Toxicity Assessment Of The Antimicrobial Triclocarban Using Sub-Lethal Behaviour And ...

214 Pages·2014·1.8 MB·English

by  Melanie Raby

Ryerson University
Digital Commons @ Ryerson
Theses and dissertations
1-1-2013
Toxicity Assessment Of The Antimicrobial
Triclocarban Using Sub-Lethal Behaviour And
Reproduction Endpoints
Melanie Raby
Ryerson University
Follow this and additional works at:http://digitalcommons.ryerson.ca/dissertations
Part of theEnvironmental Sciences Commons
Recommended Citation
Raby, Melanie, "Toxicity Assessment Of The Antimicrobial Triclocarban Using Sub-Lethal Behaviour And Reproduction Endpoints"
(2013).Theses and dissertations.Paper 2017.
This Thesis is brought to you for free and open access by Digital Commons @ Ryerson. It has been accepted for inclusion in Theses and dissertations by
an authorized administrator of Digital Commons @ Ryerson. For more information, please [email protected].
TOXICITY ASSESSMENT OF THE ANTIMICROBIAL TRICLOCARBAN USING
SUB-LETHAL BEHAVIOUR AND REPRODUCTION ENDPOINTS

by
Melanie Raby
Bachelor of Science (Honours)
University of Waterloo, 2011

A thesis
presented to Ryerson University

in partial fulfillment of the
requirement for the degree of
Master of Applied Science
in the Program of
Environmental Applied Science and Management

Toronto, Ontario, Canada, 2013

© MELANIE RABY, 2013
Author’s Declaration

I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including
any required final revisions, and accepted by my examiners.

I authorize Ryerson University to lend this thesis to other institutions or individuals for the
purpose of scholarly research.

I further authorize Ryerson University to reproduce this thesis by photocopying or by other
means, in total or in part, at the request of other institutions or individuals for the purpose of
scholarly research.

I understand that my thesis may be made electronically available to the public.

ii
Abstract
Toxicity assessment of the antimicrobial triclocarban using sub-lethal behaviour and
reproduction endpoints

Master of Applied Science, 2013
Melanie Raby
Environmental Applied Science and Management
Ryerson University

Aquatic environments have long been used as disposal sites for domestic and industrial
wastes, resulting in increasing chemical contamination, decreased water quality, and concern for
ecosystem health and drinking water sources. This study utilized bioassays, the “golden
standard” method to measure biological impact, to assess the toxicity of the widely found surface
water contaminant, the antimicrobial triclocarban. Culturing protocols were implemented to
provide healthy, age-synchronized organisms for bioassays. Behaviour and reproduction were
demonstrated as useful endpoints while refining these methods using 4-chlorophenol and were
successfully implemented in the toxicity assessment of triclocarban. While no sub-lethal
behavioural impact was seen, 10.0 ppb triclocarban was found to delay reproduction in Daphnia
magna. This delay could result in population, community, and ecosystem-level responses.

iii
Acknowledgements
This project would not have been possible without the guidance, encouragement, and
input of so many people. First, I would like to thank Dr. Lynda McCarthy who saw an
ecotoxicologist in a naive biochemist. Thank you for the opportunity and encouragement
throughout this journey. Thank you to Karen Puddephatt for teaching me the ropes, for your
friendship and for being my knitting buddy. To Dr. Jorge Loyo, my partner in crime, thank you
for helping me with “the water problem” and most importantly for your friendship and support. I
must also extend my gratitude to fellow graduate students Mark Tiley and Daniel Johnson and
research assistants Alicia Falls and Laura Taylor for their friendship and help in the lab.
Additionally, I would like to thank Dave Poirier and Richard Chong-Kit of the MOE for
their invaluable advice and their (many) gifts of Lumbriculus and Chlorella, along with Dr.
Warren Norwood of CCIW for his helpful Hyalella azteca culturing advice. I would also like to
thank Dr. Andrew Laursen for his guidance with my data analysis. Finally, I would like to thank
Dr. Michal Bardecki, Dr. Stephen Wylie, and Dr. Gideon Wolfaardt for their roles as examiners
on my defence committee.
I would like to thank my parents, Laura and Rob, my sisters, Lisa and Christine, my
grandmother Clara, as well as the Staples family for their love and support throughout this
journey. Finally, a special thank you to my partner Robbie for his patience, encouragement and
love.

