Biodegradability of Anthropogenic Organic Matter in Urbanized Rivers using Fluorescence,
UV and BDOC Measurements
Heloise G. Knapik1; Cristovão V. S. Fernandes1; Julio Cesar R. de Azevedo2; Mauricius M. dos Santos1;
Patrícia Dall’Agnol1; Darrell G. Fontane3
1
Department of Hydraulic and Sanitation, Federal University of Parana, Centro Politecnico, Bl. 5, Av. Cel
Francisco H. dos Santos, s/n, 81531-990, Curitiba - PR, Brazil
2
Department of Chemistry and Biology, Federal University of Technology - Parana, Rua Deputado Heitor
de Alencar Furtado, n° 4900, 81280-340, Curitiba- PR, Brazil.
3
Department of Civil and Environmental Engineering, Colorado State University, Fort Collins, 80523, CO USA
* Corresponding author: heloise.dhs@ufpr.br; Phone: +55 41- 33613707
Supplementary Material
1
Table S 1: Summary of modifications in BDOC analysis and examples of application
N
1
Sample water pretreatment
Sample type
Inoculumn
Nutrients
Shaking/
Aeration
Temperature (°C)
Culture type
Duration
Membrane filtration
Drinking water
Indigenous bacteria
15-25
batch
10-30 days
2
Glass fibre filtered
Raw river water, filtered water
Indigenous bacteria
and finished treated water
10-25
continuous
2 hours
3
Freeze-drying and
reconstitution
Moorland stream
15
batch
41 days
4
Membrane filtration
(0,45 μm)
5 and 15
batch ( 10 mL
glass bottles)
28 days
5
Raw and drinking water
Glass fibre filtered
8
9
N and P/
without
Membrane filtration
(0,45 μm)
10
30 rpm
Indigenous bacteria
Wastewater/ surface water
6
7
Stream waters
Indigenous bacteria/ without
Aeration
Sand fixed bacteria
Drinking water
Raw and drinking water
Suspended inoculum bacteria
River/ leachate
Ca, Fe, N, Mg,
Bacteria from activated sludge
Na, K
50 rpm
batch ( 4 L glass
bottles)
10 days
20
batch
2-7 days
Room temperature
batch
28 days
20
batch (40 mL
tubes)
28 days
20
batch (250 mL)
28 days
Stream water and samples
taken in different steps in a
water-treatment process
Indigenous bacteria (free,
Glass fibre filtered (0,4
11
Stream water and leaf leachate suspended and attached to
μm)
artificial substracta)
Ca, Fe, N, Mg,
Na, K
batch,
recirculating
batch
batch (100 mL
glass bottles
with aluminium
10 - 21 days
DOC, colony counts
Comparison of six different methods for
AOC and BDOC
DOC, UV absorbance Investigate the effect of different types of
(254 and 400 nm) bacterial inocula
150 rpm
22
batch ( 1L glass
bottles)
28 days
DOC/ BOD
25
batch ( 20 mL
glass vials)
60 days
DOC
Evaluate effluent DOM
DOC
Comparison of a bioreactor and the
traditional batch system using entrapped
microbial cells
Municipal and industrial
inoculum
13
Effluent
Soil inocula
Tap water and unchlorinated
secondary effluent
Indigenous bacteria/ activated BOD nutrients
With and without
sludge inocolum/entrapped buffer pillows aeration
microbial cells
Hach
Comparison of respirometric
biodegradation tests
20 ± 0,5
bioreactor/
batch
3 hours/ X days
3/4 hour for
bioreactor and 28
days for batch
DOC
Test of bioreactor do determine BDOC
21 days
DOC
Evaluate the refractory fraction of
effluents
15 Glass fibre filtered
Lake water
Indigenous bacteria
Plu-flow (3.5 h of
residence time)
15 - 20
continuous
bioreactor/
batch
16 Glass fibre filtered
Effluent from sewage
treatment plant
Indigenous bacteria
Slow agitation with
aeration
20
reactor (3 L)
Reference
Servais et al. (1987)
Ribas et al. (1991)
Stutter et al. (2013)
Wickland et al. (2012)
Assess the potential for transformation of
DOC, UV absorbance wastewater-derived contaminants in
