Considering ecosystem modeling by examining the creation of microcosms for Daphnia coexistence using an inorganic medium

doi:10.21203/rs.3.rs-2006187/v1

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Considering ecosystem modeling by examining the creation of microcosms for Daphnia coexistence using an inorganic medium

https://doi.org/10.21203/rs.3.rs-2006187/v1

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To conserve natural ecosystems, we require suitable experimental methods with which to investigate them. Ecosystem models, called microcosms, have the potential to predict and improve our understanding of the phenomena in natural ecosystems. However, it is known that there are discrepancies between microcosms and natural ecosystems, such as different phosphorus circulation depending on the scale of the microcosm. Therefore, it is necessary to understand why the differences appear and to consider the coexistence of species, the medium composition, and the internal structure of microcosms in order to reproduce more of the phenomena that occur in natural ecosystems. To address this, in this investigation a microcosm (DMC) was constructed in which a variety of organisms could coexist by circulating the necessary nutrients which had been biosynthesized from essential elements (31 elements) inserted into the medium as inorganic substances. The growth of Daphnia, which requires vitamins (B12, B1, B7), was then observed by changing the sediment inside the microcosm with the inorganic medium containing all the essential elements. Using quartz sand with a particle size of 0.3–0.6 mm as the sediment, resulted in the stable growth of Daphnia, from 3 to approximately 26 individuals over 35 days, due to the growth of algae such as diatoms, which are feed for Daphnia. This suggests that 0.5 mm particle spaces are suitable for anaerobic bacteria, and that the nutrients necessary for the organisms, such as vitamins, were biosynthesized from the essential elements in the space. These results indicate that the DMC, which can duplicate the cycles that occur in natural ecosystems, such as the production of nutrients from inorganic substances, and the diversification of organisms in the microcosm, will be a useful tool for understanding natural ecosystems and factors that impact them, such as the effects of chemicals.

Ecosystem model

Microcosm

Ecological risk assessment

Environmental modeling

To conserve natural ecosystems, we require effective experimental methods with which to increase our understanding of them and to predict the impacts of human and other factors. Ecosystem models called microcosms are currently used to investigate material cycles and food webs in aquatic ecosystems, and the effects of chemical substances (Beyers 1993). A microcosm is a culture of a community of organisms in a controlled environment, such as a container, which also encompasses physical, chemical, and biological factors and their interactions in the ecosystem. Furthermore, microcosms can be experimentally manipulated so that the phenomenon of interest is expressed. They are divided into three main types based on the creation method, as follows:

Gnotobiotic-type (G-type): This type involves the combination of certain species of organisms, cultivated under specific conditions, to create a stable ecosystem. Experiments that use this method are relatively easy to analyze as the species composition is known. Additionally, as ecosystems require at least one decomposer, predator, and producer species to survive (Matsui 2000), It is possible to create microcosms with various combinations of organism.
Naturally-derived-type (N-type): This type of microcosm keeps a natural ecosystem community intact. It can also show effects that are closer to those in the real world, but the reproducibility of these experiments is difficult. Many of the ecosystem models that occur in the field and build large scale ecosystems are called mesocosms.
Stress-selected-type (S-type): For this type of microcosm in which natural communities are collected and mixed from multiple locations, and the organisms are selected through repeated sub culturing under specific conditions and stabilized in a particular community. While the N-type shows effects that are closer to those in the real-world, the S-type does not involve an artificial element in the species composition, and the stable composition is more reproducible. The S-type microcosm was created by Kurihara (Kurihara microcosm) (1978) and has been in use for more than 30years (Sugiura 1992; Takamatsu 1997; Kakazu 2014), and has maintained, as it creates a stable ecosystem with high reproducibility and is easy to subculture.

