DMSO Reduction Rate in Activated Sewage Sludge

Research Articles

Determination of Microbial Activity in Activated Sewage Sludge by Dimethyl Sulphoxide Reduction - Evaluation of Method and Application Martin Sklorz, Jiirgen Binert Chair of Ecological Chemistry and Geochemistry, University of Bayreuth, Universit~itsstrafle30, D-95440 Bayreuth, Germany

Abstract A method was developed to determine the dimethyl sulphoxide (DMSO) reduction rate in activated sewage sludge at nearly natural conditions. Linearity of microbially produced dimethyl sulphide with incubation time and sample size was shown. Apart from a fast, sensitive and highly reproducible automatic analysis of dimethyl sulphide, simultaneous determination of mineralisation, respiration and phenol degradation rates was possible. The DMSO reduction rate of samplestaken from a municipal sewage plant ranged between 2 and 3 amol/(g dry matter 9 h), respiration and mineralisation rates between 30 and 80 pmol/(g.h). Added 13Ct-phenol was completely degradated after 96 h of incubation. A half-life of 14 h was calculated assuming first order decay. Dose response curves were obtained by incubating samples for 2, 6, 25, and 96 hours after addition of pentachlorophenol. At an incubation time of 6 h, the ECso values ranged from 20 mg/L (DMSO reduction) to 30 mg/L (phenol degradation) up to 180 mg/L (respiration and mineralisation). Increasing the incubation time to 96 h resulted in a lower ECs0 of 9 mg/L for DMSO reduction, whereas it increased to 500 mg/L for respiration and mineralisation.

1

Introduction

Potential degradation of organic pollutants in activated sewage plants depends on microbial activity. The degradation efficiency itself is influenced by physico-chemical parameters of the incoming water, such as: flow, residence time, temperature, content of oxygen, pH, composition of nutrients and concentration of toxic substances. Several methods are used to evaluate the acitivty of sewage sludge. Most of them are based on the determination of specific microbial processes, e.g. the dehydrogenase activity [2], or the respiration activity, such as the Sapromat-test [4, 5] and the BASF-Toximeter [6], or on measurement of the adenosine triphosphate content [3]. A new approach for the determination of bioactivity uses microbial reduction of dimethyl sulphoxide (DMSO) to dimethyl sulphide (DMS). The method was primarily developed for soils and soil aggregates by AI.EF and KLErNER [1]. Later it was applied to topsoils and humic samples [7], wastewater [8], and samples from aerated wastewater basins [9]. It this paper, the method of ALEF and KLEINER [1] was adapted to allow the determination of DMSO reduction rate in the activated sewage sludge of a municipal wastewater treatment plant. There were two main questions:

140

1. How can the reduction rate be measured under conditions as natural as possible and what are the critical methodological parameters? 2. Does the reduction rate of DMSO correlate with the respiration, the mineralisation and the transformation rate of organic pollutants, especially when microbial activity is reduced by addition of toxic substances? In the first part, the improved method for the determination of the DMSO reduction rate, its advantages and errors are described. In the second part, activated sewage sludge is poisoned with pentachlorophenol (PCP) and dose response curves were recorded for incubation times between 2 and 96 hours. The reduction rate of DMSO, oxygen consumption, and CO2 production were measured. Furthermore, transformation and mineralisation rates of added 13C6phenol were determined to evaluate the biodegradation power.

2

Principle of the Method

The determination of the DMSO reduction rate exploits the common microbial ability to reduce DMSO to DMS, proven for nearly 150 species of bacteria and fungi [1]. Many of them were identified in activated sewage sludge [10]. The co-metabolic reduction of DMSO is carried out by various enzymes under consumption of hydrogen reduction equivalents (NADH 2 or NADPH2) [11, 12]: H3C -

S -

CH 3 + 2e-

+ 2 H + --, H 3 C

-

S -

CH 3 +

H20

II O

The educt (DMSO) is a weakly toxic, high boiling, water miscible liquid. The product (DMS) is an apolar substance with a boiling point of 36.5 ~ [14]. According to its high gas/water partition ratio, it will disperse into the gas phase above the saml~le. The gaseous concentration of DMS is easily determined by injecting a headspace aliquot into a gas chromatograph. Possible abiotic sources of DMS might be the disproportionation of DMSO into sulphide and sulphone [13] and abiotic reduction. As discussed later, these reactions proved to be of minor importance under the chosen conditions. 3 3.1

