1. The Effects of Triclosan on Five 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

2. Table of Contents

Introduction

Background

Objectives

Experimental Design

Data Analysis

Results

• Effects of Triclosan on Algae Growth

• Effects of Triclosan on Algae Growth Rates

• Percent Inhibition

• EC 50 of Triclosan

Discussion

Conclusion

Special Thanks

Acknowledgments

3. Introduction

This study was conducted in order to determine the effects of triclosan on marine algae

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

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

This water may be released into the marine environment

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

4. Background Information on Triclosan

2,4,4'-trichloro-2-hydroxydiphenylether

Trade names: Irgasan Registered Trademark DP 300,Irgasan Registered Trademark PE 30,Irgasan Registered Trademark PG 60,and Irgacide Registered Trademark LP 10 5. Background Information on Triclosan Cont.

Biocidal Effects of Triclosan

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

Algicide: a chemical substance that protects the water from infestation and growth of algae

• Cell wall lysis

• Inhibits Fatty-acid synthesis

6. Background Information Cont.

Triclosan Uses:

Found in of liquid soaps and of

bar soaps

Component of:

• Antibacterial soaps, toothpaste, sponges, house cleaners, and product containers

• Hospital materials, carpeting, sports accessories, and childrens toys

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

7. Background Information Cont.

Triclosan Levels Reported in Surface Waters

Surface Water Levels (g/L) Source U.S. Rivers 0.24 - 2.7 Kolpin DW., et al. 2002 U.S. Geological Survey 0.8 (average) Tromso-Sound, Norway 0.2 - 2.4 Weigel s., et al. 2004 Bayou St. John Lake (New Orleans, Louisiana) 29 ng/L Boyd,et al.2004 Swiss Rivers 11 - 98 ng/L Balmer, 2005 8. Background Information Cont. Triclosan Levels in Waste Water Waste water Levels ( g/L) Source Primary Effluent 3.4 - 8.0McAvoy, DC et al. 2002 Final Effluent: Activated Sludge Treatment 0.20 - 0.41 Weigel S. et al. : 2004 Trickling Filter Treatment 1.6 - 2.7 Switzerland Effluents 42 - 213 mg/L Balmer 2005 9. Background Information Contd.

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

At the base of grazing food chain in the aquatic environment

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

Supply oxygen through photosynthesis

Little information is available on

triclosan effects on algae (Orviset al.2002, Balmer 2005 ,Tatarazakoet al.2004)

10. Algae Used in this Study Tetraselmis chuii Nannochloropsis oculata Chaetocerus gracilis Isochrysis galbana Rhodomonas salina 11. Algae Information Algae Class Size (m) Motile Flagella Isochrysis Prymnesiophyceae 4 8 yes 2 flagella Tetraselmis Chlorophyceae 12 -14 4 flagella Nannochloropsis Eustigmatophyceae 1.5 - 2.5 no none Rhodomonas Cryptophyceae 16 30 yes Chaetocerus Baccillariophyceae 12 - 14 no 12. The Effects of Triclosan on Various Organisms(Balmer 2005, Russel 2004, Orvoset al.2002) Organism L(E) 50 Algae Freshwater algae Anabaena flos-aquae 0.97 g/L Scenedesmus subspicatus 2. 8 g/L Selenastrum capricornutum 4.46 g/L Navicula pelliculosa 19.1 g/L Marine Algae Skeletonema costatum > 66.0 g/L Fish Danio rerioandOryzais latipes < 300 g/L Pimephales promelas 260 g/LLepomis macrochirus 370 g/L Seamonkeys Artemia 2000 g/L Bacteria Methicillin-resistant Staphylococci 0.1 2 mg/L E. coliandK. pueumoniae 5,000 g/L 13. Objectives

