Life History and Secondary Production of Cheumatopsyche Lasia Ross (Trichoptera: Hydropsychidae) with Respect to a Wastewater Tr

LIFE HISTORY AND SECONDARY PRODUCTION OF Cheumatopsyche lasia Ross

(TRICHOPTERA:HYDROPSYCHIDAE) WITH RESEPCT TO A

WASTEWATER TREATMENT FACILITY IN A NORTH

CENTRAL TEXAS URBAN STREAM

Jenny Sueanna Paul, B.S.

Thesis Prepared for the Degree of

MASTER OF SCIENCE

UNIVERSITY OF NORTH TEXAS

December 2011

APPROVED:

James H. Kennedy, Major Professor Steven J.Wolverton, Committee Member Gerard A. O’Donovan, Committee Member Art J. Goven, Chair of the Department of Biological Sciences James D. Meernik, Acting Dean of the Toulouse Graduate School Paul, Jenny Sueanna. Life History and Secondary Production of Cheumatopsyche lasia

Ross (Trichoptera: Hydropsychidae) with Respect to a Wastewater Treatment Facility in a North

Texas Urban Stream. Master of Science (Biology), December 2011, 52 pp., 5 tables, 11 figures,

references, 117 titles.

This study represents the first shift in multivoltine life history of Cheumatopsyche species

from a wastewater treatment plant (WWTP) in North America. Populations of C. lasia were examined upstream and downstream of the Denton’s Pecan Creek WWTP August 2009 through

November 2010. C. lasia is multivoltine in Pecan Creek with three cohorts observed upstream of the WWTP and four possible cohorts downstream. A fourth generation was possible downstream as thermal inputs from WWTP effluent resulted in elevated water temperatures that allowed larval development to progress through the winter producing a cohort ready to emerge in spring. Production of C. lasia was 5 times greater downstream of the WWTP with secondary production estimates of 1.3 g m-2 yr-1 and 4.88- 6.51 g m-2 yr-1, respectively. Differences in abundance were due to increased habitat availability downstream of the WWTP in addition to continuous stream flow from inputs of wastewater effluent. Results also suggest that C. lasia is important for energy transfer in semiarid urban prairie streams and may serve as a potential conduit for the transfer of energy along with emergent contaminants to terrestrial ecosystems.

These finding highlight the need for more quantitative accounts of population dynamics

(voltinism, development rates, secondary production, and P/B) of aquatic insect species to fully understand the ecology and energy dynamics of urban systems.

Jenny Sueanna Paul

ii ACKNOWLEDGMENTS

This project was associated with the University of North Texas, Institute of Applied

Sciences, and Department of Biology. Special thanks to my major advisor, Dr. Kennedy, and my committee, Drs Wolverton and O’Donovan. I appreciate the assistance and insights provided by

David Hunter and Kenneth Banks with the City of Denton. Thanks to my lab mates in the

Benthic Ecology lab for assistance and support with every aspect of this project. I am especially indebted to Kaitlin Slovak, Mike Simanonok, and Rosemary Burke for collection of field samples, particularly from November 2009 through January 2010 while I was unable to collect due to a broken ankle. I am also grateful to numerous undergraduate students who assisted with processing and picking samples. I would like to thank Dr. Venables and Benjamin Lundeen for assistance with analytical work regarding contaminants as well as David Baxter and Patrick Horn for assistance with lipid extractions. Finally, I am thankful for the support of my friends and family as they are a constant source of encouragement, love, and inspiration.

iii TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ...... iii

LIST OF TABLES ...... v

LIST OF FIGURES ...... vi

INTRODUCTION ...... 1 Background Information ...... 1 Study Organism ...... 3 Research Objectives ...... 6 Study Area ...... 6

MATERIALS AND METHODS ...... 8 Water Chemistry ...... 8 Collection of Larvae ...... 8 Laboratory Rearing ...... 9 Production ...... 10

RESULTS ...... 12 Physico-Chemical Parameters ...... 12 Pupae ...... 14 Laboratory Rearing ...... 15 Seasonal Development and Voltinism ...... 15 Biomass and Secondary Production...... 16

DISCUSSION ...... 19

APPENDIX CADDIS FLY FREQUENCY RAW DATA ...... 39

LITERATURE CITED ...... 44

iv LIST OF TABLES

Page

Table 1 Summary of water chemistry for Pecan Creek as monthly averages from August 2009 through November 2010...... 35

Table 2 Secondary production calculations for upstream (WOOD)...... 36

Table 3 Secondary production calculation for downstream (WWTP)...... 37

Table 4 Caddis fly lipids summary of results for total lipid estimates from caddis fly tissue. .... 38

v LIST OF FIGURES

Page

Fig. 1 Map of the Pecan creek watershed...... 24

Fig. 2 Discharge estimates...... 25

Fig. 3 Dissolved oxygen estimates...... 26

Fig. 4 Temperature estimates ...... 27

Fig. 5 Degree day estimates...... 28

Fig. 6 Scatter plot of head capsule measurements...... 29

Fig. 7 Illustration of larval head capsule development...... 30

Fig. 8 Illustration of pupae abdomen ...... 31

Fig. 9 Life history diagram for upstream (WOOD)...... 32

Fig. 10 Life history diagram for downstream (WWTP) ...... 33

Fig. 11 Total abundance estimates...... 34

vi INTRODUCTION

Background Information

Urbanization occurs when forests and rural areas are modified for residential, municipal, or commercial uses (Wear and Bolstad, 1998, Wear and Greis, 2002) and is particularly evident near water bodies (Feminella and Walsh, 2005). Urbanization is second only to agriculture as the leading cause of stream impairment in the United States (Paul and Meyer, 2001). Half of the world’s population (Feminella and Walsh, 2005) and over two-thirds of the U.S. population

(Paul and Meyer, 2001) live in urban centers. As this proportion continues to increase, impacts from urban areas will also increase (McGranhan and Satterthwaite, 2003).

Streams and rivers close to urbanized areas are often degraded both chemically and ecologically (Walsh et al., 2005). The pattern of degradation observed repeatedly has been termed the urban stream syndrome by Meyer et al. (2005). Symptoms include: flashier hydrographic profiles following storm events, altered channel morphology, elevated concentrations of nutrients and contaminants, and reduced biotic richness (Paul and Meyer,

2001, Meyer, Paul and Taulbee, 2005). Mechanisms driving the syndrome are complex and interactive, making it difficult to understand direct cause and effect; however, many impacts have been related to the quantity and condition of wastewater treatment plant (WWTP) effluent

(Walsh et al., 2005, Paul and Meyer, 2001, Grimm et al., 2008). Base flow conditions in urban streams may be augmented by wastewater treatment plants, the discharge from which may constitute the majority of flow during low flow periods (Paul and Meyer, 2001). This is particularly evident in arid to semi-arid regions of the United States where seasonal periods of extremely low flow, or no flow, result in an effluent-dominated stream system (Atkinson et al.,

1997, Slye et al., 2011). Although stream biota may benefit from steady stream low (Gibb and

1 Richards, 1978), changes to native flow regimes due to effluent discharge can alter physical and

water quality parameters. Urbanization can also affect water temperature (Pickett et al., 2001),

an important variable impacting key ecological processes. Water discharged from wastewater

treatment plants may be heated well over the natural temperature range (KinouchiYagi and

Miyamoto, 2007). Temperature can influence physical and chemical attributes of water, such as

dissolved oxygen concentration and the behavior of contaminants (Allan and Castillo, 2008). In

addition, crucial life history variables and the timing of life history events of aquatic insects are

often regulated by temperature (Hauer and Hill, 2007).

