Masters thesis

The Effects of Triclosan on The Effects of Triclosan on Five Marine Algae SpeciesFive Marine Algae Species

Melanie N. McHenry

Department of Biology, Marine Science Program, Jackson State University, P.O. Box

18540, 1400 J.R. Lynch Street, Jackson, Mississippi, USA

Table of ContentsTable of Contents IntroductionIntroduction BackgroundBackground ObjectivesObjectives Experimental DesignExperimental Design Data AnalysisData Analysis ResultsResults

Effects of Triclosan on Algae GrowthEffects of Triclosan on Algae Growth Effects of Triclosan on Algae Growth RatesEffects of Triclosan on Algae Growth Rates Percent InhibitionPercent Inhibition ECEC5050 of Triclosan of Triclosan

DiscussionDiscussion ConclusionConclusion Special ThanksSpecial Thanks AcknowledgmentsAcknowledgments

IntroductionIntroduction This study was conducted in order to determine the effects of This study was conducted in order to determine the effects of

triclosan on marine algaetriclosan on marine algae

Triclosan is incorporated into a number of products which will Triclosan is incorporated into a number of products which will enable triclosan to enter into the wastewater systemenable triclosan to enter into the wastewater system

After the wastewater process, the water still contains amounts After the wastewater process, the water still contains amounts of triclosanof triclosan

This water may be released into the marine environmentThis water may be released into the marine environment

This study examined how triclosan will effect the marine This study examined how triclosan will effect the marine environment, by examining a key component of this environment, by examining a key component of this environment-algaeenvironment-algae

Background Information on TriclosanBackground Information on Triclosan

2,4,4'-trichloro-2’-2,4,4'-trichloro-2’-hydroxydiphenylhydroxydiphenyl

etherether

Trade names:Trade names: Irgasan Registered Trademark DP 300, Irgasan Registered Trademark DP 300, Irgasan Registered Trademark PE 30, Irgasan Registered Trademark PE 30, Irgasan Registered Trademark PG 60, Irgasan Registered Trademark PG 60,

and Irgacide Registered Trademark LP 10and Irgacide Registered Trademark LP 10

Background Information on Triclosan Background Information on Triclosan Cont.Cont.

Biocidal Effects of TriclosanBiocidal Effects of Triclosan

Biocide: a chemical substance capable of killing Biocide: a chemical substance capable of killing different forms of living organismsdifferent forms of living organisms

Algicide: a chemical substance that protects the Algicide: a chemical substance that protects the water from infestation and growth of algaewater from infestation and growth of algae Cell wall lysisCell wall lysis Inhibits Fatty-acid synthesisInhibits Fatty-acid synthesis

Background Information Cont.Background Information Cont.

Triclosan Uses:Triclosan Uses: Found in ¾ of liquid soaps and ¼ of Found in ¾ of liquid soaps and ¼ of

bar soapsbar soaps

Component of:Component of: Antibacterial soaps, toothpaste, sponges, house Antibacterial soaps, toothpaste, sponges, house

cleaners, and product containerscleaners, and product containers Hospital materials, carpeting, sports accessories, and Hospital materials, carpeting, sports accessories, and

children’s toyschildren’s toys

Studies are currently being conducted to determine the Studies are currently being conducted to determine the effectiveness of triclosan against the causative agents of effectiveness of triclosan against the causative agents of tuberculosis, malaria, and toxoplasmosistuberculosis, malaria, and toxoplasmosis


Triclosan Levels Reported in Surface WatersTriclosan Levels Reported in Surface Waters

SurfaceSurface

WaterWaterLevels (µg/L)Levels (µg/L) SourceSource

U.S. RiversU.S. Rivers 0.24 - 2.70.24 - 2.7 Kolpin DW., et al. Kolpin DW., et al.

20022002U.S. Geological U.S. Geological SurveySurvey

0.8 (average)0.8 (average)

Tromso-Sound, Tromso-Sound, NorwayNorway

0.2 - 2.40.2 - 2.4 Weigel s., et al. 2004Weigel s., et al. 2004

Bayou St. John Lake Bayou St. John Lake (New Orleans, (New Orleans,

Louisiana)Louisiana)29 ng/L29 ng/L Boyd, Boyd, et al.et al. 2004 2004

Swiss RiversSwiss Rivers 11 - 98 ng/L11 - 98 ng/L Balmer, 2005Balmer, 2005


Waste waterWaste waterLevels Levels ((µµg/L)g/L)

SourceSource

Primary EffluentPrimary Effluent 3.4 - 8.0 3.4 - 8.0 McAvoy, DC et al. 2002McAvoy, DC et al. 2002

Final Effluent:Final Effluent:

Activated Sludge Activated Sludge TreatmentTreatment

0.20 - 0.410.20 - 0.41

Weigel S. et al. : 2004Weigel S. et al. : 2004Trickling Filter Trickling Filter

TreatmentTreatment1.6 - 2.71.6 - 2.7

SwitzerlandSwitzerland

EffluentsEffluents42 - 213 42 - 213

mg/Lmg/L Balmer 2005Balmer 2005

Triclosan Levels in Waste WaterTriclosan Levels in Waste Water

Background Information Background Information Contd.Contd.