Experimental Materials and instrumentation

Dimethyl sulphoxide (DMSO), perdeuterated dimethyl sulphide (Dt-DMS), dimethyl sulphide (DMS), phenol, and perdeuterated phenol (Dt-phenol) were purchased from

ESPR-Environ. Sci. & Pollut. Res. 1 (3) 140-145 (1994) 9 ecomedpublishers, D-86899 Landsberg,Germany

Research Articles

DMSO Reduction Rate in Activated Sewage Sludge

Aldrich-Chemie GmbH with a purity of more than 99 %. Pentachlorophenol (PCP) puriss, was obtained from Fluka Chemie AG, carbon-13 labeled phenol (13C6-phenol) from Promochem (purity 99 %, a water content of 10 +_ 5 % was determined by gas chromatography). All other reagents were purchased from Merck. The DMSO was stripped with dry nitrogen directly before use to eliminate traces of DMS. 21 mL vials, sealed with gas tight teflon coated rubber septa were used for incubation of the activated sewage samples. They were analyzed by a headspace autosampler coupled with a GC/MSD. The instrumental parameters are given in Table 1. The ions detected by the MSD and some important remarks, are listed in Table 2. Table 1: Parameters for the headspace autosampler and the GC Gas chromatograph HP 5890 He, 10 kPa headpressure Rtx 100 (Restek) 30 m length 0.32 mm i.d. 1.5/~m film thickness

Carrier gas Column

Oven program for determination of DMS phenol

isotherm 45 ~

2 min isotherm 110 ~ 10 K/min to 150 ~ 2 min final time

Table 2: Substances and ions measured by the quadruopole mass filter Ouadrupole mass filter HP 5970 Mode: single ion monitoring m/z

substance

34

oxygen

40

argon

remark detected as 180160 to reduce

excessive signal strength

45 46

used as internal standard for oxygen and carbon dioxide

13C-carbon dioxide 12C-carbon dioxide detected as 12C180160 to reduce

excessive signal strength 47 5O

DMS De-DMS

94 99

phenol Ds-phenol

100

3.2

13C6-phenol

[M - 15] + used as internal standard for DMS, [M - 18] + [M'] + used as internal standard for phenol and ~3C6-phenoi, [ M ' ] + [M" ] +

Development of the method

The aim of the experiments was to determine the DMSO reduction rate under natural conditions. Some of the parameters influencing the reduction rate were fixed, those which might be critical were optimized. Temperature, pH, concentration of suspended solid matter (dry weight), nutrient and chemical composition of the activated sewage sludge were not changed. Sample size was chosen between ESPR-Environ, Sci. & Pollut. Res. 1 (3) 1994

2 mL to 10 mL. The concentration of added DMSO, adaptation and incubation time were optimized; thereafter, the linearity of DMSO reduction with time and amount of biomass per vial was shown. 3.2.1

Procedure

After sampling, activated sludge from the municipal sewage treatment plant of Bayreuth (FRG) was immediately transported to the laboratory and transferred into the vials under stirring. During the whole experiment, the vials were shaken and thermostated at actual sampling temperature (14.3 ~ - 20.1 ~ Preliminary experiments had shown that the sample volume should exceed 2 mL, and the vials had to be adapted for more than 2 hours to obtain reproducible results. After adaptation, the vials were closed and a solution of Ds-DMS in DMSO was added to the activated sewage sludge. D6-DMS was used as internal standard for quantification of DMS produced by microorganisms. A combined addition of Ds-DMS and DMSO simplified handling and was proven to be most reproducible. The high solvation power of DMSO was additionally used when phenol and PCP were added in the experiments described in the application section below. After incubation times ranging from 10 minutes to 96 hours, microbial reduction was stopped by addition of 100 pl of a 2 N sodium hydroxide solution to reach pH 13. In later experiments, where CO 2 production was measured, the reduction was stopped with 1 ml saturated barium hydroxide solution, filtered by a 0.2/lm syringe filter directly before addition. This solution contained no detectable carbonate as proven by gas chromatography after addition of phosphoric acid. For determination of DMS and its internal standard (D6-DMS), the vials were transferred into a headspace autosampler and 1 mL of the gas phase was injected after equilibration for 1 hour at 35 ~ Abiotic production of DMS was checked by analyzing samples, which were autoclaved or sterilized with alkali before addition of DMSO and following incubation. 3.3

