3,5-dimethylbenzoyl chloride

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:

3 September 1992 - 1 October 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.6 (Degradation: Chemical Oxygen Demand)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3,5-dimethylbenzoyl chloride

EC Number:

413-010-9

EC Name:

3,5-dimethylbenzoyl chloride

Cas Number:

6613-44-1

Molecular formula:

C9H9ClO

IUPAC Name:

3,5-dimethylbenzoyl chloride

Details on test material:

DMBC
Appearance: clear liquid
Storage conditions: in darkness at room temperature

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The aeration stage of the HRC sewage treatment plant treating predominantly domestic sewage.

A sample of activated sewage sludge was collected from the sewage treatment farm on 3 September 1992. The sample was allowed to settle for approximately 30 minutes and the supernatant filtered through Whatman GFA coarse filter paper (first 200 mL discarded). The filtrate was maintained under aeration and used within 24 hours of collection.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

PREPARATION
The test and standard substances were dispersed directly in nutrient medium (see below) without the aid of auxiliary solvents or dispersing agents to give the following series:
a) Non-inoculated nutrient medium.
b) Inoculated nutrient medium.
c) 2 mg/L test substance.
d) 3 mg/L sodium benzoate.
e) 2 mg/L test substance plus 3 mg/L sodium benzoate.

Inoculation of the stock nutrient medium with activated sewage sludge filtrate (at the rate of 1 drop of inoculum per litre) was performed after preparation of series (a) and immediately prior to preparation of series (b), (c), (d) and (e).

STANDARD NUTRIENT MEDIUM
g/L
Solution 1
KH2PO4 8.5
K2HPO4 21.75
Na2HPO4.2H2O 33.3
NH4Cl 1.7

Solution 2
MgSO4.7H2O 22.5

Solution 3
CaCl2 27.5

Solution4
FeCl3.6H2O 0.25

1 mL each of solutions 1 - 4 were added to each litre of aerated reverse osmosis purified and deionised water. The nutrient medium was left at room temperature (approximately 20 °C), under gentle agitation, for approximately 24 hours prior to use.

PROCEDURE
Biochemical Oxygen Demand bottles (280 mL) of darkened glass and fitted with ground glass stoppers were filled by siphon and firmly stoppered to exclude all air bubbles. Sufficient bottles were prepared in each test and control series to allow a single oxygen determination per bottle to be made at 0, 5, 15 and 28 days (duplicate bottles at each sampling time). Sufficient bottles for series (e) were prepared to provide information on inhibitory effects on Days 0 and 28 only.

The bottles were incubated in a water bath at 20 ± 1 °C until removed for measurement of dissolved oxygen concentration by means of a Yellow Springs BOD Meter (Model 59)

EVALUATION OF DATA
Percent biodegradation is determined by comparing the oxygen depletion value with the corresponding Theoretical Oxygen Demand (see below).

The test substance is considered to have had an inhibitory effect on the bacteria if the oxygen demand in series (e) is appreciably less than the sum of oxygen demands in series (c) and (d) at 28 days (i.e. greater than 20 % inhibition).

THEORETICAL OXYGEN DEMAND (ThOD)
The theoretical oxygen demand may be calculated from:

1) Sodium benzoate. C6H6COONa. Mol.wt. = 144
ThOD = [16(14 + 2.5 + 0.5 - 2)] / 144 = 1.67 mgO2/mg

Estimated oxygen depletion for 3 mg/L = 5.01 mgO2/L


2) DMBC. C9H9OCl. Mol.wt. = 168.6
ThOD = [16(18 + 4 - 1)] / 168.6 = 1.99 mgO2/mg

Estimated oxygen depletion for 2 mg/L = 3.99 mgO2/L

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Test performance:

Oxygen depletions in the inoculated and non-inoculated control series were within the prescribed limits:

Non-inoculated control: ≤ 0.3 mgO2/L after 5 days
≤ 0.4 mgO2/L after 28 days
Inoculated control: ≤ 0.5 mgO2/L after 5 days
≤ 0.6 mgO2/L after 28 days

% Degradation

Parameter:

% degradation (O2 consumption)

Value:

80

Sampling time:

28 d

Details on results:

Dissolved oxygen measurements for the test substance and standard substance solutions and the inoculated and non-inoculated blank controls at 0, 5, 15 and 28 days are given in Table 1. Mean oxygen depletion values and percent biodegradation values (% of ThOD NO3) are given in Table 2.

