Sensitivity Comparisons of the Insect Centroptilum Triangulifer to Ceriodaphnia Dubia and Daphnia Magna Using Standard Reference Toxicants; Nacl, Kcl

Sensitivity comparisons of the insect Centroptilum triangulifer to Ceriodaphnia dubia and Daphnia magna using standard reference toxicants; NaCl, KCl

and CuSO4

A thesis submitted to the

Graduate School of the

University of Cincinnati

on 2/20/12

in partial fulfillment of the

requirements for the degree of

Masters of Science

in the Department of Biology

of the College of Arts and Sciences

Katherine Ann Hammer

B.S. University of Dayton

2003

Committee Chair: Jodi Shann, Ph.D.

Abstract

Establishing water quality criteria that is protective of all native biota is a difficult task and often aided by the use of model organisms. Common model organisms may not be the most sensitive or inclusive of all taxa. The US Environmental Protection Agency has cultured a parthenogenetic invertebrate, the insect Centroptilum triangulifer , which potentially has higher sensitivity to certain toxicants. This study established a 48 hr acute and a 14 day chronic testing procedure for C. triangulifer and compared its sensitivity to two model invertebrates, Ceriodaphnia dubia and Daphnia magna . Toxicity bioassays were conducted to determine mortality and growth effects using standard reference toxicants; NaCl, KCl and CuSO 4. Weight was at least 36% more sensitive to the toxicants than body length and head capsule width in C. triangulifer . In 48 hr acute tests, the average LC50 for the mayfly was 659 mg/L NaCl, 1957 mg/L KCl, and 11 µg/L CuSO 4. IC25 values, using weight as the endpoint, were 291 mg/L NaCl, 356 mg/L KCl and 6

µg/L CuSO 4. C. triangulifer was the most sensitive species in NaCl acute and chronic tests. KCl at concentrations tested for the two daphnid species failed to produce mortality in C. triangulifer bioassays, but the species was equally or more sensitive than C. dubia and D. magna for growth measurements. Despite possible food interactions during CuSO 4 testing, C. triangulifer was the most sensitive species during acute testing and for growth parameters in chronic tests. This study determined C. triangulifer has great potential and benefits for use in ecotoxicological studies.

iii

Acknowledgements

I would like to thank my committee members; Jodi Shann, Ph.D., Jim Lazorchak, Ph.D. and Eric Maurer, Ph.D. for their wisdom and guidance. I also owe a great deal of gratitude to my coworkers at The McConnell Group for their support throughout my study. In addition, I would like to acknowledge the late Mark Smith for his encouragement to pursue a Masters Degree and his knowledge during the creation of my proposal. Finally I would like to thank my family and friends for their continued support.

Table of Contents

Title Page i

Abstract ii

Acknowledgements iv

Introduction 1

Materials and Methods 4

Results 14

Discussion 34

Bibliography 40

Appendix 43

List of Figures and Tables

Figure 1 12

Figure 2 15

Figure 3 16, 17

Figure 4 18

Figure 5 19

Figure 6 22

Figure 7 23, 24

Figure 8 25

Figure 9 26

Figure 10 28

Figure 11 29, 30

Figure 12 31

Figure 13 32

Table of Contents (cont.)

Table 1 20

Table 2 21

Table 3 27

Table 4 27

Table 5 33

Table 6 33

Introduction

Aquatic laboratory organisms have been utilized for decades to determine the effects of chemicals in freshwater environments and to define water quality criteria. Certain model organisms have been utilized due to their life history traits, ease of culturing in a laboratory and limited variability between individuals. Common model organisms include the fishes

Pimephales promelas and Oncorhynchus mykiss , the cladocerans Ceriodaphnia dubia and

Daphnia magna , the amphipod Hyalella azteca , and the midge Chironomus tentans , among others. These aquatic species have been used extensively for acute and chronic toxicity testing and are outlined for use by the US Environmental Protection Agency

(EPA) (2002a, 2002b).

There are many benefits to using a model organism, including the ability to compare results from independent studies and literature, but there may also be disadvantages.

Developing water quality criteria that are protective of all native fauna is the main goal of aquatic ecotoxicology risk assessment studies, but the use of model organisms that are restricted to a few taxa may miss some of the more sensitive species. A study of toxicity datasets for metals showed that acute and chronic testing has under-represented aquatic insects and over-represented cladocerans (Brix et al. 2005). Further toxicity testing is needed to be conducted to determine if aquatic insects are more sensitive test organisms than those in current use.

The number of toxicity tests on aquatic insects has begun to increase, especially within the order Ephemeroptera (mayflies). A study by Echols et al. (2009) indicates that Isonychia bicolor , a mayfly species, is more sensitive than the cladoceran C. dubia to coal processing

1 effluent. The mayfly Stenonema modestum was utilized in subacute tests by Diamond et al. (1992). Toxicity testing of cadmium and lead has been conducted using the mayfly

Baetis tricaudatus . (Irving et al. 2002) (Mebane et al. 2007). B. tricaudatus has also been tested for growth, after exposure to pulp mill effluent (Lowell et al. 1995).

The EPA has identified the mayfly Centroptilum triangulifer as a prospective species for use in either mountaintop mining effluent and/or oil and gas extraction effluent studies. C. triangulifer was previously identified by McDunnough (1931) as Cloeon triangulifer and is of the Baetidae family. The species is found in marginal streams and slow flowing aquatic systems throughout eastern North America (Gibbs, 1973). Funk et al. (2006) used genetic and morphological examinations to describe differences between C. triangulifer , which is obligatory parthenogenetic, and Centroptilum alamance , which is a very similar species that reproduces sexually. C. triangulifer females produce clonal eggs and embryonic development is temperature dependent, averaging 6 days until first hatch at

25°C (Sweeney et al. 1984). C. triangulifer feeds by scraping periphyton from stream beds and other surfaces. The relative short life cycle (~30 days) and clonal reproduction makes

C. triangulifer a good candidate for toxicology studies.

Use of C. triangulifer as a test organism has increased in the last decade. Studies generally indicate this species is sensitive to certain pesticides, metals and non-metals. Work by

Sweeney et al. (1993) demonstrated larval survivorship of C. triangulifer was significantly lowered by the pesticide chlordane at a concentration of 4.3 ug/L. Further research indicated that chlordane is also transferred via lipids to mayfly eggs (Standley et al. 1994).

Conley et al. (2009) looked at maternal transfer in C. triangulifer and described the detrimental effects of selenium bioaccumulation through ingestion of contaminated

2 periphyton. In addition, bioaccumulation and the trophic transfer of the metal cadmium were examined by Xie et al. (2009). A follow-up study by Xie and Buchwalter (2011) illustrates that dietary cadmium suppressed antioxidant enzymes, leading to increased toxicity, while dissolved cadmium did not. Hassell et al. (2006) determined that survival of Centroptilum sp. was decreased when salinity (as measured by conductivity) was equal or greater than 5.0 mS cm -1 which was lower than the Cloeon sp. tested in the same study.

In the past, C. triangulifer bioassays have used field collected or laboratory cultured organisms. Studies that utilized laboratory cultured animals used a non-laboratory method for food (Conley 2009; Xie 2010; Xie 2011; Standley 1994; Sweeney 1993; Funk 2006).