surface waters
Lim et al. (2008)
DOC
Test AOC and BDOC methods
Escobar and Randall (2001)
Compare AOC and BDOC data for drinking
DOC
water
Kaplan et al. (1992)
Test AOC and BDOC methods for drinking
DOC
water
Charnock and Kjonno (2000)
Biodegradability in environmental and
DOC
anthropic samples
Labanowski and Feuillade (2009)
20 ± 0,5
Organic compounds
42 days
Applicability
Evaluate the effects of ozonation in
DOC
drinking waters
Analyze a continuous method for the
DOC
determinados of BDOC in the treatment
and distribution of drinking water
Analyze the fate of DOM in a moorland
DOC, UV absorbance
stream
DOC, UV absorbance,
EEMs, XAD resin
Evaluate seasonality
fractionation
Ocasional shaking
12
14
20-22
Test parameter
Frias et al. (1995)
Trulleyová and Rulík (2004)
Reuschenbach et al. (2003)
Saadi et al. (2006)
Khan et al. (2003)
Søndergaard and Worm (2001)
Namour and Mueller (1998)
2
Table S 2: Summary of percentage of BDOC and respective initial DOC concentration for different samples
N
Type of water
1
Stream water
2
Leaf leachate
Method of BDOC
experiment
Batch
Incubation time
42 days
42 days
Inoculumn
Indigenous bacteria (free,
suspended and attached to
Indigenous bacteria (free,
suspended and attached to
Indigenous bacteria
Indigenous bacteria
Indigenous bacteria
Indigenous bacteria
Indigenous bacteria
Indigenous bacteria
Indigenous bacteria
Indigenous suspended bacteria
Indigenous fixed bacteria
Indigenous fixed bacteria
Indigenous bacteria
DOC (mg/L)
% BDOC
Reference
3.4 ± 0.3 / 3.9 ± 0.3
34.5 ± 4.2 / 43.8 ± 2.1
12.9 ± 0.5 / 23.1 ± 0.6
52.1 ± 1.2 / 74.6 ± 0.9
12.6
1.8
8.7 / 13.1
2.7 / 5.9
6.7 / 12.5
0.8 / 10.4
1.2 / 2.4
0.5 / 1.0
2.7 / 17.3
12.4 ± 4.3
13.3 ± 4.1
43
11
17 /41
20 / 35
29 ± 11
27.4 ± 4.8
30 / 48
24 / 40
2 ± 1 / 35 ± 11
Trulleyová and Rulík (2004)
Søndergaard and Worm (2001)
Søndergaard and Worm (2001)
Servais et al. (1987)
Servais et al. (1987)
Servais et al. (1987)
Servais et al. (1987)
Lønborg (2009)
Volk et al. (1997)
Rhim (2006)
Rhim (2006)
Wickland et al. (2012)
Trulleyová and Rulík (2004)
3
4
5
6
7
8
9
10
11
12
13
Batch
Lake Fred. Slotss
Reactor
Lake Esrum
Reactor
Sewage
Batch
Forest River
Batch
Rivers
Batch
Unpolluted river and sea water
Batch
Lake Lock Creran (temperate coastal area)
2 L glass amber bottle
Stream water
Continuous Bioreactor
Influent of a biological activated carbon pilot plant BAC reactor
Efluent of a biological activated carbon pilot plant BAC reactor
Stream water (Yukon River)
CO2 detection
14
Stream water (Yukon River)
CO2 detection
28 days (15°C)
Indigenous bacteria
2.7 / 17.3
7 ± 1 / 53 ± 6
Wickland et al. (2012)
15
Stream water (BC_Vitoria River)
CO2 detection
28 days (15°C)
Indigenous bacteria
2.1 / 9.6
6 ± 2 / 51 ± 9
Wickland et al. (2012)
16
Stream water (BC_Michel River)
CO2 detection
28 days (15°C)
Indigenous bacteria
3.4 / 10
3 ± 1 / 35 ± 11
Wickland et al. (2012)
17
18
Stream water (Porcupine River)
Surface waters (unfiltered samples)
CO2 detection
Batch
28 days (15°C)
10 days
3.3 / 13.8
4.98 / 6.66
5 ± 0.4 / 43 ± 7
37
Wickland et al. (2012)
Khan et al. (2005)
19
Surface waters
Indigenous bacteria
Commercial seed
MLSS - mixed liquor suspended
solids from activated sludge
4.98 / 6.66
44
Batch
3 hours
3 hours
10-30 days (20°C)
10-30 days (20°C)
10-30 days (20°C)
10-30 days (20°C)
150 days (14°C)
1.8 hours
15 minutes
15 minutes
28 days (5°C)
10 days
Khan et al. (2005)
3
IG01
IG02
IG03
IG04
IG05
IG06
Figure S 1: Picture of sampling sites located at Iguassu River.
