667" TOXICITY OF MEON (DIETNYLENELYCOL DINITRATE) /SYNTHETIC-NC SMOKE CONSUST.. (U) JOH4NS HOPKINS UNIV

9*" SHADY SIDE NO ENVIRONMENTAL SCIENCES GROUP..

UNWCLSSIFIED DJ FISHER ET AL. 15 JAN 6? F/O 24/'4 m.

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TOXICITY OF DEGDN, SYNTHETIC-HC SMOKEo00 COMBUSTION PRODUCTS, SOLVENT YELLOW 33

AND SOLVENT GREEN 3 TO FRESHWATERAQUATIC ORGANISMS

FINAL REPORT FOR PHASE IIDaniel J. Fisher, Ph.D.Dennis T. Burton, Ph.D.Robert L. Paulson, M.S.

The Johns Hopkins UniversityApplied Physics Laboratory

Environmental Sciences GroupShady Side, MD 20764

JANUARY 1987

Supported by

U.S. Army Medical Research and Development Command

Fort Detrick, Frederick, MD 21701-5012

U.S. Navy Contract No. N00039-87-C.5301 via ARMY MIPR85MM5505The Johns Hopkins University

Applied Physics LaboratoryJohns Hopkins Road 011C

Laurel, MD 20707 D TI TEgr7NELECT '-Project Officer Major John A. Kelly

Health Effects Research Division w'N NOV 2 4 1987

U.S. Army Biomedical Research and "-Development Laboratory E

Fort Detrick, Frederick, MD 21701-5010

Approved for public release; distribution unlimitedThe findings in this report are not to be construed as an official Departmentof the Army position unless so designated by other authorized documents

,, , . , .",

NOTICE

Disclaimer

The findings in this report are not to be construed as an official Department of theArmy osition unless so designated by other authorized documents, nor does mentionof trae names or commercial products constitute endorsement or recommendation

DiS spoitionDestroy this report when it is no longer needed. Do not return it to the originator.

' .o

sEQIry CLASSIFICATION OF rwIS PAGE . -,

REPORT DOCUMENTATION PAGE&a REPORT SECURITY CLASSIFICATION lb ISR V ARIGLUnclassif ied________________________

Sa SEPITy CASIFICATION AUTHORITY 1, oISTRIUTION/AVAILAaIUrY OF REPORT

Approved for public release;2b. DECLASSIFICATION1I OWNGRADING SCH4EDULE distribution unlimitedNA

4. PERFORMING ORGANIZATION REPORT NUMBER(S) S. MONITORING ORGANIZATION REPORT NUMBER(S)

64. NAME OF PERFORMING ORGANIZATION fib. OFFICE SYMBOL 7a. NAME OF MONITORING ORGANIZATIONThe Johns Hopkins University (if aplksible) U.S. Army Biomedical Research

Applied Phyics Loratorv and Development LaboratorySt& ADDRESS (City, Stat. adZIP COW) 7b. ADDRESS (City. State. and ZIP Co*)

Environmental Sciences Group ATTN: SGRD-UBGShady Side, MD 20764 Fort Detrick, Frederick, MD .21701-5010

I&. NAME OF FUNDING I SPONSORING 8 b. OFFICE SYMBOL 9. PROCUREMENT INSTRUMENT IDENTIFICATION NUMBERORGANIZATION U.S. Army Medical Iof &A9ub e)

Research and Development Command NA 85MM5505

Bc. ADDRESS (Cmty State. and ZIP Code) 10. SOURCE OF FUNDING NUMBERS

Fort Detrick, Frederick, MD 21701-5012 PROGRAM IPROJECT TAKWORK UNITELEMENT NO. NO. 40 1ACCESSION NO.

11I. TITLE (IncLpde Security Claflcation)Toxicity of DEGDN, Synthetic-HG Smoke Combustion Products, Solvent Yellow 33 and SolventGreen 3 to Freshwater Aquatic Organisms12. PERSONAL. AUThOR(S)Daniel J. Fisher, Dennis T. Burton and Robert L. PaulsonIla. TYPE OF REPORT 13b. TIME COVERED 14. DATE OF REPORT (YeaAdlonf ) SPAGE COUNT%

FialRpotFROM 4/85 To 12/86 1987, January, 15

1G. SUPPLEMENTARY NOTATION

17. COSA1I C] 1S. SUBJECT TERMS (C 0iu on rve"?UeJInectmy and kfent*~ by bld number)FIELD GROUP , SUB-GROUP Acute toxicity So..vent yellow 33

Mixture toxicity Ao- 5lvent green 3Component toxicity DEGDN

19. ABSTRACT (Cpnlne oo/rvwrw if nwcenwy and mneily ly black number)/7 %

07 1. ai

"leacute toxicities of fourimunitions compounds to ninefreshwater aquatic organisms were'determined. The munitionswere: diethyleneglycol dinitrate (DEGDN), solvent yellow 33,solvent greon 3, and synthetic-HC, pmoke combustion products,/whi.ch_ X a c x mi~r cntaining Zn, Cd, As, Pb, Al, CC1,C72 C14)

and _ CL Fish exposea to the four materials for~hinc~ued the fathead minnow (Pimephales promelas), bluegill

(Lepomis macrochirus), channel catfish (Ictalurus punctatus) andrainbow trout (Salmo gairdneri). Invertebrates, which wereexposed for 48 h ,included the water flea (Daphnia magna),amphipod (Garnmarus pseudolimnaeus), midge larva (Paratanytarsus

20. DISTRIBUTION/IAVAILAIIUTY OF ABSTRACT 21. ABSTRACT SECURITY CLASSIFICATION. -

OUNCLASSIFIEOINUMITtO C3 SA01E AS RPT. (: OTIC USERS22a. NAME OF RESPONSIBLE INDIVIDUAL 22b. TELEPHONE anclude Alga Code) 22-C. OFFICE SYMBOL.

Ms. Virginia Miller (0)663-7325 SGRD-RMI-S

00 FORM 1473,94 mAR 83 APR edition may be used untl exhausted.SEUIYC.SFATOOPT45AGAll other editwmn are obsoet. SCRT QASFCnNO HSP~

% %

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9ECUNY CaASIICAION Of rWS PAug

19. (Cont'd)

Diethyleneglycol Zinc chloridedinitrate Lead chloride

Synthetic-HC smoke Cadmium chloridecombustion products Arsenic chloride

HCL Carbon tetrachlorideMidge larva (Paratanytarsus Tetrachloroethylene

parthogenetica) HexachloroethaneMayfly larva (Hexagenia Hexachlorobenzene

bilinata) Green alga (Selenastrumcapricornutum)

Fathead minnow (Pimephales Water flea (Daphnia magna)promelas) Amphipod (Gammarus

Rainbow trout (Salmo pseudolimnaeus)gairdneri) Channel catfish (Ictalurus

Bluegill (Lepomis punctatus)macrochirus)

20. (Cont'd)

parthogenetica) and the mayfly larva (Hexagenia bilinata).Growth of the green alga Selenastrum capricornutum was alsotested with all the compounds. j, L I |

The toxicity of DEGDN was relat ve Iyow to the ninefreshwater species tested. Toxicity values ranged from a 5-dayEC50 (growth) of 39.1 mg/L for Selenastrum capricornutum to a 96-h LC50 of 491.4 mg/L for the fathead minnow (Pimephalespromelas). The most sensitive invertebrate tested was Daphniamagna which was more sensitive than the most sensitive fishtested, Lepomis macrochirus.

The dissolved components of the synthetic-HC smokecombustion products mixture were found to be quite toxic to anumber of freshwater species, especially Selenastrumcapricornutum, Salmo gairdneri and Daphnia m . A testsolution containing only 5.6% of a stock mixture of thesecomponents caused both an algistatic and algicidal effect on thealga. The rainbow trout and the water flea had 96-h and 48-hLC50s of 2.2% and 9.3% of the stock solution, respectively.

Solvent yellow 33 and solvent green 3 were not toxic toseven of the nine freshwater species when tested at theirsolubility limits. A solubility limit solution of solvent green3 killed 50% of the rainbow trout exposed but was non-toxic whendiluted by 50%. Solvent yellow 33 was non-toxic to the rainbowtrout at its solubility limit. Both dyes caused a reduction inalga growth at solubility limits, with solvent green 3 being mostdetrimental, causing a 98 - 99% reduction after 5 days of exposure.

5CUPY C6AU914CA ON @" PA49

AD-____*/,

TOXICITY OF DEGDN, SYNTHETIC-HC SMOKECOMBUSTION PRODUCTS, SOLVENT YELLOW 33

AND SOLVENT GREEN 3 TO FRESHWATERAQUATIC ORGANISMS

FINAL REPORT FOR PHASE II -..... .

Daniel J. Fisher, Ph.D.Dennis T. Burton, Ph.D.Robert L. Paulson, M.S. ,.

The Johns Hopkins UniversityApplied Physics Laboratory

Environmental Sciences Group Dimr±:,ti ..Shady Side, MD 20764 Aval!.,11.v Codes

qv: -I fand/or:lst ~c i a 1

JANUARY 1987

Supported by

U.S. Army Medical Research and Development Command

Fort Detrick, Frederick, MD 21701-5012

U.S. Navy Contract No. N00039-87-C-5301 via ARMY MIPR85MM5505The Johns Hopkins University

Applied Physics LaboratoryJohns Hopkins Road

Laurel, MD 20707

Project Officer Major John A. KellyHealth Effects Research Division

U.S. Army Biomedical Research and 71Development Laboratory

Fort Detrick, Frederick, MD 21701-5010

Approved for public release; distribution unlimited

The findings in this report are not to be construed as an official Departmentof the Army position unless so designated by other authorized documents

EXECUTIVE SUMMARY

The U.S. Army Biomedical Research and Development Laboratory(USABRDL) has responsibility for assessing the possible healthand environmental hazards associated with munitions-unique Opprollutants released during manufacturing activities and--ployment in the field. The Health Effects Research Division ofUSABRDL has recently expressed interest in determining the acutetoxicity of four munitions to freshwater aquatic organisms: WDiethyleneglycol dinitrate (DEGDN), synthetic-HC(hexachloroethane) smoke combustion products, solvent yellow 33[2-(2'-quinolinyl)-1,3-indandione], and solvent green 3 (1,4-di-p-toluidinoanthraquinone). The acute toxicity of each munitionwas determined for the fathead minnow (Pimephales promelas),channel catfish (Ictalurus punctatus), bluegill (Lepomismacrochirus), rainbow trout (Salmo gairdneri), water flea(Daphnia magna), amphipod (Gammarus pseudolimnaeus), midge larva(Paratanytarsus parthenogeneticus), mayfly larva (Hexageniabilinata), and the green alga (Selenastrum capricornutum).

