AD-A264 408

EDGEWOO D

RESEARCH,

DEVELOPMENT &

ENGINEERING

CENTER ERDEC-TR-037

TOXICITY OF SELECTED MUNITIONS AND

MUNITION-CONTAMINATED SOIL

ON THE EARTHWORM (EISENIA FOETIDA)

Carlton T. Phillips

Ronald T. Checkai

Randall S . Wentsel

RESEARCH AND TECHNOLOGY DIRECTORAT E

D'F1C Aprii '1993

!7 Y Q

-

Y

~E,

WAY 141993

~ .

Approved for public release ; distribution is unlimited. U

: C

.S. ARMY B D

CHEMICAL ~

AND BIOLOGICAL

DEFENSE AGENCY

REPRODUCED BY '►ving Ground, Maryland

U.S 93-10812 . DEPARTMENT OF COMMERCE ~ NATIONAL TECHNICAL

INFORMATION SERVICE

SPRINGFIELD, VA 2216 1

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Disclaimer

The findings in this report are not to be construed as an official Department of the Army position unless so designated by

other authorizing documents .

A~ ::=.
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4 . TITLE AND SUBTITLE S. FUNDING NUMBER S

Toxicity of Selected Munitions and Munition-Contaminated PR-89PP9914

Soil on the Earthworm (Eisenia foetida)

6. AUTHOR(S) Sales Order No . 1HCB

Phillips, Carlton T ., Checkai, Ronald T ., and

Wentsel, Randall S .

7 . PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) B. PERFORMING ORGANIZATION

REPORT NUMBE R

DIR, ERDEC,* ATTN : SCBRD-RTL, APG, MD 21010-5423 ERDEC-TR-03 7

9 . SPONSORING/ MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSORING/ MONITORING

AGENCY REPORT NUMBER

DIR, BRDL, ATTN: SGRD-UBG-Z, Fort Detrick,

Frederick, MD 21701-5010

11 . SUPPLEMENTARY NOTE S

*When this work was conducted, ERDEC was known as the U .S . Army Chemical

Research, Development and Engineering Center, and the authors were assigned to the

Research Directorate .

12a. DISTRIBUTION I AVAILABIUTY STATEMENT 12b . DISTRIBUTION CODE

Approved for public release ; distribution is unlimited .

13 . ABSTRACT (Maximum 200 words)

The munitions HMX (cyclotetramethylenetetranitramine), RDX (cyclotrimythylenetrinitramine), and TNT (trinitrotoluene) and soils from two Army ammunition plants

(Radford, VA and Milan, TN), both contaminated with munitions or munition residues,

were tested for their toxicity to the earthworm ( Eisenia foetida ) . HMX and RDX were

tested at 0, 50, 100, 200, 400, and 500 pg/g concentrations by weight in an artificial soil mixture . TNT was tested at 0, 80, 110, 140, 170, and 200 μg/g in artificial soil and at 0, 150, 300, 400, and 500 μg/9 in a naturally-occurring forest

soil . Contaminated Radford soil ( 60 pg/g TNT and 100 Mg/g trinitrobenzene) and contaminated and fortified Radford soil (fortified with an additional 125 pg/g of 2,4-

dinstrotoluene and 40 jj g/g 2,6-dinitrotoluene) were tested for their toxicity to

earthworms . Contaminated and fortified Milan soil (initially contaminated wit h

13 pg/g HMX, 117 μg/g RDX, and 16 pg/g TNT and then fortified with 1000 μg/g of BMX,

RDX, and 2,4-DNT each and 400 Mg/g of 2,6-DNT) was also tested for its toxicity to

earthworms . The lethal and sublethal effects these munitions and munition-contaminated soils had on earthworms are discussed in terms of the no observable effects

level ( NOEL) and lowest observable effects level (LOEL) .

14. SUBJECT TERMS 15. NUMBER OF PAGES

Earthworms ( Eisenia foetida) Munitions 22

No observable effects level ( NOEL) Munition-contaminated soil 16. PRICE CODE

Lowest observable effects level (LOEL )

17 . SECURITY CLASSIFICATION 18. SECURITY CLASSIFICATION 19 . SECURITY CLASSIFICATION 20 . LIMITATION OF ABSTRACT

OF REPORT OF THIS PAGE OF ABSTRACT

UNCLASSIFIED UNCLASSIFIED UNCLASSIFIED UL

NSN 7540-01-280-5500 Stan dard Form 298 (Rev 2-89)

~ Prescribed by ANSI Sid . 1 39- 1 e

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Blank

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PREFACE

The work described in this report was authorized under

Project No . 89PP9914, Sales Order No . 1HCB . This work was

started in July 1989 and completed in February 1992 .

The use of trade names or manufacturers' names in this

report does not constitute an official endorsement of any commercial products . This report may not. be cited for purposes of

advertisement .

Reproduction of this document in whole or in part is

prohibited except with permission of the Director, U .S . Army

Edgewood Research, Development and Engineering Center (ERDEC),*

ATTN : SCBRD-RT, Aberdeen Proving Ground, MD 21010-5423 .

However, the Defense Technical Information Center and the National Technical Information Service are authorized to reproduce the

document for U .S . Government purposes .

This report has been approved for release to the

public .

Acknowledgments

The authors acknowledge Jesse Barkley and Dr . Michael

Major, BRDL, for their timely support of this research ;

Dr . Michael Simini, GeoCenters, for preparing statistics ; and

John Amos and Raphael Nwanguma, GeoCenters, for providing analytical data .

