Pollen Analysis of New Zealand Honey Science Report D

Pollen analysis of New Zealand honey Science Report D. C. Mildenhall and R. Tremain 2005/06

March 2005

Pollen analysis of New Zealand honey

D.C. Mildenhall R. Tremain

Institute of Geological & Nuclear Sciences science report 2005/06

Institute of Geological & Nuclear Sciences Limited Lower Hutt, New Zealand March 2005

Gracefield Research Centre, 69 Gracefield Road, P O Box 30368, Lower Hutt, New Zealand, Telephone: +64-4-570 1444, Facsimile: +64-4-570 4600 A Crown Research Institute

BIBLIOGRAPHIC REFERENCE

Mildenhall, D.C. and Tremain, R. 2005. Pollen analysis of New Zealand honey. Institute of Geological & Nuclear Sciences science report 2005/06: 19 p.

D.C. Mildenhall, Institute of Geological & Nuclear Sciences Limited, Wellington R. Tremain, Institute of Geological & Nuclear Sciences Limited, Wellington

CONTENTS

ABSTRACT...... ii

KEYWORDS...... ii

1. INTRODUCTION...... 1

2. POLLEN ANALYSIS...... 5

3. RESULTS ...... 5 3.1 Blackberry honey ...... 6 3.2 Borage honey...... 6 3.3 Clover honey ...... 6 3.4 Hangehange honey ...... 7 3.5 Honeydew honey...... 7 3.6 Kamahi honey...... 7 3.7 Kanuka honey...... 8 3.8 Koromiko honey...... 8 3.9 Manuka honey ...... 8 3.10 Mingimingi honey ...... 9 3.11 Penny Royal honey...... 9 3.12 Pohutukawa honey ...... 9 3.13 Rata honey...... 9 3.14 Rewarewa honey ...... 10 3.15 Tawari honey...... 10 4. DISCUSSION...... 11 4.1 Processing...... 11 4.2 Analysis...... 12 5. RECOMMENDATIONS AND CONCLUSIONS ...... 12

6. ACKNOWLEDGEMENTS...... 14

7. REFERENCES...... 14

©Institute of Geological & Pollen analysis of Nuclear Sciences Limited i New Zealand honey

ABSTRACT

This report summarises the results of a pilot study into a palynological analysis of 45 honey samples from Waikato University Honey Research Unit’s honey databank to see if the honeys were true to label based on pollen analysis. Of the 21 honeys labelled as monofloral most are accurately labelled or very close to being accurately labelled using palynological criteria. The six exceptions included two labelled as kanuka honey, which are mixed kanuka/lotus/clover honeys, one labelled as tawari honey, which is a clover honey, two labelled kamahi, which are mixed Quintinia/manuka/kamahi and clover/manuka/kamahi honeys, respectively, and one labelled as rewarewa honey, which is a clover honey. One kamahi sample, one borage sample, and one tawari honey sample did not contain enough pollen to count. All other named honeys contain enough pollen of the nominated pollen source to justify their naming and in some cases palynology showing that the honey could be called monofloral, but not necessarily on the basis of the named source species.

Modern alcohol-based processing must be used in order to obtain the maximum number and diversity of pollen grains from the honey under investigation, and in the best condition for identification. Centrifuging should be used but not crushing and straining.

KEYWORDS

Monofloral honey, pollen, pollen analysis, pollen processing techniques, ethyl alcohol.

©Institute of Geological & Pollen analysis of Nuclear Sciences Limited ii New Zealand honey

1. INTRODUCTION

The New Zealand honey industry has been given the responsibility of providing quality standards for honey, particularly manuka honey, which command higher prices both within New Zealand and externally. Premium monofloral honeys demand higher prices and authorities should always be on guard for honey that is mislabelled as premium honey, either in error or deliberately. Standards are common in most honey-exporting countries, but have yet to be established in New Zealand. Honey has been rejected from overseas markets on externally imposed and wrongly assessed criteria against which we have no defence. Neither do we have any established palynological technique to determine the accuracy of labelling on honey and associated products imported into New Zealand.

Pollen analysis is but one of a number of tests that must be used to determine the quality and source of honey. Melissopalynology, the study of pollen in honey, is undertaken in most of the major honey producing countries of the world (Johansson & Johansson, 1968), many of which impose strict laws governing importation of honey products. Almost without exception these countries require certification, including pollen analysis, to verify the floral type, quality, and precise source of honey and associated products. Other tests, done in conjunction with palynology, include analyses of the stable isotopes, colour, flavour, and aroma. If New Zealand is to export honey then it is restricted to servicing those countries that do not require certification. Similarly, if New Zealand does not require honey importers to only import certified honey then there is no accepted way of testing their typification, quality and source.

