A total of 25 taxa were sampled during the biomonitoring survey. The highest number of taxa was sampled at Site 1 and Site 3 (Table 8). Very few taxa belonging to the order Ephemeroptera, Odonata or Trichoptera were sampled during the survey. This is important as numerous studies demonstrate that the presence of taxa from the orders Ephemeroptera, Trichoptera and Odonata are often used as an indication of water quality. These taxa are often the more sensitive taxa in a community (Dickens & Graham, 2002) and their presence is a reflection of good water quality. There were no major differences between the community structure of the aquatic invertebrate communities at the reference site and the other sites further downstream. Most of the taxa present at the reference site were also sampled at one or more of the monitoring sites. Many of the taxa sampled at the monitoring site, however, belong to the order Diptera. Diptera are often indicators of poor water quality and some families prefer very fast flowing waters (Day et al., 2002). Larvae in general live in protein-rich decaying organic material where they act as scrapers although some are filter feeders.
Table 8: Results of the SASS5 index and the aquatic invertebrate diversity (x indicates presence of the particular taxa) during the survey of the Klip River.
Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 7 Site 8
Page | 36 ranged from a B/C class at the reference site to a D class at site 8. The classification system used to determine the ecological classes is indicated in Table 7. Lower SASS scores are often a reflection of alterations to habitat, while the ASPT is a good reflection of water quality. Based on this, it is evident from the results that the alterations to the aquatic invertebrate community structure are due to an alteration in habitat. Results of the habitat assessment, however, clearly indicate that habitat integrity of most of the study sites is in a good state. The only major change that is apparent is an increase in flow due to return flow from the catchment. This may be an indication that flow alteration may be responsible for most of the alteration to the invertebrate community structure. It is important to note that the ecological classes of the site below the confluence with the Rietspruit changes form a C class to a C/D and even a D class. Although the ecological integrity of the invertebrates changes from a B/C class at site 1 to a D class at site 2 the communities show some form of recovery at sites 3 and 4. This is an indication that the poor water quality from the Rietspruit is altering the community structure of the aquatic invertebrates in the Klip River. The low ASPT observed at all the study sites is an indication that the water quality of the entire Klip River is definitely degraded.
Page | 37 Figure 12 shows a RDA tri-plot showing the relationship between the sample sites, environmental variables and invertebrate species. Trends shown by the graph are those of Site 2 and 8 grouping together with similarities in dissolved oxygen and metals such as Mn, Cu, Ba and PO4. Site 1 and 7 also showed similarities and the invertebrate species occurring close to this grouping were Leptoceridae, Dixidae, Culicidae and Oligochaeta among others. Sites 5 and 6 also grouped together, showing higher pH, TH, EC and turbidity. The invertabrate species occurring close to this grouping were Coenagrionidae, Ancylidae and Chironomidae. Site 3 showed an increased amount of nutrients which is as a result of the return flow from the Olifantsvlei WWTW. There is also an indication of an increase in some metals, with the predominate invertebrates being Corixidae, Physidae, Dytiscidae and Daphniidae among others.
Figure 12: RDA Tri-plot showing the relationship between the sample sites, environmental variables and invertebrate species during the October 2009 survey of the Klip River. The triplot explains 56.3% of the total variance with 35.6 explained on the first axis and a further 20.7% explained on second axis.
Page | 38 3.3.5 Fish Health
3.3.5.1 Histopathology
The results of the fish health assessment are tabulated in Table 9 and Table 10. The selected fish species for the three selected sites was C. gariepinus. In Table 9 it can be seen that only site 6 had an adequate sample size of 10 while sites 3 and 7 did not yield enough fish for use in the assessment. The average fish weight and length increased from site 4 to site 8 from 1.5kg to 2.3kg and 59cm to 69cm respectively. The external features (skin, gills, opercula, eyes, fins) were mostly normal with some erosion of the fins occurring in some fish specimens. The only exception was the gill condition of the fish at site 8 where 50% of the specimens indicated slight discolouration.