To determine the effects of Triclosan on selected marine algae species

To estimate the EC 50values of Triclosan for five marine algae species

14. Experimental Design Treatment (ug/L) Replicate # Temperature (average) Salinity Light (lux) Control 0 3 25 o C 25ppt 2,200 1 0.2 3 25 o C 25ppt 2,200 2 1.0 3 25 o C 25ppt 2,200 3 5.0 3 25 o C 25ppt 2,200 4 25.0 3 25 o C 25ppt 2,200 5 125 3 25 o C 25ppt 2,200 6 250 3 25 o C 25ppt 2,200 7 500 3 25 o C 25ppt 2,200 Solvent Control 7.2% Ethyl Alcohol 325 o C 25ppt 2,200 15. Data Analysis

Algae growth rate was calculated using the equation:

U= (LnN t LnN o )/( t t o )

where:U= growth rate

N t = Algae #/mL at time t

N o= Algae #/mL at time 0

t= Sample time for counting algae #

t o= Initial time of the treatment.

Percent Inhibition of Algae by Triclosan was calculated as:

%Inhibition = ( U ctU tox )/U ct* 100

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

U ct= Growth rate in the control

EC 50values were estimated for each algae species

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

16. Results: Effects of Triclosan on Algae Growth

Each triclosan treatment for each algae species experienced growth

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

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

17. Isochrysis galbana 18. Tetraselmis chuii 19. Nannochloropsis oculata 20. Rhodomonas salina 21. Chaetocerus gracilis 22. Results: Effects of Triclsoan on Algae Growth Rates

