Phosphonium, tetraphenyl-, salt with 4,4'-sulfonylbis[phenol], compd. with 4,4'-sulfonylbis[phenol] (2:2:1)

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17/02/2004 - 19/3/2004

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch number UK030622B

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained on 16 February 2004 from the aeration stage of the Severn Trent Water Pie sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of 21°C and was used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the activated sewage sludge by suction through pre-weighed GF/A filter paper using a Buchner funnel. The filter paper was then dried in an oven at approximately 105°C for at least I hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids was equal to 2.8 g/1 prior to use.

Duration of test (contact time):

28 d

Initial conc.:

14.2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

The following test preparations were prepared and inoculated in 5 litre glass culture vessels each
containing 3 litres of solution:
a) A control, in duplicate, consisting of inoculated culture medium.
b) The standard material (sodium benzoate), in duplicate, in inoculated culture medium to give a
final concentration of 10 mg carbon/I.
c) The test material, in duplicate, in inoculated culture medium to give a final concentration of IO mg carbon/I.
d) The test material plus the standard material in inoculated culture medium to give a final concentration of 20 mg carbon/I to act as a toxicity control ( one vessel only).
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids ( ss )/1. The study was carried out in a temperature controlled room at 21 °C, in darkness.
Approximately 24 hours prior to addition of the test and standard materials the vessels were filled with 2400 ml of culture medium and 32.1 ml of inoculum and aerated overnight. On Day 0 the test and standard materials were added and the volume in all the vessels adjusted to 3 litres by the addition of culture medium.
The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approximately 40 ml/minute and stirred continuously by magnetic stirrer.
The CO2-free air was produced by passing compressed air through a glass column containing self-­indicating soda lime (Carbosorb®) granules.
The CO2 produced by degradation was collected in two 500 ml Dreschel bottles containing 350 ml of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water.

CO2 analysis
Samples (2 ml) were taken from the first CO2 absorber vessel on Days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, 28 and 29. The second absorber vessel was sampled on Days 0 and 29.
The samples taken on Days 0, 1, 2, 3, 6, 8, 10, 14, 16, 20, 22, 24, 27, 28 and 29 were analysed for CO2 immediately. The samples taken on Days 12 and 18 were stored at approximately -20°C. However, these samples were not analysed for CO2 as the results obtained from previous and subsequent analyses showed that the level of degradation of the test material did not significantly increase during this time and therefore additional analyses were considered to be unnecessary.
On Day 28, 1 ml of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.
The samples were analysed for CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyser and an Ionics 1555B TOC analyser. Samples (300 or 40 µl) were injected into the IC (Inorganic Carbon) channel of the TOC analyser. Inorganic carbon analysis occurs by means of the conversion of an aqueous sample by orthophosphoric acid using zero grade air or nitrogen ( oxygen free) as the carrier gas. Calibration was by standard solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.

Dissolved organic carbon (DOC) analysis
Samples (20 ml) were removed from the test material and toxicity control vessels on Day O prior to the addition of the test material in order to calculate the Inorganic Carbon/Total Carbon (IC/TC) ratio in the test media. The samples were centrifuged (3500 rpm, 15 minutes) prior to DOC analysis.
DOC analysis of the test material dispersions after dosing was not possible due to the insoluble nature of the test material in water.
On Days O and 28 samples (20 ml) were removed from the control and standard material vessels and centrifuged (3500 rpm, 15 minutes) prior to DOC analysis.
The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser. Samples (27 or 13 µl) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. Total carbon analysis is carried out at 680°C using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using standard solutions of potassium hydrogen phthalate (C8HsKO4) and sodium carbonate (Na2CO3) in deionised water. Each analysis was carried out in triplicate.

pH measurements
The pH of the test preparations was determined on Day 28, prior to acidification with hydrochloric acid, using a WTW pH 320 pH meter.

Calculation of carbon content
The theoretical amount of carbon present in the test material for SBCAT-03 (Cs4H6sO12S3P2) was calculated as follows:

No of C atoms * mol wt of C / mol wt of test material * 100%
= 84*12.011 / 1427.6 * 100 = 70.67%

Thus for a concentration of 10 mg C/1 (a total of 42.6 mg) the total organic carbon present was 30mg C.
The theoretical amount of carbon present in the standard material, sodium benzoate (C6H5COONa) was calculated as follows:

No of C atoms * mol wt of C / mol wt of sodium benzoate* 100%
= 7 * 12.011 / 144.11 * 100 = 58.34%
Thus for a 10 mg C/1 test concentration ( a total of 51 .4 mg) the total organic carbon present for sodium benzoate was 30 mg C.

Percentage degradation
The percentage degradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values given in Table 1 in the following equation:
The values of Replicates R1 and R2 are meaned for the control, test and standard materials before substitution in the equation.

%ThCO2 (=%degradation) = mgl C in test flask - mg lC in control / mg TOC as test material * 100%
The percentage degradation from the results of the DOC analysis, see Table 4, is calculated from the equation below. Replicate values are corrected for the mean control value prior to calculation of percentage degradation.

