For the Microtox test system, the calculations to estimate the EC50 and its 95% confidence limits were included in the Microtox Omni™ software provided by the manufacturer. For each test concentration, the Gamma function was calculated as (ASTM, 1995):
\({ {\Gamma }}_{t}= \frac{{I}_{c} }{{I}_{t}}\)-1 = \(\frac{{H}_{t}}{100-{H}_{t} }\) (1)
with \({I}_{c}\) = the average light reading of filtrates of the control solution, \({I}_{t}\) = the light reading of a filtrate of a particular concentration of the test material and \({H}_{t}=\)the percentage inhibition.
From the results, a linear regression between the concentration (C) and the Gamma function was computed according to the following equation (ASTM, 1995):
$$log{\Gamma }= b*log\text{C}+loga$$
2
In the above equation, log a is the intercept of the regression line with the ordinate at log\({\Gamma }\)= 0, corresponding to \({\Gamma }\) = 1. Therefore, the EC50 can be derived from the antilog of the ratio of the intercept divided by the slope.
Linear regression plots of observed \({\Gamma }\)/concentration and % effect/concentration values allowed to visualize the magnitude of toxicity change for a given compound. An example, as conducted herein, is given in Fig. 4 for values collected over the 5-15-30 min contact series for the pure toxic compound.
In the test with the Vibrio fischeri, to identify the optimal dilution range for the target, we considered a preliminary solution with 500 mg of the tested sample dispensed into 50 ml diluent and performed tests in a dilution series (1:10). The inhibition effect of the tested toxicant resulting from the preliminary bioassay with a concentration of 10 g/L was:
The test results indicated a toxicity interval between 1 g/L and 10 g/L. The resulting toxicity data within the chosen intervals of dilutions, in increasing order of toxicity and for incubation times of 15 and 30 min, are reported in Table 2.
No significant differences were observed between 15 min and 30 min exposure times, suggesting bacterial toxicity was complete after 15 min of exposure. The Microtox® test outcomes indicated in all sample’s acute toxic effects on Vibrio
fischeri at the highest test concentration. The toxicity measured in the sample of pure phosphorite was significantly higher than that of the two samples of the mixed material and sand, which shared a similar degree of toxicity.
Figure 5 shows the bioluminescence inhibition of Vibrio fischeri by the tested samples at 30 min exposure time. Significant differences of the responses of the strains to the addition of sand to the toxic sample extracts are visible from the comparison among the different compounds. In particular, 82% bioluminescence inhibition after 30 min exposure was recorded at the highest pollutant concentration. In other work (Mekki et al., 2017) reporting results on the usage of the luminescent bacteria for toxicity estimation of wastewater from the phosphate processing industry in a Mediterranean soil, a similar effect was measured.
The resultant EC50s of the analysed samples are displayed in Table 3.
Since 15 min exposure data did not permit the development of concentration-response relationship (EC50 calculated from two data points), the associated values for the 95% confidence intervals were given only for 30 min exposure time data.
Table 2
Relative toxicity of the samples expressed as a percentage of luminescence inhibition compared to the controls for the tests with the marine bacteria Vibrio fischeri
Concentration g/L | Test sample (pure) Phosphorite 10 g/L | Test sample (+) Phosphorite 10 g/L Sand 10 g/L | Test sample (++) Phosphorite 10 g/L Sand 50 g/L |
| 15 min % inhibition | 30 min % inhibition | 15 min % inhibition | 30 min % inhibition | 15 min % inhibition | 30 min % inhibition |
0.625 | -2.047 | 0.539 | -6.726 | -2.230 | -3.351 | 2.542 |
1.250 | 0.184 | 7.270 | -7.334 | -1.933 | -2.408 | 2.780 |
2.500 | 5.252 | 9.581 | -1.384 | 5.421 | -1.056 | 5.377 |
5.000 | 40.170 | 41.070 | 13.050 | 19.370 | 13.620 | 18.310 |
10.000 | 78.740 | 81.630 | 54.860 | 57.540 | 51.220 | 55.450 |
Table 3
Acute toxicity data (95% confidence interval) of target compounds on Vibrio fischeri
Compound EC50 (g/L) | Test sample (pure) Phosphorite 10 g/L | Test sample (+) Phosphorite 10 g/L Sand 10 g/L | Test sample (++) Phosphorite 10 g/L Sand 50 g/L |
15 min EC50 | 6.22 g/L | 9.38 g/L | 9.82 g/L |
30 min EC50 [95% CL] | 5.56 g/L [1.23–25.12] | 8.94 g/L [2.50–32.05] | 9.31 g/L [2.03–42.54] |
The 15 min EC50 values ranged from 6.22 g/L to 9.82 g/L and the 30 min EC50 values from 5.56 g/L to 9.31 g/L.
