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I N S I D E Vol 14 No 2 September 2014
Watsons Notes
Innovative Solutions for Difficult Problems
Y o u h a v e r e c e n t l y b e e n in A f g h a n i s t a n I p e r c e i v e
With these words was born the mos t famous t e am in detect ive f i c t ion Sher lock Ho lmes and his t rus ted comrade and b iographer D r John H Watson
I n the spir i t of Wa t son who chronic led the explo i ts of Ho lmes we have created this news le t te r named Watsons Notes
In the pages of Watsons Notes modern day scribes documen t the discover ies unusual cases and o ther news of Inves t i ga t i ve Science Inco rpo ra ted our scienti f ic consul t ing f i rm in Bur l i ng ton Ontar io Canada
Please contact us if you have c ommen t s and please read on
I N V E S T I G A T I V E S C I E N C E INCORPORATED
Scientists and Technical Consultants 1050 Cooke Blvd Uni t 2 L7T 4A8 (905)634-4200 mailinvestigativesciencecom wwwinvest igat ivesciencecom
New Substance Notif ication (NSN) Testing Requirements under the Canadian Environmental Protection Act (CEPA)
O v e r v i e w of t h e O E C D Methods R e q u i r e d by C E P A
Canadian legislators require that test ing offered in support of regulatory applications such as the introduction of new chemicals into Canada (New Substance Notification Regulations see Canada Gazette Part I I Vol 139 No 19 pg 1864-1928) must be done according to OECD methods These OECD tests are also used worldwide for chemical registration and are similar to the OPPTS tests used in the United States We also provide these testing services in compliance with the Good Laboratory Practice (GLP) for European Australian and other jurisdict ions worldwide The OECD or the Organization of Economic Cooperation and Development is an international organization of which Canada is a member
The OECD Methods are a series of analytical tests designed to assess the chemical physical and environmental properties of chemicals and polymers The methods themselves are contained in a document entit led the OECD Guidelines for Testing of Chemicals Section 1 deals with Physical-Chemical Properties Section 2 Effects on Biotic Systems is largely focused on the behaviour of chemicals in the environment and describe a range of ecotoxicity bioassays Section 3 covers Degradation and Accumulation often referred to as biodegradation while Section 4 deals with Health Effects which are largely toxic ity tests
The chemical and physical tests described in Sections 1amp3 are the focus of our work at IS I These tests include
OECD 101 OECD 102 OECD 103 OECD 104 OECD 105 OECD 106
OECD 107
OECD 108
UV-Vis Absorption Spectra Melting Point Boiling Point Vapour Pressure Water Solubil ity Adsorption-Desorption (to and from representative soils) Octanol-Water partit ion coefficient (Flask Method) Complex Formation Abil ity In Water
OECD 109 Density OECD 110 Particle Size
OECD 110 Particle Size
OECD 111 Hydrolysis as a Function of pH
OECD 112 Dissociation Constants in water
OECD 113 Screening Test for Thermal Stabil ity and Stabil ity in Air
OECD 114 Viscosity of Liquids OECD 115 Surface Tension OECD 116 Fat Solubil ity OECD 117 Octanol-Water partit ion
coefficient (HPLC Method) OECD 118 Molecular Weight by Gel
Permeation Chromatography (GPC)
OECD 119 Determination of Low Molecular Weight Content by GPC
OECD 120 SolutionExtraction Behaviour of Polymers in Water
OECD 121 Estimation of the Adsorption Coefficient on Soil and Sewage sludge by HPLC
OECD 122 (Nov 2000 Draft)Partit ion Coefficient pH-Metric Method for Ionisable Substances
OECD 122 (July 2013) Determination of pH Acidity amp Alkalinity
OECD 316 Photolysis
W h e n A r e T h e s e Methods U s e d
In practice the CEPA regulation for New Substance Notification test ing which was released in October 2005 only requires a subset of this list for regulatory submission along with various tests from OECD Sections 2-4 Which test is required depends on how much material is produced whether the new material is a chemical (typical ly discrete molecular weight no repeat units) or polymer (contains repeat units and a distr ibution of molecular weights) as well as other factors
For example for new chemical test ing Schedule 5 of the CEPA Guidelines requires Melting Point (OECD 102) Boiling Point (OECD 103) Density (OECD
109) and Water Solubil ity (OECD 105) Vapour Pressure (OECD 104) is required if the boiling point is 0 degC or greater while OctanolWater Partition Coefficient (OECD 117) is required if the water solubil ity is 5 gL or less
CEPA Schedule 6 for chemicals produced in amounts greater than 10 000 kg requires UV IR NMR or Mass Spectra (OECD 101) in addition to the Schedule 5 requirements If the water solubil ity is 200 ugL or more Adsorption-Desorption (OECD 106) and Hydrolysis (OECD 111) are also required
For polymers produced in amounts greater than 10 000 kg Schedule 10 requires Water Extractabil ity (OECD 120) Octanol-Water Partition Coefficient (OECD 117) Number Average Molecular Weight Distribution (OECD 118) and Low Molecular Weight Content of a Polymer (OECD 119) Hydrolysis (OECD 111) is required when the water extractabi l i ty is 2 or more
H o w Do t h e Methods W o r k
UV I R NMR or M a s s S p e c t r a ( 1 0 1 )
