||6 µg/m3 (1.4 ppb)
||Collection on two Florisil adsorbent tubes in
series, each of which have been pretreated with 11 mg of DL-α-tocopherol,
desorption with a solution composed of equal volumes methylene chloride and methyl
alcohol, analysis by GC with chemiluminescence detection.
|Detection limit based on
recommended air volume:
|0.4 µg/m3 (96 ppt)
|Recommended air volume
and sampling rate:
|25 L at 0.2 L/min
|Standard error of estimate
at the target concentration:
||The air sampler must be kept from light
during and after sampling.
|Status of method:
||A sampling and analytical method which has
been subjected to the established evaluation procedures of the Organic Methods Evaluation
|Date: August 1979
||Chemist: Warren Hendricks
Organic Methods Evaluation Branch
OSHA Analytical Laboratory
Salt Lake City, Utah
1. General Discussion
2. Sampling Procedure
1.2. Detection limit, precision, sensitivity and working
Sample Collection: Diethylnitrosamine (DENA) vapors
have been collected using the same methods used for dimethylnitrosamine. These methods
include cryogenic techniques that involve the use of successive cold traps, ambient
temperature KOH bubblers and Tenax GC cartridges (Ref. 5.1).
Analytical: Analytical procedures for DENA are
similar to those used for dimethylnitrosamine. The separation is usually by gas
chromatography and detection by one or more of the following techniques: mass
spectrometry, Coulson Electrolytic Conductivity Detector, Hall Electrolytic Conductivity
Detector, nitrogen selective alkali flame-ionization detector, and Thermal Energy Analysis
For several reasons, the existing sampling procedures are
not adequate for use by OSHA personnel. Some of these reasons are - poor collection
efficiency, low extraction efficiency and artifactual formation of DENA on the sampling
device. Therefore, the primary emphasis of this work has been to develop new air sampling
techniques. The analytical method utilizes separation by gas chromatography and detection
with the Thermal Energy Analyzer (TEA). The TEA detector was selected because it is both
sensitive and selective for nitrosamines.
1.1.2. Toxic effects (This section is for information only
and should not be taken as the basis of OSHA policy.)
Acute: The LD50 for DENA, administered by
intraperitoneal injection to the rat, is 216 mg/kg. The lowest published toxic dose is 100
mg/kg also given by intraperitoneal injection to rats (Ref. 5.2). The acute toxic effects
from exposure to DENA are similar to those produced by dimethylnitrosamine with serious
destruction of liver tissue as the most important result (Ref. 5.3).
Chronic: There is no correlation between acute toxic
effects and carcinogenic potential for nitrosamines. This is demonstrated by the fact that
even though DENA has only about one-sixth the acute toxicity of dimethylnitrosamine (Ref.
5.2), if administered continuously to rats, it is probably a more active liver carcinogen
DENA has been shown to be carcinogenic to the mouse, the
rat, the hamster, the guinea-pig, the rabbit, the dog, the pig, the monkey,
and to aquarium fish. The agent induces tumors primarily in the nasal cavity, trachea,
esophagus, and liver. It causes cancer after different modes of exposure which include
ingestion, inhalation, and skin painting. It is carcinogenic in single doses and following
prenatal exposure. In lifetime feeding studies with rats in which daily doses between 1
and 10 mg/kg body weight were administered, tumor yields approaching 100% have been
obtained (Ref. 5.5).
A dose-response study has been conducted using rats. DENA
was administered in drinking water and the daily exposure was between 0.075 and 14.2 mg/kg
body weight in 9 groups of animals. The total dose, until death occurred, was between 64
and 965 mg/kg body weight. The tumor induction time was between 68 and 840 days. All daily
doses higher than 0.15 mg/kg body weight gave a tumor incidence of 100%. When a dose of
0.15 mg/kg body weight per day was administered, a tumor yield of 90% was obtained. At
0.075 mg/kg body weight per day, 20 rats survived for more than 600 days and 11 of the 20
animals had tumors of the liver, esophagus, or the nasal cavity. All 4 of the animals that
lived longer than 940 days at this dose level had tumors (Ref. 5.5).
