Fish Health, Pesticides and Endocrine Disruptors

Vicki Blazer, PhD U.S. Geological Survey

National Fish Health Research Laboratory Leetown Science Center, Kearneysville, WV

Fish Health Issues ! 2003 – first indications of major fish kills in the South

Branch, Potomac ! 2004-2005 – first observations of major kills in the North

Fork and South Fork Shenandoah ! 2009 – major kill in the Monocacy

! State agencies estimated losses of >60% of the adult bass populations in affected stretches of the rivers in first few years

! Primarily centrarchids (smallmouth bass, sunfish) but also suckers, rock bass, etc.

! Still have lesions and low chronic mortality

Adult Fish in the Potomac - Findings ! Multiple bacterial pathogens, but no consistent findings ! Aeromonas hydrophila and other motile Aeromonads ! Aeromonas salmonicida ! Flavobacterium columnare

! Multiple, often heavy parasite infestations ! Leeches, trematodes, myxozoans, cestodes

! Opportunistic fungal infections ! Skin papillomas ! Largemouth Bass Virus ! High prevalence of intersex, vitellogenin in male fishes

Impaired Ecosystem Immunosuppression

Fish Health Issues Susquehanna River

! 2005 – mortalities of young-of-year ! Demonstrated population declines ! Adults have variety of skin lesions although fish kills have

not been reported ! Adult males have a high prevalence and severity of intersex

Middle Susquehanna River (Sunbury to York Haven Dam)

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YOY SMB cpue

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Organisms Observed in YOY Susquehanna

! Aeromonas hydrophila and other motile

Aeromonads ! Flavobacterium columnare ! Largemouth Bass Virus ! Trematodes ! Myxozoan parasites

Overview of Findings Biological

! Intersex is widely distributed in bass (smallmouth, largemouth) in the Potomac and Susquehanna drainages ! Prevalence and severity at many sites is

greater than we see at most other sites nationally ! Species differences (metabolic, life styles) ! Seasonal differences in intersex and Vtg ! Intersex bass had significantly less sperm,

less motile ! Prevalence of intersex was not higher

downstream of WWTP

Integrative Approach ! Biological effects in wild fishes ! Visual observations, microscopic pathology,

parasitology, plasma hormone/vitellogenin, gene expression

! In vitro, cell-based reporter assays ! BLYES – total estrogenicity, other yeast assays ! Nuclear translocation assays – NIH-NCI

! Chemical analyses – water, sediment, fish tissue ! Grab water samples, integrated passive water samplers

! GIS – land cover, land use etc. ! Telemetry – fish movement

Temporal nature of chemical exposures – climatic and other factors that influence exposure

Exposure pathways during critical life stage.

Factors to Consider

Correlations with Landuse and Chemicals PA Drainages

Chemical Contaminants or Landuse

Intersex Prevalence rho p

Intersex Severity rho p

Estrone (water) 0.6530 0.0238 0.7609 0.0055 Agricultural landuse 0.6843 0.0170 0.7044 0.0129

WWTP/sewage facilities -0.5298 0.0794 -0.8441 0.0936

Prevalence of males with vitellogenin

Estrone (water) 0.7914 0.0033

Evaluated smallmouth bass and white sucker at 16 sites in three river drainages in the summer (low flow)

Intersex and Land-use Spring Pre-spawn Study Potomac

Landuse Characteristics

Intersex Prevalence

Intersex Severity

r2 p r2 p Human population 0.39 0.10 0.42 0.08 # WWTP 0.22 0.24 0.34 0.13 WWTP flow 0.32 0.15 0.63 0.02 Percent agriculture 0.63 0.02 0.50 0.05 # Animal feeding operations 0.28 0.17 0.56 0.03 Total animal numbers 0.27 0.18 0.48 0.06 Animal density 0.49 0.05 0.58 0.03 Poultry Houses 0.27 0.18 0.50 0.05

Results - Discrete Water 39 of 200 compounds detected (20%)

Atrazine 86% herbicide Deethylatrazine 86 herbicide degradate Caffeine 86 stimulant Simazine 86 herbicide Iso-CT 71 antibiotic degradate Metolachlor 71 herbicide Epi-iso-CT 57 antibiotic degradate Galaxolide 57 fragrance Tetracycline 57 antibiotic Oxytetracycline 43 antibiotic

Results – Passive Water

41 compounds detected via passive samplers were not found in discrete water samples

Chlorpyrifos 100% insecticide Diazinon 100 insecticide Dieldrin 100 insecticide Androstenedione 80 biogenic hormone 17α-estradiol 60 biogenic hormone Estrone 60 biogenic hormone Triclosan 60 antimicrobial disinfectant 17β-estradiol 40 biogenic hormone

100 of 161 compounds detected (62%)

Results – Bed Sediment 46 of 130 compounds detected (35%)

Caffeine 100% stimulant Cholesterol 100 plant/animal sterol Indole 100 fragrance p-cresol 100 wood preservative sitosterol 100 plant sterol PCBs 86 industrial compound Stigmastanol 86 plant sterol Trans-chlordane 86 insectide Trans-nonachlor 86 insecticide Dieldrin 71 insecticide Estrone 71 biogenic hormone

