Luận văn contributions to online measurement systems for the investigation of wastewater toxicity on activated sludge

‘Ihe aim of the monitoring campaigns was to apply the online respiration inhibition respirometer NitriTox to do a case study in which extend the activated-sludge process of indusbial was

VIETNAM NATIONAL UNIVERSITY, HANOI

VNU UNIVERSITY OF SCTENCE

Ferdinand Friedrichs

CONTRIBUTIONS TO ONLINE MEASUREMENT SYSTEMS

FOR THE INVESTIGATION OF WASTEWATER TOXICITY ON

VIETNAM NATIONAL UNIVERSITY, HANOI

VNU UNIVERSITY OF SCIENCE

Supervisors: Assoc Prot Dr Do Quang ‘Trung

Pref Dr Dr Wolfgang Genthe

Ha Noi - 2018

Acknowledgement

Firat of all, T would like to thank both of my supervisors, Prof Dr Dr Gentha who gave me the chance to work for the AKIZ project in Vietnam as an employee of LAR Process Analysers AG, for giving me the chance to work on such an exciting project, any Kind of support, inspiring discussions and arranging enough time to finalize the Ph.D

th

site fiom Vietnam National University | highly appreciate for giving me the opportunity

to do my Ph.D at the Viemam National University VNU Many thanks for the support to

Many thanks to Prof Dr Do Quang Trung as my supervisor from the Vietnamese

Can ‘Tho Also many thanks to the students from VNU Mr Bac, Ms Dung and Ms Lanh

for coming to Can Tho to do research at the AKIZ, laboratory

Many thanks to my colleagues from LAR Process Analysers AG for the support

regarding technical issues related to the analysers and the interesting discussion related to the research topic Therefore I want to acknowledge Thomas, Rafael, Olga, Olaf, 'Winfied, Gerhard, and Agnos

Many thanks to the project leader Prof Dr Rudolph as project leader and Dominic,

Rene, Sandra and Mr Long for coordinating the AKTZ, Project

Last but not least, I would like to thank tamily and friends for theix patience and care, especially to mry wift Huyen, my son Nico Tri, and my daughter Lina Kim for keeping me ina good mood

Acknowledgement

First of all, 1 would like to thank both of my supervisors, Prof, Dr, Dr Genthe who

gave me the chance to work for the AKIZ project in Viemam as an employee of LAR

Process Analysers AC, for giving me the chance lo work on such ar exciting projecl, any kind of support, inspiting discussions and arranging enough time fo finalize the Ph.D thesis Many thanks to Pro Dr Do Quang Trung as my supervisor from the Vietnamese site from Victnam National University I highly appreciate for giving me the opportunity to do my Ph.D at the Vietnam National University VNU Many thanks for the support to finalizing

my Ph.D subjects, to give me the chance to work on research projects with students ftom VNU and the interesting discussion related to my Ph.D thesis, At this point I would like to acknowledge Prof Dr nmilt, Rudolph as the leader of the AKIZ project, who always supported me, especially with the publication of scientific articles

Secondly, many thanks to all my colleagnes at TAR Process Analysers, the AKIZ leam, and the members and students from VNU who supported during my lime in Vietnam

, Mr Nhan, Mr Huy and Mrs, Lanh for the support in the laboratory and the beautiful time spent together in Can

‘Tho, Also many thanks to the students from VNU Mr, Bac, Ms Dung and Ms Hanh for coming to Can'Tho to do research at the AKIZ, laboratory

doing my Ph.D thesis, Special thanks to my lsb members Ms Huy

Many thanks to my colleagues from LAR Process Analysers AG for the support regarding leclmical issues related tơ the analysers and the interesting discussion related to the research topic Therefore, {want to ucknowledge Thomas, Rafael, Olga, Olaf, Winied, Gerhard, and Agnes

Many thanks to Dominic, Rene, Sandra and Mr Long for coordinating the AKIZ Project and their support

Last but not least, 1 would like to thank family and ftiends for their patience and care, especially to my wite Huyen, my son Nico Tri, and my daughter Lina Kim for keeping me ina good moad, Many thaiks lo my parents who always supporled me during my studies

and the entire life.

Declaration

| hereby declare that | have written the present thesis independently and without the use

of others than the indicated sources

Berlin, 16" April 2018 Signature:

Abstract

Removal of nitrogen compounds and organic pollutants from wastewater 1s one of the essential issues in wastewater treatment Commonly applied for this treatment step is the activated-sludge process

monitor the inhibitory effect of toxic substances on activated-slndge bacteria ‘This is

‘The aim of the study is to increase the sensitivity of toxicants on the activated-sludge respiration test to create an adjustable biosensor ‘'o this end, the research question is as follows: Which parameters have an influence on the sensitivity of the activated-sludge respiration-inhibitions test?

The rescarch question is answered through experiments using the inlemational

slandardixed activated-sludge respiration-inhibilion lest and the two online-respirometers NitriTox and Biomonitor of LAR Process Analysers AG To influence the sensitivity of these bio assays following parameters were investigated pH, temperature, oxygen concentration in the fermenter, incubation time, nutrient limitation and biomass concentration ‘I'hese experiments were realized with using Zn(), CuQ]), Cr(VD and 3,5

DCP as foxicatils A

eries of experiments are described with this objeclive, and showed in

isilivily of the bioassay could be varied by the investigated paranncters

‘The sensitization of the lest organisms can be explained by altering the activity of the bacteria and also the speciation of the toxicants in the prescnee of the nuticnt solution and its biological degradation products, It is, therefore, possible to detect toxic pollutants in lower concentrations, which have an inhibiting effact on activated-sludge bacteria | expect that this new approach is applied to detect inhibiting substances in wastewater in lower

concentrations to protect activated-sludge bacleria in a wastewater treatment plant, more

efficiently

Additionally, a mobile laboratory was develaped and assembled to conduct wastewater

monitoring in seven industrial zones across the country Victnam with toxicity as a critical

HL

parameter ‘Ihe aim of the monitoring campaigns was to apply the online respiration inhibition respirometer NitriTox to do a case study in which extend the activated-sludge process of indusbial wastewater treatment plant are inhibited by toxic waslewalers in

