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Biodegradation of volatile chemicals in soil – Separating volatilization and degradation in improved test setup (OECD 307) Prasit Shrestha, Boris Meisterjahn, Michael Klein, Philipp Mayer, Heidi Birch, Christopher Hughes, and Dieter Hennecke Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b05079 • Publication Date (Web): 03 Dec 2018 Downloaded from http://pubs.acs.org on December 7, 2018

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Environmental Science & Technology

O2

Volatile chemicals Improved test setup (OECD 307)

Complete Mass balance + Degradation Kinetics

Tetralin 1 1

Decane

Soil ACS Paragon Plus Environment

%aR(Mineralization)

60 40 20 0 0

% aR(Volatilization)


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Decane

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120

% aR(Volatilization)

%aR(Mineralization)

Tetralin

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Biodegradation of volatile chemicals in soil – Separating

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volatilization and degradation in improved test setup

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(OECD 307)

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Prasit Shrestha1,2, Boris Meisterjahn1*, Michael Klein1, Philipp Mayer2, Heidi Birch2,

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Christopher B. Hughes.3, Dieter Hennecke.1

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1Fraunhofer

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2Department

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Lyngby, Denmark

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3Ricardo

IME-AE, Auf dem Aberg 1, 57392 Schmallenberg Germany. of Environmental Engineering, Technical University Denmark, 2800 Kgs.

Energy and Environment, Harwell, OX11 0QR, UK

ACS Paragon Plus Environment


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Abstract

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During environmental risk assessments of chemicals, higher tier biodegradation tests in soil,

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sediment and/or surface water systems are required using standard OECD 307, 308 and 309

13

guidelines, respectively. These guidelines are not suitable for testing highly volatile chemicals

14

and recommend a biometer closed-incubation setup for testing slightly volatile chemicals. In

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this setup, degradation kinetics of highly volatile chemicals can largely be influenced by

16

volatilization. Additionally, guidelines lack sufficient information on test system geometry and

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guidance on how to measure and maintain aerobic conditions during the test. Our objectives

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were 1) to design a closed test setup for biodegradation tests in soil, where maintaining and

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measuring of aerobic conditions was possible without the loss of volatile test chemicals and 2)

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to suggest data treatment measures for evaluating degradation kinetics of volatile test

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chemicals. With the new setup, full scale OECD 307 tests were performed using the volatile

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14C

23

balances were observed, and the new setup ensured that the volatilization losses were kept

24

below the mineralized fraction. Based on the obtained data an extended model was developed

25

that enabled considering volatilization in the modelling of degradation kinetics.

labelled chemicals decane and tetralin. For both test chemicals, reproducible complete mass


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1. Introduction

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During prospective environmental risk assessment of chemicals, biodegradation kinetics data

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are a key parameter in different regulatory frameworks like REACH1, Pesticide regulation2,

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Biocide regulation3 etc. These degradation kinetics data are generated using a set of tiered

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laboratory biodegradation tests with increasing levels of complexity4,5, which are performed

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using standard OECD guidelines6. The highest tiers are formed by the environmental simulation

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biodegradation tests and were designed to simulate biodegradation in various environmental

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matrices such as soil (OECD 307)7, water/sediment (OECD 308)8 or surface water (OECD

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309)9. The OECD guidelines were originally developed for pesticide regulation where they

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work well with most of the relevant chemicals. However, when those guidelines are applied to

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other regulatory frameworks it should be considered whether the chemicals regulated under

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those frameworks posess different physical-chemical properties that might make them more

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challenging to test or render the existing test guidelines unsuitable. In particular, the previously

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mentioned guidelines face serious drawbacks when it comes to the testing of volatile chemicals.

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Some of these issues are listed below:

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

Although the scope of OECD 307, 308 and 309 guidelines clearly state that the tests are

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not suitable for volatile chemicals6,7, with the exception of OECD 309 they do not define

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a threshold. In OECD 309 a threshold for the Henry´s law constant of 100 Pa m3/mol is

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stated9.

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

When testing volatile chemicals in a flow-through setup, incomplete mass balances is a

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major problem and it is often impossible to meet the validity criteria of 90%-110%

47

recovery for radiolabelled chemicals and 70% - 110% for non-labelled chemicals7-9.

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Prior work has suggested that incomplete mass balances mainly result due to inability

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of the traps to capture the volatile chemicals and hence they escape from the test setup.

