Organophosphate Esters (OPEs) as Emerging Contaminants in the

Organophosphate Esters (OPEs) as Emerging Contaminants in the Environment: Indoor Sources and Transport to Receiving Waters.

by

Jimmy W Truong

A thesis submitted in conformity with the requirements for the degree of Masters of Applied Science Department of Chemical Engineering and Applied Chemistry University of Toronto

© Copyright by Jimmy W Truong 2016

Organophosphate Esters (OPEs) as Emerging Contaminants in the Environment: Indoor Sources and Transport to Receiving Waters Jimmy W Truong Masters of Applied Science Department of Chemical Engineering and Applied Chemistry University of Toronto 2016

Abstract Organophosphate esters (OPEs) are high usage chemical additives that are of increasing concern because of growing evidence of potential toxicity and ubiquitous occurrence in the environment. This thesis summarizes the analysis, sources and environmental abundance of OPEs using Toronto as a case study. This was accomplished by documenting concentrations, loadings and factors influencing 19 OPEs in three Toronto streams during high and low flow periods, final effluent from three waste water treatment plants (WWTP), urban rain and near shore water from Lake Ontario. Tris (2-chloropropyl) phosphate (TCPP) was found at the highest concentrations in streams and WWTP effluent. Estimated mass loadings showed that WWTP discharges contributed significantly to the mass of OPEs entering into nearshore Lake Ontario, however, streams and rain could contribute equal or higher loadings during wet periods. These results suggested two major pathways to Lake Ontario: direct discharge from WWTP; and atmospheric deposition and wash-off into streams.

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Acknowledgments

I would like to acknowledge and personally thank my supportive supervisor Miriam Diamond, and co-supervisors Paul Helm and Liisa Jantunen for providing me with ideas and encouraging me to always strive for perfection. Their belief in me and guidance helped me prevail through all my ups and downs. Our mutual collaboration and constant discussions have shaped me into the man I am today. This body of work would not be possibly if not for all past and former members of the Diamond Group, who how been at times comrades, friends and mentors. I would like to thank my all my colleagues, especially, Joe Okeme and Aman Saini for all their advice on my work and helping me navigate through my degree, and Congqiao Yang for her aid in analytical chemistry. Additionally, I would like to thank my parents and family for encouraging me and believing in my success and to all my friends who have kept me going and would not let me give up. I would like to acknowledge Dano Morrison, for our mutual competition to finish our theses and publish our papers; Stephanie Vaughn, for her weekly visits, cupcakes and positive energy; Erika Dawson, for our love of adventure and her inspirational career advice; and Craig Christensen, for your love and support during my dark period – without all of you I would not be here today.

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Table of Contents Acknowledgments ..................................................................................................................... iii Table of Contents ...................................................................................................................... iv List of Tables ............................................................................................................................ vi List of Figures .......................................................................................................................... vii List of Appendices .................................................................................................................. viii Introduction ........................................................................................................................... 1 1.1 Background .................................................................................................................... 1 1.2 Organophosphate Esters .................................................................................................. 1 1.3 Measurement in Outdoor Environment: .......................................................................... 2 1.4 Transport from Indoor to Outdoor Environment: ............................................................. 3 1.5 Toxicity .......................................................................................................................... 4 1.6 Research Objectives: ....................................................................................................... 4 1.7 References ...................................................................................................................... 6 Organophosphate esters flame retardants and plasticizers in urban rain, streams, and wastewater effluent entering into Lake Ontario .....................................................................10 Abstract ................................................................................................................................10 2.1 Introduction ...................................................................................................................11 2.2 Methods .........................................................................................................................13 2.3 Results and Discussion ..................................................................................................17 2.4 Conclusion .....................................................................................................................29 2.5 References .....................................................................................................................30 Isomers of Tris(chloropropyl) Phosphate (TCPP), Replacement Flame Retardant in Technical Mixtures and Environmental Samples ...................................................................33 Abstract ................................................................................................................................33 3.1 Introduction ...................................................................................................................34 3.2 Methods .........................................................................................................................35 iv

