Environ. Sci. Techno/. 1995, 29, 478-485
Microbial T m s f o r m ~ sand Fm&iiemDeiived Benrotkiazoles Determined in industrial Wastewater THORSTEN REEMTSMA,*st OLIVER FIE",' G U N T E R KALNOWSK1,I A N D MARTIN J E K E L ' Department of Water Quality Control, Technical University of Berlin, Sekretariat KF 4, Strasse des 17, Juni 135, 0-10623 Berlin, FRG, and Department of Hygiene, Technical University of Berlin, Sekretariat ZI 3, Amrumer Strasse 12, 0-13353 Berlin, FRG
Benzothiazole (BT), 2-mercaptobenzothiazole (MBT), and 2-(methylthio)benzothiazole (MTBT) were determined as degradation products of the fungicide 2-(thiocyanomethylthio)benzothiazole (TCMTB) in tannery wastewater and are shown t o be incompletely removed (75%) in an anaerobic and aerobic wastewater treatment pilot plant. Average total concentration of these benzothiazoles is 5.7 p m o l L-l in the untreated wastewater and 1.4 pmol L-l after aerobic treatment. Aerobic batch degradation tests revealed that TCMTB is transformed to MBT. MBT is primarily methylated to MTBT, which was not further degradable. BT was aerobically degraded along unknown pathways. Potential effects of benzothiazoles entering the aquatic environment are illustrated by luminescence inhibition of Vibrio fischeri (=Photobacterium phosphoreum; EC50 between 0.03 pmol L-l for TCMTB and 32 p m o l L-l for BT) and growth inhibition of the same organism with TCMTB and MBT. MBT, BT, and MTBT a t concentrations of 0.1 - 0.2 p m o l L-l inhibit nitrification on sediment columns and mixed culture respiration determined as BOD (0.6-11 p m o l L-l). It is concluded that 2-substituted benzothiazoles employed in industrial processes are not completely removable by biological wastewater treatment and are of concern for aquatic environment due to their limited biodegradability and potential toxicity.
Introduction The fate and effects of 2-substituted benzothiazoles in biological wastewater treatment and aquatic environment have not gained much attention, although these substances are widely employed in and subsequently discharged from industrial processes. For example, the fungicide 2-(thiocyanomethylthio) benzothiazole (TCMTB) is widely employed in lumber (1, 2) and leather production (3, 4) as a substitute for chlorophenols. 2-(Mercapto)benzothiazole (MBT)serves as a biocorrosioninhibitor in industrial cooling systems (5) and in the galvanic industry (6). Other benzothiazole derivatives are used as herbicides in the agriculture industry ( 3 ,while MBT-derived compounds are being used as chemotherapeutics (8). All these applications indicate the high biological activity of benzothiazoles. Apart from this, MBT and various derivatives are well-known vulcanization accelerators (9), and benzothiazole (BT) derivatives are intermediates in dye production (10). The occurrence of benzothiazoles in river water was ascribed to the tire and rubber manufacturing industry (11, 12) or suggested as an indicator of street runoff (13). Meanwhile, benzothiazole compounds have been detected in various environmental compartments: BT (14-1 7) and 2-(methy1thio)benzothiazole(MTBT) (14, 18) have been identified as surface water pollutants. BT was also determined in ground waters (19-21) in soils or sediments (13, 22) and quite regularly in municipal wastewater effluents (23-26). It was further identified in industrial emission (27) and atmospheric deposition (28). These manifold detections of BT and MTBT emphasize that the emission of benzothiazoles is no longer, if it has ever been, restricted to the rubber industry and that biodegradation might be limited. Some toxicological data on benzothiazoles are reported: MBT was determined as a mutagen in rats (29); TCMTB apart from being acutely toxic was shown to induce morphological alterations in fish (30);LCsovalues of MBT and BT against fish are reported (I 7). MBTS and MBT were identified as allergens (31).However, little is known upon the fate and effects of benzothiazoles in aquatic environment. The most detailed studywas onlyrecently presented (16)but concentrated on physicochemical processes such as hydrolysis and photolysis of TCMTB and MBT. We here report on an additional source of benzothiazoles, their occurrence in wastewater of the tanning industry, and their behavior during biological treatment in a combined anaerobic and aerobic biological treatment pilot plant. Microbial transformation processes of benzothiazoles are more closely studied by aerobic batch tests. Biological effects of benzothiazoles in the aquatic milieu such as growth, bioluminescence, nitrification, and respiration inhibition are examined, and their implications on the biological treatment processes and the aquatic environment are discussed. * Corresponding author; FAX: (30) 314 23850; e-mail address:
[email protected]. + Department of Water Quality Control. * Department of Hygiene.