Vietnam The high necessiiy of monitoring the taxicily of industrial waslewalor can be

1⁄25 — Towicily Asseszmeni Methods to Determine the Inhibition of Pollutants on

127 Comparison of the Toxicity-Assessment Methods used to determine the

Inhibition of Pollutants on Activated-sindge Bacteria U? 1.28 Comparison of EC 50 Values of Activated Studge Inhibition-Test cel

Adaption and Alteration of the Commumity Structure of Nitvifiers cel Matrix Effects

Onygen-Producing and Oxygen-Consuming SubstanCes 1essnessnnnenstnnne 32

PH Change of Substances under Aer@li0n occu wannnsennmstnntinnssinnne 33

Offline Respiration-Inhibition Measurements

Assessing the Inhibition of Nitrification of Activated-Sludge Microorganisms 38

‘Online Respiration-Inhibiden Measurements

Biomonitr

Analytical Standard Methods

VI

NittiTox Measurement According to DIN 180 8192 31 Influence of Nutrient Solution on MitriTox Meacurements 52

Influence of Biornass Concentration on Inhibition s5 s2 eiee 3 Influence of Incubation Time in the Measurement Cell of Inhibition 53 Influence of Incubation Tirne at the Measnrernent Phase 1T on ïnhibition 8 Influence of ©; Concentration in the Fermenter on Inhibition 5° 83 Influence of the Sample Matrix Nutrients on Inhibiti0n 532 ca 3

Calculation of Theoretical Heavy-Metal Speciation by using MINTEQ 3.1

Technical Introduction of the Mobile Laboratory

2.10.2 Nam Sach - Industrial Zone

210.5 Company rox Becket cceceusssnsrinieatmnninnenanatncinananannenan vena senene OS

an Sampling Description

2.113 [oa Cam - Industiial Zone

Chapter 3: Results and Discussion

31 Studies with the Activated-SIndge Respiration Test Sensitization by

Varying Nutrient Solutions

3.1.1 Sensitization of Activated-Studge Respiration-Inhibition Testing by Varying

3.1.2 Influence of the Nutrient Solution on Heavy-Metal Speciation 70 3.13 Verifying the Results of Activated-Sindge Respiration-Inhilntion Testing using

32 Activated-Sludge Respiration Inhibition with the Online Respirometer

Biomonttor

3.24 Influencing factors of respiration inhibition with the Biomomitor 81 3.24.1 Infuenct sf NuhicntsintheEemenfer ssseceeeiee -81 3.24.2 _— Infuenctsf SMđạc ConeentratonS - se series 83 3.24.3 Influence of the ASR value on the respiration inhibilion «se 4

33 Activuted-Sludge Respiration-Inhibilion with the Ouline Respirometer

NiuiTox

VII

3.3.1.1 NitriTox in-House Round-Robin Test ve re -86 3.3.1.2 Stabulily ofResuils over [bur Yeas se series -88 3.3.1.3 Comparison of Toxioty-Asscssnenl Molhods to Dciemmine the Inhibition of Pollntants

en Adlivatcd-Sludgc Bacicria with the Resulls of the NitriTox Ánalyser 30 3.3.14 Comparison of EC 50 Valucs of Activated-Sludge Inbibition-Tcs from Lalcralure with

Nil Tox Mcasuroments c0seussennienniinunannninnunnssnssiie sai a1 3.3.1.5 Nilrifox Mcasurcmenl according lo DINISO 8192 95

33.2 Difluencing Faclorg to NHHÍTox MiCasUTEH16108 à sào so setcceeeeeercec.ĐĐ

3.3.3.1 Influlence oŸ Nutrient Sohtion in the Fementer - 96

33.2.5 Influence af Incubation Time in the Measurement Cell 108 33.2.6 Influence af Incubation Time during the Respiration Measurement 11a 3.3.2.7 Influence af the O; Concentration in the Fermenter H2 3.3.2.8 Adaplion and Alleration of the Communily Structure of Nitrifiers 114 3.3.2.9 Oplimum Conditions for the Sensilizalion of NitriTox Measurements „118

31 Application of NitriTox to Monitor Ladustrial Wastewater in Tra Noo

3.1.1.2 — Monitoring of Wastewater Canal Outlets to Hau River -124 3.1.1.3 Monitoring of Centralized Wastewater Treatment Plant 135

3.1.2 Summary of Data Collected During Toxicity Monitoring in Industrial Zones 126

Ik

List of Abbreviations

Abbreviation Meaning of Abbreviation

AOB Ammonia-oxidizing, bacteria

ASR Activeled-sludge respination

BỌC Biological Oxygen Consumption

BOD Biolugival Oxygen Deand

cop Chemical Oxygen Demand

DOUR Dissolved Oxygen Uptake Rate

EC 50 Rffective Concentration 50

EcD Electrochemical Detector

EPA Euvirumnental Protection Agency

FISH Fluorescence In Situ Hybridization

IcP Inductive Coupled Plasma

Iso Intemational Organization for Standardization

Measurement Phase

Non Dispersive Infrared Nitule Oxidizing Bacteria Namo Paaticle

Organization for Economic Co-operation and Development

Oxidation Reduction Potential

Oxygen Uptake Rate

Respiration Total Abiotic

Respiration Total Biotic

Specific Oxygen Uptake Rate

afler 15 trấn Œnzynte inlibiling, 30 ain (ATE luminescence, V

fixchen), 2 tt (Nilclivation mbibition) mu 3k Respiromety) exposure to the pollu [27] 17 Influence of pI on the nitrification rate [54]

‘The tewrelicd dishibutiou of the predouimanl chemical species ul Cr [65], pH dependent

‘Typical bacterial growth curve [71]

Influence of suspended solids in activated sludge on respiration rate inhibition /2) [35] „3i

Respiropram of NitiTox messuremeuts; for a feodic aud rơnloxic even 4“ a) Picture of the Diomoniter; b) Schematic setup of a Diomonitor online measurement

