4. Chemical analysis
4.2. Total Organic Carbon (TOC) measurement
TOC were measured by using Apollo 9000 TOC Combustion Analyzer. It calculates TOC values of the samples in mg/l. It analyses the sample completely automatically starting from sample handling to the final analysis and diagrams.
First, Carbon in the sample is converted to CO2 by the combustion furnace (from 680°C to 1000°C) for TOC analysis. Then a carrier gas sweeps the derived CO2 through a
Nondispersive Infrared (NDIR) Detector which is sensitive to the absorption frequency of CO2. The NDIR generates a non-linear signal that is proportional to the instantaneous concentration of CO2 in the carrier gas. Then, that signal is linearized and integrated over the sample analysis time. The resulting area is compared to stored calibration data and a sample concentration in parts-per-million (ppm) is calculated.
40
Results and Discussion
Assessment of toxic effects of municipal wastewater for recipient freshwater systems using different inocula
As mentioned in the Materials and Methods part, the samples were collected from the municipal treatment plant of Veszprém. Two samples were taken: raw (1) and treated (2) wastewater. Both types were than mixed an inoculum taken from a natural, intact water body, Lake Balaton (A) and from the recipient stream, Séd (B).
Figure 7 shows the toxicity changes of sample 1 (raw wastewater) and the inoculated raw wastewater samples (1A & 1B). The toxicity increased by the end of the 1st week reaching app. 99% inhibition till the 3rd week. After the 3rd week, there was a striking decrease in toxicity and the toxicity started to decrease in steady way.
Fig.7: Toxicity changes of the sample 1 in comparison with the presence of different inocula
Figure 8 shows that the toxicity of sample 2 (treated wastewater) and the inoculated treated wastewater samples (2A & 2B). There is an increase till the end of 1st week and then a sudden decrease can be experienced. By the end of the test, the toxicity of the samples behaved in a steady way.
0 20 40 60 80 100 120
0 10 20 30 40 50
% Inhibition
Time(Days)
1 1A 1B
41 Fig.8: Toxicity changes of Sample 2 in comparison with the presence of different inocula COD values of the samples decreased during the incubation time in somewhat regular pattern (Fig.9).
Fig.9: COD values of the samples during the incubation time
The raw communal wastewater exerted a high toxicity and risk to the environment even after dilution and exposure to different natural origin microbial communities. Then toxicity decreased as biodegradation proceeded, finally reaching an acceptable level (40% of bioluminescence inhibition). App. the same tendency could be observed for the
0
42 treated effluent, but in this case the high initial toxicity was much more rapidly followed by a decrease. These results might give an indication how effluents (both raw and treated) might “behave”, and perhaps might be used in cases where wastewater treatment systems are not operating efficiently.
Toxicity assessment did also show that, despite related literature, source of inoculum did not have any significant effect on the process of biodegradation and on the temporal pattern of ecotoxicity.
Assessment of toxic effects of municipal wastewater for recipient freshwater systems under different temperature regimes
Continuing the previous study, municipal wastewater of Veszprém Treatment Plant was analyzed. Based on the findings of the previous study, only one inoculum was used, taken from the recipient stream, Channel Séd. Three temperature regimes were set: one series of samples was kept at 10°C, the second at room temperature (app. 22°C) and the third at 30°C.
Samples are marked as follows:
R10: raw sample at 10°C
R22: raw sample at room temperature (22°C) R30: raw sample at 30°C
RS10: raw sample + inoculum from the recipient water body (Séd) at 10°C
RS22: raw sample + inoculum from the recipient water body (Séd) at room temperature (22°C)
RS30: raw sample + inoculum from the recipient water body (Séd) at 30°C
Figs 10 and 11 show toxicity changes from Day 0 to Day 153. The raw communal wastewater exerted a high toxicity (expressed as 80.9% bioluminescence inhibition) and risk to the environment. With no inoculum added, this toxicity even increased during the first 12 days, showing somewhat different patterns in the three exposure regimes: at room temperature (sample R22) inhibition was already as high as 91.8%, still increasing to Day 12, reaching its maximum, 97.8% inhibition. Under the other two temperature regimes,
43 10 °C (sample R10) and 30 °C (sample R30) first a slight decrease was shown, than by Day 12 the maximum toxicity appeared, reaching 94.9 and 97.3% inhibition, respectively. Afterwards, between Day 12 and Day 19 first a rapid decrease could be observed, than from Day 19 on, a steady, slower decrease, finally reaching a “tolerable”
level of app. 30% of inhibition by Day 26 in the case of R22 (34.55%), by Day 40 in the case of sample R10 (34.15% inhibition) and finally by Day 54 in the case of sample R30 (32.45% inhibition). It should be noted, however, that this sample showed an anomalous low inhibition of 26.1% on Day 19, which can be most likely attributed to test error.
