Benzene, 1,2,3-trichloro-5-(trichloromethyl)-

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

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. QA statement)

Remarks:

Italian Ministero della Salute

Test material

Test material information

Constituent 1

Reference substance name:

1,2,3-trichloro-5-(trichloromethyl)benzene

EC Number:

700-556-0

Cas Number:

66682-07-3

Molecular formula:

C7H2Cl6

IUPAC Name:

1,2,3-trichloro-5-(trichloromethyl)benzene

Details on test material:

- Name of test material : 3,4,5-trichloro-benzotrichloride
- Substance type: intermediate for synthesis
- Analytical purity: 97.7%
- Purity test date: 03 June 2010
- Lot/batch No.: 06/2010
- Expiration date of the lot/batch: June 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: the test item is kept in glass vial. Kept away from incompatible materials (oxidisers, not noble metals, amines and alcohols). Kept 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:

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 water was 7.22, so adjustment was not needed.

Since the low water solubility of test item, the test item solutions (100 mg/L) were prepared by directly weighting into the bottles, 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.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Parameter:

% degradation (CO2 evolution)

Remarks:

classic manometric method (consumption)

Value:

9.4

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 3,4,5-trichloro-benzotrichloride was calculated as 0.64 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 a maximum value of biodegradation of 9.4% at the end of the test period (mean value between two replicates). 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 3,4,5-trichloro-benzotrichloridein a Manometric Respirometry test.

Day of measurement	Reference item BOD	Reference item % biodegradation	Test item BOD	Test item % biodegradation
1	0.20	12.0	-0.02	0.0*
2	0.41	24.6	-0.02	0.0*
3	0.63	37.8	-0.02	0.0*
4	0.76	45.3	-0.03	0.0*
5	0.97	57.9	0.01	1.6
6	1.09	65.4	-0.05	0.0*
7	1.18	70.8	-0.05	0.0*
8	1.21	72.6	-0.05	0.0*
9	n.d.	n.d.	n.d.	n.d.
10	n.d.	n.d.	n.d.	n.d.
11	1.29	77.1	-0.04	0.0*
12	1.31	78.3	-0.06	0.0*
13	1.41	84.3	-0.06	9.4
14	1.37	82.2	-0.02	2.3
15	1.38	82.8	-0.15	0.0*
16	n.d.	n.d.	n.d.	n.d.
17	n.d.	n.d.	n.d.	n.d.
18	1.47	87.9	-0.02	0.0*
19	1.53	91.8	-0.02	0.0*
20	1.54	92.4	-0.02	0.0*
21	1.53	91.5	-0.13	0.0*
22	1.55	92.7	-0.16	0.0*
23	n.d.	n.d.	n.d.	n.d.
24	n.d.	n.d.	n.d.	n.d.
25	1.59	95.4	-0.03	0.0*
26	1.59	95.1	-0.05	0.0*
27	1.60	95.7	-0.06	0.0*
28	1.61	96.3	-0.08	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

Interpretation of results:

other: not ready biodegradable

Conclusions:

The test item 3,4,5-trichloro-benzotrichloride has showed to be not ready biodegradable under the conditions applied in a Manometric Respirometry test, having not reached the pass level (60% biodegradation), but showing a maximum value of degradation of 9.4% 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 3,4,5-trichloro-benzotrichloride 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 ThOD_NH3values were calculated to be equal to 1.67 mg O₂/mg for reference item and 0.64 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 (65.4%). The test item 3,4,5-trichloro-benzotrichloride was found to be not ready biodegradable under the conditions applied in a Manometric Respirometry test, having showed a maximum biodegradation of 9.4% during the test period (mean value between two replicates). 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 (25.2%).