Levels of toxaphene indicator compounds in fish meal, fish oil and fish feed

Chemosphere, Vol. 37, No. 1, pp. 1-11, 1998

Pergamon

1998 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0045-6535198 $19.00+0.00

PII: S0045-6535(98)00033-2 LEVELS OF TOXAPHENE INDICATOR COMPOUNDS IN FISH MEAL, FISH OIL AND FISH FEED

Katrin Oetjen, Horst Karl

Federal Research Centre for Fisheries, Institute for Biochemistry and Technology, Palmaille 9, D-22767 Hamburg, Germany (Received in Germany 1 October 1997; accepted 9 January 1998)

ABSTRACT

Three toxaphene indicator compounds were determined in fish oil and fish meal of different origin and raw material as well as in feed containing fish oil and fish meal. Fish oil and fish meal from South America contained no or only traces of the indicator congeners. The sum of the three compounds varied in fish oils from Europe between 13 lag/kg fat (sand eel oil) and 206 ~g/kg fat (cod oil). Varying concentrations were also determined in European fish meal and in feed. Toxaphene indicator compounds were as well detected in trout and salmon fed with toxaphene residues containing feed. Results indicate that feed can contribute to toxaphene contamination of farmed salmon from Europe. ©1998 Elsevier Science Ltd. All fights reserved

KEYWORDS: Toxaphene, fish oil, fish meal, fish feed

INTRODUCTION

Toxaphene (polychlorinated monoterpenes, ISO term: camphechlor) is a pesticide which was used worldwide and is still used in some countries. It has a broad spectrum of pesticidial activity and was applied for example to peanuts and soybeans. Beside pest control toxapbene was also used as a cattle dip and fish poison to eliminate undesirable fish from lakes. However, most of the toxaphene was applied as pest control in cotton production. It was used extensively in the southern and southeastern U S [ l ] , but also in other parts of the world. The global toxaphene usage since 1946 is estimated to be more than one million tons [2]. Toxaphene is a complex mixture of more than 200 compounds, mainly bornanes and bornenes with 5-9 chlorine atoms [1] As toxaphene is relatively persistent and highly mobile it can accumulate in biota

thousands of km away from toxaphene usage [1]. Toxaphene residues were detected in marine mammals and fish of the Arctic, Atlantic, North Sea and Baltic Sea [3 - 5] as well as in human milk [4,6] Toxaphene pattern found in biota is different to that of technical toxaphene [7,8]. In fish the three congeners

• Indicator compound 1 (Ind.1): 2-endo,3-exo, 5-endo,6-exo,8,8,10,10-octachlorobornane, • Indicator compound 2 (Ind.2):

2-endo,3-exo,5-endo,6-exo,8,8,9,10,10-nonachlorobornane and

• Indicator compound 3 (Ind.3): 2,2,5,5,8,9,9,10,10-nonachlorobornane

are found to dominate with at least 25-33% of total toxaphene [9]. In Germany these congeners are provided as indicator compounds for a maximum level oftoxaphene in fish, being 0.1 mg/kg wet weight (w.w.) for the sum of the three compounds [10].

Toxaphene was evaluated to have acute and chronical toxcity for aquatic and wildlife and may be a carcinogenic risk for humans [1]. Residues of these indicator congeners were detected in nearly all important fish species consumed in Germany [5]. A relationship between the fishing ground and the toxaphene content could not be established with the exception of differences between the residue contents of salmon from the northern and the southern hemisphere, respectively. In contrast to farmed salmon from northern Europe toxaphene residues could hardly be detected in farmed salmon from Chile. Toxaphene residues were also detected in farmed trout from northern Europe. For fish two contamination paths of organic pollutants are predominately discussed: Resorption from water through the gills and uptake via feed. The accumulation of chlororganic compounds by dietary exposure was recently demonstrated in a feeding experiment with channel catfish [11]. Salmon from northern Europe is usually reared in cages near shore in open seawater, allowing both contamination paths. Trout is normally reared in ponds or pools. Little is known about the contamination levels of toxaphene in commercial salmon and trout feed and their ingredients. Thus, various feed as well as fish meal and fish oil from northern Europe and South America as major feed ingredients were analysed for toxaphene residues. Additionally, toxaphene indicator compounds 1-3 were determined in some pelagic fish species being the raw material for fish meal and fish oil production

as well as in trout and salmon fed with three of the analysed feed.

