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

60279946-100-020AECOM

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 2013AECOM

60279946-100-020 ii February 2013 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 Concentrations 1 : 0 (solvent control), 0 (dilution water control), 1,000, and 2,000

Control / Dilution Water Reconstituted water prep ared 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 > 1,218 μg/L PETN, measured
48-hr LC 50 > 1,218 μg/L PETN, measured
72-hr LC 50 > 1,218 μg/L PETN, measured
96-hr LC 50 > 1,218 μg/L PETN, measured

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

60279946-100-020 v February 2013 

### 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 PROT OCOL AND AMENDMENTS .................................................... A-1

APPENDIX B ANALYTICAL DATA SUMMARY ................................................................. B-1

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

APPENDIX D RANGE-FINDING TEST DATA..................................................................... D-1AECOM

60279946-100-020 vi February 2013 

### 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 ....................................... 11AECOM

60279946-100-020 vii February 2013 

### LIST OF FIGURES

Figure 3-1. Graphical depiction of daily P. promelas survival during the 96-hour acute definitive exposure to PETN ...................................................................................... 9AECOM

60279946-100-020 February 2013 1 

### 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 CaCO 3 , 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 copi es 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

60279946-100-020 February 2013 2 

### 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, e ffective (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/cm 3 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 C enter 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 identif ication 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
CaCO 3 , respectively. The temperature of the water was adjusted to approximately 25°C prior to spiking and/or mixing of the test solutions.AECOM

60279946-100-020 February 2013 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 #10533. 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 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.AECOM

60279946-100-020 February 2013 4 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.AECOM

60279946-100-020 February 2013 5 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% HNO 3 ) for ~24 hours, removed, and rinsed three timeswith 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 ri nsed 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 temperat ure-controlled environmental chamber that maintained the test solution temperature at 25 ± 2°C. Lighting was controlled to provide 16hours 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 replicat e 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 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 controls and the highest PETN concentration at test initiation. Dissolved oxygen and pH were measured in each concentration daily and temp erature 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.AECOM

60279946-100-020 February 2013 6 Biological : During the test, organisms were observ ed 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
LC 50 , respectively) were determined by inspection. The LC 50 values are expressed as average measured PETN concentrations measured in new and old test solutions (μg PETN/L).AECOM

60279946-100-020 February 2013 7 

### 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
CaCO 3 ) Alkalinity (mg/L as
CaCO 3 ) pH Conductivity (μS/cm) Total
Ammonia (mg NH 3 -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 forAECOM

60279946-100-020 February 2013 8 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 summa ry is provided in Appendix B. Values for new solutions represent one measured value while three replicat es 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 dilu tion 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 tr eatments also appeared to be slightly weakened. Copies of the raw data and statistical analyses are included in Appendix C.AECOM

60279946-100-020 February 2013 9 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 C4Survival(%)



### PETN Treatments 24 h 48 h 72 h 96 hAECOM

60279946-100-020 February 2013 10 Table 3-5. Reference toxicant test results for P. promelas

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

Low High 2,523 1,422 4,156 Note: Values are expressed as mg/L chlorideAECOM

60279946-100-020 February 2013 11 

### 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 LC 50 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) LC 50 (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 Contro l) -- -- 100
100 -- 100 --
500 430 / 420 100 -- 1,000 860 -- 100 2,000 -- 60 70
GLP-056 a 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.AECOM

60279946-100-020 February 2013 12 

### 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.)AECOM

60279946-100-020 February 2013 13 

### 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.AECOM

60279946-100-020 February 2013 15 

### 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.AECOM

60279946-100-020 A-1 February 2013 

### APPENDIX A

TEST PROTOCOL AND AMENDMENTSAECOM

60279946-100-020 B-1 February 2013 

### APPENDIX B

ANALYTICAL DATA SUMMARYAECOM

60279946-100-020 C-1 February 2013 

### APPENDIX C

TEST DATAAECOM

60279946-100-020 D-1 February 2013 

### APPENDIX D

RANGE-FINDING TEST DATA