Field Observations of the Contributions of Alkyl Phenols on Fish Endocrine Disruption

Clifford P. Rice and Carys L. Mitchelmore

Overview

Introduction

Study sites, sampling regime

Chemical data

Biological parameters

Endocrine disruption?

Summary

IntroductionEndocrine Disrupting Chemicals (EDCs)

Many chemicals mimic natural estrogen

e.g. nonylphenol ethoxylates (NPEs)

Numerous effects occur detrimental to normal endocrine functioning

e.g. disrupting sex ratiosimpairing fertility - infertility (even changing behavior)

Often high levels in Rivers due to industry, agriculture & urbanization

Especially high levels found in discharges from Waste Water Treatment Plants

Use biomarkers to determine exposure to and effects of these chemicals;

e.g. presence of the female yolk protein VITELLOGENIN (VTG) in male or juvenile fish

Changes in normal steroid profiles; estrogen (E2) and 11-ketotestosterone (11-KT)

Cuyahoga River

Study site;

Cuyahoga River, famous in 1969 as oil slick left river on fire

Pivotal role for 1972 Clean Water Act

Sampled species

Common carp, Cyprinus carpioBenthic bottom feeder

Chemicals of concern

Nonylphenol ethoxylatesEspecially prevalent in WWTP dischargesKNOWN estrogenic effects on resident wildlife

Cuyahoga River

Aims and Objectives;

1. Determine levels of nonylphenol ethoxylates in (sediment,water) and carp tissuesNonylphenol (NP)NP1EONP2EOtotal NPEs (tNPEs)

2. Is there a difference between levels of NPEs in pristine (headwaters) vs. possible impacted sites (downstream)

4. Is there a correlation between biological parameters and levels of NPEs ?In particular measures of - Vitellogenin (VTG),

Estradiol (E2) and 11-ketotestosterone (11-KT)?

3. Is there a correlation in levels of NPE with possible discharge sites?

e.g. downstream of Akron Waste Water Treatment Plant (WWTP) - Site 5/6

Cuyahoga River Sampling Sites

Seven sites along a 74-mile length of the River

Site 1 - headwaters relatively pristine

Site 5/6 - downstream of Akron WWTP; Site 8 - downstream of Southerly WWTP

N

Cuyahoga River Methods

Seven sites along a 74-mile length of the River (n.b. Site 5/6 referred to as 6)

Male and female fish (n= 5-6 of each sex) sampled at each site

Levels of nonylphenol ethoxylates determined in fish tissues

General Biological and health measures determined for fish including ;

- Length - Weight (whole fish and gonad weight)- Gonadal somatic index (GSI)- Condition factor - Percentage lipid

Endocrinological parameters measured, including ;

- Circulating steroid levels - Estradiol (E2) - 11-ketotestosterone (11-KT)

- Vitellogenin levels in blood (VTG)

Chemistry - Total NPEs in carp

0

250

500

750

1000

1250

1500

0153045607590

River MileUpstream Downstream

ALL FISH

Total NPE(ng g-1 wet wt.)

Site 1

Sites 3,4

Sites 7,8

Site 6

Site 2

Summary

Lowest levels at Site 1 - uppermost site Peak at Site 6 - downstream of Akron WWTP High levels at Sites 7 & 8

(Levels in carp, water and sediment; Rice et al., 2003)

Results; Means S.D. (n=11-12) Site 1 25 20Site 2 160 93Site 3 164 76Site 4 181 74Site 6 957 260Site 7 582 217Site 8 523 203

Chemistry - Total NPEs

0200400600800

100012001400

1 2 3 4 6 7 8

Males

Site No.

0200400600800

100012001400

1 2 3 4 6 7 8

Site No.

Females

Summary

Similar levels found in both males and females Lowest levels at Site 1 - uppermost site Peak at Site 6 - downstream of Akron WWTP High levels at Sites 7 & 8

T

otal

NP

E(n

g g-1

wet

wt.

)

Results; Means S.D. (n=5-6)

0

50

100

150

200

250

300

0153045607590

Chemistry - Nonylphenol in carp

River MileUpstream Downstream

ALL FISH

NP(ng g-1 wet wt.)

Site 1

Sites 3,4

Sites 7,8

Site 6

Site 2

Summary

Lowest levels at Site 1 - uppermost site Increasing levels downstream High levels at Sites 6, 7 & 8; Very variable levels Highest at Sites 6 & 8 ; downstream of WWTP’s Similar levels to other fish studies (e.g. In UK, Lye et al., 1999 = 5-180 ng g-1) Lower cf. some other US studies (e.g. 223-1842 ng g-1; Datta et al., 2002)

Results; Means S.D. (n=11-12) Site 1 8.2 6.0Site 2 33.7 21.9Site 3 31.7 20.6Site 4 41.8 20.8Site 6 137.2 105.2Site 7 83.4 64.7Site 8 140.0 82.7

0

50

100

150

200

250

300

1 2 3 4 6 7 80

50

100

150

200

250

300

1 2 3 4 6 7 8

Chemistry - Nonylphenol in Carp

Males

Site No. Site No.

