EFFECT OF AGE ON THE SUSCEPTIBILITY OF ZEBRAFISH

Wat.Res.Vol.35,No.15,pp.3754–3757,2001#2001Elsevier Science Ltd.All rights reserved

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TECHNICAL NOTE

EFFECT OF AGE ON THE SUSCEPTIBILITY OF ZEBRAFISH

EGGS TO INDUSTRIAL WASTEWATER

GEORG GELLERT*and JAN HEINRICHSDORFF

Staatliches Umweltamt Ko ln,Postfach 130244,50496Ko ln,Germany (First received 6November 2000;accepted in revised form 31January 2001)

Abstract }Toxic e?ects of 40%(v/v)and 60%(v/v)concentrations of wastewater from the industrial

sector of chemical production on eggs of the zebra?sh (Danio rerio )were investigated.Signi?cant poisonous e?ects could be registered in test solutions containing 40%wastewater for eggs up to 0.5h of age.Solutions containing 60%wastewater a?ected the mortality of the exposed eggs up to 1h of age.Earlier stages were more sensitive to industrial wastewater than later stages.The di?erences in susceptibility must be ascribed to the ontogenetic stage of the eggs at which the experiment was started and to di?erences in the chorion permeability.The chorion is less permeable after hardening and works as a barrier to detrimental wastewater contents.This circumstance explains the higher viability of zebra?sh eggs at the age of >1h.When using early stages of the zebra?sh for ecological risk estimations,the eggs should,at best,not exceed the age of 0.5h.#2001Elsevier Science Ltd.All rights reserved Key words }zebra?sh,?sh egg,toxicity,industrial wastewater,sensitivity

INTRODUCTION

In the last 30years the static ?sh acute toxicity test performed with golden orfes (Leuciscus idus melano-tus )played an important role in the toxicological monitoring of wastewater in Germany.Golden orfes have been proven to yield reproducible results and to be as sensitive as trouts (Fischer and Gode,1978).The only problem in using this test system arises from the ethical point of view,because mortality was determined as the toxicological endpoint.Taking this argument into consideration when assessing the impact of industrial e?uents,the German Institute of Standardization intends to standardize and validate a new test system with aquatic vertebrates for all types of ?nal e?uents.For this evaluation zebra?sh (Danio rerio )embryos have been chosen as test organisms.The test period ends before hatching.Obviously,the monitoring of pollutants on the embryonic development of zebra?sh is regarded as morally less objectionable than on the lethality of juvenile golden orfes.

Worldwide,zebra?sh have proven to be attractive for toxicity evaluations because they are easy to keep and to breed in the laboratory (sexual maturity after 3months),free of pathogenic microorganisms and deliver eggs of high quality independent of the season (Bresch,1991).

It is known that toxicity results depend on numerous factors of the experimental design.In the present study,the centre of interest was to investigate how far the susceptibility of fertilized zebra?sh eggs to chemical pollutants could be a?ected by the egg age.To achieve this evaluation an e?uent sample from a chemical production plant was chosen.

MATERIALS AND METHODS

The adult zebra?sh were kept in tap water at 268C.A photoperiod of 12:12(day :night)was used.They were fed daily with freshly hatched nauplii of Artemia salina and commercial ?ake food ad libitum .The eggs were obtained according to the method described by Nagel (1986).Spawning and fertilization of the unexposed parent ?sh in a 150l aquarium was stimulated by light and by green plastic trees acting as spawning substrates.The fertilized eggs were collected in plastic dishes,which were exposed in the aquarium for 10min immediately after having switched on the light.

Approximately 30eggs were transferred from the plastic dish by glass pipets to each Petri dish (1.3cm height,5.3cm in diameter,15ml test solution)containing clean water (controls)or diluted wastewater (test solutions).The Petri dishes were covered and the solutions,adjusted to a pH of neutral range,were not aerated.Clean water (also used as dilution water)consisted of distilled water and the following ions (in mg l à1):Ca 80.0,Mg 12.2,Na 17.2,K 2.9.

The wastewater was taken as a quali?ed spot sample from the ?nal e?uent of a puri?cation plant belonging to a chemical factory producing diverse aromatic compounds,substances for pharmaceutical products,substances for pesticides,and chemicals for plastic manufacturing.The sewage plant consists of a primary settling basin,

an

*Author to whom all correspondence should be addressed.