The toxicity of DEGDN was relatively low to the ninefreshwater species tested, especially when compared to othercommonly used nitrate ester explosives such as nitroglycerin andethyleneglycol dinitrate. Toxicity values ranged from a 5-dayEC50 (standing crop) of 39.1 mg/L for Selenastrum capricornutumto a 96-h LC50 of 491.4 mg/L for the Pimephales promelas. Themost sensitive invertebrate tested was Daphnia magna which wasmore sensitive than the most sensitive fish tested, Lepomismacrochirus. Due to its high water solubility, DEGDN could causeenvironmental problems if sufficient amounts of the materialenter an aquatic ecosystem.

The dissolved components of the synthetic-HC smokecombustion products mixture were found to be quite toxic to anumber of freshwater species, especially Selenastrumcapricornutum, Salmo gairdneri and Daphnia magna. A testsolution containing only 5.6% of a stock mixture of thesecomponents caused both an algistatic and algicidal effect on thealga. LC50 values for the rainbow trout and the water flea were2.2% and 9.3% of the stock solution, respectively. Additionaltests with the water flea indicated that zinc was the major toxiccomponent of the mixture. Information concerning environmentallevels of the various components after use or disposal of themunitions is necessary in order to assess possible hazards toaquatic life.

Solvent yellow 33 and solvent green 3 were not toxic toseven of nine freshwater species when tested at their solubility 'S-V

limits. A solubility limit solution of solvent green 3 killed50% of the rainbow trout exposed for 96 h but was non-toxic whendiluted by 50%. Solvent yellow 33 was non-toxic to the rainbowtrout during 96 h of exposure at its solubility limit. Both dyescaused a reduction in alga growth at solubility limits, with "'

-I.5

soivent green 3 being most detrimental, causing a 98 - 99%reduction after 5 days of exposure. These dyes could cause aproblem if released to the environment since detrimental effectswere found at their solubility concentrations (i.e., 0.2 mg/Lsolvent yellow 33 and < 0.002 mg/L green component of solventgreen 3).

This research was conducted in accordance with GLPregulations for nonclinical laboratory studies (EPA 1983. Fed.Reg. 48: 53946-53969). These studies were inspected by theJHU/APL Quality Assurance unit, and the results in the finalreport were audited for completeness and accuracy of reporting tothe raw data.

2

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ACKNOWLEDGEMENTS 06.

We would like to thank Michael J. Lenkevich of The AppliedPhysics Laboratory for his analysis of samples during theproject. We would also like to thank Major John A. Kelly and Dr.William van der Schalie of the U.S. Army Biomedical Research andDevelopment Laboratory, Fort Detrick, MD, for their constructivesuggestions and help throughout the study. Florence H. Broski,of the same group, conducted invaluable statistical analyses ofthe Selenastrum capricornutum toxicity data. This researchproject was sponsored by the U.S. Army Medical Research andDevelopment Command, Fort Detrick, MD, under Contract No. ARMYMIPR 85 5505 through the Applied Physics Laboratory's NAVSEAContract No. N00024-83-C-5301.

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TABLE OF CONTENTS

EXECUTIVE SUMM4ARY ........................................... 1

ACKNOWLEDGEMENTS ....................................... 3

LIST OF FIGURES ........................................ 6

LIST OF TABLES .............................................. 7

1. INTRODUCTION ........................................... 8

1.1 General ........................................... 8

1.2 DEGDN ............................................. . 8

1.3 Synthetic-HC Smoke Combustion Products Mixture .... 9

1.4 Solvent Yellow 33 and Solvent Green 3 ................ 9

2. OBJECTIVES OF STUDY ........................................ 12

3. MATERIALS AND METHODS .................................. 13

3.1 Toxicant Characterization and Analytical Chemistry 13

3.1.1 DEGDN .......................................... 13

3.1.2 Synthetic-HC Smoke Combustion ProductsMixture ........................................ 13

3.1.3 Solvent Yellow 33 and Solvent Green 3 ...... 14

3.2 Test Solution Preparation ............................ 14

3.2.1 DEGDN .......................................... 14

3.2.2 Synthetic-HC Smoke Combustion ProductsMixture ........................................ 15

3.2.3 Solvent Yellow 33 and Solvent Green 3 ...... 17

3.3 Dilution Water Quality ............................... 17

3.4 Test Organisms ........................................ 17

3.5 Test Methodclogies ................................... 22

3.5.1 General ........................................ 22

3.5.2 DEGDN ...................................... 25

4o 4

TABLE OF CONTENTS (Cont'd)

Page

3.5.3 Synthetic-HC Smoke Combustion Products

Mixture ........................................ 25

3.5.4 Solvent Yellow 33 and Solvent Green 3 ...... 25

3.6 Test End Points and Data Analysis ................. 26

4. RESULTS AND DISCUSSION ................................... 28

4.1 Water Quality ..................................... 28

4.2 DEGDN ............................................. 28

4.3 Synthetic-HC Smoke Combustion Products Mixture .... 36

4.4 Solvent Yellow 33 and Solvent Green 3 ............... 52

5. CONCLUSIONS ............................................ 59

6. LITERATURE CITED ........................................... 60

APPENDIX A Dissolved Concentrations of the Componentsof the Synthetic-HC Smoke CombustionProducts Mixture in Dilutions Used DuringToxicity Tests ................................ 64

A-1 Daphnia magna ................................. 65A-2 Paratanytarsus parthenogeneticus ........... 67A-3 Salmo gairdneri ............................... 68 .A-4 Pimephales promelas, Hexagenia bilinata,

Ictalurus punctatus, Lepomis macrochirus,Gammarus pseudolimnaeus and Selenastrum-capricornutum ................................. 70 j

A-5 Daphnia magna - Component Toxicity ......... 71

APPENDIX B Additional Good Laboratory PracticeStandards Report Requirements .............. 73

Final Quality Assurance Unit Statement ..... 74

DOCUMENT DISTRIBUTION LIST ............................. 75

5 ."

LIST OF FIGURES

Page

1. The effects of DEGDN on the growth of Selenastrumcapricornutum as measured by density ................... 33

2. The effects of DEGDN on the growth of Selenastrumcapricornutum as measured by biomass ................... 35

3. The effect of the synthetic-HC smoke combustionproducts mixture on the growth of Selenastrumcapricornutum as measured by density ................... 43

4. The effect of the synthetic-HC smoke combustionproducts mixture on the growth of Selenastrum s-.capricornutum as measured by biomass ................... 44

5. 48-h LC50s for Daphnia magna and various components ofthe synthetic-HC smoke combustion products mixture -based on percentages of stock solutions ................ 47

6. 48-h LC50s for Daphnia magna and various components ofthe synthetic-HC smoke combustion products mixture -

based on measured Zn concentrations .................... 49

7. The effects of solvent yellow 33 and solvent green 3at their solubility limits on the growth of Selenastrumcapricornutum as measured by density ................... 56

8. The effects of solvent yellow 33 and solvent green 3at their solubility limits on the growth of Selenastrumcapricornutum as measured by biomass ................... 57

6

0.

LIST OF TABLES

Page

1. Percent composition, by weight, of the synthetic-HCsmoke combustion products mixture ...................... 10

2. Quantities of components of the synthetic-HC smokecombustion products mixture added to 15.5 L diluentwater during toxicity tests ............................ 16

3. Comprehensive dilution water analysis, 1986 ............ 18

4. Information on organisms used in testing ............... 20

5. Test chamber and test solution volumes ................. 24

6. Water quality data for toxicity tests .................. 29

7. Acute toxicity of DEGDN to eight freshwater aquaticorganisms in a static test system ...................... 31

8. Effects of DEGDN on the growth of Selenastrumcapricornutum: cells/mL vs test day ................... 32

9. Effects of DEGDN on the growth of Selenastrumcapricornutum: relative Chlorophyll a measurements .... 34

10. Concentrations of dissolved components in 100% stocksynthetic-HC smoke combustion products mixture forvarious toxicity tests ................................. 37

11. Acute toxicity of a synthetic-HC smoke combustionproducts mixture to eight freshwater aquatic organisms. 39

12. Average growth of Selenastrum capricornutum exposed tothe synthetic-HC smoke combustion products mixture ..... 41

13. Acute toxicity of HC combustion products mixturecomponents to Daphnia magna. Based on % of stocks ..... 46

14. Acute toxicity of HC combustion products mixturecomponents to Daphnia magna. Based on measured zincconcentrations ......................................... 48

15. Acute toxicity of solvent yellow 33 and solvent green 3to eight freshwater aquatic organisms .................. 53

16. Average growth of Selenastrum capricornutum exposedto solvent yellow 33 and solvent green 3 ............... 54

7

k LA A'-

SECTION 1

INTRODUCTION

1.1 General

The U.S. Army Biomedical Researcn and Development Laboratory(USABRDL) has responsibility for assessing the possible healthand environmental hazards associated with munitions-uniquepollutants released during manufacturing activities anddeployment in the field. The Health Effects Research Division ofUSABRDL has recently expressed interest in determining the acutetoxicity of the explosive propellant Diethyleneglycol dinitrate(DEGDN) and three smoke munitions compounds: synthetic-HC(hexachloroethane) smoke combustion products, solvent yellow 33[2-(2'-quinolinyl)-1,3-indandione], and solvent green 3 (1,4-di-p-toluidinoanthraquinone) to freshwater aquatic organisms. Theacute toxicity of each munition was determined for the fatheadminnow (Pimephales promelas), channel catfish (Ictaluruspunctatus), bluegill (Lepomis macrochirus), rainbow trout (Salmogairdneri), water flea (Daphnia magna), amphipod (Gammaruspseudolimnaeus), midge larva (Paratanytarsus parthenogeneticus),mayfly larva (Hexagenia bilinata), and the green alga(Selenastrum capricornutum).

1.2 DEGDN

DEGDN is an explosive propellant chosen for use as aplasticizer replacement for nitroglycerin in the propellantmixtures for the 120 mm shell of the Ml Abrams tank. In the pastit has been manufactured and processed at Radford Army AmmunitionPlant, Radford, VA, which discharges waste effluents to the NewRiver. It is presently manufactured at the Naval OrdinanceStation, Indian Head, l1D, which discharges waste effluents to thePotomac River. The potential for release of DEGDN into streamsand rivers means that some information on its toxicity to aquaticorganisms is needed to allow reasonable discharge standards to beestablished by regulatory authorities. The objective of thisstudy was to determine the acute toxicity of dissolved DEGDN tonine freshwater species.

The solubility of DEGDN in water has been shown to be0.4 g per 100g at 200 C, indicating the potential of high aqueousconcentrations in streams subject to DEGDN discharge (Linder1980). Environmental fate studies indicate that DEGDN is astable compound once dissolved in water (photolytic half-life of29 to 40 days; hydrolytic half-life of 144 days) with a low Kowof 9.6 and a low affinity for absorption to sediment (Spanggordet al. 1985). This same study indicated that microbialbiotransformation will be a major fate process for DEGDN when itis introduced into waters with other organic waste substrates.