1.1.3. Worker exposure
The chemical reaction, in the condensed phase, between
nitrous acid and diethylamine or triethylamine to form DENA is well known (Ref. 5.6). Any
tertiary amine that contains the diethyl moiety may react with a nitrosating agent to form
DENA. Recently, it has been shown that both amines can react with oxides of nitrogen in
the vapor phase to give DENA as a reaction product (Ref. 5.7). This means that even
though DENA is not used at a particular location, it may be formed from its precursors and
therefore be found in the occupational environment.
Exposure to DENA can occur during operations in which
diethylamine or triethylamine is utilized or produced. The exposure results because both
amines are often contaminated with the nitrosamine. Further, if the amine is used as a
chemical intermediate, DENA can possibly appear in the reaction product (Ref. 5.8).
DENA is extensively used in cancer research facilities.
Human exposure occurs when unchanged DENA is excreted by the laboratory animals (Ref.
DENA is not widely used by industry today. Uses or proposed
uses of DENA include: as a solvent in the fiber industry, as a softener for copolymers, as
an additive for lubricants, in condensers to increase the dielectric constant and for the
synthesis of 1,1-diethylhydrazine (Ref. 5.5).
Non-occupational exposure to DENA can be a result of eating
a normal western diet. It has been estimated that the weekly intake of preformed dialkyl
and heterocyclic nitrosamines per person on a normal English diet is approximately 1 and 3
µg/week respectively (Ref. 5.10). Another source of exposure to DENA is the endogenous
formation of the agent in the gastrointestinal tract. Diethylamine has been shown to react
with nitrite to form DENA in human gastric juice (Ref. 5.5). DENA has been reported to be
a component of tobacco and tobacco smoke (Ref. 5.11).
1.1.4. Number of workers that face exposure - unknown
1.1.5. Physical properties (Ref. 5.5)
N,N-diethylnitrosamine; DEN; DENA; DANA
||a yellow, volatile liquid
||177°C (760 mm Hg) 64-65°C (17 mm Hg)
||max 230 nm; log ε 3.86
||max 332 nm; log ε 1.98
||about 10% in water; soluble in organic
solvents and in lipids
1.2.1. The detection limit for the analytical procedure is
50 pg. The coefficient of variation is 0.14 at this level (Section 4.1). The detection
limit was determined using 5-µL injections from standard solutions.
1.2.2. The pooled coefficient of variation for the
analytical procedure over the range of 80 to 300 ng per sample is 0.034 (Section 4.2).
This represents an air concentration range of from 3.2 to 12.0 µg/m3 based on
the recommended sampling and analytical procedures.
1.2.3. The sensitivity of the analytical procedure over a
concentration range of 80 to 300 ng (3.2 to 12.0 µg/m3 based upon the
recommended air sampling volume of 25 L) is 61918 area units (HP-5840A) per µg/mL. The
sensitivity is determined by the slope of the calibration curve (Section 4.3). The
sensitivity will vary somewhat with the particular instrumentation used in the analysis.
1.2.4. The lower limit of the estimated working range,
assuming adequate desorption efficiency, is 0.4 µg/m3. The upper limit of the
working range is dependent on the capacity of the treated Florisil tubes.
1.3.1. The overall procedure must provide results that are
within ±25% of the true value or better at the 95% confidence interval.
1.3.2. The recovery of analyte from the collection medium
during storage must be 75% or greater.
1.3.3. The overall procedure has met the above validation
criteria (Section 4.5).
1.4.1. The sampling procedure is convenient.
1.4.2. The significance of artifactual formation of DENA
upon the sampling device has been eliminated through pretreatment of the air sampler.
1.4.3. The analytical procedure is quick, sensitive, and
1.4.4. Reanalysis of the samples is possible.
1.4.5. The samples are stable, even when stored at room
temperature for 17 days.
