materials
Article
Testing of Eluates from Waterproof Building Materials for
Potential Environmental Effects Due to the Behavior of
Enchytraeus albidus
Marya Anne von Wolff 1,2, Stephan Pflugmacher 2and Dietmar Stephan 1,*
Citation: von Wolff, M.A.;
Pflugmacher, S.; Stephan, D. Testing of
Eluates from Waterproof Building
Materials for Potential Environmental
Effects Due to the Behavior of
Enchytraeus albidus.Materials 2021,14,
294. https://doi.org/10.3390/
ma14020294
Received: 16 November 2020
Accepted: 6 January 2021
Published: 8 January 2021
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4.0/).
1Group of Building Materials and Construction Chemistry, Department of Civil Engineering,
Technische Universität Berlin, Gustav-Meyer-Allee 25, 13B, 13555 Berlin, Germany; vonwolf[email protected]
2Joint Laboratory of Applied Ecotoxicology, Environmental Safety Group, Korea Institute of Science and
Technology Europe (KIST Europe), Stuhlsatzenhausweg 97, 66123 Saarbrücken, Germany;
*Correspondence: [email protected]; Tel.: +49-30-314-72101
Abstract:
In order to determine the potential environmental impact of construction products, it
is necessary to evaluate their influence on organisms exposed to them or their eluates under en-
vironmental conditions. The behavior of the white worm Enchytraeus albidus is a useful tool for
assessing the potential environmental impact of construction products in contact with water and
soil. This study investigates the environmental effects of eluates from two construction products, a
reactive waterproofing product, and an injection resin, on the reproduction and avoidance behavior
of E. albidus. The eluates were prepared according to existing guidelines. The soil used for the tests
was moistened with the eluates of the construction products. The reproduction results of the worms
were collected after six weeks of exposure. Offsprings were counted under the microscope and
statistically analyzed. Results from the avoidance behavior were collected after 48 h of exposure, and
results were compared with the reproduction results. The eluates from both construction products
induced significant changes in the reproduction behavior of E. albidus. Undiluted or only slightly
diluted eluates of the injection resin drastically reduced the reproduction of the worms, whereas
the leaches of the reactive waterproofing product only had a minor effect. The avoidance results for
the injection resin indicates that its presence in the habitat is clearly detrimental to the survival of
E. albidus, while the avoidance results for the waterproofing resin showed an initial avoidance of the
eluates, but no harmful effects were observed. The avoidance test is a way of rapid toxicity screening
of environmental samples when time is a critical parameter to measure possible environmental effects.
This study shows that ecotoxicological tests using Enchytraeids are a valuable and important tool for
understanding the mode of action of eluates from construction products in the environment.
Keywords: Enchytraeids; waterproof building materials; ecotoxicology; biotest
1. Introduction
The environmental impacts of existing construction materials are important to consider
while developing new products. Once the construction products are exposed to weathering,
they could potentially be leached, and the resulting eluates could have a negative impact
on organisms in the environment [
1
]. The chemical composition of construction materials
and their leaching behavior are crucial for the environmental compatibility of a product [
2
].
In addition to this prospect, other stages of the building material also play a role in its
environmental compatibility. Even during the construction phase, dismantling, recycling,
and disposal [
3
,
4
], water-soluble substances can be released, especially from fine-grained
materials, which have an impact on the environment [
5
]. As all building materials have
a limited life span and weather over time, the assessment of leaching behavior and the
evaluation of the environmental relevance of the resulting eluates are of great importance
for the certification of building materials in the European market [6].
Materials 2021,14, 294. https://doi.org/10.3390/ma14020294 https://www.mdpi.com/journal/materials
Materials 2021,14, 294 2 of 14
Dynamic surface leaching test (DSLT), according to CEN/TS 16637-2:2016 [
7
], is widely
applied in Europe to determine the leaching behavior of construction materials and is
recommended by the German Environmental Agency (Umweltbundesamt, UBA- Berlin,
Germany) for their environmental impact assessment [
8
]. The DSLT is one of the standard
methods to evaluate the release of dangerous substances from building products. The test
determines the release of inorganic and non-volatile organic substances through contact
with leaching agents per unit area of the construction product under investigation as a
function of time. During the leaching process, 8 eluates are generated under specified
test conditions, which are then examined for chemical and ecotoxicological parameters.
The end of the standard experiment is the 64th day of the experiment, and the release of
substances related to the specific surface is determined.
Several studies have investigated the release behavior of inorganic ions such as Na
+
,
Al
3+
, Ca
2+
, Si
4+
, and Cu
2+
and organic substances (e.g., superplasticizers) from concrete
materials and construction waste [
8
–
11
]. However, only a few are taking the results of an
ecotoxicological analysis into account [12–14].
Currently, the most used toxicological bioassays for testing products are made using
species as Daphnia sp., Algae, and Danio rerio. Although those species are very representa-
tive for the aquatic environments, the scenarios of construction products are often in the
terrestrial environment, turning those species not always the ideal match for the necessary
bioassays. Ecological risk assessment from construction materials can be examined by
aquatic and terrestrial biomarkers [
15
], among others, the ecotoxicological tests using the
Oligochaeta species of genus Enchytraeidae. Although ecotoxicological tests using these
species have been developed and standardized only within the last two decades, it be-
came an important indicator organism for the determination of impacts on soil ecosystem
due to their sensitivity to a broad spectrum of xenobiotics, ease of maintenance in the
laboratory [15], and widely representation worldwide [16–18].
Between the possible ecotoxicological tests available, two are specifically used: the
reproduction and the avoidance behaviors. The reproduction test using Oligochaetas is
very commonly used, but it takes much more work and is very time-consuming since the
complete ecotoxicological test takes up to 6 weeks of work and constant measurements.