MATERIAL AND METHODS

Standard materials

Toxaphene indicator compounds 1-3 were obtained from Ehrenstorfer (Augsburg, Germany). They were diluted with 2,2,4-trimethylpentane to known concentrations. All solvents used were of quality ,,for residue analysis" and tested as blanks.

Samples

Crude fish oil,

fish meal and feed were obtained from different manufacturers, wholesalers and t~ed

(Oncorhynchusmykiss) and salmon (Salraosalar) were obtained from European farms. Sprat (Sprattus sprattus), herring (Clupea harengus), small sand eel (Ammodytes lancea) and greater sand eel (Hyperoplus lanceolatus) were caught in 1996 in the North Sea during the 172th cruise of the German

producers. Trout

research vessel ,,Walther Herwig III% Sprat from the Baltic Sea were obtained from the fish market in Kiel (Germany). Krill (Euphausia~lperba) was caught in 1985 in the Antarctic.

Clean up

Fish meal and feed were homogenised by an electric mill. An aliquot containing about 2g fat was mixed with water (fish meal / feed : water = 1 : 3 based on weight). Fat was extracted with methanol / dichloromethane according to a modified method of Bligh and Dyer [12]. After removing the solvent, fat content was determined by weight. The clean up of these fat extracts and of the fish oils was carried out as described by Karl and Lehmann [13]. Toxaphene compounds and other chlorinated compounds as PCBs were isolated from the fat by gel permeation chromatography with Bio-Beads SX-3 (Bio Pad Laboratories, 200-400mesh) The mobile phase consisted of cyclo-hexane / ethylacetate (1/1). After removal of the solvent the extract was fractionated by chromatography with silica gel (water content: 1%). Most of the pesticides and PCBs were eluted with 80 ml n-hexane (first fraction), toxaphene congeners were eluted together with chlordane compounds and some other pesticides such as DDT and HCHs with 100 ml dichloromethane / n-hexane (2/8) (second fraction). Solvents were removed and the residues solved in 1ml 2,2,4-trimethylpentane.

4 Determination

Determination was carried out by capillary gas chromatography. A Hewlett-Packard model 5890 equipped with a 63Ni-ECD, split/splitless injector and a SE-54 capillary column 50m x 032mm ID, 0.251am film thickness was used for the detection of the organochlorine compounds. Results were confirmed by a Varian GC-3400 gas chromatograph equipped with a Finnigan Mat IST-40 ion trap mass spectrometer in the electron ionisation mode (El/MS), split/splitless injector and a DB-5 capillary column 60m x 025mm ID, 0.25 ~tm film thickness.

Detection limit and validation

The detection limit is 1.6 iag/kg fat with regard to each indicator compound. The corresponding detection limit based on wet weight depends on the fat content of the sample. Considering feed or fish meal containing 10% fat the limit is 0.16 ~tg/kg w.w. for each indicator compound.

No standard reference material containing certified amounts of toxaphene indicator compounds 1-3 is available at the moment. Recoveries were determined from fish meal spiked with three different levels of a standard solution containing 0.04 ~tg/ml of each indicator compound. In fish meal itself none of the indicator compounds was detectable. After addition of standard the three fish meal samples contained 0.46 ~g/kg w.w., 2.3 lag/kg w.w. and 4.6 lag/kg w.w. of each indicator compound, respectively. The samples were analysed as described above. Recoveries of the three indicator compounds varied between 104 and 128% ( 0.46 lag/kg w.w), between 75 and 87% ( 2.3 ~tg/kg w.w.) and between 79 and 93% ( 4.6 lag/kg w.w.). The fish meal contained 11% fat resulting in a detection limit of 0.18 ~tg/kg w.w..

RESULTS AND DISCUSSION

Fish oil and fish meal

Fish meal and fish oil are used as raw material especially for poultry, pig, fish and shrimp feed [14]. The eight most important fish meal and fish oil producing countries are Peru, Chile, Norway, Denmark, Iceland, USA, Japan and South Africa [15,16]. In 1996 these countries produced 4,736 000 t fish meal and 1,093 000 t fish oil of which 70% and 60%, respectively, was produced by Chile and Peru [ 15,16]. For production of fish oil and fish meal various fish species such as anchoveta, sardine, sprat, herring, mackerel and capelin are used. Fish meal and fish oil are also made out offish-processing waste. Those meals and oils are in general a mixture of various fish species.

