United States Prevention, Pesticides EPA712-C-98-351
Environmental Protection and Toxic Substances January 1998
Agency (7101)
&EPA Fate, Transport and
Transformation Test
Guidelines
OPPTS 835.3160
Biodegradability in Sea
Water
-------
INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-0132 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from EPA's World Wide Web site
(http://www.epa.gov/epahome/research.htm) under the heading "Environ-
mental Test Methods and Guidelines."
-------
OPPTS 835.3160 Biodegradability in sea water.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline is OECD guideline 306, Biodegradability
in Sea Water.
(b) General introduction. (1) When the original Organization for
Economic Cooperation and Development (OECD) Test Guidelines were
developed, it was not known to what extent results from the screening
tests for ready biodegradability using fresh water and sewage effluent or
activated sludge as inoculum could be applied to the marine environment.
Variable results on this point have been reported (e.g., see paragraph (e)(l)
of this guideline).
(2) Many industrial waste waters, containing a variety of chemicals,
reach the sea either by direct discharge or via estuaries and rivers in which
the residence times are low compared with the period necessary for com-
plete biodegradation of many of the chemicals present. Because of the
growing awareness of the need to protect the marine environment against
increasing loads of chemicals and the need to estimate the probable con-
centration of chemicals in the sea, test methods for biodegradability in
sea water have been developed.
(3) The methods described here use natural sea water both as the
aqueous phase and as the source of microorganisms. In an endeavor to
conform with the methods for ready biodegradability in fresh water, the
use of ultrafiltered and centrifuged sea water was investigated, as was the
use of marine sediments as inocula. These investigations were unsuccess-
ful. The test medium therefore is natural sea water pre-treated to remove
coarse particles.
(4) This guideline consists of two test methods: the Shake Flask
Method and the Closed Bottle Method. In order to assess ultimate
biodegradability with the Shake Flask Method, relatively high concentra-
tions of the test substance must be used because of the poor sensitivity
of the dissolved organic carbon (DOC) analytical method. This in turn
necessitates the addition to the sea water of mineral nutrients (N and P),
the low concentrations of which would otherwise limit the removal of
DOC. It is also necessary to add the nutrients in the Closed Bottle Method
because of the concentration of the added test substance.
(5) Hence, the methods are not tests for ready biodegradability since
no inoculum is added in addition to the microorganisms already present
in the sea water. Neither do the tests simulate the marine environment
-------
since nutrients are added and the concentration of test substance is very
much higher than would be present in the sea.
(c) Application. (1) The methods in this guideline are intended for
application when the pattern of use and disposal of the chemical substance
in question indicates a route to the sea. If the result is positive (>70%
DOC removal; >60% ThOD—theoretical oxygen demand), it may be con-
cluded that there is a potential for biodegradation in the marine environ-
ment. However, a negative result does not preclude such potential but indi-
cates that further study is necessary, for example using as low a concentra-
tion of the test substance as possible.
(2) In either case, if a more definitive value for the rate or degree
of biodegradation in sea water at a particular site is required, other more
complex and sophisticated, and hence more costly, methods would have
to be applied. For example, a simulation test could be applied using a
concentration of test substance nearer to the likely environmental con-
centration, or non-fortified, non-pretreated sea water taken from the loca-
tion of interest could be used and primary biodegradation followed by spe-
cific chemical analysis. For ultimate biodegradability, 14C-labeled test sub-
stance would be necessary to allow rates of disappearance of soluble or-
ganic 14C and production of 14CC>2 to be measured at environmentally
realistic concentrations of that substance.
(d) Choice of methods. The selection of test method depends on a
number of factors. The following table 1 is given to help the investigator
select a test method. Whereas chemicals having water solubility below the
equivalent of about 5 mg C/L cannot be tested using the Shake Flask
Method, in principle they may be tested using the Closed Bottle Method.
-------
Table 1.—Advantages and Disadvantages of the Shake Flask and Closed Bottle Methods
Method
Advantages
Disadvantages
Shake flask
simple apparatus except for C
analyzer
60 d duration is not a problem
no interference from
nitrification
can be adapted for volatile chemi-
cals
needs C analyzer
uses 5-40 mg DOC/L; could
be inhibitory
DOC determination is difficut
at low concentrations in
sea water (chloride effect)
DOC sometimes high in
sea water
Closed bottle
simple apparatus
simple end determination
uses low concentration of test
compound (2 mg/L), thus less
chance of inhibition
easily adapted for volatile
chemicals
- can be difficut to maintain
airtightness of bottles
- wall growth of bacteria can
lead to false values
- blank 02 uptake values can be
high, especially after 28 days;
can be overcome by aging
the sea water
- possible interference from 02
uptake by nitrification
(1) Shake flask method—(i) Introduction. (A) This method is a sea
water variant of the Modified OECD Screening Test (see paragraph (e)(2)
of this guideline). It was finalized as a result of a ring test organized for
the Europeon Economic Community (EEC) by the Danish Water Quality
Institute (see paragraph (e)(3) of this guideline).
(B) In common with the accompanying sea water Closed Bottle Meth-
od, the results from this test are not to be taken as indicators of ready
biodegradability, but are to be used specifically for obtaining information
about the biodegadability of chemicals in marine environments.
(ii) Principle of the method. A predetermined amount of the test
substance is dissolved in the test medium to yield a concentration of 5-
40 mg DOC/L. If the limits of sensitivity of organic carbon analyses are
improved, the use of lower concentrations of test substance may be advan-
tageous, particularly for inhibitory substances. The solution of the test sub-
stance in the test medium is incubated with agitation in the dark or in
diffuse light under aerobic conditions at a fixed temperature (controlled
to + 1°C) which will normally be within the range 15-20°C. In cases
where the objective of the study is to simulate environmental situations,
tests may be performed at temperatures outside this range. The rec-
ommended maximum test duration is 60 days. Degradation is followed
by DOC measurements (ultimate degradation) and, in some cases, by spe-
cific analysis (primary degradation).