OECD Method 101 actually only requires UV spectra but the CEPA regulation allows any of the above spectral techniques to be used Examples of the four types of spectra for benzene are shown in Figures 1-4 The spectrum of a substance refers to a characteristic behaviour usually the absorption of radiation displayed by the substance when placed in the appropriate device For regulatory purposes the spectra can then be used to ver i fy the identity of the mater ia l and in some circumstances may verify the purity or show the presence of impurit ies start ing materials and other reactants
170 180 190 200
Wavelength (nm| NIST Chemistry WcbBook (hltpwcbboaknis laovchcmislry)
Figure 1 - UV Spectrum of Benzene
100-
80not
S
20 -
o | bull bull i i m i |
00 15 30 45 SO 75 90
mz NIST Chemistry WebBook (htlpiZwebbooknlstgovbhernlstry)
Figure 2 - Mass Spectrum of Benzene
Figure 3 - NMR Spectrum of Benzene
00 I mdashmdashbullmdashbullmdashbullmdashbullmdashmdashbullmdashbullmdashbullmdashbullmdashimdashmdashLmdash-3000 2000 1000
Wavenumber (cm-1) NIST Chemistry WcbBook (httpZAvebbooknis1govchemiBtry)
Figure 4 - IR Spectrum of Benzene
Melting Po int ( 1 0 2 ) Boi l ing Po int ( 1 0 3 ) D e n s i t y ( 1 0 9 )
These tests are relatively straightnotforward physical tests used to characterize the new material Crystals of the material are heated in a controlled fashion until they melt or boil Traditionally the sample is placed in a glass vessel along side a thermometer and heated in a hot oil bath (see Figure 5) For crystall ine material a melt ing point apparatus is used
Density is simply the mass of the sample that can be held in a container of known volume As an i l lustration a kilogram of Styrofoam chips used for packing may fill a small room A kilogram of gold is only sl ightly bigger than a pile of business cards one centimetre high The method used depends on the nature of the sample The bulk density is most commonly used for regulatory purposes Non-viscous liquids are tested using a pycnometer while solids not affected by water can be tested using a displacement method
Figure 5 - Melting amp Boiling Point Apparatus
V a p o u r P r e s s u r e ( 1 0 4 )
Vapour pressure is an indicator along with melt ing point of the volat i l i ty of a compound ISI generally employs the Effusion method using a modified Knudsen Cell The test measures the change in weight of a substance as vapour is given off under controlled conditions
W a t e r So lub i l i ty ( 1 0 5 ) a n d W a t e r E x t r a c t a b i l i t y ( 1 2 0 )
The solubil ity tests simply put measure the amount of test substance that dissolves in water buffer or other solvent There are actually a series of similar tests designed to measure solubil ity in fat (OECD 116) octanolwater mixtures (OECD 107) and water (OECD 105 for pure chemicals and OECD 120 for polymers) OECD 105 may also be used to determine solubil ity in other solvents such as octanol
The tests are similar in concept but there are subtle differences in experimental set-up and interpretat ion For example OECD 105 is used to determine the solubil ity of pure chemicals in water The test substance is stirred or shaken in a flask with water at a carefully controlled temperature until the concentration of the substance in the water has stabilized This may take a few days to a month
For polymers using OECD 120 the CEPA regulation specifies that the water must be neutral acidic or basic pH depending on the type of polymer
All of the solubil ity methods require a specific method of detecting the chemical of interest in water octanol or whatever the solvent happens to be This method must be developed as part of the job Non-specific tests
such as weight (gravimetr ic) or Total Organic Carbon (TOC) are frowned upon in the method descriptions They are typical ly used only when there is no other workable option
O c t a n o l W a t e r Par t i t ion Coe f f i c i en t by H P L C ( 1 1 7 ) or F l a s k Method ( 1 0 7 )
The octanolwater partit ion coefficient is simply the extent to which a substance partit ions between octanol and water The partit ion coefficient (POW) is the ratio of the concentration of the test substance in the octanol phase divided by the concentration in the water phase
The test was developed many years ago to estimate the extent to which a chemical dissolved in water would partit ion into a cell membrane such as the skin of a mammal Octanol was used because its polarity was thought to be closely similar to that of the inside of a cell membrane The octanolwater partit ion coefficient or its logarithm (log POW) provides a useable estimate of the tendency of a material to bioaccumulate in animals
According to the theory any chemical with a log POW of 3 or greater will bioaccumulate by building up in animal fat assuming that the chemical is not already broken down A value of 3 means essentially that the material is 1000 t imes more soluble in octanol (and theoretical ly fat) than in water I t has been shown experimental ly that there is a good correlation between log POW and bioaccumulation in fish
The original method (107) employed a flask containing octanol water and the test substance More recently High Pressure Liquid Chromatography (HPLC) using a C18 reverse phase column (OECD 117) has become the preferred method according to Health Canada This approach exploits the correlation between log POW and HPLC retention t ime (how long it takes for the unknown compound to elute from the HPLC) The HPLC is calibrated using a series of chemicals with known POW A plot of log POW versus the log of the retention t ime gives a straight line (see Figure 7) The unknown is run on the same system and from the retention t ime the POW can be determined
-10000 ^5000 00000 05000 10000 15000
bgk
Figure 7 - Chromatogram of POW Standards and Calibration Curve
