Ecotoxicology and Environmental Safety 252 (2023) 114599

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Toxicokinetics and toxicodynamics of Ag nanomaterials (NM300K) in the

soil environment–impact on Enchytraeus crypticus (Oligochaeta)
Fátima C.F. Santos a, Rudo A. Verweij b, Cornelis A.M. van Gestel b, Mónica J.B. Amorim a, *
a
Department of Biology & CESAM, University of Aveiro, 3810-193 Aveiro, Portugal
b
Amsterdam Institute for Life and Environment (A-LIFE), Faculty of Science, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, the Netherlands

A R T I C L E I N F O A B S T R A C T

Editor: Dr. Caterina Faggio Silver (Ag) is one of the most used elements in the nanomaterials (NMs) form, which upon release to the
environment can be harmful to organisms. We compared the toxicokinetics (TK) and toxicodynamics (TD) of Ag
Keywords: from AgNO3 (0, 15, 45, 135, 405 mg Ag/kg soil) and AgNM300K (0, 75, 150, 300, 600, 1200 mg Ag/kg soil) in
Bioaccumulation the model organism Enchytraeus crypticus. Organisms were exposed in LUFA 2.2 soil, and besides body Ag
Silver forms
concentrations, survival and reproduction were determined, in a time series (for 21 days). In the soil, the
Toxicity
available (CaCl2 extractable) Ag fraction from Ag NM300K increased from 0 to 21 days but did not consistently
TKTD
Soil organisms change for AgNO3. Internal concentrations reached equilibrium in most exposures to both Ag forms. The or­
ganisms were able to internalize and eliminate Ag, but less when exposed to Ag NM300K. The overall uptake rate
constants for Ag from AgNO3 and Ag NM300K exposures were 0.05 and 0.06 kg soil/kg organism/day,
respectively, the elimination rate constants 0.2 and 0.1 day− 1, respectively. For AgNO3 the median lethal con­
centrations decreased steadily with time, while for Ag NM300K they remained constant during the first 10 days
of exposure followed by a 2-fold decline in the last 7 days. The 21-d LC50s for both Ag forms were similar but the
LC50inter (based on internal concentrations) were 63 and 121 mg Ag/kg body DW (Dry Weight) for AgNO3 and
Ag NM300K, respectively, showing higher toxicity of AgNO3. These results show the importance of assessing time
to toxicity, a relevant factor in toxicity assessment, especially for NMs.

1. Introduction McGillicuddy et al., 2017; Whitley et al., 2013). Ag is a non-essential

Because of its high antimicrobial activity, silver (Ag) has been used in
a variety of applications (Courtois et al., 2021; McGillicuddy et al., 2017;
Panyala et al., 2008). Ag nanomaterials (NMs) are most often incorpo­
rated in nano-functionalized consumer products (daily care products,
clothing, sports gear, food packaging), and materials used in medicine
and electronics. (Hadrup and Lam, 2014; McGillicuddy et al., 2017;
Panyala et al., 2008). The production of AgNMs amounts to around 1000
tons per year (McGillicuddy et al., 2017), and their use may lead to
increasing release to the environment (Tourinho et al., 2016; Vance
et al., 2015) during manufacturing, transport, use and disposal (Giese
et al., 2018). Soil is a major sink, with application of sewage sludge
being one of the main sources of entry, e.g. sludge treated soil showing
between 0.5 and 24.9 mg Ag/kg soil (Giese et al., 2018; Makama et al.,
2016; McGillicuddy et al., 2017; Tourinho et al., 2016). Hence, soil
organisms are exposed to increasing levels of Ag with potential adverse
effects (Chen et al., 2017; González-Fuenzalida et al., 2018;
trace metal and higher concentrations of AgNMs are known to cause
adverse effects on various soil organisms, e.g. survival and reproduction
in enchytraeids (Bicho et al., 2016), earthworms (Gomes et al., 2015;
Jesmer et al., 2017; van der Ploeg et al., 2014), springtails (Jesmer et al.,
2017), isopods (Tourinho et al., 2016), and mealworms (Khodaparast
et al., 2021).
A toxicokinetics-toxicodynamics (TKTD) experimental design is a
highly relevant approach to assess the effects of toxicants (Ashauer and
Escher, 2010; Jager et al., 2011), especially in soil media due to its high
complexity. While toxicokinetics aids translating the actual impact of
external soil concentration into the internal concentration in the or­
ganisms within a time frame, allowing information on uptake, elimi­
nation, and metabolism, toxicodynamics evaluates the results of those
internal concentrations in terms of phenotypic effects, e.g. reduced
survival or reproduction, while also describing the time-course to
damage (Ardestani and van Gestel, 2013; Gong et al., 2021; Roeben
et al., 2020; Topuz and van Gestel, 2015; Zhang and van Gestel, 2017).

* Corresponding author.
E-mail address: mjamorim@ua.pt (M.J.B. Amorim).

