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Selective uptake of algal cells (1-2 µm) or microplastic cells (1 µm) by the Demospongiae Haliclona sp. (OTU QM2474) via spectrophotometry.
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Margot Tenison Bligh 2017
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Abstract | |
The abundance of dangerous microplastics (<5 mm) in the ocean is increasing. As highly efficient filterers, sponges (Porifera) are particularly threatened, with microplastics similarly sized to food particles. Recent studies suggest that sponges can, to some degree, selectively uptake particles. This study aimed to investigate whether the Demospongiae Haliclona sp. (OTU QM2474) can selectively uptake from micron-sized microplastic and algal cells. Cell concentration was measured via spectrophotometry. No selective uptake was detected in the study. Microplastics and algae were taken up at the same rate by Haliclona sp. (OTU QM2474), regardless of whether they were in mixed or separate solutions. This study raises important questions about the way microplastics could effect sponges. Further study in this area is needed, both on selective uptake, and post-uptake effects on sponges.
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Introduction | |
Plastic litter in the world’s oceans represents a significant, and growing issue. Despite reporting of plastics in the ocean since the 1970s, real scientific interest has only existed in recent decades (1). The massive amount of plastic debris now in the ocean, along with a growing knowledge of its ecological effects, define the topic as a high-priority research area (1). Annual global demand for plastics has continually increased over recent years- it stood at 245 million tonnes in 2011 (1). Of particular concern is the occurrence of microplastics in the oceans (12). Microplastics are defined by the National Oceanic and Atmospheric Administration as less than 5 mm in size (13). They accumulate in ocean waters via direct introduction with runoff, and degradation of larger plastics (1). A significant relationship between microplastic abundance and human population-density has been found, meaning as human population continues to increase exponentially, microplastic abundance will also most likely increase (12).
Research and media on plastics in the ocean has typically focused on entanglement of marine mammals and other species, and ingestion by marine birds (1, 12). This focus likely exists as higher organisms, particularly vertebrates, are more visible, and the public generally feels more connected to them (12). However, suspension, filter and deposit feeders, detritovores and planktivores are at least as threatened by plastics in the ocean as higher trophic, higher 'profile' animals. The size similarity between microplastics, and sediments and planktonic organisms, creates risk that microplastics could accumulate within these organisms via feeding (12). This could cause physical harm, by internal abrasions and blockages. Toxic effects could also occur from the inherent microplastic contaminants such as monomers and plastics additives, and the hydrophobic persistent organic pollutants concentrated on their surface (1, 12). Beyond the direct effects of microplastic accumulation on lower trophic organisms, the ecological place of these organisms means that there is a high likelihood of bioaccumulation up the food chain.
Sponges (Phylum Porifera) are active suspension filter feeders, with a collar sieving mechanism (8, 14). They can filter massive volumes of water- 60 to 900 times their volume per hour (14). This high filtration rate, combined with sponges place as primary filter feeding invertebrates in some marine and freshwater environments, means that sponges significantly affect benthic-pelagic coupling, and organic matter and nutrient cycling (2, 5, 13). While sponges are able to efficiently retain particles down to 0.1µm, they mainly feed on micron-sized cells (8). Sponges can discriminate food particles by size. Particles smaller than 5 µm are ingested mainly by choanocytes, while larger particles are ingested by mainly by pinacocytes (4). Until recently, it had generally been assumed sponges could not select on any other features. However, evidence is emerging to suggest sponges can actively selective preferred particles. Studies have found that sponges are able to discriminate between symbiotic and pathogenic bacteria, and selectively uptake spermatozoa for transfer to the oocyte (2, 4, 5, 11). The details of the sponge filtration mechanisms however, remain unclear (2, 5, 12). If this filtering selectivity extends to microplastics and food particles, sponges could potentially avoid problems associated with increasing concentrations of microplastics in the ocean.
