4,4',4''-(ethan-1,1,1-triyl)triphenol

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

The study was conducted according to the test guidelines in effect at the time of study conduct.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

The study was conducted according to the test guidelines in effect at the time of study conduct.

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): The aeration stage of the HRC Limited sewage treatment plant treating predominantly domestic sewage
- Preparation of inoculum for exposure: The sample was allowed to settle and the supernatant filtered through Whatman GFA filter paper (first 250 mL discarded).
- Pretreatment: not reported
- Concentration of sludge: One drop of activated sludge filtrate per liter
- Initial cell/biomass concentration: not reported

Duration of test (contact time):

0 - 28 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium:
Standard nutrient medium
Solution 1
8.5 g/L KH2PO4
21.75 g/L K2HPO4
33.3 g/L Na2HPO4.2H2O
1.7 g/L NH4Cl
Solution 2
22,5 g/L MgSO4.7H2O
Solution 3
27.5 g/L CaCl2
Solution 4
0.25 g/L FeCl3.6H2O

1 mL of each of solutions 1-4 was added to each liter of aerated reverse osmosis purified and deionized water. The "dilution water" was left at room temperature (approximately 20ºC), under gentle agitation, for 24 hours prior to use.
- Solubilising agent (type and concentration if used):
The test substance was dissolved in diethyl ether to give a stock solution of 560 mg/10 mL. Six 10 µL aliquots of stock solution were placed on Whatman GFA paper and the solvent allowed to evaporate to dryness. One piece of paper was placed in each test bottle prior to filling with inoculated medium. Filter paper blanks were prepared in the same matter using solvent only.
- Test temperature: 20 ± 1ºC
- pH: not reported
- pH adjusted: not reported
- CEC (meq/100 g): not reported
- Aeration of dilution water: gently agitated for 24 hours before use
- Suspended solids concentration: not reported
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 280 mL BOD bottles (darkened glass) with ground glass stoppers
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: BOD bottles were filled, by siphon, and firmly stoppered to exclude all air bubbles.
- Measuring equipment: Yellow Springs BOD Probe (Model 54)

SAMPLING
- Sampling frequency: 0, 5, 15, and 28 days
- Sampling method: Dissolved oxygen concentrations by means of a Yellow Springs BOD Probe (Model 54).

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Other: non-inoculated dilution water
-Other: inoculated dilution water and filter paper

Reference substance:

benzoic acid, sodium salt

Remarks:

3 mg/L

Reference substance:

aniline

Remarks:

2 mg/L

Parameter:

% degradation (O2 consumption)

Value:

8

Sampling time:

28 d

Remarks on result:

other: Not readily biodegradable

Details on results:

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

Results with reference substance:

Sodium benzoate attained 93% biodegradation within 28 days. Aniline attained 66% biodegradation within 28 days.

Oxygen depletion (28 days): 0.400 mg O₂/L

Theoretical oxygen demand (ThOD): 4.82 mgO₂/L

Table 1: Dissolved oxygen measurements (mg O₂/L)

 

Culture medium		Day
		0	5	15	28
(a) Dilution water without inoculum	R1	8.90	8.80	8.65	8.45
	R2	8.90	8.75	8.75	8.50
	Mean	8.900	8.775	8.700	8.475
(b) Dilution water with inoculum	R1	8.90	8.75	8.60	8.40
	R2	8.90	8.70	8.60	8.45
	Mean	8.900	8.725	8.600	8.425
(c) Dilution water with inoculum and filter paper	R1	8.90	8.55	8.55	8.35
	R2	8.90	8.75	8.65	8.40
	Mean	8.900	8.650	8.600	8.375
(d) Test substance (2 mg/l) plus filter paper	R1	8.90	8.60	8.20	8.00
	R2	8.90	8.45	8.15	7.95
	Mean	8.900	8.525	8.175	7.975
(e) Standard substance (3 mg/l) Sodium benzoate	R1	8.90	4.95	3.95	3.75
	R2	8.90	4.90	4.05	3.80
	Mean	8.900	4.925	4.000	3.775
(f) Standard substance (2 mg/l) Aniline plus filter paper	R1	8.90	7.20	5.25	4.40
	R2	8.90	7.50	5.35	4.25
	Mean	8.900	7.350	5.300	4.325

 R1 & R2 Replicates 1 and 2

Table 2: Mean oxygen depletion and percentage biodegradation values

 