DEGDN toxicity data are sparse. Krasovsky et al. (1973)reported LD50s for rats of 777 mg/kg (oral) and for guinea pigs

8

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and white mice of 1250 mg/kg (peroral in vegetable oil),indicating that it is less toxic than nitroglycerin orethyleneglycol dinitrate, two other commonly used nitrate esterexplosives. They set a maximum permissible level for DEGDN inreservoir waters of 1.0 mg/L based on human perception thresholdsand influences on biochemical oxygen uptake, mineralization of

organic impurities and dynamics of growth of saprophyticmicroflora. Holleman et al. (1983) stressed a need for aquatictoxicity studies on DEGDN and other nitrate esters but suggestthat DEGDN should be less toxic than nitroglycerin due to itsincreased molecular weight. Bentley et al. (1978) foundnitroglycerin to be acutely toxic to freshwater aquatic organismsin amounts ranging from 1.67 mg/L (96-h LC50 for bluegill) to55 mg/L (48-h EC50 for Chironomus tentans).

1.3 Synthetic-HC Smoke Combustion Products Mixture

Synthetic-HC smoke combustion products are the combustionproducts of the M8 grenade, M5 smoke pot and M4AI floating smokepot. The percent composition, by weight, of this mixture isgiven in Table 1. These percentages were derived by the Armyfrom published reports (Katz et al. 1980, Spanggord et al. 1985).All but aluminum and hexachloroethane are considered prioritypollutants by EPA (USEPA 1986a). These products represent acomplex chlorinated organic/metallic mixture dominated by zinc.When the above devices are used in training exercises, thesecomponents are released to the environment where they coulddamage aquatic life. Also, there is a considerable stockpile ofthese munitions on hand. In the event this stockpile has to bediscarded, the question of its potential effect on aquaticorganisms needed to be quantified.

Data for the prediction of the toxicity of this mixture arenot available. There is data concerning the toxicity of theindividual components to aquatic organisms, since most arepriority pollutants. For example, acute toxicity values for zincrange from 50.6 jLg/L for Ceriodaphnia reticulata to 86.7 mg/L fora damselfly (USEPA 1986b). This range covers 43 species offreshwater organisms. Toxicity values for the parent compound ofthis mixture, hexchloroethane, range from a 96-h LC50 of0.98 mg/L for rainbow trout and bluegill to a 48-h EC50 of8.07 mg/L for the water flea (USEPA 1980b).

The objectives of this study were twofold: first, todetermine the acute toxicity of the total mixture of dissolvedcomponents to freshwater organisms; second, to determine themajor toxic component(s) of the mixture using a series of statictests with the water flea.

1.4 Solvent Yellow 33 and Solvent Green 3

Solvent yellow 33 and solvent green 3 are components of asmoke munition. The munition contains 42% by weight of the dyes.Solvent green 3 is a 30:70 mixture of solvent yellow 33 [2-(2'-

9 a.

V

%

%

TABLE 1. PERCENT COMPOSITION, BY WEIGHT, OF THE SYNTHETIC-HCSMOKE COMBUSTION PRODUCTS MIXTURE

Hydrochloric acid (HCl) 1.828

Carbontetrachloride (CCl4 ) 5.515

Tetrachloroethylene (C2 C14 ) 14.411

Hexachloroethane (C2C16 ) 4.480

Hexachlorobenzene (C6 C16 ) 1.634

Aluminum oxide (A1203 ) 10.917

Zinc chloride (ZnCl2 ) 61.073

Lead chloride (PbCI2 ) 0.089

Cadmium chloride (CdC12 ) 0.051

Arsenic chloride (AsCl3 ) 0.001

'I

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quinolinyl)-1,3-indandione] and a green dye component (l,4-di-p-toluidinoanthraquinone). Data concerning the acute toxicity ofthese dyes to aquatic organisms are not available. Solventyellow 33 has been shown to cause hypersensitivity reactions inhumans exposed to 10 4g/g in insult patch testing (Weaver 1983).Research is presently underway to determine the inhalationtoxicity of these dyes (Henderson et al. 1984). The objective ofthis study was to determine the toxicity of these dyes to ninefreshwater species as a necessary data base for establishingwater quality criteria.

N

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Ii , I11 _o1

SECTION 2

OBJECTIVES OF STUDY

1) To determine the acute toxicity of DEGDN, synthetic-HC smoke Icombustion products mixture, solvent yellow 33 andsolvent green 3 to nine species of freshwater aquaticorganisms.

2) To determine the major toxic component(s) of thesynthetic-HC smoke combustion products mixture toDaphnia magna.

1.

.4-.

'.2

12-'.

SECTION 3

MATERIALS AND METHODS

3.1 Toxicant Characterization and Analytical Chemistry

3.1.1 DEGDN

The propellant was obtained from the Naval Ordinance Station(NOS), Indian Head, MD. Upon manufacture, the pure DEGDN wasdissolved in absolute ethanol for shipment. When dissolved inethanol, the propellant can be shipped as a flammable liquidrather than an explosive. The DEGDN received was 2.86% by weightin ethanol. HPLC analysis of the DEGDN indicated no contaminantsin this stock solution (Fisher et al. 1985). The liquid wasstored at room temperature in the dark since DEGDN becomes lessstable at lower temperatures.

Analysis of aqueous DEGDN samples was done by HPLC (Waters4BONDAPAC C18 column) using an isocratic 30% water : 70% methanolmobile phase and a detector wavelength of 215 nm. Aqueoussamples were injected directly into the HPLC after filtration to0.45 m. This analytical technique allowed for a detection limitof 0.286 mg/L. Fisher et al. (1985) showed no loss of thecompound in dilution water over 48 h at 220 C. The details of theanalytical technique, including precision and accuracy data alongwith solubility and stability information are described in Fisheret al. (1985). Holleman et al. (1983) describe the chemical andphysical properties of this munition.

3.1.2 Synthetic-HC Smoke Combustion Products Mixture

Synthetic-HC smoke combustion products are a complex mixturewhose composition is given in Table 1. The stock test solutionwas mixed prior to each test using these percentages. In thisway, we avoided combusting munitions and collecting individualcomponents for the tests. HPLC grade solvents were used for theliquid chlorinated organics, while ultrapure HCI and metalliccompounds were used for the inorganic portions of the mix. Thesecomponents were added to diluent well water and allowed to mixand settle for 24 and 6 h, respectively.

The chlorinated organics analytical protocol was amodification of Standard Method 509A (APHA et al. 1985) for theanalysis of organic pesticides. A pentane extraction/concen-tration (10X) procedure was used to remove the chlorinatedorganics from the water prior to injection into a gaschromatograph equipped with an electron-capture detector.Aluminum, lead, cadmium and arsenic were analyzed by EPA's atomicabsorption(AA) spectrophotometric graphite furnace Methods 202.2,239.2, 213.2 and 206.2, respectively (USEPA 1983). Arsenic wasalso analyzed by EPA's AA spectrophotometric gaseous hydrideMethod 206.3. The zinc was analyzed by EPA's direct aspirationflame AA spectrophotometric Method 289.1. The details of the

13

V.3

analytical technique, including precision and accuracy data alongwith individual compound solubility and stability, are given inFisher et al. (1985).

The analytical methods allowed for detection limits for theindividual components of: 0.01 mg/L - chlorinated organics(0.005 mg/L with increased injection volume), 0.002 mg/L - Al,0.0002 mg/L - Cd, Pb and As and 0.00008 mg/L - Zn. Due to thelarge number of individual components in the mixture, results ofthe solubility and stability experiments from Fisher et al.(1985) will not be detailed here. Concentrations of componentsin the 100% stock mixture ranged from 0.0015 mg/L for As to258 mg/L for Zn (Table 10 and Appendix A). Stability of thecomponents in the stock mixture varied with component, time andtemperature.

3.1.3 Solvent Yellow 33 and Solvent Green 3

Technical grade and purified solvent yellow 33 and solventgreen 3 were received from the Inhalation Toxicology ResearchInstitute (ITRI), Lovelace Biomedical and Environmental ResearchInstitute, Albuquerque, NM. Chemically, the technical grade dyematerials used in testing were 93 - 95% pure, with the majorcontaminants being the precursors used in synthesis or, in thecase of solvent yellow 33, an apparent artifact of the synthesisprocess in which 3 instead of 2 molecules combined (Henderson etal. 1984).

Aqueous samples were analyzed by HPLC (Waters 4BONDAPAC C1 8column) using an isocratic 10% water : 90% methanol mobile phasefor solvent yellow 33 and a linear gradient from 10% water : 90%methanol to pure methanol for solvent green 3. The UV detectorwavelength for both dyes was 254 nm. The details of theanalytical technique, including precision and accuracy data alongwith solubility and stability information are described in Fisheret al. (1985). Aqueous samples were injected directly into theHPLC following filtration to 0.22 jin. This analytical methodallowed a detection limit of 0.08 mg/L for both dyes. Use of aC18 solid phase extraction cartridge allowed for an increase insensitivity to 0.002 mg/L. Initial studies on the solubility ofthese dyes indicated that the solubility of solvent yellow 33ranged from 0.09 mg/L at 120C to 0.17 mg/L at 220 C. The yellow33 component of solvent green 3 exhibited the same solubility,although the green component of this mixture was neverdetectable, even with a detection limit of 0.002 mg/L. Both dyeswere stable once dissolved in water, with no loss of compound ina 48 h period (Fisher et al. 1985).

3.2 Test Solution Preparation

3.2.1 DEGDN

Test solutions of DEGDN for the fish and invertebrate testswere prepared by vacuum evaporation of the ethanol from the

14

shipping solution and dissolving the DEGDN in diluent well water.This solution was then brought to the proper test temperature andused as the stock. The stock solution varied in DEGDNconcentration from 600 mg/L for the invertebrates to 3500 mq/Lfor the fish. Appropriate dilutions of these stocks were madewith diluent water to achieve the range of test concentrationsnecessary. The stock solutions and individual test solutionswere analyzed for aqueous DEGDN concentration. For theSelenastrum capricornutum bioassay, the pure DEGDN was dissolvedin the algae medium described later, and dilutions were made withthis same medium.

