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Binelli* A., Pogliaghi* A., Zicchinella* M., Marazzi° F., Parolini* M., Soave* C., Mezzanotte° V.
*Department of Life Sciences, University of Milan, via Celoria 26, Milan (Italy) ° Department of Science of Environment and Territory, University of Milan-Bicocca, P.zza della Scienza 1, Milan (Italy) Introduction and objectives:
The aim of this research is the evaluation of possible abatement of some chemicals in sewage waters by the use of a biological process. In detail,
we built a pilot-plant (Fig.1) in the biggest depuration plant of Milan (Italy) in which we put tens of thousand specimens of the freshwater bivalve
Dreissena polymorpha. Mussels were attached to Plexiglas panels that force the waste to follow a zigzag pathway (Fig. 2). We exploited two
natural behaviors of this mussel: the high filtration rate (average of 200 mL/mussel/h), which transfer the contaminants from waste to the bottom of
Attachment tanks
tank by the production of faeces and pseudo-faeces, and the bioaccumulation capability of lipophilic chemicals in mussel soft tissues. Thus, by the cyclic elimination of faeces and contaminated mussels from tank, we may depurate the waste. Our project plan foresee three different steps: 1) the Pilot-plant
evaluation of the capability of D. polymorpha specimens to attach themselves to panels and to live in wastewater 2) to test the possible decrease of pollutants in re-circulation conditions; 3) to check the contaminant abatement with "open" pilot-plant (without re-circulation). We will measure the possible removal of several environmental pollutants (Tab. 1), which were found in previous studies carried out in the wastewater treatment plant of Milano-Nosedo. Re-circulation tank
outlet valves
waste inlet
Residence time at 3,500 L/h = 15 min.
atenolol, carbamazepine, cyprofloxacine, clarithromicyne, dehydro-
erythromycin, diclofenac, ibuprofen, paracetamol, furosemide, hydrochlorothiazide, ketoprofen, naproxen, ofloxacin Fig. 1 Pilot-plant located in the depuration plant of Milano-Nosedo
Pilot-plant characteristics:
cocaine, benzoylecgonine, methamphetamine, methadone In May 2011 we built the pilot-plant (800 L) by stainless steel, which collect the HEAVY METALS
waste directly from the canal came out from the sedimentation tank of the sewage Al, Cr, Fe, Mn, Ni, Pb, Cu, Cd, As treatment plant. We can also regulate the inlet pump flux (0-5,000 L/h). The bottom waste outlet
of the tank is skew to favor the collection and elimination of sediment and faeces and pseudo-faeces. We can add twenty removable Plexiglas panels in which zebra acenaphthene, acenaphthylene, anthracene, benzo(a)anthracene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(g,h,i)perylene, mussel specimens are attached. An outlet valve allows to put the waste to the sand- benzo(a)pyrene, chrysene, dibenz(a,h)anthracene, fluoranthene, fluorene, filters or to a re-circulation tank (Fig. 1). Moreover, we built also two "attachment tanks" in which Plexiglas panels are put in horizontal to allow the natural indeno(1,2,3,c,d)pyrene, phenanthrene, pyrene attachment of mussels. Finally, the "nursery" is a little tank in which panels can be HYDROCHEMICAL PARAMETERS
placed vertically before the positioning in the pilot-plant. Fig. 2 scheme of wastewater pathway in
COD, Total Suspended Solids (TSS) the pilot-plant tank
Escherichia coli Experimental Design:
After several preliminary tests (Tab. 2; Fig. 3, 4), we started to carry out the
Tab. 1 Chemical and biological parameters measured in the removal tests
preliminary clearance tests by measuring the COD decrease due to the filtration rate of mussels in comparison with controls (no mussels in the pilot-plant). The first test was conducted by adding Spirulina spp. to the final waste, since its COD resulted very low (≈10 mg O2/L). After the confirmation of the possible role >2,000 mussels
