FRESHWATER TOOL

LEECHES

FOR DETECTING

(HIRUDINEA) ORGANIC

AS A SCREENING CONTAMINANTS

IN THE ENVIRONMENT J. L. M E T C A L F E ,

M. E. F O X , and J. H. C A R E Y

National Water Research Institute, Canada Centre for Inland Waters, Environment Canada, Burlington, Ontario, Canada

(Received May 1988) Abstract. In earlier work, we found that leeches from an industrially polluted creek bioaccumulated

chlorophenols to much higher concentrations than other resident benthic invertebrates and fish. We suggested that leeches may have significant potential as biomonitors for these and other organic contaminants in the environment. In this study, we compared the bioaccumulation and depuration of 16 organic compounds, including eight chlorophenols (CPs), lindane, DDT and four derivatives, benzothiazole (BT) and 2-(Methylthio)benzothiazole (MMBT) for three species of leeches. Dina dubia had the highest bioaccumulation capacity for most contaminants, but residues persisted longest in Erpobdella punctata. Helobdella stagnalis appeared capable of degrading some compounds. Half lives of CPs, DDT and DDT derivatives were generally longer than one month. In contrast, half lives were only 1 day for lindane, 1-2.5 days for MMBT and 7 days for BT despite very high initial tissue concentrations of the latter two compounds. Bioconcentration factors for contaminants in leeches were higher than those reported for other aquatic organisms. Half lives for lindane, DDT and DDT derivatives were consistent with the literature for other organisms, but half lives for CPs were much longer. The results suggest that leeches would be excellent biomonitors of both continuous and intermittent contamination of a waterway with CPs and DDT, as they retain these compounds for long periods after exposure. Their usefulness as a screening tool for lindane and benzothiazoles would be limited to chronically contaminated environments.

1. Introduction Aquatic organisms which bioaccumulate high concentrations of certain contaminants in their tissues m a y be used as biomonitors to determine the occurrence and levels o f these contaminants in aquatic environments. This type of biomonitoring, labelled 'bioanalysis' by Butler et al. (1985), offers several advantages over traditional water and sediment monitoring surveys. Because organisms concentrate contaminants f r o m surrounding media, they can serve as early warning indicators - demonstrating the presence of contaminants long before they reach detectable levels in the water. I f contaminant levels are measureable in biota, it follows that spatial and temporal trends m a y then be quantified. Biomonitoring also provides a direct measure of the bioavailability of contaminants which cannot be determined by water or sediment analyses alone, but which is an important aspect of risk assessment. Finally, bioanalysis m a y actually be more cost-effective. Organisms integrate ambient pollution conditions over time, and are therefore more representative of environmental quality than are instantaneous samples of water or sediment. Sampling frequency can therefore be reduced without sacrificing accuracy, resulting in considerable savings in terms of time, effort and cost. Environmental Monitoring and Assessment 11: 147-169, 1988. 9 1988 Kluwer Academic Publishers. Printed in the Netherlands.

148

.1. L. METCALFE ET AL.

Organisms which eliminate contaminants slowly offer further benefits as biomonitors. Theoretically, the slower the elimination rate of the organism, the less frequently it must be sampled. In environments where contamination is intermittent, either due to spills or sporadic discharges, organisms with slow elimination rates will provide evidence of the pollution event long after the fact. In an earlier survey for the occurrence of chlorophenols in aquatic organisms from an industrially polluted creek (Metcalfe et al., 1984), we found that leeches accumulated chlorophenol concentrations that were up to two orders o f magnitude higher than those in other resident macroinvertebrates and fish. We suggested that leeches may have significant potential as biomonitors for these and other organic contaminants in the environment. In the present study, we report the bioaccumulation capacities and elimination rates for a variety of chlorophenols, neutral organochlorines and non-chlorinated neutral compounds for three leech species. Our results are compared with the literature on the bioaccumulation and elimination of organic contaminants by other aquatic organisms.

2. Materials and Methods

2.1.

LOCATION

OF STUDY AND CONTAMINANTS

INVESTIGATED

Canagagigue Creek is a minor tributary of the Grand River, which in turn empties into Lake Erie (Figure 1). Four major types of synthetic organic contaminants occur in the creek (Carey et al., 1983), all of which originate in the Town of Elmira, Ontario. Benzothiazoles, which are manufactured in Elmira and are used in the vulcanization of rubber goods and in automobile antifreeze, and lindane, which is used in the formulation of pesticides, both enter the creek via the municipal sewage treatment plant. Chlorophenols and DDT and its derivatives enter the creek as groundwater seepage from a disused chemical waste disposal area. Some of these buried wastes are related to the manufacture of 2,4-D and 2,4,5-T which were produced in Elmira until 1969. Sixteen individual compounds, which represented a wide range of the two major chemical properties which influence bioaccumulation (Table I), were investigated. The study site, known as CN-3 in earlier reports (e.g. Carey et al., 1983), was located approximately 1.5 km downstream from the town. Leeches are abundant due to nutrient enrichment from domestic sewage and very high concentrations of chlorophenols in resident leeches were previously reported (Metcalfe et al., 1984). 2.2.

EXPERIMENTAL

DESIGN

AND PROCEDURE

Contaminated leeches were collected from Canagagigue Creek site CN-3 on October 15, 1984. The stream temperature was 15.5 ~ Specimens were harvested by hand from the undersides of rocks in the quiet areas, and with the aid of a Surber sampler in the riffles. A sampling effort of eight man-hours yielded 55 Erpobdella punctata, 101 Dina dubia (both F. Erpobdellidae) and approximately 750 Helobdella

LEECHES AS A SCREENING TOOL FOR ORGANIC CONTAMINANTS

149

ONTARIO

•• /~ \,

"----"~-~,

LAKE

ONTARIO

.A,,.LToN

GL,..

~,,

\ - - - ~ - - ~ \~,~._~,~

Fig. 1.

