ISO 16221:2001 download.Water quality – Guidance for determination of biodegradability in the marine environment.
ISOITC 147 has established International Standards for testing biodegradability of substances and waste water in the aquatic environment. All these methods, which are summarized in ISO 15462, can only be used for the determination and prediction of biodegradability in fresh water. There are, however, many cases, for example, substances used off-shore, where an urgent need exists for testing biodegradability in the marine environment. ISO 16221 describes biodegradation testing in marine test systems, and is based on an established OECD Guideline and the experience gained by a working group of the Oslo and Paris Commission (OSPARCOM) which has selected suitable standardized ISO methods, adopted for marine conditions and checked in a ring test.
ISO 16221 specifies five methods for determining the ultimate aerobic biodegradability of organic compounds in the marine environment by aerobic microorganisms in static aqueous test systems. Standard degradation methods developed for testing in fresh water are modified and adapted to marine conditions. These methods are the DOC die-away test (ISO 7827), the closed bottle test (ISO 10707), the two-phase closed bottle test (ISO 10708), the CO2 evolution test (ISO 9439) and the CO2 headspace test (ISO 14593).
The methods apply to organic compounds which
a) are water-soluble under the conditions of the test used;
b) are poorly water-soluble under the conditions of the test used, in which case special measures may be necessary to achieve good dispersion of the compound (see for example, ISO 10634):
c) are volatile, provided that an appropriate test with suitable conditions is used:
d) are not inhibitory to the test microorganisms at the concentration chosen for the tests. The presence of inhibitory effects can be determined as specified in this International Standard.
NOTE The conditions described in ISO 16221 do not always correspond to the optimal conditions tar allowing the maximum degree of biodegradation to occur. For biodegradation methods in fresh water see ISO 14593 and ISO 15462, and for biodegradation at low concentrations see ISO 14592.
2 Normative references
The following normative documents contain provisions which, through reference in this text, constitute provisions of ISO 16221. For dated references, subsequent amendments to, or revisions of, any of these publications do not apply. However, parties to agreements based on ISO 16221 are encouraged to investigate the possibility of applying the most recent editions of the normative documents indicated below. For undated references, the latest edition of the normative document referred to applies. Members of ISO and IEC maintain registers of currently valid International Standards.
ISO 7827, Water quality — Evaluation in an aqueous medium of the “ultimate” aerobic biodegradability of organic compounds — Method by analysis of dissolved organic carbon (DOC).
ISO 16221 specifies five methods for determining the ultimate aerobic biodegradability of organic compounds in the marine environment by aerobic microorganisms in static aqueous test systems. Standard degradation methods developed for testing in fresh water are modified and adapted to marine conditions. These methods are the DOC die-away test (ISO 7827), the closed bottle test (ISO 10707), the two-phase closed bottle test (ISO 10708), the CO2 evolution test (ISO 9439) and the CO2 headspace test (ISO 14593).
The methods apply to organic compounds which
a) are water-soluble under the conditions of the test used;
b) are poorly water-soluble under the conditions of the test used, in which case special measures may be necessary to achieve good dispersion of the compound (see for example, ISO 10634):
c) are volatile, provided that an appropriate test with suitable conditions is used:
d) are not inhibitory to the test microorganisms at the concentration chosen for the tests. The presence of inhibitory effects can be determined as specified in this International Standard.
NOTE The conditions described in this International Standard do not always correspond to the optimal conditions tar allowing the maximum degree of biodegradation to occur. For biodegradation methods in fresh water see ISO 14593 and ISO 15462, and for biodegradation at low concentrations see ISO 14592.
2 Normative references
The following normative documents contain provisions which, through reference in this text, constitute provisions of ISO 16221. For dated references, subsequent amendments to, or revisions of, any of these publications do not apply. However, parties to agreements based on ISO 16221 are encouraged to investigate the possibility of applying the most recent editions of the normative documents indicated below. For undated references, the latest edition of the normative document referred to applies. Members of ISO and IEC maintain registers of currently valid International Standards.
ISO 7827, Water quality — Evaluation in an aqueous medium of the “ultimate” aerobic biodegradability of organic compounds — Method by analysis of dissolved organic carbon (DOC).
3.8
biochemical oxygen demand
BOD
mass concentration of dissolved oxygen consumed under specified conditions by the aerobic biological oxidation of
a chemical compound or organic matter in the water sample
NOTE It is expressed in this case as milligrams oxygen uptake per milligram (or gram) test compound.
3.9
theoretical oxygen demand mOD
theoretical amount of oxygen required to oxidize a chemical compound completely, calculated from the molecular formula
NOTE It Is expressed in this case as milligrams oxygen uptake per milligram (or gram) test compound.
3.10
theoretical amount of formed carbon dioxide
ThCO2
theoretical amount of carbon dioxide formed after oxidizing a chemical compound completely, calculated from the molecular formula
NOTE It is expressed in this case as milligrams oxygen uptake per milligram (or gram) test compound.
3.11
theoretical amount of inorganic carbon ThIC
theoretical amount of inorganic carbon formed after oxidizing a chemical compound completely, calculated from the molecular formula
NOTE It is expressed in this case as milligrams oxygen uptake per milligram (or gram) test compound.
