Joss12085 PDF

DETECTION THRESHOLDS OF 10 ODOR-ACTIVE COMPOUNDS
NATURALLY OCCURRING IN FOOD USING A REPLICATED

FORCED-CHOICE ASCENDING METHOD OF LIMITS
SARA R. JAEGER
1,3
, H. NIHAL de SILVA
1
and HARRY T. LAWLESS
2
1
Mt Albert Research Centre, Auckland Mail Centre, The New Zealand Institute for Plant & Food Research Ltd, Private Bag 92 169, Auckland 1142,
New Zealand
2
Department of Food Science, Cornell University, Ithaca, NY
3
Corresponding author.
TEL: +64-9-925-7000;
FAX: +64-9-925-7001;
EMAIL: sara.jaeger@plantandfood.co.nz
Accepted for Publication November 6, 2013
doi:10.1111/joss.12085
ABSTRACT
A series of threshold measurements for aroma detection of 10 compounds natu-
rally occurring in foods were collected on 113 individuals in quadruplicate, using
a 3-alternative forced-choice ascending method of limits. Mixed-model analyses
examined differences among compounds, subgroups of individuals tested as
cohorts, order effects and several covariates such as age, gender, propylthiouracil
sensitivity and body mass index. Differences among compounds were observed, as
expected. Women had lower thresholds (higher sensitivity) than men for three of
the 10 compounds, cis-3-hexenol, isovaleric acid and 4-methyl octanoic acid. A
consistent warm-up effect was noted for the second compound tested on any
given day. The overall panel was tested in cohorts of approximately 10 panelists
each, and signicant residual variation was noted for -ionone and
isobutyraldehyde, for which bimodal threshold distributions were observed. The
latter result suggests that testing small groups of panelists (e.g. n = 10), as sug-
gested by the ASTM E-679 procedure, may not accurately capture the population
threshold variability, nor provide an accurate estimate of the mean detection
threshold. The research contributes to guidelines regarding good practice for odor
detection testing.
PRACTICAL APPLICATIONS
This research reports on odor detection thresholds for 10 compounds. The data
were obtained using a 3-alternative forced-choice ascending method of limits
which was executed in accordance with the specications in ASTM E-679. Mean
and standard deviation ratio values obtained from 113 individuals are reported for
each odorant. For researchers studying foods, beverages and personal/household
care products where these odorants are odor-active, the data have practical
signicance. Further, the research has implications for others measuring odor
thresholds. Specically, we nd that odor threshold testing is prone to warm-up
effects and we caution that threshold testing using small groups may be prone to
misestimation of population thresholds.
INTRODUCTION
A detection threshold is the minimum concentration of a
chemical that is detected by 50% of a sample group. Thresh-
olds have long served as an indicator of the biological
potency of a compound when stimulating the chemical
senses. As such, they have long been of interest to avorists
and avor chemists as an index of the lower limit to the
effective concentration range of different substances. They
also serve as a measure of the sensitivities of different indi-
viduals. People vary widely in their sensitivities to various
aroma and avor chemicals, often for reasons of genetic dif-
ferences, age, gender and many other underlying sources.
Thus thresholds serve a dual purpose, to indicate the
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Journal of Sensory Studies ISSN 0887-8250
43 Journal of Sensory Studies 29 (2014) 4355 2014 Wiley Periodicals, Inc.
stimulatory efciency of any given chemical and to serve as
a measurement of a persons sensitivity to taste and odor
materials.
Measurement of thresholds has been attempted by many
different methods since the dawn of psychophysics in the
1800s (Gescheider 1997). A variety of specic procedures
comprises a large literature with different methodologies
(Walker et al. 2003; see also Lawless and Heymann 2010,
Chapter 6, for an overview). A practical and useful proce-
dure is the forced-choice ascending method of limits known
as ASTM method E-679 (ASTM 2008a). In this procedure, a
small group of 1025 panelists sample increasing concen-
trations (e.g. eight geometrically spaced intervals) of a
specic chemical of interest. The odorants or tastants at
each level are included in a triad of samples, two of which
do not contain the test material, but only the solvent or
diluent. The task of the individual at each concentration
step is to correctly identify which of the three containers has
the taste or smell. Specically, the instructions as worded in
the ASTM document are to choose the one sample most dif-
ferent from the other two. If the person is unsure, they must
guess, so the procedure is a form of 3-alternative forced-
choice (3-AFC) task, but with triangle test instructions.
1
The ASTM procedure was developed as a way to get a
rapid cost-efcient estimate of group thresholds for various
avor compounds (Meilgaard et al. 2006). It has been
recommended in several texts on sensory evaluation
(Meilgaard et al. 2006; Lawless and Heymann 2010; Lawless
2013). Historically, its popularity and widespread applica-
tion grew out of the use of a device known as a triangle
dilution olfactometer (Dravnieks and Prokop 1975;
Dravnieks et al. 1978). The method has also been used to
get population estimates that are projectable to the larger
consumer base (Stocking et al. 2001; USEPA 2001). In
theory, the method could also be extended to examine indi-
vidual thresholds, as it is a version of the classical psycho-
physical procedure known as the method of limits.
However, because individuals participate in evaluation of
only a single concentration series, the certainty of any indi-
vidual threshold estimate is not high. This situation is
potentially remedied by replicated testing (ASTM 2008b).
