a) For the municipal wastewater samples collected from the municipal treatment plant of Veszprém, two tests series were carried out to test the effect of different inocula, and different temperature regimes . Inoculum and the sample were mixed in a 1:1 ratio. Samples were aerated prior to toxicity testing
i) For the first test series: the tested samples were kept at room temperature and marked as follow:
(1): Raw wastewater.
(1A): Raw wastewater + Inoculum (Balaton lake water).
(1B): Raw wastewater + Inoculum (Séd stream water) . (2) Treated wastewater.
(2A) Treated wastewater + Inoculum (Balaton lake water) . (1:1).
(2B) Treated wastewater + Inoculum (Séd stream water) . (1:1).
All the test samples were incubated at 20oC under aerobic condition in darkness for 6 weeks.
ii) For the second test series: three temperature regimes were set: one series of samples was incubated at 10°C, the second at room temperature (app. 22°C) and the third at 30°C. Samples were marked during the assay as follow:
R10: raw sample at 10°C.
R22: raw sample at room temperature (22°C).
R30: raw sample at 30°C.
34 RS10: raw sample mixed with inoculum from the recipient water body (Séd) at 10°C.
RS22: raw sample mixed with inoculum from the recipient water body (Séd) at room temperature (22°C).
RS30: raw sample mixed with inoculum from the recipient water body (Séd) at 30°C.
T10: Treated wastewater at 10oC.
T22: Treated wastewater at room temperature (22oC).
T30: Treated wastewater at 30oC.
TS10: Treated wastewater mixed with inoculum from Séd stream at 10°C.
TS22: Treated wastewater mixed with inoculum from Séd stream at room temperature (22°C).
TS30: Treated wastewater mixed with inoculum from Séd stream at 30oC.
All the samples were incubated at the specified temperatures under aerobic condition in darkness for 153 days (the test duration).
b) 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)
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 under aerobic condition in darkness for 12 weeks.
35 c) 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.
i) 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:
Rp: wastewater sample from Dunapack Ltd.
Rs: wastewater sample from Dunacell Ltd Rt: wastewater sample from Dunafin Ltd.
Rm: The input wastewater (Mixed wastewater of the previous three) T: The output treated wastewater
TD: Treated wastewater mixed with inoculm (500 ml of treated wastewater + 500 ml of Danube river water).
All the samples were incubated at 22oC under aerobic condition in darkness for 10 weeks.
ii) Testing the ecotoxicological impact of the wastewater with/without treatment and to test the efficiency of using inoculum from the recipient water body.
Nine test samples were prepared and marked as follow:
DF: wastewater sample from Dunafin Ltd.
DC: wastewater sample from Dunacell Ltd DP: wastewater sample from Dunapack Ltd.
Dfi: wastewater sample from Dunafin Ltd. mixed with inoculm (500 ml of raw wastewater + 500 ml of Danube river water)
Dci: wastewater sample from Dunacell Ltd. mixed with inoculm (500 ml of raw wastewater + 500 ml of Danube river water)
Dpi: wastewater sample from Dunapack Ltd. mixed with inoculm (500 ml of raw wastewater + 500 ml of Danube river water)
OS: The input wastewater (Mixed wastewater of the previous three companies) Osi: The input wastewater mixed with inoculm (500 ml of raw wastewater + 500 ml of Danube river water)
36 T: The output treated wastewater
All the samples were incubated at 22oC under aerobic condition in darkness for 13 weeks.
3. Ecotoxicity testing 3.1. ToxAlert test
The bacterial suspension was prepared by using the reconstitution solntion provided with the kits to reconstitute the freeze-dried bacteria (Vibrio fischeri). The freeze-dried bacteria and the reconstitution solution are kept in the freezer at -18°C before the test.
