Verification of Egg Farming Systems from The Netherlands and New

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Verification of egg farming systems from the Netherlands and New Zealand using stable isotopes Karyne M. Rogers, Saskia M. van Ruth, Martin Alewijn, Andy Philips, and Pam Rogers J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01975 • Publication Date (Web): 05 Sep 2015 Downloaded from http://pubs.acs.org on September 6, 2015

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Journal of Agricultural and Food Chemistry

Verification of Egg Farming Systems from the Netherlands and New Zealand using Stable Isotopes Karyne M. Rogersa*, Saskia van Ruthb, Martin Alewijnb, Andy Philipsa, Pam Rogersa a

National Isotope Centre, GNS Science, 30 Gracefield Road, Lower Hutt 5040, New Zealand

b

RIKILT Wageningen University and Research Center, P.O. Box 230, 6700 AE Wageningen, the Netherlands

*Corresponding author Tel. +6445704636, Fax. +64 4 5704656, E-mail. [email protected]

1 ACS Paragon Plus Environment


ABSTRACT 1

Stable isotopes were used to develop authentication criteria of eggs laid under cage, barn,

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free range and organic farming regimes from the Netherlands and New Zealand. A training

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set of commercial poultry feeds and egg albumen from 49 poultry farms across the

4

Netherlands was used to determine the isotopic variability of organic and conventional feeds,

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and assess trophic effects of these corresponding feeds and barn, free range and organic

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farming regimes on corresponding egg albumen. A further 52 brands of New Zealand eggs

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were sampled from supermarket shelves in 2008 (18), 2010 (30) and 2014 (4) to characterize

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and monitor changes in caged, barn, free range and organic egg farming regimes. Stable

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carbon (δ13C) and nitrogen (δ15N) isotopes of 49 commercial poultry feeds and their

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corresponding egg albumens reveals that Dutch poultry are fed exclusively on a plant-based

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feed and that it is possible to discriminate between conventional and organic egg farming

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regimes in the Netherlands. Similarly, it is possible to discriminate between New Zealand

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organic and conventional egg farming regimes, although in the initial screening in 2008,

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results showed that some organic eggs had similar isotope values to conventional eggs,

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suggesting hens were not exclusively receiving an organic diet. Dutch and New Zealand egg

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regimes were shown to have a low isotopic correlation between both countries, because of

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different poultry feed compositions. In New Zealand, both conventional and organic egg

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whites have higher δ15N values than corresponding Dutch egg whites, due to the use of

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fishmeal or MBM (Meat and Bone Meal, which is banned in European countries). This study

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suggests that stable isotopes (specifically nitrogen), shows particular promise as a screening

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and authentication tool for organically farmed eggs. Criteria to assess truthfulness in labeling

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of organic eggs were developed and we propose that Dutch organic egg whites should have a

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minimum δ15N value of 4.8 ‰ to account for an organic plant derived diet. Monitoring of

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New Zealand egg isotopes over the last 7 years suggests that organic eggs should have a 2 ACS Paragon Plus Environment

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minimum δ15N value of 6.0 ‰, and eggs falling below this value should be investigated

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further by certification authorities.

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KEYWORDS : authenticity; barn; carbon; diet; egg albumen; free range; isotope;

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Netherlands, New Zealand; nitrogen; organic; supermarket

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INTRODUCTION

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Regulation compliance by egg farmers and corresponding feed manufacturers as well as

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correct labeling of farming regime by egg producers and distributors are key issues affecting

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the organic and free range poultry egg industry worldwide. Reports estimate that the

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Australian free range egg industry does not have sufficient free range hens to produce the

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numbers of “free range” eggs sold annually in Australia and that up to one in six eggs

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(around 37 million eggs) may be fraudulently sold as free range.1 Past egg scandals also

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include large volumes of Irish battery farm eggs sold to UK Tesco’s as free range or organic

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from 2000 to 2005. This was detected by sharp eyed inspectors using ultraviolet light scans

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which showed up cage marks due to deposition of fluorescing particles from indoor lights on

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the egg, but not where it touched the cage.2 Recently in 2013, more than 100 farms in

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Germany were alleged to have sold eggs laid by battery hens as organic or free range3, and

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almost 2.5 million falsely labeled caged eggs were sold through supermarkets as free range in

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New Zealand in 2014. These large scale fraud events highlight the need and utility of low

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cost, rapid authentication tools for higher value food products such as free range and organic

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eggs to verify added value claims4-6. Previous egg authentication studies have shown good

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potential for egg authentication of laying regimes based on stable isotopes and carotenoid

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profiles7-10, but did not investigate datasets between countries.

