AECOM



# Acute Toxicity of Pentaerythritol
Tetranitrate (PETN) to the Cladoceran ( **Daphnia magna)**
U nder Static

- Renewal Test
Conditions

**For** 

# Dyno Nobel Inc. West Jordan, Utah, USA

Prepared by

AECOM Technical Services, Inc.
Fort Collins Environmental Toxicology Lab

December 2012

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Study Title

Acute Toxicity of Pentaerythritol Tetranitrate (PETN) to the Cladoceran ( Daphnia magna ) Under
Static

- Renewal Test Conditions

Study Initiation Date : 1 February 2012

Study Completion Date : 20 Decem ber 2012

Conducted for : Dyno Nobel Inc.
3000 West 8600 South West Jordan, Utah 84088

Project Officer : Neal Olsen, DNI Environmental Affairs Manager

Performing Laboratory : AE COM Fort Collins Environmental Toxicology Laboratory
4303 West LaPorte Avenue Fort Collins, Colorado 80521

AECOM Project ID
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Final Report
December 2012AECOM

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Study Director Rami B. Naddy, Ph.D.

Test Laboratory AECOM Fort Collins Environmental Toxicology Laboratory
4303 West LaPorte Avenue Fort Collins, Colorado 80521

Analytical Laboratory Test America Laboratories, Inc.
TestAmerica Denver
4955 Yarrow Street Arvada, Colorado 80002

Location of Original Data and Final Report AECOM
4303 West LaPorte Avenue Fort Collins, Colorado 80521

or Data

- Lock Records Management 6707 McMurray Ranch Road
Bellvue , Colorado 805 12

Test Substance Pentaerythritol Tetranitrate (PETN)

AECOM Identification Number (s) GLP

- 06 5
Subject 48

- Hour Static

- Renewal Toxicity Test

Test Method OPPTS Method 850.1010 and ASTM Method E 729

- 96

Experimental Test Dates 30 May to 01 June 2012
Length of Study 48 Hours

Test Species Daphnia magna

Source of Organisms AECOM In

- house cultures
Life Stage (Age) of Organisms <24 hours old at test initiation

Test Concentrations (μ g/L as PETN) Nominal Concentratio ns: 0 (solvent control), 0 (dilution water control), 500, 800, 1,100, 1,700, 2,500

Control / Dilution Water Reconstituted water prepared from 100 % Milli

- Q ® water ; target
hardness of 170 mg/L

, target alkalinity of 115 mg/L as CaCO 3

Results 24

- hr LC 50 48

- hr LC > 943 μ g/L PETN, measured

50 > 943 μ g/L PETN, measuredAECOM

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### TABLE OF CONTENTS

1.0 INTRODUCTION

................................................................................................................. 1

2.0 MATERIALS AND METHODS

............................................................................................. 2

2.1 Basis

......................................................................................................................... 2

2.2 Test Substance

......................................................................................................... 2

2.3 Test Dilution / Control Water

..................................................................................... 2

2.4 Test Organisms

......................................................................................................... 3

2.5 Exposure System

...................................................................................................... 3

2.6 Te st Chambers

......................................................................................................... 4

2.7 Glassware Preparation

.............................................................................................. 4

2.8 Test Conditions

......................................................................................................... 5

2.9 Te st Initiation

............................................................................................................ 5

2.10 Test Monitoring

......................................................................................................... 5

2.11 Statistical Analysis

.................................................................................................... 6

3.0 RESULTS

........................................................................................................................... 7

3.1 Water Qualit y Parameters

......................................................................................... 7

3.2 Test Concentrations

.................................................................................................. 8

3.3 Organism Responses

............................................................................................... 9

3.4 Reference Toxicant Studies

..................................................................................... 10

4.0 CONCLUSIONS

................................................................................................................. 11

5.0 STUDY PERSONNEL

........................................................................................................ 12

6.0 LOCATION OF ARCHIVES

................................................................................................ 13

7.0 AECOM PROTOCOL DEVIATIONS

................................................................................... 14

8. 0 REFERENCES

.................................................................................................................. 15

