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An Investigation of the sensitivity of Ceriodaphnia affinis to City of Kyiv tap water


by Michael Hoffmann and Victor I. Rakov1)


The reliability of toxicity testing using Ceriodaphnia affinis as a test organism can be severely reduced or even eliminated when tap water is used for blanks and dilution water as recommended in the Ukrainian norm method document. In order to investigate this problem, Kyiv tap water has been physically and chemically treated to remove different groups of toxicants. Further, tests have been performed using untreated water and several other variations. The results indicate that smaller organic compounds rather than inorganic compounds are the reason for the toxicity of the Kyiv tap water to the test organisms. Further gas-chromatographic analyses should be carried out to identify those compounds, but it is clear that the Ukrainian norm document should be changed to avoid this problem.


1.  Introduction

Toxicity tests are a very effective tool for environmental investigation and protection because they can detect bio-toxicity before carrying out expensive chemical analyses of an enormous variety of inorganic and organic compounds. In recent years, the use of Ceriodaphnia sp. for toxicity testing has become common practice in many water laboratories. This organism is by far more sensitive to various toxicants than the other often used phyllopode, Daphnia magna. The Ceriodaphnia-test is used to measure acute and chronic toxicity. In the Ukraine, a test procedure has been normed and is officially recommended for use /1/. But also in other countries, like in the USA, manuals, rules or recommendations have been published. In Ukraine, C. affinis (further C.a.), in the US C. dubia is used for testing. In Ukraine, results of toxicity tests have yet to be adopted to impose sanctions or fines for waste water discharges which exceed a defined permissible level. Unfortunately, the Ceriodaphnia test procedure as described in the Ukrainian document leads to problems which greatly reduce the usability of the test.

While EPA recommends the use of an artificial standard dilution water as mentioned in chapter 3 /2/, the Ukrainian norm procedure recommends the use of tap water for cultures and as dilution water for testing. Drinking water in Kyiv however, after treatment in the water works, is known to be rather hard and still loaded with inorganic and organic matter. Reportedly, aluminum and organic matter often exceed the Ukrainian standards. The usual concentration of organics is about 3 to 5 mg/L DOC. The organic matrix originates from the Dnepr river (additionally from the distribution system) and contains humic matter, mainly fulvic acids, and smaller organic compounds, degradation products etc. By far the biggest problem is the occurrence of various disinfection byproducts in variable quantities /3, 4/. The two biggest groups, also relevant for human health, are formed by trihalomethanes (THM) and haloacetic acids (HAA) /5/. Its also worthwhile mentioning the compound MX (3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanon) that has been identified as to be responsible for more than 50 % of the mutagenity of chlorinated tap water /6/. Drinking water quality is also variable because, in a few places, ground water is added directly to the distribution network. Consequently, the concentration of organic matter and halogenorganic compounds as well as electrical conductivity is altered significantly /7/. Therefore, following the Ukrainian procedures, tap water has to be stored for one week and aerated before use, mainly to get rid of chlorine which usually is added at the water treatment plant. This is however not always sufficient. Sometimes some or even all Ceriodaphnia die in such tap water for unknown reasons.

Such problems are probably not limited to Kyiv tap water and Ceriodaphnia sp. Daphnia magna, that is less sensitive (e.g. against potassium dichromate), reacts in a similar manner. It therefore appears to be important to get an initial idea of what the problem is and eventually define the cause of the problem. An important reason to investigate the problem is to avoid stress or illness of the test organisms caused by unsuitable culture water. It also will help to get more confidence in the test results.


2.  Investigation program

To identify the class of pollutant(s) present a Toxicity Identification/Toxicity Reduction Evaluation (TIE/TRE) is suggested. First, the investigation should clarify if the focus must be on inorganic or organic compounds. Do they occur in natural water systems like fulvic and carbonic acids, phenols, hydrocarbons, or are they man made, e.g., the result of pollution or disinfection of raw and drinking water? Since the quality of Kyiv tap water is not uniform, samples were taken several times from two households and the laboratory on the right bank side of the river Dnepr in Kyiv. This water is supplied primarily from the Dnepr water works. The samples have been modified in various ways to isolate (if possible) the cause of the negative impacts on the cultures.