https://doi.org/10.1016/j.ecoenv.2023.114599
Received 12 December 2022; Received in revised form 26 January 2023; Accepted 31 January 2023
Available online 3 February 2023
0147-6513/© 2023 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
F.C.F. Santos et al.
Time has been highlighted as an important factor in toxicity evaluation
(He and van Gestel, 2013; Santos et al., 2021a; Zhang et al., 2019; Zhang
and van Gestel, 2017) but is rarely considered in traditional soil toxicity
assessments where organisms are typically exposed to a range of con­
centrations for a fixed time period (Santos et al., 2021b). In a TKTD
approach, the effects on one or more endpoints can be described in
relation to time and (dynamics of) exposure level. As the same TKTD
models can be used for different elements and species, this approach
may allow comparisons and derive patterns of toxicity that can be
helpful to the hazard and risk assessment of chemicals (Jager and
Ashauer, 2018).
Hazards depend not only on total soil concentrations of toxicants but
also on their bioavailability (Zhang and van Gestel, 2017). Tox­
icokinetics may provide an indication of the availability of elements in
exposure media as the best measure of metal bioavailability and pre­
diction of metal toxicity is the internal concentration, reflecting the
actual exposure in a specific environment (Ardestani et al., 2014b;
Ardestani and van Gestel, 2013; He and van Gestel, 2013). Besides the
biology of the test organism, important factors contributing to the in­
ternal concentration are the exposure time, concentration and the
toxicant itself (Ardestani and van Gestel, 2013; Gong et al., 2021;
Spurgeon and Hopkin, 1999; Zhang and van Gestel, 2017).
Although few metals and exposure conditions have been tested in
TKTD studies with different soil organisms, e.g. Enchytraeus crypticus
exposed to Ni, Ag and Pb in different media (He and van Gestel, 2013;
Topuz and van Gestel, 2015; Zhang et al., 2019; Zhang and van Gestel,
2017), Folsomia candida exposed to Cu and Cd (Ardestani et al., 2014a),
the TKTD of AgNO3 and Ag NM300K (nanomaterial) in soil has not been
investigated.
The aim of this study was to investigate the TKTD of Ag in
E. crypticus, comparing the effects of AgNO3 and Ag NM300K, a refer­
ence Ag NM (Klein et al., 2011). Specific aims were to assess: 1) tox­
icokinetics, Ag uptake at different Ag concentrations in the soil and at
different exposure times, 2) toxicodynamics, time to toxicity at different
Ag concentrations in soil, and 3) the link between toxicokinetics and
toxicodynamics. We hypothesized that 1) toxicokinetics will differ be­
tween salt and nano Ag exposure, so providing different uptake and
elimination rate constants, 2) time to toxicity will be different for the
two forms of tested Ag, showing distinct toxicodynamics, and 3) it will
be possible to relate toxicokinetics and toxicodynamics that should be
different for the different forms tested. Enchytraeids are widely
distributed soil invertebrates (Didden and Römbke, 2001), important for
the functioning of soil ecosystems (Jänsch et al., 2005) and E. crypticus is
a standard soil model (OECD 220 (OECD, 2016)).
2. Materials and methods
2.1. Test organisms
Enchytraeus crypticus (Enchytraeidae, Oligochaeta) cultures were
maintained in agar media composed of Bacti-Agar medium (Oxoid, Agar
No. 1) and a sterilized mixture of 4 different salt solutions at final
concentrations of 2 mM CaCl2•2 H2O, 1 mM MgSO4, 0.08 mM KCl, and
0.75 mM NaHCO3. The cultures were fed on ground autoclaved oats
twice a week and maintained at 19 ± 1 ◦ C with a 16:8-h light: dark
photoperiod.
2.2. Test soil
The natural standard LUFA 2.2 soil (Speyer, Germany) was dried at
80 ◦ C for 48 h before use (to remove any indigenous organisms) and had
the following main characteristics (provided by the supplier): pH (0.01
M CaCl2) of 5.6, 1.71% organic carbon, cation exchange capacity of 9.2
cmolc/kg, a maximum water holding capacity (WHCmax) of 44.8%, and
grain size distribution of 8.0% clay (<0.002 mm), 13.7 silt (0.002–0.05
mm) and 78.3% sand (0.05–2.0 mm).
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Ecotoxicology and Environmental Safety 252 (2023) 114599
2.3. Test substances and spiking procedures
Silver nitrate (AgNO3, purity =99.9%, Sigma-Aldrich, CAS:
7761–88–8) and the reference silver nanomaterial NM300K (Klein et al.,
2011) were used. Ag NM300K is part of the European Commission Joint
Research Centre (JRC) NM-series of materials and hence is fully char­
acterized (Klein et al., 2011). In short, Ag NM300K are spherical, con­
sisting of a colloidal dispersion with a nominal Ag content of 10.2%
w/w, dispersed in 4% w/w of polyoxyethylene glycerol trioleate and
polyoxyethylene 20 sorbitan monolaurate (Tween 20). Approximately
> 99% of the particles have a nominal size of 15 nm. Transmission
electron microscopy (TEM) showed a size (AV±SD) of 17 ± 8 nm and
smaller nanoparticles of ca. 5 nm were also present.
Nominal test concentrations were 0, 15, 45, 135 and 405 mg Ag/kg
soil DW (Dry Weight) for AgNO3 and 0, 75, 150, 300, 600 and 1200 mg
Ag/kg soil DW for Ag NM300K, selected based on the results of survival
and reproduction toxicity tests with E. crypticus exposed to soil spiked
with the same Ag materials (Bicho et al., 2016). The concentrations were
selected to include sublethal to lethal effect levels. For Ag NM300K the
dispersant was also tested alone and corresponding to the amount pre­
sent in the highest concentration of NM300K tested (1200 mg Ag/kg soil
DW).
Spiking was performed by mixing the aqueous solutions with the pre-
moistened soil. For AgNO3 an aqueous stock solution was prepared,
serially diluted, and added to soil batches, which were then mixed to
obtain a homogeneous distribution and split into replicates. For Ag
NM300K, spiking was done using the same procedure but replicate by
replicate. Soil moisture content was adjusted to 50% of the WHCmax.
Soils were aged for 14 days prior test start, under the same conditions as
used in the test (20 ± 1 ◦ C with a 16:8 h light:dark photoperiod).
2.4. Experimental procedure
The OECD bioaccumulation test guideline 317 (OECD, 2010) was
followed to assess the uptake at a range of concentrations of Ag in
E. crypticus. In short, each test vessel contained 20 g of moist soil (water
control, NM300K dispersant control, spiked Ag materials) and 15 mg of
autoclaved ground oats as food, to which 10 organisms of similar size
and well-developed clitellum were added. During the 21-day exposure,
food (15 mg) and water content were replenished weekly based on
weight loss. Three replicates were used per treatment, i.e., concentration
and sampling day. Tests ran at 20 ± 1 ◦ C and a 16:8 h light: dark
photoperiod. Organisms were sampled at days 0, 1, 2, 4, 7, 10, 14 & 21,
except day 1 for Ag NM300K, to assess survival and internal Ag con­
centration. At each sampling day, surviving animals were collected from
the soil, counted and transferred to ISO water (ISO, 2012) for 12 h to
void their guts from soil particles. After depuration and blotting dry on
filter paper, 5 organisms were introduced individually into cryotubes
and frozen at − 20 ◦ C until further analysis. The reproduction was also
assessed at day 21, i.e., after removing the adults, the juveniles were
fixated with 96% ethanol and stained with Bengal rose (1% in ethanol).
