Viktor Fihlman

Degree project for Master of Science 30 hec

Department of Biological and Environmental SciencesUniversity of Gothenburg 2013

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

1

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.

2

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.

3

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

4

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

al. 2008).

Fig. 1: Triclosan (ESIS)

5

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.

6

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

F0, the fluorescence emitted when exposed to a measuring light only, Fmax which is the maximum

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

7

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: Fm = the maximum fluorescence yield and F0 = the

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: Fm’

= the average maximum fluorescence yield during actinic light after a saturating light pulse, F0’ = the

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

8

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 log10-distribution of five concentrations within that range.

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

9

(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

10

120 µmol photons/m2*s-1. The light/dark regime was set to 14.5/9.5 hours to simulate the natural

light conditions in Sweden at this time of year.

PAM measurements

A PHYTO-PAM (Heinz-Walz, Effeltrich) with the Phyto-Win software V. 1.45 was used in this study. All

samples were analyzed with the same PAM procedure as described by Hofstraat et al. (1994) with

the exception of F0´ which was not measured. Before measurement began, a Zero offset (Zoff)

measurement to determine and eliminate “background-fluorescence” was made using a clean glass

disc and 0.2 µm filtered seawater. A rapid light curve (RLC) was also made prior to the experiment to

determine the appropriate actinic light intensity.

Variables calculated with the PAM instrument in the short-term experiment were: 1) Photochemical

yield of open Photosystem II (PSII) reaction centers. 2) The photochemical efficiency of PSII per

absorbed photon and 3) NPQ (see Introduction for definitions) for the four different wavelength

channels. Important to note about the photochemical yield of open Photosystem II (PSII) reaction

centers and NPQ measurements is that these measurements require complete dark-adaptation

before measuring. Since there was no time for long periods of dark-adaptation the samples were

only dark-adapted until the fluorescence readings were stable. Thus, these values should be

considered estimates of the “true” value.

For the long-term microcosm experiment the following variables were calculated: 1) Photochemical

yield of open Photosystem II (PSII) reaction centers. 2) The photochemical efficiency of PSII per

absorbed photon. 3) NPQ and 4) Fo which is used as an estimation of the chlorophyll a-

concentration, which in turn is an estimation of biomass as described by Schmitt-Jansen et al. (2008).

In the long-term experiment the samples were dark-adapted 40 minutes prior to measurements by

removing them from the aquaria and placing them in darkness and the same exposure

concentrations as in the aquaria.

High pressure liquid chromatography (HPLC)

For measuring pigment composition, four glass discs were sampled from each aquarium. The edges

of the discs were gently wiped with a Kleenex tissue to remove excess water and periphyton from

the edges of the glass discs. The discs were selected randomly from all positions on the racks in the

aquaria. Wiped discs were then transferred to scintillation vials containing 2 ml of a pigment

extraction medium composed of 30% methanol, 30% acetone: 30% dimethylsulfoxide and 10%

distilled water. The vials were covered with aluminum foil and immediately put on ice in order to

minimize the breakdown of pigments. The pigments were stored in a freezer at -18°C and analyzed

17 days after their removal from the aquaria.

To further enhance the extraction of pigments the samples were sonicated for 3x15 seconds and

manually shaken. The extraction medium was filtered through a 0.45 µm nylon filter and injected

into the HPLC instrument.

Calculations and statistics

Values of photochemical yield, photochemical efficiency and NPQ were calculated using the formulas

11

presented in the introduction section. The data were plotted against concentration as percent of

control.

To determine statistical significance the software SPSS (V. 21, IBM, New York, NY, USA) was used.

Significance was established using a p-value of ≤ 0.05. The no observed effect concentration (NOEC)

and the lowest observed effect concentration (LOEC) and deviations from mean were calculated by

first testing the homogeneity of variances and then performing ANOVA and Dunnets ad-hoc test to

determine which treatments deviated from the controls. The concentration which causes a 50%

response in the studied effect, the EC50, was calculated by curve fitting using the Weibull-

distribution. Parameters for constructing a Weibull-fit were obtained through the statistic-program

NLREG (V. 6.5 (Demonstration), Phillip H. Sherrod, Brentwood, TN, USA).