‘The Biomoniter measurement curve is ø loxicify measurement with the measurement phase: 1) addition of the toxicant, 2) begin of respiration inhibition and 3) replacing sample with water $0 Sigmoidal dose sesponse curve for determination of the EC50 value,

Map of the Tra Nov industrial zane and the representative meastirement point 0F thứ open stonn water system (1 —6 white) and mexsurcuent points of (he oullets of fe open slonn-

‘Map of the Nam Sach industrial zone and the representative measurement point of the sewage

1 valttes of activated-sludge respiralion-inhibition test for

DCP; using synthetic wastewater and sodium acetate as mnhient

1) Comparison of EC 40 values for the toxicams: Cr(VIp, Cu(D), 3,$ DCP and Zn}

measuring the respiration with an oxygen sensor and an ORP lectrode using synthetic wastewater or NaAc as amutrient solution The rents are presented with staudard deviation 1)

‘Comparison of respiration for reference water and 3,5 DCP (20 mg 1-1) with au oxygen sensor

and au ORP Flectrode using synthetic wastewater as @ nutvient solution TS

XI

1} Inffuence of the dilution of the sludge: green) undiluted shidae; red) dilution 1:2; magenta}

1:4: eyan: 18,1) Tnlluence of the nolrient sale: real) Sadkurn acetate; bine) Pepa 78

‘Toxicity aeustrencaly of uetivated sludge: Lue) accordug te DIN TRO B192, 1ed) Biounouiter measurement

‘Respirogram: blue) peptone ar nutrient solution, red) Sodium acetate as nutrient solution mĩ

Influence of TSS concentration ou respiration inhibition using Bionumlor 84 Influence of TSS concentration on respiration inhibition using Biomonitor

‘NitiTox results of am in-humse roicuL-rotiu Lest - ar

‘Validation of NilsiTux results over a period of 4 yours 89 Comparison of toxicity EC 50 values for 3,5 DCP, Znf), Or(V]) and CHD of NitriTox and

‘Biomonitor measurements with the standard methods V, fischerie, nitrification inhibition ($0 9509), activated-aludge respiration inhibition (80 8192) and witrification respiration

100 Bioscience [32], Activated shidge rexpiration-inbitition lest according ta DIN TSO

8192 — using aclivated <mdye uy biomass [27], 5 L Bateh Reactor [33] und Oxyinm-ER 10 34]

‘Comparison of toxicity EC 50 values for Cu(II) of NilriTox messurements with results of BI-

1000 Bioscience [32], Baroxymeter [29], Amtox TM [26], Activated-shudee respiration inhibition test according to BIN ISO 8192 using activated ndge as biomass [271, Strathtox [0], $I Hatch Reactor [33] and Oxymax-ER 10 [34] ” Comparison of toxicity EC 50 vnlues for Cu(H) of NitriTox measurements with results of BI-

1000 Bioscience [32], Strathox [30], I Batch Reactor [33], Activated-shudge respiration:

inhibition test according to DIN 180 8192 — using activated sludge as biomass DIN ISO

8192 [38], [27], Baroxymeter [29], Oxymax-ER 10 [34] and Fed Datch Reactor [36] 94

‘Toxicity measurements lo activated dodge: blue) according In DIN ISO 8194, red) NitsTox

Influence of Nutricots on MitriTox measurement using a} LAR Nitrifiers as biomass and ammonia bicarbonate and synthetic wastewater as muttients; b) activated sludge as biomass and sodium acetate aud synthetic wastewater ar a nntrieat,

XIL

1) orange dichromate solution at pIT7; 2) yellow chromate solution at pil 9 „ 102

LVIVIS spectruun of Cr(VI) at pH 6, 6.5, , 7-4, 8 in pranence of the biclagical degradation

TinHluenee off the Ganpersture ou the respiration rate; ¢) Siusultaeony 0ubme kueasurenten

curve of temperature (black) andthe respiration rate (blue) at the inlet of the WWTP in the

Influence of leanperalure om RC 50 values of NitsiTox messaurements for Zu(0, Cui], {x(VD) and3,5 DCP, with temperatures of 10, 28 and 40 °C: 105 Ðepcnđenec of tcmperalure sn chroruate'diehrornste cquilibriom „ 106

Influence of biomass concentration in the fermenter and the measurement cell on EC 50

‘values of NitiTox measurements for Zn(ID), Cu), Or(V]) and 3,5 DCP: a) with a TSS concentration af 1.8 1." in the fermenter and the ratios of 5, 10, 15%6 biomass in the measurewent cell; b) with a TSS concentration of 0.8 L* am the fermenter and the rativs ob

‘Influence of the incubation time in the measurement cell on EC £0 values of NitriTox

‘measurements for Zu(TT), Gud), CrCV1) and 3,5 DCP; with an incubation time of 0 and 30 min; ()) activated dudge, (1) nitrifiers - ws

0) Influence of expasure tise caving; the measur enent Blaise TT on RC SO values af NitiTok measurewents Lor Zu(T}, Caf), CVT) mid 3,5 DCP: a) period of weasuremeut Plowe Ti of

180, 300 and 420 s; (r) respirogram of NiftiTox measurements for reference water and Cu(II)

10 mg L-!-with a period of measurement Phase II of 420 s - - mỹ Influence of the aeration rate in the fermenter ou BC $0 values of NitriTox measurements for

Aah, Cu), Cr(V and 4,5 DCP; a) with an aeration rate of $ Leh and S41 1, hờ mà Tnfluuce of 807 concentralims in fee sonple ow RC SŨ vahtes uŸ Nïhi Tu mheasurennenlss

tor ZH), Cu), CHVT} and 3,5 DCP, with 0 and 1000 mg L! S07 us

Influence of aeration of the sample on the pH value andthe Gradieut II () simultmeous online measurement ctirve of pIT (black) and the respiration rate (blue) at the inlet of the

Influence of particles and filtration af the sample on NitriTox mensntements on FC 40 values

of NitriTox measurements for Zn(11), Cul), CVD) and 3,5 DCP; a) without kaolin; b) with 0.3 gL! kaolin before filtration; ©} with 0.5 gL! Kaolin after filtration WS