Page | 39 Table 9: A summary of fish condition variables from the three sites on the Klip River for the October 2009 survey for Clarias gariepinus.
Site
Page | 40 Table 10: A summary of fish condition variables from Site 7 on the Klip River for the November 2009 survey of Labeo capensis.
Site Fish No. Sex
Body Weight
(g)
Total Length
(cm)
Standard Length
(cm) Eyes Skin Fins Opercula Gills Bile
Mesenteric
fat (%) Liver Spleen Hindgut Kidney Parasites
7 1 M 840 44.5 35.5 0 10 0 0 0 D green <50 30 0 0 0 No
2 M 970 43 35 0 10 0 0 0 D green <50 30 0 0 0 No
3 M 880 42.7 35 0 10 0 0 0 D green <50 30 0 0 0 No
4 M 1210 44.2 35.3 0 0 0 0 0 D green <50 0 0 0 0 No
5 M 550 40.6 32.7 0 10 0 0 0 D green <50 30 0 0 0 No
6 M 770 44.6 36.8 0 10 0 0 0 D green 50 0 0 0 0 No
7 M 650 44.5 36 0 10 0 0 0 D green 50 0 0 0 0 No
8 M 600 38.5 31.5 0 30 0 0 0 D green <50 30 0 0 0 No
9 M 572 45 36.5 0 10 0 0 0 D green >50 30 0 0 0 No
10 F 1111 48.5 39.4 0 10 0 0 0 D green <50 30 0 0 0 No
11 M 646 40 33 0 10 0 0 0 L straw <50 30 0 0 0 No
Page | 41 External abnormalities identified on the fish showed skin lesions in (6/18) C. gariepinus and all L. capensis specimens. These included fin erosion which was identified in (5/18) C. gariepinus.
A pectoral fin was missing in one of the fish. Gill abnormalities were also identified in (5/18) C.
gariepinus, which included pale and slightly discoloured gill filaments. Cysts associated with parasitic infections were also identified in the gills of 11.1% of C. gariepinus. Macroscopic examination of the visceral organs showed discolouration of the livers in 44.4% C. gariepinus and 72.7% L. capensis specimens. Other abnormalities identified in C. gariepinus included fatty livers, and cysts associated with parasitic infections. The condition factors calculated for both species are presented in Table 11. L. capensis showed a slightly higher mean condition factor compared to that of C. gariepinus, averaging 1.3±1.0 and 0.8±0.8 respectively.
Table 11: Mean body mass, total length, condition factor (CF), and standard deviations, for two fish
Table 12: Percentage prevalence of histological alterations identified in the fish species studied
Target organ/
Page | 42 Liver histopathology
The percentage prevalence of histological alterations identified and the histological index results are presented in Tables 12 and 13 respectively. The livers of L. capensis were most affected in terms of the percentage prevalence of the liver histological alterations identified. This is most likely as a result of the low sample size. The liver index (IL) of both fish species was found to be in Class 1, considered to have tissue structure with slight histological alterations, scoring 4.7 and 5.5 for C. gariepinus and L. capensis respectively.
Table 13: Mean organ index and fish index values, ranges in parentheses, for the fish species studied. IL = liver index, IG = gill index, IK = kidney index, IT = testis index, IO = ovary hyaline droplet degeneration. However, the percentage prevalence of most alterations was higher in C. gariepinus than in L. capensis. Microscopic examination showed parasitic infections in both species. The gill index (IG) of both fish species was also found to be in Class 1, considered to have tissue structure with slight histological alterations, scoring 5.6 and 2.2 for C.
gariepinus and L. capensis respectively.
Kidney histopathology
The kidneys of C. gariepinus were most affected in terms of the percentage prevalence of the histological alterations identified. The kidney samples of this species showed dilation of glomerulus capillaries, hyaline droplet degeneration, nuclear alterations, and melano-macrophage centres, thickening of Bowman’s capsule membrane and infiltration by leucocytes.
