Chlorate and Chlorite Analysis in Seawater,
Chlorate Sinks,
and Toxicity to Phytoplankton
BY
Estelle Couture
Submitted in partial fulfillment of the requirements
for the degree of Master of Science
Dalhousie University
Halifax, Nova Scotia
Canada
July 6,1998
@Copyrightby Esteiie Couture, 1998
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Contents
Table of Contents
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List of Figures
.-..................................................................................................
List of T~lbles
......................................................................................................
Abbreviations and Symbois
Absîract
...............................................................................
..............................................................................................................
Acknowledgments
..............................................................................................
.
Chapter 1 General introduction.............,..,...........................................
...
1.1 Preface ...................,.,............. ...............................................................
1.2 Background
1.2.1 What is chiorate and what are its commercial uses?................
1.2.2 Sources of chlorate in the environment.....................................
1.2.3 Chlorate fiom the puip and paper industry ..................... .
.
..
1.2.4 The formation of chlorate in a bleaching plant.........................
1.2.5 The removal of chlorate fiom bleaching plant effluents............
12.6 Reporteci cases of chlorate causing damage to the environment.
1.2.7 Mechanism of chlorate toxkity................................................
1.2.8 Chlorate toxicity in the presence of ammonium........................
1.2.9 Chlorate toxicity studies on algae.............................................
1.2.1 O û v e ~ e w
of exisring methods for chlorate analysis.................
.................
.
1.3 Hypotheses and objectives of this thesis........................ .
.
Chapter 2 Colorimetric method for the measurement of chlorate
and chlorite in fresh and seawater .
batch and continuous modes
2.1 Introduction
.........................................................................................
2.3 Results and discussion ........................... ..............................................
.....................
2.3.1 Chlorate only analysis .........................
.......
2.3.2 Simultaneous chlorate and chlorite analysis:
..............
The subtraction method ........................ .......
.
,
.
Chapter 3 Chlorate sinks in the water column of a marine
47
environment A laboratory experiment
...................................
O
3.I Introduction .........................................................................................
3.2 Materials and rnethods .........................................................................
3 -3 Resuits and discussion .........................................................................
.
47
48
55
Chapter 4 Chlorate toxicity in marine phytoplankton in relation to 63
species and nitrogen nutrition
........................................................
4.1 Introduction .........................................................................................
4.2 Materials and methods ......................,. ..............................................
4.3 Results ..................................................................................................
4.4 Discussion .............................................................................. ...............
.
Chapter 5 Summary and conclusions
................................O..........e.
List of Figures
Figure L halytical manifold for the automated system ...................................... 26
Figure 2 a) Absorbante as a function of concentration for chlorate ranghg ..............30
from 0.24 to 95
(A=O.852 c3+ 1.24c2+ 1.52 c =O, ~'=0.995)
b) Absorbance as a fuaction of concentration for chlorate
ranging fÎom 0.24 to 20pM. (A = 0.0133~- 0.000509, R* is 0.999)
m.
Figure 3 Chlorate standard curves generated using various grades and brands of acid...33
Figure 4 Absorbance at 448 nm as a function of HCI concentration for 24 W...........42
chiorate and 24 p M chlorite
Figure 5 Graphic representation of the subtraction method.. .................................45
Figure 6 a)Chlorate concentration as a function of time in double distiiled water.. ....A0
b)Chlorate concentration as a function of time in artificial seawater (potential
sink = salts)
c)Chlorate concentration as a fûnction of t h e in naturai seawater filtered
through a 0.2 p M Glter (potential sink = dissolved organic matter)
d)Chiorate concentration as a ftnction of time in naturai seawater Ntered
through a 2pm filter (potential sink = bacteria)
Chlorate concentration measured in treatments 3 and 4 is lower than
the initial concentration of 18 p M because of the interference
caused by DOM found in natural seawater (See chapter 2 for M e r detail).
Figure 7 a)Chiorate and nitrate concentration and in vivo fluorescence.. ................ ..6 1
of a naîural assemblage of phytoplankton as a b c t i o n of time
for treatment 5a (CCIo3-= -0.0439t + 14.7) b) Chlorate
and nitrate concentration and in vivo fluorescenceof a nahual
assemblage of phytoplankton as a f'unction of time for treatment
Sb (Cam. = -0.043 1t + 14.9) c) Chlorate and nitrate concentration
and in vivo fiuorescence of a culture of PhneodactyZum
tricomutum as a fiulction of time for treatnient 6a (CClo3= -0.353t
+ 15.5) d) Chlorate and nitrate concentration and in vivo
fluorescence of a culture of Phneodactym tricomutum as a
f'unction of time for treatment 6b (Ccio3- = -0.268t + 15.1)
Figure 8 a) Chlorate concentration in double distilied water as a h c t i o n of.. .........62
time when SuperQ water is Uradiated with simulated sunlight
b) Chlorate concentration in naturai seawater as a fùnction of
time when natural seawater is irradiated with simulated sunlight
Figure 9 Growth curves when a) DunaIieIla tertiolecta b) Phaeodactylum............7l -72
tricomutum c) Thalassiosirapseudonmu2 d ) EmiZiamia hwleyi e) 77mZussiosiira
weissflgii f ) Chaetoceros gracilis g) Teiraselmis sp.
h) Odontella mobiliensi..are exposed to O, 1O, 100,l O00 and
10,000 FM chlorate. Each point represents the average of a tripkate.
Error bars were omitted for clbut they are refiected the
standard error of growth rates in table 7. (e,f,g,h are shown on the next page)
Figure 10 Fluorescence, ammonium concentration and chlorate concentration.. ......75-76
as a h c t i o n of time for a) control b) 10 p l V I chlorate treatment
c) 1O0
chlorate treatment d) 1000 pM chlorate treatment and
e) 10,000 chlorate treatment for the species DumZiella tertiolecta
(c,d,e are shown on the next page). As previousIy error bars have
been omitted for clarity but are rdected in the standard error of
the growth rate calculation s h o w in Table 7.
Figure 11Fluorescence, nitrate concentration and chlorate concentration as.. ........80-8 1
a fhction of time for a) control b) 10 pM chlorate treatment
c) 100 pMchlorate treatment d) 1000 pM chlorate treatment
and e) 10,000 chlorate treatment for the species D u ~ l i e l h
tertiolectu. (c,d,e are shown on the next page). The shaded
bar in d) represents the time intervai dining which the zone of
critical chlorate to nitrate ratio is reached. As before e m r bars
have been omitted for cl*
but they are reflected in the standard
error of the growth rate calculation shown in Table8.
List of Tables
Table 1 Slopes for identical sets of chlorate standards measured.. ...........................40
in water fiom various sources
Table 2 Description of the progressive treatments to detennine chlorate sinks.............5 1
Table 3 Irradiated treatments in the experiment to detennine chlorate sida ...............54
Table 4 Rate of chlorate concentration change per &y, standard emr,. .................... 56
95% confidence interval, t statistic and the number of
observations used to calculate statistics. Each observation
represents the average of six replicates for each sample day.
Table 5 Summary of the treatments in the experiment testing nitrate.. .....................68
and ammonium in relation to chlorate toxicity.
Table 6 Growth rate, points used for its dcuiation, standard enor........................ .73
taking triplkates into consideration, p value and R2for each
species tested and each treatment
Table 7 Growth rate ,standard error, p value and RZfor each chlorate. ....................77
treatment when Dunaliella tertiolecta uses nitrate or a.mmoniumas
a nitrogen source
List of abbreviations and symbols
CIO,'
chlorate
cioz-
chlorite
HCI
hydrochlonc acid
OTO
ortho-tolidine dihydrochloride
Cl8
bound octadecyl functional group
DOC
dissolved organic carbon
PP='
part per million
FM
micro molar
GFIC
glas fiber flters, size "cm (0.45 @f)
w
ultraviolet
A
absorbance
C
concentration
f72
medium for the culture of phytoplankton, (GuiIIad, 1972)
K
Enrichment medium based on f medium (Keller et al, 1987)
DCMU
[3-(3,4-dichIoropheny1)- 1, l aimethylurea]
F
Fluorescence
Cr
Specific growth rate
Abstract
An existing colorimetric method for the detection of chlorate was modified to
permit seawater measurement of chlorate at environmental concentrations in batch and
continuous modes. The method is based upon the oxidation of ortho-tolidine to a yellow
holoquinone in a strongly acidic medium. The working concentration range for chlorate
is O to 20 p M and the detection limit was determined to be 0.02 PM.This rnethod c m be
used in artificial and natural seawater but marine organic material interferes, yielding a
decrease in absorbance values. 0th- interfmng cornpounds include nitrite, sulfite, iron,
iodide and bromide. It is recommended that standard additions be used when measuring
chlorate in environmental samples. The method was dso modifïed to meanire chlorite
and chlorate when both are present in a sample by subtracting the chlorite contribution to
total absorbance using the merence in redox potential of both species in acidic solution.
A laboratory experiment was conducted to determine chlorate sinks in the
environment. The potentiai sinks tested included, double distiiled water, seawater,
dissolved organic matter, bacteria, a natural assemblage of phytoplanlcton, a laboratory
culture of Phaeodocf~lmnicomtum and simulated sunlight The redts of this
experiment showed that the oniy sink for chlorate was uptake by phytoplankton. The
experiment was qualitative in nature and consequently no empirical relationships could
be derived fiom the resuits.
The next section examines the effects of chlorate on phytoplankton. Laboratory
cultures of 8 different species were subjected to identical chlorate treatments ranging
from O to 10,000 @Mto investigate interspecies variation of chlorate toxicity. Odontdh
mobiiiensis was sensitive to chlorate at concentrations as low as 10 ph4 while the other
species required at least 100 pM to show toxic effects. Following this experiment, it was
hypothesized that a ratio of chlorate to nitrate specific to each species was responsible for
chlorate toxicity and not chlorate itself. To ve* this hypothesis, a DunuIieIlo tertidectu
culture was subjected to the same chlorate treatrnents (O to 10,000 CiM) and was given
either ammonium or nitrate as a source of nitrogen. The hypothesis that toxic effects
would not be observed in D. tertiolecta grown on ammonium, at any of the tested
chlorate concentrations was verfieci. When ceiis were grown on nitrate, a critical
chlorate to nitrate ratio of between 40 and 155 was dete&ed.
From those results came
the conclusion that most species of phytoplankton would not be affectecl by
environmental concentrations of chlorate when either nitrate is not completely depleted
(the cntical ratio would never be attained) or when ammonium is present.
Acknowledgments
First, I would Iike to thank all the mernbers of my s u p e ~ s o r cornmittee,
y
Marlon
Lewis, Bruce Johnson and Bill Miller for their help, patience and continuou support
throughout this project. I would also Like to thank them for dowing me to direct my own
project, to work at my own Pace and to let me make my own mistakes. It has been
dBcult at times but 1can honestly say that it has been the most constructive period of
my Ne.
1 would like to say speciai t h d a to Gary Maillet for teaching me everything 1
know about the AutoAnaiyzer and phytoplankton cultures. Thank you for all your
invaluable help and advice and your endless patience. 1could not have done it without
you.
Th&
to Mike Scarratt, David Slatmwhite, Tammi Richarson and Aurea Ciotti
for support and advice. Thanks to Pierre Clement of BI0 for his technical help and for
allowing me to use bis AutoAnalyzer for ammonium mea~u~ements.
I would like to thank my parents and my a m t and uncle (Marthe and Francis) for
unconditional support and understauding. Thanlcs for always believing in me no matter
what, and help me overcome obstacles during these last few years.
Finally, thanks to Darren and di my fnends for their support, understanding and
for a i l the gmd times.
Chapter 1
General introduction
1.1 Preface
Chlorate is released into marine coastai areas in the liquid efnuents of certain
industries, &y
the pulp and paper industry. Chlorate is a bleaching by-product and a
well known herbicide. In the Baltic Sea, pulp miil chlorate has caused extensive damage
to the marine ecosystem (Lehtinen et al., 1988), and as a result, aiiowable concentrations
of chlorate in pulp mill effluents are now regulated in both Sweden and Finland.
ALthough the pulp and paper industry plays a signincant role in the Canadian economy
with 144 plants (Enviromnent Canada, 1983), no comparable regdations cmently exist
to control chlorate discharges and no studies have been conducted to determine whether
chlorate adverseiy affects the CanSnian marine environment.
In Canada,severai chemical pulp miUs, an important source of chlorate discharge,
are iocated on both Atlantic and Pacifk Ocean shores (Energy, Mines and Resources
Caaada, 1983). Quantification of chlorate concentration in both effluent and in the water
bodies to which the discharge takes place is a necessary first step to evaluate the
environmental consequences of this discharge. No analytical method currently exists for
the determination of chlorate in seawater. It is therefore important that a method be
developed in order to assess the importance of chlorate discharges fiom chemical puip
mills and to establish the persistence of chlorate in marine coastal areas. In fiesh water,
ion chromatography has been adopted as the standard method for chlorate analysis
(ASTM, 1996) but the required insûumentation is costly and is not available to a l l
analysts. Furthemore, the fact that chloride concentration in seawater is several orders of
magnitude higher than the concentration of chlorate may pose a problem when both are
detected using the sarne separation column. Consequently, a need exists for a simple,
inexpensive method for chlorate d y s i s in both hshwater and seawater.
Secondly, few studies have exarnined the mechanisms of chlorate toxicity (Crafls,
1935; Hurd-Karrer?1941;Gorenfiot, 1947; Aberg, 1947; Perrier-Benoit, 1951) and none
of these studies have been conducted on marine phytoplankton. They are the likeIy path
by which chlorate am affect the marine ecosystem, since they potentially take up chlorate
(Balch, l987), and are the basis of the food web in the aquatic environment.
This thesis is focused on the initial evaluation of the potential environmental
effects of chlorate discharge to the marine environment. To accomplish this, an analytical
method for the measurement of chlorate in seawater was modified fiom one already
existing (Urone and Bonde, 1960), and a series of assays and bioassays have been canied
out to determine chlorate sinks and chlorate toxicity.
1.2 Background
1.2.1 What is chlorate and what are its cornmerciai uses?
Chlorate (C103>, an oxidized form of the chlorine atom, has an oxihtion state of
+S. making it a strong oxidant. Chlorate is usually combined with sodium or potassium
to form a salt. Over the years, sodium chlorate has become more commerciaüy
signifiant than the potassium sait because of its lower manufacturing cost and its high
solubility in water (95.7 g/ 100 ml at 20°C) (Robbins et al., 1942).