XIHI

NitsiTox meamirement of a real sample in the presence of H1C2; a) reference water, b}

sample befire HO; sareple will 0% bisnass, o) suople Define HO, sample sith 15%

'NilriTex mensurement of a sample with high sulfite concentration; abiotic respiration rate due to sulfite, while aeration at pI 2

heavy-metal concentrations and legal-limit values before and after chemical treatment,

‘Nibilicaliva iubibiGion defenmined for mexsurcsncul Point 5 of the upeu storm-waler ema system in the Tra Noc industrial zone the corresponding Cr and Zn roncentrations,

‘Nitvification respiration inhibition determined for measurement Point 6 of the open storm-

‘water canal system in the Tra Noc industrial zone and the corresponding Cr and Zn concentrations

Adeulfied dired dischargers in Tra Nac, lal: valelazeu arew of messuremea Poin! 5, right

‘Nitrification respiration inhibition determined for the outlets af the open storm-water canal system leading to the environment in the Tra Noc industrial zone

XIV

Emission of heavy metals from industrial branches [17] - u

Existing respiromcter used iu WWTP end the churactcristics of the flow regime 16

‘Comparison of bioassay sensitivity, cost, duration and relevance [27

Inhibitory ettects of Zn(I}) on activated-shudge bacteria 2

Inhibitory effects o£ Cr(VT) on activafed-slndee bacteria

Inhibitory effects of 3,5 DCP on activated-studge bacteria z4

‘Theoretical Cu(I) speciation under initial conditions

‘Theoretical Zin(T]} speciation under iuitial conditions a

‘Theoretical Cr speciation under initial pơnhous - - Ta

Dilution factor and TSS concentrations

‘Innportant parameters for activated-aludge respiration test according to DIN ISO 9182 and

Dilution factors, corresponding TSS conceatration and the resuits at the investigation af the

‘TSS concenfrations ou acypiration iubibition using Biomtomstur 83

‘Volume of nutrient colution, corresponding ASR valuss

Recults of a NitriTox in-house round-robin test conducted by LAR AG, University Stuttgart,

‘Comparison of bioassay sensitivity

‘Comparison of bioassay sensitivity and incubation time,

pil valuesin the measurement cell with 0, 5, 10 znđ 12% Biomass

‘Theoretical Cu(ID) and Zu(TT) speciation under initial condhlioms on mà Air flow rates and the corresponding 2 concentrations in the fermenter and the dosing rates

optimum and uon-optimum conditions for NitriTox messuremeuis, Us

XV

Introduction

‘The number of industrial zones continuss to increase in Vietnam, In July of 2015, there were 299 industial parks, the majority of which lack sustainable wastewater treatment The consequences are highly polluted and toxic wastewaters, which are discharged trom

factories directly into the environment or to a centralized wastewater-treatment plant

(GWWLP) One method to determine the toxicity of wastewater is the activated-sludge respiration-inhibition

The monitning of activated-sludge and mitification respiration-imbibition al WWTPs

can be justificd by detection of toxic pollution compounds, which aro not biologically degradable and are discharged trom the WWTP outlet into the environment In addition, according to the standard A QCVN 40/2011/BTNMT, the legal Limit values for total nitrogen and NH- are 20 mg L”' and 5 mg L”, respectively Dne to the discharge of toxic

wastewater ftom the factories to the wastewater treatment plant, the nitrification process

might be inhibited, in which case the legal limit valnes for total nitrogen and NII, are

Because it is needed to determine the inhibiiom of pollulanls to activaled-sludge bacteria, the procedure of the activated-sludge respiration-inhibition test is described in 1SO

8192 [2], OECD 209 [3] and TCVN 6226 [4] This method is fundamental to the lwo online respiration analysers made by LAR Process Analysers AG, which were used for the research in this thesis The field of activated-shudge respiration-inhibition testing, sutfers trom large gaps in the understanding of how to increase the sensitivity of the measurement

procedure and of which factors influence the result One of the most important variables for

improving monitoring involves improving the sensitivity of the test organisms [5]

2 Objectives

Increasing the sensitivity of activated-sludge respiration-inhibition tests ensures the detection of toxic pollutants and contaminants im lower concentrations, this procedure makes it possible to protect microorganisms wathin the activated-sludge process at the WIWIP and hence renders the biological degradation of contaminants more efficient, To monitor the inhibiting effect of wastewater discharged into a WWLP, the sensitivity of the test organisms should be higher than that of the microorganism used for the treatment

process Lo creale an sarly-wannng syslenL Ror this reason, the objective to increase the sensitivity of the activated-sludge respiration-inhibitions teal is af high relevance

In addition, neither the online nor the standard activaled-studge respiralion-test has, to

my knowledge, been uscd to assess the wastcwatcr quality in Victnam’s industrial zones

Hence, the objectives of this thesis are as follows:

© To develop a new application for the online respirometer Biomonitor to an online- activated-sludge respiration-inhibition analyser

© To increase the sensitivity of the activated sludge respization-inhibition test

* To validate the online respirometers, Biomonitor and NitriTox

© ‘Yo apply the Nitri!’ox system for wastewater monitoring in seven industrial zones

time, and oxygen concentration in the fermenter

2) Replacement of the synthetic-wastewater nutrient solution with sodium acetate increases toxicity ‘his can be explained by the formation of complexes of heavy metals wilh ingredients in the synthslic wastewater, especially peptone The heavy

metal — peptons complex has a lower toxicity than the heavy metal speciation in the presence of sodium acetate

3) An online sysiem for monitoring the toxicity of wastewater has been applied in Vietnam for the first tiane A mucasurcmont campaign in scven industrial zonos across the county has been conducted The wontloring shows that toxicities lo nitificants

have occured in five of the investigated industrial zones.

