BIODEGRADATION STUDIES OF BISPHOSPHONATES USING MANOMETRIC RESPIROMETRY METHOD
Akbar Jimah MSc Thesis MSc degree program in Environmental Biology University of Eastern Finland, Department of Environmental and Biological Sciences November 2019
Akbar Jimah: Biodegradation studies of Bisphosphonates using Manometric Respirometry method.
MSc thesis 57 pages, 1 appendix (1 page)
Supervisors: Eila Torvinen, PhD. University of Eastern Finland, Department of Environmental and Biological Sciences.
Prof. Jouko Vepsäläinen, University of Eastern Finland, Faculty of Health Sciences, School of Pharmacy.
November, 2019
Keywords: bisphosphonate, N10O, clodronate, biodegradation, OECD 301F
ABSTRACT
Standardised tests to investigate the biodegradability of chemical compounds are becoming progressively important in chemical risk assessment in order to meet regulations and limit guidelines such like REACH. Ready biodegradation testing is the first tier of test and the most stringent to show if a chemical is speedily degradable or not. Bisphosphonates are a class of chemicals noted for their P-C-P backbone. N10O is an example of a novel bisphosphonate compound that has found use in industry as a metal chelator in water purification while Clodronate has found use as a drug in managing hypercalcemia.
This study was designed to determine the extent of ready biodegradability of the bisphosphonate compounds – N10O and Clodronate. Manometric respirometry method (OECD 301F) was used to assess the biodegradation of N10O and Clodronate at a concentration of 50 mg/L. The measurement device was System Oxitop ® Control. N- Allylthiourea (ATU) was employed to inhibit nitrification. Activated sludge collected from Lehtoniemi Wastewater Treatment Plant, Kuopio, was left to settle and then used as an inoculum. Sodium acetate was selected as the readily biodegradable reference compound to test the functionality of the system and viability of the microbial inoculum. The test period was for 28 days. Each compound was tested thrice.
Biodegradation results are presented in relation to the Theoretical Oxygen Demand (ThOD) which is the total amount of oxygen required to oxidize a chemical completely. For N10O studies, Sodium acetate percentage biodegradation was between 83 % and 87 % on the 14th day. The toxicity test recorded a degradation of 34 %. The blank test registered Oxygen uptake of between 5.8 mg O2/l and 13.2 mg O2/l. With a final pH of 8, N10O registered a degradation of -3 % to 0.0 %. For studies of Clodronate, Sodium acetate measured a degradation of 71 % to 82 % on day 14. Toxicity test showed biodegradation of 74 % to 87 %. The blank test registered an oxygen uptake of between 8.45 mg O2/l and 12.5 mg O2/l. With a final pH of 7 Clodronate registered a degradation of between -28 % and -10 %. OECD 301F stipulates that the reference compound must attain at least 60 % degradation on day 14, this stipulation was met by Sodium acetate, an indication that the inoculum was viable, and the system functionality and procedures were in order. N10O and Clodronate could be regarded as non-inhibitory to microbial respiration as the guideline states that inhibition is true if toxicity series is less than 25 % in 14 days. The blank test conforms to the requirement that Oxygen uptake should not exceed 60 mg O2/l in the 28 days of testing. N10O and Clodronate are not readily biodegradable by manometric respirometry method as they both failed OECD 301F pass levels for readily biodegradation which is 60 % of ThOD in a 10-day window within the 28 days test period.
In the name of Allah, the Entirely Merciful, the Especially Merciful. All praise is due to God almighty, Allah, the Lord of the worlds.
I am grateful to the staff and students of the Department of Environmental and Biological Sciences, University of Eastern Finland for their assistance and support during my period of study.
My profound gratitude and sincere thanks goes especially to my supervisor, Eila Torvinen (Ph.D.). Same to Prof. Jouko Vepsäläinen, Laura Antikainen (of Savonia University of Applied Sciences) and Sirpa Martikainen for their technical assistance and guidance during the course of the research work and writing of this thesis. Their knowledge, patience, and experience contributed a lot in getting this work done.
A lot of appreciation and love goes to my family and friends for their chiding words of encouragement towards completing my Masters’ degree program.
ATU N-Allylthiourea
BOD Biochemical Oxygen Demand
BPs Bisphosphonates
CaCl2 Calcium chloride
CAS Chemical Abstract Services
CBOD Carbonaceous Biochemical Oxygen Demand
CO2 Carbon dioxide
COD Chemical Oxygen Demand
FeCl3·6H2O Iron (III) chloride hexahydrate K2HPO4 Potassium hydrogen phosphate KH2PO4 Potassium dihydrogen phosphate MgSO4·7H2O Magnesium sulphate heptahydrate MRT Manometric Respirometry Test
MW Molecular weight
N – BP Nitrogen containing bisphosphonate
N10O 11-amino-1-hydroxyundecylidene-1, 1-bisphosphonic acid Na2HPO4·2H2O Sodium hydrogen phosphate dihydrate
NaOH Sodium hydroxide
NBOD Nitrogenous Biochemical Oxygen Demand
NN – BP Non-nitrogen containing bisphosphonate
OECD Organisation for Economic Co-operation and Development
OUR Oxygen Uptake Rate
RBT Ready Biodegradation Test
REACH Registration, Evaluation, Authorization and Restriction of CHemicals ThOD Theoretical Oxygen Demand
CONTENTS
1. INTRODUCTION ... 6
2. LITERATURE REVIEW ... 8
2.1 CHEMICALS AND THE ENVIRONMENT ... 8
2.2 BIODEGRADATION: DEFINITION AND TYPES ... 9
2.2.1 Factors affecting biodegradation reactions ... 10
2.2.2 Measurement and estimation of biodegradation rates ... 11
2.2.3 Ready Biodegradation Test (RBT) ... 12
2.2.4 OECD 301F – Manometric Respirometry Test and Biochemical Oxygen Demand (BOD). 14 2.3 BISPHOSPHONATES ... 16
2.3.1 Structure and Chemistry ... 16
2.3.2 Classification of Bisphosphonates ... 17
2.3.3 Pharmacokinetics ... 17
2.3.4 Application of Bisphosphonates ... 18
2.4 N10O... 19
2.5 CLODRONATE ... 21
2.6 BIODEGRADATION OF BISPHOSPHONATES ... 22
3. AIM OF THE STUDY ... 24
4. MATERIALS AND METHODS ... 25
4.1 PRINCIPLE OF MRT ... 25
4.2 CLOSED RESPIROMETER ... 26
4.3 PREPARATION OF MINERAL MEDIUM FROM STOCK SOLUTION ... 27
4.4 PREPARATION OF BOD FLASKS ... 27
4.5 INOCULUM ... 28
4. 6 PROCEDURE... 29
4.7 CALCULATIONS AND EXPRESSION OF RESULTS OF BIOLOGICAL OXYGEN DEMAND, THEORETICAL OXYGEN DEMAND AND PERCENT DEGRADATION ... 30
5. RESULTS ... 32
5.1 BOD28 ATU RESULTS FOR N10O... 32
5.2 BIODEGRADATION OF N10O ... 34
5.3 BOD28 ATU RESULTS FOR CLODRONATE ... 36
6. DISCUSSION ... 43
7. CONCLUSION ... 47
REFERENCES ... 48
APPENDIX I ... 1
1. TESTING FOR MOST EFFECTIVE INOCULUM TREATMENT ... 1
1. INTRODUCTION
The high standard of living enjoyed in modern industrialised societies today is mainly as a result of the persistent efforts of chemists in their ever-increasing scale of chemical production.
