and long-term effects of the antibacterial agent triclosan on photosynthesis of marine periphyton

25 Pages·2013·1.49 MB·English

Short- and long-term effects of
the antibacterial agent triclosan
on photosynthesis of marine
periphyton communities
Viktor Fihlman
Degree project for Master of Science 30 hec
Department of Biological and Environmental Sciences
University of Gothenburg 2013
Abstract

Antibacterial agents are today used in a wide variety of products. One of the chemicals most widely
used for antibacterial purposes is triclosan. In this study, short-term and long-term effects of
triclosan to marine periphyton communities were evaluated using Pulse Amplitude Modulation
(PAM) fluorometry and High Pressure Liquid Chromatography (HPLC). In the static short-term
exposure experiments, PAM was used to investigate effects on photosynthesis after 1.25 hours and
2.5 hours of exposure respectively. In the long-term exposure experiment, periphyton communities
were continuously exposed to triclosan in a flow-through test system and effects were detected
using both PAM fluorescence and HPLC in order to investigate long-term effects on photosynthesis
and pigment composition.

Results showed that triclosan had a significant short-term negative effect on periphyton at the
highest tested concentration only (10000 nM) with a calculated EC50-value of 2390 nM. The opposite
result was seen in the long-term exposed samples where a small, statistically significant stimulation
of photosynthetic efficiency could be seen at the two highest tested concentrations (316 and 1000
nM). Also, when comparing long-term exposed samples and controls a stimulation of chlorophyll a
could be detected with HPLC at exposure levels of 100 – 1000 nM. The effects on chlorophyll a were
measured using both PAM and HPLC. PAM was shown to be less sensitive in estimating effects on
chlorophyll a content in the communities studied here.

Using HPLC analysis of pigment composition, an estimation of effects on community structure after
long-term exposures, was made. Results show that a shift in pigment composition occurs at relative
low concentrations of triclosan (31.6 nM). The effect observed here indicates a clear and sudden shift
in community structure at 31.6 nM, with no further shifts at tenfold higher exposure levels. These
results indicate that triclosan might alter community structure of algae and cyanobacteria in
periphyton, eliminating sensitive species and promoting resistant species.
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Svensk sammanfattning
Antibakteriella medel används idag i en mängd produkter. Ett av de mest använda antibakteriella
medlen är triclosan. I den här studien har korttids- och långtidseffekter av triklosan på
perifytonsamhällen undersökts med hjälp av metoderna Pulse Amplitude Modulation (PAM) och
High Pressure Liquid Chromatography (HPLC). Perifyton är en varierande och komplex
sammansättning av bakterier, cyanobakterier, mikroalger samt en- och flercelliga mindre djur som i
akvatiska miljöer växer som ett tunt lager (biofilm) på exponerade ytor. I denna studie användes
runda glasplattor som substrat för perifyton-biofilmen. I korttids-tester exponerades perifyton för
triklosan under 1,25 och 2,5 timmar varefter effekten mättes med PAM. I långtids-experimentet
användes ett testsystem bestående av 24 st. akvarier med kontinuerligt in- och utflöde av havsvatten
och triklosan. Organismer i havsvattnet fick under experimentets gång kolonisera glasplattorna som
fanns i akvarierna. Perifyton-samhällena/biofilmen undersöktes sedan med PAM-fluorometri och
HPLC för att uppskatta triklosans effekter på fotosyntes, klorofyll a-innehåll och
pigmentsammansättning. Resultaten från korttids-testet visade att triklosan har en signifikant
negativ effekt på fotosyntesen hos perifyton, men bara vid den högsta testade koncentrationen
(10000 nM). Det lägsta EC50-värdet för korttids-testet beräknades till 2390 nM. Den motsatta
effekten observerades för långtids-exponerade perifytonsamhällen där en liten, statistisk signifikant,
stimulerande effekt på fotosyntes kunde observeras i de två högsta testade koncentrationerna (316
och 1000 nM).
Vid jämförelse av klorofyll a-innehåll i långtids-studien syntes samma stimulerande effekt av
triklosan. Klorofyll a-innehåll mättes med hjälp av både PAM och HPLC men effekten syntes endast
på de mätningar som utförts med HPLC. PAM och HPLC mäter klorofyll a-koncentration på olika sätt, i
HPLC extraheras och isoleras pigmenten ifrån perifyton varefter dessa mäts enligt en kromatografisk
metod. PAM använder istället fluorescensen från intakta perifytonsamhällen vilket gör att
fluorescensen från olika organismer kan skuggas av varandra och därmed ge en felaktig bild av den
totala fluorescensen i provet.
Med hjälp av en HPLC analys av pigmentsammansättningen kunde strukturella förändringar i
sammansättningen av perifyton som exponerats under lång tid uppskattas. Resultaten visar att en
förändring i pigmentsammansättning sker vid relativt låga koncentrationer av triklosan (31.6nM).
Denna förändring verkar ske inom ett relativt snävt koncentrationsintervall för att sedan inte
förvärras nämnvärt även då triklosankoncentrationen ökas till det tiodubbla. Resultaten från detta
experiment visar att triklosan kan ha potentiella effekter på artstrukturen i perifyton efter en längre
tids exponering då känsliga arter försvinner och ersätts av mer toleranta arter.
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Contents