This method used local stream water that was pumped over acrylic plates promoting the cultivation of algal colonies. The plates were then placed into containers for feeding during culturing or testing of C. triangulifer . There are problems with this approach. The actual community or species composition and density of the algal slides may vary and utilizing stream water may introduce toxicants present in a local system into the laboratory culture. In addition this method is not practical for those without access to a native stream.

An alternate means of maintaining mayfly cultures has been developed (Weaver et al.

(unpublished). In this method, diatoms are cultured as a food for the mayfly. Three species of diatoms were selected based on size and availability: Mayamaea atomus var. permitis (Hustedt) Lange-Bertalot, Nitzschia cf. pusilla (Kützing) Grunow emend. Lange-

Bertalot, and Achnanthidium minutissimum (K ützing) Czarnecki. These diatoms are added in equal proportions to a container with etched glass microscope slides. After a minimum of 7 days, colonized microscope slides are moved into C. triangulifer culture containers for

3 grazing. This culture method helped standardize culturing techniques, making C. triangulifer more easily cultured for use in testing.

Now that culturing techniques have been standardized for this new organism, there is a need for standardized toxicity testing procedures. The establishment of a uniform testing procedure would increase the usefulness of C. triangulifer as a bioassay species. Because studies utilizing mayflies have begun to increase and a convenient culturing method has been documented, this study aims to establish standardized acute and chronic test methods for C. triangulifer .

In addition to standardizing bioassays for C. triangulifer , the benefits of using this species as a model system will be evaluated. This study will compare results of C. triangulifer acute and chronic bioassays to parallel tests conducted with the common model organisms

C. dubia and D. magna. The sensitivities of each of these three species will be examined using standard reference toxicants. Comparing the sensitivities of the mayfly to the cladoceran will help to discern the usefulness of C. triangulifer in future toxicology studies.

Materials and Methods

Test Waters

Three waters were used throughout the study which was conducted at the U.S.

Environmental Protection Agency Andrew W. Breidenbach Environmental Research

Center (AWBERC) in Cincinnati, OH. Two waters were used in food preparation and culturing of organisms as directed by an established standard operating procedure and the

4 third was used for testing according to EPA manual (2002a, 2002b). The first water was

Millipore Super-Q® system (SQ) water, produced from a de-ionizing system using 4 cartridges: an activated carbon filter, two ion exchange filters, and an Organex-Q filter.

This water was used for some food preparation. The second water was Lab-line; an in- house water which is a carbon filtered and dechlorinated tempered (24-26 °C) tap water.

Lab-line water was supplemented with liquid calcium chloride to reach a hardness of 160 –

200mg/L as measured by CaCO 3. Lab-line was used in culture water for all three species of organisms. The third water, Moderately Hard Reconstituted Water (MHRW), is SQ water enhanced with reagent grade chemicals to reach hardness of 80 – 100 mg/L and alkalinity of 60 – 70 mg/L as CaCO 3. All testing used MHRW for both control water and dilutions.

Test Animals

Centroptilum triangulifer Cultures

C. triangulifer cultures originated from eggs obtained from the Stroud Research Center

(Avondale, PA) in 2010 and cultured at AWBERC. Eggs were from C. triangulifer clone

WCC-2 isolated from White Clay Creek (Chester Country, PA). Cultures were maintained at a constant temperature of 25°C with a 16:8 H light to dark photoperiod. Animals were held at a density of 60 (+/- 15%) animals/per 10L container with 3L Lab-line that was renewed with 80% fresh water at 2-3 weeks. Lab-line was aerated at a low rate using an air stone. Containers were covered with a nylon mesh to contain emerging adults. A diatom food supply was provided on microscope slides colonized in the lab with three species of diatoms (previously listed) . Slides were removed and replaced with fresh ones

5 as needed, approximately every 3 to 4 days. Diatom culturing and slide colonization are described by Weaver et al. (unpublished).

When adults emerged (25-30 days at 25°C) they were removed from the culturing container and placed into an empty holding jar. The adults were individually weighed on a

Toledo Mettler® Scale to the nearest 0.00001 g. Baseline mass measurements were determined for quality assurance. Each adult was then held by the wings using forceps over a 25 ml vial of Lab-line and the abdomen lowered until it made contact with surface water. At that point eggs were released rapidly. Separate vials were used to collect eggs from each adult. Depending on need, C. triangulifer eggs were either stored at 25 °C for use upon hatching (2-3 days) or stored at 4°C for no more than 4 months. If stored at 4 °C, eggs were moved to a 25°C incubator three to four days prior to test initiation. Methods for collection of adults and eggs were adapted from procedures from Stroud Research

Center.

Food was added to each vial at a rate of 0.3 ml of the diatom mixture prior to hatching.

The mixture had equal volumes of each species with a cumulative concentration of 74 mg

(+/- 5%) diatoms/ 50 ml Lab-line determined by dry weight measurements. Eggs were monitored for hatching daily and nymphs were discarded when necessary to ensure animals were <24 hrs old at time of test setup. Each test used a random pool of two or three vials of eggs (from 2 or 3 adults).

Ceriodaphnia dubia Cultures

C. dubia test organisms were cultured and maintained using standard techniques at

AWBERC in 500 ml beakers of both Lab-line and MHRW (U. S. EPA 2002a, 2002b).

Beakers of all-female cultures were restarted weekly, but staggered to ensure there were adults old enough to reproduce everyday. They were fed a daily diet of 2 ml unicellular green algae, Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum ), at a concentration of 100 million cells/ml and 1 ml blended alfalfa (Nature’s Way®: 12 capsules in 1 L SQ). They were maintained at 25°C with a 16:8 light to dark photoperiod.

C. dubia reproduction was tracked weekly with a minimally acceptable average criterion of

20 young/individual over 7 days.

Less than 24 hr old C. dubia young were collected from the culture and held in 1 L

MHRW with 2 ml P. subcapitata added as a food source prior to test initiation.

Daphnia magna Cultures

D. magna was also cultured and maintained according to EPA standards at AWBERC at

25°C with a 16:8 light to dark photoperiod (U. S. EPA 2002a, 2002b). The all-female cultures were restarted every 14 days in 1 L Lab-line/SQ mixture (60:40). They were restarted at staggered intervals so each pair of beakers was a different age to ensure adequate young production everyday. Cultures were fed 3 ml P. subcapitata and 3 ml blended alfalfa daily. D. magna production was tracked monthly to ensure a minimum average reproduction of 40 young/individual over 14 days. Prior to acute or chronic test setup, D. magna young that were < 24 hrs were collected from cultures and held in 1 L

MHRW with 3 ml P. subcapitata.

Bioassays

Two salts and one metal were used as reference toxicants in this study. They were chosen because there is a large database of toxicology results using these chemicals and they are often present in aquatic systems. The two salts chosen were potassium chloride (KCl)

(Fisher Scientific, laboratory grade, CAS 7447-40-7) and sodium chloride (NaCl) (Fisher

Scientific, enzyme grade, CAS 7647-14-5). An extensive record of toxicity tests using

KCl on C. dubia and D. magna has been maintained for years at AWBERC (2002a,

2002b). The metal chosen for this study was copper (as CuSO 4) (Fluka, laboratory grade,

CAS7758998), which is used as a standard reference toxicant by the U. S. EPA (2002a,

2002b). Little data on copper toxicity currently exists for mayflies, but one study by

Brinkman and Johnston (2007) examined acute toxicity testing of the mayfly Rhithrogena hageni and reported a LC50 of 0.137 mg/L after 96 hrs at 12°C.