In this research, detection thresholds of 10 odor-active
compounds naturally occurring in food were obtained
using a replicated forced-choice ascending method of limits
with 113 individuals. Uncovering the genetic bases of odor
perception in part motivated the work and McRae et al.
(2013) provide results for the genome-wide association tests
performed on the present data. It is beyond the scope of this
paper to cover the role odor thresholds play in the search for
genetic bases of odor perception and the extent to which
such bases have been uncovered (interested readers are
referred to Newcomb et al. 2010 for an introduction and
Newcomb et al. 2012 for a recent review). Of relevance here
we note that detection thresholds are known links to genetic
factors (Bufe et al. 2005; Keller et al. 2007), and genetic
inuences have been demonstrated for compounds such as
androstenone, for which some people are anosmic (Wysocki
and Beauchamp 1988). In this paper, the focus is directed to
the opportunity afforded by this large data collection effort
to search for correlates of odor detection performance,
including warm-up and practice effects (Engen 1960; Rabin
and Cain 1986; Miyazawa et al. 2009; Lawless 2010), vari-
ance among subgroups of individuals tested at different
times and covariates for panelist and environment charac-
teristics. Such knowledge is of interest to sensory researchers
and can contribute to best practice in threshold testing.
Sensitivity to the bitter taste of propylthiouracil (PROP)
and its correlation with higher fungiform papillae counts
(among PROP-sensitive groups) has given rise to the theory
of supertasting (Hayes and Keast 2011). The so-called
supertasters have been shown to be sensitive to a wide
variety of chemical, thermal and tactile stimuli. This classi-
cation, along with its correlate in thermal tasting, has been
shown to affect the perceptions of retronasal aromas and
avors (Green and George 2004; Pickering et al. 2006).
Therefore, PROP tasting status and fungiform papillae
counts were also measured among the participants in this
study. Body mass index (BMI) has been shown to correlate
(negatively) with olfactory sensitivity (Stafford and Welbeck
2011) and was also shown to correlate with PROP taster
status in one study of Italian females (Tepper et al. 2008),
and so height and weight information were also collected
from participants in order to calculate BMI. Information
about age and gender, which often, but not always, are iden-
tied as correlates of odor detection performance (Koelega
and Koster 1974; Rabin and Cain 1986; Cain and Gent 1991;
Cometto-Muiz and Abraham 2008, 2009; Doty and
Cameron 2009) were also obtained. Environmental corre-
lates included daily temperature and relative humidity at
the time of data collection. There is evidence, albeit incon-
sistent, that perception of odor intensity and quality is
inuenced by these factors (e.g. Fang et al. 1998; Sakawi
et al. 2011a,b; Reinikainen et al. 1992), suggesting that
thresholds may also be subject to meteorological conditions.
In summary, taking a data-driven approach, our aim was
to explore procedural, environmental and panelist factors
that may systematically affect odor detection threshold esti-
mates at the individual and group levels, and to estimate the
1
The ASTM methods instructions, an oddity task, are not con-
sistent with a 3-AFC procedure, but are rather a form of the tri-
angle test. However, the stimulus presentation in the ASTM
follows the typical 3-AFC procedure in that only one item has the
target chemical. A true triangle procedure, in contrast, would
present triads of two targets and one diluent/blank on half the
trials.
DETECTION THRESHOLDS IN 10 ODOR-ACTIVE FOOD COMPOUNDS S.R. JAEGER, H.N. DE SILVA AND H.T. LAWLESS
magnitude of unaccounted variability. As opposed to focus-
ing on the threshold values per se, we focused on detecting
relationships that were robust across multiple odorants,
seeking a degree of assurance that these ndings would
extend to threshold measurements for other compounds.
MATERIALS AND METHODS
General Design of the Study
Participants attended sessions at a sensory facility for 21
consecutive working days. Each session lasted 1.52 h.
Testing took place across 300 sessions in the period Febru-
ary 2009 through May 2010. Participants attended research
sessions in groups of 1012 people, called cohorts. Data
from 11 cohorts were analyzed here. Each participant com-
pleted four replicate threshold estimates for each odorant,
completed on different days across the 21-day duration of
the study for that cohort. Odor detection thresholds were
obtained for 10 compounds, listed in Table 1. The psycho-
physical methodology used to obtain the odor detection
threshold estimates was the ascending 3-AFC method,
which is described in ASTM standard E-679 (ASTM 2008a).
Eight geometrically spaced concentrations were used for
each compound. Mean thresholds and variability estimates
were obtained at the individual, cohort and group levels. A
number of potential panelist and environmental correlates
of odor sensitivity were collected.
Participants
A sample of 113 consumers from a Caucasian population in
the area of Auckland, New Zealand, was recruited, including
59 females aged 20 to 50 (mean 36) and 54 males aged 19 to
50 (mean 30). Pregnant women and participants that self-
reported existing medical conditions that might impair
their ability to smell (such as hay-fever or chronic sinusitis)
were excluded. Participants were recruited from the greater
Auckland area by a professional recruitment company and
were selected from an existing database of people who were
interested in market research projects. Panelists were
grouped into 10 cohorts. Two cohorts had 12 and 11 panel-
ists each and all others comprised 10, for a total of 113
panelists. Some participants were withdrawn from the
cohorts because of poor sensory acuity or personality issues
(e.g. inability to comply with instructions, disruptive and
inappropriate verbal behavior) and replacement partici-
pants were recruited. Institutional review and ethical
approval was obtained from the Northern X Regional Ethics
Committee (NTX/08/11/111) and written informed consent
was obtained from all participants.