This enormous productivity has led to increased food yield due to fertilizer use, improved health and extended life resulting from pharmaceuticals (Manahan, 2006). This rapid development and growing production of new chemicals has become a matter of concern in respect to their adverse effects on the environment; through deliberate or accidental release of pollutants and toxic substances into the environment and high persistent, accumulation potential and distribution pattern of the chemicals (Vighi and Calamari, 1993).
Regulations have been developed to assess the impact of these chemicals as well as to minimise the release of persistent chemicals to the environment. Regulation, Evaluation, Authorization and Restriction of CHemicals (REACH) is one of such standards designed by the European Union. Risk assessment and comparison to persistence criteria could then be modelled from the data derived from this type of evaluations (European Commission, 2006).
Biodegradability, which according to Kaiser (1998) is a measure of how much a compound can be broken down by microbial actions under specified conditions, is an essential element in environmental fate assessment of chemicals. Determination of biodegradability based on standardized methods is usually performed experimentally using a tiered approach. The first are the Ready Biodegradation Test (RBTs). At this level, the conditions to determine biodegradability of a compound are very stringent. One such test in the RBTs, is Organisation for Economic Co-operation and Development (OECD 301F) manometric respirometry technique.
Bisphosphonates (BPs) are synthetic compounds characterised by a P-C-P backbone. The first of this type of compounds was produced in 1865 in Germany (Fleisch, 2001). Their initial use was in industries – as water treatment agents, application in detergent and bleaching formulations, anti-scaling agents etc. In recent times, their application has become more pronounced in medicine as drugs administered for post-menopausal and glucocorticoid- induced osteoporosis, tumour bone diseases amongst others (Widler et al., 2012). Clodronate which is a type of non-nitrogen containing class of bisphosphonate has use in Paget’s disease of bone and malignant hypercalcaemia (Muratore et al., 2011). N10O on the other hand is a novel bisphosphonate that has potential use in industry for metal ions removal from aqueous
solutions (Turhanen et al., 2015). Due to their vast applications (in both medicine and industry) and the fact that they are not excluded from the risk posed by chemicals to the environment, there is the need to investigate their biodegradation. Besides, there seems to be no known literature currently on first tier of biodegradation analysis of both bisphosphonate compounds.
The aim of this study was to determine the level of biodegradation of the BPs compounds - N10O and clodronate; using activated sludge as the inoculum in a Manometric Respirometry Test (MRT) based on OECD 301F guidelines.
2. LITERATURE REVIEW
2.1 CHEMICALS AND THE ENVIRONMENT
Chemicals have been very beneficial to modern society, whether in medicine (as pharmaceuticals), agriculture (in the form of fertilizers and herbicides) or industry (example being lubricants). At the same time, certain chemicals whether deliberately or by accident have been quite harmful to humans - causing sufferings or even death in some cases; or becoming a source of pollution to the environment (European Commission, 2001).
Chemical pollution of the environment (whether groundwater or soil) has become a global problem. Kümmerer (2009) reported detecting pharmaceuticals, cosmetics and personal care products in virtually all environments. The use, release and persistence of chemicals is the major source of this pollution. According to Vighi and Calamari (1993) there is the potential for chemicals to accumulate in food chain and cause harm to the biota of affected habitats.
Policies, standards and limits are being legislated to ensure a maximum level of protection for human health and the environment. It has become crucial to understand the ecotoxicological profile of chemicals for ecological risk assessment and in the event of any disasters.
Registration, Evaluation, Authorization and Restriction of CHemicals (REACH) regulations which is enforced in the European Union since July 2007 is an example of such regulations geared towards improving human health and environmental safety posed by the dangers from chemicals and their use (Brown et al., 2018).
Chemical biodegradation analysis is a central component of the REACH regulations in conducting environmental risk assessment of chemicals (Williams et al., 2009). According to Kayashima et al. (2014) biodegradation is conceived to be the major route by which chemicals are eliminated from the environment; hence it plays a significant role in telling the environmental fate of chemicals.
Even though biodegradation remains the most crucial mechanism for the disappearance of organic compounds from the environment, abiotic degradation such as photolysis, oxidation, volatilization and hydrolysis do also lead to the removal of some organic chemicals (Petruzzelli et al., 2010). Elimination of compounds from the environment could also be due to adsorption
and complexation which are physical mechanisms rather than biological or photochemical degradation (Kümmerer, 2009).
2.2 BIODEGRADATION: DEFINITION AND TYPES
Biodegradation is defined as series of steps that eventually lead to the breakdown and transformation of organic materials due to the activities of microorganisms (Pepper et al., 2015).
According to De Wilde (2013) oxygen availability decides the type of molecules that organic carbon (substrate) is broken down to. In the presence of Oxygen (aerobic conditions) carbon is converted to carbon dioxide and water, as illustrated in equation (1), whereas in anaerobic situations (where oxygen is absent) shown as equation (2), transformation of carbon leads to the formation of carbon dioxide and methane. In both conditions however, organic carbon is partly incorporated into the microbial cellular constituent to form a new biomass. There is a chance that not all organic residue undergoes mineralization; hence the production of residual carbon or mineral salts (metabolites).
C organic material CO2 + H2O + C biomass + C residual (1) C organic material CO2 + CH4 + C biomass + C residual (2) Zieliñski and Jakóbczyk-Baraniecka (2001) have described biodegradation as the most effective mechanism in eliminating organic materials from the environment. Prokkola (2015) supports this by asserting that it is “nature’s own way of material disposal and the most important indicator of how environmentally friendly a compound is”.
Biodegradation is a stepwise reaction where specific enzymes catalyse the reaction pathway.
These enzymes maybe produced from within the cell or could be extracellular enzymes which are excreted outside the cell. In the absence of an appropriate enzyme in any step of the pathway biotransformation comes to a halt, leading to the persistent of the compound under “microbial attack”. This is very peculiar to compounds that have strange structural arrangements, hence microorganisms have yet to evolve the appropriate catabolic enzyme for such biodegradation pathway (Cao et al., 2009).
Microbial actions Microbial actions
anaerobic aerobic
The mechanism of biodegradation of chemicals which is the potential of microorganisms to transform organic molecules into smaller molecules as a result of microbial activities is divided into two types: the complete biodegradation (ultimate biodegradation) and the incomplete biodegradation (or partial biodegradation).
Pepper et al. (2015) explained that complete biodegradation, which is also referred to as mineralization, is the oxidation of organic compounds to yield carbon dioxide and water;
during which carbon and energy is released and used for cellular growth and reproduction.
