Biological Consequences of Urban Stormwater Runoff on ...

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Culminating Projects in Biology Department of Biology

12-2016

Biological Consequences of Urban StormwaterRunoff on Reproduction and Survival of AquaticOrganismsBenjamin M. WesterhoffSt. Cloud State University, [email protected]

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Biological Consequences of Urban Stormwater Runoff on Reproduction and Survival of Aquatic

Organisms

by

Benjamin M. Westerhoff

A Thesis

Submitted to the Graduate Faculty of

St. Cloud State University

in Partial Fulfillment of the Requirements

for the Degree of

Master of Science in

Cell and Molecular Biology

December 2016

Thesis Committee:

Dr. Heiko Schoenfuss, Chairperson

Dr. Timothy Schuh

Dr. Cassidy Dobson

2 Acknowledgements

For my parents,

Thank you for the love, guidance, and support.

For my siblings,

I will never forget the stories we made and adventures we had.

For my advisor,

Dr. Heiko Schoenfuss,

You have given me confidence in my academic pursuits,

And provided me an opportunity to expand my knowledge,

Thank you for the mentorship and support these past two years.

3 Abstract

Best Management Practices storm water ponds (BMPs) are ways of controlling and filtering storm

water and storm sewer effluent. Urbanized areas are increasingly turning to the use of these BMPs

as a means to mitigate the affect of storm water runoff on aquatic environments. However, the

effectiveness of BMPs is not well understood and there have been few studies that look at the

biological efficacy of these filtration systems. This study looked at the effectiveness of three

BMPs in the cities of St. Paul and Minneapolis, MN. The BMPs being studied used iron filings

blended with sand as a filtration substrate. Water was collected at winter snow melt, spring rain

event, and summer rain event. Collected water was used to expose Daphnia magna and

Pimephales promelas. D. magna exposure endpoints were survival and reproduction. Daphnia

were exposed for 16-day periods to establish adult survival and neonate production. P. promelas

exposure endpoints were larval c-start performance, growth, and feeding assays. Larval P.

promelas were exposed to stormwater for 21-days before assessing their endpoints. Water

chemistry indicates that there is an improvement in water quality from the Inflow to Outflow in

each BMP. However, there were few significant differences in larval minnow or Daphnia

performance results between the Inflow and Outflow of the BMPs for all three storm water

sampling events. The only area that showed marked improvement was larval performance over

time, with minnows exposed to water collected later in the year performing better on predator

escape assays than minnows exposed to winter snow melt. The design of the BMP filtration

system may need to be improved or more filtration may be needed to achieve improvements in

biological outcomes.

4 Table of Contents

List of Tables ............................................................................................................................................... 5

List of Figures .............................................................................................................................................. 6

CHAPTER 1: INTRODUCTION TO MANAGEMENT OF STORMWATER POLLUTION IN URBAN

ENVRIONMENTS ...................................................................................................................................... 7

Stormwater Runoff in Urban Environments ..................................................................................... 7

CHAPTER 2: DEVELOPMENT AND EXECUTION OF A NOVEL ASSAY UTILIZING THE

ECO RESPONSIVE GENOME OF D. MAGNA REINFORCED WITH TRADITIONAL

REPRODUCTIVE AND SURVIVAL ASSAYS ......................................................................... 10

Introduction ........................................................................................................................ 11

Methods and Materials ....................................................................................................... 12

Discussion........................................................................................................................... 22

CHAPTER 3: EFFECTS OF URBAN STORMWATER RUNOFF AND BEST

MANAGEMENT PRACTICES ON THE GROWTH AND PREDATOR ESCAPE RESPONSE

OF PIMEPHALES PROMELAS .................................................................................................. 25

Introduction ........................................................................................................................ 25

Methods and Materials ....................................................................................................... 27

Results ................................................................................................................................ 29

Discussion........................................................................................................................... 34

CHAPTER 4: CONCLUSION....................................................................................................... 36

Key Findings ..................................................................................................................... 36

Future Directions ............................................................................................................... 38

References ...................................................................................................................................... 39

5 List of Tables

Table

2.1. Average load of 20 chemicals from each stormwater runoff sampling site in St. Paul and

Minneapolis, MN .................................................................................................................. 15

3.1. Comparison of best management practices influent and effluent waters for winter

snow melt .............................................................................................................................. 29

3.2: Comparison of best management practices influent and effluent waters for spring rain

event ...................................................................................................................................... 30

3.3. Comparison of best management practices influent and effluent waters for first summer

rain event ............................................................................................................................... 31

3.4. Seasonal changes in performance assays and feeding from winter snow melt to spring

rain event exposures .............................................................................................................. 32

3.5. Seasonal changes in performance assays and feeding from spring rain event to first

summer rain event exposures ................................................................................................ 33

3.6. Temporal improvement in performance assays from winter snow melt to first summer

rain event.. ............................................................................................................................. 33

6 List of Figures

Figures

1.1. Pathways that pollutants can use to enter surface waters………………………………………….……9

2.1. D. magna neonate production when exposed to pyriproxyfen……………………………………… .17

2.2. Sum total neonates/female/day/treatment for Daphnia exposed to stormwater effluent………………18

2.3. Survival of adult Daphnia exposed to stormwater effluent……………………………….………......21

3.1. Native range of Pimephales promelas…………………………………………………………………25

7 CHAPTER 1: INTRODUCTION TO MANAGEMENT OF STORMWATER POLLUTION IN

URBAN ENVIRONMENTS

Stormwater Runoff in Urban Environments

Stormwater runoff is often overlooked as a source of pollution by the general public. This is

understandable, given that people know water runs into storm sewers. What many individuals do not

know is that stormwater is not treated after entering a storm drain. This can lead some individuals to

dispose of chemicals down storm drains. However, intentional disposal in this manner is not the only

source of pollution in stormwater runoff.