Odor Stimuli
Table 1 lists the 10 stimuli, how they were sourced and the
purity used. The stimuli were selected because of their
natural presence in foods (and as an aside we note they are
all commercially available as food grade). It is beyond the
scope of this work to provide a detailed listing of all foods/
beverages where the compounds are found, so instead we
provided a single reference for each compound as an exem-
plar: Wang et al. (2011), Moio et al. (2000), Young and
Braggins (1998), Caporale et al. (2004), Nielsen and Poll
(2004), Fukami et al. (2002), Thierry et al. (2004),
Gassenmeier et al. (2008), Ferreira et al. (2000) and Larsen
et al. (1991).
Concentrations for the odorant compounds were estab-
lished initially from existing literature and then rened
using bench-top piloting with volunteers from Plant and
Food Research staff. During bench-top testing, the 3-AFC
procedure (outlined below) was used on an extended range
of concentrations aiming to encompass all levels of sensitiv-
ity. For some later cohorts, the range and step size was
adjusted slightly for several odorants to better bracket the
threshold range.
TABLE 1. DESCRIPTION OF ODOR COMPOUNDS
Odorant
Compound
number Source Purity CAS number
1,8-cineole F01 Aldrich #C80601 99% 470-82-6
2-heptanone F02 Sigma #537683 99% 110-43-0
Hircinoic acid (4-methyl octanoic acid) F03 Sigma #W357502 98%, FG 54947-74-9
Cis-3-hexen-1-ol F04 Aldrich #H12900 98% 928-96-1
Dipropyl disulphide F05 Aldrich #149225 98% 629-19-6
Isobutyraldehyde F06 Aldrich #418110 99.5% 78-84-2
Isovaleric acid F07 Sigma #59850 98.5% 503-74-2
Vanillin F08 Aldrich #V1104 99% 121-33-5
-damascenone F09 Sigma #W342017 1.11.3 wt. % in 190 proof ethanol 23696-85-7
-ionone F10 Aldrich #I12603 96% 79-77-6
FG, food grade.
S.R. JAEGER, H.N. DE SILVA AND H.T. LAWLESS DETECTION THRESHOLDS IN 10 ODOR-ACTIVE FOOD COMPOUNDS
Each compound had different high (set 8) and low (set 1)
concentrations, so every compound had a specic dilution
protocol. All solutions were freshly prepared on the day they
were presented to the participants. Solutions were diluted in
water produced by a Microlene (activated carbon) lter
(Davey Water Products Pty Ltd, Scoresby, Australia). Dilu-
tion volumes were measured by weighing specic volumes
of water diluents or odorant solution aliquots on a balance.
Each dilution protocol began with a small volume of a pure
odorant compound being added to water, followed by suc-
cessive steps of serial dilution. Dilution factors varied from
2.25-fold to sixfold. Two odorants, dipropyl disulde and
-ionone, were difcult to dissolve in water and so were rst
dissolved in a small volume of ethanol. This pure/dissolved
compound was then pipetted into the initial bottle contain-
ing water. A specied volume of this dilution was then
weighed into another bottle of Microlene ltered water, and
so on until the eight dilution steps were achieved. When
ethanol was required to dissolve the compound, the blank
solutions also contained an equivalent amount of ethanol to
the respective concentration set (i.e. the blanks used in set
one contained the same amount of ethanol as the set one
target). Using an automatic dispenser (Eppendorf Easypet;
Eppendorf AG, Hamburg, Germany), 10 mL aliquots of
each compound dilution were placed into 210 mL ISO
wine tasting glasses and immediately covered with watch
glasses to retain volatile compounds inside the glass. To
allow equilibration of the headspace, odorant solutions
were poured into glasses 1 h prior to the start of a threshold
testing session. Identical glasses containing only water,
and also covered with watch glasses, were used as blanks
during comparative testing. A member of staff who had not
been involved in solution preparation or pouring then
smelled the three highest concentrations to ensure the
concentrations were correct, discernable and increased
appropriately.
Procedures for Data Collection
Test Order. Each test day was structured around two
series of 3-AFC measurements, separated by a 20 min
break. In each of these, one odorant was presented in an
ascending series of eight concentrations to provide one
measurement of best estimate threshold or BET, followed
by a second, different odorant. These two series of tests are
referred to as T1 and T2. There were a few days (4%)
where a third (T3) or a fourth test (T4) was included to
enable participants who had been absent on previous days
to obtain four replicates for each odorant. On any given
cohort/day, the T1 and T2 tests were undertaken with dif-
ferent odorants so that no odorant was repeated within a
day. The exceptions were T3 and T4 data, which were col-
lected using T1 or T2 odorants.
The experimental design ensured that the presentation
order of the 10 odorants was near balanced with T1 and T2
test levels. Thus different combinations of odorants were
presented on the 20 different days of each cohort.
Test Procedure. The ASTM 3-AFC procedure involves
the simultaneous presentation of three samples where two
are the same and one is different (i.e. contains the target).
The participant is asked to identify the sample that is differ-
ent. Eight concentration levels were presented (one per set)
in an ascending concentration order.
Threshold testing was conducted in individual booths.