DMBC attained 80 % biodegradation within 28 days. The "pass" level of 60 % was reached within 10 days of exceeding the 10 % level and, consequently, the test substance may be termed as readily biodegradable.

Cultures containing both test and standard substances combined showed an oxygen depletion value 16 % higher than that anticipated on the basis of results from separate cultures. Consequently DMBC is not considered to have had an inhibitory effect on sewage bacteria under the conditions of this test.

BOD5 / COD results

Results with reference substance:

Sodium benzoate attained 71 % biodegradation within 28 days.

Any other information on results incl. tables

Table 1 Dissolved Oxygen Measurement

Culture Medium	Replicate	Day
		0	5	15	28
(a) Nutrient medium without inoculum	R1 R2 Mean	8.90 8.90 8.900	8.72 8.68 8.700	8.68 8.59 8.635	8.50 8.56 8.530
(b) Nutrient medium with inoculum	R1 R2 Mean	8.90 8.90 8.900	8.65 8.55 8.600	8.51 8.54 8.525	8.48 8.32 8.400
(c) Test substance (2 mg/L)	R1 R2 Mean	8.90 8.90 8.900	7.91 7.83 7.870	5.86 5.72 5.790	4.93 5.46 5.195
(d) Standard substance (3 mg/L) Sodium benzoate	R1 R2 Mean	8.90 8.90 8.900	5.37 5.51 5.440	4.86 4.88 4.870	4.86 4.48 4.850
(e) Test substance (2 mg/L) plus standard substance (3 mg/L)	R1 R2 Mean	8.90 8.90 8.900	n/a n/a n/a	n/a n/a n/a	0.62 0.52 0.570

 

Table 2 Mean Oxygen Depletion and Percentage Biodegradation Values

Culture Medium		Day
		5	15	28
(a) Nutrient medium without inoculum	O2 Depletion (mgO2/L)	0.200	0.265	0.370
(b) Nutrient medium with inoculum	O2 Depletion (mgO2/L)	0.300	0.375	0.500
(c) Test substance (2 mg/L)	O2 Depletion (mgO2/L) % Degradation	0.730 18	2.735 69	3.205 80
(d) Standard substance (3 mg/L) Sodium benzoate	O2 Depletion (mgO2/L) % Degradation	3.160 63	3.655 73	3.550 71
(e) Test substance (2 mg/L) plus standard substance (3 mg/L)	O2 Depletion (mgO2/L)	n/a	n/a	7.830

(i) Test and standard substances

Oxygen depletion = (a - c or d) - (a - b) from Table 1

(ii) Inoculated and non-inoculated nutrient medium

Oxygen depletion = initial O2 concentration - measured value

(iii) Inhibition check (test substance plus sodium benzoate)

Oxygen depletion = (a - e) - (a - b

(iv) % Degradation = (Oxygen depletion / (conc. of substance x ThOD NO3)) x 100

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

DMBC may be termed as readily biodegradable.

Executive summary:

A study was performed to assess the ready biodegradability of DMBC in accordance with OECD Guideline for Testing of Chemicals No. 301D and EEC Methods for the Determination of Ecotoxicity, EEC Directive 67/548 Annex V Part C.6 (841449/EEC).

 

Sealed bottles containing the test substance and inorganic nutrient medium were inoculated with activated sewage sludge bacteria and incubated for up to 28 days at 20 ± 1 °C. On Days 0, 5, 15 and 28 duplicate bottles were taken and dissolved oxygen measurements were performed electrochemically. Percentage biodegradation values were calculated by comparing the extent of oxygen depletion with the Theoretical Oxygen Demand (3.99 mgO₂/L). Additional bottles, containing both the test substance and a readily biodegradable standard substance were prepared in order to provide additional information on the inhibitory effect of the test substance.

 

DMBC attained 80 % biodegradation after 28 days. The pass level of 60 % was achieved within 10 days of passing the 10% level. DMBC was not found to be inhibitory to activated sewage sludge bacteria under the conditions of this test.

Therefore, DMBC may be termed as readily biodegradable.