When analyzing pigment peaks acquired from the HPLC, only the larger peaks with a clear visible

pattern were selected. Data for selected pigment peak areas were then treated with the Bray-Curtis

dissimilarity index:

Where nik is the value for the pigment peak area for the average control and njk is the value for the

pigment peak area of the individual sample.

The resulting index values were analyzed with the PAST software (Hammer & Harper, Oslo) version

2,17b to produce a Multidimensional scaling chart (MDS).

Eq. 5: Bray-Curtis Index (BCI)

12

0

20

40

60

80

100

120

140

0.1 10 1000

Effe

ct o

n p

ho

tosy

nth

etic

ef

fici

ency

(%

of

con

tro

ls)

Concentration [nM] 0

0

20

40

60

80

100

120

140

0.1 10 1000

Effe

ct o

n p

ho

tosy

nth

etic

ef

fici

ency

(%

of

con

tro

ls)

Concentration [nM]

0

Results Photochemical yield is described in literature as a variable especially sensitive for detecting

photoinhibition (Hofstraat et al. 1994). In these experiments it yielded roughly the same results as

the photochemical efficiency of PSII (data not shown). Since samples were not completely dark-

adapted prior to the measurements no conclusions on the sensitivity of this method can be made.

The fluorescence originating from different excitation wavelengths gave similar results (data not

shown) and therefore the average values from the four channels were used. This was true for all

yield- and NPQ measurements after both short-term and long-term exposure.

Short-term effects on photosynthesis

Triclosan had a significant negative effect on photochemical efficiency of PSII in the highest tested

concentration after both 1.25 hours and 2.5 hours.

This experiment was also designed to investigate the time-dependence in toxicity of triclosan with

two consecutive measurements after 1.25 (Fig. 2a) and 2.5 hours (Fig. 2b) respectively. No such time-

dependent toxicity could however be found. The only difference between the two exposure times is

that the variance generally increased between samples after 2.5 hours.

At the highest concentration used in the experiment (10000 nM), the photochemical efficiency of PSII

decreased to around 10-20% of controls for both exposure times. The EC50-values for 1.25 hours and

2.5 hours were 2380 nM and 3690 nM respectively. A decreasing trend could also be seen already at

1000 nM for both exposure times although this effect is not statistically significant. The lowest

observed effect concentration (LOEC) in this experiment would therefore be the highest tested

concentration, 10000 nM. In the same way the no observed effect concentration (NOEC) for this

experiment would be 1000 nM. This is because NOEC and LOEC are defined as actually tested

concentrations, which highlights why these parameters may in many cases be misleading.

NPQ is defined as “the normalised decrease of the maximal fluorescence yield with respect to the dark-adapted situation” (Hofstraat et al 1994). Dark-adaptation is therefore crucial for a correct

a

b

Fig. 2: Short-term effects on photochemical efficiency off PSII after a) 1.25 hours and b) 2.5 hours.

a

13

-200

-100

0

100

200

0.1 10 1000

No

n-p

ho

toch

emic

al q

uen

chin

g (N

PQ

) (%

of

con

tro

ls)

Concentration [nM] -100

-50

0

50

100

150

200

0.1 10 1000

No

n-p

ho

toch

emic

al q

uen

chin

g (N

PQ

)

(% o

f co

ntr

ols

)

Concentration [nM]

80

85

90

95

100

105

110

115

120

0.1 1 10 100 1000

Ph

oto

che

mic

al y

ield

Ex

pre

sse

d a

s %

of

con

tro

l

Concentration [nM]

80

85

90

95

100

105

110

115

120

0.1 1 10 100 1000

Ph

oto

che

mic

al e

ffic

ien

cy o

f P

SII (

Exp

ress

ed

as

% o

f co

ntr

ols

)

Concentration [nM]

measurement. Even so, NPQ was calculated from the short-term experiments data even though the samples were not completely dark-adapted. The aim was to see if there was any potential for detect-ing effects on non-photochemical quenching for future experiments.

