TOXICITY OF POLYCHLORINATED BIPHENYLS (AROCLOR 1254)

ON THE EARTHWORM EISENIA FOETIDA

THESIS

Presented to the Graduate School of the

University of North Texas in Partial

Fulfillment of the Requirements

For the Degree of

MASTER OF SCIENCE

By

Rain Sassani, B.S.

Denton, Texas

August, 1991

3moJ t

/ (9 f

N . 679

Sassani, Ramin, Toxicity of Polychlorinated Biphenyls

(Aroclor 1254) on the Earthworm Eisenia foetida. Master of

Science (Biology), August, 1991, 57 pp., 6 tables, 7

figures, references, 9 titles.

Objectives were to: (1) assess toxicity of

polychlorinated biphenyls on Eisenia foetida, in terms of

survival (LC5O/LD5O), and suppression of coelomocytes to

form secretory (SR) and erythrocyte rosettes (ER) with, and

to phagocytize rabbit erythrocytes; and (2) compare results

with those of Lumbricus terrestris to assess relative

sensitivities to PCB. Using 5-d filter paper contact

exposure protocol, LC50 and LD50 were 30.4 cg/cm2 and 4450

cg/g dry mass, respectively. Nominal PCB exposure

concentrations of 5.0 and 10.0 pg/cm2 resulted in tissue

levels of 1389 and 2895 pg/g dry mass causing a significant

reduction in SR formation by 18 and 52%, respectively. ER

formation and phagocytosis were reduced significantly (52

and 61%) only at the higher tissue concentration. Compared

to reported data on lethality and immunomodulation in L.

terrestris, E. foetida appears to be more resistant.

TABLE OF CONTENTS

Page

LIST OF TABLES.............. ....... .. ......... iv

LIST OF FIGURES........................".".w... ...V

Chapter

I. INTRODUCTION................. ........... ... 1

II. RELEVANT LITERATURE..................... ... 7

III. MATERIALS AND METHODS". . ....... .... 13

IV. RESULTS .....-.. . . .. . ...... ...... 19

V. DISCUSSION................................... 43

APPENDIX A...................................... 46

APPENDIX B....................................... 50

REFERENCES ............ ........... ........ ...... 52

iii

LIST OF TABLES

TABLE Page

1. Relation between nominal filter paper exposureto and whole body concentrations of PCB forthe earthworm Eisenia foetida after 5-dexposure . . . . . . . . . . . . . . . . . . . 19

2. Comparison of erythrocyte (ER) and Secretory(SR) rosette formation, and phagocytosis (PG)by coelomocytes from earthworm Eiseniafoetida exposed for 5-d to three PCBconcentrations with those from non-exposedcontrols. Expressed as actual number (SE) of cells per 100 randomly countedcoelomocytes...... .... . . . . . . . .. 29

3. Comparison of erythrocyte (ER) and Secretory(SR) rosette formation, and phagocytosis (PG)by coelomocytes from earthworm Eiseniafoetida exposed for 5-d to three PCBconcentrations with those from non-exposedcontrols. Expressed as % of normal ( SE) . 30

4. Comparison of nominal filter paper exposureand whole body concentrations of PCBs betweenthe earthworms Lumbricus terrestris (Lt) andEisenia foetida (Ef) exposed for 5-d to PCB . . 39

5. Comparison of erythrocyte (ER) and Secretory(SR) rosette formation, and phagocytosis (PG)from two species of earthworms, Eiseniafoetida and Lumbricus terrestris, exposed for5-d to three PCB concentrations on filterpaper. Expressed as actual number ( SE)of cells per 100 randomly countedcoelomocytes and as % of normal . . . . . . . . 41

6. Comparison on lethal (LC/LD5O 95% CL) andsublethal immunomodulatory effects (R se),expressed as % normal (i.e., controls), ofPCB on earthworms, Eisenia foetida andLumbricus terrestris........................42

iv

LIST OF FIGURES

FIGURE

1. Comparison of LC50 from two species of theearthworms, Lumbricus terrestris and Eiseniafoetida, exposed for 5-d to PCB.. . . . .

2. Comparison of LD50 from two species of theearthworms, Lumbricus terrestris and Eiseniafoetida, exposed for 5-d to PCB. .-. . . .

3. Relation between mortality and nominal PCBexposure concentration showing the LC50 forEisenia foetida as predicted by probitanalysis-..-.....-.-...-..................

4. Relation between mortality and PCBs bodyconcentrations showing the LD50 for Eiseniafoetida as predicted by probit analysis. . .

5. Comparison of E-rosettes produced bycoelomocytes of Eisenia foetida exposed toPCBs in a 5-d filter paper contact test.

6. Comparison of S-rosettes produced bycoelomocytes of Eisenia foetida exposed toPCBs in a 5-d filter paper contact test.

7. Comparison of phagocytic coelomocytes ofEisenia foetida exposed to PCBs in a 5-dfilter paper contact test.0-0-0 - -1-.0

. . . 32

. ." . 34

36

V

Page

21

23

25

27

CHAPTER I

INTRODUCTION

The Environmental Effects Research Group at the

University of North Texas has been developing a set of

toxicity biomarkers using the earthworm Lumbricus terrestris

(Annelida, Oligochaeta, Lumbricidae) for assessing both

acute lethal (i.e. LC and LD50s) and sublethal effects of

environmental contaminants associated with hazardous wastes

and pesticides in terrestrial ecosystems. Initial research

has focused on coelomocyte-based biomarkers (e.g. total and

differential coelomocyte counts, viability, phagocytosis,

nitroblue tetrazolium dye reduction, and formation of

erythrocyte and secretory rosettes associated with the

immune system [1-6]. Earthworms were selected for

development of a bioassay system for terrestrial xenobiotic

pollutants for a number of reasons: (1) Extensive

literature exists on their basic biology and ecology [7-10];

(2) earthworms have been used in numerous laboratory and in

situ acute toxicity and/or bioaccumulation studies [11-15];

(3) standardized laboratory exposure protocols have been

developed for earthworms [16]; (4) earthworms, as Nature's

great cultivators, process and redistribute soil ingredients

and contaminants; (5) they are important components of

1

2

terrestrial food chains; (6) they are inexpensive and

socially non-controversial research organisms, obtainable

from reliable commercial suppliers; (7) their high surface

area/volume, feeding and behavior facilitate uptake of

contaminants; and (8) for immunotoxicity studies, earthworms

are ideal because certain aspects of their immune systems

are analogous, perhaps homologous, to those of vertebrates,

including mammals [17-18]. As such, they have potential as

surrogates to screen xenobiotics for their immunotoxic

potential in important wildlife. Emphasis on immune

function is timely and relevant to contemporary public

health considerations, such as immune suppression in humans

exposed to environmental contaminants.

