Toxicity identification evaluation of wastewater effluents : effects of test organisms, metal species and effluent organic matters

유지수 고려대학교 대학원 2015 국내박사

Physicochemical analysis alone is limited to assess the ecological impacts of effluents; thus, whole effluent toxicity (WET) methods have been widely used to overcome this limitation. Along with WET, toxicity identification evaluation (TIE) methods are generally used to characterize and identify toxicity-causing substances, as not all compounds in wastewater effluents are responsible for the observed toxicity. In this study, TIE and chemical speciation modeling were applied to determine the causes of toxicity of effluents and stream waters as well as to evaluate the effect of effluent organic matters (EfOMs) on metal bioavailability and toxicity. A comprehensive toxicity monitoring study from August to October 2011 using Daphnia magna and Ulva pertusa was conducted to identify the cause of toxicity in a stream receiving industrial effluents (IEs) from a textile and leather product manufacturing complex. Acute toxicity toward both species was observed consistently in IE, which influenced toxicity of downstream (DS) water. TIE confirmed that both copper (Cu) and zinc (Zn) were key toxicants in IE, and that the calculated toxicity based on Cu and Zn concentrations well simulated the variation in the observed toxicity (r2 = 0.9216 and 0.7256 for D. magna and U. pertusa, respectively). In particular, U. pertusa was more sensitive in detectecting acute toxicity in DS water and was useful for identifying Zn as a key toxicant. Activities of catalase, superoxide dismutase, glutathione peroxidase, glutathione S-transferase and malondialdehyde were induced significantly in D. magna, although acute toxicity was not observed. In addition, higher levels of antioxidant enzymes were noted in DS than in upstream waters, likely due to Cu and Zn from IE. Overall, TIE procedures with a battery of bioassays were effective in identifying the cause of lethal and sub-lethal toxicity in effluent and stream water. A toxicity identification and speciation study using D. magna was conducted to identify the cause of toxicity of effluent from a semiconductor lead frame manufacturing factory. Acute toxicity of lead frame effluent to D. magna was found to be 22.62 TU, far exceeding the toxicity discharge limit mandated in Korea (< 1 TU). Results from TIE phases I and II suggest that a mixture of Ag, Cu and CN are likely responsible for observed toxicity, and this was also confirmed by mass balance approach (TIE phase III). In addition, Visual MINTEQ simulation suggested the presence of [Ag(CN)2]- and [Cu(CN)3]2- complexes in effluent. Therefore, TIE procedures with chemical speciation modeling were effective in identifying the cause of acute toxicity in industrial effluents. Physicochemical characterization and toxicity identification study using various EfOMs from sewage and industrial wastewater treatment plants (WWTPs) was conducted to evaluate the effect of EfOMs on bioavailability and toxicity of metals. Suwannee River natural organic matters (SR-NOMs) and EfOMs were isolated into hydrophobic, transphilic and hydrophilic fractions, and were characterized by elemental, specific ultraviolet absorbance (SUVA), fluorescence excitation emission matrix (FEEM) and Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) analyses. EfOMs had a higher hydrogen to carbon (H/C) ratio, lower SUVA and higher fluorescence index values than SR-NOMs, and presented B and T peaks in FEEM analysis. These findings indicate that SR-NOMs may have higher aromaticity than EfOMs. In addition, both SR-NOMs and EfOMs (no acute toxicity to D. magna) significantly reduced acute toxicity of Ag and Cu toward D. magna, which was largely dependent on fraction types having different polarities. According to the Freundlich isotherm of Ag and Cu, adsorption capacities (KF) of hydrophobic (HPO) fractions of SR-NOMs and EfOMs from industrial WWTPs were higher than those of HPI fractions. However, the opposite results were found for EfOMs from sewage WWTPs. Moreover, different patterns of acute toxicity to D. magna were well explained through different adsorption behaviors of SR-NOMs and EfOMs fractions. These findings suggest that EfOMs have different physicochemical and toxicological properties compared with those of NOMs, which needs to be further identified with various sources of EfOMs.