3.2.2 Synthetic-HC Smoke Combustion Products Mixture

The water-soluble fractions of the synthetic-HC smokecombustion products were obtained with a method similar to thatused by Anderson et al. (1974) to determine the water-solublefractions of crude and refined oils. Diluent water (15.5 L) wasplaced in a 22.5 L pyrex jar and brought to the appropriate testtemperature in an incubator (i.e., 24, 22, 17, or 12°C). For thealga test, 15.5 L of algal media was used instead of diluentfreshwater. The chlorinated organics, metals and HC1 were addedin the proportions shown in Table 2. Range-finding testsindicated that dilutions of this stock mixture would be toxic andallow for determinations of LC50 values. A stir bar was added,and the jar was sealed and placed on a magnetic stir plate in anincubator at the appropriate temperature. The stirring speed wasadjusted to create a vortex which extended to approximately 25%of the water depth. After 24 h of stirring, the jar was allowedto stand for 6 h to allow for settling of particulates.Appropriate dilutions were made from this 100% stock solution andused to determine LC50 values based on percentage dilutions as inan effluent test.

Water samples were taken from the stock to determineindividual component concentrations for each test. For testswith Daphnia magna and Salmo gairdneri, samples of each testdilution were analyzed at the beginning and end of each staticrenewal test period and at the beginning and end of the statictests. A new 100% stock solution was prepared for each renewal.

Further tests were conducted to determine the major toxiccomponent(s) of this mixture to Daphnia magna. The followingcomponent mixes were tested: total mix, metals only, organicsonly, Zn and HCI only and the total mix less Zn. Each componentmix was prepared in the same manner as the total stock mixturedescribed above. Each component was added in the amount shown inTable 2. LC50 values were based on the percentage dilution ofthe stock component mix. For the Zn plus HCI tests, LC50 valueswere also based on measured Zn. All the stock mixes anddilutions were analyzed for all individual components present.

15

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Technical grade formulations of the dyes were added toaerated diluent water or filter sterilized algal media andstirred for 24 h at the appropriate test temperature. Excessamounts were added in order to achieve a saturated solution of .

each dye (Fisher et al. 1985). The stock solutions were vacuumfiltered to 0.22 Wm and used to make appropriate test dilutions.Aqueous concentrations of the dyes in the stock solutions anddilutions were determined so that LC50 values could be based onmeasured dye concentrations.

3.3 Dilution Water Quality

The dilution water used in the fish and invertebrate testswas obtained from a non-chlorinated deep well. A comprehensiveanalysis of this well water is provided in Table 3.

3.4 Test Organisms

Specific information on the organisms used in testing isgiven in Table 4. The diurnal photoperiod for the fish andinvertebrates during culture and holding was 16 h light: 8 hdark. Lighting was provided by normal laboratory fluorescentlights of 50-100 foot candles intensity. During holding, therainbow trout were fed trout food pellets of appropriate sizesupplied by the National Fisheries Center - Leetown, WV. Theother fish were fed live Artemia until they were large enough totake flake food. Rainbow trout, bluegill and catfish wereobtained from fish hatcheries, held for two weeks and tested.Fathead minnow stock cultures were maintained in 76 L aquaria at250 C (± 20 C) using a recirculating system. Adult fish were fedflake food once each day ad libitum. Spawning substratesconsisted of inverted 15-cm sections of 10-cm diameter clay tileor PVC pipe cut in half longitudinally. Eggs were collected fromthe substrates and hatched in a separatory funnel under vigorousaeration. Upon hatching, larvae were transferred to a rearingtank and fed freshly-hatched Artemia less than 24 h old. Thelarvae were maintained for 2 weeks at 22*C (± 1°C) beforetesting.

A starter culture of Daphnia magna was obtained from theNational Fisheries Research Laboratory, Columbia, MO. Thisculture was used to initiate a continuous in-house culture.Twenty adult daphnids were stocked into each of seven 2.5 Lculture tanks. Aerated well water flowed through each tank at arate of 20 mL/min. Daphnids were fed Selenastrum capricornutumdaily ad libitum. Cultures were maintained at 220 C (± 10C).Neonates were removed 3 times/week or 24 h prior to theinitiation of a test.

Hexagenia bilinata nymphs were acquired from RobinsonWholesalers of Lake Geneva, WI. The nymphs, which wereapproximately 2-5 cm total length, were stocked at densities of

17

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500/200 L culture tank. These tanks contained 100 L of aeratedwell water and 4-5 cm of autoclaved biocide-free garden soil.Agricultural lime and composted hay were added to the substate toraise the pH to a range of 7-8 and increase organic material toapproximately 15%. Temperature was raised from 5°C to 22°C overa period of 9 days. Thereafter, temperature was maintained at220 C (± 10C). They were fed Tetra-min at a rate of 15 g/tanktwice/week until testing began.

Eggs of Paratanytarsus parthenogeneticus were obtained fromthe USEPA Environmental Research Laboratory in Duluth, MN. Eggswere stocked at a density of 250/40 L tank. Each tank contained7 L of aerated well water maintained at 220 C (± 10C). Afterhatching, larvae were fed a trout chow/cerophyll suspension asneeded to maintain a supply of food in the bottom of each tank.Upon emergence, adults were aspirated into a 500 mL Erlynmeyerflask containing 75 mL of aerated well water and allowed tooviposite overnight. Egg masses were collected the followingmorning and the process was repeated. Larvae used for testingpurposes were collected from 7 day old cultures by dislodgingthem from their tubes with a gentle stream of water.

Gammarus pseudolimnaeus were collected from the LittleClover River, Portage County, WS. They were shipped to APL andstocked at densities of 70 per 40 L culture tanks containing 30 Lof aerated well water. The culture tank substrate consisted ofan assortment of aged leaves collected from local streams.Cultures were maintained at 17°C (± 10C). Aerated well water wasused to replace 1/2 of the water in each tank three times/week.The amphipods were fed at a rate of 0.2 g/tank twice/week withTetra-min. Young for tests were removed from the tanks using a 4mm diameter pipette.

Selenastrum capricornutum starter culture was purchased fromNorth Texas State University, Denton, TX. Stock algal cultureswere reared in 2.5 L Pyrex culture flasks containing 1 L ofsterilized assay medium prepared according to Miller et al.(1978). Cultures were maintained under constant cool-whitefluorescent lights (4300 lux ± 10%) at a temperature of 240C(± 10 C) on a shaker table oscillating at 100 rpm (± 10%). Algalcells for testing purposes were obtained from 8 day old stockcultures.

3.5 Test Methodologies

3.5.1 General

Static and static renewal acute bioassay methods were thoserecommended by The American Society for Testing and Materials(ASTM 1980). Fish used in testing were acclimated to the wellwater for periods indicated in Table 4. Fish were not used intesting if they had any symptoms of disease within 10 days of thestart of the test, or if more than 3% died within 48 h precedingthe start of the test. Feeding was discontinued 24 h prior to

22

41S

the start of tests. All fish and tank assignments were random.Two replicates were used for each treatment with 10 fish perreplicate. Fathead minnows were transferred to the test beakersusing a fire-polisheO pipet, while other fish were transferred insmall beakers.

Test chambers and solution volumes used in testing allorganisms are given in Table 5. Temperatures were held withinI°C of the holding temperature (Table 4) by maintaining the testvessels in a constant temperature water bath. Temperature in thewater bath was monitored with a probe and chart recorder.Lighting was of the same quality and photoperiod as used duringholding for all species. The pH and dissolved oxygenconcentrations were measured daily in one replicate of eachtreatment which contained live organisms. Total hardness wasmeasured in the well water at the beginning of each test. Thetotal duration of the fish static acute tests was 96 h. Testconcentrations were renewed every 24 h where a static renewalmethod was used.

Invertebrate static and static renewal acute tests weresimilar to the fish tests except that the duration was 48 h.During the 24 h neonate collection period for daphnids, algalfood was provided. Neonates were transferred to a beakercontaining clean diluent water prior to the test. Otherinvertebrates were transferred to a common pool 24 h prior totesting and were not fed, except for Paratanytarsusparthenogeneticus, which were allowed a small amount of food tobuild tubes. Hexagenia bilinata were tested using small glasstubes of various diameters as substrates. Transfer of allinvertebrates was done using a 2 mm fire-polished pipet.Replicate beakers were used for each treatment, with 10 organismsper beaker.

Algal toxicity test methods were based on those recommendedby Payne and Hall (1979). Stock solutions were made by usingfiltered, sterilized assay media (Miller et al. 1978) instead ofdiluent well water. Test solutions were prepared by dilution ofthe stock solutions with uncontaminated filter sterilized assaymedia within a sterile transfer room. Test solutions weredispensed into Delong flasks and innoculated with Selenastrumcapricornutum cells in log growth to achieve a density of 5000cells/mL. Triplicates were prepared for each treatment. Flaskswere then capped and placed onto a shaker table in an incubatorset at the culturing conditions described earlier. Growthmeasurements (biomass and cell density) were made at 0, 24, 48,72, 96, and 120 h. Two 5 mL algal samples were frozen and, at alater time, biomass was indirectly measured by in vivochlorophyll a fluorescence with a Turner Designs Model 10Fluorometer. Algal cell density was determined from a separate 5mL sample with a Coulter Counter. Background fluorescence andcell density of a solution of AAP with the maximum amount oftoxicant added was also determined. After 120 h, all treatmentswith mean cell counts of : 6,500 cells/mL were restarted in

23

Z 23

TABLE 5. TEST CHAMBER AND TEST SOLUTION VOLUMES

Species Test Chamber Test Solution Volume(L)

Fathead minnow 0.4 L Beaker 0.35

Channel catfish 3.0 L Tank 2.75

Bluegill 3.0 L Tank 2.75

Rainbow trout 3.0 L Tank 3.00

Water flea 0.4 L Beaker 0.35

Midge larva 0.4 L Beaker 0.35

Mayfly larva 3.0 L Tank 1.50

Amphipod 0.4 L Beaker 0.35

Alga 0.28 L Delong flask 0.17

2I'"

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uncontaminated media and allowed to grow for an additional ninedays, at which time final growth measurements were recorded.

F

3.5.2 DEGDN

Initial tests with DEGDN indicated it to be very stable oncedissolved in diluent water, therefore, all acute toxicity testswere static, using test vessels with non-airtight covers.Aqueous DEGDN samples were analyzed at the beginning and end ofeach test for all treatments. This compound did exert asignificant oxygen demand during tests with fish. Aeration wasused in treatments where dissolved oxygen approached 40% ofsaturation. Aeration had no effect on aqueous DEGDNconcentrations. For example, in one aerated treatment vessel,DEGDN values averaged 178.9 mg/L at the start and 177.7 mg/L atthe end of the test.

3.5.3 Synthetic-HC Smoke Combustion Products Mixture

Initially, both static and static renewal definitive testswere to be conducted on all test species. Midway through thestudy, tests with the water flea and rainbow trout showed that nodifference in results occurred between the two methods.Therefore, the static method was used to test the remainingspecies. A full chemical analysis of all components wasconducted on the 100% stock solutions and individual treatmentsat the beginning and end of each renewal period and the beginningand end of the static tests for the daphnids and rainbow trout.These two species were found to be the most sensitive non-algalspecies. For the fathead minnows, midge larvae and mayflylarvae, chemical analyses were conducted on the 100% stocksolutions at each renewal. For the bluegill, catfish, amphipodand alga bioassays, full chemical analyses were conducted on the100% stock solutions and all treatments at the beginning of eachstatic test. All tests were conducted without aeration inclosed, but not airtight, chambers.

