The origin, effects and control of air pollution

in laboratories used for human embryo culture

Jerry Hall1, Antonia Gilligan2, Tim Schimmel3,
Michael Cecchi4 and Jacques Cohen3'5
Center for Reproductive Research and Testing, Rockville, Maryland, and
Shady Grove Fertility Centers, 2Alpha Environmental, Jersey City,
New Jersey, 3The Institute for Reproductive Medicine and Science of Saint
Barnabas, Livingston, New Jersey, and 4GenX International, Madison,
Connecticut, USA
5
To whom correspondence should be addressed at: The Institute for Reproductive
Medicine and Science of Saint Barnabas, 101 Old Short Hills Road, West Orange,
NJ 07052, USA

Testing shows that most laboratories conducting human gamete and embryo
culture have air quality and sources of contamination that exceed the levels
measured in homes, businesses and schools. The sources of these contaminants
have been shown to be either from activities outside the laboratory, or
emitted from materials used in the facility, such as compressed gas, cleaning
and sterilizing agents, plastic and stored materials. Both the laboratory
structure and the air handling systems may affect the air composition. The
significance of these findings is being validated by the accumulation of field
case studies and now by assay procedures. Products given off by road sealant
were shown to have accumulated in one of the examined laboratories,
adjacent to a large re-surfaced parking area. Aldehydes such as acrolein,
hexanal, decanal, pentanal and others were detected at elevated concentra-
tions that were statistically significant. Since it is not appropriate to add
potentially suspect chemicals to human embryos, we used a mouse-model to
study the effect of acrolein. The growth of mouse embryos was significantly
affected after acrolein was added at different concentrations to the culture
environment. The physiological effect was noted at concentrations in the low
ppm range. The testing end-point of embryo death must still be considered
to be a crude basis for evaluating toxicological effects, since it involves
addition of compounds to culture media and unprotected growth until the
blastocyst stage. The findings may, however, support observations of
decreased pregnancy rate following exposure of human embryos to aldehydes
or other adverse conditions. With proper engineering and material selection,
it is possible to reduce such contamination. The usefulness of this approach
for controlling aldehydes has been demonstrated by decreasing levels in the
laboratory to below those of the outside air.
Key words: acrolein/air sampling/aldehydes/incubatorfiltration/mouseembryo test

14o © European Society for Human Reproduction and Embryology Human Reproduction Volume 13 Supplement 4 1998
 Laboratory air

Introduction

We recently described a series of incidents and measurements, which strongly

suggested that volatile organic compounds (VOCs) could have an adverse affect
on the success rate of in-vitro fertilization (IVF) practices. Here, we show that
the air quality in IVF laboratories may be worse than that of most homes, offices
and schools. Since our initial report (Cohen et al., 1997), we have studied other
laboratories and their immediate environments and compared them with published
tables of total volatile organic compounds (TVOCs). While synonyms may vary,
the labels TVOCs and chemical air contamination (CAC) are essentially the
same. A brief summary of field samples is presented with an emphasis on outdoor
air, air supplied to the laboratory and finally the air in incubators. While the
tabulation of studies includes a variety of quantification procedures, they generally
use a charcoal or Tenex trap, followed with either gas chromatography/flame
ionization detector (GC/FID) or gas chromatography/mass spectroscopy(GC/
MS). Tenex is a family of porous polymers made from 2,6-diphenyl -/?-phenylene
oxide. It is an absorbent material, well-suited for the analysis of alcohols, glycols,
phenols, amines, aldehydes, ketones and chlorinated aromatic compounds. Our
work, as well as the cited studies, is comparable.
Significant increases in some materials in one laboratory occurred after spraying
tarmac sealant over the building's parking area. The material was an emulsion
containing water and coal-tar derived materials. It was suspected that this induced
a decline in performance. Air sampling at the location, showed statistically
significant increases in aldehydes, such as acrolein, butanal, pentanal, hexanal,
heptanal, and nonanal. These materials are likely to be the products of unsaturated
volatiles from the paving materials that have been oxidized by the air and light.
Assays of mouse embryo development in vitro were performed in the presence
of one of the elevated components, acrolein. The study showed an increase in
mouse embryo mortality at low mg/1 concentrations.

