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Microcosms-experiments to assess the potential for natural attenuation of contaminated groundwater
PII: S0043-1354(00)00315-8
Wat. Res. Vol. 35, No. 3, pp. 720–728, 2001 # 2001 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/01/$ - see front matter
MICROCOSMS-EXPERIMENTS TO ASSESS THE POTENTIAL FOR NATURAL ATTENUATION OF CONTAMINATED GROUNDWATER KATRIN ALTHOFF, MATTHIAS MUNDT, ADOLF EISENTRAEGER, WOLFGANG DOTT and JULIANE HOLLENDER* Institute of Hygiene and Environmental Medicine, RWTH Aachen,Pauwelsstrae 30, D-52074 Aachen, Germany (First received 25 February 2000; accepted in revised form 9 June 2000) Abstract}Groundwater samples from six wells of a former gas plant site were characterised using chemical, microbial and ecotoxicological methods. Degradation studies were performed in batch-culture under aerobic conditions with the groundwater samples containing their autochthonous microflora and original contaminant mixture. The highest O2-consumption (3 mmol 100 ml ÿ 1), combined with BTEX (8.3 mg l ÿ 1) and naphthalene (171.3 mg l ÿ 1) degradation, as well as formation of organic acids was found after N- and P-supplementation with the highest contaminated groundwater sample. The other highly polluted groundwater sample showed no activity obviously because of the toxicity of some compounds. The major part of the PAHs and BTEX was eliminated in the assays with the low contaminated groundwater samples. The results indicate that the microbial degradation capacity and thereby the natural attenuation capacity in each groundwater differ and cannot be assessed simply by chemical, microbial and toxicological data. Additionally activity tests with authentic groundwater samples with and without nutrient supplementation are recommended. # 2001 Elsevier Science Ltd. All rights reserved Key words}BTEX, groundwater, laboratory batch studies, microbial degradation, natural attenuation, PAHs
INTRODUCTION
At gas plant sites contamination of soils and groundwater has occurred from disposal of wastes generated during the manufacturing process of city gas as well as from tank leakage. Typical contaminations include tar residues and sludge containing various heavy metals, volatile organic compounds (VOC), monoaromatic hydrocarbons and polycyclic aromatic hydrocarbons (PAHs). Natural attenuation at such sites is getting more and more interesting, because it seems to enable the bioremediation with minimised cost, avoiding land disruption and human exposure. A number of processes determine natural attenuation of organic contaminants in soil and groundwater such as biodegradation, dispersion, sorption, dissolution and volatilisation (Cho et al., 1997; Stapelton and Sayler, 1998). Many different chemical, physical, biological and geological parameters of both kind of pollutants and aquifer influence these processes. Therefore, multidisciplinary studies and an extensive characterisation of the
*Author to whom all correspondence should be addressed. Tel.: +49-241-8088-282; fax: +49-241-8888-477; e-mail: [email protected] 720
field are necessary to provide an accurate assessment of natural attenuation capacity. In order to understand the fate of xenobiotica microcosms, columns, batch-cultures, lysimeter and field studies were performed. Each of these methods has its advantages and is appropriate to answer special questions (Ho¨hener et al., 1998). An up-scaling to real-field conditions especially by results from laboratory experiments is difficult. But the influence of many different parameters such as redox conditions, oxygen concentration, nutrient concentration on the microbial degradation can be investigated by microcosms studies. Soil or groundwater with its authentic contamination is rarely used for experiments (Dyreborg et al., 1997). Most laboratory experiments concerning the degradation of organic compounds in aquifers include aquifer sediment, because the use of groundwater alone has resulted in lower degradation rates (Albrechtsen et al., 1997; Poeton et al., 1999). The solid particles were used as biomass support material, as inoculum and as surface area for colonisation. However, from some aquifers sediment cannot easily be obtained because of deep wells of geologic settings of boulders. Besides, soil samples frequently are very heterogeneous and therefore are not representative of the site. Hot spots of
Natural attenuation potential of contaminated groundwater
contaminants occur and bioavailability is influenced strongly by sorption processes. Because of the latter it is difficult to distinguish between microbial degradation and sorption to the soil (Nielsen et al., 1995). Some investigations showed higher degradation rates with groundwater without sediment because the kinetic of the desorption of the organic compounds is slower than the degradation rate (Scow and Alexander, 1992; Zhang and Bouwer, 1997). The groundwater of contaminated sites reflects the fate of the plume, the current situation of the contamination and plays the main part in leaching, release and transport processes of pollutants. For these reasons studies with groundwater are recommended but the question is whether investigations with pure groundwater can reflect microbial degradation activity. How is the degradation capacity of free bacteria living in a low-nutrient environment such as groundwater and not adsorbed on particles inhabit organically rich microzones (Alfreider et al., 1997)? In this work authentic groundwater samples of six different wells of a contaminated site were used to study the microbial degradation capacity under aerobic conditions with and without N- and Psupplementation. A method was developed and evaluated to assess and observe the microbial activity in native groundwater with autochthonous microflora and original contaminant mixture by monitoring O2-consumption and CO2-production.
MATERIALS AND METHODS
Study site and samples The groundwater samples (GW 64 to 69) were collected from six wells distributed over the area of a former gas plant. The investigation site is located in the south-west of Germany in the Neckar valley. Below the site is a quaternary porous aquifer of medium-grained gravel intersected locally by sandy and silty lenses. The first aquifer is completed by a sequence of upper triassic rocks (Middle Keuper). Till the 1970s the gasworks produced city gas, thus, there are contaminations by products of coal gas and the following processes like tar distillation and gas washing. A heterogeneous contamination, consisting of aliphatic and aromatic hydrocarbons (BTEX, indane, indene, biphenyls, PAHs, etc.), heterocyclic aromatic compounds containing oxygen or sulphur (benzofurane, benzothiophene, etc.) in high concentrations as well as phenols and compounds containing nitrogen (quinoline, pyridine, etc.) in low concentrations was detected at this site (Annweiler et al., 1997). Besides this high contamination with organic compounds there is a pollution with inorganic compounds such as NH3, cyanides and sulphur caused by clinker and gas washing. Physico-chemical methods The pH and the conductivity were measured with a glass electrode described in DIN 38 404 part 5. Cations were determined with atomic absorption spectrometry (DIN 38 406) apart from NH+ which was detected with an 4 ammonium-selective electrode Orion 9512 (Orion Research, Boston, USA). Anions were measured using an ion chromatograph DX-100 (Dionex, Sunnyvale, USA) according to DIN 38 405 part 20. Eh was measured by platinum
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electrode (Mettler Toledo, Urdorf, Swiss). The CO2concentration in the aqueous phase was determined according to DIN 38 405 part 8. The concentrations of the 16 US Environmental Protection Agency (EPA) PAHs in the groundwater samples were determined after twice repeated extraction with pentane using 1,10 -binaphthyl as internal standard. A HewlettPackard 1050 HPLC-system with Beckmann pumps (model 126, Beckmann, Munich, Germany), a time programmable fluorescence detector and a diode array detector were used. Separation was achieved at 358C on a 250 mm 3 mm ID MZ-PAH column (particle size 5 mm) with a 12.5 mm 3 mm I.D. pre-column (MZ-Analytical, Mainz, Germany). An acetonitrile–water gradient, from 60 to 100% acetonitrile in 35 min, was used as mobile phase with a total flow rate set to 0.6 ml min ÿ 1. For the detection of polar aromatic compounds an acetonitrile–water gradient, from 30 to 100% acetonitrile within 35 min, was used. BTEX concentrations were measured by GC-MS using multiple headspace extraction (MHE). The samples were taken from the infusion bottles after cooling the batch culture to 48C to avoid loss of VOC. 20 ml glass headspace vials were filled with 5 ml of sample, sealed with Teflon-lined septa and frozen until analysis. Analyses were performed using a Hewlett-Packard HP 5890 gas chromatograph system equipped with a HP-5-MS-capillary column (30 m 0.25 mm ID) coated with chemically bonded SE-54 (df=0.25 mm; Hewlett-Packard, Waldbronn, Germany). Helium (99.9 v/v) (1.6 ml min ÿ 1) was utilised as carrier gas. The analyses were performed using the following temperature program: 408C held for 1 min, heating rate 48C min ÿ 1 up to 3108C, final temperature held for 1.5 min. Injector and detector temperatures were 250 and 3008C, respectively; detection was achieved using HP 5872 mass detector. CO2-production and O2-consumption during the degradation tests were measured by GC with thermal conductivity detector (TCD) (Perkin Elmer, U¨berlingen, Germany, model autosystem) using a CTR 1-column (Alltech GmbH, Unterhaching, Germany). The injection volume of 100 ml was taken manually with a gastight 100 ml-syringe (Hamilton, Darmstadt, Germany) from the headspace of the assays. The column temperature was set at 408C isothermal. Helium (62.5 ml min ÿ 1) was utilised as carrier gas. Injector and detector temperatures were 65 and 1408C, respectively.
