Morphological Deformities In Chironomus Spp Biology Essay

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5.1 INTRODUCTION

The larvae of non-biting aquatic midges (Chironomidae: Diptera) are considered as ideal organisms for bioassays because they spend most of their developmental time in sediments' surface where they remain exposed to different toxicants; also, they are relatively easy to culture and have a short life cycle. These criteria make them suitable organisms for ecotoxicological monitoring (Warwick 1985, Ingersoll and Nelson 1990, Vermeulen 1995). When continuously exposed to stress or pollution, late instars of some chironomid larvae frequently develop deformities in the mouthparts, especially the mentum. An existing hypothesis based on field observations is that the deformities in benthic midge larvae are a reflection of the environmental pollution due to heavy metals, radioactivity, organic pesticides, and other xenobiotics (Bird 1994, Servia et al. 1998, Watanabe et al. 2000, Vermeulen et al. 1995, Martinez et al. 2004). The occurrence of developmental deformities in chironomids appears to be a general stress response to a wide range of environmental contaminants (Vermeulen 1995), nevertheless, morphological deformities in chironomid larval populations in uncontaminated environments could also occur but are relatively less severe and less frequent than those occurring under the stressed conditions (Warwick 1985). The larval deformities could provide a useful tool for assessing aquatic pollution, specifically that relates to industrial wastes and agricultural runoff (Wiederholm 1984, Warwick 1985, Warwick and Tisdale 1988, Janssens de Bisthoven et al. 1992, Lenat 1993, Vermeulen 1995, Dickman and Rygiel 1996, Hamalainen 1999, Bhattacharya et al. 2005, MacDonlad and Taylor 2006).

Efforts to demonstrate the relationship of deformity incidence in chironomid larvae with water and sediment quality have been made (Vermeulen 1995). The application of chironomid deformities as bioindicators of pollution stress has been reviewed and illustrated primarily for bioassessment purposes (Hamalainen 1999). In this context, many researchers have developed several deformity indices based on different types of chironomid larval head capsule deformities to better understand the causes. For example, the pioneering Index of Severity of Antennal Deformities (ISAD) of Warwick (1985) was based on antennal deformities of Chironomus larvae, whereas the Toxic Score Index (TSI) proposed by Lenat (1993) was based on malformation of the mentum of Chironomus larvae. The latter author categorized the deformities of mentum into three classes: Class I includes slight deformities which are difficult to separate from the "chipped" teeth; Class II consists of larvae with more conspicuous deformities, such as extra teeth, missing teeth, large gaps, and distinct asymmetry; and in Class III are included the larvae that suffer severe deformation, including at least two Class II characters. According to Lenat (1993), the TSI can be computed as follows:

[No. Of Class I +2(No. Of Class II) + 3(No. Of Class III)] x 100 / Total Number of Larvae.

The pollution of aquatic ecosystems in Malaysia has emerged as a major ecological problem coinciding with rapid industrialization and urbanization. In 1994, Malaysian Department of Environment (DOE) classified the Juru River (situated in northeastern peninsular Malaysia) as "very polluted" based on the Water Quality Index (WQI) categorization by DOE (DOE 1994). Lim and Kiu (1995) pointed out that the Juru River is one the most polluted rivers in Malaysia, with sediments highly contaminated with non-residual heavy metals, such as Cd, Cu, Pb, and Zn. These contaminants in the river are most likely a result of discharges from the light and heavy industries in the Prai Industrial Estate (established in the early 1970s), Penang, Malaysia (Mat and Maah 1994).

In Malaysia, considerable efforts have been made in the past two decades to analyze chemical pollution in several rivers (including the Juru River basin) (e.g., Mat and Maah 1994, Lim and Kiu 1995), however, relatively much less attention is paid to utilize aquatic organisms for purposes of environmental bioassessments (Morse et al. 2007). The available studies concerning effects of contaminants on aquatic invertebrates in Malaysia at present primarily focus on diversity and abundance of benthic macroinvertebrates inhabiting contaminated rivers (Azrina et al. 2006).