Application - Influence of pentachlorophenol on the

bioactivity of activated sewage sludge Activated sewage sludge was spiked with PCP to establish dose response curves between 0 and 3 000 mg/L. For a first set (12 samples at 4 mL, in triplicate, incubated for 2 h, 6 h and 25 h), DMSO reduction, oxygen consumption, and CO 2 production were determined. A second set (15 samples at 2 mL) was incubated without repetition for 6 h and in triplicate for 96 h. 13C6-phenol was added to these samples to evaluate the influence of PCP on metabolic activity. The disappearence of 13C6-phenol and the release of 13CO 2 w e r e measured in addition to the parameters listed above. The samples were taken from the municipal sludge plant of Bayreuth on 23 Sep 93 and 5 Oct 93. Dry weight, pH, and temperature of the activated sewage sludge were 5.1 g/L, 7.24 and 20.1 ~ resp. for the first set and 4.85 g/L, 7.06 and 19.4 ~ resp. for the second.

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DMSO Reduction Rate in Activated Sewage Sludge

According to the description of the method given above, D6-DMS, 13C6-phenol and PCP dissolved in DMSO were added to the activated sewage sludge after adaptation for 3 hours. The concentration of DMSO was 5 % (vol/vol) and that of 13C6-phenol was about 20 mg/L. Oxygen and its internal standard, argon, were determined in the same run as DMS and D6-DMS (headspace bath temperature: 35 ~ Subsequently, D6-phenol was added as internal standard for 13C~-phenol and the samples were acidified with phosphoric acid, to displace phenol and carbon dioxide into the gas phase. The vials were equilibrated for 1 h at 95 ~ and CO2, 13CO2, D6-phenol and 13C6-phenol were measured by a second injection. Calibration of DMS was carried out by analyzing sterile samples (addition of Ba(OH) 2 before D6-DMS/DMSO solution) and sterile samples spiked with DMS (0.146/amol/vial and 0.731/~mol/vial, respectively); CO2 and 13CO2 were calibrated by spiking with 10/~L and 50/aL resp. of a NazCO3 solution (1 mol/L). Oxygen was quantified in relation to sterile samples, assuming contents of 150 and 170/amol Oz/vial for sample volumes of 4 and 2 mL.

4

Results and Discussion

4.1 Evaluation of the method The method of AI.EF and KLEINER[1] w a s adapted for the determination of DMSO reduction rate in activated sewage sludge. DMS production is a result of microbial activity. There was no detectable DMS without addition of DMSO to the activated sewage sludge. Abiotic reduction of DMSO to DMS as well as the disproportionation of DMSO in alkaline media were negligible at the chosen conditions. However, abiotic production of DMS was important in acidified (pH 2) activated sewage sludge: The amount of DMS increased by one to three orders of magnitude when the sample was heated to 95 aC. Because this high equilibration temperature was needed for determination of phenol by headspace gas chromatography, DMS had to be determined prior to phenol. Terminating the bioreactions by alkali allowed easy handling and automatic sampling. The DMS production by microorganisms as well as the mineralisation and the respiration stopped immediately and completely after alkalizing to pH 13. The results did not change when storing the samples up to 2 weeks. DMS production is independent of DMSO concentrations between 2 and 10 %. As shown in Fig. 1, the reduction rate of DMSO increased with increasing DMSO concentration up to 1.5 % (vol/vol). Inhibition of the DMS production was observed at DMSO concentrations of more than 15 % (vol/vol). Between 2 and 10 % (vol/vol), substrate saturation, a prerequisite for a time independent DMSO reduction rate, is reached and the reduction rate is independent of the DMSO concentration. For all following experiments, a concentration of 5 % (vol/vol) was chosen. DMS production is linear with sample volume and incubation time. Activated sewage sludge (0 to 10 mL) was diluted to a final volume of 10 mL, by addition of sterile filtered

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Research Articles

Rel. response of OMS in 7.