Microbial methods Aerobic and facultative anaerobic bacteria were counted as colony-forming units (cfu). The groundwater samples were diluted in decimal steps up to 10 ÿ 6 and plated out on R2A-Agar (Difco Laboratories, Detroit, USA). Two parallels of each dilution step were incubated for 10 days at 258C. Sulphate-reducing microorganisms were quantified by a turbidimitric most probable number (MPN) three-tube technique (Alexander, 1982) with liquid medium according to Alef (1991). The results were determined after incubation for six weeks at 258C.
Ecotoxicological methods The measuring parameter for the bioluminescence inhibition test with Vibrio fischeri is the decrease of the light emission through presence of toxic substances in the groundwater samples (DIN 38412-part 341). In the algal growth inhibition test with Scenedesmus subspicatus inhibition of the biomass production was determined (DIN 38412-part 33). In the growth inhibition tests with Pseudomonas putida (DIN 38412-part 8) and Vibrio fischeri (DIN 38412-part 37) inhibition of cell increase was measured photometrically. Both tests were modified to microplate assays (Schmitz et al., 1998). The Daphnia magna
0.01 0.01 0.01 0.01 2.05 5 0.01 5 5 5 5
0.03 0.03 0.04 0.03 1.03 0.03 0.33 0.56 0.69 0.58 109.68 0.09 0.41 0.49 1.88 2.91 213.07 0.16 1.75 17.83 3.63 12.29 25.75 0.53 0.17 5 0.001 0.47 1.06 7.46 5 0.001 0.12 0.013 0.44 2.24 8.47 5 0.001 0.12 5 0.001 0.50 1.77 1.88 5 0.001 1.23 4.60 1.04 2.69 6.25 0.53 1945–70 tar oil pit 1914–45 benzene distillation and tanks Tanks 1915–70/74 tar distillation/pitch processing 1910–70/74 tar sedimentation tank 1970/74 deposit/anthropogenic filling 64 65 66 67 68 69
0.12 13.22 1.18 4.54 1.70 5 0.001
Four-ring Three-ring Total BTEX Styrene/o-xylene m-/p-Xylene
BTEX (mg l ÿ 1)
Toluene
The location of the six investigated wells on the former gas plant is listed in Table 1. The BTEX and PAH concentrations of the six groundwater samples differ to a large extent. Well 69 is not located within the plume and therefore the groundwater was only slightly polluted. The sample of well 64 also contained only low concentrations of contaminants despite the well position next to a former tar oil pit. Well 65 is located at a former benzene distillation and therefore the groundwater was highly polluted with benzene. Well 67 was drilled near the former tar distillation and pitch processing. The groundwater sample of this well contained a broad spectrum of contaminants in high concentrations. Well 66 lies near the gas tanks and the groundwater sample contained a wide spectrum of pollutants in medium concentrations. Well 68 was originally not highly polluted, but during pumping of the groundwater an old subsurface lake of tar oil was tapped. High amount of tar oil was released and droplets of tar oil swam on the aqueous phase. Therefore, the groundwater sample was very heterogeneous and some chemical analyses and the growth inhibition tests
Benzene
Characterisation of the groundwater samples
Contamination history
RESULTS AND DISCUSSION
Well
The groundwater samples were taken in autumn 1998. They were filled in autoclaved 1 l-infusion bottles, closed gastight with viton-butylrubber stoppers, which were specially produced for these experiments by Reichelt GmbH (Heidelberg, Germany), placed in coolers during transportation to the laboratory and stored at 48C. At most after 2–3 days the groundwater samples were used for the degradation tests. Three parallel degradation assays and two abiotic controls (0.5% sodium azide) were prepared with the groundwater samples from the six wells, respectively. One hundred and twenty millilitres groundwater was filled in 150 ml infusion bottles and closed gastight with a vitonbutylrubber-stopper. Apart from additional assays with groundwater from well 68 all batch cultures were supplemented with ammonium and phosphate to a final concentration of 2.8 mM N and 0.11 mM P to avoid nutrient limitation. The assays were shaken at 120 rpm in the dark at 208C. Monitoring the CO2- and O2-concentration in the gaseous phase was performed regularly. At the beginning samples for BTEX-analysis were taken and after approx. 140–160 days samples were collected for analysis of BTEX (2 5 ml), anions and ammonium (2 ml), the CO2-content in the aqueous phase (3 5 ml) and the quantification of aerobic microorganisms (1 ml). The remaining groundwater was extracted with pentane prior to the PAH analysis. Oxygen as electron acceptor was available throughout the batch experiments.
Table 1. BTEX and PAH concentrations of the original groundwater samples of the six wells
Degradation tests
Two-ring
PAHs (mg l ÿ 1)
Five-ring
Total
survival test was based on the inhibition of the mobility (DIN 38 412-part 30). The results of the tests were expressed as G-values, which are defined as the smallest reciprocal dilution factor with an inhibition of less than 20% (for the acute luminescenceinhibition assay, the algal growth-inhibition test and the growth-inhibition tests with V. fischeri and P. putida) and 10% (for the D. magna survival test), respectively.
0.77 1.08 2.61 3.52 325.83 0.28
Katrin Althoff et al.
Ethyl-benzene
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lites of degradation processes as described by King et al. (1999). The number of microorganisms growing on R2Aagar increased in every assay. But there is no significant relation between the microbial counts and the observed microbial activity in the degradation test as described elsewhere (Kao and Borden, 1997; Carmichael and Pfaender, 1997). However, it is well known that it is difficult to quantify microorganisms from groundwater or soil samples with bacterial growing techniques and to isolate especially the degrading bacteria without a xenobiotic compound as growth substrate (Stapleton and Sayer, 1998). Monitoring the CO2- and O2-concentration in the headspace (Figs 1, 3) showed that the O2-consumption is the more reliable parameter for quantification of the microbial activity, although O2 is less exact than CO2 because of its higher partial pressure in the air. CO2 is detectable with more sensitivity but depending on the pH-value a high part of CO2 remains in the aqueous phase. So it is most suitable to measure both parameters as well as the CO2concentration in the liquid phase to get the most information. Generally, the activity of the groundwater samples determined by the O2-consumption and CO2-production was low apart from sample 68 (Fig. 1).
with Scenedesmus subspicatus could not be performed. The physical and chemical data of the groundwater samples are summarised in Table 2. The sulphate concentration was high in all samples apart from the sample of well 65. This indicates a sulphate reducing activity in this part of the aquifer, which was supported by the occurrence of sulphate reducing microorganisms in the sample of well 65 determined by the MPN method. None of the groundwater samples was contaminated with heavy metals. Therefore, toxic effects on the microbial activity caused by heavy metals could be excluded. The results of the ecotoxicity tests are shown in Table 3. The groundwater sample of well 68 with tar oil in phase showed the highest toxicity. High Gvalues were detected in the bioluminescence inhibition test with Vibrio fischeri and in the survival test with Daphnia magna. The groundwater of well 67 showed also a high toxicity probably caused by an additive effect of many different contaminants or the occurrence of substances in toxic levels which repress all microbial activity. Degradation studies The characteristic physico-chemical parameters of the degradation assays of the groundwater samples were compared with the data of the original samples and with the abiotic controls at the end of the test (Table 4). A drop of the pH-value in the test assays was a first indication for microbial activity caused by the microbial formation of organic acids as metabo-
Highly contaminated groundwater samples In the assays with groundwater from well 68 high microbial activity was found (Table 4) as shown by
Table 2. Physicochemical characteristics of the original groundwater samples of the six wells Well 64 Ph O2 (% saturation) Eh (mV) Conductivity (mS cm ÿ 1) Anions (mg l ÿ 1)
Cations (mg l ÿ 1)
Cl ÿ NO2ÿ NO3ÿ PO34 ÿ SO24 ÿ NH+ 4 Mg2+ 2+ Ca
65
7.6 4.0 ÿ 68.6 1.5 174.5 50.1 51 51 796.5 0.4 76.3 386.1
66
7.2 10.6 +181 0.9 74.8 50.1 10.9 51 17.6 0.3 42.6 183.8
7.5 1.6 ÿ 103.4 1.7 164.3 50.1 51 51 788.7 1.3 85.2 337.8
67 8.1 4.0 ÿ 99.6 1.6 149.6 50.1 51 51 614.1 1.4 45.8 356.2
68 8.1 4.9 +203.6 1.7 199.0 50.1 51 51 1062.5 1.7 81.8 501.0
69 7.2 10.8 +212 1.3 92.4 50.1 51 51 526.6 1.0 44.8 257.4
Table 3. Ecotoxicological characteristics of the original groundwater samples of the six wells Ecotoxicological test
Vibrio fischeri bioluminescence inhibition test Pseudomonas putida growth inhibition test Vibrio fischeri growth inhibition test Daphnia magna survival test Scenedesmus subspicatus growth inhibition test a
n.d.: Not determined.