No short-term effects on NPQ could be seen on periphyton after 1.25 hours. In the analysis done

after 2.5 hours an effect was clearly visible with a significant decrease at 10000 nM.

Long-term effects on photosynthesis

To assess the long-term effects on photosynthesis from triclosan exposure, photosynthetic yield,

photochemical efficiency of PSII and NPQ were determined in periphyton grown in flow-through

aquaria for 14 days. Results showed that long-term exposure to triclosan did not impair

photosynthetic activity; on the contrary, a slight increase in photosynthetic yield and efficiency could

be seen (Fig. 4a & 4b).

Figure XX: Light yield from chronically exposed samples

divided into wavelengths.

a

b

a b

0

Fig. 4: Long-term effects on photosynthetic yield and photochemical efficiency of PSII after 14 days of exposure.

a) photochemical yield and b) photochemical efficiency.

Fig. 3: Short-term effects on non-photochemical quenching (NPQ) after: a) 1.25 hours and b) 2.5

hours.

0

14

Fig. 5: Relative long-term effects on non-photochemical

quenching after 14 days of exposure.

-300

-200

-100

0

100

200

300

400

0.1 1 10 100 1000

No

n-p

ho

toch

em

ical

qu

en

chin

g af

ter

chro

nic

exp

osu

re

Exp

ress

ed

as

% o

f co

ntr

ols

Concentration [nM]

For photochemical yield, treatments with 31.6 nM, 100 nM and 316 nM had a statistically significant

higher photochemical yield than controls. At 1000 nM however there was no statistically significant

difference between controls and triclosan exposed samples. The photochemical efficiency was

similar but showed a positive and significant increase in yield at 316 nM and 1000 nM.

Since results from the short-term exposure indicated a response from non-photochemical quenching

an experiment with proper dark-adaptation was conducted to further investigate such effects. This

time samples were dark-adapted for 40 minutes, a time period which should be more than enough to

eliminate all quenching processes, meaning that all reaction centers are open.

The effects on non-photochemical quenching were once again very erratic and showed no clear

response. (Fig. 5).

15

0

20

40

60

80

100

120

140

0.1 1 10 100 1000

Fo (

% o

f co

ntr

ols

)

Concentration [nM]

0

Long-term effects on chlorophyll content In order to estimate the effect of triclosan on periphyton chlorophyll content, measurements of the

fluorescence emitted during measuring light only (F0) was made. Using this method no significant

difference in biomass could be detected between the controls and treatments (Fig. 6).

Fig. 6: F0 after 14 days of exposure, measured with PAM. Black solid line is linear regression.

To validate the estimation of chlorophyll content with PAM a corresponding analysis was done using

High Pressure Liquid Chromatography (HPLC). Since almost all fluorescence measured by PAM stems

from chlorophyll a, fluorescence measurement can be regarded as an approximation for chlorophyll

a concentration. By expressing F0 and HPLC derived concentrations of chlorophyll a as percent of

controls, these endpoints can be compared.

Fig. 7: Chlorophyll a concentration derived with HPLC after 14 days of exposure.

From this graph it is obvious that there is an increase in chlorophyll a content at the three highest

tested concentrations of triclosan (100 nM, 316 nM and 1000 nM). A statistical analysis of the results

0.1 1 10 100 1000

0

20

40

60

80

100

120

140

160

180

200

Conceantration [nM]

Ch

loro

ph

yll a

co

nte

nt

der

ived

by

HP

LC.

(% o

f co

ntr

ols

)

0

16

confirm that there is a difference between treatments and controls but ad-hoc tests to determine

which treatments deviated failed since some of the treatments are only represented with one

sample each. These results are contradictory to the biomass approximation calculated by PAM in the

previous experiment (Fig. 6).