After 24 h, the suspension was sieved through decreasing pore size
meshes (1.6, 0.5 and 0.3 mm), which allowed the separation of most of
the soil from the organisms to facilitate counting. A stereo microscope
was used to count juveniles. At days 0 and 21, three replicates of 5 g of
soil were sampled for chemical analysis.
2.5. Chemical analysis
Previously frozen animals were freeze-dried for 24 h. After weighing
on a microbalance, each animal was individually digested with 300 µL of
a mixture of HNO3 (67 % Fisher Chemical OPTIMA Grade) and HClO4
(70%; Baker ULTREX II Ultra- Pure) (7:1 v/v) in a Techne Dri-Block
heater with a heating ramp ranging from 85◦ to 180◦ C. The residue
left, after acid evaporation, was dissolved in 300 µL of 1 M HCl and Ag
concentrations in the digests were measured by graphite furnace atomic
F.C.F. Santos et al.
absorption spectrometry (AAS; PinAAcle 900Z, Perkin Elmer, Germany).
The certified reference material DOLT 4 was included in the analysis for
quality control. Average ( ± SD, n = 4) Ag concentrations measured in
DOLT 4 were 76 ± 5% of the certified values. LOD (Limit Of Detection)
for Ag analysis in organism samples was 0.051 µg Ag/kg DW.
To determine total Ag concentrations, dried soil samples (130 mg)
were digested with 2 ml of a destruction mixture of HNO3 (65%, Sigma-
Aldrich) and HCl (37%, Sigma-Aldrich) (1:4 v/v), using Teflon con­
tainers closed tightly and heated at 140 ◦ C for 7 h. After cooling down,
the Teflon containers were opened, and 8 ml of deionized water was
added. Flame AAS (AAnalyst 100; Perkin Elmer; Germany) was used to
measure total metal concentrations. The certified reference material
LGC 6181 was included in the analysis. Average ( ± SD; n = 2) Ag
concentrations measured in the reference material were 82.9 ± 6.7% of
the certified values. LOD for Ag analysis in soil samples was 0.003 mg
Ag/kg soil DW.
To measure CaCl2-extractable Ag concentrations, 5 g fresh soil were
shaken with 25 ml 0.01 M CaCl2 at 200 rpm for 2 h. The vessels were left
overnight to allow sedimentation. The next day, pH was measured, and
the supernatant filtered over a 0.45 µm nylon syringe filter conditioned
with 0.1 M Cu (NO3)2, to prevent loss of Ag due to sorption to the filters.
Flame AAS (AAnalyst 100; Perkin Elmer; Germany) was used to measure
concentrations in the CaCl2 extracts.
2.6. Data analysis
Using measured concentrations, a one-compartment model was used
to describe the development of internal concentrations with time
(Ardestani et al., 2014b):
Ku
Ct = C0 + ∗ Cexp ∗ (1 − e− Ke∗t
) (1)
Ke
where Ct is the metal concentration in the enchytraeids after t days of
exposure (mg Ag/kg body DW), C0 the background concentration in the
enchytraeids (mg Ag/kg body DW), Ku the uptake rate constant (kg soil/
kg organism/day), Ke the elimination rate constant (day− 1), Cexp the
exposure concentration (mg Ag/kg soil DW), and t the exposure time
(days).
The uptake and elimination rate constants were calculated using
both the total and available (CaCl2-extractable) concentrations as the
exposure concentration (Cexp), assuming exposure concentrations
remained constant throughout the test period.
At each exposure time, median lethal concentration was calculated.
The relationship between survival and time was described by a logistic
survival model as described by He and van Gestel (2013):
µt
e−
S(t) = Cexp b
(2)
1+ (LC50(t))
where S(t) is the survival fraction at time t, Cexp the measured total
exposure concentration (mg/kg soil DW), LC50(t) the LC50 (Lethal
Concentration killing 50% of the organisms) value in mg Ag/kg soil DW
calculated for each exposure time t, µ the natural mortality rate per day,
and b the slope parameter. This function was fitted to all survival data
from all exposure times and exposure concentrations together.
Toxicity dependent on time was explained in terms of elimination
kinetics as described by He and van Gestel (2013):
LC50∞
LC50(t) = (3)
1 − e− ke2∗t
where LC50(t) is the LC50 in mg Ag/kg soil DW after t days of exposure,
LC50∞ the ultimate LC50 in mg Ag/kg soil DW, and Ke2 the elimination
rate constant for toxicity (day− 1). This equation assumes that survival is
affected when a (time-invariant) critical body concentration is
exceeded.
3
Ecotoxicology and Environmental Safety 252 (2023) 114599
A logistic dose-response equation was used to describe the relation­
ship between survival and internal concentration:
S0
S= C0
(4)
1 + (LC50 )b
inter
where S0 is the maximum survival fraction in the control, and Co the
internal concentration in the enchytraeids. By fitting this equation using
all survival data from all treatments and exposure times, an overall
LC50inter (based on internal concentrations) and a value for the slope
parameter b could be estimated.
The bioaccumulation factor (BAF) (kg soil/kg organism), the ratio
between the concentrations in the organisms (mg Ag/kg body DW) and
in the soil (mg Ag/kg soil DW) at the steady state, was calculated as:
Ku
BAF = (5)
Ke
The half-life for elimination of Ag from the enchytraeids was calcu­
lated as:
ln(2)
DT 50 = (6)
Ke
LC50s were estimated using the trimmed Spearman Karber method
(Hamilton et al., 1978, 1977). Effect Concentration (EC) calculations
were performed using the Toxicity Relationship Analysis Program (2
parameters) (TRAP v1.22) (Erickson, 2012). One-way analysis of vari­
ance (ANOVA) followed by Dunnett´s comparison post hoc test
(p ≤ 0.05) was used to assess if there were any significant differences
between control and Ag treatments in terms of number of surviving
organisms and their reproduction (SigmaPlot 14.0, 2017).
3. Results
3.1. Exposure characterisation
Soil pH was not affected by Ag treatment (Table 1). Measured Ag
concentrations were 71.1–102% and 58.9–87.0% of the nominal ones
for AgNO3 and Ag NM300K, respectively (Table 1). The CaCl2-extract­
able concentrations increased with increasing total soil concentrations
of AgNO3 (from 0.087 to 0.13 mg Ag/kg soil DW) but were similar
across all concentrations of Ag NM300K ranging between 0.068 and
0.14 mg Ag/kg soil DW (Table 1). Extractable concentrations did not
consistently increase or decrease over the 21-day exposure period for
AgNO3, but slightly and consistently increased with time for Ag
NM300K (Table 1). All calculations used measured concentrations;
CaCl2-extractable concentrations were averaged over the 21-day expo­
sure period.
3.2. Survival and reproduction
Survival in controls after 21 days was 93% for AgNO3, 100% for Ag
NM300K exposure and 100% for dispersant. The average juvenile
numbers ( ± SE; n = 3) were 549 ± 6, 535 ± 39 and 569 ± 6 for the
water control exposure to AgNO3, Ag NM300K and Ag NM300K control
dispersant, respectively. Since no differences were observed between
control and control dispersant, data was further analysed with the water
control.
Both Ag forms affected survival and reproduction of E. crypticus
within the tested concentration ranges (Fig. 1). For AgNO3, at 322 mg
Ag/kg soil DW survival and reproduction were about 10% of the control,
while at 116 mg Ag/kg soil DW almost no reduction was seen. For Ag
NM300K, a dose-related response occurred within the tested range, with
LC50 and EC50, based on measured concentrations, of 227 and 123 mg
Ag/kg soil DW, respectively. Effect concentrations (EC) are shown in
Table 2.
F.C.F. Santos et al. Ecotoxicology and Environmental Safety 252 (2023) 114599

