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Article

Uptake Through Feeding and/or Culture Medium of 0.5% Dimethyl Sulfoxide (DMSO): Biological Response of Daphnia magna and Ceriodaphnia dubia in Ecotoxicity Tests

by
Stefania Bigi
1,†,
Karin Schlappa
2,†,
Serena Anselmi
3,
Francesca Provenza
3 and
Monia Renzi
1,2,*
1
Department of Life Science, University of Trieste, 34127 Trieste, Italy
2
CoNISMa—Consorzio Nazionale Interuniversitario per le Scienze del Mare, 00196 Roma, Italy
3
BsRC—Bioscience Research Center, 58015 Orbetello, Italy
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Water 2025, 17(2), 191; https://doi.org/10.3390/w17020191
Submission received: 7 December 2024 / Revised: 5 January 2025 / Accepted: 10 January 2025 / Published: 12 January 2025
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Abstract

:
Dimethyl sulfoxide (DMSO) is an aprotic solvent widely used in ecotoxicological assays, suitable for solubilizing a wide range of polar and non-polar substances. The aim of this study was to analyze the species-specific biological response of the model organisms Daphnia magna and Ceriodaphnia dubia to 0.5% DMSO, under feeding conditions. Both species were exposed in culture medium, with or without 0.5% DMSO, to the unicellular algae (Raphidocelis subcapitata, Tetradesmus obliquus), either individually or in combination; the algae were pretreated or not pretreated with 0.5% DMSO. The exposure was carried out in acute (24 h and 48 h) and chronic (72 h and 168 h) ecotoxicity tests. The experimental results suggest that a lower DMSO concentration limit (<0.5%) would be appropriate for C. dubia due to its greater sensitivity to the solvent. Finally, considering the ingestion of DMSO-contaminated algae, it can be concluded that, under certain experimental conditions, the solvent ingested through the diet may significantly affect the mortality of both D. magna and C. dubia.

Graphical Abstract

1. Introduction

Dimethyl sulfoxide or DMSO (C2H6OS) is an aprotic polar organic solvent commonly used in research laboratories, in the chemical and pharmaceutical industries, and as an oxidizing agent in various organic reactions [1,2].
Due to its small and compact structure, amphipathic nature, and ability to form hydrogen bonds, it can penetrate biological tissues without causing significant damage [3] and solubilize various otherwise poorly soluble polar and non-polar molecules. It is used as a solvent for drugs, as a vehicle for chemicals of interest in in vivo and in vitro tests [4], and is also known for its anti-inflammatory, radioprotective, neuroprotective, and free radical scavenging properties [5]. In an ecotoxicological context, DMSO, being miscible with water and most organic liquids, is often used as a co-solvent. This is because toxic substances of a hydrophobic nature, to be evaluated in toxicity tests on aquatic organisms, require a solvent that can solubilize them and facilitate their permeation through biological membranes [6]. The Organization for Economic Co-operation and Development (OECD) guidelines recommend that the concentration should not exceed 100 mg/L (0.1 mL/L) [7], while the United States Environmental Protection Agency (U.S. EPA) indicates that the concentration of organic solvents should not exceed 0.5 mL/L and 0.1 mL/L in basic static and basic flow-through tests, respectively [8].
Nevertheless, to achieve effective solubilization of lipophilic substances, higher concentrations of DMSO are used in ecotoxicological tests, up to 10% (v/v) [6]. The effects of the solvent on model organisms have mostly been studied in acute exposure tests (of a few minutes or hours), but it is through chronic exposure tests that sublethal effects become more evident [9].
In addition, the recommended thresholds are not representative of each single species, and the dose considered safe for some organisms may be toxic to others. Andrade-Vieira et al. [10] found negative effects of DMSO on the growth rate of the algal population (Raphidocelis subcapitata) at a lowest observed effect concentration (LOEC) of 0.125% after 72 h of exposure to DMSO, whereas significant differences in the mobility of D. magna after 24 h and 48 h of exposure were found at a concentration of 1%. Therefore, the authors suggest that the final concentrations of DMSO in experimental solutions should not exceed 0.125% for R. subcapitata and 0.5% for D. magna.
An additional aspect to be considered when referring to the use of model organisms in ecotoxicological testing is that in most cases, parameters are measured in response to exposure to one or more contaminants dissolved in the aqueous medium, without considering any resulting effects of dietary exposure [11]. Studies investigating this route of intake (dietary uptake) tend to focus on bioaccumulation, but limited attention has been paid to simultaneous aquatic and dietary exposure to a contaminant, even though this is the most likely scenario for organisms living in aquatic environments [12].
Considering the above, the purposes of this study can be summarized as follows:
  • To investigate the sensitivity to 0.5% DMSO of model species belonging to the same trophic level (Daphnia magna and Ceriodaphnia dubia), both in the context of acute exposure (24 h and 48 h) and chronic exposure (72 h and 168 h), under feeding conditions, taking into account that C. dubia, compared to D. magna, can be considered as equally sensitive [13,14,15], more sensitive [16,17], or less sensitive [18], depending on the chemicals tested, the endpoint considered, and the duration of the test;
  • To compare the differences in biological responses (sensitivity) between Daphnia magna and Ceriodaphnia dubia, both in relation to the presence of DMSO in the culture medium and its uptake via the dietary route—specifically through the ingestion of contaminated algae (Raphidocelis subcapitata and/or Tetradesmus obliquus)—while also considering potential dietary preferences.
This study intends to provide useful indications for the choice of the most appropriate model organism, between Daphnia magna and Ceriodaphnia dubia, by evaluating the potential consequences resulting from the absorption of the solvent present in the aqueous medium and/or introduced through the diet, treating it as a potential contaminant and considering that simultaneous exposure would represent a more realistic environmental contamination scenario [12], contributing to the study of a central issue of ecological importance [19].

2. Materials and Methods

2.1. Species Used in Ecotoxicity Assays

2.1.1. Daphnia magna and Ceriodaphnia dubia

To investigate the difference in sensitivity between model species belonging to the same trophic level, ecotoxicity tests were performed on Daphnia magna (Straus, 1820) and Ceriodaphnia dubia (Richard, 1894), two species of primary consumers [20,21] inhabiting shallow freshwater environments, temporary pools, or ponds, belonging to the family Daphniidae. As filter-feeding planktonic organisms [22,23], they play an important role in energy transfer along the trophic chain [24,25], and in addition to their feeding mode and ecological role, they share a reproductive strategy and several morpho-anatomical features. D. magna is characterized by a larger body size, takes longer to reach reproductive maturity (6–10 days), and has longer generation times (4–6 days). In contrast, adults of C. dubia are approximately 1/6 the body size of D. magna and reach reproductive maturity within three days. The short generation time of C. dubia allows for chronic exposure tests to be conducted over three broods within approximately 5–8 days.
D. magna and C. dubia ephippia, contained in miniature kits (Microbiotests, Kleimoer, Belgium) purchased from Ecotox LDS (Milano, Italy), were used for this experiment, with reference to the standard procedures UNI EN ISO 6341:2013 (for D. magna) [26] and APAT CNR IRSA 8040, 29/2003 (for C. dubia) [27].

2.1.2. Raphidocelis subcapitata and Tetradesmus obliquus

The unicellular green algae Raphidocelis subcapitata (Selenastraceae) and Tetradesmus obliquus (Scenedesmaceae) were selected for the experimental phase. The purpose of this selection was to test the effect of DMSO through dietary uptake, while also considering any potential food preferences of D. magna and C. dubia.
R. subcapitata is part of the freshwater phytoplankton and is commonly present in ponds, lakes, and rivers. In general, it is found in the solitary form; the individual cell has a lunate shape, a dry weight of 2–3 × 10−8 mg cell−1, and dimensions ranging from 8 to 15 µm in length and 1.9 to 4 µm in width, while the biovolume ranges from 22 to 74.5 µm3.
Because it is a sensitive and reliable indicator in ecotoxicological assays, this model species is one of the most widely used in ecotoxicological tests [28].
The genus Tetradesmus is characterized by high phenotypic plasticity and significant differentiation among strains, allowing it to adapt to various environmental conditions.
T. obliquus, a freshwater microalga, generally forms colonial structures (cenobia) [29], consisting of 2–4 (8) cells arranged alternately or in a linear pattern. The individual cells are fusiform with pointed ends, measuring between 13 and 34 µm in length and 6 and 13 µm in width [30]. Due to its characteristics such as rapid growth and resistance to adverse conditions, T. obliquus is used in various fields such as biofuel production, wastewater treatment, as food in aquaculture, as a biosensor to detect pollutants in water, and as a source of bioactive components in food [31].
The unicellular algae used in the experiment were derived from pure cultures maintained under sterile conditions in the laboratory through in-house propagation, as suggested by the standard UNI EN ISO 10253:2017 [32].