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A study of different laying regimes using New Zealand eggs demonstrated the usefulness

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of nitrogen (δ15N) isotopes of egg components to unravel dietary differences and hence



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laying regime7. Conventionally grown poultry feeds have lower δ15N values as they comprise

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of plant based components grown using synthetic fertilizers with δ15N values ranging from -2

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to 3 ‰,5 while organic poultry feeds tend to have higher δ15N values (>3 ‰) as they are

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grown using organic manures (either animal manure or plant composts). These isotopic

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differences are transferred through the poultry diet and into the eggs providing a unique

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tracer relating back to the hen’s diet. However other factors can complicate and increase δ15N

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values in poultry diets such as feeds which contain animal and fish protein

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(blood/bone/tissue) which are legally permitted in New Zealand (up to 15% in organic

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poultry feeds). Furthermore farming systems, such as free range or organic hens, which allow

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frequent outdoor access can also increase the δ15N values of egg albumen as hens can roam

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and forage freely in pasture and soils, picking up insects, seeds and grass. These dietary

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components also take on higher δ15N values due to the ongoing deposition of poultry manure

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and the use of these nutrients for pasture growth over time. When analyzed for stable isotopes,

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these higher protein dietary inputs and nutrient transfers from poultry manure may confound

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eggs sampled from conventional free range farming systems with eggs from organic farming

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systems by increasing the overall δ15N values, giving similar δ15N values to those of organic

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eggs. However, where the δ15N values of organic eggs are lower than expected, it is usually

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due to a non-organic diet, unless the hen’s diet is comprised mainly of N-fixing plants (i.e.

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lucerne, alfalfa or peas) or plants grown on green composts used to fertilize feed crops.

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New Zealand organic eggs sampled in a previous study7 showed a wide range of δ15N

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values suggesting the likelihood of fraudulently labeled eggs. Several organic egg producers

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were found to be supplying organic eggs with much lower δ15N values than other organic

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farms. Their isotope values were similar to eggs from conventional farming regimes,

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suggesting that the hens were not receiving an organic diet and/or they were not free ranging

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or had very limited outdoor access. 4 ACS Paragon Plus Environment

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Organic farming practices world-wide aim to enhance biodiversity, biological cycles and

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soil biological activity to achieve economically and ecologically sustainable systems. As yet

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there are no recognized scientific testing standards to distinguish free range and organic laid

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eggs (with outdoor access) from caged or barn laid eggs (indoor access only).11 Evaluation

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frameworks by organic bodies use compliance and auditing systems to ensure eggs and

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commercial feed comply with regulations.9 However with the high price of compliance

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systems, some producers and distributors self-regulate, and do not adopt any national

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certification standards, leaving the opportunity for misinterpretation of regulations or

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mislabeling to arise. Although past verification of egg products using stable isotopes7 or

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carotenoid profiling8, 10 has been shown to identify cases which do not entirely fit within

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normal datasets, these can be more rigorously evaluated if they meet certain specifications.

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This screening potential holds great promise for the egg industry especially if it can be shown

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to be applicable on an international, country or regional scale.8-10, 12

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We investigate for the first time a large training set of poultry feed and corresponding egg

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albumen from barn, free range and organic hens collected in the Netherlands in 2009 to

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understand isotopic feed variation, trophic transfer to egg albumen and provide guidelines for

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egg verification specific to Dutch farming systems. Data from a previous study of New

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Zealand eggs and critical feed ingrediants4 is compared to new isotope data of eggs acquired

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in 2010 and 2014 to investigate if any significant feeding changes have occurred during this

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time. Finally, stable isotope egg profiles from barn, free range and organic farmed egg

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albumen from both countries are compared to determine if isotope ranges used to verify and

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authenticate country specific farming regimes are internationally applicable.

