P R O T O C O L

READY BIODEGRADABILITY BY THE CARBON DIOXIDE EVOLUTION TEST METHOD

Organisation for Economic Cooperation and Development OECD Guideline 301B

and
Council of the European Communities Directive 67/548/EEC Annex V, Guideline C.4-C- 2 -

READY BI ODEGRADABI L I T Y BY T H E CARBON DI OX I D E EV OLUT I ON T E ST M E T HOD

SPONSOR :

SPONSOR’ S REPRESENT A T I V E :

T E ST I NG FACI LI T Y :

ST UDY DI RECT OR :

LABORAT O RY M ANAGEM E NT :

F O R LAB O RATO R Y USE O N LY Proposed Dates: Experim e ntal Start Date: ________________________ Experim e ntal T e rm ination Date: _________________ Proj ect No.: ________________________
T e st Concentr ations: _________________________________________________________

T e st Substance No.: ___________ Reference Substance No. (if applicable): ____________

PROT OCOL APPROV AL

ST UDY DI RECT OR DAT E

LABORAT O RY M ANAGEM E NT DAT E

SPONSOR’ S REPRESENT A T I V E DAT E- 3 -

I N TRO DUCTI O N
T e sts of ready biodeg r adability , by definiti on, prov ide lim ited opportunity for acclim ation and biodeg r adation to occur. A positiv e result in a test of ready biodeg r adability is an indication that the test
substance w ill underg o rapid and ultim ate biodeg r adation in the env i ronm ent. A neg a tiv e result in a test of ready biodeg r adability does not necessarily m ean that the test substance w ill not be biodeg r aded
under relev a nt env i ronm ental conditions but that additional testing m a y be needed.

OBJE C T IV E
T h e obj ectiv e of the study w ill be to m easure the am ount of carbon dioxide (C O 2 ) produced
from the biodeg r adation of a nonv olatile org a nic test substance and express it as a percentag e of the
theoretical am ount of CO 2 (TC O 2 ) that could hav e been produced assum i ng com p lete biodeg r adation of
the test substance to C O 2 and other inorg anic constituents.

EXP ERI M ENTAL DESI GN
T h e test w ill contain a blank control g r oup, a reference g r oup, and a treatm e nt g r oup. Each

g r oup w ill contain three replicate test cham bers. T h e blank control is used to m easure the back g r ound
CO 2 production of the inoculum and w ill not be dosed w ith a carbon source. T h e reference cham bers

w ill be dosed w ith sodium benz oate, a substance k now n to be biodeg r adable, at a concentration of
10 m g C / L. T h e treatm e nt g r oup w ill be used to ev aluate the test substance at 10 m g C / L.

M A TERI A LS AND M ETH O D S
T e st m e thods are based on the procedures speci fied in the OECD Guid eline for T e sting of
C h em icals, G u ideline 301B (1) and C ouncil of the European C o m m

unities, G u ideline C .4- C
, Carbon
Di ox i d e (CO 2 ) Evolution (2).

Test Subst a nce I n form ation on the characteriz ation of test, cont rol or reference substances is required by Good Laboratory Practice (GLP ) Standards and Principles. T h e Sponsor is responsible for prov iding

v e rification that the te st substance has been characteriz ed according to G L P s prior to its use in the study . I f v e rifica tion of GLP test substan ce characteriz ation is not

prov ided to ., it w ill be noted in the com p liance statem ent of the final report.

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T h e Sponsor is responsible for all inform ation re lated to the test substance and ag rees to accept
any unused test substance and/or test substance containers rem a ining at the end of the study

.

I f

the test substance is relativ ely w a ter solubl e, a stock solution of the test substance w ill be prepared at a nom inal concentration of 400 m g C / L in hig h quality w a ter. V o l u m e t r i c a d d ition of a test
substance stock solution is the m o st appropriate rout e of adm i nistration for water- soluble m a terials. The dosing v o lum e of the test substance stock solu tion w ill be calculated based on the m easured TO C

. T h e stock solution m a y be stored in a refrig e rator for a m a xim u m of three day s

.