The utility of alterations in chironomid larval head capsule morphology, such as deformation, phenodeviations, asymmetries, etc., has proven to be useful in terms of indicating aquatic pollution-related stress in many countries, such as Canada (Warwick 1985, MacDonald and Taylor 2006), Sweden (Wiederholm 1984, Janssens de Bisthoven and Gerhardt 2003), and India (Bhattacharya et al. 2005), but no such study has applied this empirical tool in Malaysian rivers.

The present study was undertaken with the objective of elucidating the incidence of deformities in Chironomus spp. larvae collected from three small rivers in the Juru River Basin. Specifically, the incidence of morphological abnormalities of the epipharyngis, antennae, mentum, and mandibles of the fourth-instar larvae of Chironomus spp. collected from these rivers that differed in their nature of contamination was studied. The severity of mentum teeth deformities was used as a criterion to evaluate effects of pollution and was scored using the TSI of Lenat (1993).

5.2 MATERIALS AND METHODS

5.2.1 Study Area and Sampling Sites

For the present study, three small rivers, [Permatang Rawa River (PRR), Kilang Ubi River (KUR), and Pasir River (PR)] in the Juru River system were selected (see Chapter 3 (3.2.1 and Figure 3.1). In each river, one permanent site for collecting chironomid larvae and water and sediment samples was established.

5.2.2 Physico-chemical Parameters of Water

See Chapter 3 (3.2.2).

5.2.3 Sediment Samples

See Chapter 3 (3.2.3).

5.2.4 Chironomid Larval Sampling

The chironomid larvae were collected from the three rivers from November 2007 to March 2008. See Chapter 4 (4.2.2).

5.2.5 Chironomid Deformities Investigation

Permanent slide mounts of chironomid larvae were prepared to examine morphological deformities in the larvae. The preserved larvae were transferred to a Petri dish containing 10% KOH solution and left in the solution for 24-48 h to digest the larval muscles. Thereafter, the permanent slide mounts of the larvae were prepared following the method of Epler (2001). The slide-mounted larvae were identified to genus using appropriate taxonomic keys (Kikuchi et al. 1985, Hasegawa and Sasa 1987, Morse et al. 1994, Merritt and Cummins 1996, Epler 2001, Cranston 2004). No attempt was made to identify the larvae to species level because of taxonomic difficulties in the identification of chironomid immature stages (Warick and Tisdale 1988). Larval head capsules of 4th instar Chironomus spp. were examined for deformities under a compound microscope, generally at 400X magnification; mentum, epipharyngis, mandibles, and antennae were examined for deformity occurrence. Examination of the antennae was carried out only for gross deformities, such as missing or extra segment, or major differences in size of segments between both antennae. According to Bird (1994), epipharyngis and mandibles are classified as deformed if they exhibit extra teeth, missing teeth including gaps, or are very asymmetrical or abnormal in shape. Damaged antennae or mandibles as a result of cleaning and mounting process of these mouth parts usually have abrupt breaks that are readily visible and easily distinguishable from deformed structures (Dermott 1991).

The mentum of Chironomus spp. has 15 dark pigmented teeth comprising a tripartite median arrangement of one large tooth flanked on either side by single smaller teeth and two larger and four smaller outer teeth (Cranston 1995). The occurrence of mentum deformities in the present study was scored according to Lenat (1993).

5.2.6 Statistical Analysis

The incidence of deformed larvae (mouth parts) from the different rivers was expressed as the proportion ± standard error (SE) of the total larvae mounted from each river. Standard errors were calculated according to the binomial theorem, i.e., SE= (pq/k)1/2 , where "p" is the proportion of deformed specimens, "q" is the proportion of non-deformed individuals, and "k" is the sample size (Hudson and Ciborowski 1996). The mean percentage of deformities from individual sampling site was compared using the non-parametric procedure (Kruskal-Wallis Test) and general linear models of the SPSS software package. The data of deformities incidence percentages were transformed using the arc-sine √x transformation prior to analysis. The Redundancy Analysis (RDA) of CANOCO program (ter Braak and Prentice 1988, ter Braak 1989) was used to investigate influence of the environmental parameters on the total deformities incidence, including mentum, mandibles, antennae, and epipharyngis deformities. Specifically, the RDA provides the dimensions of the influencing parameters, indicating the strength and direction of correlation between these parameters and incidence of the morphological deformities. In this analysis, all measured environmental parameters were included and the program performed forward selection of significant parameters.

