Algal Research 79 (2024) 103478

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Algal Research
journal homepage: www.elsevier.com/locate/algal

Review article

Analytical methods for the analysis of bromoform in red seaweed

Asparagopsis armata and Asparagopsis taxiformis – A review
Joshua L. Hutchings , Yevgeniya Grebneva , Sarah J. Dilmetz , Daniel W.M. Pincher ,
Peter Hoffmann *
University of South Australia, Clinical and Health Sciences, Australia

A R T I C L E I N F O A B S T R A C T

Keywords: Global warming is a significant risk to all species on this planet and is exacerbated by the release of greenhouse
Bromoform gases (GHG) into the atmosphere. Agricultural practices represent >20 % of the total GHG emissions with 14.6 %
Methanogenesis being ruminant livestock farming alone. The red seaweed species Asparagopsis taxiformis and Asparagopsis armata
Asparagopsis
are a significant source of bioactive substances including brominated compounds that have been shown to reduce
Algae
Gas chromatography
methane emission of livestock by up to 98 % when supplemented into livestock feeding regimes. Bromoform is a
Quantitative methods major contributor to the reduction in methanogenesis and can be found in remarkably high concentrations of
1–5 % in the dry mass of Asparagopsis species. The high toxicity of bromoform makes it essential for the emerging
seaweed industry to accurately quantify bromoform in Asparagopsis. Bromoform quantification is normally
conducted on a hyphenated gas chromatography system due to its volatility and routine quantification methods
have been established for over 60 years in water and air. The complexity of the algal matrix provides a challenge
for researchers and industry alike to extract and quantify bromoform in a manner that is safe, green, cost-
effective and reflective of the actual concentration. This review summarises gas chromatography instrumenta­
tion and methods, non-chromatography instrumentation potentially useful for quantification, sample preparation
methods, in-field analysis, and storage and stability of bromoform in seaweed.

1. Introduction
Global warming is a significant issue that threatens all species on this
planet by an increase in land and ocean temperatures, leading to more
frequent and extreme meteorological events [1]. Greenhouse gases
(GHG) contribute to global warming with the main source being human
practices such as the burning of fossil fuels, deforestation,
manufacturing and agriculture that produce N2O, CH4 and CO2 [2].
Currently, livestock farming is the source of 14.6 % of all human con­
tributions to GHG emissions producing an estimated 7.1 billion tonnes of
CO2-equivalent (CO2e) per annum [3]. This is mostly through ruminant
animals, like cattle and sheep, as they metabolise their feed through
microbial fermentation creating the gaseous waste of CH4 and CO2
[4–7]. The consumption of meats will rise with the population due to the
inherent nutritional value and cultural significance perceived by society
[8]. A growing population expected to reach 9.7 billion people by 2050
and a lack of sustainable technologies is exacerbating the problem [9].
The family of Bonnemaisoniaceae, belonging to the large phyla of
marine red algae, Rhodophyta, contains two noteworthy species of the
red seaweed Asparagopsis, A. armata and A. taxiformis. Asparagopsis spp.
have been shown to produce a wide range of halogenated metabolites
with biological activities which have been demonstrated to reduce
methane emissions whilst increasing feed efficiency when incorporated
into ruminant feed [10–12]. Implementing this technology into routine

Abbreviations: GHG, Greenhouse Gases; NMR, Nuclear Magnetic Resonance; SLE, Solid-Liquid Extraction; LLE, Liquid-Liquid Extraction; GC, Gas Chromatog­
raphy; EAE, Enzyme Assisted Extraction; MS, Mass Spectrometry; SPE, Solid Phase Extraction; HS, Headspace; SPME, Solid Phase Microextraction; SBME, Solvent Bar
Microextraction; PT, Purge & Trap; ECD, Electron Capture Detector; RSD, Relative Standard Deviation; VOC, Volatile Organic Compound; XSD, Halogen Specific
Detector; FID, Flame Ionisation Detector; m/z, Mass-to-Charge; EI, Electron Ionisation; Q, Quadrupole; MS/MS, Tandem Mass Spectrometry or Triple Quadrupole;
MRM, Multiple Reaction Monitoring; SIM, Selective Ion Monitoring; SIFT-MS, Selected Ion Flow Tube-Mass Spectrometry; LOQ, Limit of Quantification; LOD, Limit
of Detection; ISTD, Internal Standard; SL, Splitless; S, Split; FD, Freeze-Dried; FF, Fresh Frozen; LPME, Liquid Phase Microextraction; TOF, Time-Of-Flight; IR,
Infrared; PMR, Proton Magnetic Resonance; DBP, Disinfection By-Products; pptv, Parts Per Trillion Volume.
* Corresponding author.
E-mail address: Peter.Hoffmann@unisa.edu.au (P. Hoffmann).