The CuSO4 stock solution was made by adding 0.982 g CuSO4 into 250 ml SQ to create

1000 mg/L Cu++. When preparing CuSO4 testing solutions, a volume of stock solution was added to 1 L MHRW to create the desired concentration level. NaCl and KCl were added in salt form to MHRW when making testing concentrations. All test concentrations were prepared by serial dilutions of the highest concentrations. Concentrations were then dispensed into 30 ml plastic cups using a sterile syringe and placed in an environmental incubator to reach the test temperature parameter of 25°C.

The serial concentrations of NaCl used for acute testing ranged from 250.0 to 8000.0 mg/L. Concentrations for NaCl chronic testing were determined by a preliminary range finder and were from 78.1 to 1250.0 mg/L for C. triangulifer , 156.3 to 2500.0 mg/L for C. dubia and 312.5 to 5000.0 mg/L for D. magna . The treatment concentrations for acute KCl bioassays ranged from 62.5 to 2000.0 mg/L. During chronic KCl tests the concentration

8 range was 125.0 to 2000.0 mg/L for C. triangulifer and 62.5 to 1000.0 mg/L for both C. dubia and D. magna . Treatment concentrations for acute CuSO 4 bioassays ranged from

2.5 to 80.0 µg/L. Finally, in chronic CuSO4 tests the concentrations ranged from 1.3 to

80.0 µg/L for C. triangulifer , 2.5 to 80.0 µg/L for C. dubia and 5.0 to 80.0 µg/L for D. magna .

Acute Bioassays

Acute bioassays were conducted for 48 hrs. The endpoint was mortality, expressed as an

LC50 (lethal concentration for 50% of the population). When possible, tests between species were run simultaneously and with the same dilution water to limit variations in conditions. Procedures for acute testing were from U.S. EPA (2002a) acute toxicity test methods, except feeding which was modified as described below. Acute test conditions for all three species are listed in Appendices 1a, 1b and 1c.

Preliminary data indicated C. triangulifer did not meet acceptable survival criterion without feeding, even in the acute tests. Non-fed <24 hr mayflies had 11% survival after

48 hrs in MHRW, while <24 hr mayflies that were feed 0.2 ml diatom mixture [74(+/- 5%) mg/50 ml] prior to the test, but not during, had 56% survival. Only mayflies that were fed

0.1 ml diatom mixture daily surpassed the acceptable survival criterion (≥ 90%) with 94% survival after 48 hrs. To further ascertain the minimal required feeding, another 48 hr test was conducted feeding <24 hr mayflies either 0.1 ml, 0.05 ml or 0.03 ml of diatom mixture daily. Survival results were 100%, 89% and 83% respectively. Since 0.1 ml of diatom mix daily was the only feeding regime to pass survival criteria it was used in all acute testing. Results of the C. triangulifer feeding tests are presented in Appendix 2.

C. dubia and D. magna acute bioassays do not require feeding (U. S. EPA 2002a), but to eliminate the possible interaction effects of food and toxicants all acute tests were fed 0.1 ml diatom mixture regardless of the test species. Organisms in the acute tests were fed twice during the test; at initial setup (0 hr) and after water renewal (24 hr). Temperature, pH (Oakton® meter, ph 11 series), conductivity (Oakton® meter, Con 11 series) and dissolved oxygen (DO) (Orion® 830A meter) were recorded daily for initial solutions (0 hr) and for solution renewal (24 hr).

Test solutions were renewed and mortality was recorded after 24 hours. C. dubia and D. magna were counted and then carefully pipetted from the test board into cups of fresh solution on another board. Survival of C. triangulifer was determined using a dissecting stereomicroscope and an 80% water renewal was performed. Water renewal was performed by placing the tip of a 12.5 ml syringe below the surface of the water while observing under the stereomicroscope. Using a syringe, 12.5 ml of solution was slowly extracted without removing the animals. Then 12.5 ml of fresh testing water was added carefully to avoid injuring the organism.

Chronic Bioassays

Due to variations in life cycles, the duration, conditions and end point of chronic tests varied for each test species. Chronic methods for C. dubia and D. magna were already established from U.S. EPA Chronic Methods (2002b) and Lazorchak et al. (2009) respectively. As previous chronic methods for C. triangulifer were not established, test duration, feeding and endpoints were chosen based on life cycle. Sublethal endpoints were used for chronic exposures, in addition to mortality. Initial and final measurements for

10 temperature, DO, conductivity and pH were recorded daily for testing solution at each concentration. Complete test conditions for chronic exposure are listed for C. triangulifer ,

C. dubia and D. magna in Appendices 3a, 3b, and 3c, respectively.

C. triangulifer

Chronic studies for C. triangulifer were 14 days in duration. This test length was chosen because it was about 50% of their larval life cycle and should provide adequate time for growth to occur. Since C. triangulifer can be cannibalistic in limited space or nutrients

(personal observation), only one animal was placed per testing cup. Food quantity chosen was 0.2ml daily, which was double the minimal food requirement used in acute testing. As the organisms grew in size, food quantity was increased from 0.2 ml to 0.4 ml of the diatom mixture at day 10. Excess diatoms were always visible in testing cups.

At the start of the test, two or three vials (representing eggs from two or three females) of newly hatched nymphs were combined in a Petri dish. Nymphs had been monitored and maintained to make certain all were <24 hrs old. Daily water changes were conducted using the same procedure described in acute testing.

The ideal non-lethal endpoint for C. dubia (reproduction) and D. magna (growth) have already been established for chronic tests (U.S. EPA 2002b) (Lazorchak et al 2009). As the test duration did not allow sufficient time for reproduction, growth was chosen as the non-lethal endpoint. Growth of C. triangulifer was recorded using three separate criteria: head capsule width, body length and weight. Three criteria were used in order to determine which one was the most sensitive indicator of growth. After 14 days mayflies were transferred individually using a pipette to an aluminum weigh pan containing enough

11 water to keep the mayfly totally submerged and mobile. The weigh pans were placed under a 25x magnification stereomicroscope with attached digital camera. An image of each organism was taken using Image-Pro Plus® v.7.1 software. Software analysis was used to measure the head capsule (distance between outer edge of eyes) and body length

(distance from tip of head to end of abdomen) to the nearest 0.001 mm (Figure 1). After images were taken, the water was removed using a fine tip pipette and weigh pans were transferred to a drying oven (60°C). After 24 hrs, the pans were removed and organisms weighed on a Cahn® microbalance to the nearest 0.0001 mg.

Figure 1. Diagram of growth measurements of C. triangulifer

C. dubia

C. dubia chronic bioassays lasted 7 days in duration, as described in the U.S. EPA methods manual for chronic testing of fresh water invertebrates (2002b). Water was renewed and animals were fed daily throughout the 7 days. Upon completion of the test, young were tabulated for a total number of young produced per individual. Due to the variation in age when initiating test (0 to 24 hrs), only 3 broods were counted towards the total number of young produced. In cases where an individual produced a 4 th brood, these young were not included. In some cases the production of young occurred before and after water changes, leaving partial broods. These were analyzed per individual case and only those in the fourth brood were not counted.

D. magna

Methods for the 4 day chronic D. magna test were taken from Lazorchak et al. (2009). At the end of 4 days (96 hrs), D. magna from each replicate were transferred to aluminum weigh pans and water removed with a fine tip pipette. Pans were placed in a drying oven

(60°C) for 24 hrs and then weighed on a Toledo Mettler® Scale to the nearest 0 .00001 g.