Green lighting was used to prevent participant identi-
cation of target samples by any changes in color in com-
parison with blank samples. In booths, the temperature
was maintained at 20C, and a positive pressure airow
system was used to prevent odor build-up. Humitidy in
the sensory booth area was atmospheric and not con-
trolled. Data were collected at computer workstations
using Compusense software (Compusense Inc., Guelph,
Ontario, Canada). Participants were asked to sniff each
three-glass sample set in the order in which glasses
were presented, left-front to right-back, and decide which
one was the different sample. Resmelling was permitted;
participants were asked to ensure they resmelled the entire
set in the order presented. A 75-s break was enforced
between each of the eight sets to mitigate olfactory fatigue.
A policy of no talking and no reading was imposed
to promote concentration during testing. All sessions
took place in the late morning to early afternoon, 10:00
a.m1:00 p.m.
Of the 21 test sessions, the rst was used to familiarize
panelists with the 3-AFC method and required them to
complete one series of eight tests using linalool (CAS
78-70-6) (data not shown). Participant covariate measures
were also obtained on this day.
Covariate Measures. In addition to data of the 3-AFC
trial responses, two sets of covariate measurements were
recorded: panelist-specic (day 1 only) and environmental
(daily).
Height and weight measures, for BMI calculation, were
taken from participants wearing normal indoor clothing
but no shoes, using a common set of bathroom scales. Mea-
sures were taken by an experimenter in a separate room to
give privacy for participants.
PROP sensitivity measurements were obtained by asking
participants to hold a strip of lter paper impregnated with
an aqueous solution of 17.63 mM PROP on their tongues
for 30 s. Participants then rated the intensity of any bitter
taste they experienced on a generalized labeled magnitude
scale (Bartoshuk et al. 2004). Each participants generalized
labeled magnitude scale rating was recorded as a proportion
of the total line length.
Fungiform papillae density measures were obtained by
placing a small amount of blue dye on the left or right ante-
rior surface of each participants tongue and then position-
ing a white reinforcing ring on top. The reinforcing ring
(Esselte catalog number 80196EP) had an outer diameter of
14 mm and an inner diameter of 6 mm. This ring delineated
a standardized space in which to determine the density of
fungiform papillae for each person. Each participants
papillae count area was photographed. Cropped versions of
the resulting images were presented in a single montage to
three coders who counted large, unstained papillae within
the area. In cases where the coders counts differed to a large
extent (>25%), coders examined images together to deter-
mine which papillae were counted. Else, the average value
was used.
Temperature and relative humidity information was
obtained using New Zealands National Climate Database
(http://clio.niwa.co.nz/). Throughout the study, all infor-
mation were obtained from the same climate station,
located approx. 5 km from the sensory test facility. Using
hourly data spanning the time interval when tests took
place, daily averages for temperature and relative humidity
were calculated.
Statistical Analyses
Mean Threshold and Variance Estimates. Thresholds
were estimated using the recommended ASTM procedure
that calculates the threshold from the last concentration
correctly identied, when all subsequent concentrations are
correctly identied, known as the last reversal rule. The
actual threshold is the square root (i.e. geometric mean) of
the product of that concentration and the next lower step
(i.e. the last incorrect response). If the panelist identies all
targets correctly, the threshold is estimated as the rst con-
centration level, divided by the square root of the step size
multiplier. If the highest concentration in the series is incor-
rect, the threshold is estimated as the highest concentration
times the square root of the step size multiplier. These last
two rules are in effect estimating the geometric mean
between the rst (or last) step and the next one, had the
concentration series been extended down or up by one
more step.
Because of the geometric spacing (equal log steps) for the
concentration variable, a standard deviation ratio (SDR)
was employed as a variance measure, in order to transform
log thresholds back into a more meaningful concentration
measure. Specically, we used the SDR of the logarithm to
base 10 of estimated thresholds, hence SDR
SD
=10
log
where
SD
log
is the standard deviation estimated on the log scale.
The SDR provides a convenient way of calculating limits of
1 SD about the back-transformed mean value. We simply
multiply and divide the back-transformed mean by the SDR
to give the lower and upper limits of 1 SD about the mean.
That is, U(=Mean + SD) / Mean = SDR hence U = Mean
SDR and L(=Mean SD) / Mean = 1 / SDR, hence L = Mean
/ SDR. Back-transforming refers to converting from log
concentration back to actual concentration units (ppb). As
SD is always 0, SDR will be 1.
For panelist-to-panelist variation, we used the intraclass
correlation, which is generally dened as the ratio of
Between to (Between + Within) variances. Between
is in this case, the sum of panelist and panelist-within-
cohort variation.
Accounting for the Multilevel Data Structure.
Although the threshold estimate was calculated for each of
the panelists replicates, most independent variables were
measured or observed at different levels, which made the
data structure multilevel or hierarchical. For example, the
environmental covariates such as humidity and temperature
that were measured at the test day level were common to all
panelists of the cohort tested on that day. Similarly, the
covariates measured at the panelist level (e.g. age) on day 1
were common to all the threshold responses of any given
panelist. The testing of participants in groups of 10 indi-
viduals (cohorts) meant that the overall population com-
prised multiple subgroups. The hierarchical levels were
analyzed by aggregating to the higher level at which predic-
tors were measured. Therefore, different levels of data
aggregation were employed in three separate linear models,
to avoid the nonindependence problem of having such
unchanging covariates and changing xed effects in the
same model.
