Remediation of Abandoned Shipyard Soil by Organic ... - CORE

Remediation of Abandoned Shipyard Soil by Organic Amendment

Using Compost of Fungus Pleurotus pulmonarius

by

C H A N Sze Sze

A Thesis Submitted in Partial Fulfillment

of the Requirements for the

Degree of Master of Philosophy

in

Biology

• The Chinese University of Hong Kong

July, 2005

The Chinese University of Hong Kong holds the copyright of this thesis. Any person(s) intending to use a part or whole of the materials in the thesis in a proposed publication must seek copyright release from the Dean of the Graduate School.

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THESIS COMMITTEE:

Prof. S.W. Chiu (Supervisor)

Prof. N. F. Y. Tarn (External Examiner)

Prof. P. O. Ang, Jr (Internal Examiner)

Prof. P.K. Wong (Internal Examiner)

Acknowledgements

In this two-year fruitful and unforgettable M.Phil study, I wish to acknowledge the

following persons. First, I would like to express m y heartfelt gratitude to m y

supervisor Professor Siu-wai Chiu for her kind supervision and encouragement.

With her valuable and critical opinion, I could experience a meaningful project.

M y special thanks go to m y thesis committee members, Professor Put 〇.Ang, Jr. and

Professor RK. Wong, for their kind guidance and valuable suggestion throughout m y

M . Phil study. Furthermore, I would like to acknowledge m y external examiner,

Prof. N. F. Y. Tam, who examines m y thesis.

I wish to thank Ms. L. M. Wai, Mr. H. C. Ho, M s Jessie Lee, Mr. Freddie Kwok, M r

W. H. Wong and M r K. H. Man. Their technical advices and enthusiastic help

smoother! m y study. Also, I want to take this chance to thank Mr. Chiu, Mr. Ng

and Mr. Poon in Civil Engineering Development Department, The Government of

H K S A R and Mr. Andrew Kwan, Mr. Eddie Tse, Mr. Eric Wong and shipyard site

staff in Gammon Construction Ltd. for technical support and assistance.

Furthermore, I would like to acknowledge m y labmates (Ms. K. C. Chan, Ms. Polly

Fu, Ms. K.M. Ho, Mr. K.H. Lai, Ms. W. Y. Luk, Ms. S. Liang, Mr. S.T. Ting, Mr. Y.

K. Wong, Ms. S. N. Yau and Mr. M. H. Yu) for their support, continuous

encouragement and help. In addition, I sincerely thank m y friends in Andrew

Fellowship for their spiritual support and pray during m y study. I would like to

express m y genuine gratitude to Mr. K.H. Ser for his endless support. Moreover, I

would like to thank m y God who gives me strength and perseverance to continue.

Last but definitely not the least, I appreciate m y family for their unconditional love,

tolerance and support during m y M.Phil study.

i

Abstract

Polycyclic aromatic hydrocarbons (PAHs) and heavy metals such as lead, copper and

zinc were found in an abandoned shipyard site in North Tsing Yi，Hong Kong.

Total PAHs (45 士 13 mg/kg) and heavy metal levels (lead: 2700 土 370 mg/kg; copper:

1290 ± 1 7 7 mg/kg and zinc: 794 士 106 mg/kg) exceeded the international safety

standards. These originated from the storage and leakage of oil, uses of

metal-containing paints and winching of ships. Some PAHs such as chrysene,

benz[a]anthracene, benzo[a]pyrene and indeno[l,2,3-c,d]pyrene are carcinogenic and

pose threat to human health. 4- to 6- rings PAHs were detected in the soil while no

2- to 3- rings PAHs was found. Due to the strong adsorption onto soil particles and

organic matter, PAHs are recalcitrant, persistent and low bioavailability in soil and

sediment. All the PAHs determined in the site are the priority pollutants listed by

USEPA.

The first part of this study was to investigate the effectiveness of mushroom

Pleurotus pulmonarius compost added to the biopile in remediation of the

organopollutants. On site ex-situ biopile was the bioremediation method specified

by the Hong Kong Government. One ton of P. pulmonarius mushroom compost

was produced in the mushroom cultivation complex, Department of Biology, The

Chinese University of Hong Kong. The compost was then mixed with 90 tons of

soil in a biopile to form fungal treatment. The performance of fungal treatment was

compared with biopile treatment. Soil PAH contents were weekly determined by

gas chromatography mass spectrometry. Oil and grease and total petroleum

hydrocarbons (TPH) were also monitored by gravimetric methods while soil

bacterial and mold populations were determined by viable cell counts. On Day 4,

ii

most of the PAHs in fungal treatment dropped below the initial levels while PAHs in

biopile treatment were increased above the initial concentrations expressed in mg/kg.

In both biopile treatment and fungal treatment, the P A H concentrations rose above

the initial levels and reached peak values during monitoring. All peak PAH

contents in fungal treatment were significantly less than those in biopile treatment

except pyrene. It took less time for the high molecular weight (5- and 6-rings)

PAHs to completely disappear before the low molecular weight (4-ring) PAHs.

Although all detected PAHs were degraded at the end of the 109 day monitoring

period in both treatments, residual TPH and oil and grease contents in biopile

treatment (TPH: 952 士 33 mg/kg; oil and grease: 4380 士 432 mg/kg) were still higher

than those in fungal treatment (TPH: 594 士 48 mg/kg; oil and grease: 3310 士 325

mg/kg). The greater biodegradation in fungal treatment could be provided by the

larger bacterial and mold population sizes in fungal treatment than biopile treatment.

Also, immobilized laccase and manganese peroxidase in the mushroom compost

could contribute by degrading organopollutants including high molecular weight

hydrocarbons. Although the initial total nitrogen and phosphorus contents in both

types of treatment soil were not different, the final contents were greater in fungal

treated soil. Acute toxicities of the fungal treated and biopile treated soil were

assessed using three indigenous bacteria Bacillus cereus, Pseudomonas aeruginosa

and Methylobacterium sp.; four fungi Trichoderma asperellum, T. harziauum,

Fusarium solani and R pulmonarius and three in vitro seed germination tests with

plants: wheat (Triticum aestivum), ryegrass (Lolium perenne) and Chinese cabbage

{Brassica chinensis). Toxicities towards the plants and bacteria decreased

concurrently with the decrease in PAH concentrations after biopile or fungal

treatment. Nevertheless, biopile treated soil was found to be more toxic than the

fungal treated soil towards B. cereus and P. aeruginosa.

iii

Removal of the heavy metals in these treated soils was attempted through chemical,

biological and physical methods. Soil washing could remove > 55% lead; > 50%

copper and > 75% zinc using 0.5 N HCl for 6 hours at 150 ipm at 25' C. For

phytoextraction using three plants T. aestivum, L. perenne and B. chinensis,

mycoextraction using P. pulmonairus and integrated biological extraction using plant

T. aestivum and fungus P. pulmonairus combination, soil lead was excluded from

bioaccumulation in the aerial plant biomass or fungal fruiting bodies. Among them,

mycoextraction could remove the largest amount of metals (copper: 63 士 6 /xg; zinc:

270 士 32 jLtg) in its fruiting bodies. 40% (w/w) cement could stabilize all copper,

lead and zinc in soil such that their leachabilities were below the universal treatment

objectives. Glass encapsulation could limit the leachability of soil metals. In

terms of removal efficiency, soil washing was the best although it generated a large

volume of metal-containing solution for further treatment.

This study reveals the potential in applying R pulmonarius compost in

bioremediation of organopollutants. However, ultimate solution in metal removal is

still under development. More investigation using pilot scale experiments and field

trial would be appropriate for developing P. pulmonarius compost in environmental

cleanup.

iv

摘要

在香港青衣島北一個廢棄了的船塢，泥土含有多環芳香烴(Polycydic aromatic

hydrocarbons MHs)及重金屬如鉛(Lead)、銅(Copper)和鋅(Zinc)。當中的總多環

芳香烴量達每公斤45 ± 13毫克、錯：每公斤2700 ± 370毫克、銅：每公斤1290

士 177毫克及鋅：每公斤794 ± 106毫克，這些汚染物的濃度都超出國際安全標

準。漏油、採用含重金屬的油漆及絞船活動都會釋放這些污染物。多環芳香烴

中的屈(chrysene)、苯并(a)蒽(benz[a]anthracene)、苯并(a)JE(Benzo(a)pyrene)和

(1，2，3-c，d)并苗(indeno[l,2,3-c,d]pyrene)已被證實是致癌物’並對人類的健康構

成威脅。在青衣船塢的泥土中，只驗出有四至六環的多環芳香烴（PAHs)，而沒

有發現二至三環的多環芳香烴（PAHS)。由於多環芳香烴（PAHs)能強力地吸附

在泥土及沈積物微粒及有機物上，因而表現出十分頑強、持久及低生物利用度

的特性，在青衣船塢泥土中發現的多環芳香烴都屬於美國環保署(USEPA)分類爲

優先處理的污染物。

本硏究首要目的是硏究鳳尾薛堆肥物pu lmonar i us mushroom compost)

能否提升生物堆(biopile)處理有機污染物的成效。香港政府原用現場非原位生物

堆(biopile)的方法處理北青衣船塢的有機污染物’這生物堆處理(biopile treatment)

會作對照。真菌處理(fungal treatment)是將一噸在香港中文大學生物系的真菌裁

培綜合實驗室製造的鳳尾薛堆肥物，與九十噸的污泥混合，然後進行同一環境

調控。每星期生物堆處理和真菌處理泥土中的多環芳香烴(PAHS)會利用氣相色

譜質譜儀量化，而泥土的油脂(oil and grease)及總石油碳氬化合物(total petroleum

hydrocarbon)則會用測定重量的方法計算。每星期亦會利用活細胞數方法(viable

cell count)檢驗污泥中的細菌及霉菌數量。最後’真菌處理的效率會與生物堆處

理的作比較。第四天後，真菌處理中的大部份多環芳香烴的濃度已低於開始値，

但在生物堆處理的卻高於開始値。在真菌處理及生物堆處理降解過程中，多環

XV

芳香烴的含量會升至高於開始値，濃度達到高峰値後會回落，即污染物消失。

除了蓝(pyrene)外，真菌處理中的所有多環芳香烴的高峰値都比生物堆處理的

高峰値顯著低。五至六環的多環芳香烴比四環的多環芳香烴快達至完全降解。

雖然在109日後，所有多環芳香烴都被生物堆處理和真菌處理完全降解，生物

堆處理後泥土剩下的油脂和總石油碳氫化合物都明顯地比真菌處理後的泥土爲

高。由於真菌處理有較快速的細菌及霉生長，因此真菌處理有較快的生物降解

速度。而且鳳尾猫堆肥物含有漆酶(laccase)和猛過氧化物酶(manganese

peroxidase)。許多科學文獻証明它們可降解碳氧化合物。此外，鳳尾薛堆肥物更

爲泥土提供了氮和磷，真菌處理泥土中的營養含量都比生物堆處理泥土中的爲

高。細菌生長抑制測試，霉菌生長抑制測試及種子發芽率會被用作監測真菌處

理和生物堆處理後泥土的急性毒性。細菌生長抑制測試利用原位分離的蠟狀芽

？ c e r e u s ) , Ujg^^fi® (Pseudomonas aeruginosa) S. ^ SI¥M

{Methylobacterium sp.);而霉菌生長抑制J 9\試貝(1禾I[用木霉菌(Trichoderma

asperellum 禾口 T. harzianum)，鐮刀菌{Fusarium solani)fPJH®IS{Pleurotus

pulmonarius)。種子發芽實驗會利用三種植物，分別是小麥(ThY/cwm aestivum),黑

麥草力和小白菜 C B r ^ m / c a chinensis)。結果發現，當多環芳香烴的

濃度減少，泥土對於種子及細菌的毒性均減少。然而，及cereus和P. aeruginosa

反映生物堆處理後的泥土毒性比真菌處理的爲高。

本硏究的第二個目的是比較重金屬的化學、生物及物理處理方法。土壤冲洗法

(soil washing)的最佳條件是利用0.5N驢旨麦於25度室溫搖動泥土六小時，轉速爲

每分鐘150轉。於此最佳化的條件下，土壤冲洗法能去除多過55%的錯、多過

50%的銅及最少75%的鋅。結果又指出利用小麥，黑麥草和小白菜作植物萃取

(phytoextraction);鳳尾蔽作真菌萃取(mycoextraction)及綜合生物萃取(小麥和鳳

尾薛)都不能吸收並積累泥土中的錯。比較三項生物處理方法，利用鳳尾薛真菌

萃取(mycoextraction)能在其擔子果積累最多的銅(63 ± 6微克)及鋅(270 士 32微

克)。40%的水泥固化(cementation)能穩固泥土中的鉛、銅和鋅，而它們的浸出性

vi

都低於國際標準。至於玻璃封裝(glass encapsulation)，它能減低金屬的浸出性。

因此土壤冲洗法是最大清除效率的金屬處理方法，但這方法會產生大量的金屬

廢水。

本硏究完全展露了有效的鳳尾薛堆肥物對有機污染物的實地生物修復

(bioremediation)。至於重金屬的修復，其最完善的處理方法仍有待硏究。

vii

Contents Page

Acknowledgements i

Abstracts ii

摘要 V

Contents viii

List of figures xv

List of tables xix

Abbreviations xxii

1 Introduction 1

1.1 The North Tsing Yi Abandoned Shipyard area 1

1.2 Polycyclic aromatic hydrocarbons (PAHs) in the site 3

1.2.1 Characteristics of PAHs 3

1.2.2 Sources of PAHs 8

1.2.3 Environmental fates of PAHs 9

1.2.4 Biodegradation of PAHs 10

1.2.5 Toxicity of PAHs 13

1.2.6 PAHs contamination in Hong Kong 14

1.2.7 Soil decontamination assessment in Hong Kong 16

1.2.8 Environmental standards of PAHs 18

1.2.9 Remediation technology of PAHs 21

1.2.9.1 Bioremediation 22

1.3 Heavy metals in the site 28

1.3.1 Characteristics of copper, lead and zinc 29

viii

1.3.2 Sources of copper, lead and zinc 32

1.3.3 Environmental fates of copper, lead and zinc 34

1.3.4 Toxicities of copper, lead and zinc 36

1.3.5 Copper, lead and zinc contamination in Hong Kong 39

1.3.6 Environmental standards of copper, lead and zinc 40

1.3.7 Remediation technology of heavy metal 42

1.3.7.1 Chemical method 42

1.3.7.2 Biological method 43

1.3.7.3 Stabilization and Solidification 45

1.4 Aim of study 47

1.5 Objectives 47

1.6 Research Strategy 47

1.7 Significance of study 48

2 Materials and Methods 49

2.1 Soil Collection 49

2.2 Characterization of soil 49

2.2.1 Sample preparation 49

2.2.2 Soil pH, electrical conductivity & salinity 50

2.2.3 Total organic carbon contents 51

2.2.4 Soil texture 51

2.2.5 Moisture 53

2.2.6 Total nitrogen and total phosphorus 53

2.2.7 Available nitrogen 53

2.2.8 Available phosphorus 54

2.2.9 Soil bacterial and fungal population 54

2.2.10 Extraction of PAHs and organic pollutants 55

ix

2.2.10.1 Extraction procedure 55

2.2.10.2 GC-MS condition 56

2.2.10.3 Preparation of mixed PAHs stock solution 56

2.2.11 Oil and grease content 57

2.2.12 Total Petroleum Hydrocarbons (TPH) 57

2.2.13 Total heavy metal analysis 58

2.2.14 Toxicity characteristic leaching procedure (TCLP) 59

2.2.15 Extraction efficiency 59

2.3 Production of mushroom compost 60

2.4 Characterization of mushroom compost 62

2.4.1 Enzyme assay 62

2.4.1.1 Laccase assay 62

2.4.1.2 Manganese peroxidase assay 62

2.5 Addition of mushroom to soil on site 63

2.5.1 Transportation of mushroom compost to Tsing Yi 63

2.5.2 Mixing of mushroom compost and soil 64

2.6 Soil Monitoring 64

2.6.1 On site air and soil measurements 64

2.6.1.1 Air temperature and moisture 64

2.6.12 Light intensity 64

2.6.1.3 U V intensity 65

2.6.1.4 Rainfall 65

2.6.1.5 Soil temperature 65

2.6.2 Soil chemical characteristic 65

2.6.3 Relative residue pollutant (%) 65

2.7 Toxicity of treated soil 66

XV

2.7.1 Seed germination test 66

2.7.2 Indigenous bacterial toxicity test 67

2.7.3 Fungal toxicity test 68

2.7.3.1 Preparation of ergosterol standard solution 70

2.8 Soil Washing 70

2.8.1 Optimization of soil washing 70

2.8.1.1 Effect of hydrochloric acid concentration 70

2.8.1.2 Effect of incubation time 71

2.9 Phytoremediation 71

2.10 Mycoextraction 72

2.11 Integrated bioextraction 72

2.12 Cementation 73

2.13 Glass encapsulation 73

2.14 Statistical analysis 74

3 Results 75



3.2.1 Enzyme activity 78

3.2.2 Total nitrogen and total phosphorus contents 78

3.3 Soil monitoring 79

3.3.1 Initial pollutant content in biopile and fungal treatment soils 79

3.3.2 On site air and soil physical characteristics 81

3.3.2.1 Soil temperature and air temperature 81


3.3.3.1 Effect of type of treatment on total petroleum hydrocarbon

content 85

xi

3.3.3.2 Effect of type of treatment on oil and grease content 87

3.3.3.3 Soil pH 89

3.3.3.4 Moisture 91

3.3.3.5 Electrical conductivity 92

3.3.3.6 Salinity 93

3.3.3.7 Microbial population 95

3.3.3.8 Removal of organopollutant PAHs in biopile and fungal

treatment 98

3.3.3.9 Effect of type of treatment on residual PAHs at Day 4 104

3.3.3.10 Effect of type of treatment on residual PAHs at peak

levels 107

3.3.3.11 Effect of type of treatment on residual organopollutants

at the end of treatments 109

3.3.3.12 Effect of type of treatment on total nitrogen and

phosphorus contents 111

3.3.3.13 Effect of type of treatment on physical and chemical

properties of soil 113


3.4.1 Seed germination test 116

3.4.2 Indigenous bacterial toxicity test 120

3.4.3 Fungal toxicity test 125

3.5 Soil washing 129

3.5.1 Optimisation of soil washing 129

3.5.1.1 The effect of hydrochloric acid concentration 129

3.5.1.2 The effect of incubation time 134


xii

3.7 Phytoextraction and integrated bioextraction 146

3.8 Cementation 153


4 Discussion 160



4.2.1 Enzyme activity 162

4.2.2 Total nitrogen and total phosphorus contents 163

4.3 Soil monitoring 163

4.3.1 Initial pollutant content in biopile and fungal treatment soil 163

4.3.2 On site air and soil physical characteristics 164


4.3.3.1 Soil pH 164

4.3.3.2 Moisture 165

4.3.3.3 Electrical conductivity 165

4.3.3.4 Salinity 166

4.3.3.5 Microbial population in biopile and fUngal treatments 166

4.3.3.6 Removal of organopollutant PAHs in biopile and fungal

treatments 168

4.3.3.7 Effect of type of treatment on residual PAHs at peak levels 170

4.3.3.8 Effect of type of treatment on residual oil and grease and

TPH contents 171

4.3.3.9 Effect of type of treatment on total nitrogen and phosphorus

contents 172

4.3.3.10 Effect of type of treatment on physical and chemical

properties of the soil 173

xiii


4.5 Summary of Pleurotus pulmonarius mushroom compost on

organopollutant remediation 177

4.6 Soil washing 178


4.8 Phytoextraction and integrated bioextraction 182

4.9 Cementation 184


4.11 Summary of physical, chemical and biological heavy metal removal

treatments 186

4.12 Future studies 187

5 Conclusion 190

6 References 193

xiv

List of Figures

Figure 1.1 The overview of the North Tsing Yi Shipyard 2

Figure 1.2 Structures of the 16 PAHs on the EPA priority pollutant list. 7

Figure 1.3 A flowchart of essential steps of contamination assessment in Hong 17

Kong

Figure 1.4 A typical diagram of Biopile 26

Figure 2.1 The soil textural classification triangle 52

Figure 2.2 A flowchart showing the production of the mycelial compost of 61

Pleurotus pulmonarius (Pl-27)

Figure 2.3 A 3D sketch diagram of the target area of Biopile 63

Figure 3.1 The change of soil and air temperatures during the monitoring of 82

biopile and fungal treatment

Figure 3.2 The change in residue total petroleum hydrocarbon contents with 86

time

Figure 3.3 The change in residue oil and grease contents with time 88

Figure 3.4 The change in soil pHs of the biopile and fungal treatment with time 90

Figure 3.5 The change in soil moisture of the biopile and fungal treatment with 91

time

Figure 3.6 The change in soil conductivity of the biopile and fungal treatment 92

with time

Figure 3.7 The change in soil salinity (%) of the biopile and fungal treatment 93

with time

Figure 3.8 The change in total bacteria population of the biopile and fungal 96

treatment with time

Figure 3.9 The change in total mold population of the biopile and fungal 97

treatment with time

XV

Figure 3.10 The change of soil fluoranthene contents during biopile and fungal 99

treatment

Figure 3.11 The change of soil pyrene contents during biopile and fungal 100

treatment

Figure 3.12 The change of soil benz[a]anthracene contents during biopile and 100

fungal treatment

Figure 3.13 The change of soil chrysene contents during biopile and fungal 101

treatment

Figure 3.14 The change of soil beiizo[a]pyrene contents during biopile and fungal 101

treatment

Figure 3.15 The change of soil benzo[g,h,i]perylene contents during biopile and 102

fungal treatment

Figure 3.16 The change of soil indeno[ 1,2,3-cd]pyrene contents during biopile 102

and fungal treatment

Figure 3.17 The relative PAHs contents (%) at Day 4 in comparison to their initial 105

concentrations in biopile treatment and fungal treatment soil

Figure 3.18 The volatile profiles of (a) biopile treatment and (b) fungal treatment 106

soil revealed by GC-MS on Day 0 and Day 4

Figure 3.19 A comparison of the peak P A H levels during biopile treatment and 108

fungal treatment

Figure 3.20 The volatile profiles of (a) biopile treatment and (b) fungal treatment 110

soil revealed by GC-MS on Day 109

Figure 3.21 The change in total nitrogen contents at Day 0, Day 4 and Day 109 of 112

biopile treatment and fungal treatment soil

Figure 3.22 The change in total phosphorus contents at Day 0, Day 4 and Day 109 112

of biopile treatment and fungal treatment soil

Figure 3.23 Photographs of the soil before (a) biopile treatment and (b) fungal 115

treatment; and after (c) biopile treatment and (d) fungal treatment

Figure 3.24 The effects of the biopile treatment and fungal treatment on the 116

germination frequencies of wheat Triticum aestivium


germination frequencies of ryegrass Lolium perenne


germination frequencies of Chinese cabbage Brassica chinensis.

xvi

Figure 3.27 Photographs of in vitro acute toxicity test using plants: (a) Triticum 119

aestivum, (b) Lolium perenne and (c) Brassica chinensis on biopile

treated and fungal treated soils


population growth of Bacillus cereus


population growth of Pseudomonas aernginosa


population growth of Methylobacterium sp.

Figure 3.31 Photographs of the recovered colonies from the in vitro acute toxicity 124

test using the three indigenous bacteria


population growth of Trichoderma asperellum


population growth of Trichoderma harziauum


population growth of Fusarium solani


population growth of Pleurotus pulmonarius

Figure 3.36 The effect of hydrochloric acid concentrations on the removal of 131

heavy metals in the (a) biopile treated soil and (b) fungal treated soil.

Figure 3.37 The effect of hydrochloric acid concentrations on the heavy metal 132

leachability of (a) biopile treated soil and (b) fungal treated soil.

Figure 3.38 The effect of hydrochloric acid concentration on the residual total 133

heavy metal concentrations in (a) biopile treated soil and (b) fungal

treated soil.

Figure 3.39 The effect of incubation time on removal of heavy metals of (a) 135

biopile treated soil and (b) fungal treated soil.

Figure 3.40 The effect of incubation time on the heavy metal leachability of (a) 136


Figure 3.41 The effect of incubation time on the residual total heavy metal 137

concentrations in (a) biopile treated soil and (b) fungal treated soil

Figure 3.42 Photographs of the (a) biopile treated and (b) fungal treated soils after 138

soil washing

xvii

Figure 3.43 The effect of compost amounts on metal concentrations in fruiting 141

bodies of Pleurotus pulmonarius grown in (a) biopile treated soil and

(b) fungal treated soil

Figure 3.44 The effect of compost amount on metal amount in fruiting bodies of 142

Pleurotus pulmonarius grown in (a) biopile treated soil and (b) fungal

treated soil

Figure 3.45 The effect of mycoextraction on the heavy metal leachability of (a) 143

biopile treated soil and (b) fungal treated soil

Figure 3.46 The effect of mycoextraction on the residual total heavy metal 144

concentrations in (a) biopile treated soil and (b) fungal treated soil

Figure 3.47 Photographs of the effect of soil to compost ratio (w/w) on removal 145

of heavy metals from the (a) biopile treated soil and (b) fungal treated

soil

Figure 3.48 Effects of phytoextraction and integrated bioextraction of wheat 149

Triticum aestivum and compost of Pleurotus pulmonarius

on removal of metals from (a) biopile treated soil and (b) fungal

treated soil


Triticum aestivum and compost of Pleurotus pulmonarius on heavy

metal leachability of (a) biopile treated soil and (b) fungal treated


Triticum aestivum and compost of Pleurotus pulmonarius on residual

heavy metal concentration of (a) biopile treated soil and (b) fungal

treated soil.

Figure 3.51 Photographs of the effects of plant species used in extraction of heavy 152

metals from biopile treated soil and fungal treated soil with and

without integration of mycoextraction

Figure 3.52 Effects of cementation on heavy metal leachability of (a) biopile 155

treated soil and (b) fungal treated soil.

Figure 3.53 Effects of cementation on residual heavy metal concentration of (a) 156


Figure 3.54 Photographs of the 40% cementation of (a) biopile treated and (b) 157

fungal treated soil.

Figure 3.55 Photographs of glass encapsulation of the biopile treated and fungal 159

treated soils.

xviii

List of Tables

Table 1.1 The characteristics of representative PAHs 5

Table 1.2 Estimated half-lives of PAHs in various environmental media 6

Table 1.3 The European Dutch, Canadian, United Stated and Australia 19

environmental standards for selected PAHs in soil

Table 1.4 Approximate costs of different remediation technologies 22

Table 1.5 Characteristics of copper, lead and zinc 31

Table 1.6 The European Dutch, Canadian, United Stated and Australia 41

environmental standards for selected heavy metals in soil

Table 2.1 Relationship between the reading and the corresponding salt content 50

Table 2.2 Particle size distributions of sand, silt and clay in International Scale 52

Table 2.3 Composition of nutrient agar (NA) plate 55

Table 2.4 Composition of potato dextrose agar (PDA) plate 55

Table 2.5 Conditions of GC-MSD for PAHs analysis 56

Table 2.6 Temperature profile for GC-MSD for PAHs analysis 57

Table 2.7 Extraction efficiencies of PAHs and heavy metals in soil 60

Table 2.8 Extraction efficiencies of heavy metal in aerial part of wheat 60

Table 2.9 Composition of Luria Broth (LB) 67

Table 2.10 Composition of Potato Dextrose Broth 69

xix

Table 2.11 Conditions of G C - M S D for ergosterol analysis 69

Table 2.12 Temperature profile for G C - M S D for ergosterol analysis 69

Table 2.13 The amounts of glass used for embedding different amounts of soil. 74

Table 3.1 The physical and chemical properties of North Tsing Yi Abandoned 77

Shipyard soil

Table 3.2 The enzyme activities of mushroom compost 78

Table 3.3 The total nitrogen and total phosphorus contents of mushroom compost 78

Table 3.4 The initial pollutant concentration of biopile treatment and fungal 80

treatment soil

Table 3.5 The heavy metal leachability of biopile treatment and fungal treatment 80

soil.

Table 3.6 The on site physical properties during monitoring of both biopile and 83

the fungal treatment

Table 3.7 The results of the two-way A N O V A analyses on the study of total 86

petroleum hydrocarbons during the treatments of the contaminated soil

Table 3.8 The results of the two-way A N O V A analyses on the study of relative oil 88

and grease content during the treatments of the contaminated soil

Table 3.9 The results of the two-way A N O V A analyses on the studies of pH， 94

moisture, electrical conductivity and salinity during the treatments of

the contaminated soil

Table 3.10 The results of the two-way A N O V A analyses on the studies of total 97

bacterial population and total mold population during the treatments of

the contaminated soil

Table 3.11 The results of the two-way A N O V A analyses on the studies of 103

individual PAHs during the treatments of the contaminated soil

Table 3.12 The physical and chemical properties of soil after biopile treatment and 114

fungal treatment

Table 3.13 The number of wheat {Triticum aestivum), ryegrass (Lolium perenne) 116

and Chinese cabbage (Brassica chinensis) seeds germinated in garden

soil

XV

Table 3.14 The initial population sizes of Bacillus cereus, Pseudomonas 121

aeruginosa and Methylobacterium sp.

Table 3.15 A summary of population growth of Bacillus cereus, Pseudomonas 123

aeruginosa and Methylobacterium sp. in biopile treated, fungal treated

and garden soil

Table 3.16 The initial population sizes of Trichoderma asperellum, Trichoderma 126

harziauum, Fusarium solani and Pleurotus pulmonarius in terms of

ergosterol content

Table 3.17 A summary of population growth of Trichoderma asperellum, 128

Trichoderma harziauum, Fusarium solani and Pleurotus pulmonarius in

biopile treated, fungal treated and garden soil in terms of % increase in

ergosterol content

Table 3.18 The initial total heavy metal concentrations and metal leachability in 130

biopile treated soil and fungal treated soil used for the study of soil

washing

Table 3.19 The initial total heavy metal amounts and metal leachability in biopile 140

treated soil and fungal treated soil which were used for removal of

metals by mycoextraction

Table 3.20 The initial total heavy metal amounts and metal leachability in biopile 148

treated soil and fungal treated soil which were used for removal of

metals by phytoextraction and integrated bioextraction

Table 3.21 The biomass gain (aerial part dry weight) of three testing species after 148

4-week planting

Table 3.22 The initial total heavy metal concentration and metal leachability in 154

biopile treated soil and fungal treated soil which were used for removal

of metals by cementation

Table 3.23 The initial total heavy metal concentration and metal leachability in 158

biopile treated soil and fungal treated soil which were used for removal

of metals by glass encapsulation

Table 3.24 The TCLP values of glass ampoules after encapsulation 159

Table 4.1 The optimal soil properties for plant growth 162

Table 4.2 A summary of relative PAHs, microbial population and nutrient 170

contents in biopile treatment and fungal treatment on Day 4

xxi

Abbreviations A A S Atomic absorption spectrophotometer

A N O V A Analysis of variance

B E N Benzo[a]pyrene

BEN(A) Benz[a] anthracene

cfli Colony forming unit

C H R Y Chrysene

D C M Dichloromethane

EE Extraction efficiency

F L U O Fluoranthene

GC-MSD Gas chromatography- mass selective detector

GHI Benzo [g,h,i]perylene

HCl Hydrochloric acid

H K E P D Hong Kong Environmental Protection Department

H M W High molecular weight

HNO3 Nitric acid

lARC International Agency for Research on Cancer

ICP Inductively coupled plasma spectrophotometer

INDENO Indeno[ 1,2,3-cd]pyrene

Koc Organic carbon partition coefficient

Kow Octanol-water partitioning coefficient

LB Luria Broth

LD50 Median lethal dose

L M W Low molecular weight

M R L Minimum risk level

N A Nutrient agar

PAHs Polycyclic aromatic hydrocarbons

P D A Potato dextrose agar

POPs Persistent organic pollutants

P Y R Pyrene

RE Removal efficiency

TCLP Toxicity characteristic leaching procedure

TOC Total organic carbon

TPH Total petroleum hydrocarbon USEPA United States Environmental Protection Agency

xxii

Chapter 1 Introduction

1.1 The North Tsing Yi Abandoned Shipyard area

The North Tsing Yi shipyard area has been occupied by 19 shipyard sites, and it has

been abandoned since May 2000. Also, there were 19 workshops situated near the

site boundary of the reclamation project contracted by the H K S A R government.

The reclamation project involved construction of about 330 m of sloping seawall and

about 160 m vertical seawall, reclamation by public fill of about 3.8 hectares from

foreshore and seabed, and formation of about 3.2 hectares reclaimed soil from

vacated shipyards. The bare soil and concrete surfaces of buildings and shipyards

areas are contaminated. The main contaminants are expected to be oils, greases and

solvents, and metals from the paints and swarf. There may be localized areas of

high oil contamination where activities such as winching the ships or storage of

waste oil have been carried out (MEMCL, 2001). According to M E M C L (2001),

soil samples at most areas of the subject site are found contaminated with heavy

metals, total petroleum hydrocarbons (TPH), polycyclic aromatic hydrocarbons

(PAHs), phenols, polychlorobiphenyl (PCBs,) benzene, toluene, ethylbenzene and

xylenes (BTEX) and tributyltin (TBT). Also, the contents of these pollutants

exceeded the Dutch C (requires remediation) levels. Therefore, soil in North Tsing

Yi shipyard was heterogeneous and mixed with organic and inorganic pollutants.

Actually it is quite usual of mixed contaminants, about 80% of US Superfund sties

(designated by USEPA as priority sites for cleanup) contaminated with heavy metals

and organic pollutants (Ensley, 2000). There are 1,400,000 sites contaminated with

heavy metals and/or organic pollutants in Western Europe alone (ETCS, 1998). The

presence of both types of contaminants on the same site represents technical and

economic challenges for decontamination strategies.

1

Figure 1.1 The overview of the North Tsing Yi Shipyard (Source: HKCEDD).

2

1.2 Poly cyclic aromatic hydrocarbons (PAHs) in the site

1.2.1 Characteristics of PAHs

PAHs are non-polar, neutral, and hydrophobic organic compounds made up of two or

more fused benzene rings arranged in linear, angular or cluster form (Luthy et al.,

1997; ATSDR, 1995). There are more than 100 different PAHs. Being derivatives

of the benzene rings, they are thennodynamically stable due to their large negative

resonance energy (Mueller et al., 1996). As pure chemicals, PAHs generally exist

as colorless, white, or pale yellow-green solids. They can have a faint, pleasant odor.

PAHs are common, persistent and recalcitrant environmental contaminants with a

tendency to bioaccumulate (Crawford and Crawford, 1996; Harvey, 1997). Soil and

sediment are major sinks of PAHs (Wild and Jones, 1995; Durrant et al., 1999).

PAHs generally occur as complex mixtures (for example, as part of combustion

products such as soot), not as single compounds. 16 PAHs are listed as priority

pollutants by the USEPA (Giraud et al； 2000). The characteristics and molecular

structure of some common PAHs are listed in Table 1.1 and Figure 1.2 respectively.

In general, PAHs have low water solubilities, high melting and boiling points, and

low vapor pressure. Solubility decreases, melting and boiling points increase and

vapour pressure decreases with increasing molecular weight of a PAH (Miller and

Olejnik, 2001; Atagana, 2004). Transport and partitioning of PAHs in the

environment are determined to a large extent by their physicochemical properties

such as water solubility, vapor pressure, Henry's law constant, octanol-water

partition coefficient (Kow), and organic carbon partition coefficient (Koc). The

Henry's law constant is the partition coefficient that expresses the ratio of the

concentrations of a chemical in air and water at equilibrium and is used as an

indicator of the potential of the chemical to volatilize. The Koc indicates the

3

potential of the chemical to bind to organic carbon in soil and sediment. The Kow is

used to estimate the potential for an organic chemical to move from water into lipid

and has been correlated with bioconcentration in aquatic organisms. Some of the

transport and partitioning characteristics (e.g., Henry's law constant, Koc values, and

Kow, values) of the PAHs are roughly correlated to their molecular weights. So

from Table 1.1，when molecular weight increases, water solubility decreases, the Log

Kow and Log Koc increase. The higher molecular weight a PAH has, the higher

potential to bind to organic carbon in soil and higher potential to move from water

into lipid will be. As an example, Hattemer-Frey and Travis (1991) found that the

low solubility, low vapor pressure and high Kow of benzo[ajpyrene result in its

partitioning mainly between soil (82%) and sediment (17%), with roughly 1%

partitioning into water and < 1 % into air, suspended sediment and biota.

PAHs are persistent organic pollutants (POPs) in the environment, having long

half-lives in soils, sediments, air or biota. However, in practice a POP could have a

half-life of years or decades in soil/sediment and several days in the atmosphere

(Jones and Voogt, 1999). According to the definition based on the half-life of an

organic compound in soil of over 100 days, all PAHs (except naphthalene) are

deemed persistent (Park et al., 1990; Launen et al., 1995). Table 1.2 shows the

half-lives of some representative PAHs in different environmental media. The

half-lives of PAHs are different based on their molecular weight, shape, media and

other environmental parameters. The mean half lives of 4- and 5- rings PAHs are 2

years (Table 1.2). At hazardous waste sites, half-lives of PAHs may be longer since

other contaminants at the site may be toxic inhibiting to microbial growth and

biodegradation.

4

Table 1.1 The characteristics of representative PAHs (Mackay

et al., 1992; ATSDR, 1995; Majcherczyk

et al., 1998; Juhasz and Naidu, 2000).