Most of the work at UNT has been with L. terrestris,

since its size allows for harvesting large numbers of

immunoactive coelomocytes and chemical analysis of

individuals. However, the manure worm, Eisenia foetida

(Annelida, Obligochaeta, Lumbricidae), has been chosen for

several toxicity protocols by the European Economic

Community (EEC) [19], Organization for Economic Cooperation

and Development (OECD) [20], The Food and Agricultural

Organization of the United Nations (FAO), and finally the

U.S. Environmental Protection Agency [21], including: (1)

48-h filter paper contact test [12,22]; and (2) 14-d

artificial soil test [16,22]. E. foetida was selected for

the protocols for a number of reasons: (1) Large numbers of

3

manure worms can be cultured easily in a wide range of

animal or vegetable organic wastes. This means that

laboratories could easily breed their own stock if supplied

with cocoons from a central source [23], (2) E. foetida

occurs in specific habitats [24]. It is not found in large

numbers in soil although it can live in soils with

considerable organic matter. It is common in sewage beds,

particularly in trickling filters, where it is often exposed

to industrial chemicals [23], (3) considerable information

has accumulated on its growth [24], reproduction [25],

physical requirements [26], use in waste management systems

[24], and response to metals [27], (4) E. foetida is a

species with a short life cycle, reaching maturity in 7 to 8

weeks at 15-20*C, (5) it is very prolific; a single worm

produces two to five cocoons per week. Cocoons hatch in 14

to 21 d, and generally contain two to seven young [14].

Since relatively few comparative toxicity data exist

for L. terrestris and E. foetida, their respective

sensitivities to various classes of compounds are largely

unknown and consequently, there are conflicting reports

concerning their relative sensitivities and usefulness in

environmental toxicology [11]. Thus, I will take the

present study to compare effects of Aroclor 1254, a

commercial PCB mixture, on'survival (LC50-LD50) and

sublethal effects on several immune parameters with those

reported for L. terrestris [3-4].

4

Aroclor 1254 (hereon PCB) was chosen by the

Environmental Effects Research Group for several reasons:

(1) It is known to cause immunosuppression in earthworms [3-

4,6] and also in mammals [27-28]; (2) because of previous

and on-going work at UNT; (3) PCBs are stable mixtures

disseminated throughout the world; (4) PCBs can be extracted

easily via several known methods [29-30]; (5) due to its

structural stability, PCBs have a slow rate of metabolism,

which facilitates long-term evaluation during tissue

analysis (tissue analysis refers to PCB identification and

measurement in an exposed organism).

Potential toxicities of chemicals frequently are

expressed as LC50s, or concentration of a chemical in the

exposure medium which is lethal to 50% of test organisms

over a specified time period. LC value measures and/or

represents organisms survivorship to different contact

exposures. Although LC50 is used to represent potential

toxicity of chemicals, it only estimates the actual exposure

level required to produce toxicity. True potential toxicity

of chemicals is best measured by LD values, since tissue-

level dose replaces contact exposure (i.e. environmental

concentration). LD values represent organism's survivorship

in regard to the concentration of chemicals in its tissue.

LD50 is defined as concentration of a chemical within the

tested organisms capable of producing 50% mortality.

5

To compare the sublethal effects of PCB on immune

system of E. foetida with those in L. terrestris, studied by

Rodriguez [3] and Eyambe [4], I will study phagocytosis,

secretory rosette (SR) and erythrocyte rosette (ER)

formation by coelomocytes after being exposed to a sublethal

level of Aroclor 1254, determined from a definitive contact

test. Phagocytosis represents a non-specific immune

response and is a primary defense mechanism against invading

microorganisms. Phagocytosis which is phylogenetically

conserved among animals, is the most primitive type of non-

specific immune response among unicellular and multicellular

organisms. Rosette formation represents humoral and

cellular immunity against antigens by means of agglutinins

and surface receptors. SRs are those coelomocytes that bind

several layers of at least four or more antigenic red blood

cells through production and secretion of humoral factors

(e.g. agglutinins). Formation of erythrocyte rosettes (ER)

involves presence of specific surface receptors in

coelomocytes. These coelomocytes bind one layer of at least

four antigenic red blood cells. Previous work at UNT has

demonstrated that after being exposed to PCBs, coelomocytes

from L. terrestris show reduced ability to form ERs and SRs,

and to phagocytize rabbit red blood cells. Such alterations

portend dysfunction of immune response.

Comparative data determined in this study should enable

the Environmental Effects Research Group to evaluate the

6

potential use of E. foetida in standardized exposure

protocols for assessing toxicity of xenobiotics.

CHAPTER II

RELEVANT LITERATURE

Earthworms are classified as segmented worms in the

order Oligochaeta. The family Lumbricidae contains many of

the most common earthworm species of North America. Five

species are commonly found in North American soils:

Allolobophora caliginosa, the common field worm;

Allolobophora chlorica, a rather inactive, green colored

worm; Eisenia foetida, the yellow and maroon banded manure

worm; Lumbricus rubellus, the red worm; and Lumbricus

terrestris, the night crawler [14]. Three species would

seem to be most suitable for earthworm toxicity testing

[14,24]. (1) L. terrestris is a large species which feeds

on the soil surface at night so that it comes into contact

with chemicals both on the surface and within the soil. The

main disadvantage of this species for toxicity is that it is

slow growing, taking about 2 y to mature, however due to

easy maintenance, large size and living in various habitats,

L. terrestris has the potential as the animal of choice for

conducting laboratory toxicity tests and for determining the

toxicity level of various chemicals in the soil (field

studies). (2) A. caliginosa is a small, but extremely

common species which moves through the upper layers of the

soil. It reproduces faster than L. terrestris but it is

7

8

difficult to produce in large numbers. (3) E. foetida

resembles A. caliginosa in size, however it is not found in

large numbers in soil although it can live in soils with

considerable organic matter. E. foetida can be cultured in

a wide range of animal or vegetable organic wastes and

easily maintained in laboratory. It is a species with a

short life cycle, reaching maturity in 7 to 8 weeks. It is

prolific, producing two to five cocoons per week. E.

foetida has proved to be a suitable, readily obtainable and

easily bred laboratory test species with a representative

susceptibility to chemicals [23]. It has been proposed that

the two races "andrei" and "foetida" should be recognized as

separate species [31]. However, very limited biological and

ecological evidence exist to suggest a splitting of the

original species. At this time, the two races can be

separated only by color.