The avoidance behavior test is much simpler compared to the reproduction test and is also
less time-consuming since an avoidance test takes only 48 h to get results. As shown in
previous studies, avoidance behavior can be used as a first indication of the occurrence of
damage in Enchytraeids when exposed to nanoparticles [
19
], giving a very fast result when
compared to the traditional studies using reproduction tests.
However, although faster results can be obtained using test methods as avoidance
behavior, the reproduction tests with Enchytraeids seem to be the most accurate in looking
at the long term exposure scenario [
20
] and is likewise the best way to associate with real
field exposures of chemicals from anthropogenic activities [21].
Tests performed in this study intended to fill the information gap about exposures of
Oligochaetes in the presence of eluates from construction product particles. This study
aims to verify the possible effects of waterproof building materials in the environment
using the reproduction and avoidance behavior as ecotoxicological tools [
22
] to elucidate
the effects of leaches from construction materials prepared with different types of water for
the environment.
2. Materials and Methods
2.1. Construction Products
This research selected two examples of construction products that are applied directly
in contact with soil or water.
The first construction product used was a waterproof material to protect walls from
water ingress, for example. The non-commercial material consisted of 2 components, mixed
by hand with a spatula in the mass proportion of 1:1 in a beaker. The resulting paste was
placed in silicone forms and cured for 46 days until the product was completely dry.
Materials 2021,14, 294 3 of 14
The second construction product is a very fast-reacting silicate injection resin. The
injection resin also consists of 2 components, which have to be mixed within a few seconds.
These were filled in two-chamber cartridges. A static mixer was screwed to the outlet of the
cartridges, and the cartridge was placed on a compressed air gun. The components react
immediately, and the mixture was extruded with the air-gun in silicone molds, where it
hardens within a few minutes. Although the material cures very quickly due to the reaction
heat, in this study, we waited for 7 days for complete stabilization of the product after the
reaction before submerging in the water baths for leaching. Due to a long time for leaching
and constantly washing the pieces on the baths, all the eluates were tested without further
dilution.
Since the two products cover very different areas in terms of their structural applica-
tion, the results are not intended to be compared either. At this point, it must be pointed
out again that special laboratory formulations with known ingredients were deliberately
worked on and not on ecotoxicologically tested and marketable products, so that effects
can be achieved in the comparative investigations with a high probability.
2.2. Leaching Method
Leaching of the two construction materials was performed according to CEN/TS
16637-2 [
7
]. The decision to prepare the eluates with two different types of water was
taken to search for the best conditions for survival and thus for the reproduction of
the Enchytraeids. The eluates were prepared using tap water and distilled water as the
medium for leaching the components from the construction products. The pieces of
the construction products previously prepared according to item 2.1 were submerged in
baths of distilled water or tap water. The dimensions of the container used for leaching
was
31 ×23 ×9 cm3(l ×w×h)
and has a capacity of 3.5 L. Eluates from both construc-
tion products were collected after exposure times of 6 h and 1, 2, 4, 9, 16, 36, and 64 days,
and vacuum filtered using a micropore filter of 0.45
µ
m. All eluates were characterized for
pH, electric conductivity, and the following inorganic components: Al
3+
; Ca
2+
; Cu
2+
; K
+
;
Na
+
; Si
4*
; NO
3−
; SO
42−
; Cl
−
. The leaching procedure was performed in triplicates, and
results are presented further with the mean values obtained for the parameters.
2.3. Test Organisms
This study used the species Enchytraeus albidus as the test organism [
22
]. Worms were
cultured for many years in the ecotoxicological laboratory of the Technische Universität
Berlin using a bio garden soil, kept at a controlled temperature of 10
◦
C and fed at libitum
with bio rolled oats, autoclaved and finely grounded. Organisms were cleaned from soil
particles and acclimatized before starting the test procedure.
2.4. Soil
Standard soil (LUFA 2.2) was used to perform the avoidance and reproduction
tests. The soil was commercially acquired from Landwirtschaftliche Untersuchungsund
Forschungsanstalt (LUFA) Speyer, Germany [
23
]. The characteristics of the soil are: soil
type: loamy sand; dry matter of the soil: 94.8 wt.%; water content 5.4 g water/100 g soil;
maximum water holding capacity (WHC): 44.8
±
2.9 wt.%; pH: 5.6
±
0.4; cation exchange
capacity: 9.2 cmol/kg ±1.4.
2.5. Experimental Design
2.5.1. Reproduction Test
Rounded glass vessels with 100 mL were used for all the reproduction tests. In each
vessel, 50 g of soil was placed inside and moistened using the eluate until the maximum
water holding capacity of 46 wt.%. As control sets, the same conditions were placed using
three different types of water: tap water, distilled water, and reconstituted freshwater. All
the control vessels were completely free of leaching from the construction products. For
each condition tested, four replicates were placed [21].
Materials 2021,14, 294 4 of 14
Ten adult Enchytraeids individuals with a well-developed clitellum were placed in
each vessel. All vessels were covered with a lid containing small holes in order to avoid
escaping the worms. The worms in each vessel were fed with 0.2 mg of bio rolled oats
per week, distributed equally. Vessels were weighed and kept at a controlled temperature
of 20
◦
C, and water content was replaced when evaporation occurred. The experimental
plan ran for six weeks. After the first four weeks of exposure, the adult Enchytraeids were
removed, and vessels were kept at the same conditions in order to wait for the hatching
of the cocoons. After additional two weeks, organisms were fixed and colored using the
extraction method of staining with Bengal red according to ISO 16387:2014 [
21
] and counted
under the microscope. The results obtained from the tests were compared to the control
results.
2.5.2. Avoidance Test
To perform the avoidance tests, once again, rounded glass vessels with 100 mL were
used. The tests were performed according to ISO 17512 [
19
]. The vessels were divided
into two sections using a removable wall. On one side, was placed 25 g of LUFA soil 2.2,
moistened until the maximum water holding capacity of the soil was reached using the
eluate of the construction product. On the opposite side of the vessel, LUFA soil moistened
with the same corresponding type of water was placed. That means, if the eluate from the
construction product was previously prepared with tap water, the soil on the control side
was also moistened with tap water, but without any previous contact with the construction
product. This was an attempt to prove the avoidance behavior by the possible components
leached and not just by the type of water.