Abstract ................................................................................................................................................... 1
Svensk sammanfattning .......................................................................................................................... 2
Contents .................................................................................................................................................. 3
Introduction ............................................................................................................................................. 4
Periphyton communities ..................................................................................................................... 5
Pulse Amplitude Modulation ............................................................................................................... 6
High Pressure Liquid Chromatography ................................................................................................ 7
Materials and Methods ........................................................................................................................... 8
Toxicant solutions ................................................................................................................................ 8
Periphyton colonization and sampling ................................................................................................ 9
Short-term toxicity experiment ........................................................................................................... 9
Long-term microcosm experiment ...................................................................................................... 9
PAM measurements .......................................................................................................................... 10
High pressure liquid chromatography (HPLC) ................................................................................... 10
Calculations and statistics ................................................................................................................. 10
Results ................................................................................................................................................... 12
Short-term effects on photosynthesis............................................................................................... 12
Long-term effects on photosynthesis ............................................................................................... 13
Long-term effects on chlorophyll content......................................................................................... 15
Pigment composition in periphyton after long-term exposure to triclosan ..................................... 16
Discussion .............................................................................................................................................. 17
Effects on photosynthesis ................................................................................................................. 17
Effects on biomass ............................................................................................................................. 19
Effects on community structure and physiological status ................................................................ 19
Acknowledgements ............................................................................................................................... 21
References ............................................................................................................................................. 22

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Introduction
Antibacterial agents are today used in a wide variety of products. Since many of these agents are
used in relatively large amounts in a number of different products, the effects that these compounds
might exert once released into the environment have become a relevant issue. These compounds are
designed to have an effect on certain organisms and their fate and potential effects in the
environment is therefore an important area of study. One of the chemicals most widely used for
antibacterial purposes are triclosan.