PCBs include 209 compounds of the general formula C=12,

H=x, Cl=y where x=0-9 and y=10-x. PCBs are produced by

chlorinating the biphenyl compound, which has 10 positions

[29]. Although 209 possible isomers of chlorinated

biphenyls can be formed, only 100 are present in commercial

products [32]. Commercial PCBs were produced by collecting

boiling-point fractions during distillation of chlorinated

biphenyl mixtures. In the'United States, the only large

producer of PCBs was Monsanto Chemical Company, which sold

them from 1929 to 1975 under the Aroclor trademark. From

9

the beginning of production, PCBs found their way into

industries due to their characteristics, especially in

production of industrial transformers and capacitors due to

their excellent coolant and dielectric properties [33].

Eventually, PCB applications expanded into other industries

such as paint and ink, adhesives, hydraulic fluids,

plastics, pesticides, and as coating to reduce the

flammability of wood products [34]. Despite their wide

spread use in industries, in 1976 Congress banded the

manufacture, processing, distribution and use of PCB, except

in totally closed systems (Toxic Substances Control Act,

TSCA, public law 94-469) [35]. During the 45 y that PCBs

were in production and use, they were disseminated

throughout the world [29]. By 1978, U.S. landfills had

accumulated 140 kilograms and an additional 82.5 million

kilograms of PCBs had been introduced into the environment

of the U.S. in forms available for transport, transformation

and accumulation [34]. PCBs are still being used in 2.8

million capacitors and 150,000 transformers, some of which

fail each year and eventually release additional quantities

into the environment (Miller 1982). PCBs were first

reported in environmental samples in 1966 [36] and by 1974

were the most discussed of pollutants [37].

There have been two major outbreaks of PCB

contaminations and exposures in human populations. First,

the 1968 incident, which occurred in Japan, was associated

10

with PCB poisoning, and the second incident occurred in

Taiwan, in 1979 [38]. Suppression of immune system occurred

in both outbreaks among patients who were chronically

exposed to PCBs [38]. It has been suggested that the immune

function will be altered by different possible mechanisms

after being exposed to PCBs and similar compounds [39]. PCB

exposure alters normal hormonal level through induction or

suppression of certain hormone producing cells and genes.

These hormonal abnormalities would have an effect on the

immune functions indirectly. In addition, PCB exposure

alters the functions and/or the population of immune cells,

causing a direct effect on immune function. Immunotoxicity

of PCBs has been observed among vertebrates such as atrophy

of the lymphoid tissues, reduction of spleen and bursa in

weights in chickens. In addition, immunotoxicity has been

reported in guinea pigs, rabbits, rats, mice and monkeys

[40-41].

Immune System of Earthworms

The immune system of the earthworm is located within

the coelomic cavity, which contains coelomic fluid and a

group of cells called coelomocytes. Earthworm coelomocytes

act similarly to leukocytes in mammals, meaning that they

are active in (1) non-specific immune responses such as

phagocytosis and inflammation, and (2) humoral immune

responses such as production and secretion of agglutinins

11

and lytic factors. The epithelial cells lining the coelomic

cavity give rise to two groups of coelomocytes [42]: (1)

Ameobocytes which are involved in initiation of non-specific

and specific immune response [43-48]; and (2) eleocytes

which function in storage of fats and carbohydrates.

Ameobocytes are further classified into neutrophils and

basophils [42]. Neutrophils respond to any type of tissue

injury or inflammation, whereas basophils participate in

graft rejection, and synthesis and release of humoral

factors. Overall the immune system of earthworms is similar

to the mammalian immune system in several ways: (1) It can

recognize and dispose of foreign materials via non-specific

mechanism, similar to that found in mammals [42]; (2)

earthworms are capable of initiating (a) inflammatory

response to tissue damage and (b) tissue rejection in

response to tissue grafting (allo-, auto- and xenografts)

[45-46, 49]; (3) immune system of the earthworm is capable

of differentiating self from non-self, a basic element of

any immune system including mammals which would serve as the

first step in the initiation of subsequent immune responses;

(4) the coelomic fluid in earthworms is similar to mammalian

serum, containing lytic and agglutinating substances. Small

basophils produce and secrete agglutinins and lysins which

participate in immune responses. Although these factors

exhibit similar functional properties to mammalian antibody,

they are structurally different.

12

Coelomocytes synthesizing and secreting agglutinins can

be assayed by determining their ability to form SRs with

antigenic erythrocytes. Small basophils release agglutinins

in response to specific stimulation by erythrocytes,

resulting in formation of multiple layers of erythrocytes

[50]. Basophils not only secrete humoral factors but also

equipped with special surface receptors capable of binding

to erythrocytes directly. ER formation indicates presence

of these receptors. S- and E-rosette assays have been used

to evaluate humoral immune responses of exposed and

unexposed earthworms after exposure to PCBs for 5-d [3-4].

CHAPTER III

MATERIALS AND METHODS

The earthworms used in my study were purchased from a

commercial supplier (Carolina Biological Supply, Burlington,

North Carolina, USA). Stock adult earthworms were

maintained in cow manure reconstituted with water within

plastic containers (29x18x13 cm), and kept in continuous

darkness at 20 C in environmental chambers. The worms were

acclimated for 2 weeks prior to experimentation.

