Registration Dossier

Ecotoxicological information

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:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
Council Regulation EC 440/2008 (C.3)
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 concentration was chosen based on the results of a pre-test (range-finding test).
The study was carried out at the nominal concentration of 100 mg/L, moreover a negative control with algal culture medium only was also tested.
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 (0.0500 g of test item into 500 mL of algal culture medium). The obtained solution showed oily insoluble drops, 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.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The study was performed with the unicellular, fresh-water green algae Pseudokirchneriella subcapitata, 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:
yes
Total exposure duration:
72 h

Test conditions

Test temperature:
The test was incubated in a temperature-controlled room, under continuous illumination.
Temperature: the room temperature under the lights was measured twice a day by a digital thermometer. It was in the range 24.6 – 25.3 °C during the test period.
Light conditions: continuous illumination; light intensity values ranged between 4078 and 4156 Lux.
pH:
The pH of the test medium after preparation was 7.23 for test item and 7.96 for control.
Nominal and measured concentrations:
100.0 mg/L
Details on test conditions:
The treatment vessels were identified by experiment number, group and replicate number.
All the described procedures were conducted under a laminar flow bench to ensure the sterility of the algal cultures.
For the test concentrations and for the negative control, 100 mL capacity conical glass flasks capped with air permeable stoppers were used.
Three replicates for each test concentration and six for control were prepared. At the start of the test, 50 mL of test solutions were poured into the test flasks and inoculated with algae to obtain a starting cell density of around 10exp.4 cells/mL.The algal inoculum was taken from a pre-culture, started 3 days before the beginning of the test in order to ensure that the assay was performed with exponentially growing algal population. The cell density of the inoculum was around 10exp.6 cells/mL.
The test was incubated in a temperature-controlled room, under continuous illumination.



Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield rate
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 191. 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 32.9% and a coefficient of variation of average growth in replicates control cultures during the test period of 3.7%.
In the test solution the algal growth rate (r) and the yield (y) after 72 hours of exposure did not show any significant effects in comparison with negative control (Bonferroni adjusted test), on the contrary the test solution inoculum showed a growth quite higher than the control, as demonstrated by negative inhibition values.
The ExCx calculations were carried out using a linear interpolation analysis.
The EC10, the EC20 and the EC50 for each observation time and for both endpoints could not be determined, but they could be assessed as higher than the tested concentration 100 mg/L.
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 run on April 2010 (CHE-I-003/2010) with 3,5-Dichlorophenol.
Effect of 3,5-dichlorophenol on the growth of Pseudokirchneriella subcapitata:
ErC50 (mg/L): Chemservice study CHE-I-003/2010: 4.12 (3.32 -4.98)
ErC50 (mg/L): Literature data Reference ISO 8962 (2004):3.38 (2.08-4.68)
Reported statistics and error estimates:
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. The observed values were used to calculate the percentage inhibition of cell growth as yield and as growth rate in comparison to the control.

The statistical comparison with the control was assessed by Bonferroni adjusted test for all observation times and for both endpoints.
The EyCx and the ErCx values were calculated by a linear interpolation method.

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.
The CETIS v.1.026D software was used to carry out the statistical analyses, which automatically selects the most appropriated statistic analysis.

Any other information on results incl. tables

Effect of 3,4-dichlorobenzotrichloride on the growth of Pseudokirchneriella subcapitata

Nominal

test item concentration [mg/L]

replicate

Cell density (n. cells x 104/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

9.4076

8.2643

8.3027

8.4695

9.1593

8.7286

8.7220

62.6555

66.3055

71.1255

57.3325

54.6075

72.4655

64.0820

190.2024

186.9064

201.6264

184.8364

1924764

1913644

191.2354

100.0

A

B

C

D

E

F

Average value

1

1

1

1

1

1

1

4.5259

4.4439

4.7334

4.5702

5.5045

4.4335

4.7019

53.6893

44.2353

42.1873

37.1223

49.5383

24.4953

41.8780

211.8673

146.6333

222.2273

208.4543

188.2903

224.6553

200.3546

Algal growth rate and inhibition caused by 3,4-dichlorobenzotrichloride 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)

90.2

-----

86.6

-----

73.0

-----

100.0

64.4

28.6

77.2

10.8

73.5

-0.7

Algal yield and growth inhibition caused by 3,4-dichlorobenzotrichloride 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)

1912354

-----

100.0

2003546

-4.8

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The toxicity of test item 3,4-dichlorobenzotrichloride was tested on Pseudokirchneriella subcapitata.
The EC10, EC20 and EC50 for growth rate and yield were determined to be higher than the nominal tested concentration of 100.0 mg/L.
Executive summary:

The influence of the test item 3,4-dichlorobenzotrichloride on the growth of the green algal speciesPseudokirchneriella subcapitata, formerly known asSelenastrum capricornutum, was investigated in a 72-hour limit test according to the OECD Guideline No. 201, 2006 and to Council Regulation EC 440/2008 (C.3).

or this purpose, exponentially growing test algae were exposed for 72 hours to an aqueous test medium solution at the only nominal concentration of 100.0 mg/L, under defined conditions. Besides this concentration, 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.

 The pH of the test medium after preparation was 7.23 for test item and 7.96 for control.

The room temperature was in the range 24.6 – 25.3 °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 4078 - 4156 Lux (within the range 4000 - 6000 Lux).

 

In the negative control the cell density increased on average by a factor of 191. 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 (32.9%) and the coefficient of variation of average growth in replicates control cultures during the test period (3.7%).

In the test item solution, at the nominal concentration of 100.0 mg/L, no significant biological effects were shown in comparison with negative control for both the endpoints, yield and growth rate, after 72 hours of exposure (Bonferroni adjusted test).

Therefore, the 72-h EyC10,EyC20, EyC50, and 72-h ErC10,ErC20,ErC50, were determined to be higher than the nominal tested concentration (100 mg/L).