Aquatic organisms maybe exposed to toxicants through aqueous (dissolved) phase and/or through direct contact (dietary) with metal contaminated particles. It has been found that the amount of contaminant accumulated by aquatic organisms was influenced by the biological factors such as food quality and quantity, partitioning of contaminants on the food particles, digestive physiology of the organisms, contaminant characterization within the animal tissue and its associations with different geochemical fractions in food particles. Recent studies have shown that exposure to metal-contaminated diets can be harmful to various freshwater invertebrates thus, might observed reduction in survival, growth and reproduction. Therefore, there is growing evidence that dietborne metal toxicity might be important in aquatic ecosystems.

The current understanding of dietborne metal toxicity is, however insufficiently developed and, as a consequence, the dietary exposure route is generally not considered explicitly in most existing regulations or risk assessment. Regulatory assessments of metal toxicity in freshwaters are mostly based on dissolved metal concentrations, assuming that toxicity is caused by waterborne metal only. Previously, the dissolved phase was thought to be the main source of metal exposure for planktonic organisms, but recently a number of studies have demonstrated that dietary exposure is also an important route for metal accumulation.

A better understanding of the fate of sediment-associated metals and metal contaminated food are needed because if dissolved metal concentrations alone are used to predict toxicity, then environmental risk could be underestimated if there is bioavailable metal associated with these. It is presumed that organisms do not take up metal from food or food is not considered as metal source.

The main objective of this study was to investigate the effect of heavy metal distribution in the food organisms to freshwater ostracod Heterocypris incongruens in the whole sediment toxicity test. Specifically, to determine bioaccumulation and toxicity of heavy metals to ostracod from metal-contaminated microgreen algae Scenedesmus acutus, Chlorella vulgaris, and Ankistrodesmus falcatus, to evaluate the metal uptake and its subcellular distribution in different microgreen algae and to determine the potentially available fraction of heavy metals for possible trophic transfer to benthic ostracod, and to analyze the effect of different food type and light condition on heavy metal toxicity to benthic ostracod. This study will give information to understand the mechanisms of ostracod toxicity test which was recently listed in the ISO publication (ISO 14371:2012 (E)). Ostracod toxicity test has been cited in literatures but the actual exposure routes in the test were not simple thus giving a complex cause of toxicity.

Preliminary modifications of different test conditions were done from the standard ostracod toxicity test such as the use of quartz sand as the control sediment, photoperiod exposure of the test (i.e. 24-h dark and light/dark conditions) and different food sources (i.e. TetraMinR, Chlorella vulgaris and Ankistrodesmus falcatus). These fundamental investigations were necessary for further understanding the effect of different exposure routes in the sediment toxicity test. The toxicity test using quartz sand exposed both under 24-h dark and light/dark conditions showed that mortality of ostracod were relatively comparable to the reference sediment. However, the measured mean growth of ostracod was smaller than in the reference sediment (266±53 and 365±48 μm under 24-h dark and light/dark conditions, respectively). Thus, quartz sand can be used as the control sediment since measured mean growth and mortality were within the acceptable criteria based on the standard procedure. The mortality of ostracod under light/dark condition for reference sediment and quartz sand were 2% and 0%, respectively. This implied that the toxicity test can also be exposed to different photoperiod. Light/dark condition was tested since this condition simulates the actual environmental condition wherein aquatic organisms were exposed as well in light condition. In order to determine whether Chlorella vulgaris and Ankistrodesmus falcatus can be possible food for ostracod, different algal concentrations were prepared and tested both under 24-h dark and light/dark conditions. Algal suspension preparation for the new algal species followed the standard protocol (ISO 14371:2012 (E)). The following algal cell concentrations will be used based on the optimum measured mean growth and mortality of ostracod after the 6-day toxicity tests - C. vulgaris: 6.0x108 cells/well and A. falcatus: 3.0x106 cells/well. The results of the modifications of test conditions were used for the next stage of experiments.