화학 및 약물 독성 예측을 위한 설명 가능한 인공 지능 모델 개발

키타나자가나탄 전북대학교 일반대학원 2023 국내박사

Toxicity prediction is a crucial aspect of drug discovery and safety assessment, and the development of explainable artificial intelligence (AI) models for toxicity prediction is of great interest to the scientific community. Traditional methods for toxicity prediction, such as animal testing, are costly, time-consuming, and ethically questionable. Therefore, the use of AI models has emerged as a promising alternative for toxicity prediction, with the ability to process large datasets and identify complex patterns. However, AI models are often criticized for being black boxes, lacking transparency and interpretability, which limits their practical applications in the drug discovery process. This thesis aims to develop and apply explainable AI models for the toxicity prediction of chemicals and drugs. The proposed models use optimal molecular descriptors and different machine learning algorithms to predict toxicity while maintaining a high level of transparency and interpretability. The models are developed and evaluated using three separate studies, each focusing on a different type of toxicity. The first study focuses on addressing the critical concern of drug-induced liver toxicity, which poses a significant safety risk in drug development. The main objective is to develop quantitative structure-activity relationship (QSAR) models using machine learning algorithms and systematic feature selection methods for a comprehensive set of molecular descriptors. A dataset comprising 1253 diverse drug compounds was utilized to construct these models, and their performance was assessed through rigorous internal validation using 10-fold cross-validation. To enhance the predictive accuracy of the models, various feature selection techniques were employed to identify the optimal subset of descriptors. Among the different classifiers tested, the support vector machine (SVM) emerged as the most effective, yielding superior classification accuracy even with a reduced number of molecular descriptors. The final optimized model exhibited an impressive accuracy of 81.10%, a sensitivity of 84.0%, a specificity of 78.30%, and Matthew's correlation coefficient of 0.623 when evaluated against the internal validation set. Remarkably, the proposed model outperformed previous studies not only in the internal test sets but also when tested against external datasets. This achievement can be attributed to the careful selection of distinct molecular descriptors as crucial modeling features, resulting in a powerful in silico model with exceptional predictive performance. The second study focuses on addressing the significant public health concern of respiratory toxicity, which arises from the adverse effects of drugs or chemicals. It is crucial for the pharmaceutical and chemical industries to have reliable computational tools to accurately assess the respiratory toxicity of compounds. The main objective of this study was to develop robust quantitative structure-activity relationship (QSAR) models using a large dataset of chemical compounds associated with respiratory system toxicity. To enhance the efficiency of modeling, various feature selection techniques were explored to identify the optimal subset of molecular descriptors. Eight different machine learning algorithms were employed to construct respiratory toxicity prediction models. Among these models, the support vector machine (SVM) classifier demonstrated superior performance, surpassing all other optimized models during 10-fold cross-validation. It achieved an impressive prediction accuracy of 86.20% and Matthew's correlation coefficient (MCC) of 0.722 on the test set. To gain insights into the predictions made by the proposed SVM model, the SHapley Additive explanation (SHAP) approach was utilized. This approach prioritizes the identification of key modeling descriptors that influence the prediction of respiratory toxicity. By understanding the relevance of these descriptors, the model's predictions can be better comprehended and interpreted. The proposed SVM model, with its high prediction accuracy and explainability through SHAP, holds tremendous potential in the early stages of drug development. It can effectively predict and provide a deeper understanding of potential respiratory toxic compounds, offering valuable insights for decision-making processes. The third study addresses the significant issue of organ toxicity caused by chemicals, including medications, insecticides, chemical products, and cosmetics. The presence and development of chemical-induced organ damage have been linked to various adverse effects, particularly mitochondrial dysfunction. In this study, an explainable artificial intelligence (XAI) model was proposed to classify compounds as either mitochondrial toxic or non-toxic. To construct the model, the Mordred feature descriptor was carefully selected after applying feature selection techniques. These selected features were then combined with the CatBoost learning algorithm. The proposed model exhibited a remarkable prediction accuracy of 85% during 10-fold cross-validation and achieved an accuracy of 87.10% in independent testing. These results demonstrate a substantial improvement in prediction accuracy compared to existing state-of-the-art methods described in the literature. The proposed model, which utilizes a tree-based ensemble approach, provides valuable insights into the prediction of mitochondrial toxicity. Furthermore, the global model explanation offered by the XAI model aids pharmaceutical chemists in gaining a better understanding of the underlying factors influencing the prediction of mitochondrial toxicity. This enhanced understanding can contribute to more informed decision-making and support the development of safer chemical compounds in various industries. Overall, the results of this thesis demonstrate the potential of explainable artificial intelligence models for the toxicity prediction of chemicals and drugs. By providing insight into the key molecular descriptors driving toxicity prediction, these models have the potential to improve our understanding of toxicity mechanisms and aid in the early identification of potentially toxic compounds, ultimately leading to safer and more effective drugs and chemicals.

Identification of multi-level toxicity of liquid crystal display wastewater toward Daphnia magna and Moina macrocopa

김새봄 Graduate School, Korea University 2013 국내석사

Toxicity-based regulations of industrial effluent have been adopted to complement the conventional discharge limits based on chemical analyses. In this study, multi-level toxicity including acute toxicity, feeding rate inhibition and oxidative stress of effluent from a liquid crystal display (LCD) wastewater treatment plant (WWTP) to Daphnia magna (reference species) and Moina macrocopa (native species) were periodically monitored from April 2010 to April 2011. Raw wastewater was acutely toxic to both D. magna and M. macrocopa, but the toxicity reached less than 1 TU in the final effluent (FE) as treatment proceeded. Although acute toxicity was not observed in the FE, the feeding rate of daphnids was significantly inhibited. Additionally, the antioxidant enzyme activity of catalase, superoxide dismutase and glutathione peroxidase (GPx) in D. magna increased significantly when compared to the control, while only GPx activity was increased significantly in M. macrocopa (p < 0.05). A toxicity identification evaluation using D. magna showed that Cu was the key toxicant in the FE, which was not effectively removed by the coagulation/flocculation process in the LCD WWTP. In addition, Al originating from the coagulant seemed to increase toxicity of the FE. 독성에 근거한 산업폐수의 관리는 기존 개별 화학물질 배출허용기준의 문제점을 보완하기 위하여 도입되었다. 본 연구에서는 LCD 생산공장 폐수종말처리시설 방류수의 급성독성, 섭식률 저해, 산화스트레스와 같은 다양한 수준에서의 독성을 국제 표준종인 Daphnia magna와 국내 토착종인 Moina macrocopa를 이용하여 2010년 4월부터 2011년 4월까지 모니터링 하였다. 폐수종말처리장 원수는 D. magna 와 M. macrocopa 두 종 모두에서 급성독성을 유발하였으나, 처리공정을 거친 최종방류수의 독성은 1이하로 나타났다. 그리고 최종방류수에서 급성독성이 관찰되지 않은 경우에도 물벼룩에 대한 섭식률 저해는 유의하게 나타났다. 또한, D. magna에서는 catalase, superoxide dismutase, gludtathione peroxidase와 같은 항산화 효소의 활성이 대조구와 비교하여 유의하게 증가한 반면, M. macrocopa에서는 gludtathione peroxidase의 효소 활성만 유의하게 증가하였다 (p < 0.05). 한편, D. magna를 이용한 독성동정평가를 통해 구리가 최종방류수의 주된 독성원인물질로 확인되었으며, 이것은 폐수종말처리시설의 응집처리 과정에서 효과적으로 제거되지 않음을 알 수 있었다. 그리고 응집제에 포함되어있는 알루미늄이 최종방류수의 독성을 증가시키는 것으로 판단된다.

심장 독성에서의 Sinapic Acid, Cucurbitacin I, 양배추 추출물의 억제 효과

양동권 전북대학교 일반대학원 2019 국내박사

Sediment toxicity identification and evaluation (TIE) study using South Korea domestic species glyptotendipes tokunagai