In the presence of the inoculum, the maximum toxicity was lower for samples RS10 and RS22 on Day 12 than it was the case with samples without inoculum. Afterwards, a much more rapid decrease was initiated, resulting in inhibition below 30% even by Day 19. (There was a slightly higher inhibition value for RS22 on Day 54.) Sample RS30, however, did show a somewhat different pattern: toxicity increased to 94.3% of inhibition by Day 12, than a rapid and steady decrease could be observed, but finally toxicity started to increase again, showing a 40.7% of inhibition by Day 153.
Fig.10. Toxicity changes of the raw wastewater sample without inoculum, at different temperature regimes
0 20 40 60 80 100 120
0 50 100 150 200
% Inhibition
Time (Days)
Toxicity
R10 R22 R30
44 Fig.11. Toxicity changes of the raw wastewater sample with inoculum, at different
temperature regimes
Figs 12 and 13 show toxicity changes from Day 0 to Day 153 of the inoculum-free and inoculated treated wastewater. In the beginning, the treated communal wastewater showed a tolerable toxicity (expressed as 35.8% bioluminescence inhibition). With no inoculum added, at room temperature (sample T22), the inhibition changes between slight increase and decrease reaching its maximum (41% inhibition in the Day40) followed by rapid decrease reaching 25.1% in the Day 54. Under the other two temperature regimes, 10°C (sample T10) and 30°C (sample T30): T10 and T30 toxicity showed drastic decreased in the Day 19 (reaching 16.5% and 15.9% respectively), showing somewhat different pattern than T22 (36.9%). The samples showed decrease in toxicity in Day 54 under the three temperature regimes. T22 and T30 showed a steady slower decrease, finally reaching a “tolerable” level of app. 30% of inhibition by Day 153, while, T10 showed an increase reaching 53.45%.
In the presence of the inoculum, toxicity changes of the samples showed similar pattern under the three temperature regimes till Day 68. The samples reached inhibition below 30% by Day 19, then app. 40% by Day 26. Afterwards, they showed a drastic decrease in Day 54 reaching 22% inhibition. Samples TS22 and TS30 showed a steady slower decrease, finally reaching a “tolerable” level of app. 26% of inhibition by Day
45 153, however, sample TS10 did show a somewhat different pattern: toxicity increased to 49.8% of inhibition by Day 153.
Fig.12. Toxicity changes of the treated wastewater sample, at different temperature regimes
Fig.13. Toxicity changes of the treated wastewater sample with inoculum, at different temperature regimes
46 samples, with no differentiation between inoculum-free and inoculated samples. Also, different temperature regimes seemed to have no effect on the pattern of COD reduction.
Fig.14. COD changes of the raw wastewater sample with and without inoculum, at different temperature regimes
Fig.15. COD changes of the treated wastewater sample with and without inoculum, at different temperature regimes
Fig.16 and 17 show TOC changes from Day 0 to Day 153. TOC changes, similar to COD changes pattern, show first a rapid then a steady decrease for all samples, with no
47 differentiation between non- and inoculated samples. Different temperature regimes seemed to have no effect on the reduction pattern of TOC.
