THE BIOLOGICAL EFFECT OF PARTICULATE MATTER DERIVED FROM RED SLUDGE DISASTER IN HUNGARY János Fent, Blanka Emődy-Kiss, Viktor Szentgyörgyi, Sándorné Lukács, András Gachályi, Susan Lakatos Hungarian Defence Forces, dr.Radó György Military Medical Centre, Scientific Institute, Budapest, Hungary H-1553 P.O.Box1,latyakos24@gmail.com
Introduction
Light microscope study: The microscopically observable particles are small and more or less amorphous having an average equivalent diameter of 0.87 ± 0.65 µm. The roundness index is 1.39 ± 0.23.
Results
There is no exact definition of the word “nanoparticle”, but it means "a particle having one or more dimensions of the order of 100 nm or less” according to PAS 71:2005 document developed in the UK. The physical, chemical and consequently, the biological properties of nanoparticles may significantly differ from those of the bulk material. Where can nanoparticles be generated? A vast amount of nanoparticles are produced by industry, but nanoparticles can be generated by natural processes too. It is reasonable to suppose that nanoparticles are generated also from the desiccated red sludge. The red sludge disaster in Hungary
If moist red sludge is greasy, clay-like, if dried it becomes a fine but sticky reddish powder. The PBS effectively buffered the extreme pH of red sludge. At the concentration of 0.1 mg/ml sludge the pH of the samples were approximately 7.4.
In vitro cell activation In vitro the suspension of dried red sludge did not result in either platelet or granulocyte activation at concentrations up to 0.1 mg/ml. We could not detect any changes in Jurkat cell viability and activation status in the presence of sludge in the culture medium (at a concentration of 0.1 mg/ml).
CD62P expression on platelets
mean fluorescence intensity
A unique situation drew up on October 4, 2010, when the dyke of a reservoir of an aluminum plant broke and villages Devecser and Kolontár (Hungary) were flooded by red sludge. This byproduct of alum-earth has a very high alkalinity, so the primary danger was evident: many people and animals that got in touch with red sludge, suffered serious chemical burn. However, the long-term physiological effects due to any contacts with or just to the presence of the sludge still need to be revealed.
Samples
CD 63 expression on granulocytes
18
80
16
70 mean fluorescence intensity
What is nanoparticle?
14 12 10 8 6 4
60 50 40 30 20
2
10
0
0 control
Microscopic photograph of native red sludge powder (original magnification: 400x)
Heavy metal element content control
red sludge
red sludge
Activation marker expression of platelets (left) and granulocytes (right) in whole blood incubated with 0.1 mg/ml red sludge.
Cell count changes on Jurkat cell line
CD 69 expression on Jurkat cells
300
18 mean fluorescence intensity
250
%
200
150
100
50
4h
Metal
Metal element content of red sludge
12
Na
10 8
As Zn
Fe
6
Pb
Mn
4
0h
24 h
red sludge
4h
mg/g
Al
53
As
0.06
Cd
0.0009
Cr
0.19
K
Cu
Cu
0.05
Others
Cd
Fe
29.6
K
1
negative control
Ni
24 h red sludge
Cell count in the percent of inoculated cell number (left) and activation level (CD69 expression) of Jurkat cells (right) incubated with 0.1 mg/ml red sludge after 4 and 24 h. Apoptosis- AnnexinV-FITC Propidium Iodide profile after 24 hours on Jurkat cell line
Apoptosis- AnnexinV-FITC Propidium Iodide profile after 4 hours on Jurkat cell line 120
120
100
100
80
80 %
%
Samples Samples were taken from the greasy sludge which mulched the soil and from the upper 2-3 cm thick layer of dried sludge. The desiccated sample was mechanically ground using a mortar and pestle. This procedure simulates the mechanical effect of walking on the dried sludge in the fields. Light microscope study: The size distribution of dry native particles was measured by a Leica LMD light microscope without any additional solvent (magnification: 400x). A semi-automatic software (QWin Pro v.3.1.0, Leica) was used to measure the size parameters: the equivalent diameter (the diameter of a circle of equal area) and the roundness of particles. Dynamic light scattering The size distribution of insoluble small particles of ground samples was measured by a Zetasizer Nano (Malvern) instrument. Dried and ground red sludge was resuspended in distilled water at a concentration of 0.2 mg/ml and sedimented at 1g for an hour. The 0.22 micrometer filtered fraction was analyzed separately. In vitro cell activation To test the biological effect of the red sludge a semi-stable suspension was prepared according to the methods described by Bihari et al. (Part Fibre Toxicol. 