Stormwater is a mixture of water coming from multiple sources. These can include runoff from

industrial parks, recreational areas, roads, roofs, and open spaces (gardens and golf courses) (Lundy et al.,

2012). There is also potential for combined sewer overflow events when residential sewers are connected

with storm sewers, causing overflows at waste water treatment plants (Rechenburg et al. 2006; Suarez and

Puertas, 2005; Heinz et al. 2009).

8

Fig.

Figure 1.1. Pathways that pollutants can use to enter surface waters. Figure based on work by Lundy et al.

2012.

A common method of holding runoff is through detention or filtration basins and constructed

wetlands (Villarreal et al. 2004; U.S. Environmental Protection Agency, 2005). Best management

practices (BMP) filtration basins can also use sand filters to further remove pollutants before discharging

stormwater runoff (Weiss et al. 2007). These methods of holding the water reduce erosion and slow the

velocity of runoff in addition to cleaning the water.

There are several ways to describe the efficacy of BMPs. One of them is the reduction of chemical

load from influent to effluent. The efficacy varies between the type of BMP being used, with sand filters

removing 82% of total suspended solids (TSS) and 46% of phosphorus. Infiltration trenches could remove

95% of TSS and 65% of phosphorus (Weiss et al., 2007). An enhanced version of the sand filter (the

Minnesota filter) uses sand mixed with 5% iron filings. This can remove up to 88% of phosphates

(Erikson et al. 2012) which indicates that BMPs can be effective in improving water quality.

9 Many researchers have attempted to quantify the improvement in water quality that BMPs provide.

But, they have not answered the ultimate question driving attempts to clean stormwater runoff. Does

BMP filtration result in measurable improvements in biological outcomes for aquatic organisms? Without

knowing this endpoint, the question of BMP efficacy is moot.

10 CHAPTER 2: DEVELOPMENT AND EXECUTION OF A NOVEL ASSAY UTILIZING THE

ECO RESPONSIVE GENOME OF D. MAGNA REINFORCED WITH TRADITIONAL

REPRODUCTIVE AND SURVIVAL ASSAYS

Introduction

Biology of Daphnia magna

Daphnia are a group of small planktonic crustaceans that inhabit freshwater environments. They are

a widespread genus, inhabiting all continents but Antarctica. Primarily filter feeders, Daphnia are an

important interface between primary producers and secondary consumers like fish and insects (Galbraith,

2011; Krueger and Dodson, 1981). Daphnia are cyclic parthenogens. They can reproduce both sexually

and through parthenogenetic (clonal) means (Arbaciauskas and Lampert, 2003). This ability to switch

reproductive modes make them an interesting and useful model organism for studies looking at

reproduction and genetics (Larsson and Miracle, 1997).

Daphnia as a model toxicological organism

Daphnia are a widely used model organism in aquatic toxicology because of their sensitivity to

metals and endocrine disrupting compounds (EDCs) (Olmstead and Leblanc, 2000; Doa et al. 2017). The

use of Daphnia in aquatic toxicity testing (both acute and chronic) is well established, with over 500

studies conducted using Daphnia from 1996-2006 (Sarma and Nandini, 2006). Metals have been shown

to be toxic to Daphnia (Heinlaan et al. 2008; Ra et al. 2016; Adam et al. 2015). This is important, because

metals are commonly found in road runoff (Lundy et al, 2012) and can access the environment through

this runoff.

Another reason that Daphnia make good model organisms is that their genomes are being sequenced

(D. pulex in 2011 and partial sequencing of D. magna) (Colbourne et al. 2011; Orsini et al. 2016).

Sequencing of the genome allows researchers to examine these widely-used laboratory models in new

ways. Tang et al (2015) demonstrated that D. pulex showed alterations in gene expression in DNA repair

11 and stress responses to metals, while Giraudo et al (2015) showed that exposure to a flame retardant

(TBOEP) caused alterations in 101 genes of D. magna.

Given the evidence for DNA methylation and transgenerational effects when Daphnia are exposed to

toxicants (Wan et al. 2010; Vandegehuchte et al. 2010) there is a need for research into these areas. Many

of these compounds are also endocrine active. Pyriproxyfen, a juvenile hormone analog (JHA) is

commonly used as a pesticide to control fleas, ticks, and mosquitos (Ginjupalli and Baldwin, 2013).

Exposure to pyriproxyfen causes a shift in parthenogenetic reproduction from primarily female to male

offspring in a dose-dependent manner (Olmstead and Leblanc, 2003; Matsumoto et al. 2008).

Alterations in neonate reproduction are good indicators that gene expression is being influenced as a

result of exposure to toxicants. Combining this knowledge with the fact that there are Daphnia genome

libraries opens up a new area for exploration. Researchers should now be able to identify which genes are

being up or down regulated in response to chemical insults. This is the basis for the development of a new

assay to identify environmental pollutants through alterations in gene expression. The purpose of this

study is to expose Daphnia magna to pyriproxyfen, sequence the RNA of the exposed organisms and

identify alterations in genes that are specific to pyriproxyfen. The ultimate goal of this experiment is to

expose Daphnia magna to stormwater effluent and look for specific alterations in gene expression.

Our hypotheses are H1: Pyriproxyfen and stormwater will cause alterations in gene expression of D.

magna; H2: D. magna raised in BMP effluent will have greater reproduction than D. magna raised in

BMP influent; H3: D. magna raised in BMP effluent will have greater survival than D. magna raised in

BMP influent.

Methods and Materials

Materials and Experimental Design for Identification of Pyriproxyfen EC10 in Daphnia magna

A replicate 16-day exposure was conducted from 3/9/2016-3/25/2016 in the Aquatic Toxicology

Laboratory at St. Cloud State University (St. Cloud, MN). This exposure was conducted to determine the

EC10 of pyriproxyfen. The exposure was modified from the OECD reproductive testing guideline (OECD,

12 2012). Exposure length was modified from a 21-day exposure to a 16-day exposure because of high

mortality in reproductive adults.