Fig.16. TOC changes of the raw wastewater sample with and without inoculum, at different temperature regimes
Fig.17. TOC changes of the treated wastewater sample with and without inoculum, at different temperature regimes
Although pattern of toxicity changes varied amongst samples as described above, there were common features, proving our null hypothesis. During the first period, till Day 12 toxicity of all effluent samples increased, both for raw and inoculated ones and for all
48 temperature regimes, posing high environmental risk during app. two weeks. A decline in toxicity began only after this peak. The two-week period before effective biodegradation begins can be regarded as an average. Household wastewaters are very complex mixtures, for example Eriksson et al. (2002) have estimated that grey wastewater from Danish households could potentially contain more than 900 different XOCs (xenobiotic organic compounds). Only from wastewater originated from bathrooms, almost 200 different organic chemicals were identified (Eriksson et al., 2003). The very diverse nature of household wastewater makes it very difficult to follow the processes by which biodegradable substances break down and to analyze the toxicity of each intermediate product separately.
Whole effluent toxicity, as the definition implies, gives a good estimation on the aggregate environmental hazard of the effluent. Our results underline a serious environmental risk: raw municipal wastewater if untreated might undergo such degradation which results in toxic intermediates, prolonging the period during which ecosystem of the recipient freshwater might be impacted. However, it seems also reasonable that in the presence of competent, pre-adapted microbial community biodegradation can be enhanced and risk can be sooner mitigated.
Naturally, it is not feasible technologically to regularly monitor a 3-month biodegradation process. The Vibrio fischeri bioluminescence inhibition test, however, can give some indication for the predicted behaviour of selected effluent types (Lapertot et al., 2008).
49
Biodegradation assessment of liquid manure disposal
Göcsej Pig Inc. has liquid manure stabilization ponds both with and without technical protection (isolation). One of the ponds was abandoned two years ago and here no mechanical isolation was built. The groundwater was analyzed in the vicinity of the pond (Table 2) to assess the environmental impact, but no toxicity was measured. In addition, the farm has several stabilization ponds with proper technical protection, they are still in use.
Table 2: Results of groundwater analysis
Components Measured values (mg/l)(μg/l) Limit values
F1 F2 F3 A B used, one collected from a clean (reference) environment and one collected at the site.
Two liquid manure samples were collected from Göcsej Pig Inc. Ormándlak (Western Transdanubia, Hungary). One (the fresh manure sample) from the currently used pond, while the other (aged manure sample) from the reservoir which was abandoned two years ago. The soil inocula were collected at the site and from Veszprém (reference clean soil).
The six test samples were prepared and marked as follows:
1: Liquid manure from the currently used pond
1T1: Liquid manure (1) mixed with clean soil. (200 cm3 soil + 200 cm3 liquid manure)
50 1T2: Liquid manure (1) mixed with soil collected at the site. (200 cm3 soil + 200 cm3 liquid manure)
2: Liquid manure from the abandoned reservoir
2T1: Liquid manure (2) mixed with clean soil. (200 cm3 soil + 200cm3 liquid manure) 2T2: Liquid manure (2) mixed with soil collected at the site (200 cm3 soil + 200 cm3 liquid manure)
All the samples were incubated at 20oC for 12 weeks.
Figure 18, 19 and 20 show the difference in the toxicity of sample 1 (fresh liquid manure), sample 2 (aged liquid manure) and also toxicity changes of the subsamples. It is obvious that the toxicity of sample 2 is much lower than that of sample 1, because the abandoned pond has more time (about 2 years) to recover without adding any new pollutants and the microbial communities living there had been adapted to these pollutants. For both samples, toxicity did reach a sort of equilibrium by the end of the test. Toxicity of sample 1 was app. 90% by the 3rd month (Day 84), proving that the test period was not enough to reach a significant reduction in toxicity. However, sample 2 did show a striking decrease in toxicity, from the initial 65% to the rather constant 45%
inhibition.
After adding the soil inocula to sample 1 and 2, the subsamples 1T1, 1T2, 2T1 and 2T2 showed more varying trends in the toxicity changes during the degradation processes than the untreated samples. The toxicity of the subsamples is lower than the original ones.