In practice these methods have some l imitations For example many factors such as charge affect the HPLC retention t ime of a compound For this reason the HPLC method is usually restricted to neutral or weakly acidic or weakly basic compounds As well wi th a polymer one usually sees a series of peaks so the calculated P O W is the weighted average of the peaks that are detected
Soi l A d s o r p t i o n - D e s o r p t i o n ( 1 0 6 amp 1 2 1 )
The OECD 106 soil adsorption-desorption test determines how readily the test substance sticks to soil and is released by the soil The test employs 5 well-characterized local soils having a range of pH values clay silt and organic content The new chemical is dissolved in salt water and incubated with the soils The amount and rate of disappearance of the chemical from the soil is measured Once the material has adsorbed to the soil the rate of release into fresh water is measured and Kd determined This test comprises three tiers prel iminary study screening test and determinat ion of kinetics or Freundlich adsorption or desorption isotherms Typically the regulatory agencies are only interested in the adsorption phase and are satisfied with the screening portion of the test
Soil Type 3 Tier 1 Analysis
Tme ^j r s )
Figure 8 - OECD 106 Tier 1 Adsorption Rate Plot
OECD 121 is a prel iminary test for OECD 106 using HPLC to determine a KOC value the adsorption coefficient normalised to the organic carbon content of the soil This method can also be applied to sludge as well as soil I t is similar to OECD 117 but uses a different column
H y d r o l y s i s ( 1 1 1 )
As the name implies the hydrolysis test measures the rate at which a chemical breaks down in water at various pH values and temperatures found in the environment The test requires a detection method capable of distinguishing the start ing material f rom the breakdown products typical ly some type of chromatography such as HPLC or GC This test is a tiered approach generating many samples for analysis and can take up to two months to complete
The test gives a hydrolysis rate constant (Kobs) at each pH and temperature from which the half-life ( t 0 5 ) of the reaction can be calculated The half-life is the t ime required for the initial amount of the test substance to decrease by half When Kobs values are known at two or more temperatures the Arrhenius Activation Energy (E) can be determined by plott ing ln(Kobs) vs 1temperature (see Figure 9) The slope is equal to -ER
Arrhenius Plot pH 9 74min
I S I MORE THAN J U S T A T E S T I N G LABORATORY I S I of fers suppor t fo r OECD bioassays (Sect ion 2 and 3 t e s t s ) as we l l as our Sect ion 1 tes t i ng This inc ludes ana ly t i ca l me thod deve lopment preparat ion and cert i f i cat ion of stock so lut ions and analysis (chemica l con f i rmat ion and quan t i t a t i on of the tes t substance in the tes t m a t r i x ) of the samples genera ted dur ing the tests There are two opt ions for runn ing the t e s t s
1 You manage and run the tests at you r fac i l i ty and I S I takes care of the support
2 I S I manages the job and contracts the runn ing of the tests to a GLP-compl iant lab t ha t specializes in runn ing these tests I S I rout ine ly prov ides suppor t fo r these tests Our staf f can prov ide guidance and t roub leshoot ing on a l l aspects of the tes t i ng
E L E M E N T A R Y MY
DEAR W A T S O N
S h erl lt Sherlock Holmes amp Watson go to a rock museum
They wa lk around for a whi le when Watson notices t ha t there is a rock t ha t doesnt have a display name
What kind of rock is tha t he asks Sherlock
S e d i m e n t a r y m y d e a r W a t s o n s a y s H o l m e s
-10678X + 30023 R3 = 09986
ln(k) Predicted ln(k)
-L inear (ln(k))
000305 00031 000315 00032 000325 00033 000335 00034 000345 Temperature (1K)
Figure 9 - Arrhenius Plot for OECD 111
Molecu lar W e i g h t ( 1 1 8 ) And L o w Molecu lar W e i g h t C o n t e n t ( 1 1 9 ) by G P C
Molecular weight determinat ion is required only for polymers and is done by Gel Permeation Chromatography (GPC) a type of HPLC that separates molecules by their size or molecular weight The test substance usually appears on a GPC chromatogram as one or more broad humps reflecting a distr ibution of molecular weights (see Figure 10)
To determine the proportion of material at each molecular weight using OECD 118 the hump is sliced electronically at regular intervals corresponding to known molecular weights The amount of material in each slice is proportional to the area under the curve of the pr intout (chromatogram)
The CEPA regulations require that the number average molecular weight (one of many ways to report the average weight) be reported as well as the percentage of material wi th molecular weight less than 500 and 1000 Daltons
Figure 10 - GPC Chromatogram of a Polymer in THF
The low molecular weight content is typically determined by GPC using OECD 119 This is closely related to 118 but focuses the calibrations in the 500-1000 molecular weight range According to the regulations polymers having less than 5 of their weight below 1000 Daltons and less than 2 below 500 fall into a lower risk category
Di rec t P h o t o l y s i s ( O E C D 3 1 6 )
This test is designed to determine whether the test substance degrades in water when exposed to the equivalent of 30 days sunshine Actual sunlight can be used for the test but usually a temperature controlled chamber employing a lamp similar to sunlight is used to allow more control over the conditions Quartz sample containers are used to maximize exposure of the sample to light
