Arctic and Subarctic Natural Soils Emit Chloroform and Brominated

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Arctic and subarctic natural soils emit chloroform and brominated analogues by alkaline hydrolysis of trihaloacetyl compounds Christian Nyrop Albers, Ole Stig Jacobsen, Erico Marlon Moraes Flores, and Anders Risbjerg Johnsen Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 09 May 2017 Downloaded from http://pubs.acs.org on May 15, 2017

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Environmental Science & Technology

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Arctic and subarctic natural soils emit chloroform and brominated analogues

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by alkaline hydrolysis of trihaloacetyl compounds

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Christian N. Albers1,2*, Ole S. Jacobsen1, Erico M. M. Flores3, Anders R. Johnsen1

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1350 Copenhagen K, Denmark

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2

Geological Survey of Denmark and Greenland (GEUS), Department of Geochemistry, Øster Voldgade 10, DK-

Center for Permafrost (CENPERM), Department of Geosciences and Natural Resource Management, University

10

of Copenhagen, Denmark

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3

Departamento de Química, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil

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* Email: [email protected], phone +45 91333557, address: Øster Voldgade 10, DK-1350 Copenhagen K

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1 ACS Paragon Plus Environment


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Abstract

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There has been increasing recognition of the occurrence of natural, halogenated organic compounds in marine

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and terrestrial environments. Chloroform is an example of a halogenated organic compound with natural

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formation as its primary source. Chloroform emission from soil has been reported from diverse Arctic,

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temperate and (sub)tropical ecosystems. The terrestrial environment is a significant source to the atmosphere,

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but little is known about the formation pathway of chloroform in soil. Here, we present evidence that

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chloroform is formed through the hydrolysis of trichloroacetyl compounds in natural, organic-rich soils. In-situ

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emissions of chloroform from soil in nine Arctic and subarctic ecosystems were linked to soil trichloroacetyl

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turnover. The residence time from formation of the trichloroacetyl compounds in soil to the release of

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chloroform to the atmosphere varied between 1 and 116 active months in unfrozen topsoil, depending on soil

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pH. Non-specific halogenation that leads to trihaloacetyl formation does not discriminate between chloride and

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bromide, and brominated analogues were formed alongside chloroform. Soil may therefore be a previously

27

unrecognised, natural source of brominated haloforms. The formation pathway of haloforms through

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trihaloacetyl compounds can most likely be extended to other ecosystems with organic topsoils.

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Introduction

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In this article, we report on the formation mechanism of chloroform and its brominated analogues in natural

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Arctic-subarctic soil. Chloroform has received considerable attention as a frequently detected pollutant in

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groundwater1. Increasing attention has also been focused on the effects in the troposphere of short-lived

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organohalogens, such as chloroform and its brominated analogues2-4. In the last 20 years, it has been

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recognized that natural formation is the primary source of chloroform5 and that substantial amounts of

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chloroform may be emitted from soils of temperate, coniferous plantations6-9 as well as natural forest

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floors10,11. Indeed, based on atmospheric measurements in Mace Head, Ireland, the main sources of chloroform

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in Europe are the less populated areas of Scandinavia, Ireland and Scotland12. This is in contrast to other

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volatile organohalogens that have their primary European sources in industrialized areas12.

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Although most previous studies have been conducted in forest plantations and other non-natural areas, it

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seems that the formation of chloroform is a common process in many natural ecosystems. However, there is

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very limited understanding about the mechanism of chloroform formation in soil.

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Soil organic matter from chloroform-emitting forest plantations has recently been found to contain chemical

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structures that can release chloroform at high pH13. The only known organochlorine structure that shows high

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stability at low pH and hydrolyses to release chloroform at high pH is the trichloroacetyl group14,15.

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Trihaloacetyl compounds are known intermediates in the nonspecific halogenation reactions that lead to

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haloform formation during chemical water chlorination and paper pulp bleaching14. We therefore hypothesized

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that the natural formation of chloroform and brominated analogs occurs through nonspecific halogenation of

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soil organic matter leading to the formation of trihaloacetyl compounds in soil. These compounds will

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eventually undergo nucleophilic attack by hydroxide ions (alkaline hydrolysis) and release haloforms (equation

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1). The rate of hydrolysis will depend on the concentration of the hydroxide ion16, which is determined by the

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soil pH.

