Biological Alteration of Zinc Complexation Characteristics of Dissolved Organic Matter in Domestic Wastewater Treatment Plant Effluent under River Water Environment

Naturally occurring processes such as biological reaction might modify the properties of dissolved organic matter (DOM) for binding with heavy metals. Complexation of heavy metals with DOM determines their environmental and ecological impacts. In this study, biodegradation experiments were carried out separately for river water and river water spiked with DOM in wastewater treatment plant (WWTP) effluent to evaluate the biological alteration of zinc complexation characteristics of DOM under river water environment. Zinc complexation parameters, conditional stability constant and binding site concentrations were determined using anodic stripping voltammetry with Scatchard linearization. Total ambient zinc binding site concentration of river water DOM was reduced from 410 nM to 74 nM (82% decline) during two weeks of incubation. Compared to the river water DOM, 1-month incubation of DOM in WWTP effluent under the river water environment, showed only 22% decline in total ambient Zn binding sites. On the other hand, conditional stability constants, for Zn binding sites of DOM from WWTP effluent, did not vary during 1 month of incubation. The result suggests that metal (Zn) binding sites of DOM from WWTP effluents are biologically persistent in the urban river water environment.

Address correspondence to Tushara CHAMINDA G G, Environmental Science Center, The University of

Biological Alteration of Zinc Complexation Characteris-tics of Dissolved Organic Matter in Domestic Wastewa-ter Treatment Plant Effluent under River WaWastewa-ter Environment

Tushara CHAMINDA G G*, Fumiyuki NAKAJIMA*, Ikuro KASUGA**

*Environmental Science Center, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo,

113-0033, Japan

**Department of Urban Engineering, School of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo, Tokyo, 113-8656, Japan

ABSTRACT

Naturally occurring processes such as biological reaction might modify the properties of dissolved organic matter (DOM) for binding with heavy metals Complexation of heavy metals with DOM determines their environmental and ecological impacts In this study, biodegradation experiments were carried out separately for river water and river water spiked with DOM in wastewater treatment plant (WWTP) effluent to evaluate the biological alteration of zinc complexation characteristics of DOM under river water environment Zinc complexation parameters, conditional stability constant and binding site concentrations were determined using anodic stripping voltammetry with Scatchard linearization Total ambient zinc binding site concentration of river water DOM was reduced from 410 nM to 74 nM (82% decline) during two weeks of incubation Compared to the river water DOM, 1-month incubation of DOM in WWTP effluent under the river water environment, showed only 22% decline in total ambient

Zn binding sites On the other hand, conditional stability constants, for Zn binding sites of DOM from WWTP effluent, did not vary during 1 month of incubation The result suggests that metal (Zn) binding sites of DOM from WWTP effluents are biologically persistent in the urban river water environment

Keywords: biodegradation, dissolved organic matter, river water, wastewater treatment plant

effluent, zinc complexation

INTRODUCTION AND BACKGROUND

Dissolved organic matter (DOM) is important in the transport of metals in aquatic systems It also provides protection to the aquatic organisms from heavy metal stress by forming strong complexes, which results in the decline of the free metal ion concentration Many water bodies in the urban environment, such as rivers and bays, receive effluent from wastewater treatment plant (WWTP) which may contain considerable concentrations of toxic metals as well as DOM that control the metal speciation The chemical structure of DOM, which originated from biological wastewater treatment plant, is much complicated and includes organic compounds in

different groups (Ma et al., 2001) Recent studies have revealed that DOM in treated

effluent tends to bind with many metals (eg; Zn and Cu) to a greater degree than natural organic matter (NOM) does (Sarathy and Allen., 2005; Cheng and Allen., 2006;

Chaminda et al., 2008)