Further tests were conducted using the various components ofthis mixture to determine the toxic component(s) to daphnids.These were 48 h static acute tests with chemical analyses forindividual components of the 100% stock solutions and eachtreatment at the start of the test.

3.5.4 Solvent Yellow 33 and Solvent Green 3

Initial studies with these dyes indicated both to beminimally soluble in water but stable once dissolved (Fisher etal. 1985). Two range finding tests with Daphnia magna andParatanytarsus parthenogeneticus indicated no toxicity fromeither dye at its solubility limit. Therefore, only the aqueoussolubility limit was used in the remaining toxicity tests, exceptwhere toxicity was found. All tests were conducted using astatic acute protocol with covered, but not air-tight chambers.

25

Water samples were analyzed for each dye at the beginning and endof each test in all treatments.

3.6 Test End Points and Data Analyses"t.

In the tests with DEGDN and the dyes, LC50s were calculatedbased on average measured concentrations. In the tests with thesynthetic-HC smoke combustion products mixture, LC50s were basedon percentages of the 100% stock solutions used in the treatmentsrather than on individual concentrations of components, (as in aneffluent toxicity test). In some instances, LC50s were alsocalculated based on the concentration of an individual component(i.e., zinc in the Zn only tests with daphnids).

A fish was considered dead when ventilatory movements ceasedand the fish failed to respond to gentle prodding. Dead fishwere removed from test chambers twice/day, at which timemortalities were recorded. All invertebrates were similarlyrecorded dead when they failed to show any movement followinggentle stimulation. For the smaller organisms (i.e., Daphniamagna and Paratanytarsus parthenogeneticus), a microscope wasused to identify dead organisms.

LC50s and their 95% fiducial limits were determined usingthe probit method contained in a computer program developed byStephan (1978), unless otherwise noted. In all cases, thegoodness of fit probability of the data to the probit model wasgreater than 0.05.

In the algal toxicity test, the end point monitored wasgrowth, measured as density (cells/mL) and biomass (chlorophyll ain g/L). A log(10) transformation was used for all growthmeasurements. When treatment groups were compared to controls, aStudent's t test with Bonferroni's correction for simultaneouscomparisons was used. An overall significance level of 0.05 wasused in all analyses. When possible, three specific end pointswere evaluated:

1. Algistatic concentration. The minimum algistaticconcentration was determined by an inverse regression techniquedescribed by Payne and Hall (1979) and Sokal and Rohlf (1981).In this study, an algistatic effect was said to have occurred if,after the 5-day growth period, cell counts did not increasesignificantly from the initial inoculum level.

2. Algicidal concentration. This is the lowestconcentration tested which causes an apparent algistatic effectafter 5 days and which prevents cells transferred to clean mediafrom resuming logarithmic growth.

3. 5-day EC50 based on standing crop. Where possible, thealgal bioassay data were analyzed to calculate the 5-day EC50 andits 95% confidence limits. A linear model with treatment and dry

26

weight (density) both transformed to log(10) was fitted to thedata. Analysis of variance(ANOVA) was performed to test for fitto the model and for effects. Regression analysis was performedto provide estimated values for the day 5 data and for input usedto calculate the 5-day EC50 and its associated 95% confidencelimits. SAS PROC GLM computer software was used for theseanalyses (SAS 1982). The 5-day EC50 and its associated 95%confidence limits were computed using the FORTRAN CONFINTcomputer program (Feder 1981).

27

S%

SECTION 4

RESULTS AND DISCUSSION

4.1 Water Quality V

Dissolved oxygen varied between tests, with the lower valuesrecorded for the larger fish species (Table 6). The lowestdissolved oxygen values were approximately 4.0 mg/L at the end ofthe 96-h static DEGDN and dye tests with bluegill. These valueswere above the required 40% saturation limit for warm waterspecies at the test temperature (3.5 mg/L), but loading was near Vthe limit for the test vessels. As noted earlier, aeration didnot decrease the stability of the DEGDN. Values for pH wererelatively constant between tests, ranging from an average of 7.3to 8.4 at the end of the test (Table 6).

4.2 DEGDN

DEGDN proved to be relatively non-toxic to the freshwaterorganisms tested (Table 7). The invertebrate 48-h LC50s rangedfrom 90.1 to 355.3 mg/L, with the water flea being mostsensitive. All the fish species had similar sensitivities (mean96-h LC50 = 273.5 mg/L), except for the fathead minnows, whichwere somewhat more tolerant with an LC50 of 491.4 mg/L.

There was a dose-response relationship between Selenastrumcapricornutum growth and increased DEGDN concentration (Tables 8and 9; Figures 1 and 2). By day 5, the lowest concentration ofDEGDN tested (58.4 mg/L) caused a significant reduction in algalcell density and chlorophyll a when compared to control values(Student's t tests with Bonferroni's correction for simultaneouscomparisons, p = 0.0001). The 5-day minimum algistaticconcentration based on cell density was 171.6 mg/L (95%asymmetrical confidence limits = 86.9 - 335.5 mg/L). The minimumalgicidal concentration was greater than 542.4 mg/L, the highestconcentration tested. When algal cultures from this treatmentwere restarted in clean media, they resumed logarithmic grcwth(Figures 1 and 2). The 5-day EC50 for DEGDN and this alga wa39.1 mg/L based on dry weight (95% asymmetrical confidence 1imits= 28.8 - 52.9 mg/L). Thus, Selenastrum capricornutum was themost sensitive species tested with this compound.

There are no other aquatic toxicity data available forDEGDN. Krasovsky et al. (1973) reported LD50s for rats of 777mg/kg and for guinea pigs and white mice of 1250 mg/kg,indicating this compound to be relatively non-toxic compared tonitroglycerin and ethyleneglycol dinitrate, two similar explosivecompounds. Holleman et al. (1983) cited the lack of aquatictoxicology data for DEGDN but suggested it should be less toxicthan nitroglycerin due to its greater molecular weight.

Data from the present study indicates that DEGDN is less

toxic than nitroglycerin. Bentley et al. (1978) found

28

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33

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35

, &,: ',',,,,, ..- ,,. t,' . .. ' '. ',./. .,'.. ,, ,-.. . ."-"'. ' .' .","-" "-' -'

nitroglycerin to be toxic to freshwater aquatic organisms inamounts ranging from 0.4 mg/L (5-day EC50 - growth) forSelenastrum capricornutum and 2.8 mg/L (96-h LC50) for Salmogairdneri to 46 mg/L (48-h EC50) for Daphnia magna. For bothcompounds, the most sensitive species tested was Selenastrumcapricornutum.

Algal species appear to be the most sensitive species formany nitrogen containing munitions products. Liu et al. (1984)found that the freshwater algae Microcystis aeruginosa andSelenastrum capricornutum were the most sensitive species to 2,4-DNT(dinitrotoluene) and a complex condensate wastewater mixturecontaining 31 different nitrogen containing compounds. Therelative species sensitivities to nitrogen containing munitionsseem to vary depending on the compound tested. Of the 31compounds examined individually by Liu et al. (1984), Daphniamagna was more sensitive than Lepomis macrochirus in 14 cases.Bentley et al. (1978) found freshwater fish to be more sensitivethan invertebrates to nitroglycerin. Liu et al. (1984) found thesame trend of sensitivities with TNT (trinitrotoluene). Ourtoxicity studies with DEGDN indicate a more mixed sensitivitytrend, with Daphnia magna being the most sensitive animal speciesbut the rest of the invertebrates and fish showing variedsensitivities (Table 7). Fathead minnows proved to be the leastsensitive species, while rainbow trout exhibited only averagesensitivity (mean LC50 for all species = 282.5 mg/L; 96-h LC50for rainbow trout = 284.1 mg/L).

Spanggord et al. (1985) found DEGDN to be very soluble inwater (3900 mg/L). They indicated that aerobic and anaerobicbiotransformation and photolysis appear to be the major processeswhich would determine the persistence of DEGDN in aqueousenvironments. In natural waters, they found biotransformationand photolytic half-lives of 40 and 27 days, respectively. Therange of toxicities reported in this study indicate that DEGDNshould not be a problem in most environments with respect toacute toxicity. Because of its high water solubility, it couldpose a problem at production sites where the potential forspills exists.

4.3 Synthetic-HC Smoke Combustion Products Mixture

The concentrations of dissolved components in the 100% stocksolutions varied between tests (Table 10). Values for thechlorinated organics were less for tests conducted later in thestudy (i.e., last four species in Table 10). An explanation forthis anomaly is not apparent since, 1) all experimental andanalytical conditions were the same throughout the study and 2)the metal concentrations did not change over the course of thestudy. The concentration of dissolved zinc remained relativelyconstant throughout the study (mean ± S.D. = 23.43 + 2.3 mg/L).Zinc is by far the most abundant component in the stock mixture.Even after large dilutions of the stock mixture (i.e., 2 to 3%stock), the concentration of zinc still remained in the 1.0 to

36

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2.0 mg/L range (Appendix A). The relative consistency of thestock mixture over time can be seen in the identical toxicresponse of Daphnia magna tested at the beginning and end of thestudy (Table 13).

Actual measured component concentrations for all stocks anddilutions used during the study are presented in Appendix A. Forsome of the tests, analyses were conducted on the stock solutionsonly. The Appendix data shows good agreement between measuredconcentrations and concentrations predicted from dilutions of themeasured stocks, especially for the more stable compounds. Forexample, measured zinc concentrations in the dilutions for thebluegill bioassay are similar to values predicted from thedilutions. There were considerable differences between measuredand predicted values for the more unstable chlorinated organics.

The stability of the mixture varied according to thespecific component examined and the elapsed time. The most

extensive data concerning stability is from the rainbow trouttest (Appendix A-3). These data show that after 24 h, most ofthe chlorinated organics had decreased in concentration by 64 to82% in the 10% dilution. During that same period, the loss ofmetals ranged from 0 to 20%. An earlier study by Fisher et al.(1985) showed that the metals loss was due to precipitation fromthe dissolved phase. When a total sample was acidified after48 h, it was found to contain the same amount of total metals asat the start. Further loss of the chlorinated organics was foundafter 96 h. For example, only 10% of the CC1 4 remained in N.solution at the end of the test. In contrast, there was littlefurther loss of the metal components after 24 h.