Table 1 3.3. Toxicokinetics

Total (average of days 0 and 21) and 0.01 M CaCl2-extractable Ag concentra­
tions measured (days 0 and 21) of the toxicokinetics-toxicodynamics tests with Internal concentrations of Ag in E. crypticus increased with increasing
Enchytraeus crypticus in LUFA 2.2 soil spiked with AgNO3 and Ag NM300K. Also exposure concentration and time (Fig. 2A, B).
included are nominal concentrations and pH (0.01 M CaCl2) at days 0 and 21. [% For both AgNO3 and Ag NM300K the accumulation of Ag was close to
recovery]: recovery compared to nominal Ag concentrations. Values are
steady state after 21 days. For AgNO3 the highest internal concentration
expressed as average ± standard error (AV±SE). DL: Detection limit.
was 143 mg Ag/kg body DW (exposed to 322 mg Ag//kg soil DW) after
Nominal pH Total CaCl2 extractable 10 days. For Ag NM300K the highest internal concentration was 563 mg
(mg/kg soil (0.01 M (mg/kg soil (mg/kg soil DW)
Ag/kg body DW (exposed to 241 mg Ag/kg soil DW) after 21 days.
DW) CaCl2) DW) [% of total concentration]
[% recovery] Uptake and elimination rate constants are shown in Table 3. The animals
showed only small changes in weight over the 21-day exposure period,
Time (days) 0 21 0 21
AgNO3 with growth rate constants generally being negative (mass loss), ranging
0 5.7 5.1 < 0.003 (DL) < 0.003 (DL) 0.005 between − 0.054 and 0.0008 day− 1 (exponential model), and so no
15 5.8 5.4 15 ± 4 0.0017 0.013 ± 0.011 correction for growth rate was included in the toxicokinetic modelling.
[102%] ± 0.0044 [0.07%]
[0.02%]
3.4. Toxicodynamics – survival throughout time
45 5.8 5.6 32 ± 4.6 0.047 ± 0.0044 0.0033
[71.1%] [0.14%] ± 0.083
[0.01%] Impact on survival of E. crypticus by AgNO3 and Ag NM300K
135 5.9 5.6 116 ± 3.3 0.087 ± 0.015 0.13 ± 0.0029 increased with both exposure concentration and time (Fig. 2 C, D).
[85.5%] [0.07%] [0.003%]
Mortality rate µ and slope b derived from the logistic model (Eq. 2) were
405 5.8 5.6 322 ± 11.8 0.10 ± 0.019 0.098 ± 0.011
[79.4%] [0.03%] [0.03%]
0.003 per day and 4.4 for AgNO3 and 0.002 per day and 1.96 for Ag
Ag NM300K NM300K, respectively.
0 5.5 5.3 < 0.003 < 0.003 < 0.003 Table 4 shows the LC50s for the effect on the survival of E. crypticus at
Dispersant 5.6 5.5 0.2 ± 0.06 0.0083 0.0033 different time points based on measured total and available soil con­
± 0.0083 ± 0.0017
centrations, and internal concentrations. For AgNO3 a steady state was
75 5.7 5.6 63 ± 2.5 0.087 ± 0.0044 0.10 ± 0.014
[84.4%] [0.15%] [0.15%] almost reached, and by plotting LC50 versus time (Eq. 3) a final LC50 of
150 5.6 5.6 130 ± 5.2 0.10 ± 0.015 0.10 ± 0.011 201 mg Ag/kg soil DW could be estimated. For Ag NM300K, based on
[86.3%] [0.07%] [0.08%] total Ag concentrations in the soil, toxicity was similar in the first 10
300 5.6 5.6 241 ± 8.8 0.097 ± 0.021 0.14 ± 0.019
days, after which it increased by a factor of 2. Due to this pattern, for Ag
[80.5%] [0.04%] [0.06%]
600 5.6 5.6 522 ± 17.5 0.073 ± 0.012 0.12 ± 0.0076
NM300K no reliable final LC50 could be estimated using, but the
[87%] [0.01%] [0.02%] observed final LC50 values after 21 days were similar for both Ag forms
1200 5.6 5.6 707 ± 69.9 0.068 ± 0.012 0.14 ± 0.016 (Table 4).
[58.9%] [0.01%] [0.02%]

