Treatment of Organophosphorus Insecticides and Phenoxyacetic Acid Herbicides Using Membrane Bioreactor Technology

TREATMENT OF ORGANOPHOSPHORUS INSECTICIDES AND PHENOXYACETIC ACID HERBICIDES USING MEMBRANE BIOREACTOR TECHNOLOGY

AVIK GHOSHDASTIDAR

Thesis submitted in partial fulfillment of the requirements for the Degree of Master of Science (Chemistry)

Acadia University Spring Convocation 2012

This thesis by Avik Ghoshdastidar was defended successfully in an oral examination on April 18, 2012.

The examining committee for the thesis was:

______Dr. Rick Mehta, Chair

______Dr. Michael Earle, External Examiner

______Dr. Amitabh Jha, Internal Examiner

______Dr. Anthony Tong, Supervisor

______Dr. John Murimboh, Head

This thesis is accepted in its present form by the Division of Research and Graduate Studies as satisfying the thesis requirements for the degree Master of Science (Chemistry)

………………………………………….

This thesis by Avik Ghoshdastidar was defended successfully in an oral examination on April 18, 2012.

The examining committee for the thesis was:

Dr. Rick Mehta, Chair

Dr. Michael Earle, External Examiner

Dr. Amitabh Jha, Internal Examiner

Dr. Anthony Tong, Supervisor

Dr. John Murimboh, Head

This thesis is accepted in its present form by the Division of Research and Graduate Studies as satisfying the thesis requirements for the degree Master of Science (Chemistry)

I, Avik Ghoshdastidar, grant permission to the University Librarian at Acadia University to reproduce, loan or distribute copies of my thesis in microform, paper or electronic formats on a non-profit basis. I, however, retain the copyright in my thesis.

______Avik Ghoshdastidar

______Dr. Anthony Tong

______April 18, 2012

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Table of Contents Table of Contents ...... iv List of Tables ...... viii List of Figures ...... x Abstract ...... xii List of Abbreviations ...... xiii Acknowledgements ...... xvii 1 Introduction ...... 1 1.1 Introduction to Pesticides ...... 1 1.1.1 Definitions ...... 1 1.1.2 History of Pesticide Use ...... 1 1.1.3 Pesticide Usage ...... 5 1.1.4 The Economics of Pesticide Use ...... 11 1.1.5 The Chemistry of Organophosphorus Insecticides ...... 14 1.1.6 The Chemistry of PA and Benzoic Acid Herbicides ...... 19 1.1.7 PA and Benzoic Acid Metabolites ...... 22 1.1.8 Environmental Exposure and Pesticide Occurrence ...... 22 1.1.9 Fate of Pesticides in Aquatic Environments ...... 28 1.1.10 Pesticide Toxicology ...... 38 1.1.11 Pesticide Regulations and Legislation ...... 43 1.2 Water Quality ...... 45 1.2.1 Physical Properties ...... 45 1.2.2 Inorganic Non-metallics ...... 47 1.2.3 Organics ...... 49 1.2.4 Microbiological Examination ...... 50 1.3 Pesticide Treatment ...... 50 1.3.1 Conventional Wastewater Treatment ...... 50 1.3.2 Constructed Wetlands ...... 51 1.3.3 Granular Activated Carbon ...... 52 1.3.4 Ozonation ...... 53 1.3.5 Photocatalytic oxidation ...... 54 1.3.6 Fenton’s Reagent ...... 56

1.3.7 Gamma Irradiation ...... 59 1.3.8 Ultrasonic Wave Treatment ...... 59 1.3.9 Bioremediation ...... 59 1.3.10 Metabolite Treatment ...... 61 1.3.11 Pesticide Wastewater Treatment ...... 62 1.4 Membrane Bioreactor ...... 63 1.4.1 Introduction to Membrane Bioreactor ...... 63 1.4.2 The History of Membrane Bioreactors ...... 65 1.4.3 Membrane Fouling ...... 66 1.5 Objective ...... 68 2 Materials and Methods ...... 69 2.1 Membrane Bioreactor ...... 69 2.2 Experimental Setup ...... 69 2.2.1 Wastewater Tank ...... 69 2.2.2 Intermediate Tank ...... 71 2.2.3 Pesticide Tank ...... 71 2.2.4 Peristaltic Pumps and Flow Rates ...... 72 2.2.5 Aeration Spargers ...... 72 2.2.6 Temperature Controls ...... 73 2.2.7 Aeration ...... 73 2.2.8 Sampling ...... 73 2.3 Analytical Methods ...... 74 2.3.1 Solid Phase Extraction ...... 74 2.3.2 Gas Chromatography ...... 74 2.3.3 High Pressure Liquid Chromatography ...... 75 2.3.4 Mass Spectrometry ...... 75 2.3.5 Quantitation...... 75 2.4 Materials and Standards ...... 75 2.4.1 OP Pesticide Experiment ...... 75 2.4.2 PA Pesticide Experiment ...... 77 2.5 Chemical Analysis ...... 78 2.5.1 Solid Phase Extraction ...... 78 2.5.2 Gas Chromatography – Mass Spectrometry ...... 79

2.5.3 Liquid Chromatography – Mass Spectrometry ...... 80 2.5.4 Quality Assurance/Quality Control ...... 81 2.6 Spectrophotometric Analysis ...... 81 2.7 Potentiometric Analysis ...... 82 2.7.1 pH ...... 82 2.7.2 Dissolved Oxygen ...... 83 2.7.3 Conductivity and Total Dissolved Solids (TDS) ...... 83 2.8 Microbiological Analysis ...... 84 2.8.1 Heterotrophic Plate Count ...... 84 2.9 Statistical Analysis ...... 84 3 Organophosphorus Insecticides ...... 85 3.1 MBR Setup ...... 85 3.1.1 Wastewater Tank ...... 85 3.1.2 Wastewater Tank – Gaseous Flushing...... 86 3.1.3 Wastewater characteristics ...... 88 3.1.4 Pesticide Tank ...... 88 3.1.5 MBR acclimatization period ...... 88 3.1.6 MBR Trans-membrane Pressure ...... 92 3.2 Trace Pesticide Analysis Results ...... 92 3.2.1 SPE Method and Data ...... 92 3.2.2 GC/MS Method and Data ...... 93 3.3 Chemical Wastewater Results ...... 96 3.4 Microbiological Results ...... 100 3.5 General Wastewater Parameters ...... 101 3.6 Statistical Analysis ...... 104 4 Phenoxyacetic Acid Herbicides ...... 108 4.1 Trace Herbicide Analysis Results ...... 108 4.1.1 Solid Phase Extraction ...... 108 4.1.2 LC/MS Results ...... 111 4.2 Chemical Wastewater Results ...... 122 4.3 Microbiological Results ...... 125 4.4 General Wastewater Characteristics ...... 126 4.5 Statistical Analysis ...... 129

5 Future Work ...... 132 6 Conclusion ...... 133 7 References ...... 134 8 Appendices ...... 143

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List of Tables

Table 1. Target insecticide (I) and herbicide (H) use across Canada ...... 9

Table 2. Pesticide utilization in Nova Scotia in 2003 ...... 11

Table 3. Solubility of target OP insecticides and PA herbicides ...... 24

Table 4. OP organic carbon-water partition coefficient ...... 29

Table 5. PA Organic carbon-water partition coefficient ...... 30

Table 6. Hydrolysis half-lives of azinphos methyl ...... 31

Table 7. Hydrolysis half-lives of chlorpyrifos ...... 32

Table 8. Hydrolysis half-lives of diazinon...... 33

Table 9. Hydrolysis half-lives of malathion ...... 34

Table 10. Hydrolysis half-lives of phorate ...... 34

Table 11. Hydrolysis half-lives of phenoxyacetic and benzoic acid herbicides ...... 35

Table 12. Photolysis half-lives of OP insecticides ...... 36

Table 13. Photolysis half-lives of phenoxyacetic and benzoic acid herbicides ...... 36

Table 14. Volatilization of pesticides ...... 37

Table 15. Canadian drinking water quality guidelines ...... 43

Table 16. Canadian water quality guidelines for aquatic life ...... 44

Table 17. Canadian water quality guidelines for agricultural water uses ...... 45

Table 18. Wastewater solution composition ...... 70

Table 19. OP pesticide formulations, active ingredients and manufacturers ...... 76

Table 20. Concentration of herbicides in concentrated Weedex ...... 77

Table 21. OP pesticide retention times, quantifier and qualifier ions ...... 80

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Table 22. PA pesticide retention times and quantifier ions ...... 82

Table 23 . COD, nitrogen and phosphorus of wastewater mixture ...... 90

Table 24. OP Pesticide control sample recoveries ...... 93

Table 25. OP Pesticide exponential decay regressions ...... 96

Table 26. Interpretation of Spearman coefficients ...... 105

Table 27. PA pesticide influent and Weedex concentrations...... 108

Table 28. Chromabond® HR-P SPE eluent comparison ...... 109

Table 29. Salting out hydroquinones in SPE ...... 110

Table 30. PA pesticide SPE sorbent comparison ...... 111

Table 31. PA pesticide dissociation constants ...... 111

Table 32. PA pesticide exponential decay regressions ...... 122

List of Figures

Figure 1. Structures of OP insecticides ...... 16

Figure 2. PA and benzoic acid herbicides, and aerobic metabolites ...... 20

Figure 3. OP insecticide toxicities ...... 41

Figure 4. Phenoxyacetic and benzoic acid herbicide toxicities ...... 42

Figure 5. MBR setup and flow rates ...... 72

Figure 6. COD of the wastewater solution over one wastewater cycle ...... 86

Figure 7. pH and temperature prior to the OP pesticide experiment ...... 87

Figure 8. DO and temperature prior to the OP pesticide experiment ...... 91

Figure 9. Total dissolved solids prior to the OP pesticide experiment ...... 92

Figure 10. Total ion count (TIC) for 1-ppm OP pesticides ...... 94

Figure 11. GC/MS analysis of OP Pesticides in MBR Effluent ...... 95

Figure 12. OP pesticide COD MBR in effluent ...... 97

Figure 13. OP pesticide nitrate and total nitrogen MBR effluent concentrations ... 98

Figure 14. OP pesticide ammonia MBR effluent concentration ...... 99

Figure 15. OP pesticide phosphorus MBR effluent concentrations ...... 100

Figure 16. OP pesticide MBR heterotrophic bacteria population ...... 101

Figure 17. OP pesticide MBR effluent pH ...... 102

Figure 18. OP pesticide MBR effluent DO ...... 103

Figure 19. OP pesticide general water quality parameters ...... 104

Figure 20. Total Ion Count (TIC) for 1- ppm PA pesticides ...... 112

Figure 21. Persistence of dicamba in the PA pesticide MBR effluent ...... 113

Figure 22. Concentrations of 3,6-DCSA in MBR PA pesticide effluent ...... 114

Figure 23. Concentrations of 2,5-DCP in the MBR PA pesticide effluent ...... 115

Figure 24. Persistence of Mecoprop in the MBR PA pesticide effluent ...... 116

Figure 25. Concentrations of o-cresol in the MBR PA pesticide effluent ...... 117

Figure 26. Concentrations of 4-chloro-2-methylphenol in the MBR effluent ...... 118

Figure 27. Persistence of 2,4-D in the MBR PA pesticide effluent ...... 119

Figure 28. Concentrations of chlorohydroquinone in the PA pesticide effluent .... 120

Figure 29. Concentration of 2,4-DCP in the MBR PA pesticide effluent ...... 121

Figure 30. PA Pesticide COD MBR effluent concentration ...... 123

Figure 31. PA pesticide ammonia MBR effluent concentration ...... 124

Figure 32. PA pesticide nitrate MBR effluent concentration ...... 125

Figure 33. PA pesticide MBR heterotrophic bacteria population ...... 126

Figure 34. PA pesticide MBR effluent pH ...... 127

Figure 35. PA pesticide MBR effluent TDS ...... 128

Figure 36. PA pesticide MBR effluent DO ...... 129

Abstract

Organophosphorus (OP) insecticides and phenoxyacetic acid (PA) herbicides are among the most widely used chemical classes of pesticides. These pesticides can cause adverse health effects in humans and aquatic organisms, and some are considered endocrine disrupting chemicals and carcinogens. This work aimed to develop a novel method for treating pesticides in water using membrane bioreactor (MBR) technology.

A PA herbicide mix and five OP pesticide formulations were subjected to MBR treatment. Target analytes were extracted from effluent samples using solid phase extraction before separation by gas or liquid chromatography and detection with selective ion monitoring mass spectrometry.

Removal rates for OP targets ranged from 84% to 98%. The herbicide 2,4- dichlorophenoxyacetic acid (2,4-D) was reduced by 99% within ten days. Removal rates of 60% and 65% for persistent herbicides, mecoprop and dicamba, were achieved in 110 days. MBR was demonstrated as an effective, environmentally friendly and compact method pesticide wastewater treatment.

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List of Abbreviations

2,4-D – 2,4-dichlorophenoxyacetic acid

2,4-DCP – 2,4-dichlorophenol

2,4,5-T – 2,4,5-trichlorophenoxyacetic acid

2,5-DCP – 2,5-dichlorophenol

3,6-DCSA – 3,6-dichlorosalicylic acid

4-Cl-2-MP – 4-chloro-2-methylphenol

Ach – Acetylcholine

AChE – Acetylcholinesterase

APCI – Atmospheric pressure chemical ionization

BA – Benzoic acid

BuChE – Butyrylcholinesterase

CFU – Colony forming units

CHQ – Chlorohydroquinone

COD – Chemical oxygen demand

DDT – dichlorodiphenyltrichloroethane

DO – Dissolved oxygen

EC – Emulsifiable concentrate

EI – Electron impact

EPS – Extra-polymeric substances

FTU – Formazin turbidity unit

GC – Gas chromatography

GC-MS – Gas chromatography – mass spectrometer

H - Herbicide

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HPLC – High performance liquid chromatography

HPLC-MS/MS – High performance liquid chromatography - tandem mass spectrometer

HRT – Hydraulic retention time

I – Insecticide

IPM – Integrated Pest Management

IS – Internal standard kG – Kilo-grays

KOC – Organic carbon-water partition coefficient

LC-MS – Liquid chromatography – mass spectrometer

μg – Microgram

μL – Microlitre

μm – Micrometre

M – moles per litre

MAC – Maximum acceptable concentrations

MBR – Membrane Bioreactor

MCPA – 2-methyl-4-chlorophenoxyacetic acid

MCPB – 4-(4-chloro-o-tolyloxy)butyric acid

MCPP – Mecoprop, 2-(4-chloro-2-methylphenoxy) propanoic acid

MDL – Method detection limit mg – Milligram

MHQ – Methylhydroquinone mL – Millilitre

MLSS – Mixed liquor suspended solids

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MLVSS – Mixed liquor volatile suspended solids

MS – Mass spectrometry mS/m – Millisiemens per metre

N.A. – Not applicable/not available

N.D. – Not detected ng – Nanogram

NH4-N – Ammonia nitrogen

NIST – National institute of standards and technology

OC – Organochlorine

OP – Organophosphorus

PA – Phenoxyacetic acid

PAC – Powder activated carbon

PAH – Poly aromatic hydrocarbons

PCP – Pesticide control product

PCCD – polychlorinated dibenzo-p-dioxins

PMRA – Pest Management Regulatory Agency ppb – parts per billion ppm – parts per million

PTFE – Polytetrafluoroethylene, Teflon™

QA/QC – Quality assurance/quality control

ρ – Spearman coefficient

R2 – Coefficient of determination

RSD – Relative standard deviation

SIM – Selective ion monitoring

SPE – Solid phase extraction

SRT – Sludge retention time

SS – Surrogate standard

TDS – Total dissolved solids

TIC – Total ion count

TIPs – Thermally induced phase separation

TNT – Test ‘N Tube™

ULR – Ultra-low range

UV – Ultraviolet

W/m2 – Watts per metre squared

WHO – World Health Organization

WWTP – Wastewater treatment plant

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Acknowledgements

I would like to take this opportunity to thank and acknowledge the many people who have assisted in the completion of this work and who have contributed to my time here at Acadia.

First, I give thanks to the many members of the Tong Research Group for their support, encouragement and friendship: Jasmine, John and Emma for helping me gain my bearings in the lab; Kayleigh and Corbin for conducting a significant amount of work involved with experiments; Dr. Wang and Brian for their company during sampling trips; Jill and Graham for the adventures inside and outside of the lab and finally Martin and Crystal for their encouragement as I finished my thesis. I am indebted to you all.

Laboratory work has been important, but has not been the only thing that has occupied my time. For two years, I was privileged to be a Resident Assistant and later an Assistant Residence Director for a very special building on campus: War Memorial House. Thank you to Dean Martin for hiring a persistent Queen’s Don and believing that he would embody the Acadia spirit for the incoming freshmen and Michelle Johnson for her constant support and her sixth sense of knowing when I needed a big sister. To my residents, who gave me additional purpose to keep me going, and to my fellow RA staff team, my residents who later became RAs and those whose hands I left the building in; I lift my glass and toast “The House that Heroes built and Those who live there still.”

To those who had me as a Teaching Assistant during their time in undergraduate chemistry, I hope that you never forget your units! There will be a generation of Acadia chemists I am sure who will remember the name CAPA Jim; at least I will never forget it. If you were one of the senders of the 800 emails I responded to, let

xvii me say what an opportunity it was for me to learn how to teach in such a unique setting.

To the curlers at the Wolfville Curling Club with whom I rediscovered my love of the sport of curling: Thank you! Monday, Wednesday and Thursday nights will not be the same without you. To my Little Rocks who are constantly falling and bouncing on the ice, you have been a pleasure to coach.

To my 24 Prospect Roommates: Sara, Lauren, Ashley and Sarah: thank you for allowing me in your home. I owe you so much for bringing such joy and laughter into my life. It has been a pleasure for me to watch you grow over these years into such accomplished young women.

To the members of the faculty at this department and Dr. Martin Tango of the School of Engineering, thank you for your advice and support. I have learned so much from you all. My sincerest thanks to Dr. Tong for agreeing to act as my supervisor and for his guidance and support throughout my Masters. I have learned so much in my time here about how to be the best scientist I can be from his excellent example.

Finally, to my parents, Ashim and Sunanda Ghoshdastidar, who have never stopped inspiring me. Thank you for your love and support and for instilling within me a sense of purpose and of service. My accomplishments are only an extension of your own.

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1 Introduction 1.1 Introduction to Pesticides

1.1.1 Definitions

Pests are organisms which cause inconvenience or harm to people, animals or crops.1 The three major groups of pests are insects, fungal pathogens and weeds.

Other pests include: rodents, nematodes, slugs and snails, macroorganisms, viruses, bacteria, and microorganisms not in a living person or animal.2,3

Pest control, then, are those physical, chemical or biological processes designed to destroy, repel or mitigate pests without adversely affecting desired crops or livestock.3 Pesticides are chemical formulations or microorganisms used in pest control, of synthetic or natural origin, that target a wide range of pests.2

1.1.2 History of Pesticide Use

The advent of agriculture 10,000 years ago allowed for the expansion of human populations and development. While agricultural crops are intended for human consumption, insects, animals and microorganisms also thrive on them. Similarly, the nutrients available naturally to field and fibre crops or provided by farmers are attractive to other plants. With industrialization, the growth of human populations necessitated a reduction in crop loss to pests, as the survival of individuals is dependent on the yield from agricultural holdings. Industrialization and the rise of capitalism also required crop loss reductions in order for farmers to remain financially competitive. The field of pesticide science emerged in the mid-

1 nineteenth century with advances in synthetic chemistry and toxicology although it was only in the 1940s and 1950s that chemicals became the principle method of pest management.1

Insects were among the first major targets of chemical pesticide development.

