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Environmental fate & pathways

Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23/4/97 to 21/5/97
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.
according to guideline
other: OECD Guideline 306:"Biodegradability in Seawater" - Closed Bottle Test (OECD, 1992)
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
Inoculum or test system:
natural water
Details on inoculum:
Inoculum: The microorganisms present in the seawater only.

The seawater was collected at NIVA's research station at Solbergstrand. The seawater was aged by storing at the test temperature and by gentle aeration. Prior to use, coarse particles were removed from the seawater by sedimentation and decanting.
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
A pre-determined amount of the test substance is dissolved in the test medium in a concentration of usually 2-10 mg of test substance per litre (one or more concentrations may be used). The solution is kept in a filled closed bottle in the dark 20.0°C ± 2.4 for 28 days. The degradation is followed by oxygen analyses over a 28-day period.

Aged and decanted seawater with added mineral solutions as below:
The following stock solutions were prepared:
A) KH2PO4: 8.50 g
K2HPO4: 21.755 g
Na2HPO4.2H20: 33.30 g
NH4Cl: 0.50 g
The solution was dissolved and made up to 1000 ml with deionised water.

B) CaCl2.2H20: 36.45 g
The solution was dissolved and made up to 1000 ml with deionised water.

C) MgSO4: 10.99g
The solution was dissolved and made up to 1000 ml with deionised water.

D) FeCl3.6H20: 0.25 g
The solution was dissolved and made up to 1000 ml with deionised water.

1 ml of each of the stock solutions A) to D) was added to about 500 ml aged seawater and then diluted to 1 litre with aged seawater.

The test substance was added directly to the test medium and mixed on a magnetic stirrer for at least 15 minutes.

- Blank: test medium only
- Inoculum control: test medium and added reference substance (sodium benzoate)
- Test substance: test medium and added test substance
- Control of inhibitory action: test medium added test substance and reference substance.

Two parallels for each measurement.

The bottles were incubated at 20.0°C ± 2.4 for 28 days.

A WTW 320 oxygen meter was used to measure the oxygen consumption. The instrument automatically corrected for salinity, temperature, and pressure. The instrument was calibrated prior to each measurement.

The dissolved oxygen concentration in the test was determined after 0, 5, 15 and 28 days.
The bottle was placed on the stirrer without stirring. The stopper was removed and the oxygen electrode immediately put through the bottle neck, so that the stopper closed the bottle. The stirrer was started with a speed so as to prevent a vortex. All measurements were performed at the same stirring speed.

The chemical oxygen demand (COD) of the chemical was determined.

Specific gravity is needed for calculation of % biodegradation. Specific gravity was therefore determined for the chemical.

To determine possible nitrification in the test flasks, nitrite and nitrate concentrations were measured at day 28 at BUVA. Nitrate levels exceeding the background levels in the test medium are then considered as being derived from the test substance. The contributions to O2 consumption by the nitrification process can then be subtracted from the total O2 consumption in order to correctly assess the BOD.

Reference substance:
other: sodium benazoate
Test performance:
The test is considered valid if the following requirements are met:
- The blank respiration has not exceeded 30% of the oxygen in the test bottle.
- Percent degradation of the reference substance has reached the level of 50% within 5 days (the lag-phase not included).
- The temperature has been in the range 20.0°C ± 2.4°C

In this test, the control parameters yielded the following results:
- Percent (%) degradation of reference substance after 5 days: 78%
- The blank respiration (O2 mg/l), after
5 days: 0
15 days: 0.3
28 days: 0.4
i.e. the blank respiration is less than 30% of the oxygen in the test bottle.

- Temperature variation during the test: 20.0°C ± 2.4°C.

% degradation (O2 consumption)
Sampling time:
28 d
Details on results:
Table 1 presents the results from the biodegradation testing of the test substance EC6359A Active.

The raw data are presented in Appendices 1, 2, 3, 4 (see attached background material).
Results with reference substance:
- Percent (%) degradation of reference substance (sodium benzoate) after:
5 days: 78%
1 days: 86%
28 days: 86%

Table 1: Percent (%) biodegradation of the inoculum control, the test substance EC6359A Active and the control of inhibitory action


Time (days)

% Biodegradation

Inoculum control

Test Substance 

Control of inhibitory action













1 oxygen consumption due to nitrification is taken into consideration.

According to OECD 306, a test is considered valid if the percentage degradation of the reference substance (inoculum control) has reached the level of 50% within 5 days (the lag phase not included) and the blank respiration has not exceeded 30% of the oxygen in the test bottle. In the test, the activity of the inoculum was satisfactory. The inoculum control (sodium benzoate) was degraded by 78% within 5 days, i.e. the test is considered valid. Moreover, the oxygen depletion in the inoculum blank did not exceed 30% of the oxygen in the test bottle.

The biodegradation of the test substance was 12% after 28 days.

Validity criteria fulfilled:
Interpretation of results:
other: not readily biodegradable.
A test substance has a potential for biodegradation in the marine environment if it has been degraded by at least 60% within 28 days.
The test substance EC6359A Active was degraded by 12% after 28 days. Based on this, the test substance has not a potential for biodegradation in the marine environment.
Executive summary:

Biodegradation testing has been performed on the test substance EC6359A Active based on OECD guideline 306 for testing of chemicals:"Biodegradability in Seawater-Closed Bottle Test".


The test substance was degraded by 12% after 28 days based on COD calculations and measurements to account for O2consumption by nitrification.


The test substance cannot be characterized as readily biodegradable in the marine environment according to the OECD guideline 306.

biodegradation in water: screening test, other
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to Annex 3 of the CSR or IUCLID Section 13 for justification of read-across within the HEBMP category.
Reason / purpose for cross-reference:
read-across source
% degradation (O2 consumption)
Sampling time:
28 d

Description of key information

HEBMP-H is not readily biodegradable, based on 12% degradation in 28 days in a reliable screening study (OECD 306) with the read across substance, HEBMP-xNa. HEBMP-H is not inherently biodegradable, based on 25% degradation in a Zahn-Wellens test. In the same test, toxicity controls demonstrated that HEBMP-H was not inhibitory to the inoculum.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Both studies were conducted in accordance with a relevant Guideline and with GLP.

Whilst the proportion of cyclic and linear constituents was not measured for the tested sample, it is conservatively interpreted that the cyclic constituent could have been present at ca. 50% w/w.

The acid and sodium salts in the HEBMP category are freely soluble in water. The HEBMP anion can be considered fully dissociated from its sodium cation when in dilute solution. Under any given conditions, the degree of ionisation of the HEBMP species is determined by the pH of the solution. At a specific pH, the degree of ionisation is the same regardless of whether the starting material was HEBMP-H, HEBMP-xNa, or another salt of HEBMP.


Therefore, when a salt of HEBMP is introduced into test media or the environment, the following is present (separately):

  1. HEBMP is present as HEBMP-H or one of its ionised forms. The degree of ionisation depends upon the pH of the media and not whether HEBMP-xNa salt, HEBMP-H (acid form), or another salt was used for dosing.
  2. Disassociated sodium cations. The amount of sodium present depends on which salt was added.
  3. It should also be noted that divalent and trivalent cations would preferentially replace the potassium ions. These would include calcium (Ca2+), magnesium (Mg2+) and iron (Fe3+).