G-value 64
65
66
67
68
64 16 2 2 4
128 8 4 4 2
128 16 2 2 2
256 16 16 6 128
4096 16 8 96 n.d.
69 2 8 2 n.d.a n.d.
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Table 4. Results of the aerobic degradation studies after approx. 140–160 days: mean value of the three parallel assays compared to the abiotic control and the original groundwater sample of the six wells Sample
pH
CO2 (water) (mmol 100 ml ÿ 1]
NO2ÿ (mg l ÿ 1)
NO3ÿ (mg l ÿ 1)
GW 64 original GW 64 end Control GW 64 end
7.6 7.5 0.3 7.1
0.61 0.19 0.52 0.01
51 51
51 51 n.d.a
GW 65 original GW 65 end
7.2 6.0 0.2
0.49 0.12
>80b
11 15 1b
Control GW 65 end GW 66 original GW 66 end
6.8 7.5 7.5 0.1
1.03 0.08
51
0.61 0.05
51
Control GW 66 end GW 67 original GW 67 end
7.2 8.1 7.5 0.1
0.63 0.05
51
0.93 0.17
51
Control GW 67 end GW 68 original GW 68 endd GW 68 end –N-P Control GW 68 end
7.2 8.1 5.7 0.1 6.5 0.0 6.7
1.42 0.01
51
1.06 0.71 0.22 0.27 0.96 0.23
GW 69 original GW 69 end Control GW 69 end
7.2 6.2 0.1 7.0
0.36 0.08 0.94 0.01
ÿ1 NH+ ) 4 (mg l
0.4 44.3 1.2 2.1 0.3 50.1
Heterotrophic bacteria (CFU ml ÿ 1) 1 102 2 105–1 106 0 2 104 3 105–7 106
n.d.c 51 51
62 1.3 46.3 0.5
0 5 101 1 106–8 106
n.d.a 51 51
32 1.4 47.3 0.5
0 n.d.c 2 105–1 106
51 51 51
n.d.a 51 51 51 n.d.a
3.1 1.7 30.0 4.0 0.4 0.0 37
0 0 6 105–1 106 1 107 0
51 51
51 151 11 n.d.a
1 0.2 0.0 2.4
8.2 103 1 106–1 108 0
a
Not determined. Statistical data are calculated from two experiments, the third assay showed no nitrite accumulation but oxidation to nitrate. Not determinable. d Statistical data are calculated from two experiments because the third assay showed no activity at all. b c
Fig. 1. O2- and CO2-kinetics of the degradation assays with highly contaminated groundwater from well 67 and 68. (O2-consumption and CO2-production for the assays GW 68; O2-concentration and CO2concentration for the assays GW 67).
an O2-consumption of approx. 3 mmol 100 ml ÿ 1 (Fig. 1). The groundwater sample contained, as described above, tar oil in the aqueous phase and accordingly a lot of contaminants especially high PAH concentrations. No aerobic bacteria could be found on R2A-agar in the groundwater sample 68 at the beginning (Table 4) and the toxicity was very high (Table 3). Hence, a microbial activity was not expected. However, the highest ammonium- and O2-consumption, high CO2-production and a significant pH-drop were observed in two of the three assays. One assay was not active at all and was not taken into consideration. A decrease of the BTEX and PAH concentrations in contrast to the abiotic control was determined in the two assays (Tables 5 and 6). As expected, the amount of the higher molecular weight PAHs did not decrease significantly
during the aerobic incubation because these PAHs are known to be not or moderately mineralizable (Grosser et al., 1995). 8.3 mg l ÿ 1 BTEX and 171.3 mg l ÿ 1 naphthalene were transformed in the assays within 159 days. The O2–consumption of 3 mmol 100 ml ÿ 1 was sufficient for the complete mineralization of these BTEX and naphthalene concentrations. In Fig. 2 the GC-MS chromatograms (headspace extraction) of one degradation assay and the abiotic control after 159 d incubation are compared. The abiotic control (Fig. 2(a)) contained a lot of different contaminants in high concentrations. Most of the contaminants were degraded during the aerobic incubation period (Fig. 2(b)). Corresponding to these results the HPLC-FLD-chromatograms of the degradation assay showed organic acids at the test end which were not found in the control (Fig. 2(c) and
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Table 5. BTEX concentrations of the degradation assays after approx. 140–160 days compared to the abiotic control (statistical data are calculated from three experiments) BTEX (mg l ÿ 1) Sample
Benzene
Toluene
Ethyl-benzene
m-/p-Xylene
o-Xylene/styrene
a
n.d. 50.001 50.001
Total
GW 64 begin. GW 64 end Control GW 64 end
n.d. 50.001 0.075
n.d. 50.001 0.004
n.d. 50.001 0.025
n.d. 50.001 0.014
GW 65 begin. GW 65 end Control GW 65 end
2.98 0.7 50.001 6.01
50.001 50.001 50.001
50.001 50.001 0.007
0.01 0.00 50.001 0.031
50.001 50.001 0.02
2.99 0.69 50.001 6.07
GW 66 begin. GW 66 end Control GW 66 end
0.32 0.02 50.001 1.36
0.29 0.03 50.001 0.40
0.25 0.02 50.001 0.25
0.23 0.05 50.001 0.27
0.16 0.02 50.001 0.15
1.26 0.02 50.001 2.43
GW 67 begin. GW 67 end Control GW 67 end
0.99 0.08 1.37 0.17 1.81
0.59 0.03 0.53 0.16 0.83
2.97 0.17 50,001 0.34
4.36 0.22 0.36 0.06 0.55
1.54 0.07 0.22 0.04 0.32
10.45 0.51 2.81 0.74 3.86
GW 68 begin. GW 68 endb GW 68–N-P end Control GW 68 end
n.d. 0.39 0.39 1.36 0.26 0.44
n.d. 0.97 0.74 7.37 0.93 2.33
n.d. 0.5 0.28 4.5 0.68 0.92
n.d. 1.18 0.76 19.46 2.57 4.51
n.d. 0.83 0.51 16.71 0.53 3.94
} 3.87 2.67 49.39 4.97 12.14
a b
} 50.001 0.12
n.d.: Not determined. Statistical data are calculated from two experiments because the third assay showed no activity at all.
Table 6. PAH concentrations of the degradation assays after approx. 140–160 days compared to the abiotic control (statistical data are calculated from three experiments) PAHs (mg l ÿ 1) Sample
Two-ring
Three-ring
Four-ring
Five-ring
Total
GW 64 begin. GW 64 end
0.41 0.03 0.02
0.37 50.01
50.01 50.01
50.01 50.01
0.78 0.03 0.02
Control GW 64 end GW 65 begin. GW 65 end Control GW 65 end
0.33 0.49 0.02 0.01 0.34
0.15 0.59 50.01 0.32
0.02 50.01 50.01 50.01
50.01 50.01 50.01 50.01
0.50 1.08 0.02 0.01 0.66
0.69 0.04 0.00 0.22
0.04 50.01 0.01
50.01 50.01 50.01
0.04 50.01 50.01
50.01 50.01 50.01
GW 66 begin. GW 66 end Control GW 66 end
1.88 50.01 1.01
GW 67 begin. GW 67 end Control GW 67 end
2.91 2.52 0.07 3.15
0.58 0.19 0.02 0.54
GW 68 begin. GW 68 enda GW 68–N-P end Control GW 68 end
213.07 29.77 23.13 269.37 1.74 201.08
109.68 81.63 1.71 41.57 18.82 99.53
a
1.03 16.3 0.95 54.19 40.46 22.04
2.05 4.07 0.29 12.15 3.70 7.08
2.61 50.01 1.23 3.53 2.71 0.08 3.69 325.83 131.76 24.18 375.22 21.55 329.72
Statistical data are calculated from two experiments because the third assay showed no activity at all.