Inorganic chemicals were widely in use as pest control agents before being displaced by synthetic organic chemicals, and included the use of sulphur by the

Romans.2,4 Insecticides isolated from natural products like nicotine and rotenone were also important first steps in insect management.3

In the 1860s, arsenic containing Paris Green was used to kill potato beetles in the

American Midwest. Further developments in inorganic pesticides included other arsenic based compounds including calcium and lead arsenates.4 Many of these arsenical pesticides were later confirmed to be human carcinogens.5

Swiss scientist P.H. Mueller of the Geigy company discovered the insecticidal activity of DDT (dichlorodiphenyltrichloroethane) in 1939 which remains one of the most effective insecticides in eradicating disease vectors but is banned because of its bioaccumulative, persistence and impact on ecological health.4. The promise of DDT in providing cost-effective, broad-spectrum activity, for crops, livestock and human health applications encouraged insecticide investment and research in the following decade. In time, further research into dosage, application timing, pesticide type and application type ensued.1

Organochlorines grew in prominence after the discovery of hexachlorocyclohexane’s insecticidal activity in 1940. The chemistry of the Diels-

Alder reaction spurred a growth in the number of cyclodiene-based insecticides.4

The low mammalian toxicity of organochlorines and broad-spectrum of activity were favoured over OPs and carbamates which exhibited mammalian toxicity.4

Concern over pesticide use increased after the publication of Silent Spring by

Rachel Carson in the 1960s.2 Carson outlined the dangers of pesticides including their persistence or continued presence in the environment long after use, bioaccumulation or the accumulation of organic contaminants in an organism, the difficulty of detecting emerging pesticide contaminants and the impact of pesticides on microbial life and nutrient replenishment in soils. Carson cautioned against the “scorched Earth” approach to pest control and favoured less severe biotic control methods.6 The persistence and occurrence of organochlorines prompted research into pesticide fate and metabolism.7 A shift towards OP and carbamate insecticides began in the 1990s amid growing bioaccumulation and persistence concerns.8

The discovery of Physotigma venenosum cholinesterase inhibiting activity, a hydrolysis catalyzing enzyme for the choline neurotransmitter, prompted research into carbamates and OP compounds, which are two major chemical classes of pesticides. Acetylcholinesterase is responsible for degrading the neural transmitter, acetylcholine. Though organophosphorus chemistry dates back to the

3 synthesis of phosphoric acid in 1820, its toxicity was not fully realized until the

1930s. Commercial insecticides, such as tepp (1937) and dimefox (1940), were produced by IG Farbenindustrie and German scientist Gerhard Schrader.3,9

Research into cholinesterase inhibiting activity and toxic potency of organophosphorus compounds was coopted by the German Ministry of War and directed towards chemical warfare agent development resulting in sarin, tabun and soman synthesis. Research into these compounds in Germany and England during World War II resulted in the synthesis of parathion, which was widely used in Canada until a 2001 phase-out. The recognition of OP mammalian toxicity necessitated the synthesis of compounds of lower toxicities, culminating in the production of the widely used OP insecticide, malathion by American Cynamid.4

By the 1970s, over 200 OP active ingredients were marketed for use as pesticides, with over 50,000 OP compounds produced by the beginning of the 1960s.9

Physical methods of weed control included plowing and hoeing and the earliest chemical method may be the use of sodium chlorate or sodium chloride by conquering armies to prevent growth of crops by “salting the earth.”3 Herbicide chemistry flourished with the advent of 2,4-dichlorphenoxyacetic acid (2,4-D), the first plant growth regulating pesticide synthesized and has been in use for weed control since 1942.1 Along with atrazine, synthesized in 1958, these two herbicides spawned a growth of herbicides that were selective and required low application rates. Its effectiveness was best noted during the Vietnam War, where Agent

Orange, a concentrated mix of 50:50 ( 2,4,5-T (2,4,5-trichlorophenoxyacetic acid)

4 and 2,4-D was used to defoliate trees in the rainforest.10 In the past, there were concerns regarding the presence of polychlorinated dibenzo-p-dioxins (PCCD) in formulations of 2,4-D, especially those which contained carcinogenic 2,4,5-T prompting Agriculture Canada to limit the PCDD concentration in formulations to

10 ppb (parts-per-billion).10

1905 marks the first instance of manipulating genes to resist pathogenic invasion.1

Genetic modification of crops where herbicide resistance is incorporated into a crop’s genetic code allows for broad-spectrum elimination of weeds without damage to desired crops.4 A prime example is the resistance of RoundUp Ready canola to Monsanto’s glyphosate.

1.1.3 Pesticide Usage

1.1.3.1 Pesticide use worldwide

In 2006 and 2007, worldwide pesticide sales amounted to $35.8 and $39.4 billion, respectively, representing 5.6 billion pounds of pesticides sold. Most pesticides sold annually are used to protect crops from insects, fungi, bacteria and weeds, and in doing so, the livelihoods of over 1.8 billion people who work in the agricultural sector worldwide.5,11

Herbicide use occurs primarily in the agricultural sector and amounts to approximately 78% of total herbicide use. Insecticides in the U.S. are split in their use by agriculture (45%) and domestic use (38%).11 Of the three herbicides chosen

5 for study, 2,4-D, dicamaba and mecoprop, all are widely used in the U.S. and

Canada. Out of the 25 most used pesticides in the agricultural sector, 2,4-D ranks

7th with use of 25-27 million pounds.11 2,4-D is the most used active ingredient in non-agricultural sectors such as domestic, industrial/commercial and government use, accounting for 8-11 million and 19-22 million pounds used, respectively.11

Mecoprop and dicamba rank 4th and 8th in the most widely used domestic active ingredients.

Insecticide use has been decreasing in total million pounds used since the 1980s. In

1980, OPs accounted for 57% of all insecticide; this increased to 72% by 1991 before a sharp drop to 45% in 2002 due to replacement by less toxic chemical classes of insecticides. Total million pounds use has decreased from the 1980s from 131 to 80 in 1995 and then 40 in 2005.11 Of insecticides, 45% were used agriculturally and

38% for home and garden use.11 Of the most used OP insecticides in the U.S., the pesticides chosen for study, chlorpyrifos, malathion, phorate, diazinon and azinphos-methyl all rank in the top 10 and occupy the top two positions.11 The most widely used OP, Chlorpyrifos, ranks as the 14th most used pesticide agriculturally with 7-9 million pounds sold.11 In the domestic and industrial/commercial sectors, Malathion ranked 7th and 10th respectively of all pesticides.

With limited or no exposure control limits in many countries, 25 million agricultural workers are inadvertently poisoned annually. In 2001, only 60% of

6 farms across Canada used a formally trained applicator with the lowest proportion in the highest pesticide using province (by percentage) Saskatchewan. Given the risks associated with handling pesticides and applying pesticides, this is a significant concern for applicators, crops and the surrounding environment alike.12

While active ingredients possess toxicity, the formulation, which contains many other ingredients, may also be responsible for acute and chronic toxic effects in exposed workers or their families. Additionally, the U.S. Department of

Agriculture estimates that 50 million people are potentially exposed to drinking water contaminated by pesticides.5

1.1.3.2 Pesticide use in Canada

Crop Life Canada, an association of pesticide manufacturers and distribut0rs, reported pesticide sales totaling $1.8 and $1.9 billion win 2008 and 2009, respectively. Of the pesticides sold, herbicides accounted for 76% of all pesticide sales which is comparable to the proportions used in the U.S.13

Health Canada’s Pest Management Regulatory Agency (PMRA), created in 1995, is empowered to collect sales data under the Pest Control Products Act and is currently in its fourth year of collecting data; however, no public reports of the data are anticipated in the near future. The most recent publication of pesticide sales data remains a 2005 report from Environment Canada, entitled “Pesticide

Utilization in Canada: A Compilation of Current Sales and Use Data,” which lists

2003 pesticide sales for the province of Nova Scotia.14 Though the utilization will

7 have changed in a nine-year time period, most of the top selling pesticides continue to be registered active ingredients.

Canada represents approximately 3% of global pesticide sales with the majority of use occurring in the Western provinces. With over 7000 registered pesticide formulations as of 2002, 91% of pesticide usage in Canada is for agricultural purposes, with the remaining 9% for domestic, ornamental and forestry purposes.

Intensity is greatest, however, in non-field crop producing provinces such as P.E.I., where usage averages 4.87 kg of active ingredient per hectare compared to 0.79,

0.41 and 1.09 for Alberta, Manitoba and Ontario, respectively. The share of farms which used pesticides in 2001 ranges from 48% for British Columbia and Nova

Scotia to 80 and 82% in P.E.I. and Sasktachewan, respectively.12 By pesticide class, the overwhelming majority of pesticides sold are herbicides, accounting for 77% of total sales. Insecticides, fungicides and specialty products represent 8%, 9% and

6%, respectively.14 The target pesticides chosen for study are shown with their quantities sold and provincial and territorial sale occurrences in Table 1.

Table 1. Target insecticide (I) and herbicide (H) use across Canada

Provinces/Territories Rank Pesticide Type Quantity (kg) Used In* 4 2,4-D H 1,492,553.27 8/2 13 Dicamba H 356,130.62 8/2 18 Chlorpyrifos I 252,649.07 7/2 19 Mecoprop H 251,507.11 8/2 50 Diazinon I 62,039.39 6/2 65 Malathion I 30,506.89 6/1 71 Phorate I 25,373.94 4/0 Azinphos- 75 I 23,604.37 6/0 methyl

* Except Saskatchewan and Nunavut

OP usage worldwide is decreasing and the trend is also visible in Canada. In the five years between 2005 and 2010, use of diazinon decreased by 75% mainly due to an urban phase-out.15 Many organophosphorus pesticides have been subjected to phase outs in domestic use and many provinces and municipalities have instituted complete bans on domestic pesticide use due to concerns around human exposure to these compounds especially among children. Moreover, pesticide sales have been decreasing in many provinces due to lower application quantities, a move towards less pesticide intensive crops and adoption of integrated pest management techniques that require a more holistic approach to pest control through biological and physical methods in addition to chemicals.14 In 2001, only

40% of Canadian farmers set action thresholds or points at which pest control would be taken as opposed to 28% who apply insecticides upon the first sighting of

9 pests. On the application of herbicides, approximately half of Canadian farmers applied based on the growth stage of the crop in question.12

1.1.3.3 Pesticide usage in Nova Scotia

Over 500 pesticide active ingredients are registered for use in Canada, of which nearly 200 active ingredient-containing pesticides were sold or used in Nova

Scotia.14 The OP insecticides and PA herbicides chosen for study are shown with their quantities sold and proportion of total sales in Table 2. The herbicides chosen are present in individually or in mixed active ingredient formulations.16 The five

Organophosphorus pesticides, 2,4-D and dicamba have mandated concentrations in the Canadian Drinking Water Quality Guidelines and many are listed in the

Water Quality Guidelines for the Protection of Aquatic Life and Agricultural Uses.

17-19 All are currently registered for use in Nova Scotia and across the country, have been detected in surface, drinking and groundwater water across the country, remain among the most toxic pesticides used and are a concern for human and ecological health. Mecoprop, in particular, was chosen because it accounts for nearly half of all the pesticides used in the province.14

Table 2. Pesticide utilization in Nova Scotia in 2003

Rank Pesticide Type Quantity (kg) Proportion 1 Mecoprop H 206,604.75 46.78% 15 2,4-D H 4,633.37 1.05% 19 Diazinon I 3,786.27 0.86% 20 Phorate I 2,887.80 0.65% 26 Chlorpyrifos I 1,715.06 0.39% 32 Dicamba H 1042.53 0.24% 55 Malathion I 319.03 0.07% 59 Azinphos-methyl I 252.63 0.05%

1.1.4 The Economics of Pesticide Use

Pesticides have improved agricultural productivity, increased food supplies, the efficiency of crop yields and reduced harmful vectors of disease.20 Major losses to pests historically include the potato blight epidemic in 1840s Europe and the devastation of rice crop by Cochliobolus miyabeanus fungus during the Great

Bengal Famine, which caused widespread starvation and 2 million deaths. The

Black Death pandemic in Europe that wiped out nearly half of Europe was spread by oriental rat fleas carrying Yersinia pestis. Potential loss estimates, without any form of crop protection vary from crop to crop, range from 8.5% for cotton to

21.2% for potatoes, respectively.21

As of 1996, approximately 90% of pesticide active ingredients were produced by 15 manufacturers worldwide. 22 Pesticide production in the United States brings in

$9.8 billion in sales and accounts for 3.1% of farm expenditures.11 Brazil accounts for roughly half of the world’s production of herbicides with production increasing

44% between 1995 and 2005 due to growth of the country’s sugar cane industry.23

Canadian pesticide manufacturing firms are subsidiaries of larger, global companies. In general, manufacturing consists mainly of formulation manufacturing and not the synthesis of active ingredients themselves.22

In 2007, there were 12 major pesticide producers, 120-150 pesticide formulators and

150-250 major distributors in the US. Smaller producers, formulators and distributors accounted for 100, 1,550 and 13,250 companies, respectively. There are an estimated 25,600 extermination or pest control firms and 538,053 and 399,044 private and commercial applicators. As of 1996, there were over 300 and 700 distributors and retailers in Ontario and Western Canada respectively with only

25% of retailers independently run and not linked to a major formulator. Across the country, there were approximately 2900 pesticide warehouses including 15 in

Nova Scotia. Sasksatchewan and Alberta had over 950 and 650 alone. 22 Thus, the pesticide industry is a major contributor to jobs and the agricultural sector of the

American and Canadian economies.11

In 1990, Pimentel et al. estimated that pests resulted in a 37% loss of potential of crop, that pest loss would increase by 10% without the use of any pesticides at all and a $4 return in saved crops results from every dollar spent on pest control.

Despite this and a magnitude increase in insecticide use, crop loss has doubled from 7.5% in 1945 to 13% in 1989. The explanation is a shift in agricultural practices from rotating crop production to continuous crops.24 With the emergence of

12 integrated pest management (IPM), these losses from agricultural practices are being minimized. IPM stresses the a holistic approach to pest control that incorporates physical and biological methods to control in addition to the use of chemical pesticides. Thresholds for action are set in IPM where action is only taken if the impact of pests exceeds a critical point. Identification and monitoring of pests is integral to the program and when action is required, control begins with the least harmful methods of pest control first and pesticides are used only if a necessity.

Economic, environmental and health related costs associated with pesticide use include acute poisonings, carcinogenicity, livestock losses, destruction of natural pest enemies, bee losses and crop loss from drift.24 In 1989, the World Health

Organization (WHO) estimated that 20,000 of 1 million people poisoned from pesticide use died, with most poisonings occurring in developing countries where little or no exposure control measures are in place. Livestock and crop losses from pesticide residues above legal limits resulted in the destruction of 1-3% of crops annually and a 35% detection rate on foods. Livestock losses were estimated at $29 million annually in the United States alone. Inadvertent eradication of the natural enemies of pests can cause crop losses by secondary pests. With natural enemies accounting for an estimated 50 – 90% of crop pest control, this is a significant problem. For example, a $1.5 billion dollar rice crop loss in Indonesia resulted from the eradication of the brown planthopper’s natural enemies. Finally, crop loss from pesticide drift, excessive application and residual presence of herbicides can

13 adversely affect crops. With both ground and aerial equipment having a tendency to miss their target crop by up to 10 – 35% and 50 – 75%, respectively, this accounts for large losses. Additionally, only 14% of farms across Canada and in Nova Scotia calibrate sprayers which ensure accurate application between applications of different types of pesticides which is the recommended practice. Instead, half of all farms calibrate their sprayers at the beginning of the crop season.12 Other unintended economic losses include the reduction in pollination resulting in losses as high as $4 billion to the honeybee industry.24

1.1.5 The Chemistry of Organophosphorus Insecticides

OP insecticides can be generalized by the following structure: a central phosphorus atom with a double bond to oxygen or sulfur, two organic moieties and a leaving group that is displaced during phosphorylation of acetylcholinesterase. They can also be thought of as analogs of phosphoric or phosphonic acids, where hydrogens have been replaced by organic substituents and one or more of the surrounding oxygens have been replaced by either sulfur or nitrogen.9,25

The chemical structures of the OP insecticides being analyzed in this work and the surrogate and internal standards used for quality assurance and quality control

(QA/QC) are given in Figure 1.

1.1.5.1 Azinphos-methyl

Azinphos-methyl (GUTHION®, SNIPER®) is an OP insecticide and mite-control acaricide used against sucking and chewing insects on fruit, vegetables and field crops in agriculture, on forest and shade trees in forestry, and ornamentals. It is a cholinesterase inhibitor and stomach and contact poison. Formulations include wettable and dust powders and emulsifiable concentrates.26

In April 2006, the Pest Management Regulatory Agency (PMRA) announced azinphos-methyl use would be phased out because of an unacceptable risk to agricultural workers. The phase out schedule includes final sales by registrants in

December 2010, by retailers in December 2011 and last product use by December

2012.27

1.1.5.2 Chlorpyrifos

Chlorpyrifos (LORSBAN®, DURSBAN®) is a non-systemic OP insecticide used against ectoparasites, like fleas and lice, in livestock, as a foliar and soil insecticide for vegetable, fruit and cereal crops, and was once used to control household pests like mosquitoes and flies. Non systemic pesticides kill on contact and do not travel through the organism to untreated tissues. It is a cholinesterase inhibitor, and contact and stomach poison. Formulations include solutions, solids, aerosol, granulars, dust and wettable powders, and emulsifiable concentrates.26

CH3 H3C H3C O O Cl O O O H3C P N P S N S S Cl O N N Azinphos-methyl Cl Chlorpyrifos

H3C CH3

CH3 O N CH3 CH H3C N 3 O S O O CH3 H3C O P O P O S O CH3 S O Diazinon Malathion

CH3 CH3 O S O P S H C 3 S Phorate

Figure 1. Structures of OP insecticides

Chlorpyrifos underwent a re-evaluation process by the PMRA in June 1999 and domestic use of the product was suspended in December 2001. Chlorpyrifos use on tomatoes was suspended in December 2003. A proposal to discontinue use on corn, lentils, oats, pepper, sugar beet and tobacco crops was introduced in 2003 with reductions in applications per season and implementation of buffer zones and worker re-entry intervals for pesticide applicators for use on registered crops.28 A final decision on continuing registration of chlorpyrifos is pending a final assessment.29

1.1.5.3 Diazinon

Diazinon (DIAZINON 500®, BASUDIN®) is an OP insecticide, nematicide and acaricide used against soil insects, fruit, vegetable, tobacco and other field crop insects, in veterinary use against flies and ticks and formerly household pests like cockroaches, grubs and flies. It is a cholinesterase inhibitor, stomach, contact and respiratory poison, and was first introduced in 1953.30,31 Formulations include wettable and dust powders, granules, solutions and emulsifiable concentrates, slow release generators, and aerosols. Granulars and foliar sprays were phased out in

2005.30

Continued registration was granted for soil drench use for blackberry, broccoli,

Brussel sprouts, cabbage, cauliflower, loganberry, onion, raspberry, rutabaga and turnip crop and cattle ear tags on the condition of risk reduction measures.32 A phase out remains in effect for all other uses such as Christmas tree application,

17 greenhouse pepper and tomatoes, and seed treatments for beans, corn onion, peas, potatoes and sugar beet.30

1.1.5.4 Malathion

Malathion (SANEX®, CYTHION®) is a non-systemic phosphorodithioate OP insecticide and acaricide used in agricultural, horticultural, veterinary and household areas. Malathion crop use includes fruits, vegetables, nuts and berries.