(No. of r

ings)

fo

rmula

(g

/mê

) Pom

t (。

C)

ô^^

25°C

(Pa)

(m

g/L

)at2

5

C

mm

g/k

gj

cla

ssific

ation <

Nap

hth

alene (2)

Cio

Hg

128.1

8

80.2

217.9

6.5

6

31

J 2.6

6

3.3

7

1,7

80

C

Ace

nap

hth

yle

ne (2)

CnH

g

152.2

1

92.5

265

1.2

2 x

lO]

393

3.5

9

4.0

7

1,7

00

D

Ace

naphth

ene

(2)

C12

H10

154.2

1

96.2

277.5

3.3

3 x

10'

3

9

3.6

6

4.3

3

600

/

Flu

ore

ne (2)

C13

H10

166.2

1

116.5

295

1.1

2

1.8

9

3.7

6

4.1

8

2,0

00

D

Phe

nant

hren

e (3)

C14

H10

178.2

4

99.2

340

9.0

7 x

'

1.1

5

4.1

5

4.4

6

700

D

Ant

hra

cene

(3)

ChHio

178.2

4

216.2

340

2.6

1 x

10"'

0.0

7

4.2

4.4

5

1,4

70-2,4

00

D

Flu

ora

nth

ene (3)

Cig

Hio

202.6

11

1

250

9.1

1 x

0.2

6

4.6

5

5.3

3

2,0

00

D

Pyr

ene (4)

Cie

Hjo

202.6

15

3

404

8.0

0 x

0.1

4

4.7

8

5.3

2

2,7

00

D

Chr

yse

ne (4)

C18

H12

228.2

8

256

448

8.4

0x 1

0.5

0.0

02

4.8

9

5.6

1

>320

B2

Ben

z[a]

anth

race

ne (4)

C18

H12

228.2

8

158

400

6.6

7 x

10"

0.0

12

4.5

6

5.6

1

>200

B2

Benz

o[b

]flu

ora

nthene

(4)

C20

H12

252.3

2

167.5

357

6.6

7 x

10"

0.0

012

5.7

4

6.5

7

72

B2

Benz

o[k

]flu

ora

nthene

(4)

C20

H12

252.3

2

217

480

6.6

7 x

10'

0.0

0055

5.9

2

6.8

4

ND

B

2

Ben

zo[a

]pyre

ne (5)

C20

H12

252.3

2

176.5

495

6.6

7 x

'

0.0

038

6.2

6

6.3

1

50

B2

Dib

enz

[a,h

]ant

hra

cene

(5)

C22

H14

278.3

6

270

524

1.3

0x 1

0.8

0.0

005

6.2

2

5.9

7

ND

B

2

Indeno[l,2

,3-c,

d]p

yre

ne (6)

C22

H12

276.3

4

163

536

1.3

0 x

10'

0.0

62

7.2

7.6

6

ND

B

2

Benzo

[g,h

,i]pery

lene (6)

C22

H12

276.3

4

273

550

1.3

3 x

10"

0.0

0026

6.2

6

6.6

3

ND

D

a Log K

oc：

Logar

ithm

of org

anic

car

bon p

artition c

oeffic

ient;

Log

Kqw：

Logarith

m o

f oct

anol-

wat

er

part

itio

nin

g c

oeffic

ient;

"LD

SA：

Leth

al dose

mean

s the

amount

of

a c

hem

ical

it

took o

rally

to k

ill

50 %

of ra

ts;

'U

SEPA

car

cinogenic

ity c

lass

ific

atio

n:

B2 =

pro

bab

le h

um

an c

arci

nogen;

C =

poss

ible

hum

an

carc

inogen; D

= n

ot cl

assi

fiab

le a

s to c

arci

nogenic

ity.

5

Table 1.2 The estimated half-lives of 16 priority PAHs in various environmental

media (Mackay et al., 1992).

Class * Air Water Soil Sediment

priority pollutants^^^^

Naphthalene 2 4 6 7

Acenaphthylene 3 5 7 8

Fluorene 3 5 7 8

Phenanthrene 3 5 7 8

Anthracene 3 5 7 8

Fluoranthene 4 6 8 9

Pyrene 4 6 8 9

Chrysene 4 6 8 9

B enz [ a] anthracene 4 6 8 9

B enzo [k] fluoranthene 4 6 8 9

Benzo[a]pyrene 4 6 8 9

Dibenz[a,h] anthracene 4 6 8 9

where

Class* Mean half-lives (hours) Range (hours)

1 5 <10

2 17 (〜1 day) 10-30

3 55 (〜2 days) 30-100

4 170 (〜1 week) 100-300

5 550 (~3 weeks) 300- 1,000

6 1,700 (〜2 months) 1,000-3,000

7 5,500 (〜8 months) 3,000 - 10,000

8 17,000 (2 years) 10,000 - 30,000

9 55,000 (6 years) > 30,000

6

^

II I ( ^ " ^ V ^ I I I ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^

Naphthalene Acenaphthene Acenaphthylene Phenanthrene

Fluorene Anthracene Benz[a] anthracene

Chrysene Pyrene Fluoranthene

Benzo[b]fluoranthene Benzo[k] fluoranthene Benzo[a]pyrene

Indeno[l,2,3-cd]pyrene Benzo[g，h,i]perylene Dibena[a,h] anthracene

Figure 1.2. Structures of the 16 PAHs on the USEPA priority pollutant list.

7

1.2.2 Sources of PAHs

Polycyclic aromatic hydrocarbons (PAHs) are released to the environment through

natural and synthetic sources. Natural sources include emissions from volcanoes

and forest fires. Synthetic sources include incomplete combustion of fossil fuels,

petroleum refinery, manufactured gas production (Bewley et al. 1989; Tumey and

Goerlitz 1990), creosote production (Ellis et al. 1991; Hansen et al., 2004), wood

treatment facilities (Weissenfels et al 1990; Mueller et al. 1991)，coking plants

(Werner et al. 1988; Weissenfels et al. 1990), and automobile and truck emissions

(Durrant et al., 1999; W u et al., 2003; Roy, et al., 2005). Synthetic sources provide

a much greater volume release than natural sources. The principal sources of PAHs

in soils along highways and roads are vehicular exhausts and emissions from wearing

of tires and asphalt. Also, PAHs can be manufactured as individual compounds for

research purposes in contrast to the mixtures found in combustion products.

Environmental tobacco smoke, unvented radiant and convective kerosene space

heaters, and gas cooking and heating appliances are the significant sources of PAHs

in indoor air. A few PAHs are used in medicines and to make dyes, plastics, and

pesticides (ATSDR，1995). PAHs are found throughout the environment in the air,

water and soil. They can occur in the air as attached to dust particles or in soil or

sediment as solids. Thus PAHs are produced naturally and artificially as a result of

combusion. Human cannot avoid the exposure (Juhasz and Naidu, 2000).

Hazardous waste sites can be the concentrated sources of PAHs on a large scale.

PAHs have been identified in at least 600 of the 1,408 hazardous waste sites that

have been proposed for inclusion in the USEPA National Priorities List (NPL)

(HazDat 1994). Examples of PAHs contaminated sites are abandoned

wood-treatment plants (sources of creosote) and former manufactured-gas sites

8

(sources of coal tar). The anthropogenic sources of PAHs are the current focus of

many environmental cleanup programs and consequently the basis for the

development of effective bioremediation technologies (Muller et al., 1996).

1.2.3 Environmental fates of PAHs

The environmental fate of PAHs includes volatilization as well as abiotic and biotic

transformations. Volatilization is important only for 2-ring PAHs such as

naphthalene and 1 -methylanpthalene. Abiotic mechanisms account for up to 20%

of total reduction in PAHs during bioremediation. Only the biotic mechanisms are

responsible for removal of PAHs containing more than 3-rings (Park et al., 1990).

PAHs released to the atmosphere are strongly bounded to air borne organic

particulates and gases. Particle-bound PAHs are subject to short- and long-distance

transport and are removed by precipitation, wet and dry deposition onto soil, water,

and vegetation. Most of the PAHs determined in plant tissues are probably derived

from atmosphere.

PAHs can enter surface water through atmospheric deposition and from discharges of

industrial effluents (including wood-treatment plants), municipal waste water, and

improper disposal of used motor oil. In surface water, PAHs are transformed by

volatilization, photooxidation, chemical oxidation，biodegradation, sorption to

suspended particles or sediments, or accumulated in aquatic organisms (with

bioconcentration factors often in the 10- 10,000 range) (ATSDR, 1995).

PAHs in soil can volatilize, undergo abiotic degradation (photolysis and oxidation),

biodegrade, or accumulate in plants. PAHs in soil can also enter groundwater and

be transported within an aquifer. Sorption of PAHs to soil and sediments increases

9

with increasing organic carbon content and with increasing surface area of the

sorbent particles. However, in soil and sediments, microbial metabolism is the

major process for degradation of PAHs. Photolysis, hydrolysis, and oxidation

generally are not considered to be important processes for the degradation of PAHs

in soils (Sims and Overcash 1983). In most cases, when soil has been contaminated

for decades, the high molecular weight PAHs are detected in soil while smaller PAHs

have degraded (Ahtiainen et al., 2002). In sediments, PAHs can biodegrade or

accumulate in aquatic organisms. PAHs usually accumulate in organisms with high

lipid contents and poor microsomal monooxygenases system, and high molecular

weight ( H M W ) PAHs are most difficult to be excreted (Varanasi et al., 1989).

1.2.4 Biodegradation of PAHs

PAHs have low aqueous solubilities which limit bioavailabilities (Ahtiainen et al.,

2002). They are strongly adsorbed onto soil particles especially clays (Luthy et al.,

1997). 5- and 6-ring PAHs have especially very low water solubilities and are often

tightly bound to soil particles (Kotterman et al., 1998; Al-Daher et al., 2001).

Biodegradability decreases with an increase in the number of benzene rings in PAHs.

Also few reports deal with biodegradation of PAHs containing 6-rings. A general

sequence of petroleum components in the order of decreasing biodegradability is

represented as follows: n-alkanes > branched-chain alkanes > branched alkenes >

low molecular weight n-alkyl aromatics > monoaromatics > cyclic alkanes >

polynuclear aromatics » » asphaltenes (Ward and Singh, 2004a).

Bacteria

Low bioavailability of PAHs is a major rate-limiting factor in the degradation of

these compounds by bacteria. When bioavailability of oxidized PAH metabolites are

10

increased, these compounds can be more easily mineralized by bacteria (Kotterman

et al., 1998). Bacterial isolates often degrade only a narrow range of PAHs

(Bouchez et al., 1995). Also only a very limited number of bacteria have been

isolated that can grow in pure cultures on PAHs with five or more aromatic rings

(Johnsen et al., 2005). In no case is the PAH utilized as the sole carbon source by

bacteria.

Bacteria initiate PAH degradation by the action of intracellular dioxygenases. PAHs

must be taken up by the cells before degradation can take place. Bacteria oxidize

aromatic compounds by incorporating both atoms of molecular oxygen into the

aromatic substrate to form a cis-dihydrodiol (Mueller et al,, 1996; Johnsen et al.,

2005). The reaction is catalyzed by a multicomponent dioxygenase, consisting of a

flavoprotein, and iron-sulfur protein and a ferredoxin. Further oxidation of the

cis-dihydrodiol leads to the formation of catechols in NAD.-dependent

dehydrogenation reactions. Then the aromatic ring was cleaved by dioxygenase

enzymes to give aliphatic intermediates (Rosenberg and Ron, 1996). These

intermediates are channelled into central pathways of metabolism, i.e. T C A cycle,

where they are oxidized to provide cellular energy or used in biosynthesis of cellular

constituents (Mueller et al., 1996).

Fungi

The non-ligninolytic fungi degrade PAHs using intracellular detoxification processes.

PAHs are oxidized to epoxides by cytochrome P-450 monooxygenase (Cemiglia,

1984). The expoxides are then non-enzymatically rearranged to phenols, which can

be conjugated. Alternatively, the epoxides are converted to trans-dihydrodiols by

epoxide hydrolases (Cemiglia, 1984).

11

Beside the intracellular enzymatic systems, the ligninolytic fungi degrade PAHs by

non-specific radical oxidation, catalized by extracellular ligninolytic enzymes known

as laccase, lignin peroxidase and manganese peroxidase (Cemiglia and Sutherland

2001; Reddy and Mathew, 2001; Johnsen et al； 2005). Intracellular ezymes such as

hydrolase such as endoglucanase (CMC) and cellobiohydrolase (CBH) were also

found in Pleurotus pulmonarius (Velazquez-Cedeno et al., 2002). These enzymes

are non-specific and oxidize a wide variety of organic compounds. The lack of

selectivity may be due to the random structure of lignin. The ligninolytic enzymes

cleave the C-C and C-0 bonds of the lignin molecules regardless of the chiral

conformations of the lignin molecule (Fernando and Aust, 1994). This bond fission

may result partially from the free radial mechanism of lignin degradation of white rot

fungi. The free radical species generated during lignin or organopollutant

degradation process may serve as secondary oxidants, which may mediate the

oxidation of other compounds away from the active sites of the enzymes (Barr and

Aust, 1994). Laccase uses oxygen molecules to oxidize phenolic compounds to

very reactive, free radicals. The presence of primary, mediating substrates extend

the substrate range of laccases to non-phenolic aromatics by forming potent radicals

which co-oxidize non-phenolic lignin compounds and also PAHs (Pickard et al.,

1999). Some ligninolytic fungi can further metabolize the PAH quinines by

cleaving the aromatic rings, with subsequent breakdown of the PAH to carbon

dioxide (Hammel, 1995). The ligninolytic enzymes have the advantage that they

may diffuse to the highly immobile PAHs sorbed to soil. This is very different to

bacterial PAH-dioxygenases, which are generally cell-bound because they require

N A D H as a co-factor (Johnsen et al., 2005).

12

1.2.5 Toxicities of PAHs

PAHs cause a significant environmental risk and human health threat. They are

considered both mutagenic and carcinogenic, although the toxicity and

carcinogenicity of different PAHs vary widely (Table 1.1) (Roy et al., 2005). PAHs

can enter the body through lungs when air is breathed in. Cigarette smoke, wood

smoke, coal smoke, and smoke from many industrial sites may contain PAHs.

People living near hazardous waste sites can also be exposed by breathing air

containing PAHs. However, it is not known how rapidly or completely lungs

absorb PAHs. Drinking water and swallowing food, soil or dust particles that

contain PAHs are other routes for these chemicals to enter the body, but absorption is

generally slow when PAHs are swallowed. For Chinese cuisine for roasted pork or

western barbecue foods, the PAH contents in these products are regulated. Under

normal conditions of environmental exposure, PAHs could enter the body through

skin contacts with soil which contains high levels of PAHs (Sims and Overcash, 1983;

vanAgteren et al., 1998; Kelley et al” 1990).

Several of the PAHs, including benz[a] anthracene, benzo [a]pyrene,

benzo [b] fluoranthene, benzo [j ] fluoranthene, benzo [k] fluoranthene, chrysene,

dibenz [a,h] anthracene, and indeno [ 1,2,3 -c,d]pyrene, have caused tumors in

laboratory animals after inhalation, eating and long time skin contact with them.

According to the USEPA toxicity category, the oral LD50 of PAHs on rats are from

caution to warning levels (e.g. phenanthrene: 700 mg/kg and benzo[a]pyrene: 50

mg/kg) (Table 1.1.). Studies with human subjects show that individuals exposed by

breathing or skin contact for long periods to mixtures that contain PAHs can also

develop lung and bladder cancers (Mastrangelo et al., 1996).

13

USEPA has determined that benz[a] anthracene, benzo[a]pyrene,

benzo[b] fluoranthene, benzo[k] fluoranthene, chrysene, dibenz[a,h] anthracene and

indeno[l,2,3-c,d]pyrene are probable human carcinogens while acenaphthylene,

anthracene, benzo[g,h,i]perylene, fluoranthene, fluorene, phenanthrene and pyrene

are not classifiable as to human carcinogenicity. The International Agency for

Research on Cancer (lARC) has determined the following: benz[a]anthracene and

benzo[ajpyrene are probably carcinogenic to humans; benzo[b]fluoranthene,

benzo[j]fluoranthene, benzo[k]fluoranthene and indeno[l，2,3-c,d]pyrene are possibly

carcinogenic to humans; and anthracene, benzo[g,h,i]perylene, benzo[e]pyrene,

chrysene, fluoranthene, fluorene, phenanthrene, and pyrene are not classifiable as to

their carcinogenicity to humans. Acenaphthene has not been classified for

carcinogenic effects by the lARC or USEPA.

1.2.6 PAHs contamination in Hong Kong

Airborne PAHs are monitored under the Toxic Air Pollutants (TAP) Programme on a

long-term regular basis to evaluate the effectiveness of strategic air quality control

measures since 1997 (Lee et al., 2001; Sin et al., 2003). From 1984, the Hong

Kong Environmental Protection Department commenced the monitoring the

concentrations of the total PAHs and individual PAHs in marine sediments at 52

stations in ten water quality control zones and 13 stations in typhoon shelters

(Connell et al., 1998a and 1998b). Fluorene, phenanthrene, anthracene,

fluoranthene, pyrene, chrysene, benzo [k] fluoranthene, benzo[a]pyrene and

benzo[g,h,i]perylene have been reported in Hong Kong sediments. Marine bottom

sediments in Hong Kong have been seriously contaminated with PAHs, especially in

certain areas of Victoria Harbour (Hong et al., 1995, Connell et al., 1998a and Zheng

and Richardson, 1999). However, there are also reports about PAH contamination

14

in mangrove sediments. Total PAHs (from 56 to 3758 ng g—were found in surface

sediments in Yi 0，Ma Wan, Sheung Pak Nai and Ting Kok mangrove swamps.

The PAH concentrations in some mangrove swamps were higher than those in

marine bottom sediments (Tarn et al., 2001). Tarn et al. (2001) reported that surface

sediments of Mai Po and Ho Chung mangrove swamps were seriously contaminated

with the mean total PAHs concentrations of 6.19 and 1.95 /xg/g (dry wt) respectively.

Another study by Zheng et al. (2002) showed that the Mai Po sediments were less

contaminated, with total PAHs concentrations ranging from 0.18 to 0.96 /xg/g (dry

wt). The estimated half-lives of PAHs in Hong Kong marine bottom sediments

ranged from 2.9 to 10.2 years, with an average of 6.7 years (Cormell et al., 1998b).

Ke et al. (2005) suggested that the PAH contamination was the most severe between

1958 and 1979 but started to decline. This decline was most likely due to changes

in petroleum usage in urban areas and a better control of wastewater discharges from

1980 onwards.

In Hong Kong, there were PAH-contaminated sites with 15 - 200 mg/kg in coastal

soils, such as the former Kai Tak Airport and Penny's Bay in Lantau Island (HKEPD,

2002). The concentration of PAHs of these sites exceed the Dutch B and sometimes

even Dutch C level. In the target site of this study, 7 PAHs (4-6 rings) were

reported (Total PAHs = 22-12 mg/kg). The site is mixed contaminated with other

toxic organics and inorganics. But the PAHs found in soil or sediment still caused

serious health effects, damaged the marine ecosystem and posed a risk to consumers

of seafood harvested (Connell et al., 1998a and 1998b). Further, it is noteworthy

that the deleterious effects are likely to be additive to those of other co-existing

contaminants such as polychlorobiphenyl and dichlorodiphenyltrichloroethane

(Connell et al, 1998a).

15

1.2.7 Soil decontamination assessment in Hong Kong

To cope with the rapid development of Hong Kong and the great demand for land for

various purposes, sites that may have been contaminated due to their former usage

are also considered for redevelopment. In order to avoid or minimize any risks or

hazards associated with these sites, the project proponent should carry out a site

contamination assessment and implement remediation measures to clean up the land

if necessary prior to any redevelopment works. H K E P D identified a number of

industries as having potential for causing land contamination, including oil

installations; gas work, power plants, shipyards/boatyards, chemical manufacturing/

processing plants; steel mills/metal workshops and car repairing/dismantling

workshops (HKEPD, 1994 & 1999).

The project proponent should follow the guidance issued by HKEPD. Prior to the

commencement of the land contamination site investigation work, the project

proponent has to prepare and submit a Contamination Assessment Plan (CAP) to

EPD for endorsement. Based on the endorsed CAP, the project proponent should

conduct a contamination assessment and compile a Contamination Assessment

Report (CAR) to document the findings for approval by the EPD. If the findings

confirm that the site is contaminated, a Remediation Action Plan (RAP) should be

drawn up. A wide range of land remediation measures for restoration of

contaminated sites from worldwide were listed in the guidance issued by HKEPD.

These include: recovery trenches or wells; soil venting, biotreatment; immobilization;

excavation and landfilling. However, excavation and landfilling were considered as

the last resort of remediation as this would transfer much of the burden to landfills

with land a scarce and valuable resource in Hong Kong (HKEPD, 1994). The RAP

16

and C A R may be submitted as a combined report for EPD's approval referencing the

corresponding CAP prior to the commencement of clean up work on site. The

procedure of the contamination assessment process is summarized in the flow chart

in Figure 1.3.

Site appraisal (�nformatkm coHectlon)

• V , Plan & design sfte investigation

^ V Obtain endorsement of

Contamination Assessment Plan (CAP) fmm EFD {

, • • Sfte Investlgatton

(sample collection end laboratory tests) • „ — — — • - 一 ~

Result Interpretation �

Prepare Contamination Assessment Report (CAR) and submit to EfiD

^ ^ 歸 ^ Keep GAR for future record

Prepare Remecflation Action Plan (RAP) and obtain approval from EPD

」"."‘'11.”I」"」“”。W-….....-,〜、. Implement RAP

SjHiRIIHHiHRIIIillllllilllililliPMPIIIPIIiHMIilliillilliRPIiRiRniiiillliPRiiiliiliil ‘

Figure 1.3 A flowchart of essential steps of contamination assessment in Hong

Kong (redrawn from HKEPD, 1999).

17

1.2.8 Environmental standards of PAHs

There are a number of international references of safe levels for PAHs including

those from United States, Canada, the Netherlands and Australia. Since the Dutch

Indicative Index is considered more comprehensive in terms of its coverage of

parameters, this is almost the mostly commonly used reference worldwide (HKEPD,

1994). Hong Kong EPD has adopted this as a general guideline for most of the

cases. The Dutch "ABC" criteria consist of 3 levels of standard: Dutch A,

unpolluted; Dutch B, pollution present and requires further investigation or

remediation; Dutch C, significant pollution present and requires remediation

(HKEPD, 1994). H K E P D requires remediation of soil with contamination above

Dutch B level. The Dutch and other countries standards of PAHs are listed on Table

1.3.

18

Table 1.3. The European Dutch, Canadian, United Stated and Australian environmental standards for selected PAHs in soil (Wilson and Jones,

1993; Crawford and Crawford, 1996; Mueller

et al,, 1996; CCME, 2002; ADEP, 2003).

USEPA

priority

! D

utc

h S

tandar

ds

Can

adia

n E

nvironm

enta

l Q

uality

Guid

elines

USEPA

Ris

k B

ased

Aust

ralia H

ealth Invest

igation L

evels

pollu

tants

(m

g/k

g)

(mg/k

g)

Conce

ntr

atio

n (

mg/k

g)

(mg/k

g)‘

AB

C

Agricultura

l Res/

Par

k C

om

merc

ial

Indust

rial

Indust

rial

Resi

dential

AB

C

D

E

F

Nap

hth

alene

oT

^ 5

50

0.1

0.6

22

22

20000

1600

60

/ /

/ /

190

Phe

nant

hrene

0.1

10

10

0

0.1

5

50

50

/ /

/ /

/ /

丨

綱

Anth

race

ne

0.1

10

10

0

/ /

/ /

310000

23000

21900

/ _

/ /

/ 100000

Flu

ora

nth

ene

0.1

10

10

0

/ /

/ /

40880

3129

2290

_!_

/

/ /

30100

Pyre

ne

II

I 0.1

10

10

0

100

30660

2346

2310

/ /

/ /

54220

Chr

yse

ne

0.1

5

50

/ /

/ /

392

87

//

//

//

Ben

z[a]

anth

race

ne

0.1

5

50

0.1

1

10

10

3.9

2

0.8

7

/ /

/ /

/ /

Benzo

[b]f

luora

nth

ene

II

I 0.1

1

10

10

3.9

2

0.8

7

/ /

/ /

/ /

Benzo

[k]f

luora

nth

ene

0.1

5

50

0.1

1

10

10

39

9

//

//

//

Ben

zo[a

]pyre

ne

0.0

5

1

10

0.1

0.7

0.7

0.7

0.3

9

0.0

9

1

/ /

4

2

5

Dib

enz

[a,h

]ant

hra

cene

I

II

0.1

1

10

10

0.3

9

0.0

9

/ /

/ /

/ /

Indeno[l

,2,3

-c,

d]p

yre

ne

0.1

5

50

0.1

1

10

10

4

1

//

//

//

Benzo

[g,h

,i]pery

lene

0.1

10

10

0

/ /

/ /

/ /

//

//

//

Tota

l PA

Hs

10

500

1000

/

Tar

get

Inte

rvention

20

/ /

80

40

100

1

40

19

a A

ust

ralia

heal

th in

vest

igat

ion le

vels

:

A. Sta

ndar

d resi

dential

with g

arden/

acce

ssib

le s

oil (hom

e g

row

n p

roduce

contr

ibuting le

ss than

10%

of vegeta

ble

and fru

it inta

ke; no

poultry

); this

cat

egory

incl

udes c

hild

ren's

day

care

cent

res,

kin

derg

arte

ns,

pre

-sc

hools

and p

rim

ary

sch

ools

.

B. Resi

dential

with s

ubst

antial

vegeta

ble

gar

den (co

ntr

ibuting 1

0%

or m

ore

of vegeta

ble

and fru

it in

take) an

d/o

r poultry

pro

vid

ing a

ny

egg o

r poultry

meat

die

tary

inta

ke.

C. Resi

dential

with s

ubst

antial

vegeta

ble

gar

den (co

ntr

ibuting 1

0%

or m

ore

of vegeta

ble

and fru

it in

take);

poultry

excl

uded.

D. Resi

dential

with m

inim

al opport

unitie

s for

soil a

cces

s: in

cludes d

wellin

gs w

ith fully o

r perm

anently p

aved y

ard s

pace

such

as h

ighrise

apar

tmen

ts a

nd fla

ts.

E. Par

ks,

recr

eat

ional

open s

pace

and p

layin

g field

s, incl

udes s

eco

ndar

y s

chools

.

F. C

om

merc

ial/In

dust

rial

, in

cludes p

rem

ises s

uch a

s s

hops

and o

ffic

es a

s w

ell a

s fac

tories a

nd indust

rial site

s

20

1.2.9 Remediation technology of PAHs

In the US Superfund sites, 79% of common remediation technologies are established

technologies in the remediation for environmental cleanup. They are mainly

physical or chemical processes. Within all common remediation technologies, there

are 21% being innovative. Also bioremediation makes up more than half of the

innovative technologies (USEPA 2004b). The specific bioremediation process

applied in Superfund sites was (% of sites): natural attention (28%), biopiles (16%),

landfarming (7%) and bioventing (0.8%). Non-bioremediation approaches used

were: landfilling (34%), soil vapor extraction (9%), thermal desorption (3%),

incineration (2%) and soil washing (0.2%) (Ward and Singh, 2004).

During selection for a favorable type of remediation technology, several factors need

to be considered. They are: (1) contaminant type and characteristics including

properties, volume, location, exposure risk; (2) site characteristics including soil

types, permeability, surface and ground water properties, climate; (3) capital,

operating and maintenance costs; (4) remediation schedule; (5) regulatory and public

acceptance (EC, 1997). Table 1.4 shows the approximate cost of physical, chemical

and biological treatment. Biological treatment is much cheaper than physical and

chemical treatment such as solvent extraction, soil washing and incineration.

Biological treatment is more competitive than physical and chemical treatment in

terms of cost. Bioremediation is an environmental friendly and relatively

cost-effective alternative to conventional physico-chemical soil treatment techniques

(Plaza et al., 2005).

21

Table 1.4. Approximate costs of different remediation technologies (Semple et al.,

2001).

Treatment Approximate cost (£/tonne)

Physical treatment

Soil washing 25 - 150

Vapour extraction 75

Chemical treatment

Solvent extraction 50 - 600

Chemical dehalogenation 175 - 450

In situ flushing 25 - 80

Thermal treatment

Thermal desorption 25-225

Incineration 50-12000

Biological treatment

Biopiles 15-35

Land farming 10-90

Bioventing 15-75

Bioslurry 50-85

1.2.9.1 Bioremediation

Bioremediation is the use of biological treatments, for the clean-up of hazardous

chemicals in the environment (Al-Daher et al., 2001; Semple et al., 2001). It can be

regarded as a more effective and environmentally friendly strategy since it results in

the partial or complete biotransformation of organoxenobiotics to carbon dioxide,

water and microbial biomass which are stable, innocuous end-products (Colleran,

22

1997; Al-Daher et al., 2001; Gestel et al., 2003).

There are two approaches of bioremediation: biostimulation and bioaugmentation.

Biostimulation means stimulating native microbial activity by introducing nutrients,

oxygen and etc. It is of low cost. But it has disadvantages of only effective at

limited number of sites and of uncertain results. Bioaugmentation means

stimulating bioremediation with additional microorganisms. It has the advantage of

increased rate of remediation. But it has the disadvantage of being expensive

(Jorgensen et al., 2000; Watanable et al., 2001).

Petroleum-contaminated soil can be bioremediated by the addition of nutrients

(biostimulation), the addition of microbial inocula (bioaugmentation), aeration and

turning, or by a combination of these practices (Gestel et al., 2003). Some

scientists would add inorganic fertilizer, straw, wood chips, activated sludge and

manure to facilitate biodegradation of organopollutants in piles (Al-Daher et al.,

2001; Chaineau et al., 2003; Juteau et al., 2003; Atagana, 2004b; Miller et al., 2004).

Bioremediation has also been applied with reintroduction of indigenous

microorganisms isolated from a contaminated site, or inoculated with white-rot fungi,

or the addition with compost, in which some microorganisms are introduced to the

soil.

There are two main treatment formats in bioremediation, ex situ and in situ. For ex

situ treatment, there are landfartning, biopile and composting. Actually,

landfarming is quite similar to biopile. Both of them are above-ground, engineered

systems that use oxygen to stimulate the growth and reproduction or aerobic bacteria

to degrade the petroleum constituents adsorbed to soil. However, landfarms are

23

aerated by tilling or plowing, but biopiles are aerated by forcing air to move by

injection or extraction through slotted or perforated piping placed under the pile

(Biowise，2000). Composting refers to the addition of compost's primary

ingredients to contaminated soil, where the compost matures in the presence of the

contaminated soil (Semple et al.’ 2001). While for in situ applications, it includes

bioslurry, bioventing and natural attenuation (Jorgensen et al., 2000; Watanable et al.,

2001; USEPA, 2004b). One of the major obstacles in bioremediation of soils

contaminated with synthetic organic compounds is the failure of laboratory

remediation schemes to simulate the impact of field soil conditions on both the

contaminant and the microorganism (Ward and Singh, 2004). So it is important to

have field application.

Biopiles

Biopile technology involves forming petroleum contaminated soils into piles or

windrows above ground and stimulating aerobic microbial activity within the soils

through aeration (Jorgensen et al., 2000). By using microorganisms to degrade

contaminants in soil, biopile transforms hazardous/toxic materials into harmless

elements such as water, carbon dioxide and other innocuous products. The height

of a biopile is usually 2 - 4 m. Biopile may be amended with a bulking agent,

usually with straw, sawdust, bark or wood chips (Chaineau et al., 2002). It is also

very common to add compost to biopile. The compost can be sewage sludge,

mature compost and etc (Al-Daher et al., 2001; Juteau et al., 2003). Biopile

treatment of TPH and PAHs contaminated soil have been applied in field studies

(Von Fahnestock et al, 1998; Al-Daher et al., 2001; Mohn et al” 2001; Chaineau et

a!., 2003; Juteau et al., 2003).

24

Biopile in North Tsing Yi Shipyard

Figure 1.4 shows the diagram of a typical biopile. The largest biopile is around

20,000 m . The biopile base consists of impermeable liner, soil bund and leachate

collection channel. The foundation of the biopile will be graded at approximately

1% to 2 % fall towards the leachate collection channel. The soil bund serves as a

containment to divert all leachate to the collection channel. An impermeable liner

will be placed over the base. The liner is typically 0.75 m m thick plastic material

such as high-density polyethylene (HDPE). The aeration system of biopile will be

operated in the extraction mode and consists of aeration pipelines including

perforated pipe and main suction pipe, valves at the manifold branch points, air

blowers-high performance pressure blower; carbon filter units and pressure gauge.

The maximum airflow of the blower system is 20,000 m^ per hour (i.e. 12,000 cubic

feet per minute). In the Tsing Yi abandoned shipyard site, two blowers were used at

early stage of biopile operation and only one blower for continuous operation. The

off-gas treatment system is an activated carbon filter unit which is established to

remove volatile organic compound (VOC). A PVC nylon membrane which is a

waterproof plastic will be used to cover the biopile during operation. Portions of

cover will be removed to facilitate soil mixing/turning or soil sampling.

25

Treated Off-Gas

( ) Qff'Qm / treatment

B1 帽 er V ^

Ojnci nsate Collection T^nk

个 / / * ^ * Qsntaminated Soil *

I » m * '"峰, » » •*" << 1

Figure 1.4. A typical diagram of Biopile (redrawn from USEPA, 2004b).

26

Pleurotus pulmonarius mushroom compost

In the remediation of organopollutants by biopile, Pleurotus pulmonarius mushroom

compost was added to biopile soil to form the fungal treatment. Pleurotus

pulmonarius belongs to white rot fungi and has the ability to efficiently degrade

lignin, which is a naturally occurring aromatic polymer. This ability of white rot

fungi is due to the ligninolytic enzymes such as lignin peroxidase, manganese

peroxidase, laccases (Reddy, 1995; Bezalel et al., 1996; Novotny et al., 1999; Canet

et al., 2001; Rabinovich et al., 2004). These enzymes are extracellular and able to

catalyze radical formation by oxidation to destabilize the bonds in a molecule

(Kotterman et al., 1998; Ball and Jackson, 1995; Hofrichter et al., 1999). The

polyaromatic structure of both lignin and polycyclic aromatic hydrocarbons

suggested that the same fungi are able to degrade these pollutants (Bezalel et al.,

1996; Canet et al., 2001). White rot fungi could mineralize PAHs like pyrene,

fluoranthene, benzo[a]pyrene to CO2 in liquid medium (Bezalel et al., 1996) and soil

medium (Gramss et al., 1999; Novotny et al., 1999; Eggen and Sasek, 2002).

Rodriguez et al. (2004) reported that Pleurotus pulmonarius could transform 100 u M

benzo[a]pyrene up to 75% in a 2-week incubation period while Lau et al. (2003)

applied the spent compost of P. pulmonarius to degrade PAHs including

benzo [ajpyrene in soil system. Mineralization of benzo [ajpyrene in liquid cultures

was only observed with P. pulmonarius.

During soil bioremediation, white rot fungi are normally applied in the form of

mycelium grown on wood chips, sawdust, chopped straw which is then mixed with

the contaminated soil (Novotny et al., 1999). Then, the fungi grow by hyphal

extension and thus can reach pollutants in the soil in ways that other organisms

cannot (Reddy and Mathew, 2001). White rot fungi like Phanerochaete

27

chrysosporium and several fungi belonging to the genus Pleurotus were proven to be

good soil colonizers (Andersson and Henrysson, 1996; Martens and Zadrazil, 1998;

Kotterman et al., 1999). Straw mushroom compost produced in the mushroom

industry has been investigated as an inoculum in the bioremediation of

chlorophenol-contaminated soil. Laine and Jorgensen (1996) showed after a

3-month catabolic induction stage the induced mushroom straw compost of Agaricus

bisporus could mineralize up to a 56% of the added PCP amount to CO2 whereas

uninduced compost did not mineralize the chlorophenol.

The advantage of using mushroom compost is the ability to improve the poor

physical characteristics and nutrient status of restoration later (Wong and Ho, 1994).

Also inorganic fertilizers are not as good as mushroom compost because they are

known to contain various amounts of heavy metals, including Cd and Cu

(Kabata-Pendias and Pendias, 1992). Continuous and heavy application of these

soil amendments can potentially exacerbate the accumulation of heavy metals over

time. Moreover, the fungal extracellular enzyme system is constitutive or inducible

by the organic contaminants even in the presence of heavy metal contamination.

(Canet et al., 2001).

1.3 Heavy metals in the site

Unlike many organic pollutants that can be eliminated or reduced by chemical

oxidation techniques or microbial activity, heavy metals could not be degraded.

Heavy metals will continue to be an environmental concern for a long time unless

they are taken out from the ecosystems. More than 50,000 metal-contaminated sites

await remediation in the US alone (Ensley, 2000). In this project, copper, lead and

zinc were the target metals as they exceeded the Dutch Intervention levels.

28

1.3.1 Characteristics of copper, lead and zinc

The characteristics of copper, lead and zinc are summarized in Table 1.5

Copper (Cu)

Copper is a reddish-brown metal that occurs naturally in rock, soil, water, sediment

and at low levels in air. Copper possesses high electrical and thermal conductivity

and resists corrosion (WHO, 1998). It is insoluble in water with high melting and

boiling points (Table 1.5). It is an essential element for all known living organisms

including humans and other animals at low levels of intake. Copper is a metal

cofactor for many enzymes, e.g. laccase.

Lead (Pb)

Lead is a naturally occurring silvery grey metal found in small amounts in the earth's

crust. It has no characteristic taste or smell. Metallic lead does not dissolve in

water and does not bum. Lead can combine with other chemicals to form what are

usually known as lead compounds or lead salts. Some lead salts dissolve in water.

Some natural and manufactured substances contain lead but do not look like lead in

its metallic form. Some of these substances can be burnt 一 for example, organic

lead compounds in some gasolines (WHO, 1995).

Zinc (Zn)

Zinc is one of the most common elements in the earth's crust. In its pure elemental

(or metallic) form, zinc is a bluish-white, shiny metal. Powdered zinc is explosive

and may burst into flames if stored in damp places. Zinc has many uses in industry.

A common use is to coat iron or other metals to prevent rust and corrosion. Zinc is

also mixed with other metals to form alloys such as brass and bronze. A zinc and

copper alloy is used to make pennies in the United States. Zinc is also used to make

29

dry cell batteries. Zinc can also combine with other elements, such as chlorine,

oxygen, and sulfur, to form zinc compounds. The solubility of ionic zinc is pH and

anion dependent (WHO, 2001; ATSDR, 2003).

30

Table 1.5

Characteristics of heavy metals (Mason

et al., 1992; ATSDR, 1997; W

HO, 2001; ATSDR, 2003; ATSDR, 2004).

Mole

cula

r IM

cltins

Boilin

g

USEPA

H

eav

y

Vap

or pr

ess

ure

Wat

er

solu

bility

Tole

rable

daily inta

ke

Min

imum

Ris

k L

evel

..

weig

ht

poin

t poin

t carc

inogenic

ity

meta

l ^

(Pa)

(mg/L

)at2

5

(TD

I) (ug/p

ers

on/d

ay)

mg/k

g/d

ay

i .广

,a

(g/m

ole

) (°

C)

CC

) cla

ssific

ation

Copper

63 5

10

83

2324

130 (1628

Inso

luble

180

0.0

1 b

D

(C

u)

Lead

207.1

9

327.4

1740

240 (1000 °

C)

Inso

luble

75

/ B

2

(Pb)

Zin

c

65.3

7

419

907

133 (487

Inso

luble

300 -

1000

0.3

'

D

(Zn)

a USEPA

carcinogenicity classification: B2

= probable h

uman

carcinogen; C

= possible h

uman

carcinogen; D

= not classifiable as to

carcinogenicity

An acute (1-14 days )and intermediate (15-364 days ) oral M

RL

c An intermediate (15-364 days) and chronic (365 days or over) oral M

RL

31

1.3.2 Sources of copper, lead and zinc

Copper

Copper occurs naturally in all plants and animals. At much higher levels, toxic

effects can occur. It is also found in many mixtures of metals, called alloys, such as

brass and bronze. Many compounds (substances formed by joining two or more

chemicals) of copper exist. These include naturally occurring minerals as well as

manufactured chemicals. The most commonly used compound of copper is copper

sulfate. Many copper compounds can be recognized by their blue-green color

(ATSDR, 2004).

