Development

standardization

and

refinement

of

procedures

for

evaluating

effects

of

endocrine

active

compounds

on

development

and

sexual

differentiation

of

Xenopus

laevis

Xenopus

laevis

has

been

introduced

as

a

model

to

study

effects

of

endocrine-active

compounds

-LRB-

EAC

-RRB-

on

development

and

sexual

differentiation

However

variable

and

inconsistent

data

have

raised

questions

about

the

reliability

of

the

test

methods

applied

The

current

study

was

conducted

in

two

laboratories

to

develop

refine

and

standardize

procedures

and

protocols

Larvae

were

exposed

in

flow-through

systems

to

-LRB-

-RRB-

at

concentrations

from

to

μg

L

in

Experiment

and

to

μg

L

in

Experiment

In

both

studies

survival

-LRB-

-RRB-

and

percentage

of

animals

that

completed

metamorphosis

-LRB-

-RRB-

indicated

reproducible

biological

performance

Furthermore

minor

variations

in

husbandry

led

to

significant

differences

in

snout-to-vent

length

weight

and

gonad

size

In

Experiment

almost

complete

feminization

occurred

in

all

treatment

groups

whereas

a

concentration

response

was

observed

in

Experiment

resulting

in

an

of

μg

L

The

final

verified

protocol

is

suitable

for

determining

effects

of

EAC

on

development

and

sexual

differentiation

in

X.

laevis

Introduction

During

the

last

ten

years

the

number

of

chemicals

evaluated

for

endocrine-active

effects

has

increased

Potential

exposure

of

wildlife

populations

including

fish

amphibians

birds

and

mammals

to

environmental

estrogens

has

attracted

much

attention

due

to

the

feasible

impact

of

these

substances

on

survival

and

population

sustainability

-LSB-

-RSB-

Laboratory

studies

using

a

range

of

wildlife

species

have

demonstrated

that

physiological

processes

including

development

and

reproduction

are

sensitive

to

environmental

estrogens

-LSB-

-RSB-

Due

to

the

diversity

of

wildlife

certain

species

have

been

used

as

representatives

of

the

various

taxa

that

are

considered

in

eco-toxicological

hazard

assessment

One

example

of

a

well

established

standardized

animal

model

is

the

frog

embryo

teratogenesis

assay

-LRB-

FETAX

-RRB-

-LSB-

-RSB-

using

Xenopus

laevis

embryos

to

determine

impacts

of

chemicals

on

early

development

This

species

has

been

investigated

focusing

on

larval

stages

for

studying

the

hormonal

regulation

of

metamorphosis

development

sexual

differentiation

and

the

impact

of

endocrine

active

compounds

-LRB-

EAC

-RRB-

-LSB-

-RSB-

However

the

results

in

the

literature

for

basic

biological

parameters

such

as

mortality

growth

development

and

sexual

differentiation

vary

considerably

among

laboratories

-LSB-

-RSB-

making

interpretation

difficult

For

example

it

has

been

reported

that

the

herbicide

atrazine

feminizes

and

demasculinizes

amphibians

-LSB-

-RSB-

but

other

investigators

have

not

been

able

to

replicate

these

findings

-LSB-

-RSB-

The

published

data

on

potential

effects

of

atrazine

on

gonadal

development

in

laboratory

studies

and

reproductive

effects

in

wildlife

populations

were

evaluated

by

a

United

States

Environmental

Protection

Agency

-LRB-

EPA

-RRB-

Scientific

Advisory

Panel

-LRB-

SAP

-RRB-

in

The

SAP

identified

a

number

of

factors

that

adversely

affected

the

quality

and

comparability

of

data

in

the

open

literature

on

atrazine

These

factors

included

variability

in

study

design

differences

in

animal

husbandry

conditions

-LRB-

e.g.

feeding

rates

-RRB-

excess

biomass

in

treatment

chambers

and

the

effects

of

these

factors

on

water

quality

Other

factors

that

contributed

to

difficulty

in

interpreting

the

results

were

variability

in

the

response

to

the

positive

control

substance

and

the

negative

control

as

well

as

differences

in

the

use

of

gross

and

microscopic

pathology

terminology

limited

statistical

power

and

lack

of

compliance

with

Good

Laboratory

Practice

standards

In

consequence

the

SAP

published

a

White

Paper

-LSB-

-RSB-

that

highlighted

inconsistencies

and

variability

among

studies

and

by

so

doing

provided

guidance

for

the

development

of

a

standardized

study

design

to

evaluate

potential

effects

of

EAC

on

development

and

sexual

differentiation

of

X.

laevis

The

purpose

of

this

study

was

to

develop

standardize

and

refine

a

protocol

meeting

specifications

of

the

SAP

that

could

be

used

to

assess

estrogenic

effects

on

development

and

sexual

differentiation

in

X.

laevis

A

team

of

scientists

representing

endocrinology

toxicology

chemistry

pathology

and

statistics

was

involved

in

the

creation

of

the

study

design

selection

of

endpoints

and

choice

of

data

analysis

tools

The

team

concluded

that

it

was

necessary

to

conduct

two

independent

studies

consecutively

in

two

different

locations

to

develop

refine

and

validate

the

study

design

by

assessing

effects

of

on

gonadal

development

of

X.

laevis

exposed

from

NF

-LRB-

Nieuwkoop

and

Faber

-RRB-

stages

through

stage

in

a

flow-through

exposure

system

Experiment

was

conducted

at

Wildlife

International

Ltd

MD

USA

-LRB-

WLI

-RRB-

in

order

to

develop

and

standardize

conditions

for

rearing

and

handling

X.

laevis

larvae

operation

of

a

flow-through

system

and

maintaining

water

quality

and

environmental

parameters

A

concentration

range

for

of

and

μg

L

was

chosen

to

calculate

the

effective

concentration

needed

to

achieve

a

effect

-LRB-

-RRB-

for

gonadal

feminization

and

to

evaluate

abnormal

development

of

gonads

In

addition

this

preliminary

study

was

used

to

establish

the

terminology

for

classifying

gross

and

histological

gonadal

abnormalities

to

cross-train

laboratory

personnel

and

to

develop

the

appropriate

statistical

analysis

methodology

The

procedures

developed

in

Experiment

were

refined

and

implemented

in

Experiment

conducted

at

the

Leibniz-Institute

of

Freshwater

Ecology

and

Inland

Fisheries

Germany

-LRB-

IGB

-RRB-

Material

and

methods

Test

organism

Tadpoles

of

X.

laevis

were

obtained

from

the

commercial

supplier

Xenopus

I

-LRB-

Dexter

MI

USA

-RRB-

These

larvae

-LRB-

approx.

for

each

study

-RRB-

originated

from

ten

pairs

of

adult

breeders

for

each

study

Xenopus

I

reared

the

eggs

until

day

post

fertilization

-LRB-

dpf

-RRB-

and

then

shipped

them

to

each

laboratory

Rearing

Upon

arrival

on

dpf

tadpoles

were

acclimated

to

water

and

temperature

conditions

-LRB-

°C

-RRB-

over

a

six-hour

time

period

During

the

acclimation

period

larvae

were

maintained

in

two

glass

aquaria

containing

L

well

water

-LRB-

ca.

larvae/tank

Experiment

-RRB-

and

three

glass

aquaria

containing

L

dilution

water

-LRB-

ca.