https://doi.org/10.1016/j.algal.2024.103478
Received 13 September 2023; Received in revised form 27 November 2023; Accepted 13 March 2024
Available online 19 March 2024
2211-9264/© 2024 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
J.L. Hutchings et al.
ruminant agricultural practices could create more sustainable farming
by reducing GHG emissions from livestock which would be a great
benefit to the environment, farmers and consumers. A. armata and
A. taxiformis, have been shown to inhibit methane production by up to
98 % in vivo when incorporated into ruminant feed [6,10,12]. These
seaweeds are native to New Zealand and Australia but are considered
extremely invasive in European oceans making them widely distributed
throughout the ocean in temperate, sub-tropical and tropical regions
[13,14]. They can be sustainably harvested in the ocean however
aquaculture farming of A. armarta has been successfully developed,
unlike A. taxiformis [14].
The lifecycle of Asparagopsis consists of haploid stages (n), gameto­
phytes and diploid stages (2n), carposporophyte and tetrasporophyte
(Fig. 1) [15–17]. Gametophytes are macroalgae characterised by their
plumose branches and extruding barbs. The male haploid gametophytes
release gametes to fertilize the gametes produced in the female game­
tophyte cystocarps (reproductive structures within the algae). The
fertilized gametophyte (carposporophytes) releases diploid carpospores,
which are single cell units, into the surrounding water for dispersion of
the genetic material. These carpospores grow into tetrasporophytes,
pompom-like structures made up of trisiphonous filaments. They un­
dergo meiosis to form haploid spores which grow into the gametophytes.
The gametophytes and tetrasporophytes are defined as free-living algae
but differ significantly in morphology and physiology; the gametophytes
are found secured into the subtidal substrata whereas tetrasporophytes
attach to other algae through their trisiphonous filaments which con­
tributes to their ability to quickly proliferate and outcompete native
marine flora [18].
Red seaweed is known to contain numerous bioactive compounds
involved in antimicrobial and antiviral activity, enzyme inhibition and
antioxidation [19]. Halocarbons, which are small organic molecules
containing one or more halogen atoms, are major metabolites in red
seaweed and contribute to bioactivity [19]. The more abundant chlorine
and bromine containing compounds in red seaweed, particularly
Asparagospis, include tribromomethane (bromoform), dibromochloro­
methane, dibromoacetic acid, bromochloroacetic acid, dibromo­
methane, tetrabromomethane and dibromonitromethane; iodine
compounds are far less abundant [20,21]. Unlike Asparagopsis (Bonne­
maisoniaceae asparagoides), other members of the Bonnemaisoniaceae
family which include B. nootkana, B. hamlfera and Trailliela intricate are
unable to produce halomethanes and instead, produce a range of C7-C9
halogen containing ketones, alcohols and carboxylic acids [22,23]. One
of the proposed reasons for this is the lack bromoperoxidases which
oxidize bromide anions into bromonium intermediate cations required
to form halomethanes such as bromoform [24–26]. The abundance of
some of these major halogenated compounds in A. armata and
A. taxiformis can be seen in Table 1.
Fig. 1. The lifecycle of Aspargopsis armata adapted from Félix et al. 2021 [16].
Figure generated at https://www.biorender.com/.
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Algal Research 79 (2024) 103478
A variety of factors affect the concentration of bromoform and the
overall halocarbon profile in red seaweed. McConnell & Fenical 1976
observed a marked difference in the dominant halocarbons not only
between A. armata and A. taxiformis, but also within the A. taxiformis
species harvested from different geographical locations [22]. Addition­
ally, a review by Kladi, Constantinos & Roussis 2004 provides a
comprehensive list of the halogenated volatile compound profiles for the
gametophyte and tetrasporophyte life stages of both A. armata and
A. taxiformis, showing a tremendously larger profile in gametophytes
[11]. Parchemin et al. 2023 used an interesting approach to compare
temporal, spatial and interspecies differences in the metabolome
combining data from gas chromatography and liquid chromatography
mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy
of A. armata and A. taxiformis. They found that the variation of metab­
olome was significantly associated with water temperature, which is
indirectly caused by the seasonality of sampling and sampling location.
Autumn and spring months, in particular October and March compared
to other months of the year sampled from Straits of Messina in Italy,
Bretagne, Banyuls-sur-Mer and Marseille in France and Moorea in
French Polynesia, tended to show the least variation in the metabolome.
This was perhaps due to the smaller variation in day/night temperature
and less fluctuation from day to day. It was also suggested that the
shallow sampling depth of 0 to − 1 m for A. armata contributed to less
inter-group variability when compared to A. taxiformis which was
sampled at a depth of between − 5 and − 15 m [29]. It should be noted
however, that it is not clear what life stage or anatomical section of the
algae was collected for analysis and some variation in the metabolome
could be attributed to this, given different gene and protein expression
in different tissue types will alter the metabolites produced [30]. This
highlights the importance of well-planned sample collection and thor­
ough reporting of experimental methods. Regarding spatial variation for
A. taxiformis, Greff et al. 2017 selected seaweed samples from both
tropical and temperate zones and measured higher levels of bioactivity
in temperate zones which indicated a heightened defence against mi­
crobes using halogenated metabolites such as bromoform [21,31].
The production of bromoform by red seaweed increases with envi­
ronmental and oxidative stress and provides a defence mechanism from
herbivorous sea animals and pathogenic bacteria [21,32,33]. Marshall
et al. 1999 found that increases in light intensity resulted in higher
concentrations of released halocarbons, with peak bromoform produc­
tion at 12 h followed by a gradual decrease over the remaining 36 h of
their experiment [34]. Low light conditions tended to stimulate pro­
duction of dibromomethane and dibromoethylene which were inversely
correlated to bromoform production. More recently, Torres et al. 2023
supported the results of Marshall and co-workers, also finding that
production rate of bromoform tended to decrease at very high levels of
irradiance and white light containing white intermediate wavelengths
promoted bromoform formation with indoor A. taxiformis tetraspor­
ophyte cultures with long term cultures exposed to <60 μmol photons
m− 2 s− 1 as a recommendation [35]. It was determined by Paul et al.
2006 that the concentration of internal bromoform is not correlated with
bromoform released into the surrounding water, acting as a possible role
in feeding deterrence in addition to surface-mediated bacterial protec­
tion. It was also found that an absence of bromide in the culture medium
resulted in the absence of halogenated metabolites due to the loss of
refractile vesicles, leading to an increased susceptibility to surface
colonisation by bacteria. [21]. The abundant expression of bromoform
in algae due to oxidative stress has led to the high concentrations found
across the ocean. It has a reputation for depleting the ozone layer as it
reacts readily with reactive oxygen species, ozone, upon evaporation
from the ocean [36,37].
Bromoform, along with other halocarbons such as dibromochloro­
methane, are volatile constituents of red seaweed primarily responsible
for the reduction of methane production by inhibiting metalloenzymes
in the Wolfe cycle. The structural analogy with methane allows for
competitive binding of these halomethanes to target enzymes M
J.L. Hutchings et al.
Table 1
Reported concentrations of the most abundant halogenated compounds in Asparagopsis spp.
Species Compound (% dry weight)
BF DBCM
A. armata 1.45 (T) 0.02 (T)
1.67 (G) 0.03 (G)
0.10–1.04 – –
A. taxiformis 0.42 0.014
0.1723 0.00158
T, Tetrasporophyte; G, Gametophyte; BF, Bromoform; DBCM, Dibromochloromethane; BCA, Bromochloroacetic acid; DBA, Dibromoacetic acid; DBM,
Dibromomethane.
methyltransferase and methyl coenzyme M reductase [5,38]. To help
understand the complex changes that occur in ruminants when exposed
to feed containing bromoform, several authors conducted in-vitro and
in-vivo experiments. In-vitro assays conducted by O'Hara et al. 2023
used rumen medium from canulated beef heifers to investigate changes
in ruminant microbial communities when grown on a 50:50 barley
silage: barley straw substrate with or without a 2 % dry mass addition of
either seaweeds A. taxiformis, Mazzaella japonica or Palmaria mollis. A
progressive change in microbial genera over the 13-day assay occurred
only in response to A. taxiformis addition, seeing almost a complete
disappearance of methane-producing microbes as well as a reduction in
major volatile fatty acids (VFA) and fibrinolytic bacteria primarily
involved in recalcitrant lignocellulosic biomass digestion [39]. In
agreement, Machado et al. 2016 saw a decrease in VFAs and CH4 for
both A. taxiformis and green algae Oedogonium sp. in a dose-response
manner. Asparagopsis proved to be a more potent methane inhibitor
than Oedogonium, providing a > 99 % CH4 reduction with >2 % dose
compared to a 61.6 % CH4 reduction at 100 % dose, respectively [28].
An extensive in-vivo experiment carried out by Krizsan et al. 2023
used only a 0.5 % dry mass A. taxiformis supplementation on Nordic Red
dairy cows and found no significant difference in methane-producing
Archaea, lower CH4 emissions and increased H2 and CO2 emissions
when compared to the control. Increases in H2 and decreases in CH4 are
proposed to be due to redirection of propionate synthesis in Provotella
spp., which increased in proportion in the treatment group [40]. The
effect of Asparagopsis feed addition to the intestinal microflora of post-
weening ruminants has not to our knowledge been explored for bro­
moform or other halogenated compounds. However, Meale et al. 2021
investigates how supplementation of a synthetic compound, 3-nitrooxy­
propanol, from birth to 3 weeks post-weaning in calves increased the
persistence of methane reductions in calves to at least 1-year post-
weaning, with 17.5 % lower emissions than those not supplemented
from birth [41]. It was also demonstrated the early feed addition of 3-
nitrooxypropanol maintained a microbial community phenotype that
favoured H2 production and suppressed microbes associated with CH4
generation. A similar study that investigates the persisting microbial
changes and CH4 reduction with bromoform would be tremendously
beneficial to this field of research and should be considered in the future.
Abbott et al. 2020 and Glasson et al. 0.2022 extensively review the
known impacts of brown, red and green seaweeds on ruminant microbes
and discusses archaeal and bacterial biosynthetic pathways associated
with methane inhibition [4,5]. Besides the well-established action of
bromoform, Milledge, Nielsen & Harvey 2019 suggested that some
tannins and flavonoids found in Asparagopsis may also contribute to anti-
methanogenesis [42].
Bromoform is a high-density liquid at room temperature and toxic by
inhalation, skin absorption and ingestion, primarily affecting the central
nervous system [43]. Each batch of seaweed grown and harvested could
contain varying amounts of bromoform due to the conditions of growth
and life stage [21]. Therefore, to safely implement seaweed supple­
mentation in feed, accurate quantification of bromoform concentrations
is required to ensure they do not exceed safe limits and still provide an
adequate degree of methane reduction in livestock. There is currently no
Algal Research 79 (2024) 103478
Ref
BCA DBA DBM
0.14 (T) 0.12–2.6 (T) – [21]
0.08 (G) 0.02–1.1 (G) – [21]
– – [27]
– – 0.00001 [20]
0.00098 0.00009 – [28]
Australian legislation that dictates the maximum concentration of
halomethanes allowed in bovine meat or milk products however, the
Medical Research Council have long established a limit of 0.25 mg/L
individually or total trihalomethanes in drinking water [44].
Accurately quantifying the concentration of bromoform in seaweed
is important from a health and business prospective. The volatility of
bromoform presents challenges at all timepoints from collection to
feeding the livestock as there is no certainty of retention in the seaweed,
affecting dosages and assurances to the buyers. To overcome these
challenges technologies and processes could be developed to be inte­
grated into the workflow including analysis at the point of collection,
valid analytical methods, and optimised sample preparation, storage
and stability conditions. The current literature regarding these processes
will be discussed in this review, including technologies that could be
used to achieve an accurate and robust workflow for seaweed analysis
from field to lab to farms to guarantee product quality.
2. Sample preparation
Vital to any quantitative analysis of volatile compounds is a robust
sample preparation workflow to optimise analyte recovery to reflect the
true concentration in a sample in addition to high precision and sensi­
tivity [45]. When developing a suitable workflow for the emerging
seaweed industry to measure the concentration of bromoform and other
anti-methanogenic compounds, minimising the cost, the use of
dangerous or toxic solvents, workflow and analytical apparatus
complexity, and sample preparation time must be considered.
2.1. Preparation of raw material
Seaweed in the gametophyte life stage consists of various unique
structures including the leaves, stem and roots which when sampled and
analysed, can induce variation between samples of the same plant. The
use of a blender or homogeniser to liquify and blend all fresh or fresh-
frozen gametophyte structures before extraction is a viable way of
decreasing experimental variation. Alternatively, freeze-drying is an
effective way of removing moisture from fresh-frozen samples at low
temperature under vacuum while mostly retaining volatile compounds
by sublimating ice [46]. Freeze dried samples can then be ground to a
fine powder in a blender or mortar to increase homogeneity [47].
Freeze-drying is however an energy intensive process, is time-
consuming especially for large quantities of material, can produce
inconsistently dried material that is irregular in structure such as
gametophyte material and is difficult to up-scale to a commercial level
required for animal feed production [48]. The rate at which a sample is
frozen in preparation for freeze drying will also contribute to drying
time, with faster freezing creating smaller ice crystals that sublime
slower than larger crystals. Larger ice crystals, however, may penetrate
adjacent cells and release metabolites such as bromoform into the vac­
uum, so a balance in freezing rate is a consideration for preparing
samples for analysis [49]. In contrast to multi-structured gametophytes,
tetrasporophytes are homogenous in structure and metabolite distribu­
tion, and are better suited to immediate solid-liquid extraction (SLE),
3
J.L. Hutchings et al.
negating the need to freeze-dry and powder.
As an alternative to freeze-drying, edible oil submersion of the raw
product and the subsequent use of the bromoform-rich oil has been
explored by Magnusson et al. 2020 and further evaluated by Tan et al.
2022 who compared this technique to freeze-drying for the conservation
of bromoform [48,50]. This technique can be considered a SLE for the
usable product, however a further liquid-liquid extraction (LLE)
extraction is conducted for gas chromatography (GC) quantification of
bromoform using methanol. In both studies however, no internal stan­
dard was added during the initial stages of sample preparation to assess
the recovery of bromoform in the different matrices and extraction
performance. The stability of bromoform using these methods will be
discussed in a later section of this review.
2.2. Solid-liquid extraction
Analysis of seaweed samples by liquid injection GC requires the