*When this work was conducted, ERDEC was known as the U .S . Army

Chemical Research, Development and Engineering Center, and the

authors were assigned to the Research Directorate .

3

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QUALITY ASSURANCE

This study was examined for compliance with Protocol 22091000X029 and Standard

Operating Procedures governing the testing described below. The dates of all

inspections and the dates the results of those inspections were reported to the Study

Director and management were as follows:

Phase inspected P= Date reported

Soil preparation; 12 Apr 91 15 Apr 91

weighing and addition

of earthworm s

Final report and Data Audit 14 Sep 92 14 Sep 92

To the best of my knowledge, the methods described were the methods followed

during the study. The report was determined to be an accurate reflection of the raw

data obtained.

DENNIS W. JOHNS

Quality Assurance Coordinator, Research

4
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CONTENTS

Page

1 . INTRODUCTION . . . . . . . . . . . . . . . . . . 7

2 . METHODS AND MATERIALS . . . . . . . . . . . . . . 7

2 .1 Range-Finding Studies . . . . . . . . 7

2 .2 Standardized Earthworm Toxicity Studies . . 10

3 . RESULTS AND DISCUSSION . . . . . . . . . . . . . 12

3 .1 Results of Range-Finding Studies . . . . . . 12

3 .2 Results of Standardized Earthworm

Toxicity Studies . . . . . . . . . . . . . 14

4 . CONCLUSIONS . . . . . . . . . . . . . . . . . . . 18

LITERATURE CITED . . . . . . . . . . . . . . . . 19

APPENDIX - STATISTICAL DATA . . . . . . . . . . . 21

DTIC QUAL.. yTj 74,0it,D 3

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LIST OF TABLES

1 . Experimental Design of Range-Finding Studie s

on Earthworms . . . . . . . . . . . . . . . . . . . 9

2 . Experimental Design of Acetonitrile Studies

(#6 & 7) . . . . . . . . . . . . . . . . . . . . . . 10

3 . Physical and Chemical Characteristic s

of Forest Soil . . . . . . . . . . . . . . . . . . . 11

4 . Experimental Design of Standardized Earthwor m

Toxicity Studies . . . . . . . . . . . . . . . . . . 12

5 . Summary of the Results of Range-Finding Studies . . . 13

6 . Summary of the Results of Acetonitrile Studies . . . . 14

7 . Effects of Radford Contaminated and Fortified Soi l

on Earthworms . . . . . . . . . . . . . . . . . . . 15

8 . Effects of TNT in Artificial Soil on Earthworms . . . 16

9 . Effects of TNT in Forest Soil on Earthworms . . . . . 17

6

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TOXICITY OF SELECTED MUNITIONS AND MUNITION-CONTAMINATED SOIL

ON THE EARTHWORM ( EISENIA FOETIDA)

1 . INTRODUCTION

There is a need to determine the toxicity of munitions

and munition-contaminated soil on the soil ecosystem . Out-ofdate and out-of-specification munitions have commonly been

disposed of by burning and detonation on unprotected ground . 1

This practice generates a mixture of contaminants into the

immediate area at high concentrations . 2

The purpose of this study was to develop baseline

environmental toxicity data on cyclotetramethylenetetranitramine

(HMX), cyclotrimethylenetrinitramine ( RDX), trinitrotoluene (TNT)

and some of their degradation by-products ( i .e ., 2,4-dinitrotoluene [2,4-DNT] and 2,6-dinitrotoluene [2,6-DNT]) utilizing the

earthworm toxicity test . The results of these tests will help

determine at what level this contamination will adversely affect

the environment . The data generated will assist decision makers

in determining the use and disposal of these materials .

The earthworm toxicity test uses the earthworm, Eisenia

foetida , as the test species . Because of their role in maintaining the physical characteristics and processes of soil, such as

aeration, water permeability, and breakdown of organic matter,

earthworms are considered key organisms in the soil community .

Earthworms, which can number up to 250,000 individuals per acre,

increase the fertility of soil by increasing the availability of

nutrients, and they are also an important link in the food chain .

Roberts and Dorough3 published a review of the importance of

earthworms to terrestrial ecosystems and their use in assessing

the hazards of chemicals to these nontarget organisms . Dean-Ross

discussed the strengths and weaknesses of experimental methods

for testing the toxicity of chemicals to earthworms and the

sensitivity of earthworm species to various chemicals . Based on

this review, Dean-Ross recommended that Lumbricus and

Eisenia foetida be used as the test species of choice .

2 . METHODS AND MATERIALS

2 .1 Range-Finding Studies .

The method used to determine the toxicity of munitions

and munition-contaminated soil to earthworms was adapted from

Neuhauser and co-workers .5 The earthworms ( Eisenia foetida )

originally purchased from Bert's Bait Farm, Irvine, KY, were

raised in our laboratory from this initial stock . They were

housed in styrofoam coolers at ambient room temperature prior to

the start of a study .

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The range-finding study consisted of one replicate pe r

concentration of compound tested . For each replicate, 200 g test

media was placed in a 600-mL beaker . The main component of the

test medium was a nonsterile artificial soil . The use of an

artificial soil limits test variability that would otherwise

occur due to heterogeneity of soil parameters . Other advantages

of using an artificial soil mixture are ease of preparation and

comparability to other data in the literature .6 The components

of the artificial soil were 10 % finely ground sphagnum peat ,

20% kaolinite clay, 69 % fine sand, and 1% calcium carbonate .

