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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was ocnducted between 26 July 2016 and 23 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
ISO 14593:1999 (Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test))
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Version / remarks:
2014
Deviations:
no
GLP compliance:
yes (incl. certificate)
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
Preparation of Inoculum
Upon receipt in the laboratory, the sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded).
In order to reduce the inorganic carbon (IC) content of the inoculum, the filtrate was sparged with CO2-free air* for approximately 1 hour whilst maintaining its pH at 6.5 using concentrated orthophosphoric acid. After sparging, the pH was restored to its original value of 7.5 using 7 M sodium hydroxide and the inoculum allowed to settle for approximately 1 hour prior to removal of an aliquot (2 liters) of the supernatant for use in the test. The supernatant was maintained on aeration using CO2-free air until use.
Duration of test (contact time):
ca. 28 d
Initial conc.:
ca. 25.6 mg/L
Based on:
test mat.
Initial conc.:
ca. 20 mg/L
Based on:
IC (inorganic carbon)
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Preliminary Solubility Work
Information provided by the Sponsor indicated that the water solubility of the test item was 257 mg/L. Therefore preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation.

Test Item Preparation
Following preliminary solubility work and the recommendations of the International Standards Organisation (ISO, 1995) and in the published literature (Handley et al, 2002) the test item was dissolved in an auxiliary solvent prior to adsorption onto filter paper*. Using the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation.
A nominal amount of test item (1096 mg) was dissolved in 10 mL of acetone to give a 1096 mg/10 mL solvent stock solution. An aliquot (25 μL) of this solvent stock solution was dispensed onto a filter paper* and the solvent allowed to evaporate to dryness for approximately 15 minutes. The filter paper was added to inoculated mineral medium (107 mL) to give a final concentration of 25.6 mg/L, equivalent to 20 mg carbon/L. The volumetric flask containing the solvent stock solution was inverted several times to ensure homogeneity of the solution. The test vessels were then sealed using Teflon lined silicon septa and aluminum crimp caps.
A filter paper*was added to each control vessel in order to maintain consistency between the test and procedure control vessels. Acetone (25 μL) was dispensed onto each filter paper and evaporated to dryness for approximately 15 minutes. The filter paper was added to each vessel. The test vessels were then sealed using Teflon lined silicon septa and aluminum crimp caps.

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 with the aid of ultrasonication for approximately 5 minutes. An aliquot (137.2 mL) of this stock solution was dispersed with inoculum (400 mL) and mineral medium to a final volume 4 liters, to give a test concentration of 34.3 mg/L, equivalent to 20 mg carbon/L. Aliquots (107 mL) of the 34.3 mg/L test concentration were dispensed to each of 33 replicate test vessels and the vessels sealed using Teflon lined silicon septa and aluminum crimp caps.
A filter paper*was added to the procedure control vessels in order to maintain consistency between the test and procedure control vessels.
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 study.
Aliquots (107 mL) of the 34.3 mg/L reference item concentration were dispensed to 9 replicate test vessels.
An aliquot (25 μL) of the 1096 mg/10 mL test item solvent stock solution was dispensed separately on to 9 filter papers** and the solvent allowed to evaporate to dryness for approximately 15 minutes and a filter paper was added to each vessel.
The final concentration in the toxicity control vessels was 25.6 mg test item/L plus 34.3 mg reference item/L, equivalent to 40 mg carbon/L.

Preparation of Test System
The following test preparations were prepared and incubated in 125 mL glass Wheaton bottles (total volume when full 160 mL) each containing 107 mL of solution:
a) An inoculated control consisting of inoculated mineral medium, plus a filter paper**, 33 replicate vessels.
b) The procedure control containing the reference item (sodium benzoate) in inoculated mineral medium, plus a filter paper**, to give a final concentration of 20 mg carbon/L, 33 replicate vessels.
c) The test item in inoculated mineral medium, plus a filter paper**, to give a final concentration of 20 mg carbon/L, 29 replicate vessels.
d) The test item plus the reference item in inoculated mineral medium, plus a filter paper**, to give a final concentration of 40 mg carbon/L to act as a toxicity control, 9 replicate vessels.
A filter paper was added to the inoculum control and procedure control vessels in order to maintain consistency between these vessels and the test item vessels.
Test media a to d were inoculated with the prepared inoculum at a final concentration of 100 mL/L.
Aliquots (107 mL) of the test media were dispensed into replicate vessels to give a headspace to liquid ratio of 1:2. Sufficient vessels were prepared to allow a single inorganic carbon determination per vessel with triplicate vessels for the inoculum control, procedure control, test item and toxicity control at each sampling occasion (five replicates for analysis on Day 28). Additional inoculum control and procedure control vessels were prepared to provide samples for Dissolved Organic Carbon (DOC) analyses on Days 0 and 28 (duplicate vessels per sampling occasion).
All vessels were sealed using Teflon lined silicon septa and aluminum crimp caps and incubated in darkness at 20 ± 1 oC with constant shaking at approximately 125 rpm (INFORS Version 2 Multitron® Incubator).