Incomplete or partial degradation on the other hand occurs when a compound is transformed by microbial action to some other organic substance but not carbon dioxide due probably to lack of appropriate enzymes. A type of partial biodegradation is co-metabolism, where energy produced from oxidation of organic compound is not used for cellular growth. Co-metabolism occurs when the cell produces enzymes that are non-specific hence possessing ability to degrade compounds only in the presence of other compounds which act as microbial substrate.
Partial degradation may also be in the form of polymerization where the resultant intermediate metabolites from initial stages of the degradation process becomes more complex and highly reactive. This results to the formation of stable complexes that do not degrade due to the specific cellular or extracellular enzymes (Pepper et al., 2015).
2.2.1 Factors affecting biodegradation reactions
The attempt to break down organic substance by microorganisms in order to source for carbon (substrate) and energy for themselves is affected by certain factors. These factors are those related to the compound under microbial degradation and the factors relating to the environment where such microbes are found (Joutey et al., 2013; Eskander and Saleh, 2017).
A compound’s degradability depends on its molecular structure and most importantly the presence of a functional group. The structural nature of a compound i.e. steric effect has direct influence on its accessibility of material for enzyme-catalysed cleavage. Branching or functional group presence can slow down biodegradation by either changing the biodegradation chemistry or slowing down cell membrane transport. Toxicity of the compound is also a critical factor in biodegradation of chemical materials. A compound toxic to microbial population would have an inhibitory response to microbial population growth, function and activity.
Electronic effects of a compound are mainly caused by functional groups of the compound (Pepper et al., 2015).
In respect of microorganisms and their environment Madigan et al. (2010) posits that microbial survival and activity is highly dependent on the nutritional requirements of microbes. Organic matter being the main source of carbon for microorganisms, is a great influence on microbial occurrence and abundance. Various physical and chemical factors that relate to the environment could also limit or hinder biodegradation as they are essential for microbial activities. The most important of these are oxygen availability, temperature, pH, salinity and water activity. Biodegradation is more likely to occur and at a faster rate due to increased microbial activities; which in turn is determined by the optimal availability of these environmental (physical-chemical) requirements. Environmental conditions are so critical that they define the type of microorganisms inherent or inhabiting them.
2.2.2 Measurement and estimation of biodegradation rates
It is important to measure at standard conditions the rate of biodegradation of compounds in the environment. The goal of estimating the rate of biodegradation in the laboratory is to have data that would enable researchers and experts make predictions as to the probability of such a chemical degrading in a natural environment. To ensure reproducibility and comparison of such results amongst laboratories, various guidelines and standards for biodegradation analysis have been proposed, introduced and published by both national and international standardization bodies and organisations. These guidelines and standards vary in definitions, classifications, testing conditions and pass levels. These differences in text and guidelines stem from difference in geographical locations, environmental laws, economic situations and policies of the various regulatory or standardization bodies. These guidelines have occasionally undergone some modifications. (Klaus, 1998).
According to Mistriotis et al., (2014), examples of the most prominent of these references for biodegradation testing have been developed by International Organisation for Standardization (ISO), Organisation for Economic Cooperation and Development (OECD), the Japanese Ministry of International Trade and Industry (MITI), the United States Environmental Protection Agency (USEPA) and American Society for Testing and Materials (ASTM).
OECD strategy for measuring biodegradation is a tiered approach, which involves series of tests in each level. The three tiers of OECD biodegradation estimation are: Ready Biodegradation Test (RBT), Inherent Biodegradation Analysis and Simulation Tests (Mei et al., 2015). The rigorousness of a measurement decreases with an increase in complexity and environmental reality. It ensures that chemicals can be categorised based on how they degrade, either rapid ultimate degradation or possessing the potential to degrade.
RBTs are the first tier of test and the most stringent to show if a chemical is speedily degradable or not. They are referenced as OECD 301. Any compound that passes this test is assumed to undergo ultimate degradation releasing non-toxic products (Goodhead et al., 2014).
Dick et al. (2016) concluded that the RBTs do have some limitations and do not always give a correct image of biodegradation. Hence a negative result in an RBT test does not absolutely rule out biodegradation of such a compound. Rather, the compound is then tested under more relaxed conditions such as increased biomass, longer incubation periods, pre-adapted inoculums, etc. OECD refers to this level of testing as Inherent Biodegradation Test and referenced as OECD 302 guidelines. It is important to understand that, some researchers (Toräng and Nyholm, 2005; amongst others) have referred to this tier of test as “enhanced RBTs”. Inherent biodegradation test increases the chances of a compound being defined as biodegradable compared to RBT. If a chemical produces a negative result in an inherent test it is an indication of its potential for environmental persistence (Seyfried et al., 2015).
According to Pagga (1997) and Thousand et al. (2011) simulation test, which are the highest tier of biodegradation testing under OECD standards, are designed such as to make a laboratory system mimic the aerobic stage of a wastewater treatment plant (WWTP) or an environmental compartment. This level of testing is the most reliable in terms of information about the actual degradation rate of a compound under environmentally relevant conditions, although it is the most expensive and complex to set-up. Simulation testing is referenced as OECD 303. The type of simulation test to be carried out on a compound is dependent on the potential receptor environment of concern (WWTP, soil, sediment, surface water).
2.2.3 Ready Biodegradation Test (RBT)
The principle of RBTs according to OECD (1992) is that a solution or suspension of a test compound in a mineral medium is inoculated and incubated under aerobic conditions in the
dark or diffused light. Compared to the amount of organic carbon, the amount of dissolved organic carbon (DOC) in the test solution is kept at its barest minimum. Allocation is made for the endogenous activity of the inoculum by running series of blanks with inoculum only (i.e.
without the test substance). A reference compound (sodium acetate; whose ready biodegradation is already confirmed) is run parallel to ascertain the functionality of the procedures and the viability of the inoculum. Degradation is determined by various parameters (such as DOC, CO2 production or Oxygen consumption). These parameters are measured frequently at adequate intervals in order to identify the start and termination of biodegradation.
The test has a duration of 28 days.
There have been reported criticisms and limitations with the OECD 301 batch of RBTs standardizations. Kowalczyk et al. (2015) in a detailed review proposed a complete refinement of the entire OECD test technique, arguing that the methods in their current version have major flaws. They cited problems of inoculum source, concentration and preparations. They suggested better inoculum characterization and standardization as a measure to curb high variability in biodegradation results. Vázquez-Rodríguez et al., (2011) had previously highlighted these challenges with the OECD 301 tests. Chemical concentration plays a crucial role in biodegradation kinetics. Vázquez-Rodríguez et al., (2011) and Kowalczyk et al. (2015) recommend that the chemical concentration should better reflect environmental reality where observed chemicals in WWTP are usually within the range of ng/l to µg/l rather than the 10 – 40 mg of carbon/litre currently applied in OECD RBTs. As for inoculum type, noting that microbial inocula used in RBT test are the most poorly controlled variable, the use of biofilms was suggested because of its heterogenous nature. In terms of the test duration, where currently it is 28 days, within which there is the 10-day window period in which biodegradation must take place, they argued that these are arbitrary test durations that do not give full account of biodegradation in the environment. Battersby (2000) shares the same opinion.
