Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August - October 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: in accordance with guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
Principles of method if other than guideline:
OPPTS 835.3110. Ready Biodegradability. EPA fate, transport and transformation test guidelines, 1996
GLP compliance:
yes (incl. certificate)
Remarks:
Italian Ministero della Salute

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Algea Fert Solid K+
- Substance type: fertilizer ( algal extract)
- Physical state: solid, black micro-flakes
- Lot/batch No.: 00680
- Expiration date of the lot/batch: March 2012
- Stability under test conditions: Stable under suitable storage conditions.
- Storage condition of test material: original tightly-closed containers in well- ventilated place.

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
The study was performed using aerobic activated sludge from ALSI (Alto Lambro Servizi Idrici) wastewater treatment plant treating civil wastewater. The sludge was washed by centrifugation and the supernatant liquid phase decanted. A homogenized aliquot of the final sludge suspension was weighted, thereafter dried and the ratio of wet to dry weight was calculated as 8.8%.
Based on this ratio, calculated amounts of wet sludge were suspended in the test water (see next chapter) to get a concentration equivalent to 3.0 g dry material/L. During holding, the sludge was aerated at room temperature in dark conditions. Before using, the sludge inoculum was diluted with test water to obtain a final concentration of 15 mg/L (dry weight).
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Remarks:
classic manometric method (consumption)
Details on study design:
The assay on test item has been run in parallel with a ready biodegradable reference item, i.e. sodium benzoate, the inoculum control, the abiotic control containing the test item and a abioticizing agent (HgCl2) and the toxicity control, containing test item, reference compound and inoculum.
BOD flasks of 500 ml capacity, equipped with pressure sensors fitted on the bottle mouth, have been used as test units.
The test water was prepared according to the testing guidelines: salts of analytical grade were dissolved in purified water to obtain the following stock solutions (up to 1000 mL):
a) KH2PO4 8.50 g/L
K2HPO4 21.75 g/L
Na2HPO4 x 2H2O 33.40 g/L
NH4Cl 0.50 g/L
The pH of this solution should be 7.4.
b) CaCl x 2H2O 36.40 g/L
c)MgSO4 x 7H2O 22.50 g/L
d) FeCl3 x 6 H2O 0.25 g/L

10 mL of stock solution a) and 1 mL of stock solution b), c) and d) will be combined and made up to 1000 mL with purified water. The pH of test medium was 7.62, so adjustment was not needed.

The pH of test medium was 7.62, so adjustment was not needed.
A stock solution of 100 mg/L of test item was prepared by directly weighted into test medium (0.2002 g of test item into 2000 mL of test medium), while the reference item solution was prepared by diluting an appropriate volume of an aqueous stock solution of 10 g/L into test medium.
Activated sludge was added to each flask and the flasks were made up to a volume of 250 mL or 150 mL, according to the scale setting of the manometric device, with the test water.

Principle of the method:
The method is based on an electro-chemical analyses process. The biodegradation process consumes the dissolved oxygen in liquid and generates CO2. The CO2 is adsorbed by soda lime and the total pressure decreases in the test flasks. The decrease of the pressure is detected and converted into an electrical signal by means of an electrode type manometer. The consumed oxygen is replaced by electrolysis generated oxygen from copper sulfate solution.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
Parameter:
% degradation (CO2 evolution)
Remarks:
classic manometric method
Value:
45.1
Sampling time:
10 d
Remarks on result:
other: in the 10 days- window
Details on results:
The test concentration was not measured during the test, therefore all reported results are related to the nominal concentration of the test item.
The biodegradation has been followed by measuring the BOD (Biochemical Oxygen Demand) of the test item under defined conditions by the means of a manometric method.
The measured oxygen concentrations in the test solutions and in the inoculum control, the oxygen uptake of the reference item, abiotic control and toxicity control were used to calculate the BOD value and the % of biodegradation.
COD for Algea Fert Solid K+ was calculated as 0.71 mg O2/mg and 1.86 mg O2/mg for Sodium Benzoate. the 10-days window begun after one day of incubation (17.6% biodegradation of the test item, mean of duplicate values); by the end of the 10-days period (day 10 after test initiation) the test item was degraded by a maximum value of 45.1%, this result indicates that the test item is not ready biodegradable under the test conditions. However, after 17 days by the test start, the test item reached and exceeded the 60% of biodegradation achieving a maximum value of 113.4% after 26 days of exposure.
The toxicity control clearly shows that the test item does not cause inhibitory effect on the microorganism activity at the tested concentration. In fact, the degradation of the reference item in presence of the test item exceeded the 25% of biodegradation at first day of exposure .
In the abiotic control no biodegradation was observed (Table 5), indicating the absence of abiotic degradation.

