Chemosphere, Vol. 39, No. 12, pp. 2007-2018. 1999 0 1999 Elwicr Science Ltd. Ail rights reserved 0045-6535/99/S - see front matter
PII: 50045-6535(99)ooo90-9
EVALUATION
OF LABORATORY-MADE
BIODEGRADABILITY
SLUDGE
FOR AN ANAEROBIC
TEST AND ITS USE FOR ASSESSMENT
‘Kazumi Kawahara*,
OF 13 CHEMICALS
‘Yoshikuni Yakabe, 2Tomohisa Ohide and 2Kenji Kida
1. Chemicals Assessment
Center, Chemicals Inspection & Testing Institute,
19-14 Chuo-machi, Kurume-shi, Fukuoka 830-0023, Japan e-mail:
[email protected] 2: Depertament
of Applied Chemistry & Biochemistry,
2-39-l Kurokami, Kumamoto-shi
Kumamoto University
860-8555, Japan
(Received in Germany 27 December 1998; accepted 11 March 1999)
ABSTRACT Laboratory-made
sludge for a biogas based anaerobic biodegradability
digested sludge from wastewater productions
of digested
test was prepared as an alternative for
treatment plants (WWTPs). Biodegradation
activities and background
sludge from various WWTPs were found to vary significantly
source, which adversely affected test reliability. Subsequently, and sludge washing were examined with the laboratory-made without washing was determined
test conditions
biodegraded nitriphenol
tests
sludge and a sludge concentration
to be most suitable. Under these conditions,
showed
that chemicals
were
completely inhibiting methane fermentation
highly toxic
as low as 20 mg/‘L. 0
1999
tests were
bacteria were evaluated. The
with -OH and -CHzOH
to methanogenic
of 1.Og-SSIL
biodegradability
radicals
and those with -Cl, -NO*, -NHz, -SO,H and -CHs had inhibited degradation and 2,4,6-trichlorophenol
on the
such as sludge concentration
conducted for 13 select chemicals and their relative toxicities to methanogenic results of biodegradability
depending
gas
bacteria,
were readily responses.
m-
with m-nitropheol
Elsevier Science Ltd. All rights reserved
Key words: anaerobic biodegradability, digested sludge, laboratory-made sludge, biogas
INTRODUCTION To evaluate the impacts of chemical contaminants interaction
with the environment),
biodegradability
on environmental
ecosystems
tests have been established
Most of these tests are designed to evaluate biodegradability 2007
and human health (via
by OECD and others [l]
of test chemicals under aerobic conditions and
thus are not suitable to assess responses that may occur in anaerobic environments. indicate that anaerobic environments, aerobic environments
under anaerobic
conditions
anaerobic conditions,
must also need to be evaluated. However, is lacking.
In order to evaluate
biodegradability
the biodegradabilities
have been proposed
in using this procedure.
sludge
biodegradability
biodegradability
data for chemicals of chemicals
[3-71. Digested
Due to diverse characteristics
under
sludge from a
of wastewater
among WWTPs, it is expected that variation in microbial composition
may negatively influence the validity of such tests. Shelton et al. reported digested
than
plant (WWTP) is normally used as the inoculum for such tests; however, there are
involved
operating conditions
of contaminants
to assess the fate of chemicals in the environment,
various screening methodologies
waste water treatment some uncertainties
i.e. sediment, are subject to higher concentrations
[Z]. Therefore,
under anaerobic conditions
Many monitoring surveys
from
different
WWTPs
varied
significantly
[8].
and its activity
that biodegradation
On the
other
and
hand,
activity of standardized
test method for anaerobic conditions has been published by IS0 [9]. This protocol allows the
use of both digested sludge of WWTPs or laboratory-made
sludge. However, methods for cultivating the test
sludge were not defined. In the present study, in order to compare biodegradation were conducted
activity of digested sludge between WWTPs, tests
using phenol and benzyl alcohol as substrates.