싱라훌 한국외국어대학교 대학원 2025 국내박사

Sediment pollution poses significant environmental and ecological risks, particularly in regions experiencing industrial and agricultural growth. In South Korea, sediment toxicity risk assessments have traditionally relied on the "conventional" method to address risks and determine the causality of toxic sediments. Unfortunately, this approach is subject to several limitations: too much focus on primary pollutants and insufficient understanding of the more complex issues that we must face i.e., bioavailability and the interactions between mixtures of contaminants. Researchers have developed "toxicity-based" methods in which organism responses are used to connect the dots. Sediment toxicity identification evaluations (TIEs) are one such tool that involves physical and/or chemical manipulations of the sediment to perform reduction or increment in the toxicity of certain chemicals or classes of chemicals. If toxic effects on the organism are changed by manipulation, then it is likely that the manipulated toxicant is responsible for these effects. Despite its potential value, sediment TIEs remain limited to laboratory settings and background research is relatively new; official testing of these approaches only became available in the United States as recently as 2007. The sediment TIE technique, an advanced sediment toxicity testing procedure that produces more accurate results in less time, has not yet been readily adopted by the region or in fact many areas of the world but this may be due to a lack of general awareness and advocacy on its behalf rather than actual opposition and those regions would seem to benefit hugely from greater insight into regional sediments. This study refined and validated sediment Toxicity Identification Evaluation (TIE) methodologies using Glyptotendipes tokunagai, a native species of South Korea, to address contamination caused by ammonia, cadmium, copper and organic compounds such as acenaphthene, phenanthrene and cyhalothrin. Key findings indicated that removal agents like zeolite, cation exchange resin, sodium sulfide, carbonaceous resin and coconut charcoal successfully reduced concentrations of toxicants, greatly reducing mortality in contaminated sediments. Experimental results thus validated G. tokunagai sensitivity and applicability in TIE protocols, yielding information on pollutant-specific lethality thresholds and interactions within sediment matrices. The ecological implications of sediment toxicity are further discussed, with an emphasis on the role of benthic organisms in maintaining aquatic ecosystem health and nutrient cycling. Besides, some strategies for improving methodologies of TIE to make them more efficient and adaptable to diverse environmental conditions are discussed. In addition, we have assessed the effects of sediment-bound copper and ammonia on Chironomus riparius, and to identify metabolomic biomarkers of exposure and toxicity. C. riparius larvae, selected for their ecological relevance, sensitivity to contaminants, and stable life in sediment, making them ideal bioindicators. The key role that metabolomic analysis played in sediment toxicity testing is to identify several biomarkers: homoeriodictyol and isoleucine in the case of ammonia toxicity, and hectochlorin and nicotinate for copper toxicity. These biomarkers allow for deeper insight into metabolic pathways being disturbed by toxicants, highlighting stress response, energy metabolism, and detoxification mechanisms. The use of native species in this study allows for ecological relevance and increases the accuracy in regional sediment pollution assessments. The results emphasize the potential of metabolomics-informed biomonitoring programs to improve sediment toxicity management and encourage sustainable industrial and agricultural practices. This study lays the groundwork for the development of sediment toxicity assessments, integrating scientific evidence into policy to reduce the harmful effects of sediment-bound contaminants on aquatic ecosystems and human health. In conclusion, this dissertation provides a general framework for further sediment TIE protocols in South Korea, and it extends the original methods with greater robustness and applicability to different classes of sediment contaminants. It also supplements the work already done in Northern Hemisphere countries and introduces changes and methods applied to improve it as an easy, economical way of testing the whole-sediment TIE method for the surroundings of South Korea.

Dermal toxicity evaluation of poly (ethylene glycol) diglycidyl ether and poly (ethylene glycol) diglycidyl ether-crosslinked hyaluronic acid dermal filler