Scenedesmus acutus and H. incongruens were used to evaluate the effect of different food and different light condition on ostracod sensitivity under different concentrations of copper and zinc. Effect of using different food on the growth of ostracod shows that as the dosage of TetraMinR available for ostracod increased, the mean growth also increased from 289±74 to 384±71 μm under 24-h dark condition. No mortalities were observed at TetraMinR dosages of 0.5 mg/well and 1.0 mg/well but mortality of 7% was observed at 2.0 mg/well. Though better growth was observed at 2.0 mg/well TetraMinR, consequently dosage of 1.0 mg/well TetraMinR was used. Therefore, TetraMinR can be used as possible food supplement in ostracod sediment toxicity test as results met the criteria both in terms of the mortality and growth inhibition of the ostracod. Furthermore, the results of the different light conditions tests revealed that light/dark condition had no significant influenced on the ostracod sensitivity to zinc when fed with TetraMinR (T2 and T3) and S. acutus but had a significant effect when fed with C. vulgaris and A. falcatus. On the other hand, light condition had a significant influenced in copper toxicity to ostracod in all different experiments. Lastly, comparison of the determined LC50 of copper and zinc using different food type and food amount showed that ostracod were more sensitive to zinc than copper.

Heavy metal contamination can enter the aquatic food chain either through direct consumption of water or biota or through aquatic exposure. Metal in water can be uptaken by algae and distributed in several fractions in the algal cell. Thus, it is important to know the available fractions of metal that can have potential risk to higher trophic aquatic organisms. Therefore, different algae were exposed to different concentrations of metals to determine the distribution of metals in their cells and to determine the hypothetical fractions of metals available for trophic transfer to ostracod. Scenedesmus acutus, C. vulgaris and A. falcatus were exposed to different concentrations of copper and zinc. After the 10-day exposure and the subsequent metal fractionation of algal cells, the copper in the algal cells were distributed in almost similar proportion for each algal species. Scenedesmus acutus, C. vulgaris and A. falcatus accumulated most of the copper in their algal cells (1.1 to 6.9 x 102 μgCu/L). Zinc accumulation in the algal cells varies among the three species - S. acutus (7.8 to 2.9 x 102 μgZn/L), C. vulgaris (7.0 to 2.4 x 102 μgZn/L) and A. falcatus (5.2 to 1.4 x 102 μgZn/L). Additionally, most of zinc in the cells of S. acutus were internally bound (intracellular soluble: 5.9 to 2.0 x 102 μgZn/g and intracellular insoluble: 35 to 4.9 x 102 μgZn/g). Chlorella vulgaris accumulated zinc inside their cells mainly as intracellular insoluble form (46 to 1.0 x 103 μgZn/g). On the other hand, in A. falcatus most of zinc was in cell-surface exchangeable (3.5 to 3.3 x 102 μgZn/g) and intracellular insoluble forms (99 to 6.5 x 102μgZn/g). Copper in the cells of S. acutus, C. vulgaris and A. falcatus were distributed in intracellular soluble (0.4 to 1.3 x 103 μgCu/g, 4.6 to 1.4 x 103 μgCu/g, and 2.3 to 7.8 x 102 μgCu/g, respectively) and intracellular insoluble fractions (12 to 9.7 x 103 μgCu/g, 14 to 1.1 x 104 μgCu/g, and 26 to 1.0 x 104 μgCu/g, respectively). Hence, distribution of zinc incorporated in exposed algae varied among the three species while accumulated copper were mostly in intracellular soluble and intracellular insoluble fractions for the three algae. From these results, we may conclude that bioavailability of metals is dependent on the algal species.

Hypothetical trophically available metal (TAM) was determined as the sum of the cell-surface exchangeable and the intracellular soluble fractions in the algal cells. Estimated trophically available zinc varied with zinc concentration, Z1(420 μgZn/L) having the highest estimated TAM corresponding to 36% (S. acutus), 33% (C. vulgaris) and 55% (A. falcatus). Chlorella vulgaris had the highest percentage of trophically available copper in their cells around 12 to 25%, compared to S. acutus and A. falcatus which accumulated only about 4 to 15% and 5 to 8 %, respectively.