High residues of copper are found in copper or other metal mines, metal-handling

factories, landfills, and waste disposal sites. The copper in tailings represents the

portion of copper that could not be recovered from the ore and is generally in the

form of insoluble sulfides or silicates (Perwak et al. 1980). Copper can also enter

the environment through waste dumps, domestic waste water, combustion of fossil

fuels and wastes, wood production, phosphate fertilizer production, municipal refuse,

waste from electroplating, iron and steel producers, and discarded copper products

(e.g., plumbing, wiring) and natural sources (for example, windblown dust, from

native soils, volcanoes, decaying vegetation, forest fires, and sea spray). Therefore,

copper is widespread in the environment. About 1.4 billion pounds or 640 thousand

metric tonnes of copper were released into the environment by industries in year

2000 (ATSDR, 2004). Copper has been identified in at least 906 of the 1,647

hazardous waste sites that have been proposed for inclusion on the USEPA National

Priorities List (NPL) (HazDat, 2004).

Lead Major sources of lead contamination of soil include lead mining and smelting

activities, disposal of lead-based paints, and lead battery reclamation, burning of

fossil fuels use of fertilizers, pesticides and sewage sludge (Kim et al., 2003; Kos and

Lestan, 2003). Most lead used by industry comes from mined ores ("primary") or

from recycled scrap metal or batteries ("secondary"). Lead is used in a large variety

of medical equipment (radiation shields for protection against X-rays, electronic

ceramic parts of ultrasound machines, intravenous pumps, fetal monitors, and

surgical equipment). Lead is also used in scientific equipment (circuit boards for

computers and other electronic circuitry) and military equipment (jet turbine engine

32

blades, military tracking systems) (ATSDR, 1997). Lead is important in the

production of some types of batteries. It is also used in the production of

ammunition, in some kinds of metal products (such as sheet lead, solder, some brass

and bronze products, and pipes) and in ceramic glazes. Other chemicals containing

lead are used in paint and pigments. The amount of lead added to paints and

ceramic products, caulking, gasoline and solder has also been reduced in recent years

to minimize lead's harmful effects on people and animals (WHO, 1995).

Urban soils show higher lead concentration than rural soils mainly because of motor

vehicle emissions from using leaded gasoline (Davies，1988). Some chemicals

containing lead, such as tetraethyl lead and tetramethyl lead, were once used as

gasoline additives to increase octane rating. Human activities (such as the former

use of "leaded" gasoline) have spread lead and lead compounds to all parts of the

environment. Lead is also in plants and animals that people may eat. Lead has

been identified in at least 1,026 of the 1,467 current or former USEPA National

Priorities List (NPL) hazardous wastes sites (HazDat, 1998).

Zinc

Zn is released to the environment from both natural and anthropogenic sources.

Zinc is found in air, soil and water and is present in all foods. Natural inputs are

mainly due to igneous emissions and forest fires. Anthropogenic sources of zinc

are mining, purifying of zinc, lead, and cadmium ores, steel production, coal burning,

and burning of wastes (WHO, 2001; ATSDR, 2003). These activities can increase

zinc levels in the atmosphere.

Waste streams from zinc and other metal manufacturing and zinc chemical industries,

domestic waste water, and run-off from soil containing zinc can discharge zinc into

waterways. The level of zinc in soil increases mainly from disposal of zinc wastes

from metal manufacturing industries and coal ash from electric utilities. The

releases to soil are probably the greatest source of zinc in the environment. The

most important sources of zinc in soil are discharges of smelter slag and wastes, mine

tailings, coal and bottom fly ash, and the use of commercial products such as

fertilizers and wood preservatives that contain zinc. Releases of zinc from

anthropogenic sources are always greater than those from natural sources. Zinc has

been identified in at least 953 of the 1,636 hazardous waste sites that have been

33

proposed for inclusion on the USEPA National Priorities List (NPL) (HazDat, 2003).

1.3.3 Environmental fates of copper, lead and zinc

Heavy metals remain mainly in the upper layer of the soil profile or migrate on a

limited basis. The mechanical composition of soil may influence metal

concentration in soil. Wang and Li (1987) reported that heavy metals such as Cu,

Ni, Pb and Zn contents in soil profiles increased in accordance with its clay fractions.

Heavy metals are highly persistent in soil, with residence time in the order of

thousands of years (McGrath, 1987). Among many soil properties, pH is perhaps

the most important, since most heavy metals are more available under acidic

conditions than in neutral and alkaline conditions (Wang, 2000). Excessive

accumulation of heavy metals can have deleterious effects on soil fertility, affect

ecosystem functions and constitute a health risk to animals and human beings (Sun et

al., 2001).

Copper is released from smelters and ore processing plants in the form of particulate

matter or adsorbed to particular matter, and is then carried back to earth through

gravity or in rain or snow. Copper is also carried into the air on windblown

metallurgical dust. Indoor release of copper comes mainly from combustion

processes, e.g. kerosene heaters (WHO, 1998; ATSDR, 2004).

When copper and copper compounds are released into water, the copper which

dissolves can be carried in surface waters either in the form of copper compounds or

as free copper or, more likely, copper bound to particles suspended in the water.

Even though copper binds strongly to suspended particles and sediments, there is

evidence to suggest that some water-soluble copper compounds do enter groundwater.

Copper that enters water eventually is collected in the sediments of rivers, lakes, and

estuaries.

An estimated 97% of copper released from all sources into the environment is

primarily released to land (Perwak et al. 1980). When copper is released into soil,

it can become strongly attached to the organic material and other components (e.g.,

clay, sand, etc.) in the top layers of soil and may not move very far when it is

released. Soil pH, organic matter, soil redox potential, presence of oxides, cation

exchange capacity and proportions of clay to silt to sand particles influence the fate

34

of copper in soil (WHO, 1998). Most copper compounds found in air, water,

sediment, soil and rock are strongly attached to dust and dirt or imbedded in minerals.

Some copper in the environment is less tightly bound to soil or particles in water and

will be soluble in water and taken up by plants and animals. This will threaten

human health.

Most lead emissions from fixed, mobile and natural sources are deposited near the

source, although some particulate matter (2 |Lim in diameter) is transported over long

distances and causes contamination of remote sites (WHO, 1995). Airborne lead

contributes to the human exposures through the contamination of food, water and

dust, as well as through direct inhalation.

Small amounts of lead may enter rivers, lakes and streams when soil particles are

moved by rainwater. Lead may remain stuck to soil particles in water for many

years. Movement of lead from soil particles into underground water or drinking

water is unlikely unless the water is acidic or "soft." Most lead is retained strongly

in soil, and very little is transported into surface water or groundwater (USEPA,

1986).

Disposal of lead in municipal and hazardous waste dump sites increases soil lead

contamination to soil. Sources of lead in dust and soil include lead that falls to the

ground from the atmosphere, and weathering and chipping of lead-based paint from

buildings and other structures. This accumulation of high levels of lead in soil near

roadways came from car exhaust in the past. Once lead falls onto soil, it usually

sticks to soil particles (ATSDR, 1997). Movement of lead from soil will also

depend on the type of lead salt or compound and on the physical and chemical

characteristics of the soil. These processes are dependent on such factors as soil pH,

soil type, particle size, organic matter content of soil, the presence of inorganic

colloids and iron oxides, cation exchange capacity (CEC) and the amount of lead in

soil (Reddy et al., 1995). Lead is strongly sorbed to organic matter in soil, and

although not subject to leaching, it may enter surface water as a result of erosion of

lead-containing soil particulates. Clays, silts, iron and manganese oxides, and soil

organic matter can bind metals electrostatically (cation exchange) as well as

chemically (specific adsorption) (Reed et al. 1995).

35

Although the bioavailability of lead in soil to plants is limited because of the strong

absorption of lead to soil organic matter, the bioavailability increases as the pH value

and the organic matter contents of the soil are reduced. Lead may be taken up in

edible plants from the soil via the root system, by direct foliar uptake and

translocation within the plant, and by surface deposition of particulate matter. The

amount of lead in soil that is bioavailable to a vegetable plant depends on factors

such as cation exchange capacity, pH, amount of organic matter present, soil

moisture content and the type of amendments added to the soil (ATSDR, 1997).

Zinc enters the air, water, and soil as a result of both natural processes and human

activities. In air, zinc is present mostly as fine dust particles. This dust eventually

settles over land and water (USEPA, 2003). Zinc does not volatilize from water but

is deposited primarily in sediments through adsorption and precipitation (USEPA,

2003). Zinc also does not volatilize from soil. Although zinc usually remains

adsorbed to soil, leaching has been reported at waste disposal sites. Zinc occurs in

the environment mainly in the +2 oxidation state (Lindsay, 2001). Sorption is the

dominant reaction, resulting in the enrichment of zinc in suspended and bed

sediments.

Severe zinc contamination tends to be confined to areas near emission sources.

Most of the zinc in soil is bound to the soil and does not dissolve in water. The

relative mobility of zinc in soil is determined by the same factors that affect its

transport in aquatic systems (i.e., solubility of the compound, pH, and salinity).

However, depending on the type of soil, some zinc may reach groundwater, and

contamination of groundwater has occurred from hazardous waste sites.

Consequently, zinc primarily remains in recalcitrant, immobile forms in

contaminated soils (Chlopecka et al. 1996; M a and Rao 1997a; Kaminiski and

Landsberger 2000; Kabala and Singh 2001). Zinc may be taken up by animals eating

soil or drinking water containing zinc.

1.3.4 Toxicities of copper, lead and zinc

Some metals are essential for cells (e.g., Cu, Fe, Mn, Ni，Zn), but higher

concentrations are also toxic. One reason metals may become toxic is because they

may cause oxidative stress. Some metals occur in the environment as radioactive

isotopes, posing an additional health risks.

36

Long-term exposure to copper dust can irritate human nose, mouth, and eyes, and

cause headaches, dizziness, nausea, and diarrhea (ATSDR, 2004). High intakes of

copper can cause liver and kidney damage and even death. There is some evidence

from animal studies to suggest that exposure to airborne copper or high levels of

copper in drinking water can damage the immune system (ATSDR, 2004).

Cu2+件 take up or give off an electron giving rise to free radicals that cause damage

(Jones et al., 1991). Also, copper ion replaces other essential metals in pigments or

enzymes, disrupting the function of these molecules. Hg+ and Cu+ are very active

to thiol groups and can interfere with protein structure and function (Pilon-Smits and

Pilon, 2002). USEPA has classified copper in Group D, not classifiable as to human

carcinogenicity because there are inadequate human or animal cancer studies. The

lARC has classified the pesticide, copper 8-hydroxyquinoline, in Group 3,

unclassifiable as to carcinogenicity in humans.

Estimates of exposure levels posing minimal risk to humans (Minimal Risk Level,

M R L ) have been made for copper and listed in Table 1.5. An M R L is defined as an

estimate of daily human exposure to a substance which is likely to be without an

appreciable risk of adverse effects (non-carcinogenic) over a specified duration of

exposure. M R L are derived when reliable and sufficient data exist to identify the

target organ(s) of effect or the most sensitive health effect(s) for a specific duration

within a given route of exposure. M R L are based on noncancerous health effects

only and do not consider carcinogenic effects. M R L can be derived for acute,

intermediate, and chronic duration exposures for inhalation and oral routes.

Lead enters body through food consumption, soil ingestion, or dust inhalation (Kos

and Lestan, 2003). Dust and soil that contain lead may get onto the skin, but only a

small portion of the lead will pass through the skin and enter the blood if it is not

washed off. The only kinds of lead compounds that easily penetrate the skin are the

additives in leaded gasoline, which is no longer sold to the general public. Shortly

after lead gets into the human body, it travels in the blood to the "soft tissues" (such

as the liver, kidneys, lungs, brain, spleen, muscles, and heart). After several weeks,

most of the lead (73 - 94%) moves into bones and teeth. Some of the lead can stay

in bones for decades. Some might dissolve and circulate in the blood and affect

37

other organs under some circumstances, e.g. during pregnancy and periods of breast

feeding, after a bone is broken, and during advancing age (ATSDR, 1997).

The main target for lead toxicity is the nervous system, both in adults and in children.

Long-term exposure of adults to lead at work has resulted in decreased performance

in some tests that measure functions of the nervous system. Besides metabolic

disorders and neurophysiological deficits in children and adults, lead affects the

haematological and renal systems (Kos and Lestan, 2003). Lead exposure may also

cause anemia, a low number of blood cells (ATSDR, 1997). At high levels of

exposure, lead can severely damage the brain and kidneys in adults or children. In

pregnant women, high levels of exposure to lead may cause miscarriage.

High-level exposure in men can damage the organs responsible for sperm production.

But there is no oral and nor inhalation M R L derived for lead (Table 1.5).

Zinc can enter through human lungs if zinc dust or fumes from zinc-smelting or

zinc-welding operations is inhaled. Zinc can enter the body through the digestive

tract when eating or drinking. The amount of zinc that passes directly through the

skin is relatively small (ATSDR, 2003). Zinc is stored throughout the body. Zinc

increases in blood and bone most rapidly after exposure. Zinc may stay in the bone

for many days after exposure. Normally, zinc leaves the body in faeces (5-10

mg/day) (WHO, 2001).

Inhaling large amounts of zinc can cause a specific short-term disease called metal

fume fever (ATSDR, 2003). However, very little is known about the long-term

effects of breathing zinc dust or fumes. Taking too much zinc into the body through

food, water, or dietary supplements can also affect health. Zinc administration has

also resulted in reductions in leukocyte number and function (ATSDR, 2003).

Ingesting high levels of zinc for several months may cause anemia, damage the

pancreas, and decrease levels of high-density lipoprotein (HDL) cholesterol

(Goodwin et al, 1985).

Long-term consumption of excess zinc may also result in decreased iron status in

women. Other studies have also reported respiratory effects of zinc chloride

inhalation, including dyspnea, cough, pleuritic chest pain, bilateral diffuse

infiltrations, pneumothorax, and acute pneumonitis from respiratory tract irritation.

38

Zinc is not classifiable as to its human carcinogenicity according to USEPA because

of lack of information (ATSDR, 2003). An intermediate (15 - 364 days) oral M R L

of 0.3 m g zinc/kg/day has been derived for exposure to zinc (Table 1.5). It has been

also accepted as the chronic (365 days or over) oral MRL.

1.3.5 Copper, lead and zinc contamination in Hong Kong

Hong Kong has a high density of vehicular traffic. Lead contamination of soil and

roadside vegetation and street dust was detected (Lau and Wong, 1982; Tarn et al.,

1987). The level of Pb in roadside dust ranges between 107 - 915 mg/kg (dry wt)

while soil Pb level was reported to be 40 mg/kg (dry wt) (Lau and Wong, 1982; Tarn

et al., 1987). A more recent report from Lee et al. (2005) indicated the surface soils

in urban and suburban areas are enriched with metals, such as Cu, Pb, and Zn. The

Pb concentration in the urban soils was found to exceed the Dutch target value. The

mean concentration of Pb in the local urban soils (88.1 mg/kg) exceeded the target

values recommended by the Dutch target value (85 mg/kg) and the mean Zn

concentration (103 mg/kg) was close to the Dutch target value (140 mg/kg). The

heavily pollution by Pb in urban soils in Hong Kong was from gasoline combustion.

Other metals, such as Mn, Zn, Fe, Cu and Cd present in vehicles are also discharged

into the environment as a result of wear and tear, contaminating roadside ecosystem.

The water extracts of roadside soil and dust can inhibit seed germination and root

growth of vegetables (Wang et al., 1984). Some forest soils also contained elevated

levels of As, Cu and Pb (Chen et al., 1997).

Besides soil, heavy metals were found in sediment in Hong Kong. Uncontrolled

disposal of industrially polluted wastes, especially from the mid-1950s to the

presence, resulted in much contaminated Hong Kong's harbour seabed (Tanner et al.,

2000). Heavy metals in Hong Kong marine surfacial sediments have been

monitored routinely by the Environmental Protection Department (EPD) since 1986,

and the results are summarized each year (HKEPD, 1997). High concentrations of

heavy metals in sediment are found near the industrial and coastal areas as well as in

the typhoon shelters, due to the enclosed nature and pollution loadings they receive

(Tanner et al., 2000). The highest concentrations of copper (Cu, 1.66 mg/g), lead

(Pb, 0.354 mg/g), zinc (Zn, 2.2 mg/g) and chromium (Cr, 0.047 mg/g) were found in

the Fo Tan Nullah, a major tributary of the Shing Mun River (Sin et al., 2001). 18 士

1.7 jLtg /g (dry wt) copper were found in sediment in Mai Po, Hong Kong (Ye, et al.’

39

2003). Heavy metals in the contaminated sediments would accumulate over the

years on the river bed and act as secondary sources of pollution to the overlying

water column in the river.

1.3.6 Environmental standards of copper, lead and zinc

Hong Kong Environmental Protection Department has adopted Dutch standard as a

general guideline for heavy metals just like PAHs. The Dutch and other countries

standards of PAHs are listed in Table 1.6.

40

Table 1.6. The European Dutch, Canadian, United States and Australian environmental standards for selected he

avy metals in soil (van den

Berg, 1994; C

EQQ

2002; AUDEP, 2003; USEPA, 2004b). Footnotes of this table are at the bottom of Table 1.3.

S

Euro

pean

(D

utc

h) st

and^^

^^

Canadia

n

Lp^n

gr^

^^^^

^^^^

A

ust

raH

a a

ssess

ment

AB

C

Tar

get

Inte

rvention A

gricultura

R^s

./Par

k ôim

^rci

jjdust

rial

In

dust

rial

Resi

dential

A

B

C

D

E

F

Copper

(Cu)

15.6

10

0

500

36

190

63

63

91

91

40880

3129

1000

/ /

4000

2000

5000

Lead

(Pb)

50

150

600

85

530

70

140

260

600

/ /

300

/ /

1200

600

1500

Zin

c

(Zn)

51.5

500

3000

140

720

200

200

360

360

306600

23464

7000

/ /

28000

14000 35000 41

1.3.7 Remediation technology of heavy metal

Soil washing, solidification/stabilization, electrokinetics, bioremediation and

phytoremediation are the remediation technologies that are applicable to treat metal

contaminated soils (USEPA, 1995 & 1997). The development of low cost and

effective remediation methods to treat contaminated soils has been the focus of many

environmental remediation professionals during the last decade.

1.3.7.1 Chemical method

Soil washing

Soil washing is one of the most suitable ex-situ technique for remediating sites

contaminated with heavy metals (Cline and Reed, 1995; Juang and Wang, 2000;

Reddy and Chinthamreddy, 2000; Hong et al., 2002; Zeng et al., 2004). In

conventional soil washing processes, excavated soil is vigorously mixed with a

solution that separates the contaminants from the large particle size fractions. The

soil and the extracting solution are mixed thoroughly for a period of time. Also the

soil is dewatered to separate the soil and liquids. The resulting soil that meets

regulatory requirements can be backfilled at the excavated site. The recyclable

solution is then treated to remove the colloidal particles and the original

contaminants. Common washing solution includes hydrochloric acid,

ethylenediaminetetraacetic acid (EDTA), acetic acid, phosphoric acid, and calcium

chloride (Cline and Reed, 1995; Reddy and Chinthamreddy, 2000; Juang and Wang,

2000; Sunet al., 2001).

For washing with HCl, metal release was partly due to the selectivity of the soil

surface groups for H+ over the bound metal ion, like Pb^^, Cu " and Zn^^. From an

electrostatic viewpoint, negatively charged surface sites have a greater affinity for tri-

and divalent ions than monovalent ions. However, H+ ions are attracted more

strongly than any other cations (Cline and Reed, 1995). Acid washing will change

soil properties and result in a large volume of liquid that must be treated before

discharge. In addition, acid washing may be very difficult in carbonate-rich soils

(Neale et al., 1997).

Another common extractant is chelate. Chelate is a ligand that contains two or

more electron-donor groups so that more than one bond is formed between metal ion

42

and the ligand. Chelant such as E D T A can form soluble complexes with Pb2+,

reducing the quantity of metals retained by soil particles and thereby increasing

heavy metal mobility. Thus, chelating agents are well suited for removing metals

bound by soils (Cline and Reed, 1995). However, using chelates in remediation of

metal-contaminated sites are usually of high cost (Zeng et al., 2004). Furthermore,

the complexing agents complicate the treatment of industrial effluents and other

polluted waters, which would reduce the efficiency of metal removal by conventional

chemical precipitation (e.g., 0H-), ion exchange, adsorption, and other processes

(Tunay et al., 1994; Hong et al., 2002). Also, chelates such as E D T A do not

biodegrade rapidly and concerns from its effects are magnified by its persistence

(Davis and Green, 1999). Also E D T A is a non-specific chelating agent and it reacts

with other metals present in soil (Kim et al., 2003).

1.3.7.2 Biological method

Phytoremediation

Phytoremediaiton method means using various plants to extract, contain, immobilize

or degrade contaminants from soil and water. Some plants can remove

contaminants from soil by direct uptake, followed by subsequent transformation,

transport and accumulation in a non-phytotoxic form. The harvested biomass could

be reduced in volume and/or weight by composting, anaerobic digestion, low

temperature incineration and leaching. Phytoremediation technologies are most

appropriate for large areas of low and moderately contaminated soils (Ward and

Singh, 2004b). Phytoremediation is still actively being researched and

plant-microbial associations seem to be the key to enhance removal of heavy metals.

The diverse approaches of phytoremediation are phytodegradation, phytoextraction,

phytostabilization, phytovolatilization and rhizofiltration. Phytoextraction and

rhizofiltration processes have shown promise for commercialization.

Phytoextraction is an environmental friendly and cost-effective approach that uses

green plants for the in situ risk reduction for contaminated soil, sludge, sediments,

and groundwater through contaminant removal. This process has been investigated

in several field experiments (Blaylock et al., 1997; Kayser et al., 2000).

Phytoremediation has the advantage of improving the utility of lands for agriculture

and forestry. Also it is solar-energy driven cleanup technology, there is minimal

environmental disruption and in situ treatment preserves topsoil. It is useful at sites

43

with low levels of contamination. It is useful for treating a broad range of

environmental contaminants. The establishment of vegetation on a contaminated

site also reduces oil erosion by wind and water, which helps prevent the spread of

contaminants and reduces exposure of humans and animals (Macek et al., 2004). It

is inexpensive (60 - 80% or even less costly) than conventional physico-chemical

methods (Meagher, 2000; Morikawa and Erkin, 2003). Phytoextraction is an

environmental friendly and cost-effective approach that uses green plants for the in

situ risk reduction for contaminated soil, sludge, sediments, and groundwater through

contaminant removal. This process has been investigated in several field

experiments (Blaylock et al., 1997; Kayser et al., 2000 in Cui et al., 2004).

However, phytoremediation is a time-consuming process, and it may take at least

several growing seasons to cleanu a site. In phytoextraction, plants that absorb

toxic heavy metals or persistent chemicals may pose a risk to wildlife and

contaminate the food chain (Morikawa and Erkin, 2003). There are roughly 400

known species of plants characterized as metal hyperaccumulators and about 75% of

these accumulate nickel and come from ultramafic soils (Baker et al., 1994).

Attempts have been made to increase the accumulation of heavy metals in plants

producing high amount of biomass (Clemens et al； 2002). Also, there is a

significant lack of knowledge of heavy metal transport, vacuolar uptake,

ATP-binding cassette (ABC) transporters (Macek et al., 2004).

Fungi in heavy metal remediation Fungi are able to remove heavy metals from aqueous solution (Siegel et al., 1990;

Kapoor and Viraraghvan, 1995). Aspergillus sp. has been shown to biosorb heavy

metals (Huang et al., 1988; Mullen et al., 1992). Biosoiption of heavy metals on

fungi occurs as a result of ionic interactions and complex formation between metal

ions and functional groups present on the fungal cell surface. The carboxylate and

amine groups are important in metal biosoiption (Kapoor and Viraraghavan, 1997).

White-rot fungi can concentrate metals taken up from substrate in their mycelia

(Baldrian, 2003). Pleurotus ostreatus was able to accumulate 20 mg/g (dry wt)

cadmium from liquid medium containing 150 ppm cadmium with at least 20% of

accumulated cadmium deposited intracellularly (Favero et al., 1991). Biosoiption

of copper by fruit bodies of nine tropical wood-rotting fungi were tested by

Muraleedharan (1995). But no research is about using Pleurotus pulmonarius in

44

heavy metal removal in soil medium.

Biological extraction means the extraction of metals or organics by any biological

means from soil to translocate them to aboveground parts. If fungi are used, the

contaminant was uptaken by the fungi and translocated to the fruiting bodies. Then

the fruiting bodies were harvested and disposed. There is no published research of

using white rot fungi compost in remediate heavy metal contaminated soil. Also,

there is no research about integrating mycoextraction and phytoextraction.

1.3.7.3 Stabilization and Solidification

Stabilization refers to processes that reduce the risk posed by a waste by converting

the contaminants into a less soluble, mobile or toxic form (FRTR, 2004). The

stabilized matrix may be a solid impermeable mass or a more friable solidified

matrix (Vangronsveld and Cunningham, 1998). The physical nature of waste is not

necessarily changed (Anderson, 1994). Solidification refers to processes that

encapsulate the waste in a monolithic solid of high-structural integrity (FRTR, 2004).

Solidification does not necessarily involve a chemical interaction between the waste

and the solidifying reagents, but may mechanically bind the waste in the monolith.

Contaminant migration is restricted by vastly decreasing the surface area exposed to

leaching and/or by isolating the waste within an impervious capsule (Anderson,

1994).

There are many innovations in the stabilization and solidification technology. Most

of the innovations are modifications of proven processes and are directed to

encapsulation or immobilizing the harmful constituents and involve processing of the

waste or contaminated soil. Nine distinct innovative processes or groups of

processes include: (1) bituminization, (2) emulsified asphalt, (3) modified sulfur

cement, (4) polyethylene extrusion, (5) pozzolan/Portland cement, (6) radioactive

waste solidification, (7) sludge stabilization, (8) soluble phosphates, and (9)

vitrification/molten glass (FRTR, 2004).

Cementation Portland cement and Portland cement with fly ash are the most commonly used

media in the stabilization process (Carmalin and Kanchana, 2005). Portland cement

is cement-based material. This material chemically reacts with water to form a

45

solid cementious matrix which improves the handling and physical characteristics of

the waste. Cement has a high pH at which many metals form insoluble compounds

such as insoluble hydroxide or carbonate salts, thereby also chemically stabilizing

the contaminants (Visvanathan, 1996). Cement is typically appropriate for

inorganic contaminants. The effectiveness of this binding agent with organic

contaminants varies (FRTR，2004).

Stabilization using cement has been applied in mercury containing wastes (Zhang

and Bishop, 2002), municipal solid waste incineration plants fly ashes (Polettini et al.,

2001), circuit board printing factory sludge (Li et al., 2000), tailing wastes (Jang and

Kim, 2000), sewage sludge (Vails and Vazquez, 2000), foundry sludge (Coz et al.,

2004)，hospital solid waste fly ash (Lombardi et al., 1998) and electroplating waste

(Carmalin Sophia and Kanchana Swaminathan, 2005). Halim et al. (2005)

demonstrated the stabilization of lead contaminated soil with cement and buffered

phosphate techniques. Ruiz and Irabien (2004) demonstrated the stabilization of

heavy metal contaminated soil with Portland cement and different types of additives,

organophilic bentonite, line and coal fly ash (Dutre et al., 1998; Ruiz and Irabien,

2004).

Glass encapsulation Some of the technologies used for solidification/stabilization can be used for either

treatment or containment. For example, "encapsulation" of a waste in plastic drums

is source control containment (USEPA, 2004). Glass structure is a relatively strong

and durable material that is resistant to leaching. Glasses have been reported to •n I

show excellent resistance against leaching of heavy metal ions with < 0.04 ppm Cd ，

< 0.02 ppm Cr3+，< 0.04 ppm Cu�. and < 0.2 ppm Pb^^ (Park and Heo, 2002). Glass

encapsulation is economically viable if the glass product can be sold. Glass

encapsulation is different from vitrification. Vitrification involves converting the

contaminated matrix to a solid block of glass-like material. Borosilicate and

soda-lime are the principal glass formers and provide the basic matrix of the vitrified

product (FRTR, 2004). It uses a heat source which is an electrical current passed

between electrodes placed in soil. The soil is heated to 1600 - 2000°C until the

matrix melts, graphite or glass frit is often placed between the electrodes to help

conduct the electric current and initiate heating. The high temperatures destroy any

organic constituents with very few byproducts. Materials, such as heavy metals and

46

radionuclides, are then incorporated into the glass structure (Vangronsveld and

Cunningham, 1998). Vitrification is applicable for soil and sediments but expensive

(Mulligan et al., 2001). Usually, stabilization of high level radioactive or extremely

hazardous wastes would employ vitrification.

1.4 Aim of study

To examine the potential for Pleurotus pulmonarius mushroom compost in treatment

of contaminated soil

1.5 Objectives

• To examine the influence of Pleurotus pulmonarius mushroom compost on the ex

situ biopile remediation technology;

• To compare and contrast the toxicities of soils after biopile and fungal treatments

by different ecotoxicity tests;

• To evaluate the efficiencies of heavy metal removal by physical, chemical and

biological remediation technologies;

• To evaluate the reduction in leachability of residue heavy metals in treated soils;

• To evaluate the reduction in residual total heavy metals of the treated soils.

1.6 Research Strategy

The study was divided into two parts. The first part was a field investigation on the

effectiveness of the fungal treatment in contrast to the conventional biopile treatment

in the Tsing Yi abandoned shipyard area. The scale of the study was in terms of

hundred of tonnes of contaminated soil. Also one tonne of Pleurotus pulmonarius

mushroom compost was produced in campus and applied to the biopile. Weekly

monitoring was carried out to follow the residual organopollutant concentrations in

the soil. For the second part of, specified by the Hong Kong Government for the

treatment systems, the biopiled soil would then be cemented before soil reclamation.

However, in this study, the treated soil would be handled using biological, chemical

and physical methods in laboratory scale. The advantages and disadvantages of

these methods and the effectiveness of the methods in handling the heavy metal

contamination would be commented. Thus a two-phase treatment system for

handling contaminated soil of mixed pollutants will be formulated.

47

1.7 Significance of study

This would be the first field study on applying Pleurotus pulmonarius mushroom

compost on bioremediation. It is also important to see whether this type of

remediation technology is applicable to Hong Kong local contaminated soil. In

Hong Kong, shipping industry is very prosperous. There are many shipyards along

the coastal line of Hong Kong. Usually, shipyard is severely contaminated. The

waste materials frequently contain high concentrations of heavy metals. Also, this

field study could support the laboratory scale study of PAHs biodegradation using

Pleurotus pulmonarius. Field environmental conditions which may affect the

performance of Pleurotus pulmonarius mushroom compost could be investigated.

Nevertheless, the result of this study could provide information on the mushroom

compost added biopile technology.

Besides PAHs, harmful levels of toxic heavy metals were determined in the soil.

Various heavy metal remediation technologies were reported. But it is seldom to

compare among them. In this study, several methods including physical, chemical

and biological methods were performed and compared so as to find the most suitable

one in this contaminated soil. The long-term significance of this project is to

develop low cost but effective and environmentally friendly method in remediating

mixed contaminated soil.

48

Chapter 2 Materials and Methods

2.1 Soil Collection

North Tsing Yi Abandoned Shipyard Soil

The soil samples were collected from abandoned North Tsing Yi Shipyard.

Laboratory gown and face mask were worn to avoid any pollutant exposure to the

collector. Gloves were worn to avoid any potential contamination to the samples and

to the collector. Soil was collected with a cleaned spade and put into autoclaved

bags. Then the soil sample was labelled and brought to research laboratory for

immediate handling.

2.2 Characterization of soil

2.2.1 Sample preparation

One g fresh soil was aseptically taken out for microbial biota analysis (biological

characterization) in Section 2.2.9. Another sample of 5 g soil was taken out for soil

moisture analysis in Section 2.2.5. Then the soil was divided into two equal

portions: one was air-dried, and the other was oven-dried at 50。C for two days. The

air-dried soil was used for pH, electrical conductivity, salinity, soil texture, total and

available nitrogen, total and available phosphorus analysis; while the oven-dried soil

was used for total organic carbon, PAHs, oil and grease, TPH and heavy metals

analysis.

After air drying, soil was homogenized using a hammer and sieved through a 2 m m

sieve before analysis. Unless specified, all experiments for the physical and

chemical characterization of soil were done with five replicates.

49

2.2.2 Soil pH, electrical conductivity & salinity

Ten gram of air-dried soil sample were extracted with 25 ml ultra-pure water (pH =

5.5) in 50 ml conical flask. The mixture was shaken at 200 rpm for 30 minutes.

Then the suspension was filtered through Whatman no.l filter paper (Allen, 1989;

Landon, 1991). A p H electrode connected to Orion 410A+ pH meter measured the

pH value of the liquid sample. Also the electrical conductivity was measured by a

Jenway Conductivity & pH Meter (4330, Jenway). 100 ul of a liquid sample were

added into a hand held salinity refractometer with automatic temperature

compensation (Atago S-10, 0-10%) with a pipetteman. Then the salinity of the

sample was recorded from the reading in the meter. The relationship between the

readings and the salt contents of samples is shown in Table 2.1.

Table 2.1 Relationship between the reading and the corresponding salt contents.

R e a d i n g N a C l M g C l i MgSCU K2SO4 CaCl]Sucrose

(ppt) (%, w/w) (%, w/w) (%, w/w) (%, w/w) (%, w/w) (Brix*)

0 00 Ko

To LO ^ lA LS

^ lA L8 L5 ^

^ 37I ^ ^ 43 ^ 3J

^ 4I ^ 49

50 sTl 45 73 ^

^ ^ 42 5A O 7A

70 72 ^ ^ 53 ^

^ O ^ 72 1L8 ^ ^

^ ^ ^ ^ UA ^ I L O ^

• Brix is a hydrometer scale used to measure the approximate sugar content of

grape juice, sweet wines, and sugar solutions. The scale is calibrated to indicate

50

percentage by weight of sucrose.

2.2.3 Total organic carbon contents

A thin layer of oven-dried soil was placed into a preheated (900°C in oven) ceramic

boat. The weight of soil was recorded. The carbon content of the sample was

measured by a total organic carbon analyzer (Shimadzu, 5000A) and expressed on a

weight percent basis.

2.2.4 Soil texture

The Bouyoucos hydrometer method (Allen, 1989) was adopted in textural analysis

which measures the decrease in density of the suspension as particles settle. Fifty

grams of air-dried 2 m m sieved soil sample were weighed and added into a container

of a high-speed stirrer. Twenty five ml 5% Calgon solution (50 g sodium

hexametaphosphate/L (Sigma P8510, pH = 9) and 400 ml tap water were added.

Afterwards the mixture was stirred for 15 minutes and then transferred to a 1 L

cylinder, and tap water was added to IL. Then, a paddle was used to stir for 1

minute. A Bouyoucos soil hydrometer was used to commerce timing for readings.

Readings were taken at 4 min 48 seconds (for silt and clay contents) and 5 hours (for

clay content). For every degree above 19.5。C to each reading, 0.3 units were added.

The soil moisture was determined at the time of weighing. The sand, silt and clay

content were expressed as percentages and its textural class was determined

following the classification of International Society of Soil Science. Table 2.2

shows the particle size distribution in ISSS. Figure 2.1 shows the soil textural

classification triangle.

51

Table 2.2 Particle size distributions of sand, silt and clay in International Scale

(International Society of Soil Science).

Fraction Particle diameter (mm)

Sand 2.0-0.02

^ 0.02-0.002 Clay <0.002

赢 / A /sandy 'o^nX \ / slit loam \ / \ ,

1。裤 < percent sand

Figure 2.1 The soil textural classification triangle (redrawn from Soil Science

Society of America)

52

2.2.5 Moisture

About 10 g of fresh soil samples were added into a pre-weighed petri dish and then

placed in a 105°C oven until constant weight (Allen, 1989). After cooling in a

desiccator, the dried soils were weighed and the water contents of the samples were

calculated by mass difference.

2.2.6 Total nitrogen and total phosphorus

Five g of air-dried soil or freeze-dried mycelial compost were weighed into a

digestion tube. Five ml 69% nitric acid, 1 ml 37% HCl and 0.5 ml 98% sulphuric

acid were added (modified from Allen, 1989). Samples were heated in a heating

digestion block (VELP D K 42/26). The samples were cooled down and diluted to

about 10 ml with ultrapure water. Then the liquids were filtered through Whatman

no. 1 filter paper and diluted to mark in a 50 ml volumetric flask. Three sample

blanks were also carried out in the same way. The total nitrogen and phosphorus

contents were determined by AIA.

2.2.7 Available nitrogen

Available nitrogen was measured by San"^ Automated Wet Chemistry Analyzer (AIA)

(Skalar) after extraction with 2 M potassium chloride (KCl) (BDH) (Allen, 1989;

Dahnke and Johnson, 1990). Ten g of air-dried 0.2 m m sieved soil and 50 ml KCl

were put in a 200 ml conical flask. Then the mixture was shaken at 180 rpm for 15

minutes. Two blanks were run with 2 M KCl only. The suspension was allowed

to settle and filter through Whatman no. 1 filter paper before measurement.

53

2.2.8 Available phosphorus

Available phosphate was determined by AIA after extraction with Troug's reagent

((NH4)2S04 buffer, pH = 3.0) (Troug, 1930). One g of air-dried 0.2 m m sieved soil

and 50 ml Troug's reagent were put in a 200 ml conical flask. The mixture was

shaken at 180 rpm for 30 minutes. Two blanks were run without soil. The

suspension was allowed to settle and filtered through Whatman no. 1 filter paper

before measurement.

2.2.9 Soil bacterial and mold population

Aseptic techniques were practiced. One g of fresh soil sample was added to an

autoclaved test tube with 9.0 ml of sterilized 0.85% sodium chloride solution. Then

this mixture was vortex-mixed and serial dilution (10' to 10" ) was performed. One

hundred /xl of each dilution of microbial solutions were transferred as incoculum into

nutrient agar plate (Difco) for total bacterial count. Another 100 [xl of each dilution

of microbial solutions were inoculated onto potato dextrose agar P D A plate (Difco)

for total mold count. The inoculum was spread evenly on the plates with a glass

spreader. For total bacterial count, the plates were incubated at 30°C for 2 days.

The plates with colony number within countable range (30 - 200) were chosen as the

suitable dilution. As fungi usually require a longer time to grow into visible

colonies, the fungal plates were examined after incubation at 28 °C for 5 days. The

result was presented as the colony forming unit (cfu) per gram of oven-dried soil.

54

Table 2.3 Composition of nutrient agar (NA) plate (Difco).

Component Amount (g/L)

Peptone 5

Beef Extract 3

Sodium chloride 8

Agar 12

Table 2.4 Composition of potato dextrose agar (PDA) plate (Difco).