This report is the result of an investigation into the palynological analyses of 45 honey samples, encompassing 15 different honey types (Table 1), from Waikato University Honey Research Unit’s honey databank. The purpose of the research was to see if the honeys were true to label based on pollen analysis, using the results of Moar (1985) as a previously determined quality standard (Table 2) and a draft “New Zealand Honey Profiles Varieties” standard, produced by the National Beekeepers’ Association, currently under discussion amongst honey producers.

Honey can also be categorised by the number of pollen grains per 10 grams of honey (Table 3) (Maurizio, 1975). The number of grains is usually a reflection of the honey production system used, storage of pollen by the bees, season of the year, dilution of the honey by, for example, the addition of sugar or high-fructose syrup, and pollen sources. This is not further discussed here but an estimation of the number of pollen grains per 10 grams of sample is given in the results section below. These figures are calculated using the formula

Pollen count = no. of exotic Lycopodium spores added x no. of pollen grain counted no. of exotic Lycopodium spores counted x amount of honey (g) processed

©Institute of Geological & Pollen analysis of Nuclear Sciences Limited 1 New Zealand honey

The nectar from flowers of a plant is collected, modified and concentrated by the honey bee into honey. As the honey is formed it retains the pollen collected in the honey, with a little wind dispersed pollen as well, giving the pollen a characteristic assemblage dependent on the prime source or sources for the pollen. The number of individual pollen grains of a particular species may differ considerably according to the production levels of the source species. For example, a manuka monofloral honey would contain 70% or more pollen from manuka while a rewarewa monofloral honey would only contain about 10% or more rewarewa pollen.

©Institute of Geological & Pollen analysis of Nuclear Sciences Limited 2 New Zealand honey