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

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

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

Not all the growth rates for each day were significantly different

23. Isochrysis galbana 24. Isochrysisgrowth rate ANOVA table 24 hours Source of Variation Degrees ofFreedom Sum of Squares MeanSquare 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-Square CoefficientVar Root MSE YIELDMean 0.995692 13.61679 0.099503 0.730741 25. Means with the same letter are not 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 26. Isochrysisgrowth rate ANOVA table 48 hours Source of Variation Degrees ofFreedom Sum of Squares Mean Square F-Value Pr > F Model 10 8.75095556 0.87509556 198.57 F Model 10 2.49982222 0.24998222 1097.48 F Model 10 1.23659259 0.12365926 763.15 < F Model 10 10.17803704 1.01780370 81.79 < 0.0001 Error 16 0.1991037 0.01244398 Total 26 10.37714074 R-Square Coefficient Var Root MSE YIELD Mean 0.980813 55.36618 0.111553 0.201481 34. Means with the same letter are not significantly differentt Grouping Mean N Treatment A 0.68000 3 Control B A 0.60667 3 Treatment 1 B 0.51000 3 Treatment 2 B 0.46333 3 Treatment 3 C 0.24333 3 Treatment 4 C 0.14667 3 Treatment 5 D -0.15667 3 Treatment 6 E -1.36667 3 Treatment 7 A 0.68667 3 Treatment 8 35. Tetraselmisgrowth rate ANOVA table 48 hours Source of Variation Degrees of Freedom Sum of Squares Mean Square F-Value Pr > F Model 10 1.02720000 0.10272000 2.89 0.0287 Error 16 0.56946667 0.03559167 Total 26 1.59666667 R-Square Coefficient Var Root MSE YIELD Mean 0.643340 44.68205 0.188658 0.422222 36. Means with the same letter are not significantly differentt Grouping Mean N Treatment A 0.6200 3 Control A 0.5933 3 Treatment 1 B A 0.5567 3 Treatment 2 B A 0.5400 3 Treatment 3 B A 0.4567 3 Treatment 4 B 0.4667 3 Treatment 5 B C 0.2367 3 Treatment 6 C -0.0267 3 Treatment 7 A 0.3467 3 Treatment 8 37. Tetraselmisgrowth rate ANOVA table 72 hours Source of Variation Degrees of Freedom Sum of Squares Mean Square F-Value Pr > F Model 10 1.05683704 0.10568370 3.38 0.0149 Error 16 0.50061481 0.03128843 Total 26 1.55745185 R-Square Coefficient Var Root MSE YIELD Mean 0.678568 30.71321 0.176885 0.575926 38. Means with the same letter are not significantly different t Grouping Mean N Treatment A 0.8567 3 Control B A 0.7700 3 Treatment 1 B A 0.6933 3 Treatment 2 B A C 0.6300 3 Treatment 3 B A C 0.5733 3 Treatment 4 B C 0.5100 3 Treatment 5 C 0.3767 3 Treatment 6 0.1600 3 Treatment 7 A 0.6133 3 Treatment 8 39. Tetraselmisgrowth rate ANOVA table 96 hours Source of Variation Degrees of Freedom Sum of Squares Mean Square F-Value Pr > F Model 10 0.77628889 0.07762889 3.08 0.0221 Error 16 0.40377778 0.02523611 Total 26 1.18006667 R-Square Coefficient Var Root MSE YIELD Mean 0.657835 21.69543 0.158859 0.73222 40. Means with the same letter are not significantly differentt Grouping Mean N Treatment A 0.8967 3 Control A 0.8700 3 Treatment 1 A 0.8500 3 Treatment 2 A 0.8067 3 Treatment 3 A 0.7867 3 Treatment 4 A 0.7433 3 Treatment 5 A 0.6600 3 Treatment 6 A 0.3067 3 Treatment 7 B 0.6700 3 Treatment 8 