김도현 건국대학교 대학원 2024 국내박사

As the demand for minimally invasive aesthetic procedures such as dermal filling rises, the importance of developing safe and effective injectable dermal fillers intensifies. Hyaluronic acid (HA), also known as hyaluronate, is extensively utilized in dermal fillers for its structural integrity and ability to augment skin volume. HA is also prized for its biodegradability, biocompatibility, and minimal potential to provoke allergic or immunogenic reactions. However, native HA suffers from rapid degradation due to oxidative species and enzymes, and exhibits limited mechanical strength. To improve the viscoelastic properties and extend the residence time of HA gels, crosslinking agents such as 1,4-butanediol diglycidyl ether (BDDE), poly (ethylene glycol) diglycidyl ether (PEGDE), divinyl sulfone (DVS), p-phenylene bisethyl carbodiimide (BCDI), and 1, 2, 7, 8-diepoxyoctane (DEO) have been utilized. BDDE is the primary crosslinking agent used in commercial dermal fillers owing to its biodegradability and biocompatibility. PEGDE has emerged as a promising alternative, offering chemical reactivity comparable to BDDE and distinctive rheological properties with potentially lower toxicity. Although HA fillers using BDDE and other cross-linking agents are considered safe, accurate evaluations of the toxicity of these agents in hyaluronic acid fillers are lacking. Additionally, the increasing use of HA fillers in large volumes for body contouring underscores the need for extensive safety assessments. Consequently, this study aims to assess the dermal toxicity of HA fillers incorporating BDDE and PEGDE, evaluating the potential of PEGDE as a safer alternative crosslinking agent. The dermal toxicity of PEGDE was investigated in vivo. Doses ranging from 500 to 40,000 μg of PEGDE were subcutaneously injected into the paraspinal dorsum of BALB/c male mice and observed over a period of 28 days. On day 28, tissue samples from the skin, liver, kidney, and spleen were examined for local and systemic toxic responses. Mice receiving the highest dose of 40,000 μg exhibited severe toxic reactions and were euthanized. Lower doses of 10,000 and 20,000 μg induced epidermal ulcer formation and hair loss, with histological examination revealing re- epithelialized or unhealed wounds. Notably, PEGDE caused subcutaneous toxicity, particularly at doses above 10,000 μg per mouse, without affecting other organs. The cytotoxic effects of BDDE and PEGDE were also evaluated on human keratinocyte (HaCaT) and human dermal fibroblast (HDF) cell lines. Cells treated with BDDE concentrations above 100 ppm displayed significantly reduced viability, increased cytotoxicity, cell membrane damage, reactive oxygen species production, matrix metalloproteinases activity, and inflammatory responses. Conversely, cells treated with PEGDE exhibited lower cytotoxicity, oxidative stress, and inflammatory responses at equivalent concentrations, suggesting that PEGDE is potentially safer than BDDE as a crosslinking agent in HA dermal fillers. The physical properties, toxicity, and inflammatory reactions of HA fillers crosslinked with either BDDE (HA-BDDE filler) or PEGDE (HA-PEGDE filler) were further assessed through in vitro and in vivo studies. The HA-PEGDE filler demonstrated superior rheological properties, such as higher storage modulus (Gʹ), loss modulus (tan δ), and complex viscosity, as well as reduced cytotoxicity, oxidative stress, and inflammation in cell cultures compared to the HA-BDDE filler. In animal models, the HA-PEGDE filler showed favorable biocompatibility, decreased expression of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) genes, and reduced inflammatory cell infiltration, indicating its suitability as an alternative to HA-BDDE filler, especially in contexts requiring minimized inflammatory responses. In conclusion, PEGDE has demonstrated potential as a safe crosslinking agent for HA dermal fillers. The findings of this study provide evidence that HA fillers incorporating PEGDE are safer for large volume injections in both body contouring and facial applications. 최근 피부 개선을 위한 시술에서 침습을 최소화한 미용 시술에 대한 수요가 증가함에 따라 안전하고 효과적인 필러 개발의 중요성이 커지고 있다. 히알루론산은 피부 볼륨 증가에 탁월한 효과를 가진 구조와 높은 생분해성 및 생체적합성으로 인해 의료기기 및 필러의 주원료로 광범위하게 사용된다. 그러나 자연 상태의 히알루론산은 산화종과 효소에 의해 빠르게 분해되고, 제한된 물성으로 인해 필러의 재료로서 한계를 가지고 있다. 따라서, 히알루론산 겔의 물성 및 체내 잔류 시간을 개선하기 위해 1,4-부탄디올 디글리시딜 에테르(BDDE), 폴리(에틸렌 글리콜) 디글리시딜 에테르(PEGDE), 디비닐 술폰(DVS), p-페닐렌 바이에틸 카보디이미드(BCDI), 1, 2, 7, 8-다이에폭시옥탄(DEO)과 같은 가교제가 사용된다. BDDE는 높은 생분해성과 생체적합성 때문에 필러 제조에 가장 많이 사용된다. PEGDE는 BDDE와 유사한 화학적 반응성을 보이며 독특한 물성과 낮은 독성으로 인해 BDDE에 대한 대안으로 주목받고 있다. 기존 필러들은 안전하다고 알려져 있지만, 필러에 사용되는 가교제의 독성 평가는 부족한 실정이다. 또한 최근 아름다운 몸매에 대한 관심증가에 따라 신체 시술을 위해 필러의 대용량 사용이 증가하고 있어 대용량 사용시 가교제와 필러에 대한 독성 평가 또한 필요하다. 따라서 본 연구는 기존에 주로 사용되는 가교제(BDDE)와 신규 가교제(PEGDE)를 포함하는 히알루론산 필러의 피부 독성을 평가하여 대용량 사용시 더 안전한 대체 가교제로서 PEGDE의 잠재력을 평가하고자 한다. PEGDE의 피부 독성은 동물 실험 모델을 통해 평가되었다. PEGDE는 마우스 당 500-40,000 μg의 용량으로 BALB/c 수컷 쥐의 척추 옆 부위에 피하주사 되었고, 주사 후 28일간 관찰되었다. 주사 후 28일째에 피부, 간, 신장, 비장 조직 샘플을 분석하여 PEGDE의 국소 및 전신 독성 반응을 평가하였다. 40,000 μg이 주사된 받은 마우스는 심각한 독성 반응을 보여 안락사 되었다. 10,000 μg 과 20,000 μg이 주사된 마우스에서 표피 궤양 형성과 탈모가 관찰되었으며, 조직 검사에서 흉터 또는 치유되지 않은 상처가 관찰되었다. 특히 10,000 μg 이상의 용량에서 PEGDE는 다른 장기에 영향을 주지 않으면서 피하 독성을 유발했다. BDDE 및 PEGDE의 세포독성 효과는 인간 각질 세포(HaCaT) 및 인간 피부 섬유아세포(HDF)를 이용하여 평가되었다. 100 ppm 이상의 BDDE가 처리된 세포는 낮은 세포 생존율과 높은 세포독성, 세포막 손상, 활성 산소종 생성 및 염증 반응을 보였다. 반면에 같은 농도의 PEGDE가 처리된 세포에서 세포독성, 산화 스트레스 및 염증 반응이 낮게 나타났다. 따라서, PEGDE가 BDDE보다 안전한 가교제로서 잠재성이 있음을 세포 수준에서 검증하였다. BDDE(HA-BDDE 필러) 또는 PEGDE(HA-PEGDE 필러)로 가교된 히알루론산 필러의 물리적 특성, 독성 및 염증 반응이 세포 및 동물 실험을 통해 평가되었다. HA-PEGDE 필러는 HA-BDDE 필러보다 높은 G', tan δ, 복소 점도를 보였다. 또한, HA-PEGDE 필러가 처리된 HaCaT 및 HDF 세포는 HA-BDDE 필러가 처리된 세포보다 낮은 수준의 세포독성, 산화 스트레스 및 염증 반응을 보였다. 동물 실험에서는 HA-PEGDE 필러가 주입된 SKH1 마우스의 피부조직이 더 낮은 염증 반응을 보였다. 결과적으로, HA-PEGDE 필러는 HA-BDDE 필러의 대체재로서 적합하다고 판단된다. 최종적으로, 본 연구를 통해 히알루론산 필러에서 PEGDE가 안전한 가교제임을 증명하였다. 이러한 결과로 대용량 사용시 더 안전한 필러로써 PEGDE로 가교한 히알루론산 필러의 이용이 가능할 것으로 사료된다.

Molecular response and biotransformation of arsenic compounds in aquatic invertebrates

