(A) bioassay-based approach for the determination of benzalkonium ion distribution coefficients between water and soil components

다국어 초록 (Multilingual Abstract)

Chemical risk assessment requires reliable models to describe the environmental fate of chemicals. Special attention should be given to those chemicals known to be bioactive and also have high production volumes. Benzalkonium compounds are one such group of chemicals, designed to be biocidal and also produced in large volumes due to their widespread use in industrial, domestic and agricultural applications. Although benzalkonium compounds present a risk to terrestrial and aquatic ecosystems, there are no suitable models to describe their environmental fate. One of the reasons for the absence of reliable models is the unavailability of suitable analytical techniques to measure free concentrations of benzalkonium compounds at environmentally relevant levels. Since benzalkonium compounds are solids at environmental temperatures, their distribution will be confined to water and soils. Novel approaches are therefore, required to determine benzalkonium ion distribution coefficients between water and soil components.
This study reports on a bioassay-based method for the determination of benzalkonium ion distribution coefficients between water and soil components. A standardized algal growth inhibition test using Pseudokirchneriella subcapitata was modified by including sorbents in the culture medium. The nominal median inhibitory concentrations obtained in sorbent free medium were subtracted from those obtained from media with sorbents in order to estimate the sorbed concentrations. A toxicokinetic model was then used to compute free concentrations after accounting for the chemical sorbed to algal cells. Sorption to soil components accounted for over 80% of the initial spiked amount. The algal growth inhibition assay was sufficiently sensitive to allow distribution coefficients to be determined at free concentrations below 1 µmol L-1 and sorbed concentrations that were below 10% of the sorbent cationic exchange capacity. These values are within the range of reported environmental concentrations.
The developed method was used to determine distribution coefficients for peat, kaolinite and montmorillonite. When distribution coefficients were corrected for each sorbent’s cation exchange capacity, the computed distribution coefficients for each compound were all within one order of magnitude. It was also noted that at low sorbate loadings, van der Waals interactions between the neutral organic surfaces on peat and the alkyl chains of benzalkonium ions did not make a significant contribution to sorption. Therefore, at environmentally relevant concentrations sorption to the tested soil components was attributed to charge-charge interactions between the cationic sorbate and negatively charged sorbent surfaces. However, there was a strong positive correlation between the computed distribution coefficients and the length of the alkyl chain of the benzalkonium ion. This correlation was attributed to the increase in hydration energy with molecular weight of the ion. Higher hydration energies favor the partitioning of cations away from the aqueous phase. This was further explained by computing sorption energies of each ion and comparing them with hydration energies of a homologous series of alkanes.
This study also developed a method to calculate the distribution coefficients for soils of a known composition. Since the magnitude of the distribution coefficient was determined by both length of the alkyl chain and the sorbent cation exchange capacity, the model considers the two parameters in predicting sorption coefficients. Although the developed model is not suitable for the 16 and 18 carbon alkyl chain homologues, it does cater for the most popular homologues (C12 and C14) in most commercial formulations and the environment.

국문 초록 (Abstract)

화학물질의 위해성평가를 위하여 화학물질의 거동에 대한 정확한
정보가 필요하다. 벤잘코늄 화합물은 세정제 및 살생물제로 널리 사용되고
있으며 수계와 토양으로 배출된다. 이로 인해 거의 모든 환경매체에서
벤잘코늄 화합물이 검출된 바 있다. 벤잘코늄은 살생제로 널리 쓰이는
물질이므로 환경중으로 배출되었을 때 본질적으로 악영향을 미칠 우려가
있다. 그럼에도 아직까지 벤잘코늄 이온의 환경 중 분배를 설명하기에
적합한 신뢰성이 있는 모델이 개발되어 있지 못하다. 특히 환경 중
잔류수준에서의 자유농도를 측정하기 위한 적절한 분석기법이 개발되지
못한 것도 한 원인이다. 그러므로 물과 토양 성분 사이의 벤잘코늄 이온의
평형 분배계수를 결정하는 새로운 접근법이 필요하다.

본 연구는 물과 토양 성분 사이의 벤잘코늄 이온의 분배 계수를
결정하기 위한 새로운 생물학적 기법을 제시한다. Pseudokirchneriella
subcapitata를 이용한 표준 조류성장저해 시험에 벤잘코늄 이온을 수착할 수
있는 매질을 배지에 넣는 방법을 통해 분배계수를 얻을 수 있도록
변형하였다. 수착된 농도를 계산하기 위하여 수착매질이 없는 배지에서
얻어진 반수영향농도(EC50)와 수착매질이 포함된 배지에서 얻어진 EC50
값의 차이를 구하였다. 이 차이와 독성동태학적 모델 (toxicokinetic model)을
이용하여 조류 세포에 수착된 벤잘코늄 이온의 양을 고려한 후 자유농도를
계산하였다. 새로운 생물검정법은 민감도가 매우 높아 1 μmol L-1 이하의
자유농도와 수착제 양이온 교환 용량의 10% 미만이 수착된 수준에서 분배
계수를 결정할 수 있었다.