41. Nannochloropsis oculata 42. Nannochloropsisgrowth rate ANOVA table 24 hours Source of Variation Degrees of Freedom Sum of Squares Mean Square F-Value Pr > F Model 10 5.89219259 0.58921926 45.75 < 0.0001 Error 16 0.20605926 0.0128787 Total 26 6.09825185 R-Square Coefficient Var Root MSE YIELD Mean 0.966210 17.30140 0.113484 0.655926 43. Means with the same letter are not significantly differentt Grouping Mean N Treatment A 1.32667 3 Control B 0.92667 3 Treatment 1 C B 0.85000 3 Treatment 2 C 0.71000 3 Treatment 3 D 0.51333 3 Treatment 4 E 0.29333 3 Treatment 5 F -0.08333 3 Treatment 6 F 0.09333 3 Treatment 7 A 1.27333 3 Treatment 8 44. Nannochloropsisgrowth rate ANOVA table 48 hours Source of Variation Degrees of Freedom Sum of Squares Mean Square F-Value Pr > F Model 10 4.89813333 0.48981333 4.00 0.0069 Error 16 1.95913333 0.12244583 Total 26 6.85726667 R-Square Coefficient Var Root MSE YIELD Mean 0.714298 32.77111 0.349923 1.067778 45. Means with the same letter are not significantly differentt Grouping Mean N Treatment A 1.6300 3 Control B A 1.5067 3 Treatment 1 B A 1.3900 3 Treatment 2 B A C 1.1600 3 Treatment 3 B A C 1.1033 3 Treatment 4 B C 0.9500 3 Treatment 5 C 0.5600 3 Treatment 6 0.2100 3 Treatment 7 A 1.1000 3 Treatment 8 46. Nannochloropsisgrowth rate ANOVA table 76 hours Source of Variation Degrees of Freedom Sum of Squares Mean Square F-Value Pr > F Model 10 4.89813333 0.48981333 4.00 0.0069 Error 16 1.95913333 0.12244583 Total 26 6.85726667 R-Square Coefficient Var Root MSE YIELD Mean 0.714298 32.77111 0.349923 1.067778 47. Means with the same letter are not significantly different t Grouping Mean N Treatment 1.63000 3 Control B 1.5067 3 Treatment 1 B 1.3900 3 Treatment 2 B C 1.1600 3 Treatment 3 B C 1.1033 3 Treatment 4 B C 1.1000 3 Treatment 5 D C 0.9500 3 Treatment 6 D 0.5600 3 Treatment 7 B A C 0.2100 3 Treatment 8 48. Nannochloropsisgrowth rate ANOVA table 96 hours Source of Variation Degrees of Freedom Sum of Squares Mean Square F-Value Pr > F Model 10 2.04006667 0.20400667 20.12 F Model 10 3.28800000 0.32880000 31.99 F Model 10 1.68814815 0.16881481 2.29 0.0675 Error 16 1.17988148 0.07374259 Total 26 2.86802963 R-Square Coefficient Var Root MSE YIELD Mean 0.588609 35.55775 0.271556 0.763704 54. Means with the same letter are not significantly different t Grouping Mean N Treatment A 1.1267 3 Control A 1.05333 3 Treatment 1 A 0.7267 3 Treatment 2 B A 0.8233 3 Treatment 3 B A 0.7600 3 Treatment 4 B A 0.7267 3 Treatment 5 B C 0.5567 3 Treatment 6 C 0.2333 3 Treatment 7 B A 0.8667 3 Treatment 8 55. Rhodomonasgrowth rate ANOVA table 72 hours Source of Variation Degrees of Freedom Sum of Squares Mean Square F-Value Pr > F Model 10 1.36481481 0.13648148 3.80 0.0087 Error 16 0.57394815 0.03587175 Total 26 1.93876296 R-Square Coefficient Var Root MSE YIELD Mean 0.703962 27.97460 0.189398 0.677037 56. Means with the same letter are not significantly different t Grouping Mean N Treatment A 0.9300 3 Control A 0.8933 3 Treatment 1 A 0.8367 3 Treatment 2 A 0.8100 3 Treatment 3 B A 0.7167 3 Treatment 4 B A 0.6300 3 Treatment 5 B C 0.4567 3 Treatment 6 C 0.1800 3 Treatment 7 B A 0.6400 