Canada is an important manufacturer of sodium chlorate and most of its
production is used in the pulp and paper industry. In 1983, Canada produced 296.5
kilotonnes of sodium chlorate (Environment Canada, 1985). Of this total, 64% was sold
to the Canadian pulp and papa industry and 37% was exported or used for other pinposes
(Environment Canada, 1985). Since 1983, the demand for sodium chlorate has grown
due to the trend in the pulp and papa industry to substiMe chlorine dioxide for molecuiar
chlorine during the bleaching process (Piccione, 1991). Sodium chlorate is a necessary
chernical reagent for the on-site production of chlorine dioxide (Pdeologou et ai., 1994,
Ginkel et ai, 1995). In 1990,Canadian mil1 consumption of sodium chlorate had grown
to 295 kilotonnes, and to meet a nsing demand, four new sodium chlorate plants opened
in 199 1, producing an additional 177 kilotonnedyear (Piccione, 1991).
Chlorate has dso been commonly employed in agriculture. Chlorate was used
extensively as a herbicide and as a defoliant especidy between the years 1930 and 1950.
The first attempt to use chlorate as a herbicide occurred in Austraiia at the tum of the 2ûth
century, but without much success (Anonymous, 1901). It was not untill92 1 in
SwitzerIand that work began again on the use of chlorate as a herbicide (Neuweiler,
1930). Initially, sodium chlorate was used only on road sides, ditches and waste areas
but, by the late 1920s, sodium chlorate began to be used on arable land (Aberg, 1947).
Sodium chlorate was considerd unique among chernical herbicides because its
toxic effects couid be produced in a number of ways (Robbins et al. 1942). It could act as
a contact poison on plant tissue, kiuùig plants by direct root absorption. Under humid
atmospheric conditions, it could serve as a translocated herbicide i.e. the herbicide is
appiied to leaves for transport to the mots (Robbins et ai. 1942). The herbicidal action of
sodium chlorate has k e n attrïbuted to the CIO3-anion, and consequently, humidity in the
soi1or in the aîmosphere is required for sodium chlorate to dissociate and to penetrate the
plant.
The use of chlorate as a herbicide has decreased in the last 30 years because of its
inefficiency in fertile soils and because of its explosive properties (Isensee et al., 1973).
It represents an extreme fire hazard when spilled on clothes, wood or papet, igniting by
fiiction or heat This led to its replacement by more effective and less hazardous
use in the manufacture of
chernicals. More recently, chlorate has found colll~~lercial
matches and fireworks (Envitonment Canada, 1985)Although the use of sodium chlorate a s a herbicide has decreased significantly, it
bas not completely ceased. In 1988,4 million pounds of chlorate were sprayed over
Cotton fields in California in order to defoliate before barvest (Stimmann and Ferguson,
IWO).
1.2.2 Sources of chlorate in the environment
Chlorate is not known to occur nahirally in the environment. Most often, chlorate
is discharged in liquid effluents as a by-product of various industrial processes. For
example, chtomte is found in effluents h m the pulp and papa industry, fiom the textile
and wool indutry and fiom the flou.and food product industry as a result of the use of
chlorine dioxide as a bleaching agent (Cosson and Ernst, 1994). In the chlor-aikali
industry, membrane ce11 technology has replaced mercury-based processes in order to
eliminate toxicity caused by mercury. With the new process however, the effluents can
contai.significant quantities of chlorate (van Wijk and Hutchison, 1995).
Chlorate can also enter the environment as a by-product of disinfection. When
hypochlorite is released in water, it decomposes to chlorate and chlonde (van Ginkel et
al., 1995). In drinking water, chlorine dioxide has begun to replace chlorine as the
principal disinfecthg agent (Aieta and Berg, 1986). Chlorine diosde dws not react with
humic substances as c h i o ~ does
e to form trihaiomethanes and it does not react with
ammonia to fonn chioramines (Aieta and Berg, 1986, Lykins et ai., 1990) but it fan form
chlorite and chlorate as by-products (Gordon et al., 1990).
One of the trihaiomethanes is chioroform. Chloroform has been linked to the
increased occurrence of tumors in test animais (L.W.Condie, 1986) and consequently
trihalomethanes have ken strongiy regdated (Federal Register, 1986) in driaking waterAs for chlorine dioxide by-products, exposure to chlonte is of concern but chlorate
toxicity remaius controversiai (Lykins et al. 1990). Further studies of these oltidants are
needed in order to understand how they can affect human heaith. Because of this
uncertainty, the US EPA has recommended a Iimit of 1 mg/l of total residual oxidants of
chlorine dioxide in drinking water (EPA, 1983).
123 Chlorate from the pulp and paper industry
Chlorate discharges corne primarily fkom bleaching plants of kraft pulp mills.
Kraft is the name of a chemical pulping process. In pulping, the main objective is to
loosen and separate wood fibers from each other, and this can be achieved either
mechanically or chemically.
Mechanical pulping is accomplished by crushing and grinding wood logs or chips
in the presence of steam or water near the boiling point Wiih this method, most of the
lignin temains, resuiting in weaker paper that is used mostly as newsprint (Environment
Canada, 1983 and Laasonen, 1985).
The two most common chemical pulping processes use sulnte which operates
under acidic conditions, and krafî, also d e d sulfate, which operates under allcaline
conditions. Delignification and fibre sepmtion take place in a high pressure and high
temperature digester to which wood chips and chemicals (NaOH for kraft and S 4 for
sulnte) are added. Since chernical delignification is more thorough than mechanical
pulping, paper fiom chemical processes is strong and of good quality . In Canadg the
kraft process is more prevalent than the sulfite process because of its versatility and
fiexibiliîy (Environment Canada,1983).
The pulp generated by kraft mills is dark due to raidual phenolic groups of lignin.
Most of these residuals are eliminated in the pre-bleaching stage but M e r bleaching is
required to achieve the desired standard of brightness (Environment Canada, 1983). In
most cases, chlorine compounds are used because they both dissolve lignin and bleach
pulp (Laasonen, 1984). Oxygen, ozone and hydrogen peroxide are also used as bleaching
agents in some mills (PPC, 1996). Although the present trend in kraft plants is away
h m chlorine, chlorine dioxide is Likely to remain in use for some tirne in North America
(PPC, 1996).
Until the early 80's, molecular chlorine was the most common bleaching agent-
In the pre-bleaching stage, chlorine would combine with lignin to f o m hydrophobie,
persistent and highly toxic by-products such as organochlorines and AOX (adsorbable
organicdy bound halogens) (Environment Canada, 1983). To produce l e s toxic
effluents, there bas been a trend in North Amerka and in Scandinavia to substitute
chlorine dioxide for part of the molecular chlorine (Axegard, 1986). Chlorine dioxide
does not resdt in the formation of organochiorines Like dioxiris and furans because
chlorine dioxide breaks down the aromatic rings of Lignin. However, one of the byproducts of the use of chlorine dioxide is chlorate. Although chlorate is toxic to plants, it
is thought to be not as persistent as other chlorine compounds so it has not k e n
considered a problem for toxicity . Chlorate's high solubility d e s it an unlikely
compouud to adsorb on food particles or bioaccumulate in aquatic animals (van Wijk and
Hutchison, 1995). There are currently no data available on the persistence of chlorate in
the water column (van Wijk and Hutchison, 1995).
12.4 The Formation of chlorate in a bleaching plant
Chlorate discharges Vary fkom miil to mil1 and depend on a number of operating
parameters including pH, the percentage of chlorine dioxide substitution, the order of
addition and the time between CIO2 and Cl2 addition (Bergnor et al., 1987, Liebergott et
al., 1990, Ni et al., 1993). When chlorine dioxide is used as a bleaching agent, it is
partially oxidized and the yield to chlorate can be as much as 50 mole % of the chlorine
dioxide used (Genngard et al., 1981). In a mixture of chlorine and chlorine dioxide,
chiorate formation decreases with decreasing pH (Reeve and Rapson, 198 1). Adding
chlorine dioxide before chlorine and addhg them sequentiaily instead of simultaneously
ais0 reduces chlorate formation (Liebergott et al., 1990). However, adopting conditions
that minimize chlorate formation is not a good solution because such conditions ofien do
not provide effective bleaching of the pulp (Bergnor et al., 1987). It is very difficult to
i
predict chlorate discharges because bleaching parameters vary periodicaily and fkom d
to mill (Solomon et al, 1987).
12.5 The removal of chlorate from bleaching plnnt effluents
Chlorate fornation in the pulping process is unavoidable and thus, the most
efficient way to reduce its concentration is to treat the effluents before they are discharged
into the environment (Gerxngard, 1989). Effluents can be treated either chemically or
bioiogically. One of the chemical methods to remove chlorate is reduction to chloride ion
by sulfur dioxide. The drawback with this method is that for every mole of chlorate
removed, three moles of sulfuric acid are produced, quiring m e r treatment to
n e u t d k the acid (Bergnor et al., 1987). Another method for removal of chlorate, as
Gonce and Voudrias (1 994) showed in a bench scale experiment, is to pass the effluent
through a column packed with granular aaivated carbon which physically and reversibly
adsorbes chlorate.
BioIogicai treatments are by far the most commonly employed methods for
secondary treatment of effluents. In Canada, aerated lagoons have been adopted almost
universally by the pulp and paper industry (Environment Canada, 1983). Efnuents are
retained in basins for approximately 5 to 10 days (Laasonen, 1984) where the main
objective is to reduce the concentraîion of dissolveci organic material using naturaIIy
occiimng micro-organisms (Environment Canada, 1983). Aerators a~ o f h used to keep
the oxygen levels hi&. (Laasonen, 1984). However, chlorate removal can ody be
accomplished under anaerobic conditions (Gemigard, Berglund, 1987) or under low
oxygen concentration (less than 0.5 mg/I) (MFG Akhiellt, 1988). Because of this
problem, k m g a r d (1989) suggested a two stage scheme where the first part of the
lagoon is not aerated in order to achieve anaerobic conditions.
In Canada, chlorate discharges are not regdateci (V. Li, pers. comm.,1995)
whereas in Sweden and Finland, where problems related to chiorate toxicity have been
observed, strict regdations now exist (J. Tana, pers comm., 1995). As a direct
consequence, most of the research efforts aimed at reducing chlorate discharges have
been conducted and applied in Finland and Sweden only. Environment Canada currently
does not require miils to supply any data regarding chlorate concentration in effluents (Li,
V., pers. communication). It is therefore not known whether chlorate discharges are king
reduced during secondary treatment.
1.2.6 Reported cases of chlorate causing damage to the environment
There are at least two reported cases where puip mil1 chlorate has k e n identified
as having caused extensive damage to plants. In South f i a i , the gras of pasture land
sustained substantial damage after it had k e n im'gated with the effluents of the kraft puip
mill of Ngodwana which was reported to contai.as much a s 1800pM chlorate.
(Raubenheimer et al., 1989)
The only well documented report of damage to marine M e occurred in the Bdtic
Sea. In Sweden, chlorate discharges nom pulp milis ranged h m 12 to 840 p M
(SFIWAPRF,1985) and effluents fiom one pulp mil1 caused the disappearance of the
algae Funcr vesiculosu~in an area of 12 km2(Lehtinen et al., 1988). This created a great
deal of concem since Fucus vesiculosus plays a key role in the Baltic Sea ecosystem
(Lehtinen et al., 1988). The same author reported that the disappearance of the algae
caused a net shift h m autotrophy/heterotrophy to detritus eaters in the benthic
cornmunity and indirectiy affected the recniitment of pelagic fish populations.
12.7 Mechanism of chlorate toxicity
There have been several reports on the physiological rnechanism of the toxic
action of chlorate on higher plants. For example, Crafts (1935) and Latschaw and
Zahnley (1927) found that the roots of weeds treated with sodium chlorate were rapidly
depleted of theh carbohydrates, specincaily their starch reserves. Gorenflot (1 947) found
that sodium chlorate inhibited photoqmthesis in Elodea canadenris and Hofstra ( 1 977)
reported that chlorite causes general oxïdative breakdown of proteins in tomato plants.
Aberg (1 947) studied young wheat plants after they had been treated with chlorate and
found a severe inhibition of growth in mots and bleaching of leaves. He also found that
toxicity is enhanced by light and high temperatures He poshilated that it is the reduced
forms, chlorite and hypochlorite, rather than chlorate iîself that is toxic, and that the
reduction process was enzymatic. In a subsequent study, Liljestrbm and Aberg (1966)
proposed that nitrate reductase, the same enzyme that reduces nitrate to nitrite, is
responsible for the reduction of chlorate to chlorite. This hypothesis was later
investigated and confirmed with studies on Escherichia coli (Goksoyr, 1952), on the
green alga Chlorella vulgmis (Solomonson and Vemesland, (1972) and on tomato plants
(Hofsira, 1977).
Chlorate can provide a substrate for the enzyme nitrate reductase (Solomonson
and Venneslmd, 1972, Hofstra, 1977, Siddiqi et al., 1992) but it cannot induce nitrate
reductase activity (Hofstra, 1977, Siddiqi at al., 1992). The affhity of nitrate reductase is
considerably lower for chlorate than it is for nitrate. In f a Solomonson and Vennesland
(1972) found a K, vdue of 1200 p M for chlorate and 84 p M for nitrate in Chiorella
vuigmis while H o f m ( 1 977) found K , values of 4000 pM and 150 pM in tomato plants
for chlorate and nitrate respectively.
Chlorate is a nitrate analogue (Deane-Dnunmond and Glass, 1982), competing for
the same transport site (Balch, 1987). This fact was put to use in genetic studies of the
higher plant Arabidobsis t h a i i m where chlorate was used to meen for the presence or
the absence of nitrate reductase in mutants. The lack of toxicity would indicate a mutant
without the enzyme and vice-versa (Oostindier-Braaksma and Feenstra, 1973). Balch
.
(1987) showed that chlorate codd also be used as a tool to measure nitrate transport in
marine diatoms such as Skeletonema costatum and Nitzschia clostetium but that two
dinoflagellate species, GonyauIaxpoZye&a and Scrippsiella tmichoidea do not take up
detectable amounts of chlorate (Balch, 1985). In higher plants, D e a n e - b o n d (1982)
reported that cells do not discriminate against chlorate and that chlorate and nitrate are
taken up at the same rate. In contras, Bdch (1987) found that in marine diatoms, nitrate
transport is a fiinction of growth phase and that it can be transported up to 10,000 times
faster than chlorate when the cells are in logarithmic phase. This preference for nitrate
may be due to slight différences in molecular structure Le. chlorate is pyramidal while
nitrate is planar (Zachariasen, 1931).
Chlorate and nitrate act as cornpetitive inhibitors of each other at the transport site
(Balch, 1987) and at the reduction site (Solomonson and Vennesland; 1972, H o f m
1977). The reduction product of chlorate, chlorite, directly oxidizes nitrate reductase and
causes heparable damage (Solornoason and Vennesland, 1972). Chiorite causes more
damage to cells than chlorate because it is a stronger oxidant (Cotnam and Gendron,
1988). In fact, chlorite is ofien used as a bleaching agent in various industrial processes.