Fig. 1. Survival and reproduction of Enchytraeus crypticus after 21 days exposure in LUFA 2.2 soil to (A) AgNO3 and (B) Ag NM300K. Results are expressed as average
± standard error (AV±SE) and plotted against measured Ag concentrations in the test soil. Lines represent the model fit to data. *p < 0.05 (Dunnett´s).

Table 2
Summary of the effect concentrations (ECx) for Enchytraeus crypticus exposed for 21 days to AgNO3 and Ag NM300K in LUFA 2.2 soil. Results show ECx estimates and
corresponding 95% confidence intervals (in brackets) based on measured concentrations expressed in mg Ag/kg soil DW. Log 2 Par: logistic 2 parameters; S: slope; Y0:
intercept.
Endpoint EC10 EC20 EC50 EC90 Model & parameters

AgNO3
Survival 273 282 298 323 Log 2 par
(-) (-) (-) (-) S:7.3E-03; Y0:9.3
Reproduction 85 125 192 299 Log 2 par
(53–117) (97–152) (159–225) (237–360) S:5.3 E-03; Y0:571
Ag NM300K
Survival 41 110 227 412 Log 2 par
(− 68 to 151) (37–182) (166–288) (248–576) S:1.9E-03; Y0:10
Reproduction 46 74 123 201 Log 2 par
(6–85)) (48–101) (106–140) (155–247) S:2.4E-03; Y0:527

4
F.C.F. Santos et al. Ecotoxicology and Environmental Safety 252 (2023) 114599

Fig. 2. Toxicokinetics-Toxicodynamics results for Enchytraeus crypticus exposed for 21 days in LUFA 2.2 soil to AgNO3 and Ag NM300K at different concentrations
(total values are shown in mg Ag/kg soil DW). A and B (Toxicokinetics) show the relationship between internal concentration and exposure time for AgNO3 and
NM300K, respectively. Each dot represents an individual enchytraeid replicate; solid lines show the one-compartment model (Eq. 1) fit to data, based on measured
total soil concentrations. C and D (Toxicodynamics) show the survival fraction when exposed to AgNO3 and Ag NM300K, respectively. Each dot represents total
number of survivors; solid lines show the model (Eq. 2) fit to data. E and F show the survival fraction as a function of internal Ag concentrations for AgNO3 and Ag
NM300K exposures, respectively. Data points represent observed data; solid lines show the model (Eq. 4) fit to all data together.

3.5. Linking survival and reproduction to internal concentrations
E. crypticus mortality increased with increasing internal Ag concen­
trations (Fig. 2 E, F). Overall LC50inter ( ± SE) values were 63.2
( ± 0.007) and 121 ( ± 0.6) mg Ag/kg body DW for AgNO3 and Ag
NM300K, respectively, after fitting Eq. 4 to all data.
The 21-day reproduction EC10 and EC50 based on internal concen­
trations were 42.1 (35.1–49.1) and 64.9 (59.5–70.4) (Logistic Eq. 2
parameters; S: 1.94 E-02; Y0: 571) mg Ag/kg body DW for AgNO3 and
60.4 (49.7–71.1) and 68.7 (63.6–73.7) (Logistic Eq. 2 parameters; S:
1.26 E-01; Y0: 527) mg Ag/kg body DW for Ag NM300K, respectively.
4. Discussion
4.1. Exposure characterisation
For AgNO3, the available (CaCl2-extractable) Ag fraction increased
with increasing soil concentrations while for Ag NM300K it was similar
across the range of concentrations. Similarly, other studies reported such
a pattern of higher available concentrations in soils spiked with ionic
(salt) compared to nano Ag forms (Diez-Ortiz et al., 2015a, 2015b; van
der Ploeg et al., 2014). Diez-Ortiz et al. (2015a) also reported similar
available concentrations in soils spiked with increasing concentrations
of Ag NMs, with porewater Ag concentrations ranging between 0.2 and
0.3 mg Ag/L after 1 week incubation of soils spiked up to 4395 mg Ag
NMs/kg soil DW. In another study of Diez-Ortiz et al. (2015b), the Ag
available concentrations for soil spiked with AgNO3 (20 and 100 mg
Ag/kg) and Ag NPs (100 and 500 mg Ag/kg) were 0.003 and 0.26 mg
Ag/L, and 0.024 and 0.044 mg Ag/L, respectively. As shown, Ag (bio)
availability and toxicity in AgNO3 or AgNM spiked soils depends on
characteristics and properties of the nanomaterials and on the exposure
conditions that influence their transformations (aggregation, agglom­
eration, dissolution, surface modification, metal speciation and solid
phase binding of dissolved ions) (Diez-Ortiz et al., 2015a; Santos et al.,
2021a). For Ag NM300K it is likely that the higher the concentration the
higher the agglomeration of particles leading to less surface-active sites
and lower bioavailability, compared to lower concentrations. On the
other hand, the Ag became more available with time for Ag NM300K,
with CaCl2-extractable concentrations increasing 2-fold during the 21
days exposure, e.g., from 0.068 to 0.14 mg/kg soil DW. This was not the
case for AgNO3, where overall the available fraction fluctuated without
a consistent pattern of decrease or increase within the 21 days. This
highlights the importance of assessing the hazards of Ag NM300K in
longer term exposures studies by taking into account changes in its (bio)

