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Biodegradation in water and sediment: simulation tests

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biodegradation in water: simulation testing on ultimate degradation in surface water
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 April - 6 May 2011
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 306 (Biodegradability in Seawater)
according to guideline
other: EPA OPPTS 835.3160.
GLP compliance:

Test material

Constituent 1
Reference substance name:
Test material form:
solid: crystalline
Details on test material:
Isopropylhydroxylamine, batch number CEC-201002707-61, was used. Test material had a purity of 99.7% based on gas chromatographic area percent determination. The amount of water present was 0.23% using the Karl Fischer titration method. Test material confirmation was confirmed by nuclear magnetic resonance and mass spectrometry.
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Study design

Oxygen conditions:
Inoculum or test system:
natural water
Details on source and properties of surface water:
A mixed population of marine micro-organisms in sea water was obtained on 31 March 2011 from Huttoft on the East Coast of the United Kingdom.

The characteristics of the sea water collected for use in the study are as follows:
Date of sampling: 31 March 2011
Depth of sampling: approximately 30 cm
Appearance of sample: sandy brown dispersion
Temperature of sample on collection: 13.3°C
Temperature of sample on arrival at laboratory: 16.8°C
Salinity of sample: 32.2%0
Delay between collection of the sample and use in the study: 8 days
Ammonium concentration in the seawater: 0.000 mg/l
Phosphate concentration in the seawater: 0.083 mg/l
Nitrate concentration in the seawater: 2.10 mg/l
Total microbial viable count: too many to count
Dissolved organic carbon concentration after ageing for 8 days: 3.53 mg/l (mean of two values)
Details on source and properties of sediment:
Not applicable
Details on inoculum:
The sample of sea water was allowed to settle prior to filtration through coarse filter paper to remove any particulate matter and the filtrate maintained on continuous aeration in a temperature controlled room at approximately 21°C for 8 days prior to the start of the test in order to reduce the basal Biological Oxygen Demand (BOD) of the sea water.

Whilst the Test Guidelines suggest to age the sea water prior to pre-treatment to remove any particulate matter, experience has shown that pre-treatment prior to ageing results in lower basal BOD values and less variability between replicate vessles.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
1.5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
For the purpose of the test, the test item was dissolved directly in test medium.

An amount of test item (100 mg) was dissolved in test medium and the volume adjusted to 100 ml to give a 1000 mg/l stock solution. An aliquot (9 ml) of this stock solution was dispersed in a final volume of 6 litres of test medium to give a concentration of 1.5 mg/I.

The volumetric flask containing the stock solution was inverted several times to ensure homogeneity of the solution.

A test concentration of 1.5 mg/l was employed in the study as the Theoretical Oxygen Demand (ThOD) of the test item was calculated to be 2.56 mg 02/mg. Hence if complete degradation of the test item occurred, the oxygen depletion in the test vessels would be 3.84 mg O2/1 and as such deoxygenation of the test media would not occur.

No analysis was conducted to determine the concentration, homogeneity or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
Key result
% Degr.:
O2 consumption
Sampling time:
28 d
Transformation products:
not measured
Details on results:
The oxygen depletion in the control vessels was 3% of the initial dissolved oxygen concentration and the residual oxygen concentration remained at 3.70 mg 0 2/1 or greater in all test vessels, thereby satisfying the validation criteria for oxygen depletion.

The test item attained 19% degradation after 28 days.

Variation in degradation rates on different sampling days was considered to be due to normal biological variation in respiration rates between control and test vessels. The toxicity control attained 39% degradation after 28 days. The oxygen depletion value was greater than that expected based on the sum of the oxygen depletion values from the test and reference item thereby confirming that the test item was not toxic to the marine micro-organisms used in the test.

Results with reference substance:
The reference item, sodium benzoate, attained 71 % degradation after 14 days and 77% degradation after 28 days thereby confirming the suitability of the test method and culture conditions.

Examination of the biodegradation curve for the reference item showed the lag phase to be less than 1 day and the time taken to achieve 50% degradation to be approximately 4 days. These values were in line with those obtained in an EEC ring-test of this method where the lag phase and time taken to achieve 50% degradation were 0 to 2 days and 1 to 4 days respectively for sodium benzoate.

Any other information on results incl. tables

Mean Percentage Biodegradation Values

 Test Series                          Day
   3  5  7  11  14  18  20  25  28
 Sodium Benzoate (2.0 mg/L) in Test Medium  45  60  56  62  71  71  65  69  77
 Test Item (1.5 mg/L) in Test Medium  2  4  -1  3  9 17  13  10  19
 Test Item (1.5 mg/L) plus Sodium O2 Depletion (mg O2/L)Benzoate (1.0 mg/L) in Test Medium  -  18  -  33  -  -  -  39

Applicant's summary and conclusion

Validity criteria
Validity criteria fulfilled:
The test item attained 19% degradation after 28 days.
Executive summary:

A study was performed to assess the biodegradability of the test item when exposed to marine micro-organisms in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 306, “Biodegradability in Sea Water; Closed Bottle Test” and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3160.

Methods. The test item, at a concentration of 1.5 mg/l, was exposed to marine microorganisms with test medium in sealed culture vessels in the dark at a temperature of approximately 19ºC for 28 days. The degradation of the test item was assessed by the determination of the amount of oxygen consumed. Control solutions containing the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

Results. The test item attained 19% degradation after 28 days.

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