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Comment on Influence of Serving Temperature and Wine Type on Perception
of Ethyl Acetate and 4-Ethyl Phenol in Wine
Sierra Rayne a
a
Ecologica Research, Penticton, British Columbia, Canada
Online Publication Date: 01 July 2009

To cite this Article Rayne, Sierra(2009)'Comment on Influence of Serving Temperature and Wine Type on Perception of Ethyl Acetate

and 4-Ethyl Phenol in Wine',Journal of Wine Research,20:2,159 — 166
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URL: http://dx.doi.org/10.1080/09571260903319945

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Journal of Wine Research, 2009, Vol. 20, No. 2,
pp. 159–166

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Comment on Influence of Serving Temperature and
Wine Type on Perception of Ethyl Acetate and 4-Ethyl
Phenol in Wine

SIERRA RAYNE

In their article, Cliff and King (Journal of Wine Research, 2009, Volume 20, Number 1,
pp. 45– 52) present a study examining the influence of serving temperature and wine
type on perception of ethyl acetate and 4-ethylphenol in a blended red wine sample
of unspecified composition, a blended white wine sample of unspecified composition,
a Gewurztraminer white wine, and a Chardonnay white wine. This Comment will
address a number of scientific concerns in the manuscript that require discussion.
Throughout their manuscript, Cliff and King (2009) have not discussed any relevant
studies regarding the reported levels of ethyl acetate or 4-ethylphenol in commercial
wines. Such integration of these well established fields of research is required to both contextualize and integrate Cliff and King’s (2009) findings into the broader scientific literature. With respect to 4-ethylphenol, it is important to note that published peer
reviewed surveys of this compound in commercial wines have reported average concentrations of 3 mg L21 (range: 0 to 28 mg L21) and 440 mg L21 (range: 1 to 6,000 mg L21) in
white and red wines, respectively, from France (Chatonnet et al., 1992), an average concentration of 795 mg L21 (range: 2 to 2,700 mg L21) in red wines from Australia (Pollnitz
et al., 2000a,b), and average/median concentrations of 56/29 mg L21 (range: ,0.5 to
544 mg L21; .99.5% of all samples had concentrations ,500 mg L21 and .97% of
all samples had concentrations ,100 mg L21), respectively, in red wines from the
Okanagan Valley of British Columbia, Canada (Rayne and Eggers, 2007, 2008). In
addition, the sensory impacts of Brettanomyces derived organoleptic defaults, of which
4-ethylphenol is one contributing compound, have been the subject of much recent
attention since the early work of Chatonnet (1993) that is the only citation provided
by Cliff and King (2009) with regard to 4-ethylphenol and/or Brettanomyces. These
post-1993 works warranted integration (see recent reviews by Snowdon et al. (2006),
Renouf et al. (2007), Suarez et al. (2007), and Oelofse et al. (2008), the sensory work
by Romano et al. (2009), and references in these publications therein).
Similarly, a significant quantity of research has been conducted on ethyl acetate concentrations in wine and associated sensory impacts. For example, wine rejection levels
of ,200 mg L21 have been developed for ethyl acetate (Corison et al., 1979), and concentrations of this compound can range widely over several orders of magnitude from
Sierra Rayne Ecologica Research, 412-3311 Wilson Street, Penticton, British Columbia, Canada, V2A 8J3
(E-mail: rayne.sierra@gmail.com)
ISSN 0957-1264 print/ISSN 1469-9672 online/09/020159-8 # 2009 Taylor & Francis
DOI: 10.1080/09571260903319945