analyte to be extracted from the solid seaweed matrix and into a suitable
solvent, a process known as SLE or leaching [51]. Solubility of the
metabolite and solvent need to be complementary to effectively recover
the metabolite of interest and minimise the extraction of interfering
compounds into the liquid matrix. Commonly used solvents for bro­
moform extraction include methanol, hexane, pentane and dichloro­
methane, all being volatile and directly compatible with GC analysis. In
many protocols, methanol is used to extract bromoform however there
are cases were bromoform that has been released into fresh or salt water,
LLE with pentane is used [52,53]. Methanol has long been used as an
extraction solvent for a wide range of compounds, being cheap and
effective but also toxic and highly flammable, leading researchers and
industry to investigate alternative options. Green solvent alternatives,
which have been detailed extensively by Clarke et al. 2018, are assessed
on five main factors and should be renewable, sustainable, non-toxic,
recyclable and scalable [54]. Green solvents used for high value com­
pound extraction from agricultural food waste in particular has been
thoroughly reviewed by Torres-Valenzuela, Ballesteros-Gómez & Rubio
2019. While not all of the explored solvents discussed by Torres-
Valenzuela and co-workers are compatible with GC due to their low
volatility or are relevant to seaweed, it showcases technologies and
solvents that have potential for effective extraction of small molecules
such as bromoform [55]. Ethyl acetate, which can be produced by some
yeasts from sugar, is an environmentally friendly and non-toxic alter­
native to methanol which has been previously used to extract bromo­
form for GC analysis from treated water [56], soil [57] and meat
products [58]. The extraction solvent-to-sample ratio, time and tem­
perature play key roles in obtaining results that are representative of the
absolute concentration of bromoform within the sample.
A detailed method of bromoform extraction and analysis from red
seaweed published by Romanazzi et al. 2021 involved a double solvent
extraction in methanol of either fresh or freeze-dried seaweed at a ratio
of 1:10 or 1:100, respectively, for 30 min at 0 ◦ C with sonication [59].
Results concluded that the first extraction recovered >80 % of the
bromoform in the sample and the second extraction increased the re­
covery to 94 % and 99 % for freeze-dried and fresh seaweed, respec­
tively. Subsequent extractions provided negligible increases in recovery.
Other published methods followed similar protocols but with varying
extraction multiples, solvent ratios, extraction times and temperatures
[21,28,52,53]. The inconsistency between protocols indicates there is an
opportunity to optimise the standard protocol to achieve more repre­
sentative results.
2.3. Liquid-liquid extraction
Liquid-liquid extraction requires the analyte to be in a liquid matrix
which is then thoroughly mixed with a miscible or partially miscible
solvent where the analyte will partition between each phase as equi­
librium is established once agitation has ceased. Complete coalescence
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Algal Research 79 (2024) 103478
of common liquids then allows sampling from each phase [60]. The
molar ratio of the analyte in the organic to aqueous phase at equilibrium
is called the partition coefficient and is a determining factor when
selecting a suitable LLE solvent [61]. SLE can be conducted prior to LLE
to provide a liquid matrix for the addition of the partitioning solvent. For
bromoform emissions from seaweed samples, extracting from a seawater
aqueous phase is a common approach. Authors conducting analysis of
bromoform in water sources frequently use LLE in combination with
liquid or headspace GC techniques which are well-established methods,
employing n-hexane [61–63] and pentane [63,64]. Separating the
organic and aqueous phases in LLE can be challenging if preparing a
liquid injection, especially if the phases are partially miscible which can
induce inconsistencies in data, and normally requires a large volume of
solvent. Headspace methods negate the separation of phases and extract
volatiles from the organic top layer, providing that the analyte partitions
by the majority to the organic phase.
2.4. Enzyme-assisted extraction
Compounds of interest may be present in various forms within the
matrices, either free or bound to structural constituents. Free com­
pounds can be readily analysed following a simple solvent extraction,
however, bound compounds must be released from the matrix using
various methods such as thermal treatment, acid or alkaline digestion,
chemical treatment or enzyme-assisted extraction (EAE) [65]. Macro­
algae such as Asparagopsis contain a diversity of oligo- and poly­
saccharides, providing structure to the cell walls and influencing the
composition of symbiotic, epiphytic bacteria on the algal surface [66].
Sulphated polysaccharides, specifically sulphated galactans, are major
water-soluble components of the Rhodophycean outer cell wall which
form gels when hydrated which create a physical barrier that reduces
enzymatic access to target sites [67,68]. Work by Rodríguez Sánchez
et al. 2023 builds on previous literature to determine the fine structure
of sulphated polysaccharides in A. taxiformis using NMR spectroscopy,
reinforcing findings of unusual sulfation patterns and several different
branched agaran and carrageenan species [69]. Additionally, other
complex saccharide species such as lignans and carrageenan which may
hinder enzymatic hydrolysis are also present and their proportions vary
depending on the algal life stage and growing conditions [70]. Paul, Nys
& Steinberg 2006 demonstrated that many of the potentially autotoxic
secondary metabolites including bromoform are contained within gland
cells, inside parent cells and thus would require disruption of the cell
walls to release the metabolites, whether that be enzymatic digestion or
other methods [21]. Higher extraction efficiency, greener processes,
high selectivity and low toxicity are key advantages of using EAE when
compared to acid or alkaline treatments, which can hydrolyse bonds
between extremely resistant plant cell wall components such as lignans,
pectin, cellulose and hemicellulose, thereby permitting release of cell
contents for analysis and increasing the efficiency of a subsequent
extraction [71]. Factors including temperature, time, pH, particle size
and substrate type can affect the efficiency of enzymes, therefore it is
essential to select an enzyme that is optimised for a condition likely to
retain the compounds of interest [72,73]. For example, an enzyme that
acts faster at lower temperatures might be more appropriate when
investigating highly volatile haloforms or thermally unstable
compounds.
EAE has been previously applied to workflows and reviewed for the
analysis of polyphenolics [74], lipids, pigments, polysaccharides and
peptides in plants [74–78]. Given these examples of EAE being suc­
cessfully employed to extract valuable constituents from seaweed, it is
clear that EAE is a valuable tool in the biotechnology space. The
extraction of various bioactive saccharides in seaweed has been subject
to extraction on an industrial scale using alkaline solvents and novel
technologies, including EAE [79]. Sánchez-Camargo et al. 2016 used the
carbohydrase Viscozyme L and protease Alcalase 2.4 L FG to hydrolyse
the brown algae Sargassum muticum for the extraction of phlorotannins
J.L. Hutchings et al.
and measurement of the extract antioxidant capacity [71]. A higher
extraction yield of 13.6 ± 1.4 % and 20.6 ± 0.1 % total phenols was
observed after 2 h using Alcalase and Viscozyme, respectively, when
compared to a 2.5 h extraction using a heated 1 M aqueous NaOH so­
lution. The extraction of fatty acids from the microalgae Nannochloropsis
spp. using various extraction methods has been extensively reviewed by
Brennan & Regan 2020, finding that a mixture of the enzymes cellulase
and hemicellulase gave a higher fatty acid recovery when compared to
the individual use of the enzymes [80]. The volume of enzymes required
for large-scale extractions was noted to be costly but was argued by
Wang et al. 2017 that Novozyme enzymes could be reused several times,
consequently increasing the value for money of EAE [81].
The extraction of small molecular weight volatile metabolites using
EAE has not been thoroughly documented [82]. This is especially true
for algal sources, however, volatile oils from various terrestrial plant
materials have been investigated. A review by Wijesinghe & Jeon 2012
evaluated the extraction of industrially important metabolites from
seaweed, including bioactive polysaccharides and peptides, carotenoids
and polyphenols but gave no mention of volatile components [83]. They
reinforced the importance of EAE, noting a range of different enzymes
that target mainly structural carbohydrates and significant to this study,
the optimal temperature for the enzymes which ranged between 40 and
60 ◦ C. Chacko & Vasudevan 2016 compared 4 common enzymes for the
treatment of cardamon spice followed by the GC–MS analysis of volatile
flavour compounds, finding that Viscozyme gave the highest oil yield in
almost all assessed incubation temperatures, pH and enzyme concen­
tration conditions when compared to other common enzymes. Inter­
estingly, when compared to the control of no enzyme pre-treatment, the
GC–MS spectra for many of the flavour compounds showed no signifi­
cant difference for enzyme-treated cardamon which is in agreement
with Sowbhagya et al. 2010 who investigated volatile oil components in
cumin seeds [84,85]. Further research should investigate whether EAE is
beneficial for the extraction of bromoform and other volatile brominated
compounds from seaweed, and what is the most effective enzymatic
regime to achieve higher efficiency extraction. Given the complexity of
the cell wall polymers in Asparagopsis, a stepwise approach utilising
endohydrolytic enzymes, for instance, glucanases, cellulases, gal­
actanases and proteases independently or in combination is advised
[19,21,68].
3. Gas chromatography (GC)
Gas chromatography is a popular choice for separating and purifying
volatile and semi-volatile compounds [86]. A gas or liquid sample is
introduced through a heated injection port by a gas-tight syringe and
mixed with an inert gas mobile phase, frequently He, N2 or H2 [87].
Components in a sample are separated based on their boiling point and
polarity as they pass over a column and are retained to varying degrees,
passing through an ionisation source, then eluting onto a detector at
different timepoints which can be displayed on a time vs intensity
chromatogram for identification and quantification [86]. Two
frequently used ionisation modes; electron ionisation (EI), a type of hard
ionisation that bombards the eluent with electrons to cause high frag­
mentation of the analyte, and chemical ionisation which is a soft ion­
isation using a reagent gas that becomes protonated by an electron beam
before transferring a proton to the analyte [88]. Most GC systems use a
split mode where the sample is diluted post-injection by redirecting a
defined ratio to waste. Split mode is useful for quantifying samples with
a high analyte concentration, so to prevent overloading of the column
and negating the need for dilution during sample preparation and
improving peak shape [89,90].
3.1. Sample introduction
3.1.1. Liquid injection
A traditional method for GC analysis is a liquid injection which
5
Algal Research 79 (2024) 103478
draws up a solvent-extracted sample with a syringe and delivers it to a
heated injection port, vaporising the sample which can then be mixed
with a carrier gas. Solid phase extraction (SPE) is sometimes conducted
prior to liquid injection to concentrate analytes and remove interfering
compounds which have been co-extracted by the solvent but can result
in sample loss [91].
3.1.2. Headspace sampling techniques
Headspace (HS) refers to the gaseous phase above a liquid or solid
phase sample in a sealed vial and can be used as a sampling space for
volatile compounds released as the system equilibrates [92]. Headspace
is advantageous in that no solvents and little sample preparation are
required for analysis but has relatively low sensitivity when compared to
liquid injection with SPE [93]. Three main types of HS sampling are
headspace solid phase microextraction (HS-SPME), solvent bar micro­
extraction (SBME) and purge and trap (PT). Headspace can also be
performed without any additional concentration step. A major challenge
when attempting to quantify bromoform in seaweed using HS-GC is the
tendency for bromoform to remain in the matrix, rather than partition
into the headspace or onto the adsorbent SPME fibre. Several authors do
quantify bromoform of fresh seaweed using GC–MS without any pre-
treatment to measure bromoform emissions into the surrounding
seawater or air, however modern analytical methods aim to disrupt the
cell wall [34,94,95]. Mechanical, chemical or enzymatic processing
methods are required to release the higher concentration of bromoform
contained within gland cells or vesicles, which are protected by tough
cell walls of a parent cell and an inner membrane. Paul, Nys & Steinberg
2006 determined the ratio of released: internal bromoform and dibro­
momethane to be a large ratio of 1:77 and 1:83, respectively. This
cellular disruption is required before it can be analysed by headspace
methods which otherwise would only measure emitted bromoform
released by physiological means [21]. Processing methods for accessing
bromoform from algal cells will be discussed later in this review but is an
important consideration if headspace sampling is used to quantify
bromoform.
The use of HS sampling for bromoform quantification in various solid
materials other than macroalgae has been previously investigated. In a
study by Cardador & Gallego 2016, various carbohydrate-rich canned
vegetables were homogenised and the bromoform was SLE was con­
ducted using Na2SO4 and the organic modifier n-pentane for analysis by
HS-GC–MS from the same vial [96]. A LOQ of 0.3 μg/kg with an RSD of
5.2 % was achieved without any pre-concentration step. By comparison,
Calderón-Preciado & Bayona 2015 used HS-SPME to concentrate sam­
ples of leafy vegetables to quantify bromoform using GC coupled to an
electron capture detector (ECD) with a LOQ of 0.25 μg/kg and an RSD of
3.5–7.8 % [97].
3.1.3. Headspace solid-phase microextraction (HS-SPME)
Headspace sampling techniques have been popularised for quanti­
tative and qualitative analysis of volatile and semi-volatile compounds
due to the minimal sample preparation, low cost and relative speed of
analysis, negation of solvent use and simplicity of operation [98].
Solvent-free HS-SPME further simplifies traditional methods of sample
concentration and headspace analysis by removing the need for a gas
injection into the headspace by using an adsorbent fibre, concentrating
the analyte in the process. Partitioning of the analyte between the matrix
and the organic phase on the fibre permits the separation of analytes for
analysis [99]. The sample is placed unadulterated in a headspace vial
and a fused silica-coated fibre is inserted into the vial headspace that has
been given time to equilibrate at an elevated temperature, commonly
40–60 ◦ C. Free volatiles in the headspace are adsorbed to the fibre over
5–30 min before thermal desorption into the GC injector port is con­
ducted [100]. Aside from the advantages of HS-SPME, fibres can be
costly and have a short lifespan but efforts are being made to develop
sorbents to improve these attributes [101]. The data produced by HS-
SPME-GC–MS can be complex and require experienced personnel to
J.L. Hutchings et al.
interpret the results. HS-SPME-GC has been previously used by various
authors for the analysis of trihalomethanes in water samples [102–104]
and food [105–107]. HS-SPME-GC–MS has been employed for qualita­
tive volatile analysis of seaweed by several authors, however quantita­
tive studies investigating bromoform using this sampling method in
seaweed are scarce [29,108].
3.1.4. Solvent bar microextraction (SBME)
Concentrating an analyte from a liquid or slurry matrix for headspace
