[No public or meaningful name is available]

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

GLP compliance:

yes

Remarks:

[C(97)186/Final]

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): C-4000
- Physical state: liquid
- Analytical purity: 86.3 +/- 0.7%
- Purity test date: 2008-07-09
- Lot/batch No.: 51V034K7
- Expiration date of the lot/batch: 2008-10
- Storage condition of test material: refrigerated

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

- Concentrations: 10, 32, 100, 320, 1000 mg/L
- Sample storage conditions before analysis: refrigerated

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test material could not be easily weighed to
prepare test solutions on a weight to volume basis. Therefore, based on whole product, test
substance amounts were corrected for the density of C-4000 and prepared on a calculated weight
to volume basis. The test substance was added by direct volume to appropriately-labeled contact
flasks and contact flasks were transferred to the constant temperature chamber. Test substance
volumes of 4.95, 15.8, 49.5, 158, and 495 µL were added to the 10, 32, 100, 320, and 1,000 mg
whole product/L test flasks, respectively. A nominal 1,000 mg whole product/L abiotic control
was also prepared.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: The microbial inoculum used in the test was activated sludge collected August 27, 2008 from
Aeration Basin #1 at the Columbia Wastewater Treatment Plant in Columbia, Missouri.
Approximately 20 liters of activated sludge was collected.
- Preparation of inoculum for exposure: Twelve, 250 mL centrifuge bottles were filled with activated sludge and
concentrated by centrifugation at 3,000 rpm for approximately five minutes using IEC CR 6000 and Beckman Model J2-21 centrifuges. The supernatant was decanted, and the bottles were refilled with activated sludge and centrifuged three more times. After the final centrifugation, the supernatant was decanted, and the activated sludge was rinsed by adding ABC well water, shaking, and centrifuging as above. The supernatant was then decanted. The activated sludge pellet in each bottle was washed two more times in the same manner. The final supernatant was decanted, and a suspension of the sludge pellet was prepared by adding 200 mL of well water to each bottle. The microbial inoculum contained within the bottles were shaken then combined in a carboy, placed on a stir plate in an environmental chamber maintained at 20 ± 2 ºC, and aerated while stirring using compressed air. A synthetic sewage feed solution composed of peptone, beef extract, urea, NaCl, MgSO4·7H2O, CaCl2·2H2O, and K2HPO4 was used to sustain the inoculum overnight. The pH of the prepared activated sludge was 8.10.

Study design

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

1 d

Remarks on exposure duration:

Results from the range-finder indicated the respiration inhibition from the test substance was < 50% at the nominal loading rates; therefore a definitive test was not necessary.

Test conditions

Hardness:

284 (2008-08-22)
286 (2008-08-28)

Test temperature:

19.9 (2008-08-22 )
20.3 (2008-08-28 )

pH:

7.23 (2008-08-22 )
7.65 (2008-08-28 )

Salinity:

634 (2008-08-22 )
640 (2008-08-28 )

Details on test conditions:

TEST SYSTEM
- Test vessel: 1000 mL glass flask
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, 1000 mL
- Aeration: stirred and aerated using compressed air at 500 to 1000 mL/minute
- No. of organisms per vessel: 200 mL of microbial inoculum was added to each flask, except the abiotic control
- No. of vessels per concentration (replicates): one
- No. of vessels per control (replicates): one


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: well water (non-chlorinated) was used for all inoculum preparation procedures, synthetic sewage feed preparation, and test flask preparations; Reagent water was used to prepare the reference substance stock solution
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium:
- Intervals of water quality measurement:


OTHER TEST CONDITIONS
- Light intensity: ambient light


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : oxygen consumption (respiration rate) and percent inhibition


TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 10, 32, 100, 320, and 1,000 mg whole product/L
- Results used to determine the conditions for the definitive study: Based on the results, a definitive test was not required and therefore not conducted.

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol

Results and discussion

Effect concentrations

Duration:

1 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Results with reference substance (positive control):

- Results with reference substance valid? yes; The percent inhibitions for the three reference substance flasks were 0, 9.4, and 65.3% at
3,5-dichlorophenol concentrations of 3.2, 10, and 32 mg/L, respectively. The estimated EC50 was 23.3 mg/L. This EC50 was within the 5 to 30 mg/L acceptability range as recommended by the OECD Guideline 209.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Since the percent inhibition at the highest concentration tested for C-4000 was less than 50%, the EC50 was reported as >1,000 mg whole product/L. The respiration rates of control flasks as well as the estimated EC50 values of the reference toxicant testing met the acceptability criteria outlined in OECD Guideline 209.

Executive summary:

A test was conducted to determine the effects of C-4000 on microorganisms by measuring the respiration rate under defined conditions in the presence of varying concentrations of the test substance. The microbial inoculum was exposed to nominal concentrations of 0 (control), 10, 32, 100, 320, and 1,000 mg whole product/L.

The results of this test showed that the respiration rates of the two control flasks were within 15% of the mean respiration rate which satisfies the criteria for acceptability outlined in the test guideline concerning data from the control flasks.

The test substance showed 0, 0, 0, 18.1, and 0% inhibition at test concentrations of 10, 32, 100,

320, and 1,000 mg whole product/L. Under the conditions of the study, the estimated EC50 for

C-4000 was >1,000 mg whole product/L based on the nominal loading rate. Based on these

results, a definitive test was not required and therefore not conducted.

The three reference substance flasks exhibited percent inhibition values of 0.0, 9.4, and 65.3% at

3,5-dichlorophenol concentrations of 3.2, 10, and 32 mg/L, respectively. An EC50 value was

calculated to be 23.3 mg/L for 3,5-dichlorophenol, which was within the acceptable value range

of 5 – 30 mg/L for the reference substance.