Five earthworms were randomly selected, weighed as a

group, and placed in beakers containing test soil . Each beaker

was covered with nylon screen and cheesecloth held in place by a

rubber band . The soil moisture level was brought up to 25% (w/w)

by mixing 50 mL distilled water with each 200 g test medium . The

beakers were randomly placed in a tray of distilled water within

a low-temperature incubator (21 .0° C, ±0 .2) during the 14-day

test period . The cheesecloth and tray of water, which helped

keep the relative humidity high within the test chambers, prevented the test medium from drying out . After 14 days, the

earthworms were reweighed and examined for physical condition

(i .e ., color, rigidity, and motility) .

Five different treatments were tested for their toxicity to earthworms in these range-finding studies . The first

study used munition-contaminated soil from Radford Army Ammunition Plant, Radford, VA . High-performance liquid chromatography

(HPLC) analysis showed that this soil contained 100 μg/g trinitrobenzene (TNB) and 60 pg/g TNT . Mixing some of the Radford

contaminated soil with artificial soil provided treatment levels

of 0, 1 .0, 2 .5, 5 .0, and 10 .0% by weight (contaminated : artificial soil) . This mixture produced concentrations of 0, 1 .0, 2 .5,

5 .0, and 10 .0 μg/g of TNB and 0, 0 .6, 1 .5, 3 .0, and 6 .0 μg/g TNT .

The second study used Radford contaminated soil

fortified with 125 μg/g 2,4-DNT and 40 μg/g 2,6-DNT . The treatment levels and the amounts of TNB and TNT in each level were the

same as the first study except that 0, 1 .25, 3 .13, 6 .25, and

12 .50 pg/g 2,4-DNT and 0, 0 .4, . 1 .0, 2 .0, and 4 .0 μg/g 2,6-DNT

were incorporated into the mixture .

Milan contaminated and fortified soil, Milan Army

Ammunition Plant, Milan, TN, was used in the third range-finding

study . An HPLC analysis showed that this soil was contaminated

with 13 μg/g HMX, 117 μg/g RDX, and 16 μg/g TNT . This soil was

fortified with an additional 1000 μg/g HMX, RDX, and 2,4-DNT, and

400 μg/g 2,6-DNT .

The fourth and fifth studies used only RDX and HMX

mixed with artificial soil to produce concentrations of 0, 50,

100, 200, 400, and 500 μg/g by weight . The test medium was

8
P. 11

prepared by mixing varying amounts of either contaminated soil or

contaminated and fortified soil with artificial soil to obtain

the desired concentration . For tests of munitions only, the

selected munition was first dissolved in acetonitrile and then

sprinkled over sand . This was allowed to air dry overnight in a

fumehood. The sand/munition mixture was used to replace some of

the sand in the artificial soil to obtain the desired concentration . The experimental design for the range-finding studies is

summarized in Table 1 .

Table 1 . Experimental Design of Range-Finding Studies

on Earthworms

Levels Contaminants ( u g /g)*

Treatment ($) Tf18 TNT 2 .4-DNT 2 .6-DNT HMX RDX

Radford contaminated 0 - - - - - -

soil 1.0 1 .0 0 .6 - - - -

2 .5 2 .5 1 .5 - - - -

5 .0 5 .0 3 .0 - - - -

10-0 10 .0 6 . 0

Radford contaminated 0 - - - • - -

and fortified soil 1 .0 1.0 0 .6 1.25 0.4 • -

2 .5 2 .5 1 .5 3.13 1.0 - -

5 .0 5 .0 3 .0 6.25 2 .0 - -

10 .0 10 .0 6 .0 12 .50 4. 0

Milan contaminated 0 • - - - - -

and fortified 0 .50 - 0.08 5.0 2.0 5 .07 5 .59

5 .0 0:80 50 .0 20.0 50 .65 55 .8 5

RDX 0 - - • - • 0

0 .005 - • - - - 50

0 .01 - • - - - 100

0 .02 - • - • - 200

0 .04 - - - - - 400

0 .05 - 500

HM C 0 - - - • 0 -

0 .005 - - - - 50 -

0 .01 - - - - 100 -

0 .02 - - - - 200 -

0 .04 - • - - 400 -

0 .05 - 500

*The contaminants used in these studies were trinitrobenzene

(TNB), trinitrotoluene ( TNT), 2,4-dinitrotoluene ( 2,4-DNT), 2,6-

dinitrotoluene ( 2,6-DNT), cyclotetramethylenetrantitramine (HMX),

and cyclotrimethylenetrinitramine (RDX) . For soil studies, the

concentrations represent the percent of contaminated soil in the

total mixture ( contaminated and artificial soil) on a weight-toweight basis . For the other studies, the concentrations are the

amount of compound added to the total amount of test medium (w/w

basis) .

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To ensure that the solvent, acetonitrile, which wa s

used to dissolve the munitions in studies 4 and 5, has no lethal

or sublethal effects on earthworms, a separate set of studies

(#6 & 7) were conducted in conjunction with the RDX study . In

study #6, RDX was incorporated into the artificial soil by

directly mixing 0 .25g RDX with 49 .75g sand . The spiked sand was

then used to make RDX concentrations in the same manner as in

study 4 . In study #7, only acetonitrile (4 .4 mL) was sprinkled

over the sand and allowed to air dry overnight in a hood . The

spiking procedures used in study 4 were followed to obtain the

desired concentrations . The experimental design for these two

studies is summarized in Table 2 .