(iii) Information on the test substance. (A) To detemine whether
the test may be applied to a particular substance, certain properties of the
-------
substance must be known. The organic carbon content of the substance
must be established, its volatility must be such that significant losses do
not occur during the course of the test, and its solubility in water should
be greater than the equivalent of 25-40 mg C/L. Further, the test substance
should not significantly sorb to glass surfaces. Information on the purity
or the relative proportions of major components of the test substance will
be useful in interpreting the results obtained, especially when the result
lies close to the "pass" level.
(B) Information on the toxicity of the test substance to bacteria, for
example as measured in short-term respiration rate tests (see paragraph
(e)(4) of this guideline), may be useful when selecting appropriate test
concentrations, and may be essential for the correct interpretation of low
biodegradation values. However, such information is not always sufficient
for interpreting results obtained in the biodegradation test, and the proce-
dure described in paragraph (d)(l)(vi)(F)(3) of this guideline may be more
suitable.
(iv) Reference substance. (A) Suitable reference substances shall be
used to check the microbial activity of the sea water sample. Sodium ben-
zoate, sodium acetate and aniline are examples of chemicals that may be
used for this purpose. If reference substances are not degraded within a
reasonably short time, it is recommended that the test be repeated using
another sea water sample.
(B) In the EEC ring test (see paragraph (e)(3) of this guideline), the
lag phase (TL) and time to achieve 50 per cent degradation (tso) excluding
the lag phase were 1 to 4 days and 1 to 7 days, respectively, for sodium
benzoate. For aniline the TL ranged from 0 to 10 days and the t50 from
1 to 10 days.
(v) Reproducibility and sensitivity of the method. The reproducibil-
ity of the method was established in the ring test (see paragraph (e)(3)
of this guideline). The lowest concentration of test substance for which
this method can be used with DOC analysis is largely determined by the
detection limit in the organic carbon analysis (about 0.5 mg C/L, at
present) and the concentration of dissolved organic carbon in the sea water
used (usually on the order of 3-5 mg/L for water from the open ocean).
The background concentration of DOC should not exceed about 20% of
the total DOC concentration after addition of test substance. If this is not
feasible, the background concentration of DOC may sometimes be reduced
by aging the sea water prior to testing. If the method is used with specific
chemical analysis only (by which primary degradation is measured), the
investigator must document, by supplying additional information, whether
ultimate degradability can be expected. This additional information may
consist of the results from other tests for ready or inherent
biodegradability.
-------
(vi) Description of the method — (A) Apparatus. Normal laboratory
apparatus and:
(7) Shaking machine accommodating 0.5- to 2-L Erlenmeyer flasks,
either with automatic temperature control or used in a constant temperature
room at 15-20°C controlled to ±1°C.
(2) Narrow neck, 0.5- to 2-L Erlenmeyer flasks.
(3) Membrane filtration apparatus, or centrifuge.
(4) Membrane filters, 0.2-0.45(j,m.
(5) Carbon analyzer.
(6) Equipment for specific analysis (optional).
(B) Sea water. (7) Collect a sample of sea water in a thoroughly
cleaned container and transport it to the laboratory, preferably within
1-2 days of collection. During transport do not allow the temperature of
the sample to exceed significantly the temperature to be used in the test.
Identify the sampling location precisely and describe it in terms of its
pollutional and nutrient status. Especially for coastal waters, include in
this characterization a heterotrophic microbial colony count and the deter-
mination of the concentrations of dissolved nitrate, ammonium and phos-
phate.
(2) Provide the following information for the sea water sample itself:
(/) Date of collection.
(//) Depth of collection.
(///) Appearance of sample — turbid, etc.
(/v) Temperature at the time of collection.
(v) Salinity.
(v/) DOC.
(v//) Delay between collection and use in the test.
If the DOC content of the sea water sample is found to be high
(see paragraph (d)(l)(v) of this guideline), it is recommended that the sea-
water be aged for approximately one week prior to use. This is accom-
plished by storing the sample under aerobic conditions at the test tempera-
ture and in the dark or in diffuse light. If necessary, maintain aerobic con-
ditions by aerating gently. During aging, the content of easily degradable
organic material is reduced. In the ring test (see paragraph (e)(3) of this
guideline), no difference was revealed between the degradation potential
of aged and freshly collected sea water samples. Prior to use, pretreat the
-------
sea water to remove coarse particles, e.g., by filtering through a nylon
or coarse paper filter (not membrane or GF-C filters), or by ceatrifuging
gently. The procedure used must be reported. Pretreat after aging, if used.
(C) Stock solutions for mineral nutrients. (1) Prepare the following
stock solutions using analytical grade reagents:
(/) Potassium dihydrogen orthophosphate, KH2PO2. . . 8.50 g
Dipotassium hydrogen orthophosphate, K2HPO4. .. 21.75 g
Disodium hydrogen orthophosphate dihydrate,
Na2HPO4.2H2O. . . 33.30 g
Ammonium chloride, NfUCl. . . 0.50 g
Dissolve and make up to 1 L with distilled water.
(//) Calcium chloride, CaCl2. . . 27.50 g
26Dissolve and make up to 1 L with distilled water.
(Hi) Magnesium sulfate heptahydrate, MgSO4.7H2O . . . 22.50 g
Dissolve and make up to 1 L with distilled water.
(iv) Iron (III) chloride hexahydrate, FeCl3.6H2O. . . 0.25 g
Dissolve and make up to 1 L with distilled water.
(2) Precipitation in solution (see paragraph (d)(l)(vi)(C)(7) (iv) of this
guideline) may be prevented by adding one drop of concentrated HC1 or
0.4 g ethylenediaminetetraacetic acid (EOTA, disodium salt) per L. If a
precipitate forms in a stock solution, replace it with freshly made solution.
(D) Preparation of test medium. Add 1 mL of each of the stock
solutions described in paragraphs (d)(l)(vi)(C)(7)(/) through
(d)(l)(vi)(C)(7)(/v) of this guideline per L of pretreated sea water.