The samples were altered in various ways. Samples have been aerated to eliminate volatile purgeable compounds. Polar compounds, non volatile and other substances would remain in the test water. Then, the water was boiled in an attempt to eliminate less volatile and/or semi-volatile substances as well. As has been shown in earlier investigations, Kyiv tap water can be improved through boiling with respect to chlororganic compounds /7/. After 7 minutes of intensive boiling between 30% and 45% (n=6), could be purged. In this case, however, it is possible that the organic matter might also be changed. The main reason for this test was to determine if boiled tap water could be successfully altered and made suitable for use as a standard dilution water for the cultures.

In addition to the above, samples were treated chemically with different reagents. FeCl3 was used for flocculation that more easily eliminate bigger molecules /8/.

To preferentially adsorb smaller non-polar molecules /9/, activated carbon was selected.

An alternative adsorbent Al2O3 was used because its surface is polar and its absorbance characteristics are correspondingly different /10/. It should preferentially adsorb, among others, chlorinated acetic and other organic acids.

Aggressive components like possibly Cl2 or other reactive inorganic Cl-compounds as well as radicals, build in-situ following the absorption of solar radiation, have also been suspected to harm C.a. Therefore Na2SO3 has been selected to reduce those compounds as well. This reagent, however, can change or degrade some of the organic substances.

For controls and comparison, standard water has been prepared as described in the EPA-document /2/. An additional control was made using fresh tap water (and one week old water as required by the Ukrainian norm) plus the reagents necessary to prepare EPA-standard water. In this way, the possible effect of toxic metals could be reduced. The variants tested are listed in the table below. More details are given in the following chapter.


3.  Methods

For the chemical treatment, tests have been carried out using laboratory tap water that was treated through flocculation with FeCl3. Various quantities have been added resulting in different residual concentrations of organic matter and the pH was raised to 7,4.

Additional samples were prepared using 0,5 g/L special carbon (MERCK) with a particle size < 150 um, specific surface 850 m2/g and an iodine number > 1050. This carbon is normally used for the determination of AOX (shaking method). It was added to the sample and stirred for about two hours. For better adsorbance, the sample pH was lowered to 3,0 - 3,5. After mixing, sedimentation and filtration, the pH was adjusted to 6,5 7,5. Additional experiments were performed using only 0,25 g/L of activated carbon. The organic matter was not completely removed and about 50 % of the organics remained in the sample. To control the effects of the pH adjustments, the test was repeated in the same way but without changing the pH before or after mixing.

For the alternative adsorbent Al2O3, the specific surface is given with only 10 100 m2/g /10/. Therefore, a higher concentration (5 g/L) was used. Samples were prepared as described for the tests with activated carbon and the pH was not changed.

Another variant was treatment with Na2SO3. The reagent quantity added was just sufficient to decrease the oxygen level to 0,1 mg/L. After one hour, the test water was aerated up to 6 7 mg/L.

Following the EPA procedure, the standard (for blank and dilution water) was prepared using ultra-pure water. To get a moderately hard water, NaHCO3, CaSO4, MgSO4 and KCl had to be added. For details see /2/.

As a further variant, commercially available humic acids (FLUKA) were added to the EPA standard water. The selected concentrations were within the concentration range typical for raw and tap water. This artificial humic acid solution might, however, differ too much from natural aquatic humic matter. Therefore, initial tests have used water taken from the river Dnepr.

In this preliminary phase of investigation, only a few chemical standard parameters have been analyzed to briefly describe the type of water. The quantity of organic compounds and thus the adsorption efficiency of the variants, are estimated by measurements of the spectral absorption coefficient at 254 nm (SAC254 given in 1/m, DIN standard method 38404-C3, /11/). This method seems to be sufficiently relevant for this study.

The toxicity tests have been carried out following the Ukrainian standard but using EPA standard water as a reference blank. Ten test organisms were used for each test variant and after one day the number of dead animals were counted. After the second day, no significant changes were noted.


