Microscopic recognition and identification of fish meal in compound feeds

Figures & data

Table 1. Overview of material used in the study.

Order	Latin name	Common name	Set A: Barcoded	Set B: RIKILT collection
Anguilliformes	Anguilla anguilla	Eel	6
Clupeiformes	Clupea harengus	Herring	2	6909
	Sardina sp.	Sardine		1984
	Engraulis sp.	Anchovy		1999
Salmoniformes	Oncorhynchus mykiss	Rainbow trout	7
Gadiformes	Merlangius merlangus	Whiting	5	5201
	Merluccius merluccius	Hake	8
	Melanogrammus aeglefinus	Haddock	1
	Gadus morhua	Cod		5207, 6912
Perciformes	Scomber scombrus	Mackerel	9	5210, 6911
	Dicentrarchus aurata	Sea bass	13
Pleuronectiformes	Pleuronectes platessa	Plaice	12	5204, 6913
	Psetta maxima	Turbot	10	5208, 6914
	Limanda limanda	Common dab	3	5206
	Solea solea	Sole	4	5203
	Platichthys flesus	Flounder		5205
Scorpaeniformes	Chelidonichthys lucerna	Sea robin	11	5202, 6915

Figure 1. Seven types of bone fragments in teleost fish: a, b: type 1 (herring and anchovy); c: type 2 (trout); d, e: type 3 (tuna and mackerel); f: type 4 (cod); g: type 5 (plaice); h: type 6 (flounder); and i: type 7 (sea robin). Bar = 50 µm.

Figure 2. (colour online) Profiles of seven fish bone types for 17 different species. Indications of orders are arranged according to evolutionary development.

Figure 3. Filament extracted from the gill arch of sardine. Bar = 100 µm.

Figure 4. Different types of gill filament fragments: (top) anchovy, (bottom) cod. Note the presence of the distinct palisade layer (top image). Bar = 50 µm.

Figure 5. Share of cellular (types 1–5) and acellular (types 6 and 7) fish bone types for 17 different species. Indications of orders are arranged according to evolutionary development.

Table 2. Scale types and their distribution over the teleost fish orders represented in this study.

Type:	Clupoid	Clup/cycloid	Cycloid	Cycloid/gadoid	Gadoid	Ctenoid
Order
Anguilliformes					Eel
Clupeiformes	Herring, sardine	Anchovy
Osmeriformes			Capelin^a
Salmoniformes			Salmon, trout
Argentiniformes			Argentine^a			Argentine^a
Gadiformes			Hake		Cod, whiting, haddock, pollack^a, ling^a
Perciformes			Tuna, mackerel	Sea bass, weever^a, jack mackerel^a	Wolffish^a	Sea bass
Pleuronectiformes				Plaice, sole, flounder, turbot, common dab, halibut^a		Sole, common dab
Scorpaeniformes				Redfish^a	Sea robin	Redfish^a

Note: ^aFor illustrations and additional information, see Voehringer (1958).

Table 3. Recovery of fish bone fragments in mixtures of mineral:fish of 9:1 with four different fish meals. Results are shown for unstained material and material stained with alizarin. Type means share of acellular bone fragments.

			Unstained					Stained
			A priori		A posteriori			A priori		A posteriori
Source	Type	Slide	Mean	SD	Mean	SD	Recovery (%)	Mean	SD	Mean	SD	Recovery (%)
Denmark	High	A1	135	1	80.5	15.5	59.6%	149.5	10.5	101	1	67.6%
		A2	128.5	0.5	67	1	52.1%	113.5	0.5	99	4	87.2%
Norway	High	B1	199.5	7.5	114	6	57.1%	152	1	125	5	82.2%
		B2	151	9	97	4	64.2%	98	7	85	13	86.7%
Chile	Low	C1	119.5	1.5	100.5	9.5	84.1%	100.5	6.5	89	3	88.6%
		C2	116	7	101.5	5.5	87.5%	157	15	93	12	59.2%
Peru	Low	D1	116	6	108	1	93.1%	181.5	16.5	151	5	83.2%
		D2	109	3	115	3	105.5%	148.5	11.5	148.5	11.5	100.0%

