Santicizer 278®

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:

15 May 2018 - 13 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guidelines for Testing of Chemicals (1992) No. 301B "Ready
Biodegradability; CO2 Evolution Test"

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Version / remarks:

Method C.4-C of Commission Regulation (EC) No. 440/2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110
(Paragraph (m))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test Item: Benzyl 3-isobutyryloxy-1-isopropyl-2,2 dimethylpropyl phthalate
Alternative Name: Sancticizer® 278
CAS Number: 16883-83-3
Lot Number: 3057
Purity: 98.652%
Physical state/Appearance: Pale yellow viscous liquid
Expiry Date: 16 January 2020
Storage Conditions: Room temperature in the dark

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Inoculum
A mixed population of activated sewage sludge micro-organisms was obtained on 14 May 2018 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Preparation of inoculum
The activated sewage sludge sample was washed twice by settlement and re-suspension in mineral medium to remove any excessive amounts of Dissolved Organic Carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and 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 washed activated sewage sludge by suction through pre-weighed GF/A filter paper (Rinsed 3 times with 20 mL deionized reverse osmosis water prior to drying in an oven) using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 ºC for at least 1-Hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 2.4 g/L prior to use.

Duration of test (contact time):

28 d

Initial conc.:

10 mg/L

Based on:

other: carbon

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Medium: The mineral medium used in this study was that recommended in the OECD Guidelines. The mineral medium used for the test contained less than 1 mg/L Total Organic Carbon
(TOC).

Preliminary Solubility Work: Information provided by the Sponsor indicated that the test item was insoluble in water. Therefore preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation.

Test Item Preparation: The test item was dispersed directly in mineral medium. An amount of test item (42.0 mg) was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) prior to dispersal in inoculated mineral medium. The volume was adjusted to 3 liters to give a final concentration of 14.0 mg/L, equivalent to 10 mg carbon/L. A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.

Reference Item Preparation: A reference item, sodium benzoate (C6H5COONa), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium prior to the volume being adjusted to 3 liters to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

Toxicity Control: A toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test. An amount of test item (42.0 mg) was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) prior to dispersal in inoculated mineral medium. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 liters to give a final concentration of 14.0 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.

Preparation of Test System: The following test preparations were prepared and inoculated in 5 liter test culture vessels each containing 3 liters of solution:
a) An inoculated control, in duplicate, consisting of inoculated mineral medium
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
c) The test item, in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
d) The test item plus the reference item in inoculated mineral to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).

Data from the inoculum control and procedure control vessels was shared with similar concurrent studies. Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg
suspended solids (ss)/L. The test was carried out in a temperature controlled room at temperatures of between 20 and 25 °C for 28 days, in darkness.

Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 37.5 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter. The pH was adjusted to pH 7.4 ±0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all the vessels being adjusted to 3 liters by the addition of mineral medium which had been purged overnight with CO2-free air. The inoculum control vessels were prepared in a similar manner without the addition of test item or reference item.

The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/minute per vessel 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 water.

Assessments

Observations: The appearance of the test preparations was recorded on Days 0, 7, 14, 21 and 28.

pH Measurements: The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification with hydrochloric acid, using a Hach HQ40d Flexi handheld meter.

Inorganic Carbon Analysis: Samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21,28 and 29. The second absorber vessels were sampled on Days 0 and 29. All samples were analyzed for Inorganic Carbon (IC) immediately. The remainder of all samples with the exception of the Day 0 samples were frozen for further analysis if required. 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 analyzed for IC using a Shimadzu TOC-LCSH TOC analyzer. Samples (135 µL) were injected into the IC channel of the TOC analyzer. IC analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid or 2M HCl using zero grade air as the carrier gas. Calibration was by reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in at least triplicate with three replicates being used in the calculation/reported.

Inorganic Carbon/Total Carbon Ratio: Samples (30 mL) were removed from the test item vessels on Day 0 prior to the addition of the test item in order to calculate the IC content in the test media. The samples were filtered through 0.45 µm Gelman AcroCap filters (first approximate 5 mL discarded in order to pre-condition the filter) prior to DOC analysis. Samples (30 mL) were also removed from the inoculum control vessels on Day 0 and filtered through 0.45 µm Gelman AcroCap filters (first approximate 5 mL discarded in order to pre-condition the filter) prior to DOC analysis.

Inorganic Carbon/Total Carbon analysis of the test item dispersions after dosing was not possible due to the insoluble nature of the test item in water. The samples were analyzed for IC and Total Carbon (TC) using a Shimadzu TOC-VCSH TOC analyzer and a Shimadzu TOC-VCPH TOC Analyzer. Samples (50 µL) were injected into the TC and IC channels of the TOC analyzer. TC analysis is carried out at 680 °C using a platinum based catalyst and zero grade air as the carrier gas. IC analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using reference solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionized water. Each analysis was carried out in at least triplicate with three replicates being used in the calculation/reported.

Validation Criteria: The results of the degradation test are considered valid if in the same test the reference item yields ≥60% degradation (in a 10-Day window) by Day 14.

The test item may be considered to be readily biodegradable if ≥60% degradation is attained within 28 days. This level of degradation must be reached within 10 days of biodegradation exceeding 10%.
The toxicity control (test item and sodium benzoate) should attain ≥25% degradation by Day 14 for the test item to be considered as non-inhibitory.

The test is considered valid if the difference of the extremes of replicate values of production of CO2 at the time the plateau is reached, at the end of the test or at the end of the 10-Day
window, as appropriate, is less than 20%.

The total CO2 evolution in the inoculum control vessels at the end of the test should not normally exceed 40 mg/L medium (= 120 mg/3 liters, corresponding to 33 mg C per flask), however values up to 70 mg/L are acceptable. Data from studies where values in excess of 70 mg/L are obtained should be critically examined.