The synthetic-HC smoke combustion products mixture was toxic N?

to all organisms tested (Table 11). The most sensitive fishtested was the rainbow trout with a 96-h LC50 of 2.2% (95%fiducial limits = 1.9 - 2.7%) for a static test and 2.3% (2.0 -2.7%) for a static renewal test. These values are based on thepercentage of 100% stock solution in the dilutions. The mostsensitive invertebrate appeared to be the water flea, with a 48-hLC50 of 9.3% (95% fiducial limits = 7.7 - 11.3%) for a statictest and 9.6% (7.6 - 12.8%) for a static renewal test. The LC50values for the two test types (static and static renewal) werenot significantly different for either species.

The similarity between results from the two test types is -indicative of toxicity due to metals. Miller et al. (1986) foundthe same LC50 for Ephemerella grandis exposed to a complex metalsmixture whether the test was a 24-h static or a 96-h flowthrough. This is indicative of a rapid effect where most of themortalities occur early in the exposure. This rapid earlymortality was evident in the present study, regardless of thetype of test. In both the daphnid and rainbow trout tests, theconcentration of metals at the end of the tests were similar inthe static and static renewal tests indicating a rapid decline inmetals over the 24 h renewal period followed by stabilization

38

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39

thereafter (Appendix A-I and A-3). Also, the total metals in thetest system did not change over the experimental period, asdiscussed earlier.

The amphipod also appeared to be quite sensitive to themixture, although an LC50 value could not be calculated becausethe test was only designed as a range-finding bioassay. The testyielded 89% mortality at an 18% stock solution and 72% mortalityat the greatest dilution tested (10% stock solution). Thesemortality data do not meet the criteria necessary to calculate anLC50 (Stephan 1978). LC50 values for the remaining fish speciesranged from 13.6% for the fathead minnow to 45.9% for thebluegill. Values for the remaining invertebrates ranged from54.1% for the mayfly larvae to 89% for the midge larvae.

Exposure to the synthetic-HC mixture for 5 days caused adramatic reduction in Selenastrum capricornutum growth based onboth density (Table 12; Figure 3) and biomass (Table 12; Figure4). At the greatest dilution tested (5.6% stock), there was areduction from control algal growth of 99.6% based on density(cells/mL) and 99.7% based on biomass (4g/L chlorophyll a). Forall the dilutions tested (5.6, 10, 18, 32, 56 and 100% stock),the average reduction in density and biomass was 99.4% (S.D. = ± .10.34%) and 99.7% (S.D. = + 0.31%), respectively. The measuredcomponent concentrations (mg/L) at the 5.6% treatment were:0.02 - CC1 4 ; 0.06 - C2C1 4 ; 0.01 - C2C1 6 ; 5 0.005 - C6C16;2.22 - Zn; 0.004 - Pb; 0.0029 - Cd; 50.0002 As; and 0.006 - Al.The lack of significant algal growth at the greatest dilutiontested (5.6% stock) made it impossible to calculate a minimumalgistatic concentration. Since the 5.6% stock treatmentresulted in such a dramatic reduction in algal growth, it is safeto say that the algistatic concentration is well below thislevel. This indicates that Selenastrum capricornutum was themost sensitive species tested.

Algae cultures with measured densities : 6500 cells/mL after5 days exposure were restarted in fresh, uncontaminated media andallowed to recover for 9 days in an effort to determine an -Valgicidal concentration. New cultures from the 5.6, 32, 56 and100% treatments were restarted. At the end of the recoveryphase, no culture resumed logarithmic growth (Figures 3 and 4).There was a slight, but statistically significant, increase incell density in the culture from the 5.6% treatment (Table 12;Figure 3)(Student's t test, p = 0.05). In contrast, this cultureshowed no significant increase in biomass (Table 12; Figure 4).There was no measured growth in cultures from the other restarted 7,treatments, based on either density or biomass. In fact, therewas a decrease in biomass to non-detectable levels in all thesecultures. These data indicate that the greatest dilution tested(5.6% stock) had an algicidal effect on Selenastrumcapricornutum. Since there was an almost complete growth Vreduction of the alga at all dilutions tested, it was notpossible to calculate a 5-day EC50.

40 %

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43

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44

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These toxicity data show that the dissolved components ofthis mixture are very toxic to a number of freshwater aquaticspecies, especially the alga Selenastrum capricornutum. A reviewof the literature indicates that this toxicity is not surprisingconsidering the dominance of zinc in the mixture. Greene et al.(1975) found 14 day EC95 growth values of 40.4 and 68.0 iLg Zn/Lfor this algal species, while Bartlett et al. (1974) showedgrowth inhibition of the same species at zinc levels of 30 4g/L.In the present study, the 5.6% stock solution treatment had ameasured Zn concentration of 2.22 mg/L. According to thefindings of Bartlett et al. (1974), even a dilution containingonly 0.11% of the stock would have produced a measurable effect.This dilution would have been extremely dificult to obtain withany accuracy in the present study considering the test volumesused. The zinc concentration in the 5.6% treatment is well abovethe newest water quality criteria proposed by EPA (1985b) for thedilution water hardness used in the tests (criterion continuousconcentration [CCC] = 147 4g/L; criterion maximum concentration[CMC] = 162 4g/L).

The data also indicate that zinc may be the componentresponsible for the toxicity of the mixture. Tests withindividual and groups of components with daphnids seem to verifythis (Table 13; Figure 5). Component concentrations for thestock solutions and the various dilutions used in these statictests are presented in Appendix A-5. Results of these testsindicate that the metals were the major toxic group, exhibitingtoxicity equal to that of the total mixture. The tests alsoindicate that zinc was the major toxic individual component.Zinc tested alone yielded a 48-h LC50 of 12.8% (mean of twotests; S.D.s ranged from 9.3 to 17.2%). These percentages arebased on dilutions of a Zn stock mixed using the same amount ofZn as in the total mix. This toxicity is similar to the averagetoxicity value of 9.0% (S.D.s ranged from 6.3 to 12.8%) from fourtests with the total mix. When the chlorinated organics weretested as a group, they resuted in only minimal toxicity to thedaphnids. Also, when the total mix was tested without zinc, thedaphnids were much less sensitive with a 48-h LC50 of only 37.1%.The toxicity values from these component tests were also computedbased on the measured zinc concentrations. The LC50 valuescomputed in this manner were similar for the total mixture, Znonly and metals only tests, with average 48-h LC50 values of1.83, 3.21 and 2.12 mg/L Zn, respectively (Table 14; Figure 6).

The tests with Daphnia magna indicate the possibility ofsome synergistic toxicity effect when the remaining metals arepresent with Zn. The total metals mixture had a 48-h LC50 of8.1% (95% fiducial limits = 6.0 - 10.1%) while the zinc onlymixture had LC50s of 13.8% (10.4 - 17.2%) and 11.7% (9.3 - 14.1%)in two separate tests. When these LC50 values were computedbased on measured zinc concentrations (Table 14) it appeared thatthe daphnids were more sensitive to zinc in the presence of theother metals (e.g., LC50 for Zn lower when other metals present).Mixtures of metals have been shown to behave differently

45

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toxicologically than their individual component metals (Wong andBeaver 1980; Wong et alo 1978; Eaton 1973; Sprague and Ramsey1965). More information about the toxicity of the individualmetals and their mixtures would be necessary to verify theinteraction seen in the present study.

As noted above, Zn appears to be the major toxic componentof this mixture, at least for the alga and daphnids. The 48-LC50values for Daphnia magna based on measured Zn in the Zn only andtotal mix tests were 1.83 and 3.21 mg/L, respectively. Thesevalues are similar to zinc toxicity values reported in theliterature for this species. The proposed national water qualitycriteria for zinc list a range of 48-h EC50s from 280 to 799 4g/Lwhen the Zn was presented as ZnCI 2 , as in the present study(USEPA 1986b). These values were based on immobilization ratherthan death and are therefore not directly comparable. Since thetoxicity of zinc is quite variable, depending on a number ofchemical factors (i.e., calcium, magnesium, hardness, pH andionic strength), toxicity values will vary considerably betweenstudies (USEPA 1986b).

Other than zinc, the concentrations of the remaining metalsin the mixture appear to be below levels which would beconsidered acutely toxic. The national water quality critera(USEPA 1985a) for arsenic are 0.19 and 0.36 mg/L for CCC and CMC,respectively, while the average concentration in the 100% stocksolutions was only 0.0014 mg/L. The lowest species mean acutevalue (SMAV) given for lead in the national water qualitycriteria is 0.15 mg/L for Gammarus pseudolimnaeus (USEPA 1985b).Other SMAVs listed in the same document were 0.45, 2.45, 25.44and 52.31 mg/L for daphnids, rainbow trout,fathead minnow andbluegill, respectively. In contrast, the concentration of leadin the 100% stock solutions averaged only 0.008 mg/L. SMAVs forcadmium reported in the national water quality criteria rangefrom a low of 0.0036 mg/L for rainbow trout to 6.96 mg/L forbluegill (USEPA 1986c). Daphnia magna was the second mostsensitive species reported, with a SMAV of 0.013 mg/L. Since theconcentration of cadmium in the stock solutions for all testsaveraged 0.025 mg/L, there is a possibility for acute cadmiumtoxicity from the stock solutions, especially to daphnids andrainbow trout. If cadmium concentrations are examined at theLC50 dilutions though, it can be seen that they are below thereported SMAV (i.e., 0.0008 mg/L at the LC50 dilution for rainbowtrout and 0.004 mg/L for daphnids). Burrows (1977) reports toxiclevels of aluminum ranging from 0.32 mg/L for Daphnia magna to 5mg/L for Salmo gairdneri. Aluminum concentrations in the stocksolutions in the present study averaged only 0.05 mg/L.

Similarly, the chlorinated organic compounds also seem to bepresent in amounts that should not be acutely toxic, especiallyat dilutions of the stock mixtures which caused toxicity. Whenthese organics were tested as a group, they exhibited onlyminimal toxicity to Daphnia magna (Table 13; Figure 5). Carbontetrachloride does not appear to be acutely toxic to daphnids at

50

concentrations below 27.3 mg/L (USEPA 1978). In fact, a chronicno-effects level for Pimephales promelas was reported to be 3.4mg/L in this same document. In the 100% stock solutions, theaverage concentration of carbon tetrachloride was only 1.95 mg/L(S.D. = ± 0.71 mg!L) Shubat et al. (1982) found a 96-h LC50 of4.99 mg/L for Salmo gairdneri using tetrachloroethylene. BothLeBlanc (1980) and Richter et al. (1983) found a 48-h LCS0 of18.0 mg/L for this compound with Daphnia magna, while Walbridgeet al. (1983) found a 96-h LC50 o4 13.4 mg/L for the fatheadminnow. Although the average concentration oftetrachloroethylene in the 100% stocks in the present study was6.16 mg/L (S.D. = ± 1.80 mg/L), the measured concentrations werewell below acutely toxic levels at dilutions near the LC50. Forexample, the 48-h LC50 for Daphnia magna was 9.0%. The measuredconcentration of this organic compound in the 10% dilution ofthis test was 0.45 mg/L, well below levels found toxic by Leblanc(1980) and Richter et al. (1983).