Table 2 . Experimental Design of Acetonitrile Studies (#6 & 7)

Concen . spiked sand additional sand Other Components of Water

(Ogg) (g) (e) artificial soil (g) (mL)

0 0 138 62 50

50 2 136 62 50

100 4 134 62 50

200 8 130 62 50

400 16 122 62 50

500 20 118 62 50

2 .2 Standardized Earthworm Toxicity Studies .

A standardized earthworm toxicity study consisted of

three replicates for each concentration of compound tested . For

each replicate, 200 g soil (dry weight) was used . The soil

moisture level was brought up to 25% of the total amount of soil

used by adding 50 mL distilled water to the 200 g soil and mixing

in a food blender . The moist soil was placed in a 600-mL beaker .

Five earthworms were rinsed with distilled water, blotted with a

paper towel, and weighed as a group . The earthworms were added

to the beakers, covered with cheesecloth and nylon screen held in

place by a rubber band . Beakers were randomly placed in trays of

water inside a low-temperature incubator set at 21 .0 ± 0 .2°C . At

the end of the two-week study period, earthworms were removed,

examined for physical condition, and again weighed as a group .

~Average weight loss or gain were used to determine sublethal

--effects and mortality used to determine lethal effects . A

standardized toxicity test was conducted on earthworms to determine the toxicity of Radford contaminated and fortified soil, TNT

mixed with artificial soil, and TNT mixed with a forest soil .

The test medium consisted of three soil types . One

type of test medium was soil obtained from an open-burning site

at Radford Army Ammunition Plant, Radford, VA . For this study,

contaminated and fortified soil was mixed with uncontaminated

soil from the same site to produce the desired concentrations .

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The second test medium was a forest soil obtained from an uncontaminated area at the Edgewood Area of Aberdeen Proving Ground,

MD . The physical and chemical characteristics of this soil are

summarized in Table 3 . Some of the forest soil was air dried,

sieved and then spiked with a known quantity of munition to

produce a spike . The spike was then added to uncontaminated soil

to produce the desired concentration of munition in the test

medium . The third type of test medium was the artificial soil

mixture .

Table 3 . Physical and Chemical Characteristics of Forest Soil

Soil Parameters*

Mechanical Analysis Soil Analysis

% sand - 33 .5 Nog- (lb/A) - 35 .7

P2O5 (lb/A) - 31 .5

% silt - 56 .0 IC20 (lb/A) - 48. 0

Ca (lb/A) • 20 .0

% clay - 10 .5 Mg ( lb/A) - 23 .0

Mn (lb/A) . 4 . 0

% organic matter - 5 .9 Zn ( lb/A) - 9 .6

Cu (lb/A) . 3 .2

Texture - silt loam CEC (meq/100g) - 6 . 2

pH - 3.8

*Determined by the Soil Testing Laboratory, University of

Maryland ( College Park, MD )

To produce a spike, the munition was first dissolved in

acetonitrile ( approx . 20 mL) and then pipetted onto a layer of

either soil ( for the forest soil) or sand ( for the artificial

soil) at a specified amount to produce a spike . The spike was

allowed to air dry overnight in a hood which permitted the highly

volatile acetonitrile to evaporate, leaving the munition in the

soil . After drying, the munition/soil mixture was ground and

thoroughly mixed in a food blender to ensure uniformity of the

mixture . An appropriate amount of spike was mixed with additional quantities of soil to produce a spike-soil mixture of the

desired concentration. The soil moisture level of 25% was

obtained by adding 50 mL distilled water to each 200 g test

medium during mixing in a food blender . The experimental design

for these studies are summarized in Table 4 .

The statistical methods used to evaluate the data were

the Newman-Keuls pairwise comparison of means,7 and the Analysis

of Covariance ( ANCOVA)a to test the weight differences of the

earthworms . The LCm was performed on a basic LCm program . '

il

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Table 4 . Experimental Design of Standardized Earthworm

Toxicity Studies .

Levels Contaminants u8/¢-)

Treatment ($) TNB TNT 2.4-DNT 2 .6-DNT

Radford contaminated 0 - - ' -

and fortified soil 10 10 6 12 .50 4

25 25 15 31.25 10

50 50 30 62.50 20

75 75 45 93.75 30

100 100 60 125 .00 40

TNT in artificial soil 0 -

-

- '

0 .008 • 80 - -

0 .011 - 110 - •

0 .014 140 - -

0 .017 - 170 - -

0,020 200

TNT in forest soil 0 • - • '

0 .015 • 150 • -

0 .030 - 300 • -

0 .040 - 400 - -

0 050 500 -

3 . RESULTS AND DISCUSSION

3 .1 Results of Range-Finding Studies .

Results of the range-finding studies are summarized in

Table 5 . Radford contaminated soil mixed with artificial soil

produced no lethal or sublethal effects (increasing weight loss

as concentrations increased ; a change in physical condition such

as color, rigidity, texture, and motility) on earthworms . All of

the earthworms survived and gained an equal amount of weight over

the 14-day study . The results of this range-finding study

indicated that the Radford contaminated soil was neither lethal

nor sublethal to earthworms at the concentrations tested .

Analysis of this soil showed that it was contaminated with

100 μg/g TNB and 60 μg/g TNT .

Radford contaminated and fortified soil produced no

lethal or sublethal effects on earthworms in the concentrations

tested . All of the earthworms survived and gained weight over

the 14-day study . The results obtained from the Radford contaminated and fortified soil range-finding study indicated that the

small quantity of added TNT degradation by-products were not

toxic enough to produce lethal or sublethal effects on earthworms

at the concentrations tested .