In Tier 1 a UVVisible spectrum is taken to determine whether theoretical ly the material will degrade Essentially if the material displays any absorbance above 290 nm Tier 2 must be performed Figure 11 shows the results of a typical Tier 1 analysis
M o l a r D e c a d i c E x t i n c t i o n C o e f f i c i e n t V s W a v e l e n g t h
Figure 11 - UVVis Absorbance vs Wavelength
Tier 2 exposes the substance to 30 days equivalent sunlight determines the rate of hydrolysis compared to a dark control and calculates the degradation rate constants Figure 12 shows a typical kinetic plot from which f irst order rate constants are calculated
OECD 316 Irradiated Vs Dark Control
Figure 12 - Typical ln(concentration) Vs Time plot
OECD 316 also requires that the major degradation products be identif ied This requires a laboratory experienced with photochemistry analytical chemistry and structure determinat ion ISI has these qualif ications to carry out this test on pure chemicals and mixtures
iS i INVESTIGATIVE SCIENCE INCORPORATED
Scientists and Technical Consultants
109) and Water Solubil ity (OECD 105) Vapour Pressure (OECD 104) is required if the boiling point is 0 degC or greater while OctanolWater Partition Coefficient (OECD 117) is required if the water solubil ity is 5 gL or less
CEPA Schedule 6 for chemicals produced in amounts greater than 10 000 kg requires UV IR NMR or Mass Spectra (OECD 101) in addition to the Schedule 5 requirements If the water solubil ity is 200 ugL or more Adsorption-Desorption (OECD 106) and Hydrolysis (OECD 111) are also required
For polymers produced in amounts greater than 10 000 kg Schedule 10 requires Water Extractabil ity (OECD 120) Octanol-Water Partition Coefficient (OECD 117) Number Average Molecular Weight Distribution (OECD 118) and Low Molecular Weight Content of a Polymer (OECD 119) Hydrolysis (OECD 111) is required when the water extractabi l i ty is 2 or more
H o w Do t h e Methods W o r k
UV I R NMR or M a s s S p e c t r a ( 1 0 1 )
OECD Method 101 actually only requires UV spectra but the CEPA regulation allows any of the above spectral techniques to be used Examples of the four types of spectra for benzene are shown in Figures 1-4 The spectrum of a substance refers to a characteristic behaviour usually the absorption of radiation displayed by the substance when placed in the appropriate device For regulatory purposes the spectra can then be used to ver i fy the identity of the mater ia l and in some circumstances may verify the purity or show the presence of impurit ies start ing materials and other reactants
170 180 190 200
Wavelength (nm| NIST Chemistry WcbBook (hltpwcbboaknis laovchcmislry)
Figure 1 - UV Spectrum of Benzene
100-
80not
S
20 -
o | bull bull i i m i |
00 15 30 45 SO 75 90
mz NIST Chemistry WebBook (htlpiZwebbooknlstgovbhernlstry)
Figure 2 - Mass Spectrum of Benzene
Figure 3 - NMR Spectrum of Benzene
00 I mdashmdashbullmdashbullmdashbullmdashbullmdashmdashbullmdashbullmdashbullmdashbullmdashimdashmdashLmdash-3000 2000 1000
Wavenumber (cm-1) NIST Chemistry WcbBook (httpZAvebbooknis1govchemiBtry)
Figure 4 - IR Spectrum of Benzene
Melting Po int ( 1 0 2 ) Boi l ing Po int ( 1 0 3 ) D e n s i t y ( 1 0 9 )
These tests are relatively straightnotforward physical tests used to characterize the new material Crystals of the material are heated in a controlled fashion until they melt or boil Traditionally the sample is placed in a glass vessel along side a thermometer and heated in a hot oil bath (see Figure 5) For crystall ine material a melt ing point apparatus is used
Density is simply the mass of the sample that can be held in a container of known volume As an i l lustration a kilogram of Styrofoam chips used for packing may fill a small room A kilogram of gold is only sl ightly bigger than a pile of business cards one centimetre high The method used depends on the nature of the sample The bulk density is most commonly used for regulatory purposes Non-viscous liquids are tested using a pycnometer while solids not affected by water can be tested using a displacement method
Figure 5 - Melting amp Boiling Point Apparatus
V a p o u r P r e s s u r e ( 1 0 4 )
Vapour pressure is an indicator along with melt ing point of the volat i l i ty of a compound ISI generally employs the Effusion method using a modified Knudsen Cell The test measures the change in weight of a substance as vapour is given off under controlled conditions
W a t e r So lub i l i ty ( 1 0 5 ) a n d W a t e r E x t r a c t a b i l i t y ( 1 2 0 )
The solubil ity tests simply put measure the amount of test substance that dissolves in water buffer or other solvent There are actually a series of similar tests designed to measure solubil ity in fat (OECD 116) octanolwater mixtures (OECD 107) and water (OECD 105 for pure chemicals and OECD 120 for polymers) OECD 105 may also be used to determine solubil ity in other solvents such as octanol
The tests are similar in concept but there are subtle differences in experimental set-up and interpretat ion For example OECD 105 is used to determine the solubil ity of pure chemicals in water The test substance is stirred or shaken in a flask with water at a carefully controlled temperature until the concentration of the substance in the water has stabilized This may take a few days to a month
For polymers using OECD 120 the CEPA regulation specifies that the water must be neutral acidic or basic pH depending on the type of polymer
