Developmental effects on Daphnia magna induced by titanium dioxide and iron oxide mixtures

All Nanomaterials (TiO₂ and Fe₂O₃) were supplied as powders from the laboratory of chemistry (Guelma University Algeria) free of metals and organic impurities. The TiO₂ NPs (product number: 637254, particle size: 25 nm, 99.7% trace metals basis). The Fe₂O₃ were elaborated at the laboratory of chemistry; it was 26 nm in size. The elaboration of α-Fe₂O₃ nanoparticles was carried out using mechanical grinding at from the elemental powder of hematite. The milling was performed in a Pulverizing 7 planetary mill (Fritsch, Idar-Oberstein, Germany) using two steel jars (stainless steel with a volume of 45 ml). The preparation of the charge (beads and powders) was carried out in a glove box under argon atmosphere. The mass ratio beads to powders were approximately 1/20 and the grinding speed was at 500 rpm. To minimize the effects related to the temperature increase inside the jars, the grinding was done with sequences of half an hour followed by 15 min of pause and this for 3 h. Before the exposure experiments, stock solutions were sonicated for 30 min (50 W L⁻¹ at 40 kHz) and diluted with ultrapure water.

The freshwater crustacean D. magna used in this study were collected from a water pond in the Algerian park (Jardin d'essai du Hamma, Algiers; 36° 44' 53'' North, 3° 04' 34'' East). D. magna were maintained in unpolluted natural water at a constant temperature of 23.5 °C with a 16:8 h (light–dark) photoperiod at the University of Guelma, Algeria. D. magna cultures were maintained in 1 l glass beakers (20 individuals/beaker). The culture medium was renewed every 48 h, and daphnids were fed daily with Raphidocelis subcapitata at a concentration of (3 × 10⁵ cell/mL). Third or fourth generation neonates (<24 h) from the same female were collected to eliminate variation between clones. Individual neonates of similar size were exposed to different concentrations of NPs: In the immobilization test, various concentrations of (TiO₂ + Fe₂O₃) in a 1:1 fixed ratio (v/v), namely 0, 2.55, 7.75, 13, and 27.5 mg l⁻¹, were used. These concentrations were represented as control, C1, C2, C3, and C4, respectively. For the chronic test, concentrations of 50% (TiO₂ + Fe₂O₃) were employed in a 1/2:1/2 fixed ratio (v/v), specifically 0, 1.27, 3.87, 6.5, and 13.75 mg l⁻¹, which corresponded to control, C1, C2, C3, and C4, respectively.

Immobilization tests were performed based on the OECD guideline 202 (OECD 2004).Tests were carried out using sixteen replicates randomly per concentration and negative control, with eight neonates with ≤ 24 h per replicate exposed in 50 ml glass beakers. Tests were maintained at room temperature of 20 ± 1 °C and 16:8 h light: dark photoperiod and daphnids were not fed during the entire experiment. After 12, 24, and 48 h, immobilization (i.e., inability to swim within 15 s after gentle agitation of the beaker) and mortality were assessed using a Leica microscope equipped with a digital camera (Leica, Wetzlar, Germany) and then recorded (Ozmen et al 2023). The concentration that caused the immobilization of 50% of the neonates (LC50) was calculated. Nominal concentrations of single exposures of TiO₂ and Fe₂O₃ were (0.1 ; 0.5 ; 1 and 5 mgl⁻¹) and (5 ; 15 ; 25 and 50 mg ml⁻¹), respectively, were chosen according to their EC50 values and from previous modified published studies. Mixture concentrations of 100% (TiO₂ + Fe₂O₃) used were (0; 2.55 ; 7.75 ; 13 and 27.5 mg L⁻¹) of (control; C1; C2; C3; C4), respectively. Theses concentrations were chosen according to their EC50 values from the single exposures for each experimental setup.

The chronic toxicity of the mix of NPs TiO₂ + Fe₂O₃ was assessed according to an OECD standard protocol (OECD 211 1998) with modification. Based on the acute toxicity results, neonates (6–24 h old) where exposed for 21 days under the following concentrations that were chosen according to the acute test: 50% (TiO₂+ Fe₂O₃) (control); 1.27 ; 3.87 ; 6.5 and 13.75 mg l⁻¹ (figure 1).