4
60
100
IG 01
IG 02
BOD
COD
DOC
80
Concentration (mg/L)
Concentration (mg/L)
BOD
COD
DOC
40
20
60
40
20
0
140
100
BOD
COD
DOC
48
C
45
C
41
C
37
33
C
C
29
25
C
C
IG 04
BOD
COD
DOC
80
Concentration (mg/L)
Concentration (mg/L)
Field campaign
IG 03
120
21
17
Field campaign
C
C
9
13
C
C
C
4
48
C
45
41
C
C
37
C
33
C
29
C
25
21
C
C
17
9
13
C
C
C
C
4
0
100
80
60
60
40
40
20
20
C
48
C
45
C
41
C
37
C
33
48
C
45
C
41
C
37
C
33
C
29
C
25
C
21
C
17
C
13
C
9
48
C
45
41
C
C
37
33
C
C
29
25
C
C
21
C
C
C
17
0
13
0
9
20
4
40
20
Field campaign
C
29
60
C
Concentration (mg/L)
40
C
BOD
COD
DOC
80
60
4
IG 06
C
BOD
COD
DOC
C
Concentration (mg/L)
100
IG 05
80
C
25
Field campaign
Field campaign
100
C
21
C
17
C
13
C
4
C
48
C
45
C
41
C
37
C
33
C
29
C
25
C
21
C
17
C
C
13
9
C
4
C
9
0
0
Field campaign
Figure S 2: Variation of BOD, COD, and DOC concentration along the main river (IG01 is located at headwater
and IG06 is the most downstream site). Field campaign numerated from C1 to C48 (2005 to 2013).
5
60
50
50
40
40
DOC (mg/L)
BOD (mg/L)
60
30
20
10
30
20
10
0
0
IG01
IG02
IG03
IG04
IG05
IG06
IG01
IG02
12
100
10
80
8
DO (mg/L)
COD (mg/L)
120
60
IG04
IG05
IG06
IG05
IG06
6
40
4
20
2
0
0
IG01
IG02
IG03
IG04
IG05
IG01
IG06
IG02
IG03
IG04
Monitoring sites
Monitoring sites
30
3
20
2
PO4-P (mg/L)
NH3-N (mg/L)
IG03
Monitoring sites
Monitoring sites
10
0
1
0
IG01
IG02A
IG03
IG04
Monitoring sites
IG05
IG06
IG01
IG02A
IG03
IG04
IG05
IG06
Monitoring sites
Figure S 3: Box-plots showing the variability of BOD, COD, DOC, DO, NH3-N, and PO4-P concentration along
the main river (IG01 is located at headwater and IG06 is the most downstream site). Data considered from48
field campaign (2005 to 2013).
6
500
500
0.00
0.00
0.25
0.25
0.50
1.00
1.25
1.25
1.75
2.00
2.25
350
0.75
1.00
1.50
400
450
2.50
Emission (nm)
Emission (nm)
0.50
0.75
450
300
1.50
400
1.75
2.00
2.25
350
2.50
300
IG01_C42
250
IG02A_C42
250
250
300
350
400
450
500
250
300
Excitation (nm)
350
400
450
500
Excitation (nm)
500
500
0.00
0.00
0.25
0.25
0.50
0.50
0.75
1.00
1.25
1.25
1.75
2.00
2.25
350
0.75
1.00
1.50
400
450
2.50
Emission (nm)
Emission (nm)
450
300
1.50
400
1.75
2.00
2.25
350
2.50
300
IG03_C42
250
250
300
350
400
450
IG04_C42
250
500
250
300
Excitation (nm)
350
400
450
500
Excitation (nm)
500
500
0.00
0.00
0.25
0.25
0.50
0.50
450
0.75
1.00
1.25
1.25
1.50
400
1.75
2.00
2.25
350
0.75
1.00
2.50
Emission (nm)
Emission (nm)
450
1.50
400
1.75
2.00
2.25
350
2.50
300
300
IG05_C42
250
250
300
350
400
450
500
IG06_C42
250
250
Excitation (nm)
300
350
400
450
500
Excitation (nm)
Figure S 4: Example of three-dimensional excitation-emission matrices (EEMS) for all sites monitored along
main river (IG01 is located at headwater and IG06 is the most downstream site). Samples collected during field
campaign n. 42 (Nov/2012), Fluorescence intensities are in Raman units.
