1. 3. Wastewater characterization (1/6)
3.1. Generalities
3.2 Biodegradability assessment
3.3 Toxicity assessment
3.4 Chemical Characterization
3.5 General strategy for wastewater characterization before and during the treatment
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2. 3.1 Wastewater characterization (2/6)
Biodegradable
Effluents
Wastewater Characterization
 charge
 flow
 composition
 toxicity
 biodegradability
Biorecalcitrant
Effluents
Toxic and/or not
biodegradable
effluents
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3. 3.1 Wastewater characterization (3/6)
Causes of biorecalcitrance or non biodegradability
Biological causes:
Chemical causes:
Environmental causes:
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4. 3.1 Wastewater characterization (4/6)
Causes of biorecalcitrance or non biodegradability
Biological causes:
Inability of organisms to metabolise substrate, caused by:
- failure to induce enzymes
- inability to enter the cell
- failure to serve as substrate for the enzymes
- production of toxic compounds that disrupt metabolism
Absence of appropriate organisms
Predation, parasitism or poor viability
Loos of genetic potential; genetic instability
Absence of extra-cellular enzymes
Failure to involved appropriate metabolic attack
Inadequate acclimation or adaptation
Chemical causes:
Environmental causes:
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5. 3.1 Wastewater characterization (5/6)
Causes of recalcitrance
Chemical causes:
Refractory to enzymatic and/or non-enzymatic attack because of:
- molecular size, charge, shape
- presence of stable bonds or residues
- number, type and position of substituent groups
Concentration too low, too high, or too variable
Inadequate or slow solubility in water or lipids
Failure to form emulsions
Unsuitable physical state; solid, liquid or gas
Covalent or other bounds to organic or inorganic materials
Adsorption to surfaces
Biological causes:
Environmental causes:
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6. 3.1 Wastewater characterization (6/6)
Causes of biorecalcitrance or not biodegradability
Environmental causes:
Inappropriate temperature, pH, water potential, pO2, redox
potential, light or pressure
Unsuitable ionic conditions, including salinity
Presence of toxins or other inhibitory or competing compounds
Physical inaccessibility
Biological causes:
Chemical causes:
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7. 3.2 Biodegradability assessment (1/14)
Conditions 1
Ready 2
Inherent 3
Simulation
Inoculum Conc.
low
high
Acclimatisation no
possible
yes
Mineral media
natural
Target compound conc.
Other sources of organic carbon
Natural substrates (sediments, algae)
1 positive:
Chemicals rapidly biodegradable in the environment
2 positive:
Chemicals inherently biodegradable under optimal conditions
3 positive:
Chemicals biodegradable in simulated conditions
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8. 3.2 Biodegradability assessment (2/14)
Readily biodegradability test
Inherent biodegradability test
Simulation biodegradability test
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9. 3.2 Biodegradability assessment (3/14)
Readily biodegradability tests – OECD 301
CH3CH2OH + 3 O CO H20
COD O2
consumption
Respirometric
Methods
Formation
CO2
AFNOR: Evolution of DOC
STURM: Evolution of CO2
MITI: O2 consumption
FILOLE: O2 consumption
Respirometric
methods
Method
AFNOR
STURM
MITI
FIOLE
Poorly
soluble NO
YES
Volatile
Yes/NO SI
OCDE No 301 A
OCDE No 301 B
OCDE No 301 C
OCDE No 301 D
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10. 3.2 Biodegradability assessment (4/14)
Biochemical Oxygen Demand
The mass concentration of dissolved oxygen consumed under specific conditions by the biological oxidation of organic and / or inorganic matter in water.
Organic
pollutant
Micro-organisms
Mineral medium
This parameter constitutes a good indicator of the amount of biodegradable organic matter of a water.
Principle:
BOD is measured after 5 days (= BOD5), at 20°C (favourable temperature for the activity of the O2-consuming microorganisms) and in darkness (to avoid all parasitic photosynthesis).
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11. 3.2 Biodegradability assessment (5/14)
The OxiTop system is a patented method of determining biochemical oxygen demand, which offers numerous advantages over traditional methods. In addition, it meets all testing requirements of Standard Method 5210-D.
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12. 3.2 Biodegradability assessment (6/14)
Inherent Biodegradation
OECD 302B Zahn-Wellens
Biodegradability test applied in
(industrial) wastewater treatment plants.