After placing both soils on the vessel, the wall was removed, and 10 Enchytraieds per
vessel with a well-developed clitellum were introduced in the fine line that divides both
soils. The vessels were covered with a lid containing small holes to permit air exchange.
Four replicates per treatment were prepared, and vessels were left at a temperature of 20
◦
C
and period light control (16/8—light/dark) for 48 h without food.
After 48 h, a removable wall was again introduced in the division of the soils and
both sides were searched individually for the worms. Worms were counted, and results
were compared. Figure 1shows the schematic representation of the avoidance test, with all
steps.
Materials 2021, 14, x FOR PEER REVIEW 4 of 14
2.5. Experimental Design
2.5.1. Reproduction Test
Rounded glass vessels with 100 mL were used for all the reproduction tests. In each
vessel, 50 g of soil was placed inside and moistened using the eluate until the maximum
water holding capacity of 46 wt.%. As control sets, the same conditions were placed using
three different types of water: tap water, distilled water, and reconstituted freshwater. All
the control vessels were completely free of leaching from the construction products. For
each condition tested, four replicates were placed [21].
Ten adult Enchytraeids individuals with a well-developed clitellum were placed in
each vessel. All vessels were covered with a lid containing small holes in order to avoid
escaping the worms. The worms in each vessel were fed with 0.2 mg of bio rolled oats per
week, distributed equally. Vessels were weighed and kept at a controlled temperature of
20 °C, and water content was replaced when evaporation occurred. The experimental plan
ran for six weeks. After the first four weeks of exposure, the adult Enchytraeids were re-
moved, and vessels were kept at the same conditions in order to wait for the hatching of
the cocoons. After additional two weeks, organisms were fixed and colored using the ex-
traction method of staining with Bengal red according to ISO 16387:2014 [21] and counted
under the microscope. The results obtained from the tests were compared to the control
results.
2.5.2. Avoidance Test
To perform the avoidance tests, once again, rounded glass vessels with 100 mL were
used. The tests were performed according to ISO 17512
[
19
]
. The vessels were divided into
two sections using a removable wall. On one side, was placed 25 g of LUFA soil 2.2, mois-
tened until the maximum water holding capacity of the soil was reached using the eluate
of the construction product. On the opposite side of the vessel, LUFA soil moistened with
the same corresponding type of water was placed. That means, if the eluate from the con-
struction product was previously prepared with tap water, the soil on the control side was
also moistened with tap water, but without any previous contact with the construction
product. This was an attempt to prove the avoidance behavior by the possible components
leached and not just by the type of water.
After placing both soils on the vessel, the wall was removed, and 10 Enchytraieds per
vessel with a well-developed clitellum were introduced in the fine line that divides both
soils. The vessels were covered with a lid containing small holes to permit air exchange.
Four replicates per treatment were prepared, and vessels were left at a temperature of 20
°C and period light control (16/8—light/dark) for 48 h without food.
After 48 h, a removable wall was again introduced in the division of the soils and
both sides were searched individually for the worms. Worms were counted, and results
were compared. Figure 1 shows the schematic representation of the avoidance test, with
all steps.
Figure 1. Schematic representation of the experimental procedure of the Enchytraeid avoidance test: (1) inserting the mov-
able wall into the center of the test vessel; (2) introduction of the soils to be tested; (3) the movable wall is removed; (4)
placing the Enchytraeids in the middle of the soil; (5) covering the test vessel with a lid (perforated); (6) reintroduce the
wall to separate the floors and count the organisms present on each side.
Figure 1.
Schematic representation of the experimental procedure of the Enchytraeid avoidance test: (
1
) inserting the movable
wall into the center of the test vessel; (
2
) introduction of the soils to be tested; (
3
) the movable wall is removed; (
4
) placing
the Enchytraeids in the middle of the soil; (
5
) covering the test vessel with a lid (perforated); (
6
) reintroduce the wall to
separate the floors and count the organisms present on each side.
Negative control was also placed where both sides of the vessel contained the same
type of control to evidence the non-avoidance behavior when both sides contain the same
component.
3. Results and Discussion
3.1. Eluate Characteristics
The values measured for pH and electric conductivity of the eluates are represented in
Figure 2. The chart represents the mean values obtained from the triplicates made for each
day of measurement for the following eluates: Silicate resin product distilled water (DW)
Materials 2021,14, 294 5 of 14
and silicate resin product tap water (TW) and also for the waterproof product distilled
water (DW) and waterproof product tap water (TW).
Materials 2021, 14, x FOR PEER REVIEW 5 of 14
Negative control was also placed where both sides of the vessel contained the same
type of control to evidence the non-avoidance behavior when both sides contain the same
component.
3. Results and Discussion
3.1. Eluate Characteristics
The values measured for pH and electric conductivity of the eluates are represented
in Figure 2. The chart represents the mean values obtained from the triplicates made for
each day of measurement for the following eluates: Silicate resin product distilled water
(DW) and silicate resin product tap water (TW) and also for the waterproof product dis-
tilled water (DW) and waterproof product tap water (TW).
Figure 2. Graphic representation of pH and electric conductivity. Values are measured for all eluates of silicate and wa-
terproof products.
The variation of electric conductivity of the silicate injection resin for DW presented
a variation between 1.5 and 3.5 mS/cm. This variation can be explained due to the chemical
behavior of the silicate building material when exposed to constant contact with water
[24]. The range variation of the electric conductivity of eluates from the waterproof mate-
rial with TW and DW was very stable, reaching a maximum value of 1 mS/cm during the
total time of leaching.
