3.

Wastewater characterization (1/6)

3.1 Generalities

3.2 Biodegradability

3.3 Toxicity 3.1. Generalities

3.4 Chemical analysis
3.2 Biodegradability assessment
3.5 Strategy

3.3 Toxicity assessment

3.4 Chemical Characterization

3.5
General strategy for wastewater characterization before a
nd during the treatment

11/11/21 1
 3.1 Wastewater characterization (2/6)

3.1 Generalities

3.2 Biodegradability Biodegradable

Biodegradable
Effluents
Effluents
3.3 Toxicity

3.4 Chemical analysis

3.5 Strategy
Wastewater
WastewaterCharacterization
Characterization
charge
charge
flow
flow Biorecalcitrant
Biorecalcitrant
composition
composition Effluents
Effluents
toxicity
toxicity
biodegradability
biodegradability

Toxic
Toxicand/or
and/ornot
not
biodegradable
biodegradable
effluents
effluents

11/11/21 2
 3.1 Wastewater characterization (3/6)

3.1 Generalities

3.2 Biodegradability
Causes of biorecalcitrance or non biodegradability
3.3 Toxicity

3.4 Chemical analysis Biological causes:

3.5 Strategy

Chemical causes:

Environmental causes:

11/11/21 3
 3.1 Wastewater characterization (4/6)

3.1 Generalities
Causes of biorecalcitrance or non biodegradability
3.2 Biodegradability Biological causes:
3.3 Toxicity • Inability of organisms to metabolise substrate, caused by:

3.4 Chemical analysis

- failure to induce enzymes
- inability to enter the cell
3.5 Strategy
- 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:

11/11/21 4
 3.1 Wastewater characterization (5/6)

3.1 Generalities
Causes of recalcitrance
Chemical causes:
3.2 Biodegradability
• Refractory to enzymatic and/or non-enzymatic attack because of:
3.3 Toxicity
- molecular size, charge, shape
3.4 Chemical analysis
- presence of stable bonds or residues
3.5 Strategy
- 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:

11/11/21 5
 3.1 Wastewater characterization (6/6)

3.1 Generalities
Causes of biorecalcitrance or not biodegradability
3.2 Biodegradability

3.3 Toxicity
Environmental causes:
3.4 Chemical analysis
• Inappropriate temperature, pH, water potential, pO2, redox
3.5 Strategy

potential, light or pressure

• Unsuitable ionic conditions, including salinity
• Presence of toxins or other inhibitory or competing compounds
• Physical inaccessibility

Biological causes: Chemical causes:

11/11/21 6
 3.2 Biodegradability assessment (1/14)

Biodeg.
3.1 Generalities 1 2 3
Conditions
3.2 Biodegradability Ready Inherent Simulation 1 positive:
Chemicals rapidly
Inoculum
3.3 Toxicity Conc. low high low biodegradable in the
environment
3.4 Chemical analysis
Acclimatisation no possible yes
3.5 Strategy
2 positive:
Mineral media no yes natural Chemicals inherently
biodegradable under
Target compound conc. low high low optimal conditions

Other sources of organic

no possible yes
carbon 3 positive:
Chemicals biodegradable in
Natural substrates
no no yes simulated conditions
(sediments, algae)

11/11/21 7
 3.2 Biodegradability assessment (2/14)

3.1 Generalities

3.2 Biodegradability
Readily
Readilybiodegradability
biodegradabilitytest
test
3.3 Toxicity

3.4 Chemical analysis

3.5 Strategy
Inherent
Inherentbiodegradability
biodegradabilitytest
test

Simulation
Simulationbiodegradability
biodegradabilitytest
test

11/11/21 8
 3.2 Biodegradability assessment (3/14)

3.1 Generalities Readily biodegradability tests – OECD 301

3.2 Biodegradability
CH3CH2OH + 3 O2 2 CO2 + 3 H20
3.3 Toxicity

COD O2 Formation
3.4 Chemical analysis CO2
consumption
3.5 Strategy
Respirometric
Methods
AFNOR: Evolution of DOC
STURM: Evolution of CO2
Respirometric
MITI: O2 consumption
methods
FILOLE: O2 consumption