Pigment composition in periphyton after long-term exposure to triclosan

In order to examine the long-term effects of triclosan the pigment composition of periphyton was

studied with High Pressure Liquid Chromatography (HPLC). Pigment profiles were compared between

treatments and controls using the Bray-Curtis Dissimilarity Index. The dissimilarities expressed as

Bray-Curtis values were then plotted and analyzed using Non-metric Multi-Dimensional Scaling

(MDS).

Fig. 8: Non-metric multidimensional scaling chart (MDS) generated by Bray-Curtis Index values. The MDS is

based on log-transformed Bray-Curtis Dissimilarity Index values for pigment composition. Different colors

symbolize different treatments of triclosan. Distance in the chart represents dissimilarity between samples.

-0,4 -0,3 -0,2 -0,1 0,0 0,1 0,2 0,3 0,4

Coordinate 1

-0,18

-0,12

-0,06

0,00

0,06

0,12

0,18

0,24

0,30

Co

ordinate

2

- Controls

- 0.316 nM

- 1 nM

- 3.16 nM

- 10 nM

- 31.6 nM

- 100 nM

- 316 nM

- 1000 nM

17

The chart shows that periphyton exposed to triclosan concentrations of 31.6, 100 and 316 nM are

affected in a similar way, and are divergent from the controls. Another thing to note is that there are

no clear differences between the controls and the low concentrations. Also treatments 31.6 nM, 100

nM and 316 nM appear as a group with little or no internal order within the group. This indicates that

the effect of triclosan does not seem to increase substantially between 31.6 nM and 316 nM.

Additionally, a comparison in pigment compositions between the control aquaria used in the test

system and natural periphyton collected with racks from the field next to the test system water

intake was made. This comparison describes the impact of the test system on non-exposed

periphyton. The results showed that periphyton in the aquaria had a higher concentration of most

major pigments. One pigment was an exception however with high concentrations in the periphyton

collected next to the test system water intake and low concentrations in aquaria periphyton. This

pigment was not identified but had a retention time in the HPLC instrument of 8.52 minutes.

Discussion

Information about the effects triclosan might cause in the aquatic environment is needed. There is a

widespread global use of triclosan as a biocide due to its effectiveness and relative low cost.

Concerns are relevant because of the heavy use and fate of triclosan in the environment. Since

triclosan is relatively hydrophobic (log Kow = 3.5-4.8 at neutral pH) (Bedoux et al. 2012) it is expected

to attach to solids and sediments and may therefore pose a concern for aquatic organisms which

lives in or close to the sediment. Additionally, triclosan is classified as bioaccumulating (Samsø-

Petersen et al. 2003) and has been found in relatively high concentrations in fish both in the natural

environment (Remberger et al. 2002) and in fish exposed to WWTP effluents (Adolfsson-Erici et al.

2002). Coupled with the fact that triclosan has showed significant toxicity towards non-target

organisms, of which algae seem to be one of the most sensitive groups (Franz et al. 2008), additional

testing of triclosan is necessary.

Effects on photosynthesis

Triclosan had a significant and adverse negative effect on photosynthesis of periphyton in the highest

tested concentration (10000 nM) in the short-term exposure experiment (Fig. 2). This indicates that

most photosynthetic species in the periphyton community are severely affected by the toxicant at

this concentration. At this point the photosynthetic efficiency was only 10-20% of the control values.

The concentration of triclosan required for a 50% effect compared to controls (EC50) was calculated

to be 2380 nM. A slight negative trend could be seen already at 1000 nM but this was not statistically

significant. This correlates well with Franz et al. (2008) who found an EC50-value of 3100 nM using

the same method but with periphyton from a German river.