D. magna less then 24hr old were raised under a 16:8 h light: dark photoperiod. Aerated well water

was used for all treatment groups, and treatment solutions were made one day before beginning the

experiment. Treatment concentrations were 0, 29, 59, 89, 119, 179, and 239 ng/L pyriproxyfen. D. magna

were raised in 50 ml beakers containing 40 ml exposure solution. There were a total of 10

replicates/treatment with 3 D. magna/beaker. Evaporated water was replenished daily with well water.

Organisms were transferred to new treatment solution every fourth day to prevent mold growth in

beakers.

Exposure Chemical

Pyriproxyfen (Sigma-Aldrich, St. Louis, MO) exposure solutions were made one day before

exposures from super stock solutions of pyriproxyfen dissolved in 100% ethanol. Super stock solutions

were made by serial dilution. Exposure solutions were stored at 4O C in amber glass bottles until used. D.

magna were placed into clean beakers with new exposure solution every fourth day.

Biological Endpoint: Male Neonate Production

Hatched D. magna neonates were removed from the exposure solution every day and examined under

10X magnification to identify males. Males were identified by the enlarged primary antennae (Ebert,

2005). Neonates were discarded after sexual identification was complete.

Materials and experimental Design for 4-day D. magna Gene Expression Assay

A four-day exposure to wetland water and pyriproxyfen spiked wetland water was conducted at St.

Cloud State University. The purpose of this study was to generate RNA for analysis at the University of

Minnesota’s Genomics Center.

D. magna <24 hr. old were raised under 16:8 h light: dark photoperiod. Wetland water samples were

vacuum filtered with Whatman #1 filters (Sigma-Aldrich, St. Louis, MO) pore size 11µm. Water samples

were separated into pyriproxyfen spiked (119 ng/L) and non-spiked samples. A blank control and two

13 spiked control treatments (119 and 239 ng/L) were also run. The purpose of the spiked controls was to

establish baseline activity for effective concentrations. D. magna were raised in 50 ml beakers containing

40 ml exposure solution. There were a total of 7 replicates/treatment with 5 D. magna/beaker.

Evaporated water was replenished daily.

D. magna were harvested after 96 hrs because we wanted to harvest organisms that were not yet

showing compensatory action against pyriproxyfen. Harvested organisms (n=5) were removed from

exposure beakers with a wide-mouthed pipette and placed into 2 mL vials labeled with the treatment

group. Each vial contained 100 µL RNAlater® (Sigma-Aldrich, St. Louis, MO). Vials were stored at -80O

C until RNA extraction.

Whole RNA Extraction

RNA extraction was completed at SCSU. Tubes containing Daphnia were thawed and homogenized

with a sonicator for 30 seconds. A PureLink® RNA Mini Kit (Thermo Fisher Scientific, Waltham, MA)

was then used to extract whole RNA from the D. magna. Samples were analyzed with a NanoDrop 2000

(Thermo Fisher Scientific, Waltham, MA) at A260/A280. Any samples with a nucleic acid content

reading lower than 1.8 were discarded. Samples were shipped to the University of Minnesota Genomics

Center (UMGC) on dry ice. UMGC will sequence the RNA and look for alterations in gene expression

between control, pyriproxyfen free, and pyriproxyfen spiked wetland water samples.

Wetland Water Samples

Wetland water samples were collected from six sites (Woodland, Breen, KERK, KAND, DOUG, and

LESU) throughout west central Minnesota in summer 2015. Samples were collected in 1L amber glass

bottles at a depth of 5-15 cm below pond surface. Samples were then transported to St. Cloud State

University and stored at -20oC.

Materials and Experimental Design for 16-day D. magna Reproductive and Adult Survival Assay for

Stormwater Runoff Exposure

14 Two replicate 16-day exposures were conducted (4/18/16-5/4/16;5/26/16-6/11/16) in the Aquatic

Toxicology Laboratory at St. Cloud State University (St. Cloud, MN). These exposures were conducted

to determine the biological effectiveness of BMP stormwater filtration devices on reproduction of D.

magna. We also examined the reproduction and survival of D. magna exposed to untreated stormwater

runoff. The exposures were modified from the OECD reproductive testing guideline (OECD, 2012).

Exposure length was modified from a 21-day exposure to 16-day exposure because of high mortality in

reproductive adults.

D. magna <24hr old were raised under a 16:8 h light: dark photoperiod. Aerated well water was used

as a control. Treatment solutions were thawed and filtered through Whatman #1 filters (Sigma-Aldrich,

St. Louis, MO) pore size 11µm. D. magna were raised in 50 ml beakers containing 40 ml of exposure

solution. There were a total of 10 replicates/treatment with 3 D. magna/beaker. Evaporated water was

replenished daily with well water. Organisms were transferred to new treatment solution every fourth day

to prevent mold growth in beakers.

Biological Endpoints: Male and Total Neonate Production

The total number of neonates were counted every day after reproduction began. Neonates were

removed from exposure jars and counted under 10X magnification. Neonates were discarded after

identification of males.

Stormwater Runoff Collection

Stormwater runoff was collected from three best management practice (BMP) sites and three

stormwater outflow pipes within St. Paul and Minneapolis, MN. Stormwater was collected in 1L Nalgene

bottles. Water was collected directly from holding ponds containing inlet water by filling the bottles 5-15

cm below the water surface. Outlet water was collected directly from an outlet pipe at all three BMPs.

Water was transported to SCSU and stored at -20O C.

Stormwater runoff was collected at four different rain events to determine the unique chemical profile

of stormwater runoff throughout the seasons (winter snow melt, spring rain event, first summer rain event,

15 and second summer rain event). The BMPs being sampled were TBNS1_IN and OUT, TBNS2_IN and

OUT, and 37ST_IN and OUT. Stormwater outflow pipes were St. Anthony Park, Crystal Lake, and Cedar

Lake. TBNS2 was frozen at the time of winter snow melt sampling and was omitted from the first

reproductive and adult survival test.