3 Treatment 8 57. Rhodomonasgrowth rate ANOVA table 96 hours Source of Variation Degrees of Freedom Sum of Squares Mean Square F-Value Pr > F Model 10 0.97977037 0.09797704 3.17 0.0196 Error 16 0.49503704 0.03093981 Total 26 1.47480741 R-Square Coefficient Var Root MSE YIELD Mean 0.664338 29.90695 0.175897 0.599148 58. Means with the same letter are not significantly different t Grouping Mean N Treatment A 0.8600 3 Control B A 0.7567 3 Treatment 1 B A C 0.6900 3 Treatment 2 B A C 0.6567 3 Treatment 3 B A C 0.6000 3 Treatment 4 B C 0.5467 3 Treatment 5 D C 0.4167 3 Treatment 6 D 0.1667 3 Treatment 7 B A C 0.6000 3 Treatment 8 59. Chaetocerus gracilis 60. Chaetocerusgrowth rate ANOVA table 24 hours Source of Variation Degrees of Freedom Sum of Squares Mean Square F-Value Pr > F Model 10 35.12774815 3.51277481 34.51 < 0.0001 Error 16 1.62859259 0.010178704 Total 26 36.75634074 R-Square Coefficient Var Root MSE YIELD Mean 0.955692 32.94112 0.319041 0.968519 61. Means with the same letter are not significantly different t Grouping Mean N Treatment A 2.0967 3 Control B A 1.7767 3 Treatment 1 B A 1.5867 3 Treatment 2 B C 1.2633 3 Treatment 3 D C 0.9100 3 Treatment 4 D 0.6700 3 Treatment 5 E 0.0433 3 Treatment 6 F -1.6967 3 Treatment 7 A 2.0667 3 Treatment 8 62. Chaetocerusgrowth rate ANOVA table 48 hours Source of Variation Degrees of Freedom Sum of Squares Mean Square F-Value Pr > F Model 10 4.85472593 0.48547259 2.86 0.0298 Error 16 2.71705926 0.16981620 Total 26 7.57178519 R-Square Coefficient Var Root MSE YIELD Mean 0.641160 50.82853 0.412088 0.810741 63. Means with the same letter are not significantly different t Grouping Mean N Treatment A 1.3067 3 Control B A 1.1767 3 Treatment 1 B A 1.0633 3 Treatment 2 B A 0.9633 3 Treatment 3 B A 0.9000 3 Treatment 4 B A 0.8033 3 Treatment 5 B 0.5367 3 Treatment 6 C -0.2133 3 Treatment 7 B A 0.7600 3 Treatment 8 64. Chaetocerusgrowth rate ANOVA table 72 hours Source of Variation Degrees of Freedom Sum of Squares Mean Square F-Value Pr > F Model 10 1.06761482 0.10676148 3.04 0.0233 Error 16 0.56248148 0.03515509 Total 26 1.63009630 R-Square Coefficient Var Root MSE YIELD Mean 0.654940 29.31336 0.187497 0.639630 65. Means with the same letter are not significantly different t Grouping Mean N Treatment A 0.9167 3 Control B A 0.8300 3 Treatment 1 B A 0.7700 3 Treatment 2 B A 0.6967 3 Treatment 3 B A 0.6500 3 Treatment 4 B A 0.6200 3 Treatment 5 B 0.5600 3 Treatment 6 C 0.5233 3 Treatment 7 B 0.5600 3 Treatment 8 66. Chaetocerusgrowth rate ANOVA table 96 hour Source of Variation Degrees of Freedom Sum of Squares Mean Square F-Value Pr > F Model 10 0.65040000 0.06504000 2.63 0.0412 Error 16 0.39600000 0.02475000 Total 26 1.04640000 R-Square Coefficient Var Root MSE YIELD Mean 0.621560 27.76259 0.157321 0.56667 67. Means with the same letter are not significantly different t Grouping Mean N Treatment A 0.7800 3 Control B A 0.7433 3 Treatment 1 B A C 0.6733 3 Treatment 2 B A C 0.5967 3 Treatment 3 B A C 0.5533 3 Treatment 4 B D C 0.5033 3 Treatment 5 D C 0.4500 3 Treatment 6 D 0.2367 3 Treatment 7 B A C 0.5633 3 Treatment 8 68. Results: Percent Inhibition