1.4.6. Dimethylnitrosamine can be determined using the
recommended sampling and analytical techniques. The desorption efficiency must be
1.4.7. It may be possible to determine other nitrosamines
1.4.8. The effects of potential interferences are reduced
through the use of a selective detector (the TEA) and can be further reduced by proper
selection of GC parameters.
The relative humidity of the sampled air affects the
ability of the adsorbent to retain the analyte.
3. Analytical Procedure
2.1.1. An approved and calibrated personal sampling pump
whose flow can be determined to ±5% at the recommended flow rate.
2.1.2. Florisil adsorbent tubes: Glass tubes, 6-mm o.d.,
4-mm i.d., 7-cm length, containing 100-mg front and 50-mg rear (separated by a 2-mm
portion of urethane foam or silylated glass wool) sections of 20/40 mesh Florisil. SKC,
Inc. Catalog No. 226-39 or equivalent. Each tube is pretreated with 11 mg DL-α-tocopherol. (Section 4.6)
2.3 Sampling technique
2.3.1. The air sampler is composed of two treated Florisil
tubes in series. The tubes are easily connected with an end cap that has been modified by
cutting off the closed portion.
2.3.2. Connect the air sampler to the sampling pump with
flexible tubing. The 50-mg section of each tube should be positioned toward the sampling
pump. Cover each tube of the air sampler with masking tape or other suitable material to
prevent light from reaching the adsorbent.
2.3.3. The air sampler should be placed in a vertical
position during sampling to minimize channeling.
2.3.4. Sampled air should not pass through any hose or
tubing before entering the sampling device.
2.3.5. Immediately after sampling, separate the air sampler
into its component tubes, identify each tube as front or backup and seal each tube with
plastic end caps. Also, wrap each samples end to end with official OSHA seals.
2.3.6. With each batch of samples, submit at least one
blank tube from the same lot used for sampling. This tube should be subjected to exactly
the same handling as the samples (seal, transport) except that no air is drawn through it.
2.3.7. Transport the samples (and corresponding paperwork)
to the lab for analysis.
2.3.8. If bulk samples are submitted for analysis, they
should be transported in glass vials with Teflon-lined caps. The samples must be kept from
light. Bulk samples must not be put in the same mailing container used for the treated
The relative humidity of the sampled air has a significant
effect on the ability of the air sampler to retain DENA. However, laboratory studies
indicate that 25 L of air containing the target concentration of DENA, at 80% relative
humidity and 22°C, can be sampled with no loss of analyte. Breakthrough from the front to
the rear tube, when the air sampler was challenged with vapors containing 0.15 µg DENA at
these conditions, was about 30%.
No quantitative breakthrough information can be obtained
from the analysis of individual tubes from a particular air sampler, but, if the amount
found on the rear tube is 45% or greater of the total, then it is possible that some of
the analyte may have been lost.
2.5. Desorption efficiency
2.5.1. The average desorption from DL-α-tocopherol treated Florisil adsorbent tubes spiked with 80,
150, and 300 ng DENA was 95.3% (Section 4.4). The spiked tubes represent an air
concentration range of 3.0 to 12.0 µg/m3 based upon the recommended air
2.6. Recommended air volume and sampling rate
2.5.2. The desorption efficiency for DENA may vary from one
lab to another, and from one lot of DL-α-tocopherol
treated Florisil tubes to another. Therefore, it is necessary for each laboratory to
determine the desorption efficiency for each lot of DL-α-tocopherol
treated Florisil tubes.
2.6.1. The recommended sampling rate is 0.2 L/min.
2.7. Interferences (sampling)
2.6.2. The recommended air volume is 25 L.
2.7.1 Since it is possible that the precursors of DENA -
diethylamine, triethylamine, other tertiary amines with the diethyl moiety, and various
nitrosating agents (oxides of nitrogen, nitrites, etc.), are present in the environment,
it is conceivable that DENA may be formed upon the sampling device and not be present in
the sampled air.
2.8. Safety precautions (sampling)
Laboratory studies indicate that it is possible to form
DENA from its precursors on untreated Florisil tubes. Further experiments show that when
the Florisil tube is treated with 11 mg of DL-α-tocopherol,
the formation of DENA does not take place.
