METHOD FOR DETERMINATION OF VITELLOGENIN AS BIOMARKER FOR AN EXOGENOUS OESTROGENIC EFFECT ON FISH

Abstract

The invention relates to a method for the determination of vitellogenin as a biomarker or end point for an exogenous oestrogenic effect on fish. The problem addressed by the invention is the creation of a non-destructive method in which the laboratory animals do not have to be killed. The method also should not necessitate any additional use of laboratory animals for carrying out tests on oestrogens, anti-oestrogens, and endocrine disruptors. This problem is solved by a method for the determination of vitellogenin as biomarker for an exogenous oestrogenic effect on fish comprising the method steps of taking a skin mucus sample from a fish by swabbing, transferring the skin mucus sample to a reaction vessel, homogenising the skin mucus sample, and taking an aliquot of the skin mucus sample for vitellogenin determination by means of an ELISA method of detection.

Claims

1. A method for determining vitellogenin as biomarker for an exogenous estrogenic effect on fishes, the method comprising: a) taking a skin mucus sample of a fish by swabbing, b) transferring the skin mucus sample to a reaction vessel, c) homogenizing the skin mucus sample, and d) removing an aliquot of the skin mucus sample for vitellogenin determination by means of an ELISA detection method.

2. The method as claimed in claim 1, wherein, a further aliquot of the homogenized skin mucus sample is removed as parallel sample and a further vitellogenin determination by means of an ELISA detection method is carried out.

3. The method as claimed in claim 1, wherein, a further aliquot of the homogenized skin mucus sample is removed and a total protein determination is carried out in order to determine the relationship of the total protein content to the vitellogenin content of the skin mucus sample.

4. The method as claimed in claim 1, wherein, the skin mucus sample of the fish is taken by means of a forensic swab with predetermined breaking point.

5. The method as claimed in claim 1, wherein, the skin mucus sample is transferred to a micro-reaction vessel containing protein-stabilizing buffer.

6. The method as claimed in claim 4, wherein, the forensic swab remains as agitator in the micro-reaction vessel during homogenization of the skin mucus sample.

7. The method as claimed in claim 1, wherein, the skin mucus sample is taken on the dorsal body region of the fish.

8. The method as claimed in claim 1, wherein, the aliquot is transferred to a microtiter plate for further determination.

9. The method as claimed in claim 1, wherein, the fishes are Cypriniformes fishes.

10. The method as claimed in claim 1, wherein, the fishes are Perciformes fishes.

11. The method as claimed in claim 1, wherein, the fishes are Salmoniformes fishes.

12. The method as claimed in claim 9, wherein, the Cypriniformes fishes originate from the species common carp (Cyprinus carpio), goldfish (Carassius auratus), roach (Rutilus rutilus), zebrafish (Danio rerio), fathead minnow (Pimephales promelas) or Japanese rice fish (Oryzias latipes).

13. The method as claimed in claim 10, wherein, the Perciformes fishes originate from the species European perch (Perca fluviatilis), Nile tilapia (Oreochromis niloticus), three-spined stickleback (Gasterosteus aculeatus) or Australian rainbow fishes (Melanotaenia species).

14. The method as claimed in claim 11, wherein, the Salmoniformes fishes originate from the species brown trout (Salmo trutta forma fario), rainbow trout (Oncorhynchus mykiss) or Arctic char (Salvelinus alpinus).

15. The method as claimed in claim 1, wherein, a micro-reaction vessel is used as reaction vessel.

Description

[0014] Against this background, the object of the disclosed inventive method for determining vitellogenin as biomarker or end point for an exogenous estrogenic effect on fishes is to provide a nondestructive method in which the test animals need not be killed. It is also intended here that the method require no additional usage of test animals for carrying out tests for estrogenic, antiestrogenic and endocrine disruptors.

[0015] The method according to the invention for determining vitellogenin as biomarker or end point for an exogenous estrogenic effect on fishes comprises the method steps of [0016] a) taking a skin mucus sample of a fish by swabbing, [0017] b) transferring the skin mucus sample to a reaction vessel, [0018] c) homogenizing the skin mucus sample, [0019] d) removing an aliquot of the skin mucus sample for vitellogenin determination by means of an ELISA detection method.