Influence of the origin

Toxaphene indicator compounds 1-3 were determined in various fish oils and fish meals from northern Europe, in one fish oil and two fish meals from South America and in one oil of Antarctic krill. All fish meals and oils except for the krill oil were commercially produced and can be used as feed ingredients. Krill is an important part of the marine food chain. Therefore the contamination of krill can give hints on the contamination of marine fish of the southern hemisphere. Results of toxaphene determination are presented in Table 1. Levels of toxaphene indicator compounds 1-3 in fish meal were low, ranging between "below detection limit" and 7.3 lag Ind. 1-3/kg w.w.. Those fish meals which contained no detectable amounts of the indicator congeners were either produced in South America or were of unknown origin. The other fish meal of unknown origin contained only traces of toxaphene indicator compound 2. On the other hand in each fish meal from northern Europe at least two of the indicator congeners were detectable and the sum of Ind. 1-3 ranged between 1.9 and 7.3 lag/kg w.w.. Toxaphene content of fish oils of northern Europe varied considerably, between 13 and 206 lag Ind. 1-3/kg fat. In contrast to European fish oils only traces of toxaphene indicator compounds were detected in the analysed South American fish oil and none of the indicator compounds could be detected in Antarctic krill These results, i.e. no or only traces oftoxaphene indicator compounds in South American fish meal and oil in contrast to varying toxaphene concentrations in samples from northern Europe, reflect the situation found in farmed salmon on the German market.

Table 1: Toxaphene content in fish meal, fish oil, feed and fish

Fat No.

Sample

Origin, raw material

%

Toxaphene

Sum:

Ind.1

Ind.2

Ind.3

Ind. 1-3

~tg/kg

~tg/kg

~tg/kg

~g/kg wet wt.

wet wt.

wet wt.

wet wt.

1

Fish meal

unknown a)

11.8

0

0

0

0

2

Fish meal

unknown a)

13.4

0

0.2

0

0.2

3

Fish meal

Europe a)

11.1

2.1

3.5

1.7

7.3

4

Fish meal

Peru a)

11.1

0

0

0

0

5

Fish meal

Chile a)

11.1

0

0

0

0

6

Fish meal

Europe, Sprat

13.8

0.9

1.4

0

2.3

7 8

Fish meal Fish meal

Europe, Sprat Europe, Sprat

13.2 15.7

1.0 0.8

0.9 1.2

0 0

1.9 2.0

Sample

Origin, raw material

%

Sum-"

Toxaphene

Fat No.

lnd.l

Ind.2

ind.3

Ind. 1-3

~tg/kg

~gg/kg

Jtg/kg

~tg/kg

fat

fat

fat

fat

1

Crude fish oil

Europe, various fish

100

22

47

36

105

2

Crude fish oil

Europe, various fish

100

25

59

52

136

4

Crude fish oil

Europe, various fish

100

34

74

49

157

5

Crude fish oil

Baltic sea, sprat

100

15

22

8

45

6

Crude fish oil

Baltic sea, sprat

100

22

28

10

60

9

Crude fish oil

Baltic sea, sprat

100

16

11

0

27

10

Crude fish oil

Chile a)

100

3

4

0

7

11

Crude fish oil

Antarctic, krill

100

0

0

0

0

12

Crude fish oil

Germany, cod

100

54

99

53

206

13

Crude fish oil

Denmark, mackerel

100

11

18

14

43

14

Crude fish oil

Denmark, sand eel

100

6

7

0

13

~Lg/kg

~gg/kg

jtg/kg

~Lg/kg

wet wt.

wet wt.

wet wt.

wet wt.

2.0

Sprat

Baltic Sea

6.0

1.0

1.0

0

Sprat

North Sea

3.0

0.7

1.1

0

1.8

Sand eel

North Sea

2.7

0.2

0.4

0

0.6

Sand eel

North Sea

2.8

0.3

0.3

0

0.6

Herring

North Sea

0.9

0.8

1.8

0.6

3.2

Trout

Germany, farmed

4.2

0.4

0.8

0.4

1.6

Salmon

Norway, farmed

14.7

2.5

5.7

3.8

12.0

~g/kg

~g/kg

~kg

~g/kg

wetwt,

wetwt,

wetwt,

wetwt.