Additionally,
laboratory-made
sludge was
prepared using a digested sludge seed with mixed sludge (mixture of primary sludge and thickened surplus sludge) substrate. Biodegradability the laboratory-made biodegradation Furthermore,
tests of 13 chemicals having different structure were then performed using
sludge and effects of substitute position on biodegradability
was calculated
by measuring
toxicity assays were conducted
responses to the biodegradability
volume of gas produced to tinther characterize
were investigated. Extent of
with a pressure
transducer
[6].
those chemicals that showed inhibited
tests.
MATERIALS
AND METHODS
Sludge Collection times and properties of the digested sludges used for experiments Digested sludges from the secondary
are shown in Table 1.
digestion tank of each WWTP was collected in 2L plastic containers
without headspace to limit exposure to air and brought to the laboratory. The collected sludges were then stirred under anaerobic condition for 3 days at 37 % to reduce the total organic carbon (TOC) content prior to use in biodegradability The laboratory-made digested
tests.
sludge was prepared by draw and fill method at 37 “C for more than 60 days using
sludge from Kumamoto
East WWTP seed and mixed sludge (mixture
of primary sludge and
thickened surplus sludge) substrate. The AUh4B used for the inhibition tests was continuously cultivated with the acetate amended synthetic wastewater
in our laboratory.
2009 Table 1 Collection times and properties of digested sludges from WWTPs and laboratory-made Kurume
Kumamoto
Central
Southern
WWTP
WWTP
Kumamoto
sludge
Laboratory-
Origin East WWTP
made sludge
Time of collection
8, July, 97
16, July, 97
16, July, 97
-
SS (g/l)
17.4
14.3
38.1
10.6
PH (-)
8.2
8.0
5.4
74
TOC (msfl)
477.5
108.0
1331
158 6
IC (mg/l)
553.5
302.5
c10.0
223.2
vFA (mg/l)
Cl.0
Cl.0
2606.8
<1 0
SS, suspended solid; TOC, total organic carbon; IC, inorganic carbon; VFA, volatile fatty acids
Chemicals I-hexanol,
benzyl alcohol, 2,4,6-trichlorophenol,
aniline, toluene, phenol and m-nitrophenol
from Nacalai Tesque Inc. Kyoto, Japan. 5-hexene-l-01
were obtained
was obtained from Aldrich Chemical Company, IJSA.
n- and iso- valeric acid were obtained from Tokyo Kasei Kogyo Co. Ltd, Tokyo, Japan. Benzene sulfonic acid, triethylamine
and trimethylchloromethane
were obtained from Wako Pure Chemical Industries
Ltd,
Osaka, Japan. All chemicals were commercially available and of analytical grade.
MEDIUM The component
inorganic medium used for the biodegradability
test of the above test chemicals are depicted
in Table 2.
Table 2 Inorganic medium for biodegradability test (g/L) KH2po4
0.27
Na&IPOe. 12HzO
1.12
NHXI
0.53
CaC12
0.075
MgCI2
01
FeC12
02
Na2S
01
Resazurin 0.001 (Redox indicator)
2010 Cultivation of laboratory-made
sludge
Cultivation was carried out by the till and draw method using a 3L completely Marubishi Co. Ltd. Type; MD-500)
stirred tank reactor (CSTR,
as shown in Fig. 1. The seed for cultivation was middle temperature
(37 “C) digested sludge from Kumamoto
East WWTP and a mixed sludge, 1: 1 mixture of primary sludge
and thickened surplus sludge, from the same WWTP was used as substrate.
During cultivation, the sludge
was stirred at 300 rotations per minute by a magnetic stirrer at 37 “C. 300 mL of the sludge solution was removed daily and replaced with an equal amount of the substrate sludge. The biogas produced during the cultivation was collected and quantified in a gas-trapping
Substrate
tank.