2.2. Treatments

2.2.1. Pretreatment of DMSO Carrier Algae

The unicellular DMSO carrier algae used in the toxicity tests performed to evaluate the biological response to dietary uptake were subjected to a pretreatment consisting of keeping them in algal culture medium (according to UNI EN ISO ISO 8692:2012 [33]) containing 0.5% DMSO, at 6000 lux (photoperiod of 16 h light and 8 h dark), at a temperature of 24 ± 1 °C.
These conditions were maintained for four days prior to the start of the experiment. DMSO: CAS No: 67-68-5, Carlo Erba, Cornaredo, Milano, Italy.

2.2.2. Tested Conditions (Treatments)

To address the research questions, sixteen experimental conditions were established and examined in both the acute and chronic ecotoxicity assays for each model species (i.e., sixteen for D. magna and sixteen for C. dubia). The full list of treatments (or conditions), and their corresponding nomenclature, is presented in Table 1. In general, the treatments consisted of exposing D. magna and C. dubia to algae under the following conditions: algae not pretreated with 0.5% DMSO, algae pretreated with 0.5% DMSO, algae not pretreated but with 0.5% DMSO in the medium, and algae pretreated with 0.5% DMSO and 0.5% DMSO in the medium. The treatments were applied in all possible combinations, either considering the presence of one species of alga (100 µL of R. subcapitata or 100 µL of T. obliquus) or both species together (50 µL of R. subcapitata and 50 µL of T. obliquus) in the medium. The tests were conducted in six-well plates (for each species), with two conditions established for each plate (number of replicates = 3). The test volumes consisted of 10 mL of culture medium, (corresponding to 8 of 16 treatments) and 10 mL of culture medium with 0.5% DMSO (corresponding to 8 of 16 treatments).
The nutrients in the culture medium are consistent with those described by UNI EN ISO 8692:2012, with a ratio of 10:100 for group 1 nutrients and 1:100 for groups 2, 3, and 4 nutrients [33]. For each test well, five daphnids were utilized.

2.2.3. Dilution Medium

In order to identify which of the dilution media, provided in the operating procedures for D. magna and C. dubia, was compatible with optimal algal growth of R. subcapitata and T. obliquus, a preliminary test was conducted on the following media: water from the Ceriodaphtoxkit F® commercial kit (KIT) used in the test on C. dubia, APAT 8020 water (APAT) [34], and ISO 6341 water (ISO) [26] used in the tests on D. magna; Table S1 reports the content of dissolved salts in each water. The water found to be suitable for the purposes of this study corresponded to that described in the Ceriodaphtoxkit F® kit (KIT). Therefore, it was employed as the dilution medium for all treatments listed in Table 1. The commercial kit includes concentrated salt solutions (Table S1), which must be diluted in accordance with the standard operating procedures. The results of the preliminary tests for quantifying algal growth are presented in the Supplementary Materials (Tables S2 and S3, Figure S1).

2.2.4. Controls

The list of controls (and their nomenclature) established for both algae (R. subcapitata, T. obliquus) and daphnids (D. magna, C. dubia) is provided in Table 2. Since the medium used in the tests is the freshwater described by Ceriodaphtoxkit F® (KIT), a control test was performed to exclude the possibility that the biological response observed in D. magna was influenced by the type of water used in the assay (which was selected based on its suitability for algal growth). In addition, a control test with ISO 6341 water (ISO) was performed on C. dubia in order to highlight the biological response also depending on this type of water (although it does not correspond to the one selected for the test), taking into account the elements that make up the test medium (NUT and/or DMSO), and comparing it with all the treatments associated with the KIT (Ceriodaphtoxkit F®) water controls. All combinations established in the controls were configured to highlight the contributions of the elements that make up the test medium, both for algae and daphnids, excluding the food component provided for D. magna and C. dubia.

2.3. Ecotoxicity Tests

D. magna and C. dubia

After an incubation period of the ephippia (maximum 80 h at 25 ± 1 °C for C. dubia and maximum 90 h at 21 ± 1 °C for D. magna, both at 6000 lux), the hatchlings, not older than 24 h, were transferred to the test plates (set up with the corresponding treatments) and incubated at 22 ± 2 °C, with a photoperiod of 16 h light and 8 h dark.
Biological response was measured at 24 h, 48 h, 72 h, and 168 h and expressed as percent effect (reported as mean and ±SD); the endpoint considered is mortality.

2.4. Data Analysis

Experimental data were reported on Microsoft Excel spreadsheets (version 16.90). A Student’s t-test was conducted (ISPRA 2013 [35]) to determine whether the average biological response differed between species and treatments, at the same exposure time.
The t-test was also used to assess statistical significance between the following experimental conditions, separately for D. magna and C. dubia:
  • Difference in biological response, under the same feeding conditions, between assays conducted with DMSO-free culture medium (ACM) and assays conducted with DMSO-containing culture medium (ACM + DMSO);
  • Difference in biological response between treatments containing one algal species (RS or TO), pretreated or non-pretreated, evaluating separately treatments with DMSO-free culture medium (ACM) and DMSO-containing culture medium (ACM + DMSO);
  • Difference in biological response between treatments containing both algal species (RS and TO), pretreated or non-pretreated (both or individually), evaluated separately with DMSO-free culture medium (ACM) and DMSO-containing culture medium (ACM + DMSO).
The biological response (mortality %) as a function of exposure time and treatment was presented in the form of box-and-whisker plots and histograms (separately for D. magna and C. dubia) in order to make appropriate assessments in conjunction with the results obtained from t-tests.
A multivariate analysis of the data set was performed to determine the significance of the observed variability with respect to the factor of interest “species” (two levels: D. magna and C. dubia); for this purpose, the one-way ANOSIM test (number of permutations: 9999; with software PRIMER, version 7.0) was performed on the Euclidean distance matrix calculated on the normalized data. Finally, for each species, a two-way ANOSIM test was performed on the factors “time” (four levels: 24 h, 48 h, 72 h, 168 h) and “treatment” (sixteen experimental conditions or treatments), to study the contribution of these factors to the observed variance.

2.5. Quality Assurance

The experiments were carried out in the UNI CEI EN ISO/IEC 17025:2018 accredited laboratory (BsRC—Bioscience Research Center, LAB. No. 1715L); to ensure the reliability of the assay, a quality control (according to the standard reference method) was performed by setting up the positive control with potassium dichromate (K2Cr2O7; CAS No. 7778-50-9, Carlo Erba, Cornaredo, Milano, Italy). The experiment was considered valid only if the biological response detected in the negative control (% of immobile organisms less than or equal to 10%, at 24 h and 48 h) and positive control (EC50 at 24 h, with K2Cr2O7, between 0.6 and 2.1 mg/L for D. magna and 0.21 and 0.26 mg/L for C. dubia) met the reliability criteria established by the method.

3. Results

Table S4 reports the values (%) of the biological response (mortality) recorded for D. magna and C. dubia, in terms of the mean and ±SD, for each exposure time. Table S5 presents the values (%) recorded from the controls (24 h, 48 h, 72 h, and 168 h), in terms of the mean and ±SD. Table S6 reports the control values (%) observed for the algal growth of R. subcapitata and T. obliquus, in terms of the mean and ±SD (48 h, 72 h, and 168 h)

3.1. Sensitivity Differences Between D. magna and C. dubia (D. magna vs. C. dubia)

Non-metric multidimensional scaling (n-MDS) shows the distribution of mortality values of D. magna and C. dubia in response to the treatments, at the four experimental times (24 h, 48 h, 72 h, 168 h), Figure S2. The mortality values of each species form two distinct segregated groups, indicating a difference in biological response, consistent with the result of the one-way ANOSIM test (two levels: D. magna and C. dubia), where R: 0.1; significance level: 0.01%; number of permutations: 9999; number of permuted statistics greater than or equal to R: 0. The average biological response (mortality %) recorded at the four experimental times, considering all treatments, is shown in Figure 1. For both species, the lowest mortality range is observed at 24 h, while the highest is observed at 168 h. Mortality in C. dubia is higher at all exposure times except at 168 h, where the range of mortality values between the two species is quite similar. Nevertheless, the mean mortality remains higher for C. dubia and lower for D. magna (considering all treatments).
With reference to the sixteen experimental conditions (or treatments), the recorded biological response (mean and ±SD) is shown in Figure 2; comparisons performed with the t-test (Table 3, D. magna vs. C. dubia) show significant differences in the mortality between the two species in 6 out of 16 treatments, of which 5 out of 6 contain DMSO in the medium. For three conditions, there are significant differences at three of the four experimental times, for two conditions, there are significant differences at one of the experimental times, and for one condition, there are significant differences at 72 h and 168 h.

