Fatty acids, C16-18 and C18-unsatd., branched and linear, reaction products with diethanolamine

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:

Experimental Starting Date: 05 December 2012 Experimental Completion Date: 06 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted 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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Isostearamide DEA

IUPAC Name:

Isostearamide DEA

Test material form:

other: amber coloured liquid

Details on test material:

The integrity of the supplied data relating to the identity, purity and stability of the test item is the responsibility of the Sponsor.

Identification: Isostearamide DEA
Description: amber coloured liquid
Batch: OE11124 (6/202593/00)
Purity: unknown
Expiry / retest date: unknown
Storage conditions: room temperature in the dark

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Reference Item
The integrity of the supplied data relating to the identity, purity and stability of the reference item is the responsibility of the Supplier.

Identification: Sodium benzoate
Description: white solid
Batch: MKBH2299V
Purity: 99%
Expiry / retest date: 26 June 2013
Storage conditions: room temperature over silica gel

Study design

Oxygen conditions:

aerobic

Remarks:

aqueous medium

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained on 07 January 2013 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 two times by settlement and resuspension 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* 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 3.1 g/L prior to use. The study plan
states that the concentration should be 4 g/L but as the amount of sewage sludge added to the test vessels was at the correct concentration, this
has been considered to have no adverse effect on the integrity of the study.


Mineral Medium
The mineral medium used in this study (see attached Appendix 1) was that recommended in the OECD Guidelines.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

10 other: mg Carbon/L

Based on:

DOC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Remarks:

The total CO2 evolution in the inoculum control vessels on Day 28 was 35.30 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.

Details on study design:

Test Species
A mixed population of activated sewage sludge micro-organisms was obtained on 07 January 2013 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Experimental Preparation
Following the recommendations of the International Standards Organisation (ISO, 1995) and in the published literature (Handley et al, 2002) the test item was adsorbed onto silica gel prior to dispersion in mineral medium to aid dispersion of the test item in the test medium and to increase the surface area of the test item exposed to the test organisms.

An amount of test item (40.2 mg) was adsorbed onto the surface of 100 mg of granular silica gel (230-400 mesh) prior to dispersal in approximately 400 mL of mineral medium with the aid of high shear mixing (7500 rpm, 15 minutes). The test item/silica gel/mineral mineral medium dispersion was then dispersed in inoculated mineral medium and the volume adjusted to 3 liters to give a final concentration of 13.4 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.

Inoculum control vessels were prepared containing 100 mg silica gel per 3 liters of inoculated mineral medium in order to maintain consistency between the control and test item vessels.

Data from the inoculum control vessels was shared with similar concurrent studies.


Toxicity Control
For the purposes of the test, 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 (40.2 mg) was adsorbed onto the surface of 100 mg of granular silica gel (230-400 mesh) prior to dispersal in approximately 400 mL of mineral medium with the aid of high shear mixing (7500 rpm, 15 minutes). The test item/silica gel/mineral medium dispersion was then
dispersed in inoculated mineral medium and an aliquot (51.4 mL) of the sodium benzoate stock solution added. The volume was adjusted to 3 liters to give a final concentration of 13.4 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 plus 100 mg silica gel.
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium plus 100 mg silica gel to give a final concentration of 10 mg carbon/L.
c) The test item, in duplicate, in inoculated mineral medium plus 100 mg silica gel to give a final concentration of 10 mg carbon/L.
d) The test item plus the reference item in inoculated mineral medium plus 100 mg silica gel to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).

Silica gel was added to the inoculum control and procedure control vessels in order to maintain consistency between these vessels and the test item vessels.

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 22 °C ± 2°C 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 29 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 WTW pH/Oxi 340I pH and dissolved oxygen meter. If necessary 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 test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/min 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.


Sampling and Analysis
CO2 Analysis
Samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 3, 6, 9, 12, 14, 22, 28 and 29. The second absorber vessels were all
sampled on Days 0 and 29.

The samples taken on Days 0, 3, 6, 9, 12, 14, 22, 28 and 29 were analyzed for CO2 immediately.

On Day 28, 1 mL of concentrated hydrochloric acid was added to each test item vessel and the toxicity control 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 inoculum and procedural control vessels were not acidified as they were shared with a similar concurrent study which was continuing after the termination of
this study.