5.3 RESULTS

5.3.1 The Incidence of Deformity in Chironomus spp. Larvae

A total of 616 Chironomus spp. larvae collected from the three rivers was processed and examined for deformity incidence. Statistical analysis of the data revealed a significant difference between deformity incidence among the three rivers (Kruskal-Wallis, n = 15, X2= 8.66, P < 0.05). Overall, this incidence was the highest in the larvae collected from KUR, followed by PRR, and PR (Table 5.1). The observed monthly percent larval deformity in Chironomus spp. in the three study rivers is shown in Table 5.1. In KUR, only 59 larvae were successfully mounted for the deformity investigation. In this river, an overall deformity incidence of 47.17% was recorded with monthly incidence ranging from 0 % (November 2007) to 68.42% (January 2008). In November 2007, only 3 larvae were collected from this river with no deformity noticed in them. In PRR, the overall percent mean deformity amounted to 33.71% among the total 448 larvae collected, with monthly incidence ranging from 26.6% (December 2007) to 40.9% (March 2008). In PR, the overall observed deformity incidence was 30.34%. In this habitat, the lowest deformity incidence was recorded in January 2008 (15.38%) and the highest (42.3%) in March 2008.

The deformities in Chironomus spp. larval menta (Figure 5.2) amounted to 27.9, 20.87, and 30.19% in the PRR, PR, and KUR, respectively. However, incidence of deformity in mandibles (Figure 5.3) was relatively low and amounted to 3.79% (PRR), 3.48% (PR), and 3.77% (KUR) (Figure 5.6).

There was no statistically significant difference (P > 0.05) between deformities of menta or deformities of mandibles in Chironomus spp. larvae collected from the three rivers. Among the deformity traits of mentum, Kohn gap, missed teeth, fused teeth, and broken teeth were the most dominant. Although deformities in mandibles were relatively rare, but where present, were severe resulting in broken or missing teeth. The incidence of deformity in epipharyngis (Figure 6.4) was generally lower compared with mentum deformities, with the highest incidence (18.87%) occurring in KUR and the lowest (6.96 %) in PR. The highest percentage (10.43%) of antennal deformity (Figure 5.5) was recorded in the PR and the lowest (4.91%) in the PRR. Statistical analysis revealed that the incidence of deformities in the epipharynges and antennae among the three rivers varied significantly [Kruskal-Wallis, X2=21.46 (n = 15) and 10.49 (n = 15), respectively, at P < 0.05]. The distinction in the incidence of deformities among the different head capsule structures of Chironomus spp. larvae in the three rivers is summarized in Figure 5.6.

Table 5.1: Larval deformity incidence (percentage ± SE) in Chironomus spp. sampled monthly (November 2007-March 2008) from Permatang Rawa River (PRR), Pasir River (PR), and Kilang Ubi River (KUR) in the Juru River Basin, Penang, Malaysia. The total number of larvae processed and examined each month in each habitat is given in parenthesis

2007-2008

PRR

PR

KUR

November

40.9±4.61 (88)

33.33±2.16 (21)

0.0± 0.0(3)

December

26.6± 4.28 (94)

29.41±1.88 (17)

26.67±1.71 (15)

January

31.86±4.95 (113)

15.38±1.30 (13)

68.42±2.03 (19)

February

32.2±4.45 (91)

26.32±2.71 (38)

14.29±0.93 (7)

March

40.32±3.86 (62)

42.3±2.52 (26)

11.11±1.25 (15)

Overall mean

33.71±10.01 (448)

30.34±4.94(115)

47.17±3.63(59)

Table 5.2: Mean ± SE values of selected physico-chemical parameters of water and sediments measured monthly from November 2007 to March 2008 at a permanent sampling site in each of three rivers, Permatang Rawa River (PRR), Pasir River (PR), and Kilang Ubi River (KUR), in the Juru River Basin, Penang, Malaysia.