Culture medium		Day
		5	15	28
(a) Dilution water without inoculum	O2Depletion (mg O2/L)	0.125	0.200	0.425
(b) Dilution water with inoculum	O2depletion (mg O2/L)	0.175	0.300	0.475
(c) Dilution water with inoculum and filter paper	O2depletion (mg O2/L)	0.250	0.300	0.525
(d) Test substance (2 mg/l) plus filter paper	O2depletion (mg O2/L)	0.125	0.425	0.400
	% degradation	3	9	8
(e) Standard substance (3 mg/L) Sodium benzoate	O2depletion (mg O2/L)	3.800	4.600	4.650
	% degradation	76	92	93
(f) Standard substance (2 mg/L) Aniline plus filter paper	O2depletion (mg O2/L)	1.300	3.300	4.050
	% degradation	21	53	66

 

(i)      Test substance

            Oxygen depletion = (a – d) – (a – c) from Table 1

 

(ii)        Standard substance   (sodium benzoate)   

            Oxygen depletion = (a – e) – (a – b) from Table 1

 

(iii)     Standard substance (aniline)         

           Oxygen depletion = (a – f) – (a – c) from Table 1

 

(iv)      Inoculated and non-inoculated dilution water (plus filter paper blank)       

            Oxygen depletion = initial O₂concentration – measured value

 

(v)   % Degradation = Oxygen depletion/(conc. of substance x ThOD NO₃) x100

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the conditions of this study, the test substance cannot be termed as readily biodegradable.

Executive summary:

The test substance was assessed for ready biodegradability using the closed bottle test according to OECD Guideline No. 301D and EEC Directive 67/548 Annex V C.6. Incubations were performed with activated sludge, and dissolved oxygen was measured at 0, 5, 15, and 28 days. Both inoculated and non-inoculated blank controls were used, and sodium benzoate and aniline were used as reference substances. The test substance attained only 8% biodegradation after 28 days and cannot, therefore, be termed as readily biodegradable. Sodium benzoate and aniline attained 93% and 66% biodegradation, respectively, within 28 days. Oxygen depletion in the inoculated and non-inoculated control series were essentially within the prescribed limits. The limit was slightly exceeded in the non-inoculated dilution water on Day 28, but this is not considered to have a significant influence on the results or conclusions of the test.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test), except that the activated sludge was preadapted for 14 days with the substance to assess inherent biodegradation.

Deviations:

yes

Remarks:

Modified to include a pre-adaptation phase, rendering the study as an inherent biodegradation test

Qualifier:

equivalent or similar to guideline

Guideline:

other: EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test) except that the activated sludge was preadapted for 14 days with the substance to assess inherent biodegradation.

Deviations:

yes

Remarks:

Modified to include a pre-adaptation phase, rendering the study as an inherent biodegradation test

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

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

- Preparation of inoculum for exposure: The pre-adapted inoculum was thoroughly washed using nutrient media. A sub-sample of approximately 500 mL was homogenised for 2 minutes with a mechanical blender, allowed to settle for 30 minutes and the supernatant decanted to provide the inoculum for both test and control cultures.

- Pretreatment: An activated sewage sludge sample was pre-adapted to the test substance under aerobic conditions at room temperature for 14 days before study start. Three times per week, the activated sewage sludge was allowed to settle for 30 minutes and 1 litre of the supernatant decanted from the initial 2 litre volume. The sludge was then topped up with 50 mL of synthetic sewage and 950 mL of a saturated solution of the test substance (nominal concentration 50 mg/L, previously prepared by stirring in deionised water for 48 hours).

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium:
Standard nutrient medium

Solution (a):
8.50 g/L KH2PO4
21.75 g/L K2HPO4
33.40 g/L Na2HPO4.2H2O
0.50 g/L NH4Cl

Solution (b):
27.50 g/L CaCl2 (or 36.40 g/L CaCl2.2H2O)

Solution (c):
22.50 g/L MgSO4.7H2O

Solution (d):
0.25 g/L FeCl3.6H2O (If stored, one drop of conc. HCl is added per litre of stock solution)

10 mL of solution (a) was mixed with 800 mL of reverse osmosis-purified water, 1 mL each of solutions (b-d) were added and the volume made up to 1 litre.

- Test temperature: 22 ± 2ºC
- pH: Solution (a) pH = 7.4
- pH adjusted: no
- Aeration of dilution water: Reverse osmosis-purified water was aerated vigorously for approximately 20 minutes and allowed to stand with only gentle large-bubble aeration for about 20 hours at approximately 20ºC to give a dissolved oxygen concentration of approximately 9 mg O2/L.
- Suspended solids concentration: not reported
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 5 litre fermentation vessels
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Cultures were continuously mixed by magnetic stirrer and aerated with CO2-free air via sintered glass airstone at the rate of approximately 10 mL/min.
- Measuring equipment: Inorganic Carbon channel of an Ionics TC/TOC Analyser Model 555 incorporating a Horriba PIR-2000 infrared gas analyser with purified nitrogen as the carrier gas regulated to 50 psi at a flow rate of 100 cc/min.
- Test performed in open system: No; performed in a sealed system that had CO2-free air passed through it
- Details of trap for CO2 and volatile organics if used: 2 x 250 mL Dreschel bottles filled with 200 mL 0.05N NaOH. The CO2 absorbing solutions were prepared using degassed water (ion exchange and reverse osmosis-treated water, boiled for approximately 1 hour and then cooled).
NOTE:

SAMPLING
- Sampling frequency: Days 0, 4, 7, 11, 14, 18, 21, 25, and 28
- Sampling method: Air from each culture vessel passed through 2 x 250 mL Dreschel bottles filled with 200 mL 0.05N NaOH. 2 mL samples were taken from the CO2 absorbers.
- Sample storage before analysis: analysis was done on the day of collection except that on Day 28, 1 mL of HCl was added to each culture vessel to halt biodegradation and to drive off any residual CO2 from solution. Aeration was continued for a further 24 hours, and the first and second absorbers sampled on Day 29.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

Remarks on result:

other: not inherently biodegradable

Results with reference substance:

Sodium benzoate attained 63% biodegradation within 28 days, confirming the suitability of the inoculum and the culture conditions.

Measured inorganic carbon values (i.e. evolved CO2) from the blank, test, and standard substance cultures are given in Table 1 below. The percent biodegradation values are given in Table 2 below.

                                                       Table 1: CO₂evolution

Time (days)	Total carbon trapped (mg)
	Test substance		Standard substance	Blank
	20 mg/L R1	20 mg/L R2	28 mg/L
4  (1^stabsorber)	2.5	2.6	19.1	1.7
7  (1^stabsorber)	3.5	4.0	28.7	3.8
11 (1^stabsorber)	5.6	3.5	29.9	4.0
14 (1^stabsorber)	4.8	4.6	40.7	8.7
18 (1^stabsorber)	7.2	5.6	45.9	17.0
21 (1^stabsorber)	8.8	7.5	44.3	11.6
25 (1^stabsorber)	9.2	9.6	45.3	12.1
28*(1^stabsorber)	8.7	8.8	37.6	10.3
28*(2^ndabsorber)	-0.2	-0.1	3.9	0.2

 

*Biodegradation halted on Day 28 and the absorbers sampled on Day 29

R₁& R₂= Replicates 1 and 2

 

 

                                            Table 2: Percent biodegradation 

Time (days)	%Biodegradation
	Test substance		Standard substance
	20 mg/L R1	20 mg/L R2	28 mg/L
4	2	2	36
7	-1	1	51
11	3	-1	53
14	-8	-9	66
18	-21*	-24*	59*
21	-6	-9	67
25	-6	-5	68
28	-4	-4	64

*Anomalous value due to high blank on Day 18

 

% Biodegradation = (C_t or C_s– C_b/T_c) x 100

 

Where C_t= mg carbon trapped (test substance)

           C_s= mg carbon trapped (standard substance)

           C_b= mg carbon trapped (blank)

           T_c= theoretical amount of carbon added to system (i.e. 47.04 mg for the test substance and 48.69 mg for the standard substance)

 

(Values calculated using non-rounded data)

Validity criteria fulfilled:

yes

Interpretation of results:

not inherently biodegradable

Conclusions:

The test substance attained 0% biodegradation after 28 days and cannot be termed inherently biodegradable.

Executive summary:

The study was performed to assess the inherent biodegradability of the test substance. The method was based upon the EEC Methods for Determination of Ecotoxicity Annex to Directive 92/69/EEC (O.J. No. L383A, 29.12.92) Part C, Method 4-C "Determination of ready biodegradability, CO₂Evolution Test" and OECD Guideline for Testing of Chemicals No. 301 B "Ready Biodegradability: CO₂Evolution Test" modified to include a pre-adaption phase rendering the study as an inherent biodegradation test. Closed vessels containing the test substance dispersed in inorganic nutrient medium were inoculated with pre-adapted activated sewage sludge bacteria and the cultures incubated at 22 ± 2ºC for 28 days under continuous aeration with CO₂-free air. Carbon dioxide produced in the vessels was trapped by venting the exhaust air through sodium hydroxide solution. On Days 0, 4, 7, 11, 14, 18, 21, 25 and 28, samples were removed from the CO₂traps and the concentration of inorganic carbon determined using a carbon analyser. Percentage biodegradation values were calculated by comparing the amount of carbon trapped with the amount of carbon added to the vessel at the start of the study. The test substance attained 0% biodegradation at 20 mg/L after 28 days. Therefore, it may not be termed as inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The substance was not significantly biodegraded in ready or inherent biodegradation studies. In a ready biodegradation test, degradation was 8% after 28 days. In the inherent biodegradation test 0% mineralized in 28 days.