The response of stream biota to urbanization varies by assemblage group; however,

macroinvertebrates often show the highest degree of sensitivity (Wenger et al., 2009). Benthic assemblages have been widely studied in urban stream ecosystems (Chessman and Williams,

1999, Walsh et al., 2001, Morley and Karr, 2002, Stepenuck, Crunkilton and Wang, 2002, Roy et al., 2003, Wang and Lyons, 2003, Paul and Meyer, 2001). However, research regarding the secondary production of benthic macroinvertebrates (Walsh et al., 2005) and life histories of aquatic insects (Paul and Meyer, 2001) in response to urban land use has received less attention.

Studies in life history evolution for targeted species can provide insights as to particular impacts of urban development, such as that from wastewater inputs or influence of contaminants.

Life histories provide environmental and genetic information on aquatic insect populations (Butler, 1984) and are a vital component of ecological research. Life history refers to the variable events associated with the life cycle such as voltinism, development, and emergence patterns (Butler, 1984). Life cycles are species specific; however, life histories of aquatic insects often exhibit variability in response to environmental conditions (Butler, 1984, Price, 1997).

Environmental parameters such as temperature or dissolved oxygen can influence life histories

2 through increased growth rates and number of generations produced annually (Mackay and

Wiggings, 1979, Ward and Cummins, 1979, Krueger and Waters, 1983, Sweeney, 1984,

Sweeney and Vannote, 1986, Sanchez and Hendricks, 1997).

Benke (1984, 1993) noted that the secondary production of stream macroinvertebrates presents a means with which to assess changes in energy flow that may occur in response to environmental variation. Secondary production is defined as the biomass produced by an animal population over a unit of time (Benke, 1984, Rigler and Downing, 1984). It is ecologically important as it represents the energy available to higher trophic levels (Benke, 1984, Benke,

1993). The magnitude of secondary production is influenced by life history features (Waters,

1979, Benke, 1984, Huryn and Wallace, 2000) as well as environmental parameters (Krueger and Waters, 1983, Sweeney and Vannote, 1986, Robinson and Minshall, 1998). Hence, patterns of production among macroinvertebrate populations reflects the relationship between life history and the environment (Southwood, 1988).

Cheumatopsyche is ideal for investigating ecological differences attributed to wastewater.

They are widespread across much of the United States and are common to urbanized streams.

Through filter feeding, C. lasia represents an important ecological linkage and has the potential to provide toxicological information. In addition, literature regarding the production and life history of caddis flies is well documented, allowing for a strong framework with which to detect ecological impacts of wastewater discharges in a semiarid prairie stream.

Study Organism

Trichoptera, otherwise known as caddis flies, are found in most freshwater habitats and

occupy every trophic level and functional feeding group in freshwater ecosystems (Mackay,

3 1978, Mackay and Wiggings, 1979). They are holometabolous insects with a determinant number of five instars. The family Hydropsychidae is a widespread and diverse group

comprising over 1200 species, approximately 150 of which occur in North America (Wiggins,

1996); (Ross and Wallace, 1983). Hydropsychid larvae are characterized by tough sclerotization

on the dorsum of each thoracic segment combined with branched gills on the ventral surface of

the abdomen and a tuft of setae at the base of each anal proleg (Moulton and Stewart, 1996).

The family Hydropsychidae are well known for the capture nets they construct to collect food

and are typically found in turbulent, highly oxygenated water (Wiggins, 1977). Nets are strung at

the entrance of a tubular fixed retreat they construct from the substrate with silk. By feeding on

small organic particles that might otherwise be lost downstream, they retain nutrients within the

ecosystem (Wiggins, 1996). Hydropsychidae are ecologically important and can reach high

densities, especially in areas with an abundance of particulate organic matter as sometimes

occurs downstream from WWTPs and impoundments (Mackay and Waters, 1986, Parker and J.

R. Voshell, 1983, Valett and Stanford, 1987).

Cheumatopsyche is represented on every continent except South America (Wiggins,

1977). It was first established in 1891 to subdivide the large and complex genus Hydropsyche.

The genus Cheumatopsyche was erected to accommodate species with an undivided R2+3 vein in the hind wing (Marlier, 1962) and an irregularly shaped tenth tergum on the adult male (Ulmer,

1951). The larvae of Cheumatopsyche in North America have a uniformly dark head capsule,

unlike the varied color patterns of Hydropsyche, and most have a distinctive median notch on the

anterior margin of the frontoclypeal apotome (Wiggins, 1977). There are forty-three species of

Cheumatopsyche in North America (Morse, 1993) with larvae inhabiting a variety of stream types. Cheumatopsyche have been found in streams with differing water quality (Moulton and

4 Stewart, 1996) and are known to be successful in streams that are too polluted for other caddis

flies (Wiggins, 1977). They have also been found deep into the interstitial substratum of streams

(Williams and Hynes, 1974). While C. lasia has occasionally been collected in large rivers, it

prefers small streams (Ross, 1938).

Cheumatopsyche lasia was first described by Ross (Ross, 1938), and has a wide

distribution among the western and Midwestern United States (Gordon, 1974). Larvae of

Cheumatopsyche lasia are often abundant in heavily silted streams and have been collected

across the Midwest (Ross, 1944). Adults of C. lasia exhibit seasonal variability in length;

however, the species exhibits no seasonal variability in color (Rhame and Stuart, 1976). Pupal

sex ratios of C. lasia are reported as 1:1 (Rhame and Stuart, 1976); however, ratios determined

by light trapping adults of Cheumatopsyche may be heavily weighted to females (Harris, 1971,

Rhame and Stuart, 1976). Ovoposition of C. lasia occurs underwater and eggs are deposited singly in one or multiple egg masses (Rhame and Stuart, 1976).

Cheumatopsyche is listed with a tolerance value of 6 according to the Texas Commission on Environmental Quality (TCEQ, 2007). Tolerance values of benthic macroinvertebrates range from 0 to 15 with lower values associated with sensitive taxa and higher values associated with higher tolerance to poor water quality. Cheumatopsyche is an ideal organism for this study as it is listed within the middle range of values representing a moderate tolerance to pollution, such as that commonly associated with urban streams. Insight into the production and life history of this species is important for understanding the ecological processes in Pecan Creek and similar urban systems.

