • Hohlohsee (MA) 7.sl [] B&rnsee (HA) • B~rnsee (HA) 6.94 o Podsol (FA) • Podsol (FA)

6 6 6 6 40 40

raise pH lower pH raise pH lower pH raise pH lower pH

mg/I mgJl mg/I mg/l mg/l mg/I

Fluorescence

raise pH with Na= CO3 5 % lower pH with HCI 3 % + 1 % MgCI2

•

•

0

0

0

O

6.37

0

OO

5.80 O •

OI

0

•

0 O

5.23

O O

4.66

Figure 1. Normalized induced (308 nm

4.09 d~X 3.52

I

3

I

4

I

/

5

|

I

6

'

'

;

'

pH

!

excitation light) fluorescence response (FL) versus pH of natural humic and fulvic acid solutions. HA: B[irnsee (1986), Hohlohsee (1986); FA: Podsol (1986).

94

Ferrari

A sample n, 1 9

2.0

~.

8

1.8

7

(Sacca di Goro, 1989) r2 = 0.90

13 sample n. 3

(Sacca di Goro, 1989) r2 = 0.94

0 sample n. 13

(Sacca di Goro, 1989) r2 = 0.90

~

0

0[3

-

__o..o...o_,.o.-- O

-

r-~...I3-.d~

,,-0 " - " ' ' ' ' - ' - 0 ~ "

''''0"

"0-" "0"-

u

1.8

1.4

6

1,2

5

Figure 2. Normalized induced (308 -- --z~--,.~ ,

1.0

i

4

for the

,

,

5

,

,

6

,

,

7

8

9

•

,

..

•

l

sample n, 3

of fluorescence intensity was tested using a solution of HC1 3%+ 1% MgCI 2, which was found to be complete for all the specimens except the fulvic acid (Podsol), the fluorescence of which, related to its structure, appears particularly influenced by the Mg +2 ion present in sea water, as described by Willey (1984). The salinity simulation, obtained adding Mg 2÷ ions to the acid/basic solutions for changing pH, has determined a significant increase of fluorescence in fulvic acid (Podsol, Fig. 1). This can be due to an easier substitution for fulvic rather than humic acid of hydrogen with magnesium, which so add crosslinking to the structure determining an increase of the fluorescence.

tance of the pH variation on the fluorescence spectra of YS has been investigated by Smart et al. (1976), Laane (1982), Saar and Weber (1980), Stewart and Wetzel (1980), Willey (1984), and Vodacek and Philpot (1987). The relative fluorescence signal was calculated as the ratio of main peak height minus the distilled water contribution at the same wavelength to the Raman peak of distilled water. Figures 1 and 2 display the dependence relative fluorescence on pH for H / F acids and natural samples with YS. In Figure 1, humic and fulvic acid show similar type of dependence on pH with a broad conic curve with a maximum between pH 7 and 8. This behavior is not clearly evident in Figure 2, where natural samples from three different sites in Sacca di Goro are shown. The experimental points fit a straight line with regression coefficients r 2= 0.9, 0.94, and 0.9 and different slopes: 0.11, 0.05, and 0.16 for samples 1, 3, and 13 from Sacca di Goro, respectively. This reflects the composite character of dissolved organic matter in natural samples. The reversibility

O B&rnsee (HA) 17 mg/I r2 = 0.90 111 13 Hohlohsee (MA) 6 mg/I r2 = 0.97

2.5

.

10

pH

mn excitation light) fluorescence responses (FL) versus pH of riverine (Po di Volano st. N ° 13) and estuarine (st. 1 and 3) water samples collected in May 1989 in Sacca di Goro (deltaic area of the Po River), points at pH > 9.5 excluded.