Females

Summary

Similar levels found in both males and females Lowest levels at Site 1 - uppermost site High levels at Sites 6, 7 & 8 (males high at Sites 6 & 8, females peak at Site 6) Very variable levels

Results; Means S.D. (n=5-6)

NP

(ng

g-1 w

et w

t.)

0100

200300400

500600700

800900

0153045607590

Chemistry - NP1EO in carp

River MileUpstream Downstream

ALL FISH

NP1EO(ng g-1 wet wt.)

Site 1

Sites 3,4

Sites 7,8

Site 6

Site 2

Summary Lowest levels at Site 1 - uppermost site Increasing levels downstream High levels at Sites 6, 7 & 8 Highest value downstream of Akron WWTP Similar to other studies (e.g. Lye et al., 1999 = n.d. to 940 ng g-1; Snyder et al., 1999 = 242 ng g-1) Dissimilar to other studies showing no detectable levels (Bennie et al., 1998;

Keith et al., 2001).

Results; Means S.D. (n=11-12) Site 1 17 20Site 2 97 56Site 3 113 54Site 4 116 52Site 6 569 157Site 7 377 150Site 8 269 106

0100200300400500600700800

1 2 3 4 6 7 8

0100200300400500600700800

1 2 3 4 6 7 8

Chemistry - NP1EO in Carp

Males

Site No. Site No.

Females

Summary

Similar levels found in both males and females Lowest levels at Site 1 - uppermost site High levels at Sites 6, 7 & 8 ; Highest at Site 6

Results; Means S.D. (n=5-6)

N

P1E

O(n

g g-1

wet

wt.

)

0

50

100

150

200

250

300

350

0153045607590

Chemistry - NP2EO in carp

River MileUpstream Downstream

ALL FISH

NP2EO(ng g-1 wet wt.)

Site 1 n.d.

Sites 3,4

Sites 7,8

Site 6

Site 2

Summary

Increasing levels downstream Highest levels - Site 6 - downstream of WWTP High levels at Sites 6, 7 & 8 Different to many US studies that have not detected NP2EO in fish (e.g. Snyder et al., 1999; Bennie et al., 1998; Keith et al., 2001)

n.d. = not determinedResults; Means S.D. (n=11-12)

Site 1 n.d.Site 2 29 20Site 3 20 10Site 4 23 13Site 6 250 58Site 7 123 33Site 8 114 55

050

100150200250300350

1 2 3 4 6 7 8

050

100150200250300350

1 2 3 4 6 7 8

Chemistry - NP2EO in Carp

Males

Site No. Site No.

Females

Summary

Similar levels found in both males and females Levels increase in downstream direction High levels at Sites 6, 7 & 8

n.d n.d

N

P2E

O(n

g g-1

wet

wt.

)

Results; Means S.D. (n=5-6)

Summary - NPEs in carp tissues

Levels are lowest at Site 1 - upstream location

Levels increase in a downstream direction

Peak (source) at site 6 - downstream of Akron WWTP(and at 8 for NP in males - downstream of Southerly WWTP)

Correlation with biological parameters?

Highest values at Sites 6,7 & 8

Biological measures

Length and weight higher at uppermost sites, decrease in downstream direction

y = -30.952x + 679.4

R2 = 0.7581

400

500

600

700

800

1 2 3 4 6 7 8

y = -37.91x + 726.77

R2 = 0.8836

400

500

600

700

800

1 2 3 4 6 7 8

Leng

th

Males Females

y = -414.73x + 4313.7

R2 = 0.6508

0

1000

2000

3000

4000

5000

6000

1 2 3 4 6 7 8

y = -693.04x + 6003.2

R2 = 0.8529

010002000300040005000600070008000

1 2 3 4 6 7 8

Wei

ght

Site No.Site No.

Biological measures

No differences between sites in; GSI, condition factor or % lipid

No correlation of % lipid with levels of NPEs

y = 13.753x + 241.41

R2 = 0.0293

0200400600800

1000120014001600

0 5 10 15 20 25

Percentage Lipid

Tot

al N

PE

s No differences between sites in female gonad weight,

but is difference in males (bigger upstream) - impact of NPEs??

Biological measures - endocrine endpoints

Males;

0

0.04

0.08

0.12

0.16

0.2

1 2 3 4 6 7 8

VT

G (

mg

/ml)

Higher levels at Site 6 correlates with highest tNPE, NP1EO, NP2EO downstream of Akron WWTP BUT NP highest at Site 8 (below WWTP2) and then Site 6.