Table 2.1: Average load of 20 chemicals from each stormwater runoff sampling site in St. Paul and

Minneapolis, MN. Column 1 is the treatment group, column 2 is the winter snow melt; column 3 is spring

rain event; column 4 is first summer rain event.

Exposure Organism

D. magna were obtained exclusively from one biological supply company (Carolina Biological,

Burlington, NC). D. magna less than 24 hrs. old were placed into treatment solutions at the beginning of

the experiment. D. magna were fed 0.25 ml YCT food once/day. YCT food was made from fermented

trout chow, cereal leaves, and yeast at SCSU. YCT food preparation was completed using a protocol from

Environmental Protection Series (1996).

16 Water Quality

Total water hardness, free chlorine, total chlorine and alkalinity were assessed every 4 days for all

exposures using AquaChek 5-in-1 Water Quality Test Strips (Hach Company, Loveland, CO).

Results for D. magna Exposures

Pyriproxyfen EC10 Exposure in D. magna

Male neonate production was dose dependent. Increasing the dose of pyriproxyfen resulted in higher

percentages of males and a decrease in overall neonate production. The lowest effective dose was 119 ng/L,

resulting in an EC20. This LOEC (lowest observed effects concentration) for pyriproxyfen is similar to work

done by (Olmstead and Leblanc, 2003; Matsumoto et al. 2008) which suggest a EC50 range of 55-100 ng/L

for pyriproxyfen.

16%

22%

39%

73%

0

10

20

30

40

50

60

70

80

90

100

0 29 59 89 119 179 239

% M

ale

D. m

agn

a

Pyriproxyfen ng/L

(a)

17

Figure 2.1. D. magna neonate production when exposed to pyriproxyfen. (a) Percentage of male neonates

produced in response to pyriproxyfen exposure. The 59 ng/l dose was disregarded because only one

female was producing male neonates. The 119 ng/L group was the lowest pyriproxyfen concentration at

which males were consistently produced. (b) Total neonate production for all treatment concentrations.

Future work will utilize the 119 ng/L concentration as the lowest observed effects concentration

(LOEC) and the 239 ng/L concentration will be used as a positive control for gene expression assays in D.

magna.

Four-day D. magna Gene Expression Assay

Survival in the four-day assay was greater than 99% and RNA extractions were generally of high

quality. One batch of extractions totaling 12 samples was discarded due to operator error in pipetting.

Samples were shipped to UMGC and are currently being analyzed.

Sixteen-day D. magna Reproductive and Adult Survival Assay for Stormwater Exposure

Reproduction Assay

Filtration of stormwater runoff through BMPs appears to do little to improve reproduction in D.

magna. Results from the winter snow melt (WSM) were difficult to analyze because TBNS2 was omitted

from the exposure due to being frozen and 37ST_IN was not tested because several sample bottles were

0

100

200

300

400

500

600

0 29 59 89 119 179 239

To

tal

Neo

nat

e R

epro

duct

ion

Pyriproxyfen ng/L

(b)

18 not properly filled. Samples taken from stormwater pipe outflows were significantly different from

stormwater taken from BMPs.

(a)

19

Figure 2.2. Sum total neonates/female/day/treatment for winter snow melt and spring rain event (a);

reproduction for winter snow melt (b); reproduction for spring rain event; there were no significant

differences between inflow and outflow of BMPs.

There were no significant differences between the inflow and outflow of winter snow melt (WSM) or

spring rain event (SRE) exposures. There was however a general trend of higher reproduction in the

inflow versus outflow groups within BMPs. This may be related to the fact that Daphnia eat bacteria

which could be more prevalent in BMP influent. Due to lack of data in the WSM exposure it is difficult to

draw any conclusions about the influence of seasonality on the reproduction of D. magna exposed to

stormwater runoff. Control Daphnia didn’t reproduce at any time.

Adult Survival

Adult survival in stormwater was highly variable across treatment groups. Missing data from the

winter snow melt is attributable to TBNS2 being frozen and Crystal Lake and 37ST_IN water sampling

bottles not being properly filled. Controls always experienced high mortality because SCSU well water is

(b)

20 harsh (the water is hard and has a high alkalinity). The explanation for why SCSU well water was used as

a control is explained in the discussion section.

(a)

21

Figure 2.3. Survival of adult Daphnia exposed to stormwater effluent. Adult D. magna survival in

stormwater effluent from unfiltered and BMP filtered sources; (a) survival of adult D. magna raised in

winter snow melt runoff; (b) Survival of adult D. magna raised in spring rain event runoff;

Discussion

The goal of these experiments was to determine if D. magna could be utilized as a test organism in a

novel assay. Once developed, this novel assay was utilized to determine the effects of stormwater runoff

on reproduction and survival in D. magna. Many studies have already established the effectiveness of D.

magna in toxicity testing (Olmstead and Leblanc, 2003; Ginjupalli and Baldwin, 2013; Sarma and

Nandini, 2006). These experiments, however, have examined alterations in reproduction and survival, not

alterations in gene expression. The ability to use gene expression as a metric for toxicity would add

another tool in addition to those already availed (reproduction and survival assays) for the identification

of potentially harmful chemicals.

(b)

22 The pyriproxyfen exposure was performed to validate this novel assay at St. Cloud State University.

We wanted to determine if the D. magna colony maintained at St. Cloud State University responded to

pyriproxyfen in a manner consistent with published literature. Studies using pyriproxyfen suggest a fairly

narrow concentration range that approximates the EC50 value for this compound. Previous studies

(Olmstead et al. 2003; Matsumoto et al. 2008) have identified the EC50 value of pyriproxyfen as 50-100

ng/L. The pyriproxyfen concentrations (29, 89,119, 178, and 239 ng/L) used in the current study match

previous studies and show similar adverse outcomes. Figure 1 shows that the D. magna exposed to

pyriproxyfen in the SCSU Aquatic Toxicology Laboratory have a EC70 at 239 ng/L and EC20 at 119

ng/L, consistent with (Ginjupalli and Baldwin, 2013). The differences in effective concentration values

may be due to pyriproxyfen’s steep concentration curve and differences between laboratories (Wang

2005). The results of this preliminary study were used to identify the LOEC (EC20) concentration that

was used in the wetland water exposures.