I used contact test procedures described by EEC (1983)

Edwards [19-23]. In this test, glass vials (40 ml) were

lined with filter paper (Whatman No. 1, 14.4x5.5 cm) to

which PCBs were added. A glass pipette was used to spread a

constant volume (Iml) of acetone containing the appropriate

amount of PCBs over the surface of each filter paper. The

vials were sealed and laid on their side for approximately 4

min, allowing the filter paper to absorb the solution

evenly. After which, the vials were unsealed and placed

under a ventilating hood for 24 h, allowing solvent to

evaporate completely. Control vials were prepared in the

same way, but without PCBs in order to check on other

possible factors, in addition to the toxicant. Prior to

earthworm exposure, each vial was moistened with 1 ml of DDI

water to provide enough moisture for the earthworm. Each

13

14

worm was weighed and rinsed with DDI water before being

placed inside each vial. I designated 10 vials for each PCB

exposure concentration, and placed only one adult worm (300-

500 mg) into each vial, because the death of one worm in the

vial could adversely affect the well being of other worms

present. The vials were sealed and kept on their side in an

incubator. Additionally, vials were ventilated through a

small hole in the cap. Exposures were carried out under

darkness at 20 C throughout the 5-day period. Worms were

checked daily for mortality, and they were considered dead

if they did not respond to a gentle mechanical stimulus [51-

52]. At the end of the fifth day, worms were taken from

their corresponding vials and placed in aluminum cups.

Worms were killed by a brief shower of acetone which, also

rinsed PCBs present from their surface. Earthworms were

then dried in an oven at 45 C. Later, worms were weighed,

pulverized with mortar and pestle, and then transferred into

test tubes for extraction (Appendix I). A Perkin-Elmer

Sigma 3B Capillary Gas Chromatograph was used to determine

the PCBs in the extracts, following a method described by

Plumb [30]. Following is a list of the chromatographic

conditions that I used:

a) Column: 30 m Glass Capillary (0.75 mm I.D., SPB1, film thickness 1.Og).

b) Detector: Electron capture.

c) Carrier gas: 95% argon/5% methane, at 5m1/min.

15

d) Injector temperature: 300 C.

e) Initial oven temperature: 300 C.

f) Final oven temperature: 200 C.

g) Ramp rate: 5*C/min.

h) Final hold time: 20 min.

i) Injection volume: 0.2 - 0.4 AL.

Extract concentrations were determined by quantification

against an internal standard (decachlorobiphenyl) added to

all preparations. A standard curve, based on five

concentrations, related the ratio of the sum of the

principal PCB peak heights to that of the internal standard

as a function of PCB injected. Sample chromatography are

presented in Appendix II.

First, I used a series of tests to determine the range

of PCB concentration that had an adverse impact. Among the

existing methods of testing toxicity of chemicals, I

followed simple filter paper contact test procedures after

considering the following: (1) This test, developed by EEC

(1983) is used widely [19, 23, 52, 54]; (2) it enables

determination of direct effects of a chemical via contact-

only exposure; and (3) provides an efficient cost-effective

way for rapidly screening chemicals for toxicity. I

conducted this test to observe how E. foetida would respond

to a broad range of PCB concentrations. This permitted the

use of a narrow, more definitive range to determine the more

specific LC50 value. Previous work at UNT with L.

16

terrestris has shown that the concentrations of 0.10, 10.0

and 100.0 ug/cm2 were sufficient to generate a significant

response [:3]. For this reason, I selected the same

concentrations for my range-finding test. Based on results

of the range finding test, concentrations of 6.25, 12.50,

25.0, 75.0 and 100.0 g/cm2 were selected as a definitive

exposure range. Control worms were treated identically to

experimentals except for PCB exposure, were included in each

series of experiments. Results of the definitive contact

test (concentration vs. mortalities of worms to different

exposures) were analyzed by a computer program to estimate

the 5-d LC5O and LD50 values with their corresponding 95%

limits [53]. LC50s and LD50s are used in reference to

laboratory toxicity tests with chemicals that are ingested

or absorbed by animals [54]. Furthermore, by analyzing data

using simple linear correlation regression techniques, I

established an exposure concentration effect relationship

[55-57].

Assessing effects of PCB on several immune parameters

required establishment of sublethal exposure concentrations.

Thus, based upon the results of the definitive test, I chose

2.50, 5.0 and 10.0 g/cm2 as sublethal concentrations for 5-

d immunoassay experiments. Exposure procedures followed the

procedures of range finding tests and definitive tests,

except that 20 worms were used for each experimental and

control group. At the end of the fifth day, worms were

17

removed from vials, rinsed in ice-cold saline (0.85%), and

placed on a moist paper towel. The posterior end of the

worms was gently massaged to expel debris from the lower end

of the gut. Cleaned worms were placed in a 100mm x 15mm

petri dish containing 3 ml of extrusion medium developed for

collection of coelomocytes.

Extrusion medium, developed by the Environmental

Effects Research Group at UNT [5], contains 5% ethanol, 2.5

mg/ml EDTA, 10 mg/ml Guaiacol Glyceryl ether and 95% saline

for every liter, pH = 7.3. Ethanol stimulates earthworms,

resulting in muscle contractions, secretion of mucus and

extrusion of coelomocytes through integumental pores. Since

guaiacol glyceryl is a mucolytic agent, it was used to

prevent mucus secretions from the earthworm trapping the

extruded coelomocytes. EDTA was used to .prevent cells from

clumping.

Worms were bathed in this medium for 3 min. The

extruded cells were placed into test tubes containing 20m1

of ice-cold saline. Cells were washed 3x at 1000 RPM in a

refrigerated Beckman T-J6 centrifuge. After the third wash,

cells were suspended in 1 ml of cold calcium-free Lumbricus

balanced salt solution (LBSS-300) [44]. 0.1 ml of 2% RRBCs

(gluteraldehyde stabilized rabbit red blood cells, RRBCs,

reconstituted with phosphate buffer) was added to the 0.1 ml

volume of coelomocytes. Mixture of RRBC and coelomocytes

were centrifuged at 100x g for 5 min, and then kept at 4C

18

for 24 h before counting the number of coelomocytes out of

100 selected coelomocytes forming erythrocytic and secretory

rosettes (ER, SR) with phagocytizing RRBC. 0.1 ml of 1%

crystal violet in LBSS was added to the cell suspension to

stain the nuclei. Tubes were shaken gently to resuspend the

pellet, and a sub-sample was placed in a hemocytometer

chamber. The number of phagocytic cells and E and S

rosettes (ER, SR) were counted within the first sample of

100 cells.