478 1 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 29. NO. 2, 1995
0013-936X/95/0929-0478$09.00/0
0 1995 American Chemical
Society
Experimental Section Chemicals. TCMTB was received from the Dr. Eberle Company (Tubingen, FRG). BT was obtained from Aldrich (Steinheim, FRG), and MTBT was from Ferak (Berlin,FRG). MBT and all solvents were purchased from Merck (Darmstadt, FRG). 2-(Methylsulfinyl)benzothiazole(MSiBT)was prepared from MTBT by hypochlorite oxidation according to Brownlee et al. (16). Solvents were redistilled in an allglass apparatus prior to use. Double-distilled water was employed for standard solutions. Samples. Wastewaterwas obtained from the equalizing basins of two German tanneries treating cattle hides; it was transported to the laboratory at ambient temperature, acidified to pH 7, and subjected to biological treatment within 7-10 d. The biological treatment pilot plant consisted of atwo-stage anaerobic treatment and an aerobic activated sludge column (32),with average residence times of 3 and 2 d, respectively. The pilot plant was operated with tannery wastewater for 2 years prior to these investigations. Before anaerobic treatment, dissolved sulfide of the tannery wastewater was removed by the addition of FeC13. Particulate matter was removed after the anaerobic treatment by cloth filtration. Samples were taken from all stages of the pilot plant and stored frozen until analyzed. Analysis. TCMTB, MBT, BT, and MTBT were routinely analyzed from the wastewater after liquid-liquid extraction by reversed-phase HPLC with UV detection at variable wavelenghts. The detection limits were between 2 and 5 pg L-' (0.01-0.04 pmol L-l). Details are given elsewhere (33). 2-Methylbenzothiazolewas employed as the internal standard. Aerobic batch tests were analyzed by direct injection of the aqueous solutions. Acidic and alkaline extracts of the aqueous solutions and the biomass from biodegradation batch tests were analyzed by GUMS after derivatization with bis(trimethylsily1)trifluoroacetamide(BSTFA;Fluka, Buchs, Suisse) or trimethylaniliumhydroxide (TMAH; Regis, Morton Grove, IL). A HP 5890 I1 gas chromatograph equipped with a 30 m x 0.25 mm SPB5 glass capillary column (Supelco, Bellefonte, PA) was coupled to a HP 5989 quadrupole MS (Hewlett-Packard, Waldbronn, FRG). Spectra were obtained at 70 eV in the full-scan mode (45 bis 700 amu) and compared with the fragmentation of standard compounds. Sludge and biomass extracts were obtained by Soxhlet extraction with acetone for 16h. Dissolved organic carbon content of the wastewater samples was determined with an Astro LiquiTOC 2001-MB analyzer (Foss-Heraeus, Hanau, FRG) and UV absorbance on a Lambda-2 spectrophotometer (Perkin-Elmer, Uberlingen, FRG). Nitrate, nitrite, ammonium, and phosphate were determined by flow-injection analysis with the FIA-Star 5012 analyzer and dual-channel detection (FIA-Star 5042; Tecator, Uppsala, Sweden) according to the application notes given by the manufacturer. Statistical analyses were performed with the SPSS V.5.1 program (SPSS, Chicago, IL); the hypothesis of zero difference (T-test) was rejected below a two-tailed significance of 0.01. Degradation Tests. Tests on the aerobic biodegradation of TCMTB, MBT, BT, and MTBT were performed at room temperature in duplicates as 2-L batch test analogues to the IS0 9888 (34) with 115 mg L-l of yeast extract (Serva, Heidelberg, FRG) (43 mg L-l of DOC) as an additional carbon source and 3 mL of activated sludge inoculum from