7
500
500
0.00
0.00
0.25
0.25
0.50
0.50
0.75
1.00
1.25
1.25
1.75
2.00
2.25
350
0.75
1.00
1.50
400
450
2.50
Emission (nm)
Emission (nm)
450
1.50
400
1.75
2.00
2.25
350
2.50
300
300
IG02_Day 0
IG02_Day 1
250
250
250
300
350
400
450
250
500
300
350
400
450
500
Excitation (nm)
Excitation (nm)
500
500
0.00
0.00
0.25
0.25
0.50
0.50
450
0.75
1.00
1.25
1.25
1.50
400
1.75
2.00
2.25
350
0.75
1.00
2.50
Emission (nm)
Emission (nm)
450
1.50
400
1.75
2.00
2.25
350
2.50
300
300
IG02_Day 3
IG02_Day 2
250
250
250
300
350
400
450
500
250
300
Excitation (nm)
350
400
450
500
Excitation (nm)
500
500
0.00
0.00
0.25
0.25
0.50
0.50
0.75
1.00
1.25
1.25
1.75
2.00
2.25
350
2.50
300
0.75
1.00
1.50
400
450
Emission (nm)
Emission (nm)
450
1.50
400
1.75
2.00
2.25
350
2.50
300
IG02_Day 4
250
250
300
350
400
Excitation (nm)
450
500
IG02_Day 5
250
250
300
350
400
450
500
Excitation (nm)
8
500
500
0.00
0.00
0.25
0.25
0.50
1.00
1.25
1.25
1.75
2.00
2.25
350
0.75
1.00
1.50
400
450
2.50
300
Emission (nm)
Emission (nm)
0.50
0.75
450
1.50
400
1.75
2.00
2.25
350
2.50
300
IG02_Day 6
250
IG02_Day 10
250
250
300
350
400
450
500
Excitation (nm)
250
300
350
400
450
500
Excitation (nm)
Figure S 5 Full set of three-dimensional excitation-emission matrices (EEMS) for site IG02. Batch incubations
were performed for samples collected during Dec/2013, considering open system (Experiment A). Incubation
time: 10 days. Fluorescence intensities are in Raman units.
9
2,0
2,0
1,0
IG01 - Exp. 3B
22/10
23/10
24/10
25/10
27/10
28/10
29/10
30/10
31/10
01/11
22/10
23/10
24/10
25/10
27/10
28/10
29/10
30/10
31/10
01/11
1,5
Absorptivity (a.u.)
Absorptivity (a.u.)
1,5
IG01 - Exp. 3A
0,5
0,0
200
1,0
0,5
300
0,0
200
400
Wavelength (nm)
300
2,0
2,0
IG02 - Exp. 3B
IG02 - Exp. 3A
Absorptivity (a.u.)
1,0
0,5
0,0
200
22/10
23/10
24/10
25/10
27/10
28/10
29/10
30/10
31/10
01/11
1,5
Absorptivity (a.u.)
22/10
23/10
24/10
25/10
27/10
28/10
29/10
30/10
31/10
01/11
1,5
1,0
0,5
300
0,0
200
400
Wavelength (nm)
300
2,0
IG05 - Exp. 3B
IG05 - Exp. 3A
1,0
22/10
23/10
24/10
25/10
27/10
28/10
29/10
30/10
31/10
01/11
1,5
Absorptivity (a.u.)
22/10
23/10
24/10
25/10
27/10
28/10
29/10
30/10
31/10
01/11
1,5
Absorptivity (a.u.)
400
Wavelength (nm)
2,0
0,5
0,0
200
400
Wavelength (nm)
1,0
0,5
300
400
Wavelength (nm)
0,0
200
300
400
Wavelength (nm)
Figure S 6: Variation of UV absorptivity for sites monitored at Iguassu River. Batch incubations were performed
for samples collected during Oct/2013, considering open system (Experiment A) and close system (Experiment
B). Incubation time: 10 days. Absorptivity is in arbitrary units.
10
2,0
2,0
IG01 - Exp. 4B
IG01 - Exp. 4A
Absorptivity (a.u.)
1,5
1,0
0,5
0,0
200
1,0
0,5
300
0,0
200
400
Wavelength (nm)
2,0
Absorptivity (a.u.)
2,0
0,5
0,5
300
2,0
0,0
200
400
400
2,0
IG05 - Exp. 4B
06/12
07/12
08/12
09/12
10/12
11/12
12/12
16/12
1,5
Absorptivity (a.u.)
06/12
07/12
08/12
09/12
10/12
11/12
12/12
16/12
1,0
300
Wavelength (nm)
IG05 - Exp. 4A
1,5
Absorptivity (a.u.)
06/12
07/12
08/12
09/12
10/12
11/12
12/12
16/12
1,0
Wavelength (nm)
0,5
0,0
200
400
IG02 - Exp. 4B
1,5
Absorptivity (a.u.)
06/12
07/12
08/12
09/12
10/12
11/12
12/12
16/12
1,0
300
Wavelength (nm)
IG02 - Exp. 4A
1,5
0,0
200
06/12
07/12
08/12
09/12
10/12
11/12
12/12
16/12
1,5
Absorptivity (a.u.)
06/12
07/12
08/12
09/12
10/12
11/12
12/12
16/12
1,0
0,5
300
400
Wavelength (nm)
0,0
200
300
400
Wavelength (nm)
Figure S 7: Variation of UV absorptivity for sites monitored at Iguassu River. Batch incubations were performed
for samples collected during Dec/2013, considering open system (Experiment A) and close system (Experiment
B). Incubation time: 10 days. Absorptivity is in arbitrary units.
11
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