3.2. Comparison of the Biological Response Detected Between Treatments with and Without DMSO in the Medium (ACM vs. ACM + DMSO)

Differences in the biological response to DMSO were studied by performing the t-test between the mortality values recorded at the four experimental times, comparing conditions without DMSO in the culture medium (ACM) and conditions characterized by the presence of DMSO in the culture medium (ACM + DMSO), under the same dietary condition (RS and/or TO, pretreated and/or not pretreated with DMSO), separately for each species.

3.2.1. Daphnia magna: ACM vs. ACM + DMSO

Table 3 (Daphnia magna ACM vs. ACM + DMSO) reports the statistical significance obtained from the comparison performed with the t-test, considering the absence and presence of DMSO in the medium (respectively: ACM and ACM + DMSO), under the same dietary condition. Below are the significant treatments characterized by higher mortality:
  • D. magna exposed to T. obliquus + DMSO in the medium, TO + DMSO, at 168 h;
  • D. magna exposed to R. subcapitata pretreated with DMSO + T. obliquus not pretreated, RS (DMSO) + TO, at 72 h.
In all remaining conditions, the presence or absence of DMSO in the medium did not result in statistically significant differences in mortality.

3.2.2. Ceriodaphnia dubia: ACM vs. ACM + DMSO

In C. dubia, a greater number of statistically significant differences were observed. Table 3 (C. dubia ACM vs. ACM + DMSO) provides the statistical significance detected from the comparison performed using the t-test, considering the absence and presence of DMSO in the medium (respectively, ACM and ACM + DMSO) in organisms subjected to the same feeding regime. Below are the significant treatments characterized by higher mortality:
  • C. dubia exposed to R. subcapitata + DMSO in the medium, RS + DMSO, at 48 h and 72 h;
  • C. dubia exposed to R. subcapitata pretreated with DMSO + DMSO in the medium, RS(DMSO) + DMSO, at 24 h;
  • C. dubia exposed to R. subcapitata pretreated with DMSO + T. obliquus + DMSO in the medium, RS (DMSO) + TO + DMSO, at 48 h, 72 h, and 168 h;
  • C. dubia exposed to R. subcapitata + T. obliquus pretreated with DMSO + DMSO in the medium, RS + TO (DMSO) + DMSO, at 48 h, 72 h, and 168 h.

3.3. Dietary Uptake of 0.5% DMSO, Mortality, and Food Preferences

3.3.1. Daphnia magna

The ANOSIM two-way test, performed considering the sixteen treatments for each species and their exposure times (24 h, 48 h, 72 h, 168 h), indicated significant results for the factor “treatment” (Rho: 0.424; significance level: 0.01%; number of permutations: 9999; number of permuted statistics greater than or equal to the mean Rho: 0) and weakly significant for the factor “time” (Rho = 0.161; significance level: 0.5%; number of permutations: 9999; number of permuted statistics greater than or equal to the mean Rho: 47).
Figure 3 (Daphnia magna) provides the range of average percentages of dead organisms detected for each of the sixteen treatments, without distinguishing the exposure time.
In treatments characterized by non-DMSO-pretreated algae (conditions 1, 3, 5, 7, 9, 13), the average mortality remained below 20% in all cases except condition 7 (>20%), which is characterized by the presence of DMSO in the culture medium (TO + DMSO).
In treatments characterized by one algal species not pretreated with DMSO (condition 1, 3, 5, 7 of Figure 3), the mortality of D. magna was lower in cases where the algal species present was R. subcapitata, both in the absence of DMSO in the medium (condition 1 vs. condition 5) and in its presence (condition 3 vs. condition 7).
No significant differences were observed regarding the type of algal species present in the medium without DMSO (Table 3, D. magna Feeding with one alga + ACM).
Considering the treatments with single algal species and DMSO in the culture medium, significantly higher mortality was recorded in the TO + DMSO treatment at 72 h and 168 h, and in the RS (DMSO) + DMSO treatment at 168 h; see Table 3, D. magna Feeding with one alga + ACM + DMSO.
Under the conditions characterized by the presence of the two algal species not pretreated with DMSO (condition 9 and 13 of Figure 3, Daphnia magna RS + TO), the average mortality of D. magna is less than 20%, while the highest mortality values are recorded when both algal species are pretreated with DMSO, condition 12—RS (DMSO) + TO (DMSO) and condition 16—RS (DMSO) + TO (DMSO) + DMSO, see Figure 3. Under these conditions, the average mortality (considering the treatment) is close to 60% and the maximum range of variation exceeds 80%.
In treatments in which D. magna was exposed in culture medium (without DMSO) in the presence of two algal species (RS and TO), one of which was pretreated with DMSO (conditions 10 and 11 of Figure 3, Daphnia magna), higher mortality was recorded in the treatment in which the DMSO-pretreated alga was the R. subcapitata species, RS (DMSO).
Comparing treatments in which D. magna was fed both algal species (RS and TO), it appears that mortality values may be increased when DMSO intake is via the diet, with or without DMSO in the medium (Figure 2).
Specifically, considering the conditions characterized by the culture medium without DMSO, the treatments in which the mortality of D. magna was significantly higher, with respect to the comparison condition, correspond to the RS (DMSO) + TO (DMSO) and RS (DMSO) + TO treatment (Table 3, D. magna Feeding with two algae in ACM).
In the conditions characterized by the presence of DMSO in the medium, the treatment in which D. magna mortality was significantly higher, than in the comparison condition, corresponds to RS (DMSO) + TO (DMSO) + DMSO, see Table 3, D. magna Feeding with two algae in ACM + DMSO.

3.3.2. Ceriodaphnia dubia

The ANOSIM two-way test, performed considering the sixteen treatments for each species and their exposure times (24 h, 48 h, 72 h, 168 h), showed significant results for the “treatment” factor (Rho: 0.406; significance level: 0.01%; number of permutations: 9999; number of permuted statistics greater than or equal to mean Rho: 0) and for the “time” factor (Rho: 0.392; significance level: 0.01%; number of permutations: 9999; number of permuted statistics greater than or equal to mean Rho: 0).
Figure 3 (Ceriodaphnia dubia) provides the percentage ranges of the mean mortality values recorded for each of the sixteen treatments, without distinguishing the exposure time.
The following treatments exhibited an average mortality rate of less than 20%:
  • Condition 1: RS;
  • Condition 5: TO; 6: TO (DMSO); 7: TO + DMSO; 8: TO (DMSO) + DMSO;
  • Condition 9: RS + TO; 13: RS + TO + DMSO;
  • Condition 11: RS + TO (DMSO).
Average mortality values close to 80% were recorded in condition 12, corresponding to the RS (DMSO) + TO (DMSO) treatment, and condition 14, corresponding to the RS (DMSO) + TO + DMSO treatment; rather high mortality rates were also recorded in condition 16, corresponding to the RS (DMSO) + TO (DMSO) + DMSO treatment.
It should be noted that in the treatments with two algal species, one of which was pretreated with DMSO (conditions 10, 11, 14, 15), independently of the presence of DMSO in the medium, the highest mortality rates were observed in the treatments where the algal species R. subcapitata (RS) was pretreated with DMSO (conditions 10 and 14).
No statistically significant differences were observed in the biological response between treatments characterized by the presence of a single algal species (RS or TO) in a culture medium without DMSO (ACM). See Table 3: C. dubia Feeding with one alga in ACM.
Conversely, in the biological response detected among treatments characterized by the presence of one algal species (RS or TO) in culture medium with DMSO, there was significantly higher mortality in the RS + DMSO and RS (DMSO) + DMSO condition (see Table 3, C. dubia Feeding with one alga in ACM + DMSO).
Considering the treatments with both species of algae (RS and TO) in DMSO-free culture medium, the condition in which there is a significantly higher mortality of C. dubia, in relation to the comparison condition, corresponds to the RS (DMSO) + TO (DMSO) treatment (Table 3, C. dubia Feeding with two algae in ACM).
Finally, in the treatments containing both algal species (RS and TO) in culture medium containing DMSO (Table 3, C. dubia Feeding with two algae in ACM + DMSO), the conditions under which a significantly higher mortality of C. dubia was recorded, in relation to the comparison condition, are as follows:
  • RS (DMSO) + TO (DMSO);
  • RS (DMSO) + TO;
  • RS + TO (DMSO).

