Comment on Hossain et al. Exposure to Dodecamethylcyclohexasiloxane (D6) Affects the Antioxidant Response and Gene Expression of Procambarus clarkii. Sustainability 2021, 13, 3495

Hossain et al. [1] discussed oxidative stress induction and changes in gene expression in a crayfish species (Procambarus clarkii) following aqueous exposure to dodecamethylcyclohexasiloxane (D6). However, there are critical flaws in the design of the study, which limit the reliability of the presented results. These are focused on the magnitude of the test concentrations of D6 used in the assay and the conditions under which the crayfish were kept during the experiment. Specific criticisms of each of these points are discussed in further detail herein.

The test concentrations used in this study are neither appropriate based on the measured aqueous solubility of D6 nor are they environmentally relevant. The solubility of D6 in water is 0.0051 mg/L [2]. However, the test concentrations used in this study far exceeded that, ranging from 10 to 1000 mg/L, or approximately 2000- to 200,000-fold greater than the measured aqueous solubility of D6. As described in the Guidance Document of Aquatic Toxicity Testing of Difficult Substances and Mixtures [3], a solubility experiment should be performed prior to conducting any testing to determine the maximum dissolved concentration that can be achieved in the specific test system. The test concentrations cited are nominal, as no analytical verification of the actual dissolved concentrations of D6 present in the test system was conducted. Conducting aquatic toxicity tests at concentrations that exceed the limit of solubility of the test substance, particularly at the magnitude presented here, is inappropriate. At the applied concentrations used in this study, neat D6 was undoubtedly present in a separate phase, likely as a colloidal dispersion, which could certainly exert physical effects such as coating the gills or other breathing surfaces. Such physical effects are not a true measure of the toxicity of a substance in the environment. The Chemical Safety Report for D6 indicates that the highest concentration used in aquatic toxicity tests (0.0046 mg/L) was close to its functional water solubility limit [4], and it was concluded that D6 is not toxic to fish, invertebrates and algae following short- and long-term exposure at its functional water solubility limit.

Several recent publications have determined concentrations of D6 in surface waters of industrialized and urban areas, thus representing the upper end of the distribution of concentrations that are expected to be found in the environment. Maximum concentrations of D6 detected in surface waters collected from crude oil production areas in Daqing, China, ranged from 49.2 to 150 ng/L [5]. Surface waters collected from five rivers that run through industrialized areas of China had maximum concentrations of D6 ranging from 6.74 to 523 ng/L [6]. Concentrations of D6 in surface waters of eight rivers that flow into Tokyo Bay, Japan, ranged from <2 to 15 ng/L [7]. The lowest test concentration used in this crayfish testing (10 mg/L) is approximately 100,000-fold greater than the concentrations detected in the environment. Thus, the test concentrations used have no environmental relevance in addition to their technical deficiencies based on exceeding water solubility limits, as discussed above.

Furthermore, there are also concerns regarding the appropriateness of the crayfish husbandry practices used during the study. The described experimental set-up indicates that the aquaria did not include any source of cover or shelter. This particular crayfish species exhibits conspecific aggression and can be cannibalistic. Thus, it is essential that enclosures include this important feature, especially when housing more than one crayfish, to allow individuals to avoid aggressive interactions. Given the density of crayfish in each aquarium in this study (<30 crayfish per m²), combined with the lack of shelter, it is likely the test specimens were stressed from the test system itself [8]. Stress responses from the experimental set-up and the physical effects of D6 make the results of this testing difficult to properly interpret.