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Total Biogenic Hormone+Sterol, in µg/kg

ρ = 0.986, p<0.001

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Sediment Chemical Results

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Number of Herbicides Detected in Water

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Water Results – Chemicals and TO

Intersex and Chemical Contaminants Spawning Study

Chemical Contaminants

Intersex Prevalence Intersex Severity

rho P rho p

Atrazine 0.93 0.003 0.88 0.009

Deethylatrazine 0.78 0.039 0.68 0.090

Acetochlor 0.65 0.116 0.79 0.036

Metolachlor 0.87 0.011 0.81 0.028

Land-use Characteristics Estrogenicity

rho p % Forest -0.654 0.008 % Pasture/ Hay 0.629 0.012 % Crop 0.586 0.021 % Developed 0.453 0.086 Poultry Density 0.696 0.004 Beef Density 0.530 0.041 Dairy Density 0.360 0.180 WWTP (MGD) -0.006 0.974

Shenandoah Small Tributary Study

POCIS pesticides (26 total) Estrogenicity

rho p

Desethylatrazine 0.670 0.006 Metolachlor 0.631 0.011 Atrazine 0.582 0.022 Simazine 0.541 0.037

No fish data Total estrogenicity based on the estrogen equivalents using the BLYES

Correlations of Intersex and Estrogenicity with Herbicides

! Is there some type of synergy with the herbicides and co-occurring contaminants? ! Does the use of herbicides lead to high levels

of phytoestrogens or algal products in the water/sediment?

Correlations of Herbicides with Intersex and Estrogenicity

•  Atrazine has been associated with intersex in frogs – controversial

•  Atrazine is not thought to bind to the estrogen receptor •  Has been shown to alter phytoplankton and algal populations,

increase trematode infections and cause immunosuppression •  Could algal/cyanobacteria and their toxins be contributing to

endocrine disruption and fish health issues

Potential Association of Atrazine and Health

! Atrazine augments the richness and abundance of gastropods ! Intermediate hosts for trematode parasites

! Atrazine and other herbicides decrease phytoplankton which tend to increase periphytic algae

! Increase the abundance of the cercariae (infective stage of trematodes for fish and amphibians

! Low doses of atrazine have been shown to be immunosuppressive in frogs, fish and salamanders

! Exposure increased susceptibility to A. hydrophila infections in fish and viral infections in salamanders

Current Studies ! Identifying most important agricultural sources and

potential management actions ! 13 sites in the Potomac and Susquehanna with varying

intensity and types of ag ! Biological responses in adult and young-of-year bass ! Comprehensive chemical analyses of water, sediment and

fish tissue ! Coordinating with the water quality investigations in

show case watersheds to assess temporal and spatial changes in hormone activities (estrogenic, androgenic, glucocorticoid) ! Studies on association of lesions, estrogenicity with algal

populations and algal toxins

Current Studies

! Identifying pathways and timing of exposures ! Adults – water, sediment during nest-building and guarding,

food web ! Young – maternal sources through the egg, water and

sediment in the nesting areas ! Two potentially important exposure periods: ! Reproductive cycle – in late summer/early fall recrudescence begins.

Germ cells undergo division and begin development into eggs and sperm. Yolk begins to be incorporated into the egg.

! Fry as they go through sexual differentiation

! Chemical analyses of eggs immediately prior to spawn as well as young

Temporal Studies ! Understanding temporal changes within a year

and between years ! Hormone activity – passive samplers, grab water

–  South Branch (3 sites); 4 sites MD mainstem sites –  2011 and 2014

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2011 South Branch

Seneca Falls

Young of Year Smallmouth Bass Tissue Analyses

! Compared whole body contaminants ! 10 fish from LSC ponds and 16 fish from two sites on the

Susquehanna ! Suite of organic contaminants including pesticides (legacy

and current-use), PCBs, BDE, triclosan ! LSC ponds – mean # of detects was 4 (range 2-5) ! BDE 99, BDE 200, PCB 180, hexachlorobenzene

! Susquehanna – mean # of detect was 19 (range 14-28) ! BDE 47, 7 PCBs, dieldrin, chlorpyrifos, nonachlors, chlordanes,

pentachloroanisole, triclosan, pyraclostrobin

Herbicide Concentrations (ng/L) Monocacy – Big Pipe Creek

Dates Atrazine Metolachlor Simazine May 15, 2013 364 228 343 June 8, 2013 75 81 58 June 11, 2013 227 215 215 July 11, 2013 45 61 20 Aug 28, 2013 37 34 13 April 4, 2014 26 30 12 April 14, 2014 31 47 15 April 25, 2014 16 13 - April 30, 2014 3268 703 2416 May 12, 2014 201 93 112 May 16, 2014 5399 4643 76 May 30, 2014 2872 1612 1976

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Conclusions ! Hormone activity – animal and potentially

plant sterols, estrogenic chemicals as well as pesticides appear to be associated with intersex and adverse health affects in fish ! Need to better understand specific sources,

how these chemicals interact in the complex mixtures fish are exposed to, the factors affecting temporal changes in chemical concentrations and hormone activity, timing of exposure and the factors contributing to species differences in sensitivity