(d)). By comparison of retention time, UV- and FLD-spectra with reference substances substituted naphthalenes and some methyl-benzoic acids could be identified. Apparently, completely different growth conditions were predominant in the groundwater sample 68 compared to the other samples. Micelles were formed because of the tar oil in the aqueous phase and bacteria could be adsorbed at the surface of these micelles. Hence, the presence of the micelle surface probably stimulated the microbial activity. Further assays with groundwater 68 without Nand P-supplementation showed only few microbial
degradation activity as concluded from O2-consumption, CO2-production as well as the BTEX and PAH concentrations at the test ends (Tables 5, 6 and Fig. 1). The results indicate that natural attenuation at this site without N- and/or P- supplementation probably would not occur. The groundwater sample of the well 67 showed no microbial activity in the degradation assays. Neither CO2-production nor O2- or ammonium consumption was detected (Fig. 1). In addition, the difference in the PAH and the BTEX concentrations compared to the abiotic control was not significant. Possibly the microbial activity was inhibited by the contaminants,
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Fig. 2. GC-MS-chromatograms (headspace extraction) of the volatile organic carbons of one assay with the groundwater sample from well 68 (a) and the abiotic control (b) and HPLC-FLD-chromatograms of polar organic compounds of this assay (c) and the abiotic control (d) after 159 days incubation.
since the sample showed a high ecotoxicological effect. The sample contained a broad spectrum of contaminants. Therefore, it was not possible to identify the effective toxic substances. Comparing the results of groundwater sample 67 and 68 bacteria could not be found in the original groundwater samples of both wells. The contamination and the toxicity were higher in sample 68 than in sample 67. Nevertheless, the microbial activity of the assays with groundwater 68 was very high and the assays with groundwater 67 showed no activity at all. These results confirm that it is rather difficult to assess microbial processes and the fate of xenobiotica in contaminated groundwater by analysis of physical, chemical, toxicological and biological parameters of groundwater samples. Low and medium contaminated groundwater samples The groundwater sample of well 66 was moderately contaminated with BTEX and PAHs and therefore microbial degradation activity was expected. The O2-consumption was significantly lower than in the assays with the sample 68 (Fig. 3), but BTEX were consumed completely (Table 5). PAHs were nearly completely transformed, too. Only threering PAHs were found in traces in one of the assays which also showed the lowest O2-consumption of the three parallel assays. Another phenomenon of microbial activity was found in the assays with groundwater of the wells 65
and 69, which are low or not contaminated and therefore contained a low organic carbon content. O2 was consumed, the pH dropped, ammonium was used completely and nitrate or nitrite accumulated (Fig. 3, Table 4). This shows that chemolithoautotrophic nitrifyers were active. The CO2-production kinetics of the assays with groundwater of well 65 showed that in the first four weeks BTEX degradation occurred accompanied by a diauxic growth pattern. The CO2-concentration increased in two steps. This may be caused by a change of the growth substrate from toluene to benzene, which is more difficult to mineralise (Oh et al., 1994). Later on CO2 was consumed by the nitrifyers and decreased in the gaseous and aqueous phase over time. The O2kinetics show also the diauxic growth, but the O2consumption curve for the degradation of the second substrate conceded with the O2-consumption of the nitrification. Nitrite accumulated in two assays in high amounts (>80 mg l ÿ 1), whereas in one assay ammonium was completely oxidised to nitrate (254 mg l ÿ 1). The groundwater of well 69 was not polluted with BTEX and PAHs and showed only nitrifying activity. The supplied ammonium was nearly completely oxidised to nitrate and the second highest O2-consumption was observed. Neither degradation nor nitrifying activity could be detected in the assays with groundwater of well 64. The pH did not change during the incubation, ammonium and oxygen were not consumed and the CO2-concentrations in the water phase was similar as
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Fig. 3. O2- and CO2-kinetics of the degradation assays with groundwater from well 64, 65, 66 and 69: (O2-consumption and CO2-production for the assays 65, 66 and 69; O2-concentration and CO2concentration for the assay 64).
in the control assay. Only the number of aerobic bacteria increased during the incubation but, as stated above, this parameter gives no information about the degrading activity. BTEX and PAHs disappeared during incubation under aerobic conditions. Besides, the GC-MS-chromatograms show a complete decrease (except aniline) of all contaminants. In contrast, all contaminants were detected in the abiotic control (data not shown). This elimination can be partly due to degradation processes, which are too small for detection by O2 and CO2monitoring. But the disappearance of the higher molecular weight PAHs caused by degradation activity is improbable. Possibly, the produced biomass serves as biological matrix for sorption of low concentrations of hydrophobic organic contaminants as described elsewhere (Stringfellow and AlvarezCohen, 1999). CONCLUSIONS
The microbial activity of six groundwater samples from a former gas plant site with different levels of contamination was studied in batch culture by monitoring the O2- and CO2-concentrations over time and analyses of some physical and chemical parameters at the end of the experiments. One of the highly contaminated samples (68) showed high degradation activity, whereas the other was not active at all due to toxic substances (67). The contaminants were nearly completely degraded in the medium and low polluted samples (66, 65, 64). Two low-contaminated samples showed nitrifying activity (65, 69). The results show that *
The degradation capacity of the native microbial population and thereby the natural attenuation
*
*
*
potential cannot be predicted simply by chemical, microbial and ecotoxicological data of groundwater samples from contaminated sites. Additionally, it is recommended to perform degradation tests with the authentic groundwater samples with their contaminant mixture. The degradation tests cannot be characterised only by CO2 and O2 detection. To distinguish microbial degradation activity from other microbial and sorption processes it is necessary to determine parameters like the concentrations of the contaminants, electron acceptors and metabolites in the degradation assays. The counts of aerobic bacteria in the original groundwater and in the assays at test end do not relate with the observed microbial activity. Natural attenuation at this former gas plant site with just a small autochthonous microflora without ammonium and phosphate supplementation would not be successful as a remediation strategy.
Acknowledgements}This paper carries publication no. 118 of the Priority Program 546 ‘‘Geochemical processes with long-term effects in anthropogenically-affected seepage- and groundwater’’. Financial support was provided by Deutsche Forschungsgemeinschaft. The authors wish to thank M. Mo¨ller, M. Duisken, U. Kaufmann, C. Schmitz, N. Mende and K. Ehrhardt for their technical assistance.
REFERENCES
Albrechtsen H.-J., Smith P. M., Nielsen P. and Christensen T. H. (1997) Importance of sediment fines in laboratory studies on the degradation of organic chemicals in aquifers. Water Res. 31, 2287–2299. Alef K. (1991) Handbook of Methods for Soil Microbiology: Activities, Biomass, Differences. Landsberg/Lech, ecomed.
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Alexander M. (1982) Most probable number method for microbial populations. In Methods of Soil Analysis Part 2, eds A. L. Page, R. H. Miller and D. R. Keeney, pp. 815– 820. Amer. Soc. for Agron, Madison, USA. Alfreider A., Kro¨ssbacher M. and Psenner (1997) Groundwater samples do not reflect bacterial densities and activity in subsurface systems. Water Res. 31(4), 832–840. Annweiler E., Eiswirth M., Ho¨tzl H., Michaelis W., Pape T., Reichert B. and Richnow H. H. (1997) Distribution, transport and biodegradation of organic contaminants in a near surface aquifer in the area of a manufactured gas plant site. Proceedings of the EUG-Conference, March, Straburg, pp. 23–27. Carmichael L. M. and Pfaender F. K. (1997) Polynuclear aromatic hydrocarbon metabolism in soils: relationship to soil characteristics and preexposure. Environ. Toxicol. Chem. 16, 666–675. Cho J. S., Wilson J. T., DiGiulio D. C., Vardy J. A. and Choi W. (1997) Implementaion of natural attenuation at a JP-4 jet fuel release after active remediation. Biodegradation 8, 265–273. Dyreborg S., Arvin E. and Broholm K. (1997) Biodegradation of NSO-compounds under different redox-conditions. J. Contam. Hydrol. 25, 177–197. Grosser R. J., Warshawsky D. and Vestal J. R. (1995) Mineralization of polycyclic and N-heterocyclic aromatic compounds in hydrocarbon-contaminated soils. Environ. Toxicol. Chem. 14, 375–382. Ho¨hener P., Hunkeler D., Hess A., Bregnard T. and Zeyer J. (1998) Methodology for the evaluation of engineered in situ bioremediation: lessons from a case study. J. Microbiol. Methods 32, 179–192. Kao C. M. and Borden R. C. (1997) Site-specific variability in BTEX biodegradation under denitrifying conditions. Ground Water 35, 305–311.