larvae/tank

Experiment

-RRB-

derived

from

the

flow-through

system

At

the

initiation

of

treatment

twenty-five

larvae

were

randomly

assigned

to

each

glass

aquarium

-LRB-

n

larvae

per

treatment

group

-RRB-

so

that

the

biological

loading

rate

was

always

less

than

L

day

In

Experiment

exposures

were

initiated

at

dpf

NF

stage

and

in

Experiment

at

dpf

when

tadpoles

had

reached

NF

stage

as

identified

by

visible

hind

limb

buds

-LSB-

-RSB-

Exposures

continued

until

completion

of

metamorphosis

or

dpf

whichever

came

first

Throughout

the

acclimation

period

water

temperature

was

recorded

twice

daily

whereas

dissolved

oxygen

-LRB-

DO

-RRB-

concentration

and

pH

were

measured

daily

Experiment

was

conducted

in

compliance

with

the

local

animal

protection

committee

-LRB-

-RRB-

Feeding

Animals

were

fed

Sera

Micron

-LRB-

Lot

No

Sera

Heinsberg

Germany

-RRB-

a

commercially

available

food

which

was

reported

by

the

manufacturer

to

consist

of

protein

fat

fibre

and

ash

A

sample

of

Sera

Micron

was

analysed

by

Lancaster

Laboratories

-LRB-

Lancaster

PA

USA

-RRB-

for

potential

contaminants

-LRB-

metals

and

pesticides

-RRB-

and

all

tested

contaminants

were

below

their

limits

of

quantification

for

analysis

at

that

facility

A

mixture

of

diet

and

dilution

water

was

tested

for

estrogenicity

and

androgenicity

using

YES

-LRB-

yeast

estrogen

screen

-RRB-

-LSB-

-RSB-

and

YAS

-LRB-

yeast

androgen

screen

-RRB-

assays

-LSB-

-RSB-

Based

on

these

analyses

no

estrogenic

or

androgenic

activity

was

detected

During

acclimation

of

larvae

in

Experiment

g

Sera

Micron

was

added

to

each

tank

daily

This

amount

of

food

was

found

to

be

excessive

In

experiment

after

temperature

acclimation

larvae

were

transferred

to

the

acclimation

tanks

where

they

promptly

attached

to

the

glass

aquaria

walls

Therefore

no

food

was

added

on

dpf

The

following

day

some

of

the

tadpoles

were

free

swimming

and

mg

Sera

Micron

was

added

that

evening

by

rinsing

through

a

sieve

The

amount

of

feed

was

chosen

based

on

the

intake

of

food

by

the

larvae

and

care

was

taken

to

ensure

that

feeding

did

not

adversely

affect

water

quality

From

dpf

to

the

free

swimming

tadpoles

were

fed

mg

Sera

Micron

in

each

tank

twice

a

day

Throughout

the

remaining

course

of

both

studies

tadpoles

were

fed

a

suspension

of

Sera

Micron

-LRB-

mg

L

-RRB-

three

times

daily

The

total

quantity

of

food

added

per

tank

each

day

increased

from

mg/tank

on

dpf

to

mg/tank

on

dpf

The

quantity

of

food

added

to

each

tank

was

adjusted

periodically

in

Experiment

and

on

a

daily

basis

in

Experiment

to

account

for

mortality

and

removal

of

tadpoles

as

they

completed

metamorphosis

Study

design

Because

an

almost

completely

feminized

phenotype

was

observed

in

Experiment

in

all

treatment

groups

the

nominal

concentrations

in

Experiment

were

adjusted

to

and

μg

L

respectively

Mid

and

high

concentrations

were

selected

to

correspond

to

experiment

and

allow

comparison

of

results

from

the

two

laboratories

Replicates

Each

study

consisted

of

at

least

one

negative

control

group

plus

three

groups

each

comprising

eight

replicated

glass

aquaria/treatment

groups

Eight

glass

aquaria

were

arranged

in

two

clusters

of

four

tanks

each

The

clusters

of

negative

control

tanks

and

treated

tanks

were

randomly

distributed

within

the

environmental

chamber

in

Experiment

and

within

water

baths

in

Experiment

Each

cluster

was

supplied

water

via

the

same

water

flow

meter

and

mixing

chamber

Experiment

included

control

tanks

that

were

initially

divided

into

two

groups

designated

as

negative

control

and

reference

control

respectively

The

animals

from

all

treatment

groups

were

handled

in

a

blinded

fashion

so

that

the

biologists

and

histologists

involved

in

the

experiments

did

not

know

the

treatment

applied

to

frogs

being

observed

An

additional

non-blind

reference

control

group

was

included

to

provide

reference

information

on

rates

of

spontaneous

incidence

of

gonadal

abnormalities

for

blind

histological

assessment

of

gonads

from

the

treatment

groups

Histological

analysis

was

conducted

on

all

frogs

of

Experiment

At

the

end

of

Experiment

it

was

discovered

that

slightly

elevated

temperature

-LRB-

°C

-RRB-

in

one

cluster

of

four

negative

control

tanks

had

resulted

in

accelerated

growth

of

the

frogs

in

the

tanks

Therefore

measurements

of

weight

length

gonad

image

area

and

age

at

metamorphosis

from

those

tanks

were

dropped

from

analysis

and

the

four

remaining

negative

control

tanks

were

combined

with

the

reference

control

group

to

provide

these

measurements

Flow-through

system

Both

studies

were

conducted

using

continuous

flow-through

systems

operating

at

a

flow

rate

of

at

least

L

dilution

water

per

tank

per

day

This

was

equivalent

to

a

water

exchange

rate

of

approximately

seven

tank

volumes

per

day

Test

tanks

consisted

of

glass

aquaria

-LRB-

cm

-RRB-

containing

L

water

Dilution

water

for

Experiment

was

obtained

from

a

well

of

approximately

m

depth

located

on

the

WLI

site

In

Experiment

the

dilution

water

used

was

non-chlorinated

municipal

water

Dilution

water

samples

from

Experiment

were

analysed

for

possible

metal

or

pesticides

by

Lancaster

Laboratories

and

water

samples

of

Experiment

by

Environmental

Chemistry

and

Pharmanalytics

-LRB-

RCC

Ltd.

Itingen

Switzerland

-RRB-

The

flow-through

systems

in

the

two

laboratories

were

essentially

identical

Prior

to

use

the

water

was

passed

through

several

filters

-LRB-

and

μm

-RRB-

to

remove

particulate

material

a

UV-sterilizer

and

a

final

particle

filter

-LRB-

μm

-RRB-

After

being

filtered

and

temperature

conditioned

the

dilution

water

was

divided

into

streams

Each

stream

passed

through

a

flow-control

assembly

consisting

of

a

rotameter

flow

gauge

and

a

needle

valve

to

a

mixing

chamber

The

flow

from

each

mixing

chamber

was

then

split

to

supply

a

cluster

of

four

tanks

Each

tank

received

mL

min

dilution

water

-LRB-

-RRB-

Primary

solutions

The

primary

stock

solutions

were

continuously

delivered

to

each

mixing

chamber

-LRB-

mL

min

-RRB-

where

it

was

vigorously

mixed

with

the

dilution

water

Primary

stock

solutions

were

metered

into

the

mixing

chambers

by

an

eight-channel

peristaltic

pump

-LRB-

MasterflexL/S

Model

Cole

Parmer

IL

USA

-RRB-

The

flow-through

system

-LRB-

temperature

water

filter

pressure

flow-meter

-RRB-

and

peristaltic

pumps

were

calibrated

four

weeks

prior

to

study

initiation

and

inspected

visually

twice

daily

throughout

the

study

solutions

Once

every

seven

days

a

primary

stock

solution

was

prepared

in

amber

bottles

by

dissolving

mg

-LRB-

Lot

No

CAS

purity

Sigma

Aldrich

WLI

Allentown

PA

USA

IGB

Taufkirchen

Germany

-RRB-

in

alkaline

solution

-LRB-

mol

L

NaOH

-RRB-

applying

moderate

heating

and

constant

mixing

of

the

solution

overnight

similar

to

procedures

employed

by

the

Duluth

Laboratory

of

the

USEPA

Office

of

Research

and

Development

-LRB-

personal

communication

by

J.