3.3 Results and Discussion ..................................................................................................36 3.4 Conclusion .....................................................................................................................44 3.5 References .....................................................................................................................46 Is Spray Polyurethane Foam (SPF) Insulation a source of Tris(chloropropyl) phosphate (TCPP) to the Indoor Environment? ......................................................................................48 Abstract ................................................................................................................................48 4.1 Introduction ...................................................................................................................49 4.2 Methods .........................................................................................................................51 4.3 Results and Discussion ..................................................................................................53 4.4 Conclusion .....................................................................................................................58 4.5 References .....................................................................................................................59 Conclusion............................................................................................................................62 5.1 Future Work ..................................................................................................................63 Appendices................................................................................................................................64

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List of Tables

Table 3.1. TCPP1-3 concentration average and ranges measured in Toronto stream, rain and WWTPs: mean ± stdev (range) (µg/L). .................................................................................43 Table 4.2. Comparison of ∑TCPP concentrations in insulated house dust and air to reported literature values. ...................................................................................................................55

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List of Figures

Figure 2.1. Sampling locations in the Toronto, Ontario, Canada area for streams ......................14 Figure 2.4. Average relative composition profile of OPEs measured in Toronto urban water. ...23 Figure 2.5 Principle Components Analysis (PCA) on concentrations the 8 OPE compounds quantified in this study. .........................................................................................................24 Figure 2.6. Estimated instantaneous ΣOPE loadings (Kg/day) from sample locations at Etobicoke Creek, Don River, and Highland Creek, and three Waste Water Treatment Plants (WWTP). ...................................................................................................................26 Figure 3.1. Chromatogram of the TCPP isomers from AccuSTD TCPP standard ......................37 Figure 3.2. GC-MSD full scan of the AccuSTD mix. ................................................................38 Figure 3.3. Box plot showing TCPP1/TCPP2 ratios in the Sigma and AccuSTD standards, urban tributaries, WWTP effluent and rain water. .................................................................44 Figure 4.1. Box plots of TCPP1/TCPP2 isomer ratios from standards, insulation, insulated house samples and dust. ........................................................................................................56 Figure 4.2. TCPP concentrations in dust from insulated/non-insulated Vancouver homes. ........57

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List of Appendices

Appendix 1 - Supporting information for Chapter 2: Organophosphate esters flame retardants and plasticizers in urban rain, streams, and wastewater effluent entering into Lake Ontario .........................................................................................................................67 Appendix 2 – Supplementary Information for Chapter 3: Isomers of Tris(chloropropyl) Phosphate (TCPP), Replacement Flame Retardant in Technical Mixtures and Environmental Samples…………………………………………………….. ........................ 87 Appendix 3 - Supporting information for Chapter 4: Is Spray Polyurethane Foam (SPF) Insulation a source of Tris(chloropropyl) phosphate (TCPP) to the Indoor Environment? ... 106

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Introduction 1.1 Background Organophosphorus esters (OPEs), which are used as flame retardants (FRs) and plasticizers, are high production volume chemicals that have been measured at elevated levels in media ranging from indoor air and dust to Arctic air. Interest in these compounds has arisen because they are being used as alternatives to brominated flame retardants (BFRs) such as polybrominated diphenyls (PBDEs) for which new production and new uses have been controlled. Action has been taken to control all PBDE mixtures in Canada, U.S., Europe and internationally because of their behaviour as persistent organic pollutants (POP). Canada is currently assessing several OPEs under the Canadian Environmental Protection Act (CEPA) to determine if any OPEs should be classified as toxic (for listing under Schedule 1) and subject to control. This follows from controls of certain OPEs that have been implemented in some jurisdictions such as the European Union and California. This thesis addresses the lack of Canadian data for OPEs by providing data relevant to Canada and Ontario. Data are presented on the distribution, levels and sources from residential inputs of OPEs into the environment. Toronto, Canada, was used as a case study. This allowed comparison with previous research on PBDE in some of the same locations (Melymuk et al. 2014).