a municipal wastewater treatment plant in tap water. Substrate levels were 60 pmol L-' of MBT, 72 pmol L-' of BT, 50 pmol L-l of MTBT, and 42 pmol L-' of TCMTB. Abiotic controls were poisoned with2 g L-' of sodium azide. An additional batch with pure biomass was used to identify metabolic products of the biomass itself. Sediment Columns. Four polymethacrylate columns of 50 cm x 6 cm i.d. filled with quartz sand (0.5-1.0 mm) were fed in parallel with 250 mL d-' of a wastewater solution, corresponding to a trickling rate of 30 cm d-' and a column residence time of 1.7 d. The columns had been in use with diluted tannery wastewater for 4 months prior to these investigations. Effluentof the tannerywastewater treatment pilot plant was filtered over glass fiber filters (GF 92; Schleicher & Schuell, Dassel, FRG) and diluted with nine parts of tap water. The mixture was saturated with oxygen ('50 mg L-l) by aeration with pure oxygen in an ice bath for 30 min. Yeast extract (33 mg L-l) was added, and the pH was adjusted to 7.2. Stock solutions of MBT, BT, and MTBT in acetone were added to dry bottles; the solvent was allowed to evaporate and then was filled with the wastewater solutions. These column influent mixtures and the effluent bottles were stored in the dark at 1 "C, while the columns were operated at room temperature. Dissolved oxygen contents of the column effluents were in the range of 5-8 mg L-l, indicating strictly aerobic conditions within the columns. Inhibition Tests. Respiration inhibition was detected in parallel via the biological oxygen demand at substrate levels of 100, 500, and 1500 pg L-l (BT, MBT, and MTBT) with glucose (825 mg L-l) and glutamic acid (750 mg L-l) as carbon sources. The BOD was manometrically determined with a H&C Model 210 apparatus (Hoelzle and Chelius, Neu-Isenburg, FRG) according to DIN 38409 T51 (35)at 21 "C. Mixed cultures from a municipal wastewater treatment plant were used as inoculum. Oxygen consumption was measured for 14 d, since it continued after the usual 5 d. The bioluminescence of I/: fischeri was determined according to DIN 38412 T34.1 (361, with test suspensions in 2% NaCl and 30 min incubation at 15 "C. The samples were adjusted to pH 7.0 and a NaCl content of 20 g L-l and diluted in a geometrical series with 2% NaCl solution. The determination of growth inhibition followed a novel DIN proposal being issued this year. A phosphatebuffered NaCl solution (20 g L-l) with peptone (1 g L-l), yeast extract (0.5g L-l), and glycerol (3 g L-l) was employed, and growth was observed on microtiter plates with 0.1 mL of the sample and 0.1 mL of bacterial suspension. The plates were incubated at 20 "C for 20 h in a microtiter incubation reader (340 ATTC; SLT Inc.), and the optical density at 414 nm was automatically detected every 20 min.
Results and Discussion Biological Treatment of Tannery Wastewater. Wastewater of two German tanneries was treated in a pilot plant combining anaerobic and aerobic biological treatment (32). All stages were analyzed for MBT, BT, TCMTB, and MTBT (Figure 1). Average total concentration of these benzothiazoles was around 8pmol L-l(4.3-13.7pmol L-l) in tannery I and 3.4 pmol L-l (3.1-3.6 pmol L-I) in tannery I1 wastewaters (Figure 2). MBT was the dominant benzothiazole in both cases with average concentrations of 6.9 and 3.3 pmol L-l, accounting for 87 and 94% of the four benzothiazoles. BT and MTBT were also present, but remained minor constituents in the untreated wastewater. VOL. 29, NO. 2, 1995 / ENVIRONMENTAL SCIENCE &TECHNOLOGY rn 479
R
substance
substance MET (tautomer)
MTET SOCH,
MSBT
SCHzMET
MMET
FIGURE 1. structums of benzothiazole derivatives.
I
i
E
T
tannery I i
I
n I I
untreated anaerobic aerobic ntreated anaerobic aerobic
FIGURE 2 Average concentrations of benzothiazoles in tannery wastewater and eflluenta ofthe tannery wastewatertreatment pilot plant. (tannery I, n = 8: tannery 11, n = 6 total observation period, 13 weeks).