This study aimed to investigate whether the Demospongiae Haliclona sp. (OTU QM2474) can discriminate between algal and microplastic cells. According to the literature, particles of bacterial size are retained most efficiently (10). For this reason, cells were selected of the smallest size available- 1-2 µm. Changes in algal and microplastic cell concentrations were measured over time in the water surrounding sponges. As controls, changes in cell concentration of solutions with seawater, algae, and microplastics only were also measured. Changes in concentrations of each cell type in the water were observed as changes in optical density, via spectrophotometry. This was possible as the amount of scatter that occurs when light is passed through a suspension is proportional to the number of cells present in that solution (9).
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Materials and Methods |
Methods | |
This study was done using the Demospongiae Haliclona sp. (OTU QM2474) (Figure 1). Eight sponges of approximately similar volume were collected from the UQ Aquarium. The sponges were originally collected from the Heron Island region. Two dead or dying sponges, as identified by colour change (blue to brown), were excluded leaving six sponges in the study.
Four groups were used in the experiment: seawater, microplastics, algae, and a mixture of microplastics and algae. Nanno 3600™ algae (1-2 µm Nannochloropsis sp), and 1 µm Fluoresbrite® BBCarboxylate Microspheres were used. A sponge was randomised to each group using a dice. Sponges were placed in plastic tubs with 250 mL of artificial seawater (NaCl 3.5%)- enough to cover the sponges. Algae and microplastics were added to the “algae” and “microplastics” groups respectively. Both algae and microplastics were added to the “mixture” treatment in a 1:1 ratio. These groups each had a total particle concentration (i.e.[algae], [microplastics] or [algae + microplastics]) of 1.8 x 10E06 particles/mL.
Treatments lasted for an hour. Tub was stirred every 10 minutes to keep particles suspended. The experimental set up is shown in Figure 2. At 0, 20, 40 and 60 minutes, 3 cuvettes were filled per tub with 2 mL of the tubs' suspensions. The optical density (OD) of the cuvette samples were measured using a spectrophotometer, at both 600nm and 690nm. Artificial seawater (NaCl 3.5%) was used to blank the spectrophotometer. The entire project was replicated once.
Four treatments were used in the experiment: seawater only, microplastics only, algae only, and a mixture of microplastics and algae. Nanno 3600™ algae (1-2 µm Nannochloropsis sp), and 1 µm Fluoresbrite® BBCarboxylate Microspheres were used in this experiment. This was the smallest cell size available in both algae and microplastics. Sponges were randomised to treatments. Each sponge was assigned a number, and a dice was rolled to assign numbers to treatments. For each treatment, sponges were placed in plastic tubs. 250 mL of artificial seawater (NaCl 3.5%) was added to each tub- enough to cover the sponges. Algae and microplastics were added to the “algae” and “microplastics” treatments respectively. These were the positive controls. Both algae and microplastics were added to the “mixture” treatment. Algae and microplastics were added so that each treatment had a total particle concentration (i.e.[algae], [microplastics] or [algae + microplastics]) of 1.8 x 10E06 particles/mL. The total particle concentration in the "mixture" treatment was made up of equal parts algae and micro plastic cells (9 x 10 E05 particles/mL of each). Nothing was added to the “seawater” treatment- the negative control.
Treatments lasted for an hour. Each tub was stirred every 10 minutes, in order to keep particles suspended in the water column. The experimental set up is shown in Figure 1. At 0, 20, 40 and 60 minutes, 3 cuvettes were filled per tub with 2 mL of the tubs' suspensions. The optical density (OD) of the cuvette samples were measured using a spectrophotometer, at both 600nm and 690nm. Artificial seawater (NaCl 3.5%) was used to blank the spectrophotometer. The entire project was replicated once.
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| Figure 1 |
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| Figure 2 |
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Analysis of Results | |
All analysis was done on Excel. Mean OD600 and OD690 values, and the standard deviations of those means, were found for each group for all time points. OD values one or more standard deviations from the mean were excluded from analysis as outliers. The "LINEST" function was used to calculate the statistics for lines that best fit the data by using the "least squares" method.