Testing laboratory air

The methods we employed to assess the composition of laboratory air were

described in Cohen et al. (1997). The system is a modification of existing
environmental testing procedures aimed at detecting compounds at the jig/m3 or
ppb level. Routine industrial hygiene tests using charcoal tubes or Tenex traps
with gas chromatography, photoionization detector (PID) or FID detection can
be adequate for highly contaminated spaces. With longer sampling times they
can produce low detection limits. The use of tubes with gas chromatography/
mass spectroscopy widens the analytical scope and lowers the detection limits
significantly. The use of EPA TO-14 (Environmental Protection Agency, 1984,
1988) illustrated this method. Laboratories can sample for this method using an
evacuated sample vessel with laboratory analysis performed at a referral labora-
tory. Aldehyde sampling is usually done using specialized sampling tubes

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J.Hall et al.

combined with a sampling pump. Generally, field gas chromatographs, infrared

detectors, and photo-acoustic detectors do not have the low detection limits
required for this analysis.

What is typical air quality?

Since the energy crisis of the 1970s, building techniques have changed and sick
building syndrome (SBS) has become part of the modern lexicon. In accessing
the health effects of VOCs, many investigators have measured air for determining
the variety and concentrations of organic chemicals and attempted to sort out the
adverse impact of these volatile organic chemicals on health. The literature is
full of poorly conducted studies, but a recent review of 1100 selected articles
describes valid analytical estimates of common air constituents and their concen-
trations (Anderson et al., 1997). The goal of these studies was to determine
adverse effects on health. The sample of locations represented buildings which
were suspect for disproportional poor air quality. The average lower limit of air
quality was 330-2240 (ig/m3 (± 636 |Ug/m3). These studies, carried out using
accepted sampling and analytical procedures, show a wide range in the level of
airborne materials. The studies did not show that high VOC numbers alone were
indications of sick building syndrome. Adverse affects on the inhabitants were
found in a number of studies. The authors noted that highly elevated numbers
were more likely to be associated with reports of irritant or adverse reactions.
The concept of TVOC as an indicator of SBS is probably not universally true,
but the VOC composition is crucial in determining if there is an adverse affect.
In our work to date we have seen elevations in some groups of compounds that
did not appear to affect IVF success, such as alkanes. Alkanes are saturated
hydrocarbons characterized by their chemical stability.
The same principal between TVOC and SBS may apply to the relationship
between TVOC and cell tissue culture where a complex of specific physical and
chemical factors may influence the growth and health of different types of cells.
In this respect, it is important to realize that incubated cells are largely unprotected,
since they lack an immunological defence apparatus, lack a functional liver with
its complement of detoxifying pathways, and lack physical barriers such as
epithelium, excretory or pulmonary systems. It is likely, though not proven, that
such cells grown in vitro are more sensitive to certain compounds than complex
organisms.

The IVF laboratory air

After detailed sampling of seven IVF practices, the values described in Table I
were found. The data was collected from seven IVF facilities located primarily
in the Eastern seaboard of the USA. The location of the laboratories ranged from
rural suburban to urban settings in major cities with chronic air pollution
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Table I. Levels of volatile organic compounds (VOCs) in different areas of seven in-vitro
fertilization (IVF) clinics