12.8 Chlorate toxicity in the presence of ammonium
The presence of ammonium causes the repression of nitrate uptake and reduction
in marine phytoplankton (Dortch and Conway, 1984; Blasco and Conway, 1982;
CressweII and Syrett, 1979; Mac Isaac and Dugdale, 1972). Carpenter and Dunham
(1985) showed that in an estuarine phytoplankton community, ammonium was taken up
preferentiaily except when its concentration is Iess than or quai to 1.6 PM. Another
study by Paasche and Kristiansen (1982) showed that nitrate uptake was inhibited by
ammonium concentrations higher than 1 p M in the Oslofjord.
In generai, if ammonium is present in sigaificant quantities, it is taken up
preferentidly until its concentration decreases to low levels. When this happens and if
nitrate is presenk nitrate reductase activity increases dong with utilkation of nitrate
(Syrett, 1981).
The repression of nitrate reductase activity occurs because ammonium
indirectly reduces the enzyme h m its active form to its inactive form. However, this
reaction is reversible upon depletion of ammonium ( Losada et ai., 1973). Since chlorate
is a niirate analogue, it is reasonable to assume that ammonium will have s i d a r
inhibitory effects on chlorate uptake as it does on nitrate uptake in marine phytoplankton.
Results of a prelimùiary experiment indicated that ammonium inhibited chlorate toxicity
in ThaZasssissimweissjIgii (Couture and Roussel, unpublished results).
Nitrate induces the formation of nitrate reductase. Thus, for nitrate reductase to
be formed and active, cells mus&have been grown previously on nitrate (Blasco and
Conway, 1982). If cells are grown on ammonium, nitrate reductase WU
not be foimed.
1.2.9 Chlorate toxicity studies on algae
In the 40's and 50's, chlorate toxicity studies focused almost exclusively on
higher plants (Aberg, 1947, Crafts, 1935, Goredot, 1947, Perrier-Benoit, 1951, HurdKarrer, 1941) since chlorate was a widely used herbicide and there was a need to
understand its toxicity mechanism. In recent years, interest in chlorate toxicity was
renewed d e r the occurrence of the ecological disaster in the Baltic Sea in the early 80's.
In Sweden, studies that were conducted focused maialy on Fucus vesictliostls,the algae
that was most affected (Lehtinen et al, 1988 and Rosemarin et al., 1985) and on other
marine and fieshwater macroalgae (Rosernaria at al., 1994). They found that brown algae
(Phaeophyta), which includes Fucus vesicuZosus, were sipnincantly af5ected by chlorate
concentrahons as low as 6 pM and that all other groups of dgae were relatively
dected.
Chlorate toxicity studies on phytoplankton are rare. Solomonson and Vermesland
(1972) were the first to investigated chlorate in relation to a phytoplaakfon (ChloreIIla
~ I g a r i s ) .They codkmed that it was chlorite rather than chlorate that caused toxic
effects on that fkhwater species. M e r the Baltic Sea incident, Rosemarin et al. (1994)
conducted an 8 hour study on a natural phytoplankton assemblage and found that a
concentration of 60 pM only produced a slight inhibitory effect on prixnary productivity
They dso report that the assemblage tested was dominated by blue-green algae which
lack the enzyme nitrate reductase. Also foiiowing the Baltic Sea incident, P
d and
Bothwell (1992) examined the potential effects of chlorate on naturai riverine periphyton
characteristic of many western rivers in Canada. In that study they used typical Iow
nitrate concentration (0.2 PM)and chlorate concentrations of up to 6 p.M. Their results
did not show growth inhibition of the algae at the tested chlorate concentrations. Stauber
et al. (1994) examined the toxicity of total pulp and paper effluents on the marine diatom
Nitzschia closterium and found that toxicity was not correlated to chlorate. Van Wijk and
Hutchison (1995) pefiomed a senes of laboratory tests on the marine diatom
Phaeodactylum tricormrturn and their r e d t s showed that this diatom is insensitive to
chlorate. However, it is important to note that the concentration of nitrate was high in the
growth medium (590 PM).
12.10 OveMew of existing methods for chlorate anaiysis
The standard method for chlorate analysis: ion chromatography
Standard Methods for the Examination of Water and Wastewater (1989) does not
describe any method for the analysis of chlorate in water. However, The Annual Book of
ASTM Standards has designateci ion chromatography as the standard method to measure
chlorate in water (ASTM, 1996). This method is used &y
for the detection of
disinfection by-products as the result of the use of chlorine dioxide in drinking water
(PfafTand Brockhoff, 1990, Hautman and Bolyard, 1992).
Spectrophotomeric methods
Spectrophotometric methods for the analysis of chlorate in water were developed
predominantly between 1940 and 1970 for two primary reasons. First, sodium chlorate
was the most popular herbicide in use and there was a need to detennine chlorate
contamination in weii waters. Second, chlorate as an impurity in ammonium perchlorate,
considerably lowers the temperature of deflagration when ammonium perchlorate is used
in explosives and pyrotechnies (Eger, 1955).
The oxidizing properties of chlorate have been utilized in many
spectrophotometric methods. Sneli and Sneli (1949) reported a method using aniline
hydrochloride as a reagent Eger (1955) proposed brucine as a reagent in the
detennination of chlorate impurities in ammonium perchlorate and Maoz-Leyva et al.
(1984) utiiïzed chlorate oxidation of If -cyclohexanedione bisthiosemicarbazone for its
quantification. Prince (1964) developed a method based on the absorbance of femc iron
after chlorate oxidation of ferrous iton at low pH. The reaction of the 1st method was
slow and thus, the method was revised by Chen (1967) to improve the reaction time fiom
4 hours to a few minutes.
Burns (1960) developed a method to measure chlorate based on the oxidation of
bemidine. With this method, chlorate is reduced to chloriw in a strongly acidic medium
which in him oxidizes benzidine to pmduce a yellow color. Urone and Bonde (1960)
developed a version of this method using ortho-tolidine as the color producing reagent.
Mo-tolidine was commonly used as a reagent in the quantitative analysis of residual
cidorine (White,1972). Jordanov and Daiev substituted NB's'-tetramethyl-o-tolidine
for O-tolidinebecause it has a higher nonoal redox potential and is apparently more
selective and sensitive than O-tolidine.
Spectrophotomeûic rnethods are most fkquently based on the oxidation of an
organic reagent to form a colored solution but that is not dways the case. For example,
Trautwein and Guyon (1968) proposed a method based on the interference of chlorate ion
on the formation of the rhenium-a-Mdioxime cornplex. Wang et al. (1991) used the
coior produced by the ~ 1 0 ~ ~ - ~ o ( ~ ~ ) ~ h o s ~ h o acid-Ïm o l i bl,l0
d i cphenanthroline- starch
system. Kou et d.(1988) used the oxidinng properties of chlorate to discolor a DSPCF
( N , N ' - b i s ( 2 - h y d r o x y - 5 - s U l f o p h e n y l ) - C - c y ~solution
Other methods
Various titration methods have been developed to measure chlorate and other
oxychlorine species. Duty and Ward (1994) published the results of a study where a
potentiornetric titration was used to measure chlorate. In this study, sodium nitrite was
used to reduce chlorate to chloride &er which an argentometric titration was performed.
This method obviously cannot be adapted to the analysis of chlorate in seawater because
of the presence of high and variable concentration of chlotide in the medium.
A second type of titration used is the weU known and commonly employed
iodometric titration (Madec et al., 1987). Ikeda et al. (1984) describeci a method where
chlorate oxidizes iodide in a strongly acidic medium to fom iodine. ûxygen interferes
with this method and numerous precauîions must be taken. Aieta et al. (1984) described a
sequential iodometric method to measure ail O
X ~ C ~ ~ species
O M ~ based
on their oxidation
power and the required solution pH to oxidue iodide. Meites and Hofsass (1955) used
the reaction between chlorate and fmous iron in a suIfhic acid soIution and its
polarographic characteristics to quantify chlorate.
There has been some effort to h d merent methods for the detection of chlorate
in water. For example, Tromballa (1970) and Deane-Dnunmond (198 1) devised a
method to produce 36~10pwhich could be quantified with a liquid-scintillation counter.
Pacey et al. (1986) suggested a flow injection analysis method to replace the standard
iodometnc titration. This method eliminiites the air oxidation problem and could
potentiaily provide a means of kinetic enhancement However, the rnethod has not been
fully tested.
Anions are fiequeatly measured with ion chrornatography but recently there have
been an increasing number of studies testing other chromatographie methods iike hi&performance liquid chromatography (HPLC). Retention of ions in reverse phase systems
is made possible by the presence of an ion-pairing agent in the mobile phase (Wu et al.
1988). The authors reportai measuring chlorate at low concentration in fieshwater.
More recentiy, capillary electrophoresis (CE), an alternate for ion chromatography has
been used in the detection of chlorate, but tbis method has not k e n fully tested. In
capillary electrophoresis, the ions are separated based on their mobility in an electrolyte
rather than on their retention time in a stationary phase as it is the case with
chromatography (Jones and Jandik, 1991).
The ortho-toiidine method
Mer carefui review of ail the methods, the ortho-tolidine method was chosen for
this study because of its simplicity and also because the interferences in seawater were
minimal. This method also had the potential to be modifiecl for chlorite analysis and
could easily be automated.
Ortho-tolidine in an acetic acid solution was first suggested in 1909 by Phelps as a
quantitative test to measure chlorine residuals in potable water (White, 1972). This test
did not provide reproducible results because the color development varied fiom sample to
samp1e. The solution would tum either yeilow, green blue or red. Later, E l h and
Hauser (19 13) investigated this phenornenon and found that the color of the final solution
depended on its pH. They suggested using hydrochlonc acid to keep the pH below 1.3 in
orda to dways obtain a deep yellow color. As a result of their work, O-tolidinebecame
the accepted standard for the detection of chlorine residuais and the test appeared for the
f i time in the twelfth edition of Standard Methods in 1965. This method was widely
used because of its simplicity and convenience. However, numemus investigators
examined the O-tolidinereaction and found that consistency and reproducibility of results
were not aiways attainable (White, 1972). This method was subsequentiy removed fkom
Standard Methods (Jolley et d.,1983). Since then, it has been almost abandoned because
O-tolidinehas been found to cause cancer in the ininary tract (White, 1972). Some
authors also cited the lack of quantification and precision as reasons for abandonhg the
method (Gordon, 1988, White, 1972). However, much of the lack of quantification
comes from the procedure itseif. In this methocl, one ml of reagent was added to the
sample and the concentration was determined by eye through cornparison to a
commercial "Ortho-tolidine color comparator" (White, 1972). This is the same system
that is used to measure chlorine in residentid nvimmiog pools.
The method using ortho-tolidine as a reagent for chlorate detection was first
introduced by Urone and Bonde (1960) specificaiiy for the determination of chlorate in
weii waters. The spectrophotometrîcpmcedxe is based upon the color produced after
chlorate ion is converted to chlorine in a strongly acidic medium (6.3 M HCI) and the
subsequent oxidaaon of O-tolidineto a yellow holoquinone.
13 Hypotheses and objectives of tbis thesis
The fïrst objective of this thesis was to develop a simple method for the
measurement of chlorate and chlorite in seawater at environmentally relevant
concentrations. Such a rnethod is ~quiredto attempt any understanding of the
mechanisms involved in the study of chlorate toxicity. The second chapter, c'Anaiyticai
method for the measurement of chlorate and ciilonte in seawater
- batch and
continuous modes" is a description of the method used for the meamernent of chlorate
and chlorite. The method is laqely based on the one described by Urone and Bonde
(1960). Its applicability for kshwater versus artincial and natural seawater are discussed
dong with potentiai interferences fiom various sources. A description is given on the
automation of the method using a Technicon AutoAnalyzerTM,an k t n m e n t commonly
employed in marine chemistry. In addition, modifications have been made to the above
method to allow meastuement of chlorate and chlorite present in the same sample, and a
discussion of problems encountered is also included.
The second objective of this thesis was to identify chlorate sinks in the water
column of the marine environment. This step is of critical importance in the risk
assessrnent process since the persistence of a toxicant in the environment is essentiai in
determining whether a chernical is environmentally hazardous (Maki and Bishop, 1985).
To date, no data have been published on chlorate persistence in the marine water column.
To address this problem, a laboratory experiment was designed to ident* chlorate sinks
under laboratory conditions that attempt to simulate a naturai coastal marine system.
Chapter 3, "Chlorate sinks in the water column in a marine environment - A laboratory
experiment" is a description of this experiment and a presentation of the redts.
A third objective of this thesis was to determine the relative toxicity of chlorate
among various species of marine phytoplankton subjected to the same treatments and to
determine whether a correlation exists between chlorate toxicity and taxonomie class.
The few studies that exist on chlorate toxicity were conducted on one species only
(Solomonson and Vemesland, 1972; van wjk and Hutchison, 1994) or on natural
assemblages (Rosemarin et al., 1994; Perrin and Bothwell, 1992). It is not known
whether marine phytoplankton exhibit intempesies variation as is the case with marine
macroalgae (Romarin et al., 1994). The first section of Chapter 4, "Chlorate toxicity in
marine phytoplankton in relation to species and nitrogen nutrition" presents the results of
an experiment where 8 species of marine phytoplankton were exposed to identical
chlorate treatments.
The fourth objective of this thesis was to compare chlorate toxicity in a single
species of marine phytoplankton when given nitrate or ammonium as a nitrogen source.
The results of the experiment where DunaZielZu tertiolecto, a marine green alga, was
grown on either nitrate or ammonium and exposed to a series of chlorate concentrations
are presented in Chapter 4. The last objective of this thesis is to detennine a critical
chlorate to nitrate ratio at which chlorate becomes toxic. These results are also presented
in Chapter 4 with a discussion of implications for the environment.
Chapter 5 "Summary and conclusions" is a review of the hdings in the thesis and
this is followed by a list of all the references cited.
Chapter 2
Colorimetric method for the measurement of chlorate and
chIofite in fresh and seawater -batch and continuous modes
2.1 Introduction
In the early 198O9s,chlorate (CIO,') caused environmentai damage in the Baltic Sea
where the population of F u w vesicuZo~~~~,
a atidcomponent of the ecosystem,
disappeared in an area of 12 km2in the vicinity of a pulp mill. The concentration of
chlorate in that area of the Baltic Sea, at the tirne, was reported to be approhately 6 PM
(Lehtinen, 1988). Chlorate, a bleaching by-product, entered the environment via the
effluents of a kraft pulp and paper miIi that had modifïed its bleaching process by
substituthg chlorine dioxide for a hction of molecular chlorine.
Other environmental sources of chlorate include industrial processes such as the
bleaching of textiles, wool, flou and other food products (Cosson and Earnst, 1994). It
c m also enter the environment as a by-product of drinking.=ter disinfection, where
chlorine dioxide is increasingly used to replace chlorine as the principal disinfecting
agent (Aieta and Berg, 1986). In agriculture, sodium chlorate is occasionally used as a
herbicide but more recently it has been used primarily as a defoliating agent (Stimmann
and Ferguson, 1990) which can enter the environment as a result of ninoff.
