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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 July - 26 August 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, to GLP
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Waterschap de Maaskant, 's-Hertogenbosch, the Netherlands
- Laboratory culture: not applicable
- Method of cultivation: not applicable
- Storage conditions: aerated until use
- Storage length: no data
- Preparation of inoculum for exposure: Before use, the sludge was allowed to settle (30-90 minutes) and the liquid was decanted for use as inoculum.
- Pretreatment: no data
- Concentration of sludge: 10 mL/L of mineral medium
- Initial cell/biomass concentration: no data
- Water filtered: no data
- Type and size of filter used, if any: not applicable
Duration of test (contact time):
28 d
Initial conc.:
80 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: four stock solutions were used to make up the mineral medium; Solution A: KH2PO4 - 8.5 g, K2HPO4 - 21.75 g, Na2HPO4.12H2O - 67.2 g and NH4Cl - 0.5 g (dissolved in milli-Q water and made up to 1 L). Solution B: MgSO4.7 H2O - 22.5 g (dissolved in milli-Q water and made up to 1 L). Solution C: CaCl2.2 H2O - 36.4 g (dissolved in milli-Q water and made up to 1 L). Solution D: FeCl3.6 H2O - 0.25 g (dissolved in milli-Q water and made up to 1 L). Mineral medium was prepared by mixing 10 mL of solution A and 1 mL each of solutions B-D were filled up to 1000 mL with milli-RO water.
- Additional substrate: not applicable
- Solubilising agent (type and concentration if used): not applicable
- Test temperature: 21.3 - 22.2 deg C
- pH: 7.4 - 8.7
- pH adjusted: no
- CEC (meq/100 g): no data
- Aeration of dilution water: no data
- Suspended solids concentration: 4.5 g/L
- Continuous darkness: no data

TEST SYSTEM
- Culturing apparatus: brown coloured bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: connected to an air line
- Method used to create anaerobic conditions: not applicable
- Measuring equipment: no data
- Test performed in closed vessels due to significant volatility of test substance: no data
- Test performed in open system: no data
- Details of trap for CO2 and volatile organics if used: Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit line of each test bottle

SAMPLING
- Sampling frequency: days 0, 2, 5, 7, 9, 14, 19, 23, 27 and 29
- Sampling method: The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with HCl (0.05 M).
- Sterility check if applicable: not applicable
- Sample storage before analysis: used on day of sampling

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: not applicable
- Toxicity control: containing test substance, reference substance and inoculum

STATISTICAL METHODS:
Theoretical carbon dioxide (ThCO2), expressed as mg CO2/mg test substance was calculated as follows:
ThCO2 = [No. of carbon atoms in test substance x MW (CO2)]/[MW (test substance)]

The amount of 0.05 N HCl titrated is converted into mg of CO2 produced:
mg (CO2) = 44 x [0.05 x mL HCl titrated]/2
A figure of more than 10% degradation was considered significant.

If less than 25% degradation (based on ThCO2) occurred within 14 days for the toxicity control, the test substance was assumed to be inhibitory to microorganisms.
Reference substance:
acetic acid, sodium salt
Remarks:
40 mg/L added to the test medium of the positive control bottle and the toxicity control bottle
Preliminary study:
not applicable
Test performance:
no data
Parameter:
% degradation (CO2 evolution)
Value:
3
Sampling time:
2 d
Remarks on result:
other: result for bottle A
Parameter:
% degradation (CO2 evolution)
Value:
35
Sampling time:
5 d
Remarks on result:
other: result for bottle A
Parameter:
% degradation (CO2 evolution)
Value:
57
Sampling time:
9 d
Remarks on result:
other: result for bottle A
Parameter:
% degradation (CO2 evolution)
Value:
64
Sampling time:
14 d
Remarks on result:
other: result for bottle A
Parameter:
% degradation (CO2 evolution)
Value:
74
Sampling time:
29 d
Remarks on result:
other: result for bottle A
Details on results:
On day 29, the biodegradation in each bottle was 75 and 56% respectively, with a mean value of 66%. A discrepancy of 19% was significant but within the limit for validity of the test (<20%). The investigators noted that in test bottle A, biodegradation of at least 60% was reached within 10 days of biodegradation exceeding 10%. However, it is difficult to verify this criterion from the tabulated data though the graphical representation is more insightful (see attached PDF). From the graph, the biodegradation (bottle A) appears to exceed 10% on around day 3 and surpasses 60% around day 11. Therefore, the criterion for ready biodegradability was met.

