α,α,α,4-tetrachlorotoluene

Toxicity to aquatic algae and cyanobacteria

Description of key information

EC50 based on growth rate = 75.54 mg/l, nominal concentration

Key value for chemical safety assessment

EC50 for freshwater algae:

75.54 mg/L

Additional information

In the key study, the influence of the test item on the growth of the green algal species Pseudokirchneriella subcapitata, formerly known as Selenastrum capricornutum, was investigated in a 72-hour full test according to the OECD Guideline No. 201, 2006 and to the Council Regulation EC 440/2008 (C.3.).

For this purpose, exponentially growing test algae were exposed for 72 hours to an aqueous test medium containing the test item at five nominal concentrations in a geometric series, namely 10.0, 17.8, 31.6, 56.2 and 100.0 mg/L, under defined conditions. Besides the five concentrations, a negative control without the test item was also prepared to check the acceptability of the algal inoculum.

 Algal cell density was measured every 24 hours by fluorescent reading with a spectrofluorophotometer in few millilitre samples taken from each test concentration replicate and from controls.

At the beginning of the test, the pH of test item solutions was in the range 4.64 – 7.92 and the pH of algal culture medium was 8.02. At the end of the test the pH values of negative control ranged between 9.46-9.51, with a difference lower than 1.5 units as provided by OECD guideline, the pH mean values of test solutions after 72 hours were in the range 4.90-9.41.

The room temperature was in the range 24.7 °C during the test period, according to the OECD’s recommended range (24.0± 2 °C).Light intensity during the test period was in the range 5079 - 5150 Lux (within the range 4000 - 6000 Lux).

In the negative control the cell density increased on average by a factor of 251. This value complies with the validity criteria of the test according to the mentioned guidelines, such as the coefficient of variation of daily growth rates (26.6%) and the coefficient of variation of average growth in replicates control cultures during the test period (6.7%).

In the test item solutions the algal growth rate (r) after 72 hours of exposure was inhibited by a minimum value of 7.8% for the nominal concentration of 56.2 mg/L to a maximum value of 103.3% for the highest concentration tested, compared to the negative control.The three lowest concentration did not shown any inhibitory effects on growth rate.

The yield (y) decreased by a minimum value of 0.2% for the lowest tested concentration , 10.0 mg/L, to a maximum value of 100.1% for the highest tested concentration, compared to the negative control.The three lowest concentrations (10.0, 17.8 and 31.6 mg/L) did not show any significant inhibitory effects on the cells algal growth (Bonferroni adjusted test).

 

The 72-hour EC₁₀, EC₂₀and EC₅₀with their 95% confidence limits calculated on the basis of the nominal concentrations of test item, for both the end-points, are reported in the following below: 

Endpoint	72 h EC10(mg/L)	72 h EC20(mg/L)	72 h EC50(mg/L)
Yield	38.19 (4.00– 41.17)	45.25 (32.49 – 50.26)	66.02 (57.15 – 71.02)
Growth rate	57.18 (55.33 – 58.49)	61.77 (60.09 – 63.08)	75.54 (73.95 – 76.82)

In the supporting study of Lee (2009), the authors tested the influence of Similar Substance 01 (more details attached) on the final yield and growth rate of Pseudokirchneriella subcapitata usinga 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. Based on a range finding study, six to seven concentrations, ranging from 0.5 to 123 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 and LOEC values at 5% level of significance.

In these test conditions, EC50(48h) for final yield and growth rate were 6.42 mg/L and 17.29 mg/L respectively and the NOEC (48h) for both endpoints was 0.57 mg/L.