1.2 Organophosphate Esters OPEs are high production volume chemicals. The halogenated (mostly chlorinated) compounds, Cl-OPEs, tend to be used as flame retardants (FRs) and the non-halogenated (Non-Cl OPEs) compounds are mostly used as plasticizers. However, other uses include as additives to floor waxes, hydraulic fluids, lacquers, paint, glue, textiles, rubber, epoxy resins, polyurethane foam and cosmetics (REF). For example tris(2-chloroisopropyl) phosphate (TCPP) and tris(2-3dichloropropyl) phosphate (TDCPP) are widely used as flame retardants in flexible foam used for upholstered furniture and automotive seats (e.g., as a replacement for penta-BDE) and electronics (Van der Veen & de Boer 2012). TPhP is added at 18-35% by weight to LCD 1

screens, and tris(o-cresyl) phosphate (ToCP) is used in the manufacturing of lacquers, synthetic fabrics and as a waterproofing agent (Van der Veen & De Boer 2012)(Marklund et al. 2003).

The total consumption of OPEs in Europe in 2006 was estimated to be ~91,000 tonnes (Regnery & Püttmann 2010). Globally, total production in 2013 represented 30% of global flame retardant market at over 620 kilotons of OPEs (China Market Research Reports 2014). Since OPEs are typically added to polymers rather than being chemically bonded, they are subject to release into the environment via volatilization, dissolution and abrasion (e.g., Rauert et al. 2014). OPEs have vapour pressures that are orders-of-magnitude higher than most other halogenated flame retardants such as polybrominated diphenyl ethers or PBDEs (Bergman et al. 2012). Their high vapour pressures increase the likelihood of release from a product or material. As PBDEs and other brominated flame retardants have been phased out due to national and international regulations and policies and PBDE-containing products are retired (Abbasi et al. 2015), the inventory of OPE-containing products is expected to increase.

1.3 Measurement in Outdoor Environment: OPEs have been detected globally in a variety of environmental media including indoor dust and air (Reemtsma et al. 2008)(Stapleton et al. 2009), wastewater (Meyer & Bester 2004), groundwater (Fries et al. 2001)(Regnery & Püttmann 2010), surface water (Andresen et al. 2007)(Wolschke et al. 2015), and sediments (Cao et al. 2012). The presence of OPEs in air in remote locations raises concerns about their potential for long range transport. These reports include OPEs in the Norwegian and Canadian Arctic (Salamova et al. 2014)(Sühring et al. 2016), Antarctic and the North Sea (Möller et al. 2011). Sühring et al. (2016), found 14 OPEs dominated by tris(chloroethyl) phosphate (TCEP), TCPP, TDCPP in air across the Canadian Arctic. The occurrence of Cl-OPEs was reported by Laniewski et al. (1998) who found TCEP and TCPP in rainwater from Ireland and in snow from Poland and Sweden. The occurrence of OPEs in remote locations is not consistent with their estimated atmospheric half-lives, which were estimated to be less than the 2-day criterion under the Stockholm Convention and for 2

which long range atmospheric transport capability was not suggested (Zhang and Sühring et al. 2016). However, high concentrations of non-CL OPEs measured in Arctic air without a clear geographic pattern suggest that they do undergo long range atmospheric transport (Sühring et al. 2016).

Generally, OPEs are not degraded or removed in waste water treatment plants (WWTPs) (Meyer & Bester 2004)(Marklund et al. 2005a)(Schreder & La Guardia 2014). As such, effluent from WWTPs are thought to be the main sources of OPEs to receiving surface waters (Fries et al. 2001)(Andresen et al. 2004). However, Jantunen et al. (2013) reported elevated concentrations of non-Cl OPEs and Cl- OPEs in rural Ontario and urban Toronto streams that ranged from 101600 ng/L, suggesting sources of OPEs to surface waters in addition to WWTP discharges.