King M. W. G., Barker J. F., Devlin J. F. and Butler B. J. (1999) Migration and natural fate of a coal tar creosote plume 2. Mass balance and biodegradation indicators. J. Contam. Hydrol. 39, 281–307. Nielsen P. H., Bjarnado´ttir H., Winter P. L. and Christensen T. H. (1995) In situ and laboratory studies on the fate o specific organic compounds in an anaerobic landfill leachate plume, 2. Fate of aromatic and chlorinated aliphatic compounds. J. Contam. Hydrol. 20, 51–66. Oh Y.-S., Shareefdeen Z., Baltzis B.-C. and Bartha R. (1994) Interactions between benzene, toluene, and pxylene (BTX) during their biodegradation. Biotechnol. Bioengng. 44, 533–538. Poeton T. S., Stensel H. D. and Strand S. E. (1999) Biodegradation of polyaromatic hydrocarbons by marine bacteria: effect of solid phase on degradation kinetics. Water Res. 33, 868–880. Schmitz R. P. H., Eisentra¨ger A. and Dott W. (1998) Miniaturized kinetic growth inhibition assays with Vibrio fischeri and Pseudomonas putida (application, validation and comparison). J. Microbiol. Meth. 31(1), 159–166. Scow K. M. and Alexander M. (1992) Effect of diffusion on the kinetics of biodegradation: experimental results with synthetic aggregates. Soil. Sci. Soc. Am. J. 56, 128–134. Stapelton R. D. and Sayler G. S. (1998) Assessment of the microbiological potential for the natural attenuation of petroleum hydrocarbons in a shallow aquifer system. Microb. Ecol. 36, 349–361. Stringfellow W. T. and Alvarez-Cohen L. (1999) Evaluating the relationship between the sorption of PAHs to bacterial biomass and biodegradation. Water Res. 33, 2535–2544. Zhang W.-X. and Bouwer E. J. (1997) Biodegradation of benzene, toluene and naphthalene in soil-water slurry microcosms. Biodegradation 8, 167–175.
Wat. Res. Vol. 35, No. 3, pp. 720–728, 2001 # 2001 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/01/$ - see front matter
MICROCOSMS-EXPERIMENTS TO ASSESS THE POTENTIAL FOR NATURAL ATTENUATION OF CONTAMINATED GROUNDWATER KATRIN ALTHOFF, MATTHIAS MUNDT, ADOLF EISENTRAEGER, WOLFGANG DOTT and JULIANE HOLLENDER* Institute of Hygiene and Environmental Medicine, RWTH Aachen,Pauwelsstrae 30, D-52074 Aachen, Germany (First received 25 February 2000; accepted in revised form 9 June 2000) Abstract}Groundwater samples from six wells of a former gas plant site were characterised using chemical, microbial and ecotoxicological methods. Degradation studies were performed in batch-culture under aerobic conditions with the groundwater samples containing their autochthonous microflora and original contaminant mixture. The highest O2-consumption (3 mmol 100 ml ÿ 1), combined with BTEX (8.3 mg l ÿ 1) and naphthalene (171.3 mg l ÿ 1) degradation, as well as formation of organic acids was found after N- and P-supplementation with the highest contaminated groundwater sample. The other highly polluted groundwater sample showed no activity obviously because of the toxicity of some compounds. The major part of the PAHs and BTEX was eliminated in the assays with the low contaminated groundwater samples. The results indicate that the microbial degradation capacity and thereby the natural attenuation capacity in each groundwater differ and cannot be assessed simply by chemical, microbial and toxicological data. Additionally activity tests with authentic groundwater samples with and without nutrient supplementation are recommended. # 2001 Elsevier Science Ltd. All rights reserved Key words}BTEX, groundwater, laboratory batch studies, microbial degradation, natural attenuation, PAHs
INTRODUCTION
At gas plant sites contamination of soils and groundwater has occurred from disposal of wastes generated during the manufacturing process of city gas as well as from tank leakage. Typical contaminations include tar residues and sludge containing various heavy metals, volatile organic compounds (VOC), monoaromatic hydrocarbons and polycyclic aromatic hydrocarbons (PAHs). Natural attenuation at such sites is getting more and more interesting, because it seems to enable the bioremediation with minimised cost, avoiding land disruption and human exposure. A number of processes determine natural attenuation of organic contaminants in soil and groundwater such as biodegradation, dispersion, sorption, dissolution and volatilisation (Cho et al., 1997; Stapelton and Sayler, 1998). Many different chemical, physical, biological and geological parameters of both kind of pollutants and aquifer influence these processes. Therefore, multidisciplinary studies and an extensive characterisation of the
*Author to whom all correspondence should be addressed. Tel.: +49-241-8088-282; fax: +49-241-8888-477; e-mail: [email protected] 720
field are necessary to provide an accurate assessment of natural attenuation capacity. In order to understand the fate of xenobiotica microcosms, columns, batch-cultures, lysimeter and field studies were performed. Each of these methods has its advantages and is appropriate to answer special questions (Ho¨hener et al., 1998). An up-scaling to real-field conditions especially by results from laboratory experiments is difficult. But the influence of many different parameters such as redox conditions, oxygen concentration, nutrient concentration on the microbial degradation can be investigated by microcosms studies. Soil or groundwater with its authentic contamination is rarely used for experiments (Dyreborg et al., 1997). Most laboratory experiments concerning the degradation of organic compounds in aquifers include aquifer sediment, because the use of groundwater alone has resulted in lower degradation rates (Albrechtsen et al., 1997; Poeton et al., 1999). The solid particles were used as biomass support material, as inoculum and as surface area for colonisation. However, from some aquifers sediment cannot easily be obtained because of deep wells of geologic settings of boulders. Besides, soil samples frequently are very heterogeneous and therefore are not representative of the site. Hot spots of
Natural attenuation potential of contaminated groundwater
contaminants occur and bioavailability is influenced strongly by sorption processes. Because of the latter it is difficult to distinguish between microbial degradation and sorption to the soil (Nielsen et al., 1995). Some investigations showed higher degradation rates with groundwater without sediment because the kinetic of the desorption of the organic compounds is slower than the degradation rate (Scow and Alexander, 1992; Zhang and Bouwer, 1997). The groundwater of contaminated sites reflects the fate of the plume, the current situation of the contamination and plays the main part in leaching, release and transport processes of pollutants. For these reasons studies with groundwater are recommended but the question is whether investigations with pure groundwater can reflect microbial degradation activity. How is the degradation capacity of free bacteria living in a low-nutrient environment such as groundwater and not adsorbed on particles inhabit organically rich microzones (Alfreider et al., 1997)? In this work authentic groundwater samples of six different wells of a contaminated site were used to study the microbial degradation capacity under aerobic conditions with and without N- and Psupplementation. A method was developed and evaluated to assess and observe the microbial activity in native groundwater with autochthonous microflora and original contaminant mixture by monitoring O2-consumption and CO2-production.