TCMTB,originallyappliedinthepicklingstepofthetanning process, was not detectable. This is consistent with the observed rapid hydrolysis of TCMTB under alkaline conditions (tl12,3 h at pH 11; Fiehn. unpublished data). Wastewater samples were taken from the equilibration basins of the tanneries, where the total wastewater is routinely alkalinized in order to suppress the emission of hydrogen sulfide. During anaerobic treatment, the total benzothiazole concentration increased, primarily due to significantly increasing concentrations of BT, which reach an average of 1.7 and 1.4 pmol L-I, respectively (Figure 2). However, MTBT concentrations significantly decrease. The sources of BT in anaerobic treatment are not yet clear. GUMS analyses of the technical fungicide formulation provided considerableamounts of 3-(Benzothiazolyl)benzothiazole2-thione (BBT)and methylenebis(mercaptobenzothiazo1e) (MMBT). The spectrum of the first was equivalent to that given by Niessen et al. (37),while the latter was tentatively identified by its fragmentations. These benzothiazole derivatives oflowpolaritywouldenterthe pilot plant bound onto the particulate matter from which BT or MBT might be reductively liberated during anaerobic treatment. Analogously, the formation of BT and MBT from TCMTB was observed during incubation under methanogenic conditions (Fiehn, unpublished data). Subsequent aerobictreatment substantiallydiminished the concentration of benzothiazole compounds to 1.5 and 1.3 pmol L-I, according to 20 and 38% of the original concentration (Figure 2). This is due to decreasing MBT 480 m ENVIRONMENTAL SCIENCE &TECHNOLOGY I VOL. 29.
NO. 2,1995
and BT concentrations, whereas MTBT is formed during the aerobic treatment of tannery II wastewater. Finally, MBT accounts for 40% of total benzothiazoles only and is exceeded by BT in the treated tannery I wastewater (53%) and MTBT in tannery I1 wastewater (57%). Adsorption onto biomass appears not to be a major elimination process for MTBT, BT, and MBT. Contents in anaerobic sludge were around 0.02 pg g-I of MTBT, 76 pg g1of BT, and 130 pg g' of MBT. No correlations were detected between the decrease of one and the increase of another benzothiazole derivative during biological treatment, and the transformation processes are thus not clear. Despite the marked differences between the tannery1and IIwastewatertreatments (Figure 2). alterations during biological treatment coincide in several respects: (i) BTisformedfromunknown precursors in the anaerobic treatment, whereas MTBT is significantly diminished and MBT appears to be refractory against anaerobic treatment; (ii) MBT and BT are substantially eliminated by aerobic treatment, while MTBT is formed. The combined anaerobic and aerobic treatment is not feasible for completely removing the derivatives of the originally applied fungicide TCMTB. Tannery I employed an average 29.4 mol d-1 of TCMTB and produced 350 m3d-' of wastewater. The average total concentration of benzothiazoles determined in its wastewater correspondsto roughly 10%ofthe employedTCMTB. Cooper (38) determined 60% of the originally applied TCMTB in the wastewater of a sheep hide tanning process. If a n equal poriion of the fungicide was lost in tannery I, the m e emission of benzothiazoles would substantially exceed those amounts determined as MBT, BT, and MTBT in the dissolved phase. As far as TCMTB, BBT, and MMBT are considered, adsorption onto protein and fat flocs of the tannerywastewater, whichareremovedfromte equalizing basins, must be taken into account. Aer0bicBatchTest.s. Inorder to elucidatethemicrobial transformation processes occurring in the biological wastewatertreatment andintheaquaticenvironment, the aerobic biodegradation of MBT, BT, and MTBT was investigated in separate batch tests. Although not determined in the alkaline wastewater, TCMTB was included in these investigations. The pilot plant was designed for the in-plant treatment of tannery wastewater (32). In this case, the wastewater would no longer be alkalinized in the equilibration basins but neutralized only, and TCMTB would then also enter the biological treatment plant. TCMTB was aerobically degraded for about 95%within 9-12 d (Figure 3a). MBT is the main product, while about 5% of the TCMTB appears to be methylated from MBT to form MTBT. These derivatives were not determinedin the abiotic control, indicatingthatMBT formation fromTCMTB was biologically mediated and need not be a hydrolytic or photolytic process (16). Only45W oftheemployedTCMTB was recovered as MBT and MTBT in all experiments. DOC and Wdata, however, stronglyplead against adegradation of 50% of the substrate but suggest that the benzothiazole skeleton is preSeNed (Table 1). Indeed, HPIK analyses provided a considerably less polar substance, which we assume to be a coupling product of MBT, either with biogenic compounds (39, 40) or with BT to BBT. Unfortunately,attemptstowardanidentificationbyGC/MSafter fraction collection failed. Aerobic biotransformationof MBT is displayed in Figure 3b. Within 28 d, 10%of the MBT was methylated to MTBT
TCMTB
5 0 1 tTCMTB ---+major
-
+ MBT anaerobic,
BT
anaerobic
4 MTBT SH------* aerobic
I ,1
MBT
aerobic
60
@;&!-CH3+----MSiBT
1
aerob'c m : k S - - C H s
MTBT
) I aerobicx
I
1 t
aerobic
1
degradation
%
FIGURE 4. Proposed metabolic pathways (chemical transformation in parentheses).