CHAPTER IV

RESULTS

The relation between PCB exposure concentrations and

whole body-burden concentrations in E. foetida is shown in

Table 1. A simple linear correlation-regression test was

applied to the data in Table 1 to confirm a positive

correlation between PCB exposure concentrations and body-

burden concentrations.

Table 1. Relation between nominal filter paper exposure toand whole body concentrations of PCB for the earthworm Eiseniafoetida after 5-d exposure.

Exposure Body Burden Number of WormsConcentration Concentration

( g/cm2 ) (/cg/g dry mass) Exposed Survivors( SE) N %

2.5 1379 379 30 100%

survivors

5.0 1896 394 30 25 83

6.25 2967 405 10 08 80

10.0 2895 407 40 30 75

12.5 3375 523 10 07 70

25.0 3923 729 10 06 60

a b

50.0 4721 844 09 04 44

75.0 6011 920 09 03 33

100.0 5936 896 10 01 10

a LC50 = 30.4 g/cm2 (95% CL 0.014-0.061)b LD50 = 4450 gg/g dry mass (95% CL 3572-5556)

19

20

Body-burden concentrations increased significantly, R2

= 0.965, r = 0.982, p = 0.001, F = 191.84, df = 1 and a

highly significant slope 1249.60, t = 6.59, P = 0.003 in the

equation Y = a + bx, where Y is the predicted PCB body-

burden .g/g dry mass), x is the PCB exposure concentration

(g/cm 2 ), b is the slope (1250) and a the Y-intercept

(173.41), from the lowest (2.5 pgPCB/cm 2) to the highest

exposure concentration (100.0 ggPCB/cm 2), 1379 to 5936

cgPCB/g dry mass, respectively. No mortality was observed

among experimentals at 2.5 pgPCB/cm2 exposure concentration.

Mortality first occurred at exposure concentration of 5.0

pgPCB/cm2 . Mortality increased from 17% at 5.0 pgPCB/cm2 to

90% at 100.0 ggPCB/cm 2 , resulting in LC50 of 30.43 ggPCB/cm 2

( CI: 14.82-61.21) and corresponding LD50 of 4450 ggPCB/g

dry mass ( CI: 3572-5556) Tables 1, 4, and 6 Figures 1, 4.

Throughout all experiments, no mortality occurred among

controls, although they had undetectable amounts of PCB

within their bodies. Overall, as acute toxicity exposure

dose increased both the body-burden and the mortality rate

increased.

21

Fig. 1. Comparison of LC50 from two species of theearthworms, Lumbricus terrestris and Eisenia foetida,exposed for 5-d to PCB.

-

I

I

70

60

Eiseniafoetida

Lumbricusterrestris

22

700

600

500 E0)

400

300

200

100

N~

EV

0)

0

50

40

30

20

10

I I

I

23

Fig. 2. Comparison of LD50 from two species of theearthworms, Lumbricus terrestris and Eisenia foetida,exposed for 5-d to PCB.

24

6000

v, 5000

i 4000

30000

W 2000

1000

I. -~

TI

I

1

3000

2500 c

2000

15000)

1000

500

Eiseniafoetida

Lumbricusterrestris

s _

...

I

I

25

Fig. 3. Relation between mortality and nominal PCB exposureconcentration showing the LC50 for Eisenia foetida aspredicted by probit analysis.

26

o C

LOO

oc~,

-o o 0

Ai1VOe eO AiIII6VBOd

e~~~

co d doo

A.Ll~V.L}IOVI :.10A118V80}Jd

27

Fig. 4. Relation between mortality and PCBs bodyconcentrations showing the LD50 for Eisenia foetida aspredicted by probit analysis.

28

o 8 M

-J r

i.

co

0/)

- o p

r a L

A N .LOL 0 .l~LV88

oe~Oo u

DM V

N CL)

o a

00) CO0 0(00)0 0 0 0 0 0

AiIIVIUOIJ:10 AI1ISVSO~d

29

Immunoassays

Comparison of mean ER, SR and phagocytosis percentages

among manure worms exposed to 2.5, 5.0 an 10.0 pgPCB/cm2 for

5-d, and controls is shown in Tables 2 and 3, Fig. 5-7. The

Table 2. Comparison of erythrocyte (ER) and Secretory (SR)rosette formation, and phagocytosis (PG) by coelomocytes fromearthworm Eisenia foetida exposed for 5-d to three PCBconcentrations with those from non-exposed controls.Expressed as actual number ( SE) of cells per 100 randomlycounted coelomocytes.

Function Exposure Concentration (yg/cm2) Controls

2.5 5.0 10.0

ER 33.29 38.58 17.35a 35.82

(1.77) (1.78) (2.09) (2.40)

SR 32.17 30.58a 14.23a 37.11

(1.76) (1.75) (1.31) (2.16)

PG 30.88 31.17 13.58a 34.58

(1.36) (1.68) (1.36) (2.46)

a Significantly different (Dunnett's Alpha = 0.05) from

controls.

lowest exposure concentration used in the immunoassays was

2.5 ggPCB/cm2 (LC7) which yielded a body-burden

concentration of 1378 ggPCB/g dry mass with 95% CI: of 189-

2207 Table 1. There were no significant differences between

controls and experimentals in mean ER, SR and phagocytosis

percentages at 2.5 gPCB/cm2 exposure level. Exposure to

5.0 g/PCB/cm2 resulted in body-burden concentration of 1896

AgPCB/g dry mass with 95% CI: of 470-2748 Table 1. The

ability of E. foetida coelomocytes to form SR was affected

30

Table 3. Comparison of erythrocyte (ER) and Secretory (SR)rosette formation, and phagocytosis (PG) by coelomocytes fromearthworm Eisenia foetida exposed for 5-d to three PCBconcentrations with those from non-exposed controls.Expressed as % of normal (x SE).