-b
-
Fig. 1 Cultivation apparatus for laboratory-made
Performing
biodegradability
sludge
test
After filling digested sludge into a bottle that had been previously purged with nitrogen, the bottle was placed in a glove box (SANYO Co. Ltd. type: MIP-1025). of sludge was calculated and transferred
Based on suspended
solids (SS), the necessary quantity
to a centrifuge tube from the bottle, balanced and capped. Then the
tube was removed from the glove box, centrifuged at 7000 x g at 25 “C for 10 minutes. The tube was then returned
to the glove box, the supernatant
oxygen-free
removed,
and the sludge centrifugate
resuspended
into the
inorganic medium. For the removal of dissolved oxygen from the inorganic medium, a mixture of
nitrogen and carbon dioxide (8:2) gasses was passed through the deoxidization
copper column at 350 “C
and purged for 20 minutes into the medium. Subsequently, 90 mL of fresh oxygen-free
inorganic medium, 10
ml of sludge suspension and 10 mgC of each test chemical were added to a 125 mL serum bottle and tightly capped with a butyl rubber stopper and aluminum cap. The bottle was then removed from the glove box and placed in a water bath at 37 “C. Following
one-hour
of stirring, excess pressure
in the headspace
was
released by inserting a syringe needle. Serum bottles were then moved to an incubator and biodegradability tests were started at 37 !n for 8 weeks. During the cultivation measured at least once a week using the pressure transducer
period, gas volume of produced
was
(Toyoda Kouki Co. Ltd Type: AA6150) and
2011 extent biodegradability
of chemicals were determined by net gas production
(NGP). During the cultivation,
Bottles containing culture solution that changed to red (indicative of oxygen intrusion) were discarded. For examination of the effect of washing the sludge by centrifuge on gas production, 20 mL of test solution in 25 mL serum bottles was applied. The concentration
a smaller test scale using
of sludge was set at lg-SSL
for all tests. For the assessment of sludge quantity, sludge without washing at 0.1 to 5.0 g-S% the biodegradability
test as described
above. Both examination
results were evaluated by NGP and background gas production
of test conditions
was used for
and washing the sludge,
(BGP).
Toxicity test of persistent substances The inhibition of persistent
substances
on the activity of AUMB was carried out using the simple-type
bacterium activity meter (Fig. 2) as follows. First, 0.2 mL of 298.6 mg/L sodium acetate was added to a 15 mL serum bottle to a final concentration
of 70 mML
Next, test chemicals were added to final concentrations
of 50, 100 or 200 mg/L (20 mg/L was adapted for m-nitrophenol).
A 15 mm stirrer bar was added to each
serum bottle which was then tightly capped with a butyl rubber stopper and screw cap. Nitrogen purged for one minute into the headspace with a syringe needle. After connecting a gas-trapping
gas was
tank (3 mL
scalpel pipette), the bottles were placed in a water bath at 37 “C and let? for l-2 hours. 10 mL of AUh4B fluid was collected from the cultivation tank with a syringe, quickly added it to the serum bottle and the top level of the saturated
sodium chloride solution was adjusted to a zero reference
stirring at 100 rotation
per minutes, the gas production
relative gas production
volume in the presence and absence of the test substances
mark. After one hour of
volume over a time course was measured.
inhibitory effect of the chemicals on the AUMB was determined
Stirrer Fig. 2 Simple-type bacterium activity meter
was compared
The
and the
2012
Analysis methods The pHs of samples were measured
by a pH meter (TOA CO. Ltd. type: 3OSHM)
TOC and IC were
measured by a TOC analyzer (Shimadzu Co. Ltd. type: TOC-500). For carbon analysis, test solutions were centrifuged
at 10,600g for 10 minutes (Tommy Seiko CO. Ltd. type MCX-I 50; rotor type TMA-3) and
diluted to a TC concentration was measured
as follows.
below 300 mg/L with distilled water. SS concentration
An aliquot of the sludge was centrifitged
at 3,OOOxg for 10 minutes and the
precipitate was washed into a porcelain dish of known weight. AtIer evaporating precipitate
in a water bath, the dish was moved to an isothermal
MOV-102)
and dried at 100 “C overnight.
room temperature
of the digested sludge
incubator
excess moisture from the
(Sanyo Electronic
Ltd. type’
The dried sample was then moved to a desiccator,
cooled to
and weighed.