Other histological alterations identified in this species include structural alterations and inflammatory responses. The kidney index (IK) of both fish species was found to be in Class 1, considered to have tissue structure with slight histological alterations, scoring 5.7 and 2.0 for C.
gariepinus and L. capensis respectively.
Page | 43 Gonad histopathology
The developmental stages of gonads in both species ranged from early developmental to fully matured individuals. This is to be expected considering that sampling took place mid-spring when most fish species are nearing maturation. Apart from a few melano-macrophage centres, no abnormalities were identified on any of the collected fish’s gonads.
Combined histological response
The mean fish index (IFISH) calculated per species showed that C. gariepinus was the most affected in terms of the prevalence and severity of the histological alterations identified (IFISH = 16.6) (Table 13).
Results show that minor external abnormalities were identified on both species. Skin lesion and fin erosion are most likely a result of past injuries that have healed. Although cysts associated with parasitic infections were also identified and may have contributed to gill abnormalities, the pale and slight discolouration of the gills identified on some gills was most likely due to other injuries acquired during capture. Condition factor is an indication of the ratios between mass and length of the fish. C. gariepinus specimens had a slightly lower condition factor as compared to that of L. capensis (Table 11); similar results for C. gariepinus were obtained in a recent study conducted at the Vaal River (Jeffares & Green, 2010).
Significant alterations of tissue were mostly limited to the liver, gills and kidneys. Some of the more evident alterations found in the liver included nuclear alterations and vacuolation of hepatocytes. The gills are probably the most sensitive organs for toxicant exposure as they actively filter water bound oxygen. They showed alterations that included congestion, rapture of pillar cells and hyperplasia. These alterations are not toxicant specific but can be associated with pathogens, sewage effluents, and metal pollution in the water. Gill immune responses that serve to slow entry of the toxicant have the undesirable effect of threatening to suffocate the fish (Skidmore, 1964; Burton et al., 1972). The kidneys of some of the C. gariepinus specimens showed dilation of the glomerulus capillaries which is significant as it may lead to changes in blood pressure passing through the kidneys and thus affecting the kidney function. Leukocyte infiltration was also found in both species indicating inflammatory responses which may be as a result of a number of pathogens or injury.
Page | 44 The histological alterations identified were mostly associated with progressive and regressive changes. Minimal circulatory disturbances were also found in the gills and kidneys in the form of telangiectasia and dilation of glomerulus capillaries respectively. Most affected of the target organs were the livers and the kidneys, which is to be expected considering the detoxification function of the liver (Van Dyk et al., 2009b) and the kidney’s function in filtering out waste products. The organ index results of all organs of both species were within Class 1, indicating normal tissue structure with slight histological alterations. However, sample size should be considered when evaluating the index results, as a larger sample size will be more representative of the resident fish community.
3.3.5.2 Biomarkers
All biomarkers analyzed in this study were compared to those reported by Wepener et al. (2011) for risk region C. Biomarker analyses yielded non-significant differences between the sites or the fish with exception of the catalase activity, which showed significant differences between sites. Wepener et al. (2011) reported significantly higher MT levels at risk region C (includes Klip confluence with the Vaal) which is supported by a predominance of metal bioaccumulation in this region.
Acetylcholine Esterase (AChE): AChE plays an important role in the regulation of nerve impulse transmission at the cholinergic synapses. AChE hydrolyses acetylcholine, a common neurotransmitter, and thereby prevents it from accumulating in and around the synapse (Huggett et al., 1992). Among fish, AChE is predominantly localised in the brain and muscle (Huang et al., 1997). Inhibition of esterases is used as a specific indicator of stress induced by organophosphate and carbamate pesticides (Murphy, 1980). In case of acute poisoning, the parasympathetic nervous system, the neuromuscular junction, as well as the central nervous system, are severely affected (Yawetz et al., 1993). In addition to organophosphate and carbamate pesticides, a number of other contaminants including mercury and some physiological conditions; i.e. infections, anaemia, malnutrition and liver diseases are known to cause inhibition (Mayer et al., 1992).
