Does Microbial Biodegradation of Water-Soluble ... - ACS Publications

Mar 7, 2018 - Does Microbial Biodegradation of Water-Soluble Components of Oil Reduce the Toxicity to Early Life Stages of Fish? Bjørn Henrik Hansen*...
0 downloads 0 Views 4MB Size
Subscriber access provided by UNIV OF SCIENCES PHILADELPHIA

Ecotoxicology and Human Environmental Health

Does microbial biodegradation of water-soluble components of oil reduce the toxicity to early life stages of fish? Bjørn Henrik Hansen, Julia Farkas, Trond Nordtug, Dag Altin, and Odd Gunnar Brakstad Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b06408 • Publication Date (Web): 07 Mar 2018 Downloaded from http://pubs.acs.org on March 8, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

Environmental Science & Technology

1

Does microbial biodegradation of water-soluble

2

components of oil reduce the toxicity to early

3

life stages of fish?

4 5

Bjørn Henrik Hansen†*, Julia Farkas†, Trond Nordtug†, Dag Altin‡ & Odd Gunnar

6

Brakstad†

7 8



9



SINTEF Ocean AS, Postboks 4762 Torgarden, 7465 Trondheim, Norway BioTrix, 7020 Trondheim, Norway

10

*Corresponding author: Bjørn Henrik Hansen. E-mail: [email protected]. Phone: +47

11

98283892

12 13

Key words: Oil; PAH; biodegradation; ecotoxicity; cardiac toxicity; developmental toxicity

14 15

1 ACS Paragon Plus Environment

Environmental Science & Technology

16

Abstract

17

Microbial degradation following oil spills results in metabolites from the original oil.

18

Metabolites are expected to display lower bioaccumulation potential and acute toxicity to

19

marine organisms due to microbial-facilitated incorporation of chemical functional groups

20

and a general decrease in lipophilicity. The toxicity and characterization of metabolites are

21

poorly studied. The purpose of the present work was to evaluate the toxicity of degraded (0-

22

21 days) water-soluble oil components. Low-energy water accommodated fraction (LE-WAF)

23

of a weathered crude oil was prepared with nutrient amended seawater at 5°C, kept in dark,

24

and sampled at 0, 10, 14 and 21 days. Samples were extracted with dichloromethane and

25

toxicity experiments were conducted with reconstituted extracts. Toxicity experiments were

26

conducted for 4 days on developing cod (Gadus morhua) embryos during a critical period of

27

their heart development. After exposure, embryos were kept in clean seawater and observed

28

until 5 days post hatch. Survival, hatching, morphometric aberrations and cardiac function

29

was studied. The expected decrease in sub-lethal toxicity during the biodegradation period

30

was not found; indicating that metabolites formed during biodegradation likely contributed to

31

larvae toxicity.

32 33 34 35

2 ACS Paragon Plus Environment

Page 2 of 30

Page 3 of 30

Environmental Science & Technology

36

Table of Contents (TOC)/Abstract Art.

37

3 ACS Paragon Plus Environment

Environmental Science & Technology

38

Introduction

39

In the event of an oil spill at sea, oil will be affected by abiotic and biotic processes that

40

change the physical-chemical properties of the oil. These 'weathering' processes include

41

dispersion, dissolution, spreading, emulsification, UV-oxidation and microbial degradation.1

42

In cold marine environments, like the Arctic, biodegradation processes are expected to be

43

slower than in temperate environments.2,

44

seawater (SW) after oil spills is associated with oxidative processes. Aerobic n-alkane

45

degradation is associated with oxygenases and dehydrogenases, in which the alkane is

46

converted to alcohols and further to acetyl-coA4, while aromatic HCs are degraded primarily

47

by dioxygenases.5 The resulting metabolites are more water-soluble and thus are attributed

48

with lower octanol-water partitioning coefficient (Kow). Often, acute effect concentrations

49

(e.g LC50) of HCs are predicted using LogKow vs LogLC50-regressions6, resulting in

50

reduced acute toxicity estimates of metabolites compared to their original parent components.