Chlorate is a nitrate analogue, Le. once chlorate is in the marine environment, it c m
be taken up by micro and macroalgae using the same mechanisn as nitrate (Aberg, 1947).
Some higher plant species take up chlorate at the same rate as nitrate (Deane-Dnimmond,
1982) whereas marine diatoms such as Skeleronema costamm and Nitzschio closterium
show a preference for nitrate over chlorate of up to 10,000 times (Balch, 1987). When
chlorate is reduced by nitrate reductase, the resuiting chionte is highly toxic and causes
breakdown of proteins (Hofstra, 1977), starch resenres (Crafts, 1935), and can even
inhibit photosynthesis (Goredot, 1947) in higher plants..
Since chlorate potentialiy poses a threat to the marine environment, quantification
of its concentration becomes imperative. Unfortunately, no analyticai method exists to
measure chlorate and chlonte in seawater at environmentally relevant concentrations (O to
i.4
20 m. In fksh water, ion chr~matographyhas been adopted as the standard rnethod for
chlorate analysis (ASTM, 1996) but the instrumentation is costly and is not available to
all d y s t s . Consequently, the objective of this study was to modify an existing
colorimetric method (Urone and Bonde, 1960) that could be used for the detection of
chlorate and chlorite in seawater at low concentration. The method was also modified for
use in continuous mode using a Technicon AutoAnalyzerm.
2.2 Experimental
Reagents
Oriho-tolidine: In a 1 Litre volumetric f k k , 0.4 g o-tobdine dihydrochloride (Fisher
Scientinc) was dissolved in approximately 600 ml MilliporeR"Super Q water (distilled
water passed through activated charcoal, through an ion exchange resin and through a 0.2
micron filter) to which 250 ml concentrated hydrochioric acid (Fisher Scientific, ACS
grade) was added, fonning a colorless to slightly yellow solution. The solution was
cooled to room temperature, filled to the one litre mark with Super Q (concentration = 1.4
mM), and kept in a dark container at room temperature. This reagent was found to be
stable for several months. When using the continuou mode, 1 ml of surfactant (Brij-35,
Technicon) was added to the solution.
Hvdrochloric acid: The chlorate analysis required concentrateci hydrochloric acid (12 M)
while chlorite analysis required hydrochloric acid diluted to 4.8 M (or 2 5 by volume). In
this study, Fisher ACS grade was use& however, results showed that one must be careful
when choosing a brand and grade of this acid since impurities may cause interfierence.
Interference fkom impurities in HCl will be discussed later in this thesis.
Chlorate and chIoRte standard soiutions
Chlorate stock: 1.2755 g NaC103(Aldrich, 99%) was didissved in 1 litre of double
distilled water using a volumetric flask (concentration = 0.012 M). This solution can be
stored for several months.
Chlorite stock: 1.2606 g NaC102(Fluka, 80% NaC102, 20 % NaCI) was dissolved in 1
litre double distilled water using a volumetric flask (concentration = 0.012 M). This
solution must be kept in the dark and must be made fresh daily.
Chlorate and chlorite working soIutions: 10 ml chlorate or chlorite stock solutions were
transferred to a 1O0 ml volumetric flask and f2.iedto the mark with artificial seawater
prepared according to Keller et al. (1987) (concentration = 1.2 mM).
Chlorate and chlorite standards: chlorate and chlorite standards were prepared by dilution
of the working solution to concentrations of 0.48,2.4,4.8,9.6,14.4
and 19.2 pM.
Equipment
AU colorimetric measurements in batch mode were made using a Mode1 8453
Hewlett Packard diode array spectrophotometer. Colored solutions were introduced in a 1
cm path length glas cuvette and measured at 448 MI and 442 nm, the wavelengths at
which absorbante is maximal for chlorate and chlorite respectively. At the start of every
experiment, a blank measurement, which consisted of amficial seamter and both
reagents, was made. Blanks were also measured perïodically during the course of an
experiment to eliminate emr caused by instrument drift
For measurements in continuous mode, the experiments were carried out using a
Technicon AutoAoalyzer II system consisting of an automatic sampler, a proportioning
pump, a manifold, a flow-through colorimeter and a data acquisition system. The
sampler has a 40-U1 cam (40king the samphg rate per hour and 2/1 the ratio between
samphg time and rinsing time between two samples). The siainless steel probe was
replaced by a Kel-f probe (polymer tube obtained from Pulse Instnimentation, Saskatoon)
to avoid potential interference fkom iron. The analyticd circuit for the deterrnination of
chlorate is shown in Figure 1. A debubbler was added in the fhst section of the circuit to
remove interfering bubbles that are introduced to the system as the probe travels fiom the
wash to the sample and vice versa The colorimeter has a 50 mm path length flow ceil
and interference filters with a band width of 20 nm (between 440 nm and 460 nm). Two
data acquisition systems were used simultaneously to prevent loss of data in case of a
F Iow rate
(mI/min)
Debubbler
1'
To waste
CO1orimeter
0.32
To waste
1 1
0.8
To waste
Figure 1 Analfical manifold for the automated system
(bladblak)
(red-red, soIvafkx) HCI
failure of one of the systerns. A chart recorder in the peak mode was used (OnmiScribe
Recorder) dong with a data collection and analysis package (Pro-
wrïtten by Peter
Strain, BIO) designed for continuous data acquisition. The program dso calculated
concentrations based on a Miclmelis-Menten f i t of standards and check standards,
Instrument drift was corrected by the program.
Al1 glassware was washed by soaking in a SparkleenB solution for at least one
&y, rinsed with double distilled water and then immersed in a 10% HCl bath for at least
one day. Ail pipettes were calibrated periodically.
Procedure for chiorate measurement in batcb
A 4 ml sample, 1 ml of O-tolidinereagent and 5 ml concentrated hydrochloric acid
are added in that order to a 25 ml Erlenmeyer flask. The solution is gently mixed and left
to react for 5 minutes. A yellow color develops, and then absorbance is meamred at 448
nm. The solution is stable for a maximum of 30 minutes. If chlorate concentration is too
high, the sample is diluted before reaction with O-tolidine. The samples are compared to
a standard curve. The reactions involved are:
Procedure for chlorite measurement in batch
The procedure for chlorite analysis is ahost identical to chlorate analysis except
that hydrochioric acid is diluted to 4.8 M. In a 25 ml Erlenmeyer flask, a 4 ml sample, 1
ml ortho-tolidine solution and 5 ml hydrochlonc acid solution are added in that order,
stirred and lefi to react for 5 minutes and then meanired at 442 m, the wavelength at
which absorbance is maximal. The reactions invoJved are:
Statistics
Standard cuve fitting was performed using linear regression and error on slopes
was obtained with standard emr cdculstions using Excel5.0.
2.3 Results and discussion
The main goal of this study was to fïnd a method for the detection of chlorate and
chlorite in natuml seawater. In order to achieve that goal, experiments were initidy
performed using artincial seawater to detemiine if seawater salts interfered. Since they
did not interfixe, more tests were performed to identify other inorganic interferences that
may possibly corne fiom reagents or h m mitinal seawater. Finally, the method was
tested in naturai seawater and interference fiom natural organic matter was studied.
23.1 Chlorate oniy analysis
Concentration Range
Urone and Bonde (1960) reported that chlorate could be measured reliably fiom
0.1 p M to 120 @i4 in £îeshwater. To determine if the same was tnie for seawater,
chlorate standards ranging fiom 0.24 pM to 120 pM were made up in amficial seawater
and their absorbance was m
d
. The results are shown in Figure 2. Figure 2a shows
absorbances for chlorate between 0.24 and 95 pM. Above 95 pM, absorbances were too
high and the resuits obtained are not reliable. Absorbante as a fiinction of concentration
was fitted to a 3rd order polynornial equation with an R~= 0.995.
For environmental measurements of chlorate, the concentration range for chlorate
standards was chosen to be between 0.5 and 20 pM based on the 6 pM chlorate
concentration found in the Baltic Sea (Lehtinen, 1988). At this concentration range,
linearity is observed and the Beer-Lambert law is ciosely foliowed in amficial seawater
except for some deviation at the origin i.e. the linear fit has a negative intercept (Figure
2b). The detection limit, caicuiated as 3 times the standard deviation of the blank was
0.03 pM for both &cial
and natural seawater in batch mode and for the continuous
mode the limit of detection was 0.02 p M for artincial seawater and 0.3 pM for naturd
seawater. The cwature observed in figure 2a and the negative intercept in Figure 2b
0.0
20.0
40.0
60.0
80.0
100.0
16.0
20.0
Chlorate concentration (MM)
0.0
4.0
8.0
120
Chiorate concentration tuM)
Figure 2 a) Absorbance as a h c t i o n of concentration for chlorate ranging fiom 0.24 to
95 PM.(A= 0.852 c3 + 1.24 c2+ 1.52 c = O, R~= 0.995)
b) Absorbance as a hction of concentration for chlorate ranging fiom 0.24 to
20pM. (A = 0.0133~- 0.000509, R~is 0.999)
are possibly due to trace reductants in one of the reagents (ürone and Bonde, 1960). This
reagent was later determined to be the hydrochloric acid (discussed below).
Interferences
The effect of hvdrochioric acid brand and grade
Concentrated hydrochlonc acid usually contains 35% w/w hydrochloric acid in
water plus a number of organic and inorganic impurities. The concentration of these
impurities depends on the grade of the acid. As a general d e , a lower concentration of
impurities is found in higher grades of acid and vice versa However, in the trace metal
grade, for example, metal impurities are at extremely low concentration but other
impurities are not monitored, resulting in potentialiy higher concentrations of those
impurities than those found in the lowest grade.
Various grades and brand names of hydrochioric acid were tested for use with this
method. Figure 3 shows chlorate standard c w e s using 6 dinerent grades and brands of
acid. Al1 curves were obtained using identicai chlorate standards in Super Q water and Otolidine solution. The r e d t s show that the highest grade of acid tested (BDH Aristar)
resuited in a higher dope (0.071) than the Fisher ACS grade used in this study (0.013).
Both show good linearity and both have a negative intercept With Fisher trace metai
grade and Seastar acid, chlorate could not be detected below 5 PM.The two tested lots of
BDH ACS grade acid showed a different response. Chlorate could not be detected below
12 pM with one lot while the other lot had a dope tbat was very close to the Fisher ACS
grade but with a high positive intercept value (0.14 ). The most likely cause is by an
impurity present in relatively large concentration, such as chlorine, that c a w d strong
background absorbace.
For colorimetric analyses, the standard cuve should ideally follow Beer-
Lambert's law (Le. it should be linear and pass through the origin). These resuits show
deviation from Beer-Lambert's law with all the acids tested, because of either a negative
or positive intexcept or, in some cases because of non-linear behavior. However, for the
range of chlorate concentraîions (O to 20 pM) studied, the Fisher ACS grade was found to
be suitable for chlorate analysis because of the linear behavior and also because the linear
best fit passes close to the ongin. It must be noted, however, that Fisher ACS grade may
not always give the best results. From the results of the tests on two different lots of
BDH ACS grade. impwities within a given brand and grade of acid are not always in the
same concentration and may Vary nom lot to lot. Consequently, acids should be tested
prior to analyses and the same brand, grade and lot should be used throughout a set of
experiments.
The positive or negative intercepts observed in Figure 3 are likely c a w d by
impurities such as sulfite and chlorine in the acid. A negative intercept means that some
of the chlorate has been consumed by a reducing agent iike sulfite. When the intercept is
positive, as is the case with the curve obtained using the older lot of BDH acid, an
oxidant such as chlorine has interfered (Burns, 1959). Both impinities cannot exia at the
same time because they are incompatible and thus only one of these two can be present in
0.00
4.00
8.00
12.00
.
16.00
20.00
Concentration (PM)
Figure 3 Chlorate standard curves generated using various grades and brands of acid
solution (Burns, 1959). DBerences in dope values observed using two Merent acids
means that an impurïty consumed chIorine fiom reaction (1).
The reducing agents in Fisher ACS grade pose a problem at the lowest end of the
concentration range where the interfefence h m sulfite becornes more significant The
cornpetitive interaction between sulfite and chIorate causes a concave curvature of
absorbance values at low concentrations (see figure 2b) which cannot be accounted for
with a linear best fit. Consequently, at low concentrations, a iinear best fit overestimates
chlorate. Forcing the linear fit through the ongin does not improve chlorate predictions at
low concentrations and causes an underestimation of chlorate concentration. To acfiieve
better accuracy at low chlorate concentration, it is essential when using lower grades of
HCl, to choose a set of working standards in the desired concentration range in order to
more adequately mode1 the respome of the suindard c w e .
Nitrite
Nitrite interferes with the o-tolidine method. Nitrite causes an increase in total
absorbance due to the formation of a yellow diau> derivative of O-tolidine( reaction 5). If
reaction 5 occurs, the contribution to total absorbance of 1 '
j
M
nitrite is equai to the
absorbance produced by 0.5 pM chlorate (Couture, unpubfished results). Nitrite can dso
cause an underestimation of chlorate concentration due to the loss of chlorate by direct
reduction (reaction 6) (Urone and Bonde, 1960). The reactiom involved are the
following:
The reported range for nitrite concentration in CO-
areas is 0.01 to 0.4 pM
(Peter Strain, perscomm), consequently, it is not likely to be a concem in the
measurement of chlorate in the environment.
Sulfite
Sulfite interferes with the O-tolidinemethod Sulfite spontaneously reduces
chlorate, chlorine and all other o x y c h l o ~ especies to chloride following reactions 7 and
8 (Burns, 1960):
3 soi2+ c10;
+ Cl- + 3 ~ 0 4 "
s03Q+ Cl, + H20 + 2c1- + HSOi + H*
(7)
(8)
Suifite is not reported to be nahirally occurrllig in seawater however, it causes
interference since it is one of the major impurities in hydrochloric acid. Although
concentrations are relatively s m d (less than 12.5 p M in Fisher and BDH ACS Grade), it
is sufficient to cause a noticeable effect.
Ferrous and femc iron
In weli oxygemted waters of neutral or slightly alkahe pH, ferrous iron is
unstable and is easily oxidized to its more stable form, femc iron (Meitess and Hofsas,
1959). If ferrous iron is present in a sample, it intdefes by reducing chlorate to chloride
following the reaction:
If this reaction takes place in parailel with reactions 1 and 2, a second peak
appears in the spectrum at 360 mn, the wavelength at which femc iron absorbs. Femc
iron does not interfere with reactions (1) and (2) but ifpresent in signincant quantity, it
increases absorbance at 448 nm where the yeIIow hoIoquinone formed fiom O-tolidineis
meanired. Urone and Bonde (1960) reported that 1
ferric iron has the same
absorbance as 0.08 ph4 chlorate. Typical concentration values of Kon in the ocean range
fiom 0.15 to 2.5 n M (Bruiand, 1983) therefore environmental iron is not expected to be a
concem. Iron fiom reagents is more likely to interfere since the concentration can be as
high as 3.5 FM in ACS grade hydrochloric acid, however, the interference is mal1 and
constant,
and poses no serious problem.