All three sites as shown in Figure 13A show certain levels of exposure to AChE inhibitors.
Differences as expected are seen between upstream sites and those downstream, however
Page | 45 these weren’t statistically different. This comes as a result of an increasing gradient in AChE inhibitors as the river progresses downstream. Differences in species response (Figure 15A) to AChE inhibition is also seen between the target organisms and this can be attributed to different feeding behaviours.
Metallothioneins (MT): The evaluation of MT induction as a response to metal exposure may be useful as a biomarker of exposure. According to Viarengo et al. (1997), when heavy metal cations accumulate within an organism’s cells, metalloprotein neosynthesis is stimulated, thus leading to an increase in MTs that rapidly react with free metal cations present in the cytosol.
Thus, the quantification of MTs may prove useful in assessing metal exposure and predicting potentially detrimental effects induced by metals. MT site differences results (Figure 13B) show similar trends to that of AChE, thus indicating metal exposure of increasing gradients downstream. However, less significant species and site differences were seen when comparing the results with the study done at the Vaal River by Wepener et al. (2011), both species showed similar metal exposure.
Cytochrome P450 (CYP450): CYP450 is a catalytic measurement of cytochrome P4501A induction. A multitude of chemicals induce CYP450 activity in a variety of fish species, the most potent inducers being structural analogs of 2,3,7,8-tetracholordibenzo-p-dioxin. Although certain chemicals may inhibit CYP450 induction/activity, this interference is generally not a drawback to the use of CYP450 induction as a biomarker (Whyte et al., 2000). CYP450 response (Figure 13C) between sites shows no significant differences with only a minor increase in gradient downstream. No differences were seen in species (Figure 15C) showing similar exposure to organometallic pollutants. Similar results were reported for risk region C at the Vaal (Wepener et al., 2011).
Page | 46 Figure 13: Mean ± standard error of biomarkers of exposure in Clarias gariepinus
from three sites in the Klip River. Statistical significant differences are explained in the text.
Page | 47 Catalase (CAT): Catalase is a common enzyme found in nearly all living organisms that are exposed to oxygen, where it functions to catalyze the decomposition of hydrogen peroxide to water and oxygen to prevent oxidative stress and in maintaining cell homeostasis (Chelikani et al., 2004). Catalase is often induced concomitantly with the antioxidant, superoxide dismutase (SOD), as a result of oxidative stress (Di Giulio et al., 1989). Significantly different results were found for catalase response between the three sites (Figure 14D), which may indicate increased levels of stress at site 6 as compared to the other two. And higher values were recorded for C.
gariepinus (Figure 16D).
Malondialdehyde (MDA): Malondialdehyde is the organic compound with the formula CH2(CHO)2. This reactive species occurs naturally and is a marker for oxidative stress. MDA is a well characterized oxidation product of polyunsaturated fatty acids in lipoproteins (Uner et al., (2006). Slight differences (not significant) were recorded between responses in the different sites (Figure 14C) and between species (Figure 16C).
Protein Carbonyls (PC): It has been established in mammalian systems including humans that direct damage to proteins or chemical modification of amino acids in proteins during oxidative stress can give rise to protein carbonyls. Protein carbonyl induction, as a biomarker of oxidative stress has been used in laboratory studies to assess the toxic effects of pesticides in freshwater fish for a while now (Parvez & Raisuddin, 2005). Lower protein carbonyl responses were recorded for site 7 as compared to the other sites which showed similarities in PC responses (Figure 14B). No significant differences were seen between species (Figure 16B).