Not significant at P>0.05, due to low numbers of samples and variability

0

50

100

150

200

250

300

1 2 3 4 6 7 8

Site No.

N

P(n

g g-1

wet

wt.

)Site No.

No differences between sites in levels of E2 or 11-KT

Is a suggested difference in levels of VTG

Biological measures - endocrine endpoints Males;

But at Site 6 ONLY (highest VTG)Correlations with NPEs;

NP2EO = r2 8%NP1EO = r2 50% tNPEs = r2 70% NP = r2 85%

BUT NP at Site 8 r2 = 9%

y = 0.0003x + 0.0313

R2 = 0.2734

0

0.05

0.1

0.15

0.2

0.25

0.3

0 100 200 300 400

VT

G (

mg

/ml)

NP (ng g-1 wet wt.)

Overall very poor correlations NPEs and male VTG levels;tNPEs (r2=17%), NP1EO (r2=12%) and NP2EO (r2=11%)

Poor correlation between NP and VTG levels (but is highest at r2=27%)

Biological measures - endocrine endpoints Males;

0

0.02

0.04

0.06

0.08

0.1

0.12

1 2 3 4 50

50

100

150

200

250

300

VT

G (

mg

/ml)

Rank

NP

(ng g-1 w

et wt.)

Rank order (group into categories) NP levels; 1= 0-25 2= 25-503 = 50-100 ng g-1 NP4= 100-200 5 = 200+

Plot average values for VTG and NP levels in each rank

Is correlation (r2=87%) between NP and VTG levels

Biological measures - endocrine endpoints

- To increase n number (sample size), place fish into Zones (groups of Sites)

- Sites 1&2 = Zone 1; Sites 3&4 = Zone 2; Site 6 = Zone 3; Site 7= Zone 4:Site 8 = Zone 5

ZonesV

TG

(m

g/m

l)3

4

N

Zone 4

Zone 1

Zone 2

Zone 3

Zone 5

00.020.040.060.080.1

0.120.14

1 2 3 4 5

Results: means (n=5-12) ± S.E.M.

Males;

Summary

Higher levels at Zone 3 correlates with high NPEs downstream of Akron WWTP

* ; Zone 3 significantly different to Zone 2 at P>0.02, and Zone 1 at P>0.1

*

Biological measures - endocrine endpoints

Females;

11-

KT

(pg

/ml)

Are significant differences in levels of 11-KT and ratio E2 / 11-KT between sites

No correlation / patterns with levels of NPEs - other chemicals / factors ?

2

2.5

3

3.5

4

1 2 3 4 6 7 8

0

1

2

3

4

5

1 2 3 4 6 7 8

E2

/11-

KT

Site No.Site No.

no differences between sites in levels of VTG or E2

Summary and Conclusions Our levels of NPEs are comparable to other studies

Highest NPEs downstream of Akron WWTP (Site 6; except NP) and high levels downstream of Southerly WWTP (Site 8; highest NP)

NPEs especially NP have been shown to affect fish endocrine parameters, e.g.;- Elevated VTG in males ; we have weak correlation with NP?- Changes in steroid hormones for males and females ;

do not see in this study

WWTPs have also been shown to affect the fish endocrine parameters above

Clear estrogenic impact along whole length of river as all male fish have some VTG present.

Better correlations of biological parameters with water / sediment NPE levels?

No obvious impact of NPEs to females.

More data required ; increase n number, different season sampling

Water column Sediment Common carp tissue

Total NPE Total NPE Total NPE (ug/L) (ug/kg dry wt.) (ug/kg wet wt.)

Miwb* 0.024 0.109 0.191

IBI** 0.069 0.317 0.43

% Omnivores 0.114 0.031 0.205

% Top Carnivores 0.326 0.031 0.573

%DELT*** 0.069 0.261 0.002

Linear regression (R2)

Biological Variable

Roger Thoma’s Indices of the River’s Health Status versus total NPE Concentrations in Different Media.

*Modified index of well-being; **Index of Biotic Integrity; ***Percent Deformities, Eroded fins, Lesions and Tumors.

Contribution of Akron WWTP Discharge to Cuyahoga River

Flow• Sampling Period 7 July to 13 July, 2000 was a low

flow period, e.g., 35% of average flow for that year.

• Plant discharge average 70 mgd for July 2000 versus 90 mgd rated capacity of plant.

• Therefore % contribution of plant discharge for sampling period was anywhere from 40 to 60 % of River flow at that location.

Acknowledgements

•Ohio EPA, esp. Roger Thoma and Ed Link.

•Analysts with USDA-Beltsville,

Isabelle Afonso-Schmidt and Jorge Loyo-Rosales.

•USEPA Region V, esp. Peter Howe and Al Alwan.

•Stephen Smith, USGS Biological Research Division,

Reston VA.