D. magna wetland water exposures were conducted in order to provide the University of Minnesota’s

Genomic Center (UMGC) with RNA from exposed D. magna. Current analytical chemistry can detect

thousands of chemicals, but is incapable of showing if those chemicals are causing alterations in

organisms. The purpose of the current study is to identify alterations in gene expression that are linked to

the pyriproxyfen exposure. By measuring changes in gene expression, it may be possible to tie alterations

in one or several genes to an individual chemical. Studies using flame retardant (Giraudo et al. 2015) and

metals (Tang et al. 2015) have already shown that the expression of D. magna genes are altered by these

exposures. The goal of this experiment is to provide scientists with a bioassay that not only detects

alterations in D. magna but also allows them to identify the causative agent of that alteration.

The final exposure that was performed with D. magna examined the impact of stormwater runoff on

reproduction and survival. The purpose of this exposure was to examine the effectiveness of best

management practices (BMP) in improving water quality from a biological standpoint (reproduction and

survival). Previous studies have examined water chemistry results to determine effectiveness of BMPs

23 (Erickson and Gulliver, 2010; Geosyntec Consultants and Wright Water Engineers, 2014), but largely

ignore bioassays. This is critically important, because while BMPs may improve water quality from an

analytical chemistry standpoint, the most important question is if they improve it from a biological

standpoint.

These experiments used traditional reproductive and survival assays to determine biological

outcomes. Unfortunately, unforeseen circumstances when collecting stormwater resulted in missing data

from the winter snow melt exposure. Because of the missing data, it is impossible to draw any

conclusions from the exposure, other than that the control group exposed to SCSU well water experienced

very high mortality. The cause of this mortality is most likely due to SCSU well water having a high

alkalinity. Testing guidelines (OECD, 2012) recommend using reconstituted defined media for toxicity

testing. This was not possible however, because the D. magna tests were followed up with testing of

larval fathead minnows that utilized SCSU well water as a control.

Although the winter snow melt exposure was not complete, thus preventing its further use in this

study, the spring rain event exposure provided a complete data set. It was found that reproduction was not

significantly different between the inflow and outflow of the BMPs. This is very interesting, because

water quality analysis showed a decrease in chemical load in BMP effluent. This reduction in chemical

load is often used to indicate that BMPs are functional in preventing chemical pollutants from entering

riverine aquatic ecosystems (Erikson and Gulliver, 2010; Erikson et al. 2012; Weiss et al. 2007).

When examining survival of Daphnia exposed to stormwater runoff for 16 days, there was a decline

in survival of individuals exposed to BMP effluent. This was surprising and contradictory to our

assumptions. While only one BMP (TBNS2) showed a statistically significant decrease (30%) in survival

of effluent exposed organisms, there also was a reduction in survival for BMP_37ST(10%) and

BMP_TBNS1( 23.3%) sites. In addition to this, the survival of Daphnia exposed to BMP influent was

similar to that of stormwater collected from PIPE_SAP, PIPE_Cedar_Lake, and PIPE_Crystal_Lake.

24 These results suggest that Daphnia either survive better in untreated stormwater or are being

detrimentally impacted by the BMP treatment.

25 CHAPTER 3: EFFECTS OF URBAN STORMWATER RUNOFF AND BEST MANAGEMENT

PRACTICES ON THE GROWTH AND PREDATOR ESCAPE RESPONSE OF PIMEPHALES

PROMELAS

Introduction

Fathead minnows (Pimephales promelas) are a species of fish found throughout North America.

These fish are commonly used in aquatic toxicology testing to elucidate the biological effects of

environmental pollutants and wastewater effluent. Because of their widespread habitat range (Figure 1),

tolerance for laboratory conditions, short maturation period, and sensitivity to environmental contaminant

(Denny, 1987; Gieger et al. 1988; Jensen et al. 2001) the fathead minnow has been adopted as a model

organism for aquatic toxicology testing. While many experiments use adult fathead minnows (Frankel et

al. 2016; Prosser et al. 2016; Parrott and Blunt, 2005) there is also a use for larval fathead minnow

exposures in toxicological testing.

Figure 3.1: Native range of Pimephales promelas (Credit: William Heikkila)

26 Studies by Painter et al (2009) and Schoenfuss et al (2016) have demonstrated that exposure to

endocrine active compounds affects the escape performance of larval minnows. This is important,

because alterations in escape behavior could result in higher mortality of young fish. The mechanism by

which alterations in predator escape performance is measured is the C-start. C-starts are a biologically

universal escape reaction in teleost fishes that enables them to evade predators (Nissanov and Eaton,

1989). The c-start can be broken down into segments (escape velocity, escape angle, body length, latency,

and total escape response) that are measured and used to quantify escape performance (Painter et al. 2009;

McGee et al. 2009). By measuring these parameters, alterations in escape performance in response to

chemical exposures can be identified.

Another way to measure larval performance is through feeding assays. Beitinger (1990) found that

fish are very sensitive to environmental stressors, causing alterations in foraging performance. Because

feeding is such an important behavior, any alterations in foraging could have detrimental impacts on

larval survival. Environmental stressors can be chemicals within the environment. In a study by Mehrle et

al (1988) it was discovered that polychlorinated compounds affected behavior, survival and growth of

rainbow trout, and a study by Brown et al (1987) found that guppies were significantly more likely to be

eaten when exposed to pentachlorophenol. These studies reinforce the fact that environmental exposure to

chemicals can cause detrimental effects in fish.