Microcosms are especially useful when assessing the effects of chemicals on ecosystems, and the Organization for Economic Co-operation and Development (OECD) and the United States Environmental Protection Agency (EPA) provide guidelines for their creation, as follows:

OECD (2004): Guidance document on simulated freshwater lentic field tests (outdoor microcosms and mesocosms). This guideline states that the media and sediments used for micro-mesocosms should be collected from the natural environment. Micro-mesocosms created using this guideline are intended to test specific hypotheses based on information obtained from existing research, allowing for a flexible design of the micro-mesocosm.
EPA (1996): They provide the Ecological Effects Test Guidelines from the OPPTS 850.1900 Generic Freshwater Microcosm Test Laboratory. The guidelines describe how to create a standardized aquatic microcosm (SAM). The sediment in the SAM is filled with 200 g of silica sand, 0.5 g of chitin, and 0.5 g of cellulose powder. There is a T82MV medium containing 14 elements, EDTA, and 11 vitamins, and it can be used to cultivate 10 species of algae and 5 species of animals that graze on the algae or resultant detritus.

Ecosystem models range from those based on the three biological species to those at scales close to natural ecosystems. However, they must all be suitable for predicting and understanding the phenomena that occur within natural ecosystems. The importance of field studies at appropriate scales has been noted, as there are considerations that microcosms can produce different results, such as for phosphorus circulation which depends on the scale at which they are created (Carpenter 1996). However, research by Sugiura (2010), Kakazu (2014), and others has found that the Kurihara microcosm and mesocosm correlate in their toxicity test results for 11chemicals (Fig.1). The correlating equations between the mean of no-effect concentrations (mean of NOEC) in natural ecosystems (mesocosm test) and no observed adverse effect concentrations (M_NOAEC) in the Kurihara microcosm is expressed as follows:

log [mean of NOECs] = 1.08log [M_NOAEC] − 1.22

It is to be noted that a high correlation of the mesocosm test results was obtained despite various factors such as diversity and differences in the constituent species of the natural environment. However, there are some chemicals that were found to fall well outside the above correlation equation. The NOEC of γ-BHC (Lindane) in the mesocosm test was reported to be 0.2µg/L (Mitchell 1993) and using the correlating equation above, the M_NOAEC of the Kurihara microcosm was found to be 3 µg/L. However, it has been reported that even 5mg/L of γ-BHC had no effect on the constituent species of the microcosm (Sugiura 1992). Alachlor also showed that 1 mg/L had no effect on the Kurihara microcosm (Kakazu 2013), although the NOEC of the mesocosm was 10µg/L (Lina 2005) and the M_NOAEC was 100µg/L. Thus, there are chemicals that do not affect the constituent species of the Kurihara microcosm and are not correlated. This may be due to the fact that y-BHC is an insecticide and Alachlor is a herbicide, which inhibit only specific biochemical reactions. This means that if a chemical is harmless to the organisms that make up the ecosystem, it will not contribute to the correlation equation. Therefore, even if a toxicity test is performed with the Kurihara microcosm and the effect is small, it will be difficult to determine whether the chemical is not affecting the constituent species or whether it is a low toxicity chemical.

Thus, the Kurihara microcosm is a microbial ecosystem that includes only seven species, excluding bacteria, and this is not sufficient to predict the effects on natural ecosystems. However, it is impossible to construct an ecosystem model that includes all organisms, and the challenge is how to create a microcosm in which diverse species coexist and identify which kinds of species should coexist in a microcosm is appropriate.

1.1 Diversification of species coexisting in a microcosm

To diversify the species coexisting in a microcosm, the following two points must be considered:

(1) That an inorganic medium that contains all the essential elements for the organism is required.

(2) That a diverse living space for the organisms, created using bottom sediments with materials that can be reproduced, is provided.

1) Microcosms need to include all essential elements

Elements are substances that cannot be produced by living organisms themselves. For example, if an organism is deficient in phosphorus, an essential element, it cannot convert nitrogen atoms into phosphorus atoms, and consequently this deficiency results in growth inhibition. All elements that are currently known to be essential for living organisms must thus be included in the medium. All other nutrients (amino acids, vitamins, proteins, etc.) that are necessary for the growth of living things are made up of combinations of essential elements. By creating a medium that contains all the essential elements, the factors that inhibit the growth of the organisms in the microcosm due to nutritional deficiencies will depend on whether the necessary nutrients can be produced in the microcosm. Thus, it is important for the ecosystem model that all the essential elements are circulating in an ecosystem by changing their forms. This investigation has aimed to create a microcosm in inorganic medium that contains all essential elements.

