# Presentation: 499940

## Science to Achieve Results (STAR):

- Exposure Measurement Tools for Endocrine Disrupting Chemicals in Mixture
- Heiko L. Schoenfuss1, Larry B. Barber2, David O. Norris3, and Matthew L. Julius1
- 1St. Cloud State University, 2US Geological Survey and 3Colorado University

## Effects of Endocrine Disrupting Compounds on Aquatic Organisms

*Cyclotella*
*meneghiniana*

*Daphnia sp.*

*Pimephales promelas*

**Primary producer**

**Grazer**

**Consumer**

## Population

**Size**

- Parent Generation

- F1 Generation

- F2 Generation

- Food &
- Predation

**Age**

- Sexual
- Competition

- Reproductive
- Fitness

**SURVIVAL**

**REPRODUCTIVE**
**SUCCESS**

_**The Ultimate Endpoint**_
***Reproductive Fitness - ***ability to produce the greatest number of offspring.

**Proximate Endpoints**

## Project Goal

**This project evaluates the hypothesis that mixtures of estrogenic compounds will have adverse effects on ****aquatic vertebrate reproductive fitness**** that can be rapidly assessed by quantifying alterations in the neuroendocrine system of fishes. **

- Assess synergistic interactions of mixtures.
- Assemble broad, biologically relevant effects matrix.
- Develop neuroendocrine endpoints for rapid assessment.

## Approach

**Approach**

**Outcome**

**Rapid & Biologically Relevant Neurochemical Assessment Tools Correlating with Reproductive Fitness**

## Chemical Mixtures

## Endocrine Disrupting Compounds & Mixtures

**Endocrine Disrupting Compounds & Mixtures**

- _Criteria for Determining Exposure Concentrations_:
- Compound must have been found in at least one of the analyzed effluents.
- Exposure concentrations should be within one order of magnitude of published effects concentrations (if available).
- Compounds will be tested at three concentrations bracketing measured effluent concentrations.

## Boulder Creek Watershed

**Boulder Creek Watershed**
**Colorado Front Range**

**Boulder**

## Metropolitan Wastewater Treatment Plant

**Metropolitan Wastewater Treatment Plant**
**St. Paul, MN**

- Mississippi
- River

## Plant Comparison

**Boulder Creek:**
- History of estrogenicity
- Mid-size pant with secondary treatment.
- Dam prevents fish upstream movement.
- Constitutes up to 75% flow or Boulder Creek.

**Metro Plant, St. Paul**
- History of estrogenicity
- Large plant with tertiary treatment.
- 500 m natural discharge channel with 100% effluent content.
- Constitutes less than 20% of Mississippi River flow.

**Plant Comparison**

## Endocrine Disrupting Compounds & Mixtures

**Endocrine Disrupting Compounds & Mixtures**

## Endocrine Disrupting Compounds & Mixtures

_**I.**__**	**__**Natural & Synthetic Estrogens**_**:**
- Estrone (E)
- Estradiol (E2)
- Ethynylestradiol (EE2)
- Well understood interactions with estrogen receptors.
- Action along hypothalamus-pituitary-gonadal axis is mechanistically apparent.
- Wealth of previous studies as benchmarks in lab & field.

## Endocrine Disrupting Compounds & Mixtures

- Fluoxetine - Antidepressant blocking reuptake of serotonin (SSRI).
- Carbamazepin - Antiepileptic acts in a receptor mediated manner by inhibiting neurotransmitters.
- Triclosan - Anti-microbic chlorophenol suspected of being hormonally active.

**Endocrine Disrupting Compounds & Mixtures**

- Have been documented in US surface waters.
- Are known to, or likely to interact with hypothalamus-pituitary-gonadal axis.

## Endocrine Disrupting Compounds & Mixtures

_**III.**__**	**__**Alkylphenols**_**:**
- Nonylphenol & Octylphenol - Parent compounds for surfactant mixtures.
- Nonylphenol/Octylphenol Carboxylates.
- Nonylphenol/Octylphenol Ethoxylates.
- Have been documented in many US surface waters.
- Exhibit estrogenic activity.
- Appear to facilitate the uptake of other compounds into biological organisms by increasing their solubility and bridging the phospholipids bilayer of the cell.

## Endocrine Disrupting Compounds & Mixtures

_**IV.**__**	**__**Mixtures**_**:**
- Alkylphenols - used and discharged as mixtures, occur always as mixtures, shorter chained NP/OP more potent, longer chained APs with greater surfactant qualities.
- Estrogens + Alkylphenols - commonly detected in same water samples, both estrogenic.
- Pharmaceuticals + Alkylphenols - different mode of action, likely that APs will facilitate uptake of pharmaceuticals by organism.
- Estrogens + Alkylphenols + Pharmaceuticals - a “biologically realistic” mixture representing the compounds common presence in effluents.

## Mixture Concentrations

- Highest no-effect concentration for each compound used for mixtures.