The eluates with TW and DW of the silicate injection resin showed very similar be-
havior in the variation of the pH, starting with values of pH 10 and slowly stabilizing until
pH 8. The pH of the eluates from the waterproof material in TW and DW presented a
small variation of results along the 64 days of leaching alternating between pH 8 and 9.
Table 1 presents the mean values of inorganic content analyzed for the eluates of the
construction products and the blank samples. All samples were analyzed in triplicates by
inductively coupled plasma atomic emission spectroscopy ICP-AES method.
The highest variations in analogy with the correspondent blank sample occurred for
the silicate product TW, indicating that the constant baths of the pieces stimulated the
increase of components as Al3+; Ca2+, Na+, Si4+, and NO3−. The same occurred for the silicate
samples leached with DW; however, the increase was proportional to the initial presence
of inorganic components. For the waterproof product in TW and DW, inorganic compo-
nents as Ca2+ and Na+ were leached out.
Figure 2.
Graphic representation of pH and electric conductivity. Values are measured for all eluates of silicate and
waterproof products.
The variation of electric conductivity of the silicate injection resin for DW presented a
variation between 1.5 and 3.5 mS/cm. This variation can be explained due to the chemical
behavior of the silicate building material when exposed to constant contact with water [
24
].
The range variation of the electric conductivity of eluates from the waterproof material
with TW and DW was very stable, reaching a maximum value of 1 mS/cm during the total
time of leaching.
The eluates with TW and DW of the silicate injection resin showed very similar
behavior in the variation of the pH, starting with values of pH 10 and slowly stabilizing
until pH 8. The pH of the eluates from the waterproof material in TW and DW presented a
small variation of results along the 64 days of leaching alternating between pH 8 and 9.
Table 1presents the mean values of inorganic content analyzed for the eluates of the
construction products and the blank samples. All samples were analyzed in triplicates by
inductively coupled plasma atomic emission spectroscopy ICP-AES method.
Table 1. Inorganic characterization of the eluates: mean values for leachings and blanks in mg/L.
Parameter Silicate TW Silicate DW Waterproof TW Waterproof DW Blank TW Blank DW
Al3+ 0.34 0.14 0.5 0.1 0.01 0.001
Ca2+ 50.2 47.6 123.5 53.8 1.6 0.9
Cu2+ 0.6 0.5 0.9 0.1 0.1 0.1
K+11.0 10.1 32.7 7.9 1.1 0.2
Na+1193.6 771.8 232.5 58.6 181.1 9.1
Si4+ 53.8 48.6 8.2 6.8 6.2 0.2
NO3−11.3 9.3 6.4 1.2 5.7 0.3
SO42−167.5 111.4 136.5 14.6 102.4 3.4
Cl−66.8 57.8 64.8 53.7 49.8 2.2
Materials 2021,14, 294 6 of 14
The highest variations in analogy with the correspondent blank sample occurred for
the silicate product TW, indicating that the constant baths of the pieces stimulated the
increase of components as Al
3+
; Ca
2+
, Na
+
, Si
4+
, and NO
3−
. The same occurred for the
silicate samples leached with DW; however, the increase was proportional to the initial
presence of inorganic components. For the waterproof product in TW and DW, inorganic
components as Ca2+ and Na+were leached out.
The DSLT has already been used in many studies. Brameshuber et al. [
25
] applied
this to the mortar to investigate the release of organic constituents from concrete under
practice-relevant conditions, whereby the effects of organic substances in the eluates could
be classified as minor. However, contamination with sodium, sulfates, aluminum, and
some heavy metals could be proven while using contaminated concrete blocks. As part
of the selection of the processes suitable for the ecotoxicological assessment of building
materials [
13
], the DSLT was carried out on 37 representative building products that
contained mobilizable organic substances. The point of criticism of the DSLT was that
some substances could no longer be identified due to their biodegradability over the
duration of the DSLT of 64 d. This argument is also put forward by Bandow et al. [
1
]
because the conversion of organic substances cannot be excluded, especially under real
conditions. The transferability of the test results from the DSLT to real environmental and
practical conditions also appears problematic. However, in its principles for evaluating
the effects of construction products on soil and groundwater [
26
], the Deutsche Institut
für Bautechnik already states how the laboratory results from the horizontal leaching test
can be transferred to real conditions through model considerations. Scherer described the
DSLT as “the authoritative and recognized test procedure for evaluating the environmental
impact” [
27
]. Due to the frequent use of tests, test variants modified by the DIN standard
are already available. Märkl et al. [
12
] adapted the DSLT for plastic products and used
it to leach polyurethane resin during the curing phase. Within the research project, the
DSLT was used both for reactive sealing and for a silicate injection resin. As described in
DIN CEN/TS16637-2, the DSLT applies to evaluating the surface-dependent release for
monolithic, plate-like, and film-like products. The type of construction product determines
the implementation conditions and the dimensions of the test specimen. The waterproof
product was classified as a plate-like product, while the silicate injection resin was classified
as a monolithic product.
3.2. Reproduction Results
In order to determine the best control design for the reproduction behavior and the
subsequent comparison of the results with leachings of the construction products, this study
collected data of three different scenarios of control sets using tap water, distilled water,
and reconstituted freshwater as watering medium of the LUFA soil. The results presented
in Figure 3evidence the highest number of offsprings for the vessels moistened with
reconstituted freshwater, while a very similar result was achieved using tap water. Based
on these results, which were combined to simulate real construction scenario situations,
this study carried out the leaching of the construction components with tap water and
distilled water.
After the total exposure time of 6 weeks, the offsprings of E. albidus were counted,
and the results are shown in Figures 4and 5. The control represents the mean number
of Enchytraeids on the water reproduction test, where no eluates of construction products
were in contact with this group.
Materials 2021,14, 294 7 of 14
Materials 2021, 14, x FOR PEER REVIEW 7 of 14
Figure 3. Reproduction behavior of E. albidus comparing three different types of water.