All essential elements should be placed in the medium as inorganic substances and the other nutrients such as vitamins and organic substances should not be placed in the medium unless reproducing a specific environment. For example, the T82MV medium used for SMA contains 11 types of vitamins, but this is not useful if trying to see the effects of chemicals that inhibit the processes involved in vitamin production. In a natural ecosystem, the inability to produce vitamins would be fatal, and would be a factor in the collapse of an ecosystem.

2) Need for a variety of living spaces

In microcosms without sediment, the constituent organisms form colonies on the bottom and separate their living spaces (Murakami 2004). The reason why organisms form colonies is to efficiently obtain the necessary nutrients or to hide from predators. In the natural environment, the existence of living spaces with a variety of bottom materials, such as soil, sand, and stones, creates a variety of biological niches and enables a variety of species to coexist. Additionally, it has been reported that vitamin B₁₂ is continuously supplied to seawater from coastal sediments (Sugita 1994), and this sediment provides a living space for vitamin-producing bacteria and other organisms. Therefore, to produce the necessary nutrients using essential elements, bottom sediment is required, and we must understand what type of bottom space would be an ideal habitat for the bacteria that produce vitamins. One point to note is that it is possible to generate vitamins by using natural soil, but it is difficult to reproduce and to determine which factors are affecting it. This investigation has thus examined the microcosm sediments using reproducible materials including glass beads and quartz sand.

1.2 Adequacy of species coexisting in a microcosm

Identifying the kinds of species that can coexist appropriately in a microcosm was addressed, with a focus on the following points:

The human factor in selecting the microbial communities to support an ecosystem is eliminated as much as possible by using an S-type microcosm.

For this method of creating microcosms in which, natural communities from multiple locations are collected, mixed, and then organisms are selected through repeated sub culturing and the ecosystem is maintained by a specific community of organisms. The Kurihara microcosm introduced above is cultured in a Erlenmeyer flask using a TP (Taub + polypeptone) medium, and the constituent species are stable with a combination of two species of green algae and one species of blue-green algae as producers, bacteria as decomposers, one species of protozoan ciliates, two species of metazoan rotatoria, and one species of metazoan archioligochaeta as predators (Kurihara 1978). Thus, the S-type naturally settles into a stable constituent species with repeated sub culturing and is characterized by the absence of artificial factors in species composition. Therefore, the S-type was utilized to create a microcosm in this investigation.

2. It is important to consider what organisms of visible size will coexist, as they cannot be passaged unless intentionally included in the subculture.

Currently, fish, invertebrates (mainly Daphnia), and algae are used as test organisms in the ECETOC method, which predicts the effect of no-effect concentrations of chemicals on natural ecosystems by applying an assessment factor to the most sensitive of the three species types. This was adopted as the standard test method by the OECD (ECETOC 1997). The creation of a microcosm that coexists in the ecosystem with Daphnia, which is globally accepted as a test organism for chemicals, is thus required. As vitamins (B₁₂, B₁, B₇) are essential for Daphnia (Keating 1985; Elendt 1990), it can also be used to determine whether vitamins are being produced in the microcosm as explained above. Therefore, in this investigation, a microcosm in which algae and Daphnia coexisted as a first step (Hereinafter, this microcosm will be written as DMC) was developed. The creation of DMC will provide a foothold for the future creation of microcosms that include fish.

The purpose of this study was thus to find the sediment in which Daphnia can be cultured in an S-type microcosm using inorganic medium containing all essential elements. This will help us to understand what properties of the sediments that are affecting the production of vitamins and other nutrients from inorganic substances. In addition, by duplicating the nutrient production cycle of producing from inorganic substances, which also occurs in natural ecosystems, the microcosm will have a higher correlation with natural ecosystems.