20 It is a cholinesterase inhibitor of particular importance where application to mammals is desired. Formulations include: emulsifiable concentrates, aerosols, particulates, wettable powders, solution, dust or powder.26

A 2010 decision by the PMRA recommended continued registration of malathion in agricultural and non-agricultural settings except for use against mosquitoes in breeding areas and standing water, greenhouse mushrooms, ornamental greenhouse plants, seeds, and against numerous structures and human habitats, municipal dumps and residential yards.33

1.1.5.5 Phorate

Phorate (THIMET®, CYGARD®) is a soil and systemic phosphorodithioate OP insecticide used to control wireworms on potatoes only.34 Phorate is a cholinesterase inhibitor, and a stomach and contact poison. It is available in granular formulations and sold in combination with fertilizers and fungicides.26

Phorate use on corn, lettuce, beans and rutabagas was discontinued in December

2003 and restricted to wireworm control on potatoes until December 2006.35 As a result of continued wireworm issues and lack of alternatives, dates of final sale and usage were extended to May 2012 and August 2012 respectively.36

1.1.6 The Chemistry of PA and Benzoic Acid Herbicides

The three herbicides, their confirmed metabolites and manufacturing intermediates chosen for study are shown in Figure 2.40-43

1.1.6.1 2,4-D

2,4-D is a post-emergence, selective, systemic aryloxyalkanoic acid herwbicide used to control broad-leaved weeds in cereal crop, pastures and grasslands, and domestically on lawns.37,38 Systemic pesticides are absorbed by the weed and transferred throughout the plant tissue and are slower acting than contact herbicides. Post-emergence pesticides are applied after a crop has emerged. It is an auxin growth inhibitor, a regulator responsible for the balance between root and culmn growth that is absorbed by the foliage and roots of target weeds. 2,4-D is available, as sodium, ammonium and amine salts and as esters, in granules and emulsifiable concentrates. It is commonly used alongside mecoprop, dicamba, dichlorprop, MCPA and MCPB in herbicidal mixtures.26 A re-evaluation decision by the PMRA in 2008, concluded that all products containing the diethanolamine form or used in aquatic environments would be phased out.39

2,4-dichlorophenol 1,2,4-benzenetriol 2,4-dichlorophenoxyacetic acid OH OH O Cl OH O Aerobic aquatic metabolism Aerobic aquatic metabolism OH

Cl Cl Aerobic aquatic metabolism Cl OH

OH Cl

Chlorohydroquinone

OH OH OH CH3 O H3C H3C O Aerobic aquatic metabolism Aerobic aquatic metabolism OH

CH3 Cl Cl OH Mecoprop Aerobic aquatic metabolism 4-chloro-2-methylphenol Methylhydroquinone

CH3 OH

o-cresol

O OH O OH

Cl O Aquatic aerobic metabolism Cl OH CH3

Cl Cl Dicamba Possible metabolic productt 3,6-dichlorosalicylic acid

Manufacturing intermediate

Cl OH

Cl 2,5-dichlorophenol

Figure 2. PA and benzoic acid herbicides, and aerobic metabolites

1.1.6.2 Mecoprop

Mecoprop (PAR III®, Target®) is a post-emergence, systemic, selective PA acid herbicide used in the control of chickweed, cleavers, clovers and plantain in agricultural cereal crops and domestic lawn and turf.44 It is available in emulsifiable concentrates and solutions as amine and potassium salts. It is also a constituent in mixed formulations with other post-emergence herbicides such as

2,4-D and dicamba.26 In 2004, after a re-evaluation of mecoprop by the PMRA, sales of technical racemic mecoprop were voluntarily discontinued with December

2005 scheduled as the last date of sale for end-used products.45

1.1.6.3 Dicamba

Dicamba (DYCLEER®, BANVEL®, DYVEL®, PAR-III®) is a benzoic acid, selective, systemic herbicide used against broadleaf weeds like thistle and buckwheat in corn, small grain crops, non-cropland turf and pastures, and brush along railroad, highway and utility lines. Dicamba is an auxin-like growth regulator that is absorbed by roots or leaves. Formulations include suspensions and solutions, granules, pellets and wettable pellets, soluble powders and emulsifiable concentrates. It is often included in mixtures with 2,4-D, dichlorprop and mecoprop.26 In 2008, Dicamba registration was continued for use in lawn, turf and agricultural sectors. Formulations with diethanolamine were phased out.26,46

1.1.7 PA and Benzoic Acid Metabolites

Phenols are used as precursors in chemical, pharmaceutical and petrochemical industry and are ubiquitous in wastewater from these facilities.47 Chlorophenols are often produced during the chlorination of phenol wastewaters and are used as a precursor in 2,4-D production. Dichlorophenols are listed as EPA priority pollutants because of their persistence in water, effects on aquatic life and status as carcinogens.48,49 They are used in wood preservation and are by-products of pulp bleaching, water disinfection and waste combustion processes.48

1.1.8 Environmental Exposure and Pesticide Occurrence

1.1.8.1 Introduction of pesticides into the environment

Pesticides contaminate groundwater and surface water through leaching50-52, runoff,16,23,51-54, atmospheric deposition16,52, application drift16 and improper disposal of pesticides, rinsates23,51,55 and manufacturing plant discharges23,38,52.

Many pesticides worldwide are kept in storage facilities from which accidental spills and leaks may occur, including stocks that are obsolete.56 With many acidic pesticides, corrosion of storage containers may occur. Improper disposal includes pesticide burial that may contaminate underlying groundwater. While incineration is the preferred technique, costs range between $3,000 and $4,500 per tonne US and developing nations often do not have the funds necessary to do so.

To deviate pesticide containers with residues from agricultural waste, efforts are being made to collect containers and treat rinsates.57 In both 2009 and 2010, nearly

4.6 million pesticide containers were collected across Canada and 11,000 kg of obsolete pesticides were collected and safely destroyed. Additionally, a pilot program began in Prince Edward Island before expanding to Nova Scotia and New

Brunswick in which pesticide paper bags were collected. More than 130,000 paper bags have been collected since the program began in 2010.13

Partial or incomplete treatment of pesticides during treatment makes wastewater treatment plants a major point source of pesticides into aquatic environments.58

Contamination of wastewater influents can occur through the means described above with little removal occurring during the treatment process.

Leaching occurs when the amount of water soils can hold is exceeded by the water present. The result is a downward motion of water to areas where a soil-water deficit exists.59 Pesticides leach only if they are soluble in water, highly mobile and have a greater affinity to water than organic sediments (as indicated by a low organic carbon-water partition coefficient or Koc).

Finally, one and a half tonnes of wastewater is discharged for every tonne of pesticide oil produced. Wastewater from OP pesticide manufacturing facilities are high in organics (50,000 mg/L), ammonia (40,000 mg/L) and OPs (10,000 mg/L) with 90% of the organics present resistant to biodegradation.60

1.1.8.2 Factors influencing the introduction of pesticides

Many factors may influence the introduction of pesticides into aquatic environments such as the physical and chemical properties of the pesticides.

Degree of contamination of surface or ground water will depend on factors such as persistence, mobility and solubility in water.2 Phenoxyacetic and benzoic acid herbicides are both persistent and polar in character. Herbicides such as 2,4-D are highly mobile,61 and OPs are relatively soluble in water.62,63 The solubility of target

OP insecticides and PA herbicides are shown in Table 3.

Table 3. Solubility of target OP insecticides and PA herbicides

Pesticide Solubility Interpretation31 (20 °C) [mg/L] 2,4-D 23,180 High Azinphos-methyl 28 Low Chlorpyrifos 1.05 Low Diazinon 60 Moderate Dicamba 250,000 High Malathion 148 Moderate Mecoprop 250,000 High Phorate 50 Moderate

The movement of pesticides is also affected by external factors such as climate, soil characteristics and the topography of the ground.59 For example, concentrations of pesticides from runoff are expected to increase during storms53,61 and off impermeable paved surfaces in urban areas.53 Human activity will also play a role in pesticides introduction. For example, urban watersheds tend to have higher

24 concentrations of insecticides than herbicides, while the opposite is true for agricultural watersheds.61

1.1.8.3 Organophosphorus pesticides in surface water

OPs have been detected in a number of studies across urban and rural watersheds in Canada. Between 2003 and 2005, numerous samples were taken across Canada to determine the occurrence of 141 pesticides and transformation products as part of a national water quality surveillance program study.51 Diazinon, chlorpyrifos, azinphos-methyl, malathion, and phorate were all detected. The most significant detection statistics include an 80.6% detection rate for diazinon with a maximum concentration of 12.5 g/L found in surface water from British Columbia and a 77% detection rate for chlorpyrifos in samples obtained from isolated lakes in Ontario.

A study of the Yamaska watershed in Quebec from 2004 to 2005, showed detections of 13 of 53 pesticide targets including 2,4-D, mecoprop, dicamba and chlorpyrifos. Chlorpyrifos was detected at levels above the Canadian Council of

Ministers of Environment (CCME) protection of aquatic life limits.64

In the United States, a study of urban streams in Sacramento and Stockton,

California showed that 85% of samples exceeded Department of Fish and Game water quality criteria for diazinon and 80% of samples exceeded chlorpyrifos guidelines.65 Of nearly 2500 measurements from 300 sites in California from 2005 to 2010, Diazinon was detected in roughly 15% of samples with a maximum

25 concentration of 24 μg/L detected in the Salinas Valley region and 10% of samples exceeded the 100 ng/L concentration mark.15

A major study of 600 sites in seven major river watersheds in China, including the

Yangtze, Yellow Pearl and Haihe Rivers, between 2003 and 2004 showed detections of one or more OP insecticides in 91% of samples. Mean concentrations of malathion across watersheds ranged from 36.5 ng/L to 101.2 ng/L in the Huaihe and Yellow Rivers respectively with a maximum concentration of 1.29 μg/L detected in the Yellow River.66

1.1.8.4 Phenoxyacetic and benzoic acid herbicides in surface water

2,4-D has been detected in water bodies across Canada.38 A study of 34 sites across southern Ontario in 2003 – 2004, showed higher concentrations of Mecoprop in the summer than other seasons. Mecoprop was frequently detected (57% of samples) with concentrations exceeding water quality guidelines in 1.2% of samples. In both years, the highest concentrations detected were from Vineland

Creek at 1.9 μg/L and 103 μg/L respectively. The enantiomeric distribution of these samples showed 39%, 46% and 15% in the R-(+) and S-(-) enantiomers and racemic mixture, respectively.67 Since this time, racemic mecoprop has been phased out.

86 samples from storm water ponds in Regina from May to October 2007 showed

100% detection rates for 2,4-D and mecoprop [MDL: 30 ng/L and 10 ng/L, respectively]. Method detections limits (MDL) give the minimum concentration of pesticide detectable in a sample after sample preparation, detection and

26 quantitation. Average concentrations were 626 ng/L, 730 ng/L, 45 ng/L and 97 ng/L for 2,4-D, mecoprop, 4-chloro-2-methylphenol and 2,4-dichlorphenol respectively though the metabolites were much less frequently detected (< 13%).68

From April to September 2007, a study was conducted to determine herbicide concentrations at 19 sites in 16 urban watersheds across Canada. Concentrations of the three target herbicides were highest in Ontario at urban sites and after precipitation events. 2,4-D was detected in 83% of samples, with a mean concentration of 119.6 ng/L. Mecoprop had a mean concentration 59.4 ng/L and was detected in 80% of samples. Dicamba was detected in 68.5% of samples, with a mean concentration of 13.1 ng/L. Dicamba was not detected in any samples from

Atlantic Canada. The three pesticides were roughly present in the same 9:5:1 ratio that is common in herbicide formulations including the mix used in this study.53

2,4-D, mecoprop and dicamba were detected in both the Lower Fraser Valley and the Okanagan Basin with higher levels detected in the Fraser Valley. Mecoprop was detected in 75% of samples in the Lower Fraser Valley. 2,4-D was detected at a maximum 1.23 µg/L, mecoprop at 917 ng/L and dicamba at 179 ng/L.

Concentrations were higher in the Lower Fraser Valley than the Okanagan Basin.69

2,4-D, dicamba and mecoprop were all detected in Prairie wetlands at frequencies above 85% at maximum concentrations of 4.29 µg/L, 1.27µg/L and 241 ng/L, including a 100% detection rate for 2,4-D. River detections of 2,4-D and dicamba were observed in Prairie river samples at 92% and 83%. Reservoir samples had

27 frequencies of 99.5% with a maximum concentration of 1.85 µg/L for 2,4-D.

Dicamba and mecoprop detections were both above 77%.69

1.1.8.5 Pesticide wastewater

Two independent analyses of pesticide factory wastewater in China found chemical oxygen demands (COD) of about 50,000 mg/L and 33,700 mg/L with total OP concentrations of 10,000 mg/L and 2,040 mg/L, respectively.60,70

Chlorinated herbicide manufacturing is low in phosphorus and nitrogen, total solids of 104,00 mg/L, CODs and BOD5 and 8,300 and 6,300 mg/L and chloride, chlorophenols and chlorophenoxy acid concentrations of 52,000, 112 and 235 mg/L.71

1.1.9 Fate of Pesticides in Aquatic Environments

Pesticide transformations may occur through photolysis, hydrolysis or metabolism by microorganisms and plants in a variety of settings like soil, water or the air.

Transformation products may be more toxic and more persistent that the parent compound.2 Pesticides may also be removed from wastewater through adsorption to organic carbon or suspended solids.

1.1.9.1 Adsorption of OP insecticides and PA herbicides

Major differences in the half-lives of OPs in filtered and unfiltered water suggest that adsorption can be a major fate pathway depending on the structure and substituents of the pesticide, possible sorbents and water conditions such as pH, a measure of the hydrogen ion concentration in a sample.8 For example, chlorpyrifos

28 has a high sorption potential to soil and low solubility in water and is apt to sorb in aquatic environments.25 Of a series of sorbents, only goethite had significant sorption of phorate, 33% and 20% at pH 5.7 and 8.5.73

Adsorption to metal cations may be dependent on whether the OP has a phosphoryl (P=O) or thiophosphoryl (P=S) bond, as a stronger negative charge on oxygen will promote complexing. Lone-pair donations from side chain oxygen and nitrogen will also promote complex formation.73 Adsorption is very much pesticide dependent with slight and marked structural differences resulting in significant

8 changes in the phenomenon. Another indicator of adsorption potential is the KOC as shown in Table 4.

Table 4. OP organic carbon-water partition coefficient

Pesticide Log KOC Interpretation Azinphos methyl 3.05 Slightly mobile31 Chlorpyrifos 3.8, 3.91 Non-mobile31,74 Diazinon 1.6 – 2.6, 2.78 Slightly mobile7,31 Malathion 3.25 Slightly mobile31,75 Phorate 2.73, 3.22 Slight mobile31,74

In general, PA herbicides weakly sorb to soil particles.34 The absorption potential of these herbicides and some metabolites are shown in Table 5.

In a study of herbicide by Ward and Getzen, 10 mg of carbon were added to a 10-4

M solution of dicamba and shaken at pH 3.0, 7.0 and 11.0, showing 37.5%, 11.2% and

6.0% adsorption. 2,4-D showed adsorptions of 60.1%, 18.8% and 14.3%, respectively at those pHs. Both indicate greater adsorption with decreasing concentration of

29 the ionic form of the pesticide and surface carbon and hydrogen ion binding, which enhances negative anion removal from solution.76 Mecoprop adsorption is higher at lower pHs.31

Table 5. PA Organic carbon-water partition coefficient

74 Pesticide Log KOC Interpretation 2,4-D 1.94 Moderately mobile31 Dicamba N/A N/A Mecoprop 0.72 – 1.12, 1.67 Mobile31,77

1.1.9.2 Hydrolysis of OP insecticides and PA herbicides

OP insecticides are stable at low temperatures, in the dark and in anhydrous conditions. At all but low pHs, hydrolysis can occur through hydroxide attack of the P-O-C linkage. In general, P=O analogues are hydrolyzed faster than P=S

(though they are more toxic) and are slowed by increasing the size and bulk of the alkyl substituents.8 P-S-C linkages can be hydrolyzed as well with attack by a nucleophile resulting in thiocarbon bond breakage under basic conditions and the thiophosphorus bond breakage under acidic conditions. Phosphorus-nitrogen bonds undergo hydrolysis only under acidic conditions.9 A wide range of half-lives have been described for many of the OP insecticides. Variables that affect these rates include pH, metal ion concentration, microorganisms, salinity, suspended solids and their characters.25 In general, half-lives decrease with increased temperatures and pH values.8

Azinphos-methyl does not degrade under acid hydrolysis conditions and increases with temperature and pH. Hydrolysis half-lives are shown in Table 6.

Table 6. Hydrolysis half-lives of azinphos methyl

Pesticide Half-life (days) Conditions 415 6 °C, pH 6.1, distilled water8 278 6 °C, pH 7.3, filtered river water8 115 22 °C, pH 6.1, distilled water8 87 20 °C, pH 431 50 20 °C, pH 731 36.4 6 °C, pH 8.6, distilled water78 35 22 °C, pH 7.3, filtered river water8 27.9 22 °C, pH 8.6, distilled water78 Azinphos methyl 26 22 °C, sea water, 8

11 Variable temp, sea water, sunlight8 4.95 6 °C, pH 9.6, distilled water78 4 20 °C, pH 931 3.9 6 °C, pH 10.7, distilled water78 2.40 22 °C, pH 9.6, distilled water78 2.0 22 °C, pH 10.7, distilled water78 2 Natural waters26 No Degradation 6 °C, sea water, sunlight8

Hydrolysis rates of chlorpyrifos increase with increased pH in distilled water

(Table 7),79,80 and increase in the presence of ferrous ions in surface water.80

Products of the process included 3,5,6-trichloro-2-pyridinol, O-ethyl O-hydrogen

O-(3,5,6-trichloro-2-pyridyl) phosphorothioate and O, O-dihydrogen O-(3,5,6- trichloro-2-pyridiyl) phosphorothioate.25,80 A study of the chlorpyrifos half-lives in water collected from sites across Chesapeake Bay showed a range of half-lives from

24 days to 126 days. No correlation could be determined with pH but correlations with copper concentration (hydrolysis catalyst), total suspended solids and salinity were strongly negative, strongly positive and strongly negative, respectively.25

Macalady and Wolfe reported a magnitude decrease in hydrolysis of adsorbed chlorpyrifos compared to free chlorpyrifos.25,79

Table 7. Hydrolysis half-lives of chlorpyrifos

Pesticide Half-life (days) Conditions 153 25 °C, pH 5.90, distilled water79 68 20 °C, pH 531 62.7 25 °C, pH 4.7, distilled water80 Chlorpyrifos 35.3 25 °C, pH 6.9, distilled water80 25.5 20 °C, pH 731 23 20 °C, pH 831 22.8 25 °C, pH 8.1, distilled water80 10 25 °C, pH 9.77, distilled water79

Diazinon is stable to hydrolysis at neutral pH but undergoes rapid hydrolysis in acidic conditions and is faster at higher temperatures (Table 8). Products of hydrolysis include 2-isopropyl-6-methyl-4-pyrimidinol and diethyl thiophosphate.81 5 ppm concentrations of chlorpyrifos, diazinon and 10 other OC,

OP and carbamate pesticides were added to natural and sterile marsh, and sterile and non-sterile distilled water, at pH 7.8 in darkness. Degradation of diazinon was complete in sterilized distilled water and natural water within 4 and 16 weeks, and reached 85% and 80% degradation in sterilized natural water and distilled water in

16 weeks. Degradation in sterilized distilled water is attributed to reaction with sodium azide which would have reacted with salts present in the natural water.

When autoclaving was used for sterilization, rates were similar between sterilized, non-sterilized distilled water and sterilized natural water, and all were slower than unsterilized natural water.7 Diazinon concentrations are reduced to below 99.9% at pH greater than 2 via acid hydrolysis.

Table 8. Hydrolysis half-lives of diazinon

Pesticide Half-life (days) Conditions81 181 6 °C, unfiltered river water 144 6 °C, milliQ distilled water 138 20 °C, pH 7,31 132 6 °C, filtered river water 125 6 °C, saltwater 80 22 °C, unfiltered river water Diazinon 77 20 °C, pH 9,31

69 22 °C, milliQ distilled water 52 22 °C, filtered river water 50 22 °C, saltwater 47 22 °C, saltwater, sunlight 43 22 °C, filtered river water, sunlight 12 20 °C, pH 5,31

Malathion degradation under acid conditions is not significant at common aquatic environment pHs and temperatures (Table 9). Under basic conditions (pH 8), the half-life of malathion is 36 hours. Products include malathion monoacids, diethyl fumarate and O,O-dimethylphosphorodithioic acid, which indicates carboxyl ester hydrolysis and elimination reaction possibilities. These monoacids have half-lives of 24 days in alkaline water. 80

Table 9. Hydrolysis half-lives of malathion

Pesticide Half-life (days) Conditions 212 6 °C, pH 6.2, distilled water8 107 25 °C, pH 531 53 6 °C, unfiltered river water8 42 22 °C, pH 6.2, distilled water8 41 6 °C, saltwater8 Malathion 19 6 °C, filtered river water8 14 Variable temp, saltwater8 8 22 °C, filtered river water8 7 22 °C, unfiltered river water8 6.2 20 °C, pH 731 6 22 °C, saltwater8 0.49 25 °C, pH 931

Phorate hydrolysis rates are shown in Table 10. Diethyl disulfide was identified as a product of a major degradation pathway forming from ethanethiol dimerization.73 In natural waters (pH 5-9), phorate undergoes oxidation to phorate-oxon, phorate-sulfoxide, phorate-sulfone abiotically and biotically.34

These metabolites also possess animal toxicity.34

Table 10. Hydrolysis half-lives of phorate

Pesticide Half-life (days) Conditions 3.9 20°C, pH 931 3.2 20°C, pH 731 2.6 pH 9.4, distilled water73 Phorate 2.5 pH 8.5, distilled water73 2.3 - 2.8 pH 5.7, metal oxides73 2.2 pH 5.7, distilled water73 2 - 2.4 pH 8.5, metal oxides73 1.4 pH 10.25, distilled water73

All three herbicides are soluble in water and are stable to hydrolysis. (Table 11)

Table 11. Hydrolysis half-lives of phenoxyacetic and benzoic acid herbicides

Pesticide Half-life (days) Conditions 2,4-D Stable 20°C, Between pH 5 and pH 931 Mecoprop Stable 20 °C to 70 °C, Between pH 5 and pH 9 31 Dicamba Stable 20°C, Between pH 7 and pH 931

1.1.9.3 Photolysis of OP insecticides and PA herbicides

Photolysis occurs when light is absorbed, the molecule is excited and energy is lost through decomposition. However, there are many other pathways for energy to take, including fluorescence or excitation of other molecules. In general, irradiation with light decreases molecular half-lives.8

UV degradation of OP insecticides in the presence of oxygen, nitric acid or peroxyacids will result in oxidation of the thio-phosphorus bond to the oxon via a three membered phosphorus-oxygen-sulfur ring. The phosphorus-thio-carbon linkage may be oxidized to ester sulfoxides that rapidly break down. UV irradiation of the C-O-P=S bond induces a rearrangement to the C-S-P=O. The mechanisms of these reactions are not entirely clear, but the transformation products have greater toxicities than the parent pesticides.9 In comparison to the parent compound, transformation products are more polar, soluble in water, apt to leach and be transported but are also less persistent.81

Table 12. Photolysis half-lives of OP insecticides

Pesticide Half-life Conditions Azinphos methyl 3 pH 731 Chlorpyrifos 29.6 pH 7 Diazinon 50 20°C, pH 731 Malathion 98 20°C, pH 7, natural light31 Malathion 156 20°C, pH 7, not natural light31 Phorate 1.1 20°C, pH 7, UV light

Photolysis of phenoxyacetic and benzoic acid herbicide rates are shown in Table 13.