3.4. Controls

Table S5 provides the biological response of the organisms (mortality %, in terms of mean and ±SD) recorded from the controls established for D. magna and C. dubia. The results indicate that the Ceriodaphtoxkit F® (KIT) water, selected for its proven suitability for algal growth (refer to the Section 2.2.3), is compatible with D. magna (mean mortality at 24 and 48 h equal to 0%). In addition, considering the negative control established by the standard for each species (ISO water for D. magna and KIT water for C. dubia) confirms the validity of the test (mortality less than 10%), see Section 2.5 and Table S5. The combinations of H2O + NUT, H2O + DMSO, and H2O + DMSO + NUT provide the contribution of the individual elements that make up the different media under non-feeding conditions; it is underlined that the condition set for the test instead corresponds to a feeding condition. Considering D. magna, it can be observed that the most marked difference between ISO and KIT water is related to the DMSO + NUT treatment (the greatest biological response is recorded with ISO water, at all exposure times).
With reference to the controls of R. subcapitata and T. obliquus (Table S6), which were set up to check algal growth (considering that the test on D. magna and C. dubia was set up under feeding conditions), the values (in terms of mean and ±SD) suggest a growth condition (evaluated with respect to the control consisting of algal culture medium, ACM ISO), especially considering the treatments KIT + NUT and KIT + DMSO + NUT. Again, the contribution of the elements constituting the medium, both individually and in combination (KIT + NUT; KIT + DMSO; KIT + DMSO + NUT), is reported in Table S6.
The control results suggest that the medium selected (KIT) represents an optimal compromise for all organisms tested.

4. Discussion

DMSO is an aprotic polar solvent widely used in biological research [36] and is considered the gold standard in its category [5]; its success is due to its low toxicity, its ability to solubilize a wide range of polar and non-polar substances, its ability to permeate biological membranes without compromising their structural integrity, and its capacity to inhibit bacterial proliferation in an aquatic environment [36,37]. However, in an ecotoxicological test, the use of co-solvents can potentially alter the dose–response curve [38] and international authorities have proposed acceptable concentrations [7,8] although they are not representative of all species, and the dose considered safe for some organisms may be toxic to others [10].
The aim of the present study is to provide useful information for the selection of the model organism between Daphnia magna and Ceriodaphnia dubia for use in toxicity tests with DMSO (under feeding conditions).
D. magna and C. dubia are two closely related species of invertebrates that are typical inhabitants of freshwater environments. C. dubia has a shorter life cycle than D. magna, its use in chronic tests can reduce exposure times from twenty-one to seven days [13], and it is in chronic tests that the sublethal effects of the solvent are most evident [9].
Additionally, the study aimed to assess the potential consequences of the uptake of 0.5% DMSO present in the aqueous medium and/or introduced through the diet, considering it as a model (or a potential contaminant). This approach takes into account that simultaneous exposure to a substance represents a more realistic scenario of environmental contamination [12], contributing to both methodological and ecological perspectives [19].

4.1. D. magna vs. C. dubia: Sensitivity

D. magna is a species that has been used for decades as a model organism in various fields of research [39] while C. dubia has been less successful (compared to D. magna), but over time, the issues related to the use of this species have been progressively alleviated [22].
In the first paragraph, it was discussed how C. dubia may be equally sensitive, more sensitive, or less sensitive than D. magna, depending on the chemicals tested. Given the widespread use of DMSO as a solubilizing agent, the study investigated whether the mortality of the two species (D. magna, C. dubia) exposed to the solvent is comparable, considering a maximum exposure time of seven days (168 h) in acute and chronic toxicity tests, under feeding conditions.
The ANOSIM one-way test showed a significant difference in the detected mortality between the two species considering all exposure conditions. In particular, C. dubia was found to be overall more sensitive to DMSO than D. magna, showing significantly higher mortality values (Table S4).
An analysis of mortality trends over time (Figure 1) demonstrates that, at 24 h of exposure, the sensitivity of the two species is quite comparable, with both exhibiting mortality rates within the 20% range. At 48 h (acute exposure), C. dubia has a wide range of mortality values, but the average remains below 20%. Conversely, D. magna has a very narrow range of mortality values at 48 h. In this case, the wide range in which C. dubia mortality values are distributed depends on the mortality associated with all sixteen experimental conditions (or treatments), with conditions characterized by high toxicity and/or suboptimal feeding conditions having a strong effect on survival, but not particularly affecting the average of the sixteen treatments. After 72 h of exposure, the average mortality of C. dubia exceeds 20%, vice versa for D. magna (average mortality below 20%; see Figure 1). Finally, at 168 h (seven days), the range of mortality values covered by the two species seems to be almost overlapping. Considering the exposure times, the average mortality suggests a higher sensitivity of C. dubia to DMSO.
From the t-test comparisons, considering the RS (DMSO) + TO (DMSO) and RS (DMSO) + TO (DMSO) + DMSO treatments, for which the mean biological response of D. magna and C. dubia is among the highest recorded (see Figure 2 and Table S4), no significant differences in mortality are found. This suggests that maximum DMSO intake, either via diet and culture medium (at 0.5%) or via diet alone (via algae pretreated with 0.5% DMSO), may equally affect the mortality of D. magna and C. dubia.
Moreover, the significant differences found between the mortality values of the two species compared occurred in conditions characterized by the presence of DMSO in the medium (+DMSO), in five out of the six statistically significant conditions (see Table 3, D. magna vs. C. dubia). Overall, from these conditions, it appears that D. magna is less sensitive than C. dubia to 0.5% concentrations of DMSO in the culture medium (under feeding conditions), except in the TO + DMSO treatment where the mean mortality of D. magna is significantly higher at 72 h and 168 h.
However, C. dubia is characterized by a lower LC50 than D. magna when considering the specific case of solvents [40]. For instance, considering acute exposure to ethanol, the LC50 for C. dubia is 6942 mg/L, while that for D. magna is 9248 mg/L [18]. Similarly, in the case of chronic exposure to ethanol, using reproduction inhibition as the endpoint, the EC50 for C. dubia was found to be lower than that for D. magna, with values of 9.6 mg/L and 14 mg/L, respectively [18].
The results suggest that C. dubia exhibits a greater sensitivity than D. magna.

4.2. ACM vs. ACM + DMSO

4.2.1. Daphnia magna

Through comparisons conducted via a t-test, with and without DMSO in the medium (ACM vs. ACM + DMSO), it was observed that a 0.5% concentration of DMSO in the culture medium did not exert a significant impact on mortality in the majority of the conditions assessed, aligning with the findings reported by Andrade-Vieira et al. [10], except in the TO + DMSO treatment at 168 h (Table 3, Daphnia magna ACM vs. ACM + DMSO). This suggests that a 0.5% concentration of DMSO does not have significant effects on the mortality of D. magna, both in acute (24 h, 48 h) and chronic (168 h) exposures.
However, 168 h (seven days) is not considered a sufficient duration to fully represent chronic exposure; therefore, it cannot be excluded that a twenty-one-day exposure at this concentration might significantly impact mortality.
There were no significant differences in the mortality of D. magna related to the presence of DMSO in the culture medium, even in the conditions where only one DMSO pretreated algal species (RO or TO) was provided, as well as in the conditions where both algal species (RO and TO) pretreated in DMSO were provided. In contrast, there was a significantly higher mortality in the RS (DMSO) + TO treatment (without DMSO in the culture medium), suggesting a feeding preference for R. subcapitata and a greater sensitivity to DMSO via dietary exposure rather than to DMSO in the culture medium.
Finally, although no significant differences in mortality were observed in conditions where both algal species (RS and TO) were pretreated with DMSO, with or without DMSO in the medium, the average mortality values at the four exposure times are comparable; see Figure 2, Daphnia magna RS (DMSO) + TO (DMSO) vs. RS (DMSO) + TO (DMSO) + DMSO. Consequently, it can be hypothesized that both conditions (algae pretreated with 0.5% DMSO without DMSO in the culture medium and algae pretreated with 0.5% DMSO + DMSO in the culture medium) influence the mortality of D. magna (based on % mortality, see Table S5) but the dietary uptake of DMSO appears to be the component mainly responsible for the mortality recorded.
In conclusion, the data suggest that for Daphnia magna, in nearly all the experimental conditions analyzed, the observed mortality is primarily attributable to the ingestion of DMSO through feeding (i.e., through DMSO-pretreated algae) rather than the presence of DMSO in the culture medium.