APPENDIX A TEST PROTOCOL AND AMENDMENTS

................................................... A-1

APPENDIX B ANALYTICAL DATA SUMMARY

................................................................ B-1

APPEN DIX C TEST DATA

................................................................................................ . C-1

APPENDIX D PRELIMINARY TEST DATA & ANALYTICAL

............................................. D-1AECOM

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### LIST OF TABLES

Table 2

- 1. Test treatments and nominal PETN concentrations

.................................................. 4

Table 3

- 1. Water quality parameters measured at test initiation for the control waters
and hi ghest test treatment

........................................................................................ 7

Table 3

- 2. Ranges of dissolved oxygen, pH, conductivity, and temperature measured during the 48

- hour acute definitive exposure of D. magna to PETN

.......................... 7

Table 3

- 3. Measured PETN concentrations from the 48

- hour acute definitive study with D. magna

.................................................................................................................. 8

Table 3

- 4. Summary of D. magna survival at termination of the 48

- hour acute definitive exposure to PETN

.................................................................................................... 9

Table 3

- 5. Reference toxicant test results for D. magna

........................................................... 10

Table 4

- 1. Median lethal PETN concentrations (μg PETN/L) for D. magna

.............................. 11

Table 4

- 2. Results of PETN preliminary study using D. magna

................................................ 11AECOM

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### LIST OF FIGURES

Figure 3

- 1. Graphical depiction of daily D. magna s urvival during the 48

- hour acute definitive exposure to PETN

................................................................................... 9AECOM

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### 1.0 INTRODUCTION

This report presents the results of a s tudy conducted to determine the acute toxicity of pentaeryt h ritol tetranitrate ( PETN ) to the freshwater cladoceran

, Daphnia magna

, under static

renewal test conditions

. The toxicity of PETN was evaluated in an acute (48

- hour) static -r enewal exposure of D. magna in laboratory reconstituted water targeting certain water qual ity parameters

. The target water quality parameters were chosen to comply with the Office of Prevention, Pesticides, and Toxic Substances (OPPTS) method 850.1010, Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids (OPPTS 1996) and American Society of Testing
Materials (ASTM) method E 729

- 96, Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians (ASTM 2009). Where differences in these two guidance documents exist, the ASTM requirements were used. The target water hardness and alkalinity were 170 ± 10 and 1 15 ± 5 mg/L as CaCO 3 , respectively

.

All toxicity testing and doc umentation were carried out in compliance with EPA Good Laboratory Practice standards (USEPA 1989; see compliance statement). The organism exposure was conducted at AECOM’s Fort Collins Environmental Toxicology Laboratory (FCETL), Fort Collins, Colorado, USA. Chemical analyses for confirmation of test substance concentrations were
conducted at TestAmerica Denver in Arvada, Colorado

.

All biological and toxicological data, along with copies of analytical data related to this study are
maintained for at least five years in the FCETL archives, either on site (4303 West LaPorte Avenue, Fort Collins, Colorado) or at Data

- Lock Records Management (6707 McMurray Ranch
Road

, Bellvue , Colorado). Electronic copies of the analytical data related to this study are maintained in the TestAmerica archives (4955 Yarrow Street, Arvada, Colorado) for at least five

years.AECOM

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### 2.0 MATERIALS AND METHODS

2.1 Basis

The study was conducted according to the protocol, “ Acute T oxicity of Pentaery thritol tetranitrate (PETN) to the Cladoceran ( Daphnia magna ) Under Static

- Renewal Conditions ”

, AECOM Protocol No. DM2UT.304.001, effective (signed by the Study Director) on 01 February 2012, as amended 22 May 2012. The basis for the protocol was the Office of Prevention, Pesticides, and Toxic Substances method 850.1010, A quatic Invertebrate Acute Toxicity Test, Freshwater Daphnids (OPPTS 1996) and American Society of Testing Materials (ASTM)
method E 729

- 96 Conducting Acute Toxicity Tests on Test Material s with Fishes, Macroinvertebrates, and Amphibians (ASTM 2009). Copies of the protocol and amendments can be found in Appendix A

.