Mixed-model analyses of variance were applied to the
data in several linear modeling forms. The rst level of
aggregation was the panelist-by-day analysis:
y G I T x x
ijkl o j i j k ijkl ijkl ijkl
= + + + + + + +
( )

1 2

(1)
where y
ijkl
is the l-th replicate in k-th test position of the i-th
panelist in the j-th cohort; i = 1 to 12, j = 1 to 11, k = 1 or 2
and l = 1 to 4. The effect of test sequence position, T
k
, is
assumed to be xed, whereas cohort G
j
and the panelist-
within-cohort I
i(j)
are specied as random effects in the
model, (G
j
, I
i(j)
) N (0,
2
), i.e. with mean zero and corre-
sponding variance
2
. The
1
,
2
, . . . values are regression
coefcients corresponding to the covariates in the model.
The model as specied is univariate, i.e. analysis was done
with one odorant at a time.
The second level of aggregation was the cohort-by-day
analysis. The predictors of interest in this analysis were the
Day properties (day 221 and weekday [Monday
Friday]). For each cohort, individual thresholds were sum-
marized by sample cohort means. As the aggregate data
were at a manageable level, we specied a multivariate
mixed model, i.e. tting all odorant compounds at the same
time, the variable O referring to odorant:
y O G OG T x x
jklm o m j mj k jkl jkl jklm
= + + + + + + + +
1 2

(2)
where y
jklm
= sample mean log
10
(threshold) of the l-th repli-
cate of j-th cohort in k-th test position and the response
specied is for the m-th odorant.
The nal level of aggregation was for the panelist-specic
characteristics such as age, gender, BMI, etc. For each panel-
ist, thresholds were aggregated and summarized by panelist
means. The following univariate mixed model was specied
for the analysis;
y I x x
i i i i i
= + + + + +
0 1 1 2 2
(3)
where y
i
= sample mean log
10
(threshold) of the i-th panelist.
Panelists are generally considered to be a random, rather
than xed effect (Lawless 1998; Lea et al. 1998) and were
treated as such, while all others factors including odorants
and test order were specied as xed effects, thus requiring
a mixed model (Smith et al. 2003). All statistical analyses
were carried out using PROC Mixed in SAS software (SAS
Institute Inc. 2008).
RESULTS
Using a data-driven approach, the aim of this work was to
explore procedural, environmental and panelist factors that
may systematically affect odor detection threshold estimates
at the individual and group levels and to estimate the mag-
nitude of unaccounted variability. The following four sub-
sections presents the results as they pertain to each of these
sets of main factors.
Mean Thresholds and Variance Estimates
for Odorants
Analysis at the rst level of aggregation (Eq. 1) was focused
on odorant effects. Results are presented in the upper half of
Table 2 which shows the mean threshold values for the 10
compounds and the SDR. Not surprisingly, compound
mean thresholds differed, as expected from the literature
(ASTM 1978; Punter 1983; Amoore and Hautala 1993). The
back-transformed means estimates range from 0.0093 to
1800 ppb. We are not aware of previous reports of SDR
values, but note that these also differ (1.53.0), as would be
expected. Standard deviations for odor thresholds are also
reported to differ (Brown et al. 1968; Punter 1983). For
completeness, we note that the actual mean threshold values
and comparison of these against extant sources was not a
focus; neither were the density histogram for thresholds. For
the latter, we refer interested readers to McRae et al. (2013).
The lower part of Table 2 shows the variance components
(as percents) for the random effects of cohort, panelist-
within-cohort and residual variance. Using the sum of
TABLE 2. FITTED MEANS AND PARAMETER ESTIMATES OF THE MIXED MODEL FITTED TO INDIVIDUAL PANELISTS LOG10 (THRESHOLD) DATA,
TOGETHER WITH P-VALUES OF TESTS OF MODEL FIXED EFFECTS (FACTORS AND COVARIATES)
Factor / covariate
Odorant
F01 F02 F03 F04 F05 F06 F07 F08 F09 F10
P-value for the factor test 0.031 0.013 0.032 0.001 0.122 0.005 0.102 0.001 0.418 0.042
Back-transformed xed effect means (ppb)
Overall tted mean 3.4 58 990 94 2.4 6.0 1,800 650 0.0093 11.0
Standard deviation ratio 3.5 1.6 1.6 1.8 1.5 3.0 1.7 1.6 2.7 2.1
Test First 4.6 71 1,200 130 2.8 8.9 2,000 890 0.0120 12.0
Nonrst 2.6 47 820 68 2.0 4.0 1,600 480 0.0074 9.2
Multiplier (rst/nonrst) 1.8* 1.5* 1.5* 1.9* 1.4* 2.2* 1.3 1.9* 1.6(*) 1.3
Multiplier (rst/second) 1.8* 1.5* 1.5* 1.7* 1.4* 2.5* 1.3 1.9* 1.6* 1.3
Random effect Variance component estimates as percentages of total variance (log scale)
Cohort 0 2 4 3 10* 3 2 2 2 0
Panelist (cohort) 34* 16* 20* 30* 9* 52* 8* 5 37* 72*
Residual 66* 82* 76* 67* 81* 45* 90* 93* 61* 28*
Intraclass correlation 0.34 0.18 0.24 0.33 0.19 0.55 0.10 0.07 0.39 0.72
Note: Note that means presented (in ppb) are back-transformed values from the log scale. Refer to Table 1 for odorant identities.