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SIERRA RAYNE

the low mg L21 into the low thousands of mg L21 range (see Lambrechts and Pretorius
(2000) and Rojas et al. (2003), references in these publications therein, and related
references). While Ribereau-Gayon et al. (2000), the only citation for ethyl acetate
in Cliff and King (2009), is a key secondary reference for organoleptic defects
from this compound, there has been much additional work published in the primary
literature regarding ethyl acetate sensory impacts on wines in the decade since the
publication of Ribereau-Gayon et al. (2000).
Within this established literature context, it is clear that both 4-ethylphenol and
ethyl acetate display wide concentration ranges in commercial wines, and also have
broad matrix specific odour threshold ranges, thereby warranting more sophisticated
sensory experimental designs than were employed by Cliff and King (2009). With
reported odour thresholds ranging over two orders of magnitude from 1 to 200 mg
L21 (see Table 1 in Cliff and King (2009)), any temperature related sensory work
must examine more than just an arbitrary single spike addition of 200 mg L21 to an
unknown pre-existing ethyl acetate concentration in each of their wines. Ethyl
acetate has a perceived odour threshold that is highly subject to matrix influences,
and effectively unique for each wine (both inter- and intra-variety). Consequently,
when studying ethyl acetate concentration influences on perceived aroma defects,
the investigations need to determine what the pre-existing ethyl acetate concentrations
are in each wine (i.e., analyze the control wines for background ethyl acetate levels),
and then determine the specific ethyl acetate odour threshold for each wine using a
sequence of progressively higher standard additions. With this combined knowledge,
sufficient ethyl acetate can be added to the control wines to achieve final treatment concentrations that are just above the specific ethyl acetate odour thresholds for each wine.
If any of the wines examined by Cliff and King (2009) had ethyl acetate odour
thresholds near the lower limit of the literature range (1 mg L21), the addition of
200 mg L21 ethyl acetate to their wines could result in an excess above the odour
threshold of more than two orders of magnitude (not including the contributions
from any background concentrations in the wines), and not consistent with the
authors’ claims of spiking their analytes at “defect levels . . . chosen to be slightly
above reported threshold levels.” To simply add an arbitrary quantity of ethyl
acetate to each of the wines, and to not determine the final concentrations of analyte
being subjected to sensory analysis, weakens the value of the findings to other researchers or the wine industry.
Similarly, the influence of matrix effects on the perceived aroma type and threshold of
4-ethylphenol is well known. For example, Romano et al. (2009) found strong masking
effects of isobutyric acid and isovaleric acid on the sensory perception of 4-ethylphenol.
In wines without added isobutyric acid and isovaleric acid, an odour threshold of about
90 mg L21 was reported for 4-ethylphenol. With 1 mg L21 each of these two carboxylic
acids, the 4-ethylphenol odor threshold was raised to about 300 mg L21. Rapp (1998)
found that spiking unfaulted Silvaner white with as little as 50 mg L21 4-ethylphenol led
to a significant difference in the sensory characteristics, while a 500 mg L21 addition
produced a dramatic effect. Dufour et al. (2000) reported interactions between
various aroma compounds, including volatile phenols and anthocyanins, demonstrating the importance of quantifying matrix effects as best as practical. In addition, low
correlations between 4-ethylphenol concentrations and “Brett character” perception
may result from the corresponding presence of other simple phenols (e.g., phenol,
cresols, guaiacols and syringols) that can also be present in the wines, and whose
aroma descriptors (e.g., smoky, hospital, leather, smoky, spicy, bitumen) (Escudero
et al., 2007) could interfere with detection of similar descriptors for 4-ethylphenol.