2.2 Test Substance

The test substance, PETN, a nitric acid ester of pentaerythritol (Product name: pentaerythritol tetranitrate, a.k.a. penthrit, pentrite, nitropenta, nitropentaerytritol), was received in solvent at a nominal concentration of 5,000 mg PETN/L acetone (CAS No. 78

- 11

- 5). Solid PETN is an odorless, white crystalline substance with a molecular weight of 316.14 g/mol, density of 1.773 g/cm 3

The test substance was assumed to be 100% active ingredient for purposes of stock solution and exposure concentrations calculation and preparation. All concentrations are expressed as micrograms of PETN per liter of dilution water ( μg PETN /L). at 20 ° C and a melting point of 140 to 141 ° C. The clear colorless liquid sample of PETN was received from the United States Army Center for Health Promotion and Preventative Medicine (CHPPM) on 18 May 2012

. One glass amb er bottle with a pproximately 100 milliliters of the material, given the unique AECOM identification number (GLP

- 065 ), was received. The
stock solution was stored at approximately 4 ° C in the amber jar while not in use. The stock solution was warmed to room temperature prior to use in testing. Test substance use was recorded in a test substance usage log each time material was removed.

2.3 Test Dilution / Control Water

The dilution/control water used in this study was 100% Milli -Q ® ( ASTM type I; Millipore, Billerica MA) water (18 meg o hm ·cm resistance water)

. This water is periodically analyzed for
contaminants ; a copy of results from the most recent analyses can be found with the raw data.
The water was adjusted to a target hardness and alkalinit y of 170 ± 10 and 115 ± 5 mg/L as
CaCO 3 , respectively. The temperature of the water was adjusted to approximately 20° C prior to spiking and/or mixing of the test solutions.AECOM

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- 010 December 2012 3 Calcium sulfate ( CaSO 4 · 2H 2 O; CAS # 10101

- 41 -4 ) and magnesium sulfate ( MgSO 4 ; CAS
# 7487

- 88 -9 ) were added at a ratio of 1 :1 on a mass basis to achieve the target hardness per normal methods (ASTM 2009, USEPA 2002)

. Sodium bicarbonate (NaHCO 3 ; CAS # 144

- 55 -8 ) was added to increase the alkalinity to approximately 115 mg/L as CaCO 3

. The only other salt added was potassium chloride (KCl; CAS # 7447

- 40 -7 ), per normal methods ( ASTM 2009, USEPA 2002). Enough water was prepared to ensure sufficient volume for this study. The test water was given the reconstituted batch number RW # 10336

. Selenium (as Na 2 SeO 4 , CAS
#13410

- 01

- 0) was added to this reconstituted water per FCETL SOP # 5202 to maintain adequate organism performance (nominal concentration of 2 μg /L)

.

An additional control was used to test the toxicity of the solvent, acetone, at t he highest
concentration used in the test. Acetone ( CAS # 67

- 64

- 1) technical grade, was added to the
dilution water (RW #10336) used in the test at a rate of 0.5 ml acetone / L of dilution water. Enough of the solvent control was prepared to ensure sufficient volume for the duration of the study, and was given the reconstituted batch number RW #10345

.

2.4 Test Organisms

Test organisms were Daphnia magna neonates (<24

- hours old) obtained from AECOM FCETL
in

- house cultures

. Adult female D. magna were cultured in 3.2 -L plastic containers containing approximately 2. 3 L of hard water. Organisms were fed 16 ml of a 3:5 mixture of yeast:trout chow:cereal leaves (YTC) / algal suspension (USEPA 2002 ; FCETL SOP s # 510 6 & 51 14 ) daily per culture chamber

. Sufficient numbers of gravid adult D. magna were isolated to ensure that enough neonates could be produced that would be < 24

- h old for testing. Organisms were
cultured in an environmental chamber at 25 °C , and were moved to a 20 o C environmental
chamber at least 48 hours prior to test initiation

. Brood D. magna were also maintained in 100% dilution water for at least 48 hours prior to test initiation

.

The genus and species of s elect adult test organisms from the in

- house culture w ere confirm ed by Aquatics Associates (Fort Collins, CO); verification can be found with the raw data

.

Prior to test initiation, neonates were f ed the suspension of algae

- YTC ad libitum

. Organisms were not fed during testing.

D. magna used for the definitive toxicit y test were designated FCETL Batch number 0 52912 and were less than 24 hours old at test initiation. It was not possible to determine the sex of the organisms used for this experiment, although the intent was that all organisms were female.