,~176 1 5O

J

o~

i

i

i

,

i

i

i

5

i

I

i

i

i

i

10

~

i

i

1,5

i

i

j

i

i

i

~

i

~

i

i

i

i

~

i

i

20 25 30 Conc. of DMSO in X ( v o l / v o l )

Fig. 1: DMS production as a function of D M S O concentration, mean values and standard deviation (dashed lines), n = 3 samples

sample. The obtained linearity of the DMSO reduction with the amount of active biomass is shown in Fig. 2. To examine the linearity of DMSO reduction and incubation time, 2 mL activated sewage sludge were incubated for 0 to 4 hours (-~ Fig. 3). The response was linear, even when the incubation time was prolonged to 96 hours. A further increase of incubation time may lead to a non-linear response, caused by toxic metabolites and a deficiency of oxygen and substrate. Rel. response of DMS in X

0

~ ' ~ ' ~ l l ~ f ~ l l l l l

0

IIrl~l

2

,

I

I ~ l l l f l

4

6

lilt

I L l l l l l

8

q l l l l

10

Living activated sludge in ml

Fig. 2: Linearity of the D M S O reduction with the volume of active biomass per vial, m e a n values and standard deviation, n = 3 samples Rel. response

of OM$ in X

0

1

2

3 4 incubation time in hours

Fig. 3: Linearity of the DMSO reduction with the incubation time, mean values and standard deviation, n = 3 samples

ESPR-Environ. Sci. & Pollut. Res. 1 (3) 1994

Research Articles

DMSO Reduction Rate in Activated Sewage Sludge

The method enabled a reproducible, sensitive, and correct determination of the DMSO reduction rate. The standard deviation of the DMSO reduction rate calculated for 6 samples taken simultaneously was lower than 5 %. Detection limits for DMS were below 10 nmol/vial, corresponding to a reduction rate of 200 nmol/(g" h) (2 mL activated sewage sludge, dry weight of 5 g / L , incubation time of 6 hours). The limit can be lowered by a factor of ten by using a larger sample volume or a longer incubation time. The use of Ds-DMS as internal standard eliminated errors caused by adsorption to solid matter and by variation in gas/ liquid partition of DMS. Furthermore, scattering because of instrumental variation or leaking septa were minimized, and the possibility of replicate injections was possible. One disadvantage is the need for a more expensive mass selective detector (MSD) for the determination of the isotopically labeled internal standard. Compared with the FID used by ALEFand KLEINER [1], the sensitivity decreased, but it was still sufficient for analyses of activated sewage sludge. On the other hand, the MSD exhibited an increased selectivity and the determination of oxygen and DMS (in alkaline media), as well as of CO 2 and phenol (in acidified media) in the same vial became possible. 4.2

Determination of microbial activity

DMSO reduction, mineralisation and respiration rate of the activated sewage sludge samples are listed in Table 3. Table 3: Comparison of measured values for bioactivity in activated

might be a possible explanation for this decrease, but more probably it is caused by a lack of nutrients and organic substrate. As expected, even the highest respiration rates of (77 +_20)/amol/(g. h) and (86 _+24)/amol/(g- h) determined by a linear interpolation between 0 and 6 h, were still lower than a short term (a few minutes) respiration rate of 180/lmol/(g. h) measured with the BASF-Toximeter [16]. In other investigations, nutrients were added to the activated sewage sludge, stimulating the respiration (500 pmol/(g" h) [5] and 1000/~mol/(g-h) [6]). Phenol is easily decomposed in the activated sludge process. Removal ratios of 7 7 - 100 % in 31 activated sludge plants and 9 7 - 100 % in static flask tests were reported [17]. In our investigation, 13C6-phenol was completely ( _ 98 %) metabolized during 96 h incubation time; during the first 6 hours, 27 % were transformed. Molar ratios of 13COz produced to 13C6-phenol consumed of 2.6_+0.6 and 3.3 _+0.3 resp. were determined for an incubation time of 6 and 96 h. Assuming a first order decomposition reaction without a microbial lag phase, a transformation rate of 0.053 h -1 (resulting half-life: 13 h) was received. Values of 0.038 h 1 and 18.5 h were calculated from the production of 13CO2. UaANO and KATO [18] reported rate constants for phenol degradation of 0.037 - 0.041 h -x after a'lag phase of 15 h. Intense dilution of the sample with an inorganic nutrient medium to a suspended particulate matter concentration of 30 mg/L may be responsible for the occurrence of the lag phase they observed.

sewage sludge without addition of PCP, mean values and standard deviations are calculated from three identicalsamples and

given in/amolper g solid dry matter and hour incubation time Production and consumption rates without addition of PCP incubation time 1.