The use of triclosan has recently been questioned due to a number of scientific publications that
have discovered negative effects from triclosan on many non-target organisms such as fish, algae and
amphibians (Orvos et al. 2001, Ishibashi et al. 2003, Marlatt et al. 2013). The effect on organisms in
the aquatic environment is relevant since most triclosan eventually ends up in sewage water due to
its main use in personal care products, and is then released into rivers and coastal environments.
High levels of triclosan outside waste water treatment plants (WWTP´s) (Adolfsson-Erici et al. 2002)
also suggest that removal treatments in WWTP´s are not always efficient enough to prevent triclosan
from reaching the environment. Measured triclosan concentrations in natural waters close to
WWTP´s differ greatly between countries and even within countries. For Sweden, concentrations
vary between 0.34 nM – 0.55 nM in rivers close to WWTP effluents. (Samsø-Petersen et al. 2003,
Bendz et al. 2005). Final concentrations in Skagerrak are difficult to estimate but studies from the
North Sea close to the estuaries of rivers Elbe and Weser detected a decreasing gradient from the
estuaries and outwards into the open sea. The concentration ranged from 4.2-24 pM at the Elbe
estuary to 0.003-0.017 pM in the open sea (Xie et al. 2008). Concentrations at the Swedish west
coast are likely to be slightly lower since population density is higher around the Elbe than around
Göta älv, the largest river with an estuary in Skagerrak. In other places like Spain, concentrations can
be much higher as described by Agüera et al. (2003). In their study of WWTP effluents, triclosan
concentrations ranged between 2.8 nM – 130 nM. The differences between countries are likely due
to differences in population density and water scarcity.
Triclosan (also called Irgasan, systematic name 5-Chloro-2-(2,4-dichlorophenoxy)phenol) (Fig. 1) is an
organic, lipophilic compound used as an ingredient in many everyday personal care
products such as shampoos, perfumes, soaps and toothpastes. Besides the main
use of triclosan as an ingredient in personal care products, the compound can also
be found in other products labeled “antibacterial”, such as toys, textiles, cosmetics,
cleaning agents and many more (Bedoux et al. 2011). The use of triclosan as an
antibacterial surface coating has been increasing during the last 20 years (Levy
2001). Triclosan is commonly used as an antibacterial agent due to its relatively low
cost and effectiveness against gram-positive and gram-negative bacteria (Franz et
Fig. 1: Triclosan (ESIS)
al. 2008).

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When triclosan eventually reach the aquatic environment, the observed effects include: 1) Toxicity to
non-target organisms, including photosynthesis inhibition and endocrine disrupting properties.
(Veldhoen et al. 2006, Franz et al. 2008, Ricart et al. 2010). 2) Transformation through
photodegradation into more toxic compounds such as dioxins in ordinary wastewater (Mezcua et al.
2004). 3) Possibly promotion of antibiotic resistance (Levy 2001, Aiello et al. 2007). Triclosan is a
broad spectrum biocide and is believed to have multiple mechanisms of action depending on
organism, concentration and physio-chemical properties of the environment. For bacteria, one
mechanism of action that has been determined is the inhibition of lipid synthesis by blocking the
enoyl reductase-enzyme (McMurry et al 1998). Another study by Villalain et al. (2001) observed that:
“Triclosan is incorporated into phospholipid membranes, probably aligning itself with the
phospholipid acyl chains, interacting and affecting phospholipid membranes without any cell leakage
and inducing the formation of perturbed membrane structures”. A specific mechanism of action
towards algae has not yet been discovered (Franz et al. 2008).
Because of the negative effects of triclosan and a growing concern for the persistence and
bioaccumulation potential of triclosan, it is currently under ongoing evaluation by the European
Chemical Agency under the Community Rolling Action Plan (ECHA, 2012). Without waiting for the
ECHA evaluation, the Swedish Chemicals Agency (KemI, 2013) is already recommending users of
triclosan to phase out the compound and several companies including H&M (H&M, 2012) and Nokia
(Nokia, 2012) have voluntarily banned or restricted the compound in their own products.
Periphyton communities