played by Zebra mussels in the COD abatement (Fig. 5), now we are carrying out several tests to evaluate the clearance of the selected chemicals in the real wastes ADVANTAGES IN THE USE OF ZEBRA MUSSEL (Tab. 1). In detail, we are testing four different types of waste: 1) the treated •  ATTACHMENT OF THOUSAND OF MUSSELS/PANEL effluent which flows from the sedimentation tank of the waste water treatment plant; 2) the inlet waste of depuration plant; 3) 50% of inlet and 50% of treated •  HIGH FILTRATION RATE effluent; 4) 25% of inlet and 75% of treated effluent. We are measuring the •  TOLERABLE TO POLLUTANTS contaminant concentrations every 30 min for 4 hours, in order to obtain the typical removal slope for each measured chemical. These first tests were carried out in re- •  HANDY SIZE •  Zebra mussel is integrated in the Northern Italian circulation conditions (Fig. 1) with a waste flux of 3,500 L/h, which corresponds ecosystems since the end of 1960s. to a residence time of 15 min in the pilot-plant (Fig. 2). The time of complete re- POSSIBLE DRAWBACK •  The pilot-plant is located before the sand filters circulation was 18 min since we added always 200 L of selected waste in the re- •  ALLOCTONOUS SPECIES and disinfection system (peracetic acid). circulation tank. This experimental design can allow a longer contact of waste with mussels since it can re-circulate about 14 times in 4 hours. Each sample was •  Narrow mesh nets close every outlet. previously filtered by a bag filter (mesh=1mm) in order to eliminate the gross particulate. Preliminary results
We show only preliminary results because the first determinations of chemical removal rate comparing controls (without mussels in the tank) with
"exposed" are still in progress. Considering the advantages and drawbacks of different attachment systems (Tab. 2), we chosen the "natural" system as the ATTACHMENT
best way to attach mussels. In particular, a high mussel mortality has noticed after few weeks for the artificial systems. By contrast, the natural attachment has guaranteed a very low mortality for several months, even if we noticed a slow time of attachment (two weeks at least) and a weak adherence by the byssus. The second preliminary step has been the evaluation of possible metabolism effects on D. polymorpha due to wastewater exposure. Figure 3 AQUARIUM
shows a little decreasing trend for lipid percentage and a corresponding non-significant increase of proteins measured in a pool of mussel sampled during a period of about two months. Moreover, a lipid content of about 10% was subsequently measured after three months of placement in the pilot-plant, •  HIGH VITALITY highlighting the mussel adaptation to physical and chemical wastewater characteristics. In order to check the possible photo- and bio-degradation of ADVANTAGES
pharmaceuticals and drugs of abuse, we firstly carried out four assays with different waste concentrations without mussels in the pilot-plant (Fig. 4), protecting the tanks by photodegration with plastic dark large sheets. No differences were noticed after six hours of re-circulation. The first removal test •  HIGH DENSITY was the comparison of COD decrease with or without mussels in the pilot-plant, by adding to final waste a content of Spirulina spp. sufficient to reach about 50 mg/L of COD. Figure 5 clearly shows the higher and significant COD removal rate (two-way ANOVA, F=809.9; p<0.001) observed in the •  LOW DENSITY experiments conducted with Zebra mussel specimens in the pilot-plant in comparison with blanks (without mussels). The COD decrease (19%) measured in tests without mussels was due only to natural algal sedimentation, while the removal rate due to Zebra mussel filtration increased until a mean of 42% after four hours. Tab. 2 Comparison between artificial and natural
systems for mussel attachment to panels.
Lipid percentage 120 150 180 210 240 Fig. 5 Average COD trends measured by adding Spirulina spp. in
Fig. 4 Concentration of several pharmaceuticals and drugs measured at t=0 and after six
the pilot-plant with and without mussels. Difference
hours (t=6).
between tests is significant (Two-way ANOVA, F=809.9,
in=inlet of depuration plant; out=canal from the sedimentation tank. ate=atenolol; carb=carbamazepine; cypr=cyprofloxacine; clar=clarithromicyne; Fig. 3 Lipid percentage and protein content
dehydr=dehydroerythromycin; dicl=diclofenac; fur=furosemide; hydr=hydrochlorothiazide; (±st. dev.) measured in mussel pools
ket=ketoprofen; nap=naproxen; ofl=ofloxacin; coc=cocaine; benz=benzoylecgonine; maintained in the wastewater.  
met=metamphetamine; meth=methadone  


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