Percentage degradation = (1- mg DOC in test flask on Day 28 / mg DOC in test flask on Day 0) *100%

The total CO2 evolution in the control vessels at the end of the test is calculated from the equation
below. The inorganic carbon values for Replicates R1 and R2 on Day 28 are meaned before substitution into the equation.

Total CO2 evolution = mg lC in control * 100/ %C of CO2 * 1/ test volume

=mg lC in control * 100/27.29 * 1/3

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

3

Sampling time:

28 d

Details on results:

Inorganic carbon values for the test material, standard material, toxicity control and control vessels at each analysis occasion are given in "any other information on results". Percentage biodegradation values of the test and standard materials and the toxicity control are given in Table 2 and the biodegradation curves are presented in "illustrations". Total and Inorganic Carbon values in the culture vessels on Day 0 are given in "any other information on results", and the results of the Dissolved Organic Carbon analyses performed on Days 0 and 28 are given in "any other information on results". The pH values of the test preparations on Day 28 are given in "any other information on results". Observations on the test preparations throughout the test period are given in "any other information on results".
The total CO2 evolution in the control vessels on Day 28 was 33.68 mg/I and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC/TC ratio of the test material suspension in the mineral medium at the start of the test (see Table 3) was below 5% and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation criterion given in the OECD Test Guidelines.
The test material attained 3% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions ofOECD Guideline No 301B.
Variation in degradation rates for the test material on different sampling days was considered to be due to variation in the CO2 production rates between the replicate control and replicate test vessels.
The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels with the exception of standard material Replicate R2. These increases were considered to be due to CO2 present in solution being driven off by the addition of hydrochloric acid on Day 28 and resulted in an increase in the percentage degradation value for the test material from 3% on Day 28 to 7% on Day 29.
The toxicity control attained 35% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study. The increase in inorganic carbon in the first absorber vessel on Day 29 resulted in an increase in the percentage degradation value for the toxicity control from 35% on Day 28 to 39% on Day 29.
Sodium benzoate attained 85% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The increase in inorganic carbon in the standard material Replicate R1 first absorber vessel on Day 29 combined with the increases in inorganic carbon in both control replicate first absorber vessels on Day 29 resulted in no change in the percentage degradation value for the standard material from Day 28 to Day 29.
Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.
Analysis of the test media taken from the standard material culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), see Table 4, gave percentage degradation values of 100% and 101 % respectively for Replicates R1 and R2. The degradation rates calculated from the results of the DOC analyses were higher than those calculated from inorganic carbon analysis. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation, and hence CO2 evolution occurring. Degradation values in excess of 100% were considered to be due to sampling/analytical variation.
Observations made throughout the test period (see "any other information on results"), showed that the contents of the control vessels were light brown dispersions and the standard material vessels were light brown dispersions with no undissolved standard material visible. On Day O the test material vessels were light brown dispersions with white particles of test material visible throughout and the toxicity control vessel was a light brown dispersion with white particles of test material visible throughout with no undissolved standard material visible. However, on Days 6, 13, 20 and 27 the test material vessels were light brown dispersions with no undissolved test material visible and the toxicity control vessel was a light brown dispersion with no undissolved test material or standard material visible.

Results with reference substance:

The toxicity control attained 35% degradation after 28 days thereby confirming that the test
material was not toxic to the sewage treatment micro-organisms used in the study. The increase
in inorganic carbon in the first absorber vessel on Day 29 resulted in an increase in the percentage
degradation value for the toxicity control from 35% on Day 28 to 39% on Day 29.

Sodium benzoate attained 85% degradation after 28 days thereby confirming the suitability of the
inoculum and test conditions. The increase in inorganic carbon in the standard material Replicate
R1 first absorber vessel on Day 29 combined with the increases in inorganic carbon in both
control replicate first absorber vessels on Day 29 resulted in no change in the percentage
degradation value for the standard material from Day 28 to Day 29.
Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed
that no significant carry-over of CO2 into the second absorber vessels occurred.