Function Exposure Concentration (pg/cm2) Controls

2.5 5.0 10.0

ER 93 100 48a - 35.82

(1.77) (1.78) (2.09) (2.40)

SR 87 82a 38a 37.11

(1.76) (1.75) (1.31) (2.16)

PG 89 90 39a 34.58

(1.36) (1.68) (1.36) (2.46)

a Significantly different (Dunnett's Alpha = 0.05) fromcontrols.

by exposure to 5.0 gPCB/cm2 .

Significant differences were seen between mean

percentages ANOVA: F = 0.098, P = 0.0001. ER and SR

formations and phagocytosis by coelomocytes from manure

worms exposed for 5-d to nominal PCB concentrations of 10

gg/cm2 , body-burden of 2895 with 95% CI: of 1358-3612, were

significantly lower (Dunnett's Alpha = 0.05) than those from

non-exposed controls Table 2 and Figures 5-7. The ability

of coelomocytes to form ER, SR with and to phagocytize RRBCs

was significantly reduced after 5-d exposure to 10.0

pgPCB/cm2 ANOVA: ER P = 0.0001 F = 21.84, SR P = 0.0001 F =

31.34 and PG P = 0.0001 F = 27.64. Exposure to 10.0

jgPCB/cm2 suppressed ER, SR formations and phagocytosis by

coelomocytes Tables 2, 3 and Figures 5-7.

31

Figs. 5 - 7. Comparison of E, S-rosette, and phagocytosispercentages in the earthworm Eisenia foetida exposed to PCBson filter paper for 5-d. Open symbols representexperimental and shaded symbols represents controls.Asterisks indicate significant difference from controls.Horizontal line is the mean, rectangles are standard errorand vertical line represents 95% confidence intervals.Sample sizes are indicated by numbers above assay points.

32

Fig. 5. Comparison of E-rosettes produced by coelomocytesof Eisenia foetida exposed to PCBs in a 5-d filter papercontact test. Open symbols represent experimental andshaded symbols represent control.

* = Significantly different from control, shaded symbol,(P<O.05).

fH-Et

001

1ObiLNOO %

33

0q0

0

0U,c0j

zE

0

I-zW

00W

CD

CL)0C-xw0(M)

-o

00,

I 1

I,%% .1c ITM -- c - I

I

34

Fig. 6. Comparison of S-rosettes produced by coelomocytesof Eisenia foetida exposed to PCBs in a 5-d filter papercontact test. Open symbols represent experimental andshaded symbols represent control.

* = Significantly different from control, shaded symbol,(P<O.05).

-10

II

0LC)

1OM.LNO3 %

35

00

1

0

0L

01'r H4-H

NCE

z0

0

zw(a)00.

0.0.

00

ref * Htk-

r~ 4cT _

II

rft kTOM 4*30

36

Fig. 7. Comparison of phagocytic coelomocytes of Eiseniafoetida exposed to PCBs in a 5-d filter paper contact test.Open symbols represent experimental and shaded symbolsrepresent control.

* = Significantly different from control, shaded symbol,(P<0.05).

rH-tJ-

pz ...

0OC

10WNOO %

37

00

O

0

O*

0Y

-1o

C

zV

0

I-zW0Z00W

D.C,,0amxwa

00

r~

I

38

Comparison Between E. foetida and L. terrestris

A comparative relations among exposure concentrations,

whole body concentrations and their respective mortalities

for L. terrestris and E. foetida are shown in Table 4.

Previous work at UNT by Rodriquez [3] showed a significant

correlation (R 2 = 0.44, r = 0.67, P < 0.0001) between

nominal filter paper and entire-body PCB concentrations in

L. terrestris after 5-d. Similarly, in E. foetida existed a

positive correlation (R 2 = 0.965, r = 0.982, P < 0.0001)

between exposure concentrations and body-burden

concentrations after 5-d PCB filter paper exposure.

Doubling the exposure concentration did not double the body-

burden concentrations or mortality rates for either species.

Additionally, Rodriquez showed that 100.0 ggPCB/cm2 to be

the lowest exposure concentration (body-burden 834 ggPCB/g

dry mass) causing first mortality in L. terrestris (Table

4). However, 100.0 ggPCB/cm2 was equivalent to the highest

lethal concentration in E. foetida (body-burden 5936 pgPCB/g

dry mass). Occurrence of high mortality in E. foetida

suggested that 5-d PCB exposure had more modulatory effects

on E. foetida than L. terrestris. Furthermore, at equal

exposure concentration, E. foetida accumulated more PCB than

L. terrestris. Exposure to 2.5 AgPCB/cm2 (body-burdens of

1389 and 56.1 ggPCB/g dry tnass) caused no mortality among

experimentals of either species over the 5-d period (Table

4) . Exposure to 5.0 AgPCB/cm2 (body-burdens of 1896 and

39

Table 4.whole body

Comparison of nominal filter paper exposure andconcentrations of PCBs between the earthworms

Lumbricus terrestris (Lt)a andfor 5-d to PCB.

Eisenia foetida (Ef) exposed

Nominal Body Burden Number of WormsExposure Concentration Percent

Concentration (/g/g dry Exposed Survivors Survivors

(gg/cm2) mass)

Ef Lt Ef Lt -"Ef /Lt Ef/Lt Ef/Lt

2.5 2.5 1389 56.1 30/15 30/15 100/100

5.0 5.0 1896 76.5 30/18 25/18 84/100

6.25 --- 2967 ---- 10/-- 08/-- 80/---

10.0 10.0 2895 184.9 40/26 30/26 75/100

b c 91.2d

100.0 100.0 5936 834.0 10/12 01/09 10/75

---- 200.0 ---- 982.0 --/12 -- /06 --/50

e f

400.0 ---- 1311 -- /12 -- /03 -- /25

800.0 ---- 1426 -- /12 -- /05 -- /45

a Data on LC/LD 50 for L. terrestris from Rodriguez etal. (1989). 2

b LC50 = 30.4 gg/cmcd

ef

LD50 = 4450 jig/g dry mass.Data on LD50 for L. terrestris from Eyambe et al.(1990, 1991) [4].LC50 = 300 pg/cm2 from Rodriquez et al. (1989) [3].LD50 = 1139 pg/g dry mass from Rodriquez et al [3].(1989) .