Potato Starch 4

Dextrose 20

Agar 15

2.2.10 Extraction of PAHs and organic pollutants

2.2.10.1 Extraction procedure

The organic fraction of 5 g oven-dried soil was extracted with 10 ml

dichloromethane (DCM) (Analytical grade, BDH) shaking at 200 rpm for 2 hr

(modified Zheng and Obbard，2003). After the first extraction, it was repeated with

another 10 ml solvent for another 2 hr. The solvent was transferred in a 50 ml round

bottom flask and then concentrated and evaporated at 60°C by a rotary evaporator

with a vacuum pump (Bucih B171). One ml of acetone (HPLC grade, Labscan)

was used to redissolve the residue. The acetone solution was filtered by a 0.45 /xm

filter (Acrodisc syringe filters 4CR PTFE) and kept at -20°C before gas

chromatography-mass spectrometry (GC-MSD) measurement.

55

2.2.10.2 GC-MS condition

Separation of sample components was performed on a 0.25 m m (i.d.) x 30 m HP-1

methyl siloxane capillary column coated with a 0.25 /xm film (Hewlett Packard

HP19091Z-413). The conditions and temperature profiles for PAHs analysis are

listed in Tables 2.5 and 2.6.

2.2.10.3 Preparation of mixed PAHs stock solution

A 100 mg/L of mixed PAHs stock solution was prepared by dissolving 1 g of each,

fluoranthene (> 99% Sigma 423947), pyrene (98% Aldrich 185515.),

benzanthracene (99% Aldrich B2209), chrysene (98% Aldrich 245186),

benzo[a]pyrene (> 97% Sigma B1760), benzo[g,h,i]perylene (Sigma B6511)，

indeno[ 1,2,3-cd]pyrene (Supelco 48499) into 100 ml acetone (HPLC grade,

Lab-Scan). The stock solution was kept in darkness at -20°C.

Table 2.5 Conditions of GC-MSD for PAHs analysis.

Parameter Condition

Carrier gas High purity helium

Column flow rate 2 ml/minute

Column pressure 122.2 kPa

Oven temperature 60。C

Oven equilibrium time 1.5 minutes

Injection temperature 250°C

Interface temperature 250°C

Final temperature 300°C

Ramp temperature 8°C/minute

Hold time 10 minutes

Split mode Splitless

Solvent cut 4 minutes

56

Table 2.6 Temperature profile for GC-MSD for sample analysis.

Rate Temperature Hold time Run time Oven situation

(。C/min) (°C) (min) (min)

Initial / ^ LS LS

Ramp 8 ^ l O 4L5

Post run 1 m 05

2.2.11 Oil and grease content

The oil and grease content of soil was determined by a gravimetric method modified

from Juteau et al. (2003). One g of oven-dried soil sample was suspended in 10 ml

ultrapure water, acidified with 2-3 drops of 37% hydrochloric acid (Merck) and

extracted five times with 10 ml ethyl acetate (Analytical grade, Labscan). Extracts

were dehydrated with anhydrous sodium sulfate (Fischer Scientific) and filtered

using syringes with PTFE 0.45 (im-micro filter. The solution was placed in a

preweighed 100 ml round-bottomed flask, and the solvent was evaporated with a

rotary evaporator kept at 30°C. The flask was put into deep freezer for 2 hours.

Then it was freeze-dried under vacuum for 3 days using a lyophilizer (brandname

model no.) and then weighted. The oil and grease content of the sample was

calculated based on the weight difference of the flask.

2.2.12 Total Petroleum Hydrocarbons (TPH)

Total petroleum hydrocarbons content was analyzed by a gravimetric method

(modified from USEPA 1664 Reference Method). Five g of oven-dried soil sample

was suspended in 10 ml ultrapure water, acidified with 2-3 drops of 37%

hydrochloric acid (Merck) and extracted three times with 10 ml n-hexane (Analytical

grade, Labscan). Extract was dehydrated with anhydrous sodium sulfate (Fischer

57

Scientific) and filtered using syringes with PTFE 0.45 jLim-micro filter. Then, 5 g of

silica gel 60 (Merck) were mixed with the extract. Afterwards, the extract was

filtered using Whatman No.l filter paper to remove silica gel and soil. Next, the

solution was placed in a preweighed 50 ml round-bottomed flask, and the solvent

was evaporated with a rotary evaporator kept at 40°C. The flask was freeze-dried

by freeze-dryer (Labconco, Missouri, USA) at -43°C and reduced pressure at 64 x

10-3 MBar for days. The TPH content of the sample was calculated based on the

weight difference of the flask.

2.2.13 Total heavy metal analysis

All glassware and plastic wares were acid-treated in an acid bath made with 10%

hydrochloric acid (HCl) (Merck, Germany) for 24 hours. They were then rinsed

with deionized water and transferred to a water bath for at least 5 hours and then

oven-dried. The heavy metals were extracted from the samples by microwave

digestion system (Milestone ETHOS Microwave Digester) (modified from USEPA

3051 reference method). For every sample, 0.2 g oven-dried sample was weighed

and put into a T F M vessel and 20 ml 69% HNO3 (BDH) was added then (Zhou et al.,

1995). The T F M vessel was sealed and heated in the microwave digester.

Temperature was first increased to 170°C in 15 minutes and then increased to 180°C

in 10 minutes. After cooling in fume hood, 10 ml of ultrapure water were added.

Then, the solution was filtered through a Whatman No.l filter paper and diluted with

ultrapure water in 25 ml or 50 ml volumetric flask. The digested samples were

stored in PE bottles at 4°C until measured. The heavy metals contents were

analyzed by an inductively coupled plasma (ICP) spectrophotometer

(ATOMSCAN16 ICP-AES, Thermo Jarrell Ash) or an atomic absorption

spectrophotometer (AAS) (Polarized Zeeman Z-8100 AAS, Hitachi).

58

2.2.14 Toxicity characteristic leaching procedure (TCLP)

Toxicity characteristic leaching procedure (TCLP) was used to determine if a waste

had the characteristic of leachate toxicity and is therefore hazardous. One g of dried

soil was extracted with 20 ml of C H 3 C O O H (pH = 2.88 土 0.05) for 18 h at 30 rpm

(USEPA 1311 reference method). Then the sample was filtered with Whatman No.

1 filter paper. The extract obtained was then analyzed to determine metal

concentrations with ICP or AAS.

2.2.15 Extraction efficiency

Let EE be the percentage of target PAHs or heavy metal extracted from the soil/plant;

Ae is the amount of chemical (mg) extracted from the soil/plant, and Ai is the amount

of chemical (mg) artificially spiked into the soil/plant.

In parallel, the background amount (b) was also measured with the unspiked

soil/plant to give Ab.

Therefore,

Ae (measured with spiked sample) = EE (b + Ai)

Ab (measured with unspiked sample) = EE (b)

Therefore,

Ae 二 EE (Ab/EE + Ai)

Ae = Ab + EE (Ai)

(Ae-Ab)/Ai = EE

3 replicates were performed for each sample for determination of the extraction

efficiency.

The extraction efficiencies of 7 PAHs and 3 heavy metals are listed in the Tables 2.7

and 2.8.

59

Table 2.7 Extraction efficiencies of PAHs and heavy metals in soil. Data is

presented in Mean 土 SD of 3 replicates.

PAHs Extraction Efficiency (%)

Fluoranthene 101.25 ± 6.38

Pyrene 103.09 土 6.35

Benzanthracene 100.21 土 3.97

Chrysene 101.17 ± 9.24

Benzo[a]pyrene 101.51 土 2.58

Benzo[g,h,i]perylene 103.13 土 6.06

Indeno[l,2,3-cd]pyrene 100.12 土 1.02

Heavy metal

98.10 ± 5.82

9 8 . 0 0 土 6 . 1 2

100.00 ± 5.76

Table 2.8 Extraction efficiencies of heavy metal in aerial part of wheat Data is

presented in Mean 土 SD of 3 replicates.

Heavy metal Extraction Efficiency (%)

97.00 土 3.22

100.20 ± 1.30

1 0 1 . 3 0 土 7 . 4 7

2.3 Production of mushroom compost

Fresh mushroom compost from the cultivation of oyster mushroom Pleurotus

pulmonarius (Pl-27) was prepared and used in this study. The production of the

mushroom compost is described in Fig. 2.2. The cultivation compost of oyster

mushroom Pleurotus pulmonarius was consisted of: straw (agent: Tai W o Trading

60

Company, China), wheat bran (agent: Wing Hing Loon, Tai Po), lime (food grade,

Guangxi Metals and Minerals Import and Export Corporation, China) and sugar

(agricultural grade) mixed in a ratio of 85: 13: 1: 1 (w/w/w/w). After mixing, the

constituents were then moisturized to 60% by tap water (modified from Ching, 1997).

The well-mixed cultivation compost was then undergone fermentation for 5 days in

open area. About 2 kg of the fermented substrate were packed into an autoclavable

bag. Then the compost was sterilized by autoclaving twice at 121°C for 30 minutes.

Wheat grain culture of strain Pl-27 was used to inoculate the autoclaved compost.

The inoculated compost was then incubated at 28°C for about 6 weeks for mycelial

running in the walk-in environmental chamber in the mushroom cultivation complex,

Department of Biology, The Chinese University of Hong Kong.

Straw 85%, wheat bran 13%, lime 1%, sugar 1% + 60% water content

T 5 days fermentation

Fermented substrate

y

Sterilization

T autoclaving twice at 121°C for 30 minutes

Inoculate with Pleurotus pulmonarius (Pl-27) culture

+

Mycelia running 6 weeks)

Figure 2.2 A flowchart showing the production of the mycelial compost of

Pleurotus pulmonarius (Pl-27).

61

2.4 Characterization of mushroom compost

2.4.1 Enzyme assay

2.4.1.1 Laccase assay

The laccase activity of crude enzyme was measured by the ABTS assay modified

from Lang et al. (1997) and Galvez et al. (2000). The reaction mixture contained 1

m M 2,2'-azinobis-3-ethylbenthiazoline-6-sulfonate (ABTS), 100 m M succinic-lactic

acid buffer (pH 4.5) and 100 |LIL sample, in a total volume of 1 ml. The reaction

rate of cation radical formation was measured at 420 nm (Emax = 36,000 M'^ cm'^) at

room temperature for a total of 3 min. A diode array spectrophotometer (Milton

Roy SP3000) fitted with time scan function was used. The reaction was initiated at

the moment of sample addition and the absorbance was monitored every 15s in the

following 3 min in order to determine the slope of the rate of color change, which

indicated the rate of catalytic reaction.

2.4.1.2 Manganese peroxidase assay

Manganese peroxidase of crude enzyme was measured by the M B T H / D M A B assay

modified from Castillo et al. (1994) and Lang et al (1997). The reaction mixture

contained 0.07 m M 3-methyl-2-benzothiazolinone hydrazone (MBTH), 0.99 m M

3-(dimethylamino)-benzoic acid (DMAB), 0.3 m M MnS04, 0.05 m M H2O2, 100

m M succinic-lactic acid buffer (pH 4.5) and 100 sample, in a total volume of 1 ml.

The reaction of purple indamine dye product formation was initiated by addition of

H2O2. The absorbance of reaction mixture was measured at 37°C at 590 nm (Emax

=53,000 M—i cirfi). A spectrophotometer fitted with time scan function was used.

The first absorbance measurement was recorded after exactly 15s. Then the reaction

62

was monitored in the following 3 min.

2.5 Addition of mushroom to soil on site

2.5.1 Transportation of mushroom compost to Tsing Yi

In order to make performance comparison with fermented pig manure added to

biopile by the Gammon Construction Limited, the mushroom compost to be added to

the biopile was set at the equivalent dose of 1 to 90 (supplement: soil; w/w). 90

tonnes of contaminated soil were used for the fungal bioremediation. Fig 2.3 shows

the 3D sketch diagram of the fungal bioremediation setup in the site.

3m 3m 3m

13m X /

Fig 2.3 A 3D sketch diagram of the target area of Biopile (T: Treatment zone; SB:

Soil barrier; C: Control zone).

63

2.5.2 Mixing of mushroom compost and soil

Each bag of mushroom compost was opened and manually stretched to small pieces

to facilitate mixing. Then all the mushroom compost was thoroughly mixed with

soil in the treatment zone by excavator provided by Gammon Construction Ltd.

Sampling of soil was first done from Control and Treatment zones at Day 0. At the

same day, the air blower was turned on, and the soil pile was covered with an

impermeable plastic sheet.

2.6 Soil monitoring

From 6th May to July 2004, soil samples of Treatment and Control zones were

collected weekly. From July to 2nd Aug 2004, soil samples were collected

biweekly. Also the last soil monitoring was on Aug. On site physical

characteristic (temperature, humidity, visible and U V light intensities, temperature of

soil) and chemical characteristic (soil microbial population, PAHs, oil and grease,

total petroleum hydrocarbons and heavy metals of the biopile soil were carried out

during every soil monitoring.

2.6.1 On site air and soil measurements

2.6.1.1 Air temperature and moisture

Air temperature and moisture were recorded using a thermohygrometer (Traceable

Hygrometer S1930L). Also, data from Hong Kong Observatory, H K S A R were also

recorded for reference.

2.6.1.2 Light intensity

Light intensity was recorded using light intensity meter (Li-Cor LI-250).

64

2.6.1.3 U V intensity

U V intensity was recorded using a U V meter (Spectroline DIX Series).

2.6.1.4 Rainfall

Rainfall data were collected from Hong Kong Observatory, H K S A R for reference.

2.6.1.5 Soil temperature

Soil temperature was recorded using a thermometer. Also, data provided by

Gammon Construction Ltd were used for reference.

2.6.2 Soil chemical characteristic

Soil moisture, soil pH, salinity, electrical conductivity were measured by methods

described in Section 2.2.1 to 2.2.5. PAHs concentration, oil and grease content and

TPH content were determined by methods described as Sections 2.2.12 to 2.2.14.

Soil bacterial and fungal populations were determined by methods to be described in

Section 2.2.11.

2.6.3 Relative residue pollutant (%)

Relative residue pollutant (%) is defined as the ratio of the amount of the PAH

remained to the initial PAH amount (Day 0) in percentage. It is calculated

according to following equations:

Relative residue by degradation (%) = (Q / Ci) x 100 %

where

Ci = initial PAHs concentration (mg/ kg);

Cf= residue PAHs concentration (mg/ kg);

65

2.7 Toxicity of treated soil

The biopile treated and fungal treated Tsing Yi soil were tested whether the toxicity

of the soil had been decreased by various toxicity tests which can be used to evaluate

the effects on different trophic levels. Microbial tests are simple, rapid, sensitive and

inexpensive toxicity tests (Arfsten et al., 1994; Bitton et al., 1994; Bemand et al.,

1996; Juvonen et al., 2000). Besides this, seed germination test using several species

of plants is often used as a biological endpoint to test the effects of environmental

pollutants (Crowe et al., 2002). Plant tests are cost effective, relatively easy to

perform and are suitable for turbid sample testing (Wang, 1991). Wheat {Triticum

aestivum), perennial ryegrass {Lolium perenne) and Chinese white cabbage {Brassica

chinensis) were selected. Perennial ryegrass was selected because grasses have

fibrous root systems which can provide higher surface areas that could increase the

soil microbial population and maximize bioremediation in the rhizosphere (Siddiqui

and Adams, 2002). All seeds were stored in desiccators at room temperature until

use.

2.7.1 Seed germination test

All the seeds were surface sterilized by soaking in 1% hypochlorite solution for 15

min and washed with distilled water for 30 min. Ten g of biopile treated or fungal

treated soils were placed in plastic petri dishes (90mm Sterilin Petri dish, U.K.).

Four ml of ultra pure water were added to provide water for seed germination. Five

replicates were used per sample and 25 seeds per plate. Control using garden soil

provided from Greenhouse, Department of Biology, C U H K was done in parallel.

Seeds were maintained in a controlled temperature incubator (Thermoline FR285C,

Australia, Refrigerated Incubator) in darkness at 24 土 0.5 °C and were kept moist

with 2 - 4 ml of ultra pure water (Crowe et al, 2002). A 1-mm radicle emergence

66

from seeds was considered germination (Munzuroglu et al., 2002). The results are

expressed as a percentage of the mean of the garden soil (relative seed germination,

R S G %).

2.7.2 Indigenous bacterial toxicity test

Three species of bacteria isolated from the Tsing Yi soil samples were used,

including a Methylobacterium sp., a Pseudomonas sp. and a Bacillus sp. (Ho et al.,

2004). The bacterium was first grown in Luria Broth (LB) medium at 30°C for 24

hours and the culture were aerated by shaking at 200 rpm. The composition of the

LB medium is shown in Table 2.9. Then, the cultures were centrifuged and was

washed with 0.85% NaCl for three times to remove residual nutrients. One ml of

each bacterial suspension was then inoculated into 2 g of autoclaved biopile treated

or fungal treated soil and incubated at 30°C for 3 days. The bacterial count on LB

agar plate was done before and after the incubation by spread plate method. The

difference between the changes in the number of bacterial colonies of control and

treatment were used as an indicator of soil toxicity. The colonies were counted on

dilution plates with 20 - 300 colonies. Bacterial colony forming units (cfu) were

calculated per g of soil. The change of cfu in biopile treated and fungal treated soil

indicates the toxicity towards the bacterium if any.

Table 2.9 Composition of Luria Broth (LB).


Tryptone (Difco) 10

Yeast extract (LabM) 5

Sodium chloride (BDH) 5

67

2.7.3 Fungal toxicity test

Pleurotus pulmonarius and three indigenous species of fungi isolated from the Tsing

Yi soil samples were used, including a Trichoderma asperellum, Trichoderma

harzianum and Fusarium solani (Ho et al., 2004). The fungi were first grown in

P D A broth (Difco) at 28°C for 5 days, and the cultures were aerated by shaking at

200 rpm. Then the biomass was collected by filtration aseptically. Then the

biomass was washed by autoclaved deionised water for three times to remove

residual nutrients. The biomass was put into a sterilized blender with double volumes

of autoclaved ultra pure water and the biomass was blended into mycelial

homogenate as an inoculum. One ml of inoculum was then inoculated into 2 g of

autoclaved biopile treated or fungal treated soil，respectively and incubated at 28°C

for one week. Then the sample was extracted with 10 ml methanol (Analytical

grade, Lab-Scan) shaking at 200 rpm for 2 hrs. After the first extraction, it was

repeated with another 10 ml methanol for another 2 hours. The solvent was

transferred in a 50 ml round bottom flask and then concentrated and evaporated at

60°C by a rotary evaporator with a vacuum pump. One ml of methanol (HPLC grade,

Fisher Scientific) was used to redissolve the sample after evaporation. The methanol

was filtered by a 0.45 fim filter (Acrodisc syringe filters 4CR PTFE) and kept

at -20°C for gas chromatography-mass spectrometry (GC-MSD) measurement.

The ergo sterol content was measured before and after the incubation. The

ergo sterol content of 1 ml inoculum and the change in the ergosterol contents of

biopile treated and fungal treated soil were used as a measure of soil toxicity in

inhibiting fungal growth.

68

Table 2.10 Composition of Potato Dextrose Broth (Difco).


Potato starch 4

Dextrose 20

Table 2.11 Conditions of GC-MSD for ergosterol analysis.

Parameter Condition

Carrier gas High purity helium

Column flow rate 1 ml/minute

Column pressure 9.4 psi

Oven temperature 60°C

Oven equilibrium time 1.5 minutes

Injection temperature 250°C

Interface temperature 250°C

Final temperature 290°C

Ramp temperature 10°C/miniite

Hold time 5 minutes

Split mode Splitless

Solvent delay 4 minutes

Table 2.12 Temperature profile for GC-MSD for ergosterol analysis.

Rate Temperature Hold time Run time Oven situation

(。C/min) (。C) (min) (min)

Initial 1 ^ LS L 5

Ramp 10 ^ 5 ^

Post run / ^ 05 41

69

2.7.3.1 Preparation of ergosterol standard solution

A 100 mg/1 of ergosterol stock solution was prepared by dissolving 1.33 g of

ergosterol (> 75% purity, Sigma) into 100 ml methanol (HPLC grade Fisher

Scientific). The stock solution was kept in darkness at —20°C.

2.8 Soil Washing

Soil washing is a popular ex-situ technique for remediating sites contaminated with

heavy metals (Cline et al., 1995; Sun et al., 2001; Priego-Lopez and Castro, 2002).

In conventional soil washing process, chelating agents or acids are used to enhance

heavy metal removal. Hydrochloric acid was selected because of inexpensive

nature and strong extraction ability for different heavy metals.

2.8.1 Optimization of soil washing

The optimal conditions of soil washing using hydrochloric acid were determined

under the constant conditions with only one factor varying at one time.

Performance of the hydrochloric acid towards heavy metal removal of the soil was

assessed by RE (Removal efficiency). Five replicates were done unless specified.

Removal Efficiency (RE) = metal concentration in effluent (mg/1) / initial metal

concentration in soil (mg/kg) x 100%

2.8.1.1 The effect of hydrochloric acid concentration

Five g of biopile treated or fungal treated soil was measured and put into 50 ml

conical flask. 25 ml hydrochloric acid (Fisher Scientific) was added. The solution

was shaken at 150 rpm at 25°C for 6 hrs. Different concentrations of HCl were

used: 0.2N，0.5N, IN, 2N，3N and 4N. The soil extract was harvested by filtration

70

with Whatman No. 1 filter paper. The filtrate was stored in PE bottles at 4°C until

ICP or A A S measurement. For the soil remained, it was rinsed with ultrapure water

and dried at 105°C for 2 days. The total metal remained in soil was determined as

described in Section 2.2.13. To study the effect of soil washing on metal

leachability, final TCLP of soil were measured as described in Section 2.2.14.

2.8.1.2 The effect of incubation time

From the result of Section 2.8.1.1, the optimal HCl concentration is 0.5N.

Therefore, another set with the same conditions described in Section 2.8.1.1 was

carried out except changing the incubation time into 2 h, 4 h, 6 h, 12h, 24h and 48h.

For the soil remained, it was rinsed with ultrapure water and dried at 105°C for 2

days. The total metal remained in soil was determined as described in Section

2.2.13. To study the effect of soil washing on metal leachability, final TCLP of soil

were measured as described in Section 2.2.14.

2.9 Phytoremediation

200 g of non-autoclaved biopile treated or fungal treated soil were placed in a plastic

cup (Sigma P-5682). 80 ml of ultrapure water were poured into the soil to moist the

soil. Then, 25 germinated seeds of one of the following plants, wheat, ryegrass or

Chinese white cabbage, were separately placed into a soil layer of 0.5 cm depth with

forceps. The pots were put in Department greenhouse for plants to grow for 4

weeks. The plants were irrigated daily with 10 ml ultrapure water. After 4 weeks,

the aerial part was collected and rinsed with ultrapure water. Then the aerial part

and remained soil were oven dried at 105°C for 2 day. The oven dry weights of the

aerial part and soil were recorded. Both aerial part and soil were undergone

microwave digestion described in Section 2.2.13 before A A S measurement for total

71

metal analysis.

The metal uptake in aerial part = metal concentration (mg/kg) x biomass (kg)

=metal uptake (mg)

To study the effect of phytoremediation on metal leachability, final TCLP of soil

were carried out as described in Section 2.2.14.

2.10 Mycoextraction

100 g of non-autoclaved biopile treated or fungal treated soil were placed into a

plastic cup (Sigma P-5557). Different amounts of mycelia compost were added to

and mixed with the soil to achieve the following soil to compost ratio: 1:0.01，1:0.25,

1:0.5, 1:1，1:2 and 1:5. Then, the pots were placed in an incubator of 28°C

(Lab-Line Imperial II Incubator) to allow mycelial running for 4 weeks. Afterwards,

the pots were transferred to an environmental chamber in Mushroom House,

Department of Biology, The Chinese University of Hong Kong. During fruiting,

any fruiting body emerged out was collected and rinsed with ultrapure water. Then

the fruiting body and remained soil were oven dried at 105°C for 2 day. The dry

weights of fruiting body and soil were recorded. Then, the fruiting body and soil

were undergone microwave digestion as described in Section 2.2.13 for total metal

analysis.

The metal uptake in fruiting body = metal concentration (mg/kg) x biomass (kg)

=metal uptake (mg)

To study the effect of mycoextractionn on metal leachability, final TCLP of soil were

carried out as described in Section 2.2.14.

2.11 Integrated bioextraction

200 g of non-autoclaved biopile treated or fungal treated soil were placed in a plastic

72

cup (Sigma P-5682). 40 ml of ultrapure water were poured into the soil to moisture

the soil. 100 g of mycelia compost were thoroughly mixed with soil. Then, 25

germinated wheat seeds were separately placed into 0.5 cm subsoil with forceps.

The pots were put in incubated in Greenhouse, Department of Biology, The Chinese

University of Hong Kong, for plants to grow for 4 weeks. The plants were watered

daily to keep the moisture. After 4 weeks, the aerial part was collected and rinsed

with ultrapure water. Then the aerial part and remained soil were oven dried at

105°C for 2 day. The dry weights of aerial part and soil were recorded. Both

aerial part and soil were undergone microwave digestion described in Section 2.2.13

before A A S measurement for total metal analysis.

The metal uptake in aerial part = metal concentration (mg/kg) x biomass (kg)

=metal uptake (mg)

To study the effect of compost added phytoremediation on metal leachability, final

TCLP of soil were carried out as described in Section 2.2.14.

2.12 Cementation

For 4, 8, 16, 30 and 40% cementation of soil, 100 g of biopile-treated or fungal

bioremediated- soil was mixed with different amounts of Portland Cement. Then

15 ml of ultrapure water were added to obtain 15% moisture. Then the mixture was

poured to Blender (Waring commercial blender) to mix for 10 minutes. After one

hour, the cemented soil was oven-dried at 105°C for 1 day. The soil was undergone

microwave digestion for total heavy metal analysis described in Section 2.2.13. At

the same time, TCLP test described in Section 2.2.14 was carried out.

2.13 Glass encapsulation

One g，2g，5g and lOg of soil were put separately into different sizes of glass tubes.

73

Then the glass tubes were sealed by Blowing Glass Service, Department of

Chemistry to form glass ampoules. Amount of glass used to embed different

amount of soil was listed in Table 2.13. TCLP test described in Section 2.2.14 was

carried out to determine leaching of heavy metal if any.

Table 2.13 The amounts of glass used for embedding different amounts of soil.

Data presented in mean + SD of 5 replicates.

Amount of soil ( g ) A m o u n t of glass used (g)

0.6518 ±0.1325

2 4.2034 ±0.6109

5 7.0359 ±0.6118

To 14.9914 ±0.2923

2.14 Statistical analysis

Data were presented in mean 土 standard deviation. If only two sets of data were

compared, Student T test was used. One-way analysis of variance (ANOVA) was

used to detect any significant difference between the Biopile treatement and the

Fungal treatment if there were a number of cases. Ranking of the groups was

performed with Tukey test (p = 0.05) in the case of significant difference was found

among the group and letters a, b, c represent the ranking from the highest to the

lowest. Two-way analysis of variances (ANOVA) was applied to determine the

effect of time and/or type of treatment. The significance of differences between

mean values was determined by post-hoc comparison performed by the Tukey test.

A probability value of p = 0.05 was considered as significant. All statistical

programmes were provided in SPSS (Version 11.5) software.

74

Chapter 3 Results


North Tsing Yi Abandoned Shipyard Soil

The physical and chemical properties of North Tsing Yi abandoned shipyard soil

were determined and are listed in Table 3.1 The abandoned shipyard soil was

slightly alkaline (pH 7.76 士 0.08) with low moisture (12 士 3 %) and low salinity

(0.05 士 0.05 %) in nature. The soil contained 71 ± 5 % of sand and the soil texture

was classified as loamy sandy according to the soil textural classification triangle in

Figure 2.1. The organic carbon was 2.380 士 0.448 % (23800 士 4480 mg/kg). Also,

it had low nutrient levels for both nitrogen (total nitrogen: 21 士 9 mg/kg; available

nitrogen: 3 ± 1 mg/kg) and phosphorus (355 ± 42 mg/kg). The average electrical

conductivity was 0.485 士 0.077 mS/cm in soil. According to the contamination

assessment report (MEMCL, 2001), organopollutants such as polycyclic aromatic

hydrocarbons (PAHs), phenols, polychlorobiphenyl (PCBs,) benzene, toluene,

ethylbenzene and xylenes (BTEX) and tributyltin (TBT) were found in the soil.

PAHs were selected as the target organopollutant. Other pollutants such as oil and

grease and total petroleum hydrocarbon (TPH) and heavy metals were found.

Seven 4- to 6- ring PAHs (fluoranthene, pyrene, benz[a]anthracene, chrysene,

benzo[a]pyrene, benzo[g,h,i]perylene and indeno[l,2,3-cd]pyrene) were found in the

soil. No 2- to 3- rings PAHs were detected in the soil. The total PAHs content (45

±13 mg/kg) exceeded the Dutch A level (10 mg/kg) and the new Dutch Intervention

level (40 mg/kg). The TPH content (1719 士 524 mg/kg) exceeded the safety

standard (1000 mg/kg) of Association for Environmental Health and Sciences, US.

The oil and grease content was 12200 士 2308 mg/kg, but there is no international

standard for oil and grease.

For heavy metals, lead (2700 士 370 mg/kg), copper (1290 土 177 mg/kg) and zinc

(794 ±106 mg/kg) exceeded the new Dutch Intervention Level (Pb: 530 mg/kg; Cu:

190 mg/kg; Zn: 720 mg/kg). Lead and copper also exceeded the Dutch C levels (Pb:

600 mg/kg; Cu: 500 mg/kg) and Zn exceeded the Dutch B level (500 mg/kg). For

less toxic metals like aluminum, manganese and iron, they did not exceed the USEPA

risk based concentration (Al: 1600000 mg/kg; Mn: 20000 mg/kg; Fe: 310000 mg/kg).

75

Although lead, copper, aluminum, zinc, manganese and iron were also detected in

shipyard soil, only lead, copper and zinc were selected as target metals based on their

high toxicity properties to human beings.

The large deviation of minimum and maximum values of the organopollutants and

heavy metals indicated the heterogeneity of the soil. For example, the minimum

TPH and total PAHs contents were 1100 mg/kg and 22 mg/kg respectively. But

their maximum values were 2740 mg/kg and 72 mg/kg respectively. In general, the

shipyard soil was highly heterogeneous and contaminated with organopollutants and

heavy metals. Therefore, treatment of the both organopollutants and heavy metals

was required.

76

Table 3.1 The physical and chemical properties of North Tsing Yi Abandoned

Shipyard soil. Data are presented in mean + SD of 5 replicates.

Parameters Mean 士 SD Min Max

^ 7 . 7 6 ± 0 . 0 8 ^ 5 7

Moisture (%) 12 ± 3 ^ 15.72

Salinity (%) 0.05 ± 0.05 0 ol

Conductivity (mS/cm) a 0 . 4 8 5 ± 0.077 0.255 0.642

Organic Carbon (%) 2.380 ±0.448 2.112 2.897

Clay (%) 12 ± 0 / 1

Silt (%) 17±5 12 ^

Sand (%) 71 ± 5 ^ ^

Total N (mg/kg) 21 ± 9 13 ^

NO3-N (mg/kg) ^ 2 4

Total P (mg/kg) 355 ±42 m

K (mg/kg) 1550 ±364 U ^ ^

Oil & Grease (mg/kg) 12200 ±2308 ^ 16100

TPH (mg/kg) 1719 ±524 U ^

Total PAHs (mg/kg)b 45 ± 13 ^ Tl

^ 2700 ± 370

^ 1290 ± 177 ni5

Al 8000 ± 844 r m

^ 794 ± 106 ^ ^

M n 1690 ±515 UTO

F^ 143000±176 125000 160547

a mS/cm = milliSiemen/cm = /xmho/cm

includes fluoranthene, pyrene, benz[a]anthracene, chrysene, benzo[a]pyrene,

benzo[g,h,i]perylene and indeno[ 1,2,3-cd]pyrene and any PAH putatively identified

by GCMS.

77


3.2.1 Enzyme activity

The laccase and manganese peroxidase activities of Pleurotus pulmonarius

mushroom compost were 23.54 士 1.44 and 2.38 士 0.56 jitmole/min/g compost

respectively (Table 3.2). As one ton of mushroom compost were added, total 23.54

士 1.44 mole/min laccase and 2.38 土 0.56 mole/min manganese peroxidase activities

were immobilized in the compost.

Table 3.2 The enzyme activities of mushroom compost (U: |Limol /min). Data are

presented in mean + SD of 5 replicates. Experimental condition: 3 g of mushroom

compost was shaken with 24 ml deionized water at 4 for 4 hours

Immobilized enzyme U/g compost

Laccase 23.54 土 1.44

Manganese peroxidase 2.38 ± 0.56

3.2.2 Total nitrogen and total phosphorus contents

The total nitrogen and total phosphorus contents are listed in Table 3.3. The total

nitrogen and total phosphorus contents of mushroom compost were 1387 ± 36 mg/kg

and 2875 ±30 mg/kg respectively. They were much higher than those in shipyard

soil (Table 3.1). For one ton of mushroom compost, there were 1.387 士 0.036 kg

total nitrogen and 2.875 士 0.03 kg total phosphorus. The mushroom compost was

rich in nutrient.

Table 3.3 The total nitrogen and total phosphorus contents of mushroom compost.

Data are presented in mean 土 SD of 5 replicates.

Concentration (mg/kg)

Total N 1387 士 36

Total P 2875 士 30

78

3.3 Soil monitoring

3.3.1 Initial pollutant content in biopile and fungal treatment soils

From Table 3.4, oil and grease, total petroleum hydrocarbons and PAHs like

fluoranthene, pyrene, benz [a] anthracene, chrysene, benzo[a]pyrene,

benzo[g,h,i]perylene and indeno[l,2,3-cd]pyrene were found in both biopile

treatment and fungal treatment soils. Although the shipyard soil was highly

heterogeneous, the initial fluoranthene, pyrene, benz[a]anthracene, chrysene,

benzo[a]pyrene, benzo[g，h，i]perylene and oil and grease contents in biopile treatment

and fungal treatment were not significantly different except indeno[l,2,3-cd]pyrene

and TPH. The initial benz[a] anthracene, chrysene, benzo [ajpyrene,

benzo[g,h,i]perylene indeno[l,2，3-cd]pyrene levels in biopile and fungal treatment

soil were above the Dutch B level. The initial fluoranthene and pyrene

concentrations were above the Dutch A levels. The total PAHs concentrations of

biopile treatment soil and fungal treatment soil were 41.88 ±5.21 mg/kg and 37.15 士

6.68 mg/kg which were highly above the new Dutch Target level (1 mg/kg). The

total TPH contents in both biopile and fungal treatment exceeded the U S standard

(1000 mg/kg). Oil and grease contents in biopile treatment (12160 士 2120 mg/kg)

and fungal treatment (12020 士 2654 mg/kg) were very high. There was no 2- and

3-ring PAHs detected in biopile and fungal treatment soil.

Besides organopollutants, toxic heavy metals such as Pb, Cu and Zn were also

determined in both biopile and fungal treatment soils. Their contents were

statistically the same. From Table 3.5，Pb, Cu and Zn were leached out from both

biopile treatment and fungal treatment soils after shaking with C H 3 C O O H for 18

hours. The leached Pb, Cu and Zn contents were highly above the USEPA

Universal Treatment Objectives.

79

Table 3.4 The initial pollutant concentration of biopile treatment and fungal

treatment soil. Data are presented in mean 土 SD of 5 replicates. Data indicated

with * shows significant difference at 5% levels after Student t test.

Pollutant biopile treatment (mg/kg) fungal treatment (mg/kg)

PAHs

Fluoranthene 5.12 ±0.57 5.22 ± 1.02

Pyrene 5.03 士 0.82 4.41 士 1.69

Benz[a]anthracene 4.70 士 0.64 3.56 ± 1.94

Chrysene 4.75 ± 0.65 4.40 ± 0.622

Benzo[a]pyrene 7.48 ± 1.02 6.61 ±0.84

Benzo[g,h,i]perylene 6.06 ±0.85 5.56 ±0.53

Indeno[l,2,3-cd]pyrene 8.73 士 1.09* 7.39 士 0.85

Total PAHs 41.88 ±5.21 37.15 ±6.68

Oil and grease 12160 ±2120 12020 ±2654

Total petroleum hydrocarbons 1784 ±241 * 1320 士 169

Heavy metal

^ 2563 ±234 2702 ± 268

^ 1285 ±90 1292 ± 177

^ 807 ± 95 794 ±106

Table 3.5 The heavy metal leachability of biopile treatment and fungal treatment

soils. Data are presented in mean 土 SD of 5 replicates. Data indicated with *

shows significant difference at 5% levels after Student t test.

Heavy Universal Treatment biopile treatment (mg/1) fungal treatment (mg/1)

metal Objectives (mg/1)

^ 87 ±23 66 士 23 075

Cu 79 士 11 81 士 10

Zn 112 士 21 120 ±21 43

‘(USEPA)

80

3.3.2 On site air and soil physical characteristics

3.3.2.1 Soil temperature, air temperature

The soil and air temperatures on site plotted against time are presented in Figure 3.1.

The soil temperature and air temperature fluctuated during monitoring but followed

similar patterns between them. The lowest and highest soil temperatures during

monitoring were 29。C and 34.5。C respectively. Therefore, the soil temperatures

were still within the optimal temperature range (SO 'C to 40°C) for biodegradation of

petroleum (Bossert and Bartha, 1984). The minimum and maximum on site air

temperatures during soil monitoring were 27.54°C and 41.0rC respectively. Also

the minimum and maximum air temperatures measured by Hong Kong Observatory

were 25°C and 33°C respectively. The on site air temperatures were always higher

than the record of Hong Kong Observatory. Table 3.6 shows the on site physical

properties during monitoring. It is found that the weather during soil monitoring

was always sunny with high light intensity except rainfall found only on Day 46, 60

and 74. So the soil temperature and air temperature were also lower in these days

(Figure 3.1). From Table 3.6，the short wavelength U V (254 nm) was undetectable

but long wavelength U V (365 nm) was very high during sunny day. Also the CO2

(Detection range: 0.1-2.6%), SO2 (Detection range: 1 - 60 ppm) and H2S (Detection

range: 0.2%) gases were below the detection limits of the gas tubes with only < 0.1%,

< Ippm and < 0.2% in the air.

81

45 Soil temperature Air Temperature Air Temperature (HK Observatory)

20 1 1 1 1 1 1

0 20 40 60 80 100 120

Time (Day)

Figure 3.1 The change of soil and air temperatures (on site and reference from

Hong Kong Observatory) during the monitoring of biopile and fungal treatments.

82

Table 3. 6. The on site physical properties during monitoring of both biopile and the fungal treatment. Data are presented in mean + SD of

3

replicates.