iv
Table of Contents

1.0 INTRODUCTION ........................................................................................................................ 1
1.1 Overview ...................................................................................................................................... 1
1.2 Objectives ..................................................................................................................................... 2
1.3 Importance of Bioassays............................................................................................................... 2
1.3.1 Review of Bioassay Organisms in WWTP Effluent Toxicity Research ............................... 3
1.3.2 Single-Species Bioassays ...................................................................................................... 4
1.4 Protocol Development .................................................................................................................. 4
1.4.1 Reference Toxicants .............................................................................................................. 5
1.5 Bioassay Endpoints ...................................................................................................................... 9
1.5.1 Rationale for Selection of Test Endpoints ............................................................................. 9
1.5.2 Behaviour ............................................................................................................................ 10
1.5.3 Reproduction ....................................................................................................................... 19
1.6 Bioassay Organisms ................................................................................................................... 19
1.6.1 Rationale for Selection of Bioassay Organisms .................................................................. 19
1.6.2 Daphnia magna ................................................................................................................... 20
1.6.3 Hyalella azteca .................................................................................................................... 27
1.6.4 Lumbriculus variegatus ....................................................................................................... 33
1.6.5 Lemna minor ........................................................................................................................ 37
1.7 Triclocarban ................................................................................................................................ 41
1.7.1 Environmental Impact of Triclocarban ................................................................................ 42
2.0 METHODS ..................................................................................................................................... 45
2.1 General ....................................................................................................................................... 45
2.1.1 Dechlorination of Municipal Drinking Water ..................................................................... 45
2.1.2 Glassware Washing Procedure ............................................................................................ 46
2.1.3 General Bioassay Conditions............................................................................................... 46
2.2 Culturing and Age Synchronization ........................................................................................... 46
2.2.1 Daphnia magna ................................................................................................................... 47
2.2.2 Hyalella azteca .................................................................................................................... 52
2.2.3 Lumbriculus variegatus ....................................................................................................... 56
2.2.4 Lemna minor ........................................................................................................................ 58
2.3 Positive Toxicant ........................................................................................................................ 60
v
2.3.1 Behaviour Development ...................................................................................................... 60
2.3.2 Behaviour ............................................................................................................................ 62
2.3.3 Life Cycle ............................................................................................................................ 63
2.4 Triclocarban ................................................................................................................................ 65
2.4.1 Behaviour ............................................................................................................................ 65
2.4.2 Life Cycle ............................................................................................................................ 65
2.5 Statistical Analysis ..................................................................................................................... 65
3.0 RESULTS AND DISCUSSION ..................................................................................................... 67
3.1 Culturing ..................................................................................................................................... 67
3.1.1 Dechlorination of Municipal Drinking Water ..................................................................... 67
3.1.2 Daphnia magna ................................................................................................................... 69
3.1.3 Hyalella azteca .................................................................................................................... 73
3.1.4 Lumbriculus variegatus ....................................................................................................... 75
3.1.5 Lemna minor ........................................................................................................................ 76
3.2 Bioassay Method Refinement ..................................................................................................... 76
3.2.1 Behaviour ............................................................................................................................ 77
3.2.2 Life Cycle .......................................................................................................................... 100
3.2.3 Summary: Behaviour and Reproduction ........................................................................... 104
3.3 Triclocarban .............................................................................................................................. 106
3.3.1 Behaviour .......................................................................................................................... 106
3.3.2 Life Cycle .......................................................................................................................... 115
3.3.3 Summary: TCC .................................................................................................................. 119
4.0 OVERALL SUMMARY AND FUTURE DIRECTIONS ........................................................... 121
4.1 Overall Summary ..................................................................................................................... 121
4.2 Future Directions ...................................................................................................................... 122
REFERENCES ................................................................................................................................... 171

vi
List of Tables
Table 1 Recently measured TCC concentrations in surface and wastewaters ............................................ 43
Table 2 Select toxicity data for freshwater organisms ................................................................................ 44
Table 3 Mean Hyalella azteca juvenile produced: surviving adult ratio for each brood stock, A-D .......... 74
Table 4 Daphnia magna reference versus stress behaviour for three behaviour categories ....................... 78
Table 5 Hyalella azteca reference versus stress behaviour for three behaviour categories ........................ 85
Table 6 Lumbriculus variegatus reference versus stress behaviour for three behaviour categories ........... 92
Table 7 Lemna minor reference versus stress appearances for three apperance categories ........................ 96
Table 8 L. minor growth rate and % growth inhibition after 7 days in 4-chlorophenol............................ 103
Table 9 Lemna minor growth rate and % growth inhibition after 7 days in TCC .................................... 118