Algae was chosen to evaluate the toxicity of Algae was chosen to evaluate the toxicity of Triclosan b/c they are:Triclosan b/c they are:

At the base of grazing food chain in the aquatic environmentAt the base of grazing food chain in the aquatic environment

Major food source for rotifers, clams, oysters, and larval Major food source for rotifers, clams, oysters, and larval shrimpshrimp

Supply oxygen through photosynthesisSupply oxygen through photosynthesis

Little information is available on Little information is available on triclosan effects on algae (Orvis triclosan effects on algae (Orvis et al.et al. 2002, Balmer 2005 2002, Balmer 2005, , Tatarazako Tatarazako et al. et al. 2004)2004)

Algae Used in this StudyAlgae Used in this StudyTetraselmis chuii

Nannochloropsis oculata

Chaetocerus gracilis

Isochrysis galbana

Rhodomonas salina

AlgaeAlgae ClassClassSize Size (µm)(µm)

MotileMotile FlagellaFlagella

IsochrysisIsochrysis PrymnesiophyceaePrymnesiophyceae 4 – 84 – 8yesyes

2 flagella2 flagella

TetraselmisTetraselmis ChlorophyceaeChlorophyceae 12 - 1412 - 14 4 flagella4 flagella

NannochloropsisNannochloropsisEustigmatophyceaeEustigmatophyceae

1.5 - 2.51.5 - 2.5 nono

nonenoneRhodomonasRhodomonas CryptophyceaeCryptophyceae 16 – 3016 – 30 yesyes

ChaetocerusChaetocerus BaccillariophyceaeBaccillariophyceae 12 - 1412 - 14 nono

Algae Information

The Effects of Triclosan on The Effects of Triclosan on Various Organisms Various Organisms

(Balmer 2005, Russel 2004, Orvos (Balmer 2005, Russel 2004, Orvos et al.et al. 2002) 2002)

Organism L(E)50

Algae

Freshwater algae

Anabaena flos-aquae 0.97 µg/L

Scenedesmus subspicatus 2. 8 µg/L

Selenastrum capricornutum 4.46 µg/L

Navicula pelliculosa 19.1 µg/L

Marine Algae Skeletonema costatum > 66.0 µg/L

Fish

Danio rerio and Oryzais latipes < 300 µg/L

Pimephales promelas 260 µg/L

Lepomis macrochirus 370 µg/L

Seamonkeys Artemia 2000 µg/L

BacteriaMethicillin-resistant Staphylococci 0.1 – 2 mg/L

E. coli and K. pueumoniae 5,000 µg/L

ObjectivesObjectives

To determine the effects of Triclosan on To determine the effects of Triclosan on selected marine algae speciesselected marine algae species

To estimate the ECTo estimate the EC5050 values of Triclosan values of Triclosan for five marine algae speciesfor five marine algae species

Experimental DesignExperimental DesignTreatmentTreatment

(ug/L)(ug/L)Replicate #Replicate #

TemperatureTemperature

(average)(average)SalinitySalinity

LightLight

(lux)(lux)

ControlControl 00 33 2525ooCC 25ppt25ppt 2,2002,200

11 0.20.2 33 2525ooCC 25ppt25ppt 2,2002,200

22 1.01.0 33 2525ooCC 25ppt25ppt 2,2002,200

33 5.05.0 33 2525ooCC 25ppt25ppt 2,2002,200

44 25.025.0 33 2525ooCC 25ppt25ppt 2,2002,200

55 125125 33 2525ooCC 25ppt25ppt 2,2002,200

66 250250 33 2525ooCC 25ppt25ppt 2,2002,200

77 500500 33 2525ooCC 25ppt25ppt 2,2002,200

SolventSolvent

ControlControl7.2% Ethyl 7.2% Ethyl

AlcoholAlcohol3 3

2525ooCC 25ppt25ppt 2,200 2,200

Data AnalysisData Analysis Algae growth rate was calculated using the equation:Algae growth rate was calculated using the equation:

UU = (Ln = (Ln NNtt – Ln – Ln NNoo)/()/(t – tt – too))

where: where: UU = growth rate = growth rate

NNtt = Algae #/mL at time t= Algae #/mL at time t

NNoo = Algae #/mL at time 0 = Algae #/mL at time 0

tt = Sample time for counting algae # = Sample time for counting algae #

ttoo = Initial time of the treatment. = Initial time of the treatment. Percent Inhibition of Algae by Triclosan was calculated as:Percent Inhibition of Algae by Triclosan was calculated as:

%Inhibition = (%Inhibition = (UUct ct – – UUtoxtox)/U)/Uctct * 100 * 100

where: where: UUtox tox = Growth rate in the presence of Triclosan= Growth rate in the presence of Triclosan

UUct ct = Growth rate in the control= Growth rate in the control ECEC5050 values were estimated for each algae species values were estimated for each algae species

by applying a non-linear regression to the plot of Percent by applying a non-linear regression to the plot of Percent Inhibition against natural log of Triclosan Concentration.Inhibition against natural log of Triclosan Concentration.

Results: Effects of Triclosan Results: Effects of Triclosan on Algae Growthon Algae Growth

Each triclosan treatment for each algae Each triclosan treatment for each algae species experienced growthspecies experienced growth

As the time period increased, the more As the time period increased, the more growth each species experiencedgrowth each species experienced

The higher the amount of triclosan in the The higher the amount of triclosan in the treatment, the lower the algae species treatment, the lower the algae species growthgrowth

Isochrysis galbanaIsochrysis galbana THe Effect of Triclosan on Isochrysis Algae

0.00

500,000.00

1,000,000.00

1,500,000.00

2,000,000.00

2,500,000.00

3,000,000.00

Initial Day 1 Day 2 Day 3 Day 4

Time Period

Alg

ae D

en

sit

y (

#/m

L)

Control (0.0 ug/L)

Treatment 1 (0.2 ug/L)







Alcohol Control

Tetraselmis chuiiTetraselmis chuii

The Effect of Triclosan on Tetraselmis Algae

0.00

1,000,000.00

2,000,000.00

3,000,000.00

4,000,000.00

5,000,000.00

1 2 3 4 5

Time Period

Am

ount

of A

lgae

(#/m

L)