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In this experiment, eight neonate (aged 6–24 h) were placed in each glass beakers containing 50 ml of test solution. A total of 16 randomly placed replicates of each concentration. Test containers were monitored every 24 h to record deaths or any anomaly. Daphnids were fed daily using Raphidocelis subcapitata at a concentration of (3 × 10⁵ cell mL). Each adult (and offspring if present) was carefully separated from beaker and test solutions were replaced each three days. The incubation was carried out at a temperature of 23 °C, under the same photoperiod conditions as those used in the culture experiment. The chronic effect endpoints recorded were used to evaluate the reproduction of D. magna including the age of maturation, and the number of eggs per female. The number of mortality of adults in each treatment was recorded during the exposure period and life span.

Growth rate was defined by the average increase in body length of individuals (8 ind of D. magna) in each measurement for each cup from the beginning of the experiment to the 21 days. The body length was measured from the anterior part of the head to the end of the body, excluding the caudal spine. It was calculated from photos taken every five days using a photo processing software image J.

The mortality rate (i.e., number of individuals dying at a specific time) was determined as the slope of the number of individuals dying versus time over the 21-day duration of the study. However, the life span was defined by the number of days of life of each individual in each cup throughout the period of the experiment.

Throughout the experiments, mature females were individually placed in separate cups, with one female per cup. These cups maintained the same conditions as the solution concentration, temperature, and daily feeding with Raphidocelis subcapitata. When the eggs hatched, we recorded the first brood's date and the number of eggs.

2.8.1. Swimming performance

2.8.2. Heart beat rate

Statistical analyses were carried out with R 4.0.2 (R Core Team 2021). The normality, homogeneity assumptions, and residual patterns in the data were checked (Zuur et al 2007). A linear mixed-effects models was used to compare the growth rate of the species between concentration treatments including concentration treatments as fixed effect and cups as random intercept, Tukey's post hoc test with glht function was applied using multcomp package (Hothorn et al 2008) to reveal potential differences in the growth rate between the different treatments. A separate generalised linear mixed effect model with Poisson error distribution with Log link function was applied to assess the difference of all traits: life span (the number of days of life of each individual in each cup), age at maturation (time in days until first brood of each individual), eggs number at the first brood, and mortality of the species between the treatments concentrations. This was followed by Tukey post hoc analyses for pairwise comparison of the different nanoparticle concentration exposures. The models included trait as a response variable, treatments concentrations as fixed effect and females ID or cups (to account for over-dispersion) as random effect. All models were fitted using the R package lme4 (Bates et al 2015). ANOVA analyses were conducted to determine the effects of the nanoparticle concentrations treatments on the swimming performance and heart beats count. To evaluate which levels of the nanoparticle treatment differed we additionally carried out Tukey pair-wise tests. The data was presented as mean ± standard error (SE). The minimum threshold of significance retained is p < 0.05.

Growth rate was defined by the average growth rate of body length of individuals in each measurement for each cup from the beginning of the experiment (21 days). ANOVA analysis of a general linear mixed effects model (LMER) showed a highly significant effect of treatment on growth rate of the species compared to the null model (χ2 = 637.78, Df = 4, p < 0.0001) (table 1 and figure 2). However, growth rate between the control group and the different nanoparticle concentration exposed groups showed statistically significant differences (table S1). Subsequent Tukey tests for pairwise comparisons of growth rate of length showed that the four concentrations of mixed nanoparticles exposures (C1, C2, C3, and C4) significantly affected the growth of length of D. magna compared to the control treatment, and the growth rate was higher at the control treatment. Individuals from the control group had an average growth rate of 0.0137 ± 0.0002 mm, as compared with the experimental group growth rates of 0.007 ± 0.0002 mm, 0.006 ± 0.0002 mm, 0.005 ± 0.0003 mm, 0.00453 ± 0.0004 mm, for C1, C2, C3, C4, respectively.

