Aquatic leeches (Hirudinea) as bioindicators of organic chemical contaminants in freshwater ecosystems

Chemosphere, Vol.13, No.l, Printed in G r e a t B r i t a i n

pp

143

-

AQUATIC LEECHES

150,.1984

(HIRUDINEA)

OF ORGANIC CHEMICAL CONTAMINANTS J.L. Metcalfe*,

0045-6535/84 $ 3 . 0 0 + .OO ©1984 Pergamon Press Ltd.

AS BIOINDICATORS IN FRESHWATER

ECOSYSTEMS

M.E. Fox and J.H. Carey

Canada Centre for Inland Waters 867 Lakeshore Burlington,

Road,

P.O. Box 5050

Ontario,

Canada

LTR 4A6

Abstract Freshwater leeches from an industrially polluted creek were found to contain very high residues of chlorophenols, when compared with fish, tadpoles and most other benthic invertebrates. Leeches are recommended as possible bloindlcators of aquatic organic contamination.

Introduction A wide variety levels

of biological

of aquatic environmental

indicator

freshwater ~ and marine z contaminant served

as bioindlcators

contaminants as

the

age,

temperature,

size,

sex,

the

contamination

collected

water

the bioavailabillty

residue

an

and

aquatic

levels

of

synthetic

"early warning"

Materials

and Methods

before to water

organic

an

creek.

Based

indicators

domestic

study

and industrial

from a disused

of chlorophenol of

compounds

on the

Creek is a minor tributary

intensive

to estimate

by natural

organism

However, is

as

is that

also

levels

variables

well

organisms

detectable

surveys

have

to monitor

as

of

such

season,

can be used

in

to

conventionally

they do not

reflect

chemical

we

A few reports

suggest

that

this

for these compounds.

in leeches

suggest

that

of the Grand River which

waste

dump.

the

effects

and

observed

sewage effluent

of the creek.

fate

leeches 5,/,8,9, potential

results,

contamination

Stream ecology and the identification

in

organisms.

and other leeches

biota

may

from

serve

as

of aquatic organic contamination.

of Study Area

source

the

it

past

fish and macroalgae been used

as indicator

bloaccumulation

Canagagigue

well as leachates

of

drawback

Description

It receives

content

pollutants ~ ,3.

the levels of chlorophenols

polluted

excellent

Erie.

in the

are complicated

leeches have never been proposed

This paper describes industrially

have

ecosystem

class of annelids may have a considerable

an

used

have been recommended for both

teleost

eggs

surveys

lipid

of several

An additional

Certain their

been

molluscs

of contaminants.

To our knowledge, on

in

samples.

and

Indicator

mobility,

coexistence

identify

gulls

have

Bivalve

monitoring.

and herring

in the Great Lakes 3 .

and

organisms

contamination.

of

latter

The results

synthetic

of the contaminants

143

The

in turn empties

from the town of Elmira, appears

presented

organic

as

to be the principal came to light during

contaminants

are discussed

into Lake Ontario,

in

the

creek.

in detail elsewhere ~U .

144

Collection Fish, were

of Biota and Water Samples

crayfish and bullfrog

weighed

fresh,

then

were made in May, July,

tadpoles

frozen

September,

Benthos were harvested

where

they were

fied and sorted live. in concentrated dues.

HCI

and November,

1981.

medium.

analysis.

They

Collections

1980, at six sampling sites along the creek.

were placed

steel forceps,

from the under-

in glass Jars and transported

at 4°C.

The following

morning

on ice to

they were

identi-

mL acid)

for storage

prior to analysis

for chlorophenol

resi-

1980, and the rest between May

Several sites were sampled on each occasion. sampled several days prior to each biota collection

In 1980,

then acidified

that 2½ L of water

electroshocker.

for later

of benthos were made - one in November,

! L amber glass

pellets of potassium hydroxide 4°C,

foil

Samples of each type were weighed wet to the nearest 0.01 g and dissolved

Water was generally turbed

Organisms

held overnight

(I g tlssue/7

Eight collections

and December,

using a portable

aluminum

by hand, or with the aid of stainless

sides of rocks on the creek bed. the laboratory

were collected

in pre-cleaned

to pH i-2 just were

bottles with

had been previously

collected

prior and

teflon lined screw caps were used.

added to each bottle.

to analysis.