Periphyton communities typically consist of different groups of organisms such as algae, bacteria and
associated animals. These communities play an important role in ecosystems as primary producers,
habitat for larger organisms and the foundation of several aquatic food webs (Stevenson & Bahls,
1999).
Periphyton has a long history in ecological and environmental research and monitoring (Cooke 1956,
Blanck 1985, Franz et al. 2008). Experiments with periphyton have been done since at least the early
1900´s. An experimental setup much like the one used in this study was for example used in the
canals of Hamburg in 1916 where periphyton or “aufwuchs” was grown on submerged glass plates
(Cooke 1956). One typical definition of periphyton comes from Young (1945): “By periphyton is
meant that assemblage of organisms growing upon free surfaces of submerged objects in water, and
covering them with a slimy coat.” There are many other definitions and names for periphyton, the
above mentioned aufwuchs is one of them, but in this study the definition stated by Young is used.
The use of periphyton in ecotoxicological experiments has both advantages and disadvantages. Since
periphyton consists of both algae, bacteria and associated animals such as nematodes, there are
always a large number of ecological interactions between different species present in periphyton
(Hansson, L.-A., 1988). This gives the experiment a high level of ecological realism when compared to
single species test. However it also makes the experiment more complex and difficult to interpret.
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Pulse Amplitude Modulation

A suitable method for measuring photosynthetic activity in periphyton is Pulse Amplitude
Modulation (PAM). PAM measures the fluorescence emitted from in vivo periphyton when exposed
to a controlled source of light. The intensity of fluorescence is influenced by a number of factors
including “pollutant stress, light intensity and temperature” (Hofstraat et al. 1994). By keeping all
parameters except pollutant stress constant, an approximation of the pollutants effect on
photosynthesis can be made. Fluorescence can also give additional information like photosynthetic
efficiency, biomass and various other photochemical processes in photosystem II for instance
photochemical and non-photochemical quenching.
A Phyto-PAM instrument consists of Light Emitting Diodes (LED) which emits light of four alternating
wavelengths; (blue (470 nm), green (520 nm), light red (645 nm) and dark red (665 nm). When the
light hits the antenna pigments they enter an excited state. To return to a normal state, one out of
four things can happen; the energy can be converted via electron transport to photosystem I, as is
the usual case in photosynthesis. It can also form a triplet version of chlorophyll a which in turn can
form singlet oxygen. The excited state can also return to a non-excited state through heat dissipation
or through fluorescence dissipation (Hofstraat et al. 1994). Due to effective competition from
photochemistry in photosystem I almost all the fluorescence detected with PAM will stem from
photosystem II (Hofstraat et al. 1994). This means that values acquired from fluorescence
measurements can only be compared with other values acquired in the same way since they are not
absolute.
The Phyto-PAM detects the fluorescence after the excitations from the four wavelengths in turn.
These four fluorescence signals are useful for discerning between the light harvesting pigments used
by different algal and cyanobacterial groups (Schmitt-Janssen and Altenburger 2008).
In this study the PAM instrument has been used in the way described by Hofstraat et al. (1994) called
saturating pulse fluorescence. The basis for this technique is to subject the sample to a series of short
light pulses with varying background light. This scheme is designed to find certain parameters such as
F , the fluorescence emitted when exposed to a measuring light only, Fmax which is the maximum
0
fluorescence yielded by a sample previously only exposed to measuring light and F’max which is the
maximum fluorescence after the sample has been exposed to actinic light, a light source with a
higher intensity than the measuring light. The intensity of the measuring light is set to such a low
intensity that it cannot be used to drive photosynthesis. The actinic light is high enough to drive
photosynthesis but not high enough to cause photo-damage.
In order to determine a suitable intensity of the actinic light, a rapid light curve (RLC) must be
calculated. The RLC consists of a series of light periods of increasing actinic light intensity. The
relative electron transport rate (ETR) after each period is plotted against the actinic light intensities
used. The ETR is defined as “an approximation of the rate of electrons pumped through the
photosynthetic chain” (Beer et al. 2001) and is given by multiplying the effective quantum yield with
the actinic light intensity (PAR). This is done automatically by the Phyto-Win software used together
with the PAM instrument. After a certain number of light periods the intensity becomes high enough
to saturate the electron transport from photosystem II to photosystem I. This gives a hyperbolic
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curve that reaches a plateau and might eventually drop as photo-inhibition sets in to avoid photo-
damage. From the RLC-curve an appropriate actinic light intensity can be chosen to avoid
photoinhibition.
The fluorescence signals after various illumination regimes and light pulses can be used to calculate
various parameters.
The photochemical yield: This parameter is referred to as “the photochemical yield of open PSII
reaction centers” by Hofstraat et al. 1994 and is typically used to assess photoinhibition. This
parameter requires prolonged dark adaptation to remove the effects of non-photochemical
quenching. The factors used in the calculation are: F = the maximum fluorescence yield and F = the
m 0
minimum fluorescence yield when the sample is only illuminated by measuring light. The formula for
the photochemical yield is usually written as:
Eq. 1 (Hofstraat et al. 1994)