Parameter

PRR

PR

KUR

Water depth (cm)

19.07±2.03

25.87±1.83

20.43±1.45

River width (m)

3.36±0.08

4.65±0.18

3.42±0.09

pH

7.10±0.03

6.90±0.01

6.97±0.03

DO (mg/l)

3.30±0.36

2.75±0.20

1.14±0.29

Temperature (°C)

30.15±0.26

27.01±0.15

27.86±0.16

TOM (%)

8.57±1.38

6.21±0.75

3.29±0.82

TSS (mg/l)

81.07±1.18

22.57±2.53

68.57±1.36

Phosphate-P (mg/l)

2.15±0.09

2.67±0.35

3.97±0.41

Ammonium-N (mg/l)

3.83±0.31

3.30±0.22

5.62±0.50

Nitrate-N (mg/l)

1.50±0.30

0.85±0.14

0.96±0.11

Sulphate (mg/l)

13.29±2.03

6.38±0.44

27.69±3.16

Chloride (mg/l)

5.45±0.84

3.25±0.21

4.24±0.20

Aluminum (mg/l)

0.22±0.04

0.08±0.01

0.17±0.03

Sediment Zn (ppm)

38.83±4.76

24.81±1.11

44.72±4.17

Sediment Mn (ppm)

50.54±6.19

16.78±3.12

17.91±1.22

Sediment Cu (ppm)

3.13±0.70

1.98±0.91

1.64±0.57

Sediment Ni (ppm)

2.92±2.23

3.01±2.04

ND

n = 15, ND = not detected

5.3.2 Possible Influence of Selected Environmental Parameters on Chironomid Deformities

Data concerning various water and sediments physico-chemical parameters measured during the study period are summarized in Table 5.2. The mean water depth at the sampling locations at PRR, PR, and KUR was 19.07, 25.87, and 20.43 cm, respectively, indicating these sites to be relatively shallow. Similarly, mean river width at sampling locations at PRR, PR, and KUR measured 3.36, 4.65, and 3.42 m, respectively. The mean value of water pH in three rivers ranged from 6.90 (PR) to 7.10 (PRR). As expected, the mean value of dissolved oxygen level was rather low in all three habitats, with KR showing the lowest mean value of 1.14 mg/l. Water temperature generally remained 2-3 °C higher in the PRR (mean 30.15 °C), compared to the other two habitats. Total organic matter showing a mean value of 8.57% in the PRR was 2-3 fold higher in this habitat than KUR where a mean value of 3.29 was recorded; in the PR mean value of total organic matter amounted to 6.21%. Total suspended solids were the highest in PRR (mean value of 81.07 mg/l), followed by KUR (mean value of 68.57 mg/l), and PR (mean value of 22.57 mg/l). The phosphate-P content of water in the three rivers ranged from 2.15 mg/l (PRR) to 3.97 mg/l (KUR); in PR, the mean value amounted to 2.67 mg/l. Ammonium-N content in water of PRR and PR had mean values of 3.83 and 3.30 mg/l, respectively, while the highest mean value (5.62 mg/l) of this parameter was recorded in KUR. In the PRR, mean nitrate-N value was 1.5 mg/l, whereas in PR and KUR, the mean value of nitrate-N was 0.85 and 0.96 mg/l, respectively. The sulphate content in the three rivers showed a large variation, with the lowest mean value of 6.38 mg/l recorded in PR and the highest 27.69 mg/l in KUR. In PRR water, the mean value of sulphate concentration was 13.29 mg/l. The mean value of chloride in the PRR, PR, and KUR was 5.45, 3.25, and 4.24 mg/l, respectively. The aluminum concentration in samples of water from the three rivers ranged from 0.08 to 0.22 mg/l, with the highest concentration occurring in PRR and the lowest in PR; in KUR, aluminum concentration amounted to 0.17 mg/l. The concentration of this metal was nearly three fold higher in PRR compared to PR.