Absorbance All the solutions, natural and artificial, display some dependence of light absorption on pH. Ionization, resulting from the change in pH, alters the energetic orbital state of excitable electrons, modifying the light absorption capacity. The beam ab-

(

3

~:~,.....,.---'0~

,.o

#

# 8L 1.5 1.0 0.5-

7 6 Z~

5 ^ _ - - -

4

+

Figure 3. Absorption coefficient at

3

0,0. for Podsol (FA)

2 1

=

51

I

6/

i

7t

=

8=

I

91

'l

110

=

pH

1=1

375 nm ( m - l) versus pH of natural humic anf fulvic acid solutions. HA: B~irnsee (1986), Hohlohsee (1986); FA: Podsol (1986), points at p H > 9.5 excluded.

Absorption and Fluorescence of Yellow Substance 95

6A

O sample n. 1

(Sacca di Goro, 1989) r2 = 0.88

[3 sample n. 3

(Sacca di Goro, 1989) r2 = 0.87

A sample n. 13

(Sacca di Goro, 1989) r2 = 0.64

0 C

]

~

°

00,AmO ¢0

5

¢~

"o" 4-

. ~ "~~'~" ~ ~ A ~ " "~ ~" ~ A " "

1

3-

I

6 for the sample n. 13

I

I

"" ' ' ~ ~" " ~ ' ~ "~ "r,

I

7

I

8

I

9

Table 4. P e r c e n t o f F l u o r e s c e n c e tions at Constant pH" Pb (tzglL)

I

10

DH

sorption coefficient at 375 nm increases linearly with increasing pH (Figs. 3 and 4). Similar results have been obtained by Laane (1982) in samples from the Ems-Dollart estuary. An a priori kind of direct physical dependence between light absorption and pH cannot be established, due presumably to the molecular differences between the different organic matter investigated. However, a certain homogeneity can be found by examining the slopes of the interpolation straight lines, which, excluding the fulvic acid, are in the range 0.2-0.5 with an average of 0.37 and standard deviation of 0.12. The natural

Humic Acid (mglL)

I

Figure 4.

&

Absorption coefficient at 375 nm (m -1) versus pH of riverine (st. 1 3 ) a n d e s t u a r i n e (st. 1 a n d 3 ) water samples collected in May 1989 in Sacca di Goro (deltaic area of the Po River), points at pH > 9.5 excluded.

pH excursion in the estuarine environment (e.g., Sacca di Goro) being from 6 to 9.5, the equations were derived using only data in this range.

Heavy Metal Quenching Fluorescence Different numbers and/or positions of functional groups in the molecular structure of HS are responsible for the complexing capacity with respect to metal cations such as Cu +2, Pb +2, and Cd +2 present in solution. The complexation reduces the

I n t e n s i t y V a r i a t i o n s a f t e r A d d i n g M e t a l I o n s ( P b 2+, C u 2 + ) a t F o u r D i f f e r e n t C o n c e n t r a -

FL%

Cu (l~glL)

FL%

0.68 3.42 6.84 13.68

- 3.4 - 3.1 - 4.0 - 10.8

Goro (April 1990) [Ay(375) m -~]

Pb (btglL)

2.78 2.78 2.78

1.74 8.68 17.36

6.57 6.67 6.57 6.57

1.74 8.68 17.36 34.72

( Biirnsee) -

3.1 4.1 8.4 9.7

FL%

Cu (l~glL)

FL%

Station 9

2.7 2.7 2.7 2.7

1.74 8.68 17.36 34.72

0.6 0.6 0.6 0.6

1.73 8.68 17.36 34.72

-

0.4 1.4 1.1 0.6

0.68 3.42 6.84 13.68

-

3.1 3.1 3.1 3.1

0.87 1.73 8.68 17.36

- 0.8 -0.8 - 3.1 - 3.8

0.68 3.42 6.84 13.68

- 1.6 -3.1 - 3. I - 4.7

8.65 8.65 8.65

1.74 8.68 17.36

12.2

1.74

- 1.0

0.68

+ 0.3

8.65

34.72

12.2 12.2 12.2

1.74 17.36 34.72

-0.5 - 4.1 - 4.6

3.42 6.84 13.68

- 1.7 - 5.6 - 5.3

( Hohlohsee)