5 Research Objectives

As life history and secondary production of aquatic insects reflects the relationship

between ecological processes and the environment, differences between caddis fly populations

upstream and downstream of a WWTP gives insight towards ecological impacts of wastewater

on urbanized prairie systems. The purpose of this research is to describe ecological aspects of

Cheumatopsyche lasia in reference to a WWTP. Specific objectives were to determine the number of generations produced annually, abundance, development, emergence, biomass and secondary production at both sites.

Study Area

Pecan Creek is typical of streams in the north central Texas region and offers a context for studying the impacts of wastewater discharges in semi-arid urbanized systems. Pecan Creek is a 3rd order, intermittent stream that flows through the city of Denton, TX (Figure 1), and

eventually feeds into Lake Lewisville, a reservoir providing drinking water to the cities of

Denton, Dallas, and Lewisville. The basin is characterized as an urban watershed encompassing

63.5 km2 (Taylor, 2002) with agricultural, industrial, residential, and recreational land uses

(Appel, 2000). Pecan Creek receives nonpoint source pollution from urban sources, in addition to

discharges from the Pecan Creek Water Reclamation Plant, a municipal water treatment facility.

The water reclamation plant treats an average of 21 million gallons of water a day and has a peak

flow capacity of 46 million gallons (Denton, 2009).

Two study sites were established in riffles for collecting samples and environmental data

from Pecan Creek (Figure 2). The upstream site (WOOD) is located approximately 3 km

upstream from the water treatment facility. The upstream site is approximately 12 m long and 2

6 m wide, consisting primarily of sand and gravel with a few rocks unevenly dispersed with a mean annual discharge of 0.07 m3s-1. The downstream site (WWTP) is located approximately

150 m below the wastewater treatment facility outfall. A portion of the stream channel at this site has been paved with concrete and rocks were introduced creating a riffle area approximately 12 m wide and 6.5 m long with a mean annual discharge of 0.98 m3s-1. Both sites are monitored by the City of Denton Watershed Protection Program.

7 MATERIALS AND METHODS

Water Chemistry

Selected physical and chemical measures of water quality were monitored throughout the study period. A Hydrolab® Datasonde maintained by the City of Denton was deployed at each site. The sondes recorded water parameters including temperature (°C), dissolved oxygen

(mg/L), pH, and conductivity (µS/cm, every 30 minutes throughout the study. Discharge was calculated as m3/s, utilizing a combination of field and historical data (Taylor, 2002). Particulate organic carbon (POC) was assessed for each site August 2010 through February 2011 at approximately two-week intervals using a loss by ignition technique (Dean, 1974).

Collection of Larvae

Quantitative samples for life history and production were taken from August 2009 to

November 2010, at approximately two week intervals. Both sampling sites were mapped into a square meter grid based on a central transect. All square meters containing suitable habitat were identified and assigned a number. A list of quadrants for each site was compiled randomly, and on each sampling date, the top numbers were sampled and crossed off the list. No quadrant within either grid was re-sampled until the list had been completed, to ensure adequate recovery time. On each sampling date, rocks ranging from approximately 10 to 50 cm were sampled from each site. A fine mesh Surber sampler was placed downstream to collect any debris that was dislodged. Rocks were placed into a five gallon bucket containing stream water and gently scoured to remove debris and organisms. The contents of the bucket were poured through a 180

µm sieve and stored in 70% ethanol.

The surface area of each rock suitable for larvae colonization was estimated with heavy

8 weight aluminum foil based on a simple linear regression of foil surface area on foil mass

(BottBrockDunn et al., 1985, Johnson and Kennedy, 2003). Each rock sampled was covered with

heavy duty aluminum foil, taking care to minimize overlap from irregularities such as bumps or

crevices. The mass of each aluminum sample was divided by the average mass of one square centimeter of aluminum foil to give an estimate of surface area in cm2.

Laboratory Rearing

An artificial stream lined with gravel and rocks no larger than 5 cm was set up in

laboratory to rear C. lasia. First instar larvae, collected from the field, were brought back to the

laboratory in stream water and placed in the rearing set up. The temperature in the artificial

stream was maintained at 22 - 29 °C under a 12:12 photoperiod. Temperature was monitored

continuously with a HOBO® U22 Pro v2 onset data logger. The number of degree days needed

to complete development to the imago stage was estimated based on the accumulated

temperature during the rearing period (Markarian, 1979).

Abundance and Life History

In the lab, larvae and pupae were sorted from the substrate with the aid of an Olympus®

SZH compound microscope and preserved in 70% ethanol. Head capsule lengths and widths

were measured with a Tucsen® TCA5.0 camera mounted on an Olympus® SZH compound

microscope coupled with TSView® image processing software. Larval instars were determined

by plotting the size frequency distribution of head capsule width against head capsule length to

obtain size ranges for instars I through V (Mackay, 1978). Distinguishing characteristics of each

instar were evaluated and described in detail. Larval counts of each instar were determined by

9 head capsule widths and standardized to surface area of rocks sampled. The abundance of each instar based on head capsule widths was plotted over the study period. Life history diagrams were used to track the growth and development of subsequent generations and identify life history events.

Production

Dry mass of C. lasia was estimated using live larvae brought back to the laboratory in stream water and sorted by instar. Larvae were placed on clean, pre-fired ceramic crucibles, dried for 24 h at 60°C and weighed to the nearest 1µg with a Cahn® C-34 electrobalance. First and second instars were placed in groups on pre-fired ceramic crucible shards and a mean dry mass was calculated. Ash free dry mass, which is used to estimate production, was determined using a loss by ignition technique (Dean, 1974).

Estimates of C. lasia standing stock biomass, annual secondary production, cohort production/biomass ratio (P/B) and annual P/B were determined from HCW measurements of field preserved specimens from each sampling date. Annual secondary production was estimated using the size frequency method described by Hynes (1961), Hynes and Coleman (1968) and modified by Hamilton (1969) and Benke (1979). The cohort production interval (CPI) required to calculate annual secondary production was estimated from instar frequency distributions of field collected specimens combined with laboratory reared specimens. The CPI is the mean length of time an aquatic insect spends as a larva. The annual production was calculated by multiplying the average cohort production by the number of generations per year. The annual secondary production divided by the mean biomass over the year approximated the rate of biomass turnover.

10 In addition, total caddis fly lipid concentrations were compared for instars IV and V

between sites. Larvae were brought back to the lab in stream water, placed in pre-weighed aluminum weight boats, dried for 24 h at 60°C and weighed to the nearest µg. Lipids were

estimated using Time-Domain NMR on a Bruker® Minispec TD-NMR analyzer. NMR

frequencies were translated to mg lipids by regressing %NMR and known caddis fly lipids

extracted with a standard Folch procedure (FolchLees and Sloane Stanley, 1957) to produce a

standard curve.