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MATERIALS AND METHODS

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Dutch training set. A training set of eggs and corresponding feed were collected from 49

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Dutch layer hen farms in May 2009. Organic (24), free range (12) and barn (13) farms were

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selected with help of the Dutch poultry and egg product board and the Dutch organic produce

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certification body SKAL. The selection was balanced with regard to sampling a range of

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geographical locations (north, east, south, west) and farm size per production system.

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Organic farm groups consisted of farm sizes 50,000 hens. The differences in categories between organic and

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conventional productions reflect the particular farm populations, where organic farms are

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usually smaller sized than conventional farms. Information on the breed, age of the laying

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hen flock, as well feed samples (type and supplier) was collected along with eggs. Fresh eggs

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and feed were stored at 4º C until analysis.

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New Zealand test set. Thirty different brands of (7 caged, 7 barn, 12 free range and 4

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organic) eggs were purchased from local poultry farmers and several grocery stores in Lower

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Hutt, New Zealand in 2010. They were integrated with published results from 18 brand of

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eggs (5 caged, 3 barn, 6 free range, 4 organic) sampled in 20087 to monitor changes within

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the egg industry during this time. A further 4 brands of organic eggs were sampled in 2014 to

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provide a final check on organic labeling practices. The eggs represented a range of

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producers from small cottage industries with 20-30 hens to large industrial operations with

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20,000-50,000 hens.

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Sample preparation and analyzes. Nine eggs from each farm or egg brand were sampled

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for isotope analyzes. Albumen from three eggs were separated from yolks, pooled in

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triplicate in order to avoid single anomalous results (i.e. if a particular hen was less free

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ranging than others), and freeze-dried. Egg albumen and feed samples were homogenized,


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ground and weighed in duplicate. Approximately 1.5 mg of albumen and 3-10 mg of feed

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were transferred into 6x4 mm tin capsules. Carbon and nitrogen content and isotopic

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composition of Dutch and New Zealand egg albumen and feed samples were analyzed at the

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GNS Science Stable Isotope Laboratory, Lower Hutt, New Zealand, using a Europa Geo

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20/20 (PDZ Europa Ltd. U.K.) isotope ratio mass spectrometer, interfaced to an ANCA-SL

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elemental analyzer (PDZ Europa Ltd. U.K.) in continuous flow mode (EA-IRMS). The

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carbon dioxide gas was resolved from nitrogen gas using gas chromatographic separation on

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a column at 65° C, and analyzed simultaneously for isotopic abundance as well as total

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organic carbon and nitrogen. International and working reference standards (NIST-N1,

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IAEA-CH6, leucine, wheat flour and beet sugar) and blanks were included during each run

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for calibration.

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Isotopic ratios (13C/12C and 15N/14N) are expressed as isotopic deviations δ defined as: δ(‰) =

Rs – RRef x 1000 RRef

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where Rs is the isotopic ratio measured for the sample and RRef that of the international

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standards. The δ13C value is relative to the international Vienna Pee Dee Belemnite (VPDB)

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standard, and the δ15N value is relative to atmospheric air. Results are expressed in δ (‰)

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versus the specific reference. Analytical precision (standard deviation or SD) of standards

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and samples are within ±0.1 ‰ for carbon and ±0.2 ‰ for nitrogen (1σ).

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Statistical analysis. Nitrogen and carbon isotopic data of Dutch and New Zealand eggs

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were subjected to multi-factor analysis of variance (MANOVA; including country, farming

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regime, farm, replicate analyses, year factors) to investigate the effects of two or more

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independent variables for significant effects on two or more metric dependents (in this case

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nitrogen and carbon isotope ratio values). It allows a joint analysis of each dependent rather

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than performing several univariate tests, thus avoiding multiple testing risks. Fisher’s least

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significant difference (LSD) tests were carried out to determine significant differences among



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groups using XLSTAT 2014.3.02 (Addinsoft, Paris, France). A significance level of P

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2.7 ‰. Poor isotopic separation between some free range and organic feeds suggested that

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some free range farms may be using organic feeds, even though their farming system is not

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labeled organic, and/or there was some other significant source of 15N enriched protein (plant,

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animal or inorganic supplement) component included in feeds.5, 13-18

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Carbon isotopes gave an indication of the level of C3 and C4 feed components18, with