No matter how strong the complexations of metals are with DOM, naturally occurring processes such as biological degradation and photochemical reaction might modify the

properties of DOM in binding with heavy metals Although the photodegradation

effects of DOM on heavy metal speciation have been investigated (Shiller et al., 2006; Brooks et al., 2007) to some extent, biodegradation effects of DOM have not been well

understood The microbial degradation of organic ligands in natural water bodies may release free metal ions and subsequently increase the metal toxicity

In this study, biodegradation experiments were carried out for river water and river water spiked with DOM from WWTP effluent to evaluate the biological alteration of zinc complexation characteristics of DOM under river water environment Zinc complexation parameters (conditional stability constant and binding site concentration), which characterize the nature of DOM responsible for complexing with Zn, were determined using the square wave anodic stripping voltammetry (SWASV)

MATERIALS AND METHODS

Sampling

The effluent used in this study was collected from a WWTP, where domestic wastewater was treated using conventional activated sludge process and disinfected by chlorination, in the late morning of a dry day in April, 2009 (pH = 7.0; dissolved organic carbon (DOC) = 8.1 mg/L; average ambient temp = 19°C) River water samples for preliminary study and biodegradation study were collected from the Edogawa River (+35° 45' 56.3", +139° 52' 46.1") in January, 2009 (pH = 7.2; DOC = 2.1 mg/L; average ambient temp = 7°C) and April, 2009 (pH = 7.1; DOC = 0.9 mg/L; average ambient temp = 19°C), respectively

River water samples were immediately filtered by 10 µm PTFE filter (Millipore) to remove large particles and organisms and were stored in a refrigerator for biodegradation experiments A portion of river water samples was also filtered through 0.5 µm PTFE filter (Millipore) to separate the suspended particles and was kept in a refrigerator at 4oC in the laboratory for dissolved and labile Zn analysis Collected WWTP effluent was filtered through 0.5 µm PTFE filter (Millipore) and stored in a freezer for further analysis and biodegradation experiments

Biodegradation experiment

Preliminary experiments were conducted using the river water samples collected in January, 2009 to find whether the metal (Zn) binding sites in river water DOM are biodegradable The river water sample (filtered by 10 µm PTFE filter) was transferred

to 1 L bottles which were covered to avoid exposure to light Then, two separate bottles (bottle 1 and bottle 2) were subjected to incubation at controlled temperature under 20°C for 6 weeks The bottles were stopped with air permeable silicon caps to allow the passage of air and to prevent the system from contamination and from becoming anaerobic Headspaces were also left in the bottles in such a way that air exchange could take place during the incubation Throughout the incubation, the bottles were shaken manually at 1 - 2 day intervals Water samples were withdrawn after two (from bottle 1) and six (from bottle 2) weeks and analyzed for Zn complexation parameters (conditional

stability constant, K' and binding site concentration, [L]) using voltammetry Water

samples were also withdrawn in 3-7 day intervals for DOC, UV absorbance at 254 nm (UV254), SUVA (specific UV absorbance) and dissolved and labile Zn analysis

Although the pH was not maintained at a constant value, the results showed that the variation of pH (7.0 - 7.2) was not significant during the incubation

Cations in the filtered WWTP effluent samples (< 0.5 µm) were removed by cation exchange resin AGMP-50 (H+ form, 100 - 200 mesh, Bio Rad) in a batch mode and then DOC was concentrated by freeze drying (FDU 540, EYELA) Subsequently, the concentrated DOM of WWTP effluent was spiked into river water sample which was collected in April and pre-incubated for two weeks Two weeks were chosen, because the preliminary results, which will be discussed in the succeeding section of the paper, suggested that Zn binding sites in river water DOM seemed to be persistent after two-week incubation The mixture of DOM of WWTP effluent and the pre-incubated river water (DOC in the mixture = 4.9 mg/L) was incubated, in three separate bottles (bottle 1, bottle 2 and bottle 3), for one month under the same condition as explained in the preliminary experiment Samples were withdrawn after 4 days (bottle 1), 7 days (bottle 2) and 1 month (bottle 3) to determine the Zn complexation parameters