The photochemical efficiency: Also calculated by PAM software, this parameter is referred to as “The
photochemical efficiency of PSII per absorbed photon- , or photon yield” (Hofstraat et al. 1994). This
parameter is also an estimation of photosynthetic efficiency. Factors used in this formula include: F ’
m
= the average maximum fluorescence yield during actinic light after a saturating light pulse, F ’ = the
0
fluorescence yield after the actinic light source have been switched off and a far-red illumination
switched on and F = the steady state when actinic light is activated. The formula for this parameter is:
Eq. 2 (Hofstraat et al. 1994)
Non-photochemical quenching (NPQ) is a mechanism that algae use to protect themselves from high
intensity radiation. The excess light energy absorbed by pigments is dissipated via molecular
vibrations as heat thus protecting the organism from singlet oxygen damage. This mechanism is
known to be sensitive to different stress factors, including triclosan (Ricart et al. 2010). The formula
for NPQ is normally written as:
Eq. 3 (Hofstraat et al. 1994)
In this experiment however, a different formula was used as described by Bilger & Björkman 1990.
They used the Stern-Vollmer relationship to simplify the formula above and write it as:

Eq. 4 (Bilger & Björkman 1990)

High Pressure Liquid Chromatography

Biodiversity and community structure in algal communities is often difficult to determine. Abundance
of different species needs to be examined, ideally in a light microscope by an expert in the field, to
determine diversity and community structure. This is both expensive and time-consuming, yet
species composition is an essential endpoint when examining toxic effects on communities. One
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solution to this problem is to use pigment composition as an indicator for species composition on
algae and cyanobacteria which is determined by identifying and quantifying pigment peaks in
chromatograms acquired through a high pressure liquid chromatography instrument (HPLC) (Mackey
et al. 1996, Porsbring et al. 2007). This approach relies on the observation that different algal and
cyanobacterial species have different pigment compositions. Hence, a shift in pigment composition
might indicate a shift in species composition within the community. It needs to be noted however,
that physiological changes in a single species can also alter its pigment composition. Thus, a shift in
pigment composition is dependent both on which species are selected for, and on their physiology.
This method can naturally not give the same amount of detail as a species composition determined
by light microscopy; it can however identify major groups of algae and cyanobacteria (Gieskes &
Kraay 1983). One way of estimating the change in community structure induced by a toxicant is to
measure the change in pigment composition between controls and exposed samples. This change
can be described using a similarity or a dissimilarity index, such as the Bray-Curtis index.
Aim and objectives
The aim of the present study was to investigate the short-term (1.25 h & 2.5 h) and long-term (14
days) effects of triclosan to photosynthesis and pigment composition in marine periphyton
communities.
Materials and Methods

The study consisted of three separate experiments, two short-term exposure experiments , in which
the effects of 1.25 and 2.5 hours of triclosan exposure on photosynthesis was investigated, and one
long-term exposure in which the effects of triclosan on photosynthesis, biomass and pigment
composition were examined after 14 days. The experiments were performed at the Sven Lovén
Centre for Marine Sciences - Kristineberg at the Swedish west coast between September and October
2012.
Toxicant solutions

