PAPER
Muqi Xu,*a Hong Cao,a Ping Xie,*b Daogui Deng,b Weisong Feng*b and Jian Xuc
www.rsc.org/jem
Use of PFU protozoan community structural and functional
characteristics in assessment of water quality in a large, highly
polluted freshwater lake in China
a
Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology,
Chinese Academy of Sciences, Beijing, China. E-mail: [email protected]
b
Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China.
E-mail: [email protected]@ihb.ac.cn
c
Centre for Environmental Monitoring, Hefei, China
Received 29th March 2005, Accepted 10th May 2005
First published as an Advance Article on the web 14th June 2005
Structural and functional parameters of protozoan communities colonizing on PFU (polyurethane foam
unit) artificial substrate were assessed as indicators of water quality in the Chaohu Lake, a large, shallow
and highly polluted freshwater lake in China. Protozoan communities were sampled 1, 3, 6, 9 and 14 days
after exposure of PFU artificial substrate in the lake during October 2003. Four study stations with the
different water quality gradient changes along the lake were distinguishable in terms of differences in the
community’s structural (species richness, individual abundance, etc.) and functional parameters (protozoan
colonization rates on PFU). The concentrations of TP, TN, COD and BOD as the main chemical indicators
of pollution at the four sampling sites were also obtained each year during 2002–2003 for comparison with
biological parameters. The results showed that the species richness and PFU colonization rate decreased as
pollution intensity increased and that the Margalef diversity index values calculated at four sampling sites
also related to water quality. The three functional parameters based on the PFU colonization process, that
is, Seq, G and T90%, were strongly related to the pollution status of the water. The number of protozoan
species colonizing on PFU after exposure of 1 to 3 days was found to give a clear comparative indication of
the water quality at the four sampling stations. The research provides further evidence that the protozoan
community may be utilized effectively in the assessment of water quality and that the PFU method furnishes
rapid, cost-effective and reliable information that may be useful for measuring responses to pollution stress
in aquatic ecosystems.
DOI: 10.1039/b504396b
Introduction
670
Biological information is essential to the assessment of pollution stress because biological monitoring, the orderly and
systematic gathering of biological data to determine whether
the environment is favorable to living organisms, can be used
to ensure that management of hazardous wastewater discharge
meets society’s expectations.1,2
Biological monitoring of freshwater environments is being
used widely in North American and European countries to
assess water quality, since pollution affects the structure and
function of aquatic ecosystems.3 However there have been
many difficulties with the indicator species concept and the
use of such organisms to evaluate biological water quality is
not commonly accepted everywhere.4,5 Structural and functional characteristics of entire communities of organisms have
been found to be much more reliable for evaluating environmental conditions.2,5–11
Protozoans, which usually are considered to include autotrophic and heterotrophic flagellates, amoebae and ciliates, are
important and integral components of aquatic ecosystems.
These organisms play a key role in energy flow and mineral
cycling in aquatic food webs. Because of their small size, rapid
generation times, short life cycles, high species diversity, quick
response to environmental changes and cosmopolitan distribution, protozoa have been increasingly recognized as good
indicators of water quality, particularly with regard to organic
pollution in lakes, rivers, reservoirs and oceans.2,5,9,11–16
PFU (polyurethane foam unit) protozoan communities have
been shown to form complex species assemblages which exhibit
many of the characteristics of structural communities.2,9,17
J. Environ. Monit., 2005, 7, 670–674
Cairns et al.18 have expounded that the rate at which protozoans colonize on PFU artificial substrates in lentic waters (a
functional characteristic which is observed by looking at
changes in structure through time) is correlated with the degree
of the lake’s eutrophication. Since the 1970s, Cairns and his
colleagues have developed the PFU method to monitor pollution levels of freshwater.5,8,17–22 The PFU method was introduced to China by Shen in the 1980s and has been widely used
to evaluate the environmental quality of natural waters in
China and other countries.2,9–11,23,24
The Chaohu Lake, located along one of the tributaries of the
Yangtze River, is one of the five largest freshwater lakes in
China (longitude 1171 16 0 4600 –1171 51 0 5400 , latitude 301 43 0 2800 –
311 25 0 2800 ), with a catchment area of about 9200 km2 and
surface area of 753–774 km2. It contains approximately 32.3 108 m3 of water in the rainy season but 17.2 108 m3 of water
in the dry season and its depth is variable according to the
hydrologic conditions, usually below four metres (maximum
depth 6 m, mean 3 m). Situated centrally in the southern China
plain with subtropical climate, it plays an important part in not
only being intensively utilized for commercial fishing but also
widely used for the water supply, irrigation, navigation, tourism and recreation as well as being of great conservation value
for its wildlife and wetland system. However, now this lake has
been greatly impacted by the increasingly serious pollution
caused by wastewater discharge from Hefei City (a capital of
Anhui Province) through the Nanfei River into the western
lake zone. Over the past more than 10 years the Chaohu Lake
has been extensively studied for a variety of limnological
purposes and related problems.25,26 However, to the best of
our knowledge, no previous research on freshwater protozoan
This journal is & The Royal Society of Chemistry 2005
communities has been carried out for the assessment of pollution
status. The purpose of this research was to use protozoan communities established on PFU artificial substrates to evaluate the
changing gradient in water quality along the lake, and to provide
basic biological information to engineering plans for pollution
control developed by the central and local governments.
