AECOM

Acute Toxicity of Pentaerythritol 

Tetranitrate (PETN) to the 

Cladoceran (Daphnia magna) 

Under 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

60225262-304-010
AECOM

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 December 2012

Conducted for: 

Dyno Nobel Inc.

3000 West 8600 South 

West Jordan, Utah 84088

Project Officer:

Neal Olsen, DNI Environmental Affairs Manager

Performing Laboratory: 

AECOM Fort Collins Environmental Toxicology Laboratory

4303 West LaPorte Avenue

Fort Collins, Colorado 80521

AECOM Project ID

60225262-304-010

Final Report

December 2012

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SUMMARY

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

TestAmerica 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 80512 

Test Substance Pentaerythritol Tetranitrate (PETN)

AECOM Identification Number(s) GLP-065 

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 

(μg/L as PETN)

Nominal Concentrations:

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 CaCO3 

Results 24-hr LC50

48-hr LC

 > 943 μg/L PETN, measured

50 > 943 μg/L PETN, measured

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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 Test Chambers ......................................................................................................... 4

2.7 Glassware Preparation.............................................................................................. 4

2.8 Test Conditions......................................................................................................... 5

2.9 Test Initiation ............................................................................................................ 5

2.10 Test Monitoring ......................................................................................................... 5

2.11 Statistical Analysis .................................................................................................... 6

3.0 RESULTS ........................................................................................................................... 7

3.1 Water Quality 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

APPENDIX C TEST DATA................................................................................................. C-1

APPENDIX D PRELIMINARY TEST DATA & ANALYTICAL ............................................. D-1

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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 highest 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 ................................................11

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LIST OF FIGURES

Figure 3-1. Graphical depiction of daily D. magna survival during the 48-hour acute

 definitive exposure to PETN ................................................................................... 9

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1.0 INTRODUCTION

This report presents the results of a study conducted to determine the acute toxicity of 

pentaerythritol tetranitrate (PETN) to the freshwater cladoceran, Daphnia magna, under staticrenewal test conditions. The toxicity of PETN was evaluated in an acute (48-hour) static-renewal

exposure of D. magna in laboratory reconstituted water targeting certain water quality 

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 115 ± 5 mg/L as CaCO3, respectively. 

All toxicity testing and documentation 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.

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2.0 MATERIALS AND METHODS

2.1 Basis

The study was conducted according to the protocol, “Acute Toxicity of Pentaerythritol 

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, 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). 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/cm3

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 amber bottle with approximately 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 megohm·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 alkalinity of 170 ± 10 and 115 ± 5 mg/L as 

CaCO3, respectively. The temperature of the water was adjusted to approximately 20°C prior to 

spiking and/or mixing of the test solutions. 

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Calcium sulfate (CaSO4·2H2O; CAS #10101-41-4) and magnesium sulfate (MgSO4; 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 (NaHCO3; CAS #144-55-8) 

was added to increase the alkalinity to approximately 115 mg/L as CaCO3. 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 Na2SeO4, 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 the 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 SOPs #5106 & 5114) 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 20oC 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 select adult test organisms from the in-house culture were confirmed 

by Aquatics Associates (Fort Collins, CO); verification can be found with the raw data. 

 

Prior to test initiation, neonates were fed the suspension of algae-YTC ad libitum. Organisms 

were not fed during testing. 

D. magna used for the definitive toxicity test were designated FCETL Batch number 052912 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 Exposure System 

The highest test concentration, 2.5 mg PETN/L (nominal), was prepared by addition of the 

appropriate volume of the PETN stock solution in acetone (GLP-065) to 10 L of dilution water. 

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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 4oC 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 dilution 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 measurements, 

and analytical sampling. 

Table 2-1. Test treatments and nominal PETN concentrations

Treatment Nominal PETN 

concentration (μg/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 ~200 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 20oC. 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 soaked in an acid bath (1% HNO3) for ~24 hours, removed, and rinsed three 

times with tap water. Plasticware / glassware was then washed using laboratory detergent 

(MICRO-90® obtained from International Products Corporation, Burlington, New Jersey). 