All of the solubil ity methods require a specific method of detecting the chemical of interest in water octanol or whatever the solvent happens to be This method must be developed as part of the job Non-specific tests
such as weight (gravimetr ic) or Total Organic Carbon (TOC) are frowned upon in the method descriptions They are typical ly used only when there is no other workable option
O c t a n o l W a t e r Par t i t ion Coe f f i c i en t by H P L C ( 1 1 7 ) or F l a s k Method ( 1 0 7 )
The octanolwater partit ion coefficient is simply the extent to which a substance partit ions between octanol and water The partit ion coefficient (POW) is the ratio of the concentration of the test substance in the octanol phase divided by the concentration in the water phase
The test was developed many years ago to estimate the extent to which a chemical dissolved in water would partit ion into a cell membrane such as the skin of a mammal Octanol was used because its polarity was thought to be closely similar to that of the inside of a cell membrane The octanolwater partit ion coefficient or its logarithm (log POW) provides a useable estimate of the tendency of a material to bioaccumulate in animals
According to the theory any chemical with a log POW of 3 or greater will bioaccumulate by building up in animal fat assuming that the chemical is not already broken down A value of 3 means essentially that the material is 1000 t imes more soluble in octanol (and theoretical ly fat) than in water I t has been shown experimental ly that there is a good correlation between log POW and bioaccumulation in fish
The original method (107) employed a flask containing octanol water and the test substance More recently High Pressure Liquid Chromatography (HPLC) using a C18 reverse phase column (OECD 117) has become the preferred method according to Health Canada This approach exploits the correlation between log POW and HPLC retention t ime (how long it takes for the unknown compound to elute from the HPLC) The HPLC is calibrated using a series of chemicals with known POW A plot of log POW versus the log of the retention t ime gives a straight line (see Figure 7) The unknown is run on the same system and from the retention t ime the POW can be determined
-10000 ^5000 00000 05000 10000 15000
bgk
Figure 7 - Chromatogram of POW Standards and Calibration Curve
In practice these methods have some l imitations For example many factors such as charge affect the HPLC retention t ime of a compound For this reason the HPLC method is usually restricted to neutral or weakly acidic or weakly basic compounds As well wi th a polymer one usually sees a series of peaks so the calculated P O W is the weighted average of the peaks that are detected
Soi l A d s o r p t i o n - D e s o r p t i o n ( 1 0 6 amp 1 2 1 )
The OECD 106 soil adsorption-desorption test determines how readily the test substance sticks to soil and is released by the soil The test employs 5 well-characterized local soils having a range of pH values clay silt and organic content The new chemical is dissolved in salt water and incubated with the soils The amount and rate of disappearance of the chemical from the soil is measured Once the material has adsorbed to the soil the rate of release into fresh water is measured and Kd determined This test comprises three tiers prel iminary study screening test and determinat ion of kinetics or Freundlich adsorption or desorption isotherms Typically the regulatory agencies are only interested in the adsorption phase and are satisfied with the screening portion of the test
Soil Type 3 Tier 1 Analysis
Tme ^j r s )
Figure 8 - OECD 106 Tier 1 Adsorption Rate Plot
OECD 121 is a prel iminary test for OECD 106 using HPLC to determine a KOC value the adsorption coefficient normalised to the organic carbon content of the soil This method can also be applied to sludge as well as soil I t is similar to OECD 117 but uses a different column
H y d r o l y s i s ( 1 1 1 )
As the name implies the hydrolysis test measures the rate at which a chemical breaks down in water at various pH values and temperatures found in the environment The test requires a detection method capable of distinguishing the start ing material f rom the breakdown products typical ly some type of chromatography such as HPLC or GC This test is a tiered approach generating many samples for analysis and can take up to two months to complete
The test gives a hydrolysis rate constant (Kobs) at each pH and temperature from which the half-life ( t 0 5 ) of the reaction can be calculated The half-life is the t ime required for the initial amount of the test substance to decrease by half When Kobs values are known at two or more temperatures the Arrhenius Activation Energy (E) can be determined by plott ing ln(Kobs) vs 1temperature (see Figure 9) The slope is equal to -ER
Arrhenius Plot pH 9 74min
I S I MORE THAN J U S T A T E S T I N G LABORATORY I S I of fers suppor t fo r OECD bioassays (Sect ion 2 and 3 t e s t s ) as we l l as our Sect ion 1 tes t i ng This inc ludes ana ly t i ca l me thod deve lopment preparat ion and cert i f i cat ion of stock so lut ions and analysis (chemica l con f i rmat ion and quan t i t a t i on of the tes t substance in the tes t m a t r i x ) of the samples genera ted dur ing the tests There are two opt ions for runn ing the t e s t s
1 You manage and run the tests at you r fac i l i ty and I S I takes care of the support
