Benzene, 1,2,3-trichloro-5-(trichloromethyl)-

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July - October 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: in accordance with guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Italian Ministero della Salute

Test material

Test material information

Constituent 1

Reference substance name:

1,2,3-trichloro-5-(trichloromethyl)benzene

EC Number:

700-556-0

Cas Number:

66682-07-3

Molecular formula:

C7H2Cl6

IUPAC Name:

1,2,3-trichloro-5-(trichloromethyl)benzene

Details on test material:

- Name of test material : 3,4,5-trichloro-benzotrichloride
- Substance type: intermediate for synthesis
- Analytical purity: 97.7%
- Purity test date: 03 June 2010
- Lot/batch No.: 06/2010
- Expiration date of the lot/batch: June 2012
- Stability under test conditions: Stable away from water and heat, on iron, at temperature < 50°C. Moreover if mixed with sulphuric acid a violent hydrolysis occurs.
- Storage condition of test material: the test item is kept in glass vial. Kept away from incompatible materials (oxidisers, not noble metals, amines and alcohols). Kept away from heat and ignition sources.

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

The test medium was prepared as follows: prior to the start of the test, a stock solution of 100.0 mg/L was prepared by direct weighing into algal culture medium (0.0504 g of test item into 500 mL of algal culture medium). The obtained solution showed oily insoluble drops on the surface, therefore it was mixed by a magnetic stirrer in dark conditions for about 3 days to achieve the maximum solubilisation of test item; then, it was filtered by a cartridge filter (pore diameter: 0.45 µm) to obtain a clear solution.
The test concentrations were prepared by taking adequate aliquots of stock solution and diluting them into the algal medium.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The study was performed with the unicellular, fresh-water green algae Pseudokirchneriella subcapitata strain, Sele-IRSA, cultured in the ChemService laboratory and originally obtained from the IRSA-CNR (Water Research Institute-Italian Research Council), Brugherio, Milano.
The algae have been cultured in a temperature controlled room at 24.0 °C ± 2.0 °C under continuous uniform illumination of approximately 6000 Lux in the spectral range 400-700 nm. The culture medium was the same of the test medium; the stock culture were weekly transferred to fresh medium and maintained in continuous shaking to ensure the necessary amount of CO2 and to keep algae in suspension. Cultures containing deformed or abnormal cells were discarded. Only exponentially growing algal cultures have been used to start the test.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

The room temperature was in the range 25.2 – 25.6 °C during the test period, according to the OECD’s recommended range (24.0 ± 2.0 °C).

pH:

At the beginning of the test, the pH of test item solutions was in the range 7.11 – 7.81 and the pH of algal culture medium was 7.85. At the end of the test the pH values of control ranged between 9.30-9.32, 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 8.39-9.28.

Nominal and measured concentrations:

Five nominal concentrations in a geometric series, namely 10.0, 17.8, 31.6, 56.2 and 100.0 mg/L

Details on test conditions:

The test medium was prepared as follows: prior to the start of the test, a stock solution of 100.0 mg/L was prepared by direct weighing into algal culture medium (0.0504 g of test item into 500 mL of algal culture medium). The obtained solution showed oily insoluble drops on the surface, therefore it was mixed by a magnetic stirrer in dark conditions for about 3 days to achieve the maximum solubilisation of test item; then, it was filtered by a cartridge filter (pore diameter: 0.45 m) to obtain a clear solution.
The test concentrations were prepared by taking adequate aliquots of stock solution and diluting them into the algal medium

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

24.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: range 10.90– 33.32

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

34.63 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: range 25.79 – 41.98

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

57.97 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: range 51.69 – 64.43

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

47.71 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: range 42.61 – 53.12

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

67.76 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: range 64.07 – 71.37

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

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 end of the test the cell density in the negative control was increased on average by a factor of 254. This value complies with the validity criteria of the test, according to the mentioned guidelines, which indicate a minimum increase factor of 16.
The negative control met also the other validity criteria, with a coefficient of variation of daily growth rates of 29.8% and a coefficient of variation of average growth in replicates control cultures during the test period of 4.0%.
In the test item solution the algal growth rate (r) after 72 hours of exposure was inhibited by a minimum value of 1.0% for the nominal concentration of 17.8 mg/L to a maximum value of 39.7% for the highest concentration tested, compared to the negative control.
The lowest concentration of 10.0 mg/L did not shown any inhibitory effect, being the algal growth rate higher than the control (mean % inhibition : negative value).
The ErC values were determined by a linear interpolation analysis.
The effect due to the two lowest concentrations (10.0 and 17.8 mg/L as product) after 72 hours of exposure was assessed to be not significant by Bonferroni adjusted test.The yield (y) decreased by a minimum value of 5.6% for the nominal concentration of 17.8 mg/L, to a maximum value of 89.1% for the highest concentration, compared to the negative control.
The lowest concentration of 10.0 mg/L did not shown any inhibitory effect, being the algal growth rate higher than the control (mean % inhibition : negative value).
The EyCx calculation was carried out using a non linear regression analysis.
The effect due to the two lowest concentrations (10.0 and 17.8 mg/L as product) after 72 hours of exposure was determined to be not significant by Bonferroni adjusted test.

Results with reference substance (positive control):

The results of the most recently performed test with a reference substance are reported and compared with literature data to demonstrate that the algal strain used has got an adequate level of sensitivity to that substance. In the current case the results refer to a test performed on April 2010 (CHE-I-003/2010) with 3,5-Dichlorophenol as reference substance.