The present findings documented the toxic nature of all the analysed samples, showing that potentially harmful effects are reduced by the presence of sand addition. It is also worth emphasizing that the adverse effects of all compounds to luminescent bacteria is significant, as evidenced by about 51% inhibition of luminescence at 15 min exposure to the less toxic mixture.
The above results indicate toxicity for concentrations above 100 mg/L, thus representing in principle a low contamination risk for biodiversity with respect to several known pollutants. Anyway, concentration levels in the waste material hosted in the study area are comparable to and even greater than the measured thresholds.
The above data are in accordance with other work (Mekki et al., 2017) using fish for the analysis of acute toxicity test of phosphate compounds, taking into account the fish and Vibrio fischeri interspecies correlations in the toxicity analysis of numerous chemicals (Wang et al., 2016).
For the Daphnia magna acute toxicity test, the Log-normal model in the REGTOX software (Vindimian, 2005) for Microsoft Excel was used for the calculation of the dose-response parameters. The REGTOX software program is based on non-linear regression with Hill model to calculate the values. Effect concentrations (EC50) and their confidence intervals were estimated using the non-parametric bootstrap method.
For a preliminary comparison between bioindicators, equal exposure concentrations of toxicants were considered during pre-tests. The results indicated that Daphnia magna was much less sensitive to toxic effects than Vibrio fischeri in all five reference toxicant concentrations. Henceforth, a test sample strongly enriched in phosphorite, up to a concentration of 100.000 mg/L, was deemed to be used for this bioassay. The compound samples were tested for toxicity at the following concentrations: 6.25%, 12.5%, 25%, 50% and 100%. For the five effect doses the parameter values with their confidence intervals were given with two different significance levels, according to the user’s choice. The resulting % of immobilization for the 24- and 48-hours’ measurements (Table 4) provided evidence of a dose-response effect in the highest-concentration sample. The obtained EC50 at 48h for immobilization was 94.27 g/L.
This result is well above 100 mg/L and is consistent with previous work (Kim et al., 2013), where no Daphnia magna immobility was observed up to 100 mg/L in the toxicity assessment of phosphate compounds.
Table 4
Mean immobilization (% of total organisms) of Daphnia magna for exposure to toxicant concentrations up to 100 g/L
| Daphnia Magna Mean Immobilization |
Toxicant concentration (mg/L) | 24 h exposure | 48 h exposure |
6250 | 0 | 0 |
12.500 | 5% | 10% |
25.000 | 5% | 10% |
50.000 | 10% | 15% |
100.000 | 35% | 55% |
Bioassay sensitivity testing
For both the first two bioassays, Vibrio fischeri and Daphnia magna, the performance of a reference test was advised in order to validate the correct execution of the laboratory procedure and the sensitivity of the test organisms (Buikema et al., 1982). The reference toxicant used in this study as a positive control was potassium dichromate, whose toxicity is well-known (Diamantino et al., 2000).
Standard response of Daphnia magna to toxicant (positive control) was verified as EC50 after 24 h exposure. The optimal conditions for this procedure are an illumination of at least 6000 luces and a temperature range of 20–25 0C. Results were compared to the expected EC50 according to the literature.
Following the acceptability criteria reported by OECD guideline (OECD, 2004), tests are considered valid if:
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the mortality in negative controls does not exceed 10% after 24 h of exposure without feeding,
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the EC50 recorded value of tested organisms in the quality control test is within the range 0.6–2.1 mg/L.