Location of study. (Shaded areas are population centres.)

stagnalis (F. Glossiphoniidae). Leeches were placed in glass jars containing creek water, and transported on ice to the laboratory where they were held overnight at 4 ~ to clear their gut contents. No mortality occurred. To compare the initial tissue concentrations of contaminants in leeches with exposure levels in their environment, creek water was also sampled. For the analysis of chlorophenols, lindane and D D T and its derivatives, triplicate 1L filtered water samples were collected; contaminantfree potassium hydroxide pellets were added to the samples on site to preserve them at a p H of 11.0 prior to extraction and analysis. For benzothiazole analysis, three 4.28 L filtered water samples were collected and passed through Sep-Paks (C18 concentration cartridges, Water Associates) on site, then held at 4 ~ until they could be processed. For logistic reasons, water sampling was conducted two weeks later than biota sampling. Although concentrations of contaminants in the water are known to vary seasonally, with m a x i m u m concentrations occurring in the spring, changes over the two week period were not expected to be significant. Chlorophenol concentrations reported in this study were within an order or two of those observed for creek water from site C N - 3 in September, 1980 (Carey et al., 1983) and September, 1981 (Carey et al., 1984) and are therefore considered to be representative of the fall season. Concentrations of benzothiazoles varied within an order or two

J. L. METCALFEET AL.

150

TABLE I Chemical properties of the contaminants investigated Compound

Abbreviation Used in Text

Solubility in Water (ppm)

Benzothiazole derivatives: 2-(Methylthio)benzothiazole Benzothiazole

MMBT BT

Chlorophenols: 2,6-dichlorophenol 2,4-dichlorophenol 3,4-dichlorophenol 2,4,6-trichlorophenol 2,3,6-trichlorophenol 2,4,5-trichlorophenol 2,3,4,6-tetrachlorophenol Pentachlorophenol

2,6-DCP 2,4-DCP 3,4-DCP 2,4,6-TCP 2,3,6-TCP 2,4,5-TCP 2,3,4,6-TECP PCP

Lindane

Lindane

DDT and its derivatives: DDT DDD DDE

p, p'-DDT

.00556

p', p'-DDD; o, p'-DDD p',p'-DDE; o, p'-DDE

.0206 .0146

1001 > 1001

Octanol/Water Partition Coefficient (Kow)

10002 1002

62003 4503

49004

9503 2003 103

52004

7.86

102 0004' 52007

1000 0005,8 1050 0005 5500005,8

1 R. F. Platford (pers. comm.); 2 Brownlee et al. (1981); 3 Jones (1981); 4 Leo et al. (1971); 5 Kenaga and Goring (1978); 6 Weil et al. (1974); 7 K. L. E. Kaiser (pers. comm.); 8 Chiou et al. (1977).

to t w e n t y f r o m values r e p o r t e d f o r S e p t e m b e r a n d N o v e m b e r , 1980 ( C a r e y et al., 1983). T h e r e are no fall d a t a a v a i l a b l e f r o m p r e v i o u s y e a r s with which to c o m p a r e t h e l i n d a n e a n d D D T c o n c e n t r a t i o n s r e p o r t e d in this study. D e p u r a t i o n e x p e r i m e n t s were c o n d u c t e d in d u p l i c a t e for each species. Six 10 L glass a q u a r i a , each c o v e r e d with a t i g h t l y - f i t t i n g lid which e x c l u d e d light, were used. C o n t a m i n a n t - f r e e water was s u p p l i e d to each a q u a r i u m at a flow rate o f 500 m L m i n - t , allowing 99~ r e p l a c e m e n t in a p p r o x i m a t e l y 1.5 h. T e m p e r a t u r e r a n g e d f r o m 13.0 to 14.0 ~ for the d u r a t i o n o f the e x p e r i m e n t , p H was 7.9 a n d dissolved o x y g e n c o n c e n t r a t i o n s were at s a t u r a t i o n levels. Several pieces o f clean l a b o r a t o r y g l a s s w a r e were p l a c e d in the a q u a r i a to p r o v i d e a t t a c h m e n t sites for the leeches. T h e e x p e r i m e n t c o m m e n c e d a p p r o x i m a t e l y 24 h a f t e r the leeches were collected. D . d u b i a specimens were weighed i n d i v i d u a l l y , d i v i d e d into two e q u a l g r o u p s , a n d p l a c e d in d u p l i c a t e a q u a r i a . C a r e was t a k e n to ensure a similar size d i s t r i b u t i o n o f a n i m a l s in each d u p l i c a t e . Nine very small D. d u b i a were n o t used. E. p u n c t a t a s p e c i m e n s were d i s t r i b u t e d in the s a m e m a n n e r using all 55 i n d i v i d u a l s . A l l 750 H . s t a g n a l i s specimens were e q u a l l y d i s t r i b u t e d b e t w e e n their t w o a q u a r i a in r a n d o m b a t c h e s o f ten, as t h e r e was little size v a r i a t i o n a m o n g individuals; t w e n t y leeches o f this species f r o m each a q u a r i u m were weighed i n d i v i d u a l l y for c o m p a r i s o n with

LEECHES AS A SCREENING TOOL FOR ORGANIC CONTAMINANTS

151

TABLE II Initial live weights of leeches Species

Aquarium

Sample Size

Mean Weight (g)

Range

s.d. as ~ of Mean

Dina dubia

DD1 DD2

46 46

0.071 0.072

0.017-0.156 0.024--0.233

41% 54o70

Erpobdella punctata

EPI EP2

28 27

0.191 0.209

0.0424).517 0.050-0.445

62~ 56%

Helobdella stagnalis

HS1 HS2

20* 20*

0.0068 0.0085

0.0047-0.0091 0.0043-0.0132

19% 28%

* Subsample of approximately 375 specimens per aquarium.

the other two species. The initial live weights for each species are presented in Table II. Leeches were allowed to depurate their body burdens of organic contaminants for a period of 27 d in the laboratory. They were not fed. Samples of leeches for contaminant analysis were taken on days 0, l, 2, 5, 8, 13, 21 and 27, each sample consisting of 6 D. dubia, 3 E. punctata or 40 H. stagnalis from the appropriate duplicate aquarium. These numbers were determined by the minimum amount of material required for analytical accuracy and precision (ideally 0.5 g, not less than 0.15 g). A range of organism sizes, determined visually, was selected on each occasion. Initial (Day 0) samples were taken prior to distributing the test organisms among the aquaria. The organisms in each sample were weighed individually (with the exception of H. stagnalis which were weighed in batches of 10) before being combined, wrapped in pre-cleaned aluminium foil, and stored frozen prior to analysis. 2.3.