analysis can also be achieved using solvent bar microextraction (SBME).
An aliquot of organic solvent is dispensed into a hollow fibre which is
most commonly polypropylene due to its compatibility with a wide
range of organic solvents and small pore size [109]. The fibre is then
sealed using a flame and pliers, placed into a sample headspace vial and
magnetically tumbled in the matrix [101]. This facilitates the movement
of the analyte from the aqueous phase into the organic phase, leaving
behind particulates and hydrophilic compounds which is especially
useful for “dirty” matrices such as soil or wine [110,111]. SBME pro­
vides a high level of enrichment and reproducibility with very little
organic solvent, however, preparation of solvent bars for each sample,
manual trimming and withdrawing an aliquot of analyte-enriched sol­
vent for injection into a GC is time-consuming [112]. Contamination
between samples can occur if the bar is not replaced between
extractions.
Previous authors have used SBME to quantify various halogen-
containing compounds in water, wine and soil. Cardador & Gallego
2010 reported a 10 times higher sensitivity when compared to HS-SPME
for the quantification of haloacetic acids in water, but found the optimal
extraction time was 60 min, compared to 25 min and 20 min for HS-
SPME and HS-GC–MS, respectively [109]. It was also suggested that
the addition of sodium sulphate prevents the decomposition of tri­
bromoacetic acid to bromoform, which could otherwise result in an
overestimation of bromoform in a homogenised seaweed matrix. Simi­
larly, with water samples, Correa et al. 2015 achieved 5.8 % relative
standard deviation (RSD) and 91 ± 2 % recovery using SBME-GC-ECD
for water samples spiked with trace amounts of bromoform ranging
between 10 and 900 ppb (ng/mL) [113]. The authors concluded that the
method exhibited good performance parameters, considering that bro­
moform is volatile. As such, the migration of bromoform into a different
phase such as the headspace or a different solvent does not just depend
on its volatility but more on the affinity for the original matrix phase
[114]. Feasibly, the use of industrialised SBME fibres would improve the
precision of the method as well as the application of different extraction
solvents. In another study by Wang et al. 2012, the speed of solvent bar
preparation was increased by using a hand-held sealing machine instead
of pliers and immersing the solvent bar in organic solvent rather than
dispensing it with a pipette [110]. Optimisation and analysis of four
chlorobenzenes in soil using the modified solvent bar preparation
demonstrated that they were well sealed and able to achieve an RSD of
1.6–6.3 % for a 100 ng/g matrix spike and a recovery of 93–105 %,
compared to Soxhlet extraction which achieved a lesser 79–85 % re­
covery and 7.9–9.3 % RSD. It was concluded by Chia & Huang 2005 that
SBME had similar precision and a much higher sensitivity to hollow fibre
liquid phase microextraction when quantifying organochlorine pesti­
cides in wine [111].
3.1.5. Purge and trap (PT)
Purge and trap (PT) is a method of sampling volatiles that has been in
use since the 1970s and since, numerous design changes have been
developed to improve the technique for a range of matrices [115].
Traditional PT only analysed liquid samples by bubbling an inert gas
through a syringe into a liquid sample, then using a sorbent trap to hold
and concentrate volatiles from the headspace and thermally desorbed
onto a column [116]. Nowadays, setups work to remove water vapour
with condensers or permeators to prevent response degradation and
potential damage to the mass spectrometer or electron capture detectors
6
Algal Research 79 (2024) 103478
and are commonly used for water analysis. Several authors have re­
ported good repeatability and reproducibility of volatile organic com­
pound (VOC) quantification with PT but note the expense of glass
sparging tubes and low throughput when compared to SPME methods
[117,118]. Literature detailing PT-GC for the analysis of bromoform
emissions from seaweeds is abundant, however this is not the case for
cell-bound bromoform in seaweed requiring extraction methodology.
Abe, Okuda & Hasimoto 2021 recently investigated the effect of light
intensity on the emission of halomethanes by the marine diatom Ditylum
brightwellii, achieving a detection limit of <5 pmol/L and a RSD of <5 %
[119]. Keng et al. 2021 also measured bromoform and other halo­
methane emissions from tropical seaweeds with a PT-GC–MS method
with detection limits of 10 pmol/L, however did not report any other
quality control parameters [120].
3.2. Detectors
Selecting an appropriate detector is essential for analyte quantifi­
cation in order to achieve the required sensitivity, reproducibility and
accuracy. The analyte structure and electronegativity of components,
ionisation potential, molecular mass, predicted concentration and ma­
trix composition will help determine the type of detector that is used for
analysis. Most GC detectors fall under the class of gas-ionisation phase
detectors and this review will focus on mass spectrometers (MS), elec­
tron capture detectors (ECD), halogen-specific detectors (XSD) and
flame-ionisationhalogen-specific detectors (FID) for the quantification
of halomethanes primarily in seaweed, but also in water and oil.
3.2.1. Mass spectrometer (MS)
Gas chromatography mass spectrometry (GC–MS) analysis has been
employed across a multitude of research fields and is based on the
unique mass-to-charge ratio (m/z) of ionised compounds [121]. The GC
column-separated analytes are introduced into an MS via a heated
transfer line where they first undergo ionisation by, most commonly,
bombardment with an electron beam generated by a filament known as
electron ionisation (EI) [122]. Ions enter the quadrupole (Q1), consist­
ing of four parallel rods that generate an alternating polarity electrical
field which causes oscillation of ions to filter out neutral species and
allow through only ions with the m/z determined by the user. The
quadrupole can then be scanned over a certain m/z range and detect all
m/z ions produced during ionisation in that m/z range. Single quadru­
poles report permitted ions to a detector that generates an m/z vs in­
tensity spectrum. Coeluting species of the same m/z may be impossible
to differentiate on a single quadrupole and can be overcome by the use
of a triple quadrupole (MS/MS) or isotopically labelled compounds
[123]. A MS/MS consists of the Q1, followed by a collision cell (Q2)
filled with an inert gas that precursor ions collide with and fragment into
product ions. Product ions are filtered in a second quadrupole (Q3) to
select ions of interest and the most intense ion is selected as the quan­
tifier ion. The intensity or peak area of the quantifier can be compared to
a standard curve of the analyte. Other characteristic but less intense
product ions may be selected as qualifier ions and are used to confirm
the identity of a compound [124].
To date, the most notable advantage of MS/MS over other methods of
detection is the ability to rapidly and simultaneously detect multiple
analytes in a sample in a mode known as multiple reaction monitoring
(MRM) [125]. MRM alternates the m/z scan windows between analytes,
searching for only the precursor ions and corresponding product ions of
interest to detect them in a single run. When there is only one analyte of
interest, selective ion monitoring (SIM) increases the sensitivity by
1000-fold by scanning for only a handful of m/z characteristic to the
analyte, rather than spending additional time conducting a full m/z
scan. SIM is not confined to MS and can be used in other detector types
for single compounds analysis [126].
For the analysis of metabolites in complex matrices, GC–MS or
GC–MS/MS have served as indispensable tools both in the laboratory
J.L. Hutchings et al.
and in the field, subsequently GC–MS has been used by several authors
for bromoform analysis in seaweed which is summarised in Table 2
[20,50,59]. GC–MS does however come with its disadvantages [127].
The initial and upkeep cost of an MS-based system is considerably higher
than that of gas-ionisation phase detectors, requiring before-use and
scheduled maintenance for optimal performance. A GC–MS provides a
relatively easy platform for users to operate and maintain, however
additional operator training may be required for the use and interpre­
tation of data generated by a triple quadrupole.
Bromine has two naturally occurring isotopes, m/z 79 and 81, which
when analysed by EI-GC–MS (Fig. 2), appear as ions of approximately
equal abundance [126]. A loss of one bromine radical from bromoform
with an m/z 253 shows the highest abundance triplet at m/z 171, 173
and 175. An m/z 158, 160 and 162 clusters with a low relative abun­
dance is characteristic of two bromine radicals. Loss of H79Br+ 2 ⋅ and
H81Br+ 79 + 81 +
2 ⋅ to give C Br2 and C Br2 at m/z 91 and 93, respectively, can be
observed as the second highest abundance cluster [128].
3.2.2. Selected ion flow tube mass spectrometry (SIFT-MS)
A variation of the typical quadrupole configuration is SIFT-MS, a
technology commercially introduced in the 1990's which has primarily
been used for volatile analysis of food, human breath and air [129–133].
In contrast to a triple quadrupole, air and water are ionised using a
microwave plasma source, producing a number of positively or nega­
tively charged ions, known as reagent ions which are introduced into
and selected by the Q1. Carrier gas and gaseous sample from a head­
space vial, mouthpiece or airflow meter via heated line is introduced
into the Q2 and analyte molecules react with selected reagent ions to
produce product ions and neutral products that can be filtered by the Q3.
Signals from product ions are amplified by a particle multiplier for
computational processing [134].
Quantitative analysis using SIFT-MS has been employed for a variety
of compounds but has not been thoroughly investigated for trihalo­
methane targets or algal matrices. Using HS-SIFT-MS, Perkins & Lang­
ford quantified bromoform in drinking water using O+ 2 ⋅ reagent ions to
achieve an LOQ of 2 μg/L which was almost 2 orders of magnitude
higher than a compared PT-GC/MS method at 0.021 μg/L and >4 times
higher than the compared HS-GC-ECD at 0.47 μg/L [135]. It was noted,
however that the throughput was at least 3 times higher in HS-SIFT-MS
than HS-GC-ECD and showed precision and accuracy of 2.9–7.3 % RSD
and 6.0–13.5 % RSD, respectively, but these parameters were not
directly compared to other quantification methods. In another study,
quantification of chloroform produced from NaOCl interaction with
various matrices was conducted using SIFT-MS [136]. Quantification
was achieved using relative ion ratios, however, validation data was not
reported. Nonetheless, approximately 1 ppb of chloroform was quanti­
fied using O+2 ⋅ reagent ions and identifying peaks that match isotopic
ratios characteristic of chlorine isotopes. In a complex seaweed/
seawater matrix that varies in metabolite composition and ratio between
batches, overlapping peaks from isobaric species would increase the
difficulty in both identification of bromoform and accurate
quantification.
3.2.3. Electron capture detector (ECD)
Electron capture detectors are widely used for detecting compounds
with a high electron affinity, most notably halogens like bromine or
bromine-containing compounds. The inert carrier gas is bombarded
with alpha or beta particles from the radionucleotide (commonly 63Ni)
to form free electrons, forming a background level of current flow. When
an eluent is introduced with the carrier gas it is negatively ionised by
capturing electrons as it passes over the grid, reducing the flow of
electrons which can be detected as a decrease in current [137]. More
recently, non-radioactive electron sources such as thermionic emission
in a vacuum from silicon nitride membrane-wrappeda hot filaments or
pulsed helium discharge ionisation have been developed in a bid to
reduce the upkeep and risk associated with ionising radiation sources
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Algal Research 79 (2024) 103478
[138,139]. While ECD provides good sensitivity, precision and accuracy
for halogens, the selectivity for electronegative species also captures
noise and negative peaks for co-eluting hydrocarbons from the matrix
[140]. Additionally, saturation occurs at much lower analyte concen­
trations when compared to a quadrupole which causes a drop-off in
analyte linearity and an inaccurate value [141,142]. A summary of
quantitative GC-ECD methods for bromoform can be seen in Table 3.
3.2.4. Halogen specific detector (XSD)
The requirement for higher selectivity and specificity of halogens
than what has been provided by ECD gave rise to the XSD [143]. The
principle of operation between XSD and ECD is similar but presents some
distinct differences which allow for exclusion of coeluting compounds
that are absent of halogens [144]. The GC eluent and air or oxygen enter
the heated reactor core which combusts organic compounds and as they
move towards a platinum cathode surface, are sensitised by an alkali
metal such as potassium. Halogen atoms adsorb to the cathode surface
and due to the high electron affinity of the halogen compared to the
potassium, causes negative surface ionisation and a consequent increase
in electron emission and local surface temperature. Some electrons,
potassium halide ions and halide ions strike the anode, which is sup­
ported by an alkali glass ceramic rod, and causes measurable change in
voltage at the detector [145]. Andersson et al. 2019 used a GC-XSD to
quantify a range of halogenated disinfection by-products in water and
observed a similar sensitivity to other authors reviewed in the literature
using GC-ECD, with a limit of quantification (LOQ) of 0.05 μg/L [140].
This was only slightly higher sensitivity obtained by Nikolaou et al. 2002
who achieved a LOQ of 0.03 μg/L for bromoform in drinking water using
GC-ECD [144]. Given these results, there appears to be little difference
in sensitivity when using XSD when compared to ECD for bromoform
quantification.
3.2.5. Flame ionisation detector (FID)
Flame ionisation detectors typically use a hydrogen flame and high
voltage anode to combust and positively ionise gaseous sample intro­
duced by a GC, which passes through the flame chamber and strikes a
negatively charged detector. The movement of ions to the cathode
generates a current where the magnitude can be related to the mass of
carbons in the sample, which can be amplified and converted to a
spectrum [146,147]. FID provides a wide dynamic range and high
sensitivity to volatile organic compounds, especially hydrocarbons such
as petrochemicals [148]. While there is a requirement for a high-purity
hydrogen source, FID is generally considered robust and portable [149].
Like ECD, FID detects compounds only on the basis of retention time and
therefore coeluting compounds cannot be differentiated. While GC-FID
is a less common tool for bromoform quantification than ECD or MS,
it has been used for wastewater [150], emissions from seaweed [151],
and desalinated water [152].
3.3. Detection of bromoform using GC
The requirement for detection and quantification of bromoform
emerged long before the discovery of bromoform-rich seaweed, but as a
toxic by-product of water chlorination processes in swimming pools and
drinking water designed to kill and inhibit the growth of bacteria [153].
Consequently, researchers have investigated numerous means of quan­
tifying bromoform in water to ensure that safe concentration limits
dictated by state legislation are not exceeded [154]. A seaweed matrix is
far more complex than water, containing structural polysaccharides and
lignans, minerals, polyphenols, a high concentration of salt and similarly
to treated water, the presence of other trihalomethanes that may
interfere with the extraction and quantification of bromoform [155].
Given this, specificity and precision are key factors to consider when
selecting a detector whereas sensitivity is perhaps of lesser importance
with concentrations of bromoform in Asparagopsis spp. several folds
higher than that of disinfected water sources [19]. Quantification
J.L. Hutchings et al. Algal Research 79 (2024) 103478