For this experiment, NOEC was determined to 1000 nM and LOEC to 10000 nM. The values of NOEC

and LOEC are in this case correct but might be misleading and there is a risk of underestimating the

18

toxicity of triclosan. This is because even though 10000 nM is the lowest observed effect

concentration, effects might occur at much lower concentrations and LOEC could be lower if more

exposure concentrations had tested between 1000 and 10000 nM.

The photosynthetic yield of the long-term exposed samples was completely different from the short-

term. For the long-term exposure a significant positive effect could be seen with a peak around 100

nM (Fig. 4a). This correlates well with the treatments that diverge from the controls in the MDS-plot

when investigating changes in pigment concentrations. The short-term exposure was completely

different from the long-term and saw a decrease in yield at 10000 nM. If the same concentration had

been used also in the long-term experiments, a decrease in photosynthetic yield might have been

observed also in the long-term exposure. This is however far from certain since organisms in the

long-term exposure would have been selected for their ability to tolerate triclosan and as a result,

might be less affected by it. For the light-adapted yield a similar increase in efficiency could be

observed although slightly shifted towards higher concentrations with significant positive increases

at 316 and 1000 nM.

Non-photochemical quenching measurements were performed in both short-term and long-term

exposures. Short-term exposure showed a significant deviation from controls only in the highest

tested concentration for the longest exposure time meaning that there is a possibility of time-

dependent toxicity acting specifically on non-photochemical quenching processes. This time-

dependent effect seems to only affect the NPQ response and not the photosynthetic efficiency

where no time-dependent toxicity could be seen. Since the values for NPQ calculated here were not

dark-adapted, as they should be according to definition, no solid conclusions can however be drawn

from this. It is however possible to speculate that the effects on NPQ that can be observed here are

the same effects that Ricart et al (2010) found in their 48 hour triclosan exposure to freshwater

periphyton.

In the long-term experiment, samples were fully dark-adapted to allow NPQ to be calculated in a

correct way. The long-term exposed samples varied greatly however and no significant response

could be obtained. This is surprising since Ricart et al. (2010) found NPQ to be the most sensitive

endpoint with an EC50-value of 380 nM of triclosan. One likely explanation is that since the

organisms in this study were exposed for a longer time in the long-term experiment, all organisms

were selected for their ability to tolerate triclosan. This would not have been the case in the study by

Ricart et al. (2010), who exposed river biofilms to 48 hours only. Because of the sensitivity of NPQ to

triclosan exposure, Ricart et al. (2010) speculates that triclosan may cause damage in the

photosynthetic apparatus. If this is true, this damage must occur sometime between 2.5 – 48 hours

since no effect on NPQ could be observed after 1.25 hours and only a weak effect after 2.5 hours.

After 14 days of exposure this effect would likely have resulted in a loss of fitness in affected

organisms leading to them being replaced by more tolerant ones, according to the concept of

Pollution-Induced Community Tolerance (PICT). Interestingly, in the long-term experiment reported

here Eriksson detected PICT to triclosan at exposure levels 100 – 1000 nM (unpublished). In

Eriksson’s study PICT was detected using incorporation of 14C-labelled carbonate, thus measuring

another photosynthesis-related endpoint. The exposure levels that induced triclosan community

tolerance coincides with the observations of elevated photosynthetic efficiency (Fig 4a and 4b),

19

increased concentrations of chlorophyll a (Fig. 7) and the changed pigment composition (Fig. 8) in

this study.

Measurements to determine if there was any difference between the four excitation channels used

in the experiment were also performed but these analyses showed no significant difference between

the channels in either the short-term or long-term exposures. This indicates that triclosan is equally

toxic to most groups of organisms found in periphyton, or that the PHYTO-PAM measurements are

not sensitive enough to detect such differences.

Effects on biomass

An interesting difference could be seen when measuring chlorophyll a content with the PAM

instrument and HPLC instrument. Since photochemical yield and efficiency had increased in some

medium to high concentrations, it was expected that there would be an increase in chlorophyll a as

well. The PAM method of determining chlorophyll a content showed no such increase however.