Iodide and bromide
Denis and Masschelein (1983) used the O-tolidinemethod to measure chlorate in
drinking water and investigated iodide and bromide for potential interference. They
fond that both interfere significantly in concentrations as low as 1 pM, with iodide
causing slightly more interference than bromide. They fomd that for both species,
absorbance decreases proportiody to iodide or bromide concentrations ranging nom O
to 65 PM. Beyond 65 FM the interference remains constant, The reactions involved are:
C103-+61-+6H'+ 3 1 ~ + C 1 - + 3~~0
(10)
CIO; + 6 B i + 6 ~ +
+ Br2 + Cr + 3 H20
(1 1)
In weil oxygenated waters, iodide concentration is approximately 0.2 p M and
increases to 0.5 p M in anoxic water due to the reduction of iodate to iodide (Bruiand,
1983). In reagents, no iodide concentration is reported. Thus, the interference from
iodide should be minimal. Bromide, on the other han& is a major constituent of seawater
and has a concentration of 0.84 m M at a salinity of 35%0. Its concentration in HCI is
reported to be as hi& as 0.63 m M in ACS grade. Bromide interferes but its interference
is constant since bromide is a consemative ion (concentration always constant relative to
salinity) in seawater. Therefore, the method described here can be usexi in the presence of
a hi@ concentration of bromide.
Artificid versus natuml seawater: The effect of marine orpanics
Artificial seawater dif5ers nom naturai seawater because the latter comprises a
wider range of inorganic compounds in trace amounts and a broad variety of marine
organic compounds that are not included in Keller's (1987) amficial seawater recipe. In
nahiral seawater, the concentration of total dissolved organic carbon @OC) is 1-5 mg CI1
and is related to primary productivity which is a h c t i o n of nutrient input,
hydrodynamics and other factors (Slauenwhite, 1991). Characterized organic compounds
such as carbohydrates, metallo-organic compounds, aldehydes, ketones, sterols, vitamins,
amines and halogenated compounds only make up about 10 % of DOC (Wiiliams, 1971).
The remaining 90% of DOC consists of complex organic compounds of unkuown
chemical character that have k e n biologicaüy produced and transfonned (Slauenwhite,
1991). They are referred to as humic substances. Humic substances may potentidy
interfere with this method because they can be oxidized by molecdar chlorine.
Freshwater humic substances are known to be the major precursors of trihalomethanes
when drinking water is treated with chlorine (Stevens et al., 1976).
Hurnic material is found in naturai waters, patïcularly in coastal regions and it is
believed to play an important role in chemical and microbiological professes in seawater.
There have k e n a growing number of studies on the subject and a number of methods for
extraction have been examine& Hydrophobie solid-phase e-ction
P
using C-18 Sep
W d d g e s (Waters, Inc.) was chosen for this study because of its simplicity, and its
higher efficiency (between 22 and 84%) for DOM removal relative to other methods like
XAD and ultmfltration (Amador et al., 1990). C-18 SepPak TM only removes up to 80%
of humic substances. The 80% is the hydrophobie fiaction of humic material and the
other 20% consists of the hydrophïlic cornponent that m o t be removed with SepPaks
(Amador et al., 1990).
Experiments were conducted to determine if DOC causes interference with the
analytical method for chlorate. Standards ranghg nom 0.5 to 20 pM chlorate were made
up in filtered naîural seawater fkom various sources in the Nova Scotia area (e.g. North
West Arm, Saody Cove, Bedford Basin and Scotian dope). Standards were also
measured in double distilled water, in artincial seawater, in filtered waste water fiom a
continuous culture of ThaZasssissirapseudo~naand in North West k m water passed
through three C-18 SepPaks
connected in series. The waste water f?om the
continuous T.pseudo~naculture consisted of filtaed K medium (Keller et ai., 1987)
with nutrients in V 2 concentration (Gdard, 1972). For the C-18 Sep-PaksTb<
experiment, with the aid of a vacuum pump, 500 ml of North West Arm water that was
previously acidified to pH 2.6 with 0.3 M HCI was passeci at a rate of 8 ml per minute
through two C- 18 Sep-Paks
comected in series. The Sep-PaksTM were preconditioned
following the procedure described by Mills at al. (1987). Bnefly,this procedure
consisted of passing 10 ml double distilled water through the Sep-PaksN, followed by 10
ml of a 0.3 mM HCl solution, 10 ml of methano1 and W
y 10 ml double distilled water.
The results of this experiment (table 1) indicate that slopes obtained in a l l natual
seawater samples tested are reduced by approximately 10-15% compared to the slopes for
artincial seawater. Slopes for artificial seawater and Super Q water are comparable, and
theretore the difference in ionic composition between natural and artificial seawater was
rejected as a potentid explanation for the dope difference. Waste water h m the
continuous culture was also tested to determine if algd exudstes can interfere with the
method, but the slope obtained for this experiment was simüar to the one obtained in
artificid seawater. M e r DOM was removed with the SepPakçm, the dope increased by
6% relative to the same water prior to this treatment. This resulk coupled with the resuit
of the continuous culture waste, indicates that humic material causes interference.
However, the investigation was not thorough enough to conclude that humic material is
the only interference in natural seawater. In order to assure that the C-18 SepPaks TM did
not cause the observed dope difference, control treatments were performed by comparing
1 Water source
Slope
super Q
Artificid seawater
Continuous culture water waste
0.0135t0.0001
0-0135t 0.0001
0.0 133 t 0.0002
North West Ami
Sandy Cove
Bedford Basin
Scotian Slope
0.0 1 19 t 0-0001
0.0118 10.0001
0.0120 f 0.0001
0.01 16 t 0.0002
North West Ami
0.0128t 0.0002
(passed through C 18 SepPaksy
(~18)
(n=17)
(FI 7)
(FI 8)
(FI 8)
(~18)
(n=iû)
(a=t8)
Table 1 Slopes for identical sets of chlorate standards measured
in water fiom various sources.
the slope of a set of standards made with amficial seawater passed through C-18 Sep
Paks TM, with the dope of the same set of standards made in artificial seawater that was
not passed through the Sep-Paksm. The resuits were identical.
Since DOM concentration in naturd seawater may Vary spatially and temporaily,
the interference may equaily Vary, causing emrs in chlorate determinations. For accurate
results, standard additions should be use& which consist of adding a set of known
chlorate concentrations to the sample and measuring absorbance d e r each addition. The
original concentration can then be deterrnined with this standard cuwe.
23.2 Simaltaneous chlorate and chlorite analysis simple: The sabtraction method.
The method descrïbed above is no? specific to chlorate, therefore a stronger
oxidant like chlonte interferes by causing an increase in the formation of the colored
product. In order to measure chlorate and chlonte separately, the contribution of chionte
to the total absorbance must be subtracted fiom chlorate reactïons. The method described
below is based on the ciifference in redox potential of both species in acidic solutions.
Chlorate and chlorite in separate solutions were m
d and plotted as a
function of HCI concentration (figure 4). This figure shows that color development with
chlorite is maximal between 2.1 and 2.7 M HCI, after which it decreases as HCl
concentration increases to 6.3 M. When chlorate is being mea~u~ed,
there is no color
production up to approximately 3 M HCl after which the absorbance cuve rises sharply
to its maximum value at 6.3M HCI. Maximal color development with chlorite occurs at
an HCl concentration range at which color does not develop with chlorate. To subtract
Figure 4 Absorbante at 448 nm as a h c t i o n of HCI conceneation for 24 pM chlorate
and 24 pM chlorite
chionte contribution fiom total absorbance at 6.3 M HCl, the sample is measured at lower
acid concentration (between 2.1 and 2.7 M FICI), detecting chlorite only. The absorbance
of chlorite at the lower concentration range of HCI is then translated to an absorbance
value at 6.3 M HCI using Figure 5 and subtracted fiom the total absorbance of both
chlorate and chlorite. For the rernainder of this discussion, the maximal working HCI
concentration for chlorite analysis was set at 2.7 M.
Steps followed to subtract chiorite from total absorbance at 6 3 M HCL
1. -Three standard cuwes were generated: one for chlorate (6.3 M HCI) and two
for chlorite (one at 2.7 M HCl and one at 6.3 M HCI)and a best fit on al1 the
curves was cdculated.
2. -Absorbance of the sample containhg both chlorite and chlorate was measured
twice, once with 2.7 M HCl at 442 nm,and once with 6.3 M HCl at 448 nm. At
this point, chiorite concentration was determined simply by using the chlorite
standard curve made with 2.7 M HCI.
3. -The corresponding absorbance at 448 nm due to chlorite using 6.3 M HCl was
determined using the relationship:
G.7= C63
And accordingly,
Finally
was detennined as the only unknown in equation (14) where,
CL7= chlorite concentration predicted by chiorite 2.7 M HCl best fit
Cs3 = chlorite concentration predicted by chlorite 6.3 M HCI best fit
x,
= absorbance meanued
with 2.7 M HCI
b = intercept of chiorite 2.7 M HCI best fit
q3= corresponding absorbance predicted by 6.3 M HCI best fit
e = intercept of chiorite 6 3 M HCI best fit
(this step is illustrated in Figure 5 for more clarity)
4. - The corresponding chlorite absorbance at 6.3M HCl ( x ~ ~obtained
),
in the previous
step was subtracted fkom the total sample absorbance reading at 6.3M HCI (this
step permits the subtraction of the interfence caused by chlorite fkom the chlorate
reading).
5. - The chlorate standard c w e was then used to detennine the chlorate concentration
fkom the chlorate absorbance value obtahed in step 4.
1
2.7 M HCI
'
Figure 5 Graphic representation ofthe mbtraction method
In nimmary, this chapter desmis a simple colorimetric method to masure
seawater samples contaking, chlorate, chlorite or both. The method can be used either in
batch or in continuou mode using a Technicon AutoAnalyzerfM.Interferences include
nitrite, sulfite, femc and ferrous iron, iodide and bromide. Humic substances in coastal
seawater also interfite, causing a decrease in the standard cuve dope of about 10-15%.
Since DOM,varies spatiaiiy and tempordy, standard additions should be used when
analyzing environmental samples with this method. The subtraction method for
simultaneous detection of b t h chlorate and chiorite is based on the ciifference in redox
potential of both species in acidic solution where chlorite interference is subtracted fiom
fiom the total absorbante of the samp1e.
Chapter 3
Chlorate sinks in the water column of a marine environment A laboratow ex~eriment.
3.1 Introduction
Knowledge of transport, distribution, transformation and fate of a toxicant is
essential when assessing its risk to the environment. In order to detennine if a chemical
poses a threat to an aquatic environment it is necessary to understand the chernicd
(hydroiysis, oxidation and photolysis), physical (molecular structure, solubility, volatility,
etc...) and biological (biotransfonnation) factors that can &ect environmental
concentrations (Rand and Petrocelfi, 1985).
Several chemical, physical and biological p r o p d e s are known for chlorate. For
example, chlorate is a strong oxidant and is therefore not expected to persist in the
presence of material that can easily be oxidized. Chlorate is highly soluble in water (95.7
g/100 mi at 2 0 ' ~ )(Robbins et al., 1942 ) and will therefore be unifody distributed in
the water column and be readiiy available to aquatic organisms (Randand Petrocelli,
1985). Chlorate is converted to toxic chlorite by biological processes because its
molecular structure resembles nitrate.
Studies to evaluate the persisteme of chlorate in the water column in a marine
environment are rare. Axegard et al. (1986) simulateci Swedish receiving water
conditions (13'~) and used natural sediments in an experiment to detennine chlorate
persistence. They found tbat about two months are requued for 235-470 p.MCIO< to be
degraded.
The present laboratory study was designed to identfi chemicd a d o r biological
sinks for chlorate in the marine water column. The potential sinks tested in this snidy
included water, seawater, dissolved marine orgaoic materid, bacteria, a mtural
assemblage of phytoplankton, a laboratory culture of Phaeodactyium îricornuizim and
simulated sunlight The experiment was progressive in that each potential sink was
successively added until natural seawater was reconstituted. Such an experimental design
permits the determination of which components of naturai seawater act as a si& for
chlorate. Treatments where water sarnples were irradiated with simulated sunlight were
also included to assess the role of photodegradation as a potential sink for chlorate in the
environment.
3.2 Materiais and methods
Pnor to the experimenS ail glassware was washed and soaked in a 2% Mirom
soap solution for at least a &y, then rinsed with Nano Purem water (Bamstead system
that passes distilled water through advated charcoai, a macroreticular min, ion
exchange resins, 0.2 micron filter and an ultrafilter), tramferreci to a 10% HCl bath for at
least a &y, and rinsed again with Nano mirem water.
Preparation of the various media for the progressive treatmeats
Treatment 1: This treatment consisted of Nano Purem water oniy.
Treatment 2: This treatment consisted of artificial seawater (Kmedium, Kelier et al.,
1987) containing nutrients, vitam& and trace metals in f72 concentrations (Guillard,
1972).
For treatments 3 to 6, d a c e water was collected fkom Bedford Basin, Nova
Scotia, Canada on November 14,1996 and was immediately prefiltered ushg a 2 0 p
fiiter to remove large size moplankton that could gaze upon phytoplankton.
~ to
Treatrnent 3: Prefiltered Bedford Basin water was nItered through a 0 . 2 filter
remove phytoplankton and bacteria, presumably leaving only dissolved organic and
inorganic material.
Treatment 4: Prefiltered Bedford Basin water was fiitered through a 2pm filter to remove
most phytoplankton and presumably leave bacteria, dissolved organic matter and
potentially smail phytoplankton in the filtrate.
Treatment 5: Prefiltered (20 pm) Bedford Basin water which included dissolved organic
matter, bacteria and the full size range of the naturai phytoplankton assemblage. It should
be noted that this natural assemblage is likely not entirely natural. Some species of
phytoplankton are sensitive and do not survive the sampling process andor the filtration
process or some do not grow in a "semi-continuous"culture environment.
Treatment 6: It consisted of prefiltered (20 pm) Bedford Basin water to which a
Iaboratory grown inocculum of Phaeoductylum 0-icornutumwas added. This treatment
was included to insure that at least one of the treatments would contain phytoplankton in
case the naturai assemblage died a s a result of laboratory conditions.
Procedure
Two litres of each medium desctibed above was tranfe~dto 4-litreErlenmeyer
flasks after which chlorate was added to a concentration of 18 p M (see table 2 for
summaq of treatment 1 to 6).
