Full text of DOI:10.4319/lo.2000.45.2.0520

520
Comments
Limnol. Oceanogr., 45(2), 2000, 520–522
᭧
2000, by the American Society of Limnology and Oceanography, Inc.
Evidence of the natural production of trichloroethylene (Reply to the comment by
Marshall et al.)
Since the publication of the article dealing with the natural
formation of trichloroethylene and perchloroethylene (Abra-
hamsson et al. 1995), at least one investigation has been
made in order to confirm our findings. Marshall et al. (2000)
conducted a number of experiments with several laboratory
grown Falkenbergia phases and found no production of tri-
chloroethylene and perchloroethylene. In this comment, we
would like to point out three major differences between the
investigations made by us and those by Marshall et al.
(2000) and show evidence indicating a marine source of tri-
chloroethylene. We should emphasize to the readers that the
ocean is of minor importance as a producer of perchloroeth-
ylene compared to anthropogenic sources, whereas the con-
tribution of anthropogenic and marine sources of trichloro-
ethylene are probably of equal magnitude.
ues are calculated from 11 incubated seawater blanks (in-
cubation period of 6 to 12 h), and the detection limit was
calculated as three times the standard deviation of the blanks.
All of the subtropical algae investigated in Abrahamsson
et al. (1995) were collected at the same occasion, and the
experiments were performed within 2 h after collection. No
signs of contamination could be seen in any of the experi-
ments. Therefore, we do not agree with Marshall et al.
(2000) that contamination is the cause of the disagreeing
results.
In the investigation by Abrahamsson et al. (1995), the
identity of the individual compounds was ascertained
through spiking of samples to determine the relative reten-
tion times of the compounds. Even if degassed seawater was
used to prepare the standard solutions, trace amounts of tri-
chloroethylene could be found, and consequently a standard
addition was performed. Marshall et al. (2000) claimed that
since the mass spectrometer was not used in the investigation
presented by Abrahamsson et al. (1995), the identity of tri-
chloroethylene could not be established. In order to optimize
resolution, Abrahamsson et al. (1995) used a dual-column
system. Two capillary columns with different polarities were
connected with glass-fit connectors, thus improving the se-
lectivity of the chromatographic system (Abrahamsson and
Klick (1990). Even if the identification of the individual
compounds was done based on careful determination of rel-
ative retention times only, we have found no reason to doubt
the identity of trichloroethylene based on the relative reten-
tion times found during the investigation.
The following discussion will deal with the differences
between the investigations regarding analytical procedure,
cultivation and physiology of algae, and incubation of algae.
The fourth issue will be to present some supportive results.
Analytical procedure—The method for the determination
of halocarbons that has been used by Abrahamsson et al.
(1995) is described in detail by Ekdahl and Abrahamsson
(1997). In the latter paper comparisons were made between
the method used by Abrahamsson et al. (1995) that uses a
relatively large trap and a megabore gas chromatographic
column, versus an improved method with a microtrap and a
capillary column. The main differences between the two
methods are due to basic chromatographic theory. The de-
tection in Ekdahl and Abrahamsson (1997) was made both
with electron capture detection and mass spectrometry. Ek-
dahl and Abrahamsson (1997) found a shift in relative re-
tention times between separations performed with a mega-
bore column and the capillary column due to differences in
polarity between the two stationary phases. On the other
hand, the elution order of trichloroethylene or perchloroeth-
ylene was not changed.
The method used by Abrahamsson et al. (1995) and Ek-
dahl and Abrahamsson (1997) was designed to minimize
sample handling, and thereby the risk of contamination of
the samples. The analytical method has been thoroughly
evaluated according to traditional analytical procedures. As
described in Abrahamsson et al. (1995), seawater blanks
were monitored continuously during the investigation, and
no contamination from the incubation media or the labora-
tory could be observed. The overall detection limit, the pre-
cision of the incubation procedure, and the analytical pro-
cedure were calculated for data presented in Abrahamsson
et al. (1995). The detection limit for trichloroethylene and
perchloroethylene was 8.4 pmol LϪ1 and 1.2 pmol LϪ1, re-
spectively, and the relative standard deviations were 14% for
trichloroethylene and 26% for perchloroethylene. These val-
Cultivation and physiology of algae—In Abrahamsson et
al. (1995) the analytical equipment was brought to the Ca-
nary Islands in order to be able to investigate freshly col-
lected algae within 2 h after collection. The approach of
Marshall et al. (2000) was to investigate cultivated strains.
Even if they collected species of Falkenbergia at the Canary
Islands, these species were transported to Ireland and kept
in culture for an unknown period of time before the incu-
bation experiments were performed.
Cultivated algae differ from freshly collected ones in that
they are adapted to laboratory conditions, as has been de-
scribed in a textbook by Cole and Sheath (1990). It is there-
fore not surprising that they also differ in production of hal-
ocarbons.