I
C
0
'3
E
40-
w
C
g 208
0
7
14 time (d)
21
28
FIGURE 3. Aerobic transformation of (a) TCMTB, (b) MBT, and (c) BT by mixed cultures. TABLE 1
Aerobic Biodegradation of MBT, BT, and MTBT; DOC Contents and UV254 Absorbances after 25 d benzothiazole derivative'
blank TCMTB MBT
substrate DOC* ( m g L-l) BT-skeleton DOC* ( m g 1-l) final D O C (mg L-l) 6.1 A DOCb (rng L-l) 0 final UV264 ( c m - l ) 0.09
4.54 4.93 3.53 4.93 9.9 12.0 3.8 5.9 0.28 0.38
BT
MTBT
5.85 4.85 5.85 4.24 6.7 10.1 0.6 4.0 0.10 0.34
* A n asterisk ( * )indicates calculated values. *Difference in final DOC between substrate and blank.
by the bacteria, while 87%remained unaltered. Adsorption onto the biomass is negligible ('0.02% of MBT, '0.004% as MTBT). Trace amounts of BT were determined by GCI MS analysis of the final solution. The final DOC content and ITv254 absorption correspond to the preservation of the BT structure (Table 1). No alteration of MBT was observable in the abiotic control. Methylation of aromatic thiols by bacterial S-methyltransferase is reported and was suggested as a method of detoxification,because methylthio
ethers are generally considered less toxic (41). This aerobic batch test provided no insight into the process underlying MBT removal in the aerobic stage of the wastewater treatment pilot plant (Figure 2). Conjugation of MBT with glucuronic acid and glutathione is reported for mammals (39, 40, 42) and might be a relevant process in the pilot plant with comparatively high amounts of biomass. However, it was not observed in the batch test. BT was completely degraded after a 8-d lag phase within another 8 d, as monitored by HPLC analysis (Figure 312). DOC content and U V 2 5 4 absorbance decreased to those values determined in the control with pure biomass (Table 1)and support the suggested complete degradation of BT. However,the degradative pathway could not be elucidated. Neither HPLC nor G U M S analyses revealed any degradation products or intermediates. No BT or other benzothiazole derivatives were detectable in the biomass. Wilson et al. (43)observed the oxidative opening of the heterocyclic system of BT in guinea pig, primarily yielding 2-(methy1mercapto)aniline. Further oxidation products such as 2-methylsulfinylaniline and the corresponding hydroxylamine are expected to be degradable by bacteria. This pathway seems also likely for the aerobic microbial degradation of BT. Only traces of MTBT were altered by aerobic treatment over 25 d, as determined by HPLC analysis (not shown) and the final sum parameters (Table 1). A total of 0.04% (40.3 nmol) of MTBT was finally detected in the biomass, together with 0.003% as MBT (3.4 nmol). GCIMS analysis of the final solution of one series provided traces of MSiBT as a metabolite. Its mass spectrum was identical with the fragmentation of MSiBT prepared according to Brownlee et al. (169. However, MSiBT was not detected in the second batch. S-Demethylation by rat liver enzymes has been reported, and the oxidation of MTBT to the sulfoxide as the initial step of the methylthio displacement by glutathione was suggested (39). Both transformations were observed in these experiments, but they did not affect a relevant amount of the MTBT. If benzothiazole was substituted with the sterically demanding urea group in the 2-position (as in benzthiazuron), hydroxylation of the benzene system by soil fungi was observed ( 4 4 , 4 5 ) . No benzene-hydroxylated adduct was detected by means of HPLC and GCIMS. Thus, MTBT appears to be hardly degradable in aerobic biological wastewater treatment. VOL. 29, NO. 2 , 1 9 9 5 /ENVIRONMENTAL SCIENCE &TECHNOLOGY 1481
Figure 4 summarizes the transformation processes observed in the batch experiments: The fungicide TCMTB is hydrolytically and microbially transformed under aerobic conditions to MBT, while MBT and BT are formed under anaerobic conditions. MBT is primarily methylated to MTBT under aerobic conditions. This process is not always complete. Traces of MBT were converted to BT. MBT is additionally suggested to be eliminated by adduct formation with biogenic compounds; a process that was reported with digestive enzymes of mammals (39, 40, 42). In accordance with these transformation processes, MBT has never been detected in the environment far-off its pollution sources. This might also be due to analytical difficulties connected with MBT it is difficult to extract and poorly