Fig.18: Toxicity changes of sample 1 and sample 2 during the test
0
51 Fig.19: Toxicity changes of sample 1 in comparison with the presence of different
inocula
Fig.20: Toxicity changes of sample 2 in comparison with the presence of different inocula
When using inocula (either from clean place or from the impacted site), toxicity changes show somewhat similar patterns in comparison to the raw sample. It should be noted, however, that minimum toxicity is achieved in week 7, after that toxicity increases again.
It can be due to the fact that adsorption capacity of the soils used as inocula is exhausted.
0
52 COD measurements showed somewhat more regular pattern. It can be seen in figures 21, 22 and 23 that during the incubation period, COD of the fresh and aged liquid manure samples and treated samples decreased. The aged liquid manure finally reached a constant value of app. 500 mg/l.
Fig.21: COD values of sample 1 and sample 2 during the incubation time
Fig.22: COD values of sample 1 and its subsamples during the incubation time
0 500 1000 1500 2000 2500
0 20 40 60 80 100
COD (mg/l)
Time (Days)
1 2
0 500 1000 1500 2000 2500
0 20 40 60 80 100
COD (mg/l)
Time (Days)
1 1T1 1T2
53 Fig.23: COD values of sample 2 and its subsamples during the incubation time
We can conclude that, in the optimal case, toxicity decreases as biodegradation goes on. However, it often happens that such intermediate products are formed which are more toxic than the original compound was, as what happened with sample 1 and subsamples 1T1 and 1T2.
Adding soil as inocula increases and enhances the biodegradation rate significantly. In contrary to our original hypothesis, the source of inocula did not have an unambiguous effect on the rate of biodegradation. In case of sample 1 the subsample treated with soil collected at the site did show significantly lower toxicity in comparison to the untreated sample and the sample treated with clean (reference) soil (Fig. 19), but in case of sample 2 no such difference can be stated (Fig. 20). On the contrary, decrease of COD values did reflect the biodegradation processes in a more obvious way.
The final conclusion can be that liquid manure has a high toxicity. It is very important, as up to now no regulation has prescribed the need for toxicity measurements in case of liquid manure usage and disposal. The fact that the liquid manure sample collected from the stabilization pond abandoned two years ago showed an initial toxicity (bioluminescence inhibition) of 65% reveals that liquid manure might pose a long-term environmental risk.
0 200 400 600 800 1000 1200 1400
0 20 40 60 80 100
COD (mg/l)
Time (Days)
2 2T1 2T2
54
Biodegradation studies on paper mill effluent
The samples were collected from the wastewater treatment plant of Dunaújváros.
Capacity of the plant is 15,000m3/day. The input wastewater from Dunafin Ltd., Dunacell Ltd. and Dunapack Ltd. mix before the start of the treatment.
The inoculum was collected from Danube River (recipient water).
Two tests series were carried out to test the ecotoxicological impact of the pulp and paper industrial wastewater with/without treatment on Danube river environment and to test the efficiency of using inoculum.
A) Testing the ecotoxicological impact of the pulp and paper industrial wastewater with/without treatment on Danube river.
The six test samples were prepared and marked as follow:
1- Rp: wastewater sample from Dunapack Ltd.
2- Rs: wastewater sample from Dunacell Ltd 3- Rt: wastewater sample from Dunafin Ltd.
4- Rm: The input wastewater (Mixed wastewater of the previous three) 5- T: The output treated wastewater
6- TD: Treated wastewater + Inoculm (500 ml of treated wastewater + 500 ml of Danube river water).
All the samples were incubated at 22oC for 10 weeks.
B) Testing the ecotoxicological impact of the wastewater with/without treatment and to test the efficiency of using inoculum from the recipient water body.
The nine test samples were prepared and marked as follow:
1- DF: wastewater sample from Dunafin Ltd.
2- DC: wastewater sample from Dunacell Ltd 3- DP: wastewater sample from Dunapack Ltd.
4- Dfi: wastewater + Inoculm (500 ml of raw wastewater + 500 ml of Danube river water)
5- Dci: wastewater + Inoculm (500 ml of raw wastewater + 500 ml of Danube river water)
55 6- Dpi: wastewater + Inoculm (500 ml of raw wastewater + 500 ml of Danube river water)
7- OS: The input wastewater
8- Osi: The input wastewater + Inoculm (500 ml of raw wastewater + 500 ml of Danube river water)
9- T: The output treated wastewater
All the samples were incubated at 22oC for 13 weeks.