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more positive δ13C values (< -18 ‰) suggesting a higher proportion of C4 plants (ie. maize)

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in the feed, where;

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δ13C feed = X (δ13C C3 plants + additives) + Y (δ13C C4 plants)

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where X +Y = 1, δ13C C3 plants ~ -25 to -28 ‰, and δ13C C4 plants ~ -10 to -12 ‰. The feeds

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all showed a wide range of δ13C values from -16.3 to -22.3 ‰, and using the above mixing

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model suggests feeds comprise between 20-70% C4 plants (ie. maize). Although barn feeds

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had the widest range of values of the three groups of feed, barn and free range feeds are 8 ACS Paragon Plus Environment

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farmed conventionally suggesting the δ13C isotopic feed differences are mostly determined

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by different combinations of feed. Where feeds had more positive δ13C values, it is likely that

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there was a higher C4 plant component (i.e. maize) contained in the feed and a lower reliance

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on C3 plant components (i.e. wheat and soy)18, 19, although the most positive feed δ13C value

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was an omega-3 enriched barn feed at -16.3 ‰. Given that another omega-3 enriched feed

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had a δ13C value of -20.2 ‰, the variation in δ13C values is most likely due to a variation in

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C3 and C4 plant feed components than the dominance of specific omega-3 enriched additives.

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The δ13C value of conventional and organic feeds will most likely vary seasonally as the use

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of C3 and C4 plants in poultry feed is usually indexed to the price of these feed components in

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any one year. Barn and free range eggs sell for lower prices than organic eggs and are likely

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to use the cheapest ingredients obtainable at the time while retaining a balanced diet.

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Dutch egg albumen. The carbon and nitrogen isotope values of egg whites from 49 farms

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(13 barn – including 2 omega-3 enriched egg farms, 12 free range and 24 organic farms)

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were investigated. Values ranged from -22.5 to -17.3 ‰ for δ13C, and 3.4 to 6.2 ‰ for δ15N

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(Figure 2, Table 1). Isotopic discrimination between organic and barn egg whites was

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feasible using δ15N values, although discrimination between all three groups (organic, barn

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and free range egg whites) or between free range and organic eggs was not clear (Table 1).

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A histogram was constructed to demonstrate the frequency distribution of Dutch egg white

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δ15N values from different farming regimes (Figure 3). All Dutch organic egg whites tested

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in this study had δ15N values > 4.8 ‰. This contrasted with barn and free range egg whites

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where the majority had δ15N values below 4.8 ‰, apart from four barn and three free range

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farms which had slightly higher values ranging between 4.8 and 5.2 ‰. Higher δ15N values

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in egg whites from the three free range farms may be due to use of organic feeds, increased

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15

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usually included in conventional feeds. The wide range of free range egg δ15N values (from

N from foraging in manured fields or some other higher protein feed supplement not



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3.6 to 5.2 ‰) may indicate poultry feed composition differences, but based on feedback from

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farmers, would more likely indicate that some hens are experiencing more opportunity to free

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range than others.

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Two barn farms supplemented their feed with omega-3 products to produce omega-3

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enriched eggs. These egg whites also had a tight range of δ15N values (c. 5.1 ‰) although in

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this training set, only two farms (six composites of 3 eggs) were sampled. These values are

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above the usual range for barn raised eggs possibly due to the added omega-3 supplement

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which is usually derived from flaxseed or fish oil (increased 15N from protein). However their

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corresponding feeds do not indicate any noticeable enrichment in

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for hens laying omega-3 eggs were 2.5 and 2.4 ‰ respectively) values expected with fish oil,

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so either there is a further omega-3 supplement included in their diet which was not tested, or

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these hens are acquiring enriched dietary nitrogen from an unknown source, not typically

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used in conventional feeds7. The inclusion of barn omega-3 eggs in the dataset (Table 1)

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skews the barn egg white δ15N values from 4 ‰ (without omega-3 eggs) to 4.3 ‰ (with

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omega-3 eggs), also providing a higher SD than the other farming regimes.

15

N (δ15N values of feeds

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It was not possible to discriminate between farming regimes based on δ13C values alone.

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Barn and free range egg whites had a narrower range of δ13C values than organic egg whites.