The weight per pan was divided by the number of surviving animals to produce a weight/individual. When performing statistical analysis for the IC25 the weight per pan was divided by the number of original animals per pan.

Statistical Analysis

For all tests and species, the LC50 was established using the Trimmed Spearman-Karber

(TSK) method (version 1.5), which adjusts for control mortality. Animals that were accidentally killed or lost (via spillage or cracked cup) were not included in TSK analysis.

The IC25, or inhibitory concentration via a 25% reduction in a biological function, was calculated for all chronic tests using the Linear Interpolation Method for Sublethal

Toxicity (version 2.0). The IC25 was established using weight ( D. magna and C. triangulifer ), length and head capsule width ( C. triangulifer ) or offspring production ( C. dubia ).

For each test, survival, growth and/or reproductive endpoint differences within a species were analyzed using an ANOVA with Post-Tukey comparisons (p ≤0.05) in Systat 11

(Systat® Software, San Jose CA). Mean LC50s (for acute tests) and IC25s (for chronic tests) were compared between species for each toxicant by ANOVA with Post-Tukey comparisons (p ≤0.05) as well.

Results

Acute and chronic C. triangulifer bioassays produced over 90% survival in the controls for all tests, surpassing the test criteria and validating the methods. While the chronic test duration was not a full life study, 14 days was ample time for differential growth effects to occur when present. A full life cycle test (25-30 days) requires additional labor but could be done if adult emergence or fecundity data was required.

NaCl

Acute Tests

Three acute tests were conducted for each species using NaCl. Results from the NaCl acute tests are displayed in Figure 2. A between species ANOVA indicated that the mean

LC50 from each species was significantly different from the other (F{2} =89.753, p=0.003,). Post-Tukey comparisons indicate that C. triangulifer ’s LC50 is significantly lower than C. dubia ’s LC50 (p=0.003) and D. magna ’s LC50 is significantly higher than both C. dubia and C. triangulifer (p=0.001) [NaCl acute mortality endpoint sensitivity: C. triangulifer > C. dubia > D. magna ].

7000 C 6000

5000

4000 B Test 1 3000 Test 2

2000 Test 3 A LC50 (NaCl mg/L) (NaCl LC50 1000

0 C. triangulifer C. dubia D. magna Species

Figure 2. Acute 48 hr results for NaCl. Error bars represent 95% confidence intervals. Mean NaCl LC50s for C. triangulifer , C. dubia , and D. magna were 658.7, 2504.3, and 4868.7 mg/L respectively.

Results from the NaCl acutes tests are also displayed for each species as dose-response curves in

Figure 3. These curves indicate there was an immediate increase in mortality for C. triangulifer as the concentration increased from control to 500 mg/L. However, C. dubia and D. magna did not show an increase in mortality until a concentration of 1000 mg/L NaCl. The slope of their dose-

15 response curve was relatively flat until 2000 to 4000 mg/L in C. dubia and 4000 to 8000 mg/L in

D. magna at which point the slope became very steep.

1.2 a) C. triangulifer 1

0.8

0.6 Test 1 Test 2 0.4 Test 3 Percent Mortality Percent 0.2

0 0 500 1000 2000 4000 8000 NaCl concentration (mg/L)

1.2 b) C. dubia 1

0.8

0.6 Test 1 Test 2 0.4 Test 3

Percent Mortality Percent 0.2

0 0 500 1000 2000 4000 8000 -0.2 NaCl concentration (mg/L)

1.2 c) D. magna 1

0.8 Test 1 0.6 Test 2 0.4 Test 3 Percent Mortality Percent 0.2

0 0 500 1000 2000 4000 8000 D. magna NaCl concentration (mg/L)

Figure 3a,b,c. Dose response of C. triangulifer , C. dubia and D. magna to NaCl concentrations.

Chronic Tests

Chronic testing with NaCl was repeated 3 times for each species. Figure 4 displays the

LC50 chronic results for NaCl. The average chronic LC50 value for C. triangulifer was determined by two tests, as the third test did not produce sufficient mortality. ANOVA results between species indicated there were significant differences between the mean

LC50s (F{2}=108.726, p<0.001). Further post-Tukey comparisons show that C. triangulifer and C. dubia chronic LC50s were not significantly different from each other

(p=0.153) but were different from D. magna (p=0.000) [NaCl chronic mortality endpoint sensitivity: C. triangulifer = C. dubia > D. magna ].

6000 B 5000

4000

3000 A A 2000

1000 LC50 (NaCl mg/L) (NaCl LC50

0 C.triangulifer C. dubia D. magna Species

Figure 4. Chronic mean LC50 results for NaCl. Error bars represent 95% confidence intervals. Mean for C. triangulifer was the average of two tests, mean for C. dubia and D. magna was average from three tests. Mean LC50s for C. triangulifer , C. dubia and D. magna were 994.5, 1571.3, and 4312.0 mg/L respectively.

Growth data from the mayfly chronic tests indicated that weight was more sensitive than head capsule width or body length. The average IC25 for weight was 290.5 (+/- 189.2) mg/L, head capsule width was 464.9 (+/- 61.9) mg/L and body length was 453.1 (+/- 78.0) mg/L. Although not statistically significant, weight was consistently the most sensitive growth parameter for C. triangulifer . The average IC25 for weight was at least 36% lower than the second lowest parameter in all toxicant tests. For the duration of the test, weight was used as the IC25 for analysis between species for all three toxicants.

The average IC25 results for NaCl are presented in Figure 5. Mean IC25s for each species differed significantly from each other in an ANOVA (F{2}=83.695, p<0.001). C. dubia mean IC25was significantly greater than C. triangulifer mean IC25 in post-Tukey comparisons (p=0.027). D. magna mean IC25 value was significantly greater than both C.

18 triangulifer and C. dubia (p<0.001) [NaCl growth/reproduction endpoint sensitivity: C. triangulifer (weight) > C. dubia (reproduction) > D. magna (weight)].

3500 C 3000

2500

2000 B 1500 A 1000

IC25 (NaCl mg/L) (NaCl IC25 500

0 Head Cap Length Weight # Young Weight C. triangulifer C. dubia D. magna Species

Figure 5. Chronic IC25 results for NaCl. Error bars represent 95% confidence intervals. The mean IC25s for C. triangulifer for head capsule width, body length and weight were 464.9, 453.1, and 290.5 mg/L respectively. The mean IC25s for C. dubia and D. magna were 989.0 and 2747.2 mg/L respectively.

Testing Conditions

There were modest variations between DO, pH and temperature measurements throughout

NaCl acute and chronic tests. The lowest and highest values within acute and chronic tests are displayed for each species in Table 1. These values represent the range within NaCl tests.

a) Acute pH D.O. Temp (°C) Species low high low high low high C. triangulifer 7.58 8.13 8.1 9.3 23.3 26.1 C. dubia 7.40 8.24 7.9 9.4 23.3 24.6 D. magna 7.50 8.11 7.9 9.3 23.5 25.0

b) Chronic pH D.O. Temp (°C) Species low high low high low high C. triangulifer 7.59 8.01 8.0 9.3 23.5 26.2 C. dubia 7.48 8.04 7.8 9.4 24.2 27.1 D. magna 7.51 7.92 7.8 9.2 23.0 26.9

Table 1a and1b. These tables contain the range of pH, dissolved oxygen and temperature for acute (a) and chronic (b) tests using NaCl.