2.7.2. At the present time, it is unknown if any compound
would severely interfere with the collection of DENA on treated Florisil tubes. In
general, the presence of other compounds will reduce the breakthrough volume for a
2.7.3. Any compound which is suspected of interfering with
the collection or analysis should be listed on the sampling data sheet.
2.7.4. Light will decompose DENA (Ref. 5.5). The air
sampler must be protected from light during and after sampling.
2.8.1. Observe due care when working with the sharp ends of
the air sampler.
2.8.2. Attach the sampling equipment to the worker in such
a manner that it will not interfere with work performance.
2.8.3. Follow all safety practices that apply to the work
area being sampled.
4. Backup Data Section
3.1.1. A gas chromatograph interfaced to a Thermal Energy
3.1.2. A GC column capable of resolving DENA from the
desorption solvent and potential interferences. The column used in this work was a 10-ft
× 1/8-in. stainless steel column containing 10% Carbowax 20M with TPA on 80/100 mesh
Chromosorb W AW.
3.1.3. An electronic integrator or other suitable method to
measure peak area.
3.1.4. An analytical balance capable of accurately weighing
to 5 decimal places.
3.1.5. Vials. 2-mL vials with Teflon-lined caps.
3.1.6. Microliter syringes. 5-µL syringes for sample
injections and other convenient sizes.
3.1.7. Pipets. Convenient sizes for diluting standards. A
1-mL dispenser pipet for desorbing solvent.
3.1.8. Volumetric flasks. Convenient sizes for diluting
3.1.9. Dewar flasks. Convenient sizes for liquid nitrogen.
3.2.1. DENA, authentic primary standard, 98% minimum.
3.3. Sample preparation
3.2.2. Methyl alcohol, chromatographic grade.
3.2.3. Methylene chloride, chromatographic grade.
3.2.4. Isopropyl alcohol, chromatographic grade.
3.2.5. Ethyl alcohol, U.S.P., 95%.
3.2.6. Gases, purified GC grade helium and medical grade
3.2.7. Nitrogen, liquid.
3.3.1. The status of the OSHA seal on each sample is noted
and recorded as intact, broken, or none.
3.4. Standard preparation
3.3.2. The field and laboratory identification numbers on
each seal are checked against those on the sample identification sheets.
3.3.3. Avoid exposure of the samples to light. DENA will
3.3.4. The front and rear tubes from each sampler are
transferred to separate 2-mL vials.
3.3.5. The contents of each vial are desorbed with 1.0 mL
of desorbing solution. The desorbing solution is composed of equal parts by volume methyl
alcohol and methylene chloride.
3.3.6. The vials are sealed immediately with Teflon-lined
caps and desorbed for 30 min with intermittent shaking.
3.4.1. Stock standards are prepared by diluting a weighed
amount of DENA with isopropyl alcohol. The stock standard is diluted to the working range
with isopropyl alcohol (Section 3.8 Safety Precautions).
3.4.2. A solution composed of 0.15 µg/mL DENA in isopropyl
alcohol is equivalent to an air concentration of 6.0 µg/m3 for a 25-L air
sample desorbed with 1.0 mL of desorbing solution. This amount is not corrected for the
3.4.3. Standards are stored in dark bottles under
3.5.1. GC conditions
|helium (carrier gas) flow rate:
|TEA transfer line temperature:
3.5.2. TEA conditions
|GC pyrolyzer furnace temperature:
||1.8 mm Hg
|cold trap temperature:
||-130°C (ethyl alcohol and liquid nitrogen)
Complete instructions for the TEA are found in its manual.
3.5.3. Chromatogram (Section 4.3)
3.5.4. Peak areas are measured by an electronic integrator
or other suitable means.
3.5.5. An external standard procedure is used to prepare a
calibration curve from the analysis of at least three different standard solutions. The
calibration curve is prepared daily. The integrator is calibrated to report results in
micrograms per milliliter after correction for desorption efficiency.