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In addition, our own experience and other studies indicate that volatile test chemicals



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are also being sorbed to the plastic or silicone materials present in the fittings of the test

52

setup.10

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

As an alternative, OECD 307, 308 and 309 allow to apply static biometer-type

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incubation setups7,8. However, these guidelines give only limited information on how

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to handle the headspace in the biometer type test setup for testing volatile chemicals.

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Several studies suggest that the headspace volume is quite important to keep the system

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aerobic in a static test setup. On the other hand too much headspace might lead to higher

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mass distribution into the headspace volume leading to a lowered fraction in the water,

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sediment or soil available for degradation, and a corresponding effect on degradation

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kinetics10,11.

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

The guideline does not stipulate any data requirements to prove whether the test system

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remains aerobic. This is a general problem with static test setups, especially if a co-

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solvent is used for the application of test chemicals. In a normal case the co-solvent

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should be evaporated after test substance application but during the application of

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volatile chemicals the evaporation step has to be avoided as loss of test chemicals may

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occur. However, our own studies with closed biometer type test setups have shown rapid

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oxygen depletion after solvent application. This can have a major impact on the

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degradation kinetics, but to monitor the oxygen content in the system containing volatile

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chemicals is a challenging task.

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

As suggested by Birch et al. 201711, reductions in bioavailability as a result of

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partitioning to headspace can largely influence the degradation kinetics. Therefore, new

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data treatment methods are needed for generating meaningful degradation kinetics in

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tests where volatile chemicals are used.

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To address these drawbacks, our objectives were: (1) to develop an incubation test for testing

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degradation of volatile chemicals in soil; (2) to demonstrate the usefulness of the developed test

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setup in a full scale OECD 307; and, (3) to suggest methods on how to treat the data obtained

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from such tests for evaluating degradation kinetics. The first objective was obtained during a

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series of pre-tests with different test setups. Maintainance of aerobic conditions inside the flask

80

was also taken into consideration and was done by external oxygen measurements thereby

81

minimizing test chemical losses due to opening of the vessel. Secondly, with the new test setup,

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a full scale OECD 307 test was performed using two chemicals with different volatilization and

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degradation potential and four different soils, varying in terms of texture, organic carbon (OC)

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content and microbial activity. The data obtained were used to demonstrate the reproducibility

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and reliability of the test setup in terms of mass balance. Thirdly, the data from the tests were

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used to improve our understanding of different processess such as volatilization and sorption

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processes taking place during the test and its influence on the final biodegradation data

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obtained. Furthermore, data treatment methods were demonstrated for generating meaningful

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degradation kinetics for the volatile chemicals in the new test setup. The first hypothesis of the

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study was that improved methodology was necessary to determine the biodegradation kinetics

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of hydrophobic volatile chemicals in soils. The second hypothesis was that careful experimental

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testing and data analysis can facilitate the separation of degradation and evaporative losses.

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2. Materials and methods

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2.1. Test chemicals

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Two 14C labelled chemicals were used for the degradation tests covered within this project: 1)

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tetralin (1,2,3,4-tetrahydronaphthalene, C10H12, CAS no:119-64-2, Item no. 17445740, >98%

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purity, Hartmann Analytics), and 2) decane (n-decane, C10H22, CAS no: 124-18-5, Item no.



99

17539210, > 98% purity Hartmann Analytics). The test chemicals were selected based on their

100

differences in air-water partitioning and sorption properties with tetralin being slightly volatile

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(KH 138 Pa m3 mol-1, VP 0.37 mmHg at 25°C and Koc 1068 L/kg)13 and decane being highly

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volatile but theoretically more sorbing than tetralin (KH 522,000 Pa m3 mol-1, VP 1.43 mmHg

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at 25°C and Koc 22,270 L/kg)12. For the optimisation of specific chemical analytics and also as

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a quality control, cold standards from Sigma Aldrich with purity > 99% were used.

