306
Adopted:
17.07.92
OECD  GU ID ELIN E  FOR  TESTIN G  OF  CHEMICALS
Adopted by the Council on 17th July 1992
Biodegradability in Seawater
GEN ERAL IN TRODUCTION
OECD Test Guidelines were developed, it was not known to what extent original
1. When
the
results from the screening tests for ready biodegradability using freshwater, 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. (1)).
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 complete 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 concentration of chemicals in the sea, test methods for biodegradability
in seawater have been developed.
3.The methods described here use natural seawater both as the aqueous phase and as the source
of micro-organisms. In an endeavour to conform with the methods for ready biodegradability in freshwater, the use of ultra-filtered and centrifuged seawater was investigated, as was the use of marine sediments as inocula. These investigations were unsuccess ful. The test medium therefore is natural seawater pre-treated to remove coarse particles.
4.In order to assess ultimate biodegradability with the Shake Flask Method, relatively high concentrations of the test substance have to be used because of the poor sensitivity of the dissolved organic carbon (DOC) analytical method. This in turn necessitates the addition to the seawater 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 n utrients 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 micro-organisms already present in the seawater. 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. For these reasons the methods are proposed under a new subsection "Biodegradability in Seawater".
APPLICATION
6.The results of the tests, which would be applied because the pattern of use and disposal of the chemical in question indicated a route to the sea, give a first impression of biodegradability in seawater. If the result is positive (>70% DOC removal; >60% ThOD - theoretical oxygen demand),
it may be concluded that there is a potential for biodegradation in the marine environment. However,
306OCD E / OECD
a negative result does not preclude such a potential but indicates that further study is necessary, for
example, using as low a concentration of the test compound as possible.
7. In either case, if a more definitive value for the rate or degree of biodegradation in seawater
at a particular site is required, other more complex and sophis ticated, 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 concentration. Also, non-fortified, non-pre-treated seawater taken from the location of interest could be used and primary biodegradation could be followed by specific chemical analysis. For ultimate biodegradability, 14C-labelled chemicals would be necessary in order that the rates of the disappearance of soluble organic 14C and the production of 14CO
at environmentally realistic concentrations could be measured.
2
CHOICE OF METHOD S
8.The selection of which method to use depends on a number of factors; the following Table
is given to help the selection. While chemicals of water solubility below the equivalent of about 5 mg C/l cannot be tested in the Shake Flask Method, at least, in principle, poorly soluble chemicals may be tested in the Closed Bottle Method.
TAB LE:ADVAN TAGES  AN D  D ISADVAN TAGES  OF  THE  SHAKE  FLAS K  AN D CLOS ED B OTTLE TES T
OCD E / OECD306
SHAKE FLAS K METHOD
IN TRODUCTION
1.This method is a seawater variant of the Modified OECD Screening Test (2). It was finalised
as a result of a ring test organized for the EEC by the Danish Water Quality Institute (3).
2.In common with the accompanying marine Closed Bottle Method, 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.
PRIN CIPLE OF THE METHOD
3.  A pre-determined amount of the test substance is dissolved in the test medium to yield a concentration of 5-40 mg dissolved organic carbon (DOC)/l. If the limits of sensitivity of organic carbon analyses are improved, the use of lower concentrations of test substance may be advantage
ous, particularly for inhibitory compounds. The solution of the test substance in the test medium is incubated under agitation in the dark or in diffuse light under aerobic conditions at a fixed temperature (controlled to ± 2°C) which will normally be within the range 15-20°C. In cases where the objective
of the study is to simulate environmental s ituations, tests may be carried out beyond this normal temperature range. The recommended maximum test duration is about 60 days. Degradation is followed by DOC meas urements (ultimate deg radation) and, in some cases, by specific analysis (primary deg radation).
IN FORMATION ON THE TES T SUBSTAN CE
4. In order to know whether the test may be applied to a particular substance, some of its properties 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. Also, the test substance should not significantly adsorb onto glass surfaces. In formation on the purity or the relative proportions of major components of the test material is required in order that the results obtained can
be interpreted, especially when the result lies close to the "pass" level.
5.Information on the toxicity of the test substance to bacteria, for example as measured in
short-term respiration rate tests (4), 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 procedure described in paragraph 18 is more suitable.
REFEREN CE COMPOUNDS
6.Suitable reference compounds must be used to check the microbial activity of the seawater sample. Sodium benzoate, sodium acetate and aniline are examples of chemicals which may be used
for this purpose. The reference compounds must be degraded within a reasonably short time span, otherwise it is recommended that the test be repeated using another seawater sample.