Control (0.0 ug/L)




Treatment 4 (25 ug/L)




Nannochloropsis oculataNannochloropsis oculata

The Effects of Triclosan on Nannochloropsis Algae

0.00

1,000,000.00

2,000,000.00

3,000,000.00

4,000,000.00

5,000,000.00

6,000,000.00

7,000,000.00


Time Period

Am

ou

nt

of

Alg

ae

(#

/mL

)

Control (0.0 ug/L)








Rhodomonas salinaRhodomonas salina

The Effect of Triclosan on Rhodomas Algae

0.00

500,000.00

1,000,000.00

1,500,000.00

2,000,000.00

2,500,000.00

3,000,000.00

3,500,000.00


Time Period

Am

ou

nt

of

Alg

ae (

#/m

L)

Control (0.0 ug/L)








Chaetocerus gracilisChaetocerus gracilis

The Effect of Triclosan on Chaetocerus Algae

0.00

500,000.00

1,000,000.00

1,500,000.00

2,000,000.00

2,500,000.00

3,000,000.00


Time Period

Am

ou

nt

of

Alg

ae

#/m

L

Control (0.0 ug/L)








Results: Effects of Triclsoan on Results: Effects of Triclsoan on Algae Growth RatesAlgae Growth Rates

There is an inverse relationship between the There is an inverse relationship between the triclosan treatment and the growth ratetriclosan treatment and the growth rate

There is an inverse relationship between the time There is an inverse relationship between the time period and the growth rateperiod and the growth rate

SAS was run each day for each algae species to SAS was run each day for each algae species to determine the significant difference of each determine the significant difference of each growth rate growth rate

Not all the growth rates for each day were Not all the growth rates for each day were significantly differentsignificantly different

96hr

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

-100.00 0.00 100.00 200.00 300.00 400.00 500.00 600.00

Triclosan Concentration (ug/L)

Alga

e Gro

wth

Rate

(#/d

ay)

72hr

-0.20

0.00

0.20

0.40

0.60

0.80

1.00

1.20

-100.00 0.00 100.00 200.00 300.00 400.00 500.00 600.00


Alga

e Gr

owth

Rat

e (#

/day

)

48hr

-1.00

-0.50

0.00

0.50

1.00

1.50

-100.00 0.00 100.00 200.00 300.00 400.00 500.00 600.00


Alga

e Gro

wth R

ate (#

/day)

Isochrysis galbanaIsochrysis galbana 24hr

-3.00

-2.00

-1.00

0.00

1.00

2.00

3.00

-100.00 0.00 100.00 200.00 300.00 400.00 500.00 600.00


Alga

e Gro

wth

Rate

(#/d

ay)

IsochrysisIsochrysis growth rate ANOVA growth rate ANOVA table 24 hourstable 24 hours

Source of Variation

Degrees of Freedom

Sum ofSquares

Mean Square

F-Value Pr > F

Model 10 36.61077037 3.66107704 369.77 < 0.0001

Error 16 0.15841481 0.00990093

Total 26 36.76918519

R-SquareCoefficient

VarRoot MSE

YIELD Mean

0.995692 13.61679 0.099503 0.730741

Means with the same letter are Means with the same letter are not significantly differentnot significantly different

t Grouping Mean N Treatment

A 1.85667 3 Control

B 1.65000 3 Treatment 1

C 1.29333 3 Treatment 2

D 1.02333 3 Treatment 3

E 0.61333 3 Treatment 4

F 0.31000 3 Treatment 5

G -0.01000 3 Treatment 6

H -203333 3 Treatment 7

A 1.87333 3 Treatment 8


Source of Source of VariationVariation

Degrees of Degrees of FreedomFreedom

Sum of Sum of SquaresSquares

Mean Mean SquareSquare

F-ValueF-Value Pr > FPr > F

ModelModel 1010 8.750955568.75095556 0.875095560.87509556 198.57198.57 < 0.0001< 0.0001

ErrorError 1616 0.070511110.07051111 0.00440694 0.00440694

TotalTotal 2626 8.821466678.82146667

R-SquareR-SquareCoefficient Coefficient

VarVarRoot MSERoot MSE

YIELD YIELD MeanMean

0.9920070.992007 12.1189312.11893 0.0663850.066385 0.5477780.547778


t Groupingt Grouping MeanMean NN TreatmentTreatment

AA 1.166671.16667 33 ControlControl

BB 0.910000.91000 33 Treatment 1Treatment 1

CC 0.720000.72000 33 Treatment 2Treatment 2

DD 0.576670.57667 33 Treatment 3Treatment 3

EE DD 0.490000.49000 33 Treatment 4Treatment 4

EE 0.423330.42333 33 Treatment 5Treatment 5

FF 0.236670.23667 33 Treatment 6Treatment 6

GG -0.8000-0.8000 33 Treatment 7Treatment 7

AA 1.206671.20667 33 Treatment 8Treatment 8







ModelModel 1010 2.499822222.49982222 0.249982220.24998222 1097.481097.48 <0.0001<0.0001

ErrorError 1616 0.003644440.00364444 0.000227780.00022778

TotalTotal 2626 2.503466672.50346667



YIELDYIELDMeanMean

0.9985440.998544 2.5388932.538893 0.0150920.015092 0.5944440.594444









GG 0.486670.48667 33 Treatment 6Treatment 6

HH 0.316670.31667 33 Treatment 7Treatment 7

II -0.08333-0.08333 33 Treatment 8Treatment 8







ModelModel 1010 1.236592591.23659259 0.123659260.12365926 763.15763.15 < <0.0001< <0.0001