There is a case for the use of high throughput techniques as an emerging tool for biodegradation. The disadvantage of this is the rapidly changing nature of the ‘omics’ field which will require constant review and development of standards which is quite a costly process (Arora and Shi, 2010; Arora and Bae, 2014).
François et al. (2016) observed that the data obtained from RBTs was limited to a specific type of environment and this gives a narrow and “variable” view of biodegradation. The quantity and complexity of environmental variables are not addressed in RBT level of testing. They
went on to propose the ProbaBio approach (Thousand et al, 2011) claiming that makes biodegradation testing possible under more realistic environmental conditions.
RBTs which are the preliminary tests for estimating biodegradation have been the central foundation of regulatory frameworks for long. They are noted for their ease of use and rather simple interpretation. Four analytical methods have been used by OECD to develop its battery of six RBT tests which are set under the OECD 301(A – F) protocol for testing ready biodegradation. The choice of method to be used in determining the ready biodegradability of a compound depends on the physicochemical properties, especially solubility, volatility and adsorptivity of that chemical (OECD, 1992) (Table 1).
Table 1: Appropriateness of a test method (OECD, 1992)
Test Analytical
method
Suitable for compounds that are
Reference Poorly
soluble Volatile Adsorbing DOC Die-Away OECD 301 A Dissolved organic
carbon - + +/-
CO2 Evolution OECD 301 B Respirometry:
CO2 evolution + - +
MITI (I) OECD 301 C Respirometry:
oxygen consumption
+ +/- +
Closed Bottle OECD 301 D Respirometry:
dissolved oxygen +/- + +
Modified OECD screening
OECD 301 E Dissolved organic
carbon - - +/-
Manometric Respirometry
OECD 301 F Oxygen
consumption + +/- +
2.2.4 OECD 301F – Manometric Respirometry Test and Biochemical Oxygen Demand (BOD).
Manometric Respirometry Test (MRT) is based on the premise that a measured volume of mineral medium containing a specified concentration of the substance under investigation as the only source of organic carbon is inoculated and kept stirring in a closed flask at a constant temperature for 28 days. During this period, oxygen is consumed while evolving carbon dioxide by the microbial respiration. The released carbon dioxide is absorbed in a solution of potassium hydroxide or a similar salt e.g. Sodium hydroxide. The quantity of oxygen consumed
is determined by measuring the drop-in pressure or volume required to maintain constant gas volume. The rate of biodegradation of the test substance is obtained by subtracting the endogenous oxygen uptake, which is a parallel batch of the test, and then expressed in ratio to the theoretical oxygen demand (ThOD) or less preferably Chemical Oxygen Demand (COD) of the compound under study (OECD, 1992).
Respirometry is a measurement and interpretation of the biological oxygen consumption under well-defined experimental conditions. The respiration rate is usually measured with a respirometer. This measurement is usually manometric or volumetric. Manometric method measures the pressure change in the headspace of a reaction vessel. On the other hand, volumetric method measures the weight or volume of the displaced liquid then uses it as an indication of the amount of gas produced (Spanjers and Vanrolleghem, 2016).
The pressure changes are measured by a manometer and converted to oxygen consumption by the device to estimate the Biochemical Oxygen Demand (BOD) value. There are two classes of manometric systems based on how change in pressure is measured: those that use mercury barometer on a graduated scale (such as Velp Scientifica) and those that use pressure sensor (such as OxiTop ®, BOD EVO and Sapromat). These manometric devices are noted for their simplicity and ability to allow measurement of large levels of carbon compounds without dilution (Jouanneau et al, 2014).
Oxitop ® kit manufactured by WTW (Weilheim, Germany) is reported by Brown et al (2018) to be the most popular of the manometric respirometry equipment in use by contract research organisation. It determines the oxygen consumption in the closed respirometers via semi continuous pressure measurements.
Manometric Respirometry test which is a widely used RBT is noted for its reproducibility, precision and accuracy (Reuschenbach, Pagga and Strotmann, 2003). Stasinakis et al. (2008) credits this technique over other RBTs because it measures a direct biological parameter of aerobic degradation rather than indirect conclusions such as measuring dissolved organic carbon. There is a possibility of providing big data as 360 values in 28 days are collected in a spreadsheet format which can be used to calculate the first order kinetics in the biodegradation phase as proposed by Boethling and Lynch (2007).
BOD is a measure of the dissolved oxygen consumed by microbes during oxidation of reduced substances in water and waste, at a fixed temperature and over a period. The typical sources of BOD are organic carbon that is Carbonaceous Biochemical Oxygen Demand – CBOD and Nitrogenous Biochemical Oxygen Demand – NBOD (Penn, Pauer and Mihelcic, 2009;
Roppola, 2009). According to Jouanneau et al (2014) one of the applications of BOD is that it gives an indication of the biodegradable fraction of the effluent in a WWTP by obtaining the ration between BOD and COD. In addition, it is one of the most widely used criteria for water quality assessment and a crucial environmental index for monitoring organic pollutants or pollution in wastewaters. Microbial (activated sludge) respiration has a direct relationship with BOD and rate of biodegradation (Roppola, 2009).
2.3 BISPHOSPHONATES 2.3.1 Structure and Chemistry
Bisphosphonates (BPs) are chemical compounds that have two phosphonates (PO(OH)2) groups and are renowned for the common P–C–P “backbone” they possess. They are reported to have been first manufactured in Germany at about 1865. They have also been referred to as gem-diphosphonates (Abdou and Shaddy, 2009) albeit erroneously, according to Fleisch (2000).
Bisphosphonates are the synthetic analogues of pyrophosphate which is a naturally occurring compound. The difference is that bisphosphonates are chemically and enzymatically stable, and rather than the P–O–P structure of pyrophosphate, they have the P–C–P skeleton without any alkyl side chains (Figure 1) (Aderibigbe et al., 2016).
Figure 1: Structure of bisphosphonate (left) and pyrophosphate (right) O = P C P = O
HO OH
HO OH
R1
R2
O =P O P = O
OH OH
OH OH
The stability of BPs is attributed to the two phosphonate groups (PO3) that are linked to the central carbon atom. Attached to the central carbon atom are two covalently bonded groups referred to as side chains. The short side chain (R1) sometimes called “the hook” and the long side chain (R2) (Papapoulos, 2006). The stereochemistry of the side chains and the phosphonate groups determines resistance, chemical properties, mode of action, efficacy and biological activity of bisphosphonate compounds (Ballantyne, 2015).
The nature of the P–C–P skeleton has made it possible for this chemical group to undergo a lot of alterations. These alterations are usually mostly a change in the lateral chains on the carbon or esterification of the phosphonate groups. This high level of possibility to change the stereochemistry of BPs has led to possible variations in types and ability to create novel bisphosphonate compounds. Alterations in their structural configuration leads to a disparity as regard the physicochemical, biological, therapeutic and toxicity properties of bisphosphonate compounds. This variability in properties of BPs is responsible for their wide range of applications and use. Another angle to this variability is that since each bisphosphonate compound has a unique physicochemical and biological characteristic, it is somewhat dangerous to extrapolate the results of one compound to another, especially in terms of mode of action (Fleisch, 2001).