Two-tailed, unpaired t-tests were done comparing the slopes and y-intercepts of the lines within each group, and across some groups. The test statistic was "t= x1-x2 / (s2x1 + s2x2)½ ~ T (n1 + n2 - 4)", where "x" represents slope/intercept, and "sx" represents the standard error of that slope/intercept. Two-tailed t-tests were done to test the significance of the "seawater" slopes and intercepts. The test statistic was "t= b / sb ~ T (n - 2)", where "b" represents slope/intercept and "sb" represents the standard error of that slope/intercept. A significance level (α) of 0.05 was used.
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Sponge Identification | |
A thin longitudinal section was made of the
sponge, pictured in Figure 3 under a Nikon fluorescence microscope (10x magnification). Spicule structure and arrangement could be clearly seen under the compound microscope,
allowing sponge identification as Demospongiae Haliclona sp. (OTU QM2474) (7).
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| Figure 3 |
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Results | |
The OD values of the "seawater" tub were measured over time as a negative control. Negative linear trends were found for both OD600 (OD600= -1.00 x 10E-05 minutes + 0.0013, R2= 0.600) and OD690 (OD690= -1.50 x 10E-05 minutes + 0.0017, R2= 0.100) (Figure 4). Table 1 shows the results of statistical anlyses on these lines. Neither slope was found to be significant (p > 0.05,). The intercepts did not significantly differ from one another (p > 0.05). The OD600 intercept was not significantly different from zero (p > 0.05), while the OD690 intercept was (p < 0.05).
Table 1. Table summarising statistical analyses of the negative control "seawater" results. Two-tailed t-tests were done to find the difference between intercepts, and the significance of the intercepts and slopes. The values of the intercepts or slopes (i.e. paremeters) and the p-values returned from these tests are shown in the table. Using a significance level (α) of 0.05, no significant difference between intercepts was found. Neither slope was found to be significance. OD600 intercept was not found to be significant, but OD690 intercept was.
| Test |
Wavelength (nm) |
Parameter value |
P value |
| Difference between intercepts |
600 |
0.0013 |
0.670 |
| 690 |
0.0017 |
| Intercept significance |
600 |
0.0013 |
0.243 |
| 690 |
0.0017 |
0.034 |
| Slope significance |
600 |
-1.0 x 10E-05 |
0.684 |
| 690 |
-1.5 x 10E-05 |
0.225 |
Negative linear trends between OD and time were observed for the positive controls ("algae", "microplastics", Figure 4) and treatment ("mixture", Figure 5) at both wavelengths. For “microplastics”, OD600= -1.15 x 10E-04 minutes + 0.0187 (R2= 0.808) and OD690= -1.35 x10E-04 minutes + 0.0123 (R2= 0.748). For “algae”, OD600= -7.00 x 10E-05 minutes + 0.0281 (R2= 0.980) and OD690= -6.50 x 10E-05 minutes + 0.0312 (R2= 0.966). For "microplastics", the OD600 line was above the OD690 line, and vice versa for "algae". Within controls, the slopes appeared to be the same regardless of wavelength. The treatment trends were found as OD600= -1.50 x 10E-04 minutes + 0.0207 (R2= 0.833) and OD690= -1.25 x 10E-04 minutes + 0.0191 (R2= 0.877). Slopes appeared identical, and the OD600 line appeared to be slightly above the OD690 line, but with much overlapping of error bars. Table 2 summarises statisical anlyses of differences within groups. Within controls there were no significant differences between slopes (p > 0.05), but there were between intercepts (p < 0.05). Within the treatment, neither the intercepts or slopes were significantly different (p > 0.05)
Table 2. Table summarising statistical analyses of differences within positive controls ("microplastics", "algae") and treatment ("mixture"). Unpaired two-tailed t-tests were done to find the differences between intercepts and slopes. The p-values returned from these tests are shown in the table. Using a significance level (α) of 0.05, no significant differences between slopes were found within any group. Significantly different intercepts were found within the positive controls. No significant difference was found between "mixture" intercepts.