- 1.2 - 0.4 - 3.0

0.68 3.42 6.84

0.0 - 3.1 - 4.5

0.68 3.42 6.84 13.68

0.0 -0.8 - 1.7 - 3.0

+0.1 + 0.1 - 1.i

0.68 3.42 6.84

0.0 - 1.6 - 3.7

- 1.9

13.61

- 7.5

Station 6 0.4 1.3 6.7 7.5

- 1.8 -3.6 - 4.8 - 6.4 Station 1

a T h e i n d u c e d f l u o r e s c e n c e values w e r e c o r r e c t e d b y radiation absorption a n d n o r m a l i z e d to t h e intensity o f t h e R a m a n p e a k o f distilled w a t e r ( F e r r a r i a n d Tassan, 1990). T h e d a t e relative to C d ~+ a d d i t i o n a n d t h o s e to t h e fulvie a c i d (Pedsol) a r e not presented b e c a u s e t h e variations a r e n o t significant.

96

Ferrari

fluorescence of YS due to interaction of the quenching material (transition metal cations) with the fluorescing centers of the molecule. For example, a functional group binding a cation directly attached to an aromatic ring may quench fluorescence by deexcitation of the shared electron. On the other hand, when the functional complexed group is not directly linked to an aromatic ring, the fluorescence is altered less (Saar and Weber, 1980). On the basis of the stability constant between Cu 2+, Pb 2÷, and Cd 2+ and humic acids from diverse sources, the complexes with Cu 2÷ and Pb 2÷ were considerably more stable than those of Cd 2+ (Stevenson, 1976). According to this author, the empirical stability constants of the complexes derived at different ionic strength follow the order: Cu 2+ > Pb 2+ >> Cd 2+, which is also in agreement with the findings of Schnitzer (1969). The results from the experiment described in the Material and Methods section seem to show how the high stability constants of copper and lead complexes are reflected in greater quenching of fluorescence than by cadmium, especially for the complexes formed with humic acids (Biirnsee and Hohlohsee). The samples of the Sacca di Goro, chosen at different YS concentrations, as measured by the absorption coefficient at 375 nm, show a similar behavior mainly expressed by the low reactivity of the cadmium. The samples of the fulvic acid (Podsol), probably due to the relatively limited complexing capacity of the molecules, do not display any fluorescence quenching phenomena. The results are summarized in Table 4, where humic acids are used in concentrations similar to those found in a natural estuarine environment. The HA metal complexes fluorescence quenching is masked by the pH changes affect, at least as far as the metal concentrations used are concerned. In other words, since for humic matter the pH increase gives rise to an increase in fluorescence response (as discussed in the previous paragraph), the metal cation complexes are in too low a concentration to cause a significant depressing effect distinguishable from that due to the pH increase. Given an error on the Raman peak estimate of distilled water of 1.3% (20 observations) and of 2.3% for the main peak at 420 nm and of 0.9% for the Raman peak of humic substance, the fluorescence decrease at constant pH appears to be in the order of the experimental error, excluding a quenching effect due to the lead and copper

HA complexes at high metal concentration. Moreover, the metal level added to the samples reaches values 10 times those measured in the deltaic area of the North Adriatic Sea (Ferrari and Ferrario, 1989), although occasionally such high levels can be found in particular pollution-stress situations. This is the case of lead at 34.72 ~ g / L , where it seems to cause a significant depression of fluorescence response ( ~ - 1 0 % ) of a 2.7 m g / L HA solution (Biirnsee). Absorbance Although cases of absorbance changes due to Cu, Pb, and Cd complexed at the low concentrations used in this experiment are not reported in the literature, a series of beam absorption measurements has been attempted on the same samples that had been tested for fluorescence quenching. Considering that the experimental error in the absorbance estimate of a standard solution of HA had been found to be in the order of 3% (20 observations), no changes in the absorbance at 375 nm, in the presence of the metal cations Cu 2+, Pb 2÷, and Cd 2+ at the low concentration cited above, were observed. Correction Factors for the YS Estimate