11 RESULTS

Physico-Chemical Parameters

Water chemistry measured for Pecan Creek are listed in Table 1. A fourteen-fold increase in mean discharge was observed downstream from the wastewater treatment facility. Seasonally, stream flow in Pecan Creek was steady upstream with an average discharge of 0.07 m3 s-1;

however, flow downstream vacillitated around 1 m3 s-1 with several peaks on the hydrograph, the

greatest of which reaching 3.5 m3 s-1 (Fig. 2). Oxygen concentration was typically 1-2 mg/L

higher downstream of the WWTP than upstream (Fig. 3). Dissolved oxygen concentrations

ranged from daily minimum of 4.65 mg/l to daily maximum of 6.38 mg/l upstream compared to

5.7 and 6.91 mg/l for the downstream site. Annual stream temperatures ranged from 2.4- 29.7 °C

upstream and 6.9-30.2 °C downstream. Over the winter months, temperatures were 10-15 °C

higher downstream (Fig. 4). An increase in the accumulation of degree days (DD) was observed

downstream (Fig. 5). Lesser differences were observed for pH and Conductivity. A two-factor

analysis of variance found significant seasonal differences in POC F (1,81) = 3.2, P < 0.005.

Although mean concentrations of POC were slightly higher below the wastewater outfall, no

significant difference was detected between sites, F(1.10)= 0.41, P= 0.52). There were

significant interactions between site and date, F(1,51)= 3.27, P = 0.012).

Larval Descriptions

Keys for the species identification of immature Cheumatopsyche are unavailable;

however, all Cheumatopsyche adults collected from the stream during this study were identified

as C. lasia. Therefore, Cheumatopsyche larvae analyzed for this study were presumed to be that

of C. lasia. Instars were clearly separated by plotting head capsule width against head capsule

12 length. There was no overlapping of head capsule widths or head capsule lengths between

instars, as head capsule measurements were tightly clustered together (Fig. 7).

First instar larvae are small, lightly sclerotized and relatively unpigmented. They lack gills and have an overall body length of approximately 1mm. Second instar larvae are similar to first instar larvae; however, abdominal gills are present on the ventral surface of the abdomen.

They have an overall body length of approximately 2 mm. The median notch on the anterior margin of the frontoclypeal apotome is absent in first and second instars (Fig. 7), making it difficult to decipher from early instars of Hydropsyche.

Although Mackay (1978) reported that early instars of Cheumatopsyche can be distinguished from early instars of Hydropsyche by observing a long, lanceolate setae on the anterolateral border of the pronotum instead of a short one, the difference was difficult to see and

the high number of early instars made this technique very time consuming. However,

Hydropsyche were rarely present when compared to the number of Cheumatopsyche collected.

Therefore, first and second instars of Hydropsyche were considered a negligible contributor to

the community structure and not separated from first and second instars of C. lasia. Another

caddis fly larval family present in the study area sometimes mistaken for early Hydropsychidae

was the micro caddis fly Hydroptilidae. Preserved first instar larvae Cheumatopsyche were

easily separated from early instar Hydroptilidae larvae based on body shape. C. lasia were often

curled into a “C” shape as opposed to a rigid, strait body formation found in Hydroptilidae

larvae. In addition, the prologs of Hydroptilidae are short, stout and lack setae.

Third instar larvae of C. lasia are robust and strongly resemble later instars with a relative

body length of 3 mm to 4mm. A shallow medial notch on the anterior margin of the

frontoclypeal apotome is visible (Fig. 7). The head capsule is pigmented with four long tapering

13 setae on either side. The plates on abdominal segments VIII and IX are developed. A dense tuft

of setae at the posterior apex of each anal proleg is visible. Fourth and fifth instar larvae are very

similar, with body lengths ranging from 4 mm to 5 mm and 6 mm to 8 mm, respectively. The

head capsule is uniformly dark brown with light patches surrounding the eyes. The sclerites on

the ventral surface of abdominal segments VIII and IX are well defined with a thick brush of

setae on the posterior apex of each proleg. The abdomen of some individuals of fifth instar larvae

are pigmented green.

Pupae

Pupae of C. lasia range from approximately 4.5 – 5mm (Fig. 8). The mandibles are

lanceolate with the base broader than the tip. The inner margins are serrate with four well-

defined teeth. The abdomen contains a row of long setae along the posterior margin of the dorsal

surface of segment IV extending past three-fourths of segment V. Hook plates are present on

segments III – VIII. Two pairs of hook plates are present on segment III with the anterior pair

containing 9 to 11 denticles and the posterior pair containing 18 to 20 denticles. Two smaller

pairs of hook plates are present on segment IV with the anterior pair consisting of four flat lobes

and the posterior hook plate consisting of 7 to 9 denticles. A single pair of hook plates are

present on segments V-VII consisting of three sharp points. A single pair of plates are present on

segment VIII consisting of 7 to 9 sharp denticles. Sex ratio of C. lasia pupae collected

quantitatively was not significantly different between upstream and downstream sites (paired t-

test, P = 0.22). Sex ratio of C. lasia was approximately 1:1 at both sites. The ratio upstream was

estimated at 1.13, with 180 m-2 males and 131 m-2 females. Below the wastewater outfall, the ratio was estimated at 1.39, with 340 m-2 males and 299 m-2 females.

14 Laboratory Rearing

Despite numerous attempts to induce oviposition in females collected from light trapping in the field, fertilized eggs were not obtained. Larvae took a total of 41 days, or1205

accumulated degree days (DD), to develop from first instar larvae to adult. These results are very similar to the degree days estimated by Rhame and Stuart (1976), who reported 61 days at 20 °C to24 °C, or approximately 1220 DD for C. lasia.

Seasonal Development and Voltinism

Frequency patterns of field-collected larvae, peaks in pupal abundance, and cohort lines derived from laboratory DD estimates applied to field temperature data substantiate a multivoltine life cycle for C. lasia in Pecan Creek with three overlapping generations upstream of the wastewater treatment facility and possibly four overlapping generations downstream.

Based on laboratory rearing, first instar larvae of C. lasia require 1205 degree days (DD) for development into an adult. Larval developmental rates based upon DD estimates were used in addition to instar frequency data to estimate cohort lines. All five instars were present for at least

9 months out of the year at both locations. C. lasia overwinters in the terminal instar. First instar larvae were completely absent from the downstream site late December through April; however, first instars were collected through January from the upstream site and were then absent through

April.

Proposed cohort lines based on instar frequency analysis agree with larval developmental

rates from DD estimates. Three clearly discernable cohorts were observed upstream (Fig. 9).

Cohort 1 was recruited in August, likely as the result of eggs laid in late June/early July. This

cohort developed into instars IV and V by September and emerged in October. A second cohort

15 began development in mid October as the result of a late September emergence. This cohort

developed into the terminal instar by January with an emergence in early April. A third cohort

was recruited from eggs laid in April and completed development in late July. A second

overwintering cohort began recruitment from eggs laid in August.