Location Range of VOC (|ig/m3) Mean VOC levels (|ig/m3) SD

Outside air 128-1830 533 705

Air supply 303-2797 1152 1110
IVF laboratory 310-4404 2862 1818
Incubator 717-9485 2769 3054
Adjacent to IVF 473-7141 4372 2419

concerns. The majority of the facilities were associated with hospitals or buildings
with medical practices. The impact of poor air was usually not determined by
the geographical location, but the immediate area adjacent to the laboratory.
There are issues that cannot be easily determined. Modern construction such as
computer networks and environmental systems often combines ventilation systems
allowing pollution from one area to migrate internally. Solid walls are often
punctuated for plumbing and cables, providing another avenue for the transfer
of materials. Air lock systems are, in general, not used in IVF facilities.
The laboratories we investigated were diverse in their layouts but had the
following common features. The IVF laboratory was always separated from the
procedure room and ancillary laboratories. The IVF laboratories were all equipped
with high efficiency particulate air filtration (HEPA) filtration and over-pressure
for particulate control. The sizes of the laboratories varied greatly from 3X4 m
with normal ceiling height to large spaces >100 m2 with 3.5 m ceiling height.
The ventilation was in general dedicated to the IVF facility and all laboratories
used standard incubators with 5% CO2 in air culture. All but one facility used
oil overlay as part of their culture system.
The air contaminants were measured using the US Environmental Protection
Agency procedure TO-14 (1984, 1988), from the compendium of methods for
the determination of toxic organic compounds in ambient air (Cohen et al,
1997). This procedure uses a stainless steel sampling canister with analysis using
cryo-concentration followed by gas chromatography and mass spectroscopy. The
sum of all the materials found was used to calculate the VOCs. While the sum
of this analysis will eliminate certain low molecular weight organics, for ease of
reference it will be referred to as total volatile organic compounds. Additionally,
the data may combine materials with overlapping gas chromatography character-
istics and similar mass spectra. This rare phenomenon does not affect the total
VOC calculations. The areas of categorization were: (i) outside air, which should
be the cleanest in terms of chemical contaminants; (ii) air supplied to the IVF
practice, this is the air as it leaves the supply duct; (iii) air in the IVF laboratory;
(iv) air in the incubator; and (v) air in areas adjacent to the IVF laboratory, such
as a doctor's examining room or hallway. The last area is generally a low-
pressure area relative to the laboratory. The sample size is small, but the
trend illustrated in Table I demonstrates that the existing engineering steps of
compartmental ventilation have reduced much of the air contamination found in

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J.Hall et al.

the laboratory environment compared with that found in areas adjacent to the
IVF facility.
The data shows the mean value for the various areas as the sum of all the
VOCs as determined by the method described by the US Environmental Protection
Agency TO-14 procedure. The extreme variability in the numbers generated the
high values of standard variation. The extreme levels were often due to a uniquely
high level of one material. Alcohols in particular could vary dramatically from
one laboratory to another. They could also vary inside the same facility depending
on the location. The level of isopropanol could drop from the exterior level in
the laboratory to inside the incubator. In this case, we postulate that the water
pan for humidifying the incubator is the sink for the alcohols.
The data indicates that air quality deteriorates as it passes from the exterior
of a building to inside the laboratory and deteriorates again inside the incubator.
The range of numbers is from an average low of 533 fig/m3 for outside air VOCs
to an average of 2769 )ug/m3 in the incubators. This 5-6-fold increase in VOC
concentration may present concerns regarding the possible impairment of growth
of embryos. Considering the limited amount of information available at this time,
it is unclear which levels and contaminants produce adverse effects.

Particular pollutants

Having reviewed the total VOC data from IVF facilities and found that it equals
or exceeds the literature references for epidemiological VOC studies, it is
appropriate to review the data for individual materials. In doing these studies,
we have noticed unusual numbers that may suggest a potential decline in IVF
outcome. The following study from one particular location, identified as number
7 (collaborators from facility 7 are not identified), shows that aldehydes as a
group correlate closely with being both unusually high concurrently with the
facility experiencing a decrease in pregnancy outcome.
The aldehyde data, from outside air samples from seven institutions, is
presented in Table II. It is evident that individual aldehydes, as well as their total
concentrations, were elevated in locations 5 and 7. There is a fair degree of
variability, but the exterior air level of 57.6 fig/m3 for location number 7 exceeds
the next highest level by 27.6 (ig/m3. At the 95% confidence level, the maximum
aldehyde level in facility number 7 should have been 53.1 (ig/m3. The confidence
level was calculated based on the mean + 3X SD. The mean (± SD) for outside
air was 14.9 (± 24.1) |ig/m3.
Aldehydes in the incubators of the facilities are shown in Table III. The
concentrations of aldehydes in the incubators sampled in locations 3, 5 and 7
appear to be raised. The concentration in the incubator of the seventh facility
was again higher, in comparison with the data in other locations. The mean
(± SD) of all facilities except for the seventh location was 2.9 (± 6.4) jig/m3.
The maximum level at the 95% confidence level should be 67.8 (ig/m3, indicating
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 Laboratory air
3
Table II. Levels (|ig/m ) of aldehydes in outside air at seven in-vitro fertilization (IVF) clinics