2.1 Medium composition of DMC

A microcosm that is based in medium containing essential elements can be utilized to culture a wide variety of organisms, and result in a great increase in the number of biochemical reactions that occur. The medium composition was thus designed to contain all the essential elements (31 elements) for living organisms that are currently known (Sakurai 2006; Schrauzer 2002). Since it has been reported that the effects of chemicals on organisms varies depending on the elemental concentrations in the medium (Vernberg 1974), It was aimed to keep them close to their natural concentrations. Thus, the DMC medium was created by adjusting the reagents based on the elemental concentrations in the river, as compiled by Huheey (1993). However, the concentration of the elements in the river water is lower than that in the lakes and reservoirs, and the concentration of the elements in the water decreased as the elements were absorbed by organisms during the transition phase of the microcosm. Therefore, in this experiment, the elemental concentration was set to 1.5 times^*1 that of the river water to achieve a moderate community composition as an ecosystem model (Table1). Each substance was dissolved in pure water to prepare the DMC medium. FeSO₄-7H₂O was dissolved in Na₂EDTA before use.

*1 It would be better to adjust the medium concentration for future research to the average elemental concentrations in the lakes and marshes.

Table1 Composition of the DMC medium and the concentrations of the essential elements in a river (Huheey 1993).

The DMC medium was prepared by dissolving each substance in water at the concentrations listed in the table to achieve 1.5 times the elemental concentration of the river water. FeSO₄•7H₂O was dissolved in Na₂EDTA before use

Substance	DMC medium (mg/L)	Essential elements	River (mg/L)
Na₂CO₃	28.2	Na	9
		Cl	8
MgCl₂	9.4	Mg	4.1
Na₂SiO₃•9H₂O	60.7	Si	4
MgSO₄・7H₂O	38.1	S	3.7
KCl	5.9	K	2.3
CaCl₂・2H₂O	8.3	Ca	1.5
		C	1.2
FeSO₄・7H₂O	5.00	Fe	0.67
Na₂EDTA・2H₂O	2.73	Fe	0.67
NaNO₃	1.01	N	0.25
KF	0.46	F	0.1
SrCl₂•6H₂O	0.23	Sr	0.05
KH₂PO₄	0.13	P	0.02
NaBr	0.04	Br	0.02
H₃BO₄	0.11	B	0.01
ZnSO₄•7H₂O	0.07	Zn	0.01
MnCl₂•4H₂O	0.027	Mn	0.005
CuSO₄•5H₂O	0.029	Cu	0.005
KI	0.010	I	0.005
PbCl₂	0.006	Pb	0.003
NH₄VO₃	0.003	V	0.001
CrCl₃•6H₂O	0.008	Cr	0.001
C₉H₉AsN₆O	0.006	As	0.001
RbCl	0.002	Rb	0.001
Na₂MoO₄•2H₂O	0.004	Mo	0.001
NiCl₆H₂O	0.0018	Ni	0.0003
Co(NO₃)₂・6H₂O	0.0015	Co	0.0002
Na₂SeO₄	0.0007	Se	0.0002
SnCl₂•2H₂O	0.00011	Sn	0.00004
LiCl	0.027	Li	0.003
AgCl	0.0006	Ag	0.0003

2.2 Sediments for the DMC

As a preliminary experiment, the following two types of glass bead (GB; Glass beads BZ-1, BZ-2, As One Corporation, Osaka, Japan) with different diameters were used as the sediment to assess the differences in particle size of the sediment.

(1) GB with a diameter of 1.5–2.5 mm (L-GB): 200g

(2) GB with a diameter of 0.991–1.397 mm (S-GB): 200g

Quartz sand (Albany27 (SiO 99.8%, Particle size 0.3 ~ 0.6 mm exists in 87%), Tochu Corporation, Aichi, Japan) was used as the sediment for the main experiment.

(3) Quartz sand: 275g

2.3 Collecting locations of the constituent organisms

Microorganisms were collected using plankton nets from lakes and rivers, mainly in eastern Japan (Abukuma River, and Lake Kasumigaura, etc.), and they were mixed into the DMC containing the above medium and sediments, and the culture was started. Subsequently, the Subculture was repeated while removing the soil and other impurities, and the transition of the organisms was observed. Daphnia were cultured in a pond near Fukushima University (GoogleMap:37°41'12.4"N 140°27'17.1"E) (Photo.1).