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Thus, the odour threshold of 4-ethylphenol spans from about 50 mg L21 to an upper
limit of about 700 mg L21 as reported by Chatonnet et al. (1992), or more than an
order of magnitude, depending on the specific wine.
Consequently, if the 4-ethylphenol odour thresholds in any of the wines studied by
Cliff and King (2009) were ,100 mg L21 (a very reasonable occurrence, particularly
for the white wines), their arbitrary 1,000 mg L21 addition could result in final concentrations more than an order of magnitude above the wine specific odor threshold.
Again, this would not be consistent with the claims of Cliff and King (2009) using
4-ethylphenol levels “slightly above reported threshold levels.” As well, 4-ethylphenol
is produced in oak barreled wines via the sequential decarboxylation and reduction of
p-coumaric acid primarily by Brettanomyces/Dekkera spp., although other microorganisms such as lactic acid bacteria can be minor contributors (Heresztyn, 1986;
Cavin et al., 1993; Chatonnet et al., 1992, 1993, 1995, 1997; Rapp, 1998). Since p-coumaric acid concentrations in white wines are generally about four-fold lower than in red
wines (Goldberg et al., 1998, 1999; Rapp, 1998), with concentrations often highest in
the Bordeaux varieties, there is a well established gradient of 4-ethylphenol production
potentials among different types of wines. Even though some white wines are lightly
oaked, the combination of lower 4-ethylphenol precursor levels and the generally
reduced oaking of these varieties compared to many red wines results in general agreement among the wine research community and wine industry that red wines are most at
risk from 4-ethylphenol contamination. Cliff and King (2009) should have noted these
issues, particularly since they chose to devote three out of the four wines in their experiments to white varieties. Based on the established maximum 4-ethylphenol concentration ranges reported in red wines from around the world, it appears highly
unlikely that the 1,000 mg L21 levels of 4-ethylphenol used by Cliff and King (2009)
for their three white wines are of practical relevance. Even red wines are now unlikely
to reach 1,000 mg L21 levels of this compound, as evidenced by the surveys cited above.
It is also important to note that some low levels of 4-ethylphenol are thought to enhance
the complexity of red wines, giving them positive “Old World” aromas. Therefore, the
sensory research needs are on 4-ethylphenol levels of practical and widespread utility,
most likely in the range from 50 to 500 mg L21, depending on the variety.
Furthermore, the odour threshold of a compound displays both a temperature
dependent discontinuity in the sensory response (i.e., near the odour threshold, there
is a rapid change in sensory impact with increasing concentration), as well as temperature dependent responses above and below the odour threshold. Thus, the odour
threshold of a compound is temperature dependent, since the odour threshold (given
in concentration units for an analyte in a solution) is a surrogate for the often more difficult to determine, but more physiologically relevant, concentration of the analyte in
the headspace above the solution (which is what the olfactory senses respond to). The
air-wine partitioning constant (Kair-wine) determines the relationship between the
respective concentrations of an aroma compound in the wine and in the headspace
above the wine, and is a more relevant molecular descriptor for aroma compounds
than the molecular weight, boiling point, and vapour pressure data given in Table 1
of Cliff and King (2009). Diban et al. (2008) have reported a Kair-wine for ethyl
acetate of 0.092 atm L mol21, in excellent agreement with the range of experimental
air-water partitioning coefficients for this compound (Kair-water; 0.11 to 0.21 atm
L mol21; Sander (1999); also see Buttery et al. (1971) for a classic paper on Kair-water
relevance to food sensory studies). 4-Ethylphenol does not have a published Kair-wine,
but its Kair-water of 0.00077 atm L mol21 (Nirmalakhandan et al., 1997) is almost
300-fold lower than the corresponding values for ethyl acetate.

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In addition to the synergistic/antagonistic relationships between different aroma
compounds in the headspace towards olfactory receptors, the Kair-wine also plays a
major role in the matrix specific odour thresholds for wine aroma contributors. In
other words, different wines have different pH values, ethanol contents, and compositions of other major and minor constituents that modify the Kair-wine for each
aroma compound both within and between different wine samples. As well, even if a
particular analyte has the same Kair-wine for different wines, the odour thresholds
may still differ since the other minor/major constituents in the headspace above the
wine may interact with olfactory sensors to enhance or inhibit the perception of the
target analyte. These complexities explain the broad ranges in odour thresholds
reported for single compounds (such as ethyl acetate and 4-ethylphenol) across different wines in the literature, and attest to the requirement for sensory work to determine
the temperature dependent wine specific odor thresholds for a particular compound
prior to any standard additions analysis.
As an example, if any of the wines examined by Cliff and King (2009) had a background ethyl acetate concentration of 20 mg L21 and a wine specific odor threshold of
177 mg L21 at room temperature (the upper limit of the range quoted by these
authors), then the post-addition ethyl acetate concentration of 220 mg L21 would be
only marginally above the 208C wine specific odour threshold (a factor of only 1.25).
Upon cooling their samples to 108C, and especially to 08C, the temperature dependence of both Kair-wine and the odour threshold could possibly result in headspace concentrations of ethyl acetate below the olfactory threshold in one wine (representing an
experimental discontinuity), but perhaps not in another wine (due to differing matrix
effects). Raising the temperature where analyte concentrations are already above the
odour threshold will, all other factors being equal (i.e., ignoring temperature dependent synergistic/antagonistic influences from other aroma compounds), increase the
intensity in a relative continuous manner (the relationship may be linear or curved,
depending on matrix effects). However, lowering the temperature may reduce the
headspace concentrations of an analyte to below the olfactory threshold, representing
a discontinuity in the sensory response.
As an example, the Kair-wine for ethyl acetate appears to be approximately equal to
the Kair-water in some wines. The SPARC software program (http://ibmlc2.chem.uga.
edu/sparc/; August 2007 release w4.0.1219-s4.0.1219) accurately estimates the experimentally determined 208C Kair-water for this compound at 0.19 atm L mol21. Upon
decreasing the solution temperature from 208C to 108C and 08C, as Cliff and King
(2009) did, the Kair-water is expected to decline by factors of 1.7 and 3.0, respectively.
Thus, the (unknown) final concentrations of ethyl acetate in the wines studied by
Cliff and King (2009) would have needed to be greater than three-fold higher than
the wine specific odour threshold (which is also unknown; it could have likely
ranged somewhere between 1 and 200 mg L21) to avoid the sensory discontinuity,
whereby simply cooling the samples to 08C is sufficient, in the absence of any other
effects, to lower the headspace concentrations of this analyte to below the olfactory
threshold. This would not have yielded any particularly informative scientific findings,
since basic gas-solution phase physicochemical properties and equilibrium partitioning
behaviour could have been used to predict that cooling a wine sample substantially can
lower the headspace concentration of an aroma compound to such an extent that it is
sufficiently involatile to elicit an olfactory response. Alternatively, if the final ethyl
acetate concentrations were much greater than three-fold the wine specific odour
thresholds (this is unknown), then any resulting changes (or lack thereof) in sensory
impacts between 208C and 08C would have been due to the changing aroma intensity