Falkenbergia hillebrandii and Asparagopsis taxiformis
have so-called vesicle cells. These cells contain high
amounts of bromine and iodine, which have been localized
by X-ray microanalysis (Wolk 1968), and they are very frag-
ile. If the vesicle cells break, free iodine is released (Kylin
1928). Iodine, together with hydrogen peroxide and the per-
oxidases of the alga, gives rise to many possible chemical
interactions and consequently different halogenated organic

Comments
521
Table 1. The halocarbon production rates of two macroalgae de-
termined by GC-MS. The rates are given in ng per g fresh weight
compounds, compared to reactions made only by peroxidas-
es and hydrogen peroxide.
Ϫ1
(FW) and hour (ng g FWϪ1 h ). (Abrahamsson and Ekdahl, unpubl.
Marshall et al. (2000) stated: ‘‘However, if the biosyn-
thetic trait for trichloroethylene and perchloroethylene pro-
duction is established in this species to the extent suggested
by the high rates of release recorded by Abrahamsson et al.
(1995), it is difficult to explain why production of neither
compound was detectable in any of the genetically diverse
collection of laboratory-cultured isolates examined at rates
several orders of magnitude less than those reported by
Abrahamsson et al. (1995).’’ We do not agree with this state-
ment. The algae change their metabolism according to the
environmental conditions and their life cycle. It is therefore
incorrect to claim that if the algae under certain conditions
produce toxic volatile halocarbons, they should always do
so under any environmental condition.
data.)
Meristiella
gelidium
Gracilariopsis
lemaniformis
Compound
Methyliodide
2
2
*
20
30
3
*
40
*
0.8
0.2
0.4
0.1
2
Ethyliodide
Tetrachloromethane
Trichloroethene
Dibromomethane
Chloroiodomethane
2-iodobuthane
Dibromochloromethane
1-iodobuthane
Bromoform
*
1
2
0.1
100
*
2 500
40
Diiodomethane
Marshall et al. (2000) suggest that trichloroethylene and
perchloroethylene could possibly be derived from biosyn-
thesis of tetrachloroethane or pentachloroethane by algae, or
by abiotic dehydrohalogenation. In bacteria, fungi, algae,
and plants, ethylene is produced and readily released from
all tissues to the surroundings. The concentration of ethylene
is generally estimated to 4.4 nmoles LϪ1 in the organisms.
The rate of production depends on the age of the organism
and the environmental conditions. Ethylene production is
usually related to physiological stresses. Any type of wound-
ing will induce ethylene biosynthesis, as well as all kinds of
stress. Ethylene is synthesized by plants from S-adenosyl-
methionine, which is synthesized from methionine and aden-
osine 5Ј-triphosphate (ATP) (ATP is mainly formed in light),
and is an intermediate in the ethylene biosynthetic pathway.
The immediate precursor of ethylene is 1-aminocyclopro-
pane-1-carboxylic acid (ACC), which is converted to ethyl-
ene by the enzyme ACC oxidase (Taiz and Zeiger 1998). It
is possible that both trichloroethylene and perchloroethylene
are formed by halogenating reactions from ethylene pro-
duced during stress by the algae, and not only from the con-
version of tetrachloroethanes and pentachloroethanes.
* Not detected.
photosynthesis. The uptake of inorganic carbon at pH 8.2 or
higher depends on the ability of the algae to convert
HCOϪ3 to CO2. Carbon acquisition mechanisms of marine
macroalgae are described in the textbook by Falkowski and
Raven (1997).
We believe that a large headspace such as that used by
Marshall et al. (2000) will change the chemical environment
in an unpredictable way, unless otherwise explained.
The different results shown by the two investigations raise
the question of which method mimics best the natural en-
vironment. We have compared the production rates measured
in our incubation experiments with production rates mea-
sured in a natural environment at the Canary Islands (Ekdahl
et al. 1998). The production rates derived in the laboratory
experiments are in agreement to the in situ measurements.
Unfortunately, Falkenbergia hillebrandii was not present in
the investigated pool.
Supportive results—Identification of naturally produced
halocarbons by two red macroalgae, Meristiella gelidium and
Gracilariopsis lemaniformis, was achieved by using the
method described in Ekdahl and Abrahamsson (1997), and
the results are presented in Table 1. The incubations were
made in duplicates, with a corresponding blank consisting
of only seawater. The algae (0.6 g fresh weight) were in-
Incubation of algae—Another difference between the two
investigations is that the incubation setup used by Marshall
et al. (2000) is not identical to ours. Marshall et al. (2000)
performed their experiments with a headspace of air, whereas
we took care to avoid headspace. There are several reasons
why such a volume of air should be avoided. Marshall et al.
(2000) comment on the losses of halocarbons to the air in-
side the incubation chamber, and suggest that the losses
should be of minor importance due to the relatively low
Henry’s law constants. However, other parameters might be
changed using headspace, owing to the presence of organic
compounds, as well as an alga.
The headspace present in the incubation experiments per-
formed by Marshall et al. (2000) may well have changed
factors initiating oxidative stress, thereby changing the pro-
duction of hydrogen peroxide, and consequently the produc-
tion of trichloroethylene and perchloroethylene by the algae.