As stated in the introduction, for the remote sensing application, yellow substance is estimated as absorption coefficient at 375 nm through a direct measurement or by means of conversion of the fluorescence which is well correlated with the absorbance. A new problem is that of introducing another correction factor for the absorbance and fluorescence responses due to pH and heavy metal concentration changes. The factors to correct, for pH change, the absorption and fluorescence values were obtained by the ratio between the absorption or fluorescence values computed at pH 7 and those at pH varying from 4 to 10 using the empirical equation in Table 5 as a result of the regression curves shown in Figures 2, 3, and 4. For fluorescence, the pH factors (Fig. 5) were computed using the regression line [Eq. (9) in Table 5] shown in Figure 2 relative to sample of the station 1 which displays intermediate slope and intercept among the three. A similar statistical fit has not been attempted for humic and fulvic acid (Fig. 1), where it seems difficult to identify a linear depen-

Absorption and Fluorescence of Yellow Substance 97

Table 5. Empirical Equations with Statistical Parameters Showing the Relationship b e t w e e n p H and Fluorescence and Absorption Coefficients at 375 n m of H u m i c and Fulvic Acids and Natural Samples Collected in Sacca di Goro (Deltaic Area of the River Po) in April 1989 Absorption 1) Humic acid (B~imsee, September 1986) 17 mg/L: Ay(375) = 5.68+0.59 pH, r 2 = 0.90, S(Y/X)= 0.36, 2) Humic acid (Hohlohsee, July 1986) 6 rag/L: Ay(375) = 1.60+0.53 pH, r 2 = 0.97, S(Y/X)= 0.14, 3) Fulvic acid (Podsol, 1986) 12.36 mg/L: Ay(375) = 0.23+0.04 pH, r 2 = 0.73, S(Y/X) = 0.05, 4) Station 13 (Goro, 1989): Ay(375)= 1.24+0.33 pH, r 2 = 0.64, S(Y/X)= 0.27, 5) Station 3 (Goro, 1989): A y ( 3 7 5 ) = - 0 . 4 + 0 . 3 0 p H , r2=0.87, S(Y/X)=O.14, 6) Station 1 (Goro, 1989): Ay(375) = 0.38+0.20 pH, r 2 = 0.88, S(Y/X)= 0.1,

F = 43.63 F = 101.05 F = 10.7 F = 14.1 F=21.3 F = 38.1

Fluorescence 7) Station 13 (Goro, 1989): F L = 7 . 0 4 + 0 . 1 6 p H , r2=0.90, S(Y/X)=O.1, F = 46.07 8) Station 3 (Goro, 1989): FL = 1.38+0.05 pH, r 2 = 0.94, S(Y/X)= 0.02, F = 122.1 9) Station 1 (Goro, 1989): FL = 4.17+0.11 pH, r z = 0.90, S(Y/X)= 0.06, F = 63.1

1.3 ¸

Sacca di Goro 1989 O sample n. 1

1.2

FLf= 4.94/(4.17 + 0.11 pH) 1.1

1.0

~-----.~-------~----~.----`-~-~--..~..~-.-.~------~----7.~------~-~-----~-------~ -

0.9 0.8 0.7

Figure 5. Fluorescence correction factor I

I

I

I

5

I

I

6

I

I

7

I

I

8

I

I

9

10 pH

1.6

( F L f ) at varying p H derived from the empirical equation (9) for the water sample 1, (Sacca di Goro, May 1989).