m m u i a t a liu a s ar o o ul if op r c m t ) i

c RESULT OF ANALYSES

e s r g c e ( a c u s

e p g t

HONEY TYPES a (Honey identification using / des . ls a y i

h r p e lig um s s e o -t i c ig a e

c pollen) ia o ns e i m on fa x sp e m a g n r m l ex a a er

a n numbe e e o u e ia e a w t d ma t cu i a i e p m c c a ia pog s n s n le t m lin i a e m ha n gr o o o t xa p r liu os h s i e a ba br e ago n a in e o l tr nt r n i l rce ac mpl r lix m ig hiu t r is tu uc ptos her if e e r t a o r a B Copr Ec G G Ix Kn Le Le Lo Me Me Po Qu Ranun Ros Sa T T We O Pe Sa Po S As 1 Blackberry BL 1 x x x 20 7 18 x 11286x 28 27100 1 2003 Mixed clover/tawari/lotus 2 Tawari D 1 5 2 14 x 11 2 5361 14100 5 13,349 Clover unifloral 3 Clover Cl.4/4 10 13 x 3 x x 76.5 x 6 100 1.2 593 Clover unifloral 4 Manuka D 104/94/81 x x 1 85 2111x 141299 0.5 25,426 Manuka unifloral 5 Clover U CL28.396 1 x x 5 x x x 1 89 2199 1 22,818 Clover unifloral 6 Rewarewa RR2/100 2 x 8 22 x 10 x 3 x 1 4 2411 6100 0.4 12,303 Rewarewa unifloral 7 Rewarewa 3/2/97 1 x x 18 7 x 1 x x 2 x 64 44100 1 1344 Rewarewa unifloral 8 Rewarewa RE.7 1 x 9 x 51 62165 28100 1.3 10,243 Mixed rewarewa/clover 9 Rewarewa U RE10.396 1 37 37 1 x 11x 2759100 1.8 4184 Rewarewa unifloral 10 Rewarewa U RE.22/96 2 x 1 27 x 10 3 1 x 823526102100 1.5 2537 Rewarewa unifloral 11 Koromiko D 3 x x 10 x 14 118 1 3122 11 25 100 5 626 Mixed clover/lotus/koromiko 12 Rata Sthn D.4.196 1 94 1 x 121100 5 9206 Rata unifloral 13 Manuka D SG 16+ 1 x x x 10 21 6 x x x 12256 1 100 1 31,044 Clover unifloral 14 Borage D x 11x x x x 88 x 10 100 4 27,242 Clover unifloral 15 Rata Sthn D x 2 x 84 x 1 x 166100 5 6890 Rata unifloral 16 Rewarewa U TL 24 1 x 7 15 x 11x 3 x 64 2599 0.8 1460 Rewarewa unifloral 17 Mingimingi 2 1 x 2 620 8169x 40 14100 5 24,835 Mixed clover/lotus 18 Kamahi U KA22.496 0 0.8 0 Sample too small to analyse 19 Kanuka D.K12 x 18 28 41x x x x x 1 40 8 100 5 25,426 Mixed clover/lotus/kanuka 20 Kamahi U KA23.96 x 11 x x x x 79 8 98 1.4 734 Kamahi unifloral 21 Manuka D SG.B1 1 x x 1 71 4 11 1 124x 13 100 3 16,180 Manuka unifloral 22 Borage D. Waikaki 1 x 40 11 x 2248 14100 0.9 3828 Mixed vipers bugloss/clover 23 Honeydew U HD9.296 x x 21 x x 211540 x 48 100 1.2 4328 Honeydew with beech/clover 24 Manuka D MB/B+H 1 x 1 x x 71 42x 1 x x 19 10100 1.1 32,146 Manuka unifloral 25 Penny Royal D BW 46 1 x x x 3 x 27 1 x 11 1157 x 6 100 0.8 24,359 Mixed clover/lotus 26 Kanuka U.K33/9 x x 40 24 8 x 26 2 100 5 38,139 Mixed kanuka/clover/lotus 27 Hangehange U HH1.1096 x 30 25 38 x 2 x x 3199 5 15,658 Hangehange unifloral 28 Rata (Stn) D RAT 5/96 x x 28 32 271 1 21716 2 99 0.9 10,987 Mixed lotus/manuka/kamahi 29 Borage U B3.296 0 0.8 0 Sample too small to analyse 30 Kanuka U K 28/196 x x 25 48 61 1x 1 x 5112100 1.5 37,869 Mixed lotus/kanuka 31 Tawari U 3/2/97 0 1 0 Sample too small to analyse 32 Tawari U.TA32 1 13 15 14 1 x 2 x 211241 11 4 99 5 1441 Tawari unifloral 33 Tawari U.TA33 x 3 x x 31x 1170 18 3 100 5 3657 Clover unifloral 34 Kanuka U.K25.290 x x 87 4 x 1 x x x 7 x x 99 5 27,382 Kanuka unifloral 35 Kamahi U.KA14 18 4 x x 30 x x 1518 23 99 5 19,416 Mixed Quintinia/manuka/kamahi 36 Kamahi U.KM/7-10-11/00 8 x 1123 41x 12534 10 9 99 5 39,552 Mixed clover/manuka/kamahi 37 Rewarewa U.RE35 x 1129 11x x 1 x 59 x 24 11101 5 59,328 Rewarewa unifloral 38 Rewarewa U.RE32 x 1 11 x x x x x 22 x 52 8 5 99 5 11,608 Rewarewa unifloral 39 Rewarewa U.RE15-1195 1 x x x 6 22x x 1 x 41x 1 71 3698 1 13,055 Clover unifloral 40 Pohutukawa U.PO40/SMNZPO x x 1 x x x x 11048 x 9 x x x 8184 99 5 15,256 Pohutukawa unifloral 41 Clover D.Woodlands x 1 x x 1 x x 1 x x 1184 1898 5 7216 Clover unifloral 42 Clover D.Mosspos 3 x 1 20 21 10 1 x x x 4247 2699 5 6,173 Mixed clover/tawari/lotus 43 Clover D.PS8 x x 11611x x 1 x 2 76 2 100 5 56,205 Clover unifloral 44 Manuka D.Honeyvillage x 87 x 2 x x 10 x 99 2 102,683 Manuka unifloral 45 Manuka D.Mosspos B3/26100 x 1 x x 73 10 x 11 x x 1161398 3 48,104 Manuka unifloral

Table 1. Summarised pollen counts. The counts for the key taxa in each sample are in bold. x = less than 1%, and figures are rounded, hence they do not always add up to 100%. The honey types, as labelled by the Waikato University Honey Research Unit, are in the left column-hand and the suggested honey types, determined palynologically, are given in the right-hand column. U = designated monofloral.

©Institute of Geological & Pollen analysis of Nuclear Sciences Limited 3 New Zealand honey

Honey type Minimum percentage of Representation nominated pollen in honey Citrus honey 10-20% under-represented Clover honey 45% mixed representation Kamaki honey 60-70% over-represented Lotus honey * mixed representation Manuka honey 70% over-represented Matagouri honey * over-represented Rata honey 45% * Rewarewa honey 10% under-represented Thyme honey 20% under-represented Viper’s bugloss honey 45% *

Table 2. Percentage pollen for determining monofloral (= unifloral) honey found in selected honeys from Moar (1985). * not determined.

Categories Pollen content Possible reasons for such content 1 20,000/10 g or fewer honeydew honey, adulteration, sugar fed bees, pressure filtered honey, poor pollen sources 2 20,000-100,000/10 g from normal floral sources 3 100,000-500,000/10 g high pollen producers, pure pollen storage cells in honey 4 5000,000-1,000,000/10 g rich rich pollen sources 5 1,000,000/10 g or more rich pollen sources

Table 3. Honey categories based on number of pollen grains per 10 grams of honey (Maurizio 1975).