Tietge

-RRB-

In

Experiment

the

working

stock

solutions

were

prepared

at

nominal

concentrations

of

and

μg

L

and

in

Experiment

at

and

μg

L

The

quantity

of

NaOH

in

test

solutions

delivered

to

each

tank

was

negligible

and

therefore

no

NaOH

was

added

to

the

negative

control

Analysis

of

concentration

Sampling

In

Experiment

water

samples

were

collected

from

each

tank

in

all

treatment

groups

to

confirm

the

operation

of

the

diluter

two

days

prior

to

study

initiation

In

Experiment

concentrations

were

monitored

twice

weekly

for

a

two-week

period

prior

to

study

initiation

After

study

initiation

water

sampling

was

carried

out

twice

weekly

from

alternating

tanks

in

two

tanks

per

cluster

until

study

termination

Throughout

the

studies

water

samples

were

collected

routinely

two

days

after

the

freshly

prepared

solution

was

connected

to

the

flow-through

system

and

a

second

time

in

the

same

week

At

mid-depth

from

each

test

tank

mL

water

was

collected

using

a

glass

pipette

and

mL

methanol

-LRB-

MeOH

-RRB-

was

added

Of

this

solution

mL

was

decanted

into

a

mL

amber

scintillation

vial

for

immunosorbent

assay

-LRB-

ELISA

-RRB-

analysis

The

remaining

volume

of

mL

was

stored

as

backup

at

°C

until

analysis

was

completed

Water

samples

from

Experiment

were

shipped

to

WLI

for

analysis

The

stability

of

was

confirmed

by

shipping

samples

of

-LRB-

μg

L

-RRB-

that

were

prepared

and

analysed

in

parallel

with

the

water

samples

Sample

analysis

Rapid

analysis

was

performed

using

ELISA

kits

-LRB-

Ecologiena

Abraxis

LLC

Warminster

PA

USA

-RRB-

to

ensure

the

absence

of

the

test

substance

in

the

negative

control

tanks

and

to

verify

the

proper

concentration

in

each

tank

The

ELISA

method

was

based

upon

the

kit

manual

provided

by

the

manufacturer

The

method

detection

range

of

this

assay

was

μg

L

Parallel

analyses

of

water

samples

for

concentration

were

performed

by

direct

aqueous

injection

using

an

Agilent

Series

High-Performance

Liquid

Chromatograph

coupled

with

a

MDS

Sciex

API

tandem

mass

spectrometer

-LRB-

LC

MS

MS

-RRB-

-LRB-

Applied

Biosystems

Foster

City

CA

USA

-RRB-

and

MDS

Sciex

API

heated

nebulizer

ion

source

-LRB-

Applied

Biosystems

-RRB-

operated

in

the

multi-reaction

mode

-LRB-

MRM

-RRB-

Chromatographic

separations

were

achieved

using

a

Keystone

Betasil

column

-LRB-

mm

mm

μm

particle

size

-RRB-

-LRB-

Thermo

Hypersil

Madison

WI

USA

-RRB-

The

method

limit

of

quantitation

-LRB-

LOQ

-RRB-

for

HPLC

MS

MS

estradiol

analyses

was

defined

as

μg

L

Environmental

conditions

Temperature

In

Experiment

all

glass

aquaria

were

situated

in

a

walk-in

environmental

chamber

-LRB-

°C

-RRB-

In

Experiment

glass

aquaria

were

placed

in

temperature

controlled

water

baths

-LRB-

°C

-RRB-

In

both

experiments

water

temperatures

were

continuously

recorded

and

temperatures

in

all

glass

aquaria

were

measured

weekly

using

a

liquid-in-glass

thermometer

Light

The

target

light

intensity

at

tank

level

was

lux

and

was

measured

weekly

using

a

Sper

Scientific

-LRB-

AZ

USA

-RRB-

light

meter

A

photoperiod

of

h

of

light

and

h

of

dark

was

used

in

Experiment

and

was

adjusted

to

a

h

light

h

dark

cycle

in

Experiment

A

low

light

-LRB-

around

intensity

of

full

light

-RRB-

transition

period

was

installed

in

both

laboratories

Water

quality

Water

quality

parameters

were

assessed

according

to

ASTM

-LRB-

-RRB-

-LSB-

-RSB-

and

US

EPA

guidelines

Water

hardness

pH

conductivity

and

the

concentrations

of

nitrate

and

ammonia

were

measured

weekly

in

one

tank

from

each

cluster

throughout

the

experiments

Hardness

and

alkalinity

were

measured

by

titration

based

on

procedures

described

in

Standard

Methods

for

the

Examination

of

Water

and

Wastewater

-LSB-

-RSB-

Nitrate

concentrations

were

analysed

with

a

Hach

colorimeter

-LRB-

Hach

Company

Colorado

USA

-RRB-

and

specific

conductance

was

detected

using

a

YSI

conductivity

meter

-LRB-

WLI

Yellow

Springs

Instruments

Model

IGB

Yellow

Springs

OH

USA

-RRB-

pH

and

ammonia

concentrations

were

measured

with

an

Orion

pH/ISE

Meter

-LRB-

Thermo

Electron

USA

-RRB-

DO

concentrations

were

measured

in

alternate

tanks

per

treatment

three

times

a

week

with

an

Orion

Model

dissolved

oxygen

meter

-LRB-

Thermo

Electron

-RRB-

Commencing

at

dpf

in

Experiment

and

at

dpf

in

Experiment

gentle

external

aeration

was

applied

by

a

glass

pipette

in

each

test

tank

to

ensure

maintenance

of

an

adequate

DO

concentration

Maintaining

water

quality

The

tanks

required

daily

cleaning

to

minimize

microbial

growth

and

to

maintain

adequate

water

quality

Bio-film

that

accumulated

on

the

inner

walls

and

bottom

of

the

tanks

was

scraped

off

daily

and

detritus

was

siphoned

from

the

tanks

Daily

cleaning

proved

inadequate

to

maintain

appropriate

water

quality

throughout

the

entire

exposure

period

Hence

in

Experiment

all

tanks

were

replaced

with

clean

tanks

weekly

and

in

Experiment

twice

throughout

the

experiment

when

siphoning

did

not

lead

to

satisfactory

results

In-life

assessment

of

tadpoles

Tadpoles

were

monitored

for

changes

in

general

health

swimming

behaviour

and

morphological

appearance

daily

The

numbers

of

dead

or

moribund

tadpoles

and

tadpoles

completing

metamorphosis

were

recorded

daily

for

each

tank

Snout-to-vent

length

and

weight

Each

tadpole

that

had

either

completed

metamorphosis

by

NF

stage

or

had

failed

to

achieve

metamorphosis

by

dpf

was

removed

from

the

test

tank

and

euthanized

by

immersion

in

MS

-LRB-

tricaine

methanesulfonate

-RRB-

-LRB-

WIL

Sigma

Aldrich

Allentown

PA

USA

IGB

Sigma

Aldrich

Taufkirchen

Germany

-RRB-

-LRB-

g

L

buffered

to

pH

with

-RRB-

Immediately

thereafter

the

snout-to-vent

length

was

measured

to

the

nearest

millimetre

Each

frog

was

blotted

dry

and

then

weighed

to

the

nearest

milligram

A

unique

identifier

was

assigned

to

each

frog

that

included

study

number

in-life

laboratory

ID

colour

code

replicate

and

animal

number

Gross

pathology

The

pleuroperitoneal

cavity

was

opened

to

expose

the

visceral

organs

The

liver

stomach

and

intestine

were

examined

using

a

dissecting

stereo

microscope

-LRB-

WIL

Olympus

Olympus

America

Melville

NY

USA

IGB

Olympus

Olympus

Hamburg

Germany

-RRB-

and

unusual

variations

in

size

colour

and/or

abnormal

structure

were

recorded

The

gastrointestinal

tract

was

excised

to

expose

the

ventral

surfaces

of

the

kidney

and

gonads

which

were

similarly

assessed

for

gross

abnormalities

Tadpoles

that

did

not

complete

metamorphosis

at

study

termination

-LRB-

Experiment

Experiment

-RRB-

were

staged

according

to

NF

and

the

gonad

morphology

was

assessed

-LRB-

if

possible

-RRB-

and

the

carcasses

were

archived

in

fixative

Data

derived

from

such

animals

were

excluded

from

further

analysis

Assessment

of

gonad

morphology

Using

a

dissecting

stereo

microscope

gross

evaluations

of

gonad

morphology

were

performed

on

all

animals

that

reached

NF

stage

To

enhance

visualization

of

the

gonads

which

presented

as

thin

pale-tan

strips

of

tissue

on

the

ventromedial

margin

of

the

kidney

several

drops

of

Bouin

's

solution

-LRB-

WIL

Sigma

Aldrich

Allentown

PA

USA

IGB

Sigma

Aldrich

Taufkirchen

Germany

-RRB-

were

applied

to

partially

fix

these

organs

Based

on

gross

observation

the

gonads

of

each

animal

were

identified

as

testes

ovaries

or

malformed

Examples

of

observed

malformations

included

mixed

sex

intersex

pearling

and

segmental

aplasia

Mixed

sex

was

defined

as

the

co-occurrence

of

both

ovarian

and

testicular

tissue

in

a

single

gonad

Intersex

was

assessed

as

ovarian

and

testicular

tissue

in

the

same

individual

as

separate

gonads

-LRB-

left/right

-RRB-

The

term

pearling

was

characterized

by

the

presence

of

multiple

prominent

segmental

enlargements

and/or

attenuations

along

the

length

of

one

or

both

gonads

whereas

gonads

that

presented

as

nodular

islands

of

testicular

or

ovarian

tissue

with

either

intervening

membranous

connections

or

no

connections

at

all

were

classified

as

segmental

aplasia

Gross

findings

for

the

gonads

of

each

frog

were

recorded

separately

for

the

left

and

right

gonads

The

gonadal

findings

for

each

frog

were

verified

independently

by

a

second

biologist

If

the

two

biologists

differed

in

their

interpretations

the

disparate

opinions

were

discussed

and

a

consensus

finding

was

recorded

Prior

to

the

initiation

of

the

first

experiment

a

list

of

standardized

terms

for

changes

of

potential

morphological

gonadal

observations

was

developed

Subsequent

to

the

completion

of

both

experiments

the

applied

terminology

was

refined

in

order

to

better

characterize

and

group

different

types

of

gonad

changes

observed

and

which

in

turn

could

be

analysed

For

example

the

subcategories

narrow

slightly

narrow

truncated

slightly

truncated

and

margin

entire

were

integrated

into

the

main

category

hypoplasia

This

term

describes

all

gonads

which

appear

smaller

than

those

characterised

as

typical/normal

Since

the

term

typical/normal

is

rather

general

further

sub-categories

allow

the

biological

variability

of

gonadal

morphology

to

be

described

However

during

the

investigations

it

was

revealed

that

too

few

observations

in

each

sub-category

ruled

out

reasonable

statistical

analyses

Therefore

a

refined

list

of

qualitative

descriptive

terminology

for

gonadal

features

was

created

as

presented

in

Table

Table

of

terms

used

to

describe

features

observed

during

gross

morphological

examination

of

gonads

in

X.

laevis

NF

stage

-LRB-

other

tissue

-RRB-

Gonads

joined

to

other

abdominal

-LRB-

agenesis

-RRB-

Complete

lack

of

gonad

AplasiaGonad

is

longitudinally

discontinuous

Tissue

separationOne

or

more

areas

along

the

length

of

the

gonad

is

poorly

developed

attenuated

with

an

essentially

complete

lack

of

gonadal

tissue

Extraneous

gonadal

tissueA

small

disjunct

cluster

of

gonad-like

of

the

gonad

oriented

longitudinally

along

the

cranial

caudal

axis

Protuberance

-LRB-

s

-RRB-

Small

projection

-LRB-

s

-RRB-

of

the

gonad

along

the

margins

SymmetricA

Y

shaped

single

gonad

with

relatively

equal

deformityOne

or

more

bends

in

the

gonad

to

an

excessive

degree

Gonad

foldedThe

gonad

is

bent

over

or

doubled

up

so

that

one

part

lies

on

gonad

or

a

section

of

it

is

not

located

in

the

typical

location

Displaced

laterallyThe

gonad

or

a

section

of

it

is

not

located

in

the

typical

location

Instead

it

is

located

further

away

from

the

frog

's

midline

Displaced

mediallyThe

gonad

or

a

section

of

it

is

not

located

in

the

typical

location

Instead

it

is

located

closer

to

or

crosses

over

the

frog

's

midline

Displaced

craniallyThe

gonad

is

not

located

in

the

typical

location

Instead

it

is

located

more

towards

the

anterior

Displaced

caudallyThe

gonad

is

not

located

in

the

typical

location

Instead

it

is

located

more

towards

the

and

right

gonads

joined

to

varying

degrees

at

one

or

more

locations

along

cranial

caudal

of

the

gonad

is

larger

than

typical

WideGonad

appears

broad

Lateral

width

is

large

relative

to

the

length

ElongatedGonad

appears

long

-LRB-

cranial

caudal

-RRB-

Length

is

large

relative

to

the

length

of

the

kidneys

and

the

torso

of

the

frog

ThickGonad

relatively

increased

in

the

distance

between

the

dorsal

and

ventral

surfaces

EnlargedA

generalized

increase

in

the

size

of

the

gonad

along

all

hypertrophyA

gonad

where

one

or

more

areas

are

excessively

large

Enlargement

-LRB-

mass

-RRB-

A

section

of

the

gonad

substantially

bigger

in

width

and/or

thickness

than

the

remaining

tissue

PearlingPronounced

multiple

segmental

enlargement

of

the

gonad

Partly

thickA

section

or

sections

of

the

gonad

is

relatively

increased

in

the

distance

between

the

dorsal

and

ventral

of

the

gonad

is

decreased

NarrowGonad

appears

strap-like

Lateral

width

is

markedly

reduced

relative

to

the

length

Slightly

narrowLateral

width

is

somewhat

reduced

relative

to

the

length

TruncatedGonad

appears

short

-LRB-

cranial

caudal

-RRB-

Length

is

small

relative

to

the

length

of

the

kidneys

and

the

torso

of

the

frog

Slightly

truncatedGonad

appears

somewhat

short

-LRB-

rostal

caudal

-RRB-

Length

is

somewhat

reduced

relative

to

the

length

of

the

kidneys

and

the

torso

of

the

frog

ThinGonad

relatively

reduced

in

distance

between

the

dorsal

and

ventral

surfaces

Margin

entireAll

of

the

ovary

or

indeterminate

gonad

has

a

smooth

edge

no

scalloping

or

hypoplasiaA

gonad

where

one

or

more

areas

are

excessively

reduced

attenuated

or

poorly

developed

but

not

separated

Partly

narrowA

section

or

sections

of

the

gonad

has

a

small

lateral

width

relative

to

the

length

and

the

width

of

the

other

sections

Partly

thinA

section

or

sections

of

the

gonad

is

relatively

reduced

in

distance

between

the

dorsal

and

ventral

surfaces

Margin

slightly

sinuateGonad

has

only

shallow

waved

or

scalloped

edge

-LRB-

typically

associated

with

a

narrow

or

partly

narrow

ovary

-RRB-

Margin

partially

entireA

section

or

sections

of

the

gonad

has

a

smooth

edge

-LRB-

typically

associated

with

a

narrow

or

partly

narrow

ovary

-RRB-

PearlingPronounced

multiple

segmental

attenuation

or

narrowing

of

the

and

testicular

tissue

present

as

separate

structures

-LRB-

i.e.