Byeon, Eunjin Sungkyunkwan University 2023 국내박사

비소는 환경에 널리 분포하는 준금속으로 잔류성과 축적이 잘 되는 특성 때문에 독성물질로 여겨진다. 현재까지 수생환경에서 비소의 발생, 분포 및 생물학적 영향에 대해 광범위하게 연구되어 왔으며, 비소에 대한 급성 및 만성 독성은 개체에서 분자 수준까지 치명적인 영향과 관련이 있다고 알려져있다. 수생생물에서 비소의 독성은 비소의 종류와 농도에 따라 달라진다. 수생환경에서 무기비소는 가장 지배적인 형태이며 3가 비소는 5가 비소에 비해 더 큰 독성을 가지고 있다. 무기비소는 수생생물에서 생체 내 변환을 통해 다양한 형태로 나타날 수 있다. 생체 내 변환 기작과 비소의 종 분화는 광범위하게 연구되어 왔지만, 생물 내에서의 화학적 종 분화, 독성 및 생물 이용성 사이의 관계를 다루는 연구는 아직 부족하다. 따라서 본 연구에서는 수생생물에서 생체 내 변환 기작과 비소 독성의 작용방식을 이해하기 위해 해양 및 담수 무척추동물에서 비소에 대한 반응기작을 중심으로 연구를 진행하였다. (1) 해양 윤충류 (Brachionus plicatilis) 및 요각류 (Paracyclopina nana)에서 비소 화합물의 종간 생체 내 변환 및 해독 기작 연구, (2) 미소 및 미세 플라스틱과 결합된 비소 노출이 윤충류 B. plicatilis에 미치는 영향 연구, (3) Glutathione S-tranferase omega 2 (GST-O2) 유전자 표적 담수 물벼룩 Daphnia magna 돌연변이에서 비소에 대한 차등 감수성 연구. 본 연구에서, 두 종의 동물플랑크톤 사이에서 비소에 대한 종 의존적인 민감도가 나타났다. 두 유기체 모두에서 3가 비소 (AsIII)는 5가 비소 (AsV)보다 독성이 더 강하게 나타났으며, 윤충류 B. plicatilis는 요각류 P. nana보다 더 강한 내성을 보였다. 또한 비소 노출 시 생물 특이적인 생체 내 변환과 항산화 반응이 관찰되었다. 게다가, 윤충류 B. plicatilis에서 플라스틱 입자의 크기에 따라 비소 독성 및 생물 내 축적이 다르게 나타날 수 있음이 관찰되었다. 미소 플라스틱 (nanoplastic)과 비소의 결합 노출 시, 다중 생체이물 내성 (MXR)의 억제로 인한 비소의 생체 농도 증가로 인해 더욱 높은 독성을 유도한 반면, 미세 플라스틱 (microplastic)은 생물에 미치는 비소 독성을 완화시켰다. 마지막으로, CRISPR/Cas9 시스템에 의해 생산된 야생형 및 GST-O2 표적 돌연변이 물벼룩을 이용하여 D. magna의 비소 생체변환 및 해독과 관련된 GST-O2 유전자의 역할을 조사하였다. GST-O2 유전자의 부재는 비소해독 능력과, AsV를 AsIII로의 환원율을 감소시켰으며, GST-O2가 D. magna의 비소 대사 기능에 중요한 역할을 한다는 것을 시사했다. 본 연구는 수생 유기체에 미치는 비소 독성 및 종간 해독 기작에 대한 이해와, 비소 및 환경 오염물질의 작용방식에 대한 심층적인 이해에 도움을 줄 것이다. Arsenic is a toxic metalloid that is widely distributed in the aquatic environment. Acute and chronic arsenic intoxications are associated with fatal effects at the individual and molecular levels. The toxicity of arsenic to aquatic organisms depends on its speciation and concentration. In aquatic environments, inorganic arsenic is the dominant form, and can assume a variety of forms through biotransformation in aquatic organisms. Biotransformation mechanisms and speciation of arsenic have been studied extensively, but only a few reports have addressed the relationships among speciation, toxicity, and bioavailability in biological systems. Therefore, to understand the modes of action of arsenic toxicity along with its mechanism of biotransformation in aquatic organisms, I have focused on studying the response to arsenic in aquatic invertebrates through: (1) interspecific biotransformation and detoxification of arsenic compounds in marine rotifer and copepod; (2) arsenic exposure combined with nano- or microplastic induces different effects in the marine rotifer Brachionus plicatilis; (3) differential susceptibility to arsenic in glutathione S-transferase omega 2 (GST-O2)-targeted freshwater water flea Daphnia magna mutants. In my studies, species-specific sensitivity to arsenic was found between Brachionus plicatilis and Paracyclopina nana. The rotifer B. plicatilis exhibited stronger tolerance to arsenic, compared to the P. nana. Lineage-specific biotransformation and antioxidant responses upon arsenic exposure were shown. Moreover, I observed that arsenic toxicity and bioaccumulation could be different depending on the size of plastic particles in the rotifer B. plicatilis. Exposure to arsenic combined with nanoplastic (NP) induced higher toxicity due to the increased bioconcentration of arsenic that is possibly associated with multixenobiotic resistance (MXR) inhibition, while microplastic (MP) alleviated arsenic toxicity. Finally, using the GST-O2-targeted mutant fleas D. magna produced by CRISPR/Cas9, the role of GST-O2 genes was investigated in the biotransformation and detoxification. The absence of the GST-O2 gene decreased the ability of detoxification and pentavalent arsenic reduction rate, suggesting that GST-O2 plays an important role in arsenic metabolic functions in D. magna. Overall, these findings will be helpful for a better understanding of arsenic toxicity and interspecific-detoxification mechanisms in aquatic invertebrates, as well as an in-depth insight into the effects of arsenic and environmental pollutants.

Evaluation of Developmental Toxicity of Thiabendazole, Pyridaben, and Fenoxycarb Using a Zebrafish (Danio rerio) Animal Model

