Reaction mass of carbon tetrachloride and chloroform and 1,2-dichloroethane

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, OECD 201, with provisions for such a volatile compound ensuring growth and substance recovery

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Storage of samples: none.
Samples treatment: dilution with acetonitrile.

Vehicle:

no

Details on test solutions:

Medium according to Annex 3 of OECD 201 with HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ) buffered medium containing sodium hydrogenocarbonate as test is done in closed vials

Test organisms (species):

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

Details on test organisms:

Pseudokirchneriella subcapitata, CCAP 278/4 stock (previously named Raphidocelis subcapitata and Selenastrum capricornutum) are obtained from the Culture Centre of Algae and Protozoa (Ambleside, UK).
The conditions used for culturing algae are described in Annex 2 of the OECD 201 guideline. Two flasks, each containing approximately 100 mL of axenic stock culture of algae are incubated at 23 ± 1 °C under lighting (photoperiod: 16 hours of illumination, 8 hours of darkness), slowly continuously shaken.
These stock cultures are renewed every week, using two new cultures.
The quality of the stock culture was verified for the absence of micro-organisms and deformed cells under microscopic observation before use.
Three days before the beginning of the study two pre-cultures were prepared by inoculating each stock suspension of algae (5 mL) into sterile dilution water (500 mL). The pre-cultures were incubated under the same conditions as those used for the stock cultures.
At the beginning of the test, the cell concentration of the pre-culture was determined. The result was used to calculate the volume to be introduced into each test flask in order to get an initial cell concentration of 104 cells/mL.

The cell density of the pre-culture was about 1.58 x 106 cells/ml for the preliminary test and about 1.11 x 106 cells/ml for the definitive test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

NA

Test temperature:

23 +/- 1 °C

pH:

T=0h: between 7.33 and 7.44
T=72h: between 7.35 and 7.81

Dissolved oxygen:

T=0h: between 8.2 and 8.6 mg/L
T=72h: between 8.8 and 34.8 mg/L

Salinity:

NA

Nominal and measured concentrations:

Concentrations of CARBON TETRACHLORIDE

Nominal Concentration Measured in non-inoculated solutions
Initial Final Final/Initial
(mg/L) (mg/L) (mg/L) %
0 (T) < DL < DL -
5 2.21 2.21 100
8.5 3.35 3.56 106.3
14.6 6.88 7.32 106.4
25 14.0 13.9 99.3
42.7 24.9 23.7 95.2
73.1 47.0 48.9 104
125 114 117 102.6

Details on test conditions:

TEST SYSTEM
- Test vessel: 120 mL glass bottles hermetically closed with PTFE caps
- Fill volume: 50 mL in the range finding test; 120 mL in the definitive test (no headspace).
- Aeration: no
- Initial cells density: 10E4 cells/mL.
- Control end cells density: 1.55 10E7 cells/mL.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used: According to OECD GL 201 with addition of HEPES 1M buffer:
Add to 8 L of ultrapure water:
• 100 ml of stock solution 1 (10 mL/L)
• 10 ml of stock solution 2 (1 mL/L)
• 10 ml of stock solution 3 (1 mL/L)
• 10 ml of stock solution 4 (1 mL/L)
• 3 g of NaHCO3 (0.3 g/L final)
• 60 mL of HEPES buffer (6 mM final)

Make up to 10 L with ultrapure water.


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod and Light intensity and quality: constantly illuminated by 8 fluorescent tubes between 6,000 and 10,000 lux (Mazdafluor, white industry 33)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Determination of algae concentration was carried out using cytofluorimetry with a Cytofluor 2350 device in the range-finding test. In the definitive test, cells were numbered with a cell counter (Beckman Coulter Z2).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.71
- Range finding study
- Test concentrations: 100, 50, 10, 5, 1, 0.1 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 13-37 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 3.1-10 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL: 6.1-15 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL: 1.3-4.9

Details on results:

Calculations done with nominal concentrations as measured concentrations (both intial and final) were in the range: 95 - 106 % of nominal

Results with reference substance (positive control):

The sensitivity of the test system and the methodology are evaluated every two months by performing an algal growth inhibition test on potassium dichromate. The nearest values of ErC50 and EbC50 obtained on September 30 2010 were respectively 1.2 mg/L and 0.57 mg/L.
For information, ISO 8692 reports the following results for an inter-laboratory exercise on potassium dichromate: ErC50: 0.60 to 1.03 mg/L EbC50: 0.20 to 0.75 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

Growth inhibition of freshwater algae Pseudokirchneriella subcapitata by carbon tetrachloride was measured according to OECD Guideline 201. The following results are obtained: ErC50-72 = 20 mg/L, ErC10-72 = 6.3 mg/L and NOECr = 2.2 mg/L

Executive summary:

Growth inhibition of freshwater algae Pseudokirchneriella subcapitata by carbon tetrachloride was measured according to OECD Guideline 201. In order to take account of the volatility of the substance, the experiment was carried out in stoppered flasks with no headspace, and using a medium buffered with HEPES in order to avoid pH drift. Good recovery of the test substance was demonstrated through analytical measurement (GC/MS) and other validity criteria were met.

Consequently the values obtained in this study can be retained as key information about algae toxicity: ErC50-72 = 20 mg/L, ErC10-72 = 6.3 mg/L and NOECr = 2.2 mg/L

Description of key information

OECD 201 test (GLP) on carbon tetrachloride (Gancet, 2011):

EC50 (72h)=20 mg/L

NOEC (72h)=2,2 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

20 mg/L

EC10 or NOEC for freshwater algae:

2.2 mg/L

Additional information

No experimental study was conducted on the multiconstituent substance (Flux1). Instead, a constituent-based, Weight-of-Evidence approach, was performed.

The three major constituents were targeted, representing ca 95% of a typical composition (carbon tetrachloride (CAS n° 56-23-5),1,2-Dichloroethane (CAS n°107 -06 -2) and chloroform (CAS n° 67-66 -3)). Numerous published data were available, and a single consensus value was selected for each, from a reliable source.

No average key value was derived for the multiconstituent substance: as the Risk Assessment is conducted per constituent, individual values are the key data.

Growth inhibition of freshwater algae Pseudokirchneriella subcapitata by carbon tetrachloride was measured according to OECD Guideline 201 (Gancet, 2011). In order to take account of the volatility of the substance, the experiment was carried out in stoppered flasks with no headspace, and using a medium buffered with HEPES in order to avoid pH drift. Good recovery of the test substance was demonstrated through analytical measurement (GC/MS) and other validity criteria were met. Calculations done with nominal concentrations as measured concentrations (both initial and final) were in the range: 95 - 106 % of nominal

Consequently, the values obtained in this study can be retained as key information concerning algae toxicity of carbon tetrachloride: ErC50-72 = 20 mg/L and NOECr = 2.2 mg/L.