ErrorError 1616 0.002592590.00259259 0.000162040.00016204

TotalTotal 2626 1.239185191.23918519

R-SquareR-SquareCoefficientCoefficient


YIELDYIELDMeanMean

0.9979080.997908 2.1975272.197527 0.0127290.012729 0.5792590.579259










GG 0.433330.43333 33 Treatment 7Treatment 7

HH 0.11000.1100 33 Treatment 8Treatment 8

96hr

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

-100.00 0.00 100.00 200.00 300.00 400.00 500.00 600.00


Gro

wth

Rat

e (#

/day

)

72hr

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

-100.00 0.00 100.00 200.00 300.00 400.00 500.00 600.00


Gro

wth

Rat

e (#

/day

)

48hr

-1.40

-1.20

-1.00

-0.80

-0.60

-0.40

-0.20

0.00

0.20

0.40

0.60

0.80

-100.00 0.00 100.00 200.00 300.00 400.00 500.00 600.00


Gro

wth

Rat

e (#

/day

)

Tetraselmis chuiiTetraselmis chuii24hr

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

-100.00 0.00 100.00 200.00 300.00 400.00 500.00 600.00


Gro

wth

Rat

e (#

/day

)

TetraselmisTetraselmis growth rate growth rate ANOVA table 24 hoursANOVA table 24 hours




Mean SquareMean Square F-ValueF-Value Pr > FPr > F

ModelModel 1010 10.1780370410.17803704 1.017803701.01780370 81.7981.79 < 0.0001< 0.0001

ErrorError 1616 0.19910370.1991037 0.012443980.01244398

TotalTotal 2626 10.3771407410.37714074


VarVarRoot MSERoot MSE YIELD MeanYIELD Mean

0.9808130.980813 55.3661855.36618 0.1115530.111553 0.2014810.201481

Means with the same letter are Means with the same letter are not significantly different not significantly different



BB AA 0.606670.60667 33 Treatment 1Treatment 1





DD -0.15667-0.15667 33 Treatment 6Treatment 6

EE -1.36667-1.36667 33 Treatment 7Treatment 7







ModelModel 1010 1.027200001.02720000 0.102720000.10272000 2.892.89 0.02870.0287

ErrorError 1616 0.569466670.56946667 0.035591670.03559167

TotalTotal 2626 1.596666671.59666667



0.6433400.643340 44.6820544.68205 0.1886580.188658 0.4222220.422222









BB CC 0.23670.2367 33 Treatment 6Treatment 6

CC -0.0267-0.0267 33 Treatment 7Treatment 7







ModelModel 1010 1.056837041.05683704 0.105683700.10568370 3.383.38 0.01490.0149

ErrorError 1616 0.500614810.50061481 0.031288430.03128843

TotalTotal 2626 1.557451851.55745185



0.6785680.678568 30.7132130.71321 0.1768850.176885 0.5759260.575926






BB AA CC 0.63000.6300 33 Treatment 3Treatment 3




0.16000.1600 33 Treatment 7Treatment 7







ModelModel 1010 0.776288890.77628889 0.077628890.07762889 3.083.08 0.02210.0221

ErrorError 1616 0.403777780.40377778 0.025236110.02523611

TotalTotal 2626 1.180066671.18006667



0.6578350.657835 21.6954321.69543 0.1588590.158859 0.732220.73222












96h r

0

0.2

0.4

0.6

0.8

1

1.2

0 100 200 300 400 500 600

Triclosa n Conce ntra tion (ug /L )

Gro

wth

Ra

te (

U/d

ay

)

72h r

0

0.20.4

0.6

0.8

11.2

1.4

1.6

0 100 200 300 400 500 600


Gro

wth

Ra

te (

U/d

ay

)

48h r

00.20.40.60.8

11.21.41.61.8

0 100 200 300 400 500 600


Gro

wth

Ra

te (

U/d

ay

)

Nannochloropsis oculataNannochloropsis oculata 24h r

-0.20

0.20.40.6

0.81

1.2

1.41.6

0 100 200 300 400 500 600


Gro

wth

Ra

te (

U/d

ay

)

NannochloropsisNannochloropsis growth rate growth rate ANOVA table 24 hoursANOVA table 24 hours





ModelModel 1010 5.892192595.89219259 0.589219260.58921926 45.7545.75 < 0.0001< 0.0001

ErrorError 1616 0.206059260.20605926 0.01287870.0128787

TotalTotal 2626 6.098251856.09825185



0.9662100.966210 17.3014017.30140 0.1134840.113484 0.6559260.655926





CC BB 0.850000.85000 33 Treatment 2Treatment 2




FF -0.08333-0.08333 33 Treatment 6Treatment 6








ModelModel 1010 4.898133334.89813333 0.489813330.48981333 4.004.00 0.00690.0069

ErrorError 1616 1.959133331.95913333 0.122445830.12244583

TotalTotal 2626 6.857266676.85726667



0.7142980.714298 32.7711132.77111 0.3499230.349923 1.0677781.067778










0.21000.2100 33 Treatment 7Treatment 7


Nannochloropsis Nannochloropsis growth rate growth rate ANOVA table 76 hoursANOVA table 76 hours




Mean SquareMean SquareF-F-

ValueValuePr > FPr > F

ModelModel 1010 4.898133334.89813333 0.489813330.48981333 4.004.00 0.00690.0069

ErrorError 1616 1.959133331.95913333 0.122445830.12244583

TotalTotal 2626 6.857266676.85726667



0.7142980.714298 32.7711132.77111 0.3499230.349923 1.0677781.067778



1.630001.63000 33 ControlControl






DD CC 0.95000.9500 33 Treatment 6Treatment 6








ModelModel 1010 2.040066672.04006667 0.204006670.20400667 20.1220.12 <0.0001<0.0001