(E) Inoculum. Do not add any inoculum in addition to the microorga-
nisms already present in the sea water. Optionally, determine the number
of colony-forming heterotrophs in the sea water test medium (and pref-
erably also in the original sea water samples) using a suitable method,
such as plate counts with marine agar. This is particularly desirable for
samples from coastal or polluted sites. Check the heterotrophic microbial
activity in the sea water by using a reference compound.
(F) Preparation of flasks. (1) Ensure that all glassware is scru-
pulously clean (e.g., using alcoholic hydrochloric acid), rinsed and dried
-------
before use in order to avoid contamination with residues from previous
tests. Flasks must also be cleaned before they are used for the first time.
(2) Evaluate test substances in duplicate flasks simultaneously, to-
gether with a single flask for the reference compound. Carry out a blank
test, in duplicate, with neither test nor reference substance, for the deter-
mination of analytical blanks. Dissolve the test substance in the test me-
dium—it may be conveniently added via a concentrated stock solution—
to give the desired starting concentration of 5-40 mg DOC/L. Test the
reference substance at a starting concentration corresponding to 20 mg
DOC/L. If stock solutions of test and/or reference substances are used,
ensure that the salinity of the sea water medium is not greatly altered.
(3) If toxic effects can be expected or cannot be ruled out, it may
be advisable to include an inhibition experiment, in duplicate, in the test
design. Add the test and reference substances to the same vessel, the con-
centration of the reference compound being normally the same as in the
control test (i.e., 20 mg DOC/L) in order to allow comparison.
(4) Dispense adequate amounts of test solutions into the Erlenmeyer
flasks (up to about half the flask volume is a convenient amount) and
subsequently provide each flask with a loose cover (e.g., aluminium foil)
that makes gas exchange between the flask and the surrounding air pos-
sible. (Cotton wool plugs are unsuitable if DOC analysis is used). Place
the vessels on the shaker and shake continuously at a gentle rate (e.g.,
100 rpm) throughout the test. Control the temperature (15-20°C and within
±2°C), and shield the vessels from light in order to avoid growth of algae.
Ensure that the air is free of toxic materials.
(G) Physical-chemical control test (optional). If abiotic degradation
or loss mechanisms are suspected, such as hydrolysis (a problem with spe-
cific analysis only), volatilization, or sorption, it is advisable to perform
a physical-chemical control. This can be done by adding mercury (II) chlo-
ride (HgCL:)1 (50-100 mg/L) to vessels with test substance in order to
inhibit microbial activity. A significant decrease in DOC or specific
compound concentration in the physical-chemical control test indicates abi-
otic removal mechanisms. (If mercury chloride is used, attention should
be paid to interferences or catalyst poisoning in DOC analysis).
(H) Number of flasks. In a typical experiment, the following flasks
are used:
(7) Flasks 1 & 2—containing test substance.
(2) Flasks 3 & 4—containing sea water only (blank).
iMercury (II) chloride (HgCb) is a very toxic substance that should be handled
with suitable precautions. Aqueous wastes containing this chemical should be disposed
of appropriately; they should not be discharged down the drain.
-------
(3) Flask 5—containing reference substance.
(4) Flask 6—containing test and reference substance (toxicity con-
trol)—optional.
(5) Flask 7—containing test substance and sterilizing agent (abiotic
sterile control)—optional.
(I) DOC analysis. In the course of the test, withdraw samples at suit-
able intervals for DOC analysis as described in paragraphs
(d)(l)(vi)(I)(7)(/) through (d)(l)(vi)(I)(7)(jc) of this guideline. Always take
samples at the start of the test (day 0) and at day 60. A minimum of
five samples in total are required to describe the time-course of degrada-
tion. No fixed time schedule for sampling can be stated since the rate
of biodegradation varies. Carry out the DOC determination in duplicate
on each sample.
(7) Determination of organic carbon in sea water. (/) For the deter-
mination of organic carbon of a water sample, the organic compounds in
the sample are oxidized to carbon dioxide using one of the following three
techniques:
(a) Wet oxidation by persulfate/UV-irradiation.
(b) Wet oxidation by persulfate/elevated temperature (116-130°C).
(c) Combustion.
(//) Evolved CO2 is then quantified employing infrared spectrometry
or titrimetry. Alternatively, CO2 is reduced to methane, which is quantified
using a flame ionization detector (FID).
(///) The persulfate/UV-method is commonly used for the analysis of
"clean" water with a low content of particulate matter. The persulfate/
elevated temperature and combustion methods can be applied to most
kinds of water samples, the former being most suitable for samples with
low levels of nonvolatile organic carbon (NVOC), and the combustion
method being applicable to samples with NVOC content well above 1 mg
C/L.
(2) Interferences. (/) All three methods depend on eliminating or
compensating for inorganic carbon (1C) present in the sample. Purging of
CO2 from the acidified sample is the most frequently used method to
eliminate 1C, although this also results in a loss of volatile organic com-
pounds (see paragraph (e)(5) of this guideline). Complete elimination or
compensation of 1C must be ensured for each sample matrix, and it may
be necessary to determine volatile organic carbon (VOC) separately from
NVOC.
8
-------
(//) High chloride concentrations result in decreased oxidation effi-
ciency using the persulfate/UV-method (see paragraph (e)(6) of this guide-
line). Application of an oxidation reagent modified by the addition of mer-
cury (II) nitrate may remove this interference. It is recommended that the
maximum tolerable sample volume be used to evaluate each type of chlo-
ride-containing sample. High salt concentrations in samples analyzed using
the combustion method can cause salt coating of the catalyst and excessive
corrosion of the combustion tube. Precautions should be taken according
to the manufacturer's manual.
(///) Highly turbid samples as well as samples containing particulate
matter may be incompletely oxidized by the persulfate/UV-method.