Chapter 1: Literature Review

The purpose of wastewater treatment is to remove pollutants that can harm the aquatic

environment if they are discharged into it Because of the hannful ettects of low dissolved

oxygen (DQ) concentrations in waters, wastewater-treatment engineers have historically

focused on the removal of pollutants that would cause depletion of the DO concentration in

the aquatic system ‘I’hese demanding oxygen pollutants exert their effects by serving as a food source for microorganisms, which consume oxygen for biological oxidation Most oxygerrdemanding pollutants are organic compounds: armanania and nitrale are important inorganic ones Hence, WWTPs are designed to remove these organic and inorganic

contaminants Another problem with dischargmg nutrients such as nitrogen compounds into

the receiving water bodies is eutrophication, which is the accelerated aging of a lake dus to enhanced algae growth WW'TP operators have become concemed about the discharge of toxic chemicals into the treatment plant that would inhibit the biological degradation of the

organic and inorganic pollutants [6]

Before focusing on activated-slndge- or more specifically nitrificatiominlibition, i is

important to have a general idea of the complete process undurlaken by a “standard”

WWTP The basic scheme of a WWTP consists of the following tree main sleps:

© Primary treatment

In primary treatment, sedimentation and skimmume typically remove 40% to 70% of suspended solids The BOD (Biological Oxygen Demand) removals by the primary treatment nonmally range from 15% to 70% Primary treatment reduces the load on the secondary treatment system and allows the microorganisms to work on the dissolved

la remove large, Mloaling objects such as cloth and sticks that might block pipes Afler the

sewage has been sereencd, it flows into a grit chamber, where cinders, sand, and small

stones settle down to the bottom 'I'his treated wastewater still contains suspended solids,

and a sedimentation tank can remove them from the sewage [7]

© Secondary treatment

Secondary wastewater treatment is also called biological wastewater treatment because

it uses living inicroorganistes 1o degrade or oxidive residual conlaminants: primarily soluble organic compounds, Microbes achieve this by consuming the organic matter as food, and converting it to waler, carbon dioxide, and cnergy for their reproduction and growth, Al the biological-treatment stage, the sewage Icaves the scttling tank and is pumped into an aeration tank, After being mixed with activated sludge, it is aerated for several hours During this tme, the bacteria break down the organic matter into hannless by-products The activated sudge is retumed to the aeration tank for mixing with new skidge during aeration

Tl can be reused Afler featinent in the aeration tank, the pre-treated

wage is pumped loa snbscquonl sedimentation lank lo rortove excess bacteria [7] The secondary treatment can remove up to 85% BOD and suspended solids, Environmentat conditions within treatment systems can haye a profound impact on the diversity and complexity of the bacterial population, Factors that affect microbial growth and survival have been studied extensively, They include temperature, pH, oxygen concentration, organic loading and specific toxicants

In addition to the removal of organic compounds, the secondary treatment is responsible for the microbial elimination of nitrogen (N) by nitrification and denitrification

‘Vis thesis mainly focuses on the inhibition of nitrifiers ‘Ihe nitification process is described in detail in Chapter 1.1.2 “Nitrification”,

«Tertiary treatment

Tertiary treatment provides a final slop of wastewater treatment to inercase the

cfflucut’s quality before it is discharged into the reeciving body of watcr The technologies applied in the tertiary treatment are filtration, ultrafiltration and activated-carbon filtration,

To reduce the number of micro-organisms in the water, disinfection is always the final treatment step Methods commonly used for disinfection include chlonnation, ultraviolet light, and ozons In addition, lagoons and constructed wetlands belong to the tertiary

treatment [7]

1.11 Activated-Sludge Process

Activated-sludge systems handle wastewaters that contain high concentrations of BOD and COD (Chemical Oxygen Demand) ‘hey have been important wastewater processing systems for many decades Activated sludge is a suspended-growth process that relies on natural bio-oxidation mechanisms Organic contaminants within the waste stream exhibit rapid biodegradability The activated sludge converls organi

taller lo energy and new

cellular material, as shown im the formula below:

Organics (BOD)+ O, + Nutrients par? CO, +H,0+ Microbes + Energy wy

Other aerobic and anaerobic by-products of these systems include water, inert materials, and gasses (¢.g., carbon dioxide, methane, hydrogen suliide, nitrogen, etc.) The standard activated-sludge process is mainly aerobic An external source of air or oxygen and mechanical mixing are needed to help artificially replenish dissolved oxygen levels that have beon depleted in the bio-oxidation reaction, The microurganisms found in the aeration basin can be comprised of the primary groups This study is restricted ia various active, metabolizing bacteria:

© active, metabolizing baetena that are primarily responsible for metabolizing soluble

wastes

© filamentous bacteria that are mainly responsible for providing, structural support to growing colonies of bacteria (referred to as flock)

= profevoans thal assist in metabolizing wastes, reducing cfflucnt turbidity, ad

building bacterial flocks.

112 Nitntication

Nitrification is necessary, because it is the conventional process by which anumonia is removed in waste-treatment systems (by being converted to nitrate) Nitrification is the bacterial oxidation of NIL to NOs It can be deseribed by the following equation

In the first stib-reaction, NIJ, is oxidized to NO; by the bacteria Nitrosomonas:

Heterotrophic bacteria (BOD degraders) ean divide in 20-30 min [8]

Nitritiers are extremely sensitive to certain organics in low concentrations, including phenols, thiourea, methanol, aniline compounds, isothiocyanate compounds, carbamates, cyanide and many others Sensitivity to heavy metals in low concentrations is typical,

including Cr(¥1, Cr(ILN, Lg, Cu, Ni, Zn (at > 0.25 mg L *) and Pb (at >0.5 mg L*) Grady [9] zeported that the most potent and specific inhibitors of nitrification are compounds that chelate metals and contain amine groups, some of which are capable of decreasing the

mitificalion rate by 50% al concentralions of less than 1.0 mg [Halogenated

hydrocarbons arc also belicved to be mainly inhibitory.

y Monitoring

1.2.1 Inhibition and Toxicity

Toricily is the degree to which a substance ca lanh ant organism such as humans, arizmals

= an initial flagellate bloom, followed by subsequent complete die-off of protozoa and

higher life forms (diagnosed with routine microscopic examination);

«significant reduction in respiration rate, or specific oxygen uptake rate (activatcd-sludge

