Leonard W. D. van Raamsdonk

Microplastics have become ubiquitous in the environment and are increasingly found in a variety of matrices, including in animal feed. At the same time, the presence of plastics from packaging materials in animal feed is still prohibited in the European Union. This is a major barrier preventing certain organic waste streams from being repurposed as feed. However, issues associated with the presence of plastic particles in feed for ‘conventional’ livestock such as poultry and pigs (e.g. accumulation in stomach/other tissues) may not pose a problem to reared insects such as black soldier fly larvae (BSFL). In the hypothetical absence of such transfer, insects reared on feed contaminated with plastics could potentially be repurposed as feed. Such reuse would be a major opportunity for a more circular food chain. The aim of this exploratory study was to develop a method to determine the potential transfer of microplastics from the substrate to biomass of reared BSFL. Tested plastics consisted of low-density polyethylene (LDPE) of three different sizes, and polyethylene terephthalate (PET); to mimic the types of synthetic polymers commonly used for food packaging. The results showed no significant effects of dietary exposure of tested (micro-)plastics on larval yield and survival. We benchmarked fluorescence microscopy through Nile Red staining against a newly developed technique utilizing polarised light optical microscopy to identify plastic particles in BSFL. Tested LDPE particles were virtually absent from the post-exposure larvae. PET was recovered from the larvae, although it is unclear what the exact mechanism for this transfer was. More research is needed to validate these experimental findings with plastics of different types and particle sizes.

Visual examination of visually recognisable substances, including microscopy, focus on targets or contaminants such as particles of animal origin, plant seeds, spore bodies of moulds, sclerotia, packaging material, microplastic and ‘Besatz’ (everything that differs from the norm). The two principal results are counts (numbers) and weights for macroscopic methods, or presence/absence for microscopic methods. The level of detection equals at least the size of one unit, usually with a weight exceeding 1 mg, which is in the range of parts per million (ppm). These parameters do not follow a normal distribution but Poisson (counts), lognormal (weights) or binomial (Booleans) distributions, with effect on the interpretation of validation parameters. As for other domains, examination methods for visual monitoring need to be properly validated and quality control during actual application is needed. In most cases procedures for validation of visual methods are based on principles adopted from other domains, such as chemical analysis. A series of examples from publications show inconsistent or not correct implementations of these validation procedures, which stress the need for dedicated validation procedures. Identification of legal ingredients and composition analysis in the domain of visual examination relies on the expertise of the laboratory staff, therefore validation of a method usually includes the validation of the expert. In the view of these specific circumstances, a Guidance for quality assurance and control of visual methods has been developed, which are being presented and discussed in this paper. The general framework of the Guidance is adopted from ISO standards (17023, 17043, 13528). Part 1 of the Guidance includes the general background, theory and principles. Part 2 presents the actual validation procedures with experimental designs and equations for calculating the relevant parameters, and can be used as blueprint for a SOP in a quality management system. An EURL and NRL network for physical hazards is strongly recommended. Graphical Abstract

In the view of a circular economy, there is an increasing need for (re-)using animal by-products that have a wide range of applications and sufficient safety. Hydrolysates of animal proteins (HPs) are frequently used as feed ingredients. Nevertheless, clear criteria for legal use and methods for monitoring feed applications are not available. Here, a range of methods have been used and evaluated for characterizing a set of 26 samples of hydrolysed proteins, ‘hydrolysed’ feather meals and processed animal proteins (PAPs), with verification based on an additional set of eight samples. Methods included determination of ash content, sediment (mineral fraction) content, protein content, species identity, solubility, protein solubility, size exclusion chromatography and polyacrylamide gel electrophoresis (SDS-PAGE). After a comparison of results obtained with water and SDS, water was chosen as the solvent for environmental and occupational reasons. Typical HP samples have a protein content higher than 60%, a solubility exceeding 50% and a virtual absence of a mineral fraction. The first discrimination between HPs and PAPs could be based on the absence or presence, respectively, of a mineral fraction. An approach for HP characterization is designed using a Hydrolysation Index (HI) based on the fraction of peptides smaller than 10 kDa, the solubility of the sample and the fraction of soluble proteins. A simplified version (HI_s), exclusively based on the fraction of peptides smaller than 10 kDa and the solubility of the sample, shows a trend among the samples highly comparable to HI. Values for HI and HI_s exceeding 60% would characterise HPs. Feather meals, which are heat treated instead of treatment by a chemical process of hydrolysation, range among the PAPs and should not be indicated as “hydrolysed.” The HI_s can be used as an easy parameter for classifying HPs and for legal enforcement.

The presence is regulated of visually detectable seeds from a selection of toxic plants and fungi mycelium bodies (sclerotia) in feed (Directive 2002/32/EC) and in food (Regulation (EC) 1881/2006). Homogenisation as typical for chemical analyses is not applicable, and dedicated approaches are needed for visual examination methods. Visual methods require two parameters to characterise measurement uncertainties for both unit counts and unit weights. A new approach is to divide approximately 2 kg of sample material into four subsamples of approximately 500 g and to separately examine the four subsamples for numbers and particle weights of seeds or sclerotia. This study is the first to produce datasets on inhomogeneity among subsamples of a sample for visually detectable undesirable substances. Analytical thresholds were calculated from a simulation model and bootstrap procedures based on our data. The analytical thresholds assuring a controlled false-negative rate of 5% for decisions in compliance with legal limits depend on the diversity of the unit counts and weights, the level of the legal limit and the amount of material examined initially in the step-wise approach, either one or two subsamples. A procedure is proposed for examination in practice where only two subsamples, or alternatively even only one subsample, would be examined. If the resulting level of contamination exceeds the relevant threshold additional subsamples need to be examined as well. In most of the investigated cases, analytical thresholds could be established for the examination of just one subsample (500 g) taken from a sample of 2 kg. However, for ergot sclerotia in food with a legal limit of 200 mg kg⁻¹, at least two subsamples (1000 g) need to be examined in the first step. Other groups of visually detectable undesirable substances exist which need further attention. Graphical Abstract

Description of the subject. Specific densities of 13 samples of eight vegetable feed ingredients and of blood meal have been established. Objectives. The aim of the study was to use the parameter specific density for classification of a selection of feed materials. The range of specific densities for each of the tested materials could support the identification of ingredients in a compound feed. Ingredients in mixtures could be separated when different specific densities apply to these ingredients. Method. The share of the flotate and of the sediment of each of the tested materials has been established in duplicate along a range of increased specific density of the solvent mixture, from 1.22 g·cm-3 up to 1.52 g·cm-3, initially in four steps. Intermediate densities have been used where appropriate for collecting more detailed ranges. The specific densities which resulted in approximately 100% sediment and in approximately 100% flotate are used as indicators for the range of the specific density of that material. Results. The materials used appeared to show three different ranges of specific density: corn gluten and blood meal (1.12 – 1.32 g·cm-3), corn distillers’ grain, rapeseed kernel meal, soya kernel meal and wheat middlings (1.25 – 1.42 g·cm-3), and palm kernel meal, citrus pulp and beet pulp (1.35 – 1.55 g·cm-3). The specific densities range from those found for vertebrate muscles and insect fragments (below 1.3 g·cm-3) up to the specific density of bone fragments (exceeding 1.62 g·cm-3). Four mixed samples, each existing of two different ingredients at a ratio of 1:1 (w/w) were tested with solutions at different specific densities. The ingredients of each mixed sample appeared to separate in the expected fractions, predominantly in either the flotate or in the sediment. Conclusions. The experiments show that separation based on specific density can assist in the identification of feed ingredients in one or more combinations of sediment and flotate fractions.