Determination of dissolved and labile Zn, DOC, UV absorbance and SUVA

Dissolved organic carbon concentration was measured as non-purgeable organic carbon with a TOC analyzer (TOC-VCSH, Shimadzu, Japan) Absorbance at 254 nm (UV254) was obtained by a Hitachi U-2000 UV spectrophotometer using a 1.0 cm quartz cuvette Specific UV absorbance is defined as the UV254 divided by the DOC concentration Dissolved Zn and labile Zn concentrations were analyzed by an ICP-MS (HP 4500,

Yokogawa, Japan), in accordance with the methods described by Chaminda et al

(2008) The labile Zn was operationally defined by fractionation using a chelating disk

cartridge (Empore) (Chaminda et al., 2008)

Zinc complexation analysis using voltammetry

The filtered (< 0.5 µm) samples of the WWTP effluent and river water were subjected

to SWASV to determine the conditional stability constant (K') and binding site

concentration ([L]) of Zn All the samples were either concentrated by freeze drying or diluted by Milli-Q water to maintain an equal DOC concentration (3.5 mg/L) and then subjected to zinc titration with Nano-BandTM Explorer portable system (TraceDetect), equipped with a thin film mercury electrode Zinc titration against DOM in water samples were conducted by the addition of known increments of Zn followed by the measurement of electrochemical labile Zn after a certain equilibrium period The Zn titration data were then linearly transformed using the Scatchard equation, as explained

in Chaminda et al (2010) and in the Appendix of this paper

RESULTS AND DISCUSSION

Biodegradability of zinc binding sites in river water DOM

Fig 1 represents the variation of DOC, UV254 and SUVA during the river water incubation for 6 weeks in the preliminary study During the first 4 days of incubation, DOC concentration declined by 54% No further significant DOC reduction was observed up to 6 weeks of incubation as shown in Fig 1 Similar to the trend in DOC, there was a momentous change in UV254 and SUVA, which is a good indicator of aromaticity, during the first 4 days and no significant changes were observed afterwards The original river water and the withdrawn samples after 2 weeks and 6 weeks of

0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 0.040

0.0

1.0

2.0

3.0

4.0

5.0

-1 m

-1 )

Incubation period, days

Note:

Error bars represent the standard deviation of triplicate measurements

DOC, mg L -1

SUVA₂₅₄, L mg -1 m -1

UV₂₅₄, cm -1

Fig 1 - Variation of DOC, UV and SUVA during the incubation of river water

0.0

0.5

1.0

1.5

2.0

Strong type binding sites, L₁

Weak type binding sites, L₂

Note: [Zn'] is the SWASV labile

Zn concentration, [ZnL] is the complexed Zn concentration and

K' is the conditional stability

constant (with respect to SWASV labile Zn) in the Scatchard linearization equation;

(Chaminda et al, 2010).

L]

[ -[L ] [ L]

[

' K Zn

Zn =

'

Fig 2 - Scatchard linearization results on Zn titration for DOM in river water in the

original sample, after 2 weeks, and after 6 weeks of incubation

incubation were titrated separately with Zn to determine the conditional stability

constant K' and total binding site concentration [LT] As shown in Fig 2 and Table 1, Zn titration data obtained for DOM in the original river water sample seems to fit into two linear portions indicating two classes of binding sites

In contrast, DOMs in the samples after 2 and 6 weeks fitted to a single line, signifying only one type of binding site It seems that the weak type of binding site in the river water DOM might have been degraded by biological activities On the other hand, total ambient binding site concentration was reduced from 410 nM to 74 nM (82% decline)

during 2 weeks of incubation, while there was no significant changes, either in K' or