2.5 Expo sure System

The highest test concentration, 2.5 mg PETN/L (n ominal ) , was prepared by addition of the appropriate volume of the PETN stock solution in acetone (GLP -0 65 ) to 10 L of dilution water

.AECOM

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- 010 December 2012 4 This was sufficient volume for the entire duration of the study. An appropriate volume of the highest concentration was then diluted to 1.4 L to prepare all other test solutions daily

. This was sufficient volume for biological and analytical sampling. The remainder of the highest test concentration was stored in a cubitainer without headspace at approximately 4 o C in the dark. This solution was warmed to test temperature prior to use each day.

D. magna were exposed in a 48

- hour static

- renewal test design; test solutions were renewed daily

. D. magna (five organisms per cup; 20 per treatment ) were exposed to five concentrations
of PETN (using a dilu tion factor of approximately 1.5X ), a solvent control (0.5 ml acetone/L dilution water), and a dilution water control. The nominal test concentrations and treatment nomenclature for this study are presented below (Table 2 -1 ). There were four replicates for each treatment; all replicates were used for biological observations, chemistry measurement s, and analytical sampling

.

Table 2 -1 . Test treatments and nominal PETN concentrations

Treatment Nominal PETN concentration ( μ g/L)

C1 0 (Solvent Control)
C2 0 (Dilution Water Control)
C3 500
C4 800
C5 1,100
C6 1,700
C7 2,500

2. 6 Test Chambers

Test chambers were 500 -ml (nominal volume) plastic cups containing ~20 0 ml of test solution

. Test chambers were placed on Styrofoam ® board according to a computer

- generated random design and placed in an environmental chamber at 20 oC

. Test chambers were covered during the test to minimize evaporation and possible air

- borne contamination.

2. 7 Glassware Preparation

Prior to use, all test plasticware (i.e., mixing containers but not test chambers) / glassware and
utensils were s oak ed in an acid bath (1% HNO 3 ) for ~24 hours , removed, and rinsed three times with tap water

. Plasticware / glassware was the n washed using laboratory detergent ( MICRO

- 90 ® obtained from International Products Corporation, Burlington, New Jersey ).
Follow ing washing, the plasticware / glassware was rinsed with technical

- grade acetone and
1 0% (v/v) hydrochloric acid. Between washing and the acetone and acid rinses, the plasticware / glassware was rinsed with tap water. Following the final tap water rinse, all containers w ere rinsed five times with Milli -Q ® water, and then allowed to air dry

. Test chambers used for theAECOM

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to use

. Each test chamber was labeled with the nominal test concentration and replicate letter (e.g., A, B, C, etc)

, and the board was labeled with the study number

.

2. 8 Test Conditions

The exposure chambers were housed in a temperature

- controlled environmental chamber that
maintained the water temperature at 20 ± 2 ° C. Lighting was controlled to provide 16 hours of light per 24

- hour period; light intensity was ambient (approximately 50 - 100 foot

- candles). The exposure chamber volume was selected based upon OPPTS (1996) and ASTM (2009) guidance and in order to provide adequate surface area to maintain sufficient dissolved oxygen during the study.

2. 9 Test Initiation

The definitive test was initiated on 30 May 2012, when five D. magna neonates were impartially assigned to each of fou r replicate chambers within each treatment (20 organisms per treatment ; 14 0 organisms total ).

2.1 0 Test Monitoring

Biological observations and measurements of water quality were made and recorded as
described below.

Water Quality : On the day of test initiation, temperature (Standard Method 2550 B ; APHA 1998 )
was measured and recorded for each test chamber. pH (Standard Method 4500 -H + B)

, dissolved oxygen (D .O. ; Standard Method 4500 O G), and conductivity (EPA method 12 0.1 ) were measured an d recorded for each test treatment

. Hardness (Standard Method 2340 C) , alkalinity ( Standard Method 2320 B), both as mg CaCO 3 /L

, total ammonia ( ASTM D1426

- 98A, mg/L as N), and total residual chlorine (Standard Method 4500 Cl G) were also measured in both
control s and the highest PETN concentration at test initiation. D.O. and pH were measured in
each concentration daily and temperature was measured in each test chamber daily. These water quality parameters were measured following FCETL SOPs which were based on the standard analytical methods listed.