2.4 Culture method of DMC

A 1000 mL Erlenmeyer flask was filled with 700 mL of the DMC medium and the sediment, covered with a silicone cap, and sterilized in an autoclave. Then, 30 mL of the DMCs, which had sub-cultured (Take microorganisms inside the sediment as well), were added to the medium and incubated at 20°C (OECD recommend 18–22℃ (1998)), 2,000 lux (light 14.5 h, dark 9.5 h)^*², and static conditions. A fluorescent lamp (National FL20SS-W/18) was used as the light source. Then, after 14 days of culture, three eggless Daphnia were transplanted. For each subculture, three replications were conducted (Photo.2).

*2 Light-dark cycle: The OECD test method for cultivating Daphnia is 16 hours light and 8 hours dark (OECD 1998), and considering the long light period, the cycle was set to 14.5 hours light and 9.5 hours dark to match the average daylight hours in Japan during the summer solstice.

2.5 Measurement method

The dissolved oxygen concentration and temperature in the DMC culture mediums were measured using an Optical Dissolved Oxygen instrument (YSI Professional Optical Dissolved Oxygen (Pro ODO) DO + Temperature + barometer). The Daphnia in the population were counted using visual inspection. In addition, photographs of the main organisms in the DMC were taken using a light microscope (Photo.3).

3.1 Results and discussion of the preliminary experiment

As a preliminary experiment, GBs with different diameters were used to assess the differences in the sediment particle size. Algae including green algae and cyanobacteria were the main proliferators and the number of species was limited when the sub-culture was continued in DMC with L-GB as the sediment. Furthermore, there was no growth of the Daphnia. However, when S-GB was used, diatoms (Ulnaria, Pinnularia, etc.) also began to proliferate, the phenomenon of an increasing number of algae species was observed, and the Daphnia began to reproduce. As explained above, it is known that Daphnia require essential vitamins (B₁₂, B₁, B₇) (Keating 1985; Elendt 1990), and many species of algae also require vitamins (B₁₂, B₁, B₇) (Croft 2006). Therefore, it was thought that vitamins were produced in the S-GB, and this resulted in an increase in the algal species and growth of the Daphnia. There are many species of algae that require vitamin B₁₂ (155 of the 306 species of algae surveyed) (Croft 2006), and it is thought that vitamin B₁₂ can only be biosynthesized by prokaryotes (Heterotrophic bacteria, Cyanobacteria, Archaea) (Grossman 2016). Sugita (1994) reported that most of the species producing vitamin B₁₂ in ponds are anaerobic bacteria and are present in the sediment not the water.

The measurements of the dissolved oxygen concentrations in each GB showed that in the L-GB they were approximately 1.1 mg/L but decreased to less than 0.1 mg/L in the S-GB (Fig. 2). It is thus thought that because the inside of the S-GB became anaerobic, the vitamin-producing bacteria proliferated, and the algae were able to obtain vitamins. It is thought that the Daphnia reproduced by preying on algae and obtaining vitamin B₁₂(Watanabe 2018).

It has also been shown by Suzuki et al. that the degradation performance of polychlorinated biphenyls (PCBs) differs depending on the particle size of the GB. In this study, PCBs were added to a liquid medium under anaerobic conditions containing paddy soil or GB (particle size 2, 0.5, and 0.05 mm), and its residues were measured. The results show that the culture using GB0.5 mm as an inorganic sediment had a predominantly higher degradation rate than the soil culture system, and that it degraded many homologues and isomers. In addition, 16s rRNA gene sequencing analysis showed the presence of species different from the previously reported PCB-degrading bacteria. Furthermore, the degradation activity remained unchanged even after heating to 100°C, suggesting that the constituent species of the GB0.5 mm culture are formed from species that are not yet well known (Suzuki 2013). Based on these results and the results of the preliminary experiments, spaces constructed by particles with diameters from ≥ 0.05 mm to ≤ 2 mm are considered suitable spaces for the survival of anaerobic bacteria.

As a result, this investigation was conducted using quartz sand^*3 with a particle size of about 0.5 mm as the sediment.

*3 If the GB was used as the sediment, light penetrates to the bottom of the sediment and algae growth is observed even under the sediment, which is a peculiar environment that differs from the real sediment, and quartz sand is also used as a sediment for OECD tests (OECD 2010), so this experiment used quartz sand.

3.2 Results of the main experiment

This experiment was carried out using a DMC that had been sub-cultured for approximately one year. The DMC was started by combining S-GB DMC culture medium and water collected from the natural environment with quartz sand as the sediment.