An example of a suitable method. (/) Nonvolatile organic carbon
is determined by oxidation with persulfate/UV-irradiation and subsequent
quantification of evolved CO2 employing non-dispersive infrared spec-
trometry.
(//) The oxidation reagent is modified in accordance with the sugges-
tions given in paragraph (e)(6) of this guideline, as described in the manu-
facturer's manual:
(a) 8.2 g of HgCl2 and 9.6 g of Hg(NO3)2.H2O are dissolved in sev-
eral hundred mL of low-carbon concentration reagent water.
(b) 20 g of K2S2O8 are dissolved in the mercuric salt solution.
(c) 5 mL of concentrated HNOs are added to the mixture.
(d) The reagent is diluted to 1000 mL.
(Hi) The interference from chloride is removed using a 40-(iL sample
volume for 10% chloride and 200-(iL sample volume for 1.9% chloride.
Samples with high chloride concentrations and/or larger sample volumes
can be analyzed using this method provided that build-up of chloride in
the oxidation vessel is prevented. Determination of volatile organic carbon
can subsequently be performed, if relevant, for the sample type in question.
(iv) Automated systems have also been described in the literature (see
paragraph (e)(7) of this guideline).
(J) Sampling. (1) The required volume of the samples depends upon
the analytical method (specific analysis), the carbon analyzer used, and
the procedure (membrane filtration or centrifugation) selected for sample
treatment before carbon determination (see paragraphs (d)(l)(vi)(J)(3) and
(d)(l)(vi)(J)(/) of this guideline). Before sampling ensure that the test me-
dium is mixed well and that any material adhering to the wall of the flask
is dissolved or suspended.
-------
(2) Membrane filter or centrifuge immediately after sampling. If nec-
essary, store the filtered or centrifuged samples at 2-4°C for up to 48
hours or below -18°C for longer periods. If it is known that the substance
will remain unaffected by acidification, acidify to pH 2 before storing.
Membrane filters (0.2-0.45 (im) are suitable if it is ensured that
they neither release carbon nor sorb the substance in the filtration step.
Polycarbonate filters are generally suitable. Some membrane filters are im-
pregnated with surfactants for hydrophilization and may release consider-
able quantities of dissolved carbon. Prepare such filters by boiling in
deionized water for three consecutive periods, each of one hour. After boil-
ing, store the filters in deionized water. Discard the first 20 mL of the
filtrate.
(4) Centrifugation of the samples may be an acceptable alternative
to membrane filtration. Centrifuge at 40,000 m.s-2 (-4000 g) for 15 min-
utes, preferably in a refrigerated centrifuge.
Note: The differentiation of Total Organic Carbon (TOC) from DOC by
centrifugation at very low concentrations does not seem to work, since either
not all bacteria are removed, or carbon as part of the bacterial plasma is redis-
solved. At higher test concentrations (> 10 mg C/L) the centrifugation error
seems to be comparatively small.
(K) Frequency of sampling. (7) If analyses are performed imme-
diately after sampling, determine the next sampling time by considering
the result of the analytical determination.
(2) If samples are preserved (see paragraph (d)(l)(vi)(J)(2) of this
guideline) for analysis at a later time, take more samples than the required
minimum number of five. Analyze the last samples first, and by a step-
wise "backwards "selection of appropriate samples for analysis, it is pos-
sible to obtain a good description of the biodegradation curve with a rel-
atively small number of analytical determinations. If no degradation has
taken place by the end of the test, no further samples need to be analyzed,
and in this situation, the "backwards" strategy may save considerable ana-
lytical costs.
(3) If a plateau on the degradation curve is observed before the 60th
day, end the test. If degradation has obviously started by day 60, but has
not reached a plateau, extend the experiment for a further period.
10
-------
(vii) Data and reporting—(A) Treatment of results. (7) Record the
results on the data sheet below, and calculate the biodegradation values
for both test and reference substances from the equation:
1-
-C
t H
xlOO
where:
Dt = degradation in percentage DOC or specific compound removal at
time t;
C0 = starting concentration of DOC or specific compound in the test
medium;
Ct = concentration of DOC or specific compound in the test medium
at time t;
= starting concentration of DOC or specific compound in the
blank;
= concentration of DOC or specific compound in the blank at
time t.
11
-------
BIODEGRADATION IN SEA WATER—SHAKE FLASK METHOD
DATA SHEET
LABORATORY:
DATE AT START OF TEST:
TEST SUBSTANCE:
Name:
Stock solution concentration (mg/L as chemical):
Initial concentration in medium, t0 (mg/L as chemical):.
thus (mg DOC/L):
1. SEA WATER:
Source:
Date of collection:
Depth of collection:.
Appearance at time of collection (e.g., turbid, etc.):.
Salinity at collection (%):
Temperature at collection (°C):
DOC "x"hours after collection (mg/L):.
Pretreatment prior to testing (e.g., filtration, sedimentation, aging,
etc.):
Microbial colony count of original sample (colonies/mL):
At start of test (colonies/mL):
Other characteristics:
12
-------
2. CARBON DETERMINATIONS:
Carbon analyzer:
Test: nutrient-fortified
sea water with test
substance
Blank: nutrient-for-
tified as waterout
test substance
Flask no.
1
2
1
2
ai
a2
mean, Ca(t)
bi
b2
mean, Cb(t>
Cl
C2
mean, CC(t)
di
d2
mean, Cd(t)
mean (Cc(t) + Cdm)/2
DOC after n days (mg/L)
0
ni
n2
n3
nx
13
-------
3. EVAULATION OF RAW DATA
Flask No.
1
2
Mean (*)
Calculation of results
D j Qt) ~ Cbl(D x 10Q
"i A r - r
'-o uW(o)
D j CKO ~ cww „ 100
^2 ' p _ ,-,
uo cW(o)
„ _ D, + D2
' 2
% Degradation after n days
0
0
0
0
n,
n,
n.
n,
* Dj and D2 should not be averaged if there is a considerable difference.
Note: Similar formats may be used when degradation is followed by specific analysis and for the
reference compound and toxicity controls.