ErrorError 1616 0.162200000.16220000 0.010137500.01013750

TotalTotal 2626 2.202266672.20226667



0.9263490.926349 19.5294519.52945 0.1006850.100685 0.5155560.515556










EE -0.06333-0.06333 33 Treatment 7Treatment 7


72hr

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

-100.00 0.00 100.00 200.00 300.00 400.00 500.00 600.00


Gro

wth

Rat

e (#

/day

)

72hr y = 8.7879e0.3397x

R2 = 0.9674

0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00

100.00

-3.00 -2.00 -1.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00

Natural Log of Triclosan Concentration (ug/L)

Th

e A

vera

ge

Inh

ibit

ion

of

Alg

ae

Gro

wth

Rat

e (%

)

48hr

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

-100.00 0.00 100.00 200.00 300.00 400.00 500.00 600.00


Gro

wth

Rat

e (#

/day

)

Rhodomonas salinaRhodomonas salina 24hr

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

-100.00 0.00 100.00 200.00 300.00 400.00 500.00 600.00


Gro

wth

Rate

(#/d

ay)

RhodomonasRhodomonas growth rate growth rate ANOVA table 24 hoursANOVA table 24 hours





ModelModel 1010 3.288000003.28800000 0.328800000.32880000 31.9931.99 <0.0001<0.0001

ErrorError 1616 0.164466670.16446667 0.010279170.01027917

TotalTotal 2626 3.452466673.45246667



0.9523630.952363 12.4825712.48257 0.1013860.101386 0.8122220.812222

















ModelModel 1010 1.688148151.68814815 0.168814810.16881481 2.292.29 0.06750.0675

ErrorError 1616 1.179881481.17988148 0.073742590.07374259

TotalTotal 2626 2.868029632.86802963



0.5886090.588609 35.5577535.55775 0.2715560.271556 0.7637040.763704


















ModelModel 1010 1.364814811.36481481 0.136481480.13648148 3.803.80 0.00870.0087

ErrorError 1616 0.573948150.57394815 0.035871750.03587175

TotalTotal 2626 1.938762961.93876296




0.7039620.703962 27.9746027.97460 0.1893980.189398 0.6770370.677037


















ModelModel 1010 0.979770370.97977037 0.097977040.09797704 3.173.17 0.01960.0196

ErrorError 1616 0.495037040.49503704 0.030939810.03093981

TotalTotal 2626 1.474807411.47480741




0.6643380.664338 29.9069529.90695 0.1758970.175897 0.5991480.599148












96hr

0.000.100.200.300.400.500.600.700.800.90

-100.00 0.00 100.00 200.00 300.00 400.00 500.00 600.00


Gro

wth

Rat

e (#

/day

)

72hr

0.000.100.200.300.400.500.600.700.800.901.00

-100.00 0.00 100.00 200.00 300.00 400.00 500.00 600.00


Gro

wth

Rat

e (#

/day

)

48hr

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

-100.00 0.00 100.00 200.00 300.00 400.00 500.00 600.00


Gro

wth

Rat

e (#

/day

)

Chaetocerus gracilisChaetocerus gracilis24hr

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

-100.00 0.00 100.00 200.00 300.00 400.00 500.00 600.00


Gro

wth

Rat

e (#

/day

)

ChaetocerusChaetocerus growth rate growth rate ANOVA table 24 hoursANOVA table 24 hours






ModelModel 1010 35.1277481535.12774815 3.512774813.51277481 34.5134.51 < 0.0001< 0.0001

ErrorError 1616 1.628592591.62859259 0.0101787040.010178704

TotalTotal 2626 36.7563407436.75634074




0.9556920.955692 32.9411232.94112 0.3190410.319041 0.9685190.968519










FF -1.6967-1.6967 33 Treatment 7Treatment 7








ModelModel 1010 4.854725934.85472593 0.485472590.48547259 2.862.86 0.02980.0298

ErrorError 1616 2.717059262.71705926 0.169816200.16981620

TotalTotal 2626 7.571785197.57178519




0.6411600.641160 50.8285350.82853 0.4120880.412088 0.8107410.810741










CC -0.2133-0.2133 33 Treatment 7Treatment 7








ModelModel 1010 1.067614821.06761482 0.106761480.10676148 3.043.04 0.02330.0233

ErrorError 1616 0.562481480.56248148 0.035155090.03515509

TotalTotal 2626 1.630096301.63009630




0.6549400.654940 29.3133629.31336 0.1874970.187497 0.6396300.639630












ChaetocerusChaetocerus growth rate growth rate ANOVA table 96 hourANOVA table 96 hour




Mean SquareMean SquareF-F-

ValueValuePr > FPr > F

ModelModel 1010 0.650400000.65040000 0.065040000.06504000 2.632.63 0.04120.0412

ErrorError 1616 0.396000000.39600000 0.024750000.02475000

TotalTotal 2626 1.046400001.04640000



0.6215600.621560 27.7625927.76259 0.1573210.157321 0.566670.56667








BB DD CC 0.50330.5033 33 Treatment 5Treatment 5




Results: Percent InhibitionResults: Percent Inhibition

The Percent Inhibition decreased as The Percent Inhibition decreased as the time period increasedthe time period increased

However, this trend had noticeable However, this trend had noticeable exceptions in concentrations with exceptions in concentrations with lower amounts of triclosanlower amounts of triclosan

Percent Inhibition: Percent Inhibition: IsochrysisIsochrysis

Percent InhibitionPercent Inhibition InitialInitial Day 1Day 1 Day 2Day 2 Day 3Day 3 Day 4Day 4