Junho Park 고려대학교 대학원 2025 국내박사

Environmental contaminants, including heavy metals, plastics, particulate matter, and pesticides, are being detected in ever greater numbers, raising the risk of exposure for living organisms, including humans. According to the World Health Organization (WHO), around 7 million deaths per year are attributable to pollution, and the links between pollutants and various diseases are continually being documented. For instance, pesticides have been consistently used to improve agricultural production by controlling harmful pests; however, with population growth, the significance of agricultural production has risen, resulting in a continuous increase in pesticide use. Global pesticide consumption was reported to have reached 4.1 million tons in 2019. Consequently, pesticides have been detected across multiple environments as they circulate through ecosystems, posing risks not only to human health but also to biodiversity due to their diverse toxic effects. For example, pesticides can exert toxic effects on the liver, heart, nervous system, reproductive system, and other organs in the human body, prompting ongoing research into their harmful impacts. Given the growing emphasis on environmental contaminants, numerous toxicity studies using various animal models are being conducted, with extensive use being made of zebrafish (Danio rerio) embryos because of the significant advantages they offer. For instance, as vertebrate animal models, zebrafish are smaller in size than other models and incur lower maintenance expenses. Moreover, the zebrafish exhibits morphology and physiology comparable to that of humans in its cardiovascular, nervous, and digestive systems, with approximately 70% genetic similarity. A single pair of zebrafish can lay 200-300 eggs per mating, and their transparent embryos facilitate easy observation of the developmental process. Furthermore, zebrafish undergo rapid development, with the majority of organs, such as the brain, heart, liver, and pancreas, reaching full maturity within five days post-fertilization (dpf). The structural anomalies in different organs can be readily observed using transgenic zebrafish models, whose specific markers for each organ are labeled with fluorescence. In this study, the developmental toxicity of the widely used pesticides thiabendazole, pyridaben, and fenoxycarb was evaluated using zebrafish embryos. The results revealed that thiabendazole downregulated the PI3K/Akt and MAPK pathways, induced excessive apoptosis, and increased the production of reactive oxygen species (ROS), leading to a range of organ toxicities. Pyridaben disrupted Ca2+ homeostasis and inhibited phosphorylation of the PI3K/Akt pathway, resulting in cell cycle arrest and increased apoptosis in zebrafish liver cells (ZFL). These changes (in vitro) caused a reduction in liver size among zebrafish larvae (in vivo). Fenoxycarb increased the number of macrophages and induced changes in the expression of inflammatory cytokines, which led to cardiovascular toxicity, hepatotoxicity, and pancreatic toxicity. These results highlight the mechanisms responsible for toxicant-induced organ toxicity during development and could serve as a crucial foundation for gaining a more comprehensive understanding of the attendant environmental risks. Moreover, they will also facilitate the prediction of toxicity in humans as well as other aquatic organisms. 세계적으로 중금속, 플라스틱, 미세먼지 및 농약을 포함한 환경 오염 물질들이 점차 많이 검출되고 있으며, 이는 인간을 포함한 비표적 생물체의 노출 위험을 증가시키고 있다. 세계보건기구(WHO)에 따르면 매년 약 700만 명의 사망이 오염에 기인하며, 오염 물질과 다양한 질병 간의 연관성도 지속적으로 보고되고 있다. 특히, 농약은 농업 생산량 증대를 위해 꾸준히 사용되어 왔으며, 인구 증가와 함께 농업 생산의 중요성이 더욱 부각되면서 그 사용이 최근까지도 지속적으로 증가하고 있다. 실제로 2019년, 전 세계 농약 소비량은 410만 톤에 달한 것으로 보고되었다. 농약은 생태계를 순환하며 여러 환경에서 검출되고 있으며, 다양한 독성 효과로 인해 인간의 건강뿐만 아니라 생물 다양성에도 위험을 초래하고 있다. 농약은 간, 심장, 신경계, 생식계 및 기타 기관에 독성 영향을 미칠 수 있어, 그 유해성에 대한 연구가 활발히 진행되고 있다. 환경 오염 물질에 대한 관심이 커짐에 따라 다양한 동물 모델을 이용한 독성 연구가 활발히 진행되고 있으며, 특히 제브라피쉬 (Danio rerio) 배아는 여러 장점 덕분에 광범위하게 사용되고 있다. 제브라피쉬는 척추동물 모델로, 다른 모델에 비해 크기가 작고 유지 비용이 낮다는 이점을 가지고 있다. 제브라피쉬는 심혈관, 신경계, 소화계에서 인간과 유사한 형태와 생리적 특성을 보이며, 약 70%의 유전적 유사성을 공유한다. 제브라피쉬 한 쌍은 교배 시 200-300개의 알을 산란할 수 있으며, 투명한 배아를 가져 발달 과정의 관찰이 용이하다. 또한, 제브라피쉬는 발달 과정이 빠르게 진행되며 수정 후 5일 이내에 뇌, 심장, 간, 췌장 등 대부분의 기관이 완전히 형성된다. 특히 각 기관에 특정 형광이 표지 된 형질전환 제브라피쉬 모델을 활용하여 다양한 기관의 구조적 이상을 쉽게 관찰할 수 있다 이 연구에서는 널리 사용되는 농약인 thiabendazole, pyridaben 및 fenoxycarb의 발달독성을 제브라피쉬 배아를 이용하여 평가하였다. Thiabendazole은 PI3K/Akt와 MAPK 경로를 억제하고, 과도한 세포사멸을 유도하며, 활성산소의 생성을 증가시켜 다양한 기관 독성을 초래하였다. Pyridaben은 제브라피쉬 간 세포주 (ZFL)의 Ca²⁺ 항상성을 붕괴시키고 PI3K/Akt 경로의 인산화를 억제하여 세포 주기를 정지시키고 세포사멸을 증가시켰다. 이러한 세포주에서의 변화는 실제 제브라피쉬 유충에서의 간독성으로 이어졌다. Fenoxycarb는 염증성 사이토카인의 발현 변화와 함께 대식세포의 수를 증가시켜 심혈관 독성, 간독성 및 췌장독성을 유발하였다. 이러한 결과는 발달 과정 중 독성 물질에 의한 기관 독성의 기전을 제시하며, 환경 위험성을 보다 포괄적으로 이해하는 데 중요한 기초 자료가 될 수 있다. 또한, 이를 통해 인간 및 기타 수생 생물에서의 독성을 예측할 수 있을 것이다.

생활화학제품이 폐 상피세포에 미치는 혼합영향

정혜진 경성대학교 일반대학원 2022 국내석사

Household chemicals are frequently used in daily life for purposes such as cleaning, air freshening, and disinfection, greatly enhancing convenience and improving our daily lives. These products often release particles into the air in the form of aerosols or sprays, which can cause respiratory toxicity. The compositions of these chemicals often include not only a single substance but also combinations of two or more compounds. Therefore, it is crucial to accurately understand the information on the toxicity of these chemical substances. This study aims to investigate the individual and mixture toxicity of benzalkonium chloride (BKC) and dodecyl dimethyl ammonium chloride (DDAC), which are both quaternary ammonium compounds (QACs). In addition, we conducted research to determine the mixed toxicity effects of polyethylene glycol (PEG), which is used as a stabilizer in our products. Cytotoxicity was assessed by using lactate dehydrogenase (LDH) and water-soluble tetrazolium salt 1 (WST-1) assays, and both submerged and air-liquid interface ALI cultures were utilized as cell culture methods. We used the MRA Toolbox prediction model to determine the effect of mixed toxicity. The toxicity of the mixture tends to display an additive effect when mixed and exposed, as indicated by the EC50 value of each individual material. In addition, no significant effect was observed on the toxicity of BKC and DDAC when treated with the PEG mixture, regardless of whether it was enhanced or weakened. There was a significant difference in toxicity results between the submerged and ALI cultures. In particular, we observed that the toxicity of the substances was lower in the ALI culture method compared to the submerged method. Differences in culture methods seemed to have caused the variations in virulence. In this study, we investigated the mixture effects of QACs, as well as the effects on PEG. We believe that our study can contribute to the understanding of the mixture effect of household chemicals. We highly recommend using the results of this study as additional support for future investigations on the mechanism of action of mixture toxicity and other toxicity endpoints.