51

The toxicity of oil to marine species depends on the characteristics of the oil, and for pelagic

52

organisms, dissolved oil components are expected to display the highest bioavailability and

53

subsequently have the highest contribution to toxicity7-10. Biodegradation likely reduces the

54

toxicity of oil spills over time due to the rate of biodegradation of dissolved oil components.

55

Oil spill models are often utilized to perform risk assessments and assessments of

56

environmental harm. The overall fate of the oil is predicted using empirical data of different

57

weathering processes, including biodegradation rates of known oil components or component

58

groups.11 Metabolites resulting from microbial degradation are rarely accounted for when

59

predicting damage to natural resources. Unfortunately, metabolized oil components are often

60

omitted from modelled assessments as knowledge pertaining to their fate and effects is

61

limited. Metabolites are generally not expected to contribute to overall toxicity since they

3

Biodegradation of oil hydrocarbons (HCs) in

4 ACS Paragon Plus Environment

Page 4 of 30

Page 5 of 30

Environmental Science & Technology

62

display a lower potential for bioaccumulation compared to their more lipophilic parent

63

compounds. This, however, lacks verification.

64

Relations between biodegradation and acute toxicity have been investigated in several studies,

65

but mainly in soil or groundwater 12-15 or using bacterial cultures16, 17. Only a few studies have

66

systematically investigated the relationships between biodegradation and ecotoxicity to

67

marine organisms18, 19 and in general, toxicity data are limited for Arctic species.20

68

In recent years, the sensitivity of early life stages (ELS) of fish to oil exposure has received

69

increased attention, particularly after the Deepwater Horizon incident in the Gulf of Mexico in

70

2010.21-23 It has been shown that exposure to low concentrations of weathered crude oils may

71

cause craniofacial and jaw deformations and cardiotoxicity manifested as pericardial oedema,

72

reduced heart rate (bradycardia), arrhythmia, contractility defects, reduced stroke volume and

73

reduced cardiac output.21,

74

larvae following acute spills.26 It has been argued that these effects are PAH-dependent27,

75

however, it should not be ruled out that these effects might be mediated by the presence of

76

unknown components like metabolites.

77

The purpose of the current work was to study how biodegradation impacted the toxicity of

78

WAF to ELS fish. We hypothesised that toxicity decreases as a function of WAF

79

biodegradation. We hope that these results will generate relevant information and empirical

80

data for improving risk and damage assessment processes following acute oil spills.

23-25

Such effects have even been observed in field-collected fish

81 82

Materials and Methods

83

Water supply and WAF biodegradation. Natural seawater (SW) was used as microbial

84

inoculum in the experiment. The SW was collected from a depth of 80 m (below thermocline)

85

in a non-polluted Norwegian fjord (Trondheimsfjord; 63°26'N, 10°23'E), supplied by a 5 ACS Paragon Plus Environment

Environmental Science & Technology

86

pipeline system from the source to our laboratories (salinity of 34 ‰). The SW was

87

acclimated to 5°C three days before start of the experiments, aerated with filter-sterilized air

88

(0.22 µm), and fortified with mineral nutrients.28 The oil used in this experiment was Troll oil;

89

a naphthenic oil that has been the subject of numerous laboratory experiments.7-9, 29, 30 Two 10

90

L low energy water accommodated fractions (LE-WAFs) of crude oil were generated in glass

91

bottles as described previously31 with an oil-to-water ratio of 1:100 using nutrient-enriched

92

seawater.32. Slow stirring was used to prevent the formation of oil droplets and stirring was

93

performed for 72 hours at 5°C. WAF preparations were collected from the glass port located

94

at the bottom of the bottle. The two WAFs (100% stock solutions) were pooled and

95

homogenized prior to sampling. Samples (800 ml) were acidified, liquid-liquid-extracted