First, the dissolved oxygen concentration in the culture medium was continuously measured for 1488 hours (62 days) after the start of the DMC culture (Fig. 3). In the early stage of the culture, photosynthetic activity was lower than that of respiration, and the dissolved oxygen concentration decreased, but from the fifth day, approximately, oxygen production by photosynthesis increased, and the dissolved oxygen concentration increased. After approximately 14 days, the cycle of consuming the oxygen produced during the 14.5 hours of light was repeated during the 9 hours of dark, and the ecosystem was shown to be stable. Therefore, it was decided to transplant three Daphnia on the 14th day to the start of the stable phase.

The temperature in the DMC was also measured in the same way as the dissolved oxygen concentration (Fig.4). The temperature was approximately 20.7°C. It increased during the light hours and decreased during the dark hours. In the early stages of the culture, the range of temperature increase was considered to be larger, probably due to the high activity of the organisms.

Subculture I: Daphnia was transplanted on the 14th day of the starting culture or the DMCs that had been repeatedly subculture, and the population change was measured. As a result, the population of Daphnia increased as shown in Figure 5, up to a maximum of 28 individuals. Then, on the 36th day, 30 mL of culture medium was taken from each culture system by breaking up the sediment and transplanting it into the DMCs (3 replication) of subcultures II. Two weeks later, three eggless Daphnia were transplanted in each replication.

Subculture Ⅱ: Daphnia transplanted from Subculture I on the 14th day of culture increased as shown in Figure 6. Offspring were born six days after the start of the culture and reached their peak at day 42, approximately. The population grew to a maximum of 23 individuals and then declined (Fig. 6). The reason for this was peculated to be that the Daphnia had eaten all the algae it. Subculture II was then broken down to the sediment structure and transplanted into DMCs of subculture III, 30 mL each. Then, on the 14th day of culture, three eggless Daphnia in each replication were transplanted into subculture III.

Subculture Ⅲ: On the fourth day after the Daphnia were transplanted, as in subculture II, offspring began to be born, and they peaked on the 32nd day approximately at 27, and then decreased, like Subculture II (Fig.7).

The results of this experiment showed that the Daphnia could be successfully cultured in a microcosm created with inorganic medium using sediment with a particle size of approximately 0.5 mm. This indicates that vitamins B₁₂, B₁, and B₇, which are essential for Daphnia, are produced in the sediment of the DMC and then utilized by the Daphnia, which leads to growth. It is believed that the sediment produced by the materials with a particle size of approximately 0.5 mm creates an ideal living space for anaerobic bacteria and plays an important role in ecosystem composition of the ecosystem.

4.1 Correlation between DMC and natural ecosystems

The purpose of these experiment utilizing microcosms is to improve our ability to understand the phenomena and effects impacting on natural ecosystems. Although the Kurihara microcosm correlated with the mesocosm test in the toxicity tests for 11 chemicals, it is expected that the correlation will be higher in the DMC than the Kurihara microcosm for the following three reasons, but verification is still required:

Correlation is expected to be higher due to the increase in the number of species and the diversity of the biochemical reactions that occur in the microcosm. The Kurihara microcosm is cultured using TP medium (Sugiura 1992; Takamatsu 1997; Kakazu 2014), which consists of 18 elements (excluding oxygen and hydrogen), but species that lack essential elements cannot grow. As an example, TP medium does not contain silicon, so diatoms do not grow, and only green algae and blue-green algae grow. Also, looking at the medium composition of Daphnia in the OECD test method (OECD 1998), there are eight elements that are not included in the TP medium, indicating that larger microorganisms such as Daphnia require more elements than the constituent species of the Kurihara microcosm, and that more complex biochemical reactions are occurring in the body. This would make it possible to detect more chemical effects, including those that inhibit specific biochemical reactions.
The presence of trace essential elements and the correlation of chemicals that have effect at low concentrations are expected to be higher. Trace essential elements in humans are known to include zinc (Zn), copper (Cu), selenium (Se), chromium (Cr), cobalt (Co), iodine (I), manganese (Mn), and molybdenum (Mo), but the percentage of these elements in the total body weight is 0.02%. However, it has been known that various symptoms can occur if there are deficiencies(Wada 2004). This shows that trace essential elements play an important role in life, despite their low amounts. For example, the element with the lowest concentration in the DMC medium is Sn at 40 ng/L, but for organisms that require Sn, even 40 ng/L will have an effect if there is an inhibition in the uptake. In other words, biochemical reactions involving trace essential elements may be affected by the inhibition of chemicals at low concentrations. Therefore, it is necessary to include trace essential elements to clarify the correlations of ng to several μg/L in natural ecosystems and the microcosm.
The correlation is thought to be higher because of the creation of a cycle of essential elements in the ecosystem, including the sediment. The DMC medium is an inorganic medium, meaning that essential organic matter and other nutrients have been biosynthesized by the organisms and circulated within the ecosystem. Importantly, vitamins, which are essential for the growth of Daphnia, are thought to be produced in the sediment. This means that toxicity assessments can be conducted using the nature of the phenomena that occur in natural ecosystems, such as inhibition of pathways involved in the production of vitamins or their transfer to Daphnia after production. Therefore, the correlation with natural ecosystems is expected to be higher.