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A large majority of organic egg whites had more positive δ13C values than barn and free

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range egg whites due to a larger maize (C4) component in the feeds. Maize is a commonly

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grown organic crop, and is frequently diverted into animal feeds. Two organic farms had egg

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whites with significantly more negative δ13C values than all other samples. The hens which

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laid these eggs may have had a higher proportion of C3 components in their feed (i.e. wheat

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or soy beans), or significant access to fresh vegetation (grass or other green crops).5, 7

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Trophic effects (TE) or ∆15N (egg-feed) are examined across different Dutch farming

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regimes (Figures 4 and 5) to investigate differences between feeds and feeding regimes on 10 ACS Paragon Plus Environment

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the resultant egg products from each farm. Barn, free range and organic raised eggs have an

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average TE of 2.1, 1.9 and 1.8 ‰ respectively (Table 1), consistent with strict herbivores

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which have TE values < 2 ‰.20, 21, 22 The slightly elevated TE in barn raised eggs (from farm

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Size ‘E’, Table 2) is due to the skew from the two barn omega-3 egg farms, which have TE’s

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of 2.5 and 2.8 ‰ respectively. When the omega-3 egg data are removed, barn eggs have an

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average TE of 2.0 ‰. Higher TE values (TE > 2 ‰) in omega-3 eggs may be due to added

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dietary omega-3 derived protein (such as algae or flax oil,) rather than foraged worms and

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insects, which would only contribute only a fraction of the daily protein intake.

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Elevated TE values occur in all farming regimes (Table 2) although 60% of farms

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producing barn raised eggs had a TE > 2‰, while free range and organic farms only had 25

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and 17 % respectively. Higher TE may be caused by cannibalism, which can occur more

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frequently in barn raised poultry due to their enclosed habitat, lack of outside foraging

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opportunities and the obvious lack of animal protein in their diet. In this study, cannibalism is

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a more plausible explanation for higher TE’s than the inclusion of added animal protein in

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feed due to strict regulations around MBM in European (EU) animal feeds. A meat product

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ban in EU poultry feed was instigated in the mid 1990’s due to the BSE (Bovine spongiform

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encephalopathy) or ‘mad cow disease” crisis in Europe where more than 200 people (mainly

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in the UK) died of Creutzfeldt-Jacob disease.

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It is also interesting to note that the dietary feed difference of 1.2 ‰ between average δ15N

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(organic-barn feed) is almost fully retained in the egg whites, as the average δ15N (organic-

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barn egg whites) is 1.0 ‰. Based on these similarities between TE’s, it is possible to confirm

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δ15N values of egg whites from its corresponding feed (excluding omega-3 feeds, Figure 5).

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New Zealand eggs. Egg whites from four egg farming regimes studied in New Zealand in

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20087 were compared with a new suite of eggs collected in New Zealand in 2010 and 2014 to

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monitor changes of egg white δ15N values. The previous 2008 study suggested that some 11 ACS Paragon Plus Environment


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hens producing free range and organic eggs had diets that were no different to cage or barn

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hens on the basis of δ15N values, and the consumer was being misled. These conclusions

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were based on the small (1.4 ‰) difference between δ15N (average cage-average organic) egg

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values, although cage and organic eggs farming regimes had much bigger SD’s (Table 3).

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An evaluation of eggs from 2010 showed the difference between mean cage and organic

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eggs δ15N values had only increased slightly from 1.4 ‰ (in 2008) to 1.6 ‰ (in 2010, Table

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3). However a stepwise change in feed composition had occurred over this time, and each

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farming regime was more enriched relative to their 2008 mean values (up to 1.2 ‰ for caged

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eggs, 1.7 ‰ for barn eggs, 0.1 ‰ for free range eggs and 1.3 ‰ for organic eggs; Table 3,

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Figure 6), reflecting a significant dietary composition shift of New Zealand poultry feeds.