Effect of 3,5-dichlorophenol on the growth of Pseudokirchneriella subcapitata:
Chemservice study CHE-I-003/2010, ErC50 (mg/L)= 4.12 (3.32 -4.98)
Literature data ISO 8962 (2004), ErC50 (mg/L)= 3.38 (2.08-4.68)

Reported statistics and error estimates:

The CETIS v.1.026D software was used to carry out the statistical analysis. The statistical comparison with the control at each observation time was determined by Bonferroni adjusted test for both the end-points, growth rate and yield, after analyzing the normality of distribution and the homogeneity of variance by Shapiro-Wilk and Barlett’s test respectively.

The EyCx and the ErCx values were determined, respectively, by a non linear regression analysis and by a linear interpolation analysis, chosen by the PC software as more appropriate.
The negative control was considered to check that the growth of the algal population used in the test meets the validity criteria.
As input data nominal concentrations were used.

Any other information on results incl. tables

Effects of 3,4,5-trichloro-benzotrichloride on the growth ofPseudokirchneriella subcapitata

Nominal test item concentration [mg/L]	replicate	Cell density (n. cells x 10^4/ml)
		0 h	24 h	48 h	72 h
Negative control	A B C D E F Average value	1 1 1 1 1 1 1	5.2748 5.6119 5.2796 5.8411 6.6736 5.6251 5.7177	63.6086 62.9536 67.2056 61.9706 75.2096 61.8406 65.4648	256.2697 237.3697 243.7697 254.1897 253.9697 276.4497 253.6697
10.0	A B C Average value	1 1 1 1	4.0128 3.9312 3.6985 3.8808	49.0453 41.8813 46.7183 45.8816	264.3271 278.4871 261.0271 267.9471
17.8	A B C Average value	1 1 1 1	5.3781 4.7015 3.2559 4.4452	45.2132 54.1132 33.5172 44.2812	240.5286 234.6866 243.1466 239.4539
31.6	A B C Average value	1 1 1 1	2.9056 2.8468 2.4497 2.7340	29.4884 30.8584 27.5704 29.3057	214.7393 202.2793 188.0293 201.6826
56.2	A B C Average value	1 1 1 1	2.5409 2.8204 3.0435 2.8016	13.5762 18.6932 20.3762 17.5485	121.2493 134.9273 128.2113 128.1293
100.0	A B C Average value	1 1 1 1	1.9578 1.3760 1.6603 1.6647	7.2627 4.9066 4.7410 5.6368	33.4315 28.8665 23.1575 28.4852

Algal growth rate and inhibition caused by 3,4,5-trichloro-benzotrichloride over 72 hours of exposure in comparison with control

Nominal test item concentration [mg/L]	24 h		48 h		72h
	Mean growth rate (x10^-3)	Mean inhibition %	Mean growth rate (x10^-3)	Mean inhibition %	Mean growth rate (x10^-3)	Mean inhibition %
0 (negative control)	72.5	-----	87.1	-----	76.9	-----
10.0	56.5	22.1	79.7	8.5	77.6	-1.0*
17.8	61.3	15.5	78.6	9.8	76.1	1.0
31.6	41.8	42.4	70.3	19.2	73.7	4.1
56.2	42.8	41.0	59.4	31.8	67.4	12.3
100.0	20.8	71.3	35.6	59.1	46.4	39.7

* the cells growth rate was higher than the negative control

Algal yield and growth inhibition caused by 3,4,5-trichloro-benzotrichloride over 72 hours of exposure in comparison with negative control

Nominal test item concentration (mg/L)	Mean biomass at 72h (cell/ml)	Mean growth inhibition %
0 (Negative control)	2536697	-----
10.0	2679471	-5.7*
17.8	2394539	5.6
31.6	2016826	20.6
56.2	1281293	49.7
100.0	284852	89.1

* the cells growth was higher than the negative control

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of test item 3,4,5-trichloro-benzotrichloride was tested on Pseudokirchneriella subcapitata. The EC10, EC20 and EC50 with their confidence limits (LCL, Lower Confidence Level and UCL, Upper Confidence Level) for both the end-points after 72 hours, calculated based on the nominal concentrations were as follows:

Endpoint 72 h EC10 (mg/L) 72 h EC20 (mg/L) 72 h EC50 (mg/L)
Yield 24.10 (10.90– 33.32) 34.63 (25.79 – 41.98) 57.97 (51.69 – 64.43)
Growth rate 47.71 (42.61 – 53.12) 67.76 (64.07 – 71.37) > 100.0

Executive summary:

The influence of the test item 3,4,5-trichloro-benzotrichloride on the growth of the green algal speciesPseudokirchneriella subcapitata, formerly known asSelenastrum capricornutum, was investigated in a 72-hour full test according to the OECD Guideline No. 201, 2006.

 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 7.11 – 7.81 and the pH of algal culture medium was 7.85. At the end of the test the pH values of control ranged between 9.30-9.32, 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 8.39-9.28.

 Light intensity during the test period was in the range 4871 - 5105 Lux (within the range 4000 - 6000 Lux).

  The room temperature was in the range 25.2 –25.6 °C during the test period.

In the negative control the cell density increased on average by a factor of 254. 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 (29.8%) and the coefficient of variation of average growth in replicates control cultures during the test period (4.0%).

 In the test item solution the algal growth rate (r) after 72 hours of exposure was inhibited by a minimum value of 1.0% for the nominal concentration of 17.8 mg/L to a maximum value of 39.7% for the highest concentration tested, compared to the negative control.

The yield (y) decreased by a minimum value of 5.6% for the nominal concentration of 17.8 mg/L, to a maximum value of 89.1% for the highest concentration tested, compared to the negative control.

After 72 hours of exposure, the lowest concentration (10.0 mg/L) did not show any inhibitory effects on the algal growth.

At the end of test period the two lowest concentrations (10.0 and 17.8 mg/L) did not shown any significant biological effects due to test item (Bonferroni adjusted test).