SOM-R 54

HOX

O SOM-C-CX3

OH- hydrolysis

O CHX3 + SOM-C-O-

Trihaloacetyl compounds

(1)

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where SOM-R is soil organic matter with a reactive group, such as a phenol, quinone, ketone etc. X is a halogen

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atom, such as chlorine, bromine and possibly iodine.

57 58

At present, known halogenation mechanisms in the terrestrial environment include enzymatic reactions with

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flavin-dependent halogenases, α-ketoglutarate dependent halogenases, methyl halide transferases or heme-

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and non-heme haloperoxidases17,18 and non-enzymatic Fenton-like reactions19,20. Of these, only extra-cellular

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chloro- or bromoperoxidases and Fenton-like reactions have the capacity to form free reactive chlorine or

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bromine species such as HOCl and HOBr, and hence in theory lead to the formation of trihaloacetyl-groups in

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soil organic matter. Using carbon and chlorine stable isotopes analysis, it was recently demonstrated in a lab-

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experiment, that soil humic acid can be chlorinated by both hypochlorite and a commercial chloroperoxidase,

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which led to pH dependent formation of chloroform, most likely through hydrolysis of trichloroacetyl

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intermediates15,21. Indeed, most previous research on this subject has been conducted in the laboratory using

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chemical chlorination with hypochlorite, which is extremely artificial compared to natural processes.

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Trichloroacetyl compounds were detected in a limited study of three temperate, coniferous plantations13, but

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data on the general occurrence of trichloroacetyl compounds in natural environments as well as the natural

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occurrence of brominated analogs has not been reported previously. Empirical support of the trihaloacetyl

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hypothesis based on analyses of environmental samples is therefore strongly needed.



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The trihaloacetyl hypothesis has two testable predictions:

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1. Trihaloacetyl compounds will be formed in haloform-producing soils, so that there will be a quasi-steady

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state between trihaloacetyl formation and hydrolysis to haloforms. Trihaloacetyl compounds will hydrolyze

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faster at higher pH, and these compounds will therefore show a shorter residence time in soils of higher pH.

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2. The trihaloacetyl formation mechanism will be nonspecific and will not discriminate between chloride and

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bromide. Brominated analogues of chloroform will therefore be formed if bromide is present in the soil.

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We carried out a study to test these predictions for nine Arctic and subarctic ecosystems, in which chloroform

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emissions have recently been reported11 (Fig. S1).

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Experimental

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Study sites

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Four Arctic and five subarctic sites with different vegetation and soil properties were chosen for the study (Fig.

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S1, Table 1). At each site, a 12-m permanent transect was established, with a distance of three metres between

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the individual 0.06 m2 flux chamber bases. A detailed description of the transects and the study sites has

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previously been published11.

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Haloform analyses

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Emissions of chloroform and brominated analogues were determined as described previously11. Briefly, 20-L

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flux chambers were fitted onto the chamber bases for 65 minutes. Haloforms in the chamber headspace air

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were trapped and stored in custom-made thermal desorption tubes (89×6.35 mm, containing Carbotrap B (22

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mm), Carboxen 1003 (16 mm) and Carboxen 1000 (16 mm), Supelco, USA) for subsequent GC-ECD/MS analysis.

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Before entering the thermal desorption tubes, the air was dried by passage through a Nafion™ desiccant

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membrane dryer (DM-110-24, Perma Pure, NJ, USA). Each haloform emission was calculated based on four 1-L

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samples. The chloroform emissions in Table 1 are the mean of two (Kan and Nar sites) or four (Abi and Dis

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sites) sampling campaigns during the active season from late May to early September.

99 100

On 29 August 2014, 35 additional emission measurements were performed at site Kan-C, to better establish

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the observed correlation between chloroform and bromodichloromethane emissions. Mobile chamber bases


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instead of permanently installed bases were used for these samples, to allow the high number of

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measurements to be performed. Concentrations of CFC11, which is neither consumed nor produced in the soil

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and therefore should decrease in the chamber after each sampling, were used as a check that the system was

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not leaking11. In addition, only analyses showing emissions of more than 15 ng m-2 h-1 chloroform or 0.2 ng m-2 h-

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1

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uncertain. Out of 35 samples, 18 fulfilled these criteria.

bromodichloromethane were used for further data analysis, as emissions lower than these values were highly

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To sample soil air, a custom made probe comprising a 17-mm brass filter (Propartner, Denmark) connected to a

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4-mm inner diameter polyamide tube (MB-LONGLIFE™ PA-12, Propartner, Denmark) was pushed to a depth of

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20 cm. Soil air was then drawn using a membrane pump at 100 mL min-1 (210-1003MTX Twin Port Pocket

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Pump, SKC, UK). The first 200 mL air was discarded to clean the probe and the following 400 mL was sampled in

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thermal desorption tubes. A 0.45 µm PTFE filter (Midisart 2000, Sartorius, Germany) and a Nafion™ desiccant

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membrane dryer were placed between the probe and the pump to remove dust particles and water vapour.