After the total exposure time of 6 weeks, the offsprings of E. albidus were counted,
and the results are shown in Figures 4 and 5. The control represents the mean number of
Enchytraeids on the water reproduction test, where no eluates of construction products
were in contact with this group.
An analysis of Figure 4a shows a low decrease in Enchytraeids reproduction behavior.
Nevertheless, after 16 days of leaching, an apparent decrease in the number of offsprings
can be seen, with less than 15 worms counted. The same decrease was perceived for the
eluates of the waterproof material with DW after 16 days, (Figure 4b), however with a
total amount of 17 Enchytraeids. The most significant results are emphasized with an (*)
point.
(a) (b)
Figure 4. (a) Enchytraeid reproduction results for eluates from the waterproof material using tap water. (b) Enchytraeid
reproduction results for eluates from the waterproof material using distilled water. Significant results are marked with an
(*).
The reproduction tests were performed for all the eluates. When observing the repro-
duction results for both eluates, after 64 days, a stabilization of the reproduction behavior
was noticed at the end of the test, indicating a reduction of the toxicity and decrease in the
presence of active ingredients from the waterproof material when exposed to water for a
longer time [28]. The drop in the number of offsprings was not very alarming for eluates
of the waterproof material. However, effects on the Enchytraeid populations can be no-
ticed. An alarming situation would be configurated when the component tested affects
0
5
10
15
20
25
30
Control Fresh
water
Control Tap
water
Control D.
Water
Number of Offsprings
Reproduction E. albidus in different
controls
Figure 3. Reproduction behavior of E. albidus comparing three different types of water.
Materials 2021, 14, x FOR PEER REVIEW 7 of 14
Figure 3. Reproduction behavior of E. albidus comparing three different types of water.
After the total exposure time of 6 weeks, the offsprings of E. albidus were counted,
and the results are shown in Figures 4 and 5. The control represents the mean number of
Enchytraeids on the water reproduction test, where no eluates of construction products
were in contact with this group.
An analysis of Figure 4a shows a low decrease in Enchytraeids reproduction behavior.
Nevertheless, after 16 days of leaching, an apparent decrease in the number of offsprings
can be seen, with less than 15 worms counted. The same decrease was perceived for the
eluates of the waterproof material with DW after 16 days, (Figure 4b), however with a
total amount of 17 Enchytraeids. The most significant results are emphasized with an (*)
point.
(a) (b)
Figure 4. (a) Enchytraeid reproduction results for eluates from the waterproof material using tap water. (b) Enchytraeid
reproduction results for eluates from the waterproof material using distilled water. Significant results are marked with an
(*).
The reproduction tests were performed for all the eluates. When observing the repro-
duction results for both eluates, after 64 days, a stabilization of the reproduction behavior
was noticed at the end of the test, indicating a reduction of the toxicity and decrease in the
presence of active ingredients from the waterproof material when exposed to water for a
longer time [28]. The drop in the number of offsprings was not very alarming for eluates
of the waterproof material. However, effects on the Enchytraeid populations can be no-
ticed. An alarming situation would be configurated when the component tested affects
0
5
10
15
20
25
30
Control Fresh
water
Control Tap
water
Control D.
Water
Number of Offsprings
Reproduction E. albidus in different
controls
Figure 4.
(
a
)Enchytraeid reproduction results for eluates from the waterproof material using tap water. (
b
)Enchytraeid
reproduction results for eluates from the waterproof material using distilled water. Significant results are marked with an (*).
Materials 2021, 14, x FOR PEER REVIEW 8 of 14
the reproduction behavior in an exposed concentration that affects fifty percent of the
population. The different results over time represent typical leaching behavior. In the be-
ginning, there was a first wash-off followed by reduced diffusion-controlled release.
Both types of eluates (TW and DW) of the waterproof material contained a low vari-
ation in the quantities of ions when compared to the corresponding blank samples. Ions
of Na
+
and Ca
2+
increased with the leaching. However, it is already proven that sodium
and calcium have a small influence on toxicological effects for Enchytraeids [29], and the
presence of these minerals on the leachings can help to clarify the reduction of offsprings
in the results.
Analyzing the results of the eluates of the silicate resin are plotted in Figure 5a,b. A
high impact of the eluates from the silicate product on the reproduction behavior of the
Enchytraeids can be seen. In all phases of the leaching process, small numbers of offsprings
were counted. The silicate eluates prepared with distilled water presented the highest im-
pact on the population, bringing special attention to the reproduction test from day 9 sil-
icate DW, where a mean number close to 0 was achieved. For both reproduction tests of
silicate leachings from day 64, the Enchytraeids presented a tendency to recover the popu-
lation of worms, and a higher number of offsprings was counted; however, the number of
offsprings is still under fifty percent of the population when compared to the control
group. The most significant results are emphasized with an (*) point.
(a) (b)
Figure 5. (a) Enchytraeid reproduction results for the silicate leach using tap water. (b) Enchytraeid reproduction results for
the silicate leach using distilled water. Significant results are marked with an (*).
The silicate leaching for DW on day 16 presented a slight recovery of the results com-
pared to the previous reading on the same test. This effect can sometimes occur once the
Enchytraeids are biological indicators, and the sensibility can change according to the ex-
posure [30]. Few other details were also visible during the reproduction test days, e.g.,
some of the silicate eluates revealed the presence of a few eggs, but these were too weak
to hatch and did not hatch until the end of the test. In other situations, the initially intro-
duced adult worms used died at the beginning of the test or even one week before the end
of the test. Besides, the absence of adult worms had a direct effect on the number of off-
springs.
Besides the increased concentration of sodium, chlorides, and sulfates in the eluates
from the silicate component, it is impossible to confirm with certainty what causes the
strong toxicity influence from the eluates from the silicate resin for the Enchytraeids. The
very small number of offsprings, in one factor, can be explained by the sensitive sensors
on the body of the Enchytraeids, which perceive limited survival conditions early on and
will avoid habitats [31].