4. ABIOTIC DEGRADATION (optional)
DOC cone. (mg/L) in sterile control
Time (days)
0
Cs(0)
t
C,(.)
C —C
% abiotic degradation = -^ S^L x 100
Cs(o}
(2) State degradation as the percentage DOC removal (ultimate deg-
radation) or specific compound removal (primary degradation) at time t.
Calculate the DOC concentrations to the nearest 0.1 mg/L, and round up
the means of the Dt values to the nearest whole per cent.
(3) Illustrate the time course of degradation graphically in a diagram
as shown in the figure in paragraph (d)(l)(vii)(C)(3) of this guideline. If
there are sufficient data, calculate from the curve the lag phase (TL) and
the time to reach 50 per cent removal from the end of the lag phase (tso).
(B) Test report. (7) The test report shall contain the following infor-
mation:
(/) Test substance, (a) Physical nature and, where relevant, physical/
chemical properties.
(b) Identification of the substance.
14
-------
(//) Test conditions, (a) Location and description of the sampling site;
pollutional and nutrient status (colony counts, and nitrate, ammonium and
phosphate levels if appropriate).
(b) Characteristics of the sample (date of sampling, depth, appearance,
temperature, salinity, DOC (optional), delay between collection and use
in the test.
(c) Method used (if any) for aging the sea water.
(d) Method used for pretreatment (filtration/sedimentation) of the sea
water.
(e) Method used for DOC determination.
(f) Method used for specific analysis (optional).
(g) Method used for determining the number of heterotrophs in the
sea water (plate count method or alternative procedure) (optional).
(h) Other methods (optional) used to characterise the sea water (ATP
measurements, etc.).
(Hi) Results, (a) Analytical data reported on a data sheet as shown
in paragraph (d)(l)(vii)(A)(7) of this guideline.
(b) The course of degradation represented graphically in a diagram
showing the lag phase (TL), slope, and time (starting from the end of the
lag phase) to reach 50% removal (tso). The lag phase may be estimated
graphically as shown in the figure in paragraph (d)(l)(vii)(C)(3) of this
guideline or conveniently taken as the time needed for 10% degradation.
(c) Percentage degradation measured after 60 days, or at end of test.
(iv) Discussion of results.
(C) Validity and interpretation of results. (1) The results obtained
with the reference compounds (e.g., sodium benzoate, sodium acetate or
aniline) should be comparable to results obtained in the ring test (see para-
graphs (d)(l)(iv) and (e)(3) of this guideline. If results obtained with ref-
erence compounds are atypical, the test should be repeated using another
sea water sample. Although results of inhibition tests may not always be
straightforward to interpret because of the contribution of DOC by the
test material, a significant reduction of the total DOC removal rate, com-
pared with that of the control, is a positive sign of toxic effects.
(2) Owing to the relatively high test concentrations used as compared
with most natural systems and consequently an unfavorable ratio between
the concentrations of test substances and other carbon sources, the method
is to be regarded as a preliminary test that can be used to indicate whether
or not a substance is easily biodegradable in sea water. Accordingly a
15
-------
low result does not necessarily mean that the test substance is not bio-
degradable in marine environments, but indicates that more work will be
necessary to establish this.
(3) An example of graphical presentation of data and estimation of
the values of TL (length of lag phase) and t50 (time, starting at tL, needed
to reach 50% removal) is given in the following Figure 1.
FIGURE i.—TYPICAL BIODEGRADATION CURVE FOR THE SHAKE FLASK
METHOD
100-
20-
(2) Closed bottle method—(i) Introduction. (A) This method is a
sea water variant of the Closed Bottle Test (see paragraph (e)(8) of this
guideline) and was finalized as a result of a ring test organised for the
EEC by the Danish Water Quality Institute (see paragraph (e)(3) of this
guideline).
(B) In common with the accompanying sea water Shake Flask Meth-
od, results of this test are not to be taken as indications of ready
biodegradability, but are to be used specifically for obtaining information
about the biodegradability of chemicals in marine environments.
(ii) Principle of the method. (A) A predetermined amount of the
test substance is dissolved in the test medium at a typical concentration
of 2 to 10 mg of test substance/L. One or more concentrations may be
16
-------
used. The solution is kept in a filled closed bottle in the dark in a constant
temperature bath or enclosure controlled to ±1°C within the range 15-
20°C. In those cases where the objective of the study is to simulate envi-
ronmental situations, tests may be performed at temperatures outside this
range provided that the temperature is properly controlled. Degradation
is followed by oxygen analyses over a 28-day period.
(B) The ring test showed that if the test was extended beyond 28
days no useful information could be gathered, in most cases, due to severe
interferences. The blank biological oxygen demand (BOD) values were
excessively high probably due to wall growth, caused by lack of agitation,
and to nitrification. Thus, the recommended duration is 28 days, but if
the blank BOD value remains within the 30 per cent limit (see paragraph
(d)(2)(vi)(B)(/) of this guideline) the test could be prolonged.
(iii) Information on the test substance. (A) To determine whether
the test may be applied to a particular substance, certain properties of the
substance must be known. The empirical formula is required so that the
theoretical oxygen demand (ThOD) may be calculated as described in
paragraphs (d)(2)(iii)(A)(7)(/) through (vf) of this guideline; otherwise the
chemical oxygen demand (COD) of the substance must be determined to
serve as a surrogate for the ThOD. The use of COD is less satisfactory
since some chemicals are not fully oxidized in the COD test.
(7) Calulation of the theoretical oxygen demand. (/) The ThOD
of the substance CcHhClciNnNanaOoPpSs of molecular weight MW is cal-
culated as follows:
16 2c + —(h- cl- 3n) + 3s+-p + -na - 0
2 2
MW
(if) This calculation implies that C is mineralized to CO2, H to H2O,
P to P2Os and Na to Na2O. Halogen is eliminated as hydrogen halide
and nitrogen as ammonia.