The IC content of the test item suspension in the mineral medium at the beginning of the test should be <5% of the TC.

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

ca. 89

Sampling time:

28 d

Details on results:

The total CO2 evolution in the inoculum control vessels on Day 28 was 32.83 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.

The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.

The number of colony forming units in the replicate inoculum control vessels were calculated to be an average of 5000/mL in inoculum control Replicate 1 and 5500/mL in inoculum control Replicate 2.

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.

Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item. The results of the inorganic carbon analysis of samples from the first absorber vessels on
Day 29 showed an increase in all replicate vessels.

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.

The test item attained 89% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Results with reference substance:

Sodium benzoate attained 65% biodegradation after 14 days with greater than 60% degradation being attained in a 10-Day window. After 28 days 113% biodegradation was attained. Biodegradation values in excess of 100% were considered to be due to sampling/analytical variation. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines.

Table 1 Inorganic Carbon Values on Each Analysis Occasion

Day	Inorganic Carbon (mg IC)
	Inoculum Control				Procedure Control				Test Item				Toxicity Control
	R1		R2		R1		R2		R1		R2		R1
	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2
0	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.87	1.75	1.75	1.75	1.75	1.75	1.87
2	9.40	-	8	-	21.58	-	24.36	-	9.16	-	7.54	-	5.92	-
6	15.80	-	14.3	-	28.83	-	35.06	-	19.14	-	16.26	-	11.65	-
8	18.23	-	15.94	-	33.48	-	42.43	-	23.74	-	21.21	-	31.88	-
10	21.20	-	19.95	-	37.05	-	45.37	-	33.17	-	30.89	-	48.56	-
14	23.35	-	20.63	-	37.06	-	45.79	-	39.67	-	33.66	-	50.55	-
21	27.94	-	27.72	-	59.60	-	59.60	-	51.60	-	49.80	-	70.87	-
28	27.55	-	26.21	-	57.57	-	54.21	-	55.55	-	51.30	-	77.95	-
29	31.06	3.48	29.5	3.48	66.24	3.48	62.23	3.48	56.67	3.48	57.34	3.48	85.12	3.48

R = Replicate
Abs = CO2 absorber vessel

 

 

Table 2 Percentage Biodegradation Values

Day	Biodegradation (%)
	Procedure Control**	Test Item**	Toxicity Control
0	0	0	0
2	48	0	0
6	56	9	0
8	70	18	25
10	69	38	47
14	65	49	48
21	106	76	72
28	97	89	85
29*	113	89	91

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

 

 

Table 3 Total and Inorganic Carbon Values in the Culture Vessels on Day 0

Test vessel	Test Carbon* (mg/L)	Inorganic Carbon* (mg/L)	IC Content
Test Item 10 mg C/L R1	9.99***/ 10.22**	9.96***/ 0.16	100*** / 2
Test Item 10 mg C/L R2	9.73***/ 10.54**	10.62***/ 0.08	109***/ 1

R = Replicate
∗ Corrected for control values
**TC value given is the sum of the TC value obtained from analysis and the nominal TC contribution of the test item
*** Samples re-analyzed due to poor IC calibration

 

 

 

Table 4 pH Values of the Test Preparations on Days 0 and 28

Test Vessel	pH
	Day 0	Day 0	Day 28
	Pre-Adjustment	Post-Adjustment
Inoculum Control R1	7.7	7.4	7.3
Inoculum Control R2	7.7	7.4	7.3
Procedure Control R1	7.7	7.4	74
Procedure Control R2	7.7	7.4	7.4
Test Item R1	7.7	7.4	7.3
Test Item R2	7.8	7.4	7.3
Toxicity Control	7.7	7.4	7.4

R = Replicate

 

 

Table 5 Observations on the Test Preparations Throughout the Test Period

Test Vessel		Observations on Test Preparations
		Day 0	Day 7	Day 14	Day b 21	Day 28
Inoculum Control	R1	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
	R2	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
Procedure Control	R1	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible
	R2	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible
Test Item	R1	Light brown cloudy dispersion	Light brown cloudy dispersion	Light brown cloudy dispersion	Light brown cloudy dispersion	Light brown cloudy dispersion
	R2	Light brown cloudy dispersion	Light brown cloudy dispersion	Light brown cloudy dispersion	Light brown cloudy dispersion	Light brown cloudy dispersion
Toxicity Control		Light brown cloudy dispersion, no undissolved reference item visible	Light brown cloudy dispersion, no undissolved reference item visible	Light brown cloudy dispersion, no undissolved reference item visible	Light brown cloudy dispersion, no undissolved reference item visible	Light brown cloudy dispersion, no undissolved reference item visible

R = Replicate

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The results of the test showed that the test item attained 89% biodegradation after 28 days and satisfied the 10 -Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%.  The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Executive summary:

The ready biodegradability of the Benzyl 3-isobutyryloxy-1-isopropyl-2,2dimethylpropyl phthalate was determined in a 28 -day biodegradation study performed in accordance with OECD 301B, "Ready Biodegradability; CO2 Evolution Test". The biodegradation of the test item was assessed by the determination of carbon dioxide produced.  Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes. The results of the test showed that the test item attained 89% biodegradation after 28 days and satisfied the 10 -Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%.  The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Description of key information

The ready biodegradability of the Benzyl 3-isobutyryloxy-1-isopropyl-2,2dimethylpropyl phthalate was determined in a 28 -day biodegradation study performed in accordance with OECD 301B, "Ready Biodegradability; CO2 Evolution Test". The biodegradation of the test item was assessed by the determination of carbon dioxide produced.  Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes. The results of the test showed that the test item attained 89% biodegradation after 28 days and satisfied the 10 -Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%.  The test item can therefore 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:

readily biodegradable