Development of novel toxicity assessment methods using thiosulfate utilizing denitrifying and sulfur-oxidizing bacteria

Ashun, Ebenezer 강원대학교 대학원 2022 국내박사

The development of simple and rapid toxicity testing procedures has been instrumental in effective pollution monitoring and control. Consequently, this study reports for the first-time the possibility of deploying gas production by thiosulfate utilizing denitrifying bacteria (TUDB) as a proxy to evaluate water toxicity. The test relies on gas production by TUDB due to inhibited metabolic activity in the presence of toxicants. Gas production was measured using a bubble-type respirometer. Optimization studies indicated that 300 mg NO_3^--N/L, 0.5 mL acclimated culture, and 2,100 mg S2O32-/L were the ideal conditions facilitating the necessary volume of gas production for sensitive data generation. Determined EC50 values of the selected heavy metals were: Cr6+, 0.51 mg/L; Ag+, 2.90 mg/L; Cu2+, 2.90 mg/L; Ni2+, 3.60 mg/L; As3+, 4.10 mg/L; Cd2+, 5.56 mg/L; Hg2+, 8.06 mg/L; and Pb2+, 19.3 mg/L. The advantages of this method include operational simplicity through the elimination of cumbersome preprocessing procedures which are used to eliminate interferences caused by turbidity when the toxicity of turbid samples is determined via spectrophotometry. A direct contact bioassay of thiosulfate utilising denitrifying bacteria (TUDB) based on gas production inhibition was deployed to assess the toxicity of laboratory soils artificially contaminated with heavy metals and naturally contaminated field soils. 1 g soil sample, 80 RPM and 48 h reaction time were the optimal conditions responsible to the maximum gas production. The determined half maximal concentration responsible for 50% reduction in gas production by TUDB were 3.01 mg/kg Cr6+; 15.30 mg/kg Ni2+;15.50 mg/kg Cu2+;16.60 mg/kg Ag+; 20.60 mg/kg As3+; 32.80 mg/kg Hg2+; 54.70 mg/kg Cd2+; and 74.0 mg/kg Pb2+. Since soils toxicity is usually influenced by the various physicochemical characteristics, ten reference soils were used to determine the toxicity threshold and used to evaluate the toxicity of naturally contaminated field soils. All the 8 contaminated soils were toxic to TUDB as their levels of inhibition ranged between 72% to 100% and exceeded the determined toxicity threshold of 10%. The obtained results affirm the suitability of the developed assay for soil toxicity assessments. The high sensitivity of nitrifying bacteria (NB) to inhibition by various toxicants has positioned it as an alternative bioassay for water toxicity assessment. The technique is based on inhibition of oxygen consumption resulting from the inhibition of nitrification in the presence of toxicants. Changes in oxygen consumption was used as the proxy for toxicity analysis. Oxygen consumption measurements were obtained using the glass syringe methodology and used as a proxy for toxicity detection. The concentration of heavy metals causing 50% decrease in the oxygen consumption activity of NB was 2.36 mg/L Ag+, 3.35 mg/L As3+, 25.1 mg/L Cd2+, 1.11 mg/L Cr6+, 4.25 mg/L Cu2+, 1.94 Hg2+, and 19.6 mg/L Pb2+. The decreasing order of inhibitory effect on the NB bioassay was Cr6+; Hg2+, Ag+, As3+, Cu2+, Ni2+, Pb2+ and Cd2+ respectively. Because of its simplicity, short processing time, and the highly reproducible data obtained, the compared to conventional nitrification assays, the developed procedure can be adapted for field toxicity testing and can replace contention nitrification assays that demand complex instrumentation and test procedures. The current study tested the employment of sulfur-oxidizing bacteria (SOB) in a direct contact bioassay to evaluate toxicity of soil contaminated with heavy metals. SOB was directly exposed to soil spiked with arsenic, nickel, hexavalent chromium, zinc, mercury, copper, lead, or cadmium for 12 h in a kit-type bioassay. Oxygen consumption by SOB was used as the end-point measurement for toxicity evaluation. The results demonstrate that oxygen consumption by SOB decreased as the doses of spiked heavy metals increased. Particularly, oxygen consumption in the tests spiked with arsenic, nickel, hexavalent chromium, or zinc was less than that spiked with mercury, copper, lead, or cadmium at the same concentrations. The 12 h half-maximum effective concentrations (EC50) of SOB for arsenic, nickel, hexavalent chromium, zinc, mercury, copper, lead, and