The structural and functional parameters measured were,
respectively, changes of species richness, individual abundance,
diversity index and the colonization rates of PFU protozoan
communities.
Materials and methods
The investigations were conducted during October 2003. Four
sampling stations along the lake were selected for the PFU
protozoan collection and the comparative chemical–physical
analyses. Site 1 was close to the mouth of the Nanfei River
which directly receives industrial wastewater and domestic
sewage from Hefei City and then discharges into the lake. Sites
2, 3 and 4 were arranged at different distances away from the
stress of pollution sources (Fig. 1).
Polyurethane foam units (PFUs)—a commercial bedding
material with an aperture of about 250–300 mm—were used
as artificial substrates for the establishment of protozoan
communities at each of the four sampling stations throughout
the study. PFUs with dimensions of 5 6.5 7.5 cm were
anchored approximately 30 cm under the surface of the water
near the littoral zones (Fig. 2). PFU samples were harvested at
time intervals of 1, 3, 6, 9, 14 days, respectively during the
periods of exposure in water, Plafkin et al.7 having demonstrated that species numbers show negligible increase after this
time interval. Each time, two PFU blocks were collected as
parallel samples for analysis.
Protozoan species from PFUs were obtained by manually
squeezing as much water as possible from the substrate into a
clean glass beaker (200 ml). Protozoan taxon richness was
determined from living material by pipetting two or three
drops of well-mixed deposit material in the bottom of the
beaker and examining the whole field at 100–400 magnification. Identification was made by using standard protozoological keys,9,27–31 and the examination of all samples was usually
completed within a few hours of collection at a temporary
laboratory called the Zhongmiao Working Station on the
shore of the lake (Fig. 1). A much more detailed description
of the step regarding the PFU sampling method was described
by Xu and Wood.32
The colonization of introduced PFU artificial substrates
provides a means of evaluating the diversity and dispersal ability
Fig. 2 PFU systems applied in this study: (a) a single unit; (b) PFUs
hanging in the water body.
of microbial species. Although the PFU substrates used in this
study also collect bacteria, fungi, algae, and some micrometazoa, we have restricted our taxonomic analyses to protozoa.
For quantitative analyses of protozoan populations, one litre
mixed water samples from upper-mid-bottom layers of the water
column were taken with an integrating organic glass sampler for
each site. Samples were fixed with acidified Lugol’s iodine
(which would have inevitably rendered any remaining small-size
species such as Bodo, Amoeba and Monas unidentifiable and
uncountable). All samples were precipitated for 48 hours to be
concentrated to 30 ml. Four subsamples (0.1 ml) of the final
concentrate were placed in a perspex counting chamber and
counted under a light microscope at a magnification of 400.
The four replicate sub-samples from each water sample yielded
SE o 8% of the mean values of counts. Chemical and physical
data on the annual values during 2002–2003 at the four sampling
stations were obtained for comparison with the colonization
parameters of PFU protozoan communities. All chemical and
physical analyses were performed using standard techniques.33
The protozoan communities of the four sampling stations
were also compared using the Margalef Index of Diversity
(1957), D value:
D ¼ S 1/ln N
Where S ¼ number of species, and N ¼ total number of
individuals. Low D values indicate a lack of diversity and
therefore possibly poor water quality.