Location Acrolein Acetaldehydea Butanal Pentanal Hexanal

1 0.0 0.0 0.0 0.0 0.0

2 1.6 0.0 0.0 0.0 0.0
3 0.0 0.0 0.0 0.0 0.0
4 0.0 0.0 0.0 0.0 0.0
5 0.0 20.0 6.0 2.0 2.0
7 3.6 20.0 8.0 3.0 4.0

Location Heptanal Octanal Nonanal Total

1 0.0 0.0 0.0 0.0

2 0.0 0.0 0.0 1.6
3 0.0 0.0 0.0 0.0
4 0.0 0.0 0.0 0.0
5 0.0 0.0 0.0 30.0
7 3.0 6.0 10.0 57.6
a
May contain trailing peaks from isobutane.

Table III. Levels of aldehydes in incubators from seven in-vitro fertilization (IVF) clinics

Location Acrolein Acetaldehyde Butanal Pentanal Hexanal

1 0.0 0.0 0.0 0.0 0.0

1 0.0 0.0 0.0 0.0 0.0
1 0.0 0.0 0.0 0.0 0.0
1 0.0 0.0 0.0 0.0 0.0
3 1.1 0.0 8.0 0.0 8.0
4 0.0 0.0 0.0 0.0 0.0
5 3.5 0.0 0.0 0.0 0.0
7 3.7 20.0 4.0 2.0 5.0

Location Heptanal Octanal Nonanal Decanal Total

1 0.0 0.0 0.0 0.0 0.0

1 0.0 0.0 0.0 0.0 0.0
1 0.0 0.0 0.0 0.0 0.0
1 0.0 0.0 0.0 0.0 0.0
3 0.0 0.0 0.0 0.0 17.1
4 0.0 0.0 0.0 0.0 0.0
5 0.0 0.0 0.0 0.0 3.5
7 4.0 7.0 30.0 3.0 75.7

an unusually high accumulation of aldehydes for number 3 and especially number

7. The mean (± SD) levels of aldehydes in incubators was 12 (± 26.4) (ig/m3.
The source for these aldehydes is believed to be an unusual chain of events
leading to their synthesis. Prior to the decline at site 7, the parking lot was
resurfaced with a commercial product containing coal tar derived materials. This
operation was carried out in early summer in an area with elevated ozone
concentrations. The low molecular weight components of the coal tar volatized.
These materials include alkanes, alkenes, polycyclic aromatics and other complex
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organics. These then reacted with ozone and possibly nitrogen oxides to
yield various products. The unsaturated alkenes and aromatics were subject to
incomplete oxidation to produce a variety of carbonyl compounds including
aldehydes. The conjecture is based upon the well-known presence of aldehydes
in smog. The more reactive species produced by this process were not detected,
but this is thought to be a result of the sampling and analysis procedure. The
sample and analysis plan did not allow for the analysis of formaldehyde in this
case. It is reasonable to conjecture this to be elevated (Wolkoff et al, 1997).