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2. POLLEN ANALYSIS

All samples were processed in the same way following the ethyl alcohol method recommended by Jones & Bryant (1998; 2001a). This included heating the sample and stirring to ensure a thorough and uniform mixing of pollen, dissolving a measured amount of honey using a minimum amount of warm water, adding a Lycopodium tablet and ethyl alcohol, centrifuging, acetolysis, washing, staining, and mounting on slides. The spiking of honey samples with exotic Lycopodium tablets permits calculation of spore and pollen (palynomorph) concentrations. The sample numbers given in the text below refer to the sample numbers in table 1.

Sample weights were taken before processing. Due to the small size of the material sampled only one Lycopodium tablet was added in order to determine the number of pollen grains per gram of sample. The normal recommended number is five tablets per 10 grams of honey.

A total of 500 grains were counted in most samples. Three of the samples were not counted as pollen recovery was too low. Two samples were counted to 200 (sample 3 clover) and 450 (sample 7 rewarewa) (see Table 1), but in both cases the low count was unlikely to affect the pollen classification of the honeys.

Pollen identification of the dominant and characteristic pollen types were made, but identification of rare pollen types in the honeys was not so rigorous and counting and identifications were done quickly to come within the allocated funded time limit. Some pollen types are not distinctive enough to be positively identified to species. These conservative pollen species, like those of the Myrtaceae (e.g. individual pollen grains of pohutukawa and rata [Metrosideros]; manuka [Leptospermum] and kanuka [Kunzea]), were not always distinguished in this exercise, and some identified taxa, missing from our reference collection, could not be checked. However, all samples labelled as manuka, kanuka, rata, and pohutukawa contain these pollen types by close comparison of some grains with reference collections and published descriptions (McIntyre, 1963). While the different species of clover (red and white) were not differentiated it was noted that white clover was clearly the most common in every sample, reflecting its wide-spread distribution throughout New Zealand.

3. RESULTS

From a palynological perspective, most of the 45 samples appeared to be true to label or very close to it, although a few significant differences were identified. The main results are discussed below in alphabetical order of the common name for the honey investigated.

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3.1 Blackberry honey (sample 1)

This sample appeared to be more like a mixed clover/tawari/lotus honey. Blackberry produces a relatively large grain and probably about 10% blackberry (Rosa) could justify a ranking of blackberry monofloral. This honey sample contains about 8% Rosa pollen so is very close to being monofloral although the honey was not labelled as such. At 20% tawari pollen it would justify being called tawari monofloral. Clover forms 28%. The very small size of this sample (1 gram) probably resulted in an inaccurate pollen assessment of this honey as the absolute pollen count was a low 20,000 grains per 10 grams of honey.

3.2 Borage honey (samples 14, 22, 29)

Too few pollen grains were recovered from honey sample 29 (0.8 grams) to warrant analysis. This was the only borage sample noted as being monofloral. Only 3 borage (Borago) pollen grains were counted in sample 14, which should more accurately be called a clover (88%) honey.

Sample 22 also only had 3 borage pollen grains in the count to 500 and is best called a mixed clover/viper’s bugloss (Echium) honey with 48% and 40% pollen respectively. Since both borage and viper’s bugloss have small blue flowers the two plants are occasionally misidentified in the field by bee keepers. No pollen standard for this type of honey has been set in New Zealand, but pollen production of borage is low and a very small percentage of pollen, possibly 8-10%, may justify a monofloral label.

Samples 14 (4 grams) and 22 (0.9 grams) contain an absolute pollen count of between 38,500 and 272,500 grain per 10 grams with the lesser number from the smaller honey sample 22.

3.3 Clover honey (samples 3, 5, 41-43)

Five samples were analysed, four of which contain more than 65% clover (Trifolium) pollen and thus are true to label although only sample 5 was listed as monofloral. The percentages of clover pollen in these four samples are 77% (sample 3, with 10% tawari pollen, probably enough to also call this honey tawari monofloral, although many of the grains are under- developed), 89% (sample 5), 84% (sample 41), and 76% (sample 43, with a little lotus pollen - 16%).

Sample 42 is a mixed clover/tawari/lotus honey with 47%, 20% and 10% respectively - with enough under-developed tawari pollen to justify calling this a monofloral tawari honey.

Moar (1985) recorded between 16,000 and 120,000 pollen grains per 10 grams of monofloral clover honey. In this study they range quite widely in round figures per 10 grams - 228,000 pollen grains in labelled monofloral sample (5), 6000 (monofloral sample 3, and sample 42 which was not monofloral on palynological grounds) 72,000 (monofloral sample 41) and 562,000 (monofloral sample 43).