Tespirahon-inhrbition test);

© increase in reactor-dissolved oxygen levels (online DO monitoring preferably with

logging),

© increase in extracellular ATP (special testing needed);

© biomass deflocculation, offen accompanied by foaming and high effluent TSS

(microscopic, visual and water quality monitoring),

= Joss of BOD rzmoval in extreme cases (waler quality monitoring); and

© filamentous bulking upon process recovery (tnicroscopic and settle ability cbservations)

Deciding whether toxicity exists in a system cam involve extensive detective work Toxicity

is an indicator of inhibiting substances If'a toxic event is detected, a subsequent analysis via inductively coupled plasma (ICP), high-performance liquid chromatography (ILPLC) or mass spectrometry (MS) is needed to detect the individual compound The composition of

the wastewater needs to he known to draw a conclusion about further treatment Common

treatments for detoxification of organic pollutants include advanced oxidation processes (AOP) and, for heavy metals, flocculation and precipitation

Monitoring methods thal can contribute to determnininy the presence of toxicity or inhibition include dissolved oxygen tronds, sludge sutlling abilily, Ircatability Lesting, micrascopy, oxygen uptake or proprietary methods such as MicroTox™ or olluas based on ATP, Sue of these are discussed in Chapter 1.2.5 “Toxicity Assessment Methods to Detennine the Inhibition of Pollutants on Activated-Sludge Bacteria’

1.22 — Necessity of Toxicity Monitoring

In order Lo protect water bodies from algas blooms, authorilics have imposed strict

regulations on the waler quality of WWTP cflucnis, especially for uitrogen compounds Commonly centralized WWTPs process industrial wastewater in industrial zones The

common treatment of industial wastewater and domestic wastewater is also practiced in

urban areas As a consequence, there is a possible threat that inhibitmg substances including

organic compounds, heavy metals, nanoparticles, and high salt loads are discharged from a factory to a WWTP [10] The discharge of loxic wastewaler to a WWTP is a general mublem

The need to monitor the inhibition of loxic wastewater Lo activated sludge can be seen

in the fact that 45 of 109 tested municipal wastewater treatment plants have received

inhibiting wastewater [11] In addition, it has been reported that the inhibition of

nitrification has been experienced at several Swedish wastewater treatment plants that were

performing nitrogen removal [12], and a study of 38 Danish municipal wastewater types

showed that about one-third were inhibitory to nitrification to a considerable extent [13]

Another example is reported by Grau et al [1-1] Ile states that wastewater with a high load

of phenol hindered a wastewater treatment im Brazil for six months The latest incident occuted in 2016, where a mass death af fish ovewred along the contal coasts of Vielnamn

assoeiated with toxis industrist wastewater discharges, thereby proving once more the

growing importance of industrial wastewater monitoring in Vietnam

The protection of nitrifying bacteria is especially relevant because two distinct properties characterize them: sensitivity to inhibitory substances and slow growth rates [1] Therefore, if the nitifiers are exposed to inhibitory compounds, their ability to oxidize

9

ammonia will decrease Moreover, due to the slow growth rate of the autotrophic bacteria, it takes considerable time to return them to their nermal activity compared to heterotrophic

batleria Towever, when loxic substances are added to the wastewater, th m tubibil the

a critical parameter,

1.23 Sum-Parameters in Water Monitoring

A large number of chemical substances are present in every water body Due to a large

number of chemical compounds, a quantitative determination of each compound is not possible This reason makes it easy to understand why wastewater technology requires

analytical methods, such as sum parameters, that are easy to use Kor this purpose, cost-

ellective methods for COD, BODs, TOC (Total Organic Carbon) and toxicity were

developed to cover a large number of chemical compounds wilh one analytical method The analytical method provides dala aboul a particular properly of the analysed samples For

example, TOC and COD offer information about the pollution of the wastewater with organic compounds A toximeter measures the inhibition of’ a sample on an appropriate

organism Therefore, it is not necessary to analyse each compound, which is time- consuming and expensive

‘The method most often used to analyse the inhibition of toxicants to activated-sludge is

the activated-sindge respiration-inhibition test It provides direct information about the rate

of biedegradation by activated-sludge bacteria Toxic wastes inhibit hoth respiration and

Iiodegradation The respivation rate provides information about the toxicity of the

wastewater, and iL is also import to process optimization

10

1.24 _ Sources of Heavy Metal Pollution

The occurrence of heavy metals in water bodies can be from anthropogenic or natural sources Tixcessive metal levels in surface water are hanmfnl to humans and to the environment Arsenic, which occurs in many minerals, is an example of a natnral contaminant [15] The natural pollution

of a water boy by Arsenic (As) happens due to the natural presence of As in the bedrack, which is

a Widespread problem around the world: e.g., in West Rengal, regions af China and in northern regions of Vielwan, The As can be releazed info the enviroment by exusion or pl changes In Tbangladesh, the groundwater af 2 population of 120 million people is contaminated with natural As Mining comstitules an anthropogenic source of As pollution

The antiopogenic soutes can be distinguished into point and nonpuint sources Point sources

we industrial cfflucits, chemical or petrulewo spills, und dunps ad smokestacks Non-pou

soees inchide common agrochemicals (pesticides and ferlilizers), cars, atmospheric deposition,

and desorption or leaching, from large arcas The presence of toxic heavy metals in industrial

eflucats is vw of the most sexivus tucats to the enviroment Primary zources uf heavy-metal

pollutants incinde atmospheric pollution from the petroleum of moter vehicles, the combustion of fossil fuels, agricultural fertilizers and pesticides organic manures, urban and industrial wastes, metallurgical industries mining and smelting of non-ferrons metals Heavy metals such as

Cadmium, Chromium, Lead, Nickel, Zinc, Mercury, Copper and Arsenic are found in the effluents

of foundries, electroplating plants, petrochemical plants, battery manufacturers, tauneries, fertilizer

plauts, dying factories, textile factonies, metallurgical aud metal-fiushing plants [16] Table 1-1

provides an overview of the emission of heavy metals (Cu(II), Zn(II), aud Cr(VD) in plants

associated with various industnes that are relevant to this thesis

‘Table 1-1: Emission of heavy metals trom industrial branches [17]