In the sediments of PRR, concentrations of non-residual metals, Zn, Mn, Cu, and Ni were 38.83, 50.54, 3.13, and 2.92 ppm, respectively; Mn was highest in concentration and was the main contaminant in this habitat, followed by Zn. In PR sediments, concentration of Zn at 24.81 ppm was the highest, followed by Mn, Cu, and Ni, with mean values of 16.78, 1.98, and 3.01 ppm, respectively. In KUR, Ni was below the detection level of the instrument used but Zn, Mn, and Cu concentrations amounted to 44.72, 17.91, and 1.64 ppm, respectively.

Table 5.3 shows correlation coefficient values between deformed mentum, epipharyngis, antennae, and mandibles of Chironomus spp. larvae and various physico-chemical parameters of water and sediments encountered in the three rivers. Among statistically significant correlations with mentum, total organic matter, and total phosphate-P were inversely correlated, while sediment Mn and Cu concentrations showed a positive relationship with mentum deformities. The epipharyngis deformities were negatively correlated with phosphate-P, ammonium-N, and sulphate concentrations in water, and positively correlated with dissolved oxygen, and Mn in the sediments. The antennal deformities had a negative correlation with phosphate-P, and positive correlation with total organic matter, and concentrations of Mn and Cu in the sediments. The only statistically significant correlations between mandible deformities were a positive correlation with total organic matter, and inverse relationship with phosphate-P and sulphate in water.

The calculated TSI for the three rivers based on the encountered frequency of Chironomus spp. larval mentum deformity is presented in Table 5.4. The data show that the highest TSI mean value of 59.16 was recorded for PRR, followed by 46.15 for PR, and 44.05 for KUR.

Table 5.5 summarizes linear positive or negative relationship between TSI and various environmental parameters in the three rivers. Among the statistically significant correlation coefficient values, in PRR, water pH, DO, and sediment Ni showed inverse relationship, while sediment Zn showed a positive relationship. Similarly, in PR, water pH, TOM, phosphate-P, and ammonium-N were negatively correlated with TSI, and DO had a positive relationship with TSI; the sediment Cu was also positively correlated with TSI in this habitat. In the KUR, the only significant correlation was noted for sediment Mn having a positive correlation.

As shown in Figure 5.7, the RDA selected (forward selection) TSS, sediment Zn, Mn, Cu, and Ni, and water pH, dissolved oxygen, water temperature, total organic matter, nitrate-N, chloride, phosphate-P, ammonium-N, sulphate, and aluminum as parameters that significantly affected some proportion of deformities. The first axis explained 75.1% and the second axis 15.5% of the variance and Montrcarlo permutation test (499 permutations) was significant (P < 0.05).

The total deformities correlated closely with deformities of mentum but only weakly with deformities in other parts of head. The total deformity incidence was strongly correlated with high contents of sediment Mn and Ni. The mentum and epipharyngis deformities incidence was highly correlated with increase of TSS, total aluminum, and ammonium-N, and decrease in pH and dissolved oxygen.

Table 5.3: Non-parametric correlation (correlation coefficient values) between different types of deformities (data transformed using arsin√x2) in head capsule mouth parts of Chironomus spp. larvae and water and sediment parameters (sampled from November 2007 to March 2008) in Permatang Rawa River (PRR), Pasir River (PR), and Kilang Ubi River (KUR) in the Juru River Basin, Penang, Malaysia.