©Institute of Geological & Pollen analysis of Nuclear Sciences Limited 6 New Zealand honey

3.4 Hangehange honey (sample 27)

Although this monofloral sample only has 25% hangehange (Geniostoma) pollen it is probably enough for it to be called monofloral. A New Zealand pollen standard for hangehange monofloral honey has not been set. This sample has a lot of pollen from other native New Zealand plants including Griselinia and Coprosma, which together form 68% of total pollen. This sample contains an absolute pollen count of 156,500 grains per 10 grams of honey.

3.5 Honeydew honey (sample 23)

Honeydew honey is made from plant secretions, other than that from flowers. In New Zealand honeydew honey often comes from native beeches (Nothofagus) and usually contains a high percentage of pollen from beech, dandelion (Taraxacum), gorse (Ulex), and grass (Poaceae) (Moar, 1985). The monofloral sample analysed has a diverse range of wind and insect dispersed pollen types and could justifiably be called a honeydew monofloral honey. It has plenty of clover (40%), beech (7%), gorse (12%), dandelion-type pollen (5%), and only a trace of grass pollen. No pollen standard has been set for New Zealand honeydew honey and it is uncertain whether a high percentage of gorse pollen should be included in the honeydew statistics (Moar, 1985). This sample contains an absolute pollen count of 43,500 pollen grains per 10 grams of honey.

3.6 Kamahi honey (samples 18, 20, 35, 36)

Too few pollen grains were recovered from monofloral sample 18 (0.8 grams) to warrant analysis.

Sample 20 (1.4 grams) was kamahi monofloral with 79% kamahi (Weinmannia) pollen, well within the minimum 60% kamahi pollen level to justify calling it monofloral (Moar 1985), and c. 7500 grains per 10 grams (Category 1), far less than the standard expected number of c.140,000 (Category 3).

Sample 35, labelled as monofloral, is a mixed Quintinia/manuka/kamahi honey at 30%, 18%, and 18% respectively. It is possibly close to being a monofloral Quintinia honey, but kamahi percentage is considerably lower than the New Zealand pollen standard for monofloral kamahi honey. No pollen standard has been set in New Zealand for Quintinia honey.

Monofloral sample 36 is a mixed clover/manuka honey with 34% and 23% respectively and only 10% kamahi pollen, far too low to justify calling it monofloral kamahi on pollen evidence.

The absolute pollen counts from samples 20, 35, and 36 were highly variable, being 7,500, 19,500 and 395,500 respectively.

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3.7 Kanuka honey (samples 19, 26, 30, 34)

It is difficult to distinguish kanuka (Kunzea) and manuka (Leptospermum) pollen and this has not been attempted in these samples so the assumption here is that the Myrtaceae pollen is all kanuka. Only sample 34 contains enough kanuka pollen (87%) to warrant its monofloral label. Sample 19 only contains 18% kanuka pollen, but it would require about 70% kanuka pollen to justify this appellation. Enough to justify mentioning it as a major constituent, justifying its listing as not monofloral. The dominant pollen types are clover (40%) and lotus (28%) and it is a mixed clover/lotus/kanuka honey on pollen evidence.

Sample 26 is a mixed kanuka/clover/lotus honey at 40%, 26% and 24% respectively, but it would require about 70% kanuka pollen to justify being called monofloral on palynological criteria.

Sample 30 was labelled as monofloral but it is a mixed lotus/kanuka honey with 48% lotus and only 25% kanuka pollen.

Absolute pollen counts for this group ranged between 254,000 and 381,500 grains per 10 grams, typical of manuka and kanuka.

3.8 Koromiko honey (sample 11)

This sample is better called a mixed clover/lotus/koromiko honey at 22%, 14% and 16% respectively, on pollen evidence. A pollen standard for New Zealand koromiko (Hebe) monofloral honey has not been set and 16% may be an adequate percentage to justify calling it monofloral, although it was not labelled as such. A very low absolute pollen count from this sample of 6260 grains per 10 grams, suggests that this sample needs further investigation. It did not dissolve fully during processing and many pollen grains were probably not recovered from the honey.

3.9 Manuka honey (samples 4, 13, 21, 24, 44, 45)

All these samples do contain manuka pollen and all small Myrtaceae pollen grains were therefore deemed to be from manuka although not all grains were thoroughly examined. Six samples were analysed, five of which contain more than 70% manuka (Leptospermum scoparium) pollen and could justifiably be called monofloral on palynological grounds. One sample (13) was clover monofloral with only 10% manuka pollen. This percentage was probably high enough to indicate the presence of manuka honey in the sample, which was not labelled as monofloral. The percentages of manuka pollen in the other five samples are 85% (sample 4), 71% (sample 21), 71% (sample 24), 87% (sample 44, with a little clover pollen at 10%), and 73% (sample 45, with a little lotus at 10% and clover pollen at 6%).