All DO values for both acute and chronic NaCl tests were above the acceptable value of

4.0. The temperature values did exceed +/- 1°C from 25°C, at +/- 2.1°C. This variation is likely due to inconsistencies in the incubator. The pH values were from 7.40 to 8.24, which are within the EPA National Recommended Water Quality Criteria

(http://water.epa.gov/scitech/swguidance/standards/current/index.cfm ) of 6.5 to 9 and within the normal standard reference toxicity testing range. Low pH values can have an effect on hatch success and the 96 hr LC50 of small nymphs is 4.75 for pH as determined by Tabak et al. (1991).

Conductivity values increased across NaCl concentrations as expected. A range of conductivities would not be pertinent; therefore values were averaged for the control and each concentration per species. The averages per species for all tests are in Table 2.

a) Acute Average Conductivity (µS) Concentration Control 500.0 1000.0 2000.0 4000.0 8000.0 C. triangulifer 340 1265 2270 4132 7662 14516 C. dubia 348 1279 2269 4097 7625 14323 D. magna 334 1268 2246 4078 7605 14354

b) Chronic Average Conductivity (µS) Species Control 78.1 156.3 312.5 625.0 1250.0 2500.0 5000.0 C. triangulifer 327 484 643 951 1547 2752 C. dubia 329 641 948 1546 2758 5100 D. magna 342 946 1540 2784 5061 9510

Table 2a and2b. These tables contain the average conductivity per concentration of NaCl for acute (a) and chronic (b) tests.

KCl

Acute Tests

Three KCl acute tests were conducted using C. triangulifer and four using D. magna and

C. dubia . Acute LC50 results for KCl are presented in Figure 6. ANOVA analysis of mean LC50s indicated significant differences between species (F{2}=44.608, p<0.001).

The mean C. triangulifer LC50 was significantly higher than both C. dubia and D. magna by post-Tukey’s comparison (p<0.001). The mean C. dubia and D. magna mean LC50 do not differ significantly (p=0.699) [KCl acute mortality endpoint sensitivity: C. triangulifer

< C. dubia = D. magna ].

3000 A 2500

2000

Test 1 1500 Test 2 B B 1000 Test 3 Test 4 LC50 (KCl mg/L) (KCl LC50 500

0 C. triangulifer C. dubia D. magna Species

Figure 6. Acute 48 hr results for KCl. Error bars represent 95% confidence intervals. Mean KCl LC50s for C. triangulifer , C.dubia and D. magna were 1956.7, 579.3 and 699.8 mg/L respectively.

Dose-response curves from acute KCl tests are displayed in Figure 7. Examination of the

C. triangulifer dose-response curve showed variable mortality from 250 to 2000 mg/L.

Even though C. dubia and D. magna had significantly lower LC50s, if the lethal concentration endpoint had been decreased to 25% (LC25) C. triangulifer would have been equally as sensitive to KCl for that endpoint.

1.2 a) C. triangulifer 1

0.8

0.6 Test 1 Test 2 0.4 Test 3 Percent Mortality Percent 0.2

0 0 125 250 500 1000 2000 4000 KCl concentration (mg/L)

1.2 b) C. dubia 1

0.8

0.6 Test 1 Test 2 0.4 Test 3 Percent Mortality Percent 0.2

0 0 125 250 500 1000 2000 KCl concentration (mg/L)

1.2 c) D. magna 1

0.8

0.6 Test 1 Test 2 0.4 Test 3 Percent Mortality Percent 0.2

0 0 125 250 500 1000 2000 KCl concentrations (mg/L)

Figure 7a, b, c. Dose response of C. triangulifer , C. dubia and D. magna to KCl concentrations.

Chronic Tests

Three chronic KCl tests were conducted with each species. Chronic LC50s for C. triangulifer could not be estimated, as there was not enough mortality at the highest concentration (2000mg/L). Figure 8 displays LC50 results from the chronic testing of

KCl. An ANOVA analysis of mean chronic LC50s between species showed significant differences (F{2}=590.183, p<0.001). Post-Tukey comparisons show that the C. triangulifer mean chronic LC50 is significantly higher than the other species (p<0.001). C. dubia and D. magna did not differ significantly from each other (p=0.727). [KCl chronic mortality endpoint sensitivity: C. triangulifer < C. dubia = D. magna ].

2500 A * 2000

1500 B B 1000

LC50 (KCl mg/L) (KCl LC50 500

0 C.triangulifer C. dubia D. magna Species

Figure 8. Chronic LC50 results for KCl. Error bars represent 95% confidence intervals. Mean KCl LC50s for C. dubia and D. magna were 638.3 and 673.7 mg/L respectively. *All LC50s for C. triangulifer were greater than the highest concentration (2000 mg/L).

Results of the growth/reproduction chronic tests are presented in Figure 9. An ANOVA between all three species showed no significant difference (F{2}=3.509, p=0.098).

However a post-Tukey comparison produced a trend (p= 0.147) that C. triangulifer had a lower IC25 than D. magna , but it was not significant due to the large confidence interval of

C. dubia . Removing C. dubia and performing a pair-wise comparison between C. triangulifer and D. magna resulted in a significant difference (p=0.003) [KCl growth endpoint sensitivity: C. triangulifer {weight} > D. magna {weight}].

900 800 700 600 500 400 300

IC25 (KCl mg/L) (KCl IC25 200 100 0 Head Cap Length Weight # Young Weight C. triangulifer C. dubia D. magna Species

Figure 9. Chronic IC25 results for KCl. Error bars represent 95% confidence intervals. The mean KCl IC25s for C. triangulifer for head capsule width, body length and weight were 637.3, 569.0, and 356.2 mg/L respectively. The mean KCl IC25s for C. dubia and D. magna were 338.8 and 615.6 mg/L respectively.

Testing Conditions

The range of chemistry values for DO, pH and temperature during acute and chronic KCl tests are depicted in Tables 3a and 3b. DO and pH values were within acceptable limits.

Temperatures varied by +1.1/-2.6°C from 25°C due to fluctuations in the incubator.

a) Acute pH D.O. Temp (°C) Species low high low high low high C. triangulifer 7.48 8.08 8.3 9.2 22.4 26.1 C. dubia 7.48 8.19 8.4 9.3 22.7 25.2 D. magna 7.48 8.02 7.9 9.5 22.7 25

b) Chronic pH D.O. Temp (°C) Species low high low high low high C. triangulifer 7.60 8.03 8.0 11.1 23.5 25.3 C. dubia 7.48 8.15 7.8 11.1 23.5 25.8 D. magna 7.51 8.32 7.9 11.1 23.4 26.1

Table 3a and 3b. These tables contain the range of pH, dissolved oxygen and temperature for acute (a) and chronic (b) tests using KCl.

Similar to the NaCl tests and as expected, conductivity values increased when the KCl concentrations increased. The average for each concentration for each species in acute and chronic testing is displayed in Tables 4a and 4b.

a) Acute Conductivity (average) (µS) Species Control 62.5 125.0 250.0 500.0 1000.0 2000.0 4000.0 C. triangulifer 327 766 1208 2102 3736 7230 C. dubia 321 433 548 776 1175 2064 3808 D. magna 323 434 549 771 1175 2073 3815

b) Chronic Conductivity (average) (µS) Species Control 62.5 125.0 250.0 500.0 1000.0 2000.0 C. triangulifer 319 558 794 1257 2169 3949 C. dubia 309 428 544 777 1222 2120 D. magna 311 433 551 784 1240 2167

Table 4a and 4b. These tables contain the average conductivity per concentration of KCl for acute (a) and chronic (b) tests.