not

contained

in

the

same

gonad

-RRB-

-LRB-

left/right

-RRB-

Mixed

sexOvarian

and

testicular

tissue

present

in

the

same

appears

not

so

dense

light

able

to

pass

through

diffusely

Tissue

slightly

translucentTo

a

small

extent

the

gonad

appears

not

so

dense

a

small

amount

of

light

able

to

pass

through

translucenceA

section

or

sections

of

the

gonad

appears

not

so

dense

light

able

to

pass

through

decreasedApparently

fewer

pigment-containing

cells

than

typically

seen

Applies

only

to

females

mixed

sex

frogs

and

frogs

of

undetermined

sex

ApigmentationNo

melanophores

in

the

internal

tissue

Applies

only

to

females

mixed

sex

frogs

and

frogs

of

undetermined

melanophoresPigment-containing

cells

within

the

gonadal

tissue

Applies

only

to

males

mixed

sex

frogs

and

frogs

of

undetermined

finding

not

listed

Describe

as

needed

For

any

other

finding

that

study

personnel

consider

not

incidental

notify

the

Study

Director

and

other

laboratories

as

soon

as

possible

via

email

including

a

description

and

photograph

Photography

After

gross

gonad

inspection

was

complete

each

gonad

was

photographed

in

situ

using

a

digital

camera

-LRB-

WIL

Olympus

Olympus

America

Melville

NY

USA

IGB

Olympus

Hamburg

Germany

-RRB-

attached

to

the

stereo

microscope

Each

digitalized

image

included

a

millimetre

measurement

scale

placed

adjacent

to

the

gonads

to

permit

gonad

size

to

be

determined

from

the

photograph

Following

photography

each

frog

was

placed

in

a

labelled

individual

container

of

mL

of

Bouin

's

solution

for

approximately

h

At

the

end

of

the

fixation

period

each

carcass

was

rinsed

several

times

in

ethanol

and

was

placed

in

mL

neutral

buffered

formalin

-LRB-

WIL

VWR

Westchester

PA

USA

IGB

Histofix

Roth

Karlsruhe

Germany

-RRB-

A

complete

gonadal

histological

evaluation

was

performed

on

each

frog

of

experiment

and

results

are

presented

in

a

separate

paper

-LRB-

Wolf

et

al.

in

preparation

-RRB-

In

some

cases

the

growth

and

histological

findings

differ

and

a

better

understanding

of

results

is

gained

by

considering

both

types

of

observation

Gonad

measurement

The

biological

variability

of

morphological

features

of

the

gonads

and

the

subjective

nature

of

the

assessment

prompted

us

to

develop

a

more

quantitative

metric

of

gonad

size

Thus

individual

measurements

of

gonad

image

area

were

derived

from

photographs

of

the

gonads

Gonad

image

areas

were

obtained

using

image-processing

software

-LRB-

Image

Pro

Plus

Version

Media

Cybernetics

Silver

Spring

MD

USA

-RRB-

that

calculated

the

combined

area

of

the

left

and

right

gonad

whose

outline

had

been

manually

traced

around

the

digital

image

The

program

was

calibrated

by

the

millimetre

measurement

scale

prior

to

measurement

for

the

gonad

of

each

tadpole

Statistical

analysis

For

both

experiments

observations

were

collected

for

each

animal

However

consistent

with

the

experimental

design

the

tank

is

considered

the

primary

experimental

unit

When

tank

differences

are

present

statistical

analysis

should

accommodate

this

experimental

structure

directly

using

nested

random

effects

analyses

or

indirectly

by

analysing

tank

means

or

tank

percentages

Neither

approach

is

completely

satisfactory

for

all

endpoints

Nested

models

have

specific

distributional

requirements

and

in

the

case

of

incidence

data

become

unstable

when

frequencies

are

at

or

near

zero

Analyses

of

tank

means

can

also

be

problematic

for

these

experiments

Estradiol-induced

feminization

causes

the

number

of

males

per

tank

to

be

smaller

for

treated

groups

than

for

the

control

group

This

in

turn

results

in

greater

variation

between

estradiol-treated

tanks

than

between

control

tanks

Most

parametric

and

non-parametric

statistical

procedures

however

require

equal

variability

between

tanks

within

every

group

When

tank

differences

are

small

or

absent

however

the

individual

data

from

all

tanks

within

the

same

treatment

group

can

be

pooled

and

analysed

more

simply

using

the

frog

as

the

basic

experimental

unit

For

Experiments

and

endpoints

were

tested

for

differences

between

tanks

using

a

test

of

homogeneity

A

chi-square

homogeneity

test

was

used

for

frequency

endpoints

-LRB-

males

females

mixed

sex

-RRB-

and

a

Kruskal

Wallis

test

was

used

for

measurement

endpoints

-LRB-

snout-to-vent

length

body

weight

age

at

completion

of

metamorphosis

gonad

image

area

-RRB-

This

homogeneity

test

was

calculated

for

each

experimental

group

and

the

p-values

combined

using

Tippett

's

minimum-p

method

Tank

differences

were

found

for

several

frequency

endpoints

in

Experiment

In

addition

there

was

evidence

that

the

presence

of

a

humidifier

caused

a

temperature-induced

impact

on

measurement

endpoints

for

two

clusters

of

tanks

In

contrast

Experiment

did

not

reveal

any

statistically

significant

-LRB-

p

-RRB-

tank

effects

Consequently

all

subsequent

statistical

analyses

for

Experiment

can

be

conducted

on

individual

animals

with

all

tanks

pooled

Thus

analyses

of

Experiment

are

quite

complex

and

varied

while

Experiment

results

can

be

analysed

rather

simply

In

order

to

summarize

the

results

consistently

to

allow

comparisons

of

both

experiments

only

animal-level

means

and

standard

deviations

are

presented

The

means

and

standard

deviations

given

in

this

report

for

both

experiments

were

calculated

from

individual

animals

using

Excel

Version

For

Experiment

these

simple

statistics

are

identical

with

those

obtained

from

the

statistical

analyses

described

below

For

Experiment

these

means

-LRB-

and

standard

deviations

-RRB-

should

be

considered

only

approximate

They

may

differ

slightly

from

means

obtained

using

more

sophisticated

analyses

that

incorporate

a

tank

structure

In

Experiment

the

estradiol-treated

groups

were

compared

with

controls

in

a

step-wise

manner

that

preserved

power

and

protected

against

excessive

false

positives

An

overall

test

of

group

differences

and

a

test

for

trend

with

dose

were

both

conducted

If

either

of

these

two

overall

tests

showed

statistical

significance

at

the

level

-LRB-

i.e.

p

-RRB-

then

comparisons

of

each

dose

group

to

the

control

were

made

For

frequency

endpoints

a

chi-square

homogeneity

test

and

a

Cochran

Armitage

test

for

trend

were

used

for

the

overall

homogeneity

and

trend

tests

respectively

Comparisons

of

dose

groups

to

control

were

conducted

using

Fisher

's

exact

test

For

measurement

data

essentially

all

endpoints

showed

statistically

significant

deviations

from

normality

-LRB-

p

Shapiro

Wilk

test

-RRB-

Consequently

a

Kruskal

Wallis

nonparametric

test

was

used

for

group

homogeneity

and

a

Jonckheere

Terpstra

test

was

used

for

trend

Comparisons

of

dose

groups

to

control

were

conducted

using

the

nonparametric

Wilcoxon

Mann

Whitney

U-test

All

statistical

analyses

of

Experiment

were

calculated

using

Release

of

the

SAS

statistical

package

-LRB-

-RRB-

Results

It

is

emphasized

that

Experiment

was

performed

first

in

order

to

standardize

conditions

for

rearing

X.

laevis

and

for

operation

of

the

flow-through

system

Improvements

of

these

procedures

were

implemented

in

Experiment

resulting

in

optimised

growth

and

development

conditions

for

X.

laevis

tadpoles

Those

parameters

that

were

successfully

achieved

during

Experiment

are

summarised

in

Table

and

may

be

used

as

guidance

for

similar

experiments

The

following

sections

focus

on

results

obtained

in

Experiment

presenting

results

from

Experiment

as

needed

to

allow

comparison

of

the

two

experiments

Table

study

design

specifications

Brief

summary

of

the

most

important

parameters

for

exposing

X.