Inorganic Carbon Values on Each Analysis Occasion

Day	Control (mg IC)				Sodium Benzoate (mg IC)				Test Material (mg IC)				Test Material plus Sodium Benzoate Toxicity Control (mg IC)
	R1		R2		R1		R2		R1		R2		R1
	Abs1	Abs2	Abs1	Abs2	Abs1	Abs2	Abs1	Abs2	Abs1	Abs2	Abs1	Abs2	Abs1	Abs2
0	1.40	1.40	1.40	1.17	1.28	1.17	1.05	1.17	1.28	1.28	1.28	1.28	1.17	1.28
1	2.78	-	5.33	-	8.12	-	9.74	-	4.76	-	5.11	-	5.11	-
2	5.54	-	7.84	-	17.07	-	20.99	-	9.34	-	7.84	-	11.07	-
3	6.65	-	11.81	-	21.33	-	23.97	-	10.89	-	10.43	-	14.33	-
6	6.95	-	12.31	-	25.99	-	31.23	-	13.23	-	12.31	-	16.07	-
8	13.15	-	13.15	-	28.90	-	37.06	-	16.77	-	16.32	-	27.20	-
10	14.98	-	16.56	-	36.95	-	36.95	-	18.48	-	18.59	-	31.10	-
14	19.04	-	18.26	-	37.41	-	40.97	-	19.26	-	20.26	-	37.97	-
16	20.81	-	21.25	-	39.73	-	43.27	-	22.02	-	22.91	-	43.49	-
20	19.57	-	20.34	-	44.17	-	42.97	-	21.10	-	22.74	-	46.03	-
22	22.06	-	21.73	-	44.34	-	48.57	-	23.58	-	26.08	-	46.51	-
24	22.03	-	22.46	-	44.82	-	47.20	-	23.00	-	26.14	-	45.79	-
27	23.93	-	24.58	-	50.34	-	51.52	-	24.26	-	27.91	-	47.23	-
28	27.73	-	27.41	-	52.05	-	54.19	-	26.03	-	30.72	-	48.85	-
29	27.98	2.90	27.88	2.55	53.64	2.67	53.11	2.78	27.13	2.78	32.54	2.78	50.88	3.13

 

Percentage Biodegradation Values

Day	% Degradation Sodium Benzoate	% Degradation Test Material	% Degradation Test Material plus Sodium Benzoate Toxicity Control
0	0	0	0
1	16	3	2
2	41	6	7
3	45	5	9
6	63	10	11
8	66	11	23
10	71	9	26
14	68	4	32
16	68	5	37
20	79	7	43
22	82	10	41
24	79	8	39
27	89	6	38
28	85	3	35
29^*	85	7	39

* Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2

 

Total and Inorganic Carbon Values in the Culture Vessels on Day O

Test vessel	Total Carbon^*(mg/l)	Inorganic Carbon^* (mg/l)	IC/TC Ratio (%)
Sodium Benzoate 10mg C/l R1	7.76	-2.24	0
Sodium Benzoate 10mg C/l R2	8.04	-2.25	0
Test Material 10 mg C/l R1	11.28^**	0.28	2
Test Material 10 mg C/l R2	10.61^**	-0.01	0
Test Material plus Sodium Benzoate Toxicity Control 20 mg C/1	20.00^**	0.17	1

R₁ - R₂ - Replicates 1 and 2

*Corrected for control values. Negative values are due to measured concentration values being less than control values

^**Total carbon value given is the sum of the TC value obtained from analysis and the nominal TC contribution of the test material and sodium benzoate where applicable

 

Dissolved Organic Carbon (DOC) Values in the Culture Vessels on
Days O and 28

DOC^*Concentration
Test vessel	Day 0		Day 28
	mg C/l	% of Nominal Carbon Content	mg C/l	% of Initial Carbon Concentration	% Degradation
Sodium Benzoate 10 mg C/1 R1	10.00	100	0.04	0	100
Sodium Benzoate 10 mg C/1 R2	10.29	103	-0.08	-1	101

R1 - R2 - Replicates 1 and 2
^*Corrected for control values. Negative values are due to measured concentration values being less than control values

 

pH Values of the Test Preparations on Day 28

Test vessel	pH
Control R1	7.7
Control R2	7.7
Sodium Benzoate 10 mg C/l R1	7.8
Sodium Benzoate 10 mg C/l R2	7.8
Test Material 10 mg C/l R1	7.8
Test Material 10 mg C/l R2	7.8
Test Material plus Sodium Benzoate Toxicity Control 20 mg C/l	7.8

R1 -R2 = Replicates 1 and 2

Observations on the Test Preparations Throughout the Test Period

Test vessel	Observations on Test Preparations
	Day 0	Day 6	Day 13	Day 20	Day 27
Control R1	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
Control R2	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
Standard Material R1	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible
Standard Material R2	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible
Test Material R1	Light brown dispersion, white particles of test material visible throughout	Light brown dispersion, no undissolved test material visible	Light brown dispersion, no undissolved test material visible	Light brown dispersion, no undissolved test material visible	Light brown dispersion, no undissolved test material visible
Test Material R2	Light brown dispersion, white particles of test material visible throughout	Light brown dispersion, no undissolved test material visible	Light brown dispersion, no undissolved test material visible	Light brown dispersion, no undissolved test material visible	Light brown dispersion, no undissolved test material visible
Toxicity control	Light brown dispersion, white particles of test material visible throughout, no undissolved standard material visible	Light brown dispersion, no undissolved test material or standard material visible	Light brown dispersion, no undissolved test material or standard material visible	Light brown dispersion, no undissolved test material or standard material visible	Light brown dispersion, no undissolved test material or standard material visible

 

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test material attained 3% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Executive summary:

Introduction. A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC), and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 Paragraph (m).
Methods. The test material, at a concentration of 10 mg C/1, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at 21 °C for 28 days.
The degradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.
Results. The test material attained 3% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Description of key information

Introduction. A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC), and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 Paragraph (m).
Methods. The test material, at a concentration of 10 mg C/1, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at 21 °C for 28 days.
The degradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.
Results. The test material attained 3% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater