Processi biologici per la rimozione dell`atrazina nelle falde

Università degli Studi di Napoli Federico II
Facoltà di Ingegneria
Corso di Laurea in Ingegneria per l’Ambiente ed il Territorio
Elaborato di Laurea
Processi biologici per la rimozione
dell’atrazina nelle falde contaminate
Anno Accademico 2014/2015
Relatore:Prof.Massimiliano Fabbricino
Candidato:Valerio Volpecina
1 Sommario
•  Inquinamento falde
•  Che cosa è l’atrazina e i sui effetti sull’ambiente
•  Analisi di diversi studi sui processi biologici per la rimozione
dell’atrazina e dei fattori principali da cui sono influenzati
•  Conclusioni
2 Inquinamento falde
Cause
•  Pesticidi e fertilizzanti usati in agricoltura
•  Discariche di rifiuti non adeguatamente
impermeabilizzate(discariche non controllate)
•  Scarichi industriali
•  Scarichi civili,perdite sistema fognario,ecc….
3 Atrazina
Gruppo s-triazine
Struttura atrazina
4 should be present in drinking water [23]. As such no guidelines exist till now on the
permissible limit of atrazine concentration on effluent discharged into water bodies.
TOXICITY OF ATRAZINE
In most of the cases, toxicity effect of atrazine was observed at higher concentrations or
doses, but detrimental effects on several species of algae were observed even at an atrazine
concentration as low as 20 ppb [24]. Although it is less toxic to mammal, birds and fishes,
many aquatic organisms are susceptible to atrazine at low levels [13]. Although atrazine is
placed in toxicity class III by USEPA, which means it is slightly toxic, it has been classified as
restricted use pesticide (RUP) due to its ground water contamination potential. Also exposure
to atrazine may cause detrimental effects and irritation to eyes, nose and throat [25]. Due to
the toxicity behavior of atrazine, German Government banned all atrazine-containing products
in 1991 [26].
Effetti dell’atrazina sugli esseri viventi
•  Tossicità acuta e cronica
Acute Toxicity
toxicity is the ability of a substance to cause harmful effects soon after a single
• Acute
Cancerogenicità
exposure or dose or any severe poisonous effect resulting from a single short-term exposure
a toxic substance. LD (lethal dose 50) is defined as the dose that kills 50% of a
• topopulation
Mutagenicità
edoraleffetti
sulla toriproduzione(studi
condotti da
be 1869-3080, 1750-3992 and 750
of test animals. The
LD was observed
mg of technical grade atrazine/kg for rats, mice and rabbits respectively [13]. The 1-hour and
Concentration)
was observed to be greater than 0.7 mg l and
4-hour
inhalation
LC (Lethal
Tyron
Hayes
sugli
anfibi)
5.2 mg l respectively in rats [25, 27]. Table 1 shows the LD of atrazine on various test
50
50
-1
-1
50
50
species. It had been observed that after consuming a large oral dose, rat exhibits muscular
weakness, breathing difficulty, prostration, convulsions and death [21].
LD50:dose letale perTable
il 50%1.della
di cavieon
testata.
value of atrazine
various test species
LD popolazione
50
Type
LD50
LD50
LD50
LD50
LD50
LD50
LD50
LD50
mode
oral
intraperitoneal
oral
oral
skin
oral
inhalation
intraperitoneal
Species
Rat
Rat
Mouse
Rabbit
Rabbit
Humane
Rat
Mouse
Amount
672
235
850
750
7500
1000
5200
626
Units
mg kg-1
mg kg-1
mg kg-1
mg kg-1
mg kg-1
mg kg-1
mg m-3 4hr-1
mg kg-1
5 Tecniche di rimozione
•  Trattamenti chimici
•  Trattamenti termici(Incenerimento)
•  Trattamenti fisici(Adsorbimento)
•  Trattamenti biologici Utilizzo di macrofiti(Fitorimediazione)
Utilizzo di biomasse microbiche
6 the control of atrazine, biological method is the only method that can mineralize atrazine.
eudomonas preferentially utilize isopropyl side chain than ethyl side chain. Next
alkylation was deamination in which Cyanuric acid was formed. Consequent
stepspathway
Biodegradation
conversion of cyanuric acid to biuret, decomposiotn of biuret to urea
by urease
Understanding
the biodegradation pathway of any pollutant in a specific condition is useful for
degradation
and the degradation end products were CO2 and NH3 [120]. Completebetter
application and control. Atrazine degradation can occur via biotic and abiotic
e was observed through continued hydroxylation of the triazine ring
and Atrazine
the biodegradation had been reviewed extensively by Kaufman and Kearney9
processes.
of anniline, ammelide and cyanuric acid prior to ring cleavage, and finally
to CO
2 Lee [110] whereas, the abiotic detoxification and degradation had been
and Erickson
and
[117]. Considering all the above results a proposed pathwayreviewed
of atrazine
by Jordan et al. [111]. Structure of various intermediates, which could be produced
ation is shown in figure 2.
during the biodegradation process, is described in Figure 1.
