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Ecotoxicological information

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:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Principles of method if other than guideline:
Council Regulation EC 440/2008 (C.2)
GLP compliance:
yes (incl. certificate)
Remarks:
Italian Ministero della Salute

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 3,4-dichlorobenzotrichloride
- Substance type: intermediate for synthesis
- Physical state: from colourless to slightly yellow liquid with characteristic aroma
- Analytical purity: 99.8%
- Purity test date: March 10, 2010
- Lot/batch No.: 03/2010
- Expiration date of the lot/batch: March 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: Keep away from incompatible materials (oxidisers, not noble metals, amines and alcohols). Keep away from heat and ignition sources.

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
The test concentrations were chosen based on the results of a pre-test (range-finding test). 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 solution showed oily insoluble drops on 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.

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 the mean of a digital thermometer. The measured values ranged between 21.3 and 21.7 °C during the test period. A 16 hours light and 8 hours darkness daily photoperiod, with light intensity values in the range 382 -394 Lux was that of the test.
pH:
The pH of the control (test medium without test item) after preparation was 6.88 while the pH of stock test solution (100 mg/L) was 3.06, therefore it was corrected before the beginning of the test by adding 9 drops of a NaOH solution (1N) obtaining a pH value of 6.83 (within the provided range 6-9).
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 meeting the OECD guideline 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)
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.
Nominal and measured concentrations:
10.0, 17.8, 31.6, 56.2 and 100.0 mg/L.
Details on test conditions:
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 higher than one daphnid per 2 mL of test solution.
The daphnids were observed for immobility after 24 and 48 hours of exposure (those organisms not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile).
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
56.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
IC50
Effect conc.:
87.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: range 75.7 – 101.3
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
31.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
56.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
IC50
Effect conc.:
48.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: range 41.4 – 57.3
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 24 and 48h-IC50 values were calculated by a Trimmed Spearman-Karber method.
No remarkable observations were made concerning the appearance of the test medium.
The test medium was a clear solution throughout the whole test duration.
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 run on May 2010 (CHE-I-004/2010) with potassium dichromate as reference substance.
Effect of potassium dichromate on Daphnia magna:
24 h IC50 (mg/L) in ChemService study CHE-I-004/2010: 0.68, Confidence interval (95%, mg/L) : 0.53 – 0.82
24 h IC50 (mg/L) in Reference data UNI EN ISO 6341/99: 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 Trimmed Spearman-Karber method for both observation times.
As input data the nominal concentrations of product were used.

Any other information on results incl. tables

Lethal effect of 3,4-dichlorobenzotrichloride to Daphnia 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

10.0

20

0

0

0

0

17.8

20

0

0

0

0

31.6

20

0

2

0

10

56.2

20

0

14

0

70

100.0

20

13

18

65

90

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of test item 3,4-dichlorobenzotrichloride on Daphnia magna was tested under static conditions.
The NOEC, LOEC and IC50 values after 24 and 48 hours of exposure, calculated based on nominal concentrations were as follows:
24 h NOEC: 56.2 mg/L
24 h LOEC: 100.0 mg/L
24 h IC50: 87.5 mg/L (75.7 mg/L – 101.3 mg/L)
48 h NOEC: 31.6 mg/L
48 h LOEC: 56.2 mg/L
48 h IC50: 48.7 (41.4 mg/L – 57.3 mg/L)
Executive summary:

The acute toxicity of the test item 3,4-dichlorobenzotrichloride toDaphnia magnawas determined in a 48‑hour static test according to the OECD Guideline for Testing of Chemicals, No. 202 (2004) and to Council Regulation EC 440/2008 (C.2).

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 10.0, 17.8, 31.6, 56.2 and 100.0 mg/L.The exposed organisms were checked for immobilization 24 and 48 hours after test initiation.

The pH of the control (test medium without test item) after preparation was 6.88 while the pH of stock test solution (100 mg/L) was 3.06, therefore it was corrected before the beginning of the test by adding 9 drops of a NaOH solution (1N) obtaining a pH value of 6.83 (within the provided range 6-9). 

The room temperature was in the range 21.3 –during the test period, according to the OECD’s recommended range (20 ± 2 °C).

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 hours. The results are as reported below:

Time

NOEC (mg/L)

LOEC (mg/L)

24 h

56.2

100.0

48 h

31.6

56.2

 

Time

IC50(mg/L)

95% Confidence range (mg/L)

24 h

87.5

75.7 – 101.3

48 h

48.7

41.4 – 57.3