Results and discussion
Biodegraakbilig test using anaerobic digested sIudgefrom dljjferent WWTPs When using unaltered digested sludge from WWTPs for anaerobic biodegradability or collection biodegradation
time may have a significant potential
influence on the test results. Therefore,
among different suldges, digested
WWTPs and biodegradability
test, operating conditions
sludge was collected
in order to compare
from the three different
tests of phenol and benzyl alcohol (based on gas production)
were carried out
(Fig. 3). At the same time, gas production of a blank test was also measured.
a) Kumamoto 25
east WWTP
b) Kumamoto
east WWTP, 2-week strage
-
0
10
20
30
40
50
0
60
10
20
30
c)
0
d) Kumamoto
Kurume Central WWTP
A
10
20
-1AJ..__
50
60
Southern WWTP
1__L.
30
40
50
60
0
10
20
Day
Fig. 3 Gas production
40
Day
Day
30
______
40
50
1
60
Day
curve of phenol and benzyl alcohol by digested sludge from different WWTPs (-3- , control; -A-, benzyl alcohol; -X -, phenol)
2013 Digested
sludge from Kumamoto
degradation
Eastern and Kurume Central WWTPs had relatively low BGP and the
curve plateaued within 2-4 weeks. However, the biodegradation
lag periods for the two sludges. Conversely, the sludge from Kumamoto high background
gas production
and biodegradation
but did not reach a plateau within the 8-week Kumamoto
curve of phenol had different
Southern WWTP had a relatively
of phenol was observed following a 6-week lag period cultivation
Southern WWTP may be caused by unsuitable
period. operating
This phenomenon conditions
suggested from the low pH value of 5.4 (see Table 1) and the high concentration
for anaerobic
Considering
concluded
not
biodegradation
Examination
using
digested
sludges
of test conditions by laboratory-made
sludges from WWTPs.
concentration
from
WWTPs
was
suitable
these results, it was for biogas
based
test and storage of sludge had better avoid if it is possible.
The applicability of laboratory-made digested
directly
digestion,
of volatile fatty acids Afier
a 2-week storage at 4 “C, lag period of phenol became long somewhat. that
by sludge from
sludge for the biodegradability
test was examined as an alternative to
The effect and need for washing the sludge and recommended
were also evaluated.
bottles using laboratory-made
sludge
Biodegradability
test were performed
sludge. As shown in Fig. 4, biodegradation
sludge
using phenol in 25 mL serum
of phenol was observed following a
IO-15 days of lag period and reached a plateau within 20 days at any number of sludge washings.
Gas
production could not be compared directly to the results of Fig. 3 because the sizes of bottles used for testing were different, the biodegradation
activity of laboratory-made
sludge was not inferior to that of digested
sludges from WWTPs (see Fig 3). From this result, it was found that laboratory
made sludge instead of
digested sludge from WWTPs can be used as an inoculum for biogas based anaerobic biodegradation
_--_-. 0
10
20
_I
30
L.
40
50
__~_!
-L-
60
0
10
Bay
1; -a-,
_~I__
20
1
30
40
~~
50
60
Day
Fig. 4 Effect of washings on NGP
Washing number = -0-,
_~~
test
2 and -Cl-, 3
Fig.5 Effect of washings on BGP
Washing number = -3-,
1; -A-, 2 and -O-, 3
Effects of sludge washings were identified by NGP and BGP curves. The number of washings had little effect on gas production
as shown in Fig. 4 and 5. With the biodegradation
of phenol, almost the same NGP curve
2014 was observed with both washed and un-washed sludge, besides, BGPs were also not different. This may have been due to the laboratory-made The laboratory-made
sludge and digested sludge from Kumamoto
TOCX, respectively significantly
(see Table l), and therefore gas production
decreased.
biodegradation
sludge fluid containing lower TOC than the digested sludges from WWTP. East WWTP contained
159 and 478 mg-
from the sludge fluid itself probably was not
In the same time, sludge washing was not making negative effect on anaerobic
activity at least laboratory-made
sludge. Also, it was found that the laboratory-made
sludge
did not experience an autolysis easily when other energy sources were consumed.
