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Toxicity to microorganisms

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toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Valid and conclusive non-GLP test according to a standard protocol which is in evaluation of a OECD guideline standard.
according to guideline
other: TETRATOX assay
equivalent or similar to guideline
other: Draft for an OECD protozoan test / Tetrahymena assay
GLP compliance:
Analytical monitoring:
Details on test solutions:
- Chemical name of vehicle: DMSO (CAS 67-68-5)
- Concentration of vehicle in test medium: <375 µL per 50 mL (<0.75%), which is considered not toxic the the test organisms
- Controls: Inoculated control without test material and true blank
Test organisms (species):
Tetrahymena pyriformis
Details on inoculum:
- Strain:GL-C obtained from John R. Kennedy, Department of Biochemistry, Cell and Molecular Biology, The University of Tennessee - Knoxville, U.S.A. More recently (1992), a second culture was obtained from Jorgen Larsen, Environmental Technology, Danish Technological Institute (P. O. Box 141, DK-2630, Taasturp, Denmark).
- Laboratory culture: Yes, since 1975
- Preparation of inoculum for exposure: Dilution in order to obtain a log-growth-phase
Test type:
Water media type:
Limit test:
Total exposure duration:
40 h
Test temperature:
buffered to pH 7.40
Details on test conditions:
- Test vessel: Erlenmeyer flasks
- Type: open, i.e. foam-stoppered
- Fill volume: 50 mL
- No. of organisms per vessel: Initial density of ca. 2500 (range 1000 to 5000) cells/mL
- No. of vessels per concentration (replicates): 3 times 2 (three independend experiments with two replicates each)
- No. of vessels per control (replicates): 3 times 2 inoculated (three independend experiments with two replicates each) and 3 times one true blank
- Sterile conditions: Yes

Distilled H2O 1000 mL
Proteose peptone 5 g
D-Glucose 5 g
Yeast extract 1 g
Tris - HCl 1.2114 g

10 mL each of the following salt solutions and pH to 7.35 with saturated NaOH

Salt 1 (chlorides):
100 mL H2O
0.5 g CaCl2.2H2O
0.05 g CuCl2.2H2O
0.0125 g FeCl3.6H2O

Salt 2 (sulfates):
100 mL H2O
1 g MgSO4.7H2O
0.25 g Fe(NH4)2(SO4)2.6H2O
0.005 g MnCl2.6H2O
0.0005 g ZnCl2

Spectrophotometry at 540 nm at the end of the test (in some cases, e.g. dyes electronic particle counting was employed)

According to range finding study the three main experiments were conduicted with a range from no effect to complete growth inhibition
Reference substance (positive control):
not required
- as a series of 250 compounds was tested
40 h
Dose descriptor:
Effect conc.:
1.74 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks on result:
other: Reported as log (IC50^-1) = -0.24
Details on results:
The authors conclude on other than baseline toxicity (i.e. specific mode of action)
Reported statistics and error estimates:
The IGC50 (i.e. the 50% inhibitory growth concentration) was calculated using probit analysis (dependent variable = normalized absorbance, independent variable = test item concentration in mg/L)
- Finney DJ (1971) Probit Analysis, 3d ed. Cambridge, UK. Cambridge University Press 333 p
Validity criteria fulfilled:
not applicable
Under the conditions of the test an IC50 of 1.74 mg/L for ciliate growth inhibition was determined. The authors conclude on a specific toxic mode of action, but there are no generally accepted rules for interpretation. Accordingly the mode of action considerations may be subject to further evaluation together with other information.
Executive summary:

A common freshwater cilliate (Tetrahymena pyriformis), which is considered representative for the protozoan community, was exposed during 40 h at 27±1 °C to 250 substances in order to determine the 50% inhibitory growth concentration (IGC50) according to the TETRATOX assay. The protocol is similar to the recent draft OECD guideline.

Description of key information

1.74 mg/L, 40h, Tetratox, Schulz 1997
47 mg/L, no inhibition, toxicity control, OECD 301A, Eisner 2010

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
47 mg/L

Additional information

Section R. of the Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7b, states that data from ciliate growth inhibition tests, preferably Tetrahymena, are relevant for the risk assessment for sewage treatment plants. As ciliates are mainly responsible for floc formation and settling properties, rather than for degradation processes, toxicity data on ciliates are considered to be supplementary to the data on activated sludge or specific bacterial strains, i.e. no correlation exists between activated sludge and ciliate test results, neither are ciliates consistently more sensitive. Data are also available from the toxicity control of a ready biodegradability test using activated sludge (Eisner 2010, see section 5.2.1).

As indicated in Table R.10-6 of the Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7b, both the tetrahymena pyriformis and ready biodegradability toxicity control test data can be used to derive a PNECstp along with suitable assessment factors.