Table 2
Summary of GC–MS methods for bromoform quantification.
Instrument GC program Parameters Matrix LOD LOQ Precision Recovery (%) Ref
(RSD %)

HS-GC–MS 35 ◦ C for 5 min, ramped at 10 ◦ C/ Carrier gas: He Oven-dried red – – – – [167]

min to 230 ◦ C for 5 min. Detector temperature: seaweed
200 ◦ C
ISTD: fluorobenzene
GC–MS (SL) 40 ◦ C ramped at 16 ◦ C/min to Carrier gas: H2 FD red seaweed – – – – [159]
250 ◦ C. Gas flow rate: 1.5 mL/min
Injection volume: 1 μL
Injector temperature:
180 ◦ C
Ion source: positive mode
Dwell time: 50 ms
HS-SPME- 40 ◦ C for 10 min, ramped at 4 ◦ C Carrier gas: He Cryogenically – – – – [173]
GC–MS (SL) to 140 ◦ C, ramped at 10 ◦ C to Gas flow rate: 0.8 mL/min homogenised brown
200 ◦ C, ramped at 30 ◦ C/min to Transfer line temperature: seaweed
250 ◦ C and held for 5 min. 280 ◦ C
Source temperature: 230 ◦ C
Quadrupole temperature:
150 ◦ C
Mass range (m/z): 40–450
GC–MS (S) 40 ◦ C for 1 min, ramped at 20 ◦ C/ Carrier gas: He FD red seaweed – – – – [170]
min to 250 ◦ C for 0.5 min. Gas flow rate: 1 mL/min
Injection volume: 1 μL
Split ratio: 50:1
Injection temperature:
280 ◦ C
Electron impact ionisation:
70 eV
ISTD: Naphthalene
GC–MS (SL) 40 ◦ C for 1 min, ramped at 16 ◦ C/ Carrier gas: He FD red seaweed and 0.06 mg/g 0.18 – – [50]
min to 250 ◦ C and held for 2 min. Injection volume: 1 μL canola oil containing mg/g
Inlet pressure: 8 psi fresh red seaweed
Injection port temperature:
250 ◦ C
Interface temperature:
300 ◦ C
Qualifier ions (m/z):
172–174, 127–129 (ISTD)
ISTD: Naphthalene
GC–MS (S) 40 ◦ C for 5 min, ramped at 20 ◦ C/ Carrier gas: He FD seaweed 1 μg/mL – 2.0–5.1 88–112 [20]
min to 160 ◦ C, ramped at 60 ◦ C/ Gas flow rate: 1.2 mL/min
min to 280 ◦ C and held for 4 min. Injection volume: 2 μL
Inlet temperature: 250 ◦ C
Transfer line temperature:
280 ◦ C
Mass range (m/z): 50–460
(SIM)
ISTD: Diiodomethane-d2
GC–MS (SL) Split vent time 0.5 min. Carrier gas: H2 FF and FD red seaweed 0.08 mg/g 0.26 5.3 98.4–100.5 [59]
40 ◦ C for 1 min, ramped at 16 ◦ C/ Gas flow rate: 1.5 mL/min mg/g
min to 250 ◦ C and held for 2 min. Injection volume: 1 μL
Injector temperature:
180 ◦ C
Positive mode
50 ms dwell time (SIM)
Ion source electrode
temperature: 230 ◦ C
Quadrupole temperature:
150 ◦ C
Quantifier ion (m/z): 172.8
Qualifier ions (m/z): 170.8,
174.8, 251.8 and 253.8
ISTD: Not used
GC–MS (Pulsed 40 ◦ C for 1 min, ramped at 16 ◦ C/ Carrier gas: He FD red seaweed – – – – [27]
SL) min to 250 ◦ C and held for 2 min. Injection volume: 1 μL
Injection port temperature:
180 ◦ C
Transfer line temperature:
280 ◦ C
Pulse pressure: 9.8 psi
HS-GC–MS (S) 40 ◦ C for 1 min, ramped at 16 ◦ C/ Refers to the method by FD red seaweed – – – – [6]
min to 250 ◦ C and held for 2 min. Paul, Nys & Steinberg 2006
[21]
(continued on next page)

8
J.L. Hutchings et al. Algal Research 79 (2024) 103478

Table 2 (continued )
Instrument GC program Parameters Matrix LOD LOQ Precision Recovery (%) Ref
(RSD %)

GC–MS (SL) 40 ◦ C for 1 min, ramped at 16 ◦ C/ Carrier gas: He FD red seaweed and – – – – [48]
min to 250 ◦ C and held for 2 min. Gas flow rate: 2 mL/min red seaweed in blended
Injection volume: 1 μL vegetable oil
pulsed at 35 psi
Injection port temperature:
250 ◦ C
GC–MS interface
temperature: 300 ◦ C
ISTD: Naphthalene
GC–MS 40 ◦ C for 3 min, ramped at 10 ◦ C/ Carrier gas: He FD red seaweed – – – – [37]
min to 200 ◦ C for 1 min. Gas flow rate: 0.9 mL/min
Injection port temperature:
250 ◦ C
Interface temperature:
300 ◦ C
Ion source temperature:
230 ◦ C
GC–MS/XSD 27 ◦ C for 1.3 min, ramped at Carrier gas: He Drinking water – 0.05 – 81 [140]
(SL) 7 ◦ C/min to 80 ◦ C, ramped MS/XSD split ratio: 1:9 μg/L
30 ◦ C/min to 250 ◦ C and held for Electron ionisation:
5 min. 70 eV
Mass range (m/z): 40–550
Surrogate ISTD:
1,2-dibromopropane
ISTD: 1-chlorodecane
GC–MS (SL) 40 ◦ C for 1 min, ramped at 16 ◦ C/ Carrier gas: He FD red seaweed – – – – [171]
min to 250 ◦ C for 2 min. Gas flow rate: 2 mL/min
Injection volume: 1 μL
Pulsed pressure: 35 psi
Injection port temperature:
240 ◦ C
Interface temperature:
250 ◦ C
ISTD: Naphthalene
GC–MS (SL) 40 ◦ C for 1 min, ramped at 16 ◦ C/ Carrier gas: He Seawater – – – – [172]
min to 250 ◦ C and held for 2 min. Gas flow rate: 1 mL/min
Injection volume: 1 μL
Inlet pressure: 8 psi
Injection port temperature:
240 ◦ C
Interface temperature:
250 ◦ C
ISTD: Naphthalene
PT-GC–MS 36 ◦ C for 5 min, ramped at 20 ◦ C/ Carrier gas: He Seawater 10 pmol/L – – – [168]
min to 200 ◦ C, ramped at 40 ◦ C/ Transfer line temperature:
min to 240 ◦ C. 95 ◦ C
ISTD: d-methyl iodide,
d-diidomethane
SPME-GC–MS 35 ◦ C for 3 min, ramped at 30 ◦ C/ Carrier gas: He Drinking water 0.1 ng/mL – 3.9 (SPME) 101 ± 4 [174]
(SL), PT- min to 125 ◦ C for 1 min, ramped Gas flow rate: 1 mL/min (SPME) (SPME)
GC–MS (SL) at 50 ◦ C/min to 270 ◦ C for 5 min. Electron ionisation: 4.7
70 eV 0.04 ng/ (PT) 101 ± 4 (PT)
Purge trap temperature: mL (PT)
250 ◦ C
Manifold temperature:
50 ◦ C
Transfer line temperature:
280 ◦ C
Scan time: 0.6 scan/s
GC–MS (SL) 40 ◦ C for 1 min, ramped at 16 ◦ C/ Carrier gas: He FD red seaweed – – – – [21]
min to 250 ◦ C for 2 min. Injection volume: 2 μL
Inlet pressure: 8 psi
Injection port temperature:
250 ◦ C
Interface temperature:
300 ◦ C
ISTD: Naphthalene
Several 31 ◦ C for 1 min, ramped at 1 ◦ C/ Carrier gas: He (PT) Drinking water 0.1 μg/L – 0.1–28 126.7 [169]
min to 40 ◦ C, ramped at 80 ◦ C/ Injection volume: 1 μL (LLE- (LLE- (HS-GC–MS)
min to 200 ◦ C. (LLE), 3 μL (PT), 500 μL GC–MS) GC–MS)
(HS)
ISTD: halothane 1 μg/L 2.63–20.35
(PT- (PT-GC–MS)
GC–MS)

(continued on next page)

9
J.L. Hutchings et al. Algal Research 79 (2024) 103478

Table 2 (continued )
Instrument GC program Parameters Matrix LOD LOQ Precision Recovery (%) Ref
(RSD %)