96

using dichloromethane (DCM), and analysed for C10-C36-total extractable material (C10-C36-

97

TEM) using Gas Chromatography – Flame Ionization Detector (GC-FID). Semi-volatile

98

organic components (SVOC), including PAHs and phenols, were also analysed using gas

99

chromatography – mass spectrometry (GC-MS). The concentration of unknown components

100

in the GC-FID chromatograms, here regarded as 'unresolved complex mixtures' (UCM) in the

101

WAFs were estimated by subtracting the identified and quantified components (SVOC) from

102

the TEM. Samples (40 ml) was also taken for analyses of volatile organic components (VOC)

103

using Purge & Trap GC-MS using standard methodology.31 A complete list of target

104

compounds are given in Supplementary Information (Table S1 and Table S2). Water samples

105

were also analysed for oxygen concentrations using an oxygen meter (Model 59 with 5905

106

BOD probe, YSI Inc., OH, USA). An aliquot of the 100% WAF (800 ml) was separately

107

extracted with dichloromethane (DCM) for subsequent reconstitution for the toxicity tests

108

(day 0, T0). Finally, the remaining 100% WAF was distributed into separate 2 L glass bottles

109

filled to the rim and capped. These bottles were kept at 5°C until they were sampled at 10

110

(T10), 14 (T14) and 21 (T21) days. Each time point was analysed for oxygen content, TEM,

6 ACS Paragon Plus Environment

Page 6 of 30

Page 7 of 30

Environmental Science & Technology

111

SVOC and VOC as described above. In addition, an aliquot of these biodegraded WAF

112

samples was obtained at each time point and extracted using DCM for subsequent

113

reconstitution and toxicity testing (800 ml),.

114 115

Preparation of exposure solutions. When fish embryos were available, the biodegraded

116

WAF extracts (t=0, t=10, t=14 and t=21 days) were reconstituted into filtered seawater to

117

prepare the representative exposure solutions at each time point. Each DCM-extract, extracted

118

from biodegraded WAFs (800 ml), was added to the bottom of a glass bottle (1 L) and flushed

119

using nitrogen gas for 60 min to remove the DCM. Using filtered seawater (0.22 µm), the

120

dried extract was reconstituted to the same volume of seawater as it was initially extracted

121

(800 ml). Extracts were resolubilized into seawater using ultrasonication for 30 min. The

122

reconstituted WAFs from each time point (t=0, t=10, t=14 and t=21 days) were subsequently

123

diluted in filtered seawater to three nominal concentrations (10%, 50% and 100%

124

(undiluted)). 'Solvent controls' (DCM controls) were prepared following the same

125

reconstitution procedure as oiled-extracts, but contained clean DCM instead of extracts.

126

Negative controls contained filtered seawater only. All prepared exposure solutions were

127

cooled to 5°C and aeriated with filtered ambient air for 30 min to increase oxygen saturation.

128

Aliquots of all reconstituted and diluted samples were extracted and analysed for TEM and

129

SVOC as described above for exposure verification. Exposure solutions were then transferred

130

into glass jars (80 ml) for the fish embryo exposure experiment.

131 132

Fish exposure. A complete time line of the exposure experiment is given in Supplemental

133

Information (Table S3). Fish embryos (day 0 post fertilization, 0 dpf) were obtained from the

134

Austevoll Research Station, Norway. Embryos were shipped directly to our laboratory in a

7 ACS Paragon Plus Environment

Environmental Science & Technology

135

cooling container using airfreight. Embryos arrived less than 12 hours after fertilization and

136

were immediately transferred to flow-through tanks (250 L) containing filtered seawater (1

137

mm, 6°C). The flow-through rate ensured that the entire tank (250 L) of seawater was

138

exchanged every 24 hours. Gentle aeration kept embryos moving continuously in the tanks.

139

Dead embryos were removed from the tank daily.