Reduced glutathione content (GSH): Glutathione (GSH) is a tripeptide. It contains an unusual peptide linkage between the amine group of cysteine and the carboxyl group of the glutamate side chain. Glutathione, an antioxidant, helps protect cells from reactive oxygen species such as free radicals and peroxides. It is also important as a hydrophilic molecule that is added to lipophilic toxins and waste in the liver during biotransformation before they can become part of the bile. Glutathione is also needed for the detoxification of methylglyoxal, a toxin produced as a by-product of metabolism (Pompella et al., 2003). No significant differences were recorded between sites (Figure 14A) and only minor differences were seen between species (Figure 16A).
Page | 48 Superoxide dismutase (SOD): Superoxide dismutases (SOD) are a class of enzymes that catalyze the dismutation of superoxide into oxygen and hydrogen peroxide. As such, they are an important antioxidant defense in nearly all cells exposed to oxygen. Simply put, SOD out competes damaging reactions of superoxide, thus protecting the cell from superoxide toxicity.
SOD is related to CAT in such ways that both serve to reduce oxidative stress, and may be induced in tandem (McCord & Fridovich, 1988). Similar to the CAT results a gradual increase in gradient was found between sites (Figure 14) and C. gariepinus again recorded the higher response as compared to L. capensis (Figure 16), however these were not significantly different.
Page | 49 Figure 14: Mean ± standard error of biomarkers of effect in Clarias gariepinus from
three sites in the Klip River. Statistical significant differences are explained in the text.
Page | 50 Figure 15: Mean ± standard error of biomarkers of exposure in Clarias gariepinus and
Labeo capensis from site 7 in the Klip River. Statistical significant differences are explained in the text.
Page | 51 Figure 16: Mean ± standard error of biomarkers of effect in Clarias gariepinus and
Labeo capensis from site 7 in the Klip River. Statistical significant differences are explained in the text.
Page | 52 Figure 17 below shows a PCA biplot for the average biomarker response from the Klip River and Vaal River (A-F). A PCA biplot was completed to indicate differences between sites with regards to biomarker responses in the fish. Site groupings indicate a clear difference in biomarker response between the Klip and the Vaal River. All six Vaal samples showed similarities and are far removed from the Klip samples. PC, MDA, CYP450 and AChE were all much higher in the Klip River. CAT, was slightly different from the other biomarkers but also seems higher in the Klip when compared to the Vaal. MT indicated a much higher response in the Vaal river, this may be attributed to a higher metal bioaccumulation in the Vaal river. It must however, be kept in mind that the fish species used in the Vaal was L. capensis and the Klip study utilized C. gariepinus in Site 3 and 6, while only 50% of the sampled fish in site 7 were L.
capensis.
Figure 17: PCA biplot for the average biomarker responses in Labeo capensis from the Klip River (3, 6, 7) and Vaal River (A-F). The biplot explains variance with 96.2% explained on the first axis and a further 3.2 % explained on the second axis.
3.3.5.3 Bioaccumulation
The results of the metal analysis in the muscle tissue of fish from the selected sites are presented graphically in Figure 18. Only the metals shown (strontium, zinc, copper, manganese, cobalt and chromium) were in concentrations above the instrument detection levels. The results of cobalt indicated a slight decrease in concentration in the C. gariepinus from site 3 to site 7
Page | 53 while the L. capensis concentrations were the lowest. The chromium concentration in muscle tissue at site 6 was higher than the other sites. However, these values are lower than values reported in the Olifants River by Coetzee et al. (2002), where the chromium concentrations ranged from 11 – 56 μg/g in C. gariepinus and Labeo umbratus.
The copper concentration in the muscle tissue increased from site 3 to site 7 for the C.
gariepinus while the L. capensis concentrations were the highest. Kotze (1999) also completed bioaccumulation of metals for specimens of C. gariepinus and Labeobarbus aeneus at the
gariepinus while the L. capensis concentrations were the highest. Kotze (1999) also completed bioaccumulation of metals for specimens of C. gariepinus and Labeobarbus aeneus at the