Fig. 6 and 7 show the time course of NGP and BGP curve from phenol biodegrading concentrations
5
42 = 1 33 40
of laboratory-made
with respect to different
sludge
zic
a
32 =1
0 ,/-
k 0
10
20
30
40
50
60
0
10
20
DW
5
30
40
50
60
0
10
20
5r
d
30
40
50
60
40
50
60
Day
Day
e
42 =1
OU’
33 4 0 ir-0
10
20
30
40
50
60
0
10
20
DW
30
40
50
60
0
10
0
10
20
30
20
30 DW
Day
40
50
60
Day
Fig. 6 NGP curve of phenol at different sludge concentration Sludge concentration
At all sludge concentrations, with concentration following
= a, 0.lg& b, 0.3g/L; c, O.&/L; d, O.Sg/L; e, 1.Og/L; f, 3.OgL and g, S.OgL
biodegradation
of phenol was observed, however NGP shows different patterns
of the sludge. At 0.1, 0.3, and 0.5 g/L of sludge concentration,
NGP curves rose gently
lo-20 days of lag period and reached plateau about the 40th day. At sludge concentration
of 0.8
2015 and 1.0, NGP curves rose promptly and reached plateau in the 20th day. On the other hand, more than 3 0 g/L of sludge concentration, together
with increase
in sludge
sensitivity of biodegradation to be 0.8 to 1.O g-S%. influenced negatively considering
NGP curves winded due to excess BGP. As for BGP curves, BGP increased concentration,
From the above
and low background
When the sludge concentration
gas production, was determined
was higher than this level, test reliability may be
and when the sludge concentration
sludge. The concentration
considering
levels, the optimum sludge concentration
the above results, following biodegradability
laboratory-made
results,
was lower, biodegradation tests were conducted
was prolonged.
With
using 1 .O g/L of un-washed
of test chemicals was set at 100 mg-TOUL
for all tests except
the toxicity test.
-0.3g/L ~-0,59/L *0.8g/L
-
1 .Og/L
--A-3.Og/L --m-5.09/L
Fig. 7 BGP curve at different sludge concentration
The biodegradability From the results biodegradable,
tests of 13 selected chemicals of NGP
curves,
13 tested
chemicals
were
divided
into three
classes,
e.g. readily
persistent and inhibitory as summarized in Table 3. Fig. 8 shows NGP curves for some typical
readily biodegradable
chemicals. The NGP curves of all readily biodegradable
two to three weeks, but gas production
chemicals reached a plateau in
of the persistent chemicals showed almost the same response as that
of the control. Some chemicals had an inhibitory effect on the sludge and gas production blank. Although phenol was classified as readily biodegradable WWTPs) the rate of its initial gas production
(as with results using digested sludge from
was lower than other readily biodegradable
respect to chemical structure, it was found that among aliphatic compounds, acids were promptly biodegraded reported that biodegradability number of unsaturated
and halogenated
of hydrocarbons
bonds; however,
under methanogenic
chemicals. With
alcohol derivatives and organic
alkanes and trimethylchloromethane
5-hexene-l-01
was less than the
were not. Schink
conditions decreased with an increase in
(an unsaturated
alcohol) was easily biodegraded
well as the saturated alcohol 1-hexanol [IO] For seven tested aromatic compounds
as
(phenol, aniline, toluene,
2016 benzyl alcohol, m-nitrophenol,
2,4,6-trichlorophenol
and benzene sulfonic acid), the effect of each substitute
was classified into two categories as shown in Table 4. When the effect of different substitutes is considered, the results can be roughly classified as either positive or negative with respect to anaerobic biodegradability.
-+Benzylalcohol
15
i*HHaxanol
1
10
0
20
30
40
50
60
Day
Fig. 8 NGP curves of readily biodegradable
Table 3 Biodegradability
chemicals
of 13 chemicals
Readily biodegradable Persistent chemicals
Strongly inhibited chemicals
chemicals 1-hexanol
Trimethylchloromethane
5-hexene- l-01
Triethyamine
Benzyl alcohol
Aniline
m-nitrophenol Toluene n- and iso-valeric acid
2,4,6-trichlorophenol
Phenol
Benzene sulphonic acid
Table 4 Effect of benzene substitute on biodegradability Positive
Negative
I -Cl,
-NO*, -NHz,
-CHzOH, -OH -SOjH, -CHx
According -COOH,
to a report described by Kameya et al., benzene compounds -OH and -CH2CH(NHz)COOH
persistence in an anaerobic biodegradation
were easily biodegraded test[ 1 I].