Control (0µg/L)Control (0µg/L) n/an/a n/an/a n/an/a n/an/a n/an/a

Treatment 1 (0.2µg/L) Treatment 1 (0.2µg/L) n/an/a 11.2711.27 22.1022.10 11.6211.62 12.2812.28



Treatment 4 (25µg/L)Treatment 4 (25µg/L) n/an/a 66.7366.73 58.2258.22 40.9840.98 36.5936.59




Treatment 8 (ethyl alcohol Treatment 8 (ethyl alcohol control)control)

n/an/a -0.65-0.65 -3.10-3.10 14.0914.09 0.720.72

Percent Inhibition: Percent Inhibition: TetraselmisTetraselmis











n/an/a -1.59-1.59 -0.54-0.54 -2.60-2.60 0.570.57

Percent Inhibition:Percent Inhibition:NannochloropsisNannochloropsis



Treatment 1 (0.2µg/L)Treatment 1 (0.2µg/L) n/an/a 30.7030.70 10.4610.46 4.954.95 2.542.54








n/an/a -49.15-49.15 -21.24-21.24 -9.21-9.21 -9.08-9.08

Percent Inhibition:Percent Inhibition:RhodomonasRhodomonas










Treatment 8 (ethyl Treatment 8 (ethyl alcohol control)alcohol control)

n/an/a -13.41-13.41 -9.31-9.31 1.151.15 -1.61-1.61

Percent Inhibition: Percent Inhibition: ChaetocerusChaetocerus










Treatment 8 (ethyl Treatment 8 (ethyl alcohol control)alcohol control)

n/an/a -15.51-15.51 0.300.30 8.338.33 -1.95-1.95

ECEC5050 of Triclosan of Triclosan Overall, as the time period increased, Overall, as the time period increased,

the ECthe EC5050 increased for each algae increased for each algae speciesspecies

This was due to the reduction in This was due to the reduction in triclosan’s strength as time went ontriclosan’s strength as time went on

More triclosan was required for the More triclosan was required for the same effectsame effect

96hry = 15.619e0.2286x

R2 = 0.9169

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

-3.00 -2.00 -1.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00


Th

e A

vera

ge

Inh

ibit

ion

of

Alg

ae G

row

th R

ate

(%)

72hry = 15.091e0.2305x

R2 = 0.8865

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

-3.00 -2.00 -1.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00


Th

e A

vera

ge

Inh

ibit

ion

of

Alg

ae G

row

th R

ate

(%)

48hry = 17.438e0.2499x

R2 = 0.894

0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

-3.00 -2.00 -1.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00


Th

e A

vera

ge

Inh

ibit

ion

of

Alg

ae G

row

th R

ate

(%)

Isochrysis galbanaIsochrysis galbana

24h r y = 25.168e0.2901x

R2 = 0.974

0

50

100

150

200

-4 -2 0 2 4 6 8

Na tura l Log of Triclosa n Conce ntra tion (ug /L )

Th

e A

ve

rag

e

Inh

ibit

ion

of

Alg

ae

G

row

th R

ate

(%

)

The time Period and its The time Period and its corresponding ECcorresponding EC5050 value value

Time PeriodTime Period ECEC5050 value value

Day 1Day 1 2.10 μg/mL2.10 μg/mL




96hry = 6.4552e0.3125x

R2 = 0.8985

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

-3.00 -2.00 -1.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00


Th

e A

vera

ge

Inh

ibit

ion

of

Alg

ae

Gro

wth

(%

)

72hr y = 20.256e0.2038x

R2 = 0.9466

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

-3.00 -2.00 -1.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00


Th

e A

vera

ge

Inh

ibit

ion

of

Alg

ae

Gro

wth

(%

)

48hr y = 7.2124e0.4446x

R2 = 0.8608

0.00

50.00

100.00

150.00

200.00

250.00

300.00

350.00

-3.00 -2.00 -1.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00


Th

e A

vera

ge

Inh

ibit

ion

of

Alg

ae

Gro

wth

(%

)

Tetraselmis chuiiTetraselmis chuii

24hr y = 21.347e0.3686x

R2 = 0.8878

0.00

50.00

100.00

150.00

200.00

250.00

300.00

350.00

400.00

450.00

500.00

-3.00 -2.00 -1.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00


Th

e A

vera

ge

Inh

ibit

ion

of

Alg

ae

Gro

wth

(%

)

The time period and its The time period and its corresponding ECcorresponding EC5050 value value





Day 4Day 4 >500 μg/mL>500 μg/mL

96hry = 6.4552e0.3125x

R2 = 0.8985

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

-3.00 -2.00 -1.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00


Th

e A

vera

ge

Inh

ibit

ion

of

Alg

ae

Gro

wth

(%

)

72hr y = 20.256e0.2038x

R2 = 0.9466

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

-3.00 -2.00 -1.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00


Th

e A

vera

ge

Inh

ibit

ion

of

Alg

ae

Gro

wth

(%

)

48hr y = 7.2124e0.4446x

R2 = 0.8608

0.00

50.00

100.00

150.00

200.00

250.00

300.00

350.00

-3.00 -2.00 -1.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00


Th

e A

vera

ge

Inh

ibit

ion

of

Alg

ae

Gro

wth

(%

)

Nannochloropsis oculataNannochloropsis oculata

24hr y = 21.347e0.3686x

R2 = 0.8878

0.00

50.00

100.00

150.00

200.00

250.00

300.00

350.00

400.00

450.00

500.00

-3.00 -2.00 -1.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00


Th

e A

vera

ge

Inh

ibit

ion

of

Alg

ae

Gro

wth

(%

)



Day 1Day 1 2.10 2.10 μg/mL μg/mL




96hry = 18.519e0.2048x

R2 = 0.901

0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00

100.00

-3.00 -2.00 -1.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00


Th

e A

vera

ge

Inh

ibit

ion

of

Alg

ae

Gro

wth

Rat

e (%

)