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In 2008, New Zealand poultry feeds would have consisted of mostly plant derived

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products (similar to the Netherlands), yet by 2010 this study shows the isotopic composition

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of New Zealand eggs from cage, barn and organic hens had both higher δ15N and δ13C values

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than those from 2008 due to a trophic level increase from leguminous proteins23 to animal

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protein18 (where estimated δ15N and δ13C values of grass-fed beef is c. 8 ‰22 and c. -20 ‰24

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respectively). Feed manufacturers have confirmed that up to 15% MBM (from

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conventionally raised animal origin) has been included in New Zealand organic poultry feeds

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since 2010. The addition of MBM can be attributed to economic and practical changes

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affecting New Zealand poultry farmers and feed growers over this time. Anecdotally,

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cannibalism frequently occurs in flocks which suffer protein deficiency and personal

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accounts from several New Zealand farmers indicated the need for poultry feed

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manufacturers to include more animal protein in feeds to counteract this unwanted trait in

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non-caged farming systems.

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A further four brands of organic eggs were rechecked in 2014 to confirm the 15N increase

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found in 2010. The δ15N values of organic eggs sampled in 2014 were found to be very 12 ACS Paragon Plus Environment

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similar to those from 2010, although the δ13C values were found to be slightly more positive

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(-19.6 ‰) than both the 2008 and 2010 values (-21.3 and -21.2 ‰ respectively). The variance

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(SD) of all δ13C organic egg values decreased from 3.0 ‰ and 2.9 ‰ (in 2008 and 2010, n=4

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for each year) to 1.2 ‰ (in 2014, n=4), suggesting a more uniform feed is now being used by

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egg producers. Nonetheless, as organic plant-only poultry feeds are significantly more

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expensive than conventional feed, the use of non-organic dietary MBM included in New

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Zealand organic feeds conveniently lowers feed costs for organic poultry farmers, although

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the undisclosed use of MBM in organic poultry feed may be of interest to organic vegetarian

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egg consumers.

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When taking the three New Zealand sampling events between 2008 and 2014 into

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account (Table 4), key trends between farming regimes were observed for the δ15N values but

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not for the δ13C values. Organic eggs presented highest δ15N values, followed by barn and

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free range eggs which did not differ significantly, and finally cage eggs.

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In 2008, organic egg whites had a δ15N value of 5.8 and an SD of 1.7 ‰. Even though

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only four organic brands were sampled at this time, it was noted that two organic egg farms

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had considerably lower δ15N values than the others. We compare these values with the

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average δ15N value and SD of organic egg whites sampled in 2010 and 2014: 7.1 ‰ in 2010

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and 6.9 ‰ in 2014, compared to 5.7 ‰ in 2008; and 1.0 ‰ in 2010 and 1.2 ‰ in 2014,

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compared to 1.7 ‰ in 2008. Organic eggs sampled in 2010 and 2014 have δ15N values which

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are consistently higher and their SD’s are smaller than 2008 values, confirming discrepancies

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in the integrity of some organic eggs sampled in 2008 and indicating potentially fraudulent

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labeling of organic eggs.

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This observation was further supported by MANOVA test of the organic egg white data

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(Table 3) with conventional (cage, barn and free range) egg whites. Fisher LSD test showed

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only small differences between δ15N values of conventional (cage, barn and free range) egg 13 ACS Paragon Plus Environment


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whites from 2008 compared to 2010. Fisher LSD test results also showed a complete overlap

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between δ15N values of organic and free range egg whites sampled in 2008 (Table 3).

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Comparison between δ15N values of Dutch and New Zealand egg whites.

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A comparison between the δ13C and δ15N isotopic compositions of Dutch and New

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Zealand eggs under different farming systems was undertaken using MANOVA. Statistical

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analyses focused on the data per country. For the Dutch egg whites a significant difference in

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δ15N values was observed between organic eggs, and barn/free range eggs (Table 1). The

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δ13C values of the Dutch egg whites showed significant differences between free range eggs

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and organic/barn eggs (Table 1).

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A comparison between δ15N values of egg whites from the Netherlands and New Zealand

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was made, including the three sets of New Zealand eggs sampled in 2008, 2010 and 2014

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(Figure 7). Dutch egg whites (sampled in 2009) corresponded more closely with New

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Zealand egg whites sampled from 2008 (Figure 7, Table 5) rather than those from 2010 and

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2014. Dutch barn and organic egg whites did not differ significantly with their respective

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New Zealand counterparts sampled in 2008 (P

Verification of Egg Farming Systems from The Netherlands and New

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