2.3.2 Classification of Bisphosphonates
There are two classes of bisphosphonate compounds; the Non-nitrogen containing bisphosphonates (NN-BP) which include e.g. clodronate, etidronate and tiludronate. The other class is the Nitrogen containing bisphosphonates (N-BP) e.g. N10O, zoledronate, ibandronate and risedronate. While the N-BP have at least one nitrogen atom in their R1 or R2 groups, the NN-BPs possess none. This seemingly minute difference is responsible for the difference pertaining to their mechanism of action, chemical structure, potency, delivery, dosage, bioavailability and half-life (Cremers and Papapoulos, 2011; Ballantyne, 2015).
2.3.3 Pharmacokinetics
Lin (1996) reported that the elimination of BPs from plasma (when taken in form of an intravenous drug) is very rapid; with a half-life of 1-2 hours. Oral absorption of bisphosphonate drugs on the other hand is usually about 1 – 3%, which is quite low. This is due to BP
hydrophilicity being a negatively charged ion at physiological pH (6 – 8), large molecular sizes and chelation with Ca2+ (Giger et al., 2013).
Drake et al. (2008) have explained that the unabsorbed BP has a very high affinity to the bone tissues and is usually absorbed on bone surface. However, 50% of the bisphosphonate that is taken in whether orally or intravenously is excreted by the kidneys unchanged, i.e. without being metabolised.
2.3.4 Application of Bisphosphonates
Until the 1960s, BP compounds were not used in the field of medicine for which they have become popular today, rather they had their use in industry as corrosion inhibitors and complexing agents in textile, fertilizers and oil industries. They were also used as “water softeners”, i.e. to prevent scaling in industrial and domestic water installations (Russell, 2006).
Bisphosphonates became a major class of pharmaceuticals and are still rapidly growing after it was discovered that unlike its natural analogue pyrophosphate it was metabolically stable and could also inhibit calcification in bones (Abdou and Shaddy, 2009; Chmielewska and Kafarski, 2016).
Medically, for the treatment of bone disease, BPs have reported profound results. As reported by Chapurlat and Delmas (2006) Osteoporosis is an asymptomatic condition involving weakening of the bones and making them fragile and therefore more likely to break. BPs drugs have become the most common drug for this condition which is considered a major health challenge affecting mainly postmenopausal women.
BPs are the primary drugs used for Paget’s disease of the bone. They are sometimes used in combination with analgesics. Paget’s disease of the bone is a disease that can cause pain, fractures and skeletal deformities in some patients and exhibit no symptoms in others as new bone tissues gradually replace old ones (Reid and Hosking, 2011).
Bisphosphonates drugs have been reported to have both direct and indirect anti-cancer activity.
By direct effects, BPs have been found to inhibit proliferation and adhesion of tumour cell, and have induced apoptosis in myeloma, prostate and breast cancers whereas indirectly they have exhibited inhibition of angiogenesis (Gnant and Clézardin, 2012). This anticancer activity has made BPs very important and reliable in treating cancer conditions related to bones. Mhaskar
et al. (2010) reports that clodronate is the standard drug for osteolytic bone disease involving multiple myeloma (a type of cancer). Since the 1980s, BPs have been used to treat bone metastasis resulting from breast and prostate cancer (Costa and Major, 2008). Hypercalcemia of malignancy is a condition that affects 30% of cancer patients where increased bone resorption by osteoclasts leads to excessive release of calcium to the blood. BPs have been found to be the most effective drugs in managing this condition (Stewart, 2005).
Another medical significance of bisphosphonates is their anti-inflammatory effects. Iannitti et al. (2013) reports that BPs can affect the production of cytokines and change the molecular expression associated with the immune processes and anti-inflammatory response. There are conflicting data on this effect (Alanne, 2014).
Bone scintigraphy, a method for bone disease diagnosis (especially for metastatic analysis and therapy) that involves scanning bones with radiochemical is another medical field where BPs have found a niche in their application. Palma et al. (2011), reports that BP have been used as radiopharmaceuticals for single photon emission computed tomography (SPECT) imaging for over 30 years.
Martin et al. (2001) have reported some findings of BP use in the treatment of protozoan parasitic diseases. Risedronate (a N – BP) was found to have been effective against Plasmodium falciparum, Trypanosoma cruzi, and Toxoplasma gondii. However, this research path into BP use in medicine is still very much at a developmental stage.
As for the non-medical use of BP, the chelation capability of BP has been exploited positively to chelate metal ions thereby producing stable complexes (Matczak-Jon and Videnova- Adrabińska, 2005). This understanding is very useful in mineral purification, wastewater purification, and other industrial process demanding complexing of metal ions (Turhanen et al., 2015). There has been renewed interest to investigate the use of BPs in agriculture as a form of herbicide (Kafarski et al., 2000).
2.4 N10O
11-amino-1-hydroxyundecylidene-1,1-bisphophonic acid, N10O for short with the molecular formula of C11H27NO7P2·H2O (Figure 2) is one of ten aminobisphosphonates compounds
synthesized in 2012 in the University of Eastern Finland, under the supervision of Prof. Jouko Vepsäläinen (Alanne, 2014).
Figure 2: N10O. Structural formula (left). Zwitterionic form (right)
N10O is registered with Chemical Abstract Services (CAS) with Registry Number: 97815-71- 9. It is a bisphosphonate compound with a carbon chain length of 10. It has an aqueous solubility of 58 mg/l at 21 ºC which makes it a poorly soluble compound (Alanne et al., 2012).
The chain length is responsible for its hydrophobicity hence it holds no potential as a drug, although it is proving to be very promising for industrial application and this is due to its high chelating and complexing abilities.
In a study conducted by Alanne et al. (2013a), N10O proved most effective (amongst other BP compounds) in the removal of chromium (III) ions from water solutions and tannery effluents by its absorption of the metal ions.
Hydrogels are products that are made of polymeric materials, whose hydrophilic structure affords them the capacity to hold large amount of water in their three-dimensional networks.
They have found application in hygienic products as sealing, drug delivery systems, coal dewatering, biosensors etc. (Ahmed, 2015). Alanne et al. (2013b) reported that N10O was found to have potentials as a good candidate acting as an hydrogelator.
N10O enhanced to a large extent the plant shoot nickel ion removal thereby increasing the shoot biomass. It was further able to enhance shoot zinc removal in a zinc spiked soil as well as lead ions removal in a clean soil, although the researchers could not understand the mechanism of action (Alanne et al., 2014).
The ability of N10O to be applied in water treatment for chelation and metal ions removal was also tested (Turhanen et al., 2015). N10O was able to complex and collect metal cations from
ground water and mining site and it proved effective in the chelation of solutions with high magnesium and calcium contents.
The applicability of N10O in agricultural field was recently investigated by Salam et al. (2019).