17
-------
Example:
Glucose C6H1206, MW = 180
16(2 x 6 + - x 12 - 6)
ThOD = = 1.07 mg O2 / mg glucose
180
(///) Molecular weights of salts other than those of the alkali metals
are calculated on the assumption that the salts have been hydrolyzed.
(iv) Sulfur is assumed to be oxidized to the state of +6.
Example:
Sodium dodecylbenzenesulfonate C18H29S03Na, MW = 348
29 1
16(36 + — + 3 + --3)
ThOD= - - - - - = 2.34 mg 0? / mg substance
348 2
(v) In the case of nitrogen-containing substances the nitrogen may
be eliminated as ammonia, nitrite, or nitrate, and each corresponds to a
different theoretical oxygen demand.
For nitrite,
, .L 1 „ ^ o 3 5 1 1
16 2c + — (h-cl) + 3s + — n + — p + na - o
2 222
ThODNO? =— ^ - - - - - - - - - ^
2 MW
For nitrate,
16
ThODxjn =
2c + (h-cl) + 3s + —n + —p + na - o
L 2 222.
18
-------
(v/) Suppose full nitrate formation had been observed by analysis in
the case of a secondary amine: (C 12^.25)2 NH, MW = 353. Then
16(48 + 51/2 + 5/2)
ThODNO = = 3.44 mg O2 /mg substance
3 353
(B) The solubility of the substance should be at least 2 mg/L, though
in principle less soluble compounds could be tested (e.g., after sonication)
as could volatile compounds. Information on the purity or the relative pro-
portions of major components of the test substance will be useful in inter-
preting the results obtained, especially when the result lies close to the
"pass" level.
(C) Information on the toxicity of the substance to bacteria, for exam-
ple as measured in short-term respiration tests (see paragraph (e)(4) of
this guideline), may be useful when selecting appropriate test concentra-
tions, and may be essential for the correct interpretation of low biodegrada-
tion values. However, such information is not always sufficient for inter-
preting results obtained in the biodegradation test, and the procedure de-
scribed in paragraph (d)(2)(vi)(F)(7) of this guideline may be more suit-
able.
(iv) Reference substance. (A) Suitable reference substances shall be
used to check the microbial activity of the sea water sample. Aniline, so-
dium acetate and sodium benzoate are examples of substances that may
be used for this purpose. Degradation of these compounds must reach at
least 60 per cent with respect to their ThOD within a reasonably short
time span; otherwise it is recommended that the test be repeated using
another sea water sample.
(B) In the EEC ring test (see paragraph (e)(3) of this guideline), the
lag phase (TL) and the time to achieve 50 per cent degradation (tso) exclud-
ing the lag phase were 0 to 2 days and 1 to 4 days, respectively, for
sodium benzoate. For aniline the TL and tso values were 0 to 7 and 2 to
12 days, respectively.
(v) Reproducibility. The reproducibility of the method was estab-
lished in the EEC ring test (see paragraph (e)(3) of this guideline).
(vi) Description of the method—(A) Apparatus. Normal laboratory
equipment and:
(7) 250- to 300-mL BOD bottles with glass stoppers or narrow neck
250-mL bottles with glass stoppers.
(2) Several 2-, 3- and 4-L bottles for the preparation of the experiment
and for the filling of BOD bottles.
19
-------
(3) Waterbath or constant temperature room for keeping the bottles
at constant temperature (+ 1°C)) with the exclusion of light.
(4) Equipment for analysis of dissolved oxygen.
(5) Membrane filters, 0.2 to 0.45 (im (optional).
(6) Equipment for specific analysis (optional).
(B) Sea Water. (7) Collect a sea water sample and provide informa-
tion on the sampling location and sample as described in paragraphs
(d)(l)(vi)(B)(7) and (d)(l)(vi)(B)(2) of this guideline.
(2) If the DOC content of the sample is found to be high or if it
is thought that the blank BOD after 28 days may be more than 30% of
that of the reference substance, it is recommended that the sea water be
aged for about a week prior to use. Follow the procedures described in
paragraph (d)(l)(vi)(B)(3) of this guideline.
(C) Stock solutions for mineral nutrients. Prepare stock solutions
as described in paragraph (d)(l)(vi)(C) of this guideline.
(D) Preparation of test medium. Add 1 mL of each of the stock
solutions described in paragraph (d)(l)(iv)(C) of this guideline per L of
sea water. Saturate the test medium with air at the test temperature by
aerating with clean compressed air for approximately 20 minutes. Deter-
mine the concentration of dissolved oxygen for control purposes. The satu-
rated concentration of dissolved oxygen as a function of salinity and tem-
perature may be read from the nomogram in the following Figure 2.
20
-------
FIGURE 2.—NOMOGRAM OF SATURATION CONCENTRATION OF OXYGEN
vs. TEMPERATURE AT VARIOUS SALINITIES
21
-------
(E) Inoculum. See paragraph (d)(l)(vi)(E) of this guideline.
(F) Preparation of test bottles. (7) Perform all necessary manipula-
tions including aging and pretreatment of the sea water at the chosen test
temperature in the range of 15 to 20°C, ensuring cleanliness but not steril-
ity of all glassware.
(2) Prepare groups of BOD bottles for the determination of the BOD
of the test and reference substances in simultaneous experimental series.
Perform all analyses on duplicate bottles (i.e., duplicate blanks, duplicate
reference substance bottles and duplicate test substance bottles). A mini-
mum of five sampling times in total are required to describe the time
course of degradation. For oxygen analyses, five sampling times require
a total of3x2x5 = 30 bottles (blank, reference substance and test sub-
stance), and thus about 10 L of test medium.
(3) Prepare separate solutions of test and reference substances in large
bottles of sufficient volume (see paragraph (d)(2)(vi)(A)(2) of this guide-
line) by first adding test and reference substances either directly or by
adding a concentrated stock solution to the partly filled large bottles. Add
further test medium to give the final desired concentrations. If stock solu-
tions of test and/or reference substances are used, ensure that the salinity
of the sea water medium is not significantly altered.