140

When embryos reached 9 dpf, they were transferred into the glass jars (80 ml) containing

141

exposure solutions (472±63 embryos per jar). Embryos were exposed for 4 days until 13 dpf,

142

and surviving embryos were transferred into clean seawater and monitored for an additional 9

143

days (until 22 dpf). During the exposure (days 9-13 post fertilization), dead eggs were

144

counted in all jars daily. During the post-exposure monitoring period (days 14-22 post

145

fertilization), dead eggs were counted almost every day (days 16-18, 20 and 22 post

146

fertilization). At the same time points, hatching and larvae mortality was monitored. The

147

experiment was terminated at 5 days post hatch (dph).

148

Seawater control groups included 8 replicates (N=8), and all treatments contained 4 replicates

149

(N=4), with on average 472±63 embryos per replicate. Replicates were kept separate until

150

being pooled at hatch. In the morning at largest hatch, newly hatched larvae from replicates

151

were transferred into a new glass jar (80 ml) in order to provide a sufficient number of larvae

152

at the exact same age to be sampled at the different time points. Oxygen saturation in the

153

exposure containers were monitored throughout the experiment with a phase fluorometer

154

(NeoFox with FOXY R-sensor, OceanOptics Inc., FL, US).

155 156

Biological endpoints. The heart rates (HR) of individual embryos/larvae were monitored with

157

automated video analyses. Videos of embryos (17 dpf) and individual larvae (1, 3 and 5 dph)

158

were taken through a microscope (Eclipse 80i, Nikon Inc., Japan) equipped with a CMOS

8 ACS Paragon Plus Environment

Page 8 of 30

Page 9 of 30

Environmental Science & Technology

159

camera (MC170HD, Leica Microsystems, Germany). Microscopy images were also taken of

160

larvae at 1, 3 and 5 dph for biometric analyses using Image J33, 34 and blinded deformation

161

ranking analysis. Care was taken to make sure that the larvae from all groups were identical in

162

age, so newly hatched larvae were transferred into new glass jars in the morning (being 99%) after

201

incubation at 5°C for 21 days. The TEM fraction was also depleted, but to a lower extent

202

(25%), while the aromatic fraction of the extractable material (sum of decalines, naphthalenes, 11 ACS Paragon Plus Environment

Environmental Science & Technology

203

2- to 6-ring PAH and phenols) were depleted by 98% (Supplemental Information Table S4).

204

The TEM depletion was well in line with the oxygen consumption (24%), and both the

205

oxygen consumption and TEM depletion could therefore be used as measures of

206

mineralization. Since the experiment was performed in closed flasks without headspace,

207

avoiding evaporation, biodegradation is likely the cause of the depletion. This was further

208

substantiated by the temporary increase in the phenol concentrations, expected to be products

209

of BTEX and PAH biodegradation.18,

210

groups were mainly in agreement with recent results, comparing biodegradation of LE-WAF

211

and gas compounds in natural SW at 5°C 37.

212

Concentrations of the unresolved components from the GC-FID chromatograms (UCM

213

fraction) was also reduced by almost 25% at T21, but the fraction of UCM increased, which

214

includes undegraded unresolved components as well as metabolites (Fig. 2B). This is caused

215

by an almost complete removal of the identified SVOC components (Fig 2C and D),

216

particularly decalins (100% loss), naphthalenes (100% loss) and 2-3-ring PAHs (86.7% loss).

217

Concentrations for 4-6 ring PAHs were very close to levels of quantification in all initial

218

WAFs. As mentioned above, phenols increased at days 10 (38.5% increase) and day 14

219

(164.2%) compared to the initial WAF, and returned to below T0 levels at day 21. This

220

suggests that the biotransformation of cyclic organic oil components includes OH-substitution

221

to generate phenols, and that these are readily broken down by the end of the three-week

222

degradation period. The identified and quantified SVOC components represent a small

223

fraction (