[LT], observed afterwards These results agreed with the SUVA results between 2 and 6 weeks of incubation This further implied that the weak Zn binding sites in the river water DOM were significantly altered and more likely removed by biodegradation during the first 4 days of incubation period and became persistent This degradation of binding sites was also in line with the increment of labile Zn concentration during the first 4 days of incubation Consequently, labile Zn concentration increased from 3.7 ± 0.4 µg/L to 5.4 ± 0.3 µg/L during the first 4 days of incubation From this preliminary experiment, it was concluded that Zn binding sites in DOM in river water became persistent within or less than two weeks

Biodegradability of zinc binding sites in DOM of WWTP effluent in river water

Fig 3 shows the variation of DOC, UV254 and SUVA during the incubation of DOM

in WWTP effluent with river water, where river water was pre-incubated for two

DOC, mg/L SUVA 254 , L/mg/m

UV 254 , 1/cm

(Chaminda et al, 2010)

Table 1 - Conditional stability constants, K', and the binding site concentrations, [LT] for

Zn during the incubation of river water DOM and DOM of WWTP effluents in river water

Normalized binding site concentration Ambient binding site concentration

log

K 1 ' log K 2 '

[L 1,T ] [L 2,T ] [L T ] = [L 1,T ] + [L 2,T ] [L 1,T ] [L 2,T ] [L T ] = [L 1,T ] + [L 2,T ] Sample

Original

sample 6.9 6.1 0.79 1.6 2.4 140 ± 10 270 ± 20 410 ± 22 After 2

weeks 6.8 ND 1.0 ND 1.0 74 ± 17 ND 74 ± 17

After 6

weeks 6.7 ND 1.1 ND 1.1 72 ± 8 ND 72 ± 8 Original

sample 7.2 6.3 0.85 1.5 2.4 350 ± 7 610 ± 12 960 ± 14 After 4

days 7.2 6.3 0.84 1.4 2.2 340 ± 5 550 ± 8 890 ± 9 After 7

days 7.2 6.3 0.86 1.4 2.3 350 ± 7 550 ± 12 900 ± 14

After 1

month 7.2 6.3 0.83 1.3 2.1 290 ± 14 460 ± 22 750 ± 26

Note: K' = conditional stability constant with respect to electrochemical labile Zn (L/mol), [L1,T ] and [L 2,T ] represent two types of binding sites: strong and weak, respectively; [L T ] = total binding site concentration; ND = not detected; Ambient binding site concentration = Normalized binding site concentration × DOC concentration (Titration condition: DOC = 3.5 mg/L; pH≈7; ionic strength = 0.02 M; temperature = 25ºC)

weeks inadvance It was observed that the initial DOC concentration in river water decreased from 0.9 mg/L to 0.4 mg/L during the two weeks of pre-incubation period, indicating the availability and activities of microorganism in river water However,

as shown in Fig 3, there was no considerable change of DOC in the mixture of DOM of WWTP effluents and pre-incubated river water within the first week of incubation Slight DOC reduction (14%) was observed after one month of incubation

In contrast to the results of the preliminary experiment for river water, there was also no significant variation of SUVA in the incubation of DOM in WWTP effluent

The results for the conditional stability constants, K', and the concentrations of the

binding sites, [LT] for Zn are listed in Table 1 Scatchard linearization for the Zn titration data of four samples (original sample, after 4-days, 7-days and 1-month

incubation) indicated the existence of two classes of binding sites: strong type, K 1 '

and weak type, K 2 ' (Fig 4) As shown in Fig 4, titration plots of four samples are close to each other, indicating no considerable difference in the conditional stability constants, which were deduced from the slopes of the lines in a particular binding site class Conditional stability constant for the strong type of binding site remained

at log K 1 ' = 7.2 while that in weak type remained at log K 2 ' = 6.3 for all the four

samples throughout the incubation period The conditional stability constant for Zn

in this study was consistent with the values reported (log K 1 ' = 7.1 - 7.4 and log K 2 '

= 6.2 - 6.4) for the effluents from three different WWTPs (Chaminda et al 2009)

Moreover, the normalized binding site concentrations ([LT] divided by DOC concentration), as listed in Table 1, show no significant changes (2.1 - 2.4 nM/ µM