The effect of lime and N10O amendment of soil on growth attributes and copper, nickel, and zinc accumulation in plant tissues were studied. The researchers found that the addition of lime and N10O improved growth parameter values and photosynthesis parameters and concluded that their results indicated that N10O may have fertilizing properties.
Based on these studies, and with such promising results so far, the prospect of N10O as a candidate for a variety of industrial application is not in doubt.
2.5 CLODRONATE
Clodronate has the molecular formula of CH2Cl2Na2O6P2·4H2O. It is chemically known as dichloromethylene-bisphosphonate disodium tetrahydrate (Figure 3). Clodronate has a CAS Registration Number: 88416-50-6 (European Chemical Agency, 2019). It is miscible in water with a solubility value of 20.8 mg/l (Santa Cruz Biotechnology, 2011). Clodronate is a first- generation non-nitrogen containing bisphosphonate compound (NN-BP). Frediani and Bertoldi (2015) referred to it as the father of bisphosphonates highlighting the position it holds amongst this class of compounds and that it is a widely and thoroughly studied BP that has been used as a drug since the 1960s.
Figure 3: Structure of Clodronate
Clodronate is majorly used as a medicine. It is the primary drug for osteoporosis treatment due to its anti-resorptive activity. Clodronate mechanism of action in tackling osteoporosis is by
producing a certain ATP analogue that blocks mitochondrial oxygen utilization hence leading to apoptosis (Frith et al., 2001; Lehenkari et al., 2002). It is also noted for its anti-cancer chemotherapeutic and anti-angiogenic activities (Soltau et al., 2008).
2.6 BIODEGRADATION OF BISPHOSPHONATES
The vast usage of bisphosphonate compounds which have been previously highlighted and the pharmacokinetics (being that 50% is excreted unchanged) should be good motivation to understand bisphosphonate biodegradation profile.
Phosphonates when naturally formed are simple compounds with a stable carbon – phosphorous (C – P) bond. In nature bacteria plays a key function in biodegradation of phosphonates. Phosphonates are found in different organisms, from plants to prokaryotes, bacteria, fungi and insects. Bacteria have evolved the ability to breakdown this naturally occurring phosphonates by cleaving the C – P bond and then utilizing phosphonates as a source of phosphorous for growth. Aminophosphonates can also be used as a nitrogen source by some bacteria (Nowack, 2003).
For these simple and partly naturally occurring phosphonates, there exist several studies on the biodegradability of their C – P bonds. As for their industrial counterparts which are more complex, carry a negative charge and are much larger only a few studies are available. The reason for this besides the difference in structure is that microbes have only been in contact with these complex chelating compounds for such a short time that they are yet to evolve the appropriate enzyme needed to efficiently degrade these industrially synthesized compounds (Rott et al., 2018).
According to Rott et al. (2018) 1-hydroxyethane 1,1-diphosphonic acid (HEDP) and 2- phosphonobutane1,2,4-tricarboxylic acid (PBTC) are the most important polyphosphonates compounds. Biodegradation tests using sludge from municipal sewage treatment plants did not indicate any biodegradation on HEDP (Nowack, 2003). Using OECD 301F test method no consumption of oxygen was reported for PBTC and HEDP (Rott et al., 2018).
That notwithstanding, Fleisch, (2001) points out that BP structural activity and classification are unique for each compound and their chemistry is a function of this structural arrangement.
It behoves us still to study N10O (which is a N – BP with prospect in industrial application)
and clodronate (an NN – BP of enormous medical relevance), especially as there is a dearth of literature on their ready biodegradation status.
3. AIM OF THE STUDY
The main aim of the study was to investigate the biodegradation of bisphosphonates.
The detailed aim was by using OECD 301Fmanometric respirometry method to analyse the ready biodegradability of bisphosphonates compounds – N10O and Clodronate employing settled activated sludge as the source of inoculum.
4. MATERIALS AND METHODS
4.1 PRINCIPLE OF MRT
OECD 301F: Manometric Respirometry Test (OECD, 1992) was selected as the most appropriate method of the RBTs due to the solubility status of the test compounds under investigation.
MRT operates on the principle that a measured volume of inoculated mineral medium, containing a known concentration of the test substance as the only source of organic carbon, is stirred at a constant temperature for 28 days. The microbes from the inoculum consume oxygen in the flask, which leads to carbon dioxide being released. The released carbon dioxide is absorbed by NaOH pellets. The oxygen consumption leads to a change in pressure which is required to maintain constant gas volume in the respirometer. The change in pressure in the flask is converted to BOD using the equation (3):
𝐵𝑂𝐷 = 𝑀(𝑂2)
𝑅×𝑇𝑚 × (𝑉𝑡𝑜𝑡− 𝑉𝑖
𝑉𝑖 + 𝛼𝑇𝑚
𝑇0) × ∆𝜌(𝑂2) (3) Where
M(O2) = Molecular weight of oxygen (32000 mg/mol) R = Gas constant (83.144 L·mbar/mol·K)
T0 = reference temperature (0 OC = 273.15 K) Tm = measuring temperature (20 OC ±2 = 293.15 K) Vtot = bottle volume (theoretical volume) [mL]
Vi = volume of sample
α = Bunsen absorption coefficient (0.03103)
Δρ(O2) = the difference of the partial oxygen pressure [hPa]
4.2 CLOSED RESPIROMETER
Manometric respirometry tests were carried out using Oxitop ® Control System (WTW, Weilheim, Germany). This system is composed of an Oxitop ® OC110 Controller (Figures 4 and 5) which is the interface between the measuring heads and the computer system, Oxitop
®-C measuring heads which are fitted to the BOD flasks contain the respirometer and measures the values automatically and a stirrer IS 12 which holds the BOD flasks in the incubator and make sure they allow for continuous and automated stirring of the content of the bottles with the magnets inserted.
Figure 4. The Oxitop ® OC 110 controller and Oxitop ® - C measuring heads.
Figure 5. BOD bottle fitted with Oxitop® - C measuring heads (left) and BOD bottles fitted with Oxitop ® - C measuring heads placed on the stirrers IS 12 in the incubator (right).
4.3 PREPARATION OF MINERAL MEDIUM FROM STOCK SOLUTION
Based on the guideline of OECD 301F (OECD, 1992), mineral medium was prepared from four stock solutions (A, B, C and D) using analytical grade chemicals.
To a flask filled with 800 ml of deionized water, Solution A (KH2PO4, K2HPO4, Na2HPO4·2H2O, NH4Cl), Solution B (CaCl2), Solution C (MgSO4·7H2O) and Solution D (FeCl3·6H2O) were added (Table 2), and the volume was then adjusted to 1000 ml with deionized water.