chromatographed by GC (33);thus, it would most likely not have been detected in the usual GUMS screening procedures. MTBT, formed by the methylation of MBT, appears to be hardly degradable. Oxidation to MSiBT and S-demethylation to MBT are observed, but are of minor importance. This corresponds with reports on the occurrence of MTBT in municipal wastewater effluent (23,24, 26) and surface waters (14, 18). BT, formed under anaerobic conditions, is completely degraded by mixed cultures in aerobic milieu. However, BT was detected in surface water (14-161 and in groundwater wells (191, indicating that its biodegradation need neither be complete nor fast; BT was also determined in the effluent of the wastewater treatment pilot plant (Figure 2). Transformations on Model Sediment Columns. The results from the biological treatment pilot plant suggest that benzothiazole derivatives (MBT, BT, and MTBT) are not completely removable by biological treatment and will, thus, be discharged into surface waters. We, therefore, studied the behavior of these three metabolites at significantly lower concentrations, simulating the mixture of industrial effluent with surface water (0.1-0.2 pmol L-' level) on model sediment columns within the diluted wastewater matrix. Apart from lower concentrations, these experiments might provide results differing from the batch tests since sediment columns favor biofilm-formingbacteria rather than free-living species. While, initially, small amounts of MBT were detected in its column effluent, organisms rapidly adapted to the S-methylation of MBT (Figure 5a). MTBT, however, was adsorbed for a 20-d period, but was then displaced by other organic matter. Consequently, effluent concentrations exceeded the influent concentrations for some weeks, with a steady state obtained after 70-d of MBT addition. In contrast to the batch test, MBT was completely methylated within the column. This is in accordance with Brownlee et al. (16),who observed rapid and complete methylation after mixing MBT with sedimentary matter. BT was not detectable in the effluent of its column. BT exhibits the lowest KO, value of the four benzothiazoles (around 100;16),and adsorption of BT is thus veryunlikely. Soxhlet extraction of the upper 50 g of the column material provided no BT. Analogous to the aerobic batch test, BT was completely degraded within the sediment column. The elution pattern of MTBT was similar to that obtained with MBT (Figure 5b), but adsorption of MTBT appears to be less distinct on this column. A steady state is achieved after 60 d (35 bed volumes). Other benzothiazole compounds, namely, MSiBT,were not detectable in the column effluent. MTBT appears to be refractory even under substantially diluted conditions and with adapted bacteria. 482 1 ENVIRONMENTAL SCIENCE &TECHNOLOGY / VOL.
29, NO. 2, 1995
c
t
8 .03 s -
0 0
20
40 60 time (d)
80
FIGURE 5. Effluent concentrationsof model sediment columns: (a) MTB and (b) MTBT.
160 -A-
i3n
l+
control MBT
+ MTBT n
0
3
6 9 time (d)
12
15
FIGURE 6. Effect of MBT (0.6pmol L-l), BT (11pmol L-l), and MTBT (2.8pm0lL-~)on the oxygen consumptionof mixed culturesdegrading a glucose/glutamic acid mixture.
BiologicalEffects. Evaluating the environmental hazard potential of a chemical requires information on its biodegradability and its influences on aquatic organisms or biological processes (46).Furthermore, biocides within a wastewater might directly interfere with the proper operation of a biological treatment process. We, therefore, investigated respiration and nitrification inhibition of mixed cultures and luminescence and growth inhibition of V. fischeri by the four benzothiazole compounds. Respiratory Inhibition. All three metabolites MBT, BT, and MTBT interfered with the degradation of easily degradable organic matter (glucose/glutamic acid) as determined by BOD measurements (Figure 6). A 40-50% inhibition after 10 d (BODlo)was observed with MBT at 0.6 pmol L-l, whichwas the most effective metabolite, followed
I
r 4 i
TAWE 3 EC50 Values of luminescence and Growth Inhibition of Vibrio fischeri (pmol 1") luminescence
a
Ii
,
0
20
80
100
TABLE 2
Average Ammonium Contents of Model Sediment Column Effluents (n = 12; period, 95 d) blank
MBT
BT
MTBT
effluent NH4+ 3.6 (1.4) 8.5 (3.8) 9.6 (4.5) 8.9 (4.1) (mgL-')* A NH4+ (mg L-l)b 10.5 5.6 4.5 5.2 A'4" (%)* 74 40 32 37 a Standard deviation in parentheses. influents and effluents.