3.5.6. Bracket the samples with analytical standards.
3.6.1. Any compound that has the same GC retention as DENA
and will elicit a response from the TEA detector is an interference. Possible
interferences are listed on the sample data sheets.
3.6.2. GC parameters may be changed to circumvent most
3.6.3. Retention time on a single GC column is not proof of
chemical identity. Samples should be confirmed by GC/Mass Spectrometry or other suitable
means when required.
3.7.1. The integrator value in micrograms per milliliter
(corrected for desorption efficiency) is used for reference only. More reliable results
are obtained by use of the calibration curve. The peak area, for each standard, is
compared to its concentration in micrograms per milliliter (corrected for desorption
efficiency) and the equation for the best straight line through the data points is
determined by linear regression.
3.7.2. The concentration in micrograms per milliliter
(corrected for desorption efficiency) for a particular sample is determined by comparing
its area to the calibration curve.
3.7.3. Analytical results from the two tubes that compose
an air sampler are added together.
3.7.4. The air concentration for a sample result is
calculated by the following equation:
DENA, µg/m3 = (A)(B)(1000)/C
||µg/mL from 3.7.3.
||air volume in liters
3.7.5. To convert micrograms per cubic meter
to parts per billion (ppb), the following relationship is used:
DENA, ppb = (µg/m3)(24.46)/102.1
||result from 3.7.4.
||molar volume at 25°C and 760 mm Hg
||molecular weight of DENA
3.8. Safety precautions (analytical)
3.8.1. DENA is an extremely potent carcinogen and utmost
care must be exercised when working with this compound.
3.8.2. Avoid skin contact with liquid nitrogen and the
3.8.3. Confine the use of solvents to a fume hood.
3.8.4. Wear safety glasses in all laboratory areas.
3.8.5. Check to be sure that the TEA exhaust is connected
to a fume hood.
4.1. Detection limit
The following data were generated by replicate 5-µL
injections of a standard solution whose concentration was 0.01 µg/mL. The detection limit
was determined to be 50 pg which is equivalent to 0.4 µg/m3 based on the
recommended air volume. Peak heights were used because integrated data were unreliable at
Detection Limit Data
||peak height, mm
SD = 10.74
CV = 0.14
These data represent multiple injections of standard
solutions. The injection volume was 5 µL and the concentrations of the standards were
0.08 µg/mL, 0.15 µg/mL and 0.30 µg/mL. Peaks were integrated by a Hewlett-Packard 5840A
|× target conc.
A typical calibration curve is shown in Figure 4.3.1. The
slope of the curve indicates the sensitivity of the method.
A representative chromatogram is shown in Figure 4.3.2.
Samples representing 3.2, 6.0 and 12.0 µg/m3
based on 25-L air volumes were prepared by injecting liquid standards on treated Florisil
|overall average =
Samples were generated by the liquid injection of DENA on
Polar Partition tubes containing about 150 mg of adsorbent. The tubes were allowed to
equilibrate overnight and then were placed in front of the treated Florisil tubes.
Twenty-five liters of air, at 80% relative humidity and 22°C, were drawn through the
sampling train. The DENA on the Polar Partition tube was desorbed by the humid air and was
deposited on the air sampler. Studies conducted at ambient relative humidity and
temperature indicated that desorption was essentially complete after 1 L of air had passed
through the Polar Partition tube (Figure 4.5.1). In these studies the spiked Polar
Partition tube was connected directly to the TEA.
The data in Table 4.5. represent the effects of storage at
ambient (21-26°C) and reduced (-20°C) temperatures on DENA collected using
treated Florisil tubes. The recoveries for the six samples run on the designated days are
not corrected for desorption efficiency. A graphical representation of the data may be
found in Figures 4.5.2 and 4.5.3.
4.6. Preparation of sampling tubes
Methylene chloride, chromatographic grade.
(Vitamin E), 90% minimum - available from ICN Pharmaceutical Inc., K & K Labs
Division, Life Sciences Group (1979 Catalog No. 18787). Prepare two solutions containing
50 and 100 mg/mL in methylene chloride. Store under refrigeration in dark bottles.
