2,4-dinitrophenol

Toxicity to terrestrial arthropods

Description of key information

Dinoseb, the pesticide used in this study, is a herbicide and insecticide belonging to the dinitrophenol family. Structural formulas of 2,4-dinitrophenol and Dinoseb are similar.Dinitrophenols may also originate as impurities in certain pesticides such as Dinoseb (Wegman RCC, Wammes JI. 1983. Determination of nitrophenols in water and sediment samples by high-performance liquid chromatography. Med Fat Landbouwwet Rijksuniv Gent 48:961-969).

No effect on adult survival was observed after 28 days at the hightest tested concentration of 20 µg/kg soil dw (NOEC);

The most conservative data obtained from reproduction test is the EC50 of 14.4 µg dinoseb/kg soil dw.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil dwelling arthropods:

14.4 µg/kg soil dw

Long-term EC10, LC10 or NOEC for soil dwelling arthropods:

20 µg/kg soil dw

Additional information

Dinoseb, the pesticide used in this study, is a herbicide and insecticide belonging to the dinitrophenol family. Structural formulas of 2,4-dinitrophenol and Dinoseb are similar. Dinitrophenols may also originate as impurities in certain pesticides such as Dinoseb (Wegman RCC, Wammes JI. 1983. Determination of nitrophenols in water and sediment samples by high-performance liquid chromatography. Med Fat Landbouwwet Rijksuniv Gent 48:961-969).

The effect of dinoseb on the energy reserves (lipid and protein content) of the Collembola Folsomia candida was investigated to test the hypothesis that dinoseb exposure reduces energy reserves before an effect can be detected on life cycle parameters (growth, reproduction and survival).

Folsomia candida was exposed to dinoseb in artificial soil, while survival, reproduction, weight, lipid and protein content were determined at different time intervals in order to determine the relative sensitivity of those parameters. The observed effects at different levels of biological organization were analyzed and compared over time (temporal analysis) as well as after certain time intervals (fixed time analysis).

A reproduction test was conducted according to the ISO 11267 standard (ISO, 1999).

Results are explained here below:

Survival

No significant effect on survival was recorded after 28 days at the highest tested concentration of 20 µg/g soil dry soil. In chronic toxicity test conducted on a compressed soil with 30 Collembola per mini Petri dish a highly significant decrease of survival was observed after 21 days of exposure at 20 µg/g dry soil. However, as an important mortality was observed in the control at the beginning of the experiment, no LC50 could be estimated. This mortality was probably due to overpopulation. In the second test conducted with one Collembola per box, the toxicity is higher than for the experiment conducted according to the ISO standard (LC50 17 µg/g dry soil).

Compression of soil seems to have an impact on the sensitivity of Folsomia candida (Rundgren and Van Gestel, 1998). On a compressed soil, a water film appears on the soil surface. The ventral tube of Folsomia candida that enables them to regulate their water content (Rundgren and Van Gestel, 1998) is in these conditions constantly in contact with the water surface. As soil pore water is probably the most important route of uptake of dinoseb, it can be can hypothized that the degree of exposure is more important in this test that in the ISO standard test with loose soil. Furthermore, as Collembola are aggregative organisms, contact between individuals is necessary (Green, 1964; Verhoef and Nagelkerke, 1977; Joosse and Veltkamp, 1970). Thus, isolation could also be responsible for the observed increase of sensitivity because it can provoke additional stress. According to Green (1964), optimal conditions correspond to 1.2 cm2 per individual. Thus, in these experiments we should work with approximately four organisms per mini Petri dish.

The analysis of dinoseb concentrations in artificial soil from a previous study (Staempfli et al., 2002) showed that degradation is observed during the first 14 days. This phase is followed by a stationary phase, showing a relative stability of the compound. The quantity of dinoseb degraded over time (4 µg/g dry soil in 21 days) was not correlated with the initial concentrations (10 and 20 µg/g dry soil), which indicates that degradation is probably due to microbial processes.

Dinoseb mode of action

The final weight, length and the total lipid content of adults after 21 days was affected by dinoseb concentrations of 25 and 30 µg/g dry soil. These effects can be linked to the mode of action of dinoseb. Dinoseb uncouples ATPsynthesis by canceling the membrane potential, thus disturbing the transformation of nutritive macromolecules such as glucose or triglycerides into useful energy. The energy, instead of being used to form ATP, is dissipated as heat (Escher, 1995). The decrease of energy available for maintenance could be responsible for the lower size and weight reached by the organisms after 3 weeks of exposure. The energy left for storage also decreases, which can explain the decrease of the total lipid content (lipids being the main form of energy reserves in invertebrates). Jager et al. (2004), who tested the toxicity of triphenyltin, which is also an ATP synthesis inhibitor, onFolsomia candidafound that this compound decreased the growth and reproduction rate of the exposed organisms over time but increased their lifespan. It has been observed such a lifespan increase, but our results on weight, length, and lipid content are in agreement with their results.

