The best evidence for establishing the level of eutrophy of a water-body is its algal production which makes it possible to identify the type and the intensity of the eutrophication according to the kind and number of algal species present: when the number of algae exceeds half a million per litre then one speaks o an ”algal bloom”. The scope of the present research aims to verify if the alga Selenastrum capricornutum can be used as a test alga under our culture conditions and to determine the eutrophic level of the secondary effluent of a modern plant for the treatment of domestic discharge and to investigate the eventual ”limiting factors”. Finally this paper aims to study the effect on the secondary effluent of tertiary treatment carried out artificially in the laboratory

Bernagozzi, M.

Prosperi, A.

Romano, G. C.

Tonelli, E.

Aquatic Commons

Freshwater Biological Association 
FBA Translation (New Series) No. 96 
Title: Application of algal bioassays in the determination of 
eutrophic power of waste water. 
Author(s): PROSPERI A., BERNAGOZZI M., ROMANO G.C. & TONELLI E. 
Reference: 1g. Sanita pubbl. 31, 257-267 
Original language: Italian 
Date of publication of original: 1975 
Translator: W.J.P. SMYLY 
Date of publication of translation: 1977 
Number of pages of translation: 7 pages 
FBA Translations are to be considered as "provisional". As a rule 
they have not been prepared by expert translators, nor have they 
been edited by the original authors. 
They are available from The Librarian, 
Freshwater Biological Association, 
The Ferry House, 
Far Sawrey, 
AMBLESIDE, 
Westmorland, 
at the current rate for xerox copying. 
F.B.A. Translation (New Series) No. 96 
Application of algal bioassays in the determination 
of eutrophic power of waste water. 
PROSPERI A., BERNAGOZZI M., ROMANO G.C. & TONELLI E. 
1g. Sanita pubbl. 31, 257-267 
Translated by W.J.P. Smyly. 
The terra "eutrophic" which traditionally refers to a natural and optimal 
condition of a water-body, nowadays has come to mean instead a state of 
pollution due to an excess of nutritive substances accompanied by an increased 
production of algal microflora. Eutrophication is a process tied up with 
transfer of nutrients, natural or artificial in the surface water which, may 
exert a beneficial effect when within the limits suited to the biological 
capacity of the receiving water-body whereas, in excess, can cause a pollution, 
(Genovese 1973). 
The best evidence for establishing the level of eutrophy of a water-body 
is its algal production which makes it possible to identify the type and the 
intensity of the eutrophication according to the kind and number of algal species 
present: when the number of algae exceeds half a million per litre then one speaks of 
an "algal bloom". (Fruh, 1967). 
Eutrophication causes a notable increase in the total amount of algae, 
represented however by few species: on the contrary in oligotrophic waters, 
that is, those low in nutrients one observes a relatively modest growth of 
aquatic microorganisms but with many kinds of species. 
An increase in productivity is thus secondary to an increase of nutrients. 
These, indeed, among which we note especially nitrogen and phosphorus, are 
considered to be the essential cause, and fundamental in acceleration, of 
eutrophication (Sawyer 1947, Weiss 1969, Gerloff et al 1957). 
Phosphorus in particular has been shown from numerous experiments to be 
the most important element. At the start of the process other substances 
also participate such as potassium, magnesium, sulphate, traces of metals and 
numerous organic micro nutrients. (Provasoli 1961: Goldmann 1965). 
Some workers take 2.8 μg/l. of phosphorus as the maximum amount in 
unpolluted water (Ketchum 1969) : others record that algal blooms occur when the 
mean annual concentrations of phosphorus and nitrogen are 0.015 and 0.3 mg/l 
respectively ( Sawyer et al 1944). 
Some authors think that carbon, in the form of "carbonic anhydride" produced 
in bacterial decomposition of organic waste (Kuentzel 1969) or in the oxidative 
metabolism of heterotrophic populations (Kerr et al 1970), can play an important 
part in promoting development of noxious algae, thus relegating direct and active 
absorption of phosphorus and nitrogen to a secondary role. Moreover, whether or 
not both are nutrient limiting factors, there is good reason to believe that in 
reality the situation is even more complex than these views suggest and that in 
fact numerous elements interact to determine the productivity of a water-body 
and its trophic state. 
Among the factors that influence the enrichment by nutrients of the 
harvest in superficial waters, one should consider some geological factors; 
nutritional transport in some cases may depend on the geochemistry of the 
drainage basin (possibilities for percolation and washing of the soil and 
rocks), the size of the basin and of its hydrology (Brezonik 1972). 
It is necessary to record also that the productivity of a water-body 
may be affected by factors other than the concentration of nutrients, factors, 
for the most part unknown, which condition the distribution, the availability 
and the use of the same nutrients. 
For these reasons, the traditional chemical, physical and bacteriological 
indices are not sufficient for a true evaluation of the trophic capacity of a 
water and in fact we find that instead the most effective method is the 