In the toxicity control, containing both platinum (2 +) tetraammine diacetate and sodium acetate, 34% biodegradation occurred by day 14. Therefore, the test substance was assumed to exert no inhibitory effect on the microorganisms.

The pH in bottle A reached 8.7 on day 28, whereas the pH values for all other flasks at the start and end of the test were between 7.4 and 7.8.
Results with reference substance:
The reference compound, sodium acetate, was biodegraded by 77% within 14 days of exposure, thus confirming the suitability of the activated sludge.

Table 1: Percentage biodegradation of platinum (2 +) tetraammine diacetate in the presence of activated sludge (comparison of the test substances in bottles A and B)

 Day     Biodegradation (%)         
 Bottle A  Bottle B  Mean (A and B)  Difference between A and B
 0  0  0  0  0
 2  3  0  2  3
 5  35  30  33  5
 7  51  43  47  8
 9  57  50  54  7
 14  64  54  59  10
 19  66  56  61  10
 23  68  56  62  12
 27  69  56  63  13
 29  73  56  64  17
 29  74  56  65  18
 29  75  56  66  19

Table 2: CO2 production and percentage biodegradation of the positive control

 Day     HCl (0.05 N) titrated (mL)     Produced CO2 (mL HCl)     Produced CO2 (mg)     Cumulative CO2 (mg)     Degradation (%)   
 Blank (mean)  Positive control
 0  -  -  -  -  -  -
 2  45.89  44.33  1.56  1.7  1.7  2
 5  45.57  20.03  25.54  28.1  29.8  35
 7  45.38  28.43  16.95  18.6  48.4  56
 9  45.82  36.53  9.29  10.2  58.7  68
 14  45.26  38.32  6.94  7.6  66.3  77
 19  43.57  41.09  2.48  2.7  69.0  80
 23  42.25  41.26  0.99  1.1  70.1  81
 27  45.54  46.08  0.00  0.0  70.1  81
 29  46.25  45.50  0.75  0.8  70.9  82
 29  48.01  47.62  0.39  0.4  71.4  83
 29  49.17  49.03  0.14  0.2  71.5  83

Table 3: CO2 production and percentage biodegradation of the toxicity control

 Day     HCl (0.05 N) titrated (mL)     Produced CO2(mL HCl)     ProducedCO2 (mg)     CumulativeCO2 (mg)     Degradation(%)   
 Blank (mean)  Positive control
 0  -  -  -  -  -  -
 2  45.89  44.33  0.00  0.0  0.0  0
 5  45.57  22.50  23.07  25.4  25.4  16
 7  45.38  31.59  13.79  15.2  40.5  25
 9  45.82  38.60  7.22  7.9  48.5  30
 14  45.26  40.38  4.87  5.4  53.9  34
 19  43.57  40.51  3.06  3.4  57.2  36
 23  42.25  41.74  0.51  0.6  57.8  36
 27  45.54  44.71  0.82  0.9  58.7  37
 29  46.25  45.31  0.94  1.0  59.7  37
 29  48.01  47.50  0.51  0.6  60.3  38
 29  49.17  48.85  0.32  0.4  60.6  38

Table 4: pH values for the different test media

   Start of the test  Day 28
 Blank control (A)  7.5  7.4
 Blank control (B)  7.5  7.4
 Positive control  7.5  7.8
 Test substance (A)  7.5  8.7
 Test substance (B)  7.5  7.8
 Toxicity control  7.5  7.8
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
In a guideline GLP study, platinum (2+) tetraammine diacetate was readily biodegradable within the 28 day exposure period, in the presence of activated sludge. A mean biodegradation of 66% was observed on day 29.
Executive summary:

In a guideline study (OECD TG 301B), conducted according to GLP, the ready biodegradability of platinum (2 +) tetraammine diacetate was assessed in a modified Sturm test. The overall biodegradation on day 29 was 66% as a mean of two bottles, though there was a significant discrepancy between the bottles (19% on day 29). In test bottle A, biodegradation of at least 60% was reached within 10 days of biodegradation exceeding 10%. Therefore, the criterion for ready biodegradability was met. The pH in bottle A reached 8.7 on day 28, whereas the pH values for all other flasks at the start and end of the test were between 7.4 and 7.8.

The reference compound, sodium acetate, was biodegraded by 77% by day 14, thus confirming the suitability of the activated sludge. In the toxicity control, containing both platinum (2 +) tetraammine diacetate and sodium acetate, 34% biodegradation occurred by day 14. Therefore, the test substance was assumed to exert no inhibitory effect on the microorganisms.

Description of key information

not relevant for inorganic salt

Key value for chemical safety assessment

Additional information