12 pellets

to ensure an undis-

Procedures

of sodium

Five

Samples were stored at

in 1981 were similar

hydroxide

were

except

used as a preserva-

tive.

Analysis

of Chlorophenol

Residues

in Water and Biota

Water The acidified water samples were extracted with 40 mL pesticide a separatory with

funnel.

The

2x30 mL toluene.

toluene

layer was

The combined

40, 30 and 30 mL of 0.I M K2CO 3 made up with were

placed

grade was

in a 125 mL erlenmeyer

hexane

shaken

and

the addition

acetic

The hexane

final volume

organic

the aqueous

were

anhydride

layer was extracted

then back extracted

free water. lined

were

The

added

and

grade

centrifuge

twice

times with

KzCO ~ extracts

Ten mL of pesticide

the tightly

capped

flask

pipet and evaporated

tube with a stream

Iso octane as a keeper.

three

combined

screw cap.

layer was removed using a pasteur

in a graduated

of 4 mL of pesticide

and

flask with a teflon

1 mL of redlstilled

for one hour.

an appropriate

removed

toluene extracts

grade toluene by shaking in

of dry nitrogen

This is a modification

to

after of the

method of Chau and Coburn i~ . The

acetylated

chromatograph lary

column

analyses,

extracts

were

using a Dani 33S ~ N i and

appropriate

where required,

analysed

for

chlorophenols

on

a Hewlett

Packard

EC detector and a 25 m x 0.2 mm ID HP OVI fused

standards.

Reproducibility

were performed

was

+10%

or

better

and

5700

gas

SiO 2 capilconfirmatory

on an OVl01 column.

Biota The hexane

acidified

(volume

equal volume

of organic

up and analyzed to remove dried

biota

depending

llplds

by passing

solutions on sample

free water.

as for the water which

were size),

Extracts

with

pesticide

three

and the combined

samples.

interferred

it through

extracted

times

10-20

extracts

mL

pesticide

were washed

were back extracted with 0.I M K~CO~,

Some biota samples the analysis.

grade

with

hexane

required

In these

Na2SO ~ previously

cases,

fired

a further

then worked

clean-up

the washed

at 500°C.

grade

with an

The

extract

step was

dry hexane

145

extract was passed through a disposable mini column consisting of an 8 mm I.D. diameter pasteur pipet packed with 8 cm of 40% H~SO~ on precleaned

and dried

chromatographic

silica gel.

The

eluant was collected and a further 30 mL of hexane was passed through the column and added to the eluant.

Oxidized lipid material, which remained on the column, was discarded.

The combined

eluant and hexane rinse were then back extracted with 0.i M K2CO ~ and the procedure continued as described above.

Results Although

many

biota

collections

were

obtained

during

1980

and

1981,

not

(including leeches) were represented at each site on each sampling occasion.

all

organisms

Therefore, we have

presented a selection of data which provides comparisons among the widest range of leech species and other aquatic organisms. The following chlorophenols were identified in the water and/or biota: (2,6-DCP);

2,4-dichlorophenol

(2,4,6-TCP);

2,4,5-trichlorophenol

pentachlorophenol in

poor

(PCP).

recoveries

Therefore,

(2,4-DCP);

of

3,4-dichlorophenol

(2,4,5-TCP);

(3,4-DCP);

2,6-dichlorophenol

2,4,6-trichlorophenol

2,3,4,6-tetrachlorophenol

(2,3,4,6-TTCP),

and

The lipid removal procedure, required for some biota samples, resulted 2,6-DCP

and

2,4-DCP

(50-60%)

and virtually

complete

loss

of

3,4-DCP.

data for these three chlorophenols will not be reported for those samples subjected

to lipid removal. Table i shows that leeches from site CN-3 (1.7 km below the contaminant source) accumulated chlorophenols

to levels

water concentrations.

ranging

from 40,000 x (2,4-DCP)

By contrast,

to 140,000 x (2,4,5-TCP)

levels in other organisms

maximum of 3000x the average water concentration for 2,6-DCP in rock bass. that

several

other

fish

species,

darters were also analyzed. Table

2 presents

including

white

sucker,

the average

ranged from not detectable

common

It should be noted

shiner,

longnose

dace,

Levels of chlorophenols

Leeches

accumulated

at site

CN-5,

7.4 km below

the contaminant

in the water here are approximately one-third of those at CN-3.

