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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

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 202 (Daphnia sp. Acute Immobilisation 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 reconstituted water (0.0500 g of test item into 500 mL of reconstituted water). The obtained suspension showed oily insoluble drops on the surface, therefore it was mixed by a magnetic stirrer in dark conditions for about 48 hours 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 filtered stock solution and diluting them in aerated reconstituted water. A negative control without test item was tested.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The study was performed on the freshwater crustacean Daphnia magna Straus. A strain of this species was originally supplied by the Italian Health Department (Istituto Superiore di Sanità) in September 2004. Since that time, the clone has been bred in ChemService laboratories in reconstituted water with the same characteristic to the water used in the tests (as for pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests (see below). The daphnia breeding was daily fed with a fixed amount (300.000 cells/mL) of green algae Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) cultivated in the ChemService Laboratory and with a suspension of the yeast Saccharomyces cerevisiae.

At the start of the test, the organisms used in the test were 624 hours old and were not first brood progeny.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

Water Hardness: 140 - 250 mg/L as CaCO3

Test temperature:

The incubation temperature was recorded twice a day during the course of the study by a digital thermometer. The recorded values ranged between 21.3 and 21.7 °C.
(LIGHT:) a 16 hours light and 8 hours darkness daily photoperiod with a light intensity within the range 382 -394 Lux.

pH:

The pH of the control (test medium without test item) after preparation was 6.88. The pH of stock test solution (100 mg/L) immediately after preparation was 3.69, therefore it was corrected before the beginning of the test period by adding 10 drops of a NaOH solution (1N) achieving a pH equal to 6.34, within the range provided by OECD guideline

Dissolved oxygen:

The dissolved oxygen concentrations and pH were determined in the test medium and the test solutions at the start and at the end of the test period. During the test period, the dissolved oxygen concentrations in the test medium and the test solutions were always higher than 6.0 mg/L , thus according to validity criterion that provided a minimum oxygen concentration of 3.0 mg/L.

Salinity:

Reconstituted test water: analytical grade salts were dissolved in purified water to obtain the following nominal concentrations:
CaCl2 x 2H2O :2.0 mmol/L (= 294 mg/L)
MgSO4 x 7H2O:0.5 mmol/L (= 123 mg/L)
NaHCO3:0.75 mmol/L (= 65 mg/L)
KCl:0.075 mmol/L (= 5.8 mg/L)
Alkalinity:0.8 mmol/L
Ratio of Ca : Mg = 4 : 1 (based on molarity), Na : K= 10 : 1 (based on molarity)

Nominal and measured concentrations:

Five different concentrations in a geometric series, namely 1.0, 3.2, 10.0, 31.6 and 100.0 mg/L.

Details on test conditions:

The test concentrations were chosen based on the results of a pre-test (range-finding test), which was not performed in compliance with the GLP Regulations. Therefore, it is excluded from the Statement of Compliance in the test report, however the Raw Data are archived under the code of the present study.
The main test included five different concentrations of test item, namely 1.0, 3.2, 10.0, 31.6 and 100.0 mg/L.
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 reconstituted water (0.0500 g of test item into 500 mL of reconstituted water). The obtained suspension showed oily insoluble drops on the surface, therefore it was mixed by a magnetic stirrer in dark conditions for about 48 hours 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 filtered stock solution and diluting them in aerated reconstituted water. A negative control without test item was tested.
The test was performed in 50 mL glass beakers filled with 40 mL of test medium. The beakers were covered with glass plates to reduce the loss of water and to avoid the entry of dust into the solutions. The test vessels were labelled with the ChemService study number and all necessary additional information to ensure unmistakable identification.
For each test concentrations and for the control, 20 daphnids were used divided into four replicates of five daphnids each. The daphnids were randomly distributed to the test vessels at initiation of the test. The daphnid’s loading rate was greater than one daphnid per 2 mL of test solution.
The test water was aerated prior to the start of the study until oxygen saturation was reached. During the test period, test water was not aerated.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

24 h

Dose descriptor:

IC50

Effect conc.:

41.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% Confidence range (mg/L): 37.1 – 45.6

Effect concentrations 4

Duration:

48 h

Dose descriptor:

IC50

Effect conc.:

20.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% Confidence range (mg/L): 19.4 – 21.5

Effect concentrations 5

Duration:

24 h

Dose descriptor:

LOEC

Effect conc.:

31.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 6

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

31.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

The daphnids were observed for immobility after 24 and 48 hours of exposure. In the negative control no immobilization was observed at the end of the test period and no daphnids were found to be trapped on the surface water. These values comply with the validity criterion of the test, that immobilization and/or a maximum number of daphnids trapped in to water surface should not exceed 10% in the control medium at the end of the test.
The NOEC and the LOEC values were determined from the Raw Data by statistical comparison with the control (Fisher’s Exact test with α = 0.05); CETIS v.1.026D software was used to carry out the statistical analyses. The 24 and 48h-IC50 were calculated by a non linear regression analysis. No remarkable observations were made concerning the appearance of the test medium. The test medium was a clear solution throughout the whole test duration. Light intensity was measured once a day during the test period, the values recorded were in the range 382-394 Lux.