76.5 gPCB/g dry mass) only caused mortality among Ej.

foetida, reducing their survivorship to 84%. Exposure to

10.0 gPCB/cm2 decreased the survivorship of E. foetida even

further to 75%, yet no mortality occurred among L.

terrestris (Table 4).

40

Comparative Immunoassay

A direct comparison of ER and SR formation and

phagocytosis by coelomocytes from two species of earthworms,

E. foetida and L. terrestris, is shown in Tables 5 and 6.

Previous works at UNT [3-4] showed that 5-d exposure to 10

gPCB/cm 2 substantially reduced the ability of L. terrestris

coelomocytes to form ER and SR and to phagocytize RRBC's.

Additionally, decreased SR in L. terrestris after 5-d

exposure to 5.0 pgPCB/cm2 was reported by Rodriquez [3]. ER

and SR formation and phagocytosis by coelomocytes from E.

foetida exposed for 5-d to nominal PCB concentrations of

10.0 gPCB/cm 2 were significantly lower (Dunnett's Alpha =

0.05, P = 0.0001) than those from non-exposed controls.

Except for SR formation by coelomocytes from the group

exposed to 5.0 ggPCB/cm2 , there were no significant

differences between controls and other exposed groups.

In either species, exposure to 10.0 AgPCB/cm 2 decreased

mean SR, ER and phagocytosis percentages significantly.

Furthermore, either species showed a decrease in SR

formation by coelomocytes after 5-d exposure to 5.0

ggPCB/cm 2.

41

Table 5. Comparison of erythrocyte (ER) and Secretory (SR)rosette formation, and phagocytosis (PG) from two species ofearthworms, Eisenia foetida and Lumbricus terrestris, exposedfor 5-d to three PCB concentrations on filter paper.Expressed as actual number (7 SE) of cells per 100 randomlycounted coelomocytes and as % of normal.1

Earthworms

E. foetida L. terrestris

Nominal FunctionConc.

(pgPCB/cm 2) ER SR PG ER SR PG(5 se) ( se)

2.5 33.29 32.17 30.88 12.93(1.8) (1.8) (1.4) (0.8)

5.0 38.58 30.58a 31.17 9.44ab(1.8) (1.8) (1.7) (0.7)

10.0 17.35a 14.23a 13.58a 6 .2 3 ab(2.1) (1.3) (1.4) (0.8)

10.0 4.5ac 3 .9 ac 4 . 6 0 ac(0.3) (0.2) (0.3)

Expressed as % of normal

2.5 93 87 89-110b(1.8) (1.8) (1.4) (0.8)

5.0 100 82a 90 7 7 ab(1.8) (1.8) (1.7) (0.7)

10.0 48a 38a 39a 45ab(2.1) (1.3) (1.4) (0.8)

10 .0 36ac 38ac 34ac(0.3) (0.2) (0.3)

a Significantly different (Dunnett's Alpha = 0.05)controls.

b Data on SR, ER, and PG for L. terrestris fromRodriguez et al. (1989) [3].

C Data on SR, ER, and PG for L. terrestris from Eyet al. (1990, 1991) [4].

f rom

ambe

42

Table 6. Comparison on lethal (LC/LD50 95% CL) andsublethal immunomodulatory effects (5? se), expressed as %normal (i.e., controls), of PCB on earthworms, Eisenia foetidaand Lumbricus terrestris.

Earthworms

Parameter Eisenia foetida Lumbricus terrestris

Lethality a b

LC50 (pg/cm 2) 30.4 300(14.8-61.2) (180-590)

LD50 (gPCB/g dry 4450.0 1139mass) (3572-5556) (1000-1368)

Immunomoduatory'

Body Burdens 2895 184.9 91.2(gg/g dry mass)

Secretory 38 1.31 45 0.87 38 0.20rosettes (37.11) (13.79) (10.2)

Erthrocyte 48 2.09rosettes (35.82)

Phagocytosis 39 1.36 48 0.41 34 0.22(34.58) (6.77) (13.3)

a Data on LC/LD50s, S and E rosettes, .and phagocytosisfor 1. terrestris from Rodriquez et al. (1989).

b Data on S and E rosettes, and phagocytosis for L.terrestris from Eyambe et al. (1990, 1991).

C Rosetting and phagocytosing coelomocytes presented as% of normal (i.e. controls) after 5-d exposure tonominal concentration of 10.0 ggPCB/cm2 on filterpaper. Values for respective controls, as actualnumber per 100 randomly-counted coelomocytes, aregiven in parenthesis

CHAPTER V

DISCUSSION

Direct filter paper exposure to PCB using the ring

method developed for the EEC [19] and recently adopted by

the US EPA [16] was extremely effective for dosing E.

Foetida. Compared to results presented for _L. terrestris by

Rodriguez [3], the relation between nominal filter paper

concentrations and tissue body burdens of PCB was higher in

E. foetida (R 2 = 0.0965 vs. 0.44, respectively) . Exposure

of E. foetida to nominal PCB concentrations of 2.5, 5.0,

10.0 and 100.0 Ag/cm2 yielded body burdens of 1389, 1896,

2895 arid 5936 Ag/g dry mass, respectively. However,

Rodriguez [3] reported lower body burdens (56.1, 76.1, 184.9

and 834.0 Ag/g dry mass) in _L. terrestris at identical

exposure concentrations. Furthermore, Eyambe [4] reported

body burdens of only 91.2 gg/g dry mass in L. terrestris at

10.0 gg/cm 2 exposure concentration.

Differences in body burdens and coefficients of

determination (R 2) can be explained in terms of (1) exposure

techniques, (2) ranges of PCB exposure concentrations and

(3) differences between the two earthworm species.

Rodriguez [3] exposed L. terrestris to PCB using filter

paper discs (63.58 cm2), placed at the bottom of 0.47 liter

glass jars, following methods described by Roberts and

43

44

Dorough [14]. In their method, worms were exposed to

smaller surface area than were E. foetida using the ring

method (79.2 cm2). L. terrestris was exposed to a range of

nominal PCB concentrations that extended to 800.0 g/cm2 ,

whereas the range of nominal exposure concentrations for E.