MATERIALS AND METHODS
Study site and samples The groundwater samples (GW 64 to 69) were collected from six wells distributed over the area of a former gas plant. The investigation site is located in the south-west of Germany in the Neckar valley. Below the site is a quaternary porous aquifer of medium-grained gravel intersected locally by sandy and silty lenses. The first aquifer is completed by a sequence of upper triassic rocks (Middle Keuper). Till the 1970s the gasworks produced city gas, thus, there are contaminations by products of coal gas and the following processes like tar distillation and gas washing. A heterogeneous contamination, consisting of aliphatic and aromatic hydrocarbons (BTEX, indane, indene, biphenyls, PAHs, etc.), heterocyclic aromatic compounds containing oxygen or sulphur (benzofurane, benzothiophene, etc.) in high concentrations as well as phenols and compounds containing nitrogen (quinoline, pyridine, etc.) in low concentrations was detected at this site (Annweiler et al., 1997). Besides this high contamination with organic compounds there is a pollution with inorganic compounds such as NH3, cyanides and sulphur caused by clinker and gas washing. Physico-chemical methods The pH and the conductivity were measured with a glass electrode described in DIN 38 404 part 5. Cations were determined with atomic absorption spectrometry (DIN 38 406) apart from NH+ which was detected with an 4 ammonium-selective electrode Orion 9512 (Orion Research, Boston, USA). Anions were measured using an ion chromatograph DX-100 (Dionex, Sunnyvale, USA) according to DIN 38 405 part 20. Eh was measured by platinum
721
electrode (Mettler Toledo, Urdorf, Swiss). The CO2concentration in the aqueous phase was determined according to DIN 38 405 part 8. The concentrations of the 16 US Environmental Protection Agency (EPA) PAHs in the groundwater samples were determined after twice repeated extraction with pentane using 1,10 -binaphthyl as internal standard. A HewlettPackard 1050 HPLC-system with Beckmann pumps (model 126, Beckmann, Munich, Germany), a time programmable fluorescence detector and a diode array detector were used. Separation was achieved at 358C on a 250 mm 3 mm ID MZ-PAH column (particle size 5 mm) with a 12.5 mm 3 mm I.D. pre-column (MZ-Analytical, Mainz, Germany). An acetonitrile–water gradient, from 60 to 100% acetonitrile in 35 min, was used as mobile phase with a total flow rate set to 0.6 ml min ÿ 1. For the detection of polar aromatic compounds an acetonitrile–water gradient, from 30 to 100% acetonitrile within 35 min, was used. BTEX concentrations were measured by GC-MS using multiple headspace extraction (MHE). The samples were taken from the infusion bottles after cooling the batch culture to 48C to avoid loss of VOC. 20 ml glass headspace vials were filled with 5 ml of sample, sealed with Teflon-lined septa and frozen until analysis. Analyses were performed using a Hewlett-Packard HP 5890 gas chromatograph system equipped with a HP-5-MS-capillary column (30 m 0.25 mm ID) coated with chemically bonded SE-54 (df=0.25 mm; Hewlett-Packard, Waldbronn, Germany). Helium (99.9 v/v) (1.6 ml min ÿ 1) was utilised as carrier gas. The analyses were performed using the following temperature program: 408C held for 1 min, heating rate 48C min ÿ 1 up to 3108C, final temperature held for 1.5 min. Injector and detector temperatures were 250 and 3008C, respectively; detection was achieved using HP 5872 mass detector. CO2-production and O2-consumption during the degradation tests were measured by GC with thermal conductivity detector (TCD) (Perkin Elmer, U¨berlingen, Germany, model autosystem) using a CTR 1-column (Alltech GmbH, Unterhaching, Germany). The injection volume of 100 ml was taken manually with a gastight 100 ml-syringe (Hamilton, Darmstadt, Germany) from the headspace of the assays. The column temperature was set at 408C isothermal. Helium (62.5 ml min ÿ 1) was utilised as carrier gas. Injector and detector temperatures were 65 and 1408C, respectively.
Microbial methods Aerobic and facultative anaerobic bacteria were counted as colony-forming units (cfu). The groundwater samples were diluted in decimal steps up to 10 ÿ 6 and plated out on R2A-Agar (Difco Laboratories, Detroit, USA). Two parallels of each dilution step were incubated for 10 days at 258C. Sulphate-reducing microorganisms were quantified by a turbidimitric most probable number (MPN) three-tube technique (Alexander, 1982) with liquid medium according to Alef (1991). The results were determined after incubation for six weeks at 258C.
Ecotoxicological methods The measuring parameter for the bioluminescence inhibition test with Vibrio fischeri is the decrease of the light emission through presence of toxic substances in the groundwater samples (DIN 38412-part 341). In the algal growth inhibition test with Scenedesmus subspicatus inhibition of the biomass production was determined (DIN 38412-part 33). In the growth inhibition tests with Pseudomonas putida (DIN 38412-part 8) and Vibrio fischeri (DIN 38412-part 37) inhibition of cell increase was measured photometrically. Both tests were modified to microplate assays (Schmitz et al., 1998). The Daphnia magna
0.01 0.01 0.01 0.01 2.05 5 0.01 5 5 5 5
0.03 0.03 0.04 0.03 1.03 0.03 0.33 0.56 0.69 0.58 109.68 0.09 0.41 0.49 1.88 2.91 213.07 0.16 1.75 17.83 3.63 12.29 25.75 0.53 0.17 5 0.001 0.47 1.06 7.46 5 0.001 0.12 0.013 0.44 2.24 8.47 5 0.001 0.12 5 0.001 0.50 1.77 1.88 5 0.001 1.23 4.60 1.04 2.69 6.25 0.53 1945–70 tar oil pit 1914–45 benzene distillation and tanks Tanks 1915–70/74 tar distillation/pitch processing 1910–70/74 tar sedimentation tank 1970/74 deposit/anthropogenic filling 64 65 66 67 68 69
0.12 13.22 1.18 4.54 1.70 5 0.001
Four-ring Three-ring Total BTEX Styrene/o-xylene m-/p-Xylene
BTEX (mg l ÿ 1)
Toluene
The location of the six investigated wells on the former gas plant is listed in Table 1. The BTEX and PAH concentrations of the six groundwater samples differ to a large extent. Well 69 is not located within the plume and therefore the groundwater was only slightly polluted. The sample of well 64 also contained only low concentrations of contaminants despite the well position next to a former tar oil pit. Well 65 is located at a former benzene distillation and therefore the groundwater was highly polluted with benzene. Well 67 was drilled near the former tar distillation and pitch processing. The groundwater sample of this well contained a broad spectrum of contaminants in high concentrations. Well 66 lies near the gas tanks and the groundwater sample contained a wide spectrum of pollutants in medium concentrations. Well 68 was originally not highly polluted, but during pumping of the groundwater an old subsurface lake of tar oil was tapped. High amount of tar oil was released and droplets of tar oil swam on the aqueous phase. Therefore, the groundwater sample was very heterogeneous and some chemical analyses and the growth inhibition tests
Benzene
Characterisation of the groundwater samples
Contamination history
RESULTS AND DISCUSSION
Well
The groundwater samples were taken in autumn 1998. They were filled in autoclaved 1 l-infusion bottles, closed gastight with viton-butylrubber stoppers, which were specially produced for these experiments by Reichelt GmbH (Heidelberg, Germany), placed in coolers during transportation to the laboratory and stored at 48C. At most after 2–3 days the groundwater samples were used for the degradation tests. Three parallel degradation assays and two abiotic controls (0.5% sodium azide) were prepared with the groundwater samples from the six wells, respectively. One hundred and twenty millilitres groundwater was filled in 150 ml infusion bottles and closed gastight with a vitonbutylrubber-stopper. Apart from additional assays with groundwater from well 68 all batch cultures were supplemented with ammonium and phosphate to a final concentration of 2.8 mM N and 0.11 mM P to avoid nutrient limitation. The assays were shaken at 120 rpm in the dark at 208C. Monitoring the CO2- and O2-concentration in the gaseous phase was performed regularly. At the beginning samples for BTEX-analysis were taken and after approx. 140–160 days samples were collected for analysis of BTEX (2 5 ml), anions and ammonium (2 ml), the CO2-content in the aqueous phase (3 5 ml) and the quantification of aerobic microorganisms (1 ml). The remaining groundwater was extracted with pentane prior to the PAH analysis. Oxygen as electron acceptor was available throughout the batch experiments.
Table 1. BTEX and PAH concentrations of the original groundwater samples of the six wells
Degradation tests
Two-ring
PAHs (mg l ÿ 1)
Five-ring
Total
survival test was based on the inhibition of the mobility (DIN 38 412-part 30). The results of the tests were expressed as G-values, which are defined as the smallest reciprocal dilution factor with an inhibition of less than 20% (for the acute luminescenceinhibition assay, the algal growth-inhibition test and the growth-inhibition tests with V. fischeri and P. putida) and 10% (for the D. magna survival test), respectively.
0.77 1.08 2.61 3.52 325.83 0.28
Katrin Althoff et al.