| Group |
Difference between slopes
(p-value) |
Difference between intercepts
(p-value) |
| Microplastics |
0.779 |
0.046 |
| Algae |
0.670 |
< 0.001 |
| Mixture |
0.906 |
0.578 |
As summarised in Table 3, several differences and similarities were found between goups. Both "algae" trend lines appeared to be above the "microplastics" lines, with a slightly different slope (Figure 4). The observed difference in intercepts between controls was significant (p < 0.05), but the difference in slopes was not (p > 0.05). The "mixture" intercepts and slopes appeared similar to those of the positive controls. (Figures 4 and 5). No significant differences were found between the "mixture" and "microplastics" OD600 intercepts, or "mixture" and "algae" OD690 and OD600 slopes (Table 3, p > 0.05).
Table 3. Table summarising statistical analyses of differences between positive controls ("microplastics", "algae") and treatment ("mixture"). Unpaired two-tailed t-tests were done to find the differences between intercepts and slopes of different groups. The difference analysed (i.e. "test"), and the p-values returned from these tests are shown in the table. Using a significance level (α) of 0.05, no significant differences were found between positive control slopes, or "mixture" and positive control slopes or intercepts. A significant difference was found between the positive control intercepts.
| Group |
Wavelength (nm) |
Test |
P-value |
| Microplastics |
600 |
Difference between slopes |
0.307 |
| Algae |
600 |
| Mixture |
690 |
Difference between slopes |
0.389 |
| Algae |
600 |
| Microplastics |
600 |
Difference between intercepts |
< 0.001 |
| Algae |
600 |
| Mixture |
600 |
Difference between intercepts |
0.519 |
| Microplastics |
600 |
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| Figure 4 |
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| Figure 5 |
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Discussion |
Optical density | |
This study investigated the selectivity of algal and microplastic cell uptake by the Demospongiae Haliclona sp. (OTU QM2474). OD of the water column was used as a measure of cell concentration. This was possible as, within a specific range, light scattering increases with cell concentration (9). Clearance rates were therefore measured as changes in OD values over time. OD was measured at 600nm and 690nm for all samples, as chosen by experimental investigation. It was found that algae have higher OD than microplastics both 600nm and 600nm (Figure 4, Table 3). Importantly, it was also observed that microplastic OD is significanlty higher at 600nm than it is at 690nm, while the opposite is true for algae (Figure 4, Table 2). In this way, when OD600 and OD690 of “mixture” samples where measured over time, differences in rates of change between wavelengths were indicative of greater uptake of one particle type over the other.
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Positive and negative controls | |
The “seawater” results matched expected negative control results. It was expected that OD, and thus cell concentration, would always be zero. While results showed apparent negative linear trends for both wavelengths (Figure 4), neither slope was found to be significant (Table 1, p > 0.05). The intercepts were not significantly different from one another (Table 1, p > 0.05). Therefore OD, and thus cell concentration. was the same across wavelengths as expected. The OD600 intercept was not significant (Table 1, p > 0.05). However unexpectedly, the OD690 intercept was (Table 1, p < 0.05). This does not make sense, considering that the intercepts are not significantly different. The R2 values of the lines may offer explanation. The OD600 line had a much higher R2 (0.600) than OD690 (0.100), indicating the former better reflected the data. We can therefore have more confidence in the OD600 trend, and assume that OD was indeed not significantly different from zero for “seawater”, as expected. The similarity of observed and expected negative control results allows us to analyse the positive control and treatment results without taking into account background effects.