During the incubation, we have a high production of hydro-
gen peroxide in the closed vials and other stress-related com-
pounds, as the algae become deficient in total inorganic car-
cubated in 60 ml of seawater at a light intensity of 300 ␮mol
photons mϪ2 sϪ1 for 3 h. The identification and quantification
were performed with a mass spectrometer as detector. The
measured production rates are comparable with the results
presented by Abrahamsson et al. (1995), and Ekdahl et al.
(1998). Also, tetrachloromethane, an even more controver-
sial compound, was produced by one of the algae.
Earlier we have shown that there are large diurnal varia-
tions in the production of halocarbons (Ekdahl et al. 1998)
in a tidal pool. Not only the traditionally expected halocar-
bons showed diurnal variations, but also trichloroethylene
and perchloroethylene. Comparisons were made with the an-
thropogenic 1,1,1-trichloroethane, which did not vary over
a period of 24 h. The highest concentrations were measured
at the time of day with maximum photosynthesis.
bon and the pH increases to high levels (pH
ϳ 10) due to

522
Comments
for the ability of algae to produce halocarbons. The results
from these two studies prove the need to standardize the
methods used for production studies of halocarbons, in order
to be able to make relevant comparisons.
Katarina Abrahamsson
Department of Analytical and Marine Chemistry
Chalmers University of Technology
SE-412 96 Go¨teborg, Sweden
Marianne Pederse´n
Department of Plant Physiology
Stockholm University
SE 116 11 Stockholm, Sweden
Fig. 1. Diurnal variation of the concentrations of trichloroeth-
ylene (dashed line, left axis), diiodomethane (dotted line, right axis),
and PAR (straight line, right axis). Samples were taken every hour
References
´
´
ABRAHAMSSON, K., A. EKDAHL, J. COLLEN, AND M. PEDERSEN.
1995. Marine algae—a source of trichloroethylene and per-
chloroethylene. Limnol. Oceanogr. 40: 1321–1326.
or every 2 h during a 24-h period at a station located at 60
Њ23ЈS,
6Њ
00 E in the Southern Atlantic Ocean (Abrahamsson and Lore´n,
Ј
,
AND S. KLICK. 1990. Determination of biogenic and an-
unpubl. data). The determinations were made according to Ekdahl
and Abrahamsson (1997).
thropogenic volatile halocarbons in sea water by liquid-liquid
extraction and capillary gas chromatography. J. Chromatogr.
513: 39–45.
C
OLE, K. M., AND R. G. SHEATH. 1990. Biology of red algae. Cam-
Similar results were obtained in the open ocean. The de-
termination was made according to Ekdahl and Abrahams-
son (1997). The low concentrations in oceanic seawater do
not permit the use of a mass spectrometer as detector. How-
ever, a gas chromatography–mass spectrometry system, with
an identical chromatographic capillary column, confirmed
the retention times and the elution order. As can be seen in
Fig. 1, the concentrations of trichloroethylene and diiodo-
methane, measured at chlorophyll maximum at 25 m, reach
their maxima during midday and again indicate the relation-
ship between the production of trichloroethylene and pho-
tosynthesis. A second maximum was observed in the even-
ing, which probably is caused by hydrogen peroxide
produced during respiration. The levels of tetrachlorometh-
ane and 1,1,1-trichloroethane did not vary over this 24-h
period.
bridge Univ. Press.
EKDAHL, A., AND K. ABRAHAMSSON. 1997. A simple and sensitive
method for the determination of volatile halogenated organic
Ϫ1
Ϫ1
compounds in sea water in the amol l to pmol l range.
Anal. Chim. Acta 357: 197–209.
´
, M. PEDERSEN
,
AND K. ABRAHAMSSON. 1998. A study of
the diurnal variation of biogenic volatile halocarbons. Mar.
Chem. 63: 1–8.
FALKOWSKI, G. P., AND J. A. RAVEN. 1997. Aquatic photosynthesis.
Blackwell.
¨
K
YLIN, H. 1928. Uber Falkenbergia hillebrandii und ihre Bezie-
hung zur Abspaltung von Iod. Bot. Notiser 233–254.
M
ARSHALL, R. A., J. T. G. HAMILTON, M. J. DRING
, AND D. G.
HARPER. 2000. The red alga Asparagopsis taxiformis/Falken-
bergia hillebrandii—a possible source of trichloroethylene and
perchloroethylene? Limnol. Oceanogr. 45: 516–519.
TAIZ, L., AND E. ZEIGER. 1998. Plant physiology, 2nd ed. Sinauer.
W
OLK, C. P. 1968. Role of bromine in the formation of the refractile
inclusions of the vesicle cells of the Bonnemaisoniaceae (Rho-
dophyta). Planta 78: 371–375.
Conclusions—The results obtained by Marshall et al.
(2000) do not contradict the findings of Abrahamsson et al.
(1995), but they clearly demonstrate that the history of an
algae, and the pretreatment of the algae, are crucial factors
Received: 18 June 1999
Accepted: 23 September 1999