Ethyl-benzene
722
Natural attenuation potential of contaminated groundwater
723
lites of degradation processes as described by King et al. (1999). The number of microorganisms growing on R2Aagar increased in every assay. But there is no significant relation between the microbial counts and the observed microbial activity in the degradation test as described elsewhere (Kao and Borden, 1997; Carmichael and Pfaender, 1997). However, it is well known that it is difficult to quantify microorganisms from groundwater or soil samples with bacterial growing techniques and to isolate especially the degrading bacteria without a xenobiotic compound as growth substrate (Stapleton and Sayer, 1998). Monitoring the CO2- and O2-concentration in the headspace (Figs 1, 3) showed that the O2-consumption is the more reliable parameter for quantification of the microbial activity, although O2 is less exact than CO2 because of its higher partial pressure in the air. CO2 is detectable with more sensitivity but depending on the pH-value a high part of CO2 remains in the aqueous phase. So it is most suitable to measure both parameters as well as the CO2concentration in the liquid phase to get the most information. Generally, the activity of the groundwater samples determined by the O2-consumption and CO2-production was low apart from sample 68 (Fig. 1).
with Scenedesmus subspicatus could not be performed. The physical and chemical data of the groundwater samples are summarised in Table 2. The sulphate concentration was high in all samples apart from the sample of well 65. This indicates a sulphate reducing activity in this part of the aquifer, which was supported by the occurrence of sulphate reducing microorganisms in the sample of well 65 determined by the MPN method. None of the groundwater samples was contaminated with heavy metals. Therefore, toxic effects on the microbial activity caused by heavy metals could be excluded. The results of the ecotoxicity tests are shown in Table 3. The groundwater sample of well 68 with tar oil in phase showed the highest toxicity. High Gvalues were detected in the bioluminescence inhibition test with Vibrio fischeri and in the survival test with Daphnia magna. The groundwater of well 67 showed also a high toxicity probably caused by an additive effect of many different contaminants or the occurrence of substances in toxic levels which repress all microbial activity. Degradation studies The characteristic physico-chemical parameters of the degradation assays of the groundwater samples were compared with the data of the original samples and with the abiotic controls at the end of the test (Table 4). A drop of the pH-value in the test assays was a first indication for microbial activity caused by the microbial formation of organic acids as metabo-
Highly contaminated groundwater samples In the assays with groundwater from well 68 high microbial activity was found (Table 4) as shown by
Table 2. Physicochemical characteristics of the original groundwater samples of the six wells Well 64 Ph O2 (% saturation) Eh (mV) Conductivity (mS cm ÿ 1) Anions (mg l ÿ 1)
Cations (mg l ÿ 1)
Cl ÿ NO2ÿ NO3ÿ PO34 ÿ SO24 ÿ NH+ 4 Mg2+ 2+ Ca
65
7.6 4.0 ÿ 68.6 1.5 174.5 50.1 51 51 796.5 0.4 76.3 386.1
66
7.2 10.6 +181 0.9 74.8 50.1 10.9 51 17.6 0.3 42.6 183.8
7.5 1.6 ÿ 103.4 1.7 164.3 50.1 51 51 788.7 1.3 85.2 337.8
67 8.1 4.0 ÿ 99.6 1.6 149.6 50.1 51 51 614.1 1.4 45.8 356.2
68 8.1 4.9 +203.6 1.7 199.0 50.1 51 51 1062.5 1.7 81.8 501.0
69 7.2 10.8 +212 1.3 92.4 50.1 51 51 526.6 1.0 44.8 257.4
Table 3. Ecotoxicological characteristics of the original groundwater samples of the six wells Ecotoxicological test
Vibrio fischeri bioluminescence inhibition test Pseudomonas putida growth inhibition test Vibrio fischeri growth inhibition test Daphnia magna survival test Scenedesmus subspicatus growth inhibition test a
n.d.: Not determined.
G-value 64
65
66
67
68
64 16 2 2 4
128 8 4 4 2
128 16 2 2 2
256 16 16 6 128
4096 16 8 96 n.d.
69 2 8 2 n.d.a n.d.
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Katrin Althoff et al.
Table 4. Results of the aerobic degradation studies after approx. 140–160 days: mean value of the three parallel assays compared to the abiotic control and the original groundwater sample of the six wells Sample
pH
CO2 (water) (mmol 100 ml ÿ 1]
NO2ÿ (mg l ÿ 1)
NO3ÿ (mg l ÿ 1)
GW 64 original GW 64 end Control GW 64 end
7.6 7.5 0.3 7.1
0.61 0.19 0.52 0.01
51 51
51 51 n.d.a
GW 65 original GW 65 end
7.2 6.0 0.2
0.49 0.12
>80b
11 15 1b
Control GW 65 end GW 66 original GW 66 end
6.8 7.5 7.5 0.1
1.03 0.08
51
0.61 0.05
51
Control GW 66 end GW 67 original GW 67 end
7.2 8.1 7.5 0.1
0.63 0.05
51
0.93 0.17
51
Control GW 67 end GW 68 original GW 68 endd GW 68 end –N-P Control GW 68 end
7.2 8.1 5.7 0.1 6.5 0.0 6.7
1.42 0.01
51
1.06 0.71 0.22 0.27 0.96 0.23
GW 69 original GW 69 end Control GW 69 end
7.2 6.2 0.1 7.0
0.36 0.08 0.94 0.01
ÿ1 NH+ ) 4 (mg l
0.4 44.3 1.2 2.1 0.3 50.1
Heterotrophic bacteria (CFU ml ÿ 1) 1 102 2 105–1 106 0 2 104 3 105–7 106
n.d.c 51 51
62 1.3 46.3 0.5
0 5 101 1 106–8 106
n.d.a 51 51
32 1.4 47.3 0.5
0 n.d.c 2 105–1 106
51 51 51
n.d.a 51 51 51 n.d.a
3.1 1.7 30.0 4.0 0.4 0.0 37
0 0 6 105–1 106 1 107 0
51 51
51 151 11 n.d.a
1 0.2 0.0 2.4
8.2 103 1 106–1 108 0
a
Not determined. Statistical data are calculated from two experiments, the third assay showed no nitrite accumulation but oxidation to nitrate. Not determinable. d Statistical data are calculated from two experiments because the third assay showed no activity at all. b c
Fig. 1. O2- and CO2-kinetics of the degradation assays with highly contaminated groundwater from well 67 and 68. (O2-consumption and CO2-production for the assays GW 68; O2-concentration and CO2concentration for the assays GW 67).
an O2-consumption of approx. 3 mmol 100 ml ÿ 1 (Fig. 1). The groundwater sample contained, as described above, tar oil in the aqueous phase and accordingly a lot of contaminants especially high PAH concentrations. No aerobic bacteria could be found on R2A-agar in the groundwater sample 68 at the beginning (Table 4) and the toxicity was very high (Table 3). Hence, a microbial activity was not expected. However, the highest ammonium- and O2-consumption, high CO2-production and a significant pH-drop were observed in two of the three assays. One assay was not active at all and was not taken into consideration. A decrease of the BTEX and PAH concentrations in contrast to the abiotic control was determined in the two assays (Tables 5 and 6). As expected, the amount of the higher molecular weight PAHs did not decrease significantly
during the aerobic incubation because these PAHs are known to be not or moderately mineralizable (Grosser et al., 1995). 8.3 mg l ÿ 1 BTEX and 171.3 mg l ÿ 1 naphthalene were transformed in the assays within 159 days. The O2–consumption of 3 mmol 100 ml ÿ 1 was sufficient for the complete mineralization of these BTEX and naphthalene concentrations. In Fig. 2 the GC-MS chromatograms (headspace extraction) of one degradation assay and the abiotic control after 159 d incubation are compared. The abiotic control (Fig. 2(a)) contained a lot of different contaminants in high concentrations. Most of the contaminants were degraded during the aerobic incubation period (Fig. 2(b)). Corresponding to these results the HPLC-FLD-chromatograms of the degradation assay showed organic acids at the test end which were not found in the control (Fig. 2(c) and
Natural attenuation potential of contaminated groundwater
725
Table 5. BTEX concentrations of the degradation assays after approx. 140–160 days compared to the abiotic control (statistical data are calculated from three experiments) BTEX (mg l ÿ 1) Sample
Benzene
Toluene
Ethyl-benzene
m-/p-Xylene
o-Xylene/styrene
a
n.d. 50.001 50.001
Total
GW 64 begin. GW 64 end Control GW 64 end
n.d. 50.001 0.075
n.d. 50.001 0.004
n.d. 50.001 0.025
n.d. 50.001 0.014
GW 65 begin. GW 65 end Control GW 65 end
2.98 0.7 50.001 6.01
50.001 50.001 50.001
50.001 50.001 0.007
0.01 0.00 50.001 0.031
50.001 50.001 0.02
2.99 0.69 50.001 6.07
GW 66 begin. GW 66 end Control GW 66 end
0.32 0.02 50.001 1.36
0.29 0.03 50.001 0.40
0.25 0.02 50.001 0.25
0.23 0.05 50.001 0.27
0.16 0.02 50.001 0.15
1.26 0.02 50.001 2.43
GW 67 begin. GW 67 end Control GW 67 end
0.99 0.08 1.37 0.17 1.81
0.59 0.03 0.53 0.16 0.83
2.97 0.17 50,001 0.34
4.36 0.22 0.36 0.06 0.55
1.54 0.07 0.22 0.04 0.32
10.45 0.51 2.81 0.74 3.86
GW 68 begin. GW 68 endb GW 68–N-P end Control GW 68 end
n.d. 0.39 0.39 1.36 0.26 0.44
n.d. 0.97 0.74 7.37 0.93 2.33
n.d. 0.5 0.28 4.5 0.68 0.92
n.d. 1.18 0.76 19.46 2.57 4.51
n.d. 0.83 0.51 16.71 0.53 3.94
} 3.87 2.67 49.39 4.97 12.14
a b
} 50.001 0.12
n.d.: Not determined. Statistical data are calculated from two experiments because the third assay showed no activity at all.