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of this compound, as well as changes in the relative masking/intensifying influences
of other antagonistic/synergistic aroma compounds. Similarly, the Kair-water of
4-ethylphenol is expected to decline by up to three-fold between 208C and 08C,
necessitating an analogous determination of what the wine specific aroma thresholds
for this compound were.
Consequently, since Cliff and King (2009) did not determine room temperature wine
specific odour thresholds for either ethyl acetate or 4-ethylphenol, and certainly did not
determine temperature dependent wine specific odour thresholds for these two analytes
at each of the three experimental temperatures for each of the four wines, it is impossible to derive any significant (and generalizable) findings from their study. Their results
are not likely to offer predictive value towards other varieties of wine or other samples of
the same varieties these authors investigated, and (using the example above) their findings may not even be reproducible at other analyte concentrations within the same
samples that Cliff and King (2009) studied. Consequently, the insufficient experimental design leads to the conclusion that the results of Cliff and King (2009) represent
single isolated datapoints that are specific to the particular wine/temperature/aroma
compound concentration combinations they chose to study. It is difficult to see how
the wine research community derives benefit from such work, since it provides no
general quantitative or qualitative predictive value nor presents any deeper understanding of the specific aroma compound/matrix/temperature effects that may
explain the different results obtained between their samples.
In their forthcoming Addendum, it is this author’s understanding that Cliff and King
have chosen to only correct their inconsistently quoted experimental serving temperatures within the Abstract and the Materials and Methods sections of their original
article to yield “new” values of 0, 10, and 208C (and not elsewhere in the work), such
that these “new” Abstract/Materials and Methods values of 0, 10, and 208C now do
not coincide with the values given in Figures 1, 2, and 3, or in the Results and Discussion
section, of Cliff and King (2009). As such, it appears the authors have now corrected
their intended temperatures such that the regression equations shown in Figure 1,
and any associated statistical tests, are no longer applicable, and are inconsistent
with the revised temperatures intended for the remainder of the manuscript. One
must also question the relevance of doing wine aroma sensory studies at 08C for
either red or white wines, or at 108C for red wines. In their Introduction, the authors
state that “[a]ccepted wine serving temperatures (white 8-128C; red 18-228C) are
based on optimising wine flavour” (Cliff and King, 2009). By claiming that they
intended to mean 08C rather than 58C throughout the manuscript, Cliff and King
have carried through work on an experimental design using a wine temperature of
08C that is not a serving temperature, and is effectively irrelevant. Within the
context of the wine serving temperature ranges for red and white varieties quoted by
Cliff and King, it would have been more useful to conduct the lower temperature
end member sensory studies at 88C. Given the different serving temperature ranges
for white and red wines, it is also unclear why Cliff and King chose to use the same
three temperatures to examine both white and red varieties. For example, in another
study regarding serving temperature effects on the sensory properties of red and
white wines, Ross and Weller (2008) used different temperatures for the white (4, 10,
and 188C) and red (14, 18, and 238C) wines, making their work of direct practical
relevance to the research community and wine industry.
There is dubious value in the authors performing linear regression on three data
points, and subsequently quoting five significant figures in the y-intercepts (perceived
fruit or defect intensity axis) and three significant figures in the associated slopes