In general, photolysis half-lives increase with pH.

Table 13. Photolysis half-lives of phenoxyacetic and benzoic acid herbicides

Pesticide Half-life Conditions 2,4-D 13 pH 731 Dicamba 50.3 pH 731 Mecoprop 44 pH 7 Mecoprop 42 pH 5 Mecoprop 32 pH 9

1.1.9.4 Volatilization of pesticides

In a study of the biodegradation of 12 pesticides, malathion, diazinon and oxo- diazinon by activated sludge microbes under aerobic and anaerobic aquatic conditions, less than 0.1% were lost to volatilization.82 However, volatilization is a major pathway for chlorpyrifos removal from water. Vapour pressures for OP and

PA herbicides are shown in Table 14

Table 14. Volatilization of pesticides

Pesticide Vapour Pressure 25 °C (mPa) Interpretation Azinphos methyl 0.0005 Not Volatile31 2,4-D 0.0187 Not Volatile31 Chlorpyrifos 1.43 Volatile31 Diazinon 11.97 Volatile31 Malathion 3.1 Volatile31 Mecoprop 1.6 Volatile31 Phorate 112 Volatile31 Dicamba 1.67 Volatile31

1.1.9.5 Biodegradation and transformation of pesticides

In a study of activated sludge biodegradation, malathion, oxo-diazinon and diazinon were shown to have greater rates of biodegradation under aerobic conditions than anaerobic conditions. Degradation in the presence of the pesticide alone (for the three pesticides considered here) was greater than the control, suggesting biodegradation contribution to removal. In reactors where glucose and peptone were present in addition to the pesticide, degradation rates were higher because of higher microbial populations and little difference in preference between the pesticides and glucose and peptone.82

2,4-D undergoes biodegradation readily at concentrations below 10 ppb but is poorly biodegradable at concentrations above 1 ppm.37,83 Detection of 2,4- dichlorphenol suggests degradation by Pseudomonas cepacia, Alcaligenes eutrophus and Arthobacter spp.38 Biodegradation of mecoprop enantiomers in soils may result in preferential degradation of one enantiomer over the other as well as

37 inter-conversion between the two. For example, at soil pH above 7.2, the R-(+) enantiomer is preferentially degraded.67

Phenol concentrations can inhibit microorganisms in wastewater treatment thereby reducing the degradation efficiency of readily biodegradable compounds.

At concentrations below 200 ppm, phenols can inhibit microbial function and growth. Organisms that are able to degrade phenols will thrive in these conditions.

Phenols can be degraded by the following microorganisms: Aeromonas,

Pseudomonas, Flavomonas oryzihabitans, Chryseomonas luteola, Alacaligenes spp.,

Sarcinas spp., Desulovibrio spp., Bacillus alkaligenes, Acinetobacter spp. and T. cutaneum. The highest potential for phenol degradation by microorganisms is from Rhodocci spp. and Pseudomonas putida.47

1.1.10 Pesticide Toxicology

1.1.10.1 Introduction

While individual concentrations of pesticides in surface water are seldom above

CCME maximum acceptable concentrations, as shown in Table 15 and Table 16, the presence of multiple pesticides in samples is a concern. In samples taken from the

Kisco River, New York, seven herbicides, four insecticides and two fungicides were detected at levels greater than 100 ng/L.61 Also, transformation products are often more toxic than their parent compounds.68 For example, some metabolites of diazinon have higher acetylcholinesterase activity than the parent.81

1.1.10.2 Organophosphorus insecticide toxicity

Acetylcholinesterases (AChE) and Butyrylcholinesterases (BuChE) are two main classes of cholinesterases that break down acetylcholine (ACh) in red blood cells and butyrylcholine (BuCh) in the plasma respectively. Cholinesterases are highly homologous in their amino acid sequences across broad classes of organisms.84

AChE can be present in two forms: asymmetric (long-tailed) or globular. Recovery of cholinesterase differs between BuChE and AChE because of the inability of red blood cells to synthesize protein. As a result, recovery is based on the life cycle of the erythrocyte (red blood cell) though brain AChE esterase activity recovers more quickly than in erythrocytes, it remains slower than in plasma BuChE.

The mechanism of acetylcholinesterases with acetylcholine can be modeled using a reversible Michaelis-Menton scheme as shown below. Acetylcholine binds with the acetylcholinesterase enzyme at two sites; the quaternary nitrogen of choline and the carbonyl group bind at the anionic and a serine residue at the esteratic sites on the enzyme, respectively. Acetylation occurs at the serine site, releasing the choline group before releasing acetate as a product. The enzyme is reactivated upon hydrolysis.85

k1 k 2 k 3 E +AChEAChEA  ChE  A Ch k1

The mechanism between OP pesticides and acetylcholinesterase is similar to the  enzyme with acetylcholine. The OP pesticide binds to the acetylcholinesterase and

39 releases a leaving group before the enzyme is phosphorylated at the serine residue.

The rate of dephophosphorylation is much slower (on the order of hours and days) than the rate of acetylation as with an acetylcholine substrate (on the order of microseconds), therefore acetylcholine builds up at the receptor sites in the body.

85 The result of acetylcholine build up is prolonged muscle contraction and spasms, neuropathy, and stimulation of glands and the central nervous system. OP insecticide and PA herbicide toxicities are shown in Figure 3 and Figure 4.31

Most OP insecticides are neurotoxicants due to acetylcholine build up. Metabolites of chlorpyrifos are suspected to have reproductive and developmental effects on children. Diazinon has been identified as a possible mutagen or endocrine disruptor. Malathion may be a possible liver and adrenal gland toxicant.31 Phorate and azinphos-methyl have the greatest acute toxicity and malathion the least. The

LD50, or the dosage required to kill 50% of a population, of rats administered orally and human ADI, or acceptable daily intakes, are shown in Figure 3.85

2500 0

2000 0.01

1500 0.02

1000 0.03 Rat LD50 oral(mg/kg)

500 0.04 Human ADI (mg/kgb.w.)

0 0.05 Azinphos- Chlorpyrifos Diazinon Malathion Phorate methyl Rat LD50 oral 4.4 135 300 1375 1.6 Human ADI 0.005 0.01 0.002 0.02 0.0001 Organophosphorus Insecticides

Figure 3. OP insecticide toxicities

Metabolites of OP insecticides vary in whether they are greater than the parent.

Benzazimide, a metabolite of azinphos methyl, has an oral LD50 of 50 mg/kg,

3,5,6-tirchloro-2-pyridinol, a chlorpyrifos metabolite has an oral LD50 of 794 mg/kg and phorate sulfone and sulfoxides have LD50s of 2.5 and 2.4 mg/kg, all of which are less toxic than their parents. Chlorpyrifos-oxon and malaoxon has LD50 oral rats of 82 and 158 mg/kg, respectively and diazoxon is 10,000 more potent as an acetylcholinesterase inhibitor.

1.1.10.3 Phenoxyacetic and Benzoic acid toxicity

All three herbicides are auxin-like growth regulators. Auxins regulate the balance between root and culmn growth with high and low auxin concentrations inhibiting and stimulating root growth, respectively. The mechanism of action is not clearly

41 understood, but is known to adversely affect cell division and elongation. 2,4-D is known to cause reproductive and developmental effects and is a neurotoxicant.

Mecoprop has one chiral center; the R-(+) enantiomer has herbicidal activity whereas the S-(-) enantiomer is an anti-auxin. Mecoprop is a known mutagen. The acute toxicities of the three herbicides are shown in Figure 4.31

2500 0 0.1

2000 0.2

0.3 1500 0.4 0.5 1000 0.6

0.7 Rat LD50 oral(mg/kg)

500 0.8 Human ADI (mg/kgb.w.) 0.9 0 1 2,4-D Dicamba Mecoprop Rat LD50 oral 469 1581 1166 Human ADI 0.05 0.3 0.01 Phenoxyacetic acid Herbicides

Figure 4. Phenoxyacetic and benzoic acid herbicide toxicities

Toxicities of target PA herbicide metabolites are comparable or slightly less than the parent. 2,4- and 2,5-dichlorophenol, o-cresol and 4-chloromethylphenol oral rat LD50s are 430, 580, 1350, 1190 mg/kg, respectively.

1.1.11 Pesticide Regulations and Legislation

The Canadian Council of Ministers of the Environment has implemented water, sediment and tissue residue standards for the protection of aquatic life and drinking water quality. All target pesticides save Mecoprop have been listed with maximum acceptable concentrations (MAC) for drinking water (Table 15).

Table 15. Canadian drinking water quality guidelines

Pesticide Maximum Acceptable Year of Approval Concentration (MAC) [ng/L] 2,4-D 100 1991 Azinphos-methyl 20 1989 (2005) Chlorpyrifos 90 1986 Diazinon 20 1986 (2005) Dicamba 120 1987 (2005) Malathion 190 1986 (2005) Phorate 2 1986 (2005) Metabolites Maximum Acceptable Year of Approval Concentration (MAC) [ng/L] 2,4-Dichlorophenol 900 1987 (2005) Parameters Guidelines (mg/L) Year of Approval Turbidity 1.0 NTU 2004 Colour 15 Nitrates 45 1987 pH 6.5 – 8.5 1995 TDS ≤500 1991

Freshwater and marine maximum acceptable concentrations for five target pesticides and metabolites are listed in the Canadian water quality standards for the protection of aquatic life (Table 16).

Table 16. Canadian water quality guidelines for aquatic life

Pesticide Freshwater MAC Marine MAC Year of Approval [ng/L] [ng/L] Chlorpyrifos 35 20 1997 Dicamba 10,000 N/A 1993 Malathion 1986 (2005) Phenoxy herbicides 4000 N/A 1987 Metabolites Freshwater MAC Marine MAC Year of Approval [ng/L] [ng/L] Monochlorophenols 7000 N/A 1992 Dichlorophenols 200 N/A 1992 Phenols (mono- and 4000 N/A 1999 dihydric0 Water Quality Freshwater MAC Marine MAC Year of Approval Parameters [µg/L] [ng/L] Ammonia (total) 19 No guideline Colour Narrative Narrative 1999 >8000 and Dissolved Oxygen 5500–9500 1999, 1996 Narrative Nitrate 13000 16000 2003 Nitrite 60 N/A 1987 7.0 – 8.7 and pH 6.5 –9 1987, 1996 Narrative Guideline Phosphorus framework Turbidity Narrative Narrative 1999

Freshwater and marine maximum acceptable concentrations for four target pesticides are listed in the Canadian water quality standards for the protection of agricultural water use (Table 17).

Table 17. Canadian water quality guidelines for agricultural water uses

Pesticide Livestock MAC Irrigation MAC Year of Approval [ng/L] [ng/L] Chlorpyrifos 24,000 Insufficient data 1997 Dicamba 122 6 1993 Phenoxy herbicides 100 N/A 1987 Water Quality Livestock MAC Irrigation MAC Year of Approval Parameters [ng/L] [ng/L] Colour Narrative 1999 Nitrate + Nitrite 100000 N/A 1987 Nitrite 10000 N/A 1987 Total dissolved 3000000 500000-3500000 1987 solids

None of the pesticides or their confirmed or possible metabolites appear in the

Canadian sediment quality guidelines for the protection of aquatic life86 or the

Canadian Tissue Residue Guidelines for the Protection of Aquatic Biota.87

1.2 Water Quality

1.2.1 Physical Properties

1.2.1.1 Colour

Colour arises from metallic ions (iron and manganese), humus, plankton and industrial wastewaters. Apparent colour differs from true colour in that suspended solids, which contribute to colour, are filtered out, or removed, through centrifugation. Colour may be reported through visual comparison with standards of coloured solutions or instrumentally using spectrophotometric methods. Colour is measured in PtCo units where 1 unit is equivalent to the colour produced by 1 mg/L chloroplatinate ion. Colour is pH dependent and increases with pH and is an important aesthetic quality for drinking water.88

1.2.1.2 Turbidity

Water clarity is an important indicator of the condition and productivity of aquatic systems. Turbidity is the degree to which light is scattered and absorbed by a sample rather than transmitted through the medium. It is caused by the presence of suspended solids, clays, soluble organics and microorganisms. These particulates will differ in their light scattering properties a result of size, shape and refractive index differences. Turbidity is measured with respect to a formazin- polymer reference standard in FTU (or formazin turbidity units).88

1.2.1.3 Conductivity and Total Dissolved Solids

Conductivity is a measure of the electric current carrying capacity of an aqueous solution in mS/m. Due to absorption of carbon dioxide and ammonia, distilled water has a conductivity of 0.05 to 0.2 mS/m. Drinking water has a conductivity of

5 to 150 mS/m and wastewater above 1000 mS/m. Conductivity increases with the number of ions, charge on the ions and temperature of the sample and is an informal measure of the concentration of inorganic acids, bases and salts in solutions. Total dissolved solids can be obtained from conductivity measurements by multiplying by a factor which ranges from 0.55 to 9 depending on temperature.88 Total dissolved solids are the solids left after a sample is passed through a 40 – 60 μm filter and evaporated at 180°C.88

1.2.1.4 Total suspended solids

The total suspended solids are those solids retained by a filter with pores 40-60 μm in diameter after drying at 103 – 105 °C.88

1.2.2 Inorganic Non-metallics

1.2.2.1 pH value

pH is a measure of the hydrogen ion concentration of a sample; the acidity or basicity of a sample. Many parameters in water quality are pH dependent. Natural waters have pHs between 4 and 9 with most being slightly basic because of the presence of carbonates and bicarbonates.88

1.2.2.2 Ammonia nitrogen

Eutrophication, or depletion of dissolved oxygen due to algal blooms, is caused by nitrogen and phosphorus depending on which is a limiting nutrient in an aquatic system. Ammonia is present in surface and wastewaters (30 mg/L) and at trace levels in groundwater (>10 μg/L) because of adsorption to soil and clays. It is the product of urea hydrolysis and deamination of organic nitrogen.88

1.2.2.3 Nitrite nitrogen

Nitrite is the product of ammonia oxidation or nitrate reduction and in acid can form nitrous acid, which reacts with secondary amines to form carcinogenic

47 nitrosamines. It is a common industrial anti-corrosive and is ubiquitous in industrial wastewaters.88

1.2.2.4 Nitrate nitrogen

Nitrate is present in surface water in trace amounts but is present in wastewater effluents at concentrates up to 30 mg/L because of nitrifying bacteria used in secondary treatment. Nitrate is an essential nutrient for photosynthetic autotrophs, or bacteria whose energy source is inorganic or fixed from other organisms, and is a limiting nutrient in ocean environments. It has been linked to methemoglobinemia in babies and infants where hemoglobin is oxidized into methemoglobin, which has a reduced capacity for oxygen and can lead to death.

Nitrate is usually regulated to levels below 10 mg/L.88,89

In aquatic systems, nitrogen occurs mainly as inorganic nitrate because of its high solubility in water and low potential to sorb to suspended solids and soils.

Ammonia is converted to nitrite and then nitrate. These compounds are also precursors to nitrosamines, which can be mutagenic, teratogenic and carcinogenic.89

1.2.2.5 Dissolved oxygen

Saturation of dissolved oxygen (DO0 water depends on temperature, chlorinity and atmospheric pressure. As temperature and chlorinity decrease and atmospheric pressure increases, DO also increases. DO is a measure of the capacity

48 of microorganisms to break down organics and a measure of the ability to support macroorganisms like fish. At anoxic DO levels below 2 mg/L, fish can no longer survive.88

1.2.2.6 Phosphorus

Phosphorus is a limiting nutrient in many aquatic environments and is found in both natural surface and wastewaters as orthophosphates, condensed phosphates and organic phosphorus. Phosphorus may be introduced into wastewater through cleaning detergents and runoff from use agriculturally as fertilizers. Discharge of effluent rich in phosphorus may cause algal blooms which consume the DO in the receiving waters.88

1.2.3 Organics

1.2.3.1 Chemical oxygen demand (COD)

COD is a measure of the organics within a sample of water or wastewater. Unlike biolological oxygen demand (BOD), which is a measure of the organics that are biodegradable, COD measures organics that can be broken down by a harsh chemical oxidant. Effluent with a high level of COD requires microorganisms to break these compounds down in receiving waters and result in DO depletion.88

1.2.4 Microbiological Examination

1.2.4.1 Heterotrophic plate count

Heterotrophic bacteria are bacteria that use organic molecules as a source of energy; their population may be indicative of the concentration of biodegradable organic compounds introduced to the MBR and the capacity of the MBR system for decomposing organic chemicals.88

1.3 Pesticide Treatment

1.3.1 Conventional Wastewater Treatment

The wastewater treatment plant (WWTP) in the Greek city of Agrinio (pop.

90,000) uses solid and grit removal for pre-treatment, anoxic treatment followed by an activated sludge secondary treatment, sand filtration and chlorination for tertiary treatment. Mean concentrations of chlorpyrifos, diazinon and malaoxon in the influent were 35.7 ng/L, 341 ng/L and 428 ng/L. These concentrations were reduced to 3.52 ng/L, 173 ng/L and 122 ng/L after secondary treatment, and to 0.21 ng/L, 13.6 ng/L and 84.6 ng/L after tertiary treatment. Maximum concentrations in influent were 1.86 μg/L, 1.48 μg/L and 292 ng/L for malaoxon, diazinon and chlorpyrifos respectively. Chlorpyrifos and diazinon both seem to be removed via sedimentation and sorption to suspended solids. Activated sludge degradation for chlorpyrifos and malathion were 26.2% and 91.5%. Chlorination of OPs results in the formation of oxons that resist further attack. Tertiary treatment removals were

21% for malaoxon while 91% and 80% removals were achieved for chlorpyrifos and diazinon.58

Activated sludge is commonly used in the treatment of domestic sewage as it contains fatty acids and low weight carbohydrates which are readily metabolized by bacteria.20 Malathion was degraded in an activated sludge reactor with 4 mg/L

DO. Malathion degrading Pseudomonas spp. was isolated for its metabolic activity, oxidative nature and aerobicity and degraded a 1000 mg/L solution of malathion by 90% within 29 hours of treatment.