Moar (1985) recorded over 100,000 pollen grains per 10 grams of monofloral manuka honey. In this study they range, in round figures per 10 grams, from 162,000-321,500 pollen grains in

©Institute of Geological & Pollen analysis of Nuclear Sciences Limited 8 New Zealand honey

the four labelled samples and between 162,000 and 1,027,000 pollen grains in all samples palynologically determined as manuka monofloral.

3.10 Mingimingi honey (sample 17)

This sample was not listed as monofloral. Mingimingi can be either Coprosma or Cyathodes, neither of which occurs in this sample. If mingimingi refers to Leucopogon fasciculata, as it often does, then this sample contains 6% mingimingi pollen. This seems a rather small percentage to justify calling it monofloral and on pollen the honey is better called a mixed clover (40%)/lotus (20%) honey. There is no pollen standard for mingimingi honey in New Zealand and since the plant produces relatively few pollen grains 6% may justify its selection as monofloral. The absolute pollen abundance from this sample is 248,000 pollen grains per 10 grams.

3.11 Penny Royal honey (sample 25)

This sample, not listed as monofloral, is a mixed clover/lotus honey with 57% and 27% pollen respectively, on pollen evidence. Mentha (penny royal) pollen only forms 1% of the total pollen. A pollen standard for Mentha honey has not been set in New Zealand. An absolute pollen count of 243,500 grains per 10 grams was calculated for this sample, even though it was a very small sample (0.8 grams).

3.12 Pohutukawa honey (sample 40)

It is not always easy to distinguish pohutukawa and rata pollen, both coming from the genus Metrosideros, but this sample does contain pohutukawa pollen although not all grains were thoroughly examined. This monofloral sample contains 48% Myrtaceae pollen, at the lower end of monofloral pohutukawa honey, with 18% kamahi and 10% lotus pollen. The pollen standard for pohutukawa honey would be the same as that set for rata honey at 45% (Moar, 1985). The number of pollen grains per 10 grams in this sample is 152,500, within the normal range for such honey.

3.13 Rata honey (samples 12, 15, 28)

These samples do contain rata pollen and all pollen was deemed to be from rata although not all grains were thoroughly examined. None of the samples were listed as monofloral. Honey sample 12 has 94% Metrosideros pollen, presumably rata, well above the minimum required level for monofloral rata honey.

Sample 15 has 84% rata pollen also well above the minimum required level for monofloral honey.

Sample 28 has only 7% rata pollen which is probably just enough to mention that rata honey is in the sample but it contains far more pollen from manuka (28%), lotus (32%), and kamahi (16%).

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Rata honeys have about 100,000 grains per 10 grams (Moar 1985). These three samples have 92,000, 69,000 and 110,000 pollen grains per gram respectively.

3.14 Rewarewa honey (samples 6-10, 16, 37-39)

Eight of the nine samples had 9% or more rewarewa (Knightia excelsa) pollen and could justifiably be called monofloral. Sample 8, which was not labelled as monofloral, contains 9% rewarewa pollen, just short of being monofloral (10% is the minimum according to Moar 1985), but only 1.3 grams of honey was analysed and a bigger sample may result in a slightly different count.

Monofloral samples 6 (not labelled as monofloral), 9 and 10 had 22%, 37% and 27% rewarewa pollen, respectively over twice the usual percentage of rewarewa pollen.

Monofloral honey sample 37 contains 9% rewarewa pollen and is a mixed willow (59%), rewarewa, and clover (24%) honey. The identification of a reticulate pollen grain in this sample as a species of willow (Salix) needs confirmation with reference material that we do not currently have access to. This sample is probably justified in being called monofloral rewarewa at 9%.

Honey samples 7 and 16 could be called monofloral clover honeys as well as monofloral rewarewa, since they both had 64% clover pollen, at the lower end of the limit for monofloral clover, with 18% and 15% rewarewa pollen respectively. Sample 7 was not listed as being monofloral.

Honey sample 38 with 11% rewarewa pollen, 52% clover and 22% blackberry pollen is monofloral rewarewa.

Sample 39 with 71% clover and a little rewarewa pollen at 6% is monofloral clover, not monofloral rewarewa, but only 1 gram was analysed and again a bigger sample may result in a higher rewarewa count.

Absolute pollen counts fluctuate greatly in rewarewa honey and cannot be used for characterising this honey type (Moar 1985). This is confirmed by the range of all nine rewarewa honeys of between 14,600 and 593,500 pollen grains per 10 grams, with the higher numbers coming from the bigger samples.