5
F.C.F. Santos et al. Ecotoxicology and Environmental Safety 252 (2023) 114599

Table 3
Toxicokinetic model parameters estimated for the uptake and elimination of Ag in Enchytraeus crypticus exposed to a concentration range of AgNO3 and Ag NM300K in
LUFA 2.2 natural soil. Parameters were estimated for each exposure concentration and for all data together. Ku: uptake rate constant, Ke: elimination rate constant,
brackets [] show the 95% confidence intervals, Cexp: metal exposure concentration in spiked soil, BAF: bioaccumulation factor, DT50: time needed to eliminate 50%.
Values were calculated based on measured total and 0.01 M CaCl2-extractable concentrations in the soil.
Nominal concentration Base Cexp Ku Ke BAF DT50 (days)
(mg/kg soil DW) (mg/kg soil DW) (kg soil/kg organism/day) (day− 1)

AgNO3
15 Total 15.3 0.04 [0.02–0.06] 0.00001 [-] 4039 69315
CaCl2 0.01 82 [47.5–117] 0.00001 [-] 8239513 69315
45 Total 32.0 0.04 [0.02–0.07] 0.00001 [-] 4227 69315
CaCl2 0.03 54 [− 605–713] 0.00001 [-] 5410244 69315
135 Total 115 0.06 [0.03–0.09] 0.08 [0.01–0.2] 0.8 8.8
CaCl2 0.11 65.8 [36–95] 0.08 [0.01–0.2] 833 8.8
405 Total 322 0.05 [0.03–0.08] 0.2 [0.08–0.4] 0.2 2.9
CaCl2 0.10 103 [8.0–199] 0.2 [− 0.06–0.5] 477 3.2
Kinetics together Total 0.05 0.2 0.3 4.12
CaCl2 112 0.2 703 4.35
Ag NM300K
75 Total 63.3 0.3 [0.09–0.5] 0.41 [0.08–0.7] 0.8 1.7
CaCl2 0.09 208 [59.8–356] 0.41 [0.08–0.7] 507 1.7
150 Total 130 0.15 [− 0.06–0.4] 0.2 [− 0.2–0.6] 0.7 3.3
CaCl2 0.10 192 [65.6–318] 0.2 [0.03–0.4] 913 3.3
300 Total 241 0.06 [0.007–0.1] 0.02 [− 0.09–0.1] 2.9 33.8
CaCl2 0.12 122 [14–229] 0.02 [− 0.09–0.1] 5928 33.8
600 Total 522 0.03 [0.02–0.03] 1E− 07 [-] n.d. n.d.
CaCl2 0.09 140 [106–174] 1E− 07[-] n.d. n.d.
1200 Total 707 0.1 [− 0.1–0.4] 0.6 [− 0.7–1.9] 0.2 1.2
CaCl2 0.10 1048 [− 742–2838] 0.6 [− 0.5–1.7] 1785 1.2
Kinetics together Total - 0.06 0.1 0.4 5.1
CaCl2 - 134 0.1 966 5.0

aging time (Diez-Ortiz et al., 2015a; Santos et al., 2021a; van der Ploeg
Table 4
et al., 2014). The similar results for Ag NM300K of Santos et al. (2021a)
LC50 values for the effects on the survival of Enchytraeus crypticus when exposed
indicate that 3- and 14-days soil aging did not induce significant vari­
for 21 days to AgNO3 and Ag NM300K in LUFA 2.2 soil. LC50 are based on
ation in toxicity for this nanomaterial.
measured total and 0.01 M CaCl2-extractable soil concentrations, and internal
concentrations in the animals and estimated using the trimmed Spearman
Karber method (Hamilton et al., 1978, 1977). In between brackets, 95% confi­
4.3. Toxicokinetics
dence intervals are given. – no LC50 could be estimated. * data fitted to Eq. 4
( ± Standard Error).
The one-compartment model successfully described the uptake for
Time LC50 based on:
both Ag forms. Although significant variation was observed, there was a
(days)
Total measured Extractable Internal (LC50inter) tendency to steady state during the 21-day exposure period. Previous
concentration (mg concentration (mg concentration (mg Ag/ studies also reported variation in internal concentrations (Santos et al.,
Ag/kg soil DW) Ag/kg Soil DW) kg body DW)
2021a, 2021b; Zhang and van Gestel, 2017). This variation can be
AgNO3 related with the heterogeneous distribution of the metal in the soil and
4 > 321.5 > 0.1 > 36.2
the biological variation in the way organisms handle the element
7 234 (200–274) 0.103 (0.10–0.11) 52.2 (47.3–57.7)
10 216 (191–246) 0.104 (0.10–0.11) 71.9 (66.9–77.3) (Spurgeon et al., 2011). Another interesting fact was that during our
14 211 (189–235) 0.104 (0.10–0.11) 60.9 (60.5–61.2) experiment some organisms were observed to appear on top of the soil or
21 205 (191–220) 0.104 (0.10–0.11) 67.2 (66.6–67.9) in the food, seemingly avoiding the spiked soil, for both Ag forms.
All data* 63.2 ( ± 0.007) Although it is normal for enchytraeids to be observed in the food, this
Ag NM300K
2 526 (453–498) 0.103 (0.10–0.11) 42.3 (37.4–47.2)
may also have contributed to the observed variation.
4 506 (524–587) - 175 (128–231) The previous results from comparable conditions (material, con­
7 586 (503–669) - 172 (133–210) centration, time) (Santos et al., 2021a) confirmed similar uptake: for
10 538 (404–672) 0.103 (0.10–0.11) 266 (12.0–520) AgNO3, exposure to 45 mg Ag/kg soil DW (aged 14 days in LUFA 2.2
14 286 (199–338) - 94.0 (77.0–111)
soil) led to an uptake of around 20 mg Ag/kg body DW at day 14, while
21 226 (166–288) 0.103 (0.10–0.10) 267 (145–390)
All data* 121 ( ± 0.6) for Ag NM300K, exposure to 60 mg Ag/kg soil DW (aged 14 days in
LUFA 2.2 soil) gave an uptake of around 40 mg Ag/kg body DW at day
14.
availability. In the current study, the worms exposed to AgNO3 (15.3–322 mg Ag/
kg soil DW) showed the highest uptake rate constants at the highest
4.2. Survival and reproduction concentrations (both available and total). The same trend was observed
in other studies with soil organisms exposed to metal salts, e.g.,
Both Ag forms affected enchytraeid survival and reproduction at E. crypticus exposed to AgNO3 in inert sand medium (Topuz and van
similar levels. Results from Bicho et al. (2016) where E. crypticus was Gestel, 2015), and in Eisenia fetida exposed to a field soil historically
exposed to the same Ag materials showed similar results for Ag NM300K contaminated with chromated copper arsenate (CCA) mixture with total
but higher toxicity of AgNO3. The difference between these results can concentrations of Cu between 5.1 and 79 mg Cu/kg soil DW (Kilpi-Koski
be due to the fact that soil was aged for 3 days in Bicho et al. (2016) et al., 2019). Other studies reported increasing available concentrations
whereas we aged the soil for 14 days. It has been shown that aging time with increasing total concentrations, but the metal uptake rate constants
reduces availability of the Ag salt, leading to lower toxicity after longer did not increase in the same way (Diez-Ortiz et al., 2015b; Giska et al.,