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2.2. Soils

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In total four soils were used to cover a broad range of soil texture, organic carbon (OC) content

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and microbial biomass as required by the OECD guideline 307. Two soils with low OC content

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01A (loamy sand, OC 0.80%) , 02A (silty loam, OC 0.98%) and two with higher OC content

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03G (silty loam, OC 3.05%), 04A (loamy sand, OC 2.79%) were used. The detailed soil

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properties are listed in Table S1. The soil texture was determined using the standard DIN ISO

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1127713 and OC content was determined using DIN EN 1593614. As per the guideline

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requirement, the soil was sampled from the upper 20 cm layer and was sieved using a 2 mm

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sieve. The soils were maintained at 50% water holding capacity (WHC) for preincubation under

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test conditions (at 20°C in the dark) for a period of 2-3 weeks before test start.

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2.3. Test chemical application and sample incubation conditions

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The 14C labelled test chemical application solution was prepared using the co-solvent acetone

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for tetralin and methanol for decane. The application solution was then spiked directly on the

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soil and the sample bottle was immediately closed using an insert cap. The starting test

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substance concentration was 1 mg/kg (dw soil) for all tests conducted within this project. The

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amount of applied radioactivity per soil sample of 50 g was 203 kBq in 219 µL acetone for

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tetralin and 195 kBq in 139 µL methanol for decane. After application of the test chemical the

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soil samples were incubated at 20 ± 2°C in the dark.


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2.4. Pre-tests

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In order to develop a biodegradation test setup for volatile chemicals, a series of preliminary

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tests were conducted using 14C labelled tetralin. The first tests comprised of a soil degradation

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study using a flow-through setup as suggested by OECD 307 with slight modifications. The

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primary objective of this experiment was to check for a complete mass balance of tetralin during

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the incubation period. These tests were followed by other tests using closed flask test setups

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where maintaining and measuring aerobic conditions in the test setup was considered along

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with the complete mass balance of the test chemical.

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2.4.1.

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50 g dry weight (dw) of preincubated soil was weighed in a 100 mL sample flask and applied

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with 14C labelled tetralin (see section 2.3). A flow-through setup consisted of a sample flask

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connected with absorption traps containing 100 mL of 2N NaOH and ethylene glycol flasks to

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trap mineralized 14CO2 and volatile parent/intermediate, respectively. A schematic diagram of

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the flow-through setup used is given in the Figure S1. Behind the traps a tube furnace operating

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at 850°C was connected, filled with a CuO catalyst in order to combust any substance in the

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flow-through, which could not be captured in the prior traps. To trap the resulting 14CO2, one

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further sample flask with 100 mL 2N NaOH was connected behind the oven. At specific

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sampling points (0, 3, 7, 14 days after application) duplicate soil samples along with their traps

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were sampled, processed and taken for chemical analysis (See section 2.6 and 2.7).

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2.4.2.

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A biometer type closed test setup was designed consisting of a 500 mL bottle with 50 g soil

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(dW), 0.5 g tenax (for absorbing the volatiles) and 4 g of soda lime (for absorbing 14CO2). The

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flask was closed using a stainless steel, plastic-free lock system (Swagelok connections, Hamlet

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valves and fitting) (see Figure S2). The test was conducted exemplarily with just one soil (02-

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A) in duplicates. The test chemical application and sample incubation were done as described

Flow-through setup (OECD 307) with volatiles combustion

Closed flask test setup 1



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in section 2.3, incubation period was 14 days. At each sampling date, the air in the headspace

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was stripped out of the sample bottles through the tenax and was passed through 2N NaOH and

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ethylene glycol traps using a vacuum pump. The soil sample, traps and the tenax was taken for

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further analysis (See section 2.6 and 2.7).

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2.4.3.

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Unlike test setup 1, this new test setup comprised of a smaller test vessel 100 mL flask, instead

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of 500 mL to lower the headspace volume and the soda lime was replaced with 6 mL of 2N

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NaOH. Instead of self-prepared tenax inserts a tenax-adsorption tube (CAT no. 226-35 SKC

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Inc.) was permanently connected to the flask in order to passively trap the volatilized fraction.

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The same stainless steel, plastic free lock systems used in setup 1 were used to completely close

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the test setup. The test chemical application and sample incubation were done as described in

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section 2.3 and samples were taken in duplicates at 0, 7 and 14 days.

Closed flask test setup 2

Figure 1. Closed flask incubation test setup 2 used for testing the biodegradation of 14C labelled tetralin and decane in soil using guideline OECD 307. The test design consisted of 100 mL glass flask with 50 g (dm) soil with an internal CO2 absorbing flask and a permanently hanging tenax tube for passively trapping the volatile fraction. The entire test design was closed using a plastic free stainless steel lock system.