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The system for collecting soil air was tested in the laboratory and found to neither emit nor adsorb measurable

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levels of haloforms.

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Soil samples

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Soil was sampled from the transects by removing living mosses and lichens and then hammering a steel core (Ø

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= 6 cm) to a depth of 10 cm. Two sets of soil samples were used in this study. The first set was sampled at the

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nine sites during 2012 before the emission measurements. The 2012 samples were sampled 0.8 m from the

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center of the chamber bases and used to determine total organic halogen. The second set, used for all other

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measurements, was sampled in 2014 at the center of each flux chamber after the emission measurements

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were terminated. The soil for analysis of total halogen and trichloroacetyl compounds was frozen on the day of

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sampling and stored frozen at -18 °C. Before analysis, the soil was freeze dried and ground to fine powder. In

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2014 an additional soil core was taken within each chamber base and stored at 5 °C until homogenization and

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shaking with water (1:2.5, soil:water) to determine pH and perform the analysis of chloride and bromide by ion

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chromatography (LC50-CD50, Dionex, CA, USA). Soil organic matter was determined as loss on ignition (550 °C,

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2 hours).

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Analysis of trichloroacetyl compounds



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The concentration of trichloroacetyl compounds in soil from the nine sites was quantified after alkaline

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hydrolysis followed by gas chromatographic detection of the formed chloroform as previously described13.

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Briefly, 0.5 g freeze-dried and homogenized soil was put into a 120-mL serum bottle and then 2.5 mL 0.6M

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NaOH was added and the bottle closed with crimp-caps containing an alumina-coated septum (Mikrolab

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Aarhus, Denmark). After overnight shaking, a 9-mL headspace sample was taken with an airtight syringe and

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injected into thermal desorption tubes for subsequent GC-ECD/MS analysis11. The chloroform in the headspace

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(>80% of total chloroform) was corrected for sorption to organic matter and dissolution into water based on

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partitioning experiments with different amounts of water and soil organic matter (supplementary methods).

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The concentration of trichloroacetyl compounds was calculated as chloroform equivalents. The weight-based

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trichloroacetyl concentrations were recalculated to area-based trichloroacetyl concentrations at each sampling

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point based on the weight of the 10 cm soil core. The area-based trichloroacetyl concentration was then

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divided by the mean chloroform emission at the same sampling point to obtain the mean residence time of

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trichloroacetyl compounds in units of “months with non-frozen topsoil” (active months) for that specific

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sampling point. Trichloroacetyl mean residence times at site Nar-B could not be reasonably calculated due to

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chloroform emissions being too low and hence uncertain.

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To test the pH-stability of trihaloacetyl compounds 0.2 g freeze dried and powdered Abi-A soil was weighed

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into 120-mL serum bottles and pH was adjusted with 5 mL of a 1M citrate buffer (pH 4.7-5.8) or phosphate

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buffer (pH 6.2-7.9) or just added 5 mL water (pH 4.3) and shaken gently for 24 hours at 23 °C.

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Analysis of total organic halogen in soil

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Total organic Cl and Br were determined using microwave-induced combustion and subsequent halogen-

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specific detection, as previously described22,23. Briefly, inorganic halogen was removed from the powdered

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samples by sequential washing (3 x 0.02M HNO3/0.2M KNO3 and 1 x water). The low pH during washing

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ensured minimal loss of soil organic matter and ensured that trihaloacetyl compounds were not hydrolyzed.