The 24 h reference test with potassium dichromate carried out in this study provided an EC50 value of [0.65; 1.25]mg/L 95% CL. The observed percentage of immobilization in the negative control was 5%. Both recorded values validated the procedure.
For the luminescent bacteria analysis using potassium dichromate as reference compound, the control test is valid if the reference substance causes 20–80% inhibition after a 30 min contact times at a concentration < 6 mg/L (ISO 11348, 2007). The positive control with potassium dichromate run alongside Vibrio fischeri tests gave EC50 = 3,4 mg/L with a percentage inhibition effect of 48%.
In this research, consistent results were achieved for each test control in accordance with the criteria for validity of the respective test guideline. A summary of the endpoint values of the different test models is reported in Table 5.
Table 5
Results of the reference tests performed on potassium dichromate with the ecotoxicological essays used in this analysis
Organism group | Organism | Endpoint | Reference substance, validity range | Mean values (and SDs) |
Water flea | Daphnia magna | EC50 | Potassium dichromate, 0.6–2.1 mg/L | 0.95 ± 0.30 mg/L |
Luminescent bacteria | Vibrio fischeri | % Immobilization (&& concentration) | Potassium dichromate, 20–80% < 6 mg/L | 48% 3.4\(\pm 0.9\)mg/L |
For the seed germination and root elongation test, all experiments were performed in four replicates. Averages and standard deviations of the growth inhibition were calculated and fitted to the regression analysis. The averages of growth inhibition were compared by T-test and p-values were determined to evaluate the differences among treatments. The effects of phosphorite residues on the seedling germination and growth were observed at the highest examined concentration of 100 g/L.
The germination test results are presented as average number of germinated seeds and standard deviation for each treatment. The response of the toxicity of the compound is summarized in Table 6. Based on these values, the toxicity test outcomes showed similar trend but different magnitude of plant responses.
Results showed that the compound significantly inhibited root elongation (p < 0.05, marked with *) of the Lepidium sativum sample. Moreover, the compost toxicity appeared to have a major influence on the root length response variable.
According to (Zucconi et al., 1985), GI lower than 50% indicate high phytotoxicity, values between 50% and 80% indicate moderate phytotoxicity, and values above 80% mean no phytotoxic material. In line with this, phosphorite at the tested concentration can be considered as highly phytotoxic for the Sorghum saccharatum seed and moderate phytotoxic for the Lepidium sativum tested seed.
Table 6
Effect of toxicant at a concentration of 100 g/L on germination and early growth in Sorghum saccharatum and Lepidium sativum compared to the control. Results with SDs were from four replicated dishes, each containing ten seeds. Double-distilled water was used as control medium
Bioassay response | Sorghum saccharatum | Lepidium sativum |
Control | Sample | Control | Sample |
Average number of germinated seeds | 9.3\(\pm 0.5\) | 9.3\(\pm 0.5\) | 9.9\(\pm 0.1\) | 9.8 \(\pm\) 0.8 |
Root length (mm) | 85.9\(\pm 11.7\) | 40.0\(\pm 13.3\) | 77.1\(\pm 3.4\) | 55.5 \(\pm 0.3\) * |
GI% | 46.5% | 71.3% |
The visual rating of the analysed compound toxicity on the tested plants is available in Fig. 6 for the Sorghum and in Fig. 7 for the Lepidium plant, respectively. Figure 8 illustrates the germination percentage of Lepidium and Sorghum seeds, respectively, exposed to the tested toxic sample of phosphorite with a concentration of 100 g/L.
As general conclusions, the sediment did not prove to be toxic for the plant species germination up to a concentration of 100 g/L. At this level of content, the sample altered the root elongation of both the plant species to various degrees, inducing a statistically significant bio suppression (response lower than in negative control) in Lepidium sativum growth. The germination indices of both Sorghum saccharatum and Lepidium sativum were inhibited by the sediment material.
Anyway, much experimental concerns on the results of phytotoxicity assays are mandatory, due to the rather high variability affecting analysis of data, such as (the level and rapidity of) seed germination. Therefore, these indications should preferably be confirmed testing more sediment samples, with a larger range of concentrations of these chemicals.