CuSO 4

Acute Tests

Three C. triangulifer acute tests and four C. dubia and D. magna acute tests were conducted using CuSO 4. Acute 48 hr results are displayed in Figure 10. A between species ANOVA indicated a significant difference in LC50s (F{2}=73.539, p =0.001).

Comparisons by post-Tukey analysis showed that C. triangulifer had the lowest mean 48 hr LC50 followed by C. dubia with the second lowest LC50 (p=0.004). D. magna had the highest mean LC50 (p<0.001) [CuSO 4 acute mortality endpoint sensitivity: C. triangulifer

> C. dubia > D. magna ].

80 C 70

µg/L) 50 4 B Test 1 40 Test 2 30 Test 3 A Test 4

LC50 (CuSO LC50 20

0 C. triangulifer C. dubia D. magna Species

Figure 10. Acute 48 hr results for CuSO 4. Error bars represent 95% confidence intervals. Mean averages for C. triangulifer , C. dubia and D. magna are 10.7, 27.6, and 54.5 µg/L respectively.

Results from the acute CuSO 4 bioassays are displayed as dose-response curves in Figure

11. The shape of the curve was relatively consistent across all three species, however the curve began near 5, 20 and 40 µg/L for C. triangulifer , C. dubia and D. magna respectively. The steep slope present in all three species indicated a rapid increase in lethal effects as the concentration of CuSO 4 increased.

1.2 a) C. triangulifer 1

0.8

0.6 Test 1 Test 2 0.4 Test 3 Percent Mortality Percent 0.2

0 0 2.5 5 10 20 40 80 CuSO 4 concentrations ( µg/L)

1.2 b) C. dubia 1

0.8

0.6 Test 1 Test 2 0.4 Test 3 Percent Mortality Percent 0.2

0 0 2.5 5 10 20 40 80 CuSO 4 concentrations ( µg/L)

1.2 c) D. magna 1

0.8 Test 1 0.6 Test 2 0.4 Test 3

Percent Mortality Percent Test 4 0.2

0 0 5 10 20 40 80 CuSO 4 concentrations ( µg/L)

Figure 11a,b,c. Dose response of D. magna to CuSO 4 concentrations.

Chronic Tests

The average chronic LC50 for C. triangulifer and C. dubia was determined by only two tests each, as LC50s could not be generated from the third tests due to lack of mortality.

Chronic mortality results are displayed in Figure 12. ANOVA results indicate the mean

LC50s did not differ significantly from each other across species (F{2}=0.405, p=0.691).

[CuSO 4 chronic mortality endpoint sensitivity: C. triangulifer = C. dubia = D. magna ].

70 A A 60 A 50

4 µg/L) 4 40

CuSO 30

20 LC50 ( LC50

0 C.triangulifer C. dubia D. magna Species

Figure 12. Chronic mean LC50 results for CuSO4. Error bars represent 95% confidence intervals. Mean for C. triangulifer and C. dubia was the average of two tests, mean for D. magna was average from three tests. Mean LC50s for C. triangulifer , C. dubia and D. magna were 40.3, 43.2 and 34.2 µg/L respectively.

IC25 results from chronic CuSO 4 tests are displayed in Figure 13. Once again, in CuSO 4 chronic tests weight was the most sensitive parameter of growth for C. triangulifer and was used for between species comparisons. ANOVA comparison of all three species yielded a trend that IC25s were different, but due to the large variation in C. dubia it was not significant (F{2}=4.253, p=0.071). However, a pair-wise comparison of C. triangulifer and D. magna results yielded a significant difference (F{1}=19.190, p=0.012) [CuSO 4 growth endpoint sensitivity: C. triangulifer {weight}> D. magna {weight}]. A pair-wise comparison of C. triangulifer and C. dubia showed a trend (F{1}=5.885, p= 0.072), but no significance [CuSO 4 growth/reproduction endpoint sensitivity: C. triangulifer {weight} =

C. dubia {reproduction}].

50 A 45 40 35 µg/L) A 4 30 25 20 15 A 10 IC25 (CuSO IC25 5 0 Head Cap Length Weight # Young Weight C. triangulifer C. dubia D. magna Species

Figure 13. Chronic IC25 results for CuSO 4. Error bars represent 95% confidence intervals. The mean CuSO 4 IC25s for C. triangulifer for head capsule width, body length and weight were 13.8, 13.0, and 6.0 µg/L respectively. The mean CuSO 4 IC25s for C. dubia and D. magna were 28.7 and 22.4 µg/L respectively.

Testing Conditions

DO, pH and temperature values for acute and chronic CuSO4 tests varied minimally and were within acceptable ranges. The range of DO, pH and temperature measured for each species during testing is listed in Tables 5a and 5b.

a) Acute pH D.O. Temp (°C) Species low high low high low high C. triangulifer 7.58 7.88 8.1 9.3 23.2 25.8 C. dubia 7.57 7.85 8.0 9.4 23.1 24.9 D. magna 7.57 7.8 8.2 9.1 23.1 25.0

b) Chronic pH D.O. Temp (°C) Species low high low high low high C. triangulifer 7.56 8.12 7.8 11.1 23.7 25.1 C. dubia 7.53 7.96 8.0 9.7 23.5 26.3 D. magna 7.54 7.89 7.7 9.4 23.9 26.4

Table 5a and 5b. These tables contain the range of pH, dissolved oxygen and temperature for acute (a) and chronic (b) tests using CuSO 4.

Unlike NaCl and KCl, conductivity for CuSO4 tests did not vary. The average control value and the lowest and highest values are displayed in Tables 6a and 6b.

a) Acute Conductivity (µS) Species Control (avg.) low high C. triangulifer 320 302 356 C. dubia 317 302 353 D. magna 316 302 351

b) Chronic Conductivity (µS) Species Control (avg.) low high C. triangulifer 348 310 412 C. dubia 341 310 381 D. magna 346 319 386

Table 6a and 6b. These tables contain the range of conductivity measured during CuSO4 acute (a) and chronic (b) tests.

Discussion

Overall, this study was able to develop and validate acute and chronic toxicity procedures for the mayfly C. triangulifer . These test procedures were highly reproducible and could be easily followed in different laboratories. C. triangulifer response to reference toxicants differed from that of other commonly used ecotoxicogical models. This suggests a role for

C. triangulifer as a test species that may reflect chemical impacts on under-represented aquatic groups.

NaCl and KCl

C. triangulifer has proven to be equally or more sensitive than tested model organisms for

NaCl and further use of this species in NaCl studies is warranted. C. triangulifer is less sensitive than the model organisms with KCl as the toxicant when LC50 is the endpoint, but equally or more sensitive during growth/reproduction studies. If the endpoint had been a more conservative value, like a LC10 or LC25, KCl would have been equally as sensitive as the other species. In addition, during KCl chronic studies there was a large variation in the number C. dubia offspring, which masked any significant difference in mean IC25s between species. The large variation in offspring production was likely due to the 0 to 24 hrs age range of individuals at the start of the test. Replicates with animals closer to 0 hrs in age may have taken longer to reproduce than those with organisms close to 24 hrs. To decrease variability in the future, a smaller age window for C. dubia young is recommended.