chlorophenols

to levels generally

one

to two orders

than all other benthic invertebrates, except aquatic worms.

of magnitude

greater

Of the seven chlorophenols present,

2,4-DCP, 2,4,5-TCP and 2,4,6-TCP tended to accumulate to the highest levels in the biota. also

present

at

Table 3 compares CN-31 tion.

and CN-32,

and

All had residue levels lower than those in rock bass ~0.

data from four collections

input.

were

to a

the

highest

the residue

which are

1.7,

concentrations

levels of four species

in

the

water

of leeches

3.3 and 4.2 km, respectively,

on

most

collected

They

occasions.

at sites

CN-3,

below the source of contamina-

Dine dubia accumulated the highest levels of all chlorophenols on all occasions with the

exception of 2,6-DCP in June and PCP in November. the lowest levels.

Glossiphonia complanata generally accumulated

Again residues of 2,4-DCP, 2,4,5-TCP and 2,4,6-TCP were the highest.

Discussion This study shows that leeches accumulated industrially polluted creek.

very high levels of seven chlorophenols from an

These levels were one to two orders of magnitude greater than the

levels found in thirteen other benthic invertebrates as well as fish and tadpoles. oligochaetes,

which

are

a

class

of

annelids

closely

related

to

leeches,

had

Only aquatic comparable

1g.58 3.80

B u l l f r o g tadpole (Rane cetesbeiana) .306 (.495)

ND4

5

116

12267

2,4-DCP

.133 [,211)

ND

1

NO

6064

.194 (.209)

4 ND - not detectable

10

10

7

13588

2,4,6-TCP

.087 (.128)

31

3

48

11883

2,4,5-TCP

Chlorophenol concentration (ppb) l 3,4-0CP

Dine dubla (predominant), Glosslphonia complanata~ Helobdeila stagnalis

.082 (.116)

84

292

258

5350

2e6-OCP

3 average of four samples collected on May 23p July 8, September 10, and November 12p 1980

2 several species combined:

ng/g wet weight f o r biota; ~g/L for water

Water - annual mean3 ( s . d . )

25°66

Rock Bass ( A m b l o p I I t ~ r u p e s t r l s )

Crayfish (Orconectes proplnquus)

2.40

Wet wgt. (g)

Chlorophenol levels In biota and water at s l t e CN-3 on November 13, 1980

Leeches2 (Hlrudinea)

Sample

Table 1°

.020 (.024)

ND

5

9

1380

2,3,4,6-TTCP

.036 (.031)

19

1

10

2671

PCP

==

Chlorophenol

levels

ND1 - not d e t e c t a b l e NA2 data not a v a i l a b l e ;

5.37 .45 .17 .40 .42 4.01 1.95 2.28

.21 2,12

June 2,

1981

355 561 119 5 10 ND 29 8 118 ND 14 18 29 19 303 185 11 I 76 90 .037 .067

$25 175 1288 270

430 485

710 1110

lipid

removal procedure

NA NA NA 16 14 NA NA NA 21 5 NA NA NA 18 8 NA NA NA 30 9 NA NA NA ND ND NA NA NA 1t 7 NA NA NA ND 2 NA NA NA 2 ND .OlD .119 .027 ,053 .041

50 512

250 10 3 NA NA NA NA NO ND I0 .016

4524

September 30, 1981

8~ 588 ND ~ 41 ND ND NA~ NA NA NA NA NA NA NA 109 124 ND 14 NO 38 ,025 ,015

1515 2456 855 5715

6

6 I0 14 33 ND 5 16 ND ,006

66 96

9 24 59 20 7 4 19 ND .004

18 51

45 8 68 .006

33

120 I 51 .004

$14 25 6 18 34 15

834

37 119 15 t39 77 $6

583

Chloropbenol c o n c e n t r a t i o n (ppb) 2t6 2e43,42~4,62t4r52t3f4t6F~5P

samples subjected t o

A q u a t i c Worms (Ollgochaeta) Snails (Physa s p . ) MayfJy larvae (Ephemeroptera) Dragonfly larvae ( A n i s o p t e r a ) Oamselfly larvae (Zygoptera) F i s h f l y larvae ( N J g r o n l a s p . ) C a d d i s f l y larvae (Hydropsych|dae) Water pennies (Psephenidae) C r a n e f l y larvae ( T i p u l l d a e ) Water