The colonization process of protozoan communities can be
followed by sampling replicate PFU artificial substrates over
time to follow the rate and magnitude of the species accrual
process. Data can then be fitted to the colonization equilibrium
model developed by MacArthur and Wilson:34
St ¼ Seq (1 eGt)
Three functional parameters, i.e. Seq, G, and T90% were
obtained by a computer program (M-W or M-W-W). Where
St is the number of species at time t, Seq is the estimated
equilibrium species number of colonization, G is the constant
value of colonization rate, and T90% is the time taken for
reaching 90% Seq. Fitness tests of the sampling stations were
done by a computer program in order to confirm whether the
species numbers observed according to days fit with the
MacArthur–Wilson Model at the 0.05% significance level.
Results and discussion
1.
Fig. 1 Map of the Chaohu Lake showing protozoan sampling sites.
J—PFU protozoan collection sites.
Main chemical and physical variables at the four stations
At present the Chaohu Lake is a main receiving pollutant
repository for Hefei City, the capital of Anhui Province. The
biggest pollutant influx to the lake is the Nanfei River which
discharges a large amount of untreated domestic sewage and
industrial wastewater with a total of 1.8 108 tons per year
from Hefei City into the lake. Annually the quantities of
sewage discharged into the lake from the city are about
J. Environ. Monit., 2005, 7, 670–674
671
Table 1
Chemical–physical parameters of the four PFU sampling stations in the Chaohu Lake during 2002–2003 (mean values for two years)a
Stations
Parameters
1
Temp/1C
pH
DO/mg L1
BOD5/mg L1
CODmn/mg L1
TP/mg L1
TN/mg L1
NH41–N/mg L1
20.14
7.38
3.56
4.81
24.9
0.47
4.88
0.86
a
2
7.83
0.50
2.17
1.34
1.33
0.28
3.90
0.52
19.3
7.83
6.72
3.94
12.8
0.18
2.62
0.64
9.40
0.87
1.58
0.91
5.21
0.10
1.20
0.86
20.4
7.93
6.98
1.81
8.56
0.12
1.65
0.32
4
10.3
0.66
1.49
0.67
6.15
0.03
0.76
0.14
21.1
7.69
7.39
1.80
7.35
0.11
1.56
0.29
7.9
0.45
1.54
0.50
4.95
0.04
0.65
0.09
Stations 1–4 have been selected as representatives of 12 regular sampling sites for the analyses of chemical–physical parameters in the whole lake.
18 360 tons of total nitrogen (TN) and 1050 tons of total
phosphorus (TP). The chemical data from the investigations in
the past two years indicate that the Chaohu Lake was both
organically polluted by COD, BOD and highly enriched by N
and P. The average concentrations of COD, BOD, TP and TN
in the lake have greatly exceeded the National Grade III
Standards of Surface Water in recent years (National Standard
issued by EPA of China—GB 3838-1988).
The ranges of water chemical and physical parameters at the
four sampling stations for two recent years are summarized in
Table 1. Among these variables, temperature and pH showed
minor differences at all sampling stations during the two year
period. However, the dissolved oxygen concentrations of the
four sites were commonly below the saturated value with a mean
value of 6.16 mg L1. There was distinct oxygen depletion with
an average content of 3.56 mg L1 at site 1, close to the pollution
source, and then the oxygen concentration increased gradually
toward the farther stations along the lake. The highest oxygen
concentration occurred at site 4, which was far away from
pollution source stress. The data in Table 1 also indicate that
stations 1 and 2 displayed higher concentrations of BOD, COD,
N and P, particularly at site 1. The major changes in the degree
of pollution are the obvious reductions in pollutant concentrations at sites 3 and 4, located far from the wastewater discharges.
The improvement in water quality at sites 3 and 4 is probably
enhanced by the lake’s self-purification processes such as dilution, dispersion, sedimentation and biological degradation.
2. Taxonomic composition and species number collected on
PFU
Ciliates, sarcodines, and autotrophic and heterotrophic flagellates comprised the major taxonomic groups of protozoa in the
Chaohu Lake. Table 2 lists the taxa composition and total
number of protozoan species collected from PFUs at the four
sampling stations during 14-day colonization experiments. A
total of 102 protozoan species have been found from the
examination of colonization on approximately 40 PFU artificial substrates through the period of investigation. The 102
protozoan taxa were comprised of 28 flagellates (Phytomastigophorans, 22 and Zoomastigophorans, 6), 20 Sarcodines and
Table 2 Species composition and numbers of protozoans found on
the PFU systems at the four sites in the Chaohu Lake during the period
of investigation
Station
672
3
Taxa
1
2
3
4
Total
Phytomastigophorans
Zoomastigophorans
Sarcodines
Ciliates
Total species
10
4
5
6
25
21
5
16
34
76
17
6
19
53
95
16
6
18
52
92
22
6
20
54
102
J. Environ. Monit., 2005, 7, 670–674
54 Ciliates. There was great variation in the distribution of
species in the three major taxonomic groups (flagellates, ciliates, and sarcodines) from site 1 to site 4. At stations 1 and 2,
which had poorer water quality, especially in station 1, flagellates and sarcodines possessed greater species composition, and
species such as Euglena, Anisonema, Oikomonas, Petalomonas,
Cryptomonas, Bodo, Actinophrys, Mayorella and Amoeba, etc.,
which are commonly considered as indicators of tolerant forms
to heavy organic pollution, were the dominant species groups.
