Evaluation of laboratory-made sludge for an anaerobic biodegradability test and its use for assessment of 13 chemicals

Evaluation of laboratory-made sludge for an anaerobic biodegradability test and its use for assessment of 13 chemicals

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...

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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.

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