1.25 Toxicity Assessment Methods to Determine the Inhibition of Pollutants on

These methods are described in this chapter

International Standard ISO 8192, 2007, Water-quality test for inhibition of oxygen

consumption by activated sludge for carbonaceous and ammonium oxidation (ISO 5192)

This method was specifically designed to assess the total inhibition effect of a given

subslance aver the respiration ais of the activated-sludge microorganisins The respiration

ratc of reference wa 110 a loxivity of 0%, and the total imbibition of the respiration

rate to 100% The nitrification inhibition can be calculated by subtracting the heterotrophic respiration-inhibition (obtained by specifically inhibiting all nitvificanon through the addition of ATU) diom the total respiration inhibiton,

Euternational Standard ISO 11348-3, 2007 Water-quality determination of the inhibitory

effect af water samples on the light emission of Vibrio fischer! (Laminescent bacteria test) -

Method using freese-dried bacteria (ISO 11348-3):

Determination of the mlubitory efitect of water samples on the light emission of Vibrio fischeri is used to determine the inhibiting effect of wastewater, according to DIN EN ISO

11348, ‘Thus, the ability of bacteria to emit light is used ‘The light intensity is measured before the addition of the wastewater samples and 30 minutes after the addition of the test substance The intensity of the emitted light is a grade of the inhibiting effect

ueruaHonal Standard ISO 9509, 2006 Water-quality toxicity test for assessing the

inhibition of nitrification of activated-sludge micraorganisms (ISO) 9509):

‘The percentage of nitrification inhibition is determined by measuring the biological degradation products of ammonia by the nitritication process, The nitrifiers convert

12

ammonia to nitrite and nitrate If the nitrification is inhibited, the biological degradation of ammonia to nitrite and nitrate is also inhibited

The uitrite and mitrale salls that are consequently formed are delermined afer am

incubation of four hours under standardized conditions The eonucutrations of the oxadized

mitregen compounds are determined with and without uitrogen inhibitor tv calculate the percentage of inhibition This calculation can also be done by measuring the levels of

ammonia concentrations betore and after incubation

The disadvantages of this method are the long incubation time and the time-consuming

chemical analysis of nitrate and nitrite

Determination of the inhibitory effect of water samples on the ATP (Adenosine

triphosphate) luminescence of activated-sludge bacteria

The method is based on ATP {a measure of active biomass) reduction by the effect on the toxicant Toxicants are added to domestic activated sludge, and the sample is incubated

for 30 min After the incubation, the A'l'P concentration is determined with a luminometer

Comparison of toxicity asvessment methods

‘Table 1-2: Comparison uf loxicity-assesaneut methods

of Nitrate and Nibite

concentration

13

1.2.6 Activated-Sludge Respiration-Inhibition Test

1.26.1 Offline Respiration-Tnhibition Measurements

The activated-sludge respiration-inhibition test is fimdamental to this study The biological test most commonly used to determine the inhibiting etfect of wastewater on the biological treatment is the activated-sludge respiration-inhibition test It is an appropriate method for determining the inhibition by pollutants, as it uses microorganisms directly from

the WWIP

‘The activated-sludge respiration-inhibition test is based on the dissolved oxygen uptake rate (DOUR) It was developed as a method for assessing the potentiat impact of chemicals

on wastewatcr-(realment systems Because iL is ne

pollutants to activated-siudge bacteria, the procedure of this biological test is described in

DIN 1808192 [2] and OECD 209 [3]

‘The respiration tate is primarily caused by the biological oxidation of the organic

ssary for determining the inhibition of

pollutant by the activated-sludge heterotrophs and the nitrifying autotrophs for ammonia removal, which reqnires oxygen If the biological oxidation is inhibited, the respiration rate

decreases,

This relationship makes il possible to determine the ibibiting effect of toxicants on activatcd-sludge bactcria by measuring the respiration rate of refercnee water and the toxic sample The oxygen consumption of activated-sludge bacteria in the presence of reference water is set to the toxicity of 0%, 100% toxicity means ne respiration

1.2.6.2 Online Respiration-inhibition Measurements

online toxicity measurements of wastewater treatment plants and is merely a long-term

are based on

performance evaluation instrument, Therefors, online measurement dev

respiration amcasurements, which allow a muasuroment interval of LS min Several rospiromelers were dovelopod belwecn 1970 and 1990 They can be classified in accord

4

with two criteria: 1) measurement of the respiration in the liquid or the gaseous phase, and 2) the flow regime of the liquid and gaseous phase (either flowing or static) Table 1-3

‘biomass and the sample are added to a measurement cell and the oxygen consumption is

recorded 1o calculate the respiralion rate [18-20] The other type is based on a continuous

flow approach, in which the biomass and ihe sample are delivered continuously to the

measurement coll and the respiration rale is calculated by the difference of lwo DO readings

at a particular retention time [21-23] The disadvantage of this method for toxicity

measurements is that activated sludge is used continuously for analysis and cannot be

retumed to the fermenter in the case of toxic samples To do so would cause the poisoning

of the total biomass For a continuous flow regime the online analyser needs to be provided

with activated sludge directly from the WWTP

18

fable 1-3: Existing respirometer tinedin WWTP and the charaderistics of the flaw repime

The online analysers used in the frame of this study are the NitriTex and the

Biomonitor NitiTox measurements are based on the stopped-tlow batch-wise procedure

The novelty of the NitriTox is that is uses a nitrifier culture, which is characterized by a

high sensilivily to the toxin and a high measurement slability Using the NilriTox analysis qrocedure, we have for the first time succeeded in developing an onling tespiration- inhibition analyser that yields sable and reproducible resulls aver a specified time The

other novelty is the technical impicmentation of the online analyser The analyscr comes

equipped with modem technology, which allows for operation via touch screen

The Biomonitor is based on a continuous-flow procedure The novelty of the Biomonitor is that it uses a measurement cell for respiration measurement and a cascade

consisting of four vessels that allow a faster biodegradation of the organic pollution by the

activated-sludge bacteria In contrast to the NitriTox measurements, the respiration is

measured in the gaseous phase and not in the liquid phase A detailed description of the