Different metal contaminated algal food were prepared and used in the toxicity test to determine the possible dietary metal effect to ostracod. Based on the dose response relationship, the total zinc content increased almost 19 times (43 to 8.0 x 102 μgZn/g) but the mortality of ostracod did not significantly increased (33 to 52 %). On the other hand, as the total zinc accumulated in the algal cells of C. vulgaris increased, the toxicity to ostracod increased (23 to 83%) more clearly than the case of S. acutus. In C1 and C2 tests, significant mortality was observed for ostracod fed with Cu-exposed S. acutus and C. vulgaris. On the other hand, similar results were observed with Zn-exposed and Cu-exposed A. falcatus, high mortality was observed at control (clean algal cells) (68% and 57%, respectively) condition and maybe due to food insufficiency. Additional experiments were suggested to determine the possible source of high toxicity.

Based on dose response relationship when ostracod was exposed to different zinc and copper concentration through aqueous exposure (Chapter 5), the measured zinc and copper in the overlying water was not high enough to cause lethal toxicity to ostracod, thus the observed mortality should be considered as the result of the metal-exposed food (dietary metal).

There was almost no difference among the different fractions of metals to determine the possible toxic effect of dietary metal to ostracod especially when exposed to copper based on the dose response curves thus, total metal or trophically available metal may be used to discuss the toxicity of contaminated algae to ostracod.

To conclude, the current study provides significant implications for understanding the dietary toxicity of copper and zinc in the benthic ostracod. The study highlights the importance of dietborne metal and suggests that dietary exposure should be incorporated when assessing the ecological effects of contaminants in the sediments where contaminated food is one of the potential exposure pathways. This recently standardized toxicity test was believed to be utilized by more researcher for evaluation of sediment toxicity thus, the developed solid-phase dose response curves can be give fundamental information for the interpretation of future ostracod toxicity test results. This study also indicates that a clearer understanding of the effects of both aqueous and dietary exposure routes as well as of interactions between them is necessary for further refinement of the chronic metal toxicity test for H. incongruens.

都市周辺の水域生態系の保全を考えるに当たり、水質のみならず底質についても考慮が必要である。底質は有害物質の蓄積の場となり、底生生物への影響を介して生態系全体へ影響をもたらすことが懸念されている。特に重金属については全亜鉛に関する水生生物保全のための水質環境基準が平成15年に設定され、銅やその他の元素についても基準の検討が予定されているところであるが、底質については未だ基準に関する考え方も定まっていないのが現状である。底質そのものの有害性評価には生物を用いた手法が適用されるが、中でも2012年にISOにて標準化作業が完了した淡水性カイミジンコを用いた底質毒性試験法は、従来のユスリカ幼虫やヨコエビを用いた試験法よりもコンパクトな試験系を達成しただけでなく、必要なときに乾燥卵から孵化させて実験可能であるため日常の試験生物飼育の手間もなく、今後のより広範な利用が期待されている。しかしながら、底質毒性試験の結果を理解し、環境管理に役立てるためにはその機構に関する知見の集積が必要であり、特に固形物が多量にある環境に存在する底生生物が、汚染底質自体からの摂食曝露と、周辺汚染水からの水系曝露とのどちらからより大きな影響を受けているかの理解は基本をなすものである。しかしながら、その点について未だ体系的な知見は不十分である。

本論文は、「Effect of Heavy Metal Distribution in the Food Organisms to Freshwater Ostracod (Heterocypris incongruens) in Whole Sediment Toxicity Test(全底質毒性試験における淡水カイミジンコ(Heterocypris incongruens)に対する餌生物中の重金属分布の影響)」と題し、淡水性カイミジンコH. incongruensを用いた底質毒性試験において、餌生物中の重金属の分布がどのような影響を与えるかを明らかにすることを目的としている。より具体的には、重金属汚染された微細緑藻類Scenedesmus acutus、Chlorella vulgaris、Ankistrodesmus falcatus の捕食者移行性の重金属の定量を行い、それらを用いたカイミジンコへの重金属の摂食曝露による毒性影響を明らかにし、水系曝露との比較を行うものである。さらに、カイミジンコへの重金属毒性に与える、餌の種類や光条件についても解析をしている。論文は8つの章で構成され、結果に関する章は4~7章であり、特に6、7章が主たる結果となっている。