determination in the laboratory of algal growth applying the procedures 
of the biological sages propounded in the "National Eutrophication Research 
Program" (1971). 
These methods enable one to observe directly the influence of the water 
in tests on the development either of indigenous algae or of selected 
"experimental" algae (Selenastrum capricornutum. Microcystis aeruginosa, 
Anabaena flos-aquae) thus determining biologically the availability of 
limiting nutrients for the growing algae. 
Moreover, by artificially increasing those nutrients in reduced concentrations, 
one can use the test algae to identify which are the limiting factors and what 
concentrations are needed to produce an algal bloom. In this way it is possible 
to forecast with some precision the potential effect on the development of the 
algae on addition of nutrient. This last possibility is of great value because 
one can indicate, even in ways not wholly exact, that a water-body is becoming 
progressively more eutrophic and to what extent. For an evaluation of this 
question continuous and periodic uniform bioassays will be necessary as well 
as other routine chemical and physical tests. 
Sanitary authorities and many students of the problem are interested in 
eutrophication because the phenomenon in question is related to the serious 
deterioration of the quality of water (disagreeable tastes and odours, 
particle colouring, intense turbidity etc.) thus rendering it unfit for other 
uses, domestic, industrial, agricultural, and recreational; and afterwards very 
difficult and inconvenient to eliminate with modern and expensive treatment 
(Fruh 1967). 
Among the more important causes of the progressive enrichment in nutrient 
substances of the water are in particular the indiscriminate use of phosphate 
detergents, intensive agriculture use of fertilisers, industrialisation, and 
urbanisation of populations; consequently through direct discharge of sewage, 
one has a strong and continuous contamination of the superficial water-table. 
Primary and secondary treatment, to which domestic discharges are normally 
subjected, if on the one hand they get rid of hazardous decompositions and also 
to some extent those which are infective, on the other they increase the 
capacity for eutrophication in many forms especially oxides of nitrogen and 
phosphorus which are the most efficient stimulants of the growth of algae 
(Middlebrooks et al 1971). 
2. 
It becomes necessary therefore in most instances to adopt a third kind of 
treatment which, amongst various methods (Bernard 1972) is capable of removing 
almost all the content of fertilizer substances (Miller et al 1971). 
The scope of the present research aims to: verify if the Selenastrum 
capricornutum can be used as a test alga under our culture conditions * :to 
determine the eutrophic level of the secondary effluent of a modern plant for the 
treatment of domestic discharge and to investigate the eventual "limiting 
factors"; and to study the effect on the secondary effluent of tertiary treatment 
carried out artificially in the laboratory. 
Material and methods. 
Water samples for experiment were taken from the outflows of a purification 
station in our province. The installation, of recent design and working, purifies 
water of exclusively domestic origin and is based upon our traditional system 
of primary and secondary treatment. This last is formed from an activated 
sludge treatment plant. 
The effluent was collected at four distinct times of years:- winter (Feb. 1974) 
spring (May 1974), summer (July 1974) and autumn (September 1974). On the spot, 
at the moment of sampling, we have measured dissolved oxygen and the temperature 
of the water-samples and of the air. We have taken note of the prevailing 
atmospheric conditions and those for the preceding days. 
On each sample we have made chemical tests and biological trials for algae 
(see table). 
Chemical analysis was carried out according to the methods suggested in 
"Standard methods for the examination of water and wastewater" (1971), the test 
water being previously filtered through filter paper Whatman no. 1. This was 
done with all samples and has been repeated also in the laboratory after 
application of the third treatment. 
These last have been carried out on the second, third and fourth samplings 
using respectively ammonium sulphate (200mg./l) calcium oxide (700 mg/l)and 
aluminium sulphate (200 mg./l) with successive filtrations through activated 
charcoal (Bernard 1972). 
Each sample, after chemical estimations has been subjected to algal bioassay 
according to the proceedings outlined in the "Provisional algal assay 
Procedure" (National Eutrophication Research Program, 1971) using exclusively 
as test species the unicellular green alga, Selenastrum capricornutum Prinz. 
At the beginning of the bioassays, all samples were filtered through membrane 
filters millipore of 0.46μ to retain all algae present, and then stopped and 
inoculated at pH 7. The tests were made in 250 ml glass-bottles holding 60 ml 
of the test water. All the samples subjected to third treatment have been made 
up to, either singly or in phosphorus combination (as K2H PO4), to a concentration 
* We thank the National Eutrophication Research Program, Pacific North West 
Water Laboratory, Corvallis, Oregon (U.S.A.) for having kindly supplied this 
laboratory with the unicellular green alga Selenastrum capricornutum Prinz. 
3. 