Percorso di degradazione dell’atrazina proposto
da Erickson e Lee
X
Deethyldeisopropylatrazine
Deethylatrazine
N
N
Deisopropylatrazine
Atrazine
R-1 (4)
R-2 (6)
N
Hydroxyatrazine
Deethylhydroxyatrazine
Deisopropylhydroxyatrazine
Deethyldeisopropylhydroxyatrazine
Cyanuric Acid
Biuret
Urea
CO2 + NH3
X (2), R-1(4) and R-2 (6) are the substituted groups in s-triazine ring in 2, 4 and 6 positions
respectively.
Common Name
X
R-1
R-2
Atrazine
Cl
C2H5NH
CH3CHCH3NH
Hydroxyatrazine
OH
C2H5NH
CH3CHCH3NH
Deethylatrazine
Deisopropylatrazine
Deethyldeisopropylatrazine
Deethylhydroxyatrazine
Deisopropylhydroxyatrazine
Cl
Cl
Cl
OH
OH
NH2
C2H5NH
NH2
NH2
C2H5NH
CH3CHCH3NH
NH2
NH2
CH3CHCH3NH
NH2
Deethyldeisopropylhydroxyatrazine
OH
NH2
NH2
Cyanuric acid
OH
OH
OH
Urea
Biuret
Figure 1. Structure of atrazine and its transformed products
Structure of different components has been illustrated in Figure 1.
Figure 2. Atrazine biodegradation pathway
7 Principali fattori che influenzano il tasso di
degradazione dell’atrazina
•  Fonti di ossigeno esterne
Studi di Armstrong sulla
dipendenza del tasso di
degradazione dalla presenza di
materia organica assimilabile
•  Fonti di azoto esterne
Studi di Gebendinger con
l’uso del batterio M91-3
•  Contenuto d’acqua del suolo
Studi di Hurle e Kibler sulla
dipendenza dell’emivita
dell’atrazina dal contenuto
d’acqua nei terreni sabbiosi
ghiaiosi
8 The 23 isolates that showed variable growth stimulation in the
resistance/sensitivity test were individually enriched in 2X RD
Atrazine amended-liquid cultures for 10 days to select the most
promising acclimatized bacteria for biodegradation of Atrazine
in the contaminated soils (Table 6). Results revealed the
following:
Based on the Atrazine-enrichment test and the visual bioassay
screening, 20 indigenous and 3 exogenous purified isolates
were classified into genera and/or species whenever possible.
All Atrazine-resistant isolates from the tested soils were
Gram-negative rods expect 8 Gram-positive rods (R2, R5,
R12, R13, R16, R17, R21 and R23). They shaped long, short
or puffy bacilli. Genomic DNA was prepared from the selected
isolates, the PCR products were purified and the two strands
of each amplicon were sequenced using chain terminator method (Bioneer Company, Korea). The 16S rDNA gene sequences
of the isolates were submitted to GeneBank sequencing data
and aligned against the 16S rDNA sequences of Ribosomal
127Table 7 compiles GeneBank accession numDatabase project.
bers of the highest sequence similarity as well as the closest
neighbor(s) to the 16S rDNA gene partial sequences of the
Batteri resistenti all’atrazina
Isolation, identification and acclimatization of Atrazine-resistant
1. Applying Atrazine at its highest dose produced the most
resistant isolates which possess the highest degradative
capabilities. Seven isolates (R1, R9, R16, R20, R21, R22
and R23) exhibited remarkable stimulation (S: 70.7soil 88.7%)
bacteria
in their growth and considered acclimatized and
highly Atrazine-resistant, therefore, were selected for bioremediation experiment.
Studio condotto da El-Bestawy,J.Sabir,A.H.Mansy,N.Zabermawi
Table 7
Similarity percentages to the nearest neighbors of the selected isolates.
Isolate No.
Nearest neighbor(s)
Table 6 Stimulation/inhibition
total viable
count (TVC) of the indigenous
and %
exogenous bacteria in Atrazine-enrichment test.