Trout feed

unknown a)

12.7

0.9

1.3

0

2.2

Trout feed

unknown a)

15.3

1.5

1.6

1.2

4.3

Trout feed

unknown a)

14.1

1.4

1.7

1.0

4.1

Trout feed

unknown a)

21.9

3.2

4.2

1.9

9.3

Trout feed Trout feed Trout feed

unknown a) unknown a)

16.5 20.3

0.4 0.5

0.6 0.9

0 0

1.0 1.4

unknown a) Norway

18.6 31.3 8.1

0.7 2.1

1.3 3.5

0 2.0

2.0 7.6

0

0

0

0

Salmon feed Pig feed b)

unknown a)

a) = market samples

b) = include fish meal

0 = below detection limit

Influence of the fish species used as raw material

European fish oils and meals of different raw material were analysed to study the influence of fish species on toxaphene content. Information on raw material and the results oftoxaphene analysis are given in Table 1. The analysed oils can be divided into three groups, i.e. oils with relatively high toxaphene content~ oils with moderate content and oils with low content. A cod oil and three oils produced from processing waste of various fish species showed the highest contents of Ind. l-3, being in the range of 105 to 206lag Indl-3/kg fat. Three sprat oils and a mackerel oil were moderately contaminated, ranging between 27 and 60 lag Ind. l-3/kg fat. The lowest content was found in a sand eel oil, being 13 lag Ind.l-3/kg fat. Toxaphene concentraion of the fish meals were at least one magnitude lower and differed in the same order. A meal produced from processing waste of mixed fish species contained 7.3 lag Ind. 1-3/kg w.w. being more contaminated than three sprat meals containing 1,9 - 2,3 lag Ind. l-3/kg w.w.. Although the number of analysed samples is low, the results indicate that toxaphene contamination of fish meal and oil depends on fish species used as raw material.

Comparison of fish (raw material) and fish oil

Considering that toxaphene content of fish oil and meal depends on fish species used as raw material, five pooled samples of sprat, sand eel, and herring which are widely used for fish meal and fish oil production in northern Europe were analysed. Each sample contained between 20 and 50 individuals. The results are presented in Table 1. Concentrations of toxaphene indicator compounds were low and varied between 0.6 and 3.2 lag Ind. 1-3/kg w.w,..

Table 2 gives a comparison oftoxaphene contents of fish oils with those of corresponding fish, calculated on fat base. Additionally, toxaphene content of the edible part of redfish and mackerel as found by Karl and Alder [9] were added in Table 2. Redfish was added in the table because the raw material used for oil production contained redfish offal. The sum of Ind. 1-3 of fish differed with regard to fish species in the same order as shown by fish oils, i.e. redfish showed the highest contamination level, mackerel and sprat were moderately contaminated and the lowest content was found in two sand eel samples. For each fish species the sum o f l n d 1-3 offish and corresponding fish oil were comparable. Thus toxaphene content of fish oil reflects the toxaphene content found in fish fat showing that toxaphene concentration in fish oil depends on fish species used as raw material.

8

Table 2: Comparison of toxaphene content of fish and corresponding fish oil

Fish species

Redfish

Sum Ind. 1-3

Sum Ind. 1-3

(~g/kg fat)

(p.g/kg fat)

fish

fish oil

114-786 (n=18)

1

105-157 (n=3)

Mackerel

15-82

(n = 7)

l

43

(n=l)

Sprat

33-57

(n = 2)

2

27-60

(n=3)

(n = 2)

2

13

(n=l)

Sand eel

23

3

i edible part 2 : whole fish 3 : fish oil produced from processing waste including redfish offal