[0020] Therefore, the method is a nondestructive method in which the test animals need not be killed and also no additional usage of test animals for carrying out tests for estrogenic, antiestrogenic and endocrine disruptors is required.

[0021] After an exposure time of 4-7 days, it is possible to record the efficacy of the exogenous noxious agents (harmful substances) at the time of maximal induction of vitellogenin and hence of optimal species-specific sensitivity.

[0022] Estrogenic efficacy can be tested as an additional end point, i.e., coupled to subchronic long-term tests such as OECD 215, or in sublethal concentrations of the acute test OECD 203. No additional experimental effort is required therefore.

[0023] The matrix disclosed in the disclosed method according to the invention is well defined in contrast to conventional assays. The samples do not need to be separated from non-target tissues such as blood, lymph, epithelia and others.

[0024] Also, complicated sample preparation procedures are not necessary.

[0025] Appropriately, in an expansion of the method, a further aliquot of the homogenized skin mucus sample is removed as parallel sample and a further vitellogenin determination by means of an ELISA detection method is carried out. In this way, the first detection is checked on the basis of said parallel sample and a greater reliability is achieved with regard to the results of the detection method.

[0026] In an advantageous expansion of the method, a further aliquot of the homogenized skin mucus sample can be removed and a total protein determination can be carried out, since this makes possible a determination of the relationship of the total protein content to the previously determined vitellogenin content of the skin mucus sample, which relationship gives further information. Here, said total protein determination can be carried out both in a method as claimed in the main claim and in a method in combination with the parallel sample as claimed in claim 2.

[0027] The skin mucus sample of the fish can be usefully taken here by means of a forensic swab with predetermined breaking point. Here, the use of the swab with predetermined breaking point has advantages especially in combination with a method workflow in which the forensic swab remains as agitator in the reaction vessel during homogenization of the skin mucus sample. A micro-reaction vessel can be appropriately used here.

[0028] Moreover, it has been found to be very advantageous for the method when the skin mucus sample is transferred to a reaction vessel containing protein-stabilizing buffer. The use of suitable buffers prevents the protein from breaking down, making it possible to apply the assay to a larger number of species (taxonomic groups) such as Cypriniformes fishes for example.

[0029] A further appropriate aspect of the method is that the skin mucus sample is taken on the dorsal body region of the fish. It has been found that said body region of the fish is particularly suited for the removal of the skin mucus sample.

[0030] To carry out the detection method after the homogenization of the sample, it has been found to be advantageous to transfer the aliquot to a microtiter plate for further determination. A standardized evaluation in the laboratory is thus easily possible. This applies especially to the discussed ELISA method as antibody-based detection method or immunoassay method that is based on an enzymatic color reaction.

[0031] The method according to the invention has been tested and is especially suitable for Cypriniformes fishes, for Perciformes fishes and for Salmoniformes fishes, and in the case of the Cypriniformes fishes those of the species common carp (Cyprinus carpio), goldfish (Carassius auratus), roach (Rutilus rutilus), zebrafish (Danio rerio), fathead minnow (Pimephales promelas) or Japanese rice fish (Oryzias latipes) are disclosed.

[0032] In the case of the Perciformes fishes, fishes of the species European perch (Perca fluviatilis), Nile tilapia (Oreochromis niloticus), three-spined stickleback (Gasterosteus aculeatus) or Australian rainbow fishes (Melanotaenia species) were advantageously used, and in the case of the Salmoniformes fishes, those of the species brown trout (Salmo trutta forma fario), rainbow trout (Oncorhynchus mykiss) or Arctic char (Salvelinus alpinus) were advantageously used as suitable test animals.

[0033] The disclosed method according to the invention can be used, inter alia, for the detection of estrogenic effects in laboratory experiments as an additional end point in standard methods, for determining the spawning maturity of female Cypriniformes fishes and for recording the exposure to estrogenically effective noxious environmental agents outdoors. It is also easily possible with this method to record a disturbance in sexual maturity due to antiestrogenic noxious environmental agents outdoors.