When measuring concentrations of chlorophyll a using HPLC a significant increase was found at the

higher concentrations of triclosan. Since some treatments were only represented by one sample,

ANOVA showed that there was a difference between treatments but was unable to specifically show

which treatments deviated from controls. By observing the graph however (figure 7), 100 nM, 316

nM and 1000 nM seem to have a clearly higher concentration of chlorophyll a.

The concentrations of chlorophyll a determined in the HPLC instrument should be proportional to

the fluorescence measured by the PAM instrument since almost all fluorescence stems from

chlorophyll a. The reason that these two results differ is likely due to quenching of the measuring

light and/or the fluorescence for the organisms. The periphyton biofilm on the glass discs is so thick

that the measuring light might not reach all cells and/or the fluorescence emitted by algae and

bacteria at the bottom of the biofilm get quenched by organisms higher up in the biofilm.

These results demonstrate that PAM might not be a good method for estimating biomass in thick

biofilms.

Effects on community structure and physiological status

An analysis of pigment composition determined by HPLC represents a cheaper and quicker way of

estimating community structure and functional response to toxicants than determining and counting

species observed in a microscope. HPLC derived pigment composition identifies and quantifies all

pigments present in a community. This information represents a physiological response in photosyn-

thetic organisms since organisms can, to some extent; change their own composition of different

pigments. Additionally, different classes of algae and cyanobacteria differ in pigment composition,

thus these pigments can to some extent represent certain groups of organisms (Mackey et al. 1996,

Porsbring et al. 2007).

A clear effect of triclosan could be seen in the MDS-plot for communities exposed to 31.6, 100 and

316 nM of triclosan. These are the only treatments that can be said to form a distinct group, separat-

20

ed from the controls. The communities in this group seem to be affected in a similar way since they

are located at the same place in the MDS-graph, while most of the controls are located together with

the lowest concentrations in their own group. The 1000 nM community is located far away from the

31.6-316 nM grouping; since this treatment is only represented by one sample it is hard to draw any

conclusions from this. The 1000 nM community is located in what might be described as a smaller

third group. This group is composed of one control, two low concentrations communities and the

highest concentration used in the experiment. The explanation for the placement of the community

exposed to 1000 nM in this smaller group might be coincidence, an experimental error which affect-

ed these three different communities in a similar way. It is also plausible that different triclosan tol-

erance mechanisms are effective at different exposure levels and that the community exposed to

1000 nM use a non-photosynthetic mechanism to overcome triclosan exposure. Another explanation

might be that a disturbance in some ecological process in the community, e.g. toxicity to and reduc-

tion of grazers, renders the tolerance mechanism used at lower exposure obsolete. Something to

note in the MDS-plot is that almost all samples from the 31.6, 100 and 316 nM treatments are locat-

ed in a one group with no apparent order within the group. This indicates that whatever effect is

causing this difference occur somewhere between 10 and 31.6 nM and that even if the concentration

is increased by 10 times to 316 nM the effect does not seem to increase. Furthermore, since there is

no clear trajectory leading from the control group towards the group of 31.6, 100 and 316 nM treat-

ments it is clear that the effect is not step-wise but rather confined to this concentration interval.

Possible explanations for this non concentration-dependent change in pigment composition are that

one or several species are severely affected by the triclosan already at 31.6 nM-316 nM and disap-

pear from the community. The species that replace them are likely slightly better at tolerating

triclosan exposure. These new species might have different pigment compositions compared to the

sensitive ones and as a result, the total pigment composition of the community would change. This

explanation is validated by the observations of community tolerance to triclosan in the same com-

munities by Eriksson (unpublished) as outlined above. This hypothesis does not explain the odd

placement of the 1000 nM community however as this treatment showed an even higher degree of

tolerance than the 100-316 nM communities. If the tolerance mechanism is the same at the expo-

sure 100-1000 nM exposure levels then these communities should have the same or at least similar

concentrations of pigments. The fact that the 1000 nM treatment diverges from the other tolerant

communities indicates that some other pigment associated change has occurred. Possible explana-

tions to this could be a second tolerance mechanism or that the 1000 nM treatment is simply an out-

lier. In a similar experiment (Backhaus et al. 2011), conducted at the same location with the same

test organisms but using a different method, EC50-values for triclosan were calculated for several

pigments. In this 96-hour incubation system, EC50-values for individual pigments ranged from 1166-