Tel.:+49-228-5386-804;fax:+49-228-23-0337;e-mail:poststelle@stua-k.nrw.de

3754

activated sludge tank and an installation for chemical precipitation.

The e?uent had the following global characteristics:pH (6.73),conductivity(2960ms mà1),Cl(3.7g là1),SO4 (0.44mg là1),NO3-N(0.09mg là1),NH4-N(20.8mg là1), total P(1.69mg là1),TOC(33.1mg là1)and AOX (0.6mg là1).

The routinely performed test with golden orfes,under-taken by dilution of the sample with clean water to determine the limit at which it loses lethal e?ects,showed, as test result,a dilution factor of1:2(1volume unit clean water:2volume units wastewater).

The sample(10l)was handled as follows:?rst,it was split into10subsamples and stored at–188C.Before use,the subsamples were warmed up in a waterbath at408C and subsequently agitated.To avoid interference by suspended solid particles,the subsamples were then left for1h for sedimentation.

The survival of the eggs in each experiment was determined daily.The eggs were examined with a10?–80?dissecting microscope.The toxicological endpoint was embryo mortality.Criteria to establish death were opaque and white egg membranes and immobilized embryos. Immobilization was de?ned as the inability of the eggs to move when touched lightly by tweezers.Dead eggs were removed after24h from the test solutions to minimize fungal growth.

Each experiment consisted of one control and two di?erent test solutions containing40%(v/v)or60%(v/v) wastewater.In screening tests these dilution rates proved suitable to measure di?erent toxic e?ects from minor to 100%mortality.During the experiments the medium was not changed.Exposures were started0.25,0.5,1,2,and4h after the plastic dish had been exposed.Therefore,the egg age of the experiments,which were for instance started 0.25h(or0.5h)after the dish had been placed in the aquarium,was theoretically between15and5min or between30and20min,respectively.

The toxic response was measured as the number of live eggs in%calculated from the total number of eggs after24 and48h minus all the dead and unfertilized eggs.To allow statistical treatment of the data,the experiments were replicated nine times.Each of the replications was started on a di?erent day.The tests were only considered as valid when at least90%of the embryo solutions in the control were viable after48h.This criterion demanded a high percentage of fertilized eggs,over90%.

All experiments were performed in a laboratory contain-ing an air-conditioning system,which guaranteed a room temperature of268C.The test solutions had a dissolved oxygen content(measured with a WTW Microprocessor Oximeter,Model OXI196)after48h of at least3.7mg là1, corresponding to a saturation of42.5%.The pH level was found at7.5(recorded with a WTW Microprocessor Oximeter,Model OXI196).

Data are expressed as means(?SD)of percentage

mortality in24h and48h of continuous exposure respec-tively.The normality hypothesis for replicated treatments was tested by the Kolmogorrof–Smirnov test and the homogeneity of variances by the Cochran test.The multiple comparison of each treatment group versus the control followed the Dunn-test.Signi?cance of di?erences in the viability between di?erent e?uent concentrations was tested using the pairwise Mann and Whitney U-test.The Dunn-and the U-test do not require normal distribution and homogeneity of variance of the test results.Rejection level of all statistical treatments was p>0:05.

RESULTS

Figures1and2show the sequence of mortality of ?sh eggs at di?erent ages in the chosen wastewater concentrations after exposures of24and48h.First, it can be stated that the average percentage of viable embryos in the control solutions was over90%and did not change within48h.The diluted wastewater caused an increase in egg mortality.In Fig.1it is shown that in experiments performed with40% wastewater the egg viability did not exceed50% when the eggs were between0.25and0.5h old,before the tests had been started.Eggs at the age of1h and older responded weakly to40%wastewater concen-tration.

In Fig.2the toxicity response obtained with60% wastewater is presented.In these experiments the test solutions exhibited higher toxicity.For eggs up

to Fig.1.Percentage of surviving zebra?sh eggs exposed to a 40%(v/v)wastewater sample from a chemical plant at di?erent ages(n?9).Asterisk indicates statistical di?erence

from the control(p50:

05).

Fig.2.Percentage of surviving zebra?sh eggs exposed to a 60%(v/v)wastewater sample from a chemical plant at di?erent ages(n?9).Symbols are the same as in Fig.1.