6
F.C.F. Santos et al.
2014; Waalewijn-Kool et al., 2014). Hence, differences in the uptake
rate constants may arise due to different species and exposure
conditions.
For Ag NM300K, the worms showed the highest uptake rate con­
stants at the lowest/intermediate exposure levels of 63.3 and 130 mg
Ag/kg soil DW (Ku of 0.3 and 0.15 kg soil/kg organism/day, respec­
tively). A similar pattern has been reported, e.g. for Lumbricus rubellus
exposed for 4 weeks to NM300K (van der Ploeg et al., 2014), where
internal concentration was higher at 15.4 mg Ag/kg soil DW than at
154 mg Ag/kg soil DW. A study exposing E. fetida to Ag NPs reported
higher accumulation of Ag following exposure to 45 and 112 mg Ag/kg,
the lowest concentrations tested (Diez-Ortiz et al., 2015a). In terms of
the toxicity, results showed that when exposing E. crypticus to a range of
concentrations of Ag NM300K in a full life cycle test (Bicho et al., 2016),
the lowest tested concentration (20 mg Ag/kg) was as toxic as the
highest exposure concentration (170 mg Ag/kg). All these data show
evidence of agglomeration/aggregation of the NMs at the higher total
concentrations, hence lower bioavailability, lower uptake rate constants
and consequently lower toxicity.
Internalization of Ag can occur by the ingestion of soil and also
through dermal absorption of dissolved ions (Courtois et al., 2021;
Vijver et al., 2003). Nanomaterials can act as a continuous source of Ag
ions to the medium (Diez-Ortiz et al., 2015a; van der Ploeg et al., 2014)
and at lower concentrations there seems to be an optimum window for
higher dissolution and toxicity (Santos et al., 2021a). There is also a
possibility that nano-particulate Ag is being taken up by enchytraeids –
E. crypticus mouth size is ca. 100 µm (diameter in the opening)(West­
heide and Graefe, 1992) – especially at the lowest exposure concentra­
tions of Ag NM300K, where higher dispersion is expected.
Differences in the way Ag is dealt with by the enchytraeids could also
be observed through the elimination patterns found for the different
exposures: for AgNO3 the Ke increased with increasing concentration
(for 15.3 and 322 mg Ag/kg soil DW, Ke of 0.0001 and 0.2 day− 1,
respectively). For Ag NM300K, the Ke decreased with increasing con­
centration (between 63.3 and 522 mg Ag/kg soil DW), but was higher at
the highest concentration tested (707 mg Ag/kg soil DW,
Ke=0.6 day− 1). Lower Ag elimination has been observed for Ag NM300K
in a full toxicokinetic study, i.e. 14d uptake plus 14d elimination period,
compared to AgNO3 (Santos et al., 2021a). Our study only had an uptake
phase; without an elimination phase, which admittedly leads to less
certain Ke values, as can be seen from the rather wide 95% confidence
intervals in Table 3. We used values obtained in our previous study with
elimination phase (Santos et al., 2021a) as the starting point when
fitting the models to our data, which helped estimating the Ke values. We
therefore are confident our study shows the potentially larger impacts of
Ag NM300K compared to AgNO3.
4.4. Toxicodynamics
The LC50s based on total soil concentrations showed increased
toxicity with increased exposure time, for both AgNO3 (>322 mg/kg soil
DW at day 4 and 205 mg/kg soil DW at day 21) and Ag NM300K
(526 mg/kg soil DW at day 4 and 226 mg/kg soil DW at day 21). For Ag
NM300K, the LC50s based on total soil concentration remained fairly
constant during the first 10 days but decreased to about half after 14 and
21 days. It seems that the response to Ag NM300K is more variable than
to AgNO3. In any case, at day 21, the LC50s for both Ag forms were
similar, 205 and 226 mg Ag/kg soil DW for AgNO3 and NM300K,
respectively, suggesting similar toxicity. The LC50s based on CaCl2-
extractable concentrations after 21 days of exposure did not vary much
and were similar for both Ag forms. Topuz and van Gestel (2015) also
found that LC50s based on available concentrations did not differ be­
tween Ag forms (or soil type). In the work of Topuz and van Gestel
(2017), the LC50 based on total concentrations after 21 days of exposure
to soil spiked with AgNO3 was 111 mg Ag/kg soil DW, a value lower
than the one found in the present study. One likely explanation is the
7
Ecotoxicology and Environmental Safety 252 (2023) 114599
difference in aging period prior test start, which was only 1 day, while
we aged the soil for 14 days. This is in agreement with previous studies
(Diez-Ortiz et al., 2015a; Santos et al., 2021a; van der Ploeg et al., 2014),
where the bioavailability and toxicity of Ag salt decreased with aging.
The LC50inter was higher for Ag NM300K compared to AgNO3, i.e.,
toxicity occurred faster and required a lower internal Ag concentration