Leeches (Dine dubla) Leeches (l~'~o-sslphonla complanata) Leeches (Haemopsls 9 r a n d l s )

.53 .80 ,35 .40 .16 ,44 I.07 5.50 .20 1.70

.50

Wet wgt° (~)

in b i o t a and water a t s i t e CN-5 on f o u r occasions

A q u a t i c Worms (Oligochaeta) Limpets ( F e r r l s s l a s p . ) F i n g e r n a i l Clams ( 5 p h a e r l l d a e ) Dragonfly larvae ( A n l s o p t e r a ) Damselfly larvae (Zygoptera) F l s h f l y larvae ( N l q r o n l a s p , ) C a d d l s f l y larvae~l~y~-cr~6-psycbldee) C a d d i s f l y larvae (Pycnopsyche s p . ) D i v i n g b e e t l e s (Agabus s p . ) C r a n e f l y larvae (TIp-p-~ldae) Water

Leeches (Dine dubla) Leeches (~-p-o-b~-eTTa-puncteta)

Sample

Table 2. July 9,

1981

1356 1046

NA

1128 7 25 ND 24

894 36 27 2 46 I I .015

NA

NA NA NA NA NA

26

NA

4.65 1.97 3,72

NA NA NA NA NA

22

NA NA NA NA NA

.54 5.11 ,68 1.64 1,53

4917 640

t981

1529 221

November 4, 4817 550 226 26

NA NA NA NO NA NA NA ND .007 .017 .O06 .014

160 50

.036

756 1107

.013 .005 .004 .011

NA

267 545

34 NA

869 932

67

NA

152 968

ND I .003

3

124 2 4 ND 24

119 21

.001

25

122 113

IO I .003

4

180 I 12 64 3

35 1

,002

15

149 i23

Chlorophenol c o n c e n t r a t i o n (ppb) 2,6 2f4 3t4 2 e 4 f 6 2 f 4 f 5 2 e 3 e 4 f 6 PCP

.08 1.22

.64

.27 4.20

Wet wgt. (~)

Water

Glosslphonla complanata

.21

.19

Helobdella stegnells

Erpobdella punctata

.10

Wet *91. (9)

Dine duble

Leech species

Water

.227 .182

.112

645 .035

2298

3,4

.055

7

117

.166 .048

902

NO SAMPLE

ND 1562 336

22

.039 .075

158

1086 934

1.56

.83

.45

2.59

.065

639

371

.085

1688

2461

19

72

188

.007 .005

100

140

508

.34

.45

1.14

705

102

1188

4559

.168

.362

1339 4140

834

250

~

903

172 .255 1.52 .321

153

NO SAMPLE

ND 1829 418

168 4299 584

2,4

.015

335

151

290

.034

167

258

1275

1313

.443

467

356

.723

1781

2253

1225 3297

.035

45

79

105

.024

5

39

36

21416 2~415 21314p6 FCP

CN-3 November 4, 1981

.122 .083 .111

36

790 1162 637

2112 1947 748

720

2r416 2r415 213r4r6 PCP

958 8196 2488 3620 8545

2w4 ~

CN-3t June 2, 1981

214r6 2r415 2p31416 R3P Wet wgt. (g) 216

4915 1331 2201 10262

2,4

CN-3 September 30p 1981

249

2,6

.144

156

806

173

Glosslphonle complanata

4.92

NO SAMPLE

Erpobdella punctata

1519 10619 3410 6674 15308 NO SAMPLE

1.05

Chlorophenol concentration {ppb) 2r6 214 3r4 214r6 2r415 2131416 PCP Wet w~t. (g) 2,6

Helobdelle stagnalls

Dine dubla

Wet wgt. (q)

CN-32 May 5, 1981

Chlorophenol levels in leeches and water at s i t e s CN-3, CN-31 and CN-32

Leech species

Table 3.

p= ¢= co

149

residues.

Annelids

excreting leech

these

Dina

dubia

chlorophenols another post

are

primitive

compounds.