laevis

tadpoles

in

a

flow-through

exposure

systemParameterCharacteristicWater-quality

parameters

Supply

water

filtered

contaminant

freeFiltered

and

UV

sterilized

tested

for

chemical

contaminants

Dissolved

oxygen

of

saturation

to

to

mg

L

to

mg

L

to

mg

L

to

mg

L

Specific

to

μg

S

cm

Supplemental

aerationInitiated

not

later

than

dpf

husbandry

Animal

supplyXenopus

Dexter

MI

USA

Shipped

from

SupplierDay

post

fertilization

-LRB-

dpf

-RRB-

Acclimationdpf

Treatment

perioddpf

Feed

Sera

MicronSera

Micron

-LRB-

contaminants

and

estrogenic

potential

evaluated

-RRB-

Contents

protein

fat

fibre

ash

Feeding

rate

acclimationdpf

per

larvae

mg

if

free

swimming

dpf

mg

twice

daily

-LRB-

based

on

the

intake

of

food

by

the

larvae

-RRB-

Feeding

rate

NF

stages

mg

L

fed

three

times

daily

mg/tank

from

dpf

increasing

to

mg/tank

at

dpf

then

adjusted

during

experiment

as

animals

were

removed

-LRB-

dead

froglets

-RRB-

Exposure

chamber

cleaningDaily

Exposure

chamber

replacementAs

needed

Water-quality

parametersHardness

ammonia

nitrate

pH

specific

conductivity

twice

a

week

DO

three

times

a

week

°C

-LRB-

verified

twice

daily

measured

once

a

week

-RRB-

Light

lux

-LRB-

measured

once

weekly

at

level

of

surface

of

water

-RRB-

Light

dark

transition

-LRB-

of

full

light

-RRB-

Experimental

design

Performed

in

compliance

with

Good

Laboratory

Practice

StandardYes

Colour

coded

-LRB-

blind

-RRB-

treatmentYes

Randomization

of

tadpolesTadpoles

distributed

in

rotation

one

or

two

at

a

time

to

transport

vessels

to

contain

then

vessels

were

randomly

assigned

to

test

tanks

Randomization

of

tank

clustersCompletely

randomized

placement

one

cluster

consists

of

four

tanks

Negative

controlWater

-LRB-

eight

replicates

two

clusters

-RRB-

Treatment

μg

L

eight

replicates

two

clusters

for

each

group

Tank

L

-LRB-

water

volume

L

-RRB-

tanks

covered

Tank

flow

L

day

Number

of

larvae

per

treatment

group

Animal

load

densityLess

than

g

L

water

larvae/tank

HPLC

MS

MS

verificationdpf

all

tanks

dpf

weekly

-LRB-

alternating

tanks

-RRB-

Environmental

conditions

In

both

experiments

experimental

conditions

generally

met

the

criteria

listed

in

Table

In

Experiment

the

mean

and

SD

for

hardness

and

alkalinity

were

mg

and

mg

L

respectively

Specific

conductance

was

μS

cm

pH

nitrate

mg

L

and

ammonia

mg

L

The

measurements

from

negative

control

tanks

were

not

significantly

different

from

those

from

the

tanks

Technical

problems

in

Experiment

prevented

accurate

determination

of

DO

concentration

however

based

on

subsequent

studies

using

the

same

procedures

DO

was

consistently

above

saturation

exposure

verification

In

each

experiment

was

not

present

in

any

of

the

negative

control

tanks

Measured

concentrations

of

stock

solutions

ranged

from

to

in

Experiment

and

between

and

in

Experiment

In

Experiment

the

mean

concentration

of

for

three

treatment

groups

began

to

decline

after

experiment

initiation

and

fell

below

of

nominal

concentration

by

dpf

Between

exposure

dpf

and

mean

concentration

of

decreased

to

minima

of

and

of

nominal

concentrations

of

in

the

and

μg

L

treatment

groups

respectively

After

dpf

concentrations

of

began

to

increase

in

all

treatment

groups

and

exceeded

of

nominal

concentration

on

dpf

-LRB-

Fig.

-RRB-

In

Experiment

concentrations

of

declined

after

study

initiation

and

dropped

below

of

nominal

concentration

by

dpf

On

dpf

concentrations

of

fell

to

and

in

the

and

μg

L

treatment

groups

respectively

The

concentration

of

increased

to

of

nominal

concentration

on

dpf

-LRB-

except

the

μg

L

with

-RRB-

-LRB-

Fig.

-RRB-

Fig.

of

measured

mean

concentrations

in

water

samples

of

treatments

in

the

flow-through

system

Mean

values

represent

the

concentrations

of

of

weekly

sampling

intervals

from

dpf

or

through

the

last

sampling

interval

on

termination

of

the

experiments

on

dpf

a

concentrations

of

Experiment

b

concentrations

of

Experiment

The

limit

of

quantification

was

μg

L

calculated

as

the

product

of

the

concentration

of

the

lowest

calibration

standard

-LRB-

μg

L

-RRB-

and

the

dilution

factor

of

the

matrix

blank

samples

-LRB-

μg

L

-RRB-

In-life

assessment

of

tadpoles

Appearance

and

behaviour

No

effects

of

treatment

on

tadpoles

condition

behaviour

or

external

appearance

was

observed

in

any

of

the

treatment

groups

at

any

time

throughout

the

entire

course

of

Experiment

or

Experiment

Survival

Excellent

survival

was

achieved

in

both

experiments

in

controls

as

well

as

in

treated

groups

The

total

survival

was

in

Experiment

and

in

Experiment

B

In

Experiment

survival

in

the

combined

negative

control

and

μg

L

treatment

groups

was

and

respectively

In

Experiment

survival

rates

were

in

the

negative

control

and

and

in

the

and

μg

L

exposure

groups

respectively

Snout-to-vent

length

and

weight

In

Experiment

for

females

the

average

length

values

were

and

mm

in

the

and

μg

L

treatment

group

compared

with

mm

in

the

combined

control

group

Males

treated

with

had

an

average

snout-to-vent

length

of

and

mm

in

the

combined

control

and

μg

L

treatment

group

respectively

The

increase

in

the

mean

weight

of

males

was

more

pronounced

than

the

increase

in

the

mean

weight

of

females

-LRB-

Fig.

-RRB-

Fig.

weight

-LRB-

means

SD

-RRB-

of

a

females

and

b

males

after

exposure

to

from

dpf

or

in

a

flow-through

system

until

completion

of

metamorphosis

or

day

post-fertilization

μg

L

was

not

used

in

Experiment

and

μg

L

was

not

used

in

Experiment

Neg

Ctrl

negative

control

group

In

Experiment

no

statistically

significant

differences

between

the

snout-to-vent

length

for

females

and

males

were

observed

The

snout-to-vent

length

for

females

and

males

in

the

negative

control

group

was

and

mm

respectively

For

females

and

males

treated

with

μg

L

snout-to-vent

length

was

and

mm

respectively

No

statistically

significant

treatment-related

effect

was

observed

for

body

weight

either

in

females

or

in

males

At

completion

of

metamorphosis

frogs

from

Experiment

weighed

more

than

twice

as

much

as

frogs

from

Experiment

regardless

of

the

gender

-LRB-

Fig.

-RRB-

Time

course

of

metamorphosis

On

average

the

process

of

metamorphosis

for

tadpoles

took

longer

in

Experiment

than

in

Experiment

In

Experiment

the

age

at

which

the

first

frog

in

the

experiment

completed

metamorphosis

was

delayed

by

days

-LRB-

dpf

-RRB-

in

each

treatment

group

compared

with

animals

in

Experiment

-LRB-

dpf

-RRB-

-LRB-

Fig.

-RRB-

In

Experiment

exposure

of

females

to

and

μg

L

resulted

in

a

slight

delay

of

metamorphosis

In

Experiment

the

time

course

of

metamorphosis

for

females

in

the

μg

L

treatment

group

was

similar

to

that

for

females

in

the

negative

control

group

but

exposure

to

and

μg

L

caused

a

delay

of

completion

of

metamorphosis

-LRB-

Fig.

b

-RRB-

A

comparable

picture

for

the

cumulative

time

course

of

metamorphosis

of

males

is

shown

in

Fig.

d

In

Experiment

males

treated

with

and

μg

L

had

a

delay

in

completing

metamorphosis

of

approximately

ten

days

compared

with

males

in

the

negative

control

group

In

Experiment

metamorphosis

of

male

frogs

in

the

μg

L

treatment

group

was

slightly

postponed

whereas

for

males

treated

with

μg

L

completion

of

metamorphosis

was

delayed

by

three

days

The

lowest

concentration

used

obviously

did

not

affect

the

mean

time

to

complete

metamorphosis

Fig.

portion

of

X.

laevis

frogs

completing

metamorphosis

after

exposure

to

different

concentrations

of

from

day

or

post-fertilization

in

a

flow-through

system

until

completion

of

metamorphosis

or

day

post-fertilization

The

number

of

individuals

per

treatment

group

-LRB-

n

-RRB-

is

given

in

the

legend

a

portion

of

females

in

Experiment

b

portion

of

females

in

Experiment

c

portion

of

males

in

Experiment

-LRB-

just

one

male

was

observed

in

the

μg

L

group

-RRB-

d

portion

of

males

in

Experiment

Neg

Ctrl

negative

control

group

Exposure

to

had

no

effect

upon

the

percentage

of

surviving

tadpoles

that

completed

metamorphosis

on

dpf

-LRB-

in

Experiment

in

Experiment

-RRB-

By

dpf

-LRB-

out

of

-RRB-

and

-LRB-

out

of

-RRB-

individuals

failed

to

complete

metamorphosis

in

Experiment

and

Experiment

respectively

and

no

treatment-related

effect

was

observed

In

Experiment

and

individuals

failed

to

complete

metamorphosis

by

dpf

in

the

negative

control

and

μg

L

treatment

groups

respectively

Mean

age

at

completion

of

metamorphosis

The

differences

in

the

time

course

of

metamorphosis

described

in

the

previous

section

are

reflected

in

the

mean

ages

of

frogs

at

completion

of

metamorphosis

in

each

of

the

treatment

groups

In

Experiment

the

mean

ages

at

completion

of

metamorphosis

of

females

treated

with

and

μg

L

were

dpf

and

respectively

and

substantially

greater

than

the

mean

age

in

the

negative

control

group

-LRB-

dpf

-RRB-

-LRB-

Fig.