3.15 Tawari honey (samples 2, 31, 32, 33)

Monofloral honey sample 31 was too small (1 gram) to analyse since so few pollen grains were recovered. A characteristic of tawari honey is the scarcity of pollen.

Sample 2, not listed as monofloral, only had 5% tawari (Ixerba brexioides) pollen and is better called a monofloral clover (61% – just short of the minimum set pollen standard, but probably close enough to justify calling it a monofloral clover honey). No pollen standard has been set in New Zealand for tawari monofloral honey but 5% tawari pollen seems a bit low to justify calling this monofloral, but probably high enough to note its presence.

Monofloral sample 32 is a mixed clover/lotus/tawari honey at 41%, 14% and 13% respectively, but at 13% tawari is probably high enough to justify labelling as monofloral tawari.

Sample 33 contains 70% clover with a little kamahi pollen at 18%. Tawari pollen at 3% is almost certainly too low to justify calling it monofloral tawari; it is a monofloral clover honey.

The absolute pollen count per 10 grams for samples 2, 32 and 33 were 14,500, 36,500 and 133, 500 respectively.

4. DISCUSSION

4.1 Processing

The internationally recommended size of each honey sample is 10 grams (Maurizio, 1975). This permits a representative pollen count, an accurate indication of pollen diversity, and ensures ease of counting. Only small amounts of honey were available for analysis in this study so the samples varied from 0.4 to 5 grams in size. The reliability of the count in the case of samples of less than c. 3 grams is suspect (Jones & Bryant 1998) as pollen recovery from such small samples is less efficient. In this study far fewer pollen grains per gram were recovered from many of the samples of 3 grams or less than from larger samples. Counting 500 pollen grains in smaller samples took up to three hours while counting of samples of 5 grams took as little as 20 minutes to complete.

One problem found in processing such a small amount was that all the recovered spores and pollen had to be placed on one slide. For an unknown reason the spread of pollen on some of the slides was not even and where pollen numbers were low they tended to migrate to the edges of the slides. Since the larger grains tended to concentrate at the margins, including the Lycopodium spores, these spores may be over-represented in the count of some samples where 500 grains were counted in less than one traverse of the slide. This can be circumvented by random counts from different parts of the slide.

We understand that most honey samples analysed in New Zealand by honey producers and scientists have used a water dilution system (Moar, 1985). This does not allow for the accurate representation of pollen from honey and a controlled alcohol-based dilution system with short centrifuging time is recommended (Jones & Bryant, 1998, 2001a, 2001b). The

problem with the water-based processing is that honey bees collect fresh pollen. This pollen can have a specific gravity of less than one and float on any water/honey medium. This seriously hampers the recovery of pollen from a centrifugal operation. If a liquid, like alcohol, with a specific gravity of less than one can be used then pollen recovery from honey should be greatly improved as all pollen will sink. The recommended easily available, non- toxic and inexpensive alcohol to use is ethyl alcohol or ETOH (Jones & Bryant, 1998).

An additional advantage in obtaining a higher percentage of pollen from honey is that a lower pollen count, 400-500 rather than 500 or more, can be done saving time. This is because the pollen recovered is more diverse and more accurately reflects the sources visited by the honey bee.

4.2 Analysis

It is well known that palynology alone cannot accurately determine monofloral honey. Jones & Bryant (1996) illustrated this by showing that a monofloral fireweed (Epilobium) honey sample in all other respects, taste, colour, aroma and chemical composition was a canola (Brassica) honey. It is clear from some of the pollen analyses on manuka honey that some samples have plenty of pollen and little nectar while others have appear to have a manuka flavour and colour but relatively little manuka pollen. Thus it is important that palynology be used along with other analytical techniques. Palynology, however, can be used quickly to form an initial determination as pollen is always an indicator of the flowers utilised by the honey bee.

5. RECOMMENDATIONS AND CONCLUSIONS

Of the 21 honeys labelled as monofloral (U in Table 1) most are accurately labelled or very close to being accurately labelled using palynological criteria. The six exceptions are

• 26, 30 (both labelled kanuka honey, but are mixed kanuka/lotus/clover honeys), • 33 (labelled tawari, but is a monofloral clover honey), • 35, 36 (both labelled kamahi, but are mixed Quintinia/manuka/kamahi and clover/manuka/ kamahi honeys, respectively), and • 39 (labelled rewarewa, but is a monofloral clover honey).

One kamahi monofloral, one borage monofloral and one tawari monofloral sample did not contain enough pollen to count.

All other named honeys probably contain enough of the nominated pollen type to justify its labelling as a being a prime pollen source, with some cases palynology showing that the honey could be called monofloral (Table 1), but not necessarily on the basis of the named source species.

1. Misidentified honeys should be further investigated to see if other quality standards were also suggestive of misidentification, or whether palynology has not accurately picked up the prime nectar source.