0.1 μg/L 25.49

(HS- (HS-GC–MS)
GC–MS)
SPME-GC–MS 35 ◦ C for 4 min, ramped at 6 ◦ C/ Injector temperature: Drinking water 0.116 μg/ – 9 85 [180]
min to 160 ◦ C for 5 min. 220 ◦ C L
ISTD: fluorobenzene
Surrogate STD:
4-bromofluorobenzene
Several 40 ◦ C for 3 min, ramped at 1 ◦ C/ Carrier gas: He Drinking water 0.03 – 4.1–37.9 92–127.6 [160]
min to 150 ◦ C. Ramped at 6 ◦ C/ Gas flow rate: 1.3 mL/min μg/L (LLE- (LLE-GC–MS)
min to 175 ◦ C/min. Split ratio: 1:25 (LLE- GC–MS)
Oven temperature: 35 ◦ C GC–MS) 88–102
Injector temperature: 0.7–9.5 (PT-GC–MS)
175 ◦ C 0.01 μg/L (PT-GC–MS)
Detector temperature: (PT- 82–112
300 ◦ C GC–MS) 5.4–39.1 (HS-GC–MS)
Solvent delay: 14 min (HS-GC–MS)
Source temperature: 280 ◦ C 0.1 μg/L
(HS-
EMV: 2200 V GC–MS)
Acquisition mode: SIM
Dwell time: 100 ms
PT-GC–MS 40 ◦ C for 1 min, ramped at 10 ◦ C/ Carrier gas: He Fresh red seaweed 5 μg/L – 35 – [34]
min to 230 ◦ C for 5 min. Transfer line temperature:
250 ◦ C
ISTD: 1,2-dibromoethane
PT-GC-ECD/ 35 ◦ C for 4 min, ramped at 5 ◦ C/ Carrier gas: He Seawater 2 pmol/L – 2 92 [161]
MS min to 60 ◦ C for 2 min, ramped at Flow rate: 1.5 mL/min (small
15 ◦ C/min to 130 ◦ C for 3 min (small trap) trap)
(megabore column)

35 ◦ C for 3 min, ramped 5 ◦ C/

min to 60 ◦ C for 2 min, ramped at
15 ◦ C/min to 180 for 3 min
(narrow bore column).

Fig. 2. Chemical structure of bromoform and MS spectrum adapted from NIST Chemistry WebBook 2014 [128].

methods for trihalomethanes using the range of detectors are summar­
ised in Tables 2–4 and a detailed description of the operating principles,
performance characteristics and the response mechanisms of detectors
have been previously reviewed [156].
Gas chromatography operating conditions, precision, recovery, LOD
and LOQ are poorly reported across the literature and highlight the need
for a robust standardised method that can be easily implemented. When
selecting a suitable analytical method from the literature, the lack of
method information and instrument parameters provided by authors
raises questions as to whether the results and method performance that
they present are valid and reliable. Comparison of LOD and LOQ is
difficult with authors using a mixture of different units to present these
values.
The operating conditions for a GC system are a defining feature of the
data quality and vary between authors. GC oven programs are generally
similar but are noted to have between 1 and 3 temperature ramps with

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J.L. Hutchings et al. Algal Research 79 (2024) 103478

Table 3
Summary of GC-ECD methods for bromoform quantification.
Instrument GC program Parameters Matrix LOD LOQ Precision Recovery Ref
(RSD %) (%)

GC-ECD (S) 35 ◦ C for 20 min, ramped at 20 ◦ C/min to Carrier gas: He Drinking water 0.35 μg/L 4 μg/ 4.84 92.1–105 [157]
225 ◦ C and held for 2 min. Gas flow rate: L
1.3 mL/min
Split ratio: 1:25
Injector temperature:
220 ◦ C
Detector temperature:
300 ◦ C
GC-ECD 35 ◦ C for 22 min, ramped at 20 ◦ C/min to Carrier gas: He Chlorinated sea – – – – [166]
145 ◦ C and held 2 min, ramped at 20 ◦ C/min to Gas flow rate: 1 mL/min water
225 ◦ C and held for 15 min, ramped at 10 ◦ C/ Injector temperature:
min to 280 ◦ C and held for 2 min. 200 ◦ C
Detector temperature:
310 ◦ C
ISTD:
1,2-dibromopropane
PT-GC-ECD 45 ◦ C for 7 min, ramped at 5 ◦ C/min to 90 ◦ C, Carrier gas: N2 Sea water 0.5 pmol/ – 15 – [162]
ramped at 10 ◦ C/min to 240 ◦ C, held at 200 ◦ C L
for 5 min.
GC-ECD (S) 45 ◦ C for 3 min, ramped at 15 ◦ C/min to 100 ◦ C. Carrier gas: N2 Water 0.12 μg/L 0.36 6.06 (intra- – [163]
Ramped at 25 ◦ C/min to 230 ◦ C. Gas flow rate: 1 mL/min μg/L day)
Split ratio: 1:25
Injector temperature: 6.59 (inter-
250 ◦ C day)
Detector temperature:
280 ◦ C
LPME-GC- 40 ◦ C for 4 min, ramped at 4 ◦ C/min to 130 ◦ C. Carrier gas: N2 Tap water 0.25 μg/L – – 70–73 [164]
ECD (SL) Ramped at 15 ◦ C/min to 220 ◦ C and held for 2 Gas flow rate:
min. 1.5 mL/min
Injection volume: 1 μL
Makeup gas flow rate:
43.5 mL/min
Injector temperature:
280 ◦ C
Detector temperature:
300 ◦ C
PT-GC-ECD 35 ◦ C for 3 min, ramped at 5 ◦ C/min to 60 ◦ C, Carrier gas: N2 Seawater – – – – [165]
ramped at 15 ◦ C/min to 180 ◦ C for 3 min. Gas flow rate: 6 mL/min
LLE-GC-ECD 40 ◦ C for 3 min, ramped at 1 ◦ C/min to 150 ◦ C. Carrier gas: N2 Drinking water 0.01 μg/L – 0.5–8.6 94.7–112.8 [160]
Ramped at 6 ◦ C/min to 175 ◦ C/min. Gas flow rate:
1.6 mL/min
Split ratio: 1:25
Oven temperature:
39 ◦ C,
Injector temperature:
175 ◦ C
Detector temperature:
300 ◦ C
Solvent delay: 14 min
PT-GC-ECD/ 35 ◦ C for 4 min, ramped at 5 ◦ C/min to 60 ◦ C for Carrier gas: N2 Seawater 30 fmol/L – 4–6 97 [161]
MS 2 min, ramped at 15 ◦ C/min to 130 ◦ C for 3 min Gas flow rate: (small
(megabore column) 13 mL/min (large trap); trap)
10 mL/min (small trap)
35 ◦ C for 3 min, ramped 5 ◦ C/min to 60 ◦ C for 2 200 fmol/
min, ramped at 15 ◦ C/min to 180 for 3 min L (large
(narrow bore column). trap)
HS-GC-ECD 40 ◦ C for 2.5 min, ramped at 15 ◦ C/min to Carrier gas: He Seawater 50 ng/L – 2–10 75–100 [94]
(SL) 165 ◦ C, ramped at 70 ◦ C/min to 220 ◦ C for 4 Injection volume: 100
min. μL
Transfer line
temperature: 160 ◦ C
Detector temperature:
300 ◦ C
PT-GC-ECD 5 ◦ C for 5 min, ramped at 5 ◦ C/min to 220 ◦ C for Carrier gas: He Fresh red, brown – – – – [95]
41 min. Flow velocity: 14.5 cm/ and green
s seaweed

starting oven temperatures of 35–50 ◦ C, with the exception of Andersson
et al. 2019 who starts at 27 ◦ C to capture halogen-containing compounds
other than bromoform and with a higher volatility [140]. Romanazzi
et al. 2021 uses a GC oven program derived from Paul, Hys & Steinberg
2006 to achieve exceptional recovery of bromoform from A. armata
biomass, finding splitless mode injections gave better precision of RSD 2
% when compared to split mode at RSD 5 % [21,59]. In agreement,
authors that use split mode to quantify bromoform in seaweed and
drinking water achieved a precision of >2 % and consequently, most use
splitless mode and achieve better precision [6,20,157].

