Frank M. PRIESNITZ, Manfred SECKMANN, Erich ZEECK
Institute for Chemistry and Biology of the Marine Environment, (ICBM),
C.v.O.-University of Oldenburg, 26111 Oldenburg, Germany.
Tanker-disasters during the last few years demonstrate the catastrophic consequences on the marine eco system. Here we report the effects of volatile organic substances (VOS) of the EKO-FISK crude oil on the reproductive behaviour of the two nereid species, Platynereis dumerilii and Nereis succinea and the identification of the responsible substances.
Like many nereid species, Platynereis dumerilii and Nereis succinea undergo a metamorphosis before reproduction as epitokous heteronereids. Controlled by temperature and lunar cycles, ripe heteronereids of both species assemble near the water surface during summer nights and show a characteristic swarming behaviour, the nuptial dance. Once the sexes have met they swim in circles of decreasing diameter near to each other until the males release a small amount of sperm. This is followed by spawning of the eggs by the female and finally males release the total amount of sperm to fertilize the eggs. As typical for semelparous nereids, after reproduction heteronereids of both species die within a few hours. The reproduction behaviour is strictly synchronized by several pheromones.
In order to study bioassay of behavioural effects, samples were added to isolated males which had already started the swarming behaviour. These animals had already left their dy ailing tubes and started to swim at the water surface which is the typical searching behaviour prior to contact with the female. Bioassays were carried out in 50 ml of fresh pasteurized seawater in glass dishes (1 worm per dish). In all experiments a positive reaction was only stated when gametes were released and all worms were used for bioassay only once. As a positive control of releasing ability of the males which had not responded within 60 min after adding the samples under investigation, coelomic fluid of the females was added. The number of males which then released gametes in the presence of coelomic fluid were counted as negative bioassays.
A sample of 550 g of crude oil (EKO-FISK, North Sea) was rectified under reduced pressure. The analysis of the resulting 46 rectified fractions was carried out by coupled gas chromatography-mass spectrometry with a Finnigan MAGNUM GCMS system in order to identify the VOS from the oil-fractions. One fraction (1.69 9) containing VOS in the volatility range between n-undecane and n-dodecane (SAC, 17 mbar) induced sperm releasing of males of both nereid species The separation of aromatic hydrocarbons from alkanes and polar N. S and O compounds was performed with liquid chromatography (LC) using activated silica gel type 60 and spectrograde n-hexane. In bioassays on males of Platynereis dumenlli as well as Nereis succ~nea only the aromatic eluate fractions induced the sperm release.The main activity containing n-hexane eluate was separated by GC and the aromatic hydrocarbons eluting between n-undecane and n-dodecane were recondensed in 0.3 ml acetone (nanograde) using an effluent splitter (peak-trapping). In bioassays on ripe male heteronereids of both species one trapped ffaction induced the characteristic nuptial dance and resulted in the release of gametes into the free water.
The analysis of this active ffaction was performed with GC-MS also
and showed a small and a wide peak. One of the volatile aromatic
hydrocarbons was pentylbenzene, verified by comparison of mass spectral
and gas chromatographic retention times with the commerdaly available
substance (m/z(%)): 91(100), 92(53), 148(20), 105(15), 119(5), 133(4)).
In case of the second peak in the active fraction three substances with
different mass spectra were observed: 1.) 105(100), 106(23), 148(25),
91(13), 119(9), 133(3), a x-butyl-y-methylbenzene isomere; 2.)
133(100), 119(56), 105(38), 148(36), 91(27), a x,y-diethyl-z-methylbenzene
isomers; 3.) 119(100), 91(15), 148(12),120(10), 105(7), 133(1),
maybe a x,y-dimethyl-z-propylbenzene isomere. Founded on these data
without doubt it is a question of the isomeres of C5-alkylbenzenes
(C
In biosassays the calculated amount of 2.7x10-9 g (1.8x10-11 mol) C5-alkylbenzenes in the active fraction induces the charateristic swarming behaviour and results in the sperm release of ripe males Platynereis dumerlii and Nereis succinea. The identification of the unknown isomeres is still under investigation.