Figure 6. Simplified evolutionary tree of teleost fish, based on Near et al. (2012) and Broughton et al. (2013). Rectangle sizes indicate the relative portion of species per group, with the share of cellular species (left, dashed) and acellular species (right, blank) after Kranenbarg (2005). The Ostariophysi is a paraphyletic superorder according to molecular evidence, but is maintained here for its relation with data on (a) cellular bone occurrence.

Table 4. Specificity (column indicated with 0%) and sensitivity (columns indicated with percentages above 0%) related to fish meal detection in proficiency tests over a period of six years, organised by EURL AP or RIKILT on behalf of IAG section feed microscopy. EURL results are recalculated to a ‘zero tolerance’ approach. EURL and IAG reports are referenced by their first author, Veys and van Raamsdonk, respectively.

Spike level of target:		0%	0.1%	0.5%	1%	2%
Target	Source	SP	SE
Fishmeal, in compound feed	van Raamsdonk et al. (2016)	0.98
Fishmeal, in compound feed	Veys et al. (2016)	1.0	1.0
Fishmeal, in compound feed	van Raamsdonk et al. (2015)	0.88
Fishmeal Denmark, in compound feed	van Raamsdonk et al. (2015)		0.95
Fishmeal Chile, in compound feed	van Raamsdonk et al. (2015)		1.0
Fishmeal, in compound feed	Veys et al. (2015)	1.0
		0.96
Fishmeal, in compound feed	van Raamsdonk et al. (2014)	0.92
		0.96
Fishmeal, in compound feed	Veys et al. (2014)	0.93
		0.93
Fishmeal, in compound feed	van Raamsdonk et al. (2013)	0.96				0.98
Fishmeal, in compound feed	Veys and Berben (2013)	0.96
		0.93
		0.91
Fishmeal, in compound feed	van Raamsdonk et al. (2012b)	0.91
		0.96
Fishmeal, in compound feed	van Raamsdonk et al. (2011)	0.98
Fishmeal (salmon), in compound feed	Veys et al. (2016)			1.0
Fishmeal, in compound feed with 0.1% MBM	van Raamsdonk et al. (2016)		0.84			0.98 1.0
Fishmeal, in compound feed with 0.05% MBM	Veys et al. (2014)		1.0
Fishmeal, in compound feed with 0.1% MBM	van Raamsdonk et al. (2012b)					1.0
Fishmeal, in compound feed with 0.1% MBM	van Raamsdonk et al. (2013)	0.94
Fishmeal, in poultry MBM containing feathers	Veys & Berben (2011)	0.75
Fishmeal, in compound feed with 1% feather	van Raamsdonk et al. (2015)	0.88
Spike level of target:		0%	0.1%	0.5%	1%	2%
Target	Source	SP	SE
MBM, in compound feed with 0.1% fish	van Raamsdonk et al. (2015)	0.76
		0.93
MBM, in fish feed containing fish meal	Veys et al. (2013)		0.93
MBM, in compound feed with 2% fish	van Raamsdonk et al. (2011)	0.98
MBM, in compound feed with 0.5% salmon	Veys et al. (2015)	0.65
MM, in fishmeal^a	Veys et al. (2012b)	0.87
MBM, in fishmeal with 10% salmon	van Raamsdonk et al. (2012b)	0.70
MBM, in fishmeal	van Raamsdonk et al. (2011)				0.95
Feather meal, in fish feed containing fish meal	Veys et al. (2014)		0.22
Feather meal, in fish feed containing fish meal	Veys et al. (2012)			0.31

Note: ^aA bovine meat meal without bone fragments was used in the presence of fish meal; four participants reported terrestrial animal material, three of them feathers.

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