NaCl
organic
TCMTB MBT BT MTBT
0.03 0.70 32 12.0
0.05 0.93 37 17.6
E&:
growth organic > > >
5.0 28a 46b 2gb
24.1 ymol L-l. bGrowth inhibition questionable (see text).
I
40 60 time (d)
FIGURE 7. Ammonium removal on model sediment columns with and without the addition of benzothiazoles (as percent of influent ammonium).
benrothiazole derivative
medium
Differences betweencolumn
by MTBT (2.8 pmol L-l) and BT (11 pmol L-I), Similar effects were obtained with TCMTB at 4 pmol L-' (not shown). Nitrification Inhibition. Influents and effluents of the model sediment columns were analyzed for ammonium, nitrate, and nitrite. Effluent ammonium contents of the three columns spiked with MBT, BT, or MTBT ran parallel, and their average ammonium removal is displayed in Figure 7. Two phases have to be distinguished phase I with benzothiazole concentrations of 0.09-0.19pmol L-l, which comes up to 50% of the concentrations of pilot plant effluents, and phase I1 with as much as 1.5pmol L-l of each benzothiazole. After aperiod of adaptation (14 d) necessary for the columns enriched with benzothiazoles, nitrification was obtained on all columns during phase I. However, nitrification on the spiked columns remained less stable, as visible toward the end of phase I (40-60 d). The addition of 1.5pmolL-' ofbenzothiazole derivatives (phase11)further reduced the extent of ammonium oxidation. While an average of 74%of the ammonium was oxidized on the blank column throughout the whole period (Table 21, between 32 and 40% was oxidized on the columns spiked with one of the three benzothiazole derivatives. In contrast to one ofthe reports ( 4 3 ,first stage nitrification (nitrite formation) by Nitrosomonas was inhibited in these experiments rather than nitrite oxidation by Nitrobacter. Consequently, effluent nitrite concentrations were constantly lowwith 0.10.2 mg L-I, while an average of 8.8 mg L-l was determined in the column influent. Due to the aerobic conditions, denitrification was not measurable. The concentrations of benzothiazoles inhibiting nitrification on the model sediment columns are at least 1order
of magnitude lower than those previously reported: effects of BT concentrations exceeding 2200 pmol L-' and MBT at 60 pmol L-l were reported in an laboratory aeration tank (47), while an E G 5 value of 18 pmol L-' was determined for MBT in an activated sludge process (48). This might be due to a substantially lower amount ofbiomass (48)present on the model sediment columns compared to an activated sludge process. Nevertheless, the concentrations of benzothiazole derivatives determined in the (anaerobically treated) tannery wastewater (Figure 2) might contribute to the observed incomplete nitrification within the pilot plant (32). Acute Aquatic Toxicity. Acute toxicity against aquatic organisms was monitored with V.fischeri, previouslyknown as P.phosphoreum, in the luminescence inhibition test. All four benzothiazoles caused luminescence inhibition (Table 31, with TCMTB being most active (EC50, 0.03 pmol L-l). MBT is about 1order of magnitude less effective (0.7pmol L-l), followed byMTBT (12pm0lL-~)andBT (32pmolL-l). Thus, the observed biomethylation of MBT to MTBT during aerobic treatment might be a useful strategy of toxicity reduction, although it appeared to be misleading with respect to biodegradation (see above). Literature data for luminescence inhibition of MBT and BT (EC50,4.5and 14.8 pmol L-l; 49) are slightlylower than those determined here. The EC5o value determined for TCMTB is in the range of LC50 data reported for juvenile salmonid (0.05-0.21 pmol L-l; 30);EC50 of MBT and MTBT are, however, much lower than the LC50 data reported for red killifish (25 and 500 pmol L-I; 17). This is not astonishing since the luminescence test monitors physiological activity rather than mortality. Growth inhibition of Kfischeri by the four benzothiazoles was much less pronounced than luminescence inhibition. The EC5o of TCMTB was 5.0 pmol L-' (Table 3); the initial concentrations of MBT, BT, and MTBT (30-40 pmol L-l) were too low to obtain 50% growth inhibition. However, 20% inhibition occurred with 24.1 pmol L-I of the second active derivative, MBT. Concentration-dependent effects were not discernible for BT and MTBT. The weaker effects of the benzothiazole derivatives on the growth of V.jischeri are not due to the presence of organic substrates in the growth medium applied in this test. Results of luminescence inhibition employing this growth medium in the same concentration are comparable to those obtained with the original NaCl solution (Table 3). Thus, these benzothiazoles appear to selectively effect the enzymatic processes underlying the luminescence rather than to generally affect the physical status of the bacteria. From those biological effects investigated, luminescence inhibition of V. jischeri was the strongest, followed by nitrification inhibition, respiratory inhibition, and finally VOL. 29, NO. 2, 1995 / ENVIRONMENTAL SCIENCE &TECHNOLOGY
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growth inhibition. Out of the four benzothiazoles, TCMTB generally exhibited highest activity, followed by MBT, MTBT, and BT. However, the concentrations of MBT detected in tannery wastewaters (4-7 pmol L-1) are well above the determined limits of respiration, nitrification, and luminescence inhibition and might, hence, interfere with the biological treatment of these effluents.
