2-chlorotoluene

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to microorganisms, other

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

ISO 10712 (Water quality – Pseudomonas putida growth inhibition test (Pseudomonas cell multiplication inhibition test

Principles of method if other than guideline:

Cell multiplication inhibition test

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Prior to preparation of dilution series, the test item stock solution of known concentration was neutralized to pH 7.0.
Four parallel dulution series were prepared in 300 ml Erlenmayer flasks, stoppered with cotton-lined plastic caps. Each of the dilution contained one part (v/v) of the pollutant solution in 2^0 to 2^14 parts (v/v) of the mixture. The dilution series was prepared as follows: The first flask of each solution series was filled with 160 ml test substance stock solution, which was prepared with double-distilled water. From the first flask 80 ml stock solution was transferred to the second flask and 80 ml double-distilled water was added. The dilution scheme was applied to the following flasks.

Three of the four parallel dilution series (one was kept as control) were inoculated with 10 ml adjusted bacterial suspension (see details on test inoculum); 5 ml of stock solution I and 5 ml of stock solution II were added (for stock solution I and II see any other additional information on materials and methods). Controls were filled with 10 ml saline, 5 ml stock solution I and 5 ml stock solution II (for saline see any other additional information on materials and methods). The final volume of all flasks was 100 ml.

Test organisms (species):

Pseudomonas putida

Details on inoculum:

Laboratory stock cultures of Pseudomonas putida were kept on the nutrient medium in agar slant tubes (for nutrient medium see any other information on materials and methods). For the cell multiplication inhibition test, stock cultures of Pseudomonas putida were incubated at 25 °C for 24 h. After 24 h, the cell material was washed off with sterile saline. The extinction value of monochromatic radiation at 436 nm for a 10 mm layer was photoelectrically measured. By onward dilution with saline the extinction value was finally adjusted to a turbidity corresponding to the extinction value of a Formazin standard suspension TE/F/436 nm = 10. The adjusted bacterial solution was used as test inoculum.

Details on cultivation of stock cultures:
Stock cultures and preliminary cultures were kept on the same nutrient in agar slant tubes (change interval: 1 week). Inoculated stock cultures are incubated at 25 °C for 24 h.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

16 h

Test temperature:

25°C

pH:

7.0 (neutralized)

Details on test conditions:

TEST PRINCIPLE
The concentration of bacterial suspensions was determined by photometric measurements and expressed by the extinction value of the primary light of monochromatic radiation at 436 nm for a layer of 10 mm thickness. The concentration at which an inhibitory effect of the test substance occurred (TTC = toxicity threshold concentration) was derived on the basis of a calculated extinction value that was 3% below the mean value of extinction for all non-toxic dilutions in the test.

TEST SYSTEM
- Test vessel: 300-mL Erlenmeyer flask
- Type (delete if not applicable): stoppered with cotton lined plastic caps
- Fill volume: 100 mL
- Aeration: no, by shaking
- No. of organisms per vessel: cell density as extinction at 436 nm: the extinction value of the inoculum solution (see section "Details on inoculum") was finally adjusted to a turbidity corresponding to the extinction value of a Formazin standard suspension TE/F/436 nm = 10. 10 mL of this adjusted bacterial suspension was used as test inoculum.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 1

TEST PROCEDURE
The inoculated dilution series and the controls were incubated for 16 hours at 25°C. Afterwards, the extinction of monochromatic radiation at 436 nm in a 10 mm layer was measured for the inoculated dilution series. In case discolouration or turbidity occurred, respective controls were used as photometric blank value.

EVALUATION
Graphical evaluation of effect concentration were performed after the test using means of measured extinction values, i.e. the extinction mean of all non-toxic test solutions A (SD of < 3%) and the mean of the lowest observed effect concentration B. A horizontal line was plotted for the mean extinction value A and B on a semi-logarithmic scale. The coordinate of the highest observed non-toxic concentration P-NOEC and lowest observed toxic concentration P-LOEC were marked on the respective lines. A regression line was drawn from P-NOEC to P-LOEC. In order to determine the TTC (toxicity threshold concentration, adverse effect of ca. 3%) a horizontal line was plotted at the extinction value A-3%. From the intersection point between the horizontal line A-3% and the regression line from P-NOEC to P-LOEC the TTC was derived.

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Photoperiod: no data
- Light intensity: no data

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- growth (cell multiplication; determined turbidimetrically)

Key result

Duration:

16 h

Dose descriptor:

other: TT

Remarks:

TT = toxicity threshold concentration (approx. EC3)

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

TT = Toxicity threshold; determined at 3 % effect compared to control

Validity criteria fulfilled:

not applicable

Remarks:

Pre-guideline study

Conclusions:

A toxic threshold value (TT, ca. =EC3) of 15 mg/L was determined in a 16 h Pseudomonas putida growth inhibition test performed equivalent to ISO 10712.

Executive summary:

The effect of 2-chlorotoluene solutions on Pseudomonas putida was tested in a cell multiplication inhibition test over a test period of 16 hours (Bringmann 1977, 1979, 1980; equivalent to ISO 10712). The concentration of bacterial suspensions was determined by photometric measurements and expressed by the extinction value of the primary light of monochromatic radiation at 436 nm for a layer of 10 mm thickness. The concentration at which an inhibitory effect of the test substance occurred (TT = toxicity threshold concentration) was derived on the basis of a calculated extinction value that was 3% below the mean value of extinction for all non-toxic dilutions in the performed test. The derived TTC (16 h) for the test substance was 15 mg/L. In conclusion, any inhibition of the degradation activity of activated sludge microorganisms by 2-chlorotoluene is not anticipated when introduced in concentrations below this value.

Description of key information

The toxicity of 1 -chloro-2 -methylbenzene towards microorganisms was tested using Pseudomonas putida as test species. An EC3(16 h) of 15 mg/L was observed having the inhibition of cell multiplication inhibition as endpoint (Bringmann 1977, 1979, 1980; equivalent to ISO 10712).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

15 mg/L

Additional information

In the valid and reliable key study (Bringmann 1977, 1979, 1980) the effect of 2-chlorotoluene solutions on Pseudomonas putida was tested in a cell multiplication inhibition test over a test period of 16 hours (equivalent to ISO 10712). The concentration of bacterial suspensions was determined by photometric measurements and expressed by the extinction value of the primary light of monochromatic radiation at 436 nm for a layer of 10 mm thickness. The concentration at which an inhibitory effect of the test substance occurred (TT = toxicity threshold concentration) was derived on the basis of a calculated extinction value that was 3% below the mean value of extinction for all non-toxic dilutions in the performed test. The derived TT (16 h) for the test substance was 15 mg/L. In conclusion, any inhibition of the degradation activity of activated sludge microorganisms by 2-chlorotoluene is not anticipated when introduced in concentrations below this value.
Several further studies investigating adverse effects towards different protozoa are available reporting results either above 40 mg/L or above 80 mg/L (Bringmann 1978, 1980). However, those studies did not follow any accepted testing protocol, such that the reliability of the study outcome is not assessable. Further, the relevance of those test organisms for the activated sludge process is questionable. E.g., Blum & Speece (1991) investigated the effects towards methanogenic bacteria. The use of the inhibition of biogas production to estimate the risk of a substance to STPs with biological nutrient removal and thus its relevance for microbial processes in a STP is still under development (Guidance Document R.7b, ECHA, 2008). Hence, results of such tests solely may serve as supporting / additional information but cannot be used for the environmental hazard and risk assessment for the activated sludge process of sewage treatment plants.