A N A L Y T I C A L METHODS

2.3.1. Leeches 2.3.1.1. Sample preparation, extraction, clean-up and fractionation. Each frozen leech sample was ground with approximately five times its weight of previously fired (to 400 ~ anhydrous sodium sulphate to a uniform consistency in a mortar. This mixture was extracted for 2.5 h with 230 mL o f residue-free dichloromethane in a Soxhlet extractor. The extract was then concentrated to 1-2 mL on a rotary evaporator at 25 ~ Lipids and other high molecular weight co-extractives were removed by gel permeation chromatography using a 35 x 2.5 cm gravity flow column filled with BioBeads SX-3, with cyclohexane/dichloromethane in a l:l ratio as the eluant. Fraction 1 (0-110 mL), which contained the lipids, was discarded. Fraction 2 (110-240 mL), which contained the contaminants of interest, was placed in a 1 L separatory funnel with 30 mL cyclohexane and sequentially extracted with 40, 30 and 30 mL aliquots of 0.1 M K E C O 3. The aqueous phase containing the acid fraction

152

J.L.

METCALFE ET AL.

was set aside for chlorophenol analysis. The solvent phase containing the neutral fraction was washed with 50 mL organic-free water, dried through anhydrous sodium sulphate, concentrated to 1-2 mL and transferred to a 10 mL Kuderna Danish tube. Two mL of iso-octane were added and the solution concentrated to 1.0 mL at 25 ~ under a stream of dry nitrogen. The extract was divided into two 0.5 mL portions for analysis of neutral organochlorines and non-chlorinated neutral compounds, respectively. 2.3.1.2. Analysis of chlorophenols. One mL of residue-flee acetic anhydride and 10 mL o f hexane were added to the acid fraction in a 125 mL Erlenmyer flask with a teflon-lined screw cap. The mixture was shaken for 1 h, then the hexane layer was transferred to a 10 mL Kuderna Danish tube. Two mL of iso-octane were added and the solution was concentrated to 1.0 mL under dry nitrogen. The final extract was analyzed by electron capture gas chromatography using two 30 m fused silica columns (DB5 and DB17) for quantitation and confirmation. The temperature program was 70 to 270 ~ at 4 ~ m i n - 1 with a 5 min hold at 270 ~ The injector was held at 230 ~ (split 10:1) and the detector at 350~ 2.3.1.3. Analysis o f neutral organochlorines. One of the 0.5 mL portions of the neutral fraction was passed through a mini clean-up column consisting of a Pasteur pipet packed with 2 cm o f 40~ sulphuric acid on silica gel and 0.5 cm anhydrous sodium sulphate. Three 1 mL rinses of residue-free hexane were then added to the column, allowing each one to penetrate the bed completely. The eluant was collected in a 10 m L Kuderna Danish tube. Two mL of iso-octane were added and the eluant was concentrated to 0.5 mL under dry nitrogen. The final volume was adjusted to 0.5 mL, then analyzed for lindane and DDT and its derivatives by electron capture gas chromatography. The temperature program was 122-138 ~ at 1 ~ m i n - 1 ; 138-250 ~ at 30 ~ m i n - l ; final hold at 250 ~ for 2 min. A minor component of total DDT in this study was o, p ' - D D T . This c o m p o u n d co-elutes with, and therefore cannot be separated from, p , p ' - D D D . 2.3.1.4. Analysis of non-chlorinated neutral compounds. The second 0.5 mL portion of the neutral fraction was analyzed for benzothiazole and its derivative MMBT by flame ionization gas chromatography using a 30 m DB5 column programmed from 90-250 ~ at 4 ~ m i n - i. The injector (split 10/1) and detector were held at 250 ~ 2.3.2. Water 2.3.2.1. Analysis of chlorophenols and neutral organochlorines. The 1 L water samples were acidified to pH 1-2 with concentrated HCI and extracted sequentially with 40, 30 and 30 mL of residue-free toluene. The toluene extract was treated identically to Fraction 2 of the leech samples from this point onward, except that

L EECHES AS A SCREENING T O O L FOR O RG A N I C C O N T A M I N A N T S

153

the neutral fraction was not split into two portions because separate water samples had been collected for the analysis of benzothiazole derivatives. 2.3.2.2. Analysis o f non-chlorinated neutral compounds. Sep-Paks containing the residues from the 4.28 L water samples were eluted with 5 mL acetone. One mL of iso-octane and sufficient methanol to produce a single phase was then added. The extract was concentrated to a final volume of 1.0 mL and analyzed by flame ionization gas chromatography as previously described for the leech samples.