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Additionally, two reference samples in duplicates (See section S5) were prepared according to

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test setup 2, without the tenax and NaOH flask but instead including an oxygen sensor spot

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(“Redflash technology”, Pyroscience) attached to the inner wall of the test vessel in the

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headspace. One of these references samples were applied with the same amount of co-solvent

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without test chemical as in the test samples applied with 14C tetralin, whereas the other reference

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sample was incubated under test conditions without solvent application. Every 2-3 days the

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oxygen saturation in the headspace of these reference samples was measured with a Fiber optic

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oxygen meter (Firesting O2, Pyroscience) without the need to open the vessel (For details see

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SI section S7). In case the oxygen concentration in the headspace was found 5% of the applied radioactivity (aR) were taken for specific

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chemical analysis. The extracts were centrifuged at 12300G for 5 min in 2 mL vial (Eppendorf

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Germany) using a mini centrifuge (Microstar 12, VWR). The supernatant was taken for specific

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chemical analysis using radio-HPLC. The tenax extracts were analysed without any clean-up

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using radio-HPLC.

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2.7.1

Radio-HPLC

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For radio-HPLC analysis a Dionex Ultimate 3000 was used coupled with a UV detector (PDA-

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100 photodiode Array detector) and a

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separation was achieved by a Luna C18 (2) 3 μm 150 x 2 mm (MZ) column (Phenomenex,

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Germany), with the column temperature maintained at 30°C. (For details on radio-HPLC

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method used see SI section S11)

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14C

detector (Ramona Raytest). The chromatographic

2.8 Data treatment

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The starting point for fitting the degradation data was the degradation kinetics recommended

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by FOCUS (FOrum for the Coordination of pesticide fate models and their USe)15. FOCUS

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has developed various guidance documents originally to be used in pesticide registration.

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However, many documents were later adopted by other regulations e.g., for biocides or

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veterinary compounds. FOCUS recommended a single first order (SFO = Single First Order)

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together with three different biphasic kinetic models (DFOP = Double First Order in Parallel,

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HS: Hockey Stick and FOMC = First Order Multi Compartment). The fitting of the ACS Paragon Plus Environment


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experimental residues was done with using the software CAKE version 3.2 running on R

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version 3.0.0. CAKE is a freely available software tool, which completely follows the

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recommendations of FOCUS (2006/2014).

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The recommendation for this type of data treatment is given for studies where volatilization is

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not a significant process. Thus, in order to address the processes in these studies adequately,

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the volatilization losses were considered as an additional product that neitherdecline nor

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repartition into the soil. The volatilization is thus treated as a separate sink for the parent

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compound, rather than as a partitioning process that can bring the parent compound back into

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the soil11. The volatilization loss was considered to occur in parallel to the biodegradation.

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Therefore, in this extended model the degradation and the volatilization of the compound were

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considered as two processes and separated so that individual rate constants could be calculated

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for the volatilization process as well as the degradation process. The calculation of DegT50 and

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DegT90 values was based on the fraction of radioactivity in extracts, which could be identified

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as parent substance. In general, the model assumes first order kinetics with k as an overall

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dissipation rate and c the concentration of test chemical according to following equation: dc = - kc dt

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(1)

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However, for the extended model it is assumed that k consists of two rate constants kV

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(volatilization rate) and kTt (transformation rate).

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(2)

k = kT + kV

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To describe the ratio of the two parallel processes the model internally uses “fractions”

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FV(volatilization fraction) and FT (transformation fraction) which can be calculated based on

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the individual rates for volatilization and transformation together with the overall decline rate

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as follows:

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FT =

kT k

( 3a)

FV =

kV k

(3b)


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For the structure of the standard and the extended model see Figure S12-13.

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3. Results and Discussion

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3.1. Pre-tests

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The results from the biodegradation tests carried out with 14C tetralin using different test setups

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after a 14 day incubation period in 02-A soil is shown in Figure S3. An incomplete mass balance

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was obtained for the soil degradation test carried out in a flow-through setup despite using a

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combustion oven to trap the volatile fraction in the form of completely oxidised 14CO2. This

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suggested that the volatilized fraction was either escaping or being adsorbed to the fittings used

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to connect the flow-through setup.

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modification could not be utilised to attain a complete mass balance, which is a validation

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criteria for the test. In contrast, a complete mass balance was obtained using test setup 1.