Figure 5.
(
a
)Enchytraeid reproduction results for the silicate leach using tap water. (
b
)Enchytraeid reproduction results for
the silicate leach using distilled water. Significant results are marked with an (*).
Materials 2021,14, 294 8 of 14
An analysis of Figure 4a shows a low decrease in Enchytraeids reproduction behavior.
Nevertheless, after 16 days of leaching, an apparent decrease in the number of offsprings
can be seen, with less than 15 worms counted. The same decrease was perceived for the
eluates of the waterproof material with DW after 16 days, (Figure 4b), however with a total
amount of 17 Enchytraeids. The most significant results are emphasized with an (*) point.
The reproduction tests were performed for all the eluates. When observing the
reproduction results for both eluates, after 64 days, a stabilization of the reproduction
behavior was noticed at the end of the test, indicating a reduction of the toxicity and
decrease in the presence of active ingredients from the waterproof material when exposed
to water for a longer time [
28
]. The drop in the number of offsprings was not very alarming
for eluates of the waterproof material. However, effects on the Enchytraeid populations
can be noticed. An alarming situation would be configurated when the component tested
affects the reproduction behavior in an exposed concentration that affects fifty percent of
the population. The different results over time represent typical leaching behavior. In the
beginning, there was a first wash-off followed by reduced diffusion-controlled release.
Both types of eluates (TW and DW) of the waterproof material contained a low
variation in the quantities of ions when compared to the corresponding blank samples.
Ions of Na
+
and Ca
2+
increased with the leaching. However, it is already proven that
sodium and calcium have a small influence on toxicological effects for Enchytraeids [
29
],
and the presence of these minerals on the leachings can help to clarify the reduction of
offsprings in the results.
Analyzing the results of the eluates of the silicate resin are plotted in Figure 5a,b. A
high impact of the eluates from the silicate product on the reproduction behavior of the
Enchytraeids can be seen. In all phases of the leaching process, small numbers of offsprings
were counted. The silicate eluates prepared with distilled water presented the highest
impact on the population, bringing special attention to the reproduction test from day
9 silicate DW, where a mean number close to 0 was achieved. For both reproduction
tests of silicate leachings from day 64, the Enchytraeids presented a tendency to recover
the population of worms, and a higher number of offsprings was counted; however, the
number of offsprings is still under fifty percent of the population when compared to the
control group. The most significant results are emphasized with an (*) point.
The silicate leaching for DW on day 16 presented a slight recovery of the results
compared to the previous reading on the same test. This effect can sometimes occur once
the Enchytraeids are biological indicators, and the sensibility can change according to the
exposure [
30
]. Few other details were also visible during the reproduction test days, e.g.,
some of the silicate eluates revealed the presence of a few eggs, but these were too weak to
hatch and did not hatch until the end of the test. In other situations, the initially introduced
adult worms used died at the beginning of the test or even one week before the end of the
test. Besides, the absence of adult worms had a direct effect on the number of offsprings.
Besides the increased concentration of sodium, chlorides, and sulfates in the eluates
from the silicate component, it is impossible to confirm with certainty what causes the
strong toxicity influence from the eluates from the silicate resin for the Enchytraeids. The
very small number of offsprings, in one factor, can be explained by the sensitive sensors on
the body of the Enchytraeids, which perceive limited survival conditions early on and will
avoid habitats [31].
3.3. Avoidance Results
The same eluates were tested for the avoidance behavior of the Enchytraeids, and results
are represented in the following graphics. Figure 6represents the avoidance behavior of
the Enchytraeids using the same treatment on both sides (negative control).
Materials 2021,14, 294 9 of 14
Materials 2021, 14, x FOR PEER REVIEW 9 of 14
3.3. Avoidance Results
The same eluates were tested for the avoidance behavior of the Enchytraeids, and re-
sults are represented in the following graphics. Figure 6 represents the avoidance behav-
ior of the Enchytraeids using the same treatment on both sides (negative control).
Figure 6. Enchytraeid avoidance distribution for both sides with control soil.
Figure 6 shows the graphic proportion of worms that prefer to stay on each side of
the soil. On both sides, the same control conditions were used, and it was possible to see
that when both sides contain a control soil free of toxicants, the worms do not show one
preferred side. The same average distribution was observed for both sides, being one side
with 49% of the population and the other side with 51% of the population of Enchytraeids.
According to the ISO 17512 (2008), the control is considered valid if the proportions stay
between 40–60% in the distribution hack.
The avoidance results for the waterproof leachings are plotted in Figure 7a,b. The
green line represents the number of Enchytraeids that chose the control side, while the red
line represents the number of Enchytraeids that preferred the eluate side.
(a) (b)
Figure 7. (a) Enchytraeid avoidance results for the waterproof leach using tap water. (b) Enchytraeid avoidance results for
the waterproof leach using distilled water.
Analyzing the avoidance behavior of the Enchytraeids testing the waterproof leaches,
it was possible to see that the worms tend to prefer mostly the side of the control, but for
Avoidance Control versus
Control
Control A Control B
Figure 6. Enchytraeid avoidance distribution for both sides with control soil.
Figure 6shows the graphic proportion of worms that prefer to stay on each side of
the soil. On both sides, the same control conditions were used, and it was possible to see
that when both sides contain a control soil free of toxicants, the worms do not show one
preferred side. The same average distribution was observed for both sides, being one side
with 49% of the population and the other side with 51% of the population of Enchytraeids.
According to the ISO 17512 (2008), the control is considered valid if the proportions stay
between 40–60% in the distribution hack.
The avoidance results for the waterproof leachings are plotted in Figure 7a,b. The
green line represents the number of Enchytraeids that chose the control side, while the red
line represents the number of Enchytraeids that preferred the eluate side.