3. Results 3.1. BIOACCUMULATION CAPACITIES OF LEECHES FOR ORGANIC CONTAMINANTS Leeches from Canagagigue Creek contained benzothiazoles, chlorophenols, lindane and DDT and its derivatives, although only benzothiazoles and chlorophenols were detected in creek water (Table III). D. dubia appeared to have the greatest bioconcentration capacity among the three species, as it accumulated the highest tissue concentrations for 11 of the 16 compounds. E. punctata accumulated the highest concentration for one compound (2,6-DCP) and was generally intermediate, while concentrations in H. stagnalis were lowest for nine compounds and never highest. Concentrations of 2,4,6-TCP and 2,3,6-TCP accumulated by D. dub& and E. punctata were similar, while concentrations of 2,3,4,6-TECP and P C P were approximately the same in all three species. H. stagnalis differed from the other two species in that it did not accumulate detectable residues of benzothiazole, 2,6-DCP or lindane. In general, contaminants were bioaccumulated in proportion to their relative occurrence in the water; that is, concentrations in both leeches and water were highest for benzothiazoles, intermediate for chlorophenols and lowest for lindane and DDT and its derivatives. Among the eight chlorophenols investigated, however, this general trend was not consistent (Figure 2). The dominant chlorophenol isomer in the water was 2,4-DCP, accounting for 38~ of the total chlorophenols present. It was also a dominant isomer in leeches, representing 31-46~ of the total chlorophenols accumulated. However, 2,4,5-TCP accounted for higher proportions in leeches (28-4407o) than in water (15070), while proportionately less 3,4-DCP was bioaccumulated (3-507o) than would be expected from its relative occurrence in water (1607o). Less 2,6-DCP was also accumulated by D. dubia (2070) and H. stagnalis (0) but not by E. punctata (1407o) than would be anticipated (12~ in water). Pentachlorophenol, 2,3,4,6,-TECP and 2,3,6-TCP were proportionately low in both leeches and water. Bioconcentration factors (BCFs), which refer to the ratio between tissue and water concentration, are presented for benzothiazoles and chlorophenols in Table IV. For MMBT and BT, BCFs were low, ranging from 100-400 X. For chlorophenols, BCFs were higher and quite variable, ranging from 600-16,700 X depending upon the species and isomer. No clear pattern relating the degree of chlorination and chemical

0.002 0.002 0.002 0.001 0.001 0.001 0.001 0.001

0.001

0.001 0.001 0.001 0.001 0.002

2,6-DCP 2,4-DCP 3,4-DCP 2,4,6-TCP 2,3,6-TCP 2,4,5-TCP 2,3,4,6-TECP PCP

Lindane

p, p ' - D D T p, p ' - D D D p, p ' - D D E o, p ' - D D D o, p ' - D D E

1.0 1.0 1.0 1.0 1.0

0.5

2.0 2.0 2.0 1.0 1.0 1.0 0.5 1.0

100.0 100.0

Leeches (ng g-~)

* Mean (and s.d.) of triplicate water samples. ND = not detectable

0.1 0.1

Water (/tg L ])

Analytical Detection Limits

MMBT BT

Compound

T A B L E II1

ND ND ND ND ND

ND

0.039 0.128 0.053 0.052 0.002 0.049 0.004 0.008

(0.006) (0.013) (0.006) (0.013) ( < 0.001) (0.013) (0.002) (0.005)

23.27 (3.59) 3.02 (1.17)

Concentration in Creek Water (/tg L - ]) *

28 75 67 63 15

8

42 852 91 170 26 651 19 24

10900 1400

22 80 68 68 16

11

61 1336 146 263 40 984 24 32

7200 600

D. dubia DD1 DD2

7 28 26 20 5

3

140 335 33 180 29 317 16 32

5400 700

5 16 17 13 3

3

162 332 31 188 26 297 16 36

4600 700

E. punctata EP1 EP2

6 7 16 9 4

ND

ND 202 33 59 14 274 24 25

2200 ND

3 8 22 12 5

ND

ND 207 32 61 14 280 20 22

2300 ND

H. stagnalis HS1 HS2

Concentration in Leech Tissues (ng g-~ live weight)

Concentrations of organic contaminants in water and leeches from Canagagigue Creek at the beginning of the experiment

LEECHES

AS A SCREENING

TOOL

FOR ORGANIC

155

CONTAMINANTS

40-

WATER

:30-

20o z w ~ o

10-

o

q LEECHES

~40-

[]

Dina dubia

[]

Erpobdella i~nctata

i

Helobdella stagnalis

2010-

3,4-0CP

24-OCP

2,4,6-TCP

2,4.5-TCP

2.6-OCP

PCP

2,3,4,6"TECP

2,3,6- TCP

CHLOROPHENOL ISOMER Fig. 2.

Relative proportions of chlorophenol isomers in creek water and leeches.

TABLE IV Average

bioconcentration factors (BCFs) for chlorophenols in leeches

Compound

MMBT BT 2,6-DCP 2,4-DCP 3,4-DCP 2,4,6--TCP 2,3,6-TCP 2,4,5-TCP 2,3,4,6-TECP PCP

benzothiazoles

and

Average BCFs

D. dubia

E. punctata

H. stagnalis

400 350 1300 8500 2200 4200 16500 16700 5500 3500

200 250 3900 11900 600 3500 14000 6300 4000 4300

100 1600 600 1200 7000 5700 5500 3000

X X X X X X X X X X

X X X X X X X X X X

X

X X X X X X X

properties of the various isomers (Table I) to their bioaccumulation potentials in leeches was evident. BCFs could not be calculated for lindane or DDT and its derivatives because these compounds were not detectable in the water.

156

J. L. M E T C A L F E E T A L .

3.2. ELIMINATION RATES OF ORGANIC CONTAMINANTS BY LEECHES Elimination

rates of organic contaminants

points from each aquarium

by leeches were determined using data

as d u p l i c a t e s . A s i n g l e c o m p a r t m e n t

model adequately

d e s c r i b e d t h e e l i m i n a t i o n p r o c e s s ; t h a t is, p l o t s o f t h e n a t u r a l l o g a r i t h m (In) o f t i s s u e concentration

vs. t i m e i n d a y s g a v e e s s e n t i a l l y s t r a i g h t l i n e s ( F i g u r e s 3, ~ a n d 5).