For poorly soluble m a terials, the test substa nce w ill be adm i nistered by direct w e ig ht
addition. D i rect w e ig ht addition of a test substance that is relativ ely insoluble in w a ter is the m o st appropriate route of adm i nistrati on. D o sing am ounts w ill be based on the carbon content of the test substance. I f the carbon content of the test s ubstance is not prov ided by the Sponsor, the carbon

content w ill be determ ined by elem ental analy s is and w ill be perform ed by Q u antitativ e T echnolog ies
I n c. (Whitehouse, N J ). T h e carbon analy s is w ill not be conducted in accordance w ith G ood Laboratory Practice Standards.

Ref erence Substance
A stock solution of sodium benz oate w ill be pr epared at a nom inal concentration of 400 m g C / L in hig h quality w a ter. The TO C of the stock solution m u st w ithin 15% of the nom inal concentration to
be acceptable. I f the T O C is not acceptable, a new stock solution w ill be prepared. T h e dosing v o lum e of the reference substance stock solution w ill be calculated based on the m easured TO C

. The stock
solution m a y be stored in a refrig e rator for a m a xim u m of three day s

.

T e st Medium
The test m e dium is a m odified biochem i cal oxy g e n dem a nd (B O D ) test dilution w a ter and w ill be prepared using hig h quality w a ter as described in A ppendix I

.

Test Apparat u s and Condi t i ons The test cham bers w ill be am ber 4- liter bottles. The air entering the cham bers w ill be passed throug h D r ierite to rem o v e am bient m o isture and then throug h A s carite to produce C O 2 free air. T h e air

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exiting the test cham bers w ill be passed throug h a se ries of three g a s w a shing bottles each containing approxim a tely 100 m Lof 0.5 N K OH to trap the CO 2 that has ev olv e d w ithin the cham ber. A n additional set of g a s w a shing bottles that are not connected to a cham ber w ill be m a intained concurrently with the traps connected to the cham bers. T h e am ount of CO 2 detected in these traps w ill
be subtracted from the blank control traps to determ ine the am ount of CO 2 produced by the blank
control. T h e test w ill be incubated at 20±3° C

. T e st tem p eratures w ill be m easured each w o rk ing day

u s i n g a m i n

/ m a x t h e r m o m e t e r

. Mag n etic stirrers w ill be em ploy ed to m i x the contents of the
cham bers. The stirrers w ill be cy cled on and off approxim a tely ev ery 15 m i nutes to prev ent the

t r a n s f e r o f h e a t f r o m t h e s t i r r e r m o t o r s t o t h e t e s t c h a m b e r s

. Test cham bers w ill be identified by proj ect num ber, test substance I D , te st concentration, and v e ssels num ber.

T e st Inoculum
A c tiv ated sludg e w ill be collected from D e nton Wastew a ter T r eatm e nt Facility
, D e nton, Mary land. T h e sludg e w ill be siev ed using a 2 m m sc reen and then aerated for approxim a tely four
hours. A f ter the aeration period, the sludg e w ill be hom og eniz ed in a blender at m edium speed for approxim a tely 2 m i nutes and then allow e d to settle for approxim ately 30 m i nutes. I f in the opinion of
the S t udy D i rector the supernatant abov e the settled solids contains hig h lev els of suspended sludg e solids after 30 m i nutes of settling
, the sludg e m a y be settled for an additional 30 m i nutes. The supernatant w ill be used as the inoculum the sam e day that it is prepared. A total suspended solids

m easurem ent and standard plate count w ill be perform ed on the inoculum . Plates w ill be incubated at 20 ± 3° C for approxim a tely 48 hours.