A laboratory activated sludge setup consisted of an aeration chamber, denitrification chamber and clarifier. Mecoprop experienced a 2-week long lag time in the activated sludge treatment before a strong increase in biodegradation until nearly complete removal was achieved. With short application periods, high- loads of mecoprop did not undergo biodegradation by microorganisms that were struggling to adapt, and simply were discharged into receiving waters.52

1.3.2 Constructed Wetlands

Constructed wetlands initially rose to prominence because of their ability to treat point source pollution like waste leachates but are now gaining favour as a means of treating non-point source wastewater. A constructed wetland was built along a tributary of the Lourens River in South Africa between December 1998 and June

1999. Pesticides are removed through adsorption to particles, decomposition and metabolism by microorganisms. 77 to 93% of azinphos methyl was retained and no

51 detection of chlorpyrifos was made in the outflow effluent or sediment. Inlet sediment samples showed decreases in azinphos-methyl concentrations from 43

µg/kg to below detection limits. In addition to pesticides, total suspended solids, orthophosphate and nitrates were reduced by 78%, 75% and 84% respectively.90

1.3.3 Granular Activated Carbon

Granular activated carbon entrains pesticides through sorption but does not degrade them.91 Other substrates include soils, clay and microorganisms but due to its high porosity, large surface area and surface structure, activated carbon is used most widely. 100 mg of 6-16, H-type commercial granular activated carbon with point of zero charge, or the point where charge density on the surface of a particle is zero, pH at 8.2 was added to 2,4-D solutions of varying concentrations, pHs and temperatures. Adsorption decreased with increasing pH due to lowering of the surface charge on the activated carbon and changes in the degree of ionization of 2,4-D and slightly increased with temperature perhaps due to increases in pore size or the creation of new sites with bond rupture. Adsorption also increased with increasing 2,4-D concentration. Equilibrium was achieved within 4 to 6 days. Varying pH, temperature and initial 2,4-D concentration, the highest 2,4-D uptake capacity achieved was 518 mg/g at 45 °C under acidic conditions (pH 2.0).37

1.3.4 Ozonation

Ozone can be used to break down organic contaminants in wastewater. It can oxidize organics directly by forming radicals when used in conjunction with hydrogen peroxide. At pH 7 and below, direct oxidation dominates. For aromatic compounds, the breakup of the aromatic ring occurs readily before the oxidation of aliphatic substituents.92 Ozone produced from an ozonator was bubbled through a 100 μM solution of mecoprop. Increases in pH and ozone partial pressure result in a higher rate of oxidation while temperature increases result in a decrease in ozone solubility. At 20°C, pH 12 and an ozone pressure of 550 Pa, mecoprop was degraded in 6 minutes.93 Rates of 2,4-D ozonolysis also increased with the pH of the solution, the concentration of ozone, 2,4-D concentrations and when applied with UV irradiation. 2,4-D was completely degraded within two hours.92

Ozone has a number of advantages in treating contaminants in wastewater including: its capacity to oxidize organics quickly, bactericidal activity and its transformation to oxygen in water, though the transformation products of contaminant ozonolysis may be more toxic than the parent.92

Ozone can be formed from UV irradiation of oxygen or corona discharge where a current accelerates electrons into oxygen molecules breaking the O2 double bond resulting in an oxygen atom reacting with another molecule of O2. Ozone production increases with pressure, gas flow rate, lower temperature and when

53 oxygen is used as a feedstock instead of air.92 Disadvantages of ozone wastewater treatment include the cost associated with ozone production.92,94

Photolytic ozonation involves the application of ozone to directly degrade organics or through the formation of radicals in addition to irradiation with UV light. Costs associated with the generation of ozone and to power UV lamps are high. Ozone was bubbled through a 20 mg/L phorate solution at 50 L/hour and irradiated with

254 nm UV light at 31.2 W/m2. Within 20 minutes, phorate was degraded by 95% and this result was independent of pH conditions. The degradation rate did increase with ozone concentration in the bubbled gas stream.62

1.3.5 Photocatalytic oxidation

Photocatalytic oxidation involves the photo-excitation of valence electrons producing free electron holes.95

→

Resulting conduction band electrons and positive free electron holes generate radicals, the majority of which are a result of free electron hole oxidation at the surface of the titanium oxide.95

→

Reactions are limited by the necessity of the contaminant to bind to the TiO2 particle and recombination of the free electron hole and the photo-excited

95 electrons. The advantages of using TiO2 in photocatalytic reactions include its photo-stability, low toxicity and low expense.96

20 mg/L solutions of diazinon and diazoxon with 500 mg/L TiO2 (pH 6) were irradiated with 300-400 nm radiation. Within 30 minutes, complete degradation of diazinon was achieved and 10% mineralization, or complete conversion to inorganics and minerals, of the parent. Complete mineralization occurred after 5 hours. Controls of diazinon and TiO2 without irradiation did not result in a change in diazinon concentration.81

22 μg/L solutions of dicamba were added to 5 mg/L solutions of TiO2 and irradiated with UV lamps at 300 or 350 nm upon addition of hydrogen peroxide.

Photolysis rates were 3-5 times higher when TiO2 was added compared to irradiation alone and is due to radical induced free electron hole oxidation rather than adsorption to TiO2 particles. Rates increase with increasing pH largely due to greater hydroxyl concentrations and electrostatic attraction between TiO2 and dicamba that dissipates above the zero charge of TiO2 above pH 6. The addition of hydrogen peroxide generates hydroxyl radicals through direct photolysis by UV irradiation and additionally through reaction with photo-excited electrons. This addition can increase the reaction rate up to an optimal concentration before scavenging occurs.95

A 20 W black light blue fluorescent tube irradiated a photoreactor containing 175 mg of TiO2 and 1 L solution (pH 4) of 2,4-D at 10 mg/L. Mineralization was 96.8% within seven hours and the half-life for 2,4-D was calculated to be 1.36 hours.

Microtox toxicity tests measuring Photobacterium phosphoreum inhibition

55 indicated the formation of toxic transformation products increased after irradiation began peaking at the hour mark before steadily decreasing.97

1.3.6 Fenton’s Reagent

Fenton’s Reagent is the combination of ferrous ions and hydrogen peroxide that in acidic conditions produces highly oxidative hydroxyl radicals.70

→

Advanced oxidation processes readily mineralize organic contaminants of all classes.23 Hydroxyl radicals reduce COD and enhance biodegradability potential by indiscriminately breaking down organic molecules. The use of Fenton’s Reagent is advantageous because of its low power consumption compared to other advanced oxidative processes and the use of non-toxic materials in hydrogen peroxide and iron. Acidic conditions are required for the formation of radicals and would otherwise require use of large volumes of acid but for the acidity of the pesticide wastewater itself.70

1.3.6.1 Fenton’s reagent & enzymatic treatment

Fenton’s Reagent can be coupled with enzymatic treatment where OP hydrolase isolated from E. coli breaks down residual concentrations of OPs. Optimal Fenton conditions included an addition of 2.7 mM hydrogen peroxide, 4:1 ratio of ferrous ions to hydrogen peroxide and a pH of 3.5 which resulted in an 80% removal within 15 minutes. Pure hydrolase is difficult to isolate but "whole cell enzymes" that required transport of parathion across the cell membrane resulted in a 25% degradation efficiency after 14 minutes. 90% degradation was achieved using

56 enzymes from lysed cells in the same time period. The overall removal efficiency was 95%.94

1.3.6.2 Photo-Fenton reaction

Photo-Fenton oxidation enhances the efficiency of Fenton degradation by reducing ferric ions to ferrous ions that are then available to react with additional hydrogen peroxide. The use of ferrioxalate instead of ferrous ions allows for Fenton oxidation to occur with solar radiation up to 550 nm thereby reducing power consumption associated with ultraviolet light. The photolytic contribution to degradation is minimal in comparison to hydroxyl radical degradation.55 Light penetrates further with longer wavelengths compared to UV irradiation. Low pH is necessary for catalysis and iron removal is required.

1.3.6.3 Electrochemical Fenton’s oxidation

Electro-Fenton oxidation involves the breakdown of organics using ferrous addition combined with a Pt (anode) and polytetrafluoroethylene (PTFE) O2

(cathode) electrolytic cell which produces hydrogen peroxide. Fenton’s Reagent generates hydroxyl radicals. Ferrous ions are regenerated by cathodic reduction.

Electrochemical Fenton oxidation produces ferrous ions and hydrogen peroxide but requires neutral conditions, which limits its degradation efficiency.55

1.3.6.4 Photo-electrochemical Fenton’s oxidation

Photo-electroFenton oxidation uses UV irradiation to regenerate ferrous ions and photo-degenerate organo-ferric complexes.

→

Ferrous ions were added to a 230 ppm solution of 2,4-D at a concentration of 1 mM. The solution was subjected to electrolysis at 450 mA and UV irradiation. In

20 minutes, 2,4-D was degraded. Intermediates included 2,4-dichlorophenol, chlorohydroquinone ,4,6-dichlororesorcinol and chlorobenzoquinone and breakdown after dechlorination into glycolic, maleic, fumaric and oxalic acids.83

Mecoprop of 100, 200, 375, and 640 mg/L concentrations with 0.5mM ferrous ions at pH 3.0 and solar light irradiation using photoelectron-Fenton oxidation degraded completely in 15, 20, 30 and 50 minutes. The photoreactor consisted of a

Boron doped diamond anode and carbon-PTFE O2 diffusion cathode fed with O2 to produce hydrogen peroxide.44

1.3.6.5 Cathodic and anodic Fenton’s oxidation

In anodic Fenton oxidation, a current was applied between iron (anode) and graphite (cathode) electrodes connected by a salt bridge. 2,4-D was added to the anodic cell (which is under acidic conditions) and ferrous ions were donated from the electrode upon application of the current. Hydrogen peroxide was added (10:1

H2O2:Fe) to the anodic cell forming Fenton’s Reagent. Under a 0.010A current, a

200 μM 2,4-D solution was degraded by 90% within 10 minutes via an sigmoidal curve. As the temperature of the reaction vessels was increased, so to was the degradation rate.55

1.3.7 Gamma Irradiation

Basfar et al. used gamma-ray irradiation from radioactive cobalt to degrade ppb level concentrations of diazinon finding 90% degradation with acetic and formic acids as final by-products.98

50 – 2000 μM concentrations of 2,4-D were gamma-irradiated using a 60Co gamma source. 90% conversion of organic chlorine to chloride was achieved using 10 kGy

(kilograys) dose. With ozone bubbling, the same result was achieved at a lower 2.7 kGy dose.99

1.3.8 Ultrasonic Wave Treatment

A novel method that is beyond chemical oxidation and photocatalytic methods was reported by Matouq et al. in 2008.100 Using high frequency ultrasonic waves,

800  1800 ppm solutions of diazinon in simulated industrial wastewater was degraded by 70% within 600 seconds.

1.3.9 Bioremediation

Some pesticides and their metabolites may deactivate microorganisms and decrease biodegradation;94 however, enzymes from bacteria that are able to metabolize OPs can be used to treat them. Organophosphorus hydrolase from

Pseudomonas diminuta or Flavobacterium spp. can degrade a broad spectrum of

OPs.

1.3.9.1 Activated sludge

Biologic treatment is advantageous because it is inexpensive, no chemicals are added and the diversity of bacteria can degrade organic compounds of various classes and types. Species found in activated sludge include Pseudomonas,

Flavobacterium Achromobacter, Rhomobacterium, Azobacter, Micrococcus, Bacillus alkaligenes, Arthrobacter, Ycobacterium Aeronomonas, Nocardia and

Lophomonas.47 However, the presence of more than one carbon source may enhance or repress degradation of target compounds.38

1.3.9.2 Sequencing batch reactors

Two sequencing batch reactors were seeded with phenol degrading microorganisms and WWTP active sludge and fed phenol and dextrose respectively for two months. After this acclimatization period, 2,4-D was added to the two reactors at 40 mg/L concentrations. Phenol and dextrose degradation rates decreased before complete removal was again observed on Day 20. No degradation of 2,4-D was observed until Day 80 when rates of 50% were observed for 10 days.

The hydraulic retention time (HRT), or the average time that the soluble compounds remain in a bioreactor, was increased from 16 h to 48 h and within three days and 5 weeks respectively, degradation in the dextrose and phenol reactors approached 99% 2,4-D removal. A third reactor was seeded with sludge from reactors 1 and 2 and 2,4-D was added the sole carbon source and complete degradation with no lag time was observed.38

1.3.10 Metabolite Treatment

Chlorophenols can be degraded biotically and abiotically through advanced oxidation processes such as Fenton’s oxidation, ozone/UV processes and photocatalytic oxidation.47,49 2,4-dichlorophenol is degraded anaerobically to 4- chlorophenol which in turn is degraded to benzoate, phenol and acetic acids. The process takes days to months to complete.49

Acclimated activated sludge is more effective at degrading phenolic compounds than pure strains by 10 – 100 times. Immobilized cells such as with a fluidized bed bioreactor perform 66% better than freely suspended cells. Phenol degradation increases with temperature to an optimum near 30 °C. pH significantly affects decomposition; degradation is optimal at pH 7 but chlorophenol metabolite concentrations will cause the pH to decrease. The surface of bacteria carry a negative charge over a pH range from 3 to 10 and when contaminants are negatively ionized the electrostatic attraction between cells and contaminants is lost. To acclimatize activated MBR sludge to phenols, phenol concentrations were gradually increased over seven days and glucose decreased until phenol was the only carbon source. The MBR activated sludge (10 g/L) was acclimated to 3 g/L phenol concentrations over a four month period. Inorganic minerals were added during the acclimation step.47

1.3.11 Pesticide Wastewater Treatment

Pesticide wastewaters are high in COD and low in BOD. 70 Biological treatment is inexpensive but ineffective because of the low biodegradability of the influent.

Wastewater from a Bayer 93820 OP pesticide factory was acidified using sulfuric acid to pH 0.8 and refluxed for an hour. Under acidic conditions, a nitrogen atom is protonated promoting nucleophilic attack of the central phosphorus and ejection of ammonia followed by two other substituents about the central atom.

The final product is thiophosphorusic acid. 97% removal of the OP pesticide was achieved in an hour in addition to 93% removal of sulfides and 56% reduction of

COD. To remove the 80% residual ammonia and the inorganic phosphates, 15% calcium hydroxide was added and the solution was refluxed for 25 minutes. The result was a more biodegradable wastewater, COD below 22,000 mg/L, OP concentrations below 260 mg/L and ammonia below 1400 mg/L. A disadvantage was the severity of treatment conditions.60

Pesticide wastewater, from a phorate manufacturing facility in Hebei province,

China, was subjected to Fenton’s Reagent treatment coupled to moving-bed biofilm reactor (MBBR). MBBR involves biomass growth on bio-carriers that circulate through the reactor through mechanical stirring or aeration. COD is reduced and biodegradability is enhanced during Fenton’s treatment allowing for a more efficient biological treatment step.70 For example, the pH of an MBBR was adjusted using calcium hydroxide to neutral conditions for bacterial growth

62 inducing the precipitation of ferric and phosphate ions. The wastewater had a

COD of 33,700 mg/L, a BOD5/COD ratio of 0.18 and OP concentrations of 2,040 mg/L. Hydroxyl radicals attack the thiosulfyl double bond forming sulfate ions and phosphate ions form after the oxidation and mineralization of intermediate organic molecules.70 After Fenton’s oxidation, the COD R to 12,000 mg/L, the OP reduced by 98% and biodegradability enhanced to a BOD5/COD of 0.47. After

MBBR treatment, COD was reduced to 500 mg/L, an overall reduction of 85%.70

1.4 Membrane Bioreactor

1.4.1 Introduction to Membrane Bioreactor

Membrane Bioreactors (MBR) combine the degradation potential of microorganisms with the filtration ability of membranes.101 Membranes can be immersed in the bioreactor (submerged) or be exterior (crossflow) where liquor is circulated to the membranes.101,102 Power consumption for crossflow reactors is generally higher than for submerged reactors because of the necessity to recirculate MBR liquor to the membranes and back to the bioreactor.101

Higher biomass concentrations can be achieved in MBR because of membrane separation.103 Biomass in conventional wastewater treatment is limited to biomass concentrations of 5 g/L while in MBR, biomass can be up to six times higher. The higher biomass concentration and retention of suspended solids eliminates the need for a settling tank and reduces bioreactor size.104 As suspended solids are completely retained, organic substances tend to accumulate on the sludge and

63 bacteria, and acclimatize to the compounds in the influent yielding more efficient mineralization of compounds.103 MBRs offer high sludge retention times (SRTs) which are correlated with higher rates of degradation as compared to conventional wastewater treatment.103

MBR bioreactor sludge has several advantages over sludge from conventional wastewater treatment. The sludge is better able to acclimate to contaminants in the influent because of a higher SRT, it has greater biodiversity in microbial species and there is more sludge to begin with. Removal of contaminants in the

MBR occurs through adsorption, biodegradation or both. In many cases, hydrolysis, volatilization and photolysis are too slow to occur given the hydraulic retention time (HRT).105 Biodegradation increases with sludge age with biodegradation peaking for some select pharmaceuticals with 60 – 80 day old sludge.105

MBR offers several advantages, including a separation of HRT and SRT, longer SRT resulting in more specialization, lower expenses due to operation at lower trans- membrane pressures, slow-growing microorganisms that are able to remove otherwise recalcitrant organics and greater consistency of effluent despite fluctuations in influent contaminants. Disadvantages include the expense associated with membrane purchase and maintenance, relatively low permeate flux, monitoring costs in addition to pressure, and pH limitations.103,104

Ultrafiltration membranes are able to limit bacteria and remove many viruses producing bacteria-free effluent. Merlin detected 95% removal of a MS-2 indicator virus that has a diameter of 25 nm. Pore sizes may be reduced by the formation of cake layers through influence of the membranes on the elimination of the pesticides is insignificant.102

1.4.2 The History of Membrane Bioreactors

The first application of MBR to wastewater treatment was by the Dorr-Oliver

Corporation in the United States as a substitute for other methods of tertiary water treatment. Membrane manufacturing costs and the inconvenience of maintenance resulted in stunted commercial growth of MBR until the early 1990s. Fouling, where flux is reduced by organics and microorganisms adhered on to the membranes, was reduced with the advent of submerged or immersed MBR by

Japanese group Yamamoto et al. Canadian company Zenon developed its first submerged MBR in 1992. The thermally induced phase separation (TIPs) method for membrane manufacture was applied by Zeng et. al in 2007 significantly reducing MBR membrane maintenance requirements and doubling permeate flux rates to 80 L/m2 h.106

Installations of pilot MBR plants began in the late 1990s with plants in England,

San Diego and Shanghai, with a record 80 installed by 2002. The largest MBR- based municipal wastewater treatment plant is in Kaarst Germany102 MBR brought in 40 million euros in 2005, and is growing by 9% annually. There are currently

65 nine major manufacturers in Europe and over 60 distributors. Growing markets include stadiums and shopping centres, especially in areas where water stress exists.102

1.4.3 Membrane Fouling

With size selective pores, bacteria, colloids and macrosolutes such as extracellular polymeric substances (EPS), all remain in the MBR and are active contributors to membrane fouling and the formation of cake layers.107 Cake layers are formed by bacteria colonies, at the interface between the mixed MBR liquor and the membrane surface, and the accumulation of EPS, which provides support.108 Trans- membrane pressure is monitored closely as an indicator of the degree of membrane fouling. Increased fouling produces sharp increases in the trans- membrane pressure required to maintain constant flow/flux conditions.107 52 - 60% of resistance caused by fouling can be attributed to dissolved matter, with colloids accounting for 25% and particulates for 23%.104

Aeration can be used to scour the formation of cake layers on the membrane surface.101 These cake layers are responsible for increases in trans-membrane pressure and require chemical cleaning or backflow to dislodge fouling.101 Cake layer removal is accelerated with increasing aeration flow to an optimal value and reduction of aeration has significant impacts on trans-membrane pressure.

Concentrating airflow and the membranes over a small area increases aeration intensity without increasing flow, and can also improve fouling prevention

66 efficiency.101 Fouling reduces the filtrate flux and increases membrane area requirements.109

The advantages of an aerobic MBR besides the higher DO concentrations in the effluent include higher COD removal rates and slower membrane fouling rates.

Comparing two MBRs operating at 6.0 mg/L and <0.3 mg/L of DO, COD removal rates were 94.8% and 27% respectively. Membrane fouling occurred five times faster in the anaerobic bioreactor despite scouring using N2 and a lower suspended solids concentration. The structure of the cake layers formed is also affected by

DO levels. Aerobic biofilms tend to have stronger microbial cohesive forces than adhesive forces to the membrane. In anaerobic systems, the opposite is true. The result is a more compact cake layer in anaerobic films, which is a less porous and more evenly spread out film.108

Many measures can be put in place to prevent or slow membrane fouling such as backwashing, air scouring, flux operation below critical levels, chemical washing and coagulant addition.109,110 Fixing microorganisms on support media and decreasing microorganisms in the mixed liquor is another such proposal.109 With an 8 h HRT, COD was reduced by 99% in the attached growth MBR and 98% in the suspended growth MBR. Similarly, ammonia was reduced by 95% and 99% respectively. While both systems were effective at treating wastewater, attached growth systems reached critical trans-membrane pressures seven times faster than with suspended growth systems.109

The presence and release of EPS from floc breakage can be offset by adsorption to powdered activated carbon (PAC). PAC addition to submerged MBRs can reduce cake layer formation and can extend operating times by a factor of three.110

Activated carbon also removes small and less biodegradable organic compounds that would otherwise be able to permeate untreated through membrane pores.

