Maissey Flashcards
Aims of Toxicity ID Evaluation
improve control of chronic as well as acute testing
properly document effluent toxicity
ID the causes of toxicity
provide info to enable reduction or elimination of effluent toxicity
gives fantastic data for ecological relevnce
considers bioavailability
LIMITED in ability to ID toxicant
In vivo testing
Uses whole living organisms to assess toxicological effect
EDA
uses biological effects tp narrow down potential toxicants
• EDA extracts right from the outset (organic solvent extract, solid
phases extraction, etc.) with a focus on organic compounds
• This is to the detriment of potential non organic toxicants.
• By utilising pre-concentrated and purified samples, the whole
sophisticated array of instrumentation available to analytical
chemistry can be utilised.
• This Neglects bioavailability, data of minor ecological relevance.
•In vitro testing used to narrow the range of possible compounds
(quicker and cheaper testing).
In vitro testng
Outside of the living organism Looks at various known biological outcomes of toxic materials e.g. • Endocrine disruption • Estrogenic activity • Androgenic activity • Dioxin and dioxin-like compound activity • Cytochrome P-450 activity • Mutagenicity • Bioluminescence • Growth factors
TIE:
What chemicals are causing the observed toxicity?: maintain sample integrity by using
three phases: characterisation, identification and confirmation.
EDA :
What biological effects can we observe to narrow down the potential list of toxicants:
Extract using organic solvents to isolate the likely toxicants and then performs biotesting.
Bioavailability
what is it and what is it dependent on
What fraction of the toxicant exists in a form that is able to be
absorbed, metabolised or otherwise integrated into an organism
Bioavailability is significantly dependant on:
• Polarity
• Speciation
• pH
• Ionic strength
• Sample matrix (what other chemical species exist in the sample)
• Temperature
• Chelation
Phase 1 testing - Typical Fractionation steps
• Filtration (removes particulates > 0.45 mm)
• Treatment of filtrate with C18 or XAD resin (removes non-polar organics)
• Organic-free fraction subdivided and treated with some of the following treatments…
• EDTA
• cation-exchange resin
• anion exchange resin
• zeolite column
• aeration
• pH adjustment
• treatment with thiosulfate (reacts with halogens, bleaches, etc.)
Remove non-polar organics (resin or chromatography column)
• Remove metals (add EDTA)
•Exchange cations or anions (swap metal cations plus NH4
+
for
something like sodium)
•Remove volatiles (e.g. hydrocarbons, ammonia, by bubbling air
through)
•Remove acidic or basic species (e.g. change pH)
•Remove small cations, ammonia, some reactive organics (zeolite
column)
• Remove oxidising materials like halogens and bleaches , etc.
(add thiosulfate )
Phase 2 testing - examples of follow-up from phase 1
If organics are implicated
• e.g. toxicity reduced after chromatography / inorganics not implicated
• Phase II : the stuff from the chromatography column can be eluted and
a reconstituted organics solution made up & tested separately
If zeolite, aeration and pH 2 treatment all reduce toxicity
• implies a volatile, basic species is the cause
• Phase II: test specifically for NH3 or for organic amines if no ammonia
found
Purpose of chemical
analysis in toxicology
Separate complex mixtures
• Identify individual chemical constituents
• Identify secondary pollutants
• Secondary pollutants are derivatives of
primary pollutants by either
decomposition or chemical reaction.
• Quantify individual and groups of chemical
constituents.
Fit for
Purpose?
• What will the chemical information be used for? This question is crucial in
determining which chemical tests could and can be applied.
• Timing considerations? Is a result required immediately, in a few hours or
days?
• What degree of confidence in the result is required? Different contexts
require varying degrees of quality assurance (QA) and quality control (QC) .
• What is the nature of the sample available?
• Is the sample divisible?
• Is the sample homogenous making a sub-sample representative or do multiple tests
need to be performed?
• Is there sufficient sample for multiple tests to performed?
• Can some or all of the sample be destroyed?
Overview of Chromatographic Techniques
• The mobile phase (which can be liquid or gas) carries the
sample across or through the stationary phase (solid or
viscous liquid).
• The attraction of each component of the mixture to either
the mobile or stationary phase will dictate how long it
takes to pass through the system, thus separating the
components of a mixture.
Solid phase extraction
Solid phase extraction is based on the need to partition a
liquid sample into two categories.
• One which contains a target group or individual
analyte – this remains on the stationary phase.
One that does not contain the target group/individual
analyte – this portion remains in the mobile phase.
• The species on the stationary phase can then be
extracted and analysed or used in toxicity tests
(usually EDA analysis)
• The mobile phase can then be used in toxicity tests
(usually TIE analysis)
Solid phase extraction (SPE) capsules
SPE columns are used for rapid solid phase
extraction.
Some example categories extracted with SPE:
• non-polar extractions (Lichrolut® RP18 and RP18e)
• polar extractions (Lichrolut® Si and Lichrolut® CN)
• Cation Exchange extraction (Lichrolut® SCX)
• mixed mode extraction (Lichrolut® TSC)
• non-polar extractions on a polymer phase
(Lichrolut® EN) which is especially well suited for
the extraction of pesticides and phenols in water
or drugs in body fluids.”
What is ion exchange?
The exchange of ions of the same charge between
an insoluble solid and a solution in contact with it:
enables cation and anions to be separated and
examined.
Anion-exchange resin: mobile anions (in sample) are
retained by cations bonded to the stationary phase
and replaced with single, known anion.
Converse mode of exchange is also available (cationexchange
resin).
Ion exchange resins
• Micro beads of an organic polymer substrate
• Targeted ions are trapped in pores triggering release of replacement
ions of equivalent charge.
Ion Chromatography
Stationary phase : anion-exchange or cation-exchange resin (polymer) Mobile phase : Aqueous solution of an electrolyte : ions in the mobile phase compete with analyte ions for ion-exchange sites on the resin this enhances separation.
Mobile Phases- ion chromatography
The aim of chromatography is to separate the minor constituents of the
sample. The mobile phase can help facilitate this.
• Aqueous solution of an electrolyte
• e.g. for separation of anions, using Na2CO3
(aq) as CO3
2-
ions compete with
sample anions for sites on the anion-exchange resin
• e.g. for separation of cations, using HCl (aq) as H+
ions compete with sample
cations for sites on the cation-exchange resin
Mobile Phases- ion chromatography
Retention depends on
• The charge and size of the ions (more highly charged ions retained longer)
• The type and concentration of the competing ions in the mobile phase (can
be varied)
• For some ions, pH will be very important e.g. carbonate, phosphate,
carboxylates
Gas Chromatography - GC
• Mobile Phase is a carrier gas. • Stationary Phase is a solid or liquid (supported on a surface). • Stationary phase is located in a (usually) long and narrow column. • Sample introduced near beginning of column where it is vapourised. • Detector senses compounds eluting from the column. • GC is most important analytical method for volatile compounds.