•, sample n. 1 Goro1989 A y f = 1.83/(0.38 + 0.2 pH) x Hohlohsee - 6 mg/I

1.4

,'~ B&rnsee - 17 mg/I

1.3

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

1.2 1.1 1.0

0.9

Figure 6. Absorption correction factor (Ayf)

0.8 0.7 0,6

I

5

I

I

6

I

I

7

,1

I

8

I

I

I

9

I

10 pH

at varying p H for natural h u m i c acids: B~irnsee (1986), Hohlohsee (1986) a n d for water sample 1, collected in Sacca di Goro (May 1989).

98

Ferrari

10

8 O3

a (o)

Ay = 0.138 + 0.88 FL r2 = 0.98, S(Y/X) = 0.26, F = 1015

a _

b ( ~ ) Ay = -0.08 + 0.87 FL r2 = 0.99, S(Y/X) = 0.20, F = 1681

/

7

j.&~..~"~" \b

/

~

f

6

.po

3 2!

°-

Figure 7. Absorption versus fluorescence

1 Gq / ~

" ~~ I ~ 1

~ ~

, 3

2

,

, 4

,

, 5

~

t 6

,

i 7

t

I 8

i

i 9

i 10 FL

10

a (o)

Ay = 0.7 + 0.77 FL r2 = 0.99, S(Y/X) = 0.23, F = 2466

relationship for the 1989 sample campaign in Sacca di Goro: (a) regression line of the data without correction; (b) regression line with corrections due to the pH changes.

,,,~=,~

b (ix) Ay = 0.47 + 0.79 FL r2 = 0.99, S(Y/X) = 0.20, F = 3654

- - ~ / ~I ' - ~ /

I

I

1

1

I

2

I

I

3

I

I

4

I

Figure 8. The same of Figure 7 relative to

I

5

6

7

8

1

11 FL

dence of the fluorescence on pH in the range 6-9.5. The pH correction factors for absorption (Fig. 6) were computed for all the natural and artificial samples investigated with the exclusion of fulvic acid (Podsol), for which the pH dependence appears to be small, but, of all the curves, the curve relative to the sample of the station 1 is chosen for its intermediate trend as shown in Figure 6. Applying these factors to correct the experimental data of the Goro 1989 and 1990 campaigns, showing the strict dependence of the fluorescence versus absorption coefficient at 375 nm, a significant difference in the intercepts is obtained and no difference appears between the slopes of the straight lines relative to the values with and without pH correction. The statistical check was performed with the SAS-ANOCVA procedures through a test of parallelism and an analysis of covariance between the intercepts after assuming parallelism. Although significantly different, the interpolation lines in Figure 8 appear closer, because of the limited pH excursion, with respect to that found in 1989, as shown in Figure 7. As far as heavy metals are concerned, no correction factor was introduced because of the insignificant heavy

the data of the campaign performed in Sacca di Goro in April 1990.

metal quenching at the concentrations found in even moderately polluted systems (Ferrari and Ferrario, 1989). However, this does not mean that such a correction factor need not be introduced in high contamination conditions. 4.

CONCLUSIONS

The results of this work indicate that: 1. An important effect on fluorescence and absorption measurements is due to pH variations typical of fresh-saline waters mixing. To determine the absorption from fluorescence measurements the following procedure is suggested: a. Normalize the fluorescence measurement to pH 7 multiplying the real value for the factors obtained by the equation FLj. = 4.94/(4.17 + 0.11 pH) (Fig. 5). b. Obtain the corresponding absorption value at pH 7, using the regression fluorescence vs absorption [Fig. 7 or 8, Eq. (b)]. c. Convert the absorption value to actual pH dividing by the factor obtained by the equation Ay = 1.83/(0.38 + 0.2 pH) (Fig. 6).