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(Figure 1 in Cliff and King (2009)). One must also question the appropriateness of the
linear model. There is no reason to believe that the perceived fruit or defect intensities
in these samples will display linear responses as functions of temperature (Kair-water
values do not have linear temperature dependencies as well). Indeed, a closer examination of the four regressions given in this figure shows that the regression lines always
overestimate the 08C and 228C experimental datapoints, and always underestimate
the 108C datapoints. This consistent bias strongly suggests an exponential/power
relationship may be a more accurate representation of the sensory descriptor versus
temperature relationships. With only three datapoints, however, it is more reasonable
to just present the data as was done in Figure 3, rather than imply a greater level of
trending insight (interpolation accuracy) than the three point linear regressions with
clear endpoint biases are able to do. If the authors had wanted to develop a useful
quantitative relation between the perceived intensity descriptors and temperature,
they should have analyzed at more than just three temperatures.
On page 50 of Cliff and King (2009), the authors state that “[e]thyl acetate was
perceived more intensely in the white and red wines (228C) (Figure 2a). This suggests
that Gewurztraminer and Chardonnay wines contain constituents that masked the perception of ethyl acetate.” In the caption to Figure 2, the authors state “[f]or a given
wine, bar charts marked with different subscripts were significantly different at
(p 0.05).” As Figure 2a should thus be correctly interpreted, there is no significant
difference in the ethyl acetate intensity between the white wine, the Gewurztraminer,
and the Chardonnay, or between the red wine, the white wine, and the Chardonnay.
The only significant difference in the ethyl acetate intensity is between the red wine and
the Gewurztraminer. Also on page 50, the authors state that “[i]n contrast, 4-ethyl
phenol was perceived more intensely in the white and Gewurztraminer wines (228C)
(Figure 2b), compared to the red and Chardonnay wines.” As can be seen in Figure
2b, there is no significant difference between the red wine and the Gewurztraminer.
Furthermore, referring to Figure 3, the authors state that “[t]he magnitude of the
effect was largest for the white and Gewurztraminer wines at 228C”. In Figure 3, the
caption states that “[f]or a given wine, bar charts marked with different subscripts
were significantly different at (p 0.05).” As shown clearly in Figure 3, there is no significant difference in the change in fruit intensity for Gewurztraminer wines at 108C
and 228C. Cliff and King’s use of non-significant relative size descriptors (e.g., more,
largest) is nonsensical in light of their corresponding statistical tests.
The authors conducted their ANOVA tests to determine if mean values of different
treatments were different. If the statistical test indicated the means were not significantly different (i.e., p . 0.05) at their chosen a ¼ 0.05, then one cannot reasonably
say they are different. How can one state there is a difference between the terms “different” and “significantly different”? Since no two of any measurements presented in Cliff
and King (2009) are exactly the same, this reasoning implies that “not significantly
different” is equivalent to “different”, and that each measurement reported by Cliff
and King (2009) is “different” from every other measurement. The natural extension
to this reasoning is that Cliff and King must describe Figure 2a in their article as
follows: The perceived ethyl acetate intensity was higher in the red wine than the
white wine, higher in the white wine than the Chardonnay, and higher in the Chardonnay than the Gewurztraminer, but only the red wine and the Gewurztraminer were
significantly different at a ¼ 0.05. If a statistical test is conducted that indicates two
values are not significantly different, and then the authors say the two values are still
different, one must question the point of the statistical test, and at what p-value
above 0.05 do we transition from “not significantly different” to “significantly the

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same”? By further extension, what is the meaning of “not significantly different” as
implied by such an approach to interpreting data and the results of statistical
testing? If the authors want to pursue this highly unusual data interpretation approach,
then they must do so consistently throughout their article, thereby necessitating a complete re-write of Cliff and King (2009) to express each data point as either “significantly
different” and/or “not significantly different” and/or “not significantly the same” and/
or “significantly the same” from every other data point.
Cliff and King (2009) do not give any description of the varietal compositions of the
blended white and red wines carrying the British Columbia Vintners Quality Alliance
(BC VQA) designation. Such an omission does not allow the scientific community to
gain any meaningful insights from the results for these wines. As discussed above,
aroma compound effects are well known to be highly sensitive to matrix effects.
Thus, omitting blend compositions renders any conclusions from such studies of no
value, as readers must question whether the unspecified blends are representative of
any other possible blends. Additional wine sample descriptors such as all details
about winemaking, vintage, storage time and conditions prior to the study, basic
chemical compositions (e.g., pH, ethanol content, total reducing sugars, volatile
acidity, total phenolics, etc.), etc., should also have been specified by the authors to
ensure relevance to the scientific community. A good guide for the enology community
in this regard is the Guide to Authors for the American Journal of Enology and Viticulture
(available at http://ajevonline.org/misc/guidetoaus_2008.pdf), which lays out reporting requirements for both wine samples and sensory experiments.
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