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Nevertheless, the test setup was still considered inappropriate as 98% of the applied

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radioactivity was volatilized and trapped in the tenax and thus unavailable for microorganism

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in the soil. This made biodegradation measurements impossible, which is the primary goal of

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the test. Additionally, the soda lime used as internal

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considered impractical for direct analysis of mineralization as it had to be ground and taken for

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combustion for radioactive analysis. The high amount of radioactivity in the tenax was

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attributed to a higher headspace volume of the sample bottle, a higher amount of tenax filled in

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the inserts, its dimensions and position within the sample bottle. A higher headspace volume

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will result in a greater mass fraction of the test substance partitioning into the headspace10,11,

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which could subsequently be trapped in the tenax thus making it unavailable for degradation.

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Considering these factors, a test using setup 2 with smaller headspace, a different type and

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positioning of the tenax absorber, NaOH as CO2 absorber and with regular oxygen monitoring

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was performed. The results observed using test setup 2 displayed a complete mass balance with

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only 20% of the applied radioactivity trapped on the tenax. Setup 2 was therefore considered

Therefore, the flow-through setup even with this

14CO

2


trap in the test setup 1 was also


280

operationally superior to setup 1, as it would permit measurement of biodegradation with less

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volatile losses. These results reveal the relevance of headspace and amount, dimensions and

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position of tenax in a closed test setup for the distribution and the fate of a volatile chemical in

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the test system.

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3.2. Main test

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3.2.1.

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Figure 2 shows the oxygen saturation in the headspace of the reference samples of test setup 2

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during the initial 28 days of the tetralin main study. The results showed almost no oxygen

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depletion in the samples applied without co-solvent during the incubation period. In contrast,

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for soil samples applied with co-solvent, different oxygen depletion trends were observed, with

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the organic carbon rich silty loam (03-G) showing the most rapid oxygen depletion relative to

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the other soils. In general, rapid depletion of oxygen saturation took place between day 0 and

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14 in all soil samples applied with co-solvent. If the oxygen saturation measured was below

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15% the samples were re-oxygenated with oxygen rich air to 18-20% saturation. Despite of

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oxygenation every 2-3 days, continuous depletion of oxygen saturation was observed between

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day 0 and 14 for all soils. This oxygen depletion could be attributed to the oxygen consumption

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due to the degradation of the co-solvent.

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These results suggest that the conditions in a closed biometer test setup can rapidly turn

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anaerobic without regular oxygen supply, if a co-solvent is not evaporated after the application

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of the test chemical. In all cases where the co-solvent can be evaporated without extensive

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losses of test chemical this is therefore preferable. In cases where test chemical losses are too

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high, and co-solvents cannot be avoided, such as when testing highly volatile chemicals,

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changes from aerobic to anaerobic test conditions may influence the degradation kinetics due

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to effects on the microbial communities. Therefore, we strongly recommend monitoring the O2

Oxygen monitoring in reference samples


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saturation during tests conducted in a closed biometer test setup if no evaporation of co-solvent

305

is performed. If the O2 saturation drops, supplement of additional oxygen is needed.

Figure 2 Oxygen saturation in the headspace of the reference samples for all four soils during the tetralin main study using test setup 2. Two reference samples were used, one being applied with and the other without solvent. The samples applied with solvent were oxygenated when oxygen saturation was < 15%, continuous depletion in the oxygen saturation was observed between 0-14 days. The oxygen measurements were done without the need to open the test vessel using an optical oxygen meter (Firesting, Pyroscience). 306

Mass balance

307

3.2.2.

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In line with the results observed in the preliminary tests, the average overall observed recovery

309

was 99.9% ± SD of 10.6 (N=90) for decane and 104.8% ± SD of 5.5 (N=90) for tetralin in these

310

studies. A mass balance of 100 ± 15% was observed in 88.9% (N=90), and 100% (N=90) of the

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test samples for decane and tetralin, respectively. Thus results from the main study confirm ACS Paragon Plus Environment


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that the test setup and approach that was developed to test volatile chemicals was highly

313

effective and reproducible in terms of obtaining a complete mass balance. The complete

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degradation time series for all soil types for both test chemicals from the main study are shown

315

in Figure S8-11.

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3.2.3.