While there have been many studies looking at the impact of wastewater and the pharmaceuticals

contained therein, there are no studies, to our knowledge, that have examined the effects of stormwater

runoff on larval minnows. The goal of this study is to determine the effect of stormwater runoff on larval

fathead minnows and the potentially beneficial impact of best management practices on filtering

stormwater runoff. If best management practices work as stated, they should reduce the chemical load in

surface water runoff. This reduction in chemical load has been observed through chemicals analysis

(Geosyntec Consultants and Wright Water Engineers, 2014). However, just because this decrease in

chemical load is occurring does not mean that there is a biological improvement.

27 The goal of this research is to identify any alterations in larval fathead minnow escape performance

when exposed to stormwater runoff. The hypotheses that we will be examining are that filtration of

stormwater runoff by best management practice sites will improve biological outcomes. H1: BMP

effluent exposed larval minnows will perform better on escape assays by having lower latency, higher

escape velocity, greater escape angle, and greater total escape response than BMP influent exposed

minnows; H2: BMP effluent exposed larval minnows will forage more than BMP influent exposed

minnows.

Methods and Materials

Experimental Design

In the present study, larval fathead minnows were exposed to stormwater runoff collected from urban

areas in St. Paul and Minneapolis, MN. Minnows were obtained from Environmental Testing and

Consulting, Superior, WI. All testing was conducted starting with larvae <24 h post hatch and lasted 21-

days. Larvae were maintained in the St. Cloud State University Aquatic Toxicology Lab in a 16:8 h light:

dark photoperiod; temperatures were maintained at 23.84±0.98[mean ± standard deviation]; 4.44±0.59

dissolved oxygen; 69.81±30.14 alkalinity; 138.25±68.12 total hardness; 8.52±0.17 pH;) and fed twice

daily ad libitum with Artemia. Exposures were conducted concurrently throughout 2016. All maintenance

of experimental organisms was carried out in accordance with St. Cloud State University’s IACUC

policies.

Stormwater Runoff Larval Fish Exposures

Larval fish were randomly distributed to 1L treatment jars (n=20 larvae/jar with 3 jars/treatment). Jars

were filled with 2/3 L stormwater. Each day stormwater was thawed, vacuum filtered with Whatman #1

filter paper (Sigma-Aldrich, St. Louis, MO) and used to do 50% renewals of each treatment jar. A control

treatment used SCSU well water. At the end of the 21-day exposure period larvae were evaluated with a

predator escape assay.

28 Four exposures were conducted with stormwater runoff collected during winter snow melt (WSM),

spring rain event (SRE), first summer rain event (FSRE), and second summer rain event (SSRE).

Exposures were conducted concurrently in 2016.

C-start Predator Escape Response

Predator escape performances are evaluated after exposures with C-start responses. C-starts were

recorded with a RedLake MotionScope M1 at 1000 frames per second. Larvae were placed into a 5 cm

dish containing 10 ml water and placed on top of a 1 mm grid pattern. An electronic buzzer under the dish

was activated to simulate a predator. The response of the larvae were recorded and stored as a AVI file.

Larvae (3/treatment jar) were tested in this manner, with treatment jars being randomly tested until all

were analyzed.

AVI video files were analyzed with ImageJ software to determine several endpoints. These endpoints

included latency (delay after stimulus is applied), escape velocity (body length/ms), total escape response

(body length/ms), body length (mm), and escape angle (degrees). Larval performance assays were based

on work by Painter et al (2009).

Statistical Analysis

An ordinary one-way ANOVA was conducted to determine differences between treatment groups. A

Dunnett's multiple comparisons test was used to check for variance in latency, escape velocity, total

escape response, body length, and escape angle. A student’s t-test was used to determine p-values when

comparing the inflow and outflow of BMP stormwater sites.

Stormwater Runoff Collection

Stormwater runoff was collected from three best management practice (BMP) sites and three

stormwater outflow pipes within St. Paul and Minneapolis, MN. Stormwater was collected in 1L Nalgene

bottles. Water was collected directly from holding ponds containing inlet water by filling the bottles 5-15

cm below the water surface. Outlet water was collected directly from an outlet pipe at all three BMPs.

Water was transported to SCSU and stored at -20O C.

29 Stormwater runoff was collected at four different rain events to determine the unique chemical profile

of stormwater runoff throughout the seasons (winter snow melt, spring rain event, first summer rain event,

and second summer rain event). The BMPs being sampled were TBNS1_IN and OUT, TBNS2_IN and

OUT, and 37ST_IN and OUT. Stormwater outflow pipes were St. Anthony Park, Crystal Lake, and Cedar

Lake. TBNS2 was frozen at the time of winter snow melt sampling and was omitted from larval testing.

Two replacement samples were collected later in the year to replace the missing samples from the winter

snow melt event.

Results

Larval Escape Performance and Feeding

Larval fathead minnows exposed to the influent and effluent of best management practice sites

showed little improvement in five key areas relating to larval survival. Analysis showed significant

differences in several of the performance assays and feeding trials for winter snow melt, spring rain event,

and the first summer rain event exposures.

Table 3.1: Comparison of best management practices influent and effluent waters for winter snow melt.

There was a decrease in escape velocity and increase in escape angle for larval fathead minnows exposed

to BMP_TBNS1 effluent.

Winter Snow Melt p-values (α=0.05)

Treatment Latency

Body

Length

Escape

Velocity

Total Escape

Response

Escape

angle Feeding

BMP_37ST_OUT vs

BMP_37ST_IN 0.140 0.199 0.400 0.429 0.420 0.460

BMP_TBNS1_OUT vs

BMP_TBNS1_IN 0.336 0.117 0.039 0.334 0.018 0.419

BMP_TBNS2_OUT vs

BMP_TBNS2_IN NA NA NA NA NA NA

30 Escape velocity (body lengths/millisecond) was altered along with escape angles (degrees). All

measurements are given as (mean± standard deviation) Escape velocity (TBNS1_IN 0.041±0.015;

TBNS1_OUT 0.033±0.014). Escape angle (TBNS1_IN 88.9±14.5; TBNS1_OUT 117.8±27.7) for the

winter snow melt.