-RRB-

In

contrast

in

Experiment

females

treated

with

the

different

concentrations

showed

a

dose

response

with

a

significant

increase

noted

in

the

μg

L

group

-LRB-

dpf

-RRB-

and

a

greater

effect

-LRB-

dpf

-RRB-

in

the

μg

L

treatment

group

compared

with

dpf

for

females

in

the

negative

control

group

For

males

in

Experiment

the

mean

age

at

completion

of

metamorphosis

was

increased

from

dpf

in

the

negative

control

to

dpf

and

dpf

in

the

and

μg

L

treatment

groups

respectively

In

Experiment

the

mean

age

at

completion

of

metamorphosis

of

males

was

statistically

significant

greater

in

the

μg

L

-LRB-

dpf

-RRB-

and

μg

L

-LRB-

dpf

-RRB-

treatment

groups

than

in

the

negative

control

group

-LRB-

dpf

-RRB-

-LRB-

Fig.

-RRB-

Fig.

age

at

completion

of

metamorphosis

-LRB-

mean

SD

-RRB-

by

treatment

group

for

female

a

and

male

b

frogs

at

completion

of

metamorphosis

or

on

day

post-fertilization

μg

L

was

not

used

in

Experiment

and

μg

L

was

not

used

in

Experiment

Only

one

male

frog

was

observed

in

the

μg

L

group

in

Experiment

Neg

Ctrl

negative

control

group

For

Experiment

significant

differences

are

marked

by

asterisks

-LRB-

p

p

p

two-sided

Wilcoxon

Mann

Whitney

test

of

equality

with

negative

control

group

-RRB-

A

Kruskal

Wallis

test

of

overall

group

differences

was

significant

-LRB-

females

p

males

p

-RRB-

and

a

Jonckheere

Terpstra

test

of

trend

was

significant

-LRB-

females

p

males

p

-RRB-

Appearance

and

examination

of

non-gonadal

organs

No

externally

visible

morphologic

abnormalities

were

observed

in

each

treatment

group

in

either

experiment

Likewise

inspection

of

the

liver

stomach

intestine

kidney

for

tumours

lesions

or

any

other

remarkable

features

had

no

effect

Determination

of

sex

percent

female

male

and

mixed

sex

by

gross

examination

The

percentages

of

male

female

and

mixed-sexed

animals

were

calculated

based

on

frogs

that

completed

metamorphosis

-LRB-

NF

stage

-RRB-

among

all

animals

In

Experiment

the

percentage

of

female

phenotype

was

increased

in

all

treated

groups

In

the

and

μg

L

treatment

group

and

of

frogs

showed

female

phenotype

In

Experiment

the

incidence

of

female

phenotype

in

the

treated

groups

displayed

a

statistically

significant

distinct

dose-responsive

increase

with

a

high

percentage

of

females

observed

for

the

-LRB-

-RRB-

and

μg

L

-LRB-

-RRB-

treatment

groups

respectively

-LRB-

Fig.

-RRB-

The

concentration

that

results

in

an

increase

of

female

phenotype

to

is

defined

as

the

effective

concentration

-LRB-

-RRB-

Based

on

the

feminization

effect

observed

in

Experiment

the

was

calculated

to

be

μg

Fig.

bars

represent

frequencies

of

sex

of

X.

laevis

frogs

among

all

animals

according

to

treatment

group

based

on

gross

morphological

evaluation

the

total

number

of

frogs

-LRB-

n

-RRB-

that

completed

metamorphosis

is

given

on

top

of

the

bars

Tadpoles

were

exposed

to

in

a

flow-through

system

from

day

or

post-fertilization

through

completion

of

metamorphosis

or

day

post-fertilization

The

was

calculated

from

the

results

of

Experiment

and

was

μg

The

numbers

of

frogs

in

the

negative

control

in

Experiment

was

higher

for

evaluation

of

gender

because

the

animals

which

were

excluded

from

calculation

of

weight

and

length

were

included

in

calculation

of

the

portion

of

males

females

and

mixed

sex

Neg

Ctrl

negative

control

group

For

Experiment

asterisks

denote

significant

differences

-LRB-

p

p

two-sided

Fisher

's

exact

test

for

comparison

to

negative

control

-RRB-

Both

an

overall

exact

chi-square

test

of

equality

of

all

experimental

groups

and

a

Cochran

Armitage

trend

test

were

statistically

significant

-LRB-

p

-RRB-

in

every

case

In

Experiment

the

percentage

of

mixed

sex

individuals

were

-LRB-

-RRB-

-LRB-

-RRB-

and

-LRB-

-RRB-

in

the

and

μg

L

treatment

groups

respectively

In

Experiment

the

incidences

of

mixed-sex

animals

were

statistically

significant

with

-LRB-

-RRB-

and

-LRB-

-RRB-

in

the

and

μg

L

treatment

groups

respectively

-LRB-

Fig.

-RRB-

In

both

experiments

the

percentage

of

frogs

displaying

mixed

sex

gonads

was

higher

in

groups

than

in

the

negative

control

groups

When

results

from

the

two

experiments

are

compared

the

percentage

of

mixed

sex

animals

was

seen

to

be

similar

in

equal

concentrations

Segmental

aplasia

was

detected

in

Experiment

in

one

of

nine

male

individuals

out

of

the

μg

L

treatment

group

In

Experiment

segmental

aplasia

was

observed

in

four

of

-LRB-

-RRB-

nine

of

-LRB-

-RRB-

eighteen

of

-LRB-

-RRB-

and

four

of

seven

-LRB-

-RRB-

male

frogs

in

the

and

μg

L

treatment

groups

respectively

Thus

the

frequency

of

segmental

aplasia

in

the

and

μg

L

groups

was

significantly

elevated

Pearling

was

observed

in

two

of

nine

and

one

of

five

male

frogs

the

and

μg

L

groups

respectively

in

Experiment

In

Experiment

the

occurrence

of

pearling

was

determined

in

two

frogs

out

of

within

the

μg

L

treatment

group

Intersex

was

not

found

in

any

of

the

Xenopus

evaluated

for

gonad

morphology

in

both

studies

Figure

b

demonstrate

ordinary

developed

ovarian

and

testes

tissue

that

was

assumed

to

be

normal

gonad

morphology

for

gross

morphological

evaluation

Figure

to

e

illustrate

gonads

showing

mixed

sex

pearling

and

segmental

aplasia

and

clarify

the

terminology

which

was

applied

in

these

experiments

to

describe

morphological

changes

in

gonadal

appearance

of

X.

laevis

due

to

treatment

To

allow

the

variability

of

the

morphological

appearance

of

gonads

to

be

described

an

extensive

terminology

to

include

the

biological

variability

was

developed

and

used

in

the

two

experiments

-LRB-

Table

-RRB-

In

Experiment

there

were

apparent

differences

in

the

occurrence

of

ovaries

that

were

truncated

narrow

entire

margin

or

had

enlarged

masses

However

no

differences

in

morphological

characteristics

were

observed

between

and

control

frogs

in

Experiment

Fig.

Ovary

of

X.

laevis

revealing

segmented

lobular

structure

and

clearly

visible

internal

melanocytes

in

the

tissue

the

length

of

the

ovary

is

slightly

shorter

than

the

length

of

the

kidney

b

In

contrast

a

testis

is

shorter

relative

to

the

length

of

the

kidney

-LRB-

half

of

the

kidney

-RRB-

straight

shaped

and

does

not

contain

internal

melanocytes

In

general

testis

tissue

appears

much

denser

than

ovary

tissue

c.

Mixed

sex

gonads

were

defined

as

the

co-occurrence

of

both

ovarian

and

testicular

tissue

in

a

single

gonad

The

ovary

tissue

in

the

presented

gonad

shows

a

reduced

lobular

structure

just

a

few

melanophores

whereas

the

testis

tissue

appears

bulbous

Arrows

indicate

the

different

gonad

structures

f

ovary-like

tissue

and

m

male

like

tissue

d.

Pearling

-LRB-

p

-RRB-

was

characterized

by

the

presence

of

multiple

prominent

segmental

enlargements

and/or

attenuations

along

the

length

of

one

or

both

gonads

e.

Segmental

aplasia

-LRB-

segA

-RRB-

was

classified

as

nodular

islands

of

testicular

or

ovarian

tissue

with

either

intervening

membranous

connections

or

no

connections

The

kidney

and

the

attached

gonad

tissue

were

fixed

with

Bouin

's

solution

Gonadal

measurement

Because

of

the

subjective

nature

of

gross

gonad

morphological

assessment

quantitative

measurements

of

gonad

area

were

performed

on

digital

images

In

Experiment

mean

gonad

areas

of

female

and

male

frogs

were

approximately

twice

as

large

as

in

animals

of

the

corresponding

gender

in

Experiment

The

mean

gonad

image

areas

for

females

in

Experiment

were

and

in

the

combined

negative

control

and

μg

L

treatment

groups

respectively

In

Experiment

for

females

treated

with

and

μg

L

mean

gonad

image

areas

were

and

respectively

compared

with

in

the

negative

control

group

Mean

gonad

areas

in

females

treated

with

μg

L

were

significantly

increased

-LRB-

p

-RRB-

In

Experiment

the

measured

mean

gonad

areas

of

males

in

the

combined

control

and

L

were

and

respectively

In

Experiment

males

treated

with

and

μg

L

had

a

slight

increase

of

mean

gonad

areas

and

respectively

compared

with

the

negative

control

-LRB-

-RRB-

A

Kruskal

Wallis

test

of

overall

group

differences

was

marginally

non-significant

-LRB-

p

-RRB-

but

a

Jonckheere

Terpstra

test

of

trend

was

significant

-LRB-

p

-RRB-

Discussion

Gonadal

development

in

anuran

amphibians

has

been

proposed

as

a

valuable

model

to

evaluate

the

physiological

consequences

of

exposure

to

EAC

with

-LRB-

anti

-RRB-

estrogenic

and/or

-LRB-

anti

-RRB-

androgenic

activity

-LSB-

-RSB-

It

has

long

been

known

that

treatment

of

tadpoles

with

gonadal

steroids

can

disrupt

normal

gonadal

development

in

several

anuran

species

and

might

even

lead

to

complete

sex

reversal

-LSB-

-RSB-

However

studies

on

the

potential

effects

of

EAC

on

anuran

gonadal

development

have

produced

variable

and

in

some

cases

inconsistent

results

From

analysis

of

published

studies

it

appears

that

some

of

the

variability

between

EAC

studies

may

be

because

of

differences

between

the

experimental

conditions

employed

One

example

to

illustrate

this

situation

is

the

outcome

of

three

different

exposure

studies

assessing

the

effects

of

bisphenol

A

-LRB-

BPA

-RRB-

on

gonadal

development

in

X.