* statistically signicant at 5%.
(*) statistically signicant at 10%.
Estimates are rounded off to two signicant digits.
All covariates in the model (panelist and environmental) were consistently nonsignicant across all the compounds; hence P-values are not
reported.
Standard deviation ratio divide / multiply the mean by standard deviation ratio to give lower and upper bounds corresponding to 1 standard
deviation about mean.
Based on analysis of subset of data excluding T3 and T4 tests.
variance for cohort and panelist-within-cohort as an overall
estimate of the panelist-to-panelist variance, we calculate
intraclass correlations as the ratio of the between-panelist to
total variance. Reecting differences in the variance compo-
nent estimates these values vary considerably (0.070.72).
For vanillin (F08), the residual variance component
estimate is 93%. Conversely, two of the odorants have
high intraclass correlations (-ionone [F10] and isobuty-
raldehyde [F06]), pointing to large systematic panelist
variation (52% and 72%). Figure 1 shows the density histo-
gram for thresholds for these two odorants and bimodal
patterns are evident, perhaps to be expected in light of spe-
cic anosmias having been previously reported (Amoore
et al. 1976; Plotto et al. 2006).
Procedural Inuences on
Threshold Estimates
To further understanding regarding procedural inuences
on threshold estimates, the data were next explored for
effects linked to warm-up and practice effects, and other
factors linked to procedural aspects of data collection.
Analysis at the rst level of aggregation (Eq. 1) revealed a
signicant test order effect (Test in the model). The top
and middle parts of Table 2 presents P-values for Test,
which was signicant for seven of the 10 tested odorants.
The middle part of Table 2 shows that the rst threshold
estimate was uniformly lower than subsequent runs on a
given day. The term nonrst is used because on a few
occasions, panelists performed make-up sessions on the
same day with a third or fourth test series because of missed
sessions. A stricter analysis of rst versus second test series
showed the same effect. Analysis at the second level of
aggregation (Eq. 2) which was performed at the cohort level
of aggregation and included all 10 odorants in a single
analysis also revealed the test order effect, and for cohort
means it was estimated that the rst test mean on average
was 25% higher (Table 3).
Signicant effects relating to effect of day of threshold
testing are reported in Table 3 (factor Cohort-day).
However, there was no consistent trend across the 20 days of
testing for cohort means or within-cohort variance to
decrease, as one might expect if there was a practice effect
(Fig. 2). Instead, as seen from Fig. 2, there was random vari-
ance in variation in cohort mean and within-cohort-
Mean detection threshold
A
E
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1
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3
0
.
4
0
.
5
0
.
6
0.01 0.1 1 10 100 1000 10000
Mean detection threshold
E
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p
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.1 1 10 100 1000
FIG. 1. ODOR DETECTION THRESHOLD DISTRIBUTIONS FOR -IONONE (A) AND ISOBUTYRALDEHYDE (B) DISTRIBUTION OF PANELLIST MEAN
THRESHOLDS (LOG10) AS SHOWN BY A SMOOTHED KERNEL DENSITY PLOT OVERLAID ON A HISTOGRAM
TABLE 3. TEST OF FIXED EFFECTS ON COHORT MEAN THRESHOLDS
AND WITHIN-COHORT VARIANCES
Fixed effects
P-value
Cohort mean
Within-cohort
variance
Odorant 0.000 0.000
Cohort 0.000 0.013
Odorant Cohort 0.000 0.001
Test (T1T4) 0.005 0.220
Cohort day (d2d21) 0.034 0.018
Weekday (MondayFriday) 0.443 0.309
Relative humidity 0.038 0.448
Temperature 0.803 0.923
variance estimates. Finally, it was found that the weekday on
which threshold testing took place did not systematically
inuence the cohort mean or variance estimates.
Cohort Inuences on Threshold Estimates
In the analyses performed at the second level of aggregation
(Eq. 2: cohort-by-day), an important effect was noted in a
signicant cohort-by-odorant interaction for both the
cohort mean and within-cohort variance. This is consistent
with a difference in the rank ordering of mean odorant
thresholds by different cohorts. This result is intuitive, as
cohorts were subsamples of only 10 or so individuals, and
the kind of bimodality observed for -ionone indicates that
such a small sample will not give a very stable estimate of
the group threshold. Table 4 shows the mean within-cohort-
variance estimates as SDR, and again the high variability of
-ionone (F10) and isobutyraldehyde (F06) threshold esti-
mates relative to the other eight odorants is evident.
Covariate Inuences on Threshold Estimates
The third level of aggregation enabled estimation for the
panelist-specic characteristics such as age, gender, BMI,
etc. As seen in the top part of Table 5, the gender effect was
signicant (P < .05) for 4-methyl octanoic acid (F03), cis-3-
hexenol (F04) and isovaleric acid (F07). A gender difference
in threshold estimates was observed with women having
lower thresholds than men for all odorants except -ionone
(men lower but not signicantly) and 2-heptanone (equal
mean thresholds). No effect was observed for any of the
other panelist covariates, including age, height, BMI, PROP
rating and fungiform papillae count (data not presented).
The lack of an age effect was expected, as the upper limit
was set at 50 years for participants.
The environmental correlates were investigated when
tting Eq. (2) (Table 3) to reveal no effect for temperature,
but a signicant effect for relative humidity, with detection
thresholds lower at higher humidity (regression coef-
cient = 0.005).
