AECOM 

Acute Toxicity of 

Pentaerythritol Tetranitrate 

(PETN) to the Fathead Minnow 

(Pimephales promelas) Under 

Static-Renewal Test Conditions

For 

 Dyno Nobel Inc. 

 West Jordan, Utah, USA

Prepared by 

AECOM Technical Services, Inc. 

Fort Collins Environmental Toxicology Lab

February 2013 

60279946-100-020 
AECOM 

Study Title 

Acute Toxicity of Pentaerythritol Tetranitrate (PETN) to the Fathead Minnow (Pimephales 

promelas) Under Static-Renewal Test Conditions 

Study Initiation Date: 

1 June 2012 

Study Completion Date: 

4 February 2013 

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 

60279946-100-020 

Final Report 

February 2013 

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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 96-Hour Static-Renewal Toxicity Test 

Test Method OPPTS Method 850.1075 and ASTM Method E 729-96 

Experimental Test Dates 12 to 16 November 2012 

Length of Study 96 Hours 

Test Species Pimephales promelas 

Source of Organisms AECOM In-house cultures 

Life Stage (Age) of Organisms 14 days old at test initiation 

Test Concentrations 

(μg/L as PETN) 

Nominal Concentrations1

: 

0 (solvent control), 0 (dilution water control), 1,000, and 2,000 

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 > 1,218 μg/L PETN, measured 

48-hr LC50 > 1,218 μg/L PETN, measured 

72-hr LC50 > 1,218 μg/L PETN, measured 

96-hr LC50 > 1,218 μg/L PETN, measured 

 

1

 Preliminary studies at the water solubility indicated no toxicity so a full dilution study was not performed. 

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

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

2.7 Glassware Preparation ................................................................................................ 5

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

3.3 Organism Responses .................................................................................................. 8

3.4 Reference Toxicant Studies ........................................................................................ 9

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 RANGE-FINDING TEST DATA..................................................................... 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 solvent control, 

dilution water, and highest test treatment .................................................................. 7 

Table 3-2. Ranges of dissolved oxygen, pH, conductivity, and temperature measured during 

the 96-hour acute definitive exposure of P. promelas to PETN ................................. 7 

Table 3-3. Measured PETN concentrations from the 96-hour acute definitive exposure of P. 

promelas to PETN ..................................................................................................... 8 

Table 3-4. Summary of P. promelas survival at termination of the 96-hour acute definitive 

exposure of P. promelas to PETN ............................................................................. 9 

Table 3-5. Reference toxicant test results for P. promelas ......................................................... 10 

Table 4-1. Median lethal PETN concentrations (μg PETN/L) for P. promelas ............................ 11 

Table 4-2. Results of PETN preliminary experiment with P. promelas ....................................... 11 

 

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LIST OF FIGURES

 Figure 3-1. Graphical depiction of daily P. promelas survival during the 96-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 fathead minnow, Pimephales promelas, under staticrenewal test conditions. The toxicity of PETN was evaluated in an acute (96-hour) static-renewal 

exposure of P. promelas in reconstituted laboratory water. The target water hardness and 

alkalinity were 170 ± 10 and 115 ± 5 mg/L as CaCO3, respectively. The study method followed 

the Office of Prevention, Pesticides, and Toxic Substances (OPPTS) method 850.1075, Fish 

Acute Toxicity Test, Freshwater and Marine (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). 

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 Fathead Minnow (Pimephales promelas) Under Static-Renewal 

Conditions”, AECOM Protocol No. PP2UT304.002, effective (signed by the Study Director) on 1 

June 2012. The basis for the protocol was the Office of Prevention, Pesticides, and Toxic 

Substances method 850.1075, Fish Acute Toxicity Test, Freshwater and Marine (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). A copy 

of the protocol 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

 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. 

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). 

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 25°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 #10533. 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 #10533) used in the test at a rate of 0.4 ml acetone / L of dilution water. The 

solvent control was prepared daily prior to test initiation or renewal. 

2.4 Test Organisms 

Test organisms were 14 day old juvenile Pimephales promelas at test initiation, targeting fish 

that were less than 3 grams (wet weight, blotted dry) (OPPTS 1996), obtained from AECOM 

FCETL in-house cultures. Pre-test fish lengths and weights were obtained for ten representative 

P. promelas from the same batch of fish used in testing. These ten fish were sacrificed 

(euthanized) prior to measurements (Appendix C). 