vii
List of Figures
Figure 1 Example of a mean chart or warning chart (Environment Canada, 1990)...................................... 6
Figure 2 Structure of 4-chlorophenol (Sigma-Aldrich, 2013) ...................................................................... 7
Figure 3 Interrelationships governing behaviour ........................................................................................ 10
Figure 4 Behavioural ecotoxicology and other disciplines (Dell’Omo, 2002) ........................................... 13
Figure 5 Stereoscope image of Daphnia magna (Puddephatt, 2013) ......................................................... 21
Figure 6 An idealized freshwater food web (Dodson and Hanazato, 1995) ............................................... 22
Figure 7 Anatomy of Female Daphnia spp. (Environment Canada, 1990) ............................................... 23
Figure 8 Sexual and asexual (parthenogenic) life cycle of a Daphnia (Ebert, 2005) ................................. 24
Figure 9 Stereoscope image of Hyalella azteca (Puddephatt, 2013) .......................................................... 27
Figure 10 Anatomy of Hyalella azteca (Environment Canada, 1997) ........................................................ 28
Figure 11 Stereoscope image of Lumbriculus variegatus (M. Raby, 2013) ............................................... 33
Figure 12 Lumbriculus variegatus feeding position in paper towel substrate (M. Raby, 2013) ................. 35
Figure 13 Image of Lemna minor (M. Raby, 2013) .................................................................................... 37
Figure 14 Chemical structure of triclocarban (TCC) (Snyder et al., 2010b) .............................................. 41
Figure 15 Daphnia magna Culturing and Toxicity Bioassay Flow-Chart .................................................. 47
Figure 16 Interlocking screens constructed of PVC piping, Nitex screening, and aquarium glue ............. 51
Figure 17 Hyalella azteca Culturing and Toxicity Bioassays Flow Chart .................................................. 53
Figure 18 Lumbriculus variegatus Culturing and Toxicity Bioassay Flow Chart ...................................... 56
Figure 19 Lemna minor Culturing and Toxicity Bioassay Flow Chart ....................................................... 59
Figure 20 Selection of Behaviours and Appearances ................................................................................. 61
Figure 21 Daphnia magna survival at 0, 24 & 48 hours in aerated, filtered & aerated, unfiltered water ... 69
Figure 22 Average Daphnia magna brood size over time .......................................................................... 71
Figure 23 Hyalella azteca ratio juveniles produced: surviving adults in culturing brood stocks over time74
Figure 24 Daphnia magna ethogram .......................................................................................................... 77
Figure 25 Daphnia magna mean swimming score in 4-chlorophenol over time ........................................ 79
Figure 26 Daphnia magna mean number of threshold crossings in 4-chlorophenol over time .................. 81
Figure 27 Daphnia magna mean percent mobilization in 4-chlorophenol over time ................................. 82
Figure 28 Daphnia magna loss in mobilization due to mortality at 24 hours in 4-chlorophenol ............... 83
Figure 29 Hyalella azteca ethogram ........................................................................................................... 84
Figure 30 Hyalella azteca mean locomotion score (/2) in 4-chlorophenol over time ................................. 86
Figure 31 Hyalella azteca mean body score (/2) in 4-chlorophenol over time ........................................... 87
Figure 32 Hyalella azteca mean percent mobilization in 4-chlorophenol over time .................................. 89
viii
Figure 33 Hyalella azteca loss in mobilization due to mortality at 24 hours in 4-chlorophenol ................ 90
Figure 34 Lumbriculus variegatus ethogram .............................................................................................. 91
Figure 35 Lumbriculus variegatus mean locomotion score (/2) in 4-chlorophenol over time.................... 93
Figure 36 Lumbriculus variegatus mean body score (/2) in 4-chlorophenol over time .............................. 94
Figure 37 Lumbriculus variegatus mean percent mobilization in 4-chlorophenol over time ..................... 95
Figure 38 Lemna minor mean chlorosis score (/2) in 4-chlorophenol over time ....................................... 97
Figure 39 Lemna minor mean necrosis score (/2) in 4-chlorophenol over time ......................................... 98
Figure 40 Lemna minor mean colony break-up score (/2) in 4-chlorophenol over time ............................ 99
Figure 41 Daphnia magna mean time-to-first brood (days) for reference and 4-chlorophenol ................ 101
Figure 42 Daphnia magna mean first brood size per surviving adult for reference and 4-chlorophenol . 102
Figure 43 Growth of Lemna minor in 4-chlorophenol over time .............................................................. 103
Figure 44 Daphnia magna mean swimming score in TCC over time ...................................................... 106
Figure 45 Daphnia magna mean number of threshold crossings in TCC over time ................................ 108
Figure 46 Hyalella azteca mean locomotion score (/2) in TCC over time ............................................... 110
Figure 47 Hyalella azteca mean body score (/2) in TCC over time ......................................................... 111
Figure 48 Hyalella azteca mean percent mobilization in TCC over time ................................................. 112
Figure 49 Lumbriculus variegatus mean locomotion score (/2) in TCC over time .................................. 113
Figure 50 Lumbriculus variegatus mean body score (/2) in TCC over time ............................................ 114
Figure 51 Daphnia magna mean brood size (neonates) for treatments with surviving adults .................. 116
Figure 52 Daphnia magna mean time-to-first brood for treatments with surviving adults ...................... 117
Figure 53 Growth of Lemna minor in TCC over time .............................................................................. 118
ix