This study found that algal (1-2 µm) and microplastic cells (1 µm) are cleared at the same rate by Haliclona sp. (OTU QM2474), when in separate solutions (Figure 4). It was unclear from the literature what results would be expected. Sponges sort particles based on size (4), and to some extent cell character (2, 4, 5, 11). The mechanism and extent of this selectivity is unknown. Negative linear OD600 and OD690 trends were found for both positive controls (Figure 4), with no significant difference between OD600 slopes found across groups (Table 3, p > 0.05). As slopes were not different with control groups (p > 0.05, Table 2), this finding could be extended to mean algae and microplastic particles were taken up at the same rate when in separate solutions. These findings, combined with the OD and negative control results, mean that any difference detected in rate of OD change across wavelengths for “mixture” would most likely be due to selective uptake of a preferred particle.
It was expected that all sponge clearance rates would follow negative exponential curves, as in research by Turon et al (10). As filter feeders, sponges’ feeding rates are proportional to particle concentration rates in the surrounding water. However linear trends were found to closely fit the data, evident in relatively high R2 values (Figures 4 and 5). This likely occurred because volume decreased over time, as samples were removed for OD anlysis. Water turnover is higher with lower volumes. In this way, volume decreases could partially compensate for the effect of concentration decreases, resulting in linear trend lines.
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Selective uptake of algal or microplastic cells | |
This study found that Haliclona sp. (OTU QM2474) does not selectively uptake algal (1-2 µm) and microplastic (1µm) from the same solution. It was unclear from the literature what results would be expected, for the same reasons as the controls. Negative linear trends were found for across both wavelengths for "mixture" (Figure 5). There was no significant difference between the slopes (p > 0.05, Table 2), suggesting particle uptake was not selective. The considerably high R2 values of the trend lines (0.833, 0.870) means lines were relatively reliable. The "mixture” and "algae" OD690 and OD600 slopes did not differ significantly (p > 0.05, Table 3). These slopes were compared as the most representative of the data, as per their high R2 values.(0.870, 0.980). With no significant slope differences within or across positive control groups, it can be inferred that uptake of algal and microplastic cells was the same regardless of mixed or separate solutions. Therefore, this study found that the Haliclona sp. (OTU QM2474) does not selectively uptake either micron-sized algal or microplastic cells.
Unexpectedly, no significant difference was found between "mixture" intercepts. From the OD findings, it was expected both "mixture" lines would be between “microplastics” and “algae”, with a higher OD690 line. However, intercepts were not significantly different within "mixture" (p > 0.05, Table 2), or in comparison to the “microplastics” OD600 intercept (p > 0.05, Table 3). This suggests human and/or spectrophotometer error occurred. Spectrophotometers carry inherent inaccuracy in their limited wavelength accuracy- 1nm shifts can affect results (3). To minimize error in future studies, uptake should be measured directly by cell count using a haemocytometer. In this study, direct cell counts were not feasible, as cells could not be reliably differentiated with the available microscopes. Future studies should use more powerful microscopes. As replication was limited in this study, human error had a large effect. Further studies should involve more replication, thereby increasing statistical power (6).
The topic investigated in this study carries serious environmental and ecological implications. If sponges cannot selectively uptake food particles over microplastics, as suggested here, there is a high risk of harmful microplastic accumulation in sponges (1, 12). Sponge health is critical to the health of marine, especially reef, ecosystems (2, 4, 11). Further study of this topic is therefore necessary, in order to assess what response is need to minimise microplastic damage. Similar research to this study but with direct cell counts and more replication is recommended. I suggest further study should also be done on the post-uptake effect of microplastics, as long-term microplastic effects remain unclear.
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Conclusion | |
This
study found that theDemospongiae Haliclona sp. (OTU QM2474) does not selectively uptake micron-sized
microplastic or algal cells. Both cell types were found to be taken up at the
same rate, regardless of whether they were in mixed or separate solutions.
Further research is needed to confirm this result, and to further elucidate the
effect of microplastics on sponges.
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Acknowledgements | |
The author wishes to acknowledge the wonderful course coordinators and lab tutors. Acknowledgement is also made to Mathias Jonsson for his generosity in sharing his microplastics and advice, amd to Dr. Kathryn Hall for her help with sponge identification.
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References | |
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