W h e r e t h e s l o p e o f a l i n e w a s s i g n i f i c a n t l y d i f f e r e n t f r o m z e r o a t t h e p < .05 level, rate constants were determined

a n d h a l f lives w e r e c a l c u l a t e d ( T a b l e V ) .

Leeches depurated chlorophenols and DDT derivatives slowly from their systems. H a l f lives f o r c h l o r o p h e n o l s

in leech tissues were in most cases longer than one

m o n t h . T h e e l i m i n a t i o n p r o c e s s f o r t w o r e p r e s e n t a t i v e i s o m e r s is s h o w n i n F i g u r e 3. Chlorophenols

p e r s i s t e d l o n g e s t i n E. punctata, w h e r e n o s i g n i f i c a n t d e p u r a t i o n w a s

observed for any compound

e x c e p t P C P d u r i n g t h e 27 d e x p e r i m e n t . A l l t h r e e l e e c h

species eliminated p,p'-DDD

and o,p'-DDD

more rapidly than p,p'-DDE

and

as i l l u s t r a t e d f o r t h e p,p'-derivatives i n F i g u r e 4. W h i l e n o s i g n i f i c a n t

o,p'-DDE,

elimination was observed for p, p'-DDT

in erpobdellid leeches, this compound

a h a l f life o f o n l y 5.5 d i n H. stagnalis. A s w a s t h e c a s e f o r c h l o r o p h e n o l s ,

had DDT

d e r i v a t i v e s a p p e a r e d t o p e r s i s t l o n g e s t i n E. punctata.

TABLE V Depuration rate constants and half lives for organic contaminants in leeches Compound

D. dubia Rate Constant dKr (day- 1)

E. punctata Half Life (days)

Rate Constant dKT (day- 1)

MMBT BT

.437 .073

1.5 9.5

.281 .102

2,6-DCP 2,4-DCP 3,4-DCP 2,4,6-TCP 2,3,6-TCP 2,4,5-TCP 2,3,4,6-TECP PCP

.021 .015 .022 .027 NSD NSD NSD NSD

34 46 31 25 -

NSD NSD NSD NSD NSD NSD NSD .025

1.199

.5

NC

NSD .070 NSD .054 NSD

10 13 -

NSD .033 NSD .022 NSD

Lindane p, p'-DDT p, p'-DDD p, p'-DDE o, p'-DDD o, p'-DDE

NSD = No significant depuration observed at p_< .05. NC = Not calculated due to insufficient data points. < D L = Not detectable.

H. stagnal& Half Life (days)

Rate Constant dKT (day- 1)

Half Life (days)

NC < DL

< 1 -

28

< DL .017 .008 .020 NSD .023 NSD NSD

41 87 34 30 -

1

< DL

2.5 7

21 32 -

.125 .053 NSD .115 .012

5.5 13 6 58

LEECHES AS A SCREENING TOOL FOR ORGANIC CONTAMINANTS

157

2,4,6- T R I C H L O R O P H E N O L 8-

tC(~ e 1/2 =200 25 dng/g

l

C(o)=60ng/g te 1/2- 34 d

C(~ 2OOng/g

x

.

-. 9

4-

e.c

3

=o

O (3 2,4- DICHLOROPHENOL C(o ) -

1100 ng/g

C(o)= 300 ng/g

C(o)= 200 ng/g te 1/2= 41d

e_J

O

Time in Days Erpobdella punctata

Dina dubia

Helobdella stagnalis

D e p u r a t i o n of chlorophenols by leeches. C(o) = initial tissue concentration; 9 = single data point; x = two data points with the same value.

Fig. 3.

P,P'

6-

-

:3-

half life;

DDD

C(o ) - 8Ong/g t e 112= 1Od

5-

tel~2 =

C(o)= 20 ng/g te 1/2 = 21 d

C(o) = 10 ng/g tel/2 " 13d

9 ~ |

2-

8

c O

p,p'-DDE 6-

03 69

_5

C(o ) . 70 ng/g

5v

4- "o e

9

9

32-

C(o )= 20 ng/g

[..x. L

C(o )- 20 ng/g

,

;

1-

~

; ;

1;

2~

2;

O12 5 ~]

1'3

21

2F7

o,2

5 8

G

~1

Time in Days Erpobdella punctata

Dina dubia

Fig. 4.

H e l o b d e l l a stagnali_s

D e p u r a t i o n of D D T derivatives by leeches.

~,

158

J. L. METCALFE

ET AL.

2 - (METHYLTHIO) BENZOTH IAZOLE lO9=

C(o)- 9000ng/g

C(o)- 5000 ng/g te 1/2= 2.5d

tel/~ ~1.5 d

l X

C(o) = 2000 ng/g te 1/2- ": ld

9

o

0 o

g 5

BENZOTH I A ZOLE 9]

C(o) . 1000 ng/g

C(o) . 700ng/g

84

te 1/2= 9.5 d

te I/2= 7d

C(o)" ND

7 oe

3012

5

8

Dina dubia

Fig. 5.

13

21

o'I~ 's ~ ,'3

27

Time in Days Erpobdella punctata

~i

~7

Helobdella stagnalis

Depuration of benzothiazole derivatives by leeches, o = single data point and o = two data points with concentrations less than the detection limit.