Method Poorly Volatile

soluble

AFNOR NO NO OCDE No 301 A

STURM YES NO OCDE No 301 B
MITI YES Yes/NO OCDE No 301 C
FIOLE NO SI OCDE No 301 D

11/11/21 9
 3.2 Biodegradability assessment (4/14)

3.1 Generalities
Biochemical Oxygen Demand
The mass concentration of dissolved oxygen
3.2 Biodegradability
consumed under specific conditions by the biological
3.3 Toxicity oxidation of organic and / or inorganic matter in
water.
3.4 Chemical analysis

3.5 Strategy

This parameter constitutes a good indicator of the amount of Organic

biodegradable organic matter of a water. pollutant

Principle: Micro-organisms

• BOD is measured after 5 days (= BOD5), at 20°C Mineral

(favourable temperature for the activity of the O2- medium
consuming microorganisms) and in darkness (to avoid all
parasitic photosynthesis).

11/11/21 10
 3.2 Biodegradability assessment (5/14)

3.1 Generalities

3.2 Biodegradability

3.3 Toxicity

3.4 Chemical analysis

3.5 Strategy

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.

11/11/21 11
 3.2 Biodegradability assessment (6/14)

3.1 Generalities
Inherent Biodegradation
3.2 Biodegradability

3.3 Toxicity

3.4 Chemical analysis

OECD
OECD302B
302BZahn-Wellens
Zahn-Wellens Semicontinuous
Semicontinuousactivated
activatedsludge
sludge
(SCAS)
(SCAS)
3.5 Strategy Biodegradability
Biodegradabilitytest
testapplied
appliedinin
(industrial)
(industrial)wastewater
wastewatertreatment
treatment Biodegradability
Biodegradabilityininthe
the
plants.
plants. environment)
environment)

••Mineral
Mineralmedium
medium(synthetic)
(synthetic) ••Mineral
Mineralmedium
mediumnatural
natural
(domestic
(domesticwastewater)
wastewater)
••Target
Targetsubstances
substancesas
asonly
onlysource
sourceofof
carbon
carbon ••Others
Otherssources
sourcesofofcarbon
carbon

••Target
Targetsubstance
substanceatatlow
low
concentration
concentration
(COD:
(COD:20 20mg/l)
mg/l)

11/11/21 12
 3.2 Biodegradability assessment (7/14)

3.1 Generalities

3.2 Biodegradability

3.3 Toxicity

3.4 Chemical analysis

3.5 Strategy

OECD, Guidelines for testing of Chemicals

Vol 2, test 302B. 1996

Organization for Economic Cooperation and Development

11/11/21 13
 3.2 Biodegradability assessment (8/14)

3.1 Generalities
Zahn-Wellens-Biodegradability test
3.2 Biodegradability

3.3 Toxicity

3.4 Chemical analysis

3.5 Strategy

11/11/21 14
 3.2 Biodegradability assessment (9/14)

3.1 Generalities Zahn – Wellens biodegradability test

3.2 Biodegradability

3.3 Toxicity 25° C + 3° C

3.4 Chemical analysis Thermometer

Air
3.5 Strategy
Temperature Aeration

microscopic
Observations

pO2
pH Agitator
Agitator

11/11/21 15
 3.2 Biodegradability assessment (10/14)

3.1 Generalities Zahn – Wellens biodegradability test

3.2 Biodegradability
In this method, the measure of the concentration of dissolved
3.3 Toxicity organic carbon or the chemical oxygen
3.4 Chemical analysis
demand is used to assess the ultimate biodegradability of the test
substance.
3.5 Strategy

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.