Biomonitor and NitriTox analysers is given in Chapter 2.2 “Online Respiration-Inbibition

Measurements”

16

1.2.7 Comparson of the Toxicity-Assessment Methods used to determine the

Inhibition of Pollutants on Activated-sludge Bacteria

Dalzell [27] compared five toxicity assessment methods for determining the inhibition

of pollutants on activated sludge The authors determined the EC 50 values for toxicity tests

using the toxicants Cr, Cu, Zn and 3,5 DCP with following biological tests: nitrification

inhibition (similar to ISO 9509), vibrio fishery (ISO 11348-3), respirometry (ISO $192), ATP luminescence (according to Arretexe [28]) and enzyme inhibition The results of the study are shown in Figure 1-1 Each test was ranked for sensitivity by the authors, as shown

in Table 1-4 (A score of one was given to the most sensitive, and five to the least sensitive Therefore, the lower the ranked score, R, the more sensitive the test.) The test with the

highest sensitivity is the Vibrio Fischeri bioassay Nitrification inhibition is the test with the

highest sensitivity to toxicants, as it uses activated-sludge bacteria for the biological test

These test organisms are also used for the online respirometer Nit Tox, which was used for

the experiments in this study, ATP luminescence and enzyme inhibition are the bioassays

with the lowest sensitivity

100000

HBB Nitrifcation inhibition

Respirometry ATP Luminescence HEB Vibrio Fisheri

Figure 1-1: Comparison of toxicity data for Cd, Cr, Cu and Zn using five rapid toxicity bioassays Values presented are

EC values after 15 min (Enzyme inhibition), 30 min (ATP luminescence, V fischeri), 2 h (Nitrification inhibition) and 3

hh (Respirometry) exposure to the pollutant [27].

Iathermore, Table 1-4 eompares each test with regard to cost of investment, eost per

test, working time, relevance, and ease of use

In general, Vibrio fischeri is the test, with the highest, sensitivity Il can be therefore used as a surecning test to highlight possible problem areas, BuLil doos not give information about a potential effect of an unknown toxicant on activated sludge, because the inbibitory effect of an unknown substance might be completely different from that of activated-sludge bacteria Thus, it might be that the Vibrio fischeri test shows a wastewater toxicity of 100%

‘but that the substance does not have an inhibiting efffect on the wastewater treatment with activated-sludge bacteria In conclusion, due to the high sensitivity of the Vibrio fischeri test, the result could overestimate the possible inhibiting effect of the tested sample

‘Table 1-4: Comparison of bicasay sensitivity, cos, duration and relevance [27]

Parameter Inhibition Respizometry luminescence ¥ fiselicri_ Inhibition

4) Tame for alndge espesare (30min) noc mchuded ex aworksris met crired daring this period

18

1.2.8 Comparison of EC 50 Valucs of Activated-Sludge Inhibition-Test

Much conilicting data has been published on EC 50 values measured by the activated-siudge respiration-inhibition test Inhibitory data from different studies for the same compounds can range over three orders of magnitude or more, making it difficult to predict what will be inhibitcry

in any particular system This chapter summarizes published data for 3,5 DCP, Zn({), Cu) and

CxCVD These compounds were also chosen for the experiments of this s

uly Tnany case, with respect

io the sutunariaxd EC 50 values in this dapler, note that the respiration was prlorrmodd under di (Meret conditions by the several aulhors Relevant paramictas of the activated-sludge respiration lest such as biomass, incubation time, and the ratio of sludge, muirients solution and sample differ in the published studies, which makes t chificult to compare the results of several studies

2700 times greater than the value reported by Hayes [26], was published by Tzoris [29] Also, Ilartmann [30] determined a very high TC 30 value of 625 mg 1.1 ‘The authors followed the provedure according to DIN ISO 8192 by using the Strathtex rospiroi

tr Dalvell [27] used the same method according lo DIN 180 8192 but detennined a much lower EC 50 value of 28 mg L'' This shows onec again how large the variation of the activaled-sludge respiration-inlubition test can be Ochoa-Herrera et al |31] published an

EC 50 value of 4.6 ng L*t of the degradation of glucose by activated sludge heterotrophs The aim of the studies of Ochoa-Herrera et al is to investigate the inhibition effect of Cu(I)

on the removal of nutrients in wastewater by several microbial trophic groups Other KC! 50 values of 32.07, 59.9 and 8.17 were published by Gutidrey [32], Madoui [33] and Goyeu

[34]

19

'Table 1-5: Inhibiloey effects of Cu(IT} ơn acivated-sincdae bacteria

39.9 activated sludge 1h offline respiration Madoni

Oxymax-ER 1U Gas Respirometer by Columbus Instnments

(USA)

44H activated sludge = 10 oin offline — Raroxymeter 'Tzmis

putida

determine an EC 50 values of 4.31 mg L~ using nitrifying sludge as biomass.

'Table 1-6: Inhibiloey effects of Zn( IT) om activated-sindge hactaria

3578 activated sludge 3h offing — Fleetrolytic Guticrre

respiromelsr Model [32]

BI-1000 Rioscicness,

Ine,, Bethlehem, PA,

[37] reports 19.36 after 3h of the addition of the loxiesnl Au RC 50 value of 60 mg 1.7 aller

an incubation time of 30 tin to acclimatizd activated sludge is reported by Cokgor [38]

As cxpected, this finding is controversial, as acclimatized activated sludge should indicate a higher tolerance to chromium addition compared to non-acclimatized activated sludge Cheng [39] reports that 5 mg L*' Cr(V1) decreased the efficiency of NHs removal from 97%

to 58% and of COD trom 93% to 76% Cokgor et al [38] used the OECD 209 nutrient

solution with an incubation time of 30 min to determine an EC50 valne of 60 mg L.'

arimann [30] used the Strathtox respirometer following the procedure according to DIN