1.4 Transport from Indoor to Outdoor Environment: OPEs have been measured in indoor air and or dust in the US, Europe (Brommer et al. 2012)(Marklund et al. 2003) (Sjödin et al. 2001), Japan (Tajima et al. 2014)(Kanazawa et al. 2010), and to a limited extent in Canada (Shoeib et al. 2012) . Some of the highest concentrations of flame retardants measured indoors and outdoors are those of TDCPP (Abbasi et al. 2016) and TCPP (Brommer et al. 2012)(Stapleton et al. 2009). Some of these concentrations can be orders of magnitudes higher than PBDEs. Evidence has been lacking regarding the sources of these compounds into the indoor environment and the pathway from indoors to outdoors. TCPP has been shown to be in polyurethane foam in couches (Stapleton et al. 2012), and emission of TCPP from spray polyurethane foam has been demonstrated in chamber studies (Poppendieck et al. 2014). However, other than circumstantial evidence, no conclusive evidence as of yet has linked the occurrence of OPEs indoors and outdoors to these products. Schreder et al (2015) suggest that laundry waste water is an efficient conduit for the transfer of OPEs from the indoor environment into the waste water stream. Saini et al. (2016) substantiated this contention by showing that fabric could accumulate OPEs from indoor air followed by released of more than 80% into laundry water. Thus, OPEs accumulated by clothing from indoor 3

air and then released through laundering could be a major source of OPEs to receiving waters. Furthermore, many studies show evidence OPEs partitioning onto particles in the urban environment (Marklund et al. 2005b)(Regnery & Püttmann 2009)(Shoeib et al. 2014). This urban particulate matter could be transported long distances by streams via runoff or wet deposition, similarly to other SVOCs (Csiszar et al. 2014)(Melymuk et al. 2014).

1.5 Toxicity The acute toxic effects of OPEs are well-documented and related to their neurotoxicity due to binding to the acetylcholine esterase enzyme (Van der Veen & de Boer 2012). For example, triphenyl phosphate (TPhP) is a suspected neurotoxin and the most acutely toxic OPE (Verbruggen 2005). TPhP is thought to behave similarly to organophosphate ester pesticides (IPCS 1991). However, acute toxicity is of limited relevance to environmental exposures. Documentation of OPE toxicity at more environmentally relevant chronic low doses is sparse. Cl-OPEs such as TDCPP and TCEP have been shown to exert development and carcinogenic effects in organisms such as, daphnia, algae, zebrafish and rats (Van der Veen 2012)(National Research Council 2000). TCPP, TCEP, TPhP, TDCPP and have also been shown to cause endocrine disruption by effecting steroidogenesis and metabolism in zebrafish and MVLN cell lines (Liu et al. 2012). Recent evidence shows that these compounds impair zebrafish swimming behaviour (Sun et al. 2016)(Dishaw et al. 2014)(Wang et al. 2013), and have developmental effects on zebrafish embryos (Dishaw et al. 2014). However, knowledge is incomplete regarding the effects of chronic, long-term, low dose exposure on aquatic organisms, and effects due to exposure to mixtures, which is the reality of environmental exposures.

1.6 Research Objectives: Given the uncertainty in toxicity data and their widespread use, there is a need to evaluate the levels of OPEs in Toronto and Canada with the aim of assessing their risk and to identify factors that influence their input to the aquatic environment. The goal of this thesis was to provide data and insights to enable the evaluation of OPEs. This was accomplished by measuring levels and 4

loadings of OPEs in the urban aquatic environment, and investigating a potentially large source of the most abundant OPE, TCPP. In this thesis, my research is presented in the form of three research papers from Chapters 2 -5 as follows. Ch. 2: Organophosphate esters flame retardants and plasticizers in urban rain, streams, and wastewater effluent entering nearshore Lake Ontario Aims: To monitor the concentrations of 19 OPEs in the urban aquatic environment through different pathways (streams, WWTP effluent, rain), and conditions (wet and dry periods), and to approximate loadings into Lake Ontario.

Ch.3: Isomers of Tris(chloropropyl) Phosphate (TCPP) in Technical Mixtures and Environmental Samples. Formatted for submission to Journal of Analytical and Bioanalytical chemistry Aim: To evaluate, verify and adapt analytical methods for measuring TCPP in environmental samples. This included clarifying the ambiguity in the literature regarding TCPP identification and quantification, and verifying the identity and developing quantification methods for measuring TCPP and its isomers. Ch4. Is Spray Polyurethane Foam (SPF) Insulation a source of Tris(chloropropyl) phosphate (TCPP) to the Indoor Environment? Aim: To investigate the source of the most highly detected OPE (TCPP) in indoor air and dust by linking TCPP in SPF insulation to indoor levels using concentrations and ratios of TCPP isomers.