Biological : During the test, organisms were observed for survival daily; the number of live organisms present in each replicate was recorded daily. Organisms were considered dead (immob ilized) if no movement or visible response was observed in response to gentle prodding. The exposure continued for 2 days and was terminated on 01 June 2012.

Analytical : Samples for PETN analysis were collected from bulk solutions at test initiation

, after distributing the test solutions to the test chambers (see Protocol Deviation) , from each fre shly prepared test treatment , the solvent control, and the dilution water. On day 1, samples were
collected from bulk solutions of all freshly prepared test t reatments after distributing to testAECOM

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- 010 December 2012 6 chambers, as well as, from all replicates of old test solutions after organisms had been transferred to new solutions. At test termination, each replicate of the old test solutions was sampled

. All samples were poured directly into certified clean 50 0- ml glass amber bottles. The samples were refrigerated until shipped (on ice) to TestAmerica Denver , an independent laboratory, located in Arvada, Colorado

.

EPA Method 8330 A was used following USEPA (1986) guidelines to analyze for nitroaromatics and nitramines by HPLC

.

Analytical data from TestAmerica Denver were reviewed by AECOM . All data were determined
to be valid and usable.

2.1 1 Statistical Analysis

Given that there was no mortality observed in any treatment of this study, statistical analyses were not required. The concentrations estimated to cause death (or immobility) of 50% of the test organisms after 24 and 48 hours of exposure (the 24 - and 48

- hour EC 50 , respectively) were
determined by inspection. The EC 50 values are expressed as average measured PETN
concentration s measured in new and old test solutions (μ g PETN /L)

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### 3.0 RESULTS

3.1 Water Quality Parameters

Hardness, alkalinity, pH, conductivity

, total ammonia (mg/L as N), and total residual chlorine (mg/L) measured in the solvent control, dilution water

, and highest test concentration during the test are presented in Table 3-1.

Table 3

- 1. Water quality parameters measured at test initiation
for the control water s and highest test treatment

Nominal
Treatment (μg PETN /L) Hardness (mg/L as
CaCO 3 ) Alkalinity (mg/L as
CaCO 3 ) pH Conductivity (μ S/cm) Total
Ammonia (mg NH 3 - N/L) Total
Residual
Chlorine

(mg/L) C1 / 0 ( Solvent
Cont. ) 172 117 8.6 575 <1.0 <0.02

C2 / 0 ( Dilution

Water Cont.) 172 116 8.5 579 <1.0 <0.02

C7 / 2,500 170 117 8.5 572 <1.0 <0.02

The physical test conditions (dissolved oxygen, pH, conductivity, and temperature) monitored during the test were within acceptable ranges for the survival of D. magna and were in compliance with the protocol and ASTM requirements (Table 3

- 2)

.

Table 3

- 2. Ranges of dissolved oxygen, pH, conductivity, and t emperature measured during the 48

- hour acute definitive exposure of **D. magna** to PETN

Nominal Treatment (μ g PETN /L) Dissolved Oxygen (mg/L) pH ( s.u.) Conductivity (μ S/cm) Temperature (°C)

C1 / 0 ( Solvent Cont.) 7.1

– 7.3 8.5

– 8.6 563

– 579 19

– 22
C2 / 0 (Dilution Water Cont.) 7.0

– 7.2 8.5 565

– 581 20

– 22
C3 / 500 7.0 – 7.2 8.5 – 8.6 574 – 581 19 – 22
C4 / 800 7.0

– 7.1 8.5

– 8.6 570

– 585 19

– 22
C5 / 1,100 6.9

– 7.1 8.5

– 8.6 569

– 585 20

– 22
C6 / 1,700 7.0 – 7.2 8.5 – 8.6 572 – 585 20 – 22 C7 / 2,500 7.1 8.5

– 8.6 572

– 582 20

– 22

Note: All parameters were measured daily in each test concentration; temperature was measured in each test chamber daily

.AECOM

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Samples were collected and analyzed for total PETN from new (freshly prepared bulk test solutions) test solutions at test initiation and 24

- hour renewal, and all replicates of old test
solutions at the 24

- hour renewal and test termination. Presented below are values measured in new test solutions on day s 0 and 1 and an average of values measured in all replicates in old
test solutions on day s 1 and 2 (Table 3

- 3)

. An overall average is presented which is an average
of PETN concentrations measured in new and old test solutions. All endpoints were calculated
based on the overall average (includes new and old test solutions )

.