Table: Variants of tested tap water (tw.) and range of spectral absorption coefficients at 254 nm (SAC254)



water test variant

SAC 254








EPA standard (as blank and for dilution)






unchanged or diluted tap water



fresh tw.

15 - 25


fresh tw. (1:3)



fresh tw. (1:5)






physically treated



tw. after 60 min aeration

15 - 21


tw. stored one week and aerated



tw. strongly boiled for 5 min

15 - 26





chemically treated



tw. + 0,25 g/L act. c., pH 3

11 - 12


tw. + 0,50 g/L act. c., pH 3

1 6


tw. as before but pH unchanged



tw. + 5 g/L Al2O3

11 - 16


tw  + FeCl3

7 - 16


tw. + Na2SO3 + aeration



tw. + EPA reagents



tw. 1 week old + EPA reagents






other variants



EPA standard + humic acid

19 - 44


Dnepr water (500 m upstream water work)

28 - 33


4.  Results

The results of the tests with unaltered tap water have shown that a correlation exists between the amount of organic matter (determined as SAC 254) and the mortality of C.a. (r=0,93; p=0,05; n=9). EC and pH are not shown to be correlated to mortality.

If all tap water test variants are included into the calculation, including the different matrices and pretreatments, the correlation is diminished (as to be expected) but is still significant.

The physical treatment by aeration results in only a small decrease in mortality. The tests with boiled tap water were more effective but mortality could only partly be eliminated. Some of the remaining compounds are obviously still toxic to the test organisms, apparently depending on the concentration of remaining organic matter.

The various flocculation and adsorption experiments produced rather clear results. After treatment, mortality is related to the reagent used and the remaining concentration of organics (SAC 254 in the figure). As the concentration of organics


Figure: Comparison of the organic load (SAC 254) of the test variants (smaller columns) and the quotient dead organisms related to the concentration of organics (Ym/SAC 254), striped columns; abbreviations: ct = standard, kp = unchanged tap water, ae = tap water after 1 hour of intensive aeration, KM = tap water after 5 minutes of strong boiling, AL = treated with Al2O3, 25 = treated with 0,25 g/L activated carbon, 50 = treated with 0,5 g/L activated carbon, Fe = treated with 0,5 g/L FeCl3


has been always different, mortality was related to the amount of organic matter determined as SAC254 (Ym / SAC 254) as shown in the figure. This procedure could and should not, of course, eliminate the influence of the changing qualities of organic compounds.

The effect of flocculation was rather weak. Mortality after flocculation was still quite high compared to the other forms of treatment with approximately the same amount of organics.

Adsorption reduced mortality more effectively. The use of 0,5 g/L of activated carbon removed nearly all of the organics and correspondingly lowered the mortality. Half the quantity (0,25 g/L) was not sufficient for complete removal of organics and mortality. After treatment with Al2O3, the organic concentration of the test water, measured as UV-absorption (SAC 254), was similar or even higher than in the second activated carbon variant (compare figure). Nevertheless, mortality of C.a. was always less in this case. Obviously, Al2O3 specifically eliminates more toxic (polar) compounds than activated carbon.

Tap water treated with Na2SO3 did not show any significant difference compared to the untreated tap water.

EPA reagents did also not diminish toxicity. In fresh tap water as well as in one week old tap water (plus EPA reagents) all C.a. died after the first day. EPA standard water containing artificial humic acids in two different concentrations did not show any toxicity effects. The same is true for tests with raw water taken from the river Dnepr. This last test, however, has to be repeated during the summer because the type and concentration of organics changes significantly over the year. Further, Dnepr water itself is not free from chlororganic compounds /12, 13/. Chlororganics found in river water are usually different from those in tap water /14/ and could even be more problematic.  