Parameter

Mentum

Epipharyngis

Antennae

Mandible

Water depth

0.01

0.038

0.097

-0.137

River width

0.075

0.147

0.091

-0.171

pH

-0.025

0.004

0.163

0.216

DO

-0.037

0.268*

0.060

0.123

Temperature

0.026

0.042

-0.028

0.187

TOM

-0.376**

0.127

0.263*

0.411**

TSS

0.046

-0.033

0.161

0.181

Phosphate-P

-0.258*

-0.318**

-0.304**

-0.482**

Ammonium-N

0.071

-0.390**

-0.151

-0.118

Nitrate-N

-0.213

-0.136

-0.154

0.074

Sulphate

-0.008

-0.377**

-0.190

-0.240*

Chloride

-0.135

0.038

-0.056

0.157

Aluminum

0.191

0.136

0.011

0.027

Sediment Zn

-0.024

-0.042

0.052

0.033

Sediment Mn

0.284**

0.316**

0.282**

0.189

Sediment Cu

0.240*

0.190

0.231*

0.007

Sediment Ni

0.076

-0.059

0.098

0.109

*P < 0.05; **P < 0.01

Table 5.4: Calculated Lenat's Toxic Score Index values (Lenat 1993) based on frequency of mentum deformities in Chironomus spp. larvae sampled in Permatang Rawa River (PRR), Pasir River (PR), and Kilang Ubi River (KUR) in the Juru River Basin, Penang, Malaysia.

2007-2008

PRR

PR

KUR

November

57.95

47.62

0.00

December

37.23

64.71

13.33

January

37.17

30.77

89.47

February

74.73

18.42

28.57

March

88.71

69.23

88.89

Overall mean

59.16

46.15

44.05

Table 5.5: Correlation coefficient values between Lenat's Toxic Score Index (Lenat 1993) values based on frequency of mentum deformities in Chironomus spp. larvae and prevailing selected environmental physico-chemical parameters in Permatang Rawa River (PRR), Pasir River (PR), and Kilang Ubi River (KUR) in the Juru River Basin, Penang, Malaysia.

Parameter

PRR

PR

KUR

pH

-0.882*

-0.553*

0.247

DO

-0.671*

0.694*

0.106

Temperature

-0.318

-0.318

-0.224

TOM

0.247

-0.741*

-0.365

TSS

-0.268

-0.268

-0.437

Phosphate-P

0.318

-0.671*

0.247

Ammonium-N

-0.412

-0.718

0.365

Nitrate-N

0.437

-0.296

-0.352

Sulphate

0.374

-0.600

0.106

Chloride

0.412

-0.035

-0.318

Aluminum

ND

ND

0.070

Sediment Zn

0.765*

0.388

ND

Sediment Mn

0.153

0.341

0.482*

Sediment Cu

0.294

0.647*

0.014

Sediment Ni

-0.585*

0.097

ND

ND = Not detected

*P < 0.05

A

B

C

D

E

F

Figure 5.2: Deformities of mentum in Chironomus spp. larvae collected from three rivers in the Juru River Basin: A: Normal teeth, B: Slightly broken median-lateral tooth, C: Severely broken median tooth, D: Kohn gap, E: Fused median teeth, F: Missing median-lateral tooth.

A

B

C

20 μm

Figure 5.3: Deformities of larval mandible of Chironomus spp. collected from three rivers in the Juru River Basin: A: Normal mandible, B and C: Deformed mandible.

10 μm

A

B

Figure 5.4: Deformities of pectin epipharyngis of Chironomus spp. larvae collected from three rivers in the Juru River Basin: A: Normal epipharyngis, B: Deformed epipharyngis.

A

B

Figure 5.5: Deformities of antennae of Chironomus spp. larvae collected from three rivers in the Juru River Basin: A: Normal antennae, B: Deformed antennae.

Figure 5.6: Percentage (± SE) of deformity incidence in different structures of head capsule of Chironomus spp. larvae collected monthly (November 2007 to March 2008) from Permatang Rawa River (PRR), Pasir River (PR), and Kilang Ubi River (KUR) in the Juru River Basin, Penang, Malaysia.