Stock solutions used in the short-term experiments were made with triclosan (97%) (Sigma-Aldrich,
St. Louis, USA) and acetone (99,9%). The solutions were stored in air-tight vials at -8 °C. The test
solutions used in the experiment were made by mixing 15 µl stock solution with 14.985 ml filtered
(Whatman GF/F) seawater collected from the sampling site, giving a 1000-fold dilution of the stock
solutions and 0.1‰ acetone in the test solutions. The exposure concentration range was 1 nM –
10000 nM with a log -distribution of five concentrations within that range.
10
For the long-term experiment, triclosan stock solutions were and stored in the same way as for the
short-term experiments. Test solutions were made by a 1000-fold dilution of the stock solutions with
deionized water. Exposure concentrations in the flow-through aquaria were made by setting up a
constant inflow of seawater and test solution. The final exposure solutions used in the microcosm
experiment were 0.316, 1, 3.16, 10, 31.6, 100, 316 and 1000 nM. For the untreated controls equal
amounts of deionized water containing acetone was added.
Additionally, in order to get triclosan to dissolve properly in the deionized water, sodium hydroxide
8
(NaOH) was added to the test solutions in amounts that resulted in a 0.3 mM concentration of NaOH
in the test solutions. These amounts of NaOH were calculated not to alter pH in the aquaria more
than 0.1 units.
Periphyton colonization and sampling

For the short-term experiments, a periphyton sampling rack with 170 circular glass discs (Blanck &
Wängberg, 1988) was deployed in a relatively secluded area of the Gullmarsfjord outside the Sven
Lovén Centre for Marine Sciences – Kristineberg. The glass discs had a surface area of 1.5 cm2 and
had been rinsed with ethanol prior to deployment into the sea. With the help of weights and a buoy
the rack was held in a horizontal position at approximately 1.5 m depth for about 2 weeks until a
biofilm of suitable thickness had formed on the glass discs. The rack was recovered and the glass
discs were immediately put in a sealed box with water from the site to protect the discs from
dryness, temperature changes and direct sunlight.
In the long-term mesocosm experiment seawater from the Gullmar fjord was pumped into 22 liter
aquaria from approximately 1.5 meters depth. A 1 mm mesh was used to stop large organisms from
entering the aquaria. Organisms present in the seawater were allowed to colonize the same types of
glass discs that were used in the short-term experiment. After 14 days of colonization and growth,
sampling and measurements were made.
Short-term toxicity experiment

After sampling, approximately 90 discs were sorted to obtain 63 discs with an even and undamaged
biofilm and without large organisms such as barnacles and sea stars. These discs were put into beak-
ers containing filtered seawater from the sampling site. Every beaker contained two discs to allow for
two separate measurements to determine time-dependent toxicity. The samples were incubated at
15.2 C°, which was the in situ temperature in the aquaria. Fluorescent tubes (Osram Lumilux Daylight
L18W/11) with a photon flux density of ca. 125 µmol photons m-2 * s-1 were used as the light source. .
The experiment started when 15 µl stock solution was added to the beakers. The same amount of
acetone was added to the controls. PAM measurements were done after 1.25 hours and 2.5 hours.
The order in which measurements were made was randomized and all measurements were done
using a stopwatch to ensure that measurement time was identical for all samples.
Long-term microcosm experiment

In the microcosm experiment, test solution and seawater was pumped individually into each
aquarium. The flow rates of test solution and seawater were measured and adjusted every day to
make sure the exposure concentrations were as stable and close to the desired concentrations as
possible.

All aquaria had identical glass disc holders, stirrers and light sources. The stirrers moved back and
forth to simulate natural water movement in the sea and to ensure a homogenous triclosan exposure
for all discs. To eliminate differences in light and temperature, the room was sealed from outdoor
light during the entire experiment. The light intensity from the fluorescent tubes were approximately
9