Zebra?sh eggs and industrial wastewater3755

0.5h of age only a viability rate of up to15%could be observed.Even1h old eggs showed a mortality rate of60%.A viability rate over50%was recorded only for eggs at the age of2h and older.

In all wastewater dilutions the standard deviations from the survival means for eggs between the ages of 0.25and1h di?ered to a larger extent than for eggs of the controls and of the experiments performed with eggs older than1h(Figs1and2).The variability for survival was generally higher for eggs exposed to60%wastewater than for those exposed to40%wastewater.

Statistical analysis(Dunn-test)revealed that the lower percentage of viable embryos in the diluted wastewater in comparison with that of the control solutions is only signi?cant when the eggs are not older than0.5h(in40%wastewater)and1h(in60% wastewater).

The e?uent concentration a?ected the mortality of embryos.Mortality was higher in a60%wastewater solution than in a40%wastewater solution.Follow-ing the results of the U-test of Mann and Whitney there were signi?cant di?erences in mortality only for eggs not older than0.5h.

DISCUSSION

The toxic response of zebra?sh eggs after exposure to diluted wastewater at the age of51h was statistically di?erent from that elicited by exposure of eggs older than1h.No statistically signi?cant di?erences in survival were found between the means of eggs older than1h and the control.

The greater variability in survival means of the experiments conducted on eggs between0.25and1h of age may be caused by their higher susceptibility at this life-span to the complex composition of waste-water.

The greater variability of embryonic viability at higher concentrations of wastewater corresponds to the evaluations of Dave et al.(1987).They found that the variability of the hatching of zebra?sh eggs increased at higher concentrations of the toxicants, chromium and zinc.

Obviously,not later than1h after spawning the eggs already became relatively insensitive to the tested complex chemical mixture.Di?erences in susceptibility may be ascribed to the ontogenetic stage at which the experiment was started.This phenomenon may also be due to the egg membrane. According to Hagedorn et al.(1997)membrane permeability of the zebra?sh embryo changes during development.Ensenbach and Nagel(1995)noticed that the egg hatchability of zebra?sh(Danio rerio) was not a?ected by lindane or3,4-dichloroaniline. They assumed that the chorion works as a barrier. Also,according to Herrmann(1993)chorion hard-ening explains the higher egg viability of zebra?sh, because early stages were three times more sensitive than later stages to the anticonvulsant drug valporic and related substances.

Similar observations were made with other?sh species.Van Leeuwen et al.(1985)found that freshly fertilized eggs of rainbow trouts(Salmo g airdneri) were less sensitive to tested single chemicals than early life stages.He assumed that egg membrane permeability decreases during early embryo genesis. The chorion appears to slow down the intrusion of toxicants.According to Terner(1968)the chorion of eggs of rainbow trouts is more permeable before hardening.Eggs of the desert pup?sh(Cyprinodon n. ne v adensis)were more susceptible to acid exposure immediately after exposure(Lee and Gerking,1981). Vuorinen and Vuorinen(1987)demonstrated that two-night old embryos of the brown trout(Salmo trutta)were much more resistant to bleached kraft mill e?uent than newly hatched sac fry.They assumed that the process of swelling and hardening of the chorion had been terminated at this age, making the eggs more resistant.

The toxicity test with fertilized zebra?sh eggs can be regarded as an alternative to other?sh acute toxicity tests in routine wastewater control.Follow-ing Friccius et al.(1995)the?sh embryo test is suitable to assess acute toxicity of industrial waste-water.They describe this method as more sensitive than the golden orfes test for industrial e?uents. Tests determining the non-acute-poisonous e?ect(by dilution limits)of wastewater from11di?erent sewage plants showed that zebra?sh eggs were as sensitive as juvenile golden orfes for wastewater from six sewage plants and more sensitive for wastewater from?ve sewage plants.

CONCLUSION

The objective of this study was to explore the extent to whict the egg age of Danio rerio a?ects the toxicity level of industrial wastewater.The sample in the tested dilutions produced a signi?cant toxic response only for eggs1h old.This phenomenon can be connected mainly with the changing perme-ability of the chorion,depending on the age.When establishing this test as a useful tool in environmental supervision the age of the used zebra?sh eggs must inevitably be restricted to0.5h in order to achieve a satisfactory sensitivity.

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