Feed

Poultry and pigs consume a big part of the world's fish meal production, but the amount offish oil and meal in poultry and pig feed is much lower than the amount in trout or salmon feed. Trout feed usually contains 30% fish meal and 10% fish oil, but also feed enriched with about 15% fish oil resulting in feed with a total fat content of up to 25 % are available [17]. Salmon feed usually contains 50 % fish meal and 15 % fish oil. High energy feed for salmon contains more fish oil resulting in a total fat content of up to 35% [17]. Four salmon feed, seven trout feed and one pig feed, all commercially produced in northern Europe, were analysed. All feed contained fish meal and various amounts of fish oil except for the pig feed resulting in different fat content. None of the three indicator compounds could be detected in a pig feed sample (Table 1). All the other feed contained toxaphene with the sum of Ind. 1-3 varying between 1.0 and 9.3 ~tg/kg w.w.. Although most of the fat in trout and salmon feed originates from fish oil and fish meal no relation between fat content and toxaphene content could be established (Figure 1). However, the varying toxaphene contents of the feed samples can be explained by the addition of differently contaminated fish oil and meal. Considering the results obtained for fish meal and fish oil, the toxaphene levels of feed depends mainly on the fish species, used as raw material for both ingredients.

Figure 1: Toxaphene and fat content of various feed

~'

10 9

35

; .'r..C~.,,=.,,E~

30

\

8

A

25 ~

15 Q.'~

3

m x

2

1ou

10 u. m ~

0 .

0

5

"5

5

5

"5

5

£ I---

£ I--

£ I---

£ I---

£ I---

£ I---

__

,.

.

¢o

"5 £

(U

F--

E --

5

O3

Transfer

of contaminants

from feed to fish

To get first information about possible accumulation of toxaphene from dietary exposure the edible part of salmon and trout fed with three of the analysed feed (sample-no.2, 3 and 8 in tablel) in commercial European fish farms were analysed for toxaphene residues. The analysed salmon was reared in a fjord, the trout in a roofed over pool with a well water flow through. The salmon feed contained 7.6 lag Ind. 1-3/kg w.w. and the trout feed

on an average 4.2 lag Ind.l-3/kg w.w., respectively. Pooled samples of three salmons and five

trouts, respectively, contained 12.0 lag Indl-3/kg w.w. and 1.6 lag Ind.l-3/kg w.w. indicating toxaphene resorption from the feed. As the salmon has been reared in open water additional toxaphene intake from water can not be excluded. However, the presence of toxaphene in the trout sample clearly indicates that toxaphene was resorpted from the feed, since toxaphene has never been used in the region of the trout farm.

CONCLUSION

Varying concentrations of toxaphene indicator compounds 1-3 were determined in fish meal and fish oil as well as in salmon and trout feed containing fish meal and fish oil. Toxaphene content of fish meal and fish oil of South America was low in contrast to content of samples from northern Europe. Toxaphene content of European fish oils ranged between 13 lag ind. l-3/kg fat (sand eel oil) and 206 lag ind. l-3/kg fat (cod oil) and depended on fish species used as raw material. Toxaphene content of commercial fish oils corresponded to toxaphene content of fish fat. For feed no relationship between toxaphene content and fat content could be

10 established. Varying toxaphene contents of feed can be explained by use offish meal and especially fish oil of varying contamination levels. Toxaphene was also detectable in trout and salmon fed with toxaphene containing feed. The results indicate that feed can contribute to the toxaphene found in farmed salmon from northern Europe, provided that European fish meal and fish oil is used for feed production. Otherwise the results indicate that South American feed can transfer only little toxaphene if at all into farmed salmon, provided that South American fish meal and fish oil is used for feed production.

REFERENCES

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11 [12] J. Oehlenschlager, Eine universell verwendbare Methode zur Bestimmung des Fettgehaltes in Fischen und anderen Meerestieren, Infit Fischw. 33, 188-190 (1986) [l 3] H Karl, I Lebmann and K Oetjen, Levels of chlordane compounds in fish muscle, -meal, -oil and -feed, publication submitted [ 14] A.G.J. Tacon, Dependence of intensive aquaculture systems on fishmeal and other fishery resources,

Aquaculture Newsletter 6, 10-16 (1994) [15] Anon., Fishmeal boom in last quater, Globefish Highlights 1, 17-18 (1997) [16] Anon., Lower fish oil output, Globefish Highlights 1, 19 (1997) [17] GW. Chamberlain, Aquaculture trends and feed projections, WorldAquaculttlre 24, 19-29 (1993)