laevis

-LSB-

-RSB-

By

using

a

semi-static

exposure

system

feminization

of

tadpoles

due

to

BPA

treatment

was

reported

by

Kloas

-LSB-

-RSB-

and

Levy

-LSB-

-RSB-

Attempts

to

replicate

these

findings

in

a

flow-through

exposure

system

by

Pickford

-LSB-

-RSB-

were

not

successful

A

more

recent

example

includes

inconsistent

results

observed

in

different

studies

on

the

putative

effects

of

atrazine

on

survival

growth

metamorphosis

and

sexual

development

of

X.

laevis

-LSB-

-RSB-

The

failure

to

reproduce

the

original

findings

leaves

regulatory

authorities

with

many

uncertainties

and

in

consequence

may

prevent

sound

assessment

of

potential

risks

associated

with

the

use

of

the

chemicals

of

concern

Contradictory

results

from

several

studies

of

atrazine

and

the

criticism

associated

with

each

of

these

studies

made

it

impossible

for

the

US

EPA

to

perform

an

unambiguous

evaluation

of

the

actual

impact

of

atrazine

on

development

and

sexual

differentiation

in

X.

laevis

The

US

EPA

therefore

requested

further

independent

studies

to

be

conducted

using

a

standardized

test

protocol

including

validated

positive

control

treatments

and

an

optimized

study

design

-LSB-

-RSB-

To

address

uncertainties

resulting

from

inconsistencies

of

previously

reported

data

the

current

multi-site

study

was

undertaken

in

order

to

develop

and

refine

an

optimized

testing

protocol

for

the

use

in

developmental

studies

with

X.

laevis

tadpoles

The

current

paper

presents

the

results

of

the

two

independent

exposure

experiments

that

were

performed

by

two

different

laboratories

A

first

experiment

-LRB-

Experiment

-RRB-

served

as

a

pilot

study

to

identify

appropriate

testing

conditions

for

the

development

of

a

standardized

testing

protocol

Accordingly

the

test

protocol

used

in

the

follow-up

experiment

-LRB-

Experiment

-RRB-

included

some

modifications

reflecting

the

experience

gained

in

Experiment

In

combination

the

current

multi-site

study

aimed

at

a

characterization

of

the

general

performance

of

the

tadpoles

using

the

optimized

testing

protocol

and

a

detailed

evaluation

of

concentration-dependent

effects

of

on

tadpole

growth

larval

development

and

sexual

differentiation

The

testing

protocol

under

investigation

specified

the

use

of

a

flow-through

exposure

system

to

achieve

exposure

of

tadpoles

The

main

advantages

of

flow-through

systems

versus

static

renewal

systems

are

the

maintenance

of

constant

testing

conditions

-LRB-

e.g.

water

quality

-RRB-

and

the

continuous

delivery

of

the

test

substance

to

the

exposure

vessels

to

achieve

stable

exposure

of

the

test

species

to

the

test

substance

In

the

two

exposure

experiments

with

however

difficulties

were

encountered

with

regard

to

the

maintenance

of

the

target

test

concentration

of

even

under

flow-through

conditions

Although

concentrations

in

the

stock

solutions

were

within

acceptable

ranges

in

both

exposure

studies

and

the

quantity

of

metered

into

the

tanks

was

correct

actual

concentrations

in

the

exposure

tanks

decreased

to

levels

well

below

nominal

test

concentrations

during

the

entire

course

of

the

experiments

-LRB-

Fig.

-RRB-

These

observations

clearly

indicate

that

in

future

studies

care

must

be

taken

to

maintain

constant

nominal

concentrations

of

the

test

compound

throughout

exposure

It

is

recommended

the

flow

rate

of

the

stock

solutions

be

adjusted

on

a

daily

basis

according

to

the

actual

test

substance

concentration

present

in

the

tanks

Comparison

of

the

time-course

of

concentration

changes

with

estimates

of

the

biomass

present

in

treatment

tanks

at

different

times

during

Experiments

and

suggests

that

concentrations

were

inversely

related

to

changes

in

total

biomass

Aqueous

concentrations

decreased

concurrent

with

the

increase

in

body

size

and

body

weight

of

tadpoles

during

premetamorphosis

-LRB-

NF

stages

up

to

-RRB-

prometamorphosis

-LRB-

NF

stages

to

-RRB-

and

climax

-LRB-

to

-RRB-

Concentrations

of

began

to

increase

as

tadpoles

that

completed

metamorphosis

were

removed

from

the

test

tanks

and

as

remaining

tadpoles

began

to

lose

weight

during

tail

resorption

Figure

illustrates

that

concentrations

were

inversely

related

to

changes

in

total

biomass

of

the

tadpoles

Furthermore

it

should

be

noted

that

the

biomass

was

greater

in

Experiment

than

in

Experiment

and

the

decline

in

aqueous

concentration

was

more

marked

in

Experiment

These

observations

combined

with

the

relatively

high

octanol

water

partition

coefficient

of

-LRB-

log

Kow

-RRB-

suggest

that

additional

measures

-LRB-

e.g.

gradual

increase

of

amounts

of

delivered

to

the

test

tanks

-RRB-

are

required

to

maintain

a

constant

exposure

to

in

terms

of

actual

aqueous

concentrations

In

semi-static

systems

disappears

quickly

which

suggests

the

animal

may

absorb

the

Only

few

studies

are

available

that

have

addressed

this

aspect

Results

from

Hayes

and

Licht

-LSB-

-RSB-

suggest

that

exogenous

is

readily

metabolized

by

X.

laevis

and

other

anuran

species

and

there

was

no

evidence

of

bioaccumulation

of

in

anuran

tadpoles

However

metabolic

enzymes

and

the

sites

of

metabolism

in

anuran

tadpoles

are

largely

unknown

Possible

candidate

metabolites

might

include

various

conjugates

but

neither

the

test

solutions

nor

the

test

animals

have

been

examined

analytically

for

the

presence

of

such

metabolites

To

the

best

of

our

knowledge

there

are

also

no

data

available

regarding

possible

developmental

profiles

of

capacities

that

might

explain

the

transient

reduction

of

aqueous

concentrations

in

the

present

studies

It

is

further

not

known

whether

any

of

the

putative

metabolites

still

possess

estrogenic

or

other

hormonal

activity

Finally

it

is

possible

that

factors

other

than

changes

in

tadpole

biomass

may

have

contributed

to

the

observed

changes

of

concentrations

For

example

a

small

fraction

of

may

have

been

absorbed

either

in

the

biofilm

or

directly

on

the

surface

of

the

glass

tanks

Another

possibility

could

be

that

partition

from

water

to

fat

in

the

administered

Sera

Micron

feed

-LRB-

fat

-RRB-

occurred

Notably

daily

food

amounts

administered

to

each

tank

were

adjusted

to

total

larvae

biomass

Therefore

it

is

an

interesting

possibility

that

at

least

a

portion

of

the

dose

may

reach

biological

targets

in

the

larvae

as

a

result

of

oral

ingestion

of

the

feed

Fig.

of

changes

in

concentration

and

the

biomass

of

growing

tadpoles

during

the

exposure

period

in

Experiments

and

An

important

objective

underlying

the

development

of

a

standardized

testing

protocol

for

exposure

of

X.

laevis

tadpoles

was

to

maintain

appropriate

water

quality

throughout

the

exposure

experiments

Thus

special

care

was

taken

to

standardize

and

refine

water

quality

parameters

to

ensure

optimal

growth

and

development

of

X.

laevis

tadpoles

Except

for

a

transient

decline

of

DO

concentration

below

desirable

levels

all

water-quality

parameters

were

within

acceptable

limits

The

decrease

of

DO

concentrations

through

dpf

in

Experiment

and

inability

to

maintain

the

DO

concentration

above

may

have

been

due

to

insufficient

cleaning

Based

on

this

observation

the

cleaning

procedure

was

enhanced

and

additional

aeration

was

provided

by

gently

bubbling

air

through

glass

pipettes

in

each

tank

An

interesting

finding

in

these

two

experiments

was

the

effects

on

growth

and

development

of

X.

laevis

tadpoles

that

occurred

due

to

slight

differences

in

rearing

procedures

between

Experiment

and

Experiment

Tadpoles

in

Experiment

required

more

time

to

complete

metamorphosis

and

the

metamorphic

froglets

-LRB-

NF

stage

-RRB-

were

larger

and

heavier

than

those

in

Experiment

Whereas

the

relationship

between

age

and

size

is

in

accordance

with

established

amphibian

life-history

models

-LSB-

-RSB-

the

factors

that

are

responsible

for

the

inter-laboratory

differences

are

not

entirely

clear

in

this

case

In

Experiment

the

photoperiod

was

hours

light

hours

dark

compared

with

hours

light

hours

dark

in

Experiment

The

mean

temperature

was

approximately

°C

lower

and

more

variable

-LRB-

°C

-RRB-

in

Experiment

The

differences

in

photoperiod

and

water

temperature

are

certainly

prime

candidates

to

explain

the

slightly

slower

development

in

Experiment

On

average

tadpoles

in

Experiment

consumed

food

days

longer

than

those

in

Experiment

Subsequently

the

longer

food

consumption

of

tadpoles

in

Experiment

led

to

greater

weight

compared

with

that

of

the

froglets

in

Experiment

Based

on

the

results

achieved

in

experiments

and

and

to

consider

the

reported

differences

on

growth

and

development

of

X.

laevis

in

the

literature

it

is

obviously

important

to

standardize

the

test

systems

Results

from

Experiment

indicated

that

most

of

the

tadpoles

-LRB-

-RRB-

from

the

negative

control

group

completed

metamorphosis

within

a

relatively

short

time

-LRB-

days

-RRB-

whereas

metamorphosis

was

delayed

-LRB-

days

-RRB-

in

a

few

tadpoles

only

-LRB-

-RRB-

In

contrast

time

to

metamorphosis

in

Experiment

was

much

more

variable

because

-LRB-

-RRB-

of

the

tadpoles

completed

metamorphosis

within

twenty-two

days

and

-LRB-

-RRB-

in

thirteen

days

Furthermore

tadpoles

in

Experiment

completed

metamorphosis

later

than

in

Experiment

-LRB-

Fig.