0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10

0.0

1.0

2.0

3.0

4.0

5.0

6.0

-1 ), SUVA

-1 m

-1 )

Incubation period, days

DOC, mg L -1

SUVA₂₅₄, L mg -1 m -1

UV₂₅₄, cm -1

Note:

Error bars represent the standard deviation of triplicate measurements

Fig 3 - Variation of DOC, UV and SUVA during the incubation of DOM of WWTP

effluent with river water

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Strong type

Note: [Zn'] is the SWASV labile

Zn concentration, [ZnL] is the complexed Zn concentration and

K' is the conditional stability

constant (with respect to SWASV labile Zn) in the Scatchard linearization equation;

(Chaminda et al, 2010).

L]

[ -[L ] [ L]

[

' K Zn

Zn =

'

Fig 4 - Scatchard linearization results on Zn titration for DOM in WWTP effluent

in the original sample, after 4 days, after 7 days and after 1 month of incubation

C) As the bottles were covered to avoid exposure to light and as the incubation was done in a controlled temperature, it was assumed that the changes in metal complexation parameters were only due to the activities of indigenous microbes in the river water Therefore, it can be hypothesized that, unlike binding sites in DOM

of the river water, binding sites in DOM of WWTP effluents were less biodegradable In other words, treatment of wastewater by activated sludge process has shifted the DOM composition to more biologically recalcitrant compounds in terms of zinc complexation

On the other hand, a further examination of the variation of labile Zn during the incubation revealed a slight increment of the labile Zn concentration (from 2.1 µg/L

to 2.5 µg/L) in the sample after 1 month of incubation Decline of total ambient binding site concentration (from 960 nM to 750 nM) by 22% after one month of incubation, might have caused this slight increase of labile Zn concentration However, the residence time of urban rivers is usually shorter than one month and hence, it can be suggested that the DOM from WWTP effluent seems persistent in receiving waters of urban environment It may be important to study the long-term biological alteration of organic matter for the prediction of heavy metal speciation in bay or seawater It is also similarly important to study in details the microbial community involved in the biodegradation of metal binding sites to further understand the fate of metal speciation in the water environment

DOC, mg/L SUVA 254 , L/mg/m

UV 254 , 1/cm

(Chaminda et al, 2010)

CONCLUSIONS

Biodegradation experiments revealed that 82% of Zn binding sites of river water DOM was degraded during 2 weeks of incubation In contrast, Zn binding sites of DOM in WWTP effluent as well as their conditional stability constants showed no significant changes during 1 month of incubation and hence, no significant effects

on Zn complexation This implies that metal (Zn) binding sites in the WWTP effluent were biologically resistant under the river water environment

ACKNOWLEDGEMENT

This study was funded by Grants-in-Aid for Scientific Research (B) (20360239) of

Japan Society for the Promotion of Science (JSPS)

REFERENCES

Brooks M L., Meyer J S and Boese C J (2007) Toxicity of copper to larval

Pimephales promelas in the presence of photodegraded natural dissolved organic

matter, Can J Fish Aquat Sci 64, 391-401.

Chaminda G G T., Nakajima F and Furumai H (2008) Heavy metal (Zn and Cu) complexation and molecular size distribution in wastewater treatment plant effluent,

Water Sci and Technol.,58 (6), 1207-1213

Chaminda G G T., Nakajima F., Furumai H., Kasuga I and Kurisu F (2009) Zn and Cu complexation with DOM in wastewater treatment plant effluent, Proceeding of SETAC Europe 19th Annual Meeting, Göteborg, 40-41

Chaminda G G T., Nakajima F., Furumai H., Kasuga I and Kurisu F (2010) Comparison of Metal (Zn and Cu) Complexation Characteristics of DOM in

Different Urban Wastewaters, Water Sci and Technol., (in press)