The Percent Inhibition decreased as the time period increased

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

69. Percent Inhibition:Isochrysis Percent Inhibition Initial Day 1 Day 2 Day 3 Day 4 Control (0g/L) n/a n/a n/a n/a n/a Treatment 1 (0.2g/L)n/a 11.27 22.10 11.62 12.28 Treatment 2 (1.0g/L)n/a 30.40 38.49 21.35 19.11 Treatment 3 (5.0g/L)n/a 44.80 50.69 29.52 28.48 Treatment 4 (25g/L) n/a 66.73 58.22 40.98 36.59 Treatment 5 (125g/L) n/a 83.19 63.75 49.47 41.86 Treatment 6 (250g/L) n/a 100.60 79.30 67.29 47.90 Treatment 7 (500g/L) n/a 208.44 166.54 108.72 86.96 Treatment 8 (ethyl alcohol control) n/a -0.65 -3.10 14.09 0.72 70. Percent Inhibition:Tetraselmis Percent Inhibition Initial Day 1 Day 2 Day 3 Day 4 Control (0g/L) n/a n/a n/a n/a n/a Treatment 1 (0.2g/L)n/a 12.55 4.13 14.98 4.20 Treatment 2 (1.0g/L)n/a 26.30 7.89 20.47 7.27 Treatment 3 (5.0g/L)n/a 33.20 14.67 30.24 10.73 Treatment 4 (25g/L) n/a 64.64 25.66 35.64 14.12 Treatment 5 (125g/L) n/a 79.14 26.39 42.91 18.79 Treatment 6 (250g/L) n/a 122.51 71.78 61.49 33.29 Treatment 7 (500g/L) n/a 432.19 288.66 90.46 78.05 Treatment 8 (ethyl alcohol control) n/a -1.59 -0.54 -2.60 0.57 71. Percent Inhibition: Nannochloropsis Percent Inhibition Initial Day 1 Day 2 Day 3 Day 4 Control (0g/L) n/a n/a n/a n/a n/a Treatment 1 (0.2g/L) n/a 30.70 10.46 4.95 2.54 Treatment 2 (1.0g/L) n/a 35.82 16.51 7.85 4.46 Treatment 3 (5.0g/L) n/a 46.84 30.71 12.78 6.76 Treatment 4 (25g/L) n/a 60.74 34.51 20.63 13.38 Treatment 5 (125g/L) n/a 77.83 44.57 43.61 20.90 Treatment 6 (250g/L) n/a 106.07 75.01 55.89 39.63 Treatment 7 (500g/L) n/a 89.70 93.71 95.22 95.37 Treatment 8 (ethyl alcohol control) n/a -49.15 -21.24 -9.21 -9.08 72. Percent Inhibition: Rhodomonas Percent Inhibition Initial Day 1 Day 2 Day 3 Day 4 Control (0g/L) n/a n/a n/a n/a n/a Treatment 1 (0.2g/L)n/a 4.10 22.20 4.82 14.02 Treatment 2 (1.0g/L)n/a 16.60 28.75 10.38 21.23 Treatment 3 (5.0g/L)n/a 20.02 30.98 14.93 22.55 Treatment 4 (25g/L) n/a 31.18 34.81 25.45 33.28 Treatment 5 (125g/L) n/a 39.50 36.92 32.12 36.87 Treatment 6 (250g/L) n/a 68.58 57.83 60.78 56.97 Treatment 7 (500g/L) n/a 87.38 92.06 90.54 91.51 Treatment 8 (ethyl alcohol control) n/a -13.41 -9.31 1.15 -1.61 73. Percent Inhibition:Chaetocerus Percent Inhibition Initial Day 1 Day 2 Day 3 Day 4 Control (0g/L) n/a n/a n/a n/a n/a Treatment 1 (0.2g/L)n/a 0.73 11.37 10.10 9.02 Treatment 2 (1.0g/L)n/a 11.43 21.11 16.12 18.73 Treatment 3 (5.0g/L)n/a 29.20 27.17 25.50 26.78 Treatment 4 (25g/L) n/a 49.11 31.30 28.84 32.09 Treatment 5 (125g/L) n/a 62.57 41.25 31.98 37.08 Treatment 6 (250g/L) n/a 96.98 65.26 46.86 46.52 Treatment 7 (500g/L) n/a 181.22 125.25 95.00 83.40 Treatment 8 (ethyl alcohol control) n/a -15.51 0.30 8.33 -1.95 74. EC 50 of Triclosan

Overall, as the time period increased, the EC 50increased for each algae species

This was due to the reduction in triclosans strength as time went on

More triclosan was required for the same effect

75. Isochrysis galbana 76. The time Period and itscorresponding EC 50value Time Period EC 50value Day 1 2.10 g/mL Day 2 2.44 g/mL Day 3 3.97 g/mL Day 4 4.81 g/mL 77. Tetraselmis chuii 78. The time period and itscorresponding EC 50value Time Period EC 50value Day 1 10.0 g/mL Day 2 77.7 g/mL Day 3 83.8 g/mL Day 4 >500 g/mL 79. Nannochloropsis oculata 80. The time period and itscorresponding EC 50value Time Period EC 50value Day 1 2.10g/mL Day 2 2.44g/mL Day 3 3.97g/mL Day 4 4.81g/mL 81. Rhodomonas salina 82. The time period and itscorresponding EC 50value Time Period EC 50value Control (0g/L) 109 g/mL Treatment 1 (0.2g/L)98 g/mL Treatment 2 (1.0g/L)168 g/mL Treatment 3 (5.0g/L)128 g/mL 83. Chaetocerus gracilis 84. The time period and itscorresponding EC 50value Time Period EC 50value Control (0g/L) 46.2 g/mL Treatment 1 (0.2g/L)68 g/mL Treatment 2 (1.0g/L)183 g/mL Treatment 3 (5.0g/L)162 g/mL 85. Discussion: Compare Marine to Freshwater Algae Time Period Marine Algae Species EC 50values Isochrysis Nannochloropsis Rhodomonas Chaetocerus Tetraselmis 24hr 10.8 6.3 109 46.2 10.0 48hr 68.5 72 98 68 77.7 72hr 182 165 168 183 83.8 96hr 164 360 128 162 >500 Freshwater Algae Species L(E) 50values Navicula pelliculosa Selenastrum capricornutum Scenedesmus subspicatus Skeletonema costatum Anabaena flosaquae 96hr 19.1 g/L 4.46 g/L 2. 8 g/L > 66.0 g/L 0.97 g/L 86. Discussion: Comparison of Algae, cont. (Orvoset. al2002).