from AgNO3 to cause toxicity. This is related with the higher solubility
and availability of Ag+ from AgNO3.
However, the reproduction EC50 based on internal concentrations
was similar for AgNO3 (64.9 mg Ag/kg body DW) and Ag NM300K
(68.7 mg Ag/kg body DW). For AgNO3 this was in accordance with the
value found by Topuz and van Gestel (2015) (75.2 mg Ag/kg body DW).
4.5. Toxicokinetics and toxicodynamics
The estimated LC50inter of AgNO3 varied between 36.2 and 71.9 mg
Ag/kg body DW and the LC50inter based on all data was 63.2 mg Ag/kg
body DW. It is of interest to observe that the internal body concentration
in organisms exposed to AgNO3 reached a maximum concentration of
140 mg Ag/kg body DW (exposure to 322 mg Ag/kg soil DW, 10 days)
exceeding the LC50inter values. This suggests enchytraeids do not regu­
late Ag and can also accumulate high levels before dying. The enchy­
traeids were able to survive at concentrations in soil of 116 mg Ag/kg
soil DW, and their reproduction showed only minor signs of impact. The
uptake of Ag reached a maximum of 80 mg Ag/kg soil DW by day 7, after
which a decrease or regulation was observed.
For Ag NM300K, the LC50inter varied within the exposure period,
being 42.3 mg Ag/kg body DW at day 2 and 267 mg Ag/kg body DW at
day 21. The LC50inter based on all data was 121 mg Ag/kg body DW. The
body concentration of organisms exposed to Ag NM300K reached a
maximum concentration of 550 mg Ag/kg (exposure to 241 and 522 mg
Ag/kg soil DW, 21 days) exceeding by far the LC50inter values. This
explains the mortality observed at these concentrations and onwards.
Moreover, also reproduction was severely affected with an EC50 of
158 mg Ag/kg soil DW. Exposure to 130 mg Ag/kg soil DW of Ag
NM300K – close to EC50 reproduction – induced an uptake up to 300 mg
Ag/kg body DW at day 10, hence enchytraeids can accumulate high
levels of Ag NM300K before reproduction starts to decrease.
Topuz and van Gestel (2015) exposing E. crypticus to different forms
of Ag reported LC50inter values of 21.8 mg Ag/kg body DW for AgNO3
and Ag NP-PVP and 57.6 mg Ag/kg body DW for Ag NP-Cit. These lower
LC50inter must be related to the testing in an inert sand matrix, the
unfavourable conditions in this test medium making the animals
perhaps more sensitive. Because LC50inter values account for concen­
tration and time, they allow an in-depth interpretation of the results
when merged with toxicokinetics.
5. Conclusions
The toxicokinetics and toxicodynamics of AgNO3 and Ag NM300K in
the soil enchytraeid Enchytraeus crypticus were adequately described by
a one-compartment model. The uptake rates were comparable between
Ag forms, but Ag was less eliminated when E. crypticus was exposed to
Ag NM300K. Further, the available fraction of Ag NM300K increased
from 0 to 21 days, while for AgNO3 it did not consistently fluctuate. This
highlights the importance of assessing the hazards of Ag NM300K in
longer term exposures studies (>21 days). The LC50inter (based on in­
ternal Ag concentrations) was higher for the nano exposure than for the
salt exposure, showing a higher toxicity of AgNO3. Nevertheless, the
LC50s (day 21) based on total soil concentrations were similar for both
Ag forms. This study shows the importance of measuring effects
throughout time to fully understand toxicity especially for
nanomaterials.
F.C.F. Santos et al.
CRediT authorship contribution statement
Conceptualization, C.A.M.van.G. and M.J.B.A.; methodology, F.C.F.
S.; formal analysis, F.C.F.S., R.A.V., C.A.M.van.G., and M.J.B.A.; inves­
tigation, F.C.F.S.; resources, C.A.M.van.G. and M.J.B.A.; data curation,
C.A.M.van.G. and M.J.B.A.; writing - original draft, F.C.F.S., C.A.M.van.
G., and M.J.B.A.; writing - review & editing, F.C.F.S., R.A.V., C.A.M.van.
G., and M.J.B.A.; supervision, C.A.M.van.G. and M.J.B.A.; funding
acquisition, C.A.M.van.G. and M.J.B.A. All authors have read and agreed
to the published version of the manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgements
This study was supported by the European Commission Project
NanoInformaTIX (H2020-NMBP-14–2018, GA No. 814426), BIORIMA
(H2020-NMBP-2017, GA No. 760928), and NANORIGO (H2020-NMBP-
13–2018, GA No. 814530). Further support by FCT/MCTES through
national funds (PIDDAC), and the co-funding by the FEDER, within the
PT2020 Partnership Agreement and Compete 2020 via CESAM (UIDB/
50017/2020+ UIDP/50017/2020+ LA/P/0094/2020) and BEAUTY:
Big gEnome wide Applications for an ecotoxicology soil model – a
knowledge base to Unravel mechanisms (nanopesTY_cides)” (PTDC/
CTA-AMB/3970/2020). Thanks are due to FCT via a PhD grant to FCFS
(SFRH/BD/118294/2016) and the exceptional period (COVID/BD/
151720/2021).
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