AU treatments were perfomied in duplicate. The flash
were capped with a cotton plug covered with foi1 to mhimke contamination and placed
in a room where the temperature was kept constant at SOC. The room was subjected to a
1ight:dark cycle of 14:1O where light was provided by cool white fluorescent tubes
yielding an irradiance of 6 1.2 p o l photons m-2 s-1 . Fluorescence was measured in
treatment 4, where Bedford Basin water was nItered through a 2 pM filter, as a check on
dgal content in the filtrate. Their absence was codkned again when nutrient
concentrations were measured and s h o w to remain constant throughout the experiment
The natural assemblage of phytoplankton (treatment 5 ) and Pheaodactylm ~ i c o m r ~ r n
(treatment 6) were grown for two weeks. Mer two weeks, P. tricornutum numericdy
dominated the natural assemblage in treatment 6 flasks. Both treatment flasks were
replenished with phosphate and silicate in fï4 concentration. Nitrate was aliowed to
decrease to low levels (<5 pM) before chlorate was added.
Treatment
#
4
Initiai chlorate
concentration
(Fm
Water type
Potential sink
18
double distilled
water
Nano Purem water
18
artificial seawater
seawater d t s
18
natuml seawater
dissolved organic
matter
18
naturaj seawater
mainly bacteria
18
na&
18
naturai seawater
seawater
naturai assemblage
of phytoplankton
Phaeodrrciylum
ZricornurUnr
Table 2 Description of the progressive treatments to determine chlorate sinks
Every two days for 32 days, samples varyhg f h m 20 to 40 ml were taken h m
each flask usuig an automatic pipette with acid cleaned plastic tips. Sampling took place
in a W stedized transfer hood to minimize contamination. Approximateiy 10 mi of the
samples containing phytoplankton were meanired for in vivo fluorescence using a Tumer
Designs fluorometer after which the photosynthetic inhibitor DCMU was added, to verie
the relative photosynthetic efficiency of the cultures. Another subsample of
approximately 20 mi of treatments 5 and 6 was filtered using Whatman GFfC nIters. The
filtrate of these two samples and the samples fkom treatments 1 to 4 were placed in acid
cleaned plastic tubes and were immediately fiozen for later analyses of chlorate and
nitrateVitamins, trace metals and nutrients except nitrate were replenished weelcly in
treatments containing phytoplankton to avoid depletion. Nitrate concentration was kept
under 8 ph4 to simulate natural concentrations and to minimize inhibition of chlorate
uptake. Nitrate was added every 3-4 days to a total concentration of approxhately 8 W.
Nitrate and chlorate were SLnalyzed in triplicate on a Technicon AutoAnalyzer IITH
following the Technicon AutoAnalyzer II Industrial Method No. 158-71W for nitrate and
the method described in chapter 2 for chlorate. Stock solution and standards were made
fkshprior to every set of analysis.
Bacteria from each flask were sampled and stained weekly with acridhe orange
on a glass slide to qualitatively estimate the amount of bacteria present in each flask and
treatments 5 to 6 were measured for pH as an hdicator of CQ depletion.
Sunlight experiments
Treatment 7: One beaker containing 300 ml double distilled water with 18 pM added
chlorate was piaced under a Hereaus Suntest CPS solar simuiator with a 1000 watt xenon
lamp at an intensity of 2.2 sunç for 3 hours. This is approximately equivalent to 7 hours
of midday sunlight measiwd in late January at latitude 34% (Miller and Zepp, 1995).
Two 4-mi samples were taken every 30 minutes and immediately meanired for chlorate
accordhg to the method described in Chapter 2.
Treatment 8: Two M e r s containing 300 ml of Bedford Basin water fltered through a
20 pWhatman GFIC filter to which chlorate was added in a final concentration of 18
p M were placed in the solar simuiator described above for 3 hours. Two 4-ml samples
were taken every 15 minutes and immediately measined for chlorate according to the
method described in Chapter 2. The two treatments described here are summarized in
Table 3.
Statistics
Microsoft Excel5.0 was used to calculate t-values, linear regression parameters
and 95% confidence intervais. These statistical values were used as a tool to determine
whether chlorate decay had occurred over the time course of the experiment.
Treatment
#
Initial chlorate
concentration
Water type
Component added
fkom previous
treatment
double distiUed
water
sunlight
Natural seawater
suniight
[PM)
7
18
Table 3 Irradiated treatments in the experiments to determine chlorate sinks
3.3 Resuits and discussion
Treatments 1 to 4
The results of these experiments show that chlorate concentration does not
decrease over a period of 32 days in double distiiied water, in artificial seawater, in filter
sterilized natural seawater and in natural seawater with bacteria (figure 6). The rate of
change in chlorate concentration over the duration of the experiment was calculated for
each treatment and the results are shown in table 5. For treatments 12 and 4 the rate of
change is O at a 95% confidence level however treatment 3 shows a slight i n m e (ttest) in concentration but this increase becomes insignincant at the 99% level. This slight
increase, is believed to be due to systematic errors that occurred during chlorate analyses.
Samples were measured for nitrate and chlorate approximtely every week, each time
with a new chlorate stock solution. In assessing the variability of data in Figure 6, there
may have been errors introduced in the preparation of chlorate standards especially in the
last b a t h (last 6 points of every graph) where the points seem higher than expected. To
m h i m k this effect, a correction was applied to each point of every graph by
recalcuiating chlorate concentrations using chlorate standards fiom the first analysis day.
These voltage values were copied and pasteci replacing the standards of the subsequent
analyses, the base line was readjusted and the concentrations for the samples were
recalculated using the program. Even after the correction was applied, there was d l a
smd1 positive trend in treatments 3 at the 95 % level. Since errors have presumably been
introduced in the prialyses and dthough t-tests indicate that the increase is significant, it is
difncult to evaiuate the meaning of this slightly positive trend (see table 4 and figure 6).
Treatment
dC/dt
Wday
Standard
error
95%
CI.for
dC/dt
t
n
statisüc
2
ASW
3
Filtered natural
seawater (0.2~)
4
Filtered natural
seawater (2p)
Sa
natural seawater +
naturai assemblage
phytoplankton
5b
naturai seawater +
nahual assemblage
phytoplankton
6a
natural seawater +
P. tricornutum
6b
naturai seawater +
P. tricornutum
Table 4 Rate of chlorate concentration change per day, standard error, 95% confidence
interval, t statistic suid the number of observations used to calculate statistics.
Each observation represents the average of six replicates for each sample per
day.
Therefore, 1 conclude that chlorate concentration remaineci constant throughout the
experiment for treatments 1 to 4. Bacteria were expected to take up chlorate (Goksoyr,
1951) but no decrease in chlorate concentration was observed. However, it must be noted
that the treatment testing bactena as a potentid sink did not receive any nutritionai
supplement Therefore those bacteria couid have been severely h;unpered,which couid
explain the lack of chlorate concentration d-
over the duration of the experiment
Treatments 5 and 6
Treatments 5 and 6 showed a decrease in chlorate concentration. For these
treatments, the rate of decay appears to be related to the concentration of phytoplankton
in the samples. Treatments Sa and 5b (nahual assemblage of phytoplankton) both
showed a decrease in chlorate concentration of 0.04 p.bf per day and their average in vivo
fluorescence (fluororneter oufput) throughout the experiment was 0.070 and 0.039 units
of fluorescence (volts) respectively. Chlorate concentration for treatments 6a and 6b
(Phaeodactyhm tricornutwn) decreased by 0.35 pM and 0.27 pM per &y (table 5) and
the average in vivo fluorescence (fl uorometer output) throughout the experiment was 0.23
and 0.22 units of fluorescence respectively (Figure 7). in other words, an approximately
5-fold increase of fluorescence resulted in an approximately 8 fold increase in chlorate
decay rate.
Although the results seem to indicate that higher phytoplankton concentration
means higher chlorate decay rate, one must be carefùl when drawing conclusions f?om an
experiment such as this one. This experiment was designeci only to determine if
phytoplanldon act as a sink for chlorate in seamter. This experimental design gives
d t s that are quaiitative rather thao quantitative. It would be difficult to determine an
empirical relationship between rate of decay of chlorate concentration and in vivo
fluorescence because of an insufncient number of experimental treatments with
phytoplankton and dso because other variables must be considered (e-g. nitrate and
ammonium concentration, species of phytopianldon in the sample, initial chlorate
concentration, etc). Varying one of those parameters may substantially alter the results.
Consequentiy, the only conciusion that can be drawn for treatments 5 and 6 is that
phytoplankton are biologicai sinks of chlorate under the experimental conditions
imposed.
Treaûnents 7 and 8
Simulated sunlight did not cause the photodegradation of chlorate in distilled
water as shown by the results of Figure 8a In naturai seawater, W light may potentially
cataiyze a reaction between organic and/or inorganic chernical compounds and chlorate.
However, the results showed no degradation of chlorate over the duration of the
experiment. However Figure 8b shows a slight hcrease of chlorate concentration in the
£îrst 70 minutes of the experiment. A possible explimation for that phenornenon is the
organic interference is eliminated as the sample is irradiated. As for the s m d decrease
fiom 70 to 200 minutes, a conclusion can not be drawn since more tests wodd be
required to determine if it is signincant From these d t s , photodegradation does not
appear to be an important sink for chlorate in the marine environment.
In summary, uptake by phytoplankton was the oniy sink for chlorate and
the rate of decay seemed to be related to the concentration of phytoplankton. Two
biological parameters were tested (eg. bacteria and phytoplankton) but decrease in
chlorate concentration was obsemed only in the sample containing phytoplankton.
Dining the month long experimenf phytoplankton caused a demase in chlorate of up to
50 O
h of the onginal concentration. Other potentid sinks tested involved double distiUed
water, &cial
seawater and dissoived organic matter. According to the results, none of
these other potential sinks seemed to affect chlorate concentration over a period of one
month. However, in the water coiumn, dissolved organic matter concentration is low
relative to organic matter in sediments. Therefore, to fdiy assas the roie of organic
matter as a potentid sink, a treatment with organic rich sediments shouid be included in
any fùture determination of the fate of chlorate in the marine environment.
Photodegradation of chlorate by shulated sunlight was also tested and the resdts did not
show any signincant loss of chlorate over the duration of the experiment.
Figure 6 a) Chlorate concentration as a h c t i o n of t h e in double distiued water
b) Chlorate concentration as a fiinction of tirne in artificial seawater (potential
sink = salts)
c) Chlorate concentration a s a h c t i o n of tune in nahiral seawater filtered
filter@otentid
l
sink = dissolved organic matter)
through a 0.2 @
d) Chlorate concentration as a function of time in naturai seawater fïitered
through a 2 p filter botentiai sink = bacteria)
Chlorate concentration measured in treatments 3 and 4 is lower than the initial
concentration of 18 p M because of the interference caused by DOM found in
natural seawater (See chapter 2 for M e r detail).
*
Ni-
Figure 7 a) Chlorate and nitrate concentration and in vivo fluorescence of a naturai
assemblage of phytoplankton as a fûnction of tirne for treatment Sa
(CClo3-= -0.043% + 14.7)
b) Chlorate and nitrate concentration and in vivo fluorescence of a naturaI
assemblage of phytoplankton as a fùnction of time for treatment Sb
(Cc,,- = -0.043 1t + 14.9)
c) Chlorate and nitrate concentration and in vivo fluorescence of a culture of
Phaeodactyium tricomurum as a function of time for treatment 6a
(Cclo3. = -0.353t + 15.5)
d) Chlorate and nitrate concentration and in vivo fluorescence of a culture of
Phaeodactylum hicomuturn as a fùnction of time for treatment 6b
(Cclo3-= -0.268t + 15.1)
O
40
80
120
160
200
Tirne (min)
O
40
80
120
160
200
Time (minutes)
Figure 8 a) Chlorate concentration in double distilled water as a fiuiction of fime when
Super Q water is irradiated with simulated sunlight
b) Chlorate concentration in natural seawater as a fûnction of time when natural
seawater is irradiated with simulated ninlight
Chapter 4
Chlorate toxicitv in marine ph
s~eciesand nitrogen nutrition
4.1 Introduction
Sodium chlorate was successfiilly used as a herbicide in the 1930's to 1960's and
studies of its toxicity have focused primarily on terrestriai plants. It has only ken in
ment years that chlorate toxicity in the marine environment raised interest in the
scientinc community. In the mid 803, chlorate began to be considered an environmental
hazard when chlorate caused the disappearance of the macro dgae Fucus vesicuZosus, a
species believed to be the base of the ecosystem of the Baltic Sea (Lehtinea, 1988).
Chlorate had entered the marine environment via the effluents of a pulp mil1 foliowing a
change in the bleaching process where chlorine dioxide was substituted for molecular
chlorine in order to reduce the formation of organochlorines.
The mechanism of chlorate toxicity in plants is not well understood. Sîudies on
higher plants indicated that it is chlonte rather than chlorate that causes toxic effects
(Aberg, 1947). When chlorate is transported into celis, it is transformed to chionte via
nitrate reductase (Aberg and Liljestrom, 1966) which in hirn oxidizes the ce11 nom
within. Chlorate's molecular structure resembles nitrate and thus chlorate has been used
as a nitrate analogue for the study of nitrate uptake in marine phytoplankton (Balch,
1987). Chlorate is not necessarïly taken up at the same rate as nitnite when both are
present. While Deane-Drummond (1982) reporteci that ceus of barley plants do not
discriminate against chlorate, Balch (1987) report4 a preference for nitrate of up to
10,000 times above chlorate in Skeletonema costaha and Nitzschia closterium depending
on growth phase.
The objectives of this chapter are 1) to examine relative chlorate toxicity in eight
species of phytoplankton nom various taxonomie classes and determine whether there is
a conelation between toxicity and class of marine phytoplankton, 2) To test the n d
hypothesis that the use of ammonium as a nitrogen source will eliminate toxicity due to
chlorate and 3) to determine the chlorate to nitrate ratio at which chlorate toxicity can be
obsewed in the green dga Dunaliella tertioiecta
4.2 Materials and methods
Relative toxicitv in various s m i e s
Stock cultures of Thaic~ssiosira
pseudonana, ïïtul~~~siosira
weissjlgii, DunalieIZu
tertiolectu, Tetraselmis sp., Emilionia huxleyi, Phaeodcctyium hicornuîum, Chaetoceros
gracilis and Udonteila mobiliensis were grown in 500 ml botties containhg K medium
(Keller et al., 1987) with tir nuûients (Guillard, 1972)except that nitrate was 100pM.
Cuitures were transferred twice to aiiow acclimation to their new medium.