Absorption and Fluorescence of Yellow Substance 99

This procedure, which allows us to evaluate the correct absorption parameters to introduce in the remote sensing retrieval model for chlorophyll, has yielded a mean absorption reduction of 5% with standard deviation 6.9 with min = 0%, max = -18%, relative to the 15 samples collected in Sacca di Goro, 1989. In this case the error appears low but this does not preclude that the error can be larger where the pH excursion and YS concentration are different. Neglecting this correction, the contribution of YS in the remote sensing of chlorophyll-like pigment (Tassan, 1988) appears to be overestimated, leading to underestimation of pigment concentration. 2. A quenching effect caused by Cu e+ and Pb 2+ ions on the fluorescence response of humic acids either isolated (mainly B~irnsee) or in natural mixtures (Goro, 1990) has been found only for high metal concentrations in the dissolved phase ( > 15 ~ g / L Pb and > 10/.~g/L Cu) and for middle concentrations of humie acid ( ~ 3 m g / L ) (Table 4). Only in similar eases, quite rare but possible in estuarine systems, could a correction of the fluorescence value be suggested.

Bristow, M. P. F., Bundy, D. H., Edmonds, C. M., Ponto, P. E., Frey, B. E., and Small, L. F. (1985), Airborne laser fluorosensor survey of the Columbia and Snake rivers: simultaneous measurements of chlorophyll, dissolved organics and optical attenuation, Int. J. Remote Sens. 6:1707-1734.

I wish to express my thanks to D. Van der Linde for his help during the absorbance and fluorescence measurements; to Dr. D. Kotzias and Mrs. R. Beyerle-Pfniir who supplied humic and fulvic acids; and to the "'Assessorato Ambiente" of the Amministrazione Provinciale of Ferrara which proved the research vessel HYDRA, the staff of which was very helpful during the water sampling in Sacca di Goro. I would like to acknowledge the help of Dr. Stelvio Tassan for his assistance and his useful advice. Particular thanks are addressed to Dr. Anthony Vodacek, visiting scientist in Ispra, for his constructive ideas and critical suggestions. I am indebted also to B. Hosgood for his patience in the revision of the English text.

H6jerslev, N. K. (1979), On the origin of yellow substances in the marine environment, in Workshop on the Eurasep Ocean Color Scanner Experiments (B. M611er SSrensen, Ed.), Proe. Joint Research Centre Ispra, pp. 13-28.

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Carder, K. L., Steward, R. G., Harvey, G. R., and Ortner, P. B. (1989), Marine humie and fulvie acids: their effects on remote sensing of ocean chlorophyll, Limnol. Oceanogr. 34(1):68-81. Cline, J. T., and Holland, J. F. (1977), Fluorometrie evidence for reaction of heavy metals with pore-water organics, in Proc. of the 15th Hanford Life Sciences Syrup., Riehland, WA, 1975, Teeh. Infor. Center, Energy Res. and Development Admin., pp. 264-279. Duursma, E. K. (1974), The fluorescence of dissolved organic matter in the sea, in Optical Aspects of Oceanography (N. G. Jerlov and E. Steeman Nielsen, Eds.), Academic, London, pp. 237-255. Ferrari, G. M., and Ferrario, P. (1989), Behavior of Cd, Pb, and Cu in marine deltaic area of the Po River (North Adriatic Sea), Water Air Soil Pollution 43:232-343. Ferrari, G. M., and Tassan, S. (1991), On the accuracy of the determination of light absorption by "yellow substance" through induced fluorescence measurement, Limnol. Oceanogr., forthcoming. Hama, T., and Handa, N. (1980), Molecular weight distribution and characterization of dissolved organic matter from lake waters, Arch. Hydrobiol. 90:106-120.