Acrolein bioassay

Acrolein is an ubiquitous aldehyde found in both outside and inside air,

presumably as a result of industrial activity, and photochemical action on organic
materials produced by biota (Waldbott, 1978). It has also been detected in tobacco
smoke (Racowsky et al, 1989). Specific accumulation of this agent from outside
air brought into the laboratory air-conditioning system as illustrated above, may
be the result of drastic environmental changes, such as new construction sites
and road resurfacing. In light of the possible relevance of acrolein on gamete
and embryo culture, we studied the consequences of different concentrations of
acrolein in culture medium. For this purpose, we exposed preimplantation mouse
embryos to acrolein and followed development in vitro as well as postimplantation
development after embryo transfer. Embryos were obtained from 5-6 week old
B6C3F1 mice. Early on day 2, 36 h after ovulation stimulation, 2-cell stage
embryos were collected from the oviducts and pooled into one dish of culture
medium (Whittingham; 1971). Embryos were allocated to dishes containing 0.01,
0.025, 0.0375, 0.05, 0.1 or 1.0 mM acrolein or to acrolein-free control dishes.
The 2-cell embryos were assessed 24 h later for development to the eight-cell
stage and left in culture until 85 h post-ovulation for assessment of early
blastocyst formation. Embryos exposed to 0.01 mM acrolein and non-exposed
embryos were evaluated for their potential to develop to term by transferring
them to recipient mice. Recipient CD-I females were made pseudo-pregnant by
mating with vasectomized CD-I males. Embryos were transferred via a flank
incision to the uterine horns on day three of pseudo-pregnancy. The number of
embryos that had developed to live to term or were absorbing as fetuses were
recorded for each pregnant animal. Other embryos not transferred were analysed
for cell number (Tarkowski, 1966).
The results showed that embryo development was inversely correlated with
the concentration of acrolein. Both development to the blastocyst (Table IV) as
well as the mean cell number (Table V) were clearly affected. Comparisons were
calculated using %2 analyses. The percentage of embryos that developed to the
8-cell stage in the group treated with 0.01 mM acrolein was comparable with
that for the control group (94.6 versus 96.1% respectively; not significant). In
the groups where the concentration of acrolein was increased to 0.025 and 0.0375
mM, embryo development was reduced when compared with the untreated group
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 Laboratory air

Table IV. Effect of acrolein on mouse 2-cell embryo development in vitro. Comparisons carried
out using the %2 test

Treatment Group m M Development state a

(ppm) N o . of embryos Percentage 8-cell b Percentage blastocyst c

Untreated (control) 625 96. l d 87.9 k

0.01 (0.58) 638 94.6 e 80.1 1
0.025 (1.40) 521 89.0 f 41.3m
0.0375 (2.10) 502 85.08 3.0 n
0.05 (2.80) 425 7.7 h 0.0°
0.1 (5.60) 468 O.O1 0.0P
1.0 (56.00) 192 0.0J 0.01
a
Percentage of total cultured.
b
Evaluated on day 3 post-ovulation.
c
Evaluated on day 4 post-ovulation.
d
Difference not significant in comparison with e.
d
Significantly different (P < 0.05) compared with f and g.
k
Signiflcantly different (P < 0.01) compared with 1.
k
Significantly different (P < 0.001) compared with m.
k
Significantly different (P < 0.0001) compared with n, o, p, and q.

Table V. Cell number after 48 h exposure of 2-cell mouse embryos to acrolein

Treatment group m M (ppm) No. of embryos evaluated Mean cell no.

Untreated (control) 170 43.4

0.01 (0.58) 215 37.3
0.025 (1.40) 115 30.9
0.0375 (2.10) 41 9.5
0.05 (2.80) 248 4.7
0.1 (5.60) 50 2.0
1.0 (56.00) 50 2.0

(89.0 and 85.0% respectively; P < 0.05). A highly significant negative effect of
0.05 mM acrolein and higher on early embryo cleavage in relation to the control
group (P < 0.0001) was evident. Only 8% of 2-cell embryos treated with 0.05
mM acrolein developed to the 8-cell stage; no 8-cell embryos were obtained at
higher concentrations. The adverse impact on embryos exposed to 0.025 mM
acrolein was significant, compared with the control group, as only 41.3%
developed to the blastocyst stage (P < 0.01). In 0.0375 mM acrolein, only 3%
of the embryos became blastocysts and at 0.05 mM and higher, embryos arrested.
Embryos transferred to recipient mice and cultured at 0.01 mM implanted as
well as control embryos, however significantly (P < 0.05) fewer developed to
term (75 compared with 55%).
It can only be speculated on how acrolein and possibly other aldehydes reduce
the growth of embryos (Fabro, 1978). Incubation of isolated rat hepatocytes with
acrolein may cause a decrease of reduced glutathione (GSH) that is reversible
up to concentrations of 0.25 mM. Concentrtaions of 0.5 mM and above irreversibly
deplete cells of GSH (Zitting and Heinonen, 1980). The toxicological significance
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J.Hall et al.