DISCUSSION
Implications for Odor Threshold
Testing Practice
We discuss in this section the results with a view to implica-
tions for threshold testing practice. We found that odor
detection thresholds are susceptible to a warm-up effect on
a given day, and this aligns with previous reports of perfor-
mance in difference testing increasing when participants are
warmed-up (OMahony and Goldstein 1986; OMahony
et al. 1988; Thieme and OMahony 1990). Warm-up refers
to a short-term effect observed at the start of a testing
session whereby panelist performance rapidly increases. It is
distinct from a practice effect which is an improvement in
2 4 6 8 10 12 14 16 18 20
0.7
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
1.6
Cohort mean
Within-cohort variance
S
c
a
l
e
d

e
s
t
i
m
a
t
e
Cohort Day
FIG. 2. PLOT OF THE SCALED ESTIMATE OF COHORT MEAN AND
WITHIN COHORT VARIANCE ACROSS DAYS. THE DATA ARE SCALED
ACROSS ALL 10 ODORANTS
Note: Cohort Day recorded as 120, correspond to days 221
TABLE 4. BACK-TRANSFORMED FITTED VALUE OF WITHIN-COHORT
MEAN VARIANCES, PRESENTED AS STANDARD DEVIATION RATIOS
Odorant
F01 F02 F03 F04 F05 F06 F07 F08 F09 F10
6.2 1.7 1.8 2.3 1.5 10.2 1.7 1.6 4.6 13.4
Note: Refer to Table 1 for odorant identities.
TABLE 5. ANALYSIS OF GENDER EFFECTS ON
MEAN THRESHOLDS BACK-TRANSFORMED
FITTED VALUE OF WITHIN-COHORT MEAN
VARIANCES, PRESENTED AS STANDARD
DEVIATION RATIOS
P-values by odorant
F01 F02 F03 F04 F05 F06 F07 F08 F09 F10
0.196 0.440 0.020 0.010 0.223 0.256 0.036 0.665 0.121 0.728
Back-transformed xed effect means (ppb)
Female 3.0 58 770 71 2.0 5.3 1,500 630 0.0075 13.0
Male 4.3 58 1,300 140 2.8 7.1 2,200 670 0.0110 8.3
Note: Refer to Table 1 for odorant identities.
performance that generally takes a long time to achieve and
is relatively permanent. OMahony et al. have reported how
warm-up can be purposefully achieved by a series of rapid
tasting of alternate samples, but note that the effect has not
been systematically studied and important questions remain
such as how many samples are needed to achieve the effect.
Further, Thieme and OMahony (1990) note that warm-up
protocols may not always be appropriate, given that they
make participants more sensitive than they would be under
normal conditions. Overall, warm-up effects occur haphaz-
ardly at the start of experimental sessions and cannot be
avoided. However, they can be managed by deliberate use of
warm-up protocols, which may ensure that differences in
panelist performance are due to sensitivity rather than
stimulus confusion. We advocate that researchers detail any
pretesting protocols used prior to threshold testing, so that
informed comparison of reported values is possible.
One important nding of this study suggests that testing
thresholds using small groups may be prone to misestima-
tion of population thresholds. Our analysis by different
cohorts showed that the within- and between-cohort vari-
ance can be quite high for some odors, such as -ionone
and isobutyraldehyde. That is, the luck of the draw for any
randomly selected group of 10 or so people may give a dif-
ferent mean threshold value from the next 10. This is
important as the published examples of threshold data in
the appendices to ASTM E-679 (ASTM 2008a) show tables
for groups of only ve (replicated), nine and 16 panelists,
and Meilgaard et al.s (2006) example shows 25 panelists. In
any study where genetic factors or other sources of
interindividual variability are likely to be in play, a larger
sample of panelists and close inspection of the variance pat-
terns is warranted. In the data of Stocking et al. (2001), the
group threshold is near the middle of the tested concentra-
tion series. However, 10 of the 57 panelists were able to cor-
rectly identify the target sample at all concentrations,
indicating a subgroup of highly sensitive individuals to the
odor of the drinking water pollutant, methyl tertiary butyl
ether. Although the group average threshold for methyl ter-
tiary butyl ether is about 15 ppb, fully one-sixth of their
panel had individual BETs in the range of 12 ppb. Had
Stocking et al. tested fewer panelists, as done in the ASTM
E-679 examples, this important nding might potentially be
missed.
The absence of signicant effects of panelist characteris-
tics suggested that there are no obvious attributes other
than the general inclusion criteria needed to screen partici-
pants when recruiting for threshold panels. Effects of panel-
ists height, BMI, PROP rating and fungi form papillae
count were not established. Given the exclusion of partici-
pants aged 50 or above, it was not surprising that age effects
were nonsignicant. With regard to gender, our ndings
align with Doty and Cameron (2009) who concluded that
while women on average are more sensitive than men, the
differences are not always large, and they do not exist for all
compounds. Doty and Cameron (2009) note that the fre-
quency of specic anosmics within male and female groups
can be a contributing factor to the discrepancies observed.
They also point to odor exposure as a factor in causing
variation insensitivity and one which can may be of signi-
cance in men compared with women (Dalton et al. 2002;
Boulkroune et al. 2007). Overall, in odor threshold testing,
we suggest that a gender-balanced sample is warranted.