第1章では、研究の背景と目的、および論文の構成を述べている。

第2章では、文献レビューとして、底質毒性試験、水環境中の重金属、藻類への重金属摂取および毒性に与える因子、水生生物の重金属蓄積、藻類細胞中の重金属分画について、先行研究をまとめている。

第3章では、試験手法および化学分析の方法について示している。

第4章では、以降の章の基礎となる試験手法の変更の可能性について検討をしている。変更の可否判定には、ISO標準試験手法における妥当性確認の基準を用いている。その結果、control底質として試薬として購入可能な石英砂が成長に影響は与えるものの許容範囲内であったこと、より現実の環境に近い16時間明/8時間暗条件でも基準を満たすこと、藻類C. vulgarisおよびA. falcatus、市販魚餌でも投与量を調整すれば利用可能であること、が示されている。

第5章では、前章での予備的検討を踏まえ、餌の種類と光条件がカイミジンコの銅・亜鉛の水系曝露による毒性に与える影響を明らかにしている。標準の餌藻類であるS. acutusや市販魚餌を投与した場合にはカイミジンコの亜鉛に対する感受性は光条件の変更によって影響を受けないのに対し、他の二種の緑藻類C. vulgarisおよびA. falcatusの場合には毒性影響が明らかに変化した。一方、銅の場合にはS. acutus以外の餌では光条件が大きく影響を与えた。さらに、これら全ての実験で得られたLC50を総合し、カイミジンコH. incongruensが銅よりも亜鉛に感受性が高いと結論している。

第6章では、上述の3種の藻類を異なる重金属濃度の培養液で10日間培養し、細胞内への銅・亜鉛の分布(細胞表面交換態、細胞内溶存態、細胞内懸濁態)を定量している。S. acutusが摂取した亜鉛の大半は細胞内に存在(溶存態:5.9~200 μg/g、懸濁態:35~490 μg/g)し、C. vulgarisの場合には大半が細胞内懸濁態(46~1000 μg/g)であった。A. falcatus の場合は細胞表面交換態(3.5~330 μg/g)や細胞内懸濁態(99~650 μg/g)が多く存在した。銅は三種の藻類において主として細胞内溶存態および細胞内懸濁態として存在した。これらの結果から、亜鉛については種による分布の差が認められたのに対し、銅については類似した分布となったと結論し、捕食者への影響が餌に依存する可能性を示唆している。さらに、仮想的な捕食者移行性画分として、細胞表面交換態と細胞内溶存態の和を提案し、この考え方に従うと、亜鉛については420μg/Lの培養条件において36% (S. acutus)、33% (C. vulgaris)、55% (A. falcatus)が移行性となるのに対し、銅の移行性画分はC. vulgarisで12~25%と比較的高いが、S. acutusやA. falcatusでは4~15%、5~8%と低くなることを示唆している。

第7章では、第6章で調整された汚染餌をカイミジンコに投与しその用量応答関係を明らかにしている。S. acutusを用いた際には亜鉛含量の増加に対してカイミジンコの致死率に大きな増大が見られないのに対し、C. vulgarisではより明確に毒性が増加したとしている。銅については、どちらの藻類を投与した場合でも、カイミジンコに対する致死毒性が明確に認められている。第5章の結果と照合すると、この汚染藻類による致死毒性の結果は、試験系の上澄みに存在する溶存態の銅や亜鉛の毒性では説明できず、明らかに摂食曝露に由来するものと結論づけられる。

第8章では、上記の研究成果から導かれる結論と今後の課題や展望が述べられている。

以上のように、本論文は、従来知見が大幅に不足していた底質毒性試験種の重金属の摂食曝露による影響に関する、室内実験に基づく貴重なデータを提供している。これは水系曝露のみが考慮されている現在の水環境管理に対し、重要な知見を与えるものである。また、固形物由来の銅・亜鉛濃度の影響を定量的に示す結果は、今後の当該毒性試験手法の結果を解釈する上で極めて有用な情報となると考えられる。本論文の成果は、今後の都市環境工学の学術の進展に大きく寄与することが期待される。

よって本論文は博士(工学)の学位請求論文として合格と認められる。