Foetida extended only to 100.0 gg/cm2 . Data showed that PCB

body burdens in L. terrestris began to decrease relative to

nominal exposure concentrations above 100.0 Ag/cm2 , which

reduced the strength of R2 reported by Rodriguez [3]. In

addition, Eyambe [4] reported lower body concentration in L.

terrestris at equal PCB exposure concentrations, using the

ring method. Differences in size results in E. foetida

having a higher surface area to body mass ratio than L.

terrestris which should have enhanced PCB uptake. Moreover,

E. foetida appears to have a qualitatively different lipid

composition than L. terrestris which may have influenced

their respective uptake of lipophilic PCB. Analysis of

lipids in both species will be conducted at UNT. Moratti

[58] reported higher tissue levels of both hydrophilic

cadmium and lipophilic chlordane in E. foetida than in L.

terrestris after 5-d exposure via filter paper ring contact

test and 14-d artificial soil test [19]. In addition,

Gilman and Vardanis [59] reported higher tissue

concentrations of carbofuran in E. foetida than in L.

terrestris after exposure in artificial soil.

45

Potential toxicities of chemicals usually are expressed

as LC50s, although LD50 values are more meaningful. LC50

for PCB in E. foetida was lower than reported for L.

terrestris by Rodriguez [3], but the LD50 was reversed.

Gilman and Vardanis [59] reported higher LC50 and LD50 for

carbofuran in E. foetida than for L. terrestris. Moratti

[58] reported higher LC50 and LD50 for cadmium, and LC50 for

chlordane in E. foetida than for L. terrestris. These

comparative data indicate that E. foetida may be more

tolerant to organics, including PCB and at least one metal

than L. terrestris.

In E. foetida, both suppression of SR formation and

mortality first occurred at 5.0 gPCB/cm2 tissue exposure

concentration of 1896 pg/g dry mass). Whereas, Rodriguez

[3] reported suppression of SR formation at a much lower

tissue concentration of PCB (76 gg/g dry mass) than that

producing lethality (100.0 gg/cm2 ; 834.2 pg/g dry mass) in

L. terrestris. Although immunosuppression was shown in SR,

ER and phagocytosis in E. foetida, it occurred at tissue

levels of PCB which also producted mortality. Thus, for PCB

and the three immune parameters, L. terrestris is more

sensitive than E. foetida for measuring sublethal toxicity.

It also appears to be more resistent, in general, to

toxicity than L. terrestris; suggesting that the common

night crawler may be a more sensitive bioindicator of

terrestrial contamination.

APPENDIX A

EARTHWORM TISSUE EXTRACTION PROCEDURES

46

47

Purpose:

Removal of organic compounds for instrumental analysis.

Safety Notes:

The entire process must be carried out under a hood

having proper ventilation. In addition, Latex gloves

and safety goggles are highly recommended.

Materials:

Sonifier Cell Disruptor, electric oven, test tubes,

forceps, 50% hexene/benzene solution, glass pipettes,

aluminum foil and cups.

1. First, be certain that the operating surface is covered

by aluminum foil to minimize chemical contamination.

Gently shake the vial toward the table for 45 seconds

or until the tissue becomes disengaged from the filter

paper. If the tissue did not appear use a micro glass

pipette to loosen its position on the filter paper and

then repeat the previous step. After removal of the

worm tissue, use small forceps to hold it up 10 cm

above a petri dish. Then give the worm acetone shower

(simply pour 5 ml of acetone over its entire body).

48

Use forceps to take the contaminated filter paper out

of the vial. Later, the filter paper can be destroyed

properly by placing it in an incinerator.

2. Place the worm on a pre-weighed aluminum cup and place

the cup in an electric oven for 5-d. Set the oven as

the following: a) power selection switch on low b)

temperature switch at 450C.

3. After the fifth day, remove the worm from the oven and

weigh it. Then transfer it into a pestle and pulverize

it using a mortar. Pulverization process must be

performed within a large plastic bag in order to

prevent the ingestion and/or inhalation of the dried

pulverized material.

4. Transfer the contents of the pestle into a coded (e.g.

#1) test tube and then add 10 ml of 50% hexene/benzene

solution. Vortex the mixture for about 2 minutes.

5. Use a stand and a clamp to hold the test tube 30 cm

above the work surface. Position the probe of a

Sonifier Cell Disruptor 3 cm below the solution line.

Throughout the sonifi6ation process, the probe must

remain secure in its position with the help of another

large stand and clamp. The tip of the probe should not

49

touch the bottom of the test tube at any time, to avoid

shattering the test tube. Set the output of the

sonifier at 6 and set its timer for 3 minutes.

6. Remove the probe and centrifuge the test tube at 1000

rpm for 5 min. With a glass pipette, transfer the

supernatant into another coded test tube. Add another

10 ml of 50% hexene/benzene to the first test tube,

vortex the sediment and twice repeat #4 and 5. After

the third wash, the second test tube should contain at

least 30 ml of clear supernatant. If the sediments

appeared in the supernatant, centrifuge the second test

tube and transfer the supernatant into another test

tube. Save the supernatant and discard the sediments

at this time.

7. Place the corresponding test tube under a ventilating

hood or inside an electric oven (low temperature) for 7

days to evaporate the solvent.

APPENDIX B

(1) Chromatograph of an extract from an earthworm

exposed to 0.0625 pg/cm2 of Aroclor 1254

(Attenuation: 128).

(2) Chromatograph of an extract from an earthworm

exposed to 0.075 jg/cm 2 of Aroclor 1254

(Attenuation: 128).

(3) Chromatograph of an extract from a control

earthworm (Attenuation: 128).

50

51

Jl

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2. Fitzpatrick, L. C., A. J. Goven, B. J. Venables, and E.L. Cooper. 1990. Earthworm immunoassays for evaluatingbiological effects of exposure to hazardous chemicals.In: S. Sandhu et al. (Ed.), In Situ Evaluation ofBiological Hazards of Environmental Pollutants. PlenumPress, New York. 119-129.

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