Fathead minnows were cultured by maintaining brood stock under flow-through conditions 

within 10-gallon aquaria. Each aquarium received laboratory process water from Horsetooth 

Reservoir maintained at 25 ± 2oC. Each brood tank contained two males and five female 

fathead minnows and three to four spawning substrates. On a daily basis, spawning substrates 

containing eggs were removed from the brood stock tanks and transferred to bins containing 

moderately hard laboratory process water (prepared according to FCETL SOP #5202). The 

freshly harvested eggs were termed “pre-eyed” and were treated with Fungal Guard® until the 

eggs reached the eyed stage. Juvenile fish were held in laboratory process moderately hard 

water for 9 days. Approximately 400 organisms were then transferred to a bin containing the 

test dilution water five days prior to test initiation. During this time, the batch of ~400 fish were 

fed approximately 1 to 1.5 ml of a concentrated suspension of newly hatched brine shrimp 

(Artemia sp.; cultured according to FCETL SOP #5104) per ~8 L of test dilution water three 

times daily. Organisms were not fed during the test or the 48 hour period prior to test initiation. 

Juvenile fathead minnows were held at 25 ± 2oC and ≥80% D.O. saturation (5.4 mg/L at local 

elevation) for at least 7 days prior to test initiation. Organism food is periodically analyzed for 

contaminants; the results of the latest contaminant analysis are included with the raw data 

package. 

 

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The genus and species of select adult test organisms from the in-house culture were verified by 

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

package. 

 

P. promelas used for the definitive toxicity test were designated FCETL Batch number 102912 

and were 14 days old at test initiation. This age cohort was chosen to meet the weight 

requirements required for testing (< 3 g per fish and loading rate of ≤ 0.5 g of organism per liter 

of test solution). 

2.5 Exposure System 

Each day, the highest test concentration, 2,000 μg PETN/L (nominal), was prepared by addition 

of 0.8 ml of the PETN stock solution in acetone (5,000 mg PETN/L, GLP-065) to 2 L of dilution 

water. A 650 ml aliquot of the highest concentration was then diluted to 1.3 L to prepare the 

1,000 μg PETN/L (nominal) treatment daily. This was sufficient volume for biological and 

analytical sampling. 

P. promelas were exposed in a 96-hour static-renewal test design; test solutions were renewed 

daily. P. promelas (ten organisms per test chamber; 30 per treatment) were exposed to two 

concentrations of PETN (using a dilution factor of 2X), a solvent control (0.4 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 three 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 PETN/L) 

C1 0 (Solvent Control) 

C2 0 (Dilution Water Control) 

C3 1,000 

C4 2,000 

2.6 Test Chambers 

Test chambers were new, 500-ml (nominal volume) plastic cups containing ~250 ml of test 

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

computer-generated random design and placed in an environmental chamber at 25oC. Test 

chambers were covered during the test to minimize evaporation and possible air-borne 

contamination. 

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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 (MICRO90® 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 

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), 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 test solution temperature at 25 ± 2°C. Lighting was controlled to provide 16 

hours of light per 24-hour period; light intensity was ambient (approximately 50 - 100 footcandles). 

2.9 Test Initiation 

The definitive test was initiated on 12 November 2012, when ten P. promelas juveniles were 

impartially assigned to each of three replicate chambers within each treatment (30 organisms 

per treatment; 120 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. Dissolved oxygen and pH were 

measured in each concentration daily and temperature was measured in each test chamber 

daily (new and old test solutions). These water quality parameters were measured following 

FCETL SOPs which were based on the standard analytical methods listed. 

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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 4 days and was terminated on 16 November 2012. 

Post-test weight and length measurements were obtained for each surviving fish tested in the 

solvent control and dilution water control, and length measurements were obtained for surviving 

fish tested in the 1,000 and 2,000 μg/L PETN (nominal) treatments, following euthanization at 

test termination (Appendix C). 