개발된 기법을 활용하여 토탄, 카올리나이트 및 몬트모릴로나이트에
대한 분배계수를 결정하였다. 각각의 수착제에 대한 분배계수를 양이온
교환용량으로 보정하면, 개별 화합물에 대한 얻어진 분배계수는 모두 큰
차이가 없는 것으로 나타났다. 또한 토탄 분배계수는 점토 분배계수와
비슷한 것으로 나타났다. 이는 환경 농도 수준에서 토탄의 소수성 표면이
수착과정에서 중요한 역할을 하지 않음을 의미하며 토양 성분에 대한
수착은 주로 양이온 교환으로 인한 것으로 생각된다. 알킬사슬의 길이에
따라 분배계수가 증가하는 것으로 나타났으며, 이는 알킬사슬의 길이
증가에 따라 요구되는 수화에너지가 커지는 것에 기인한 것으로 볼 수 있다.
이를 바탕으로 벤잘코늄 이온의 알킬사슬 길이와 토양구성성분을 고려한
분배계수 예측모델을 제안하였다. 이 모델은 알킬사슬의 길이가 16 및 18인
동족체에는 적합하지 않지만 대부분의 상업용 제제 및 환경에서 가장 많이
검출되는 동족체 (C12 및 C14)의 분배 계수를 설명하기에 충분하였다.

목차 (Table of Contents)

• Chapter 1. General Introduction. 1
• 1.1 General Introduction . 2
• 1.2 Research objectives. 5
• Chapter 2. Literature review: sorption of benzalkonium
• compounds to soils and soil components 6
• 2.1 Introduction. 7
• 2.2 Methods for sorption coefficient determination . 9
• 2.2.1 Batch sorption tests 9
• 2.2.2 Column chromatography . 10
• 2.2.3 Models 12
• 2.3 Sorption to clay minerals 13
• 2.4 Organic matter 16
• 2.5 Whole soils 18
• 2.6 Conclusions. 22
• Chapter 3. Application of an algal growth inhibition assay to
• determine distribution coefficients of benzalkonium ions
• between kaolinite and water 23
• 3.1. Introduction 25
• 3.2. Materials and methods . 29
• 3.2.1. Materials. 29
• 3.2.2. Methods. 30
• 3.2.2.1. Determination of cation exchange capacity 30
• 3.2.2.2. Algal growth inhibition test . 31
• 3.2.2.3. Batch sorption tests 34
• 3.2.3.4. Analysis of benzalkonium compounds . 36
• 3.3. Theory 37
• 3.4. Results and discussion . 39
• 3.4.1 Experimental determination of EC50 39
• 3.4.2 Bioassay-based distribution coefficients 43
• 3.4.3 Batch sorption tests 46
• 3.4.4 Equilibrium assumptions in growing algal cultures 49
• 3.4.5 Potentials and limitations of the bioassay-based method 52
• 3.5. Conclusion . 54
• Chapter 4. Benzalkonium ion sorption to peat and clays: relative
• contributions of charge-charge interactions and the
• molar volume effect 55
• 4.1. Introduction 56
• 4.2. Materials and methods . 59
• 4.2.1 Materials 59
• 4.2.2 Methods 60
• 4.2.2.1 Formulation of artificial soil 60
• 4.2.2.2 Determination of cation exchange capacity . 60
• 4.2.2.3 Determination of distribution coefficients 61
• 4.2.2.4 Calculation of free energy of sorption 62
• 4.3 Results and discussion 64
• 4.3.1 Algal growth inhibition test 64
• 4.3.2 Cation exchange capacity of sorbents . 65
• 4.3.3 Determination of distribution coefficients . 66
• 4.3.4 Cationic exchange capacity corrected distribution
• coefficients 69
• 4.3.5 Role of hydrophobicity in sorption. 72
• 4.3.6 Prediction of benzalkonium ion sorption to soils. 76
• 4.4 Conclusion 80
• Chapter 5. Concluding Remarks. 81
• 5. Conclusion 82
• Appendix 1. 84
• Appendix 2. 87
• References 90
• Abstract in Korean 114