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1.7 References Abbasi, G. et al., 2016. Product screening for sources of halogenated flame retardants in Canadian house and office dust. Science of The Total Environment, 545-546, pp.299–307. Andresen, J. A, Grundmann, A. & Bester, K., 2004. Organophosphorus flame retardants and plasticisers in surface waters. The Science of the total environment, 332(1-3), pp.155–66. Andresen, J.A. et al., 2007. Emerging pollutants in the North Sea in comparison to Lake Ontario, Canada, data. Environmental toxicology and chemistry / SETAC, 26(6), pp.1081–9. Bergman, Å. et al., 2012. A novel abbreviation standard for organobromine, organochlorine and organophosphorus flame retardants and some characteristics of the chemicals. Environment International, 49, pp.57–82. Brommer, S. et al., 2012. Concentrations of organophosphate esters and brominated flame retardants in German indoor dust samples. Journal of environmental monitoring : JEM, 14(9), pp.2482–7. Cao, S. et al., 2012. Levels and distributions of organophosphate flame retardants and plasticizers in sediment from Taihu Lake, China. Environmental toxicology and chemistry / SETAC, 31(7), pp.1478–84. China Market Research Reports. Global and China flame retardant industry report, 2014. Research In China at China Market Research Reports http://www.chinamarketresearchreports.com/114859.html (accessed June 30, 2016) Csiszar, S.A., Diamond, M.L. & Daggupaty, S.M., 2014. The magnitude and spatial range of current-use urban PCB and PBDE emissions estimated using a coupled multimedia and air transport model. Environmental science & technology, 48, pp. 1075-1083 Dishaw, L. V et al., 2014. Developmental exposure to organophosphate flame retardants elicits vvert toxicity and alters behavior in early life stage zebrafish. Society of Toxicology, pp.1– 10. Fries, E. & Puttnam, W., 2001. Occurrence of organophosphate esters in surface water and ground water in Germany. J. Environ. Monit., 5, 346–352, pp.621–626. International Panel on Chemical Safety (IPCS), 1991. Environment Health Criteria (EHC) 111 Triphenyl Phosphate. United Nations Environment Programme, and World Health Organisation, Geneva. Jantunen, L. et al., 2012. Organophosphate flame retardants in southern Ontario tributaries and precipitation. Poster presentated at the Eastern Canada Trace Organic Workshop Kanazawa, a et al., 2010. Association between indoor exposure to semi-volatile organic compounds and building-related symptoms among the occupants of residential dwellings. Indoor air, 20(1), pp.72–84. 6

Liu, X., Ji, K. & Choi, K., 2012. Endocrine disruption potentials of organophosphate flame retardants and related mechanisms in H295R and MVLN cell lines and in zebrafish. Aquatic Toxicology, 114-115, pp.173–181. Marklund, A., Andersson, B. & Haglund, P., 2003. Screening of organophosphorus compounds and their distribution in various indoor environments. Chemosphere, 53(9), pp.1137–46. Marklund, A., Andersson, B. & Haglund, P., 2005a. Organophosphorus flame retardants and plasticizers in Swedish sewage treatment plants. Environmental science & technology, 39(19), pp.7423–9. Marklund, A., Andersson, B. & Haglund, P., 2005b. Traffic as a source of organophosphorus flame retardants and plasticizers in snow. Environmental science & technology, 39(10), pp.3555– 62. Melymuk, L. et al., 2014. From the city to the lake: loadings of PCBs, PBDEs, PAHs and PCMs from Toronto to Lake Ontario. Environ. Sci. Technol., 48, pp. 3732−3741 Meyer, J. & Bester, K., 2004. Organophosphate flame retardants and plasticisers in wastewater treatment plants. Journal of environmental monitoring : JEM, 6(7), pp.599–605. Möller, A. et al., 2011. Organophosphorus flame retardants and plasticizers in the atmosphere of the North Sea. Environmental pollution, 159(12), pp.3660 National Research Council, 2000. Toxicological Risks of Selected Flame-Retardant Chemicals, National Academy Press, N.W. Washington D.C Poppendieck, D. et al., 2014. Long Term Emission from Spray Polyurethane Foam Insulation. Proceedings of 13th International Conference on Indoor Air Quality and Climate, Indoor Air, pp.HP0126 Rauert, C. et al., 2014. A review of chamber experiments for determining specific emission rates and investigating migration pathways of flame retardants. Atmospheric Environment, 82, pp.44–55. Reemtsma, T. et al., 2008. Organophosphorus flame retardants and plasticizers in water and air I. Occurrence and fate. Trends in Analytical Chemistry, 27(9), pp.727–737. Regnery, J. & Püttmann, W., 2009. Organophosphorus flame retardants and plasticizers in rain and snow from middle Germany. CLEAN - Soil, Air, Water, 37(4-5), pp.334–342. Regnery, J. & Püttmann, W., 2010. Seasonal fluctuations of organophosphate concentrations in precipitation and storm water runoff. Chemosphere, 78(8), pp.958–64. Salamova, A., Hermanson, M.H. & Hites, R. a, 2014. Organophosphate and halogenated flame retardants in atmospheric particles from a European Arctic site. Environmental science & technology, 48(11), pp.6133–40. 7