11
J.L. Hutchings et al.
Table 4
Summary of other GC methods for bromoform quantification.
Instrument GC program
SPME-GCxGC- – Scan speed: 500 scans/s
TOF
GC–MS/XSD 27 ◦ C for 1.3 min, ramped at 7 ◦ C/min to 80 ◦ C,
(SL) ramped 30 ◦ C/min to 250 ◦ C and held for 5 min.
Relative standard deviation (RSD); limit of detection (LOD); limit of quantification (LOQ); fresh frozen (FF); freeze dried (FD); headspace (HS); mass spectrometry
(MS); liquid-liquid extraction (LLE); split mode (S); split-less mode (SL); selected ion monitoring (SIM), internal standard (ISTD); liquid phase microextraction (LPME);
solid phase microextraction (SPME); halogen selective detector (XSD) electron capture detection (ECD); electron multiplier voltage (EMV); purge and trap (PT).
Selection of a suitable GC column ensures that the analyte and in­
ternal standard are retained to provide adequate peak separation but
also can be desorbed for analysis in a reasonable timeframe to reduce
instrument runtime [158]. Columns used for bromoform analysis are
generally suitable for retention of polar analytes and high temperatures,
including popular weak anion exchange (WAX) columns
[21,48,59,159], but also include low polarity [20] and non-polar sta­
tionary phase materials [140]. Reproducible results and sensitivity
adequate to the studies conducted were achieved using a wide range of
column brands and sorbent types and selection ultimately comes down
to the cost and availability of the column. The choice of carrier gas for
bromoform quantification varies across the literature between N2
[160–165], He [20,21,27,34,37,48,50,94,95,140,157,160,161,
166–174] and H2 [59,159]. Methods can be developed to suit most of
these carrier gasses depending on what is available to the user, however
it has been established that the use of nitrogen in GC–MS greatly reduces
the sensitivity with EI by a factor of >20 and literature detailing bro­
moform quantification is limited to non-MS detectors [175].
The option of positive or negative chemical ionisation on a GC–MS
allows the user to select a polarity that the precursor ion is better ionised
when passing through the ionisation source and one mode may provide
greatly improved resolution and sensitivity over the other [88]. Mostly
protonated species are formed and only literature using positive mode
was identified for bromoform analysis, it is evident that positive
chemical ionisation or EI are the preferred mode for quantification
[59,140,159,170,174].
3.4. Data standardisation for GC
Methods of standardisation for data generated by GC-coupled sys­
tems in a bid to provide results reflective of the actual analyte concen­
tration have been of much debate. Matrix effects, loss of analyte through
sample preparation, evaporation, degradation, conversion of precursor
compounds to the analyte and injection volume contribute to variations
in the calculated concentration of the analyte [176]. Isotopically
labelled internal standards (ISTD) are structurally identical and share
binding characteristics to their unlabelled counterparts with the addi­
tion of heavy carbon, oxygen, hydrogen, or nitrogen constituents to give
a net increase of at least 3 mass units [177]. The ISTD synchronously
elute from a column with the analyte but is distinguishable by slightly
different molecular weights and fragmentation patterns in MS/MS and
should not enhance or suppress the intensity of analyte peaks. The
addition of an ISTD in a known concentration immediately before
analysis allows for easier identification of the analyte in a spectrum and
provides a quick means of analyte concentration estimation. Addition­
ally, the ratio of the ISTD to the analyte provides a means of stand­
ardisation and correction when conducting absolute quantification
using the ISTD and the analyte. Isotopically labelled bromoform has
been employed by Muizelaar et al. 2021 for quantification of bromoform
Algal Research 79 (2024) 103478
Parameters Matrix LOD LOQ Precision Recovery Ref
(RSD %) (%)
FD red 50 μg/ – – – [178]
ISTD: Isotopically labelled seaweed kg
bromoform
Carrier gas: He Drinking – 0.05 – 81 [140]
MS/XSD split ratio: 1:9 water μg/L
Electron ionisation: 70 eV
Mass range (m/z): 40–550
Surrogate ISTD:
1,2-dibromopropane
ISTD: 1-chlorodecane
on two-dimensional GC–MS (GCxGC–MS) which resulted in acceptable
recovery on all days of analysis except one, demonstrating almost
identical on-column characteristics even in a complex GCxGC setup
[178,179]. GC-ECD systems are unable to benefit from ISTDs due to
coelution with the analyte and no third dimension of separation to
differentiate. Romanazzi et al. 2021 argued that using an ISTD for
normalisation did not improve instrument or method performance when
quantifying bromoform in seaweed by GC–MS [59]. Additionally,
several other authors have omitted the use of ISTDs for the analysis of
bromoform and other trihalomethanes which are shown in Tables 2–4
without a listed ISTD in their method.
In cases where an isotopically labelled ISTD is not available, there is
scope to use an unlabelled compound with a similar retention time and
absence in the sample, sometimes referred to as a surrogate ISTD. For
bromoform analysis, surrogate ISTDs including fluorobenzene [167],
bromofluorobenzene [180], d2-diiodomethane [20,168], halothane
[169], 1-chlorodecane [140], 1,2-dibromoethane [34], dibromopropane
[166] and naphthalene [21,48,50,170–172] have been previously used.
Surrogate ISTDs are used for non-specific detectors such as FID or ECD
due to different retention times compared to the analyte. To our
knowledge, there has been no comparison of surrogate ISTD for bro­
moform quantification and it would be prudent to recommend a specific
compound, other than selecting a compound with a similar retention
time to bromoform for the column of choice, due to the variation in
methods and instrumentation.
4. Non-chromatographic quantification methods
While the majority of reported bromoform quantification studies
have been performed using GC–MS or GC-ECD, examples of quantifi­
cation by spectroscopic methods have also been described. Infrared,
fluorescence and colourimetric spectroscopic methods and NMR have
been used. While these methods have lower sensitivity than CG-MS and
GC-ECD, they require minimal sample preparation and have additional
application in the identification of unknown compounds through the
elucidation of the compound chemical structure [181–186].
4.1. Infrared (IR)
Infrared spectroscopy is an analytical technique in which molecules
absorb infrared light at resonant frequencies characteristic of their
structure. As such, infrared spectroscopy can be used to quantify
brominated hydrocarbons with their characteristic bromine‑carbon
signal appearing at a wavenumber of 690–515 cm− 1 [187,188]. As this
bromine‑carbon signal appears at a similar wavenumber for all bromi­
nated alkanes, to quantify an individual species either a preliminary
separatory step must be performed or knowledge of the desired target
concentration proportional to that of other species must be known
[189,190].
12
J.L. Hutchings et al.
One method of separation used in conjunction with infrared spec­
troscopy is gas chromatography (GC-IR). This method was used by
Cavanagh et al. 1992 and Richardson et al. 1999 for the identification of
brominated organics in ozonated water [191,192]. In the study by
Cavanagh et al. GC-IR was used to quantify the concentration of bro­
mohydrins in ozonated water. The samples in this study used bromo­
form, dibromoacetonitrile, and bromoacetone to calibrate their
instrument while 1,2-dibromopropane was used as an internal standard.
Subsequently, in the study by Richardson et al. 1999 GC-IR was also
used to detect the brominated organics; bromoform, tri­
bromoacetaldehyde, tetrabromopropanone, dibromoacetonitrile, and
tribromonitromethane that had been spiked into samples of river water.
As this study investigated the synthesis of a range of unknown bromi­
nated alkanes, further analysis by GC–MS and NMR was required to
confirm the identity of each brominated alkane before their respective
retention times could be determined.
4.2. Fluorescence spectroscopy
Fluorescence spectroscopy is an analytical method that measures the
emitted fluorescent light of molecules whose electrons have been
excited by light of a higher wavelength [193]. As the fluorescence in­
tensity is typically proportional to the concentration of the fluorescing
molecule, referred to as a fluorophore, this method can be used to
determine the concentration of the fluorophore in a pure sample. As
fluorescence spectra typically have broad peaks this method is unsuit­
able for the quantification of single analytes unless either their pro­
portion in regard to other fluorophores is known or the analyte is
modified to fluoresce under conditions specific to it [183].
Despite this limitation, fluorescence spectroscopy has been used to
quantify bromoform concentration, typically in drinking water. Pal et al.
2001 sensitised bromoform through the addition of diphenylamine in
aqueous TX-100 medium and irradiation with ultraviolet light to create
a fluorescent compound. Following excitation at 400 nm the sensitised
bromoform analyte was found to fluoresce with emission at a wave­
length of 480 nm [194]. Both bromoform and dibromomethane were
found to produce fluorescent compounds under the reaction conditions
used in this experiment, as such this method could not be used to solely
quantify bromoform unless its concentration relative to dibromo­
methane was known. The interference of other haloalkanes was inves­
tigated with the use of trichloroacetic acid, dichloroacetic acid,
monochloroacetic acid, tetrachloromethane and chloroform. Under the
same reaction conditions, none of these resulted in a fluorescent
compound.
Trueman et al. 2016 similarly used fluorescence spectroscopy to
quantify disinfection
by-products (DBP); haloacetic acids and trihalomethanes (and spe­
cifically bromoform), in treated drinking water [195]. A fluorescence
excitation-emission matrix was correlated with data from a GC-ECD to
train a model that would predict DBP concentration from fluorescence
spectroscopy data. As fluorescence spectra of these compounds overlap,
this method could not be used to solely quantify bromoform unless the
ratio of these DBP's is known, again requiring additional analysis
including a means of separation.
4.3. Colourimetry
Bromoform has also been quantified through colourimetric analysis.
Colourimetric analysis is a method that uses the Beer-Lambert law to
determine the concentration of a coloured substance in a solution. While
bromoform is relatively colourless, Svensson 2019 produced a prototype
nanofibre membrane which changed colour in the presence of tri­
halomethanes, such as bromoform, through the process of a Fujiwara-
Moritani reaction [182]. After a period of 30 min in the presence of
bromoform at a concentration of 80 ppm, the membrane was observed
to change colour from white to pink, while at concentrations of 80 and
13
Algal Research 79 (2024) 103478
250 ppb, the membrane was seen to change colour to brown. Never­
theless, no colour change was observed at a bromoform concentration of
8 ppb.
4.4. Nuclear magnetic resonance (NMR)
NMR has been used for the relative quantification of bromoform in
an oil fraction extracted from A. taxiformis. Burreson and co-workers
reported a method in which volatile compounds, including bromo­
form, and water were extracted from A. taxiformis under vacuum and
collected by a dry ice-cooled condenser [196,197]. The extracted vola­
tile compounds were further extracted from water through liquid/liquid
extraction with the resulting crude oil fractionated by column chroma­
tography on silica gel. The fractions were subsequently analysed with
the major constituents of each fraction identified and quantified by 1H
NMR, 13C NMR, and GC–MS [197]. Bromoform was identified with a
signal at pmr δ 6.80 which matched a reference material, with integra­
tion of the signal peak used to quantify bromoform in comparison to
other signals. Of the oil collected using this method, bromoform was the
major constituent and estimated to be 80 % by weight. It should be noted
though that the author of the paper stated that this method of extraction
prevented them from collecting a number of compounds, including
chloroform and carbon tetrachloride, while the fractionation process led
to a further loss of compounds, including iodoform, that had been
identified in the crude oil.
Overall, compared to IR, fluorescence and colourimetry, gas chro­
matography methods are superior for the quantification of bromoform
due to its volatility and presence within the complex seaweed matrix.
While the quantification of bromoform with spectroscopic and colouri­
metric methods has been reported, the low concentration of bromoform
and the presence of other brominated hydrocarbons in the complex
seaweed matrix would make these methods unsuitable for bromoform
quantification [198–201]. As such, without either a means of separation,
working with a pure solution of bromoform or knowledge of the ratio
that bromoform is present in relation to other fluorescent compounds,
spectroscopic analysis is considered unsuitable for bromoform quanti­
fication, while with their greater sensitivity GC–MS and GC-ECD are
considered the more suitable methods for bromoform quantification in
seaweed samples.
5. In-field sampling and analysis
The requirement for point-of-collection analysis has pushed the
boundaries of in-field analysis over the last 25 years and has been
applied to food safety, space material, environmental samples and
toxicology. Specifically, for seaweed analysis the lack of lab proximity
represents one of the major limitations for companies working on bro­
moform in seaweed preservation due to the difficulties in maintaining
suitable transport conditions. In-field analysis presents different chal­
lenges when compared to laboratory analysis, however, literature de­
tailing approaches and challenges for targeted in-field GC analysis is
scarce [202]. In-field analysis of material allows for a fast turnaround of
results and an almost immediate adjustment to a targeted sampling
routine, ultimately saving time and money [203]. For organisations
looking to make their processes “green”, one of the strategies would be
minimising the physical distance between sampling and analysis.
Volatile compounds are subject to evaporation, degradation or con­
version to different compounds between sampling and analysis time
which can be as a result of light or air exposure, large headspace or
temperature fluctuations [204]. These environmental variations are the
most obvious challenge of in-field analysis and include changes in hu­
midity, air pressure, wind speed, air quality and particulates, back­
ground noise and vibration [205]. Other factors to consider may include
training for non-scientific users, servicing and maintenance, storage and
disposal of hazardous chemicals, contamination and simplicity of sam­
