"We got them from a lake", "What lake?", "I dunno, a lake somewhere..."

Introduction

Excess runoff, like trace metals, can be exuded into the environment by industrial activity. Some of these metals can have a toxic side effect, such as cadmium (Cd), which can disrupt key cellular processes. Increased contamination may lead to imbalance in the ecosystem and lead to eutrophication, which increases the likelihood of cyanobacterial blooms. This, in turn, may influence an increase in dissolved organic matter (DOM) which can lead to a decrease in the overall efficacy of the microbial loop. Cyanobacterial blooms of Cylindrospermopsis raciborskii have shown an increase in frequency and also distribution, indicating a high level of environmental tolerance factors. Its exudates can bind with free Cd²⁺ ions and, whilst making the Cd less toxic, could be transferred through the food web. Nogueira, et al. (2014) investigated these Cd-C. raciborskii-exudate complexes, examining their toxicity, and influence to the trophodynamics of a plankton food chain.

Methods

Cylindrospermopsis raciborskii exudates
A culture of C. raciborskii (BB048) was grown using ASM-1 medium buffered with 500mgL^-1 TRIS. The medium was then inoculated with the culture once it had reached exponential growth. Exudates were separated from the culture and freeze dried.

Bacterioplankton and zooplankton
A sample of a natural bacterioplankton community was taken via filtration of 500mL of freshwater through 1.2µm glass fibre filters to remove any bacterivores. The water was taken from a ‘lake’ but no information was given as to the whereabouts of the lake. The sample was centrifuged and the supernatant discarded. The zooplankton obtained were collected from a ‘eutrophic freshwater lake’ but it is unknown if it is the same lake which the bacterioplankton community was collected from. Ciliate and copepod stock cultures were kept in 25°C freshwater. No species names were mentioned in this section, which leads to confusion later on in the experimental design as it is the first time (aside from the abstract) that their species names are given.

Cadmium complexing properties of Cylindrospermopsis raciborskii exudates
Cadmium titration was done to ensure the Cd would bind with the exudates which determined the conditional stability constant (K’) and ligand concentration (CL) of the organic material. Reconstituted freshwater, without C. raciborskii exudates, was used as the procedural blank. Cd was found using a Cd ion selective electrode with an Analion double-junction glass reference electrode. Scatchard plots were used to obtain K’ and CL values. The mean CL value was used as a definition of the total Cd concentration to be added at the beginning of the experiment.

Experimental design
18mgL^-1 total organic carbon (TOC) of C. raciborskii exudates were used and measured in a TOC-5000 Analyzer. The amount of Cd was used at a consistent concentration of exudates; 1 x 10^-5 molL^-1. Three different treatments were done (with three replicates of each) consisting of the Cd treatment (Cd), exudate treatment without Cd (EPS) and a treatment with both Cd and exudates (EPS + Cd). The bacterioplankton were exposed to the Cd-exudate complexes whilst the zooplankton (being higher on the food chain) were exposed to organisms fed with Cd and a medium containing Cd from lower trophic level cultures. Organisms were harvested after incubation and the supernatant filtered through 0.2µm acetate membrane to be incubated with the next trophic level. Organisms were harvested again and suspended in reconstituted freshwater to be used for predation experiments and determination of particulate Cd.

For the bacterioplankton experiment, 5mL aliquots were used as population inocula. Population density was found by light absorption at 781nm with a spectrophotometer. DOC levels were determined in 10 mL aliquots of culture obtained at 0, 48, 72, 96, 384 and 552 h of incubation and filtered through a 0.2 mm membrane filter for DOM quantitation. DOC was measured using a TOC-5000 carbon Analyzer. Ciliate growth was checked by counting preserved organisms under a stereomicroscope. Mortality of the protozoa, Paramecium caudatum, was the endpoint of Cd toxicity, given as the LC₅₀. This is where they mention the species names for the first time in the full paper, which is odd considering they do not give the full genus name. The copepod cultures were incubated for 72h and then harvested for particulate Cd determination. No genus name was given at all for the copepods, only in the abstract which is Mesocyclops longisetus.

Results

Bacterioplankton density indicated that high levels of free Cd inhibited growth of the community, but in the treatments with exudates there was the highest level of density observed at 1-2days post-incubation. However, the EPS + Cd treatment saw a fall in density after 72h. Therefore, free Cd has a higher toxic effect than complexed Cd. Cd accumulation in ciliates via waterborne Cd show >50% mortality whereas for dietborne Cd, no mortality was observed, only Cd accumulation. Cd accumulation by the copepods show accumulation in the dietborne Cd (EPS + Cd treatment) and the waterborne Cd (Cd treatment). Mortality was found only in the waterborne experiments possibly due to the higher dissolved Cd concentration.

Discussion

The results show a decrease in DOC concentration and an increase in bacterioplankton density in the EPS treatment, indicative of heterotrophic bacterial activity on the exudate. This is expected as around half of this activity can be supported by algal extracellular products that are released during the growth of phytoplankton. However, there is also evidence of a reduction of DOC in bacterioplankton communities due to DOM adsorption onto the bacterial cell surface. Cd toxicity was also affected by the exudate, which is again an expected result as free metal ions have a high toxicity level than their complexes. Complexes also diffuse a lot slower across cell walls, resulting in a decrease of bioavailability and thus toxicity. However, this does mean the accumulation can occur, as seen in the copepod experiments whereas high mortality rates are found upon exposure to free ions. This indicates that dissolved Cd is much more toxic than food-based Cd. This is most likely due to the fact the free ions are metabolically available so are capable of disrupting metal ion homeostasis and alter signal transduction across cellular membranes. Gut passage time can also influence the toxicity of a metal. For a smaller tract, there will be less time for assimilation to occur meaning the toxicity effects would be less than for a longer one, for example in larger organisms.

Conclusion

By simulation of a microbial loop across multiple trophic levels, demonstration of the toxicity of Cd was achieved by comparison of free ions versus its complexed form. The conclusion the authors give is brief, but fails to report on any future studies that can come from their report.

If anything, the abstract is the most essential part of the entire report because it explains key information that isn’t provided in the actual paper, such as the organism names. Also key information is left out such as where any of the organisms were obtained from! The highest level of detail that is achieved is a ‘eutrophic freshwater lake’ but the reader does not know if it is the same lake that the bacterioplankton community was sampled from. All in all, the paper raises some key and important issues regarding toxic metals seeping into the environment by industrial runoff but the paper itself is very difficult to understand, leading the reader to look to outside information for understanding.

Ref: Nogueira, P. F. M., Nogueira, M. M. & Lombardi, A. T. (2014) Influence of the microbial loop on trophodynamics and toxicity of cadmium complexed by cyanobacterium exudates. Environmental Science: Processes and Impacts. 16(5), 1029-1034. doi: 10.1039/c3em00550j.