Schreder, E.D. & Guardia, M.J. La, 2014. Flame retardant transfers from U.S. households dust and laundry wastewater to the aquatic environment..Environment Science and Technology, 48, 11575-11583 Shoeib, M. et al., 2014. Concentrations in air of organobromine, organochlorine and organophosphate flame retardants in Toronto, Canada. Atmospheric Environment, 99, pp.140–147. Shoeib, M. et al., 2012. Legacy and current-use flame retardants in house dust from Vancouver, Canada. Environmental Pollution, 169, pp.175–182. Sjödin, a et al., 2001. Flame retardants in indoor air at an electronics recycling plant and at other work environments. Environmental science & technology, 35(3), pp.448–54. Stapleton, H.M. et al., 2009. Detection of organophosphate flame retardants in furniture foam and U.S. house dust. Environmental science & technology, 43(19), pp.7490–5. Stapleton, H.M. et al., 2012. Novel and high volume use flame retardants in US couches reflective of the 2005 PentaBDE phase out. Environmental Science and Technology, 46(24), pp.13432–13439. Sühring, R. et al., 2016. Organophosphate esters in Canadian Arctic air : occurrence, levels and trends. Environ. Sci. Technol., 50 (14), pp. 7409–7415. Sun, L. et al., 2016. Neurotoxicology and teratology developmental exposure of zebra fish larvae to organophosphate flame retardants causes neurotoxicity. Neurotoxicology and Teratology 55, pp.16–22. Tajima, S. et al., 2014. Science of the Total Environment Detection and intake assessment of organophosphate fl ame retardants in house dust in Japanese dwellings. Science of the Total Environment, The, 478, pp.190–199. Van der Veen, I. & de Boer, J., 2012. Phosphorus flame retardants: properties, production, environmental occurrence, toxicity and analysis. Chemosphere, 88(10), pp.1119–53. Verrbruggen, E.M., et al., 2005. Environmental Risk Limits for Several Phosphate esters, with possible application as flame Retardant. RIVM Report 601501024/2005. Wang, Q. et al., 2013. Exposure of zebrafish embryos / larvae to TDCPP alters concentrations of thyroid hormones and transcriptions of genes involved in the hypothalamic – pituitary – thyroid axis. Aquatic Toxicology, 126, pp.207–213. Wolschke, H. et al., 2015. Organophosphorus fl ame retardants and plasticizers in the aquatic environment : A case study of the Elbe River , Germany. Environmental Pollution, 206, pp.488–493. Zhang, X. et al., 2016. Chemosphere novel flame retardants : Estimating the physical – chemical properties and environmental fate of 94 halogenated and organophosphate PBDE 8

replacements. Chemosphere, 144, pp.2401–2407.