4.2.2. C. dubia

From the comparison t-test, for the same exposure time and under the same feeding conditions, considering the presence or absence of DMSO in the algal culture medium, significant differences in the mortality of C. dubia were observed (Table 3, C. dubia ACM vs. ACM + DMSO). These results suggest that, in these cases, the presence of 0.5% DMSO in the culture medium can significantly increase mortality.
Additionally, at 24 h, exposure to RS (DMSO) alone did not impact the survival of C. dubia (0% mortality). In contrast, the addition of DMSO to the culture medium, RS (DMSO) + DMSO, resulted in an average mortality of 47% at 24 h (see Figure 2 and Table S4, Ceriodaphnia dubia).
Significant differences in mortality, based on the presence of DMSO in the culture medium, were also observed when comparing conditions with both algal species (RS and TO) and only one species pretreated with DMSO. These differences were observed as early as 48 h into the test and persisted up to 168 h (see Table 3, C. dubia, Feeding with two algae in ACM + DMSO). This result suggests that moderate dietary exposure to DMSO (understood as the availability of pretreated and untreated algae) in combination with 0.5% DMSO in the culture medium is potentially capable of increasing mortality levels.
In the literature, there are no specific EC50 values for DMSO in C. dubia. However, according to Cowgill and Milazzo [18], EC50 values for other common solvents are generally lower for C. dubia than for D. magna, indicating a greater sensitivity of the first species.
In this case, while for D. magna it is recommended not to exceed a 0.5% DMSO concentration in the preparation of solutions for ecotoxicological assays [10], for C. dubia, based on the results obtained, the “safe” concentration should be lower (considering the experimental conditions of this study).

4.3. Evaluation of the Effects of DMSO Ingestion by the Dietary Route and Food Preferences

4.3.1. Daphnia magna

The highest average mortality rates (90–100%) were observed in the condition where both algal species (R. subcapitata and T. obliquus) pretreated with DMSO were provided as food, RS (DMSO) + TO (DMSO), regardless of the presence of DMSO in the culture medium (Table S4, D. magna). The mortality values in the RS (DMSO) + TO (DMSO) and RS (DMSO) + TO (DMSO) + DMSO treatments were comparable, considering equivalent exposure times. This suggests that, irrespective of the solvent’s presence in the medium, dietary exposure to DMSO (modeled as a potential contaminant) is the primary route that affects survival. Conversely, DMSO ingested through one of the two algal species in the medium (one of them pretreated with DMSO) does not cause the same effects on mean mortality and may be a consequence of dietary selection in favor of the algae not pretreated (or not contaminated) with DMSO (Figure 2, Daphnia magna).
The observation of higher mortality rates in the presence of both pretreated algal species, compared to treatments with only one algal species pretreated with DMSO, suggests that the initial dietary intake is greater when both pretreated (contaminated) species are available. This increased intake may be responsible for the higher mortality observed.
It should be noted that there was a higher mortality in the RS (DMSO) + TO treatment than in the RS + TO (DMSO) treatment, although the difference was not statistically significant, see Table S4, D. magna. However, this result suggests that among the two algal species, R. subcapitata may be a preferential food and for this reason it can be selected, even if contaminated (pretreated with DMSO).
In terms of food preferences and in the absence of contamination, there were no statistically significant differences between organisms fed one type of alga rather than the other (RS or TO).
Conversely, there was a significantly lower mean mortality (at 72 h and 168 h) in organisms exposed to the RS + DMSO treatment (see Table 3, D. magna Feeding with one alga + ACM + DMSO). This result suggests that feeding with one algal species associated with an improved metabolic status may be crucial.
In the comparison between the RS (DMSO) vs. TO (DMSO) treatments at 168 h, lower mortality is observed in the TO (DMSO) treatment (see Table 3, D. magna Feeding with one alga + ACM + DMSO). Indeed, if R. subcapitata represents a dietary preference, its presence would likely result in higher mortality rates when the algae are pretreated with DMSO.
Although no studies specifically examining the feeding preference of Daphnia magna for the algal species Raphidocelis subcapitata and Tetradesmus obliquus are available in the literature, standardized ecotoxicity testing protocols suggest R. subcapitata as the preferred algal species for feeding D. magna. In fact, both the U.S. EPA/600/8–87/011 protocol [41] and the APAT 8020 protocol [34] recommend R. subcapitata as the algal species to be used as food for D. magna.
According to Yin et al., 2010 [42], the feeding preferences of Daphnia magna may depend on factors such as algal cell size, the search time associated with a specific food source, handling time, nutritional content, and assimilation efficiency. Moreover, the dietary choices of D. magna could represent a trade-off between small algae, which are quickly captured, and larger algae, which require a longer handling time but offer higher energetic gains per unit of food.

4.3.2. Ceriodaphnia dubia

As in the case of D. magna, high mortality was observed in the RS (DMSO) + TO (DMSO) treatment, with 100% mortality at 168 h, both compared to the RS + TO treatment and compared to treatments characterized by two algal species, one of which was pretreated with DMSO (see Figure 2, Ceriodaphnia dubia); statistically significant differences are observed at all experimental times (Table 3, C. dubia Feeding with two algae in ACM).
These results suggest some degree of sensitivity associated with dietary exposure to DMSO.
Also, for C. dubia, the high mortality recorded in the RS (DMSO) + TO (DMSO) treatment, compared to conditions characterized by only one algal species (RS or TO) pretreated in DMSO, suggests that in the presence of a contaminated algae, C. dubia could respond by reducing its feeding rate after ingesting a quantity of contaminated algae.
Again, when two pretreated (contaminated) algal species are present in the culture medium, the initial food intake is higher and could be responsible for the increased mortality.
Considering the treatments characterized by DMSO in the culture medium and the presence of two algal species (Table 3, C. dubia Feeding with two algae in ACM + DMSO), the results suggest that DMSO uptake through one of the two pretreated algae was sufficient to significantly affect the mortality of the organisms, suggesting a higher sensitivity to DMSO associated with C. dubia (compared to D. magna), under experimental conditions.
When examining the mortality percentages recorded in the RS or TO treatments (exposure of C. dubia to a single algal species), Table S4, no significant differences in organism mortality were observed for C. dubia. This suggests that feeding Raphidocelis subcapitata or Tetradesmus obliquus does not have a significant effect on survival.
Furthermore, at 168 h, with or without DMSO in the medium, the presence of two algal species (one pretreated with DMSO) resulted in higher mortality (though not statistically significant) when the DMSO-contaminated species was Raphidocelis subcapitata (Figure 2; Table S4). This observation suggests that when both R. subcapitata and T. obliquus are present, C. dubia shows a preference for R. subcapitata, even when it is contaminated with DMSO. The higher feeding rate and subsequent increased mortality of C. dubia in the presence of R. subcapitata suggest a feeding preference.
Indeed, Patterson et al. [43] identified Raphidocelis subcapitata as one of the two best dietary regimes for C. dubia, along with a yeast-based diet. Additionally, the APAT 8040 method [27] designates R. subcapitata as a suitable algal species for feeding C. dubia.
As with D. magna, factors that may explain the dietary preference for one algal species over another include algal size, handling time, nutritional value, and assimilation efficiency [42]. It is likely that the trade-off between handling time and energy gain favors R. subcapitata over T. obliquus (considering D. magna and C. dubia).
However, further studies could be valuable for better understanding the dynamics of potential dietary preferences.