Three clearly discernable cohorts were observed downstream of the WWTP with a

possible fourth cohort (Fig. 10). Cohort 1 was recruited in mid August, presumably from eggs

laid in late July/early August, and emerged in late September/early October. A second cohort

began recruitment in late October and developed into instar V by December. This cohort

overwintered and emerged in early March. Although the data are sparse, a possible spring cohort

was recruited in late March/early April from eggs laid by the overwintering generation and

emerged in late May. A fourth cohort was recruited in the summer with emergence in late

July/early August. A second fall cohort began recruitment in late July/early August with

emergence in October, corresponding to Cohort 1. A second overwintering cohort began

recruitment in late September and presumably developed until the following spring.

Biomass and Secondary Production

Estimates of annual secondary production for C. lasia in Pecan Creek were 1.3 g m-2 yr-1

and 4.88- 6.51 g m-2 yr-1 for sites upstream and downstream respectively. Standing stock biomass

for the two sites were 105.11 mg m-2 y-1 for the upstream and 423.21 mg m-2 y-1 downstream.

The cohort production: biomass ratio and annual cohort: production biomass ratios for the two sites were 4.12 /yr and 12.36/yr upstream and 3.84 /yr and 11.52- 15.63 /yr downstream.

Annual secondary production, calculated using the Hynes method, is shown in Table 2 for the upstream site and Table 3 for downstream. Each instar was considered a size category

16 Secondary production was calculated by summing the losses from each developmental

class. Loss was calculated as: Loss = (number lost) x (mean weight at loss) x (loss factor). Loss

from one instar to the next was estimated by subtracting the mean number of individual for one

instar from the mean number of individuals of the previous instar. Typically, there is a decrease

in abundance with progressively later instars resulting in positive values. Theoretically, it is

impossible for there to be an increase in number from one generation to the next; however,

smaller instars are more difficult to sample and develop quickly compared to the time spent in

the later instars. However, negative values sometimes occur and were not factored into the final

calculation of this study. Weight at loss was assumed to be the midpoint between two

developmental classes. The loss factor was five as an expression of the number of distinct size

classes. The losses were then summed and the total multiplied by a cohort production interval

(CPI) of four as C. lasia was determined to produce at least four cohorts in Pecan Creek.

Estimates for the possible fifth cohort downstream were also calculated. The mean numbers of

organisms collected were 413 m -2and 3,369 m -2, upstream and downstream respectively (Fig.

11).

Densities were significantly different between the upstream population and the

population downstream of the wastewater outfall (one-way ANOVA, F(1,41)= 11.05 p =0.002).

Although mean lipid percentages were slightly higher downstream of the WWTP, a two-way analysis of variance revealed no significant differences in the percentage of total lipids per gram of caddis fly tissue between sites, F(1,10)= 9.3, P > 0.05. Seasonal differences were not

significant, F(1,76)= 40.6, P > 0.05); however, significant interactions occurred between site and

season F(1, 33)= 60.7, P = 0.09. A summary of caddis fly lipid analyses is listed in Table 4.

Results from time-domain NMR analysis of caddis fly tissue are given in Figure 18. To produce

17 a standard curve simple linear regression of % NMR frequencies and lipids (g) indicated a strong relationship (F = 102.27, r2 = 0.98, p < 0.0001) between the NMR signal and known lipid weights (Figure 18). The linear relationship can be explained by the following equation: (%

NMR Frequency) = -2.13 + 10779.42 (grams of caddis fly lipids). The 95% confidence intervals for the slope were 9423 to 11340.

18 DISCUSSION

Pecan Creek is representative of many urban streams across the region as it is intermittent

upstream of the wastewater treatment plant yet permanently flowing downstream. The hydraulic regime downstream is dominated by wastewater treatment plant (WWTP) discharge as effluent typically comprises 90% of total flow and at least 50% during base flow conditions in (Taylor,

2002). The City of Denton values water quality in Pecan Creek and manages treatment at the wastewater facility to ensure that effluent discharged into the creek meets or exceeds TCEQ water quality standards established for the facility (Taylor, 2002). Increased water flow and water quality downstream of the WWTP permits the development of an enriched aquatic community compared to upstream site. The results from this study indicate that shifts in life history and changes in the production of these benthic macroinvertebrates occur due to changes in habitat such as altered temperature regimes and increased flow that result from the inputs of wastewater effluent.

Cheumatopsyche lasia occurs in Pecan Creek both upstream and downstream of the

WWTP. However, a fourth generation of C. lasia was possible below the outfall as elevated water temperatures allowed larval development to progress throughout the winter months, producing a cohort ready to emerge in the early spring. Alexander and Smock (2005) reported three distinct generations, with a possible fourth generation, for Cheumatopsyche analis in a third order urban stream in Virginia with mean water temperature of 15.7 °C. Mean temperatures in Pecan creek ranged from 18°C upstreamsite and 22°C downstream. The estimated threshold for completion of a cohort for C. lasia was approximately 1200 cumulative degree days, as determined through laboratory rearing and field data. From early November through March, nearly 2500 degree days were accumulated, double that required to complete a cohort. Over the

19 same time period, the upstream site accumulated roughly 1500 degree days, enough to complete

a single cohort. WWTPs have been identified as the primary source of anthropogenic heat inputs into urban streams (Kinouchi et al., 2007, Hogg and Williams, 1996), (Miyamoto and Kinouchi,

2005, CanobbioValeriaSanfilippo et al., 2009, Nelson, 2011). Given the magnitude of temperature increases observed in Pecan Creek, and subsequent alteration in both timing and number of cohorts, these findings demonstrate the impact of wastewater-mediated thermal inputs on the life history of benthic invertebrates like C. lasia.

Abundance below the wastewater treatment plant was 8 times greater, likely due to habitat availability as well as stream flow. In other studies, high densities of Cheumatopsyche have been linked to the availability of stable habitat (Sanchez and Hendricks, 1997, Mackay and

Waters, 1986). An abundance of Cheumatopsyche was observed on rock weirs when compared to snag or streambed habitats in the Cache River of southern Illinois (Walther and Whiles, 2008).

High densities of caddis flies were observed on concrete structures designed for erosion control in the Wash outside of Las Vegas (Nelson, 2011). An in-stream structure composed of cement and rock was installed in Pecan Creek to oxygenate water post-treatment through turbulence as it hits the structure at high velocities. The structure creates prime habitat for C. lasia by providing stable surface for constructing nets and fixed retreats. In addition, the abundance of

Cheumatopsyche observed downstream was possible due to the continuous flow. Hydropsychids rely on stream flow to deliver food, remove waste products, and provide dissolved oxygen.