Materials 2021, 14, x FOR PEER REVIEW 9 of 14
3.3. Avoidance Results
The same eluates were tested for the avoidance behavior of the Enchytraeids, and re-
sults are represented in the following graphics. Figure 6 represents the avoidance behav-
ior of the Enchytraeids using the same treatment on both sides (negative control).
Figure 6. Enchytraeid avoidance distribution for both sides with control soil.
Figure 6 shows the graphic proportion of worms that prefer to stay on each side of
the soil. On both sides, the same control conditions were used, and it was possible to see
that when both sides contain a control soil free of toxicants, the worms do not show one
preferred side. The same average distribution was observed for both sides, being one side
with 49% of the population and the other side with 51% of the population of Enchytraeids.
According to the ISO 17512 (2008), the control is considered valid if the proportions stay
between 40–60% in the distribution hack.
The avoidance results for the waterproof leachings are plotted in Figure 7a,b. The
green line represents the number of Enchytraeids that chose the control side, while the red
line represents the number of Enchytraeids that preferred the eluate side.
(a) (b)
Figure 7. (a) Enchytraeid avoidance results for the waterproof leach using tap water. (b) Enchytraeid avoidance results for
the waterproof leach using distilled water.
Analyzing the avoidance behavior of the Enchytraeids testing the waterproof leaches,
it was possible to see that the worms tend to prefer mostly the side of the control, but for
Avoidance Control versus
Control
Control A Control B
Figure 7.
(
a
)Enchytraeid avoidance results for the waterproof leach using tap water. (
b
)Enchytraeid avoidance results for the
waterproof leach using distilled water.
Analyzing the avoidance behavior of the Enchytraeids testing the waterproof leaches, it
was possible to see that the worms tend to prefer mostly the side of the control, but for few
leaches, the difference in the number of worms in each side is small. A less poor scenario in
the number of organisms on each side is achieved in the vessels of waterproof TW 64 days.
The statistical avoidance percentage was calculated as represented in Table 2.
Materials 2021,14, 294 10 of 14
Table 2.
Avoidance results percentages (%) for E. albidus, calculated for all eluates of waterproof and
silicate.
Time Waterproof TW Waterproof DW Silicate TW Silicate DW
6 h 65 75 85 85
1 day 65 75 85 85
2 days 70 50 55 70
4 days 60 25 70 50
9 days 65 30 85 80
16 days 55 30 40 40
36 days 40 40 40 45
64 days 30 50 30 50
Figure 8a,b represents the avoidance behavior of the Enchytraeids for the eluates of the
silicate. The result indicates that one more time, the Enchytraeids tend to move mostly to the
side of the control soil, where no contaminations of chemicals are detected. The avoidance
was similar in both tests, with few variations in numbers for 9 and 64 days.
Materials 2021, 14, x FOR PEER REVIEW 10 of 14
few leaches, the difference in the number of worms in each side is small. A less poor sce-
nario in the number of organisms on each side is achieved in the vessels of waterproof
TW 64 days. The statistical avoidance percentage was calculated as represented in
Table 2.
Figure 8a,b represents the avoidance behavior of the Enchytraeids for the eluates of
the silicate. The result indicates that one more time, the Enchytraeids tend to move mostly
to the side of the control soil, where no contaminations of chemicals are detected. The
avoidance was similar in both tests, with few variations in numbers for 9 and 64 days.
(a) (b)
Figure 8. (a) Enchytraeid avoidance results for the silicate leach using tap water. (b) Enchytraeid avoidance results for the
silicate leach using distilled water.
A statistical analysis was carried out to calculate the percentage of worms affected.
Avoidance was calculated according to the following Equation (1):
𝑥=
𝑛−𝑛
𝑁×100 (1)
where:
x is avoidance, expressed as a percentage;
nc is the number of worms in the control soil (either per vessel or in the control soil of
all replicates);
nt is the number of worms in the test soil (either per vessel or in the test soil of all rep-
licates);
N is the total number of worms (usually 10; either per vessel or in the control soil of all
replicates).
Using the results collected on the avoidance tests, the percentage of avoidance was
calculated and the results are represented in Table 2.
Figure 8.
(
a
)Enchytraeid avoidance results for the silicate leach using tap water. (
b
)Enchytraeid avoidance results for the
silicate leach using distilled water.
A statistical analysis was carried out to calculate the percentage of worms affected.
Avoidance was calculated according to the following Equation (1):
x=nc−nt
N×100 (1)
where:
xis avoidance, expressed as a percentage;
nc is the number of worms in the control soil (either per vessel or in the control soil of
all replicates);
nt is the number of worms in the test soil (either per vessel or in the test soil of all replicates);
Nis the total number of worms (usually 10; either per vessel or in the control soil of
all replicates).
Materials 2021,14, 294 11 of 14
Using the results collected on the avoidance tests, the percentage of avoidance was
calculated and the results are represented in Table 2.
According to ISO 17512, when the avoidance results are
≥
80%, a limited survival
habitat function is configurated. If an attraction of >80% by the test soil is observed, the
presence of chemical substances cannot be excluded. The result indicates an impact on the
behavior of the organisms.
The limited survival habitat was determined on the following eluates: silicate TW (6 h,
1 day, 9 days) and silicate DW (6 h, 1 day, 9 days). Although eluates from waterproof did
not represent a limited habitat function, in few eluates, the avoidance percentage reaches
75% being very close to this value and indicating the possible presence of chemicals.
The concentration of harmful substances consequently seems to be significantly in-
creased in the first leaching steps since many components that can be leached out at the
beginning of a DSLT can be released through washing. With the length of the leaching
times, the number of Enchytraeids that migrate to the eluate side increases, indicating a
decreased release of harmful substances with the time of leaching.
