SUPPLEMENTAL DATA
1
2
Section S1: Sampling sites description
3
Table S1: Description of sampling sites for sediment and water
Sites
Wivenhoe Dam
Expected levels
of contaminants
low
Oxley Creek
high
Brisbane River
high
Port of Brisbane
moderate
Expected types of contaminants
Sites description and land use
Very low levels, possibly some herbicides from
agricultural runoff [1]
PAHs from road runoff and stormwater,
pesticides, pharmaceuticals and personal care
products from wastewater treatment plant
effluent [2]
As for Oxley Creek
Drinking water source for Brisbane; grazing,
agriculture, rural and residential use
Rural residential, industry, sand extraction and
bank erosion
Petroleum hydrocarbons, low levels of
above-mentioned groups [3]
4
5
6
7
8
9
10
11
SI-1
Downstream of high density urban
development; high levels of boat traffic
Industrial; high levels of shipping activity and
subject to occasional oil spills but also strong
tidal influence with dilution by seawater
12
Section S2: Additional information on extraction methods for surface water with SPE and
13
sediment with ASE
14
In brief, surface water samples were extracted using 1g OASIS® HLB solid phase cartridges
15
(Waters, Australia) following filtration with a glass fiber filter (GF/AWhatman). The solid phase
16
cartridges were conditioned in a Visiprep vacuum manifold (Supelco, Australia) with 10 mL
17
methanol and 20 mL of 5 mM HCl in MilliQ water. A known volume of surface water (3 L)
18
sample was percolated under vacuum and the cartridges were dried for 2-3 h before eluting with
19
10 mL methanol and 10 mL hexane:acetone (1:1). All eluates were evaporated to approximately
20
1 mL under purified nitrogen gas and were solvent exchanged to methanol at a final volume of
21
1mL. The extracts were stored at -20C until testing.
22
The current ASE protocol followed the standard method by US EPA [4-6]. Three static cycles,
23
each lasting 5min, were run at 100 °C and 1500 psi using hexane and acetone as solvent (1:1,
24
V/V). Flush volume and purge time was 60% and 90 s, respectively. This method allows good
25
extraction efficiency (80-120%) for chlorinated pesticides, semi-volatile organics and PCBs.
26
The typical recovery rate for PCDD/Fs using this protocol is ~60% (experience in our Centre),
27
which falls within the acceptable range of 60-120%. It would be better to use toluene to extract
28
PCDD/Fs for target chemical analysis but toluene extracts were not amenable to bioanalytical
29
screening of the chemical mixture as a whole.
30
31
32
33
SI-2
34
Section S3: Total organic carbon content (TOC), dissolved organic carbon (DOC) and
35
water content measurement for sediment
36
Organic compounds were oxidized to carbon dioxide at 680 C with pure oxygen and a
37
platinum catalyst (Analytik Jena Multi N/C 2100S). The formed carbon dioxide was measured
38
with a near infrared detector (NIRD). Total Organic Carbon (TOC) in sediment samples was
39
calculated by measuring total carbon (TC) and total inorganic carbon (TIC), i.e., TOC = TC –
40
TIC. Dissolved Organic Carbon (DOC) in surface samples corresponds to the TOC of filtered
41
samples with 0.45 µm filter. In the following, when we refer to OC, we used the TOC measure.
42
Water content of sediment samples (H2O%) was determined based on the weight loss of the
43
samples before and after freeze-drying.
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
SI-3
59
Section S4: Sediment weight calculation for non-depletive passive sampling system
60
Polydimethylsiloxane (PDMS) disk were used as passive samplers to assess the bioavailable
61
fraction in the sediment experiments. The volume and the weight of the PDMS disks were
62
15.07 µLand 17.6 mg, respectively. The minimum sediment mass was the most important factor
63
in the sediment matrix equilibrium passive sampling to satisfy the criteria of <5% depletion in
64
relation to the entire sediment. The extracted amount of chemicals from PDMS had to be kept
65
substantially below the total amount of chemicals in the entire sample matrix. Therefore, the
66
following condition must be fulfilled into:
CPDMS ×mPDMS
67
CPDMS ×mPDMS +Cfree ×Vwater +Cfree ×KOC−water ×fOC ×msed
≪1
(S1)
and
68
mPDMS
69
mPDMS +
K
+ OC−water ×mOC
KPDMS−water KPDMS−water
Vwater
≪1
70
(S2)
71
Vwater/KPDMS-waterand mPDMScould be neglected because the value is very small compared with
72
the last term in denominator. The above equation was further simplified:
KPDMS−water
73
KOC−water
×
mPDMS
mOC
≪1
74
(S3)
75
Partition coefficients of KPDMS-water and KOC-water are in the same order of magnitude, so if the 5%
76
depletion rate was taken, mPDMS/mOC should be less than 5%. The required sediment weight (as
77
dry weight (dw)) is listed in the Table SI-2. We used more sediment than the minimum amount
78
needed according to these theoretical considerations (see Table SI-2) in order to ensure the
79
non-depletive conditions for the system of sediment, DOC and pore water.