Compared to stations 1 and 2, stations 3 and 4 were characterized by higher levels of ciliate species composition, equalling
the proportion of resident flagellates and sarcodines, which
coincided well with the improved water conditions (Tables 1
and 2). Generally, ciliates would be present with greater species
diversity in a normal body of water.
Station 1, with only 25 total species, was clearly less diverse
than station 2 (76), with a further dramatic increase in the
number of species at stations 3 and 4 (95 and 92, respectively).
The low species richness at station 1 was probably due to the
very high levels of pollution as evidenced by the high CODmn,
BOD5, total phosphate and total nitrogen values, and low
dissolved oxygen concentration. The species recovery at station
2 and great increase at stations 3 and 4 were associated with a
decrease in the pollution stress caused by discharge of high
levels of municipal wastewater. It is widely accepted that
species diversity and richness decrease in an aquatic community under stress conditions. Generally, low levels of nutrient
enrichment in microbial communities are related to increases in
the numbers of extant protozoan species, and oppositely,
severe stress, whether caused by heavy metals, extreme organic
pollution, or sharp changes in any environmental factor such
as pH or temperature, usually reduces the species richness of
the community and increases the individual abundance of
tolerant forms.2,5,19,20,35
3.
Individual abundance and diversity index
In total, the abundance of the main protozoan populations in
the Chaohu Lake showed that this lake now has a high
eutrophic status according to the common standard of lake
eutrophication categories.36 The variations in the individual
abundances and diversity index values of protozoan communities from water samples and PFU blocks at the four sampling
stations are presented in Fig. 3. Generally, tolerant species
form the dominant population and develop higher individual
abundance under the condition of organic pollution. Species of
flagellates mentioned above were numerically dominant, and
made up the great bulk of the population density, although
some tolerant species of ciliates such as Halteria, Strobilidium,
Cinetochilum, Cyclidium and Vorticella, also contributed higher abundances at the stations 1 and 2 (total 49 000 and 46 000
ind. L1, respectively). Total numbers of individuals then
decreased with water quality improvement at the stations 3
and 4 (29 000 and 21 000 ind. L1, respectively).
Table 3 The protozoan colonization parameters on the PFU system
at the four sampling stations in the Chaohu Lakea
Parameters
Stations
Seq
G
T90%
Fitness
1
2
3
4
24.33
76.32
77.12
91.02
0.27
0.22
0.51
0.42
8.43
10.49
4.49
5.51
Conformation
Conformation
Conformation
Conformation
a
The PFU systems were set up in the stations during the period of
October 11 through 25, 2003.
Fig. 3 Variations of individual abundance and diversity index
(D value) at the four sampling stations.
Another characteristic of the protozoan community’s
changes as a result of the impact of pollution stress was also
evident in assessment of the Margalef diversity index (D value).
D values calculated for this study generally agreed with the
water quality of the four stations. Station 1 showed the lowest
diversity value (2.22), and then increased in station 2 (6.97),
with a further rise in stations 3 and 4 (9.25 and 9.14, respectively). The species diversity index generally indicated that
protozoa might give a comprehensive evaluation of the pollution status at the community level and is a vital parameter in
environmental quality assessment.37 Species diversity usually
responds to pollution through the loss of some species, particularly some rare species, and increasing individual abundance
of tolerant species. Margalef species diversity is a simple
ecological index, which summarizes both the number of species
and the abundance of the community involved, and not only
makes community structure analyses simple but also classifies
the environmental conditions well.11,37–39
4. Colonization process of protozoan communities on PFU
system
Colonization curves by protozoan communities on PFU substrates exposed for 1, 3, 6, 9 and 14 days at the four stations are
illustrated in Fig. 4. The colonization rates expressed by the
number of species over time obtained from this study were
markedly different, both temporally and spatially. In Fig. 4, the
colonization rate for station 1, which is believed to be the most
heavily polluted site with least species richness, was significantly slow in species accumulation with time. The colonization rate at station 2 was much faster than that at station 1 and
then went up more quickly at stations 3 and 4 due to the
reduction of toxic stress, indicating that the colonization rates
increased with the decrease in pollution intensity. The differences in the colonization rates of protozoan communities at
each site were apparent at the beginning of exposure on the 1st
Fig. 4 Colonization curves of protozoan communities on the PFUs at
the four stations of the Chaohu Lake.