The national water quality criteria for hexachloroethaneindicates a most sensitive species SMAV of 0.98 mg/L for bothrainbow trout and bluegill (USEPA 1980b). At dilutions near theLC50 values for these species though, hexachloroethaneconcentrations were found to be only 0.023 mg/L for rainbow troutand 0.24 mg/L for bluegills. In contrast to the otherchlorinated organics, hexachlorobenzene was never detected in thestock mixtures, even when the detection limit was as low as 0.002mg/L. Calamari et al. (1983) found only a 12% growth inhibitionof Selenastrum capricornutum at 0.03 mg/L hexachlorobenzene, andno toxicity to Daphnia magna or Salmo gairdneri at the sameconcentration. Laska et al. (1978) found no mortality incrayfish (Procambarus clarki) or largemouth bass (Micropterussalmoides) after 10 days of exposure to 0.027 mg/L and 0.026 rr'/Lhexachlorobenzene, respectively.

Species sensitivities to the synthetic-HC mixture were alsoconsistent with zinc toxicity. The alga was the most sensitivespecies, followed by the rainbow trout and the daphnids. This isthe same general sensitivity trend reported in the national waterquality criteria for zinc (USEPA 1986b). For tests conducted atsimilar water hardnesses, this document reports a 7-day growthinhibition value for Selenastrum capricornutum of 30 Lg/L Zn, a96-h LC50 for swim-up stage rainbow trout of 93 - 136 4g/L and a48-h EC50 for Daphnia magna of 334 4g/L.

The results of the present study indicate that, at thecomponent concentrations tested, the water soluble fraction ofthe synthetic-HC smoke combustion products would be a seriousthreat to freshwater aquatic species. Every indication is thatzinc is the major toxic component of this mixture. It must bestressed that these component concentrations are artificiallyderived levels which were used in an effort to determine LC50values for the various species. At present, there is no dataconcerning the actual amounts of these components which could endup in the aquatic environment after use of the grenades and smoke

51

V

pots or after disposal of the stockpiled munitions. These typeof data are essential for determining potential hazards toaquatic organisms.

4.4 Solvent Yellow 33 and Solvent Green 3

Fisher et al. (1985) found the solubility of solvent yellow33 to range from 0.09 mg/L at 120 C to 0.17 mg/L at 220 C, whetherit was alone or part of the solvent green 3 mixture. The greencomponent of the solvent green 3 was never detected in watersamples, even when detection limits were reduced to 0.002 mg/Lusing a C1 8 solid phase extraction cartridge for concentration.

During the toxicity tests, the average concentrations of solventyellow 33 at its solubility limits were 0.20 mg/L (S.D. = ± 0.013mg/L), 0.16 mg/L (± 0.031), 0.12 mg/L (± 0.009) and 0.09 mg/L (±0.009) in tests conducted at 24, 22, 17 and 120 C, respectively(Table 15). For the yellow 33 component of the solvent green 3,concentrations at the same test temperatures averaged 0.20 mg/L(S.D. = ± 0.030 mg/L), 0.16 mg/L (± 0.031), 0.10 mg/L (± 0.001)and 0.08 mg/L (± 0.004), respectively. The green component ofthis dye mixture was never detected during the toxicity tests(detection limit = 0.002 mg/L). During the rainbow trout test,the 50% dilution of the solvent green 3 solubility stock wasfound to contain 0.055 mg/L (S.D. = ± 0.0005 mg/L) of the yellow33 component and non-detectable amounts of the green component.

All of the vertebrate and invertebrate species were testedat the solubility limit of each dye. Only during the rainbowtrout test was there significant mortality compared to thecontrols (Table 15). The solvent green 3 killed 50% of the troutexposed at the solubility limit in two separate tests. Therewere no mortalities at a 50% dilution of this mix. Due to thelack of both sufficient partial kills and a complete kill, it wasimpossible to calculate an LC50. The solvent yellow 33 did notcause any mortality to the rainbow trout. Concentrations abovethe solubility limit were not tested (i.e., no carrier solvents).

There was a significant effect when Selenastrumcapricornutum was exposed to either dye. Following 5 daysexposure to solvent yellow 33 at its solubility limit, celldensity was reduced 68% from control values, while biomass wasreduced 75% (Table 16; Figures 7 and 8). After the same exposureperiod, solvent green 3 caused a 98% reduction in cell densityand a 99% reduction in biomass compared to controls. For bothdyes, cell density values after 5 days were significantly greaterthan values at the start, indicating that neither an algistaticor algicidal concentration had been tested (Table 16). Since thealgae were tested only at the solubility limit of the dyes, noEC50 could be calculated. Because of its dramatic effect, it isobvious that the solvent green 3 EC50 for the alga is well belowits solubility limit. Thus, Selenastrum capricornutum is themost sensitive species tested with the dyes. In fact, this alga

52

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56

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57

was the only freshwater species tested which was affected by the 'solvent yellow 33.

There appears to be a potential environmental problem with Vthese dyes, especially solvent green 3. Although there was noacute toxicity caused by either dye to seven of the nine speciestested, there was an effect on the rainbow trout and the alga.The effect of solvent green 3 on Selenastrum capricornutum wasespecially dramatic considering the aqueous concentration at thesolubility limit. Whenever a compound causes mortalities in thelow ppm (0.2 mg/L yellow 33 component) and ppb (:5 0.002 mg/L)range, even to only a few species, concern must be expressed.Usage, production and disposal patterns should be examined todetermine both the existing levels of environmental contaminationand the possibility of environmental release of these dyes.

Technical formulations of both dyes contained one majorimpurity which could possibly be the cause of the toxicity seen.Henderson et al. (1984) examined the dyes and found that thisimpurity made up 2.2%, by weight, of the solvent yellow 33 and0.7% of the solvent green 3. They identified it as acondensation of one molecule of quinaldine with two molecules ofphthalic anhydride. If this impurity was the cause of thedramatic alga effect seen with the solvent green 3, a similareffect should have been seen with the solvent yellow 33 since theimpurity is present in even greater amounts. Also, this impuritydid not seem to show a differentially greater solubility than theactual dye components in our HPLC samples. Therefore, it appearsthat the possible toxic effects of this impurity are minimal.

58

.. . . . .. . . ... . . . , . .

oP

SECTION 5

CONCLUSIONS

The toxicity of DEGDN was relatively low to the ninefreshwater species tested, especially when compared to othercommonly used nitrate ester explosives such as nitroglycerin andethyleneglycol dinitrate. Toxicity values ranged from a 5-dayEC50 (standing crop) of 39.1 mg/L for the green alga Selenastrumcapricornutum, to a 96-h LC50 of 491.4 mg/L for the fatheadminnow (Pimephales promelas). The most sensitive invertebratetested was Daphnia magna which was more sensitive than the mostsensitive fish tested, Lepomis macrochirus. Due to its highwater solubility, DEGDN could cause environmental problemsfollowing significant introductions to receiving streams atproduction facilities.

The dissolved components of the synthetic-HC smokecombustion products mixture were found to be quite toxic to anumber of freshwater species, especially Selenastrumcapricornutum, Salmo gairdneri and Daphnia magna. A testsolution containing only 5.6% of a stock mixture of thesecomponents caused both an algistatic and algicidal effect on thealga. LC50 values for the trout and the water flea were 2.2% and9.3% of the stock solution, respectively. Additional tests withthe water flea indicate that zinc was the major toxic componentof the mixture. Information concerning environmental concentra-tions of the various components after use or disposal of themunitions is necessary in order to assess possible hazards toaquatic life.

Solvent yellow 33 and solvent green 3 were not toxic toseven of nine freshwater species when tested at their solubilitylimits. A solubility limit solution of solvent green 3 killed50% of the rainbow trout exposed but was non-toxic when dilutedby 50%. Solvent yellow 33 was non-toxic to the trout at itssolubility limit. Both dyes caused a reduction in alga growth atsolubility limits, with solvent green 3 being most detrimental,causing a 98 - 99% reduction after 5 days of exposure. Thesedyes could cause a problem if released to the environment sincean effect was noted at their low solubility concentrations (i.e.,0.2 mg/L solvent yellow 33 and < 0.002 mg/L green component ofsolvent green 3).

59

SECTION 6

LITERATURE CITED

Anderson, J. W., J. M. Neff, B. A. Cox, H. E. Tatem and G. M.Hightower. 1974. Characteristics of dispersions and watersoluble extracts of crude and refined oils and theirtoxicity to estuarine crustaceans and fish. Mar. Biol. 27:75-88.

APHA (American Public Health Association, American Water WorksAssociation and Water Pollution Control Federation). 1985.Standard Methods for the Examination of Water andWastewater, 16th ed. APHA, Washington, DC.

ASTM (American Society for Testing and Materials). 1980.Standard practice for conducting toxicity tests with fishes,macroinvertebrates and amphibians. Pages 272-296 in ASTMannual book of standards, ASTM Designation E 729-80.Amer. Soc. Testing Materials, Philidelphia, PA.

Bartlett, L., F. W. Rake and W. H. Fink. 1974. Effects ofcopper, zinc and cadmium on Selenastrum capricornutum.Water Res. 8:179-185.

Bentley, R. E., J. W. Dean, S. J. Ells, G. A. LeBlanc, S. Sauter,K. S. Buxton and B. H. Light. 1978. Laboratory evaluationof the toxicity of nitroglycerin to aquatic organisms.Final Report. AD A061739. EG&G Bionomics, Wareham, MA.DAMD 17-74-C-4101.

Burrows, W. D. 1977. Aquatic aluminum: chemistry, toxicologyand environmental prevalence. CRC Crit. Rev. Environ.Control 7:167-216.

Calamari, D., S. Galassi, F. Setti and M. Vighi. 1983. Toxicityof selected chlorobenzenes to aquatic organisms.Chemosphere 12:253-262.

Eaton, J. G. 1973. Chronic toxicity of a copper, cadmium andzinc mixture to the fathead minnow (Pimephales promelasRafinesque). Water Res. 7:1723-1736.

Feder, P. I. 1981. Design and analysis of chronic aquatic testswith Daphnia magna. Final Report. DAMDI7-80-C-0165.Applied Statistics Group, Batelle Columbus Laboratories,Columbus, OH.

Fisher, D. J., D. T. Burton, M. J. Lenkevich, and K. R. Cooper.1985. Analytical methods for determining the concentrationof solvent yellow 33, solvent green 3, synthetic-HC smokecombustion products and DEGDN in freshwater. Final Report -Phase I. AD-A167875. The Johns Hopkins University, Applied

60 ,

600

L5S -'-5-.ft

Physics Laboratory, Shady Side, MD.