SI-4
80
81
82
Table S2: Sediment characteristics and the calculated weight for non-depletive system
Sampling sites
WivenhoeDam
Oxley Creek
Brisbane River
Port of Brisbane
OC (%)
1.58
7.74
12.86
7.44
H2O (%)
22.3
52.4
53.3
51.4
DOC in slurry (mg/L)
24
63
67
58
Required amount of
22.28
4.55
2.74
4.73
31.08
7.14
7.01
7.78
sediment (gdw)
Sediment in experiment(gdw)
83
84
85
86
87
SI-5
88
Section S5: Additional information on the derivation of KOC-PDMS
89
Bioanalytical equivalent concentrations (BEQ) are given in concentration units and they could
90
theoretically be converted from water to sediment and other phases if the partition coefficients
91
between those phases were constant and independent of hydrophobicity. This is not the case for
92
sediment- or OC-water and PDMS-water partitioning as these partition coefficients are highly
93
dependent on the hydrophobicity of the compounds (Figure S1).
94
However, both correlations between logKPDMS-water and logKow (Jin et al. [7], Eqn. S4) and
95
logKOC-water and logKow (logKOC-water taken from reviews by Nguyen et al. [8] and Sabljicet al.
96
[9], Eqn. S5) have a slope close to 1 and were fixed to 1 in the regression equations S4 and S5.
97
logKPDMS-water = logKow-0.87
n=68, R2=0.93
(S4)
98
logKOC-water = logKow-0.55
n=64, R2=0.96
(S5)
99
The regression analyses were based on compounds across a wide hydrophobicity range (logKow
100
2-8; data for logKow<2 excluded due to a leveling-off effect and irrelevant for sediment
101
toxicants), including herbicides, pesticides, PAHs, PCBs, PBDEs and PCDDs. If multiple
102
values were available for a chemical, a geometric mean was taken.
103
The difference in intercept between the two parallel lines represents the theoretical value of
104
logKOC-PDMS (0.32, thus KOC-PDMS～2.0).
105
logKOC-PDMS= logKOC-water - logKPDMS-water = 0.32
SI-6
(S6)
106
107
Figure S1: logKPDMS-water(empty triangles) and logKOC-water(empty circles) plotted against hydrophobicity
108
expressed as logKow.
109
Thus the measured BEQPDMS can be converted to BEQs related to sediment-OC, BEQPDMS,OC,
110
by equation S7.
111
BEQPDMS,OC = K OC−PDMS × BEQPDMS
112
The units of BEQPDMS,OC and BEQPDM are related to OC content in sediment and PDMS weight,
113
respectively.
(S7)
114
SI-7
115
Section S6: Equilibrium time determination
116
Time series method: One PDMS disk was added to each of the 9 replicate amber glass jars
117
containing 15.6±0.2 g wet sediment. Every 24 h, the PDMS disk was collected from one jar for
118
AhR-CAFLUX measurement. The TCDDEQ increased over time as is shown in Figure S2 on
119
the example of Brisbane River sediment.
120
121
Figure S2: Time series method for equilibrium time confirmation with Brisbane River sediment.
122
A/V ratios method: The principle of this method was explained in detail in Reichenberg et al.
123
[10]. PDMS disks with the same diameter and different thickness were applied in the same
124
sediment sample, i.e. 0.075mm, 0.125mm and 0.25mm, to vary the A/V ratios of samplers. The
125
other procedure was similar to the time series method. The same TCDDEQ concentration in all
126
samplers or the proportionality between TCDDEQ and PDMS mass could confirm the
127
establishment of equilibrium partitioning between sediment and PDMS. Except for the
128
sediment samples from Wivenhoe Dam that required a longer period of 18 days to reach
129
equilibrium, the equilibrium was reached within 8 days for sediment from the other three sites.