Stable substrate is often limited in semi-arid prairie streams and has been indicated as a major factor for secondary production of macroinvertebrates within the region (Johnson and Kennedy,

2003). However; the abundance of C. lasia downstream of the WWTP would not be possible without the addition of wastewater effluent into the system.

20 Production of C. lasia was five times greater downstream of the wastewater treatment plant. Secondary production estimates for Pecan Creek were within the range of values reported for other Cheumatopsyche species. Values of 18.2 g m-2 yr-1 were reported for C. analis below an

impoundment, when compared to 7.2 g m-2 yr-1 upstream in an urban Virginia stream (Alexander

and Smock, 2005). Lower values of 2.0 and 3.0 g m-2 yr-1 were reported for members of

Cheumatopsyche in an Appalachian stream (Sanchez and Hendricks, 1997). Production values

ranging from 15.7 to 40.3 g m-2 yr-1 were reported for C. lasia on the Brazos river in central

Texas (Malas, 1984). The highest known values of production for Cheumatopsyche were

reported from below an impoundment at 56.8 g m-2 yr-1, compared to 0.07 – 7.5 g m-2 yr-1 at

downstream reference sites (Parker and J. R. Voshell, 1983).

The difference in annual production to biomass (P/B) obtained for C. lasia in Pecan

Creek correspond to the observed difference in voltinism. Annual P/B reflect the biomass turnover rate (Benke, 1984) and typically ranges from 5 to 10 for univoltine and bivoltine populations with higher P/B ratios reported for benthic populations with multiple generations a year (Benke and Huryn, 2006). Annual P/B ratios of 11.54 and 7.15 have been reported for bivoltine populations of Cheumatopsyche (Sanchez and Hendricks, 1997). P/B ratios of 8.31 and

10.53 were reported for bivoltine Cheumatopsyche in the Savannah River (Cudney and Wallace,

1980). The values of 12.36 and 11.52- 15.63 reported for C. lasia in this study reflect the shortened developmental period for the site receiving wastewater effluent. Sanchez and

Hendricks (1997) detected a greater accumulation of lipids in mature, overwintering

Cheumatopsyche larvae that developed quickly compared to reference populations. However, no such differences were observed in this study, possibly due to high variability between lipid concentrations of IV and V instar larvae used for analysis.

21 Through filter-feeding, C. lasia serves a critical trophic linkage by retaining energy

otherwise lost downstream. Production values of C. lasia observed in Pecan Creek indicate this

organism is important for energy dynamics of Pecan Creek. In a separate study, Cheumatopsyche

accounted for over 60% of collector-filterers in water receiving wastewater effluent, compared to

14% at the upstream reference site (ShiehWard and Kondratieff, 2003). Filtering-collectors

Cheumatopsyche, Hydropscyehe, and Simulium accounted for 46% of total production

downstream of wastewater inputs as compared to only 7% upstream (Shieh, Ward and

Kondratieff, 2002). Wastewater effluent is often associated with negative impacts to stream

biota. However, in semi-arid regions improvements in WWTP waste processing technology

often result in effluents that create better habitat conditions with higher community diversity and

secondary production downstream of the discharge (Slye et al., 2011). This condition occurs

downstream of the WWTP in Pecan Creek which support greater biomass and production of C.

lasia when compared to the upstream site.

However, many contaminants, such as pharmaceuticals and personal care products, are

not removed through current water treatment processes. In a separate study, noticeably higher

levels of the antimicrobial Triclosan® and its metabolite methyl-Triclosan were detected in the

tissue of C. lasia collected from the downstream site (Lundeen et al., 2010) when compared to upstream. Similar results have been reported for algae and gastropod species in Pecan Creek

(Coogan et al., 2007, Coogan and La Point, 2008). As caddis flies are consumed by aquatic predators in the larval and pupal stages followed by terrestrial predators while adults, there is potential for bioaccumulation of these compounds through consumption of C. lasia. Given the abundance and production of C. lasia downstream of the WWTP, they may represent a significant source of trophic transfer of these compounds. Through emergence, C. lasia deposit

22 concentrations of Triclosan and methyl-Triclosan to the riparian area regardless of predatory consumption. Further research is needed to determine the full extent with which C. lasia and other benthic invertebrates contributes to the bioaccumulation of contaminants from wastewater to the surrounding ecosystem.

Autoecology studies, including life histories and secondary production of aquatic insects, are fundamental to the understanding of stream dynamics in urban systems. This study represents the first shift in multivoltine life history of Cheumatopsyche species from a WWTP in North

America. These results demonstrate the flexibility of life histories between populations of the same species in response to changing environmental parameters within urban streams. Results also suggest that C. lasia is important for energy transfer in semiarid urban prairie streams and may serve as a potential conduit for the transfer of energy along with emergent contaminants to terrestrial ecosystems. These finding highlight the need for more quantitative accounts of population dynamics (voltinism, development rates, secondary production, and P/B) of aquatic insect species to fully understand the ecology and energy dynamics of urban systems

23 WOOD WWTP

Fig. 1 Map of the Pecan creek watershed showing the density of human population in Denton, TX as of the 2000 census.

8 WOOD WWTP

6 / sec) /

3 4

2 Discharge(M

-2

Aug Oct Dec Feb Apr Jun Aug Oct Dec 2009 2010

Fig. 2 Discharge estimates of daily mean flow from August 2009 through November 2010.

14 WOOD WWTP 12

Dissolved Oxygen (mg/ L) (mg/ Oxygen Dissolved 2

-2

Aug Oct Dec Feb Apr Jun Aug Oct Dec 2009 2010

Fig. 3 Dissolved oxygen estimates of average daily concentrations from August 2009 through November 2010.

35 WOOD WTTP 30

25 C) o 20

15 Temperature ( Temperature 10

0 Aug Oct Dec Feb Apr Jun Aug Oct Dec 2009 2010

Fig. 4 Temperature estimates of average daily stream temperature from August 2009 through November 2010

1000 WOOD WTTP

800

600

400

200 Cumulative Degree Days (by month) (by Degree Days Cumulative

0 Aug Oct Dec Feb Apr Jun Aug Oct Dec

2009 2010

Fig. 5 Degree day estimates of cumulative degree days by month from August 2009 through November 2010. The numbers in legend represent the total DD accumulated over the study period.

Fig. 6 Scatter plot of head capsule measurements representing size criteria for larval instars.

B A

D E C

Fig. 7 Illustration of larval head capsule development, shown: dorsal (left) and ventral (right) for instars V (A) through I (E).

A I

III

C V I

1 mm VII

VIII

Fig. 8 Illustration of pupae abdomen, shown: abdomen (A), abdomnial plates (B), and head capsule (C).

Fig. 9 Life history diagram for upstream (WOOD).Thickness of bars represents the percentage of each instar within the population, lines represent proposed cohorts and arrows represent proposed emergence.

Fig. 10 Life history diagram for downstream (WWTP). Thickness of bars represents the percentage of each instar within the population, lines represent proposed cohorts and arrows represent proposed emergence.