In

collected

within

annelid,

from

a two-week

the marine

exposure.

In

organisms

preliminary site

CN-3

period

the

may

be

did

not

life

for

we

eliminate

of

found any

Similarly

worm Nereis virens,

half

incapable

tests,

in clean water.

polychaete

contrast,

and

laboratory

metabolizing

that

of

their

McLeese

did not excrete

pentachlorophenol

in

and/or

specimens body

of

the

burden

et al ~2

found

of that

PCBs during 26 days'

goldfish

is

about

i0

hours ~B Leeches synthetic

have

Erpobdella

from

punctata

been

found

compounds.

punctata

collected

were

areas

tissues

tissues

also

organic

among

treated

and

copepods.

Basin

laboratory

tests, Glossiphonia

had 8.1

maximum

accumulation

factors

for the

of 2.4.

There

level

an

Phillips b .

However,

This

14.8

other

Leeches

present can

reasonable

0.20

for

are

easily

under

promising

They are both

high

cally

as a

adequate

We

have

data.

hardy

analyzed

Leeches

and

fish

of DDT in E.

Glossiphonia

DDT concentration

the

other

no residues were found in the

leech

water 9.

In

atrazine

comparison,

Prodlamesa

sta~nalis,

sp.

from

the

of the clam Pisidium.

to 200 ppb of the herbicide in

for

invertebrates

the presence

In

reached a

concentration

olivacea were

2.6 and

also had a low concentration potential

to

consider,

purposes.

besides

These

of leeches

varies

without

tissue

zones

conditions.

sensitive

areas. and

samples

as

small

as

g

A

not

two

to

easily

three

man-hour

damaged

during

effort

has

handling.

by

high

being killed.

In compari-

and therefore,

representa-

Most

for

when

review

they accumulate

of streams,

The average

0.15

in a

lakes,

Sawyer TM states

species.

for analysis.

potential,

addressed

reasons:

sedentary

in the littoral polluted

clean-water

been

for the following

they are relatively

and

bioaccumulatlon

have

leech

and ponds

that

no

species

are

sizes of Canagagigue

leech of

a

Creek

- 0.01 g, and G. complanata chlorophenols,

are easy to sample as they are most abundant

species

and Helobdella

study

bioindieators

has

Dina dubla appears

potential about

and

obtained

in the shallowest,

yielded

50

Therefore,

most

to

200

specimens.

they

are

ideal

to be the best bloindicator

chlorophenols. little

is

are all common

However,

known.

that

contaminantion.

involving other contaminants

aquatic

leeches

Their

limitations

and other previously

deserve will

accepted

on the basis

it is an uncommon,

Erpobdella

and widespread

among areas which are widely separated

demonstrated

of organic

for

which

st a~nalis

use would permit comparisons

studies

potential

of mirex in the leech

for

uptake and depuration experiments.

restricted

This

the

D. dubia - 0.I0 g, E. punetata - 0.15 g, H. sta~nalls

bloaceumulation

complanata

in

that the bioaccumulation

abundant

pristine

Of the four leech species, its

that

Helobdella

candidates

insects

yielding

are

laboratory

found

In contrast,

found

such as DDT, mirex and chlorophenols,

classified

accessible

Leeches

factors

biomonltoring

be

g.

meaningful

ppm)

and the chirouomid

leech,

our finding

size,

leeches are: -

1.76

Webster 7 reported

that

fluviatilis

important

organism

tive of the study area.

species

to

tissue

times

another

son with fish and most aquatic

are

D'Eliscu 8

the average

supports

levels of contaminants

and

(up

chemical.

eomplanata exposed

of

bioaccumulation

to species. are

selecting

times

limpet Anc[lus

respectively.

from species

the

three months after aerial spraying.

of amphipods

factor

a considerable

to Naqvl and de la Cruz 5, levels

the highest

with

Lake Tahoe

5.5,

to have

According

punctata,

of

geographi-

Glossiphonia

in North America

TM

and their

as

potential

geographically. consideration be determined sentinel

by comparative

organisms.

150

Acknowledgements The technical assistance provided by P.A. Coletta is gratefully acknowledged.

J. Madill of

the Invertebrate Zoology Division, Museum of Natural Sciences, National Museums Canada, kindly identified the leech species.

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