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Figure 3 shows the mineralization of tetralin and decane, measured in the CO2 traps, the

318

volatilization, measured in the tenax tubes, and the NER formation, measured as residues in the

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soil after extraction. The 03-G and 02-A soils showed faster mineralization of tetralin than the

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other two soils. In particular, very rapid mineralization of tetralin was observed for the 03-G

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soil, in spite of the higher OC content in this soil compared to soil 01-A and 02-A. Slightly

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faster mineralization of decane was also observed in the 03-G soil. The higher mineralization

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rates in the 03-G soil can be explained by a combination of two factors. Firstly, the 03-G soil

324

had a higher amount of microbial biomass (See Table S6 and S7), and a higher microbial

325

activity as revealed by the higher oxygen depletion in the reference samples of this soil (Figure

326

2). Secondly, the volatilization was reduced due to the higher OC content, relative to the other

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soils, leading to a higher fraction of test substance remaining in the soil available for

328

degradation.

Competing processes (sorption, volatilization and degradation)


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Figure 3 Volatilization, Mineralization and NER formation of tetralin (left) and decane (right) in four different soils (See Section 2.2). Volatilization, Mineralization, and NER were calculated as a percentage of the initial applied radioactivity (%aR). The figure shows that volatilization is higher for low OC content soils (01-A, 02-A) in comparison to the high OC soils (03-G, 04-A). In contrast, fastest mineralization and NER formation was observed in 03G soil with the highest OC content. 329

The radioactivity trapped in the tenax in both sterile and non-sterile samples was identified as

330

100% parent compound from the specific chemicals analysis data. As it can be observed in

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Figure 3, a higher amount of test chemical was observed in the tenax for the soils with lower

332

OC relative to the soils with a higher OC suggesting greater volatilization from soils with lower

333

OC content. In the sterile samples containing tetralin and decane, the amount of test chemical

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trapped in the tenax was also higher in the soils with lower OC carbon content (See Table S4).

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These results illustrate how sorption of the test chemical to organic matter in the soil reduces



336

its volatilization19-23 and, with the extended residence time, extent of biodegradation improves

337

in a closed flask test setup.

338

The results show that the volatilized fraction of the test chemical gradually or rapidly increased

339

and reached a plateau (see Figure 3). No considerable drop in the volatilized fraction was

340

observed along the incubation period. Slight drops observed at some sampling points might

341

have been due to the effect of overall lowered radioactivity recovery. This confirmed that in

342

our closed incubation test setup, the volatilized fraction was not re-mobilized and the tenax

343

traps acted as intended, as a contaminant sink24,10 rather than a partitioning reservoir25,11.

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Although the KH value of decane is much higher than that of tetralin, the final fraction of

345

volatilized decane at the test end was slightly lower than for tetralin. However, for the sterile

346

samples the distribution into the tenax was in line with the KH values (Table S4). These

347

differences in mass distribution can be explained by the faster degradation rate of decane in the

348

non-sterile samples relative to its volatilization rate. Furthermore, decane has a higher Koc value

349

which improves its sorption to the organic fraction of the soil.

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A considerable portion of the applied radioactivity was recovered as NER in both the tetralin

351

and decane studies. The rate of NER formation was faster and higher for decane compared to

352

tetralin. Similar to the mineralization, highest and rapid NER formation was observed in the

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03-G soil for tetralin, whereas in the decane study the NER formation and mineralization at the

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end of study was more or less the same in all the soils. Studies on NER26-28 suggest that the

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formation of bio-NER is linked to mineralization. This is also apparent in our study. Recently

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a microbial to biomass turnover (MTB) method was developed for predicting the fraction of

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bio-NER29-30. This MTB method was applied using required inputs from our test and literature

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data to predict the bio-NER fraction (see Table S8). The short term bio-NER prediction was

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done exemplarily until 28 days for tetralin and until end of the test (12d for soil 01A, 02A and

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04A and 7d for soil 03G) for decane. The results showed that the predicted bio-NER was higher ACS Paragon Plus Environment

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than the total NER observed in the test by a factor of 1.19-1.77 for decane whereas for tetralin

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it was slightly lower by a factor 0.65-0.77 (for details see Table S9). Thus, MTB method also

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indicates high bio-NER formation for these test chemicals. This argument was also supported

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by the results observed in the sterile sample where almost no NER formation was observed (

Biodegradation of Volatile Chemicals in Soil - ACS Publications

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