Fig. 11 Total abundance estimates from mean number collected from August 2009 through November 2010 plus or minus a standard deviation.

Table 1 Summary of water chemistry for Pecan Creek as monthly averages from August 2009 through November 2010.

Temp °C DO mg/l pH Cond µS/cm Discharge m3/s POC (mg/L)

WOOD WWTP WOOD WWTP WOOD WWTP WOOD WWTP WOOD WWTP WOOD WWTP A 25.99 27.48 2.84 6.88 7.45 7.17 447.40 699.09 0.06 0.13 J 1.46 0.83 S 22.41 25.93 3.69 6.65 7.48 7.36 401.96 589.51 0.10 0.22 A 1.84 2.04 O 16.69 21.13 7.41 2.92 7.76 7.62 371.92 389.58 0.14 0.31 S 1.39 1.66 2009

N 14.59 21.02 4.70 6.32 7.45 7.20 673.66 180.68 0.09 0.20 2010 O 1.15 1.23 D 6.16 15.18 10.89 8.38 8.17 7.00 699.14 471.73 0.10 0.21 N 1.16 0.75 J 7.75 15.21 10.41 5.33 8.11 7.27 621.48 376.15 0.01 0.02 D 0.00 1.15

F 6.55 12.64 9.48 9.07 7.54 7.31 678.37 729.19 0.10 0.22 J 1.27 1.92

M 12.29 15.65 7.37 8.43 7.64 8.22 663.46 716.98 0.07 0.16 2011 F 1.18 1.11 A 18.05 19.81 3.82 7.92 7.48 7.37 866.41 755.67 0.14 0.3 M 24.49 21.37 5.43 7.50 7.49 7.44 958.73 789.00 0.01 0.08

J 27.16 26.81 3.73 3.80 7.41 7.27 825.85 689.68 0.03 0.07 2010 J 27.82 27.87 5.88 5.57 7.71 7.41 865.45 612.67 0.01 0.05 A 27.84 29.56 2.95 6.72 7.46 7.25 479.80 644.07 0.05 0.11 S 23.09 27.47 4.14 6.59 7.79 7.25 453.04 542.58 0.08 0.20 O 17.64 23.77 3.38 3.86 7.46 7.37 664.11 545.84 0.05 0.15 N 13.21 22.25 3.83 4.79 7.09 7.43 445.60 678.01 0.11 0.26

Table 2 Secondary production calculations for upstream (WOOD).

Instar na DMb Bc Δ in nd DM at losse DM lossf x 5g

I 33 0.003 0.083 3 0.010 0.030 0.152 II 30 0.015 0.457 -11 0.042 -0.462 -2.312 III 41 0.054 2.198 -10 0.207 -2.073 -10.364 IV 51 0.307 15.673 -27 0.863 -23.301 -116.505 V 78 1.111 86.688 78 1.111 86.688 433.438 433.590mg Total Annual Secondary Production= 1.3007 g m-2 y-1

Biomass= 105.11 Cohort P/B= 4.12 Annual P/B= 12.36

Table 3 Secondary production calculation for downstream (WWTP).

Instar na DMb Bc Δ in nd DM at losse DM lossf x 5g

I 413 0.003 -296 0.010 -2.996 -14.982 II 296 0.015 4.504 -24 0.042 -1.009 -5.043 III 320 0.054 17.159 33 0.207 6.840 34.200 IV 287 0.307 88.197 6 0.863 5.178 25.890 V 281 1.111 312.298 281 1.111 312.298 1561.488

1627.50mg

Total Annual Secondary Production= 4.885- 6.510 g m-2 y-1

Biomass= 423.21 Cohort P/B= 3.84 Annual P/B= 11.52- 15.63

Table 4 Caddis fly lipids summary of results for total lipid estimates from caddis fly tissue.

Sample Site Collection Number of Sample Wt. NMR % Total Lipids % Date Ind. (g) freq (g) Lipids 1 WWTP 10/8/10 187 0.184 58.1 0.006 3.037 2 WWTP 10/8/10 114 0.117 33.6 0.003 2.833 3 WWTP 10/15/10 66 0.089 30.3 0.003 3.380 4 WOOD 10/15/10 60 0.125 36.6 0.004 2.874 5 WOOD 11/9/10 50 0.06 28.3 0.003 4.705 6 WWTP 11/9/10 165 0.175 76.2 0.007 4.152 7 WWTP 11/22/10 56 0.091 32.7 0.003 3.551 8 WWTP 12/24/10 34 0.046 14.8 0.002 3.414 9 WWTP 1/13/11 34 0.099 50.3 0.005 4.913 10 WOOD 1/31/11 10 0.045 24.7 0.002 5.531

11 WWTP 1/31/11 29 0.094 35.7 0.004 3.733 12 WWTP 2/18/11 22 0.079 40.5 0.004 5.006 13 WOOD 2/18/11 12 0.047 13.4 0.001 3.065

APPENDIX

CADDIS FLY FREQUENCY RAW DATA

S. Area Date Site I II III IV V P Total (cm) 25-VIII-09 WOOD 0 3 5 2 4 1 15 156.52 25-VIII-09 WOOD 0 4 3 3 27 0 37 481.01 25-VIII-09 WOOD 1 0 1 2 3 0 7 742.56 25-VIII-09 WWTP 55 72 98 120 114 11 470 565.45 25-VIII-09 WWTP 932 152 164 129 119 6 1502 366.59 8-IX-09 WOOD 0 0 0 3 6 4 13 944.39 8-IX-09 WOOD 0 0 1 5 13 1 20 930.89 8-IX-09 WOOD 0 1 0 5 11 2 19 1051.26 8-IX-09 WOOD 1 2 3 3 1 10 971.40 8-IX-09 WWTP 143 65 26 13 8 0 255 802.29 8-IX-09 WWTP 172 59 51 49 35 1 367 531.12 8-IX-09 WWTP 555 384 197 167 139 5 1447 563.84 24-IX-09 WWTP 70 69 45 22 34 4 244 342.11 24-IX-09 WWTP 270 171 81 82 78 4 686 771.40 24-IX-09 WOOD 1 1 0 1 1 1 5 720.37 24-IX-09 WOOD 0 0 0 0 0 0 0 626.54 24-IX-09 WOOD 0 1 4 3 1 2 11 477.12