Mineral medium (synthetic)
Target substances as only source of carbon
Semicontinuous activated sludge (SCAS)
Biodegradability in the environment)
Mineral medium natural
(domestic wastewater)
Others sources of carbon
Target substance at low concentration
(COD: 20 mg/l)
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13. 3.2 Biodegradability assessment (7/14)
OECD, Guidelines for testing of Chemicals
Vol 2, test 302B. 1996
Organization for Economic Cooperation and Development
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14. 3.2 Biodegradability assessment (8/14)
Zahn-Wellens-Biodegradability test
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15. 3.2 Biodegradability assessment (9/14)
Zahn – Wellens biodegradability test
Aeration
Thermometer
Agitator
25° C + 3° C
Air
Temperature
microscopic
Observations
pO2 pH
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16. 3.2 Biodegradability assessment (10/14)
Zahn – Wellens biodegradability test
In this method, the measure of the concentration of dissolved organic carbon or the chemical oxygen
demand is used to assess the ultimate biodegradability of the test substance.
The method is applicable only to those organic test substances which, at the concentration used in the
test:
-are soluble in water under the test conditions,
-have negligible vapor pressure under the test conditions,
-are not inhibitory to bacteria,
-are adsorbed within the test system only to a limited extent,
-are not lost by foaming from the test solution.
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17. 3.2 Biodegradability assessment (11/14)
Zahn – Wellens biodegradability test
The amount of degradation attained at the end of the test is reported as the 'Biodegradability in the
Zahn - Wellens test':
Dt(%) = (1 – (Ct – CB) / CA) *100
where:
Dt = biodegradation (%) at time T,
CA = DOC (or COD) values in the test mixture measured three hours after the beginning of the test (mg/l) (DOC = Dissolved Organic Carbon, COD = Chemical Oxygen Demand),
CT = DOC or COD values in the test mixture at time of sampling (mg/l),
CB = DOC or COD value of the blank at time of sampling (mg/l),
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18. 3.2 Biodegradability assessment (12/14)
Zahn – Wellens biodegradability test
Favorable Conditions
Air flow
Agitation
Room Temp.
Nutritious
Substances
High biomass concentration (1-2 g/l)
Zanh-wellens test
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19. 3.2 Biodegradability assessment (13/14)
Simulation tests
Wastewater Treatment Plant
Natural Ecosystems
rivers soils oceans
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20. 3.2 Biodegradability assessment (14/14)
Comparison BOD5 and Z-W
Compound
BOD5 (%COD)
Z-W (%COD)
Adipic acid 83
100
2-Aminobenzoic acid 90 97
3-Aminobenzoic acid 11
Aniline
tert-Butanol 96
Citric acid 85
2-Chlorobenzoic acid 94
2-Chloroaniline
n-Dodecylsulfate
Dyethilene glycol 80 95
EDTA 3 37
3-Nitrophenol 2 84
4-Nitrobenzoic acid 48
Triethanolamine 9 82
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21. 3.3 Toxicity assessment (1/7)
Toxic action
Organisms?
Selection of the most appropriate biological organism.
Ecological representativity
Biological criteria
LD50 (lethal dose-50)
Definition of work conditions: pH
Temperature
Light
Oxygen concentration
Alimentation of used microorganisms
Contest ?
Acute or chronic?
Appropriate test ?
Chemical Hazards Handbook
Introduction to applied toxicology.
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22. 3.3 Toxicity assessment (2/7)
In order to evaluate the impact of the toxic and biorefractory compounds on municipal activated sludge plants, various toxicity screening procedures have been proposed in the specialized literature and are based on the detection of the following variables indicative of the bacterial activity:
growth rate (viability of cells, substrate uptake, etc.),
enzymatic activity (dehydrogenase, adenosine triphosphate, etc.),
bacterial luminescence (Microtox® test),
metabolic heat production (microcalorimetric techniques), and
respiration rate.
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23. 3.3 Toxicity assessment (3/7)
Small fish
Daphnia
Protozoa (Colpidium)
Acute
Bacteria
Growth
Specific enzymatic activities
Cellular Energy
Bioluminescence
Toxicity
Testing
Algae (growth inhibition)
Chronic
Daphnia and fish (reproduction, growth, survival and accumulation effects)
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24. 3.3 Toxicity assessment (4/7)
Microtox test
The Microtox Acute Toxicity Test is a 15 minute exposure metabolic inhibition test which uses
freeze-dried luminescent bacteria (Vibrio fischeri)
to assess the acute toxicity of water, soil or sediment samples.
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25. 3.3 Toxicity assessment (5/7)
Respirometic test
OECD method 209.
Respirometer:
A closed batch reactor characterized by a high liquid/gas volume ratio (VL/VG=5) operating without liquid flow and with negligible oxygen mass transfer: according to the definition proposed by the Task Group IAWQ [10] it is an LSS respirometer (liquid phase principle, static gas, static liquid).
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26. 3.3 Toxicity assessment (6/7)
Respirometic test
OECD method 209.
The respirometer consists of a glass respirometric cell (1 l working volume) equipped with a water jacket connected to a thermostat to maintain a constant reaction temperature of 20±1°C.
The content of the cell is thoroughly stirred by a magnetic mixer. A glass diffuser feeds pure oxygen.