5. Conclusions

In conclusion, considering the experimental conditions described, this study suggests that there is a difference in sensitivity to 0.5% DMSO between the model organisms D. magna and C. dubia, both in acute exposures (at 48 h) and in chronic exposures (up to 168 h). Most of the significant differences in the mean mortality (%) between the two species, given the same time exposure and the same experimental condition, were observed in the treatments in which DMSO was present in the culture medium (see Table 3, D. magna vs. C. dubia). The presence of 0.5% DMSO concentration in the culture medium was generally not found to significantly affect D. magna, while significant differences in mean mortality were recorded for C. dubia, attributed to the presence of 0.5% DMSO in the culture medium in about half of the experimental conditions (or treatments).
Considering dietary exposure to DMSO, C. dubia has generally higher mortality rates compared to D. magna.
Furthermore, ingestion of DMSO-contaminated algae (as a model of a potential contaminant) was demonstrated to have significant effects on the mortality of both D. magna and C. dubia under certain experimental conditions. In particular, the dietary intake of two DMSO-contaminated (pretreated) algal species was found to be particularly harmful to both daphnids.
This study also suggests a degree of feeding preference for R. subcapitata in both D. magna and C. dubia, as there appears to be some selection when the alga is pretreated with 0.5% DMSO. However, under DMSO-free conditions, no significant difference in mortality between the ingested algal species (R. subcapitata, T. obliquus) was observed for either D. magna or C. dubia.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/w17020191/s1, Table S1: mineral salt composition of the waters compared; Table S2: n. of cells; cell density; n. of cells/mL and optical density; Table S3: optical density (0–168 h); Figure S1: algal growth and waters; Table S4: biological response (mortality %) recorded for D. magna and C. dubia; Table S5: controls (D. magna, C.dubia); Table S6: controls (R. subcapitata, T. obliquus); Figure S2: non-metric multidimensional scaling (n-MDS).

Author Contributions

Conceptualization and formal analysis, M.R.; methodology and software, S.A.; validation, K.S.; investigation, S.B. and F.P.; resources, M.R.; data curation, S.A., S.B. and K.S.; writing—original draft preparation, K.S.; writing—review and editing, S.A., K.S. and M.R.; visualization, supervision, project administration, funding acquisition, M.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding and was supported by the Bioscience Research Center (grant number RG0022024, grant holder Monia Renzi).

Data Availability Statement

Data are reported in the Supplementary Materials.