Light Intensity

UV

UV

UV

Ra

in

fa

ll

Air relative Ultra-violet Air pollution

^ SO^

H^^

Day

(klux)

(254mn)

(300nm)

(365

nm)

(mm)

humidity %

(HKEPD)

index

(0.1-2.6%) (l-60ppm)

(0.2%)

(HKEPD)

(HKEPD)

(HKEPD)

"O

104.53 ±0

0 60 ±0

500 ±0

^ ^

4 56

<0.1%

<lppm

<0.2%

1.75684 ±0

0 40 ±0

500 ±0

^ ^

7 ^

<0.1%

<lppm

<0.2%

~Ti

28.84 ±0

0 60 ±0

600 ±0

^ 77^

9 ^

<0.1%

<lppm

<0.2%

~T81.85497 ±0

0 50 ±

0600

±0

^ ^

6 47

<0.1%

<lppm

<0.2%

~25

1.8479 ±0

0 60 ±0

600 ±0

^ ^

6 Vx

<0.1%

<lppm

<0.2%

31.22 ±0

0 10±0

100 ±0

^ ^

3 ^

<oI%

<lppm

<0.2%

1.8593 ±0

0 60 ±

0630

±0

^ ^

6 ^

<0.2%

1.8704 ±0

0 10±0160

±0

Yl

^ 4

^ <0.2%

53

1.8846 ±0

0 70±0700

±0

^ ^

5 ^

<0.2%

60 """1.84±0

0 40±0620±0

1 ^

5 ^

^T^

<0.2%

i 1.8379

0 320 ±0

% 3

^ <0.2%

1.8579 ±0

0 80 ±

0690

±0

^ Ts

13

^ ^T^

109 I 1.8478 ±0

0 90 土

0

680 ±0

^ TO

9 ^

^T^

83


3.3.3.1 Effect of type of treatment on total petroleum hydrocarbon content

A relative scale was used to indicate both residual TPH contents and oil and grease

contents to the initial contents measured at Day 0 listed in Table 3.4. It is because

their initial contents in biopile treatment and fungal treatment were not the same, it is

better to use a relative scale to compare between them. The changes of TPH and oil

and grease contents are shown in Figure 3.2 and Figure 3.3 respectively. A one-way

analysis of variance (ANOVA) was carried out to show the relation of residual TPH

in each soil sampling date in both biopile and fungal treatment. A two-way analysis

of variance (ANOVA) was used to determine whether the relative TPH contents or

oil and grease (%) were affected by type of treatment and/or time. The F and p

values of different subjects of TPH contents are listed in Table 3.7. A probability

value of/7 < 0.05 was considered as significant.

From Figure 3.2，at Day 4，there were 34 士 9。/。TPH removal for fungal treatment

and 18 士 3o/o removal for biopile treatment. So, there was a sharp drop in TPH

content in both biopile and fungal treatment. The TPH in fungal treatment zone

were 868 士 124 mg/kg which was already below the 1000 mg/kg at Day 4. For the

fungal treatment, the TPH contents from Day 4 to Day 109 were already below the

standard level and showed decreasing values with increasing incubation time. But the

TPH in biopile treatment at Day 4 was 1460 ±55 mg/kg which was still higher than

the U S safety standard of 1000 mg/kg. From the one way A N O V A and Tukey test,

TPH contents of Day 11 to Day 108 were of no significant difference. The mean

TPH of Day 11 to Day 39 were around 920 -1860 mg/kg while those of Day 46 to

108 were 840 mg/kg to 1540 mg/kg in biopile treatment. Biopile treatment took

longer time to reduce to the safety reference level. Finally, the TPH in fungal

treatment (594 士 48 mg/kg) was significantly lower than that in biopile treatment

(952 士 33 mg/kg). In terms of the relative scale as shown in Figure 3.2, the

percentage decrease in TPH content was greater with fungal treatment than biopile

treatment.

From Table 3.7，the two-way A N O V A study showed a significant effect of time on

relative residue of TPH (F = 24.288; p < 0.050). Also it indicates a significant

84

effect of type of treatment on relative residue of TPH (F = 40.555; p < 0.050).

However, type of treatment and time had no significant interaction on relative

residue of TPH {p = 0.600). Also the F value of type of treatment was larger than

that of time. Therefore, type of treatment showed a higher significant value than

time.

85

100 ^ A • -|a B C B C -D— Biopile treatment

90 T ^ T T B C B C Fungal treatment

g 严 C B C B C

I 60 - C £ 50 - bcde ^ 40 - de de e >

• T—l

I 30 -

2 0 -

1 0 -Q 1 1 1 1 1 1 i I I I I I

0 10 20 30 40 50 60 70 80 90 100 110 120

Time (Day)

Figure 3.2 The change in residue total petroleum hydrocarbon contents with time.

Data are presented as a relative scale to the initial content as shown in Table 3.4.

Data are presented in mean 土 SD of 5 replicates. Means with the same letter are

statistically similar (one way A N O V A with Tukey test,/? < 0.05).

Table 3.7 The results of the two-way A N O V A analyses on the study of total

petroleum hydrocarbons during the treatments of the contaminated soil. A

probability value ofp< 0.05 is considered as significant.

Mean square F p value

Time 2274.397 24.288 0.000

Type of Treatment 3797.664 40.555 0.000

Time x Type of Treatment 79.547 0.849 0.600

Type of Treatment: biopile treatment versus fungal treatment

86

3.3.3.2 Effect of type of treatment on oil and grease content

From Figure 3.3, compost addition significantly removed more oil and grease than

biopile only. There was a sharp drop in oil and grease content in fUngal treatment

soil on Day 4. At Day 4, 23 士 lO。/。removal for fungal treatment and 3 士 9o/o

removal for biopile treatment only were observed. From Day 11, the oil and grease

content fluctuated and decreased slowly. At the end of treatment, 4380 士 432

mg/kg oil and grease remained in biopile treatment with only 64 士 4o/o removal,

while 3310 士 325 mg/kg oil and grease remained in fungal treatment with a 72 土 3o/o

removal. More oil and grease was removed in fungal treatment.

From Table 3.8，the two-way A N O V A study revealed a significant effect of time on

removal of oil and grease (F = 118.649; p < 0.050). Also there was a significant

effect of type of treatment on removal of oil and grease (F = 88.232; p < 0.050).

Both factors time and type of treatment had significant interaction on relative oil and

grease content (p < 0.050). The F value of time was larger than that of type of

treatment. Therefore, time shows a higher significant value than type of treatment.

87

100 Ell -Or- Biopile treatment

90 \ \ Fungal treatment

I 70 A \ I T

I 丨 ^ 20 -

1 0 -

0 1 1 1 1 1 I I I I I I

0 10 20 30 40 50 60 70 80 90 100 110

Time (Day)

Figure 3.3 The change in residue oil and grease contents with time. Data are

presented as a relative scale to the initial content as shown in Table 3.4. Data are

presented in mean + SD of 5 replicates.

Table 3.8 The results of the two-way A N O V A analyses on the study of relative oil

and grease content during the treatments of the contaminated soil. A probability

value o{p< 0.05 is considered as significant.

Mean square F p value

Time 4560.066 118.649 0.000

Type of Treatment 3391.026 88.232 0.000

Time x Type of Treatment 211.170 5.494 0.000

Type of Treatment: biopile treatment versus fungal treatment

88

3.3.3.3 Soil pH

The soil pH is a measure of the hydrogen ion activity and is defined as the negative

logarithm of H+ activity (moles per liter) in soil solution. The change in soil pH

with time is plotted in Figure 3.4. In general, the soil pH was within the range pH

7.7 to pH 8.2. Therefore, the soils of biopile treatment and fungal treatment soil

were alkaline. The pH values of both biopile and fungal treatment soil increased

from Day 0 (biopile treatment: pH 7.80 士 0.07; fungal treatment: pH 7.77 士 0.06) to

Day 11 (biopile treatment: pH 8.02 士 0.03; fungal treatment: pH 7.98 士 0.05). Also

then there is a sharp drop on Day 18 (biopile treatment: pH 7.76 士 0.01; fungal

treatment: pH 7.75 士 0.07). The pH of both biopile treatment and fungal treatment

fluctuated from Day 18 to Day 88. Also finally the pH of biopile treatment and

fungal treatment on Day 109 were 7.82 士 0.06 and 7.82 士 0.04 respectively. The

fluctuation of pHs of biopile treatment and fungal treatment soil was similar with

time. The optimal pH range for biodegradation of petroleum is 5 - 7.8 (Dibble and

Bartha, 1979). Therefore, the pH of biopile and fungal treatment soil were slightly

above the optimal pH range.

From Table 3.9, a two-way A N O V A analysis demonstrates significant effects of time

and type of treatment on pH values ([time] F = 8.34, p < 0.05; [type of treatment] F =

11.08，p < 0.05) but no significant interaction between time and type of treatment is

detected ([time x type of treatment] F = 0.625，p = 0.819). The F value of type of

treatment was larger than that of time. Therefore, type of treatment shows a higher

significant value than time.

89

8.20 r ； ^ Hlf- Biopile treatment

8 15 - T ‘ Fungal treatment

8.10 -

8.05 -

8.00 - W | i

7.75 T y

7.70 ‘ ‘ ‘ ‘ 1 1 0 20 40 60 80 100 120

Time (Day)

Figure 3.4 The change in soil pHs of the biopile and fungal treatment with time.

Data are presented in mean + SD of 5 replicates.

90

3.3.3.4 Moisture

The soil moisture is plotted in Figure 3.5. The initial soil moisture of biopile and

fUngal treatment soil were 13 士 2 % and 12 士 3o/o respectively. After the addition of

mushroom compost, the soil moisture in biopile and fungal treatment soil were 13 土

2 % and 16 士 2 % respectively on Day 4. Then there were fluctuations of soil

moisture during soil monitoring. Finally, the biopile and fungal treatment soil

moisture were 14 士 P/o and 14 士 2o/o respectively. Both average biopile treatment

and fungal treatment soil moisture were 14 士 3o/o. The optimal moisture range for

stimulating biodegradation of petroleum hydrocarbons in soil was 50 % to 80%

(Bossert and Bartha, 1984). From Table 3.9, a two-way A N O V A showed significant

effects of time and type of treatment on soil moisture ([time] F = 6.62, p < 0.05;

type of treatment] F = 19.388，< 0.05) but no significant interaction between time

and type of treatment was detected ([time x type of treatment] F = 0.674，；？ = 0.775).

The F value of type of treatment was larger than that of time. Therefore, type of

treatment shows a higher significant value than time.

22 r ： - a - Biopile

2q t treatment

T - A - Fungal treatment

1 0 -

g I I I I I I

0 20 40 60 80 100 120

Time (Day)

Figure 3.5 The change in soil moisture of the biopile and fungal treatment with

time. Data are presented in mean + SD of 5 replicates.

91

3.3.3.5 Electrical conductivity

The soil conductivity is plotted in Figure 3.6. The unit of conductivity is mS/cm

which means milliSiemen per centimetre and is equal to one /xmho/cm. Greater

value of mS/cm means more soluble salts were found in soil. The initial

conductivities of biopile and fungal treatment were 0.55 士 0.05 mS/cm and 0.43 士

0.07 mS/cmr respectively. The conductivity increased from Day 0 to Day 18

(biopile treatment: 1.11 士 0.03 mS/cm and fungal treatment: 1.11 士 0.46 mS/cm) and

then fluctuated. There was a lower conductivity in fungal treatment from Day 60

onwards. But the conductivity in biopile treatment remained high from Day 60.

The final conductivity of biopile and fungal treatment were 1.21 士 0.09 mS/cm and

0.53 士 0.14 mS/cm respectively. From Table 3.9, by two-way A N O V A analysis,

there were significant effects of time and type of treatment on electrical conductivity

([time] F = 14.98,< 0.05; [type of treatment] F = 109299, p < 0.05), and there was

significant interaction between time and type of treatment ([time x type of treatment:

F = 6.713,/? < 0.05). The F value of type of treatment was larger than that of time.

Therefore, type of treatment shows a higher significant value than time.

1.700 -O— Biopile treatment — 丁

1 500 Fungal treatment

5: WWvi 0.500 ¥ ^ ^ 0.300 ‘ ‘ ‘ ‘ ‘ ‘

0 20 40 60 80 100 120 Time (Day)

Figure 3.6. The change in soil conductivity of the biopile and fungal treatment with

time. Data are presented in mean 土 SD of 5 replicates.

92

3.3.3.6 Salinity

The soil salinity is listed in Figure 3.7. The initial salinity of biopile and fungal

treatment soil were 0.06 士 0.05o/o and 0.05 士 0.05o/o respectively. During treatment,

the salinities of biopile and fungal treatment soils fluctuated in a small range. The

general soil salinities in both biopile treatment and fungal treatment soil were about

0-0.25% and remained at such low levels. Finally, the salinities of biopile and

fungal treatment soil were 0.08 士 0.08o/o and 0.05 士 0.05o/o respectively. From

Table 3.9, a two-way A N O V A analysis revealed that there was significant effect of

time on soil salinity ([time] F = 3.09, p < 0.05), but there was no significant effect of

type of treatment on salinity ([type of treatment] F = 0.76, p = 0.384) and no

interaction between time and type of treatment ([time x type of treatment] F = 1.271，

p = 0.24).

0.30「 HIH Biopile treatment

Fungal treatment 0.25 -

g 0.20 - T

1 。 . 1 5 - A j •• 1 1

0-03 Y Y itiJ ^ 0.00 ‘ ‘ ‘ ‘ ‘ ‘

0 20 40 60 80 100 120 Time (Day)

Figure 3.7 The change in soil salinity (%) of the biopile and fungal treatment with

time. Data are presented in mean + SD of 5 replicates.

93

Table 3.9 The results of the two-way A N O V A analyses on the studies of pH,

moisture, electrical conductivity and salinity during the treatments of the

contaminated soil. A probability value of/> < 0.05 is considered as significant.

Mean square F value p value

pH

Time 0 . 0 6 3 5 9 ^ 0

Type of treatment 0.08448 11.08 0.001^

Time x Type of treatment 0.004767 0.625 0.819

Moisture

Time 3 4 . 9 1 2 ^ 0

Type of treatment 102.236 19.388 0


Conductivity

Time 0.435 1 4 . 9 8 0


Time x Type of treatment 0.195 6.713 0

Salinity

Time 0 . 0 1 0 4 2 J m 0.001

Type of treatment 0.002564 0.384

Time x Type of treatment 0.004286 1.271 O ^

94

3.3.3.7 Microbial population in soils of biopile and fungal treatment

The total bacterial counts and total mold counts along time are shown in Figs 3.8 and

3.9. The initial total bacterial population sizes in both biopile treatment and fungal

treatment soil were 201 士 66 x 10" cfu/g soil and 243 土 62 x 10' cfu/g soil

respectively. On Day 4, there were 624 士 220 % and 915 士 255 % increases in total

bacterial population in biopile treatment and fungal treatment soil accordingly. The

total bacterial population in both biopile treatment and fUngal treatment soil

increased significantly from Day 0 to Day 60. On Day 60, the total bacterial

population in fungal treatment (2420 士 167 x 10" cfu/g soil) was drastically higher

than that in biopile treatment (19 ± 3 x 10' cfu/g soil). Then the bacterial

population decreased gradually from Day 60 to Day 109. At the end of monitoring,

total bacterial population in fungal treatment (1035 ± 37 x 10" cfu/g soil) was still

higher than that in biopile treatment (73 ± 5 x 10" cfu/g soil). In general, the total

bacterial count in fungal treatment soil was higher than that in biopile treatment soil.

The initial total mold population sizes in both biopile treatment and fungal treatment

soil were 153 土 17 cfii/g soil and 166 士 19 cfu/g soil respectively. On Day 4, there

were 15 士 10 o/o decrease but 268 士 47 % increase of mold population in biopile

treatment and fungal treatment soil, respectively. The total mold population in

fungal treatment soil increased significantly from Day 0 to Day 39. On Day 39，

total mold population sizes in biopile treatment and fungal treatment soil were 183 士

10 cfU/g soil and 4473 ± 435 cfu/g soil respectively Then mold population in

fungal treatment decreased gradually from Day 39 to Day 109. However, it kept

more or less constant for biopile treatment soil. At the end of monitoring, total

mold population in fungal treatment (733 士 178 cfu/g soil) was still higher than that

in biopile treatment (86 士 13 cfu/g soil). In general, the total mold population in

fungal treatment soil was significantly higher than that in biopile treatment soil.

From two way A N O V A analysis, time and type of treatment have significant effects

on both total bacterial count ([time] F = 253741.5,< 0.05; [type of treatment] F =

1011319,;? < 0.05) and mold count ([time] F = 127.749,/? < 0.05; [type of treatment]

F = 2441.139, p < 0.05) (Table 3.10). Also there was significant interaction

between time and type of treatment in both bacterial count ([time x type of treatment]

F = 247141.8, 0.05) and mold count ([time x type of treatment] F = 116.307,/? <

95

0.05) (Table 3.10). For both bacterial and mold population, the F value of type of

treatment was larger than that of time. Therefore, type of treatment shows a greater

significant effect than time.

le+13 n —O- Biopile treatment

^ 1 计 12 - Fungal treatrmit / \

I lefll - K \

lE： / \ I le^ - t \

lef5 - - \

le+4 “‘ 1 1 1 1 1 1 -n 0 20 40 60 80 100 120

Time (Day)

Figure 3.8 The change in total bacteria population (colony forming unit/g) of the

biopile and fungal treatment with time. Data are presented in mean + SD of 5

replicates.

96

e9 n ~ • B i o p i l e treatment

Fungal treatment A

/

V

e4 I I 1 ！ 1 1 1

0 20 40 60 80 100 120

Time (Day)

Figure 3.9 The change in total mold population (colony forming unit/g) of the

biopile and fungal treatment with time. Data are presented in mean + SD of 5

replicates.

Table 3.10 The results of the two-way A N O V A analyses on the studies of total

bacterial population and total mold population during the treatments of the

contaminated soil.. A probability value ofp< 0.05 is considered as significant.


Bacteria population

Time 3.248 x lO'" 253741.5 0

Type of treatment 1.295 x 10乃 1011319 0

Time x Type of treatment 3.163 x 247141.8 0

Mold population

Time 5921790.097 127.749 0

Type of treatment 113158784 2441.139 0


97

3.3.3.8 Removal of Organopollutant PAHs in biopile and fungal treatment

A relative scale was used to indicate the residual PAH contents to the initial contents

measured at Day 0 listed in Table 3.4. The changes in the pollutants fluoranthene,

pyrene, benz[a]anthracene, chrysene, benzo[a]pyrene, benzo[g,h,i]perylene and

indeno[ 1,2,3-cd]pyrene are shown in Figure 3.10 to Figure 3.16 respectively. A

two-way analysis of variance (ANOVA) was used to determine whether the relative

individual PAHs contents (%) were affected by type of treatment and / or time. The

F and values of different subjects of all individual PAHs are summarized in Table

3.11. A probability value of> < 0.05 was considered as significant.

In general, for 4-ring PAHs like fluoranthene and pyrene, their relative contents (%)

in biopile treatment increased from Day 0 and were 118 士 18o/o for fluoranthene and

114 ± 170/0 for pyrene on Day 4. Then, their contents also increased gradually from

Day 4. But all their contents in fungal treatment dropped on Day 4 (82 士 13o/o for

fluoranthene; 93 士 17o/o for pyrene) and then fluctuated at around 100% from Day 4

to Day 18 and then increased gradually. Then their relative contents in both biopile

treatment and fungal treatment soil kept rising to peak levels on Day 53 and then

dropped significantly afterwards and were undetectable on Day 109. Therefore,

their degradation completed on Day 109.

For 5- to 6-ring PAHs like chrysene, benz[a]anthracene, benzo[ajpyrene and

benzo[g,h,i]perylene, most of their relative contents (%) in biopile treatment

increased from Day 0 and were 107 士 14o/o for chrysene, 110 士 14o/o for

benz[a]anthracene, 101 士 8o/o for beiizo[a]pyrene and 90 土 8o/o for

benzo[g,h,i]perylene on Day 4. Then, their contents also increased gradually from

Day 4. But all their contents in fungal treatment dropped on Day 4 (81 士 9o/o for

chrysene, 82 士 IQo/o for benz[a]anthracene, 80 士 T A for benzo[a]pyrene and 71 士 T/q

for benzo[g,h,i]perylene) and then fluctuated at around 100% from Day 4 to Day 11

and then increased gradually. Their relative contents (%) in both biopile treatment

and fungal treatment soil kept rising to peak levels on Day 18 and then dropped

significantly afterwards and were undetectable on Day 39. The relative

indeno[ 1,2,3-cd]pyrene contents in biopile and fungal treatment were 98 士 8o/o for

and 83 士 9o/o respectively on Day 4. The relative content of indeno[ 1,2,3-cd]pyrene

98

was undetected on Day 18 but raised to a peak level on Day 25 and then dropped

significantly and were undetectable on Day 39. All the 5- to 6-ring PAHs were

completely degraded at Day 39. The higher molecular PAHs were firstly degraded

while the lower molecular PAHs required were later attacked and removed.

Table 3.11 shows the result of the two-way A N O V A on the effects of time and type

of treatment on the removal of organopollutants. It reveals that time induced a

significant effect on residual contents of all PAHs levels {p < 0.05). Also it also

reflects a significant effect of the type of treatment on relative contents of all PAHs

(p < 0.05). Also there was a significant interaction between time and type of

treatment on the relative contents of all individual PAHs (p < 0.05) except pyrene (p

=0.486). For all individual PAHs, the F value of time was larger than that of type

of treatment. Therefore, time shows a greater significant effect than type of

treatment.

300 —•— Biopile treatment

丁 Fungal treatment

？ 5。 - A

I: ^^ ^ I 50 -

0 1 丨丨丨 ^ A g 丨

0 20 40 60 80 100 120 Time (Day)

Figure 3.10 The change of soil fluoranthene contents during biopile and fungal

treatment in relation to the initial levels as reported in Table 3.4. Data are presented

in mean + SD of 5 replicates.

99

400 r ——^

- Q - Biopile treatment

350 - Fungal treatment

^ T

奢 300 - T ”

I 2��-^ 150 - / Y V

I 100 M

0 丨 1 1 1 L ^ a 1

0 20 40 60 80 100 120

Time (Day)

Figure 3.11 The change of soil pyrene contents during biopile and fungal treatment

in relation to the initial levels as reported in Table 3.4. Data are presented in mean

+ SD of 5 replicates.

3 5 0 厂：

-B— Biopile treatment

300 - Fungal treatment

¥ A

” 。 — \ i 200 - \

I 15。- A

I 、 I 50 - A

0 丨 a—o—Q—s ra~丨——0 丨—0 丨 0 20 40 60 80 100 120

Time (Day)

Figure 3.12 The change of soil beiiz[a]anthracene contents during biopile and

fungal treatment in relation to the initial levels as reported in Table 3.4. Data are


100

180「 . I —

-B— Biopile treatment

160 - 丁 -A— Fungal treatment

I 140 - i I 120 - 丁

i 80 -A

S 60 -

1 4 � - \ 2 0 - \

0 ‘ s—e—s—B ~ 0 1——0 1

0 20 40 60 80 100 120

Time (Day)

Figure 3.13 The change of soil chrysene contents during biopile and fungal



2 5 0 厂：

-•— Biopile treatment

乞 Fungal treatment

c 2 0 0 - 丁

<D 丁 I X

卜 A j I 50 - \

0 ‘ ~ m 1 r a 1

0 20 40 60 80 100 120

Time (Day)

Figure 3.14 The change of soil benzo[a]pyrene contents during biopile and fungal



101

^ 250 厂 -0— Biopile treatment

w Fungal treatment

！15。- A ^ 100 k ^ 、

i P \ r � - \

^ 0 丨 0---O———0—J——o L 四 1

0 20 40 60 80 100 120

Time (Day)

Figure 3.15 The change of soil benzo[g,h,i]perylene contents during biopile and



250 厂：

一 —B— Biopile treatment

^ Fungal treatment I 200 - 丁

• 150 - ^

rA I ^

r ^ \

0 & ra—ra——-£3—'0 丨 — — 0 丨

0 20 40 60 80 100 120

Time (Day)

Figure 3.16 The change of soil indeno[l ,2,3-cd]pyrene contents during biopile and



102

Table 3.11 The results of the two-way A N O V A analyses on the studies of

individual PAHs during the treatments of the contaminated soil. A probability value

ofp < 0.05 is considered as significant.


fluoranthene

Time 41166.461 101.65 0



pyrene

Time 62655.104 65.667 0

Type of treatment 7016.984 7.354 0.007


benz [a] anthracene

Time 70139.779 355.774 0



chrysene

Time 36286.868 438.895 _ 0



benzofa]pyrene

Time 41530.848 601.742 0

Type of treatment 973.037 14.098 0 ~ ~ ~


benzo[g, h, i]perylene

Time 52320.297 930.949 0



indeno[1，2，J-cd]pyrene

Time 50999.441 1547.555 ~ 0



Type of treatment: biopile versus fungal treatment.

103

3.3.3.9 Effect of type of treatment on residual PAHs at Day 4

The relative pollutant contents (%) of fluoranthene, pyrene, benz[a]anthracene,

chrysene, benzo[a]pyrene, benzo[g,h,i]perylene and indeno[ 1,2,3-cd]pyrene in Day 4

are plotted in Figure 3.17. In 4 days, there was degradative removal of the PAHs

leading to lower than 100% levels of the all the tested PAHs in fungal treatment.

From the Student t test, the residues of all PAHs in biopile treatment soil were

significantly higher than those in fungal treatment soil. Also, the relative residues

of all PAHs in fungal treatment were never above 100%.

Figures 3.18 (a) and (b) show the comparison of the organopollutant profiles by

GC-MSD of biopile treatment and fungal treatment soil on Day 0 and Day 4

respectively. The chromatogram of fungal treatment on Day 4 was lower than that

on Day 0. However, the chromatogram of biopile treatment on Day 4 was similar

to that on Day 0.

104

140「

*

• Biopile treatment

• •

*

*

120

- T

丁

• Fungal treatment

一

ri

JL

T

*

& 100

- r

^

*

T

扫

—

T

门

I 80 -

^

^

^

^

n.

^

CO

•：.：

)：.：

.|

妾

60 -

CLh

：；：：：；：：：

：：；：；：：；；

；：；：：：；：；

(D

；：；：；:；:；:

：；：；：；：；：

：；：；：；：；：

40 -

ii

__

I—H

•：•：•：•：•：

：：：：：•：•：

：•：•：•：•：

(D

：：：：：：：：：

:::::::::

P

；；；：；：；；；

丨：

；；；：；；；；：

20 -

__

__

__

0 丨

丨

I

I

丨：：：

：出：：

丨_I

liMiiil___I_III.

FLUO

PYR

BEN(A)

CHRY

BEN

GHI

INDENO

Different PAHs

Figure

3.17.

The relative PAHs contents (%)

at Day 4

in comparison

to their initial concentrations

in biopile treatment and fungal treatment

soil showed

in Table

3.4.

Data

are presented

in mean 土

SD

of 5 replicates. Data indicated with * shows significant difference

at 5

% levels

after Student T test.

(Key:

FLUO—fluoranthene, PYR—

pyrene, BEN(A)—benz[a]anthracene, CHRY—

chrysene, BEN—

benzo[a]pyrene,

GHI—

benzo[g,h,i]perylene and INDENO—

indeno[ 1,2,3-cd]pyrene).

105

(a) J TIC： 2 0 0 1 0 2 0 .D 二 TIC:19G1020,D(” I

5 5 0 0 0 0 - :

： s 5 0 0 0 0 0 - I

] Da.4 | F \ 3�‘ I i f

：： ^ -000 ： DayO •。。；

0 ‘ ‘ ‘ ‘ I ‘ I 1 1 1 1 I 1 1 1 1 •• _ —

2 0 . 0 0 3 0 . 0 0 4 0 0 0

. . . .

(b)

TIC: 0 3 0 1 0 0 3 .D

- TIC: 0201003,D C) 5 0 0 0 0 0 -

4 5 0 0 0 0 - I

4 0 0 0 0 0 - I

::〕 iffiV Day。

5 0 0 0 0 - _ A J ^

一 0 J , .

‘ ‘ ‘ ^ I ‘ l ‘ I 1 1 • ! 1 1 1 1 ) o m -^n nn d n m

Figure 3.18 The volatile profiles of (a) biopile treatment and (b) fungal treatment

soil revealed by GC-MS on Day 0 and Day 4.

106

3-3.3.10 Effect of type of treatment on residual PAHs at their peak levels

From Figures 3.10 to 3.16，PAHs would reach peak levels above the initial contents

during the time of treatment. The residual % at peak levels of all PAHs were

plotted in Figure 3.19 to compare the performance of biopile treatment and fungal

treatment. The residual % at peak levels of fluoranthene (biopile treatment: 241 士

32%； fungal treatment: 198 士 20o/o)，pyrene (biopile treatment: 279 士 58o/o; fungal

treatment: 250 士 52o/o), benz[a]anthracene (biopile treatment: 268 土 42o/o; fungal

treatment: 191 士 45o/o)，chrysene (biopile treatment: 135 士 23o/o; fungal treatment: 94

士 240/0)，benzo[a]pyrene (biopile treatment: 176 士 18o/o; fungal treatment: 128 土

23%), beiizo[g,h,i]perylene (biopile treatment: 200 士 17o/o; fungal treatment: 151 士

24%) and indeno[l,2，3-cd]pyrene (biopile treatment: 143 士 19o/o; fungal treatment:

182 士 16o/o) were above 100% and some of them even above 200%. Except pyrene,

significantly less residual amounts of PAHs were found in fungal treatment than in

biopile treatment.

107

350

r

T •

Biopile treatment

*

•

T El Fungal treatment

*

g 250

- JL

rXrn

*

§ 丁

*

y *

I 200

- JU,

T r-^

T o

：：：：：：：：]

W *

T

_ 150

一

丄

T i

^ I

•扫

丨丨丨丨丨丨丨丨丨

；；；；:：；：

T

：丨丨丨丨丨丨丨丨

丨丨丨

召

100

-

《

；丨

：；：：：；：:

：；：；：；：；：

：：：：：：：：：

：;：：：：；：；

50

- 丨

⑴_

丨丨丨_

__

__

__

0 ^^——‘

L^—

—I

lihiM

——

1 liMiij——I

liliiliiiil I

丨丨 I

I'

ll

FLUO

PYR

BEN(A)

CHRY

BEN

GHI

INDENO

Different PAHs

Figure 3.19

A comparison of the peak PAH levels during biopile treatment and fungal treatment. Data are presented as relative to the initial

contents as sho

wn in Table 3.4. Data are presented in mean 土

SD of 5 replicates. (Data indicated with * shows significant difference at 5%

levels after Student T test. (Key: FLUO—

fluoranthene, PY

R—py

rene

, BEN(A)—benz[a]anthracene, CHRY—

chrysene,

BEN—

benzo[a]pyrene, GHI—

benzo[g,h,i]perylene and INDENO—

indeno[ 1,2,3-cd]pyrene)

I j

108

3.3.3.11 Effect of type of treatment on residual organopollutants at the end of

treatment

Figures 3.20 (a) and (b) showed the comparison of the organopollutant profiles by

G C - M S D of biopile treatment and fungal treatment soil on Day 109. For both

biopile and fungal treatment, the chromatograms of Day 109 were shifted from the

Day 0. But the chromatogram of fungal treatment was lower than that of biopile

treatment.

109

(a) A ㈣ d e m c e TIC: 2 1 0 1 0 2 1 .D

6 0 0 0 0 0 - T iC : T 1 2 L 4 E . D

5 5 0 0 0 0 -

5 0 0 0 0 0 -

WW I I f f I I I V Wi Day 0

350。。。 I If ’

0 I ‘ ‘ ‘ ‘ 1 1 1 1 1 — 1 1 1 1 1 1 J

T i m f t - . > 1 0 . 0 0 2 0 . 0 0 3 0 . 0 0 4 0 . 0 0

(b) A b u n d a n c e TIC: 0 3 0 1 0 0 3 . D

- TIC. T12L. m S > ( n

5 0 0 0 0 0 -

4 5 0 0 0 0 -

4 0 0 0 0 0 -二 i

〜 I / —。• , V I

誦： i j f ^ \ ，一 —

0 J 1 ' 1 • . . 1 . . . . 1 , T » m e ~ > 2 0 . 0 0 3 0 . 0 0 4 0 . 0 0

Figure 3.20 The volatile profiles of (a) biopile treatment and (b) fungal treatment

soil revealed by GC-MS on Day 109.

110

3.3.3.12 Effect of type of treatment on total nitrogen and phosphorus contents

To see the effect of mushroom addition on nutrient in soil, total nitrogen and

phosphorus contents in soil at Day 0 (no compost added), Day 4 and Day 109 were

compared (Figures 3.21 and 3.22). The initial total nitrogen content was 21 士 6

mg/kg and 21 士 11 mg/kg in biopile treatment and fungal treatment soil respectively.

It is found that total nitrogen in fungal treatment significantly increased at Day 4 (42

士 10 mg/kg) but not in biopile treatment (21 ± 4 mg/kg). Also total nitrogen in

biopile treatment soil on Day 0, Day 4 and Day 109 were statistically the same.

After 109 days, total nitrogen in fungal treatment (33 士 5 mg/kg) was still higher

than biopile treatment (23 土 4 mg/kg).

The initial total phosphorus contents were 334 士 27 mg/kg and 343 士 39 mg/kg in

biopile treatment and fungal treatment soil respectively. Total phosphorus increased

significantly in fungal treatment (387 士 25 mg/kg) soil at Day 4 but not in biopile

treatment soil (326 士 20 mg/kg). Also total phosphorus in biopile treatment soil on

Day 0 (no compost added), Day 4 and Day 109 were statistically the same. The

total phosphorus in biopile treatment and fungal treatment were 384 ± 79 mg/kg and

362 士 24 mg/kg after 109 days.

I l l

的 • Biopile treatment

IA • Fungal treatment

^ 50 - T

1 40 - r^ A

^ B • T 3 0 - a T a ：:：:：;：;：:

2 T a T ::::::丨… 扫丁

8 2 0 - |：：：：1；：；| \ W M

o :：:：:：;：;：：:>：;：;：:

^ 10 - I I I I I I

0 kihiM——I I liiiMiiil——I

0 4 109

Day

Figure 3.21 The change in total nitrogen contents at Day 0, Day 4 and Day 109 of

biopile treatment and fungal treatment soil. Data are presented in mean + SD of 5

replicates. Means with the same letter are statistically similar (one way A N O V A

with Tukey test, p < 0.05).

• Biopile treatment

500「口 Fungal treatment

450 - A a丁 ④ 400 - a B T I f

^ 350 - T pJ— \

1300 -

•I 250 -

老 2 0 0 -

§ 150 -

u 100 -50 -0 丨::::;丨——I I I

0 4 109 Day

Figure 3.22 The change in total phosphorus contents at Day 0, Day 4 and Day 109

of biopile treatment and fungal treatment soil. Data are presented in mean 土 SD of

5 replicates. Means with the same letter are statistically similar (One way A N O V A

with Tukey test,/? < 0.05).

112

3.3.3.13 Effects of type of treatment on the physical and chemical properties of soil

After treatment, the physical and chemical properties of soil were analysed and are

listed in Table 3.12. Figure 3.23 shows the photographs of soil before and after

biopile and fungal treatments. After the 109 Days treatment period, the

organopollutant like PAHs and TPH were already below the safety standard. PAHs

were completely degraded but oil and grease and TPH were not completely removed.

The nutrient content in fungal treated soil was significantly higher than that in biopile

treated soil. To study whether the treated soil was safe, the toxicities of biopile

treated soil and fungal treated soil were examined. Furthermore, it is because toxic

heavy metals like Pb, Cu and Zn were still in the soil, heavy metal removal

experiments were carried out.

113

Table 3.12 The physical and chemical properties of soil after biopile treatment and

fUngal treatment. Data are presented in mean 土 SD of 5 replicates. Data indicated

with * shows significant difference at 5% levels after Student t test.

biopile treated soil fungal treated soil

pH 7.82 士 0.06 7.82 士 0.04

Moisture (%) 14士 1 14士 2

Salinity 0.08 ± 0.08 0.05 ± 0.05

Conductivity (mS) 1.210 士 0.090* 0.530 ±0.140

Organic Carbon (%) 2.040 ±0.081 1.878 ±0.073

Total N (mg/kg) 23 士 4 33 士 5 *

N03-N (mg/kg) f T o T T 6

Total P (mg/kg) 384 ± 79 362 士 24

K (mg/kg) 1729 ±213 1803 ±334

Oil & Grease (mg/kg) 4380 ± 432 * 3310 ±325

TPH (mg/kg) 952 士 33 * 594 士 48

Total PAHs (mg/kg) T m ^

Total Pb (mg/kg) 2043 ± 91 2069 ± 144

Total Cu (mg/kg) 1038 ±77 1119 士 94

Total Zn (mg/kg) 783 ± 70 812 ±85

TCLP-Pb (mg/1) 62 ± 3 62^9

TCLP-Cu (mg/1) 59 ± 5 58^6

TCLP-Zn (mg/1) 87 ± 7 90± 13

N.D.: Non detectable

114

(b)

•

I _ (d)

_ _

Figure 3.23 Photographs of the soil before (a) biopile treatment and (b) fungal treatment; and after (c) biopile treatment and (d) fiingal treatment.

115


3.4.1 Seed germination test

The % of germination for three plants wheat (Triticum aestivum), ryegrass (Lolium

perenne) and Chinese cabbage {Brassica chinensis) relative to their corresponding

germination in garden soil is plotted in Figure 3.24, Figure 3.25 and Figure 3.26

accordingly. Figure 3.27 shows the photographs of seed germination results of

different plants cultivated on biopile and fungal treated soil. The germination

performance in garden soil is listed in Table 3.13. The initial germination

frequencies of wheat in biopile treatment and fungal treatment were both 89 土 3o/o

and for ryegrass (biopile treatment: 68 士 5o/o; fungal treatment: 71 士 4o/o) and for

Chinese cabbage (biopile treatment: 42 士 7o/o; fungal treatment: 52 士 6o/o). For all

three species, relative seed germination was significantly increased in the soil after

the two types of treatment. For wheat, the relative seed germination after both

biopile treatment and fungal treatment soil were the same at 99 士 2o/o. For ryegrass,

the relative seed germination after both biopile treatment and fungal treatment soil

reached 99 土 lOo/o and 99 士 4o/o respectively. However, for Chinese cabbage, the

relative seed germination after both fungal treatment and biopile treatment soil were

the same at 71 士 TA.

Table 3.13. The number of wheat {Triticum aestivum), ryegrass {Lolium perenne)

and Chinese cabbage {Brassica chinensis) seeds germinated in garden soil for 4 days

at 24 士 O.5OC. Data are presented in mean 土 SD of 5 replicates.