Table VI. Post-implantation development of mouse embryos exposed to acrolein during pre-
implantation culture

Treatment group No. of embryos No. of pregnant No. (%) of No. (%) of term
mM (ppm) transferred recipients implants fetusesa

Untreated (control) 140 14 108 (77.1 ) b 80 (74.7)d

0.01 (0.58) • 120 12 87 (72.5)c 47 (54.6)e

Percentage of implantation sites

b
Difference not significant compared with c.
d
Significantly different (P < 0.05) compared with e.

of this finding is important since GSH serves as a substrate in detoxifying

conjugation reactions catalysed by glutathione 5-transferase and are a substrate
of glutathione peroxidase, which protect against peroxidative membrane damage
and subsequent cell lysis. Previously, Moule and Frayssinet (1971) reported that
acrolein effectively inhibited transcriptional activity of isolated rat liver nuclei
and that RNA synthesis was inhibited in bacteria as well. These potent toxic
effects of acrolein were observed to decrease reproductive potential when Little
and Mirkes (1990) demonstrated an embryotoxic concentration of acrolein on
postimplantation rat embryos at 0.075 mM, and complete embryo lethality at
0.175 mM or higher. Until now, preimplantation and postimplantation embryo
toxicity expressed after exposure of early cleavage stage embryos to acrolein has
not been extensively evaluated. With as low as 0.025 mM acrolein (1.4 p.p.m.),
we found significant detrimental effects on mouse preimplantation embryo
development to the blastocyst stage. Acrolein at a concentration of 0.1 mM (5.6
p.p.m.) completely inhibited cleavage to either 8-cells or blastocysts. The
threshold for a significant effect of acrolein on embryo development to term was
strikingly lowered to 0.01 mM or 0.58 p.p.m.. Although there was no difference
in implantation, the percentage of viable fetuses that continued to develop to
term was only two-thirds of that obtained for untreated embryos (Table VI). It
can be concluded, that acrolein is a highly toxic compound affecting the molecular
mechanisms of cell replication. We suggest that any potential environmental
hazard exposing laboratories to elevated concentrations of aldehydes be deter-
mined and subsequently controlled. In the case of our investigation, location 7
returned to pre-acrolein exposure results, once filter systems (GenX, CT, USA)
were added and aldehyde levels dropped significantly.
Some steps can be considered to limit the adverse impact of pollutants. Firstly,
avoid locations with high pollution. The rural setting has many advantages but
is clearly questionable. In urban areas, the IVF clinic should avoid locations
adjacent to solvent (dry cleaners), fuel (parking lots, gasoline service stations),
and combustion processes. However, geographical location is not tantamount to
contamination. The vast majority of materials in the IVF laboratory are derived
from human activities in and around the laboratory (Cohen et al., 1997). Secondly,
the laboratory should be pressurized relative to adjacent spaces. Thirdly, one
should recognize that activity in and adjacent to the laboratory causes contamina-
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 Laboratory air
tion. The level can be reduced by a high rate of air exchange from the outside
rather than re-circulation. Of course, HEPA filtration and pretreatment of air with
carbon filtration and oxidation using potassium permanganate may be used to
maintain cleanliness and reduce the impact of fluctuations. Fourthly, specialized
filters (e.g. GenX) can be used to clean the laboratory air using units which can
easily be moved around. Other specialized filters can be installed inside incubators
or in-line between the gas cylinder and incubator.

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