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The samples were then combusted in closed quartz vessels using 100 mg of sample in analytical triplicate. For

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soil samples containing less than ~20% organic matter, a pyrohydrolysis method was applied according to the

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conditions previously described24. Briefly, 100 mg of sample were mixed with 500 mg vanadium pentoxide in a

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quartz boat and heated (10 min at 1000 °C; water flow-rate 1.0 mL min-1; humidified air flow-rate 200 mL min-

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1

). In both methods, halogens were absorbed in 6 mL 50mM NH4OH that was diluted to 25 mL with water prior


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to analysis by ion chromatography (chlorine) and inductively coupled plasma mass spectrometry (bromine)23.

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Accuracy was evaluated using certified reference material of coal (NIST 1632c) and spiked samples were used

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to evaluate the recovery of halogens.

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Results and Discussion

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Organic halogen in Arctic and subarctic soils

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First, we determined the total organic and inorganic chlorine and bromine concentrations (Table 2) in soil from

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nine transects where chloroform emission measurements were performed. Organic chlorine was present in all

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chloroform emitting soils. These are the first such data reported for Arctic soils and interestingly, the total

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organic chlorine concentrations of 63-341 mg kg-1 (175-1457 mg kg SOM-1) were very similar to what has been

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reported for soils in warmer climates25,26. However, it is clear that chloroform emissions did not correlate with

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total organic chlorine in soil (Table 1 and 2, Fig. S2), and that total organic chlorine in soil therefore cannot be

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used as a proxy for chloroform emission. These results suggest that several chlorination mechanisms may occur

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simultaneously in soil of which only some (those that produce free reactive chlorine species) contribute to

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chloroform formation. An alternative explanation would be unspecific chlorination of different types of organic

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matter leading to chlorinated compounds with different residence times at the different study sites. Organic

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bromine was also present at all locations, ranging from 2-32 mg kg-1 (Table 2), corresponding to 6-90 mg kg

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SOM-1. Organic bromine data are very scarce in the scientific literature, but our data from Arctic-subarctic

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ecosystems are in the same range as those for temperate peatlands and coniferous plantations21,27. The organic

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chorine/bromine ratio showed a substantial variation between study sites, ranging from only 4 at site Kan-C to

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31 at site Abi-B (Table 2).

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Trichloroacetyl compounds and their hydrolysis to chloroform

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We then measured trichloroacetyl compounds in the soils of the transects and found that they were present in

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all of the 44 soil samples analyzed, but at very different concentrations (Table 1, Fig. 1). Between sites, the

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mean trichloroacetyl concentration varied 190-fold based on dry weight and 160-fold when expressed per soil

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area (Table 1). There was also considerable within-site variation, often up to tenfold between soils in the five

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haloform flux-chambers at each site. High trichloroacetyl concentrations were found only in soils with a pH

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below 5.8 (Fig. 2a), which was in line with the first prediction that trichloroacetyl compounds become

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increasingly unstable at higher soil pH. Furthermore, soils with higher pH (pH 6 to 7) showed up to 84 times



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higher chloroform emission:trichloroacetyl ratios compared to soils with lower pH (pH between 4 and 6). This

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means that a low steady state concentration of trichloroacetyl compounds may be found even when

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chloroform emissions are high. Our results are the first to demonstrate a general and natural occurrence of

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trichloroacetyl compounds, and hence to support that chloroform is formed through trichloroacetyl

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compounds in completely natural ecosystems. Furthermore, the correlation with pH has not previously been

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demonstrated for environmental samples of any kind.

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It can be speculated that transport processes in the soil column might modify the emission fluxes to an extent

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that they become independent of the formation processes, this is however unlikely for our soils.

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Physicochemical processes such as sorption/-desorption and varying porosity would not change haloform

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fluxes in a quasi-steady-state system. Degradation processes, on the other hand, may weaken the link between

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haloform production and emission. Haloform degradation via halorespiration, which may take place under

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highly reduced conditions, was probably negligible for most of our study sites that were oxidized down to the

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permafrost or at least to a depth of 70 cm. One notable exception was the Kan-C site where the soil was

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saturated with water, which may have led to locally reduced conditions, though the water was not stagnant

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and methane emissions were undetectable or negative. Alternatively, some trihalomethane may be degraded

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via microbial oxidation in the topsoil9. We did not determine the aerobic degradation potential of our soils, but

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this process may account for some of the unexplained variation in our dataset.