and 3rd days, giving a clear comparative indication of the
water quality at the four stations. Thus the PFU protozoan
communities after exposure of 1–3 days would be enough to
furnish rapid, cost-effective, and reliable information on the
monitoring of freshwater water quality at the community level.
Estimates of three functional parameters calculated for the
colonization process (species numbers during the equilibrium—
Seq, constant value of colonization rates—G, and time taken to
reach 90% Seq—T90%) based on fitting the MacArthur and
Wilson species equilibrium model are recorded in Table 3. The
Seq and G values for the PFU system were distinctly lower at
stations 1 and 2 than stations 3 and 4. Whereas, the T90% values
as a parameter being positively correlated with the G value were
higher at stations 1 and 2 compared with stations 3 and 4,
indicating worse pollution conditions in the former two sampling sites during the survey. Generally the Seq and G values are
low, while T90% values (days) are high in the polluted body of
water.9,11,39–41 At stations 1 and 2, the pollution stress of toxic
materials from the municipal wastewater effluent strongly
affected the protozoan colonization process on the PFU system.
Conclusions
This research represents one of few case studies using PFU
protozoan community’s structural and functional characteristics to assess water quality for a heavily polluted and large
freshwater lake. Our results indicate that the various components of protozoan communities in the Chaohu Lake are
strongly related to the lake’s degree of pollution. Species
richness, individual abundance, diversity index, PFU colonization rate and three functional parameters calculated for the
colonization process, Seq, G and T90%, identically showed a
predictable changing trend of water quality at the four sampling stations along the lake. Stations 1 and 2, close to the
pollution source, had poor water quality due to receiving a
large amount of wastewater containing a high concentration of
organic pollutants from Hefei City. These are reflected in the
changes in the structural and functional parameters of protozoan communities, i.e. impoverished species richness, high
abundance of tolerant species, low diversity index values and
low colonization rates exhibited by Seq, G and T90%. Great
changes in the characteristics of the protozoan communities
were evident at stations 3 and 4 compared with stations 1 and
2, and all structural and functional parameters of protozoan
communities examined during this investigation were distinguishable for comparison with the former two sampling stations (1 and 2). The improvement in water quality at stations 3
and 4 was probably due to self-purification because of the
distance from the pollution discharge and also due to dilution
by the many tributaries into the lake.
Evidence has accumulated for a large number of freshwater
ecosystems, including a number of wetlands, that microbial
community dynamics furnish rapid, cost-effective, and reliable
information that may be useful for biological monitoring, and
measurement of recovery processes in degraded ecosystems.
Colonization rates of the PFU protozoan community were
J. Environ. Monit., 2005, 7, 670–674
673
strongly related to trophic state. The structure and function of
the microbial community may be a simple and reliable index
for categorizing water types and measuring responses to stress
and perturbation.
This investigation provides further evidence that the structural and functional parameters of a PFU protozoan community are a good tool for the bioassessment of water quality.
PFU biomonitoring of freshwater ecosystems has the following
advantages: (1) it is economical compared to chemical monitoring because of its low cost and the reusability of materials;
(2) results may be obtained quickly after exposures of 1 or 3
days; and (3) it is accurate for the bioassessment of water
quality because it reflects the ecological effects of environmental stress at the community level.
15
16
17
18
19
Acknowledgements
This research is supported by a key project of the Chinese
Academy of Sciences ‘‘Studies on the mechanisms and control
of lake eutrophication in the middle and lower reaches of the
Yangtze River (Grant No. KZCX1-SW-12)’’, the National
Natural Science Foundation of China (No. 30370224) and
the Innovation Program of the Chinese Academy of Sciences
(No. KSCX2-SW-128, No. KZCX1-SW-12 and No. KSCX3IOZ-02). We are grateful to Qiong Zhou, Hua Yang and
Longgen Guo for their kind assistance in sample collection
and water quality testing.
We are also deeply indebted to Celia Chen for improvement
to the presentation of this paper.
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