1966 nM. These values are comparable to the short-term, EC50-values of photosynthetic efficiency

calculated after 1.25 and 2.5 hours in our experiment (2380 and 3690 nM respectively). The concen-

trations in the 14 days flow-through system required for a shift in pigment compositions was howev-

er considerably lower than that of Backhaus et al. 2011 indicating that some organisms might be able

to tolerate moderate amounts of triclosan (31,6 – 316 nM) for a few days before being eliminated

from the community.

Another thing that contributes to the differences in pigment composition seen in the MDS-plot is

that many of the species present in medium to high concentrations of triclosan change their internal

composition of different pigments as a possible way of coping with the stress exerted by triclosan. As

21

mentioned above, the species that are most adversely affected at these concentrations could also be

a grazer. This could lead to an increase of growth in photosynthetic organisms and would help to

explain why the chlorophyll a content is higher in triclosan exposed communities. A reason why this

effect is visible at relative low concentrations in the long-term experiment, compared to the short-

term experiment, is because during a long-term exposure even small negative effects can result in a

loss of fitness, which might cause the affected organism to be outcompeted by more tolerant ones.

This is not the case in the short-term experiment, since the exposure time is too short to allow the

acute toxicity to develop into changed species composition.

When concentrations of triclosan become high enough to impact periphyton, effects can be expected

at the physiology level and possibly also at the level of community structure. Other research groups

like Ricart et al. (2010) found NPQ to be a particularly sensitive endpoint and speculated that

triclosan might cause damage to the photosynthetic apparatus. If triclosan is damaging or blocking

the photosynthetic apparatus this could help explain the increase in chlorophyll a content found

after the long-term exposure in this study. If some pigments are blocked or damaged and therefore

unable to absorb sunlight, the algae might produce more chlorophyll a to compensate for the effect

of triclosan. The EC50-value for NPQ as calculated by Ricart et al. (2010) (383 nM) is also very close to

the concentration where chlorophyll a content is increasing (100 nM) making this a plausible theory

for explaining the triclosan induced increase in biomass.

The reason for the change in pigment composition which can be seen at relatively low exposure

levels (>10 nM) are probably that some organisms in periphyton are more sensitive than others.

Triclosan is a bactericide and cyanobacteria should for example be affected earlier than algae. This is

not supported by the experimental data however since PAM did not find any difference between

wavelength channels.

In summary, triclosan could potentially be a hazard for periphyton in heavily polluted areas and in

places with a high population density and high water scarcity. From the results of this study,

however, it can be concluded that concentrations of triclosan in natural Swedish waters simply seem

too low to impact photosynthetic processes and pigment composition in periphyton in any significant

way. Triclosan might however still be hazardous to other organisms which dwell in sediments where

concentrations are higher or to fish in which triclosan is bioaccumulated.

Acknowledgements First of all I would like to thank my supervisors Martin Eriksson, Åsa Arrhenius and Hans Blanck for

their continued support and enthusiasm throughout the experiment, without you this final report

would have looked a lot different. Additional thanks go out to Martin who invited me to take part in

the aquaria project and who helped me design the short-term part of the experimental setup. I

would also like to thank Henrik Johansson, Alexander Grehn and Triranta Sircar for valuable help in

my experiments, but most of all for their fantastic company at Kristineberg. My final thank goes to

Stina who supported me all the way by listening and discussing parts of my experiment that must

have been vaguely interesting to her and for putting up with me being away so much.

Thank you all.

22

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