The use of Clarias gariepinus and associated helminthic parasites as bio-indicators of metal pollution in a subtropical ecosystem

Abstract

Metals are naturally occurring elements that play an important role in the functioning of organisms. However, imbalances in metal concentrations in the environment are of great concern due to their potential detrimental nature. Since the start of the Anthropocene, humans have increased their impact on the environment through anthropogenic activities such as mining, over-exploitation, deforestation, gas emission and pollution. Metals are non-biodegradable and thus will bioaccumulate in organisms that are exposed to metals for extended periods of time. Metals can also biomagnify to higher levels in the food chain, with top predators potentially having the highest levels of metal concentrations. The accumulation of metals along the food chain can have serious health implications for humans consuming these organisms. For example, lead can cause renal failure, mercury and cadmium can cause kidney problems, whilst zinc and copper can lead to nephritis. Aquatic ecosystems are under constant threat of metal pollution because of runoff into these ecosystems. Lotic and lentic ecosystems act as a sink for metals with metals being adsorbed in the sediment and building up to high concentrations. Metals in aquatic ecosystems are readily available for organisms to accumulate. It is therefore of the utmost importance to detect metal pollution in aquatic ecosystems as early as possible. Scientists use a tool called biomonitoring to assess environmental exposures to synthetic or natural chemicals by testing individual organisms’ body fluids or tissue samples, i.e. using these organisms as bio-indicators. The technique uses the knowledge that chemicals leave a footprint within cells after exposure. By measuring the mark after exposure, the amount of chemicals entering the organisms from the environment can be indicated. Biomonitoring is therefore a very important tool to assess pollution levels in the environment. The bio-indicator approach involves the measurement of the response of an organism from molecular to community levels. For the purposes of this study, Clarias gariepinus and its associated endohelminth parasites were used as bio-indicators of metals in three different aquatic systems located in the Ndumo Game Reserve (NGR). The three aquatic systems consist of a freshwater lotic system: The Usuthu River, which flows along the northern border of NGR, a freshwater lentic system: Shokwe Pan and a saline lentic system: Nyamithi Pan. Clarias gariepinus is an omnivorous and bottom-dwelling fish that has a long life-span and is therefore a good species to use as a bio-indicator of metal pollution. Parasites are found in all ecosystems on Earth and can be present at high abundances. Parasites feed on their hosts and are therefore classified as occupying a higher level in the food chain than their hosts. Clarias gariepinus in NGR is infected with five different endohelminth parasites, two of which are cestodes and three nematodes. The two cestode species are the Proteocephalus sp. and Tetracampos ciliotheca located in the intestine of their hosts. The three nematode species are the Contracaecum sp. located in the body cavity of their host, Procamallanus pseudolaeviconchus located in the stomach of their hosts and Paracamallanus cyathopgharynx located in the rectum of their hosts. For this reason, the study chose C. gariepinus and its associated helminth parasites. The metals tested for in the study were the essential metals (Fe, Co, Ni, Zn and Cu) and the non-essential metals (Al, Mn, As, Cd, Pb, Se and Ag) and the biological responses tested for were cellular energy allocation (CEA), metallothioneins (MTs) and reduced glutathione (GSH). In order to determine the metal concentrations of the different samples, the sediment, host tissues and parasites were freeze dried and microwave digested in 2.5 mL nitric acid and 7.5 mL 32% hydrochloric acid and the water samples were acidified with nitric acid, whereafter the metal concentrations were measured using standard inductively coupled plasm mass spectrometry. Univariate statistics were analysed using GraphPad Prism®7 software, where homogeneity was tested using the Shapiro-Wilk test, one-way analysis of variance (ANOVA) with Tukey post-hoc analysis or a Kruskal-Wallis followed with Dunn’s multiple comparisons to determine significance. Multivariate statistics were analysed using SPSS version 18 (PAWS Statistics, IBM, USA) to perform a Discriminant Function Analysis (DFA) and Canoco version 5 (Ter Braak & Smilauer, 2012) was used to perform a redundancy analysis (RDA). The results showed that the freshwater lentic system had the highest levels of metals in the sediment whilst the saline lentic system had the highest levels of metals in the water. The freshwater lotic system had the lowest levels of metals in the environment. The results also showed that the NGR was close to a natural state with no visible human impact on the aquatic systems, and the metal concentrations were below that of other impacted areas in South Africa. After the environmental concentrations were measured, the metal concentrations in the muscle and liver tissues of C. gariepinus were compared to the metal concentrations in the environment. The study found that C. gariepinus at the three different sites accumulated metals to a higher level than present in the environment and they can therefore be used as a bio-indicators of metals in natural ecosystems. The catfish in the freshwater lentic systems accumulated metals to higher levels than the catfish in the saline lentic and freshwater lotic systems. The catfish in the saline lentic system showed the lowest amounts of accumulation. After the bioaccumulation of C. gariepinus was determined its endohelminth parasites were evaluated as bio-indicators of metals. The study found that the five endohelminth parasites had higher concentrations of all the metals than their host muscle tissues and higher than most of the liver tissues. The study therefore found that endohelminth parasites could be used as bio-indicators of metal pollution and could even be better indicators than their hosts by accumulating metals to higher levels than their hosts. The different aquatic systems played a role in the accumulation of metals in the parasites with the Nyamithi catfish parasites accumulating the least amount of metals. Of the five helminth parasites, the cestode species Tetracampos ciliotheca had the highest bioaccumulation rates while the body cavity nematode Contracaecum sp. had the lowest bioaccumulation. The study thus established that NGR was in a natural state, that C. gariepinus accumulated environmental metal concentrations to higher levels in their tissues and that endohelminth parasites accumulated metals to even higher levels. These findings could then be used to determine the effect of metals on the biological response of both the C. gariepinus as well as the endohelminth parasites in their respective environments. The study found that there was a spatial difference in the biological response of C. gariepinus to the accumulation of metals, with catfish in the lotic system having significantly higher concentrations of metallothioneins than the catfish at the two lentic sites. The study also found that the endohelminth parasites, Contracaecum sp., had lower concentrations of metallothioneins (MT) than their hosts, but significantly higher levels of Glutathione reductase (GSH) than their hosts. These findings indicate that Contracaecum sp. does not have the same ability as their hosts to detoxify and remove metals from their systems. The biomarker responses indicate that an increase in metal concentrations will have a negative impact on their hosts with a decrease in energy. The study found that parasites had an effect on the biomarker response of their hosts to metal concentrations in that the catfish with the least number of parasites had the highest concentrations of MTs and the catfish with the highest number of parasites had the lowest concentration of MTs. This is because the parasites help to remove metals from their hosts by biomagnification, and therefore their host does not have to waste energy in removing the metals. But the study found that the catfish with the highest number of parasites had the lowest available energy because of the parasites feeding off the host. It is thus a “catch-22” situation where an increase in parasites helps to deal with the effect of metals on their hosts but at a cost to their hosts’ energy budget.