Figure 24 shows the changes in toxicity of the raw samples of the different factories, the raw mixed-sample, the treated output and the treated output mixed with inoculum from the receiving water from Day 0 to Day 69.
The toxicity of the factories raw samples showed different levels and behaviour.
Rp effluent from Dunapack Ltd. showed high toxicity in the start of the test about 83.8% inhibition, then the toxicity increased to reach the maximum (99.7%) by Day 13, and continuing this high toxicity till the last day of the test to reach approximately 92.5% inhibition.
On the other hand, Rs effluent from Dunacell Ltd. Showed extremely high toxicity in Day 0 (99.5% inhibition), then it decreased to reach 66.1% by Day 6, but by Day 13, it increased again to be extremely toxic (99.5%). Afterwards, the toxicity showed a striking decrease reaching environmental tolerable level (29.9%) by Day 20, and then changed steadily in a tolerable range (43- 46%) till the end of the test.
Rt effluent from Dunafin Ltd. showed a moderate toxicity in Day 0 (59.3%), then the toxicity decreased reaching non-toxic level by day 13 (16.8%), but by Day 20 increased again to app. 63.4%, and afterwards the toxicity decreased reaching a tolerable level starting from Day 34 till the end of the test period.
The mixed effluent (Rm) exerted a high toxicity (expressed as 83.8%
bioluminescence inhibition) and risk to the environment. During the following 2 weeks the toxicity decreased reaching app. 65% inhibition and then started to sudden increase reaching an extremely high toxic level (99.6%) by Day 20. The sample showed a striking decrease reaching 41.5% by Day 34 and afterwards a steadily decrease reaching a tolerable level by the end of the test.
56 The treated output effluent (T) and the inoculated one (TD) toxicity changes showed the same behaviour pattern. At Day 0, T toxicity was 46.2% inhibition. Afterwards, between Day 13 and Day 20 first a rapid decrease reaching non-toxic level could be observed, then increase again reaching app. 36% by Day 34. From Day 34 on, a slower steadily decrease, finally reaching a “tolerable” level of app. 26.7-29 % of inhibition by the end of the test.
Fig.24: Toxicity changes of the raw and treated samples during the incubation time
Fig.25: COD values of the samples during the incubation time
0
57 On the contrary, COD and TOC changes showed a more uniform pattern, first a rapid then a steady decrease for all samples.
It should be mentioned the high COD and TOC values of the raw effluent of Dunapack Ltd., 3946mg/l and 2253 mg/l respectively. Even by the end of the test, they were above the limits. The decrease due to biodegradation processes was rapid till the Day 48 then became steady.
Fig.26: TOC values of the samples during the incubation time
The effluent from Dunapack Ltd. was characterized with extreme toxicity level even after the biodegradation time period. This may be a result of using a wide range of toxic chemicals such as chlorophenols and aromatic derivatives which are used in the paper-mill industry. So, discharging the raw effluent without treatment would harm the environment of the recipient water. Mixing the three industrial effluents before the treatment lowered the toxicity of the original effluents which make it less harmful for the wastewater treatment biological secondary tanks. This may be due to dilution or the chemical interactions in the effluent. The treated water and the inoculated treated water had low toxicity, especially the inoculated one. This gives indication that the wastewater treatment plant works efficiently and there is no hazardous impact on the environment after discharging the treated effluent.
0 500 1000 1500 2000 2500
0 7 14 21 28 35 42 49 56 63 70 77
TOC (mg/l)
Time (Days)
Rp Rs Rt Rm T TD
58 Figure 27 shows the changes in toxicity of the raw and treated wastewater with and without inoculum from the receiving water from Day 0 to Day 91.
The toxicity changes of the factories raw samples without inoculum showed different behavior.
Dc effluent from Dunacell Ltd. Showed extremely high toxicity in Day 0 (99.6%
Dc effluent from Dunacell Ltd. Showed extremely high toxicity in Day 0 (99.6%