-RRB-

Thus

the

use

of

the

improved

testing

protocol

in

Experiment

was

associated

with

a

reduction

in

the

time

needed

for

completion

of

metamorphosis

which

resulted

in

much

more

homogenous

development

of

the

test

organisms

The

latter

finding

is

particularly

important

given

the

fact

that

still

only

few

data

are

available

upon

which

one

could

build

a

set

of

performance

criteria

to

assess

the

validity

of

exposure

experiments

with

X.

laevis

tadpoles

Homogenous

growth

and

development

in

the

negative

control

group

is

certainly

one

important

aspect

that

deserves

special

attention

Generally

it

can

be

proposed

that

homogenous

rates

of

tadpole

growth

and

development

will

reflect

appropriate

culture

and

testing

conditions

Furthermore

any

reduction

in

the

inter-individual

variability

of

growth

and

development

will

enable

more

sensitive

assessment

of

the

possible

effects

of

a

test

compound

on

the

development

of

tadpoles

To

this

end

the

robust

demonstration

of

altered

rates

of

development

due

to

test

compound

exposure

will

also

provide

additional

information

for

interpretation

of

the

effects

of

the

test

compound

on

gonadal

development

-LRB-

e.g.

identification

of

systemic

toxicity

-RRB-

Fig.

of

male

and

females

frogs

-LRB-

n

-RRB-

of

the

negative

control

groups

completing

metamorphosis

during

the

course

of

the

studies

based

on

the

total

number

of

animals

surviving

in

Experiments

and

In

contrast

it

is

still

difficult

to

assess

the

validity

of

an

exposure

experiment

based

on

the

time

required

for

completion

of

metamorphosis

According

to

Nieuwkoop

and

Faber

-LSB-

-RSB-

the

average

time

to

reach

stage

is

dpf

In

Experiment

the

average

time

to

stage

was

still

shorter

being

dpf

and

for

female

and

male

frogs

respectively

This

reduction

of

the

time

needed

for

tadpoles

to

complete

metamorphosis

is

likely

a

result

of

the

various

efforts

to

optimize

an

array

of

biotic

and

abiotic

factors

known

to

affect

tadpole

growth

and

development

An

important

element

of

experimental

approaches

used

to

assess

the

estrogenic

activity

of

chemicals

is

the

choice

of

an

appropriate

positive

control

treatment

to

confirm

the

validity

of

the

experimental

system

Therefore

a

major

focus

of

the

current

multi-site

study

was

to

establish

a

concentration

response

relationship

for

treatment

on

sexual

differentiation

of

X.

laevis

The

results

from

Experiment

were

not

appropriate

to

demonstrate

this

relationship

because

all

concentrations

-LRB-

and

μg

L

-RRB-

employed

in

Experiment

caused

a

very

high

percentage

of

phenotypic

females

Based

on

these

observations

the

test

concentrations

were

modified

in

Experiment

and

the

use

of

concentrations

of

and

μg

L

revealed

a

clear

concentration-dependent

increase

in

the

number

of

phenotypic

females

The

data

from

Experiment

allowed

the

calculation

of

an

of

μg

L

resulting

in

gonadal

female

phenotype

Literature

information

about

concentration-dependent

effects

on

growth

development

and

sexual

differentiation

in

X.

laevis

is

rare

In

most

studies

single

concentrations

ranging

from

to

μg

L

were

used

to

study

effects

on

gonadal

development

-LSB-

-RSB-

and

resulted

in

percentage

of

female

phenotype

between

and

Hu

et

al.

-LSB-

-RSB-

applied

and

μg

L

and

reported

an

increase

of

female

phenotype

to

and

respectively

Rearing

X.

laevis

larvae

under

optimized

conditions

in

Experiment

demonstrated

a

distinctive

sensitivity

for

gonadal

differentiation

This

is

demonstrated

by

a

clear

concentration-responsive

effect

and

a

distinct

effective

concentration

of

μg

L

as

shown

by

the

frequency

of

female/male

individuals

and

the

occurrence

of

substantial

mixed

sex

and

other

abnormalities

Comparing

the

results

of

Experiments

and

regarding

gonadal

effects

of

and

μg

L

a

similar

effect

was

observed

indicating

high

reproducibility

of

the

testing

protocol

Based

on

the

endogenous

concentrations

measured

in

adult

X.

laevis

being

approximately

ng

mL

for

females

and

ng

mL

for

males

-LSB-

-RSB-

and

in

froglets

approximately

μg

body

weight

-LSB-

-RSB-

the

calculated

appears

to

represent

a

physiological

concentration

in

X.

laevis

Another

question

of

high

interest

is

how

estrogenic

EAC

might

impact

gonad

gross

morphology

While

determination

of

phenotypic

sex

was

a

core

endpoint

in

the

assessment

of

the

effects

of

on

sexual

differentiation

a

number

of

other

gross

abnormalities

were

also

evaluated

To

that

end

a

glossary

of

gonadal

gross

morphological

terminology

and

criteria

was

included

in

the

study

protocol

-LRB-

Table

-RRB-

The

importance

of

this

glossary

can

not

be

understated

as

a

substantial

degree

of

diagnostic

variability

within

and

among

studies

can

be

attributed

to

differences

in

terminology

-LSB-

-RSB-

For

example

the

word

intersex

is

characterized

differently

by

several

authors

and

is

often

used

interchangeably

with

the

term

mixed

sex

whereas

in

the

glossary

these

terms

have

different

meanings

Therefore

it

is

difficult

to

describe

morphological

changes

triggered

by

EAC

in

a

way

that

is

clear

and

generally

accepted

Furthermore

assessments

of

gonadal

morphology

involve

subjective

judgement

which

make

studies

difficult

to

interpret

and

compare

In

the

current

multi-site

study

the

uncertainty

arising

from

the

subjective

judgement

that

was

necessarily

involved

in

evaluation

of

the

gross

morphological

assessment

each

gonad

was

reduced

by

having

each

finding

verified

by

a

second

biologist

Additionally

digital

photographs

of

each

gonad

were

taken

to

allow

later

verification

of

assessments

and

to

allow

measurement

of

gonadal

image

area

using

image-processing

software

Measurement

of

gonad

image

area

enables

objective

verification

of

some

of

the

subjective

size-related

observations

made

during

the

gross

assessments

Trend

analysis

revealed

for

both

sexes

in

Experiment

an

increase

of

mean

gonadal

area

that

coincides

with

an

increased

age

at

completion

of

metamorphosis

In

both

experiments

gross

morphological

evaluation

of

the

gonads

revealed

the

presence

of

mixed

sex

segmental

aplasia

and

pearling

at

μg

L

It

is

noteworthy

that

intersex

characterized

as

ovarian

and

testicular

tissue

in

the

same

individual

as

separate

gonads

-LRB-

left/right

-RRB-

was

not

found

in

any

of

the

frogs

evaluated

in

these

two

experiments

Interestingly

in

both

experiments

the

highest

incidence

of

these

testicular

abnormalities

was

observed

in

tadpoles

exposed

to

μg

L

This

was

most

likely

due

to

the

greater

degree

of

complete

sex

reversal

at

the

two

higher

concentrations

as

suggested

by

the

significantly

increased

proportion

of

phenotypic

females

compared

with

phenotypic

males

in

the

and

μg

L

treatment

groups

Based

on

gross

morphological

evaluation

no

differences

for

the

terms

of

main-categories

such

as

hyper

or

hypoplasia

were

observed

compared

with

individuals

in

the

negative

control

in

Experiment

In

conclusion

this

multi-site

study

has

developed

and

established

an

optimized

study

design

and

a

standardized

test

protocol

to

determine

optimal

growth

and

development

of

X.

laevis

larvae

High

survival

rates

homogenous

rates

of

development

relatively

short

times

to

completion

of

metamorphosis

of

control

animals

and

concentration-dependent

effects

of

treatment

on

gonadal

development

were

achieved

using

appropriate

testing

conditions

Based

upon

these

results

it

is

concluded

that

the

protocol

provides

a

basis

for

evaluation

on

potential

effects

of

chronic

EAC

exposure

on

gonadal

development

in

X.

laevis

using

a

flow-through

system

Furthermore

it

is

our

opinion

that

the

protocol

could

also

be

successfully

applied

to

investigate

the

effects

of

other

types

of

EAC

such

as

-LRB-

anti

-RRB-

thyroidal

chemicals

on

the

development

of

X.

laevis

xenopus laevis
development
sexual differentiation

flow-through exposure system
standardized protocol
17β-estradiol