12
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Table 5 . Summary of the Results of Range-Finding Studie s

INITIAL EARTHWORMS FINAL EARTHWORMS Av.wt .

Levels count Tot .wt . Av.wt . count Tot .wt Av.vt . Diff . Survival

Treatment ($) (#) (S ) (g) (#) ( g) (g) (+/-) Rate (8)

Radford- 0 5 1.40 0.28 5 1 .65 0 .33 + 0 .05

contaminated 1 .0 5 1.25 0.25 5 1 .52 0 .30 + 0 .05

2 .5 5 1.35 0.27 5 1 .65 0 .33 + 0 .06 100

5 .0 5 1.12 0.22 5 1 .30 0 .26 + 0 .0 4

10 .0 5 0 .99 0 .20 5 1 .30 0 .26 + 0 .06

Radford- 0 5 1.44 0 .29 5 2 .09 0 .42 + 0 .13

contaminated 1 .0 5 1.27 0 .25 5 1.85 0 .37 + 0 .1 2

and 2.5 5 1.45 0 .29 5 1 .86 0 .37 + 0 .08 100

fortified 5 .0 5 1.46 0 .29 5 1 .75 0 .35 + 0 .0 6

10 .0 5 1 .34 0 .27 5 1 .57 0 .31 + 0 .04

Milan- 0 5 1.68 0 .34 5 2 .10 0 .42 + 0 .08

contaminated 0 .50 5 1.18 0 .24 5 1 .63 0 .33 + 0 .09 100

fortified 5O 5 1 .34 0 .27 5 1 .65 0 .33 + 0 .0 6

RDX 0 5 2.60 0 .52 4 2 .10 0 .53 + 0 .01 80

0 .005 5 2 .25 0 .45 5 1.99 0 .40 - 0 .05 100

0 .01 5 2.91 0 .58 4 2 .00 0 .50 - 0 .08 80

0 .02 5 2.94 0 .59 5 2 .53 0 .51 - 0 .08 100

0 .04 5 2.66 0 .53 5 2 .07 0 .41 - 0 .12 100

0 .05 5 3 .35 0 .67 5 2 .74 0 .55 - 0 .12 100

HIK C 0 5 2.53 0 .51 5 2 .42 0 .48 - 0 .0 3

0 .005 5 2 .61 0 .52 5 2 .46 0 .49 - 0 .0 3

0 .01 5 2.69 0 .54 5 2 .47 0 .49 - 0 .05 100

0 .02 5 2.42 0 .48 5 1 .99 0 .40 - 0 .0 8

0 .04 5 2.76 0 .55 5 2 .34 0 .47 - 0 .08

0 .05 5 2 .45 0 .49 5 2 .05 0 .41 - 0 .08

Milan contaminated and fortified soil did not produc e

any noticeable effect on earthworms . All earthworms survived and

gained weight over the 14-day study .

• When RDX alone was tested on earthworms, it-was found

that the earthworms had an increasing weight loss with increasing

concentrations . Survival was near 100 % for all concentrations

except for one missing earthworm in the control chamber and one

dead in the 100 μg/g level, which could not be attributable to

RDX .

The HMX range-finding study indicated that FIIKX caused

an increased weight loss at the higher concentrations ( i .e, 200,

400, and 500 Ng/g) . Earthworms at the 0 level had a 4 % weight

loss . Those at the 50 Ng/g level lost 6 % , while earthworms at

the 100 μg/g level lost 8 % . There was an increased weight loss

at the three higher concentrations . Earthworms at 200 /sg/g lost

18%, the ones at the 400 pg/g level lost 15% , and those at

500 μg/g lost 16% .

13

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p. 16

The results of the RDX and HMX range-finding studie s

produced no lethal effects on earthworms at the concentrations

tested . Weight loss increased as concentrations increased

indicating that higher concentrations produce some sublethal

effects . Additional studies, using higher concentrations, would

be required to determine an LC for these munitions . Concentrations at open burn/open detonation sites may exceed 1,000 μg/g

(limit for reactivity2) .

The acetonitrile studies (Table 6) indicated that

acetonitrile did not produce any toxic effects on earthworms (at

least not in the amounts used in these studies) . When RDX was

directly mixed into the artificial soil (using no acetonitrile),

the effects were similar to the study in which the RDX was first

dissolved in acetonitrile before it was incorporated into the

test medium .

In the acetonitrile/sand study, there was no indication

of toxicity to earthworms, with 100 % survival at all concentrations . There was a small weight gain (an average of 0 .015

g/earthworm) at 0, 200, 400, and 500 μg/g levels and an average

weight loss of 0 .02 g/earthworm at the 50 and 100 μg/g levels .

Table 6 . Summary of the Results of Acetonitrile Studies

INITIAL- EA_RTHWORMS FINAL EARTHWORMS Av .wt .

Levels count Tot.wt . Av .wt . count Tot.wt Av.wt . Diff .