Overall, C. dubia and D. magna performed as expected in this study based on historical data. This study’s acute 48 hr LC50 values were compared with endpoints described by

Mount et al. (1997). This study’s acute average 48 hr LC50 for C. dubia in KCl was 579 mg/L which falls very close the 580-670 mg/L range for 48 hr LC50s seen in the earlier study (Mount 1997). The average acute 48 hr LC50 for C. dubia in NaCl was slightly above Mount’s 48 hr range of 1770-2330 mg/L at 2504 mg/L. The historical KCl LC50 range at 48 hrs for D. magna is 440-880 mg/L. During this study, the average D. magna

LC50 was within range at 700 mg/L for acute testing. The D. magna 48 hr LC50 also fell within Mount’s NaCl range of 3790-5740 mg/L at 4869 for acute testing.

Chronic test results indicated that C. triangulifer growth was affected by both NaCl and

KCl, and nymphal weight decreased as salt concentration increased. The decrease in weight can be correlated to a decrease in fecundity. A study by Weaver et. al.

(unpublished) showed that mayfly weight is positively correlated to the number of eggs produced. The prediction is that if the chronic test was extended to emergence and egg collection, NaCl concentrations greater than 312.5 mg/L (no observed effect concentration

– NOEC) and KCl concentrations greater than 250 mg/L (NOEC) would cause an effect on fecundity.

CuSO4

Results from the acute CuSO4 tests show that C. triangulifer is significantly more sensitive than C. dubia or D. magna when using mortality as a 48 hr end point. However, there was some discrepancy when examining mortality in chronic tests. The LC50 for C. triangulifer averaged 11 µg/L in acute tests but increased to 40 µg/L in chronic tests. The average

LC50 for C. dubia increased slightly from 27 to 43 µg/L in 7 day tests, while the average

LC50 for D. magna decreased from 54 to 34 µg/L in 4 day tests. Previous work from

Suedel et al. (1996) indicated that C. dubia and D. magna exposed to aqueous copper sulfate reach a mortality threshold at 7 days and 4 days, respectively.

The inconsistency in C. triangulifer mortality left questions, including whether the nominal

CuSO 4 concentrations were accurate, since values had not been analytically measured during testing. The conductivities of NaCl and KCl concentrations were an indicator that the nominal values were on target; however this did not apply for CuSO4. In order to check the measured values in comparison to nominal concentrations, CuSO4 samples were collected from the second chronic test. The three highest concentrations were collected before and after 24 hrs of exposure. The samples, and a sample of CuSO4 stock solution, were analyzed by Inductively Coupled Argon Plasma Emission Spectroscopy at the

AWBERC facility. Results indicated that all initial concentrations had 81% or better recovery rate, surpassing the acceptable recovery percentage of 80%. Therefore all initial measured concentrations were on target with nominal concentrations. However, concentrations that had been exposed for 24 hrs to animals and food ranged from 52% to

85% recovery and are listed in Appendix 4. This indicates that measured CuSO4 concentrations were lower than nominal concentrations after 24 hrs due possible to biological interaction.

The loss of measured CuSO4 was likely due to either absorption into the organism via respiration or through binding with ingested food (Mastin and Rodgers, 2000). To further investigate whether food had an effect on availability of CuSO4, a 48 hr acute test was run using the feeding regime of the chronic test. Instead of 0.1 ml diatom mix, the feeding was

0.2 ml diatom mix for C. triangulifer , 0.2 ml algae and 0.1 ml FFAY (flake food, alfalfa, and yeast) for C. dubia , and 0.3 ml algae and 0.2 ml alfalfa for D. magna . The chronic

36 food regime resulted in higher LC50s. The C. triangulifer and C. dubia 48 hr LC50s surpassed the highest concentration of 80 µg/L, while the 48 hr LC50 for D. magna was 68

µg/L. Mortality results are listed in Appendix 5.

The interaction of food and CuSO4 availability did have an effect on mortality, causing C. triangulifer to have the highest LC50 in chronic tests. However, the non-lethal effects continue to show that C. triangulifer was more sensitive than C. dubia and D. magna to

CuSO4, as the IC25 of C. triangulifer was significantly lower than the other species.

While some CuSO4 may have become unavailable by interactions with food and the remaining CuSO4 was not lethal, it still produced a significant reduction in growth of C. triangulifer .

Another consideration for the discrepancy between mortality in acute and chronic CuSO4 tests of C. triangulifer is variability between mayfly generations. Even though C. triangulifer are clonal reproducers, each generation is cultured separately in Lab-line. The conductivity of Lab-line did vary during culturing, due to the conductivity of the incoming tap water. Fluctuations in culture water could account for some variability in sensitivity.

In addition, some mayfly eggs were held at 4°C to delay hatching until use. The duration of delayed hatching did vary and may also have caused some variability in results. Further analysis of the effect of delayed hatching on C. triangulifer may be beneficial.

An evaluation of acute metal exposures by Brix et al. (2011) indicated that aquatic insects are insensitive to acute metal exposure relative to other aquatic invertebrates. Results from the current study are contradictory to Brix, but Brix does indicate a need for more chronic testing of aquatic insects. While the C. triangulifer were sensitive to CuSO4 in acute

37 testing, there was also a growth effect during chronic testing at an even lower concentration, demonstrating the benefit of conducting longer-term exposures.

Future Studies

Now that this study has shown C. triangulifer to be more than or equally as sensitive as common cladoceran to a few toxicants, its use as model system in future studies should increase. Further toxicology bioassays using more toxicants should be conducted using these newly validated acute and chronic methods. Toxicology studies that utilize this mayfly species may detect detrimental effects at lower concentrations of aquatic toxicants than if tested with C. dubia and D. magna . Using a more sensitive model system has the potential to elucidate better water quality information that is protective of more native species. The utilization of this mayfly in future studies will help expand the database of toxicology information on aquatic insects. In addition, it can help to diminish the gap between over-represented cladoceran and under-represented aquatic insects.

Two growing fields of environmental concern that may also utilize C. triangulifer as a study organism are the practice of mountain top mining (MTM) and oil and gas extraction release (hydro-fracking). The EPA issued an Environmental Impact Statement (EIS)

(2005) stating that almost 7% of the study area in the Appalachia region had been affected by MTM. MTM is the practice of removing the top portion of a mountain ridge and relocating the land to a valley fill in order to obtain exposed coal. One effect of MTM, as stated in the EIS, is the high mineral content of surface water and loss of biological diversity in streams below valley fills. As C. triangulifer has demonstrated a high sensitivity to NaCl and Cu in this study, it should be utilized as a study organism in

38 chronic exposure bioassays examining the effects of high mineral content effluent from

MTM.

The effects of hydro-fracking, the process of fracturing ground shale or coal bed in order to extract natural gas, is less understood. During hydro-fracking large volumes of highly pressurized water mixed with chemicals and sand are used to fracture the ground and keep open those fissures for the movement of natural gas to developed wells for extraction. The

EPA has established a plan for studying the impacts of hydraulic fracturing effluents on drinking water resources (U.S. EPA 2011), including effects of minerals and chemicals on water quality. This is another avenue of study where the sensitivity of C. triangulifer may make it a beneficial model organism.