GenBank
Accession of the of
Nearest
Neighbor
Similarity
R1
R2
R3
R4
R5
R6
R7
R8
R9
R10
R11
R12
R13
R14
R15
R16
R17
R18
R19
R20
R21
R22
R23
Enterobacter cloacae 279-56
Bacilluscereus ATCC 14579
Pseudomonas aeruginosa PAO1
Enterobacter cloacae 279-56
Bacillus anthracis str. Ames
Ochrobactrum intermedium CCUG 24694
Pseudomonas balearica SP1402
Pseudomonas indica IMT37
Pseudomonas indica IMT37
Pseudomonas balearica SP1402
Pseudomonas aeruginosa PAO1
Bacillus cereus ATCC 14579
Bacillus cereus ATCC 14579
Providencia vermicola OP1
Providencia vermicola OP1
Bacillus anthracis str. Ames
Bacillus anthracis str. Ames
Pseudomonas otitidis MCC10330
Pseudomonas otitidis MCC10330
Pseudomonas otitidis MCC10330
Bacillus anthracis str. Ames
Providencia vermicola OP1
Bacillus cereus ATCC14579
NR-028912.1
95
C
2X RD
NR-074540.1
98 88.7a
E
1.10
9.7
–
R1
Soil
NR-074828.1
95 –
1.6
1.0
37.5d
R2
SoilE
E
b
R3
Soil
2.4
7.2
66.7
–
NR-028912.1
95
2.8
4.3
34.9c
–
R4
SoilE
NR-0744531.1
91 69.6b
1.7
5.6
–
R5
SoilE
E
c
NR-042447.1
97 38.7
1.9
3.1
–
R6
Soil
M
c
1.7
2.6
–
R7
Soil
NR-025972.1
93 34.6
M
9.6
6.6
–
31.3d
R8
Soil
M
a
NR-028801.1
96
R9
Soil
2.4
9.3
74.2
–
1.3
2.6
–
R10
SoilM
NR-028801.1
96 50.0b
2.3
0.19 · 107
–
99.9d
R11
SoilM
NR-025972.1
95
b
R12
MC-P
3.4
6.5
47.7
–
NR-074828.1
96 –
2.8 · 106
80.9d
R13
MC-P
1.46 · 107
R14
MC-P
1.3
0.01
99.2d
NR-074540.1
96 –
R15
MC-P
9.833
0.88 · 107
–
99.9d
NR-074540.1
96 70.7a
R16
MC-P
1.2
4.1
–
–
R17
MC-P
1.5
1.9
NR-042415.1
96 21.1c
1.32
0.14 · 108
–
98.9d
R18
SoilH
NR-042415.1
97 –
R19
SoilH
2.8
1.4
50.0d
NR-074453.1
97 87.4a
R20
SoilH
1.2
9.5
–
a
–
R21
PF
1.9
6.6
NR-074453.1
99 71.2a
–
R22
PS
1.3
5.9
77.9
NR-043289.1
95 77.3a
–
R23
PQ
1.5
6.6
NR-043289.1
96 Al-Shame area, Saudi Arabia; SoilE: El-Sharqia Governorate,
SoilM: Abu El-Matameer area, El-Behaira Governorate, Egypt; SoilH: Hada
NR-043289.1
Egypt; C: control; 2X RD: double recommended dose of Atrazine; MC-P:97
mixed culture of exogenous bacteria.
a
Remarkable growth.
NR-074453.1
94
b
High growth.
c
NR-042415.1
95
Medium growth.
d
Inhibited growth.
NR-074540.1
97
Bacterial isolate
Source
TVC (CFU · 109)
Growth stimulation (S%)
Growth inhibition (I%)
Alla fine di questo studio solo sette batteri appartenenti a quattro
from which these soils were collected. Salt levels have strong
tested strains. Sequences of the tested isolates were affiliated
effects not only on the microbial biodiversity, but also on
according to their
16S rDNAgene to members of 5 genera
famiglie
diverse(Enterobacter,Pseudonomas,Bacillus,Providencia)
sono
the sensitive and semi-sensitive higher plants. In addition, they
namely Enterobacter (E. cloacae), Bacillus (B. cereus and B.
also disturb the relations between plant roots and microbial
anthracis), Pseudomonas (P. aeruginosa, P. balearica, P. indica
stati
ritenuti efficaci per essere usati
nel processo di degradazione
communities. Variations in Atrazine fate depending on soil
and P. otitidis), Ochrobactrum (O. intermedium) and Providencharacteristics are consistent with those reported by Shifu
cia (P. vermicola) with similarities ranged between 91% and
and Yunzhang (2007) during photocatalytic degradation of
dell’atrazina
da
falde
contaminate.
99%.
glyphosate in aqueous solution using TiO powder.
The phylogenetic relationships of the experimental isolates
2
9 Conclusioni
•  L’atrazina è una sostanza tossica e persistente in condizioni
normali,essa è uno degli inquinanti più comuni nelle acque di
falda.
•  Anche se il percorso di Biodegradazione dell’atrazina non è
ancora perfettamente chiaro,la presenza di altra materia organica
biodegradabile,fonti di azoto e contenuto d’acqua del suolo sono i
fattori principali che controllano il suo tasso di degradazione.
•  Dagli studi analizzati si intuisce come a volte sia molto difficile
eliminare completamente il contaminante.Pertanto, si dovrebbe
dare più importanza alla prevenzione dell'inquinamento,per
prevenire a priori piuttosto che cercare di porre un rimedio a volte
impossibile.
•  Un importante rimedio può essere rappresentato dall’agricoltura
10 biologica.