Triclosan toxicity is less for marine algae than freshwater algae

• Marine algae EC 50ranges from 128 to > 500 for the 96hr time period

• Freshwater algae EC 50ranges from 0.97 to 19.1 for the 96hr time period

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

Not relating to the salinity of the environment

87. Discussion: Comparison of EC 50and L(E) 50Values of Other Organisms Time Period Marine Algae Species EC 50values Isochryis galbana Nannochloropsis oculata Rhodomonas salina Chaetocerus gracilis Tetraselmis chuii 24hr 10.8 6.3 109 46.2 10.0 48hr 68.5 72 98 68 77.7 72hr 182 165 168 183 83.8 96hr 164 360 128 162 >500 Organisms L(E) 50valuesFish Seamonkeys Bacteria Danio rerio Oryzais latipes Artemia Staphylococci Pimephales promelas Lepomis macrochirus E. coli K. pueumoniae Pseudomonas aeruginosa < 300 g/L 2000 g/L 100-300 g/L 260 g/L 370 g/L 5,000 g/L 300,000-1,000,000 g/L 88. Discussion: Comparison of Organisms, cont.

Triclosan addition will have an effect on algae species

This addition could indirectly effect algae-dependent species

The difference of the effects of triclosan may be caused by

• the size of each organism

• their physiological composition

The EC 50range of triclosan in algae species is the range of these species: water fleaDaphnia magna , and the fishDanio rerio, Oryzais latipes, Lepomismacrochirus,andPimephales promelas

The EC 50for these species were much different than algae species: brine shrimp,Artemia , and the bacteriaE. coli ,K.pueumoniae , andPseudomonas aeruginosa

89. Discussion: The Implication of Triclosan Toxicity

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

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

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

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

90. Discussion: Comparison of the EC 50Values among the Algae Species Time Period Marine Algae Species EC 50values Isochryis galbana Nannochloropsis oculata Rhodomonas salina Chaetocerus gracilis Tetraselmis chuii 24hr 10.8 6.3 109 46.2 10.0 48hr 68.5 72 98 68 77.7 72hr 182 165 168 183 83.8 96hr 164 360 128 162 >500 91. Discusssion: Visual comparison of the EC 50(g/L) values 92. Conclusion

In conclusion, this study,The Effects of Triclosan on Marine Algae , determined the effects of triclosan on the marine algae,Tetraselmis ,Nannochloropsis ,Chaetocerus ,Isochyrsis , andRhodomonasover a 96 hour (4 day) trials

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

After calculating and comparing the EC 50of triclosan on the algae species triclosan was determined to least affectTetraselmisand most affectRhodomonas

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

93. Special Thanks

Dr. Paulinus Chigbu, for taking me under his wing

Marcus Sims

Dr. Izevbigie

Dr. Hwang

Dr. Begonia

Dr. Hardy

Ms. Simpson

Ms. Sandra Hall

Alex Suchar

Eric Evans

Quanisha

Bryon Gipson

94. Acknowledgements 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.