When looking at the comparison between controls and all groups, there was a difference (p=0.020)

between the control and PIPE_CRYSTAL_LAKE in foraging efficiency (number of brine shrimp

consumed). Controls ate 24.1±4.2 compared to 15.7±7.8 for PIPE_CRYSTAL_LAKE larvae.

Table 3.2: Comparison of best management practices influent and effluent waters for spring rain event.

There were no differences in any of the five parameters for the spring rain event exposed larval fathead

minnows.

Spring Rain Event p-values

Treatment Latency

Body

Length

Escape

Velocity

Total Escape

Response

Escape

angle Feeding

BMP_37ST_OUT vs

BMP_37ST_IN 0.091 0.492 0.421 0.336 0.136 0.300

BMP_TBNS1_OUT vs

BMP_TBNS1_IN 0.434 0.387 0.150 0.124 0.073 0.115

BMP_TBNS2_OUT vs

BMP_TBNS2_IN 0.288 0.108 0.348 0.386 0.160 0.417

The only significant difference (p=0.003) in the spring rain event was the angle of escape between

controls (84.0±35.2) and TBNS1-OUT (121.8±27.5).

31

Table 3.3: Comparison of best management practices influent and effluent waters for first summer rain

event. There was an increase in escape velocity and decrease in foraging for BMP_TBNS1 effluent

exposed larval fathead minnows. There was an increasing in foraging for BMP_TBNS2 effluent exposed

larval fathead minnows.

First Summer Rain Event p-values (α=0.05)

Treatment Latency

Body

Length

Escape

Velocity

Total Escape

Response

Escape

angle Feeding

BMP_37ST_OUT vs

BMP_37ST_IN 0.134 0.076 0.274 0.104 0.088 0.206

BMP_TBNS1_OUT vs

BMP_TBNS1_IN 0.109 0.488 0.015 0.085 0.356 0.027

BMP_TBNS2_OUT vs

BMP_TBNS2_IN 0.386 0.247 0.430 0.470 0.259 0.039

There were several significant differences for larval minnows exposed to the summer rain event

runoff. There were differences in escape velocity and foraging efficiency for TBNS1 influent and

effluent. Escape velocity increased (0.032±0.020 influent vs 0.054±0.013 effluent) while foraging

decreased (25.4±4.6 influent vs 19.6±6.1 effluent) as water was filtered through the BMP. TBNS2 also

showed significant differences in foraging (24.9±5.0 influent vs 29.6±2.5 effluent) with the effluent

exposed larvae eating more.

When comparing controls to BMPs and other exposure sites, differences were observed in foraging

(TBNS1_OUT, p=0.003) and latency (PIPE_SAP, p=0.0007). Control fish foraged more than

TBNS1_OUT (27.3±2.9 vs 19.6±6.1) and latency of control fish was lower than PIPE_SAP (67.9±42.3 vs

170.9±61.6).

Temporal Changes in Larval Fathead Minnow Performance Assays

32 There were several significant differences when comparing winter snow melt versus spring rain event

versus winter snow melt. Changes are most significant the temporally further apart the exposures are

(there is a greater difference between winter snow melt and first summer rain event than there is between

winter snow melt and spring rain event).

Table 3.4: Seasonal changes in performance assays and feeding from winter snow melt to spring rain

event exposures. Green arrows indicate a statistically significant improvement in performance, red arrows

a decrease in performance, and yellow arrows indicate a nearly significant difference in performance.

There was an improvement in feeding efficiency for all BMPs from winter to spring. There were also

sporadic differences in other areas.

33 Table 3.5: Seasonal changes in performance assays and feeding from spring rain event to first summer

rain event exposures. Green arrows indicate a statistically significant improvement in performance, red

arrows a decrease in performance, and yellow arrows indicate a nearly significant difference in

performance.

Table 3.6: shows that there is a temporal improvement in performance assays as larval minnows are

exposed to winter snow melt and then first summer rain event. This improvement was also seen in the

spring rain event, although it was not as strong.

34 Discussion

Outcomes from the larval minnow exposures to BMP influent and effluent were both encouraging

and disappointing from the standpoint of best management practice efficacy. It was difficult to interpret

the result of these exposures because of contradictory performance assay results. Many time a

biologically positive improvement in one performance assay would be followed by a negative change in

another assay.

When we look at the influent vs effluent of best management practice sites for the winter snow melt,

we see that there were only two differences. There was a decrease in escape velocity for the effluent but

an increase in angle of escape for TBNS1. These are contradictory results, because the decrease in escape

velocity is a negative response while the increase in escape angle is positive. These contradictory or weak

results were also seen in the spring rain event and first summer rain events.

When comparing control to treatment groups in the winter snow melt exposure, where was only one

significant difference. A decrease in foraging efficiency was observed between control and

PIPE_Crystal_Lake. This decrease was quite large, 24.1 vs 15.5 brine shrimp consumed, on average. This

decrease in feeding efficiency would seem to be very significant, but there were no further differences in

any other measured parameter. Body length/growth, escape velocity, and total escape response were not

affected, suggesting that the treatment group was obtaining adequate nutrition to perform all bodily

functions. The difference in feeding efficiency may just be a one-time statistically, but not biologically,

significant occurrence.

When examining the spring rain event, there was an increase in escape angle in the effluent of

TBNS1 compared to the control. This would seem to be an improvement, but there was no difference

between the influent and effluent of TBNS1. This suggests that the BMP at TBNS1 did not adequately

filter stormwater runoff to achieve measurable biological improvements.

The first summer rain event provided more significant differences in larval adverse outcomes.