3.12
lag phase
time from the start of a test until adaptation and/or selection of the degrading microorganisms are achieved and the biodegradation degree of a chemical compound or organic matter has increased to about 10 % biodegradation
When natural seawater is used, normally sufficient microorganisms are available and no additional inoculation is required.
It is recommended to determine the number of colony-forming heterotrophic bacteria in the natural seawater, e.g.
by plate count using a marine agar. A suitable bacterial concentration is about 1 cells/mi in the test vessels.
When the natural seawater has too low a bacterial density, inoculate as described for artificial seawater (6.3).
Check the activity of the natural seawater by means of the reference compound.
NOTE 1 Normally the natural seawater and the inoculum should not be pre-exposed to the test compound, to allow a general prediction of the degradation behaviour in the environment. In certain circumstances, depending on the purpose of the test, pie- exposed inocula may be used, provided that this is clearly stated In the test report (e.g. percent biodegradation – x %. using preexposed inocula) and the method of pre-exposure is detailed in the test report. Pre-exposed inocula can be obtained from marine laboratory biodegradation tests conducted under a variety of conditions or from samples collected from locations where relevant environmental conditions exist (e.g contaminated areas).
NOTE 2 The amount of bacteria for the test may be increased, e.g. by centrifugation and re-suspension in a smaller seawater sample.
To reduce the concentration of DOC or BOD in the blank, preconditioning is possible. Incubate the sample in the dark or in diffused light at the test temperature, under aerobic conditions, for up to one week. When the added inoculum contains too much DCC (>10 % of the organic carbon added by the test compound), remove the surplus by washing with artificial seawater (6.3) and centrifuging. The total inorganic carbon (TIC) content of natural seawater is usually high: if so, this shall be reduced in accordance with ISO 14593. Measure the pH of the seawater sample. Sparge with C02-free air for about 1 h while maintaining the pH at 6,5 using concentrated hydrogen chloride (HCI). Finally, restore the pH to its original value with sodium hydroxide (NaOH).
Prior to use, remove coarse particles from the seawater by filtration using e.g. a coarse paper filter or by sedimentation. To obtain sufficient buffering capacity and supply of nutrient to the test solution, add as mineral nutrients (6.2.2) the usual inorganic medium of standard biodegradation tests, excepting the solutions with magnesium sulfate and calcium chloride, as these minerals are in sufficient concentration in any natural sea water.
6.2.2 Mineral nutrients
For 1000 ml of test medium, add to about 800 ml of natural seawater (6.2) 10 ml of solution a) and 1 ml of solution b) below, and make up to 1000 ml with the seawater (6.2).
When natural seawater is used, normally sufficient microorganisms are available and no additional inoculation is required.
It is recommended to determine the number of colony-forming heterotrophic bacteria in the natural seawater, e.g.
by plate count using a marine agar. A suitable bacterial concentration is about 1 cells/mi in the test vessels.
When the natural seawater has too low a bacterial density, inoculate as described for artificial seawater (6.3).
Check the activity of the natural seawater by means of the reference compound.
NOTE 1 Normally the natural seawater and the inoculum should not be pre-exposed to the test compound, to allow a general prediction of the degradation behaviour in the environment. In certain circumstances, depending on the purpose of the test, pie- exposed inocula may be used, provided that this is clearly stated In the test report (e.g. percent biodegradation – x %. using preexposed inocula) and the method of pre-exposure is detailed in the test report. Pre-exposed inocula can be obtained from marine laboratory biodegradation tests conducted under a variety of conditions or from samples collected from locations where relevant environmental conditions exist (e.g contaminated areas).
NOTE 2 The amount of bacteria for the test may be increased, e.g. by centrifugation and re-suspension in a smaller seawater sample.
To reduce the concentration of DOC or BOD in the blank, preconditioning is possible. Incubate the sample in the dark or in diffused light at the test temperature, under aerobic conditions, for up to one week. When the added inoculum contains too much DCC (>10 % of the organic carbon added by the test compound), remove the surplus by washing with artificial seawater (6.3) and centrifuging. The total inorganic carbon (TIC) content of natural seawater is usually high: if so, this shall be reduced in accordance with ISO 14593. Measure the pH of the seawater sample. Sparge with C02-free air for about 1 h while maintaining the pH at 6,5 using concentrated hydrogen chloride (HCI). Finally, restore the pH to its original value with sodium hydroxide (NaOH).
Prior to use, remove coarse particles from the seawater by filtration using e.g. a coarse paper filter or by sedimentation. To obtain sufficient buffering capacity and supply of nutrient to the test solution, add as mineral nutrients (6.2.2) the usual inorganic medium of standard biodegradation tests, excepting the solutions with magnesium sulfate and calcium chloride, as these minerals are in sufficient concentration in any natural sea water.
6.2.2 Mineral nutrients
For 1000 ml of test medium, add to about 800 ml of natural seawater (6.2) 10 ml of solution a) and 1 ml of solution b) below, and make up to 1000 ml with the seawater (6.2).