Series 2.

Series

DMSO

respiration

mineralisation

reduction in/Jmel/(g.h) in/Jmol/(g- h) in/Jmol/(g, h)

2h 6h 25 h

2.1 _+0.1 2.3_+0.1 2.5_+0.1

not available 77_+20 57-+5

64_+40 62+_6 49-+7

6h 96 h

3.1 _+0.1 3.0+0.1

86_+24 43_+6

69+20 34_+4

The reduction rate of DMSO was not influenced by the incubation time. Values of 2.3/~mol/(g'h) for the first set and 3.0/~mol/(g. h) for the second set of samples were determined. An average reduction rate of 2.2/amol/(g.h) was measured in four samples taken from the same sludge plant [15], one year ago. These values exceed the specific reduction rates determined for aerated wastewater basins (0.016 - 0.35/~mol/(g" h) [9]) and in soils ( 0 . 0 0 2 - 0 . 0 2 / l m o l / ( g . h ) [1]) by one to three orders of magnitude, but reduction rates of comparable samples of activated sewage sludge were not available. Respiration and mineralisation were significantly time dependent. With increasing incubation time ( 6 - 9 6 h), the production rate of CO z decreased by a factor of 2. After an incubation time of 96 h, about 25 % of the initial oxygen in the vial was consumed. Therefore, an oxygen deficiency ESPR-Environ. Sci. & Pollut. Res. 1 (3)1994

4.3

Microbial activity with added PCP

PCP was added to the activated sewage sludge samples covering a range between 0.01 and 3000 mg/L. Because of adsorption to suspended solid matter, the concentrations of bioavailable dissolved PCP may be 2 0 - 100 times lower. This rough approximation is based on literature data reported by BELLand TSEZOS[19]; solvent effects of DMSO, which may be important, were ignored. Some dose response curves for 6 h incubation time are given in Fig. 4 a - d. The first significant effect of PCP was detectable at concentrations of about 2 mg/L: DMSO reduction was slightly inhibited, whereas mineralisation and respiration were activated. As reported for 3,5-dichlorophenol [5, 20], low concentrations of PCP stimulate respiration by uncoupling of oxidative phosphorylation in the respiratory chain. Increasing PCP concentrations caused a further decrease in the DMSO reduction activity (EC20 = 4 mg/L, ECs0 = 21 rag/L, ECs0 = l l 0 m g / L ) and reduced 13Cs-phenol transformation (EC20 = 16 mg/L, ECs0 = 29 mg/L, ECs0 = 54 rag/L). Transformation (disappearence of 13Cs-phenol ) and mineralisation of t3C6-phenol (production of 13CO2) did not differ significantly. Respiration and CO 2 production dropped at PCP concentrations above 8 0 m g / L , ECs0 values were 2 5 0 m g / L and 185 mg/L, respectively. A direct comparison of these values with ECs0 values of PCP reported in literature is not possible: They ranged from 10 - 30 mg/L (respiration tests with added peptone or other organic nutrients to increase sensitivity and reproducibility) to 275 mg/L for nitrification [21, 22].

143

DMSO Reduction Rate in Activated Sewage Sludge

co.t..t ~~

Dimefhylsulphide

6h

Research Articles

co.t.., ~.

pmol/Vial

Oxygen

6h

lJ.mol/Vial

150 1

~- .

.1

.1

1

10

100

1000

.1

.

.

.

i

1

10

100

Conc. of PCP in mg/I Fig. 4 a: Dose response curve of DMS obtained incubation time

.co.t..,~.

Carbon

proof/Vial

at 6 h o u r s of

d i o x i d e 6h

I

1000

Conc. of PCP in m g / I

Fig. 4 c: Dose response curve of oxygen obtained at 6 h o u r s of incubation time

Phenol

, Content in

~mol/Via!