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Organophosphate esters flame retardants and plasticizers in urban rain, streams, and wastewater effluent entering nearshore Lake Ontario Abstract Organophosphate esters (OPEs) are chemical additives that can be released from products and building materials into the environment via volatilization, dissolution and abrasion. OPEs are a concern because of recent reports of high concentrations indoors, in surface waters, and their potential toxicity to aquatic biota and humans. With Toronto, Canada, as the case study, we documented concentrations of OPEs in three streams during high and low flow periods, final effluent from three waste water treatment plants (WWTP), urban rain and nearshore Lake Ontario waters. Eight of the 19 OPEs had detection frequencies above 30%: TBEP, TCPP, TCEP, TDCPP, TnBP, TPhP, TEP, TPPO. WWTP effluent had the highest range of total OPE (ΣOPE) concentrations of 1.2-12 µg/L, followed by rivers during high flow periods of 0.78 – 8.1 µg/L, rivers during low flow periods (0.47 - 4.8µg/L), and then rain (0.18-4.7 µg/L). The lowest concentrations were measured in nearshore water in Lake Ontario (0.19 – 0.69 µg/L). The most abundantly measured OPEs were Tris butoxyethyl phosphate (TBEP), Tris chloropropyl phosphate (TCPP), and Tris chloroethyl phosphate (TCEP). ΣOPE concentrations in rivers at high flow exceeded that at low flow by a factor of two (ANOVA, p0.05). ΣOPEs in stream samples ranged from 0.47-4.8 µg/L during low flow conditions and from 0.798.1 µg/L during high flow conditions across the three streams (Tables A1.6-1.8). The similarity in concentrations suggests that the sources of OPEs to rivers are diffuse due to their ubiquity in the urban environment, as was found in German rivers (Wolschke et al. 2015) (Andresen et al. 2004). ΣOPE median concentrations were significantly greater by a factor of 1.6 (Highland Creek) to 2.0 (Etobicoke Creek and Don River) higher during wet weather (KW-ANOVA, p>0.05). The OPE compounds in order of most to least abundant were TBEP, TCPP, TDCPP and TCEP, except for WWTP(B) where TCPP was found at higher concentrations than TBEP. This overall pattern reflects which OPEs are used most in this waste water catchment area as WWTP do not effectively remove chlorinated OPEs (Marklund et al. 2005)(Schreder & Guardia 2014). High concentrations of TBEP, a non-CL OPE are most likely due to its ubiquitous use and larger volume of production than other Cl-OPEs. These high concentrations in WWTP effluent are consistent with other studies (Bester 2005)(Meyer & Bester 2004)(Andresen & Bester 2006)(Marklund et al. 2005)(Andresen et al. 19

2004), where the median concentration of TCPP (3.4 µg/L), TCEP (0.95 µg/L) and TPhP (0.051 µg/L) in Toronto WWTPs sampled in 2014 were similar to those measured in Germany and Sweden in 2003 (Meyer & Bester 2004)(Marklund et al. 2005). However concentrations of TBEP and TDCPP in Toronto were double (3.0, 1.0 µg/L), and TnBP was substantially lower in Toronto than in German WWTPs (0.082 – 1.2 µg/L)(Meyer & Bester 2004), which could also represent a change in usage in the 10 years. More recently, Schreder and LaGuardia (2014) detected ΣCl-OPEs in WWTP effluent collected in 2012 from Vancouver, Washington exceeding a mean of 10 µg/L, about an order-of-magnitude higher those measured here. The most abundant compounds they measured were TCPP > TDCPP> TCEP, which is similar to this study. Nearshore waters Lake Ontario nearshore waters had the lowest detection frequencies ranging from 0-60% (Table A1.10). These samples had the lowest concentrations of all waters tested with ΣOPE median concentrations ranging from 0.19 – 0.69 µg/L. These low concentrations were expected as the lake dilutes loadings from various urban pathways. The most frequently detected OPEs in nearshore waters were TBEP, TCPP and TCEP, ranging from below the limit of detection for each to 0.30 µg/L for each compound. The other OPEs were infrequently detected (TCPP>TCEP) followed by Lake Michigan then Lake Huron. The higher concentrations of OPEs in urban waters are a source to 20

“background levels” found in open waters of the Great Lakes (Venier et al., 2014)(Andresen et al. 2007). It is interesting to note that TBEP, the highest measured OPE in lake water actually has the shortest half-life (704 hours) of all commonly measured OPEs (Zhang et al. 2016). This suggests that despite its fast degradation in water, it is still measured in high concentrations because of its pervasive use.