Fresh medium, identical to the one described above, was sterilized using a microwave oven, was capped with a Cotton plug to ensure steriiity and was then left at room
temperature for two days to d o w CO2re-equilibration. Chlorate solutions of O, 10, 100,
1000 and 10,000 p M were prepared with the sterilized medium taking the necessary
precautions to avoid conM o n . A total of 120 IO-ml tubes were soaked in a 2%
MicroTMsoap solution for at least 24 hours, transferred to a 10% HCl bath for 24 hours
and then micro-wave sterilized. Medium containing the various chlorate concentrations
was transferred to the 10 ml tubes and exponentiaily p w i n g ceUs h m each species
were inoculated in triplicate into the five chlorate treatments. Parafilmm was placed over
each tube to prevent contamination and spillage. Although Pam£ïhTM
prevented CO2
from readily difhising back into solution, necessuy nutrient concentration calculations
were performed to ensure that nitrate was the limiting growth f&r
and not carbon. The
tubes were placed in a 20 O C culture room subjected to a Iightdark cycle of 1633 where
irradiance was provided by a set of cool white tubes yielding a photosynthetidy
avaiiable quantum scaiar irradiance of 54 v o l photons m-2 s-1 .
In vivo fluorescence was measured each &y at 16h using a Tumer Design Model
10 fluororneter. Before each sample was placed into the instrument it was gently inverteci
to provide homogeneity. In vivo fluorescence provides an easy means of measuring
growth of a phytoplankton population. However, one must be carefbi when estimahg
biornass fkom fluorescence data. Although fluorescence is a good indicator of living
organism (Hdlegraeff, 1976, 1977), its magnitude per unit organic matter may vary
widely according to species composition, celi age, nutrient availability, Light intensity and
temperature (Strickland, 1960). Fluorescence and biomass are well correlated in the
exponential growth phase, but not in the stationary phase. After nitrogen has been
exhausted, chlorophyll a content rapidly decreases and celis lose buoyancy and sink.
Nitrogen depleted ceils continue to fix carbon, therefore increasing biomass (Hdlegraeff,
1977). Consequently, since fluorescence and biomass are not dways weii correlateci,
fluorometry data shouid only be regardeci qualitatively.
Since in vivo fluorescence is proportional to biomass during the exponential
growth phase, the m e a m d fluorescence F(t) at time t is:
F(t) = F(ta)e Mt-ta)
(20)
where F(h) is the fluorescence at time to of the first measurement considered Asslmllng
k=û and taking the logarithm, equation (20) yields:
WW)= Pt + Ln(R0))
(21)
which provides an esfimate of the growth rate (p)by hear regression of the logarithm of
the measured fluorescence and t h e . For each species and each treatment, the points to
use for the calculation of growth rate were determined with the control curve (O chlorate)
since any deviation wouid presumably be the result of chlorate toxicity.
T-tests were performed for each treatrnent of each species in order to determine
the concentration at which chlorate had deletenous effects on growth. The nul1
hypothesis was that the growth rate for a treatment was identical to that of the control.
The signifieance of the ciifference is measured by the ratio of the difference in growth rate
between the control and the treatment to their weighted standard deviation,
where t = t value,
= specifk growth rate of the control,
= specific growth rate of a
control treatment, sd = weighted standard deviation.
Since the standard deviation of both compared treatments is not identical, the
weighted standard deviation is,
where, s2 = variance and n = number of observations (Neville and Kennedy, 1964)
AU growth rate &dculations and aU the associated statistics were perfomed using
Excel version 5-0.
Nitrate and ammonium in relation to chlorate toxicitv
Stock cultures of Dunnliella tertiolecta grown in modifed K medium (Keller et
al., 1987) and f72 (Guiliard, 1972)nutrients were transferred to two glass 500 ml bottles.
h
tbottle contained K medium with rnodined DT nutrients (nitrate was 100 pM) and
The f
the second bottie contained K medium with modined h/2 nutrîents (Guillard, 1972)
(ammonium was 100 pM)- Nitrate was measured in the ammonium culture pnor to the
beginning of the experiment and no detectable concentration was found. DunaiieZZa
tertiolecta was inoculated into 180 IO-mi tubes. The treatments are summarized in Table
5 for clarity.
The samples were placed in a 20°C culture room subjected to a 1ight:dark cycle of
16:8 where irradiance was provided by a set of cool white tubes yielding a
photosynthetically avaiiable quantum scalar irradiance of 54 pmol photons rn-2 s-1 .
Chiorate
Concentration
Nitmgen
Form
# Replicates
OiM)
O
10
Nitrate
1O0
(100 PW
1O00
10,000
O
10
Ammonium
1O0
(100 PM)
1O00
10,000
18
I
able 5 Summary of the treatments in the expriment
testing nitrate and ammonium in relation
to chlorate toxicity.
Since the experiment o d y lasted 5 days, each sample was m e a d for in vivo
fluorescence twice daily using a Turner Design Mode1 10 fluormeter to gather enough
data for statistical d y s i s . With every sampling, one replicate of each treatment was
sacrificed by fiitering it through GFIC filters and storing it in a fkezer for later analyses
of nitrate, ammonium and chlorate. Once a day, a thVd sample was sacrificed and used to
measure the DCMU ratio. DCMU is a photosynthetic inhibitor that blocks the electron
flow in photosystem II reaction centers resulting in an increase in relative fluorescence.
Hence, the DCMU ratio, calcdated as (FI,-F)/FDwhere F is in vivo fiuorescence and FD
is in vivo fluorescence after the addition of DCMU, is a physiological index of the
relative photosynthetic efficiency of a culture (Vincent et al. 1980).
Nitrate, ammonium and chlorate were measured in duplicates on a Technicon
AutoAnaiyzer P.The Technicon AutoAnalyzer II Industrial Method No. 158-71 W was
used for nitrate and ammonium was measured with a method stU under development
(Strain and Clement, 1996) that uses a chemistry intermediate between the one specified
by Grasshoff and Johannsen (1972) and the one specified by Mostert (1988). Chlorate
was meanired with the automated method described in chapter 2.
Growth rate was calcdated using equation 2 1 and the fluorescence values
rneasured twice M y . T-tests were perfonned similar to the previous section.
Relative toxicitv in various m i e s
Among the species tested over the course of this experimenf Emilianiu h l e y i
(figure 9d) and Dunaliella terriolecta (figure 9a) were the least affected by chlorate
during the growth phase. At chlorate concentrations of up to 1000 pM, both species grew
at rates that were not significantly dflerent (t = 0.36.0.69, 1.87 for the 10, 1O0 and
1000pM chlorate treatments respectvely at a4.05) fiom their respective control.Both
species, however, did not grow when exposed to 10,000 pM chlorate (table 7). In the
case of EmiZiania hurleyi, the growth rate vaiue for both the control and the 1000 pM
chlorate treatment obtained h m triplicate sarnples had high standard em>r values and
low R2which could Iead one to beïieve that these growth rate values are not significant.
In order to ver@ their validity, a growth rate value was calcdated for each replicate
separately. The resuits for the contml showed growth rate values that were a l l very close
to the value that was calcdated using the three replicates, therefore improving R2.The
high standard error was most likely caused by the lack of unifonnity in the inocdu. of
each replicate at the beginning of the experiment. In the case of the 1000 pM chlorate
treatment, the results did not improve with a similar analysis, indicating that the high
standard error value represents scatter in the data possibly caused by chlorate.
The four species, PhaeodactyZum îricormtum (figure 9b), Thalassiosira
weissjrogii (figure 9e), Chaetocerosgrucilis (figure 9f) and Terruselmis sp. (figure 9g),
displayed similar response when exposed to chlorate. At 10 and 100 p M chlorate, these
species grew at rates that were not signincantiy different fiom their respective control (t =
~)Q.sa
*
0.40
'E
-
-
A
-
03a-
5
0.20-
H
ri
j
-1
1
aio -j
1
Figure 9 Growth curves when a) DunaZieIla tertiolecta b) PhaeodactyZum hicornutum c)
riiclassiosirapseudonuna d ) Emiliania h d e y i e) ThaZassiosira weissjogii f )
Chaetocerosgracilis g) Tetrdrnis sp. h) Odonfellrrrnobiliensis are exposed to
0,10,100,1 O00 and 10,000 pM chlorate. Each point represents the average of
a triplicate. Error bars were omitted for clarity but they are reflected in the
standard emr of growth rates in table 7.(e,f g,h are shown on the next page)
Figure 9 (continued fiom previous page)
-
SPECIES
- .-- .-
Growtb Rate
Points ased
(Ci-')
(day)
Standard
emr
1.21
1.27
1.1 1
(n=4)
1 t04
1 to4
Ito4
1 t04
0.04
0.06
0.04
0.06
121
1.14
1.15
0.84
(n=3)
O to 2
O to 2
O to 2
O to 2
0.0 1
0.03
0.04
0.04
1.O5
(~4)
Oto 3
1.23
-
-
lhzksiosira
pseudonana
Emiliania
hdeyi
control
10
100
1O00
10,000
control
IO
100
1000
10,000
1-07
0.99
0.79
-
0.6 1
0.68
0.58
0.43
-
-
-
-
-
Oto 3
oto 3
Oto 3
0.02
0.05
0.0 1
0.05
(n=4)
3 t06
3t06
3 to 6
3 to 6
0.14
0.03
0.03
O. 12
-
-
-
-
@=4)
1.10
1.10
1-09
0.9 1
0.46
Thahsiosira
we&flogü
Chaetoceros
gracilis
Tetraselmissp.
Odonteüa
mobiliemis
control
10
100
1O00
10,000
024
control
10
1O0
1O00
10,000
0.88
0.83
0.89
0.8 1
0.42
control
10
100
1O00
10,000
0.59
0.42
124
1-18
1.13
1-07
-
-
O to 3
O to 3
Ot03
Ot03
O to 3
(n=3)
O to 2
O to 2
O to 2
O to 2
O to 2
@=7)
O to 6
O to 6
O to 6
O to 6
O to 6
(n=8)
O to 7
O to 7
0.02
0.02
-
-
-
0.04
0.04
0.04
0.06
0.1 1
0.04
0.04
0.06
0.04
0.07
0.03
0.03
0.03
0.03
0.05
-
Table 6 Growth rate, points used
standard
consideration, p value and R2for each species tested and each treatment .
1.19, 1.5 5 and 1.42,O.O 1 for C.gracilis and Tehaselmis sp. respectively at a4.05)
whereas, at 1O00 and 10,000 CiM, the growth rates were significantly decreased (tr 3.15,
12.9 and 2.03 and 8.57 for C. gracilis and Tetrmelmis sp. respectivevely at a4.05). The
unexpected decrease in fluorescence values of P. Iricomtlmr during the stationary phase
for the control and the 10 pM treatment has been attributed to ceiîs sticking to the tubes.
ThaZassiosiraweissjlogii showed a relatively high growth rate at 10,000 ph4
(approxllnately 50% of the control). The peculiar response of Chaetocerosgrocils at
100 ph4 is most iikely related to the condition of the culture in which the phytoplankton
aggregated in flakes for unknown fessons. The redts are show-in table 6.
~ Z ~ ~ ~ s i o s i r u p s a t d o n(figure
m r a 9c) showed a decrease in growth rate when
cultures were exposed to 1O0 and 1O00 pM chlorate (t = 2.26 and 4.95 respectively at a=
0.05) however the cultures did not grow at ail when exposed to 10,000 p M (table 6).
Odontella mobiliensis (figure 9h) cultures were evidently the most afhted by chlorate
exposure. In fact, at a chlorate concentration of 10 pM, the growth rate is reduced by
nearly 30 % in relation to the control. This species did not grow at al1 when exposeci to
1O0 p M chlorate and beyond (table 6).
Nitrate and ammonium in relation to chlorate toxicitv
Ammonium:
Al1 cultures of DunaZielIa tertiolecta grown with ammonium as a nitrogen source
grew at growth rates that were not significantly different from the control (t = 0.2, 1.5,
1.7,0.9 for the 10, 100, 1000, 10,000 pM chlorate treatments respectively at a=0.05)
Figure 10 Fluorescence, ammonium concentration and chlorate concentration
as a h c t i o n of time for a) control b) 10 p M chlorate treatment c)
1O0 pM chlorate treatrnent d) 1 O00 p M chlorate treatment and e)
1 0,000 chlorate treatment for the species DunaliefIutertiolecta
(c,d,e are shown on the next page). As previously error bars have
been ornitted for clarity but are reflected in the standard error of the
growth rate calcdation s h o w in Table 7.
Figure 10 (continued fiom previous page)
Nitrogen
fonn
Nitrate
Ammonium
Chlorate
Conc. (pM)
Growth Rate
(d-9
Standard
Emr
Controi (O)
0.85
0.0 1
10
0.83
0-01
100
0.83
0.0 1
1O00
0.84
0.01
10,000
0.17
0.02
Control (O)
0.93
0.02
10
0.93
0.0 1
100
0.97
0.01
1O00
0.98
0.02
10,000
0.96
0.02
Table 7 Growth rate, standard enor, p value and R2for each chlorate treatment when
Dunaliella tertiolecta use nitrate or ammonium as a nitrogen source.
regardless of chlorate concentration, even in the presence of 10,000pM chlorate. The
results are presented in figure 10 and in table 7.
Nitrate:
DumIieZIa tertiolecta grown with nitrate as a nitrogen source responded to
chlorate treatments identicaily as in the previous expriment i.e. no decrease in growth
rate at concentrations up to 1000 p M and no growth at 10,000 pM. For the remainder of
the section that refers to nitrate as a nitrogen source, dl treatments will be compared to
the control treatment as it is exempt fiom any chlorate and ceils presumably grow at their
optimum considering the conditions to which they were subjected.
Growth phase
Growth rate: The growth phase in this expriment was about 98 hours. It was
determined by the linear section of the growth curve for the control trament when the y
aKis is logarithmic. The growth rates were calculated as the slope of the best fit equation
using al1 data points gathered during the growth phase. The results for each treatment
have been tabulated in table 7 dong with their respective standard erron, R~and p values.
The growth rates for chlorate treatments of 10, 100, 1000 pM when cells are grown on
nitrate, were not statistically different frorn the control and it is only at 10,000 pM that
growth rate is significantly decreased. In fact, at this concentration, there is aimost no
growth. To illustrate, each treatment is shown graphically (figure 11 a-e) where in vivo
fluorescence, chlorate concentration and nitrate concentration have been plotted as a
fiinction of t h e . Chiorate concentration as a bction of t h e has been omitted in the
10,000 pM chlorate treatment graph because the change in chlorate concentration over
time is most Iikely insignincant in cornparison to the error introduced when the sample
was diluted to the O to 20pM range which is the working range for the chlorate d y t i c a l
method described in chapter 2.
Chlorate to nitrate toxicitv ratio: Assuming that the number of cells in the inocdimi was
identical in the replicates of each treatment and assuming no t&c effects, the increase in
fluorescence should be the same for each sample. However, if toxic effects are obsewed,
this increase in fluorescence should be d e r . In order to detennine the chlorate to
nitrate ratio at which chlorate toxicity can be observed, t-tests were performed to
detennine for each light period of treatments 10, 100 and 1O00 PM, whether the increase
in fluorescence was statisticdy ciifFerat h m the control. The Light penods rnentioned
are the time interval between 12 and 26 hours, 38 and 50 hours, 62 and 74 hours and 86
and 98 hours in figure 11 a-e. During the f
h
tthree time intervals, changes in in vivo
fluorescence were statisticaily identical as the controls at the three chlorate concentrations
considered. It is only during the fourth time interval (86 to 98 hours) that deviation 60m
the control is observed. While treatments with 10 and 100 pM chlorate show the same
increase as the control, the treatment with 1000 p M shows an inctease that is statisticdy
lower than the control (see figure 1Id). The treatment with 10,000 p M chlorate was not
considered in this analysis because D.tertiolecta obviously displayed toxic effects at that
concentration.