Table 3 -3

. Measured PETN concentrations from the 48

- hour acute definitive study
with **D. magna**

Nominal
Treatment (μ g PETN /L) Day 0 / 1 Average Day 1 / 2 Average Average Overall Average % of
Nominal New Old New Old New Old

C1 / 0
S olv

. Cont) 0.41 U 1.0 U 0.42 U 1.2 U 0.42 U 1.1 U 0.76 U --

C2 / 0 (Dil. Cont.) 0.45 U 1.2 U 0.42 U 1.2 U 0.44 U 1.2 U 0.82 U --

C3 / 500 240 288 240 305 240 296 268 54%

C4 / 800 360 463 380 475 370 469 419 52%
C5 / 1,100 480 638 500 570 490 604 547 50%
C6 / 1,700 670 978 620 968 645 973 809 48%
C7 / 2,500 390 1,350 895 1,138 643 1,244 943 38% Note: the ‘U’ qualifier denotes that the analyte was below the method detection limit (MDL). TestAmerica analytical reports are included with the raw data file at AECOM; a summary is prov id ed in Appendix B. Values for new solutions represent one measured value while four replicates were measured for old test solutions.

Average of PETN solutions ranged from 38 to 54% of the nominal concentration s

. A detailed summary of the analytical results for PETN concentrations is included in Appendix B, with individual sample analysis. Analytical reports from TestAmerica w ere archived with the original test data and pr otocol.

The highest PETN concentration was 390

- 895 μ g/L in new test solutions (note: the 390 μ g/L value could not be reanalyzed as the sample volume was consumed in the original analysis) and ranged from 750 to 1,400 μ g/L in old test solutions (Appendix B)

. While the target concentration was 2,500 μ g/L , we were not able to achieve this concentration of PETN in these studies (or additional studies completed outside of this study, AECOM report files).AECOM

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D. magna survival at test termination in individual treatments and replicates is provided in Table
3-4 . There was 100% survival in all test treatments with no visible difference between organisms in the control versus those in any of the PETN concentrations . Copies of the raw data and statistical analyses are included in Appendix C.

Table 3 -4 . Summary of **D. magna** survival at termination of
the 48

- hour acute definitive exposure to PETN

Nominal
Treatment (μg PETN/ L) % Survival at 48 hours Rep

A Rep
B Rep
C Rep
D Overall
Survival Std.
Dev.
C1 / 0 (Solv

. Cont) 100 100 100 100 100 0.0
C2 / 0 (Dil. Cont.) 100 100 100 100 100 0.0

C3 / 500 100 100 100 100 100 0.0

C4 / 800 100 100 100 100 100 0.0
C5 / 1,100 100 100 100 100 100 0.0
C6 / 1,700 100 100 100 100 100 0.0
C7 / 2,500 100 100 100 100 100 0.0

The o rganism survival response is also presented graphically over the test duration for eac h test treatment (Figure 3

- 1). Survival was not affected by the PETN concentrations tested in this study.

Figure 3

- 1. Graphical depiction of daily **D. magna** s urvival during the 48

- hour acute definitive exposure to PETN

0 20 40 60 80 100

C1 (Solvent Cont.) C2 (Dilution Cont.) C3 C4 C5 C6 C7Survival (%)



### PETN Treatments 24 -h

48

- hAECOM

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- 010 December 2012 10 3.4 Reference Toxicant S tudies

Monthly reference toxicant studies a re conducted with D. magna using sodium chloride. The results of the May 2012 monthly reference toxicant study and the historical control limits (based on the last 20 test points) are presented in Table 3 -5 . Results from May 2012 were within the
historical control limits.

Table 3 -5. Reference t oxicant t est r esults for **D. magna**

48- hour LC 50 AECOM FCETL Historical 95% Control Limits

Low High 3,268 3,203 3,456 Note: Values are expressed as mg/L chlorideAECOM

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### 4.0 CONCLUSIONS

The purpose of this study was to determine the acute toxicity of PETN around its solubility level (approximately 2 mg/L) to D

. magna under static

- renewal test conditions

. The biological endpoint for this study was immobility (mo rta lity)

. The 24 - and 48

- hour E C 50 values and 95%
confidence intervals based on the average PETN concentrations measured in test solutions for
the definitive acute D. magna study are presented in Table 4- 1.