Greene, J. C., W. E. Miller, T. Shiroyama and E. Merwin. 1975.Toxicity of zinc to the green alga Selenastrum capricornutumas a function of phosphorous or ionic strength. EPA-660/3-75-034. National Technical Information Service,Springfield, VA.

Henderson, R. F., Y. S. Cheng, J. S. Dutcher, T. C. Marshall andJ. E. White. 1984. Studies on the inhalation toxicity ofdyes present in colored smoke munitions: generation andcharacterization of dye aerosol. Final Report - Phase I.AT (29-2)-2138/3807, AD A142491. Lovelace InhalationToxicology Research Institute, Albuquerque, NM. DE-AC04-76EV01013.

Holleman, J. W., R. H. Ross and Captain J. W. Carroll. 1983.Problem definition study on the health effects ofdiethyleneglycol dinitrate, triethyleneglycol dinitrateand trimethylolethane trinitrate and their respectivecombustion products. Final Report. AD A127846. OakRidge National Laboratory - Union Carbide, Oak Ridge, TN.ORNL/EIS-202.

Katz, S., A. Snelson, R. Farlow, R. Welker and S. Mainer. 1980.Physical and chemical characterization of fog oil andhexachloroethane smoke. Final Report. DAMD17-78-C-8085.IIT Research Institute, Chicago, ILL.

Krasovsky, G. N., A. A. Korolev and S. A. Shigan. 1973.Toxicological and hygienic evaluation of diethyleneglycoldinitrate in connection with its standardization in waterreservoirs. J. Hyg. Epidemiol. Microbiol. Immunol17:114-119.

Laska, A. L., C. K. Bartell, D. B. Condie, J. W. Brown, R. L.Evans and J. L. Laseter. 1978. Acute and chronic effectsof hexachlorobenzene and hexachlorobutadiene in Red SwampCrayfish (Procambarus clarki). Toxicol. Appl. Pharm. 43:1-12.

LeBlanc, G. A. 1980. Acute toxicity of priority pollutants toWater Flea (Daphnia magna). Bull. Environm. Contam.Toxicol. 24:684-691.

Linder, V. 1980. Explosives and propellants. Pages 561-671 inM. Graysom and D. Eckroth, eds. Kirk-Othmer Encyclopedia ofChemical Technology, 3rd ed. Vol. 9, John Wiley and Sons,New York.

Liu, D. H. W., R. J. Spanggord, H. C. Bailey, H. S. Javitz and D.C. L. Jones. 1984. Toxicity of TNT wastewaters to aquaticorganisms. Vol. I & II. Final Report. DAMD17-75-C-5056.SRI International, Menlo Park, CA.

61

'IWP

Miller, W. E., J. C. Greene and T. Shiroyama. 1978. TheSelenastrum capricornutum Printz algal assay bottle test.EPA-600/9-78-018. U. S. Environmental Protection Agency.Corvallis Environmental Research Laboratory, Corvallis, OR.

Miller, T. G., S. M. MeChancon and T. W. LePoint. 1986. Use ofeffluent toxicity tests in predicting the effect of metalson receiving stream invertebrate communities. Pages 265-281 in H. L. Bergman, R. A. Kimerle and A. W. Maki, eds.Environmental Hazard Assessment of Effluents. Soc. Environ.Toxicol. Chem. Spec. Publ. Ser. Pergamon Press, New York.

Payne, A. G. and R. H. Hall. 1979. A method for measuring algaltoxicity and its application to the safety assessment of newchemicals. Pages 171-180 in L. L. Marking and R. A.Kimerle, eds. Aquatic Toxicology, ASTM STP 667, Amer. Soc.Testing Materials, Philadelphia, PA.

Richter, J. E., S. F. Peterson and C. F. Kleiner. 1983. Acuteand chronic toxicity of some chlorinated benzenes,chlorinated ethanes and tetrachloroethylene to Daphniamagna. Arch. Environ. Contam. Toxicol. 12:679-684.

SAS. 1982. SAS users guide: statistics, 1982 ed. SASInstitute Inc., Cary, NC.

Shubat, P. J., S. H. Poirier, M. L. Knuth and L. T. Brooke.1982. Acute toxicity of tetrachloroethylene and tetra-chloroethylene with dimethyl-formamide to rainbow trout(Salmo gairdneri). Bull. Environm. Contam. Toxicol. 28:7-10.

Sokal, R. R. and F. J. Rohlf. 1981. Biometry. W. H. Freemanand Company, New York.

Spanggord, R. J., Tsong-Wen Chou, T. M. Mill, R. T. Podoll, J. C.Harper and D. S. Tse. 1985. Environmental fate ofnitroquanidine, diethyleneglycol dinitrate, and hexachloro-ethane smoke - Phase I. Draft Final Report. Contract No.DAMDI7-84-C-4252. SRI International, Menlo Park, CA.

Sprague, J. B. and B. A. Ramsay. 1965. Lethal levels of mixedcopper-zinc solutions for juvenile salmon. J. Fish. Res.Board Can. 22:425-432.

Stephan, C. E. 1978. LC50 program. Unpublished data. U. S.Environmental Protection Agency, Environmental ResearchLaboratory - Duluth, Duluth, MN.

USEPA. 1978. In-depth studies on health and environmentalimpacts of selected water pollutants. EPA Contract No. 68-01-4646. U. S. Environmental Protection Agency, Washington,DC.

62

-I

USEPA. 1980a. Ambient water quality criteria for chlorinatedbenzenes. EPA 440/5-80-028. U. S. Environmental ProtectionAgency. Washington, DC. *

USEPA. 1980b. Ambient water quality criteria for chlorinated

ethanes. EPA 440/5-80-029. U. S. Environmental ProtectionAgency. Washington, DC.

USEPA. 1983. Methods for chemical analysis of water and wastes.EPA-600/4-79-020 (Rev. 1983). U. S. EnvironmentalMonitoring Support Laboratory, Cincinnati, OH.

USEPA. 1985a. Ambient water quality criteria for arsenic -revised. EPA 440/5-80-021. U. S. Environmental ProtectionAgency. Washington, DC.

USEPA. 1985b. Ambient water quality criteria for lead. EPA440/5-84-027. U. S. Environmental Protection Agency.Washington, DC.

USEPA. 1985c. Ambient water quality criteria for cadmium. EPA440/5-84-032. U. S. Environmental Protection Agency.Washington, DC.

USEPA. 1986a. Quality criteria for water - 1986. EPA 440/5-86-001. U. S. Environmental Protection Agency. Washington,DC.

USEPA. 1986b. Ambient water quality criteria for zinc. Draft.(4/10/86). U. S. Environmental Protection Agency. Originalzinc criteria - EPA-440/4-80-079. U. S. EnvironmentalProtection Agency, Washington, DC.

Walbridge, C. T., J. T. Fiandt, G. L. Phipps and G. W. Holcombe.1983. Acute toxicity of ten chlorinated hydrocarbons to thefathead minnow (Pimephales promelas). Arch. Environ.Contam. Toxicol. 12:661-666.

Weaver, J. E. 1983. Dose response relationship in delayedhypersensitivity to quinoline dyes. Contact Dermatitis9:309-312.

Wong, P. T. S., Y. K. Chou and P. L. Luxon. 1978. Toxicity of a Imixture of metals on freshwater algae. J. Fish. Res. BoardCan. 35:479-481.

Wong, S. L. and J. L. Beaver. 1980. Algal bioassays todetermine toxicity of metal mixtures. Hydrobiologia 74:199-208.

63

Oft

APPENDIX A

DISSOLVED CONCENTRATIONS OF THE COMPONENTSOF THE SYNTHETIC-HC SMOKE COMBUSTION

PRODUCTS MIXTURE IN DILUTIONSUSED DURING TOXICITY TESTS

- unless otherwise stated, ND meansnon detectable, as follows:

Compound Detection limit (mg/L)

Chlorinated organics 0.01*Zn 0.00008Pb 0.0002Cd 0.0002As 0.0002Al 0.002

The purchase of a new computer/intergrator system allowed bettersensitivity and a detection limit of 0.005 mg/L in some instances

64

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APPENDIX B

ADDITIONAL GOOD LABORATORY PRACTICE STANDARDS REPORT REQUIREMENTS

U.S. EPA Good Laboratory Practice (GLP) Standards (EPA 1983.Fed. Reg. 48:53946-53969) for non-clinical studies require thefollowing additional statements be included in all final reportswhich summarize data collected under GLP Standards:

Study director: Dennis T. Burton, Ph.D.

Project scientists: Daniel J. Fisher, Ph.DRobert L. Paulson, M.S.

Location of all raw data, documentation, records, datareports, Quality Assurance Unit (QUA) reports, and finalreport:

ArchivesThe Johns Hopkins UniversityApplied Physics LaboratoryEnvironmental Sciences GroupShady Side, MD 20764

Final Quality Assurance Unit statement: Page 74 of thisreport

bennis T. Burton,Ph.D.Study Director

Date: -/-

73

4,

MEMORANDUM

To: Dr. Dennis T. BurtonStudy Director

From: Lenwood W. Hall, Jr. - QA/QC Officer

Date: January 20, 1987

Subject: Final Good Laboratory Practice (GLP) Report, ArmyProject MIPR 85 MM 5505

I have roviewed the draft of the final report for Phase II

of the project submitted to me on January 16, 1987. The report

accurately describes the methods and standard operating

procedures originally submitted for the project and accurately

reflects the raw data.

I conducted quarterly QA/QC inspections during Phase II of

the project. These inspections were conducted on February 25,

May 28, August 26, and October 23, 1986. The project completion

deadline was extended to January, 1987 with the inclusion of

extra studies requested by the sponsor and completed at no

additional charge to the contract. The study was completed on

schedule according to the revised schedule. My inspections

indicated that the intent of the GLP regulations were followed,

although a number of minor problems were reported. These

problems were corrected to my satisfaction.

Lenwood W. Hall, Jr.

74

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DOCUMENT DISTRIBUTION LIST

No. of Copies

25 CommanderUS Army Biomedical Research and j

Development LaboratoryATTN: SGRD-UBZ-CFort Detrick, Frederick, MD 21701-5010

2 CommanderUS Army Medical Research and Development CommandATTN: SGRD-RMIFort Detrick, Frederick, MD 21701-5012

DeanSchool of MedicineUniformed Services University of the

Health Services4301 Jones Bridge RoadBethesda, MD 20814-4799

1 CommandantAcademy of Health Sciences, US ArmyATTN: AHS-CDMFort Sam Houston, Houston, TX 78234-6100

HQDAATTN: DAEN-RDM/Dr. Meyer20 Massachusetts Avenue (NW)Washington, DC 20314

CommanderU.S. Army Environmental Hygiene AgencyATTN: HSHB-AD-L (Librarian)Aberdeen Proving Grounds, MD 21010

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