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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:
July-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): 3,4-dichlorobenzotrichloride
- Substance type: intermediate for synthesis
- Physical state: from colourless to slightly yellow liquid with characteristic aroma
- Analytical purity: 99.8%
- Purity test date: March 10, 2010
- Lot/batch No.: 03/2010
- Expiration date of the lot/batch: March 2012
- Stability under test conditions: Stable away from water and heat, on iron, at temperature < 50°C. Moreover if mixed with sulphuric acid a violent hydrolysis occurs.
- Storage condition of test material: Keep away from incompatible materials (oxidisers, not noble metals, amines and alcohols). Keep away from heat and ignition sources.

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 8.1%.
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
Details on study design:
TEST CONDITIONS
Temperature: 22.3 – 23.7 °C
Light conditions: darkness
Stirring: continuous

TEST SYSTEM
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 8.1%.
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).


CONTROL AND BLANK SYSTEM
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.

Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
Parameter:
% degradation (CO2 evolution)
Remarks:
classic manometric method
Value:
>= 10
Sampling time:
28 d
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. According to OECD guideline no. 301, the oxygen consumption due to nitrification was not considered since the test substance does not contain N. 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. ThODNH3 for 3,4-dichlorobenzotrichloride was calculated as 0.79 mg O2/mg and 1.67 mg O2/mg for Sodium Benzoate. The test item has not reached the 10% of biodegradation during the 28 days of test duration, having showed no biodegradation through the test period. These results indicate that the test item is not ready biodegradable. 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 third day of exposure. In the abiotic control no biodegradation was observed , thus 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 third day of exposure.

Any other information on results incl. tables

BOD values and % biodegradation for the reference item sodium benzoate and the test item 3,4-dichlorobenzotrichloride in a Manometric Respirometry test.

Day of measurement

 

Reference item

BOD
 

Reference item

% biodegradation
 

Test item

BOD
 

Test item

% biodegradation
 1  0.20 12.0   -0.09  0.0*
 2  0.41 24.6  -0.09  0.0*
 3  0.63 37.7  -0.12   0.0*
 4  0.76 45.2 -0.18   0.0*
 5  0.97 57.8 -0.19   0.0*
 6 1.09  65.3 -0.23   0.0*
 7 1.18 70.7  -0.23  0.0*
 8 1.21  72.5  -0.24   0.0*
 9 n.d.  n.d.  n.d.  n.d. 
 10 n.d.  n.d. n.d. n.d. 
 11 1.29  76.9  -0.26   0.0*
 12 1.31  78.1  -0.25   0.0* 
 13 1.39  82.9  -0.09   0.0* 
 14 1.37  82.0  -0.13   0.0* 
 15 1.37  81.7  -0.13   0.0* 
 16 n.d.  n.d.  n.d.  n.d. 
 17 n.d.  n.d. n.d.  n.d. 
 18 1.47  87.7  -0.13   0.0* 
 19 1.53  91.6  -0.11   0.0* 
 20 1.54  92.2  -0.15   0.0* 
 21 1.53  91.3  -0.18   0.0* 
 22 1.55  92.5  -0.10   0.0*
 23 n.d.  n.d.  n.d.  n.d. 
 24 n.d.  n.d.  n.d.   n.d.
 25 1.59  95.2  -0.11  0.0* 
 26 1.59  94.9  -0.11  0.0* 
 27 1.60  95.5  -0.11  0.0* 
 28 1.61  96.1  -0.10 0.0* 

n.d. not determined being not working days.

* this value was estimated as 0 being negative(< 0) as per calculation

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The oxygen uptake in the inoculum control at the end of test period was 40 mg O2/L.The validity criterion which set the reference item biodegradation threshold at 60% is met, since it was exceeded on the sixth day of incubation (65.3%).
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test item 3,4-dichlorobenzotrichloride has showed to be not ready biodegradable under the conditions applied in a Manometric Respirometry test, having showed degradation of 0% during the test period.
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 third day of test period.
Executive summary:

The ready biodegradability of the test item 3,4-dichlorobenzotrichloride 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 ThODNH3values were calculated to be equal to 1.67 mg O2/mg for reference item and 0.79 mg O2/mg for test item.

According to OECD guideline no. 301, the oxygen consumption due to nitrification was not considered since the test substance does not contain N.

The test complies with the validity criteria since the mean oxygen uptake in the inoculum control at the end of test period was 40 mg O2/L and there was not difference between duplicate values of test item degradation at the end of the test period. Moreover, the reference item reached the biodegradation pass level (60%) on the sixth day (65.3%). The test item 3,4-dichlorobenzotrichloride was found to be not ready biodegradable under the conditions applied in a Manometric Respirometry test, having not showed biodegradation during the test period. 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 third day of test period (41.9%).