Treatment ($) (#) (E) (g) (#) (¢) (¢) (+/-

R DX/ 0 5 2.61 0.52 4* 2.10 0 .53 + 0 .01

acetonitrile 0 .005 5 2 .25 0.45 5 1.99 0 .40 - 0 .05

0 .01 5 2.91 0.58 4 2.00 0 .50 - 0 .08

0 .02 5 2.94 0.59 5 2.53 0 .51 - 0 .08

0 .04 5 2.66 0.53 5 2.07 0 .41 - 0 .12

0-05 5 3 .35 0 .67 5 2.74 0 .55 - 0 .1 2

RDX/ 0 5 2.68 0.54 5 2.88 0 .58 + 0 .04

sand 0.005 5 2 .81 0.56 5 2 .47 0 .49 - 0 .07

0 .01 5 2.49 0.50 .5 2 .11 0 .42 - 0 .08

0 .02 5 2.87 0 .57 5 2 .48 0 .50 - 0 .07

0 .04 5 2.56 0 .51 5 2 .11 0 .42 - 0 .09

0 .05 5 2 .56 0.51 5 2 .01 0 .40 - 0 .1 1

sand/ 0 5 2.24 0 .45 5 2 .34 0 .47 + 0 .02

acetonitrile 0 .005 5 2 .28 0 .46 5 2 .20 0 .44 - 0 .02

0 .01 5 2 .21 0.44 5 2.12 0 .42 - 0 .02

0 .02 5 2 .45 0.49 5 2.49 0 .50 + 0 .01

0 .04 5 2 .86 0.57 5 2.95 0 .59 + 0 .02

0 .05 5 2,44 0-49 5 2.49 0 .50 + 0 .0 1

3 .2 Results of Standardized Earthworm Toxicity Studies .

In the range-finding study, Radford contaminated and

fortified soil was not lethal to earthworms at the concentration s

14
p.17

tested. The highest concentration tested was the 10 % mixture .

However, in the standardized toxicity study ( Table 7), the

results showed that this soil was lethal to 100 % of the earthworms starting at the 50% level ( 30 μg/g TNT, 50 pg/g TNB ,

62 .50 μg/g 2,4-DNT, and 20 μg/g 2 .6-DNT), while no lethal effects

were observed at the 25% level . The LCso for earthworms in this

soil was between 25 and 50 % .

The Analysis of Covariance ( ANCOVA) of weight differences between the initial and final weights indicated a significant ( p < 0 .0002) difference between treatments ( Appendix ,

Table 1) . A Newman-Keuls pairwise comparison of means indicated

a significant ( p < 0 .01) difference between the 0 and 10 % levels

and the 25% level ( Appendix, Table 2) .

Although there were no fatalities at the 0, 10, and

25% levels, there was an increasing average weight loss as

concentrations increased . These sublethal effects were produced

at the 25% level ( 15 μg/g TNT, 25 μg/g TNB, 31 .25 μg/g 2,4-DNT,

and 10 pg/g 2,6-DNT) as indicated by the Newman-Keuls test .

Therefore, the no observable effects level ( NOEL) was at the

10% level ( 6 pg/g TNT, 10 pg/g TNB, 12 .5 μg/g 2,4-DNT, and 4 μg/g

2,6-DNT) . The lowest observable effects level ( LOEL), also

indicated by the Newman-Keuls test, was at the 25 % level .

Table 7 . Effects of Radford Contaminated and Fortified soil

on Earthworms

INITIAL EARTHWORMS FINAL EARTHWORMS Av. wt .

Treatment count Tot .wt . Av.wt . count Tot .wt Av.wt . Diff . Survival

Level ( 8) (#) ($) (¢) (#) ( 8) (g) (+/-) Rate ($)

5 1.82 0 .36 5 1.64 0 .33

0 5 1.74 0 .35 5 1.61 0 .32 - 0 .03 100

5 1.48 0 .30 5 1.37 0 .2 7

5 1.97 0 .39 5 1.76 0 .3 5

10 5 1.91 0 .38 5 1.72 0 .34 - 0 .04 100

5 1.99 0.40 5 1.80 0 .3 6

5 1.70 0 .34 5 1.31 0 .2 6

25 5 1.98 0 .40 5 1.39 0 .28 - 0 .10 100

5 1.70 0.34 5 1.26 0 .2 5

5 1.95 0.39 0 - - -

50 5 1.54 0.31 0 - - - 0

5 1.91 0.38 0 - • -

5 1.86 0.37 0 - - -

75 5 1.81 0.36 0 - - - 0

5 1.71 0.34 0 - - -

5 1.88 0.38 0 - • -

100 5 1.79 0.36 0 - - - 0

5 1.92 0 .38 0

15

~f
p. 18

The relatively nutrient-poor Radford soil is probabl y

the reason the control earthworms lost weight over the two-week

study period .

Since there were several contaminants ( i .e ., TNB, TNT,

2,4-DNT, and 2,6-DNT, and metals) in the Radford contaminated and

fortified soil, it was not possible to determine from the results

of this study if one component or a combination of components

produced toxic effects on earthworms .

A second standardized experiment was conducted to

measure the toxicity of TNT in artificial soil to earthworms .

The results are presented in Table S . Earthworms treated with

TNT in artificial soil exhibited an increasing average weight

loss as concentrations increased . TNT, when incorporated into

artificial soil, did not produce lethal effects on earthworms up

to 200 μg/g. The ANCOVA of the weight differences indicated a

significant ( p < 0 .0001) difference ( Appendix, Table 3) . The

Newman-Keuls test, which showed a significant ( p < 0 .01) difference between the 0, 80, and 110 levels and the 170 and 200 μg/g

levels ( Appendix, Table 4), indicated that the LOEL for TNT

incorporated into an artificial soil mixture was 140 /, cg/g . The

NOEL for TNT in artificial soil was at the 110 pg/g level, also

indicated by the Newman-Keuls test .

Table 8 . Effects of TNT in Artificial Soil on Earthworms

INITIAL EAR=RMS FINAL EARTHWORMS Av. wt .