However, like the previous two exposures, there were both positive and negative results in the context of

35 best management practices. TBNS1 was again the site where most activity occurred. There was an

increase in escape velocity of effluent exposed larvae, but it was accompanied by a decrease in foraging

efficiency. TBNS2 exposed larvae exhibited increased foraging when exposed to effluent.

This seemingly random pattern of improvements continued when comparing control and treatment

fish. Control fish foraged more efficiently than TBNS1 effluent fish. But this is similar to the spring rain

event, where there was not a difference between influent and effluent. Control fish also had a lower

latency period than PIPE_SAP fish.

While there was little difference between control and treatment fish, or BMP influent vs effluent in

performance assays, there was a distinct pattern of improvement between exposure events. This can be

seen in Tables 4, 5, and 6. Green arrows represent an improvement in a performance assay, red a

decrease, and yellow a nearly significant difference. The improvement between the winter snow melt and

first summer rain event is indicative of a strong temporal improvement in water quality with time.

36 CHAPTER 4: CONCLUSION

Key Findings

Daphnia magna Exposures

While there was not enough data to do a proper analysis on the winter snow melt exposure, we could

draw some conclusions from the spring rain event. There was a trend of decreasing survival in Daphnia

exposed to BMP effluent. There were also no significant differences in reproduction of Daphnia exposed

BMP filtered stormwater runoff. Best management practices seem to have very little, or even a slightly

negative, impact on biological outcomes for D. magna.

H1: Genetic analysis is ongoing and we can not evaluate this hypothesis yet

H2: D. magna reproduction was not greater in BMP effluent, so we reject H2

H3: D. magna survival was lower in BMP effluent, so we reject H3

Larval Fathead Minnow Work

The results of larval fathead minnow stormwater exposures were noteworthy. There were few

differences between best management practice influent and effluent exposed fish within individual

exposures. In contrast, there were strong temporal differences between winter snow melt and first summer

rain events, which indicate that water quality was improving from a biological standpoint later in the year.

One possible explanation for this temporal improvement in larval performance is that there could be a

flushing effect associated with the winter snow melt. Average temperatures in Minnesota in winter are -

2.2 oC (U.S. climate data, 2016). This constant low temperature allows for the buildup of snow, ice, and

any pollution trapped within (Kuoppamaki et al. 2014; Novak et al. 2016). As temperatures rise in spring

we can expect melting ice to carry any pollution trapped during the winter in the snow pack to streams,

rivers, ponds, and lakes. This large rush of pollution into the environment is called a flushing event, and

can also occur after prolonged periods of dry weather (Lee et al. 2004).

Flushing events would explain why the winter snow melt fish performed worse on performance

assays than fish from the spring snow melt or summer rain event. However, chemical analysis of

37 stormwater runoff indicates an increase in total chemical load for most stormwater collection sites in the

first summer rain event (Chapter 1, Table 1). This would suggest that winter snow melt exposed fish

should perform better than the first summer rain event, but that was not observed in our exposure.

A purely speculative, but informed, explanation for the results that were obtained is that the chemical

composition of stormwater runoff is different between seasons. This would make sense, because runoff

collected in winter would not be expected to contain many fertilizers, pesticides, or herbicides that are

used on lawns or gardens in spring and summer. This difference in chemical composition may be the

deciding factor in determining the toxicity of stormwater runoff.

When examining the effectiveness of best management practices, it appears that there is little

correlation between filtration of stormwater and improved biological outcomes. Most of the differences

that were observed between influent and effluent occurred in TBNS1, and they were sporadic. There were

a mix of beneficial and detrimental results from the six endpoints that were measured. This seemingly

random pattern in biological outcomes stands in direct contrast to the water chemistry analysis. Chapter 1,

Table 1, clearly indicates that best management practice effluent has a lower chemical load than the

influent.

The discrepancy between biological outcome and chemical analysis is puzzling. Again, there are a

couple possible reasons for these results. The chemical load may be skewed by a large abundance of one

or two chemicals that are filtered out by the BMP. It is also possible that the chemical load between

influent and effluent similar are similar enough that there are no differences in performance assay results.

Similarly, the chemical composition may include more hazardous chemicals in the winter snow melt even

if absolute chemical concentrations are lower than in spring in summer.

The main objective of this research was to determine if best management practices were improving

biological outcomes. The answer seems to be no. There is no strong correlation between filtration of

stormwater runoff through BMPs and improvements in biological outcomes. This does not mean that

BMPs should not be used. An important aspect to remember is that there was a measured improvement in

38 water quality after BMP filtration. It may be that the filters themselves need to be improved to achieve the

desired biological outcome. Another point-of-note is that only three best management practice sites were

tested in the current study. This small sample size is insufficient to definitively declare best management

practices to lack biological value.

H1: Larval fathead minnows did not respond in a consistent manner to BMP influent or effluent.

Because of the variability in response to performance assays we must reject H1. BMP filtration does not

improve larval fathead escape performance.

H2: Larval fathead minnows did not respond in a consistent manner to BMP influent or effluent.

Because of the variability in response to performance assays we must reject H2. BMP filtration does not

improve foraging efficiency.

Future Directions

Daphnia magna

As a continuation of the novel Daphnia exposure assay that was developed at SCSU, we will be

conducting RNA extractions and analysis of stormwater and BMP effluent exposed organisms. This may

help us identify what is happening in BMP effluent waters. These exposures should also be conducted

with a larger BMP sample size. It is difficult to draw any conclusions from such a small number of

sample sites.

Pimephales promelas

As with the Daphnia, there are several study directions that should be explored in more detail using

larval fathead minnows. It would be interesting to assess the phenotypic outcomes in the exposed larvae,

and determine if there are any physical abnormalities. This would be particularly interesting with the

winter snow melt treatment. Animals in this treatment were exposed to high salinity from road salt. It

would also be interesting to expand the best management practice pond sample size to further elucidate

their biological drawbacks and benefits.

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