2. A normal range of 20,000 to 100,000 pollen grains per 10 grams of honey is expected (Moar 1985). For all honeys listed above, the absolute range varied greatly from 6,000 to 1,026,000, with an average of 20,000. This low number is probably the result of the size of the samples analysed and the difficulty of getting adequate pollen recovery from such small samples. The very low number of 6000 comes from a honey (sample 11) that did not dissolve fully during processing and has been excluded from the average calculation. Two other samples (26, 34) did not completely dissolve during processing also, but pollen recovery from these was excellent.

3. Modern alcohol-based processing must be used in order to obtain the maximum number and diversity of pollen grains from the honey under investigation, and in the best condition for identification (Jones & Bryant 1998, 2001a). Centrifuging should be used and not “crushing and straining” (Moar, 1985).

4. Since all previous methods of determining pollen percentages have been calculated on water-based pollen analyses, these are all suspect and need revision following the guidelines set by Bryant & Jones (2001). To do this would need close co-operation between researchers and honey producers and this is beyond the scope of this report. However, if pollen analysis is to be used as a guide to typing and analysis of monofloral honeys then it is important that this research be carried out: • to determine the accuracy of the work done by Moar (1985), and • to determine the pollen coefficients of other monofloral honeys.

5. A minimum of 10 grams of honey should be analysed to maximise the palynological results (Jones & Bryant 2001b) ensuring that a full range of pollen types are recovered. This also allows for very quick statistical analysis, a time consuming and costly part of the total cost of processing and analysing honey samples commercially.

6. While it is not recommended that palynology alone be used as a method of determining honey quality it has been well demonstrated both here and in the literature that pollen is a good indicator of the purity, quality, and source of honey.

7. No detailed attempt has been made to determine the New Zealand source of any of the honeys, but most clearly come from the North Island since so many samples contain Ixerba brexioides and Knightia excelsa pollen. This may become important if, for example, honey or other products from certain mite-infected localities needs to be monitored. According to Moar (1985) a combination of Quintinia/ kamahi/rata/Elaeocarpus is generally representative of Westland honeys and one sample (35) seems to fall into this category, although rata pollen is scarce. The borage sample (22) with plenty of viper’s bugloss pollen is also probably from the South Island.

6. ACKNOWLEDGEMENTS

The following are thanked for their support during the analysis and writing up stages of this project. The staff of Waikato University Honey Unit generously provided the various monofloral honey types. Karyne Rogers organised the receipt of the honey samples. Various versions of the manuscript have been reviewed by Richard Cook, Erica Crouch, Liz Kennedy, and Ian Raine who tried hard to get the text into a coherent format. Kirsty Hunt formatted the text into the science report. Prof. Vaughn Bryant of Texas A & M University gave generously of his time for discussions and provided reprints of his work on pollen analysis of honey.

7. REFERENCES

Bryant, V.M.; Jones, G.D., 2001: The R-values of honey: pollen coefficients. Palynology 25: 11-28. Johansson, T.S.K.; Johansson, M.P., 1968: Pollen analysis of honey – an appraisal. American bee journal 109: 57-58. Jones, G.D.; Bryant, V.M., 1996: Chapter 23D. Melissopalynology. Pp. 933-938 in: Jansonius, J.; McGregor, D.C. (eds). Palynology: principles and applications. American Association of Stratigraphic Palynologists Foundation, volume 3. Jones, G.D.; Bryant, V.M., 1998: Pollen recovery from honey. Pp. 107-114 in: Bryant, V.M.; Wrenn, J.H. (eds). New developments in palynomorph sampling, extraction, and analysis. American Association of Stratigraphic Palynologists Foundation. Jones, G.D.; Bryant, V.M., 2001a: Alcohol dilution of honey. Pp. 453-458 in: Goodman, D.K.; Clarke, R.T. (eds). Proceedings of the IX International Palynological Congress, Houston, Texas, U.S.A., 1996. American Association of Stratigraphic Palynologists Foundation. Jones, G.D.; Bryant, V.M., 2001b: Is one drop enough? Pp. 483-487 in: Goodman, D.K.; Clarke, R.T. (eds). Proceedings of the IX International Palynological Congress, Houston, Texas, U.S.A., 1996. American Association of Stratigraphic Palynologists Foundation. McIntyre, D.J., 1963: Pollen morphology of New Zealand species of Myrtaceae. Transactions of the Royal Society of New Zealand. Botany 2(7): 83-107. Maurizio, A., 1975: Microscopy of honey. Pp. 240-257 in: Crane, E. (ed.). Honey, a comprehensive survey. Crane, Russell & Co., New York. Moar, N.T., 1985: Pollen analysis of New Zealand honeys. New Zealand journal of agricultural research 28: 39-70.