Table 6. PAH concentrations of the degradation assays after approx. 140–160 days compared to the abiotic control (statistical data are calculated from three experiments) PAHs (mg l ÿ 1) Sample
Two-ring
Three-ring
Four-ring
Five-ring
Total
GW 64 begin. GW 64 end
0.41 0.03 0.02
0.37 50.01
50.01 50.01
50.01 50.01
0.78 0.03 0.02
Control GW 64 end GW 65 begin. GW 65 end Control GW 65 end
0.33 0.49 0.02 0.01 0.34
0.15 0.59 50.01 0.32
0.02 50.01 50.01 50.01
50.01 50.01 50.01 50.01
0.50 1.08 0.02 0.01 0.66
0.69 0.04 0.00 0.22
0.04 50.01 0.01
50.01 50.01 50.01
0.04 50.01 50.01
50.01 50.01 50.01
GW 66 begin. GW 66 end Control GW 66 end
1.88 50.01 1.01
GW 67 begin. GW 67 end Control GW 67 end
2.91 2.52 0.07 3.15
0.58 0.19 0.02 0.54
GW 68 begin. GW 68 enda GW 68–N-P end Control GW 68 end
213.07 29.77 23.13 269.37 1.74 201.08
109.68 81.63 1.71 41.57 18.82 99.53
a
1.03 16.3 0.95 54.19 40.46 22.04
2.05 4.07 0.29 12.15 3.70 7.08
2.61 50.01 1.23 3.53 2.71 0.08 3.69 325.83 131.76 24.18 375.22 21.55 329.72
Statistical data are calculated from two experiments because the third assay showed no activity at all.
(d)). By comparison of retention time, UV- and FLD-spectra with reference substances substituted naphthalenes and some methyl-benzoic acids could be identified. Apparently, completely different growth conditions were predominant in the groundwater sample 68 compared to the other samples. Micelles were formed because of the tar oil in the aqueous phase and bacteria could be adsorbed at the surface of these micelles. Hence, the presence of the micelle surface probably stimulated the microbial activity. Further assays with groundwater 68 without Nand P-supplementation showed only few microbial
degradation activity as concluded from O2-consumption, CO2-production as well as the BTEX and PAH concentrations at the test ends (Tables 5, 6 and Fig. 1). The results indicate that natural attenuation at this site without N- and/or P- supplementation probably would not occur. The groundwater sample of the well 67 showed no microbial activity in the degradation assays. Neither CO2-production nor O2- or ammonium consumption was detected (Fig. 1). In addition, the difference in the PAH and the BTEX concentrations compared to the abiotic control was not significant. Possibly the microbial activity was inhibited by the contaminants,
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Fig. 2. GC-MS-chromatograms (headspace extraction) of the volatile organic carbons of one assay with the groundwater sample from well 68 (a) and the abiotic control (b) and HPLC-FLD-chromatograms of polar organic compounds of this assay (c) and the abiotic control (d) after 159 days incubation.
since the sample showed a high ecotoxicological effect. The sample contained a broad spectrum of contaminants. Therefore, it was not possible to identify the effective toxic substances. Comparing the results of groundwater sample 67 and 68 bacteria could not be found in the original groundwater samples of both wells. The contamination and the toxicity were higher in sample 68 than in sample 67. Nevertheless, the microbial activity of the assays with groundwater 68 was very high and the assays with groundwater 67 showed no activity at all. These results confirm that it is rather difficult to assess microbial processes and the fate of xenobiotica in contaminated groundwater by analysis of physical, chemical, toxicological and biological parameters of groundwater samples. Low and medium contaminated groundwater samples The groundwater sample of well 66 was moderately contaminated with BTEX and PAHs and therefore microbial degradation activity was expected. The O2-consumption was significantly lower than in the assays with the sample 68 (Fig. 3), but BTEX were consumed completely (Table 5). PAHs were nearly completely transformed, too. Only threering PAHs were found in traces in one of the assays which also showed the lowest O2-consumption of the three parallel assays. Another phenomenon of microbial activity was found in the assays with groundwater of the wells 65
and 69, which are low or not contaminated and therefore contained a low organic carbon content. O2 was consumed, the pH dropped, ammonium was used completely and nitrate or nitrite accumulated (Fig. 3, Table 4). This shows that chemolithoautotrophic nitrifyers were active. The CO2-production kinetics of the assays with groundwater of well 65 showed that in the first four weeks BTEX degradation occurred accompanied by a diauxic growth pattern. The CO2-concentration increased in two steps. This may be caused by a change of the growth substrate from toluene to benzene, which is more difficult to mineralise (Oh et al., 1994). Later on CO2 was consumed by the nitrifyers and decreased in the gaseous and aqueous phase over time. The O2kinetics show also the diauxic growth, but the O2consumption curve for the degradation of the second substrate conceded with the O2-consumption of the nitrification. Nitrite accumulated in two assays in high amounts (>80 mg l ÿ 1), whereas in one assay ammonium was completely oxidised to nitrate (254 mg l ÿ 1). The groundwater of well 69 was not polluted with BTEX and PAHs and showed only nitrifying activity. The supplied ammonium was nearly completely oxidised to nitrate and the second highest O2-consumption was observed. Neither degradation nor nitrifying activity could be detected in the assays with groundwater of well 64. The pH did not change during the incubation, ammonium and oxygen were not consumed and the CO2-concentrations in the water phase was similar as
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Fig. 3. O2- and CO2-kinetics of the degradation assays with groundwater from well 64, 65, 66 and 69: (O2-consumption and CO2-production for the assays 65, 66 and 69; O2-concentration and CO2concentration for the assay 64).
in the control assay. Only the number of aerobic bacteria increased during the incubation but, as stated above, this parameter gives no information about the degrading activity. BTEX and PAHs disappeared during incubation under aerobic conditions. Besides, the GC-MS-chromatograms show a complete decrease (except aniline) of all contaminants. In contrast, all contaminants were detected in the abiotic control (data not shown). This elimination can be partly due to degradation processes, which are too small for detection by O2 and CO2monitoring. But the disappearance of the higher molecular weight PAHs caused by degradation activity is improbable. Possibly, the produced biomass serves as biological matrix for sorption of low concentrations of hydrophobic organic contaminants as described elsewhere (Stringfellow and AlvarezCohen, 1999). CONCLUSIONS
The microbial activity of six groundwater samples from a former gas plant site with different levels of contamination was studied in batch culture by monitoring the O2- and CO2-concentrations over time and analyses of some physical and chemical parameters at the end of the experiments. One of the highly contaminated samples (68) showed high degradation activity, whereas the other was not active at all due to toxic substances (67). The contaminants were nearly completely degraded in the medium and low polluted samples (66, 65, 64). Two low-contaminated samples showed nitrifying activity (65, 69). The results show that *
The degradation capacity of the native microbial population and thereby the natural attenuation
*
*
*
potential cannot be predicted simply by chemical, microbial and ecotoxicological data of groundwater samples from contaminated sites. Additionally, it is recommended to perform degradation tests with the authentic groundwater samples with their contaminant mixture. The degradation tests cannot be characterised only by CO2 and O2 detection. To distinguish microbial degradation activity from other microbial and sorption processes it is necessary to determine parameters like the concentrations of the contaminants, electron acceptors and metabolites in the degradation assays. The counts of aerobic bacteria in the original groundwater and in the assays at test end do not relate with the observed microbial activity. Natural attenuation at this former gas plant site with just a small autochthonous microflora without ammonium and phosphate supplementation would not be successful as a remediation strategy.
Acknowledgements}This paper carries publication no. 118 of the Priority Program 546 ‘‘Geochemical processes with long-term effects in anthropogenically-affected seepage- and groundwater’’. Financial support was provided by Deutsche Forschungsgemeinschaft. The authors wish to thank M. Mo¨ller, M. Duisken, U. Kaufmann, C. Schmitz, N. Mende and K. Ehrhardt for their technical assistance.
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