H6jerslev, N. K. (1988), Natural occurrence and optical effects of Gelbstoff, Geophysical Institute, Dept. of Physical Oceanography, University of Copenhagen, Report No. 50, p. 192. Jerlov, N. G. (1976), Marine Optics, Elsevier, Amsterdam, 231 pp. Kearny, P. C. (1976), Bound and Conjugated Pesticide Residues (D. Kaufrnan et al., Eds.), ACS Syrup. Set. 29, Am. Chem. Soe., Washington, DC, p. 378. Laane, R. W. P. M. (1982), Influence of pH on the fluorescence of dissolved organic matter, Mar. Chem. 11:395-401. Laane, R. W. P. M., and Koole, L. (1982), The relation between fluorescence and dissolved organic carbon in the Ems-Dollart estuary and the western Wadden Sea, Netherlands J. Sea Res. 15:217-227. Levesque, M. (1972), Fluorescence and gel filtration of humie compounds, Soil Sc/. 113:346-353. Maracei, G., Hosgood, B., Andreoli, G., Grassi, P., Ferrari, G. M., and Van der Linde, D. (1989), European Imaging Spectroscopy Aircraft Campaign (EISAC), Sea Truth Data. Po Delta (Saeea di Goro), Joint Research Centre, Ispra, Technical Note 89.110.

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Maracci, G., Hosgood, B., Andreoli, G., Ferrari, G. M., and Van der Linde, D. (1991), GORO 1990 measurement campaign, Joint Research Centre, Ispra, forthcoming. Miinster, U. (1985), Investigations about structure, distribution and dynamics of different organic substrates in the DOM of Lake Plussee, Arch. Hydrobiol. Suppl. 70:429-480. Postma, H., Manuels, M. W., and Rommets, J. W. (1976), Breakdown and production of fluorescent substances in Dutch waters, Netherlands J. Sea Res. 10:499-516. Saar, R. A., and Weber, J. H. (1980), Comparison of spectrofluorometry and ion-selective electrode potentiometry for determination of complexes between fulvic acid and heavy-metal ions, Anal. Chem. 52:2095-2100. Satoh, Y., and Abe, H. (1987), Dissolved organic matter in coloured water from mountain bog pools in Japan, II. Biological decomposability. Arch. Hydrobiol. 111:25-35. Schnitzer, M, (1969), Reactions between fulvic acid, a soil humic compound and inorganic soil constituents, Proc. Soil Sci. Soc. Am. 33:75-81. Smart, P. L., Finlayson, B. L., Rylands, W. D., and Ball, C. M. (1976), The relation of fluorescence to dissolved organic carbon in surface waters, Water Res. 10:805-811. Stevenson, F. J. (1976), Stability constant of Cu z+, Pb 2 +, and Cd 2+ complexes with humic acids, Soil Sei. Soc. Am. J. 40:665-671.

Stewart, A. J., and Wetzel, R. G. (1980), Fluorescence absorbance ratios: a molecular-weight tracer of dissolved organic matter, Limnol. Oceanogr. 25:559-564. Tassan, S. (1988), The effect of dissolved "yellow substance" on the quantitative retrieval of chlorophyll and total suspended sediment concentrations from remote measurements of water colour, Int. J. Remote Sens. 9(4):787-797. Vane, G., and Goetz, A. (1988), Terrestrial imaging spectroscopy, Remote Sens. Environ. 24(1):1-29. Vodacek, A. (1985), Laser fluoresensing for remote detection of dissolved organic carbon and aluminium in water, MSc. thesis, Cornell University, Ithaca, NY. Vodacek, A., and Philpot, W. D. (1987), Environmental effects of laser-induced fluorescence spectra of natural waters, Remote Sens. Environ. 21:83-95. Wehry, E. L. (1973), in Practical Fluorescence: Theory, Methods, and Technique (G. G. Guilbault, Ed.), Marcel Dekker, New York, pp. 79-136. Willey, J. C. (1984), The effect of seawater magnesium on natural fluorescence during estuarine mixing, and implications for tracer applications, Mar. Chem. 15:19-45, Zepp, R. G., and Schlotzhaner, P. F. (1981), Comparison of photochemical behaviour of various hmnic substances in water: III. Spectroscopic properties of humic substances, Chemosphere. 10(5):479-486.