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By combining the trichloroacetyl concentrations and chloroform emissions, we could estimate a mean

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residence time in soil for the trichloroacetyl compounds. These estimates were based on the assumptions that:

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1) most of the trichloroacetyl compounds that release chloroform are located in the topsoil and 2) emission to

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the atmosphere is the main loss route of chloroform from Arctic soils. These assumptions are a simplification,

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but they are supported by three facts: 1) almost all organic matter in the Arctic soils and in temperate

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coniferous plantations is located in the topsoil, 2) previous studies conducted in temperate coniferous

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plantations show that chloroform is primarily formed in the topsoil7,9, and 3) 90% of the net chloroform

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formation is released to the atmosphere in temperate coniferous plantations9. In Arctic ecosystems, the topsoil

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is frozen most of the year, and chloroform emissions are considered negligible during this period11. We

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therefore estimated trichloroacetyl residence times based only on the active periods when the soils were not

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frozen. The estimated mean residence times varied from three active months at site Kan-C to 59 active months

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at site Abi-B (Fig. 2b) with a full range from 1 to 116 months for individual flux chambers (Fig. S3). The


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residence time decreased with increasing soil pH (Fig. 2b). This pH-dependence strongly supports our

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hypothesis that the soil chloroform emissions originated from alkaline hydrolysis of trichloroacetyl compounds.

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The mean residence time often showed high within-site variation (Fig. 2b), which was partly caused by within-

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site variation in soil pH (Fig. S3). At site Nar-C, the organic layer extended 10 cm that were used for determining

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the trichloroacetyl concentrations. This means, that we may have underestimated the area-based

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trichloroacetyl concentrations for this site if trichloroacetyl compounds were present in significant amounts

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below 10 cm. In addition, the mean residence times may have been slightly overestimated if some of the

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chloroform was oxidized in the topsoil or leached.

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We further investigated the influence of pH on the stability of the natural trichloroacetyl compounds in

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laboratory tests with soil from the site with the highest chloroform emissions (Abi-A). The chloroform-release,

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i.e. the trichloroacetyl stability, showed strong pH dependence with increased hydrolysis at higher pH (Fig. 2c).

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Such a strong pH response has not previously been shown for any environmental sample containing natural

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trichloroacetyl compounds, but was similar to what has been reported for the hydrolysis of trichloroacetyl

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compounds formed by non-natural, chemical chlorination14,15. It should be noted that the laboratory

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experiment (Fig 2c) shows the pH dependence of trichloroacetyl hydrolysis under laboratory conditions, which

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cannot be used for prediction of in situ residence time (Fig. 2b). This is exemplified by the fact that the pH of

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the Abi-A soil on average was 4.2 (Table 1), which corresponds to 1% hydrolysis of the trichloroacetyl

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compounds in 24 hours at pH 4.3 (Fig 2c). This is faster than suggested by the in-situ mean residence time of

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38 months (Fig 2b), but these two numbers are not directly comparable. First, the hydrolysis was determined at

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23°C, which is much warmer than average in situ soil temperatures during the active months, which was 6°C11.

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Second, soil colloids probably act as strong adsorbers of the trichloroacetyl molecules. Since the negatively

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charged colloids attract hydrogen ions, the difference in acidity between colloid surfaces and the bulk solution

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may be up to two pH units. Adsorbed molecules therefore ”experience” much lower pH than molecules in the

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bulk solution28,29. As the pH dependence (Fig 2c) was determined under laboratory conditions, where the soil

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was diluted 25-fold in 1M buffer, sorption equilibrium would presumably be shifted and more of the

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trichloroacetyl compounds would be present in the bulk liquid due to dilution and high ionic strength.

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Consequently, the trichloroacetyl compounds would be more labile than in-situ even at the same bulk pH.

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Co-emission of brominated chloroform analogues



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The formation of trichloroacetyl compounds requires nonspecific halogenation by free, reactive chlorine

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species such as HOCl16, equation (1). The formation of HOCl by either chloroperoxidases or the fenton reaction

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is, however, unspecific and other halides than chloride may be oxidized by both reaction mechanisms. This

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means that HOBr (reactive bromine) would also be formed, if bromide ions were present18,19,30. Furthermore,

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HOCl may react spontaneously with bromide and convert into HOBr due to the higher standard oxidation

260

potential of bromide compared to chloride31. If the proposed reaction mechanisms were correct (equation 1),

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then brominated chloroform analogues would be formed along with chloroform when bromide is present. The

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concentration of free bromide was below the detection limit (

Arctic and Subarctic Natural Soils Emit Chloroform and Brominated

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