O
M
a
m
m 1
rie (hcin)
0 0 1 2 0 1 s D
Figure 11 Fluorescence, nitrate concentration and chlorate concentration as a h c t i o n of
t h e for a) control b) 10 p M chlorate treatment c) 100 pM chlorate treatment
d) 1000 pM chlorate treatment and e) 10,000 pM chlorate treatment for the
species DunalieZia tertiolecta. (c,d,e are shown on the next page). The shaded
bar in d) represents the time intemai during which the zone of critical chlorate
to nitrate ratio is reached As before error bars have been omitted for clarity
but they are reflected in the standard error of the growth rate calculation
shown in Table8.
am-
.
h
,
'=1
!--*:-a-.
",
'
b-*-
[
t
0.16 7
1
Pl*
4
am 7
0.04
Stationmy phare
In batch cultures, when ceUs exhaust nutrients h m their gmwth medium they
stop multiplying but they do not die, remaining in stationary phare. When a toxicant iike
chlorate is added, the stationary phase may not be maintaineci. Since chlorate is a nitrate
d o g u e , nitrogen starved ceus may take up and metaboiize chlorate at a faster rate,
resulting in ceU de&. The results show that while stationary phase ceUs (98 to 140
holus) displayed the same behavior in the control as in the treatments with 10 and 100
pM chlorate, the 1000 pM chlorate treatment showed a fluo-ence
decrease with tirne
indicating that the ceiIs did not remah in stationary phase but died instead. This is
clearly iiiustrated in figures 11 ad.
4.4 Discussion
Relative toxicitv in various mcies:
The focus of this experiment was to examine the relative tolerance to chlorate for
various species of phytoplankton. The species tested were chosen to represent cornmon
classes of phytoplaakion including Chlorophyceae (D.tertiolecta), Prasinophyceae
(Tetraseimis sp.), Prymnesiophyceae (E. htaleyi) and Bacillariophyceae (T.weissjogii,
T.pseudonana, C.gracilis, P. îricomutum and 0. mobiZiemis) and identify a correlation
between class and toxicity if one exists. Although a clear correlation codd not be
identifie4 the results for this experiment indicate that the two most sensitive species to
chlorate exposure were diatoms (Bacikiophyceae).
The response of most species tested is comparable: there was no significant
deviation fkom the control during the growth phase, when exposed to 10 and 100 plid
chlorate, a slight inhibition of growth with 1000 pM and definite inhibition of growth
when celis were exposed to 10,000 phi chlorate. The only exception was O h t e l l a
mobiliensis, which responded drasticdy to chlorate exposure with an ECsO(growth rate)
that is approximately two orders of magnitude Iowa than aiI other species testeci. In their
review, van Wijk and Hutchison (1995) concluded that since the toxïc actions of chlorate
are presumably caused by direct oxidation of the celi by a metabolite, it is uniikely that
second generation effécts wiU be observeci in affecteci celis. This was confirmed by
Notini et al. (1991) when damageci algae completely recovered d e r the source of
chlorate was removed. Consequently, one can speculate that the apparent higher
tolerance to chlorate of fast growing phytoplankton is partly due to the low regeneration
t h e , which cm have a dilution effect and at the same time c o d t u t e a built in protection
factor. OdonteIIa mobiliensis grew at the slowest rate of all the tested species and was
also the lest tolerant to chlorate exposure, which could indicate that growth rate plays a
role in chlorate toxicity. However, EmiIimia huxleyi grew at a rate comparable to O.
mobilienris but seemed to have a much greater tolerance to chlorate. If only diatoms are
considered, the relationship rnay be m e : faster growing diatoms are apparently more
resistant to chlorate than slowly growing 0.mobiliensis. As for other species of
phytoplankton tested in this experiment, one must conclude that there are other factors
involved Le. E. htaleyi may not metabolize chlorate and maybe nitrate with the same
mechanism as the otber species.
Nitrate and ammonium in relation to chlorate toxicitv:
Ammonium:
The object of this experiment was to test ammonium as an agent to circumvent
chlorate toxicity in phytoplakton Chlorate has k e n used a s a nitrate analogue to midy
nitrate transport in marine phytoplankton (Balch, 1985, 1987) as weii as in higher plants
(Deane-Dnimmond, 1982; Prieto and Feniandez, 1993). Since chlorate is believed to be
taken up like nitrate, it was hypothesized that phytoplankton would also take up
ammonium preferentidy, sparing the celis from the deleterious eff-
of chlorate. The
results of this experiment have clearly shown that chlorate did not cause hamiful effects
to the ceils at any concentration. It is interesting to compare the d t s of the 10,000 phf
chlorate treatment when celis are grown on ammonium versus when they are grown on
nitrate. While the cells grown on ammonium grew at a statistically s i d a r rate as the
control, the nitrate medium cells did not grow at ail. Thus, the hypothesis that
ammonium would spare the celis h m chlorate toxicity was venfied for DunaIiella
tertiolecfa.
The hypothesis also impiied that celis would not take up detectable arnounts of
chlorate in the presence of ammonium, however this was not found to be the case.
Figures 1Ob and 10c show that chlorate concentration decreases over the duration of the
experiment. In the 10 p M treatment, chlorate decreases to approximately 6.5 p M while
decreasing to approximately 90 pM in the 100 p M treatment (figure 11b and 1 lc). For
the 1000 p M treatment, the decrease, ifthere is one, is too small relative to the overd
concentration therefore an assessrnent of a chlorate concentration variation over time is
not possible (figure 116) for that treatment. We know h m chapter 3 of this thesis that
the only sink for chlorate under these experimental conditions is uptake h m
phytoplankton. Consequently one m u t assume that chlorate is transported up by
phytoplankton even in the presence of ammonium and stored without metabolking i&
possibly in a vacuole. However it must be noted that these decreases in chlorate
concentration seem to be associated with Iow residual ammonium concentrations and that
the decrease of chlorate may simply rdect resumeci uptake, as it is expected with nitrate
when ammonium is exhausted h m the medium (Dortch, 1991).
Nitrate:
In their review, van Wijk and Hutchison (1 995) speculated that the m o n some
species are not sensitive to chlorate is that they are capable of reducing chlorate dïrectiy
to chloride (CC) without the production of the intemediates chiorite ( ~ 1 4 ' )and
hypochlonte (CIO'). The main hypothesis tested in this chapter is that chlorate toxicity in
phytoplankton is a function of the chlorate to nitrate ratio. As long as the chlorate to
value, no toxic effects are observed. When the
nitrate ratio does not reach a critical
d
critical ratio is reached, chlorate becomes toxic. The design of this expriment was very
simple, therefore the results only dowed an approximation of this ratio.
The results indicate that during the growth phase, the 10pM and 100pM treatment
never reach that critical ratio since the fluorescence increase for each light period is
similar to the control. It is only in the 1000 and 10,000 pM chlorate treatment that the
ratio is attained (figure 1Od-e). During the fourth light period (86 to 98 hours) of the
1000 pM treatment, the increase in fluorescence is signincantly d e r than the control
(t = 3-45, a q . 0 5 ) and h m 98 to 140 hours, fluorescence decreases, indicating dennite
toxicity. In the 10,000 pM treatment, there is inhibiteci growth for the first 38 hours after
which growth completely ceases. At the end of the experiment, nitrate is completely
exhawted fiom the medium in the O to 1000 p M chlorate treatments while uptake of
nitrate completely ceases after 50 hoins, leaving 65 pM in the lO,OOO pM chlorate
treatment The critical toxicity ratio is presumab1y reached between 86 and 98 hours
which means that the ratio is between 40 and 850 if solely based on the 1000 pM
treatment results. If we consider the 10,000 pM treatment, we know that the critical ratio
cannot exceed 10,000/65=155 because when this ratio is attained, celis stop rnultiplying
completely. Therefore, the critical chlorate to nitrate ratio is most likely situated between
40 and 155 for the species Dunaliella tertiolecta.
Environmental ini~iications:
Typical concentrations of chlorate in raw effluents have been reported to be
approximately 1200 p M (Frostell et al., 1994). Assuming raw effluents containing 1ZOO
FMchlorate are released in the marine environment, chlorate may not be toxic to most
phytoplankton species even at the pipe outlet since pulp mills also release large
concentrations of nitrate in their effluents. During the summer of 1994, nitrate levels
were reportedly 30 p M in the Letang inlet, New Brunswick in the vicinity of a pulp mill
( S a and Clement, 1996). Consequently, the chlorate to nitrate ratio wouid probably
not reach the critical value for many phytoplankton species. As distance fkom the outlet
increases, chlorate concentration decreases depending on a number of factors like mixing
and water circulation in the marine environment in question. Nitrate concentration may or
rnay not demase depending on the time of year, nitrate input from the atmosphere, f b m
rivers and h m land drainage. In a more reaiistic scenario, raw effluents are treated
biologically. Although secondary treatment of most North American mills was not
designed for chlorate reduction, it has been reporteci that chlorate concentration is
l o w d after treatment especially if there are anaerobic zones in the system (van Ginkel
et al., 1995).
Since every marine inlet is different biologically, c h e m i d y and
hydrographidy, it is e c u l t to assess the real environmental impact of pulp r d
chlorate. In order to determine whether chiorate poses a threat to the environment would
need to establish a field monitoring program to gather data on nitrate concentration,
chlorate concentration, water circulation, mixing throughout the year as these parameters
vary temporally.
The resuits of the experiments described in this chapter showed that under
Iaboratory conditions, most phytoplankton species tested were relatively insensitive to
chlorate. However, one species was very sensitive to chlorate, which shows that this
pllutant has the potential to cause damage to the environment.
Chapter 5
Summaw and conclusions
The idea for this thesis topic came nom a series of articles pubfished in which
kraft d chlorate had devastating effects on the marine environment Serious problems
related to chlorate toxicity have been observed ahost exclusively in the Baltic Sea.
Therefore, a need exists to undestand if the same disastrous effects could occur in a
typical North American marine environment where nutrients are d
y available in
higher concentrations than in the nitrate depleted Baltic Sea W e a number of studies
on chlorate tolcicity have been conducteci on higher plants, very few have examined the
effects on phytoplankton Although a change in phytoplankton species composition and
abundance may not be as visible as a change in macro algae such as Fucus vesicuIo.sus in
the Baltic Sea ,it is nonetheless as important since phytoplankton are at the base of the
food chah. This thesis examined for the fïrst time chlorate toxicity variations among
various species of marine phytoplankton, chlorate toxicity in relation to nitrogen nutrition
and the fate of chlorate in the water column. A simple autornated analyticd method was
developed in order to allow sensitive, and inexpensive analyses of chiorate and chlorite in
seawater.
An extensive literature review on chiorate is presented in Chapter 1. Chapter 2
describes a simple, inexpensive and sensitive method for environmental and laboratory
measurement of chlorate using the oxidation of ortho-tolidine to a yellow holoquinone in
a strongly acidic medium. This method can either be used in batch or in continuous
mode. The batch mode only requires a spearophotometer and the continuous mode
requires a Technicon AutoAnalyzerRd,which is an instrument commoniy employed in
marine chemistry. When chlorate is measured in coastal seawater, humic material
e approximately 10-15% in absorbance readings compared
interferes, causing a d e c ~ a s of
to those seen for equivdent chlorate concentrations in amficial seawater. If humic
material concentration varies significantly over the sampling ma, it is recornmended to
use standard additions for better accuracy. Inorgatxic interferences include iodide,
bromide, nitrite, sulfite and iron, substances which are ofien present as impurities in
hydrochloric acid. This chlorate anaiytical method has also been modifiecl to separate
and quanti@ chlorite i n t e r e n c e during chlorate analysis when both are present in the
same sample by decreasing hydrochloric acid concentration in the final solution
Chapter 3 presents the results of experiments where the objective was the
detennination of sinks and therefore the fate of chlorate in the marine water column- The
potential sinks included distilled water, artincial seawater, dissolved organic matter,
bacteria, a natural assemblage of phytoplankton, a culture of Phaeodac~yZumi r i c o r n u ~ ,
and photodegradation. The results indicated that the only sink for chlorate in the water
column is plants. Chlorate has typically been believed to be short lived in the
environment because of its strong oxidizing properties. The results did not confirm decay
of chlorate in the presence of dissolved organic matter. However, in the water column,
dissolved organic matter concentration is low relative to organic matter in sediments.
Therefore, to M y assess the role of organic matter, a treaûnent with organic rich
sediments shouid be included in any fuhne assessrnent of the fate of chlorate in the
environment.
Chapter 4 is an investigation of chlorate toxicity in marine phytoplankton The
experiments in that chapter were designed to compare chIorate toxicity in 8 species of
marine phytoplankton. Most species were relatively uniiffected by chlorate
concentrations of less than 1000 p M but Odonteilu mobiliensis showed extreme
sensitivity to concentrations as low as 10 @M. Since chlorate toxicity apperns to be
related to nitrogen source and concentration, a second expriment was designed to
compare chlorate toxicity when ammonium is the p h a r y source of nitmgen rather than
nitrate. No toxic effects were observed when ammonium was present in the medium.
When nitrate is the source of nitrogen, toxicity appears to be related to the chlorate to
nitrate ratio in the medium and for the fïrst t h e , a critical chlorate to nitrate ratio (when
chlorate becomes toxic) of 40 to 155 was inferred for the marine green aiga Dunafiella
tertidecta
Throughout this investigation, one point has become increasingly apparent.
Chlorate is not iikely to pose a threat to most phytoplankton species at environmental
chlorate and nitrate concentrations and particularly if ammonium is present- Especially
since 1996, ail Canadian pulp mills are required to perform secondary treatment of the
effluentsbefore they can be discharged in the environment. Secondary treatment is not
designed for chlorate removai but there have been reports that it can remove up to 50% of
the chlorate (Stauber et al, 1995).
Even ifthe d t s of the experiments presented in this ihesis indicate Iow
sensitivity to chlorate in most species of phytoplankt~n,one should not dismiss the fact
that one species responded d r a s t i d y to chlorate treatment. Consequently, chlorate can
pose a threat to a number of phytoplankton species. One of these sensitive species may
potentidly be a criticai component of the ecosystem as it was the case in the Baltic Sea.
The work of this thesis should therefore be regarded as baseline work in the study
of chlorate which is still at its preliminary stages. The next step shutdd be to gather more
infornation on potentiai chlorate discharges in the environment and design a field
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