Reproduction and growth stimulation

For length and total lipid content a slightly stimulating effect on the growth rate coefficient b could be observed for all the treatments. It has been observed that longevity decreased with increasing dinoseb concentrations. This observation is in agreement with Ernsting and coworkers observation of the growth pattern of the springtailOrchesella cincta. They found that longevity was negatively related to the Von Bertalanffy growth coefficient (Ernsting et al., 1993; Stam et al., 1996). Results in this study indicate that shortly after exposure to dinoseb, the Collembola adopted a strategy of growth stimulation in which they reach sexual maturity more quickly and reproduce more efficiently than unexposed organisms. This phenomenon involves a decrease in longevity. The hypothesis stated above, that stress induced by dinoseb exposure can increase the reproduction rate, is confirmed by the fact that the number of eggs counted at 20µg/gdry soil was 1.5 times greater than the number of juveniles hatched in the control.

A direct comparison of the number of eggs laid between exposed and unexposed organisms is difficult because different adult Collembola behave differently. It has been observed that despite the compression of the substrate, unexposed organisms succeeded in laying their eggs at the bottom of the boxes. With increasing dinoseb concentrations the organisms made less effort to lay eggs at the bottom of the boxes.

A reduction of 50% in the number of living juveniles was observed at 15 µg/g dry soil, indicating that dinoseb affects the hatching rate or the survival of juveniles. This assumption is confirmed by the observation of deadjuveniles (93%) at a concentration of 20 µg/g dry soil.

The higher sensitivity of juveniles to dinoseb can be explained by their thinner cuticle and higher surface/volume ratio (Holmstrup and Krogh, 1996). These results are in accordance with the EC50 of 14µg/g dry soil obtained with the reproduction tests following the ISO standard (ISO, 1999).

Stimulation of protein synthesis

The increase of the protein synthesis observed in exposed organisms after 6 days may reflect the fact that Collembola have begun to respond to stress. This hypothesis is strengthened by the induction of the heat shock protein Hsp70 involved in repairing of protein unfolding observed in previous work (Staempfli et al., 2002). The highest induction was found after 5 days of exposure in organisms exposed to 20 mg dinoseb/g dry soil. The decrease of induced Hsp70 after this time speaks for a strong stress reaction, meaning that the optimum of the Hsp70 response has been surpassed and that cell damage leads to decreasing values of Hsp70 (Staempfli et al., 2002).

A biphasic concentration-response

An exposure of 6 days to dinoseb concentrations of 15–30µg/gdry soil seems to stimulate the weight, lipid,

and protein content of the exposed organisms. This stimulation cannot easily be explained by the specific mode of action of dinoseb, but is probably a general stress reaction, a theory strengthened by the induction of heat shock proteins. Over time dinoseb most likely affects energy metabolism by inhibiting ATP synthesis. Dinoseb, much like triphenyltin studied by Jager et al. (2004), could induce an increase in maintenance costs, explaining the decrease of size, weight, and lipid content observed after 21 days.

However, the protein content is equal or superior in exposed organisms compared to the control. These results might indicate that defense and repair mechanisms were activated (detoxification enzymes) (Staempfli et al., 2002).These processes are costly in terms of energy and can be in part responsible for the decrease of lipid reserves, and finally for an increase in mortality.

The biphasic concentration-response pattern observed could be linked to a hormesis phenomenon characterized by a low-dose stimulation, corresponding to a short exposure time in our case, and a high-dose inhibition, corresponding to a long exposure time. This phenomenon is usually observed in a low dose zone, below the no observed effect levels (Calabrese, 2005), but in this case it appears at an exposure concentration that induces death 10 days later. It is not clear whether this phenomenon can be considered as hormesis.

No effect on adult survival was observed after 28 days at the hightest tested concentration of 20 µg/kg soil dw (NOEC);

LC50 of 17 µg dinoseb/kg soil dw was obtained for a survival endpoint in a mini Petri dish (growth test);

EC50 of 14.4 µg dinoseb/kg soil dw in the reproduction test.

Decrease of weight, length, and lipid content in Folsomia candida have been observed at a dinoseb concentration of 25 µg/g dry soil after 21 days of exposure. Growth (weight and length), lipid content, and survival are affected to a similar degree. The decrease of growth, lipid and protein content are not more sensitive parameters than mortality, all these parameters being less sensitive than reproduction.

However, an increase in the number of eggs, the weight and the lipid and protein content is observed after 6 days of exposure to dinoseb. If we consider this stimulation as an early warning signal, it can be can concluded that these parameters are more predictive of toxicity than mortality and reproduction.