Sludge recirculation in cross-flow MBR causes reduction in floc size. As a consequence, EPS and other colloidal and soluble components are released into the mixed liquor, enhancing cake layer formation. Submerged MBRs, in which the liquor and the membranes are in the same bioreactor, address these disadvantages.110

Membrane fouling is a major contributor to MBR operating costs and is affected by aeration, flux rate and floc properties. Above critical trans-membrane pressure, membrane fouling has significant effects on permeate flux and can be irreversible, requiring chemical cleaning.110 Membrane costs account for 70% of MBR plant costs and replacement for 65% for operating costs.

1.5 Objective

The objective of this work then is to develop economical, environmentally friendly method to treat chronically occurring, low-level concentration of pesticides to limit human exposure.

2 Materials and Methods 2.1 Membrane Bioreactor

The MBR used in this work consists of five SuperUF membrane units (Superstring

MBR Technology Corp, New York) submerged in a 130 L glass tank. The membranes have pores with diameters between 20 and 30 nm, operate at trans- membrane pressures of 10 – 20 kPa and mixed liquor suspended solids of 5,000 –

15,000 mg/L.106

Ten litres of activated sludge from the Lakeside/Timberlea WWTP was added to the tank in October 2009 with no removal of sludge prior to the beginning of the experiment. Two major experiments were performed. The first was an OP pesticide experiment in May 2010 followed by a PA herbicide experiment from February 2011 to June 2011.

2.2 Experimental Setup

2.2.1 Wastewater Tank

The first iteration of the wastewater tank consisted of a 40 L glass aquarium with a removable plastic/plexiglass lid. The wastewater solution in the tank consisted of complex nutrients such as peptone, yeast extract and meat extract that cannot be defined chemically, glucose, main elements and trace elements. The constituents of the wastewater solution, the concentrations of the components and the mass introduced to the MBR are given in Table 18.

Table 18. Wastewater solution composition

Organics Formula Mass Nutrient Flow Rate Solution into MBR Concentration (mg/day) (mg/L)

Glucose C6H12O6 11.21g 373.7 1,506 Peptone Complex 5.60g 187 752 Yeast Extract Complex 44.8g 149 602 Meat Extract Complex 11.2 37.3 150

Initially, the wastewater solution was pumped through a UV filter and back into the nutrient solution continually however bacterial growth in the tank persisted.

This setup was later adapted such that the UV lamp was directly immersed in the nutrient tank solution with pumps used to circulate solution about the lamp.

Additionally, three gases were used to blow down the surface of the nutrient solution in an attempt to displace oxygen. Argon, carbon dioxide and nitrogen were all attempted before settling on an argon blowdown because of its inertness.

Additionally, the growth on the surface of the nutrient solution was removed daily to minimize bacterial growth.

Variable flow peristaltic pumps (Fisher Scientific Ltd, Ottawa, ON) were used to pump the nutrient solution into the MBR at 3.0 mL/min. The tubing about the rotor of the pump was changed weekly, and a daily back-flush of the entire length of the tubing was performed to minimize the accumulation of solids in the tubing.

For the PA pesticide experiment, the wastewater tank was changed to a 4L solvent bottle. The solution was replenished daily and concentrations adjusted to the new

70 wastewater tank volume and flow rates. The nutrient bottle was situated in a

Coleman cooler that contained water that was circulated to a chiller ensuring a constant temperature of 4 °C. Argon flushing was maintained but the UV system was removed because bacterial growth was better minimized through daily wastewater replenishment and cooling.

2.2.2 Intermediate Tank

The intermediate tank consisted of a 10 L plastic tank with a float valve. Cold water from the tap entered the intermediate tank and water was pumped to the MBR via gravity siphoning. The float valve ensured that the volume of water in the tank remained at a constant 8 L. The purpose of the intermediate tank was to dilute the concentration of pesticides to low ppm levels and replenish the water lost in the

MBR to effluent.

2.2.3 Pesticide Tank

The pesticide tank in the OP experiment consisted of a 4L glass solvent bottle. The pesticide solution was replenished every seven days. Granular Thimet 15-G and powdered Sniper® were dissolved into 200 mL of methanol and vigorously shaken.

Water was added to the pesticide tank before adding the Thimet/Sniper solution and additions of Malathion 50 EC, Diazinon 500 E and Lorsban 4E emulsifiable concentrates. The concentration of the pesticide solution was in the part-per- thousand range. The pesticide solution was pumped into the intermediate tank for dilution to a concentration of 5 mg/L for all active ingredients.

In the PA pesticide experiment, the pesticide tank was combined with the wastewater tank and pesticide concentrations were replenished daily by spiking 1 mL of Weedex® into 4L of wastewater solution. The pesticides and wastewater were pumped directly into the MBR.

2.2.4 Peristaltic Pumps and Flow Rates

The effluent outflow rate was set to 5.4 L/hour and the level in the MBR was maintained using a float valve in the intermediate tank.

The HRT for the experiment was 21 hours. Peristaltic pumps were used to pump the pesticide and wastewater solution into, and effluent out of, the MBR. The experimental set up and flow rates for the system are shown in Error! Not a valid bookmark self-reference..

Wastewater 3.0 mL/min Tank Membrane 90 mL/min Bioreactor

Pesticide 0.4 mL/min Intermediate 87 mL/min Tank Tank

Tap Water 86.6 mL/min

2.2.5 Aeration Spargers Figure 5. MBR setup and flow rates

2.2.6 Temperature Controls

Temperature in the tank was kept at constant 25 ±1 °C using an automatic titanium alloy heater immersed below the surface of the water in the MBR with an external remote thermostat. A thermometer with digital reading exterior to the MBR was also employed.

2.2.7 Aeration

A copper coarse bubble diffuser/sparger was made in-house using a series of four copper pipes, each with holes 1 cm in diameter spaced at 2 cm apart. The system was aerated at 1400 L/hour to replenish the DO required by microorganisms for metabolism and growth, and to avoid fouling of the membranes by bacteria and organic contaminants that would otherwise strongly adhere to the surface of the membranes. Air travelled from an air tap to a compressor where any liquid water present in the airlines was allowed to condense followed by an additional water filter. Water was drained from these taps every two weeks. Shear forces, or forces applied perpendicularly to a surface, induced by aeration can control cake layer formation.102

2.2.8 Sampling

Effluent samples were drawn daily from the MBR effluent flow. Tubing from the peristaltic pump that led to the effluent tank was diverted to a 1 L glass amber bottle with 0.1 mm PFTE lined polyethylene caps. Bottles were filled air-tight.

These samples were stored at 4 °C for a week and as a precaution for use when

73 reanalysis was required. Effluent samples for chemical, chemopotentiometric and spectrophotometric tests were also drawn with tests performed immediately.

2.3 Analytical Methods

2.3.1 Solid Phase Extraction

Solid phase extraction is a process used to concentrate our analytes and transfer them from an aqueous matrix to an organic solvent that is able to be injected into the GC. Sorbents are prepared by wetting it with solvents. Then a 100 mL sample of effluent, whose pH has been adjusted to ensure compounds are in their neutral unionized form, is aspirated through the column at 3 mL/minute. Analytes will adsorb onto the sorbent column because they have a greater affinity for the sorbent packing than the mobile aqueous phase. A weaker eluting solvent is used to remove some of our interferents. And finally the analytes of interest are eluted using an organic solvent leaving behind other interferents on the column. The result is a more concentrated, cleaner sample in a desirable matrix.

2.3.2 Gas Chromatography

Gas chromatography (GC) was the method used for separating all of the organophosphorus compounds in the extract that were obtained from the solid phase extraction process. A small amount of sample is drawn, injected and heated until the compounds become gases. A constant flow of Helium sweeps the particles through a 30 metre long column. Compounds are separated based on size and affinity to the column.

2.3.3 High Pressure Liquid Chromatography

For the phenoxyacetic acid compounds, high pressure liquid chromatography

(HPLC) for separation because their far more polar and tend to stick irreversibly to the GC column thereby limiting response. Compounds are separated based on their affinity for the column packing and the mobile phase.

2.3.4 Mass Spectrometry

Mass spectrometry (MS) is a way of detecting the compounds. As the compounds leave the GC or LC column, they are bombarded by high energy electrons (EI impact) or subject to a coronal discharge (APCI) which break up the molecules in a characteristic way. Each of the pesticides can be identified by the ions that they fragment.

2.3.5 Quantitation

For both experiments we used a five point calibration with a detection range from

10 ug/L to 1000 ug/L using standards with known concentrations of pesticides. We used surrogate standards to evaluate the efficiency of the extraction process and internal standards to account for variation within the instrument.

2.4 Materials and Standards

2.4.1 OP Pesticide Experiment

The five target OP pesticides chosen for study were all available in a Canadian

Drinking Water OP Pesticide Standard Mix (>95% purity) obtained by

AccuStandard (New Haven, CT.) They were chosen for their persistence, their detection in Canadian surface waters, status of registration by the PMRA and listing in the Canadian Drinking Water Quality Guideliens. EPA 8141A surrogate standards (SS), tributylphosphate and triphenylphosphate, were also obtained from AccuStandard (New Haven, CT.) Finally, an EPA 8270 Semivolatile Internal

Standards (IS) mix containing Chyrsene-d12, Perylened-12 and Phenanthrene-d10, were obtained from Supelco (Bellefonte, PA). Pesticide formulations containing the five target pesticides are shown in Table 19.

Table 19. OP pesticide formulations, active ingredients and manufacturers

Active Ingredient Formulation PCP Manufacturer United Agri Sniper Azinphos- Products Canada Azinphos methyl PCP 23323 methyl Insecticide Inc., Dorchester, ON Lorsban-4E Dow AgroScience Chlorpyrifos PCP 14879 Insecticide Canada, Calgary, AB United Agri Diazinon 500 E Products Canada Diazinon PCP 11889 Insecticide Inc., Dorchester, ON Wilson Malathion Nu-Gro Inc., Malathion PCP 16099 50 EC Insecticide Brantford, ON Thimet 15-G Soil & Amvac Chemical Phorate Systemic Granular PCP 10532 Corporation, Insecticide Toronto, ON

Ethyl acetate, methanol, hexanes and toluene used in the solid phase extraction of effluent and for gas chromatography were all HPLC grade solvents and were obtained from Caledon Laboratories (Georgetown, ON). Argon for wastewater tank blowdown was obtained from Praxair Productions Inc. (Bridgewater, NS).

2.4.2 PA Pesticide Experiment

2,4-D, mecoprop, dicamba, and 2,4-dichlorophenol (>98% purity) were obtained from Accustandard (New Haven, CT.) 3,6-dichloro-2-hydroxybenzoic acid (98% purity) was obtained from AK Scientific Inc. (Union City, CA).

Chlorohydroquinone (90%) was obtained from Acros Organics (Geel, Belgium).

2,5-dichlorophenol (99.7% purity), 4-chloro-2-methylphenol (99.4% purity), o- cresol (99.9% purity) and methylhydroquinone (99% purity) were obtained from

Sigma Aldrich (Oakville, ON). The three herbicides chosen for study were all available in a single domestic herbicide mix, Concentrated WeedEx: Weed Control for Lawns at varying concentrations (Table 20). The active ingredients were chosen for their persistence, solubility in water, ability to leach, usage across Canada and

Nova Scotia and inclusion in the CCME water quality guidelines for the protection of aquatic life and water for agricultural uses.

Table 20. Concentration of herbicides in concentrated Weedex

Active Concentrations Formulation PCP Manufacturer Ingredient 2,4-D 90 g/L Concentrated PCP Teragro Inc., Dicamba 9 g/L WeedEx 28204 Chestermere AB Mecoprop 50 g/L

Ethyl acetate and methanol used in the solid phase extraction of effluent and for liquid chromatography were both HPLC grade solvents and were obtained from

Fisher Scientific (Ottawa, ON). Ammonium hydroxide HPLC buffer was obtained from ACP Chemicals (Montreal, QC). Argon for wastewater tank blowdown and

77 nitrogen for SPE solvent evaporation were obtained from Praxair Productions Inc.

(Bridgewater, NS).

2.5 Chemical Analysis

2.5.1 Solid Phase Extraction

One hundred mL of sample were measured using a graduated cylinder (± 5 mL) and brought to a desired pH. Samples that were refrigerated were allowed to equilibrate to room temperature prior to pH adjustment. Effluent samples were extracted daily using 3 mL Chromabond® HR-P cartridges with 200 mg absorbed weight (Macherey-Nagel GmbH & Co. KG, Düren, GER). The column was packed with a polystyrene-divinylbenzene adsorbent resin and particle sizes ranging from

50 – 100 m. HR-P cartridges were conditioned with column volumes of ethyl acetate, methanol and then distilled water. Samples were aspirated at 3 mL/min and the cartridges were dried under vacuum for 30 minutes.

2.5.1.1 OP Insecticide SPE

100 mL of effluent was acidified to pH 6 using 3 M phosphate buffer, spiked with surrogate and aspirated under vacuum. OP analytes were eluted using 2x2 mL of a

50:50 (v/v) ethyl acetate:hexane mix and transferred to two GC vials.

2.5.1.2 Phenoxyacetic and Benzoic Acid Herbicide SPE

Effluent samples were acidified to pH 1.3 using 3 M sulfuric acid and aspirated by gravity. OP analytes were eluted using 2x2 mL of ethyl acetate. Eluent was evaporated to 1 mL using nitrogen blowdown at 50 °C and transferred to a GC vial.

2.5.2 Gas Chromatography – Mass Spectrometry

Analysis of the OP insecticides was performed on an Agilent GC/MS (7890/5975) using a 20m Agilent J&W non-polar ultra-inert fused silica DB-5ms (5% phenyl/dimethyl arylene siloxane) GC capillary column with a 180 μm internal diameter and a 0.18 μm film thickness. Injection proceeded via a pulsed-splitless mode with a 2 mm diameter glass liner and an inlet temperature of 280 °C. A sandwich injection mode, where 0.4 μL of extract and 0.1 μL of internal standard were drawn and separated by an air gap, were injected using an Agilent autosampler (7893A). The concentrations of the internal standards, phenanthrene-

D10 and chrysene-D12, were 500 μg/L and the desired recovery for the surrogates, tributylphosphate and triphenylphosphate, was 250 μg/L. Ultra-high purity (UHP) helium, at 0.5 mL/min flow rate, was used to sweep the analytes through the GC column. Temperature programming consisted of an initial oven temperature of 120

°C held for one minute followed by a 10 °C/min temperature gradient ramp to a final temperature of 300 °C, which was held for four minutes. The temperatures of the GC-MS interface, electron impact (EI) ion source and quadrupole were set to

275 °C, 230 °C and 150°C, respectively. Selective ion monitoring was used to improve the sensitivity and accuracy for the OP target analytes. Calibration was performed using a five-level calibration curve with 10 μg/L, 50 μg/L, 200 μg/L, 500

μg/L and 1000 μg/L concentrations. A quadratic regression was performed with a forced origin. The retention times and ions used for quantitation and qualification are shown Table 21.

Table 21. OP pesticide retention times, quantifier and qualifier ions

Compound Retention Primary Secondary Time (min) Ion Ion (m/z) (m/z)

Phenanthrene-D10 (IS) 9.197 188 189

Chrysene-D12 (IS) 15.440 240 236

Tributylphosphate (SS) 7.567 99 155

Triphenylphosphate (SS) 14.826 326 325

Phorate 8.160 260 121

Diazinon 9.173 199 179

Malathion 10.920 173 158

Chlorpyrifos 11.040 199 197

Azinphos-methyl 16.082 160 132

2.5.3 Liquid Chromatography – Mass Spectrometry

Analysis of the PA herbicides was performed on an Agilent LC/MS/MS (1200/6410) using APCI ion source on a 25 cm long Allure® C18 column with 4.6 mm internal diameter and 5 μm spherical particle and 60Å pores. The injection volume was 4

μL. The aqueous solvent (A) consisted of ultra nanopure water with 0.1% NH4OH and the organic solvent (B) was methanol. Initial solvent ratios were 65:35 A:B which was ramped to 100% B in 15 minutes. The flow rate was 0.5 mL/minute and the total run time was 20 minutes with a 5 minute post time.

The gas and vaporizer temperatures were both 350 °C, with a gas flow rate of 5

L/min and a nebulizer pressure of 50 psi. The voltage applied to the capillary was

3000 V with a current of 15 μA. The temperature of the MS was 100 °C. Primary ions, retention times and fragment voltages are shown in Table 22.

2.5.4 Quality Assurance/Quality Control

Blank samples were prepared and extracted using SPE to test for contamination of the sample preparation apparatus and solvents. Control samples were prepared and spiked with OP pesticide standards to assess the recovery of the OP analytes.

All samples were spiked with surrogate to evaluate extraction efficiency. All surrogate and control recoveries were measured within 80% to 120% after optimizing the sample preparation, separation and detection procedures or the samples were re-tested.

2.6 Spectrophotometric Analysis

Spectrophotometric testing using a HACH 3000 DR spectrophotometer and

TNTplus vials of COD (TNT821), reactive and total phosphorus (TNT843), nitrate

(TNT835), nitrite (TNT839), ammonia (TNT830), and total nitrogen (TNT826).

Turbidity, colour and suspended solid readings were obtained on a HACH 2800

DR spectrophotometer (HACH Canada, Mississauga, ON).

For turbidity, colour and suspended solids, a 25 mL glass cuvette was rinsed and filled with distilled water for use as a blank. A second cuvette was rinsed and filled to 25 mL with effluent from the MBR. The appropriate method was chosen on the

HACH spectrophotometer and the distilled water blank was zeroed followed by as a result of the sample. 88

Table 22. PA pesticide retention times and quantifier ions

Compound Retention Primary Fragmentor Time (min) Ion Voltage (m/z)

Dicamba 5.336 175 70

3,6-DCSA 7.308 205 80

CHQ 8.247 142 100

2,4-D 9.632 219 100

Mecoprop 10.904 213 100

2,5-DCP 11.310 161 100

MHQ 13.478 122.1 100

2,4-DCP 14.237 161 100

O-Cresol 16.550 107 100

4-Cl-2-MP 18.990 141 100

2.7 Potentiometric Analysis

2.7.1 pH

pH measurements for the OP experiment were taken with two pH probes: a

Milwaukee SM801 portable pH/EC/TDS Smart Combined meter and an Oakton waterproof pHTestr 30. pH measurements for the PA experiment were taken with a HACH pH probe. The pH probes were calibrated with three NIST standards on a weekly basis. pH measurements were taken once daily.

2.7.2 Dissolved Oxygen

DO measurements for the OP experiment were taken with a portable Milwaukee

SM600 dissolved oxygen (DO) meter. Zero calibration was performed once a month with a zero-oxygen solution and slope calibration was performed weekly in saturated air.

A stock solution of 500 mg/L of cobalt (II) chloride (CoCl2) in distilled water was prepared. A final zero-oxygen solution consisted of adding 100 mg of sodium

88 sulfite (Na2SO3) to 100 mL of the cobalt (II) chloride stock solution. The final solution was deactivated through oxidation due to exposure to air and was used to calibrate the DO probe within 30 minutes of preparation.

For the PA experiment, a HACH DO probe was used. For both experiments, DO measurements of the effluent were taken daily.

2.7.3 Conductivity and Total Dissolved Solids (TDS)

Conductivity and total dissolved solids TDS measurements for the OP experiment were taken with two pH probes: a Milwaukee SM801 portable pH/EC/TDS Smart

Combined. Conductivity/TDS measurements for the PA experiment were taken with a HACH TDS probe. The TDS probes were calibrated weekly using a HACH

TDS standard. TDS measurements were taken once daily.

2.8 Microbiological Analysis

2.8.1 Heterotrophic Plate Count

Plate count agar was made according to the formula presented in the Standard

Methods for the Examination of Water and Wastewater. 88 Plate count agar was made from 5.0 g of peptone, 2.5 g of yeast extract, 1.0 g of glucose and 15.0 g of agar dissolved in 1 L of distilled water. Samples from the MBR were taken, serially diluted and inoculated onto agar plates, as per the standard method. Plates were counted after a two-day incubation period at 35 °C. Aseptic techniques were adhered to for all steps in the procedure.

2.9 Statistical Analysis

Statistical analysis was performed on the data using Microsoft® Excel (Microsoft

Corporation., Redmond, WA), Minitab® 16 (Minitab, Inc., State College, PA) and

Origin® 8.6 (OriginLab Corporation, Northampton, MA) software. Exponential decay regressions were performed on the pesticide curves to determine values for half-life and baseline concentrations for treatment efficiencies using Origin® 8.6.

Correlation studies were performed to determine the significance if any of correlations between parameters in the data using Minitab®. All remaining graphs were made using Microsoft® Excel.