6h

ZLf~rlI~ dclitinn nf P~p

.4 t J5

J

t~riie .2 ~

c

10

5 0

1 .1

~ ~ PJ~H~I 1

J ~ J~r~l

L

i

i~11111

10

p

1 O0

r

~,rt~l

I

I

I

1000

Conc. of PCP in m g / I

ol

I

.1

E

i

itlllE~

i

1

i

ittllq

i

10

ll~lll

I

t

i

rliHi

I

i

i

100 1000 Conc. of PCP in mg/I

Fig. 4 b: Dose response curve of carbon dioxide obtained at 6 hours of incubation time

Fig. 4 d : D o s e response curve of phenol obtained at 6 hours of incubation time

The effect of increasing incubation time to 96 h is demonstrated in Fig. 5 a - c (-~ p. 145). Compared with shorter incubation times (2 h, 6 h and 25 h), the dose response curve for DMSO reduction was shifted towards lower PCP concentrations ( E C s 0 = 9 m g / L ) , whereas mineralisation (-" Fig. S b), respiration, 13Ct-phenol transformation (--' Fig. 5 c) and 13Ct-phenol mineralisation dropped steeply at a higher ECs0 value of about 500 mg/L. Given the current state of knowledge we propose some hypotheses:

sewage sludge, such as the oxygen demand or the dehydrogenase activity. Reproducible measurement of the DMSO reduction rate in other matrices such as soil, sediment, fresh and waste water samples is possible [1, 7, 8, 9, 23]. Systematic errors, caused by adsorption of microbially produced DMS (for example, in topsoils with a high content of organic carbon), are eliminated by the use of Dt-DMS as internal standard. The termination of microbial reduction by alkalizing eases handling and allows automatic analyses. The method promises an easy way to determine and to evaluate microbial activity of activated sewage sludge as well as changes in bioactivity caused by toxic effects, under nearly the same conditions that are present in the activated sludge plant the sample is taken from. For the design of a standardized DMSO reduction test further investigations will be necessary:

- Adsorption of PCP to solid matter is a kinetically controlled process; prolonging the incubation time leads to lower bioavailable concentrations of PCP. - Microorganisms acclimatize during 96 h incubation time; those w h o survive the first chemical shock react with a higher bioactivity (eg. by stimulated respiration). - T h e enzymes which transform D M S O to DMS are blocked irreversibly by PCP.

5

Conclusion and Future Outlook

- Does the DMSO reduction rate correlate with respiration and biodegradation when the microbial activity is reduced by addition of chemicals other than PCP? - What is the influenceof the origin and physiologicalstate of the activated sewage sludge samples on these parameters?

The determination of the DMSO reduction rate offers an additional parameter for evaluating the bioactivity in activated

~.44

ESPR-Environ. Sci. & Poilut. Res. 1 (3) 1994

Research Articles

DMSO Reduction Rate in Activated Sewage Sludge

6

Dimefhylsulphide 96h

Con,.nt~n t~mol/Vial

0 ~terilfL I

........

{

.I

.

.

.

I

.

.

.

10

.

"~

.

100

1000

Cone. of PCP in m g / l

Fig. 5 a: Dose response curve of DMS obtained at 96 hours of incubation time

Carbon dioxide 96h

Content in pmol/Vial

302010,

tnrilR 0

I

i

i iirHq

.1

,

d i~lHq

i

1

J~JHq

10

p

i

~llJlq

1O0

~

i

1000

Conc. of PCP in r n g / I

Fig. 5 b: Dose response curve of carbon dioxide obtained at 96 hours of incubation time Content in p.mol/Vial

96h

Phenol

.4 -_-

.2 C

"t nf PCP 0

l

.1

l

i

i IIHJ

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i ILIII[

~

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1O0 1000 Conc. of PeP in m g / I

Fig. 5 c: Dose response curve of phenol obtained at 96 hours of incubation time

Acknowledgement We would like to thank Heike KAUPP,Ken FROESEand Armin HAUK for reviewing the manuscript.