Table 2: Preparation of Stock solution and Mineral medium (OECD, 1992)
Solution Chemical Stock Solution Mineral medium
mass (g) volume (l) volume (ml) total volume (ml)
A
KH2PO4 8.50
1 10
1000 K2HPO4 21.75
Na2HPO4·2H2O 33.40
NH4Cl 0.50
B CaCl2 27.50 1 1
C MgSO4·7H2O 22.50 1 1
D FeCl3·6H2O 0.25 1 1
4.4 PREPARATION OF BOD FLASKS
Each analysis consisted of four set-ups: the “blank” series containing only inoculum in the mineral medium, the “reference” series containing readily biodegradable sodium acetate as the only carbon source in mineral medium with inoculum, the “toxicity control” containing the test compound and sodium acetate in mineral medium with inoculum and the actual “test” series, which was the compound under investigation as the only source of carbon in mineral medium containing inoculum.
4.4.1 Test compounds
The test compounds, - N10O and Clodronate, were provided by the research group of Professor Jouko Vepsäläinen of University of Eastern Finland.
The test compounds were dispersed directly to the final volume of mineral medium (1000 ml) to give a test concentration of 50 mg/l. Three test vessels were used for this analysis in each experiment.
The solubility of the compounds was a critical factor in selecting the type of analysis. Both compounds are poorly soluble in water. N10O has a solubility of 58 mg/l while Clodronate has a solubility value of 20.8mg/l.
4.4.2 Reference compound
Sodium acetate (C2H3NaO2) which meets the criteria for ready biodegradability was used as a reference compound to ensure the functionality of the test system, inoculum viability and procedure. The same concentration as test substance (50 mg/l) was used for the analysis. Four test vessels were used as replicates.
4.4.3 Blank series
The blank parallel was made to create allowance to check for the endogenous activity of the inoculum. This series in the test was just the inoculum in mineral medium. Two replicates were made for this series.
4.4.4 Toxicity batch
This batch of parallel was required as there was no available information regarding the toxicity of the test compound. A solution at a mass concentration of 50 mg/l was prepared. The solution was the mineral medium in which there was the test compound (25 mg/l) and reference compound (25 mg/l) mixed together and then inoculated with activated sludge. Duplicates BOD bottles were used for this batch in each test.
4.5 INOCULUM
Activated sludge which was the inoculum used for the studies was obtained from the aeration tank of Lehtoniemi Wastewater treatment plant, Kuopio. Three treatments were tested for the most effective inoculum – untreated activated sludge, filtered activated sludge and settled activated sludge (Appendix I).
Untreated activated sludge was 200 ml of freshly collected activated sludge left at room temperature in a magnetic stirrer to aerate and mix before use without any alteration. 0.5 ml of the activated sludge was dispensed into each BOD flask as an inoculum.
Filtered activated sludge was treated by passing 200 ml of freshly collected activated sludge through a Whatman® Filter paper (Grade 2). 0.5 ml of the filtrate was then introduced into each BOD bottle as the inoculum from the magnetic stirrer where it was left to aerate.
Settled activated sludge was treated by leaving 200 ml of the freshly collected activated sludge to settle for 10 minutes at room temperature. The supernatant was poured out while the residue was left on a magnetic stirrer until ready for use as an inoculum. 0.5 ml of the inoculum was dispensed into each BOD flask. This method was adopted after testing the effectiveness of various treatment on activated sludge. Fresh activated sludge was collected and prepared for use for each analysis.
4. 6 PROCEDURE
Using the 432 ml overflow measuring flask supplied by WTW, Weilheim (Germany), the various solutions were dispensed to their respective prelabelled BOD flask. A magnet bar (4 cm), to ensure proper stirring during incubation, was dropped into each bottle. In order to get more direct results of the carbonaceous demand (CBOD), nitrification was inhibited. Nine drops of allylthiourea (ATU) (5 g/L) was added to each bottle to prevent nitrification. 0.5 ml of inoculum (settled activated sludge) was then dispensed into each bottle. The quiver was inserted to each bottle. NaOH (3 pellets) were dropped into the quiver. The Oxitop ® - C measuring heads was then screwed onto each of the bottles. The Oxitop ® OC 110 controller was then used to start the experiments using the BOD28 programme. The bottles were placed on the Oxitop ® stirrer IS 12 in the incubator for 28 days at a temperature of 20±2 OC during which progress of the experiment was checked periodically. pH was measured on day 0 and day 28 using WTW pH 3210 pH meter (Weilheim, Germany). At the expiration of the 28-days test period, the measured data (BOD) were obtained from the ACHAT OC – v3.20 software for Oxitop ® control system designed by WTW (Weilheim, Germany). The analysis for N10O and Clodronates were repeated three times.
4.7 CALCULATIONS AND EXPRESSION OF RESULTS OF BIOLOGICAL OXYGEN DEMAND, THEORETICAL OXYGEN DEMAND AND PERCENT DEGRADATION
Mean values of the replicates for each batch was calculated daily from the data collected using Microsoft Excel application from Office 365 program. Biochemical Oxygen Demand (BOD) was calculated based on the mean values obtained, by subtracting the Oxygen depleted (mg O2/l) of the inoculum blank from that of the compound under investigation. The corrected depletion was then divided by the concentration (mg/l) of the compound understudy to give the specific BOD as mg Oxygen per mg test substance as in the OECD (1992) guideline (formula 4).
BOD = mg O2⁄l uptake by test substance - O2⁄l uptake by blank
mg test substance l⁄ in vessel =mg O2⁄mg test substance (4)
The theoretical oxygen demand (ThOD) of the hypothetical compound Cc Hh Clcl Nana Oo Pp Ss, of molecular weight (MW), without nitrification, is computed based on formula 5, according to OECD (1992).
ThODNH3 = 16[2c + ½(h – cl – 3n)+ 3s + 5/2p + ½na – o]mg/mg
MW (5)
Based on this information, the ThOD of the reference compound – sodium acetate and test compounds – N10O and Clodronate were calculated (Table 3).
Table 3: ThOD of compounds
Compound Molecular formula Molecular weight (g/mol) ThOD
Sodium acetate C2H3NaO2 82.034 0.78
N10O C11H27NO7P2·H2O 365.24 1.40
Clodronate CH2Cl2Na2O6P2·4H2O 360.83 0.089
OECD (1992) guidelines was referred to in calculating for the biodegradation percentage, which is the amount of oxygen uptake by the microbes (from inoculum) corrected for by deducting the oxygen uptake in the blank series, expressed as percent of the Theoretical Oxygen Demand (ThOD) as shown in formula (6):
Biodegradation (%) = 100 × BOD
ThOD (6)
Where:
BOD is biochemical oxygen demand of the compound under study (mg/L)
ThOD is the theoretical oxygen demand required to completely oxidize a compound (mg/L)
5. RESULTS
5.1 BOD28 ATU RESULTS FOR N10O
The lowest BOD value obtained in all tests conducted was by the test compound. Lowest mean value for BOD for N10O was 4.8 mg/l (figure 7) and the highest mean BOD was 10.8 mg/l (figure 6). The blank batch (only inoculum in mineral medium) had a mean BOD value of between 5.8 mg/l and 13.2 mg/l. The reference batch (sodium acetate in mineral medium) recorded a mean BOD value of between 37.3 mg/l (figure 7) and 47.4 mg/l (figure 6). The toxicity batch of the analysis (sodium acetate and N10O inoculated in mineral medium) had a mean BOD of 42.4 mg/l (figure 6) and 44.8 mg/l (figure 7).