Acknowledgments

Authors are grateful to the administrative and technical staff of BsRC.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Li, X.; Wang, X.; Li, Y.; Xiao, J.; Du, Y. Application of DMSO as a Methylthiolating Reagent in Organic Synthesis. Org. Biomol. Chem. 2022, 20, 4471–4495. [Google Scholar] [CrossRef] [PubMed]
  2. Xiang, J.; Gao, Q.; Wu, A. The Applications of DMSO. In Solvents as Reagents in Organic Synthesis; Wiley: Hoboken, NJ, USA, 2017; pp. 315–353. [Google Scholar] [CrossRef]
  3. Szmant, H.H. Physical Properties of Dimethyl Sulfoxide and Its Function in Biological Systems. Ann. N. Y. Acad. Sci. 1975, 243, 20–23. [Google Scholar] [CrossRef] [PubMed]
  4. Galvao, J.; Davis, B.; Tilley, M.; Normando, E.; Duchen, M.R.; Cordeiro, M.F. Unexpected Low-Dose Toxicity of the Universal Solvent DMSO. FASEB J. 2014, 28, 1317–1330. [Google Scholar] [CrossRef]
  5. Kaczor-Kamińska, M.; Kaszuba, K.; Bilska-Wilkosz, A.; Iciek, M.; Wróbel, M.; Kamiński, K. Dimethyl Sulfoxide (DMSO) as a Potential Source of Interference in Research Related to Sulfur Metabolism—A Preliminary Study. Antioxidants 2024, 13, 582. [Google Scholar] [CrossRef] [PubMed]
  6. Huang, Y.; Cartlidge, R.; Walpitagama, M.; Kaslin, J.; Campana, O.; Wlodkowic, D. Unsuitable Use of DMSO for Assessing Behavioral Endpoints in Aquatic Model Species. Sci. Total Environ. 2018, 615, 107–114. [Google Scholar] [CrossRef] [PubMed]
  7. OECD. Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures; OECD Series on Testing and Assessment; OECD Publishing: Paris, France, 2019. [Google Scholar] [CrossRef]
  8. Committee on Methods for Toxicity Tests with Aquatic Organisms, N.E.R.C. Methods for Acute Toxicity Tests with Fish, Macroinvertebrates, and Amphibians; EPA 660/3-75-009; United States Environmental Protection Agency: Corvallis, OR, USA, 1975.
  9. Stevens, A.; Pirotte, N.; Plusquin, M.; Willems, M.; Neyens, T.; Artois, T.; Smeets, K. Toxicity Profiles and Solvent–Toxicant Interference in the Planarian Schmidtea mediterranea after Dimethylsulfoxide (DMSO) Exposure. J. Appl. Toxicol. 2015, 35, 319–326. [Google Scholar] [CrossRef] [PubMed]
  10. Andrade-Vieira, L.F.; Bojic, C.; Santana Alvarenga, I.F.; de Carvalho, T.S.; Masfaraud, J.-F.; Cotelle, S. Ecotoxic Effects of the Vehicle Solvent Dimethyl Sulfoxide on Raphidocelis subcapitata, Daphnia magna, and Brachionus calyciflorus. Chem. Ecol. 2022, 38, 471–483. [Google Scholar] [CrossRef]
  11. Hook, S.E.; Fisher, N.S. Sublethal Effects of Silver in Zooplankton: Importance of Exposure Pathways and Implications for Toxicity Testing. Environ. Toxicol. Chem. 2001, 20, 568–574. [Google Scholar] [CrossRef]
  12. Wilding, J.; Maltby, L. Relative Toxicological Importance of Aqueous and Dietary Metal Exposure to a Freshwater Crustacean: Implications for Risk Assessment. Environ. Toxicol. Chem. 2006, 25, 1795–1801. [Google Scholar] [CrossRef]
  13. Connors, K.A.; Brill, J.L.; Norberg-King, T.; Barron, M.G.; Carr, G.; Belanger, S.E. Daphnia magna and Ceriodaphnia dubia Have Similar Sensitivity in Standard Acute and Chronic Toxicity Tests. Environ. Toxicol. Chem. 2022, 41, 134–147. [Google Scholar] [CrossRef] [PubMed]
  14. Manar, R.; Vasseur, P.; Bessi, H. Chronic Toxicity of Chlordane to Daphnia magna and Ceriodaphnia dubia: A Comparative Study. Environ. Toxicol. 2012, 27, 90–97. [Google Scholar] [CrossRef]
  15. Constantine, L.A.; Huggett, D.B. A Comparison of the Chronic Effects of Human Pharmaceuticals on Two Cladocerans, Daphnia magna and Ceriodaphnia dubia. Chemosphere 2010, 80, 1069–1074. [Google Scholar] [CrossRef] [PubMed]
  16. Raby, M.; Zhao, X.; Hao, C.; Poirier, D.G.; Sibley, P.K. Relative Chronic Sensitivity of Neonicotinoid Insecticides to Ceriodaphnia dubia and Daphnia magna. Ecotoxicol. Environ. Saf. 2018, 163, 238–244. [Google Scholar] [CrossRef] [PubMed]
  17. Hickey, C.W. Sensitivity of Four New Zealand Cladoceran Species and Daphnia magna to Aquatic Toxicants. N. Z. J. Mar. Freshwater Res. 1989, 23, 131–137. [Google Scholar] [CrossRef]
  18. Cowgill, U.M.; Milazzo, D.P. The Sensitivity of Ceriodaphnia dubia and Daphnia magna to Seven Chemicals Utilizing the Three-Brood Test. Arch. Environ. Contam. Toxicol. 1991, 20, 211–217. [Google Scholar] [CrossRef]
  19. Clements, W.H.; Rohr, J.R. Community Responses to Contaminants: Using Basic Ecological Principles to Predict Ecotoxicological Effects. Environ. Toxicol. Chem. 2009, 28, 1789–1800. [Google Scholar] [CrossRef] [PubMed]
  20. Pakrashi, S.; Dalai, S.; Chandrasekaran, N.; Mukherjee, A. Trophic Transfer Potential of Aluminium Oxide Nanoparticles Using Representative Primary Producer (Chlorella ellipsoides) and a Primary Consumer (Ceriodaphnia dubia). Aquat. Toxicol. 2014, 152, 74–81. [Google Scholar] [CrossRef]
  21. Akter, S.; Wilfert, K.; Saliu, O.R.; Schoelynck, J.; De Boeck, G. Feeding on Stressed Algae Exerts Important Effects on Life History Traits of Daphnia magna in a Multi-Stressor Environment. Aquat. Toxicol. 2024, 273, 106988. [Google Scholar] [CrossRef] [PubMed]
  22. Versteeg, D.J.; Stalmans, M.; Dyer, S.D.; Janssen, C. Ceriodaphnia and Daphnia: A Comparison of Their Sensitivity to Xenobiotics and Utility as a Test Species. Chemosphere 1997, 34, 869–892. [Google Scholar] [CrossRef]
  23. Ebert, D. Daphnia as a Versatile Model System in Ecology and Evolution. Evodevo 2022, 13, 16. [Google Scholar] [CrossRef] [PubMed]
  24. Reilly, K.; Ellis, L.-J.A.; Davoudi, H.H.; Supian, S.; Maia, M.T.; Silva, G.H.; Guo, Z.; Martinez, D.S.T.; Lynch, I. Daphnia as a Model Organism to Probe Biological Responses to Nanomaterials—From Individual to Population Effects via Adverse Outcome Pathways. Front. Toxicol. 2023, 5, 1178482. [Google Scholar] [CrossRef] [PubMed]
  25. Lauridsen, T.L.; Jeppesen, E.; Mitchell, S.F.; Lodge, D.M.; Burks, R.L. Diel Variation in Horizontal Distribution of Daphnia and Ceriodaphnia in Oligotrophic and Mesotrophic Lakes with Contrasting Fish Densities. Hydrobiologia 1999, 408, 241–250. [Google Scholar] [CrossRef]
  26. UNI EN ISO 6341:2013; Water Quality—Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea)—Acute Toxicity Test. UNI—Ente Nazionale Italiano di Unificazione: Milano, Italy, 2013.
  27. APAT; CNR–IRSA. Metodi Analitici per le Acque, Manuali e Linee Guida 29/2003, Vol. 3, 8040: Metodo di Valutazione Della Tossicità Acuta con Ceriodaphnia dubia; APAT: Rome, Italy, 2003. [Google Scholar]
  28. Machado, M.D.; Soares, E.V. Features of the Microalga Raphidocelis subcapitata: Physiology and Applications. Appl. Microbiol. Biotechnol. 2024, 108, 219. [Google Scholar] [CrossRef]
  29. do Carmo Cesário, C.; Soares, J.; Cossolin, J.F.S.; Lima, G.L.; Basso, L.A.; Azevedo, L.F.; Cavalcanti, J.H.D.; Oliveira, V.M.; Pires, J.S.; Souza, R.R. Biochemical and Morphological Characterization of Freshwater Microalga Tetradesmus obliquus (Chlorophyta: Chlorophyceae). Protoplasma 2022, 259, 937–948. [Google Scholar] [CrossRef] [PubMed]
  30. Babu, B.T. Tetradesmus obliquus. Bakthavachalam Babu Freshwater Algae. Available online: https://bakthavachalam-babu.com/freshwater-algae/Chlorophyta/Tetradesmus%20obliquus (accessed on 31 December 2024).
  31. Oliveira, C.Y.B.; Oliveira, C.D.L.; Prasad, R.; Ong, H.C.; Araujo, E.S.; Shabnam, N.; Gálvez, A.O. A Multidisciplinary Review of Tetradesmus obliquus: A Microalga Suitable for Large-Scale Biomass Production and Emerging Environmental Applications. Rev. Aquacult. 2021, 13, 1594–1618. [Google Scholar] [CrossRef]
  32. UNI EN ISO 10253:2017; Water Quality—Marine Algal Growth Inhibition Test with the Diatom Phaeodactylum tricornutum and the Green Alga Pseudokirchneriella subcapitata (Formerly Selenastrum capricornutum). UNI—Ente Nazionale Italiano di Unificazione: Milan, Italy, 2017.
  33. UNI EN ISO 8692:2012; Water Quality—Fresh Water Algal Growth Inhibition Test with Unicellular Green. UNI—Ente Nazionale Italiano di Unificazione: Milan, Italy, 2012.
  34. APAT; CNR–IRSA. Metodi Analitici per le Acque, Manuali e Linee Guida 29/2003, Vol. 3, 8020: Metodo di Valutazione Della Tossicità con Daphnia; APAT: Rome, Italy, 2003. [Google Scholar]
  35. ISPRA. Batterie di Saggi Ecotossicologici per Sedimenti e Acque Interne. I Manuali di Ecotossicologia 88/2013; ISPRA: Rome, Italy, 2013.
  36. Sum, A.K.; de Pablo, J.J. Molecular Simulation Study on the Influence of Dimethylsulfoxide on the Structure of Phospholipid Bilayers. Biophys. J. 2003, 85, 3636–3645. [Google Scholar] [CrossRef]
  37. Kais, B.; Schneider, K.E.; Keiter, S.; Henn, K.; Ackermann, C.; Braunbeck, T. DMSO Modifies the Permeability of the Zebrafish (Danio rerio) Chorion—Implications for the Fish Embryo Test (FET). Aquat. Toxicol. 2013, 140–141, 229–238. [Google Scholar] [CrossRef]
  38. Negin, S.; Gokel, M.R.; Patel, M.B.; Sedinkin, S.L.; Osborn, D.C.; Gokel, G.W. The Aqueous Medium-Dimethylsulfoxide Conundrum in Biological Studies. RSC Adv. 2015, 5, 8088–8093. [Google Scholar] [CrossRef]