Number of seeds germinated

Wheat (Triticum aestivum) 25 ± 0

Ryegrass {Lolium perenne) 23 士 1

Chinese cabbage (Brassica chinensis) 23 士 1

116

120 厂 • Before treatment

* • After treatment 茨 100 - , _ ^ _ ,

fn T I 丁

8 0 -

I a 60 -

I 40 — i S 20 -

0 1 1

Biopile treatment Fungal treatment

Figure 3.24 The effects of the biopile treatment and fungal treatment on the

germination frequencies of wheat Triticum aestivium for 4 days at 24 士 0.5°C. Data

are presented in mean + SD of 5 replicates. Data indicated with * shows significant

difference at 5% levels after Student t test.

• Before treatment 190 r

「士 E] After treatment 本 I I

^ T *

i 100 - r - H r ^

.a 80 -

(D : 6 0 -<U (U M ^ 40 -

0 I

Biopile treatment soil Fungal treatment soil


germination frequencies of ryegrass Lolium perenne for 4 days at 24 士 0.5°C. Data

are presented in mean ± SD of 5 replicates. Data indicated with * shows significant


117

90 r • Before treatment

一 80 _ * • After treatment ^ T T *

a 70 - p - L - . ^ ^ 0

6 0 -1 T g 50 - T ~ ‘ ~ <D bO 劣八

力 40 -(D <D Z 30 -I 20 -Hj ^ 10 -

0 ‘



germination frequencies of Chinese cabbage Brassica chinensis for 4 days at 24 士

0.5°C. Data are presented in mean + SD of 5 replicates. Data indicated with *

shows significant difference at 5% levels after Student t test.

118

(a) Biopile treated Fungal treated

B B (b) Biopile treated Fungal treated

國 (c) Biopile treated Fungal treated

Figure 3.27 Photographs of in vitro acute toxicity test using plants: (a) Wheat Triticum aestivum, (b) Ryegrass Lolium perenne and (c) Chinese cabbage Brassica

chinensis on biopile treated and fungal treated soils at 25°C after 3 days incubation.

119

3.4.2 Indigenous bacterial toxicity test

The population growth of Bacillus cereus, Pseudomonas aeruginosa and

Methylobacterium sp. is shown in Figure 3.28，Figure 3.29 and Figure 3.30

respectively. This was presented as a relative to their initial population as listed in

Table 3.14. Figure 3.31 shows the photographs of the recovered colonies of various

bacteria inoculated to biopile and fungal treated soil. The population growth was

indicated by percentage increase in colony forming unit (cfU/g soil). The

population growth in soil of Bacillus cereus, Pseudomonas aeruginosa and

Methylobacterium sp. in soil before biopile or fungal treatment was: 770 土 61o/o

(biopile treatment), 938 士 79o/o (fungal treatment); 104 士 15o/o (biopile treatment), 92

士 29o/o (fungal treatment); -22 士 6 % (biopile treatment), -20 士 5o/o (fungal treatment)

accordingly. For Methylobacterium sp., it has negative population growth,

indicating the reduction in cell numbers after growing in the soil. After the biopile

and fungal treatments, the percentage increases in cfu/g soil of Bacillus cereus,

Pseudomonas aeruginosa and Methylobacterium sp. were significantly higher than

soil before treatments. The comparative result of population growth of the three

bacteria in biopile treated, fungal treated and garden soil was listed in Table 3.15.

From Table 3.15 the percentage increase in population of Bacillus cereus in biopile

treated and fungal treated soil was: 1368 土 51o/o and 2025 士 145o/o respectively, while

the percentage increase in population of Pseudomonas aeruginosa in biopile treated

and fungal treated soil was 188 士 34o/o and 346 士 42o/o respectively. The percentage

increase of cfu/g soil of Methylobacterium sp. in biopile treated and fungal treated

soil was 78 士 18o/o and 90 士 31o/o, respectively. From one-way A N O V A analysis,

the population growth of Bacillus cereus and Pseudomonas aeruginosa in fungal

treated soil was significantly higher than those in biopile treated soil. However, the

population growth of Bacillus cereus, Pseudomonas aeruginosa and

Methylobacterium sp. in garden soil after three days incubation still ranked the

highest.

120

Table 3.14. The initial population sizes of Bacillus cereus, Pseudomonas

aeruginosa and Methylobacterium sp. Data are presented in mean + SD of 5

replicates.

Population size (Colony forming

unit /g soil)

Bacillus cereus (x 10 ) 223 ± 1 6

Pseudomonas aeruginosa (x 10 ) 125 ± 1 0

Methylobacterium sp. (x lO"^) 281 ± 12

一 " —

' o S) 2500 r • Before treatment

• After treatment § ^ g) 2000 - ~ ~

• 1 - H

g

^ 1500 - * o ^ o ^ a 丫000 - T 0 丁 CO OS 1 500 -

• >—I

o

扫 0 ‘ (D

右 Biopile treatment soil Fungal treatment soil PH


population growth of Bacillus cereus after three days at 30®C. Data are presented in

mean + SD of 5 replicates. Data indicated with * shows significant difference at

5% levels after Student t test.

121

^ 450 厂 • Before treatment

I 400 — 國 Afler treatment W) T

300 -

^ 250 - *

.B 200 - • T

0 r 1 15�- _ r i T

I 100 - 广 —

f 5: - I r o 0 J I ^ Biopile treatment soil Fungal treattrent soil


population growth of Pseudomonas aernginosa after three days at 30°C. Data are

presented in mean 土 SD of 5 replicates. Data indicated with * shows significant


u • Before treatment

^ 140「 •Mertreatont

'I 120 -I 100 - *

180 - pju ri a a 60 -I 40 -I 2: I___, B -20 - 违opjlp trektment soil Bjngallritment soil g § -40 L

C I h


population growth of Methylobacterium sp. after three days at 30°C. Data are

presented in mean ± SD of 5 replicates. Data indicated with * shows significant


122

Table 3.15 A summary of population growth of Bacillus cereus, Pseudomonas

aeruginosa and Methylobacterium sp. in biopile treated, fungal treated and garden

soil. Data are presented in mean ± SD of 5 replicates. Means with the same letter

are statistically similar (One way A N O V A with Tukey test,/? < 0.05).

Percentage increase in colony forming unit (%)

biopile treated soil fungal treated soil Garden soil

Bacillus cereus 1368 ±51 c 2025 士 145 b 2632 ± 194 a

Pseudomonas

188 士 34 C 346 士 42 B 569 土 106 A aeruginosa

Methylobacterium sp. 78 士 18 B, 90 士 31 B, 154 ± 38 A '

123

(a) Biopile treated Fungal treated

MM M i

(c) Biopile treated Fungal treated

Figure 3.31 Photographs of the recovered colonies from the in vitro acute toxicity test using bacteria: (a) Bacillus cereus, (b) Pseudomonas aeruginosa and (c) Methylobacterium sp. inoculated to biopile treated and flingal treated soils.

124

3.4.3 Fungal toxicity test

Fungal growth was measured after 7-day incubation using ergosterol content which

is a fungal sterol. Figures 3.32 to 3.35 show the effect of treatments on population

growth of Trichoderma asperellum, T. harziauum, Fusarium solani and Pleurotus

pulmonarius The result is presented as a relative to their initial ergosterol

concentration in Table 3.16. In comparison there were detected increases in

population sizes of 4 fungi before any treatments (Figure 3.32 to 3.35). The

increase in ergosterol contents of Trichoderma asperellum was 210 士 36o/o (biopile

treatment) and 202 士 55o/o (fungal treatment); Trichoderma harziauum was 153 士

55% (biopile treatment) and 147 士 65o/o (fungal treatment) and Fusarium solani was

58 士 28o/o (biopile treatment) and 66 士 21o/o (fungal treatment) before any treatment.

However, for Trichoderma asperellum and Fusarium solani, they showed

significantly lower population growth after fungal treatment with 125 士 36o/o and 28

士 8o/o respectively. But there was no significant difference of population growth

after biopile treatment. For Trichoderma harziauum, there was significantly lower

population growth after biopile treatment (82 士 17 %) and less population growth in

fungal treatment (91 ± 42 %). When Pleurotus pulmonarius was used in the

toxicity test, the increase in ergosterol contents in soil before biopile and fungal

treatment was 93 ± 12 % and 90 ± 14 % respectively. The population growth was

significantly lower after both biopile treatment (67 士 6 %) and fungal treatment (69 士

10 %). When comparing with garden soil, from Table 3.17，no significant

difference in population growth of Trichoderma asperellum and Fusarium solani

among biopile treated, fungal treated and garden soil. However, there was

significantly more population increase in Trichoderma harziaum in garden soil than

that in biopile treated soil. On the other hand, there was significantly more

population growth in Pleurotus pulmonarius in both biopile treated and fungal

treated soil than that in garden soil.

125

Table 3.16. The initial population sizes of Trichoderma asperellum, Trichoderma

harziauum, Fusarium solani and Pleurotus pulmonarius in terms of ergosterol

content. Data are presented in mean + SD of 5 replicates

Ergosterol concentration (mg/kg)

Trichoderma asperellum 83.24 士 13.42

Trichoderma harziauum 7.03 ±2.12

Fusarium solani 180.64 士 48.85

Pleurotus pulmonarius 42.82 士 2.98

• Before treatment 3UU 厂 „ …

^ • After treatment ^

^ 250 - *T

S 200 - ~ T ~ § T

§ 150 - — ^ T M W 100 -

•s

I 50 -M H

0 1



population growth of Trichoderma asperellum after 7 days at 28°C. Data are

presented in mean ± SD of 5 replicates. Data indicated with * shows significant


126

250「 • Before treatment

_ After treatment O * ^ ^ 2 0 0 - 丁

0

3 150 - ~ ~

玄 r n T CO ^ 100 - J ^ — H " n

0 I I I



population growth of Trichoderma harziauum after 7 days at 28°C. Data are

presented in mean + SD of 5 replicates. Data indicated with * shows significant


100 • Before treatment 7 • After treatment ^ 90 - »ic

1 80 - T T

I 70 -

a 60 - _ _ ~ (D I 50 -0 4 0 - _ _ l <D T •I - -U 1 20 -

1 0 -

0 ‘ i Biopile treatment soil Fungal treatment soil

Figure 3.34 The effects of the biopile treatment and fUngal treatment on the

population growth of Fusarium solani after 7 days at 28°C. Data are presented in

mean + SD of 5 replicates. Data indicated with * shows significant difference at

5% levels after Student t test.

127

120「 • Before treatment

* 圔 After treatment

g 100 - T TH

CD I 80 -二 T O I I

r ] I I 4。-罢 2 0 -

0 I I



population growth of Pleurotus pulmonarius after 7 days at 28°C. Data are

presented in mean + SD of 5 replicates. Data indicated with * shows significant


Table 3.17 A summary of population growth of Trichoderma asperellum,

Trichoderma harziauum, Fusarium solani and Pleurotus pulmonarius in biopile

treated, flingal treated and garden soil in terms of % increase in ergosterol content.

Data are presented in mean + SD of 5 replicates. Means with the same letter are

statistically similar (One way A N O V A with Tukey test,;? < 0.05)

Relative ergosterol content (%)

biopile treated soil fungal treated soil Garden soil

Trichoderma asperellum 153 ± 52 125 ± 36 112 ± 9

Trichoderma harziauum 82 ± 17 b 91 ± 42 ab ~ 1 5 4 ± 53 a ~

Fusarium solani 38 ± 8 28 ± 8 49 士 30

Pleurotus pulmonarius 67士 6 人 69 土 10 人 6 士 2 B

128

3.5 Soil washing

3.5.1 Optimisation of soil washing

3.5.1.1 The effect of hydrochloric acid concentration

The initial total heavy metal concentrations and metal leachability of the biopile

treated soil and fungal treated soil used in soil washing are listed in Table 3.18. The

treated soil used for soil washing contained lead (biopile treated: 2024 士 74 mg/kg;

fungal treated: 2002 士 263 mg/kg), copper (biopile treated: 628 土 113 mg/kg; fungal

treated: 611 士 106 mg/kg) and zinc (biopile treated: 603 ± 28 mg/kg; fungal treated:

684 士 44 mg/kg) above the Dutch B and Dutch Intervention Levels. The TCLP

values of lead (biopile treated: 62 ± 4 mg/1; fungal treated: 62 ± 9 mg/1), copper

(biopile treated: 59 士 5 mg/1; fungal treated: 58 ± 6 mg/1) and zinc (biopile treated:

87 士 7 mg/1; fungal treated: 90 ± 13 mg/1) in treated soil were still above the

Universal Treatment Objectives. Concentration of lead was the highest among the

three metals in soil. From Figures 3.36 (a) and (b)，the removal efficiencies of the

three metals using 0.5 N HCl were significantly greater than those of 0.2 N in both

biopile treated and fungal treated soil. When strength of HCl increased from 0.5 N

to 4 N，the mean removal efficiency reached plateau for Pb and increased slowly for

Cu and Zn. With >0.5 N HCl, more than 50%, 60% and 80% of total Cu, Pb and

Zn were removed. Using 0.5 N HCl resulted in 62 ± 5 % of Cu, 76 士 6o/o Pb and 84

士 7 % Zn removal in biopile treated soil and 55 ± 3 % of Cu, 67 ± 9 % Pb and 83 ± 8

% Zn removal in fungal treated soil. Considering the removal efficiency and

strength of acid, 0.5 N HCl acid was used for further optimization studies.

From Figures 3.37 (a) and (b), the TCLP values of all heavy metals were ranked the

highest for water. The heavy metals leached out were significantly reduced when

acid was used for soil washing in biopile treated soil. The same result was found in

fungal treated soil. Using 0.5 N HCl, 30 士 1 mg/1 lead, 34 士 1 mg/1 copper and 32 士

1 mg/1 zinc leached out in biopile and 28 士 3 mg/1 lead, 36 ± 2 mg/1 copper and 32 士

2 mg/1 zinc leached out in fungal treated soil. The amounts of heavy metals leached

out still exceeded the USEPA universal treatment objectives.

129

Figures 3.38 (a) and (b) showed the total heavy metal concentrations in biopile

treated soil and fungal treated soil after using different concentrations of HCl for

washing. The residual total heavy metal concentration remained high in 0.2 N HCl.

The residual total heavy metal concentration significantly reduced when 0.5 N HCl

was used. Total 521 ± 123 mg/kg lead, 323 士 24 mg/kg copper and 213 ± 42 mg/kg

zinc were found in biopile treated soil; and 508 士 114 mg/kg lead, 314 士 26 mg/kg

copper and 167 士 39 mg/kg zinc were found in fungal treated soil. When the

concentration of HCl increased from 0.5 N to 4 N, no significant change in residual

total heavy metal in both biopile treated soil and fUngal treated soil was observed.

The amount of heavy metals remained in soil still exceeded the Dutch Target level.

Table 3.18 The initial total heavy metal concentrations and metal leachability in

biopile treated soil and fungal treated soil used for the study of soil washing. Data

are presented in mean 土 SD of 5 replicates.

Total heavy metal (mg/kg) TCLP value (mg/1)

biopile treated fungal treated biopile treated fUngal treated

^ 2024 ± 74 2002 士 263 62 ± 4 6 2 ^

^ 628 ± 113 611 ± 106 59 ± 5 58^6

^ 603 ± 28 684 ± 44 87 ± 7 90± 13 ：

130

(a)

^ l! - o - Copper

I 30 ^ Lead

^ c /] - ^ Z i n c

0 1 2 3 4 5

Concentration ofHCl (N)

m

100「 A, A， B, AB, ^ A

90 - T B,

^ 80 - 丄 a

^ 7 0 - / 丁 “ 一 - A — — 〜〜 —

I 60 —

I 50 - / 厂 B B B

！I 40 -

I 30 C'/l + Copper

10 —/於b "^Zinc

o^m——^ ‘ ‘ ^ •

0 1 2 3 4 5 Concentration ofHCl (N)

Figure 3.36 The effect of hydrochloric acid concentrations on the removal of heavy

metals in the (a) biopile treated soil and (b) fungal treated soil. Removal efficiency

(%) was used to indicate the effectiveness. Data are presented in mean 土 SD of 5

replicates. Means with the same letter are statistically similar (One way A N O V A

with Tukey test, p < 0.05). Experimental conditions: 5 g soil shaken with 25 ml

HCl at 150 rpm at 25°C for 6 hrs.

I

131

(a)

動 N • 0 . 2 N D O . S N B I N • 2 N S S N • 4 N 励 r s: 90 - i

运 80 _ a A I ！ 70 A I I 60 — _ 1 I

g 50 - _ • B C D I

G 40 - L B V _ B B B C D _ B , B , B C，「，

i 30 - l i T ^ l - n r ^ ' 広 20 - H f e I:::: B i I::::

Q 圓 I___M 、、Hv3 I 隱 . 1 I 險 I I I • I I 丨

Pb Cu Zn

Heavy metal

(M

JQQ 四ON •0.2N DO.SN 圓 IN B I N S 3 N E M N

9 0 - j A '

蓉 8 0 、 I

旦 70 - } A _

I r i i I ： I r f e ! _ ，

: 瞧 _ _ iiii 0 1 1 旧：：：丨 I _ : : : : : 丨 I 1 x 1 _ : 丨 ,

Pb Cu Zn

Heavy metal

Figure 3.37 The effect of hydrochloric acid concentrations on the heavy metal

leachability of (a) biopile treated soil and (b) fungal treated soil. Toxicity

characteristic leaching procedure (TCLP) was used to determine the leachability.


statistically similar (One way A N O V A with Tukey test, ；7<0.05). Experimental

conditions: 1 g soil added with 20 ml of C H 3 C O O H for 18 h at 30 rpm.

132

(a)

4 5 0 0 厂 “

- D - Copper —O-Lead -t s—Zinc 4000 -

^ 3 5 0 0 - a T

直 3000 - A

I 2500b

I 2000 _ 1 5 。观。。。。。

§ eYA'W C C o C C

I I O O O B ^ ^ C, C C, C,


M , 4500 「a

T - Q - Copper -O-Lead -T^r-Zinc

f 3 5 0 0 - I

B 3 0 0 0

I 2500 J i I • f 义\ 1 1500 B / \ \ c c C C U 9 A'\ C C ^ C c 1

0 B ,


Figure 3.38 The effect of hydrochloric acid concentration on the residual total

heavy metal concentrations in (a) biopile treated soil and (b) fungal treated soil.


statistically similar (One way A N O V A with Tukey test, p < 0.05). Experimental

conditions: 0.2 g soil extracted with 20 ml 69% HNO3 in microwave digestion

system and analyzed by AAS

133

3.5.1.2 The effect of incubation time

From Figures 3.39 (a) and (b), the removal efficiency of 0.5 N HCl for 6 hours was

significantly higher than that for 4 hours. For washing time >6 hours, more than

50%, 60% and 80% of total Cu, Pb and Zn were removed. At 6 hours washing time,

62 士 5o/o of Cu, 76 士 6 % Pb and 84 ± 7 % Zn removal in biopile treated soil and 55 士

3 % of Cu, 67 士 9 % Pb and 83 土 8 % Zn removal in fungal treated soil were

obtained. Figure 3.42 shows the photographs of the biopile treated and fungal

treated soils after washing with 0.5N HCl for 6 hours. When incubation time

increased from 6 hours to 48 hours, there was no significant increase in Cu and Zn

removal in both biopile and fungal treated soil, and for Pb, the removal even

decreased.

From Figures 3.40 (a) and (b), less Pb leached out when biopile treated (17 士 2 mg/1)

and fungal treated soil (15 ± 3 mg/1) were washed with HCl for 48 hours. But Cu

leached out most when the incubation time was 6 hours for both biopile treated (34 士

1 mg/1) and fungal treated soil (36 士 2 mg/1). Zn leached out most when the

incubation time was 24 hours for both biopile treated (36 ± 2 mg/1) and fungal treated

soil (35 士 2 mg/1). There was no such a relation that when increasing the incubation

time, Cu and Zn leached out more. The same situation was found in fungal treated

soil. The amounts of heavy metals leached out still exceeded the USEPA universal

treatment objectives.

Figures 3.41 (a) and (b) show the effect of incubation time on residual heavy metal

concentrations in biopile treated and fungal treated soil respectively. There were

significantly drop of residual Pb and Zn at 6 hrs for both biopile treated (Pb: 332 士

30 mg/kg; Zn: 66 士 22 mg/kg) and fungal treated soil (Pb: 393 士 30 mg/kg; Zn: 100

士 51 mg/kg). The residual Cu after 6 hours extraction in biopile treated and fungal

treated soil were 413 士 40 mg/kg and 454 士 18 mg/kg respectively. When the

incubation time increased from 6 hrs to 48 hrs, there was no great change in soil

residual Cu and Zn. The amount of heavy metals remained in soil still exceeded the

Dutch A or B levels.

134

(a)

100 「 A’ A’ A， - ^ C u Pb

一 II ： JA’

I :: - _ J m 50 - B'l/ A A ^ 1 40 -B, j

I 3 � -f f d #

0 & 1 1 L _ 1 I

0 10 20 30 40 50 Time (hour)

m

「 A, - D - C u - O - P b 90 - T

A, A, A’

w 70 — I a b

1:: dgf 0 ‘ 1 1 I I

0 10 20 30 40 50 Time (hour)

Figure 3.39 The effect of incubation time on removal of heavy metals of (a) biopile

treated soil and (b) flingal treated soil. Removal efficiency (%) was used to indicate

the effectiveness. Data are presented in mean ± SD of 5 replicates. Means with the

same letter are statistically similar (One way A N O V A with Tukey test, p<0.05).

Experimental conditions: 5 g soil shaken with 25 ml 0.5N HCl at 150 rpm at 25°C

with different shaking times.

135

w

國 2h • 4h H12h •24h •48h

40「 —k 35 - a 丄ABCab B， | | |

蓉 30 T 1 " l ^ c f i l i c c’鬥 c， | ^’

S 25 - H： ^ lb X：：-：

g 1 5 : |i wk _ |i p ； _ _ i _ i 0 隱::::1 I _ _:] I _ _ 隱 I _

Pb Cu Zn Heavy metal

m 45「図 2h •4h •6h H 12h •24h 048h

40 - A A B A, ^ A B T A B T A B T B，

I 35 - ; a T 里 i 丁 S 30 - ab T a "^"•X B, B,r^B，_ 3 I—*—I 丁 ^\、、、、

驢 15 ft P : P ^ - |i _ |i :i |i i 5 _ _：丨：fc |i E E

Pb Cu Zn

Heavy metal

Figure 3.40 The effect of incubation time on the heavy metal leachability of (a)

biopile treated soil and (b) fungal treated soil. Toxicity characteristic leaching

procedure (TCLP) was used to determine the leachability. Data are presented in

mean 土 SD of 5 replicates. Means with the same letter are statistically similar

(One way A N O V A with Tukey test, /><0.05). Experimental conditions: 1 g soil

added with 20 ml of C H 3 C O O H for 18 h at 30 rpm.

136

(a)

1 6 0 0 厂

^ a - Q - C u - A - Z n f 1 4 0 0 - ^ a

f 1200 - M

. 卜 _ I ^ i _ -^ 600 - A A c / c ^ ^

\ 4 。。 ^ -

I 200 「 C ^ B ^ 飞 B C

0 1 1 1 1

0 10 20 30 40 50

Time (hour)

m 1600 r ~ 0 ~ C u "™0-Pb -T^r-Zn

旦 a a % 1 4 0 0 - T

f 1200 - \

I 誦 _ \ c 一 „ ^

隱800 _ 厂 I 600 - A b W /

I 400 - D D ( 鄉 a ’ A , \ a a - •

I • — c W ? - - B ^ 0 ‘ 1 1 1 1

0 10 20 30 40 50

Time (hour)

Figure 3.41 The effect of incubation time on the residual total heavy metal

concentrations in (a) biopile treated soil and (b) fungal treated soil. Data are

presented in mean 土 SD of 5 replicates. Means with the same letter are statistically

similar (One way A N O V A with Tukey test, /><0.05). Experimental conditions: 0.2 g

soil extracted with 20 ml 69% HNO3 in microwave digestion system and analyzed by

AAS.

137

(a) , (b) ‘ .:...厂 ’...i.:.: ；. ： r.. .... “

, 、‘.. ，•.，.. • ••“ ‘ . I：；： . •• ‘‘ "w： •；••

‘ • , ,< « , . ‘ , I ‘ f t , � , I f ^ , > . ' '

Figure 3.42 Photographs of the (a) biopile treated and (b) fungal treated soils after soil washing.

138

3.6 Mycoextraction

The initial total heavy metal concentration and metal leachability in biopile and

fUngal treated soils which were used for mycoextraction are listed in Table 3.19.

The soil contaminated with lead (biopile treated: 1514 士 452 mg/kg; fungal treated:

1213 ± 118 mg/kg), copper (biopile treated: 704 士 142 mg/kg; fungal treated: 621 士

38 mg/kg) and zinc (biopile treated: 696 土 58 mg/kg; fungal treated: 758 ± 54 mg/kg)

had their concentrations above the Dutch B and Dutch Intervention Levels. The

TCLP values of lead (biopile treated: 46 士 11 mg/1; fungal treated: 71 ± 22 mg/1),

copper (biopile treated: 95 士 17 mg/1; fungal treated: 90 士 8 mg/1) and zinc (biopile

treated: 86 ± 3 mg/1; fungal treated: 87 士 3 mg/1) in soil were still above the

Universal Treatment Objectives.

Scatter plots were used in Figures 3.44 (a) and (b) to show the exact metal content in

fruiting bodies of each replicate. Figure 3.47 shows the photographs of the effect of

soil to compost ratio (w/w) on removal of heavy metals from the biopile treated soil

and fungal treated soil. From Figures 3.43 (a) and (b), the concentrations of zinc in

fruiting bodies were higher than copper at all ratios. Also there was no lead

accumulated in fruiting bodies at all ratios. At 1:0.5 ratio, it accumulated the

highest zinc (biopile treated: 82 ± 55 mg/kg; fungal treated: 107 ± 74 mg/kg) and

copper concentrations (biopile treated: 68 士 48 mg/kg; fungal treated: 70 士 48 mg/kg)

among all ratios. When the compost amount increased from 50 g to 500 g, there

was no increase in Cu and Zn concentrations in fruiting bodies. These phenomena

were also found in fungal treated soil (Figure 3.43 (b)).

From Figures 3.44 (a) and (b)，the fruiting bodies in 1:5 ratio setup extracted the

highest amounts of zinc (270 士 32 /xg) and copper (63 士 6 fig) among all ratios. The

same phenomena were found in fungal treated soil (Cu: 63 ± 7 /xg; Zn: 318 士 46jitg)

(Figure 3.44 (b)). When using linear regression analysis, the R^ of zinc are 0.9394

and 0.9437 for biopile treated and fungal treated soil in a plot of heavy metal amount

versus compost amount respectively. But R^ of copper are only 0.7645 and 0.6943

for biopile treated and fungal treated soil respectively. This means that when

increasing the compost amount, there were increased metal amounts in fruiting

139

bodies.

From Figure 3.45 (a), Cu (53 士 13 mg/1) leached out from soil in 1:0.5 setup was the

lowest for biopile treated soil. But Cu leached out was the highest in 1:1 (81 士 9

mg/1) and 1:2 (92士 21 mg/1) setups. There was no significant increase in Pb and Zn

leachabilities when the amount of compost increased in biopile treated soil. For

fungal treated soil (Figure 3.45 (b))，Pb leached out the least in 1:1 setup (21 土 5 mg/1)

and most in 1:0.01 (43 士 4 mg/1) and 1:0.5 (56 士 17 mg/1) setups, while Cu leached

out least in 1:0.01 (53 士 2 mg/1), 1:0.25 (51 士 13 mg/1), 1:0.5 (56 士 17 mg/1) and 1:1

(55 士 4 mg/1) setups and Zn leached out least in 1:0.01 (72 士 3 mg/1) and 1:1 (72 士 4

mg/1) setups. Both Cu and Zn leached out the most in 1:2 (Cu: 101 ± 4 mg/1; Zn: 79

士 3 mg/1) and 1:5 (Cu: 87 ± 3 mg/1; Zn: 81 ± 2 mg/1) setups. When the amount of

compost increased, more Cu and Zn leached out in fungal treated soil.

From Figures 3.46 (a) and (b), increasing the compost amount to soil, there was no

significant reduction in residual Cu and Zn concentration in biopile treated and

fungal treated soil. The lead, copper and zinc concentrations in all setup of fungal

treated soil were of no significant difference. In 1:0.5 setup, (biopile treated: 1752

士 323 mg/kg; fungal treated: 1026 士 40 mg/kg) lead, (biopile treated: 604 士 67

mg/kg; fungal treated: 608 土 127 mg/kg) copper and (biopile treated: 716 士 28 mg/kg;

fungal treated: 636 土 80 mg/kg) zinc were still found in soil. But there was a sharp

increase in soil lead when 100 g and 25 g compost added to biopile treated soil. For

fiingal treated soil, when the compost amount was increased, the residual total metal

content was not significantly reduced.

Table 3.19 The initial total heavy metal concentration and metal leachability in

biopile treated soil and fungal treated soil which were used for removal of metals by

mycoextraction. Data are presented in mean + SD of 5 replicates.


biopile treated flingal treated biopile treated fungal treated

^ 1514 ±452 1213 士 119 46 士 11 71 ±22

Cu 704 ± 142 621 士 38 95 士 17 90 ±8

^ 696 ± 58 758 ± 54 86 ±3 87 ±3

140

(a)

140 厂 “ T ^ • Cu O Pb A Zn

鲁 120 - ^

一 W A g 100 - a A A .1 • A A

I 8 0 - 公

8 • A ^ 8 6 0 - 公

Id I 40 - & • b • S = cd 20 - n (D 么 u _

ffi e 曰 0 H-Q-n——O O 1 1 O 1

0 100 200 300 400 500 600 Compost Amount (g)

®

180「

萝 160 - •Cu O^Pb AZn ^ ^ 140 - A

I 120 - A g A

I - - a . 会 § 80 - A " S A

^ 60 -a _ A ^ 40 - 口口

> • “ 白 a Lj Lj -

ffi 20 - • 口日

0 H-Q-n O O I 1 O 1

0 100 200 300 400 500 600

Compost Amount (g)

Figure 3.43 The effect of compost amounts on metal concentrations in fruiting

bodies of Pleurotus pulmonarius grown in (a) biopile treated soil and (b) fungal

treated soil. Experimental conditions: compost was added to 100 g of soil and

incubated at 28°C for 5 weeks (4 weeks in darkness and 1 week in light-illuminated

environmental chamber in mushroom cultivation complex, Department of Biology,

The Chinese University of Hong Kong).

141

(a)

0 . 3 5 「 ~ O Pb

• Cu - ^ 2n y = 0.0006x +0.0049 A

I 0.25 - — Z n =

I _ C u ^ ^ A

I。.2 _ . Z I。.15 -y QJ y = 0.0001x +0.0087

£ 6 R2 = 0.7645

0 iSV-5—~o o I 1 o 1

0 100 200 300 400 500 600

Compost amount (g)

(b )

0.4「 O Pb

0.35 - ° Cu y = 0.0006x +0.0088 .

I 0.3 — 二： =

I 0.25 - — & ^ ^ 么

I 0.2 _ ^

S 0.15 -

t _ . y 二 O.OOOlx + 0.0118

X „ R2 = 0.6943 ^ 0.05

0 B - g - B O O 1 1 o 1

0 100 200 300 400 500 600

Compost Amount (g)

Figure 3.44 The effect of compost amount on metal amount in fruiting bodies of

Pleurotus pulmonarius grown in (a) biopile treated soil and (b) fungal treated soil.

Experimental conditions: compost was added to 100 g of soil and incubated at 28°C

for 5 weeks (4 weeks in darkness and 1 week in light-illuminated environmental

chamber in mushroom cultivation complex, Department of Biology, The Chinese

University of Hong Kong). Linear regression analysis for metal amounts in fruiting

bodies and amounts of compost added.

142

w

曰 1:0.01 • 1 : 0 . 2 5 • 1 : 0 . 5 m i A m i : 2 01:5 I 120 r

A

^ 100 - A > •• A’ A, f 80 - A B C J M ^ A ， A ， .

> ⑶ 赫 C t 驚：、隱：：

Oh 60 - a • =

^ 40 - 工 a a T :: , _ ,

20 酔 I；：；：：：彰 _ fc ••：•••： Wp; _:::••:

0 圓•....丨~• I I I I _ _ _ • I I I I I • I Pb Cu Zn

Heavy metal

m

120 「四 1:0.01 • 1:0.25 • 1:0.5 B m B l ^ 0 1 : 0 5 A

二爾 — . A ^ M ^ AB， A, 旦 on ^ B ^ A B’ B, A'

^ R T d ^ _ ^ ^ V s N V s , ^ H ^ W W S

> -I- R ^B>、、\、\ • • - • ^B.xwsv JT 60 - B T ^、\、、、 ^ ^ : : B、、、、、 H a _ E^S^- [TB：、、、、、 M-：：- ：、、、、、

； 4 0 _ p . p

2 。 - _ 丨 | i P 11 ^^.. •. • 誓 ^ ^ . , , ^ M n n v v v ^ ^ • • • • • • � ’

0 t M - . - l _ _>、、、、、i I ^ ^ • • I ^M\、、、、i I ^ ^ BB I

Pb Cu Zn

Heavy metal

Figure 3.45. The effect of mycoextraction on the heavy metal leachability of (a)

biopile treated soil and (b) fungal treated soil. Toxicity characteristic leaching

procedure (TCLP) was used to determine the leachability. Data are presented in

mean 土 SD of 5 replicates. Means with the same letter are statistically similar (One

way A N O V A with Tukey test, p<0.05). Experimental conditions: 1 g soil added

with 20 ml of CH3COOH for 18 h at 30 rpm

143

(a)

一 2000 L T T 卜 Pb ^ C u 士 Zn|

I M abc f 畏 1500 - \ 0 / \ ——O

1 1000 • I AB，

^ AB B

X 0 1 1 1 1 I

0 100 200 300 400 500 600

Compost Amount (g)

(M

2000 - a - O - P b - O - C u - A ~ Z n GO T

^ T I 1500 — L a a

豸 a a a / >

I —— 1 I A

ffi 0 1 1 1 1 1 I

0 100 200 300 400 500 600

Compost Amount (g)

Figure 3.46 The effect of mycoextraction on the residual total heavy metal

concentrations in (a) biopile treated soil and (b) fungal treated soil. Data are

presented in mean 土 SD of 5 replicates. Means with the same letter are

statistically similar (One way A N O V A with Tukey test, /?<0.05). Experimental

conditions: 0.2 g soil extrtacted with 20 ml 6 9 % HNO3 in microwave digestion

system and analyzed by AAS.

144

(a) 1:0.01 1:0.25 1:0.5

1:1 1:2 1:5

(b) 1 : 0 . 0 1 1 : 0 . 2 5 一 1:0.5

j B ^ M l f B H ^ H B H

1:1 1:2 1:5

Figure 3.47 Photographs of the effect of soil to compost ratio (w/w) on removal of heavy metals from the (a) biopile treated soil and (b) fungal treated soil.

145

3.7 Phytoextraction and integrated bioextraction

The initial total heavy metal concentration and metal leachability in biopile treated

soil and fungal treated soil which were used for phytoextraction and integrated

bioextraction are listed in Table 3.20. The soil contaminated with lead (biopile

treated: 1514 ± 452 mg/kg; fungal treated: 1213 士 118 mg/kg), copper (biopile

treated: 704 士 142 mg/kg; fungal treated: 621 ± 38 mg/kg) and zinc (biopile treated:

696 ± 58 mg/kg; fungal treated: 758 ± 54 mg/kg) had their concentrations above the

Dutch B and Dutch Intervention Levels. The TCLP values of lead (biopile treated:

46 士 11 mg/1; fungal treated: 71 ± 22 mg/1), copper (biopile treated: 95 士 17 mg/1;

fungal treated: 90 ± 8 mg/1) and zinc (biopile treated: 86 士 3 mg/1; fungal treated: 87

士 3 mg/1) were still highly above the Universal Treatment Objectives. As no lead

was accumulated in all the plant species, only copper and zinc were plotted (Figures

3.48 (a) and (b)). Figure 3.51 shows the photographs of plant species used in

extraction of heavy metals from biopile and fungal treated soil with and without

integration of mycoextraction.

When comparing among the three plant species, wheat accumulated more zinc than

others in biopile treated soil (wheat: 24 士 6 fig; ryegrass: 7 ± 1 jitg and Chinese

cabbage: 13 ± 3 /ig) and fungal treated soil (wheat: 21 ± 6 ptg; ryegrass: 9 士 2 />tg and

Chinese cabbage: 12 ± 2 /xg); but Chinese cabbage and wheat accumulated more

copper than ryegrass did in biopile treated soil (wheat: 3 ± 1 /xg; ryegrass: 0 士 0 /xg

and Chinese cabbage: 4 ± 1 /xg) and fungal treated (wheat: 2 ± 0 /xg; ryegrass: 1 士 1

Mg and Chinese cabbage: 4 ± 1 /xg) (Figures 3.48 (a) and (b)). After 4-week

planting, the aerial biomass of wheat was the highest in both biopile treated (wheat:

0.8640 士 0.0909 g; ryegrass: 0.1236 士 0.0209 g and Chinese cabbage: 0.1880 士

0.0283 g) and fungal treated soil (wheat: 0.8650 士 0.0486 g; ryegrass: 0.1324 士

0.0249 g and Chinese cabbage: 0,1925 士 0.0099 g) (Table 3.21). Therefore, wheat

was selected in integrated bioextraction.

For integrated bioextraction, when 50 g of compost were added to soil, copper

accumulated in wheat was significantly increased in both biopile treated soil (7 士 3

fig) and fungal treated (6 士 2 fig), but no change was observed in zinc. When

146

comparing with mycoextraction using Pleurotus pulmonarius (1:0.5)，1: 0.5 setup

removed the highest Cu and Zn amounts in its fruiting bodies in both biopile treated

soil (Cu: 19 士 13 jtig; Zn: 24 士 17 / g) and fungal treated soil (Cu: 25 士 17 /ig; Zn: 38

士 25 fig). Therefore, mycoextraction removed more Cu and Zn than

phytoextraction.

From Figures 3.49 (a) and (b)，less Pb was leached out from soil phytoremediated

with ryegrass for biopile treated soil (wheat: 52 士 5 mg/1; ryegrass: 28 ± 3 mg/1 and

Chinese cabbage: 49 士 12 mg/1). Also significantly less Pb was leached out in

integrated bioextraction for both biopile treated (21 士 4 mg/1) and fungal treated soil

(26 士 7 mg/1). For copper, its leachability was more or less the same in

phytoextraction and integrated bioextraction in both biopile treated (wheat: 85 士 12

mg/1; ryegrass: 69 ± 6 mg/1; Chinese cabbage: 76 士 21 mg/1 and wheat+50g compost:

88 土 19 mg/1) and fungal treated soil (wheat: 87 士 8 mg/1; ryegrass: 78 土 9 mg/1;

Chinese cabbage: 73 士 11 mg/1 and wheat+50g compost: 83 士 3 mg/1). The same

situation was also found in zinc; its leachability was similar in phytoextraction and

integrated bioextraction in both biopile treated (wheat: 85 士 3 mg/1; ryegrass: 79 土 7

mg/1; Chinese cabbage: 74 ± 5 mg/1 and wheat+50g compost: 83 ± 3 mg/1) and

fiingal treated soil (wheat: 91 士 3 mg/1; ryegrass: 83 ± 5 mg/1; Chinese cabbage: 75 士

5 mg/1 and wheat+50g compost: 83 士 4 mg/1).