Conclusions Wastewater from two German tanneries, which employ 2- (thiocyanomethylthio)benzothiazole (TCMTB) as fungicide, and the corresponding effluents of a combined anaerobic and aerobic wastewater treatment pilot plant were analyzed for TCMTB, 2-(mercapto)benzothiazole (MBT),benzothiazole (BT), and 2- (methy1thio)benzothiazole (MTBT)for 13weeks. Tanneryeffluents aredominated by MBT, which originates from the hydrolysis of TCMTB under alkaline conditions. It accounts for an average 90% of the total benzothiazole concentration of tannery wastewater, which is around 5.7 pmol L-I. BT and MTBT are of minor importance, while TCMTB was not detected. Anaerobic treatment leads to an increase in total benzothiazole concentrations,which is suggested to be due to the reductive liberation of BT, and to a minor extent of MBT, from adsorbed compounds. MBT and BT are significantly eliminated during aerobic treatment, while MTBT is formed; the final average concentrationof benzothiazolesis 1.4pmol L-l, corresponding to 75% elimination by the combined anaerobic and aerobic wastewater treatment. A metabolic scheme is derived from aerobic batch tests with the main aerobic pathway leading from TCMTB via MBT to MTBT, which was not further degradable. Minor processes observed are the transformation of MBT to BT, the S-demethylationof MTBT to MBT and the oxidation of MTBT to 2- (methylsulfiny1)benzothiazole. The process underlying MBT removal in the aerobic stage of the pilot plant remained unidentified. BT is degraded by extensive treatment under aerobic conditions. Aerobic degradation of BT, methylation of MBT to MTBT, and persistence of MTBT were also observed under diluted conditions on model sediment columns. The observed limited biodegradability of benzothiazoles is consistent with BT and MTBT being reported in various aquatic environmental compartments. Corresponding to one of their major employments as fungicides, benzothiazoles exhibit biological effects such as respiration and nitrification inhibition of mixed cultures, and luminescence and growth inhibition of V. fischeri. TCMTB is generally most active, followed by MBT, MTBT, and BT. The aerobic microbial transformation of TCMTB via MBT to MTBT might, thus, be a useN strategyfor toxicity reduction, but appears to be a dead-end in biodegradation. The MBT concentrationsdetermined in tannerywastewater cause acute inhibitory effects and might directly affect a biological wastewater treatment. The limited biodegradability and potential aquatic toxicity suggest that benzothiazoles are of higher environmental concern than previously recognized.
Acknowledgments The authors thank F. Ba6 for running the model sediment column experiments and E. Genschow and W. Hegemann for providing samples of the tannerywastewater treatment pilot plant. The financial support by the German Research 484 1 ENVIRONMENTAL SCIENCE &TECHNOLOGY / VOL. 29, NO. 2, 1995
Council (DFG,Bonn) through SFB 193 “Biologicaltreatment of industrial wastewater” project A3 is gratefully acknowledged.
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Received for review May 31, 1994. Revised manuscript received October 20, 1994. Accepted October 27, 1994.@ ES940334M
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Abstract published in AdvanceACSAbstructs, December 1,1994.
VOL. 29, NO. 2, 1995 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 1486