Cheng T and Allen H E (2006) Comparison of zinc complexation properties of

dissolved natural organic matter from different surface waters, Journal of

Environmental Management, 80, 222-229

Ma H., Allen H E and Yin Y (2001) Characterization of isolated fractions of dissolved

organic matter from natural waters and a wastewater effluent, Water Research, 35

(4), 985-996

Sarathy V and Allen H E (2005) Copper complexation by dissolved organic matter

from surface water and wastewater effluent, Ecotoxicology and Environmental

Safety, 61, 337-344

Shiller A M., Duan S., van Erp P and Bianchi T S (2006) Photo-oxidation of dissolved organic matter in river water and its effect on trace element speciation,

Limnol Oceanogr., 51 (4), 1716-1728

APPENDIX

Model and linearization technique

The metal titration data in this study were linearly transformed using Scatchard equation which was derived from the appropriate mass balance and conditional

stability constant, K', relationship Assuming 1:1 stoichiometry between electrochemical labile metal M' and binding sites L, the conditional stability

constant can be represented by:

[M']+[L i]↔[ML i] (1)

] ][

' [

] [

'

i

i i

L M

ML

K = (2)

[L i]+[ML i]=[L i ,T] i =1, 2 (3)

where [M'] is the electrochemical labile metal concentration, [MLi] is the complexed

metal concentration, [Li] is the free binding site concentration, [Li,T] is the total

available binding site concentration and K i ' is the conditional stability constant (with

respect to ASV labile metal) The Scatchard linearization equation which is derived

from the above equations (2) and (3) can be discribed as in equation (4)

]

'

[

]

[

i i

M

ML

−

= (4)

The Scatchard linearization consists of plotting the [MLi]/[M'] against [MLi] The

labile metal concentration, [M'], was electrochemically determined by ASV and

[ML] was calculated from the mass balance equation ([MT] – [M']), where [MT] was

the total metal concentration calculated from the initial concentration and the

cumulative titrated volume If the transformation results are linear (Fig A1(a)), it

indicates that the sample contains a class of ligands having one representative

conditional stability constant K' The slope of the line corresponds to the conditional

stability constants K' The y-intercept of the line gives the product of the conditional

stability constant K' and total ligand concentration [Li,T] and the x-intercept directly

gives [Li,T]

Distinctively separate linear regions within a transformation curve indicate that more

than one class of ligands are available in the tested samples Those lines can be

separately extrapolated to determine the parameters related to each ligand class Fig

A1(b) shows the idealized Scatchard plots of titration data for the two-ligand case

In this study, conditional stability constants were determined with respect to ASV

labile, or electrochemically labile, metal The ASV is sensitive to all quickly

dissociating metal, including free metal ion, inorganic complexed metal and some

weak organic complexed metals Although the relationship between the free ion,

[M2+], and [M'] can be determined for inorganic ion pairs with simple equilibrium

speciation modeling, it is not possible to correct [M'] for quickly dissociating

organics, as their concentrations or side-reaction binding strengths are not known

Therefore, in this study K' values, determined from ASV without any corrections of

[M'], were used

K' [LT]

[LT]

Slope = –K'

[ML]

K 1' [L1,T] + K 2' [L2,T]

Slope 1 = – K 1'

[ML]

Slope 2 = – K 2'

K 2' [LT]

[LT]

K 2' [L2,T]

K 1' [L1,T]

K' [LT]

[LT]

Slope = –K'

[ML]

K 1' [L1,T] + K 2' [L2,T]

Slope 1 = – K 1'

[ML]

Slope 2 = – K 2'

K 2' [LT]

[LT]

K 2' [L2,T]

K 1' [L1,T]

(a) (b)

Fig A1 - Interpretation of titration data with Scatchard linear transformation (a)

one-ligand case (b) two-one-ligand case; K 1 ' and K 2 ' represent the conditional stability

constants of strong and weak ligand, respectively; Dotted line shows the resolved component of each ligand in the two-ligand case