72hr y = 8.7879e0.3397x

R2 = 0.9674

0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00

100.00

-3.00 -2.00 -1.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00


Th

e A

vera

ge

Inh

ibit

ion

of

Alg

ae

Gro

wth

Rat

e (%

)

48hr

y = 25.96e0.1433x

R2 = 0.7789

0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00

100.00

-3.00 -2.00 -1.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00


Th

e A

vera

ge

Inh

ibit

ion

of

Alg

ae

Gro

wth

Rat

e (%

)

Rhodomonas salinaRhodomonas salina

24hr y = 10.436e0.3344x

R2 = 0.9255

0.00

20.00

40.00

60.00

80.00

100.00

-3.00 -2.00 -1.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00


Th

e A

vera

ge

Inh

ibit

ion

of

Alg

ae

Gro

wth

Rat

e (%

)



Control (0µg/L)Control (0µg/L) 109 μg/mL109 μg/mL

Treatment 1 (0.2µg/L) Treatment 1 (0.2µg/L) 98 μg/mL98 μg/mL



96hry = 15.619e0.2286x

R2 = 0.9169

0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00

-4.00 -2.00 0.00 2.00 4.00 6.00 8.00


Th

e A

vera

ge

Inh

ibit

ion

of

Alg

ae G

row

th R

ate

(%)

72hry = 15.091e0.2305x

R2 = 0.8865

0.00

20.00

40.00

60.00

80.00

100.00

-4.00 -2.00 0.00 2.00 4.00 6.00 8.00


Th

e A

vera

ge

Inh

ibit

ion

of

Alg

ae G

row

th R

ate

(%)

48hr y = 17.438e0.2499x

R2 = 0.894

0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

-4.00 -2.00 0.00 2.00 4.00 6.00 8.00


Th

e A

vera

ge

Inh

ibit

ion

of

Alg

ae G

row

th R

ate

(%)

Chaetocerus gracilisChaetocerus gracilis

24hry = 5.4188e0.5797x

R2 = 0.8696

0.00

50.00

100.00

150.00

200.00

250.00

-4.00 -2.00 0.00 2.00 4.00 6.00 8.00


Th

e A

vera

ge

Inh

ibit

ion

of

Alg

ae G

row

th R

ate

(%)



Control (0µg/L)Control (0µg/L) 46.2 μg/mL46.2 μg/mL




Discussion: Compare Discussion: Compare Marine to Freshwater AlgaeMarine to Freshwater Algae TimeTimePeriodPeriod

Marine Algae Species ECMarine Algae Species EC5050 values values

IsochrysisIsochrysis NannochloropsisNannochloropsis RhodomonasRhodomonas ChaetocerusChaetocerus TetraselmisTetraselmis

24hr24hr 10.810.8 6.36.3 109109 46.246.2 10.010.0

48hr48hr 68.568.5 7272 9898 6868 77.777.7

72hr72hr 182182 165165 168168 183183 83.883.8

96hr96hr 164164 360360 128128 162162 >500>500

Freshwater Algae Species L(E)Freshwater Algae Species L(E)5050 values values

NaviculaNaviculapelliculosapelliculosa

SelenastrumSelenastrumcapricornutumcapricornutum

ScenedesmusScenedesmussubspicatussubspicatus

SkeletonemaSkeletonemacostatumcostatum

AnabaenaAnabaenaflosaquaeflosaquae

96hr96hr 19.1 µg/L19.1 µg/L 4.46 µg/L4.46 µg/L 2. 8 µg/L2. 8 µg/L > 66.0 µg/L> 66.0 µg/L 0.97 µg/L0.97 µg/L

Discussion: Comparison of Discussion: Comparison of Algae, cont.Algae, cont.

(Orvos (Orvos et. al et. al 2002).2002).

Triclosan toxicity is less for marine algae than Triclosan toxicity is less for marine algae than freshwater algaefreshwater algae Marine algae ECMarine algae EC5050 ranges from 128 to > 500 for the 96hr time ranges from 128 to > 500 for the 96hr time

periodperiod Freshwater algae ECFreshwater algae EC5050 ranges from 0.97 to 19.1 for the 96hr ranges from 0.97 to 19.1 for the 96hr

time periodtime period

Caused by a difference in the sensitivity of marine and Caused by a difference in the sensitivity of marine and freshwater species freshwater species

Not relating to the salinity of the environmentNot relating to the salinity of the environment

Discussion: Comparison of ECDiscussion: Comparison of EC5050 and L(E)and L(E)5050 Values of Other Values of Other

OrganismsOrganisms Time Time

PeriodPeriodMarine Algae Species ECMarine Algae Species EC

5050 values values

Isochryis Isochryis galbanagalbana

Nannochloropsis Nannochloropsis oculataoculata

Rhodomonas Rhodomonas salinasalina

Chaetocerus Chaetocerus gracilisgracilis

Tetraselmis Tetraselmis chuiichuii

24hr24hr 10.810.8 6.36.3 109109 46.246.2 10.010.0

48hr48hr 68.568.5 7272 9898 6868 77.777.7

72hr72hr 182182 165165 168168 183183 83.883.8

96hr96hr 164164 360360 128128 162162 >500>500

Organisms L(E)Organisms L(E)50 50 values values

FishFish SeamonkeysSeamonkeys BacteriaBacteria

Danio Danio reriorerio

Oryzais Oryzais latipeslatipes

ArtemiaArtemia StaphylococciStaphylococci Pimephales Pimephales promelaspromelas

Lepomis Lepomis macrochirusmacrochirus

E. E. colicoli

K. K. pueumoniaepueumoniae

Pseudomonas Pseudomonas aeruginosaaeruginosa

< 300 µg/L< 300 µg/L 2000 2000 µg/Lµg/L

100-300 100-300 µg/Lµg/L

260 260 µg/Lµg/L

370 µg/L370 µg/L 5,000 µg/L5,000 µg/L 300,000-300,000-1,000,000 1,000,000

µg/Lµg/L

Discussion: Comparison of Discussion: Comparison of Organisms, cont.Organisms, cont.