* Whatman GF/A (70 mm diameter)
**Whatman GF/A (21 mm diameter) with acetone evaporated off
Reference substance:
benzoic acid, sodium salt
Remarks:
Supplier: Sigma-Aldrich Batch: SLBM8408V Purity: >99.5% Expiry Date: 11 May 2017 Storage Conditions: room temperature over silica gel
Preliminary study:
Preliminary Solubility Work
The following preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation
i) Ultrasonication and High Shear Mixing: A nominal amount of test item (50 mg) was dispersed in 1 liter of deionized reverse osmosis purified water with the aid of shaking by hand for approximately 1 minute prior to ultrasonication for 15 minutes. This formed a hazy dispersion with small oily globules of test item visible dispersed throughout and an oily slick of test item on the surface. This was then subjected to high shear mixing (approximately 7500 rpm, 15 minutes) and formed a very slightly hazy dispersion with a slight oily slick on the surface.
This work confirmed that the test item was insoluble in water. Therefore the following additional solubility work was conducted to ascertain the best method to employ in the biodegradation test.
1. Ultrasonication
2. High Shear Mixing
3. Preliminary Solution in a Volatile Solvent
From the preliminary solubility work and following the recommendations of the International Standards Organisation (ISO, 1995) it was concluded that the best testable dispersion was found to be obtained when the test item was dissolved in acetone and an aliquot added to a filter paper prior to addition to the test vessel.
Test performance:
Validation Criteria
Test items giving a result of ≥ 60% yield of ThIC within 28 days should be regarded as readily biodegradable. This level must be reached within 10 days of the biodegradation exceeding 10%.
The test is considered valid if the reference item biodegradation rate is ≥ 60% by day 14.
The toxicity control should attain ≥ 25% biodegradation by day 14 for the test item to be considered as non-inhibitory.
The TIC produced from the control bottles at the end of the test should be ≤ 15% of the TOC added initially as test item.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 1
Sampling time:
28 d
Details on results:
The test item attained 1% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 310.
The toxicity control attained 36% biodegradation after 14 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.
Sodium benzoate attained 81% biodegradation after 14 and 28 days thereby confirming the suitability of the inoculum and test conditions.
Variation in the biodegradation rates obtained on different sampling days (Table 1) was considered to be the result of normal biological variation between the respiration rates of replicate vessels. Due to the sacrificial nature of the study design, the biodegradation rates obtained on each sampling occasion were for individual replicate vessels and not the result of cumulative biodegradation values determined from a single vessel sampled on numerous occasions and as such variation in biodegradation rates on different sampling days was to be expected.
DOC analyses conducted on samples taken from the reference item vessels on Days 0 and 28 (Table 2) showed that the replicate reference item vessels attained 100% and 97% biodegradation for each replicate vessel. The biodegradation rates for the reference item were higher than those determined by IC analyses. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to biodegradation and hence CO2 evolution occurring.

Table 1: Percentage Biodegradation Values

 Day  % Biodegradation      
   Procedure Control  Test Item  Toxicity Control
 0  0  0  0
 2  63  0  -
 6  76  0  -
 8  84  0  31
 10  74  0  -
 14  81  0  36
 16  86  0  -
 21  79  0  -
 28  81  1  -

Table 2: Dissolved Organic Carbon (DOC) Values on Days 0 and Day 28

 Test Vessel     Day 0             Day 28      
     mg C/L  mg C/L Corrected for Mean Control Value  % of Nominal Carbon Content  mg C/L  mg C/L Corrected for Mean Control Value  % Biodegradation
 Inoculum Control  R1  1.27  -  -  1.00  -  -
   R2  1.15  -  -  <LOQ  -  -
 Procedure Control  R1  21.34  20.13  101  <LOQ  <LOQ  100
   R2  21.62  20.41  102  1.06  0.56  97
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item attained 1% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 310.
Executive summary:

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 OECD Guidelines for Testing of Chemicals (2014) No. 310 and the ISO Guideline No 14593 “Water quality – Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium – Method by analysis of inorganic carbon in sealed vessels (CO2 Headspace Test)”.

Methods

The test item, at a concentration of 20 mg C/L, was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in the dark at 20 ± 1 °C for 28 days.

Following the recommendations of the International Standards Organisation (ISO 1995), the test item was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper and subsequent dispersal in test media.

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.

Results

The test item attained 1% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 310.

Description of key information

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 OECD Guidelines for Testing of Chemicals (2014) No. 310 and the ISO Guideline No 14593 “Water quality – Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium – Method by analysis of inorganic carbon in sealed vessels (CO2 Headspace Test)”.

Methods

The test item, at a concentration of 20 mg C/L, was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in the dark at 20 ± 1 °C for 28 days.

Following the recommendations of the International Standards Organisation (ISO 1995), the test item was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper and subsequent dispersal in test media.

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.

Results

The test item attained 1% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 310.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information