BOD5 / COD results

Results with reference substance:
The degradation of the reference item in presence of the test item exceeded the 25% of biodegradation at first day of exposure.

Any other information on results incl. tables

BOD values and % biodegradation for the reference item sodium benzoate and the test item Algea Fert Solid K+ in a Manometric Respirometry test.

Day of measurement

 

Reference item

BOD
 

Reference item

% biodegradation
 

Test item

BOD
 

Test item

% biodegradation
 1  0.30 16.1  0.13  17.6*
 2  0.65 34.9 0.16 21.8
 3  0.92 49.5 0.18  24.6
 4  1.25 67.2 0.21  28.9
 5  1.37 73.7 0.26  35.9
 6 1.42 76.3 0.28  38.7
 7 1.43  76.6 0.28 38.7
 8 n.d. n.d. n.d.  n.d.
 9 n.d.  n.d.  n.d.  n.d. 
 10 1.51 80.9 0.32 45.1 **   
 11 1.52  81.5 0.34  47.2
 12 1.53   82.3 0.35  49.3
 13 1.54  82.5  0.36  50.7
 14 1.54  82.5 0.36 50.7
 15 n.d.   n.d. n.d.  n.d.
 16 n.d.  n.d.  n.d.  n.d. 
 17 1.44  77.2 0.76 107.0
 18 1.45  77.7 0.73  102.1 
 19 1.47  78.8 0.79  110.6
 20 1.45   78.0 0.77 107.7
 21 1.45  77.7 0.61  85.9
 22 n.d.  n.d. n.d. n.d.
 23 n.d.  n.d.  n.d.  n.d. 
 24 1.44   77.4 0.78 109.9
 25 1.47 78.8 0.80 112.7
 26 1.45  78.0 0.81 113.4
 27 1.44 77.4 0.79 110.6 
 28 1.45  77.7  0.79 111.3

n.d. not determined being not working days.

* start of 10-d window

** end of 10-d window

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
other: not ready biodegradable
Conclusions:
The test item Algea Fert Solid K+ has showed to be not ready biodegradable under the conditions applied in a Manometric Respirometry test, having not reached the pass level (60% biodegradation) during the 10-days window, but only showing a maximum value of degradation of 45.1% at the end of this period.
However, after 17 days by the test start, the test item reached and exceeded the 60% of biodegradation achieving a maximum value of 113.4% after 26 days of exposure

Moreover, the test item showed no inhibitory effects on the microorganism activity at the tested concentration, as demonstrated by the 25% of biodegradation exceeded in the toxicity control, already at the second day of test period.

Executive summary:

The ready biodegradability of the test item Algea Fert Solid K+ was assessed in a 28-day biodegradation test following the “Manometric Respirometry” method according to the following guidelines:

  • OECD guideline for testing of chemicals no., Ready Biodegradability, Manometric Respirometry test, 1992
  • OPPTS 835.3110. Ready Biodegradability. EPA fate, transport and transformation test guidelines, 1996
  • Council Regulation EC 440/2008 (C.4 Part V)

For this purpose, the test item was added to activated sludge collected from the aeration tank of a civil wastewater treatment plant and let to stay in contact with the sludge for 28 days under continuous stirring. 

The tested concentration was 100 mg/L as test item. The biodegradation was followedby measuring the BOD (Biochemical Oxygen Demand) of the test item under defined conditions by means of a manometric method.

The test item is considered to be stable at the test conditions for a time corresponding to the exposure time (28 days). Therefore, all reported results are related to the nominal concentration of the test item.

The COD values were calculated to be equal to 1.86 mg O2/mg for reference Item and 0.71 mg O2/mg for test item.

The oxygen consumption due to nitrification was measured at the begging and at the end of the test and the absence of nitrification was assessed. The test complies with the validity criteria since the mean oxygen uptake in the inoculum control at the end of test period was 26.5 mg O2/L and the difference between duplicate values of test item degradation at the end of the test period was 1.3%. Moreover, the reference item reached the biodegradation pass level (60%) on the fourth day (67.2%). The test item Algea Fert Solid K+ was found to be not ready biodegradable under the conditions applied in a Manometric Respirometry test, having showed a maximum biodegradation of 45.1% at the end of 10-windows . However, after 17 days by the test start, the test item reached and exceeded the 60% of biodegradation achieving a maximum value of 113.4% after 26 days of exposure. Even so, the test item did not show any inhibitory effects on the microorganism activity at the tested concentration as demonstrated by the 25% of biodegradation exceeded in the toxicity control already at the first day of test period (28.8%)