Figure 6: Mean BOD28 ATU curves of N10O analysis carried out 27.6 – 25.7.2017. For N10O N
= 2, for blank N =1 and Reference N =1. Error bars represent standard deviation.
-10 0 10 20 30 40 50
BOD28 ATU(mg O2/l)
Time (days)
Reference N10O Blank
Figure 7: Mean BOD28 ATU curves of N10O analysis carried out 8.2 – 8.3.2017. For N10O N = 4, for blank N =2, for toxicity N = 2 and reference N =4. Error bars represent standard deviation.
Figure 8: Mean BOD28 ATU curves of N10O analysis carried out 4.9 – 2.10.2018. For N10O N
= 4, for blank N =2, for toxicity N = 2 and reference N =4. Error bars represent standard deviation.
-10 0 10 20 30 40 50
BOD28 ATU(mg O2/l)
time (days)
N10O blank toxicity Reference
-10 0 10 20 30 40 50
BOD28 ATU(mg O2/l)
time (days)
N10O blank reference toxicity
5.2 BIODEGRADATION OF N10O
The first analysis of 27.6 – 25.7.2017 (figure 9) indicates a % degradation for the reference compound (Sodium acetate) to as much as 87 % and -3.4 % for the test chemical (N10O) over the 28 days period.
Figure 9. Mean biodegradation curves for N10O analysis 27.6 – 25.7.2017
The second experiment (8.2 – 8.3.2018) to investigate N10O biodegradation (figure 10) reveals that the reference chemical (sodium acetate) was degraded biologically up to 80 %, while the toxicity curve was up to 33.6 % whereas the chemical under investigation (N10O) degraded to -1.5 % in the period of 28 days.
-10 0 10 20 30 40 50 60 70 80 90 100
% degradation (BOD/ThOD)
time (days)
Reference N10O
Figure 10. Mean biodegradation curves for N10O analysis 8.2 – 8.3.2018
The last experiment for N10O biodegradation (4.9 – 2.10.2018) indicates a % degradation of 87.5 for Sodium acetate (reference), 0.00 % for N10O and 34 % for the toxicity batch at the end of the 28 days period (Figure 11)
Figure 11. Mean biodegradation curves for N10O analysis 4.9 – 2.10.2018
-10 0 10 20 30 40 50 60 70 80 90 100
% degradation (BOD/ThOD)
time (days)
N10O Reference Toxicity
-10 0 10 20 30 40 50 60 70 80 90 100
% degradation (BOD/ThOD)
time (days)
Reference Toxicity N10O
5.3 BOD28 ATU RESULTS FOR CLODRONATE
Two separate experiments were conducted simultaneously for the period of 26.7 – 23.8.2018 (figures 12 and 13) and the last experiment conducted during the period of 4.9 – 2.10.2018 (figure 14).
The toxicity batch of the experiment had the highest mean of BOD value of between 42.9 mg/l (figure 14) and 45.6 mg/l (figure 12). Sodium acetate batch, the reference, recorded its highest mean BOD value of 43.3 mg/l (figure 12) and its lowest (figure 14) was 41.1 mg/l. The blank batch of the test (inoculum only in mineral medium) had quite a dissimilar mean BOD value of 12.5 mg/l (figure 12), then 10.1 mg/l for the second experiment of the same period (figure 13) and the lowest being 8.5 mg/l in the last experiment (figure 14). The clodronate batch of the experiment produced the same results in both analyses conducted 26.7 – 23.8.2018, both recorded a mean BOD value of 11.7 mg/l (figures 12 and 13). However, the experiment of 4.9 – 2.10.2018 recorded a mean BOD value of 7.2 mg/l (figure 14) which was a bit far from the others.
Figure 12: Mean BOD curves of Clodronate analysis carried out 26.7 – 23.8.2018. For Clodronate N = 4, for blank N =2, for toxicity N = 2 and reference N =4. Error bars represent standard deviation.
-5 0 5 10 15 20 25 30 35 40 45 50
BOD28 ATU(mg O2/l)
time (days)
Clodronate toxicity reference blank
Figure 13: Mean BOD curves of Clodronate analysis carried out 26.7 – 23.8.2018. For Clodronate N = 4, for blank N =2, for toxicity N = 2 and reference N =4. Error bars represent standard deviation.
Figure 14: Mean BOD curves of Clodronate analysis carried out 4.9 – 2.10.2018. For Clodronate N = 4, for blank N =2, for toxicity N = 2 and reference N =4. Error bars represent standard deviation.
-5 0 5 10 15 20 25 30 35 40 45 50
BOD28 ATU(mg O2/l)
time (days)
Reference Toxicity Clodronate Blank
-5 0 5 10 15 20 25 30 35 40 45 50
BOD28 ATU(mg O2/l)
time (days)
reference toxicity clodronate blank
5.4 BIODEGRADATION OF CLODRONATE
The first result of 26.7 – 23.8.2018, (figure 15), in this series of experiments showed that the reference compound (Sodium acetate) was able to degrade to as much as 78.6 %, while the compound under investigation (clodronate) gave a result of -17.4 % and the toxicity gave a result of 75 %.
Figure 15. Mean biodegradation curves for Clodronate analysis 26.7 – 23.8.2018.
The result (figure 16) for the second biodegradation experiment for Clodronate (26.7 – 23.8.2018) showed that at the end of the 28 days period, toxicity had the highest form of biodegradation at 80.8 % then the reference (Sodium acetate) at 79.6 and the test chemical (clodronate) degraded up to -10.5 %.
-30 -15 0 15 30 45 60 75 90
% degradation (BOD/ThOD)
time (days)
clodronate toxicity reference
Figure 16. Mean biodegradation curves for Clodronate analysis 26.7 – 23.8.2018
The result (figure 17) from the final experiment (4.9 – 2.10.2018) for clodronate biodegradation indicated a degradation percent of 83.9 for sodium acetate (reference), 79.2 for toxicity check and -28.1 for Clodronate.
Figure 17. Mean biodegradation curves for Clodronate analysis 4.9 – 2.10.2018 5.5 VALIDITY CRITERIA OF TEST
The results against the validity criteria of OECD 301F (1992) are presented for N10O (Table 4) and for Clodronate (Table 5). In respect to N10O analysis, percentage degradation for
-30 -15 0 15 30 45 60 75 90
% degradation (BOD/ThOD)
time (days)
Clodronate Reference Toxicity
-30 -15 0 15 30 45 60 75 90
% degradation (BOD/ThOD)
time (days)
Reference Toxicity Clodronate
Sodium acetate on the 14th day of analysis were within range as each test passed the 60 % mark required. The N10O results failed to meet the validity criteria in all experiments conducted as they were less than the required pass level 60 %. The studies for inhibitory nature of the chemical (toxicity) test revealed that the percentage degradation met the pass requirement for validity. Oxygen uptake for the blank series of the BOD test, were between 7.5 mg O2/l and 13.2 mg O2/l.
