Phenol, paraalkylation products with C10-15 branched olefins ( C12 rich) derived from propene oligomerization, calcium salts, sulfurized, including distillates (petroleum), hydrotreated, solvent-refined, solvent-dewaxed, or catalyc dewaxed, light or heavy paraffinic C15-C50

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase conducted on 26th April 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study following GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

not specified

Details on sampling:

- Concentrations: The targeted test concentrations for the test substance were 1, 10, 100, 1,000, and 10,000 ppm (mg/L).

Vehicle:

not specified

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
No organic solvents were used to facilitate the dispersion of the test substance, which is low in water solubility. For concentrations 1,000 ppm or 10,000 ppm, the test substance was weighed directly into the test flasks with water. For lower concentrations (1 ppm, 10 ppm, and 100 ppm) the test substance was weighed onto a solid carrier (a 1"x1" Teflon coupon) and introduced to the test flask. The test mixtures were well agitated by air flow at all times, and for test flasks with 1,000 and 10,000 ppm concentrations, magnetic stirring was employed to ensure dispersion of the test substance.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

The inoculum used was the return sludge collected from a local waste water treatment plant (Mentor, Ohio). On return to the laboratory the sludge was centrifuged and the supernatant decanted. The sludge was then washed in water three times by re-suspension and centrifugation. A small amount of the washed sludge was dried and weighed. From this result one calculates the amount of wet sludge which must be suspended in water in order to obtain an activated sludge with a mixed liquor suspended solids level of 4 g/L (+ 10%). This level gives a concentration of 1.6 g/L in the test medium.

If the sludge could not be used on the day of collection, ~50 mL synthetic sewage was added to each liter of the activated sludge prepared as described above; this was then aerated overnight at room temperature. It was then kept aerated for use during the day. Before use the pH was checked to be within 6.0- 8.0 (using pH paper).

If the same batch of sludge was required to be used on subsequent days (maximum four days), a further 25-50 mL of synthetic sewage feed was added to each liter of the sludge at the end of each working day.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

No data

Test temperature:

Ambient temperature (not monitored)

pH:

The pH of the solution was in the range 7 to 8.

Dissolved oxygen:

No data

Salinity:

No data

Nominal and measured concentrations:

Measured: 1.6, 10.6, 103.2, 1040, 10,000 ppm

Details on test conditions:

The test vessels were 1 liter Erlenmeyer flasks. The water used was deionized. The air supply was 0.5-1.0 liters/minute, oil free. The measuring
vessels were 300 ml BOD bottles. An Orion dissolved oxygen probe-Orion digital analyzer was used as the oxygen meter. The nutrient solution was synthetic sewage feed. 3,5-Dichlorophenol (10, 20, 40 ppm) was employed as well as a negative control which consisted of inoculated medium
without test substance. The test material dose levels evaluated were 1.6, 10.6, 103.2, 1040, 10,000 ppm.

Each test material level was aerated for three hours. Positive control and a second untreated control mixtures were then prepared. After 3 hours each mixture was poured into a separate 300 ml BOD bottle and the respiration rate was measured by monitoring the dissolved oxygen level in each
mixture over a period of up to 10 minutes. The respiratory rates of the negative control, positive control and the test material mixtures were calculated as mg oxygen/liter hour. The inhibitory effect of the test material and positive control was then determined at each prepared concentration in
comparison to the two negative control mixtures. The NOEC was determined as the highest concentration of test material with an observed
inhibitory effect of less than or equal to 15%. The EC50 was determined by plotting percent inhibition vs concentration.

The test is considered valid if the following criteria are met:
1. The two control respiration rates are within 15% of each other.
2. The EC50 (3 hours) of 3,5-dichlorophenol is in the accepted range 5 to 30 mg/L.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 10 000 other: ppm

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 other: ppm

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

In order for the study to be considered valid the two negative control respiratory rates were required to be within 15% of each other and the 3 hour
EC50 of the positive control was required to be in the range of 5 to 30 mg/liter. The two negative control mixture respiratory rates were within 15% of each other and the 3-hour EC50 of the positive control mixture was between 10 and 20 mg/liter confirming the validity of the assay.

The test material did not exhibit significant toxicity towards the microbial population at test concentrations levels up to 1000 ppm. At 10,000 ppm
slight inhibition (26%) of microbial respiration was observed. The EC50 was estimated to be greater than 10,000 ppm and the NOEC was estimated tobe 1,000 ppm.

Results with reference substance (positive control):

The 3-hour EC50 of the positive control mixture was between 10 and 20 mg/liter.

Reported statistics and error estimates:

1. The respiration rate is calculated from the recorder trace as mg O2/L.hr. The portion of the respiration curve over which the respiration rate is measured should be linear.
2. The following equation is used to calculate the inhibitory effect of a test/ reference substance at a particular concentration
[1- 2Rs/(RC1 +RC2)] *100 = per cent inhibition where:
Rs = oxygen consumption rate at tested concentration of substance RC1 = oxygen consumption rate, Control 1 Rc2 = oxygen consumption rate, Control 2
3. Estimate the range of concentrations where EC50 value for the reference substance (3,5 dichlorophenol) resides in.
4. Determine the NOEC of the test substance as the highest test concentration with observed inhibition less than or equal to 15%.
5. Determine the EC50 of the test substance by plotting the "percent inhibition" employing a linear Y axis versus the concentration of the test substance on a logarithmic X axis. Visually fit a straight line through the apparent slope of the data points where concentration-effect was observed. (Since only two or three data points were involved in the line fitting, no statistical method was used.) The EC50 was the concentration of the test candidate that resulted in a 50% inhibition of respiration.

The test substance up to the concentration of 1,000 ppm, did not show significant toxicity toward the microbial population. At the concentration of 10,000 ppm, slight inhibition (26%) of the microbial respiration was observed. The EC50 was estimated to be higher than 10,000 ppm and the NOEC estimated to be at 1,000 ppm.

Validity criteria fulfilled:

yes

Conclusions:

The test substance up to a concentration of 1,000 ppm, is non-inhibitory to the microbial population. It slightly inhibits microbial respiration at a concentration of 10,000 ppm.

Executive summary:

The ecotoxicity of the test substance was evaluated using the activated sludge, respiration inhibition test (OECD guideline 209 cunducted to GLP). The substance, up to the concentration of 1,000 ppm, did not show significant toxicity toward the microbial population. At higher concentration, slight inhibition of microbial respiration was observed and the EC50 was estimated to be higher than 10,000 ppm.

Key value for chemical safety assessment

EC50 for microorganisms:

10 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

In the key study, the acute toxicity of the test material to activated sludge (domestic) was investigated. The study was conducted according to OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test).

This study is presented as the key information as the reliability rating for this study is 1, according to the criteria of Klimisch, 1997. The substance, up to the concentration of 1,000 ppm, did not show significant toxicity toward the microbial population. At higher concentration, slight inhibition of microbial respiration was observed and the EC50 was estimated to be higher than 10,000 ppm.

 

The supporting study (Goodrich, M & Teer, L., 1994) because only a summary was available and, therefore, it was Klimisch coded as a 4. The study was conducted according to the OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test) and GLP. The EC50 was calculated to be greater than 10,000 mg/L.