Treatment count • Tot.wt . Av.wt. count Tot .wt Av.wt . Diff. Survival

Level (Mg /¢) (#) ( gZ (a) (#) ( g) (g) (+/-) Rate C$)

5 1.62 0 .32 5 1.52 0 .30

0 5 2.19 0 .44 5 2.13 0 .43 - 0 .02 100

5 2.45 0 .49 5 2.35 0 .47

5 1 .68 0 .34 5 1.52 0.30

80 5 1.71 0 .34 5 1.62 0.32 - 0 .02 100

5 1 .91 0 .38 5 1.78 0 .3 6

5 2 .05 0 .41 5 1.93 0 .3 9

110 5 2.20 . 0 .44 5 2.10 0 .42 - 0 .02 100

5 1 .94 0 .39 5 1 .83 0 .3 7

5 1.72 0 .34 5 1 .57 0 .31

140 5 1.91 0 .38 5 1.77 0 .35 - 0 .04 100

5 1.71 0 .34 5 1.52 0 .30

5 1.89 0 .38 5 1.67 0.3 4

170 5 2.22 0 .44 5 2.01 0.40 - 0 .05 100

5 1.70 0 .34 5 1.44 0 .2 9

5 1.95 0 .39 5 1.64 0 .3 3

200 5 1.92 0 .38 5 1.64 0 .33 - 0 .06 100

5 2,07 0 .41 5 1.77 0 .3 5

16

~~
p.19

Another standardized test was conducted to determine

the toxicity of TNT to earthworms in a forest soil with a higher

organic matter content . These results are presented in Table 9 .

TNT concentrations were increased when added to the forest soil

since no earthworms died in the TNT/artificial soil mixture . The

concentrations used in this study were 0, 150, 300, 400, and 500

μg/g . Lethal effects were exhibited starting at the 150 μg/g

level . As concentrations increased, weight loss increased and

survival rates decreased to 0% at 500 pg/g . The ANCOVA of the

weight differences for 0, 150, and 300 μg/g levels indicated a

significant (p < 0 .10) difference (Appendix, Table 5) . The

ANCOVA did not include the 400 μg/g level since there were too

few survivors to include in the analysis . The Newman-Keuls

pairwise comparison of means indicated a significant (p < 0 .05)

difference between the 0 and 150 μq/g levels and the 300 μg/g

level (Appendix, Table 6) . The LOEL for TNT in forest soil was

at 150 μg/g . The LC50 was 325 μg/g (p < 0 .05) .

Table 9 . Effects of TNT in Forest Soil on Earthworms

INITIAL EARTHWORMS FINAL EARTHWORMS__ Av . wt .

Treatment count Tot .wt . Av.wt. count Tot .wt Av .wt . Diff. Survival

Level (ag/¢) (#) (10 (e) (#) (g) (a) (+/-) Rate ($)

5 2.23 0.45 5 1.94 0 .3 9

0 5 2.12 0.42 5 1.68 0 .34 - 0.07 100

5 2.17 0.43 5 1.74 0 .3 5

5 2.05 0.41 5 1.70 0 .3 4

150 5 2.23 0.45 4 1.19 0 .30 - 0.10 93

5 2.30 0.46 5 1.93 0 .3 9

5 2.18 0.44 4 1.06 0 .2 7

300 5 2.31 0.46 4 1.23 0 .31 - 0.17 87

5 2.21 0 .44 5 1.35 0 .2 7

5 2.21 0 .44 0

400 5 2.19 0 .44 3 0.91 0 .30 - 0.13 20

5 2.03 0 .41 0 - -

5 2.08 0.42 0 - -

500 5 2.19 0.44 0 - - - 0

5 1.96 0.39 0 -

The LOEL for the TNT/artificial soil study was 140 μg/g

with a NOEL of 110 μg/g . However, in the TNT/forest soil study,

the NOEL would fall between 0 and 150 μg/g, the lowest concentration tested . The LOEL in this study was 150 μg/g, thus indicating that TNT should produce sublethal effects on earthworms at or

above the 150 μg/g level . The composition of the two media used

in these studies were different (the forest soil contained higher

17

. ~9
p. 20

organic matter) . Cataldo and co-workers9 found that plan t

uptake of TNT was inversely proportional to soil organic matter

content . If TNT is sorbed to soil particles with higher organic

matter, then TNT in forest soil should not have been as toxic as

TNT in artificial soil (with lower organic matter) at equivalent

concentrations . The % soil organic matter did not drastically

alter toxicological effects of TNT in the two soil media used

here . Other factors (e .g ., pH, other soil constituents, etc .)

may have to be considered to determine TNT toxicity in different

soil types .

4 . CONCLUSIONS

Results of the range-finding studies indicated that the

contaminated and contaminated and fortified soils were not lethal

to earthworms at the concentrations used here . Similar results

were obtained in the range-finding studies on HMX and RDX . However, when the concentration of contaminants (i .e ., TNB, TNT,

2,4- and 2,6-DNT) in the Radford contaminated and fortified soil

was increased during the standardized earthworm toxicity study,

this soil did produce lethal effects on earthworms . Since this

soil contained 4 different contaminants, additional studies are

needed to determine : (1) the compound(s) producing the toxicity

to earthworms, and (2) the compound's lowest observable effects

level on earthworms .

Results of the standardized earthworm toxicity study

on TNT showed that it produced lethal effects starting at the

150 /μg/g level in forest soil . However, in a parallel study

using an artificial soil mixture with a lower organic matter

content (1 .4% for this mixture ; the forest soil contained 5 .9 % ),

TNT did not produce lethal effects on earthworms . These results

may be valid for only this soil type . TNT in other soil types

.may behave differently . Therefore, TNT toxicity to earthworms

should be investigated using a variety of soils .