(4) Select concentrations of test and reference substances by taking
into account:
(/) The solubility of dissolved oxygen in sea water at the prevailing
test temperature and salinity (see the nomogram in paragraph (d)(2)(vi)(D)
of this guideline).
(//) The blank BOD of the sea water.
(///) The expected biodegradability of the test substance.
(5) At 15°C and 20°C and 32 parts per thousand salinity (i.e., that
of ocean water), the solubility of dissolved oxygen is about 8.1 and 7.4
mg/1, respectively. The oxygen consumption of the seawater itself (blank
respiration) may be 2 mg O2/L or more if the sea water is not aged. There-
fore, in order to ensure that a significant amount of oxygen remains after
oxidation of the test substance, use a starting concentration of test
compound of approximately 2 to 3 mg/L (depending on the ThOD) for
the compounds that are expected to be completely degraded under the con-
ditions of the test, such as reference substances. Use higher initial con-
centrations for less degradable substances, up to about 10 mg/L, provided
that toxic effects do not occur. It can be advantageous to run parallel tests
with low (about 2 mg/L) and high (about 10 mg/L) concentrations of test
substance.
22
-------
(6} An oxygen blank must be determined at each time point using
parallel bottles containing neither test nor reference substance.
(7) If inhibitory effects are to be determined, prepare the following
series of solutions in separate large bottles (see paragraph (d)(2)(vi)(A)(2)
of this guideline):
(/) 2 mg/L of an easily degradable compound, e.g., any of the ref-
erence substances listed in paragraph (d)(2)(iv)(A) of this guideline.
(//) x mg/L of test substance (x is usually 2).
(///) 2 mg/L of the easily degradable compound plus x mg/L of test
substance.
(G) Physical-chemical control test (optional). If the option of using
specific analyses is used, a physical-chemical control test may be per-
formed to determine whether the test substance is removed by abiotic
mechanisms, such as hydrolysis or sorption. A physical-chemical control
test may be performed by adding mercury (II) chloride (HgCL:)2 (50 to
100 mg/L) to duplicate flasks with test substance in order to inhibit micro-
bial activity. A significant decrease in specific compound concentration
in the course of the test indicates abiotic removal.
(H) Number of BOD bottles. In a typical experiment the following
bottles are used:
(1) At least 10 containing test substance.
(2) At least 10 containing nutrient-fortified sea water only.
(3) At least 10 containing reference substance.
(4) 6 bottles containing test and reference substances (toxicity con-
trol)—optional.
(vii) Procedure. (A) After preparation, immediately siphon each solu-
tion from the lower quarter (not from the bottom) of the appropriate large
bottle, to fill the respective group of BOD bottles. Immediately analyze
the time zero samples for dissolved oxygen (see paragraph (d)(2)(vii)(D)
of this guideline), or preserve them for later chemical analysis by precipita-
tion with MnCl2 (manganese (II) chloride) and NaOH (sodium hydroxide).
(B) Incubate the remaining BOD bottles at the test temperature (15-
20°C) in the dark, and remove bottles for analysis at appropriate time inter-
vals. No fixed time schedule can be stated since the rate of biodegradation
2Mercury (II) chloride (HgCb) is a very toxic substance that should be handled
with suitable precautions. Aqueous wastes containing this chemical should be disposed
of appropriately; they should not be discharged down the drain.
23
-------
varies. Analyze for dissolved oxygen (see paragraph (d)(2)(vii)(D) of this
guideline).
(C) Membrane filter (0.2-0.45 (im) or centrifuge for 15 minutes sam-
ples for specific analyses (optional). Store these samples for up to 48 hours
at 2-4°C, or for longer periods at -18°C, if they are not analyzed imme-
diately. If it is known that the test substance will remain unaffected by
acidification, acidify to pH 2 before storing.
(D) Determine the concentration of dissolved oxygen using a chemi-
cal or electrochemical method that is recognized nationally or internation-
ally.
(viii) Data and reporting—(A) Treatment of results. (7) Record
analytical results on the data sheet below.
24
-------
BIODEGRADATION IN SEA WATER—CLOSED BOTTLE
METHOD
DATA SHEET
LABORATORY:
DATE AT START OF TEST:
TEST SUBSTANCE:
Name:
Stock solution concentration (mg/L):
Initial concentration in medium, t0 (mg/L):
ThOD or COD (mg Ch/mg test substance):
1. SEA WATER:
Source:
Date of collection:
Depth of collection:.
Appearance at time of collection (e.g., turbid, etc.):_
Salinity at collection (%):
Temperature at collection (°C):
DOC "x"hours after collection (mg/L):.
Pretreatment prior to testing (e.g., filtration, sedimentation, aging,
etc.):
Microbial colony count of original sample (colonies/mL):
At start of test (colonies/mL):
Other characteristics:
Test medium:
Temperature after aeration (°C):
25
-------
concentration after aeration and standing before start of test
(mg 02/L):
2. DO DETERMINATION:
Method: Winkler/electrode
Test: nutrient-for-
tified sea water
with test sub-
stance
Blank: nutrient-for-
tified sea water
but without test
substance
Flask no.
1
2
Mean blank
1
2
Mean test
3i
a2
rrit = (ai + a2)/2
Ci
C2
mb = (Ci + c2)/2
mg O2 after n days
0
HI
n2
n3
n4
Note: Similar format may be used for reference compound and toxicity controls.
3. DO DEPLETION: % DEGRADATION (%D):
(mb - mt)o>
%D = (mb - mt)«) x 100/test substance (mg/l) x ThOD
DO depletion after n days
ni
n2
n3
n4
This assumes that nib(0) = mt(0), where
o) = blank value at day 0,
mt(o) = test substance value at day 0.
If mb(o) does not equal mt(0), use (mt(0) - mt(x)) - (mb(0) - mb(X)), where
mb(x) = blank value at day x,
mt(x) = test substance value at day x.
