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Toxicity to aquatic algae and cyanobacteria

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Description of key information

Since benzoyl chloride reacts almost instantaneously in water forming benzoic acid and HCl, these degradation products can be considered as test surrogates. However, one study tested the stability of benzoyl chloride in the test media and chose to calculate inhibition concentrations (EC 50 (72 h) and NOEC (72 h)) based on nominal concentrations. The two other studies are based on surrogate testing data, with one study characterizing the degradation products throughout the test. This allowed the calculation of inhibition concentrations for benzoic acid based on nominal concentrations. 
* Benzoyl chloride:
Toxicity to the freshwater algae Pseudokirchneriella subcapitata (OECD guideline n°201, GLP conditions)
Based on biomass: EC 50 (72 h) = 45 mg/L.
Based on growth rate: NOEC (72h) = 21.34 mg/L; EC 50 (72h) = 96 mg/L (95% CL: 85 -110)
* Test surrogates:
- Benzoic acid:
Toxicity to the freshwater algae Pseudokirchneriella subcapitata (methodology: closed system algal toxicity test)
Based on final yield: NOEC (48h) = 4.81 mg/L (initial pH 6.5); EC50 (48h) = 36.39 mg/L (initial pH 6.5)
Based on growth rate: NOEC (48h) = 9.62 mg/L (initial pH 6.5); EC50 (48h) = 83.29 mg/L (initial pH 6.5); EC50 (48h) = 207.5 mg/L (initial pH 7.0); EC50 (48h) = 342.8 mg/L (initial pH 7.5) and EC50 (48h) = 37.1 mg/L (non-neutralized pH)
Toxicity to the freshwater green algae Scenedesmus quadricauda and Chlorella pyrenoidosa (methodology: measuring absorbance of cultures with time)
EC50 (14d) > 11.51 mg/L for growth rate and growth yield for both species
- HCl:
HCl as a degradation product of benzoyl chloride is not expected to expose the aquatic compartment due to its on-site neutralisation. Indeed, benzoyl chloride is almost instantaneously hydrolysed into benzoic acid and HCl and then HCl is completely neutrealised during the on-site wastewater treatment before release into the environment.

Key value for chemical safety assessment

EC50 for freshwater algae:
45 mg/L
EC10 or NOEC for freshwater algae:
21.34 mg/L

Additional information

Although specific data on benzoyl chloride testing is available, the test substance reacts almost instantaneously in water forming benzoic acid and HCl, therefore these degradation products can be considered test surrogates. All data, on benzoyl chloride and its test surrogates, are discussed below.

One study (Thiebaud, 2000) is available on benzoyl chloride effects on Pseudokirchneriella subcapitata. Since this study is the most relevant and contains specific and reliable data on algae growth inhibition of benzoyl chloride, it was chosen as the key study (klimisch 1; GLP guideline study).

The authors tested the potential of algae growth inhibition of benzoyl chloride on Pseudokirchneriella subcapitata using an OECD guideline n°201 test under GLP conditions. Furthermore, they tested the stability of the test substance in the test media. Five concentrations were tested in triplicates. No deviations or restrictions with respect to the available guideline at the time were observed. The growth rate was measured daily in cell counter chamber under microscopy.

The authors established that benzoyl chloride dissociates in the test solutions in HCl and benzoic acid in less than ten minutes and chose to calculate inhibition concentrations (i.e. EC 50 (72 h) and NOEC (72 h)) based on nominal concentrations. Furthermore, the negative controls exhibited a normal exponential growth and within the OECD requirements as established in the first OECD guideline n°201. Benzoyl chloride exhibited an EC 50 (72 h) of 45 and 96 (95% CL: 85 -110) mg/L based on biomass and growth rate respectively. Furthermore, the NOEC (72 h) was established at 21.34 mg/L. No abnormal development of the cells was observed. Microbial development was slowed down by the acid pH resulting from the dissociation of benzoyl chloride in the highest tested solution.

Based on this information, according to the criteria laid down in the CLP regulation n° 1272/2008/EC, benzoyl chloride should not be considered as toxic to the aquatic environment.

 

Two supporting studies are reported for the test surrogate benzoic acid of which the study by Lee and Chen (2009) was chosen as the most critical since it provides more detailed information on materials and methodology and a range finding study was performed.

The study by Lee and Chen (2009) is reliable with restrictions (klimisch 2e). The authors tested the influence of benzoic acid on the final yield and growth rate of Pseudokirchneriella subcapitata using a closed system algal toxicity test. The algal inoculum was transferred together with growth medium (US EPA OPPTS 850.5400, without EDTA) and test substance (unless control vessel) into BOD bottles. These were filled completely, leaving no headspace and a water seal was provided to ensure a closed test environment. Cultures were kept at 24 +/- 1°C and the initial pH was set at 6.5 taken into account the results obtained from the dose-response curves at different pH-values. Indeed, four different initial pH conditions (6.5, 7.0, 7.5 and non-neutralized) were tested and showed that a non-adjusted or low pH resulted in a more severe reaction, partially due to acidity which can impair the algal growth. Based on these results and considering that in most aquatic environments the pH level is between 6.0 and 9.0, an initial pH of 6.5 was chosen as it represents a conservative consideration. Seven concentrations (4.81, 9.62, 19.24, 38.48, 76.96, 153.92, 307.84 mg/L) were tested in triplicate and controls were also included. The duration of the test was 48h and algae population density was determined using an electronic particle counter. A Probit analysis was used to determine the concentration–response relationship and the median effective concentration(EC50), while the one-tail Dunnett’s procedure was used to estimate the NOEC values at 5% level of significance.

At an initial pH of 6.5 the EC50(48h) for final yield and growth rate were 36.39 mg/L and 83.29 mg/L respectively and the NOEC(48h) were 4.81 and 9.62 mg/L respectively.

The supporting study of Stratton et al. (1982) was reliable with restrictions (klimisch 2e) and tested the influence of benzoic acid on the growth yield and growth rate of two green algae (Scenedesmus quadricauda and Chlorella pyrenoidosa). Growth was assessed by measuring the absorbance of cultures with time using a Bausch and Lomb Spectronic 20 spectrophotometer. At least five concentrations of the test substance were used ranging from 0 to 10 ppm (i.e. 0 to 11.51 mg/L) and for each concentration five replicas were performed. The EC50 (14d) was higher than 11.51 mg/L for both green algae and both tested endpoints.

Based on both studies, benzoic acid cannot be classified based on the CLP regulation (EC) N° 1272/2008 since the duration of these tests was not the required 72h or 96h.

 

No studies for the test surrogate HCl are reported considering the almost instantaneously hydrolysation of benzoyl chloride into benzoic acid and HCl and the complete neutralization of HCl during the on-site wastewater treatment before release into the environment. Thus, due to its on-site neutralisation the aquatic compartment is not expected to be exposed to HCl.