Results with reference substance (positive control):

The results of the most recently performed test with a reference substance are reported and compared with published data to demonstrate that the daphnia clone used has got an adequate level of sensitivity to that substance. In the current case, the results refer to a test performed on May 2010 (CHE-I-004/2010) with potassium dichromate as reference item.

Effect of potassium dichromate on Daphnia magna:
ChemService study CHE-I -004/2010: 24 h IC50 (mg/L)=0.68 Confidence interval (95%, mg/L): 0.65 – 0.82
Reference data (UNI EN ISO 6341/99): 24 h IC50, Confidence interval (95%, mg/L): 0.6 – 2.1

Reported statistics and error estimates:

The CETIS v.1.026D software was used to carry out the statistical analysis; this programme automatically chose the more appropriate analysis for the pool of data.
The NOEC and the LOEC were determined from the Raw Data by statistical comparison with the control (Fisher’s Exact analysis with α = 0.05).
The IC50 was assessed by a non linear regression analysis for both observation times.
As input data the nominal concentrations of test item were used.

Any other information on results incl. tables

Lethal effect of 3,4, 5 trichloro-benzotrichloride toDaphnia magna

Nominaltest item concentration [mg/L]	No. of Daphnia tested	No. of immobilized Daphnia		% of immobilized Daphnia
		24 h	48 h	24 h	48 h
0 (control)	20	0	0	0	0
1.0	20	0	0	0	0
3.2	20	0	0	0	0
10.0	20	1	2	5	10
31.6	20	6	18	30	90
100.0*	20	20	-	100	-

* the test was stopped after 24 hours being all daphnids dead at that time.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of test item 3,4-dichlorobenzotrichloride was tested on Daphnia magna under static conditions.
The NOEC, LOEC and IC50 values after 24 and 48 h, calculated based on nominal concentrations were as follows:
24 h NOEC: 10.0 mg/L
24 h LOEC: 31.6 mg/L
24 h IC50: 41.3 mg/L (37.1 mg/L – 45.6 mg/L)
48 h NOEC: 10.0 mg/L
48 h LOEC: 31.6 mg/L
48 h IC50: 20.4 mg/L (19.4 mg/L – 21.5 mg/L)

Executive summary:

The acute toxicity of the test item 3,4,5-trichloro-benzotrichloride toDaphnia magnawas determined in a 48‑hour static test according tothe OECD Guideline for Testing of Chemicals, No. 202 (2004).

For this purpose, juvenile daphnids (< 24 hours old at test initiation) were exposed to an aqueous test medium containing the test item at five different concentrations in a geometric series, namely 1.0, 3.2, 10.0, 31.6 and 100.0 mg/L. The exposed organisms were checked for immobilization 24 and 48 hours after test initiation. For the highest concentration the test was stopped after 24 hours being all daphnids dead at that time. The pH of the control (test medium without test item) after preparation was 6.88. The pH of stock test solution (100 mg/L) immediately after preparation was 3.69, therefore it was corrected before the beginning of the test period by adding 10 drops of a NaOH solution (1N) achieving a pH equal to 6.34, within the range provided by OECD guideline (6-9).

The room temperature was in the range 21.3 – 21.7° C during the test period.

Light intensity during the 48 hours of test period was in the range 382-394 Lux, within the range 200-1500 Lux.

At the end of test period, in the negative control, no immobilization was observed and no daphnids were found trapped on the test water surface. These values comply with the specified validity criterion that immobilization in the negative control group should not exceed 10% at the end of the test. The obtained experimental results allowed to calculate the NOEC, the LOEC and the IC₅₀values at 24 and 48 h. The results are reported below:

 

Time	NOEC (mg/L)	LOEC (mg/L)
24 h	10.0	31.6
48 h	10.0	31.6

 

Time	IC50(mg/L)	95% Confidence range (mg/L)
24 h	41.3	37.1 – 45.6
48 h	20.4	19.4 – 21.5