substituted
with -CHzOH, -CHO,
and those -N02, -NH* and -SO3 were
2017 Toxicity test using acetate utilizing methanogenic Toxicity tests ,of m-nitrophenol,
bacteria (AVMB)
2,4,6-trichlorophenol,
aniline, triethylamine,
toluene and benzene
sulfonic
acid that are persistent or inhibitory were performed. Fig. 9 shows the relative gas production rate normalized to controls. Gas production
rate declined with an increase in concentration
was extremely inhibitory to AUMB, without any gas production 2,4,6-trichlorophenol
for all chemicals. m-nitrophenol
even at a low concentration
of 20 mgC/L
also had a strong effect to AUMB by completely inhibiting at a concentration
mgC/L. O’Connor et al as well reported that nitrophenols
and chlorophenols
of 200
showed inhibition to anaerobic
bacterium as in this study [ 121
1.4 9
E
i I
-O-m-nitrophenol
1.2
-A-
:, 1 .z 0, m 0.8 E P m 0.6 S .;
~Xl-aniline (+triethylamine I -a-toluene
0.4
5
n” 0.2
2,4.6trichlorophenol
:
)-&-benzene sulphonoic 1 L ~~actd ~ ~~~ _~~~~~
)
0
“A 0
50 Concentration
100 150 of test chemical
200
(mg/L)
Fig.9 Inhibition test results of persistent chemicals
Acknowledgment This study was funded by the Agency of Industrial Science & Technology, Ministry of International
Trade &
Industry, Japan. We thank Dr. S. Morimura for technical assistance.
References
[II Environmental Health and Safety Division, Chemicals in the environment, Environment Agency Japan (1997).
PI
OECD, Guidelines for Testing Chemicals. OECD, Paris (1981).
[31 N S. Battersby and V. Wtlson, Survey of the anaerobic biodegradation in digested sludge. Appl. Environ. Microbid.
potentials of organic chemicals
55, 433-439 (1989).
[41 R. R. Birch, R. C. Biver, W. E. Gledhill, U. Pagga, J. Steber, H. Reust, and W. J. Bontinck, Screening of chemicals for anaerobic biodegradability.
PI
Chemosphere 19, 1527-I 550 (1989)
W. F. Owen, D. C. Stucky, J. B. Hesly Jr, L Y Young, and P L. McCarty, Bioassay for monitoring biochemical methane potential and anaerobic toxicity. Wat.Res. 13, 46492
(1979).
2018 [6] U. Pagga, and D. B. Beimborn, Anaerobic biodegradation test for organic compounds. Chemosphere 27, 1499-1509 (1993). [7] U. J. Strotmann, F. Eismann, B. Hauth and W. R. Bias, An integrated test strategy for the assessment of anaerobic biodegradability of waste water. Chemosphere 26, 224 I-2254 ( 1993). [8] D. R. Shelton and J. M. Tiedje, General Method for Determining Anaerobic Biodegradation Potential. Appl. Environ. Microbial. 47, 850-857 (1984). [9] ISO. Water quality - Evaluation of the “ultimate” anaerobic biodegradability of organic compounds in digested sludge - Method by measurement of the biogas production. International Organization for Standardization. Draft international standard, ISO/DIS 11734. (1994). [lo] B. Schink, Degradation of unsaturated hydrocarbons by methanogenic enrichment cultures. FEMS Microbiology Ecology 31,69-77 (1985). [l l] T. Kameya, T. Murayama, K. Urano and M. Kitano, Biodegradation ranks of priority
organic
compounds under anaerobic conditions. Sci. Total Environ. 170, 43-5 1 (1995).
[12] 0. A. O’Connor and L. Y Young, Effects of six different functional groups and their positions on the bacterial metabolism of monosubstituted
1419-1428 (1996).
phenois under anaerobic conditions. Environ. Sci. Technol. 30