P reparation of Test Chambers
T h e follow i ng w ill be added to each cham ber: 1) 2470 m L

of hig h quality w a ter 2) 3 m L

calcium chloride solution
3 m L

of ferric chloride solution
3 m L

of m a g n esium sulfate solution
30 m L of phosphate buffer 3) 30 m L

of the activ ated sludg e inoculum

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A ll cham bers w ill be aerated w ith C O 2 free air for approxim a tely 24 hours at a rate of 50 - 100

m

L

per m i nute to purg e the sy stem s of CO 2 . After the aeration period, the flow of CO 2 free air w ill be stopped, and three CO 2 traps each containing approxim ately 100 m L of 0.5 N KO H w ill be connected to
the exit air lines of each cham ber. A v o lum e refere nce substance stock solution necessary to deliv er 10

m g C / L w ill be added to the reference g r oup cham bers. T r eatm ent g r oup test cham bers w ill be dosed

w ith sufficient test substance to deliv er 10 m g C / L

. The v o lum e w ithin all cham bers w ill be broug ht up
to 3000 m L by the addition of hig h quality w a ter.

B i odegradation Test Initiation
T h e biodeg r adation test w ill be started by bubbling C O 2 free air throug h the test m e dia at a rate
of 50- 100 m Lper m i nute. T h e CO 2 produced from the deg r adation of org a nic carbon sources within the
test cham ber is trapped as K 2 CO 3 in the K OH solution and m easured using a carbon analy z er.

S a mp le C o llection an d A n alysis
Th e C O 2 traps w ill be rem o v e d for analy s is tw ice a w eek ov er the 4- w eek test period. More or less frequent rem o v a l and analy sis of the traps m a y be conducted at the discreti on of the study director with consultation with the Sponsor. T h e CO 2 trap nearest the cham ber w ill be rem o v e d and analy z ed for inorg a nic carbon. T h e tw o rem a ining traps are pl aced one position closer to the test cham ber and a

new trap is placed on the end of the series.

T e st T ermin ation
O n the 28th day of the test an aliquot of the contents of each test cham ber w ill be rem o v e d and the pH determ ined. The contents of cham ber w ill then be acidified by the addition of 3 m L of concentrated hy drochloric acid to driv e off i norg a nic carbonate. T h e cham bers w ill be aerated

ov ernig h t and then a sam p le from each cham ber w ill be rem o v e d for dissolv ed org a nic carbon (D O C ) analy s is. The rem a ining trapping solutions closest to the test cham bers w ill be analy z ed for inorg a nic
carbon.

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C a lcu l ation s T h e results of the carbon analy s is w ill be conv erted to m g C O 2 produced and used to determ ine
the percentag e of theoretical C O 2 (%TC O 2 ) ev olv e d at each sam p ling interv al. T h e cum u lativ e %T C O 2

w ill be plotted ag ainst tim e. T h e results of the carbon analy s es w ill be conv erted to m g C O 2 using the following equation:

mg CO re s u lt mg C L x v ol of KOH L x m g C O m g C 2 2 3 6 7 = ( )

. ( )

. /

T h e cum u lativ e m g of C O 2 for the test and reference subs tances w ill be corrected for the

am ount of CO 2 ev olv e d by control, using the following equation: 

# ∑ ∑

− = control CO mean test CO mg evolved CO mg Cumulative 2 2 2

T h e percent of theoretical C O 2 produced w ill be calculated using the follow i ng equation:

% ( ) (

. / TC O mg CO pro duc e d
mg c a rb on in te s t mg CO mg Carbo n X 2 2

2 3 6 7 100 = )

R e sults
N o bias is expected in this study and statistical m e thods w ill not be used in the analy sis of the
data. I n terpretation of the results w ill be based on the follow i ng :

1. T h e test is considered v a lid if the reference substance produces at least 60% of its T C O 2 w ithin 14 day s , and the CO 2 production of the blank control does not exceed 40 m g /L.

2. T e st substances achiev i ng 60% of T C O 2 w ithin 10 day s of reaching 10% T C O 2 are reg a rded as readily biodeg r adable. I f the 60% criteria is not m e t, biodeg r adation studies conducted under

m o re fav o rable conditions should be perform ed to ev aluate the substances biodeg r adation potential.