3 Organophosphorus Insecticides 3.1 MBR Setup

3.1.1 Wastewater Tank

CODs of real wastewater influents will change over time depending on a number of conditions including rain-flow, seasonal changes, etc so changes in COD were not regarded as deleterious to the experiment. The COD of the wastewater solution was taken over one wastewater cycle to determine the change in COD over the course of a week. The mean COD over the week was 5,238 mg/L (standard deviation: 475 mg/L) and the time evolution is shown in Figure 6. Efforts to limit degradation by microbes included argon flushing, skimming the surface interface of any growth and circulation of wastewater through a UV filter. The organic content in wastewater is in constant flux in wastewater treatment systems and this is reflected in the experiment.

The simulated wastewater in this study contained similar ingredients as other studies.52 Nitschke et al. used wastewater made with 120 mg/ peptone, 82.5 mg/L meat extract, 22.5 mg/L urea, 21 mg/L K2HPO4, 5.25 mg/L NaCl and 98 mg/L

NaHCO3 corresponding to a 150 mg/L BOD. 52 Solutions were prepared weekly and sterilized with steam in an autoclave.52 Lee et. al used a synthetic wastewater formula consisting of 114 mg/L glucose, ammonia, sulfate, chloride, phosphate ions, magnesium, ferric, calcium, sodium and potassium salts.109

New Wastewater Old Wastewater 7000

6500

y = -211.43x + 6084.3 6000 R² = 0.9242

5500

5000 COD [mg/L] COD 4500

4000 0 2 4 6 8 Day

Figure 6. COD of the wastewater solution over one wastewater cycle

3.1.2 Wastewater Tank – Gaseous Flushing

A number of gases were used to flush the surface of the wastewater tank to minimize bacterial growth including nitrogen, carbon dioxide and argon. Carbon dioxide dissolved in water will react with water and form carbonic acid with an

-3 equilibrium constant KH= 1.70 x 10 . Though little of the dissolved carbon dioxide is converted to carbonic acid and the equilibrium is reached slowly with a rate constant of 0.039 s-1 for the forward reaction, it was anticipated that the pH of the solution would be affected.

CO2  H2O H2CO3

During the acclimatization period, carbon dioxide was tested for flushing purposes  and it resulted in increasing acidity of the wastewater solution and within the MBR

86 as seen in the December 2009 pH values (Figure 7). After switching to argon, the pH of the MBR effluent increased to values above pH 7.

Though the contribution from flushing is estimated to be minimal in prevented bacterial growth, growth was minimal with argon flushing, which was employed from January 2010 onward. For the PA experiment, replenishment of the wastewater solution daily coupled with a chiller significantly reduced bacterial growth and COD losses.

8.50 50

8.00 45

7.50 40 C) °

7.00 35 pH pH 6.50 Temperature 30

6.00 25 Temperature ( Temperature 5.50 20

5.00 15 10/14 11/13 12/13 1/12 2/11 3/13 4/12 5/12 6/11 Date (2009 - 2010)

Figure 7. pH and temperature prior to the OP pesticide experiment

3.1.3 Wastewater characteristics

Supplemental organic loading of the wastewater was comprised of three mixtures: enzymatic digest peptone from meat, vegetable extract and yeast extract. Chemical tests were performed on each of these mixtures to determine the COD, phosphorus and nitrogen contents (Table 23). Phosphorus levels were relatively close, with some variation in total nitrogen concentrations. Nitrite and nitrate concentrations were negligible, with ammonia comprising 1% of the mixture.

3.1.4 Pesticide Tank

Formulations of malathion, diazinon and chlorpyrifos were present at emulsifiable concentrations and relatively easy to dissolve in solution. Sniper, an azinphos- methyl formulation present in a powder, was dissolved in 200 mL of methanol, as was granular Thimet (phorate). Despite these efforts, the granules of Thimet were difficult to dissolve completely, as they are composed primarily of inert ingredients such as clays and silica. The Thimet formed a turbid solution and some granules precipitated eventually, which caused inconsistent introduction of phorate.

Concentrations of the pesticide concentrations in the influent were confirmed by

GC/MS. The herbicides, 2,4-D, dicamba and mecoprop, were all present in one herbicide mix, and 1 mL of the herbicide was added daily to the wastewater tank.

3.1.5 MBR acclimatization period

Activated carbon and 10 L activated sludge from the rotating biological contactors at the Lakeside/Timberlea WWTP were added to the MBR in October of 2009.

From this point on, regular pH measurements of the effluent were made. An initial dip in effluent pH in December 2009 was due to surface flushing by carbon dioxide. Argon flushing was used from January 2010 onward and pH was an optimum 7 to 8 for bacterial growth. Spikes in effluent temperature in February

2010 were due to breakdowns in building heating regulators that raised lab temperatures to above 25 °C.

Ammonia

N/A N/A N/A 1020 9.7 0.95% N/A N/A N/A

040%

Nitrate

1000.6 BD >0.011% 4000 1.58 0. N/A N/A N/A

Nitrite

1000.6 BD >0.00002% 9990 0.029 0.0003% N/A N/A N/A

Total Nitrogen

53 8.43 15.9% 53.6 4.15 7.7% 52.4 6.05 11.5%

Total Phosphorus

101.0 2.32 2.3% 200 4.59 2.3% 200 7.23 3.6%

COD

. COD, nitrogen and phosphorus of wastewater mixture phosphorus and wastewater of .nitrogen COD,

101.0 87.3 86.4% 98.8 83.2 84.2% 98.2 80.8 82.3%

Table Table

Sample Concentration Reading HACH Percentage Sample Concentration Reading HACH Percentage Sample Concentration Reading HACH Percentage

Nutrient (mg/L)

Peptone from from Peptone meat, enzymatic digest Vegetable Extract Extract Yeast

DO values were taken beginning in January of 2010 (Figure 8). Dips in DO correlate well with increases in temperature. As temperature increases, saturation of DO decreases.88 While saturation of DO in water was never attained during the pre-experimental acclimatization phase, levels were well above anoxic levels of 2 mg/L for fish and were well above guidelines for safe discharge into receiving waters.88

8 Dissolved oxygen 42.5 7.5

37.5

7 C) 32.5 ° 6.5

6 27.5

5.5 22.5 Temperature ( Temperature 5 17.5

Dissolved oxygen (mg/L) oxygen Dissolved 1/22 2/21 3/23 4/22 5/22 6/21 Date (2010)

Figure 8. DO and temperature prior to the OP pesticide experiment

Total dissolved solids and electrical conductivity were measured beginning in

January 2010 (Figure 9). Levels decreased for the first month before increasing to

300 ppm. Day-to-day fluctuations of TDS were minimal and trends are observed over longer periods of time. Despite fluctuations in the concentrations of organic

91 and inorganic loads in the influent, the effluent’s conductivity (not shown), a measure of the ions in solution, was stable day-to-day. 88

Total Dissolved Solids Temperature

350 40

300

35 C) 250 °

200 30

150 25 100

20 ( Temperature 50

0 15 Total Dissolved Solids (ppm) Solids Dissolved Total 1/2 2/1 3/3 4/2 5/2 6/1 Date (2010)

Figure 9. Total dissolved solids prior to the OP pesticide experiment

3.1.6 MBR Trans-membrane Pressure

Trans-membrane pressure was maintained between 5 and 10 kPa. Typically, fouling becomes an issue at 30 kPa and requires a stoppage in flux.108 Only on one occasion did a sudden increase in trans-membrane pressure, as flow rate was being increased and calibrated, require a stoppage in flow and subsequent back-washing.

3.2 Trace Pesticide Analysis Results

3.2.1 SPE Method and Data

Pesticide recoveries from control samples were averaged and are shown in Table

24 in addition to the relative standard deviations of the recovery measurement.

Table 24. OP Pesticide control sample recoveries

Pesticide (n=7) Recoveries RSD Azinphos- 97% 4% methyl Chlorpyrifos 102% 6% Diazinon 106% 12% Malathion 104% 10% Phorate 90% 6%

3.2.2 GC/MS Method and Data

A total ion count for the OP pesticide method is shown in Figure 11 with analytes, surrogates and internal standards peaks present.

A comparison of the pesticide concentrations in the effluent versus the influent allowed for the calculation of each active ingredient’s persistence. Three of the pesticides peaked on Days 2 and 3 with phorate and chlorpyrifos as exceptions.

The phorate formulation was available in granular form, which is by design slow to release the active ingredient and prolong persistence. These issues were solved by

Day 5 with the dilution of the pesticide into 200 mL of methanol. Chlorpyrifos was not detected any day except Day 13, but was detected in control samples.111

ppm OP ppmOP pesticides

. Total ion count (TIC) for for countion 1 (TIC) .Total

Figure Figure

Phorate 50% Diazinon

Malathion

40% Chlorpyrifos

Azinphos-methyl

30%

% Persistenc e Persistenc % 20%

10%

0% 0 2 4 6 8 10 12 14 Day

Figure 11. GC/MS analysis of OP Pesticides in MBR Effluent

Initial increases in effluent pesticide concentration are linear (zero order kinetics) and can be attributed to the influent pesticide flux. Acclimatization of the microorganisms or the presence of microorganisms with the capacity to metabolize OPs counters this increase. The subsequent decrease can be modeled by an exponential decay function and valuable information about equilibrium concentrations and pesticide lifetime can be determined (Table 25). R-squared values of the exponential regression show the strength of the fit with lifetimes all close to 2 days.111

Table 25. OP Pesticide exponential decay regressions

Compound Baseline Initial value Lifetime R2 (μg/L) (μg/L) (days) Azinphos-methyl 829.8 ± 0 3728 ± 427 2.75 0.976 Diazinon 502.4 ± 0 3072.0 ± 613 1.92 0.980 Malathion 253.2 ± 0 1627 ± 198 2.32 0.975 Phorate 102.6 ± 0 N/A 2.16 0.739

3.3 Chemical Wastewater Results

Effluent COD increases before peaking on day 3, the day after the peak in the pesticide persistence curves (Figure 12). COD decreases steadily to pre- experimental levels as the pesticide concentrations reach equilibrium levels. A peak in COD precedes a bump in pesticide concentrations on day 13. Wastewater

COD was 200 mg/L, which corresponds to a 93% COD reduction pre-experiment.

The initial increase in COD may be a result of some microbial inhibition due to the presence of pesticides. While the pesticides themselves will contribute to the

COD, this accounts for only a 13% increase in the overall COD. The continued increase of COD may be also be related to inhibition due to pesticide metabolites, explaining why the COD peaked a day after the pesticide persistence curve peaks.

[mg/L] 15

Chemical Oxygen Demand (COD) (COD) Demand Oxygen Chemical 0 0 2 4 6 8 10 12 14 Day

Figure 12. OP pesticide COD MBR in effluent

Concentrations of total nitrogen and nitrate show excellent correlation, suggesting that most of the influent organic nitrogen and ammonia was converted to nitrate

(Figure 13). Total nitrogen levels dropped from 10 mg/L to 2 mg/L. Though levels did recover slightly, pre-experimental levels were not achieved. In times of stress, anaerobic bacteria may utilize nitrate as the oxidant in their metabolism. However, given the levels of DO in the MBR, it is unlikely that this is the source of the drop.

The drop instead may have been as a result of the increasing phosphorus levels

(Figure 15), typically a limiting nutrient in freshwater aquatic systems, displacing nitrogen as an excess nutrient. 111 With increased phosphorus levels, more organic nitrogen was used for metabolism and growth and was incorporated into microbial biomass. pH has a significant impact on the growth of Nitrobacter and

Nitrosomonas bacteria.20 Autotrophic bacteria are responsible for the nitrification process whereby ammonia is converted to nitrite. However, given the limited pH range of the effluent, this is unlikely to have had a significant impact on the distribution of nitrogen species.

12 12

10 Total Nitrogen Nitrate 10

8 8

N] [mg/L] N] - 6 6

4 4

2 2

Total Nitrogen [mg/L] Nitrogen Total Nitrate [NO3 Nitrate

0 0 0 2 4 6 8 10 12 14 Day

Figure 13. OP pesticide nitrate and total nitrogen MBR effluent concentrations

Ammonia levels were constant throughout the experiment with Day 7 as a notable exception (Figure 14). However, the Day 7 peak was still likely not an anomaly, as the concentrations on Day 5 and 6 indicate a rising trend. Concentrations of ammonia were 0.46 mg/L and 0.03 mg/L at equilibrium and show an overall reduction in ammonia of 99%. In comparison to wastewater effluent standards, the ammonia levels exceed the 1.25 mg/L wastewater standard112 and approach the guidelines for the protection of aquatic life in freshwater (Table 16).

0.5

0.45 0.4

0.35 N] (mg/L) N]

- 0.3 0.25 0.2 0.15 0.1

Ammonia [NH3 Ammonia 0.05 0 0 2 4 6 8 10 12 14 Day

Figure 14. OP pesticide ammonia MBR effluent concentration

Nitrite was also monitored over the course of the experiment. However, nitrite concentrations were below the detection limit of 0.015 mg/L. Nitrite is absent because the relative rate of the conversion of ammonia to nitrite is much slower than that of the conversion of nitrite to nitrate.

Total phosphorus level increases after the introduction of pesticides correlates to the increase in individual target pesticide concentrations in the effluent as does the eventual drop (Figure 15). The equilibrium level suggests that very little pesticide was actually converted to the orthophosphate and the total phosphorus suggests that most of the phosphorus remained in the tank either adsorbed to suspended solids and microbial flocs or was incorporated into the biomass in the tank. The spikes in reactive phosphorus were confirmed by re-testing the samples

99 and may correspond to systemic changes that promoted the oxidation of organic and condensed phosphates to the orthophosphate form. Both spikes were preceded by increases in the heterotrophic bacterial population.111 Wastewater effluent quality guidelines for total phosphorus concentration are set at 1 mg/L which is approached by the MBR effluent.113 Total phosphorus removal was 84.9% before the introduction of pesticides and increased to 92-95% after introduction.114

Phosphorus is removed through biomass uptake and adsorption.114

Reactive Phosphorus Total Phosphorus

1.6 1.4 1.2

P] (mg/L)P] 1 - 0.8 0.6 0.4 0.2 0

Phosphorus [PO4 Phosphorus 0 2 4 6 8 10 12 14 Day

Figure 15. OP pesticide phosphorus MBR effluent concentrations

3.4 Microbiological Results

The total heterotrophic bacterial population was counted daily and is plotted in

Figure 16. Three replicates of agar plates were prepared for each day and their results were averaged. Variation in the measurement may be a result of taking

100 three independent MBR sludge samples and the overlap of bacterial colonies on the agar plate.

Overall, the heterotrophic bacteria population in the tank is relatively constant

(given the size of the standard deviation error bars) with an average of 7.4×104

CFU/mL over the course of the experiment, except small changes on Day 7 and

Day 9. However, considering the uncertainty of experimental data, the variation was statistically insignificant. The initial introduction of pesticides did not result in a decline in the microbial population.

1.4E+05 1.2E+05

1.0E+05

8.0E+04 6.0E+04

(CFU/mL) 4.0E+04 2.0E+04 0.0E+00 Heterotrophic Plate Count Count Plate Heterotrophic 0 2 4 6 8 10 12 14 Day

Figure 16. OP pesticide MBR heterotrophic bacteria population

3.5 General Wastewater Parameters

Only minor fluctuations in the pH occurred during the experimental period with pH ranging from 7.2 to 7.45. A slight increase in pH occurred following pesticide addition, which then gradually dropped to pre-experiment values. As many

101 metabolic reactions are heavily dependent on pH, with such stable pHs, changes in these other parameters can be attributed to non-pH related factors.

7.5 7.45 7.4 7.35

7.3

7.25 pH 7.2 7.15 7.1 7.05 7 0 2 4 6 8 10 12 14 Day

Figure 17. OP pesticide MBR effluent pH

DO reductions occur after replenishments of the wastewater solution on Day 0 and Day 7, although the dip in DO decreases in magnitude, suggesting acclimation to the introduction of new pesticide solutions (Figure 18). The DO recovery is not as pronounced during the second pesticide solution tank than the first. Overall, the values exceed anoxic DO conditions. 111

102

6.8

6.6

6.4

6.2

5.8

5.6 Dissolved Oxygen (mg/L) Oxygen Dissolved 5.4

5.2 0 2 4 6 8 10 12 14 Day

Figure 18. OP pesticide MBR effluent DO

Suspended solids, turbidity and colour are all measures of the aesthetic quality of the effluent and apparent colour measurements exceed drinking water quality standards. Values of suspended solids and turbidity are low because of membrane size exclusion. The colour peak on Day 7 corresponds to new wastewater solution and on Day 12 to increases pesticide concentrations across all targets. The MBR effluent meets the 25 mg/L suspended solids standards set out by Environment

Canada.113

103

Suspended Solids Turbidity Colour

4 [FTU/PtCo/mg/L] 2

0 Turbidity/Colour/Suspended Solids 0 2 4 6 8 10 12 14 -2 Day

Figure 19. OP pesticide general water quality parameters 3.6 Statistical Analysis

Spearman coefficients () computed using Origin statistical software range from -1 to 1. The strength of the correlation increases as the ends of the range are approached with a coefficient of zero indicating no correlation and values of -1 and

1 indicating a perfect correlation. For interpretation of the size of the correlation, the following guide will be used (Table 26). A 95% confidence interval (p < 0.05) will be applied to determine whether the result is statistically significant. If a p value is less than 0.05, then the Spearman correlation is deemed statistically significant; beyond this, the magnitude of the p value is irrelevant and cannot be used to make any further conclusions about the correlation.

104

Table 26. Interpretation of Spearman coefficients

115 Positive Spearman Negative Spearman Interpretation 0.5 to 1.0 -1.0 to -0.5 Strong correlation 0.3 to 0.5 -0.5 to -0.3 Moderate correlation 0.1 to 0.3 -0.3 to -0.1 Weak correlation 0 to 0.1 -0.1 to 0 No correlation

Strong, statistically significant positive correlations (0.76 <  < 0.88) were found for many combinations of OP pesticides involving azinphos methyl, diazinon and malathion. This is confirmed in the concentration data where peaks in pesticide concentration occur on roughly the same day and drops in concentration follow a similar trend. (Refer to Appendices).

Total phosphorus shows strong, statistically significant positive correlations with

COD ( = 0.66), azinphos methyl ( = 0.66), and diazinon ( = 0.80).The correlation of total phosphorus with azinphos methyl concentrations is not surprising, as azinphos methyl and diazinon were the pesticides found in greatest concentration in the effluent. Increases of diazinon and azinphos methyl will result in a greater total phosphorus reading in the effluent. Total phosphorus showed a strong, statistically significant negative correlations with total nitrogen

( = -0.92) and nitrate ( = -0.89). When increased phosphorus was introduced to the MBR, a drop in nitrate and total nitrogen resulted. As phosphorus is a limiting nutrient in freshwater systems, when it increases in concentration, a drop in nitrogen is expected as the otherwise excess nitrogen is used for metabolism and growth and incorporated into biomass.

105

DO showed a strong, statistically significant negative correlation with malathion

( = -0.60) but with no other pesticides. Correlation between DO are positive for phorate ( = -0.06), but negative for azinphos methyl ( = -0.49) and diazinon ( =

-0.43), however they were not statistically significant. After the pesticide solutions were refreshed on Day 0 and Day 7, DO subsequently decreases. Overall, this suggests that as the concentration of OPs increases, DO will decrease, as it is necessary in the metabolism of these pesticides.

Strong, statistically significant negative correlations are seen between suspended solids (-0.76 <  < -0.63) and a number of parameters including ammonia, colour, pH and the MBR bacteria count. Colour and suspended solids are both indicators of the aesthetic quality of the effluent and it would be assumed that they would co- vary, as both are measured photospectrometrically. However, suspended solid readings are very close to the detection limit and for the most part were at 0 mg/L.

Slight changes may be attributed to noise in the spectrophotometer readings as the membranes themselves should prevent any suspended solids from entering the effluent.

Statistically significant, strong correlations are also identified between bacteria, colour and pH (0.61 <  < 0.66). The pH was relatively stable during the experiment, however slight changes in pH have been known to have significant effects on bacteria populations and vice versa. Finally, a strong negative correlation

106 between DO and temperature exists ( = -0.62) which is consistent with DO saturation decreasing at higher temperatures.

107

4 Phenoxyacetic Acid Herbicides

The influent concentration of phenoxyacetic and benzoic acid herbicides in

Weedex and possible metabolites was determined in a separate experiment.

