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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
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## WAY 141993



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D Approved for public release; distribution is unlimited . U .S. ARMY 

# B CHEMICAL ~ AND BIOLOGICAL DEFENSE AGENCY

REPRODUCED BY ' ►ving Ground, Maryland 

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DisclaimerThe findings in this report are not to be construed as an official Department of the Army position unless so designated by other authorizing documents .

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## REPORT DOCUMENTATION PAGE Form Approved OMB NO. 0704•0188

rublk r~pnnmgburden for "I'S cdkctbn or information i s !atrmated to averaqe 1 hour per response . mcludlrq the time for r-wMwinq Instructions . searching e■istrnq d~ta tources. gathermg ind maintaining the dala nKded, and completing and rNrewmg Ih! c00ectlon ol iMormatlon . Send commments rt~qarding this burden estimate or any other espect ot this cdlechon of in/ormation. mdudin0 SOns Ior reducing this burden. to'Washmqton Headpuanen Services . Directorate rorinformation Operations and Reports. 12 IS Jefferson Davis Ilighway, Suite 1200 . Arlington,w ;I

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. Paperwork Reduction Project (0704-01Sa), Washington, DC 20503 . 1 . AGENCY USE ONLY ( Leave blank) 2 . REPORT DATE 3. REPORT TYPE AND DATES COVERED 1993 April Final, 89 Jul - 92 Feb 4 . TITLE AND SUBTITLE S . FUNDING NUMBERS 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 NUMBER
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 Chemica l 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 jjg/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 2 2 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 ABSTRAC T UNCLASSIFIED UNCLASSIFIED UNCLASSIFIED UL NSN 7540-01-280-5500 Sta ndard Form 298 (Rev 2-89)

~ Prescribed by ANSI Sid
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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 thisreport 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 ; andJohn 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 earthworms 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

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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
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DTIC QUAL

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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 Earthworm

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

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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 . 7



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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 helpedkeep 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 firststudy 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 wa s 8P. 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 ofthe 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 .85 RDX 0 - - • - • 0 0 .005 - • - - - 50 0 .01 - • - - - 100 0 .02 - • - • - 200 0 .04 - - - - - 400 0 .05 - 50 0 HM C 0 - - - • 0 - 0 .005 - - - - 50 - 0 .01 - - - - 100 - 0 .02 - - - - 200 - 0 .04 - • - - 400 - 0 .05 - 50 0 *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 . Astandardized 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 . 10



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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 Analysi s % 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 . '

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Table 4 . Experimental Design of Standardized Earthworm



### Toxicity Studies
.

Levels Contaminants u8/¢-) Tre atment ($) 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 3 0 100 100 60 125 .00 40 TNT in artificial soil 0 -

- - ' 0 .008 • 80 - - 0 .011 - 110 - • 0 .014 140 - - 0 .017 - 170 - - 0,020 20 0 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 .

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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 .04 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 .12 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 HI K C 0 5 2 .53 0 .51 5 2 .42 0 .48 - 0 .03

0 .005 5 2 .61 0 .52 5 2 .46 0 .49 - 0 .03 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 .08 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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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 .0 5

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 .11 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 .01

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
14p .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 p

g/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 .27 5 1 .97 0 .39 5 1 .76 0 .35 10 5 1 .91 0 .38 5 1 .72 0 .34 - 0 .04 100

5 1 .99 0 .40 5 1 .80 0 .36 5 1 .70 0 .34 5 1 .31 0 .26 25 5 1 .98 0 .40 5 1 .39 0 .28

- 0 .10 100

5 1 .70 0 .34 5 1 .26 0 .25 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

~fp. 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 .36
5 2 .05 0 .41 5 1 .93 0 .39 110 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 .34 170 5 2 .22 0 .44 5 2 .01 0 .40 - 0 .05 100

5 1 .70 0 .34 5 1 .44 0 .29
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 .27 300 5 2 .31 0 .46 4 1 .23 0 .31 - 0 .17 87

5 2 .21 0 .44 5 1 .35 0 .27
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


# . ~9p . 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 .

18p. 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 .

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20p. 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 .0000 7

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 Soi l 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 1 7
Between: 0 .04411 6 0 .00735 163 .45 p< 0 .0001
Within : 0 .00049 11 0 .0000 4


## 21 Preceding page blankp . 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 .739 2 .216 5 .908** 7 .385** 80 1 .477 5 .170** 6 .647** 140 3 .693* 5 .170** 170 1 .477 20 0 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 .0 5 **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 .0009 6

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 30 0 q values 0 1 .429 5 .511* 150 4 .082* 30 0 q(95%) 3 .46 4 .34 q(99%) 5 .24 6 .33 *Significant at p <0 .0 5

APPENDIX 22