11/11/21 16
 3.2 Biodegradability assessment (11/14)

3.1 Generalities Zahn – Wellens biodegradability test

3.2 Biodegradability
The amount of degradation attained at the end of the test is reported
3.3 Toxicity
as the 'Biodegradability in the
3.4 Chemical analysis Zahn - Wellens test':
3.5 Strategy
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),

11/11/21 17
 3.2 Biodegradability assessment (12/14)

3.1 Generalities Zahn – Wellens biodegradability test

Favorable Conditions
3.2 Biodegradability

3.3 Toxicity
• Air flow
3.4 Chemical analysis

3.5 Strategy • Agitation

• Room Temp.
• Nutritious
• Substances
• High biomass concentration (1-2 g/l)

Zanh-wellens test

11/11/21 18
 3.2 Biodegradability assessment (13/14)

3.1 Generalities

3.2 Biodegradability

3.3 Toxicity
Simulation tests
3.4 Chemical analysis

3.5 Strategy

Wastewater Treatment Plant Natural Ecosystems

rivers soils oceans

11/11/21 19
 3.2 Biodegradability assessment (14/14)

3.1 Generalities Comparison BOD5 and Z-W

3.2 Biodegradability Compound BOD5 (%COD) Z-W (%COD)
Adipic acid 83 100
3.3 Toxicity
2-Aminobenzoic acid 90 97
3.4 Chemical analysis
3-Aminobenzoic acid 11 100
3.5 Strategy Aniline 90 100
tert-Butanol 0 96
Citric acid 90 85
2-Chlorobenzoic acid 0 94
2-Chloroaniline 0 94
n-Dodecylsulfate 85 97
Dyethilene glycol 80 95
EDTA 3 37
3-Nitrophenol 2 84
4-Nitrobenzoic acid 48 96
Triethanolamine 9 82
11/11/21 20
 3.3 Toxicity assessment (1/7)

3.1 Generalities Toxic action

3.2 Biodegradability

Organisms? Selection of the most appropriate

3.3 Toxicity
biological organism.
3.4 Chemical analysis Ecological representativity
3.5 Strategy
Contest ? Biological criteria
LD50 (lethal dose-50)

Acute or chronic? Definition of work conditions:

pH
Appropriate test ? Temperature
Light
Oxygen concentration
Alimentation of used
microorganisms
Chemical Hazards Handbook
Introduction to applied toxicology.
11/11/21 21
 3.3 Toxicity assessment (2/7)

3.1 Generalities In order to evaluate the impact of the toxic and

3.2 Biodegradability biorefractory compounds on municipal activated sludge
plants, various toxicity screening procedures have been
3.3 Toxicity
proposed in the specialized literature and are based on the
3.4 Chemical analysis detection of the following variables indicative of the
3.5 Strategy
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.

11/11/21 22
 3.3 Toxicity assessment (3/7)

3.1 Generalities •Small fish

3.2 Biodegradability
•Daphnia
•Protozoa (Colpidium)
3.3 Toxicity
Acute •Bacteria
3.4 Chemical analysis
Growth
3.5 Strategy Specific enzymatic activities
Cellular Energy
Toxicity Bioluminescence
Testing

•Algae (growth inhibition)

Chronic •Daphnia and fish (reproduction, growth,

survival and accumulation effects)

11/11/21 23
 3.3 Toxicity assessment (4/7)

3.1 Generalities Microtox test

3.2 Biodegradability The Microtox Acute
3.3 Toxicity
Toxicity Test is a 15 minute
exposure metabolic
3.4 Chemical analysis
inhibition test which uses
3.5 Strategy

freeze-dried
luminescent bacteria
(Vibrio fischeri)

to assess the acute toxicity of water, soil or sediment samples.

11/11/21 24
 3.3 Toxicity assessment (5/7)

3.1 Generalities Respirometic test

3.2 Biodegradability
OECD method 209.
3.3 Toxicity

3.4 Chemical analysis

Respirometer:
3.5 Strategy 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).

11/11/21 25
 3.3 Toxicity assessment (6/7)

3.1 Generalities
Respirometic test
3.2 Biodegradability
OECD method 209.
3.3 Toxicity
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.
3.4 Chemical analysis

3.5 Strategy
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).

11/11/21 26
 3.3 Toxicity assessment (7/7)

3.1 Generalities Simplicity and realistic approach of the biotest

3.2 Biodegradability

3.3 Toxicity enzymatic

particular Ideal
Simplicity of the biotest

3.4 Chemical analysis functions

Physiologic Biotest
3.5 Strategy mono-specific
Multigenerational
ions Communities
multi-specific
Acute toxicity test artificial Communities
multi-specific
naturals
(microcosms) Artificial
Ecosystems
(mesocosms)
natural
Ecosystem
Chronic toxicity test (macrocosms)