SI-8
130
131
Figure S3: Method test of different A/V ratios for equilibrium time confirmation of all sediment samples
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
SI-9
158
Section S7: Additional information on concentration-effect curves and benchmark metrics
159
for different bioassays
160
Prior to dosing, the sample extracts were blown down to dryness under a gentle stream of
161
nitrogen, which ensures all the extraction solvents (methanol, hexane and acetone) were
162
evaporated. The extracts were reconstituted in MeOH (water extracts) or DMSO (sediment
163
extracts) and dosed into the bioassays. The final solvent concentrations were kept at 0.1%. We
164
monitored the response for solvent (DMSO or MeOH) controls and lab blanks, and they
165
showed
166
concentration-effect curves of sample extracts, solvent controls and lab blanks for each bioassay
167
in Figure S4.
168
Effect concentration (EC) can be derived from the above concentration-effect curve. For
169
specific modes of action (AhR and ER induction, photosynthesis inhibition) and general
170
cytotoxicity, the concentration-effect curves are typically sigmoidal with a maximum response
171
(which is often defined as 100% response). EC50 (the concentration that gives 50% of the
172
maximum response) is widely used as a typical benchmark. For adaptive stress responses, such
173
as Nrf2-mediated oxidative stress response (AREc32) or reactive toxicity (such as genotoxicity),
174
the induction can go as high as the concentration increases before cell cytotoxicity kicks in.
175
Therefore, there is no plateau in the typical concentration-effect curve for oxidative stress
176
response and therefore the induction ratio IR has to be used as effect-metric.
177
Concerning the detection limit of the bioassays, the amount sampled with the chosen method
178
was high enough to obtain concentration effect curves that were well above the effect threshold,
no
significant
response
in
each
bioassay.
SI-10
We
attached
representative
179
which is defined as the average of effect caused by the controls (cells only or solvent control)
180
plus three times the standard deviation of response of controls. This detection limit is typically
181
in the range of an IR of 1.5 or % effect of less than 10%, while samples generally showed 20%
182
of maximum effect in the Microtox assay, 30% in 2h PAM, 25 % for AhR CALUX, IR 3 for
183
AREc32 and 20% for E-CALUX, for detailed information see Figure SI-4. Given the low level
184
of effect the PDMS extract could also be enriched more if ever problems with detection limits
185
of the bioassays occurred. The method detection limit can be translated to the concentration of a
186
reference compound, which defines the lowest detectable mass of chemical burden in a given
187
bioassay (e.g., 0.06 pg TCDD-EQ in a 100 µL well of the CAFLUX assay).
188
These assays were all conducted in plastic 96-well plates. Regarding the potential loss of
189
chemicals associated with the plastic plate, sorption to plastic is negligible as the cell culture
190
medium contains lipids and proteins, which have the sufficiently higher sorption capacity for
191
organic contaminants to retain them in the medium [11, 12].
192
193
SI-11
194
195
Figure S4: Representative concentration-effect curves of sample extracts, solvent controls and lab blanks with different bioassays.
SI-12
196
Section S8: Additional information on reference compounds for different bioassays
197
We used phenol as a positive control for the Microtox assay but did not derive baseline-TEQ
198
against it. As for the mode of action as membrane disruption (general cytotoxicity), all
199
chemicals can act on it in a chemical hydrophobicity-dependent way. Unlike chemicals
200
targeting specific receptor-mediated effect (e.g., dioxin-like or estrogenic chemicals), a wide
201
range of compounds contributing to general cytotoxicity bear diverse structures and
202
physicochemical properties, it is therefore not appropriate to use one single chemical to
203
represent the overall chemical burden. We thus used QSAR (quantitative structure-activity
204
relationship) to derive the EC50 of a “virtual” baseline toxicant with a molecular weight of 300
205
g/mol and octanol–water partition coefficient logKow of 3 to derive baseline TEQ [13].
206
The reference compound for the phytotoxicity test 2h-IPAM was diuron and the BEQ refers to
207
diuron equivalent concentration (DEQ).Analogously we chose estradiol for the E-CALUX,
208
2,3,7,8-tetrachloro-dibenzo-dioxin (TCDD) for the AhR-CAFLUX.