2 I S I manages the job and contracts the runn ing of the tests to a GLP-compl iant lab t ha t specializes in runn ing these tests I S I rout ine ly prov ides suppor t fo r these tests Our staf f can prov ide guidance and t roub leshoot ing on a l l aspects of the tes t i ng
E L E M E N T A R Y MY
DEAR W A T S O N
S h erl lt Sherlock Holmes amp Watson go to a rock museum
They wa lk around for a whi le when Watson notices t ha t there is a rock t ha t doesnt have a display name
What kind of rock is tha t he asks Sherlock
S e d i m e n t a r y m y d e a r W a t s o n s a y s H o l m e s
-10678X + 30023 R3 = 09986
ln(k) Predicted ln(k)
-L inear (ln(k))
000305 00031 000315 00032 000325 00033 000335 00034 000345 Temperature (1K)
Figure 9 - Arrhenius Plot for OECD 111
Molecu lar W e i g h t ( 1 1 8 ) And L o w Molecu lar W e i g h t C o n t e n t ( 1 1 9 ) by G P C
Molecular weight determinat ion is required only for polymers and is done by Gel Permeation Chromatography (GPC) a type of HPLC that separates molecules by their size or molecular weight The test substance usually appears on a GPC chromatogram as one or more broad humps reflecting a distr ibution of molecular weights (see Figure 10)
To determine the proportion of material at each molecular weight using OECD 118 the hump is sliced electronically at regular intervals corresponding to known molecular weights The amount of material in each slice is proportional to the area under the curve of the pr intout (chromatogram)
The CEPA regulations require that the number average molecular weight (one of many ways to report the average weight) be reported as well as the percentage of material wi th molecular weight less than 500 and 1000 Daltons
Figure 10 - GPC Chromatogram of a Polymer in THF
The low molecular weight content is typically determined by GPC using OECD 119 This is closely related to 118 but focuses the calibrations in the 500-1000 molecular weight range According to the regulations polymers having less than 5 of their weight below 1000 Daltons and less than 2 below 500 fall into a lower risk category
Di rec t P h o t o l y s i s ( O E C D 3 1 6 )
This test is designed to determine whether the test substance degrades in water when exposed to the equivalent of 30 days sunshine Actual sunlight can be used for the test but usually a temperature controlled chamber employing a lamp similar to sunlight is used to allow more control over the conditions Quartz sample containers are used to maximize exposure of the sample to light
In Tier 1 a UVVisible spectrum is taken to determine whether theoretical ly the material will degrade Essentially if the material displays any absorbance above 290 nm Tier 2 must be performed Figure 11 shows the results of a typical Tier 1 analysis
M o l a r D e c a d i c E x t i n c t i o n C o e f f i c i e n t V s W a v e l e n g t h
Figure 11 - UVVis Absorbance vs Wavelength
Tier 2 exposes the substance to 30 days equivalent sunlight determines the rate of hydrolysis compared to a dark control and calculates the degradation rate constants Figure 12 shows a typical kinetic plot from which f irst order rate constants are calculated
OECD 316 Irradiated Vs Dark Control
Figure 12 - Typical ln(concentration) Vs Time plot
OECD 316 also requires that the major degradation products be identif ied This requires a laboratory experienced with photochemistry analytical chemistry and structure determinat ion ISI has these qualif ications to carry out this test on pure chemicals and mixtures
iS i INVESTIGATIVE SCIENCE INCORPORATED
Scientists and Technical Consultants
such as weight (gravimetr ic) or Total Organic Carbon (TOC) are frowned upon in the method descriptions They are typical ly used only when there is no other workable option
O c t a n o l W a t e r Par t i t ion Coe f f i c i en t by H P L C ( 1 1 7 ) or F l a s k Method ( 1 0 7 )
The octanolwater partit ion coefficient is simply the extent to which a substance partit ions between octanol and water The partit ion coefficient (POW) is the ratio of the concentration of the test substance in the octanol phase divided by the concentration in the water phase
The test was developed many years ago to estimate the extent to which a chemical dissolved in water would partit ion into a cell membrane such as the skin of a mammal Octanol was used because its polarity was thought to be closely similar to that of the inside of a cell membrane The octanolwater partit ion coefficient or its logarithm (log POW) provides a useable estimate of the tendency of a material to bioaccumulate in animals
According to the theory any chemical with a log POW of 3 or greater will bioaccumulate by building up in animal fat assuming that the chemical is not already broken down A value of 3 means essentially that the material is 1000 t imes more soluble in octanol (and theoretical ly fat) than in water I t has been shown experimental ly that there is a good correlation between log POW and bioaccumulation in fish
The original method (107) employed a flask containing octanol water and the test substance More recently High Pressure Liquid Chromatography (HPLC) using a C18 reverse phase column (OECD 117) has become the preferred method according to Health Canada This approach exploits the correlation between log POW and HPLC retention t ime (how long it takes for the unknown compound to elute from the HPLC) The HPLC is calibrated using a series of chemicals with known POW A plot of log POW versus the log of the retention t ime gives a straight line (see Figure 7) The unknown is run on the same system and from the retention t ime the POW can be determined