18
p. 21

LITERATURE CITED

1 . Layton, D .W ., McKone, T .E ., Hall, C .H ., Nelson,

M .A ., and Ricker, Y .E ., Demilitarization of Conventional Ordnance : Priorities for Data Base Assessments of Environment al

Contaminants , UCRL-15902, Lawrence Livermore National Laboratory ,

Livermore, CA, 1986 ( AD-A182 922) .

2 . Anon ., Phase 5 . Hazardous Waste Study 137-26-

0593-86 : Summary of AMC Open-Burning Oeen-Detonation Rounds

Evaluations March 1981-1985 , U .S . Army Environmental Hygiene

Agency, Aberdeen Proving Ground, MD, February 1986, UNCLASSIFIED

Report .

3 . Roberts, B ., and Dorough, H ., "Hazards of Chemicals

to Earthworms," Environ . Toxicol . Chem . Vol . 4, pp 307-323

(1985) .

4 . Dean-Ross, D ., "Methods for the Assessment of the

Toxicity of Environmental Chemicals to Earthworms," Rea . Toxicol .

Pharmacol . Vol . 3, pp 48-59 ( 1983) .

5 . Neuhauser, E .F ., Loehr, R .C ., Maleck, M .R ., Milligan, .D .L ., and Durkin, P .R ., "The Toxicity of Selected Organic

Chemicals to the Earthworm Eisenia fetida ," J . Environ . oual .

Vol . 14, pp 383-388 (1985) .

6 . Karnak, R ., and Hamelink, J . "A Standardized Method

for Determining Acute Toxicity of Chemicals to Earthworms,"

Ecotox . Environ . Safety Vol . 6, pp 216-222 ( 1982) .

7 . Tallarida, R .J ., and Murray, R .B ., "Pharmacologic

Calculation System" based on Manual of Pharmacoloaic Calculations

with Computer Programs , 2nd ed ., Springer-Verlag, New York, 1986 .

8 . SAS/STAT User's Guide . Release 6 .03 Edition , SAS

Institute, Inc ., Cary, NC, 1988 .

9 . Cataldo, D .A ., Harvey, S .D ., Fellows, R .J ., Bean,

R .M ., and McVeety, B .D ., An Evaluation of the Environmental Fate

and Behavior of Munitions Material (TNT . RDX) in Soil and Plant

Systems , PNL-7370, Pacific Northwest Laboratory, Richland, WA,

August 1989, UNCLASSIFIED Report .

19

.> 21
p. 22

Blank

20
p. 23

APPENDIX

STATISTICAL DATA

ANALYSIS OF COVARIANCE ( ANCOVA) OF EARTHWORM WEIGHT DIFFERENCE

NEWMAN-KEULS ANALYSIS OF TREATMENT S

Table 1 . ANCOVA of Weight Difference ( g) of Earthworms Raised

In Radford Contaminated and Fortified Soi l

Source of Sum of Degrees of Mean F Significnce

Variation Scuares Freedom Squares Value Level

Total 0.01381 8

Between: 0.01345 3 0.00448 62 .32 p < 0 .0002

Within : 0.00036 5 0.00007

Table 2 . Newman-Reuls Analysis of All Treatments, Pairwise,

and Ranked From Low to High : Final Weights (g)

of Earthworms Raised in Radford Contaminated and

Fortified Soil

Treatment 0% 10$ 25$

q values

0= 1.4412 9 .6077**

10% 8 .1665**

25%

q(95g) 3 .46 4.34

q(99%) 5 .24 6.33

**Significant at p <0 .0 1

Table 3 . ANCOVA of Weight Differences ( g) of Earthworms

In Artificial Soil Amended with TNT

Source of Sum of Degrees of Mean F Significnce

Variation uares Freedom Sauares Value Level

.

Total 0.04460 17

Between: 0 .04411 6 0.00735 163 .45 p< 0 .0001

Within : 0.00049 11 0 .00004

21 Preceding page blank
p. 24

Table 4 . Newman-Keuls Analysis of All Treatments, Pairwise ,

and Ranked From Low to High : Final Weight (g) of

Earthworms Raised in Artificial Soil Amended with .

TNT

Treatment 0 110 80 140 170 200

q values

0 1.477 2.216 3 .693 7 .385** 8 .863**

110 0

80 1

.739 2.216 5.908** 7 .385**

140 3

.477 5.170** 6 .647**

170 1

.693* 5 .170**

.477

200

q(95% ) 3.08 3 .77 4.20 4.51 4.75

q(998) 4.32 5 .04 5.50 5.84 6.10

*Significant at p <0 .05

**Significant at p <0 .0 1

Table 5 . ANCOVA of Weight Differences (g) of Earthworms Raised

In Forest Soil Amended with TN T

Source of Sum of Degrees of Mean F Significnce

Variation Squares Freedom Squares Value Level

Total 0.01629 8

Between : 0.01151 3 0.00384 4 .01 p < 0 .10

Within : 0.00478 5 0.00096

Table 6 . Newman-Keuls Analysis of All Treatments, Pairwise,

and Ranked From Low to High : Final Weights (g) of

Earthworms Raised in Forest Soil Amended with TNT

Treatment 0 150 300

q values

150 4

0 1.429 5.511*

.082*

300

q(95%) 3 .46 4.34

q(99%) 5 .24 6.33

*Significant at p <0 .05

APPENDIX 22