ple preparation [206]. For example, qualitative analysis may simply
J.L. Hutchings et al.
confirm or deny the presence of a substance and require minimal
operator knowledge and skills to deliver a valid result [205]. Quanti­
tative analysis, whether using an internal standard for relative quanti­
fication and/or an external standard for absolute quantification
increases the need for upskilling non-scientific users or deploying
experienced personnel on-site [207]. Improvements to the sensitivity
and portability of gas chromatography systems have made them useful
and robust tools for in-field analysis designed to overcome the difficult
environmental conditions they may be expected to perform and more
user-friendly for routine analysis [208].
Toxins, pollutants, drugs and chemical warfare agents have been
extensively analysed domestically and internationally by military, po­
lice and government organisations using in-field gas chromatography
platforms in order to reduce the risk of chemical exposure to personnel
or conduct investigations [209–211]. Applications of this complexity
require reproducible results and highly sensitive instrumentation in
rugged, portable GC platforms which have become core to these orga­
nisations and allowing for real-time data collection. There is almost
exclusive use of MS detectors in these environments due to their ability
to differentiate coeluting compounds and provide immediate identifi­
cation confirmation by the use of one or more qualifier ions. It must be
noted that two main types of field instrumentation exist, being those that
are portable and can be carried by an individual, and transportable
which may be identical to their laboratory-based counterparts and
mounted to vehicle, container or trailer. Leary et al. 2019 reviewed the
use of deployable GC–MS for military users in theatre, noting a key
advantage of coupling GC to an ion trap rather than a quadrupole was
the higher operating pressure and subsequent lower power requirement
from smaller, lighter turbopumps [212]. Vacuum and pumping re­
quirements are a limiting factor for the transportability or portability of
a GC–MS system [213]. Smaller ion traps boast improved performance
but limit the number of ions that can be trapped at once for analysis
which can decrease sensitivity or increase run time [214].
In-field analysis, while typically conducted with purpose-built, rug­
gedised GC, is not confined to these systems [215]. With advances in
miniaturisation of GC and mass spectrometry and foregoing the need for
exceptional grade hardware, smaller instruments with similar perfor­
mance to their laboratory counterparts are becoming available but are
still lagging behind the faster advancement of laboratory-based systems.
However, smaller GC systems have a place within analytics with the
potential for fitment to land, air or sea-based vehicle and allowing for
transport to conduct rapid point-of-collection analysis [127,216].
Modern GC instruments can operate on 240 VDC, 10 A outlets which can
be readily provided by a small generator or from an off-the-shelf vehicle
inverter, a significant advantage over other analytical systems requiring
3-phase power [217–219]. Allocation of additional power would be
required if cooling devices were used for the preservation of volatiles
both during sample preparation and while waiting for analysis on an
autosampler rack [220]. Positioning and shielding a GC from back­
ground exhaust fumes must be carefully considered, especially if struc­
turally similar compounds to those generated by the engine are being
analysed [221].
Various high-purity gases including helium, nitrogen and hydrogen
are normally required for optimal operation of a GC system which in-
field is supplied from a bottle, increasing the logistical complexity of
analysis [205]. Hydrogen is a common carrier gas used for GC systems
and comes with the risk of explosion which can cause damage to
personnel and instrumentation. On the contrary, hydrogen is highly
abundant in supply and low in cost, when compared to helium which is
at risk of depletion over the next century [222]. Safe operating pro­
cedures and engineering measures implemented in laboratories and in-
field testing sites are designed to mitigate the risk posed by hydrogen
which may include the use of a hydrogen generator, allowing for
hydrogen to be produced on demand and reducing the need for bulk
storage [223].
Analysis of volatile compounds, such as bromoform, in a laboratory
14
Algal Research 79 (2024) 103478
already presents the issue of analyte loss during sampling, transport,
storage and preparation. Vastly reduced time between sampling and
analysis, and minimising sample transport is an inherited benefit of in-
field analysis, however, warm, windy and humid environmental con­
ditions may result in increased volatile analyte loss and sub-optimal
instrument performance. Yassaa et al. 2008 analysed monoterpenes
emissions from phytoplankton using both laboratory-stationed and boat-
mounted HS-GC–MS and observed minimal differences in detection
limits at 0.5–5 parts per trillion by volume (pptv) and 1–5 pptv,
respectively [224]. A boat-mounted trap and purge GC–MS was also
employed by Andrews et al. 2015 to measure VOCs in seawater,
including bromoform and other halogenated compounds. Continuous
and automated sampling of water was conducted through inline pumps,
degassing and dewatering apparatus before GC–MS analysis. It was
suggested that good reproducibility in-field can be attributed to the
optimisation of a method that quantifies trace levels of VOCs in the
laboratory combined with system performance testing in the field [225].
A similar purge and trap GC-FID method was implemented by Lui et al.
2009 to quantify VOCs in wastewater in-field which was compared to
the laboratory system. The two systems yielded similar results with <4
% difference for structurally similar chloroform, and naphthalene, a
compound used frequently as a GC–MS internal standard for bromoform
[226]. In contrast, Marcillo et al. 2023 recently argued that mobile
GC–MS systems are largely inferior to their stationary counterparts,
showing, respectively, higher RSD of 9.7 % to 3.5 %, lower signal-to-
noise ratio and a lower sensitivity using a mixture of 18 VOCs be­
tween 3 mobile GC–MS and one laboratory GC–MS [227]. Qualley,
Hughes & Rubenstein 2020 also acknowledged the inferior reproduc­
ibility of quantitative data of portable instruments in the field and
investigated whether the addition of internal standards into field-
deployed thermal desorption tube, which are used to collect volatiles
for GC analysis, conjunction with the existing internal standards
improved the quality of the data [228]. They found that the addition of
one or several isotopic analogues as an internal standard provided data
normalisation to substantially improve data reproducibility. While this
type of GC–MS sample introduction is suited for hazardous air pollutants
and differs from what may be suitable for bromoform analysis in
seaweed samples, it gives an indication of how in-field bromoform
quantification data can be improved by accounting for analyte volati­
lisation and degraded instrument performance. In another study that
compares a portable GC–MS to a laboratory GC–MS for soil and water
contaminant identification, authors concluded that the portable instru­
ment was suitably sensitive for triaging samples for further laboratory
analysis by a fast and reliable method of field sample preparation and
analysis [229]. For operators in the field looking to select red seaweed
for harvesting with high concentrations of bromoform, a portable in­
strument similar the unit evaluated could provide a readout in as little as
5 min, excluding sample preparation time.
Quantitative bromoform analysis in-field has predominantly been
conducted in air, sea and water as a result of trihalomethane-producing
algae sources and drinking or pool water for determination of chlori­
nation by-products using a variation of headspace sampling techniques
such as SPME or SBME. Liquid sample injection onto a GC column is
seldom used in-field due to the increased complexity, use of solvent and
longer time required for sample preparation [230]. In contrast, Roma­
nazzi et al. 2021 argue that the lower volatility of bromoform compared
to other volatiles provides an advantage of direct liquid injection over
headspace sampling methods in terms of sensitivity and variability [59].
Even with a robust in-field testing method and state-of-the-art instru­
mentation, laboratory-based liquid injection methods that are not sub­
ject to severe performance degradation by environmental factors are still
used for routine confirmation of in-field performance by selecting a
percentage of random samples to run on both instruments for
comparison.
J.L. Hutchings et al.
6. Stability and storage of bromoform in seaweed
As much as In-field analysis is giving an indication of the amount of
bromoform available in seaweed at the time of the harvest, it is crucial to
ensure an unvarying concentration of bromoform during storage and
transportation [231]. So far, experiments conducted with Asparagopsis
spp. have reported that storing the freeze-dried product in a freezer or
refrigerator prevents a decrease in bromoform concentration [50,232].
Temperatures above 25 ◦ C were reported to significantly reduce bro­
moform concentration in freeze-dried samples when stored for 24 weeks
from 7.7 to 3.3 mg/g [50]. Nevertheless, the storage of the freeze-dried
samples at 4 ◦ C or − 20 ◦ C resulted in a slight decrease of 5.5 % or no
change in bromoform levels, respectively. Fluorescent light was re­
ported to not have any effect on the freeze-dried material [50]. How­
ever, storing samples for four months at a temperature of 23 ◦ C did not
result in a significant decrease of bromoform in A. taxiformis [12]. The
same study investigated additional factors, such as light and air expo­
sure. Independent from temperature (− 20 ◦ C, 4 ◦ C, and 23 ◦ C) the au­
thors from that study reported a decrease of bromoform concentration in
A. taxiformis by 75 % when stored in the dark and a decrease by 84 %
when stored under light conditions for four months. Such conflicting
results reported in these studies suggest again that sample preparation
for Asparagopsis spp. is crucial and differences in sample preparation and
sample matrix (i.e. lifecycle stage, extent of homogenisation) can lead to
inconsistent assumptions.
Furthermore, water and oil have been investigated as a storage me­
dium for red seaweed, which also led to a decrease in bromoform con­
centration [48,50]. Regarding Asparagopsis spp. in oil, exposure to
fluorescent light was more apparent compared to freeze-dried analogues
[50]. Additionally, exposure to air was reported to rapidly decrease
bromoform levels in oil-stored red algae, from 1.35 mg/g to a remaining
30 % and 56 % of this value after 4 weeks of storage at 40 ◦ C and 25 ◦ C,
respectively. Interestingly, studies conducted on oil as a storage medium
at temperatures mentioned above reported a slight increase in bromo­
form concentration after long-term storage, with an increase of up to
26.7 % [48,50]. This increase has been linked to a continuing release of
bromoform from small particles of red algae remaining in the oil during
the storage period, which could additionally contribute to the fluctua­
tion of bromoform levels in the product supplemented to the animal
[50]. Ultimately, oil supplementation may also encounter additional
regulatory compliance as it may interact with other ration ingredients
allowed in ruminant diets [233].
7. Future direction and concluding remarks
The enormous biotechnology potential of Asparagopsis spp. is met
with the need for accurate and precise quantification methods for
halogenated compounds to confirm the safety and efficacy of down­
stream products, therefore companies must be willing to adopt well-
validated methods to maintain consumer confidence. Reporting of in­
strument parameters and method validation data in original research
papers is overall poor which increases the difficulty for those working to
identify methods to quantify bromoform in macroalgae. Validation ex­
periments are sometimes outside of the time and resource constraints of
researchers however, well-described methods are achievable. Further­
more, regulation of units when reporting validation data such as LOQ
would improve the ease of cross-method comparison, extending beyond
bromoform in seaweed. GC-MS and GC-ECD still lead the way in bro­
moform analysis with non-chromatographic methods suffering from
poor selectivity in complex matrices such as seaweed. Published HS
methods tend to measure emitted bromoform from seaweed, rather than
preparing samples so that internal bromoform can be measured by
headspace. Further research should explore the utility of EAE for
releasing internalised bromoform from seaweed, and in combination
with existing sampling techniques for rapid, user-friendly and safe
analysis in-lab and on-site. Improvement of extraction techniques,
15
Algal Research 79 (2024) 103478
especially with EAE, can be extended to include other potential
methane-inhibiting compounds that are abundant in Asparagopsis. En­
zymes that can effectively degrade red alga sulphated polysaccharides,
carrageenans and agar are recommended for future work to establish
green bromoform extraction methods. Routine on-site testing is a crucial
component for employing green processes but comes with a variety of
logistical and analytical challenges. Finally, evaluation of bromoform
retention in storage conditions that mimic the environments that con­
sumers are likely to use, and packaged similarly to the final product
would provide much-needed data to companies looking to modify their
processing methods to withstand challenging environmental conditions.
Funding
The authors gratefully acknowledge financial support from the Ma­
rine Bioproducts Cooperative Research Centre, University of South
Australia and CH4 Australia under the grant number MBCRC304.
CRediT authorship contribution statement
Joshua L. Hutchings: Conceptualization, Investigation, Project
administration, Writing – original draft, Writing – review & editing.
Yevgeniya Grebneva: Investigation, Project administration, Supervi­
sion, Writing – original draft, Writing – review & editing. Sarah J.
Dilmetz: Conceptualization, Investigation, Writing – original draft.
Daniel W.M. Pincher: Investigation, Writing – original draft. Peter
Hoffmann: Funding acquisition, Supervision, Writing – review &
editing.
Declaration of competing interest
The authors declare no conflicts of interest.
Data availability
No data was used for the research described in the article.
Acknowledgements
We would like to thank CH4 Global, the National Collaborative
Research Infrastructure Strategy (NCRIS) and the University of South
Australia for support and encouragement during this review.
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