-10000 ^5000 00000 05000 10000 15000
bgk
Figure 7 - Chromatogram of POW Standards and Calibration Curve
In practice these methods have some l imitations For example many factors such as charge affect the HPLC retention t ime of a compound For this reason the HPLC method is usually restricted to neutral or weakly acidic or weakly basic compounds As well wi th a polymer one usually sees a series of peaks so the calculated P O W is the weighted average of the peaks that are detected
Soi l A d s o r p t i o n - D e s o r p t i o n ( 1 0 6 amp 1 2 1 )
The OECD 106 soil adsorption-desorption test determines how readily the test substance sticks to soil and is released by the soil The test employs 5 well-characterized local soils having a range of pH values clay silt and organic content The new chemical is dissolved in salt water and incubated with the soils The amount and rate of disappearance of the chemical from the soil is measured Once the material has adsorbed to the soil the rate of release into fresh water is measured and Kd determined This test comprises three tiers prel iminary study screening test and determinat ion of kinetics or Freundlich adsorption or desorption isotherms Typically the regulatory agencies are only interested in the adsorption phase and are satisfied with the screening portion of the test
Soil Type 3 Tier 1 Analysis
Tme ^j r s )
Figure 8 - OECD 106 Tier 1 Adsorption Rate Plot
OECD 121 is a prel iminary test for OECD 106 using HPLC to determine a KOC value the adsorption coefficient normalised to the organic carbon content of the soil This method can also be applied to sludge as well as soil I t is similar to OECD 117 but uses a different column
H y d r o l y s i s ( 1 1 1 )
As the name implies the hydrolysis test measures the rate at which a chemical breaks down in water at various pH values and temperatures found in the environment The test requires a detection method capable of distinguishing the start ing material f rom the breakdown products typical ly some type of chromatography such as HPLC or GC This test is a tiered approach generating many samples for analysis and can take up to two months to complete
The test gives a hydrolysis rate constant (Kobs) at each pH and temperature from which the half-life ( t 0 5 ) of the reaction can be calculated The half-life is the t ime required for the initial amount of the test substance to decrease by half When Kobs values are known at two or more temperatures the Arrhenius Activation Energy (E) can be determined by plott ing ln(Kobs) vs 1temperature (see Figure 9) The slope is equal to -ER
Arrhenius Plot pH 9 74min
I S I MORE THAN J U S T A T E S T I N G LABORATORY I S I of fers suppor t fo r OECD bioassays (Sect ion 2 and 3 t e s t s ) as we l l as our Sect ion 1 tes t i ng This inc ludes ana ly t i ca l me thod deve lopment preparat ion and cert i f i cat ion of stock so lut ions and analysis (chemica l con f i rmat ion and quan t i t a t i on of the tes t substance in the tes t m a t r i x ) of the samples genera ted dur ing the tests There are two opt ions for runn ing the t e s t s
1 You manage and run the tests at you r fac i l i ty and I S I takes care of the support
2 I S I manages the job and contracts the runn ing of the tests to a GLP-compl iant lab t ha t specializes in runn ing these tests I S I rout ine ly prov ides suppor t fo r these tests Our staf f can prov ide guidance and t roub leshoot ing on a l l aspects of the tes t i ng
E L E M E N T A R Y MY
DEAR W A T S O N
S h erl lt Sherlock Holmes amp Watson go to a rock museum
They wa lk around for a whi le when Watson notices t ha t there is a rock t ha t doesnt have a display name
What kind of rock is tha t he asks Sherlock
S e d i m e n t a r y m y d e a r W a t s o n s a y s H o l m e s
-10678X + 30023 R3 = 09986
ln(k) Predicted ln(k)
-L inear (ln(k))
000305 00031 000315 00032 000325 00033 000335 00034 000345 Temperature (1K)
Figure 9 - Arrhenius Plot for OECD 111
Molecu lar W e i g h t ( 1 1 8 ) And L o w Molecu lar W e i g h t C o n t e n t ( 1 1 9 ) by G P C
Molecular weight determinat ion is required only for polymers and is done by Gel Permeation Chromatography (GPC) a type of HPLC that separates molecules by their size or molecular weight The test substance usually appears on a GPC chromatogram as one or more broad humps reflecting a distr ibution of molecular weights (see Figure 10)
To determine the proportion of material at each molecular weight using OECD 118 the hump is sliced electronically at regular intervals corresponding to known molecular weights The amount of material in each slice is proportional to the area under the curve of the pr intout (chromatogram)
The CEPA regulations require that the number average molecular weight (one of many ways to report the average weight) be reported as well as the percentage of material wi th molecular weight less than 500 and 1000 Daltons
Figure 10 - GPC Chromatogram of a Polymer in THF
The low molecular weight content is typically determined by GPC using OECD 119 This is closely related to 118 but focuses the calibrations in the 500-1000 molecular weight range According to the regulations polymers having less than 5 of their weight below 1000 Daltons and less than 2 below 500 fall into a lower risk category
Di rec t P h o t o l y s i s ( O E C D 3 1 6 )