16-X-09 WWTP 9 5 2 0 1 1 18 579.86 16-X-09 WWTP 3 0 1 1 3 4 12 846.68 16-X-09 WOOD 0 0 3 2 2 2 9 405.72 16-X-09 WOOD 8 10 7 19 13 7 64 483.52 16-X-09 WOOD 0 3 0 0 2 0 5 246.00 5-XI-09 WWTP 52 33 17 11 6 2 121 623.57 5-XI-09 WWTP 35 27 13 7 4 0 86 558.58 5-XI-09 WOOD 0 0 1 1 3 2 7 668.42 5-XI-09 WOOD 1 0 6 9 7 5 28 674.14 24-XI-09 WWTP 11 121 144 339 169 7 791 969.41 24-XI-09 WWTP 35 68 76 138 75 2 394 722.52 24-XI-09 WOOD 1 1 1 1 1 0 5 1177.21 24-XI-09 WOOD 0 3 4 6 16 0 29 1081.56 24-XI-09 WOOD 6 22 10 3 11 0 52 1138.05 11-XII-09 WWTP 0 0 0 5 9 1 15 796.38 11-XII-09 WWTP 151 45 144 181 240 1 762 755.58 13-XII-09 WOOD 2 35 48 21 19 1 126 710.27 13-XII-09 WOOD 1 13 5 3 11 0 33 634.46 13-XII-09 WOOD 3 15 27 11 20 0 76 878.47 26-XII-09 WOOD 0 0 0 0 0 0 0 605.61

S. Area Date Site I II III IV V P Total (cm) 26-XII-09 WOOD 7 47 89 37 53 0 233 390.78 27-XII-09 WOOD 0 3 1 2 17 0 23 1719.59 28-XII-09 WWTP 0 0 1 5 11 0 17 841.83 28-XII-09 WWTP 0 0 0 15 287 4 306 1425.90 4-I-10 WWTP 0 0 11 44 236 0 291 1629.47 4-I-10 WWTP 0 0 4 4 66 0 74 380.23 4-I-10 WOOD 0 2 6 11 22 2 43 376.09 4-I-10 WOOD 0 2 2 4 9 0 17 697.89 4-I-10 WOOD 0 1 1 1 15 0 18 298.22 18-I-10 WOOD 1 3 20 22 35 1 82 528.26 18-I-10 WOOD 0 1 1 6 7 0 15 690.80 18-I-10 WOOD 0 0 10 8 21 39 888.83 22-I-10 WWTP 0 0 0 44 445 489 678.12 22-I-10 WWTP 0 0 0 15 309 324 676.96 5-II-10 WOOD 0 0 0 1 2 1 4 413.27 5-II-10 WOOD 0 3 0 6 7 0 16 228.65 5-II-10 WOOD 0 0 0 0 5 0 5 384.69 5-II-10 WWTP 0 0 0 0 5 0 5 517.67 5-II-10 WWTP 0 0 1 11 50 2 64 399.43 20-II-09 WOOD 0 0 0 0 1 1 2 208.47 20-II-09 WOOD 0 0 4 16 20 4 44 38976.02 20-II-09 WOOD 0 0 1 1 2 1 5 271.40 20-II-09 WOOD 0 0 0 11 1 5 17 366.13 11-III-10 WOOD 0 0 0 0 6 0 6 370.48 11-III-10 WOOD 0 0 0 0 6 1 7 434.32 11-III-10 WOOD 0 0 0 1 4 0 5 367.96 11-III-10 WWTP 0 0 0 0 6 5 11 633.64 30-III-10 WWTP 0 0 0 0 7 5 12 355.61 30-III-10 WWTP 0 0 0 0 0 4 4 499.54 30-III-10 WOOD 0 0 0 0 5 2 7 598.86 30-III-10 WOOD 0 0 0 0 2 0 2 418.08 30-III-10 WOOD 0 0 0 0 0 4 4 387.41 26-IV-10 WOOD 136 36 26 23 55 3 279 389.93 26-IV-10 WOOD 27 21 14 8 32 6 108 802.75 26-IV-10 WOOD 89 33 11 4 32 2 171 621.51 27-IV-10 WWTP 754 357 263 261 215 5 1855 970.48 27-IV-10 WWTP 987 417 797 472 319 0 2992 531.81

S. Area Date Site I II III IV V P Total (cm) 19-V-10 WOOD 88 96 121 162 239 25 731 986.36 19-V-10 WOOD 26 19 11 9 22 3 90 506.82 19-v-10 WWTP 730 171 46 106 190 30 1273 431.97 19-v-10 WWTP 94 17 6 22 37 9 185 235.45 8-VI-10 WWTP 397 703 548 320 228 15 2211 441.21 8-VI-10 WWTP 248 283 221 121 143 21 1037 445.91 9-VI-10 WOOD 145 245 316 229 220 38 1193 588.18 9-VI-10 WOOD 41 54 66 65 54 5 285 853.79 28-VI-10 WOOD 0 0 0 0 4 3 7 458.33 28-VI-10 WOOD 22 11 55 151 195 11 445 546.21 29-VI-10 WWTP 437 727 1044 892 649 41 3790 1023.94 14-VII-10 WWTP 208 130 93 104 86 8 629 474.55 14-VII-10 WWTP 824 287 188 180 173 26 1678 607.42 14-VII-10 WOOD 30 16 20 20 73 39 198 385.00 14-VII-10 WOOD 109 24 63 93 253 68 610 380.76 30-VII-10 WWTP 427 471 538 513 466 39 2454 372.27 30-VII-10 WOOD 55 59 70 129 154 33 500 3618.33 16-VIII-10 WWTP 310 349 576 497 316 21 2069 328.33 16-VIII-10 WWTP 85 151 258 228 166 8 896 357.42 16-VIII-10 WOOD 0 0 0 0 0 0 0 504.09 16-VIII-10 WOOD 1 1 9 5 0 0 16 309.55 16-VIII-10 WOOD 0 0 1 0 3 0 4 728.18 10-IX-10 WWTP 147 98 127 203 142 16 733 908.18 10-IX-10 WWTP 0 838.94 10-IX-10 WOOD 5 0 0 0 0 0 5 444.70 10-IX-10 WOOD 0 2 1 0 0 1 4 280.76 10-IX-10 WOOD 0 0 0 0 0 0 0 580.00 21-IX-10 WWTP 118 221 243 147 59 4 792 424.39 21-IX-10 WWTP 130 200 129 61 19 1 540 565.91 22-IX-10 WOOD 1 0 6 3 0 0 10 686.52 22-IX-10 WOOD 21 2 0 1 0 0 24 412.12 22-IX-10 WOOD 28 3 1 1 1 0 34 310.91 14-X-10 WWTP 97 152 198 180 184 13 824 810.15 14-X-10 WWTP 136 187 233 253 233 14 1056 708.48 15-X-10 WOOD 0 0 0 3 10 1 14 669.55 15-X-10 WOOD 3 3 10 11 39 5 71 436.97 15-X-10 WOOD 4 1 0 8 12 1 26 193.64

S. Area Date Site I II III IV V P Total (cm) 8-XI-10 WWTP 1459 1465 1024 417 285 298 4948 374.70 8-XI-10 WWTP 681 615 440 277 109 35 2157 455.91 9-XI-10 WOOD 13 5 7 6 8 0 39 956.36 9-XI-10 WOOD 6 7 17 7 3 1 41 675.61

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