Literature

[1] K. AZEF; D. KLEINER: Rapid an sensitive determination of microbial activity in soils and soil aggregates by dimethylsulphoxide reduction. Biol. Fertil. Soils 8,349 (1989) [2] M. EWALD;K. HERMANN; M. WEIDMANN:Kurzzeittest fOr die Bestimmung der Dehydrogenaseaktivit~it von Belebtschlamm. Vom Wasser 68, 165 (1987) [3] R. KANNE: Die quantitative Bestimmung yon ATP (Adenosintriphosphat) als Parameter fOr die physiologische Aktivit~it yon Belebtschl~immen. Vom Wasser 57, 277 (1981) [4] H. WELLENS;R. ZAHN: Untersuchung iiber die Toxizit~itsbestimmung yon Abwfissern und Abwasserinhaltsstoffen nach der Dehydrogenaseaktivitiit (TTC-Methode). Chemiker-Z. 95, 472 (1971) [5] B. BROECKER; R. ZAHN: The performance of activated sludge plants compared with the results of various bacterial toxicity tests - a study with 3,5-dichlorophenol. Water Res. 11,165 (1977) [6] U. PAGGA:Der Kurzzeitatmungstest - eine einfache Methode zur Bestimmung der Atmungsaktivit~it von Belebtschlamm. Vom Wasser 57, 263 (1981) [7] G. P. SPARLING;P. L. SEARLE:Dimethyl sulphoxide reduction as a sensitive indicator of microbial activity in soil: The relationship with microbial biomass and mineralisation of nitrogen and sulphur. Soil Biol. Biochem. 25,251 (1993) [8] D. KLEINER; K. ALEF: Offenlegungsschrift DE 3907164 A1, Deutsches Patentamt 1990 [9] K.K. RAJBHANDARI; H.J. LORCH; J . C . G . OTTOW: Charakterisierung der Biomasseaktivit~t im Schlamm verschiedener Reinigungsstufen einer beliifteten Abwasserteichanlage mit Hilfe der Dimethylsulphoxidreduktase- und Dehydrogenase-Aktivit~it. VDLUFA-Schriftenreihe Kongret~band 33,642 (1991) [10] F. F. DIAS; J. V. BHAT: Microbial ecology of activated sludge. II. Bacteriophages, Bdellovibrio, Coliforms, and other organisms. Appl. Microbiol. 13,257 (1964) [11] S. H. ZINDER; T. D. BROCK:Dimethyl sulphoxide reduction by microorganisms. J. Gen. Microbiol. 105, 335 (1978) [12] R. M. GIBSON;P. L. LARGE: The methionine sulphoxide reductase activity of the yeast dimethyl sulphoxide reductase system. FEMS Microbiol. Lett. 26, 95 (1985) [13] P. M. WOOD: The redox potential for dimethyl sulphoxide reduction to dimethyl sulphide. FEBS Letters 124, 11 (1981) [14] D. MARTIN; H . G . HAUTHAL: Dimethylsulfoxid. AkademieVerlag, Berlin 1971 [15] J. BINERT: Entwicklung einer Methode zur Bestimmung der Dimethylsulph~ im Belebtschlamm und deren Beeinflussung durch Zugabe von Chlorphenolen. DiplomaThesis, Chair of Ecological Chemistry and Geochemistry, University of Bayreuth (FRG) 1993 [16] U. PAGGA:Stoffprfifungen in einem Klfiranlagenmodell - Abbaubarkeitstest und Toxizitiitstests im BASF-Toximeter. Z. Wasser Abwasser-Forsch. 18, 222 (1985) [17] D. J. RICHARDS;W. K. SHIEH: Biological fate of organic priority pollutants in the aquatic environment. Water Res. 20, 1077 (I986) [18] K. URANO;Z. KATO: A method to classify biodegradabilities of organic compounds. J. Hazard. Mat. 13, 135 (1986) [19] J. B. BELL;M. TSEZOS:Removal of hazardous organic pollutants by biomass adsorption. J. Water Pollut. Control Fed. 59,191 (1987) [20] E. F. KING; B. J. DUTKA:Respirometric techniques. In: G. BITTON and B. J. DUTKA, Toxicity testing using microorganisms, Vol I, CRC Press, Boca Raton 1986, pp. 75 [21] M. T. ELNABARAWY;R. R. ROBIDEAU;S. A. BEACH:Comparison of three rapid toxicity test procedures: Microtox, Polytox, and activated sludge respiration inhibition. Tox. Asses. 3,361 (1988) [22] E. F. KING:A comparative study of methods for assessing the toxicity to bacteria of single chemicals and mixtures. In: D. LIu and B.J. DUTKA, Toxicity screening procedures using bacterial systems, Marcel Dekker, New York 1984, pp. 175 [23] S. GRANDEL:Untersuchungen zum Bioabbau von Nonylphenolen in Sedimenten. Diploma-Thesis, Chair of Ecological Chemistry and Geochemistry, University of Bayreuth, Germany, 1994 Received: December29, 1993 Accepted: February 24, 1994

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