cadmium were analyzed to be 12.7, 13.3, 19.1, 20.8, 40.1, 67.2, 101.5, and 105.4 mg/kg, respectively. These EC50 values are largely within the regulatory standards of heavy metals in soil in South Korea, suggesting that the SOB test kit has sufficient sensitivity for use in on-site soil toxicity tests. The findings from the current study present that the SOB test kit is a suitable tool for evaluating heavy metal-induced toxicity of soil. In this study, 11 low/uncontaminated (including Lufa 2.2) and 9 contaminated field soils with varying geophysical and physicochemical characteristics were evaluated for toxicities based on oxygen consumption of sulfur-oxidizing bacteria (SOB). Oxygen consumption of the low/uncontaminated soils ranged between 7.9 mL to 9.5 mL, while contaminated soils ranged between 0.4 mL to 5.4 mL. Inherent test variability (Cvi), variation due to soil natural properties (Cvns) and minimal detectable difference (MDD) values ranged 1.2% to 3.9%, 3.5% to 16.9%, and 2.1% to 4.3%, respectively. The toxicity threshold of 20% was established for soil toxicity based maximal tolerable inhibition (MTI). All the contaminated soils were found to be toxic and showed inhibition between 42% and 100% above the 20% threshold value. Increased proportions of clay and slit enhanced the of inhibitory effect of contaminants on SOB by reducing the oxygen consumption. Current study provides a suitable method for the rapid toxicity assessment of contaminated field soils with the advantages of ease of handling and rapidity without employing elutriates and sophisticated equipment’s and tools. 편리하고 신속한 독성 시험법의 개발은 효율적인 오염 모니터링 및 제어에 중요하다. 따라서 본 연구는 수중 독성을 평가하기 위한 수단으로 티오황산염을 이용하여 탈질 반응을 유도하는 황탈질미생물(TUDB)을 사용하였다. 본 연구는 독성 물질이 존재할 때 TUDB의 활성이 억제되어 유발되는 가스 생산량의 차이를 미세가스 발생량 측정장치를 사용하여 분석하였다. 최적화 실험 결과에 의하면 TUDB 주입량 0.5 mL, 300 mg NO3--N/L 및 2,100 mg S2O32-/L의 조건이 높은 민감도를 얻을 수 있는 이상적인 조건으로 나타났다. 본 연구에 사용한 중금속의 EC50 값은 Cr6+ 0.51 mg/L, Ag+ 2.90 mg/L, Cu2+ 2.90 mg/L, Ni2+ 3.60 mg/L, As3+ 4.10 mg/L, Cd2+ 5.56 mg/L, Hg2+ 8.06 mg/L 그리고 Pb2+ 19.3 mg/L로 도출되었다. 본 측정방법의 장점은 탁도가 존재하는 시료에도 전처리 없이 적용이 가능하다는 점이다. 본 연구는 중금속을 인위적으로 오염시킨 인공 토양과 실제로 오염된 현장 토양의 독성을 평가하기 위해 가스 생성량 비교를 기반으로 하는 티오황산염을 이용한 황탈질미생물(TUDB)을 직접 접촉 방식으로 연구하였다. 토양시료 1 g, 80 RPM 의 교반속도 및 48시간 노출 시간이 최대 가스 생산의 최적 조건이었다. 각 중금속의 TUDB에 대한 EC50 농도는 Cr6+ 3.01 mg/kg, Ni2+ 15.30 mg/kg, Cu2+ 15.50 mg/kg, Ag+ 16.60 mg/kg, As3+ 20.60 mg/kg, Hg2+ 32.80 mg/kg, Cd2+ 54.70 mg/kg 그리고 Pb2+ 74.0 mg/kg으로 측정되었다. 토양 독성 평가는 일반적으로 토양의 여러 물리화학적 인자에 영향을 받기 때문에 10개의 표준 토양을 사용하여 독성 임계 값을 결정하고 실제 오염된 현장 토양의 독성을 평가하는 데 사용했습니다. 8개의 오염된 토양은 모두 저해 수준이 72%에서 100% 사이이고 결정된 독성 임계 값인 10%를 초과했기 때문에 TUDB에 저해를 주었고 토양 독성 평가를 위해 개발된 분석의 적합성을 확인하였다. 질산화미생물(NB)의 높은 독성 민감도는 수생태 독성 평가를 위한 생물학적 분석방법으로 널리 사용되고 있다. 본 방법은 독성 물질이 존재하는 상태에서 질산화 반응을 억제하여 발생되는 산소 소비량 변화를 기반으로 하는 독성도 분석을 바탕으로 진행되었다. 산소 소비량은 유리 주사기를 이용한 측정법을 사용하여 측정하였고 이를 바탕으로 독성도 분석을 수행하였다. NB의 산소 소모 량을 50% 감소시키는 중금속의 농도는 Ag+ 2.36 mg/L , As3+ 3.35 mg/L, Cd2+ 25.1 mg/L, Cr6+ 1.11 mg/L, Cu2+ 4.25 mg/L, Hg2+ 1.94, 그리고Pb2+ 19.6 mg/L로 측정되었다. NB에 대한 중금속 독성 민감도 순서는 감소 순서는 Cr6+, Hg2+, Ag+, As3+, Cu2+, Ni2+, Pb2+ 그리고 Cd2+의 순서로 높은 독성 민감도를 보였다. 편리함, 짧은 시험 시간 및 결과의 높은 재현성으로 본 시험법은 기존 질산화미생물 시험법과 비교하였을 때 현장에서 사용할 수 있으며 복잡한 기기 및 시험 절차가 필요한 기존 질산화미생물 독성평가방법 대체할 수 있을 것이라 판단된다. 본 연구는 중금속으로 오염된 토양의 독성을 평가하기 위해 황산화미생물(SOB)을 이용한 직접 접촉 방식으로 오염토양의 독성도를 평가하였다. SOB는 키트 형태로 12시간 동안 비소, 니켈, 6가 크롬, 아연, 수은, 구리, 납 그리고 카드뮴에 오염된 토양에 직접 노출되었고 독성 종말점으로 SOB에 의한 산소 소비량을 사용하였다. 결과는 주입된 중금속의 농도가 증가함에 따라 SOB에 의한 산소 소비가 감소하였다. 특히 비소, 니켈, 6가 크롬 그리고 아연을 첨가한 시험에서 산소 소비량은 동일한 농도의 수은, 구리, 납 또는 카드뮴을 첨가한 시험보다 상대적으로 적었다. 비소, 니켈, 6가 크롬, 아연, 수은, 구리, 납 및 카드뮴에 대한 SOB의 12시간 반수영향농도(EC50)는 각각 12.7 mg/kg, 13.3 mg/kg, 19.1 mg/kg, 20.8 mg/kg, 40.1 mg/kg, 67.2 mg/kg, 101.5 mg/kg그리고 105.4 mg/kg으로 분석되었다. 이러한 EC50 값은 대한민국 토양 중금속 규제 기준에 대부분 부합하며, 이는 SOB 키트 시험을 현장 토양 독성 시험에 적용하기에 적합함을 시사한다. 본 연구에서는 저농도 또는 오염되지 않은(Lufa 2.2 포함) 11개의 토양과 다양한 물리화학적 특성을 갖는 9개의 오염된 현장 토양에 대해 황산화미생물(SOB)의 산소 소비량을 기반으로 독성을 평가하였다. 저농도 또는 오염되지 않은 토양의 산소 소비량은 7.9mL에서 9.5mL 사이인 반면 오염된 토양은 0.4mL에서 5.4mL 사이로 측정되었다. 고유한 시험 변동성(Cvi), 토양의 자연적 특성(Cvns)으로 인한 변동 및 최소 검출 가능 차이(MDD) 값의 범위는 각각 1.2%~3.9%, 3.5%~16.9%, 2.1%~4.3%로 도출되었고 최대 허용 저해(MTI) 기반 토양 독성에 대해 20%의 독성 임계값이 설정되었다. 오염된 모든 토양은 독성을 띄는 것으로 밝혀졌으며 20% 임계값 이상에서 42%에서 100% 사이의 저해를 보였다. 점토와 실트의 비율 증가는 산소 소모량을 줄여 SOB에 대한 오염물질의 저해 효과를 높였다. 본 연구는 오염된 현장 토양의 신속한 독성 평가에 적합한 방법이며, 세척액과 정밀 장비 및 도구를 사용하지 않고도 사용이 쉽고 신속한 장점이 있다.