Metamorphosis of Hydractinia echinata (Cnidaria) is used to determine the toxicity of simple organic molecules.

Objective
- QSAR-statements about the effects of toxic substances in marine ecosystems
- Discrimination between effects specific for the test organisms and effects which can be found in other test systems with the same order of effectivness
- development of a procedure to (estimate) calculate the toxicity of yet not tested substances 

Method
Colonies of H. echinata can be reared in a reproductive state in the laboratory throughout the year. After three days the eggs develop into larvae which are competent to undergo metamorphosis. The larvae consist of about 10.000 cells, are spindle shaped, possess cilia, have no organs, can't feed, but nerve cells are present which help the larva to find a suitable place for metamorphosis. Metamorphosis can be triggered artificially by CsCl or by seawater reduced of Mg2+ Ions. A three hours treatment is sufficient to trigger metamorphosis, but test-subsances can prevent metamorphosis if present during triggering time. Prevention takes place irrespective of of the type ofthe triggering treatment, thus it can be concluded that the test substances do not prevent the uptake of Cs+ Ions nor do they prevent the loss of Mg2+ Ions from the larva. Both has to happen simultaneously. We determined the concentration of test substances which reduce by half the rate of metamorphosis. Testing is easy, fast, reliable, reproducible, and small amounts of test substance are needed.

Results
The order of effectivness is the same in H.echinata, Tetrahymena pyriformis (Protozoa), Daphnia magna ( Crustacea), and the fish Pimephales promelas.
In contrast to other authors, we systematically investigated simple organic substances. We found a correlation between the substance class and the toxicity (i.e. alcanes, alcohols, amines...).
We developed a new method to estimate the toxicity of related substances which effects are yet unknown. We made several assumptions:
1. The toxicity of substances can be devided into parts (element specific influence parameters, ESIPs), the sum of the parts gives the toxicity of the whole.
2. The ESIPs are the same in different substances. Thus the ESIPs can be added up to give the unknown toxicity of a not yet tested substance.
3. The ESIPs are constant in one organism and test-system. The ESIPs differ in different organisms, but can be determined by the very same method.

We tested 140 substances and determined 11 ESIPs (by an automated calculating procedure). We found a good correlation of the measured and calculated toxicity values

Application to other test organisms
The method for determination of the ESIPs was applied to data published on  Tetrahymena pyriformis (Protozoa), Daphnia magna ( Crustacea), and Pimephales promelas (Fish).
We found similar ESIPs even by using data published by different authors. Thus, we calculated and predicted the tocxicity values for substances not yet tested, i.e. alkanes in T. pyriformis  and P. promelas.

In H. echinata we tested 
alcanes
cycloalcanes
alkylbenzenes
polyaromatic hydrocarbons
alkanols
polyalkanols
phenylcarbinols
aminoalkanes
diaminoalkanes
alkyl substituted aminoethanols
thiophenes

Our results are of significance for marine ecosystems endangered by contamination with oil because crude oil contains the substances we tested. There is no other investigation we know of, which addresses the effects on marine organisms systematically as we did it. 
We demonstrated that the estimation of the ESIPs can be applied succcessfully to other test systems and the order of effectivness is  the same. Therefore, our method can help in estimating the toxicity values for man as well. 

Publications

Berking, S. (1991). Effects of the anticonvulsant drug valproic acid and related substances on developmental processes in hydroids. Toxic. in Vitro 5, 109-118.
Chicu, S. A. and S. Berking, 1997. Interference with metamorpho-sis induction in the marine cnidaria Hydractinia echinata (Hydrozoa): A structure-activity relationship analysis of lower alcohols, aliphatic and aromatic hydrocarbons, thiophenes, tributyl tin and crude oil. Chemosphere 34, 1851-1866 
Chicu, S. A., K. Herrmann, S. Berking (2000). A new approach to calculate the toxicity of simple organic molecules on the basis of QSAR analysis in Hydractinia echinata (Hydrozoa, Cnidaria). QSAR 19, 227-236
 

Embryopharmacology /Toxicology     Development of test systems
 

Hydractinia echinata, Hydra vulgaris, Brachydanio rerio

We investigated developmental processes in non mammals as test systems for toxic and teratogenic effects in mammals. This appears to be applicable at least to substances and their metabolites which are able to cross the placental barrier. 

We investigated the processes of metamorphosis in Hydractinia echinata, head regeneration in Hydra vulgaris (Cnidaria), and embryonic development in the zebrafish Brachydanio rerio (Chordata). The advantages of these species are: rearing the animals is easily done at low cost; large amounts of eggs are available every day, thus, large test series are possible; development takes place outside the maternal organism and can be manipulated and scored easily; development is fast, one to three days; treatment of the eggs means simply adding the test substances to the culture medium of the eggs, only small amounts of test substances are needed. 

We developed criteria by which the effects of substances can be easily quantified. The endpoints of development are: rate of metamorphosis in H. echinata, percentage of head regenerating animals in H. vulgaris, and morphological criteria compared to 'normal' embryos in B. rerio. The experimental schedule was easily learned and reliably handled even by untrained persons, technicians etc. The effects showed to be reproducible, the developmental rate was highly synchronous in all test systems.

We tested the anticonvulsant drug valproic acid and 12 chemically related substances and additionally in fish retinoic acid and 8 related chemicals. The effects of these substances are already known in mammal test systems.
We observed retardation of development and malformations. The effects depended on the concentrations and time of application during development

In a first approach, we quantified the effects by the MATC values, the maximal acceptable toxicant concentration. The order of effectivness was similar to that found in mammals, especially found in mammals whole embryo culture in vitro (rat and mouse).

In zebrafish, we quantitated in detail the effects on various organs, such as chorda, tail, heart, blood circulation, brain, eyes, and ears by a new scoring system. Thus, in addition to the order of effectiveness it is possible to analyze the similarities and differences in detail and to get an idea of the underlying molecular mechanisms and action of the drugs. Further, it appears that the substances tested so far, retinoids and valproids, define distinct `effect-classes` with distinct properties. 
New substances tested can be valuated determining their effects as more `retinoid` or `valproic`-like by the following properties:
(a) The range of the effective concentrations. 
(b) The slope of the dose/response curves. 
(c) The type of malformations generated by the classes of substances. 
(d) The type of malformations generated by some substances, only. 

Consequently, every new substance tested can be characterized by a set of effects and can be compared to the effects of the other substances, which have already been tested in zebrafish. Eventually, new classes have to be founded.
Thereby, it will be possible, to obtain some ideas about the mechanisms of the action of the new drugs and to predict the effects in mammals. It appears that by these properties the development of the zebrafish is more than a mere supplement to other test systems. 
 

Publications

Herrmann (1993) The effects of the anticonvulsant drug valproic acid and related substances on the early development of the zebrafish (Brachydanio rerio). Toxicology in vitro 7, 41-54.

K. Herrmann, S. Berking (1994) Die Frühentwicklung des Zebrafisches als Screening Testsystem für die Teratogenität von Substanzen. In: H. Schöffl et. al. (Hrsg.): Ersatz- und Ergänzungsmethoden zu Tierversuchen in der biomedizinischen Forschung. Springer-Verlag, Wien 

K. Herrmann (1995) Teratogenic effects of retinoic acid and related substances on the early development of the zebrafish (Brachydanio rerio) as assesed by a novel test system. Toxicology in vitro 9, 267-283.