Three openings are located in the reactor cover and are used for inserting the oxygen electrode (WTW OXI cell 325), the aeration frit (located at the bottom of the cell) and for dosing the sample.
The oxygen electrode is interfaced with an IBM PC for automatic recording of data processed by specialized software (FIG P for Windows).
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27. 3.3 Toxicity assessment (7/7)
Simplicity and realistic approach of the biotest
enzymatic
Ideal
Biotest
particular functions
Physiologic
mono-specific
Multigenerational
ions
Communities
multi-specific
artificial
Simplicity of the biotest
Acute toxicity test
Communities
multi-specific
naturals
(microcosms)
Artificial
Ecosystems
(mesocosms)
natural
Ecosystem
(macrocosms)
Chronic toxicity test
Realistic approach
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28. 3.4 Chemical Characterization (1/3)
TOC/DOC
global
ions
COD
Chemical
Analysis
GC /GC-MS
specific
HPLC UV
GC/MS and flash animations of other analytical techniques
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29. 3.4 Chemical Characterization (2/3)
Chemical Oxygen Demand (COD)
The Chemical Oxygen Demand (COD) test is used to estimate the amount of organic matter in a sample.
It is a measurement of the oxygen equivalent of the materials present in the sample subject to oxidation by a strong chemical oxidant, in this case dichromate.
COD of organic matter in wastewater is measured by allowing it to react with a strong chemical oxidizing agent (potassium dichromate) in an acidic solution.
The test is carried out at high temperatures and in the presence of a catalyst (silver sulfate).
The amount of dichromate remaining at the end of the test is measured and subtracted from the amount of dichromate originally added to the wastewater.
Chemical Oxygen Demand
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30. 3.4 Chemical Characterization (2/3)
Chemical Oxygen Demand (COD)
The equivalent amount of oxygen required to oxidize the organic matter to CO2 is equal to the COD of the wastewater and is determined from the amount of dichromate consumed in the COD test.
CH2 + Cr2O7= + 8H+  CO2 + 2Cr(III) + 5H2O
yellow/orange green
440 nm nm
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31. 3.4 Chemical Characterization (3/3)
Total Organic Carbon (TOC)
Total Organic Carbon (TOC) analysis is a well-defined and commonly used methodology that measures the carbon content of dissolved and particulate organic matter present in water.
TOC analysis consists of inorganic carbon removal, oxidation of the organic carbon into CO2, and quantification of the CO2.
Total Organic Carbon (TOC)
Dissolved Organic Carbon (DOC)
Total Carbon (TC)
Inorganic Carbon (IC).
TC = TOC + IC
TOC-5000A
TOC analyzers
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32. 3.5 General strategy for characterization (1/3)
COD/TOC
HPLC
Microtox
BOD5 UV GC
ions
Zhan Wellens
Indication about oxidation state
COD/TOC
AOS = 4(TOC – COD)/TOC
BOD5/COD
Indication about biodegradability
Domestic wastewater ~0.4
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33. 3.5 General strategy for characterization (2/3)
Relation COD/TOC
Substance
COD/TOC
CH4
5.33
CH3CH2OH
4.00
CH3COCH3
3.56
C6H6
3.34
C6H5COOH
2.86
(COOH)2
0.67
AOS -4 +4
CH4
CO2
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34. 3.5 General strategy for characterization (3/3)
Environmental Analytical Water Quality Criteria
Minimal detectable signal:
S/N = 3
Quantification limit:
S/N = 10
Sensibility
Applicability
Resolution power:
R = Num. of signals *100
Types of molec.
Specificity
Quality of the
measurement
Absolute Error : Ea
Ea = xi - xt
Relative Error : Er
Er = (xi -xt)/xt *100%
Accuracy
Reliability
Repeatability
Under the same conditions of measurement
Experimental standard Deviation: s
s = √(∑(xi -x)2/(N-1))
Variance: s2
Precision
IUPAC compendium of analytical nomenclature
Reproducibility
Different conditions
Statistics for analytical chemistry
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35. 3.5 General strategy for characterization (3/3)
Environmental Analytical Water Quality Criteria
Seuil de détection:
S/N = 3
Seuil de quantification:
S/N = 10
Sensibilité
Applicabilité
Pouvoir de résolution:
R = Nb de signaux *100
Types de moléc.
Specificité
Qualité de la
mesure
Erreur absolue : Ea
Ea = xi - xt
Erreur relative : Er
Er = (xi -xt)/xt *100%
Exactitude
Fidélité
Répètabilité
Mêmes conditions expérimentales
Déviation standard: s
s = √(∑(xi -x)2/(N-1))
Variance: s2
Précision
IUPAC compendium of analytical nomenclature
Reproductibilité
Différentes conditions
Statistics for analytical chemistry
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