  39. Ahmed, S. Applications of Daphnia magna in Ecotoxicological Studies: A Review. J. Adv. Res. Biol. 2023, 6, 16–35. [Google Scholar]
  40. Hutchinson, T.H.; Shillabeer, N.; Winter, M.J.; Pickford, D.B. Acute and Chronic Effects of Carrier Solvents in Aquatic Organisms: A Critical Review. Aquat. Toxicol. 2006, 76, 69–92. [Google Scholar] [CrossRef] [PubMed]
  41. U.S. Environmental Protection Agency. Guidelines for the Health Risk Assessment of Chemical Mixtures; EPA/600/8-87/011; U.S. Environmental Protection Agency: Washington, DC, USA, 1987.
  42. Yin, X.W.; Liu, P.F.; Zhu, S.S.; Chen, X.X. Food Selectivity of the Herbivore Daphnia magna (Cladocera) and Its Impact on Competition Outcome between Two Freshwater Green Algae. Hydrobiologia 2010, 655, 15–23. [Google Scholar] [CrossRef]
  43. Patterson, P.W.; Dickson, K.L.; Waller, W.T.; Rodgers, J.H. The Effects of Nine Diet and Water Combinations on the Culture Health of Ceriodaphnia dubia. Environ. Toxicol. Chem. 1992, 11, 1023–1035. [Google Scholar] [CrossRef]
Water 17 00191 g001
Figure 1. The range of biological response (mortality %) of Daphnia magna and Ceriodaphnia dubia recorded at the four experimental times (24 h, 48 h, 72 h, 168 h), without distinction between treatments.
Figure 1. The range of biological response (mortality %) of Daphnia magna and Ceriodaphnia dubia recorded at the four experimental times (24 h, 48 h, 72 h, 168 h), without distinction between treatments.
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Figure 2. Biological response (mortality %, expressed as mean and ±SD) for each test organism (Daphnia magna, Ceriodaphnia dubia) at the experimental times (24 h, 48 h, 72 h, 168 h) of each treatment (indicated on the x-axis). Refer to Table 1 for the nomenclature of the treatments.
Figure 2. Biological response (mortality %, expressed as mean and ±SD) for each test organism (Daphnia magna, Ceriodaphnia dubia) at the experimental times (24 h, 48 h, 72 h, 168 h) of each treatment (indicated on the x-axis). Refer to Table 1 for the nomenclature of the treatments.
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Figure 3. Range of the mean biological response (mortality %) recorded for each treatment (numbered from 1 to 16), without distinguishing between exposure times.
Figure 3. Range of the mean biological response (mortality %) recorded for each treatment (numbered from 1 to 16), without distinguishing between exposure times.
Water 17 00191 g003
Table 1. The table provides the list of treatments (or conditions), and associated nomenclature, for each species (D. magna and R. subcapitata). In the nomenclature, the species acronym RS or TO, when accompanied by DMSO in parentheses, “(DMSO)”, indicates the pretreatment of DMSO carrier algae. The words “+DMSO” refer to the solvent in the culture medium. The medium consists of artificial freshwater (see Section 2.2.3 for dilution medium specifications) and nutrients (see Section 2.2.2 for nutrient specifications).
Table 1. The table provides the list of treatments (or conditions), and associated nomenclature, for each species (D. magna and R. subcapitata). In the nomenclature, the species acronym RS or TO, when accompanied by DMSO in parentheses, “(DMSO)”, indicates the pretreatment of DMSO carrier algae. The words “+DMSO” refer to the solvent in the culture medium. The medium consists of artificial freshwater (see Section 2.2.3 for dilution medium specifications) and nutrients (see Section 2.2.2 for nutrient specifications).
SpeciesTreatmentsNomenclature
Daphnia magnaCeriodaphnia dubiaR. subcapitataRS
R. subcapitata pretreated with 0.5% DMSO RS (DMSO)
R. subcapitata + 0.5% DMSORS + DMSO
R. subcapitata pretreated with 0.5% DMSO +
0.5% DMSO in the medium		RS (DMSO) + DMSO
T. obliquusTO
T. obliquus pretreated with 0.5% DMSO TO (DMSO)
T. obliquus + 0.5% DMSO in the mediumTO + DMSO
T. obliquus pretreated with 0.5% DMSO +
0.5% DMSO in the medium		TO (DMSO) + DMSO
R. subcapitata + T. obliquusRS + TO
R. subcapitata pretreated with 0.5% DMSO + T. obliquus RS (DMSO) + TO
R. subcapitata + T. obliquus pretreated with 0.5% DMSORS + TO (DMSO)
R. subcapitata pretreated with 0.5% DMSO +
T. obliquus pretreated with 0.5% DMSO		RS (DMSO) + TO (DMSO)
R. subcapitata + T. obliquus + 0.5% DMSO in the mediumRS + TO + DMSO
R. subcapitata pretreated with 0.5% DMSO + T. obliquus +
0.5% DMSO in the medium		RS (DMSO) + TO + DMSO
R. subcapitata + T. obliquus pretreated with 0.5% DMSO +
0.5% DMSO in the medium		RS + TO (DMSO) + DMSO
R. subcapitata pretreated with 0.5% DMSO +
T. obliquus pretreated with 0.5% DMSO +
0.5% DMSO in the medium		RS (DMSO) + TO (DMSO) + DMSO
Table 2. Controls set up for all organisms involved in the experiment and their nomenclature.
Table 2. Controls set up for all organisms involved in the experiment and their nomenclature.
SpeciesControlsNomenclatureSpeciesControlsNomenclature
Daphnia magnaCeriodaphnia dubiaH2O ISO 6341ISORaphidocelis subcapitataTetradesmus obliquusAlgal Culture Medium
(ISO 8692:2012)		ACM ISO
H2O ISO 6341 + NUTRIENTSISO + NUTAlgal Culture Medium
(ISO 8692:2012) + 0.5% DMSO		ACM ISO + DMSO
H2O ISO 6341 + 0.5% DMSOISO + DMSO
H2O ISO 6341+ 0.5% DMSO +
NUTRIENTS		ISO + DMSO + NUTH2O CERIOKITKIT
H2O CERIOKITKITH2O CERIOKIT + NUTRIENTSKIT + NUT
H2O CERIOKIT +
NUTRIENTS		KIT + NUT
H2O CERIOKIT + 0.5% DMSOKIT + DMSOH2O CERIOKIT + 0.5% DMSOKIT + DMSO
H2O CERIOKIT + 0.5% DMSO +
NUTRIENTS		KIT + DMSO + NUTH2O CERIOKIT + 0.5% DMSO +
NUTRIENTS		KIT + DMSO + NUT
Table 3. The table summarizes the treatments that exhibited statistically significant differences (p-values) following comparisons performed at consistent exposure times (24 h, 48 h, 72 h, and 168 h).
Table 3. The table summarizes the treatments that exhibited statistically significant differences (p-values) following comparisons performed at consistent exposure times (24 h, 48 h, 72 h, and 168 h).
Treatmentsp-Values
D. magna vs. C. dubia 24 h 48 h 72 h 168 h
RS + DMSO –0.0050.0050.019
RS (DMSO) + DMSO 0.0250.0070.007–
TO + DMSO––0.0160.000
RS (DMSO) + TO (DMSO) 0.023–––
RS (DMSO) + TO + DMSO –0.0050.0000.033
RS + TO (DMSO) + DMSO –0.013––
D. magna
ACM vs. ACM + DMSO		24 h48 h72 h168 h
TO–––0.025
RS (DMSO) + TO ––0.016–
C. dubia
ACM vs. ACM + DMSO		24 h48 h72 h168 h
RS–0.0110.039–
RS (DMSO) 0.025–––
RS (DMSO) + TO –0.0390.0020.027
RS + TO (DMSO) –0.0130.0250.025
D. magna
Feeding with one alga + ACM		24 h48 h72 h168 h
Non-significant p-values
D. magna
Feeding with one alga + ACM + DMSO		24 h48 h72 h168 h
RS vs. TO ––0.0070.007
RS (DMSO) vs. TO (DMSO) ––-0.033
TO vs. TO (DMSO) ––0.0070.001
C. dubia
Feeding with one alga + ACM		24 h48 h72 h168 h
Non-significant p-values
C. dubia
Feeding with one alga + ACM + DMSO		24 h48 h72 h168 h
RS vs. TO –0.0050.0070.007
RS (DMSO) vs. TO (DMSO) 0.0250.0160.026–
D. magna
Feeding with two algae in ACM		24 h48 h72 h168 h
RS + TO vs. RS (DMSO) + TO (DMSO) ––0.0000.000
RS + TO vs. RS (DMSO) + TO ––0.016–
RS (DMSO) + TO vs. RS (DMSO) + TO (DMSO) ––0.008–
RS + TO (DMSO) vs. RS (DMSO) + TO (DMSO)––0.0020.001
C. dubia
Feeding with two algae in ACM		24 h48 h72 h168 h
RS + TO vs. RS (DMSO) + TO (DMSO) 0.0070.0030.0010.000
RS (DMSO) + TO vs. RS (DMSO) + TO (DMSO) 0.0470.0250.0020.014
RS + TO (DMSO) vs. RS (DMSO) + TO (DMSO)0.0470.0030.0010.000
D. magna
Feeding with two algae in ACM + DMSO		24 h48 h72 h168 h
RS + TO vs. RS (DMSO) + TO (DMSO) ––0.0020.001
RS (DMSO) + TO vs. RS (DMSO) + TO (DMSO) ––0.0000.033
RS + TO (DMSO) vs. RS (DMSO) + TO (DMSO)–––0.024
C. dubia
Feeding with two algae in ACM + DMSO		24 h48 h72 h168 h
RS + TO vs. RS (DMSO) + TO (DMSO) –0.0020.0080.011
RS + TO vs. RS (DMSO) + TO–0.0050.0010.001
RS + TO vs. RS + TO (DMSO) –0.0020.0270.045
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Bigi, S.; Schlappa, K.; Anselmi, S.; Provenza, F.; Renzi, M. Uptake Through Feeding and/or Culture Medium of 0.5% Dimethyl Sulfoxide (DMSO): Biological Response of Daphnia magna and Ceriodaphnia dubia in Ecotoxicity Tests. Water 2025, 17, 191. https://doi.org/10.3390/w17020191

AMA Style

Bigi S, Schlappa K, Anselmi S, Provenza F, Renzi M. Uptake Through Feeding and/or Culture Medium of 0.5% Dimethyl Sulfoxide (DMSO): Biological Response of Daphnia magna and Ceriodaphnia dubia in Ecotoxicity Tests. Water. 2025; 17(2):191. https://doi.org/10.3390/w17020191

Chicago/Turabian Style

Bigi, Stefania, Karin Schlappa, Serena Anselmi, Francesca Provenza, and Monia Renzi. 2025. "Uptake Through Feeding and/or Culture Medium of 0.5% Dimethyl Sulfoxide (DMSO): Biological Response of Daphnia magna and Ceriodaphnia dubia in Ecotoxicity Tests" Water 17, no. 2: 191. https://doi.org/10.3390/w17020191

APA Style

Bigi, S., Schlappa, K., Anselmi, S., Provenza, F., & Renzi, M. (2025). Uptake Through Feeding and/or Culture Medium of 0.5% Dimethyl Sulfoxide (DMSO): Biological Response of Daphnia magna and Ceriodaphnia dubia in Ecotoxicity Tests. Water, 17(2), 191. https://doi.org/10.3390/w17020191

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