4. 
* Translation uncertain. 
* Translation uncertain. 
of 0.05 mg/l; nitrogen (as Na NO3) to 1.0mg/l and carbon (as NaHCO3) to 
10.0mg/l (Maloney et al 1972). 
Each bioassay has been repeated three times starting with an inoculation 
of Ix103 cells/ml: the test alga has been cultured in the lab. in modified soil 
PAAP (Murray et al 1971). 
All samples were incubated for 21 days at thermostatically controlled 
temperature of 22± 2°C, and subjected to a cold light unbroken of 4300 lux 
and continually agitated on a small table rotating at 110 oscillations per 
minute. 
The growth of the alga was found daily by direct count under the microscope 
using the -(camera Thoma contaglobuli)* . The values given in the tables and 
in the text are for time of maximum development (10° day of incubation) and are 
the mean of three observations made on each sample. 
Results 
From study of the obtained results (see Table) one can see that samples 
taken in September generate an algal development notably superior to that 
observed in the other periods, not withstanding an inferior content of 
phosphorus and nitrogen. 
The eutrophic power of the secondary effluent has been constantly reduced 
by tertiary treatment applied in the laboratory which has assured the constant 
reduction of phosphorus either as total phosphorus or other forms of 
orthophosphate in all the samples. 
In particular, addition of calcium oxide to the secondary effluent with 
drawn in summer has yielded a reduction in total nitrogen of 37% and has cut 
down total phosphorus by as much as 99%. 
With bioassays made after this kind of treatment and after stoppering the 
samples at pH 7, the resulting response of algal development was notably 
inferior to the growth-values found in the secondary effluent: one finds in 
fact from 2.4 x 104 cell/ml to 3.0 x 103 cells/ml in the water after 
treatment. 
One proceeds then to add to the tertiary effluent the principle nutrients 
in order to decide which are the limiting factors. 
Phosphorus already present at a concentration of 7 x 10-4 mg./l. in 
orthophosphates, when added artificially to the test water about 
doubles the level of algal growth. Moreover carbon and nitrogen added in 
combination with phosphorus produce a threefold increase of algae. These results 
show that phosphorus limits the growth of algae; carbon and nitrogen become 
limiting when phosphorus is present in sufficient quantity. 
The water of the secondary effluent collected in spring has been subjected 
in the laboratory to tertiary treatment by adding aluminium sulphate. As 
the literature indicates, this method reduces effectively the major elements 
concerned in eutrophication namely nitrogen, phosphorus and (oligoelements?).* 
From study of the chemical analyses, one sees that total phosphorus 
is reduced by about 1.85% + total nitrogen by 50%. The growth-response 
of algae cultured in untreated effluent was 7.6 x 104 cells/ml whereas the 
same subjected to treatment was 4.1 x 104 cells/ml. 
Also in this sample the artificial increase of nutrients has shown 
that phosphorus is the principle limiting element: in fact addition of 
phosphorus roughly doubles the rate of algal growth. 
The final type of treatment applied in the laboratory to the sampled 
water in September and consisting of the addition of aluminium sulphate and 
successive passages through activated carbon produces an improvement in the 