2008 5: 14). Briefly: 0.2 mg of ground sample was suspended in 1 ml distilled water, sonicated three times for 1 minute with a Labsonic–P (Sartorius) instrument (40% intensity, 0.3 duty cycle) in between 1.5 mg/ml human serum albumin was added. Before the last sonication the physiological osmolality was restituted with concentrated PBS. In vitro platelet and granulocyte activation was measured in the whole human blood of healthy donors. The whole citrated blood was incubated with 0.1 mg/ml (final concentration) red sludge for 10 minutes. The samples were stained with anti-CD62P (PE) / antiCD41 (FITC) or with anti-CD63 (PE) / anti-CD15 (FITC) antibodies, and were measured by a BD FACScan flow cytometer. 5,000 CD41 or CD15 positive events were collected. The activation was characterized by mean fluorescence intensities of the anti-CD62P or the anti-CD63 signals of platelets or granulocytes, respectively. Additional cell viability and activation assays were performed on Jurkat cells. For cell culture experiments 5 mg red sludge powder was suspended in 1 ml RPMI-1640 medium containing 10% fetal calf serum, and sonicated 2-times (40% intensity, 0.3 duty cycle). Cells were incubated with 0.1 mg/ml red sludge (final concentration) for 4 and 24 hours in RPMI-1640 medium supplemented with 10% fetal calf serum at 37 °C in humidified atmosphere containing 5% CO2. Activation was characterized by the staining intensity of the anti-CD69 (PerCP) and cell viability was measured with a FITCAnnexinV Apoptosis detection kit-II (Becton-Dickinson). Heavy metal element content The heavy metal element content of sludge was determined by atomic absorption spectroscopy. 1 gram of dry powder of sludge was suspended, treated with HNO3 and hydrogen-peroxide. After evaporation and appropriate dilution flame atomizer was used to determine the Cd, Cu, Fe, K, Mn, Na, Ni, Zn content of the sample, and electrothermal atomizer for Al, As, Cr and Pb. Data: All data are given as mean±SD.
14
0
negative control
Methods
16
2
0
Photographed by Gábor Gúth
Atomic absorption spectroscopy confirmed the high concentrations of Al, Fe and Na as we presumed. Furthermore the concentrations of the Mn and Ni elements in the red sludge seem to be very high compared to the Maximum Contaminant Level (MCL) of drinking water (according to Hungarian standards). (Assuming that 1 gram red sludge get mixed into 1 litre of drinking water, the Mn, Ni, As, Pb and Cr contents exceed MCL 45.6-times, 6.4-times, 6.0-times, 5.3-times and 3.8-times, respectively.)
60
40
20
20
0
0
viable cell
apoptotic cell
dead cell
viable cell
apoptotic cell
dead cell
Ann- PI-
Ann+ PI-
Ann+ PI+
Ann- PI-
Ann+ PI-
Ann+ PI+
negative control
negative control
red sludge
red sludge
Apoptosis prifile of Jurkat cells after 4h (left) and 24h (right) incubation with 0.1 mg/ml red sludge. Ann: Annexin staining, PI: propidium-iodide staining.
Dynamic light scattering Dynamic light scattering measurements revealed that smaller insoluble particles are also present in the sludge. The size distribution of the 0.22 µm filtered fraction has a large peak at 158 ± 46 nm. Peak %
Hydrodynamically equivalent diameter
Polydispersity index
Suspension supernatant (sedimented at 1g)
91±7
346±227
0.48±0.13
Filtered suspension (0.22 um)
100
158±46
0.23±0.07
Sample
Al
Left circle shows the main metal component of red sludge. Right circle shows the other measured components. The table shows the metal contents of 1 gram sludge.
2.28
Na
28.9
Ni
0.127
Pb
0.053
Zn
0.067
Conclusion
60
40
Cr
Mn
A large amount of insoluble fine and ultrafine particulate matter was generated during the desiccation of the red sludge. It is reasonable to suppose that this matter can slather even distant area and jeopardize inhabitants. It is very promising that no direct cell toxicity was observable. Fortunately, the size of these particles is in the range where the deposition in airways is minimal (Ostiguy et al: Health Effects of Nanoparticles. REPORT R-589, IRSST, 2008). Thus, only a small amount of the inhaled particles might get into the blood stream where direct activation of platelets and granulocytes can practically be excluded. In conclusion, according to our studies the most dangerous effect of these kinds of particles, such as deposition (accumulation) in various organs can be neglected up to the date.