## Phase 1 - Preliminary Results

**Analysis of Metro and Boulder Creek Treated Wastewater Effluent [ng/L]**

|  | Metro Effluent | Boulder Effluent |
| --- | --- | --- |
| Estrone (E)
Estradiol (E2)
Ethynylestradiol (EE2) | nd
nd - 7.3 | 5 - 50
2 - 5
0.8 - 1.5 |
| Nonylphenol (NP)
Octylphenol (OP)
NP1EC
NP2EC
NP1EO
NP2EO | 333 - 13,000
50 - 933
8,756 - 21,364
17,530 - 35,512
684 - 1,002
342 - 731 | 
In
Progress |
| Fluoxetine
Carbamazepin
Triclosan | 51 - 83
823 - 1360
82 - 318 | nd - 48
nd - 58
nd - 150  |

## SSRI Distribution

- 0

- 100

- 200

- 300

- 400

- 500

- 600

- 700

- 7:30 AM

- 2:30 PM

- 11:30 PM

- 1400

- 1500

- 1600

- 1700

- 1800

- 1900

- 2000

- 2100

- 2200

- Concentration (ng/L)

**SSRI Distribution**
**in Metro Effluent**

## SSRI Distribution

**SSRI Distribution**
**in Metro Effluent**

## Phase 2A - Exposure Experiments

**Food Quality**

**Survival & Reproductive Ability**

- Chlorophyll A : Lipid Ratio

- Locomotion (escape & swimming)
- Male nest holding ability
- Female fecundity
- Offspring survival

- Juvenile Fathead Feeding Performance

## Experimental Design - Diatoms

*Cyclotella*
*meneghiniana*

**Primary producer**

**Experimental Design - Diatoms**
- Expose pure culture of diatoms in static exposure to compound or mixture for 10 days in sterile culture flask.
- In regular intervals sample diatom culture for:
- Cell count
- Lipid content
- Chlorophyll content

## Phase 2A - Preliminary Results

-  An increased ratio indicates a reduced nutritional value of the diatoms within the trophic cascade.
- E2 affects diatom food value very differently than nonylphenol.

**Phase 2A - Preliminary Results**

## Experimental Design - Fathead Minnows 

*Pimephales promelas*

**Consumer**

**Experimental Design - Fathead Minnows **
- Expose male fathead minnows for 28 days to graded concentrations of compound in flow-through exposure system.
- Allow males to compete with control males for access to spawning opportunities for seven days following exposure.
- After 35 days sample all males for:
- Vitellogenin
- Relative gonad and liver size, secondary sexual characters
- Reproductive ability (nest holding, fecundity, offspring survival)
- Locomotor ability  (escape velocity & station holding ability).
- Neuroendocrine endpoints

## Competitive Spawning

**Trials**

**Well Water**

**Well Water**
**& NP**

- 28 day exposure

- 7 day
- trial

- + 2 females
- & nest site

## Phase 2B - Linking Survival & Reproduction to Upstream Neuroendocrine Endpoints

**Phase 2B - Linking Survival & Reproduction to Upstream Neuroendocrine Endpoints**

## Neuroendocrine Endpoints

- Characterize sexual dimorphism in the neuroanatomical distribution of  enzymes and receptors responsible for expression of steroid-dependent behaviors.
- Identify changes in the distribution of these enzymes and receptors following exposure to WWTP contaminants singly or in combination.
- Identify minimum exposure duration for inducing changes in neurobehavioral endpoints.

## Neuroendocrine Endpoints

**I.  Neurotransmitter Biosynthesis**
- Tyrosine-hydroxylase is the rate limiting enzyme in the biosynthesis of norepinephrine and dopamine.
- The distribution of tyrosine-hydroxylase immunoreactive neurons in the preoptic brain is sexually dimorphic and responds to circulating concentrations of reproductive steroids.
- We hypothesize that endocrine compound exposures will alter tyrosine-hydroxylase immunoreactivity in the preoptic region of the brain.
- This change should be apparent after several weeks of exposure (“baseline change”).

## Neuroendocrine Endpoints

**II.  Neurotransmitter Analysis**
- The turnover of the monoamines dopamine, norepinephrine, serotonine to their respective catabolites will be quantified in several regions of the brain.
- We hypothesize that changes in monoamine turnover rate will become apparent after exposure and after stimulation during the competitive reproductive assays.
- This change should be apparent after several weeks of exposure and should be dramatic following the competitive reproductive assay (“stimulation capacity”).

## Fish Exposure

**Competitive Spawning**

- Distribution & abundance of tyrosine-hydroxylase immunoreactive neurons
- “Baseline Alteration”

- Monoamine turnover quantification
- “Stimulation Capacity”

**Neuroendocrine Endpoints**

## Phase 3 - Field Validation of Neuroendocrine Endpoints in Exposure Experiments at Both Field Sites

**Boulder, CO**

**St. Paul, MN**

## R832741-01-0