Lindane and benzothiazole derivatives were quickly eliminated by leeches (Table V and Figure 5, respectively). The half life for lindane was a day or less; for M M B T it was 1-2.5 d and for BT, about one week. Lindane concentrations in leech tissues were very low to begin with; however, M M B T and BT concentrations were initially very high. In fact, initial tissue concentrations of M M B T were f r o m one to several orders of magnitude higher than any other c o m p o u n d , yet this contaminant was never detected in leeches after their eighth day in clean water. 3.3. MORTALITY AND WEIGHT LOSS The incidence of mortality during the 27 d depuration period was low (1-3~ Only one mortality occurred in each of DD1, DD2 and EP1, 5 in HS1, 3 in HS2 and none in EP2. Four specimens could not be accounted for in DD1, 6 in DD2, 1 in EP1 and 2 in EP2. H. stagnalis specimens remaining at the end of the experiment were not tallied. D. dubia and E. punctata appeared to lose weight during the experiment; Table VI shows that the mean weights of sampled specimens tended to decrease over time. This evidence of weight loss is inconclusive, as we could have unintentionally sampled larger specimens earlier in the experiment. Unfortunately, we have no way of measuring weight changes in specific individuals. However, by comparing the mean

L E E C H E S AS A S C R E E N I N G T O O L FOR O R G A N I C C O N T A M I N A N T S

159

weight o f all specimens initially with the mean weight of all specimens at the time of sampling, we estimated the loss of biomass over the course o f the experiment as about 10~ for both species. This translates into a loss o f approximately 1~ of the original body weight d a y - 1 for each individual. H. stagnalis remained in excellent condition throughout the experiment, with no indication that the average weights of sampled specimens decreased over time (Table VI). 4. Discussion Leeches f r o m Canagagigue Creek accumulated 16 organic contaminants in their tissues, even though only ten of these compounds were detectable in creek water. In a similar study on the occurrence of organic contaminants in rivers in southwestern New Brunswick (Metcalfe et al., 1985), 12 chlorophenol isomers were detected in resident leeches while only three were detected in river water. As the primary route of uptake for c o m p o u n d s as soluble as benzothiazoles, chlorophenols, lindane and even DDE (Bjerk and Brevik, 1980) would be directly f r o m the water, leeches are clearly capable of concentrating minute amounts of these contaminants into measurable residues in their tissues. Interspecific differences in the bioaccumulation of contaminants were apparent. D. dubia was previously shown to accumulate higher concentrations of seven chlorophenols than any other leech species in Canagagigue Creek (Metcalfe et al., 1984), and this trend apparently applies to other organic compounds as well. H. stagnalis accumulated lower concentrations of contaminants than the two erpobdellids. According to unpublished data reported by Sawyer (1974), H. stagnalis also accumulated lower concentrations of Mirex (2 ppm) than Erpobdella sp. (26 ppm) after 5 d exposure at 1.0 p p m Mirex. Leeches generally accumulated contaminants in proportion to their relative occurrence in the water, but there were exceptions. Concentrations of BT and 2 , 4 - D C P in erpobdellids were similar despite much higher concentrations of BT in the water. Also D D T and its derivatives were never detected in creek water, yet concentrations in leeches were higher than some of the chlorophenols. These findings can be attributed to differences in the chemical properties of the various compounds(Table I). For example, the lower BCFs observed for benzothiazoles than chlorophenols were expected, because benzothiazoles are more water-soluble and have lower Kow values. Bioconcentration factors for chlorophenols in leeches varied a m o n g the different isomers in apparent contradiction o f their chemical properties (Table I). For most organochlorine compounds, increasing chlorination of the molecule results in a corresponding increase in bioconcentration. This is not necessarily the case for acidic c o m p o u n d s like chlorophenols, for which the percentage of the c o m p o u n d present in the un-ionized, biologically available, f o r m is pH-dependent. For example, as the pKa values (at 25 ~ for chlorophenols range from 4.8 for P C P to 8.59 for 3 , 4 - D C P (Jones, 1981), P C P would be more dissociated and less biologically available at

.071 (n = 46)

.059(n = 41)

16.9o7o

Initial Mean Weight of all Specimens

Mean Weight of all Sampled Specimens

Estimated Loss of Biomass

ln=5;2n=3;3n=4

.082 .070 .052 .061 .065 .046 .0341 -

8.3o70

.066(n = 39)

.072(n = 46)

.073 .086 .075 .067 .062 .050 .0372

6.9o70

.178(n = 26)

.191 (n = 28)

.243 .183 .166 .198 .246 .168 .163 .1041

EPI(n=3 d -l)

D D l ( n = 6 d -I)

DD2(n=6d -l)

E. punctata

D. dubia

0 1 2 5 8 13 21 27

Sampling Day

T A B L E VI

11.6O7o

.185(n = 25)

.209(n = 27)

.260 .163 .234 .214 .151 .199 .194 .0933

EP2(n=3 d -l)

-

.0073 .0071 .0074 .0075 .0076 .0075 .0072 .0075

HSl(n=40d

H. stagnalis

Mean weights (g) o f sampled leeches and estimated loss of biomass during the experiment

i)

-

.0069 .0078 .0077 .0075 .0077 .0078 .0071 .0074

H S 2 ( n = 4 0 d -I)