-0.6

1.0

-0.6

0.8

Total Deformity

Mentum

Epipharyngis

Antennae

Mandibles

pH

DO

Water Temp.

TOM

TSS

Phosphate-P

Ammonium-N

Nitrate-N

Sulphate

Chloride

Aluminum

Zn

Mn

Cu

Ni

Figure 5.7: First two axes from Redundancy Analysis (RDA) showing the relationship between selected (forward selection) environmental parameters and morphological deformity incidence in head capsule structures in Chironomus spp. larvae collected from Permatang Rawa River (PRR), Pasir River (PR), and Kilang Ubi River (KUR) in the Juru River Basin, Penang, Malaysia.

6.4 DISCUSSION

None of the rivers investigated in this study is perceived to be pristine or even clean because of their location amid areas of high nutrient loading from various industrial, agricultural, and anthropogenic sources. These rivers are dominated by pollution-tolerant invertebrate taxa, such as Oligochaeta (Tubifex sp.) and Chironomus spp. (data not shown). The PRR is most likely contaminated with agricultural chemicals applied periodically to the surrounding paddy fields. The KUR receives industrial discharges from garment and rubber factories. The PR is polluted primarily with domestic wastes resulting from anthropogenic activities in the surrounding residential areas of this river.

Application of living organisms in biomonitoring of aquatic ecosystems has several advantages over traditional chemical analyses for water quality. Freshwater organisms live almost continuously in the water and respond to all environmental stressors, including synergistic combinations of pollutants (Morse et al. 2007). Disturbance of the aquatic communities due to pollution is clearly proven (Goodnight 1973, Kay et al. 2001). However, Hamilton and Saether (1971) suggested that investigation of morphological abnormalities in individual organisms might prove particularly useful in determining the biological effects of contaminants in aquatic ecosystems although comparatively little is known about how individual organisms react to contaminants. Petersen and Petersen (1983) stated that changes at the organismal level might be more useful than changes at the community level for purposes of environmental monitoring. Individual response occurs before community responses and thus could provide an earlier warning of pollution stress.

The larval head capsule morphological deformities in Chironomus spp. observed in this study are comparable to those reported earlier by Warwick and Tisdale (1988), and Bird (1994). The present study clearly shows that deformation of the mentum is a widespread developmental anomaly in Chironomus spp. larvae in all three sampled rivers. Concurring with Vermeulen (1995), it is assumed that Chironomus spp. midge larvae are highly susceptible organisms to morphological deformation, therefore, they are potentially important indicators of the effects of the water and sediment- bound contaminants (Hudson and Ciborowski 1996). Morphological abnormalities appear to result from the alteration in developing cells which may lead to their improper functioning and interference with differentiation, which renders their proper development (Karmin 1988). Therefore, incidences of deformities in organisms at particular sites could indicate overload of contaminants at such sites.

In the scientific literature it is mentioned that several substances alone or together may cause morphological deformation in chironomid larvae, specifically heavy metals and pesticides, but no specific substance(s) has been identified as being more causative of deformation than others (Wiederholm 1984).

In the present study, the frequency of mouth parts deformities in Chironomus spp. larvae differed among the three rivers; the source and nature of pollution in these habitats differed as well. For example, the highest incidence of deformities in larval Chironomus spp. (47.17%) observed in the KUR may have been due to the influence of some industrial discharges from the nearby rubber and garment factories. Deformities of this magnitude are much higher compared to the 1-30% reported by Bird (1994) in a polluted river in Canada.

Dermott (1991) stated that there is no distinct evidence that could determine which of the various industrial and agricultural chemicals induce deformation in the chironomid larvae. However, Warwick (1985) and Warwick and Tisdale (1988) had reported the association between deformities in Chironomus spp. larvae and contaminated sediments. The incidence of deformities in Chironomus spp. larvae and the severity of their response were related to elevated concentrations of radioactive materials, metals, and pesticides (Warwick 1985, Warwick and Tisdale 1988).