The samples were analyzed for CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyzer and a Shimadzu TOC-VCSH TOC analyzer and a Shimadzu TOC-LCSH TOC analyzer. Samples (300 or 50 µL) were injected into the IC (Inorganic Carbon) channel of the TOC analyzer. Inorganic carbon
analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid or 2M hydrochloric acid using zero grade air as the carrier gas. Calibration was by reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.


Inorganic Carbon/Total Carbon Analysis
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 Inorganic Carbon
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.

IC/TC 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 TC using a Shimadzu TOC-VCPH TOC analyzer. Samples (50 or 800 µL) were injected into the Total Carbon
(TC) and Inorganic Carbon (IC) channels of the TOC analyzer. 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 reference solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionized water. Each
analysis was carried out in triplicate.

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 WTW pH/Oxi 340I pH
and dissolved oxygen meter.


Evaluation of Data
Calculation of Carbon Content
From analysis the percentage carbon in the test item was determined to be 74.6% and so for a concentration of 10 mg C/L (a total of 40.2 mg of test
item in 3 liters) the total organic carbon present was 30 mg C.

The theoretical amount of carbon present in the reference item, sodium benzoate (C6H5COONa) was calculated as follows:

No of C atoms x mol wt of C ÷ mol wt of sodium benzoate x 100

7 x 12.011 ÷ 144.11 x 100 = 58.34%

Thus for a 10 mg C/L test concentration (a total of 51.4 mg of sodium benzoate in 3 liters) 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, into the following equation. The values of Replicates R1 and R2 are meaned for the inoculum control, test and
reference items before substitution into the following equation:

%ThCO2 (= % degradation*) = mg IC in test flask – mg IC in control flask ÷ mg TOC
added as test chemical x 100


The total CO2 evolution in the inoculum 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 C/L) = mg IC in control x 100 ÷ %C of CO2 x 1 ÷ test volume
= mg IC in control x 100 ÷ 27.29 x ⅓

Validation Criteria
The results of the degradation test are considered valid if in the same test the reference item yields ≥ 60% degradation 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.

* The conversion factor for carbon to carbon dioxide is 3.67

Reference substance

Reference substance:

other: Sodium benzoate

Results and discussion

Preliminary study:

Following the recommendations of the International Standards Organisation (ISO 1995) and the published literature (Handley et al, 2002), the test item was
adsorbed onto granular silica gel prior to dispersion in the test medium to aid dispersion of the test item in the test medium and to increase the surface area
of the test item exposed to the test organisms.

Test performance:

Inorganic carbon values for the test item, procedure control, toxicity control and inoculum control vessels at each analysis occasion are given in Table 1.
Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 2 and the biodegradation curves are presented in the attached Figure 1. Total and Inorganic Carbon values in the culture vessels on Day 0 are given in Table 3. The pH values of the test preparations on
Days 0 and 28 are given in Table 4. Observations made on the contents of the test vessels are given in Table 5.

The total CO2 evolution in the inoculum control vessels on Day 28 was 35.30 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 (see Table 3) was below 5% of the TC content 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.

Acidification of the test item and toxicity control 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.

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

ca. 49

Sampling time:

28 d

Details on results:

RESULTS
Following the recommendations of the International Standards Organisation (ISO 1995) and the published literature (Handley et al, 2002), the test item was adsorbed onto granular silica gel prior to dispersion in the test medium to aid dispersion of the test item in the test medium and to increase the surface
area of the test item exposed to the test organisms.

Inorganic carbon values for the test item, procedure control, toxicity control and inoculum control vessels at each analysis occasion are given in Table 1.
Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 2 and the biodegradation curves are presented in the attached Figure 1. Total and Inorganic Carbon values in the culture vessels on Day 0 are given in Table 3. The pH values of the test preparations on Days 0 and 28 are given in Table 4. Observations made on the contents of the test vessels are given in Table 5.

The total CO2 evolution in the inoculum control vessels on Day 28 was 35.30 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 (see Table 3) was below 5% of the TC content 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.

Acidification of the test item and toxicity control 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 a decrease in all replicate vessels with the
exception of Procedural Control R2 and Test Item R2. This decrease was considered to be due to sampling/analytical variation. 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 49% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

The toxicity control attained 65% degradation after 14 days and 70% degradation after 28 days thereby confirming that the test item was not toxic to the
sewage treatment micro-organisms used in the test.

Sodium benzoate attained 77% degradation after 14 days and 82% degradation after 28 days thereby confirming the suitability of the inoculum and test
conditions.

BOD5 / COD results

Results with reference substance:

Sodium benzoate attained 77% degradation after 14 days and 82% degradation after 28 days thereby confirming the suitability of the inoculum and
test conditions.

Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 2 and the biodegradation curves are presented in the attached Figure 1.

Any other information on results incl. tables

Tables

Table 1     Inorganic Carbon Values on Each Analysis Occasion

Day	Inoculum Control (mg IC)				Sodium Benzoate Procedure Control (mg IC)				Test Item (mg IC)				Test Item plus Sodium Benzoate Toxicity Control (mg IC)
	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	1.05	1.05	1.28	1.28	1.05	1.05	1.05	0.93	1.05	1.05	1.05	1.05	1.28	1.40
2	11.83	-	11.60	-	34.10	-	27.14	-	16.12	-	12.18	-	39.09	-
6	17.99	-	17.53	-	39.33	-	34.14	-	23.87	-	22.38	-	48.09	-
8	17.89	-	17.43	-	38.53	-	36.69	-	28.78	-	24.54	-	52.63	-
10	19.15	-	18.81	-	41.27	-	38.53	-	31.81	-	27.02	-	54.72	-
14	20.63	-	23.69	-	46.92	-	43.63	-	37.51	-	31.51	-	61.31	-
21	25.35	-	26.25	-	49.80	-	53.07	-	43.15	-	37.41	-	67.83	-
28	27.95	-	29.84	-	50.99	-	53.55	-	45.92	-	40.21	-	71.57	-
29	27.78	2.78	28.99	2.78	49.80	2.44	55.22	3.83	45.42	3.02	40.52	2.78	70.67	2.78

 

R₁ – R₂        =   Replicates 1 and 2

Abs              =  CO₂ absorber vessels

Table 2     Percentage Biodegradation Values

Day	% Degradation Sodium Benzoate Procedure Control	% Degradation Test Item	% Degradation Test Item plus Sodium Benzoate Toxicity Control
0	0	0	0
2	63	8	46
6	63	18	51
8	67	30	58
10	70	35	60
14	77	41	65
21	85	48	70
28	78	47	71
29*	82	49	70

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

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

Test vessel	Total Carbon** (mg/L)	Inorganic Carbon* (mg/L)	IC Content (% of TC)
Test Item 10 mg C/L R1	13.26	0.99	0
Test Item 10 mg C/L R2	14.18	1.61	0

 

R₁ – R₂     =  Replicates 1 and 2

*       Corrected for control values.

**    Total carbon value given is the sum of the TC value obtained from analysis and the nominal TC contribution of the test item

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

Test Vessel	pH
Inoculum Control R1	7.5	7.5
Inoculum Control R2	7.5	7.4
Procedure Control 10 mg C/L R1	7.5	7.4
Procedure Control 10 mg C/L R2	7.5	7.5
Test Item 10 mg C/L R1	7.4	7.4
Test Item 10 mg C/L R2	7.5	7.4
Toxicity Control 20 mg C/L	7.4	7.4

 

 

 R₁– R₂    =      Replicates 1 and 2

Table 5     Observations on the Test Preparations Throughout the Test Period

Test Vessel		Observations on Test Preparations
		Day 0	Day 6	Day 13	Day 20	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 dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible
	R2	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible
Toxicity Control		Light brown dispersion, no undissolved reference item or test item visible	Light brown dispersion, no undissolved reference item or test item visible	Light brown dispersion, no undissolved reference item or test item visible	Light brown dispersion, no undissolved reference item or test item visible	Light brown dispersion, no undissolved reference item or test item visible

 

R₁ – R₂ = Replicates 1 and 2

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable, not fulfilling specific criteria

Conclusions:

The test item attained 49% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B. However, from the biodegradation curves, it is apparent that the test item had not plateaued in its biodegradation by Day 28. If the study were continued further, it is possible that the test item would have continued to biodegrade

Executive summary:

SUMMARY

Introduction

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO₂ Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (m)).

 

 

Methods

The test item, at a concentration of 10 mg Carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at 22 °C ±2 °C for 28 days.

 

Following the recommendations of the International Standards Organisation (ISO 1995) and the published literature (Handley et al, 2002), the test item was adsorbed onto granular silica gel prior to dispersion in the test medium to aid dispersion of the test item in the test medium and to increase the surface area of the test item exposed to the test organisms.

 

The degradation 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.

 

 

Results

The test item attained 49% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B. However, from the biodegradation curves, it is apparent that the test item had not plateaued in its biodegradation by Day 28.  If the study were continued further, it is possible that the test item would have continued to biodegrade