From Figures 3.50 (a) and (b)，the residual total lead in biopile treated (wheat: 1208

士 96 mg/kg; ryegrass: 1162 士 157 mg/kg; Chinese cabbage: 1478 士 556 mg/kg and

wheat+50g compost: 1379 士 232 mg/kg) and fungal treated soil (wheat: 1185 ± 43

mg/kg; ryegrass: 1163 士 35 mg/kg; Chinese cabbage: 1211 士 95 mg/kg and

wheat+SOg compost: 1124 士 182 mg/kg) after phytoextraction and integrated

bioextraction were not significantly different. The residual total copper in biopile

treated were: wheat, 653 士 21 mg/kg; ryegrass, 754 士 85 mg/kg; Chinese cabbage,

722 士 100 mg/kg and wheat+50g compost, 758 士 205 mg/kg. The residual total

copper contents in fungal treated soil were: wheat, 623 土 33 mg/kg; ryegrass, 633 士

61 mg/kg; Chinese cabbage, 610 士 66 mg/kg and wheat+50g compost, 582 土 81

mg/kg. The residual total zinc in biopile treated were: wheat, 707 士 92 mg/kg;

ryegrass, 701 士 52 mg/kg; Chinese cabbage, 690 士 66 mg/kg and wheat+50g

compost, 739 士 44 mg/kg. The residual total zinc in fungal treated soil were: wheat,

147

699 士 33 mg/kg; ryegrass, 685 士 62 mg/kg; Chinese cabbage, 690 士 71 mg/kg and

wheat+50g compost, 714 士 106 mg/kg. The residual total copper and zinc after

phytoextraction and integrated bioextraction were not significantly different in

biopile treated and fungal treated soil.



phytoextraction and integrated bioextraction. Data are presented in mean ± SD of 5

replicates.


biopile treated fungal treated biopile treated fungal treated

^ 1514 ±452 1213 士 119 46 士 11 71 ±22

Cu 704 士 142 621 ± 38 95 士 17 90 ± 8

^ 696 ± 58 758 ± 54 86 ± 3 87 ± 3

Table 3.21 The biomass gain (aerial part dry weight) of three testing species after

4-week planting. Data are presented in mean + SD of 5 replicates.

biopile treated (g) fungal treated (g)

Wheat (Triticum aestivum) 0.8640 士 0.0909 0.8650 ± 0.0486^

Ryegrass (Lolium perenne) 0.1236 ± 0.0209 0.1324 ± 0.0249^

Chinese cabbage {Brassica

0.1880 士 0.0283 0.1925 士 0.0099 chinensis)

148

(a)

70 r 因 Ryegrass

• Chinese Cabbage 60 - • Wheat

^ 圓 Wheat + 50 g compost

e 50 - •1:0.5 I A，

震 40 - A T c A 3 A’ I 30 - T T A，

1: ‘ B I 塵 — Cu H 1 Zn

Heavy metal

M

7 0 「図 Ryegrass • Chinese Cabbage A,

60 - • Wheat 丁 ^ _ Wheat + 50g compost

里 50 -LiM^ I A I 40 - T

I 30 - ab, A b H

I 20 - _ A B ， r ^ " ^ H

B _ J^ •

川 I _ 1 Cu Zn

Heavy metal

Figure 3.48 Effects of phytoextraction and integrated bioextraction of wheat

Triticum aestivum and compost of Pleurotus pulmonarius on removal of metals from

(a) biopile treated soil and (b) fungal treated soil. Data are presented in mean 土 SD

of 5 replicates. Means with the same letter are statistically similar (One way

A N O V A with Tukey test, p<0.05). Experimental conditions: 25 germinated seeds

were grown in 200 g soil in greenhouse for 4 weeks. 149

(a)

1 2 0「図 Ryegrass

• Chinese Cabbage A

100 - S^JJeat A B A B T 二 • Wheat (compost added) T T A， > • 1:0.5 J L AB' , AB'

80 X ^ l f n

丨 : i i l i Pb Cu Zn

Heavy metal (B) — ‘

图 Ryegrass 120 「•Chinese Cabbage

• Wheat • Wheat (compost added)

- 1 0 0 - • 1.0 5 A A，

I i x d A ^ B ， n ^ ’ B,

%m Pb Cu Zn

Heavy metal


Triticum aestivum and compost of Pleurotus pulmonarius on heavy metal

leachability of (a) biopile treated soil and (b) fUngal treated soil. Toxicity

characteristic leaching procedure (TCLP) was used to determine the leachability.

Data are presented in mean + SD of 5 replicates. Means with the same letter are

statistically similar (One way A N O V A with Tukey test, p<0.05). Experimental

conditions: 1 g soil added with 20 ml of CH3COOH for 18 h at 30 rpm.

150

(a)

2000 L T t ^Ryegrass

• Chinese Cabbage ^ • Wheat 當 • • Wheat + 50 g compost 旦 1500 - I T • • 50 g compost g a

I 1 � � � - l i i 1 1 1 A A ^ , A' A ' ,

1 ：. I I _ _ Pb Cu Zn

Heavy metal

签 • Chinese Cabbage 容 • Wheat 旦圓 Wheat + 50 g compost ^ 1500 - • 50 g compost •2 a a

Pb Cu Zn

Heavy metal


Triticum aestivum and compost of Pleurotus pulmonarius on residual heavy metal

concentration of (a) biopile treated soil and (b) fungal treated soil. Data are

presented in mean + SD of 5 replicates. Means with the same letter are statistically

similar (One way A N O V A with Tukey test, j!7<0.05). Experimental conditions: 0.2 g

soil extrtacted with 20 ml 69% HNO3 in microwave digestion system and analyzed

by AAS.

151

�

m (b)

Figure 3.51 Photographs of the effects of plant species used in extraction of heavy metals from biopiled treated soil and fiingal treated soil with and without integration of mycoextraction. Systems: (a) Wheat Triticum aestivum; (b) Ryegrass Lolium

perenne L.; (c) Chinese cabbage Brassica chinensis and (d) integrated biological extraction setup after four weeks.

152

3.8 Cementation

The initial total heavy metals concentrations and metals leachabilities in biopile

treated soil and fungal treated soil which were used for cementation are listed in

Table 3.22. The soil was contaminated with lead (biopile treated: 1017 士 38 mg/kg;

flingal treated: 1001 士 65 mg/kg), copper (biopile treated: 655 士 60 mg/kg; fungal

treated: 719 士 109 mg/kg) and zinc (biopile treated: 785 士 35 mg/kg; fungal treated:

800 士 40 mg/kg). Their concentrations were above the Dutch B and Dutch

Intervention Levels. The TCLP values of lead (biopile treated: 23 士 1 mg/1; fungal

treated: 22 士 2 mg/1), copper (biopile treated: 32 ± 4 mg/1; fungal treated: 35 士 3 mg/1)

and zinc (biopile treated: 43 士 2 mg/1; flingal treated: 42 士 2 mg/1) in soil were still

above the Universal Treatment Objectives.

From Figures 3.52 (a) and (b)，when the % of cement added increased, the

leachabilities of lead, copper and zinc decreased. The TCLP-Pb contents were still

above Universal Treatment Objectives at 8 % cement (biopile treated: 5 士 2 mg/1;

fungal treated: 4 ± 4 mg/1). At 16% cement, the TCLP-Pb values were below the

Universal Treatment Objectives (biopile treated: 0 ± 0 mg/1; fungal treated: 0 土 0

mg/1). For zinc, at 30% cement, the TCLP values were zero in both biopile treated

and fungal treated and thus were below the Universal Treatment Objectives. For

copper, the values were below the Universal Treatment Objectives at 40% cement

(biopile treated: 6 士 1 mg/1; fungal treated: 6 士 1 mg/1). When percentage of cement

increased, all metal leachabilities reduced. The same phenomenon was found in

fungal treated soil.

The effect of cementation on residual total heavy metal concentration of biopile

treated and fungal treated soil is plotted in Figures 3.53 (a) and (b). At 16% cement,

there was still Pb remained (biopile treated: 758 士 10 mg/kg; fungal treated: 756 士 12

mg/kg). At 30% cement, there were 459 士 15 mg/kg Zn in biopile treated soil and

474 士 12 mg/kg Zn fungal treated soil. At 40% cement there were 312 士 21 mg/kg

Cu in biopile treated and 301 士 14 mg/kg Cu in fungal treated soil. It is shown that

the total residual lead, copper and zinc concentrations reduced gradually when the

percentage of cement added increased. Figure 3.54 shows the photographs of 40%

153

cementation of biopile and fungal treated soil.



cementation. Data are presented in mean + SD of 5 replicates.



^ 1017±38 1001 ±65 23 ± 1 2 2 ^

^ 655 士 60 719 ± 109 32 ± 4 3 5 ^

^ 785 ± 3 5 800士40 43 ±2 42±2

154

(a)

A’

50 - A， -O-Lead

"A, -O— Copper

二 40 t - T ^ Z i n c

t " T A

0 1 ^ 1 ^ 1

0% 10% 20% 30% 40% 50%

Percentage of cement

m

—0—Lead

50 - - O - Copper

•A, B， I Z i n c e 4 0 K T

c \ D c , 、 d ，

0 1 ^ ^ 1

0% 10% 20% 30% 40% 50%


Figure 3.52 Effects of cementation on heavy metal leachability of (a) biopile

treated soil and (b) fungal treated soil. Toxicity characteristic leaching procedure

(TCLP) was used to determine the leachability. Data are presented in mean 土 SD of


with Tukey test,/><0.05). Experimental conditions: 1 g soil added with 20 ml of

C H 3 C O O H for 18 h at 30 rpm.

155

(a)

a a X T —0— Lead 1000

如 „ A 々 C o p p e r

€ A R,\b . - ^ Z i n c I _ t ^ J 1 60。

1 . - ^ ^ ^ ^

2 B S 2 0 0 -

0 1 -J 1 1 I

0% 10% 20% 30% 40% 50% Percentage of cement

(b )

十 a a -O-Lead

誦 ^ C o p p e r

^ .A' \ b L -i5«-Zinc

I _ ^ ^ ^

I 6。。

I - -3 B C ^ ^ s 200 - ^

0 1 1 1 1 I

0% 10% 20% 30% 40% 50%


Figure 3.53 Effects of cementation on residual heavy metal concentration of (a)

biopile treated soil and (b) fungal treated soil. Data are presented in mean 土 SD of


with Tukey test,/><0.05). Experimental conditions: 0.2 g soil extracted with 20 ml

69% HNO3 in microwave digestion system and analyzed by AAS.

156

⑷ （b)

Figure 3.54 Photographs of the 40% cementation of (a) biopile treated and (b) fungal treated soil.

157


The initial total heavy metals concentrations and metal leachabilities in biopile

treated soil and fungal treated soil which were used for glass encapsulation are listed

in Table 3.23. The soil contaminated lead (biopile treated: 1514 士 452 mg/kg;

fimgal treated: 1213 士 118 mg/kg), copper (biopile treated: 704 士 142 mg/kg; fungal

treated: 621 士 38 mg/kg) and zinc (biopile treated: 696 士 58 mg/kg; fungal treated:

758 士 54 mg/kg) concentrations were above the Dutch B and Dutch Intervention

Levels. The TCLP values of lead (biopile treated: 46 ± 11 mg/1; fungal treated: 71

士 22 mg/1), copper (biopile treated: 95 ± 17 mg/1; fungal treated: 90 士 8 mg/1) and

zinc (biopile treated: 86 士 3 mg/1; fungal treated: 87 士 3 mg/1) in treated soil were

still highly above the Universal Treatment Objectives.

From Table 3.24，the TCLP values of glass ampoules were below the detection limits

(0.005 mg/1 Pb; 0.001 mg/1 Cu and 0.0001 mg/1 Zn) of the A A S and also below the

Universal Treatment Objectives. No heavy metal was leached out from the glass

ampoules. Figure 3.55 shows the photographs of glass encapsulation of the biopile

treated and fungal treated soils.



glass encapsulation. Data are presented in mean 土 SD of 5 replicates.

Total Heavy metal ( m g / k g ) T C L P value (mg/1)


n 1514±452 1213 士 118 46± 11 71 ±22

^ 704 ± 142 621 ±38 95 ± 17 9 0 ^

^ 696± 58 758 ± 54 86±3 87±1

158

Table 3.24 The TCLP values of glass ampoules after encapsulation. Experimental

condition: 1 glass ampoule added with 20 ml of CH3COOH for 18 h at 30 rpm

Heavy metal TCLP (mg/1)Universal Treatment Objectives (mg/l”

^ < 0.005 0?^

^ <0.001

^ <0.0001 4J

a (USEPA)

Detection limits of Pb, Cu and Zn of AAS are 0.005 mg/1, 0.001 mg/1 and 0.0001

mg/1 respectively

. —‘j…’ ‘ f -'tf w ^ ‘ - 1 >

… I

I

威 t t i u L i i i l i ^ i ^ i ^ ^ l i W iiIiiiibmBBBBBE^ I

1

il、, . . . I . “及 ifc. kMtii .. IM

Figure 3.55 Photograph of glass encapsulation of the biopile treated and fungal treated soils.

159

Chapter 4 Discussion


The North Tsing Yi shipyard soil was heterogeneous in both type of contaminants

and contaminant levels (Table 3.1). The soil was contaminated with both organic

and inorganic pollutants. The total PAHs exceeded the new Dutch Intervention

Level (40 mg/kg) and highly beyond the Dutch Target Level (1 mg/kg). This means

treatment was required to decrease the PAHs content to meet the target level. In

this study, it might take 108 Days to reach the Dutch Target level. The initial TPH

content was very high and exceeded the US safety standard (1000 mg/kg) (AEHS,

2005). The oil and grease level was very high with 12200 士 2308 mg/kg.

Although the presence of other organic pollutants such as BTEX, PCB and phenols

were reported, only PAHs, TPH and oil and grease were the target organopollutants

(MEMCL, 2001). Besides organopollutants, the shipyard soil was also

contaminated with heavy metals which were above the Dutch Intervention Levels

(Pb: 530 mg/kg; Cu: 190 mg/kg; Zn: 720 mg/kg). It is common that soil containing

persistent organic compounds is often contaminated by toxic levels of heavy metals

(Baldrian, 2003). The large deviation between replicates indicates the

heterogeneous characteristic in contaminant levels. All the contaminants in soil

were highly above the international safety levels. Therefore, the shipyard soil was

hazardous and remediation of organopollutants and inorganic pollutants were

necessary. The goal of remediation is to reduce the contaminant level back to safety

level leading to reduction in toxicity. In this study, the removal sequence is first to

handle the organopollutants and then the heavy metals. This is because the heavy

metal treatments usually alter the soil properties which make bioremediation of the

organopollutants difficult to be carried out.

Bioremediation was selected for remediating the organopollutants in soil.

Bioremediation is the use of biological treatments, for the clean-up of hazardous

chemicals in the environment (Al-Daher et al., 2001; Semple et al., 2001).

Important soil factors that affect biodegradation processes including soil pH, soil

temperature, soil moisture, availability of essential macro- and micro- nutrients,

nature and bioavailability of pollutants and aeration (Guerin, 2001; Huessemann,

160

2004). The pH of shipyard soil was slightly alkaline (pH between 7 to 8) but near

neutral which was advantageous for the proliferation of soil micro-organisms and

organopollutant degradation (Dibble and Bartha, 1979; Fu and Alexander 1992).

The soil moisture was 12 士 3 % which was insufficient for biodegradation.

Moisture content between 50 % to 80% was good for stimulating microbial

population subsequently resulted in increased biodegradation of petroleum

hydrocarbons in soil (Dibble and Bartha 1979; Bossert and Bartha, 1984; Alexander

1999). The average electrical conductivity was 0.485 士 0.077 mS/cm in soil. This

also showed that the soil was non-saline. It is because from Soil and Plant Analysis

Council (2000), soil is slightly saline when conductivity in soil solution is greater

than 400 mS/cm. Also there was no optimal electrical conductivity level in soil for

biodegradation. The soil was low in salinity level (0.05 士 0.05 %). The organic

carbon content (23800 士 4480 mg/kg or 2.380 士 0.448 %) was high in soil. This

may be due to the high levels of organopollutants (PAHs, TPH and oil and grease) in

soil. But the soil was poor in nutrients (Total nitrogen: 21 士 9 mg/kg; NO3-N: 3 士 1

mg/kg) which were below the optimal level (Total nitrogen: 20 mg/kg; NO3-N: 50

mg/kg) in soil for plant growth (Table 4.1) (Landon, 1991). An optimal C-N-P of

soil for biodegradation of hydrocarbon is 100:10:1 (Golueke and Diaz, 1989). But

in this shipyard soil, the C-N-P of soil was about 71:0.06:1. As a result, nitrogen

was severely inadequate in the shipyard soil. The soil was not favorable for

hydrocarbon degradation because of low moisture and low nutrient levels. Some

amendment is necessary to provide a more favorable condition for microbial

degradation.

The ex-situ bioremediation technology in this study is biopile. It has been reported

that biopile was used for treatment of petroleum hydrocarbon contaminated soil.

The excavated soil can be bioremediated by the addition of nutrients (biostimulation),

addition of microbial inocula (bioaugmentation), aeration and turning, or by a

combination of these practices (Gestel et al., 2003). Mohn et al. (2001)

demonstrated bioremediation of using biopiles with amendment like fertilizer, peat

and inoculum in polar region. Some scientists would add inorganic fertilizer, straw,

wood chips, activated sludge and manure to facilitate biodegradation of

organopollutants in piles (Al-Daher et al., 2001; Chaineau et al, 2003; Juteau et al.,

2003; Atagana, 2004b; Miller et al., 2004). Fahnestock et al (1997) reported that

161

TPH content was degraded to 590 mg/kg after 208 days from an initial concentration

of 1549 mg/kg in a biopile. Pleurotus pulmonarius mushroom compost was added

to the biopile as amendment.

Table 4.1 The optimal soil properties for plant growth (Landon，1991).

Parameters Optimum Level

pH ^

Electrical Conductivity (mS/cm) 0 - 2 (Negligible)

C:N ratio 10 - 12

Total Nitrogen (mg kg]) 20

NH4-N ( m g kg-i) ^

N03-N(mgkg-i) ^

Total P (mg kg-i) I

Available P (mg kg") -


In this study, the mushroom compost of Pleurotus pulmonarius was added to the

biopile to act as fungal treatment. The pioneer studies on the degradation of

xenobiotics by wood-rotting fungi were performed in the early 1980s. PAH

degradation by these organisms was first reported by Bumpus et al. (1985). The

white-rot fungi are considered the most effective PAHs degraders, being able to use

their non-specific lignin degradation system as well as intracellular enzymatic

mechanisms. The Pleurotus pulmonarius mushroom compost also immobilizes

various extracellular enzymes such as laccase and manganese peroxidase.

4.2.1 Enzyme activity

The laccase and manganese peroxidase activities of mushroom compost were 23.54

士 1.44 and 2.38 士 0.56 /xmole/min/g compost (Table 3.2). The laccase found in the

mushroom compost was nearly ten fold more than manganese peroxidase. It has

been reported that laccase is the dominant extracellular enzyme in liquid cultures in

laboratory for Trametes versicolor and Pleurotus ostreatus which is a related species

of Pleurotus pulmonarius (Evans and Hedger, 2001). Both these extracellular

162

enzymes help in degradation of organic pollutants (Laine and Jorgensen, 1997;

Eggen，1999; Lau et al.’ 2003). The optimum temperatures of immobilized laccase

and manganese peroxidase were and 75°C respectively (Lau et al., 2003).

Thus，these enzymes could work under high temperatures. Enzymes usually are

substrate specific but laccase and manganese peroxidase are non-specific acting on

both phenolic and non-phenolic organic compounds through the generation of cation

radicals after one-electron oxidation (Wilson and Jones, 1993; Crawford and

Crawford, 1996; Juhasz and Naidu, 2000). Therefore, these enzymes are good to

degrade a variety of organopollutants in shipyard soil.

4.2.2 Total nitrogen and total phosphorus contents

Nutrient content in mushroom compost is another highlight of this kind of

amendments. It has been reported that the mushroom compost was a good nutrient

source (Semple and Fermor, 1997). The total nitrogen and total phosphorus

contents of mushroom compost were 1387 士 36 mg/kg and 2875 士 30 mg/kg

respectively (Table 3.3). The mushroom compost was rich in nutrient. From one

ton of mushroom compost, more than 1 kg total nitrogen and 2 kg of total

phosphorus were added to the soil. Addition of mushroom compost to the soil

would introduce nutrient to the poor nutrient soil. When the nutrient condition in

soil was improved, the microbial population would increase leading to improvement

in biodegradation.

4.3 Soil monitoring

4.3.1 Initial pollutant content in biopile and fungal treatment soil

The initial PAHs in biopile treatment and fungal treatment included 4-ring PAH such

as fluoranthene, pyrene, 5-ring PAHs such as benz[a]anthracene, chrysene,

benzo[a]pyrene, 6-ring PAHs such as benzo[g,h,i]perylene and

indeno[l,2,3-cd]pyrene (Table 3.4). Not only the total PAHs value exceeded the

Dutch Target level, the individual PAHs also exceeded the Dutch A or B levels in

both biopile treatment and fungal treatment. The initial mean values of the

organopollutants were not exactly the same and for indeno[ 1,2,3-cd]pyrene and TPH,

their initial values in biopile treatment and fungal treatment were significantly

different. This is common in field samples as we can only control the initial values

163

in artificially spiked samples in laboratory. As the soil has been contaminated for

long time, 2- and 3-ring PAHs have been degraded by volatilization and thus not

detectable. Volatilization is important only for 2-ring compounds such as

naphthalene. Abiotic mechanisms account for up to 20% of the total reduction in

PAHs during bioremediation (Hansen et al., 2004). Only the biotic mechanisms are

responsible for removal of PAHs containing more than three rings (Park et al., 1990

in Hansen et al., 2004). However, oil and grease and TPH contents were still found

in biopile treatment and fungal treatment which showed they cannot be removed by

volatilization only. Although heavy metals were detected in the soil, they were

treated after the removal of organopollutants.

4.3.2 On site air and soil physical characteristics

4.3.2.1 Soil temperature, air temperature

Although the soil temperatures fluctuated during treatment, the maximum soil

temperature was 34.5°C (Figure 3.1) which was still within the optimal temperature

range (30 to 40°C) for biodegradation of petroleum (Bossert and Bartha, 1984).

The highest air temperatures measured on site was 41.5 while measured by Hong

Kong Observatory was 33。C. This showed that air temperature in North Tsing Yi

shipyard area were especially higher. Also the record from Hong Kong

Observatory cannot reflect the real situation as its monitoring station is at another

location in Tsing Yi. Therefore it is better to have own first hand information.

There was not much rainy day during soil sampling (Table 3.6). There were only

three rainy days within the 109 days. Although the long wavelength U V (365 nm)

was very high during sunny day in the site, the biopile was covered with a thick

impermeable layer. The strong U V light would not affect the biodegradation in the

soil. As there was < 0.1% CO2 (Detection range: 0.1 - 2.6%), < 1 ppm SO2

(Detection range: 1 一 60 ppm) and < 0.2% H2S (Detection range: 0.2%) gases in the

air, the mushroom compost did not induce any nuisance odor to the site (Table 3.6).


4.3.3.1 Soil pH

Although there was significant effect of the type of treatment on pH values ([type of

treatment] F = 11.08，p < 0.05)，the pH in biopile treatment and fungal treatment

(Figure 3.4) did not deviate greatly from the optimal pH range for biodegradation of

164

petroleum, i.e. 5-7.8 (Dibble and Bartha, 1979). Biodegradation of petroleum has

been reported to be the fastest at neutral or near neutral pH (Dibble and Bartha, 1979;

Fu and Alexander 1992). Though there was fluctuation of biopile and fungal

treatment soil during monitoring, the change of pH did not differ by greater than 1.

Therefore, the fluctuations were very small. From the two-way A N O V A result, type

of treatment had significant effect on pH value, but the mushroom compost did not

alter the pH to a level not suitable for biodegradation. On the other hand, for

Pleurotus pulmonarius, its immobilized enzymes could also work in pH 7 to 8

(Zhang et al., 1999; Jaouani et al., 2005). Therefore, the shipyard soil already

provided a suitable pH for enzymatic reaction and microbial action on

organopollutant biodegradation during treatments.

4.3.3.2 Moisture

Although there is fluctuation of soil moisture in biopile and fungal treatment soil, the

increase in moisture were still far below the optimal ranges. The average biopile

treatment and fungal treatment soil moistures were the same and at 14 土 3o/o (Figure

3.5) which was far below the optimal range 50 % to 80% (Dibble and Bartha 1979;

Bossert and Bartha, 1984; Alexander 1999). From US experiences, the moisture

content in soil piles was kept between 40% - 85% of field capacity throughout the

remediation process (USEPA 1995). Because it is assumed that some drying will

take place during the pile construction and operation. Vallini et al (2002)

suggested that covering the soil piles with a 10 cm layer of mature compost,

triturated straw or other available bulking agents could protects the soil from drying,

insulates it from the ambient temperature. But excessive moisture will fill the pores

in the soil pile and reduce soil permeability, making it difficult to aerate the biopile.

Soil in the Tsing Yi biopile could not provide enough water for microbial

proliferation. Even the type of treatment has significant effect on soil moisture,

addition of mushroom compost could not raise the soil moisture to 50% in fungal

treatment. The population growth of microbes and biodegradation of

organopollutant in biopile and fungal treatment may be limited by the low moisture

content.

4.3.3.3 Electrical conductivity

Electrical conductivity means the level of any soluble ion in soil solution (Dunkle

165

and Merkle, 1994; Ludwick, 1997; Maynard and Hochmuth, 1997; Shaw, 1999).

Although there was an increase in conductivity in biopile and fiingal treatment, the

increase was too low that the soil was still non-saline. At the end of monitoring,

there was higher electrical conductivity in biopile treatment (1.210 士 0.090 mS/cm)

than that in fungal treatment (0.530 士 0.140 mS/cm) (Figure 3.6). Although

electrical conductivity also provides information about soil salinity, the salinity

measured at the end of treatments were no significant different in biopile and fungal

treatment (Figure 3.7). The lead, copper and zinc leached out did not explain higher

conductivity in biopile treatment as lead, copper and zinc leached out in biopile

treated and fungal treated soil were not significant different (Table 3.12). Therefore,

the higher conductivity in biopile treated soil implies more other soluble ion soil but

not higher salinity. Increased soluble ions may be due to the other soluble cations

present.

4.3.3.4 Salinity

The soil was low in salinity level (0 - 0.25%) (Figure 3.7). Although North Tsing

Yi shipyard is at the coastal region, the soil in biopile and fungal treatment were not

saline. This is because the soil in this biopile was not excavated from the coastal

part but the inner part of the shipyard area. Also there was no significant effect of

type of treatment on salinity ([type of treatment] F = 0.76，p = 0.384). Mushroom

compost did not introduce salt to soil. Ward and Brock (1978) found that the rates

of hydrocarbon metabolism decreased with increasing levels of salinity. So the

biodegradation of organopollutants in the soil would not be inhibited by the soil

salinity level.

4.3.3.5 Microbial population in biopile and fungal treatment

It has been reported that if the indigenous microorganisms capable of degrading

target contaminant is less than 10 cfU/g soil, bioremediation will not occur at a

significant rate (Forsyth et al., 1995). Soils that have long-term history of

petroleum contamination should have around 10 - 10 hydrocarbon degraders per

gram of soil (Bossert and Bartha 1984). The initial total bacterial count in biopile

treatment and fungal treatment were 201 土 66 x lO] and 243 土 62 x 10 cfu/g soil.

Therefore, the microbial population in both treatments should be increased by

166

biostimulation or bioaugmentation method.

After 4 days, there were 624 土 220 % and 915 士 255 % increases in total bacterial

population in biopile treatment and fungal treatment soil (Table 4.2). For total mold

count, there was 15 土 10 o/。decrease but 268 士 47 % increase in biopile treatment

and fungal treatment soil respectively (Table 4.2). The biopile blower provided

fresh air with oxygen to stimulate the microbial population in both biopile and fungal

treatments. But the significantly higher bacterial and mold population growth in

fungal treatment was due to the nutrient (nitrogen and phosphorus) provided by

mushroom compost. Besides, the immobilized bacteria and fungi in mushroom

compost also contributed to the great increase of total bacterial count in fungal

treatment. The great increase in mold population demonstrates that fungi contribute

to the degradation of PAHs in soil. These fungi may include the non-ligninolytic

fungi which metabolize PAHs by cyctochrome P450 mono-oxygenase and

ligninolytic fungus, Pleurotus pulmonarius which is a ligninolytic fungus which

degrade PAHs by non-specific radical oxidation, catalyzed by the immobilized

laccase and manganese peroxidase. A synergistic effect of white rot fungi and soil

microorganisms on mineralization of pyrene by Pleurotus sp. has been reported (In

der Wiesche et al., 1996). The fungal preoxidation of PAH increases the rate of

mineralization by bacteria. Therefore, oxidation of PAH by white rot fungi to more

water-soluble products with greater bioavailability could result in rates of

mineralization of these metabolites by bacteria than the rates of mineralization of the

parent PAH compounds (Kotterman et al., 1998). Fungi also have advantages over

bacteria since fungal hyphae can penetrate contaminated soil to reach PAHs

(Novotny et al” 1999; April et al., 2000). Most fungi isolated from contaminated

soil were shown to degrade one or many PAHs efficiently (Giraud et al., 2001).

Also，they can act synergistically with bacteria to remove PAHs by biodegradation.

Greater bacterial and mold population would enhance the biodegradation of

organopollutants.

From Day 4, the bacterial population in biopile and flingal treatment kept rising until

Day 74 (Figure 3.8). This reflects the favorable condition by increasing oxygen

supply and a stable moisture soil condition to stimulate microbial growth. Even at

the bacterial population peak level, more bacteria were found in fungal treatment

167

than biopile treatment. Conditions in fungal treatment were more favorable for

bacterial proliferation. But after Day 74，the total bacterial population decreased

greatly following the peak of relative PAHs content. This may be due to the lower

level of biodegradable hydrocarbon substrates and shortage of nutrient like nitrogen,

phosphorus and carbon source (Al-Daher et al., 2001). So the bacterial population

decreased. At the end of monitoring, still more bacteria were found in fungal

treatment soil. The enhanced bacterial population due to mushroom compost

addition can sustain to Day 109, the end of monitoring period.

For mold population, it increased significantly from Day 0 to Day 39 in fungal

treatment (Figure 3.9). This may be due to the fungi and nutrient introduced by the

mushroom compost. Then the mold population decreased gradually from Day 39 to

Day 109. However, it kept more or less constant for biopile treatment soil from

Day 0 to Day 109. Therefore, biopile could not favor the mold population

proliferation. At the end of monitoring, still significantly more mold were found in

fungal treatment but not in biopile treatment. As a result, amendments like

mushroom compost are required in biopile. This mushroom compost supplies the

microbes and also the nutrients to greatly enhance the fungal growth as well as

bacterial growth.

4.3.3.6 Removal of Organopollutant PAHs in biopile and fUngal treatment

Usually, 3 - 4 ring (low molecular weight, L M W ) PAHs are firstly degraded during

the initial stages of bioremediaiotn treatment while 5-6 (high molecular weight,

H M W ) PAHs are quite resistant to degradation (Atagana, 2004a; Huesemaim, 2004).

This may be due to their low bioavailabilities. Bioavailability is one of the limiting

factors of bioremediation. Bioavailability is primarily related to a compound's

intrinsic physico-chemical properties with aqueous solubility (Alexander, 1994; Linz

and Nakles, 1997; Adriaens et al., 1999). Factors affecting bioavailability includes

amount and nature of soil organic matter, pollutant concentration and hydrophobic

nature of high molecular weight contaminants (Prince and Drake, 1999; Semple et al.,

2001; USEPA 2002). There is no specific test available to evaluate the

bioavailability of PAHs, TPH and oil and grease in soil. But from the history of soil

contamination in the shipyard area, it can be concluded that the organic pollutants in

soil were of low bioavailability. However, one surprising observation was that the

168

H M W PAHs were completely degraded on Day 39 while L M W PAHs required 109

days for complete degradation in this study. It has been shown by Kcck et al. (1989)

that 4- and 5-ring PAHs biodegraded much faster in the presence of easily

biodegradable hydrocarbons. This is the co-metabolism of more recalcitrant H M W

PAHS. Therefore, the L M W PAHs would act as co-metabolic substrates to soil

(Bauer and Capone, 1988; Sims et al.’ 1989; Huesemann et al., 2002). Therefore,

co-metabolism of 5- and 6- rings PHAs occurred in the biopile and fUngal treatment

soil. Also, H M W PAHs are considered to be more toxic and pose a threat to human

health. It is also benefit for degrading H M W PAHs first.

Table 4.2 summarizes the relative PAHs, microbial population and nutrient contents

on Day 4 so as to see their relationship among them. After 4 days, there was greater

degradative removal of the all the tested PAHs in fungal treatment but not in biopile

treatment (Figure 3.17 and Table 4.2). Most relative PAHs in biopile treatment

were above 100% but not in fungal treatment. W e can also compare the

organopollutant removal on Day 4 by GC-MSD of biopile treatment and fungal

treatment soil (Figures 3.18 (a) and (b)). These profiles show the amount of all

organopollutant extracted by the solvent dichloromethane. The significant lower

chromatorgram in fungal treatment reflected less organopollutant were determined

on Day 4. Relatively lower PAHs, TPH and oil and grease in fungal treatment in

Day 4 may be explained by the immobilized enzymes and a greater bacterial and

mold population growth when mushroom compost was added (Table 4.2). Higher

microbial population always induced higher degradation of organopollutants. Also,

the immobilized enzymes in mushroom compost could degrade the PAHs which

were not added in biopile treatment. Also, total nitrogen and phosphorus contents

in fungal treatment were higher than that in biopile treatment (Table 4.2). Higher

nutrient content in fungal treatment could support the higher microbial population.

All these factors contributed to the significant degradation of PAHs in fungal

treatment but not in biopile treatment. Immobilized enzymes, microbial reaction

and nutrient effect contributed to the enhanced biodegradation in fungal treatment.

169

Table 4.2 A summary of relative PAHs, microbial population and nutrient contents

in biopile treatment and fungal treatment on Day 4. Data are presented in mean 土

SD of 5 replicates.

biopile treatment fungal treatment

Relative PAHs (%)

Fluoranthene 118 士 18 82 士 13

Pyrene 114± 17 79 ± 4

Benz[a]anthracene 110 ± 14 76 士 6

Chrysene 107 士 14 76 ± 5

Benzo [a]pyrene 101 ± 8 77 士 6

Benzo[g,h,i]perylene 90 士 8 71 ± 7

Indeno[l,2,3-cd]pyrene 98 ± 8 83 ± 9

Oil and grease (%) 97 ± 9 77± 10

Total petroleum 82 ± 3 66 士 9

hydrocarbons (%)

Increase in total bacterial 624 士 220 915 ±255

count (%)

Increase in total mold 15 ± 10 (decreased) 268 ± 47

count (%)

Total nitrogen (mg/kg) 21 士 3 42 ± 10

Total phosphorus (mg/kg) 326 士 20 387 ±25

4.3.3.7 Effect of type of treatment on residual PAHs at their peak levels

In both biopile and fungal treatment, there was a temporary increase in

concentrations of 4-, 5- and 6-ring PAHs. The relative content of all PAHs were

greater than 100%. This may be because microorganisms initiated PAH

degradation via dioxygenase attack and this increased PAH chemical reactivity and

solubility (Harvey et al., 2002). So, there would be an increase in the residual

PAHS if the P A H degraded by the indigenous microorganisms was not at a faster rate

than it was being released. This may apply to the L M W PAHs which can be

breakdown products of H W M PAHs during degradation. However, as the soil is

under degradation, the expression of PAH contents in concentration not in absolute

170

amount might be misleading to imply the ‘increase，. Using concentration as the

measurement unit, all PAHs contents showed peak values during the treatment period.

But it is interesting that relative PAHs content at peak level in fungal treatment were

significantly lower than that in biopile treatment except pyrene (Figure 3.19).

Besides this, the relative L M W and H M W PAHs contents in fungal treatment would

not immediately increase to above 100% from Day 0. Their contents decreased in

Day 4 and then remained around or below 100% for a period of time (18 days for

L M W PAHs and 11 days for H M W PAHs). But in biopile treatment, their relative

contents immediately increased from Day 0. Thus, the PAH degraded by the

microbes was faster in fungal treatment where mushroom compost was added. The

one ton of mushroom compost added to the soil still facilitated both microbial growth

and biodegradation during mid-term of soil monitoring.

4.3.3.8 Effect of type of treatment on residual oil and grease and TPH content

Usually, the oil and grease contents measured included different hydrocarbons and

polar fraction of the petroleum. Different sites have different combination of

petroleum hydrocarbons and natural lipids which contribute to oil and grease in the

sites. The hydrocarbons present usually comprise saturates (alkanes and

cycloalkanes) and aromatics (mono- and polynuclear) while the polar fraction of the

petroleum containing nitrogen, sulfur and oxygen is comprised mostly of

ashphaltenes and resins (Gogoi, et al., 2003).

For TPH and oil and grease, the sharp drop in fungal treatment in Day 4 (Figure 3.2，

Figure 3.3 and Table 4.2) could be explained by the great increase in bacterial and

mold population, immobilized enzymatic reaction and nutrient effect. The

hydrocarbons were consumed preferentially during this period and used as carbon

sources by microorganisms. This situation was similar to PAHs degradation.

From Day 4，the TPH contents in fungal treatment soil were already below the safety

standard but not for biopile treatment. From Day 4，the TPH and oil and grease

contents fluctuated and decreased slowly in biopile and fungal treatments. Also

their residual contents did not rise to a peak level what PAHs did. This may be

explained by the relative potentials for bioremediation of the petroleum compounds

which is in the decreasing order: monoaromatics > straight chain alkanes > branched

alkanes > naphthenes > polynuclear aromatics > polars (Huesemann, 1994). Also

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alkane degrading microorganisms were more prevalent than aromatic/polyaromatic

hydrocarbon degraders (Gogoi, et al., 2003). Most of the light hydrocarbons were

already degraded at the very beginning, only the less biodegradable hydrocarbons

remained.

At the end of monitoring, TPH and oil and grease were not completely degraded.

The polar fraction of petroleum compounds such as ashphaltenes and resins

contributed to the residual oil and grease content at the end of monitoring. But it

has been reported that even in a well aerated and nutrient-rich biopile, incomplete

TPH biodegradation can occur as a result of limited bioavailabiity, inherent

recalcitrance to degradation, or both (von Fahnestock et al., 1998). Finally, more

oil and grease were removed in fungal treatment (60%) than biopile treatment (47%).