7.In the EEC ring test where seawater samples were taken at different locations and at different
times of the year (3), the lag phase (t
L ) and time to achieve 50 per cent degradation (t
50
), excluding
306OCD E / OECD
the lag phase, were 1 to 4 days and 1 to 7 days respectively for sodium benzoate. For aniline the t
L
ranged from 1 to 10 days.
ranged from 0 to 10 days, whilst the t
50
REPRODUCIB ILITY AN D S ENSITIVITY OF THE METHOD
8.The reproducibility of the method was established in the ring test (3). The lowest
concentration of test substance, for which this method can be used with DOC analysis, is largely determined by the detection limit of the organic carbon analysis (about 0.5 mg C/l, at present) and the concentration of dissolved organic carbon in the seawater used (usually of the order of 3-5 mg/l for water from the open sea). The background concentration of DOC should not exceed about 20% of the total DOC concentration after addition of test material. If this is not feasible, the background concentration of DOC may som etimes be reduced by ageing the seawater 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.
D ES CRIPTION OF TH
E METHOD
Apparatus
9.Normal laboratory apparatus and:
(a)Shaking machine accommodating 0.5-2 litre Erlenmeyer flasks, either with automatic
temperature control or used in a constant temperature room at 15-20°C controlled to
± 2°C;
neck, 0.5-2 litre Erlenmeyer flasks;
(b)Narrow
(c) Membrane filtration apparatus, or centrifuge;
filters, 0.2-0.45µm;
(d) Membrane
analyser;
(e) Carbon
for specific analysis (optional).
(f) Equipment
Seaw ater
10. Collect a sample of seawater in a thoroughly cleansed container and transport to the laboratory,
preferably within one or two 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 determination of the concentrations of dissolved nitrate, ammonium and phosphate.
following information for the seawater sample itself:
11. Provide
the
-date of collection;
-depth
collection;
of
-appearance of sample - turbid, etc.;
-temperature at the time of collection;
OCD E / OECD306 -salinity;
-DOC;
-delay between collection and use in the test.
12.If the DOC content of the seawater sample is found to be high (paragraph 8), it is recommended that the seawater be aged for about a week prior to use. Age by storing under aerobic conditions at the test temperature and in the dark or in diffuse light. If necessary, maintain aerobic conditions by gentle aeration. During ageing, the content of easily degradable organic material is reduced. In the ring test (3), no difference was revealed between the degradation potential of aged
and freshly collected seawater samples. Prior to use, pre-treat the seawater to remove coarse particles,
< by filtration through a nylon filter or coarse paper filter (not membrane or GF-C filters), or by sedimentation and decanting. The procedure used must be reported. Carry out pre-treatment after ageing, if used.
Stock solutions for mineral nutrients
13.Prepare the following stock solutions, using analytical grade reagents:
(a)Potassium dihydrogen orthophosphate, KH
2PO
4
...............8.50 g
Dipotassium hydrogen orthophosphate, K
<21.75 g
Disodium hydrogen orthophosphate dihydrate, Na
2HPO
4
.2H
2
O..33.30 g
Ammonium chloride, NH
4
<0.50 g Dissolve and make up to 1 litre with distilled water.
(b)Calcium chloride, CaCl
2
...............................27.50 g Dissolve and make up to 1 litre with distilled water.
(c)Magnesium sulphate heptahydrate, MgSO
4.7H
2
<22.50 g
Dissolve and make up to 1 litre with distilled water.
(d)Iron (III) chloride hexahydrate, FeCl
3.6H
2
<0.25 g
flask下载Dissolve and make up to 1 litre with distilled water.
Precipitation in solution (d) may be prevented by adding one drop of concentrated HCl or 0.4 g ethylenediaminetetra-acetic acid (EDTA, disodium salt) per litre. If a precipitate forms in a stock solution, replace it with freshly made solution.
Preparation of test medium
14.Add 1 ml of each of the above stock solutions per litre of pre-treated seawater.
Inoculum
15.Do not add a specific inoculum in addition to the micro-organisms already present in the seawater. Determine (optionally) the number of colony-forming heterotrophs in the seawater test medium (and preferably also in the original seawater samples) e.g. by plate count, using marine agar. This is particularly desirable for samples from coastal or polluted sites. Check the h eterotrophic microbial activity in the seawater by performing a test with a reference compound.

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