Table 4-1 . Median lethal PETN concentrations ( μg PETN /L) for **D. magna**

Time (h ours ) EC 50 (μ g measured PETN/ L) 95 % CI

24 > 943

-- 48 > 943

--

While we did not achieve the ~2 mg/L concentration of PETN with the acetone stock , a preliminary study had been performed using a PETN solution purchased from Restek (Bellefonte, PA) . This PETN stock (1,000 mg/L) was in methanol and was used to perform an initial range

- finding study. The target PETN concentration s in this study were 100, 500, and 2,000 μ g/L. Analytical samples were not taken during the actually study; however, preliminary samples were prepared for analytical purposes only using the same Restek standard with target PETN concentrations of 500 and 1,000 μ g/L The measured concentrations of these solutions
were 430 and 860 μ g/ L, respectively – relatively close to the ir target concentration s. The
survival of th e D. magna in this preliminary study is reported below. Copies of biological reports and analytical reports are included as an appendix to this report (Appendix D). These data support the finding that PETN is not toxic at these concentrations.

Table 4 -2 . Results of PETN preliminary study using **D. magna**

Nominal PETN

Conc. ( μ g/L) % Survival at
48 Hours 0 (Control) 100

100 100

500 100 2,000 100AECOM

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### 5.0 STUDY PERSONNEL

SPONSOR: Dyno Nobel, Inc.

TITLE: Acute Toxicity of Pentaerythritol tetranitrate (PETN) to the Cladoceran ( Daphnia

magna ) Under Static Renewal Conditions

AECOM Environmental Toxicology Laboratory Project Number: 60225262

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The names of key AECOM, TestAmerica , Aquatics Associates, and Dyno Nobel, Inc. scientists,

pr ofessionals and/or supervisory personnel who participated in this study include:

Study Director: Rami B. Naddy, Ph.D. (AECOM)
Technical Staff Scientists: Christina Needham (AECOM) Amanda Bidlack (AECOM) Kara Barnett (AECOM)
Taxonomist: Tami Schneck (Aqu atics Associates, Inc) Quality Assurance Officer: Anita B. Rehner (AECOM) Gina McNerney (AECOM) Senior Project Chemist: Patrick McEntee (TestAmerica Denver) Project Officer: Neal Olsen (Dyno Nobel Inc.)AECOM

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### 6.0 LOCATION OF ARCHIVES

The raw toxicity data and final report for this study will be archived at AECOM or an off

- site storage facility contracted by AECOM (e.g., Data

- Lock Records Management, 6707 McMurray Ranch Road, Bell vue , CO 805 1 2). The test substance used during testing will be maintained

on

- site at FCETL under storage conditions appropriate to maintain the integrity of the
substance. The raw data and final report will be stored at least 5 years beyond the date on which the results of the study are submitted to the agency.AECOM

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### 8.0 REFERENCES

ASTM. 2009. Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians. Method E 729

- 96 in 2009 Annual Book of ASTM Standards, Section 11, Water and Environmental Technology, Volume 11.06, Biological Effects and Environmental Fate; Biotechnology. American Society of Testing
and Materials.

OPPTS 1996. Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids. OPPTS 850.1010. Ecological Effects Test Guidelines. EPA 712 -C- 96

- 114.

USEPA. 1986. Test Methods for Evaluating Solid Waste, Physical/Chemical Methods.
Nitroaromatics and Nitramines 8330: Third Edition. SW

- 846.

USEPA. 1989. Good Laboratory Practice Standards. Final Rule. 40 CFR Part 792 (from 54 FR 34043, August 17, 1989).

USEPA. 20 02. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms. Fifth Edition. EPA

- 821 -R- 02

- 012.AECOM

60225262

- 304

- 010 A-1 December 2012 

### APPENDIX A

TEST PROTOCOL AND AMENDMENTSAECOM

60225262

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- 010 B-1 December 2012 

### APPENDIX B

ANALYTICAL DATA SUMMARYAECOM

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- 010 C-1 December 2012 

### APPENDIX C

TEST DATAAECOM

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- 010 D-1 December 2012 

### APPENDIX D

PRELIMINARY TEST DATA & ANALYTICAL