ToxAlert®100 luminometer has a separate well for the reagent vials to ensure the required temperature (15°C). The preparation started by taking 12.5 ml of well-shacked reconstitution solution (to ensure sufficient dissolved oxygen), placed into Microquant vial and kept in the liquid dried reagent well for at least 15 minutes. One vial of freeze-dried bacteria was taken from freezer and placed in the reagent well for 2 minutes. Then 0.5 ml of reconstitution solution was added into the bacterial vial and mixed. After 15 minutes, the bacterial suspension was transferred back to the Microquant vial which contained the rest of the reconstitution solution and used as the bacterial test suspension.
Fifteen minutes pre-incubation time was set. During that time, 500 µl of the bacterial test suspension was pipetted into all cuvettes, including control cuvettes (A1 & B1) as well.
Cuvette A1 was placed into the turret shortly before beginning of the contact time. At contact time t=0, RLU of the solution was measured. After that, 500 µl of NaCl solution was added to cuvette Al and mixed gently. As the inter-cuvette time was set at 30 seconds, the same procedure was applied for cuvette B1 at t=30 sec. At t=60 and 90 sec., a slightly different procedure was applied for the sample cuvettes. For the sample, 500 µl of the diluted sample was added and gently mixed after measuring RLU.
For each test, the dilution series of 6.25%, 12.5%, 25%, 50% and 100% sample was used as suggested by the WET method manuals (USEPA, 1993, USEPA, 1994). 2 % NaCl solution was used as diluent for preparing the sample dilution series.
Exposure time ended at t=30 min. Then RLU of all cuvettes was measured again, in the same sequence with keeping 30 sec. inter-cuvette time.
37 The ToxAlert®100 luminometer calculates the inhibition effect (Ht) of the samples automatically in % values. Firstly the fkt correction factor is calculated from the measured luminescence (Equation [1]).
fkt = lkt / l0 (t = 30 min in our test) [1]
where;
fkt the correction factor for the contact time
lkt luminescence intensity in the control sample measured in RLU (relative luminescence units), after the contact time
l0 luminescence intensity of the control test suspension.
Using the correction factor, than the corrected value of for every test sample cuvettes are calculated (Equation [2]).
lct = l0 x fkt [2]
where;
fkt mean of fkt of the two control samples
l0 luminescence intensity of the control test suspension
lct corrected value of for test sample cuvettes immediately before the addition of the test sample
Then the inhibitory effect Ht of the test sample is calculated (Equation [3]).
Ht = [(lct - lTt/lct)] x 100 [3]
where;
Ht the inhibitory effect of the test sample after the contact time, in%
lct corrected value of for test sample cuvettes immediately before the addition of the test sample
lTt luminescence intensity of the test sample after the contact time, in RLU.
3.2. Flash assay (kinetic determination)
The Vibrio fischeri Reagent (NRRL-B 11177) strain was reconstituted by adding the contents of one vial of +4 °C 1243-551 Reagent Diluent. The reconstituted reagent was equilibrated at +4°C for 30 minutes. Then the reagent was stabilised at +15 °C for 30 min before pipetting into the cuvettes.
38 A dilution series of 1:2 was set, using 10 concentrations. The sample dilution series was prepared by using the 1243-552 Sample Diluent. All samples and dilutions were temperated to +15 °C for 15 min. All samples and dilutions were kept at +15 °C during the whole measurement. Measurements were done in duplicates.
Toxicity of the sample was assessed using Ascent Software provided by Aboatox Co., Finland. Inhibition of each sample is calculated as follows:
KF = IC15/IC0
INH% = 100 100 x (IT15 / KF x IT0) Where:
KF = correction factor.
IC15 = luminescence intensity of control after contact time (15 min) in RLU.
IC0 = initial luminescence intensity of control sample in RLU.
IT15 = luminescence intensity of test sample after contact time (15 min) in RLU.
IT0 = initial luminescence intensity of the test sample in RLU.
Calculation of ecotoxicity results:
In case of ToxAlert tests, EC50 values were calculated using the EPA’s (Environmental Protection Agency, USA) Probit software. For Flash assays, EC50 and EC20 values were calculated using the ABOATOX software provided with the equipment.