The final TPH was significantly lower in fungal treatment (594 士 48 mg/kg) than

biopile treatment (952 士 34 mg/kg). From the organopollutant profiles of biopile

treatment and fUngal treatment soil on Day 109 (Figure 3.20 (a) and (b))，the lower

chromatogram of fungal treatment proved that less organopollutant was extracted

from the fungal treatment. The greater residual organopollutant in biopile treated

soil may due to the remained TPH and oil and grease. The conditions in fungal

treatment were better than biopile treatment for biodegradation of oil and grease and

TPH. The better conditions were due to the immobilized nutrient and enzymes in

the mushroom compost. Also, from the Figure 3.20 (a) and (b)，the chromatograms

of Day 109 were shift to the left from the Day 0. In both biopile treatment and

fungal treatment, large portion of high molecular weight organopollutants which

appeared after 30 minutes were disappeared on Day 109. But the low molecular

weight organopollutants appeared before 20 minutes were increased. This may

because the low molecular weight organopollutants were the breakdown product or

intermediates of high molecular weight organopollutants. The degradation of high

molecular weight organopollutants generated low molecular weight organopollutants

in both biopile and fungal treatments. But there was more low molecular weight

breakdown product or intermediates in biopile treatment than fungal treatment on

Day 109.

4.3.3.9 Effect of type of treatment on total nitrogen and phosphorus contents

In theory, approximately 150 mg of nitrogen and 30 mg of phosphorus are consumed

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in the conversion of 1 g of hydrocarbon to cell material (Rosenberg and Ron, 1996).

Nutrient such as nitrogen and phosphorus are required for microbes proliferation and

hydrocarbon degradation. On Day 4, total nitrogen in fungal treatment soil was

significantly increased but not in biopile treatment (Figure 3.21). This is due to the

mushroom compost added to the fungal treatment. Even after 109 days, residual

nitrogen in fungal treatment was still higher than biopile treatment. This showed

that the nutrient effect of mushroom compost sustained to the end of monitoring.

After adding mushroom compost, the C-N-P of soil (71:0.1:1) improved but still did

not reach the optimum ratio 100:10:1 (Golueke and Diaz, 1989). The total nitrogen

content in biopile treatment on Day 0，Day 4 and Day 109 were not significantly

different. It is because there was no nutrient amendment in the soil. The

mushroom compost also increased the total potassium content in fungal treatment.

But there was no change in potassium content in biopile treatment. Biopile could

not improve the nutrient condition in soil. Supplementation is necessary in order to

provide a better condition for hydrocarbon degradation. Pleurotus pulmonarius

mushroom compost can be a choice.

4.3.3.10 Effects of type of treatment on the physical and chemical properties of the

soil

After treatment, PAHs and TPH contents in both biopile and fungal treatment were

degraded to below the safety standard. But residual TPH and oil and grease were

still detected in the soil. There was no great change in soil physical properties

between biopile treated and fungal treated soil which showed that the mushroom

compost added would not induce any adverse effect on soil quality. On the other

hand, nutrient content in fungal treated soil was higher than that in biopile treated I

soil. Several toxicity tests were employed to evaluate the toxicity of treated soil.

Also the performance between biopile treated and fungal treated soil was compared

to see whether compost addition would result less toxicity in soil.

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Toxicity assessment at the end of bioremediation can provide valuable and

complementary information to the chemical analysis (Ahtiainen et al., 2002).

Several biological toxicity tests were employed to evaluate the toxicity of treated soil.

It is because chemical data alone are not sufficient to evaluate the biological effects,

because it is impossible to analyze all the compounds and synergistic effects

contributing to toxicity (Petanen and Romantschuk, 2003; Plaza et al., 2005).

One major advantage of biological toxicity tests over chemical analysis is the direct

assessment of the potential hazard to the soil ecosystem caused by residual

contaminants (Ahtiainen et al., 2002). Many researchers used soil elutriates to do

toxicity test after bioremediation (Ahtiainen et al., 2001; Joner et al., 2004; Plaza et

al., 2005). They usually used water or D M S O to perform the extraction. But

elutriates could not extract all the residual chemicals in soil. Ahtiainen et al (2002)

reported there was limited extractability of compounds in elutriate. Some

hydrophobic chemicals strongly sorbed in soil particles. As a result, treated soil

was directly used for performing toxicity test in this study.

Usually, soil quality tests using bacteria and plants are promising tools for risk-based

corrective action (Plaza et al., 2005). Ecotoxicity tests such as seed germination

were performed after bioremediation of PAHs in soil (Sasek et al., 2003). Seed

germination test, indigenous bacterial growth test and fungal growth test were used

to evaluate the ecotoxicity. Indigenous microbes were used to evaluate the

ecotoxicity of soil as they are more indicative and representative than model

organisms (Reynoldson et al” 1994; LaPoint and Waller, 2000; Preston and

Shackelford, 2002). Also, the shipyard soil was polluted by mixed contaminants of

organic and inorganic pollutants, using indigenous microbes as they have adapted to

the environment.

Seed germination test reflects both direct and indirect effects of soil. Plant tests are

cost effective, and relatively easy to perform (Wang, 1991). Triticum aestivum,

Lolium perenne and Brassica chinensis were reported to be used as evaluation of

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phytotoxicity by its germination (Siddiqui and Adams, 2002; Ye et al., 2002). The

seeds germination of the three species was totally inhibited by the initial soil

contamination (Figure 3.24 to Figure 3.26). However, after biopile treatment and

fUngal treatment, the seed germination of the three species increased significantly.

For wheat, the relative seed germination in both biopile treated and fUngal treated

soil reached 99 士 2 % and for ryegrass, the seed germination in biopile treated and

fungal treated soil reached 99 ± 10 o/o and 99 ± 4 % respectively. The seed

germination frequency of wheat and ryegrass increased to a level similar to garden

soil. Therefore, wheat and ryegrass regard the biopile and fungal treated soil as

garden soil. For Chinese cabbage, the seed germination of both biopile treated and

fungal treated soil were 71 士 7 o/。which were still much lower than germination in

garden soil. The residual pollutants such as TPH and oil and grease in treated soils

still induced growth inhibition for Chinese cabbage. Also, there were heavy metals

in the treated soil. It has been reported that toxicity of metals in plants is Hg > Pb >

Cu > Cd > Cr > Ni > Zn (Ross, 1994). Furthermore, concentrations of Pb in soil

from 100 - 400 mg/kg were considered toxic by Kabata-Pendias and Pendias (1992).

Although zinc concentration was very high, zinc is an essential element for plant

growth and its toxicity is less than Pb. The residual lead, copper and zinc may also

inhibit the Chinese cabbage germination.

Actually, phytotoxicity responses were different for the different plant species

(Chaineau et al. 2003). The initial seed germination frequencies of the three plants

were different before any treatment. From the result, Chinese cabbage had the

lowest germination percentage while wheat had the highest initial germination

percentage. Therefore, Chinese cabbage was significantly more sensitive than

wheat and ryegrass. It is proposed that the tolerance of plant seeds to contamination

followed a decreasing order: Triticum aestivum (wheat) > Lolium perenne (ryegrass)

〉Brassica chinensis (Chinese cabbage). Different plant responses to treated soils

could be related to bioavailability issues relevant to plant-soil-contaminant

interactions (Chaineau et al 2003; Plaza et al., 2005).

Isolation of Pseudomonas aeruginosa and Methylobacterium sp. from diesel oil and

PAHs contaminated soil has been reported (Gestel et al, 2003; Andreoni et al., 2004).

Microorganisms have several advantages for use in toxicity testing, like simple, rapid,

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sensitive and inexpensive (Bitton and Dutka，1986; Bemand et al., 1996). The

toxicity of the soil before treatment was high towards Bacillus cereus, Pseudomonas

aeruginosa and Methylobacterium sp. Bacillus cereus and Pseudomonas

aeruginosa still have a positive population growth but for Methylobacterium sp., it

even had a negative population growth in soil before any treatment. From this

result, their resistance to mixed contamination followed the decreasing order:

Bacillus cereus > Pseudomonas aeruginosa > Methylobacterium sp. From the

population growth of Bacillus cereus and Pseudomonas aeruginosa in biopile and

fungal treated soil, biopile treated soil still caused more inhibition on their growth

than fungal treated soil (Table 3.15). This may be owing to the higher residual TPH

and oil and grease contents than fungal treated soil. It has been reported that fungal

treatment of creosote-contaminated soil has been shown to lead to a reduction in

toxicity (Baud-Grasset et al., 1993; Baud-Grasset et al., 1994). But biopile and

fungal treated soils showed similar toxicity to Methylobacterium sp. (Table 3.15).

From the result of one way ANOVA, these soils after treatment were still toxic to the

three bacterial when compared to garden soil (Table 3.15).

Trichoderma asperellum, Trichoderma harziauum and Fusarium solani have been

isolated in PAHs contaminated land from other studies (Giraud et al., 2001; Gestel et

al., 2003). It is interesting that the fungal growth was significantly higher in

contaminated soil before any treatment (Figures 3.32 to 3.35). This indicates that

theses fungi have the ability of using the pollutants as nutrient for growth. On the

other hand, Trichoderma asperellum and Fusarium solani had the same population

growth in biopile treated, fungal treated and garden soil. Therefore, there was no

significant toxicity induced to them by the treated soil. However, biopile treated

soil still gave toxicity towards Trichoderma harziauum as there was significantly

more population growth in garden soil. Also, Pleurotus pulmonarius showed

higher population growth in both biopile treated and fungal treated soil than that in

garden soil. This showed that Pleurotus pulmonarius could tolerate the residual

TPH and oil and grease in treated soil and even used them as carbon source for

growth.

In terms of ecotoxicity, the abandoned shipyard soil was toxic to plants and

indigenous bacteria. The indigenous fungi could utilize the organopollutants as

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substrates and survived well in this type of soil. The fungal and biopile treatments

decrease the soil toxicity towards the plants and bacteria. Yet such treatment lowers

the organopollutant contents, and consequently, the fungal population growth was

retarded. By comparing the three toxicity tests, they were all simple, sensitive and

fast for use in toxicity evaluation after bioremediation.

4.5 Summary of Pleurotus pulmonarius mushroom compost on

organopollutant remediation

In the first part of study, both biopile and fungal treatments could remediate PAHs,

oil and grease and TPH in soil. The contents of organopollutants showed

significant reduction in both treatments. Although PAHs were undetectable in both

biopile and fungal treated soil, significantly more residue TPH and oil and grease

were found in biopile treated soil. Also, fungal treatment showed a significant less

PAHs in Day 4 and at peak level. The microbial population and nutrient contents in

fungal treatment was also greater than biopile treatment. Actually the

environmental condition of biopile and fungal treatment was the same except the

addition of Pleurotus pulmonarius mushroom compost. To sum up, addition of

mushroom compost can enhance the degradation of PAHs, TPH and oil and grease in

soils in biopile by increasing microbial population, nutrient contents and the

immobilized enzymes. From plant and indigenous bacterial toxicity tests, both

biopile treatment and fungal treatment could reduce the toxicity of contaminated soil.

But for B. cereus and R aeruginosa, the toxicity of fungal treated soil was less than

biopile treated soil. Even some species showed same toxicity of biopile treated and

fungal treated soil, no tested species showed that fungal treated soil was more toxic

than biopile treated soil. Inoculating mushroom compost is a cost saving method

when compared to inorganic fertilizer and is a better way than dispose waste by

landfilling. Straw compost is available in most countries, and it is produced on a

large scale for the cultivation of mushroom. It offers good nutrient source and

attachment material for bacteria (Laine and Jorgensen, 1996; Reid et al., 2002).

In both biopile and fungal treatments, biopile is used. Biopile has advantages of

relative simple design and implementation. Also because of its low operation and

maintenance requirements, it is cost competitive. Also usually contaminants can be

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degraded to innocuous end products such as CO2 and water (USEPA, 2002; Miller et

al, 2004). But the presence of extremely high heavy metal concentrations may

inhibit microbial growth therefore retarding biodegradation. Also there may be

compaction of soil reduced air diffusion causing a decrease in bioremediation rates in

biopile. Sometimes, bulking agents could be added to increase soil porosity.

Biopile requires a large land area for treatment although less than landfarming.

Also excavation is required (USEPA, 2002; Miller et al., 2004). Excavation would

increase the risk of dispersal of volatile organic compounds and increase human

exposure.

4.6 Soil washing

Unlike many organic pollutants that can be eliminated or reduced by chemical

oxidation techniques or microbial activity, heavy metals could not be degraded.

Soil washing has recently become one of the most suitable ex-situ technique for

remediating sites contaminated with heavy metals (Juang and Wang, 2000; Reddy

and Chinthamreddy, 2000; Hong et al., 2002; Zeng et al., 2004). In conventional

soil washing processes, excavated soil is vigorously mixed with a solution that

separates the contaminants from the large particle size fractions. The soil and the

extracting solution are mixed thoroughly for a period of time. Also the soil is

dewatered to separate the soil and liquids. The resulting soil that meets regulatory

requirements can be backfilled at the excavated site. The recyclable solution is then

treated to remove the colloidal particles and the original contaminants. Common

washing solution includes hydrochloric acid, ethylenediaminetetraacetic acid

(EDTA), acetic acid, phosphoric acid, and calcium chloride (Cline and Reed, 1995;

Juang and Wang, 2000; Reddy and Chinthamreddy, 2000; Sun et al, 2001). From

Cline and Reed (1995), EDTA and HCl achieved 92% and 89% removal efficiencies

of lead while acetic acid and CaCli only achieved 45% and 36% removal efficiencies

respectively. EDTA can form soluble complexes with metal ion, reducing the

quantity of metals retained by soil particles and thereby increasing heavy metal

mobility. Thus, chelating agents are well suited for removing metals bounded by

soils (Cline and Reed, 1995). But chelating agents are not cost-effective in treating

soils having a high % of clay and silt (e.g. more than 30-50%) (Anderson, 1993).

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Therefore, HCl was selected as E D T A is relatively expensive and rather difficult to

treat in effluents. E D T A may retain in contaminated soil cause difficulty in

residuals management. Also, E D T A would reduce the efficiency of metal removal

by conventional chemical precipitation (e.g., 0H-), ion exchange, adsorption, and

other processes (Tunay et al., 1994). Although soil washing is a suitable technique

in on site heavy metal removal, conditions should be optimized in order to achieve

maximum removal efficiency. Strength of HCl and incubation time were optimized

in this study.

From Figures 3.36 (a) and (b), the removal efficiencies of Pb, Cu and Zn were the

greatest using 0.5 N HCl. The metal ions on soil surface were replaced by H+ ions

which are attracted more strongly than any other cation (Cline and Reed, 1995).

However, when the strength of HCl increased from 0.5 N to 4 N，the removal

efficiency reached plateau for Pb and increased slowly for Cu and Zn. Thus, 0.5 N

HCl acid was used for optimization of time.

From Figures 3.37 (a) and (b), soil washing using HCl significant reduced metal

leachability. This is because most mobilized metal ions were already removed by

the acid. The remained metals attached on the soil surface were very difficult to

mobilize. So the amounts of metals leached out by the acetic acid of the TCLP test

were very low. However the metals leached out were still exceeding the USEPA

universal treatment objectives even using 0.5 N HCl. Also the TCLP values of all

heavy metals ranked the highest for water extraction. Because water did not

remove most of the easily mobilized metals which can be leached out using acetic

acid. Therefore, the soil washing treated soil may pollute the groundwater by

leaching of heavy metal from soil. After washing with 0.5 N HCl, the total lead and

copper in biopile treated and fungal treated soil were still above the Dutch B levels

and total zinc was still above Dutch A levels. Large amount of heavy metal were

still strongly bounded to the soil particles.

The removal efficiencies of Cu, Pb and Zn were the highest for acid washing at 6

hours (Figures 3.39 (a) and (b)). When incubation time increased to 48 hours, there

was no significant increase in Cu and Zn removal in both biopile and fungal treated

soil, and removal of Pb even decreased. Most mobile metal ions on soil surface

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were already replaced by H+ ions during 6 hrs incubation. Increasing shaking time

did not increase metal leachability (Figures 3.40 (a) and (b)). But more Pb leached

out when biopile treated and fungal treated soil were washed with HCl for 48 hours.

For Zn, it leached out the most when the incubation time was 24 hours for both

biopile treated and fungal treated soil. When incubation time increased, more

tightly bound Pb and Zn leached out in acetic acid. However, the metals leached

out were still exceeding the USEPA Universal Treatment Objectives. The cleaned

soil still had the risk of contaminating the groundwater. After shaking with HCl for

6 hours, the residual total lead and copper in biopile treated and fungal treated soil

were still above the Dutch B levels and total zinc was still above Dutch A levels.

Large amount of heavy metals were still strongly bounded in the soil.

From the result, using 0.5 N HCl shaking for 6 hrs can remove 62 士 5 % of Cu, 76 士

6 o/o lead and 84 士 7 % Zn in biopile treated soil and 55 ± 3 % of Cu，67 士 9 % lead

and 83 士 8 % Zn in fungal treated soil. Therefore, more than half of the total metals

were removed from soil. Some alkali or alkaline earth salts such as NaOH，NasCOs

or M g O can be added to recover the metals from the extracting solution (Ajmal et al.,

1995; Panswad et al” 2001). Ajmal et al. (1995) reported with the help of the

adsorbent phosphate treated sawdust, 92 % recovery of Cr (VI) achieved when using

O.OIM NaOH. Panswad (2001) used M g O at the dosage of two-fold stiochiometric

requirement and one hour of free settling, the recovery efficiency of Cr203 was

97.6%. But the settling period for NazCOs was much longer (15-20 hours). The

recovery of metal depends on the pH and settling time (Hong et al., 2002). The

removal efficiency of soil washing was high but it has disadvantages such as

decreased soil productivity and adverse changes in chemical and physical structure of

soils (Reed et al., 1996). Then the treated soil may not be suitable for backfill.

4.7 Mycoextraction

It is reported that white rot fungi can concentrate metals taken up from substrate in

their mycelia. Pleurotus ostreatus was able to accumulate 20 mg/g dry weight Cd

from liquid medium containing 150 mg/L Cd with at least 20% of accumulated Cd

deposited intracellularly (Favero et al., 1991). Pleurotus pulmonarius mushroom

compost was used in the fungal treatment in the first part of study. Therefore, the

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ability of this compost to remove metals was also analyzed.

To show the exact metal content in fruiting bodies of each replicate, scatterplots were

used in mycoextraction result. The fruiting bodies of Pleurotus pulmonarius in all

setups accumulated more Zn than Cu from soil (Figures 3.43 (a) and (b)). This may

be because Zn is essential to Pleurotus pulmonarius. Also they excluded Pb in soil.

It has been reported white rot fUngi growing on wood in nature were found to

accumulate Cd, Fe, Zn and Cu in their fruiting bodies, whereas M n and Pb were

excluded (Baldrian, 2003). Daedalea quercina has been shown to uptake metal in

the following decreasing order: Zn > Cu > Pb (Baldrian, 2003). This matches with

the result in this study. It has been reported that white rot fungi used oxalate to

immobilize metal ions (Baldrian, 2003). It seems that the fungal preference for

individual heavy metals uptake is species-specific. When at 1:0.5 soil to compost

ratio, the fungal fruiting bodies accumulated the highest concentrations of zinc and

copper. When more compost was added to the soil, there was no increase in metal

concentration in fruiting bodies. These phenomena were found in both biopile

treated and fungal treated soil (Figures 3.43 (a) and (b)). But there was more zinc

amount in fruiting bodies which may be due to the larger fruiting bodies emerged

(Figures 3.44 (a) and (b)).

For the metal leachability of soil after mycoextraction, no significant increase in Pb

and Zn leachability when the amount of compost increased in biopile treated soil, but

there was more Zn leached out in 1:2 and 1:5 setups for fungal treated soil. Also,

more Cu leached out in 1:1 and 1:2 setups for biopile treated soil. Thus, the

mushroom compost enhanced Cu and Zn leachability from soil. But Pb was still

tightly bounded in soil.

As only a small amount of metals removed by the fruiting bodies, for the residual

total metals remained in soil, there were still > 1000 mg/kg of Pb; > 450 mg/kg of Cu

and > 500 mg/kg Zn in both biopile and fungal treated soil, their concentrations were

still above the Dutch A or B levels. The amount of metals extracted in fruiting

bodies only contributed a little amount of total metal loss in soil. Therefore, in

terms of the actual amount of metals translocated to mushroom fruiting bodies, the

removal efficiency by fungal extraction was not great.

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4.8 Phytoextraction and integrated bioextraction

Phytoextraction refers to the extraction of metals or organics by plant roots from

contaminated soil and water to translocate them to aboveground shoots (Morikawa

and Erkin， 2003; Mertens et al； 2004). Phytoremediation especially

phytoextraction has recently been proposed as an effective method to remediate soils

contaminated with heavy metals. The plants used in this study were not

hyperaccumulators which may not be commercially available. Besides, the

criterion for hyperaccumulation varies for different metals, but was summarized by

Baker et al (1994) as > 1000 mg/kg and 10000 mg/kg for Pb and Zn of shoot dry

matter (Baker et al., 1994; Meagher, 2000). There is no one plant

hyperaccumulator reported to survive in a mixed contaminated soil as used in this

study and hyperaccumulate multiple heavy metals. But the main limitations of

hyperaccumulator species are their low biomass and specific growth needs (Gleba et

al., 1999). The overall efficiency in metal extraction is thus a function of plant

biomass and metal concentration in plant tissues. Plant must be able to accumulate

toxic metals and transfer to harvestable aboveground parts. This degree of

efficiency will determine the number of sequential harvests needed to extract the

mass of metal necessary to reach target levels and in turn, the number of harvests

will determine the total cost of the operation, including biomass disposal through

landfill, incineration, or composting (Salt et al., 1998; Ensley, 2000). Also, plants

removing heavy metals must grow fast in contaminated environment, to be resistant

to the metals. Plants able to remove more than one pollutants are especially useful,

because contamination is usually caused by a mixture of more toxic compounds

(Macek et al., 2004). The bioavailability of heavy metals is an important factor in

the process of phytoextraction by non-hyperaccumulators, especially in neutral or

calcareous soils. There are two main approaches to increase the bioavailability of

heavy metals in soils: lowering soil pH, adding synthetic chelates such as EDTA，

N T A and DTPA (Huang et al., 1997 in Kos and Lestan, 2003; Roy et al., 2005).

This induces translocation of chelate-heavy metal complexes from roots to green

parts. However, these chelates are not specific to heavy metals and are subject to

numerous interferences with other cations present in soil at much higher

concentrations. Many synthetic chelates and their complexes with heavy metals are

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toxic and poorly photo-, chemo- and biodegradable in soil (Nortemann, 1999 in Kos

and lestan, 2003). As a result, no synthetic chelates were added in phytoextraction

in this study.

Lead was also excluded in wheat, ryegrass and Chinese cabbage which was found in

mycoextraction. It may be because Pb is not an essential element in metabolic

processes in plants or animals. Also Pb has very limited solubility in soils and thus

availability for plant uptake due to complexation with soil organic matter, sorption on

oxides and clay minerals and precipitation as carbonates, hydroxides and phosphates

(Lindsay, 2001 in Kos and Lestan, 2003; McBride, 1994). Pb is retained by many

plants in their roots via sorption and precipitation, with only minimal transport to the

aboveground harvestable plant portions (Brennan and Shelley, 1999). Zn was

accumulated in mycoextraction and phytoextraction, it is because Zn is an essential

element in plant and animal metabolic processes. But it can also be accumulated to

toxic level in plants (Lock and Janssen, 2001 in Cui et al., 2004). In this study,

wheat accumulated more Zn than Cu in biopile treated soil and fungal treated soil,

but Chinese cabbage accumulated more Cu than Zn in biopile treated soil and fungal

treated (Figures 3.1.48 (a) and (b)). The heavy metal uptake was species-specific.

But the amounts of Zn and Cu accumulated in aerial part of wheat and Chinese

cabbage were within 30 fig, thus the amount was very small. From Table 3.21，as

wheat has the highest aerial biomass after 4-week planting, it is selected to

investigate the integrated bioextraction. After 50 g of mushroom compost were

added to soil, Cu accumulation was significantly increased in both biopile treated soil

and fungal treated. Also Pb was still excluded in the wheat. Thus, the integrated

bioextraction setup could uptake more Cu than phytoextraction only. The aim of

integrated bioextraction is combining the advantages of both mycoextraction and

phytoexlraction. Also it has been proven that addition of mushroom compost and

pig manure together could improve the revegetation of Pb and Zn mine tailings (Ye

et al., 2000). However, when we compare mycoextraction, phytoextraction and

integrated bioextraction, mycoextraction accumulated more Cu and Zn in their

fruiting bodies.

For metal leachability, less Pb was leached out in ryegrass phytoextraction (Figures

3.1.49 (a) and (b)). Also, less lead was leached out in integrated bioextraction for

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biopile treated and fungal treated soil. It seems that Pb was immobilised in the soil

by ryegrass and integrated bioextraction which is called phytostabilisation.

Phytostabilisation is the fixation of heavy metals in soil by immobilisation or by

preventing migration (Vangronsveld et al., 1995; Mertens et al., 2004). However,

leachabilities of Cu and Zn were more or less the same in phytoextraction and

integrated bioextraction in both biopile treated and fungal treated soil.

Phytoextraction and integrated bioextraction could not immobilize Cu and Zn in soil.

The residual total lead, copper and zinc after phytoextraction and integrated

bioextraction were not significantly different. The heavy metal extracted from plant

or the fungus did not reduce the residual heavy metal in soil in a large extent. The

concentrations of lead, copper and zinc were still above the Dutch A or B levels. In

brief, biological extraction was too low in efficiency in removal of heavy metals in

soil.

4.9 Cementation

The initial metals leachabilities of soil used in cementation were lower than that of

soil used for soil washing and biological extractions (Table 3.18，Table 3.19 and

Table 3.22). It is because the soil used for cementation was about 6 months after

the end of biopile and fungal treatments. The leachabilities of metals decreased

when time increased. Metals were more strongly bound to the soil particles when

aging.

When the amount of cement mixed with soil increased, less Pb, Cu and Zn were

leached out. However, the amount of cement required to stabilize different metals

to a safety level was different. For example, 16% of cement was enough for

stabilizing Pb, but 30% and 40% of cement were required for Zn and Cu respectively

(Figures 3.52 (a) and (b)). As soil was contaminated with several heavy metals, the

highest percentage of cement was adopted and added in order to stabilize all heavy

metals. The total residual Pb, Cu and Zn concentrations reduced gradually when

the percentage of cement increased. This is due to the dilution effect caused by

cement. At 16% cement, total Pb was still above Dutch C level, and at 30% cement,

total Zn was above Dutch A level. At 40% cement, total Cu was also above Dutch

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B level. Therefore, cementation could not decrease the total heavy metals

concentrations in soil to safety level and therefore did not solve the heavy metal

problem completely.

Without doubt, immobilization of heavy metals is not a permanent solution. It is

because heavy metals were still there. Site reuse of soils is limited and long-term

monitoring is generally required (Hong et al., 2002). Also, weathering may

facilitate the leaching of immobilized elements (Schmid et al., 2000). Also some

types of microbial activity may increase leaching of metals from cement due to

active transport (Idachaba et al., 2003 & 2004). Another requirement of

cementation is the moisture content which should be less than 50% (Mulligan et al.,

2001a). Therefore, cementation was not suitable in stabilizing soil sludge and

sediment. However, cementation is still the most commonly used stabilization

method it is because adding material is low cost, readily available, and the process is

simple in operation (Visvanathan, 1996; Vangronsveld and Cunningham, 1998).

Also, the technique is well developed.


The soil used for glass encapsulation still contained high level of metal

concentrations and its metal leachability was still greater than the universal treatment

objectives. For the making of glass encapsulation, the weight of soil in each glass

ampoules was fixed, but the soil to glass ratio was not fixed (Table 2.13). It is

because sometimes the Blowing Glass Service technician sealed a large bead that soil

in it was just half-full, so more glass was used. Also，different sizes of old glass

tubes were used as materials to make the ampoules. The thickness of glass tube

wall was not the same resulting different amount of glass used. After the glass

encapsulation, no heavy metal was leached out again. But the total metal

concentraion was no change as there was no removal or leached out of metals.

Therefore, glass encapsulation could not decrease the total heavy metals

concentrations in soil to safety level but reduce the metal leachability.

Containment in glasses is depending on the durability of glass. Such amorphous

glasses are subject to corrosion, which may free trace elements (Perret et al” 2003).

185

Thus, this would eliminate the problem of polluting groundwater. But the heavy

metal was still retained in the soil. Special storage of the encapsulated soil is

necessary. Therefore, long-term monitoring was required. If there is leakage,

contaminated soil will leach out and pose threat to human. Also the soil could not

be reused again and heavy metal could not be recovered.

Another developed encapsulation is thermoplastic encapsulation. Asphalt, bitumen,

polyethylene and polypropylene and nylon can be used to create a coating or jacket

over the wastes (Visvanathan, 1996). These thermoplastic materials are generally

organic plastics which are capable of reversibly softening and hardening upon

heating and cooling. However, the materials used are expensive and flammable.

Thus special care is required during operation. Also this technique is the same as

glass encapsulation with the consumption of electricity.

One common heavy metal treatment technology is incineration. Incineration cannot

be carried out due to the lack of this facility in laboratory. But it is discussed here.

It is a fast but high investment cost treatment technology. Incineration is a thermal

oxidation process, in which the hazardous wastes are converted using the oxygen

present in the air, into gases and incombustible solid residue (Visvanathan, 1996).

Therefore, volume and weight are reduced. The gaseous products are released into

the atmosphere and solid residue which is still toxic will be landfilled. The

by-product gases are contaminated with trace quantities of hazardous organic

compounds. Thus, special care should be taken to avoid transfer of wastes from

solid or liquid phase to gaseous phase, by installing proper gas cleaning equipment.

In general, incineration is advocated for wastes which are resistant to biodegradation,

and persistent in the environment and contain organically bounded halogens, lead,

mercury, cadmium, zinc, nitrogen, phosphorus or sulphur (Visvanathan, 1996).

4.11 Summary of physical, chemical and biological heavy metal

removal treatments

It can be found that soil washing had the highest removal efficiencies of total metals

in soil. Phytoremediation, mycoextraction and integrated biological extraction only

removed a trace amount of total metals. Cementation did not remove but it had the

186

dilution effect. Glass encapsulation and incineration did not remove the total metals

in soil. Soil washing reduced the metal leachability but the TCLP values were still

above the Universal Treatment objectives. It removed residual metals in soil but

they still exceeded the Dutch A or B levels. For mycoextraction, more copper and

zinc leached out when compost amount increased. When increasing mushroom

compost, the metal leachability did not reduced to a safety level. For

phytoextraction and integrated bioextraction, they could not reduce metal

leachability and the TCLP values were still highly above the objectives. Residual

total metals concentrations were not reduced to Dutch Levels after mycoextraction,

phytoextraction and integrated bioextraction. For metal leachability, only

cementation and glass encapsulation could reduce it to below the objectives.

Stabilization/Solidification could work well in immobilizing the metal ion in soil.

For total metal in soil, none of the above methods could decrease the metal

concentration below the Dutch A or Target Level.

4.12 Future studies

The biodegradation of PAHs, oil and grease and TPH by mushroom compost and

biopile was performed. With only 1% of mushroom compost was added, the result

showed that mushroom compost enhanced biodegradation of organopollutants and

the performance was better than biopile only. This method is an environmental

friendly and sustainable technology. However, field environmental conditions are

difficult to control, and these are important to the biodegradation. Also,

biodegradation is very site specific and no two contaminated soil environment are

exactly alike. Environmental conditions like temperature, moisture and oxygen

supply should be controlled and monitored well. Another limiting factor is the low

moisture content with only 14 士 3%. To improve applicability, the optimization of

the amount of the mushroom compost such as 5 % and 10 % should be studied in

order to maximize the function of mushroom compost on biodegradation. Also,

moisture should be increased to at least 40% for better biodegradation. Although

the production of mushroom compost was easy and cheap, one way to reduce

production costs is to use waste fungal mycelium from industry e.g. spent mushroom

compost. So, spent mushroom compost may try in future application. But of

course, mushroom compost immobilises more extracellular enzymes than spent

187

mushroom compost. Also it has been known that some toxic metabolites may

release during biodegradation. So, any bioremediation intermediates can be

monitored. In this study, the Pleurotus pulmonarius mushroom compost introduced

extracellular enzymes, nutrient, microbes and acted as bulking agent to soil. All

these factors determine the result in fungal treatment soil. But their relationship and

contribution were not analysed. Their interrelation can also be analysed. What is

more, the further study of co-metabolism of PAHs by native microflora and

introduced Pleurotus pulmonarius is necessary. Sometimes the indigenous

microbes would compete with Pleurotus pulmonarius. Moreover the growth of

Pleurotus pulmonarius in contaminated soil represents the toxicity of soil to the

fungus. The growth rate can be measured by hyphal extension and phospholipid

fatty acid content in autoclaved soil (Andersson et al., 2000; Andersson, et al., 2001).

Hyphal extension has the limitation that not many fungi bear macroscopic hyphal

strands. In this study, an alternative approach was adopted; the fungal sterol,

ergosterol, is used as a routine measure of fungal growth. Moreover, CO2 is the end

product of biodegradation. It has been reported that CO2 released can be measured

by a respirometer to check whether the organopollutant was completely mineralized.

Or the i4c02 evolution from " C radiolabeled PAHs can also be evaluated in

laboratory scale (Carlstrom and Tuovinen, 2003). These studies can be further

investigated in pilot scale before going to field. But field trial is the ultimate goal to

confirm the performance and put laboratory theory into real application.

For heavy metal removal, although physical, chemical and biological methods were

applied and compared in this project. There was no perfect method in heavy metal

removal. They have different advantages and limitations. Soil washing is the best

option in terms of its highest removal efficiencies leading to the least TCLP-metal

contents. In future studies, biosurfactant such as surfactin, rhamnolipid and

sophorolipid from various bacteria and a plant-based surfactant, saponin, can be

added to further enhance the bioavailability of heavy metals thus less acidic solution

could be used so as reducing the treatment cost and impact of effluent (Mulligan et

al., 2001b; Hong et al., 2002). Sequential extraction may be tested to enhance the

removal efficiencies of various heavy metals. Phytoremediation and

mycoextraction and integrated biological extraction were carried out also. But the

amount of metal removed was not great compared with soil washing. In future,

188

more plant species may be tested to select a superplant which can accumulate more

heavy metals. From the result, wheat has the advantage of great aerial biomass and

high tolerance to the contaminated soil. To improve, surfactants may be added to

enhance the availability of metals so as to increase metal uptake. Also the

cultivation time could be extended as wheat was cultivated for four weeks in this

study. Phytoremediation using transgenic plants may also be tried so as to improve

metal uptake. But it may have the risk of uncontrolled spread of the transgenic

plants due to higher metal tolerance and the interbreeding with populations of wild

relatives (Pilon-Smits and Pilon, 2002). For mycoextraction, it accumulated more

metals than phytoremediation. As the integrated biological can significant reduce

the lead leachability of soil, the stabilization mechanism should be further examined.

The role of fungi and plant root in rhizosphere on stabilizing metal should be

investigated. For cementation, it is a well-developed technology. It has the

problem of different metals requiring different amount of cement for stabilization.

The addition of other additives such as fly ash and lime can be studied in stabilizing

metals. For glass encapsulation, the soil can be encapsulated in different shape so

as to increase markets purchase. Also, unbreakable glass could be used in

encapsulation so as to reduce the risk of leaching of contaminated soil.

189

Chapter 5 Conclusion

Biodegradation of PAHs, oil and grease and TPH can be enhanced by the addition of

Pleurotus pulmonarius mushroom compost to biopile as a fungal treatment.

Significantly lower residual PAHs at Day 4 and at their peak levels in fungal

treatment soil than biopile treatment soil were detected. Also, less oil and grease

and TPH at Day 4 and at the end of monitoring were found in fungal treatment soil.

The better result in biodegradation in fungal treatment soil was due to greater

microbial population, immobilized laccase and manganese peroxidases and nutrient

contents in fungal treatment soil. All these are important in biodegradation of

organopollutants. This observation agrees with other studies where compost has

been added to contaminated soils, resulting in enhanced levels of degradation (Reid

et al., 2002). No adverse effect was determined in mushroom compost application.

From different ecotoxicity assays, biopile treated and fungal treated soil reduced

toxicities to plants and bacteria. The fungal treated soil even caused less toxicity

than biopile treated soil in some tested bacteria. This demonstrates the potential of

combination of Pleurotus pulmonarius mushroom compost and biopile in

bioremediating orgnaopollutant contaminated soil. This can develop into an

environmental friendly and sustainable technology. Further investigation at pilot or

field scale and optimization of compost amount and moisture contents are required to

expand the potential.

For heavy metal removal, soil washing, biological extraction and

stabilization/solidification were applied and compared in this project. There was no

perfect method in heavy metal removal. They have different advantages and

limitations. The optimized soil washing conditions were: shaking the soil at a ratio

190

1:5 (soil to 0.5 N HCl) for 6 hours at 150 rpm at 25''C. It is the best option in terms

of its highest removal efficiencies (lead: > 55%; copper: > 50% and zinc: > 75%)

leading to the least TCLP-metal contents. Soil washing is a well developed

technology and it can achieve the result in a short time. However, it induces several

problems like acidic effluent generated at a volume of 25 ml from 5 g soil washed

and destructive soil quality. Thus, it is not an environmental friendly and

sustainable technology. For phytoextraction, mycoextraction and integrated

bioextraction, very few amounts of metals were removed when compared with soil

washing. Mycoextraction removed the largest amount of metals (copper: 63 ± 6 /xg;

zinc: 270 士 32 /xg) among the three methods. Also integrated bioextraction

significantly reduced the lead leachability of soil from 46 ± 11 mg/1 to 21 士 4 mg/1 in

biopile treatment and 71 ± 22 mg/1 to 26 ± 7 mg/1 in fungal treatment. But,

biological extraction methods were time consuming requiring several weeks.

Stabilization/solidification by cementation or glass are fast methods to reduce the

lead, copper and zinc leachabilities to safety levels. Copper, lead and zinc required

different amounts of cement for stabilizing. 40% cement (w/w) were required to

stabilize all three metals. This would increase the waste volume and capital input.

For glass encapsulation, leachabilities of all three metals were greatly reduced and

below the safety levels. It would be applicable if the product could be sold to

market and cover the high investment cost. But these stabilization/solidification

methods limit the reuse of soil. To sum up, only cementation and glass

encapsulation could reduce metal leachabilities to safety levels. Also none of the

tested methods could reduce the soil total metal concentration below the Dutch A or

Target Levels. Due to the non-degradable characteristic of metals, metal removal is

still a challenging issue. Human beings should avoid the release of heavy metals to

the environment.

191

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