(2) Calculate the BOD as the difference in the oxygen depletion between
the blank and a solution of test substance under the conditions of the test. Divide
the net oxygen depletion by the concentration (w/v) of the substance in order
to express the BOD as mg BOD/mg test substance. Extent of degradation is
defined as the ratio of the BOD to either the ThOD or the COD, but preferably
the former, and is expressed as a percentage (see paragraph (d)(2)(viii)(A)(3)
of this guideline).
26
-------
(3) Calculate the extent of biodegradation for each sampling time, for both
test and reference substances, using one of the following equations:
mg O, / mg tested substance
% biodegradation = x 100
mg ThOD / mg tested substance
mg O, / mg tested substance
% biodegradation = x 100
mg COD / mg tested substance
where:
ThOD = theoretical oxygen demand (for method of calculation, see
paragraph (d)(2)(iii)(A) of this guideline).
COD = chemical oxygen demand, determined experimentally.
NOTE: Sometimes the two methods of calculation (percentage of the ThOD
and percentage of the COD) do not give the same results. It is preferable to
use ThOD because some chemicals are not fully oxidized in the COD test.
(4) Illustrate the time course of degradation graphically by means of
a diagram; see the example in paragraph (d)(2)(viii)(C)(<5) of this guide-
line. If there are sufficient data, calculate the lag phase (TL) and the time
from the end of the lag phase that is required to reach 50% degradation
(tso).
(5) If specific analysis is used (optional), express the percentage of
primary degradation as the percentage of specific substance removed with-
in the test period, corrected for analytical blanks.
(B) Test report. (7) The test report must contain the following infor-
mation:
(/) Test substance:
(a) Physical nature and, where relevant, physical/chemical properties.
(b) Identification of the substance.
(//) Test conditions:
(a) Location and description of the sampling site: pollutional and nu-
trient status (colony counts, and nitrate, ammonium and phosphate levels
if appropriate).
27
-------
(b) Characteristics of the sample: date of sampling, depth, appearance,
temperature, salinity, DOC (optional), delay between collection and use
in the test.
(c) Method used (if any) for aging the sea water.
(d) Method used for pretreatment (filtration/sedimentation) of the sea
water.
(e) Method used for COD determination (if performed).
(f) Method used for oxygen measurements.
(g) Method of dispersion for substances that are poorly soluble under
the test conditions.
(h) Method used for determining the number of heterotrophs in the
sea water (plate count method or alternative procedure).
(/) Method used for determining DOC in sea water (optional).
(/) Method used for specific analysis (optional).
(k) Other optional methods used to characterize the sea water (ATP
measurements, etc.).
(Hi) Results:
(a) Analytical data reported on a data sheet (see paragraph
(d)(2)(viii)(A)(7) of this guideline).
(b) The time course of degradation represented graphically in a dia-
gram showing the lag phase, (ti_), slope, and time starting from the end
of the lag phase to reach 50% of the final oxygen uptake caused by oxida-
tion of the test compound (tso). The lag phase may be estimated graphi-
cally as shown in Figure 3 in paragraph (d)(2)(viii)(C)(<5) of this guideline,
or conveniently in taken as the time needed for 10% degradation.
(c) % degradation measured after 28 days.
(iv) Discussion of results.
(C) Validity and interpretation of results. (1) The blank respiration
should not exceed 30% of the oxygen in the test bottle. If it is not possible
to meet this criterion using freshly collected sea water, the seawater must
be aged (stabilized) before use.
(2) The possibility that nitrogen-containing compounds may affect the
results should be considered.
(3) Results obtained with the reference substances sodium benzoate
and aniline should be comparable to the results obtained in the ring test
28
-------
(see paragraphs (d)(2)(iv)(B) and (e)(3) of this guideline). If results ob-
tained with reference compounds are atypical, the test should be repeated
using another sea water sample.
(4) The test substance can be considered to be inhibitory to bacteria
at the concentration used if the BOD of the mixture of reference and test
substances is less than the sum of the BOD of the separate solutions of
the two substances.
(5) Owing to the relatively high test concentrations as compared with
most natural systems, and consequently an unfavorable ratio between the
concentrations of test substance and other carbon sources, the method is
regarded as a preliminary test that can be used to indicate whether or not
a substance is easily biodegradable in sea water. Accordingly, a low result
does not necessarily mean that the test substance is not biodegradable in
marine environments, but indicates that more work will be necessary to
establish this.
(6) An example of graphical presentation of data and estimation of
the values of TL (length of lag phase) and tso (time, starting at TL, needed
to reach 50% of the final oxygen uptake caused by oxidation of the test
substance), is given in the following Figure 3:
29
-------
FIGURE 3.—TYPICAL BIODEGRADATON CURVE FOR THE CLOSED BOTTLE
METHOD
100
80
60
40
20
oxygen consumption
% of final
days
"tog phase"
(e) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) de Kreuk J.F. and Hanstveit A.O. 1981. Determination of the
biodegradability of the organic fraction of chemical wastes. Chemosphere
10 (6): 561-573.
(2) OECD, Paris. 1992. Test Guideline 301 E.
(3) Nyholm N. and Kristensen P. 1987. Screening Test Methods for
Assessment of Biodegradability of Chemical Substances in Seawater. Final
Report of the ring test programme 1984-1985, March 1987, Commission
of the European Communities.
(4) OECD, Paris. 1984. Test Guideline 209.
(5) International Standards Organization (ISO). 1986. Water quality—
determination of total organic carbon. Draft International Standard ISO/
DIS 8245, January 16.
(6) American Public Health Association. 1985. Standard Methods for
the Examination of Water and Wastewater, 16th ed.
30
-------
(7) Schreurs W. 1978. An automated colorimetric method for the de-
termination of dissolved organic carbon in seawater by UV destruction.
Hydrobiological Bulletin 12: 137-142.
(8) OECD, Paris. 1992. Test Guideline 301 D.
31
------- |