>

>

K m

LEECHES

AS A SCREENING

TOOL

FOR ORGANIC

CONTAMINANTS

161

neutral pH than 3,4-DCP. In support of this, Call et al. (1980) determined that 2,4,5-TCP had a greater bioconcentration potential in fish than P C P and suggested that this is because P C P has a lower p K a than TCP. Kaila and Saarikoski (1977) found 2,3,6-TCP to be more toxic than P C P to the crayfish, Astacus fluviatilis, in an aqueous medium, but less so when the compounds were injected directly into the tissues. They believe that waterborne 2,3,6--TCP penetrates crayfish tissues more easily than P C P because o f its higher pKa. Ernst and Weber (1978) reported that BCFs for chlorophenols in the polychaete, L. conchilega, increased towards the lower chlorinated isomers. Values calculated from their data are as follows: PCP-3000 X, 2,3,4,6/2,3,5,6-TECP-11 000 X; 2,3,4,5-TECP-17500 X; 2 , 4 , 6 - T C P - 2 0 000 X; 2 , 4 , 5 - T C P - 2 4 000 X. Our results for leeches also demonstrated higher BCFs for 2,4,5-TCP and 2,3,6-TCP than for 2,3,4,6-TECP and PCP, but 2,4,6-TCP deviated from this pattern. Jacob (1986) exposed the leech Haemopsis marmorata to a mixture of five chlorophenols at 10 ppb each and found, as we did, lower BCFs for 2,4,6-TCP (350 X) than for 2,4,5-TCP (2280 X). We observed similar BCFs for 2,3,4,6-TECP and P C P in leeches. Paasivirta et al. (1985) reported slightly higher BCFs for the 2,3,4,6-TECP than P C P in two species of freshwater fish, while Folke et al. (1983) observed the opposite in marine mussels. In both cases, the differences were small. Paasivirta et al. (1985) did not find any evidence of food chain enrichment of chlorophenols in a contaminated lake chain in Finland. However, they did note that habitat and feeding habits influenced the pattern of uptake. For example, filter-feeders accumulated the more water soluble isomers such as 2 , 4 - D C P and 3,4-DCP, while sediment-associated organisms accumulated the less water soluble, material-bound compounds. The pattern of chlorophenol bioconcentration we observed for leeches in Canagagigue Creek may be influenced by many factors. These include the chemical properties of the isomers (where the effects of increasing chlorination, increasing lipid solubilities and decreasing water solubilities are offset by decreasing dissociation constants), the degree to which leeches are in contact with dissolved vs. sediment-bound compounds, and the unique metabolic capabilities of the organisms themselves. There have been few studies which directly compare contaminant residues in leeches with those in other aquatic organisms. The only information available concerns the persistent organochlorine pesticides, DDT and Mirex. Webster (1967) reported the presence of DDT in E. punctata tissues three months after the aerial spraying of a marsh, while no residues were found in amphipods or copepods. Meeks (1968) experimentally sprayed radiolabelled DDT over a marsh and followed its uptake and depuration by all components of the food web over a period of 5 months. DDT residues in the leech, E. punctata, were higher (up to 12.6 ppm) than those in molluscs, crustaceans and insects. In fact, the only biological samples having higher concentrations o f DDT than leeches were the fat of a carnivorous water snake and one species of bird. In laboratory experiments, de la Cruz and Naqvi (1973) determined that freshwater shrimp and crayfish accumulated twice as much Mirex as three species of leeches after 48 h exposure at 2.0 ppm Mirex. In contrast, the same

2,4,6-TCP

BT

MMBT

Compound

.052

3.02

1200-4200 x

250-350 x (erpobdellids)

100---400 x

23.27

64 x

27 x

9x

A

.110

45 x

(mussel)

A n o d o n t a piscinalis

(roach)

Leuciscus rutilus

(pike)

Es ox lucius

(burbot)

L ot a vulgaris

(marine polychaete)

Lanice conchilega

A

.001

Poecilia reticulata

(pond snail)

Lymnaea stagnalis

N. cornutus R. cataractae O. propinquus

(crayfish)

Orconectes propinquus

(Longnose dace)

Rhinichthys cataractae

(Common shiner)

Notropis cornutus

20000 x

C, 36 d

B, 7 d

B, 7 d

Organism

(guppy)

.05

9.68

30.40

Exposure Regime *, Duration

Tganisms

7000 x (c~) 12000 x ( Q )

3000 x

5x

10 x 10 x

15 •

150 •

150 x

BCF

BCF

Exposure Concentration (~g L t)

Exposure Concentration (~tg L ~)

Other Aquatic

Leeches (this study)

Paasivirta et al. (1985)

Ernst and Weber (1978)

Virtanen and Hattula (1982)

Metcalfe et al. (unpublished data)

Metcalfe et aL (unpublished data)

Reference

Bioaccumulation of benzothiazoles and chlorophenols by leeches vs. other aquatic organisms (wet weight basis)

TABLE VII

I-J

.004

.008

2,3,4,6-TECP

PCP

3000~300 x

4000-5500 •

5700-16700 x

.040

70-180 x 2600-8500 x

.035 .660

200x 240 x

.090

133 x 33 x 21Ix 44 x 44 • 33x .002-.003

.006

11000 x

170 x

.0(0-.008

.008

4.8 49.3

60 x

24000 x

1900x 1800 x

A

C, 115 d

B, 70 d

A

A

B, 70 d

A

C, 28 d

Exposure Regime *, Duration

* A = natural population; B = on site exposure; C = continuous flow, laboratory.

.049

182 x

73 x

Exposure Concentration (~/g L -~)

BCF

Exposure Concentration (#g L ~)

BCF

Other Aquatic Organisms

Leeches (this study)

2,4,5-TCP

Compound

Folke et aL (1983)

Mytilus edulis

Folke et al. (1983) Niimi and McFadden (1982)

M. edulis Salmo gairdneri

L. conchilega

(sea anemone)

Sagartia troglodytes

Ernst and Weber (1978)

Paasivirta et al. (1985)

(Rainbow trout)

Ernst and Weber (1978)

L. conchilega L. vulgaris E. lucius Leuciscus idus (Ide) L. rutilus Chironomus sp. S. lagustris

(marine mussel)

Ernst and Weber (1978)

Call et al. (1980)

Reference

L. conchilega

(fathead minnow)

Pimephales promelas

(sponge)

Spongilla lagustris

(blackfly larvae)

Simuliidae

Organism

Bioaccumulation of benzothiazoles and chlorophenols by leeches vs. other aquatic organisms (wet weight basis)

TABLE VII (Continued)

164

J.L.