We did not nd that atmospheric temperature inuenced
threshold estimates. Because we collected the data under
regulated temperature conditions, this is not surprising. We
did nd a signicant effect of relative humidity, but caution
that this result needs to be replicated. We are not aware of
others who have studied whether humidity inuences odor
detection thresholds, and note the limitation of having
obtained our data from a weather station located some dis-
tance from the sensory testing facility. In the future, we
would obtain on-site measures. Nonetheless, our data
suggest that detection threshold decreased with higher
humidity, and this aligns with work on perceived
odor intensity, which has also been found to increase as
humidity rises (Sakawi et al. 2011a,b). Acknowledging that
atmospheric humidity cannot be readily controlled by
experimenters, the implication of this result, if replicated,
could be to determine how to control for its inuence after
data collection.
The preceding paragraphs have discussed our ndings
and pointed to implications for odor threshold testing prac-
tice. In general, we note that data generated by standard
procedures in human odor threshold detection are noisy
because of the presence of many factors, either unknown or
that cannot be controlled, which affect the human response.
Added to this are limitations on the number of replicates
that can be handled, and consequently the statistical tests
may lack power to detect signicant effects unless they are
sufciently large. In spite of these limitations, this study,
which followed ASTM E-679, was able to establish with
some consistency the effects of gender and test position
on odor threshold, as well as pick up the bimodal distribu-
tions for -ionone and isobutyraldehyde previously
published.
Limitations and Suggestions for
Future Research
Our sample size of 113 participants was larger than is
typical in odor detection threshold testing. While we repli-
cated the observation of individual differences in thresholds
for compounds with known specic anosmias (i.e. -ionone
and isobutyraldehyde), we note that one other odorant in
our battery of compounds, isovaleric acid, has a known spe-
cic anosmia (Amoore et al. 1968) and marked individual
differences have been observed to similar small branched-
chain fatty acids (Brennand et al. 1989). The simplest expla-
nation for a lack of variance pattern similar to -ionone is
that the incidence of anosmia to isovaleric acid is quite low,
possibly 3% (Amoore et al. 1968; Wysocki and Labows
1984). Therefore, it would require a larger panel than 100 or
so participants in order to get a clear picture of the variance
pattern for such a low-incidence anosmia.
The data collection effort was anchored in the ASTM
rapid method, E-679, and accordingly we used the rule of
last reversal to estimate the individual BETs. However,
issues with this approach which may lead to biased thresh-
old estimates have recently been highlighted and alternative
methods proposed. In light of inadequate accounting for
the possibility of a correct guess because of chance in the
E-679 last reversal method, Lawless (2010) suggested
using the total proportions correct for each concentration
level, and then interpolating the group threshold by using
the well-known correction for guessing or Abbotts formula,
also examined in the USEPA report (USEPA 2001). Peng
et al. (2012) studied different analyses of E-679 data based
on various stopping rules. That is, one might consider dif-
ferent levels in the nal run of correct choices (e.g. rst
correct choice, second or third) in order to account in part
for the chance guessing probability. Peng et al. (2013) have
also suggested that psychometric functions can be tted to
individual level E-679 data by assuming a xed slope and
estimating only the intercept. Depending on the level of
analysis performed, the work by these authors allows for
future analysis of the current data to explore effects of
threshold estimation method. This would enable a compari-
son of results across threshold estimation methods with
regard to procedural, environmental and panelist factors
which may systematically affect odor detection threshold
estimates. As an aside, we note that different ways of analyz-
ing threshold data exist, including an approach based on
survival analysis (Hough et al. 2013).
Variance patterns such as those we observed for -ionone
and isobutyraldehyde suggest opportunities for examina-
tion of suprathreshold responses to these odorants.
Although Frijters (1978) stated that threshold measures do
not necessarily predict responses above threshold, supra-
threshold responses can in some cases show even clearer
patterns of individual differences, i.e. better group separa-
tion, than detection measures (Lawless 1980; Lawless et al.
1994). This is of interest in regard to the search for genetic
factors to odor sensitivity that in part motivated this
research. Tentatively, genetic factors explaining differences
in thresholds may also explain suprathreshold responses to
single odorants and in turn odorant mixtures and actual
product aromas. This may contribute understanding to
preference heterogeneity.
The 10 compounds studied in this research were
food active. Thresholds were determined for the pure
compounds, but foods and beverages are complex and com-
pounds are found in concert with one another. Interaction
among compounds may change thresholds (e.g., Yonder
et al. 2012) and warrants further research to further the
practical relevance for threshold data in food sensory
research.
ACKNOWLEDGMENTS
Thanks are due to the following collaborators at The New
Zealand Institute for Plant & Food Research Limited for
their input and assistance with planning, collecting and pro-
cessing data: B. Pineau, J. F. McRae, K. R. Atkinson, L. G.
Axten, C. M. Roigard, M. K. Beresford, M. Peng, S. L.
Chheang, J. Gamble, F. R. Harker, Y. Jin, R. D. Newcomb, A.
G. Paisley, S. Rouse and M. W. Wohlers. Mei Peng and
Duncan Hedderley are thanked for helpful advice on an
earlier version. Financial support from the New Zealand
Ministry of Business, Innovation and Employment is
acknowledged.
Author Contributions
SRJ conceived and planned the study. NDS analyzed the
data. All authors wrote the paper.
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