This test is designed to determine whether the test substance degrades in water when exposed to the equivalent of 30 days sunshine Actual sunlight can be used for the test but usually a temperature controlled chamber employing a lamp similar to sunlight is used to allow more control over the conditions Quartz sample containers are used to maximize exposure of the sample to light
In Tier 1 a UVVisible spectrum is taken to determine whether theoretical ly the material will degrade Essentially if the material displays any absorbance above 290 nm Tier 2 must be performed Figure 11 shows the results of a typical Tier 1 analysis
M o l a r D e c a d i c E x t i n c t i o n C o e f f i c i e n t V s W a v e l e n g t h
Figure 11 - UVVis Absorbance vs Wavelength
Tier 2 exposes the substance to 30 days equivalent sunlight determines the rate of hydrolysis compared to a dark control and calculates the degradation rate constants Figure 12 shows a typical kinetic plot from which f irst order rate constants are calculated
OECD 316 Irradiated Vs Dark Control
Figure 12 - Typical ln(concentration) Vs Time plot
OECD 316 also requires that the major degradation products be identif ied This requires a laboratory experienced with photochemistry analytical chemistry and structure determinat ion ISI has these qualif ications to carry out this test on pure chemicals and mixtures
iS i INVESTIGATIVE SCIENCE INCORPORATED
Scientists and Technical Consultants
Arrhenius Plot pH 9 74min
I S I MORE THAN J U S T A T E S T I N G LABORATORY I S I of fers suppor t fo r OECD bioassays (Sect ion 2 and 3 t e s t s ) as we l l as our Sect ion 1 tes t i ng This inc ludes ana ly t i ca l me thod deve lopment preparat ion and cert i f i cat ion of stock so lut ions and analysis (chemica l con f i rmat ion and quan t i t a t i on of the tes t substance in the tes t m a t r i x ) of the samples genera ted dur ing the tests There are two opt ions for runn ing the t e s t s
1 You manage and run the tests at you r fac i l i ty and I S I takes care of the support
2 I S I manages the job and contracts the runn ing of the tests to a GLP-compl iant lab t ha t specializes in runn ing these tests I S I rout ine ly prov ides suppor t fo r these tests Our staf f can prov ide guidance and t roub leshoot ing on a l l aspects of the tes t i ng
E L E M E N T A R Y MY
DEAR W A T S O N
S h erl lt Sherlock Holmes amp Watson go to a rock museum
They wa lk around for a whi le when Watson notices t ha t there is a rock t ha t doesnt have a display name
What kind of rock is tha t he asks Sherlock
S e d i m e n t a r y m y d e a r W a t s o n s a y s H o l m e s
-10678X + 30023 R3 = 09986
ln(k) Predicted ln(k)
-L inear (ln(k))
000305 00031 000315 00032 000325 00033 000335 00034 000345 Temperature (1K)
Figure 9 - Arrhenius Plot for OECD 111
Molecu lar W e i g h t ( 1 1 8 ) And L o w Molecu lar W e i g h t C o n t e n t ( 1 1 9 ) by G P C
Molecular weight determinat ion is required only for polymers and is done by Gel Permeation Chromatography (GPC) a type of HPLC that separates molecules by their size or molecular weight The test substance usually appears on a GPC chromatogram as one or more broad humps reflecting a distr ibution of molecular weights (see Figure 10)
To determine the proportion of material at each molecular weight using OECD 118 the hump is sliced electronically at regular intervals corresponding to known molecular weights The amount of material in each slice is proportional to the area under the curve of the pr intout (chromatogram)
The CEPA regulations require that the number average molecular weight (one of many ways to report the average weight) be reported as well as the percentage of material wi th molecular weight less than 500 and 1000 Daltons
Figure 10 - GPC Chromatogram of a Polymer in THF
The low molecular weight content is typically determined by GPC using OECD 119 This is closely related to 118 but focuses the calibrations in the 500-1000 molecular weight range According to the regulations polymers having less than 5 of their weight below 1000 Daltons and less than 2 below 500 fall into a lower risk category
Di rec t P h o t o l y s i s ( O E C D 3 1 6 )
This test is designed to determine whether the test substance degrades in water when exposed to the equivalent of 30 days sunshine Actual sunlight can be used for the test but usually a temperature controlled chamber employing a lamp similar to sunlight is used to allow more control over the conditions Quartz sample containers are used to maximize exposure of the sample to light
In Tier 1 a UVVisible spectrum is taken to determine whether theoretical ly the material will degrade Essentially if the material displays any absorbance above 290 nm Tier 2 must be performed Figure 11 shows the results of a typical Tier 1 analysis
M o l a r D e c a d i c E x t i n c t i o n C o e f f i c i e n t V s W a v e l e n g t h
Figure 11 - UVVis Absorbance vs Wavelength
Tier 2 exposes the substance to 30 days equivalent sunlight determines the rate of hydrolysis compared to a dark control and calculates the degradation rate constants Figure 12 shows a typical kinetic plot from which f irst order rate constants are calculated
OECD 316 Irradiated Vs Dark Control
Figure 12 - Typical ln(concentration) Vs Time plot
OECD 316 also requires that the major degradation products be identif ied This requires a laboratory experienced with photochemistry analytical chemistry and structure determinat ion ISI has these qualif ications to carry out this test on pure chemicals and mixtures
iS i INVESTIGATIVE SCIENCE INCORPORATED
Scientists and Technical Consultants
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