chemical character of the treated effluent referable in particular to the 
orthophosphate which in the end is reduced by 99%, total nitrogen by 42% and 
organic substances by 44%. 
Most sensitive to this general abatement are those algae which reached a 
growth level of 7.0 x 103 cells/ml against 2.1 x 105 cells/ml developed in the 
effluent treated first. 
Analogously to the preceding samples the added phosphorus shows a very 
clear increase in the development of Selenastrum capricornutum (1.1 x 105 cells/ 
ml.) when the phosphorus and the nitrogen are added together. The nitrogen then 
is not limiting the growth in the treated effluent except after the addition of 
a quantity in excess of phosphorus. 
Conclusions. 
The results presented do not always show a correlation between development 
of algae and concentration of nutrient substances. Indeed, the samples obtained 
in September gave rise to an algal growth (2.1 x 105 cells/ml) notably superior 
to samples taken in February (3.4 x 104 cells/ml.), May (7.6 x 104 cells/ml) and 
July (2.4 x 104 cells/ml) though being inferior in content of phosphorus and 
nitrogen. 
Other factors also limiting and correlated with algal multiplication such 
as dissolved oxygen and C.O.D. do not seem to have influence. 
In the reduction of total phosphorus, aluminium sulphate (200 mg/l) has 
been shown to be the least effective among the three systems of tertiary 
treatment with an efficiency of 85%, inferior to that of calcium oxide 
(700 mg/l) and also to aluminium sulphate in conjunction with post-filtration 
through activated carbon: these last systems have indeed eliminated 98 - 99% 
of total phosphorus. 
Total nitrogen was reduced to much the same extent by all three methods. 
Algal bioassay, carried out before and after the third treatment indicates the 
extent of reduction of the eutrophic powers of the secondary liquid in noteworthy 
manner: in this respect, it is interesting to note how the two treatments which 
brought about the almost total elimination of phosphorus are also the most 
effective in reduction of eutrophic power. Indeed algal productivity is reduced 
by 7 and 40 times respectively by the calcium oxide and by the aluminium sulphate 
with post-filtration, whilst it is only halved by aluminium sulphate. 
Finally, artificial addition of phosphorus, nitrogen and carbon, alone or 
in combination, to the tertiary liquid has made it possible to single out 
phosphorus as the limiting factor: nitrogen can become limiting only in the 
presence of phosphorus in excess. 
5. 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Notice 
 
 
Please note that these translations were produced to assist the scientific staff of the 
FBA (Freshwater Biological Association) in their research. These translations were 
done by scientific staff with relevant language skills and not by professional 
translators. 
 
 
 


Application of algal bioassays in the determination of eutrophic power of waste water (”Conclusions” only) [Translation from: 1g.Sanita pubbl. 31 257-267, 1975]

http://aquaticcommons.org/4880/1/96_1975_pros_appl.pdf

Application of algal bioassays in the determination of eutrophic power of waste water (”Conclusions” only) [Translation from: 1g.Sanita pubbl. 31 257-267, 1975]

Abstract

Similar works

Full text

Available Versions

Aquatic Commons