Triclosan addition will have an effect on algae speciesTriclosan addition will have an effect on algae species

This addition could indirectly effect algae-dependent This addition could indirectly effect algae-dependent speciesspecies

The difference of the effects of triclosan may be caused The difference of the effects of triclosan may be caused by by the size of each organism the size of each organism their physiological compositiontheir physiological composition

The ECThe EC5050 range of triclosan in algae species is the range of range of triclosan in algae species is the range of these species: water flea these species: water flea Daphnia magnaDaphnia magna, and the fish , and the fish

Danio rerio, Oryzais latipes, Lepomis Danio rerio, Oryzais latipes, Lepomis macrochirus, macrochirus, andand Pimephales Pimephales

promelaspromelas

The ECThe EC5050 for these species were much different than algae for these species were much different than algae species: brine shrimp, species: brine shrimp, ArtemiaArtemia, and the bacteria , and the bacteria E. coliE. coli, , K. K.

pueumoniae pueumoniae, and , and Pseudomonas aeruginosaPseudomonas aeruginosa

Discussion: The Implication Discussion: The Implication of Triclosan Toxicityof Triclosan Toxicity

The addition of triclosan has been proven to be The addition of triclosan has been proven to be toxic to marine and freshwater algae food webstoxic to marine and freshwater algae food webs

The addition of triclosan will greatly reduce the The addition of triclosan will greatly reduce the amount of algae in the aquatic systems. amount of algae in the aquatic systems.

This will lower the amount of oxygen supplied as This will lower the amount of oxygen supplied as well as the species that rely on algae for dietary well as the species that rely on algae for dietary purposes will have a reduction in their food sourcepurposes will have a reduction in their food source

This will have a ripple effect through out the food This will have a ripple effect through out the food webs and result in a reduced number of specieswebs and result in a reduced number of species

Discussion: Comparison of the Discussion: Comparison of the ECEC5050 Values among the Algae Values among the Algae

SpeciesSpecies TimeTime

PeriodPeriodMarine Algae Species ECMarine Algae Species EC

5050 values values

IsochryisIsochryisgalbanagalbana

NannochloropsisNannochloropsisoculataoculata

RhodomonasRhodomonassalinasalina

ChaetocerusChaetocerusgracilisgracilis

TetraselmisTetraselmischuiichuii

24hr24hr 10.810.8 6.36.3 109109 46.246.2 10.010.0

48hr48hr 68.568.5 7272 9898 6868 77.777.7

72hr72hr 182182 165165 168168 183183 83.883.8

96hr96hr 164164 360360 128128 162162 >500>500

Discusssion: Visual Discusssion: Visual comparison of the ECcomparison of the EC5050

(µg/L) values(µg/L) values The EC50 (ug/L)of Selected Marine Algae Species

050

100

150200250300

350400

24hr 48hr 72hr 96hr

Time Period

EC

50 (u

g/L

) V

alu

e Isochrysis

Nannochloropsis

Rhodomonas

Chaetocerus

Tetraselmis

ConclusionConclusion In conclusion, this study, In conclusion, this study, The Effects of Triclosan on Marine The Effects of Triclosan on Marine

AlgaeAlgae, determined the effects of triclosan on the marine , determined the effects of triclosan on the marine algae, algae, TetraselmisTetraselmis, , NannochloropsisNannochloropsis, , ChaetocerusChaetocerus, , IsochyrsisIsochyrsis, , and and RhodomonasRhodomonas over a 96 hour (4 day) trials over a 96 hour (4 day) trials

Triclosan was shown to inhibit the growth of marine algae Triclosan was shown to inhibit the growth of marine algae species thereby affecting the total marine environmentspecies thereby affecting the total marine environment

After calculating and comparing the ECAfter calculating and comparing the EC5050 of triclosan on the of triclosan on the algae species triclosan was determined to least affect algae species triclosan was determined to least affect TetraselmisTetraselmis and most affect and most affect RhodomonasRhodomonas

As this study progressed, the effect of the triclosan As this study progressed, the effect of the triclosan diminished to a degree suggesting that triclosan has the most diminished to a degree suggesting that triclosan has the most drastic effect after it is first introduced into the marine drastic effect after it is first introduced into the marine environment.environment.

Special ThanksSpecial Thanks

Dr. Paulinus Chigbu, for Dr. Paulinus Chigbu, for taking me under his taking me under his wingwing

Marcus SimsMarcus Sims Dr. IzevbigieDr. Izevbigie Dr. HwangDr. Hwang Dr. BegoniaDr. Begonia Dr. HardyDr. Hardy Ms. SimpsonMs. Simpson Ms. Sandra HallMs. Sandra Hall Alex SucharAlex Suchar Eric EvansEric Evans QuanishaQuanisha Bryon GipsonBryon Gipson

AcknowledgementsAcknowledgements

This research was supported, in part, by a grant from the National Oceanic and Atmospheric Administration grant # NA17AE1626, Subcontract # 27-0629-017, through the Environmental Cooperative Science Center at Florida A&M University to Jackson State University. M. McHenry was supported through the National Institutes of Health funded Research Initiative for Scientific Enhancement (RISE) grant awarded to Jackson State University.

Documents

Masters thesis