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

to distributing the test solutions to the test chambers, from each freshly prepared test treatment, 

the solvent control, and the dilution water. On days 1, 2, and 3, samples were collected from 

bulk solutions of all freshly prepared test treatments after distributing to test chambers, as well 

as from all replicates of old test solutions for treatments after organisms had been transferred to 

new solutions. Only one replicate of old test solutions was analyzed on days 1 and 3 from 

treatment C3 (total of 2 samples analyzed from old test solutions). None of the day 2 old test 

solution samples from C3 were analyzed. Each replicate of the old test solutions was sampled 

from all treatments at test termination after removing the test organisms. None of the day 4 old 

test solution samples from C3 were analyzed. 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 not ≥50% 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, 48, 72, and 96 hours of exposure (the 24-, 48-, 72-, and 96-hour 

LC50, respectively) were determined by inspection. The LC50 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 solvent control, dilution water, 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.) 176 115 8.3 582 <1.0 <0.02 

C2 / 0 (Dilution 

Water Cont.) 174 115 8.3 593 <1.0 <0.02 

C4 / 2,000 174 114 8.3 592 <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 P. promelas 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 96-hour acute definitive exposure of P. promelas to PETN 

Nominal Treatment 

 (μg PETN/L)

Dissolved 

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

Temperature 

(°C)

C1 / 0 (Solvent Cont.) 6.4 – 6.9 8.2 – 8.4 572 – 616 23.1 – 24.9 

C2 / 0 (Dilution Water Cont.) 6.3 – 6.8 8.3 – 8.4 593 – 626 23.1 – 25.1 

C3 / 1,000 6.2 – 6.8 8.3 – 8.4 588 – 631 23.2 – 24.9 

C4 / 2,000 6.2 – 6.8 8.3 – 8.4 592 - 614 23.0 – 25.1 

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

chamber daily. 

3.2 Test Concentrations 

Presented below are values measured in new test solutions on days 0, 1, 2, and 3 and an 

average of values measured in all replicates of treatments C1, C2, and C4 in old test solutions 

on days 1, 2, 3, and 4 (Table 3-3). The average for old test solutions on days 1 and 3 for 

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treatment C3 represents the value of just one replicate analyzed on these days; old solutions 

were not analyzed from this treatment on days 2 and 4. 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. 

Table 3-3. Measured PETN concentrations from the 96-hour acute definitive exposure of 

P. promelas to PETN 

Nominal 

Treatment 

(μg PETN/L) 

Day 0 / 1 

Averages 

Day 1 / 2 

Averages 

Day 2 / 3 

Averages 

Day 3 / 4 

Averages 

Overall 

Averages 

Overall 

Average

(New/Old)

% of 

Nominal

New Old New Old New Old New Old New Old 

C1 / 0 

(Solv. Cont.) 0.69 U 0.93 U 0.43 U 0.97 U 0.45 U 0.88 U 0.45 U 0.94 U 0.50 U 0.93 U 0.72 U -- 

C2 / 0 

 (Dil. Cont.) 0.53 U 0.92 U 0.43 U 0.92 U 0.45 U 0.91 U 0.45 U 0.98 U 0.46 U 0.93 U 0.70 U -- 

C3 / 1,000 530 570 500 NA 790 1,000 820 NA 660 785 723 72% 

C4 / 2,000 1,100 1,167 1,000 973 1,000 1,533 1,400 1,567 1,125 1,310 1,218 61% 

NA = Analytical samples not taken on this day 

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 three replicates were measured for most old test solutions. 

Average of PETN solutions were 72 and 61% of the nominal concentrations for C3 and C4, 

respectively. 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 PETN concentration ranged from 1,000 to 1,400 μg/L PETN in new test solutions and 

averaged from 973 to 1,567 μg/L PETN in old test solutions (Appendix B). While the target 

concentration was 2,000 μg/L PETN, we were not able to achieve this concentration of PETN in 

these studies (or in additional range-finding studies, AECOM report files). 

3.3 Organism Responses 

P. promelas survival at test termination in individual treatments and replicates is provided in 

Table 3-4. There was 100% survival in the dilution water control and 96.7% survival in the 

solvent control, meeting the test acceptability criterion of ≥ 90% survival. Organisms in the 1,000 

and 2,000 μg PETN/L (nominal) treatments appeared weak with organisms in the latter showing 

more weakness. Organisms in the control treatments also appeared to be slightly weakened. 

Copies of the raw data and statistical analyses are included in Appendix C. 

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Table 3-4. Summary of P. promelas survival at termination of 

the 96-hour acute definitive exposure of P. promelas to PETN 

Nominal 

Treatment 

(μg PETN/L) 

% Survival at 96 hours 

Rep 

A 

Rep 

B 

Rep 

C 

Overall 

Survival 

Std. 

Dev. 