4.2 Future prospects of DMC

As the DMC is an ecosystem that contains all the essential elements that are considered necessary for living things, and there are algae and Daphnia to feed on, it is thought that the microcosms could be constructed to include fish can also coexist. In the future, the coexistence of fish in DMCs will make them an even better tool with which to predict the effects on natural ecosystems. As the fish cultures used in current toxicity tests are propagated by feeding fish in the tanks with natural water, it is expected that algae and Daphnia-feeding fish can also be cultured in DMC. However, if fish are simply added to DMC ecosystems, it is thought that the Daphnia will be eaten up, so it will first be necessary to devise an internal structure as a space where the Daphnia can hide. It will also need to be larger in size to ensure adequate living space. In nature, vitamin B₁₂ is produced by certain bacteria and archaea and taken up by algae, which are then eaten by zooplankton, which are then eaten by fish, resulting in the concentration of vitamin B₁₂ in fish bodies(Grossman 2016). Therefore, by constructing a fish coexistence microcosm, it is expected to be possible to observe the chemical concentration processes that occur in an ecosystem.

Since the microcosm in which Daphnia is cultured could be constructed using quartz sand with a 0.3–0.6 mm particle size, the possibility that vitamins (B₁₂, B₁, B₇) were produced in the sediment and algae such as diatoms that were diversified was considered. Thus, DMC will be a useful tool to help understand the natural ecosystems and predict the effects of chemicals and others, as it duplicates more of the cycles that produce nutrients from inorganic substances, which also occurs in natural ecosystems, and diversifies the organisms in the microcosm.

The current test method calculates the predicted no-effect concentration on natural ecosystems by applying an assessment factor to the results of single species tests. However, chemicals are dynamic in ecosystems, due to factors such as the decomposition by bacteria, intake by algae, concentration by food webs, and adsorption to sediments. Therefore, it is necessary to use a microcosm as an ecosystem model, to predict the effects on natural ecosystems. At the very least, DMC will be a more useful test method for predicting realistic no-effect concentrations on natural ecosystems than the ECETOC method, as it assesses the effects of Daphnia and algae on ecosystems at the same time.

Acknowledgements

I would like to thank Hideto Usui and Yuki Kanzo for their careful proofreading of the manuscript. I am also grateful to Katsura Sugiura for teaching me about microcosm study and Yuhei Inamori for the use of research materials. I would like to thank Enago (ww.enago.jp) for the English language review.

Declaration of interests

The author declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability statement

The datasets generated during and analysed during the current study are available from the corresponding author on reasonable request.

Funding statement

No funding was received for conducting this study.

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Photo 1, 2 and 3 is available in supplementary section.

No competing interests reported.

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Photo1: Daphniasp. cultured in DMC.
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Photo.2 Daphniacoexisting microcosm (DMC)
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Photo3 Main organisms that coexist in DMC

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Considering ecosystem modeling by examining the creation of microcosms for Daphnia coexistence using an inorganic medium

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1. Introduction

2. Experimental Methods

3. Experimental Results

4. Discussion

5. Conclusion

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