RECO RDS TO B E M A I N TAI N ED
R ecords to be m a intained w ill incl ude, but not lim ited to, the follow i ng : 1. A copy of the sig n ed protocol.
2. I d entification and characteriz ation of th e test substance as prov ided by Sponsor.

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3. S t udy initiation and term ination dates.
4. E xperim e ntal initiation and term ination dates.
5. Stock solution concentration cal culations and solution preparation.
6. I noculum source a nd pretreatm ent data.
7. Results of CO 2 analy s is.
8. T e m p erature rang e recorded during test period.
9. Copy of final report.

F I NAL REP O RT
A final report of the results of the study w ill be prepared by The report is to include, but is not lim ite d to, the following , when applicable:
1. N am e and address of facility perform ing the study

. 2. D a tes on w h ich the study w a s initiated and com p leted.
3. A statem ent of com p liance sig n ed by the St udy Director addressing any exceptions to Good Laboratory Practice Standards.
4. O b j ectiv es and procedures stated in the approv ed protocol, including any chang e s in the orig inal protocol.
5. I d entification and characteriz a tion of the test substance as prov ided by Sponsor including nam e , CAS num ber, percent activ e, percent carbon, theo retical T O C, and other characteristics, if

prov ided by the Sponsor.
6. A description of the transform ations and calculations perform ed on the data.
7. Results of the T O C analy s is perform ed on test and reference substance stock solutions.
8. A description of the test sy stem

. 9. A description of the preparati on of the test solutions, the testing concentrations, the route of
adm i nistration, and the duration of the test.
10. A description of all circum stances that m a y of affected the quality or integ r ity of the data.
11. T h e nam e of the study director, the nam e s of ot her scientists or professionals, and the nam e s of all superv isory personnel, inv o lv ed in the study

. 12. T h e sig n ed and dated reports of each of the indi v i dual scientists or other professionals inv o lv ed in the study , if applicable.
13. T h e location where the raw data a nd final report are to be stored.

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14. A statem ent prepared by the Q u ality A ssurance U n it listing the dates that study inspections and
audits were m a de and finding s reported to the Study Direct or and Manag e m e nt.

CH ANGES TO TH E F I NAL REP O RT
I f

it is necessary to m a k

e corrections or add itions to the final report after it has been accepted, such chang e s shall be m a de in the form of an am e ndm ent issued by the Study Director. T h e am endm ent shall clearly identify the part of the study that is being am ended and the reasons for the alteration.
Am endm ents shall be sig n ed and dated by the Study Director and Laboratory QA.

C H A N G E S T O PR OT OC OL
P l anned chang e s to the protocol w ill be in th e form of w r itten am endm ents sig n ed by the Study D i rector and approv ed by the Sponsor’s R e pr esentativ e. A m endm ents w ill be considered as part of the protocol and w ill be attached to the fi nal protocol. A n y other chang e s w ill be in the form
of w r itten dev i ations sig n ed by the S t udy D i rector and filed w ith the raw data. A ll chang e s to the protocol w ill be indicated in the final report.

GO O D LAB O RATO R Y P R ACTI C ES
T h is study w ill be conducted in accordance w ith G ood Laboratory Practice Standards for EPA (40 CFR Part 792) and OECD (E NV /MC/CHEM (98) 17). Each study conducted by

s routinely exam ined by the Q u ality A ssurance U n it for

com p liance with Good Laboratory Prac tices, Standard Operating Procedur es and the specified protocol.
A statem ent of com p liance w ith G ood Laboratory Practi ces w ill be prepared for all portions of the study conducted by T h e Sponsor w ill be responsible for com p liance w ith G ood Laboratory Practices for procedures perform ed by other laboratories (e.g ., residue analy s es or patholog y ) . Raw data for all work perform ed at . and a copy of the final report w ill be filed by proj ect num ber in archiv es locat ed on the . site or at an
alternativ e location to be sp ecified in the final report.

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