209
In the AREc32 assay, t-BHQ was previously used in our work for water quality assessment [14].
210
However, this compound is of low hydrophobicity (logKow=2.57) and therefore not
211
representative of micropollutants present in sediment, which are usually much more hydrophobic.
212
Instead, p,p’-DDT (logKow=6.79) was chosen as the reference chemical for AREc32 as it is
213
active (dose-response curve in Figure S5) and could be present in sediment samples.
214
The BEQ is a concept of mixture effect burden, representing the effects that would be caused by
215
the corresponding equivalent concentration of the reference compound for each endpoint; it
216
does not imply that the reference compound is the sole causative agent or even present.
SI-13
217
218
219
220
221
222
223
Figure S5: Combined concentration-effect curve of p,p’-DDT from four independent replicates.
(A) Full concentration-response curves for cell viability (filled diamonds) and induction (empty
triangle) and (B) linear range of the concentration-effect curve at low effect levels and derivation
of the effect concentration that induces an induction ratio (IR) of 1.5, ECIR1.5.
SI-14
Section S9: Summary of the bioanalytical results expressed as mean and SD of EC50 or ECIR1.5
Table S3: Mean and SD of EC50 or ECIR1.5 of different bioassays in REF.
Sites
Microtox
IPAM 2h
AhR-CAFLUX
E-CALUX
EC50
EC50
EC50
EC50
Mean
SD
Mean
SD
Mean
SD
Mean
SD
SPE
Wivenhoe Dam
42
4.9
69
4.4
696
14
24
5.31
Oxley Creek
46
1.0
29
0.81
213
39
12
2.81
Brisbane River
50
1.9
22
0.33
295
19
11
3.94
Port of Brisbane
98
7.8
54
0.06
347
30
6.5
2.95
ASE
Wivenhoe Dam
0.17
0.008
0.61
0.079
0.85
0.13
>4.7
Oxley Creek
0.14
0.002
0.19
0.055
0.012
0.001
>4.7
Brisbane River
0.066
0.013
0.28
0.036
0.003
0.001
>4.7
Port of Brisbane
0.012
0.001
0.17
0.004
0.038
0.014
>4.7
PDMSa Wivenhoe Dam
254
18
119
45
109
6.7
>56
Oxley Creek
161
24
24
3.2
5.7
0.34
>56
Brisbane River
85
0.34
23
3.1
3.0
0.09
>56
Port of Brisbane
6.4
0.74
26
1.3
3.5
0.28
>56
b
PDMS Wivenhoe Dam
0.14
0.023
0.068
0.021
0.062
0.003
>0.25
Oxley Creek
0.39
0.008
0.060
0.009
0.014
0.001
>0.25
Brisbane River
0.21
0.003
0.059
0.007
0.008
0.001
>0.25
Port of Brisbane
0.015
0.001
0.060
0.003
0.008
0.001
>0.25
Tenax Wivenhoe Dam
0.15
0.008
2.4
0.51
1.2
0.39
>4.4
Oxley Creek
0.42
0.108
0.59
0.092
0.17
0.030
>4.4
Brisbane River
0.097
0.011
0.58
0.16
0.28
0.018
>4.4
Port of Brisbane
0.010
0.002
0.61
0.12
0.52
0.039
>4.4
a
b
Units: REFw(L/L) for SPE,REFsed(kgdw/L) for ASE and Tenax, and REFPDMS (kgdw/L and kgPDMS/L ) for PDMS.
SI-15
AREc32
ECIR1.5
Mean
14
12
15
18
0.15
0.044
0.021
0.060
12
1.4
1.5
1.1
0.0072
0.0036
0.0040
0.0026
0.25
0.10
0.10
0.13
SD
0.10
0.51
0.13
2.4
0.006
0.005
0.002
0.009
1.1
0.03
0.25
0.02
0.0006
0.0001
0.0006
0.0001
0.039
0.026
0.032
0.003
Section S10: Non-depletion confirmation of PDMS extraction
Figure S6: TCDD-EQPDMS/TCDD-EQASE ratio variation with time series using the Brisbane River
sediment.
Figure S7: BEQPDMS/BEQASE ratios of different in vitro bioassays for sediment samples.
SI-16
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SI-17