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Appendix

Appendix 1a. Test Conditions for C. triangulifer Acute Reference Toxicity Test

TEST PARAMETER CONDITION Test Organism C. triangulifer

Test Type static-renewal

Test Duration 48 hrs

Temperature 25 0C (±1 0C)

Photoperiod 16 h light: 8 h dark

Test Chamber Size 30ml

Test Solution Volume 15ml

Renewal of Test Solution Daily

Age of Test Organisms <24 hrs

No. Organisms/Test Chamber 3

No. Replicate Test Chambers 8

No. Organisms/concentration 24

Feeding Regime 0.1 ml diatom mixture

Control and/or Dilution Water Moderately Hard Reconstituted Water

Endpoint Mortality (LC50)

Test Acceptability 90% or greater control survival

Appendix 1b. Test Conditions for C. dubia Acute Reference Toxicity Test

TEST PARAMETER CONDITION Test Organism C. dubia

Test Type static-renewal

Test Duration 48 hrs

Temperature 25 0C (±1 0C)

Photoperiod 16 h light: 8 h dark

Test Chamber Size 30ml

Test Solution Volume 15ml

Renewal of Test Solution Daily

Age of Test Organisms <24 hrs

No. Organisms/Test Chamber 5

No. Replicate Test Chambers 4

No. Organisms/concentration 20

Feeding Regime 0.1 ml diatom mixture

Control and/or Dilution Water Moderately Hard Reconstituted Water

Endpoint Mortality (LC50)

Test Acceptability 90% or greater control survival

Appendix 1c. Test Conditions for D. magna Acute Reference Toxicity Test

TEST PARAMETER CONDITION Test Organism D. magna

Test Type static-renewal

Test Duration 48 hrs

Temperature 25 0C (±1 0C)

Photoperiod 16 h light: 8 h dark

Test Chamber Size 30ml

Test Solution Volume 20ml

Renewal of Test Solution Daily

Age of Test Organisms <24 hrs

No. Organisms/Test Chamber 5

No. Replicate Test Chambers 4

No. Organisms/concentration 20

Feeding Regime 0.1 ml diatom mixture

Control and/or Dilution Water Moderately Hard Reconstituted Water

Endpoint Mortality (LC50)

Test Acceptability 90% or greater control survival

Appendix 2. Results of preliminary C. triangulifer feeding tests in MHRW

48 hr Test Rep # animals start # animals survive Non-Fed 1 3 2 2 3 0 3 3 0 4 3 0 5 3 0 6 3 0 Total 18 2 Fed 0.2ml 1 3 1 prior to test 2 3 2 3 3 1 4 3 3 5 3 1 6 3 2 Total 18 10 Fed 0.1ml 1 3 2 Daily 2 3 3 3 3 3 4 3 3 5 3 3 6 3 3 Total 18 17

48 hr Test Rep # animals start # animals survive Fed 0.1ml 1 3 3 Daily 2 3 3 3 3 3 4 3 3 5 3 3 6 3 3 Total 18 18 Fed 0.05ml 1 3 3 Daily 2 3 2 3 3 3 4 3 2 5 3 3 6 3 3 Total 18 16 Fed 0.03ml 1 3 2 Daily 2 3 3 3 3 3 4 3 2 5 3 3 6 3 2 Total 18 15

Appendix 3a. Test Conditions for C. triangulifer Chronic Test

TEST PARAMETER CONDITION Test Organism C. triangulifer

Test Type static-renewal

Test Duration 14 day

Temperature 25 0C (±1 0C)

Photoperiod 16 h light: 8 h dark

Test Chamber Size 30ml

Test Solution Volume 15ml

Renewal of Test Solution Daily

Age of Test Organisms <24 hrs

No. Organisms/Test Chamber 1

No. Replicate Test Chambers 10

No. Organisms/concentration 10

Feeding Regime 0.2 ml Mixed diatom Day 0 -9, 0.4 ml Mixed diatoms Day 10-14

Control and/or Dilution Water Moderately Hard Reconstituted Water

Endpoint Head capsule length, body length, weight

Test Acceptability 90% or greater control survival

Appendix 3b. Test Conditions for C .dubia Chronic Test

TEST PARAMETER CONDITION Test Organism C. dubia

Test Type static-renewal

Test Duration 7 day

Temperature 25 0C (±1 0C)

Photoperiod 16 h light: 8 h dark

Test Chamber Size 30ml

Test Solution Volume 15ml

Renewal of Test Solution Daily

Age of Test Organisms <24 hrs

No. Organisms/Test Chamber 1

No. Replicate Test Chambers 10

No. Organisms/concentration 10

Feeding Regime 0.2 ml green algae + 0.1 ml FFAY daily

Control and/or Dilution Water Moderately Hard Reconstituted Water

Endpoint Survival and Reproduction

Test Acceptability 90% or greater control survival, average of 15 or more young/surviving female in the control

Appendix 3c. Test Conditions for D. magna Chronic Test

TEST PARAMETER CONDITION Test Organism D. magna

Test Type static-renewal

Test Duration 4 day

Temperature 25 0C (±1 0C)

Photoperiod 16 h light: 8 h dark

Test Chamber Size 60ml

Test Solution Volume 50ml

Renewal of Test Solution Daily

Age of Test Organisms <24 hrs

No. Organisms/Test Chamber 5

No. Replicate Test Chambers 4

No. Organisms/concentration 20

Feeding Regime 0.3 ml green algae + 0.2 ml alfalfa daily

Control and/or Dilution Water Moderately Hard Reconstituted Water

Endpoint Survival and Growth

Test Acceptability 90% or greater control survival, mean dry weight of is 10x greater than mean dry weight of initial <24hr test animals

Appendix 4. Results of analytical Cu samples

Species Rep 1 Rep 2 Rep 3 Observed Expected Percent Food Exposure Exposure (ug/L) (ug/L) (ug/L) Avg (ug/L) (ug/L) Recovery Initial (0H) n/a 1018.0 1015.0 1019.0 1017.3 1000.0 101.7 Initial (0H) n/a 76.1 76.5 76.0 76.2 80.0 95.2 Initial (0H) n/a 35.0 35.1 37.9 36.0 40.0 90.0 Initial (0H) n/a 18.4 14.7 15.7 16.2 20.0 81.2 0.2 ml Diatom C. triangulifer 69.8 68.4 70.2 69.5 80.0 86.8 mix 0.2 ml Diatom C. triangulifer 33.8 33.9 34.7 34.1 40.0 85.3 mix 0.2 ml Diatom C. triangulifer 15.2 17.1 16.3 16.2 20.0 80.9 mix 0.2 ml algae + C. dubia 41.6 42.7 42.6 42.3 80.0 52.9 0.1 ml FFAY 0.2 ml algae + C. dubia 32.8 30.9 31.6 31.8 40.0 79.4 0.1 ml FFAY 0.2 ml algae + C. dubia 14.8 14.3 14.0 14.4 20.0 71.8 0.1 ml FFAY 0.3 ml algae + D. magna 55.3 54.8 54.1 54.7 80.0 68.4 0.2 ml alfalfa 0.3 ml algae + D. magna 25.0 26.0 24.0 25.0 40.0 62.5 0.2 ml alfalfa 0.3 ml algae + 10.6 10.6 11.2 D. magna 10.8 20.0 54.0 0.2 ml alfalfa

Appendix 5. Mortality results of acute CuSO 4 test using chronic test feeding method

Species C. triangulifer C. dubia D. magna Animals/cup 24 20 20 Mortality at: Control 0 0 0 5 ug/L 0 0 0 10ug/L 1 0 0 20 ug/L 0 1 0 40 ug/L 0 0 0 80 ug/L 8 9 13 LC50 >80 >80 68.17