METCALFE

ET AL.

authors (Naqvi and de la Cruz, 1973) found that levels of Mirex in the leech, E. punctata, f r o m a creek recently sprayed with the chemical, were higher than in 14 other invertebrates (including shrimp and crayfish) and a frog. Only water boatmen accumulated slightly higher concentrations than leeches. It is difficult to compare the BCFs we observed for leeches with those reported for other organisms, mainly because few studies have been conducted under environmentally realistic exposure regimes such as ours. It should also be noted that BCFs generated from field studies, in which integrated values (concentrations in organisms) are compared with instantaneous values of unknown variability (concentrations in water), will be less precise than those determined under controlled laboratory conditions. Table VII presents the relevant comparisons. In all but two instances, BCFs for benzothiazoles and chlorophenols in leeches were much higher than those in other aquatic organisms. Virtanen and Hattula (1982) reported greater BCFs for 2 , 4 , 6 - T C P in guppies than we observed in leeches. However, the concentrations of 2 , 4 , 6 - T C P in their microcosm decreased f r o m 0.50 to 0.05 ~g L - 1 after 36 d due to an undetermined factor despite continuous dosing at the higher concentration. The exact exposure level under these circumstances is difficult to estimate. The marine polychaete, Lanice conchilega, appears to have a BCF for chlorophenols as high as that of leeches. According to Ernst and Weber (1978) these BCFs greatly exceed those reported in the literature for fish and mussels. Metcalfe et al. (1984) noted that among 15 taxa o f stream invertebrates collected from Canagagigue Creek, only oligochaetes accumulated chlorophenol residues comparable in magnitude to those in leeches. This information suggests that annelids in general may have unusually high bioconcentration capacities for chlorophenols. We were unable to calculate BCFs for lindane in leeches because this pesticide was not detectable in creek water. However, D. dubia and E. punctata accumulated residues of lindane (3-11 ng g - 1) which are similar to those reported by Schimmel et al. (1977) in pink shrimp, Penaeus duorarum (10 ng g - 1) exposed to 0.13 ~g L - l lindane in sea water. As their exposure levels were at least two orders of magnitude higher than ours, it appears that erpobdellid leeches have a higher bioconcentration potential for lindane than shrimp. Leeches depurated D D T and its derivatives rather slowly f r o m their systems, while lindane was eliminated very rapidly. This is consistent with the literature on the elimination rates of these compounds by other aquatic organisms (Table VIII). There is some direct evidence that leeches may eliminate D D T more slowly than other aquatic invertebrates. In a field survey, Meeks (1968) found that D D T residues were more persistent in the leech, E. punctata, than in molluscs, crustaceans or insects, exceeding 1.5 p p m 12 months after aerial spraying vs. 0.5 p p m or less in other invertebrates. H. stagnalis eliminated DDD, DDE and particularly D D T more rapidly than the two erpobdellids. This observation is not without precedent. According to an unpublished study described by Sawyer (1974), H. stagnalis from an area heavily sprayed with D D T contained significantly greater amounts of D D D and DDE than DDT, therefore appearing to dechlorinate DDT. Another unpublish-

LEECHESAS A SCREENINGTOOLFOR ORGANICCONTAMINANTS

165

TABLE VIII Half lives for lindane, DDT and DDT derivatives in leeches vs. other aquatic organisms Compound Half Life in Leeches Half Lives in Other Organisms (this study) Organism Half Life Reference Lindane

DDD DDT

< 1 day

6--32 days 51/2 to ~. 30 days

M. edulis Lepomis macrochirus (bluegill); Carassius auratus (goldfish) P. reticulata Venerupis japonica (Marine short-necked clam) M. edulis L. macrochirus; C. auratus

< 1 day Ernst(1977) 32 days Gakstatter and Weiss (1967)

ed study from the same source compared the uptake of Mirex by H . stagnalis and E. p u n c t a t a after exposure to 1 ppm Mirex in water. H . stagnalis accumulated 5 ppm after 24 h, then tissue concentrations declined to 1 ppm after 5 d. In contrast, uptake by E. p u n c t a t a was directly proportional to exposure time, increasing from 2 after 24 h to 26 ppm after 5 d. These results suggest that H . stagnalis is capable of metabolizing Mirex while E. p u n c t a t a is not. The ability o f H . stagnalis to degrade organic contaminants may be a general feature of the species or Family, and could explain why certain compounds in the present study (i.e. BT, 2 , 6 - D C P , lindane) were not detected in H . stagnalis. Chlorophenols are eliminated very rapidly by most aquatic organisms (Table IX). The long half lives we observed for these compounds in leeches are in striking contrast to those reported for fish and mussels. Elimination rates for chlorophenols did not differ markedly among leech species, although most compounds appeared to persist the longest in E. p u n c t a t a . As the data for this species was the most variable, probably due to smaller sample sizes and a greater size range of specimens, it is possible that significant depuration did occur for some compounds but we were unable to detect it. There is no information available on the depuration of benzothiazoles by aquatic organisms. D. dubia and E. p u n c t a t a lost weight during the depuration study at an estimated 1~ body weight d a y - 1. In contrast, H . stagnalis did not appear to suffer any weight loss. All three species feed primarily on chironomids and oligochaetes (Barton and Metcalfe, 1986; Davies et al., 1979), although their feeding mechanisms differ. Erpobdellids, which possess a simple gut, swallow their prey whole. The more primitive glossiphonids, some o f which are sanguivorous, suck the body fluids of their prey and store them in a diverticulated crop prior to digestion. We speculate that the diverticulated gut o f / / . stagnalis allows it to consume larger meals and store food for longer periods of time, thus remaining in better condition than D. dubia

166

J. L. METCALFE

ET AL.

TABLE IX Half lives for chlorophenols in leeches vs. other aquatic organisms Chlorophenol Half Life in Isomer Leeches (this study)

Half Lives in Other Organisms* Organism

Half Life

6.6--9.2 days Call et al. (1980) < 10 days Landneret aL (1977) (liver, fat weight basis) 4.7 days Trujilloet al. (1982)

2,4,5-TCP 2,4,6-TCP

-30 days _>25 days

P. promelas S. gairdneri

PCP

->28 days

Fundulus similis

Reference

(marine killfish) L. macrochirus

S. gairdneri

C. auratus M. edulis