Influent concentrations of these herbicides and their metabolites are shown in

Table 27. Herbicide concentrations are on the order of magnitude of activated sludge biodegradation studies by Nitschke et. al, in which influent mecoprop concentrations were 1 mg/L.52

Table 27. PA pesticide influent and Weedex concentrations

Weedex Relative Influent Concentration Standard Concentration (g/L) Deviation (µg/L) Dicamba 8.33 8% 310 3,6-DCSA 0.66 11 % 25 CHQ 0.44 20 % 16 2,4-D 96.12 4 % 3580 2,4-DCP 0.25 2 % 9 Mecoprop 53.35 3% 1990 2,5-DCP N.D.* N.A. N.A. MHQ N.D. N.A. N.A. o-cresol N.D. N.A. N.A. 4-chloro-2- 0.13 9% 5 methylphenol * Not detected 4.1 Trace Herbicide Analysis Results

4.1.1 Solid Phase Extraction

Solid phase extractions were performed using HR-P cartridges eluted with methanol; however, mecoprop values were low due to formation of the mecoprop methyl ester. A switch to ethyl acetate provided better results for the three primary

108 herbicides but with limited success for the hydroquinones. Experiments were conducted to find a more suitable eluent (Table 28).

Table 28. Chromabond® HR-P SPE eluent comparison

Acetone Ethyl 50/50 (v/v) MeOH Ethanol IPO Acetate IPO/Acetone Dicamba 98 % 100 % 96 % 90 % 86 % 67 % 3,6-DCSA 138 % 119 % 124 % 110 % 105 % 62 % CHQ N.D. 11 % 20 % 11 % 31 % 32 % 2,4-D 30 % 97 % 97 % 30 % 53 % 42 % 2,4-DCP 108 % 100 % 99 % 92 % 93 % 82 % Mecoprop 104 % 98 % 78 % 40 % 67 % 49 % 2,5-DCP 110 % 103 % 102 % 94 % 95 % 75 % MHQ 7 % N.D. 6 % 5 % 12 % 4 % o-cresol 94 % 94 % 88 % 82 % 94 % 74 % 4-chloro-2- 104 % 105 % 105 % 106 % 105 % 84 % methylphenol

Ethyl acetate produced the best overall results and isopropanol the worst.

Methylhydroquinone (MHQ) and chlorohydroquinone (CHQ) showed poor extraction efficiencies for all solvents. All but two compounds showed acceptable results when ethyl acetate was used as an eluent. Poor recoveries of MHQ and

CHQ in extraction using organic solvents is well documented,116,117 and to improve

MHQ and CHQ extraction, a salting out procedure was employed using NaCl and

MgSO4 salts.117

109

Table 29. Salting out hydroquinones in SPE

No Salt 10 % NaCl Excess Excess NaCl MgSO4 Dicamba 81 % 97 % 84 %0 90 % 3,6-DCSA 94 % 32 % 30 % 74 % CHQ 37 % 11 % 21 % 37 % 2,4-D 97 % 153 % 161 % 121 % 2,4-DCP 93 % 94 % 95 % 94 % Mecoprop 87 % Interferents* Interferents* 93 % 2,5-DCP 95 % 102 % 100 % 97 % MHQ 6 % 14 % 13 % 12 % o-cresol 97 % 120 % 120 % 112 % 4-chloro-2- 94 % 118 % 161 % 96 % methylphenol

* Interference due to matrix effects

The use of salts produced some modest improvements in MHQ’s extraction efficiency and no improvement for CHQ and resulted in the presence of interferents for mecoprop, 4-chloro-2-methylphenol and 2,4-D. Salting out procedures were abandoned for subsequent extractions. A final attempt to improve MHQ and CHQ efficiencies involved the use of different cartridges.

CHQ showed acceptable recoveries for the HR-P and Easy cartridges, which might indicate a possible instability in the HR-P method. MHQ was only extracted using the Oasis HLB cartridge. HR-P remains the most effective cartridge for extracting a majority of the compounds (Table 30).

110

Table 30. PA pesticide SPE sorbent comparison

Chromabond Chromabond Oasis HLB Chromabond HR-P Easy Hydra Dicamba 92 % 74 % 102 % 108 % 3,6-DCSA 96 % 0 % 15 % 85 % CHQ 84 % 88 % 0 % 0 % 2,4-D 107 % 104 % 103 % 97 % 2,4-DCP 98 % 98 % 101 % 97 % Mecoprop 96 % 95 % 102 % 95 % 2,5-DCP 102 % 100 % 111 % 99 % MHQ 9 % 15 % 86 % 46 % o-cresol 118 % 118 % 154 % 31 % 4-chloro-2- 110 % 111 % 120 % 114 % methylphenol

To maximize the non-ionic forms of these herbicides, the pH of each sample was reduced to pH 1.30. (Table 31). Chromabond HR-P cartridges are stable between pHs of 1 and 12; to lower the pH any further would risk deterioration of the SPE cartridges themselves.

Table 31. PA pesticide dissociation constants

Pesticide Dissociation Interpretation31,118 Constant [pKa] 2,4-D 2.8 Weak acid Dicamba 1.9 Strong acid Mecoprop 3.11, 3.2 Weak acid

4.1.2 LC/MS Results

A total ion count for the PA pesticide method is shown in Figure 20.

111

methylphenol

DCP

2,5

oro

chl

DCP

2,4

cresol

Mecoprop

Methylhydroquinone

DCSA

3,6

2,4

Dicamba Chlorohydroquinone

Figure 20. Total Ion Count (TIC) for 1- ppm PA pesticides

The ratio of influent to effluent concentrations yields the persistence value. The persistence values for dicamba are shown in Figure 21. Dicamba concentrations

112 peak on Day 15 before steadily declining over the next hundred days. While the decline can be modeled by an exponential regression, fluctuations occur about this regression. At the end of the experiment, dicamba removal was approximately

65%. 119

100%

90%

80%

70%

60%

50%

40% % Persistence % 30%

20%

10%

0% 0 20 40 60 80 100 120 Day

Figure 21. Persistence of dicamba in the PA pesticide MBR effluent

3,6-DCSA concentrations are shown in Figure 22. Peaks in 3,6-DCSA around Day 15 and Day 80 correspond to peaks in dicamba concentrations. DCSA concentrations in the influent were calculated to be 25 μg/L. Dicamba degradation will also contribute to these concentrations, and with concentrations of 3,6-DCSA below 10

113

μg/L, biodegradation and adsorption of DCSA are likely responsible for the difference.

g/L) μ 8

Concentration ( Concentration 4

0 0 20 40 60 80 100 120 Experiment Day

Figure 22. Concentrations of 3,6-DCSA in MBR PA pesticide effluent

2,5-DCP concentrations remain low during the initial phase of the experiment with one significant peak on Day 44 that corresponds to a local maximum in the dicamba concentration (Figure 23). In general, after Day 60, 2,5-DCP was rarely detected.

114

1.8

1.6

1.4

1.2

0.8

0.6 Concentration (ug/L) Concentration 0.4

0.2

0 0 20 40 60 80 100 120 Experiment Day

Figure 23. Concentrations of 2,5-DCP in the MBR PA pesticide effluent

Mecoprop concentrations peak on Day 14 and decay can be modeled by an exponential decay function. Deviations from the regression include fluctuations between Day 60 to 80 and from Day 100 onward. The persistence increases above

100% on Day 13 and Day 14 because of a membrane breakage on Day 13 where sludge permeated into the effluent. Outflow was shut down without stopping the influent, resulting in values above 100%. Percent removal of mecoprop at the end of the experiment was approximately 60%.119

115

140%

120%

100%

80%

60% % Persistence %

40%

20%

0% 0 20 40 60 80 100 120 Experiment Day

Figure 24. Persistence of Mecoprop in the MBR PA pesticide effluent

O-cresol was not detected in Weedex and was less than 1 μg/L in the effluent.

Concentrations of o-cresol were all below 1 μg/L and therefore no conclusions can be made as to the origin of the compound. The detection limit for o-cresol was 100 ng/L, with most of the o-cresol measurements at concentrations below 200 ng/L.

116

0.9

0.8

0.7

0.6

0.5

0.4

0.3 Concentration (ug/L) Concentration

0.2

0.1

0 0 20 40 60 80 100 120 Experiment Day

Figure 25. Concentrations of o-cresol in the MBR PA pesticide effluent

Concentrations of 4-chloro-2-methylphenol in the influent were calculated to be 5

μg/L. Effluent concentrations were all below this initial value and barring gross fluctuations of concentration in the first thirty days, follow an exponential decay.

No determination can be made as to the origin of the 4-chloro-2-methylphenol, whether it be from mecoprop degradation or initial influent concentration.

117

3.5

2.5

1.5

Concentration (ug/L) Concentration 1

0.5

0 0 20 40 60 80 100 120 Experiment Day

Figure 26. Concentrations of 4-chloro-2-methylphenol in the MBR effluent

2,4-D showed an impressive degradation to nearly 99% removal within 10 days of pesticide introduction.119 Given an initial concentration of 3.5 ppm, this is an impressive result. The decay fits an exponential decay with R2 value of 0.985. The initial inhibition of 2,4-D may be due to the presence of its dichlorophenol metabolite.120

118

60%

50%

40%

30%

% Persistence % 20%

10%

0% 0 20 40 60 80 100 120 Experiment Day

Figure 27. Persistence of 2,4-D in the MBR PA pesticide effluent

Chlorohydroquinone concentrations peak on Day 7 before plummeting on Day 10.

Chlorohydroquinone concentrations remain below detectable levels for most of the experiment with trace sub-5 µg/L levels. As chlorohydroquinone was detected in the influent, levels above 16 µg/L suggest contributions from pesticide transformation.

119

120

100

40 Concentration (ug/L) Concentration

0 0 20 40 60 80 100 120 Experiment Day

Figure 28. Concentrations of chlorohydroquinone in the PA pesticide effluent

Concentrations of 2,4-DCP peak on Day 3 before exponentially decaying. Aside from periodic detections, 2,4-DCP is not detected after Day 15. Phenols mandates in wastewater are 20 µg/L and are met by the MBR effluent.113

120

4.5

3.5

2.5

1.5 Concentration (ug/L) Concentration

0.5

0 0 20 40 60 80 100 120 Experiment Day

Figure 29. Concentration of 2,4-DCP in the MBR PA pesticide effluent

Those pesticides and metabolites that underwent decays were fit to an exponential decay regression. In general, lifetimes were on the order of a few days with the exception of dicamba and 4-chloro-2-methylphenol. R2 values were low except for

2,4-D which exhibited an excellent exponential decay fit.

121

Table 32. PA pesticide exponential decay regressions

Compound Baseline Initial value Lifetime R2 (μg/L) (μg/L) (days) 2,4-D 3.30 ± 2.87 4767 ± 178 2.75 0.985 Dicamba 91.3 ± 14.9 243 ± 19.3 40.90 0.710 Mecoprop 658 ± 33 3335 ± 553 15.19 0.718 2,4-DCP 0.00 ± 0.14 9.31 ± 1.70 4.08 0.858 4-chloro-2-methylphenol 0.00 ± 0.42 2.36 ± 0.32 57.14 0.477 Ammonia [NH3-N] 0.016 ± 0.008 0.59 ± 0.05 2.91 0.918

4.2 Chemical Wastewater Results

COD initially decreased in the first few days after the introduction of pesticides.

The addition of pesticides accounts for less than 1% of the total COD of the chemical wastewater, so a significant change was not expected. Two spikes in the first twenty days of the experiment occur on Days 4 and 13. The spike in COD on

Day 13 can be attributed to a breakage in the membrane which allowed for activated sludge to flow into the effluent. The activated sludge was allowed to settle, and COD tests were conducted on the effluent’s supernatant or the liquid layer above the sludge. A significant drop of COD occurs after Day 14, to levels below 10 mg/L. Higher values of COD occur between Days 40 and 60, near Day 80,

90 and 105. 119 With BOD5 of wastewater effluent to receiving waters mandated to be 20 mg/L, the COD in many cases exceeds this standard.113 Membrane separation contributes to 1.8% to 12.6% COD removal.121

122

(COD) [mg/L] (COD) 10

5 Chemical Oxygen Demand Oxygen Chemical 0 0 20 40 60 80 100 120 Day

Figure 30. PA Pesticide COD MBR effluent concentration

Ammonia levels increased after the influx of pesticides into the MBR, peaking on

Day 3 before decreasing to baseline levels around 0.02 mg/L.119 Nitrogen removal is achieved through biomass accumulation and denitrification to N2. In the presence of excess organics, autotrophic bacteria are unable to compete with heterotrophs for oxygen. However, nitrification and denitrification processes can occur simultaneously at low DO levels (0.4 – 2.0 mg/L) resulting in total nitrogen loss through N2 and N2O emissions. Using organics as reducing agents, denitrification occurs when at least 4.2 g COD exists for every g of N.121 While the mixed MBR liquor has high DO levels, layers of bacterial growth attached to the glass walls of the aquarium may be under anoxic conditions.

123

0.3

0.25

0.2

N] (mg/L) N] - 0.15

0.1

0.05 Ammonia [NH3 Ammonia

0 0 20 40 60 80 100 120 Day

Figure 31. PA pesticide ammonia MBR effluent concentration

Nitrate concentrations (Figure 32) remained consistent over the course of the experiment. A slight increase in nitrate may be due to metabolism of phenols that may inhibit microbial function. Fluctuations in nitrate levels increase after Day 60.

The concentration of nitrates is 45 mg/L in the Canadian Drinking Water Quality

Guidelines.19 At high temperatures (35 – 40 C), ammonia oxidizers have higher growth rates than nitrite oxidizing bacteria and in general are less affected by low oxygen concentrations than nitrite oxidizers. Lower temperatures and high oxygen concentrations may explain why nitrite values are so low and why nitrite oxidizers are able to thrive.122

Nitrogen can be removed through aerobic autotrophic nitrification in tandem with anaerobic heterotrophic denitrification to nitrogen gas.114 This process can also

124 occur simultaneously at low DO concentration. Optimal pH values between 7.9 and 8.2 for ammonia oxidizers and 7.2 and 7.6 for nitrite oxidizers.122 Throughout the experiment, the pH was in the optimum range of nitrite oxidizers thereby explaining the significant conversion of organic nitrogen to nitrate. Nitrification takes place in anoxic conditions such as at deeper layers of the biofilm and it is here where residual nitrate and nitrite can be removed.114

N] (mg/L) N] - 8

4 Nitrate [NO3 Nitrate 2

0 0 20 40 60 80 100 120 Day

Figure 32. PA pesticide nitrate MBR effluent concentration

4.3 Microbiological Results

Heterotrophic bacteria counts remained consistent over the course of the experiment; that is, within one order of magnitude with standard deviation error bars large enough that significant overlap is shown. The population fluctuated about 500,000 CFUs/mL, with maximum populations around 900,000 CFU/mL and minimum populations around 200,000 CFUs/mL. 119

125

1.2E+06

1.0E+06

8.0E+05

6.0E+05 CFU/mL 4.0E+05

2.0E+05

0.0E+00 0 20 40 60 80 100 120 Experiment Day

Figure 33. PA pesticide MBR heterotrophic bacteria population

4.4 General Wastewater Characteristics

pH values showed fluctuations in the first fifteen days of the experiment before holding steady at about pH 7.60 for the next 65 days. 119 Slight decreases in pH to

7.30 occurred over the subsequent 20 days. In general, pH values remained between pH 7 and 8. Adsorption of contaminants, especially chlorophenols, to activated sludge occur at these pH values with desorption occurring at higher pHs.102

126

8.25 40

8.05 pH Temperature 38

7.85 36

7.65 34 C) °

7.45 32

7.25 30 pH 7.05 28

6.85 26 Temperature ( Temperature 6.65 24 6.45 22 6.25 20 0 20 40 60 80 100 120 Day

Figure 34. PA pesticide MBR effluent pH

Total dissolved solids fell after the introduction of pesticides and stabilized around

250 mg/L for the remainder of the experiment. Decreases in dissolved solids may be an indication of microbial inhibition and inability to mineralize organics into inorganic acids, bases and salts.

127

350 40

330 38 TDS Temperature

310 36

290 34 C) ° 270 32 250 30 230 28

210 26 Temperature ( Temperature 190 24

Total Dissolved Solids (mg/L) Solids Dissolved Total 170 22 150 20 0 20 40 60 80 100 120 Day

Figure 35. PA pesticide MBR effluent TDS

DO levels remained between 7 mg/L and 8 mg/L, which are well above anoxic conditions. 119 Two moles of oxygen are required for the oxidation of ammonia to nitrate 122 Removal efficiencies of NH4-N at DO levels of 1, 3 and 6 mg/L are 70%,

92% and 98%, respectively; however, total nitrogen removal rates are 68%, 88% and 65%. This reflects the balance between the nitrification and denitrification processes. 114 At DO levels near 7 mg/L, it can be assumed that ammonia removal predominates and nitrification to N2 contributes little to total nitrogen removal.

128

8 40

7.8 DO Temperature 38

7.6 36

7.4 34 C) ° 7.2 32 7 30 6.8 28

6.6 26 Temperature ( Temperature

6.4 24 Dissolved Oyxgen (mg/L) Oyxgen Dissolved 6.2 22 6 20 0 20 40 60 80 100 120 Experimental Day

Figure 36. PA pesticide MBR effluent DO 4.5 Statistical Analysis

Refer to the discussion on Spearman coefficient (p. 104) for interpretation of the data.

There were many statistically significant correlations in the PA pesticide experiment. This can be attributed to the larger number of data points for the various parameters. Also, not all the correlations were strong; many were weak and moderate.

All three pesticides correlate positively with one another and these values are all statistically significant. Dicamba and mecoprop show a strong correlation ( =

0.78) and 2,4-D shows moderate correlations with dicamba and mecoprop ( =

0.38, 0.45).

129

2,4-D showed a strong correlation with each of its metabolites, 2,4-DCP ( = 0.73) and CHQ ( = 0.59). Mecoprop showed a moderate correlation with its metabolite

4-chloro-2-methylphenol ( = 0.49) and the correlation with o-cresol was not significant. This may be explained by the low concentrations of o-cresol detected and its relatively noisy concentration curve. Finally, Dicamba showed a moderate positive correlation with 2,5-DCP ( = 0.44) and 3,6-DCSA ( = 0.42). The herbicides also have moderate and weak correlations with metabolites of other herbicides; as the herbicides correlate with one another and the herbicides in general correlate with their metabolites, it is expected that the herbicides would correlate with the other metabolites.

All three pesticides show statistically significant positive correlations with pH (0.21

<  <0.33) and two, 2,4-D ( = -0.24) and dicamba( =-0.19), with DO. The pH during the experiment was relatively constant, and thus the correlations are weak, while decreases in DO may reduce the efficiency of herbicide breakdown by bacteria.

Ammonia concentrations were weakly correlated with turbidity, colour, barometric pressure and weakly negatively correlated with heterotrophic bacteria.

This may be due to consistently low concentrations of ammonia throughout the experiment while bacterial populations were routinely in flux. This may also be the explanation for the weak correlation between pH and bacteria. Finally, pressure

130 and dissolved oxygen were moderately correlated, which is consistent with higher saturations of DO at higher barometric pressures.

131

5 Future Work

Future work in testing the effectiveness of MBR in treating contaminants should include other chemical classes, such as organochlorines and triazines, that are both persistent and bioaccumulative. Further applications of MBR could be extended to the petroleum industry for the treatment of hydrocarbons and PAH contamination in produced water and trace levels of pharmaceuticals in domestic sewage.

The effectiveness of the MBR at treating ultra-trace sub-ppb levels of pesticides and other contaminants should also be tested. This is of particular importance, as technology for detecting trace level contamination continues to push detection limits to the ng/L and sub ng/L levels, which are most common for contaminants.

Isolating and subsequently identifying the bacterial species of contaminants is an important step in determining the metabolic pathways that contaminants follow in aquatic environments. This is of particular importance, as the majority of currently available information on bacterial metabolism of pesticides is in soil environments. Continuing to assess potential and confirmed metabolites of target contaminants is an essential aspect of the experiment, especially considering that many metabolites have insecticidal or herbicidal activity, and in some cases present a greater acute toxicity to humans.

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6 Conclusion

MBR has proven to be an effective, environmentally friendly and compact method of treating pesticide contaminants in wastewater. Two classes of pesticides, OP and PA/BA, were studied; they have varying degrees of toxicity, chemical characters and persistence and occurrence in the environment. Metabolites of phenoxyacetic and benzoic acid herbicides were also investigated to confirm their presence in aquatic metabolism pathways. Two analytical methods were developed using GC/MS and LC/MS for target compounds that varied greatly in terms of functional groups; chromatography and solid phase extraction procedures were optimized for detection limits and recoveries. Numerous water quality parameters were performed as part of daily analysis. OP insecticide removal rates ranged from

84% to 98% and the system reached equilibrium within six days. PA herbicides, which are more persistent, were removed 60% to 99% over the course of a 110-day experiment.

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