Each Florisil tube is treated with 11 mg DL-α-tocopherol. This is accomplished by first breaking open both
ends of each tube and adding 200 µL of the 50 mg/mL DL-α-tocopherol
solution. Both sections of the tube should be uniformly wetted by the solution. The
methylene chloride is allowed to evaporate in a fume hood overnight. The tube is further
treated by placing 10 µL of the 100-mg/mL DL-α-tocopherol
solution on the head of the front section of Florisil. The methylene chloride is again
evaporated and then both ends of the tube are sealed with the plastic end caps supplied
with the tubes. The tubes should be stored in a freezer with suitable precautions taken to
prevent their contamination. The tubes are stable, when stored in a freezer, for at least
Figure 4.3.1. Calibration curve for
Figure 4.3.2. A typical chromatogram for
Figure 4.5.1. The rate at which
diethylnitrosamine is desorbed from Polar Partition adsorbent by air at ambient
temperature and relative humidity.
Figure 4.5.2. Ambient temperature storage
test for diethylnitrosamine.
Figure 4.5.3. Refigerated temperature test
5.1. W. Hendricks, Dimethylnitrosamine (Method 06, Organic
Methods Evaluation Branch, OSHA Analytical Laboratory, Salt Lake City, Utah), Unpublished.
5.2. Registry of Toxic Effects of Chemical Substances, 1976
Edition. (H.E. Christensen, E.J. Fairchild, Eds., B.S. Carroll, R.J. Lewis, Project
Coordinators) U.S. Department of Health, Education, and Welfare, Public Health Service,
Center for Disease Control, National Institute for Occupational Safety and Health, U.S.
Government Printing Office, Washington, D.C. (1976).
5.3. P.N. Magee and J.M. Barnes, Carcinogenic Nitroso
Compounds, Advances in Cancer Research 10, 163-247 (1967).
5.4. D. Druckey, R. Preussmann, S. Ivankovic and D.
Schmahl, Organotrope Carcinogene Wirkany bei 65 Verschiedenen N-Nitroso-ver-bindungen and
BD-Ratten, Z. Krebsforsch, 69(103), 103-201 (1967).
5.5. IARC Monographs on the Evaluation of Carcinogenic Risk
of Chemicals to Man, Vol. 1, International Agency for Research on Cancer, Lyon, France,
World Health Organization, 107-124 (1971).
5.6. J. March, Advanced Organic Chemistry: Reactions,
Mechanisms and Structure, McGraw-Hill Book Company, New York, 486 (1968).
5.7. J.N. Pitts, P. Grosjean, K.V. Cauwenberghe, J.P.
Schmid, and D.R. Fitz, Photooxidation of Aliphatic Amines Under Simulated Atmospheric
Conditions: Formation of Nitrosamines, Nitramines, Amides and Photochemical Oxidant, Environmental
Science and Technology, 12(8) 946-953 (1978).
5.8. B. Spiegelhander, G. Eisenbrand and R. Preussman,
Contamination of Amines With N-Nitrosoamines, Angew. Chem. Int. Ed. Engl.,
17(5) 367-368 (1978).
5.9. P. Issenberg and H. Sornson, A Monitoring Method for
Volatile Nitrosamine Levels in Laboratory Atmospheres, Environmental N-Nitroso Compounds
Analysis and Formation, E.A. Walker and P. Bogovski, Eds., Lyon, France, IARC Scientific
Publication No. 14 (1976).
5.10. T. Kakizoe, T.T. Wang, VW.S. Eng., R. Farrer, P. Dion
and R. Bruce, Volatile N-Nitrosoamines in the Urine of Normal Donors and of
Bladder Cancer Patients, Cancer Research 39, 829-832 (1979).
5.11. K. D. Brunnemann, L. Ye and D. Hoffmann, Assessment
of Carcinogenic Volatile N-Nitrosamines in Tobacco and in Mainstream and
Sidestream Smoke from Cigarettes, Cancer Research 37, 3218-3333 (1977).