5.  Conclusion

The test results presented here can only be considered as a first step towards understanding the problems of using tap water for toxicity tests with C.a. The tests make clear that inorganic, aggressive substances like chlorine (Cl 2) are not the cause of the problem. Toxic metals as the primary reason is also not probable. The significant correlation between the concentration of organic matter and mortality using tap water indicates that further investigation into the nature of the changing pool of organics within the water distribution system as well as the changes over time are necessary. One assumption is that these problem organics are anthropogenic in origin. Therefore, the future focus should be on substances appearing after the treatment in the water works. Whether these compounds are only halogenated compounds is not known at this time. Within this context, no correlation exists between the amount of organics (as SAC 254) and adsorbable organic halogens (AOX) in Kyiv tap water. This is based on samples(n=80) taken at various places and times (HOFFMANN, unpublished data from 1997 2000). Further tests with accompanying analyses of halogenated organic substances are therefore necessary to proceed further.

Nevertheless, a few additional details seem evident concerning the type of toxicants involved. Some (but not all) toxicants are semi-volatile and can be adsorbed on activated carbon, others are polar and adsorbable on aluminum oxide. They should have a rather small molecular size that can not (or almost not) be eliminated through flocculation. Living organisms have adsorptive body surfaces that can adsorb reactive elements and hydrophobic non-polar substances /10/. Halogenated acetic acid (which is sometimes used as a pesticide), MX and chloroform could be three of those compounds. The type of influence and possible interactions between the various toxicants remain a subject for future investigations.

In conclusion, storage and aeration of Kyiv tap water does not guarantee that the test organisms will be safe and fit for testing. It is therefore recommended using standard water as recommended by EPA or in the French standard /15/. The Ukrainian standard should be changed correspondingly.


6.  Literature

1. ̲Ͳ ί (1997): Ceriodaphnia affinis Lilljeborg. - KND, Kiev

 2. US Environmental Protection Agency (1994): Short term methods for estimating the chronic toxicity of effluents and receiving water to freshwater organisms 3rd edition. EPA-600-4-91-002

 3. , .., , .. (1993): - .. - ; 15, 6,  p. 424 435

 4. HOFFMANN, M. (1995): Ueber Herkunft und Vorkommen toxischer Stoffe im Trinkwasser ukrainischer Grossstaedte. - gwf Wasser/Abwasser, 136 (2), pp. 85 90, (German)

 5. STEVENS, A.A., MOORE, L.A., and MILTNER, R.J. (1989): Formation and Control of Non-Trihalomethane Disinfection By-products - J. AWWA p.54-60 

 6. HUCK, P.M., ANDREWS, R.C., and DAIGNAULT, Susan A. (1990): Removal of Mutagenic Compound 'MX' from Drinking Water by Activated Carbon. - Vom Wasser 74, S. 245-259

 7. , .  M, .. (1994): . . - ; 16, 5, p. 472 - 479

 8. FETTIG, J. und STEINERT, C. (1995): Untersuchungen zum Adsorptionsverhalten von unterschiedlich vorbehandeltem Deponiesickerwasser. Vom Wasser 85; p. 167-181

 9. JOHANNSEN, C., ASSENMACHER, M., KLEISER, M., ABBT-BRAUN, G, SONTHEIMER, H. and FRIMMEL, F. (1993):  Einfluss der Molekuelgroesse auf die Adsorbierbarkeit von Huminstoffen. Vom Wasser 83; p. 79-87

 10. SIGG, Laura and STUMM, W. (1991): Aquatische Chemie - 2.ed. Teubner, Stuttgart and Verl. d. Fachvereine, Zuerich; p. xxx

 11. Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung (DEV)(1998): - Beuth Verl. Berlin 

12. , .  , . (1998):   , . - ; 20, 2,  p. 154 167

 13. Vasenko, O.G. (1998): Environmental Situation in the Lower Dnipro River Basin, Water Qual. Res. J. Canada 33 (4), pp. 457- 487

 14. YAMAMOTO, K., FUKUSHIMA, M. and KURODA, K. (1992): Total Organic Halogen: Chemical Pollution Parameter in Urban River Waters.- Wat. Sci. Tech. Vol.25, No.11, p. 25-32

 15. , .. (2000): Daphnia magna Str. ( ). , 36 5 . 50 70


1) Water Laboratory of the Ecological Inspectorate (MEP), Kiev, Ukraine



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