Realistic approach
11/11/21 27
 3.4 Chemical Characterization (1/3)

3.1 Generalities
TOC/DOC
3.2 Biodegradability

3.3 Toxicity global ions

3.4 Chemical analysis

3.5 Strategy
COD
Chemical
Analysis
GC /GC-MS

specific HPLC

U
GC/MS and flash animations of other analytical techniques
V
11/11/21 28
 3.4 Chemical Characterization (2/3)

3.1 Generalities Chemical Oxygen Demand (COD)

3.2 Biodegradability
The Chemical Oxygen Demand (COD) test is used to estimate the
3.3 Toxicity amount of organic matter in a sample.

3.4 Chemical analysis 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
3.5 Strategy
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

11/11/21 29
 3.4 Chemical Characterization (2/3)

3.1 Generalities Chemical Oxygen Demand (COD)

3.2 Biodegradability

3.3 Toxicity
The equivalent amount of
3.4 Chemical analysis
oxygen required to oxidize the
3.5 Strategy 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 600 nm

11/11/21 30
 3.4 Chemical Characterization (3/3)

3.1 Generalities Total Organic Carbon (TOC)

3.2 Biodegradability Total Organic Carbon (TOC) analysis is a well-defined and
3.3 Toxicity commonly used methodology that measures the carbon content of
dissolved and particulate organic matter present in water.
3.4 Chemical analysis

3.5 Strategy 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
11/11/21 31
 3.5 General strategy for characterization (1/3)

3.1 Generalities

3.2 Biodegradability
COD/TOC HPLC Microtox
BOD5
3.3 Toxicity

3.4 Chemical analysis ions UV GC

3.5 Strategy Zhan Wellens

COD/TOC Indication about oxidation state

AOS = 4(TOC – COD)/TOC

BOD5/COD Indication about biodegradability

Domestic wastewater ~0.4

11/11/21 32
 3.5 General strategy for characterization (2/3)

3.1 Generalities

3.2 Biodegradability

3.3 Toxicity Relation COD/TOC

3.4 Chemical analysis
Substance COD/TOC
3.5 Strategy
CH4 5.33 AOS
CH3CH2OH 4.00
CH3COCH3 3.56 -4 +4
C6H6 3.34 CH4 CO2
C6H5COOH 2.86
(COOH)2 0.67

11/11/21 33
 3.5 General strategy for characterization (3/3)

3.1 Generalities Environmental Analytical Water Quality Criteria

Minimal detectable
3.2 Biodegradability signal:
S/N = 3
3.3 Toxicity
Sensibility Quantification limit:
3.4 Chemical analysis S/N = 10
Applicability
3.5 Strategy Resolution power:
Specificity R=
Num. of signals *100
Quality of the Types of molec.
measurement Absolute Error : Ea
Ea = x i - xt
Accuracy
Relative Error : Er
Reliability Repeatability Er = (xi -xt)/xt *100%
Under the same
conditions of Experimental standard
Precision measurement Deviation: s

IUPAC compendium of analytical nomenclature Reproducibility s = √(∑(xi -x)2/(N-1))

Statistics for analytical chemistry Different conditions Variance: s2

11/11/21 34
 3.5 General strategy for characterization (3/3)

3.1 Generalities Environmental Analytical Water Quality Criteria

Seuil de détection:
3.2 Biodegradability S/N = 3

3.3 Toxicity Seuil de quantification:

Sensibilité S/N = 10
3.4 Chemical analysis
Applicabilité
3.5 Strategy Pouvoir de résolution:
Specificité R=
Nb de signaux *100
Qualité de la Types de moléc.
mesure Erreur absolue : Ea
Ea = x i - xt
Exactitude
Erreur relative : Er
Fidélité Répètabilité Er = (xi -xt)/xt *100%
Mêmes conditions
expérimentales Déviation standard: s
Précision
s = √(∑(xi -x)2/(N-1))
IUPAC compendium of analytical nomenclature Reproductibilité
Variance: s2
Statistics for analytical chemistry Différentes conditions

11/11/21 35