A better distribution of the number of organisms on both sides of the vessels for all
leachings is noted in leaches of 64 days when an avoidance rate of 30% and 50% is reached.
3.4. Evaluation of Enchytraeid Results—Reproduction Versus Avoidance
Comparing the results of the reproduction tests with the results of the avoidance tests,
a certain similarity can be observed in the sensitivity of both test results.
The initial eluates of the silicate product caused an impacting reduction on the number
of Enchytraeids in the reproduction test, while in the avoidance test, the same eluates
indicated an avoidance of 85% of the population.
Besides, the eluates from waterproof for reproduction tests did not present high
toxicity for the organisms, but a high avoidance percentage was observed for the same
components. It is important to remember that the reproduction test conditions supply
constant contact with the soil + eluate for a chronic exposure time, allowing the leaches to
stabilize for a longer period of time [32].
For all eluates of 64 days, in the reproduction test, an increase in the number of
offsprings was observed. At the same time, the avoidance test also shows better survival
habitat conditions with smaller avoidance behavior, being both test results an indicator of
stabilization of chemicals in the soil.
The reproduction test is applied to detect effects resulting from sublethal concentra-
tions and long term scenario exposures. The avoidance test exists to investigate the habitat
function of soil with earthworms as representatives of the soil biocenosis. The endpoints
are determined to obtain information on the environmental effects. The reproduction test
is very labor-intensive and time-consuming, needing long incubation periods and results
being collected after 6 weeks of exposure and constant work, turning the reproduction tests
more expensive. In contrast, the avoidance test presents very fast results of environmental
effects and a high level of sensitivity. However, the avoidance test is not intended to replace
the reproduction test but to provide faster screenings of environmental effects in different
levels of sensitivity. For a complete and better understanding of the effects of a toxicant
on the soil, it is possible to use the avoidance test as the first information screen and the
reproduction test to verify the chronic sublethal effects.
4. Conclusions
The reproduction behavior of Enchytraeus albidus presented the highest numbers when
worms were in soil moistened with the reconstituted freshwater as a control, followed
by tap water and, in last, the demineralized water. The type of water used to leach the
components influenced the reproduction behavior of E. albidus. The best performance
in the number of juveniles is achieved when the standard soil is moistened with liquids
containing physic-chemical conditions more similar to those found in the environment.
Materials 2021,14, 294 12 of 14
Evaluating the reproduction results, it can be concluded that the eluates from the
waterproof material did not present high toxicity to the reproduction behavior of E. albidus;
besides, a decrease in the number of juveniles was measured. The leachings from water-
proof TW presented a higher number of juveniles in comparison to waterproof DW. The
leachings from silicate TW and silicate DW were toxic for the reproduction behavior of
E. albidus. Eluates from silicate DW had the lowest number of juveniles and inhibited more
than 50% of the population of E. albidus.
The avoidance results for all the eluates presented a similar sensitivity for E. albidus
when compared to the reproduction results. The organisms avoided soils containing high
concentrations of chemicals. Eluates of the silicate resin product in soil presented a limited
habitat function with avoidance results >80%.
Looking at these results, it can be concluded that it is important to track biomarkers
for Enchytraeids in order to assess the possible hazard effects of construction products and
understand the mechanism of action in the environment.
5. Final Comments
The time and effort required for the preparation of the leachings in accordance with
DIN CEN/TS 16637-2 should also be emphasized: it is extensive and intensive work, and
therefore this study recommends for screening the use of other methods of leaching as the
EN 12457-4 [33], which lead to similar results from a biological point of view.
It is important to note that a difference in the final products and consequently in the
eluates may occur due to different handling of the starting products. The air gun and
mixers used in the laboratories are prepared to handle a much smaller sample volume
than those used in the construction industry. For this reason, it is recommended here to
collect samples from real applications in order to clarify whether the products have possible
toxicity. Another problem is the solubility of these components in the environment. The
laboratory tests try to consider as far as possible the aspects from a real scenario situation,
but it is still likely that this will not give the same results as in the real environment and
that a much higher dilution of these components would occur and, therefore, lower toxicity
would be detected.
The organisms used in these tests may never come into contact with this type of
component in a real scenario. Nevertheless, it is the responsibility of ecotoxicology to
identify the possible causes and influences that could occur in case of such an event. This
study does not condemn the use of construction products, nor does it attempt to restrict or
prevent their use. The sole purpose is to clarify human activities and their consequences for
the environment and to find sustainable solutions that would help to focus on satisfying
the needs of the present without compromising the ability of future generations to meet
their needs.
Author Contributions:
Conceptualization, D.S. and S.P.; methodology, M.A.v.W.; formal analysis,
M.A.v.W.; investigation, M.A.v.W.; resources, D.S.; data curation, S.P.; writing—original draft prepa-
ration, M.A.v.W.; writing—review and editing, M.A.v.W., S.P. and D.S.; supervision, D.S.; project
administration, D.S.; funding acquisition, D.S. All authors have read and agreed to the published
version of the manuscript.
Funding:
This research was funded by “Forschungsinitiative Zukunft Bau des Bundesinstitutes für
Bau-, Stadt- und Raumforschung”, grant number SWD-10.08.18.7-17.49.
Institutional Review Board Statement: Not Applicable.
Informed Consent Statement: Not Applicable.
Data Availability Statement: Data availability within this article.
Acknowledgments:
The authors thank Daria Blizniukova for helping in the cultivation of Enchy-
traeids and the collection of leachings data.
Conflicts of Interest: The authors declare no conflict of interest.
Materials 2021,14, 294 13 of 14
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für Materialien mit einer Korngröße unter 10 mm (ohne oder mit Korngrößenreduzierung) Deutsche Fassung EN 12457-4:2002
Ausgabe 2003-01. 2002. Available online: https://www.din.de/de/mitwirken/normenausschuesse/naw/wdc-beuth:din21:
52065766 (accessed on 8 January 2021).