In the PRR, the total percent deformities in Chironomus spp. larvae amounted to 33.71%. Presumably the chironomid population in this river is exposed to runoff of agricultural chemicals, including fertilizers, fungicides, herbicides, and insecticides that are routinely applied to the surrounding Permatang Rawa rice fields. Dickman et al. (1992) reported that increase in frequency of deformities was correlated with high levels of metal, coal tar, urban or agricultural runoff, and pesticides. Madden et al. (1992) reported a significant correlation between the antennal and mouth parts deformities in larval chironomids (Chironomus spp., Dicrotendipes conjunctus, and Procladius paludicola) and the concentrations of DDT and the herbicide, Dacthal.

It is known that heavy metals contamination is a major cause of deformities in the chironomid larvae. For example, in a heavily polluted river in India, Bhattacharya et al. (2005) reported 35.88% antennal deformities in Chironomus circumdatus under sediment Zn and Cu concentrations of 165.79 and 51.33 ppm, respectively. Janssens de Bisthoven and Gerhardt (2003) in their investigation reported 14% deformed larvae of Procladius choreus and interpreted this deformation of mouth parts as an effect of metal-related pollution; the concentration of some metals in water with acidic environment (pH 6.4) in their study was 0.01, 0.02, and 0.03 mg/l for Cu, Pb, and Zn, respectively.

In a study to assess sediment toxicity and sublethal effects on chironomid larvae, Meregalli et al. (2000) reported high levels of morphological deformities reaching 40, 10, and 20% in menta, mandibles, and epipharyngis, respectively, under Zn and Cu concentrations of 212 and 28 ppm, respectively. By comparison, percent deformity in those three structures of Chironomus spp. larvae in the present study was much lower under the highest sediment Zn, Ni, and Cu concentrations of 44.72, 3.01, and 3.13 ppm, respectively. Nevertheless, such concentration levels of these metals probably induce some morphological deformities in Chironomus spp. as observed in the present study.

The occurrence of head capsule morphological deformities in Chironomus spp. larvae expressed as percent in the present study was inadequate to quantify the environmental stress. Therefore, it was necessary to score deformities of mentum for each habitat by using TSI of Lenat (1993). According to Lenat (1993), a mean toxic score of 49 classifies the habitat as "Toxic Fair" in terms of water quality. Based on Lenat's TSI, the studied rivers fall under (or near) the "Toxic Fair" group as this score's calculated mean value amounted to 59.16 (range: 37.17-88.71), 46.15 (range: 18.42-69.23), and 44.05 (range: 0.0-89.47) for PRR, PR, and KUR, respectively. Although Lenat (1993) designed TSI to be applied in situations of organic loading, the present study shows that it is also useful in heavy metals contaminated rivers.

Ordination technique facilitated identification of the influence of various environmental parameters on deformities in Chironomus spp. larvae. Concentration of metals, particularly Ni and Mn were highly correlated with larval deformities. There were significant non-parametric correlations between several environmental parameters and mentum, epipharyngis, antennae, and mandible deformities (Table 5.3). Previously, the number and severity of chironomid larval head capsule deformities have been shown to be correlated with high levels of Zn and Cu in the sediments (e.g., Warwick 1985, Warwick and Tisdale 1988, Hudson and Ciborowski 1996, Meregalli et al. 2000, Bhattacharya et al. 2005).

In conclusion, the present study demonstrates the possible influence of industrial and anthropogenic contaminants in terms of deformities in various head capsule structures of Chironomus spp. larvae inhabiting the studied Malaysian rivers. The deformity incidence in larval menta in the investigated rivers is relatively high compared to some similar earlier studies reported from temperate regions (e. g., Bird 1994, Dickman and Rygiel 1996). The identified deformities are indicative of certain environmental stresses on the studied habitats and could serve as an empirical tool for their assessment. The study also provides baseline data on some physico-chemical conditions prevailing in the investigated rivers for future reference. Based on biomonitoring assessment, the study identifies some pollutants in the rivers that are detrimental to inhabiting organisms, requiring appropriate water quality improvements.

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