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Life span was defined by the number of days of life of each individual in each cup throughout the period of experiment. ANOVA analysis of a general linear mixed effects model (GLMER) showed a highly significant effect of treatment on life span of the species compared to the null model (χ2 = 1229.8, Df = 4, p < 0.0001) (table 2 and figure 3(a)). The Tukey post-hoc analysis (table S2) indicated that the number of days in which the organism was in life decreased significantly from an average of 20.1 ± 0.188 days in the control treatment to 10.7 ± 0.503, 9.58 ± 0.514, 7.45 ± 0.418, 7.19 ± 0.440 days in C1, C2, C3, and C4, respectively.

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Mortality rate (number of individual deaths in specific time) was obtained as the slope of number of died individuals versus time over the 21-day experimental period. Mortality of D. magna occurred in all exposure treatments including the control treatment with (20.48%). The mortality rate of D. magna increased significantly with the increase of mixed nonoparticles concentrations (χ2 = 62.32, Df = 4, p < 0.0001) (table 2 and figure 3(b)). The assessment of all pairwise combinations using the Tukey contrast test (table S3) revealed that the control group was the only group with a statistically significant difference in mortality rate compared to the four concentrations groups. Individuals from the control group had an average mortality of 1 ± 0.365 individual, as compared with the experimental group mortality of 6.94 ± 0.193, 7.19 ± 0.188, 7.62 ± 0.125, 7.62 ± 0.155 individual, in C1, C2, C3, and C4, respectively.

The concentration of nanoparticle affected the age at which the species became mature (χ2 = 81.15, Df = 4, p < 0.0001). The age of maturation differed between treatments (table 2 and figure 3(c)). Subsequent Tukey tests for pairwise comparisons (table S4) of age at maturation showed that the four concentration of mixed nanoparticles exposures (C1, C2, C3, C4) significantly impacted the date of the first brood compared to the control, increasing it to (14.1 ± 0.438, 15.8 ± 0.521, 18 ± 1.05, and 20.2 ± 0.479 days), respectively. The Tukey test revealed that the age at maturation was significantly greater in concentration C4 when compared to concentrations C1 and C2.

The concentration of nanoparticle affected egg number at the first brood of D. magna (χ2 = 141.91, Df = 4, p < 0.0001) (table 2 and figure 3(d)). The average number of eggs at the first brood in the control treatment was 11.6 ± 0.80 eggs. The Tukey post-hoc analysis (table S5) indicated that the four concentration of mixed nanoparticles exposures (C1, C2, C3, C4) significantly impacted the number of eggs at the first brood of D. magna compared to the control, decreasing it by (77%, 83%, 85%, and 88%), respectively. Notably, no difference of number of eggs was found between the four concentrations (table S5).

The mixed nanoparticle affected the swimming performance (F_{4, 35} = 112.9, df = 4, p < 0.0001) (figure 4(a)). Swimming speed differed significantly between the control treatment and the four concentrations treatments, the swimming speed decreasing from 0.942 ± 0.0178 mm min⁻¹ of the control treatment to (0.569 ± 0.0346, 0.504 ± 0.0125, 0.417 ± 0.0258, and 0.285 ± 0.0190 mm/min) for (C1, C2, C3, C4 treatment), respectively. There was a significant difference in swimming speed between the concentration C4 and the other three treatments concentration (C1, C2, C4), and it was lower in C4 (table S6).

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Six individuals were randomly selected from each group for heart rate testing. Although the mixed nanoparticle affected the heart beat rate (F_{4, 25} = 19.37, df = 4, p < 0.0001) (figure 4(b)).

Interestingly, the heart rate did not show significant changes at low concentrations in C1 and C2 compared to the control (319 ± 0.40, 321 ± 0.63, 305 ± 0.65 beats/min), respectively (table S7). There was a significant difference between C4 and all treatments, it was significantly higher in C4 with 79 beats/min more than control, and higher with (60, 58, and 30 beats/min) more than C1, C2, and C3, respectively.