Following washing, the plasticware / glassware was rinsed with technical-grade acetone and

10% (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 were

rinsed five times with Milli-Q® water, and then allowed to air dry. Test chambers used for the 

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study were not cleaned, as they were new; however, they were rinsed with Milli-Q® water prior 

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 four replicate chambers within each treatment (20 organisms per treatment; 

140 organisms total). 

2.10 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 120.1) were 

measured and recorded for each test treatment. Hardness (Standard Method 2340 C), alkalinity 

(Standard Method 2320 B), both as mg CaCO3/L, total ammonia (ASTM D1426-98A, mg/L as 

N), and total residual chlorine (Standard Method 4500 Cl G) were also measured in both

controls 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

(immobilized) 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 freshly 

prepared test treatment, the solvent control, and the dilution water. On day 1, samples were 

collected from bulk solutions of all freshly prepared test treatments after distributing to test 

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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 500-ml glass amber bottles. The 

samples were refrigerated until shipped (on ice) to TestAmerica Denver, an independent 

laboratory, located in Arvada, Colorado. 

EPA Method 8330A 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.

2.11 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 EC50, respectively) were 

determined by inspection. The EC50 values are expressed as average measured PETN

concentrations measured in new and old test solutions (μg 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 waters and highest test treatment

Nominal 

Treatment 

(μg PETN/L)

Hardness

(mg/L as 

CaCO3) 

Alkalinity

(mg/L as 

CaCO3) 

pH Conductivity 

(μS/cm)

Total 

Ammonia 

(mg NH3-

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 temperature 

measured during the 48-hour acute definitive exposure of D. magna to PETN

Nominal Treatment

 (μg PETN/L)

Dissolved

Oxygen (mg/L) pH (s.u.) Conductivity(μS/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. 

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3.2 Test Concentrations

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 days 0 and 1 and an average of values measured in all replicates in old 

test solutions on days 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 

(μg PETN/L)

Day 0 / 1 

Average

Day 1 / 2 

Average Average Overall 

Average

% of 

Nominal New Old New Old New Old

C1 / 0 

Solv. 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 provided 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 concentrations. A detailed 

summary of the analytical results for PETN concentrations is included in Appendix B, with

individual sample analysis. Analytical reports from TestAmerica were archived with the original 

test data and protocol.

The highest PETN concentration was 390-895 μg/L in new test solutions (note: the 390 μg/L 

value could not be reanalyzed as the sample volume was consumed in the original analysis) 

and ranged from 750 to 1,400 μg/L in old test solutions (Appendix B). While the target 

concentration was 2,500 μg/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). 

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3.3 Organism Responses

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 organism survival response is also presented graphically over the test duration for each 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 survival 

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 C7

Survival (%)

PETN Treatments

24-h 

48-h

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3.4 Reference Toxicant Studies

Monthly reference toxicant studies are 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 toxicant test results for D. magna

48-hour LC50

AECOM FCETL Historical 95% Control Limits

Low High

3,268 3,203 3,456

Note: Values are expressed as mg/L chloride

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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 (mortality). The 24- and 48-hour EC50 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 (hours) EC50 (μg 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 concentrations in this study were 100, 500, and 

2,000 μg/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 μg/L The measured concentrations of these solutions 

were 430 and 860 μg/L, respectively – relatively close to their target concentrations. The

survival of the 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. (μg/L)

% Survival at 

48 Hours

0 (Control) 100

100 100

500 100

2,000 100

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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: 60225262-304-010

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

professionals 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 (Aquatics 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.)

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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, Bellvue, CO 80512). 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. 

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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. 2002. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to 

Freshwater and Marine Organisms. Fifth Edition. EPA-821-R-02-012.

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APPENDIX A

TEST PROTOCOL AND AMENDMENTS

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APPENDIX B

ANALYTICAL DATA SUMMARY 

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APPENDIX C 

TEST DATA 

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APPENDIX D 

PRELIMINARY TEST DATA & ANALYTICAL

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