Exposure and potential for impacts to aquatic biota It is important to put the levels reported here into the context of ecotoxicological impacts for aquatic species. Past toxicology studies for the frequently detected OPEs in Toronto waters focused on acute toxic effects, with thresholds for effects in the range of approximately 1-100 mg/L related to neurotoxicity (Verbruggen 2005)(Van der Veen & de Boer 2012). TPhP and TDCPP are the most acutely toxic, with the lowest LC50 values of 0.4-1 mg/L. Cl-OPEs such as TDCPP and TCEP have been shown to be developmental and carcinogenic toxicants (Van der Veen & de Boer)(National Research Council 2000). Recent toxicology testing has focused on more subtle endpoints related to endocrine disruption and behaviour. For example, Liu et al. (2012) found that TCPP, TCEP, TPhP and TDCPP exhibited endocrine disrupting potential with altering steroidogeneses and metabolism of estrogen in zebra fish and MVLN cell lines at concentrations of 10-100 μg/L. These concentrations can be within a factor of 10 of those reported here. Concentrations >625 µg/L impaired zebra fish locomotor behaviour in free swimming and photomotor response (Sun et al. 2016)(Dishaw et al. 2014). Cristale et al. (2013) found that acute lethal toxicity of OPE compounds was additive for Daphnia magna. This suggests that the current approach of evaluating individual OPEs for their toxicity may be underestimating their toxicity. Therefore, there is merit in further assessing the abundant OPEs (namely TCPP, TCEP and TDCPP) and ΣOPE concentrations for their potential to impair aquatic ecosystem health. OPE composition in urban waters Average compound profiles in each of the waters are depicted in Figure 2.3. The profiles were fairly consistent across streams and WWTPs. In particular, TCPP contributed, on average, 3021

51% of ΣOPEs, TBEP at 20-44%, and TCEP at 6-10% of ΣOPEs. TnBP contributed 8-11% to ΣOPEs in Etobicoke Creek samples. On average, the three chlorinated OPEs TCPP, TCEP, and TDCPP accounted for 47-62% of ΣOPEs in each of the urban waters. The highest concentrations of individual OPEs in streams were measured in the Don River during low and high flow samples. The highest concentration was for TBEP (5.2 µg/L), then TCPP (4.9 µg/L), followed by TCEP (0.70 µg/L) and TDCPP (0.35 µg/L). As noted above, the sampling site on the Don River was located downstream of a WWTP and the higher maximum concentrations at this site were likely influenced by discharges from the plant. As discussed below, the highest concentrations of OPEs are typically measured in WWTP effluent (Marklund et al. 2005) and thus receiving waters with low dilution factors, such as the Don River, are expected to have high levels (Cristale et al. 2013). However, the highest concentration of TnBP was found at Etobicoke Creek (2.2 µg/L), which is not influenced by WWTP discharges. The profiles of OPEs in rain and nearshore lake waters were more varied than stream and WWTPs. Rain generally had fewer compounds detected but had higher proportions of TCEP (20%), and was the only medium in which TPPP was detected (20%). The nearshore water profile had a relatively greater proportion of TCEP of ΣOPEs (30%), which has the greatest water solubility (794.3mg/L) of all OPEs studied except for TEP.

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Percent Composition (%)

100%

TPPP TPPO TEHP EHDPP TBEP TPhP TDCPP TPP TCPP TCEP TBPO TnBP TEP

90% 80%

70% 60% 50% 40% 30% 20% 10% 0%

Figure 2.3 Average relative composition profile of OPEs measured in Toronto urban water.

PCA A principle components analysis was undertaken to identify the similar and differences amongst OPEs in each water-type sampled and factors that accounted for the most variability (Figure 2.4). Analysis was performed on log transformed concentrations for every sample type for the 8 OPEs with greater than 30% detection frequency. Missing values were replaced with the LOD. Principle components (PC) 1 (x-axis) and 2 (y-axis) accounted for 52.3% and 12.7% of the variation, respectively, whereas other PCs each accounted for