C1 / 0 (Solv. Cont) 100 90 100 96.7 5.77 

C2 / 0 (Dil. Cont.) 100 100 100 100 0.00 

C3 / 1,000 100 90 100 96.7 5.77 

C4 / 2,000 70 80 70 73.3 5.77 

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

treatment (Figure 3-1). Survival was slightly affected by PETN at 2,000 μg PETN/L (nominal) by 

96 hours; however the median lethal test concentration was determined to be greater than the 

highest test concentration tested (i.e., > 2,000 μg nominal PETN/L or >1,218 μg PETN/L). 

Figure 3-1. Graphical depiction of daily P. promelas survival 

during the 96-hour acute definitive exposure to PETN 

3.4 Reference Toxicant Studies 

Monthly reference toxicant studies are conducted with P. promelas using sodium chloride. The 

results of the November 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 November 2012 

were within the historical control limits. 

 

0

20

40

60

80

100

C1 (Solvent Cont.) C2 (Dilution Cont.) C3 C4

Survival (%)

PETN Treatments

24 h 48 h 72 h 96 h

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Table 3-5. Reference toxicant test results for P. promelas

96-hour LC50

AECOM FCETL Historical 95% Control Limits 

Low High 

2,523 1,422 4,156 

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 to P. promelas under 

static-renewal test conditions. Due to problems getting the PETN in solution to its determined 

water solubility (~2.0 mg/L), this study was performed using a solvent carrier. The biological 

endpoint for this study was mortality (or survival). The 24-, 48-, 72-, and 96-hour LC50 values 

based on the average PETN concentrations measured in test solutions for the definitive acute 

P. promelas study are presented below (Table 4-1). The 95% confidence intervals could not be 

determined since mortality did not exceed 50% in any of the test treatments. The highest 

measured average concentration of PETN in these studies (using acetone as the solvent 

carrier) was 1,218 μg PETN/L, lower than the reported water solubility of ~2,000 μg/L. 

Table 4-1. Median lethal PETN concentrations (μg PETN/L) for P. promelas

Time (hours) LC50 (μg measured PETN/L) 95 % CI 

24 > 1,218 -- 

48 > 1,218 -- 

72 > 1,218 -- 

96 > 1,218 -- 

Two preliminary studies (Nov. 2011 and Nov. 2012) were performed using a PETN stock 

solution (1,000 mg/L) in methanol (Restek, Bellefonte, PA). A summary of the target PETN 

concentrations, the 96-h survival results of P. promelas, and measured analytical results are 

reported below (Table 4-2; raw data is included in Appendix D). Analytical samples were not 

taken from the solutions used in the actual toxicity studies, but were collected on samples 

prepared using the same Restek standard following the same preparation technique. 

Table 4-2. Results of PETN preliminary experiments with P. promelas 

Initiation Date Nov. 22, 2011 Nov. 22, 2011 Nov. 6, 2012 

Nominal PETN 

Conc. (μg/L) 

Measured 

PETN (μg/L) 

% Survival at 

96 Hours 

% Survival at 

96 Hours 

0 (Control) -- 100 100 

0 (Solvent Control) -- -- 100 

100 -- 100 -- 

500 430 / 420 100 -- 

1,000 860 -- 100 

2,000 -- 60 70 

GLP-056a 70 / 57 100 -- a

 PETN solution prepared without use of a solvent carrier. 

The range-finding data using a different solvent confirm that the 96-h LC50 for the fathead 

minnows was greater than the nominal concentration of 2,000 μg/L of PETN. 

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5.0 STUDY PERSONNEL 

SPONSOR: Dyno Nobel, Inc. 

TITLE: Acute Toxicity of Pentaerythritol tetranitrate (PETN) to the Fathead Minnow 

(Pimephales promelas) Under Static-Renewal Conditions 

AECOM Environmental Toxicology Laboratory Project Number: 60279946-100-020 

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) 

Taxonomist: Tami Schneck (Aquatics Associates, Inc.) 

Quality Assurance Officer: Anita B. Rehner (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. Fish Acute Toxicity Test, Freshwater and Marine. OPPTS 850.1075. Ecological 

Effects Test Guidelines. EPA 712-C-96-118. 

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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60279946-100-020 C-1 February 2013 

APPENDIX C 

TEST DATA 

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60279946-100-020 D-1 February 2013 

APPENDIX D 

RANGE-FINDING TEST DATA 

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